Background: Autophagy is classified as a form of programmed cell death. Nevertheless, besides the death-inducing function, autophagy enables removal of damaged organelles, energy savings, and thus cell survival. This applies in particular to cells with poor renewal capabilities, such as chondroblasts. Autophagy is regulated by a complex molecular network, including proteases and their substrates. In autopodium, autophagy-related proteases have been examined particularly within the context of the elimination of the interdigital tissue. However, the death-inducing effects of their expression/activation have not been specified yet. This work focuses on autophagy-associated proteases (cathepsins, matrix metalloproteinases, and caspases) involved in phalangeal cartilage of the mouse autopodium.

Methods: PCR Array, Real Time PCR, and immunohistochemistry were used to follow the expression of autophagy-associated genesin vivo at two developmental stages prenatal/embryonic (E)12 vs. E14. Real Time PCR was then applied to investigate the influence of rapamycin (an inductor of autophagy) on the expression of autophagy-associated proteases in chondroblasts in vitro using micromass culture.

Results: Several proteases showed increased expression levels during the transition of pre-chondrogenic cells into chondroblastsin vivo. The most significant increases were observed for Ctsb (fold regulation 2.22), Ctsd (fold regulation 2.37), Ctss (fold regulation 2.92), Mmp9 (up to 445%), and Casp8 (up to 250%). The transition was associated also with high expression of crucial autophagic inducers, such as Atgs. The in vitro treatment of chondroblasts by autophagy inductor rapamycin showed significantly decreased expression of cathepsins, a mild increase in expression of metalloproteinases, and no effect in caspase expression.

Conclusions: The present data provide a screening of autophagy-associated proteases accompanying the formation of cartilage in vivo and specify their expression under rapamycin treatment in vitro. Notably, the selected proteases are assigned to osteoarthritis, therefore their regulation might be used in clinically oriented studies.

Keywords: autophagy; cartilage; caspases; cathepsins; chondrogenesis; metalloproteinases; proteases.

Follicular dynamics and the expression of candidate genes using real-time polymerase chain reaction (PCR) were compared during the oestrous cycle of pig breeds with high (commercial line; n=24) and low (local Brazilian Piau; n=21) ovulation rates and prolificacy. Gilts were killed on Days 0, 4, 10 and 18 of the oestrous cycle and visible ovarian follicles were classified by follicular diameter. Recovered cumulus-oocyte complexes were classified as normal or atretic and frozen in liquid nitrogen until RNA extraction. Low ovulation rates and/or prolificacy in Piau gilts was associated with a different pattern of follicle development, with lower numbers of small follicles on Day 18, fewer large follicles on Days 0 and 18 (P≤0.05) and a higher proportion of atretic follicles on Days 0 and 18 (P≤0.05). Compared with commercial line gilts, less-prolific Piau gilts exhibited higher expression of apoptotic genes during luteolysis (CASP3 and FASL; P≤0.05), decreased expression of TGFBR2 and BAX mRNA in the corpus luteum (P≤0.05), higher expression of apoptotic genes (FAS, BCL2 and CASP8; P≤0.05) in granulosa cells and a greater abundance (P≤0.05) of genes controlling oocyte-secreted factors (GDF9, BMP15 and BMP6), suggesting underlying mechanisms controlling differences in follicular development, ovulation rate and inherent prolificacy in this pig breed.

The strategy for evaluating template-based models submitted to CASP has continuously evolved from CASP1 to CASP5, leading to a standard procedure that has been used in all subsequent editions. The established approach includes methods for calculating the quality of each individual model, for assigning scores based on the distribution of the results for each target and for computing the statistical significance of the differences in scores between prediction methods. These data are made available to the assessor of the template-based modeling category, who uses them as a starting point for further evaluations and analyses. This article describes the detailed workflow of the procedure, provides justifications for a number of choices that are customarily made for CASP data evaluation, and reports the results of the analysis of template-based predictions at CASP8.

Background: Optimizing the therapeutic ratio for radiation therapy (RT) in head and neck squamous cell carcinoma (HNSCC) is uniquely challenging owing to high rates of early and late toxicity involving nearby organs at risk. These toxicities have a profound impact on treatment compliance and quality of life. Emerging evidence suggests that RT dose alone cannot fully account for the variable severity of RT-related adverse events (rtAEs) observed in HNSCC patients. Next-generation sequencing has become an increasingly valuable tool with widespread use in the oncology field and is being robustly explored for predicting rtAEs beyond dosimetric data.

Methods: Patients who had Foundation Medicine sequencing data and received RT for primary or locally recurrent HNSCC were selected for this study. Early and late toxicity data were collected and reported based on Common Terminology Criteria for Adverse Events version 5.0. Dosimetric parameters were collected for pertinent structures.

Results: A total of HNSCC 37 patients were analyzed in this study. Genetic alterations in BRCA2, ERBB3, NOTCH1 and CCND1 were all associated with higher mean grade of toxicity with BRCA2 alteration implicated in all toxicity parameters evaluated including mucositis, early dysphagia, xerostomia and to a lesser extent, late dysphagia. Interestingly, patients who exhibited alterations in both BRCA2 and ERBB3 experienced a twofold or greater increase in early dysphagia, early xerostomia and late dysphagia compared to ERBB3 alteration alone. Furthermore, several gene alterations were associated with improved toxicity outcomes. Within an RT supersensitive patient subset, alterations were found in TNFAIP3, HNF1A, SPTA1 and CASP8. All of these alterations were not found in the RT insensitive patient subset. We found 17 gene alterations in the RT insensitive patient subset that were not found in the RT supersensitive patient subset.

Conclusion: Despite consistent RT dosimetric parameters, patients with HNSCC experience heterogeneous patterns of rtAEs. Identifying factors associated with toxicity outcomes offers a new avenue for personalized precision RT therapy and prophylactic management. Here, next-generation sequencing in a population of HNSCC patients correlates several genetic alterations with severity of rtAEs. Further analysis is urgently needed to identify genetic patterns associated with rtAEs in order to reduce harmful outcomes in this challenging population.

Keywords: BRCA2; CASP8; ERBB3; HNF1A; Head and neck squamous cell carcinoma; Predictive biomarkers of radiation toxicity; RT dosimetric data; Radiogenomics; SPTA1; TNFAIP3.

A growing body of literature suggests that pharmaceutical contamination poses an increasing risk to marine ecosystems. Paracetamol or acetaminophen is the most widely used medicine in the world and has recently been detected in seawater. Here, we present the results of 7 days' exposure of blue mussel adults to 40 ng/L, 250 ng/L and 100 μg/L of paracetamol. Histopathology shows that haemocytic infiltration is the most observed condition in the exposed mussels. The mRNA expression of VTG, V9, ER2, HSP70, CASP8, BCL2 and FAS in mussel gonads present different patterns of downregulation. VTG and CASP8 mRNA expression show downregulation in all exposed mussels, irrespective of sex. The V9, HSP70, BCL2 and FAS transcripts follow a concentration-dependent variation in gene expression and may therefore be considered good biomarker candidates. ER2 mRNA expression shows a downregulated trend, with a clearer dose-response relationship in males. In conclusion, this study suggests that paracetamol has the potential to alter the expression of several genes related to processes occurring in the reproductive system and may therefore impair reproduction in blue mussels.

As an important transcription factor, SOX2 involves in embryogenesis, maintenance of stem cells and proliferation of primordial germ cell (PGC). However, little was known about its function in mature gonads. Herein, we investigated the SOX2 gene profiles in testis of scallop, Chlamys farreri. The level of C. farreri SOX2 (Cf-SOX2) mRNA increased gradually along with gonadal development and reached the peak at mature stage, and was located in all germ cells, including spermatogonia, spermatocytes, spermatids and spermatozoa. Knockdown of Cf-SOX2 using RNAi leaded to a mass of germ cells lost, and only a few spermatogonia retained in the nearly empty testicular acini after 21 days. TUNEL assay showed that apoptosis occurred in spermatocytes. Furthermore, transcriptome profiles of the testes were compared between Cf-SOX2 knockdown and normal scallops, 131,340 unigenes were obtained and 2,067 differential expression genes (DEGs) were identified. GO and KEGG analysis showed that most DEGs were related to cell apoptosis (casp2, casp3, casp8), cell proliferation (samd9, crebzf, iqsec1) and spermatogenesis (htt, tusc3, zmynd10, nipbl, mfge8), and enriched in p53, TNF and apoptosis pathways. Our study revealed Cf-SOX2 is essential in spermatogenesis and testis development of C. farreri and provided important clues for better understanding of SOX2 regulatory mechanisms in bivalve testis.

The caspase 8 variants CASP8 -652 6N InsDel and Asp302His have previously been identified to promote survival of T-lymphocytes and to indicate reduced breast cancer susceptibility. Besides some preliminary findings, prognostic relevance of these polymorphisms in patients with existing breast cancer has not been investigated. Considering an immunomodulatory role of these polymorphisms, we genotyped 785 early breast cancer patients and correlated caspase 8 variants with disease-free survival (DFS) and the presence of tumor infiltrating lymphocytes (TILs). Early breast cancer specimens were collected as part of the multicenter prospective PiA study. Genotyping was performed by pyrosequencing, TILs status was assessed using hematoxylin & eosin staining. The CASP8 -652Del variant was significantly associated with improved DFS in an allele-dose dependent manner (p = 0.027). Homozygosity for the -652Del variant was an independent predictor for improved DFS (HR = 0.36; 95% CI = 0.174-0.726; p = 0.005). In patients with the 302HisHis genotype, there was no event of recurrence during observation time. Combined analysis of diplotypes revealed an influence of both polymorphisms on DFS (p = 0.029). Interestingly, patients with the 302HisHis variant among the unstratified patient cohort (and among the luminal-like subtype, by trend) had tumors with lower lymphocyte infiltration (p = 0.025). We propose a prognostically favorable role of the -652Del and the 302His variant in primary breast cancer and suggest for the first time an association between polymorphisms in apoptosis-related genes and the immunophenotype in breast cancer. Our findings encourage further investigation of caspase 8 polymorphisms as biomarkers for prognostic and immunotherapeutic considerations.

Checkpoint inhibitors have been approved for the treatment of non-small cell lung cancer (NSCLC). However, only a minority of patients demonstrate a durable clinical response. PD-L1 scoring is currently the only biomarker measure routinely used to select patients for immunotherapy, but its predictive accuracy is modest. The aim of our study was to evaluate a proteomic assay for the analysis of patient plasma in the context of immunotherapy. Pretreatment plasma samples from 43 NSCLC patients who received anti-PD-(L)1 therapy were analyzed using a proximity extension assay (PEA) to quantify 92 different immune oncology-related proteins. The plasma protein levels were associated with clinical and histopathological parameters, as well as therapy response and survival. Unsupervised hierarchical cluster analysis revealed two patient groups with distinct protein profiles associated with high and low immune protein levels, designated as "hot" and "cold". Further supervised cluster analysis based on T-cell activation markers showed that higher levels of T-cell activation markers were associated with longer progression-free survival (PFS) (p < 0.01). The analysis of single proteins revealed that high plasma levels of CXCL9 and CXCL10 and low ADA levels were associated with better response and prolonged PFS (p < 0.05). Moreover, in an explorative response prediction model, the combination of protein markers (CXCL9, CXCL10, IL-15, CASP8, and ADA) resulted in higher accuracy in predicting response than tumor PD-L1 expression or each protein assayed individually. Our findings demonstrate a proof of concept for the use of multiplex plasma protein levels as a tool for anti-PD-(L)1 response prediction in NSCLC. Additionally, we identified protein signatures that could predict the response to anti-PD-(L)1 therapy.

Keywords: PD-L1; biomarkers; immune checkpoint inhibitors; liquid biopsy; lung cancer.

Several single nucleotide polymorphisms (SNPs) influencing DNA repair capacity and apoptotic status may confer genetic predisposition to Philadelphia‑chromosome negative myeloproliferative neoplasms (PN‑MPNs), and influence therapeutic response and the clinical course. In the present study, whether SNPs in genes involved in apoptosis and the base excision repair (BER) pathway was evaluated. In addition, some known risk factors in PN‑MPNs that may influence survival and therapeutic response to hydroxyurea (HU) were analyzed, taking into account three items: Disease progression, predisposition to new non‑myeloid neoplasms and thrombotic events. The present study involved a total of 133 Caucasian Portuguese PN‑MPNs patients treated with HU, whereby 17 cases showed progression to myelofibrosis/leukemia, 11 developed new non‑myeloid neoplasms and 22 presented with thrombotic events. Progression to secondary myelofibrosis/leukemia is influenced by exposure to cytoreductive agents, and caspase and BER polymorphisms {globally, CASP8 3'untranslated region [odds ratio (OR)=0.24; 95% confidence interval (CI), 0.08‑0.69], XRCC1 Arg194Trp [OR=3.58; 95% CI, 0.98‑13.01]; for essential thrombocythemia patients CASP9 Arg173His [OR=11.27; 95% CI, 1.13‑112.28], APEX1 Asp148Glu [OR=0.28; 95% CI, 0.74‑1.03], and XRCC1 Arg194Trp [OR=6.60; 95% CI, 1.60‑27.06]}. Moreover, globally caspase and BER polymorphisms influenced the development of new nonmyeloid malignancies [CASP8 Asp270His (OR=5.90; 95% CI, 1.42‑24.62) and XRCC1 Arg399Gln (OR=0.27; 95% CI, 0.07‑1.03)]. On the other hand, only the BER pathway had a role in the presence of thrombotic events [XRCC1 Gln399Arg (OR=0.35; 95% CI, 0.14‑0.88)]. JAK2 mutation had no influence on these complications. Larger studies are required to confirm these results, and to provide conclusive evidence of association between these and other variants with PN‑MPNs therapeutic response and clinical evolution. However, this study may allow the development of drugs more directly targeted to the pathophysiology of the disease, with high efficacy, fewer adverse effects, contributing to compliance of patients with treatments. The clinical indication for classical drugs, including HU, may be guided by variant genes, which may provide additional beneficial effects.

The purpose of this study was to identify novel autoantibodies against tumor-associated antigens (TAAbs) and explore the optimal diagnosis model based on the protein chip for detecting esophageal squamous cell carcinoma (ESCC). The human protein chip based on cancer-driving genes was customized to discover candidate TAAbs. Enzyme-linked immunosorbent assay was applied to verify and validate the expression levels of candidate TAAbs in the training cohort (130 ESCC and 130 normal controls) and the validation cohort (125 ESCC and 125 normal controls). Logistic regression analysis was adopted to construct the diagnostic model based on the expression levels of autoantibodies with diagnostic value. Twelve candidate autoantibodies were identified based on the protein chip according to the corresponding statistical methods. In both the training cohort and validation cohort, the expression levels of 10 TAAbs (GNA11, PTEN, P53, SRSF2, GNAS, ACVR1B, CASP8, DAXX, PDGFRA, and MEN1) in ESCC patients were higher than that in normal controls. The panel consisting of GNA11, ACVR1B and P53 demonstrated favorable diagnostic power. The sensitivity, specificity and accuracy of the model in the train cohort and the validation cohort were 71.5%, 93.8%, 79.6% and 77.6%, 81.6%, 70.8%, respectively. In either cohort, there was no correlation between positive rate of the autoantibody panel and clinicopathologic features for ESCC patients. Protein chip technology is an effective method to identify novel TAAbs, and the panel of 3 TAAbs (GNA11, ACVR1B, and P53) is promising for distinguishing ESCC patients from normal individuals.

Keywords: Protein chip; autoantibodies; cancer-driving genes; detection; esophageal squamous cell carcinoma.

The aim of the study was to determine how a high-fat diet supplemented with various forms of chromium affects hematological and immune parameters of the blood of rats. The rats received a standard diet or a high-fat diet supplemented with chromium at 0.3 mg/kg body weight (BW) in the form of chromium(III) picolinate, chromium(III)-methionine or nano-sized chromium. Selected hematological parameters were determined in the blood of the rats, including total white blood cell (WBC) count, leukogram, red blood cell (RBC) count, hemoglobin level (HGB), hematocrit (HCT), platelet count (PLT) and platelet percentage (PCT), as well as immune parameters: levels of immunoglobulins A and E (IgA and IgE), interleukin-6 (IL-6), interleukin-2 (IL-2), and tumor necrosis factor α (TNF-α); activity of ceruloplasmin (Cp); and levels of caspase 3 and 8 (Casp3 and Casp8). Feeding rats a high-fat diet increased blood markers of induction of inflammation, ie pro-inflammatory cytokines IL-6 and TNF-α, and also significantly increased IgE. The diet had no effect on the blood count, except for an increase in the number of neutrophils. The chromium compounds tested, particularly Cr-Met and Cr-NPs, stimulated the immune system of the rats, as indicated by increased concentrations of IgA, IgE, IL-2, IL-6, TNF-α, and Cp. Given the increase in inflammatory mediators induced by chromium, it should not be used to mitigate the effects of a high-fat diet. Moreover, chromium picolinate and chromium nanoparticles were shown to increase the content of caspase 3 and 8 in the blood of rats, which indicates a pro-apoptotic effect. The effects of the use of chromium nanoparticles include reductions in the WBC count and in the thrombocyte count (leuko- and thrombopenia). Taking account these data the use of chromium as dietary supplement should be reconsidered.

Keywords: chromium nanoparticles; chromium picolinate; chromium(III)-methionine; cytokine; hematology; immunity; rat.

BACKGROUND

Molecular investigation of breast tumors has permitted better understanding about interaction of genes and pathways involved in tumor progression.

OBJECTIVE

The aim of this study was to evaluate the association between genes belonging to the pathway of apoptosis with tumor response to photodynamic therapy.

METHODS

The mammary tumors were induced in twenty-four Spraguey-Dawley female rats by oral gavage of 7,12-dimethylbenz(a)anthracene (8mg/Kg body weight). Animals were divided into three groups: G1 (normal tissue), G2 (tumors without treatment), G3 (animals euthanized 48h after treatment). The photosensitizer used was a chlorin, 5,15-bis-(2-bromo-5-hydroxyphenyl) chlorin in the dose of 8mg/kg for each animal. Light source of diode laser at a wavelength of 660nm, fluence rate of 100mW/cm, and light dose of 100J/cm was delivery to lesions for treatment. A sample from each animal was investigated by quantitative real time PCR using Rat Apoptosis RT(2) Profiler™ PCR Array platform.

RESULTS

Pro-apoptotic BAK1, CARD6, CASP8, CIDEA, CIDEB, DAPK1, TNF, TNFRSF10B, FASLG, LOC687813, and TP73 genes showed increased expression, and CD40 anti-apoptotic gene showed decreased expression in the group who underwent PDT (G3) in relation to G2.

CONCLUSIONS

The results indicated that these genes are involved more directly with cellular apoptosis induced by PDT using the Chlorin photosensitizer.

Purpose: Head and neck squamous cell carcinoma (HNSCC) affects various anatomical sites, which often dictates whether the cancer is managed with primary surgery or radiation. This study aimed to assess differences in single nucleotide variation (SNV), copy number, mRNA abundance, methylation, and tumor microenvironment (TME) between HPV-negative oral cavity (OC), oropharyngeal (OPC), hypopharyngeal (HPC), and laryngeal (LC) cancers within The Cancer Genome Atlas (TCGA).

Methods: We downloaded the clinical information and molecular data for the TCGA HNSCC cohort from the data portal and published literature. The TME was estimated using mRNA abundance data. We conducted our analyses within the Bioconductor statistical framework in the R environment. CNA and mRNA abundance results were correlated and grouped with SNV results for downstream pathway analysis.

Results: LC had a higher mutational burden than OC and OPC (p <10^-4). LC tumors were enriched in CSMD3, NSD1, DCHS2 and ANK2 SNVs, while OC tumors were enriched in CASP8 SNVs (FDR < 0.1). LCs were enriched for neuronal and glycosylation pathways, while OCs were enriched for extracellular matrix pathways. B cells and endothelial cells were more abundant in LC while monocytes were more abundant in OC (FDR < 0.1). OPC was the most hypoxic, followed by OC then LC (FDR < 0.05). OC had greater methylation of Hox genes than LC. Subsite analysis revealed that oral tongue cancers had fewer CASP8 and FBN2 mutations and higher dendritic cell abundance than other oral cavity cancers.

Conclusions: We identified significant genomic, transcriptional, and microenvironmental differences between HPV-negative HNSCC. Further study is warranted to determine if these findings portend differential response to specific treatment modalities.

Keywords: Genomics; HNSCC; HPV-negative; Hypoxia; Immune landscape; Larynx cancer; Mutational status; Oral cancer; Oral cavity; Pathway analysis; mRNA abundance.

Prostaglandin F_2α (PGF_2α) may differentially affect viability of luteal cells by inducing either proliferation or cell death (via apoptosis or necroptosis). The diverse effects of PGF_2α may depend on its local vs. systemic actions. In our study, we determined changes in expression of genes related to: (i) apoptosis: caspase (CASP) 3, CASP8, BCL2 associated X (BAX), B-cell lymphoma 2 (BCL2) and (ii) necroptosis: receptor-interacting protein kinase (RIPK) 1, RIPK3, cylindromatosis (CYLD), and mixed lineage kinase domain-like (MLKL) in the early and mid-stage corpus luteum (CL) that accompany local (intra-CL) vs. systemic (i.m.) analogue of PGF_2α (aPGF_2α) actions. Cows at day 4 (n = 24) or day 10 (n = 24) of the estrous cycle were treated by injections as follows: (1) systemic saline, (2) systemic aPGF_2α (25 mg; Dinoprost), (3) local saline, (4) local aPGF_2α (2.5 mg; Dinoprost). After 4 h, CLs were collected by ovariectomy. Expression levels of mRNA and protein were investigated by RT-q PCR, Western blotting and immunohistochemistry, respectively.

We found that local and systemic administration of aPGF_2α in the early-stage CL resulted in decreased expression of CASP3 (P < 0.01), but CASP8 mRNA expression was up-regulated (P < 0.05). However, the expression of CASP3 was up-regulated after local aPGF_2α treatment in the middle-stage CL, whereas systemic aPGF_2α administration increased both CASP3 and CASP8 expression (P < 0.01). Moreover, we observed that both local and systemic aPGF_2α injections increased RIPK1, RIPK3 and MLKL expression in the middle-stage CL (P < 0.05) while CYLD expression was markedly higher after i.m. aPGF_2α injections (P < 0.001). Moreover, we investigated the localization of necroptotic factors (RIPK1, RIPK3, CYLD and MLKL) in bovine CL tissue after local and systemic aPGF_2α injections in the bovine CL.

Our results demonstrated for the first time that genes related to cell death pathways exhibit stage-specific responses to PGF_2α administration depending on its local or systemic actions. Locally-acting PGF_2α plays a luteoprotective role by inhibiting apoptosis and necroptosis in the early CL. Necroptosis is a potent mechanism responsible for structural CL regression during PGF_2α-induced luteolysis in cattle.

Oral squamous cell carcinoma (OSCC) is a common cancer of the oral cavity in India. Cigarette smoking and chewing tobacco are known risk factors associated with OSCC. However, genomic alterations in OSCC with varied tobacco consumption history are not well-characterized. In this study, we carried out whole-exome sequencing to characterize the mutational landscape of OSCC tumors from subjects with different tobacco consumption habits. We identified several frequently mutated genes, including TP53, NOTCH1, CASP8, RYR2, LRP2, CDKN2A, and ATM. TP53 and HRAS exhibited mutually exclusive mutation patterns. We identified recurrent amplifications in the 1q31, 7q35, 14q11, 22q11, and 22q13 regions and observed amplification of EGFR in 25% of samples with tobacco consumption history. We observed genomic alterations in several genes associated with PTK6 signaling. We observed alterations in clinically actionable targets including ERBB4, HRAS, EGFR, NOTCH1, NOTCH4, and NOTCH3. We observed enrichment of signature 29 in 40% of OSCC samples from tobacco chewers. Signature 15 associated with defective DNA mismatch repair was enriched in 80% of OSCC samples. NOTCH1 was mutated in 36% of samples and harbored truncating as well as missense variants. We observed copy number alterations in 67% of OSCC samples. Several genes associated with non-receptor tyrosine kinase signaling were affected in OSCC. These molecules can serve as potential candidates for therapeutic targeting in OSCC.

Keywords: HNSCC; M-class cancer; genome aberration; mutational signature; oral cancer.

In an attempt to improve in vitro embryo production, we investigated the effect of FGF10 and 0.1 mM of cilostamide, cAMP modulator, during in vitro prematuration (PIVM) and in vitro maturation (IVM) on the developmental capacity of bovine oocytes. Five treatments (T) were used as follows: T1) IVM (control): COCs were matured for 22 h; T2) PIVM/IVM: COCs were submitted to 22 h of PIVM and 22 h of IVM; T3) PIVM + FGF10/IVM: COCs were submitted to PIVM for 22 h in the presence of FGF10 and matured for 22 h; T4) PIVM/IVM + FGF10: COCs were submitted to PIVM for 22 h and matured for 22 h in the presence of FGF10; and T5) PIVM + FGF10/IVM + FGF10: COCs were submitted to 22 h of PIVM and 22 h of IVM, both in the presence of FGF10. COCs were evaluated for CC expansion and nucleus configuration at 0 h, 22 h of PIVM and 22 h after IVM. At those time points, CCs were used for expression analysis of PTGS2, TNFAIP6, PTX3, HAS2, CASP8, CASP3, SLC2A1, SLC2A3 and FGFR2 genes. Then, embryo production was evaluated on D2 for cleavage and at D6, D7 and D8 for embryo development. Embryo quality was measured by the speed of development and by expression of KRT8, PLAC8, CD9, PAG2, PAG2, HSPB1, MSH6 genes. The percentage of COC that remained at GV after 22 h PIVM was lower (P < 0.05) in the treated group than in the control group, regardless of the addition of FGF10. Nevertheless at 22 h of maturation, none of the treatments affected the final rate of MII oocytes. No expansion of CCs was observed during the PIVM period. After 22 h of maturation expansion was similar (P>> 0.05) for all groups but they all had lower expansion (P < 0.05) than control group. Except for PTGS2 and SLC2A1 which were unchanged during PIVM, all other genes changed their expression during PIVM. When we evaluated the changes in gene expression during IVM on the different groups, we noted a change in the expression of four genes (PTGS2, CASP8, PTX3 and TNFAIP6). No differences (P>> 0.05) in the cleavage and blastocyst rates at D6 were observed among treatment groups. However, the blastocyst rates at D7 were lower (P < 0.05) than the control groups in which FGF10 was present either during PIVM or during IVM. When the speed of embryo development was evaluated on D7, embryos from groups PIVM-IVM and PIVM - IVM + FGF10 developed faster than the other groups. However, at D8, the hatching rate was similar (P>> 0.05) for all groups. Of all the analysed genes, only PLAC8 was shown to be overexpressed in PIVM/IVM + FGF10, and MSH6 was less expressed (P < 0.05) in PIVM + FGF10/IVM + FGF10 group when compared with the control. According to these findings, the PIVM of COCs in the presence of FGF10 affected the molecular profile and the expansion of CCs without affecting the nuclear maturation and embryo production rates. Although the presence of FGF10 changed the expression of genes related to embryo quality it did not show a great impact when added either or both during PIVM and IVM.

Background: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality world wide and constitutes the third most common malignancy in women. The RAIDs consortium (http://www.raids-fp7.eu/) conducted a prospective European study [BioRAIDs (NCT02428842)] with the objective to stratify CC patients for innovative treatments. A "metagene" of genomic markers in the PI3K pathway and epigenetic regulators had been previously associated with poor outcome [2].

Methods: To detect new, more specific, targets for treatment of patients who resist standard chemo-radiation, a high-dimensional Cox model was applied to define dominant molecular variants, copy number variations, and reverse phase protein arrays (RPPA).

Findings: Survival analysis on 89 patients with all omics data available, suggested loss-of-function (LOF) or activating molecular alterations in nine genes to be candidate biomarkers for worse prognosis in patients treated by chemo-radiation while LOF of ATRX, MED13 as well as CASP8 were associated with better prognosis. When protein expression data by RPPA were factored in, the supposedly low molecular weight and nuclear form, of beta-catenin, phosphorylated in Ser552 (pβ-Cat552), ranked highest for good prognosis, while pβ-Cat675 was associated with worse prognosis.

Interpretation: These findings call for molecularly targeted treatments involving p53, Wnt pathway, PI3K pathway, and epigenetic regulator genes. Pβ-Cat552 and pβ-Cat675 may be useful biomarkers to predict outcome to chemo-radiation, which targets the DNA repair axis.

Funding: European Union's Seventh Program for research, technological development and demonstration (agreement N°304,810), the Fondation ARC pour la recherche contre le cancer.

Keywords: Beta-catenin pβ-cat552 and pβ-cat675; Cervical cancer; Molecular and protein biomarkers for chemo-radiation efficiency; Molecular landscape.

The aim of the study was detailed clinicopathological investigation of SMARCB1/INI1-deficient sinonasal carcinomas, including molecular genetic analysis of mutational status and DNA methylation of selected protooncogenes and tumor suppressor genes by means of next generation sequencing (NGS) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). A total of 4/56 (7%) cases of SMARCB1/INI1-deficient carcinomas were detected among 56 sinonasal carcinomas diagnosed over a 19year period using immunohistochemical screening. The series comprised 3 males and 1 female, aged 27-76 years (median 64 years). All tumors arose in the nasal cavity. Three neoplasms were diagnosed in advanced stage pT4. During the follow-up period (range 14-111 months (median 72 months)), three tumors recurred locally, but none of the patients developed regional or distant metastases. Ultimately, two patients died due to the tumor. Microscopically, all tumors consisted of infiltrating nests of polygonal basaloid cells with a variable component of rhabdoid cells with eosinophilic cytoplasm. Immunohistochemically, there was almost diffuse expression of cytokeratins (CK), p16, p40 and p63 in all cases, while expression of CK5/6, CK7 and vimentin was only focal or absent. The detection of NUT gave negative results. In three cases, the absence of SMARCB1/INI1 expression was due to deletion of SMARCB1/INI1 gene. Methylation of SMARCB1/INI1 gene was not found. One tumor harbored HPV18 E6/E7 mRNA. All 12 genes (BRAF, BRCA1, BRCA2, KIT, EGFR, KRAS, NRAS, PDGFRA, PIK3CA, PTEN, RET, and ROS1) tested for mutations using NGS were wild-type. Regarding DNA methylation, all four SMARCB1/INI1-deficient tumors showed methylation of RASSF1 gene by means of MS-MLPA. There was a statistically significant difference in RASSF1 gene methylation between SMARCB1/INI1-deficient and SMARCB1/INI1-positive tumors (p=0.0095). All other examined genes (ATM, BRCA1, BRCA2, CADM1, CASP8, CD44, CDKN1B, CDKN2A, CDKN2B, CHFR, DAPK1, ESR1, FHIT, GSTP1, HIC1, KLLN, MLH1a, MLH1b, RARB, and VLH) were unmethylated. In summary, we described four cases of SMARCB1/INI1-deficient sinonasal carcinoma with detailed clinicopathological data indicating that these tumors can be regarded as a distinct entity with aggressive behaviour. For the first time, we performed analysis of DNA methylation in SMARCB1/INI1-deficient sinonasal carcinomas, reporting on significantly higher methylation of RASSF1 gene in this neoplasm.

The present study demonstrates apoptosis-inducing potential and mechanism of action of Tribulus terristris alkaloid extract in Jurkat E6-1 cancer cell line. Liquid Chromatography-Mass Spectrometry and High Resolution-Mass Spectrometry analysis identified the presence of four N-feruloyltyramine derivatives, namely trans-N-feruloyl-3-hydroxytyramine (1), trans-N-coumaroyltyramine (2), trans-N-feruloyltyramine (3) and trans-N-feruloyl-3-ethoxytyramine (4) in the alkaloid extract. Compounds 2 and 3 have not been yet reported in the alkaloid extract of T. terristris. In silico analysis revealed therapeutic potential of N-feruloyltyramine derivatives and strong binding efficiency to both chains of Tumor Necrosis Factor Receptor 1. Treatment of alkaloids extract to Jurkat E6-1 clone induced dose-dependent cytotoxicity (LC50 140.4 μg mL-1). Jurkat cells treated with alkaloids extract at sub-lethal concentration showed DNA fragmentation, enhancement in caspase-3 activity and phosphatidylserine translocation (apoptosis indicator) compared to control cells. Gene expression analysis using Human Apoptosis RT2 Profiler PCR Array analysis upon alkaloid treatment was found to significantly alter expression of critical genes such as TNFR1, FADD, AIFM, CASP8, TP53, DFFA and NFKB1. These genes are predicted to mediate apoptotic cell death via both intrinsic and extrinsic apoptosis pathway. In summary, we report the identification of new N-feruloyltyramine derivatives from alkaloid extract of T. terristris fruit with probable anti-leukemic and pharmacological potential.

Hepatocellular carcinoma is the most frequent primary liver cancer. Macroautophagy/autophagy inhibitors have been extensively studied in cancer but, to date, none has reached efficacy in clinical trials. In this study, we demonstrated that GNS561, a new autophagy inhibitor, whose anticancer activity was previously linked to lysosomal cell death, displayed high liver tropism and potent antitumor activity against a panel of human cancer cell lines and in two hepatocellular carcinoma in vivo models. We showed that due to its lysosomotropic properties, GNS561 could reach and specifically inhibited its enzyme target, PPT1 (palmitoyl-protein thioesterase 1), resulting in lysosomal unbound Zn²⁺ accumulation, impairment of cathepsin activity, blockage of autophagic flux, altered location of MTOR (mechanistic target of rapamycin kinase), lysosomal membrane permeabilization, caspase activation and cell death. Accordingly, GNS561, for which a global phase 1b clinical trial in liver cancers was just successfully achieved, represents a promising new drug candidate and a hopeful therapeutic strategy in cancer treatment.Abbreviations: ANXA5:annexin A5; ATCC: American type culture collection; BafA1: bafilomycin A₁; BSA: bovine serum albumin; CASP3: caspase 3; CASP7: caspase 7; CASP8: caspase 8; CCND1: cyclin D1; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; CQ: chloroquine; iCCA: intrahepatic cholangiocarcinoma; DEN: diethylnitrosamine; DMEM: Dulbelcco's modified Eagle medium; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCC: hepatocellular carcinoma; HCQ: hydroxychloroquine; HDSF: hexadecylsulfonylfluoride; IC₅₀: mean half-maximal inhibitory concentration; LAMP: lysosomal associated membrane protein; LC3-II: phosphatidylethanolamine-conjugated form of MAP1LC3; LMP: lysosomal membrane permeabilization; MALDI: matrix assisted laser desorption ionization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKI67: marker of proliferation Ki-67; MTOR: mechanistic target of rapamycin kinase; MRI: magnetic resonance imaging; NH₄Cl: ammonium chloride; NtBuHA: N-tert-butylhydroxylamine; PARP: poly(ADP-ribose) polymerase; PBS: phosphate-buffered saline; PPT1: palmitoyl-protein thioesterase 1; SD: standard deviation; SEM: standard error mean; vs, versus; Zn²⁺: zinc ion; Z-Phe: Z-Phe-Tyt(tBu)-diazomethylketone; Z-VAD-FMK: carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone.

Keywords: Antitumor; PPT1; autophagy; liver cancer; lysosome; mtor.

Environmental allergens, including fungi, insects and mites, trigger type 2 immunity; however, the innate sensing mechanisms and initial signaling events remain unclear. Herein, we demonstrate that allergens trigger RIPK1-caspase 8 ripoptosome activation in epithelial cells. The active caspase 8 subsequently engages caspases 3 and 7, which directly mediate intracellular maturation and release of IL-33, a pro-atopy, innate immunity, alarmin cytokine. Mature IL-33 maintained functional interaction with the cognate ST2 receptor and elicited potent pro-atopy inflammatory activity in vitro and in vivo. Inhibiting caspase 8 pharmacologically and deleting murine Il33 and Casp8 each attenuated allergic inflammation in vivo. Clinical data substantiated ripoptosome activation and IL-33 maturation as likely contributors to human allergic inflammation. Our findings reveal an epithelial barrier, allergen-sensing mechanism that converges on the ripoptosome as an intracellular molecular signaling platform, triggering type 2 innate immune responses. These findings have significant implications for understanding and treating human allergic diseases.

Background: Yisui Qinghuang powder (YSQHP) is an effective traditional Chinese medicinal formulation used for the treatment of myelodysplastic syndromes (MDS). However, its pharmacological mechanism of action is unclear.

Materials and methods: In this study, the active compounds of YSQHP were screened using the traditional Chinese medicine systems pharmacology (TCMSP) and HerDing databases, and the putative target genes of YSQHP were predicted using the STITCH and DrugBank databases. Then, we further screened the correlative biotargets of YSQHP and MDS. Finally, the compound-target-disease (C-T-D) network was conducted using Cytoscape, while GO and KEGG analyses were conducted using R software. Furthermore, DDI-CPI, a web molecular docking analysis tool, was used to verify potential targets and pathways. Finally, binding site analysis was performed to identify core targets using MOE software.

Results: Our results identified 19 active compounds and 273 putative target genes of YSQHP. The findings of the C-T-D network revealed that Rb1, CASP3, BCL2, and MAPK3 showed the most number of interactions, whereas indirubin, tryptanthrin, G-Rg1, G-Rb1, and G-Rh2 showed the most number of potential targets. The GO analysis showed that 17 proteins were related with STPK activity, PUP ligase binding, and kinase regulator activity. The KEGG analysis showed that PI3K/AKT, apoptosis, and the p53 pathways were the main pathways involved. DDI-CPI identified the top 25 proteins related with PI3K/AKT, apoptosis, and the p53 pathways. CASP8, GSK3B, PRKCA, and VEGFR2 were identified as the correlative biotargets of DDI-CPI and PPI, and their binding sites were found to be indirubin, G-Rh2, and G-Rf.

Conclusion: Taken together, our results revealed that YSQHP likely exerts its antitumor effects by binding to CASP8, GSK3B, PRKCA, and VEGFR2 and by regulating the apoptosis, p53, and PI3K/AKT pathways.

Japanese encephalitis virus (JEV) is the main cause of viral encephalitis resulting in more than 68 000 clinical cases every year with case fatality rate as high as 30-40% for which no specific treatments are available. We have recently exhibited belladonna may be widely applicable for the treatment of various neurological disorders. Therefore, we developed a hydroalcoholic formulation of belladonna (B200) consisting of atropine and scopolamine and showed its antiviral efficacy against JEV infection. B200 treatment increases neuronal cell survival by reducing JEV induced cytopathic effects which were evident from significant reduction in necrotic cell population by flow-cytometry analysis and caspase 3 and 8 enzymatic activities. B200 treatment was found to reduce the intracellular JEV level observed by significant reduction in JEV-fluorescein isothiocyanate (FITC) expression in both neurons and microglia. Because microglia plays a crucial role in JEV pathogenesis, we further investigated the anti-JEV effects of B200 on human microglia cells and elucidated the mechanism of action by performing whole-transcriptome sequencing. Gene expression analysis revealed that B200 reduces the pro-apoptotic and inflammatory gene expression observed by significant reduction in BAD, BAX, CASP3, CASP8, IL1B, and CXCL10 and increase in IL10 responsive gene expression. Interestingly, our molecular docking analysis revealed that atropine and scopolamine interact with the His288 residue of NS3 protein, a crucial residue for RNA unwinding and ATPase activity that was further confirmed by degradation of NS3 protein. Drug likeness, ADME (absorption, distribution, metabolism, and excretion), and toxicity analysis further suggests that atropine and scopolamine both cross the blood-brain barrier, which is crucial for effective treatment of Japanese encephalitis (JE).

Keywords: Japanese encephalitis virus; NS3 NTPase/helicase; antiviral; atropine; belladonna; scopolamine.

Husbandry conditions often expose fish to several stressors, compromising organism's global homeostasis, which has consequences on aquaculture production. In order to depict intestinal homeostatic responses to deleterious conditions and the potential beneficial effects of functional diets in aquaculture fish, the effects of chronic suboptimal density condition on the neuro-immune endocrine system of rainbow trout was investigated through a transcriptomic survey. After 30 days under high stocking density, 67% of genes involved in homeostasis were found down-regulated whereas when fed with a diet supplemented with the probiotic Saccharomyces cerevisiae, <1% of these genes were found down-regulated. Genes involved in apoptotic mechanisms were found to be particularly responsive to both density and diets. At high density, several genes involved in the intrinsic and extrinsic apoptotic pathway (Fadd, Fas, Bcl-2 and Bax) as well as caspases (Casp8 and Casp3) were found down-regulated (Fold change <-7). However, fish fed with a diet supplemented with the probiotic yeast had an opposite response, indicating mitigation of stress effects. Overall, these results indicate the activation of homeostatic machinery and particularly genes involved in the apoptotic pathway as a result of probiotic feeding, counteracting apoptotic molecular repression derived from chronic stress.