Faecalibacterium prausnitzii-derived extracellular vesicles alleviate chronic colitis-related intestinal fibrosis by macrophage metabolic reprogramming.

Faecalibacterium prausnitzii (F. prausnitzii) has been recognized for its various intestinal and extraintestinal benefits to human. And reduction of F. prausnitzii has been linked to an increased risk of intestinal fibrosis in patients of Crohn's disease (CD). In this study, oral administration of either live F. prausnitzii or its extracellular vesicles (FEVs) can markedly mitigate the severity of fibrosis in mice induced by repetitive administration of DSS. In vitro experiment revealed that FEVs were capable of directing the polarization of peripheral blood mononuclear cells (PBMCs) towards an M2b macrophage phenotype, which has been associated with anti-fibrotic activities. This effect of FEV was found to be stable under various conditions that promote the development of pro-fibrotic M1/M2a/M2c macrophages. Proteomics and RNA sequencing were performed to uncover the molecular modulation of macrophages by FEVs. Notably, we found that FEVs reprogramed every metabolism of macrophages by damaging the mitochondria, and inhibited oxidative phosphorylation and glycolysis. Moreover, FEV-treated macrophages showed a decreased expression of PPARγ and an altered lipid processing phenotype characterized by decreased cholesterol efflux, which may promote energy reprogramming. Taken together, these findings identify FEV as a driver of macrophage reprogramming, suggesting that triggering M2b macrophage polarization by oral admiration of FEV may serve as strategy to alleviate hyperfibrotic intestine conditions in CD.

The GPR40 novel agonist SZZ15-11 improves non-alcoholic fatty liver disease by activating the AMPK pathway and restores metabolic homeostasis in diet-induced obese mice.

AIM: Non-alcoholic fatty liver is the most common cause of chronic liver disease. GPR40 is a potential therapeutic target for energy metabolic disorders. GPR40 is a potential therapeutic target for energy metabolic disorders. SZZ15-11 is a newly synthesized GPR40 agonist. In this study, we estimate the potency of SZZ15-11 in fatty liver treatment. METHODS: In vivo, diet-induced obese (DIO) mice received SZZ15-11 (50 mg/kg) and TAK875 (50 mg/kg) for 6 weeks. Blood glucose and lipid, hepatocyte lipid and liver morphology were analysed. In vitro, HepG2 cells and GPR40-knockdown HepG2 cells induced with 0.3 mM oleic acid were treated with SZZ15-11. Triglyceride and total cholesterol of cells were measured. At the same time, the AMPK pathway regulating triglycerides and cholesterol esters synthesis was investigated via western blot and quantitative polymerase chain reaction in both liver tissue and HepG2 cells. RESULTS: SZZ15-11 was found to not only attenuate hyperglycaemia and hyperlipidaemia but also ameliorate fatty liver disease in DIO mice. At the same time, SZZ15-11 decreased triglyceride and total cholesterol content in HepG2 cells. Whether examined in the liver of DIO mice or in HepG2 cells, SZZ15-11 upregulated AMPKα phosphorylation and then downregulated the expression of the cholesterogenic key enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase and inhibited acetyl-CoA carboxylase activity. Furthermore, SZZ15-11 promotes AMPK activity via [cAMP]i accumulation. CONCLUSION: This study confirmed that SZZ15-11, a novel GPR40 agonist, improves hyperlipidaemia and fatty liver, partially via Gs signalling and the AMPK pathway in hepatocytes.

Safety, tolerability, pharmacokinetic, pharmacodynamic and immunogenicity profiles of Exendin-4-IgG4-Fc in healthy subjects: A phase 1, single-centre, randomized, double-blind, dose escalation study.

AIM: Novel long-acting drugs for type 2 diabetes mellitus may optimize patient compliance and glycaemic control. Exendin-4-IgG4-Fc (E4F4) is a long-acting glucagon-like peptide-1 receptor agonist. This first-in-human study investigated the safety, tolerability, pharmacokinetic, pharmacodynamic and immunogenicity profiles of a single subcutaneous injection of E4F4 in healthy subjects. METHODS: This single-centre, randomized, double-blind, placebo-controlled phase 1 clinical trial included 96 subjects in 10 sequential cohorts that were provided successively higher doses of E4F4 (0.45, 0.9, 1.8, 3.15, 4.5, 6.3, 8.1, 10.35, 12.6 and 14.85 mg) or placebo (ChinaDrugTrials.org.cn: ChiCTR2100049732). The primary endpoint was safety and tolerability of E4F4. Secondary endpoints were pharmacokinetic, pharmacodynamic and immunogenicity profiles of E4F4. Safety data to day 15 after the final subject in a cohort had been dosed were reviewed before commencing the next dose level. RESULTS: E4F4 was safe and well tolerated among healthy Chinese participants in this study. There was no obvious dose-dependent relationship between frequency, severity or causality of treatment-emergent adverse events. Cmax and area under the curve of E4F4 were dose proportional over the 0.45-14.85 mg dose range. Median Tmax and t1/2 ranged from 146 to 210 h and 199 to 252 h, respectively, across E4F4 doses, with no dose-dependent trends. For the intravenous glucose tolerance test, area under the curve of glucose in plasma from time 0 to 180 min showed a dose-response relationship in the 1.8-10.35 mg dose range, with an increased response at the higher doses. CONCLUSION: E4F4 exhibited an acceptable safety profile and linear pharmacokinetics in healthy subjects. The recommended phase 2 dose is 4.5-10.35 mg once every 2 weeks.

KYNA Ameliorates Glutamate Toxicity of HAND by Enhancing Glutamate Uptake in A2 Astrocytes.

Reactive astrocytes are key players in HIV-associated neurocognitive disorders (HAND), and different types of reactive astrocytes play opposing roles in the neuropathologic progression of HAND. A recent study by our group found that gp120 mediates A1 astrocytes (neurotoxicity), which secrete proinflammatory factors and promote HAND disease progression. Here, by comparing the expression of A2 astrocyte (neuroprotective) markers in the brains of gp120 tgm mice and gp120+/α7nAChR-/- mice, we found that inhibition of alpha 7 nicotinic acetylcholine receptor (α7nAChR) promotes A2 astrocyte generation. Notably, kynurenine acid (KYNA) is an antagonist of α7nAChR, and is able to promote the formation of A2 astrocytes, the secretion of neurotrophic factors, and the enhancement of glutamate uptake through blocking the activation of α7nAChR/NF-κB signaling. In addition, learning, memory and mood disorders were significantly improved in gp120 tgm mice by intraperitoneal injection of kynurenine (KYN) and probenecid (PROB). Meanwhile, the number of A2 astrocytes in the mouse brain was significantly increased and glutamate toxicity was reduced. Taken together, KYNA was able to promote A2 astrocyte production and neurotrophic factor secretion, reduce glutamate toxicity, and ameliorate gp120-induced neuropathological deficits. These findings contribute to our understanding of the role that reactive astrocytes play in the development of HAND pathology and provide new evidence for the treatment of HAND via the tryptophan pathway.

Development, validation, and evaluation of a risk assessment tool for personalized screening of gastric cancer in Chinese populations.

BACKGROUND: Effective risk prediction models are lacking for personalized endoscopic screening of gastric cancer (GC). We aimed to develop, validate, and evaluate a questionnaire-based GC risk assessment tool for risk prediction and stratification in the Chinese population. METHODS: In this three-stage multicenter study, we first selected eligible variables by Cox regression models and constructed a GC risk score (GCRS) based on regression coefficients in 416,343 subjects (aged 40-75 years) from the China Kadoorie Biobank (CKB, development cohort). In the same age range, we validated the GCRS effectiveness in 13,982 subjects from another independent Changzhou cohort (validation cohort) as well as in 5348 subjects from an endoscopy screening program in Yangzhou. Finally, we categorized participants into low (bottom 20%), intermediate (20-80%), and high risk (top 20%) groups by the GCRS distribution in the development cohort. RESULTS: The GCRS using 11 questionnaire-based variables demonstrated a Harrell's C-index of 0.754 (95% CI, 0.745-0.762) and 0.736 (95% CI, 0.710-0.761) in the two cohorts, respectively. In the validation cohort, the 10-year risk was 0.34%, 1.05%, and 4.32% for individuals with a low (≤ 13.6), intermediate (13.7~30.6), and high (≥ 30.7) GCRS, respectively. In the endoscopic screening program, the detection rate of GC varied from 0.00% in low-GCRS individuals, 0.27% with intermediate GCRS, to 2.59% with high GCRS. A proportion of 81.6% of all GC cases was identified from the high-GCRS group, which represented 28.9% of all the screened participants. CONCLUSIONS: The GCRS can be an effective risk assessment tool for tailored endoscopic screening of GC in China. Risk Evaluation for Stomach Cancer by Yourself (RESCUE), an online tool was developed to aid the use of GCRS.

Kynurenic acid blunts A1 astrocyte activation against neurodegeneration in HIV-associated neurocognitive disorders.

Despite extensive astrocyte activation in patients suffering from HIV-associated neurocognitive disorders (HAND), little is known about the contribution of astrocytes to HAND neuropathology. Here, we report that the robust activation of neurotoxic astrocytes (A1 astrocytes) in the CNS promotes neuron damage and cognitive deficits in HIV-1 gp120 transgenic mice. Notably, knockout of α7 nicotinic acetylcholine receptors (α7nAChR) blunted A1 astrocyte responses, ultimately facilitating neuronal and cognitive improvement in the gp120tg mice. Furthermore, we provide evidence that Kynurenic acid (KYNA), a tryptophan metabolite with α7nAChR inhibitory properties, attenuates gp120-induced A1 astrocyte formation through the blockade of α7nAChR/JAK2/STAT3 signaling activation. Meanwhile, compared with gp120tg mice, mice fed with tryptophan showed dramatic improvement in cognitive performance, which was related to the inhibition of A1 astrocyte responses. These initial and determinant findings mark a turning point in our understanding of the role of α7nAChR in gp120-mediated A1 astrocyte activation, opening up new opportunities to control neurotoxic astrocyte generation through KYNA and tryptophan administration.

Co-occurrence of gut microbiota dysbiosis and bile acid metabolism alteration is associated with psychological disorders in Crohn's disease.

This study aims to elucidate the relationships between gut microbiota, bile acid metabolism, and psychological comorbidity in Crohn's disease (CD). We profiled the fecal microbiota composition and quantified the bile acid pool of 39 CD patients and 14 healthy controls using 16S rRNA gene sequencing and liquid chromatography-tandem mass spectrometry, respectively. Significant reductions in the secondary bile acids, LCA and DCA, were found in both the feces and serum samples of CD patients, while the concentration of 7-DHCA was particularly higher in the serum of CD patients with psychological disorders. The fecal levels of HDCA and 12-DHCA of the CD patients were inversely correlated with their Self-Rated Depression Scale (SDS) scores, whereas the serum level of 7-DHCA was positively correlated with the SDS scores. In addition, the fecal levels of TDCA, TLCA, and TßMCA showed a positive correlation with the Self-Rated Anxiety Scale (SAS) scores. The fecal microbiota biodiversity was particularly declined in CD patients with psychological disorders. An enrichment of Ruminococcus gnavus in CD patients may cause psychological disorders by affecting the microbiota-gut-brain axis via its ability to degrade the gut barrier, regulate the tryptophan-kynurenine metabolism, and modulate bile acid metabolism. In addition, the overabundant Enterobacteriaceae and Lachnospiraceae in CD patients may contribute to psychological comorbidity via dysregulating their bile acids metabolism. Taken together, changes in the gut microbiota composition may cooperate with alterations in the bile acid metabolism that are involved in the development of psychological disorders in CD.

CD40 monoclonal antibody and OK432 synergistically promote the activation of dendritic cells in immunotherapy.

BACKGROUND: Colorectal cancer (CRC) with pulmonary metastasis usually indicates a poor prognosis, whereas patients may benefit from adoptive cell therapy. Tumor-specific cytotoxic T lymphocytes (CTLs) have been reported as a promising treatment for CRC. However, the antitumor effect of CTLs remains limited partially due to insufficient production of effector cells via the activation by antigen-presenting dendritic cells (DCs). METHOD: This study showed that a combination of CD40 mAb and Picibanil (OK-432) could significantly enhance the activation of CTLs by DCs, both in vitro and in vivo. Flow cytometry, colon cancer mouse model, and pathological staining were employed to demonstrate the specific functions. RESULTS: This approach promoted the maturation of DCs, augmented the production of stimulatory cytokines, and suppressed the secretion of inhibitory cytokines. Additionally, it facilitated the killing efficiency of CTLs via stimulating their proliferation while restraining the number of Tregs, concomitantly with the positive regulation of corresponding cytokines. Furthermore, the combined unit could hurdle the expansion of tumor cells on metastatic lungs in the colon cancer mouse model. CONCLUSION: Collectively, the combination of CD40-mAb and OK-432 facilitated the maturation of DCs and enhanced the cytotoxicity of T cells, promising therapeutic approach against CRC.

Discovery of 12ß-oxygenated oleanolic acid alkyl esters as potent and selective FXR modulators exhibiting hyperglycemia amelioration in vivo.

Farnesoid X receptor (FXR) ligands have been actively pursued to treat metabolic disorders, liver and bile diseases, among others. Starting from a widely occurring natural product, oleanolic acid (OA), we discovered potent and selective FXR modulator from the 12ß-oxygenated OA alkyl esters, with the assistance of molecular modeling. The representative compound 7b modulated some FXR downstream genes involved in glucose and lipid metabolism in cells, and significantly improved hyperglycemia in KKay fat mice fed with high fat diet, through the reduction of mRNA expression of gluconeogenesis genes PEPCK and G6Pase. This study provides a new series of selective FXR modulator, as well as the in vitro and in vivo evidence for their potential to improve hyperglycemia in diabetic mice through FXR antagonism.

Genome-wide association study of three litter traits in Yorkshire pigs.

Litter traits are important in the development of production and economic profits of pigs. The object of this study was to perform genome-wide association studies (GWASs) for the total number born (TNB), number born alive (NBA) and number born healthy (NBH) traits in a Yorkshire population. By mixed model, we conducted GWASs with gcta software, and in total identified 3, 7 and 12 significant SNPs associated with TNB, NBA and NBH respectively: one on Sus scrofa chromosome (SSC) 2 (p = 7.03 × 10-5 ), one on SSC4 (p = 8.53 × 10-5 ), two on SSC 6 (p = 9.27 × 10-5 to 9.64 × 10-5 ), nine on SSC 8 (p = 1.53 × 10-5 to 8.52 × 10-5 ), four on SSC 14 (p = 2.17 × 10-5 to 6.79 × 10-5 ), and one on SSC 15 (p = 9.30 × 10-5 ). We screened for flanking regions ±1 Mb nearby or within the significant SNPs, and found 203 candidate genes, including 53 for TNB, 71 for NBA and 112 for NBH. In conclusion, our findings show the significant SNPs with candidate genes for the TNB, NBA and NBH traits in Yorkshire pigs.

Voglibose Regulates the Secretion of GLP-1 Accompanied by Amelioration of Ileal Inflammatory Damage and Endoplasmic Reticulum Stress in Diabetic KKAy Mice.

Voglibose is an α-glycosidase inhibitor that improves postprandial hyperglycemia and increases glucagon-like peptide-1 (GLP-1) secretion in patients with type 2 diabetes. Recently, there has been increasing interest in the anti-inflammatory effects of voglibose on the intestine, but the underlying mechanism is not clear. This study evaluated the effects and mechanisms of voglibose on glycemic control and intestinal inflammation. Type 2 diabetic KKAy mice were treated with voglibose (1 mg/kg) by oral gavage once daily. After 8 weeks, glucose metabolism, levels of short-chain fatty acids (SCFAs), systematic inflammatory factors, intestinal integrity and inflammation were evaluated using hematoxylin and eosin staining, immunohistochemistry, immunofluorescence and Western blot analysis. Voglibose ameliorated glucose metabolism by enhancing basal- and glucose-dependent GLP-1 secretion. Several beneficial SCFAs, such as acetic acid and propionic acid, were increased by voglibose in the fecal sample. Additionally, voglibose notably decreased the proportion of pro-inflammatory macrophages and the expression of nuclear factor kappa B but increased the expression of tight junction proteins in the ileum, thus markedly improving intestinal inflammatory damage and reducing the systematic inflammatory factors. Ileal genomics and protein validation suggested that voglibose attenuated inositol-requiring protein 1α-X-box binding protein 1-mediated endoplasmic reticulum stress (ERS). Together, these results showed that voglibose enhanced the secretion of GLP-1, which contributed to the glycemic control in KKAy mice at least in part by regulating intestinal inflammation and the expression of ERS factors.

Phosphorylation of ERK-Dependent NF-κB Triggers NLRP3 Inflammasome Mediated by Vimentin in EV71-Infected Glioblastoma Cells.

Enterovirus 71 (EV71) is a dominant pathogenic agent that may cause severe central nervous system (CNS) diseases among infants and young children in the Asia-pacific. The inflammasome is closely implicated in EV71-induced CNS injuries through a series of signaling pathways. However, the activation pathway of NLRP3 inflammasome involved in EV71-mediated CNS injuries remains poorly defined. In the studies, EV71 infection, ERK1/2 phosphorylation, and activation of NLRP3 are abolished in glioblastoma cells with low vimentin expression by CRISPR/Cas9-mediated knockdown. PD098059, an inhibitor of p-ERK, remarkably blocks the vimentin-mediated ERK1/2 phosphorylation in EV71-infected cells. Nuclear translocation of NF-κB p65 is dependent on p-ERK in a time-dependent manner. Moreover, NLRP3 activation and caspase-1 production are limited in EV71-infected cells upon the caffeic acid phenethyl ester (CAPE) administration, an inhibitor of NF-κB, which contributes to the inflammasome regulation. In conclusion, these results suggest that EV71-mediated NLRP3 inflammasome could be activated via the VIM-ERK-NF-κB pathway, and the treatment of the dephosphorylation of ERK and NF-κB inhibitors is beneficial to host defense in EV71-infected CNS.

The fecal microbiota of patients with pancreatic ductal adenocarcinoma and autoimmune pancreatitis characterized by metagenomic sequencing.

BACKGROUND: The fecal microbiota in pancreatic ductal adenocarcinoma (PDAC) and in autoimmune pancreatitis (AIP) patients remains largely unknown. We aimed to characterize the fecal microbiota in patients with PDAC and AIP, and explore the possibility of fecal microbial biomarkers for distinguishing PDAC and AIP. METHODS: 32 patients with PDAC, 32 patients with AIP and 32 age- and sex-matched healthy controls (HC) were recruited and the fecal microbiotas were analyzed through high-throughput metagenomic sequencing. Alterations of fecal short-chain fatty acids were measured using gas chromatographic method. RESULTS: Principal coordinate analysis (PCoA) revealed that microbial compositions differed significantly between PDAC and HC samples; whereas, AIP and HC individuals tended to cluster together. Significant reduction of phylum Firmicutes (especially butyrate-producing bacteria, including Eubacterium rectale, Faecalibacterium prausnitzii and Roseburia intestinalis) and significant increase of phylum Proteobacteria (especially Gammaproteobacteria) were observed only among PDAC samples. At species level, when compared with HC samples, we revealed 24 and 12 differently enriched bacteria in PDAC and AIP, respectively. Functional analysis showed a depletion of short-chain fatty acids synthesis associated KO modules (e.g. Wood-Ljungdahl pathway) and an increase of KO modules associated with bacterial virulence (e.g. type II general secretion pathway). Consistent with the downregulation of butyrate-producing bacteria, gas chromatographic analysis showed fecal butyrate content was significantly decreased in PDAC group. Eubacterium rectale, Eubacterium ventrisum and Odoribacter splanchnicus were among the most important biomarkers in distinguishing PDAC from HC and from AIP individuals. Receiver Operating Characteristic analysis showed areas under the curve of 90.74% (95% confidence interval [CI] 86.47-100%), 88.89% (95% CI 73.49-100%), and 76.54% (95% CI 52.5-100%) for PDAC/HC, PDAC/AIP and AIP/HC, respectively. CONCLUSIONS: In conclusion, alterations in fecal microbiota and butyrate of patients with PDAC suggest an underlying role of gut microbiota for the pathogenesis of PDAC. Fecal microbial and butyrate as potential biomarkers may facilitate to distinguish patients with PDAC from patients with AIP and HCs which worth further validation.

The role of ARHGAP9: clinical implication and potential function in acute myeloid leukemia.

BACKGROUND: Rho GTPase activating protein 9 (ARHGAP9) is expressed in various types of cancers and can inactivate Rho GTPases that mainly regulate cytoskeletal dynamics. However, the exact role of ARHGAP9 in acute myeloid leukemia (AML) has yet to be clarified. METHODS: We compared the transcriptional expression, prognosis, differentially expressed genes, functional enrichment, and hub genes in AML patients on the basis of the data published in the following databases: UALCAN, GEPIA, Gene Expression Omnibus, the Human Protein Atlas, Cancer Cell Line Encyclopedia, LinkedOmics, Metascape, and String. Data from the Cancer Genome Atlas database was used to evaluate the correlations between ARHGAP9 expression and various clinicopathological parameters, as well as the significantly different genes associated with ARHGAP9 expression. RESULTS: We found that ARHGAP9 expression was higher in the tissues and cell lines extracted from patients with AML than corresponding control tissues and other cancer types. ARHGAP9 overexpression was associated with decreased overall survival (OS) in AML. Compared with the ARHGAP9low group, the ARHGAP9high group, which received only chemotherapy, showed significantly worse OS and event-free survival (EFS); however, no significant difference was observed after treatment with autologous or allogeneic hematopoietic stem cell transplantation (auto/allo-HSCT). The ARHGAP9high patients undergoing auto/allo-HSCT also had a significantly better prognosis with respect to OS and EFS than those receiving only chemotherapy. Most overlapping genes of the significantly different genes and co-expression genes exhibited enriched immune functions, suggesting the immune regulation potential of ARHGAP9 in AML. A total of 32 hub genes were identified from the differentially expressed genes, within which the KIF20A had a significant prognostic value for AML. CONCLUSIONS: ARHGAP9 overexpression was associated with poor OS in AML patients and can be used as a prognostic biomarker. AML patients with ARHGAP9 overexpression can benefit from auto/allo-HSCT rather than chemotherapy.

A novel epigenetic drug conjugating flavonoid and HDAC inhibitor confer suppression of acute myeloid leukemogenesis.

Epigenetic dysregulation has long been identified as a key driver of leukemogenesis in acute myeloid leukemia (AML). However, epigenetic drugs such as histone deacetylase inhibitors (HDACis) targeting epigenetic alterations in AML have obtained only limited clinical efficiency without clear mechanism. Fortunately, we screened out a novel epigenetic agent named Apigenin-Vorinostat-Conjugate (AVC), which provides us a possibility to handle the heterogeneous malignancy. Its inhibition on HDACs was presented by HDACs expression, enzyme activity, and histone acetylation level. Its efficacy against AML was detected by cell viability assay and tumor progression of AML mouse model. Apoptosis is the major way causing cell death. We found that AVC efficiently suppresses leukemogenesis while sparing the normal human cells. Kasumi-1 cells are at least 20-fold higher sensitive to AVC (IC50 = 0.024 µM) than vorinostat (IC50 = 0.513 µM) and Ara-C (IC50 = 0.4366 µM). Furthermore, it can efficiently regress the tumorigenesis in AML mouse model while keeping the pivotal organs safe, demonstrating a feasibility and favorable safety profile in treatment of AML. Collectively, these preclinical data suggest a promising potential utilizing flavonoid-HDACi-conjugate as a next-generation epigenetic drug for clinical therapy against AML.

Depicting the composition of gut microbiota in children with tic disorders: an exploratory study.

BACKGROUND: Symptom improvement in children with tic disorder (TD) following fecal microbiota transplantation led us to investigate the gut microbiota in TD. This exploratory study aims to depict the gut microbial profile in patients with TD and explore the impact of dopamine receptor antagonist (DRA) drugs on the composition and metabolic function of the gut microbiota. METHODS: The gut microbiota were profiled in fecal samples of 49 children with TD and 50 matched healthy controls (HC) using shotgun metagenomic sequencing. A random forest (RF) model was constructed using the gut bacterial species to distinguish TD from HC. Associations between clinical metadata and microbial abundance or function were analyzed using MaAsLin2 and Spearman correlation. RESULTS: The gut microbiota in children with TD was featured by higher abundances of Bacteroides plebeius and Ruminococcus lactaris (a potential pro-inflammatory taxon) and lower abundances of Prevotella stercorea and Streptococcus lutetiensis compared to HC. The constructed RF model accurately distinguished TD from HC based on the gut microbiota profile, resulting in an AUC of 0.884. Significant correlations were observed between tic symptom severity and the abundances of multiple bacterial species and gut microbiota metabolic functions. Multivariate analysis identified an upregulation of 4-aminobutanoate (GABA) degradation in the gut microbiota associated with TD status. The gut microbiota of DRA-treated TD children showed a distinct gut microbiota compared to the treatment-naïve group, represented by an increase in some potential enteric pathogens such as Escherichia coli, a decline in several species including Akkermansia muciniphila, and alterations in various metabolic functions. CONCLUSIONS: Bacterial species promoting inflammatory responses and those modulating neurotransmitters such as GABA may be involved in the pathogenesis of TD. The use of DRA drugs is likely to induce overgrowth of some enteric pathogens and alter the gut microbiota metabolism.

Epigenetic silencing of miR564 contributes to the leukemogenesis of t(8;21) acute myeloid leukemia.

The AML1-ETO oncoprotein, which results from t(8;21) translocation, is considered an initial event of t(8;21) acute myeloid leukemia (AML). However, the precise mechanisms of the oncogenic activity of AML1-ETO is yet to be fully determined. The present study demonstrates that AML1-ETO triggers the heterochromatic silencing of microRNA-564 (miR564) by binding at the AML1 binding site along the miR564 promoter region and recruiting chromatin-remodeling enzymes. Suppression of miR564 enhances the oncogenic activity of the AML1-ETO oncoprotein by directly inhibiting the expression of CCND1 and the DNMT3A genes. Ectopic expression of miR564 can induce retardation of G1/S transition, reperform differentiation, promote apoptosis, as well as inhibit the proliferation and colony formation of AML1-ETO+ leukemia cells in vitro. Enhanced miR564 levels can significantly inhibit the tumor proliferation of t(8;21)AML in vivo. We first identify an unexpected and important epigenetic circuitry of AML1-ETO/miR564/CCND1/DNMT3A that contributes to the leukemogenesis in vitro/vivo of AML1-ETO+ leukemia, indicating that miR564 enhancement could provide a potential therapeutic method for AML1-ETO+ leukemia.

Reversible Shielding between Dual Ligands for Enhanced Tumor Accumulation of ZnPc-Loaded Micelles.

Herein, we report a ligand-reversible-shielding strategy based on the mutual shielding of dual ligands tethered to the surface of nanoparticles. To exemplify this concept, phenylboronic acid-functionalized poly(ethylene glycol)- b-poly(ε-caprolactone) (PBA-PEG-PCL) and galactose-functionalized diblock polymer (Gal-PEG-PCL) were mixed to form dual-ligand micelles (PBA/Gal). PBA and Gal residues could form a complex at pH 7.4 and mutually shield their targeting function. At pH 6.8, the binding affinity between PBA and Gal weakened, and PBA preferred to bind with the sialic acid residues on the tumor cell surface rather than to Gal on the micellar surface; furthermore, the unbound Gal recovered its targeting ability toward the asialoglycoprotein receptor. When the pH decreased from 7.4 to 6.8, enzyme-linked immunosorbent assays exhibited that the percentage of exposed Gal on the micellar surface increased 1.9-fold, and flow cytometry showed that HepG2 cellular uptake increased 4.3-fold. More importantly, this process was reversible, confirming the reversible shielding and deshielding of dual ligands. With the encapsulation of a photosensitizer, zinc phthalocyanine (ZnPc), the reversible-shielding micelles showed a 48% improvement in the half-life ( t1/2) in blood circulation, a 54% decrease in liver capture, a 40% increase in tumor accumulation, and a 10.3% improvement in the tumor inhibition rate compared to the Gal-coated irreversible micelles. This dual-ligand mutual-shielding strategy provides a new perspective on reversible tumor targeting.

Development and Validation of Quantitative Real-Time PCR for the Detection of Residual CHO Host Cell DNA and Optimization of Sample Pretreatment Method in Biopharmaceutical Products.

BACKGROUND: The presence of residual DNA carried by biological products in the body may lead to an increased oncogenicity, infectivity, and immunomodulatory risk. Therefore, current agencies including WHO, EU, and the FDA limited the accepted amounts of residual DNA (less than 10 ng or 100 pg/dose). Among the methods of detecting residual DNA, qPCR is considered to be the most practical for residual DNA quantitation due to its sensitivity, accuracy, precision, and time-saving. RESULTS: In this study, the detection capacity of this method was determined by comparing the detected concentration of the commercial kit and the self-designed primer/probe set after the same treatment of the extraction method. Then, a universal sample pretreatment method based on a co-precipitant was optimized. The validation results demonstrated that the method has appropriate specificity, sensitivity, accuracy, and precision according to ICH guidelines. The limit of detection and quantitation reached 3 fg/ul and 0.3 pg/reaction respectively, which satisfies the requirement of limit of residual DNA detection in biologics. Spike recovery (82.3-105.7%) showed that the proposed qPCR assay was accurate and has good extraction efficiency. Moreover, the precision of the method based on intra- and inter-assay was 0.065-0.452% and 0.471-1.312%, respectively. CONCLUSIONS: These results all indicated that the method for determination of residual DNA in biological products expressed from CHO cells is sensitive, accurate and robust.

Helicobacter pylori infection alters gastric and tongue coating microbial communities.

OBJECTIVE: Infection with Helicobacter pylori (H pylori), especially cytotoxin-associated gene A-positive (CagA+) strains, has been associated with various gastrointestinal and extragastric diseases. The aim of this study was to characterize H pylori-induced alterations in the gastric and tongue coating microbiota and evaluate their potential impacts on human health. DESIGN: The gastric mucosa and tongue coating specimens were collected from 80 patients with chronic gastritis, and microbiota profiles were generated by 16S rRNA gene sequencing. Samples were grouped as H pylori negative (n = 32), CagA-negative H pylori infection (n = 13), and CagA-positive H pylori infection (n=35). The comparison of bacterial relative abundance was made using a generalized linear model. Functional profiling of microbial communities was predicted with PICRUSt and BugBase. Microbial correlation networks were produced by utilizing SparCC method. RESULTS: Significant alterations of the gastric microbiota were found in the H pylori+/CagA+ samples, represented by a decrease in bacterial diversity, a reduced abundance of Roseburia, and increased abundances of Helicobacter and Haemophilus genera. At the community level, functions involved in biofilm forming, mobile element content, and facultative anaerobiosis were significantly decreased in gastric microbiome of the H pylori+ subjects. The presence of CagA gene was linked to an increased proportion of Gram-negative bacteria in the stomach, thereby contributing to an upregulation of lipopolysaccharide (LPS) biosynthesis. The number of bacterial interactions was greatly reduced in networks of both tongue coating and gastric microbiota of the H pylori+/CagA+ subject, and the cooperative bacterial interactions dominated the tongue coating microbiome. CONCLUSIONS: Infection with H pylori strains possessing CagA may increase the risk of various diseases, by upregulating LPS biosynthesis in the stomach and weakening the defense of oral microbiota against microorganisms with pathogenic potential.