Concurrent depletion of Vps37 proteins evokes ESCRT-I destabilization and profound cellular stress responses.

Molecular details of how endocytosis contributes to oncogenesis remain elusive. Our in silico analysis of colorectal cancer (CRC) patients revealed stage-dependent alterations in the expression of 112 endocytosis-related genes. Among them, transcription of the endosomal sorting complex required for transport (ESCRT)-I component VPS37B was decreased in the advanced stages of CRC. Expression of other ESCRT-I core subunits remained unchanged in the investigated dataset. We analyzed an independent cohort of CRC patients, which also showed reduced VPS37A mRNA and protein abundance. Transcriptomic profiling of CRC cells revealed non-redundant functions of Vps37 proteins. Knockdown of VPS37A and VPS37B triggered p21 (CDKN1A)-mediated inhibition of cell proliferation and sterile inflammatory response driven by the nuclear factor (NF)-κB transcription factor and associated with mitogen-activated protein kinase signaling. Co-silencing of VPS37C further potentiated activation of these independently induced processes. The type and magnitude of transcriptional alterations correlated with the differential ESCRT-I stability upon individual and concurrent Vps37 depletion. Our study provides novel insights into cancer cell biology by describing cellular stress responses that are associated with ESCRT-I destabilization.

The intratumour microbiota and neutrophilic inflammation in squamous cell vulvar carcinoma microenvironment.

BACKGROUND: A causal link between microbiota composition (dysbiosis) and oncogenesis has been demonstrated for several types of cancer. Neutrophils play a role in both immune protection against bacterial threats and carcinogenesis. This study aimed to characterise intratumoral bacteria in vulvar squamous cell carcinoma (VSCC) and their putative effect on neutrophil recruitment and cancer progression. METHODS: Clinical material was obtained from 89 patients with VSCC. Next-generation sequencing (NGS) of 16S rRNA and quantitative polymerase chain reaction (qPCR) were used to detect bacterial species in VSCC. To verify neutrophil activation, CD66b expression in tumour specimens was analysed by immunohistochemistry (IHC). Subsequently, IHC was applied to detect the main neutrophil serine proteases (NSPs), cathepsin G (CTSG), neutrophil elastase (ELANE), and proteinase 3 (PRTN3) in VSCC. RESULTS: Fusobacterium nucleatum and Pseudomonas aeruginosa were identified as tumour-promoting bacteria, and their presence was found to be associated with a shorter time to progression in VSCC patients. Furthermore, high abundance of CD66b, the neutrophil activation marker, in VSCC samples, was found to relate to poor survival of patients with VSCC. The selected NSPs were shown to be expressed in vulvar tumours, also within microabscess. The increased numbers of microabscesess were correlated with poor survival in VSCC patients. CONCLUSIONS: Our results show that neutrophilic inflammation seem to be permissive for tumour-promoting bacteria growth in VSCC. The findings provide new therapeutic opportunities, such as based on shifting the balance of neutrophil populations to those with antitumorigenic activity and on targeting NSPs produced by activated neutrophils at the inflammation sites.

Genetic associations with lymphomas in Polish patients: A pooled-DNA genome-wide association analysis.

Several single nucleotide polymorphisms (SNPs) associated with susceptibility to Hodgkin lymphoma (HL) and diffuse large B-cell lymphoma (DLBCL) have been identified. The aim of this study was to identify susceptibility loci for HL and DLBCL in Polish patients. Altogether, DLBCL (n = 218 and HL patients (n = 224) and healthy individuals (n = 1181) were recruited. Lymphoma diagnosis was based on standard criteria. Genome-wide association study (GWAS) was performed using pooled-DNA samples on llumina Infinium Omni2.5 Exome-8 v1.3, and selected loci were replicated by TaqMan SNP genotyping of individuals. GWAS detected thirteen and seven SNPs associated with DLBCL and HL, respectively. In the replication study, six and seven SNPs reached significance after correction for multiple testing in the DLBCL and HL cohorts, respectively. One and four SNPs associated with DLBCL and HL, respectively, were localized within, and two SNPs-near the major histocompatibility complex (MHC) region. In conclusion, the majority of loci associated with HL and DLBCL aetiology in previous studies have potential roles in immune function. Our pooled-DNA GWAS enabled the identification of several susceptibility loci for DLBCL and HL in the Polish population; some of them were mapped within or adjacent to the MHC, and other associated SNPs were located outside the MHC.

RNA-Seq Analysis of UPM-Exposed Epithelium Co-Cultivated with Macrophages and Dendritic Cells in Obstructive Lung Diseases.

BACKGROUND: Elevated concentrations of airborne pollutants are correlated with an enlarged rate of obstructive lung disease morbidity as well as acute disease exacerbations. This study aimed to analyze the epithelium mRNA profile in response to airborne particulate matter in the control, asthma, and COPD groups. RESULTS: A triple co-culture of nasal epithelium, monocyte-derived macrophages, and monocyte-derived dendritic cells obtained from the controls, asthma, and COPD were exposed to urban particulate matter (UPM) for 24 h. RNA-Seq analysis found differences in seven (CYP1B1, CYP1B1-AS1, NCF1, ME1, LINC02029, BPIFA2, EEF1A2), five (CYP1B1, ARC, ENPEP, RASD1, CYP1B1-AS1), and six (CYP1B1, CYP1B1-AS1, IRF4, ATP1B2, TIPARP, CCL22) differentially expressed genes between UPM exposed and unexposed triple co-cultured epithelium in the control, asthma, and COPD groups, respectively. PCR analysis showed that mRNA expression of BPIFA2 and ENPEP was upregulated in both asthma and COPD, while the expression of CYP1B1-AS1 and TIPARP was increased in the epithelium from COPD patients only. Biological processes changed in UPM exposed triple co-cultured epithelium were associated with epidermis development and epidermal cell differentiation in asthma and with response to toxic substances in COPD. CONCLUSIONS: The biochemical processes associated with pathophysiology of asthma and COPD impairs the airway epithelial response to UPM.

Selective Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibition by the SCH772984 Compound Attenuates In Vitro and In Vivo Inflammatory Responses and Prolongs Survival in Murine Sepsis Models.

Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome.

Mutational Mosaics of Cell-Free DNA from Pancreatic Cyst Fluids.

BACKGROUND: Pancreatic cyst fluids (PCFs) enriched in tumor-derived DNA are a potential source of new biomarkers. The study aimed to analyze germinal variants and mutational profiles of cell-free (cf)DNA shed into the cavity of pancreatic cysts. METHODS: The study cohort consisted of 71 patients who underwent endoscopic ultrasound fine-needle aspiration of PCF. Five malignant cysts, 19 intraductal papillary mucinous neoplasms (IPMNs), 11 mucinous cystic neoplasms (MCNs), eight serous cystic neoplasms (SCNs), and 28 pseudocysts were identified. The sequencing of 409 genes included in Comprehensive Cancer Panel was performed using Ion Proton System. The mutation rate of the KRAS and GNAS canonical loci was additionally determined using digital PCR. RESULTS: The number of mutations detected with NGS varied from 0 to 22 per gene, and genes with the most mutations were: TP53, KRAS, PIK3CA, GNAS, ADGRA2, and APC. The frequencies of the majority of mutations did not differ between non-malignant cystic neoplasms and pseudocysts. NGS detected KRAS mutations in malignant cysts (60%), IPMNs (32%), MCNs (64%), SCNs (13%), and pseudocysts (14%), with GNAS mutations in 20%, 26%, 27%, 13%, and 21% of samples, respectively. Digital PCR-based testing increased KRAS (68%) and GNAS (52%) mutations detection level in IPMNs, but not other cyst types. CONCLUSIONS: We demonstrate relatively high rates of somatic mutations of cancer-related genes, including KRAS and GNAS, in cfDNA isolated from PCFs irrespectively of the pancreatic cyst type. Further studies on molecular mechanisms of pancreatic cysts malignant transformation in relation to their mutational profiles are required.

mRNA expression profile of bronchoalveolar lavage fluid cells from patients with idiopathic pulmonary fibrosis and sarcoidosis.

BACKGROUND: Sarcoidosis and idiopathic pulmonary fibrosis (IPF) are two most frequent forms of interstitial lung diseases (ILDs). Cellular and biochemical composition of bronchoalveolar lavage fluid (BALf) was shown to reflect the fibrotic changes in the lung. However, the usefulness of BALf cellular profile evaluation in the diagnosis of ILDs is limited. The aim of the study was a multivariate, molecular analysis of BALf cells from IPF and sarcoidosis patients. METHODS: Transcriptomic measurements were carried out using Affymetrix Human Gene 2.1 ST ArrayStrip in 21 samples: 9 IPF and 12 sarcoidosis. The mRNA expression for the most significantly differentiating genes was evaluated by real-time PCR in 32 samples (11 IPF and 21 sarcoidosis). RESULTS: The number of genes differentially expressed between IPF and sarcoidosis groups was 4832 (13359 probesets). Cluster analysis indicated that sarcoidosis BALf cells are characterized by increased mRNA expression of genes associated with ribosome biogenesis. Clusters formed by genes with changed mRNA expression in IPF samples were implicated in the processes of cell adhesion and migration, metalloproteinase expression and negative regulation of cell proliferation. The GO analysis indicated that predominant biological processes associated with the differential mRNA gene expression of BALf cells were upregulation of neutrophils in IPF and lymphocytes in sarcoidosis. CONCLUSIONS: Analysis of BALf from sarcoidosis and IPF showed highly different mRNA profile of cells. The most important biological processes observed at the molecular level in BALf cells were associated with ribosome biogenesis and proteasome apparatus in sarcoidosis and neutrophilic dysfunction in IPF.

Candidate diagnostic miRNAs that can detect cancer in prostate biopsy.

BACKGROUND: While histopathological evaluation remains the gold standard for diagnosis of prostate cancer (PCa), sampling errors remain a frequent problem; therefore, use of tissue biomarkers that can distinguish between benign and malignant prostate disease is a potentially beneficial diagnostic strategy. METHODS: Deep sequencing of the miRNA transcriptome of 14 benign prostatic hyperplasia (BPH) and 60 cancerous and non-cancerous prostate samples extracted from 34 cancer-bearing prostates removed by prostatectomy was performed; of the latter 60 samples, 16, 21, and 23 samples contained <10%, >30%, and no dysplastic cells, respectively. The predictive value of selected miRNAs was then tested by quantitative reverse-transcribed PCR (qRT-PCR), using two separate chemistries, Exiqon and Taqman, to evaluate the tissue samples obtained by prostatectomy. Validation experiments were also performed for a subset of miRNAs by qRT-PCR of 87 prostate core biopsies. RESULTS: We identified 123 miRNAs significantly dysregulated in PCa (adjusted P-values <0.05); 110 and 13 miRNAs were dysregulated only in cancerous samples and non-cancerous samples extracted from cancer-bearing prostates, respectively, while 31 were dysregulated regardless of the dysplastic cell content of the studied specimens. The clinical utility of eight selected miRNAs was analyzed using the same sample set with two qRT-PCR chemistries. Measurable qRT-PCR signals were obtained for seven and six miRNAs using the Exiqon and Taqman chemistries, respectively, and expression levels of six and four of these miRNAs differed significantly between BPH and PCa samples, regardless of dysplastic cell content. Validation experiments on core biopsies using qRT-PCR confirmed differential expression between BPH and PCa of four miRNAs (miR-187-3p, miR-183-5p, miR-32-5p, and miR-141-5p) using the Exiqon and one miRNA (miR-187-3p) with the Taqman chemistry. CONCLUSIONS: Our sequencing analyses identified several candidate diagnostic miRNAs and confirmed some which have previously been reported as diagnostic in prostate malignancy. The results of this study suggest also that some of selected miRNAs can differentiate between non-malignant and malignant prostates even when neoplastic cells are missing from the studied specimen.

GWAS links variants in neuronal development and actin remodeling related loci with pseudoexfoliation syndrome without glaucoma.

Pseudoexfoliation syndrome (PEXS) is an age-related elastosis, strongly associated with the development of secondary glaucoma. It is clearly suggested that PEXS has a genetic component, but this has not been extensively studied. Here, a genome-wide association study (GWAS) using a DNA-pooling approach was conducted to explore the potential association of genetic variants with PEXS in a Polish population, including 103 PEXS patients without glaucoma and 106 perfectly (age- and gender-) matched controls. Individual sample TaqMan genotyping was used to validate GWAS-selected single-nucleotide polymorphism (SNP) associations. Multivariate binary logistic regression analysis was applied to develop a prediction model for PEXS. In total, 15 SNPs representing independent PEXS susceptibility loci were selected for further validation in individual samples. For 14 of these variants, significant differences in the allele and genotype frequencies between cases and controls were identified, of which 12 remained significant after Benjamini-Hochberg adjustment. The minor allele of five SNPs was associated with an increased risk of PEXS development, while for nine SNPs, it showed a protective effect. Beyond the known LOXL1 variant rs2165241, nine other SNPs were located within gene regions, including in OR11L1, CD80, TNIK, CADM2, SORBS2, RNF180, FGF14, FMN1, and RBFOX1 genes. None of these associations with PEXS has previously been reported. Selected SNPs were found to explain nearly 69% of the total risk of PEXS development. The overall risk prediction accuracy for PEXS, expressed by the area under the ROC curve (AUC) value, increased by 0.218, from 0.672 for LOXL1 rs2165241 alone to 0.89 when seven additional SNPs were included in the proposed 8-SNP prediction model. In conclusion, several new susceptibility loci for PEXS without glaucoma suggested that neuronal development and actin remodeling are potentially involved in either PEXS onset or inhibition or delay of its conversion to glaucoma.

Exome scale map of genetic alterations promoting metastasis in colorectal cancer.

BACKGROUND: Approximately 90% of colorectal cancer (CRC) deaths are caused by tumors ability to migrate into the adjacent tissues and metastase into distant organs. More than 40 genes have been causally linked to the development of CRC but no mutations have been associated with metastasis yet. To identify molecular basis of CRC metastasis we performed whole-exome and genome-scale transcriptome sequencing of 7 liver metastases along with their matched primary tumours and normal tissue. Multiple, spatially separated fragments of primary tumours were analyzed in each case. Uniformly malignant tissue specimen were selected with macrodissection, for three samples followed with laser microdissection. RESULTS: > 100 sequencing coverage allowed for detection of genetic alterations in subpopulation of tumour cells. Mutations in KRAS, APC, POLE, and PTPRT, previously associated with CRC development, were detected in most patients. Several new associations were identified, including PLXND1, CELSR3, BAHD1 and PNPLA6. CONCLUSIONS: We confirm the essential role of inflammation in CRC progression but question the mechanism of matrix metalloproteinases activation described in other work. Comprehensive sequencing data made it possible to associate genome-scale mutation distribution with gene expression patterns. To our knowledge, this is the first work to report such link in CRC metastasis context.

Somatic mutation profiling of vulvar cancer: Exploring therapeutic targets.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) constitutes over 90% of vulvar cancer. Its pathogenesis can follow two different pathways; high risk human papillomavirus (hrHPV)-dependent and HPV-independent. Due to the rarity of VSCC, molecular mechanisms underlying VSCC development remain largely unknown. The study aimed to identify pathogenic mutations implicated in the two pathways of VSCC development. METHODS: Using next generation sequencing, 81 VSCC tumors, 52 hrHPV(+) and 29 hrHPV(-), were screened for hotspot mutations in 50 genes covered by the Ion AmpliSeq Cancer Hotspot Panel v2 Kit (Thermo Fisher Scientific). RESULTS: Mutations of TP53 (46% and 41%, of hrHPV(+) and hrHPV(-) cases respectively) and CDKN2A (p16) (25% and 21%, of hrHPV(+) and hrHPV(-) cases respectively) were the most common genetic alterations identified in VSCC tumors. Further mutations were identified in PIK3CA, FBXW7, HRAS, FGFR3, STK11, AKT1, SMAD4, FLT3, JAK3, GNAQ, and PTEN, albeit at low frequencies. Some of the identified mutations may activate the PI3K/AKT/mTOR pathway. The activation of mTOR was confirmed in the vast majority of VSCC samples by immunohistochemical staining. CONCLUSIONS: Detecting pathogenic mutations in 13/50 genes examined at comparable frequencies in hrHPV(+) and hrHPV(-) tumors suggest that genetic mechanisms of the two routes of VSCC pathogenesis may be similar, despite being initiated from different premalignant lesions. Importantly, our data provide a rationale for new anti-VSCC therapies targeting the PI3K/AKT/mTOR pathway.

Telomere length and TERT abnormalities in pituitary adenomas.

OBJECTIVES: Pituitary adenomas (PAs) are among the most frequent intracranial tumors in humans. Abnormal telomerase activity and telomere lengthening are features of tumor cells. They may result from mutations in TERT promoter region, gene amplification or aberrant DNA methylation pattern. Such changes were found in variety of tumors including those of brain. Aim of the study was to evaluate the incidence of TERT abnormalities and to assess their role in telomere lengthening in PAs. METHODS: Study involved 101 patients with PA including both nonfunctioning and functioning subtypes. Telomerase length as well as TERT mRNA level and gene amplification were estimated using quantitative PCR (qPCR). Promoter mutations were assessed using Sanger sequencing. The results from genome-wide DNA methylation profiling with HumanMethylation 450K (Illumina) were used for the analysis of TERT locus. RESULTS: Variable telomere length was observed in patients, however no relationship with clinicopathological features was found. We observed a missense variant in TERT promoter in one patient only whereas increased TERT copy number were identified in 6 patients (5.6%). However no relationship between these results and telomere length or TERT expression was found. DNA methylation at TERT locus was not found to be changed when adenoma samples and normal tissue sections were compared. CONCLUSION: The results indicate that telomerase abnormalities do not play a role in pathogenesis of pituitary tumors.

Eosinophilic and Neutrophilic Airway Inflammation in the Phenotyping of Mild-to-Moderate Asthma and Chronic Obstructive Pulmonary Disease.

Asthma and chronic obstructive pulmonary disease (COPD) are heterogeneous diseases with different inflammatory phenotypes. Various inflammatory mediators play a role in these diseases. The aim of this study was to analyze the neutrophilic and eosinophilic airway and systemic inflammation as the phenotypic characterization of patients with asthma and COPD. Twenty-four patients with asthma and 33 patients with COPD were enrolled in the study. All the patients were in mild-to-moderate stage of disease, and none of them were treated with inhaled corticosteroids. Concentrations of IL-6, neutrophil elastase (NE), matrix metalloproteinase 9 (MMP-9), eosinophil cationic protein (ECP), and IL-33 and IL-17 in serum and induced sputum (IS) were measured by enzyme-linked immunosorbent assay (ELISA). The cellular composition of blood and IS was evaluated. Hierarchical clustering of patients was performed for the combination of selected clinical features and mediators. Asthma and COPD can be differentiated based on eosinophilic/neutrophilic systemic or airway inflammation with unsatisfactory efficiency. Hierarchical clustering of patients based on blood eosinophil percentage and clinical data revealed two asthma clusters differing in the number of positive skin prick tests and one COPD cluster with two subclusters characterized by low and high blood eosinophil concentrations. Clustering of patients according to IS measurements and clinical data showed two main clusters: pure asthma characterized by high eosinophil/atopy status and mixed asthma and COPD cluster with low eosinophil/atopy status. The neutrophilic phenotype of COPD was associated with more severe airway obstruction and hyperinflation.

miR expression in MYC-negative DLBCL/BL with partial trisomy 11 is similar to classical Burkitt lymphoma and different from diffuse large B-cell lymphoma.

Fast and reliable differential diagnosis of Burkitt lymphoma (BL) vs. diffuse large B cell lymphoma (DLBCL) is of major importance for therapeutic decisions and patient outcome. Aggressive B cell non-Hodgkin lymphomas (B-NHLs) that do not belong to the abovementioned entities were categorized by the current WHO lymphoma classification as "B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL" (DLBCL/BL). We have recently described a DLBCL/BL subgroup with recurrent chromosome 11q aberrations, resembling BL (B-NHLs[11q]). Here, we analyzed 102 prospectively collected fine needle aspirates from patients with aggressive B-NHLs in order to investigate the potential of microRNA (miR)-155, its precursor BIC, as well as miR-21 and miR-26a to differentiate BL from DLBCL, and from DLBCL/BL that include B-NHLs[11q]. Both BL and DLBCL/BL cases, including B-NHLs[11q], demonstrated significantly lower expression levels of miR-155/BIC, miR-21, and miR-26a compared to primary DLBCL. In conclusion, the miRs expression in B-NHLs[11q] provides a new suggestion, in addition to pathomorphological and clinical similarities between classical, i.e., MYC translocation-positive BL, and B-NHLs[11q], to recognize the B-NHLs[11q] subgroup of DLBCL/BL category as a MYC translocation-negative variant of BL in most cases, and points to the potential utility of miR-155/BIC/miR-21/miR-26a for the differential diagnosis of a heterogeneous category of DLBCL/BL.

Limited predictive value of achieving beneficial plasma (Z)-endoxifen threshold level by CYP2D6 genotyping in tamoxifen-treated Polish women with breast cancer.

BACKGROUND: Tamoxifen, the most frequently used drug for treating estrogen receptor-positive breast cancer, must be converted into active metabolites to exert its therapeutic efficacy, mainly through CYP2D6 enzymes. The objective of this study was to investigate the impact of CYP2D6 polymorphisms on (Z)-endoxifen-directed tamoxifen metabolism and to assess the usefulness of CYP2D6 genotyping for identifying patients who are likely to have insufficient (Z)-endoxifen concentrations to benefit from standard therapy. METHODS: Blood samples from 279 Polish women with breast cancer receiving tamoxifen 20 mg daily were analyzed for CYP2D6 genotype and drug metabolite concentration. Steady-state plasma levels of tamoxifen and its 14 metabolites were measured by using the ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. RESULTS: In nearly 60 % of patients, including over 30 % of patients with fully functional CYP2D6, (Z)-endoxifen concentration was below the predefined threshold of therapeutic efficacy. The most frequently observed CYP2D6 genotype was EM/PM (34.8 %), among which 83.5 % of patients had a combination of wild-type and *4 alleles. Plasma concentration of five metabolites was significantly correlated with CYP2D6 genotype. For the first time, we identified an association between decreased (E/Z)-4-OH-N-desmethyl-tamoxifen-ß-D-glucuronide levels (r (2) = 0.23; p < 10(-16)) and increased CYP2D6 functional impairment. The strongest correlation was observed for (Z)-endoxifen, whose concentration was significantly lower in groups of patients carrying at least one CYP2D6 null allele, compared with EM/EM patients. The CYP2D6 genotype accounted for plasma level variability of (Z)-endoxifen by 27 % (p < 10(-16)) and for the variability of metabolic ratio indicating (Z)-endoxifen-directed metabolism of tamoxifen by 51 % (p < 10(-43)). CONCLUSIONS: The majority of breast cancer patients in Poland may not achieve a therapeutic level of (Z)-endoxifen upon receiving a standard dose of tamoxifen. This finding emphasizes the limited value of CYP2D6 genotyping in routine clinical practice for identifying patients who might not benefit from the therapy. In its place, direct monitoring of plasma steady-state (Z)-endoxifen concentration should be performed to personalize and optimize the treatment.

Extracellular matrix and cytochrome P450 gene expression can distinguish steatohepatitis from steatosis in mice.

One of the main questions regarding nonalcoholic fatty liver disease is the molecular background of the transition from simple steatosis (SS) to the inflammatory and fibrogenic condition of steatohepatitis (NASH). We examined the gene expression changes during progression from histologically normal liver to SS and NASH in models of obesity caused by hyperphagia or a high-fat diet. Microarray-based analysis revealed that the expression of 1445 and 264 probe sets was changed exclusively in SS and NASH samples, respectively, and 1577 probe sets were commonly altered in SS and NASH samples. Functional annotations indicated that transcriptome alterations that were common for NASH and SS, as well as exclusive for NASH, involved extracellular matrix (ECM)-related processes, although they differed in the type of matrix structure change. The expression of 80 genes was significantly changed in all three comparisons: SS versus control, NASH versus control and NASH versus SS. Of these genes, epithelial membrane protein 1, IKBKB interacting protein and decorin were progressively up-regulated in both SS and NASH compared to normal tissue. The molecular context of interactions of encoded 80 proteins revealed that they are highly interconnected and significantly enriched for processes involving metabolism by cytochrome P450. Validation of 10 selected mRNAs encoding genes related to ECM and cytochrome P450 with quantitative RT-PCR analysis showed consistent changes in their expression during NASH development. The expression profile of these genes has the potential to distinguish NASH from SS and normal tissue and may possibly be beneficial in the clinical diagnosis of NASH.

Heterogeneous nuclear ribonucleoprotein (HnRNP) K genome-wide binding survey reveals its role in regulating 3'-end RNA processing and transcription termination at the early growth response 1 (EGR1) gene through XRN2 exonuclease.

The heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a nucleic acid-binding protein that acts as a docking platform integrating signal transduction pathways to nucleic acid-related processes. Given that hnRNPK could be involved in other steps that compose gene expression the definition of its genome-wide occupancy is important to better understand its role in transcription and co-transcriptional processes. Here, we used chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) to analyze the genome-wide hnRNPK-DNA interaction in colon cancer cell line HCT116. 9.1/3.6 and 7.0/3.4 million tags were sequenced/mapped, then 1809 and 642 hnRNPK binding sites were detected in quiescent and 30-min serum-stimulated cells, respectively. The inspection of sequencing tracks revealed inducible hnRNPK recruitment along a number of immediate early gene loci, including EGR1 and ZFP36, with the highest densities present at the transcription termination sites. Strikingly, hnRNPK knockdown with siRNA resulted in increased pre-RNA levels transcribed downstream of the EGR1 polyadenylation (A) site suggesting altered 3'-end pre-RNA degradation. Further ChIP survey of hnRNPK knockdown uncovered decreased recruitment of the 5'-3' exonuclease XRN2 along EGR1 and downstream of the poly(A) signal without altering RNA polymerase II density at these sites. Immunoprecipitation of hnRNPK and XRN2 from intact and RNase A-treated nuclear extracts followed by shotgun mass spectrometry revealed the presence of hnRNPK and XRN2 in the same complexes along with other spliceosome-related proteins. Our data suggest that hnRNPK may play a role in recruitment of XRN2 to gene loci thus regulating coupling 3'-end pre-mRNA processing to transcription termination.

Mitochondrial-related proteomic changes during obesity and fasting in mice are greater in the liver than skeletal muscles.

Although mitochondrial dysfunction is implicated in the pathogenesis of obesity, the molecular mechanisms underlying obesity-related metabolic abnormalities are not well established. We performed mitochondrial quantitative proteomic and whole transcriptome analysis followed by functional annotations within liver and skeletal muscles, using fasted and non-fasted 16- and 48-week-old high-fat diet (HFD)-fed and normal diet-fed (control group) wild-type C56BL/6J mice, and hyperphagic ob/ob and db/db obese mice. Our study identified 1,675 and 704 mitochondria-associated proteins with at least two peptides in liver and muscle, respectively. Of these, 221 liver and 44 muscle proteins were differentially expressed (adjusted p values ≤ 0.05) between control and all obese mice, while overnight fasting altered expression of 107 liver and 35 muscle proteins. In the liver, we distinguished a network of 27 proteins exhibiting opposite direction of expression changes in HFD-fed and hyperphagic mice when compared to control. The network centered on cytochromes P450 3a11 (Cyp3a11) and 4a14 (Cyp4a14), and fructose-bisphosphate aldolase B (Aldob) proteins which bridged proteins cluster involved in Metabolism of xenobiotics with proteins engaged in Fatty acid metabolism and PPAR signaling pathways. Functional annotations revealed that most of the hepatic molecular alterations, which characterized both obesity and fasting, related to different aspects of energy metabolism (such as Fatty acid metabolism, Peroxisome, and PPAR signaling); however, only a limited number of functional annotations could be selected from skeletal muscle data sets. Thus, our comprehensive molecular overview revealed that both obesity and fasting states induce more pronounced mitochondrial proteome changes in the liver than in the muscles.

Effects of Soluble Dextrin Fiber from Potato Starch on Body Weight and Associated Gut Dysbiosis Are Evident in Western Diet-Fed Mice but Not in Overweight/Obese Children.

BACKGROUND: The study investigated the impact of starch degradation products (SDexF) as prebiotics on obesity management in mice and overweight/obese children. METHODS: A total of 48 mice on a normal diet (ND) and 48 on a Western diet (WD) were divided into subgroups with or without 5% SDexF supplementation for 28 weeks. In a human study, 100 overweight/obese children were randomly assigned to prebiotic and control groups, consuming fruit and vegetable mousse with or without 10 g of SDexF for 24 weeks. Stool samples were analyzed for microbiota using 16S rRNA gene sequencing, and short-chain fatty acids (SCFA) and amino acids (AA) were assessed. RESULTS: Results showed SDexF slowed weight gain in female mice on both diets but only temporarily in males. It altered bacterial diversity and specific taxa abundances in mouse feces. In humans, SDexF did not influence weight loss or gut microbiota composition, showing minimal changes in individual taxa. The anti-obesity effect observed in mice with WD-induced obesity was not replicated in children undergoing a weight-loss program. CONCLUSIONS: SDexF exhibited sex-specific effects in mice but did not impact weight loss or microbiota composition in overweight/obese children.

microRNAs in uterine sarcomas and mixed epithelial-mesenchymal uterine tumors: a preliminary report.

Uterine sarcomas and mixed epithelial-mesenchymal uterine tumors are a heterogeneous group of rare tumors for which there are very few diagnostic markers available. As aberrant microRNA (miRNA) expression patterns represent putative diagnostic cancer markers, we aimed to identify miRNA expression profiles of the major uterine sarcoma subtypes and mixed epithelial-mesenchymal tumors of the uterus. Eighty-eight miRNAs were assessed by quantitative RT-PCR in cancerous and non-cancerous tissue samples collected from 29 patients with endometrial sarcoma, leiomyosarcoma, and mixed epithelial-mesenchymal tumors. Tumor and control samples significantly (P < 0.05) differed in the expression of miR-23b, miR-1, let-7f, and let-7c in endometrial sarcomas, and miR-1, let-7c, miR-133b, let-7b, miR-143, let-7a, let-7d, let-7e, let-7g, miR-222, let-7i, and miR-214 in mixed epithelial-mesenchymal tumors. All the significantly changed miRNAs were down-regulated in the malignant tissues as compared to their normal counterparts. This may suggest their tumor suppressor role in these malignancies. No statistically significant changes in miRNA expression levels were found between leiomyosarcoma tumors and controls. The identified miRNAs warrant further studies as valuable candidate markers for the differential diagnosis of uterine sarcomas from benign uterine lesions and between uterine sarcoma subtypes.