@article {1447, title = {Dietary supplementation of extracts of red sea weed (Kappaphycus alvarezii) improves growth, intestinal morphology, expression of intestinal genes and immune responses in broiler chickens [Sea6Energy Pvt. Ltd, a C-CAMP Startup]}, journal = {Journal of the Science of Food and Agriculture}, year = {2020}, month = {08, 2020}, type = {Research Article}, abstract = {

BACKGROUND

Effects of supplementation of dried alkaline (referred to as MVP1) and aqueous (referred to as PBD1) extracts of\ K. alvarezii\ , were evaluated in broiler (Vencobb 400) chickens (1{\textendash}35 d post-hatch). In experiment I, each of the seven diets (basal diet with three levels (0.5, 1.5 or 5.0 g kg-1\ diet) of MVP1 or PBD1 and a negative control) was fed to twelve pen replicates containing five birds in each. In experiment II, each of three diets (a negative control, and PBD1 at two levels (1.0 or 1.5 g kg-1\ diet)) was fed to sixteen pen replicates of five chicks in each.

RESULTS

Concentrations of total phenolics, phycobillins and free radical scavenging activity were higher (P\<0.01) whereas carrageenan was lower in PBD1 than in MVP1. In the experiment I, PBD1 at 1.5 g kg-1\ diet improved (P\<0.05) body weight (7.11\% higher). In the experiment II, both the treatments improved (P\<0.01) BW (9.18\% and 8.47\%, respectively) as compared to control. The group fed with PBD1@ 1.0 g kg-1\ had higher (P\<0.05) HI titre, expression of intestinal claudin 2, TLR2A, NOD1, avian beta defensin 4, interleukin 2 and 6 genes than control. Treatments did not influence feed efficiency or levels of most of the antioxidant enzymes. Villus width and crypt depth were significantly higher in the group fed with 1.5 g kg-1\ of PBD1.

CONCLUSION

Supplementing dried aqueous extract of\ Kappaphycus alvarezii\ at 1 g kg-1\ diet may be an effective strategy to increase growth and immunity in broiler chicken.

}, doi = {https://doi.org/10.1002/jsfa.10708}, url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/jsfa.10708}, author = {Paul, Shyam Sundar and Venkata, Hemanth Giri Rao Vantharam and Raju, MVLN and Rama Rao, SV and Nori, Sri Sailaja and Suryanarayan, Shrikumar and Kumar, Vikas and Perveen, Zeba and Srinivas Prasad, Cadaba} } @article {1161, title = {Chronic Pressure Overload Results in Deficiency of Mitochondrial Membrane Transporter ABCB7 Which Contributes to Iron Overload, Mitochondrial Dysfunction, Metabolic Shift and Worsens Cardiac Function [Mass Spectrometry - Metabolomics Facility].}, journal = {Sci Rep}, volume = {9}, year = {2019}, month = {2019 Sep 11}, pages = {13170}, abstract = {

We examined the hitherto unexplored role of mitochondrial transporters and iron metabolism in advancing metabolic and mitochondrial dysfunction in the heart during long term pressure overload. We also investigated the link between mitochondrial dysfunction and fluctuation in mitochondrial transporters associated with pressure overload cardiac hypertrophy. Left ventricular hypertrophy (LVH) was induced in 3-month-old male Wistar rats by constriction of the aorta using titanium clips. After sacrifice at the end of 6 and 15 months after constriction, tissues from the left ventricle (LV) from all animals were collected for histology, biochemical studies, proteomic and metabolic profiling, and gene and protein expression studies. LV tissues from rats with LVH had a significant decrease in the expression of ABCB7 and mitochondrial oxidative phosphorylation (mt-OXPHOS) enzymes, an increased level of lipid metabolites, decrease in the level of intermediate metabolites of pentose phosphate pathway and elevated levels of cytoplasmic and mitochondrial iron, reactive oxygen species (ROS) and autophagy-related proteins. Knockdown of ABCB7 in H9C2 cells and stimulation with angiotensin II resulted in increased ROS levels, ferritin, and transferrin receptor expression and iron overload in both mitochondria and cytoplasm. A decrease in mRNA and protein levels of mt-OXPHOS specific enzymes, mt-dynamics and autophagy clearance and activation of IGF-1 signaling were also seen in these cells. ABCB7 overexpression rescued all these changes. ABCB7 was found to interact with mitochondrial complexes IV and V. We conclude that in chronic pressure overload, ABCB7 deficiency results in iron overload and mitochondrial dysfunction, contributing to heart failure.

}, issn = {2045-2322}, doi = {10.1038/s41598-019-49666-0}, author = {Kumar, Vikas and A, Aneesh Kumar and Sanawar, Rahul and Jaleel, Abdul and Santhosh Kumar, T R and Kartha, C C} } @article {1194, title = {Deciphering the in vivo redox behavior of human peroxiredoxins I and II by expressing in budding yeast [Mass Spectrometry - Proteomics].}, journal = {Free Radic Biol Med}, volume = {145}, year = {2019}, month = {2019 Sep 30}, pages = {321-329}, abstract = {

Peroxiredoxins (Prxs), scavenge cellular peroxides by forming recyclable disulfides but under high oxidative stress, hyperoxidation of their active-site Cys residue results in loss of their peroxidase activity. Saccharomyces cerevisiae deficient in human Prx (hPrx) orthologue TSA1 show growth defects under oxidative stress. They can be complemented with hPRXI but not by hPRXII, but it is not clear how the disulfide and hyperoxidation states of the hPrx vary in yeast under oxidative stress. To understand this, we used oxidative-stress sensitive tsa1tsa2Δ yeast strain to express hPRXI or hPRXII. We found that hPrxI in yeast exists as a mixture of disulfide-linked dimer and reduced monomer but becomes hyperoxidized upon elevated oxidative stress as analyzed under denaturing conditions (SDS-PAGE). In contrast, hPrxII was present predominantly as the disulfide in unstressed cells and readily converted to its hyperoxidized, peroxidase-inactive form even with mild oxidative stress. Interestingly, we found that plant extracts containing polyphenol antioxidants provided further protection against the growth defects of the tsa1tsa2Δ strain expressing hPrx and preserved the peroxidase-active forms of the Prxs. The extracts also helped to protect against hyperoxidation of hPrxs in HeLa cells. Based on these findings we can conclude that resistance to oxidative stress of yeast cells expressing individual hPrxs requires the hPrx to be maintained in a redox state that permits redox cycling and peroxidase activity. Peroxidase activity decreases as the hPrx becomes hyperoxidized and the limited protection by hPrxII compared with hPrxI can be explained by its greater sensitivity to hyperoxidation.

}, issn = {1873-4596}, doi = {10.1016/j.freeradbiomed.2019.09.034}, author = {Kumar, Rakesh and Mohammad, Ashu and Saini, Reena V and Chahal, Anterpreet and Wong, Chi-Ming and Sharma, Deepak and Kaur, Sukhvir and Kumar, Vikas and Winterbourn, Christine C and Saini, Adesh K} } @article {1193, title = {Molecular Engineering of Adeno-Associated Virus Capsid Improves Its Therapeutic Gene Transfer in Murine Models of Hemophilia and Retinal Degeneration [Mass Spectrometry - Proteomics Facility]}, journal = {Mol Pharm}, year = {2019}, month = {2019 Oct 22}, abstract = {

Recombinant adeno-associated virus (AAV)-based gene therapy has been promising, but several host-related transduction or immune challenges remain. For this mode of therapy to be widely applicable, it is crucial to develop high transduction and permeating vectors that infect the target at significantly low doses. Because glycosylation of capsid proteins is known to be rate limiting in the life cycle of many viruses, we reasoned that perturbation of glycosylation sites in AAV2 capsid will enhance gene delivery. In our first set experiments, pharmacological modulation of the glycosylation status in host cells, modestly decreased (1-fold) AAV2 packaging efficacy while it improved their gene expression (\~{}74\%) in vitro. We then generated 24 mutant AAV2 vectors modified to potentially create or disrupt a glycosylation site in its capsid. Three of them demonstrated a 1.3-2.5-fold increase in transgene expression in multiple cell lines (HeLa, Huh7, and ARPE-19). Hepatic gene transfer of these vectors in hemophilia B mice, resulted in a 2-fold increase in human coagulation factor (F)IX levels, while its T/B-cell immunogenic response was unaltered. Subsequently, intravitreal gene transfer of glycosylation site-modified vectors in C57BL6/J mice demonstrated an increase in green fluorescence protein expression (\~{}2- to 4-fold) and enhanced permeation across retina. Subretinal administration of these modified vectors containing RPE65 gene further rescued the photoreceptor response in a murine model of Leber congenital amarousis. Our studies highlight the translational potential of glycosylation site-modified AAV2 vectors for hepatic and ocular gene therapy applications.

}, issn = {1543-8392}, doi = {10.1021/acs.molpharmaceut.9b00959}, author = {Mary, Bertin and Maurya, Shubham and Kumar, Mohit and Bammidi, Sridhar and Kumar, Vikas and Jayandharan, Giridhara R} } @article {1139, title = {Post-translational modifications in capsid proteins of recombinant adeno-associated virus (AAV) 1-rh10 serotypes [Mass Spectrometry Facility - Proteomics \& Glycomics].}, journal = {FEBS J}, year = {2019}, month = {2019 Jul 22}, abstract = {

Post-translational modifications in viral capsids are known to fine-tune and regulate several aspects of the infective life cycle of several viruses in the host. Recombinant viruses that are generated in a specific producer cell line are likely to inherit unique post-translational modifications during intra-cellular maturation of its capsid proteins. Data on such post-translational modifications in the capsid of recombinant adeno-associated virus serotypes (AAV1-rh10) is limited. We have employed liquid chromatography and mass spectrometry analysis to characterize post-translational modifications in AAV1-rh10 capsid protein. Our analysis revealed a total of 52 post-translational modifications in AAV2-AAVrh10 capsids, including ubiquitination (17\%), glycosylation (36\%), phosphorylation (21\%), SUMOylation (13\%) and acetylation (11\%). While AAV1 had no detectable post-translational modification, at least four AAV serotypes had \>7 post-translational modifications in their capsid protein. About 82\% of these post-translational modifications are novel. A limited validation of AAV2 capsids by MALDI-TOF and western blot analysis demonstrated minimal glycosylation and ubiquitination of AAV2 capsids. To further validate this, we disrupted a glycosylation site identified in AAV2 capsid (AAV2-N253Q), which severely compromised its packaging efficiency (~100-fold vs AAV2 wildtype vectors). In order to confirm other post-translational modifications detected such as SUMOylation, mutagenesis of a SUMOylation site(K258Q) in AAV2 was performed. This mutant vector demonstrated reduced levels of SUMO-1/2/3 proteins and negligible transduction, two weeks after ocular gene transfer. Our study underscores the heterogeneity of post-translational modifications in AAV vectors. The data presented here, should facilitate further studies to understand the biological relevance of post-translational modifications in AAV life cycle and the development of novel bioengineered AAV vectors for gene therapy applications. This article is protected by copyright. All rights reserved.

}, issn = {1742-4658}, doi = {10.1111/febs.15013}, author = {Mary, Bertin and Maurya, Shubham and Arumugam, Sathyathithan and Kumar, Vikas and Jayandharan, Giridhara R} } @article {685, title = {S-Glutathionylation of p47phox sustains superoxide generation in activated neutrophils. [Mass Spectrometry Facility - Proteomics]}, journal = {Biochim Biophys Acta}, volume = {1865}, year = {2018}, month = {2018 Feb}, pages = {444-454}, abstract = {

Post-translational modifications (PTMs) induced conformational changes of proteins can cause their activation or inactivation. Neutrophils clear pathogen through phagocytosis and oxidative burst generation, while participate in inflammation through sustained and uncontrolled generation of ROS. In activated PMNs, cytosolic NOX-2 subunit p47phox following phosphorylation interacts with p67phox, p40phox and along with Rac2 translocate to the membrane. Phosphorylation of p47phox subunit occurs in both short spurts as well as sustained ROS generation, suggesting towards the unidentified molecular mechanism(s) driving these two diverse outcomes by various stimuli. The present study demonstrates that in PMA or NO treated neutrophils a subunit of NOX2, p47phox gets glutathionylated to sustain ROS generation along with a decrease in catalase, Grx-1 activity and change in GSH/GSSG ratio. Surprisingly, fMLP treated cells neither showed sustained ROS production nor glutathionylation of p47phox. S-Glutathionylation was always secondary to phosphorylation of p47phox and inhibition of glutathionylation did not alter phosphorylation but specifically impaired sustained ROS production. Interestingly, forced S-glutathionylation of p47phox converted the fMLP induced ROS generation into sustained release of ROS. We then identified the glutathionylation susceptible cysteine residues of p47phox by LC-MS/MS with IAM switch mapping. Site-directed mutagenesis of cysteine residues further mitigated p47phox S-glutathionylation. Thus, we demonstrate that p47phox S-glutathionylation plays an essential key role in the sustained ROS generation by human neutrophils.

}, issn = {0006-3002}, doi = {10.1016/j.bbamcr.2017.11.014}, author = {Nagarkoti, Sheela and Dubey, Megha and Awasthi, Deepika and Kumar, Vikas and Chandra, Tulika and Kumar, Sachin and Dikshit, Madhu} } @article {520, title = {Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods. (Mass spectrometry - Proteomics)}, journal = {PLoS One}, volume = {10}, year = {2015}, month = {2015}, pages = {e0145686}, abstract = {

BACKGROUND: Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described.

AIM: Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC).

METHODS AND RESULTS: Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4{\textdegree}C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4{\textdegree}C, or UC performed at 37{\textdegree}C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin.

CONCLUSION: Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

}, keywords = {Animals, Chromatography, Gel, Exosomes, Male, Plasma, Rats, Wistar, Ultracentrifugation}, issn = {1932-6203}, doi = {10.1371/journal.pone.0145686}, author = {Baranyai, Tam{\'a}s and Herczeg, Kata and On{\'o}di, Zs{\'o}fia and Voszka, Istv{\'a}n and M{\'o}dos, K{\'a}roly and Marton, Nikolett and Nagy, Gy{\"o}rgy and M{\"a}ger, Imre and Wood, Matthew J and El Andaloussi, Samir and P{\'a}link{\'a}s, Zolt{\'a}n and Kumar, Vikas and Nagy, P{\'e}ter and Kittel, {\'A}gnes and Buz{\'a}s, Edit Ir{\'e}n and Ferdinandy, P{\'e}ter and Giricz, Zolt{\'a}n} } @article {513, title = {L-Plastin S-glutathionylation promotes reduced binding to β-actin and affects neutrophil functions. (Mass Spectrometry)}, journal = {Free Radic Biol Med}, volume = {86}, year = {2015}, month = {2015 Sep}, pages = {1-15}, abstract = {

Posttranslational modifications (PTMs) of cytoskeleton proteins due to oxidative stress associated with several pathological conditions often lead to alterations in cell function. The current study evaluates the effect of nitric oxide (DETA-NO)-induced oxidative stress-related S-glutathionylation of cytoskeleton proteins in human PMNs. By using in vitro and genetic approaches, we showed that S-glutathionylation of L-plastin (LPL) and β-actin promotes reduced chemotaxis, polarization, bactericidal activity, and phagocytosis. We identified Cys-206, Cys-283, and Cys-460as S-thiolated residues in the β-actin-binding domain of LPL, where cys-460 had the maximum score. Site-directed mutagenesis of LPL Cys-460 further confirmed the role in the redox regulation of LPL. S-Thiolation diminished binding as well as the bundling activity of LPL. The presence of S-thiolated LPL was detected in neutrophils from both diabetic patients and db/db mice with impaired PMN functions. Thus, enhanced nitroxidative stress may results in LPL S-glutathionylation leading to impaired chemotaxis, polarization, and bactericidal activity of human PMNs, providing a mechanistic basis for their impaired functions in diabetes mellitus.

}, keywords = {Actins, Adult, Amino Acid Sequence, Animals, Case-Control Studies, Cell Polarity, Chemotaxis, Diabetes Mellitus, Female, Glutathione, HEK293 Cells, Humans, Male, Mice, Inbred C57BL, Mice, Obese, Microfilament Proteins, Middle Aged, Molecular Sequence Data, Neutrophils, Nitric Oxide, Oxidative Stress, Protein Binding, Protein Processing, Post-Translational, Young Adult}, issn = {1873-4596}, doi = {10.1016/j.freeradbiomed.2015.04.008}, author = {Dubey, Megha and Singh, Abhishek K and Awasthi, Deepika and Nagarkoti, Sheela and Kumar, Sachin and Ali, Wahid and Chandra, Tulika and Kumar, Vikas and Barthwal, Manoj K and Jagavelu, Kumaravelu and S{\'a}nchez-G{\'o}mez, Francisco J and Lamas, Santiago and Dikshit, Madhu} } @article {536, title = {Does reversible cysteine oxidation link the Western diet to cardiac dysfunction?}, journal = {FASEB J}, volume = {28}, year = {2014}, month = {2014 May}, pages = {1975-87}, abstract = {

Using a novel cysteine thiol labeling strategy coupled with mass spectrometric analysis, we identified and quantified the changes in global reversible cysteine oxidation of proteins in the left ventricle of hearts from mice with metabolic syndrome-associated diastolic dysfunction. This phenotype was induced by feeding a high-fat, high-sucrose, type-2 diabetogenic diet to C57BL/6J mice for 8 mo. The extent of reversible thiol oxidation in relationship to the total available (free and reducible) level of each cysteine could be confidently determined for 173 proteins, of which 98 contained cysteines differentially modified >=1.5-fold by the diet. Our findings suggest that the metabolic syndrome leads to potentially deleterious changes in the oxidative modification of metabolically active proteins. These alterations may adversely regulate energy substrate flux through glycolysis, β-oxidation, citric acid (TCA) cycle, and oxidative phosphorylation (oxphos), thereby contributing to maladaptive tissue remodeling that is associated with, and possibly contributing to, diastolic left ventricular dysfunction.

}, keywords = {Animals, Chromatography, Liquid, Citric Acid Cycle, Cysteine, Diet, Fatty Acids, Glycolysis, Heart Diseases, Male, Mice, Mice, Inbred C57BL, Myocardial Contraction, Myocardium, Obesity, Oxidative Phosphorylation, Oxygen, Phenotype, Protein Processing, Post-Translational, Proteomics, Reactive Nitrogen Species, Reactive Oxygen Species, Sulfhydryl Compounds, Tandem Mass Spectrometry}, issn = {1530-6860}, doi = {10.1096/fj.13-233445}, author = {Behring, Jessica B and Kumar, Vikas and Whelan, Stephen A and Chauhan, Pratibha and Siwik, Deborah A and Costello, Catherine E and Colucci, Wilson S and Cohen, Richard A and McComb, Mark E and Bachschmid, Markus M} } @article {537, title = {Redox proteomics of thiol proteins in mouse heart during ischemia/reperfusion using ICAT reagents and mass spectrometry.}, journal = {Free Radic Biol Med}, volume = {58}, year = {2013}, month = {2013 May}, pages = {109-17}, abstract = {

There is strong evidence for the involvement of reactive oxygen species in ischemia/reperfusion injury. Although oxidation of individual thiol proteins has been reported, more extensive redox proteomics of hearts subjected to ischemia/reperfusion has not been performed. We have carried out an exploratory study using mass spectrometry with isotope-coded affinity tags (ICAT) aimed at identifying reversible oxidative changes to protein thiols in Langendorff perfused isolated mouse hearts subjected to 20 min ischemia with or without aerobic reperfusion for 5 or 30 min. Reduced thiols were blocked by adding N-ethylmaleimide during protein extraction, then reversibly oxidized thiols in extracts of control perfused and treated hearts were reduced and labeled with the light and heavy ICAT reagents, respectively. Protein extracts were mixed in equal amounts and relative proportions of the isotope-labeled peaks were used to quantify oxidative changes between the control and the treated groups. Approximately 300 peptides with ICAT signatures were reliably identified in each sample, with 181 peptides from 118 proteins common to all treatments. A proportion showed elevated ICAT ratios, consistent with reversible thiol oxidation. This was most evident after early reperfusion, with apparent reversal after longer reperfusion. In comparison, there was gradual accumulation of protein carbonyls and loss of GSH with longer reperfusion. Many of the thiol changes were in mitochondrial proteins, including components of electron transport complexes and enzymes involved in lipid metabolism. The results are consistent with mitochondria being a major site of oxidant generation during early cardiac reperfusion and mitochondrial thiol proteins being targets for oxidation.

}, keywords = {Animals, Isotope Labeling, Mass Spectrometry, Mice, Mitochondrial Proteins, Myocardium, Oxidation-Reduction, Proteomics, Reactive Oxygen Species, Reperfusion Injury, Sulfhydryl Compounds}, issn = {1873-4596}, doi = {10.1016/j.freeradbiomed.2013.01.021}, author = {Kumar, Vikas and Kleffmann, Torsten and Hampton, Mark B and Cannell, Mark B and Winterbourn, Christine C} }