Emergence of highly profibrotic and proinflammatory Lrat+Fbln2+ HSC subpopulation in alcoholic hepatitis.

BACKGROUND AND AIMS: Relative roles of HSCs and portal fibroblasts in alcoholic hepatitis (AH) are unknown. We aimed to identify subpopulations of collagen type 1 alpha 1 (Col1a1)-expressing cells in a mouse AH model by single-cell RNA sequencing (scRNA-seq) and filtering the cells with the HSC (lecithin retinol acyltransferase [Lrat]) and portal fibroblast (Thy-1 cell surface antigen [Thy1] and fibulin 2 [Fbln2]) markers and vitamin A (VitA) storage. APPROACH AND RESULTS: Col1a1-green fluorescent protein (GFP) mice underwent AH, CCl 4 , and bile duct ligation (BDL) procedures to have comparable F1-F2 liver fibrosis. Col1a1-expressing cells were sorted via FACS by VitA autofluorescence and GFP for single-cell RNA sequencing. In AH, approximately 80% of Lrat+Thy1-Fbln2- activated HSCs were VitA-depleted (vs. ~13% in BDL and CCl 4 ). Supervised clustering identified a subset co-expressing Lrat and Fbln2 (Lrat+Fbln2+), which expanded 44-fold, 17-fold, and 1.3-fold in AH, BDL, and CCl 4 . Lrat+Fbln2+ cells had 3-15-times inductions of profibrotic, myofibroblastic, and immunoregulatory genes versus Lrat+Fbln2- cells, but 2-4-times repressed HSC-selective genes. AH activated HSCs had up-regulated inflammatory (chemokine [C-X-C motif] ligand 2 [Cxcl2], chemokine [C-C motif] ligand 2), antimicrobial (Il-33, Zc3h12a), and antigen presentation (H2-Q6, H2-T23) genes versus BDL and CCl 4 . Computational deconvolution of AH versus normal human bulk-liver RNA-sequencing data supported an expansion of LRAT+FBLN2+ cells in AH; AH patient liver immunohistochemistry showed FBLN2 staining along fibrotic septa enriched with LRAT+ cells; and in situ hybridization confirmed co-expression of FBLN2 with CXCL2 and/or human leukocyte antigen E in patient AH. Finally, HSC tracing in Lrat-Cre;Rosa26mTmG mice detected GFP+FBLN2+ cells in AH. CONCLUSION: A highly profibrotic, inflammatory, and immunoregulatory Lrat+Fbln2+ subpopulation emerges from HSCs in AH and may contribute to the inflammatory and immunoreactive nature of AH.

BI-2865, a pan-KRAS inhibitor, reverses the P-glycoprotein induced multidrug resistance in vitro and in vivo.

BACKGROUND: Multidrug resistance (MDR) limits successful cancer chemotherapy. P-glycoprotein (P-gp), BCRP and MRP1 are the key triggers of MDR. Unfortunately, no MDR modulator was approved by FDA to date. Here, we will investigate the effect of BI-2865, a pan-KRAS inhibitor, on reversing MDR induced by P-gp, BCRP and MRP1 in vitro and in vivo, and its reversal mechanisms will be explored. METHODS: The cytotoxicity of BI-2865 and its MDR removal effect in vitro were tested by MTT assays, and the corresponding reversal function in vivo was assessed through the P-gp mediated KBv200 xenografts in mice. BI-2865 induced alterations of drug discharge and reservation in cells were estimated by experiments of Flow cytometry with fluorescent doxorubicin, and the chemo-drug accumulation in xenografts' tumor were analyzed through LC-MS. Mechanisms of BI-2865 inhibiting P-gp substrate's efflux were analyzed through the vanadate-sensitive ATPase assay, [125I]-IAAP-photolabeling assay and computer molecular docking. The effects of BI-2865 on P-gp expression and KRAS-downstream signaling were detected via Western blotting, Flow cytometry and/or qRT-PCR. Subcellular localization of P-gp was visualized by Immunofluorescence. RESULTS: We found BI-2865 notably fortified response of P-gp-driven MDR cancer cells to the administration of chemo-drugs including paclitaxel, vincristine and doxorubicin, while such an effect was not observed in their parental sensitive cells and BCRP or MRP1-driven MDR cells. Importantly, the mice vivo combination study has verified that BI-2865 effectively improved the anti-tumor action of paclitaxel without toxic injury. In mechanism, BI-2865 prompted doxorubicin accumulating in carcinoma cells by directly blocking the efflux function of P-gp, which more specifically, was achieved by BI-2865 competitively binding to the drug-binding sites of P-gp. What's more, at the effective MDR reversal concentrations, BI-2865 neither varied the expression and location of P-gp nor reduced its downstream AKT or ERK1/2 signaling activity. CONCLUSIONS: This study uncovered a new application of BI-2865 as a MDR modulator, which might be used to effectively, safely and specifically improve chemotherapeutic efficacy in the clinical P-gp mediated MDR refractory cancers.

Cr(VI) Reduction and Sequestration by FeS Nanoparticles Formed in situ as Aquifer Material Coating to Create a Regenerable Reactive Zone.

Remediation of large and dilute plumes of groundwater contaminated by oxidized pollutants such as chromate is a common and difficult challenge. Herein, we show that in situ formation of FeS nanoparticles (using dissolved Fe(II), S(-II), and natural organic matter as a nucleating template) results in uniform coating of aquifer material to create a regenerable reactive zone that mitigates Cr(VI) migration. Flow-through columns packed with quartz sand are amended first with an Fe2+ solution and then with a HS- solution to form a nano-FeS coating on the sand, which does not hinder permeability. This nano-FeS coating effectively reduces and immobilizes Cr(VI), forming Fe(III)-Cr(III) coprecipitates with negligible detachment from the sand grains. Preconditioning the sand with humic or fulvic acid (used as model natural organic matter (NOM)) further enhances Cr(VI) sequestration, as NOM provides additional binding sites of Fe2+ and mediates both nucleation and growth of FeS nanoparticles, as verified with spectroscopic and microscopic evidence. Reactivity can be easily replenished by repeating the procedures used to form the reactive coating. These findings demonstrate that such enhancement of attenuation capacity can be an effective option to mitigate Cr(VI) plume migration and exposure, particularly when tackling contaminant rebound post source remediation.

Characteristics, Comparative Analysis, and Phylogenetic Relationships of Chloroplast Genomes of Cultivars and Wild Relatives of Eggplant (Solanum melongena).

The eggplant (Solanum melongena) is a popular vegetable around the world. However, the origin and evolution of eggplant has long been considered complex and unclear, which has become the barrier to improvements in eggplant breeding. Sequencing and comparative analyses of 13 complete chloroplast (cp) genomes of seven Solanum species were performed. Genome sizes were between 154,942 and 156,004 bp, the smallest genome was from S. torvum and the largest from S. macrocapon. Thirteen cp genomes showed highly conserved sequences and GC contents, particularly at the subgenus level. All genes in the 13 genomes were annotated. The cp genomes in this study comprised 130 genes (i.e., 80 protein-coding genes, 8 rRNA genes, and 42 tRNA genes), apart from S. sisymbriifolium, which had 129 (79 protein-coding genes, 8 rRNA genes, and 42 tRNA genes.). The rps16 was absent from the cp genome of S. sisymbriifolium, resulting in a nonsense mutation. Twelve hotspot regions of the cp genome were identified, which showed a series of sequence variations and differed significantly in the inverted repeat/single-copy boundary regions. Furthermore, phylogenetic analysis was conducted using 46 cp genomic sequences to determine interspecific genetic and phylogenetic relationships in Solanum species. All species formed two branches, one of which contained all cultivars of the subgenus Leptostemonum. The cp genome data and phylogenetic analysis provides molecular evidence revealing the origin and evolutionary relationships of S. melongena and its wild relatives. Our findings suggest precise intra- and interspecies relatedness within the subgenus Leptostemonum, which has positive implications for work on improvements in eggplant breeding, particularly in producing heterosis, expanding the source of species variation, and breeding new varieties.

Ciclopirox olamine sensitizes leukemia cells to natural killer cell-mediated cytolysis by upregulating NKG2DLs via the Akt signaling pathway.

The activating receptor natural killer group 2D (NKG2D) expressed by Natural killer (NK) cells functions as a "master-switch" in governing the awakening status of NK cells. The NKG2D-mediated cytotoxicity has been declared to be related with the expression levels of NKG2D ligands (NKG2DLs) expressed on tumor cells. Therefore, selective induction of NKG2DLs could be a reliable approach to enhance the efficacy of NK cell-mediated immunotherapy. Our existing study demonstrated that Ciclopirox Olamine (CPX), an off-patent antifungal agent, effectively elevated the expression of NKG2DLs on leukemia cells and sensitized leukemia cells to NK-cell mediated cytolysis. Induction of ROS production and AKT phosphorylation by CPX is essential for the up-regulation of NKG2DLs expressions. Inhibition of AKT by using AKT inhibitor MK2206 decreased both NKG2DLs expressions and NK cell cytotoxicity. These data indicated that increased sensitivity of CPX-treated leukemia cells to NK cell cytolysis was attributed to higher NKG2DLs expressions, resulting from activated AKT signaling pathway. Our findings support the ongoing development of CPX as an anti-tumor agent and suggest its promising immunotherapeutic value in the medication of leukemia.

The Genome of Fusarium oxysporum f. sp. phaseoli Provides Insight into the Evolution of Genomes and Effectors of Fusarium oxysporum Species.

Fusarium oxysporum f. sp. phaseoli, the causal agent of cowpea fusarium wilt, is a serious threat to cowpea production in China. In this study, a sample of cowpea fusarium wilt was identified as Fusarium oxysporum f. sp. phaseoli using the methods of morphological characters and molecular detection. We further reported the first genome assembly for Fusarium oxysporum f. sp. phaseoli, with 53.7 Mb genome sequence comprising 14,694 genes. Comparative genomic analysis among five Fusarium oxysporum genomes showed that four accessory chromosomes in the five Fusarium oxysporum display similar characteristics, with low sequence similarity (55.35%, vs. overall average of 81.76%), low gene density (2.18 genes/10 kb vs. 3.02 genes/Mb) and highly transposable element density (TEs) (15.01/100 kb vs. 4.89/100 kb), indicating that variable accessory chromosomes are the main source of Fusarium oxysporum evolution. We identified a total of 100 Fusarium oxysporum f. sp. phaseoli-specific effectors in the genome and found 13 specific effector genes located in large insertion or deletion regions, suggesting that insertion or deletion events can cause the emergence of species-specific effectors in Fusarium oxysporum. Our genome assembly of Fusarium oxysporum f. sp. phaseoli provides a valuable resource for the study of cowpea fusarium wilt, and the comparative genomic study of Fusarium oxysporum could contribute to the knowledge of genome and effector-associated pathogenicity evolution in Fusarium oxysporum study.

A novel regulatory complex mediated by Lanata (Ln) controls multicellular trichome formation in tomato.

Trichomes that originate from plant aerial epidermis act as mechanical and chemical barriers against herbivores. Although several regulators have recently been identified, the regulatory pathway underlying multicellular trichome formation remains largely unknown in tomato. Here, we report a novel HD-ZIP IV transcription factor, Lanata (Ln), a missense mutation which caused the hairy phenotype. Biochemical analyses demonstrate that Ln separately interacts with two trichome regulators, Woolly (Wo) and Hair (H). Genetic and molecular evidence demonstrates that Ln directly regulates the expression of H. The interaction between Ln and Wo can increase trichome density by enhancing the expression of SlCycB2 and SlCycB3, which we previously showed are involved in tomato trichome formation. Furthermore, SlCycB2 represses the transactivation of the SlCycB3 gene by Ln and vice versa. Our findings provide new insights into the novel regulatory network controlling multicellular trichome formation in tomato.

Parthenolide inhibits ubiquitin-specific peptidase 7 (USP7), Wnt signaling, and colorectal cancer cell growth.

It has been well-established that the deubiquitinating enzyme ubiquitin-specific peptidase 7 (USP7) supports cancer growth by up-regulating multiple cellular pathways, including Wnt/ß-catenin signaling. Therefore, considerable efforts are directed at identifying and developing USP7 inhibitors. Here, we report that sesquiterpene lactone parthenolide (PTL) inhibits USP7 activity, assessed with deubiquitinating enzyme activity assays, including fluorogenic Ub-AMC/Ub-Rho110, Ub-VME/PA labeling, and Di-Ub hydrolysis assays. Further investigations using cellular thermal shift (CETSA), surface plasmon resonance (SPR), and mass spectrum (MS) assays revealed that PTL directly interacts with USP7. Consistent with the role of USP7 in stimulating Wnt signaling and carcinogenesis, PTL treatment inhibited the activity of Wnt signaling partly by destabilizing ß-catenin. Moreover, using cell viability assays, we found that PTL suppresses the proliferation of colorectal cancer cells and induces apoptosis in these cells. Additionally, we examined the effects of two other sesquiterpene lactones (costunolide and α-santonin) on USP7 and Wnt signaling and found that α-methylene-γ-butyrolactone may provide a scaffold for future USP7 inhibitors. In summary, our findings reveal that PTL inhibits USP7 activity, identifying a potential mechanism by which PTL suppresses Wnt/ß-catenin signaling. We further suggest that sesquiterpene lactones might represent a suitable scaffold for developing USP7 inhibitors and indicate that PTL holds promise as an anticancer agent targeting aberrant USP7/Wnt signaling.

WOOLLY, interacting with MYB transcription factor MYB31, regulates cuticular wax biosynthesis by modulating CER6 expression in tomato.

Cuticular waxes play a crucial role not only in plant defense against biotic and abiotic stresses, but also in the quality and storability of fruits, such as the tomato (Solanum lycopersicum). Although the biosynthetic pathways of waxes have been extensively characterized, the regulatory mechanisms underlying wax biosynthesis in tomato remain largely unclear. Here, we show that Woolly (Wo), a multicellular trichome regulator, is involved in modulating wax biosynthesis in tomato. Wo enhances the expression of the wax biosynthetic genes SlCER6, SlKCR1, and SlPAS2, and the wax transporter gene SlLTP, and thereby promotes wax accumulation. Furthermore, Wo directly binds to the L1-box in the promoter of SlCER6, an essential element of the very-long-chain fatty acid elongase complex. Intriguingly, overexpression (OE) or knock-down of SlMYB31, an MYB transcription factor that physically interacts with Wo in vivo and in vitro, produces marked changes in wax composition, and whereas Wo knock-down inhibits wax accumulation in SlMYB31-OE lines, SlMYB31 knock-down inhibits wax accumulation in Wo-OE lines, implying that these two genes function in the same pathway. Lastly, SlCER6 expression is induced by abscisic acid in a manner that is partially dependent on Wo. These results demonstrate that Wo and SlMYB31 cooperatively control tomato cuticular wax biosynthesis by regulating the expression of SlCER6.

USP47 maintains the stemness of colorectal cancer cells and is inhibited by parthenolide.

Colorectal cancer stem cells (CCSCs) are implicated in colorectal tumor initiation, invasion, recurrence and treatment resistance, so elucidation of the mechanism underlying the cancer stem cells induction and development of drugs targeting CCSCs are vital for cancer treatment. Growing evidence shows that dysregulated deubiquitinase (DUBs) expression is frequently associated with stemness and maintenance of cancer stem cells (CSCs). In the current study, we found that upregulation of USP47 is associated with tumorigenesis and poor prognosis in clinical patients with colorectal cancer (CRC). Besides, USP47 was highly expressed in CCSCs enriched by serum-free culture. Further investigation showed that USP47 is closely involved in the maintenance of the stemness of CCSCs. USP47 silencing reduces proliferation and migration of colorectal cancer cells and suppresses the self-renewal of CCSCs by downregulating the expression of cancer stem cell markers, including CD44, CD133, CD166, OCT4 and NANOG. Furthermore, we identified Parthenolide (PTL), a natural sesquiterpene lactone, as a novel USP47 inhibitor. PTL diminishes CCSCs self-renewal and induces apoptosis of CCSCs. Taken together, our findings highlighted a novel DUB involved in the modulation of CCSCs stemness and the potential of PTL in the CRC treatment by targeting CCSCs as the USP47 inhibitor.

Antimicrobial Peptide Brevinin-1RL1 from Frog Skin Secretion Induces Apoptosis and Necrosis of Tumor Cells.

Cancer has always been one of the most common malignant diseases in the world. Therefore, there is an urgent need to find potent agents with selective antitumor activity against cancer cells. It has been reported that antimicrobial peptides (AMPs) can selectively target tumor cells. In this study, we focused on the anti-tumor activity and mechanism of Brevinin-1RL1, a cationic α-helical AMP isolated from frog Rana limnocharis skin secretions. We found that Brevinin-1RL1 preferentially inhibits tumor cells rather than non-tumor cells with slight hemolytic activity. Cell viability assay demonstrated the intermolecular disulfide bridge contributes to the inhibitory activity of the peptide as the antitumor activity was abolished when the disulfide bridge reduced. Further mechanism studies revealed that both necrosis and apoptosis are involved in Brevinin-1RL1 mediated tumor cells death. Moreover, Brevinin-1RL1 induced extrinsic and mitochondria intrinsic apoptosis is caspases dependent, as the pan-caspase inhibitor z-VAD-FMK rescued Brevinin-1RL1 induced tumor cell proliferative inhibition. Immunohistology staining showed Brevinin-1RL1 mainly aggregated on the surface of the tumor cells. These results together suggested that Brevinin-1RL1 preferentially converges on the cancer cells to trigger necrosis and caspase-dependent apoptosis and Brevinin-1RL1 could be considered as a pharmacological candidate for further development as anti-cancer agent.

Moderate alcohol intake promotes pancreatic ductal adenocarcinoma development in mice expressing oncogenic Kras.

Kras mutations are associated with pancreatic ductal adenocarcinoma (PDAC). Although tobacco smoking, pancreatitis, and obesity are known environmental risk factors for PDAC, the contribution of moderate alcohol intake to PDAC remains elusive. In the present study, we tested whether a combination of risk factors or moderate alcohol intake induces PDAC development in mice. Control Pdx1Cre and Pdx1Cre;LSL-KrasG12D mutant mice were fed a Western alcohol diet containing high levels of cholesterol and saturated fat, 3.5% alcohol, and lipopolysaccharide for 5 mo. In addition, mice were treated with cerulein, for induction of pancreatitis, and nicotine every month. Treatment with all of these risk factors promoted development of advanced pancreatic neoplasia and PDAC in the Pdx1Cre;LSL-KrasG12D mice but not in the control Pdx1Cre mice. Moderate alcohol intake or Western diet feeding also significantly promoted advanced neoplasia and PDAC development in Pdx1Cre;LSL-KrasG12D mice compared with mice fed a regular chow. Alcohol, but not Western diet, increased tumor development in the liver in the Pdx1Cre;LSL-KrasG12D mice, but its origin remained elusive due to leakiness of Pdx1Cre in hepatocytes. RNA-seq analysis revealed that alcohol feeding increases expression of markers for tumors (Epcam, Krt19, Prom1, Wt1, and Wwtr1), stroma (Dcn, Fn1, and Tnc), and cytokines (Tgfb1 and Tnf) and decreases expression of Fgf21 and Il6 in the pancreatic tumor tissues. Immunostaining showed heterogeneous expression of nephronectin, S100 calcium-binding protein A6, and vascular cell adhesion molecule 1 in pancreatic tumors surrounded by podoplanin-positive stromal cells. Our data indicate that moderate alcohol drinking is a risk factor for development of PDAC.NEW & NOTEWORTHY Heavy alcohol intake has been suspected to be a risk factor of pancreatic ductal adenocarcinoma (PDAC) in humans. However, the contribution of moderate alcohol intake to PDAC development remains elusive. In the present study, we experimentally show that moderate alcohol feeding significantly induces advanced stages of pancreatic intraepithelial neoplasia development and invasive PDAC in Pdx1Cre;LSL-KrasG12D mutant mice. Our data indicate that moderate alcohol drinking is a risk factor for PDAC.

The HD-Zip IV transcription factor SlHDZIV8 controls multicellular trichome morphology by regulating the expression of Hairless-2.

Trichomes are specialized epidermal appendages that serve as excellent models to study cell morphogenesis. Although the molecular mechanism underlying trichome morphogenesis in Arabidopsis has been well characterized, most of the regulators essential for multicellular trichome morphology remain unknown in tomato. In this study, we determined that the recessive hairless-2 (hl-2) mutation in tomato causes severe distortion of all trichome types, along with increased stem fragility. Using map-based cloning, we found that the hl-2 phenotype was associated with a 100 bp insertion in the coding region of Nck-associated protein 1, a component of the SCAR/WAVE complex. Direct protein-protein interaction was detected between Hl-2 and Hl (SRA1, specifically Rac1-associated protein) using yeast two-hybrid and co-immunoprecipitation assays, suggesting that these proteins may work together during trichome formation. In addition, knock-down of a HD-Zip IV transcription factor, HDZIPIV8, distorted trichomes similar to the hl-2 mutant. HDZIPIV8 regulates the expression of Hl-2 by binding to the L1-box in the Hl-2 promoter region, and is involved in organizing actin filaments. The brittleness of hl-2 stems was found to result from decreased cellulose content. Taken together, these findings suggest that the Hl-2 gene plays an important role in controlling multicellular trichome morphogenesis and mechanical properties of stems in tomato plants.

Hair, encoding a single C2H2 zinc-finger protein, regulates multicellular trichome formation in tomato.

Trichomes originate from the epidermal cells of nearly all terrestrial plants, which are specialized unicellular or multicellular structures. Although the molecular mechanism regulating unicellular trichome formation has been extensively characterized, most of the genes essential for multicellular trichome formation remain unknown. In this study, we identified an associated locus on the long arm of chromosome 10 using a genome-wide association study (GWAS) on type-I trichomes of 180 diverse Solanum lycopersicum (tomato) accessions. Using map-based cloning we then cloned the key gene controlling the initiation of this type of trichome, named Hair (H), which encodes a single C2H2 zinc-finger protein. Transgenic experiments showed that hair-absent phenotype is caused by the deletion of the entire coding region of H. We identified three alleles of H containing several missense mutations and a nucleotide deletion, which result in amino acid substitutions and a reading frame shift, respectively. In addition, knockdown of H or Woolly (Wo) represses the formation of type-I trichomes, suggesting that both regulators may function as a heterodimer. Direct protein-protein interaction between them was further detected through pull-down and yeast two-hybrid assays. In addition, ectopic expression of H in Nicotiana tabacum (tobacco) and expression of its homologs from Capsicum annuum (pepper) and tobacco in tomato can trigger trichome formation. Taken together, these findings suggest that the H gene may be functionally conserved in multicellular trichome formation in Solanaceae species.

The tomato WV gene encoding a thioredoxin protein is essential for chloroplast development at low temperature and high light intensity.

BACKGROUND: Chloroplast biogenesis, a complex process in higher plants, is the key to photoautotrophic growth in plants. White virescent (wv) mutants have been used to unfold the molecular mechanisms underlying the regulation of chloroplast development and chloroplast gene expression in plants. However, most of genes controlling white virescent phenotype still remain unknown. RESULTS: In this study, we identified a temperature- and light intensity-sensitive mutant, named as wv. The content of chlorophyll was dramatically decreased in the immature leaves of wv mutant under the conditions of low temperature and high-light intensity. TEM observation showed that the chloroplasts in the young leaves of wv mutant lacked an organized thylakoid membrane, whereas crescent-shaped chloroplasts with well-developed stromal and stacked grana thylakoids in the mature leaves were developed. Immunoblot analyses suggested that proteins of photosynthetic complexes were decreased substantially in wv mutants. Based on map-based cloning and transgenic analysis, we determined that the wv phenotype was caused by single base mutation in the first intron of WV gene, which encoded a thioredoxin protein with 365 amino acids. qRT-PCR analysis revealed that the expression of WV gene was significantly down-regulated in wv mutant. In addition, knockdown of WV gene through RNAi also resulted in white virescent young leaves, suggesting that the mutation possibly blocks the differentiation of chloroplasts through inhibiting the expression of WV gene. Furthermore, the expression of WV peaked in apical buds and gradually decreased along with the developmental stage, which was consistent with the wv mutant phenotype. Expression analysis of chloroplast-encoded genes by qRT-PCR showed that the wv mutation affected the expression pattern of chloroplast-encoded PEP dependent genes. CONCLUSION: Our results suggested that wv mutant was sensitive to low temperature and light intensity. WV gene was essential for chloroplast differentiation. A single base mutation in the first intron resulted in down-regulation of WV gene expression, which inhibited the expression of chloroplast-encoded genes, thereby blocking chloroplast formation and chlorophyll synthesis.

Pyroptosis by caspase11/4-gasdermin-D pathway in alcoholic hepatitis in mice and patients.

Alcoholic hepatitis (AH) continues to be a disease with high mortality and no efficacious medical treatment. Although severe AH is presented as acute on chronic liver failure, what underlies this transition from chronic alcoholic steatohepatitis (ASH) to AH is largely unknown. To address this question, unbiased RNA sequencing and proteomic analyses were performed on livers of the recently developed AH mouse model, which exhibits the shift to AH from chronic ASH upon weekly alcohol binge, and these results are compared to gene expression profiling data from AH patients. This cross-analysis has identified Casp11 (CASP4 in humans) as a commonly up-regulated gene known to be involved in the noncanonical inflammasome pathway. Immunoblotting confirms CASP11/4 activation in AH mice and patients, but not in chronic ASH mice and healthy human livers. Gasdermin-D (GSDMD), which induces pyroptosis (lytic cell death caused by bacterial infection) downstream of CASP11/4 activation, is also activated in AH livers in mice and patients. CASP11 deficiency reduces GSDMD activation, bacterial load in the liver, and severity of AH in the mouse model. Conversely, the deficiency of interleukin-18, the key antimicrobial cytokine, aggravates hepatic bacterial load, GSDMD activation, and AH. Furthermore, hepatocyte-specific expression of constitutively active GSDMD worsens hepatocellular lytic death and polymorphonuclear leukocyte inflammation. CONCLUSION: These results implicate pyroptosis induced by the CASP11/4-GSDMD pathway in the pathogenesis of AH. (Hepatology 2018;67:1737-1753).

Exposure to low-level metalaxyl impacts the cardiac development and function of zebrafish embryos.

Metalaxyl is an anilide pesticide that is widely used to control plant diseases caused by Peronosporales species. In order to study the toxic effects, zebrafish embryos were exposed to metalaxyl at nominal concentrations of 5, 50 and 500 ng/L for 72 hr, and the cardiac development and functioning of larvae were observed. The results showed that metalaxyl exposure resulted in increased rates of pericardial edema, heart hemorrhage and cardiac malformation. The distance between the sinus venosus and bulbus arteriosus, stroke volume, cardiac output and heart rate were significantly increased in larvae exposed to 50 and 500 ng/L metalaxyl compared to solvent control larvae. Significant upregulation in the transcription of tbx5, gata4 and myh6 was observed in the 50 and 500 ng/L treatments, and that of nkx2.5 and myl7 was observed in the 5, 50 and 500 ng/L groups. These disturbances may be related to cardiac developmental and functional defects in the larvae. The activity of Na+/K+-ATPase and Ca2+-ATPase was significantly increased in zebrafish embryos exposed to 500 ng/L metalaxyl, and the mRNA levels of genes related to ATPase (atp2a11, atp1b2b, and atp1a3b) (in the 50 and 500 ng/L groups) and calcium channels (cacna1ab) (in the 500 ng/L group) were significantly downregulated; these changes might be associated with heart arrhythmia and functional failure.

Fenbuconazole exposure impacts the development of zebrafish embryos.

Fenbuconazole (FBZ), a triazole-containing fungicide, is widely used in agriculture and horticulture. In the present study, the development and cardiac functioning were observed and determined in zebrafish embryos exposed to FBZ at 5, 50 and 500 ng/L nominal concentrations for 72 h. The results showed that 500 ng/L FBZ significantly increased pericardial edema rate, spine curvature rate, disturbed cardiac function, and led a shortened lower jaw. The transcription of genes such as tbx5, nkx2.5, tnnt2, gata4, bmp2b, myl7 was altered, which might be responsible for the cardiac developmental and functioning defects in the larvae. The deformation in bone development might be related with the impaired transcription levels of shh and bmp2b. The transcription of cyp26a1 (encoding retinoic acid metabolism enzyme) was significantly up-regulated in the 500 ng/L group, which might be a reason causing the teratogenic effect of FBZ. These results suggest that FBZ could have toxic effects on embryonic development, which should be considered in the risk evaluation of FBZ application.

WITHDRAWN: Specific antigen vaccination modulates memory B cell activities.

The manuscript was withdrawn by the Author.

Antigen-specific immunotherapy regulates B cell activities in the intestine.

Mature B cells (BCs) express CD23 and B cell receptors. Whether activation of CD23 and B cell receptors has different effects on BC activities is unclear. This study aims to investigate the mechanism by which the specific antigen immunotherapy regulates the activation of BCs in the skewed Th2 responses. Mice were sensitized to ovalbumin. The specific antigen vaccination (SAV) at graded doses was employed to modulate the activities of BCs in which the expression of IL-10, IgE, matrix metalloproteinase-9 (MMP9), CD23, and serum soluble CD23 by BCs was evaluated. The immune regulatory effect of BCs primed by lower or higher SAV doses was observed with an adoptive transfer mouse experiment. SAV activated CD23 to produce IL-10 in BCs at lower doses. The higher doses of SAV increased the expression of MMP9 in BCs that reduced the amounts of CD23 in BCs and increased the serum levels of soluble CD23, which was abrogated by the pretreatment with MMP9 inhibitor. Adoptively transfer with BCs primed by lower doses of SAV inhibited the ongoing antigen-specific Th2 responses whereas the BCs primed by higher doses of SAV exacerbated the ongoing Th2 responses. Exposure to specific antigens at optimal doses can activate BCs to produce IL-10 to suppress the skewed antigen-specific Th2 responses. The antigen doses of SAV higher than the optimal doses may promote the production of soluble CD23 to exacerbate the ongoing immune responses.