Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry. Recent advances in next-generation sequencing technologies have facilitated the elucidation of the microbial community composition with higher accuracy and greater throughput than was previously achievable; however, the short sequencing reads often limit the microbial composition analysis at the species level due to the high similarity of 16S rRNA amplicon sequences. To overcome this limitation, we used the nanopore sequencing platform to sequence full-length 16S rRNA amplicon libraries prepared from the mouse gut microbiota. A comparison of the nanopore and short-read sequencing data showed that there were no significant differences in major taxonomic units (89%) except one phylotype and three taxonomic units. Moreover, both sequencing data were highly similar at all taxonomic resolutions except the species level. At the species level, nanopore sequencing allowed identification of more species than short-read sequencing, facilitating the accurate classification of the bacterial community composition. Therefore, this method of full-length 16S rRNA amplicon sequencing will be useful for rapid, accurate and efficient detection of microbial diversity in various biological and clinical samples.

This study demonstrates the capacity of HM-γ-PGA treatment to significantly protect murine macrophage cells (RAW 264.7 cells) against NDV infection. Such protection can be explained by the induction of antiviral state of HM-γ-PGA in RAW 264.7 cells via TLR4-mediated IRF-3, IRF-7, IFN-β, and IFN-related gene induction as shown in time-dependent changes in mRNA expression confirmed by polymerase chain reaction (PCR). Moreover, the present research also showed that HM-γ-PGA can induce proinflammatory cytokine secretion in RAW 264.7 as measured by enzyme-linked immunosorbent assay (ELISA). Therefore, our findings suggest that HM-γ-PGA can be a potential antiviral substance that can inhibit NDV infection through its stimulation of antiviral state on RAW 264.7 cells. These results have been consistent with the previous studies showing that HM-γ-PGA can protect RAW 264.7 cells and mice against influenza infection. However, it should be noted that although murine macrophage cells are susceptible to NDV, they are not the natural host cells of the virus; thus further in vivo and in vitro studies involving chicken and chicken immune cells are needed to fully assess the efficacy and applicability of HM-γ-PGA in the poultry industry.

The synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-C28-methyl ester (CDDO-Me) is considered a promising anti-tumorigenic compound. In this study, we show that treatment with CDDO-Me induces progressive endoplasmic reticulum (ER)-derived vacuolation in various breast cancer cells and ultimately kills these cells by inducing apoptosis. We found that CDDO-Me-induced increases in intracellular Ca2+ levels, reflecting influx from the extracellular milieu, make a critical contribution to ER-derived vacuolation and subsequent cell death. In parallel with increasing Ca2+ levels, CDDO-Me markedly increased the generation of reactive oxygen species (ROS). Interestingly, there exists a reciprocal positive-regulatory loop between Ca2+ influx and ROS generation that triggers ER stress and ER dilation in response to CDDO-Me. In addition, CDDO-Me rapidly reduced the protein levels of c-FLIPL (cellular FLICE-inhibitory protein) and overexpression of c-FLIPL blocked CDDO-Me-induced cell death, but not vacuolation. These results suggest that c-FLIPL downregulation is a key contributor to CDDO-Me-induced apoptotic cell death, independent of ER-derived vacuolation. Taken together, our results show that ER-derived vacuolation via Ca2+ influx and ROS generation as well as caspase activation via c-FLIPL downregulation are responsible for the potent anticancer effects of CDDO-Me on breast cancer cells.

Piperlongumine (PL), a biologically active compound from the Piper species, has been shown to exert various pharmacological effects in a number of conditions, including tumours, diabetes, pain, psychiatric disorders and neurodegenerative disease. In this study, we evaluated the therapeutic effects of PL on hippocampal function and cognition decline in aged mice. PL (50 mg/kg/day) was intragastrically administrated to 23‑month‑old female C57BL/6J mice for 8 weeks. Novel object recognition and nest building behaviour tests were used to assess cognitive and social functions. Additionally, immunohistochemistry and western blot analysis were performed to examine the effects of PL on the hippocampus. We found that the oral administration of PL significantly improved novel object recognition and nest building behaviour in aged mice. Although neither the percentage area occupied by astrocytes and microglia nor the level of 4‑hydroxynonenal protein, a specific marker of lipid peroxidation, were altered by PL treatment, the phosphorylation levels of N‑methyl‑D‑aspartate receptor subtype 2B (NR2B), calmodulin‑dependent protein kinase II alpha (CaMKIIα) and extracellular signal‑regulated kinase 1/2 (ERK1/2) were markedly increased in the hippocampus of aged mice following the administration of PL. We also found that PL treatment resulted in a CA3‑specific increase in the phosphorylation level of cyclic AMP response element binding protein, which is recognized as a potent marker of neuronal plasticity, learning and memory. Moreover, the number of doublecortin‑positive cells, a specific marker of neurogenesis, was significantly increased following PL treatment in the dentate gyrus of the hippocampus. On the whole, these data demonstrate that PL treatment may be a potential novel approach in the treatment of age‑related cognitive impairment and hippocampal changes.

The orphan nuclear receptor estrogen-related receptor-γ (ERRγ) is a constitutively active transcription factor regulating genes involved in several important cellular processes, including hepatic glucose metabolism, alcohol metabolism, and the endoplasmic reticulum (ER) stress response. cAMP responsive element-binding protein H (CREBH) is an ER-bound bZIP family transcription factor that is activated upon ER stress and regulates genes encoding acute-phase proteins whose expression is increased in response to inflammation. Here, we report that ERRγ directly regulates CREBH gene expression in response to ER stress. ERRγ bound to the ERRγ response element (ERRE) in the CREBH promoter. Overexpression of ERRγ by adenovirus significantly increased expression of CREBH as well as C-reactive protein (CRP), whereas either knockdown of ERRγ or inhibition of ERRγ by ERRγ specific inverse agonist, GSK5182, substantially inhibited ER stress-mediated induction of CREBH and CRP. The transcriptional coactivator PGC1α was required for ERRγ mediated induction of the CREBH gene as demonstrated by the chromatin immunoprecipitation (ChIP) assay showing binding of both ERRγ and PGC1α on the CREBH promoter. The ChIP assay also revealed that histone H3 and H4 acetylation occurred at the ERRγ and PGC1α binding site. Moreover, chronic alcoholic hepatosteatosis, as well as the diabetic obese condition significantly increased CRP gene expression, and this increase was significantly attenuated by GSK5182 treatment. We suggest that orphan nuclear receptor ERRγ directly regulates the ER-bound transcription factor CREBH in response to ER stress and other metabolic conditions.

Next-generation sequencing (NGS) technology has improved enough to discover mutations associated with genetic diseases. Our study evaluated the feasibility of targeted NGS as a primary screening tool to detect causal variants and subsequently predict genetic diseases. We performed parallel computations on 3.7-megabase-targeted regions to detect disease-causing mutations in 103 participants consisting of 81 patients and 22 controls. Data analysis of the participants took about 6 hours using local databases and 200 nodes of a supercomputer. All variants in the selected genes led on average to 3.6 putative diseases for each patient while variants restricted to disease-causing genes identified the correct disease. Notably, only 12% of predicted causal variants were recorded as causal mutations in public databases: 88% had no or insufficient records. In this study, most genetic diseases were caused by rare mutations and public records were inadequate. Most rare variants, however, were not associated with genetic diseases. These data implied that novel, rare variants should not be ignored but interpreted in conjunction with additional clinical data. This step is needed so appropriate advice can be given to primary doctors and parents, thus fulfilling the purpose of this method as a primary screen for rare genetic diseases.

The plasma jet has been proposed as a novel therapeutic method for anticancer treatment. However, its biological effects and mechanism of action remain elusive. Here, we investigated its cell death effects and underlying molecular mechanisms, using air and N₂ plasma jets from a micro nozzle array. Treatment with air or N₂ plasma jets caused apoptotic death in human cervical cancer HeLa cells, simultaneously with depolarization of mitochondrial membrane potential. In addition, the plasma jets were able to generate reactive oxygen species (ROS), which function as surrogate apoptotic signals by targeting the mitochondrial membrane potential. Antioxidants or caspase inhibitors ameliorated the apoptotic cell death induced by the air and N₂ plasma jets, suggesting that the plasma jet may generate ROS as a proapoptotic cue, thus initiating mitochondria-mediated apoptosis. Taken together, our data suggest the potential employment of plasma jets as a novel therapy for cancer.

Cisplatin is a widely used chemotherapeutic agent for the treatment of various tumors. In addition to its antitumor activity, cisplatin affects normal cells and may induce adverse effects, such as ototoxicity, nephrotoxicity, and neuropathy. Various mechanisms, such as DNA adduct formation, mitochondrial dysfunction, oxidative stress, and inflammatory responses, are critically involved in cisplatin-induced adverse effects. As NAD(+) is a cofactor for various enzymes associated with cellular homeostasis, we studied the effects of increased NAD(+) levels by means of

Acetyltransferase, p300 is a transcriptional cofactor of signal-responsive transcriptional regulation. The surveillance kinase ataxia-telangiectasia mutated (ATM) plays a central role in regulation of a wide range of cellular DNA damage responses. Here, we investigated whether and how ATM mediates phosphorylation of p300 in response to DNA damage and how p300 phosphorylation is functionally linked to DNA damage. ATM-phosphorylated p300 in vitro and in vivo, in response to DNA damage. Phosphorylation of p300 proteins was observed upon gamma-irradiation in ATM(+) cells but not ATM(-) cells. Importantly, expression of nonphosphorylatable serine to alanine form of p300 (S106A) destabilized both p300 and NBS1 proteins, after DNA damage. These data demonstrate that ATM transduces a DNA damage signal to p300, and that ATM-dependent phosphorylation of p300 is required for stabilization of NBS1 proteins in response to DNA damage.

Oxidative functions of adipose tissue macrophages control the polarization of M1-like and M2-like phenotypes, but whether reduced macrophage oxidative function causes systemic insulin resistance in vivo is not clear. Here, we show that mice with reduced mitochondrial oxidative phosphorylation (OxPhos) due to myeloid-specific deletion of CR6-interacting factor 1 (Crif1), an essential mitoribosomal factor involved in biogenesis of OxPhos subunits, have M1-like polarization of macrophages and systemic insulin resistance with adipose inflammation. Macrophage GDF15 expression is reduced in mice with impaired oxidative function, but induced upon stimulation with rosiglitazone and IL-4. GDF15 upregulates the oxidative function of macrophages, leading to M2-like polarization, and reverses insulin resistance in ob/ob mice and HFD-fed mice with myeloid-specific deletion of Crif1. Thus, reduced macrophage oxidative function controls systemic insulin resistance and adipose inflammation, which can be reversed with GDF15 and leads to improved oxidative function of macrophages.

We evaluated the relationship between myocardial infarct size and inflammatory response using cardiac magnetic resonance imaging (CMR) in an acute myocardial infarction (AMI) mouse model. Myocardial infarction (MI) was induced in 14 mice by permanent ligation of the left anterior descending artery. Late gadolinium enhancement (LGE), manganese-enhanced MRI (MEMRI), and magnetofluorescent nanoparticle MRI (MNP-MRI) were performed 1, 2, and 3 days after MI, respectively. The size of the enhanced lesion was quantitatively determined using Otsu's thresholding method in area-based and sector-based approaches and was compared statistically. Linear correlation between the enhanced lesion sizes was evaluated by Pearson's correlation coefficients. Differences were compared using Bland-Altman analysis. The size of the inflammatory area determined by MNP-MRI (57.1 ± 10.1%) was significantly larger than that of the infarct area measured by LGE (40.8 ± 11.7%, P < 0.0001) and MEMRI (44.1 ± 14.9%, P < 0.0001). There were significant correlations between the sizes of the infarct and inflammatory lesions (MNP-MRI versus LGE: r = 0.3418, P = 0.0099; MNP-MRI versus MEMRI: r = 0.4764, P = 0.0002). MNP-MRI provides information about inflammatory responses in a mouse model of AMI. Thus, MNP-MRI associated with LGE and MEMRI may play an important role in monitoring the disease progression in MI.

We aimed to investigate the effect of Sicyos angulatus (SA) ethanolic extracts as antioxidants and potential treatments for liver disease.

Obesity is a medical condition in which excess body fat has accumulated to a serious extent. It is a chronic disease that can lead to dyslipidemia, insulin resistance, and type 2 diabetes. In the present study, we investigated the anti-obesity effects of Sicyos angulatus (SA) extract on a high-fat diet (HFD)-induced C57BL/6J obese mice. The mice were divided into vehicle and three SA groups (25, 50, and 100 mg/kg body weight). The mice were fed a HFD with or without SA for 12 weeks. The oral administration of SA reduced body and adipose tissue weight in HFD-fed mice compared to those in the vehicle group (p<0.05). Adipocyte size and inflammation significantly decreased in the SA-administered groups in a dose-dependent manner. In particular, adipocytes larger than 5000 µm2 were remarkably reduced by around 50% in the SA-treated groups (p<0.05). In addition, SA contributes towards reducing insulin resistance (measured as the HOMA-IR index) and glucose intolerance in HFD-induced obese mice (p<0.05; Vehicle 21.5±3.1 vs. SA100 4.7±0.4). These beneficial effects of SA on obesity may be linked to the suppression of lipogenesis and stimulating energy metabolism in white adipose tissue and muscle. In white adipose tissue and muscle, the administration of SA activated AMPK pathway, leading to the inhibition of the development of pathophysiological conditions associated with obesity, including lipogenesis and inflammation. These findings suggest that SA may prevent obesity through inhibiting fat accumulation in HFD-induced obese mice. Therefore, SA is able to exert metabolic benefits in the prevention of obesity and insulin resistance.

Tumor cells can vary epigenetically during ionizing irradiation (IR) treatment. These epigenetic variegations can influence IR response and shape tumor aggressiveness. However, epigenetic disturbance of histones after IR, implicating in IR responsiveness, has been elusive. Here, we investigate whether altered histone modification after IR can influence radiation responsiveness. The oncogenic CXCL12 mRNA and protein were more highly expressed in residual cancer cells from a hepatoma heterotopic murine tumor microenvironment and coculture of human hepatoma Huh7 and normal IMR90 cells after radiation. H3K4 methylation was also enriched and H3K9 methylation was decreased at its promoter region. Accordingly, invasiveness and the subpopulation of aggressive CD133+/CD24- cells increased after IR. Histone demethylase inhibitor IOX1 attenuated CXCL12 expression and the malignant subpopulation, suggesting that responses to IR can be partially mediated via histone modifications. Taken together, radiation-induced histone alterations at the CXCL12 promoter in hepatoma cells are linked to CXCL12 upregulation and increased aggressiveness in the tumor microenvironment.

Fas-associated factor 1 is a death-promoting protein that induces apoptosis by interacting with the Fas receptor. Until now, FAF1 was reported to interact potentially with diverse proteins and to function as a negative and/or positive regulator of several cellular possesses. However, the role of FAF1 in defense against bacterial infection remains unclear. Here, we show that FAF1 plays a pivotal role in activating NADPH oxidase in macrophages during Listeria monocytogenes infection. Upon infection by L. monocytogenes, FAF1 interacts with p67phox (an activator of the NADPH oxidase complex), thereby facilitating its stabilization and increasing the activity of NADPH oxidase. Consequently, knockdown or ectopic expression of FAF1 had a marked effect on production of ROS, proinflammatory cytokines, and antibacterial activity, in macrophages upon stimulation of TLR2 or after infection with L. monocytogenes. Consistent with this, FAF1gt/gt mice, which are knocked down in FAF1, showed weaker inflammatory responses than wild-type mice; these weaker responses led to increased replication of L. monocytogenes. Collectively, these findings suggest that FAF1 positively regulates NADPH oxidase-mediated ROS production and antibacterial defenses.

Non-structural protein 1 (NS1) of influenza virus has been shown to inhibit the innate immune response by blocking the induction of interferon (IFN). In this study, we isolated two single-stranded RNA aptamers specific to NS1 with K d values of 1.62 ± 0.30 nM and 1.97 ± 0.27 nM, respectively, using a systematic evolution of ligand by exponential enrichment (SELEX) procedure. The selected aptamers were able to inhibit the interaction of NS1 with tripartite motif-containing protein 25 (TRIM25), and suppression of NS1 enabled retinoic acid inducible gene I (RIG-I) to be ubiquitinated regularly by TRIM25. Additional luciferase reporter assay and quantitative real-time PCR (RT-PCR) experiments demonstrated that suppression of NS1 by the selected aptamers induced IFN production. It is noted that viral replication was also inhibited through IFN induction in the presence of the selected aptamers. These results suggest that the isolated aptamers are strongly expected to be new therapeutic agents against influenza infection.

N-Myc downstream regulated gene 3 (NDRG3) is a unique pro-tumorigenic member among NDRG family genes, mediating growth signals. Here, we investigated the pathophysiological roles of NDRG3 in relation to cell metabolism by disrupting its functions in liver. Mice with liver-specific KO of NDRG3 (Ndrg3 LKO) exhibited glycogen storage disease (GSD) phenotypes including excessive hepatic glycogen accumulation, hypoglycemia, elevated liver triglyceride content, and several signs of liver injury. They suffered from impaired hepatic glucose homeostasis, due to the suppression of fasting-associated glycogenolysis and gluconeogenesis. Consistently, the expression of glycogen phosphorylase (PYGL) and glucose-6-phosphate transporter (G6PT) was significantly down-regulated in an Ndrg3 LKO-dependent manner. Transcriptomic and metabolomic analyses revealed that NDRG3 depletion significantly perturbed the methionine cycle, redirecting its flux towards branch pathways to upregulate several metabolites known to have hepatoprotective functions. Mechanistically, Ndrg3 LKO-dependent downregulation of glycine N-methyltransferase in the methionine cycle and the resultant elevation of the S-adenosylmethionine level appears to play a critical role in the restructuring of the methionine metabolism, eventually leading to the manifestation of GSD phenotypes in Ndrg3 LKO mice. Our results indicate that NDRG3 is required for the homeostasis of liver cell metabolism upstream of the glucose-glycogen flux and methionine cycle and suggest therapeutic values for regulating NDRG3 in disorders with malfunctions in these pathways.

Genetic variations in mitoribosomal subunits and mitochondrial transcription factors are related to type 2 diabetes. However, the role of islet mitoribosomes in the development of type 2 diabetes has not been determined. We investigated the effects of the mitoribosomal gene on β-cell function and glucose homeostasis. Mitoribosomal gene expression was analyzed in datasets from the NCBI GEO website (GSE25724, GSE76894, and GSE76895) and the European Nucleotide Archive (ERP017126), which contain the transcriptomes of type 2 diabetic and nondiabetic organ donors. We found deregulation of most mitoribosomal genes in islets from individuals with type 2 diabetes, including partial downregulation of CRIF1. The phenotypes of haploinsufficiency in a single mitoribosomal gene were examined using β-cell-specific Crif1 (Mrpl59) heterozygous-deficient mice. Crif1beta+/- mice had normal glucose tolerance, but their islets showed a loss of first-phase glucose-stimulated insulin secretion. They also showed increased β-cell mass associated with higher expression of Reg family genes. However, Crif1beta+/- mice showed earlier islet failure in response to high-fat feeding, which was exacerbated by aging. Haploinsufficiency of a single mitoribosomal gene predisposes rodents to glucose intolerance, which resembles the early stages of type 2 diabetes in humans.

Activation of hepatocyte cannabinoid receptor-1 (CB1R) by hepatic stellate cell (HSC)-derived 2-arachidonoylglycerol (2-AG) drives de novo lipogenesis in alcoholic liver disease (ALD). How alcohol stimulates 2-AG production in HSCs is unknown. Here, we report that chronic alcohol consumption induced hepatic cysteine deficiency and subsequent glutathione depletion by impaired transsulfuration pathway. A compensatory increase in hepatic cystine-glutamate anti-porter xCT boosted extracellular glutamate levels coupled to cystine uptake both in mice and in patients with ALD. Alcohol also induced the selective expression of metabotropic glutamate receptor-5 (mGluR5) in HSCs where mGluR5 activation stimulated 2-AG production. Consistently, genetic or pharmacologic inhibition of mGluR5 or xCT attenuated alcoholic steatosis in mice via the suppression of 2-AG production and subsequent CB1R-mediated de novo lipogenesis. We conclude that a bidirectional signaling operates at a metabolic synapse between hepatocytes and HSCs through xCT-mediated glutamate-mGluR5 signaling to produce 2-AG, which induces CB1R-mediated alcoholic steatosis.

Foot-and-mouth disease (FMD) is a notifiable contagious disease of cloven-hoofed mammals. A high potency vaccine that stimulates the host immune response is the foremost strategy used to prevent disease persistence in endemic regions. FMD vaccines comprise inactivated virus antigens whose immunogenicity is potentiated by immunogenic adjuvants. Oil-based adjuvants have clear advantages over traditional adjuvant vaccines; however, there is potential to develop novel adjuvants to increase the potency of FMD vaccines. Thus, we aimed to evaluate the efficacy of a novel water-in-oil emulsion, called CAvant®SOE, as a novel vaccine adjuvant for use with inactivated FMD vaccines. In this study, we found that inactivated A22 Iraq virus plus CAvant®SOE (iA22 Iraq-CAvant®SOE) induced effective antigen-specific humoral (IgG, IgG1, and IgG2a) and cell-mediated immune responses (IFN-γ and IL-4) in mice. Immunization of pigs with a single dose of iA22 Iraq-CAvant®SOE also elicited effective protection, with no detectable clinical symptoms against challenge with heterologous A/SKR/GP/2018 FMDV. Levels of protection are strongly in line with vaccine-induced neutralizing antibody titers. Collectively, these results indicate that CAvant®SOE-adjuvanted vaccine is a promising candidate for control of FMD in pigs.