The present study was conducted to investigate the expression of apoptosis-related factors in peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B and their correlation to clinical prognosis. Sixty-two patients that were diagnosed with chronic hepatitis B were admitted to Xuzhou Hospital from March 2015 to February 2016 and were enrolled as the observation group, while 60 healthy subjects who were examined in the health examination center were selected as the control group. The PBMC of patients were collected, and mRNA expression levels of the apoptotic molecules (FAS, CASP3, CASP8 and CASP9) were measured using real-time fluorescence quantitative PCR. The protein expression levels of apoptosis-related genes in the plasma were detected using enzyme-linked immunosorbent assay (ELISA) and the serum HBV-DNA was quantitatively measured using real-time fluorescence quantitative PCR. The mRNA and protein expression levels of FAS, CASP3, CASP8 and CASP9 in the observation group were significantly higher than those in the control group (P<0.05). The positive rate and log value of the copy amount of HBV DNA in the observation group were significantly higher than those in the control group (P<0.05). The Pearson correlation coefficient analysis showed that FAS, CASP3, CASP8 and CASP9 were positively correlated with HBV DNA (P<0.05). The mRNA expression levels of FAS, CASP3, CASP8 and CASP9 in patients with negative HBV-DNA were significantly lower than those with positive HBV-DNA (P<0.05). Apoptosis of PBMCs play an important role in the occurrence and development of chronic hepatitis B, and is closely correlated to the level of serum virus replication and prognosis.

Background and objectives: Laryngeal squamous cell carcinoma (LSCC) is one of the most common head and neck tumors. The molecular mechanism of LSCC remains unclear. The aim of this study was to evaluate the prevalence of Human papillomavirus (HPV) and single nucleotide polymorphisms (SNPs) of TP53, MDM2, MDM4, MTHFR, CASP8, and CCR5 genes in LSCC, and to assess their correlations with patient survival. Materials and Methods: 49 LSCC patients were enrolled in this study. PCR and qRT-PCR were used to detect, identify, and quantify HPV. SNPs were genotyped using PCR and PCR-RFLP. Results: By analyzing the interactions of the SNPs of the genes with clinical parameters, the majority of patients with lymph node status (N1,2) were identified as carriers of MDM2T/G, CASP8ins/del, CCR5wt/wt SNP. Cluster analysis showed that patients with MDM2T/T SNP survive longer than patients identified as CASP8ins/ins, MTHFRC/C, and MDM4A/A variant carriers; meanwhile, LSCC patients with MDM2T/T polymorphic variant had the best survival. Multivariate analysis showed that HPV-positive patients without metastasis in regional lymph nodes (N0) and harboring CASP8ins/del variant had the best survival. Meanwhile, HPV-negative patients with identified metastasis in lymph nodes (N1 and N2) and CASP8ins/del variant had poor survival. Conclusions: This finding suggests patients survival prognosis and tumor behavior are different according HPV status, SNP variants, and clinical characteristics of the LSCC.

BACKGROUND

Novel derivatives of benzo[b]furan were found to be highly toxic towards human chronic myelogenous (K562), acute myelogenous (HL-60) and acute lymphoblastic (MOLT-4) leukemia cells.

OBJECTIVE

Characterization of the biological activity of novel benzofurans (influence on apoptosis, mitogen activated protein kinases and on the cell cycle). Identification of cellular protein(s) targeted by test benzofurans and defining the mechanism of action.

METHODS

chemical synthesis, fluorescence assays, flow cytometry, gene expression by DNA microarray and real time RT-PCR, western blotting, cytotoxicity assays, pull-down assay, mass spectroscopy, in vitro polymerization of tubulin, molecular docking.

RESULTS

1,1'-[3-(bromomethyl)-5,6- dimethoxy-1-benzofuran-2,7-diyl]diethanone (1) and methyl 4-bromo-6-(dibromoacetyl)-5-hydroxy-2-methyl-1-benzofuran-3-carboxylate (2) induced apoptosis in K562 and MOLT-4 cells. The profiling of gene expression revealed that 1 and 2 increased the expression of proapoptotic genes involved in both receptor (TNFRSF 10A, TNFRSF 10B, CASP8) and mitochondrial (BAX, BID, NOXA, APAF1) pathways of apoptosis. Test benzo[b]furans activated c-Jun N-terminal kinase (JNK) and p38 kinase in K562 cells. Tubulin was identified as a protein target for benzo[b]furans in pull-down experiments with biotinylated 2. Test benzo[b]furans inhibited polymerization of tubulin monomers in vitro, decreased the level of cellular microtubules and arrested cells in a G2/M phase. Molecular docking suggests that benzo[b]furans 1 and 2 bind to tubulin via colchicine binding pocket and the complex is stabilized mainly by hydrophobic interactions.

CONCLUSIONS

Novel benzo[b]furans with anti-microtubule activity were identified. They induce apoptosis in cancer cells and cause G2/M cell cycle arrest. Biological activity of 1 and 2 makes them potential lead compounds for development as anticancer drugs.

OBJECTIVE

Various genetic variants have been reported to be linked to an increased risk of meningioma. However, no confirmed conclusion has been obtained. The purpose of the study was to investigate potential meningioma-associated gene polymorphisms, based on published evidence.

METHODS

An updated meta-analysis was performed in September 2016. After electronic database searching and study screening, we selected eligible case-control studies and extracted data for meta-analysis, using Mantel-Haenszel statistics. P-values, pooled odds ratios (ORs), and 95% confidence intervals were calculated.

RESULTS

We finally selected eight genes with ten polymorphisms: MLLT10 rs12770228, CASP8 rs1045485, XRCC1 rs1799782, rs25487, MTHFR rs1801133, rs1801131, MTRR rs1801394, MTR rs1805087, GSTM1 null/present, and GSTT1 null/present. Results of meta-analyses showed that there was increased meningioma risk in case groups under all models of MLLT10 rs12770228 (all OR >1, P<0.001), compared with control groups. Similar results were observed under the allele, homozygote, dominant, and recessive models of MTRR rs1801394 (all OR >1, P<0.05), and the heterozygote and dominant models of MTHFR rs1801131 in the Caucasian population (all OR >1, P<0.05). However, no significantly increased meningioma risks were observed for CASP8 rs1045485, XRCC1 rs25487, rs1799782, MTHFR rs1801133, MTR rs1805087, or GSTM1/GSTT1 null mutations.

CONCLUSIONS

Our updated meta-analysis provided statistical evidence for the role of MLLT10 rs12770228, MTRR rs1801394, and MTHFR rs1801131 in increased susceptibility to meningioma.

The RNA quality of tissue biobank is crucial for translational research; however, the effects of the ex vivo ischemia time on RNA integrity and expression of genes related to hypoxia, stress, apoptosis and autophagy remains elusive. A total of 18 carcinoma tissues were stored at room temperature for 15 min, 30 min, 1, 2, 4, 8 and 24 h. The integrity and purity of isolated RNA were analyzed. Furthermore, the gene expression of mTOR, hypoxia‑inducible factor 1α, phosphatidylinositol 4,5‑bisphosphate 3‑kinase catalytic subunit β isoform (PI3KCB), threonine kinase 1 (AKT1), NF‑κB, protein kinase AMP‑activated catalytic subunit α1 (AMPKα1), caspase 8 (CASP8), unc‑51 like autophagy activating kinase 1 and Fas cell surface death receptor were analyzed using reverse transcription‑quantitative PCR. The results demonstrated that RNA integrity numbers (RINs) remained stable in carcinoma tissues following ex vivo ischemia for 2 h at room temperature and that degradation began at 4 h (P<0.001). Additionally, the expression of PI3KCB, AKT1, AMPKα1 and CASP8 decreased at time points 8‑24 h following ex vivo ischemia and delayed processing (P<0.001). In conclusion, >2 h of ex vivo ischemia and delayed processing induced RNA degradation and RIN, and the gene expressions of PI3KCB, AKT, AMPKα1 and CASP8 may be considered as markers to evaluate tissue quality at the gene expression level, providing a method for the standard processing and assessment of tissue specimen.

Background: The incidence of gout and hyperuricemia is increasing year by year in the world. Plantain is a traditional natural medicine commonly used in the treatment of gout and hyperuricemia, but the molecular mechanism of its active compounds is still unclear. Based on network pharmacology, this article predicts the targets and pathways of effective components of plantain for gout and hyperuricemia and provides effective reference for clinical medication.

Method: Traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) and SymMap databases were used to screen out the active compounds and their targets in plantain. GeneCards, Therapeutic Target Database (TTD), and Online Mendelian Inheritance in Man (OMIM) databases were used to find the targets corresponding to gout and hyperuricemia. Venn diagram was used to obtain the intersection targets of plantain and diseases. The interaction network of the plantain active compounds-targets-pathways-diseases was constructed by using Cytoscape 3.7.2 software. Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out.

Result: Seven active compounds were identified by network pharmacological analysis, including dinatin, baicalein, baicalin, sitosterol, 6-OH-luteolin, stigmasterol, and luteolin. Plantain plays a role in gout and hyperuricemia diseases by regulating various biological processes, cellular components, and molecular functions. The core targets of plantain for treating gout are MAPK1, RELA, TNF, NFKBIA, and IFNG, and the key pathways are pathways in cancer, hypoxia-inducible factor-1 (HIF-1) signaling pathway, interleukin (IL)-17 signaling pathway, Chagas disease (American trypanosomiasis), and relaxin signaling pathway. The core targets of plantain for hyperuricemia are RELA, MAPK1, NFKBIA, CASP3, CASP8, and TNF, and the main pathways are pathways in cancer, apoptosis, hepatitis B, IL-17 signaling pathway, and toxoplasmosis.

Conclusion: This study explored the related targets and mechanisms of plantain for the treatment of gout and hyperuricemia from the perspective of network pharmacological analysis, reflecting the characteristics of multiple components, multiple targets, and multiple pathways, and it provides a good theoretical basis for the clinical application of plantain.

Dry period heat stress impairs subsequent milk yield. Our objective was to evaluate the effect of heat stress or cooling during the early and late dry period on mammary gland gene expression and microstructure. Cows were dried off ~45 d before expected parturition and randomly assigned to 1 of 2 treatments: heat stress (HT, n = 39) or cooling (CL, n = 39) during the first 21 d of the dry period. On d 22, cows were switched or remained on HT and CL and this yielded 4 treatments: heat stress during the entire dry period (HTHT, n = 18); cooling during the entire dry period (CLCL, n = 20); HT for the first 21 d dry, then CL until calving (HTCL, n = 21); or CL for the first 21 d dry, then HT until calving (CLHT, n = 19). Data were analyzed in 2 periods: first 21 d dry (early dry period) and from 22 d until calving (late dry period) and analyzed using PROC MIXED or GLM in SAS (SAS Institute Inc., Cary, NC). Mammary biopsies (5-8 cows/treatment) were collected at -3, 3, 7, 14, and 25 d relative to dry-off to evaluate mammary gland gene expression and histology [i.e., cellular apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling) and proliferation (Ki67)]. Mammary alveoli number and connective tissue were visualized by hematoxylin and eosin and Mason's trichrome staining, respectively. During the early dry period, CL upregulated expression of CASP3, IGF1R, HSP90, HSF1, BECN1, ATG3, ATG5, and PRLR-LF relative to HT. However, in the late dry period, CLHT treatment upregulated expression of CASP3, CASP8, HSP70, HSP90, PRLR-LF, STAT5, β-casein, and ATG3 relative to CLCL. During the early dry period, cows exposed to HT had reduced mammary and stroma cell apoptosis and proliferation relative to CL. In addition to these findings, cows exposed to HT had lower connective tissue 3 d after dry-off relative to CL. However, in the late dry period, HTHT cows had higher connective tissue relative to CLCL. Also, in the early dry period, cows exposed to HT had greater alveoli number relative to CL, and HT decreased expression of genes related to autophagy and apoptosis in the early dry period, consistent with a delay in involution with HT. Thus, cows exposed to HT have extended involution with delayed apoptosis and autophagy signaling. Also, HT compromises mammary gland cell proliferation and leads to higher connective tissue later in the dry period. These results provide evidence that heat stress impairs overall mammary gland turnover during the dry period, which then affects secretory activity and productivity in the next lactation.

Keywords: apoptosis; gene expression; proliferation.

Lung cancer is the leading cause of cancer deaths in China, and about 60% of the cases are diagnosed with histological adenocarcinoma. The caspase 8 (CASP8) gene is a critical initiator of the extrinsic apoptosis pathway. To explore the relationship between tagSNPs or haplotypes of CASP8 and the efficacy of platinum-based chemotherapy in advanced lung adenocarcinoma patients of China, we recruited 555 advanced adenocarcinoma patients. We extracted the genomic DNA from patients' peripheral blood samples and sequenced tagSNPs of CASP8. We calculated the individual haplotype of CASP8 frequencies using the PHASE 2.0 program. The association between CASP8 tagSNPs and overall survival (OS) was calculated by univariate and multivariate Cox regression analysis. A univariate logistic regression analysis was done to analyze the CASP8 tagSNPs and the toxicity of platinum-based chemotherapy. The same statistical methods were used for exploring haplotypes of CASP8. Rs3769821 and rs1045494 of CASP8 were independent prognosis factors for overall survival (OS) using multivariate Cox's regression models. For the haplotype of the 7 tagSNPs, haplotype AGGAAAGA was correlated with the efficacy of platinum-based chemotherapy. The polymorphisms of CASP8, rs7608692, and haplotype AGAACAG correlated with neutropenia toxicity. The haplotype GGGGAAA was associated with thrombocytopenia toxicity. We conclude that the polymorphisms of CASP8 contribute to the prognosis of advanced lung adenocarcinoma and influence the quality of life and survival.

We present an outline of the Critical Assessment of Protein Structure Prediction (CASP) infrastructure implemented at the University of California, Davis, Protein Structure Prediction Center. The infrastructure supports selection and validation of prediction targets, collection of predictions, standard evaluation of submitted predictions, and presentation of results. The Center also supports information exchange relating to CASP experiments and structure prediction in general. Technical aspects of conducting the CASP8 experiment and relevant statistics are also provided.

Purpose: To identify a gene signature for the prognosis of breast cancer using high-throughput analysis.

Methods: RNASeq, single nucleotide polymorphism (SNP), copy number variation (CNV) data and clinical follow-up information were downloaded from The Cancer Genome Atlas (TCGA), and randomly divided into training set or verification set. Genes related to breast cancer prognosis and differentially expressed genes (DEGs) with CNV or SNP were screened from training set, then integrated together for feature selection of identify robust biomarkers using RandomForest. Finally, a gene-related prognostic model was established and its performance was verified in TCGA test set, Gene Expression Omnibus (GEO) validation set and breast cancer subtypes.

Results: A total of 2287 prognosis-related genes, 131 genes with amplified copy numbers, 724 gens with copy number deletions, and 280 genes with significant mutations screened from Genomic Variants were closely correlated with the development of breast cancer. A total of 120 candidate genes were obtained by integrating genes from Genomic Variants and those related to prognosis, then 6 characteristic genes (CD24, PRRG1, IQSEC3, MRGPRX, RCC2, and CASP8) were top-ranked by RandomForest for feature selection, noticeably, several of these have been previously reported to be associated with the progression of breast cancer. Cox regression analysis was performed to establish a 6-gene signature, which can stratify the risk of samples from training set, test set and external validation set, moreover, the five-year survival AUC of the model in the training set and validation set was both higher than 0.65. Thus, the 6-gene signature developed in the current study could serve as an independent prognostic factor for breast cancer patients.

Conclusion: This study constructed a 6-gene signature as a novel prognostic marker for predicting the survival of breast cancer patients, providing new diagnostic/prognostic biomarkers and therapeutic targets for breast cancer patients.

Pharmaceuticals pose a major threat to the marine environment, and several studies have recently described their negative effects on marine organisms. Pharmaceutical compounds are constantly being released into aquatic ecosystems, and chronic exposure, even at low concentrations, may have a major impact on marine organisms. The purpose of the present study is to evaluate the biological changes induced by one of the most widely used pharmaceuticals-paracetamol-in the blue mussel Mytilus edulis, after a long-term exposure at environmentally relevant concentrations. We present our data alongside and in comparison with results from a previous short-term exposure, to demonstrate the significance of exposure period on the effects of paracetamol in adult blue mussels. After 24 days of laboratory exposure, seven potential target genes were selected to examine toxicological effects in mussels' gonads and possible disruptive effects on reproductive processes. The results show the modulation of some important reproduction-related genes: estrogen receptor-2 (ER2), vitelline envelope zona pellucida domain-9 (V9), and vitellogenin (VTG). Variations in mRNA expression of four other genes involved in apoptosis (HSP70, CASP8, BCL2, and FAS) are also highlighted. Histopathological alterations caused by paracetamol, together with neutral red retention time response in mussels' hemocytes, are presented herein. Overall, this study highlights the exacerbated effects of low concentration of paracetamol after chronic exposure, similar to the damage induced by higher concentrations in a short exposure scenario, thus emphasizing the importance of length of exposure period when studying the effects of this substance. Additionally, this study also discusses the potential of paracetamol to inflict several major changes in the reproductive system of mussels and thus possibly affect the survival of populations.

Keywords: Bivalves; Blue mussels; Histology; Marine pollution; Paracetamol; Pharmaceuticals; Reproduction; Transcriptomics.

BACKGROUND

Leprosy is an insidious infectious disease caused by the obligate intracellular bacteria Mycobacterium leprae, and host genetic factors can modulate the immune response and generate distinct categories of leprosy susceptibility that are also influenced by genetic ancestry.

RESULTS

We investigated the possible effects of CYP19A1 [rs11575899], NFKβ1 [rs28362491], IL1α [rs3783553], CASP8 [rs3834129], UGT1A1 [rs8175347], PAR1 [rs11267092], CYP2E1 [INDEL 96pb] and IL4 [rs79071878] genes in a group of 141 leprosy patients and 180 healthy individuals. The INDELs were typed by PCR Multiplex in ABI PRISM 3130 and analyzed with GeneMapper ID v3.2. The NFKβ1, CASP8, PAR1 and IL4 INDELs were associated with leprosy susceptibility, while NFKβ1, CASP8, PAR1 and CYP19A1 were associated with the MB (Multibacilary) clinical form of leprosy.

CONCLUSIONS

NFKβ1 [rs28362491], CASP8 [rs3834129], PAR1 [rs11267092] and IL4 [rs79071878] genes are potential markers for susceptibility to leprosy development, while the INDELs in NFKβ1, CASP8, PAR1 and CYP19A1 (rs11575899) are potential markers for the severe clinical form MB. Moreover, all of these markers are influenced by genetic ancestry, and European contribution increases the risk to leprosy development, in other hand an increase in African contribution generates protection against leprosy.

Blocking interferon-function by therapeutic intervention of the JAK-STAT-axis is a novel promising treatment option for inflammatory bowel disease (IBD). Although JAK inhibitors have proven efficacy in patients with active ulcerative colitis (UC), they failed to induce clinical remission in patients with Crohn's disease (CD). This finding strongly implicates a differential contribution of JAK signaling in both entities. Here, we dissected the contribution of different STAT members downstream of JAK to inflammation and barrier dysfunction in a mouse model of Crohn's disease like ileitis and colitis (Casp8 ^ΔIEC mice). Deletion of STAT1 in Casp8 ^ΔIEC mice was associated with reduced cell death and a partial rescue of Paneth cell function in the small intestine. Likewise, organoids derived from the small intestine of these mice were less sensitive to cell death triggered by IBD-key cytokines such as TNFα or IFNs. Further functional in vitro and in vivo analyses revealed the impairment of MLKL-mediated necrosis as a result of deficient STAT1 function, which was in turn associated with improved cell survival. However, a decrease in inflammatory cell death was still associated with mild inflammation in the small intestine. The impact of STAT1 signaling on gastrointestinal inflammation dependent on the localization of inflammation, as STAT1 is essential for intestinal epithelial cell death regulation in the small intestine, whereas it is not the key factor for intestinal epithelial cell death in the context of colitis. Of note, additional deletion of STAT2 was not sufficient to restore Paneth cell function but strongly ameliorated ileitis. In summary, we provide here compelling molecular evidence that STAT1 and STAT2, both contribute to intestinal homeostasis, but have non-redundant functions. Our results further demonstrate that STATs individually affect the distinct pathophysiology of inflammation in the ileum and colon, respectively, which might explain the diverse outcome of JAK inhibitors on inflammatory bowel diseases.

Keywords: Paneth cells; STAT signaling; cell death; inflammation; inflammatory bowel disease.

Purpose: Dry eye disease (DED) is a common and multifactor-induced autoimmune ocular surface disease. Environmental factors, such as desiccating stress (DS) and hyperosmolarity, affect the corneal epithelium to induce ocular surface inflammation in DED. We aimed to explore the potential mechanisms by which innate immunity and pyroptosis are initiated in the mucosal epithelium in response to environmental stress.

Methods: Experimental dry eye was established in C57BL/6J mice and genetic mice on the background of C57BL/6J mice by subcutaneous injection of scopolamine and exposure to a desiccating environment. SDHCEC cell line was subjected to hyperosmolarity stress (450 mOsM). The phenol red thread tear test and corneal epithelial defects evaluation were used as assessments of severity of DED. RNA-sequencing, quantitative real-time PCR, western blotting and immunofluorescence staining were performed in this study.

Results: Loss-of-function studies indicated that genetic deletion of GSDMD alleviates DS-induced corneal epithelium defects, and GSDMD is needed for IL-33 processing. We further found that NLRP12 collaborates with NLRC4 inflammasome to initiate GSDMD-dependent pyroptosis, which requires TLR4-induced caspase-8 (CASP8) activation in the mucosal corneal epithelium in response to DS.

Conclusions: These findings provide compelling evidence that GSDMD-dependent pyroptosis plays a pivotal role in DED. A novel mechanism involving NLRP12 and NLRC4 inflammasomes-induced GSDMD-dependent pyroptosis, accompanied by IL-33 processing is responsible for ocular surface epithelial defects in response to environmental stress. GSDMD is required for IL-33 processing and the subsequent amplification of inflammatory cascades. These findings reveal novel therapeutic targets for treating DED.

Keywords: Autoimmunity; Desiccating stress; Dry eye disease; Gasdermin D; Inflammasome; Interleukin-33.

The sea cucumber Apostichopus japonicus (Selenka) commonly undergoes aestivation in response to high water temperatures. This process is accompanied by tissue regression and body mass reduction. Previous studies have suggested that apoptosis may play a role in the tissue remodeling that occurs during aestivation, although this has not definitively been shown. To investigate this hypothesis, the present study used A. japonicus as a model organism to examine cell loss through apoptosis in intestinal degeneration during aestivation. Apostichopus japonicus individuals were collected from Yellow Sea (N 36° 05' 44.87″, E 120° 31' 58.51″), China in April 2016 and split into two groups. Aestivation was induced in the experimental group by incubation at 25°C. This resulted in a significant decrease in body mass and increased evidence of intestinal degeneration in hematoxylin and eosin, Hoechst 33342, and in situ TUNEL analyses of tissue sections. Along with further Hoechst 33342 analysis using intestinal cell smears, these results showed that A. japonicus intestinal cell apoptosis occurred soon after the initial temperature increase, with most apoptotic events completing within 20days. Transcriptional quantification of the Ajcaspase-8 (CASP8) and Ajcaspase-3 (CASP3) apoptotic genes demonstrated that their expression was significantly elevated at the beginning of the experiment but was decreased at later stages of aestivation. The results of this study strongly suggest that apoptosis is involved in the intestinal regression of A. japonicus during aestivation, and play important role in understanding fundamental cellular events in tissue regression under environmental stress.

Current understanding is that receptor interacting serine/threonine protein kinase 1 (RIPK1) can lead to two distinct forms of cell death: RIPK3-mediated necroptosis or caspase 8 (Casp8)-mediated apoptosis. Here, we report that RIPK1 signaling is indispensable for protection from hepatocellular injury in a steatotic liver undergoing ischemia reperfusion injury (IRI) but not in the lean liver. In lean liver IRI, RIPK1-mediated cell death is operational, leading to protection in RIP1 kinase-dead knock-in (RIPK1^K45A) mice and necrostatin-1s (Nec1s)-treated lean wild-type (WT) mice. However, when fed a high-fat diet (HFD), RIPK1^K45A-treated and Nec1s-treated WT mice undergoing IRI demonstrate exacerbated hepatocellular injury along with decreased RIPK1 ubiquitylation. Furthermore, we demonstrate that HFD-fed RIPK3^-/-/Casp8^-/- mice show protection from IRI, but HFD-fed RIPK3^-/-/Casp8^-/+ mice do not. We also show that blockade of RIPK1 leads to increased Casp8 activity and decreases mitochondrial viability. Conclusion: Although more studies are required, we provide important proof of concept for RIPK1 inhibition leading to distinctive outcomes in lean and steatotic liver undergoing IRI. Considering the rising incidence of nonalcoholic fatty liver disease (NAFLD) in the general population, it will be imperative to address this critical difference when treating patients with RIPK1 inhibitors. This study also presents a new target for drug therapy to prevent hepatocellular injury in NAFLD.

Background: Yersinia ruckeri is a pathogen that can cause enteric redmouth disease in salmonid species, damaging global production of economically important fish including rainbow trout (Oncorhynchus mykiss). Herein, we conducted the transcriptomic profiling of spleen samples from rainbow trout at 24 h post-Y. ruckeri infection via RNA-seq in an effort to more fully understand their immunological responses.

Results: We identified 2498 differentially expressed genes (DEGs), of which 2083 and 415 were up- and down-regulated, respectively. We then conducted a more in-depth assessment of 78 DEGs associated with the immune system including CCR9, CXCL11, IL-1β, CARD9, IFN, TNF, CASP8, NF-κB, NOD1, TLR8α2, HSP90, and MAPK11, revealing these genes to be associated with 20 different immunological KEGG pathways including the Cytokine-cytokine receptor interaction, Toll-like receptor signaling, RIG-I-like receptor signaling, NOD-like receptor signaling, and MAPK signaling pathways. Additionally, the differential expression of 8 of these DEGs was validated by a qRT-PCR approach and their immunological importance was then discussed.

Conclusions: Our findings provide preliminary insight on molecular mechanism underlying the immune responses of rainbow trout following Y. ruckeri infection and the base for future studies of host-pathogen interactions in rainbow trout.

Keywords: Immune response; Rainbow trout; Spleen; Transcriptome; Yersinia ruckeri.

Multi-omics strategy contributes as an indispensable and efficient approach for the investigation of the innate immunity in vertebrates. To explore the crucial genes and pathways involved in the antiviral innate immunity of black carp (Mylopharyngodon piceus), the comparative phosphoproteomics and transcriptomics of Mylopharyngodon piceus kidney (MPK) cells with/without GCRV infection were performed in this manuscript. In phosphoproteomics analysis, 2637 phosphosites corresponding to 1532 proteins were identified and quantified, in which 1372 proteins were identified as differentially expressed proteins (DEPs) with 683 upregulated and 689 downregulated in GCRV infected cells. Functional annotation, enrichment analysis and pathway analysis highlighted that a large number of DEPs were enriched in immune related pathways including TLR pathway and NLR pathway. In transcriptomics analysis, a total of 2936 genes were identified as differentially expressed genes (DEGs), in which 2290 and 646 genes were upregulated and downregulated respectively after GCRV infection. As expected, pathway analysis based on DEGs also showed that a large proportion of DEGs were enriched in immune related pathways including TLR and RLR pathway. A combined list of DEPs and DEGs that enriched in above pathways were imported in Cytoscape for network analysis, reconstruction and visualization. The integrative study suggested that several significant DEPs and DEGs, such as MAP3K7 (TAK1), JUN, MAP2K2, CASP8, IL8 and IRF7 might be functionally crucial in host antiviral innate immunity. Thus, this study contributes as an indispensable reference map for the further investigation of the innate immune system of black carp.

Keywords: Bioinformatics; Black carp; GCRV; Innate immunity; Phosphoproteomics; Transcriptomics.

Multiple research groups have demonstrated that caspase-8 (CASP8)-mediated gasdermin D (GSDMD) cleavage drives pyroptotic cell death. Here, we discuss a novel role for the enzymatically inactive homolog of CASP8, the long isoform of cellular FLICE-like inhibitory protein (cFLIP_L), in the regulation of this process. Specifically, cFLIP-deficiency provides a model in which to study the mechanisms regulating CASP8-mediated activation of cell death and inflammatory signaling.

N-acetylcysteine (NAC) is a widely used anti-inflammatory agent and antioxidant in vivo and in vitro. As a nutritional supplement, NAC can improve production and reproductive performances in animals through enhancing placental function and regulating hormone production. Trophoblast proliferation and steroid hormone production are two major functions in the placenta. We hypothesized that the effects of NAC on placental function is due to its direct and indirect effects on gene expression in placental trophoblast cells (pTr). To evaluate this hypothesis, we investigated the effects of NAC on steroidogenesis, gene expression, and cell proliferation in porcine pTr in vitro. pTr were treated with NAC in serum-free medium for 24 h with different concentrations (0, 0.1 μM, 1.0 μM, 10.0 μM, 0.1 mM, 1.0 mM, and 10.0 mM). Low-dose NAC (1 μM) stimulated pTr proliferation and decreased progesterone production, while increasing estradiol production (P < 0.05). High-dose NAC (10 mM) suppressed cell proliferation (P < 0.05), but had no effect on steroidogenesis. Low-dose NAC increased CCDN1 and decreased CASP3 and CASP8 mRNA levels (P < 0.05), whereas high-dose NAC decreased CDK4 and CCDN1 and increased CASP3 mRNA levels (P < 0.05). NAC had no effect on the mRNA abundance of StAR and HSD3B. Low-dose NAC upregulated CYP19A1 mRNA expression, and high-dose NAC downregulated CYP11A1 mRNA abundance (P < 0.05). Only low-dose NAC increased NOS3 mRNA abundance and tetrahydrobiopterin reduction (BH4/BH2 ratio). We conclude that NAC may act directly and indirectly on pTr with a dose-dependent manner and may regulate placental function by affecting pTr differentiation via regulating pTr steroid synthesis, cell proliferation, and apoptosis in sows.

Keywords: Cell differentiation; N-acetylcysteine; Sow; Steroidogenesis; Trophoblast.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis is a safe method for the treatment of various types of cancer. However, TRAIL therapy is less effective in certain types of cancer, including cervical cancer. To address this problem, a combinatorial approach was employed to sensitize cervical cancer at low dosages. YM155, a survivin inhibitor, was used at low dosages along with TRAIL to induce apoptosis in HeLa cells. The effects of the individual treatment with TRAIL and YM155 on apoptosis were assessed by propidium iodide assay. In addition, to validate the DNA damage exhibited by the combination treatment, the phosphorylation status of γH2A histone family member X was investigated by immunofluorescence and western blot analysis. TRAIL or YM155 alone had no significant effect on DNA damage and apoptosis. However, the TRAIL/YM155 combination triggered a synergistic pro-apoptotic stimulus in HeLa cells. The mRNA and protein levels of CASP8- and FADD-like apoptosis regulator (cFLIP), death receptor 5 (DR5) and survivin were monitored using RT-PCR and western blot analysis, respectively. This combinatorial approach downregulated both mRNA and protein expression levels of cFLIP and survivin. Further experimental results suggested that the combination treatment significantly reduced cell viability, invasion and migration of HeLa cells. Overall, the present findings indicated that the low dosage of YM155 sensitized HeLa cells to TRAIL-induced apoptosis via a mechanism involving downregulation of cFLIP and survivin. The results indicated the importance of combination drug treatment and reveal an effective therapeutic alternative for TRAIL therapy in human cervical cancer.

Keywords: CASP8- and FADD-like apoptosis regulator; HeLa cells; YM155; apoptosis; death receptor 5; survivin; tumor necrosis factor-related apoptosis inducing ligand therapy.

Of late, many studies are attempting to find new molecules with anticancer properties, especially those with the capability to inhibit cell growth. The aim of this work was to evaluate nerolidol, a plant-based compound, as its cytotoxicity, genotoxicity, antiproliferative and apoptotic induction, cell cycle, mitochondrial membrane potential and RT-qPCR of transcripts related to those pathways in the human hepatocellular carcinoma cell line (HepG2/C3A). Only cis-nerolidol (C-NER) demonstrated cytotoxicity (100-250 μM) activity and was selected to conduct the following experiments. C-NER did not show genotoxic activity, but altered the mitochondrial membrane potential, reduced cell proliferation by arresting cell cycle in G1 phase and induced cell death. RT-qPCR showed that C-NER down-regulated genes related to apoptosis (BAK1, BAX, CAPN1, CASP8, CASP9, PARP1 and TP53), cell cycle (CCND1, CCNE1, CDK1 and CDK2), xenobiotic metabolism (CYP2D6 and CYP3A4) and paraptosis (IGF1R receptor). Up-regulation was seen in case of genes related to cell survival (BBC3 and MYC) and reticulum stress protein response (EIF2AK3 and ERN1) and xenobiotic metabolism (CYP1A2 and CYP2C19). We deduced that the antiproliferative activity of C-NER is attributable to its modulation of the cyclins and cyclin-dependent kinases as these proteins are necessary for G1/S phase transition. EIF2AK3, ERN1, CYP2C19 and CYP1A2 up-regulation suggests that endoplasmic reticulum stress was induced owing to the increased activity of cytochrome P450 enzymes. Caspase-independent cell death was also observed, indicating that another type of cell death, paraptosis, was triggered. Our results indicate that C-NER has considerable potential in anticancer therapy because it modulates important molecular targets of cell survival and proliferation.

The dibenzylbutyrolactone lignan (-)-cubebin, which is extracted from the seeds of the pepper Piper cubeba, has shown promise as an anti-inflammatory, analgesic, leishmanicidal, antiproliferative, and trypanocidal compound. Given the therapeutic potential of (-)-cubebin, this study aimed to investigate its safety profile by analyzing cytotoxicity, mutagenicity, cell proliferation kinetics, induction of apoptosis, and expression of pro-apoptotic genes in human colon adenocarcinoma cells (HT29) exposed to (-)-cubebin. MTT cytotoxicity assays demonstrated that (-)-cubebin was cytotoxic only at 280 µM, whereas it was not cytotoxic at 2.8, 14, or 28 µM. Data demonstrated that (-)-cubebin was not mutagenic as evidenced by a micronucleus (MN) assay, did not alter cell-growth kinetics over 4 d, and showed absence of induced apoptosis after 24 h. Further, CASP8 and CASP9 gene expression was not markedly changed in HT29 cells exposed to 28 µM or 70 µM (-)-cubebin for 12 h. Based on our observations, (-)-cubebin was cytotoxic at a concentration of 280 µM, suggesting that the use of this concentration should be avoided. However, lower concentrations exerted no apparent damaging effects, indicating that this lignan is safe to use for pharmacological purposes at certain concentrations.

While oncogenes promote tumorigenesis, they also induce deleterious cellular stresses, such as apoptosis, that cancer cells must combat by coopting adaptive responses. Whether tumor suppressor gene haploinsufficiency leads to such phenomena and their mechanistic basis is unclear. Here, we demonstrate that elevated levels of the anti-apoptotic factor, CASP8 and FADD-like apoptosis regulator (CFLAR), promotes apoptosis evasion in acute myeloid leukemia (AML) cells haploinsufficient for the cut-like homeobox 1 (CUX1) transcription factor, whose loss is associated with dismal clinical prognosis. Genome-wide CRISPR/Cas9 screening identifies CFLAR as a selective, acquired vulnerability in CUX1-deficient AML, which can be mimicked therapeutically using inhibitor of apoptosis (IAP) antagonists in murine and human AML cells. Mechanistically, CUX1 deficiency directly alleviates CUX1 repression of the CFLAR promoter to drive CFLAR expression and leukemia survival. These data establish how haploinsufficiency of a tumor suppressor is sufficient to induce advantageous anti-apoptosis cell survival pathways and concurrently nominate CFLAR as potential therapeutic target in these poor-prognosis leukemias.