",isbn:"978-1-80356-495-1",printIsbn:"978-1-80356-494-4",pdfIsbn:"978-1-80356-496-8",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,isSalesforceBook:!1,isNomenclature:!1,hash:"2d409a285bea682efb34a817b0651aba",bookSignature:"Dr. Saeed El-Ashram, Dr. Guillermo Téllez and Dr. Firas Alali",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/11635.jpg",keywords:"PCR, Genotyping, ELISA, Cell Lines, 2D Culture, 3D Culture, PRRs, CD4 Responses, CD8 Responses, Behavior Manipulation, Parasite Cysts, Psychiatric Disorders",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"March 4th 2022",dateEndSecondStepPublish:"May 6th 2022",dateEndThirdStepPublish:"July 5th 2022",dateEndFourthStepPublish:"September 23rd 2022",dateEndFifthStepPublish:"November 22nd 2022",dateConfirmationOfParticipation:null,remainingDaysToSecondStep:"2 months",secondStepPassed:!0,areRegistrationsClosed:!1,currentStepOfPublishingProcess:3,editedByType:null,kuFlag:!1,biosketch:"Dr. El-Ashram's research focuses on apicomplexan parasites, such as Toxoplasma and Eimeria. He has more than 96 SCI publications, he acted as an academic editor, reviewer, and he holds several registered patents.",coeditorOneBiosketch:"Researcher in enteric health, most notably probiotics and their relationship to nutrition and disease protection in poultry as well as the design of avian enteric inflammation models for the study of the impact of diet and microbiome on growth and development.",coeditorTwoBiosketch:"My research focuses mainly on apicomplexan parasites, such as Toxoplasma Cryptosporidium, Eimeria, and minor on nematodes. Prof.Alali has more than 30 publications and he acts as a reviewer in many journals.",coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"209746",title:"Dr.",name:"Saeed",middleName:null,surname:"El-Ashram",slug:"saeed-el-ashram",fullName:"Saeed El-Ashram",profilePictureURL:"https://mts.intechopen.com/storage/users/209746/images/system/209746.png",biography:"Dr. Saeed El-Ashram is a professor at Foshan University, China, and Kafrelsheikh University, Egypt, and a research professor at Zhaoqing Dahuanong Biology Medicine Co., Ltd., China. Dr. El-Ashram\\'s research focuses on parasitic diseases. He has more than 100 journal publications to his credit. He is currently an academic editor and reviewer and holds several registered patents. The primary focus of his research is to understand how the animal immune system recognizes and responds to parasitic infections with and/or without a microbial community. Some are the causative agents of significant diseases in humans, such as toxoplasmosis, cryptosporidiosis, alveolar echinococcosis, and fascioliasis. Others are a substantial financial burden to food producers because of the effects these parasites have on domestic animals, for example, coccidiosis and cryptosporidiosis (livestock and poultry).",institutionString:"Foshan University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Foshan University",institutionURL:null,country:{name:"China"}}}],coeditorOne:{id:"73465",title:"Dr.",name:"Guillermo",middleName:null,surname:"Téllez",slug:"guillermo-tellez",fullName:"Guillermo Téllez",profilePictureURL:"https://mts.intechopen.com/storage/users/73465/images/system/73465.jpg",biography:"Guillermo Tellez-Isaias received his DVM and MS in Veterinary Sciences from the National Autonomous University of Mexico (UNAM), and his Ph.D. from Texas A&M University. He worked as a professor at UNAM for sixteen years, eight as head of the Avian Medicine Department, College of Veterinary Medicine. Dr. Tellez was president of the National Poultry Science Association of Mexico and is a member of the Mexican Veterinary Academy and the Mexican National Research System. Currently, he works as a research professor at the Center of Excellence in Poultry Science, University of Arkansas. 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1. Introduction
Snake venoms are a complex mixture of compounds with a wide range of biological and pharmacological activities, which more than 90% of their dry weight is composed by proteins, comprising a variety of enzymes, such as proteases (metalo and serine), phospholipases A2, L-aminoacid oxidases, esterases, and others [1-5]. A great number of proteins were purified and characterized from snake venoms [1, 2]. Some of these proteins exhibit enzymatic activity, while many others are non-enzymatic proteins and peptides. Based on their structures, they can be grouped into a small number of super-families based on remarkable similarities in their primary, secondary and tertiary structures, however showing distinct pharmacologic effects [3].
One of the most important protein super-families present in snake venoms are the phospholipases A2 (PLA2, E.C. 3.1.1.4), a class of heat-stable and highly homologous enzymes, which catalyse the hydrolysis of the 2-acyl bond of cell membrane phospholipids releasing arachidonic acid and lysophospholipids (Figure 1). These proteins are found in a wide range of cells, tissues and biological fluids, such as macrophages, platelets, spleen, smooth muscle, placenta, synovial fluid, inflammatory exudate and animal venoms. There is a high medical and scientific interest in these enzymes due to their involvement in a variety of inflammatory diseases and accidents caused by venomous animals. Since the first PLA2 activity was observed in Naja snake venom, PLA2s were characterized as the major component of snake venoms, being responsible for several pathophysiological effects caused by snake envenomation, such as neurotoxic, cardiotoxic, myotoxic, cytotoxic, hypotensive and anti-coagulant activities [1-10].
Phospholipases constitute a diverse subgroup of lipolytic enzymes that share the ability to hydrolyse one or more ester linkages in phospholipids, with phosphodiesterase as well as acyl hydrolase activity. The amphipathic nature of phospholipids creates obstacles for the enzymes, as the substrates are assembled into bilayers or micelles and are not present in significant amounts as a single soluble substrate [11]. According to Waite [12], all phospholipases target phospholipids as substrates, they vary in the site of action on the phospholipid molecule, their function and mode of action, and their regulation. Phospholipases function in various roles, ranging from the digestion of nutrients to the formation of bioactive molecules. This diversity of function suggests that phospholipases are relevant for life; the continuous remodelling of cell membranes requires the action of one or more phospholipases. The most common phospholipids in mammalian cells are phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). The plasma membrane of most eukaryotic cells contains predominantly PC and sphingomyelin in the outer leaflet, and PI, PE and PS in the inner leaflet [11].
Figure 1.
Phospholipase hydrolysis specificity sites in a 1,2-diacylglycerolphospholipid molecule (structure design from the ACD/l Lab. via Chem. Sketch – Freeware Version 1994 – 2009 software).
Phospholipases are classified according to their site of action in the phospholipid molecule. Thus, a phospholipase A1 (PLA1) hydrolyzes the 1-acyl group of a phospholipid, the bond between the fatty acid and the glycerine residue at the 1-position of the phospholipid. A phospholipase A2 (PLA2) hydrolyzes the 2-acyl, or central acyl, group and phospholipases C (PLC) and D (PLD), which are also known as phosphodiesterases, cleave on different sides of the phosphodiester linkage (Figure 1). The hydrolysis of a phospholipid by a PLA1 or a PLA2 results in the production of a lysophospholipid. The phospholipase metabolites are involved in diverse cellular processes including signal transduction, host defense (including antibacterial effects), formation of platelet activating cofactor, membrane remodeling and general lipid metabolism [12-14].
According to the latest classification [6], these proteins constitute a superfamily of different enzymes belonging to 15 groups and their subgroups including five distinct types of enzymes: the ones called secreted PLA2 (sPLA2), the cytosolic (cPLA2), the Ca2+ independent (iPLA2), the acetyl-hydrolases from platelet activating factors (PAF-AH) and the liposomal. The classification system groups these enzymes considering characteristics such as their origin, aminoacid sequence and catalytic mechanisms, among others.
The sPLA2s have a Mr. varying from 13,000 to 18,000, usually containing from 5 to 8 disulphide bond. They are enzymes that have a histidine in the active site and require the presence of the Ca2+ ion for the catalysis. Phospholipases A2 from the IA, IB, IIA, IIB, IIC, IID, IIE, IIF, III, V, IX, X, XIA, XIB, XII, XIII, XIV groups are representative of the sPLA2s. The cPLA2s are proteins with Mr between 61,000 to 114,000 that also use a serine in the catalytic site (groups IVA, IVB, IVC, IVD, IVE, IVF). The iPLA2s are enzymes which also use a serine for catalysis (groups VIA-1, VIA-2, VIB, VIC, VID, VIE, VIF). The PAF-AH are phospholipases A2 with serine in the catalytic site that hydrolyze the acetyl group from the sn-2 position of the platelet activating factors (PAF), whose representative groups are VIIA, VIIB, VIIIA, VIIB. The liposomal PLA2s are assembled in group XV and are enzymes with an optimum pH close to 4.5 that have preserved histidine and aspartate residues, suggesting the presence of the catalytic triad Ser/His/Asp and also a supposed sequence N-terminal sign and N-bond glycosylation sites [6].
With the discovery of a great variety of phospholipase A2 in the last decade and the present expansion of the research in the area, more PLA2s should be discovered yet. Phospholipase A2 found in snake venoms (svPLA2s) are classified into groups I and II. The phospholipase A2 from group I have two to three amino acids inserted in the 52-65 regions, called “elapid loop”, being isolated from the snake venoms of the Elapidae family (subfamily: Elapinae and Hydrophiinae). The ones from group II are characterized by the lack of the Cys11-Cys77 bond which is substituted by a disulphide bond between the Cys51-Cys133, and besides that had five to seven amino acids extending the C-terminal regions, being bound in snake venoms of the Viperidae family (subfamily Viperinae and Crotalinae) [15,16].
The myotoxic PLA2s of the IIA class have been subdivided in two main groups: The Asp49, catalytically active; and the Lys49, catalytically inactive. The essential co-factor for the phospholipase A2 catalysis Ca2+. The phospholipase A2 Asp49 require calcium to stabilize the catalytic conformation, presenting a calcium bond site that is constituted by the β-carboxylic group of Asp49 and the C=O carbonylic groups of the Tyr28, Gly30 and Gly32. The presence of two water molecules structurally preserved complete the coordination sphere of Ca2+ forming a pentagonal pyramid [9,15].
The catalytic mechanism of the PLA2-phospholipid involves the nucleophilic attack of a water molecule to the sn-2 bond of the phospholipid substrate (Figure 2). In the proposed model, the proton from position 3 of the imidazole ring of the His48 residue involved in a strong interaction with the carboxylate group of the Asp49 prevents the imidazole ring rotation to occur and keeps the nitrogen at position 1 of this ring, in an appropriate special position. A water molecule then promotes the nucleophilic attack to the carbon of the ester group of the substrate and, at this moment, the imidazole ring of the His48 receives a proton from the water molecule, favoring the reaction. Subsequently to the acyl-ester bond hydrolysis at the sn-2 position of the phospholipid, this proton is donated by the imidazole ring to the oxygen, which then forms the alcohol group of the lysophospholipid to be released together with the fatty acid [15,17].
The Ca2+ ion, coordinated by the Asp49 residue, a water molecule and the oxygen atoms from the Gly30, Trp31 and Gly32 (not shown), are responsible for the stabilization of the reactive intermediary [15].
Figure 2.
Schematic representation of the catalysis mechanism proposed for the PLA2s. Interaction of the residues from the catalytic site of sPLA2s and the calcium ion with the transition state of the catalytic reaction in which a water molecule polarized by the His48 and Asp99 residues binds to the carbonyl group of the substrate [18].
The substitution of the Asp49 residue by the Lys49 significantly alters the binding site of Ca2+ in the phospholipase A2, preventing its binding and resulting in low or inexistent catalytic activity. Thus, the Asp49 residue is of fundamental importance for the catalytic mechanism of the phospholipase A2. It is likely that this occurs due to its capability of binding and orienting the calcium ion, however, there is no relevant difference between Asp49 and Lys49 in relation to the structural conformation stability of these enzymes [9,15,19].
The absence of catalytic activity does not affect myotoxicity. Most snake PLA2s from the Bothrops genus already described are basic proteins, with isoelectric point between 7 to 10, showing the presence or absence of catalytic, myotoxic, edematogenic and anticoagulating activities [9,20].
On the other hand, acid PLA2s present in Bothrops snake venoms were not studied as well as basic PLA2s, resulting in little knowledge regarding the action mechanism of these enzymes [21-25].
PLA2s catalytic activity represents a key role in envenomation pathophysiology, however, recent studies have shown that some effects are independent of PLA2s catalytic activity, such as myotoxicity [19,26]. The absence of a tight correlation between PLA2 catalytic and non-catalytic activities, together with the diversity of biological effects produced by these proteins increases the scientific interest in the understanding of the structural basis of PLA2 mechanisms of action.
Evidences suggest that these activities can be mediated by interactions between PLA2s and endogen acceptors on the target cell membrane [27-29].
2. PLA2 purification
Snake venom components, obtained with high degree of purity, could be used for the understanding of the role of these components in the physiopathological processes resulted from poisoning, as well as biotechnological/nanotechnological applications. Hence, many purified PLA2s from snake venoms, as well as epitopes of these molecules, are being mapped in order to identify determinants responsible for the deleterious actions seen, as well as possible applications in biotechnological models.
New advances in materials and equipments have contributed with protein purification processes, allowing the obtaining of samples with high degree of purity and quantity. These advances have allowed process optimization, providing reduction of steps, reagents use and thus avoiding the unnecessary exposure to agents that may, in some way, alter the sample’s functionality or physical-chemical stability.
Thus, the selection of adequate techniques and chromatographic methods oriented by physical chemical properties and biological/functional characteristics, are of fundamental importance to obtain satisfactory results. The information pertinent to protein structure, such as the homology to others already purified, should be taken into consideration and could make the purification processes easier.
Ion exchange chromatography was introduced in 1930 [30] and still one of the main techniques used for protein purification. It has been extensively used in single step processes as well as associated to other chromatographic techniques. Ion exchange chromatography allows the separation of proteins based on their charge due to amino acid composition that are ionized as a function of pH.
Proteins with positive net charge, in a certain pH (bellow their isoelectric point), can be separated with the use of a cation exchange resin and on the other hand, proteins with negative net charge in a pH value above their isoelectric point, can be separated with an anion exchange resin.
Scientific publications have shown that the use of cation-exchange resins is a very efficient method to obtain PLA2s from bothropic venoms, particularly those with alkaline pH (Table 1). The versatility of this technique can be observed in the work done by Andriao-Escarso et al. [21] who compared the fractioning of many bothropic venoms. In this work, the venoms were fractioned in a column containing CM-Sepharose® (2 x 20 cm), equilibrated with ammonium bicarbonate 50 mM pH 8.0 and eluted with a saline gradient of 50 to 500 mM of the same reagent. Under these conditions, MjTX-I and MjTX-II from B. moojeni snake venom were co-purified (isoforms of PLA2 with pIs of 8.1 and 8.2 values, respectively). The same occurs with B. jararacussu venom, where the BthTX-I and BthTX-II were purified. However, the most expressive result was observed with B. pirajai venom, from which 3 isoforms of myotoxins, called as PrTX-I (pI 8.50), PrTX-II (pI 9.03) and PrTX-III (pI 9.16) were purified. In the above cases, it is important to note that the protein elution occurs always following pIs increasing value. In our lab we used this technique routinely in order to isolate myotoxins from bothropic venoms, which can be observed in the chromatograms shown in Figure 3.
Figure 3.
Chromatographic profile using CM-sepharose® Column 1ml (Hitrap) equilibrated with Tris 50 mM buffer (buffer A) and eluted with a linear gradient of Tris 50 mM/NaCl 1 M (buffer B) in pH 8.0.A. Chromatography of the crude venom from Bothrops brazili B. Chromatography of the crude venom from Bothrops moojeni C. Chromatography of the crude venom from Bothrops jararacussu. Absorbance read at 280 nm.,2,3,4,5 and 6 marks indicate the fractions corresponding to the PLA2s of each venom.
Table 1.
PLA2s isolated from American snake venoms and respective chromatographic methods used.
Some authors have proposed changes to the methodology described above. Spencer et al. [31] described the purification of BthTX-I with the use of Resourse S® (methyl-sulphonate functional group), equilibrated in pH 7.8 (phosphate buffer 25 mM). Sample elution was done in increasing ionic strength conditions (NaCl 0 to 2 M), under 2.5 ml/min flow. In this model, the BthTX-I was eluted in NaCl 0.42M with a high degree of purity. However, the chromatographic profile in the conditions tested differs significantly from the observed in other works that describe the fractioning of this venom. This difference is due to the resin composition. This is corroborated with data obtained in experiments performed in our lab, where the effect of pH in the separation of myotoxin isoforms from B. jararacussu venom was used, as shown in Figures 4. SDS-PAGE showed that fractions corresponding to myotoxins showed protein bands with apparent molecular mass compatible with PLA2s class II (Figure 5).
Figure 4.
Chromatographic profile of the B.jararacussu venom in CM-sepharose® column 1 ml (Hitrap) equilibrated with Tris 50 mM buffer (buffer A) and eluted with a linear gradient of Tris 50 mM/NaCl 1M (buffer B) in different pH conditions. A. pH 5.0 B. pH 6.0 C. pH 7.0 D. pH 8.0. Absorbance was read at 280 nm. Fractions numbered (1 to 8) indicate the fractions selected for SDS-PAGE analysis in order to confirm the presence of PLA2s (BthTx I e BthTx II).
Figure 5.
SDS Page analysis. Lines 1 and 2 (pH 5.0); 3 and 4 (pH 6.0); 5 and 6 (pH 7.0); 7 and 8 (pH 8.0). BthTx I was obtained in high degree of purity with pHs 5.0, 6.0, and 8.0. BthTx II was obtained with pH 7.0.
Resolution differences were also observed by other authors. As performed by Lomonte et al. [26], the isolation of two basic myotoxins, MjTX-I e MjTX-II, from the B. moojeni venom was obtained using CM-Sephadex C-25 equilibrated with Tris-HCl 50 mM pH 7.0 and eluted in saline gradient up to 0.75 M of Tris-HCl. Also, Soares et al. [33] described the isolation of MjTX-II with high purity using the combination of CM-Sepharose resin and ammonium bicarbonate buffer. According to the authors, the increase of pH to 8.0 has favored the elution of several fractions, allowing MjTX-II to be eluted separately with ionic strength equal to 0.35 M of ammonium bicarbonate. Moreover, the use of CM-Sepharose® seems to have also contributed a lot in the increasing of resolution for this chromatographic separation.
The combination of chromatographic techniques has also been used to purify these toxins. The association of the Ion-exchange chromatography and molecular exclusion has been one of the most recurrent in isolation and purification of phospholipases from bothropic venoms. Gel filtration chromatography is a technique based in particle size to obtain the separation. In this type of separation there is no physical or chemical interaction between the molecules of the analyte and the stationary phase, being currently used for separation of molecules with high molecular mass. The sample is introduced in a column, filled with a matrix constituted by small sized silica particles (5 to 10 µm) or a polymer containing a uniform net pores of which solvent and solute molecules diffuse. The retention time in the column depends on the effective size of the analyte molecules, the higher sized being the first ones to be eluted. Different from the higher molecules, the smaller penetrate the pores being retained and eluted later. Between the higher and lower molecules, there are the intermediary sized molecules, whose penetration capacity in the pores depends on their diameter. In addition to that, this technique has also some very important characteristics, such as operational simplicity, physical chemical stability, inertia (absence of reactivity and adsorptive properties) and versatility, since it allows the separation of small molecules with mass under 100 Da as well as extremely big molecules with various kDa.
The work performed by Homsi-Brandeburgo et al. [34] is a example of combination of different chromatographic techniques for the isolation of myotoxins with PLA2 structure. It describes for the first time the BthTX-I purification using the combination of molecular exclusion chromatography in Sephadex G-75® resin followed by Ionic exchange chromatography in SP-Sephadex C-25®. In the first step, four fractions were obtained, called SI, SII, SIII and SIV. The Functional analysis of these fractions showed that the proteolytic activity over casein and fibrinogen was detected on fraction SI, while the phospholipase activity was concentrated in fraction SIII. The apparent molecular mass profile of this fraction showed that it was composed by proteins between 12,900 and 28,800 Da, compatible with the mass profile of the class II PLA2s.
On the second step, SIII fraction was submitted to ionic exchange chromatography and five fractions were obtained, identified as SIIISPI to SIIISPIV. The pIs and apparent molecular mass evaluation showed the following profile: SIIIPI (pI 4.2 and 22.400 Da), SIIIPII (pI 4.8 and 15.500 Da), SIIIPIII (pI 6.9 and dimeric structure, each monomer with a molecular mass of 13.900 Da), SIIIPIV (pI 7.7 and 13.200 Da) e SIIIPV called BthTX-I that presented pI 8,2 and 12.880 Da. Pereira et al. [35] obtained the complete sequence of BthTX-II, a myotoxin homologous to the BthTX-I, which corresponds to the SIIISPIV fraction described by Homsi-Brandeburgo et al. [34].
Another chromatographic technique regularly used in PLA2s purification procedures is the Reverse-phase associated with High performance liquid chromatography (RP- HPLC). This technique is characterized by its high resolution capacity and is normally used in a more refined step of the purification process, being very useful in separating isoforms. The retention principle of reverse-phase chromatography is based in hydrophobicity and is mainly due to the interactions between hydrophobic domains of the proteins and the stationary phase. This technique has many advantages, such as: use of less toxic mobile phases together with lower costs, such as methanol and water; stable stationary phases; fast column equilibrium after mobile phase change; easy to use gradient elution; faster analysis and good reproducibility.
Rodrigues et al. [36] described the isolation of two PLA2s isoforms from the B. neuwiedi pauloensis venom using the combination of ion (cation) exchange chromatography and molecular exclusion setting up a preparative phase. Subsequently, a reverse-phase chromatography was used for the analytical phase of the procedure. Initially, the venom was fractioned in a column containing CM-Sepharose® equilibrated with ammonium acetate solution 0.05 M, pH 5.5 and eluted in linear gradient up to 1 M of the same buffer, resulting in six fractions. The pH, more acid than the ones used in the work previously mentioned, has increased the surface residual charge, intensifying the interaction force between the protein and the resin, thus altering the elution profile when compared to the performed by Rodrigues et al. [37]. Proceeding with purification, the sample with phospholipase activity (S-5) was submitted to a new fractioning in a Sephadex G-50® column yielding 3 fractions, of which the denominated S-5-SG-2 showed catalytic activity. It was then submitted to RP- HPLC in C18 column to obtain toxins with high purity degree.
Also, with the use of a multiple step procedure [38] successfully isolated two isoforms of PLA2s from B. leucurus venom. After a first molecular exclusion chromatography using Sephacryl S-200®, 7 fractions were obtained, from which the named “P6” showed to be composed by proteins with apparent molecular mass bellow 30 kDa, and a major fraction of approximately 14 kDa concentrated the phospholipase activity. This fraction was re-chromatographed in a Q-Sepharose® resin (ion exchange) and equilibrated with Tris-HCl 20 mM pH 8.0, yielding 6 fractions. The fraction corresponding to the negatively charged fraction was eluted without significant interaction with the resin, hence with a positive residual charge (basic pI) was selected, showing to be a homogeneous fraction of 14 kDa and presenting phospholipase activity. This fraction was submitted to a RP- HPLC in C4 column, yielding as result two major fractions with close hydrophobicity (eluted with 33% and 36% acetonitrile) and apparent molecular mass of 14 kDa.
Myotoxins with PLA2s structure from bothropic venoms that have acid pI have being more difficult to isolate. Different from cation exchange resins (CM Sepharose®, Resource S® and CM Sephadex®), anion exchange resins have not been so efficient in the separation of components from bothropic venoms, which requires, complementary steps to obtain these toxins with a satisfactory purity degree, as shown in Table 1.
Daniele et al. [32] described the fractioning of the B. neuwiedii venom using a combination of double molecular exclusion chromatography followed by anion exchange chromatography. The first step of the molecular exclusion chromatography was done using Sephadex G-50® where a single fraction with PLA2s activity was eluted. This fraction was re-chromatographed in Sephacryl S-200® resin, yielding 2 active fractions. The first fraction was re-chromatographed in Mono Q® column (functional group quaternary ammonium) yielding a PLA2s named P-3. From the second fraction, submitted to the same chromatographic procedure, two other PLA2s isoforms were isolated, named P-1 and P-2. Although showing different behavior over the molecular exclusion resin, the three isoforms showed very close apparent molecular mass (15 kDa) when assayed by SDS-PAGE. This difference could be resulted from differential interactions of aromatic residues located on the protein surface with the stationary phase [40, 41] and can be also verified in other acid PLA2s, like the one obtained from B. jararacussu venom by Homsi-Brandeburgo et al. [34].
Other procedures used hydrophobic interaction chromatography to isolate these PLA2s. This is a method that separates the proteins by means of their hydrophobicity: the hydrophobic domains of the proteins bind to the hydrophobic functional groups (phenyl and aryl) of the stationary phase. Proteins should be submitted to the presence of a high saline concentration, which stabilize then and increases water entropy, thus amplifying hydrophobic interactions. In the presence of high salt concentrations, the matrix functional groups interact and retain the proteins that have surface hydrophobic domains. Hence, elution and protein separations can be controlled altering the salt or reducing its concentration.
Santos-Filho et al. [42], working with B. moojeni venom, applied three sequential steps to obtain BmooTX-I, a PLA2 with apparent molecular mass of 15 kDa and pl 4.2. In this work, the crude venom was chromatographed in DEAE-Sepharose® (Dietylaminoetyl) resin, equilibrated with ammonium bicarbonate 50mM, pH 7.8 and brought to a saline gradient of 0.3M of the same salt. A fraction named E3 showed phospholipase activity, being then submitted to molecular exclusion chromatography in Sephadex G-75® resin. Three fractions were obtained, from which one named S2G3 was submitted to hydrophobic interaction chromatography in Phenyl-Sepharose® resin, the BmooTX-I being eluted in the end of the process.
In a work published in 2011, Nunes et al. [43] described the isolation of an acid phospholipase named BL-PLA2, obtained from Bothrops\n\t\t\t\tleucurus through two sequential chromatographic steps. On the first step, the acid proteins were separated from the others with the use of a cation exchange column (CM-Sepharose®) equilibrated with ammonium bicarbonate, pH 7.8. The acid fraction (eluted without interaction with the resin) was lyophilized and applied to a Phenyl-Sepharose CL-4B® column (1 x 10 cm), previously equilibrated with a Tris-HCl 10mM buffer, NaCl 4M, pH 8.5. The elution occurred under decreasing NaCl gradient in a buffered environment (Tris-HCl 10 mM, pH 8.5), concluding the process in an electrolyte free environment. An enzymatically active fraction (BL-PLA2), (with pI 5.4 and apparent molecular mass of approximately 15 kDa) was obtained at the end of the process.
The bioaffinity chromatography differs from others chromatographic methods because it is based in biological or functional interactions between the protein and the ligand. The nature of these interactions varies, being the most used those which are based on the interactions between: enzymes and substrate analogous and inhibitors; antigens and antibodies; lectins and glycoconjugates; metals and proteins fused with histamine tails. The high selectivity, the easiness of performance together with the diversity of ligands that can be immobilized in a chromatographic matrix make this method a useful tool for the purification of phospholipases. Based on the neutralization of myotoxic effects of the venom from B. jararacussu by heparin [44-46], the use of a column containing Agarose-heparin® could be used for the purification of myotoxins. They also ratify the interactions between heparin and myotoxin through the reduction of many biological effects, such as: edema induction, myotoxicity (in\n\t\t\t\tvivo) and cytotoxicity over mice myoblasts culture (L.6 – ATCC CRL 14581) and endothelial cells.
Following this strategy, Soares et al. [26] described the purification of BnSP-7, a myotoxin Lys-49 from B. neuwiedi pauloensis, with the use of chromatographic process based in this heparin functionality, which corroborates previous results obtained by Lomonte et al. [46], that showed the efficient inhibitory activity of heparin against myotoxicity and edema induced by myotoxin II, a lysine 49 phospholipase A2 from Bothrops asper. Also in this study, it was possible to infer the participation of the C-terminal region of the protein in the damaging effects on the cytoplasmic membrane.
Snake venom components share many similar antigenic epitopes that can induce to a crossed recognition by antibodies produces against a determined toxin. In this context, Stabeli et al. [47] showed that antibodies that recognize a peptide (Ile1-Hse11) from Bm-LAAO present crossed immunoreactivity with components not related to the LAAOs group present in venoms from Bothrops, Crotalus, Micrurus e Lachesis snake venoms. Also, Beghini et al. [48] showed that the serum produced against crotoxin and phospholipase A2 from Crotalus durissus cascavella was able to neutralize the neurotoxic activity produced by B. jararacussu venom and BthTX-I.
Based on this information, pertinent to the crossed immunoreactivity existent between venom components, Gomes et al. [49] described the co-purification of a lectin (BJcuL) and a phospholipase A2 (BthTX-1) using a immunoaffinity resin containing antibodies produced against the crotoxin. 20 mg of crotoxin was solubilized in coupling buffer (sodium bicarbonate 100 mM, NaCl mM, pH 8.3) and incubated overnight at 4 °C with 1 g of Sepharose® activated by cyanogen bromide (CNBr). After washing with the same buffer, the resin was blocked with Tris-HCl 100 mM buffer. This resin was packed and thoroughly washed with saline phosphate buffer (PBS) pH 7.4. Crotalic counter-venom hiperimune horse plasma (20 mg) was applied over the resin at a flow of 10 mL/hr and re-circulated overnight through the column. Then, it was washed until the absorbance went back to basal levels, showing that the material was retained (IgG anti-Ctx), then eluted with glycin-HCl 100 mM pH 2.8. The IgG anti-Ctx was then immobilized in CNBr activated Sepharose® resin through a procedure analogous to the above cited, generating a new resin called Sepharose-Bound Anti-CtxIgG. 20 mg of the crude venom from B. jararacussu was applied over this resin, yielding two fractions: the first, composed by proteins that were not recognized by the immobilized antibodies and a second fraction composed by components of venom from B. jararacussu that reacted crosswise with the Anti-Ctx antibodies, called Bj-F. A posterior analysis of this fraction, done by mass spectrometry, amino-terminal sequencing by Edman degradation and search by homology in the NCBI protein data bank, showed that it was composed by lectin and BthTX-I.
Different authors used substrate analogous or reversible inhibitors coupled to the chromatographic resin. Rock and Snyder [50] were the first ones to use phospholipid analogous to build a bioaffinity matrix [Rac-1-(9-carboxy)-nonil-2-exadecilglycero-3-phosphocoline]. In addition to them, Dijkman [51] described the synthesis of an analogous of acylamino phospholipid[(R)-1-deoxy-1-thio-(ω-carboxy-undecyl)-2-deoxy- (n-decanoylamino)-3-glycerophosphocholine] which was coupled to a Sepharose 6B® resin containing a spacer arm. With the use of this resin it was possible to purify phospholipases from horse pancreas, and venoms from Naja melanonleuca and Crotallus adamanteus.
3. Characterization
Venomic can be defined as an analysis in large scale of the components present in the venom of a certain species. In this context, the proteomic approach has allowed a better understanding of the venom components, through the application of many instruments that enables the analysis of their expression, structure, pos-traductional modifications and classification by homology or function. An approach developed by Calvete [52] for the analysis of snake venomic consists in an initial fractioning step of the crude venom using RP - HPLC, followed by characterization of each fraction by a combination of amino-terminal sequencing, SDS-PAGE, IEF or 2DE and mass spectrometry to determine molecular mass and cysteine content. Additionally, the modern venomic analysis use techniques such as Peptide Mass Fingerprint and the search for sequence similarity in data banks.
SDS-PAGE is a method related to the migration of charged particles in a medium under the influence of a continuous electric field [53]. From the electrophoretic point of view, the most important properties of the proteins are molar mass, charge and conformation. Mono dimensional polyacrylamide gel electrophoresis permit the analysis of the protein in its native or denatured form. In the first case, there are no alterations in conformation, biological activity and between protein subunits. This system is called non-dissociating or native, which proteins are separated based on their charge, using the isoelectric focusing method (IEF), or else, in vertical gel without SDS. During the IEF, a pH gradient is formed and the charged species move through the gel until they reach a specific pH. In this pH, the proteins have no effective charge (known as protein pI). The IEF shows high resolution, being able to separate macromolecules with pI differences of just 0.001 pH units [54, 55]. In dissociating or denaturing systems, the proteins are solubilized in buffer containing the reagent used to promote protein denaturation. SDS-PAGE, originally described by Laemmli [56], is an electrophoresis technique in polyacrylamide gel (PAGE) that used SDS as a denaturing agent, with interacts with the proteins giving them negative charges, allowing them to migrate, through a polyacrylamide gel towards a positive electrode a be separated by the differences related to their mass
Teixeira et al [25] described the purification of an acid phospholipase from B. pirajai (BpirPLA2I). As a biochemical characterization step, polyacrylamide gel electrophoresis in denaturing conditions (SDS-PAGE) was done. Using this approach, carried out in reducing and non-reducing conditions, the author could infer that the purified protein had the form of a monomer with apparent molecular mass of 14 kDa, both in reducing conditions as well as in non-reducing conditions the proteins presented the same mass, being confirmed afterwards by mass spectrometry.
Moreover, Torres [57] fractionated B. marajoensis venom using a cationic ion exchange column followed by an analytical phase in RP- HPLC, obtaining a phospholipase BmarPLA2 that was submitted to SDS-PAGE in reducing conditions showing apparent molecular mass of 14 kDa. However, in non-reducing conditions, the author observed the appearance of a single band at 28 kDa, concluding that BmarPLA2 was a dimeric structured protein joined by disulphide bridges. Thus, the above-cited examples demonstrate the importance of this procedure (SDS-PAGE) as a protein characterization step.
The determination of the isoelectric point is another important biochemical characterization of phospholipases A2. Previous studies involving phospholipases from snake venoms have shown that the acid phospholipases are catalytically more active than their basic isoforms [22, 42, 58]. Therefore, many authors have included, as a biochemical characterization parameter, the determination of the isoelectric point of the by isoelectric focusing. Due to pI determination importance, Teixeira [25] used the methodology proposed by Vesterberg and Eriksson [59] to evaluate pI of BpirPLA2-1. In order to obtain the pI value, a 7% polyacrylamide gel was prepared and polymerized over a glass plate of 12 x 14 cm using a U shaped rubber as support. A millimeter plate was previously greased with glycerin for better refrigeration of the gel. Two strips of Pharmacia Biotech were used to connect the gel and the platinum electrodes. The cathode was in contact with NaOH 1 M solution and the anode was in phosphoric acid 1 M. The platinum electrodes were centered over the paper strips and the system was then closed. The high voltage source was adjusted to the maximum values of 500 V, 10 mA, 3 watts and 30 minutes for a pre-run. Following, the samples were applied always in the intersection of two blue lines, exactly over the more central line of the gel. Then the source was programed for 1500 V, 15 mA, 10 watts and 5 h. The end of the run was determined when the source showed a high voltage and low amperage (around 1 mA). After isoelectric focusing, about 1 cm width (lengthwise) were sliced from each extremity of the gel and placed in test tubes containing 200 µL of distilled water for the pH reading after 2 hours of rest. Next, the pH gradient determination graph was plotted. The remaining gel containing the samples was fixed in solution of trichloroacetic acid for 30 minutes, followed by silver staining.
Another important technique as a step to characterize components from snake venoms is the bidimensional electrophoresis (2D). This one was initially developed by O\'Farrell [60]. The original methodology consisted of the preparation of polyacrylamide cylindrical gels, in which a pH gradient was established through a pre-run with specific amphoterics (also called ampholytes), that present high buffering capability in pHs close to their isoelectric points (pIs). The proteins were then submitted to an isoelectric focusing (IEF) and subsequently to an electrophoresis in the presence of SDS by a conventional system described by Laemmli [56]. Then, proteins were separated in the first dimension according to their pIs (IEF) and in the second dimension based on their molecular mass (SDS-PAGE).
Bidimensional electrophoresis is laborious, time consuming and difficult to be reproduced in different laboratories and depended on the ability of the researcher to obtain consistent results. Nowadays, many of these problems were solved with the development of new technologies. An important advance which has contributed to the increase of the 2D electrophoresis reproducibility was developed by Gorg [61] of the strip form gels with immobilized pH gradient (IPG - immobilized pH gel). The strips are made by the copolymerization of acrylamide with the Immobiline® (Amershan Biosciences/GE Heathcare) reagent, which contains acid and alkaline buffering groups. Another important technological progress was the improvement of the protein samples preparation methods, together with the discovery of new non-ionic detergents, such as CHAPS surfactants and SB 3-10, used with reducing agents adequate for IEF, like Dithiothreitol (DTT) and Tributyl Phosphine (TBP). Studies performed by Herbert [62] demonstrated that these advances had strongly contributed to the solubilization of a greater number of proteins to be analyzed in bidimensional electrophoresis.
The proteomic analysis of snake components has made use of the 2D electrophoresis as a tool, due to its high-resolution capability that allows, in a single process, the determination of apparent molecular mass and isoelectric point of the venom constituents. Fernandez et al. [22] described the determination of the isoelectric point and apparent molecular mass of Basp-PLA2-II using this technique. In order to do it, the protein was focused in IPG Immobiline® Dry Strip of 7 cm and pH 3-10, under a 200 V tension for 1 min, followed by a second stage of 3500 V for 120 minutes. The second dimension was done in SDS-PAGE 12% and then subsequently dyed with Coomassie blue. It was demonstrated that Basp-PLA2 –II had a pI of 4.9, which is close to the theoretical isoelectric point value (pI 5.05) defined by the primary sequence, evaluated using the Compute pI/MW tool (www.expasy.ch/tools) software and apparent molecular weight between 15 and 16 kDa, consistent with the molecular weight (MW 14,212±6 Da) obtained by ESI/MS (Electrospray Ionization/Mass Spectrometry).
The advantage of this technique is the high resolution. Alape-Giron [63] working with B. asper venom, performed an ontogenic analysis and an analysis based on the snake’s capture location in different regions of Costa Rica. Using tryptic digestion, MALDI-TOF mass fingerprinting analysis and aminoacid sequencing by MALDI-TOF submitted to similarity search by BLAST, the author showed the intra-specific variability in venom composition. It was hence evidenced that among the venoms obtained from adult species collected in the Caribe area and the Pacific area, there are around 30 proteins that are found in a snake group from a place which find no correspondents in the other.
In our lab, this technique has been used as follows: The proteins are separated by the isoelectric point in 13 cm strips with pH values varying between 3 and 10 in a nonlinear form. These strips contain polyacrylamide gel, where the gradient pH is formed by the presence of ampholytes. To re-hydrate the strips, 250 μL of sample [400 μg of proteins plus re-hydration solution (7 M of urea, 2 M of Thiourea, 2% of Triton X-100 (v/v/), 1% of IPG Buffer® (v/v) and DTT)] is applied in a channel of the apparatus over which the strips are set. The strip’s gel is re-hydrated at room temperature for about 12 hours. After this period, the strips are taken to the focusing system in the following conditions: (1) 500 V step until accumulates 500 Vh; (2) 500 to 1000 V gradient until it accumulates 800 Vh; (3) 1000 to 8000 V gradient until it accumulates 11300 Vh and (4) 8000 V step until it accumulates 3000 Vh. In average, the program run during 5.5 hours, but the time of the final step can be lengthened, if the sample does not reach to the end of the strip during the running according the initial program, it could be confirmed by a bromophenol blue line. At the end of focusing, the strips are equilibrated in two steps. On the first, 10 mL of the solution containing 6 M of urea, 2% of SDS (m/v), 30% of glycerol (v/v), 75 mM of Tris-HCl (pH 8,8), 0,002% of bromophenol blue and 1% DTT (m/v) for each strip is used. In the second, the same solution is used, but DTT is replaced by 2.5% of iodoacetamide (m/v). Each strip equilibrium step run during 15 minutes, under light stirring. Following that, the strips are applied on 10 % polyacrylamide gels previously prepared on 180 X 160 X 1.0 mm plates. After each strip and the standard stay appropriately accommodated in the polyacrylamide gel, a 0,5% agarose (m/v) heated (40 °C) solution is added. The agarose polymerization, provides an effective contact between the strip and the gel, thus avoiding the appearance of air bubbles. Protein
Figure 6.
Electrophoretic profile in 2D-PAGE 10%, 13 cm strip pH 3-10 non-linear of proteins from crude venom from Bothrops moojeni. Molecular weight (MW) –Color Plus Prestained Protein Marker – Broad Range (7-175 kDa) (P7709S). Coomassie G-250.
separation, according to molecular mass, is done by applying 25 mA per gel and 100 W during approximately 5.5 hours. After this period, the gel is washed with deionized water. Then, the proteins are fixed using a solution containing acetic acid 10% (v/v) and ethanol 40% (v/v) during one hour. Then, the fixing solution is removed and the gel is washed again with deionized water 3 times during 10 minutes. The proteins present in the gel are exposed using traditional methods for protein coloring, such as Coomassie blue or Silver nitrate. An example of the practical application of this methodology can be seen in Figure 6.
4. Functional characterization
Many biological activities are related to myotoxins with PLA2 structure obtained from snake venoms. In bothropic snake bite accidents and in experimental models with the use of these venoms, the noxious activity induced by these toxins on the striated muscles is striking [64]. The detection of the myotoxic activity associated to the phospholipase activity detection (in the case of Snake venom PLA2 Asp49) is used as an important auxiliary biological marker in the purification procedures, monitoring its presence.
The myotoxic activity assay can be done in two ways: in vivo and in vitro. The analysis can be done through the quantification of the released intracellular enzymes activity to the periphery blood or to the supernatant of the culture medium of cellular lineages. There are two main enzymes used to this end:
Creatine Kinase (EC 2.7.3.2): is a dimeric protein formed by the combination of subunits (B and M) and in its cytosolic form is found in many tissues, especially in skeletal muscle tissue (CK-MM), cardiac (CK-MB) and in the brain (CK-BB).
Lactate dehydrogenase (EC 1.1.1.27): is an enzyme widely distributed in many tissues and organisms. It is presented in the form of homo or hetero tetramers of subunits M and H, being present in muscular tissue in the homotetrameric form of subunit M.
In vivo, the CK activity quantification in murine models has been the most used to assay the presence of myotoxic PLA2, especially due to their low cost, ease of performance and high specificity as skeletal muscular tissue lesion markers when exposed to myotoxins.
As for the In vitro assays, myoblast lineages C2C12 (ATCC CRL-1772), differentiated until the formation of myotubules, have been used as models to assay the cellular toxicity, through the quantification of LDH levels in the supernatant of cell cultures exposed to toxins.
Regarding the phospholipase activity detections, it can be done by direct and indirect methods. Directly, it is possible to detect the presence of PLA2s with the use of chromogenic substrates, such as 4N3OBA(4-nitro-3-octanoyloxybenzoic acid) that induce the formation of detectable product at 425 nm [65] and fluorescent substrates (NBD) coupled to phospholipids that are used to quantitively and qualitatively survey the PLA2s activity isolated from snake venom [23].
Indirectly, the approach used consists in the potentiometric assay of the fatty acids released after the enzymatic hydrolysis of the phospholipids, through the quantification with standard alkaline solution [66]. Moreover, fatty acids released by the enzymatic degradation can be quantified through the alteration of the optical density of the pH indicator solution, such as phenol red [67], brilliant yellow [68] and bromothymol blue [69]. Another indirect method to assay PLA2 activity present in samples consists in the detection of hemolysis induced by lysophospholipids derived from phospholipids submitted to enzymatic digestion. This can be done through the quantification of hemoglobin present in solution or through the visualization of hemolytic halo in agarose matrix with immobilized red blood cells.
4.1. In vivo assay of the myotoxic activity
Mice is used for the in vivo assay of the myotoxic activity. Swiss males weighing between 18 g and 22 g, kept in controlled environment (12 h in the light and 12 h in the dark), with food and water ad libitum up to the moment of use. PBS solubilized sample and control (PBS) are filtered through 44 µm pores immediately prior to use. Reagents for CK activity dosage are prepared and used according to manufacturer’s instructions.
A Sample (50 µL) or control (50 µL) will be injected in mice gastrocnemic muscle using adequate device in order to guarantee a precise volume control. After a time lap (3 and/or 6 h), blood sample is collected in heparinized tubes and centrifuged to separate plasma. CK concentration is determined according to manufacturer’s instructions and expressed in U/L, where one unit corresponds to the production of 1 mmol of NADH per minute [26,70-72].
4.2. In vitro myotoxic activity assay
In order to assay myotoxic in vitro activity, myoblast lineage cells are used, such as murine skeletal muscle C2C12 myoblasts (ATCC CRL-1772) as described by Lomonte et al. [73], cultivated in modified Dubelco Eagle medium, supplemented with 1% bovine fetal serum. PBS solubilized sample, negative control (PBS) and positive control (Triton X-100) should be filtered through 22 µm pore filters immediately prior to use. Reagents for LDH activity dosage are prepared and used according to manufacturer’s instructions.
In 96 well plate, 2X105 cells/150 µL are set, sample and/or control (50 μL) are incubated in humid atmosphere at 37 °C and 5% CO2 for a 3 hour period. Afterwards, collect supernatant aliquot and quantify LDH activity released by cells with cytoplasmic membrane integrity compromised, according to manufacturer’s instructions and expressed in U/I, where one unit corresponds to the production of 1 mmol of lactate per minute.
5. Phospholipasic activity
5.1. 4N3OBA Substrate enzymatic hydrolysis
Phospholipase A2 activity can be measured according to the technique described by Holzer and Mackessy [65], modified for 96 wells plate [74].
Prepare aliquots of 100 µL of 4N3OBA 0.1% solution in acetonitrile and lyophilize. Keep the aliquots at -20° C until ready to use. The color reagent is prepared solubilizing the contents of one aliquot of 4N3OBA in 1ml of reagent containing Tris 10 mM, CaCl2 10 mM, NaCl 100 mM, and pH 8.0. For the test in micro plates, add 180 µL of color reagent and 20 µL of sample or water (blank), incubate the mixture at 37 °C for 5 minutes, measuring the optical density at 425 nm and 600 nm (to correct sample turbidity) at 30 second intervals. The activity will be expressed according to the equation (1) where 1 unit of phospholipase activity corresponds to the production of 1 µmol of 4-nitro-3-hydroxy-benzoic acid per minute.
5.2. Enzymatic hydrolysis of fluorescent substrates (NBD)
The phospholipase activity can also be assayed with the used of chromogenic substrates, using acyl-NBD reagents: NBD-PC (Phosphatidylcholine), NBDPG (phosphatidylglycerol), NBD-PE (phosphatidylethanolamine) or NBD-PA (phosphatidic acid). A solution of fluorescent lipids should be previously prepared in a 1 mg/ml concentration in chloroform. 100 µL aliquots are distributed and then dried under nitrogen flow. The dried lipid will be solubilized in 1 ml of NaCl 0.15 M and sonicated until the obtention of a limpid solution. For the test, the lipids should be diluted in a solution containing Tris-HCl 50 mM, CaCl2 1 mM pH 7.5. Initially, incubate the solution at 37 °C and, after 2 minutes, make an initial reading, configuring the equipment for excitation at 460 nm and emission at 534 nm. Following, apply the sample and make a second reading after 12 minutes. The change in fluorescence intensity is converted to nanomoles of product per minute (nmoles/min) using a calibration curve, prepared by hydrolyzing completely a substrate solution through sodium hydroxide treatment. The fluorescence intensity unit was converted to nmoles/min [33].
5.3. Potentiometric titration of fatty acids
The phospholipase activity can be assayed by potentiometric titration as described by de Haas [75], using as substrate an egg yolk emulsion in the presence of sodium deoxycholate 0.03 M and CaCl2 0.6 M. Fatty acids released enzymatically are titrated with a standard solution of NaOH 0.1 N at pH 8.0 at room temperature. The phospholipase activity is generally done with different concentrations of toxin, and calculated per amount of microequivalents of alkali consumed per minute, by mg of protein. One unit of phospholipase activity can be defined as the quantity of enzyme that releases 1 μmol of fatty acid per minute, in the reaction conditions.
5.4. Phenol red
The spectrophotometric detections of phenol red solution, induced by the increase of free fatty acids concentration can also be used to assay the phospholipase activity in samples, as described by Radvanyi [67].
In order to use this technique, prepare the reagent solution containing Phosphatidylcholine 0.25% (w/v) TritonX-100 0.4% (v/v), phenol chloride 32 mM. In a thermostatic cuvette at37 °C, add 1mL of reagent solution and 10 µL of sample. After stabilization for 20 seconds, determine the optical density measuring at 558 nm for 3 minutes, in kinetic intervals of 15 seconds. One unit of phospholipase can be defined as the quantity of enzyme necessary to convert 0.001 UA 558 nm per minute.
5.5. Indirect hemolysis
In this test, phospholipids (from egg yolk, soy lecithin or other sources) are used as substrates, with the production of fatty acid and corresponding lysophospholipids.These lysophospholipids have membrane activity over red blood cells, producing hemolysis that can be detected through the quantification of hemoglobin present in solution or through a hemolysis halo present in agarose gel containing intact red blood cells [76].
For the test, collect blood in a heparinized tube, wash the red blood cells with PBS, centrifuging at 800 xg for 5 minutes and prepare the suspension at 3%. Prepare solution containing phosphate buffer 20 mM, sodium chloride 100 mM and CaCl2 10 mM, erythrocyte suspension 3% (1:30 v/v) and egg yolk solution 0.1% (1:30 v/v). Add 10 µL of sample or PBS (control 0%) or Triton X-100 0.1% (control 100%) and incubate at 37 °C for 30 minutes. Then, centrifuge, collect the supernatant and determine the optical density at 405 nm, using PBS as blank. The results will be expressed in % of hemolysis compared with the positive control.
Hemoglobin dosage present in solution with the use of the Drabkin reagent (potassium ferrocyanide in buffered environment) [77] can be done by comparing the optical density of the samples with the standard curve made with the hemoglobin cyanide solution, according to manufacturer instructions.
To assay the hemolytic activity in agarose gel, carefully heat the suspension containing agarose 2% in PBS until complete fusion. After partial cooling (45 °C), add an equal volume of PBS containing CaCl2 0.02 M; egg yolk suspension (1:30 m/v), erythrocytes washed in PBS (1:30 m/v), pouring over Petri plate until the formation of a layer approximately 2 mm thick. After solidification of the gel, orifices of uniform diameter (0.2 cm diameter) to apply the sample are made. The gel is incubated for 12 hours, at 37 °C and humid environment. The formation of a translucid halo around the gel application point is indicative of phospholipase activity, contrasting with the rest of the gel which remains with a reddish tone due to the presence of integral red blood cells retained in the gel net.
The authors are grateful to Professor Luiz Hildebrando Pereira da Silva, George A. Oliveira, Marjorie J. M. Nascimento and Rafaela D. Souza by the assistance and to the Ministry of Science and Technology (MCT), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/MCT), Financiadora de Estudos e Projetos (FINEP/MCT), Fundação de Tecnologia do Acre/Fundo de Desenvolvimento Científico e Tecnológico (Funtac/FDCT), Secretary of Development of the Rondônia State (PRONEX/CNPq), Instituto Nacional para Pesquisa Translacional em Saúde e Ambiente na Região Amazônica (INCTINPeTAm/CNPq/MCT), and Rede de Biodiversidade e Biotecnologia da Amazônia Legal (Rede Bionorte/MCT) for the financial support.
\n',keywords:null,chapterPDFUrl:"https://cdn.intechopen.com/pdfs/40385.pdf",chapterXML:"https://mts.intechopen.com/source/xml/40385.xml",downloadPdfUrl:"/chapter/pdf-download/40385",previewPdfUrl:"/chapter/pdf-preview/40385",totalDownloads:4438,totalViews:654,totalCrossrefCites:0,totalDimensionsCites:10,totalAltmetricsMentions:0,impactScore:3,impactScorePercentile:89,impactScoreQuartile:4,hasAltmetrics:0,dateSubmitted:"December 21st 2011",dateReviewed:"September 7th 2012",datePrePublished:null,datePublished:"October 24th 2012",dateFinished:"October 19th 2012",readingETA:"0",abstract:null,reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/40385",risUrl:"/chapter/ris/40385",book:{id:"2327",slug:"chromatography-the-most-versatile-method-of-chemical-analysis"},signatures:"Rodrigo G. Stábeli, Rodrigo Simões-Silva, Anderson M. Kayano, Gizeli S. Gimenez, Andrea A. Moura, Cleópatra A. S. Caldeira, Antonio Coutinho-Neto, Kayena D. Zaqueo, Juliana P. Zuliani, Leonardo A. 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Kayano",slug:"anderson-m.-kayano",email:"akayano@gmail.com",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:null},{id:"177382",title:"Dr.",name:"Leonardo de Azevedo",middleName:null,surname:"Calderon",fullName:"Leonardo de Azevedo Calderon",slug:"leonardo-de-azevedo-calderon",email:"leonardo.calderon@fiocruz.br",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/177382/images/system/177382.jpg",institution:{name:"Oswaldo Cruz Foundation",institutionURL:null,country:{name:"Brazil"}}}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. PLA2 purification",level:"1"},{id:"sec_3",title:"3. Characterization",level:"1"},{id:"sec_4",title:"4. Functional characterization",level:"1"},{id:"sec_4_2",title:"4.1. In vivo assay of the myotoxic activity",level:"2"},{id:"sec_5_2",title:"4.2. In vitro myotoxic activity assay",level:"2"},{id:"sec_7",title:"5. Phospholipasic activity",level:"1"},{id:"sec_7_2",title:"5.1. 4N3OBA Substrate enzymatic hydrolysis",level:"2"},{id:"sec_8_2",title:"5.2. Enzymatic hydrolysis of fluorescent substrates (NBD)",level:"2"},{id:"sec_9_2",title:"5.3. Potentiometric titration of fatty acids",level:"2"},{id:"sec_10_2",title:"5.4. Phenol red",level:"2"},{id:"sec_11_2",title:"5.5. Indirect hemolysis",level:"2"},{id:"sec_13",title:"Acknowledgement",level:"1"}],chapterReferences:[{id:"B1",body:'KordisD.GubensekF.2000Adaptive evolution of animal toxin multigene familiesGene14352'},{id:"B2",body:'MenezA.1998Functional architectures of animal toxins: a clue to drug design Toxicon 1115571572'},{id:"B3",body:'KangT. S.GeorgievaD.GenovN.MurakamiM. T.SinhaM.KumarR. P.KaurP.KumarS.DeyS.SharmaS.VrielinkA.BetzelC.TakedaS.ArniR. K.SinghT. P.KiniR. M.2011Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis.FEBS J. 2345444576'},{id:"B4",body:'CalveteJ. J.JuarezP.SanzL.2007Snake venomics. 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1. Introduction
Organic fertilizers are a highly diverse family of products used in agriculture for soil improvement and to provide nutrients. Their characteristics and benefits will depend on their origin and processing, as on how they are used or combined in particular contexts [1, 2, 3, 4, 5]. The main common denominator is therefore that organic fertilizers provide a sustainable option to avoid the negative impacts of chemical fertilizers for long term soil fertility [6], decrease vulnerability to climate stress and weather variability, while reducing the impacts of agriculture on the environment [7, 8].
The term ‘organic fertilizers’ refers to a very wide range of products, as do the terms chemical, inorganic or synthetic fertilizers. It is therefore exceedingly difficult to make sweeping generalisations concerning the respective benefits or characteristics of these types of fertilizers. The task becomes all the more challenging, since outcomes will depend on numerous factors. These include how the fertilizer matches soil characteristics, crops, climatic and topographical questions, landscape characteristics, but also irrigation and tilling practices, time and manner of application of the fertilizer, as well as details concerning source and manner of producing the fertilizer. Undesirable effects may result from inappropriate fertilizer production processes, and the presence of metals and other contaminants in source materials is a major concern [9, 10]. There are also challenges linked to the overall or local availability of source materials.
Using organic matter to improve soils is not only related to fertility, but also to effects on physical, chemical and biological soil properties, including aeration, permeability, water-holding capacity and nutrient preserving capacity [11]. Benefits will depend on the exact type of organic fertilizer used, as well as on soil characteristics [7, 11]. Organic fertilizers can be used alone, or in combination with other fertilizers. For instance, a study under experimental conditions suggests that under deficit irrigation conditions, a combination of chemical fertilizer with vermicompost produced better results than chemical fertilizer alone [12]. The use of organic fertilizers appears particularly interesting in conditions of stress and weather variability, while a tailored combination with micro-nutrients suitable for crop and soil enhances yields (see e.g., Parmar et al. [13]). However, much of the literature on fertilizers reduces outcome to the question of crop yield rather than resilience, and more specifically short-term gains in crop yield under normal circumstances.
The use of synthetic fertilizers was generalised as part of the so-called green revolution [14, 15], which stood for a vision of modernising agriculture through use of agricultural machinery, synthetic fertilizers, pesticides, and systematic improvement of crop varieties. The ambition was to dramatically increase food production, and thereby alleviate hunger globally, so the focus on short term crop yield is therefore not surprising. The vision of the green revolution was also very much part of an industrial paradigm, with a simplified vision of agriculture as resembling other industrial production processes, with a flow consisting of input and output, controlled process, and output, where success was measured in production units. Today, however, we have come to a realisation that this oversimplification brought with it a very high cost to the environment, human health, as well as a degradation of planetary conditions necessary for food production in the long term. Crop yields remain important, of course, but there are other implications of our choice of agricultural practices that equally need to be considered. While much of agronomical research investigates linear correlations between a small set of isolated factors under relatively stable conditions, Hou et al. [16] argue for the need to consider soil health holistically, dynamically and from an interdisciplinary perspective.
Besides the narrow focus on productivity, the industrial paradigm within which agriculture was placed has tended to favour a comparatively linear and mechanistic understanding, while disregarding the complexity of ecosystems below ground, above ground, and in water bodies. Soil exchanges gases and chemical substances with air, and aerosols from erosion, burning and vegetation affect cloud formation, precipitation and greenhouse effects [17, 18, 19]. Also, as farmers have always known, weather is highly unpredictable, and far from the controlled conditions that industrial production supposes. In view of current rapid climate change [20], farmers are facing increasing weather variability, a greater number of extreme events, and a greater extent of uncertainty with respect to future developments [21, 22]. The use of organic fertilizers alone is not sufficient to address these challenges but can, in combination with other sustainable agricultural practices, constitute an important ingredient in farmers’ climate adaptation and mitigation strategies.
2. Agriculture in the Middle East and North Africa
Soil types, crops and trade patterns vary considerably across the Middle East and North Africa (MENA) region [23], but all countries are affected by water scarcity. The region comprises arid, semi-arid and hyper-arid areas, but even comparatively water-rich countries are affected by severe water stress [24], caused in part by economic incentives to cultivate water-intensive crops. Crop choice therefore plays an important role [25]. The water crisis is aggravated by deterioration of water quality caused by pesticides and nutrient runoff [26, 27], while groundwater is impacted by leaching and excessive pumping [28, 29]. Rural flight and decline of rural populations in several countries, such as Iran and Turkey [30] can reflect reduced need for labour due to mechanisation but may also reflect insecure livelihoods and difficult conditions of farmers [31, 32], while rural populations are also affected by displacement caused by disasters related to extreme weather, including forest fires, flooding and crop failure. The region is heavily dependent on imports of cereals. Both price fluctuations and transitions away from hydrocarbons globally will lead to decline in hydrocarbons exports on which many states of the region depend, affecting their ability to ensure food security through imports [23]. However, vested interests in exploiting hydrocarbons for the production of petrochemicals for agricultural use, as well as the existence of major phosphate deposits are likely to influence national economic diversification policies.
Large parts of the Middle East and North Africa are affected by protracted conflicts, internally displaced populations, and high volatility [33, 34]. Political and economic crises are affecting access to food, clean water and energy for large population groups [35], while agriculture is impacted by rising costs of fertilizers, pesticides, fuel and machinery, combined with disruptions to infrastructure and processing, storage and distribution systems for agricultural produce. These challenges will increasingly be aggravated by climate change [36, 37, 38, 39, 40, 41] and environmental degradation. Consequently, resilient food systems and food security will become issues of major concern for the region [42, 43], highlighting the question of climate adaptation strategies for farmers [31, 44, 45, 46].
Research on organic fertilizers in the MENA region from an environmental perspective is as yet relatively limited. Thus, a Scopus search on October 14, 2021, with the search term ‘organic fertilizers’ yielded 517 articles and reviews in English concerning agricultural sciences in the MENA region for the period 2017–2021, compared to 6558 worldwide for the same period. Publications in this field were dominated by Iran, Iraq, Egypt and Turkey (92%). Only 102 (20%) of the 517 MENA publications related to environmental or earth and planetary sciences. Within these 102, a mere 5 directly dealt with water-related issues, (including keywords such as irrigation, water quality, water stress, arid regions or groundwater), and none of the overall 517 publications on organic fertilizers mentioned climate adaptation or mitigation. In view of the interrelated urgent challenges that climate change and food security pose for the region, I will therefore draw on the international literature, to situate the use of organic fertilizers with respect to these challenges.
3. Environmental impacts of agriculture
Climate and environmental impacts of fertilizer use and soil management practices include not only emissions and pollution from production of fertilizer [47], but also those linked to the mechanised and chemical-intensive agricultural production systems they are associated with, impacts of nutrient runoff and chemicals [48, 49] on receiving water bodies, as well as impacts connected to food processing, storage, transport and waste. Effects on the world’s oceans are concerning. Unsustainable land use poses a threat for climate and biodiversity [20, 36, 50]. Agricultural land use and soil management practices are from a climate and environmental perspective of relevance for carbon storage [51], but also with respect to nutrient runoff, and persistent chemicals, and to emissions of N2O and CH4 [52]. According to the IPCC, the use of fertilizers has increased nine-fold since 1961 [53], and soil management accounts for half of greenhouse gas (GHG) emissions of the agricultural sector [54].
3.1 IPCC estimates of climate impacts and mitigation potentials
No global data are available specifically for agricultural CO2 emissions, and there is considerable uncertainty concerning net balance of CO2 land-atmosphere exchanges. However, land is an overall carbon sink, with a net land-atmosphere flux from response of vegetation and soils of −6 ± 3.7 GtCo2yr (averages for 2007–2016). The capacity of land to act as a carbon sink is expected to decrease as an effect of global warming. The major impacts of agricultural land use (food, fibre and biomass production) on CO2 (5.2 ± 2.6 GtCo2yr) are connected to deforestation, drainage of soils and biomass burning rather than to the net flux balance directly caused by different fertilization practices. Numbers regarding CO2 emissions from land use can be compared to net global anthropogenic CO2 emissions, which are estimated at 39.1 ± 3.2 GtCo2yr. In addition to land use impacts, agriculture causes CO2 emissions in the order of 2.6–5.2 GtCo2yr through activities in the global food system, including grain drying, international trade, synthesis of inorganic fertilizers, heating in greenhouses, manufacturing of farm inputs, and agri-food processing [55].
Agricultural land use directly represents 40% (4.0 ± 1.2 GtCo2eq yr) of total net global anthropogenic CH4 emissions, and represents 79% (2.2 ± 0.7 GtCo2eq yr) of total net global N2O emissions. CH4 emissions are mainly caused by ruminants and rice cultivation. Half of N2O emissions are caused by livestock, and the rest mainly by N fertilization (including inefficiencies). Total average net global GHG emissions (CO2, CH4 and N2O) for all sectors 2007-2016 are estimated at 52.0 ± 4.5 GtCo2eq yr, of which agriculture directly contributes with 17-22% (not including impacts of agriculture on land available for forests), or 21-37% (including agricultural land expansion and other contributions of the food system) [55]. Importantly, agricultural soil carbon stock change is not included in these statistics. Irrigation and agricultural land management contribute to making forests vulnerable to fires, while desertification [37] amplifies global warming through release of CO2, but such emissions as well as impacts from runoff on net fluxes from wetlands, water bodies and oceans are not included in the above figures.
Although net GHG emissions are often converted to CO2 equivalents for accounting purposes, different gases remain in the atmosphere for different periods of time and will consequently have different impacts on the progression of global warming. The specific proportions of GHG will affect the likelihood of crossing critical thresholds and tipping points, setting off cascades (cf. Lenton et al. [56]) with ecosystem collapse and mass extinctions, while driving biophysical processes that further aggravate the dynamics. Effects of mitigation measures also have varying timelines.
The creation of reactive N in agriculture has significant environmental impacts [57], and excessive application of nitrogen can increase nitrous oxide emissions without improving crop yields [54]. On average, only 50% of N is used, but in countries with heavy N fertilization the efficiency can be much lower, and the potential for mitigation therefore increases [7, 36]. Use of fertilizer is responsible for more than 80% of N2O emissions increase since the preindustrial era [58]. Ruminant livestock is the overall main source of CH4 from agricultural practices [55, 59], and among organic fertilizers cattle manure has therefore been widely studied. Rice cultivation makes the greatest contribution to CH4 emissions from agricultural soils [60]. Both water logging and soil compaction also contribute to CH4 emissions [61].
4. Climate mitigation potentials in agriculture
In view of the imminent threat to planetary life systems posed by climate change [20, 56, 62], research has in recent years accelerated on potentials for carbon offsetting and impacts on GHG emissions of different land use and management systems [63, 64, 65, 66, 67], as well as with respect to climate adaptation [68] and food security [69, 70]. Large areas of the MENA-region are exposed to desertification, including relatively water rich countries. For instance, at least half of Turkey is affected [37]. Desertification amplifies global warming through the release of CO2 linked with the decrease in vegetation cover, GHG fluxes, sand and dust. In dry areas, net carbon uptake is about 27% lower than elsewhere, reducing the capacity of land to act as a carbon sink. A rise in temperatures accelerates decomposition, at the same time that moisture is insufficient for plant productivity. Further SOC is lost due to soil erosion. An estimated 241–470 GtC is stored in the top 1 m of dryland soils [37]. In 2011, semi-arid ecosystems in the southern hemisphere represented half of the global net carbon sink [37].
Integrated sustainable practices are essential for climate adaptation, but estimates with respect to mitigation potentials vary. The chapter on interlinkages in the IPCC special report on climate change, desertification, land degradation, sustainable land management, food security, and greenhouse gas fluxes in terrestrial ecosystems [8] considers technical and economic feasibility of possible mitigation measures, as well as impacts on livelihoods and human health. Some measures that specifically concern cropland and soil management are summarized in Table 1.
There is some overlap in the categories listed in Table 1, since different interventions could be envisaged for the same land, and the integrated measures discussed by Smith et al. [8] notably result in increased carbon storage in soils. The category ‘improved cropland management’ includes practices such as reduced tillage, cover crops, perennials, water management and nutrient management.
4.1 Uncertainties in estimates and critical issues
The type of management system that farmers adopt, will substantially determine the capacity of soil to act as a carbon sink, and the extent to which agricultural land will contribute to GHG emissions. However, estimates regarding the potential of agricultural soil management practices to mitigate climate change vary considerably, and have been calculated in various manners. While Minasny et al. [71] estimate that raising soil organic matter could offset 20–35% of total GHG emissions, Schlesinger and Amundson [72] believe that the combined use of biochar and enhanced silicate weathering on agricultural land will not offset more than 5% of emissions. Differences in what is included in calculations, as well as in assumptions regarding anticipated conditions and future projections naturally affect conclusions. Biochar has attracted considerable interest for its ability to improve soil fertility and immobilize pollutants, while offering potential for long term storage of carbon [51]. However, the stability of biochar and its long-term impacts will ultimately depend on conditions that affect biochar aging [73]. With respect to upscaling enhanced silicate weathering as a climate mitigation strategy, uncertainties and possible negative environmental impacts need to be taken into account [74, 75].
Types of organic fertilizer that contain organic matter will directly contribute to soil organic carbon (SOC) content, but fungi and microbes contained in certain types of organic fertilizer, as well as impacts of pH and the proportions of other nutrients and micro-nutrients, will all affect the dynamics of soil biota and ecosystems. This leads to indirect positive or negative affects not only on fertility, water retention and resilience, but also on net GHG emissions (see e.g., Galic et al. [7], Walling et al. [47], Xu et al. [52]). Among other factors, annual precipitation significantly affects SOC dynamics [37, 76], and must be considered in arid and semi-arid regions.
4.2 Carbon sequestration
Carbon stocks in agricultural soils have been depleted worldwide, affecting productivity (see Droste et al. [77]). However, these losses do not all necessarily correspond to release of CO2 into the atmosphere, and Chenu [78] therefore makes the distinction between carbon sequestration, which aims to counteract global warming, and carbon storage in soils. Numerous approaches are developed to enhance carbon sequestration. In New Zealand, for instance, ‘flipping’ is used for podzolized sandy soils with pasture grassland, to avoid water logging. Burying topsoil led to long term SOC preservation, while new organic matter could accumulate in the surface soil under these conditions [79]. However, as for most practices, impacts will be dependent on local circumstances, since disrupting soil ecosystems will alter SOC dynamics, thereby carbon contained in above-ground vegetation or root systems, while exposure of topsoil can lead to erosion. Madigan et al. [80] compare different approaches to managing pasture and argue that full-inversion tillage (FIT) during pasture renewal has potential in an Irish context, particularly when combined with re-seeding.
While many of the approaches aiming at carbon sequestration and reduction of GHG emissions [65] bring benefits for agriculture through soil improvement, increasing water retention, reducing agricultural runoff and effects of heat stress, as well as conserving ecosystems, there are nevertheless risks associated with the need to rapidly offset GHG emissions produced by the burning of fossil fuels. From the point of view of agricultural production, organic matter is urgently needed to counter loss of topsoil and soil degradation, while equally urgent ambitions to rapidly achieve long term sequestration of carbon at a large scale, will reduce the amount of organic material available. Some approaches to carbon sequestration keep soil organic matter (SOM) in soil layers and forms that remain available to vegetation, while others such as flipping [79] bury the SOM in lower layers in order to slow down metabolic processes. However, yet others aim to bind carbon in forms that are not bioavailable or bury it in deep sediment or geological layers that remove both carbon and nutrients contained in organic waste from biological cycles.
Soil microbial activity is beneficial to crops and supports agricultural productivity but can also result in a net increase of GHG emissions, depending on balance and conditions. The use of agricultural lime to improve acidic soils can either lead to increased release of CO2 in the atmosphere, or to carbon sequestration. For instance, Bramble, Gouveia and Ramnarine [81] found that combining the application of agricultural lime with poultry litter prevented CO2 emissions. Finally, it is important to also consider energy conservation in climate mitigation strategies. Soil organic content substantially affects energy requirements, and Hercher-Pasteur et al. [82] therefore argue that this should be included when calculating optimal uses for biomass.
5. Sustainable agricultural practices
Choice of fertilizer cannot be understood in isolation, but as part of overall soil and land management practices in agriculture. In the following, some examples of sustainable practices are given, that are supported by the use of organic fertilizers, but which can also enhance their benefits. Combinations of approaches lead to synergies, not only with respect to bioavailability of nutrients, but also with respect to water balance, prevention of erosion [37], pest and pathogen control, and resilience to other stressors. For instance, improving tillage practices and incorporating residue was found to increase water-use efficiency by 30%, rice–wheat yields by 5–37%, income by 28–40%, while reducing and GHG emissions by 16–25% [8]. Further options of interest include perennial crops [83, 84, 85], polyculture [86], mosaic landscapes [87] and the use of pollinator strips or other habitat [88, 89], which support crop productivity through ecological intensification [90].
5.1 The role of soil health and microbial activity
Loss of soil health exposes crops to various diseases [54]. Among the numerous challenges for soil health in arid and semi-arid regions is the risk of salinization [37, 54, 91], which is driven not only by evaporation and low precipitation, but also by use of synthetic fertilizers and reduced moisture retention in soils with low content of organic materials. Soil organisms are essential for soil fertility, by making nutrients available to crops. A healthy soil ecosystem decomposes organic matter, makes nutrients available, prevents nutrient leaching and fixes nitrogen. It also protects plants from pathogens [54], improves soil structure and promotes well-functioning root systems. However, microbial activity can contribute to GHG emissions, and net effects under different conditions therefore need to be carefully considered.
The fungal to bacteria biomass ration (F/B) is one of the important indicators of soil health. Optimal F/B ratios depend on intended use. While grains and vegetables require bacterial dominance or a balance between fungi and bacteria, orchard trees need a dominance of fungi, which are more effective at immobilizing nutrients, preventing leaching. For grasslands, higher F/B ratios are an indication of more sustainable systems, with less environmental impacts [92]. It should be noted that biomass in itself is not a complete indicator for fungal and microbial activity [92] and that the distribution across various depths is also important for fertility and GHG flux dynamics.
Fiodor et al. [54] point to the potential use of specific plant growth promoting microbes (PGPM) that protect against a wide range of stressors and pathogens, and which can be applied by methods such as inoculation. Although microbial communities can in many respects be interchangeable from a functional point of view, unique strains of PGPM that mitigate effects of biotic and abiotic stressors are especially relevant in the light of rapid climate change. Research on how organic fertilizers can support such microbes is therefore called for, as is research soil ecosystems and plant-microbial symbiotic relationships (see e.g., Porter and Sachs [93]). Impacts of antibiotic residues in organic fertilizer [10] also require attention.
5.2 Conservation agriculture
Soil conservation practices are needed for sustainable productivity [94]. Conservation agriculture (CA) conserves soil moisture and reduces both erosion and runoff, improving water quality, as well as promoting biodiversity and above-ground ecosystems [95], with potentials for pest control and pollination. CA has been found to reduce water use substantially, as well as decreasing energy inputs [96]. It is of particular interest under extreme climatic conditions, due to its ability to mitigate heat and water stress, thereby increasing crop yields [96] and resilience.
In an Indian context, Battacharya et al. [94] compared performance of CA practices with farms applying conventional tillage over a nine-year period, using a wide range of measurements for soil health and sustainability. In this Indian study, conservation agriculture was shown to increase SOC, while requiring low input. However, Palm et al. [95] underline that CA will not necessarily increase soil carbon sequestration in all contexts. In studies they reviewed, only about half reported increased sequestration with no-till practices. Furthermore, in Sub Saharan Africa, Palm et al. [95] found that lack of residues was a significant obstacle to implementing CA for smallholder farmers. Use of organic residues for soil amendment in these contexts competed with other uses that had higher values, primarily as fodder for livestock. They conclude that it is important to distinguish between high-input CA systems applied in large-scale mechanised farms, and which require large inputs of herbicides to control weeds, with conditions for smallholder systems in the tropics and subtropics.
5.3 Tillage practices
No-tillage systems and suitable cover crop management can improve SOC, total N, available P, exchangeable K-Mg, CEC, bulk density, soil penetration resistance, and substrate-induced respiration, as exemplified in a Japanese study concerning Andosols [97]. Inversely, tillage will increase microbial activity that contributes to emissions, accelerate decomposition, but the disturbance will reduce microbial communities over time [97]. However, according to the review made by Palm et al. [95], no-till systems in cooler and wetter climates are more likely to result in lower soil carbon and reduced crop yields.
5.4 Cover crops
Cover crops conserve water, moderate soil temperature, and help to control weeds. Cover crops can further increase fungal biomass and improve the biological structure of soil [92]. Long-term use of cover crops improves soil fertility through the accumulation of SOM [92]. Disrupting soils through tillage kills fungi, and therefore shifts the balance towards bacteria. Legume intercrops or cover crops can lead to higher soil carbon storage and slower decomposition in no-till rotation systems [95]. Palm et al. [95] found that while quality of organic inputs affected short-term carbon dynamics, it did not appear to substantially affect long-term storage. Quality could be modified by addition of lignin. Materials with a high carbon to N ratio could result in reduced crop yields, while residues with a lower C:N ratio, as in the case of legume residues and legume cover crops, increased N availability. Legumes are not only of interest for their N-fixing properties, but for other facilitation effects as well [98, 99, 100].
5.5 Agroforestry
Agroforestry brings benefits for soil fauna and generally improves soil quality [101, 102, 103], and soil organic carbon sequestration [51, 104]. Depending on conditions, reduced light can affect yields of crops that are grown with trees, but agroforestry is also deliberately used to provide shade and create beneficial microclimates to mitigate heat stress and loss of water through evapotranspiration, as well as to adjust for lower or more variable rainfall [105], which is highly relevant for arid and semi-arid regions. With global warming, weather systems will contain more energy, and agroforestry therefore can play a role in preventing erosion and loss of soil from wind [37], as well as from extreme rainfall. Agroforestry systems can offer valuable habitat for pollinators and fauna essential for pest control, but trees should be selected for climate resilience and the precise combinations of species of orchards, crops or other vegetation in these systems needs to be considered, as well as spacing, orientation and adjustment to topography.
6. Water conservation and pollution prevention
Major landscape changes, with loss and deterioration of wetlands [26, 106], mean that nutrient flows from agriculture rapidly move on into the oceans, destabilizing ecosystems [107]. Drainage, to claim land for agriculture or other purposes, and extensive irrigation in agriculture cause wetlands to dry [108], while other drivers of wetland loss are urbanisation and surface sealing for road networks, industrial use of water and large dams. With climate change, water is no longer released gradually over the year through snow smelting, and forest fires [41], use of woodlands for fuel or commercial logging create additional disruptions in the water systems on which wetlands depend [109]. The amount of carbon stored in wetlands and peatlands constitutes in the order of 30–40% of terrestrial carbon [110, 111].
According to UN Water, 72% of all water withdrawals globally are used by agriculture [112]. Besides practices such as no-till, reduced till, cover crops or terracing and contour farming to retain water and reduce erosion [37], leaving crop residue on the surface also serves these purposes [113]. Importantly, demand for water can be further reduced by supporting complex agricultural landscapes that include trees and other vegetation, and by shifting to crops and cultivars that require less water. Alongside conventional approaches to water conservation such as drip irrigation, such approaches are necessary to address the water crisis, which will in many regions be aggravated by climate change [39, 40, 41]. For arid and semi-arid regions in particular, conservation agriculture and other sustainable practices are crucial for their role in preserving soil moisture and reducing irrigation needs. Both organic fertilizers and other methods of increasing SOM play a role in reclaiming land and combatting desertification [8, 37, 59, 99, 114, 115, 116]. Several solutions to the issue of polluted water [26, 106] have been suggested, including phytoremediation or the use of agricultural waste to serve as biosorbants [117, 118, 119].
Bhattacharyya et al. [120] suggest nutrient budgeting as an effective approach to preventing soil-water-air contamination from crop-livestock systems. Excess nutrients do not only impact rivers, lakes and coastal waters, but also affect groundwater quality [28, 29]. Nutrient surpluses are linked to use of fertilizers and manure, as well as to low nutrient utilization efficiency of plants. Leaching, runoff and erosion are therefore all significant for sustainable agricultural practices. In this respect, a slower release of nutrients and improvements in soil structure are important potential benefits of organic fertilizers, compared to chemical fertilizers. Contributions to soil and ecosystem health of sustainable practices reduce the need for pesticides to control pests and pathogens, thereby increasing availability of good quality water [49] and protecting the world’s oceans [121, 122].
The various interlinkages and trade-offs that need to be considered in use of water resources are acknowledged in European policy on the water, energy, food, and ecosystems (WEFE) nexus [123], as well as in recent research in this field [124, 125, 126, 127]. Both general conflicts in demands concerning use of land and resources, and water scarcity, in particular, affect the arid and semi-arid regions of the MENA region. For these regions, land management must pay greater attention to how soil health and quality affects water retention. Degraded soils have poor water retention capacity, demand more fertilizer, and are less able to contribute to carbon sequestration. A more holistic view of land and soil management can also mitigate effects of stress caused by heat, extreme weather events and increased climate variability.
7. Transition issues
Conservation agriculture can lead to yield benefits, but improvements may not be noticeable in the initial years [94]. In a Swedish context, examining various sites over a period of 54 years, Droste et al. [77] find that increasing SOC leads to long-term yield stability and resilience, which is important in view of accelerating climate change. However, adopting sustainable management practices can come at the cost of short-term productivity. Policy changes to support the transition are therefore recommended [77, 128]. To minimise initial economic impacts for farmers of conversion, Yigezu et al. [46] and Tu et al. [129] recommend transition strategies that involve gradually reducing conventional inputs.
Sustainable agricultural practices achieve control of pests and pathogens without damaging the environment, but these practices are also largely dependent on healthy soil biota and rich ecosystems in the agricultural landscape. Agricultural soils have been affected by numerous sources of pollution [130]. Soil management practices and use of chemicals will have negative effects on many soil invertebrates and microbes [131, 132] but will favour others. The net effect is therefore not only loss of important strains of soil biota or total mass, but the creation of imbalances in microbial communities that can have detrimental effects for plant health and crop yields.
Since soil health and ecosystems have been damaged by prior unsustainable practices, including use of synthetic fertilizers and pesticides, restoring health takes time, and processes of remediation and restoration are therefore crucial [59, 77, 132, 133, 134]. The ability of new cultivars to benefit from plant-microbial symbiosis has been affected by selection of cultivars for other traits, and by reduced dependence on this symbiosis through the use of synthetic fertilizers [93]. Transition to sustainable farming with organic fertilizers should therefore also consider the choice of suitable cultivars and heritage varieties that retain the ability to fully benefit from improved soil health.
8. Smallholder farming and sustainable agriculture
It is difficult to evaluate the magnitude of smallholder and subsistence farming world-wide, since it is frequently undertaken in regions with limited statistics, on fragmented or mixed-use plots where land-use can be difficult to identify from satellite images. In many contexts, it is not necessarily the primary occupation of the farmer. Despite its marginal position in debates on agricultural productivity, smallholder farming plays a vital role for biodiversity, food security, human health, equity and climate resilience, since value is not lost in the distribution chain but stays with producers and their communities. Locally sourced food reduces community vulnerability to disruptions in the food supply chain, due to disasters, logistics failures, financial crises, or armed conflict. The latter consideration is significant for the MENA region, where several countries are affected by conflict or volatility [33]. Food systems worldwide are exposed to numerous disruptions, which will increase as a result of climate change and environmental degradation [69]. Smallholder farmers are particularly vulnerable to such shocks and have difficulties making adequate choices in the face of uncertainty [21, 22, 31]. To address such challenges, Kim et al. [70] suggest a land-water-nutrient nexus (LWNN) approach (see also Jat et al. [96] for strategies from an Indian context). Crop diversification can be a strategy to meet the double uncertainty of price fluctuations and crop failures [135], and polycultures also have environmental benefits. However, food processing industries and international markets tend to be oriented towards monocultures, and smallholder farmers can be obligated by contracts to produce particular crops.
Low-input smallholder production systems are one of the dominant food production systems globally [136]. In an Ethiopian case, Baudron et al. [136] observe that complex agricultural landscapes that incorporate trees offer better overall livelihoods for farmers, lead to better carbon balances, as well as being more resilient both to fluctuation in input prices and to climate stress. They further underline that low-input farming with resource-saving practices can increase profitability for farmers more than yield optimization, while yield stability is another important consideration for smallholders.
Baudron et al. [136] therefore argue for an increased attention to agricultural practices that support synergies between agriculture and biodiversity, rather than presenting the situation as an irreducible choice between ‘land sparing’—aiming to reduce demand for land through intensification— and ‘land sharing’, assuming loss in yields, as a consequence of practices that are more favourable to wildlife and biodiversity. Baudron et al. emphasize the reliance of low-input smallholder agricultural production on ecosystem services provided by biodiverse ecosystems, and further point to the crucial role of ecosystem services to maintain soil fertility, pollination, and for pest and disease control [136, 137].
Despite the benefits that low-input farming can bring [138], barriers include lack of locally relevant expertise, and the time needed to rehabilitate soils degraded by use of synthetic fertilizers and pesticides. Subsidy systems may support heavily mechanised and chemical-intensive agriculture [3, 14], with questionable benefits for smallholder farmers. Further barriers in transitioning to sustainable agricultural practices are access to markets, and global food systems structured to favour monoculture of particular crops and cultivars.
9. Conclusions
In view of the numerous factors that influence outcomes for the use of organic fertilizers, locally tailored strategies that combine approaches to enhance soil health and sustainable land management would be recommended. However, sufficiently detailed data is still lacking on how different management practices affect yields and environmental impacts depending on local conditions, particularly in the global South. Citizen science has the potential to offer a better evidence base for farmers’ choices, but the structure of many citizen science projects rarely supports longer term collaboration and dialogue with smallholder farmers in the global South [139, 140]. In addition, smallholder farmers may not be able to afford individualised consulting, and agronomists may lack expertise applicable to low-input agriculture. Transitioning to sustainable practices is knowledge intensive [44], and this is therefore an area where international networking with academic institutions could play a significant role in supporting climate adaptation and mitigation efforts. Exchange of knowledge among farmers [141] and farmers’ organizations can also play a role for mobilizing resources and expertise, but such potential contributions will depend on the orientation of the organization [142].
Among other implications of the current climate crisis, a narrow focus on crop yields is not sufficient, since outcomes of fertilizer application are usually estimated under optimal or normal growing conditions. Increased weather variability and the ensuing risk of crop failure, means that greater attention must be devoted to resilience, and the capacity to cultivate under unpredictable and less than optimal conditions. This in turn means, for instance, that effects on root growth, the capacity of root systems to absorb water and nutrients under extreme conditions, as well as the capacity of the soil to retain water and nutrients over longer periods of time all become critical factors. Also, rather than considering fertilizer application merely from the view of inputs and short-term yields, and besides measures such as C:N ratios, we need to take on a more holistic view, looking at how choice of fertilizer relates to nutrient absorption efficiency, drought resistance of root systems [143], soil health, land degradation, water management and ecological intensification. Future shortages of P [144, 145, 146], loss of arable land [37], decline in soil carbon [147], as well as widespread decline in soil fertility driven by industrial practices in agriculture, point to the important role of organic fertilizers. However, availability of organic material is constrained by competing demands on biomass and land for industrial and carbon sequestration purposes, while contamination of organic waste and wastewater [10, 118, 148] poses an issue for possible circular approaches. To generalise the use of organic fertilizers, redesign of food systems and policy changes are therefore required, adopting a more comprehensive approach to the complex interlinkages that are involved.
Acknowledgments
The Swedish Research Council for Sustainable Development, FORMAS (project number 2017-01375) has contributed to APC for this publication.
\n',keywords:"organic fertilizers, sustainable agriculture, transition pathways, smallholder farmers, semi-arid regions, low-input agriculture, soil health, soil carbon, GHG emissions, conservation agriculture, water management, climate adaptation and mitigation",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/79868.pdf",chapterXML:"https://mts.intechopen.com/source/xml/79868.xml",downloadPdfUrl:"/chapter/pdf-download/79868",previewPdfUrl:"/chapter/pdf-preview/79868",totalDownloads:130,totalViews:0,totalCrossrefCites:0,dateSubmitted:"October 13th 2021",dateReviewed:"October 26th 2021",datePrePublished:"December 31st 2021",datePublished:null,dateFinished:"December 31st 2021",readingETA:"0",abstract:"Organic fertilizers can serve as an element of transitions to sustainable low-input agriculture in semi-arid regions of the MENA region. They play a key role in supporting soil biota and soil fertility. Yield improvements, availability and relatively low costs make organic fertilizers an attractive alternative for farmers. In semi-arid regions, important considerations are improved soil quality, which in turn affects soil water retention, while better root development helps crops resist heat and water stress. Organic fertilizers thus support climate adaptation and regional food security. Soil quality is crucial for carbon sequestration, at the same time that increased nutrient retention reduces impacts of agricultural runoff on groundwater and water bodies. Factors that impede the generalised use of organic fertilizers include lack of expertise, subsidy structures, constraints of the wider food and agricultural systems, and difficulties in transitioning from conventional agriculture. Such obstacles are aggravated in countries affected by security issues, financial volatility or restrictions in access to market. Against the background of both general and local constraints, the chapter examines possible pathways to benefit from organic fertilizers, in particular synergies with other sustainable agricultural practices, as well as improved access to expertise.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/79868",risUrl:"/chapter/ris/79868",signatures:"Helen Avery",book:{id:"10989",type:"book",title:"New Generation of Organic Fertilizers",subtitle:null,fullTitle:"New Generation of Organic Fertilizers",slug:null,publishedDate:null,bookSignature:"Prof. Metin Turan and Prof. Ertan Yildirim",coverURL:"https://cdn.intechopen.com/books/images_new/10989.jpg",licenceType:"CC BY 3.0",editedByType:null,isbn:"978-1-83969-213-0",printIsbn:"978-1-83969-212-3",pdfIsbn:"978-1-83969-938-2",isAvailableForWebshopOrdering:!0,editors:[{id:"140612",title:"Prof.",name:"Metin",middleName:null,surname:"Turan",slug:"metin-turan",fullName:"Metin Turan"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Agriculture in the Middle East and North Africa",level:"1"},{id:"sec_3",title:"3. Environmental impacts of agriculture",level:"1"},{id:"sec_3_2",title:"3.1 IPCC estimates of climate impacts and mitigation potentials",level:"2"},{id:"sec_5",title:"4. Climate mitigation potentials in agriculture",level:"1"},{id:"sec_5_2",title:"4.1 Uncertainties in estimates and critical issues",level:"2"},{id:"sec_6_2",title:"4.2 Carbon sequestration",level:"2"},{id:"sec_8",title:"5. Sustainable agricultural practices",level:"1"},{id:"sec_8_2",title:"5.1 The role of soil health and microbial activity",level:"2"},{id:"sec_9_2",title:"5.2 Conservation agriculture",level:"2"},{id:"sec_10_2",title:"5.3 Tillage practices",level:"2"},{id:"sec_11_2",title:"5.4 Cover crops",level:"2"},{id:"sec_12_2",title:"5.5 Agroforestry",level:"2"},{id:"sec_14",title:"6. Water conservation and pollution prevention",level:"1"},{id:"sec_15",title:"7. Transition issues",level:"1"},{id:"sec_16",title:"8. Smallholder farming and sustainable agriculture",level:"1"},{id:"sec_17",title:"9. Conclusions",level:"1"},{id:"sec_18",title:"Acknowledgments",level:"1"}],chapterReferences:[{id:"B1",body:'Sradnick A, Feller C. A typological concept to predict the nitrogen release from organic fertilizers in farming systems. Agronomy. 2020;10(9):1448'},{id:"B2",body:'Rayne N, Aula L. Livestock manure and the impacts on soil health: A review. Soil Systems. 2020;4(4):64. DOI: 103390/soilsystems4040064'},{id:"B3",body:'Jain M, Solomon D, Capnerhurst H, Arnold A, Elliott A, Kinzer AT, et al. How much can sustainable intensification increase yields across South Asia? A systematic review of the evidence. Environmental Research Letters. 2020;15(8):083004'},{id:"B4",body:'Mengqi Z, Shi A, Ajmal M, Ye L, Awais M. Comprehensive review on agricultural waste utilization and high-temperature fermentation and composting. 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DOI: 101080/0306731920201786547'},{id:"B148",body:'Tabatabaei SH, Nourmahnad N, Kermani SG, Tabatabaei SA, Najafi P, Heidarpour M. Urban wastewater reuse in agriculture for irrigation in arid and semi-arid regions—A review. International Journal of Recycling of Organic Waste in Agriculture. 2020;9(2):193-220'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Helen Avery",address:"helen.avery@cme.lu.se",affiliation:'
Centre for Environmental and Climate Science/Centre for Advanced Middle Eastern Studies, Lund University, Lund, Sweden
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Open Access publication costs can often be designated directly in the grants or in specific budgets allocated for that purpose. Many of the most important funding organisations encourage, and even request, that the projects they fund are made available at no cost to the wider public. IntechOpen strives to maintain excellent relationships with these funders and ensures compliance with mandates.
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Please be aware that you must be a member, or grantee, of the institutions/funders listed in order to apply for their Open Access publication funds.
Open Access publication costs can often be designated directly in the grants or in specific budgets allocated for that purpose. Many of the most important funding organisations encourage, and even request, that the projects they fund are made available at no cost to the wider public. IntechOpen strives to maintain excellent relationships with these funders and ensures compliance with mandates.
\n\n
In order to help Authors identify appropriate funding agencies and institutions, we have created a list, based on extensive research on various OA resources (including ROARMAP and SHERPA/JULIET) of organizations that have funds available. Before consulting our list we encourage you to petition your own institution or organization for Open Access funds or check the specifications of your grant with your funder to ascertain if publication costs are included. Where you are in receipt of a grant you should clarify:
\n\n
\n\t
Does your institution already have a budget for covering Open Access publication costs?
\n\t
Does your grant list Open Access publication fees as legitimate direct/indirect costs?
\n
\n\n
If you are associated with any of the institutions in our list below, you can apply to receive OA publication funds by following the instructions provided in the links. Please consult the Open Access policies or grant Terms and Conditions of any institution with which you are linked to explore ways to cover your publication costs (also accessible by clicking on the link in their title).
\n\n
Please note that this list is not a definitive one and is updated regularly. To suggest possible modifications or the inclusion of your institution/funder, please contact us at funders@intechopen.com
\n\n
Please be aware that you must be a member, or grantee, of the institutions/funders listed in order to apply for their Open Access publication funds.
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Public health nurses are to perform health promotion and disease prevention work on an individual and population level. By identifying how features of different discourses are constructed and maintained, combining linguistics tools and social science perspectives, the purpose was to provide an understanding of the health promotion and disease prevention discourse in the public health nursing curriculum to reveal governmental strategies for public health nursing education in a time of transition. Fairclough’s three‐dimensional model of critical discourse analysis that consists of the analytical dimensions social events, social practices, and social structures was carried out. There is a linguistic‐discursive dialectic between the dimensions. The analysis revealed four discourses in the curriculum text: a contradictory health promotion and disease prevention discourse; a paternalistic meta‐discourse; a hegemonic individual discourse; and a hegemonic discourse for interdisciplinary collaboration. The results indicate a hegemonic disease prevention discourse, while the health promotion discourse being more disguised. The analysis revealed how language functions ideologically, and in line with the sociolinguistics, how the role of the language in the curriculum text can have consequences for the social work of public health nurses.",book:{id:"5726",slug:"sociolinguistics-interdisciplinary-perspectives",title:"Sociolinguistics",fullTitle:"Sociolinguistics - Interdisciplinary Perspectives"},signatures:"Berit Misund Dahl",authors:[{id:"195508",title:"Dr.",name:"Berit Misund",middleName:null,surname:"Dahl",slug:"berit-misund-dahl",fullName:"Berit Misund Dahl"}]},{id:"65233",doi:"10.5772/intechopen.82823",title:"Introductory Chapter: Discourse and Discourse Analysis. A Retrospective Approach",slug:"introductory-chapter-discourse-and-discourse-analysis-a-retrospective-approach",totalDownloads:2388,totalCrossrefCites:1,totalDimensionsCites:2,abstract:null,book:{id:"8632",slug:"advances-in-discourse-analysis",title:"Advances in Discourse Analysis",fullTitle:"Advances in Discourse Analysis"},signatures:"Lavinia Suciu",authors:[{id:"202159",title:"Ph.D.",name:"Lavinia",middleName:null,surname:"Suciu",slug:"lavinia-suciu",fullName:"Lavinia Suciu"}]},{id:"54482",doi:"10.5772/67729",title:"Semiotic Analysis of Computer Visualization",slug:"semiotic-analysis-of-computer-visualization",totalDownloads:1264,totalCrossrefCites:2,totalDimensionsCites:2,abstract:"The purpose of this chapter is to discuss the semiotic approach to form theory of computer visualization. Such theory should be the foundation of design, development, and evaluations of visualization systems. The “direct” semiotic analysis of visualization is defined and the scheme of the analysis is considered. This analysis reveals “who is who” in the process of the visualization semiosis and helps in design and development of the real visualization systems. The analysis allows to describe the problems arising at developments of specialized systems in terms of the semiotics and showing how this analysis can serve as a tool for the visualization systems design. It is important to analyze the sign nature of the human‐computer interface and the visualization. Such conceptions as computer metaphor, metaphor action, and metaphor formula are defined. The properties of metaphors are analyzed with a view to possible usage of metaphors for specific applications. The properties are considered by the example of the hierarchical sequence of the natural Room‐Building‐City (Landscape) metaphors. Also the properties of the molecule metaphor are considered in the context of software visualization systems. In conclusion, some approaches to the theory of computer visualization are outlined.",book:{id:"5702",slug:"interdisciplinary-approaches-to-semiotics",title:"Interdisciplinary Approaches to Semiotics",fullTitle:"Interdisciplinary Approaches to Semiotics"},signatures:"Vladimir L. Averbukh",authors:[{id:"135209",title:"Prof.",name:"Vladimir",middleName:null,surname:"Averbukh",slug:"vladimir-averbukh",fullName:"Vladimir Averbukh"}]},{id:"54495",doi:"10.5772/67860",title:"Music and Semiotics: An Experiential Approach to Musical Sense-Making",slug:"music-and-semiotics-an-experiential-approach-to-musical-sense-making",totalDownloads:1919,totalCrossrefCites:0,totalDimensionsCites:2,abstract:"This chapter sketches recent evolutions of semiotics as applied to music. Rather than providing merely a historical overview, it focuses mainly on the pragmatic turn in semiotics and the role of sensory experience in the process of musical sense-making. In order to elaborate on this experience, it delves into theoretical groundings of second-order cybernetics, biosemiotics, and ecological psychology, which are then applied to the field of music. Much effort is made to provide a broader framework to illustrate the transition from a disembodied to an embodied approach to musical semiotics. Special emphasis is laid on the concept of affordance and the role of interactions with the sounds.",book:{id:"5702",slug:"interdisciplinary-approaches-to-semiotics",title:"Interdisciplinary Approaches to Semiotics",fullTitle:"Interdisciplinary Approaches to Semiotics"},signatures:"Mark Reybrouck",authors:[{id:"196698",title:"Prof.",name:"Mark",middleName:null,surname:"Reybrouck",slug:"mark-reybrouck",fullName:"Mark Reybrouck"}]},{id:"59744",doi:"10.5772/intechopen.74625",title:"Advantages of Bilingualism and Multilingualism: Multidimensional Research Findings",slug:"advantages-of-bilingualism-and-multilingualism-multidimensional-research-findings",totalDownloads:3562,totalCrossrefCites:1,totalDimensionsCites:2,abstract:"Bilingualism and multilingualism are often perceived and considered as a problem or a major challenge to individual and/or societal development. In most instances, the only advantage recognized for the bilingual individual is the ability to use two or more languages. Beyond that, monolingualism seems more attractive, and monolinguals especially those speaking a language of wider communication seem quite content with their lot, often adopting a condescending attitude toward minority native speakers of a mother tongue who in addition have to acquire their language. Adepts of the ideology of monolingual habitus (one nation, one language) have tended to consider multilingualism and linguistic diversity as a curse and an obstacle to nation building. This chapter argues against the above ideology through a compendium of empirical evidence of advantages of individual bilingualism, societal multilingualism, and linguistic diversity of nations that emerge from research findings in the last several decades.",book:{id:"6201",slug:"multilingualism-and-bilingualism",title:"Multilingualism and Bilingualism",fullTitle:"Multilingualism and Bilingualism"},signatures:"Evelyn Fogwe Chibaka",authors:[{id:"220564",title:"Dr.",name:"Fogwe Evelyn",middleName:null,surname:"Chibaka",slug:"fogwe-evelyn-chibaka",fullName:"Fogwe Evelyn Chibaka"}]}],mostDownloadedChaptersLast30Days:[{id:"54872",title:"The Characteristics of Language Policy and Planning Research: An Overview",slug:"the-characteristics-of-language-policy-and-planning-research-an-overview",totalDownloads:3564,totalCrossrefCites:0,totalDimensionsCites:0,abstract:"This chapter has been compiled to provide an overview of the language policing and planning (LPP) field, particularly for new researchers who would like to pursue their MA or PhD. It aims to explore the following: the genesis of LPP from the 1950s to date, type of research questions pertinent to the field, methodology that can be applied, substantial literature review and case studies that have been carried out in LPP, ethnography of language policy and planning, the historical analysis approach and authorities in the field of LPP such as Hornberger, Johnson and Ricento.",book:{id:"5726",slug:"sociolinguistics-interdisciplinary-perspectives",title:"Sociolinguistics",fullTitle:"Sociolinguistics - Interdisciplinary Perspectives"},signatures:"Prashneel Ravisan Goundar",authors:[{id:"195526",title:"Mr.",name:"Prashneel",middleName:"Ravisan",surname:"Goundar",slug:"prashneel-goundar",fullName:"Prashneel Goundar"}]},{id:"59744",title:"Advantages of Bilingualism and Multilingualism: Multidimensional Research Findings",slug:"advantages-of-bilingualism-and-multilingualism-multidimensional-research-findings",totalDownloads:3562,totalCrossrefCites:1,totalDimensionsCites:2,abstract:"Bilingualism and multilingualism are often perceived and considered as a problem or a major challenge to individual and/or societal development. In most instances, the only advantage recognized for the bilingual individual is the ability to use two or more languages. Beyond that, monolingualism seems more attractive, and monolinguals especially those speaking a language of wider communication seem quite content with their lot, often adopting a condescending attitude toward minority native speakers of a mother tongue who in addition have to acquire their language. Adepts of the ideology of monolingual habitus (one nation, one language) have tended to consider multilingualism and linguistic diversity as a curse and an obstacle to nation building. This chapter argues against the above ideology through a compendium of empirical evidence of advantages of individual bilingualism, societal multilingualism, and linguistic diversity of nations that emerge from research findings in the last several decades.",book:{id:"6201",slug:"multilingualism-and-bilingualism",title:"Multilingualism and Bilingualism",fullTitle:"Multilingualism and Bilingualism"},signatures:"Evelyn Fogwe Chibaka",authors:[{id:"220564",title:"Dr.",name:"Fogwe Evelyn",middleName:null,surname:"Chibaka",slug:"fogwe-evelyn-chibaka",fullName:"Fogwe Evelyn Chibaka"}]},{id:"54552",title:"Language Evolution, Acquisition, Adaptation and Change",slug:"language-evolution-acquisition-adaptation-and-change",totalDownloads:1988,totalCrossrefCites:1,totalDimensionsCites:1,abstract:"In the twenty‐first century, there are between 6000 and 8000 different languages spoken in the world, all of which are in a continuous state of evolving, by inter‐mixing or stagnating, growing or contracting. This occurs through changes in the population size of the people who use them, the frequency and form of their use in different media, through migration and through inter‐mixing with other languages. As Stadler et al. argue, human languages are a ‘culturally evolving trait’ and when it occurs language change is both sporadic and robust (faithfully replicated) and the main established variants are replaced by new variants. Only about 200 of these disparate languages are in written as well as spoken form, and most, except the popular ones like Mandarin, Spanish, English, Hindi, Arabic, Portuguese, Bengali, and Russian, are in decline of use. But how did language itself evolve and come to be the most important innate tool possessed by people? The complex issue of language evolution continues to perplex because of its associations with culture, social behaviour and the development of the human mind.",book:{id:"5726",slug:"sociolinguistics-interdisciplinary-perspectives",title:"Sociolinguistics",fullTitle:"Sociolinguistics - Interdisciplinary Perspectives"},signatures:"Luke Strongman",authors:[{id:"189739",title:"Dr.",name:"Luke",middleName:null,surname:"Strongman",slug:"luke-strongman",fullName:"Luke Strongman"}]},{id:"57928",title:"Aspects and Dimensions of Bilingualism and Multilingualism in Europe",slug:"aspects-and-dimensions-of-bilingualism-and-multilingualism-in-europe",totalDownloads:1213,totalCrossrefCites:0,totalDimensionsCites:0,abstract:"This chapter aims to explore certain aspects and dimensions of bilingualism and multilingualism, with a focus on Europe. The issues analyzed are the following: languages coming into contact due to conquest or colonization, bilingualism and multilingualism as a reflection of political trends and contemporary lifestyles, official languages, and heritage languages. The field of language education is also treated, when it comes to the benefits of being bilingual and multilingual, which are also analyzed from the perspective of evolutionary psychology, with the claim that knowledge of several languages ensures survival and better living conditions. The conclusions are that bilingualism and multilingualism are a necessity and an inevitable phenomenon in today’s Europe, especially due to migration and due to the need of adapting to and accepting other cultures. What is more, there is a universality of bilingualism and multilingualism throughout history.",book:{id:"6201",slug:"multilingualism-and-bilingualism",title:"Multilingualism and Bilingualism",fullTitle:"Multilingualism and Bilingualism"},signatures:"Irina-Ana Drobot",authors:[{id:"209184",title:"Ph.D.",name:"Irina-Ana",middleName:null,surname:"Drobot",slug:"irina-ana-drobot",fullName:"Irina-Ana Drobot"}]},{id:"54479",title:"Grounding Functional Requirements Classification in Organizational Semiotics",slug:"grounding-functional-requirements-classification-in-organizational-semiotics",totalDownloads:1516,totalCrossrefCites:0,totalDimensionsCites:0,abstract:"An information system has its requirements rooted in organizational policies and behaviour, the complexity of which is governed by the hierarchy and the dependencies of the activities within the organization. This complexity makes requirements analysis for an envisioned information system an intricately challenging task. The absence of well‐defined body of knowledge clearly specifying which requirements must be looked for further deepens the challenge of requirements analysis. Though requirements are broadly classified as functional and non‐functional, a special concern is required for functional requirements as the information system is expected to meet the behaviour of the organization. We explore the role of organizational semiotics in extracting and analysing functional requirements for an envisioned information system. We also report the results of supervised learning to automatically extract the functional requirements from the existing available documentation.",book:{id:"5702",slug:"interdisciplinary-approaches-to-semiotics",title:"Interdisciplinary Approaches to Semiotics",fullTitle:"Interdisciplinary Approaches to Semiotics"},signatures:"Richa Sharma",authors:[{id:"195986",title:"Dr.",name:"Richa",middleName:null,surname:"Sharma",slug:"richa-sharma",fullName:"Richa Sharma"}]}],onlineFirstChaptersFilter:{topicId:"280",limit:6,offset:0},onlineFirstChaptersCollection:[],onlineFirstChaptersTotal:0},preDownload:{success:null,errors:{}},subscriptionForm:{success:null,errors:{}},aboutIntechopen:{},privacyPolicy:{},peerReviewing:{},howOpenAccessPublishingWithIntechopenWorks:{},sponsorshipBooks:{sponsorshipBooks:[],offset:8,limit:8,total:0},allSeries:{pteSeriesList:[{id:"14",title:"Artificial Intelligence",numberOfPublishedBooks:9,numberOfPublishedChapters:89,numberOfOpenTopics:6,numberOfUpcomingTopics:0,issn:"2633-1403",doi:"10.5772/intechopen.79920",isOpenForSubmission:!0},{id:"7",title:"Biomedical Engineering",numberOfPublishedBooks:12,numberOfPublishedChapters:104,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2631-5343",doi:"10.5772/intechopen.71985",isOpenForSubmission:!0}],lsSeriesList:[{id:"11",title:"Biochemistry",numberOfPublishedBooks:31,numberOfPublishedChapters:314,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2632-0983",doi:"10.5772/intechopen.72877",isOpenForSubmission:!0},{id:"25",title:"Environmental Sciences",numberOfPublishedBooks:1,numberOfPublishedChapters:11,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2754-6713",doi:"10.5772/intechopen.100362",isOpenForSubmission:!0},{id:"10",title:"Physiology",numberOfPublishedBooks:11,numberOfPublishedChapters:141,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2631-8261",doi:"10.5772/intechopen.72796",isOpenForSubmission:!0}],hsSeriesList:[{id:"3",title:"Dentistry",numberOfPublishedBooks:8,numberOfPublishedChapters:129,numberOfOpenTopics:2,numberOfUpcomingTopics:0,issn:"2631-6218",doi:"10.5772/intechopen.71199",isOpenForSubmission:!0},{id:"6",title:"Infectious Diseases",numberOfPublishedBooks:13,numberOfPublishedChapters:113,numberOfOpenTopics:3,numberOfUpcomingTopics:1,issn:"2631-6188",doi:"10.5772/intechopen.71852",isOpenForSubmission:!0},{id:"13",title:"Veterinary Medicine and Science",numberOfPublishedBooks:11,numberOfPublishedChapters:105,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2632-0517",doi:"10.5772/intechopen.73681",isOpenForSubmission:!0}],sshSeriesList:[{id:"22",title:"Business, Management and Economics",numberOfPublishedBooks:1,numberOfPublishedChapters:18,numberOfOpenTopics:2,numberOfUpcomingTopics:1,issn:"2753-894X",doi:"10.5772/intechopen.100359",isOpenForSubmission:!0},{id:"23",title:"Education and Human Development",numberOfPublishedBooks:0,numberOfPublishedChapters:5,numberOfOpenTopics:1,numberOfUpcomingTopics:1,issn:null,doi:"10.5772/intechopen.100360",isOpenForSubmission:!0},{id:"24",title:"Sustainable Development",numberOfPublishedBooks:0,numberOfPublishedChapters:14,numberOfOpenTopics:5,numberOfUpcomingTopics:0,issn:null,doi:"10.5772/intechopen.100361",isOpenForSubmission:!0}],testimonialsList:[{id:"6",text:"It is great to work with the IntechOpen to produce a worthwhile collection of research that also becomes a great educational resource and guide for future research endeavors.",author:{id:"259298",name:"Edward",surname:"Narayan",institutionString:null,profilePictureURL:"https://mts.intechopen.com/storage/users/259298/images/system/259298.jpeg",slug:"edward-narayan",institution:{id:"3",name:"University of Queensland",country:{id:null,name:"Australia"}}}},{id:"13",text:"The collaboration with and support of the technical staff of IntechOpen is fantastic. 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From\r\n1964 to 1974, he worked as Assistant in Biochemistry at the School of MedicineUniversidad Nacional de La Plata, Argentina. From 1974 to 1976, he was a Fellowof the National Institutes of Health (NIH) at the University of Connecticut, Health Center, USA. From 1985 to 2004, he served as a Full Professor oBiochemistry at the Universidad Nacional de La Plata, Argentina. He is Member ofthe National Research Council (CONICET), Argentina, and Argentine Society foBiochemistry and Molecular Biology (SAIB). His laboratory has been interested for manyears in the lipid peroxidation of biological membranes from various tissues and different species. Professor Catalá has directed twelve doctoral theses, publishedover 100 papers in peer reviewed journals, several chapters in books andtwelve edited books. Angel Catalá received awards at the 40th InternationaConference Biochemistry of Lipids 1999: Dijon (France). 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He also obtained an MSc in Molecular and Genetic Medicine, and a Ph.D. in Clinical Immunology and Human Genetics from the University of Sheffield, UK. He also completed a short-term fellowship in Pediatric Clinical Immunology and Bone Marrow Transplantation at Newcastle General Hospital, England. Dr. Rezaei is a Full Professor of Immunology and Vice Dean of International Affairs and Research, at the School of Medicine, Tehran University of Medical Sciences, and the co-founder and head of the Research Center for Immunodeficiencies. He is also the founding president of the Universal Scientific Education and Research Network (USERN). Dr. Rezaei has directed more than 100 research projects and has designed and participated in several international collaborative projects. He is an editor, editorial assistant, or editorial board member of more than forty international journals. He has edited more than 50 international books, presented more than 500 lectures/posters in congresses/meetings, and published more than 1,100 scientific papers in international journals.",institutionString:"Tehran University of Medical Sciences",institution:{name:"Tehran University of Medical Sciences",country:{name:"Iran"}}},{id:"180733",title:"Dr.",name:"Jean",middleName:null,surname:"Engohang-Ndong",slug:"jean-engohang-ndong",fullName:"Jean Engohang-Ndong",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/180733/images/system/180733.png",biography:"Dr. Jean Engohang-Ndong was born and raised in Gabon. After obtaining his Associate Degree of Science at the University of Science and Technology of Masuku, Gabon, he continued his education in France where he obtained his BS, MS, and Ph.D. in Medical Microbiology. He worked as a post-doctoral fellow at the Public Health Research Institute (PHRI), Newark, NJ for four years before accepting a three-year faculty position at Brigham Young University-Hawaii. Dr. Engohang-Ndong is a tenured faculty member with the academic rank of Full Professor at Kent State University, Ohio, where he teaches a wide range of biological science courses and pursues his research in medical and environmental microbiology. Recently, he expanded his research interest to epidemiology and biostatistics of chronic diseases in Gabon.",institutionString:"Kent State University",institution:{name:"Kent State University",country:{name:"United States of America"}}},{id:"188773",title:"Prof.",name:"Emmanuel",middleName:null,surname:"Drouet",slug:"emmanuel-drouet",fullName:"Emmanuel Drouet",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/188773/images/system/188773.png",biography:"Emmanuel Drouet, PharmD, is a Professor of Virology at the Faculty of Pharmacy, the University Grenoble-Alpes, France. As a head scientist at the Institute of Structural Biology in Grenoble, Dr. Drouet’s research investigates persisting viruses in humans (RNA and DNA viruses) and the balance with our host immune system. He focuses on these viruses’ effects on humans (both their impact on pathology and their symbiotic relationships in humans). He has an excellent track record in the herpesvirus field, and his group is engaged in clinical research in the field of Epstein-Barr virus diseases. He is the editor of the online Encyclopedia of Environment and he coordinates the Universal Health Coverage education program for the BioHealth Computing Schools of the European Institute of Science.",institutionString:null,institution:{name:"Grenoble Alpes University",country:{name:"France"}}},{id:"131400",title:"Prof.",name:"Alfonso J.",middleName:null,surname:"Rodriguez-Morales",slug:"alfonso-j.-rodriguez-morales",fullName:"Alfonso J. Rodriguez-Morales",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/131400/images/system/131400.png",biography:"Dr. Rodriguez-Morales is an expert in tropical and emerging diseases, particularly zoonotic and vector-borne diseases (especially arboviral diseases). He is the president of the Travel Medicine Committee of the Pan-American Infectious Diseases Association (API), as well as the president of the Colombian Association of Infectious Diseases (ACIN). He is a member of the Committee on Tropical Medicine, Zoonoses, and Travel Medicine of ACIN. He is a vice-president of the Latin American Society for Travel Medicine (SLAMVI) and a Member of the Council of the International Society for Infectious Diseases (ISID). Since 2014, he has been recognized as a Senior Researcher, at the Ministry of Science of Colombia. He is a professor at the Faculty of Medicine of the Fundacion Universitaria Autonoma de las Americas, in Pereira, Risaralda, Colombia. He is an External Professor, Master in Research on Tropical Medicine and International Health, Universitat de Barcelona, Spain. He is also a professor at the Master in Clinical Epidemiology and Biostatistics, Universidad Científica del Sur, Lima, Peru. In 2021 he has been awarded the “Raul Isturiz Award” Medal of the API. Also, in 2021, he was awarded with the “Jose Felix Patiño” Asclepius Staff Medal of the Colombian Medical College, due to his scientific contributions to COVID-19 during the pandemic. He is currently the Editor in Chief of the journal Travel Medicine and Infectious Diseases. His Scopus H index is 47 (Google Scholar H index, 68).",institutionString:"Institución Universitaria Visión de las Américas, Colombia",institution:null},{id:"332819",title:"Dr.",name:"Chukwudi Michael",middleName:"Michael",surname:"Egbuche",slug:"chukwudi-michael-egbuche",fullName:"Chukwudi Michael Egbuche",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/332819/images/14624_n.jpg",biography:"I an Dr. Chukwudi Michael Egbuche. I am a Senior Lecturer in the Department of Parasitology and Entomology, Nnamdi Azikiwe University, Awka.",institutionString:null,institution:{name:"Nnamdi Azikiwe University",country:{name:"Nigeria"}}},{id:"284232",title:"Mr.",name:"Nikunj",middleName:"U",surname:"Tandel",slug:"nikunj-tandel",fullName:"Nikunj Tandel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/284232/images/8275_n.jpg",biography:'Mr. Nikunj Tandel has completed his Master\'s degree in Biotechnology from VIT University, India in the year of 2012. He is having 8 years of research experience especially in the field of malaria epidemiology, immunology, and nanoparticle-based drug delivery system against the infectious diseases, autoimmune disorders and cancer. He has worked for the NIH funded-International Center of Excellence in Malaria Research project "Center for the study of complex malaria in India (CSCMi)" in collaboration with New York University. The preliminary objectives of the study are to understand and develop the evidence-based tools and interventions for the control and prevention of malaria in different sites of the INDIA. Alongside, with the help of next-generation genomics study, the team has studied the antimalarial drug resistance in India. Further, he has extended his research in the development of Humanized mice for the study of liver-stage malaria and identification of molecular marker(s) for the Artemisinin resistance. At present, his research focuses on understanding the role of B cells in the activation of CD8+ T cells in malaria. Received the CSIR-SRF (Senior Research Fellow) award-2018, FIMSA (Federation of Immunological Societies of Asia-Oceania) Travel Bursary award to attend the IUIS-IIS-FIMSA Immunology course-2019',institutionString:"Nirma University",institution:{name:"Nirma University",country:{name:"India"}}},{id:"334383",title:"Ph.D.",name:"Simone",middleName:"Ulrich",surname:"Ulrich Picoli",slug:"simone-ulrich-picoli",fullName:"Simone Ulrich Picoli",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/334383/images/15919_n.jpg",biography:"Graduated in Pharmacy from Universidade Luterana do Brasil (1999), Master in Agricultural and Environmental Microbiology from Federal University of Rio Grande do Sul (2002), Specialization in Clinical Microbiology from Universidade de São Paulo, USP (2007) and PhD in Sciences in Gastroenterology and Hepatology (2012). She is currently an Adjunct Professor at Feevale University in Medicine and Biomedicine courses and a permanent professor of the Academic Master\\'s Degree in Virology. She has experience in the field of Microbiology, with an emphasis on Bacteriology, working mainly on the following topics: bacteriophages, bacterial resistance, clinical microbiology and food microbiology.",institutionString:null,institution:{name:"Universidade Feevale",country:{name:"Brazil"}}},{id:"229220",title:"Dr.",name:"Amjad",middleName:"Islam",surname:"Aqib",slug:"amjad-aqib",fullName:"Amjad Aqib",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229220/images/system/229220.png",biography:"Dr. Amjad Islam Aqib obtained a DVM and MSc (Hons) from University of Agriculture Faisalabad (UAF), Pakistan, and a PhD from the University of Veterinary and Animal Sciences Lahore, Pakistan. Dr. Aqib joined the Department of Clinical Medicine and Surgery at UAF for one year as an assistant professor where he developed a research laboratory designated for pathogenic bacteria. Since 2018, he has been Assistant Professor/Officer in-charge, Department of Medicine, Manager Research Operations and Development-ORIC, and President One Health Club at Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan. He has nearly 100 publications to his credit. His research interests include epidemiological patterns and molecular analysis of antimicrobial resistance and modulation and vaccine development against animal pathogens of public health concern.",institutionString:"Cholistan University of Veterinary and Animal Sciences",institution:null},{id:"62900",title:"Prof.",name:"Fethi",middleName:null,surname:"Derbel",slug:"fethi-derbel",fullName:"Fethi Derbel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62900/images/system/62900.jpeg",biography:"Professor Fethi Derbel was born in 1960 in Tunisia. He received his medical degree from the Sousse Faculty of Medicine at Sousse, University of Sousse, Tunisia. He completed his surgical residency in General Surgery at the University Hospital Farhat Hached of Sousse and was a member of the Unit of Liver Transplantation in the University of Rennes, France. He then worked in the Department of Surgery at the Sahloul University Hospital in Sousse. Professor Derbel is presently working at the Clinique les Oliviers, Sousse, Tunisia. His hospital activities are mostly concerned with laparoscopic, colorectal, pancreatic, hepatobiliary, and gastric surgery. He is also very interested in hernia surgery and performs ventral hernia repairs and inguinal hernia repairs. He has been a member of the GREPA and Tunisian Hernia Society (THS). During his residency, he managed patients suffering from diabetic foot, and he was very interested in this pathology. For this reason, he decided to coordinate a book project dealing with the diabetic foot. Professor Derbel has published many articles in journals and collaborates intensively with IntechOpen Access Publisher as an editor.",institutionString:"Clinique les Oliviers",institution:null},{id:"300144",title:"Dr.",name:"Meriem",middleName:null,surname:"Braiki",slug:"meriem-braiki",fullName:"Meriem Braiki",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/300144/images/system/300144.jpg",biography:"Dr. Meriem Braiki is a specialist in pediatric surgeon from Tunisia. She was born in 1985. She received her medical degree from the University of Medicine at Sousse, Tunisia. She achieved her surgical residency training periods in Pediatric Surgery departments at University Hospitals in Monastir, Tunis and France.\r\nShe is currently working at the Pediatric surgery department, Sidi Bouzid Hospital, Tunisia. Her hospital activities are mostly concerned with laparoscopic, parietal, urological and digestive surgery. She has published several articles in diffrent journals.",institutionString:"Sidi Bouzid Regional Hospital",institution:null},{id:"229481",title:"Dr.",name:"Erika M.",middleName:"Martins",surname:"de Carvalho",slug:"erika-m.-de-carvalho",fullName:"Erika M. de Carvalho",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229481/images/6397_n.jpg",biography:null,institutionString:null,institution:{name:"Oswaldo Cruz Foundation",country:{name:"Brazil"}}},{id:"186537",title:"Prof.",name:"Tonay",middleName:null,surname:"Inceboz",slug:"tonay-inceboz",fullName:"Tonay Inceboz",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/186537/images/system/186537.jfif",biography:"I was graduated from Ege University of Medical Faculty (Turkey) in 1988 and completed his Med. PhD degree in Medical Parasitology at the same university. I became an Associate Professor in 2008 and Professor in 2014. I am currently working as a Professor at the Department of Medical Parasitology at Dokuz Eylul University, Izmir, Turkey.\n\nI have given many lectures, presentations in different academic meetings. I have more than 60 articles in peer-reviewed journals, 18 book chapters, 1 book editorship.\n\nMy research interests are Echinococcus granulosus, Echinococcus multilocularis (diagnosis, life cycle, in vitro and in vivo cultivation), and Trichomonas vaginalis (diagnosis, PCR, and in vitro cultivation).",institutionString:"Dokuz Eylül University",institution:{name:"Dokuz Eylül University",country:{name:"Turkey"}}},{id:"71812",title:"Prof.",name:"Hanem Fathy",middleName:"Fathy",surname:"Khater",slug:"hanem-fathy-khater",fullName:"Hanem Fathy Khater",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/71812/images/1167_n.jpg",biography:"Prof. Khater is a Professor of Parasitology at Benha University, Egypt. She studied for her doctoral degree, at the Department of Entomology, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, USA. She has completed her Ph.D. degrees in Parasitology in Egypt, from where she got the award for “the best scientific Ph.D. dissertation”. She worked at the School of Biological Sciences, Bristol, England, the UK in controlling insects of medical and veterinary importance as a grant from Newton Mosharafa, the British Council. Her research is focused on searching of pesticides against mosquitoes, house flies, lice, green bottle fly, camel nasal botfly, soft and hard ticks, mites, and the diamondback moth as well as control of several parasites using safe and natural materials to avoid drug resistances and environmental contamination.",institutionString:null,institution:{name:"Banha University",country:{name:"Egypt"}}},{id:"99780",title:"Prof.",name:"Omolade",middleName:"Olayinka",surname:"Okwa",slug:"omolade-okwa",fullName:"Omolade Okwa",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/99780/images/system/99780.jpg",biography:"Omolade Olayinka Okwa is presently a Professor of Parasitology at Lagos State University, Nigeria. She has a PhD in Parasitology (1997), an MSc in Cellular Parasitology (1992), and a BSc (Hons) Zoology (1990) all from the University of Ibadan, Nigeria. She teaches parasitology at the undergraduate and postgraduate levels. She was a recipient of a Commonwealth fellowship supported by British Council tenable at the Centre for Entomology and Parasitology (CAEP), Keele University, United Kingdom between 2004 and 2005. She was awarded an Honorary Visiting Research Fellow at the same university from 2005 to 2007. \nShe has been an external examiner to the Department of Veterinary Microbiology and Parasitology, University of Ibadan, MSc programme between 2010 and 2012. She is a member of the Nigerian Society of Experimental Biology (NISEB), Parasitology and Public Health Society of Nigeria (PPSN), Science Association of Nigeria (SAN), Zoological Society of Nigeria (ZSN), and is Vice Chairperson of the Organisation of Women in Science (OWSG), LASU chapter. She served as Head of Department of Zoology and Environmental Biology, Lagos State University from 2007 to 2010 and 2014 to 2016. She is a reviewer for several local and international journals such as Unilag Journal of Science, Libyan Journal of Medicine, Journal of Medicine and Medical Sciences, and Annual Research and Review in Science. \nShe has authored 45 scientific research publications in local and international journals, 8 scientific reviews, 4 books, and 3 book chapters, which includes the books “Malaria Parasites” and “Malaria” which are IntechOpen access publications.",institutionString:"Lagos State University",institution:{name:"Lagos State University",country:{name:"Nigeria"}}},{id:"273100",title:"Dr.",name:"Vijay",middleName:null,surname:"Gayam",slug:"vijay-gayam",fullName:"Vijay Gayam",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/273100/images/system/273100.jpeg",biography:"Dr. Vijay Bhaskar Reddy Gayam is currently practicing as an internist at Interfaith Medical Center in Brooklyn, New York, USA. He is also a Clinical Assistant Professor at the SUNY Downstate University Hospital and Adjunct Professor of Medicine at the American University of Antigua. He is a holder of an M.B.B.S. degree bestowed to him by Osmania Medical College and received his M.D. at Interfaith Medical Center. His career goals thus far have heavily focused on direct patient care, medical education, and clinical research. He currently serves in two leadership capacities; Assistant Program Director of Medicine at Interfaith Medical Center and as a Councilor for the American\r\nFederation for Medical Research. As a true academician and researcher, he has more than 50 papers indexed in international peer-reviewed journals. He has also presented numerous papers in multiple national and international scientific conferences. His areas of research interest include general internal medicine, gastroenterology and hepatology. He serves as an editor, editorial board member and reviewer for multiple international journals. His research on Hepatitis C has been very successful and has led to multiple research awards, including the 'Equity in Prevention and Treatment Award” from the New York Department of Health Viral Hepatitis Symposium (2018) and the 'Presidential Poster Award” awarded to him by the American College of Gastroenterology (2018). He was also awarded 'Outstanding Clinician in General Medicine” by Venus International Foundation for his extensive research expertise and services, perform over and above the standard expected in the advancement of healthcare, patient safety and quality of care.",institutionString:"Interfaith Medical Center",institution:{name:"Interfaith Medical Center",country:{name:"United States of America"}}},{id:"93517",title:"Dr.",name:"Clement",middleName:"Adebajo",surname:"Meseko",slug:"clement-meseko",fullName:"Clement Meseko",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/93517/images/system/93517.jpg",biography:"Dr. Clement Meseko obtained DVM and PhD degree in Veterinary Medicine and Virology respectively. He has worked for over 20 years in both private and public sectors including the academia, contributing to knowledge and control of infectious disease. Through the application of epidemiological skill, classical and molecular virological skills, he investigates viruses of economic and public health importance for the mitigation of the negative impact on people, animal and the environment in the context of Onehealth. \r\nDr. Meseko’s field experience on animal and zoonotic diseases and pathogen dynamics at the human-animal interface over the years shaped his carrier in research and scientific inquiries. He has been part of the investigation of Highly Pathogenic Avian Influenza incursions in sub Saharan Africa and monitors swine Influenza (Pandemic influenza Virus) agro-ecology and potential for interspecies transmission. He has authored and reviewed a number of journal articles and book chapters.",institutionString:"National Veterinary Research Institute",institution:{name:"National Veterinary Research Institute",country:{name:"Nigeria"}}},{id:"158026",title:"Prof.",name:"Shailendra K.",middleName:null,surname:"Saxena",slug:"shailendra-k.-saxena",fullName:"Shailendra K. Saxena",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRET3QAO/Profile_Picture_2022-05-10T10:10:26.jpeg",biography:"Professor Dr. Shailendra K. Saxena is a vice dean and professor at King George's Medical University, Lucknow, India. His research interests involve understanding the molecular mechanisms of host defense during human viral infections and developing new predictive, preventive, and therapeutic strategies for them using Japanese encephalitis virus (JEV), HIV, and emerging viruses as a model via stem cell and cell culture technologies. His research work has been published in various high-impact factor journals (Science, PNAS, Nature Medicine) with a high number of citations. He has received many awards and honors in India and abroad including various Young Scientist Awards, BBSRC India Partnering Award, and Dr. JC Bose National Award of Department of Biotechnology, Min. of Science and Technology, Govt. of India. Dr. Saxena is a fellow of various international societies/academies including the Royal College of Pathologists, United Kingdom; Royal Society of Medicine, London; Royal Society of Biology, United Kingdom; Royal Society of Chemistry, London; and Academy of Translational Medicine Professionals, Austria. He was named a Global Leader in Science by The Scientist. He is also an international opinion leader/expert in vaccination for Japanese encephalitis by IPIC (UK).",institutionString:"King George's Medical University",institution:{name:"King George's Medical University",country:{name:"India"}}},{id:"94928",title:"Dr.",name:"Takuo",middleName:null,surname:"Mizukami",slug:"takuo-mizukami",fullName:"Takuo Mizukami",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/94928/images/6402_n.jpg",biography:null,institutionString:null,institution:{name:"National Institute of Infectious Diseases",country:{name:"Japan"}}},{id:"233433",title:"Dr.",name:"Yulia",middleName:null,surname:"Desheva",slug:"yulia-desheva",fullName:"Yulia Desheva",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/233433/images/system/233433.png",biography:"Dr. Yulia Desheva is a leading researcher at the Institute of Experimental Medicine, St. Petersburg, Russia. She is a professor in the Stomatology Faculty, St. Petersburg State University. She has expertise in the development and evaluation of a wide range of live mucosal vaccines against influenza and bacterial complications. Her research interests include immunity against influenza and COVID-19 and the development of immunization schemes for high-risk individuals.",institutionString:'Federal State Budgetary Scientific Institution "Institute of Experimental Medicine"',institution:null},{id:"238958",title:"Mr.",name:"Atamjit",middleName:null,surname:"Singh",slug:"atamjit-singh",fullName:"Atamjit Singh",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/238958/images/6575_n.jpg",biography:null,institutionString:null,institution:null},{id:"333753",title:"Dr.",name:"Rais",middleName:null,surname:"Ahmed",slug:"rais-ahmed",fullName:"Rais Ahmed",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/333753/images/20168_n.jpg",biography:null,institutionString:null,institution:null},{id:"252058",title:"M.Sc.",name:"Juan",middleName:null,surname:"Sulca",slug:"juan-sulca",fullName:"Juan Sulca",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/252058/images/12834_n.jpg",biography:null,institutionString:null,institution:null},{id:"191392",title:"Dr.",name:"Marimuthu",middleName:null,surname:"Govindarajan",slug:"marimuthu-govindarajan",fullName:"Marimuthu Govindarajan",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/191392/images/5828_n.jpg",biography:"Dr. M. Govindarajan completed his BSc degree in Zoology at Government Arts College (Autonomous), Kumbakonam, and MSc, MPhil, and PhD degrees at Annamalai University, Annamalai Nagar, Tamil Nadu, India. He is serving as an assistant professor at the Department of Zoology, Annamalai University. His research interests include isolation, identification, and characterization of biologically active molecules from plants and microbes. He has identified more than 20 pure compounds with high mosquitocidal activity and also conducted high-quality research on photochemistry and nanosynthesis. He has published more than 150 studies in journals with impact factor and 2 books in Lambert Academic Publishing, Germany. He serves as an editorial board member in various national and international scientific journals.",institutionString:null,institution:null},{id:"274660",title:"Dr.",name:"Damodar",middleName:null,surname:"Paudel",slug:"damodar-paudel",fullName:"Damodar Paudel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/274660/images/8176_n.jpg",biography:"I am DrDamodar Paudel,currently working as consultant Physician in Nepal police Hospital.",institutionString:null,institution:null},{id:"241562",title:"Dr.",name:"Melvin",middleName:null,surname:"Sanicas",slug:"melvin-sanicas",fullName:"Melvin Sanicas",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/241562/images/6699_n.jpg",biography:null,institutionString:null,institution:null},{id:"337446",title:"Dr.",name:"Maria",middleName:null,surname:"Zavala-Colon",slug:"maria-zavala-colon",fullName:"Maria Zavala-Colon",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Puerto Rico, Medical Sciences Campus",country:{name:"United States of America"}}},{id:"338856",title:"Mrs.",name:"Nur Alvira",middleName:null,surname:"Pascawati",slug:"nur-alvira-pascawati",fullName:"Nur Alvira Pascawati",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Universitas Respati Yogyakarta",country:{name:"Indonesia"}}},{id:"441116",title:"Dr.",name:"Jovanka M.",middleName:null,surname:"Voyich",slug:"jovanka-m.-voyich",fullName:"Jovanka M. Voyich",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Montana State University",country:{name:"United States of America"}}},{id:"330412",title:"Dr.",name:"Muhammad",middleName:null,surname:"Farhab",slug:"muhammad-farhab",fullName:"Muhammad Farhab",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Agriculture Faisalabad",country:{name:"Pakistan"}}},{id:"349495",title:"Dr.",name:"Muhammad",middleName:null,surname:"Ijaz",slug:"muhammad-ijaz",fullName:"Muhammad Ijaz",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Veterinary and Animal Sciences",country:{name:"Pakistan"}}}]}},subseries:{item:{id:"93",type:"subseries",title:"Inclusivity and Social Equity",keywords:"Social contract, SDG, Human rights, Inclusiveness, Equity, Democracy, Personal learning, Collaboration, Glocalization",scope:"
\r\n\tThis topic is dedicated to the efforts and promotion of UNESCO SDG4, the UNESCO initiative on the future of education, and the need for a new social contract for education. It aims to disseminate knowledge on policies, strategies, methods, and technologies that increase the resilience and sustainability of the development of the future of education and the new social contract for education. It will also consider the global challenges such as globalization, demographic change, digital transformation, climate change, environment and the social pillars of sustainable development.
\r\n
\r\n\tResponses to the pandemic and the widespread discontent that preceded it must be based on a new social contract and a New Global Deal for education that ensures equal opportunities for all and respects all people’s rights and freedoms (UNESCO; 2021). Such a new social contract, as proposed by UNESCO, must be based on the general principles underlying human rights - inclusion and equality, cooperation and solidarity, and collective responsibility and interconnectedness - and be guided by the following fundamental principle: Ensure that everyone has access to quality education throughout their lives.
\r\n
\r\n\tWe face the dual challenge of delivering on the unfulfilled promise of ensuring the right to quality education for every child, youth, and adult, as well as fully realizing the transformative potential of education as a pathway to a more sustainable collective future. To achieve this, we need a new social contract for education that eliminates inequities while transforming the future. This new social contract must be based on human rights and the principles of non-discrimination, social justice, respect for life, human dignity, and cultural diversity. It must include an ethic of care, reciprocity and solidarity. The new social contract builds on inclusiveness, equity, lifelong learning, SDG, collaboration and personal learning in a global context for democracy.
\r\n
\r\n\tAt an international level, the adoption of the Open Educational Resources recommendation and the Open Science recommendation represents an important step towards building more open and inclusive knowledge societies as well as the achievement of the UN 2030 Agenda. Indeed, implementing the recommendations will help to achieve at least five more Sustainable Development Goals (SDGs) that are intertwined with the topic of this book series, namely SDG 5 (Gender equality), SDG 9 (Industry, innovation and infrastructure), SDG 10 (Reduced inequalities within and across countries), SDG 16 (Peace, justice and strong institutions) and SDG 17 (Partnerships for the goals).
",coverUrl:"https://cdn.intechopen.com/series_topics/covers/93.jpg",hasOnlineFirst:!1,hasPublishedBooks:!1,annualVolume:11977,editor:{id:"210060",title:"Prof. Dr.",name:"Ebba",middleName:null,surname:"Ossiannilsson",slug:"ebba-ossiannilsson",fullName:"Ebba Ossiannilsson",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002g6LkBQAU/Profile_Picture_2022-02-28T13:31:48.png",biography:"Professor Dr. Ebba Ossiannilsson is an independent researcher, expert, consultant, quality auditor and influencer in the fields of open, flexible online and distance learning (OFDL) and the 'new normal'. Her focus is on quality, innovation, leadership, and personalised learning. She works primarily at the strategic and policy levels, both nationally and internationally, and with key international organisations. She is committed to promoting and improving OFDL in the context of SDG4 and the future of education. Ossiannilsson has more than 20 years of experience in her current field, but more than 40 years in the education sector. She works as a reviewer and expert for the European Commission and collaborates with the Joint Research Centre for Quality in Open Education. Ossiannilsson also collaborates with ITCILO and ICoBC (International Council on Badges and Credentials). She is a member of the ICDE Board of Directors and has previously served on the boards of EDEN and EUCEN. Ossiannilsson is a quality expert and reviewer for ICDE, EDEN and the EADTU. She chairs the ICDE OER Advocacy Committee and is a member of the ICDE Quality Network. She is regularly invited as a keynote speaker at conferences. She is a guest editor for several special issues and a member of the editorial board of several scientific journals. She has published more than 200 articles and is currently working on book projects in the field of OFDL. Ossiannilsson is a visiting professor at several international universities and was recently appointed Professor and Research Fellow at Victoria University of Wellington, NZ. Ossiannilsson has been awarded the following fellowships: EDEN Fellows, EDEN Council of Fellows, and Open Education Europe. She is a ICDE OER Ambassador, Open Education Europe Ambassador, GIZ Ambassador for Quality in Digital Learning, and part of the Globe-Community of Digital Learning and Champion of SPARC Europe. On a national level, she is a quality developer at the Swedish Institute for Standards (SIS) and for ISO. She is a member of the Digital Skills and Jobs Coalition Sweden and Vice President of the Swedish Association for Distance Education. She is currently working on a government initiative on quality in distance education at the National Council for Higher Education. She holds a Ph.D. from the University of Oulu, Finland.",institutionString:"Swedish Association for Distance Education, Sweden",institution:null},editorTwo:null,editorThree:null,series:{id:"24",title:"Sustainable Development",doi:"10.5772/intechopen.100361",issn:null},editorialBoard:[{id:"320585",title:"Ph.D.",name:"Deborah",middleName:null,surname:"Young",slug:"deborah-young",fullName:"Deborah Young",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y00002vZLcTQAW/Profile_Picture_2022-05-10T08:30:47.jpg",institutionString:"Empowering Communities Globally",institution:null},{id:"348038",title:"Associate Prof.",name:"Feyza",middleName:null,surname:"Bhatti",slug:"feyza-bhatti",fullName:"Feyza Bhatti",profilePictureURL:"https://mts.intechopen.com/storage/users/348038/images/system/348038.jpg",institutionString:"Girne American University",institution:{name:"Girne American University",institutionURL:null,country:{name:"Cyprus"}}},{id:"302382",title:"Dr.",name:"Gina",middleName:null,surname:"Alvarado",slug:"gina-alvarado",fullName:"Gina Alvarado",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002mZoL9QAK/Profile_Picture_2022-05-26T08:14:10.jpg",institutionString:"Landesa, Seattle",institution:null},{id:"128665",title:"Prof.",name:"Man-Chung",middleName:null,surname:"Chiu",slug:"man-chung-chiu",fullName:"Man-Chung Chiu",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bR9OrQAK/Profile_Picture_2022-03-09T08:36:59.JPG",institutionString:null,institution:{name:"Beijing Normal University",institutionURL:null,country:{name:"China"}}}]},onlineFirstChapters:{paginationCount:0,paginationItems:[]},publishedBooks:{paginationCount:1,paginationItems:[{type:"book",id:"10843",title:"Persistent Organic Pollutants (POPs)",subtitle:"Monitoring, Impact and Treatment",coverURL:"https://cdn.intechopen.com/books/images_new/10843.jpg",slug:"persistent-organic-pollutants-pops-monitoring-impact-and-treatment",publishedDate:"April 13th 2022",editedByType:"Edited by",bookSignature:"Mohamed Nageeb Rashed",hash:"f5b1589f0a990b6114fef2dadc735dd9",volumeInSeries:1,fullTitle:"Persistent Organic Pollutants (POPs) - 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\r\n\tIn general, the harsher the environmental conditions in an ecosystem, the lower the biodiversity. Changes in the environment caused by human activity accelerate the impoverishment of biodiversity.
\r\n
\r\n\tBiodiversity refers to “the variability of living organisms from any source, including terrestrial, marine and other aquatic ecosystems and the ecological complexes of which they are part; it includes diversity within each species, between species, and that of ecosystems”.
\r\n
\r\n\tBiodiversity provides food security and constitutes a gene pool for biotechnology, especially in the field of agriculture and medicine, and promotes the development of ecotourism.
\r\n
\r\n\tCurrently, biologists admit that we are witnessing the first phases of the seventh mass extinction caused by human intervention. It is estimated that the current rate of extinction is between a hundred and a thousand times faster than it was when man first appeared. The disappearance of species is caused not only by an accelerated rate of extinction, but also by a decrease in the rate of emergence of new species as human activities degrade the natural environment. The conservation of biological diversity is "a common concern of humanity" and an integral part of the development process. Its objectives are “the conservation of biological diversity, the sustainable use of its components, and the fair and equitable sharing of the benefits resulting from the use of genetic resources”.
\r\n
\r\n\tThe following are the main causes of biodiversity loss:
\r\n
\r\n\t• The destruction of natural habitats to expand urban and agricultural areas and to obtain timber, minerals and other natural resources.
\r\n
\r\n\t• The introduction of alien species into a habitat, whether intentionally or unintentionally which has an impact on the fauna and flora of the area, and as a result, they are reduced or become extinct.
\r\n
\r\n\t• Pollution from industrial and agricultural products, which devastate the fauna and flora, especially those in fresh water.
\r\n
\r\n\t• Global warming, which is seen as a threat to biological diversity, and will become increasingly important in the future.
",coverUrl:"https://cdn.intechopen.com/series_topics/covers/40.jpg",keywords:"Ecosystems, Biodiversity, Fauna, Taxonomy, Invasive species, Destruction of habitats, Overexploitation of natural resources, Pollution, Global warming, Conservation of natural spaces, Bioremediation"},{id:"39",title:"Environmental Resilience and Management",scope:"
\r\n\tThe environment is subject to severe anthropic effects. Among them are those associated with pollution, resource extraction and overexploitation, loss of biodiversity, soil degradation, disorderly land occupation and planning, and many others. These anthropic effects could potentially be caused by any inadequate management of the environment. However, ecosystems have a resilience that makes them react to disturbances which mitigate the negative effects. It is critical to understand how ecosystems, natural and anthropized, including urban environments, respond to actions that have a negative influence and how they are managed. It is also important to establish when the limits marked by the resilience and the breaking point are achieved and when no return is possible. The main focus for the chapters is to cover the subjects such as understanding how the environment resilience works, the mechanisms involved, and how to manage them in order to improve our interactions with the environment and promote the use of adequate management practices such as those outlined in the United Nations’ Sustainable Development Goals.
\r\n\tPollution is caused by a wide variety of human activities and occurs in diverse forms, for example biological, chemical, et cetera. In recent years, significant efforts have been made to ensure that the environment is clean, that rigorous rules are implemented, and old laws are updated to reduce the risks towards humans and ecosystems. However, rapid industrialization and the need for more cultivable sources or habitable lands, for an increasing population, as well as fewer alternatives for waste disposal, make the pollution control tasks more challenging. Therefore, this topic will focus on assessing and managing environmental pollution. It will cover various subjects, including risk assessment due to the pollution of ecosystems, transport and fate of pollutants, restoration or remediation of polluted matrices, and efforts towards sustainable solutions to minimize environmental pollution.
\r\n\tWater is not only a crucial substance needed for biological life on Earth, but it is also a basic requirement for the existence and development of the human society. Owing to the importance of water to life on Earth, early researchers conducted numerous studies and analyses on the liquid form of water from the perspectives of chemistry, physics, earth science, and biology, and concluded that Earth is a "water polo". Water covers approximately 71% of Earth's surface. However, 97.2% of this water is seawater, 21.5% is icebergs and glaciers, and only 0.65% is freshwater that can be used directly by humans. As a result, the amount of water reserves available for human consumption is limited. The development, utilization, and protection of freshwater resources has become the focus of water science research for the continued improvement of human livelihoods and society.
\r\n
\r\n\tWater exists as solid, liquid, and gas within Earth’s atmosphere, lithosphere, and biosphere. Liquid water is used for a variety of purposes besides drinking, including power generation, ecology, landscaping, and shipping. Because water is involved in various environmental hydrological processes as well as numerous aspects of the economy and human society, the study of various phenomena in the hydrosphere, the laws governing their occurrence and development, the relationship between the hydrosphere and other spheres of Earth, and the relationship between water and social development, are all part of water science. Knowledge systems for water science are improving continuously. Water science has become a specialized field concerned with the identification of its physical, chemical, and biological properties. In addition, it reveals the laws of water distribution, movement, and circulation, and proposes methods and tools for water development, utilization, planning, management, and protection. Currently, the field of water science covers research related to topics such as hydrology, water resources and water environment. It also includes research on water related issues such as safety, engineering, economy, law, culture, information, and education.
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Biochemistry also studies small signaling molecules, coenzymes, inhibitors, vitamins, and hormones, which play roles in life processes. Biochemical experimentation, besides coopting classical chemistry methods, e.g., chromatography, adopted new techniques, e.g., X-ray diffraction, electron microscopy, NMR, radioisotopes, and developed sophisticated microbial genetic tools, e.g., auxotroph mutants and their revertants, fermentation, etc. More recently, biochemistry embraced the ‘big data’ omics systems. Initial biochemical studies have been exclusively analytic: dissecting, purifying, and examining individual components of a biological system; in the apt words of Efraim Racker (1913 –1991), “Don’t waste clean thinking on dirty enzymes.” Today, however, biochemistry is becoming more agglomerative and comprehensive, setting out to integrate and describe entirely particular biological systems. The ‘big data’ metabolomics can define the complement of small molecules, e.g., in a soil or biofilm sample; proteomics can distinguish all the comprising proteins, e.g., serum; metagenomics can identify all the genes in a complex environment, e.g., the bovine rumen. This Biochemistry Series will address the current research on biomolecules and the emerging trends with great promise.",coverUrl:"https://cdn.intechopen.com/series/covers/11.jpg",latestPublicationDate:"June 24th, 2022",hasOnlineFirst:!0,numberOfOpenTopics:4,numberOfPublishedChapters:314,numberOfPublishedBooks:31,editor:{id:"31610",title:"Dr.",name:"Miroslav",middleName:null,surname:"Blumenberg",fullName:"Miroslav Blumenberg",profilePictureURL:"https://mts.intechopen.com/storage/users/31610/images/system/31610.jpg",biography:"Miroslav Blumenberg, Ph.D., was born in Subotica and received his BSc in Belgrade, Yugoslavia. He completed his Ph.D. at MIT in Organic Chemistry; he followed up his Ph.D. with two postdoctoral study periods at Stanford University. Since 1983, he has been a faculty member of the RO Perelman Department of Dermatology, NYU School of Medicine, where he is codirector of a training grant in cutaneous biology. Dr. Blumenberg’s research is focused on the epidermis, expression of keratin genes, transcription profiling, keratinocyte differentiation, inflammatory diseases and cancers, and most recently the effects of the microbiome on the skin. 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Behind these definitions are hidden all the aspects of normal and pathological functioning of all processes that the topic ‘Metabolism’ will cover within the Biochemistry Series. 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Thus proteomics, an area of research that detects all protein forms expressed in an organism, including splice isoforms and post-translational modifications, is more suitable than genomics for a comprehensive understanding of the biochemical processes that govern life. The most common proteomics applications are currently in the clinical field for the identification, in a variety of biological matrices, of biomarkers for diagnosis and therapeutic intervention of disorders. From the comparison of proteomic profiles of control and disease or different physiological states, which may emerge, changes in protein expression can provide new insights into the roles played by some proteins in human pathologies. Understanding how proteins function and interact with each other is another goal of proteomics that makes this approach even more intriguing. Specialized technology and expertise are required to assess the proteome of any biological sample. Currently, proteomics relies mainly on mass spectrometry (MS) combined with electrophoretic (1 or 2-DE-MS) and/or chromatographic techniques (LC-MS/MS). MS is an excellent tool that has gained popularity in proteomics because of its ability to gather a complex body of information such as cataloging protein expression, identifying protein modification sites, and defining protein interactions. 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