Gut microbiota, dietary intakes and intestinal permeability reflected by serum zonulin in women.

PURPOSE: Increased gut permeability causes the trespass of antigens into the blood stream which leads to inflammation. Gut permeability reflected by serum zonulin and diversity of the gut microbiome were investigated in this cross-sectional study involving female study participants with different activity and BMI levels. METHODS: 102 women were included (BMI range 13.24-46.89 kg m-2): Anorexia nervosa patients (n = 17), athletes (n = 20), normal weight (n = 25), overweight (n = 21) and obese women (n = 19). DNA was extracted from stool samples and subjected to 16S rRNA gene analysis (V1-V2). Quantitative Insights Into Microbial Ecology (QIIME) was used to analyze data. Zonulin was measured with ELISA. Nutrient intake was assessed by repeated 24-h dietary recalls. We used the median of serum zonulin concentration to divide our participants into a "high-zonulin" (> 53.64 ng/ml) and "low-zonulin" (< 53.64 ng/ml) group. RESULTS: The alpha-diversity (Shannon Index, Simpson Index, equitability) and beta-diversity (unweighted and weighted UniFrac distances) of the gut microbiome were not significantly different between the groups. Zonulin concentrations correlated significantly with total calorie-, protein-, carbohydrate-, sodium- and vitamin B12 intake. Linear discriminant analysis effect size (LEfSe) identified Ruminococcaceae (LDA = 4.163, p = 0.003) and Faecalibacterium (LDA = 4.151, p = 0.0002) as significantly more abundant in the low zonulin group. CONCLUSION: Butyrate-producing gut bacteria such as Faecalibacteria could decrease gut permeability and lower inflammation. The diversity of the gut microbiota in women does not seem to be correlated with the serum zonulin concentration. Further interventional studies are needed to investigate gut mucosal permeability and the gut microbiome in the context of dietary factors.

Beyond the BRAFV600E hotspot: biology and clinical implications of rare BRAF gene mutations in melanoma patients.

BRAF gene mutations can be found in approximately 50% of melanomas, but the most common BRAF mutation leads to substitution at residue 600 of the protein, from valine to glutamic acid. BRAFV600E occurs in up to 95% of all melanoma cases and can be successfully blocked by using a combination of BRAF- and MEK inhibitors. The wider availability of next-generation sequencing is revealing more non-V600 BRAF mutations, and the clinical implications of these mutations are widely unknown. In this review, we will discuss the biology of the MAPK pathway and the different types of BRAF mutations as well as their effect on MEK activation. Current literature will be reviewed including in vitro data, case reports and case series.

Peritoneal papillary mesothelioma in situ: BAP1 mutation with indolent behavior for 15 years.

Papillary mesothelioma in situ (PMIS) is a rare and enigmatic disease. Most instances manifest as lesions of the peritoneal serosa. The pathogenesis and behavior of peritoneal PMIS are still poorly understood, and separation from benign well differentiated peritoneal mesothelial tumors (WDPMT) may be challenging. We describe the 15-year long course of a PMIS in an adult male in which inactivating mutations of BAP1, encoding BRCA1 associated protein 1 (BAP1), were identified. Tumor samples were obtained on 2 occasions more than 8y apart. In both samples, the tumor cells were bland, with occasional focal infiltration into the stalks of larger papillary lesions. However, no invasion into subserosal adipose tissue was identified. In both samples the tumor cells did not express nuclear BAP1. Comprehensive genomic analysis of the initial tumor sample revealed a somatic inactivating mutation in BAP1 (predicted effect, Y223*) and a somatic variant of IRS2 (A701_V702insAA). An additional inactivating mutation in BAP1 (predicted effect, T69fs*5) was detected in the later sample. The patient did not receive any treatment and is still alive 15 years after initial presentation. Our experience supports the view that peritoneal PMIS may follow an indolent course for many years and prompts the question whether these tumors should uniformly be treated aggressively.

Real-world EGFR testing practices for non-small-cell lung cancer by thoracic pathology laboratories across Europe.

BACKGROUND: Testing for epidermal growth factor receptor (EGFR) mutations is an essential recommendation in guidelines for metastatic non-squamous non-small-cell lung cancer, and is considered mandatory in European countries. However, in practice, challenges are often faced when carrying out routine biomarker testing, including access to testing, inadequate tissue samples and long turnaround times (TATs). MATERIALS AND METHODS: To evaluate the real-world EGFR testing practices of European pathology laboratories, an online survey was set up and validated by the Pulmonary Pathology Working Group of the European Society of Pathology and distributed to 64 expert testing laboratories. The retrospective survey focussed on laboratory organisation and daily EGFR testing practice of pathologists and molecular biologists between 2018 and 2021. RESULTS: TATs varied greatly both between and within countries. These discrepancies may be partly due to reflex testing practices, as 20.8% of laboratories carried out EGFR testing only at the request of the clinician. Many laboratories across Europe still favour single-test sequencing as a primary method of EGFR mutation identification; 32.7% indicated that they only used targeted techniques and 45.1% used single-gene testing followed by next-generation sequencing (NGS), depending on the case. Reported testing rates were consistent over time with no significant decrease in the number of EGFR tests carried out in 2020, despite the increased pressure faced by testing facilities during the COVID-19 pandemic. ISO 15189 accreditation was reported by 42.0% of molecular biology laboratories for single-test sequencing, and by 42.3% for NGS. 92.5% of laboratories indicated they regularly participate in an external quality assessment scheme. CONCLUSIONS: These results highlight the strong heterogeneity of EGFR testing that still occurs within thoracic pathology and molecular biology laboratories across Europe. Even among expert testing facilities there is variability in testing capabilities, TAT, reflex testing practice and laboratory accreditation, stressing the need to harmonise reimbursement technologies and decision-making algorithms in Europe.

First presentation of a frameshift mutation in the SETD2 gene of a juvenile psammomatoid ossifying fibroma (JPOF) associated with an aneurysmal bone cyst.

BACKGROUND: The rarity of juvenile psammomatoid ossifying fibroma (JPOF) and lack of cytogenetic studies prompted us to report a novel SETD2 gene mutation in a benign odontogenic tumour. CASE PRESENTATION: A 21-year-old man presented with a hard, expanded mandibular cortex. Computed tomography revealed multilocular radiopacity in the mandible; this was reconstructed via segmental mandibulectomy using a vascularised iliac crest flap. Based on the clinical and histological findings, we diagnosed JPOF associated with an aneurysmal bone cyst. Microscopically, the solid area was characterised by many rounded or angular ossicles in a cellular fibrous stroma. The stromal cells were spindle-like or stellate. Next-generation sequencing detected a frame shift mutation of the SETD2 gene, while the copy number was normal. CONCLUSIONS: Our findings suggest further genetic studies should be performed to assess whether this mutation is related to tumour genesis. .

Low-dose antibiotic prophylaxis has no significant impact on the stability of the intestinal microbiome in children with urogenital tract malformations under 1 year of age.

INTRODUCTION: Accumulating data point at potentially lasting effects of early childhood therapeutic antibiotic exposure on the intestinal microbial. Little is known on the impact of low-dose longterm antibiotic prophylaxis on the developing intestinal microbiota in children during their first year of life. OBJECTIVE: To investigate compositional changes of the intestinal microbiota by next generation sequencing based microbiome analysis and bacterial metabolites in longitudinally collected fecal samples. STUDY DESIGN: Twelve patients were analyzed in this prospective, longitudinal pilot study during a period of 70 days (sampling on days 0,7,14,30,70). Only transvaginally and term born babies, breastfed with no prior antibiotic exposure with urogenital malformation (vesicoureteral reflux and/or upper urinary tract dilatation) were included into the study. Seven patients received antibiotic longterm prophylaxis with a second-generation cephalosporin and five did not. Sequencing of bacterial 16 S rRNA allowed for an analysis of the microbiome composition. The Principal coordinate analysis was performed for the evaluation of compositional profile. Furthermore, quantitative measurement of short chain fatty acids served as a proxy for the metabolic activity of the individual microbiome over the study time. RESULTS: Analysis of observed species, Shannon Index and weighted Unifrac distances between timepoints revealed neither significant difference comparing the prophylaxis group versus the control group over the study period, nor significant changes within the groups over time. Principal coordinate analysis (PCoA) was performed for the evaluation of compositional profile. Also, no differences regarding the fecal SCFA content were found between the two groups (>0.05 at each tested point, Mann-Whitney Test). DISCUSSION: Although there were interindividual compositional differences of the microbiome (cluster of bacterial composition) at the beginning of the observation, we did not observe significant longitudinal changes regarding both bacterial diversity and SCFAs in neither group. Over the study period, the patient's microbiome remained stable and resilient to the antibiotic exposure in terms of bacterial abundance and metabolism. Limitations to the study are the low number of patients included and the use of one single antibiotic (cefaclor). CONCLUSION: This is the first pilot study to demonstrate that long term low-dose antibiotic administration in children under one year of age does neither seem to influence the composition of the intestinal microbiota nor the quantities of bacterial fermentation products compared to untreated controls.

Epigenetics of the molecular clock and bacterial diversity in bipolar disorder.

Objectives The gut microbiome harbors substantially more genetic material than our body cells and has an impact on a huge variety of physiological mechanisms including the production of neurotransmitters and the interaction with brain functions through the gut-brain-axis. Products of microbiota can affect methylation according to preclinical studies. The current investigation aimed at analyzing the correlation between gut microbiome diversity and the methylation of the clock gene ARNTL in individuals with Bipolar Disorder (BD). Methods Genomic DNA was isolated from fasting blood of study participants with BD (n = 32). The methylation analysis of the ARNTL CG site cg05733463 was performed by bisulfite treatment of genomic DNA with the Epitect kit, PCR and pyrosequencing. Additionally, DNA was extracted from stool samples and subjected to 16S rRNA sequencing. QIIME was used to analyze microbiome data. Results Methylation status of the ARNTL CpG position cg05733463 correlated significantly with bacterial diversity (Simpson index: r= -0.389, p = 0.0238) and evenness (Simpson evenness index: r= -0.358, p = 0.044). Furthermore, bacterial diversity differed significantly between euthymia and depression (F(1,30) = 4.695, p = 0.039). Discussion The results of our pilot study show that bacterial diversity differs between euthymia and depression. Interestingly, gut microbiome diversity and evenness correlate negatively with methylation of ARNTL, which is known to regulate monoamine oxidase A transcription. We propose that alterations in overall diversity of the gut microbiome represent an internal environmental factor that has an epigenetic impact on the clock gene ARNTL which is thought to be involved in BD pathogenesis.

A stress-induced early innate response causes multidrug tolerance in melanoma.

Acquired drug resistance constitutes a major challenge for effective cancer therapies with melanoma being no exception. The dynamics leading to permanent resistance are poorly understood but are important to design better treatments. Here we show that drug exposure, hypoxia or nutrient starvation leads to an early innate cell response in melanoma cells resulting in multidrug resistance, termed induced drug-tolerant cells (IDTCs). Transition into the IDTC state seems to be an inherent stress reaction for survival toward unfavorable environmental conditions or drug exposure. The response comprises chromatin remodeling, activation of signaling cascades and markers implicated in cancer stemness with higher angiogenic potential and tumorigenicity. These changes are characterized by a common increase in CD271 expression concomitantly with loss of differentiation markers such as melan-A and tyrosinase, enhanced aldehyde dehydrogenase (ALDH) activity and upregulation of histone demethylases. Accordingly, IDTCs show a loss of H3K4me3, H3K27me3 and gain of H3K9me3 suggesting activation and repression of differential genes. Drug holidays at the IDTC state allow for reversion into parental cells re-sensitizing them to the drug they were primarily exposed to. However, upon continuous drug exposure IDTCs eventually transform into permanent and irreversible drug-resistant cells. Knockdown of CD271 or KDM5B decreases transition into the IDTC state substantially but does not prevent it. Targeting IDTCs would be crucial for sustainable disease management and prevention of acquired drug resistance.

A stress-induced early innate response causes multidrug tolerance in melanoma.

Correction to: Oncogene (2015) 34, 4448­4459; doi:10.1038/onc.2014.372; published online 24 November 2014. In this article, published online 24 November 2014, the authors have noticed that the latest supplementary information was not used. The corrected supplementary information (Supplementary Materials) appears online together with this corrigendum. The authors would like to apologise for any inconvenience this may cause

The disease relevance of human hepatocellular xenograft models: molecular characterization and review of the literature.

In recent years a number of new therapeutics has been developed that were not general toxins and inhibitors of cell division like classical chemotherapeutics, but were designed to target a specific pathway. A prerequisite for this development was the comprehensive characterization of molecular alterations occurring in human hepatocellular carcinoma (HCC). However, while much knowledge of the molecular pathogenesis of human HCC has been gained, the model systems used to test the functional relevance of these alterations and applied for preclinical evaluation of drug candidates are still poorly characterized. In this paper, we reviewed the literature about several commonly used HCC cell lines and xenotransplantation models and present our own data on the molecular characterization of these. Results obtained demonstrate that it is important to have a sound knowledge of the specific molecular constitution of the experimental model and to carefully evaluate the functional status of the pathway of interest. For this reason, we make the gene expression profiles publicly available to help researchers making an informed decision about which model to use.

Gene therapy progress and prospects: stem cell plasticity.

With the identification of stem cell plasticity several years ago, multiple reports raised hopes that tissue repair by stem cell transplantation could be within reach in the near future. Krause et al reported that a single purified hematopoietic stem cell not only repopulated the bone marrow of a host animal, but also integrated into unrelated tissues. Lagasse et al demonstrated that in a genetic model of liver disease, purified hematopoietic stem cells can give rise to hepatocytes and rescue fatal liver damage. More recent work by Jiang et al demonstrated that cultured cells can retain their stem cell potential. There are a number of possible mechanisms that could explain these phenomena, and recent experiments have raised controversy about which mechanism is prevalent. One possibility is transdifferentiation of a committed cell directly into another cell type as a response to environmental cues. Transdifferentiation has been shown mainly in vitro, but some in vivo data also support this mechanism. Direct transdifferentiation would clinically be limited by the number of cells that can be introduced into an organ without removal of resident cells. If bone marrow cells could on the other hand give rise to stem cells of another tissue, then they could in theory repopulate whole organs from a few starting cells. This model of dedifferentiation is consistent with recent data from animal models. Genetic analysis of cells of donor origin in vivo and in vitro has brought to light another possible mechanism. The fusion of host and donor cells can give rise to mature tissue cells without trans- or dedifferentiation. The resulting heterokaryons are able to cure a lethal genetic defect and do not seem to be prone to give rise to cancer. All these models will clinically face the problem of accessibility of healthy primary cells for transplantation. This underlines the importance of the recent identification of a population of mesenchymal stem cells (MSCs) with stem cell properties similar to embryonic stem (ES) cells. These cells can be cultured and expanded in vitro without losing their stem cell potential making them an attractive target for cell therapy. Finally, it is still not clear if stem cells for various tissues are present in peripheral blood, or bone marrow and thus can be directly purified from these sources. Identification of putative tissue stem cells would be necessary before purification strategies can be devised. In this review, we discuss the evidence for these models, and the conflicting results obtained to date.