DMSO/SOCl2-Enabled Synthesis of 3-Chloroindoles via Desulfonylative Chlorocyclization of N,N-Disubstituted 2-Alkynylanilines.

The application of the DMSO/SOCl2 system enabled the intramolecular cyclization/chlorination of N,N-disubstituted 2-alkynylanilines, leading to the synthesis of a series of 3-chloroindoles with moderate to good yields. Differing from the previously reported interrupted Pummerer reaction featuring the introduction of SMe moiety, the current approach adopted an alternative pathway that realized the incorporation of chlorine atom to the indole skeleton via a desulfonylative chlorocyclization process.

Comparative genomic analyses of Klebsiella pneumoniae K57 capsule serotypes isolated from bovine mastitis in China.

Klebsiella pneumoniae can cause severe clinical mastitis in dairy cows, with K. pneumoniae type K57 (K57-KP) being the most common capsular serotype. To identify virulence factors and antimicrobial-resistance (AMR) genes of K57-KP with varying virulence, Galleria mellonella (greater wax moth) larvae were infected as a screening model to characterize virulence of 90 K57-KP strains, with 10 and 11 strains defined as virulent or attenuated, respectively, based on larval survival rates. Next, virulence of these 21 isolates was subsequently confirmed in adhesion and lactate dehydrogenase release assays, using bovine mammary epithelial cells cultured in vitro. Finally, genes associated with virulence and AMR were characterize with whole-genome sequencing. These 21 K57-KP strains were designated into 16 sequence types based on multi-locus sequence typing and allocated in phylogenetic analysis based on single nucleotide polymorphisms. We found great genetic diversity among isolates. In addition, adhesion-associated genes (e.g., fimA, sfaA, and focA) aminoglycoside-resistance genes (aph(6)-Id, strAB) were associated with virulence. This study provided new knowledge regarding virulence of K57-KP associated with bovine mastitis, which may inform development of novel diagnostic tools and prevention strategies for bovine mastitis.

Streptococcus uberis induced expressions of pro-inflammatory IL-6, TNF-α, and IFN-γ in bovine mammary epithelial cells associated with inhibited autophagy and autophagy flux formation.

Autophagy is a highly conserved cellular defensive mechanism that can eliminate bacterial pathogens such as Streptococcus uberis, that causes mastitis in cows. However, S. uberis induced autophagy is still unclear. In this study, we tested if certain inflammatory cytokines such as IL-6, TNF-α, and IFN-γ, critical in mastitis due to S. uberis infection, regulate autophagy activation in bovine mammary epithelial cells (bMECs). Using Western blot and laser scanning confocal microscope in bMECs challenged by S. uberis, showed that the expression of IL-6, TNF-α, IFN-γ oscillated with the expressions of autophagic Atg5, ULK1, PTEN, P62, and LC3ӀӀ/LC3Ӏ. S. uberis infection induced autophagosomes and LC3 puncta in bMECs with upregulation of Atg5, ULK1, PTEN, LC3ӀӀ/LC3Ӏ, and downregulation of P62. The levels of IL-6, TNF-α, and IFN-γ increased during autophagy flux formation to decrease during autophagy induction. Autophagy inhibition increased the expression of IL-6, TNF-α, and IFN-γ and increased S. uberis burden. This study indicates autophagy is induced during S. uberis infection and IL-6, TNF-α, and IFN-γ contribute to autophagy and autophagy flux formation.

MicroRNA miR-223 modulates NLRP3 and Keap1, mitigating lipopolysaccharide-induced inflammation and oxidative stress in bovine mammary epithelial cells and murine mammary glands.

Bovine mastitis, the most prevalent and costly disease in dairy cows worldwide, decreases milk quality and quantity, and increases cow culling. However, involvement of microRNAs (miRNAs) in mastitis is not well characterized. The objective was to determine the role of microRNA-223 (miR-223) in regulation of the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome and kelch like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) oxidative stress pathway in mastitis models induced by lipopolysaccharide (LPS) treatment of immortalized bovine mammary epithelial cells (bMECs) and murine mammary glands. In bMECs cultured in vitro, LPS-induced inflammation downregulated bta-miR-223; the latter interacted directly with the 3' untranslated region (3' UTR) of NLRP3 and Keap1. Overexpression of bta-miR-223 in bMECs decreased LPS and Adenosine 5'-triphosphate (ATP)-induced NLRP3 and its mediation of caspase 1 and IL-1ß, and inhibited LPS-induced Keap1 and Nrf2 mediated oxidative stress, whereas inhibition of bta-miR-223 had opposite effects. In an in vivo murine model of LPS-induced mastitis, increased miR-223 mitigated pathology in the murine mammary gland, whereas decreased miR-223 increased inflammatory changes and oxidative stress. In conclusion, bta-miR-223 mitigated inflammation and oxidative injury by downregulating the NLRP3 inflammasome and Keap1/Nrf2 signaling pathway. This study implicated bta-miR-223 in regulation of inflammatory responses, with potential as a novel target for treating bovine mastitis and other diseases.

Synthesis of Unnatural α-Amino Acids from (Pyridin-2-yl) Carbamate via CIPE-Induced Carbonyl Migration.

Synthetic methods of unnatural α-amino acids have always been the focus of extensive research due to their significant bioactivities. However, convenient transition-metal-free catalyzed methods are still in demand. Herein, we report a novel strategy for the construction of an unnatural α-amino acid skeleton via intramolecular rearrangement of carbamates, which are readily available from amines and their common protecting groups. This rearrangement could afford a variety of amino ester products in up to 98% yield, even in gram-scale reaction. The reaction mechanism was studied in detail through experiments and theoretical calculations. The complex-induced proximity effect (CIPE) from the 2-pyridyl group is shown to be indispensable for this transformation.

Klebsiella pneumoniae infection causes mitochondrial damage and dysfunction in bovine mammary epithelial cells.

Klebsiella pneumoniae, an important cause of bovine mastitis worldwide, is strongly pathogenic to bovine mammary epithelial cells (bMECs). Our objective was to determine the role of mitochondrial damage in the pathogenicity of K. pneumoniae on bMECs, by assessing several classical indicators of mitochondrial dysfunction, as well as differentially expressed genes (DEGs). Two K. pneumoniae strains (HLJ-D2 and HB-AF5), isolated from cows with clinical mastitis (CM), were used to infect bMECs (MAC-T line) cultured in vitro. In whole-transcriptome analysis of bMECs at 6 h post-infection (hpi), there were 3453 up-regulated and 3470 down-regulated genes for HLJ-D2, whereas for HB-AF5, there were 2891 up-regulated and 3278 down-regulated genes (P < 0.05). Based on GO term enrichment of differentially expressed genes (DEGs), relative to the controls, the primary categories altered in K. pneumoniae-infected bMECs included cellular macromolecule metabolism, metabolic process, binding, molecular function, etc. Infections increased (P < 0.05) malondialdehyde concentrations and formation of reactive oxygen species in bMECs. Additionally, both bacterial strains decreased (P < 0.05) total antioxidant capacity in bMECs at 6 and 12 hpi. Furthermore, infections decreased (P < 0.05) mitochondrial membrane potential and increased (P < 0.01) mitochondrial calcium concentrations. Finally, severe mitochondrial swelling and vacuolation, as well as mitochondrial rupture and cristae degeneration, were detected in infected bMECs. In conclusion, K. pneumoniae infections induced profound mitochondrial damage and dysfunction in bMECs; we inferred that this caused cellular damage and contributes to the pathogenesis of K. pneumoniae-induced CM in dairy cows.

Synthesis of gem-Dimethylcyclopentane-Fused Arenes with Various Topologies via TBD-Mediated Dehydro-Diels-Alder Reaction.

The development of efficient methods for the synthesis of substituted polycyclic arenes with various topologies is in high demand due to their excellent electrical and optical properties. In this work, a series of gem-dimethylcyclopentane-fused arenes with more than ten topologies were synthesized via a 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD)-mediated dehydro-Diels-Alder reaction with moderate to good yields. The introduction of the near-planar gem-dimethylcyclopentane moiety not only impacts the molecular conjugative system but also regulates the intermolecular π-π interactions and crystal packing, which are critical for the photoelectric performance of arenes. The photophysical properties, molecular geometry, molecular packing of these compounds, and electrochemical properties were investigated by UV-vis absorption, fluorescence emission spectra, DFT calculations, single-crystal X-ray structure analysis, and cyclic voltammetry study.

Virulence profiles of Klebsiella pneumoniae isolated from 2 large dairy farms in China.

We recently reported on the diversity of Klebsiella pneumoniae isolated from dairy herds in China. In our previous work, isolates from subclinical mastitis (SCM) had lower indices of diversity when compared with bacteria from other sources, possibly due to a contagious-like spread of udder adapted strains. Here we explored the virulence profile and capsular types of K. pneumoniae isolated from different sources on 2 dairy farms in China. Our overarching goal was to gain insights on the role of virulence genes toward the severity of mastitis caused by K. pneumoniae. A total of 1,484 samples were collected from clinical mastitis (CM; n = 355), SCM (n = 561), bulk tank milk (BTM; n = 130), and environmental and extramammary (EE) sites (n = 438). From those, 431 K. pneumoniae isolates were obtained, including 129, 77, 66, and 159 isolates from CM, SCM, BTM, and EE samples, respectively. Polymerase chain reactions were used to determine the capsular types and to detect potential virulence genes in all isolates. No significant farm effects were observed when comparing the distribution of most virulence genes in K. pneumoniae isolated from each source. K57 was the most prevalent capsular type in K. pneumoniae from all sources, but with increased detection rate in isolates from CM. entB, kfu, fimH1, mrkD, and ß-d-lacZ were frequently detected in K. pneumoniae from all sources. ß-d-lacZ, entB, and ituA were more prevalent in isolates from CM, whereas kfu, allS, and nif were more frequently detected in isolates from SCM. ybtS, aerobactin, and rpmA had increased prevalence in K. pneumoniae from BTM when compared with bacteria from other sources. No association was detected between virulence genes and the severity of CM. K57 and the nif gene had the highest discriminatory power to classify isolates from CM and SCM, respectively. Based on our findings, it is likely that K57 is the dominant capsular type in K. pneumoniae causing CM in large Chinese dairy herds. Likewise, we demonstrated that ß-d-lacZ is disseminated in K. pneumoniae isolated from large Chinese dairy farms, irrespectively of the source of bacteria. Our results also suggest a low contribution of the virulence profile of K. pneumoniae toward CM severity. Finally, the role of nif in increasing the adaptability to the udder and promoting a contagious-like spread of K. pneumoniae warrants further investigation.

Bacteriophage has beneficial effects in a murine model of Klebsiella pneumoniae mastitis.

Bovine mastitis caused by Klebsiella pneumoniae is usually treated with antibiotics, thereby potentially increasing antimicrobial resistance. The objective of this study was to evaluate efficacy of a bacteriophage, isolated from dairy farm wastewater, as a treatment for a murine model of K. pneumoniae mastitis. A lytic bacteriophage CM8-1 was isolated, morphological and biological characteristics were assessed with transmission electron microscopy and double-layer plate, and its genome was sequenced and analyzed. Furthermore, effectiveness of this bacteriophage for treatment of a murine model of K. pneumoniae mastitis was evaluated based on the following mammary gland characteristics: morphological changes; number of K. pneumoniae; and mRNA and protein expression of pro-inflammatory factors TNF-α, IL-1ß, IL-6, and IL-8. Bacteriophage CM8-1 had an incubation period of 30 min and a burst time of 20 min. Its viability and adsorption were stable at 30 to 50°C, but decreased significantly at >60°C, with no significant change in viability or infectivity at pH 6 to 10. In a murine model of K. pneumoniae mastitis, injecting bacteriophage CM8-1 into the mammary gland 2 h after inoculation with K. pneumoniae resulted in reductions in bacterial counts in the murine mammary gland, improvements in mammary gland tissue morphology, and reductions in mRNA and protein expression of pro-inflammatory factors. Bacteriophage CM8-1 had stable biological characteristics and suppressed K. pneumoniae mastitis when injected into the mammary gland 2 h latera in mice bacterial inoculation.

Selenomethionine activates selenoprotein S, suppresses Fas/FasL and the mitochondrial pathway, and reduces Escherichia coli-induced apoptosis of bovine mammary epithelial cells.

Escherichia coli is a major environmental pathogen causing bovine mastitis, characterized by cell death and mammary tissue damage. Apoptosis, a form of cell death, has an important role in the pathogenesis of mastitis. Selenium, an essential trace element, protects against mastitis by acting through several biochemical pathways, potentially including prevention of apoptosis. Our objective was to investigate whether selenomethionine (SeMet) attenuated E. coli-induced apoptosis in bovine mammary epithelial cells (bMEC). These cells were cultured in vitro and treated with 0, 5, 10, 20, and 40 µM SeMet for 12 h, with or without E. coli (multiplicity of infection of 5) for 8 h. Treatment with SeMet/Z-IE(OMe)TD(OMe)-FMK (ZIK)/Z-LE(OMe)HD(OMe)-FMK (ZLK, specific inhibitors of caspase-8 and -9, respectively) significantly counteracted effects of E. coli on bMEC. Specifically, SeMet upregulated selenoprotein S (SeS) and increased mitochondrial membrane potential and the ratio of Bcl-2 and Bax. Furthermore, it decreased protein expressions of Fas, FasL, FADD, cleaved caspase-8, cytochrome c, cleaved caspase-9, and cleaved caspase-3, namely, decreasing protein expression of the Fas/FasL and mitochondrial pathways. Furthermore, it downregulated total apoptosis indexes in E. coli-infected bMEC. Although ZIK and ZLK (specific inhibitors of caspases 8 and 9, respectively) significantly inhibited Fas/FasL and the mitochondrial apoptotic pathway and apoptosis indexes, respectively, substantial apoptosis still occurred. In conclusion, SeMet attenuated E. coli-induced apoptosis in bMEC by activating SeS, associated with Fas/FasL and mitochondrial pathways.

Effects of mechanical vibration on cell morphology, proliferation, apoptosis, and cytokine expression/secretion in osteocyte-like MLO-Y4 cells exposed to high glucose.

Diabetic patients exhibit significant bone deterioration. Our recent findings demonstrate that mechanical vibration is capable of resisting diabetic bone loss, whereas the relevant mechanism remains unclear. We herein examined the effects of mechanical vibration on the activities and functions of osteocytes (the most abundant and well-recognized mechanosensitive cells in the bone) exposed to high glucose (HG). The osteocytic MLO-Y4 cells were incubated with 50 mM HG for 24 h, and then stimulated with 1 h/day mechanical vibration (0.5 g, 45 Hz) for 3 days. We found that mechanical vibration significantly increased the proliferation and viability of MLO-Y4 cells under the HG environment via the MTT, BrdU, and Cell Viability Analyzer assays. The apoptosis detection showed that HG-induced apoptosis in MLO-Y4 cells was inhibited by mechanical vibration. Moreover, increased cellular area, microfilament density, and anisotropy in HG-incubated MLO-Y4 cells were observed after mechanical vibration via the F-actin fluorescence staining. The real-time polymerase chain reaction and western blotting results demonstrated that mechanical vibration significantly upregulated the gene and protein expression of Wnt3a, ß-catenin, and osteoprotegerin (OPG) and decreased the sclerostin, DKK1, and receptor activator for nuclear factor-κB ligand (RANKL) expression in osteocytes exposed to HG. The enzyme-linked immunosorbent assay assays showed that mechanical vibration promoted the secretion of prostaglandin E2 and OPG, and inhibited the secretion of tumor necrosis factor-α and RANKL in the supernatant of HG-treated MLO-Y4 cells. Together, this study demonstrates that mechanical vibration improves osteocytic architecture and viability, and regulates cytokine expression and secretion in the HG environment, and implies the potential great contribution of the modulation of osteocytic activities in resisting diabetic osteopenia/osteoporosis by mechanical vibration.

[Composition and analysis of fatty acids in freshwater products in Hunan Province in 2014].

OBJECTIVE: To investigate the nutritional composition of fatty acids in freshwater products in Hunan Province. METHODS: The edible parts of freshwater products were detected by gas chromatography, and fatty acid fingerprints were obtained by statistical analysis. RESULTS: A total of 18 freshwater products were monitored and 14-26 fatty acids were detected in each freshwater product. Among them, 12 were saturated fatty acids(SFA), ranging from 0. 74 to 3143 mg/100 g, 9 were monounsaturated fatty acids(MUFA), ranging from 1. 23 to 2790 mg/100 g, and 10 were polyunsaturated fatty acids(PUFA), ranging from 1. 75 to 2832 mg/100 g. The ratio of n-6 to n-3 in polyunsaturated fatty acids ranged from 0. 24∶1 to 15. 7∶1. CONCLUSION: The composition of fatty acids in freshwater products in Hunan Province is mainly unsaturated fatty acids. Most freshwater products are rich in n-3 PUFA, and the ratio of n-6 PUFA to n-3 PUFA is less than 6, which is beneficial to the nutritional balance. The composition and content of fatty acids have ideal nutritional value.

Gold-nanobranched-shell based drug vehicles with ultrahigh photothermal efficiency for chemo-photothermal therapy.

Development of combined chemo-photothermal nanoplatform is of great interest for enhancing antitumor efficacy. Herein, a multifunctional drug delivery system was synthesized based on gold-nanobranched coated betulinic acid liposomes (GNBS-BA-Lips) for chemo-photothermal synergistic therapy. In this system, GNBS-BA-Lips exhibited broad near-infrared (NIR) absorption, preferable photothermal response and good photostability under NIR irradiation. Importantly, the gold-nanobranched nanostructure possessed high photothermal conversion efficiency (η = 55.7%), and the temperature change (ΔT) reached 43.2 °C after laser irradiation for 5 min. Upon NIR irradiation, the nanocarriers apparently endowed higher cell uptake, resulting in an enhanced intracellular drug accumulation. Furthermore, the tumor growth inhibition ratio achieved from chemo-photothermal therapy of GNBS-BA-Lips was 86.9 ±â€¯1.1%, which was higher than that of the chemotherapy or photothermal therapy alone, showing an outstanding synergistic anticancer effect. Our data suggested that the nanoplatform should be considered as a critical platform in the development of cancer multi-mode therapies.

Effects of carbon sources, COD/NO2--N ratios and temperature on the nitrogen removal performance of the simultaneous partial nitrification, anammox and denitrification (SNAD) biofilm.

The aim of the present work was to evaluate the effects of carbon sources and chemical oxygen demand (COD)/NO2--N ratios on the anammox-denitrification coupling process of the simultaneous partial nitrification, anammox and denitrification (SNAD) biofilm. Also, the anammox activities of the SNAD biofilm were investigated under different temperature. Kaldnes rings taken from the SNAD biofilm reactor were operated in batch tests to determine the nitrogen removal rates. As a result, with the carbon source of sodium acetate, the appropriate COD/NO2--N ratios for the anammox-denitrification coupling process were 1 and 2. With the COD/NO2--N ratios of 1, 2, 3, 4 and 5, the corresponding NO2--N consumption via anammox was 87.1%, 52.2%, 29.3%, 23.7% and 16.3%, respectively. However, with the carbon source of sodium propionate and glucose, the anammox bacteria was found to perform higher nitrite competitive ability than denitrifiers at the COD/NO2--N ratio of 5. Also, the SNAD biofilm could perform anammox activity at 15 °C with the nitrogen removal rate of 0.071 kg total inorganic nitrogen per kg volatile suspended solids per day. These results indicated that the SNAD biofilm process might be feasible for the treatment of municipal wastewater at normal temperature.

Total Synthesis of the Aglycone of IB-00208.

A total synthesis of the aglycone of IB-00208 was accomplished in 22 steps using a newly developed approach towards polycyclic 1,4-dioxygenated xanthones from benzocyclobutenones. The generality of this entry to xanthones was initially established on several model systems before it was successfully applied to the construction of the hexacyclic core of the natural product. A new and potentially general approach towards angularly-fused benzocyclobutenones using ring-closing metathesis (RCM) was also developed.

IL-17 gene polymorphism is associated with susceptibility to gastric cancer.

We conducted a case-control study to investigate the role of three common IL-17A and IL-17F single nucleotide polymorphisms (SNPs) on the susceptibility to gastric cancer. A case-control study was conducted using a Chinese study population of 462 gastric cancer subjects and 462 controls. Polymerase chain reaction restriction fragment length of polymorphism (PCR-RFLP) was taken to genotype rs2275913, rs763780, and rs3748067 within the IL-17 gene. When comparing demographic characteristics of gastric cancer between gastric cancer cases and control groups, cancer cases were more likely to be cigarette smokers and alcohol drinkers, have cancer history in the first relatives, and have higher infection rate of Helicobacter pylori. By conditional regression analysis, individuals carrying IL-17 rs2275913 GA, AA genotype, and A allele were associated with an increased gastric cancer risk. Those carrying rs3748067 CC genotype and C allele had a significantly increased risk for the development of gastric cancer. Moreover, rs2275913 and rs3748067 variations had association with cigarette smoking, alcohol drinking, and H. pylori infection on the risk of gastric cancer. These results suggest that rs2275913 and rs3748067 variations significantly increase gastric cancer risk in a Chinese population.

Genetic proxy of lipid-lowering drugs and calcific aortic valve stenosis: A Mendelian randomization study.

Background: Lipid metabolism plays an important role in the pathogenesis and development of calcific aortic valve stenosis. Our aim was to evaluate the causal effect of lipid-lowering drugs, such as low-density lipoprotein cholesterol (LDL-C) lowering and triglyceride lowering drugs, on the outcome of aortic valve stenosis using a two-sample Mendelian randomization (MR) study. Methods: We used two genetic tools to represent the exposure of lipid-lowering drugs, including expression quantitative trait loci for the expression of drug target genes, and genetic variants within or near drug target genes that are associated with LDL-C and triglyceride concentrations from Genome-Wide Association Studies (GWAS). Effect estimates were calculated using summary-data-based MR (SMR) and inverse-variance-weighted MR (IVW-MR) analysis. Results: Based on the results of SMR and IVW-MR analysis, LDL-C-lowering PCSK9 inhibitors have potential in reducing the risk of aortic valve stenosis (for SMR, OR: 1.044; 95%CI: 1.002-1.404; P = 0.047; for IVW-MR, OR: 1.647, 95%CI: 1.316-2.062, P < 0.001). However, no significant association was observed between triglyceride target gene expression, as well as triglyceride-lowering drugs, and aortic valve stenosis. Conclusion: This two-sample drug-targeted MR study suggests a potential causal relationship between PCSK9 inhibitors and the reduction of calcific aortic valve stenosis risk.

An isocratic HPLC-UV analytical procedure for assessment of glutathione and its related substances.

Reduced glutathione (GSH) is an endogenous tripeptide antioxidant which plays a crucial role in a variety of physiological and pathological activities. Although GSH is not present in any FDA-approved drug product, GSH dietary supplement products and compounded GSH drugs are available to patients in the US. Several incidents of toxicity have occurred in recent years due to endotoxin or otherwise contaminated GSH in compounded drugs. Efficient and sensitive analytical methods are needed for assessing and ensuring the quality of GSH substance and associated drug or dietary supplement products. Impurities A (L-cysteinylglycine), B (cysteine), C (oxidized L-glutathione) and D (γ-L-glutamyl-L-cysteine) are the main related impurities for GSH drug substance which have been detected and quantified by capillary electrophoresis and qNMR analytical procedures. However, there are no reported HPLC methods for detecting or quantifying the three main related impurities A, B and D even though numerous HPLC analytical methods have been reported for analyzing GSH and impurity C. In this report, an isocratic HPLC-UV analytical procedure was developed and validated for separating and identifying GSH and related impurities A-D as well as a newly identified degradant, L-pyroglutamic acid (pGlu), within 10 minutes with resolution (RS) more than 3. The LOD and LOQ were determined to be 0.02 % w/w and 0.05 % w/w, respectively, for impurities A-D and pGlu. Importantly, the optimized HPLC analytical procedure for GSH assay does not have interference from impurities A, B and D, providing highly specific results compared to the commonly used iodine titration method. The newly validated analytical procedure was applied to assess different commercial GSH bulk substance samples. The results suggest that the analytical procedure described in this work is suitable for quality assessment of GSH samples.

Qualitative and quantitative analysis of glutathione and related impurities in pharmaceuticals by qNMR.

In this study, an alternative method to compendial analytical procedures with enhanced detection and separation capabilities was validated for the quality assessment of glutathione (GSH) drug substance. The related impurities A, B, C, and D present in GSH drug substance were characterized using a one-dimension proton nuclear magnetic resonance (1D 1H NMR) method on a 600 MHz spectrometer equipped with a liquid nitrogen cryoprobe. Two sample preparations at different pH were optimized to ensure the unambiguous identification of different impurities in the GSH samples. Specifically, impurities A and C in a GSH sample can be tested at pH 3.0, while pH 7.4 is more suitable for testing impurities B and D. The quantitative NMR (qNMR) method was validated following International Council for Harmonisation (ICH) guidelines. The limit of detection (LOD) was less than 0.1% wt for an individual impurity, and the limit of quantitation (LOQ) ranged from 0.14 to 0.24% wt, using about 14 min experimental time per spectrum. Following validation, the qNMR method was applied to assess different commercial GSH bulk substance samples, an in-house compounded GSH drug product, and a GSH dietary supplement product. The method was also applied to monitor GSH degradation (hydrolysis and oxidation) over time to provide quantitative information on GSH degradation and stability. The results suggest that the qNMR method can serve as a highly specific and efficient orthogonal tool for assessing the quality of GSH pharmaceuticals, providing both qualitative and quantitative information on GSH and its related impurities A-D.

Association between regulatory T cells and ischemic heart disease: a Mendelian randomization study.

Background: An imbalance of innate and acquired immune responses is significantly involved in the pathophysiology of coronary atherosclerosis and the occurrence of ischemic heart disease (IHD). Regulatory T cells (Tregs) play an essential regulatory role in atherosclerotic plaque formation and maintenance; therefore, dysfunction of Tregs triggers the formation of atherosclerotic plaques and accelerates their progression. However, due to the inherent limitations of observational research, clinical evidence is limited concerning the relationship between the variation in peripheral Tregs and the risk of IHD, and the cause-and-effect relationship between these factors is unclear. Mendelian randomization (MR) uses genetic variation as a proxy for exposure and can be used to inferentially determine the causal effect of exposure on outcomes. We thus used MR analysis to investigate whether there is a causal relationship between the biomarkers of Tregs and IHD. Methods: Selected genetic variants (P<5.00E-08) from the summary data of a genome-wide association study (GWAS) were used to conduct a two-sample bidirectional MR analysis. The analysis included 51 extensive Treg subtypes involving 3,757 individuals from the general population. Summary statistics of IHD were obtained from the IEU open GWAS project, which contains 30,952 cases and 187,845 controls. The populations in both GWAS studies were of European ancestry. Results: We identified a set of 197 single-nucleotide polymorphisms (SNPs) that served as instrumental variables (IVs) for evaluating 51 Treg subtypes. Thirteen significant variables were found to be potentially associated with IHD. After false-discovery rate (FDR) adjustment, we identified four Treg subtypes to be causally protective for IHD risk: CD28 on activated & secreting CD4 Tregs [odds ratio (OR) =0.89; 95% confidence interval (CI): 0.82-0.96; P=3.10E-03; adjusted P=0.04], CD28 on activated CD4 Tregs (OR =0.87; 95% CI: 0.80-0.95; P=3.10E-03; adjusted P=0.04), CD28 on CD4 Tregs (OR =0.87; 95% CI: 0.80-0.96; P=3.41E-03; adjusted P=0.04), and CD28 on resting CD4 Treg cell (OR =0.91; 95% CI: 0.85-0.97; P=3.48E-03; adjusted P=0.04). Reverse MR analysis found eight potential causal variables, but these associations were nonsignificant after FDR correction (all adjusted P values >0.05). Conclusions: This study identified the significance of elevated CD28 expression on CD4 Tregs as a novel molecular modifier that may influence IHD occurrence, suggesting that targeting CD28 expression on CD4 Tregs could offer a promising therapeutic approach for IHD.