NCoR1 controls immune tolerance in conventional dendritic cells by fine-tuning glycolysis and fatty acid oxidation.

Dendritic cells (DCs) undergo rapid metabolic reprogramming to generate signal-specific immune responses. The fine control of cellular metabolism underlying DC immune tolerance remains elusive. We have recently reported that NCoR1 ablation generates immune-tolerant DCs through enhanced IL-10, IL-27 and SOCS3 expression. In this study, we did comprehensive metabolic profiling of these tolerogenic DCs and identified that they meet their energy requirements through enhanced glycolysis and oxidative phosphorylation (OXPHOS), supported by fatty acid oxidation-driven oxygen consumption. In addition, the reduced pyruvate and glutamine oxidation with a broken TCA cycle maintains the tolerogenic state of the cells. Mechanistically, the AKT-mTOR-HIF-1α-axis mediated glycolysis and CPT1a-driven ß-oxidation were enhanced in these tolerogenic DCs. To confirm these observations, we used synthetic metabolic inhibitors and found that the combined inhibition of HIF-1α and CPT1a using KC7F2 and etomoxir, respectively, compromised the overall transcriptional signature of immunological tolerance including the regulatory cytokines IL-10 and IL-27. Functionally, treatment of tolerogenic DCs with dual KC7F2 and etomoxir treatment perturbed the polarization of co-cultured naïve CD4+ T helper (Th) cells towards Th1 than Tregs, ex vivo and in vivo. Physiologically, the Mycobacterium tuberculosis (Mtb) infection model depicted significantly reduced bacterial burden in BMcDC1 ex vivo and in CD103+ lung DCs in Mtb infected NCoR1DC-/-mice. The spleen of these infected animals also showed increased Th1-mediated responses in the inhibitor-treated group. These findings suggested strong involvement of NCoR1 in immune tolerance. Our validation in primary human monocyte-derived DCs (moDCs) showed diminished NCOR1 expression in dexamethasone-derived tolerogenic moDCs along with suppression of CD4+T cell proliferation and Th1 polarization. Furthermore, the combined KC7F2 and etomoxir treatment rescued the decreased T cell proliferative capacity and the Th1 phenotype. Overall, for the first time, we demonstrated here that NCoR1 mediated control of glycolysis and fatty acid oxidation fine-tunes immune tolerance versus inflammation balance in murine and human DCs.

Acute radiofrequency electromagnetic radiation exposure impairs neurogenesis and causes neuronal DNA damage in the young rat brain.

A mobile phone is now a commonly used device for digital media and communication among all age groups. Young adolescents use it for longer durations, which exposes them to radiofrequency electromagnetic radiation (RF-EMR). This exposure can lead to neuropsychiatric changes. The underlying cellular mechanism behind these changes requires detailed investigation. In the present study, we investigated the effect of RF-EMR emitted from mobile phones on young adolescent rat brains. Wistar rats (5 weeks, male) were exposed to RF-EMR signal (2115 MHz) at a head average specific absorption rate (SAR) of 1.51 W/kg continuously for 8 h. Higher level of lipid peroxidation, carbon-centered lipid radicals, and single-strand DNA damage was observed in the brain of rat exposed to RF-EMR. The number of BrdU-positive cells in the dentate gyrus (DG) decreased in RF-EMR-exposed rats, indicating reduced neurogenesis. RF-EMR exposure also induced degenerative changes and neuronal loss in DG neurons but had no effect on the CA3 and CA1 neurons of the hippocampus and cerebral cortex. The activity of Pro-caspase3 did not increase upon exposure in any of the brain regions, pointing out that degeneration observed in the DG region is not dependent on caspase activation. Results indicate that short-term acute exposure to RF-EMR induced the generation of carbon-centered lipid radicals and nuclear DNA damage, both of which likely played a role in the impaired neurogenesis and neuronal degeneration seen in the young brain's hippocampus region. The understanding of RF-EMR-induced alteration in the brain at the cellular level will help develop appropriate interventions for reducing its adverse impact.

Phytochemical rich Himalayan Rhododendron arboreum petals inhibit SARS-CoV-2 infection in vitro.

Phytochemicals with potential to competitively bind to the host receptors or inhibit SARS-CoV-2 replication, may prove to be useful as adjunct therapeutics for COVID-19. We profiled and investigated the phytochemicals of Rhododendron arboreum petals sourced from Himalayan flora, undertook in vitro studies and found it as a promising candidate against SARS-CoV-2. The phytochemicals were reported in various scientific investigations to act against a range of virus in vitro and in vivo, which prompted us to test against SARS-CoV-2. In vitro assays of R. arboreum petals hot aqueous extract confirmed dose dependent reduction in SARS-CoV-2 viral load in infected Vero E6 cells (80% inhibition at 1 mg/ml; IC50 = 173 µg/ml) and phytochemicals profiled were subjected to molecular docking studies against SARS CoV-2 target proteins. The molecules 5-O-Feruloyl-quinic acid, 3-Caffeoyl-quinic acid, 5-O-Coumaroyl-D-quinic acid, Epicatechin and Catechin showed promising binding affinity with SARS-CoV-2 Main protease (MPro; PDB ID: 6LU7; responsible for viral replication) and Human Angiotensin Converting Enzyme-2 (ACE2; PDB ID: 1R4L; mediate viral entry in the host). Molecular dynamics (MD) simulation of 5-O-Feruloyl-quinic acid, an abundant molecule in the extract complexed with the target proteins showed stable interactions. Taken together, the phytochemical profiling, in silico analysis and in vitro anti-viral assay revealed that the petals extract act upon MPro and may be inhibiting SARS-CoV-2 replication. This is the first report highlighting R. arboreum petals as a reservoir of antiviral phytochemicals with potential anti-SARS-CoV-2 activity using an in vitro system.

Troxerutin-Mediated Complement Pathway Inhibition is a Disease-Modifying Treatment for Inflammatory Arthritis.

Troxerutin (TXR) is a phytochemical reported to possess anti-inflammatory and hepatoprotective effects. In this study, we aimed to exploit the antiarthritic properties of TXR using an adjuvant-induced arthritic (AIA) rat model. AIA-induced rats showed the highest arthritis score at the disease onset and by oral administration of TXR (50, 100, and 200 mg/kg body weight), reduced to basal level in a dose-dependent manner. Isobaric tags for relative and absolute quantitative (iTRAQ) proteomics tool were employed to identify deregulated joint homogenate proteins in AIA and TXR-treated rats to decipher the probable mechanism of TXR action in arthritis. iTRAQ analysis identified a set of 434 proteins with 65 deregulated proteins (log2 case/control≥1.5) in AIA. Expressions of a set of important proteins (AAT, T-kininogen, vimentin, desmin, and nucleophosmin) that could classify AIA from the healthy ones were validated using Western blot analysis. The Western blot data corroborated proteomics findings. In silico protein-protein interaction study of tissue-proteome revealed that complement component 9 (C9), the major building blocks of the membrane attack complex (MAC) responsible for sterile inflammation, get perturbed in AIA. Our dosimetry study suggests that a TXR dose of 200 mg/kg body weight for 15 days is sufficient to bring the arthritis score to basal levels in AIA rats. We have shown the importance of TXR as an antiarthritic agent in the AIA model and after additional investigation, its arthritic ameliorating properties could be exploited for clinical usability.

Effects of mobile phone electromagnetic radiation on rat hippocampus proteome.

Worldwide, the number of mobile phone users has increased from 5.57 billion in 2011 to 6.8 billion in 2019. However, short- and long-term impact of the electromagnetic radiation emitting from mobile phones on tissue homeostasis with particular to brain proteome composition needs further investigation. In this study, we attempted a global proteome profiling study of rat hippocampus exposed to mobile phone radiation for 20 weeks (for 3 h/day for 5 days/week) to identify deregulated proteins and western blot analysis for validation. As a result, we identified 358 hippocampus proteins, of which 16 showed deregulation (log2 (exposed/sham) ≥ ±1.0, p-value <.05). Majority of these deregulated proteins grouped into three clusters sharing similar molecular pathways. A set of four proteins (Succinate-semialdehyde dehydrogenase: Aldh5a1, Na+ K+ transporting ATPase: Atp1b2, plasma membrane calcium transporting ATPase: PMCA and protein S100B) presenting each functional pathway were selected for validation. Western blot analysis of these proteins, in an independent sample set, corroborated the mass spectrometry findings. Aldh5a1 involve in cellular energy metabolism, both Atp1b2 and PMCA responsible for membrane transport and protein S100B have a neuroprotective role. In conclusion, we present a deregulated hippocampus proteome upon mobile phone radiation exposure, which might influence the healthy functioning of the brain.

Low circulatory Fe and Se levels with a higher IL-6/IL-10 ratio provide nutritional immunity in tuberculosis.

Tuberculosis (TB) patients show dysregulated immunity, iron metabolism, and anemia. In this study, circulatory cytokines, trace metals, and iron-related proteins (hepcidin, ferroportin, transferrin, Dmt1, Nramp1, ferritin, ceruloplasmin, hemojuvelin, aconitase, and transferrin receptor) were monitored in case (active tuberculosis patients: ATB) and control (non-tuberculosis: NTB and healthy) study populations (n = 72, male: 100%, mean age, 42.94 years; range, 17-83 years). Using serum elemental and cytokine levels, a partial least square discriminate analysis model (PLS-DA) was built, which clustered ATB patients away from NTB and healthy controls. Based on the PLS-DA variable importance in projection (VIP) score and analysis of variance (ANOVA), 13 variables were selected as important biosignatures [IL-18, IL-10, IL-13, IFN-γ, TNF-α, IL-5, IL-12 (p70), IL-1ß, copper, zinc, selenium, iron, and aluminum]. Interestingly, low iron and selenium levels and high copper and aluminum levels were observed in ATB subjects. Low circulatory levels of transferrin, ferroportin, and hemojuvelin with higher ferritin and ceruloplasmin levels observed in ATB subjects demonstrate an altered iron metabolism, which partially resolved upon 6 months of anti-TB therapy. The identified biosignature in TB patients demonstrated perturbed iron homeostasis with anemia of inflammation, which could be useful targets for the development of host-directed adjunct therapeutics.

Zinc2+ ion inhibits SARS-CoV-2 main protease and viral replication in vitro.

Zinc deficiency is linked to poor prognosis in COVID-19 patients while clinical trials with zinc demonstrate better clinical outcomes. The molecular targets and mechanistic details of the anti-coronaviral activity of zinc remain obscure. We show that zinc not only inhibits the SARS-CoV-2 main protease (Mpro) with nanomolar affinity, but also viral replication. We present the first crystal structure of the Mpro-Zn2+ complex at 1.9 Å and provide the structural basis of viral replication inhibition. We show that Zn2+ coordinates with the catalytic dyad at the enzyme active site along with two previously unknown water molecules in a tetrahedral geometry to form a stable inhibited Mpro-Zn2+ complex. Further, the natural ionophore quercetin increases the anti-viral potency of Zn2+. As the catalytic dyad is highly conserved across SARS-CoV, MERS-CoV and all variants of SARS-CoV-2, Zn2+ mediated inhibition of Mpro may have wider implications.

Night Migratory Songbirds Exhibit Metabolic Ability to Support High Aerobic Capacity during Migration.

Aerobic metabolism in night migratory songbirds exhibit seasonal plasticity, which depends not only on annual life history stages (LHSs), viz., migratory/nonmigratory or breeding/nonbreeding, but also on the time of the day. Initially, we studied daily changes in behavior/physiology alongside aerobic metabolism intermediates using gas chromatography-mass spectrometry-based chemometric analyses of serum of migratory male redheaded buntings during low-energy wintering, that is, the nonmigrating LHS. Then, the metabolic phenotype of nonmigrating birds was compared with that of photostimulated migrating buntings, the latter representing the high-energy LHS. Diurnal changes such as daytime feeding and activity were reflected by increased fatty acid (FA, viz., palmitic, oleic, and linoleic acids) levels and protein catabolites, whereas higher night-time levels of short-chain FAs indicated lipolysis in night-fasted birds. High night-time levels of taurine, a sulfur amino acid, suggested the endogenous metabolite rendering an adaptive advantage to hyperglycaemic night migratory songbirds during the LHS with low daily energy expenditure. Conversely, migrating birds, largely night-active, exhibited higher circulatory FA, its mobilization, and increased aerobic catabolism, and the adipocyte-secreted lipid, palmitoylethanolamide (PEA), capable of activating the peroxisome proliferator-activated receptor α-PGCα axis, showed elevated levels throughout the day. PEA is known for anti-inflammatory and cannabinomimetic properties, and we show, for the first time, circadian changes in PEA levels in any migrating bird. Significantly higher levels of pyridoxal phosphate also suggested the bird's protective ability to combat metabolic stress through high aerobic capacity during migration. This study elucidates putative "serum biomarkers" with a protective role in stress accrued by enhanced aerobic capacity requirements at the organismal level.

Whole-Genome Sequence of Drug-Resistant Mycobacterium tuberculosis Strain S7, Isolated from a Patient with Pulmonary Tuberculosis.

Over the past decades, drug-resistant Mycobacterium tuberculosis strains have presented a significant challenge, with inadequate diagnosis of tuberculosis (TB) cases. Here, we report the draft whole-genome sequence of drug-resistant M. tuberculosis strain S7, which was isolated from a patient from Tripura, India, who was diagnosed with pulmonary TB.

Serum Small Extracellular Vesicles Proteome of Tuberculosis Patients Demonstrated Deregulated Immune Response.

PURPOSE: Detailed understanding of host pathogen interaction in tuberculosis is an important avenue for identifying novel therapeutic targets. Small extracellular vesicles (EVs) like exosomes that are rich in proteins, nucleic acids and lipids, act as messengers and may show altered composition in disease conditions. EXPERIMENTAL DESIGN: In this case control study, small EVs are isolated from serum of 58 subjects (all male, 33 (15-70) in years) including drug naïve active tuberculosis (ATB: n = 22), non-tuberculosis (NTB: n = 18), and healthy subjects (n = 18). Serum small EVs proteome analysis is carried out using isobaric tag for relative and absolute quantification (iTRAQ) experiments and an independent sample (n = 36) is used for validation. RESULTS: A set of 132 and 68 proteins are identified in iTRAQ-I (ATB/Healthy) and iTRAQ-II (ATB/NTB) experiments, respectively. Four proteins (KYAT3, SERPINA1, HP, and APOC3) show deregulation (log2 -fold change > ±0.48, p < 0.05) in ATB with respect to healthy controls and Western blot data corroborated mass spectrometry findings. CONCLUSIONS AND CLINICAL RELEVANCE: These important proteins, involved in neutrophil degranulation, plasma heme scavenging, kynurenine, and lipid metabolism, show deregulation in ATB patients. Identification of such a protein panel in circulating small EVs besides providing novel insights into their role in tuberculosis may prove to be useful targets to develop host-directed therapeutic intervention.

The phytochemical bergenin as an adjunct immunotherapy for tuberculosis in mice.

The widespread availability and use of modern synthetic therapeutic agents have led to a massive decline in ethnomedical therapies. However, these synthetic agents often possess toxicity leading to various adverse effects. For instance, anti-tubercular treatment (ATT) is toxic, lengthy, and severely impairs host immunity, resulting in posttreatment vulnerability to reinfection and reactivation of tuberculosis (TB). Incomplete ATT enhances the risk for the generation of multidrug- or extensively drug-resistant (MDR or XDR, respectively) variants of Mycobacterium tuberculosis (M. tb), the TB-causing microbe. Therefore, a new therapeutic approach that minimizes these risks is urgently needed to combat this deadly disease and prevent future TB epidemics. Previously, we have shown that the phytochemical bergenin induces T helper 1 (Th1)- and Th17 cell-based protective immune responses and potently inhibits mycobacterial growth in a murine model of M. tb infection, suggesting bergenin as a potential adjunct agent to TB therapy. Here, we combined ATT therapy with bergenin and found that this combination reduces immune impairment and the length of treatment in mice. We observed that co-treatment with the anti-TB drug isoniazid and bergenin produces additive effects and significantly reduces bacterial loads compared with isoniazid treatment alone. The bergenin co-treatment also reduced isoniazid-induced immune impairment; promoted long-lasting, antigen-specific central memory T cell responses; and acted as a self-propelled vaccine. Of note, bergenin treatment significantly reduced the bacterial burden of a multidrug-resistant TB strain. These observations suggest that bergenin is a potent immunomodulatory agent that could be further explored as a potential adjunct to TB therapy.

Network-based approach to identify molecular signatures and therapeutic agents in Alzheimer's disease.

Alzheimer's disease (AD) is a dynamic degeneration of the brain with progressive dementia. Considering the uncertainties in its molecular mechanism, in the present study, we employed network-based integrative analyses, and aimed to explore the key molecules and their associations with small drugs to identify potential biomarkers and therapeutic agents for the AD. First of all, we studied a transcriptome dataset and identified 1521 differentially expressed genes (DEGs). Integration of transcriptome data with protein-protein and transcriptional regulatory interactions resulted with central (hub) proteins (UBA52, RAC1, CREBBP, AR, RPS11, SMAD3, RPS6, RPL12, RPL15, and UBC), regulatory transcription factors (FOXC1, GATA2, YY1, FOXL1, NFIC, E2F1, USF2, SRF, PPARG, and JUN) and microRNAs (mir-335-5p, mir-26b-5p, mir-93-5p, mir-124-3p, mir-17-5p, mir-16-5p, mir-20a-5p, mir-92a-3p, mir-106b-5p, and mir-192-5p) as key signaling and regulatory molecules associated with transcriptional changes for the AD. Considering these key molecules as potential therapeutic targets and Connectivity Map (CMap) architecture, candidate small molecular agents (such as STOCK1N-35696) were identified as novel potential therapeutics for the AD. This study presents molecular signatures at RNA and protein levels which might be useful in increasing discernment of the molecular mechanisms, and potential drug targets and therapeutics to design effective treatment strategies for the AD.

Biofluid metabotyping of occupationally exposed subjects to air pollution demonstrates high oxidative stress and deregulated amino acid metabolism.

Occupational exposure to air pollution induces oxidative stress and prolonged exposure increases susceptibility to cardiovascular and respiratory diseases in several working groups. Biofluid of these subjects may reflect perturbed metabolic phenotypes. In this study we carried out a comparative molecular profiling study using parallel biofluids collected from subjects (n = 85) belonging to auto rickshaw drivers (ARD), traffic cops (TC) and office workers (OW). Higher levels of oxidative stress and inflammation markers in serum of ARD subjects were observed as compared to OW and TC. Uni and multivariate analyses of metabolites identified in urine by 1H NMR revealed 11 deregulated molecules in ARD subjects and involved in phenylalanine, histidine, arginine and proline metabolism. Despite contribution of confounding factors like exposure period, dietary factors including smoking and alcohol status, our results demonstrate existence of exposure specific metabotypes in biofluids of ARD, OW and TC groups. Monitoring serum oxidative stress and inflammation markers and urine metabolites by NMR may be useful to characterize perturbed metabolic phenotypes in populations exposed to urban traffic air pollution.

Global Urine Metabolomics in Patients Treated with First-Line Tuberculosis Drugs and Identification of a Novel Metabolite of Ethambutol.

Population level variation of drug metabolism phenotype (DMP) has great implications in treatment outcome, drug-related side effects, and resistance development. In this study, we used a gas chromatography-time of flight-mass spectrometry (GC-TOF-MS)-based untargeted urine metabolomics approach to understand the DMP of a tuberculosis (TB) patient cohort (n= 20) from Tripura, a state in the northeastern part of India. Urine samples collected at different postdose time points (2 h, 6 h, 12 h, 24 h, 36 h, and 48 h) from these newly diagnosed TB patients receiving first-line anti-TB drugs were analyzed, and we have successfully detected three of the four first-line drugs,viz, isoniazid (INH), ethambutol (ETB), and pyrazinamide (PZA). The majority of their known metabolites, acetyl-isoniazid (AcINH), isonicotinic acid (INA), isonicotinuric acid (INTA), 2,2'-(ethylenediimino)-dibutyric acid (EDBA), 5-hydroxypyrazinamide (5OH-PZA), pyrazinoic acid (POA), and 5-hydroxypyrazinoic acid (5OH-POA), were also detected. Analyzing the variation in abundances of drugs and their known metabolites and calculating the metabolic ratios in these samples, we offer comprehensive DMP information on this small patient cohort that represents Tripura, India. The majority (75%) of these patients are found to be slow acetylators of INH. The average metabolic ratios of POA/PZA and 5OH-POA/POA are 3.16 ± 3.03 and 6.09 ± 6.15, respectively. Employing correlation analysis of the metabolomics metadata and a manual prediction of drug catabolism, we have proposed 2-aminobutyric acid (AABA) as a novel metabolite of ETB. These observations indicate the usefulness of GC-MS-based metabolomics to characterize the DMP at a population level and also to identify novel drug metabolites.

Deregulated tyrosine-phenylalanine metabolism in pulmonary tuberculosis patients.

Metabolic profiling of biofluids from tuberculosis (TB) patients would help us in understanding the disease pathophysiology and may also be useful for the development of novel diagnostics and host-directed therapy. In this pilot study we have compared the urine metabolic profiles of two groups of subjects having similar TB symptoms and categorized as active TB (ATB, n = 21) and non-TB (NTB, n = 21) based on GeneXpert test results. Silylation, gas chromatography mass spectrometry, and standard chemometric methods were employed to identify the important molecules and deregulated metabolic pathways. Eleven active TB patients were followed up on longitudinally for comparative urine metabolic profiling with healthy controls (n = 11). A set of 42 features qualified to have a variable importance parameter score of > 1.5 of a partial least-squares discriminate analysis model and fold change of > 1.5 at p value < 0.05 between ATB and NTB. Using these variables, a receiver operating characteristics curve was plotted and the area under the curve was calculated to be 0.85 (95% CI: 0.72-0.96). Several of these variables that represent norepinephrine, gentisic acid, 4-hydroxybenzoic acid, hydroquinone, and 4-hydroxyhippuric acid are part of the tyrosine-phenylalanine metabolic pathway. In the longitudinal study we observed a treatment-dependent trend in the urine metabolome of follow-up samples, and subjects declared as clinically cured showed similar metabolic profile as those of asymptomatic healthy subjects. The deregulated tyrosine-phenylalanine axis reveals a potential target for diagnostics and intervention in TB.

Use of urine volatile organic compounds to discriminate tuberculosis patients from healthy subjects.

Development of noninvasive methods for tuberculosis (TB) diagnosis, with the potential to be administered in field situations, remains as an unmet challenge. A wide array of molecules are present in urine and reflect the pathophysiological condition of a subject. With infection, an alteration in the molecular constituents is anticipated, characterization of which may form a basis for TB diagnosis. In the present study volatile organic compounds (VOCs) in human urine derived from TB patients and healthy controls were identified and quantified using headspace gas chromatography/mass spectrometry (GC/MS). We found significant (p < 0.05) increase in the abundance of o-xylene (6.37) and isopropyl acetate (2.07) and decreased level of 3-pentanol (0.59), dimethylstyrene (0.37), and cymol (0.42) in TB patients compared to controls. These markers could discriminate TB from healthy controls and related diseases like lung cancer and chronic obstructive pulmonary disorder. This study suggests a possibility of using urinary VOCs for the diagnosis of human TB.

Strain improvement for tannase production from co-culture of Aspergillus foetidus and Rhizopus oryzae.

Spores from the co-culture of Aspergillus foetidus and Rhizopus oryzae were subjected to UV, heat and NTG (3-nitro,5-methylguanidine) mutagenesis. A few colonies were screened from the selected media for tannase study. Amongst all, the best mutant isolated from the heat treatment (60 degrees C for 60 min) was SCPR 337. The maximum yield of gallic acid and tannase in case of mutant strain was 95.2% and 53.6 U/ml with an incubation period of 30 h as compared to wild strain where the incubation period was 48 h with an enzyme activity of 44.2 U/ml and gallic acid yield of 94%, respectively. The mutant was sensitive to tetracycline and was also an over-producer of protease and amylase.