\r\n\tThis book intends to provide the reader with a comprehensive overview of the current state-of-the-art novel imaging techniques by focusing on the most important evidence-based developments in this area.
",isbn:null,printIsbn:null,pdfIsbn:null,doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,isSalesforceBook:!1,isNomenclature:!1,hash:"d9159ce31733bf78cc2a79b18c225994",bookSignature:"Dr. Gabriel Cismaru",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/11867.jpg",keywords:"Hypertrophic Cardiomyopathy, Dilated Cardiomyopathy, Restrictive Cardiomyopathy, Transesophageal Echocardiography, Intracardiac Echocardiography, 3-Dimensional Echocardiography, Adult Congenital Heart Disease, Tetralogy of Fallot, Transposition of the Great Vessels, Coronary Artery Disease, Risk Stratification, Revascularization",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"April 21st 2022",dateEndSecondStepPublish:"May 19th 2022",dateEndThirdStepPublish:"July 18th 2022",dateEndFourthStepPublish:"October 6th 2022",dateEndFifthStepPublish:"December 5th 2022",dateConfirmationOfParticipation:null,remainingDaysToSecondStep:"3 months",secondStepPassed:!0,areRegistrationsClosed:!0,currentStepOfPublishingProcess:4,editedByType:null,kuFlag:!1,biosketch:"Dr. Cismaru Gabriel is an Assistant Professor at the University of Medicine and Pharmacy Cluj-Napoca, certified in Cardiology. After completing his certification in cardiology, Dr. Cismaru began his electrophysiology fellowship at the Institut Lorrain du Coeur et des Vaisseaux Louis Mathieu. He has authored or co-authored peer-reviewed articles and book chapters in the field of cardiac pacing, defibrillation, electrophysiological study, and catheter ablation.",coeditorOneBiosketch:"Raluca Tomoaia is an MD, Ph.D. in novel techniques in Echocardiography at the University of Medicine and Pharmacy in Cluj-Napoca, Romania., assistant professor, and a researcher in echocardiography and cardiovascular imaging.",coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"191888",title:"Dr.",name:"Gabriel",middleName:null,surname:"Cismaru",slug:"gabriel-cismaru",fullName:"Gabriel Cismaru",profilePictureURL:"https://mts.intechopen.com/storage/users/191888/images/system/191888.png",biography:"Dr. Cismaru Gabriel is an assistant professor at the Cluj-Napoca University of Medicine and Pharmacy, Romania, where he has been qualified in cardiology since 2011. He obtained his Ph.D. in medicine with a research thesis on electrophysiology and pro-arrhythmic drugs in 2016. Dr. Cismaru began his electrophysiology fellowship at the Institut Lorrain du Coeur et des Vaisseaux Louis Mathieu, France, after finishing his cardiology certification with stages in Clermont-Ferrand and Dinan, France. He began working at the Rehabilitation Hospital\\'s Electrophysiology Laboratory in Cluj-Napoca in 2011. He is an experienced operator who can implant pacemakers, CRTs, and ICDs, as well as perform catheter ablation of supraventricular and ventricular arrhythmias such as ventricular tachycardia and ventricular fibrillation. He has been qualified in pediatric cardiology since 2022, and he regularly performs device implantation and catheter ablation in children. Dr. Cismaru has authored or co-authored peer-reviewed publications and book chapters on cardiac pacing, defibrillation, electrophysiological studies, and catheter ablation.",institutionString:"Iuliu Hațieganu University of Medicine and Pharmacy",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"7",totalChapterViews:"0",totalEditedBooks:"2",institution:{name:"Iuliu Hațieganu University of Medicine and Pharmacy",institutionURL:null,country:{name:"Romania"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"16",title:"Medicine",slug:"medicine"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:null},relatedBooks:[{type:"book",id:"5970",title:"Bedside Procedures",subtitle:null,isOpenForSubmission:!1,hash:"ba56d3036ac823a7155f40e4a02c030d",slug:"bedside-procedures",bookSignature:"Gabriel Cismaru",coverURL:"https://cdn.intechopen.com/books/images_new/5970.jpg",editedByType:"Edited by",editors:[{id:"191888",title:"Dr.",name:"Gabriel",surname:"Cismaru",slug:"gabriel-cismaru",fullName:"Gabriel Cismaru"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"9064",title:"Epidemiology and Treatment of Atrial Fibrillation",subtitle:null,isOpenForSubmission:!1,hash:"1cd6bf2b3181eb82446347fbe478a2bc",slug:"epidemiology-and-treatment-of-atrial-fibrillation",bookSignature:"Gabriel Cismaru and Keith Andrew Chan",coverURL:"https://cdn.intechopen.com/books/images_new/9064.jpg",editedByType:"Edited by",editors:[{id:"191888",title:"Dr.",name:"Gabriel",surname:"Cismaru",slug:"gabriel-cismaru",fullName:"Gabriel Cismaru"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"6550",title:"Cohort Studies in Health Sciences",subtitle:null,isOpenForSubmission:!1,hash:"01df5aba4fff1a84b37a2fdafa809660",slug:"cohort-studies-in-health-sciences",bookSignature:"R. 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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"2270",title:"Fourier Transform",subtitle:"Materials Analysis",isOpenForSubmission:!1,hash:"5e094b066da527193e878e160b4772af",slug:"fourier-transform-materials-analysis",bookSignature:"Salih Mohammed Salih",coverURL:"https://cdn.intechopen.com/books/images_new/2270.jpg",editedByType:"Edited by",editors:[{id:"111691",title:"Dr.Ing.",name:"Salih",surname:"Salih",slug:"salih-salih",fullName:"Salih Salih"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"17779",title:"Fluorescence Cross-Correlation Spectroscopy for Real-Time Monitoring of Exogenous DNA Behavior in Living Cells",doi:"10.5772/18970",slug:"fluorescence-cross-correlation-spectroscopy-for-real-time-monitoring-of-exogenous-dna-behavior-in-li",body:'Many kind of DNA transfection techniques have been developed and widely used in the field of biochemical assay (Boussif et al., 1995, Chu et al., 1987, Felgner et al., 1987). Since recent development of fluorescent proteins such as green fluorescent protein (GFP), its variants and bio sensors, DNA transfection techniques play a greater role in the field of cell biology especially in bioimaging studies. DNA transfection is also used as a key technology to gene therapy (Selkirk, 2004, Schaffert et al., 2008). Thus the development of gene delivery vector with high efficiency is expected. Gene delivery systems with artificial nonviral vectors are widely used in the research field of cell biology and also in the clinical field as gene therapy to promote exogenous gene expression or inhibit production of a target protein. Nonviral vectors have advantages such as a low immune response and no risk of a disorder, which might be caused by native viral vector genome integration (Thomas et al., 2003). However, nonviral vectors need to overcome the disadvantage of low gene expression efficiency compared with native viral vectors (Luo et al., 2000). To increase the expression efficiency, it is needed to know gene delivery mechanisms. In general, complexes of exogenous DNAs and a cationic polymer or cationic lipid, commonly used as nonviral vectors, are internalized to target cells by endocytosis. Then a sequential process progresses spontaneously with escape from endosomes, dissociation of complexes, and diffusion of naked DNAs in the cytoplasm to the nucleus (Fig. 1) (Elouahabi et al., 2005).
However, such dynamic properties of transfected DNAs are not yet clearly distinguished because of the lack of suitable technique to observe intracellular DNA behavior. Fluorescence correlation spectroscopy (FCS) is a method based on observation of fluorescence intensity fluctuations that is the result of single fluorescent molecules diffuse in and out of small detection area (Fig. 2A, B) (Eigen et al., 1994). FCS technique can be applied for quantification of the absolute number of fluorescently labeled particles and measuring the molecular weight or size with extremely high sensitivity in a small sample volume, and a physical separation procedure is not needed. Therefore, FCS has currently employed to investigate molecular size or interactions
Schematic diagram of intracellular gene delivery pathway.
Schematic diagram of FCS. (A) Schematic of observation area of FCS (confocal volume). (B) Obtained fluctuation of fluorescence intensity. (C) Autocorrelation functions (ACFs).
When the two target molecules are interacting, they will move together through the observation area and have synchronized fluorescence fluctuations in both two channels (Fig. 3A). On the other hand, the fluctuations are not correlated when these molecules move independently of one another (Fig. 3B). In this chapter, We describe the advantages of FCCS technique for the research field of gene therapy. In our research, FCCS technique was applied to study i) the behavior of exogenous DNA in living cells (Sasaki et al., 2010) and ii) the formation of DNA/carrier complex. In these cases, FCCS revealed the DNA degradation by nucleases and DNA-cationic polymer interactions to form DNA/carrier complex in real-time.
Schematic diagram of FCCS. Auto and Cross-correlation functions (CCFs) of two fluorescent probes are connected (A) or separated (B).
The FCCS setup consists of excitation, detection, and data processing systems similar to that of a typical laser-scanning confocal fluorescence microscope with high numerical-aperture (NA) objective.
Laser beams are focused on the coverslip via a confocal optic system to subfemto-liter size of observation area is generated. Emitted fluorescence signal is detected two avalanche photodiode (APD) through dichroic mirror (DM) and filter sets (barrier filter; BF) for each color channels (Fig. 4).
The FCS data are interpreted in terms of autocorrelation functions (G(τ)) (Fig. 2C), that give information on the diffusion time (DT) and the number of molecules (N) in observation area. Diffusion constant can be estimated from DT and size of observation area (Eigen et al., 1994).
The fluorescence autocorrelation functions of the red and green channels, Gr(τ) and Gg(τ), and the fluorescence cross-correlation function, Gc(τ), are calculated by
where τ denotes the time delay. Ii is the fluorescence intensity of the red channel (i = r) or green channel (i = g), Gr(τ) and Gg(τ) denote the autocorrelation functions of red (i = j = x = r), and green (i = j = x = g) respectively, or Gc(τ) denotes cross-correlation function (i = r, j = g, and x = c). Acquired G(τ) were generally fitted by a one- (i = 1) or two-component model (i = 2) as
where Fi and τi are the fraction and diffusion time of component i, respectively. N is the average number of fluorescent particles in the observation area defined by radius ω and length 2z, and s is the structural parameter representing the ratio s = z/ω. The diffusion time (τi) corresponds to the average time for diffusion of fluorescent particles across the detection area, which reflects the size of particles. f is the average fraction of triplet state molecules and τt is triplet relaxation time (Widengren et al., 1995, Rigler et al., 1993). The distribution histogram of diffusion times was constructed using the CONTIN algorithm (Provencher, 1982, Björling et al., 1998).
Experimental setup for FCS and FCCS.
Instead of evaluating discrete diffusion processes with distinct species and their characteristic diffusion times, the dynamic motion of DNAs in the cell can be distributed with regard to their diffusion times using the distribution function P(τDi). The average numbers of red fluorescent particles (Nr), green fluorescent particles (Ng), and particles that have both red and green fluorescence (Nc) can be calculated by
respectively. When Nr and Ng are constant, (Gc(0)–1) is directly proportional to Nc. For quantitative evaluation of cross-correlations among various samples, the cross-correlation amplitude is normalized by autocorrelation amplitude (relative cross-correlation amplitude; RCA). RCA can be calculated by
respectively. The ratio of interacted molecule in red or green molecules can be obtained from equations 3 and 4 as
respectively. RCAg shows the ratio of interacted molecules in the red molecules (Nc/Nr) (Eqs. 5 and 7) and RCAr shows the ratio of interacted molecules in the green molecules (Nc/Ng) (Eqs. 6 and 8). FCCS measurement also gives the parameter of photon counts per molecule (CPM) of each fluorescent species. CPM is represented by count rate divided by number of particles.
Physical approaches such as microinjection (Zhang et al., 2008) and electroporation (Somiari et al., 2000) are performed to incorporate naked DNAs directly into cytoplasm and/or nucleus across the plasma membrane. On the other hand, DNAs are released into cytoplasm as naked shape and then the DNAs diffuse in the cytoplasm to the nucleus in the case of using gene carrier. The diffusion of exogenous DNAs in cytoplasm is relatively slow compared with that in solution (Dauty et al., 2005), and it is hard that large DNA goes cross the nuclear membrane in short time. Under such conditions in cytoplasm, the existence of nuclease degradation in cytoplasm is suggested (Pollard et al., 2001, Lechardeur et al., 1999, Fisher et al., 1993). It is presumed that the translocation and nuclear uptake of transfected DNA compete with digestion. Therefore, it is important to investigate behavior of naked exogenous DNAs in cytoplasm to achieve efficient gene delivery and expression. The cytoplasmic degradation for exogenous DNAs can work as a barrier against efficient gene delivery. However, little is known about the degradation mechanism because it is difficult to characterize the degradation process of exogenous DNA in living cells. The purpose of this section was to investigate the mechanism of exogenous DNA degradation in situ, in cytoplasm, by analyses of diffusion properties of DNAs and to determine the effects of cytoplasmic degradation on the gene expression rate. Thus we employed FCS to measure the diffusion properties of DNAs, and FCCS to monitor the degradation of DNAs by cytoplasmic nucleases at the single molecule level. Furthermore, we predicted that exonucleases would work as the main barrier in cytoplasm, so a capped or redundant structure was attached to the transfected DNA to enhance the expression of the protein EGFP.
A series of linear DNAs from 23 bp to 500 bp that were labeled with rhodamine green (RG) and Cy5 on both 5’ends (RG-DNA-Cy5) were synthesized by PCR. Autocorrelation curves of the green fluorescence channel (Gg(τ)) and red fluorescence channel (Gr(τ)), and a cross-correlation curve (Gc(τ)) were obtained by FCCS measurements of RG-DNA-Cy5 in solution (Fig. 5A-D).
Characterization of double-fluorescent-labeled DNAs in solution. Auto- and cross-correlation curves of (A) RG-23bp-Cy5 (B) RG-100bp-Cy5 (C) RG-200bp-Cy5 (D) RG-500bp-Cy5 DNA in 10 mM Tris-HCl (pH 7.4). (Insets) The fluorescence intensities in the red (red line) and green (blue line) channels during FCCS measurement. Blue, red and black lines represent Gg(τ), Gr(τ) and Gc(τ), respectively. (E) Auto- and cross-correlation curves of a mixture of RG-primer and Cy5-primer (23 mer each) as a negative control. (F) Distributions of diffusion times P(τDi) of various sizes of RG-DNA-Cy5 calculated from autocorrelation curves of the green channel by CONTIN algorithm. (Inset) Normalized autocorrelation curves Gg(τ)/Gg(0) (dot) with fitted curves (line) of DNAs in 10 mM Tris-HCl (pH 7.4) at room temperature.
To assess the degree of cross-correlation amplitude, relative cross-correlation amplitude (RCA) was calculated by dividing Gc(0)–1 by Gr(0)–1. The RCA value was 0.908±0.020 (mean ± S.D.) for the 500 bp DNA, which had double-fluorescent labels, and 0.104±0.023 for the mixture of fluorescent primers that were used in the PCR reactions (Fig. 5E). The RCA value represents the fraction of DNAs that have RG and Cy5 fluorophores at both DNA ends, thus intact DNAs. To extract information concerning the relative abundance and length of the DNA from the autocorrelation curve, we used the CONTIN algorithm. The algorithm can determine the distribution of the correlation time with multiple peaks and its probability without any prior assumption about the number of peaks. Figure 5F shows the distributions of diffusion times that were calculated from the autocorrelation curve of the green channel (inset) by using the CONTIN algorithm. The spectra of diffusion times of RG-DNA-Cy5 were separated from each other. This result suggested that the length of the DNA could be estimated with sufficient resolution in this range and length.
To confirm whether FCS and FCCS could identify the degradation mechanisms of different kind of nucleases, we monitored the DNA degradation with DNase I (an endonuclease that acts randomly), exonuclease III (an exonuclease that acts in the 3’ to 5’ direction) and BAL31 nuclease (an exonuclease that acts in both 5’ to 3’ and 3’to 5’ directions) in solution. The nucleases were added to RG-500bp-Cy5 solution in a droplet (50 μl) on a coverslip and FCCS measurements were performed as a function of time (Fig. 6).
Relative cross-correlation amplitudes, (Gc(0)–1)/(Gr(0)–1) after addition of (A) 0.0001 U μl–1 DNase I, (B) 0.02 U μl–1 exonuclease III, and (C) 0.001 U μl–1 BAL31 nuclease. Distributions of diffusion times P(τDi) calculated by CONTIN algorithm from Gg(τ) at several time points after the addition of DNase I (D), exonuclease III (E) and BAL31 nuclease (F).
Figure 6A, B and C show the RCA changes of cross-correlation curves of RG-500bp-Cy5 with the three different type of nucleases. With DNase I and BAL31 nuclease, RCA values rapidly decreased after the nuclease addition. In contrast, with exonuclease III, the RCA value did not decrease until 11 min. This indicated that the fluorophores did not separate in the initial phase of degradation (Fig. 6E insets). The RCA value was not changed without addition of a nuclease in the measurement time (data not shown). We regarded 11 min as the initial phase, 31 min as the transient phase and 84–97 min as the stable phase of enzymatic reaction in Figure 6A, B and C. We performed CONTIN analysis and compared distributions of diffusion times in each phase (Fig. 6D–F). Changing of distribution of diffusion time and RCA show some kind of fingerprint of enzyme activity. The distribution peak became wider and shifted to the left with addition of DNase I (Fig. 6D). The peak width reflects the various lengths of DNA fragments generated by DNase I random degradation. In the case of exonuclease III, the enzyme generates two kinds of single-stranded DNA with the same sizes as the final products. The diffusion time shifted to the left at 31 min after nuclease addition without widening of the peak (Fig. 6E). Interestingly, two distribution peaks were detected with the BAL31 nuclease (Fig. 6F). A faster peak of diffusion times represents small fragments of DNA, with a level of diffusion time comparable to that of the RG fluorophore and a slower peak represents intact DNA. This result is reasonable since the BAL31 nuclease degrades the DNA from both the 5’ and 3’ends where the fluorophore is attached. Thus, we could identify the different enzymatic behaviors by using FCCS and distribution analysis of FCS without any separation method such as gel electrophoresis or gel filtration.
To classify nuclease activity in the cytoplasm of living cells, fluorescent-labeled DNAs were directly introduced into the cytoplasm of living cells by the bead-loading method (Manders et al., 1999, Nagaya et al., 2002). RG-23bp-Cy5 localized largely in the nucleus (Figs. 7A, B, C) though, RG-100bp-Cy5 did not distribute into the nucleus (Figs. 7E, F, G, 8A and C).
LSM images of DNA loaded HeLa cells. LSM images of cells 30 min after loading of RG-23bp-Cy5 (A–D) or RG-100bp-Cy5 (E–H). Green (A, E), red (B, F), merged fluorescence images (C, G) and transmitted images (D, H), respectively. Scale bar represents 10 μm.
Auto- and cross-correlation curves of RG-100bp-Cy5 were obtained at the crosshair point in the image of laser scanning microscopy (LSM) of a living cell in which DNAs were incorporated into the cytoplasm (Fig. 8A, C). High cross-correlation amplitude was detected within 10 min after loading (Fig. 8B). However, cross-correlation amplitude was reduced after 45-min incubation at 37ºC in the same cell (Fig. 8D). This reduction indicated that exogenous DNA was degraded in cytoplasm. DNA diffusion properties were also analyzed by distributions of diffusion times from autocorrelation curves using the CONTIN algorithm. At 45 min after loading of DNA, a faster peak (around 0.2 ms) appeared in distributions of diffusion times (Fig. 8D inset).
To determine the origin of each peak in the distributions of diffusion times, autocorrelation curves Gg(τ)s of different DNA sizes that were obtained for the cytoplasm of HeLa cells, and were averaged (N = 4–8) and analyzed using the CONTIN algorithm (Fig. 9). Two peaks of diffusion times were detected in each measurement; faster peaks were of the same order of diffusion time as the free RG fluorophore.
Nuclease degradation of double-fluorescent-labeled DNAs in cytoplasm of a living COS7 cell. LSM images of (A) 10 min and (C) 45 min after loading of DNAs in the same cell. Auto- and cross-correlation curves of RG-100bp-Cy5 DNA at the crosshairs in the LSM image of (B) panel A (D) panel C. Scale bar represents 10 μm. RG, Cy5 and Cross represent Gg(τ), Gr(τ) and Gc(τ), respectively. Distributions of diffusion times P(τDi) of DNAs calculated by CONTIN algorithm (B, D insets).
On the other hand, the diffusion times of the slower peaks shifted to the right according to the increase in size of incorporated DNA (Fig. 9B). The diffusion times of DNAs in cytoplasm were 5–10 times slower than those in solution. This ratio was in good agreement with the previously reported value (Dauty et al., 2005) and this result agreed well with the 5’ to 3’ exonuclease degradation models in solution (Fig. 6F). Finally, the appearance of 5’ to 3’ exonuclease activity was expected as barrier activity in cytoplasm by comparison of incorporated exogenous DNAs using the distribution pattern of diffusion times. Distribution analysis of diffusion times using CONTIN algorithm clearly showed different peak patterns for different enzymatic activities. The distributions of diffusion times had two peaks in cytoplasm. The diffusion time of the faster peak was almost the same as that of fluorescent dye alone, whereas the other, slower peak depended on the size of the DNA that was incorporated. Moreover, moderately sized DNA fragments were not detected in the cytoplasm. Taken together, these findings helped us to determine that (i) how, (ii) when, and (iii) where incorporated DNAs were degraded in the living cell. (i) The main mechanism of exogenous DNA degradation in cytoplasm was 5’ to 3’ exonuclease activity rather than endonucleases. (ii) The time scale of degradation (<45 min) was determined by real-time measurement of optical method without invasive treatment. (iii) The location of DNA degradation was not in small compartments but in cytoplasm because the monitored DNAs were distributed homogenously and had free diffusion values.
Relationship between DNA size and diffusion time in living HeLa cells. (A) The average of Gg(τ) curves in HeLa cytoplasm at 30 min after RG-23bp-Cy5, RG-100bp-Cy5 and RG-200bp-Cy5 loading. (B) Distributions of diffusion times P(τDi) of DNAs calculated from the average of Gg(τ) curves using CONTIN algorithm. RG dye was also tested as a reference.
To confirm the enzymatic activity of the 5’ to 3’ exonuclease in cytoplasm, we carried out an inhibition experiment for the enzyme using end-capped or redundant DNAs and monitoring the expression rate of EGFP. If exonuclease degradation works as a barrier against DNA transfection, the use of longer DNA and/or end-capped-DNA that protects the coding regions from each terminal (Fig. 10) should increase the expression efficiency. We synthesized linear DNAs of various redundancy lengths and capped on both DNA ends by a hairpin-shape oligonucleotide (Zanta et al., 1999, van der Aa et al., 2005). The end-capped DNA remained intact after incubation with exonuclease III (data not shown). Fig. 11 shows
Schematic diagram of various sizes of DNA expressing EGFP and end-capped DNA.
Effect of DNA protection against exonucleases on expression efficiency with a cationic lipid. (A) HeLa, (B) COS7, (C) MEF and (D) 293 cells were transfected with 1 μg of uncapped or capped linear DNA per well. Complexes were formed with LipofectAmine 2000 reagent. Expression efficiency (E/Ep) is expressed as the mean ± S.D. of 4–5 different experiments. To normalize the values from each individual experiment, the expression rate of DNA was divided by the rate using the pEGFP-C1 plasmid. *, P<0.05; **, P<0.01; ***, P<0.001 (Student’s t test). Striped bars, uncapped linear DNA; brack bars, end-capped DNA.
the expression efficiencies of DNAs in HeLa, COS7, MEF and 293 cells, respectively. The expression efficiency was based on the EGFP expression per copy of transfected DNA (N M/W), the value was divided by the expression of pEGFP-C1 plasmid for normalization (Np Mp/Wp). The final expression efficiency was calculated by following equation,
where N, W, and M represent the number of EGFPs obtained from FCS measurement, the transfected DNA weight and molecular weight of transfected DNA, respectively. Np, Wp and Mp represent the values with the pEGFP-C1 plasmid as a reference.
Expression efficiencies of short DNAs such as that with a 1.6 kbp length were lower than those of other lengths in each cell line. When the expression efficiencies of non-capped PCR products and capped DNAs were compared, it was clear that DNA capping significantly increased the expression efficiency of 4 kbp DNA in HeLa, COS7 and MEF cells. However, the end capping did not enhance the expression rate for short DNA lengths. In MEF cells, uncapped DNAs of all lengths were little expressed and capped DNAs exhibited ~10-fold greater gene expression than the uncapped DNAs. The effect of DNA capping was not observed in 293 cells. Although there were differences in the cell lines, the results obtained for HeLa, COS7 and MEF cells confirmed the effect of exonuclease degradation on DNA transfection. The nuclease activity was inhibited by capped structures on the 3’ and 5’ ends of the DNA. Interestingly, the end-capping effect on the expression rate was different in the cell lines. In MEF cells, which are generally said to be hard to transfect (Ewert et al., 2006), the end-capping effect was significant (Fig. 11C). On the other hand, in 293 cells, which are generally used in transfection assays because of their high expression efficiency, no end-capping effect was observed (Fig. 11D). These results agreed with previous reports that the transfected plasmids were more stable in 293 cells than in COS, NIH-3T3, HeLa cells (Alwine, 1985). In summary, our results show that exonuclease activity is related to transfection efficiency, though the level of exonuclease activitity in cytoplasm might be different depending on the cell line.
Here we propose new concept of nuclease characterization by monitoring the degradation patterns of oligonucleotides based on the fragment size and direction of degradation in living cells. The concept is similar to conventional ones such as an analysis by using radioisotope-labeled oligonucleotides and a size determination by gel electrophoresis. The relationship between the exogenous DNA expression level and DNA stability by extension of DNA fragments was studied using the luciferase assay in vitro and in vivo (Hirata et al., 2007); however, this assay in a cell homogenate only provides information on bulk DNA stability, not on the individual DNA degradation mechanism. Our approach is, therefore, replacement of the conventional concept by diffusion measurement using a fluorescent tag and coincident analysis using FCS and FCCS so that sensitivity is enhanced to the single molecule level and a physical separation procedure is not needed. Therefore, our method can be employed for measurements in single living cells.
The association and dissociation between DNA and gene carriers are important
Quantum dots (QDs) are nanometer-sized semiconductor crystals that emit visible to infrared fluorescence depending on their composition and diameter. QDs have several advantages such as high resistance to photobleaching, high brightness and narrow emission wavelengths with broad excitation spectra. QDs are comprised of an inorganic core, inorganic shell and an organic coating to solubilize them to the water phase. Since the surface to volume ratio of a QD is relatively high, QDs can be used as scaffold particles for carrying ligands, therapeutic agents and gene delivery carriers.
Cationic polymers can condense with DNA by electrostatic interaction to form complexes that promote internalization of exogenous DNA into cells. Polyethyleneimine (PEI) is an efficient gene carrier, that has a proton-sponge effect (Boussif et al., 1995). PEI solubilizes QDs to water so it is also useful for surface coating of QDs (Duan et al., 2007, Zhu et al., 2005).
Diagram of PEI-QD. PEI-coated QD forms a complex with DNA.
CdSe/CdZnS QD cores that emit 605 nm fluorescence were synthesized (Jin et al., 2010) and PEI (average molecular weight about 10000) was added and heated to 60C in tetrahydrofuran. The resulting precipitates of PEI-coated QDs were dissolved in water (PEI-QDs). The PEI-QD solution was then mixed with a plasmid that coded red fluorescent protein, mKate2 (pmKate2-N). To visualize the localization of the plasmid, a sample plasmid was stained with YOYO-1 iodide. Then the plasmid/PEI-QD complex was added to cultured HeLa cells. After 24-hour incubation, fluorescence emission of expressed mKate2 was detected (Fig. 13). The result showed that PEI-QD had the potential to introduce plasmid vectors into cells. Fluorescence of QDs and YOYO-1 colocalized at large complexes, though free molecules or small complexes were not visible by the conventional microscopy, so methods that can detect their diffusion at the single molecule level, such as FCS and FCCS, are needed.
LSM images of DNA transfected HeLa cells. LSM images of cells 24 hours after addition of PEI-QD/pmKate2-N. QDs (A), YOYO-1 (B), mKate2 (C) and merged fluorescence images (D) respectively. Scale bar represents 10 μm.
The efficiency of gene delivery by DNA/carrier complexes depends on the size of the complex and the dissociation kinetics in cells.
Normalized autocorrelation curves of plasmid DNA (blue line), PEI-QDs (red line) and a mixture of the plasmid and PEI-QD (black line) in solution.
To develop an efficient gene carrier, it is necessary to characterize the properties of the DNA/carrier complexes. Previously the characterization of complexes was carried out by gel electrophoresis (Eastman et al., 1997).
Currently, there are several FCS studies of DNA/carrier association and dissociation (Van Rompaey et al., 2001, Braeckmans et al., 2010). By using FCS, the size and the ratio of the DNA to the gene carrier are evaluated in solution from the values of the diffusion time and CPM, which are obtained by FCS measurements (see section 2). A 4 kbp circular-shaped plasmid was mixed the PEI-QDs and FCS measurements were carried out. The diffusion time of the plasmid decreased due to the compaction of DNA by the interaction with the cationic gene carrier (Fig. 14).
On the other hand, FCCS can monitor the interaction of DNA with a gene carrier directly. FCS monitors the change of the diffusion time (or molecular size) though the interaction cannot be distinguished if the diffusion time is decreased or not changed due to the change of the molecular shape. For demonstration, PEI-QDs and rhodamine green-labeled 500 bp linear DNA (RG-500bp) were mixed at various ratios, and then FCCS measurements were carried out with single-laser excitation at 488 nm (Fujii et al., 2007). Fluorescence emissions were collected from two channels for rhodamine green and QD. The increase of autocorrelation amplitude of the green channel (RG-500bp) made observation of the DNA complex possible (Fig. 15). The cross-correlation amplitude was also increased (Fig. 15). This high cross-correlation amplitude directly shows the incorporation of RG-500bp and the PEI-QDs.
Cross-correlation analysis of RG-500bp and PEI-QDs. The correlation curves of various RG-500bp/PEI-QD ratios (A; 0.5, B; 1, C; 2, D; 4, E; 8). Blue, red and black lines represent Gg(τ), Gr(τ) and Gc(τ), respectively.
The RCAr value was increased according to the increase of the PEI-QD concentration (Fig. 16A). At a low DNA/QD ratio, the number of PEI-QDs in solution was larger than the number of RG-500bp so there were many QDs that did not interact with DNA. On the other hand, RCAg was decreased at once by the decrease of PEI-QD because of the relative decrease of interacting partners (Fig. 16C). The numbers of RG-500bp and PEI-QDs contained in the complexes could be estimated from the value of CPM. The CPMs of the green channel were 4 to 8 times larger than the value of a single RG-500bp and the CPMs of the red channel were comparable to the value of a single PEI-QD (Fig. 16B). These results indicated that 4 to 8 RG-500bp molecules and a single PEI-QD particle were contained in the complexes under the tested conditions. At high ratios of DNA/QDs, DNA and QDs may aggregate so that RCAr and CPMg are increased. The FCCS technique has strong advantages to investigate the properties of DNA carriers such as the strength of binding between DNA and the carrier, the size of the complex and the amount of DNA and number of carriers contained in the complex.
RCA values (A) and normalized CPM (B) of RG-500bp/PEI-QD complexes. CPM values were normalized by the CPM of a single RG-500bp or PEI-QD. Note that RCAg shows the ratio of interacting molecules among the red molecules (Nc/Nr). RCAr shows the ratio of interacting molecules among the green molecules (Nc/Ng) (see
It is necessary to ascertain the fate of exogenous DNA for the development of nonviral gene therapy. FCS and FCCS, which provide spatiotemporal information on diffusion properties and interactions of biomolecules, were employed for analyses in living cells. FCCS experiments revealed rapid DNA degradation in cytoplasm. In addition, the DNA degradation mechanism in cytoplasm due to 5’ to 3’ exonuclease was estimated by measuring DNA diffusion properties at the single molecule level. The methods using noninvasive monitoring of the diffusion properties of DNA provide information on the fate of intracellular exogenous DNA and the efficiency of DNA integration in living cells. Understanding of these intracellular events should help with the development of novel efficient gene delivery methods.
FCS and FCCS are rapidly becoming widely used to detect movements and interactions of biomolecules in living cells (Chenette, 2009, Lidke et al., 2009), and various related applications such as two-focus FCS (Yu et al., 2009), raster image correlation spectroscopy (RICS) (Digman et al., 2009) and multi-point total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) (Ohsugi et al., 2006, 2009) have been developed. FCS, FCCS and these techniques will shed new light on cell biology and gene therapy.
The authors thank Professor Takashi Jin (Osaka University) for providing various types of quantum dot cores. Our research was partly supported by Grants-in-Aid for Scientific Research (A) 18207010 and (S)21221006, by Grants-in-Aid for Exploratory Research (20657035) from the JSPS, and No. 19058001 in Priority Area “Protein Community.”
For sustainable food production, it is an absolute requirement that nutrients removed with the harvest of crops are replaced to prevent nutrient depletion and soil degradation. Achievement and maintenance of high nutrient use efficiency (NUE) together with high crop productivity have become a major challenge in both developed and developing countries with an increasing growing population, depletion of natural resources, and deteriorating environmental conditions. This is occurring at the same time as society becomes ever more concerned about resource management practices and the environment, especially when it comes to nutrient management [1]. Fertilizer nutrients applied that are not taken up by the crop are also vulnerable to losses from leaching, erosion, and denitrification or volatilization in the case of N, or they could be temporarily immobilized in soil organic matter to be released at a later time, all of which impact apparent use efficiency [2].
Improving nutrient use efficiency (NUE) in plants is vital to enhance the yield and quality of crops, reduce nutrient input cost and improve soil, water, and air quality [3]. Higher NUE by plants could reduce fertilizer input costs, decrease the rate of nutrient losses, and enhance crop yields. Improving crop nutrient use efficiency ideally requires an understanding of the whole system, from the macro (agro-ecosystem) to the molecular level [4]. Nutrient uptake and their internal utilization efficiencies are the two central cores for improving crop NUE [5]. This can be achieved through optimizing agronomic strategies (soil-rhizosphere management) and breeding nutrient-efficient cultivars. Plant genetics and physiological mechanisms and their interaction with best agronomic practice are also a tool that can be used to increase efficiency of cropping systems [3]. Thus, it needs involvement of integrated nutrient management strategies that take into consideration improved fertilizer along with soil and crop management practices are necessary [6]. Sustainable nutrient management must be both efficient and effective to deliver anticipated economic, social, and environmental benefits.
Plants experience nutrient deficiency when soil nutrient availability is either an inherently low amount or low mobility of nutrients in the soil, or poor solubility of certain chemical forms of soil nutrients [7]. Of the various nutrients essential for plants, nitrogen (N), phosphorus (P), and potassium (K) are required in the largest quantities, and their deficiency severely limits crop yield [8]. The dynamic nature of N and P in soil-plant systems creates a unique and challenging environment with nitrate and phosphate contamination of surface and/or groundwater, which can be attributed in large part to low efficiency in plant nutrient uptake. The main challenge for improving P and K use efficiency at the farm level is to apply the existing knowledge in a practical manner [9]. Hence, the best management practice for N, P, and K must consider the specific characteristics of crops, cropping systems, environments, and soils is application of 4R nutrient stewardship. Therefore, this chapter tries to summarize the concept of NUE and recent strategies for enhancing use efficiency of N, P, and K. These approaches consider economic, social, and environmental dimensions essential to sustainable agricultural systems and afford a suitable context for specific NUE indicators.
The variations in defining nutrient efficient plants and methods used in calculating nutrient use efficiency make it difficult to compare results of different studies [10, 11, 12, 13]. Understanding the terminology and the context in which it is used is critical to prevent misinterpretation and misunderstanding and determination of NUE in crop plants is an important approach to evaluate the fate of applied chemical fertilizers and their role in improving crop yields. In order to develop a common framework for NUE, scientists started to formulate concepts and definitions that should serve as a basis for comparison and discussion of research. Nutrient use efficiency in its broadest sense indicates how effectively a plant is able to capture and utilize nutrients to produce biomass. It is simply a measure of how well plants use the available mineral nutrients [10]. The earlier definition of NUE by [14] is simply increment of yield per applied nutrient (Eq. (1)).
While the most recent and complicated one used in crop modeling formula is (Eq. (2)) [12].
where
Generally, nutrient use efficiency comprises both yield as a function of inputs and percentage of nutrient recovered respectively, contributing to yield and quality [15]. The NUE is based on (a) uptake efficiency (acquire from soil, influx rate into roots, influx kinetics, radial transport in roots are based on root parameters per weight or length, and uptake is also related to the amounts of the particular nutrient applied or present in soil), (b) incorporation efficiency (transports to shoot and leaves are based on shoot parameters), and (c) utilization efficiency (based on remobilization, whole plant, i.e., root and shoot parameters) [4].
Phosphorus use efficiency can be divided into (i) P acquisition efficiency [the capacity of a cultivar to extract P from soil] and (ii) P internal utilization efficiency [the capacity of a cultivar to transform the acquired P into biomass/grain yield] [16, 17, 18].
Phosphorus uptake or acquisition efficiency (PACE)
Uptake efficiency or the ability of the plant to extract the nutrient from the soil is calculated as [19] (Eq. (3)).
Phosphorus utilization efficiency (PUTE)
Phosphorus utilization efficiency is defined as a crop’s ability to convert the absorbed P into grain yield [19] (Eq. (4)) can be calculated as:
Utilization efficiency can also be calculates as suggested by [20], (Eqs. (5) and (6)) and expressed as follows:
Generally, if P supply is limited or in more acidic and calcareous soil, P acquisition could be more important than P utilization and high fertilizer application necessary in order to provide sufficient plant-available P. On the other hand, with adequate P supply, PUTE could be considered more important than PACE for crop P efficiency [17]. Therefore, the improvement of both PACE and PUTE in the given species under different P supply conditions in the different soil types seems to be the perfect breeding approach (Figure 1) [17].
Schematic representation of the possible mechanisms of P acquisition and utilization for better growth of modern crops grown in intensive cropping systems
Hence, Nutrient use efficiency = Uptake efficiency × Utilization efficiency. All unit dry weights are in g m−2 [19].
For nitrogen use efficiency in their various definitions and components (Figure 2) [21].
Illustration of nutrient use efficiency parameters exemplified by NUE in wheat. Key process contributing to the NUE trait: nitrogen uptake efficiency, NUpE; nitrogen utilization efficiency, NUtE; nitrogen harvest index, NHI (adopted form [
Apparent recovery efficiency is one of the more complex forms of nutrient use efficiency (NUE) expressions and is most commonly defined as the difference in nutrient uptake in above-ground parts of the plant between the fertilized and unfertilized crop relative to the quantity of nutrient applied. It is often the preferred NUE expression by scientists studying the nutrient response of the crop [22]. Reference [23] proposed that the balance method be used to assess fertilizer P efficiency (Eq. (7)). The balance method is described mathematically as:
Phosphorus use efficiency has become burning issues in recent times due to several reasons [24]. Unlike N, the amount of P is less-abundant, finite resource, less-available, and poor mobility in the soil, being one of the most inaccessible elements for plants. Its deficiency is a major constraint to agricultural production, and it affects an area of over 2 billion hectares worldwide that is on about 70% of the world’s arable land [25]. Remarkably, usually only about 10–30% of the P fertilizer applied in the first year is taken up by the roots, with a substantial part accumulated in the soil as residual P not readily available for plants [26]. This may be due to nature of P that can bound to calcium in alkaline soils and readily complexed to charged Al and Fe oxides and groups hydroxyls on clay surfaces in acidic soils [23]. In addition, agricultural phosphorus (P) run-off is a primary factor in the eutrophication of aquatic and marine ecosystems and has also led to blooms of toxic cyanobacteria [27] and can contain heavy metals such as cadmium that may accumulate in arable soils. Moreover, organic material present in the soil (e.g., from manure or crop residues) can also bind phosphate ions as well as phytate (inositol compounds). In order to avoid a future food-related crisis, phosphorus scarcity needs to be recognized and addressed in contemporary discussions on global environmental change and food security, alongside water, energy, and nitrogen [28].
Selection and breeding nutrient-efficient species or genotypes within a species are justified in terms of reduction in fertilizer input cost of crop production and also reduced risk of contamination of soil and water. Many plants have evolved morphological, physiological, biochemical, and molecular adaptive systems to cope with P-deficiency stress, such as altered root architecture to explore more soil volume and increased carboxylate exudation containing phosphatases, nucleases, and various organic acids [29]. These mechanisms and strategies are necessary to liberate or solubilize Pi from organic and other insoluble pools [30], enhance Pi uptake capacity [31], recycle internal Pi remobilize/retranslocate P from mature to young developing organs [32, 33], and reprioritize metabolic P utilization [34]. Under the current situation, farmers need P-efficient genotypes that perform better than other genotypes with equivalent P inputs. Therefore, selection/identification of cultivars that can absorb and use P efficiently is a promising strategy to cope with environments deficient in bio-available P. Due to the diverse functional and structural roles of P in plants, P-use efficiency (PUE) is a complex trait to dissect [24].
The root morphological factors such as length, thickness, surface area, and volume have profound effects on the plant’s ability to acquire and absorb nutrients in soil [35]. These parameters are influencing the ability of the roots to penetrate high density soil layers, to extremes tolerate temperature, moisture, toxicities, and deficiencies of elements. Additionally, they have the ability to modify the rhizosphere pH and the nutrient uptake kinetics. Efficient acquisition will depend first on root architecture in terms of transporters and exudates and often the presence of symbiotic associations such as mycorrhiza. Hence, improving early root establishment, high-affinity transporter systems, association of microorganisms (mycorrizha), proliferation of roots, and enhanced mechanisms for increasing bio-availability of nutrients and then enhancing NUE [5]. Improvement of transporters plays essential roles particularly in conjunction with effective root proliferation in contributing to nutrient use efficiency. The other important attribute for uptake efficiency is having adequate sinks to store acquired nutrients, which will prevent negative feedback regulation on the initial acquisition/assimilatory processes and should provide important remobilizable storage [5]. The second component of uptake efficiency is root physiological activity such as differing uptake kinetics, i.e., maximum net influx (Imax), affinity of the transporter
A recent study further showed that root tips also play an important role and, despite their small size, accounted for approximately 20% of the total seedling Pi uptake [37], mainly increasing organic acid exudation strategies [38]. Plants increase total soil exploration by increasing root length, increasing root branching, increasing specific root length (i.e., roots with smaller diameter), and modifying branching angle [39, 40, 41]. The findings of Bates and Lynch [39] suggested that increased root growth is associated with improved plant performance under low P by exploring a larger volume of soil. Consequently, root: shoot ratio increases significantly in low-P environments and is an excellent index for partitioning photosynthesized carbon between above- and below-ground plant parts. Root density and root: shoot ratio generally increased under P deficiency, thus favoring P acquisition by plants [29].
Genetic variation for root hair traits, particularly root hair length, can be exploited in breeding for improved P uptake efficiency and P fertilizer use efficiency in crops. Moreover, a deeper root with more aerenchyma tissues in the cortex of the roots can also be an important trait that contributes to efficient N uptake with lower carbon input in root growth [42]. This root architecture may also be efficient in the uptake of deep water and therefore help to increase drought resistance [43]. However, Miguel et al. [44] showed in field trials that shallow and hairy root traits are synergistic in their effects on Pi uptake by bean. However, modifying root growth in response to nutrient deficiency, it is a challenge and complex to identify key regulators that are sufficiently upstream and robust to be suitable for developing plants with optimized root systems for nutrient uptake [8].
Levels of fertilizer applications influence the total dry matter accumulation, thereby affecting the nutrient demand (uptake/utilization) [9]. Improved nutrient utilization efficiency from agrochemicals through PGPR and (or) AMF can contribute to the protection of water resources against agro-pollution and reduce the growing cost of fertilizers [10]. After inorganic phosphate (Pi) acquisition from rhizosphere, Pi should be efficiently transported to shoot for the requirement of plant growth by phosphate transporters (
Another promising area for improvement of crop NUE is to enhance the efficiency of nutrient remobilization from senescing organs to young, developing organs, particularly immature leaves, and developing seeds [47]. The senescence process, that is, the dying-off of vegetative plant parts during seed maturation, is at the core of the nutrient use efficiency issue, as the nutrients need to be remobilized from these parts and translocated into the developing seed [48]. Maximizing the effectiveness of P-remobilization from senescing organs could make an important contribution to the development of crops that can tolerate Pi deficiency, because senescing organs of most “modern” crop varieties exhibit low P-remobilization efficiencies of <50% [30]. An integral understanding of P remobilization would facilitate development of effective biotechnological strategies to improve crop PUE, thereby reducing the rate of depletion of nonrenewable rock P reserves [30, 47]. Therefore, mobilization and redistribution of P from the old tissues to the young tissues will also contribute to high P use efficiency. Better distribution of nutrients in parts of plant (root, shoot, and grain) reflects their use efficiency [11].
In the plant, uptake and utilization efficiency of nutrients are governed by different physiological mechanisms and their response to deficiency, tolerance, and toxicity of element(s) and climatic variables [49]. Efficient internal utilization of nutrient is generally attributed because of high photosynthetic activity per unit of nutrient (P) and more efficient P remobilization from older to young leaves [47]. Acid phosphatase contributes to the increased P utilization efficiency in bean through P remobilization from old leaves [50]. Therefore, improving higher total chlorophyll concentration [51], enhancing phosphorylase stimulation [52], and improving partitioning of carbon between glycolytic and pentose phosphate pathways [53] also provide an effective approach to improve phosphorus use efficiency and crop productivity simultaneously.
P-utilization efficient cultivars produce high yield per unit of absorbed P under P deficient conditions, since they have low internal P demand for normal metabolic activities and growth. Hence, they have low requirement for mineral P fertilizer inputs to produce reasonably high yield. Moreover, they remove less P from soil during growth and therefore the quantity of P removed along with the harvestable parts of the crop would obviously be less, consequently reducing the quantity of mineral P fertilizer inputs required for maintenance fertilization [54].
Agronomic practices can change soil physicochemical properties and biological characteristics. As a result, a number of agronomic practices have been proposed to enhance nutrient availability under diverse climatic conditions [55, 56]. The rhizosphere (root-soil interface) is the most important area for plant–soil-microorganism interactions and is the hub for controlling nutrient transformation and plant uptake [7]. This modification is paramount to increase nutrient availability and to minimize losses in surface runoff. Possible management strategies options for improving NUE through optimizing agronomic practice or rhizosphere modification [57] are the following:
The 4R Nutrient Stewardship framework promotes the application of nutrients using the right source (or product) at the right rate, right time, and right place. The framework was established to help convey how fertilizer application can be managed to ensure alignment with economic, social, and environmental goals [58]. Nutrient Stewardship defines the right source, rate, time, and place for fertilizer application as those producing the economic, social, and environmental outcomes desired by all stakeholders of the plant ecosystem (Figure 3). This 4R techniques applies (1) right rate—supplying growing crops with the right amount of nutrients for healthy growth and development based on experimentation under various environmental conditions; (2) right time—matching nutrient availability to with the timing of plant peak nutrient uptake and demand; (3) right placement adding nutrients to the soil at a place where crops can easily access them related to volume of roots.; (4) right source—applying the correct fertilizer and organic resources that provide growing crops with all nutrients required for good growth and maturity [58]. The 4R concept was established to help convey how fertilizer application can be managed to ensure alignment with economic, environmental, and social goals [22, 59].
The 4R nutrient stewardship concept (adopted from [
Soil testing remains one of the most powerful tools available for determining the nutrient supplying capacity of the soil, but to be useful for making appropriate fertilizer recommendations good calibration data is also necessary [2]. As P is less mobile, less soluble, and highly prone to soil fixation; effectiveness of applied P depends on the properties of soil being fertilized, fertilizer itself, and time and method of its application [60]. To enhance phosphorus use efficiency (PUE) of applied P fertilizer, time and method of its application are critically important, because different P application methods differ in PUE [61]. In highly sandy soils, P may need to be managed like N, by splitting applications and applying small amounts at sowing and topdressing later in the crop growth cycle [62]. Studies of Jing et al. [63] suggested that localized supply of superphosphate combined with ammonium-N (NH4+-N) significantly stimulated root proliferation, especially of fine roots, and thus improved maize growth in a calcareous soil. Further studies indicated that localized supply of P and NH4+-N at both seeding and later growth stages increased maize yield by 8–10%, P uptake by 39–48%, and localized increases in root density and length of 50% [64]. Rehim et al. [65] also reported that the fixation of broadcasted P is much greater than the fertilizer applied in bands because of less contact with P fixing ingredients. At higher P application, the adsorption of P increased because the plants readily utilize only 8–33% of applied P in the first growing season and remaining portion remained fixed that consequently resulted in higher Olsen P. So, at higher P application rates, plants used smaller proportion of fertilizer P that resulted in low PUE [61].
In principle, N deficiency increases root growth, resulting in longer axial roots (primary roots, seminal roots, and nodal roots), and this helps maize roots to explore a larger soil volume and thus increases the spatial N availability [66]; however, long-term N deficiency stunts root growth due to insufficient N. But also, root elongation can be inhibited if the N supply is too high. Excessive application of N-P fertilizers may lead to high concentrations of soluble nutrients in the root zone, which can also restrict root growth and rhizosphere efficiency [67], even small amounts of P lost can be a cause of the adverse effects of eutrophication of surface waters. Therefore, judicious application of fertilizer best management practices (BMP) [22] that includes the right rate [68], right time [69], right source, right place, and balanced fertilization (4RB) is the best management practice for achieving optimum nutrient efficiency [2, 22].
Cereal-legume intercropping is a crop production system utilized to improve productivity and sustainability under diverse environmental conditions. It can also improve nutrient use efficiency and crop productivity [7]. Intermingling of maize and faba-bean roots increased N acquisition by both crop species by about 20% compared with complete or partial separation of the root systems. Further studies indicate that N2 fixation can be improved by yield maximization in the intercropping system. The improved productivity observed in this production system has been associated with increased levels of available phosphorus (P) in the root rhizosphere. Hinsinger et al. [70] reported more stable yield, superior land resource utilization or conservation, and enhanced pest or weed control [71, 72, 73]. Furthermore, cereal-legume intercropping can also enhance the phosphatase enzyme activity and available P in the soil due to rhizosphere acidification by the legumes in the cropping system [74].
The possible mechanism that increases PUE in intercropping is the increased rhizosphere soil acid phosphatase (RS-APase) activity observed in intercropping due to the fact that large amounts of acid phosphatase are known to be released from their roots into the root rhizosphere. The (RS-APase) activity was significantly higher (26–46%) in the intercropping and occurred concomitant with a significant increase in available phosphorus (RS-Pavailable) in the rhizosphere on podzols in cool climate boreal ecosystem [75]. Another mechanism could be secreting H+ into the soil that acidifiies the rhizosphere [57, 76] and improves dissolution of phosphorus and then enhances P-availability [70]. Additional possible mechanism that improves of plant growth and P uptake in mixed planting was due to root interspecific complementation or facilitation. The complementarity between root morphological and physiological traits of neighboring plants underpins the interactive facilitation, which was the main underlying mechanism improving nutrient-use efficiency, particularly of P, in mixed cropping system [77, 78]. The complementary niches of maize and faba bean significantly reduce interspecific nutrient competition and thus improve nutrient-use efficiency [79]. The presence of maize increased the secretion of carboxylates from alfalfa roots, suggesting that the root interactions between maize and alfalfa are crucial for improving P-use efficiency and productivity in intercropping [80]. Subsequently, Sun et al. [76] reported that decreasing rhizosphere pH and increasing organic anion exudation played key roles in soil P mobilization of maize and alfalfa, with little contribution of acid phosphatase.
The mycorrhizal symbiosis particularly, arbuscular mycorrhizal fungi (AMF), is arguably the most important symbiosis on earth [81]. AMF colonize the roots of many agriculturally important food and bioenergy crops form (approximately 80–90% of all known land plant species) [81] and could serve as “biofertilizers and bioprotectors” in environmentally sustainable agriculture [82]. In AMF associations, two pathways for plant P uptake exist: the direct pathway (P uptake by roots) and the AM fungal pathway [83]. AMF facilitates the uptake and transfer of mineral nutrients, such as phosphorus, nitrogen, sulfur, potassium, calcium, copper, and zinc, from the soil to their host plants by means of the extraradical mycelium extending from colonized roots into the soil [84]. The contribution of AMF to P uptake reaches up to 77% under low P supply compared with only 49% under high P supply [85]. Furthermore, the commercial inoculum Mycobiol, consisting of Glomus spp.,
Various mechanisms have been suggested for the increase in the plant uptake of P. These include: exploration of larger soil volume; faster movement of P into mycorrhizal hyphae; and solubilization of soil phosphorus [88]. Exploration of larger soil volume by mycorrhizal plants is achieved by decreasing the distance that P ions must diffuse to plant roots and by increasing the surface area for absorption. Faster movement of P into mycorrhizal hyphae is achieved by increasing the affinity for P ions and by decreasing the threshold concentration required for absorption of P [88]. Solubilization of soil P is achieved by rhizospheric modifications through the release of organic acids, phosphatase enzymes, and some specialized metabolites such as siderophores [55].
The composition and amount of root exudates affect the composition of microbes in the rhizosphere and the structure of the rhizosphere microbiome, affecting plant growth and nutrient uptake [81]. For precision rhizosphere management, plant-microbe interactions must be finely tuned to improve P use efficiency by crops [57]. Figure 4 illustrates the main structural differences between AM (more for P absorption) and ectomycorrhizal (more for N and few for P absorption) associations of angiosperms or gymnosperms [81].
Phosphorus acquisition efficiency related traits of wheat and barley roots affected by arbuscular mycorrhizal symbiosis in comparison to a non-colonized counterpart (adopted from [
Among the soil bacterial communities, ectorhizospheric strains from Pseudomonas and Bacilli and endosymbiotic rhizobia have been described as effective phosphate solubilizers [90]. Phosphate-solubilizing bacteria (PSB) are also capable of making P available to plants from both inorganic sources and organic ones and increasing P-fertilizer-use efficiency by different mechanisms [91]. They are rhizobacteria that convert insoluble phosphates into soluble forms through acidification, chelation, exchange reactions, and the production of organic acids [92]. Therefore, combined application of AMF and P solubilizers [93] and N fixers are the best inoculants. AM fungi together with PSMs could be much more effective in supplementing soil P. Understanding AM-plant symbiosis, developing AM fungi that could be cultured in vitro, and developing P-solubilizing AM will help realize their potential as phosphate biofertilizer [94].
Soil pH is one of the most important chemical properties influencing nutrient solubility and hence availability to plants. Large amount of P applied as fertilizer enters in to the immobile pools through precipitation reaction (fixation) with highly reactive Al3+ and Fe3+ in acidic and Ca2+ in calcareous or normal soils [94]. Acidic, highly weathered, iron (Fe)-rich soils rapidly bind phosphates at mineral surfaces, limiting access to plant roots. Furthermore, applied Pi (inorganic P) is quickly fixed into insoluble inorganic or organic forms due to its high reactivity and microbial action [95].
Soil pH markedly limits plant growth and P chemistry in soils through its effect on P adsorption and through interactions that affect precipitation of P into solid forms in soil [62]. Consequently, about 80–90% soil P becomes unavailable depending on soil composition and pH [96], 50–70% of the total applied conventional fertilizers are lost to the environment. This level of loss in agricultural nutrients not only leads to the loss of valuable resources but also causes the severe reduction of yield [97]. The pH of a calcareous soil is reduced by the presence of gypsum (CaSO4·2H2O) due to the concentration of Ca2+, which would be expected to decrease the sorption of P, if followed by leaching to removed much of the soluble Na+ and Ca2+ [98]. Thus, adjusting soil pH and base saturation are methods to reduce the amount of P that is bound by Al, Fe, and Ca, further reducing the effects of Al toxicity to plants, which can inhibit uptake, and use of P by the plant (Figure 5) [23, 99].
Soil P availability as affected by soil pH (adopted from Havlin et al. 1999).
Lime acidic soil is widely used in agriculture to create and maintain a soil pH optimal for plant growth in acid soils. Lime reduces toxic effects of hydrogen, aluminum, and manganese, improves soil biological activities, cation exchange capacity (CEC), P, Ca, and Mg availability and soil structure, promotes N2 fixation, stimulates nitrification, and decreases availability of K, Mn, Zn, Fe, boron (B), and Cu [11]. An increase in soil pH, as a result of liming, was due to an increase in hydroxide ions, which increases microbial activity and communities, hence, increasing decomposition of soil organic matter and release of Fe and Al [100]. The decrease in Al-P and Fe-P could be due to their precipitation as insoluble Al(OH)3 and Fe(OH)3 after increased addition of liming material [101]. In addition, Al and Fe oxides become more negatively charged with an increase in pH contributing to an increase in available P [102].
Liming, gypsum application, or mixing of both is an effective practice to improve pH, improve Ca content, and control Al toxicity. Lime has very low mobility in soil, and when surface applied, it does not reduce the acidity of subsurface soil horizons. Contrary to lime, gypsum (CaSO4) has a greater downward movement, and when applied to the surface, it can still impact and reduce the acidity of the subsoil [4]. The pH of a calcareous soil is reduced by the presence of gypsum (CaSO4·2H2O) due to the concentration of Ca2+, which would be expected to decrease the sorption of P, if followed by leaching to removed much of the soluble Na+ and Ca2+. The uptake of nutrients by plants, content of nutrients in plants and in soil were substantially positively influenced by both the wood ash, especially by FGD gypsum [103]. Gypsum application can ameliorate saline-sodic soil, thereby increasing crop yield and NUE [104].
Apart from traditional methods, new techniques have been developed such as site-specific/real-time nitrogen management, slow release/controlled release fertilizer (SR/CRF), site-specific precision nutrient management, and urease/nitrification inhibitor. Those techniques play an important role in decreasing fertilizer loss and increasing NUE [105]. The remote sensing is quicker than the previous two methods, and it obtains continuous data rather than spot data, which is more advantageous. It is becoming the major means of obtaining data for precision farming. GIS (geographic information system) establishes the field management information system by processing, analyzing, and trimming the data of soil and crops [105]. Another approach to synchronize release of N from fertilizers with crop need is the use of N stabilizers and controlled release fertilizers. Nitrogen stabilizers (e.g., nitrapyrin, DCD [dicyandiamide], NBPT [n-butyl-thiophosphoric triamide]) inhibit nitrification or urease activity, thereby slowing the conversion of the fertilizer to nitrate. The most promising for widespread agricultural use are polymer-coated products, which can be designed to release nutrients in a controlled manner.
Agronomic management strategies such as precision P fertilization, polymer coated P-fertilizers, and recycling of P from domestic, agricultural, and industrial wastes can be helpful in improving P use at farm level [106]. Modern concepts for tactical N management should involve a combination of anticipatory (before planting) and responsive (during the growing season) decisions [9]. On soils with moderate P and K levels and little fixation, management must focus on balancing inputs and outputs at field and farm scales to maximize profit, avoid excessive accumulation, and minimize risk of P losses. Improving the internal, on-farm and field recycling is the most important K management issue worldwide. As for N, the primary determinants for REP and REK are the size of the crop sink, soil supply, soil characteristics, and fertilizer rate.
Control release fertilizers with polymer coatings are commonly applied to crops to increase efficiency of nutrients [96]. One way of improving the P availability to crop plant is by coating diammonium phosphate (DAP) with polymer that allows a steady but controlled discharge of phosphorus from the granules for crop plant uptake and improved P recovery percentage. Thus, by the use of polymer, availability of P to plant increased because it has high cation exchange capacity, which holds the divalent calcium (Ca+2) and trivalent cations iron and aluminum (Fe+3 and Al+3) and stop P fixation with these cations. Moreover, polymer absorbs water efficiently and holds more water and keeps P in available form that enhanced the plant growth and yield-contributing factors [97]. This is because polymer-coated diammonium phosphate (DAP) absorbs water many times of its original weight, which increases the availability of phosphorus for longer period of time [107] and creates a diffusion shell around the grain of DAP and directly reduces the fixation and precipitation by reducing the availability of calcium and magnesium (Ca+2/Mg+2) cations [108]. As the result of this mechanism, availability of phosphorus to plants increases and leads to more P uptake, and this uptake indirectly influences the other nutrient absorption by crop plants.
Considering the wide variety of soil types, cropping patterns, and farmers’ resources, several management practices are adopted to reduce the magnitude of soil fertility degradation. Integrated Plant Nutrient Management System (IPNMS) is defined as the package of practices for the manipulation of the plant growth environment to supply essential nutrients to a crop in an adequate amount and proportion for optimum production without degrading the natural resources [3]. Many authors have reported that combining organic and inorganic P can improve and sustain crop yields in low fertility soils [109, 110, 111]. Best management practices (BMPs) such as use of fertilizer and amendment (lime), proper crop rotations, increases in organic matter content, and control of erosion, insects, diseases, and weeds can significantly improve crop yields and optimize nutrient use efficiency [11]. Integrated use of organic manures and fertilizers not only improves efficiency of crops but also significantly increases the availability of P [112, 113].
Organic amendment improves the structure and fertility of the soil by adding nutrients and organic matter and consequently promotes soil microbial biomass and activity. Blockage of P sorption sites by organic acids, as well as complexation of exchangeable Al and Fe in the soil, is potential cause of this mobilization [114]. Organic materials can reduce P fixation by masking the fixation sites on the soil colloids and by forming organic complexes or chelates with Al, Fe, and Mn ions, thereby improving P uptake efficiency of crop plants. Decomposition of organic matter produces organic anions that interact with soil to reduce P sorption via (1) complexation/competition for soil P binding sites such as Fe and Al oxyhydroxides or (2) increased soil PH. Organic materials also increase agronomic efficiency by improving availability of P by promoting soil aggregation, increased soil PH, microbial biomass, and parameters controlling soil-P-sorption [115]. The integration of biochar FYM, poultry manure, and inorganic P sources increases in PUE under both wheat and maize crops, and there is a concomitant increase in crop yields compared with the unamended soil [112, 113]. This increase in PUE with biochar addition could also be the result of the additional nutrients made available by biochar [112]. Similarly, FYM applications increase soil P bioavailability more than applications of triple supper phosphate that enhance P Uptake Efficiency. FYM is also a source of other nutrients used by crops via mineralization, which promotes root development and root area interception and thus increases nutrient uptake including P uptake [116].
Rotating a legume with a cereal can enhance P acquisition by cereals through indirect feedback interactions [117]. A legume crop modifies the rhizosphere through biological and chemical processes, thereby increasing P uptake by the following cereal crop. As reported by [77], legumes are able to mobilize P that is not initially available to cereal species, thereby improving the availability of P for the following crop. The biological processes include the promotion of symbiotic mutualists such as nitrogen-fixing rhizobacteria and mycorrhizal fungi, while the chemical processes are acidification of the rhizosphere and secretion of organic anions [79].
Achievement and maintenance of high nutrient use efficiency (NUE) together with high crop productivity have become a major challenge in both developed and developing countries with an increasing growing population, depletion of natural resources, and deteriorating environmental conditions. Improving nutrient use efficiency (NUE) in plants is vital to enhance the yield and quality of crops, reduce nutrient input cost and improve soil, water, and air quality [3]. Higher NUE by plants could reduce fertilizer input costs, decrease the rate of nutrient losses, and enhance crop yields. Improving crop nutrient use efficiency ideally requires an understanding of the whole system, from the macro (agro-ecosystem) to the molecular level.
The development of nutrient-efficient crop varieties that can grow and yield better with low supply is a key to improving crop production. A prerequisite for nutrient use efficiency for any germplasm will be the optimization of agronomic practice for any given environment and season. Judicious application of fertilizer that includes the right rate, right time, right source, right place, and balanced fertilization (4RB) is the best management practice for achieving optimum nutrient efficiency. By the coordination of the acquisition, root-to-shoot translocation, utilization, and remobilization of internal Pi can be achieved through genetic breeding. Selection and breeding nutrient efficient species or genotypes within a species are justified in terms of reduction in fertilizer input cost of crop production and also reduced risk of contamination of soil and water. Overall NUE in plant is a function of capacity of soil to supply adequate levels of nutrients and ability of plant to acquire, transport in roots and shoot, and remobilize to other parts of the plant. Improvement in NUE will ultimately come from integrating a range of different approaches to develop a more efficient farming system. Use of nutrient efficient crop species or genotypes within species in combination with other improved crop production practices offers the best option for meeting the future food requirements of expanding world populations. Modern tools and resources available to plant scientists and the agronomy and breeding communities should aid further improvements in NUE and hence crop production. Therefore, integrated strategy that seeks to increase phosphorus use efficiency and simultaneously seeks to recover unavoidable phosphorus losses. The nutrient inputs in the intensive farming system should be optimized to achieve both high crop productivity and high nutrient use efficiency through maximizing root/rhizosphere efficiency in nutrient mobilization and acquisition.
The authors are highly thankful to researchers whose findings are included directly or indirectly in preparing this manuscript.
The authors declare no conflict of interest.
The authors received no direct funding for this research.
All data generated are included in this article reference’s part.
AMF | arbuscular mycorrhizal fungi |
BMP | best management practice |
DAP | diammonium phosphate |
FYM | farm-yard manure |
NUE | nutrient use efficiency |
PACE | phosphorus acquisition efficiency |
PSB | phosphate solubilizing bacteria |
PUE | phosphorus use efficiency |
PUTE | phosphorus utilization efficiency |
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Dr. Saxena is a fellow of various international societies/academies including the Royal College of Pathologists, United Kingdom; Royal Society of Medicine, London; Royal Society of Biology, United Kingdom; Royal Society of Chemistry, London; and Academy of Translational Medicine Professionals, Austria. He was named a Global Leader in Science by The Scientist. He is also an international opinion leader/expert in vaccination for Japanese encephalitis by IPIC (UK).",institutionString:"King George's Medical University",institution:{name:"King George's Medical University",institutionURL:null,country:{name:"India"}}}]},{type:"book",id:"7123",title:"Current Topics in Neglected Tropical Diseases",subtitle:null,coverURL:"https://cdn.intechopen.com/books/images_new/7123.jpg",slug:"current-topics-in-neglected-tropical-diseases",publishedDate:"December 4th 2019",editedByType:"Edited by",bookSignature:"Alfonso J. 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He has edited more than 50 international books, presented more than 500 lectures/posters in congresses/meetings, and published more than 1,100 scientific papers in international journals.",institutionString:"Tehran University of Medical Sciences",institution:{name:"Tehran University of Medical Sciences",country:{name:"Iran"}}},{id:"180733",title:"Dr.",name:"Jean",middleName:null,surname:"Engohang-Ndong",slug:"jean-engohang-ndong",fullName:"Jean Engohang-Ndong",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/180733/images/system/180733.png",biography:"Dr. Jean Engohang-Ndong was born and raised in Gabon. After obtaining his Associate Degree of Science at the University of Science and Technology of Masuku, Gabon, he continued his education in France where he obtained his BS, MS, and Ph.D. in Medical Microbiology. He worked as a post-doctoral fellow at the Public Health Research Institute (PHRI), Newark, NJ for four years before accepting a three-year faculty position at Brigham Young University-Hawaii. Dr. Engohang-Ndong is a tenured faculty member with the academic rank of Full Professor at Kent State University, Ohio, where he teaches a wide range of biological science courses and pursues his research in medical and environmental microbiology. Recently, he expanded his research interest to epidemiology and biostatistics of chronic diseases in Gabon.",institutionString:"Kent State University",institution:{name:"Kent State University",country:{name:"United States of America"}}},{id:"188773",title:"Prof.",name:"Emmanuel",middleName:null,surname:"Drouet",slug:"emmanuel-drouet",fullName:"Emmanuel Drouet",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/188773/images/system/188773.png",biography:"Emmanuel Drouet, PharmD, is a Professor of Virology at the Faculty of Pharmacy, the University Grenoble-Alpes, France. As a head scientist at the Institute of Structural Biology in Grenoble, Dr. Drouet’s research investigates persisting viruses in humans (RNA and DNA viruses) and the balance with our host immune system. He focuses on these viruses’ effects on humans (both their impact on pathology and their symbiotic relationships in humans). He has an excellent track record in the herpesvirus field, and his group is engaged in clinical research in the field of Epstein-Barr virus diseases. He is the editor of the online Encyclopedia of Environment and he coordinates the Universal Health Coverage education program for the BioHealth Computing Schools of the European Institute of Science.",institutionString:null,institution:{name:"Grenoble Alpes University",country:{name:"France"}}},{id:"131400",title:"Prof.",name:"Alfonso J.",middleName:null,surname:"Rodriguez-Morales",slug:"alfonso-j.-rodriguez-morales",fullName:"Alfonso J. Rodriguez-Morales",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/131400/images/system/131400.png",biography:"Dr. Rodriguez-Morales is an expert in tropical and emerging diseases, particularly zoonotic and vector-borne diseases (especially arboviral diseases). He is the president of the Travel Medicine Committee of the Pan-American Infectious Diseases Association (API), as well as the president of the Colombian Association of Infectious Diseases (ACIN). He is a member of the Committee on Tropical Medicine, Zoonoses, and Travel Medicine of ACIN. He is a vice-president of the Latin American Society for Travel Medicine (SLAMVI) and a Member of the Council of the International Society for Infectious Diseases (ISID). Since 2014, he has been recognized as a Senior Researcher, at the Ministry of Science of Colombia. He is a professor at the Faculty of Medicine of the Fundacion Universitaria Autonoma de las Americas, in Pereira, Risaralda, Colombia. He is an External Professor, Master in Research on Tropical Medicine and International Health, Universitat de Barcelona, Spain. He is also a professor at the Master in Clinical Epidemiology and Biostatistics, Universidad Científica del Sur, Lima, Peru. In 2021 he has been awarded the “Raul Isturiz Award” Medal of the API. Also, in 2021, he was awarded with the “Jose Felix Patiño” Asclepius Staff Medal of the Colombian Medical College, due to his scientific contributions to COVID-19 during the pandemic. He is currently the Editor in Chief of the journal Travel Medicine and Infectious Diseases. His Scopus H index is 47 (Google Scholar H index, 68).",institutionString:"Institución Universitaria Visión de las Américas, Colombia",institution:null},{id:"332819",title:"Dr.",name:"Chukwudi Michael",middleName:"Michael",surname:"Egbuche",slug:"chukwudi-michael-egbuche",fullName:"Chukwudi Michael Egbuche",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/332819/images/14624_n.jpg",biography:"I an Dr. Chukwudi Michael Egbuche. I am a Senior Lecturer in the Department of Parasitology and Entomology, Nnamdi Azikiwe University, Awka.",institutionString:null,institution:{name:"Nnamdi Azikiwe University",country:{name:"Nigeria"}}},{id:"284232",title:"Mr.",name:"Nikunj",middleName:"U",surname:"Tandel",slug:"nikunj-tandel",fullName:"Nikunj Tandel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/284232/images/8275_n.jpg",biography:'Mr. Nikunj Tandel has completed his Master\'s degree in Biotechnology from VIT University, India in the year of 2012. He is having 8 years of research experience especially in the field of malaria epidemiology, immunology, and nanoparticle-based drug delivery system against the infectious diseases, autoimmune disorders and cancer. He has worked for the NIH funded-International Center of Excellence in Malaria Research project "Center for the study of complex malaria in India (CSCMi)" in collaboration with New York University. The preliminary objectives of the study are to understand and develop the evidence-based tools and interventions for the control and prevention of malaria in different sites of the INDIA. Alongside, with the help of next-generation genomics study, the team has studied the antimalarial drug resistance in India. Further, he has extended his research in the development of Humanized mice for the study of liver-stage malaria and identification of molecular marker(s) for the Artemisinin resistance. At present, his research focuses on understanding the role of B cells in the activation of CD8+ T cells in malaria. Received the CSIR-SRF (Senior Research Fellow) award-2018, FIMSA (Federation of Immunological Societies of Asia-Oceania) Travel Bursary award to attend the IUIS-IIS-FIMSA Immunology course-2019',institutionString:"Nirma University",institution:{name:"Nirma University",country:{name:"India"}}},{id:"334383",title:"Ph.D.",name:"Simone",middleName:"Ulrich",surname:"Ulrich Picoli",slug:"simone-ulrich-picoli",fullName:"Simone Ulrich Picoli",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/334383/images/15919_n.jpg",biography:"Graduated in Pharmacy from Universidade Luterana do Brasil (1999), Master in Agricultural and Environmental Microbiology from Federal University of Rio Grande do Sul (2002), Specialization in Clinical Microbiology from Universidade de São Paulo, USP (2007) and PhD in Sciences in Gastroenterology and Hepatology (2012). She is currently an Adjunct Professor at Feevale University in Medicine and Biomedicine courses and a permanent professor of the Academic Master\\'s Degree in Virology. She has experience in the field of Microbiology, with an emphasis on Bacteriology, working mainly on the following topics: bacteriophages, bacterial resistance, clinical microbiology and food microbiology.",institutionString:null,institution:{name:"Universidade Feevale",country:{name:"Brazil"}}},{id:"229220",title:"Dr.",name:"Amjad",middleName:"Islam",surname:"Aqib",slug:"amjad-aqib",fullName:"Amjad Aqib",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229220/images/system/229220.png",biography:"Dr. Amjad Islam Aqib obtained a DVM and MSc (Hons) from University of Agriculture Faisalabad (UAF), Pakistan, and a PhD from the University of Veterinary and Animal Sciences Lahore, Pakistan. Dr. Aqib joined the Department of Clinical Medicine and Surgery at UAF for one year as an assistant professor where he developed a research laboratory designated for pathogenic bacteria. Since 2018, he has been Assistant Professor/Officer in-charge, Department of Medicine, Manager Research Operations and Development-ORIC, and President One Health Club at Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan. He has nearly 100 publications to his credit. His research interests include epidemiological patterns and molecular analysis of antimicrobial resistance and modulation and vaccine development against animal pathogens of public health concern.",institutionString:"Cholistan University of Veterinary and Animal Sciences",institution:{name:"University of Agriculture Faisalabad",country:{name:"Pakistan"}}},{id:"333753",title:"Dr.",name:"Rais",middleName:null,surname:"Ahmed",slug:"rais-ahmed",fullName:"Rais Ahmed",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/333753/images/20168_n.jpg",biography:null,institutionString:null,institution:{name:"University of Agriculture Faisalabad",country:{name:"Pakistan"}}},{id:"62900",title:"Prof.",name:"Fethi",middleName:null,surname:"Derbel",slug:"fethi-derbel",fullName:"Fethi Derbel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62900/images/system/62900.jpeg",biography:"Professor Fethi Derbel was born in 1960 in Tunisia. He received his medical degree from the Sousse Faculty of Medicine at Sousse, University of Sousse, Tunisia. He completed his surgical residency in General Surgery at the University Hospital Farhat Hached of Sousse and was a member of the Unit of Liver Transplantation in the University of Rennes, France. He then worked in the Department of Surgery at the Sahloul University Hospital in Sousse. Professor Derbel is presently working at the Clinique les Oliviers, Sousse, Tunisia. His hospital activities are mostly concerned with laparoscopic, colorectal, pancreatic, hepatobiliary, and gastric surgery. He is also very interested in hernia surgery and performs ventral hernia repairs and inguinal hernia repairs. He has been a member of the GREPA and Tunisian Hernia Society (THS). During his residency, he managed patients suffering from diabetic foot, and he was very interested in this pathology. For this reason, he decided to coordinate a book project dealing with the diabetic foot. Professor Derbel has published many articles in journals and collaborates intensively with IntechOpen Access Publisher as an editor.",institutionString:"Clinique les Oliviers",institution:null},{id:"300144",title:"Dr.",name:"Meriem",middleName:null,surname:"Braiki",slug:"meriem-braiki",fullName:"Meriem Braiki",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/300144/images/system/300144.jpg",biography:"Dr. Meriem Braiki is a specialist in pediatric surgeon from Tunisia. She was born in 1985. She received her medical degree from the University of Medicine at Sousse, Tunisia. She achieved her surgical residency training periods in Pediatric Surgery departments at University Hospitals in Monastir, Tunis and France.\r\nShe is currently working at the Pediatric surgery department, Sidi Bouzid Hospital, Tunisia. Her hospital activities are mostly concerned with laparoscopic, parietal, urological and digestive surgery. She has published several articles in diffrent journals.",institutionString:"Sidi Bouzid Regional Hospital",institution:null},{id:"229481",title:"Dr.",name:"Erika M.",middleName:"Martins",surname:"de Carvalho",slug:"erika-m.-de-carvalho",fullName:"Erika M. de Carvalho",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229481/images/6397_n.jpg",biography:null,institutionString:null,institution:{name:"Oswaldo Cruz Foundation",country:{name:"Brazil"}}},{id:"186537",title:"Prof.",name:"Tonay",middleName:null,surname:"Inceboz",slug:"tonay-inceboz",fullName:"Tonay Inceboz",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/186537/images/system/186537.jfif",biography:"I was graduated from Ege University of Medical Faculty (Turkey) in 1988 and completed his Med. PhD degree in Medical Parasitology at the same university. I became an Associate Professor in 2008 and Professor in 2014. I am currently working as a Professor at the Department of Medical Parasitology at Dokuz Eylul University, Izmir, Turkey.\n\nI have given many lectures, presentations in different academic meetings. I have more than 60 articles in peer-reviewed journals, 18 book chapters, 1 book editorship.\n\nMy research interests are Echinococcus granulosus, Echinococcus multilocularis (diagnosis, life cycle, in vitro and in vivo cultivation), and Trichomonas vaginalis (diagnosis, PCR, and in vitro cultivation).",institutionString:"Dokuz Eylül University",institution:{name:"Dokuz Eylül University",country:{name:"Turkey"}}},{id:"71812",title:"Prof.",name:"Hanem Fathy",middleName:"Fathy",surname:"Khater",slug:"hanem-fathy-khater",fullName:"Hanem Fathy Khater",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/71812/images/1167_n.jpg",biography:"Prof. Khater is a Professor of Parasitology at Benha University, Egypt. She studied for her doctoral degree, at the Department of Entomology, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, USA. She has completed her Ph.D. degrees in Parasitology in Egypt, from where she got the award for “the best scientific Ph.D. dissertation”. She worked at the School of Biological Sciences, Bristol, England, the UK in controlling insects of medical and veterinary importance as a grant from Newton Mosharafa, the British Council. Her research is focused on searching of pesticides against mosquitoes, house flies, lice, green bottle fly, camel nasal botfly, soft and hard ticks, mites, and the diamondback moth as well as control of several parasites using safe and natural materials to avoid drug resistances and environmental contamination.",institutionString:null,institution:{name:"Banha University",country:{name:"Egypt"}}},{id:"99780",title:"Prof.",name:"Omolade",middleName:"Olayinka",surname:"Okwa",slug:"omolade-okwa",fullName:"Omolade Okwa",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/99780/images/system/99780.jpg",biography:"Omolade Olayinka Okwa is presently a Professor of Parasitology at Lagos State University, Nigeria. She has a PhD in Parasitology (1997), an MSc in Cellular Parasitology (1992), and a BSc (Hons) Zoology (1990) all from the University of Ibadan, Nigeria. She teaches parasitology at the undergraduate and postgraduate levels. She was a recipient of a Commonwealth fellowship supported by British Council tenable at the Centre for Entomology and Parasitology (CAEP), Keele University, United Kingdom between 2004 and 2005. She was awarded an Honorary Visiting Research Fellow at the same university from 2005 to 2007. \nShe has been an external examiner to the Department of Veterinary Microbiology and Parasitology, University of Ibadan, MSc programme between 2010 and 2012. She is a member of the Nigerian Society of Experimental Biology (NISEB), Parasitology and Public Health Society of Nigeria (PPSN), Science Association of Nigeria (SAN), Zoological Society of Nigeria (ZSN), and is Vice Chairperson of the Organisation of Women in Science (OWSG), LASU chapter. She served as Head of Department of Zoology and Environmental Biology, Lagos State University from 2007 to 2010 and 2014 to 2016. She is a reviewer for several local and international journals such as Unilag Journal of Science, Libyan Journal of Medicine, Journal of Medicine and Medical Sciences, and Annual Research and Review in Science. \nShe has authored 45 scientific research publications in local and international journals, 8 scientific reviews, 4 books, and 3 book chapters, which includes the books “Malaria Parasites” and “Malaria” which are IntechOpen access publications.",institutionString:"Lagos State University",institution:{name:"Lagos State University",country:{name:"Nigeria"}}},{id:"273100",title:"Dr.",name:"Vijay",middleName:null,surname:"Gayam",slug:"vijay-gayam",fullName:"Vijay Gayam",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/273100/images/system/273100.jpeg",biography:"Dr. Vijay Bhaskar Reddy Gayam is currently practicing as an internist at Interfaith Medical Center in Brooklyn, New York, USA. He is also a Clinical Assistant Professor at the SUNY Downstate University Hospital and Adjunct Professor of Medicine at the American University of Antigua. He is a holder of an M.B.B.S. degree bestowed to him by Osmania Medical College and received his M.D. at Interfaith Medical Center. His career goals thus far have heavily focused on direct patient care, medical education, and clinical research. He currently serves in two leadership capacities; Assistant Program Director of Medicine at Interfaith Medical Center and as a Councilor for the American\r\nFederation for Medical Research. As a true academician and researcher, he has more than 50 papers indexed in international peer-reviewed journals. He has also presented numerous papers in multiple national and international scientific conferences. His areas of research interest include general internal medicine, gastroenterology and hepatology. He serves as an editor, editorial board member and reviewer for multiple international journals. His research on Hepatitis C has been very successful and has led to multiple research awards, including the 'Equity in Prevention and Treatment Award” from the New York Department of Health Viral Hepatitis Symposium (2018) and the 'Presidential Poster Award” awarded to him by the American College of Gastroenterology (2018). He was also awarded 'Outstanding Clinician in General Medicine” by Venus International Foundation for his extensive research expertise and services, perform over and above the standard expected in the advancement of healthcare, patient safety and quality of care.",institutionString:"Interfaith Medical Center",institution:{name:"Interfaith Medical Center",country:{name:"United States of America"}}},{id:"93517",title:"Dr.",name:"Clement",middleName:"Adebajo",surname:"Meseko",slug:"clement-meseko",fullName:"Clement Meseko",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/93517/images/system/93517.jpg",biography:"Dr. Clement Meseko obtained DVM and PhD degree in Veterinary Medicine and Virology respectively. He has worked for over 20 years in both private and public sectors including the academia, contributing to knowledge and control of infectious disease. Through the application of epidemiological skill, classical and molecular virological skills, he investigates viruses of economic and public health importance for the mitigation of the negative impact on people, animal and the environment in the context of Onehealth. \r\nDr. Meseko’s field experience on animal and zoonotic diseases and pathogen dynamics at the human-animal interface over the years shaped his carrier in research and scientific inquiries. He has been part of the investigation of Highly Pathogenic Avian Influenza incursions in sub Saharan Africa and monitors swine Influenza (Pandemic influenza Virus) agro-ecology and potential for interspecies transmission. He has authored and reviewed a number of journal articles and book chapters.",institutionString:"National Veterinary Research Institute",institution:{name:"National Veterinary Research Institute",country:{name:"Nigeria"}}},{id:"158026",title:"Prof.",name:"Shailendra K.",middleName:null,surname:"Saxena",slug:"shailendra-k.-saxena",fullName:"Shailendra K. Saxena",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRET3QAO/Profile_Picture_2022-05-10T10:10:26.jpeg",biography:"Professor Dr. Shailendra K. Saxena is a vice dean and professor at King George's Medical University, Lucknow, India. His research interests involve understanding the molecular mechanisms of host defense during human viral infections and developing new predictive, preventive, and therapeutic strategies for them using Japanese encephalitis virus (JEV), HIV, and emerging viruses as a model via stem cell and cell culture technologies. His research work has been published in various high-impact factor journals (Science, PNAS, Nature Medicine) with a high number of citations. He has received many awards and honors in India and abroad including various Young Scientist Awards, BBSRC India Partnering Award, and Dr. JC Bose National Award of Department of Biotechnology, Min. of Science and Technology, Govt. of India. Dr. Saxena is a fellow of various international societies/academies including the Royal College of Pathologists, United Kingdom; Royal Society of Medicine, London; Royal Society of Biology, United Kingdom; Royal Society of Chemistry, London; and Academy of Translational Medicine Professionals, Austria. He was named a Global Leader in Science by The Scientist. He is also an international opinion leader/expert in vaccination for Japanese encephalitis by IPIC (UK).",institutionString:"King George's Medical University",institution:{name:"King George's Medical University",country:{name:"India"}}},{id:"94928",title:"Dr.",name:"Takuo",middleName:null,surname:"Mizukami",slug:"takuo-mizukami",fullName:"Takuo Mizukami",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/94928/images/6402_n.jpg",biography:null,institutionString:null,institution:{name:"National Institute of Infectious Diseases",country:{name:"Japan"}}},{id:"233433",title:"Dr.",name:"Yulia",middleName:null,surname:"Desheva",slug:"yulia-desheva",fullName:"Yulia Desheva",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/233433/images/system/233433.png",biography:"Dr. Yulia Desheva is a leading researcher at the Institute of Experimental Medicine, St. Petersburg, Russia. She is a professor in the Stomatology Faculty, St. Petersburg State University. She has expertise in the development and evaluation of a wide range of live mucosal vaccines against influenza and bacterial complications. Her research interests include immunity against influenza and COVID-19 and the development of immunization schemes for high-risk individuals.",institutionString:'Federal State Budgetary Scientific Institution "Institute of Experimental Medicine"',institution:null},{id:"238958",title:"Mr.",name:"Atamjit",middleName:null,surname:"Singh",slug:"atamjit-singh",fullName:"Atamjit Singh",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/238958/images/6575_n.jpg",biography:null,institutionString:null,institution:null},{id:"252058",title:"M.Sc.",name:"Juan",middleName:null,surname:"Sulca",slug:"juan-sulca",fullName:"Juan Sulca",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/252058/images/12834_n.jpg",biography:null,institutionString:null,institution:null},{id:"191392",title:"Dr.",name:"Marimuthu",middleName:null,surname:"Govindarajan",slug:"marimuthu-govindarajan",fullName:"Marimuthu Govindarajan",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/191392/images/5828_n.jpg",biography:"Dr. M. Govindarajan completed his BSc degree in Zoology at Government Arts College (Autonomous), Kumbakonam, and MSc, MPhil, and PhD degrees at Annamalai University, Annamalai Nagar, Tamil Nadu, India. He is serving as an assistant professor at the Department of Zoology, Annamalai University. His research interests include isolation, identification, and characterization of biologically active molecules from plants and microbes. He has identified more than 20 pure compounds with high mosquitocidal activity and also conducted high-quality research on photochemistry and nanosynthesis. He has published more than 150 studies in journals with impact factor and 2 books in Lambert Academic Publishing, Germany. He serves as an editorial board member in various national and international scientific journals.",institutionString:null,institution:null},{id:"274660",title:"Dr.",name:"Damodar",middleName:null,surname:"Paudel",slug:"damodar-paudel",fullName:"Damodar Paudel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/274660/images/8176_n.jpg",biography:"I am DrDamodar Paudel,currently working as consultant Physician in Nepal police Hospital.",institutionString:null,institution:null},{id:"241562",title:"Dr.",name:"Melvin",middleName:null,surname:"Sanicas",slug:"melvin-sanicas",fullName:"Melvin Sanicas",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/241562/images/6699_n.jpg",biography:null,institutionString:null,institution:null},{id:"117248",title:"Dr.",name:"Andrew",middleName:null,surname:"Macnab",slug:"andrew-macnab",fullName:"Andrew Macnab",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of British Columbia",country:{name:"Canada"}}},{id:"322007",title:"Dr.",name:"Maria Elizbeth",middleName:null,surname:"Alvarez-Sánchez",slug:"maria-elizbeth-alvarez-sanchez",fullName:"Maria Elizbeth Alvarez-Sánchez",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Universidad Autónoma de la Ciudad de México",country:{name:"Mexico"}}},{id:"337443",title:"Dr.",name:"Juan",middleName:null,surname:"A. Gonzalez-Sanchez",slug:"juan-a.-gonzalez-sanchez",fullName:"Juan A. Gonzalez-Sanchez",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Puerto Rico System",country:{name:"United States of America"}}},{id:"337446",title:"Dr.",name:"Maria",middleName:null,surname:"Zavala-Colon",slug:"maria-zavala-colon",fullName:"Maria Zavala-Colon",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Puerto Rico, Medical Sciences Campus",country:{name:"United States of America"}}}]}},subseries:{item:{id:"2",type:"subseries",title:"Prosthodontics and Implant Dentistry",keywords:"Osseointegration, Hard Tissue, Peri-implant Soft Tissue, Restorative Materials, Prosthesis Design, Prosthesis, Patient Satisfaction, Rehabilitation",scope:"