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Pediatric tuberculosis (TB) remains a major global health problem. Improved pediatric diagnostics using readily available biosources are urgently needed. We used liquid chromatography-mass spectrometry to analyze plasma metabolite profiles of Indian children with active TB (n = 16) and age- and sex-matched, Mycobacterium tuberculosis-exposed but uninfected household contacts (n = 32). Metabolomic data were integrated with whole blood transcriptomic data for each participant at diagnosis and throughout treatment for drug-susceptible TB. A decision tree algorithm identified 3 metabolites that correctly identified TB status at distinct times during treatment. N-acetylneuraminate achieved an area under the receiver operating characteristic curve (AUC) of 0.66 at diagnosis. Quinolinate achieved an AUC of 0.77 after 1 month of treatment, and pyridoxate achieved an AUC of 0.87 after successful treatment completion. A set of 4 metabolites (gamma-glutamylalanine, gamma-glutamylglycine, glutamine, and pyridoxate) identified treatment response with an AUC of 0.86. Pathway enrichment analyses of these metabolites and corresponding transcriptional data correlated N-acetylneuraminate with immunoregulatory interactions between lymphoid and non-lymphoid cells, and correlated pyridoxate with p53-regulated metabolic genes and mitochondrial translation. Our findings shed new light on metabolic dysregulation in children with TB and pave the way for new diagnostic and treatment response markers in pediatric TB.

Nitriles are organic compounds consisting of −C≡N group. They are frequently known to occur in nature and as intermediate by-products and waste products of various chemical, pharmaceutical, and agricultural industries. They are also found in fruit pits, cabbage, cauliflower, and sprouts, which are released upon hydrolysis. Nitrile converting enzymes like nitrilases have been extracted from microorganisms and plants. Nitrilase-mediated biocatalysis reactions have continuously aroused widespread interest to scientists and entrepreneurs in organic synthesis. Nitrile converting biocatalysts (Nitrilases) are now of substantial industrial interest from the perspective of treating toxic nitrile and cyanide-containing compounds. Nitrile degrading enzymes generally consist of nitrilases and amidases. The aim of the current review is to summarize the recent advancements on regioselective nitrilases concerning their fundamental researches and their application in the synthesis of series of high-value fine chemicals and pharmaceuticals. The present review also focuses on the utility of nitrile converting enzyme, sources, properties, classification, structure, and applications as well.

Epidemiological studies suggest that individuals in the Mediterranean region with a loss-of-function, nonsynonymous single nucleotide polymorphism (S188F), in glucose-6-phosphate dehydrogenase ( G6pd ) are less susceptible to vascular diseases. However, this association has not yet been experimentally proven. Here, we set out to determine whether the Mediterranean mutation confers protection from vascular diseases and to discover the underlying protective mechanism. We generated a rat model with the Mediterranean single nucleotide polymorphism (G6PD S188F ) using CRISPR-Cas9 genome editing. In rats carrying the mutation, G6PD activity, but not expression, was reduced to 20% of wild-type (WT) littermates. Additionally, unbiased metabolomics analysis revealed that the pentose phosphate pathway and other ancillary metabolic pathways connected to the pentose phosphate pathway were reduced ( P S188F versus WT rats. Intriguingly, G6PD S188F mutants, as compared with WT rats, developed less large arterial stiffness and hypertension evoked by high-fat diet and nitric oxide synthase inhibition with L-N G -nitroarginine methyl ester. Intravenous injection of a voltage-gated L-type Ca 2+ channel agonist (methyl 2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate; Bay K8644) acutely increased blood pressure in WT but not in G6PD S188F rats. Finally, our results suggested that (1) lower resting membrane potential of smooth muscle caused by increased expression of K + channel proteins and (2) decreased voltage-gated Ca 2+ channel activity in smooth muscle contributed to reduced hypertension and arterial stiffness evoked by L-N G -nitroarginine methyl ester and high-fat diet to G6PD S188F mutants as compared with WT rats. In summary, a mutation resulting in the replacement of a single amino acid (S188F) in G6PD, the rate-limiting enzyme in the pentose phosphate pathway, ascribed properties to the vascular smooth muscle that shields the organism from risk factors associated with vascular diseases.

Pyridine nucleotides, such as NADPH and NADH, are emerging as critical players in the regulation of heart and vascular function. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, is the primary source and regulator of cellular NADPH. In the current study, we have identified two isoforms of G6PD (slow and fast migrating) and functionally characterized the slow migrating isoform of G6PD (G6PD(545)) in bovine and human arteries. We found that G6PD(545) is eluted in the caveolae fraction of vascular smooth muscle (VSM) and has a higher maximum rate of reaction (V(max): 1.65-fold) than its fast migrating isoform (G6PD(515)). Interestingly, caveolae G6PD forms a complex with the pore-forming α(1C)-subunit of the L-type Ca(2+) channel, Ca(v)1.2, as demonstrated by a proximity ligation assay in fixed VSMCs. Additionally, Förster resonance energy transfer (FRET) analysis of HEK293-17T cells cotransfected with red fluorescent protein (RFP)-tagged G6PD(545) (C-G6PD(545)) and green fluorescent protein (GFP)-tagged Ca(v)1.2-(Ca(v)1.2-GFP) demonstrated strong FRET signals as compared with cells cotransfected with Ca(v)1.2-GFP and C-G6PD(515). Furthermore, L-type Ca(2+) channel conductance was larger and the voltage-independent component of availability (c(1)) was augmented in C-G6PD(545) and Ca(v)1.2-GFP cotransfectants compared with those expressing Ca(v)1.2-GFP alone. Surprisingly, epiandrosterone, a G6PD inhibitor, disrupted the G6PD-Ca(v)1.2 complex, also decreasing the amplitude of L-type Ca(2+) currents and window currents, thereby reducing the availability of the c(1) component. Moreover, overexpression of adeno-G6PD(545)-GFP augmented the KCl-induced contraction in coronary arteries compared with control. To determine whether overexpression of G6PD had any clinical implication, we investigated its activity in arteries from patients and rats with metabolic syndrome and found that G6PD activity was high in this disease condition. Interestingly, epiandrosterone treatment reduced elevated mean arterial blood pressure and peripheral vascular resistance in metabolic syndrome rats, suggesting that the increased activity of G6PD augmented vascular contraction and blood pressure in the metabolic syndrome. These data suggest that the novel G6PD-Ca(v)1.2 interaction, in the caveolae fraction, reduces intrinsic voltage-dependent inactivation of the channel and contributes to regulate VSM L-type Ca(2+) channel function and Ca(2+) signaling, thereby playing a significant role in modulating vascular function in physiological/pathophysiological conditions. NEW & NOTEWORTHY In this study we have identified a novel isozyme of glucose-6-phosphate dehydrogenase (G6PD), a metabolic enzyme, that interacts with and contributes to regulate smooth muscle cell l-type Ca(2+) ion channel function, which plays a crucial role in vascular function in physiology and pathophysiology. Furthermore, we demonstrate that expression and activity of this novel G6PD isoform are increased in arteries of individuals with metabolic syndrome and in inhibition of G6PD activity in rats of metabolic syndrome reduced blood pressure.

Aim: To evaluate and compare the staining propensity of SDF, SDF with Potassium iodide and SDF + Glutathione biomolecule on demineralized enamel. Study Design: Forty-five teeth were selected and stratified to receive either 38% SDF; 38% SDF + KI or 38% SDF + 20% GSH. The analysis for staining (DE) was done using a UV- Vis Spectrophotometer and was recorded at different intervals for one month. Data analysis was done using Kruskal- Wallis test, Post-hoc analysis (Dunn) anda repeat-measures analysis of variance (RM-ANOVA). Results: SDF caused maximum staining. SDF + KI staining was significantly less (P

Pulmonary hypertension (PH) is a disease of hyperplasia of pulmonary vascular cells. The pentose phosphate pathway (PPP)—a fundamental glucose metabolism pathway—is vital for cell growth. Because treatment of PH is inadequate, our goal was to determine whether inhibition of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, prevents maladaptive gene expression that promotes smooth muscle cell (SMC) growth, reduces pulmonary artery remodeling, and normalizes hemodynamics in experimental models of PH. PH was induced in mice by exposure to 10% oxygen (Hx) or weekly injection of vascular endothelial growth factor receptor blocker [Sugen5416 (SU); 20 mg kg−1] during exposure to hypoxia (Hx + SU). A novel G6PD inhibitor (N-[(3β,5α)-17-oxoandrostan-3-yl]sulfamide; 1.5 mg kg−1) was injected daily during exposure to Hx. We measured right ventricle (RV) pressure and left ventricle pressure-volume relationships and gene expression in lungs of normoxic, Hx, and Hx + SU and G6PD inhibitor–treated mice. RV systolic and end-diastolic pressures were higher in Hx and Hx + SU than normoxic control mice. Hx and Hx + SU decreased expression of epigenetic modifiers (writers and erasers), increased hypomethylation of the DNA, and induced aberrant gene expression in lungs. G6PD inhibition decreased maladaptive expression of genes and SMC growth, reduced pulmonary vascular remodeling, and decreased right ventricle pressures compared with untreated PH groups. Pharmacologic inhibition of G6PD activity, by normalizing activity of epigenetic modifiers and DNA methylation, efficaciously reduces RV pressure overload in Hx and Hx + SU mice and preclinical models of PH and appears to be a safe pharmacotherapeutic strategy. SIGNIFICANCE STATEMENT The results of this study demonstrated that inhibition of a metabolic enzyme efficaciously reduces pulmonary hypertension. For the first time, this study shows that a novel inhibitor of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in the fundamental pentose phosphate pathway, modulates DNA methylation and alleviates pulmonary artery remodeling and dilates pulmonary artery to reduce pulmonary hypertension.

Aims The diagnosis of joint infections is an inexact science using combinations of blood inflammatory markers and microscopy, culture, and sensitivity of synovial fluid (SF). There is potential for small molecule metabolites in infected SF to act as infection markers that could improve accuracy and speed of detection. The objective of this study was to use nuclear magnetic resonance (NMR) spectroscopy to identify small molecule differences between infected and noninfected human SF. Methods In all, 16 SF samples (eight infected native and prosthetic joints plus eight noninfected joints requiring arthroplasty for end-stage osteoarthritis) were collected from patients. NMR spectroscopy was used to analyze the metabolites present in each sample. Principal component analysis and univariate statistical analysis were undertaken to investigate metabolic differences between the two groups. Results A total of 16 metabolites were found in significantly different concentrations between the groups. Three were in higher relative concentrations (lipids, cholesterol, and N-acetylated molecules) and 13 in lower relative concentrations in the infected group (citrate, glycine, glycosaminoglycans, creatinine, histidine, lysine, formate, glucose, proline, valine, dimethylsulfone, mannose, and glutamine). Conclusion Metabolites found in significantly greater concentrations in the infected cohort are markers of inflammation and infection. They play a role in lipid metabolism and the inflammatory response. Those found in significantly reduced concentrations were involved in carbohydrate metabolism, nucleoside metabolism, the glutamate metabolic pathway, increased oxidative stress in the diseased state, and reduced articular cartilage breakdown. This is the first study to demonstrate differences in the metabolic profile of infected and noninfected human SF, using a noninfected matched cohort, and may represent putative biomarkers that form the basis of new diagnostic tests for infected SF. Cite this article: Bone Joint Res 2021;10(1):85–95.

The extraction of natural dye using an aqueous method is a concept of green chemistry to avoid environmental pollution and toxicity. The present study was performed to improve the color strength an...

Purpose Neutropenic fever in a pediatric oncology patient necessitates emergent initiation of efficacious broad spectrum antibiotics. We have utilized cefepime+/-vancomycin in this context for over two decades, and sough to evaluate the efficacy of this combination at our institution and, if necessary, to identify an optimal alternative regimen. Methods Blood culture isolates from pediatric oncology patients were reviewed over 12 years. Antibiotic susceptibilities for all unique isolates were analyzed via minimum inhibitory concentration breakpoints and interpretive categories as per the Clinical and Laboratory Standards Institute M100/M45 manuals. Percentage susceptibility was calculated, antibiograms generated, and intraspecies antibiotic susceptibility comparisons made. Billing data was analyzed to identify the number of oncology patients admitted for fever during this interval. Results 697 unique isolates were identified: 188 Gram-negative and 509 Gram-positive (155 Gram-positive and 343 total isolates, excluding coagulase negative Staphylococcal (CONS) species). Cefepime and meropenem each covered 95.7% of tested Gram-negatives. Stenotrophomonas (Xanthomonas) maltophilia was the most resistant Gram-negative organism; no antibiotic covered all Gram-negatives. No antibiotic offered significantly improved coverage compared to cefepime. 96.0% of total tested Gram-positives were vancomycin-susceptible (including all CONS); resistant organisms included Enterococcus faecium and Enterococcus faecalis. Linezolid did not offer significantly improved coverage, while daptomycin did. Including and excluding CONS, 46.3% and 72.5% of total Gram-positive isolates were cefepime-sensitive. Blood cultures were positive in 33.7% of admissions. Central venous catheters were routinely utilized in this patient population. Prophylactic antibacterial agents were not utilized. Conclusions Cefepime+/-vancomycin remains efficacious as first-line empiric therapy for febrile neutropenia, with notable minor exceptions. Unit-specific surveillance of antibiotic resistance is warranted to ensure ongoing appropriate empiric antibiotic usage.

Short-course preventive therapy with 12 doses of once-weekly rifapentine (900 mg) plus isoniazid (900 mg) could greatly improve tuberculosis control, especially in areas with high co-endemicity with HIV. However, a small previous trial of such therapy with dolutegravir in healthy, HIV-negative adults was halted early after two of the four patients developed serious adverse events. Because of the potential use of this therapy, and variable safety outcomes of tuberculosis drugs seen in patients with and without HIV, we aimed to characterise safety, pharmacokinetics, and virological suppression in adults who are HIV positive.DOLPHIN was a phase 1/2, single-arm trial done at The Aurum Institute (Tembisa Clinical Research Site, Tembisa, South Africa), with pharmacokinetic visits done at VxPharma (Pretoria, South Africa). Adults (≥18 years) with HIV infection and undetectable viral load (40 copies per mL) after at least 8 weeks of efavirenz-based or dolutegravir-based regimens were recruited in three consecutive groups, subject to approval by the independent safety monitoring committee. Participants received 50 mg of daily dolutegravir in place of efavirenz for 8 weeks, then began once-weekly rifapentine (900 mg)-isoniazid (900 mg) for 12 weeks. Groups 1A (n=12) and 1B (n=18) had intensive dolutegravir pharmacokinetic sampling at week 8 (before rifapentine-isoniazid), at week 11 (after the third dose of rifapentine)-isoniazid and at week 16 after the eighth dose. Group 2 (n=30) were treated with the same schedule and had sparse dolutegravir pharmacokinetic sampling at weeks 8, 11, and 16. Participants were followed 4 weeks after completion of prophylactic tuberculosis treatment. HIV viral loads were measured at baseline and at weeks 11 and 24. Primary endpoints were adverse events (grade 3 or higher) and dolutegravir population pharmacokinetics, assessed in participants who began rifapentine-isoniazid. This trial was registered at ClinicalTrials.gov, NCT03435146.Between Jan 24, 2018, and Nov 25, 2018, 61 participants were enrolled into three groups; one participant withdrew (from group 1A). 43 (70%) of 60 participants were women and all participants were black African. Median age was 40 years (IQR 35-48), CD4 cell count was 683 cells per μL (447-935), and body-mass index was 28·9 kg/mOur results suggest 12 doses of once-weekly rifapentine-isoniazid can be given for tuberculosis prophylaxis to patients with HIV taking dolutegravir-based antiretroviral therapy, without dose adjustments. Further exploration of the pharmacokinetics, safety, and efficacy in children and pharmacodynamics in individuals naive to antiretroviral therapy is needed.UNITAID.

BACKGROUND: Approximately 10% of incident TB cases worldwide are attributable to alcohol. However, evidence associating alcohol with unfavorable TB treatment outcomes is weak.METHODS: We prospectively evaluated men (≥18 years) with pulmonary TB in India for up to 24 months to investigate the association between alcohol use and treatment outcomes. Unhealthy alcohol use was defined as a score of ≥4 on the Alcohol Use Disorders Identification Test-Concise (AUDIT-C) scale at entry. Unfavorable TB treatment outcomes included failure, recurrence, and all-cause mortality, analyzed as composite and independent endpoints.RESULTS: Among 751 men, we identified unhealthy alcohol use in 302 (40%). Median age was 39 years (IQR 28–50); 415 (55%) were underweight (defined as a body mass index [BMI] 2); and 198 (26%) experienced an unfavorable outcome. Unhealthy alcohol use was an independent risk factor for the composite unfavorable outcome (adjusted incidence rate ratio [aIRR] 1.47, 95% CI 1.05–2.06; P = 0.03) and death (aIRR 1.90, 95% CI 1.08–3.34; P = 0.03), specifically. We found significant interaction between AUDIT-C and BMI; underweight men with unhealthy alcohol use had increased risk of unfavorable outcomes (aIRR 2.22, 95% CI 1.44–3.44; P < 0.001) compared to men with BMI ≥18.5 kg/m2 and AUDIT-C CONCLUSION: Unhealthy alcohol use was independently associated with unfavorable TB treatment outcomes, highlighting the need for integrating effective alcohol interventions into TB care.

Background:Implant wear in joint replacements is influenced by the chemical and physical properties of human synovial fluid (HSF). In vitro testing for implant wear uses 25% weight bovine calf serum (25BCS) to substitute for HSF, due to similar rheology and total protein content. However, previous studies have shown differences in the macromolecular composition. We aimed to evaluate the differences in small molecule composition between fluids and correlate these differences with their effects on implant material wear.Methods:HSF was harvested from osteoarthritis patients undergoing primary knee replacement (n=14). Nuclear magnetic resonance (NMR) spectroscopy with linear regression modelling analysed the metabolites present in HSF and commercially sourced 25BCS and investigated the differences. Wear properties of the fluids were evaluated using a validated quantitative laboratory bench-test utilising a cobalt/chromium/molybdenum (CoCrMo) ball oscillating against a CoCrMo disc and analysing the resulting wear scar. The variation in metabolite levels in both HSF and 25BCS was correlated to the wear properties of the fluids.Results:Differences in the levels of metabolites, lipids, and glycosaminoglycans (GAG) were observed between HSF and 25BCS: significance was confirmed by O-PLS-DA (pConclusions:The small molecule concentration differences between the fluids questions the validity of 25BCS as a model for wear analysis. The demonstration of variable metabolites present in HSF which correlate with material wear has implications for implant failure and targeted therapeutic manipulation of these metabolites. Trial Registration:Ethical approval was granted by the NRES Committee London, Chelsea, UK on the 12th May 2015. The study was performed in accordance with the ethical standards in the 1964 Declaration of Helsinki.

SETTING The optimal timing of screening for diabetes mellitus (DM) among tuberculosis (TB) cases is unclear due to the possibility of stress hyperglycemia. DESIGN We evaluated adult (18 years) pulmonary TB cases at treatment initiation as well as at 3 months, 6 months and 12 months. DM was identified by self-report (known DM) or glycated hemoglobin (HbA1c)  6.5% (new DM). Trends in HbA1c levels during treatment were assessed using non-parametric tests. RESULTS Of the 392 participants enrolled, 75 (19%) had DM, 30 (40%) of whom had new DM. Of the 45 participants with known DM, respectively 37 (82%) and 40 (89%) received medication to lower glucose levels at treatment initiation and completion; one participant with new DM initiated glucose-lowering medication during follow-up. The median HbA1c level in participants with known, new and no DM was respectively 10.1% (interquartile range [IQR] 8.3-11.6), 8.5% (IQR 6.7-11.5) and 5.6% (IQR 5.3-5.9) at treatment initiation, and 8.7% (IQR 6.8-11.3), 7.1% (IQR 5.8-9.5) and 5.3% (IQR 5.1-5.6) at treatment completion (P < 0.001). Overall, 5 (12%) with known and 13 (43%) with new DM at treatment initiation had reverted to HbA1c < 6.5% by treatment completion (P = 0.003); the majority of reversions occurred during the first 3 months, with no significant reversions beyond 6 months. CONCLUSION HbA1c levels declined with anti-tuberculosis treatment. Repeat HbA1c testing at treatment completion could reduce the risk of misdiagnosis of DM.

A major subset of IL-10 secreting B-cells, commonly characterized as CD19+/38hi/24hi, encompass B-regulatory cells (B-regs). We report effects of changes in stimulating molecule concentration on population of Interleukin-10 secreting B-regs of human origin. B-regs may be obtained from in-vitro co-culture of adipose tissue derived mesenchymal stromal cells (AD-MSC) and peripheral blood mononuclear cells (PBMC) for potential cell therapy. Lipopolysaccharide–E.coli-K12 strain (LPS-EK) was added in varying concentrations in the co-culture of AD-MSC and peripheral blood mononuclear cells (PBMC). At each time point, IL-10 concentration of cell supernatant was estimated by quantitative ELISA. Morphology, sterility, total count, viability and immune-phenotype of cells were determined. We analyzed the data to determine the time interval at which maximum B-reg population and optimum secretion of IL-10 was obtained. The present study provides precious information about the in vitro dynamics of IL-10 secretion by human B-regs generated from AD-MSC and PBMC. The present study focuses on in vitro generation of B-regs from adipose tissue derived mesenchymal stromal cells (AD-MSC) and peripheral blood mononuclear cells (PBMC), as well as on partial characterization of B-regs in terms of amount of IL-10 secreted, time taken and amount of stimulating molecule (i.e. LPS-EK) required for optimum IL-10 secretion. It has been reported that B-regs secrete cytokine in trace quantities. The medium which is added for feeding the cells further dilutes the secreted cytokines by many folds. In our study, the samples were appropriately concentrated, so that the cytokines could be detected by quantitative Enzyme Linked Immunosorbent Assay (ELISA).

STAT1α is a key transcription factor driving pro-inflammatory responses in macrophages. We found that the interferon gamma (IFNγ)-regulated transcriptional program in macrophages is controlled by ADP-ribosylation (ADPRylation) of STAT1α, a post-translational modification resulting in the site-specific covalent attachment of ADP-ribose moieties. PARP-1, the major nuclear poly(ADP-ribose) polymerase (PARP), supports IFNγ-stimulated enhancer formation by regulating the genome-wide binding and IFNγ-dependent transcriptional activation of STAT1α. It does so by ADPRylating STAT1α on specific residues in its DNA-binding domain (DBD) and transcription activation (TA) domain. ADPRylation of the DBD controls STAT1α binding to its cognate DNA elements, whereas ADPRylation of the TA domain regulates enhancer activation by modulating STAT1α phosphorylation and p300 acetyltransferase activity. Loss of ADPRylation at either site leads to diminished IFNγ-dependent transcription and downstream pro-inflammatory responses. We conclude that PARP-1-mediated ADPRylation of STAT1α drives distinct enhancer activation mechanisms and is a critical regulator of inflammatory responses in macrophages., STAT1a is required for pro-inflammatory responses in macrophages. Here the authors reveal that post-translational modification of STAT1a, ADPribosylation, plays a critical role in enhancer formation and activation, thus modulating IFNγ-stimulated inflammatory responses in macrophages.

Banana pseudostem, a cellulose-rich by-product, is regarded as an important agricultural waste during the process of banana production. Microcrystalline cellulose was successfully prepared from banana pseudostem using acid hydrolysis method. Microcrystalline cellulose was characterized through various techniques such as XRD, TGA, SEM, FTIR and antioxidant activity to explore the possible applications in the pharmaceutical industries especially as a drug delivery vehicle. The investigation revealed that the derived microcrystalline cellulose is non-aggregated, short rods with high crystallinity index 67% and stable up to 347 °C. FTIR spectroscopy showed that hydrolysis treatments are efficient for the removal of lignin and hemicellulose content. Microcrystalline cellulose exhibited good antioxidant activity 90.29% at 100 μg/ml. In vitro studies for the drug release were carried out in simulated intestinal fluid (SIF) using Isoniazid drug. The study proves that microcrystalline cellulose can be directly obtained from banana pseudostem which is not only beneficial to reduce the cost of traditional microcrystalline cellulose but is also conducive to the value-added utilization of the pseudostem.

The basic media for cell culture are α-MEM and DMEM. α-MEM has lesser and rather optimal glucose and amino acid concentration as compared to DMEM. In our current study we compared the individual effect of these two media on quantity, viability and immunophenotyping of AD-MSC. Adipose tissue was procured from anterior abdominal wall of healthy willing donors. It was divided equally into two tissue culture flasks with culture medium, one containing α-MEM and the other containing DMEM. It was incubated in collagenase-I. Pellet was re-suspended in centrifuge tubes with phosphate buffered saline and divided into 6 equal parts. Each set was incubated separately for in-vitro generation in 6-well plates. Media were replenished on alternate days and no passaging was done. One well from each set was harvested by trypsinization, every 3rd day. Sterility, quantity, viability and immunophenotyping (CD45-/90+/73+) by flow cytometry were checked. We analyzed the data to determine maximum total cell count, viability, CD45-CD90+ and CD45-CD73+ population in α-MEM and compared it with that in DMEM.

Diabetes is considered as one of the comorbidities with COVID-19 infection and indicated to influence the severity and the rate of recovery in COVID-19 patients. However, it is not known whether the lack of diabetes management may magnify COVID-19 severity and the rate of recovery in patients with diabetes. Purpose: The purpose of this study was to determine the effect of diabetes management on the severity and length of hospitalization in hospitalized COVID-19 patients. Methods: This retrospective study used medical records from patients admitted to the University Medical Center, El Paso, TX with COVID-19 (n=369; Age 60.0 ± 0.8 years; BMI 30.3 ± 0.4 Kg/m2). Glycemia was assessed by glycated hemoglobin (A1c) level at the time of hospitalization. The severity of the COVID-19 outcome was assessed by quick sepsis-related organ failure assessment (qSOFA) and length of hospitalization was determined by the number of days spent in the hospital. Patients were categorized into normoglycemic (N: A1c Results: There was no significant difference among groups in severity of COVID-19 infection (qSOFA: N 0.24 ± 0.08, Pre 0.38 ± 0.07, D 0.28 ± 0.03; p>0.05) and length of hospitalization (N 5.6 ± 0.8, Pre 9.3 ± 1.0, DM 8.9 ± 0.6 days; p>0.05). However, patients with unmanaged diabetes (no medication) showed significantly greater severity (qSOFA: 0.44 ± 0.08 vs. 0.22 ± 0.03; p Conclusion: Unmanaged diabetes shows worsened severity and the rate of recovery in COVID-19 patients. Diabetes management should be considered in the treatment of COVID-19 patients. Disclosure A. Mossayebi: None. S. Bajpeyi: None. H. Kreit: None. S. V. Cherukuri: None. R. A. Mandania: None. J. B. Concha: None. H. Jung: None. A. E. Wagler: None. A. A. Gupte: None. A. Deoker: None.

Drive train electrification in automotive industry has become the general trend in recent years. As crucial components in the encapsulation and isolation of future wide-bandgap semiconductor-based power cards, epoxy resin and its composites face great challenges from the high voltage operation. This work provides an exploration into employing a group of polycyclic aromatic hydrocarbon molecules with varied number of aromatic rings, namely naphthalene, anthracene and pyrene, as voltage stabilizers in epoxy resin. The conjugated $\pi$ system can be excited by the incoming high energy electrons, thus absorbing their kinetic energy which endangers the polymer integrity. The UV-Vis spectra of the compounds were recorded to illustrate the energy absorbing behavior, and the effects of these additives on epoxy curing, glass transition temperature and dielectric properties are presented. In the study, pyrene was found to enhance the breakdown voltage of the epoxy film.

Summary Background & aims Nutritional deficiency and inflammation may impact CD4+ T cell recovery during combination antiretroviral therapy (cART), particularly in resource-limited settings where malnutrition is prevalent. The aim of this study was to investigate the relationship of micronutrient and inflammation biomarkers to CD4 recovery after cART initiation. Methods We conducted a secondary analysis of a random sub-cohort sample (n = 270) from a multinational randomized trial of cART regimen efficacy among 1571 cART-naive adults. We measured pre-cART serum levels of micronutrients (Vitamin A, B6, B12, D, total carotenoids, selenium, and iron) and inflammation (C-reactive protein, soluble CD14 (sCD14), IFNγ, TNFα, Interleukin-6, and C-X-C motif chemokine 10 (CXCL10/IP10), EndoCab (IgM)) biomarkers. Biomarker status (i.e. micronutrient deficiency vs. sufficiency and elevated vs. low inflammation) was defined using established cutoffs or quartiles. Mixed-effects linear regression models were used to determine the association of baseline (pre-cART) concentrations of individual biomarkers with CD4 recovery through 96 weeks post-cART initiation. Results In models adjusting for time-dependent viral load and baseline CD4 count, age, sex, body mass index, country, treatment regimen, anemia and hypoalbuminemia status, pre-cART vitamin D deficiency was associated with lower CD4 recovery (−14.9 cells/mm3, 95% CI: −27.9, −1.8) compared to sufficiency. In contrast, baseline selenium deficiency (20.8 cells/mm3, 95% CI: 3.3, 38.3), vitamin A deficiency (35.9 cells/mm3, 95% CI: 17.6, 54.3) and high sCD14 (23.4 cells/mm3, 95% CI: 8.9, 37.8) were associated with higher CD4 recovery compared to sufficient/low inflammation status. Conclusions In summary, baseline vitamin D deficiency was associated with diminished CD4 recovery after cART initiation; impaired CD4 recovery may contribute to the poor clinical outcomes recently observed in individuals with vitamin D deficiency. Vitamin A, selenium and sCD14 were associated with CD4 recovery but future studies are needed to further explore these relationships.

Summary Objective To perform a systematic review of the small molecule metabolism studies of osteoarthritis utilising nuclear magnetic resonance (NMR) or mass spectroscopy (MS) analysis (viz., metabolomics or metabonomics), thereby providing coherent conclusions and reference material for future study. Method We applied PRISMA guidelines (PROSPERO 95068) with the following MESH terms: 1. “osteoarthritis” AND (“metabolic” OR “metabonomic” OR “metabolomic” OR “metabolism”) 2. (“synovial fluid” OR “cartilage” OR “synovium” OR “serum” OR “plasma” OR “urine”) AND (“NMR” or “Mass Spectroscopy”). Databases searched were “Medline” and “Embase”. Studies were searched in English and excluded review articles not containing original research. Study outcomes were significant or notable metabolites, species (human or animal) and the Newcastle–Ottawa Score. Results In the 27 studies meeting the inclusion criteria, there was a shift towards anaerobic and fatty acid metabolism in OA disease, although whether this represents the inflammatory state remains unclear. Lipid structure and composition was altered within disease subclasses including phosphatidyl choline (PC) and the sphingomyelins. Macromolecular proteoglycan destruction was described, but the correlation to disease factors was not demonstrated. Collated results suggested arachidonate signalling pathways and androgen sex hormones as future metabolic pathways for investigation. Conclusion Our meta-analysis demonstrates significant small molecule differences between sample types, between species (such as human and bovine), with potential OA biomarkers and targets for local or systemic therapies. Studies were limited by numbers and a lack of disease correlation. Future studies should use NMR and MS analysis to further investigate large population subgroups including inflammatory arthropathy, OA subclasses, age and joint differences.

Evaluating the biomass degradation using fast, validate and sensitive techniques for exploratory purposes of biofuel production has been developed since last decade. Thus, we assessed the degradation of two Indian hardwoods using FTIR and chemometric approaches. Two white rot fungi, namely Pseudolagarobasidium acaciicola AGST3 and Tricholoma giganteum AGDR1, were selected among twenty-one fungal isolates for higher hardwood degradation. In the screening, P. acaciicola AGST3 and T. giganteum AGDR1 depicted the dry woody mass loss of 20.51% and 22.38%, respectively. Cellulose crystallinity of P. acaciicola AGST3 treated hardwoods was 4-fold lower than untreated hardwoods, showing the higher cellulose degradation efficiency. P. acaciicola AGST3 treated samples exhibited maximum deviation of guaiacyl units of lignin, cellulose and hemicelluloses. T. giganteum AGDR1 treated hardwoods showed maximum deviation of guaiacyl- and syringyl- units of lignin and hemicelluloses. Multivariate approach revealed the degradation patterns and preferences are varied based on the fungi and hardwood. The approach used in the present study can certainly distinguish the variations among the different biomass samples that having similar composition. Additionally, higher lignin degradability of these fungi can be used in biomass pretreatment, which significantly utilized to produce second-generation biofuels.

In the U.S., 30% of adults suffer joint pain, most commonly in the knee, which severely limits mobility and is often attributed to injury of cartilage and underlying bone in the joint. Current treatment methods such as microfracture result in less resilient fibrocartilage with eventual failure; autografting can cause donor site morbidity and poor integration. To overcome drawbacks in treatment, tissue engineers can design cell-instructive biomimetic scaffolds using biocompatible materials as alternate therapies for osteochondral defects. Nanofibrous poly (l-lactic acid) (PLLA) scaffolds of uniform, spherical, interconnected and well-defined pore sizes that are fabricated using a thermally-induced phase separation and sugar porogen template method create an extracellular matrix-like environment which facilitates cell adhesion and proliferation. Herein we report that chondrogenesis and endochondral ossification of rabbit and human bone marrow stromal cells (BMSCs) can be controlled by scaffold pore architecture, particularly pore size. Small-pore scaffolds support enhanced chondrogenic differentiation in vitro and cartilage formation in vivo compared to large-pore scaffolds. Endochondral ossification is prevented in scaffolds with very small pore sizes; pore interconnectivity is critical to promote capillary ingrowth for mature bone formation. These results provide a novel strategy to control tissue regenerative processes by tunable architecture of macroporous nanofibrous scaffolds. STATEMENT OF SIGNIFICANCE: Progress in understanding the relationship between cell fate and architectural features of tissue engineering scaffolds is critical for engineering physiologically functional tissues. Sugar porogen template scaffolds have uniform, spherical, highly interconnected macropores. Tunable pore-size guides the fate of bone marrow stromal cells (BMSCs) towards chondrogenesis and endochondral ossification, and is a critical design parameter to mediate neotissue vascularization. Preventing vascularization favors a chondrogenic cell fate while allowing vascularization results in endochondral ossification and mineralized bone formation. These results provide a novel strategy to control tissue regenerative processes by tunable architecture of macroporous nanofibrous scaffolds.

BackgroundVitamins B12 and folate participate in the one-carbon metabolism cycle and hence regulate fetal growth. Though vitamin B12 deficiency is widely prevalent, the current public health policy in India is to supplement only iron and folic acid for the prevention of anaemia. Prompted by our research findings of the importance of maternal vitamin B12 status for a healthy pregnancy, birth and offspring health outcomes, we evaluated available literature evidence using a systematic review approach, to inform policy.MethodsA systematic search was performed for relevant Indian studies in the MEDLINE/PubMed and IndMed databases. We selected studies reporting maternal vitamin B12 status (dietary intake or blood concentrations), and/or metabolic markers of vitamin B12 deficiency (homocysteine, methylmalonic acid) or haematological indices during pregnancy and their associations with outcomes of pregnancy, infancy or in later life. Intervention trials of vitamin B12 during pregnancy were also included. Quality of evidence was assessed on the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement.ResultsOf the 635 articles identified, 46 studies met the inclusion criteria (cohort studies-26, case-control studies-13, RCT’s -7). There is a high prevalence of vitamin B12 deficiency in Indian women during pregnancy (40-70%) (3 studies). Observational studies support associations (adjusted for potential sociodemographic confounders, maternal body size, postnatal factors) of lower maternal B12, higher homocysteine or an imbalance between vitamin B12-folate status with a higher risk of NTDs (6 studies), pregnancy complications (recurrent pregnancy losses, gestational diabetes, pre-eclampsia) (9 studies), lower birth weight (10 studies) and adverse longer-term health outcomes in the offspring (cognitive functions, adiposity, insulin resistance) (11 studies). Vitamin B12 supplementation (7 RCT’s) in pregnancy showed a beneficial effect on offspring neurocognitive development and an effect on birth weight was inconclusive. There is a high quality evidence to support the role of low maternal vitamin B12 in higher risk for NTD and low birth weight and moderate-quality evidence for higher risk of gestational diabetes and later life adverse health outcomes (cognitive functions, risk for diabetes) in offspring.ConclusionIn the Indian population low maternal vitaminB12 status, is associated with adverse maternal and child health outcomes. The level of evidence supports adding vitamin B12 to existing nutritional programs in India for extended benefits on outcomes in pregnancy and offspring health besides control of anaemia.Systematic Review Registration[website], identifier [registration number]

Background Increasing numbers of successful paediatric liver transplantation (PLT) with improved survival rates reaching 90%at 10 years resulted in more children being listed for PLT but this was not associated with matching expansion in graft pool resulting in graft shortage. Reports including our published early experience showed promising short and intermediate term outcomes from DCD grafts in children. Aim To compare long-term outcomes of full size and reduced DCD grafts in terms of incidence of complications as well as survival of both recipients and grafts. Methods This is a retrospective review of PLTs using DCD grafts. Patients were divided into those who received full or reduced grafts. Data was collected for comparison of pre-transplant recipient parameters, donor parameters, operative parameters, post-transplant recipient parameters and outcomes. Laboratory markers were checked at discharge, 1- and 5-years post-transplant. Results 14 PLTs from DCD donors between 2005 and 2018 were identified; 9 full size and 5 reduced grafts. Donors of both groups were Maastricht category III donors. Functional warm ischemia time did not show significant difference while cold ischemia time was significantly longer in reduced graft group. Recipients of full grafts were significantly older, heavier in weight and tend to wait significantly longer on transplant waiting list. The main indications for PLT were biliary atresia and Alpha-1 anti-trypsin deficiency. Recipients of reduced grafts were sicker pre-transplant. Post-transplant hospital and PICU stay were longer in the reduced group but this was not significant. Only significant laboratory difference was at 1-year post-transplant as albumen and creatinine were significantly higher in the full graft group while ALT was significantly higher in the reduced graft group. There was no significant difference between two groups in terms of long-term complications. 3 patients in each group survived more than 5 years post-transplant. one child was re-transplanted in the reduced group due to portal vein thrombosis, this patient received another DBD graft then unfortunately died due to liver failure. One child from full graft group also died from a non-graft related cause (figures 1, 2). Summary 14 PLTs from DCD grafts (9 full and 5 reduced) were identified. Recipients of full grafts were older and heavier while reduced graft recipients were sicker in the pre-transplant period. Both groups demonstrated comparable graft and patient survival as well as long-term complications rates. Conclusion Selected DCD grafts are potential source to widen the graft pool in children

Melanoma is one of the most malignant skin cancers that require comprehensive therapies, including chemotherapy. A plant-derived drug, plumbagin (PLB), exhibits an anticancer property in several cancers. We compared the cytotoxic and metabolic roles of PLB in A375 and SK-MEL-28 cells, each with different aggressiveness. In our results, they were observed to have distinctive mitochondrial respiratory functions. The primary reactive oxygen species (ROS) source of A375 can be robustly attenuated by cell membrane permeabilization. A375 cell viability and proliferation, migration, and apoptosis induction are more sensitive to PLB treatment. PLB induced metabolic alternations in SK-MEL-28 cells, which included increasing mitochondrial oxidative phosphorylation (OXPHOS), mitochondrial ATP production, and mitochondrial mass. Decreasing mitochondrial OXPHOS and total ATP production with elevated mitochondrial membrane potential (MMP) were observed in PLB-induced A375 cells. PLB also induced ROS production and increased proton leak and non-mitochondria respiration in both cells. This study reveals the relationship between metabolism and cytotoxic effects of PLB in melanoma. PLB displays stronger cytotoxic effects on A375 cells, which exhibit lower respiratory function than SK-MEL-28 cells with higher respiratory function, and triggers cell-specific metabolic changes in accordance with its cytotoxic effects. These findings indicate that PLB might serve as a promising anticancer drug, targeting metabolism.

Classical pharmacological models have incorporated an "intrinsic efficacy" parameter to capture system-independent effects of G protein-coupled receptor (GPCR) ligands. However, the nonlinear serial amplification of downstream signaling limits quantitation of ligand intrinsic efficacy. A recent biophysical study has characterized a ligand "molecular efficacy" that quantifies the influence of ligand-dependent receptor conformation on G protein activation. Nonetheless, the structural translation of ligand molecular efficacy into G protein activation remains unclear and forms the focus of this study. We first establish a robust, accessible, and sensitive assay to probe GPCR interaction with G protein and the Gα C terminus (G-peptide), an established structural determinant of G protein selectivity. We circumvent the need for extensive purification protocols by the single-step incorporation of receptor and G protein elements into giant plasma membrane vesicles (GPMVs). We use previously established SPASM FRET sensors to control the stoichiometry and effective concentration of receptor-G protein interactions. We demonstrate that GPMV-incorporated sensors (v-SPASM sensors) provide enhanced dynamic range, expression-insensitive readout, and a reagent level assay that yields single point measurements of ligand molecular efficacy. Leveraging this technology, we establish the receptor-G-peptide interaction as a sufficient structural determinant of this receptor-level parameter. Combining v-SPASM measurements with molecular dynamics (MD) simulations, we elucidate a two-stage receptor activation mechanism, wherein receptor-G-peptide interactions in an intermediate orientation alter the receptor conformational landscape to facilitate engagement of a fully coupled orientation that tunes G protein activation.

Background and objective: Administration of some additives with local anesthetics can prolong pain free period post-operatively, thereby reducing need for post-operative analgesics and improving patient comfort. Potassium chloride was found to increase duration and quality of anesthesia in various studies on brachial plexus blockade. This study was designed to evaluate and compare the effect of 2% lignocaine with 1.5% potassium chloride, 2% lignocaine with adrenaline and 2% lignocaine (plain) in pterygomandibular nerve blocks. Materials and methods: A triple blind randomized controlled study was conducted on 120 adults, aged 18–45 years in ASA-I category, requiring surgical extraction of impacted mandibular third molars. The subjects were divided equally into 3 groups randomly by computer generated sequence; Group 1: 2% lignocaine plus 1.5% solution of potassium chloride, group 2: 2% lignocaine with 1:80,000 adrenaline and group 3: 2% plain lignocaine. Onset, duration, depth (pain) of anesthesia, patient satisfaction, systolic and diastolic blood pressures, heart rate and oxygen saturation, were evaluated and compared. Results: Onset was shortest for group1 and longest for grp3, statistically highly significant difference between the 3 groups (p < 0.01). Statistically significant difference (p < 0.05) was found in duration of surgery, duration of analgesia and VAS scores between groups 1 & 3. Duration and depth of anesthesia were comparable for groups 1 & 2. There was no statistically significant difference seen for total amount of dose used, SBP, DBP, HR and SpO2 between the 3 groups (p > 0.05). Conclusion: Potassium chloride, a physiological salt is inert and causes no local/systemic adverse effects when injected with lignocaine in physiologically permissible amounts. The combination achieves satisfactory onset, duration, depth of anesthesia without altering hemodynamic variables. Hence, it may be considered as a safe and effective additive.

The impact of metabolism upon the altered pathology of joint disease is rapidly becoming recognized as an important area of study. Synovial joint fluid is an attractive and representative biofluid of joint disease. A systemic review revealed little evidence of the metabolic stability of synovial joint fluid collection, handling or storage, despite recent reports characterizing the metabolic phenotype in joint disease. We aim to report the changes in small molecule detection within human synovial fluid (HSF) using nuclear magnetic resonance (NMR) spectroscopy at varying storage temperatures, durations and conditions. HSF was harvested by arthrocentesis from patients with isolated monoarthropathy or undergoing joint replacement (n = 30). Short-term storage (0-12 h, 4°C18°C) and the effect of repeated freeze-thaw cycles (-80°C to 18°C) was assessed. Long-term storage was evaluated by early (-80°C,21days) and late analysis (-80°C, 10-12 months). 1D NMR spectroscopy experiments, NOESYGPPR1D and CPMG identified metabolites and semi-quantification was performed. Samples demonstrated broad stability to freeze-thaw cycling and refrigeration of4 h. Short-term room temperature or refrigerated storage showed significant variation in 2-ketoisovalerate, valine, dimethylamine, succinate, 2-hydroxybutyrate, and acetaminophen glucuronide. Lipid and macromolecule detection was variable. Long-term storage demonstrated significant changes in: acetate, acetoacetate, creatine, N,N-dimethylglycine, dimethylsulfone, 3-hydroxybutyrate and succinate. Changeable metabolites during short-term storage appeared to be energy-synthesis intermediates. Most metabolites were stable for the first four hours at room temperature or refrigeration, with notable exceptions. We therefore recommend that HSF samples should be kept refrigerated for no more than 4 hours prior to freezing at -80°C. Furthermore, storage of HSF samples for 10-12 months before analysis can affect the detection of selected metabolites.

ER/K α-helices are a subset of single alpha helical (SAH) domains, which exhibit unusual stability as isolated protein secondary structures. They adopt an elongated structural conformation, while regulating the frequency of interactions between proteins or polypeptides fused to their ends. Here we review recent advances on the structure, stability and function of ER/K α-helices as linkers (ER/K linkers) in native proteins. We describe methodological considerations in the molecular cloning, protein expression and measurement of interaction strengths, using sensors incorporating ER/K linkers. We highlight biological insights obtained over the last decade by leveraging distinct biophysical features of ER/K-linked sensors. We conclude with the outlook for the use of ER/K linkers in the selective modulation of dynamic cellular interactions.

Immobilization of enzyme with nanostructures enhances its ideal characteristics, which may allow the enzyme to become more stable and resistant. The present investigation deals with the formulation of laccase nanosilica conjugates to overcome the problems associated with its stability and reusability. Synthesized nanosilica and laccase nanoparticles were spherical shaped, with the mean size of 220 and 615 nm, respectively. Laccase nanoparticles had an optimum temperature of 55 °C and pH 4.0 for the oxidation of ABTS. Laccase nanoparticle retained 79% of residual activity till 20th cycle. It also showed 91% of its initial activity at lower temperatures even after 60 days. Laccase nanoparticles were applied for Reactive Violet 1 degradation wherein 96.76% of decolourization was obtained at pH 5.0 and 30 °C within 12 h. Toxicity studies on microbes and plants suggested that the degraded metabolites were less toxic than control dye. Thus, the method applied for immobilization increased storage stability and reusability of laccase, and therefore, it can be utilized for efficient degradation of azo dyes. Graphical Abstract

Hydrocarbon pollution is a perennial problem not only in India but throughout the globe. A plethora of microorganisms have been reported to be efficient degraders of these recalcitrant pollutants. One of the major concerns of environmental problem is the presence of hydrocarbons due to the various anthropogenic activities. PAHs are ubiquitous in naturei.e.present in soil, water and air. Presence of PAHs in environment creates problem as their presence have deleterious effect on human and animals. They also have the ability to cause the tumors in human and animals. Some of the microorganisms are capable of transforming and degrading these PAHs and remove them from the environment. The present review describes about the sources, structure, fate and toxicity of PAHs as well as different bioremediation techniques involved in the removing of contaminants from the environment which are efficient and cost-effective. The conventional approaches used for removal of PAH are not only environment friendly but also are able to reduce the risk to human and ecosystem.

Mycoremediation is a technique wherein various fungi are used in the remediation of hazardous contaminants like polycyclic aromatic hydrocarbons, dyes, hazardous phenolics, heavy metals, and several others. Fungi are the ubiquitous and diverse group of organisms that produce a wide array of enzymes and metabolites (organic acids, exopolysaccharides, etc.). By synergic action of these metabolites, fungi can efficiently degrade or transform the hazardous contaminants. Additionally, live or dead fungal biomass has the higher sorption capability of contaminants as compared to the other microorganisms. In this chapter, application of fungi in degrading dyes and polycyclic aromatic hydrocarbons, remediation of heavy metals, and other contaminants is discussed, wherein fungi degrade the organics and aid in remediation of metals using a variety of strategies.

While allosteric modulation of GPCR signaling has gained prominence to address the need for receptor specificity, efforts have mainly focused on allosteric sites adjacent to the orthosteric ligand‐binding pocket and lipophilic molecules that target transmembrane helices. In this study, we examined the allosteric influence of native peptides derived from the C‐terminus of the Gα subunit (G‐peptides) on signaling from two Gi‐coupled receptors, adenosine A1 receptor (A1R) and cannabinoid receptor 1 (CB1). We expressed A1R and CB1 fusions with G‐peptides derived from Gαs, Gαi, and Gαq in HEK 293 cells using systematic protein affinity strength modulation (SPASM) and monitored the impact on downstream signaling in the cell compared to a construct lacking G‐peptides. We used agonists N6‐Cyclopentyladenosine (CPA) and 5’‐N‐Ethylcarboxamidoadenosine (NECA) for A1R and 2‐Arachidonoylglycerol (2‐AG) and WIN 55,212‐2 mesylate (WN) for CB1. G‐peptides derived from Gαi and Gαq enhance agonist‐dependent cAMP inhibition, demonstrating their effect as positive allosteric modulators of Gi‐coupled signaling. In contrast, both G‐peptides suppress agonist‐dependent IP1 levels suggesting that they differentially function as negative allosteric modulators of Gq‐coupled signaling. Taken together with our previous studies on Gs‐coupled receptors, this study provides an extended model for the allosteric effects of G‐peptides on GPCR signaling, and highlights their potential as probe molecules to enhance receptor specificity., Gα peptides differentially impact Gs and Gi signaling in Cannabinoid (CB1) receptors.

To mimic the bone matrix of mineralized collagen and to impart microporous structure to facilitate cell migration and bone regeneration, we developed a nanofibrous polymer scaffold with highly interconnected pores and three-dimensional calcium phosphate coating utilizing an electrodeposition technique. The mineral content, morphology, crystal structure and chemical composition could be tailored by adjusting the deposition temperature, voltage and duration. A higher voltage and a higher temperature led to a greater rate of mineralization. Furthermore, nearly linear calcium releasing kinetics was achieved from the mineralized 3D scaffolds. The releasing rate was controlled by varying the initial electrodeposition conditions. The higher deposition voltage and temperature led to slower calcium release, which was associated with the highly crystalline and stoichiometric hydroxyapatite content. This pre-mineralized nanofibrous scaffold enhanced bone regeneration over the control scaffold in a subcutaneous implantation model, which was associated with released calcium ions in facilitating osteogenic cell proliferation.

Biosorption of copper(II) and cadmium(II) was studied using Bacillus cereus sys1 cells. The biosorption capacity of the cells has been studied as a function of pH, initial metal concentration, cell concentration and contact time. Batch studies showed that the B. cereus sys1 cells could adsorb up to 90% of both metals. For copper, total contact time of 90 min was required with 100 ppm initial metal concentration at pH 5 using 10 mg/ml cells, whereas 180 min was required for 30 ppm cadmium at pH 7. The study demonstrated correlation with Langmuir model with different concentrations of both Cu(II) and Cd(II). The adsorption capacity was found to be 225 mg/g for copper and 34.67 mg/g for cadmium. As per the Freundlich isotherm, value of 1/n, which shows the strength of adsorption, was found to be 0.5114 for copper and 0.2492 for cadmium indicating a normal and favourable adsorption intensity. The surface adsorption of metals was confirmed using SEM with EDS. It revealed that the cells treated with oil could adsorb 7.55% copper as compared to untreated cells which showed 7.29% adsorption. Similarly, for cadmium oil-treated cells showed 9.44% adsorption compared to 2.63% of untreated cells. This indicated that the cells grown in the presence of oil have a better capacity for metal ion adsorption.

Shorter, more potent regimens are needed for tuberculosis. The nitroimidazole pretomanid was recently approved for extensively drug-resistant tuberculosis in combination with bedaquiline and linezolid. Pretomanid may also have benefit as a treatment-shortening agent for drug-sensitive tuberculosis. It is unclear how and whether it can be used together with rifamycins, which are key sterilizing first-line drugs. In this analysis, data were pooled from two studies: the Assessing Pretomanid for Tuberculosis (APT) trial, in which patients with drug-sensitive pulmonary TB received pretomanid, isoniazid, and pyrazinamide plus either rifampin or rifabutin versus standard of care under fed conditions, and the AIDS Clinical Trials Group 5306 (A5306) trial, a phase I study in healthy volunteers receiving pretomanid alone or in combination with rifampin under fasting conditions. In our population pharmacokinetic (PK) model, participants taking rifampin had 44.4 and 59.3% reductions in pretomanid AUC (area under the concentration-time curve) compared to those taking rifabutin or pretomanid alone (due to 80 or 146% faster clearance) in the APT and A5306 trials, respectively. Median maximum concentrations (C(max)) in the rifampin and rifabutin arms were 2.14 and 3.35 mg/liter, while median AUC(0–24) values were 30.1 and 59.5 mg·h/liter, respectively. Though pretomanid exposure in APT was significantly reduced with rifampin, AUC(0–24) values were similar to those associated with effective treatment in registrational trials, likely because APT participants were fed with dosing, enhancing pretomanid relative bioavailability and exposures. Pretomanid concentrations with rifabutin were high but in range with prior observations. While pretomanid exposures with rifampin are unlikely to impair efficacy, our data suggest that pretomanid should be taken with food if prescribed with rifampin. (This study has been registered at ClinicalTrials.gov under identifier NCT02256696.)

Ball Grid Array (BGA) package designs are increasingly used in surface mount applications while Land Grid Array (LGA) designs are predominantly used in socketing. The need to converge to a single package design has been driving the need to enable socketable BGAs. BGA spheres with a noble metal surface provide a stable mechanical contact interface with the socket paddles. These noble contact interfaces, however, have to remain intact through the socketing life of the product, considering accelerated testing temperatures of 100-120 °C. Under such conditions, it has been reported that the solder from the ball-attach joints undergoes solid-state wicking along the surface of the ball, leading to complete Au dissolution and potential undesirable intermetallic formation with the socket paddles, along with a drop in ball shear strength due to depletion of the solder from the joints. This paper discusses the use of polymer collars to address this challenge and improve the thermal aging reliability of packages with surface-modified BGA interconnections. The polymer collars were spin-coated on the package, which was aged alongside a reference package with no collars, at accelerated test temperatures of 100 °C and 120 °C, respectively. XPS studies showed that after 650 h of aging, no Au signal but a strong Sn signal was observed in the package without collars, which confirmed complete solder wicking to the top of the ball, while the Au signal remained for the package with the collars, confirming that polymer collars are effective in inhibiting solid-state solder wicking from the ball-attach joints. The joints with polymer collars also showed mechanical stability throughout thermal aging with a 3X improvement in joint shear strength.

Metabolic reprogramming is considered important in the pathogenesis of the occlusive vasculopathy observed in pulmonary hypertension (PH). However, the mechanisms that link reprogrammed metabolism to aberrant expression of genes, which modulate functional phenotypes of cells in PH, remain enigmatic. Herein, we demonstrate that, in mice, hypoxia-induced PH was prevented by glucose-6-phosphate dehydrogenase deficiency (G6PDDef), and further show that established severe PH in Cyp2c44−/−mice was attenuated by knockdown with G6PD shRNA or by G6PD inhibition with an inhibitor (N-ethyl-N′-[(3β,5α)-17-oxoandrostan-3-yl]urea, NEOU). Mechanistically, G6PDDef, knockdown and inhibition in lungs: 1) reduced hypoxia-induced changes in cytoplasmic and mitochondrial metabolism, 2) increased expression of Tet methylcytosine dioxygenase 2 ( Tet2) gene, and 3) upregulated expression of the coding genes and long noncoding (lnc) RNA Pint, which inhibits cell growth, by hypomethylating the promoter flanking region downstream of the transcription start site. These results suggest functional TET2 is required for G6PD inhibition to increase gene expression and to reverse hypoxia-induced PH in mice. Furthermore, the inhibitor of G6PD activity (NEOU) decreased metabolic reprogramming, upregulated TET2 and lncPINT, and inhibited growth of control and diseased smooth muscle cells isolated from pulmonary arteries of normal individuals and idiopathic-PAH patients, respectively. Collectively, these findings demonstrate a previously unrecognized function for G6PD as a regulator of DNA methylation. These findings further suggest that G6PD acts as a link between reprogrammed metabolism and aberrant gene regulation and plays a crucial role in regulating the phenotype of cells implicated in the pathogenesis of PH, a debilitating disorder with a high mortality rate.

RNA protein interactions (RPI) play a pivotal role in the regulation of various biological processes. Experimental validation of RPI has been time-consuming, paving the way for computational prediction methods. The major limiting factor of these methods has been the accuracy and confidence of the predictions, and our in-house experiments show that they fail to accurately predict RPI involving short RNA sequences such as TERRA RNA. Here, we present a data-driven model for RPI prediction using a gradient boosting classifier. Amino acids and nucleotides are classified based on the high-resolution structural data of RNA protein complexes. The minimum structural unit consisting of five residues is used as the descriptor. Comparative analysis of existing methods shows the consistently higher performance of our method irrespective of the length of RNA present in the RPI. The method has been successfully applied to map RPI networks involving both long noncoding RNA as well as TERRA RNA. The method is also shown to successfully predict RNA and protein hubs present in RPI networks of four different organisms. The robustness of this method will provide a way for predicting RPI networks of yet unknown interactions for both long noncoding RNA and microRNA.

BACKGROUND: The leaves of Cissus sicyoides has been utilized in traditional medicine. The berries however are underutilized and do not appear to be widely consumed. OBJECTIVE: This study was undertaken to further evaluate the antioxidant properties of C. sicyoides berries. METHODS: The free radical scavenging activity and IC50 value (inhibitory concentration) of Cissus sicyoides berries was determined utilizing the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Aqueous two-phase partitioning was utilized for preliminary purification of phenylalanine ammonia-lyase (PAL). PAL activity was determined using an endpoint spectrophotometric assay. The cytotoxicity of the berries was assessed by use of the Brine shrimp assay. RESULTS: C. sicyoides berries exhibit excellent free radical scavenging activity with an IC50 value of 0.99 mg/mL which is intermediate to that of raspberries and blackberries. The berries are a potential source of PAL with enzyme activity of 0.10±0.01 U/mg protein. CONCLUSION: Preliminary investigations suggest that the fruits are non toxic and may be utilized as a source of antioxidants. It is evident that the plant is a good source of bioactive compounds with significant potential for food, beverage and medical applications.