Comprehensive assessment of 11 de novo HiFi assemblers on complex eukaryotic genomes and metagenomes.

Pacific Biosciences (PacBio) HiFi sequencing technology generates long reads (>10 kbp) with very high accuracy (<0.01% sequencing error). Although several de novo assembly tools are available for HiFi reads, there are no comprehensive studies on the evaluation of these assemblers. We evaluated the performance of 11 de novo HiFi assemblers on (1) real data for three eukaryotic genomes; (2) 34 synthetic data sets with different ploidy, sequencing coverage levels, heterozygosity rates, and sequencing error rates; (3) one real metagenomic data set; and (4) five synthetic metagenomic data sets with different composition abundance and heterozygosity rates. The 11 assemblers were evaluated using quality assessment tool (QUAST) and benchmarking universal single-copy ortholog (BUSCO). We also used several additional criteria, namely, completion rate, single-copy completion rate, duplicated completion rate, average proportion of largest category, average distance difference, quality value, run-time, and memory utilization. Results show that hifiasm and hifiasm-meta should be the first choice for assembling eukaryotic genomes and metagenomes with HiFi data. We performed a comprehensive benchmarking study of commonly used assemblers on complex eukaryotic genomes and metagenomes. Our study will help the research community to choose the most appropriate assembler for their data and identify possible improvements in assembly algorithms.

The Pure Extraperitoneal Approach for Sacrocolpopexy in Transvaginal Natural-Orifice Transluminal Endoscopic Surgery.

Objective: The goal of this research was to investigate the feasibility, safety, and short-term clinical outcome of pure extraperitoneal sacrocolpopexy with transvaginal natural orifice transluminal endoscopic surgery (V-NOTES) for treating central pelvic defects. Material and Methods: A total of 9 patients with central pelvic prolapse underwent extraperitoneal sacrocolpopexy with V-NOTES, at the Chengdu Women's and Children's Central Hospital, Chengdu, Sichuan, China, between December 2020 and June 2022. The patients' demographic characteristics, perioperative parameters, and clinical outcomes were analyzed retrospectively. Each patient had the following major surgical procedures: (1) Establishing a platform for an extraperitoneal approach with V-NOTES; (2) separating the extraperitoneal path to the sacral promontory region; (3) suturing the long arm of the mesh to the anterior longitudinal ligament S1; and (4) suturing and fixating the short arm of the mesh at the top of the vagina. Results: The median patient age was 55, the median operative time was 145 minutes, and the median intraoperative blood loss was 150 mL. The operations were successful for all 9 cases, with a median preoperative Pelvic Organ Prolapse-Quantification score of C: +4, and a 3-months postoperative score of C: -6. There were no recurrences during a follow-up of 3-11 months, and no complications occurred, such as mesh erosion, exposure, and infection. Conclusion: As a new surgical approach, extraperitoneal sacrocolpopexy with V-NOTES is safe and feasible. (J GYNECOL SURG 39:108).

Prevalence of acquired drug resistance mutations in antiretroviral- experiencing subjects from 2012 to 2017 in Hunan Province of central South China.

BACKGROUND: There are few data on the prevalence of acquired drug resistance mutations (ADRs) in Hunan Province, China, that could affect the effectiveness of antiretroviral therapy (ART). OBJECTIVES: The main objectives of this study were to determine the prevalence of acquired drug resistance (ADR) the epidemic characteristics of HIV-1-resistant strains among ART-failed HIV patients in Hunan Province, China. METHODS: ART-experienced and virus suppression failure subjects in Hunan between 2012 and 2017 were evaluated by genotyping analysis and mutations were scored using the HIVdb.stanford.edu algorithm to infer drug susceptibility. RESULTS: The prevalence of HIV-1 ADR were 2.76, 2.30, 2.98, 2.62, 2.23and 2.17%, respectively, from 2012 to 2017. Overall 2295 sequences were completed from 2932 ART-failure patients, and 914 of these sequences were found to have drug resistance mutation. The most common subtype was AE (64.14%), followed by BC (17.91%) and B (11.50%). Among those 914 patients with drug resistance mutations,93.11% had NNRTI-associated drug resistance mutations, 74.40% had NRTI drug resistance mutations (DRMs) and 6.89% had PI DRMs. Dual-class mutations were observed in 591 (64.66%) cases, and triple-class mutations were observed in 43 (4.70%) cases. M184V (62.04%), K103N (41.90%) and I54L (3.83%) were the most common observed mutations, respectively, in NRTI-, NNRTI- and PI-associated drug resistance. 93.76% subjects who had DRMs received the ART first-line regimens. CD4 count, symptoms in the past 3 months, and ART adherence were found to be associated with HIV-1 DR. CONCLUSIONS: This study showed that although the prevalence of HIV-acquired resistance in Hunan Province is at a low-level, the long-term and continuous surveillance of HIV ADR in antiretroviral drugs (ARVs) patients is necessary.

Endoscopic Variceal Sequential Ligation Does Not Increase Risk of Gastroesophageal Reflux Disease in Cirrhosis Patients.

BACKGROUND: Endoscopic variceal sequential ligation (EVSL) is currently endorsed in our hospital, as the preferred endoscopic treatment for prevention of variceal rebleeding and achieving adequate hemostasis. There is currently a lack of consensus surrounding EVSL-induced changes in esophageal motor function and abnormal reflux. AIMS: To explore alterations in esophageal motor function and risk of abnormal gastroesophageal reflux in liver cirrhosis patients with esophageal varices, after EVSL. METHODS: Twenty-one liver cirrhosis patients with esophageal varices were studied using manometry and 24-h pH monitoring 1 day prior to and 1 month following EVSL. The EVSL consisted of performing esophageal variceal ligation using a multi-band ligator, which was repeated every 4 weeks until the varices were eradicated. RESULTS: The amplitude and duration of peristaltic contraction waves and the percentage of abnormal esophageal contraction waveforms were unaltered in both the proximal (P > 0.05) and the distal (P > 0.05) esophagus after EVSL. However, the lower esophageal sphincter pressure was decreased following EVSL (16.1 ± 7.9 mmHg vs 21.1 ± 6.3 mmHg (P < 0.05)). Various quantitative parameters including percentage of total monitoring time with pH < 4.0, total number of reflux episodes, number of reflux episodes > 5 min, and DeMeester scores were not increased in post-EVSL patients. Abnormal reflux monitored by 24-h pH monitoring occurred in ten (47.6%) pre-EVSL patients and 11 (52.4%) post-EVSL patients. CONCLUSIONS: Although EVSL affects esophageal motility by relatively decreasing LES pressure, it does not induce substantial motor abnormalities nor increase risk of abnormal gastroesophageal reflux disease in cirrhosis patients.

HBV, HCV, and HBV/HCV co-infection among HIV-positive patients in Hunan province, China: Regimen selection, hepatotoxicity, and antiretroviral therapy outcome.

Co-infection with hepatitis B (HBV) and C (HCV) is common among people living with HIV (PLHIV). This study investigates the impacts of hepatitis co-infection on antiretroviral therapy (ART) outcomes and hepatotoxicity in PLHIV. The cohort study included 1984 PLHIV. Hepatotoxicity was defined by elevated alanine aminotransferase (ALT) levels. ART outcomes were measured by CD4 cell counts, viral load, and mortality rate in patients. Among 1984 PLHIV, 184 (9.3%) were co-infected with HBV and 198 (10.0%) with HCV and 54 (2.7%) were co-infected with HBV and HCV. Of these patients, 156 (7.9%) had ALT elevation ≥ grade 1 at baseline. During the course of ART, the mortality rate and its adjusted hazard ratio (AHR) in PLHIV who were co-infected with HCV (2.6/100 person-years [py], AHR = 2.3, 95%CI 1.1-4.7) was higher than for patients with mono-infected HIV, as it was for those with an elevated ALT (4.4/100 py, AHR = 3.8, [1.7-8.2]) at baseline compared to those with normal ALT. After 6-12 months of ART, the incidence of hepatotoxicity among all the patients was 3.7/100 py. The risk of hepatotoxicity was higher in HCV co-infected (18.6/100 py, adjusted odds ratio [AOR] = 12.4, [8.1-18.2]) than HIV mono-infected patients, and for all regimens (nevirapine: 30.0/100 py, 34.2, 7.3-47.9; zidovudine/stavudine: 24.7/100 py, 22.1, 7.1-25.5; efavirenz: 14.5/100 py, 9.4, 3.5-19.2; lopinavir/ritonavir: 40.1/100 py, 52.2, 9.5-88.2) except tenofovir (4.3/100 py, 4.9, 0.8-9.5). Patients with HBV/HCV co-infected had high hepatotoxicity (10.0/100 py, 6.3, 1.2-23.3) over the same period. Patients with HCV co-infection and HBV/HCV co-infection demonstrated higher hepatotoxicity rate compared with HIV mono-infected patients in China.

Association between low-normal thyroid function and advanced liver fibrosis in metabolic dysfunction-associated fatty liver disease patients: a retrospective cohort study.

Background: Recent studies have found that thyroid function may be associated with the occurrence and development of advanced liver fibrosis in patients with metabolic dysfunction-associated fatty liver disease (MAFLD). However, the majority of such research has consisted of cross-sectional studies. This retrospective cohort study aimed to investigate the effect of low-normal thyroid function on advanced liver fibrosis in MAFLD patients over a 5-year period. Methods: This retrospective cohort study enrolled 825 outpatients and inpatients with MAFLD who attended the Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, China) between January 2011 and December 2018. Based on plasma thyroid hormone and thyroid-stimulating hormone levels, these patients were divided into two groups, namely a low-normal thyroid function group and a strict-normal thyroid function group. The fibrosis-4 score was used to assess advanced liver fibrosis. A chi-square test was conducted to compare the occurrence of advanced fibrosis between the groups. Results: Among the 825 MAFLD patients, 117 and 708 were defined as having low-normal thyroid function and strict-normal thyroid function, respectively. Follow-up data were available for 767 patients (93.0%) during a 5-year period. Eight (7.5%) MAFLD patients with low-normal thyroid function and 26 (3.9%) with strict-normal thyroid function developed advanced liver fibrosis and the cumulative incidence was not significantly different (P = 0.163). Stratification analysis showed that the lean MAFLD patients (body mass index ≤ 23 kg/m2) with low-normal thyroid function had a higher risk of advanced liver fibrosis than the lean MAFLD patients with strict-normal thyroid function (P < 0.05). Conclusion: Low-normal thyroid function is associated with advanced liver fibrosis among lean MAFLD patients.

Genome-wide identification of the AOMT gene family in wax apple and functional characterization of SsAOMTs to anthocyanin methylation.

Introduction: Anthocyanins are major pigments in the peels of red-series wax apple fruits, and two principal components of them, namely, the cyanin and the peonidin, are non-methoxylated and methoxylated anthocyanins, respectively. Anthocyanin O-methyltransferases (AOMTs) are an important group of enzymes that have the ability to catalyze anthocyanins methylation to promote the solubility, stability, and bioactivity of anthocyanins. Although AOMT genes have been studied in a variety of plants, the function of them in wax apple is generally not well understood. Methods: The anthocyanin composition in peels of two wax apple cultivars was determined by High Performance Liquid Chromatography Tandem Mass Spectrometry (HPLS-MS). The genome-wide analysis of the AOMT genes was performed with bioinformatics technology, and the expression patterns of different plant tissues, cultivars, fruit ripening stages, and exogenous abscisic acid (ABA) treatments were analyzed by transcriptome sequencing analysis and real-time quantitative PCR verification. An initial functional evaluation was carried out in vitro using recombinant the Anthocyanin O-methyltransferase Gene 5 of S. samarangense (SsAOMT5) protein. Results: Only two main compositions of anthocyanin were found in peels of two wax apple cultivars, and it was worth noting that Tub Ting Jiang cultivar contained non-methoxylated anthocyanin (Cy3G) only, whereas Daye cultivar contained both non-methoxylated and methoxylated (Pn3G) anthocyanins. A total of six SsAOMT genes were identified in the whole genome of wax apple, randomly distributing on three chromosomes. A phylogenic analysis of the protein sequences divided the SsAOMT gene family into three subgroups, and all SsAOMTs had highly conserved domains of AOMT family. In total, four types of stress- related and five types of hormone- related cis-elements were discovered in the promoter region of the SsAOMTs. Expression pattern analysis showed that SsAOMT5 and SsAOMT6 were expressed in all tissues to varying degrees; notably, the expression of SsAOMT5 was high in the flower and fruit and significantly higher in Daye peels than those of other cultivars in the fruit ripening period. Exogenous ABA treatment significantly increased anthocyanin accumulation, but the increase of methoxylated anthocyanin content did not reach significant level compared with those without ABA treatment, whereas the expression of SsAOMT5 upregulated under ABA treatment. We identified two homologous SsAOMT5 genes from Daye cultivar (DSsAOMT5) and Tub Ting Jiang cultivar (TSsAOMT5); the results of functional analyses to two SsAOMT5 recombinant proteins in vitro demonstrated that DSsAOMT5 showed methylation modification activity, but TSsAOMT5 did not. Conclusion: In conclusion, SsAOMT5 was responsible for methylated anthocyanin accumulation in the peels of wax apple and played an important role in red coloration in wax apple peels.

Intravital microscopy of satellite cell dynamics and their interaction with myeloid cells during skeletal muscle regeneration.

Skeletal muscle regeneration requires the highly coordinated cooperation of muscle satellite cells (MuSCs) with other cellular components. Upon injury, myeloid cells populate the wound site, concomitant with MuSC activation. However, detailed analysis of MuSC-myeloid cell interaction is hindered by the lack of suitable live animal imaging technology. Here, we developed a dual-laser multimodal nonlinear optical microscope platform to study the dynamics of MuSCs and their interaction with nonmyogenic cells during muscle regeneration. Using three-dimensional time-lapse imaging on live reporter mice and taking advantages of the autofluorescence of reduced nicotinamide adenine dinucleotide (NADH), we studied the spatiotemporal interaction between nonmyogenic cells and muscle stem/progenitor cells during MuSC activation and proliferation. We discovered that their cell-cell contact was transient in nature. Moreover, MuSCs could activate with notably reduced infiltration of neutrophils and macrophages, and their proliferation, although dependent on macrophages, did not require constant contact with them. These findings provide a fresh perspective on myeloid cells' role during muscle regeneration.

Muscle denervation promotes functional interactions between glial and mesenchymal cells through NGFR and NGF.

We performed scRNA-seq/snATAC-seq of skeletal muscles post sciatic nerve transection to delineate cell type-specific patterns of gene expression/chromatin accessibility at different time points post-denervation. Unlike myotrauma, denervation selectively activates glial cells and Thy1/CD90-expressing mesenchymal cells. Glial cells expressed Ngf receptor (Ngfr) and were located near neuromuscular junctions (NMJs), close to Thy1/CD90-expressing cells, which provided the main cellular source of NGF post-denervation. Functional communication between these cells was mediated by NGF/NGFR, as either recombinant NGF or co-culture with Thy1/CD90-expressing cells could increase glial cell number ex vivo. Pseudo-time analysis in glial cells revealed an initial bifurcation into processes related to either cellular de-differentiation/commitment to specialized cell types (e.g., Schwann cells), or failure to promote nerve regeneration, leading to extracellular matrix remodeling toward fibrosis. Thus, interactions between denervation-activated Thy1/CD90-expressing and glial cells represent an early abortive process toward NMJs repair, ensued by the conversion of denervated muscles into an environment hostile for NMJ repair.

Berberine alleviates non-alcoholic hepatic steatosis partially by promoting SIRT1 deacetylation of CPT1A in mice.

Background: Berberine effectively alleviates non-alcoholic fatty liver disease (NAFLD). Nevertheless, the mechanism is incompletely comprehended. It has been reported that SIRT1 mediates lipid metabolism in liver and berberine promotes the expression of SIRT1 in hepatocytes. We hypothesized that SIRT1 mediated the effect of berberine on NAFLD. Methods: The effects of berberine on NAFLD were evaluated in C57BL/6J mice fed a high-fat diet (HFD) and in mouse primary hepatocytes and cell lines exposed to palmitate. The change of fatty acid oxidation (FAO) and the activity of CPT1A were observed in HepG2 cells. Quantitative real-time polymerase chain reaction and Western blot were employed to observe the expression of SIRT1 and lipid metabolism-related molecules. The interaction between SIRT1 and CPT1A was investigated by using co-immunoprecipitation assay in HEK293T cells. Results: Berberine treatment attenuated hepatic steatosis, reduced triglyceride (190.1 ± 11.2 µmol/g liver vs 113.6 ± 7.6 µmol/g liver, P < 0.001) and cholesterol (11.3 ± 2.5 µmol/g liver vs 6.3 ± 0.4 µmol/g liver, P < 0.001) concentration in the liver, and improved lipid and glucose metabolism disorders compared with the HFD group. The expression of SIRT1 was reduced in the liver of NAFLD patients and mouse models. Berberine increased the expression of SIRT1 and promoted the protein level of CPT1A and its activity in HepG2 cells. SIRT1 overexpression mimicked the effect of berberine on reducing triglyceride levels in HepG2 cells, whereas SIRT1 knock-down attenuated the effect of berberine. Mechanistically, berberine increased the expression of SIRT1. SIRT1 deacetylated CPT1A at the Lys675 site, which suppressed its ubiquitin-dependent degradation, thereby promoting FAO and alleviating non-alcoholic liver steatosis. Conclusions: Berberine promoted SIRT1 deacetylation of CPT1A at the Lys675 site, which reduced the ubiquitin-dependent degradation of CPT1A and ameliorated non-alcoholic liver steatosis.

Single-cell chromatin accessibility profiling reveals a self-renewing muscle satellite cell state.

A balance between self-renewal and differentiation is critical for the regenerative capacity of tissue-resident stem cells. In skeletal muscle, successful regeneration requires the orchestrated activation, proliferation, and differentiation of muscle satellite cells (MuSCs) that are normally quiescent. A subset of MuSCs undergoes self-renewal to replenish the stem cell pool, but the features that identify and define self-renewing MuSCs remain to be elucidated. Here, through single-cell chromatin accessibility analysis, we reveal the self-renewal versus differentiation trajectories of MuSCs over the course of regeneration in vivo. We identify Betaglycan as a unique marker of self-renewing MuSCs that can be purified and efficiently contributes to regeneration after transplantation. We also show that SMAD4 and downstream genes are genetically required for self-renewal in vivo by restricting differentiation. Our study unveils the identity and mechanisms of self-renewing MuSCs, while providing a key resource for comprehensive analysis of muscle regeneration.

The haplotype-resolved autotetraploid genome assembly provides insights into the genomic evolution and fruit divergence in wax apple (Syzygium samarangense (Blume) Merr. and Perry).

Wax apple (Syzygium samarangense) is an economically important fruit crop with great potential value to human health because of its richness in antioxidant substances. Here, we present a haplotype-resolved autotetraploid genome assembly of the wax apple with a size of 1.59 Gb. Comparative genomic analysis revealed three rounds of whole-genome duplication (WGD) events, including two independent WGDs after WGT-γ. Resequencing analysis of 35 accessions partitioned these individuals into two distinct groups, including 28 landraces and seven cultivated species, and several genes subject to selective sweeps possibly contributed to fruit growth, including the KRP1-like, IAA17-like, GME-like, and FLACCA-like genes. Transcriptome analysis of three different varieties during flower and fruit development identified key genes related to fruit size, sugar content, and male sterility. We found that AP2 also affected fruit size by regulating sepal development in wax apples. The expression of sugar transport-related genes (SWEETs and SUTs) was high in 'ZY', likely contributing to its high sugar content. Male sterility in 'Tub' was associated with tapetal abnormalities due to the decreased expression of DYT1, TDF1, and AMS, which affected early tapetum development. The chromosome-scale genome and large-scale transcriptome data presented in this study offer new valuable resources for biological research on S. samarangense and shed new light on fruit size control, sugar metabolism, and male sterility regulatory metabolism in wax apple.

Complete chloroplast genome sequence of Syzygium samarangense (Myrtaceae) and phylogenetic analysis.

Syzygium samarangense (Blume) Merr. et Perry, 1938, commonly known as wax apple, is a Myrtaceae species that is known for its unique fruit shape, flavorful and colorful fruits, medicinal value and increasing economic relevance. In this study, we reported the complete chloroplast genome sequence of S. samarangense. The complete genome is 159,109 bp in length with a quadripartite structure containing two single copy regions, a Large Single Copy region (LSC, 88,155 bp) and a Small Single Copy region (SSC, 18,796 bp) separated by Inverted Repeat regions (IRs, 26,079 bp). The GC content was 37.0%. It encoded 126 genes, including 81 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes. The phylogenetic relationships of 20 species inferred that all Syzygium species formed a single cluster belonging to Syzygieae tribe. Our results offer insights into the evolutionary relationship of S. samarangense within Myrtaceae, indicating a closer relationship between S. samarangense and S. forrestii.

Cell fate determining molecular switches and signaling pathways in Pax7-expressing somitic mesoderm.

During development, different cell types originate from a common progenitor at well-defined time points. Previous lineage-tracing of Pax7+ progenitors from the somitic mesoderm has established its developmental trajectory towards the dermis, brown adipocytes, and skeletal muscle in the dorsal trunk; yet the molecular switches and mechanisms guiding the differentiation into different lineages remain unknown. We performed lineage-tracing of Pax7-expressing cells in mouse embryos at E9.5 and profiled the transcriptomes of Pax7-progenies on E12.5, E14.5, and E16.5 at single-cell level. Analysis of single-cell transcriptomic data at multiple time points showed temporal-specific differentiation events toward muscle, dermis, and brown adipocyte, identified marker genes for putative progenitors and revealed transcription factors that could drive lineage-specific differentiation. We then utilized a combination of surface markers identified in the single-cell data, Pdgfra, Thy1, and Cd36, to enrich brown adipocytes, dermal fibroblasts, and progenitors specific for these two cell types at E14.5 and E16.5. These enriched cell populations were then used for further culture and functional assays in vitro, in which Wnt5a and Rgcc are shown to be important factors that could alter lineage decisions during embryogenesis. Notably, we found a bipotent progenitor population at E14.5, having lineage potentials towards both dermal fibroblasts and brown adipocytes. They were termed eFAPs (embryonic fibro/adipogenic progenitors) as they functionally resemble adult fibro/adipogenic progenitors. Overall, this study provides further understanding of the Pax7 lineage during embryonic development using a combination of lineage tracing with temporally sampled single-cell transcriptomics.

Chromosome-level reference genome assembly provides insights into aroma biosynthesis in passion fruit (Passiflora edulis).

Passion fruit, native to tropical America, is an agriculturally, economically and ornamentally important fruit plant that is well known for its acid pulp, rich aroma and distinctive flavour. Here, we present a chromosome-level genome assembly of passion fruit by incorporating PacBio long HiFi reads and Hi-C technology. The assembled reference genome is 1.28 Gb size with a scaffold N50 of 126.4 Mb and 99.22% sequences anchored onto nine pseudochromosomes. This genome is highly repetitive, accounting for 86.61% of the assembled genome. A total of 39,309 protein-coding genes were predicted with 93.48% of those being functionally annotated in the public databases. Genome evolution analysis revealed a core eudicot-common γ whole-genome triplication event and a more recent whole-genome duplication event, possibly contributing to the expansion of certain gene families. The 33 rapidly expanded gene families were significantly enriched in the pathways of isoflavone biosynthesis, galactose metabolism, diterpene biosynthesis and fatty acid metabolism, which might be responsible for the formation of featured flavours in the passion fruit. Transcriptome analysis revealed that genes related to ester and ethylene biosynthesis were significantly upregulated in the mature fruit and the expression levels of those genes were consistent with the accumulation of volatile lipid compounds. The passion fruit genome analysis improves our understanding of the genome evolution of this species and sheds new lights into the molecular mechanism of aroma biosynthesis in passion fruit.

Sodium butyrate protects against lipopolysaccharide-induced liver injury partially via the GPR43/ ß-arrestin-2/NF-κB network.

BACKGROUND: Butyrate acts as a regulator in multiple inflammatory organ injuries. However, the role of butyrate in acute liver injury has not yet been fully explored. In the present study, we aimed to investigate the association between butyrate and lipopolysaccharide (LPS)-induced acute liver injury and the signaling pathways involved. METHODS: LPS-induced acute liver injury was induced by intraperitoneal injection of LPS (5 mg/kg) in G-protein-coupled receptor 43 (GPR43)-knockout (KO) and wild-type female C57BL/6 mice. Sodium butyrate (500mg/kg) was administered intraperitoneally 30 min prior to LPS exposure. Liver injury was detected by serum markers, tissue morphology, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Pro-inflammatory-factor levels were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (RT-PCR). Cell models were first treated with sodium butyrate (4 µmol/mL), followed by LPS (1 µg/mL) half an hour later in GPR43 small interfering RNA (siRNA)-transfected or control RAW264.7 cells. Cell-inflammation status was evaluated through detecting pro-inflammatory-factor expression by RT-PCR and also through checking toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB)-element levels including TLR4, TRAF6, IKKß, IкBα, phospho-IкBα, p65, and phospho-p65 by Western blot. The interaction between GPR43 and ß-arrestin-2 was tested by co-immunoprecipitation. RESULTS: Sodium butyrate reversed the LPS-induced tissue-morphology changes and high levels of serum alanine aminotransferase, aspartate transaminase, myeloperoxidase, TUNEL, and pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-6. The ameliorating effect of sodium butyrate was weakened in GPR43-KO mice and GPR43 siRNA RAW264.7 cells, compared with those of GPR43-positive controls. Sodium butyrate downregulated some elements of the TLR4/NF-κB pathway, including phospho-IκBα and phospho-p65, in RAW264.7 cells. Increased interactions between GPR43 and ß-arrestin-2, and between ß-arrestin-2 and IкBα were observed. CONCLUSION: Sodium butyrate significantly attenuated LPS-induced liver injury by reducing the inflammatory response partially via the GPR43/ß-arrestin-2/NF-κB signaling pathway.

Impact of variceal eradication on rebleeding and prognosis in cirrhotic patients undergoing secondary prophylaxis.

BACKGROUND: Endoscopic therapy has been widely applied to prevent variceal rebleeding, but data addressing the effect of endoscopic variceal eradication (VE) are lacking. We aimed to clarify the clinical impact of VE and reveal the long-term incidence and mortality of gastrointestinal rebleeding. METHODS: This prospective study included 228 cirrhotic patients who underwent secondary prophylaxis for variceal bleeding and achieved VE through a systematic procedure we proposed as endoscopic sequential therapy (EST). Rebleeding rates before and after VE were compared and cumulative incidence of rebleeding and mortality were calculated using the Kaplan-Meier method. A logistic regression model and P for trend were used to investigate the optimal time limit for VE. RESULTS: During a median (interquartile range) follow-up duration of 33.0 (23.0-48.75) months, rebleeding was identified in 28 patients (12.3%) after VE and in 27 patients (11.8%) during endoscopic sessions. The cumulative incidence of rebleeding before and after VE was 8.4% and 1.8% at 6 months, and 14.9% and 4.0% at 1 year respectively (P<0.001). The long-term incidence of all-cause/variceal rebleeding following VE was 10.4%/9.1%, and 31.5%/23.5% at 2 and 5 years respectively. Eleven patients (4.8%) died and the 5-year mortality was 9.3%. VE achieved within 6 months was associated with fewer rebleeding events compared to VE achieved after 6 months (5.5% vs. 20.0%, P=0.002), while logistic regression revealed an overall increasing trend in the odds ratio of rebleeding (vs. patients with VE time ≤6 months) for patients with 6< VE time ≤12 months and VE time >12 months (P for trend <0.001). CONCLUSIONS: VE further reduces rebleeding based on routine endoscopic prophylaxis and improves long-term prognosis. VE within 6 months seems to be the optimal timing and should therefore be advocated.

In vivo study of metabolic dynamics and heterogeneity in brown and beige fat by label-free multiphoton redox and fluorescence lifetime microscopy.

In this work, the metabolic characteristics of adipose tissues in live mouse model were investigated using a multiphoton redox ratio and fluorescence lifetime imaging technology. By analyzing the intrinsic fluorescence of metabolic coenzymes, we measured the optical redox ratios of adipocytes in vivo and studied their responses to thermogenesis. The fluorescence lifetime imaging further revealed changes in protein bindings of metabolic coenzymes in the adipocytes during thermogenesis. Our study uncovered significant heterogeneity in the cellular structures and metabolic characteristics of thermogenic adipocytes in brown and beige fat. Subgroups of brown and beige adipocytes were identified based on the distinct lipid size distributions, redox ratios, fluorescence lifetimes and thermogenic capacities. The results of our study show that this label-free imaging technique can shed new light on in vivo study of metabolic dynamics and heterogeneity of adipose tissues in live organisms.

The significance of Toll-like receptor 4 (TLR4) expression in patients with chronic hepatitis B.

PURPOSE: To investigate the importance of Toll-like receptor 4 (TLR4) expression on hepatocytes obtained from Chronic Hepatitis B patients as well as on hepatocellular carcinoma HepG2 and HepG2.2.15 cell lines. METHODS: Expression of TLR4 in liver tissues was determined by immunohistochemistry in 75 patients with CHB and in10 healthy controls. The protein and mRNA 1eve1s of TLR4 in hepatocellular carcinoma HepG2 and HepG2.2.15 cells were measured by flow cytometry (FCM) and real-time quantitative PCR (RQ-PCR), and endotoxin triggered TNF-alpha secretion in HepG2 and HepG2.2.15 cells was evaluated by ELISA. RESULTS: TLR4 expressed mainly in the cytoplasm and some on cell membrane in hepatocytes. The staining scores of TLR4 expression in the liver tissues of patients with CHB were significantly higher than that of healthy controls. The liver tissues from patients with severe CHB expressed higher level of TLR4 than those from patients with mild CHB. Furthermore, the staining scores of TLR4 expression in the liver tissues of patients with CHB were positively correlated with the grading scores. Our results also showed that the mean fluorescence intensity and TNF-alpha secretion induced by endotoxin as well as the protein and mRNA 1eve1s of TLR4 in HepG2.2.15 cells were all significantly higher than those in HepG2 cells. CONCLUSION: TLR4 was up-regulated in the hepatocytes in patients with CHB. This indicates a potentially important interaction between TLR4 expression and the pathogenesis of CHB.