Microarray analysis of
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More than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\\n\\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\\n\\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\\n\\nAdditionally, each book published by IntechOpen contains original content and research findings.
\\n\\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\\n\\n\\n\\n
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Simba Information has released its Open Access Book Publishing 2020 - 2024 report and has again identified IntechOpen as the world’s largest Open Access book publisher by title count.
\n\nSimba Information is a leading provider for market intelligence and forecasts in the media and publishing industry. The report, published every year, provides an overview and financial outlook for the global professional e-book publishing market.
\n\nIntechOpen, De Gruyter, and Frontiers are the largest OA book publishers by title count, with IntechOpen coming in at first place with 5,101 OA books published, a good 1,782 titles ahead of the nearest competitor.
\n\nSince the first Open Access Book Publishing report published in 2016, IntechOpen has held the top stop each year.
\n\n\n\nMore than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\n\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\n\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\n\nAdditionally, each book published by IntechOpen contains original content and research findings.
\n\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\n\n\n\n
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\r\n\tThe goal of this book is to give the reader an overview of a field related to various applications in chemistry, chemical engineering, and nanotechnology. This book aims to provide information about the design of ion exchangers, their application in environmental technologies, and in biotechnology and pharmaceutical applications. This book will be written by authors in the field of experimental methods and critical reviews from multi-disciplines such as chemistry, membranes, and materials science. Among others, some of the topics covered will be Structure of ion exchangers, Synthesis of ion exchangers, Synthesis of inorganic ion exchangers, Properties of ion exchangers, Ion exchange voltammetry, Ion exchange as a separations method, Ion exchange in analytical chemistry, Ion exchange and extraction, Ion exchange membranes, Preparation of organic-inorganic hybrid ion exchangers, Application in environmental technologies, Application in biotechnology and pharmaceutical applications.
\r\n\r\n\tIn this book, the authors will focus on recent studies, applications, and new technological developments on the fundamental properties of ion exchangers.
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The polysaccharide capsule often constitutes the outermost layer of the cell, and therefore is often involved in mediating direct interactions between the bacteria and its environment. It is due to these interactions that polysaccharide capsules have been implicated as important virulence factors for many bacterial pathogens.
Bacterial extracellular polysaccharides (EPS) may be classified as either capsular polysaccharides (CPS), where the polysaccharide is intimately associated with the cell surface, or as slime polysaccharides, where the polysaccharide is loosely associated with the cell [1]. Differentiation between these forms is difficult since CPS may be released from the cell, giving the appearance of a slime polysaccharide [1]. In turn, distinguishing between CPS and other cell surface polysaccharides, such as O-antigenic moieties of lipopolysaccharide (LPS), may also be difficult, since CPS may be found associated with LPS [1].
Capsular polysaccharides are highly hydrated molecules that are over 95% water [2]. They are often linked to the cell surface of the bacterium via covalent attachments to either phospholipid or lipid-A molecules, although some CPS may be associated with the cell in the absence of a membrane anchor [1, 3]. Capsular polysaccharides can be either homo- or heteropolymers composed of repeating monosaccharides joined by glycosidic linkages [4]. The multiple hydroxyl groups present within each monosaccharide may be involved in the formation of the glycosidic bond, therefore, any two monosaccharides may be joined in a number of configurations, which leads to large structural diversity among CPS types. In the case of human pathogens, a large number of different capsule serotypes have been identified, and certain CPS or K-antigens have been associated with specific infections [4]. For example, the
The genetic loci necessary for the production of bacterial capsules are primarily clustered at a single chromosomal locus, which allows for the coordinate regulation of a large number of genes that may be involved in both the biosynthesis and export of capsular polysaccharides [4, 1]. In most bacterial species, the capsule gene clusters demonstrate conserved genetic organization. The capsules of
The A+T composition of capsule gene clusters is often significantly higher than the rest of the chromosome, suggesting a common ancestry of capsule genes in gram-negative bacteria [9]. It is likely that these A+T rich regions have been horizontally transferred between bacterial species. In addition, the A+T ratio of region 2 DNA of group 2
The production of a polysaccharide capsule is widespread in pathogenic bacteria. A number of functions have been assigned to bacterial capsules including: prevention of dessication, adherence, resistance to non-specific host immunity, resistance to specific host immunity, and mediating the diffusion of molecules through to the cell surface [4, 1].
Capsules may form a hydrated gel around the surface of the bacterial cell, which may protect the bacteria from the harmful effects of dessication [10]. This may increase the survival of encapsulated bacteria outside of the host, promoting the transmission of pathogenic bacteria from one host to another [4]. Mucoid isolates of
Capsular polysaccharides may promote adherence of bacteria to both surfaces and other bacterial cells, which may facilitate colonization of a particular niche and may lead to the formation of biofilms [13]. Cell-surface polysaccharides have been shown to mediate the attachment of bacterial cells to one another, leading to biofilm formation and persistence of the organisms during colonization [1, 14].
Capsular polysaccharides are one of the components responsible for resistance to the non-specific immunity of the host. The presence of a capsule is thought to confer resistance to non-specific host defense mechanisms such as complement and complement-mediated opsonophagocytosis [4]. Bacterial capsules may resist complement-mediated killing by providing a permeability barrier to complement components, which masks the underlying cell surface structures that activate complement [15]. The capsule may also act in concert with O-antigens to confer resistance to complement-mediated killing [16]. As a result, a combination of cell surface structures is responsible for conferring resistance to killing by the complement cascade [4]. Finally, capsules are responsible for resistance to complement-mediated opsonophagocytosis. This resistance may be due to steric effects, which results in the capsule acting as a barrier between the C3b deposited on the bacterial surface and the C3b receptors present on phagocytes [4]. Alternatively, the resistance to opsonophagocytosis may be due to the net negative charge of the polysaccharide capsule [17].
Capsules may also confer resistance to the specific immune response of the host. Although most capsular polysaccharides can elicit an immune response, some capsules are poorly immunogenic [4]. Examples of such capsules include those containing NeuNAc, such as the
At the time our studies were initiated some cell-associated antigens had been identified and characterized in
To identify the genetic determinants that confer enhanced virulence in
The 373-bp DNA insert from pDD1015 was cloned into a mobilizable suicide vector, pSKM11 [44]. The resulting plasmid, pSR1015, was mobilized into wildtype
Immunogold electron microscopy of
Two methods were used to clone the genes involved in the production and export of type I O-PS. The DNA flanking the insertion of pSR1015 was cloned from SR1015 and sequenced. We also used transposon mutagenesis to clone the genes involved in production of the polysaccharide; this was done to obtain any unlinked genes that may be involved in polysaccharide production. Approximately 1,300 transposon mutants were screened for loss of type I O-PS by ELISA. Six mutants were identified, and the DNA flanking the transposon insertion was cloned and sequenced. The Tn
The polysaccharide with the structure -3)-2-
The role of CPS I in the pathogenicity of
To establish a correlation between CPS I production and clinical infection a number of strains of
Syrian golden hamsters were inoculated intraperitoneally with 101 to 105 cells of either wild type
To further demonstrate the role of the capsule in infection by
Differences in tissue distribution between
To define the role of the capsule for persistence in the blood, serum bactericidal assays were performed with the addition of purified capsule to determine if capsule had an effect on the survival of serum-sensitive strains of
Since capsule mutants of
Immunofluorescence microscopy analysis was also performed to demonstrate the difference in C3b deposition between the capsule mutant and the wild type. The same experiment described above was performed, and samples were reacted with the mouse monoclonal antibody to human complement factor C3b, except that the samples were reacted with a secondary antibody conjugated to Cy3 and stained with DAPI for visualization of bacterial cells. As shown in Figure 3, the
Immunofluorescence microscopy analysis of decreased complement factor C3b deposition in 10% normal human serum by
The capsule mutant SR1015 was phagocytosed more significantly by PMNL than the wild-type strain. The proportion of wild-type
The
Sequence analysis of the completed genome of
Organization of the chromosomal regions containing the genes comprising the
Comparative analysis of the genomes of three
In order to assess the role of CPS III in virulence a mutant in the CPS III operon was tested for virulence compared to wild type
A
The expression of the
Microarray analysis of capsule expression was performed using a low-density DNA microarray. RNA was isolated from the livers and lungs of hamsters infected with
Fold Change ( | ||
BPSS1825 | Glycosyltransferase | 1.757155 |
BPSS1826 | Glycosyltransferase | 0.093565 |
BPSS1827 | Glucose-6-phosphate isomerase | 2.337866 |
BPSS1828 | Glycosyltransferase | -0.66607 |
BPSS1829 | Glycosyltransferase | -0.18061 |
BPSS1830 | Capsule export, tyrosine-protein kinase | -0.30655 |
BPSS 1831 | Capsule export, outer membrane protein | 0.725849 |
BPSS1832 | Transport, tyrosine-protein phosphatase | 1.52665 |
BPSS1833 | UDP-glucose-6-dehydrogenase | -0.71411 |
BPSS1834 | Sugar transferase | -0.07963 |
BPSS 1835 | Mannose-1-phosphate guanyltransferase | -0.75075 |
Microarray analysis of
Glycosyl composition analysis was performed on the purified capsule by combined gas chromatography/mass spectrometry (GC/MS). GC/MS results indicated that CPS III is composed of galactose, glucose, mannose, xylose, and rhamnose residues, with the highest proportion of carbohydrate being galactose and glucose. Glycosyl linkage analysis was also performed [55]. The predominant glycosyl residue detected was a terminally-linked heptopyranosyl (t-Hep) at a percentage of 23.2. Other residues detected were a terminally-linked and a 4-linked glucopyranosyl (t-Glcp) (4-Glcp) at percentages of 14.6 and 10.8, respectively [55].
Although significant advances have been made in the field, melioidosis continues to be a public health concern in many regions of the world [59]. Completion of the sequencing of the
To obtain virulence determinants unique to
The polysaccharide with the structure -3)-2-
Virulence genes of a number of pathogenic bacteria are located on pathogenicity islands (PAIs), regions on the bacterial chromosome that are present in the genome of pathogenic strains but rarely present in those of nonpathogenic strains. The PAIs may range in size from about 30 kb to 200 kb and often differ in G+C content from the remaining bacterial genome; the PAIs are often associated with the carriage of many virulence genes. These genetic units are often flanked by direct repeats and may be associated with tRNA genes or insertion sequence (IS) elements at their boundaries. They may also be associated with the presence of mobility genes, such as IS elements, integrases, transposases, and origins of plasmid replication. These DNA regions are considered to be unstable in that they may be subject to deletion with high frequency or undergo duplications and amplifications [7]. A number of PAIs have been described for both gram-positive and gram-negative bacteria, and the application of subtraction hybridization has been used to successfully identify such genetic elements [7]. The subtractive hybridization that was carried out between
Capsule production has been correlated with virulence in many bacteria, particularly those causing serious invasive infections of humans [61]. Our studies demonstrated that CPS I is critical for the virulence of
To establish a correlation between capsule production and clinical infection a number of
CPS I production by
CPS I production was shown to be responsible for persistence in the blood by evasion of the complement cascade and the mechanism for this was determined to be through the reduction of C3b deposition and opsonophagocytosis. The addition of purified CPS I to serum bactericidal assays showed that the capsule contributes to increased resistance of serum sensitive strains lacking the O-polysaccharide moiety (O-PS) of LPS to the bactericidal effects of normal human serum. However, CPS I mutants themselves were not found to be serum sensitive because they still produced O-PS, which was previously shown to be responsible for serum resistance, because it prevents lysis by the MAC complex [36]. This led us to postulate that CPS I was affecting the complement cascade through some other mechanism and it was found that this mechanism was through the reduction of C3b deposition and opsonization [50]. Both Western blot analysis and immunofluorescence microscopy experiments using a mouse monoclonal antibody to human C3b demonstrated the inhibition of C3b deposition by CPS I. In both experiments C3b deposition was more pronounced on the surface of the CPS I mutant compared to wild type. Also evident was that some C3b deposition occurred in the wild type, but this was expected since bacterial capsules are known to allow the diffusion of some C3b to the bacterial surface and
The expression of CPS I in the presence of normal human serum was found to be significantly elevated, also confirming that this capsule contributes to survival in the host. The presence of CPS I enables
Sequence analysis of the genome of
Three other putative capsule operons were identified by sequence analysis and all of these operons were found to be present in
CPS III, located on chromosome 2, was found to contain 11 genes involved in the biosynthesis of a polysaccharide and was shown to be present in the genomes of
Previous studies have demonstrated that
Studies by another laboratory have also focused on the presence of these capsule clusters in
A study was recently published which outlines the identification of another capsule produced by
The author would like to thank Dr. Donald Woods and all colleagues in the
Emerging infectious diseases such as COVID-19, acquired or hereditary genetic defects causing cancer and other illnesses fuel the need for fast and cost-effective diagnostic tools. Gold standard for many types of disease detection is the real-time polymerase chain reaction (PCR) due to its robustness and sensitivity toward molecular biomarkers associated with diseases. Especially, quantitative PCR (qPCR) and reverse transcriptase qPCR have been staples of infectious disease diagnostics to determine the presence of pathogens and viral loads [1, 2], but have also proven their efficacy in tumor diagnostics due to high sensitivity and low input requirements [3, 4]. Many next-generation sequencing (NGS) approaches are also used as diagnostic tools. Whole exome and genome sequencing is used for the investigation of many molecular markers. The benefits of NGS include the ability to screen a large amount of possible target genes in tandem for comparatively low cost [5]. Furthermore, NGS can be used for unbiased detection and species level determination of pathogens in septic patients. This removes the need for time-consuming blood cultures [6]. Other well-established methods in diagnostics are based on antigen-antibody interaction, such as the Enzyme-Linked Immunosorbent Assay (ELISA) or paper-based lateral flow assays [7, 8].
A new addition to the diagnostic toolbox is clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics. CRISPR-associated (Cas) proteins are RNA-guided endonucleases originally part of the adaptive immune system of prokaryotes to ward off invading nucleic acids. Several types of CRISPR/Cas systems have been discovered, and some have been used for diagnostic applications such as Cas12 and Cas13 for methods such as DNA endonuclease-targeted CRISPR
CRISPR RNAs (crRNA) provide the targeting mechanism for the Cas9 nuclease activity. crRNAs are hybridized with trans-activating crRNA (tracrRNA), providing a stem-loop structure that anchors the RNA-complex to the Cas9 protein. The crRNA can be engineered to target a wide array of sequences rendering CRISPR/Cas9 a powerful tool for targeted gene editing and recognition. Cas9 proteins are characterized by two nuclease domains forming the active center, HNH and RuvC [10]. HNH is a single nuclease domain responsible for cleaving the DNA strand complementary to the RNA guide. RuvC is split into three subdomains, with RuvC I at the N-terminus of the protein and RuvC II/III flanking the HNH domain near the center of the amino acid sequence [11]. The catalytic residues D10 (in RuvC I) and H840 (in HNH) can be substituted to either limit nuclease activity in case of a single-site inactivation to create a Cas9 nickase or to generate a catalytically inactive/dead Cas9 (dCas9) variant in case of a double-site inactivation [12]. In addition to the nuclease domains, Cas9 possesses a protospacer adjacent motif (PAM)-interacting (PI) domain. The PAM is a short nucleic acid sequence downstream of the crRNA conferred target sequence, required for nuclease activity and target sequence interrogation [13]. It is thought to have originated in prokaryotes so as not to target their own DNA and thus to prevent an autoimmune response [14]. While the sequence to be cut can be easily defined via crRNA, the obligatory requirement of a protospacer adjacent motif (PAM) sequence next to the target sequence [15, 16] limits the applications of Cas9 in clinical diagnostics. Due to this limitation, regions of interest without matching PAM-site cannot be cleaved and subsequently analyzed. Several variants of Cas9 enzymes have been generated to partially circumvent those limitations with a relaxation of the PAM-site requirement. The Cas9 from
In the area of molecular diagnostics, CRISPR/Cas9 systems have proven to be effective tools in distinguishing between different Zika virus strains. Pardee et al. (2016) [20] used nucleic acid sequence-based amplification (NASBA) in combination with Zika strain-specific sgRNA/Cas9 and toehold switches to create a colorimetric assay to detect and differentiate African and American Zika virus strains. A toehold switch is an RNA molecule combining a sensor and a reporter sequence. Without the presence of the trigger, an RNA molecule complementary to the sensor sequence, a hairpin structure is formed. It limits access to the ribosomal binding site and therefore inhibits translation of the reporter. Due to strand displacement upon hybridization with the trigger RNA, the hairpin structure is resolved, allowing the translation of the reporter [21]. The toehold switch was designed to regulate
CRISPR-Cas9 nickase (SpCas9H840A nickase) strand displacement amplification (CRISDA) is an ultrasensitive method to detect target DNA with single-nucleotide accuracy and attomolar sensitivity. A pair of SpCas9 nickase ribonucleoproteins (RNPs) introduce nicks in the flanking areas next to the region of interest. Initial primers anneal to the nicked strands from where strand displacement amplification begins. Biotin and Cy5-labeled peptide nucleic acid (PNAs) probes are introduced to the amplification mix to detect and quantify amplicons. The PNA binds to the amplicons and enables a pulldown using streptavidin-coated magnetic beads. Fluorescence measurement of the pulled-down DNA allows for quantification of the generated amplicons [22].
Another nucleic acid detection strategy is CRISPR/Cas9-triggered isothermal exponential amplification reaction (CAS-EXPAR). It is based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase)-mediated nucleic acids amplification. Cas9 cleavage of the target produces a primer for the CAS-EXPAR reaction, wherein the target “X” hybridizes with a construct containing two sequences complementary to the target (“X’”), which are connected via a PAMmer. Upon extension of the double strand, Cas9 cleaves off the newly synthesized DNA, which in turn acts as a primer itself. This strategy was shown to have a detection limit of 0.82 amol and high specificity, discriminating single-base mismatches [23].
Lateral flow assays are state of the art in point-of-care diagnostics. CRISPR/Cas9-mediated lateral flow nucleic acid assay (CASLFA) combines the sensitivity of Cas9 endonuclease with the ease of use of lateral flow assays. CASLFA was developed for the identification of
FnCas9 editor-linked uniform detection assay (FELUDA) is a diagnostic tool combining preamplification of a target sequence using biotinylated primers with inactive FnCas9 to detect target sequences. The used tracrRNA is FAM-labeled and can be recognized via antibodies, and the capture of target sequences is paired with a lateral flow readout. The biotinylated amplicons bind to the test region via streptavidin interaction. If FnCas9 binds to the amplicon DNA, it will be retained in the test region, allowing for antibody-based detection in the form of a visible band. dFnCas9 was used for this assay, as it exhibits lower affinity toward sequences with single-nucleotide mismatches to the crRNA used than SpCas9 [25].
Finding Low Abundance Sequences by Hybridization (FLASH) is a method that combines Cas9 digestion, PCR, and Illumina sequencing to detect and identify antimicrobial resistance in microbial DNA samples. Isolated DNA is dephosphorylated before Cas9 digestion of target sequences. The double-strand breaks introduced by Cas9 remain phosphorylated and are subsequently dA-tailed. Adapters are ligated to the dA-tailed target sequences, which are then amplified via PCR. The resulting library can be sequenced via Illumina sequencing and achieve sub-attomolar sensitivity [26].
Next to infectious disease detection, another field of interest for targeted Cas9 diagnostics is cancer, one of the world’s leading causes of premature death [27]. As cancerous unregulated cell growth can be caused by a combination of genetic defects, it is vital for prognosis and treatment to accurately diagnose its molecular cause. Cancer diagnostics currently is often based on histological analysis of tumor tissue. Because histology is predetermined by genetics, research efforts to quickly identify aberrant tumor marker genes on the molecular level are being pursued.
One application to potentially target this challenge is CRISPR-Chip. This method utilizes dCas9 immobilized on a graphene surface, acting as a conductor. The dCas9 is paired with a specifically designed sgRNA to recognize its target. Upon binding target DNA, the conductivity of the immobilized dCas9 changes, which can be measured via the graphene surface. This allows for detection limits of 1.7 fM gDNA. Though it was demonstrated with target sequences associated with Duchenne muscular dystrophy, it could be used for any sequences as long as a suitable PAM-site is flanking the region of interest [28].
Another route to follow in molecular tumor diagnosis is the sequencing of tumor marker genes. With the advent of second- and third-generation sequencing, the feasibility of sequencing approaches in standard diagnostics is increasing due to lower costs and shorter sequencing times. However, a combination with CRISPR/Cas technology allows for a specific sequencing of the regions of interest, boosting the output of relevant regions, and thus enabling a faster and very specific and sensitive sequencing approach.
To maximize utility of Cas9-targeted sequencing, biomarkers such as mutations or methylation patterns with defined locations are favorable. Because sequencing times are determined by target sequence length, biomarkers such as defined SNPs allow for higher throughput, as flanks of the targeted sequence can be chosen in close proximity to the region of interest. In our research we developed an amplification-independent workflow to assess the tumor marker status of six relevant genes/regions in brain tumors following the 2021 WHO Classification of Tumors of the Central Nervous System [29]. These genes/regions, their function, and glioma-relevant mutations are described in the following.
Isocitrate dehydrogenases 1 and 2 (IDH1, IDH2) are crucial enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate during the Krebs cycle. Common mutations associated with glioma formation are related to codons 132 for IDH1 and 172 for IDH2 causing aberrant enzymatic activity and in turn the accumulation of 2-hydroxyglutarate, which inhibits many α-ketoglutarate dependent enzymes such as DNA-demethylases, leading to DNA hypermethylation [30].
Additionally, the promoter of telomerase reverse transcriptase (pTERT) represents a clinically relevant target due to its close association with oncogenesis and immortalization of cell lines [31]. The mutations C228T and C250T are commonly associated with aberrant expression patterns as these mutations create
As mutation detection via sequencing is of interest, it is crucial to be aware of the benefits and drawbacks of the used sequencing technologies for the development of a medical diagnostic application.
Currently, the most widely used next-generation sequencing technologies on the market are Illumina short-read sequencing, PacBio (also referred to as Single-Molecule Real-Time (SMRT) sequencing), 454 pyrosequencing, ion-torrent/proton sequencing, and nanopore sequencing by Oxford Nanopore Technologies (ONT) [35]. Illumina, 454, and ion-torrent sequencing technologies are referred to as second-generation sequencing technologies. They deliver short reads of about 50–1000 bp in length and their parallelization in sequencing reaction results in a high read throughput (0.7–15 M reads per run) and an amount of sequence information of about 0.5–8.5 Gb per run [36]. PacBio and nanopore sequencing are referred to as third-generation sequencing technologies. They usually deliver tens of kb per read up to several Mb, but far fewer reads in total. For nanopore sequencing, the amount of sequence information is strongly dependent on the flow cell. Depending on the nucleic acid library preparation and its quality, up to 2.8 Gb per run is theoretically achievable on a Flongle flow cell, 10–15 Gb on a MinION flow cell [37], and up to 153 Gb per run was reported by using the PromethION flow cell [38]. PacBio sequencing utilizes SMRT cells for sequencing, usually generating 55,000–365,000 reads per run with an average read length of 10–16 kb [39] and 15–96 Gb per run [40].
Nanopore and PacBio sequencing allow for real-time sequencing with parallel base calling of the steadily increasing raw sequencing information allowing direct usage of the results during the run. In addition, both techniques allow detection of epigenetic information of each nucleotide sequenced [41], which can be a piece of important additional information in clinical cancer diagnostics and treatment [42, 43, 44]. While methylation of a base directly impacts the raw signal of the nanopore sequencing and thus can be distinguished from an unmodified nucleotide, PacBio detects methylation by a change in DNA-polymerase kinetics during synthesis. Due to the “sequencing by synthesis” technology of second-generation sequencing techniques, they cannot detect epigenetic modification directly, but only via a pretreatment step such as bisulfite treatment, endonuclease digestion, or affinity enrichment [45].
In summary, the selection of the sequencing technology used for clinical diagnostics will be strongly dependent on requirements such as mode of analysis and time to results. The characteristics in this respect of each mentioned sequencing technology are summarized in Table 1.
Sequencing technology | Real-time | Average sequence length | Typical number of reads | Amount sequence data per run | Methylation status detection |
---|---|---|---|---|---|
Illumina | no | 2 × 150 bp (HiSeq 4000) [39] | 5 billion (HiSeq 4000) [39] | 1300–1500 Gb* | no |
2 × 300 bp (MiSeq) [39] | 25 million (MiSeq) [39] | 4.5–5.1 Gb* | |||
454 pyro-sequencing | no | 400–1000 bp [46] | > 1 million [32] | 35–450 Mb [46] | no |
Ion-torrent | no | 200–600 bp* | 2–130 million* | 0.3–50 Gb* | no |
PacBio | yes | 10–16 kb [39] | 55,000–365,000 [25] | 15 Gb (Sequel) [40] | yes |
96 Gb (Sequel II) [40] | |||||
Oxford Nanopore | yes | 10–30 kb (all flow cells) record 2.6 Mb [47] | 200,000 (Flongle) [48] | Up to 2.8 Gb (Flongle)* | yes |
1,200,000 (MinION) [48] | 10–15 Gb (MinION) [37] | ||||
Up to 250 million (PromethION)* | Up to 153 Gb (PromethION) [38] |
Most widely used sequencing technologies and their characteristics.
Specifications obtained from the respective company´s website (as of 01.06.2022).
Next to Illumina sequencing-based methods such as FLASH, third-generation sequencing can also be paired with Cas9 enrichment of target sequences. PacBio uses a generic SMRT sequencing library, which is digested by Cas9. The digested sequences are then ligated to a second set of adapters, which is used for magnetic-bead-based separation of the targeted sequences, allowing for target sequence enrichment [49].
For ONT’s Cas9-targeted sequencing process (nCATS), DNA is dephosphorylated before Cas9 digestion. Like FLASH, the phosphorylated ends of the cleaved DNA are dA-tailed and ligated to sequencing adapters, allowing for selective sequencing [50]. A similar approach was pursued in the following experimental section to develop a CRISPR/Cas, third-generation sequencing assay for diagnosis of. Utilizing the promising properties of nanopore and Cas9-dependent target enrichment, we developed an amplification-independent workflow to assess glioma biomarkers.
To test the feasibility of nanopore sequencing in brain tumor marker detection, we used pUC57 vectors containing 2 kb target sequence of a given tumor marker as either wild-type or containing a clinically relevant mutation. Cas9-RNP populations were prepared to cleave the DNA upstream and downstream of a given mutation site. The excised double-stranded DNA was used for sequencing library preparation using the SQK-CS9109 Cas9 sequencing Kit from ONT. Flongle flow cells (version R.9.4.1) were used for sequencing. Sequences were assessed using a minimap2 [51] alignment followed by custom SNP calling using python scripts. As tumor treatment is very time-sensitive [52], the possibility of intra-surgical diagnostics could alleviate an unmet clinical need. Therefore, we evaluated the results not only by accuracy but also regarding time to results. In addition to the complete sequencing data acquired a subset generated during the first 15 min of sequencing was also used for analysis.
Alt-R® S.p. Cas9 Nuclease V3 including tracrRNA and crRNAs were purchased from Integrated DNA Technologies (Coralville, IA, USA). The crRNAs were designed to target at least 200 bp upstream and downstream of each mutation site resulting in at least 1000 bp of excised dsDNA in total. crRNAs were designed for
Target | crRNA Seq fw (5’-3’) | crRNA Seq rv (5’-3’) | Fragment size [bp] |
---|---|---|---|
IDH1 | ATGTTTAATACAATCTTTGG | GCTTCCCATTACAAGAGGAG | 1062 |
IDH2 | AGTGCACACGATGTTTCTGC | TCGTCCTCACGACAACACTT | 1601 |
pTERT | CTCCCTGACGCTATGGTTCC | GTCAAGGAGCCCAAGTCGCG | 1443 |
H3F3A | AATTTGACTCGACCTTCCAG | TATTTGCGGAGGCTAAGTCT | 1081 |
Hist1H3B | GCATTCCTAACTATCTTGAA | CATAGTCTAATGCTTTCCGG | 1334 |
BRAF | GACCCTCTAAAACGGTGTGA | GCATGCATGTATAGGAGAGC | 1584 |
List of crRNAs used for enzymatic excision of tumor marker DNA from pUC57 vectors containing target gene fragments.
The pUC57 vectors containing tumor marker sequences used as target DNA for Cas9 digestion were purchased from GenScript (Piscataway, NJ, USA). For DNA digestion and library preparation, the SQK-CS9109 Cas9 sequencing kit from ONT (Oxford, UK). DNA digestion was set up by adding template plasmids to Cas9 RNPs, reaction buffer, dATP, and Taq DNA-polymerase. One digestion was performed with a mixture of six plasmids (80 ng each), each containing a different marker, in order to test multiplexing. Another digestion was set up with a mixture of two plasmids (160 ng each) containing different mutations of the same marker,
Digestion 1 | Digestion 2 | |
---|---|---|
pUC57::IDH1_Wt (80 ng/μL) | — | 2 μL |
pUC57::IDH1_R132C (80 ng/μL) | 1 μL | — |
pUC57::IDH1_R132H (80 ng/μL) | — | 2 μL |
pUC57::IDH2_Wt (80 ng/μL) | 1 μL | — |
pUC57::pTERT_Wt (80 ng/μL) | 1 μL | — |
pUC57::H3F3A_Wt (80 ng/μL) | 1 μL | — |
pUC57::Hist1H3B_Wt (80 ng/μL) | 1 μL | — |
pUC57::BRAF_Wt (80 ng/μL) | 1 μL | — |
ddH2O | 21 μL | 23 μL |
Cas9 RNPs (Population 1) (10 μM) | 10 μL | — |
Cas9 RNPs (Population 2) (10 μM) | — | 10 μL |
Reaction Buffer | 3 μL | 3 μL |
dATP | 1 μL | 1 μL |
Taq Polymerase | 1 μL | 1 μL |
Setup for digestion of different pUC57 plasmid mixtures using different Cas9-RNP populations.
The reaction mixtures were incubated for 30 min at 37°C for Cas9 cleavage. Subsequently it was incubated at 72°C for 5 min for Taq Polymerase facilitated dA-tailing of cleaved fragments.
For sequencing adapter ligation to the dA-tailed fragments, the digested and dA-tailed DNA was added to a mixture of 20 μL Ligation Buffer, 3 μL nuclease-free water, 10 μL T4 Ligase, and 5 μL Adapter Mix. The ligation components were mixed by flicking, spun down, and incubated at RT for 10 min. DNA was purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA). In total, 48 μL of magnetic beads was added to each sample and mixed by inversion. The samples were incubated for 10 min at RT without agitation. Afterward, samples were spun down, and beads were separated magnetically. The supernatant was discarded, and the pellet washed twice with 250 μL Short Fragment Buffer (SFB). The wash step consisted of resuspension in SFB and subsequent magnetic separation of the washed beads. After the supernatant was removed, the pellet was resuspended in 13 μL 50 °C Elution Buffer. The elution mixture was incubated at 50 °C and 1000 rpm in a heater shaker for 10 min. After magnetic separation, 13 μL of the eluate was removed, and DNA content and purity were analyzed via Nanodrop. Sequencing libraries were created by combining 37.5 μL Sequencing Buffer, 25.5 μL Loading Beads, and 12 μL DNA Library.
In total, 37.5 μL of each library was used for sequencing on a Flongle flow Cell (R9.4.1). DNA contents were 48.6 ng for sample 1, containing fragments of all six plasmids, and 17.4 ng for sample 2, containing fragments of two plasmids, resembling two
To analyze the possible mutation sites bioinformatically, alignment references of the tumor marker sequences were created. In the reference sequences, the possible mutation site was deleted; therefore, alignment of each generated sequence produced an insertion mutation during variant calling. The bases recognized as insertions were used to distinguish wild-type and mutated sequences. Each generated sequence was aligned with all possible target references using minimap2 [51]. Subsequently, paftools. js was used for variant-calling [51] and custom scripts accumulated the numbers of wild-type and mutant reads in real time. To ensure the highest possible accuracy, matches between generated sequences and references were split into
Creating a sequencing library from a mixture of six plasmids containing different tumor marker genes enabled us to identify each target with high accuracy, as seen in Table 4. These results were achieved after 18 h of sequencing with a library containing 48.6 ng of plasmid DNA. Overall yield of sequences was high with >39000 generated reads for all markers.
Target | Inspected variation | Mapped generated Sequences | Sequences with tumor marker information | Correctly Identified Sequence variants [%] |
---|---|---|---|---|
IDH1_R132C | R132 | 128416 | 74002 | 99.57 |
IDH2_Wt | R172 | 196040 | 151329 | 97.59 |
pTERT_Wt | C228 | 48932 | 32613 | 99.45 |
C250 | 34689 | 99.61 | ||
H3F3A_Wt | K27 | 39232 | 21899 | 99.11 |
G34 | 22308 | 97.50 | ||
Hist1H3B_Wt | K27 | 45560 | 19458 | 99.66 |
BRAF_Wt | V600 | 52387 | 35354 | 98.36 |
A sequencing library was prepared from tumor marker DNA excised from synthetic plasmids. Equal amounts of plasmid were used for each target.
Accuracy for simulated homozygosity and coverage of each marker after 18 h was very high. As time-to-result is a central parameter in clinical diagnostics, these results were examined regarding the coverage and accuracy after 15 min of sequencing time. This subset revealed a coverage for each marker of >200x after 15 min. Ratios of sequences with tumor marker information out of all generated sequences were similar after 15 min as compared with 18 h. They ranged from 48 to 84%, depending on the observed marker. Accuracy regarding the identification of the tumor marker sequence was also comparably high in this data subset. Between 96.99 and 100% of sequences with tumor marker information were annotated correctly, as shown in Table 5.
Target | Inspected variation | Mapped generated Sequences | Sequences with tumor marker information | Correctly Identified Sequence variants [%] |
---|---|---|---|---|
IDH1_R132C | R132 | 914 | 604 | 99.50 |
IDH2_Wt | R172 | 894 | 757 | 98.94 |
pTERT_Wt | C228 | 348 | 257 | 100 |
C250 | 269 | 100 | ||
H3F3A_Wt | K27 | 239 | 157 | 100 |
G34 | 166 | 96.99 | ||
Hist1H3B_Wt | K27 | 292 | 143 | 100 |
BRAF_Wt | V600 | 246 | 185 | 100 |
A sequencing library was prepared from tumor marker DNA excised from synthetic plasmids. Equal amounts of plasmid were used for each target.
As these results were achieved with one variant per marker, no conclusions regarding heterozygosity detection were possible. Most mutations are heterozygous. Therefore, a subsequent experiment was performed to assess the analysis accuracy when including a simulated heterozygous mutation. For this experiment, a mixture of equal amounts of pUC57::IDH1_Wt and pUC57::IDH1_R132H was used for Cas9 sequencing. In total, 17.4 ng of plasmid DNA contained in the library was loaded onto the Flongle flow cell. Results shown in Table 6 were achieved after 18 h of sequencing, with a subset of sequences generated during the first 15 min of sequencing being evaluated separately. In total, 625 reads were generated after 15 min of which 490 (72%) yielded tumor marker information. After 18 h, 29072 reads were generated and 20875 (78%) yielded tumor marker information. It was found that both
Sequencing time 15 min | Sequencing time 18 h | |
---|---|---|
Mapped generated sequences | 625 | 29072 |
Sequences with tumor marker information | 490 | 20875 |
Expected ratio (Wt/R132H/other IDH1) [%] | 50/50/0 | 50/50/0 |
Achieved ratio (Wt/R132H/other IDH1) [%] | 40.2/59.59/0.21 | 38.49/61.4/0.01 |
A sequencing library was prepared from tumor marker DNA excised from synthetic plasmids. Equal amounts of plasmid were used for each target.
Although Cas9 selective sequencing focuses on the sequence of interest and thus may lead to faster and more accurate results as compared with a whole genome sequencing approach, there exist some challenges. In a direct sequencing approach, there is no amplification step involved when analyzing only a single mutated base or area. Thus, one complete genome delivers only one read of the desired area, which in case of carcinoma mutations is mainly haploidic. Therefore, a huge amount of highly pure, high molecular weight, genomic DNA must be prepared and used, which depending on the amount of sample and method of nucleic acid preparation may be a limitation. Typically, the usage of 1–10 μg of human genomic DNA for Cas9 digestion is suggested (nCATS, [50]), corresponding to 150.000–1.500.000 copies of a diploid (female) genome, ideally resulting in the same number of reads. However, one must consider inefficiencies in Cas9 digestion, nucleic acid purification, and library preparation together with possible off-target digestion effects. Further on, there exists an intrinsic error rate of each sequencing method used and in case of cancerous tissue, it may consist of a mixture of wild-type and mutated cells depending on tumor heterogeneity and the general quality of tissue sampling. This may result in a general low number of reads, possibly beyond the coverage needed to safely identify a mutation.
Despite these drawbacks, we believe an nCATS-based approach to intra-surgical determination of a molecular tumor marker panel is justified, as it allows for live detection of marker variants including epigenetic information [50]. Preamplification of the targets might alleviate the high input DNA requirements but removes the ability to determine epigenetic properties of the sequences. That would render the effective analysis of markers such as MGMT methylation status, a predictive biomarker for efficacy of chemotherapy [53], impossible. PCR-based approaches such as qPCR would be very sensitive, as even a few copies of target DNA can produce a positive signal [54], but primer sets that incorporate the putative mutation site would be necessary to distinguish between wild-type and mutant sequences. This is a drawback in comparison to the chosen nCATS approach as this only detects anticipated mutations. Immunodetection of possible auto-antibodies (e.g., with ELISA) has been reported to be prone to false positives [55] and even though nanopore sequencing itself is prone to sequencing errors, they are distributed across the sequence, which leads to high consensus accuracy [56]. Drawbacks are the low resolution of homopolymers, which are prone to sequencing errors with the current flow cell generations. Second-generation sequencing would allow for high sensitivity and accuracy and is well-established but delivers only short sequences. Due to its sequencing by synthesis approach, epigenetic information is lost in this case as well [57]. A comparison between mode of action, advantages and disadvantages, accuracy and sensitivity of those diagnostic tools is shown in Table 7 below.
Diagnostic tool | Mode of action | Advantages | Challenges | Accuracy | Sensitivity |
---|---|---|---|---|---|
nCATS [50] | Excision of target sequences from genome and subsequent sequencing | Native sequencing—epigenetic profile accessible, real-time basecalling | Large input DNA requirements, possible off-target sequencing | Medium, off-target digestions possible [58]; Nanopore sequencing, high intrinsic error rate [56] | Medium |
(Reverse transcriptase) real-time PCR [3, 4] | Detection by amplification | Quantitative sequencing, well-established method | Reliant on primer specificity, affected by mutations in binding regions of primer probes | Very high | Very high |
ELISA [7, 59] | Immuno-detection | Can detect a wide range of antigens and antibodies | Prone to false positives [55] | Low–medium | Medium |
2nd Gen. Sequencing [57, 60] | Sequencing by synthesis | Established methods, high read count | Short read sequencing, high number of reads needed for contig generation | Very high | Very high |
3rd Gen. Sequencing [37, 61] | ONT: electrical current changes PacBio: Fluorescence detection | Long read sequencing, easy assembly of long contigs | High intrinsic error rate (single sequence), low homopolymer resolution | Medium | Medium |
Comparison of current diagnostic tools with nCATS.
Considering the number of reads generated in 15 min using Flongle flow cells, the process might be sped up with the use of MinION flow cells. This would increase cost but cut sequencing time in a trade-off to be considered on a case-by-case basis. We demonstrated that the defined plasmid sequences could be analyzed via the described workflow. As the described experiments represent a work in progress, the use of isolated gDNA with significantly lower target sequence density as a template must be demonstrated. Previous works used either amplification-independent whole-genome sequencing (WGS) or amplicon sequencing to assess brain tumor marker variants. The WGS alternative is significantly slower due to the excess of non-target sequences generated but yielded additional epigenetic information for the regions of interest [62]. Enrichment of target sequences via PCR yields more favorable library compositions but eliminates epigenetic information [62]. As shown here, our CRISPR/Cas9-based approach to enrich native target sequences might be able to combine the advantages of both previous strategies.
The results of the simulated heterozygosity were ~10% off from the expected 50% distribution of IDH1 Wt/IDH1 R132H, as shown in Table 6, but the fact that negligible amounts of other IDH1 mutations and no other tumor marker sequences were found is promising toward applications in clinical environments.
Ultimately, the goal of such a workflow should be a time-to-result below the time required for a neurosurgical tumor resection via craniotomy. This way, neurosurgeons could make informed decisions about the extent of the ongoing surgery and initiate personalized therapeutic modalities based on clinically actionable prognostic biomarkers [63]. Provided the RNPs are prepared in advance, the workflow described using the ONT Cas9 Sequencing kit, including 15 min time required for sequencing, would take 1:45 h altogether. Assuming gDNA extraction and preparative dephosphorylation add another 30–45 min [64], the total time-to-results may be as low as 2.5 h. Considering the lower abundancy of target sequences in gDNA compared with synthetic plasmids, sequencing times would likely increase to generate the same coverage, which must be accounted for. But this could partly be mitigated by the usage of a MinION flow cell instead of a Flongle flow cell. The analyzed marker panel can be amended by adding or subtracting crRNAs to target different sequences. A prerequisite for this approach is the presence of PAM sites in the vicinity of the new target genes. Mutations in AT-rich regions might be hard to access via SpCas9 because of its PAM-site requirement of 5’-NGG-3’. For this reason, different Cas9 proteins might be suitable candidates for amended workflows, such as xCas9, ScCas9-SC++, or an engineered FnCas9 [17, 18, 19]. In summary, these proof-of-concept results suggest that Cas9-aided targeted sequencing can generate diagnostically relevant tumor marker information in a short period of time and therefore might be a feasible diagnostic method for intra-surgical tumor diagnostics.
The authors gratefully thank Prof. Dr. Bruno Moerschbacher (University of Münster) and Prof. Dr. Wolfgang Streit (University of Hamburg) for their in-depth discussions and scientific input. We would also like to thank Sabina Schaal for the project management and scientific input. We gratefully acknowledge the financial support of the Federal Ministry of Education and Research (Funding indicator: 13GW0347A).
IntechOpen implements a robust policy to minimize and deal with instances of fraud or misconduct. As part of our general commitment to transparency and openness, and in order to maintain high scientific standards, we have a well-defined editorial policy regarding Retractions and Corrections.
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\\n\\n1. RETRACTIONS
\\n\\nA Retraction of a Chapter will be issued by the Academic Editor, either following an Author’s request to do so or when there is a 3rd party report of scientific misconduct. Upon receipt of a report by a 3rd party, the Academic Editor will investigate any allegations of scientific misconduct, working in cooperation with the Author(s) and their institution(s).
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\\n\\nPublishing of a Retraction Notice will adhere to the following guidelines:
\\n\\n1.2. REMOVALS AND CANCELLATIONS
\\n\\n2. STATEMENTS OF CONCERN
\\n\\nA Statement of Concern detailing alleged misconduct will be issued by the Academic Editor or publisher following a 3rd party report of scientific misconduct when:
\\n\\nIntechOpen believes that the number of occasions on which a Statement of Concern is issued will be very few in number. In all cases when such a decision has been taken by the Academic Editor the decision will be reviewed by another editor to whom the author can make representations.
\\n\\n3. CORRECTIONS
\\n\\nA Correction will be issued by the Academic Editor when:
\\n\\n3.1. ERRATUM
\\n\\nAn Erratum will be issued by the Academic Editor when it is determined that a mistake in a Chapter originates from the production process handled by the publisher.
\\n\\nA published Erratum will adhere to the Retraction Notice publishing guidelines outlined above.
\\n\\n3.2. CORRIGENDUM
\\n\\nA Corrigendum will be issued by the Academic Editor when it is determined that a mistake in a Chapter is a result of an Author’s miscalculation or oversight. A published Corrigendum will adhere to the Retraction Notice publishing guidelines outlined above.
\\n\\n4. FINAL REMARKS
\\n\\nIntechOpen wishes to emphasize that the final decision on whether a Retraction, Statement of Concern, or a Correction will be issued rests with the Academic Editor. The publisher is obliged to act upon any reports of scientific misconduct in its publications and to make a reasonable effort to facilitate any subsequent investigation of such claims.
\\n\\nIn the case of Retraction or removal of the Work, the publisher will be under no obligation to refund the APC.
\\n\\nThe general principles set out above apply to Retractions and Corrections issued in all IntechOpen publications.
\\n\\nAny suggestions or comments on this Policy are welcome and may be sent to permissions@intechopen.com.
\\n\\nPolicy last updated: 2017-09-11
\\n"}]'},components:[{type:"htmlEditorComponent",content:'IntechOpen’s Retraction and Correction Policy has been developed in accordance with the Committee on Publication Ethics (COPE) publication guidelines relating to scientific misconduct and research ethics:
\n\n1. RETRACTIONS
\n\nA Retraction of a Chapter will be issued by the Academic Editor, either following an Author’s request to do so or when there is a 3rd party report of scientific misconduct. Upon receipt of a report by a 3rd party, the Academic Editor will investigate any allegations of scientific misconduct, working in cooperation with the Author(s) and their institution(s).
\n\nA formal Retraction will be issued when there is clear and conclusive evidence of any of the following:
\n\nPublishing of a Retraction Notice will adhere to the following guidelines:
\n\n1.2. REMOVALS AND CANCELLATIONS
\n\n2. STATEMENTS OF CONCERN
\n\nA Statement of Concern detailing alleged misconduct will be issued by the Academic Editor or publisher following a 3rd party report of scientific misconduct when:
\n\nIntechOpen believes that the number of occasions on which a Statement of Concern is issued will be very few in number. In all cases when such a decision has been taken by the Academic Editor the decision will be reviewed by another editor to whom the author can make representations.
\n\n3. CORRECTIONS
\n\nA Correction will be issued by the Academic Editor when:
\n\n3.1. ERRATUM
\n\nAn Erratum will be issued by the Academic Editor when it is determined that a mistake in a Chapter originates from the production process handled by the publisher.
\n\nA published Erratum will adhere to the Retraction Notice publishing guidelines outlined above.
\n\n3.2. CORRIGENDUM
\n\nA Corrigendum will be issued by the Academic Editor when it is determined that a mistake in a Chapter is a result of an Author’s miscalculation or oversight. A published Corrigendum will adhere to the Retraction Notice publishing guidelines outlined above.
\n\n4. FINAL REMARKS
\n\nIntechOpen wishes to emphasize that the final decision on whether a Retraction, Statement of Concern, or a Correction will be issued rests with the Academic Editor. The publisher is obliged to act upon any reports of scientific misconduct in its publications and to make a reasonable effort to facilitate any subsequent investigation of such claims.
\n\nIn the case of Retraction or removal of the Work, the publisher will be under no obligation to refund the APC.
\n\nThe general principles set out above apply to Retractions and Corrections issued in all IntechOpen publications.
\n\nAny suggestions or comments on this Policy are welcome and may be sent to permissions@intechopen.com.
\n\nPolicy last updated: 2017-09-11
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Since their discovery, liposomes have been subject to extensive evolution, in terms of composition, manufacturing and applications, which led to several openings in both basic and applied life sciences. However, most of the advances in liposome research have been more devoted to launching new developments than improving the existing technology for potential implementation. For instance, the evolution of the conventional lipid hydration methods to novel microfluidic technologies has permitted upscale production, but with increase in manufacturing cost and persistent use of organic solvents. This chapter intends to present general concepts in liposome technology, highlighting some longstanding bottlenecks that remain challenging to the preparation, characterization and applications of liposomal systems. This would enhance the understanding of the gaps in the field and, hence, provide directions for future research and developments.",book:{id:"8095",slug:"liposomes-advances-and-perspectives",title:"Liposomes",fullTitle:"Liposomes - Advances and Perspectives"},signatures:"Christian Isalomboto Nkanga, Alain Murhimalika Bapolisi, Nnamdi Ikemefuna Okafor and Rui Werner Maçedo Krause",authors:[{id:"284670",title:"Prof.",name:"Rui",middleName:null,surname:"Krause",slug:"rui-krause",fullName:"Rui Krause"},{id:"284672",title:"Mr.",name:"Alain",middleName:null,surname:"Bapolisi",slug:"alain-bapolisi",fullName:"Alain Bapolisi"},{id:"284673",title:"MSc.",name:"Christian",middleName:null,surname:"Nkanga",slug:"christian-nkanga",fullName:"Christian Nkanga"},{id:"284675",title:"Mr.",name:"Okafor",middleName:null,surname:"Nnamdi",slug:"okafor-nnamdi",fullName:"Okafor Nnamdi"}]},{id:"52680",doi:"10.5772/65715",title:"Endogenous Antioxidants: A Review of their Role in Oxidative Stress",slug:"endogenous-antioxidants-a-review-of-their-role-in-oxidative-stress",totalDownloads:4096,totalCrossrefCites:14,totalDimensionsCites:33,abstract:"Oxidative stress (OxS) constitutes a disturbance caused by an imbalance between the generation of free radicals and antioxidant system, which causes damage to biomolecules. This, in turn, may lead the body to the occurrence of many chronic degenerative diseases. Therefore, it is very important to know the functioning of those endogenous (and exogenous) antioxidants systems to prevent such diseases. Due to evolutionary conditions in living beings, among other functions have been developed and selected defense systems against the deleterious action of free radicals. Such systems are intrinsic in cells (at level intracellular and extracellular) and act together with the dietary exogenous antioxidants. All these antioxidant systems have very important role in preserving the oxide/reduction equilibrium in the cell. To understand the role of the transcription factor Nrf2 in regulating the processes of antioxidant defense, it must also know the role of many of the endogenous antioxidants that occur because of its activation. Therefore, this chapter makes a literature review of the most important general aspects of endogenous antioxidant systems, which will provide another point of view from which to approach the study and treatment of many chronic degenerative diseases, such as diabetes, hypertension, and Parkinson.",book:{id:"5407",slug:"a-master-regulator-of-oxidative-stress-the-transcription-factor-nrf2",title:"The Transcription Factor Nrf2",fullTitle:"A Master Regulator of Oxidative Stress - The Transcription Factor Nrf2"},signatures:"Tomás Alejandro Fregoso Aguilar, Brenda Carolina Hernández\nNavarro and Jorge Alberto Mendoza Pérez",authors:[{id:"154732",title:"Dr.",name:"Jorge A.",middleName:null,surname:"Mendoza-Pérez",slug:"jorge-a.-mendoza-perez",fullName:"Jorge A. Mendoza-Pérez"},{id:"154908",title:"Dr.",name:"Tomás A.",middleName:null,surname:"Fregoso-Aguilar",slug:"tomas-a.-fregoso-aguilar",fullName:"Tomás A. 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In most of the cases, the structures of drugs or drug candidates and the interacting residues on the target proteins are also presented. In addition, a few successful examples of drug repurposing using molecular docking are mentioned in this chapter. It should provide us with confidence that the docking will be extensively employed in the industry and basic research. Moreover, we should actively apply molecular docking and related technology to create new therapies for diseases.",book:{id:"6365",slug:"molecular-docking",title:"Molecular Docking",fullTitle:"Molecular Docking"},signatures:"Mark Andrew Phillips, Marisa A. Stewart, Darby L. Woodling and\nZhong-Ru Xie",authors:[{id:"214567",title:"Prof.",name:"Zhong-Ru",middleName:null,surname:"Xie",slug:"zhong-ru-xie",fullName:"Zhong-Ru Xie"},{id:"223007",title:"Ms.",name:"Marisa A.",middleName:null,surname:"Stewart",slug:"marisa-a.-stewart",fullName:"Marisa A. Stewart"},{id:"223009",title:"Mr.",name:"Darby L.",middleName:null,surname:"Woodling",slug:"darby-l.-woodling",fullName:"Darby L. Woodling"},{id:"223013",title:"Mr.",name:"Mark Andrew",middleName:null,surname:"Phillips",slug:"mark-andrew-phillips",fullName:"Mark Andrew Phillips"}]}],mostDownloadedChaptersLast30Days:[{id:"69775",title:"Principles of Chromatography Method Development",slug:"principles-of-chromatography-method-development",totalDownloads:4294,totalCrossrefCites:5,totalDimensionsCites:11,abstract:"This chapter aims to explain the key parameters of analytical method development using the chromatography techniques which are used for the identification, separation, purification, and quantitative estimation of complex mixtures of organic compounds. Mainly, the versatile techniques of ultra−/high-performance liquid chromatography (UPLC/HPLC) are in use for the analysis of assay and organic impurities/related substances/degradation products of a drug substance or drug product or intermediate or raw material of pharmaceuticals. A suitable analytical method is developed only after evaluating the major and critical separation parameters of chromatography (examples for UPLC/HPLC are selection of diluent, wavelength, detector, stationary phase, column temperature, flow rate, solvent system, elution mode, and injection volume, etc.). The analytical method development is a process of proving the developed analytical method is suitable for its intended use for the quantitative estimation of the targeted analyte present in pharmaceutical drugs. And it mostly plays a vital role in the development and manufacture of pharmaceuticals drugs.",book:{id:"8912",slug:"biochemical-analysis-tools-methods-for-bio-molecules-studies",title:"Biochemical Analysis Tools",fullTitle:"Biochemical Analysis Tools - Methods for Bio-Molecules Studies"},signatures:"Narasimha S. Lakka and Chandrasekar Kuppan",authors:[{id:"304950",title:"Prof.",name:"Chandrasekar",middleName:null,surname:"Kuppan",slug:"chandrasekar-kuppan",fullName:"Chandrasekar Kuppan"},{id:"309984",title:"Mr.",name:"Narasimha S",middleName:null,surname:"Lakka",slug:"narasimha-s-lakka",fullName:"Narasimha S Lakka"}]},{id:"72074",title:"The Chemistry Behind Plant DNA Isolation Protocols",slug:"the-chemistry-behind-plant-dna-isolation-protocols",totalDownloads:3797,totalCrossrefCites:4,totalDimensionsCites:7,abstract:"Various plant species are biochemically heterogeneous in nature, a single deoxyribose nucleic acid (DNA) isolation protocol may not be suitable. There have been continuous modification and standardization in DNA isolation protocols. Most of the plant DNA isolation protocols used today are modified versions of hexadecyltrimethyl-ammonium bromide (CTAB) extraction procedure. Modification is usually performed in the concentration of chemicals used during the extraction procedure according to the plant species and plant part used. Thus, understanding the role of each chemical (viz. CTAB, NaCl, PVP, ethanol, and isopropanol) used during the DNA extraction procedure will benefit to set or modify protocols for more precisions. A review of the chemicals used in the CTAB method of DNA extraction and their probable functions on the highly evolved yet complex to students and researchers has been summarized.",book:{id:"8912",slug:"biochemical-analysis-tools-methods-for-bio-molecules-studies",title:"Biochemical Analysis Tools",fullTitle:"Biochemical Analysis Tools - Methods for Bio-Molecules Studies"},signatures:"Jina Heikrujam, Rajkumar Kishor and Pranab Behari Mazumder",authors:[{id:"74521",title:"Dr.",name:"Rajkumar",middleName:null,surname:"Kishor",slug:"rajkumar-kishor",fullName:"Rajkumar Kishor"},{id:"309357",title:"Prof.",name:"Pranab Behari",middleName:null,surname:"Mazumder",slug:"pranab-behari-mazumder",fullName:"Pranab Behari Mazumder"},{id:"318351",title:"Ph.D. Student",name:"Jina",middleName:null,surname:"Heikrujam",slug:"jina-heikrujam",fullName:"Jina Heikrujam"}]},{id:"64549",title:"Plant Lipid Metabolism",slug:"plant-lipid-metabolism",totalDownloads:2677,totalCrossrefCites:8,totalDimensionsCites:14,abstract:"In plants, the synthesis of fatty acids takes place in the chloroplast and the fatty acid synthase is prokaryotic type. In plants, the structure of membrane lipids is different from that of eukaryotic cells. The membranes of the chloroplasts are essentially formed of galatolipids. This chapter will also focus on the structure and biosynthesis of fatty acids and membrane lipids in plants. Lipids of seeds are essentially composed of TAG; it would be interesting to describe their synthesis during the maturation of the seeds. Some plants contain in their reserve lipids unconventional fatty acids such as gamma linolenic acid in Borrago officinalis L., short-chain fatty acids C: 12 and C: 10, fatty acids with very long chains, and fatty acids that are cyclical. All of these fatty acids can have industrial and/or pharmaceutical applications.",book:{id:"7036",slug:"advances-in-lipid-metabolism",title:"Advances in Lipid Metabolism",fullTitle:"Advances in Lipid Metabolism"},signatures:"Fatiha AID",authors:[{id:"256576",title:"Prof.",name:"Fatiha",middleName:null,surname:"Aid",slug:"fatiha-aid",fullName:"Fatiha Aid"}]},{id:"66369",title:"General Perception of Liposomes: Formation, Manufacturing and Applications",slug:"general-perception-of-liposomes-formation-manufacturing-and-applications",totalDownloads:3320,totalCrossrefCites:17,totalDimensionsCites:40,abstract:"Liposomes are currently part of the most reputed carriers for various molecular species, from small and simple to large and complex molecules. Since their discovery, liposomes have been subject to extensive evolution, in terms of composition, manufacturing and applications, which led to several openings in both basic and applied life sciences. However, most of the advances in liposome research have been more devoted to launching new developments than improving the existing technology for potential implementation. For instance, the evolution of the conventional lipid hydration methods to novel microfluidic technologies has permitted upscale production, but with increase in manufacturing cost and persistent use of organic solvents. This chapter intends to present general concepts in liposome technology, highlighting some longstanding bottlenecks that remain challenging to the preparation, characterization and applications of liposomal systems. This would enhance the understanding of the gaps in the field and, hence, provide directions for future research and developments.",book:{id:"8095",slug:"liposomes-advances-and-perspectives",title:"Liposomes",fullTitle:"Liposomes - Advances and Perspectives"},signatures:"Christian Isalomboto Nkanga, Alain Murhimalika Bapolisi, Nnamdi Ikemefuna Okafor and Rui Werner Maçedo Krause",authors:[{id:"284670",title:"Prof.",name:"Rui",middleName:null,surname:"Krause",slug:"rui-krause",fullName:"Rui Krause"},{id:"284672",title:"Mr.",name:"Alain",middleName:null,surname:"Bapolisi",slug:"alain-bapolisi",fullName:"Alain Bapolisi"},{id:"284673",title:"MSc.",name:"Christian",middleName:null,surname:"Nkanga",slug:"christian-nkanga",fullName:"Christian Nkanga"},{id:"284675",title:"Mr.",name:"Okafor",middleName:null,surname:"Nnamdi",slug:"okafor-nnamdi",fullName:"Okafor Nnamdi"}]},{id:"61865",title:"A Click Chemistry Approach to Tetrazoles: Recent Advances",slug:"a-click-chemistry-approach-to-tetrazoles-recent-advances",totalDownloads:2687,totalCrossrefCites:2,totalDimensionsCites:4,abstract:"Introduction to tetrazole and click chemistry approaches was briefed in a concise way in order to help the readers have a basic understanding. Tetrazole and its derivatives play very important role in medicinal and pharmaceutical applications. The synthesis of tetrazole derivatives can be approached in ecofriendly approaches such as the use of water as solvent, moderate conditions, nontoxic, easy extractions, easy setup, low cost, etc. with good to excellent yields.",book:{id:"6365",slug:"molecular-docking",title:"Molecular Docking",fullTitle:"Molecular Docking"},signatures:"Ravi Varala and Bollikolla Hari Babu",authors:[{id:"212519",title:"Dr.",name:"Varala",middleName:null,surname:"Ravi",slug:"varala-ravi",fullName:"Varala Ravi"},{id:"221476",title:"Dr.",name:"Bollikolla",middleName:null,surname:"Hari Babu",slug:"bollikolla-hari-babu",fullName:"Bollikolla Hari Babu"}]}],onlineFirstChaptersFilter:{topicId:"43",limit:6,offset:0},onlineFirstChaptersCollection:[{id:"82531",title:"Abnormal Iron Metabolism and Its Effect on Dentistry",slug:"abnormal-iron-metabolism-and-its-effect-on-dentistry",totalDownloads:12,totalDimensionsCites:0,doi:"10.5772/intechopen.104502",abstract:"Iron is a necessary micro-nutrient for proper functioning of the erythropoietic, oxidative and cellular metabolism. The iron balance in the body adversely affects the normal physiologic functioning of the body and structures in the oral cavity. Various abnormalities develop owing to improper iron metabolism in the body which reflects in the oral cavity. The toxicity of iron has to be well understood to immediately identify the hazardous effects which arise owing to it and to manage it. It has been very well mentioned in the chapter. The manifestations of defects of iron metabolism in the oral cavity should be carefully studied to improve the prognosis of the treatment of the same. 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Filling this knowledge gap will help us to improve and encourage sustainable weed control practices in agriculture.",book:{id:"11331",title:"Secondary Metabolites - Trends and Reviews",coverURL:"https://cdn.intechopen.com/books/images_new/11331.jpg"},signatures:"Ziming Yue, Varsha Singh, Josiane Argenta, Worlanyo Segbefia, Alyssa Miller and Te Ming Tseng"},{id:"81728",title:"Plant Secondary Metabolites: Therapeutic Potential and Pharmacological Properties",slug:"plant-secondary-metabolites-therapeutic-potential-and-pharmacological-properties",totalDownloads:30,totalDimensionsCites:0,doi:"10.5772/intechopen.103698",abstract:"Plants are an essential source for discovering novel medical compounds for drug development, and secondary metabolites are sources of medicines from plants. Secondary metabolites include alkaloids, flavonoids, terpenoids, tannins, coumarins, quinones, carotenoids, and steroids. Each year, several new secondary metabolites are extracted from plants, providing a source of possibilities to investigate against malignant illnesses, despite certain natural chemicals having distinct anticancer activities according to their physicochemical features. Secondary metabolites found in plants are frequently great leads for therapeutic development. However, changes in the molecular structure of these compounds are improving their anticancer activity and selectivity and their absorption, distribution, metabolism, and excretion capacities while minimizing their toxicity and side effects. In this section, we will discuss the most significant breakthroughs in the field of plant secondary metabolites, some of which are currently in clinical use and others that are in clinical trials as anticancer drugs. This study gives an up-to-date and thorough summary of secondary plant metabolites and their antioxidant, antibacterial, and anticancer effects. Furthermore, antioxidant and antibacterial, and anticancer effects of secondary metabolites are addressed. As a result, this article will serve as a thorough, quick reference for people interested in secondary metabolite antioxidants, anticancer, and antibacterial properties.",book:{id:"11331",title:"Secondary Metabolites - Trends and Reviews",coverURL:"https://cdn.intechopen.com/books/images_new/11331.jpg"},signatures:"Muhammad Zeeshan Bhatti, Hammad Ismail and Waqas Khan Kayani"},{id:"80495",title:"Iron in Cell Metabolism and Disease",slug:"iron-in-cell-metabolism-and-disease",totalDownloads:22,totalDimensionsCites:0,doi:"10.5772/intechopen.101908",abstract:"Iron is the trace element. We get the iron from the dietary sources. The enterocytes lining the upper duodenal of the intestine absorb the dietary iron through a divalent metal transporter (DMT1). The absorbed ferrous iron is oxidized to ferric iron in the body. 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This chapter on iron introduces the readers basic information of iron, cellular uptake, metabolism, and its role cellular physiology and provides the readers with the scope and importance of research on iron that hold the great benefit for health care and personalized medicine or diseases specific treatment strategies, blood transfusions and considerations.",book:{id:"10842",title:"Iron Metabolism - A Double-Edged Sword",coverURL:"https://cdn.intechopen.com/books/images_new/10842.jpg"},signatures:"Eeka Prabhakar"},{id:"81233",title:"Secondary Metabolites of Fruits and Vegetables with Antioxidant Potential",slug:"secondary-metabolites-of-fruits-and-vegetables-with-antioxidant-potential",totalDownloads:43,totalDimensionsCites:1,doi:"10.5772/intechopen.103707",abstract:"An antioxidant is of great interest among researchers, scientists, nutritionists, and the public because of its ability to prevent oxidative damage, as indicated by various studies. This chapter mainly focuses on the free radicals and their types; antioxidants and their mode of action against free radicals; fruits, vegetables, and their byproducts as a source of antioxidants; and various analytical methods employed for assessing antioxidant activity. Antioxidants discussed in this chapter are ascorbic acid, Vitamin E, carotenoids and polyphenols, and their mechanism of action. Different antioxidant activity assay techniques have been reported. Fruits and vegetables are abundant sources of these secondary metabolites. The waste generated during processing has many bioactive materials, which possibly be used in value-added by-products.",book:{id:"11331",title:"Secondary Metabolites - Trends and Reviews",coverURL:"https://cdn.intechopen.com/books/images_new/11331.jpg"},signatures:"Ravneet Kaur, Shubhra Shekhar and Kamlesh Prasad"},{id:"81044",title:"Metabolomics and Genetic Engineering for Secondary Metabolites Discovery",slug:"metabolomics-and-genetic-engineering-for-secondary-metabolites-discovery",totalDownloads:7,totalDimensionsCites:0,doi:"10.5772/intechopen.102838",abstract:"Since 1940s, microbial secondary metabolites (SMs) have attracted the attention of the scientific community. As a result, intensive researches have been conducted in order to discover and identify novel microbial secondary metabolites. Since, the discovery of novel secondary metabolites has been decreasing significantly due to many factors such as 1) unculturable microbes 2) traditional detection techniques 3) not all SMs expressed in the lab. As a result, searching for new techniques which can overcome the previous challenges was one of the most priority objectives. Therefore, the development of omics-based techniques such as genomics and metabolomic have revealed the potential of discovering novel SMs which were coded in the microorganisms’ DNA but not expressed in the lab or might be produced in undetectable amount by detecting the biosynthesis gene clusters (BGCs) that are associated with the biosynthesis of secondary metabolites. Nowadays, the integration of metabolomics and gene editing techniques such as CRISPR-Cas9 provide a successful platform for the detection and identification of known and unknown secondary metabolites also to increase secondary metabolites production.",book:{id:"11331",title:"Secondary Metabolites - Trends and Reviews",coverURL:"https://cdn.intechopen.com/books/images_new/11331.jpg"},signatures:"Ahmed M. Shuikan, Wael N. Hozzein, Rakan M. Alshuwaykan and Ibrahim A. 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He received his post-doctoral training in oncology and cancer proteomics for two years at the Cancer Research Institute of Human Medical University in China. In 2001, he went to the University of Tennessee Health Science Center (UTHSC) in USA, where he was a post-doctoral researcher and focused on mass spectrometry and cancer proteomics. Then, he was appointed as an Assistant Professor of Neurology, UTHSC in 2005. He moved to the Cleveland Clinic in USA as a Project Scientist/Staff in 2006 where he focused on the studies of eye disease proteomics and biomarkers. He returned to UTHSC as an Assistant Professor of Neurology in the end of 2007, engaging in proteomics and biomarker studies of lung diseases and brain tumors, and initiating the studies of predictive, preventive, and personalized medicine (PPPM) in cancer. In 2010, he was promoted to Associate Professor of Neurology, UTHSC. Currently, he is a Professor at Xiangya Hospital of Central South University in China, Fellow of Royal Society of Medicine (FRSM), the European EPMA National Representative in China, Regular Member of American Association for the Advancement of Science (AAAS), European Cooperation of Science and Technology (e-COST) grant evaluator, Associate Editors of BMC Genomics, BMC Medical Genomics, EPMA Journal, and Frontiers in Endocrinology, Executive Editor-in-Chief of Med One. He has\npublished 116 peer-reviewed research articles, 16 book chapters, 2 books, and 2 US patents. His current main research interest focuses on the studies of cancer proteomics and biomarkers, and the use of modern omics techniques and systems biology for PPPM in cancer, and on the development and use of 2DE-LC/MS for the large-scale study of human proteoforms.",institutionString:null,institution:{name:"Xiangya Hospital Central South University",country:{name:"China"}}},{id:"40482",title:null,name:"Rizwan",middleName:null,surname:"Ahmad",slug:"rizwan-ahmad",fullName:"Rizwan Ahmad",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/40482/images/system/40482.jpeg",biography:"Dr. Rizwan Ahmad is a University Professor and Coordinator, Quality and Development, College of Medicine, Imam Abdulrahman bin Faisal University, Saudi Arabia. Previously, he was Associate Professor of Human Function, Oman Medical College, Oman, and SBS University, Dehradun. Dr. Ahmad completed his education at Aligarh Muslim University, Aligarh. He has published several articles in peer-reviewed journals, chapters, and edited books. His area of specialization is free radical biochemistry and autoimmune diseases.",institutionString:"Imam Abdulrahman Bin Faisal University",institution:{name:"Imam Abdulrahman Bin Faisal University",country:{name:"Saudi Arabia"}}},{id:"41865",title:"Prof.",name:"Farid A.",middleName:null,surname:"Badria",slug:"farid-a.-badria",fullName:"Farid A. Badria",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/41865/images/system/41865.jpg",biography:"Farid A. Badria, Ph.D., is the recipient of several awards, including The World Academy of Sciences (TWAS) Prize for Public Understanding of Science; the World Intellectual Property Organization (WIPO) Gold Medal for best invention; Outstanding Arab Scholar, Kuwait; and the Khwarizmi International Award, Iran. He has 250 publications, 12 books, 20 patents, and several marketed pharmaceutical products to his credit. He continues to lead research projects on developing new therapies for liver, skin disorders, and cancer. Dr. Badria was listed among the world’s top 2% of scientists in medicinal and biomolecular chemistry in 2019 and 2020. He is a member of the Arab Development Fund, Kuwait; International Cell Research Organization–United Nations Educational, Scientific and Cultural Organization (ICRO–UNESCO), Chile; and UNESCO Biotechnology France",institutionString:"Mansoura University",institution:{name:"Mansoura University",country:{name:"Egypt"}}},{id:"329385",title:"Dr.",name:"Rajesh K.",middleName:"Kumar",surname:"Singh",slug:"rajesh-k.-singh",fullName:"Rajesh K. Singh",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/329385/images/system/329385.png",biography:"Dr. Singh received a BPharm (2003) and MPharm (2005) from Panjab University, Chandigarh, India, and a Ph.D. (2013) from Punjab Technical University (PTU), Jalandhar, India. He has more than sixteen years of teaching experience and has supervised numerous postgraduate and Ph.D. students. He has to his credit more than seventy papers in SCI- and SCOPUS-indexed journals, fifty-five conference proceedings, four books, six Best Paper Awards, and five projects from different government agencies. He is currently an editorial board member of eight international journals and a reviewer for more than fifty scientific journals. He received Top Reviewer and Excellent Peer Reviewer Awards from Publons in 2016 and 2017, respectively. He is also on the panel of The International Reviewer for reviewing research proposals for grants from the Royal Society. He also serves as a Publons Academy mentor and Bentham brand ambassador.",institutionString:"Punjab Technical University",institution:{name:"Punjab Technical University",country:{name:"India"}}},{id:"142388",title:"Dr.",name:"Thiago",middleName:"Gomes",surname:"Gomes Heck",slug:"thiago-gomes-heck",fullName:"Thiago Gomes Heck",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/142388/images/7259_n.jpg",biography:null,institutionString:null,institution:{name:"Universidade Regional do Noroeste do Estado do Rio Grande do Sul",country:{name:"Brazil"}}},{id:"336273",title:"Assistant Prof.",name:"Janja",middleName:null,surname:"Zupan",slug:"janja-zupan",fullName:"Janja Zupan",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/336273/images/14853_n.jpeg",biography:"Janja Zupan graduated in 2005 at the Department of Clinical Biochemistry (superviser prof. dr. Janja Marc) in the field of genetics of osteoporosis. Since November 2009 she is working as a Teaching Assistant at the Faculty of Pharmacy, Department of Clinical Biochemistry. In 2011 she completed part of her research and PhD work at Institute of Genetics and Molecular Medicine, University of Edinburgh. She finished her PhD entitled The influence of the proinflammatory cytokines on the RANK/RANKL/OPG in bone tissue of osteoporotic and osteoarthritic patients in 2012. From 2014-2016 she worked at the Institute of Biomedical Sciences, University of Aberdeen as a postdoctoral research fellow on UK Arthritis research project where she gained knowledge in mesenchymal stem cells and regenerative medicine. She returned back to University of Ljubljana, Faculty of Pharmacy in 2016. She is currently leading project entitled Mesenchymal stem cells-the keepers of tissue endogenous regenerative capacity facing up to aging of the musculoskeletal system funded by Slovenian Research Agency.",institutionString:null,institution:{name:"University of Ljubljana",country:{name:"Slovenia"}}},{id:"357453",title:"Dr.",name:"Radheshyam",middleName:null,surname:"Maurya",slug:"radheshyam-maurya",fullName:"Radheshyam Maurya",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/357453/images/16535_n.jpg",biography:null,institutionString:null,institution:{name:"University of Hyderabad",country:{name:"India"}}},{id:"418340",title:"Dr.",name:"Jyotirmoi",middleName:null,surname:"Aich",slug:"jyotirmoi-aich",fullName:"Jyotirmoi Aich",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y000038Ugi5QAC/Profile_Picture_2022-04-15T07:48:28.png",biography:"Biotechnologist with 15 years of research including 6 years of teaching experience. Demonstrated record of scientific achievements through consistent publication record (H index = 13, with 874 citations) in high impact journals such as Nature Communications, Oncotarget, Annals of Oncology, PNAS, and AJRCCM, etc. Strong research professional with a post-doctorate from ACTREC where I gained experimental oncology experience in clinical settings and a doctorate from IGIB where I gained expertise in asthma pathophysiology. A well-trained biotechnologist with diverse experience on the bench across different research themes ranging from asthma to cancer and other infectious diseases. An individual with a strong commitment and innovative mindset. Have the ability to work on diverse projects such as regenerative and molecular medicine with an overall mindset of improving healthcare.",institutionString:"DY Patil Deemed to Be University",institution:null},{id:"349288",title:"Prof.",name:"Soumya",middleName:null,surname:"Basu",slug:"soumya-basu",fullName:"Soumya Basu",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y000035QxIDQA0/Profile_Picture_2022-04-15T07:47:01.jpg",biography:"Soumya Basu, Ph.D., is currently working as an Associate Professor at Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune, Maharashtra, India. With 16+ years of trans-disciplinary research experience in Drug Design, development, and pre-clinical validation; 20+ research article publications in journals of repute, 9+ years of teaching experience, trained with cross-disciplinary education, Dr. Basu is a life-long learner and always thrives for new challenges.\r\nHer research area is the design and synthesis of small molecule partial agonists of PPAR-γ in lung cancer. She is also using artificial intelligence and deep learning methods to understand the exosomal miRNA’s role in cancer metastasis. Dr. Basu is the recipient of many awards including the Early Career Research Award from the Department of Science and Technology, Govt. of India. She is a reviewer of many journals like Molecular Biology Reports, Frontiers in Oncology, RSC Advances, PLOS ONE, Journal of Biomolecular Structure & Dynamics, Journal of Molecular Graphics and Modelling, etc. She has edited and authored/co-authored 21 journal papers, 3 book chapters, and 15 abstracts. She is a Board of Studies member at her university. She is a life member of 'The Cytometry Society”-in India and 'All India Cell Biology Society”- in India.",institutionString:"Dr. D.Y. Patil Vidyapeeth, Pune",institution:{name:"Dr. D.Y. Patil Vidyapeeth, Pune",country:{name:"India"}}},{id:"354817",title:"Dr.",name:"Anubhab",middleName:null,surname:"Mukherjee",slug:"anubhab-mukherjee",fullName:"Anubhab Mukherjee",position:null,profilePictureURL:"https://intech-files.s3.amazonaws.com/0033Y0000365PbRQAU/ProfilePicture%202022-04-15%2005%3A11%3A18.480",biography:"A former member of Laboratory of Nanomedicine, Brigham and Women’s Hospital, Harvard University, Boston, USA, Dr. Anubhab Mukherjee is an ardent votary of science who strives to make an impact in the lives of those afflicted with cancer and other chronic/acute ailments. He completed his Ph.D. from CSIR-Indian Institute of Chemical Technology, Hyderabad, India, having been skilled with RNAi, liposomal drug delivery, preclinical cell and animal studies. He pursued post-doctoral research at College of Pharmacy, Health Science Center, Texas A & M University and was involved in another postdoctoral research at Department of Translational Neurosciences and Neurotherapeutics, John Wayne Cancer Institute, Santa Monica, California. In 2015, he worked in Harvard-MIT Health Sciences & Technology as a visiting scientist. He has substantial experience in nanotechnology-based formulation development and successfully served various Indian organizations to develop pharmaceuticals and nutraceutical products. He is an inventor in many US patents and an author in many peer-reviewed articles, book chapters and books published in various media of international repute. Dr. Mukherjee is currently serving as Principal Scientist, R&D at Esperer Onco Nutrition (EON) Pvt. Ltd. and heads the Hyderabad R&D center of the organization.",institutionString:"Esperer Onco Nutrition Pvt Ltd.",institution:null},{id:"319365",title:"Assistant Prof.",name:"Manash K.",middleName:null,surname:"Paul",slug:"manash-k.-paul",fullName:"Manash K. Paul",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/319365/images/system/319365.png",biography:"Manash K. Paul is a Principal Investigator and Scientist at the University of California Los Angeles. He has contributed significantly to the fields of stem cell biology, regenerative medicine, and lung cancer. His research focuses on various signaling processes involved in maintaining stem cell homeostasis during the injury-repair process, deciphering lung stem cell niche, pulmonary disease modeling, immuno-oncology, and drug discovery. He is currently investigating the role of extracellular vesicles in premalignant lung cell migration and detecting the metastatic phenotype of lung cancer via machine-learning-based analyses of exosomal signatures. Dr. Paul has published in more than fifty peer-reviewed international journals and is highly cited. He is the recipient of many awards, including the UCLA Vice Chancellor’s award, a senior member of the Institute of Electrical and Electronics Engineers (IEEE), and an editorial board member for several international journals.",institutionString:"University of California Los Angeles",institution:{name:"University of California Los Angeles",country:{name:"United States of America"}}},{id:"311457",title:"Dr.",name:"Júlia",middleName:null,surname:"Scherer Santos",slug:"julia-scherer-santos",fullName:"Júlia Scherer Santos",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/311457/images/system/311457.jpg",biography:"Dr. Júlia Scherer Santos works in the areas of cosmetology, nanotechnology, pharmaceutical technology, beauty, and aesthetics. Dr. Santos also has experience as a professor of graduate courses. Graduated in Pharmacy, specialization in Cosmetology and Cosmeceuticals applied to aesthetics, specialization in Aesthetic and Cosmetic Health, and a doctorate in Pharmaceutical Nanotechnology. Teaching experience in Pharmacy and Aesthetics and Cosmetics courses. She works mainly on the following subjects: nanotechnology, cosmetology, pharmaceutical technology, aesthetics.",institutionString:"Universidade Federal de Juiz de Fora",institution:{name:"Universidade Federal de Juiz de Fora",country:{name:"Brazil"}}},{id:"219081",title:"Dr.",name:"Abdulsamed",middleName:null,surname:"Kükürt",slug:"abdulsamed-kukurt",fullName:"Abdulsamed Kükürt",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/219081/images/system/219081.png",biography:"Dr. Kükürt graduated from Uludağ University in Turkey. He started his academic career as a Research Assistant in the Department of Biochemistry at Kafkas University. In 2019, he completed his Ph.D. program in the Department of Biochemistry at the Institute of Health Sciences. He is currently working at the Department of Biochemistry, Kafkas University. He has 27 published research articles in academic journals, 11 book chapters, and 37 papers. He took part in 10 academic projects. He served as a reviewer for many articles. He still serves as a member of the review board in many academic journals. He is currently working on the protective activity of phenolic compounds in disorders associated with oxidative stress and inflammation.",institutionString:null,institution:{name:"Kafkas University",country:{name:"Turkey"}}},{id:"178366",title:"Dr.",name:"Volkan",middleName:null,surname:"Gelen",slug:"volkan-gelen",fullName:"Volkan Gelen",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/178366/images/system/178366.jpg",biography:"Volkan Gelen is a Physiology specialist who received his veterinary degree from Kafkas University in 2011. Between 2011-2015, he worked as an assistant at Atatürk University, Faculty of Veterinary Medicine, Department of Physiology. In 2016, he joined Kafkas University, Faculty of Veterinary Medicine, Department of Physiology as an assistant professor. Dr. Gelen has been engaged in various academic activities at Kafkas University since 2016. There he completed 5 projects and has 3 ongoing projects. He has 60 articles published in scientific journals and 20 poster presentations in scientific congresses. His research interests include physiology, endocrine system, cancer, diabetes, cardiovascular system diseases, and isolated organ bath system studies.",institutionString:"Kafkas University",institution:{name:"Kafkas University",country:{name:"Turkey"}}},{id:"418963",title:"Dr.",name:"Augustine Ododo",middleName:"Augustine",surname:"Osagie",slug:"augustine-ododo-osagie",fullName:"Augustine Ododo Osagie",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/418963/images/16900_n.jpg",biography:"Born into the family of Osagie, a prince of the Benin Kingdom. I am currently an academic in the Department of Medical Biochemistry, University of Benin. Part of the duties are to teach undergraduate students and conduct academic research.",institutionString:null,institution:{name:"University of Benin",country:{name:"Nigeria"}}},{id:"192992",title:"Prof.",name:"Shagufta",middleName:null,surname:"Perveen",slug:"shagufta-perveen",fullName:"Shagufta Perveen",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/192992/images/system/192992.png",biography:"Prof. Shagufta Perveen is a Distinguish Professor in the Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. Dr. Perveen has acted as the principal investigator of major research projects funded by the research unit of King Saud University. She has more than ninety original research papers in peer-reviewed journals of international repute to her credit. She is a fellow member of the Royal Society of Chemistry UK and the American Chemical Society of the United States.",institutionString:"King Saud University",institution:{name:"King Saud University",country:{name:"Saudi Arabia"}}},{id:"49848",title:"Dr.",name:"Wen-Long",middleName:null,surname:"Hu",slug:"wen-long-hu",fullName:"Wen-Long Hu",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/49848/images/system/49848.jpg",biography:"Wen-Long Hu is Chief of the Division of Acupuncture, Department of Chinese Medicine at Kaohsiung Chang Gung Memorial Hospital, as well as an adjunct associate professor at Fooyin University and Kaohsiung Medical University. Wen-Long is President of Taiwan Traditional Chinese Medicine Medical Association. He has 28 years of experience in clinical practice in laser acupuncture therapy and 34 years in acupuncture. He is an invited speaker for lectures and workshops in laser acupuncture at many symposiums held by medical associations. He owns the patent for herbal preparation and producing, and for the supercritical fluid-treated needle. Dr. Hu has published three books, 12 book chapters, and more than 30 papers in reputed journals, besides serving as an editorial board member of repute.",institutionString:"Kaohsiung Chang Gung Memorial Hospital",institution:{name:"Kaohsiung Chang Gung Memorial Hospital",country:{name:"Taiwan"}}},{id:"298472",title:"Prof.",name:"Andrey V.",middleName:null,surname:"Grechko",slug:"andrey-v.-grechko",fullName:"Andrey V. Grechko",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/298472/images/system/298472.png",biography:"Andrey Vyacheslavovich Grechko, Ph.D., Professor, is a Corresponding Member of the Russian Academy of Sciences. He graduated from the Semashko Moscow Medical Institute (Semashko National Research Institute of Public Health) with a degree in Medicine (1998), the Clinical Department of Dermatovenerology (2000), and received a second higher education in Psychology (2009). Professor A.V. Grechko held the position of Сhief Physician of the Central Clinical Hospital in Moscow. He worked as a professor at the faculty and was engaged in scientific research at the Medical University. Starting in 2013, he has been the initiator of the creation of the Federal Scientific and Clinical Center for Intensive Care and Rehabilitology, Moscow, Russian Federation, where he also serves as Director since 2015. He has many years of experience in research and teaching in various fields of medicine, is an author/co-author of more than 200 scientific publications, 13 patents, 15 medical books/chapters, including Chapter in Book «Metabolomics», IntechOpen, 2020 «Metabolomic Discovery of Microbiota Dysfunction as the Cause of Pathology».",institutionString:"Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology",institution:null},{id:"199461",title:"Prof.",name:"Natalia V.",middleName:null,surname:"Beloborodova",slug:"natalia-v.-beloborodova",fullName:"Natalia V. Beloborodova",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/199461/images/system/199461.jpg",biography:'Natalia Vladimirovna Beloborodova was educated at the Pirogov Russian National Research Medical University, with a degree in pediatrics in 1980, a Ph.D. in 1987, and a specialization in Clinical Microbiology from First Moscow State Medical University in 2004. She has been a Professor since 1996. Currently, she is the Head of the Laboratory of Metabolism, a division of the Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, Moscow, Russian Federation. N.V. Beloborodova has many years of clinical experience in the field of intensive care and surgery. She studies infectious complications and sepsis. She initiated a series of interdisciplinary clinical and experimental studies based on the concept of integrating human metabolism and its microbiota. Her scientific achievements are widely known: she is the recipient of the Marie E. Coates Award \\"Best lecturer-scientist\\" Gustafsson Fund, Karolinska Institutes, Stockholm, Sweden, and the International Sepsis Forum Award, Pasteur Institute, Paris, France (2014), etc. Professor N.V. Beloborodova wrote 210 papers, five books, 10 chapters and has edited four books.',institutionString:"Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology",institution:null},{id:"354260",title:"Ph.D.",name:"Tércio Elyan",middleName:"Azevedo",surname:"Azevedo Martins",slug:"tercio-elyan-azevedo-martins",fullName:"Tércio Elyan Azevedo Martins",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/354260/images/16241_n.jpg",biography:"Graduated in Pharmacy from the Federal University of Ceará with the modality in Industrial Pharmacy, Specialist in Production and Control of Medicines from the University of São Paulo (USP), Master in Pharmaceuticals and Medicines from the University of São Paulo (USP) and Doctor of Science in the program of Pharmaceuticals and Medicines by the University of São Paulo. Professor at Universidade Paulista (UNIP) in the areas of chemistry, cosmetology and trichology. Assistant Coordinator of the Higher Course in Aesthetic and Cosmetic Technology at Universidade Paulista Campus Chácara Santo Antônio. Experience in the Pharmacy area, with emphasis on Pharmacotechnics, Pharmaceutical Technology, Research and Development of Cosmetics, acting mainly on topics such as cosmetology, antioxidant activity, aesthetics, photoprotection, cyclodextrin and thermal analysis.",institutionString:null,institution:{name:"University of Sao Paulo",country:{name:"Brazil"}}},{id:"334285",title:"Ph.D. Student",name:"Sameer",middleName:"Kumar",surname:"Jagirdar",slug:"sameer-jagirdar",fullName:"Sameer Jagirdar",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/334285/images/14691_n.jpg",biography:"I\\'m a graduate student at the center for biosystems science and engineering at the Indian Institute of Science, Bangalore, India. I am interested in studying host-pathogen interactions at the biomaterial interface.",institutionString:null,institution:{name:"Indian Institute of Science Bangalore",country:{name:"India"}}},{id:"329248",title:"Dr.",name:"Md. Faheem",middleName:null,surname:"Haider",slug:"md.-faheem-haider",fullName:"Md. Faheem Haider",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/329248/images/system/329248.jpg",biography:"Dr. Md. Faheem Haider completed his BPharm in 2012 at Integral University, Lucknow, India. In 2014, he completed his MPharm with specialization in Pharmaceutics at Babasaheb Bhimrao Ambedkar University, Lucknow, India. He received his Ph.D. degree from Jamia Hamdard University, New Delhi, India, in 2018. He was selected for the GPAT six times and his best All India Rank was 34. Currently, he is an assistant professor at Integral University. Previously he was an assistant professor at IIMT University, Meerut, India. He has experience teaching DPharm, Pharm.D, BPharm, and MPharm students. He has more than five publications in reputed journals to his credit. Dr. Faheem’s research area is the development and characterization of nanoformulation for the delivery of drugs to various organs.",institutionString:"Integral University",institution:{name:"Integral University",country:{name:"India"}}},{id:"329795",title:"Dr.",name:"Mohd Aftab",middleName:"Aftab",surname:"Siddiqui",slug:"mohd-aftab-siddiqui",fullName:"Mohd Aftab Siddiqui",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/329795/images/system/329795.png",biography:"Dr. Mohd Aftab Siddiqui is an assistant professor in the Faculty of Pharmacy, Integral University, Lucknow, India, where he obtained a Ph.D. in Pharmacology in 2020. He also obtained a BPharm and MPharm from the same university in 2013 and 2015, respectively. His area of research is the pharmacological screening of herbal drugs/natural products in liver cancer and cardiac diseases. He is a member of many professional bodies and has guided many MPharm and PharmD research projects. Dr. Siddiqui has many national and international publications and one German patent to his credit.",institutionString:"Integral University",institution:null}]}},subseries:{item:{id:"91",type:"subseries",title:"Sustainable Economy and Fair Society",keywords:"Sustainable, Society, Economy, Digitalization, KPIs, Decision Making, Business, Digital Footprint",scope:"\r\n\tGlobally, the ecological footprint is growing at a faster rate than GDP. This phenomenon has been studied by scientists for many years. However, clear strategies and actions are needed now more than ever. Every day, humanity, from individuals to businesses (public and private) and governments, are called to change their mindset in order to pursue a virtuous combination for sustainable development. Reasoning in a sustainable way entails, first and foremost, managing the available resources efficiently and strategically, whether they are natural, financial, human or relational. In this way, value is generated by contributing to the growth, improvement and socio-economic development of the communities and of all the players that make up its value chain. In the coming decades, we will need to be able to transition from a society in which economic well-being and health are measured by the growth of production and material consumption, to a society in which we live better while consuming less. In this context, digitization has the potential to disrupt processes, with significant implications for the environment and sustainable development. There are numerous challenges associated with sustainability and digitization, the need to consider new business models capable of extracting value, data ownership and sharing and integration, as well as collaboration across the entire supply chain of a product. In order to generate value, effectively developing a complex system based on sustainability principles is a challenge that requires a deep commitment to both technological factors, such as data and platforms, and human dimensions, such as trust and collaboration. Regular study, research and implementation must be part of the road to sustainable solutions. Consequently, this topic will analyze growth models and techniques aimed at achieving intergenerational equity in terms of economic, social and environmental well-being. It will also cover various subjects, including risk assessment in the context of sustainable economy and a just society.
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