Basal MET phosphorylation is an indicator of hepatocyte dysregulation in liver disease.

Chronic liver diseases are worldwide on the rise. Due to the rapidly increasing incidence, in particular in Western countries, metabolic dysfunction-associated steatotic liver disease (MASLD) is gaining importance as the disease can develop into hepatocellular carcinoma. Lipid accumulation in hepatocytes has been identified as the characteristic structural change in MASLD development, but molecular mechanisms responsible for disease progression remained unresolved. Here, we uncover in primary hepatocytes from a preclinical model fed with a Western diet (WD) an increased basal MET phosphorylation and a strong downregulation of the PI3K-AKT pathway. Dynamic pathway modeling of hepatocyte growth factor (HGF) signal transduction combined with global proteomics identifies that an elevated basal MET phosphorylation rate is the main driver of altered signaling leading to increased proliferation of WD-hepatocytes. Model-adaptation to patient-derived hepatocytes reveal patient-specific variability in basal MET phosphorylation, which correlates with patient outcome after liver surgery. Thus, dysregulated basal MET phosphorylation could be an indicator for the health status of the liver and thereby inform on the risk of a patient to suffer from liver failure after surgery.

Tumour cells can escape antiproliferative pressure by interferon-ß through immunoediting of interferon receptor expression.

Type I interferons (IFNs) play a central role not only in innate immunity against viral infection, but also in the antitumour response, e.g. through a direct impact on cell proliferation. Particularly for cancer arising in the context of chronic inflammation, constant exposure to IFNs may constitute a strong selective pressure during tumour evolution. Expansion of neoplastic subclones resistant to the antiproliferative effects of IFNs may contribute to immunoediting of tumours, leading to more aggressive disease. Experimental evidence for this development of IFN-insensitivity has been scarce and its molecular mechanism is unclear. In this study we demonstrate that six weeks exposure of cells to IFN-ß in vitro reduces their sensitivity to its antiproliferative effects, and that this phenotype was stable for up to four weeks. Furthermore, we observed substantial differences in cellular sensitivity to growth inhibition by IFN-ß in a panel of ten different liver cancer cell lines, most prominently in a pair of highly dedifferentiated cell lines, and least in cells from well-differentiated tumours. In both, long-term IFN selection and in dedifferentiated tumour cell lines, we found IFNAR2 expression to be substantially reduced, suggesting the receptor complex to be a sensitive target amenable to immunoediting. Beyond new insights into possible molecular processes in tumour evolution, these findings might prove valuable for the development of biomarkers allowing to stratify tumours for their sensitivity to IFN treatment in the context of patient tailored therapies.

Heart Murmurs in Children: Evaluation and Management.

Up to 8.6% of infants and 80% of children have a heart murmur during their early years of life. The presence of a murmur can indicate conditions ranging from no discernable pathology to acquired or congenital heart disease. In infants with a murmur, physicians should review the obstetric and family histories to detect the possibility of congenital heart pathologies. Evaluation by a pediatric cardiologist is indicated for newborns with a murmur because studies show that neonatal murmurs have higher rates of pathology than in older children, and neonatal murmur characteristics are more difficult to evaluate during examination; referral is preferred over echocardiography. All infants, with or without a murmur, should have pulse oximetry screening to detect underlying critical congenital heart disease. In older children, most murmurs are innocent and can be followed with serial examinations if there are no findings of concern. Findings in older children that warrant referral include diastolic murmurs, loud or harsh-sounding murmurs, holosystolic murmurs, murmurs that radiate to the back or neck, or signs or symptoms of cardiac disease. Referral to a pediatric cardiologist is indicated when a pathologic murmur is suspected. Electrocardiography, chest radiography, and other tests should not be reflexively performed as part of all murmur evaluations because these tests can misclassify a murmur as innocent or pathologic, and they are not cost-effective. Emerging technologies include phonocardiography interpretation of murmurs and artificial intelligence algorithms for differentiating innocent from pathologic murmurs.

Identification of Novel Micropeptides Derived from Hepatocellular Carcinoma-Specific Long Noncoding RNA.

Identification of cancer-specific target molecules and biomarkers may be useful in the development of novel treatment and immunotherapeutic strategies. We have recently demonstrated that the expression of long noncoding (lnc) RNAs can be cancer-type specific due to abnormal chromatin remodeling and alternative splicing. Furthermore, we identified and determined that the functional small protein C20orf204-189AA encoded by long intergenic noncoding RNA Linc00176 that is expressed predominantly in hepatocellular carcinoma (HCC), enhances transcription of ribosomal RNAs and supports growth of HCC. In this study we combined RNA-sequencing and polysome profiling to identify novel micropeptides that originate from HCC-specific lncRNAs. We identified nine lncRNAs that are expressed exclusively in HCC cells but not in the liver or other normal tissues. Here, DNase-sequencing data revealed that the altered chromatin structure plays a key role in the HCC-specific expression of lncRNAs. Three out of nine HCC-specific lncRNAs contain at least one open reading frame (ORF) longer than 50 amino acid (aa) and enriched in the polysome fraction, suggesting that they are translated. We generated a peptide specific antibody to characterize one candidate, NONHSAT013026.2/Linc013026. We show that Linc013026 encodes a 68 amino acid micropeptide that is mainly localized at the perinuclear region. Linc013026-68AA is expressed in a subset of HCC cells and plays a role in cell proliferation, suggesting that Linc013026-68AA may be used as a HCC-specific target molecule. Our finding also sheds light on the role of the previously ignored 'dark proteome', that originates from noncoding regions in the maintenance of cancer.

Metagenomic analysis of the gastric microbiota cultivable from a patient with gastritis concomitant with Barrett's esophagus.

Barrett's esophagus is a distal metaplasia characterized by the transformation of squamous mucosa into columnar mucosa. This esophageal phenotype is a product not only of the chronic reflux of gastric acids, but also by microorganisms that colonize the oral cavity and stomach. Two classes of microbiota can be identified in Barrett's esophagus; microbiota type I is associated with the normal esophagus and type II with an inflamed esophagus. The present study describes the gastric microbiota of a patient with antral gastritis concomitant with Barrett's esophagus absent infection with Helicobacter pylori. Gastric biopsies were obtained following the protocol of Sydney and following ethical practices. The isolates were cultivated under microaerophilic conditions on Columbia Agar supplemented with IsoVitaleX™ and 7% sterile blood. Extracted DNA was sequenced using 454-GS and the results analyzed on the MG-RAST server. Gram negative isolates were found and bacteria resistant to levofloxacin, amoxicillin, tetracycline, erythromycin, and clarithromycin. The phyla Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria, the genus Bacteroides and the species group Bacteroides fragilis were most abundant. Functionally, the metabolism of carbohydrates, amino acids, and to a lesser extent, the metabolism of cofactors and vitamins were most dominant, and of which the enzymes ß-glucosidase (EC 3.2.1.21), ß-galactosidase (EC 3.2.1.23) and ß-N-acetylhexosaminidase (EC 3.2.1.52) were most dominant. The findings of this study, because they are of only one case may probably suggest a possible pathogenic role, previously undescribed for Bacteroides fragilis, associated with human gastritis when concomitant esophageal pathology exists.

Experiences of Deployed Physicians in Support of Operation Allies Refuge/Operation Allies Welcome: Lessons to Inform Improvements in Training.

INTRODUCTION: Stability operations, including humanitarian assistance and disaster relief missions, are key functions of U.S. Military medicine and the Military Medical Humanitarian Assistance Course (MMHAC) is a 2-day course widely used to prepare military medical personnel for such missions. It focuses on caring for those most vulnerable in the wake of disasters, particularly children. The large-scale humanitarian deployment of military medical providers in support of Operation Allies Welcome/Operation Allies Refuge (OAW/OAR) presents an opportunity to evaluate the preparedness of these providers to care for the needs of the Afghan travelers, so we explored the experiences of military medical providers deployed in support of OAW/OAR to inform improvements in the MMHAC. MATERIALS AND METHODS: We conducted a qualitative study of military medical providers who were deployed in support of OAW/OAR using a series of three virtual focus groups. Focus group questions were structured around the main topics covered in the MMHAC (patient care, ethical considerations, logistical concerns, and preventive and public health) and explicitly asked about adaptive leadership challenges faced and strategies used to overcome them. We analyzed transcripts using inductive thematic analysis within a constructivist paradigm, with adaptive leadership as a sensitizing concept. The study was approved by the Institutional Review Board of Uniformed Services University. RESULTS: We constructed 4 themes from participant responses, each addressing challenges that medical providers faced during their mission: (1) Medical providers navigated tension between medical and public health priorities and military mission priorities; (2) Chronic and complex care needs posed unique challenges for medical personnel; (3) Challenges in patient care were compounded by logistical and system-based barriers; and (4) Cultural barriers led to ethical dilemmas that physicians felt inadequately prepared to handle, most notably with respect to gender-related concerns. Within each theme, participants described which aspects of MMHAC training were most helpful and which areas were inadequate. CONCLUSIONS: Physicians found the OAR/OAW mission meaningful but also identified challenges related to medical care provision, public health, logistics, and ethical dilemmas that hindered their ability to carry out their medical mission. Lessons learned from OAW/OAR highlight several areas in which the MMHAC training could be augmented and improved to further mitigate these challenges.

Mapping alternative polyadenylation in human cells using direct RNA sequencing technology.

Alternative cleavage and polyadenylation (APA) is a widespread mechanism to generate mRNA isoforms with alternative 3' untranslated regions. Here, we detail a protocol for detecting APA genome wide using direct RNA sequencing technology including computational analysis. We describe steps for RNA sample and library preparation, nanopore sequencing, and data analysis. Experiments and data analysis can be performed over a period of 6-8 days and require molecular biology and bioinformatics skills. For complete details on the use and execution of this protocol, please refer to Polenkowski et al.1.

THOC5 complexes with DDX5, DDX17, and CDK12 to regulate R loop structures and transcription elongation rate.

THOC5, a member of the THO complex, is essential for the 3'processing of some inducible genes, the export of a subset of mRNAs and stem cell survival. Here we show that THOC5 depletion results in altered 3'cleavage of >50% of mRNAs and changes in RNA polymerase II binding across genes. THOC5 is recruited close to high-density polymerase II sites, suggesting that THOC5 is involved in transcriptional elongation. Indeed, measurement of elongation rates in vivo demonstrated decreased rates in THOC5-depleted cells. Furthermore, THOC5 is preferentially recruited to its target genes in slow polymerase II cells compared with fast polymerase II cells. Importantly chromatin-associated THOC5 interacts with CDK12 (a modulator of transcription elongation) and RNA helicases DDX5, DDX17, and THOC6 only in slow polymerase II cells. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate a novel function of THOC5 in transcription elongation.

C20orf204, a hepatocellular carcinoma-specific protein interacts with nucleolin and promotes cell proliferation.

In most human cancers, a large number of proteins with driver mutations are involved in tumor development, implying that multiple fine tuners are involved in cancer formation and/or maintenance. A useful strategy for cancer therapy may therefore be to target multiple cancer type-specific fine tuners. Furthermore, genome-wide association studies of tumor samples have identified a large number of long noncoding (lnc)RNA associated with various types of tumor. In this context we have previously found that C20orf204 (a splice variant of Linc00176) RNA contains a 189 amino acid (AA) long open reading frame (C20orf204-189AA) that is expressed predominantly in hepatocellular carcinoma (HCC). We report here that a protein, C20orf204-189AA, was detected in the nucleus of 14 out of 20 primary HCC, but not in control livers. Strikingly, overexpression of C20orf204-189AA enhanced cell proliferation and ribosomal RNA transcription. C20orf204-189AA is co-localized, and interacted with nucleolin via the C-terminal and with ribosomal RNA via the N-terminal domain. Furthermore, the expression of C20orf204-189AA upregulates the protein level of nucleolin. Nucleolin and C20orf204 mRNA levels in HCC are correlated with tumor differentiation grade and patient survival, suggesting that C20orf204-189AA is a cancer type-specific fine tuner in some HCC that presents itself for potential targeting therapy and cancer biomarker. Thus, cancer cells exhibit remarkable transcriptome alterations partly by adopting cancer-specific splicing isoforms of noncoding RNAs and may participate in tumor development.

Metagenomic analysis of the gastric microbiota cultivable from a patient with gastritis concomitant with Barrett´s esophagus / Análisis metagenómico de la microbiota gástrica cultivable a partir de un paciente con gastritis concomitante con esófago de Barrett

Barrett’s esophagus is a distal metaplasia characterized by the transformation of squamous mucosa into columnar mucosa. This esophageal phenotype is a product not only of the chronic reflux of gastric acids, but also by microorganisms that colonize the oral cavity and stomach. Two classes of microbiota can be identified in Barrett’s esophagus; microbiota type I is associated with the normal esophagus and type II with an inflamed esophagus. The present study describes the gastric microbiota of a patient with antral gastritis concomitant with Barrett’s esophagus absent infection with Helicobacter pylori. Gastric biopsies were obtained following the protocol of Sydney and following ethical practices. The isolates were cultivated under microaerophilic conditions on Columbia Agar supplemented with IsoVitaleX™ and 7% sterile blood. Extracted DNA was sequenced using 454-GS and the results analyzed on the MG-RAST server. Gram negative isolates were found and bacteria resistant to levofloxacin, amoxicillin, tetracycline, erythromycin, and clarithromycin. The phyla Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria, the genus Bacteroides and the species group Bacteroides fragilis were most abundant. Functionally, the metabolism of carbohydrates, amino acids, and to a lesser extent, the metabolism of cofactors and vitamins were most dominant, and of which the enzymes β-glucosidase (EC 3.2.1.21), β-galactosidase (EC 3.2.1.23) and β-N-acetylhexosaminidase (EC 3.2.1.52) were most dominant. The findings of this study, because they are of only one case may probably suggest a possible pathogenic role, previously undescribed for Bacteroides fragilis, associated with human gastritis when concomitant esophageal pathology exists.

El esófago de Barrett es una metaplasia distal caracterizada por la transformación de la mucosa escamosa a mucosa columnar. Este fenotipo esofágico es producto no solo de la exposición crónica al reflujo de ácidos gástricos sino también a microbios colonizantes de la cavidad oral y del estómago. El esófago Barrett presenta 2 clases de microbiotas; la microbiota tipo I asociada con esófago normal y la tipo II a fenotipos esofágicos inflamatorios. En el presente estudio se describió la microbiota gástrica de una paciente con gastritis antral concomitante con esófago de Barrett sin infección por Helicobacter pylori y se obtuvieron biopsias gástricas siguiendo el protocolo de Sydney y estándares bioéticos. Los cultivos se hicieron en condiciones microaerofílicas en agar Columbia suplementados con isovitalex y sangre estéril al 7%. El ADN extraído fue sometido a secuenciación empleando 454 GS y las lecturas fueron analizadas en el servidor MG-RAST. Se obtuvieron aislamientos gram-negativos y resistentes a levofloxacina, amoxicilina, tetraciclina, eritromicina y claritromicina. Los Phylum Bacteroidetes, Firmicutes, Fusobacteria y Proteobacteria, el género Bacteroides y las especies de grupo Bacteroides fragilis fueron los más abundantes. Funcionalmente, el metabolismo de carbohidratos, aminoácidos y en menor grado el metabolismo de cofactores y vitaminas fueron los más dominantes; de los cuales las enzimas la β-glicosidasa (EC 3.2.1.21), β-galactosidasa (EC 3.2.1.23) y la β-N-acetilhexosaminidasa (EC 3.2.1.52) fueron las más dominantes. Estos resultados, por ser de un solo caso, solo podrían sugerir un posible papel patogénico no descrito para Bacterioides fragilis asociado con gastritis humana cuando existe patología esofágica concomitante.