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Background Sequence-based identification is one of the most effective methods for species-level identification of nontuberculous mycobacteria (NTM). However, it is time-consuming because of the bioinformatics processes involved, including sequence trimming, consensus sequence generation, and public database searches. We developed a simple and fully automated software that enabled species-level identification of NTM from trace files, SnackNTM (https://github.com/Young-gonKim/SnackNTM). Methods JAVA programing language was used for software development. The SnackNTM diagnostic algorithm utilized 16S rRNA gene sequences, according to the Clinical & Laboratory Standards Institute guidelines, and an rpoB gene region was adjunctively utilized to narrow down the species. The software performance was validated using trace files of 234 clinical cases, comprising 217 consecutive cases and 17 additionally selected cases of unique species. Results SnackNTM could analyze multiple cases at once, and all the bioinformatics processes required for sequence-based NTM identification were automatically performed with a single mouse click. SnackNTM successfully identified 95.9% (208/217) of consecutive clinical cases, and the results showed 99.0% (206/208) agreement with manual classification results. SnackNTM successfully identified all 17 cases of unique species. In a processing time comparison test, the analysis and reporting of 30 cases, which took 150 minutes manually, took only 40 minutes with SnackNTM. Conclusions SnackNTM is expected to reduce the workload for NTM identification, especially in clinical laboratories that process large numbers of cases.

Constant remodeling is necessary for bacterial cell growth and bacterial morphogenesis; peptidoglycan (PG) is a crucial component in this process. Murein DD-endopeptidase (MepS), initially annotated as Spr from E. coli K12, is a NlpC/P60 family endopeptidase, which cleaves the meso-diaminopimelate (DAP)-D-Ala peptide bond of PG. The Cys68, His119, His131 triad form the active site residues. MepS has autolytic activity, which is strictly regulated by a periplasmic degradation system comprising the NlpI/Prc protease complex. MepS is essential for maintaining the cell viability, and therefore, it is a potential target for developing antibiotics. This study aimed to understand the structural basis of substrate recognition and degradation. We determined the high-resolution structures of MepS, after mutating Cys68 to serine (MepS-C68S) to improve stability. We further found that citrate and L-malate molecules bind to the active site of MepS-C68S; this is in line with the recurrent observation of organic acids binding to PG endopeptidases. The presence of conserved residues on the surface revealed the potential peptide binding sites of MepS. We modelled a cross-linked peptide model of meso-DAP-D-Ala-meso-DAP, bound to the active site groove of MepS-C68S. Two conserved tyrosine residues, Tyr56 and Tyr147 appeared to be essential for the recognition of peptides. Our structural discoveries could provide insights for the design of novel antibiotics targeting MepS.

A central goal of microbiome research is to understand the factors that balance gut-associated microbial communities, thereby creating longitudinal and cross-sectional heterogeneity in their composition and density. Whereas the diet dictates taxa dominance, microbial communities are linked intimately to host physiology through digestive and absorptive functions that generate longitudinal heterogeneity in nutrient availability. Additionally, the host differentially controls the access to electron acceptors along the longitudinal axis of the intestine to drive the development of microbial communities that are dominated by facultatively anaerobic bacteria in the small intestine or obligately anaerobic bacteria in the large intestine. By secreting mucus and antimicrobials, the host further constructs microhabitats that generate cross-sectional heterogeneity in the colonic microbiota composition. Here we will review how understanding the host factors involved in generating longitudinal and cross-sectional microbiota heterogeneity helps define physiological states that are characteristic of or appropriate to a homeostatic microbiome.

In the wake of the SARS-CoV-2 pandemic, inactivating bioaerosols became a pivotal issue which helps to prevent the transmittance of SARS-CoV-2. Thus, the current study was conducted to investigate a potential inactivating method using both ozone (O3) and ultraviolet C (UVC). Individual and integrated effects of O3 and UVC were compared. A solution containing approximately 4⁓7.3 × 106 CFU/mL of Salmonella typhimurium bacteria was used to produce bacteria droplets. These droplets were exposed to O3 and UVC to determine the reduction rate of bacteria. The exposure times were set as 1 and 30 minutes. Ozone concentrations were 100 and 200 ppmv. UVC-LEDs were used as a UVC source. Peak wavelength of the UVC-LED was 275 nm and the irradiation dose was 0.77 mW/cm2. In terms of O3 and UVC-LED interaction, 194 ppmv styrene was used as a target compound to be removed. Considering the O3 and UVC-LED interaction, the presence of O3 could reduce the performance of the UVC-LED, and UVC-LED could also reduce significant amount of O3. The sequence of O3 and UVC-LED treatment was as follows: O3 was exposed at first, then UVC-LED, and this order showed the best reduction ratio (≻99.9%). Therefore, if O3 and UVC-LED is used to disinfect Salmonella typhimurium bacteria contained in droplets, bacteria should be separately exposed to O3 and UVC-LED in order to improve the inactivation efficiency. © 2021. by Asian Association for Atmospheric Environment This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.x.

Background The catabolic activity of the microbiota contributes to health by aiding in nutrition, immune education, and niche protection against pathogens. However, the nutrients consumed by common taxa within the gut microbiota remain incompletely understood. Methods Here we combined microbiota profiling with an un-targeted metabolomics approach to determine whether depletion of small metabolites in the cecum of mice correlated with the presence of specific bacterial taxa. Causality was investigated by engrafting germ-free or antibiotic-treated mice with complex or defined microbial communities. Results We noted that a depletion of Clostridia and Erysipelotrichia from the gut microbiota triggered by antibiotic treatment was associated with an increase in the cecal concentration of sugar acids and sugar alcohols (polyols). Notably, when we inoculated germ-free mice with a defined microbial community of 14 Clostridia and 3 Erysipelotrichia isolates, we observed the inverse, with a marked decrease in the concentrations of sugar acids and polyols in cecal contents. The carbohydrate footprint produced by the defined microbial community was similar to that observed in gnotobiotic mice receiving a cecal microbiota transplant from conventional mice. Supplementation with sorbitol, a polyol used as artificial sweetener, increased cecal sorbitol concentrations in antibiotic-treated mice, which was abrogated after inoculation with a Clostridia isolate able to grow on sorbitol in vitro. Conclusions We conclude that consumption of sugar alcohols by Clostridia and Erysipelotrichia species depletes these metabolites from the intestinal lumen during homeostasis.

Glioblastoma (GBM) is an aggressive brain tumor with a strong tendency of relapse and resistance to chemotherapy, but we currently lack non-toxic agents that effectively treat GBM. In this study, high-throughput screening of FDA-approved drugs was performed to identify safe and effective molecules and test their effect on GBM cell lines, LN229, U87 and T98G. Cough suppressants, oxelaidin and butamirate inhibited GBM growth. A Ras family GTPase, Ras-related associated with diabetes (RRAD), contributes to activation of STAT3, which is essential for survival and growth of many cancer types. Interestingly, oxelaidin and butamirate did not affect proliferation in RRAD negative GBM cells. Docking simulation analyses revealed selective interactions between oxelaidin and RRAD. The mechanism by which butamirate and oxelaidin inhibits GBM cell growth involves the suppression of STAT3 transcriptional activity, leading to down-regulation of cyclin D1 and survivin. In addition, components of RRAD-associated signaling cascades, including p-EGFR, p-Akt, and p-STAT3, were inhibited upon oxelaidin treatment. Intraperitoneal administration of oxelaidin or butamirate markedly suppressed tumor growth in a glioblastoma xenograft mouse model without significant adverse effects. Our collective findings indicate that oxelaidin and butamirate exert anti-tumor effects in glioblastoma, supporting its utility as a novel therapeutic candidate for glioblastoma.

It was demonstrated herein that the adhesive property of catechol-functionalized nanocomposite hydrogel can be enhanced by tuning the cohesive strength due to the secondary crosslinking between catechol and synthetic bioactive nanosilicate, viz. Laponite (LP). The nanocomposite hydrogel consists of the natural anionic poly(γ-glutamic acid) (γ-PGA), which was functionalized with catechol moiety, and incorporated with disk-structured LP. The dual-crosslinked hydrogel was fabricated by enzymatic chemical crosslinking of catechol in the presence of horseradish peroxidase (HRP) and H2O2, and physical crosslinking between γ-PGA-catechol conjugate and LP. The PGADA/LP nanocomposite hydrogels with catechol moieties showed strong adhesiveness to various tissue layers and demonstrated an excellent hemostatic properties. These PGADA/LP nanocomposite hydrogels are potentially applied for injectable tissue engineering hydrogels, tissue adhesives, and hemostatic materials. Statement of significance Recently, many attempts have been performed to manufacture high-performance tissue adhesives using synthetic and natural polymer-based materials. In order to apply in various biological substrates, commercially available tissue adhesives should have an improved adhesive property in wet conditions. Here, we designed a mussel-inspired dual crosslinked tissue adhesive that meets most of conditions as an ideal tissue adhesive. The designed tissue adhesive is composed of poly(γ-glutamic acid)-dopamine conjugate (PGADA)-gluing macromer, horseradish peroxidase (HRP)/hydrogen peroxide (H2O2)-enzymatic crosslinker, and Laponite (LP)-additional physical crosslinking nanomaterial. The PGADA hydrogel has tunable physicochemical properties by controlling the LP concentration. Furthermore, this dual crosslinked hydrogel shows strong tissue adhesive property, regardless of the tissue types. Specially the PGADA hydrogel has tissue adhesive strength four times higher than commercial bioadhesive. This dual crosslinked PGADA hydrogel with improved tissue adhesion property is a promising biological tissue adhesive for various tissue type in surgical operation.

Background: Atopic dermatitis (AD) is characterized by chronic, relapsing skin inflammation (eczema) with itchy sensation. Keratinocytes, which are located at the outermost part of our body, are supposed to play important roles at the early phase of type 2 inflammation including AD pathogenesis. Objective: The purpose of this study was to evaluate whether keratinocytes-derived reactive oxygen species (ROS) could be produced by the allergens or non-allergens, and the keratinocytes- derived ROS could modulate a set of biomarkers for type 2 inflammation of the skin. Methods: Normal human epidermal keratinocytes (NHEKs) were treated with an allergen of house dust mites (HDM) or a non-allergen of compound 48/80 (C48/80). Then, biomarkers for type 2 inflammation of the skin including those for neurogenic inflammation were checked by reverse transcriptase-polymerase chain reaction and western immunoblot experiments. Results: HDM or C48/80 was found to upregulate expression levels of our tested biomarkers, including type 2 T helper-driving pathway (KLK5, PAR2, and NFκB), epithelial-cell-derived cytokines (thymic stromal lymphopoietin, interleukin [IL]-25, IL-33), and neurogenic inflammation (NGF, CGRP). The HDMor C-48/80-induced expression levels of the biomarkers could be blocked by an antioxidant treatment with 5 mM N-acetyl- cysteine. In contrast, pro-oxidant treatment with 1 mM H2O2 could upregulate expression levels of the tested biomarkers in NHEKs. Conclusion: Our results reveal that keratinocytes- derived ROS, irrespective to their origins from allergens or non-allergens, have a potential to induce type 2 inflammation of AD skin. (Ann Dermatol 33(1) 26∼36, 2021)

Although DNA damage responses (DDRs) are reported to be involved in nitric oxide (NO) production in response to genotoxic stresses, the precise mechanism of DDR-mediated NO production has not been fully understood. Using a genotoxic agent aphidicolin, we investigated how DDRs regulate NO production in bovine aortic endothelial cells. Prolonged (over 24 h) treatment with aphidicolin increased NO production and endothelial NO synthase (eNOS) protein expression, which was accompanied by increased eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and eNOS mRNA expression. A promoter assay using 5’-serially deleted eNOS promoters revealed that Tax-responsive element site, located at −962 to −873 of the eNOS promoter, was responsible for aphidicolin-stimulated eNOS gene expression. Aphidicolin increased CREB activity and ectopic expression of dominant-negative inhibitor of CREB, A-CREB, repressed the stimulatory effects of aphidicolin on eNOS gene expression and its promoter activity. Co-treatment with LY294002 decreased the aphidicolin-stimulated increase in p-CREB-Ser133 level, eNOS expression, and NO production. Furthermore, ectopic expression of dominant-negative Akt construct attenuated aphidicolin-stimulated NO production. Aphidicolin increased p-ATM-Ser1981 and the knockdown of ATM using siRNA attenuated all stimulatory effects of aphidicolin on p-Akt-Ser473, p-CREB-Ser133, eNOS expression, and NO production. Additionally, these stimulatory effects of aphidicolin were similarly observed in human umbilical vein endothelial cells. Lastly, aphidicolin increased acetylcholine-induced vessel relaxation in rat aortas, which was accompanied by increased p-ATM-Ser1981, p-Akt-Ser473, p-CREB-Ser133, and eNOS expression. In conclusion, our results demonstrate that in response to aphidicolin, activation of ATM/Akt/CREB/eNOS signaling cascade mediates increase of NO production and vessel relaxation in endothelial cells and rat aortas.

Significance The ubiquitin–proteasome system and autophagy are two major intracellular proteolytic pathways, and both remove misfolded and proteotoxic proteins from eukaryotic cells. This study describes the detailed regulatory pathway of proteasome degradation by autophagy for its own quality control. We discovered that a portion of inhibited proteasomes is actively sequestered into the aggresome, an insoluble fraction of the mammalian cell. The aggresome functions as a triage point for proteasome recovery and autophagic degradation. This mainly distinguishes proteasome quality control in mammals from that in other organisms. STUB1/CHIP E3 Ub ligase has a critical role in targeting inhibited proteasomes into the aggresome. These results provide strong insights into protein catabolism in various pathological conditions originating from impaired proteasomes., The 26S proteasome, a self-compartmentalized protease complex, plays a crucial role in protein quality control. Multiple levels of regulatory systems modulate proteasomal activity for substrate hydrolysis. However, the destruction mechanism of mammalian proteasomes is poorly understood. We found that inhibited proteasomes are sequestered into the insoluble aggresome via HDAC6- and dynein-mediated transport. These proteasomes colocalized with the autophagic receptor SQSTM1 and cleared through selective macroautophagy, linking aggresomal segregation to autophagic degradation. This proteaphagic pathway was counterbalanced with the recovery of proteasomal activity and was critical for reducing cellular proteasomal stress. Changes in associated proteins and polyubiquitylation on inhibited 26S proteasomes participated in the targeting mechanism to the aggresome and autophagosome. The STUB1 E3 Ub ligase specifically ubiquitylated purified human proteasomes in vitro, mainly via Lys63-linked chains. Genetic and chemical inhibition of STUB1 activity significantly impaired proteasome processing and reduced resistance to proteasomal stress. These data demonstrate that aggresomal sequestration is the crucial upstream event for proteasome quality control and overall protein homeostasis in mammals.

Background α-Synuclein (α-syn) is a major component of Lewy bodies, a pathologic marker of Parkinson's disease (PD) in post-mortem studies. The use of α-syn as a practical PD biomarker has been investigated by numerous researchers. However, reports of differences in α-syn levels in biofluids, such as cerebrospinal fluid, plasma, and saliva, between PD patients and controls are inconsistent. Recently, the measurement of α-syn oligomer levels has emerged as a novel approach to diagnose PD. Objective Lysates and culture media from two different types of dopaminergic neuronal cells or urine samples from 11 non-PD and 21 PD patients were collected and analyzed. Methods We developed and performed an enzyme-linked immuno-absorbent assay (ELISA) to detect various oligomeric α-syn using distinct pairs of antibodies. Results We validated our ELISA using rotenone-induced alterations of α-syn levels in human dopaminergic neurons. Total urinary α-syn levels, measured using our ELISA method, showed no difference between PD and non-PD individuals, but a higher level of α-syn oligomer recognized by MJFR-14-6-5-2 in PD urine samples was observed. Levels of distinct oligomeric α-syn detected by ASyO5 were lower in PD urine samples. Three different α-syn ELISA results were analyzed with respect to the severity of PD, but only the correlation between total α-syn levels and PD index was significant. Conclusion Our findings suggest that detection of distinct oligomeric formations of α-syn and measurement of their levels in urine might be feasible for use in PD diagnostics.

We tested the interactive effects of increased temperature and nutrient (ammonium, NH4+) levels on physiological properties such as photosynthetic rates, NH4+ uptake rates, relative growth rates, chlorophyll fluorescence, and tissue nutrient contents in Ulva linza Linnaeus. The experiments were conducted at four temperatures (LT, low temperature (15 °C); MT, medium temperature (20 °C); CT, control temperature (25 °C); and HT, high temperature (30 °C)) and three NH4+ concentrations (LN, low nutrient (4 μM); MN, medium nutrient (60 μM); and HN, high nutrient (120 μM)). The interaction between temperature and NH4+ levels influenced the photosynthetic rates, NH4+ uptake rates, relative growth rates, photosynthetic efficiency, tissue nitrogen contents, and C:N ratios in algal tissues. Temperature strongly affected the photosynthetic rates, NH4+ uptake rates, and photosynthetic efficiency. Nutrient enrichment increased the photosynthetic rates, nutrient uptake rates, relative growth rates, photosynthetic efficiency, tissue nitrogen contents, and tissue C:N ratios. Our study results could help understand the physiological responses of U. linza under future ocean environmental conditions such as ocean warming and eutrophication.

Since the coronavirus disease outbreak in 2019, several antibody therapeutics have been developed to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Antibody therapeutics are effective in neutralizing the virus and reducing hospitalization in patients with mild and moderate infections. These therapeutics target the spike protein of SARS-CoV-2; however, emerging mutations in this protein reduce their efficiency. In this study, we developed a universal SARS-CoV-2 neutralizing antibody. We generated a humanized monoclonal antibody, MG1141A, against the receptor-binding domain of the spike protein through traditional mouse immunization. We confirmed that MG1141A could effectively neutralize live viruses, with an EC50 of 92 pM, and that it exhibited effective Fc-mediated functions. Additionally, it retained its neutralizing activity against the alpha (UK), beta (South Africa), and gamma (Brazil) variants of SARS-CoV-2. Taken together, our study contributes to the development of a novel antibody therapeutic approach, which can effectively combat emerging SARS-CoV-2 mutations.

The phagophore expands into autophagosomes in close proximity to endoplasmic reticulum (ER) exit sites (ERESs). Here, we propose that a single-pass ER transmembrane protein, SHISA5/SCOTIN, acts as an autophagy suppressor under basal condition by blocking the contact between the phagophore and ERES. HeLa cells lacking SHISA5 displayed higher levels of macroautophagy/autophagy. The enhanced autophagy in SHISA5 KO cells requires class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) activity and functional assembly of ERES, but not ULK1 activity. A proximity ligation assay (PLA) of SEC16A (Sec16 homolog A, endoplasmic reticulum export factor)-WIPI2 (WD repeat domain, phosphoinositide interacting 2) and SEC31A (Sec31 homolog A, COPII coat complex component)-MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) demonstrated that contact between the ERES and phagophore increased in SHISA5 KO cells, and the cytosolic domain of SHISA5 was sufficient to rescue this phenotype. Close proximity between ERES and phagophore in SHISA5 KO cells was also visualized by performing an ultrastructure correlative image analysis of SEC31A associated with LC3-positive membranes. Furthermore, we observed that SHISA5 was located near ERES under basal conditions, but displaced away from ERES under autophagy-inducing conditions. These data suggest that SHISA5 functions to block spontaneous contact between ERES and phagophore, and the blockage effect of SHISA5 should be relieved for the proper induction of autophagy. ABBREVIATIONS: ATG2: autophagy related 2; BECN1: beclin 1; COPII: coat protein II; DMSO: dimethyl sulfoxide; EBSS: Earle’s balanced salt solution; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERES: ER exit site(s); GFP: green fluorescent protein; H89: H-89 dihydrochloride hydrate; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NS5A: nonstructural protein 5A; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLA: proximity ligation assay; PtdIns3P: phosphatidylionositol-3-phosphate; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RFP: red fluorescent protein; RPS6KB1/S6K: ribosomal protein S6 kinase B1; SBP: streptavidin binding protein; SEC16A: SEC16 homolog A, endoplasmic reticulum export factor; SEC31A: SEC31 homolog A, COPII coat complex component; siRNA: small interfering RNA; Str: streptavidin; ULK1: unc-51-like autophagy activating kinase 1; VSVG: vesicular stomatitis virus glycoprotein; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type

Obesity is a major public health problem related to various chronic health conditions. Lactobacillus species has been reported in obese individuals; however, its role is unknown. We compared the abundance and composition of Lactobacillus species by analyzing feces from 64 healthy control subjects and 88 obese subjects. We isolated one Lactobacillus strain from the feces of a subject with obesity and further analyzed its genetic and molecular features. We found that an increased abundance and higher prevalence of Lactobacillus sakei distinguished the fecal microbiota of the obese group from that of healthy subjects and that it was related to the increased levels of reactive oxygen species (ROS) induced by higher fat intake. The L. sakei ob4.1 strain, isolated from the feces of a subject with obesity, showed high catalase activity, which was regulated by oxidative stress at the gene transcription level. L. sakei ob4.1 maintained colon epithelial cell adhesion ability under ROS stimulation, and treatment with saturated fatty acid increased colon epithelial ROS levels in a dose-dependent manner; however, L. sakei ob4.1 did not change the level of fat-induced colon epithelial ROS. Exposing mice to a high-fat diet revealed that high-fat-diet–induced colon ROS was associated with the increased colonization of L. sakei ob4.1 through catalase activity. Four-week supplementation with this strain in mice fed a high-fat diet did not change their body weights or ROS levels. A high-fat diet induces changes in the colon environment by increasing ROS levels, which provides a colonization benefit to an L. sakei strain with high catalase activity. IMPORTANCE Lactobacillus provides many health benefits; its various species are widely used as probiotics. However, an increased abundance of Lactobacillus has been reported in obesity, and the role of Lactobacillus strains in obesity remains unknown. We found a high abundance of the Lactobacillus sakei species in a group of obese subjects and examined its relationship with a high-fat diet and reactive oxygen species (ROS) in the feces. To find the underlying mechanism, we analyzed and characterized an L. sakei strain isolated from a severely obese individual. We found that higher gut oxidative stress could link high-fat-diet–induced obesity and L. sakei. This translational research identifies the roles of the host gut environment in the colonization and survival of L. sakei.

Clinical trials suggested that the vascular system can remember episodes of poor glycemic control through a phenomenon known as hyperglycemic memory (HGM). HGM is associated with long-term diabetic vascular complications in type 1 and type 2 diabetes, although the molecular mechanism of that association is not clearly understood. We hypothesized that transglutaminase 2 (TGase2) and intracellular reactive oxygen species (ROS) play a key role in HGM-induced vascular dysfunction. We found that hyperglycemia induced persistent oxidative stress, expression of inflammatory adhesion molecules, and apoptosis in the aortic endothelium of HGM mice whose blood glucose levels had been normalized by insulin supplementation. TGase2 activation and ROS generation were in a vicious cycle in the aortic endothelium of HGM mice and also in human aortic endothelial cells after glucose normalization, which played a key role in the sustained expression of inflammatory adhesion molecules and apoptosis. Our findings suggest that the TGase2-ROS vicious cycle plays an important role in HGM-induced endothelial dysfunction.—Lee, J.-Y., Lee, Y.-J., Jeon, H.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The vicious cycle between transglutaminase 2 and reactive oxygen species in hyperglycemic memory–induced endothelial dysfunction.

In the present study, several decursin analogues (KC1-3) were synthesized and evaluated in terms of their anti-septic activities on high mobility group box 1 (HMGB1)-mediated septic responses and survival rate in a mouse model of sepsis. KC1 and KC3, but not KC2, significantly reduced HMGB1 release in lipopolysaccharide (LPS)-activated human umbilical vein endothelial cells (HUVECs) and attenuated the cecal ligation and puncture (CLP)-induced release of HMGB1. Additionally, in vitro analyses revealed that KC1 and KC3 both alleviated HMGB1-mediated vascular disruptions and inhibited hyperpermeability in mice, and in vivo analyses revealed that KC1 and KC3 reduced sepsis-related mortality and tissue injury. Taken together, the present results suggest that KC1 and KC3 both reduced HMGB1 release and septic mortality and, thus, may be useful for the treatment of sepsis.

Objectives: Inflammation is a major mechanism underlying coronary heart disease (CHD) and C-reactive protein (CRP) is a marker of inflammation. When administered soon after menopause, menopausal hormone therapy (MHT) prevents CHD. This study was conducted to examine the impact of estrogen by administration route on CRP in postmenopausal Korean women using micronized progesterone (MP4) for endometrial protection. Methods: This retrospective cohort study included 129 healthy women without CHD risk factors. Eighty-nine women took oral estrogen (conjugated equine estrogen, 0.625 mg/day or equivalent), and 40 women applied a 1.5-mg/day 0.1% percutaneous estradiol gel. MP4 was added in 82 women with an intact uterus. The CRP level was measured at baseline and three and six months after initiation of MHT. Results: The baseline characteristics were comparable between the MHT groups except current age and age at menopause. After controlling for age, menopausal age, body mass index, and basal CRP, no significant change in CRP was observed in the oral estrogen group (n = 29). Follow-up CRP levels were also similar to the baseline in the percutaneous estrogen group (n = 18). However, three-month CRP was significantly lower than six-month CRP, and there was a significant time trend within the percutaneous estrogen group. However, the group difference did not reach statistical significance. CRP also did not differ by addition of MP4 in either group. Conclusions: In postmenopausal Korean women, no change in CRP was observed with oral estrogen, while percutaneous estrogen might decrease CRP. The estrogenic impacts were not influenced by adding MP4. (J Menopausal Med 2019;25:49-54)

The tau protein is a highly soluble and natively unfolded protein. Under pathological conditions, tau undergoes multiple post-translational modifications (PTMs) and conformational changes to form insoluble filaments, which are the proteinaceous signatures of tauopathies. To dissect the crosstalk among tau PTMs during the aggregation process, we phosphorylated and ubiquitylated recombinant tau in vitro using GSK3β and CHIP, respectively. The resulting phospho–ub-tau contained conventional polyubiquitin chains with lysine 48 linkages, sufficient for proteasomal degradation, whereas unphosphorylated ub-tau species retained only one–three ubiquitin moieties. Mass-spectrometric analysis of in vitro reconstituted phospho–ub-tau revealed seven additional ubiquitylation sites, some of which are known to stabilize tau protofilament stacking in the human brain with tauopathy. When the ubiquitylation reaction was prolonged, phospho–ub-tau transformed into insoluble hyperubiquitylated tau species featuring fibrillar morphology and in vitro seeding activity. We developed a small-molecule inhibitor of CHIP through biophysical screening; this effectively suppressed tau ubiquitylation in vitro and delayed its aggregation in cultured cells including primary cultured neurons. Our biochemical findings point to a “multiple-hit model,” where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process, thus indicating that targeting tau ubiquitylation may be an effective strategy to alleviate the course of tauopathies., Multiple-hit model for tau aggregation, where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process.

Rotor syndrome is caused by digenic loss-of-function variants in SLCO1B1 and SLCO1B3 but only a few studies have reported co-occurring inactivating variants from both genes. A rotor syndrome-causing long interspersed element-1 (LINE-1) insertion in SLCO1B3 had been reported to be highly prevalent in the Japanese population but there has been no additional report. In spite of its known association with various human diseases, LINE-1 is hard to detect with current sequencing technologies. In this study, we aimed to devise a method to screen the LINE-1 insertion variant and investigate the frequency of this variant in various populations. A chimeric sequence, that was generated by concatenating the reference sequence at the junction and a part of inserted LINE-1 sequence, was searched from 725 raw sequencing data files. In cases containing the chimeric sequence, confirmatory long-range PCR and gap-PCR were performed. In total, 95 (13.1%) of 725 patients were positive for the chimeric sequence, and all were confirmed to have the SLCO1B3 LINE-1 insertion by PCR-based tests. The same chimeric sequence was searched from the 1000 Genomes Project data repository and the carrier frequency was remarkably high in the East Asian populations (10.1%), especially in Southern Han Chinese (18.5%), but almost absent in other populations. This SLCO1B3 LINE-1 insertion should be screened in a population-specific manner under suspicion of Rotor syndrome and the methods proposed in this study would enable this in a simple way.

Food allergy is an emerging epidemic, and the underlying mechanisms are not well defined partly due to the lack of robust adjuvant free experimental models of dietary antigen sensitization. As housing mice at thermoneutrality (Tn) - the temperature of metabolic homeostasis (26–30°C) – has been shown to improve modeling various human diseases involved in inflammation, we tested the impact of Tn housing on an experimental model of food sensitization. Here we demonstrate that WT BALB/c mice housed under standard temperature (18–20°C, Ts) conditions translocated the luminal antigens in the small intestine (SI) across the epithelium via goblet cell antigen passages (GAPs). In contrast, food allergy sensitive Il4raF709 mice housed under standard temperature conditions translocated the luminal antigens in the SI across the epithelium via secretory antigen passages (SAPs). Activation of SI antigen passages and oral challenge of Il4raF709 mice with egg allergens at standard temperature predisposed Il4raF709 mice to develop an anaphylactic reaction. Housing Il4raF709 mice at Tn altered systemic type 2 cytokine, IL-4, and the landscape of SI antigen passage patterning (villus and crypt involvement). Activation of SI antigen passages and oral challenge of Il4raF709 mice with egg antigen under Tn conditions led to the robust induction of egg-specific IgE and development of food-induced mast cell activation and hypovolemic shock. Similarly, Tn housing of WT BALB/c mice altered the cellular patterning of SI antigen passage (GAPs to SAPs). Activation of SI antigen passages and the oral challenge of WT BALB/c mice with egg antigen led to systemic reactivity to egg and mast cell activation. Together these data demonstrate that Tn housing alters antigen passage cellular patterning and landscape, and concurrent oral exposure of egg antigens and SAP activation is sufficient to induce oral antigen sensitization.

Gastrointestinal disorders such as colorectal cancer or inflammatory bowel disease are linked to gut dysbiosis, an unbalanced gut microbiota. This early manifestation of the disease alters colon epithelial metabolism influencing the gut autofluorescence emission, which is susceptible to carry diagnostic value. We analyzed the fluorescence properties of healthy and dysbiotic ex vivo murine colons with an intraluminal fiber-based fluorescence lifetime imaging (FLIm) instrument. The results indicate that fluorescence lifetime reacts to inflammation in a spectrally dependant manner, and the full-length colon images allow to localize specific areas of activity. Imaging results were correlated to biochemical metabolic readouts (i.e. intracellular NADH, lactate) to establish the diagnostic potential of intraluminal FLIm.

Enzymatic cross-linking of polymer-catechol conjugates in the presence of horseradish peroxidase (HRP) and H2O2 has emerged as an important method to fabricate in situ-forming, injectable hydrogels. Subsequently, tissue adhesion studies using catechol-containing polymers were extensively reported. However, because of the presence of numerous variables such as polymer concentration, oxidizing agent/enzyme, and stoichiometry, the design of the polymer with optimized tissue adhesive property is still challenging. In this study, a poly(γ-glutamic acid) (γ-PGA)-dopamine (PGADA) conjugate was synthesized, and in situ hydrogels were fabricated via enzymatic cross-linking of a catechol moiety. To optimize the tissue adhesive property of the PGADA hydrogel, the effect of various factors, such as polymer concentration, catechol substitution degree (DS), HRP concentration, and H2O2 content, on the gelation behavior and mechanical strength was investigated. The gelation behavior of PGADA hydrogels was characterized using a rheometer and rotational viscometer. Also, the possibility of its use as a tissue adhesive was examined by evaluating the tissue adhesion strength in vitro and ex vivo.

Aging is a complex biological process that occurs in all living organisms. Aging is initiated by the gradual accumulation of biomolecular damage in cells leading to the loss of cellular function and ultimately death. Cellular senescence is one such pathway that leads to aging. The accumulation of nucleic acid damage and genetic alterations that activate permanent cell-cycle arrest triggers the process of senescence. Cellular senescence can result from telomere erosion and ribosomal DNA instability. In this review, we summarize the molecular mechanisms of telomere length homeostasis and ribosomal DNA stability, and describe how these mechanisms are linked to cellular senescence and longevity through lessons learned from budding yeast.

Aphidicolin represses DNA replication by inhibiting DNA polymerase α and δ, which leads to cell cycle arrest and cell damage. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) plays an essential role in maintenance of endothelial integrity including endothelial cell (EC) survival. Previously, we reported that aphidicolin increases NO production in bovine aortic ECs (BAECs). However, the role of aphidicolin-induced NO on EC viability and its molecular mechanism remain to be elucidated. Treatment with 20 μM aphidicolin for 24 h reduced BAEC viability by ~40%, which was accompanied by increased NO production, phosphorylation of eNOS at Ser1179 (p-eNOS-Ser1179), and eNOS protein expression. The aphidicolin-increased eNOS expression and p-eNOS-Ser1179 were not altered by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), a cell permeable and specific intracellular Ca2+ chelator. Co-treatment with 2-phenyl-4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), an NO scavenger, or Nω-Nitro- l -arginine methyl ester hydrochloride ( l -NAME), a NOS inhibitor, exacerbated aphidicolin-stimulated BAEC death. Knockdown of eNOS gene expression using siRNA aggravated aphidicolin-induced BAEC death. However, exogenous NO donors including S-nitroso- l -glutathione (GSNO) or diethylenetriamine NONOate (DETA NO) had no effect on aphidicolin-decreased BAEC viability and aggravated BAEC viability at higher doses. Interestingly, aphidicolin accumulated eNOS protein in the active form, p-eNOS-Ser1179, in the nucleus. When cells were ectopically transfected with a wild-type (WT)-eNOS gene, aphidicolin induced significant localization of the protein product in the nucleus. Additionally, aphidicolin-elicited cell death was significantly reversed in WT-eNOS gene-transfected BAECs. Furthermore, overexpression of the eNOS gene containing nuclear localization signal (NLS) but not nuclear export signal (NES) significantly attenuated aphidicolin-induced BAEC death. When G2A-eNOS mutant lacking myristoylation at Gly2 was transfected, its intracellular distribution became diffuse and included the nucleus. Finally, expression of N-myristoyltransferase 2 (NMT2) but not NMT1 significantly decreased in aphidicolin-treated BAECs. Taken together, our results suggest that aphidicolin attenuates BAEC death in part by increasing nuclear eNOS localization and NO production.

Background: Obesity is one of the major public health problems related to various chronic health conditions, with steadily increasing prevalence worldwide. Lactobacillus provides various benefits to the host body; however, its role in obesity is unknown. Results: In this study, we found higher colonisation of Lactobacillus sakei species in obesity group, which in turn was related to increased reactive oxygen species (ROS) levels induced by higher fat intake. We isolated L. sakei ob4.1 strain from the faeces of one subject with obesity and compared its genetic and molecular features with those of L. sakei DSM 20017 strain. L. sakei ob4.1 showed higher catalase activity, which was regulated by oxidative stress at gene transcriptional levels. L. sakei ob4.1 maintained colon epithelial cell-adhesion ability under ROS stimulation, and live bacteria could decrease colon epithelial inflammation in a dose-dependent manner. Establishing a mouse model revealed high-fat diet-induced colon ROS to be associated with increased colonisation of L. sakei ob4.1 through catalase activity. Four-week supplementation with this strain could reduce colon inflammation effectively, though not so for body weight or ROS levels in high fat-fed mice. Conclusion: We, therefore, concluded that changes in host gut-oxidative stress levels could link high fat-induced obesity to increased colonisation of L. sakei ob4.1 , and this strain could be potent as anti-inflammatory probiotic in obese population with gut inflammation. Keywords Lactobacillus sakei; Obesity; Gut microbiome; Reactive oxygen stress; Inflammation; High fat diet

Enzymatic crosslinking of polymer-catechol conjugate in the presence of horseradish peroxidase (HRP) and H2O2 has emerged as an important method to fabricate in situ-forming, injectable hydrogels. Subsequently, tissue adhesion studies using catechol-containing polymers were extensively reported. However, due to the presence of numerous variables such as polymer concentration, oxidizing agent/enzyme, and stoichiometry, the design of polymer with optimized tissue adhesive property is still challenging. In this study, poly(γ-glutamic acid) (γ-PGA)-dopamine conjugate (PGADA) was synthesized, and in situ hydrogels were fabricated via enzymatic crosslinking of catechol moiety. To optimize the tissue adhesive property of the PGADA hydrogel, the effect of various factors, such as polymer concentration, catechol substitution degree (DS), HRP concentration and H2O2 content, on the gelation behavior and mechanical strength was investigated. The gelation behavior of PGADA hydrogels was characterized by a rheometer and rotational viscometer. Also, the possibility of its use as a tissue adhesive was examined by evaluating the tissue adhesion strength in vitro and in vivo.

Background Food-induced anaphylaxis is a serious allergic reaction caused by Fce-receptor activation on mast cells (MCs). The exact mechanisms breaking oral tolerance and the effector pathways driving food allergy remain elusive. As complement is activated in food-induced anaphylaxis, we aimed to assess the role of C5a in disease pathogenesis. Methods Oral antigen-induced food-induced anaphylaxis was induced in BALB/c wild-type (wt) and C5ar1-/- mice. Readouts included diarrhea development, changes in rectal temperature, hematocrit, antigen-specific serum IgE, MCPT-1, and intestinal MC numbers, as well as FceR1-mediated MC functions including C5a receptor 1 (C5aR1) regulation. Further, histamine-mediated hypothermia and regulation of endothelial tight junctions were determined. Results Repeated oral OVA challenge resulted in diarrhea, hypothermia, increased hematocrit, high OVA-specific serum IgE, and MCPT-1 levels in wt mice. Male C5ar1-/- mice were completely whereas female C5ar1-/- mice were partially protected from anaphylaxis development. Serum MCPT-1 levels were reduced gender-independent, whereas IgE levels were reduced in male but not in female C5ar1-/- mice. Mechanistically, IgE-mediated degranulation and IL-6 production from C5ar1-/- BMMCs of both sexes were significantly reduced. Importantly, FceR1 cross-linking strongly upregulated C5aR1 MC expression in vitro and in vivo. Finally, C5ar1-/- male mice were largely protected from histamine-induced hypovolemic shock, which was associated with protection from histamine-induced barrier dysfunction in vitro following C5aR targeting. Conclusions Our findings identify C5aR1 activation as an important driver of IgE-mediated food allergy through regulation of allergen-specific IgE production, FceR1-mediated MC degranulation, and histamine-driven effector functions preferentially in male mice.

Protein ubiquitination is an important means of post-translational modification which plays an essential role in the regulation of various aspects of leukocyte development and function. The specificity of ubiquitin tagging to a protein substrate is determined by E3 ubiquitin ligases via defined E3-substrate interactions. In this review, we will focus on two E3 ligases, VHL and Itch, to discuss the latest progress in understanding their roles in the differentiation and function of CD4+ T helper cell subsets, the stability of regulatory T cells, effector function of CD8+ T cells, as well as the development and maturation of innate lymphoid cells. The biological implications of these E3 ubiquitin ligases will be highlighted in the context of normal and dysregulated immune responses including the control of homeostasis, inflammation, auto-immune responses and anti-tumor immunity. Further elucidation of the ubiquitin system in immune cells will help in the design of new therapeutic interventions for human immunological diseases and cancer.

Inhibition of high mobility group box 1 (HMGB1) and restoration of endothelial integrity are emerging as attractive therapeutic strategies for the management of severe vascular inflammatory diseases. Recently, we found that JH-4, a synthesized decursin derivative, exhibited a strong anti-Hutchinson-Gilford progeria syndrome by efficiently blocking progerin-lamin A/C binding. In this study, we examined the effects of JH-4 on HMGB1-mediated septic responses and the survival rate in a mouse sepsis model. The anti-inflammatory activities of JH-4 were monitored based on its effects on lipopolysaccharide- or cecal ligation and puncture (CLP)-mediated release of HMGB1. The antiseptic activities of JH-4 were determined by measuring permeability, leukocyte adhesion, migration, and the activation of proinflammatory proteins in HMGB1-activated human umbilical vein endothelial cells and mice. JH-4 inhibited the release of HMGB1 and downregulated HMGB1-dependent inflammatory responses in human endothelial cells. JH-4 also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with JH-4 reduced CLP-induced release of HMGB1, sepsis-related mortality, and pulmonary injury in vivo. Our results indicate that JH-4 is a possible therapeutic agent to treat various severe vascular inflammatory diseases via the inhibition of the HMGB1 signaling pathway.

Summary: The ubiquitin-proteasome system and the autophagy-lysosome system are two major intracellular proteolytic pathways in eukaryotes. Although several biochemical mechanisms underlying the crosstalk between them have been suggested, little is known about the effect of enhanced proteasome activity on autophagic flux. Here, we found that upregulation of proteasome activity, which was achieved through the inhibition of USP14, significantly impaired cellular autophagic flux, especially at the autophagosome-lysosome fusion step. UVRAG appeared to function as a crucial checkpoint for the proper progression of autophagic flux. Although proteasome activation through USP14 inhibition facilitated the clearance of microtubule-associated protein tau (MAPT) and reduced the amount of its oligomeric forms, the same conditions increased the formation of inclusion bodies from nonproteasomal substrates such as huntingtin with long polyglutamine repeats. Our results collectively indicate that USP14 may function as a common denominator in the compensatory negative feedback between the two major proteolytic processes in the cell. : Kim et al. present evidence that the ubiquitin-proteasome system and autophagy are in a compensatory negative-feedback connection through USP14, a proteasome-associated deubiquitinating enzyme. USP14 inhibition results in elevation of proteasome activity and facilitation of tau degradation in the cell, while it delays the cellular autophagic flux. Keywords: USP14, proteasome, ubiquitin-proteasome system, autophagy, autophagic flux, negative feedback, UVRAG, proteostasis, tau, huntingtin

The mechanisms by which the sensitivity of naive CD4+ T cells to stimulation by the cognate antigen via the T cell antigen receptor (TCR) determines their differentiation into distinct helper T cell subsets remain elusive. Here we demonstrate functional collaboration of the ubiquitin E3 ligases Itch and WWP2 in regulating the strength of the TCR signal. Mice lacking both Itch and WWP2 in T cells showed spontaneous autoimmunity and lung inflammation. CD4+ T cells deficient in Itch and WWP2 exhibited hypo-responsiveness to TCR stimulation and a bias toward differentiation into the TH2 subset of helper T cells. Itch and WWP2 formed a complex and cooperated to enhance TCR-proximal signaling by catalyzing the conjugation of atypical ubiquitin chains to the phosphatase SHP-1 and reducing the association of SHP-1 with the tyrosine kinase Lck. These findings indicate that targeted ubiquitination regulates the strength of the TCR signal and differentiation toward the TH2 lineage.

Originally annotated as the initiator of fatty acid synthesis (FAS), β-ketoacyl-acyl carrier protein synthase III (KAS III) is a unique component of the bacterial FAS system. Novel variants of KAS III have been identified that promote the de novo use of additional extracellular fatty acids by FAS. These KAS III variants prefer longer acyl-groups, notably octanoyl-CoA. Acinetobacter baumannii, a clinically important nosocomial pathogen, contains such a multifunctional KAS III (AbKAS III). To characterize the structural basis of its substrate specificity, we determined the crystal structures of AbKAS III in the presence of different substrates. The acyl-group binding cavity of AbKAS III and co-crystal structure of AbKAS III and octanoyl-CoA confirmed that the cavity can accommodate acyl groups with longer alkyl chains. Interestingly, Cys264 formed a disulfide bond with residual CoA used in the crystallization, which distorted helices at the putative interface with acyl-carrier proteins. The crystal structure of KAS III in the alternate conformation can also be utilized for designing novel antibiotics.

BAFF, a member of the TNF superfamily, has been recognized as a good target for autoimmune diseases. Belimumab, an anti-BAFF monoclonal antibody, was approved by the FDA for use in treating systemic lupus erythematosus. However, the molecular basis of BAFF neutralization by belimumab remains unclear. Here our crystal structure of the BAFF–belimumab Fab complex shows the precise epitope and the BAFF-neutralizing mechanism of belimumab, and demonstrates that the therapeutic activity of belimumab involves not only antagonizing the BAFF–receptor interaction, but also disrupting the formation of the more active BAFF 60-mer to favor the induction of the less active BAFF trimer through interaction with the flap region of BAFF. In addition, the belimumab HCDR3 loop mimics the DxL(V/L) motif of BAFF receptors, thereby binding to BAFF in a similar manner as endogenous BAFF receptors. Our data thus provides insights for the design of new drugs targeting BAFF for the treatment of autoimmune diseases., Blocking B-cell activating factor (BAFF), an important soluble factor for B-cell responses, with specific antibodies is approved for treating autoimmune disorders. Here the authors show, with structural data, that antibody-BAFF interactions not only interrupt BAFF–receptor-binding, but also induce the formation of a less active BAFF polymer.

Certain plant extracts (PEs) contain bioactive compounds that have antioxidant and lifespan-extending activities on organisms. These PEs play different roles in cellular processes, such as enhancing stress resistance and modulating longevity-defined signaling pathways that contribute to longevity. Here, we report the discovery of PEs that extended chronological life span (CLS) in budding yeast from a screen of 222 PEs. We identified two PEs, the leaf extracts of Manihot esculenta and Wodyetia bifurcata that extended CLS in a dose-dependent manner. The CLS-extending PEs also conferred oxidative stress tolerance, suggesting that these PEs might extend yeast CLS through the upregulation of stress response pathways.

BACKGROUND: Type 2 immunity can be modulated by regulatory T cell (Treg) activity. It has been suggested that the deubiquitinase CYLD plays a role in the development or function of Treg cells implying it could be important for normal protective immunity where type 2 responses are prevalent. OBJECTIVE: We sought to investigate the role of CYLD in Treg function and T helper type (Th2) immune responses under steady state conditions and during helminth infection. METHODS: Foxp3-restricted CYLD conditional knockout mice (cKO) were examined in mouse models of allergen-induced airway inflammation and Nippostrongylus brasiliensis infection. We performed multiplex magnetic bead assays, flow cytometry and qPCR to understand how a lack of CYLD affected cytokine production, homing and suppression in Tregs. Target genes regulated by CYLD were identified and validated by microarray analysis, co-immunoprecipitation, shRNA knockdown, and transfection assays. RESULTS: Treg-specific CYLD knockout mice showed severe spontaneous pulmonary inflammation with increased migration of Treg cells into the lung. CYLD-deficient Treg cells furthermore produced high levels of interleukin-4 (IL-4) and failed to suppress allergen-induced lung inflammation. Supporting this, the cKO mice displayed enhanced protection against N. brasiliensis infection by contributing to type 2 immunity. Treg conversion into IL-4 producing cells was due to augmented MAPK and NF-κB signaling. Moreover, Scinderin, a member of the actin-binding gelsolin family, was highly upregulated in CYLD-deficient Treg cells, and controlled IL-4 production through forming complexes with MEK/ERK. Correspondingly, both excessive IL-4 production in vivo and the protective role of CYLD-deficient Treg cells against N. Brasiliensis were reversed by Scinderin ablation. CONCLUSIONS: Our findings indicate that CYLD controls type 2 immune responses by regulating Treg conversion into Th2-like effector cells, which potentiates parasite resistance. CAPSULE SUMMARY: Deubiquitinating enzyme CYLD plays a role in maintaining Treg function on type 2 immunity, which will be beneficial for therapeutic approaches to autoimmune disease or pathogen infection.

PURPOSE We investigated whether polymorphisms of the endothelial NO synthase (eNOS) gene are associated with normal tension glaucoma (NTG). We also investigated whether the eNOS polymorphisms are associated with NTG subgroups [NTG with and without optic disc hemorrhage (DH)]. METHODS A total of 251 patients with NTG and 245 healthy volunteers were enrolled in this study. DNA from peripheral blood leukocytes was extracted, and the genotypes of 4 polymorphisms (rs2070744, rs1549758, rs1799983, and rs2566514) in the eNOS gene were determined using restriction fragment length polymorphism and the SNaPshot method. The primary outcome was to investigate the relation between eNOS polymorphisms and NTG. The secondary outcome was to compare the frequencies of the polymorphic genotypes among the NTG subgroups. Bonferroni correction was used to adjust for type I error. RESULTS In all subjects, the genotype distribution was in accordance with Hardy-Weinberg equilibrium. None of the 4 polymorphisms showed any significant difference in the frequencies of alleles or genotypes between the NTG patients and controls. In the further analysis comparing the genotypic frequencies between NTG with DH and normal controls, the CC/CT genotype of rs2070744 was significantly associated with DH in NTG patients (genotypic association test, P-value=0.0041). On the multiple logistic regression analysis adjusted for covariates such as sex and age, the NTG with DH was associated with polymorphic genotypes of rs2070744 with a borderline significance (additive genetic model, P=0.0070). CONCLUSIONS Our results indicates that eNOS rs2070744 can be associated with NTG patients with DH. This finding suggests that the eNOS polymorphism may be a genetic risk factor in the development of DH in NTG patients.

Background Tubuloreticular inclusions (TRIs) are anastomosing networks of microtubules that are frequently found in autoimmune diseases and viral infections. In renal allografts, TRIs have been reported in glomerular endothelial cells in association with viral infections and donor specific antibodies (DSAs), but their presence in peritubular capillaries has not been explored. Methods We collected seven cases with TRIs out of 148 consecutive renal allograft biopsies taken from Dec. 2015 to Dec. 2016. Results TRIs were present in peritubular capillaries in seven cases and were concomitantly present in glomerular endothelial cells in two cases. The diagnoses included polyomavirus nephropathy (n = 2), acute T cell-mediated rejection (ACR) (n = 1), combined ACR and antibody-mediated rejection (AMR) (n = 1), suspicious for ACR (n = 1), chronic active AMR (n = 1), and moderate tubular atrophy and interstitial fibrosis (n = 1). Six patients had recent or current viral infections (BK polyomavirus, hepatitis B virus, herpes simplex virus, and cytomegalovirus in two, two, one, and one case, respectively). DSA was positive in one case. Five cases had moderate to severe interstitial inflammation and four cases had peritubular capillaritis. Conclusion TRIs are not rare in peritubular capillaries. They are associated with various viral infections and their appearance seems to be related to peritubular capillary injury.

It has been shown that epigenetic regulation plays an important role in skin wound healing. We previously found that histone H3K27me3 demethylase JMJD3 regulates inflammation and cell migration in keratinocyte wound healing. In this study, we identified Notch1 as a direct target of JMJD3 and NF-κB in wounded keratinocytes using in vitro cell and in vivo animal models. We found that Notch1 is up-regulated in the wound edge and its expression is dependent on JMJD3 and NF-κB in wounded keratinocytes. We also found that Notch1 activates the expression of RhoU and PLAU gene, which are critical regulators of cell migration. Consistently, depletion or inactivation of Notch1 resulted in decreased filopodia formation, increased focal adhesion and actin stress fiber, leading to reduced keratinocyte migration and skin wound healing. Thus, our findings provide the molecular mechanism involving JMJD3/NF-κB-Notch pathway in keratinocyte wound healing.

Bacillus subtilis sups. chungkookjang produces a higher molecular mass poly-γ-glutamic acid (γ-PGA). Recently, previous studies have demonstrated immune stimulation and an antitumor effect of the high molecular mass γ-PGA using various mouse models although these effects have not been shown in other species of animals. Therefore, the current study was conducted to determine the effect of γ-PGA in pigs with and without PRRSV infection. PRRS-negative pigs were intramuscularly injected with 1, 3, or 5 ml of 20 mg/mll γ-PGA, and one group of pigs served as a non-treatment (NT) group. All groups treated with γ-PGA had significantly higher weight gains, and pigs treated with 5 ml of γ-PGA exhibited higher tumor necrosis factor (TNF)-α, interferon (IFN)-α and IFN-β expression levels compared with the NT group. According to the preliminary results, an animal challenge study was conducted with a highly virulent PRRSV strain, MN184, along with γ-PGA treatment at different time points. Pigs treated with γ-PGA had lower levels of viral loads in the sera and in lungs and gained significantly more weight (p

Background: Highly specific assays for measuring antiphospholipid antibodies (aPLs) are required for accurate assessment of thrombotic risk. aPLs against β2-glycoprotein I domain I (anti-β2GPIdI) and against prothrombin complexed with phosphatidylserine (anti-PS/PT) have been recently identified as being associated with a hypercoagulable state. This study evaluated the synergism between anti-β2GPIdI and anti-PS/PT for predicting thrombotic events. Methods: A total of 180 patients with clinical suspicion of hypercoagulability were evaluated. The plasma levels of lupus anticoagulant (LA) and antibodies against anticardiolipin (anti-CL) (IgG and IgM), β2GPI (IgG and IgM), PS/PT (IgG and IgM), and β2GPI dI (IgG) were measured. Results: IgG anti-β2GPIdI and LA were highly associated with thrombosis. Mean values and positivity rates of IgG anti-β2GPI dI and IgG anti-PS/PT were significantly higher in the triple-positive group (LA+, IgG anti-CL+, IgG anti-β2GPI+) than in the other groups. Interestingly, the thrombotic risk [odds ratio (OR) 24.400, 95% confidence interval (CI) 1.976–63.273, p Conclusions: Our data suggest that simultaneous measurement of IgG anti-β2GPIdI and IgG anti-PS/PT may improve clinical decision-making for aPL-positive patients.

Background During two pivotal clinical trials of the infliximab biosimilar CT-P13 (PLANETAS and PLANETRA), antidrug antibodies (ADAs) and neutralising antibodies (NAbs) were detected in the sera of patients treated with CT-P13 and the reference product (RP; Remicade). Objective The aim was to assess the comparability of Remicade- and CT-P13-tagged immunoassays for the detection of ADAs and NAbs using data from these trials, in order to determine the cross-reactivity of CT-P13 and RP ADAs. Methods Sera from patients with rheumatoid arthritis and ankylosing spondylitis were analysed using an electrochemiluminescence (ECL) bridging assay or Gyros immunoassay, tagged with Remicade or CT-P13 at screening, weeks 14, 30 and 54, and the end of study visit. NAb titre was compared at screening and weeks 14 and 30. The proportion of cross-reactive samples was determined and an inter-rater agreement analysis performed to assess the concordance of results between assays. Results In PLANETAS, 93.1% (94/101) of RP ADA-positive samples and 93.0% (93/100) of RP NAb-positive samples cross-reacted with CT-P13; 99.0% (103/104) of CT-P13 ADA-positive and 98.0% (98/100) of CT-P13 NAb-positive samples cross-reacted with the RP. In PLANETRA, 94.7% (426/450) of RP ADA-positive samples and 94.3% (415/440) of RP NAb-positive samples cross-reacted with CT-P13, and 96.6% (458/474) of CT-P13 ADA-positive and 96.4% (452/469) of CT-P13 NAb-positive samples cross-reacted with the RP. In both studies, there was strong agreement in outcome between assays at all post-screening time points (PLANETAS: Cohen’s κ 0.89–0.98 for ADA, 0.86–0.98 for NAb; PLANETRA: 0.92–0.94 for both ADA and NAb, all p

Highly selective cytochrome P450 CYP2J2 (CYP2J2) inhibitors suitable for reaction phenotyping are currently not available. (7S)-(+)-(4-Nitro-phenyl)-acrylic acid, 8,8-dimethyl-2-oxo-6,7-dihydro-2H,8H-pyrano[3,2-g]chromen-7-yl-ester (LKY-047), a decursin derivative, was synthesized, and its inhibitor potencies toward CYP2J2 as well as other cytochrome P450 (P450) enzymes in human liver microsomes (HLM) were evaluated. LKY-047 was demonstrated to be a strong competitive inhibitor of CYP2J2-mediated astemizole O-demethylase and terfenadine hydroxylase activity, with Ki values of 0.96 and 2.61 μM, respectively. It also acted as an uncompetitive inhibitor of CYP2J2-mediated ebastine hydroxylation with a Ki value of 3.61 μM. Preincubation of LKY-047 with HLMs and NADPH did not alter inhibition potency, indicating that it is not a mechanism-based inhibitor. LKY-047 was found to be a selective CYP2J2 inhibitor with no inhibitory effect on other human P450s, such as CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A (IC50 > 50 μM). These in vitro data support the use of LKY-047 as a selective CYP2J2 inhibitor with potential application in the identification of P450 isoforms responsible for drug metabolism in reaction phenotyping assays.

Ethnopharmacological relevance Cordyceps militaris is an ingredient of traditional Chinese medicine and have been widely used for inflammatory diseases and cancer. Cordycepin is one of the major bioactive components of Cordyceps militaris, and has been known to have anti-inflammatory and anti-oxidant effects. Aim of this study In the present study, we examined whether WIB-801C, a standardized and cordycepin-enriched extract of caterpillar fungus (Cordyceps militaris), would attenuate blood-spinal cord barrier (BSCB) disruption by inhibiting matrix metalloprotease (MMP)−9 activity, leading to improvement of functional outcomes after spinal cord injury (SCI). Materials and methods Male Sprague-Dawley rats were subjected to contusive SCI using a New York University (NYU) impactor, and WIB-801C (50 mg/kg) was administered at 2 h and 8 h after injury orally and further treated once a day for indicated time points. BSCB disruption, MMP-9 activity, blood infiltration, inflammation, neuronal apoptosis, axonal loss, demyelination, and neurological deficit were evaluated. Results We found that WIB-801C significantly attenuated BSCB disruption by inhibiting MMP-9 expression and activation after injury. The infiltration of neutrophils at 1 d and macrophage at 5 d after SCI was also ameliorated by WIB-801C as compared with vehicle control. In addition, the expression of inflammatory cytokines and mediators such as Tnf-α, IL-1β, IL-6, Cox-2, and inos as well as chemokines such as Gro-α and Mip-2α was significantly inhibited by WIB-801C. Furthermore, WIB-801C inhibits p38MAPK activation and proNGF production in microglia after injury. These events eventually led to the inhibition of apoptotic cell death of neurons and oligodendrocytes, improved functional recovery and attenuated demyelination and axon loss after SCI. Conclusion Our results suggest that WIB-801C can be used as a therapeutic agent after SCI by attenuating BSCB disruption followed inflammation.