Comparison of bond length with experimental values in different basis sets.
\r\n\t
",isbn:null,printIsbn:"979-953-307-X-X",pdfIsbn:null,doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,isSalesforceBook:!1,hash:"287b982cd93fa41c914d5d42c3cb6895",bookSignature:"Associate Prof. Binh Ngo",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/11089.jpg",keywords:"Diet, Foraging Mode, Behavior, Life History, Microhabitat Use, Reproduction, Skink Ecology, Feeding Ecology, Reproductive Biology, Skinks, Lizards, Reptiles",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"September 30th 2021",dateEndSecondStepPublish:"October 28th 2021",dateEndThirdStepPublish:"December 27th 2021",dateEndFourthStepPublish:"March 17th 2022",dateEndFifthStepPublish:"May 16th 2022",remainingDaysToSecondStep:"7 months",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:"Dr. Ngo has received his Ph.D. in Zoology from National Cheng Kung University, Taiwan (2015), and has over five years of experience teaching Zoology, Ecology, and Animal Behavior. Dr. Ngo's research focuses primarily on behavioral ecology, bioacoustics, population biology, community ecology, conservation ecology, log-normal distributions, and site occupancy of amphibians and reptiles living in tropical regions.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"416568",title:"Associate Prof.",name:"Binh",middleName:null,surname:"V. Ngo",slug:"binh-v.-ngo",fullName:"Binh V. Ngo",profilePictureURL:"https://mts.intechopen.com/storage/users/416568/images/system/416568.jpg",biography:"BINH V. NGO is an Associate Professor of Biology and a senior lecturer at the College of Education, Hue University, Vietnam, where he has taught Zoology, Ecology, and Animal Behavior over the last 6 y. He received his Ph.D. in Zoology from National Cheng Kung University, Taiwan (2015). 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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:"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:"314",title:"Regenerative Medicine and Tissue Engineering",subtitle:"Cells and Biomaterials",isOpenForSubmission:!1,hash:"bb67e80e480c86bb8315458012d65686",slug:"regenerative-medicine-and-tissue-engineering-cells-and-biomaterials",bookSignature:"Daniel Eberli",coverURL:"https://cdn.intechopen.com/books/images_new/314.jpg",editedByType:"Edited by",editors:[{id:"6495",title:"Dr.",name:"Daniel",surname:"Eberli",slug:"daniel-eberli",fullName:"Daniel Eberli"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"57",title:"Physics and Applications of Graphene",subtitle:"Experiments",isOpenForSubmission:!1,hash:"0e6622a71cf4f02f45bfdd5691e1189a",slug:"physics-and-applications-of-graphene-experiments",bookSignature:"Sergey Mikhailov",coverURL:"https://cdn.intechopen.com/books/images_new/57.jpg",editedByType:"Edited by",editors:[{id:"16042",title:"Dr.",name:"Sergey",surname:"Mikhailov",slug:"sergey-mikhailov",fullName:"Sergey Mikhailov"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1373",title:"Ionic Liquids",subtitle:"Applications and Perspectives",isOpenForSubmission:!1,hash:"5e9ae5ae9167cde4b344e499a792c41c",slug:"ionic-liquids-applications-and-perspectives",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/1373.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"}}]},chapter:{item:{type:"chapter",id:"67340",title:"Modulating the T Lymphocyte Immune Response via Secretome Produced miRNA: From Tolerance Induction to the Enhancement of the Anticancer Response",doi:"10.5772/intechopen.86598",slug:"modulating-the-t-lymphocyte-immune-response-via-secretome-produced-mirna-from-tolerance-induction-to",body:'\nBiologically, and clinically, the concept of “self” is of crucial importance in protection against foreign biologicals (e.g., viruses and bacteria), abnormal autologous cells (e.g., cancers) and more recently developed “diseases” (i.e., the purposeful introduction of “nonself”) such as enzyme-replacement therapy and transfusion and transplantation medicine. The immune system is tasked with preserving “self” and rejecting “nonself” and has multiple components-any of which will be of variable importance depending on the context of the immunological assault. Immunological “self” of most tissues is imparted by the major histocompatibility complex (MHC) which encodes a variety of proteins that provide a means for identifying, targeting, and eliminating foreign invaders and diseased cells while preserving normal “self” tissue. The MHC proteins themselves consist of three classes. MHC Class I molecules are expressed on virtually all nucleated cells while Class II molecules are expressed exclusively on antigen presenting cells (APC; e.g., monocytes, macrophages, dendritic cell, B lymphocytes, and endothelial cells) and activated T lymphocytes. MHC Class III genes encode components of the complement system. The human MHC is referred to as the Human Leukocyte Antigen (HLA) complex while the murine equivalent is referred to as the Histocompatibility-2 (H2) complex. In the context of MHC-mediated immune recognition, the T lymphocyte (T cell) is of particular importance. T cells themselves consist of a diverse array of subsets that fall into two general categories: 1) Regulatory T cells (Treg) which modulate the strength of an immune response and maintain “self”; and effector T cells (Teff) that mediate the inflammatory response and consists, in part, of Th1, Th17 and Th2 subsets. Hence, the functional ratio of Treg to Teff (Treg:Teff) cells is critical and an imbalance of this ratio from the norm can induce either an autoimmune (excess Teff or decreased Treg) state or impaired response to “nonself” (e.g., cancer) consequent to biologically ill-advised tolerance (too many Treg or weak Teff response). Indeed, the T cell response plays a (the) central role in autoimmune diseases, transplant rejection, graft versus host disease (GVHD), graft versus leukemia (GVL), cancer and, more recently, cancer therapy. Hence, consequent to the central role of T cells as a key cellular component in the development of autoimmune diseases, graft tolerance or rejection, and the anticancer response, the T cell response has been a major focus in the development of clinical therapies (Figure 1A) [1].
\nImmune modulation via pharmacologic and immunocamouflage therapy. (A) Current pharmacologic therapy almost exclusively targets T cell activation and proliferation consequent to allorecognition. Response to nonself is in large part mediated by cell-cell interactions between antigen presenting cells (APC; e.g., dendritic cells) and naive T cells. This cell-cell interaction is characterized by essential adhesion, allorecognition and co-stimulation events. Consequent to allorecognition, a proliferation of proinflammatory T cells (e.g., cytotoxic T lymphocyte, CTL; Th17, IL-17+; Th1, IFN-γ+; and IL-2+ populations) and decrease in regulatory T cells (Treg, Foxp3+ and CD25+) is observed. Current therapeutic agents are primarily cytotoxic agents preventing T cell activation (e.g., cyclosporine and rapamycin) or T cell proliferation (e.g., methotrexate, corticosteroids and azathioprine). Additionally, blocking antibodies have been investigated. Gray text indicates current techniques to prevent/limit alloimmune responses. (B) In contrast, immunocamouflage of donor cells by methoxy(polyethylene) glycol (mPEG) results in the disruption of the essential cell-cell interactions decreasing T cell proliferation and altering differentiation patterns (decreased Th17 and increased Treg). In aggregate, the polymer induced changes induces a tolerogenic/anergic state both
Autoimmune diseases arise when the immune system recognizes the individual’s own tissues or organs as “foreign” and targets them for destruction. Autoimmune diseases can affect virtually all tissues and organ systems and encompass such diverse diseases as Type 1 Diabetes (T1D; pancreas), Idiopathic Thrombocytopenic Purpura (ITP; platelet destruction), Crohn’s disease (CD; bowel), Multiple Sclerosis (MS; brain) and Rheumatoid Arthritis (RA; joints). Despite the diversity of tissues affected, extensive research has demonstrated that Treg are downregulated while Teff are upregulated (i.e., leading to a reduced Treg:Teff ratio) leading to a chronic proinflammatory state. Current therapeutic approaches to managing autoimmune diseases are typically focused on symptom relief and the use of immunosuppressive agents capable of inhibiting the proinflammatory response arising from “self-recognition.” Most commonly, treatment for chronic autoimmune disease is via administration of systemic steroids (e.g., dexamethasone), cytotoxic anti-proliferative/activation agents (e.g., cyclosporine) that induce a general immunosuppression, and/or IVIG (pooled, polyvalent, IgG purified from the plasma of >1000 blood donors) [2, 3, 4, 5, 6]. Other experimental approaches to the treatment of autoimmune diseases include blocking monoclonal antibodies directed against the TCR, CD4, costimulatory ligands and receptors, adhesion molecules, and cytokine receptors [7, 8, 9]. A more recent approach has been to interrupt the cytokine signals necessary for the activation and proliferation of autoreactive T cells. The current gold standard for this approach is Enbrel® (etanercept), a solubilized TNF-α receptor fragment that intercepts and sequesters the TNF-α cytokine thereby inhibiting the proliferation of proinflammatory T cells [10, 11, 12, 13, 14, 15]. However, Enbrel® has been given a USA FDA “Black Box” warning due to significantly increased risks of serious infections that may lead to hospitalization or death [16, 17, 18, 19, 20, 21, 22]. Common to all of these approaches is an attempt to increase the Treg:Teff ratio by either directly increasing Treg or selectively decreasing Teff populations. However, despite their importance in clinical medicine, many of these agents have been plagued by both significant toxicity/adverse events and an inability to adequately eliminate or inhibit reactive T cells [8].
\nIn contrast to autoimmune diseases, an insufficient/inefficient immune response may underlie the proliferation and dissemination of abnormal cells (i.e., cancer cells). While this may occur for a number of reasons, immunosuppression is a known risk factor. Indeed, acquired or inherited T cell defects as well as long-term therapy with immunosuppressive drugs are clearly associated with an increased risk of neoplasia. The impaired immune response to cancer cells can arise, at least in part, from an increase in the Treg:Teff ratio (too many Treg and/or insufficient Teff cell production). To address this imbalance in the Treg:Teff ratio, experimental therapies are currently focused on the
Perhaps most importantly, current tolerogenic or proinflammatory therapeutic approaches fail to persistently reorient the systemic T cell immune response thus necessitating continual therapy. Moreover, despite the importance of the Treg:Teff ratio, in both autoimmune diseases and cancer, there are a paucity of pharmacologic tools that can directly, and in tandem, target the regulation of both the Treg and Teff subsets. Hence, to diminish or overcome the need for chronic administration of immunotherapeutic agents, new approaches capable of persistently reorienting the endogenous immune (Treg:Teff) response would be of value.
\nPrevious studies from our laboratory demonstrated that a persistent and systemic reorientation of the animal (murine; or
Since their discovery in 1996, the role of circulating (cell-free) miRNA in disease processes has become an active research area and recent findings suggest that they may be biomarkers, or possibly mediators, of cancers as well as autoimmune diseases such as T1D [44, 45, 46]. To understand mechanistically how the TA1 and IA1 miRNA biologics function, an appreciation of the biological role and regulatory complexity of miRNA is needed. Recent studies have demonstrated that miRNA are key epigenetic regulators of cellular processes including immune responses, inflammation, proliferation, survival, and cellular differentiation [47, 48]. miRNA are short (~22 nucleotides) single-stranded RNA molecules found in all eukaryotes and it is estimated that ~60% of mammalian genes are targeted by one or more miRNA [49, 50]. Moreover, because of their evolutionary importance in gene regulation, miRNA and their sequence and processing are highly conserved between mammalian species (e.g., mouse and human) [49]. While miRNA are most commonly found intracellularly, significant amounts of stable miRNA are also found in the serum of mammals suggesting an important messenger/regulatory role. While the nomenclature of miRNAs is relatively straightforward it is important to note that similarities in miRNA number designation is not indicative of similarity in functionality (Figure 2). Moreover, the literature is replete with conflicting claims for the specific actions of a single miRNA.
\nmiRNA nomenclature explained. An important concept to understand is that the miRNA number (e.g., 123 as shown) has no relationship to function or structure. For example, “hsa-miR-123” has no implied structural or functional similarity to “hsa-miR-128.” However, because of the highly conserved nature of miRNA, human “hsa-miR-123” has very similar structure and function to murine “mmu-miR-123.”
Indeed, there is a significant lack of clarity regarding the function of a single miRNA. This lack of functional clarity likely arises consequent to the complexity and low fidelity of the miRNA bioregulatory process. Of note, a single miRNA can potentially affect tens to hundreds of genes and individual genes can be regulated by multiple miRNA [50]. Hence, the effect of modifying the expression of a single miRNA on protein regulation and bioregulatory networks is unpredictable. Because of this regulatory complexity, most studies have focused on miRNA as disease biomarkers, not as therapeutic agents as there is a low probability that altered expression of a single, or even a few, miRNA would exert a potent and definitive biological response [51, 52, 53, 54]. From a bioregulatory approach, it is more probable that multiple miRNA control protein expression, proliferation and differentiation and it is this “pattern of miRNA expression” (encompassing increased, decreased and static levels) that must be mimicked to achieve pharmacologically effective miRNA-based therapeutics. To achieve this goal our laboratory approach has been purposefully chosen to biologically manufacture relatively complex miRNA preparations mimicking normal biology in order to achieve maximal biological functionality.
\nUsing a Mixed Lymphocyte Reaction (MLR) production model the T cell centric proinflammatory IA1 and tolerogenic TA1 therapeutics can be reproducibly manufactured using the control-MLR and mPEG-MLR (respectively; Figure 1). As demonstrated, the allogeneic PBMC populations within the control- and mPEG-MLR express significantly different patterns of miRNA expression relative to resting PBMC as evidenced via clustergram (Figure 3A), volcano plot (Figure 3B) and Log2 Fold (Figure 3C) miRNA expression analyses. Importantly, as shown in Figure 3C, the control- and mPEG-MLRs show unique patterns of expression. While there are some similarities in the pattern of expression there are significant disparity in miRNAs expressed as well (not shown are the miRNA unchanged from resting cells).
\nPartial qPCR characterization of the miRNA expression in the Control- and mPEG-MLR. (A–C) Clustergram (A), Volcano Plot (B) and Log2 Fold (C) analyses of the miRNA expression in the mPEG-MLR and Control-MLR relative to resting cells. (C) Because of the complexity of miRNA regulation of genes, we have consciously chosen to produce a relatively complex miRNA preparations mimicking normal biology in order to achieve maximal biological functionality. Multiple miRNA changes are noted in the hTA1 miRNA compared to either resting cells (green dashed line = 0) or the proinflammatory hIA1 miRNA preparation. Using miRNA expressing a net ΔLog2 Fold change, significantly different “patterns of expression” are noted between the hTA1 and hIA1 miRNA. This pattern of expression, comprising both INCREASED and DECREASED miRNA species is essential for effective immunomodulation of recipient animals. Values derived from a minimum of 3 independent biological replicates. Unpublished data.
Importantly, the differences in miRNA expression between the Control- and mPEG-MLR leukocyte yield secretomes that exert dramatically different effects when used to treat resting human PBMC or murine splenocytes. Collection of the secretome produced (Figure 4A) during the control and polymer modified allorecognition-based MLR yields a reproducible, acellular, miRNA-rich, material that is stable and can be frozen and thawed with minimal decrement to its activity. As schematically presented (Figure 4B), TA1 upregulates regulatory T cell populations (e.g., Treg) while simultaneously downregulating Teff (e.g., Th17 and Th1) cells. In contrast, the proinflammatory IA1 increases Teff while decreasing Treg cells. Of note, the secretome from resting cells (SYN) has minimal to no effect on human or mouse immune cells. Moreover, due to the conserved nature of mammalian miRNA, cross species efficacy is observed with both TA1 and IA1. As shown in Figure 4C, murine splenocyte produced TA1 and IA1 exerted dose-dependent effects on a human MLR with murine-sourced TA1 reducing CD3+CD4+ T cell proliferation and the murine IA1 enhancing CD3+CD4+ T cell proliferation. Hence, a polymer-based, alloresponse manufacturing system may provide a unique avenue for more effectively, and safely, modulating the Treg:Teff cell ratio via the production of therapeutically effective TA1 and IA1 miRNA-based therapeutics [32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43]. Importantly, the effects of TA1 and IA1 immunotherapy was persistent. In murine studies, a single dosing of TA1 to mice resulted in significant increase in Treg cells within the spleen of normal mice that persisted to ≥270 days post treatment (Figure 4D).
\nDifferential effects of TA1 and IA1 on the immune system. (A and B): Secretome production (A) of SYN (resting), IA1 (MLR) and TA1 (mPEG-MLR) gave rise to unique immunomodulatory activity (B). While IA1 enhanced proinflammatory subsets and reduced Treg cells, TA1 enhanced Treg while reducing Teff subpopulations.
Autoimmune destruction of pancreatic islets gives rise to T1D and occurs via T cell dependent pathways [55, 56, 57]. Elucidation of the role of T cells in T1D has been most effectively examined in the nonobese diabetic (NOD) mouse model. In the NOD mouse, evidence suggests that a deficit in Treg control over diabetogenic Teff cells leads to the development of insulitis and disease [56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66]. Indeed, changes in the Treg:Teff ratio (i.e., balance) can be observed as early as 3–4 weeks of age and becomes more pronounced with disease progression (Figure 5) [56]. Human studies have similarly demonstrated that T1D Treg exhibit an impaired ability to suppress Teff [67]. Thus, the emergence of an aggressive diabetogenic lymphocyte response in NOD mice, and likely humans, is dependent upon a change in the Treg:Teff ratio.
\nThe autoimmune disease of T1D process is mediated by a decrease in the Treg:Teff ratio and can be prevented by TA1 administration (top). Treatment with the TA1 miRNA product prevents the decrease and, in fact, significantly increases the Treg:Teff ratio. The increased Treg:Teff ratio is protective as evidenced by the finding that the majority of TA1 treated animals remained normoglycemic. Shown in the blue bars are the Treg:Teff ratio for TA1 treated mice who were diabetic (ratio of 70) and nondiabetic (ratio of 255). Mechanistically, TA1 immunotherapeutic significantly altered the expression of multiple proinflammatory (A) and tolerogenic/anergic (B) T cell subsets. These changes were systemic in nature as shown by changes in not only the pancreatic lymph node but in other immune tissues (spleen and brachial lymph node). Diabetic tissues were harvested at time of conversion, nondiabetic tissues were harvested at week 30. Diabetic values are the mean ± SD of 12 saline and 6 TA1 treated NOD mice. Nondiabetic results are the mean ± SD of 4 saline and 9 TA1 treated NOD mice. Derived from Ref. [
As demonstrated in Figure 5, the Treg:Teff ratio (defined as the ratio of Foxp3+ to Th17+ T cells) in control (saline treated) NOD mice decreased with disease progression from 103 in nondiabetic 7 week old mice to only 4.7 in diabetic mice at time of sacrifice (15–30 week). Moreover, control NOD mice exhibited a rapid onset of diabetes with 75% (12 of 16) of the mice becoming diabetic by week 19. Subsequent to week 19, no additional mice became diabetic. In contrast, a single dosing (3 injections at 2 days intervals) of the TA1 therapeutic at 7 weeks of age dramatically altered both the incidence and rate of progression of the T1D in the NOD mouse. By week 19 only 13% (2 of 15) of the TA1 treated mice became diabetic with an additional 4 mice becoming diabetic between weeks 21 and 23 (total diabetic 6/15; 40%). Mechanistically, these findings were associated with a systemic alteration of the immune system as noted in Figure 5. In control NOD mice, the progression to diabetes was characterized by significantly elevated levels of most proinflammatory Teff (e.g., INF-γ+, Th17+, and IL-2+) lymphocytes and a corresponding decrease in regulatory subsets. In contrast, TA1 therapy dramatically and significantly blunted the expansion of Teff cells (as exemplified by INF-γ+, Th17+, and IL-2+ lymphocytes; Figure 5A) relative to diabetic or nondiabetic control NOD mice coupled with a simultaneous increase in a broad range of tolerogenic/anergic regulatory T cell subsets (e.g., foxp3+, IL-10+, TGF-β+; Figure 5B) in the pancreatic lymph node. These studies also demonstrated that TA1 treated NOD mice had significant numbers of histologically normal pancreatic islets while no normal islets were identified in the untreated mice [40]. It is worth noting that all diabetic mice (control and TA1-treated) exhibited significantly lower levels of these tolerogenic cells than did the 30 week old nondiabetic (control or TA1) mice. Moreover, the effects of TA1-miRNA therapy were not localized to the pancreatic lymph node microenvironment. Analyses of the T cell subsets present in the spleen and brachial lymph node of control and TA1 treated NOD mice (diabetic and nondiabetic) similarly demonstrated dramatic changes in the Teff cell populations (Figure 5A, right) and tolerogenic T cells (Figure 5B, right). These findings demonstrate that miRNA-based TA1 therapeutic, directly targets the Treg:Teff ratio yielding a systemic protolerogenic state both
T cells plays a critical role in the anticancer inflammatory responses. An effective anticancer proinflammatory T cell response is dependent upon the activation of Teff cells. Normally, T cells are activated upon ligation of their antigen receptors with specific cognate antigens [68]. However, because of the low frequency of cancer antigen-specific lymphocytes, the immune response to cancers can be initially, and all too often remains, weak. While previous studies have attempted to enhance the anticancer T cell response using pan T cell mitogens (e.g., phytohemagglutinin; PHA), cytokines (e.g., IL-2), or monoclonal antibodies (e.g., anti-CD3 and anti-CD28) the overly robust T cell response arising from these approaches often induced significant systemic toxicity leading to the suspension or abrogation of multiple clinical trials [69, 70, 71, 72, 73, 74]. In contrast, in an allorecognition response only 1–10% of T cells are alloreactive [75]. Hence, the IA1 therapeutic, derived from a bioreactor allorecognition response (MLR), is expected to activate endogenous T cells in a more controlled manner, with less toxicity.
\nTo assess IA1’s ability to enhance the anticancer activity of resting PBMC, cells were treated for 24 hours with IA1 and overlaid on HeLa and SH-4 cancers cells. Cancer cell proliferation was then followed for 168 hours. Importantly, IA1 exerted no toxicity to resting PBMC but, as shown in Figure 4, induced significant activation (e.g., proliferation) of resting CD3+ (CD4+ and CD8+) skewed towards proinflammatory subsets thus decreasing the Teff:Treg ratio. However, as predicted by the biology of the alloresponse, IA1-mediated T cell proliferation was much more restrained than that induced by the anti-CD3/anti-CD28 or PHA stimulation [43]. This finding suggests that the systemic toxicity, relative to pan T cell activators, should be greatly reduced. Crucially, IA1-activated PBMC demonstrated a potent inhibition of cancer cell (HeLa and SH-4 melanoma) proliferation relative to the resting PBMC (Figure 6). The anti-proliferation effect of IA1-activated PBMC was noted within ~12 hours vs. 4–5 days for resting cells. These findings demonstrate that miRNA-enriched therapeutics can be biomanufactured from the secretome and can induce a potent proinflammatory, anticancer, effect on resting lymphocytes.
\nSchematic presentation of use and efficacy of the IA1 secretome therapeutic. Left panels: the enhanced efficacy of treated PBMC is supported by photomicrographs of allogenic PBMC responding to HeLa cells. As shown, after 72 hours incubation, resting (weak responders; left) PBMC show limited interaction when overlaid on HeLa cells. In contrast, the same PBMC, when treated for 24 hours with IA1, show a robust enhanced interaction (right) with the HeLa cell monolayer. Moreover, when IA1-treated PBMC are overlaid on SH-4 melanoma cells a greatly enhanced anti-cancer effect is noted relative to untreated PBMC. Shown are the growth profiles (as measured by electrical impedance) of SH-4 treated with either the SYN (derived from the secretome of resting PBMC) or IA1therapeutics. PBMC:SH-4 ratios included 50:1, 25:1 and 10:1. Right panels: bioreactor production of IA1 secretome is readily accomplished using an allogeneic MLR. Potential source materials include PBMC donors (A and B), autologous cells (dotted arrow), lymphocytic cell lines, or leukoreduction filters from blood collection bags. The secretome is collected at day 5 for processing into IA1 (
The potential utility and use of IA1 in Adoptive Cell Therapy (ACT) is diagrammatically shown in Figure 6. The bioproduction of IA1 is both inexpensive and rapid (5 days) and the IA1 can be stored for long periods (several months frozen in the laboratory; data not shown). Moreover, neither IA1 or TA1 production actually requires donor specific tissues (PBMC) making these secretome-based therapeutics an “off-the-shelf” immune adjuvant. Most importantly for patient care,
The immunomodulation of the endogenous immune system has become a major focus in treating a broad range of clinical conditions ranging from tissue/organ engraftment, autoimmune disease and cancer therapy. While significant clinical advancements have been made in immunotherapy, substantial challenges remain. One target of interest is the biologic/clinical desire to induce a persistent systemic immunological reset that could reduce both the need for chronic therapy and reduce the potential toxicities associated with current immunomodulatory approaches. Recent studies have demonstrated that miRNA are key regulators of cellular processes involved in both tolerogenic and proinflammatory immune responses and mediate immune cell proliferation and differentiation. Using an alloresponse bioreactor secretome system we have demonstrated that miRNA-based therapeutics can be reproducibly manufactured that can systemically reorient the immune system to either a tolerogenic or proinflammatory state by simultaneously modulating both regulatory and effector T cell subsets thus skewing the Treg:Teff cell ratio to favor tolerance or inflammation. The tolerogenic TA1 therapeutic is derived from polymer-mediated immunocamouflage of the alloresponse reaction while the inflammatory IA1 preparation is derived from the alloresponse itself. The secretomes from these reactions are processed to maintain the miRNA within the secretome. In contrast to most miRNA therapeutic tactics, our approach has been to mimic the “complex pattern of miRNA expression” seen in protolerogenic or proinflammatory states. This “complex” approach was predicated by the inherent nature of miRNA bioregulation in that there is a low probability that altered expression of a single, or even a few, miRNA would exert a potent and definitive biological response. As shown, this approach successfully results in significant and, in mice, systemic and persistent changes to the immune system. The tolerogenic TA1 proved useful in reducing the onset and incidence of autoimmune diabetes in the NOD mouse while the proinflammatory IA1 therapeutic greatly enhanced the efficacy of human T cells to recognize and kill cancer cells without inducing the systemic inflammatory response seen with mitogens or monoclonal antibody (e.g., anti-CD3/CD28) therapies. Moreover, this approach can simultaneously modulate both regulatory and effect T cell subtype. The successful development of this miRNA-immunomodulatory approach may prove useful in facilitating organ engraftment, treating autoimmune disease and enhancing the endogenous anticancer response.
\nThis work was supported by grants from the Canadian Institutes of Health Research (Grant no. 123317; MDS), Canadian Blood Services (MDS) and Health Canada (MDS). The views expressed herein do not necessarily represent the view of the federal government of Canada. We thank the Canada Foundation for Innovation and the Michael Smith Foundation for Health Research for infrastructure funding at the University of British Columbia Centre for Blood Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
\nCanadian Blood Services is pursuing patents related to the production and utilization of the described acellular immunomodulatory agents. Canadian Blood Services, a not-for-profit organization responsible for collecting, manufacturing and distributing blood and blood products to all Canadians (except Quebec), is the assignee for relevant patents. MDS, DW and WMT are inventors on these patents. XY has no conflicts of interest beyond being paid by Canadian Blood Services.
Dietary polyphenols have interesting spectrum biological properties, including radical scavenging activity [1]. Anthocyanins are one of essential classes in the polyphenol family [2]. These are the plant pigments responsible for the bright red-orange to blue-violet colors of many fruits and vegetables [3]. Since these are natural colored compounds, many pieces of literature come with their application as coloring or coloring material, especially in the food industry [4, 5, 6, 7, 8, 9]. Naturally, these compounds found in glycoside form can collectively be called anthocyanins. At the same time, their aglycon forms are commonly called anthocyanidins [10]. The common aglycon forms are cyanidin, delphinidin, peonidin, petunidin, malvidin, and pelargonidin. The color pigments are most abundant in berries like black currants, elderberries, blueberries, strawberries, etc., red and purple grapes, red wine, sweet cherries, eggplants, black plums, and red cabbage, etc.
The basic structure of anthocyanins is the flavylium cationic ring in which oxygen carries a positive charge. The positively charged species exhibit several equilibrium structures due to different transformations like proton transfer, isomerization, and tautomerization under various pH conditions [11]. All molecules of a group of anthocyanins have their absorption range in the visible spectrum due to effective π conjugation within the molecule. These are the largest group of water-soluble pigments in the plant kingdom. Experimental and theoretical reports show an exponential increase in findings related to its properties, color, co-pigmentation, pH effect, antiradical properties, etc. [12, 13, 14, 15, 16, 17].
The delphinidin (
Structures of compound considered for the study.
Gallic acid (3,4,5- Trihydroxy benzoic acid) and its derivatives have been found in various natural sources like nuts, tea, grapes, gallnut, oak bark, etc. Biological studies show that gallic acid has variable effects, including antiviral, antioxidant, and anticancer activities [18, 19, 20]. Due to its potent antioxidant activity against free radicals is used in food, cosmetics, and pharmaceutical industries and as a source material for ink and paints [21]. GA possesses the most robust antiradical property than Trolox, and hence, in many cases, this molecule is widely used as a reference compound for antioxidant studies. Green tea contains the highest concentration of GA-based compounds responsible for the plant’s antioxidant capacity [22, 23].
Once the compound possesses potent antioxidant activity and a specific range of absorbance in the visible spectrum can be effectively used for various purposes, especially in the food industry, they seek less hazardous, highly protective materials for coloring purposes. Hence these are colored compounds, and this work concludes the antiradical property of delphinidin and its derivatives with the help of computational methods.
The present work has utilized a theoretical approach to study the structure and properties of all compounds under consideration. The molecular forms of
The present work was carried out using density functional theory (DFT) because it is based on electron density, and antioxidant activity is mainly influenced by electron density [28, 29, 30, 31, 32]. In DFT calculation, 6–311+ G (d, p) basis set with B3LYP (Becke’s exchange functional in conjunction [33] with Lee-Yang–Parr [34]) correlational functional has been used for geometry optimization, computation of harmonic vibrational frequencies, BDE, IP, PDE, PA and ETE calculations. To obtain antioxidant parameters BDE, IP, PDE, PA, and ETE, the geometry optimization of neutral, radicals, anions, and radical cation structures of all the studied molecules are conducted in the ground state both in the gas phase and aqueous phases. Solvent effects on the calculated systems were investigated with the self-consistent reaction field (SCRF) method via the integral equation formalism polarized continuum model (IEF-PCM).
Frontier molecular orbital theory is an application of MO theory that explains HOMO/LUMO interactions. HOMO is the highest occupied molecular orbital, and LUMO is the lowest unoccupied molecular orbital. Frontier molecular orbital analysis is fundamental because HOMO or LUMO energies and bandgap energies are the key factors that drive the antiradical property of molecules. Since HOMO is in the highest energy state, so easier to remove an electron from this orbital. So in a chemical reaction or bond formation, HOMO is donating electrons, or it acts as a Lewis base or undergoes oxidation. LUMO is the lower-lying orbital; it is empty, so it is easier for LUMO to accept electrons into its orbital or acts as a Lewis acid or undergoes reduction. Reactivity becomes lower when the molecule has a higher bandgap. The distribution of HOMO orbitals and energies is calculated using the DFT-B3LYP/6–311 + G (d, p) level of theory from the optimized structures of
Several mechanisms have theoretically explained the radical scavenging mechanism of phenolic compounds. The widely used mechanisms are hydrogen atom transfer (HAT) mechanism (Eq. (1)), single-electron transfer followed by proton transfer (SET-PT) mechanism (Eqs. (2)–(4)), and sequential proton loss electron transfer (SPLET) mechanism (Eqs. (5) and (6)) [35, 36, 37, 38, 39, 40]. These mechanisms have been briefly addressed here.
HAT (hydrogen atom transfer) mechanism
By transferring the hydrogen atom of the –OH group to the radical species, the antioxidant (ArOH) scavenges the free radical (X
SET (single electron transfer) mechanism
Here, the reactive free radicals are neutralized by transferring electrons to them, resulting in anions. The most reactive hydroxyl group in antioxidant compounds provides these electrons and finally becomes a radical cation. The descriptor associated with this mechanism is AIP (adiabatic ionization potential).
SET-PT (single electron transfer followed by proton transfer) mechanism
The first process involves an electron transfer from the antioxidant (Eq. (2)), and the second step consists of a proton transfer from the radical cation (Eq. (4)) generated in the first step. Proton dissociation enthalpy is the descriptor connected with the second phase (PDE).
SPLET (sequential proton loss electron transfer) mechanism
This is also a two-stage mechanism, with the dissociation of the antioxidant into phenoxide anion and proton as the first step (Eq. (5)). The first-step phenoxide anion then interacts with free radicals at a certain pH (Eq. (6)); the compounds generated are similar to those developed in the HAT mechanism. Proton affinity (PA) is the regulating descriptor for the first stage, and electron transfer enthalpy is the driving descriptor for the second stage (ETE).
The Eqs. (7)–(11) are used for analyzing the type of mechanism involved by the compound
Thus, in the present study, BDE, IP, PDE, PA, and ETE values were used as the primary molecular descriptors to elucidate the radical scavenging activity of the investigated compounds. The enthalpies of hydrogen radicals in the water and gas phase are calculated by G09 software using the DFT/B3LYP/6–311 + G (d, p) level of theory. The enthalpies of the electron (e−) and proton in the gas phase are taken from the commonly accepted values 0.00236 Hartree for proton and 0.00120 Hartree for electron. In contrast, for water, these corresponding values are calculated with the help of the DFT/B3LYP/6–311 + G(d, p) level of theory. The enthalpies of electron and proton in solvent water were computed using the same level of theory with methodology suggested by Markovic et al. (Eqs. (12) and (13)) [41, 42].
Where Ssol is the solvent molecule solvated by the same kind of molecule,
To elucidate the reactivity of the compounds towards free radicals, the conformational and geometrical features of compounds are very significant. The structures of
The lowest energy conformer obtained after the last scan was then subjected to geometry optimization, and B3LYP/6–311 + G(d, p) level of theory with a correlation coefficient of 0.89281 are selected for the study. The crystal structure data of cyanidin are considered for experimental validation of results obtained from the computational analysis since these are not available for delphinidin molecules and tabulated in Table 1 [43]. But the basic structural unit of
Bond | Experimental bond length (AO) | Theoretical | |||
---|---|---|---|---|---|
B3LYP 6–31 G (d) | B3LYP 6–31+ G (d, p) | B3LYP 6–311+ G (d, p) | B3LYP 6–311++ G (d, p) | ||
C 2-C1’ | 1.453 | 1.44380 | 1.44380 | 1.44173 | 1.44176 |
C10-C5 | 1.432 | 1.43007 | 1.43002 | 1.42770 | 1.42769 |
C1’-C2’ | 1.409 | 1.41731 | 1.41735 | 1.41422 | 1.41423 |
C6 - C7 | 1.413 | 1.41392 | 1.41390 | 1.41107 | 1.41106 |
C2 –C3 | 1.396 | 1.42173 | 1.42177 | 1.41918 | 1.41920 |
C3’-C4’ | 1.400 | 1.40858 | 1.40863 | 1.40599 | 1.40602 |
C4–C10 | 1.382 | 1.40615 | 1.40614 | 1.40553 | 1.40351 |
C6’-C1’ | 1.404 | 1.41542 | 1.41539 | 1.41216 | 1.41218 |
C9–C10 | 1.408 | 1.41223 | 1.41222 | 1.40886 | 1.40888 |
C7 – C8 | 1.387 | 1.39860 | 1.39860 | 1.39517 | 1.39517 |
Comparison of bond length with experimental values in different basis sets.
The obtained optimized structures of the compounds using B3LYP /6–311 + G(d, p) level of theory (delphinidin (1a), delphinidin-3-O-glucoside (1b), delphinidin-3-O-gallate (1c), delphinidin-4’-O-glucoside (1d), and delphinidin-4’-O-gallate (1e)).
Frontier molecular orbital analysis gives a detailed account of HOMO/LUMO interactions in the molecule. It is very useful in describing optical and electronic properties as well as the reactivity of a molecule. The HOMO value of the molecule is strongly influenced by radical scavenging activity. The higher the HOMO energy, the easier the electron is being excited and acts substantial donor of the electron. The chemical reactivity of the molecule can be described by knowing the HOMO–LUMO gap. Like that, the distribution of orbitals among the molecule reveals probable sites for the attack of free radicals. The HOMO distribution among molecule
The increasing order of bandgap energies in the gas phase at
Gas (eV) | Water(eV) | ||||||
---|---|---|---|---|---|---|---|
EHOMO | ELUMO | Band gap (ΔEgas) | EHOMO | ELUMO | Band gap (ΔEwater) | ΔEwater - ΔEgas | |
−9.27 | −6.63 | 2.64 | −6.46 | −3.65 | 2.81 | 0.17 | |
−8.80 | −6.10 | 2.70 | −6.47 | −3.57 | 2.9 | 0.2 | |
−8.91 | −6.36 | 2.55 | −6.62 | −3.69 | 2.93 | 0.38 | |
−9.10 | −6.56 | 2.54 | −6.71 | −3.74 | 2.97 | 0.43 | |
−8.60 | −6.55 | 2.05 | −6.64 | −3.78 | 2.86 | 0.81 |
FMO analysis of studied compounds in gas and water media at B3LYP/6–311 + G(d, p) level of theory.
FMO of
The antioxidant activities of the compounds are studied using the antioxidant mechanism described in Section 2.2.2. The BDE, IP, PA, PDE, and ETE values obtained from the corresponding mechanism are used to analyze the activity of compounds. Among the five parameters, one with the lowest value and the corresponding mechanism is followed by the compound. The parameters of antioxidant activities are represented in the gas phase and aqueous phase, respectively, in Tables 3 and 4.
1a | |||||
---|---|---|---|---|---|
BDE | IP | PDE | PA | ETE | |
3-OH | 81.56 | 242.73 | 153.33 | 245.94 | 150.12 |
5-OH | 88.99 | 160.77 | 246.47 | 156.03 | |
7-OH | 90.99 | 162.76 | 246.89 | 158.61 | |
3’-OH | 92.17 | 163.94 | 269.82 | 136.85 | |
4’-OH | 83.19 | 154.96 | 246.50 | 151.19 | |
5’-OH | 84.51 | 156.28 | 258.11 | 140.90 | |
5-OH | 87.97 | 228.97 | 173.50 | 227.47 | 175.01 |
7-OH | 90.53 | 176.05 | 253.17 | 151.86 | |
3’-OH | 90.25 | 175.78 | 278.86 | 125.89 | |
4’-OH | 82.15 | 167.69 | 256.71 | 139.96 | |
5’-OH | 89.41 | 174.94 | 267.13 | 136.79 | |
5-OH | 89.80 | 174.95 | 229.35 | 249.97 | 154.33 |
7-OH | 91.99 | 231.55 | 248.88 | 157.61 | |
3’-OH | 91.49 | 231.04 | 273.24 | 132.76 | |
4’-OH | 83.12 | 222.67 | 249.81 | 147.81 | |
5’-OH | 84.18 | 223.73 | 260.56 | 138.12 | |
5”-OH | 89.16 | 228.71 | 256.98 | 166.80 | |
6”-OH | 83.09 | 222.64 | 266.51 | 131.08 | |
7”-OH | 83.60 | 223.15 | 270.08 | 128.02 | |
5-OH | 88.91 | 232.11 | 171.30 | 248.31 | 155.10 |
7-OH | 91.05 | 173.45 | 248.11 | 157.45 | |
3’-OH | 83.84 | 166.23 | 249.38 | 148.97 | |
3’-OH | 92.94 | 175.34 | 267.53 | 139.92 | |
5’-OH | 91.55 | 173.95 | 269.50 | 136.56 | |
3-OH | 88.81 | 227.74 | 175.57 | 248.48 | 154.83 |
5-OH | 90.88 | 177.65 | 248.24 | 157.15 | |
7-OH | 90.30 | 177.06 | 270.75 | 134.06 | |
3’-OH | 83.90 | 170.66 | 249.64 | 148.76 | |
5’-OH | 91.04 | 177.80 | 272.33 | 133.22 | |
5”-OH | 89.60 | 176.36 | 282.61 | 121.50 | |
6”OH | 82.80 | 169.56 | 267.24 | 130.06 | |
7”-OH | 83.22 | 169.98 | 271.13 | 126.59 |
The antioxidant mechanism study of compounds
1a | |||||
---|---|---|---|---|---|
BDE | IP | PDE | PA | ETE | |
3-OH | 80.87 | 201.36 | 13.64 | 37.88 | 177.12 |
5-OH | 85.05 | 17.83 | 37.43 | 181.76 | |
7-OH | 86.13 | 18.91 | 36.98 | 183.28 | |
3’-OH | 85.33 | 18.10 | 49.18 | 170.29 | |
4’-OH | 77.52 | 10.30 | 35.27 | 176.39 | |
5’-OH | 81.79 | 14.57 | 43.17 | 172.76 | |
5-OH | 86.30 | 200.34 | 20.10 | 37.08 | 183.36 |
7-OH | 87.77 | 21.57 | 36.41 | 185.49 | |
3’-OH | 84.02 | 17.82 | 50.14 | 168.02 | |
4’-OH | 77.42 | 11.22 | 37.11 | 174.45 | |
5’-OH | 86.08 | 19.87 | 46.66 | 173.55 | |
5-OH | 87.22 | 206.11 | 13.83 | 35.85 | 185.50 |
7-OH | 89.19 | 15.88 | 35.10 | 188.22 | |
3’-OH | 85.39 | 15.16 | 42.42 | 177.10 | |
4’-OH | 78.65 | 11.53 | 34.39 | 178.38 | |
5’-OH | 82.09 | 14.16 | 42.42 | 173.80 | |
5”-OH | 84.08 | 13.04 | 51.41 | 166.81 | |
6”-OH | 78.66 | 7.47 | 42.22 | 170.58 | |
7”-OH | 81.34 | 10.13 | 45.07 | 170.40 | |
5-OH | 85.72 | 204.22 | 15.64 | 36.36 | 155.10 |
7-OH | 87.71 | 17.62 | 35.80 | 157.45 | |
3’-OH | 81.34 | 11.26 | 35.21 | 148.97 | |
3’-OH | 88.12 | 18.04 | 49.73 | 139.92 | |
5’-OH | 88.99 | 18.91 | 42.69 | 136.56 | |
3-OH | 85.82 | 206.11 | 13.83 | 36.73 | 183.22 |
5-OH | 87.86 | 15.88 | 36.07 | 185.93 | |
7-OH | 87.14 | 15.16 | 47.37 | 173.90 | |
3’-OH | 83.51 | 11.53 | 36.78 | 180.86 | |
5’-OH | 86.15 | 14.16 | 46.43 | 173.85 | |
5”-OH | 85.02 | 13.04 | 41.67 | 167.55 | |
6”-OH | 79.45 | 7.47 | 45.24 | 171.92 | |
7”-OH | 82.11 | 10.13 | 51.60 | 171.00 |
The antioxidant mechanism study of compounds
Bond | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|
5 OH | 7 OH | 3 OH | 3’ OH | 4’ OH | 5’ OH | 5” OH | 6”OH | 7”OH | ||
SD | 0.027 | 0.022 | 0.025 | 0.061 | 0.018 | 0.032 | — | — | — | |
DI | 1.889 | 1.911 | 1.855 | 1.831 | 1.937 | 1.825 | — | — | — | |
SD | 0.027 | 0.022 | — | 0.033 | 0.019 | 0.032 | — | — | — | |
DI | 1.885 | 1.903 | — | 1.843 | 1.923 | 1.819 | — | — | — | |
SD | 0.027 | 0.023 | — | 0.032 | 0.020 | 0.032 | 0.0317 | 0.025 | 0.032 | |
DI | 1.888 | 1.901 | — | 1.845 | 1.923 | 1.825 | 1.847 | 1.884 | 1.832 | |
SD | 0.026 | 0.022 | 0.026 | 0.032 | — | 0.033 | — | — | — | |
DI | 1.892 | 1.910 | 1.860 | 1.829 | — | 1.813 | — | — | — | |
SD | 0.026 | 0.022 | 0.026 | 0.031 | — | 0.032 | 0.031 | 0.025 | 0.032 | |
DI | 1.891 | 1.910 | 1.861 | 1.839 | — | 1.824 | 1.850 | 1.888 | 1.830 |
The SD distribution for the O-radical and delocalization index (DI) of C-O bond computed for
In the gas phase, all studied molecules follow the HAT mechanism because of its lower BDE values. Hence, all compounds tending to form radicals by donating hydrogen atoms in respective positions are higher in the gas phase. In the case of
The glucose substituted derivatives of
As from Table 3 the antioxidant activity of compound
To explain the differences in BDE and consequently the differences in the reactivity of OH sites, the spin density distribution of radicals was calculated and presented in Table 5. The stability of radicals formed can be explained with the help of SD values; more delocalized SD means to be more stable is the radical formed. Moreover, the delocalization index is also a supporting parameter for explaining the stability of radicals created. The more stable the radical formed from an -OH bond, the more the corresponding C-O bond will be the delocalization index. The DI of C-O bonds in each radical site are calculated using Fuzzy atomic space analysis [44, 45, 46]. The SD contours of
Electronic spin density distributions and optimized structures of
In the SET-PT mechanism, the parameters involved are IP and PDE. In all derivative IP is found to be higher in both media. The PDE values are higher in the gas phase but lower in the water medium. But the lower value in the water medium cannot be used for final judgment about contribution in antioxidant activity. In the SET-PT mechanism, the PDE comes from the second step of this mechanism, whereas the first step is the IP. Since all cases have the highest values for IP in the water medium, they have to overcome this large energy barrier of IP to reach the second step.
In water, the parameters BDE, IP, and ETE having higher enthalpy than PA and are represented in Table 4. Hence SPLET is the mechanism followed by each molecule in the water medium. In the SPLET mechanism, the heterolytic bond cleavage of the phenolic hydroxyl group is considered, and the neutral molecule is split into an anion and a proton. The numerical parameter associated with this step is PA. The anion produced donates one electron to free radical species, and the free radical receives one electron and forms an anion. The anion of free radical react with proton forms a neutral compound by leaving the anion starting compound as radical. The numerical parameter associated with this step is ETE. The values of PA are found to be lower than BDE and other parameters in all studied cases when the solvent was aqueous. In the case of
In the case of
A theoretical study on the antioxidant activity of natural pigment delphinidin and its derivative has been evaluated. The antiradical properties of all studied molecules are finalized through an antioxidant mechanism. All compounds follow the HAT mechanism in the gas phase and the SPLET mechanism in the aqueous medium. In gas-phase gallic acid, substituted compounds possess considerable enhancement in activities by providing more hydroxyl groups of near BDEs. In the water medium,
The author, Sumayya Pottachola, expresses sincere gratitude to UGC-MANF for financial support and the central sophisticated instrumentation facility (CSIF) of the University of Calicut for Gaussian 09 software support.
The authors declare no conflict of interest.
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In this chapter, I will discuss the nature of protein aggregation of signature proteins and the status of protein proteolytic systems such as proteasome and autophagosome in Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Huntington’s disease, and prion disease under the light of recent studies including our new findings.",book:{id:"7480",slug:"neurochemical-basis-of-brain-function-and-dysfunction",title:"Neurochemical Basis of Brain Function and Dysfunction",fullTitle:"Neurochemical Basis of Brain Function and Dysfunction"},signatures:"Abdulbaki Agbas",authors:[{id:"250609",title:"Prof.",name:"Abdulbaki",middleName:null,surname:"Agbas",slug:"abdulbaki-agbas",fullName:"Abdulbaki Agbas"}]},{id:"55884",doi:"10.5772/intechopen.69111",title:"Production and Function of Serotonin in Cardiac Cells",slug:"production-and-function-of-serotonin-in-cardiac-cells",totalDownloads:1550,totalCrossrefCites:4,totalDimensionsCites:10,abstract:"Serotonin [5-hydroxy-tryptamine (5-HT)] exerts a number of effects in the mammalian heart: increase in heart rate, increase in force of contraction, fibrosis of cardiac valves, coronary constriction, arrhythmias and thrombosis. These effects are, in part, mediated by 5-HT-receptors, in part, directly by 5-HT action on intracellular proteins. In the beginning, 5-HT was thought to be only produced in the gut and then transported into the heart via platelets, because platelets can take up 5-HT in the gut and enter the capillaries and thus the mammalian heart. 5-HT is to a large extent metabolized in the liver and excreted via the urine. Here, we will also overview data that argue for additional pathways, namely production and degradation of 5-HT in the cells of the heart itself.",book:{id:"5780",slug:"serotonin-a-chemical-messenger-between-all-types-of-living-cells",title:"Serotonin",fullTitle:"Serotonin - A Chemical Messenger Between All Types of Living Cells"},signatures:"Joachim Neumann, Britt Hofmann and Ulrich Gergs",authors:[{id:"198376",title:"Prof.",name:"Joachim",middleName:null,surname:"Neumann",slug:"joachim-neumann",fullName:"Joachim Neumann"},{id:"205353",title:"Dr.",name:"Britt",middleName:null,surname:"Hofmann",slug:"britt-hofmann",fullName:"Britt Hofmann"},{id:"205354",title:"Dr.",name:"Ulrich",middleName:null,surname:"Gergs",slug:"ulrich-gergs",fullName:"Ulrich Gergs"}]}],mostDownloadedChaptersLast30Days:[{id:"57103",title:"GABA and Glutamate: Their Transmitter Role in the CNS and Pancreatic Islets",slug:"gaba-and-glutamate-their-transmitter-role-in-the-cns-and-pancreatic-islets",totalDownloads:3471,totalCrossrefCites:3,totalDimensionsCites:9,abstract:"Glutamate and gamma-aminobutyric acid (GABA) are the major neurotransmitters in the mammalian brain. Inhibitory GABA and excitatory glutamate work together to control many processes, including the brain’s overall level of excitation. The contributions of GABA and glutamate in extra-neuronal signaling are by far less widely recognized. In this chapter, we first discuss the role of both neurotransmitters during development, emphasizing the importance of the shift from excitatory to inhibitory GABAergic neurotransmission. The second part summarizes the biosynthesis and role of GABA and glutamate in neurotransmission in the mature brain, and major neurological disorders associated with glutamate and GABA receptors and GABA release mechanisms. The final part focuses on extra-neuronal glutamatergic and GABAergic signaling in pancreatic islets of Langerhans, and possible associations with type 1 diabetes mellitus.",book:{id:"6237",slug:"gaba-and-glutamate-new-developments-in-neurotransmission-research",title:"GABA And Glutamate",fullTitle:"GABA And Glutamate - New Developments In Neurotransmission Research"},signatures:"Christiane S. Hampe, Hiroshi Mitoma and Mario Manto",authors:[{id:"210220",title:"Prof.",name:"Christiane",middleName:null,surname:"Hampe",slug:"christiane-hampe",fullName:"Christiane Hampe"},{id:"210485",title:"Prof.",name:"Mario",middleName:null,surname:"Manto",slug:"mario-manto",fullName:"Mario Manto"},{id:"210486",title:"Prof.",name:"Hiroshi",middleName:null,surname:"Mitoma",slug:"hiroshi-mitoma",fullName:"Hiroshi Mitoma"}]},{id:"58817",title:"Clinical Application of MR Spectroscopy in Identifying Biochemical Composition of the Intracranial Pathologies",slug:"clinical-application-of-mr-spectroscopy-in-identifying-biochemical-composition-of-the-intracranial-p",totalDownloads:2020,totalCrossrefCites:0,totalDimensionsCites:5,abstract:"Magnetic resonance spectroscopy (MRS) provides useful information regarding metabolic composition in the tissues, and advanced spectroscopic methods are used to quantify markers of tumor membrane turnover and proliferation (e.g., choline (Cho)), energy homoeostasis (e.g., creatine (Cr)), intact glioneuronal structures (e.g., N-acetylaspartate (NAA)), and necrosis (e.g., lactate (Lac) or lipids). Results are usually expressed as metabolite ratios rather than absolute metabolite concentrations. Because glial tumors have some specific metabolic characteristics that differ according to the grade of tumor, there is a potential for MR spectroscopy to increase the sensitivity of routinely used diagnostic imaging. MRS also has many diagnostic applications in neurosciences to support the diagnosis in conditions like demyelination, infections, and dementia and in postradiotherapy cases. Biochemical changes in the metabolism of tumor cells related to malignant transformation are reflected in changes of particular metabolite concentration in the tumor tissue. Our prospective study aimed to analyze the usefulness of proton MR spectroscopy in grading of glioma and to correlate various metabolite ratios like choline/creatine, choline/N-acetylaspartate, N-acetylaspartate/creatine, and lactate/creatine with the histopathological grades of glioma.",book:{id:"6237",slug:"gaba-and-glutamate-new-developments-in-neurotransmission-research",title:"GABA And Glutamate",fullTitle:"GABA And Glutamate - New Developments In Neurotransmission Research"},signatures:"B C Hamsini, Bhavana Nagabhushana Reddy, Sankar Neelakantan\nand Sunitha Palasamudram Kumaran",authors:[{id:"211054",title:"Dr.",name:"Sunitha",middleName:null,surname:"P Kumaran",slug:"sunitha-p-kumaran",fullName:"Sunitha P Kumaran"},{id:"221485",title:"Dr.",name:"Sankar",middleName:null,surname:"Neelakantan",slug:"sankar-neelakantan",fullName:"Sankar Neelakantan"},{id:"398223",title:"Dr.",name:"B C",middleName:null,surname:"Hamsini",slug:"b-c-hamsini",fullName:"B C Hamsini"},{id:"398224",title:"Dr.",name:"Bhavana",middleName:null,surname:"Nagabhushana Reddy",slug:"bhavana-nagabhushana-reddy",fullName:"Bhavana Nagabhushana Reddy"}]},{id:"62431",title:"The United Chemicals of Cannabis: Beneficial Effects of Cannabis Phytochemicals on the Brain and Cognition",slug:"the-united-chemicals-of-cannabis-beneficial-effects-of-cannabis-phytochemicals-on-the-brain-and-cogn",totalDownloads:1768,totalCrossrefCites:3,totalDimensionsCites:10,abstract:"‘Medicinal cannabis’ can be defined as pharmaceutical grade cannabis-based products used for the treatment of illness. Beneficial treatment effects of cannabidiol (CBD), a major non-intoxicating compound isolated from the cannabis plant, have been shown in multiple states of cognitive impairment, including neurodegenerative (Alzheimer’s, Huntington’s and Parkinson’s disease), neuroinflammatory (sepsis-induced encephalopathy) and neurological disorders (ischemic brain injury). CBD can also treat some of the symptoms of schizophrenia, including cognitive deficits (impairments in learning and memory), which is a major symptom domain of the illness that is largely resistant to existing antipsychotic medications. However, empirical evidence suggests the presence of an ‘entourage effect’ in cannabis; that is, observations that medicinal cannabis seems to work better in some instances when administered as a whole-plant extract. While scientific evidence highlights isolated CBD as a strong candidate for treating cognitive impairment, the entourage effect suggests that the co-operation of other plant molecules could provide further benefits. This chapter explores the scientific evidence surrounding the benefits of CBD and other specific key phytochemicals in cannabis: linalool, α-pinene, β-caryophyllene, flavonoids and anthocyanin, on brain health and cognition.",book:{id:"7040",slug:"recent-advances-in-cannabinoid-research",title:"Recent Advances in Cannabinoid Research",fullTitle:"Recent Advances in Cannabinoid Research"},signatures:"Katrina Weston-Green",authors:null},{id:"68776",title:"Introductory Chapter: The Chemical Basis of Neural Function and Dysfunction",slug:"introductory-chapter-the-chemical-basis-of-neural-function-and-dysfunction",totalDownloads:1093,totalCrossrefCites:1,totalDimensionsCites:2,abstract:null,book:{id:"7480",slug:"neurochemical-basis-of-brain-function-and-dysfunction",title:"Neurochemical Basis of Brain Function and Dysfunction",fullTitle:"Neurochemical Basis of Brain Function and Dysfunction"},signatures:"Thomas Heinbockel and Antonei B. Csoka",authors:[{id:"70569",title:"Dr.",name:"Thomas",middleName:null,surname:"Heinbockel",slug:"thomas-heinbockel",fullName:"Thomas Heinbockel"},{id:"245650",title:"Dr.",name:"Antonei B.",middleName:null,surname:"Csoka",slug:"antonei-b.-csoka",fullName:"Antonei B. Csoka"}]},{id:"68712",title:"Synaptic Transmission and Amino Acid Neurotransmitters",slug:"synaptic-transmission-and-amino-acid-neurotransmitters",totalDownloads:1337,totalCrossrefCites:5,totalDimensionsCites:6,abstract:"Amino acids are the most abundant neurotransmitters in the brain. Neurotransmitters are synthesized and stored in presynaptic terminals, released from terminals upon stimulation with specific receptors on the postsynaptic cells. Chemical and electrical synapses are specialized biological structures found in the nervous system; they connect neurons together and transmit signals across the neurons. The process of synaptic transmission generates or inhibits electrical impulses in a network of neurons for the processing of information. Glutamate is the primary excitatory neurotransmitter in the brain, while GABA is the principal inhibitory neurotransmitter. The balance of glutamatergic and GABAergic tone is crucial to normal neurologic function. Through synaptic transmission, this information is communicated from the presynaptic cell to the postsynaptic cell. Amino acid neurotransmitters primarily glutamic acid, GABA, aspartic acid, and glycine are single amino acid residues released from presynaptic nerve terminals in response to an action potential and cross the synaptic cleft to bind with specific receptor on the postsynaptic membrane. The integral role of amino acid neurotransmitters is important on the normal functioning of the brain. The presynaptic and postsynaptic events in chemical synapses are subject to use dependent and highly regulated as per the changes in synaptic neurotransmitter release and function.",book:{id:"7480",slug:"neurochemical-basis-of-brain-function-and-dysfunction",title:"Neurochemical Basis of Brain Function and Dysfunction",fullTitle:"Neurochemical Basis of Brain Function and Dysfunction"},signatures:"Manorama Patri",authors:[{id:"196763",title:"Dr.",name:"Manorama",middleName:null,surname:"Patri",slug:"manorama-patri",fullName:"Manorama Patri"}]}],onlineFirstChaptersFilter:{topicId:"212",limit:6,offset:0},onlineFirstChaptersCollection:[],onlineFirstChaptersTotal:0},preDownload:{success:null,errors:{}},subscriptionForm:{success:null,errors:{}},aboutIntechopen:{},privacyPolicy:{},peerReviewing:{},howOpenAccessPublishingWithIntechopenWorks:{},sponsorshipBooks:{sponsorshipBooks:[],offset:8,limit:8,total:0},allSeries:{pteSeriesList:[{id:"14",title:"Artificial Intelligence",numberOfPublishedBooks:8,numberOfPublishedChapters:87,numberOfOpenTopics:6,numberOfUpcomingTopics:0,issn:"2633-1403",doi:"10.5772/intechopen.79920",isOpenForSubmission:!0},{id:"7",title:"Biomedical Engineering",numberOfPublishedBooks:12,numberOfPublishedChapters:98,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2631-5343",doi:"10.5772/intechopen.71985",isOpenForSubmission:!0}],lsSeriesList:[{id:"11",title:"Biochemistry",numberOfPublishedBooks:27,numberOfPublishedChapters:286,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2632-0983",doi:"10.5772/intechopen.72877",isOpenForSubmission:!0},{id:"25",title:"Environmental Sciences",numberOfPublishedBooks:1,numberOfPublishedChapters:9,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2754-6713",doi:"10.5772/intechopen.100362",isOpenForSubmission:!0},{id:"10",title:"Physiology",numberOfPublishedBooks:11,numberOfPublishedChapters:139,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2631-8261",doi:"10.5772/intechopen.72796",isOpenForSubmission:!0}],hsSeriesList:[{id:"3",title:"Dentistry",numberOfPublishedBooks:8,numberOfPublishedChapters:129,numberOfOpenTopics:0,numberOfUpcomingTopics:2,issn:"2631-6218",doi:"10.5772/intechopen.71199",isOpenForSubmission:!1},{id:"6",title:"Infectious Diseases",numberOfPublishedBooks:13,numberOfPublishedChapters:106,numberOfOpenTopics:3,numberOfUpcomingTopics:1,issn:"2631-6188",doi:"10.5772/intechopen.71852",isOpenForSubmission:!0},{id:"13",title:"Veterinary Medicine and Science",numberOfPublishedBooks:9,numberOfPublishedChapters:101,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2632-0517",doi:"10.5772/intechopen.73681",isOpenForSubmission:!0}],sshSeriesList:[{id:"22",title:"Business, Management and Economics",numberOfPublishedBooks:1,numberOfPublishedChapters:11,numberOfOpenTopics:2,numberOfUpcomingTopics:1,issn:null,doi:"10.5772/intechopen.100359",isOpenForSubmission:!0},{id:"23",title:"Education and Human Development",numberOfPublishedBooks:0,numberOfPublishedChapters:0,numberOfOpenTopics:2,numberOfUpcomingTopics:0,issn:null,doi:"10.5772/intechopen.100360",isOpenForSubmission:!1},{id:"24",title:"Sustainable Development",numberOfPublishedBooks:0,numberOfPublishedChapters:9,numberOfOpenTopics:4,numberOfUpcomingTopics:1,issn:null,doi:"10.5772/intechopen.100361",isOpenForSubmission:!0}],testimonialsList:[{id:"6",text:"It is great to work with the IntechOpen to produce a worthwhile collection of research that also becomes a great educational resource and guide for future research endeavors.",author:{id:"259298",name:"Edward",surname:"Narayan",institutionString:null,profilePictureURL:"https://mts.intechopen.com/storage/users/259298/images/system/259298.jpeg",slug:"edward-narayan",institution:{id:"3",name:"University of Queensland",country:{id:null,name:"Australia"}}}},{id:"13",text:"The collaboration with and support of the technical staff of IntechOpen is fantastic. The whole process of submitting an article and editing of the submitted article goes extremely smooth and fast, the number of reads and downloads of chapters is high, and the contributions are also frequently cited.",author:{id:"55578",name:"Antonio",surname:"Jurado-Navas",institutionString:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRisIQAS/Profile_Picture_1626166543950",slug:"antonio-jurado-navas",institution:{id:"720",name:"University of Malaga",country:{id:null,name:"Spain"}}}}]},series:{item:{id:"22",title:"Business, Management and Economics",doi:"10.5772/intechopen.100359",issn:null,scope:"\r\n\tThis series will provide a comprehensive overview of recent research trends in business and management, economics, and marketing. 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",coverUrl:"https://cdn.intechopen.com/series/covers/22.jpg",latestPublicationDate:"May 18th, 2022",hasOnlineFirst:!0,numberOfPublishedBooks:1,editor:{id:"356540",title:"Prof.",name:"Taufiq",middleName:null,surname:"Choudhry",slug:"taufiq-choudhry",fullName:"Taufiq Choudhry",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y000036X2hvQAC/Profile_Picture_2022-03-14T08:58:03.jpg",biography:"Prof. Choudhry holds a BSc degree in Economics from the University of Iowa, as well as a Masters and Ph.D. in Applied Economics from Clemson University, USA. In January 2006, he became a Professor of Finance at the University of Southampton Business School. He was previously a Professor of Finance at the University of Bradford Management School. He has over 80 articles published in international finance and economics journals. His research interests and specialties include financial econometrics, financial economics, international economics and finance, housing markets, financial markets, among others.",institutionString:null,institution:{name:"University of Southampton",institutionURL:null,country:{name:"United Kingdom"}}},editorTwo:null,editorThree:null},subseries:{paginationCount:3,paginationItems:[{id:"19",title:"Animal Science",coverUrl:"https://cdn.intechopen.com/series_topics/covers/19.jpg",isOpenForSubmission:!0,annualVolume:11415,editor:{id:"259298",title:"Dr.",name:"Edward",middleName:null,surname:"Narayan",slug:"edward-narayan",fullName:"Edward Narayan",profilePictureURL:"https://mts.intechopen.com/storage/users/259298/images/system/259298.jpeg",biography:"Dr. Edward Narayan graduated with Ph.D. degree in Biology from the University of the South Pacific and pioneered non-invasive reproductive and stress endocrinology tools for amphibians - the novel development and validation of non-invasive enzyme immunoassays for the evaluation of reproductive hormonal cycle and stress hormone responses to environmental stressors. \nDr. Narayan leads the Stress Lab (Comparative Physiology and Endocrinology) at the University of Queensland. A dynamic career research platform which is based on the thematic areas of comparative vertebrate physiology, stress endocrinology, reproductive endocrinology, animal health and welfare, and conservation biology. \nEdward has supervised 40 research students and published over 60 peer reviewed research.",institutionString:null,institution:{name:"University of Queensland",institutionURL:null,country:{name:"Australia"}}},editorTwo:null,editorThree:null},{id:"20",title:"Animal Nutrition",coverUrl:"https://cdn.intechopen.com/series_topics/covers/20.jpg",isOpenForSubmission:!0,annualVolume:11416,editor:{id:"175967",title:"Dr.",name:"Manuel",middleName:null,surname:"Gonzalez Ronquillo",slug:"manuel-gonzalez-ronquillo",fullName:"Manuel Gonzalez Ronquillo",profilePictureURL:"https://mts.intechopen.com/storage/users/175967/images/system/175967.png",biography:"Dr. Manuel González Ronquillo obtained his doctorate degree from the University of Zaragoza, Spain, in 2001. He is a research professor at the Faculty of Veterinary Medicine and Animal Husbandry, Autonomous University of the State of Mexico. He is also a level-2 researcher. He received a Fulbright-Garcia Robles fellowship for a postdoctoral stay at the US Dairy Forage Research Center, Madison, Wisconsin, USA in 2008–2009. He received grants from Alianza del Pacifico for a stay at the University of Magallanes, Chile, in 2014, and from Consejo Nacional de Ciencia y Tecnología (CONACyT) to work in the Food and Agriculture Organization’s Animal Production and Health Division (AGA), Rome, Italy, in 2014–2015. He has collaborated with researchers from different countries and published ninety-eight journal articles. He teaches various degree courses in zootechnics, sheep production, and agricultural sciences and natural resources.\n\nDr. Ronquillo’s research focuses on the evaluation of sustainable animal diets (StAnD), using native resources of the region, decreasing carbon footprint, and applying meta-analysis and mathematical models for a better understanding of animal production.",institutionString:null,institution:{name:"Universidad Autónoma del Estado de México",institutionURL:null,country:{name:"Mexico"}}},editorTwo:null,editorThree:null},{id:"28",title:"Animal Reproductive Biology and Technology",coverUrl:"https://cdn.intechopen.com/series_topics/covers/28.jpg",isOpenForSubmission:!0,annualVolume:11417,editor:{id:"177225",title:"Prof.",name:"Rosa Maria Lino Neto",middleName:null,surname:"Pereira",slug:"rosa-maria-lino-neto-pereira",fullName:"Rosa Maria Lino Neto Pereira",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bS9wkQAC/Profile_Picture_1624519982291",biography:"Rosa Maria Lino Neto Pereira (DVM, MsC, PhD and) is currently a researcher at the Genetic Resources and Biotechnology Unit of the National Institute of Agrarian and Veterinarian Research (INIAV, Portugal). She is the head of the Reproduction and Embryology Laboratories and was lecturer of Reproduction and Reproductive Biotechnologies at Veterinary Medicine Faculty. She has over 25 years of experience working in reproductive biology and biotechnology areas with a special emphasis on embryo and gamete cryopreservation, for research and animal genetic resources conservation, leading research projects with several peer-reviewed papers. Rosa Pereira is member of the ERFP-FAO Ex situ Working Group and of the Management Commission of the Portuguese Animal Germplasm Bank.",institutionString:"The National Institute for Agricultural and Veterinary Research. Portugal",institution:null},editorTwo:null,editorThree:null}]},overviewPageOFChapters:{paginationCount:26,paginationItems:[{id:"81791",title:"Self-Supervised Contrastive Representation Learning in Computer Vision",doi:"10.5772/intechopen.104785",signatures:"Yalin Bastanlar and Semih Orhan",slug:"self-supervised-contrastive-representation-learning-in-computer-vision",totalDownloads:3,totalCrossrefCites:0,totalDimensionsCites:0,authors:null,book:{title:"Pattern Recognition - New Insights",coverURL:"https://cdn.intechopen.com/books/images_new/11442.jpg",subseries:{id:"26",title:"Machine Learning and Data Mining"}}},{id:"79345",title:"Application of Jump Diffusion Models in Insurance Claim Estimation",doi:"10.5772/intechopen.99853",signatures:"Leonard Mushunje, Chiedza Elvina Mashiri, Edina Chandiwana and Maxwell Mashasha",slug:"application-of-jump-diffusion-models-in-insurance-claim-estimation-1",totalDownloads:2,totalCrossrefCites:0,totalDimensionsCites:0,authors:null,book:{title:"Data Clustering",coverURL:"https://cdn.intechopen.com/books/images_new/10820.jpg",subseries:{id:"26",title:"Machine Learning and Data Mining"}}},{id:"81557",title:"Object Tracking Using Adapted Optical Flow",doi:"10.5772/intechopen.102863",signatures:"Ronaldo Ferreira, Joaquim José de Castro Ferreira and António José Ribeiro Neves",slug:"object-tracking-using-adapted-optical-flow",totalDownloads:10,totalCrossrefCites:0,totalDimensionsCites:0,authors:null,book:{title:"Information Extraction and Object Tracking in Digital Video",coverURL:"https://cdn.intechopen.com/books/images_new/10652.jpg",subseries:{id:"24",title:"Computer Vision"}}},{id:"81558",title:"Thresholding Image Techniques for Plant Segmentation",doi:"10.5772/intechopen.104587",signatures:"Miguel Ángel Castillo-Martínez, Francisco Javier Gallegos-Funes, Blanca E. Carvajal-Gámez, Guillermo Urriolagoitia-Sosa and Alberto J. 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Thus proteomics, an area of research that detects all protein forms expressed in an organism, including splice isoforms and post-translational modifications, is more suitable than genomics for a comprehensive understanding of the biochemical processes that govern life. The most common proteomics applications are currently in the clinical field for the identification, in a variety of biological matrices, of biomarkers for diagnosis and therapeutic intervention of disorders. From the comparison of proteomic profiles of control and disease or different physiological states, which may emerge, changes in protein expression can provide new insights into the roles played by some proteins in human pathologies. Understanding how proteins function and interact with each other is another goal of proteomics that makes this approach even more intriguing. Specialized technology and expertise are required to assess the proteome of any biological sample. Currently, proteomics relies mainly on mass spectrometry (MS) combined with electrophoretic (1 or 2-DE-MS) and/or chromatographic techniques (LC-MS/MS). MS is an excellent tool that has gained popularity in proteomics because of its ability to gather a complex body of information such as cataloging protein expression, identifying protein modification sites, and defining protein interactions. 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