Nontuberculous mycobacterial lung disease (NTM), including Mycobacterium avium complex (MAC), is a growing health problem in North America and worldwide. Little is known about the molecular alterations occurring in the tissue microenvironment during NTM pathogenesis. Utilizing next generation sequencing, we sequenced sputum and matched lymphocyte DNA in 15 MAC patients for a panel of 19 genes known to harbor cancer susceptibility associated mutations. Thirteen of 15 NTM subjects had a diagnosis of breast cancer (BCa) before or after NTM infection. Thirty three percent (4/12) of these NTM-BCa cases exhibited at least 3 somatic mutations in sputa compared to matched lymphocytes. Twenty four somatic mutations were detected with at least one mutation in ATM, ERBB2, BARD1, BRCA1, BRCA2, AR, TP53, PALB2, CASP8, BRIP1, NBN and TGFB1 genes. All four NTM-BCa patients harboring somatic mutations also exhibited 15 germ line BRCA1 and BRCA2 mutations. The two NTM subjects without BCa exhibited twenty somatic mutations spanning BRCA1, BRCA1, BARD1, BRIP1, CHEK2, ERBB2, TP53, ATM, PALB2, TGFB1 and 3 germ line mutations in BRCA1 and BRCA2 genes. A single copy loss of STK11 and AR gene was noted in NTM-BCa subjects. Periodic screening of sputa may aid to develop risk assessment biomarkers for neoplastic diseases in NTM patients.

OBJECTIVE

The present study aimed to compare the gene-expression profiling of CD133(+) and CD133(-) D10 cells.

METHODS

Cancer stem cell-like properties and gene-expression profiling of CD133(+) D10 cells versus CD133(-) cells were evaluated.

RESULTS

The CD133(+) D10 cells showed significantly higher clonogenic and spheroid forming potential, also higher expression of stemness genes NANOG and OCT4A compared with the CD133(-) cells. Gene-expression profiling of CD133(+) versus CD133(-) D10 cells revealed that 130 genes including ABC transporter superfamily (ABCC1, ABCG2 and ABCC6) were upregulated, while 61 genes including apoptosis modifying genes (CASP8 and TNFRSF4) were downregulated.

CONCLUSIONS

We conclude that many genes involved in drug resistance and tumor aggressiveness are upregulated in CD133(+) D10 cells and targeting them might be an efficient strategy for treatment of melanoma.

<AbstractText>Abnormal apoptosis of keratinocytes is one of the pathological changes of psoriasis. Caspases (CASPs) are the central engines of apoptosis. Studies to date have shown that some SNPs alter the expression of related genes and lead to changes in disease risk. However, no studies have investigated the associations between gene polymorphisms and the risk of psoriasis in Han population in northeast China. Therefore, we conducted a case-control study to explore this question in Han population of northeastern China.</AbstractText><AbstractText>540 patients with PsV and 612 healthy age- and sex-matched controls were enrolled in this study. We determined the genotypes of 17 single nucleotide polymorphisms (SNPs) from 11 genes of caspase family by the improved multiplex ligation detection reaction (iMLDR) method. A model-based single SNP frequentist test and haplotype association studies were performed to evaluate the association between SNPs and PsV.</AbstractText><p><div><b>Results</b></div>In the single SNP tests, rs6704688 in <i><em>CASP8</em></i> was significantly associated with psoriasis vulgaris (PsV) in Han population of northeastern China (P = 0.0169, P' = 0.0179 under the additive model; <i>P</i> = 0.0126, <i>P'</i> = 0.0149 under the heterozygous model). In haplotype analyses, the <i>CASP7</i> haplotype GC was found to be associated with PsV risk (case group versus control group, 47.2% versus 54.4%, respectively, <i>p</i> = 0.0149).</p><p><div><b>Conclusions</b></div>Our study presented that the gene polymorphisms of <i>CASP7</i> and <i><em>CASP8</em></i> were significantly associated with PsV in Han population of northeastern China, which implied the functional relationship between PsV and caspase genes. <i><em>CASP8</em></i> and <i>CASP7</i> SNPs could be new potential biomarkers for risk stratification and prevention of PsV.</p>

Neuropathic pain is absent from the early stages of periodontal disease possibly due to neurite retraction. Butyric acid (BA) is a periodontopathic metabolite that activates several stress-related signals and, likewise, induce neurite retraction. Neuronal cell death is associated to neurite retraction which would suggest that BA-induced neurite retraction is ascribable to neuronal cell death. However, the underlying mechanism of BA-related cell death signaling remains unknown. In this study, we exposed NGF-treated PC12 cells to varying BA concentrations [0 (control), 0.5, 1.0, 5.0 mM] and determined selected stress-related (H2O2, glutathione reductase, calcium (Ca(2+)), plasma membrane Ca(2+) ATPase (PMCA), and GADD153/CHOPS) and cell death-associated (extrinsic: FasL, TNF-α, TWEAK, and TRAIL; intrinsic: cytochrome C (CytC), NF-kB, CASP8, CASP9, CASP10, and CASP3) signals. Similarly, we confirmed cell death execution by chromatin condensation. Our results showed that low (0.5 mM) and high (1.0 and 5.0 mM) BA levels differ in stress and cell death signaling. Moreover, at periodontal disease-level BA concentration (5 mM), we observed that only FasL amounts were affected and occurred concurrently with chromatin condensation insinuating that cells have fully committed to neurodegeneration. Thus, we believe that both stress and cell death signaling in NGF-treated PC12 cells are affected differently depending on BA concentration. In a periodontal disease scenario, we hypothesize that during the early stages, low BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurite non-proliferation, whereas, during the later stages, high BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurodegeneration. More importantly, we propose that neuropathic pain absence at any stage of periodontal disease progression is ascribable to BA accumulation regardless of amount.

Mitochondrial diabetes (MD) is a heterogeneous disorder characterized by a chronic hyperglycemia and is maternally transmitted. Syndromic MD is a subgroup of MD including diabetic microangiopathy and macroangiopathy, in addition to extrapancreatic disorder. MD is caused by genetic mutations and deletions affecting mitochondrial DNA. This mitochondrial damage initiates apoptosis. In this study, we hypothesized that functional polymorphisms in genes involved in apoptotic pathway could be associated with the development of apoptosis in MD disease and increased its risk. Detection of apoptosis was confirmed on muscle biopsies taken from MD patients using the TUNEL method and the Cytochrome c protein expression level. We genotyped then 11 published SNPs from intrinsic and extrinsic apoptotic pathway and assessed the signification of these polymorphisms in 43 MD patients and 100 healthy controls. We found 10 selected polymorphisms (p53 (rs1042522 and rs17878362), BCL2 (rs2279115), BAX (rs1805419), BAK1 (rs210132 and rs2227925), CASP3 (rs1405937), CASP7 (rs2227310), CASP8 (rs1045485) and CASP10 (rs13006529)) with a potential apoptosis effect in MD patients compared to control population. Specifically, SNPs involved in the intrinsic pathway (p53, BCL2, BAK1 and CASP3) presented the highest risk of apoptosis. Our result proved that apoptosis initiated by mtDNA mutations, can be emphasized by a functional apoptotic polymorphisms associated with high expression of cytochrome c protein and more myofibers with apoptosis in syndromic MD subgroup compared with non-syndromic MD subgroup.

Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness, tissue remodeling, and airflow obstruction. The pathogenesis of asthma is only partly understood, and there is an urgent need for improved therapeutic strategies for this disease. The protein-protein interaction (PPI) network has considerable promise as a tool for discovery of novel asthma therapeutic targets and their relationship. Among the genes that have been identified by PPI studies, APP, CDKN1B, and SP3 displayed up-regulated expression. Further study depicted that CDKN1B localized in the nucleus or cytoplasm could interact with GRB2 and CASP8, but SP3 localized in the nucleus could interact with histone deacetylase 1, SP1, and E2F1. We anticipate that these types of analyses will provide considerable insight into asthma pathogenesis and will provide a wealth of new molecules for downstream analyses.

Allo-SCT using unrelated donors is a curative treatment for patients with hematological disorders. The best donor is one matched for 10/10 HLA alleles, however studies have shown an additional survival benefit when considering other genetic factors. It has been shown that a six-nucleotide insertion/deletion polymorphism in the CASP8 gene promoter results in reduced susceptibility of T lymphocytes to undergo apoptosis. In 186 SCT recipients, we found a significantly better OS in those who received a transplant from a WT/WT donor compared with donors with a deletion (3 years: 52 vs 34%; P=0.03; multivariate analysis; RR 0.61; 95% CI 0.38-0.98, P=0.04). This was more marked when both the patient and the donor had a deletion (3 years OS: 62% compared with 36%, P=0.01). As the majority of these patients received Alemtuzumab during conditioning, we went on to analyze the in vitro effect of the polymorphism on Alemtuzumab-induced apoptosis. We showed statistically significantly higher percentages of apoptotic naïve CD4 (P<0.0005) and CD8 (P<0.0005) T cells in WT/WT donors in comparison with donors with a deletion. These data imply an unrecognized role for the CASP8 promoter polymorphism on survival following unrelated SCT particularly in the context of T-cell depletion with Alemtuzumab.

BACKGROUND

The present study evaluates molecular markers for patients at risk of poor or no response to medulloblastoma. The aim of the study is to optimize therapy stratification.

METHODS

69 snap-frozen medulloblastoma samples were examined. C-MYC amplification was determined by fluorescence in situ hybridisation (FISH) analysis. Methylation specific PCR revealed the level of promoter methylation status of the tumor suppressor genes CASP8 (Caspase 8), TIMP3, CDH1 (E-Cadherin), CDKN2A (p16) and MGMT. Expression of GAS7 was evaluated by RT-PCR.

RESULTS

C-MYC amplification: 4/69 tumors displayed high level amplification; 53/69 tumors displayed low level (< 4 copies) or no amplification; 12/69 samples were not predictive. In patients with c-MYC amplification a tendency towards unfavorable outcome was observed (p = 0.3). Promoter methylation status: CASP8: in 36/40 tumors methylated; TIMP3: 1/38 methylated; MGMT 0/44 methylated; CDKN2A: 1/46 methylated; E-cadherin 3/36 methylated; no association between methylation status and clinical outcome. GAS7: Detection of specific RNA in 20/29 medulloblastoma samples.

CONCLUSIONS

No significant association between amplification of c-MYC and clinical outcome was observed. Promoter methylation of tumor suppressor genes is non-randomly distributed with a high level of methylation of CASP8. Recent studies show that silencing of CASP8 by methylation could be overcome by interferon gamma providing a possible therapeutic mechanism. GAS7 was shown to be a marker of mature neuronal cells with potential antitumorigenic capacity in neuronal tumors. In medulloblastoma 20/29 of the tumors examined express GAS7. Therefore a tumor suppressing function of GAS7 is improbable.

S-nitrosoglutathione (GSNO) is an endogenously produced S-nitrosylating compound that controls the function of various proteins. While a number of rodent cell lines have been used to study GSNO-induced apoptosis, the mechanisms of action remain to be evaluated in human cells and in parallel with other common apoptosis-inducing agents. In this study, we compared the pro-apoptotic effects of GSNO and staurosporine (STS) on human neural progenitors (NT2, hNP1) and neuroblasts (SH-SY5Y). We show that these cells exhibit comparable levels of susceptibility to GSNO- and STS-induced apoptotic cell death, as demonstrated by condensed nuclei and CASP3 activation. Mechanistic differences in apoptotic responses were observed as differential patterns of DNA fragmentation and levels of BAX, BCL-XL, CASP8, and p-ERK in response to GSNO and STS treatment. Mitochondrial membrane potential analysis revealed that NT2 and hNP1 cells, but not SH-SY5Y cells, undergo mitochondrial hyperpolarization in response to short-term exposure to STS prior to undergoing subsequent depolarization. This is the first study to report differences in apoptotic responses to GSNO and STS in 3 complementary human neural cell lines. Furthermore, these cells represent useful tools in cell pharmacological paradigms in which susceptibility to apoptosis-inducing agents needs to be assessed at different stages of neural cell fate commitment and differentiation.

The aim of this study was a detailed clinicopathological investigation of sinonasal NUT midline carcinoma (NMC), including analysis of DNA methylation and microRNA (miRNA) expression. Three (5%) cases of NMC were detected among 56 sinonasal carcinomas using immunohistochemical screening and confirmed by fluorescence in situ hybridization. The series comprised 2 males and 1 female, aged 46, 60, and 65 years. Two tumors arose in the nasal cavity and one in the maxillary sinus. The neoplasms were staged pT1, pT3, and pT4a (all cN0M0). All patients were treated by radical resection with adjuvant radiotherapy. Two patients died 3 and 8 months after operation, but one patient (pT1 stage; R0 resection) experienced no evidence of disease at 108 months. Microscopically, all tumors consisted of infiltrating nests of polygonal cells with vesicular nuclei, prominent nucleoli and basophilic cytoplasm. Abrupt keratinization was present in only one case. Immunohistochemically, there was a diffuse expression of cytokeratin (CK) cocktail, CK7, p40, p63, and SMARCB1/INI1. All NMCs tested negative for EBV and HPV infection. Two NMCs showed methylation of RASSF1 gene. All other genes (APC, ATM, BRCA1, BRCA2, CADM1, CASP8, CD44, CDH13, CDKN1B, CDKN2A, CDKN2B, CHFR, DAPK1, ESR1, FHIT, GSTP1, HIC1, KLLN, MLH1a, MLH1b, RARB, TIMP3, and VHL) were unmethylated. All NMCs showed upregulation of miR-9 and downregulation of miR-99a and miR-145 and two cases featured also upregulation of miR-21, miR-143, and miR-484. In summary, we described three cases of sinonasal NMCs with novel findings on DNA methylation and miRNA expression, which might be important for new therapeutic strategies in the future.

The corpus luteum (CL) of the pig lacks luteolytic sensitivity (LS) to prostaglandin (PG) F-2α until after day 12 of the oestrous cycle, but the mechanisms underlying this phenomenon are poorly understood. As luteolysis involves apoptosis, we hypothesized that critical apoptotic proteins may be deficient in CLs that lack LS. The specific aim of these studies was to examine mRNA expression and protein levels of apoptosis genes/proteins (BAX/Bax, BCLX/Bcl-x, CASP3/Caspase-3, CASP8/Caspase-8, NFΚB1/NFκB, TP53/p53) in porcine CLs collected at different stages of the oestrous cycle. CLs were collected surgically, mRNA and protein extracted, and expression/levels analyzed by semi-quantitative (SQ) PCR and Western blots, respectively. At the mRNA expression level, only BAX (maximal on day 4) and TP53 (maximal on day 7) showed significant variations during the oestrous cycle. At the protein level, only Bcl-x and Caspase-3 showed significant changes during the cycle; Bcl-x decreased on day 13 and Caspase-3 increased on day 13. It is concluded that apoptosis-associated proteins (i.e. Bcl-x and Caspase 3) may play a critical role in luteolytic sensitivity in the pig.

CASP8 rs3834129 polymorphism (-652 6N insertion/deletion) is a genetic alteration which might affect the apoptosis pathway caspase enzyme. The impaired caspase enzyme would lead to the change of cancer risk. By now, the role of CASP8 rs3834129 polymorphism has been widely investigated. However, the relationship of this genetic variant on colorectal cancer (CRC) susceptibility still remains inconsistent. Therefore, we further investigated the role of rs3834129 polymorphism on CRC risk. Eligible published studies were retrieved from EMBASE, PubMed, CNKI and WANFANG database updates to March 2018. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the relationship strengths. In general, we successfully retrieved 13 studies (8 publications) involving 13058 cases and 14418 controls. The meta-analysis results demonstrated that rs3834129 polymorphism was associated with a decreased CRC risk in heterozygous model (ID vs. II: OR = 0.94, 95% CI = 0.88-0.99), but not the homozygous and allele models. Furthermore, significantly decreased risk was also found among Asian (ID vs. II: OR = 0.86, 95% CI = 0.76-0.98), and high quality score group (ID vs. II: OR = 0.90, 95% CI = 0.81-1.00) in the stratified analyses. Taken together, we showed that CASP8 rs3834129 polymorphism influences CRC susceptibility in a weak impact manner. More case-control studies are warranted to validate such relationship.

UNASSIGNED

Epigenetic alterations may play a role in the development and behaviour of Pituitary Neuroendocrine Tumours (PitNETs).

UNASSIGNED

To evaluate the effect of methylation of Tumour Suppressor Genes (TSGs) in their gene expression and in the behaviour of PitNETs.

UNASSIGNED

We use MS-MLPA and q-RT-PCR techniques to analyse the DNA-promoter hypermethylation and the gene expression of 35 TSGs in 105 PitNETs. We define functionality, size and invasiveness of tumours according to their clinical manifestations, Hardy's classification and MRI invasion of the cavernous sinus, respectively.

UNASSIGNED

We observed different methylation patterns among PitNET subtypes. CASP8 was the most hypermethylated gene in all PitNET subtypes. The methylation status of TP73 correlated negatively with their gene expression in the overall series (p=0.013) and in some subtypes. MSH6 and CADM1 showed higher methylation frequency in macro than in microadenomas in the overall series and in corticotroph PitNETs (all p≤0.053). ESR1 and RASSF1 were higher methylated in non-invasive than in invasive tumours in the overall series (p=0.054 and p=0.031, respectively) and in the gonadotroph subtype (sGT) (p=0.055 and p=0.050, respectively). ESR1 and CASP8 appeared more hypermethylated in functioning than in sCT (p=0.034 and p=0.034, respectively).

UNASSIGNED

DNA-methylation of TSGs has a selective effect on their gene expression and on the growth and invasiveness of PitNETs. Its involvement in their functionality is biased because all silent operated tumours are macroadenomas while all operated microadenomas are functioning ones. Therefore, the subtypes of PitNETs should be considered as different entities.

Ammonia is one of the major pollutants associated with the main river basins due to ammonification of uneaten food and animal excretion, which usually brings detrimental health effects to aquatic invertebrate. However, the mechanisms of ammonia toxicity in aquatic invertebrate have rarely been reported. In this study, C. fluminea was exposed to different levels of ammonia (control group, 10 mg/L, and 25 mg/L) for 24 h and 48 h, and digestive gland and gill were collected to explore toxic effects on oxidative stress, DNA damage and apoptosis under ammonia stress. The results showed that ammonia poisoning could increase the activity of oxidative stress enzyme (SOD and CAT), inducing differentially expressed genes (DRAM2, GADD45, P53, BAX, BCL2, CASP8, CASP9, CASP3, HSP70 and HSP90) and different cytokines (IL-1 beta, IL-8, IL-17 and TNF-alpha) of DNA damage and apoptosis. The difference of toxic effects induced by ammonia among digestive gland and gill were also observed by real-time PCR and TUNEL staining. Our results will be helpful to understand the mechanism of aquatic toxicology induced by ammonia in C. fluminea.

The objective of this research was to assess the association of genetic polymorphisms related to intrinsic apoptosis pathway CASP8 rs3834129 and CASP3 rs4647601 with the risk, clinical and pathological aspects, and survival of oropharynx squamous cell carcinoma (OPSCC) patients that received cisplatin and radiotherapy. The genotypes were identified in 198 patients with OPSCC and 200 controls using polymerase chain reaction methods. Chi square or Fisher's exact test and logistic regression were applied for the detection of differences between groups. Patients' genotypes were statistically evaluated considering the event-free survival and overall analysis using Kaplan-Meier estimate and Cox regression. CASP3 rs4647601 GG genotype (44.4% vs. 30.0%, p = 0.03) and G allele (63.9% vs. 55.5%, p = 0.04) were more common in patients with OPSCC than in controls. Carriers of GG genotype and G allele were under 1.78-fold and 1.40-fold increased risk of OPSCC than others, respectively. The frequency of CASP8 rs3834129 DD genotype was higher in patients with OPSCC with poorly differentiated or undifferentiated tumors when compared to others (34.5% vs. 16.1%, p = 0.02). No influence of CASP8 and CASP3 polymorphisms on OPSCC patients' survival was seen in this study. Our results indicate that inherited genetic variants in the intrinsic apoptosis pathway related to CASP3 rs4647601 and CASP8 rs3834129 polymorphisms may be an important determinant of OPSCC risk and tumor cell differentiation.

Hypoxia-induced cardiomyocyte apoptosis is one of the leading causes of heart failure. Nuclear respiratory factor 1 (NRF-1) was suggested as a protector against cell apoptosis. however, the mechanism is not clear. Therefore, the aim of this study was to elucidate the role of NRF-1 in hypoxia-induced H9C2 cardiomyocyte apoptosis, and to explore its effect on regulating the death receptor pathway and mitochondrial pathways. NRF-1 was overexpressed or knocked-down in H9C2 cells, which were then exposed to a hypoxia condition for 0, 3, 6, 12, and 24 h. Changes in cell proliferation, cell viability, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) were investigated. The activities of caspase-3, -8, and -9, apoptosis rate, and the gene and protein expression levels of the death receptor pathway and mitochondrial pathway were analyzed. Under hypoxia exposure, NRF-1 overexpression improved the proliferation and viability of H9C2 cells, and decreased ROS generation, MMP loss, caspase activities, and the apoptosis rate. However, the NRF-1 knockdown group showed the opposite results. Additionally, NRF-1 upregulated the expression of anti-apoptotic molecules involved in the death receptor and mitochondrial pathways, such as CASP8 and FADD-like apoptosis regulator, B-cell lymphoma 2, B-cell lymphoma-extra-large, and cytochrome-C. Conversely, the expression of pro-apoptotic molecules such as caspase-8, BH3-interacting domain death agonist, Bcl-2-associated X protein, caspase-9, and caspase-3 was down-regulated by NRF-1 overexpression in hypoxia-induced H9C2 cells. These results suggest that NRF-1 functions as an anti-apoptotic factor in the death receptor and mitochondrial pathways to mitigate hypoxia-induced apoptosis in H9C2 cardiomyocytes. This article is protected by copyright. All rights reserved.

Keywords: H9C2 cardiomyocytes; apoptosis pathway; cell viability; hypoxia; nuclear respiratory factor 1; reactive oxygen species.

Moringin [4-(α-L-rhamnosyloxy) benzyl isothiocyanate] is an isothiocyanate extracted from Moringa oleifera seeds. It is an antioxidant known for several biological properties useful in the treatment of neurodegenerative diseases. Several neurodegenerative disorders such as Parkinson's and Alzheimer's diseases are linked to dysfunctional mitochondria due to the resulting increase of Reactive Oxygen Species (ROS). Stem cell-based therapeutic treatments in neurodegenerative diseases provide an alternative strategy aimed to replace the impaired tissue. In this study were investigated the deregulated genes involved in mitophagy in the human periodontal ligament stem cells pretreated with moringin. The RNA-seq study reveals the downregulation of PINK1, with a fold change (FC) of -0.56, such as the genes involved in the phagophore formation (MAP1LC3B FC: -0.73, GABARAP FC: -0.52, GABARAPL1 FC: -0.70, GABARAPL2 FC: -0.39). The moringin pretreatment downregulates the pro-apoptotic gene BAX (-0.66) and upregulates the anti-apoptotic genes BCL2L12 (FC: 1.35) and MCL1 (FC: 0.36). The downregulation of the most of the caspases (CASP1 FC: -1.43, CASP4 FC: -0.18, CASP6 FC: -1.34, CASP7 FC: -0.46, CASP8 FC: -0.65) implies the inactivation of the apoptotic process. Our results suggest that mitochondrial dysfunctions induced by oxidative stress can be inhibited by moringin pretreatment in human periodontal ligament stem cells (hPDLSCs).

The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G2/M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G2/M cell cycle regulators, ABT-751 induced G2/M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria.

Erythropoiesis has been extensively studied using in vitro and in vivo animal models. Despite this, there is still limited data about the gene expression profiles (GEP) of primary (ex vivo) normal human bone marrow (BM) erythroid maturation. We investigated the GEP of nucleated red blood cell (NRBC) precursors during normal human BM erythropoiesis. Three maturation-associated populations of NRBC were identified and purified from (fresh) normal human BM by flow cytometry and the GEP of each purified cell population directly analyzed using DNA-oligonucleotide microarrays. Overall, 6569 genes (19% of the genes investigated) were expressed in ≥1 stage of BM erythropoiesis at stable (e.g., genes involved in DNA process, cell signaling, protein organization and hemoglobin production) or variable amounts (e.g., genes related to cell differentiation, apoptosis, metabolism), the latter showing a tendency to either decrease from stage 1 to 3 (genes associated with regulation of erythroid differentiation and survival, e.g., SPI1, STAT5A) or increase from stage 2 to stage 3 (genes associated with autophagy, erythroid functions such as heme production, e.g., ALAS1, ALAS2), iron metabolism (e.g., ISCA1, SLC11A2), protection from oxidative stress (e.g., UCP2, PARK7), and NRBC enucleation (e.g., ID2, RB1). Interestingly, genes involved in apoptosis (e.g., CASP8, P2RX1) and immune response (e.g., FOXO3, TRAF6) were also upregulated in the last stage (stage 3) of maturation of NRBC precursors. Our results confirm and extend on previous observations and providing a frame of reference for better understanding the critical steps of human erythroid maturation and its potential alteration in patients with different clonal and non-clonal erythropoietic disorders.

Death Receptor 5 (DR5) agonists demonstrate anti-tumor activity in preclinical models but have yet to demonstrate robust clinical responses. A key limitation may be the lack of patient selection strategies to identify those most likely to respond to treatment. To overcome this limitation, we screened a DR5 agonist Nanobody across >600 cell lines representing 21 tumor lineages and assessed molecular features associated with response. High expression of DR5 and Casp8 were significantly associated with sensitivity, but their expression thresholds were difficult to translate due to low dynamic ranges. To address the translational challenge of establishing thresholds of gene expression, we developed a classifier based on ratios of genes that predicted response across lineages. The ratio classifier outperformed the DR5+Casp8 classifier, as well as standard approaches for feature selection and classification using genes, instead of ratios. This classifier was independently validated using 11 primary patient-derived pancreatic xenograft models showing perfect predictions as well as a striking linearity between prediction probability and anti-tumor response. A network analysis of the genes in the ratio classifier captured important biological relationships mediating drug response, specifically identifying key positive and negative regulators of DR5 mediated apoptosis, including DR5, CASP8, BID, cFLIP, XIAP and PEA15. Importantly, the ratio classifier shows translatability across gene expression platforms (from Affymetrix microarrays to RNA-seq) and across model systems (in vitro to in vivo). Our approach of using gene expression ratios presents a robust and novel method for constructing translatable biomarkers of compound response, which can also probe the underlying biology of treatment response.

Opposing activities of Notch and Wnt signaling regulate mucosal barrier homeostasis and differentiation of intestinal epithelial cells. Specifically, Wnt activity is essential for differentiation of secretory cells including Wnt3-producing Paneth cells, whereas Notch signaling strongly promotes generation of absorptive cells. Loss of caspase-8 in intestinal epithelium (casp8∆int) is associated with fulminant epithelial necroptosis, severe Paneth cell death, secondary intestinal inflammation, and an increase in Notch activity. Here, we found that pharmacological Notch inhibition with dibenzazepine (DBZ) is able to essentially rescue the loss of Paneth cells, deescalate the inflammatory phenotype, and reduce intestinal permeability in casp8∆int mice. The secretory cell metaplasia in DBZ-treated casp8∆int animals is proliferative, indicating for Notch activities partially insensitive to gamma-secretase inhibition in a casp8∆int background. Our data suggest that casp8 acts in the intestinal Notch network.

Achilles tendon pathology (ATP) is a degenerative condition which exhibits excessive tenocyte apoptosis. Tumour necrosis factor receptor 1 (TNFR1), caspase-3 (CASP3) and caspase-8 (CASP8) are important regulators of apoptosis. To date, the effects of variation within the genes for TNFR1 and CASP3 as risk factors for ATP have not been described. There is evidence that two single nucleotide polymorphisms (SNPs) within the CASP8 gene are associated with ATP, but only in populations from the Southern Hemisphere. The primary aim of this study was to determine whether SNPs within the TNFRSF1A and CASP3 genes were associated with ATP in British Caucasians. We additionally sought to determine whether copy number variation (CNV) within the CASP8 gene was associated with ATP. We recruited 262 (131 ATP cases and 131 asymptomatic controls) Caucasian participants for this genetic association study and used quantitative PCR with chi-squared (χ(2)) tests and ANOVA to detect significant associations. For our entire cohort, we found no association between the TNFRSF1A rs4149577 (p=0.561), CASP3 rs1049253 (p=0.643) and CASP8 variants (p=0.219) and ATP. Likewise, when we tested potential interactions between gender, genotype and the risk of ATP, we found no association with the variants investigated. In conclusion, the TNFRSF1A, CASP3 and CASP8 gene variants were not associated with ATP in British Caucasians.

Background: RFA is a well-established treatment for symptomatic patients with AF. However, the success rate of a single procedure is low. We aimed to investigate the association between the risk of recurrence of atrial fibrillation (AF) after a single radiofrequency ablation (RFA) procedure and cardiac neurohormonal function, left atrial (LA) mechanical function as well as proteins related to inflammation, fibrosis, and apoptosis. Methods and Results: We studied 189 patients undergoing RFA between January 2012 and April 2014, with a follow-up period of 12 months. A logistic regression analysis was performed to investigate the association between pre-ablation LA emptying fraction (LAEF), MR-proANP, Caspase-8 (CASP8), Neurotrophin-3 (NT3), and the risk for recurrence of AF after a single RFA procedure. 119 (63.0%) patients had a recurrence during a mean follow-up of 402 ± 73 days. An increased risk of recurrence was associated with: Elevated MR-proANP (fourth quartile vs. first quartile: HR, 2.80 (95% CI, 1.14-6.90]; P = 0.025); Low LAEF (fourth quartile vs. first quartile: hazard ratio [HR], 2.41 [95% CI, 1.01-5.79]; P = 0.045); Elevated CASP8 (fourth quartile vs. first quartile: HR 12.198 95% CI 2.216-67.129; P = 0.004); Elevated NT-3 (fourth quartile vs. first quartile: HR 7.485 95% CI 1.353-41.402; P = 0.021). In a receiver operating characteristic curve analysis, the combination of MR-proANP, CASP8, and NT3 produced an area under the curve of 0.819; CI 95% (0.710-0.928). Conclusions: Patients with better LA mechanical function and lower levels of atrial neurohormones as well as of proteins related to fibrosis and apoptosis, have a better outcome after an RFA procedure. Unique identifier: No. NCT01553045 (https://clinicaltrials.gov/ct2/show/NCT01553045?term=NCT01553045&rank=1).

Due to the limitations in the range of antibodies recognising avian viruses, quantitative real-time PCR (RT-qPCR) is still the most widely used method to evaluate the expression of immunologically related genes in avian viruses. The objective of this study was to identify suitable reference genes for mRNA expression analysis in chicken intraepithelial lymphocyte natural killer (IEL-NK) cells after infection with very-virulent infectious bursal disease virus (vvIBDV). Fifteen potential reference genes were selected based on the references available. The coefficient of variation percentage (CV%) and average count of these 15 genes were determined by NanoString technology for control and infected samples. The M and V values for shortlisted reference genes (ACTB, GAPDH, HMBS, HPRT1, SDHA, TUBB1 and YWHAZ) were calculated using geNorm and NormFinder. GAPDH, YWHAZ and HMBS were the most stably expressed genes. The expression levels of three innate immune response related target genes, CASP8, IL22 and TLR3, agreed in the NanoString and RNA sequencing (RNA-Seq) results using one or two reference genes for normalisation (not HMBS). In conclusion, GAPDH and YWHAZ could be used as reference genes for the normalisation of chicken IEL-NK cell gene responses to infection with vvIBDV.