BACKGROUND

The FAS/FASLG system has been proposed to play a key role in germ cell apoptosis. To elucidate the role of the genetic variants of cell death pathway genes in male infertility, we genotyped the polymorphisms of FAS, FASLG and caspase-8 (CASP8) genes and evaluated their effects on sperm apoptosis and semen quality in infertile men.

METHODS

The genotypes of FAS, FASLG and CASP8 were determined in 620 infertile men. Sperm apoptosis rates were measured by terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end labelling (TUNEL) assay, and the semen quality analysis was performed using computer assisted sperm analysis.

RESULTS

We found that polymorphisms of FAS-670A/G (rs1800682: A>G) and CASP8-652 6N ins/del (rs3834129: -/CTTACT) were associated with sperm apoptosis and semen quality. Individuals with FAS-670GG showed low apoptosis rate and decreased sperm concentration, compared with the FAS-670AA genotype. Similarly, in comparison with the CASP8-652 6N ins/ins genotype, the CASP8-652 6N (ins/del+del/del) genotypes also showed significantly decreased sperm apoptosis rate and poor sperm motility. Other polymorphisms investigated did not appear to affect sperm apoptosis and semen quality.

CONCLUSIONS

This study showed for the first time that functional variants of FAS and CASP8 might contribute to the dysfunctional apoptotic mechanism in germ cells, which could result in poor semen quality of ejaculated sperm.

The multikinase inhibitor sunitinib (S) seems to have promising potential in the treatment of thyroid cancer. We focused on the impact of S and/or irradiation (R) on mechanisms of apoptosis in follicular thyroid cancer cells. The effects of R, S and their combination were evaluated 2 and 4 days after treatment, using the human thyroid cancer cell line CGTH W-1. The transcription of genes involved in the regulation of apoptosis was investigated using quantitative real-time PCR. Western blot analyses of caspases and survivin were also performed. S elevated BAX (day 4), CASP9, CASP3, BIRC5 (day 4) and PRKACA (day 4) gene expression, whereas the mRNAs of BCL2, CASP8, PRKCA, ERK1, and ERK2 were not significantly changed. S, R and R+S clearly induced caspase-9 protein and elevated caspase-3 activity. Survivin was down-regulated at day 4 in control cells and the expression was blunted by S treatment. R+S induced survivin expression at day 2 followed by a reduction at day 4 of treatment. Sunitinib and the combined application with radiation induced apoptosis in follicular thyroid cancer cells via the intrinsic pathway of apoptosis. In addition, sunitinib might induce apoptosis via decreased expression of the anti-apoptotic protein survivin. These findings suggest the potential use of sunitinib for the treatment of poorly differentiated follicular thyroid carcinomas.

Ewing sarcoma is the second most common bone cancer in children and adolescents, with poor prognosis and outcome in ~70% of initial diagnoses and 10-15% of relapses. Hydrophobic triterpene acids and hydrophilic lectins and viscotoxins from European mistletoe (Viscum album L.) demonstrate anticancer properties, but have not yet been investigated for Ewing sarcoma. Commercial Viscum album L. extracts are aqueous, excluding the insoluble triterpenes. We recreated a total mistletoe effect by combining an aqueous extract (viscum) and a triterpene extract (TT) solubilized with cyclodextrins. Ewing sarcoma cells were treated with viscum, TT and viscumTT in vitro, ex vivo and in vivo. In vitro and ex vivo treatment of Ewing sarcoma cells with viscum inhibited proliferation and induced apoptosis in a dose-dependent fashion, while viscumTT combination treatment generated a synergistic effect. Apoptosis occurred via intrinsic and extrinsic apoptotic pathways, evidenced by activation of both CASP8 and CASP9. We show that viscumTT treatment shifts the balance of apoptotic regulatory proteins towards apoptosis, mainly via CLSPN, MCL1, BIRC5 and XIAP downregulation. ViscumTT also demonstrated strong antitumor activity in a cell line- and patient-derived mouse model, and may be considered an adjuvant therapy option for pediatric patients with Ewing sarcoma.

In vivo electroporation of plasmid DNA (DNA-EP) is an efficient and safe method for vaccines. It results in increased DNA uptake, enhances protein expression, and augments immune responses to the target antigen in a variety of species. To further improve the efficacy of DNA-EP, we evaluated small interfering RNA (siRNA) sequences targeting apoptotic genes as an adjuvant to cancer vaccine. Bak1 or Casp8 siRNA was coadministered with plasmid DNA encoding the extracellular and transmembrane domains of rat HER2 ECD.TM to BALB-neuT mice, which spontaneously develop HER2/neu-positive mammary tumors. The combination regimen significantly reduced spontaneous tumor progression in BALB-neuT mice, in an advanced disease setting, when compared with DNA-EP alone. The antitumor effect was associated with a noteworthy antibody isotype switch from IgG1 to IgG2a, when siRNA was coadministered with DNA-EP. CD8+ T cell responses increased significantly, as did the number of responders to vaccination. Coimmunization of siRNA and DNA-EP at the same physical location was essential for the enhanced therapeutic effect. Silencing of the targeted genes was confirmed by in vitro Western blots. siRNA sequences targeting apoptotic genes Bax and Fas did not improve tumor protection in this mouse model when compared with DNA-EP alone. These data demonstrate that some siRNA sequences can act in concert with DNA-EP to control HER2/neu-positive mammary carcinoma. These observations emphasize the potential of siRNA as adjuvant for therapeutic DNA vaccines.

Fenretinide is significantly more effective in inducing apoptosis in cancer cells than all-trans retinoic acid (ATRA). The current study uses a genome-wide approach to understand the differential role fenretinide and ATRA have in inducing apoptosis in Huh7 cells. Fenretinide and ATRA-induced gene expressions and DNA bindings were profiled using microarray and chromatin immunoprecipitation with anti-RXRα antibody. The data showed that fenretinide was not a strong transcription regulator. Fenretinide only changed the expressions of 1 093 genes, approximately three times less than the number of genes regulated by ATRA (2 811). Biological function annotation demonstrated that both fenretinide and ATRA participated in pathways that determine cell fate and metabolic processes. However, fenretinide specifically induced Fas/TNFα-mediated apoptosis by increasing the expression of pro-apoptotic genes i.e., DEDD2, CASP8, CASP4, and HSPA1A/B; whereas, ATRA induced the expression of BIRC3 and TNFAIP3, which inhibit apoptosis by interacting with TRAF2. In addition, fenretinide inhibited the expression of the genes involved in RAS/RAF/ERK-mediated survival pathway. In contrast, ATRA increased the expression of SOSC2, BRAF, MEK, and ERK genes. Most genes regulated by fenretinide and ATRA were bound by RXRα, suggesting a direct effect. This study revealed that by regulating fewer genes, the effects of fenretinide become more specific and thus has fewer side effects than ATRA. The data also suggested that fenretinide induces apoptosis via death receptor effector and by inhibiting the RAS/RAF/ERK pathway. It provides insight on how retinoid efficacy can be improved and how side effects in cancer therapy can be reduced.

BACKGROUND

Because of the dramatically different clinical course of aggressive and indolent prostate carcinoma (PCa), markers that distinguish between these phenotypes are of critical importance. Apoptosis is an important protective mechanism for unrestrained cellular growth and metastasis. Therefore, dysfunction in this pathway is a key step in cancer progression. As such, genetic variants in apoptosis genes are potential markers of aggressive PCa. Recent work in breast carcinoma has implicated the histidine variant of CASP8 D302H (rs1045485) as a protective risk allele.

METHODS

We tested the hypothesis that the H variant was protective for aggressive PCa in a pooled analysis of 796 aggressive cases and 2,060 controls.

RESULTS

The H allele was associated with a reduced risk of aggressive PCa (OR(per allele) = 0.67, 95% CI: 0.54-0.83, P(trend) = 0.0003). The results were similar for European-Americans (OR(per allele) = 0.68; 95% CI: 0.54-0.86) and African-Americans (OR(per allele) = 0.61; 95% CI: 0.34-1.10). We further determined from the full series of 1,160 cases and 1,166 controls in the Prostate, Lung, Colorectal, Ovarian (PLCO) population that the protective effect of the H allele tended to be limited to high-grade and advanced PCa (all cases OR(per allele) = 0.94; 95% CI: 0.79-1.11; localized, low-grade disease OR(per allele) = 0.98; 95% CI: 0.79-1.23; and aggressive disease OR(per allele) = 0.73; 95% CI: 0.50-1.07).

CONCLUSIONS

These results suggest that histidine variant of CASP8 D302H is a protective allele for aggressive PCa with potential utility for identification of patients at differential risk for this clinically significant phenotype.

OBJECTIVE

Locally advanced oral squamous cell carcinoma (OSCC) shows lower locoregional control and disease specific survival rates than laryngeal and pharyngeal squamous cell carcinoma (L/P-SCC) after definitive chemoradiotherapy treatment. Despite clinical factors, this can point towards a different tumor biology that could impact chemoradiotherapy response rates. This prompted us to compare the mutational profiles of OSCC with L/P-SCC.

METHODS

We performed target capture DNA sequencing on 111 HPV-negative HNSCC samples (NKI dataset), 55 oral and 56 laryngeal/pharyngeal, and identified somatic point mutations and copy number aberrations. We next expanded our analysis with 276 OSCC and 134 L/P-SCC sample data from The Cancer Genome Atlas (TCGA dataset). We focused our analyses on genes that are frequently mutated in HNSCC.

RESULTS

The mutational profiles of OSCC and L/P-SCC showed many similarities. However, OSCC was significantly enriched for CASP8 (NKI: 15% vs 0%; TCGA: 17% vs 2%) and HRAS (TCGA: 10% vs 1%) mutations. LAMA2 (TCGA: 5% vs 19%) and NSD1 (TCGA: 7% vs 25%) mutations were enriched in L/P-SCC. Overall, we find that OSCC had fewer somatic point mutations and copy number aberrations than L/P-SCC. Interestingly, L/P-SCC scored higher in mutational and genomic scar signatures associated with homologous recombination DNA repair defects.

CONCLUSIONS

Despite showing a similar mutational profile, our comparative genomic analysis revealed distinctive features in OSCC and L/P-SCC. Some of these genes and cellular processes are likely to affect the cellular response to radiation or cisplatin. Genomic characterizations may guide or enable personalized treatment in the future.

The aim of this study was to analyse various gene expression profiles of muscle tissue during normoxia, ischaemia and after reperfusion in human muscle free flaps, to gain an understanding of the occurring regulatory, inflammatory and apoptotic processes on a cellular and molecular basis. Eleven Caucasian patients with soft tissue defects needing coverage with microsurgical free muscle flaps were included in this study. In all patients, the muscle samples were taken from free myocutaneous flaps. The first sample was taken before induction of ischaemia in normoxia (I), another one after ischaemia (II), and the last one was taken after reperfusion (III). The samples were analysed using DNA-microarray, real-time-quantitative-PCR and immunohistochemistry. DNA-microarray analysis detected multiple, differentially regulated genes when comparing the different groups (I-III) with statistical significance. Comparing ischaemia (II) versus normoxia (I) educed 13 genes and comparing reperfusion (III) versus ischaemia (II) educed 19 genes. The comparison of reperfusion (III) versus normoxia (I) yielded 100 differentially regulated genes. Real-time-quantitative-PCR confirmed the results of the DNA-microarrays for a subset of four genes (CASP8, IL8, PLAUR and S100A8). This study shows that ischaemia and reperfusion induces alterations on the gene expression level in human muscle free flaps. Data may suggest that the four genes CASP8, IL8, PLAUR and S100A8 are of great importance in this context. We could not confirm the DNA-microarry and real-time-quantitative-PCR results on the protein level. Finally, these findings correspond with the surgeon's clinical experience that the accepted times of ischaemia, generally up to 90 min., are not sufficient to induce pathophysiological processes, which can ultimately lead to flap loss. When inflammatory and apoptotic proteins are expressed at high levels, flap damage might occur and flap loss is likely. The sole expression on mRNA level might explain why flap loss is unlikely.

BACKGROUND

In recurrent or metastatic (R/M) skin squamous cell cancer (sSCC) not amenable to radiotherapy (RT) or surgery, chemotherapy (CT) has a palliative intent and limited clinical responses. The role of oral pan-HER inhibitor dacomitinib in this setting was investigated within a clinical trial.

METHODS

Patients with diagnosis of R/M sSCC were treated. Dacomitinib was started at a dose of 30 mg daily (QD) for 15 d, followed by 45 mg QD. Primary end-point was response rate (RR). Tumour samples were analysed through next-generation sequencing using a custom panel targeting 36 genes associated with sSCC.

RESULTS

Forty-two patients (33 men; median age 77 years) were treated. Most (86%) received previous treatments consisting in surgery (86%), RT (50%) and CT (14%). RR was 28% (2% complete response; 26% partial response), disease control rate was 86%. Median progression-free survival and overall survival were 6 and 11 months, respectively. Most patients (93%) experienced at least one adverse event (AE): diarrhoea, skin rash (71% each), fatigue (36%) and mucositis (31%); AEs grade 3-4 occurred in 36% of pts. In 16% of cases, treatment was discontinued because of drug-related toxicity. TP53, NOTCH1/2, KMT2C/D, FAT1 and HER4 were the most frequently mutated genes. BRAF, NRAS and HRAS mutations were more frequent in non-responders, and KMT2C and CASP8 mutations were restricted to this subgroup.

CONCLUSIONS

In sSCC, dacomitinib showed activity similar to what was observed with anti-epidermal growth factor receptor agents, and durable clinical benefit was observed. Safety profile was comparable to previous experiences in other cancers. Molecular pt selection could improve therapeutic ratio.

Pregnancy and early life are critical periods during which environmental factors such as nutrition can affect development. Rumen-protected methionine (Met; RPM) supplementation during the prepartum period improves not only performance but immune responses in dairy cows. We investigated the effects of enhanced maternal supply of Met via feeding RPM on whole-blood in vitro lipopolysaccharide (LPS; 0, 0.01, or 5 μg/mL of blood) challenge and targeted microRNA and mRNA abundance in calf blood polymorphonuclear leukocytes (PMNL). Calves (n = 12/maternal diet) born to cows fed RPM at 0.08% of diet dry matter (DM)/d (MET) for the last 21 ± 2 d before calving or fed a control diet with no added Met (CON) were used. The PMNL were isolated at birth (before colostrum feeding) and d 1 (24 h after colostrum intake), 14, 28, and 50 of age. Maternal blood was collected at -10 ± 1.3 d relative to calving. Cows in the MET group had greater DM intake and lower prepartal haptoglobin concentration. In CON cows, haptoglobin was positively correlated with proinflammatory and host-defense mRNA abundance in CON calves. Except for NOS2 and NFE2L2, abundance of CASP8, MPO, ZBP1, and TNF was lower at birth in MET calves. Interleukin 1β concentration in response to LPS challenge in CON and MET calves was greatest at birth, underscoring the role of this cytokine for lymphocyte activation. Compared with 1 d of age, the interleukin-1β response to incremental doses of LPS was greater at 14 through 28 d, suggesting that the neonatal calf can mount a robust response to inflammatory stimuli. Greater abundance in CON calves of NOS2, CADM1, and TLR2 coupled with lower SELL from 1 through 50 d of age suggested a chronic activation of the PMNL. There was a marked upregulation over time of MIR125b, MIR146a, MIR155, and MIR9 in both CON and MET calves, suggesting that these microRNA could affect gene transcription associated with differentiation and inflammatory function in PMNL. Regardless of maternal diet, the gradual downregulation of MIR223 (the most abundant microRNA in PMNL) is in line with the progressive increase over time in the proinflammatory signature of the PMNL. Data revealed the potential for maternal supply of Met during late pregnancy through either greater DM intake or Met to elicit some changes in PMNL function during early postnatal life, partly through changes in mRNA expression encompassing cell adhesion and chemotaxis, oxidative stress, Toll-like receptor signaling, and Met metabolism.

Although induction of host cell death is a pivotal step during bacteria-induced gastroenteritis, the molecular regulation remains to be fully characterized. To expand our knowledge, we investigated the role of the central cell death regulator Caspase-8 in response to Salmonella Typhimurium. Here, we uncovered that intestinal salmonellosis was associated with strong upregulation of members of the host cell death machinery in intestinal epithelial cells (IECs) as an early event, suggesting that elimination of infected IECs represents a host defense strategy. Indeed, Casp8∆IEC mice displayed severe tissue damage and high lethality after infection. Additional deletion of Ripk3 or Mlkl rescued epithelial cell death and lethality of Casp8∆IEC mice, demonstrating the crucial role of Caspase-8 as a negative regulator of necroptosis. While Casp8∆IECTnfr1-/- mice showed improved survival after infection, tissue destruction was similar to Casp8∆IEC mice, indicating that necroptosis partially depends on TNF-α signaling. Although there was no impairment in antimicrobial peptide secretion during the early phase of infection, functional Caspase-8 seems to be required to control pathogen colonization. Collectively, these results demonstrate that Caspase-8 is essential to prevent Salmonella Typhimurium induced enteritis and to ensure host survival by two different mechanisms: maintenance of intestinal barrier function and restriction of pathogen colonization.

BACKGROUND

Acute myeloid leukemia (AML) is the most common type of blood cancer in adults. Emerging evidence is establishing a connection between AML and aberrant alternative splicing of pre-mRNA, which may result from aberrant expression of splicing factors, the mediators of splicing reactions.

METHODS

Using quantitative real-time polymerase chain reaction, we measured mRNA expression of 7 splicing factors belonging to the serine/arginine-rich (SR) protein family, SRSF1 (SF2/ASF), SRSF2 (SC35), SRSF3 (SRp20), SRSF4 (SRp75), SRSF5 (SRp40), SRSF6 (SRp55), and SRSF7 (9G8), and 1 non-SR factor, heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), in peripheral blood mononuclear cells of 26 patients with newly diagnosed AML and 26 healthy controls. In addition, the relationship between splicing factors and the mRNA splicing patterns of the caspase-8 gene (CASP8) was investigated.

RESULTS

Compared to healthy controls, the expression of splicing factors was obviously aberrant in newly diagnosed AML patients. The expression of SRSF1, SRSF3 and SRSF4 mRNAs was significantly decreased. Moreover, a significant correlation was observed between several splicing factors and caspase-8 pre-mRNA splicing in AML patients, but not in control subjects.

CONCLUSIONS

These data suggest that aberrant expression of splicing factors in AML may potentially connect with abnormal expression of oncogenes and be useful for early diagnosis, prognosis, and therapy of AML.

One of the major challenges with protein template-free modeling is an efficient sampling algorithm that can explore a huge conformation space quickly. The popular fragment assembly method constructs a conformation by stringing together short fragments extracted from the Protein Data Base (PDB). The discrete nature of this method may limit generated conformations to a subspace in which the native fold does not belong. Another worry is that a protein with really new fold may contain some fragments not in the PDB. This article presents a probabilistic model of protein conformational space to overcome the above two limitations. This probabilistic model employs directional statistics to model the distribution of backbone angles and 2(nd)-order Conditional Random Fields (CRFs) to describe sequence-angle relationship. Using this probabilistic model, we can sample protein conformations in a continuous space, as opposed to the widely used fragment assembly and lattice model methods that work in a discrete space. We show that when coupled with a simple energy function, this probabilistic method compares favorably with the fragment assembly method in the blind CASP8 evaluation, especially on alpha or small beta proteins. To our knowledge, this is the first probabilistic method that can search conformations in a continuous space and achieves favorable performance. Our method also generated three-dimensional (3D) models better than template-based methods for a couple of CASP8 hard targets. The method described in this article can also be applied to protein loop modeling, model refinement, and even RNA tertiary structure prediction.

Protein Tyrosine Kinase 6 (PTK6) is a non-receptor-type tyrosine kinase known to be expressed in various cancers, including pancreatic cancer. The role of PTK6 in cancer chemoresistance remains unclear. Therefore, it was hypothesized that PTK6 mechanistically regulates gemcitabine resistance in pancreatic cancer. Gemcitabine treatment stimulated endogenous PTK6 overexpression in MIAPaCa2 and Panc1 cells. PTK6 gene silencing increased cell survival after gemcitabine treatment and decreased apoptosis, whereas PTK6 overexpression decreased cell survival and increased apoptosis. Selection for gemcitabine resistance revealed substantially lower PTK6 expression in the gemcitabine-resistant subclones compared with the parental lines, while restoring PTK6 rescued gemcitabine sensitivity. Gemcitabine induced phosphorylation of H2AX (γ-H2AX) and ataxia-telangiectasia mutated kinase (pATM), specific markers for DNA double-strand breaks. Both gemcitabine-induced phosphorylation of H2AX and ATM were reduced by PTK6 knockdown and increased by PTK6 overexpression. PTK6 overexpression also increased the S-phase fraction 48 hours after gemcitabine treatment. Although gemcitabine activated both caspase-8 (CASP8) and caspase-9 (CASP9), the effect of PTK6 on gemcitabine-induced apoptosis required CASP8 but not CASP9. In mouse xenografts, PTK6 overexpression in subcutaneous tumors attenuated tumor growth after gemcitabine treatment. In conclusion, PTK6 prolongs S-phase and increases the ability of gemcitabine to cause DNA damage in vitro and in vivo.

CONCLUSIONS

PTK6 affects cell cycle and DNA damage, thus making it an important therapeutic target to improve the outcomes of patients with pancreatic cancer.

To gain insight into the molecular pathogenesis of patients with mantle cell lymphoma (MCL), next-generation whole-exome sequencing of 16 MCL patients was performed. We identified recurrent mutations in genes that are well known to be functionally relevant in MCL, including ATM (37.5%), TP53 (31.3%), WHSC1 (31.3%), CCND1 (18.8%), NOTCH2 (6.3%), and CDKN2A (6.3%). We also identified somatic mutations in genes for which a functional role in MCL has not been previously suspected. These genes included CCDC15, APC, CDH1, S1PR1, ATRX, BRCA2, CASP8, and NOTCH3. Further, we investigated the prognostic factors associated with MCL from clinical, pathological, and genetic mutations. Mutations of TP53 (P = 0.021) was a significant prognostic factor with shorter overall survival (OS). Although there was no statistical difference, the median survival time of patients with WHSC1 mutations was shorter than those without mutations (P = 0.070). Mutations in ATM and CCND1 had no prognostic value (P = 0.552, 0.566). When adjusted for MCL International Prognostic Index (MIPI) or combined MCL-International Prognostic Index (MIPI-c), TP53 and WHSC1 mutations were the most important prognostic factors in MCL (P < 0.05). Our data provide an unbiased view of the landscape of mutations in MCL and commend that all patients benefit from mutations of TP53 and WHSC1 at diagnosis, in addition to MIPI and MIPI-c score.

The study sought to identify genetic aberrations driving oral squamous cell carcinoma (OSCC) development among users of shammah, an Arabian preparation of smokeless tobacco. Twenty archival OSCC samples, 15 of which with a history of shammah exposure, were whole-exome sequenced at an average depth of 127×. Somatic mutations were identified using a novel, matched controls-independent filtration algorithm. CODEX and Exomedepth coupled with a novel, Database of Genomic Variant-based filter were employed to call somatic gene-copy number variations. Significantly mutated genes were identified with Oncodrive FM and the Youn and Simon's method. Candidate driver genes were nominated based on Gene Set Enrichment Analysis. The observed mutational spectrum was similar to that reported by the TCGA project. In addition to confirming known genes of OSCC (TP53, CDKNA2, CASP8, PIK3CA, HRAS, FAT1, TP63, CCND1 and FADD) the analysis identified several candidate novel driver events including mutations of NOTCH3, CSMD3, CRB1, CLTCL1, OSMR and TRPM2, amplification of the proto-oncogenes FOSL1, RELA, TRAF6, MDM2, FRS2 and BAG1, and deletion of the recently described tumor suppressor SMARCC1. Analysis also revealed significantly altered pathways not previously implicated in OSCC including Oncostatin-M signalling pathway, AP-1 and C-MYB transcription networks and endocytosis. There was a trend for higher number of mutations, amplifications and driver events in samples with history of shammah exposure particularly those that tested EBV positive, suggesting an interaction between tobacco exposure and EBV. The work provides further evidence for the genetic heterogeneity of oral cancer and suggests shammah-associated OSCC is characterized by extensive amplification of oncogenes.

The role of various inflammatory mechanisms and oxidative stress in the development of atherosclerosis and arterial hypertension (AH) has been increasingly acknowledged during recent years. Hypertension per se or factors that cause hypertension along with other complications lead to infiltration of activated leukocytes in the vascular wall, where these cells contribute to the development of vascular injury by releasing cytokines, oxygen radicals, and other toxic mediators. However, molecular mechanisms underlying leukocyte activation at transcriptional level in AH are still far from being clear. To solve this problem we employed cDNA microarray technology to reveal the differences in gene expression in peripheral blood leukocytes from patients with AH compared with healthy individuals. The microarray data were verified by a semi-quantitative RT-PCR method. We found 25 genes with differential expression in leukocytes from AH patients among which 21 genes were upregulated and 4 genes were downregulated. These genes are implicated in apoptosis (CASP2, CASP4, and CASP8, p53, UBID4, NAT1, and Fte-1), inflammatory response (CAGC, CXCR4, and CX3CR1), control of MAP kinase function (PYST1, PAC1, RAF1, and RAFB1), vesicular trafficking of molecules among cellular organelles (GDI-1 and GDI-2), cell redox homeostasis (GLRX), cellular stress (HSPA8 and HSP40), and other processes. Gene expression pattern of the majority of genes was similar in AH patients independent of the disease stage and used hypotensive therapy, but was clearly different from that of normotensive subjects.

Aberrations in the apoptotic mechanisms that cause excessive or deficient programmed cell death have been linked to a wide array of pathological conditions. In this study, using real-time reverse transcriptase-PCR, we analyzed the expression of apoptosis-regulating miRNAs (miR-15a, miR-16, miR-17-5p, miR-20a, miR-21, miR-29a, and miR-34a) in 20 oral squamous cell carcinoma and 5 normal oral mucosa tissue samples. Bioinformatic algorithms were used to identify the target genes of these miRNAs (BCL2, CASP2, CASP7, CASP8, DIABLO). The expression transcript levels of the target genes were measured in 50 oral squamous cell carcinoma and 10 normal oral mucosa tissue samples. We observed downregulation of miR-15a, miR-29a, and miR-34a in 50, 75, and 70% of samples, respectively. miR-16, miR-17-5p, miR-20a, and miR-21 expression was normal in 80, 75, 90, and 60% of samples, respectively. BCL2 transcripts were downregulated in 60% of samples, and normal-like expression was observed for CASP2, CASP7, CASP8, and DIABLO transcripts in 66, 82, 68, and 60% of samples, respectively. BCL2 expression was negative to weak, and that of proteins CASP2, CASP7, CASP8, and DIABLO was moderate to strong. Our study provides evidence of alterations in the expression of apoptosis-regulating miRNAs and genes in the apoptotic pathway, demonstrating that regulation of apoptosis is a hallmark of oral squamous cell carcinoma pathogenesis.

The prediction of the correct β-sheet topology for pure β and mixed α/β proteins is a critical intermediate step toward the three dimensional protein structure prediction. The predicted beta sheet topology provides distance constraints between sequentially separated residues, which reduces the three dimensional search space for a protein structure prediction algorithm. Here, we present a novel mixed integer linear optimization based framework for the prediction of β-sheet topology in β and mixed α/β proteins. The objective is to maximize the total strand-to-strand contact potential of the protein. A large number of physical constraints are applied to provide biologically meaningful topology results. The formulation permits the creation of a rank-ordered list of preferred β-sheet arrangements. Finally, the generated topologies are re-ranked using a fully atomistic approach involving torsion angle dynamics and clustering. For a large, non-redundant data set of 2102 β and mixed α/β proteins with at least 3 strands taken from the PDB, the proposed approach provides the top 5 solutions with average precision and recall greater than 78%. Consistent results are obtained in the β-sheet topology prediction for blind targets provided during the CASP8 and CASP9 experiments, as well as for actual and predicted secondary structures. The β-sheet topology prediction algorithm, BeST, is available to the scientific community at http://selene.princeton.edu/BeST/.

BACKGROUND

Template-based modeling, the most successful approach for predicting protein 3D structure, often requires detecting distant evolutionary relationships between the target sequence and proteins of known structure. Developed for this purpose, fold recognition methods use elaborate strategies to exploit evolutionary information, mainly by encoding amino acid sequence into profiles. Since protein structure is more conserved than sequence, the inclusion of structural information can improve the detection of remote homology.

RESULTS

Here, we present ORION, a new fold recognition method based on the pairwise comparison of hybrid profiles that contain evolutionary information from both protein sequence and structure. Our method uses the 16-state structural alphabet Protein Blocks, which provides an accurate 1D description of protein structure local conformations. ORION systematically outperforms PSI-BLAST and HHsearch on several benchmarks, including target sequences from the modeling competitions CASP8, 9 and 10, and detects ∼10% more templates at fold and superfamily SCOP levels.

BACKGROUND

Software freely available for download at http://www.dsimb.inserm.fr/orion/.

BACKGROUND

jean-christophe.gelly@univ-paris-diderot.fr.

BACKGROUND

Supplementary data are available at Bioinformatics online.

BACKGROUND

Non-alcoholic fatty hepatitis (NASH) is highly prevalent, mitochondria damage is the main pathophysiological characteristic of NASH. However, treatment for mitochondria damage is rarely reported.

METHODS

NASH model was established in rats, the protective effects of curcumin were evaluated by histological observation; structure and function assessments of mitochondria; and apoptotic genes expression.

RESULTS

NASH rats treated with curcumin displayed relatively slight liver damage when compared with NASH livers. The average mitochondrial length and width of NASH (12.0 ± 3.2 and 5.1 ± 1.1 micrometers) were significantly longer than that of normal (6.2 ± 2.1 and 2.1 ± 1.5 micrometers) and NASH treated with curcumin (7.4 ± 1.2 and 3.2 ± 1.5 micrometers) rats. The average malondialdehyde (MDA) and 4-hydroxy nonyl alcohol (HNE) levels in liver homogenates of NASH rats (4.23 ± 0.22 and 19.23 ± 2.3 nmol/Ml) were significantly higher than these in normal (1.32 ± 0.12 and 3.52 ± 0.43 nmol/mL) and NASH treated with curcumin (1.74 ± 0.11 and 4.66 ± 0.99 nmol/mL) rats. The expression levels of CytC, Casp3 and Casp8 of the NASH livers were significantly higher than normal and NASH treated with curcumin rats livers.

CONCLUSIONS

Our data demonstrated that curcumin prevents the NASH by mitochondria protection and apoptosis reduction and provided a possible novel treatment for NASH.

We report on a patient with an interstitial deletion of the long arm of chromosome 2 at 2q31.2q33.2. She had prenatal and postnatal growth retardation, microcephaly, facial dysmorphism, cleft palate, camptodactyly, bilateral talipes equinovarus, severe intellectual disability, and ectodermal anomalies. She showed thin, atrophic skin, sparse, brittle, slowly growing hair, oligodontia with abnormally shaped teeth, normal sweating, and normal fingernails, consistent with a diagnosis of ectodermal dysplasia. Array CGH analysis (Agilent 44K) showed the deletion to span 26 Mb, between cytogenetic bands 2q31.2 and 2q33. The deletion leads to hemizygosity for the HOXD cluster and its regulatory elements, COL3A1/COL5A2, GTF3C3, CASP8, CASP10, and SABT2 could perhaps interfere with long range control of DLX1 and DLX2 expression. This girl confirms the existence of a clinically recognizable 2q32 microdeletion syndrome, as recently delineated by Van Buggenhout et al. and confirms a novel putative locus for ectodermal dysplasia on chromosome 2q31q33. We recommend considering cytogenetic and/or molecular screening for del(2q32) in patients with developmental disability and ectodermal dysplasia-like phenotype, including thin skin, oligodontia, dysplastic teeth, and sparse hair.

Caspase-8 and caspase-10 play crucial roles in both cancer development and chemotherapy efficacy. In this study, we aimed to comprehensively assess single nucleotide polymorphisms (SNPs) of the caspase-8 (CASP8) and caspase-10 (CASP10) genes in relation to toxicity outcomes with first-line platinum-based chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). We genotyped 13 tag SNPs of CASP8 and CASP10 in 663 patients with advanced NSCLC treated with platinum-based chemotherapy regimens. Associations between SNPs and chemotherapy toxicity outcomes were identified in a discovery set of 279 patients and then validated in an independent set of 384 patients. In both the discovery and validation sets, variant homozygotes of CASP8 rs12990906 and heterozygotes of CASP8 rs3769827 and CASP10 rs11674246 and rs3731714 had a significantly lower risk for severe toxicity overall. However, only the association with the rs12990906 variant was replicated in the validation set for hematological toxicity risk. In a stratified analysis, we found that some other SNPs, including rs3769821, rs3769825, rs7608692, and rs12613347, were significantly associated with severe toxicity risk in some subgroups, such as in nonsmoking patients, patients with adenocarcinoma, and patients treated with cisplatin combinations. Consistent results were also found in haplotype analyses. Our results provide novel evidence that polymorphisms in CASP8 and CASP10 may modulate toxicity outcomes in patients with advanced NSCLC treated with platinum-based chemotherapy. If validated, the findings will facilitate the genotype-based selection of platinum-based chemotherapy regimens.

Coal workers' pneumoconiosis (CWP) is a chronic interstitial lung disease with a complex etiology that can occur after cumulative dust exposure. A case-control study was conducted to test the hypothesis that single-nucleotide polymorphisms (SNP) within CASPASE-8 (CASP8) promoter involved in resolution of inflammatory processes modulate the risk of CWP development. The study population consisted of 619 underground coal miners in the 5 coal mines of Xuzhou Mining Business Group Co. Ltd., China, of whom 315 were diagnosed with CWP. The association study between CASP8 -652 6N ins/del polymorphism with CWP by multiple logistic regression analysis showed a significant association of the genotype del/del with CWP compared with to ins/ins genotypes, and showed that the risk was significantly higher for stage I CWP. Further analysis showed that in subjects with the del/del genotype there was significantly increased risk for CWP occurrence among younger individuals (<66 yr) or those with longer duration dust exposure >>or=26 yr). These findings suggested that CASP8-652 6N ins/del polymorphism may contribute to the genetic susceptibility for CWP development.