Single-cell bacterial transcription measurements reveal the importance of dimethylsulfoniopropionate (DMSP) hotspots in ocean sulfur cycling.

Dimethylsulfoniopropionate (DMSP) is a pivotal compound in marine biogeochemical cycles and a key chemical currency in microbial interactions. Marine bacteria transform DMSP via two competing pathways with considerably different biogeochemical implications: demethylation channels sulfur into the microbial food web, whereas cleavage releases sulfur into the atmosphere. Here, we present single-cell measurements of the expression of these two pathways using engineered fluorescent reporter strains of Ruegeria pomeroyi DSS-3, and find that external DMSP concentration dictates the relative expression of the two pathways. DMSP induces an upregulation of both pathways, but only at high concentrations (>1 μM for demethylation; >35 nM for cleavage), characteristic of microscale hotspots such as the vicinity of phytoplankton cells. Co-incubations between DMSP-producing microalgae and bacteria revealed an increase in cleavage pathway expression close to the microalgae's surface. These results indicate that bacterial utilization of microscale DMSP hotspots is an important determinant of the fate of sulfur in the ocean

Sterol metabolism modulates susceptibility to HIV-1 Infection

Background: 25-hydroxylase (CH25H) is an Interferon stimulated gene (ISG), which catalyzes the synthesis of 25-Hydroxycholesterol (25HC). 25HC intervenes in metabolic and infectious processes as controls cholesterol homeostasis and influences viral entry into host cells.We verified whether natural resistance to HIV-1 infection in HIV-1-exposed seronegative (HESN) individuals is at least partially mediated by particularities in sterol biosynthesis. Methods: Peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) isolated from 15 sexually-exposed HESN and 15 healthy controls (HC) were in vitro HIV-1-infected and analyzed for: 1) percentage of IFN\u3b1-producing plasmacytoid Dendritic Cells (pDCs); 2) Cholesterol signaling and inflammatory response RNA expression; 3) resistance to HIV-1 infection. MDMs from 5 HC were in vitro HIV-1-infected in the absence/presence of exogenously added 25HC. Results: IFN\u3b1-producing pDCs were augmented in HESN compared to HCs both in unstimulated and in in vitro HIV-1-infected PBMCs (p<0.001). An increased expression of CH25H and of a number of genes involved in cholesterol metabolism (ABCA1, ABCG1, CYP7B1, LXR\u3b1, OSBP, PPAR\u3b3, SCARB1) was observed as well; this, was associated with a reduced susceptibility to in vitro HIV-1-infection of PBMCs and MDMs (p<0.01). Notably, addition of 25HC to MDMs resulted in increased cholesterol efflux and augmented resistance to in vitro HIV-1-infection. Conclusions: Results herein show that in HESN sterol metabolism might be particularly efficient. This could be related to the activation of the IFN\u3b1 pathway and results into a reduced susceptibility to in vitro HIV-1 infection. These results suggest a possible basis for therapeutic interventions to modulate HIV-1 infection

HPV in sperm is efficiently removed by washing: A suitable approach for assisted reproduction

Research question: Is it possible, by sperm-washing spermatozoa from clinically HPV-positive men, to obtain spermatozoa free of human papillomavirus (HPV) to be employed in assisted reproduction? Design: This was an observational study performed on HPV-positive men. Freshly ejaculated semen was collected and readily processed by gradient separation followed by swim-up from the washed pellet. The resulting fractions were seminal plasma, cell pellet, round cells, non-motile spermatozoa and motile spermatozoa. All fractions were then tested for the presence of HPV DNA. Results: Of the 15 clinically HPV-positive subjects, 67% were positive in at least one of the seminal fractions. If any postivity was detected, the plasma was always HPV positive. No consistent pattern was observed throughout different samples in the cell pellet, round cell and non-motile spermatozoa fractions. However, after the sperm-wash procedure, the fraction of motile spermatozoa was never found to be HPV-positive. Conclusions: The sperm-washing technique, which was previously successfully used to remove human immunodeficiency virus, can efficiently remove HPV from spermatozoa. However, the present study was conducted on a small population so a larger follow-up study is recommended. HPV screening should be performed in sperm samples and, upon HPV positivity, sperm-washing should be considered before assisted reproduction techniques are used

Endoplasmic Reticulum Associated Aminopeptidase 2 (ERAP2) is released in the secretome of activated MDMs and reduces in vitro HIV-1 infection

Background: Haplotype-specific alternative splicing of the endoplasmic reticulum (ER) aminopeptidase type 2 (ERAP2) gene results in either full-length (FL, haplotype A) or alternatively spliced (AS, haplotype B) mRNA. HapA/HapA homozygous (HomoA) subjects show a reduced susceptibility to HIV-1 infection, probably secondary to the modulation of the antigen processing/presenting machinery. ERAP1 was recently shown to be secreted from the plasma membrane in response to activation; we investigated whether ERAP2 can be released as well and if the secreted form of this enzyme retains its antiviral function. Methods: Human monocyte derived macrophages (MDMs) were differentiated from peripheral blood mononuclear cells (PBMCs) isolated from 6 HomoA healthy controls and stimulated with IFN\u3b3 and LPS. ERAP2-FL secretion was evaluated by mass spectrometry. PBMCs (14 HomoA and 16 HomoB) and CD8-depleted PBMCs (CD8-PBMCs) (4 HomoA and 4 HomoB) were in vitro HIV-infected in the absence/presence of recombinant human ERAP2-FL (rhERAP2) protein; p24 viral antigen quantification was used to assess viral replication. IFN\u3b3 and CD69 mRNA expression, as well as the percentage of perforin-producing CD8+ T Lymphocytes, were analyzed 3 and 7-days post in vitro HIV-1-infection, respectively. The effect of rhERAP2 addition in cell cultures on T cell apoptosis, proliferation, activation, and maturation was evaluated as well on 24 h-stimulated PBMCs. Results: ERAP2 can be secreted from human MDMs in response to IFN\u3b3/LPS stimulation. Notably, the addition of rhERAP2 to PBMC and CD8-PBMC cultures resulted in the reduction of viral replication, though these differences were statistically significant only in PBMCs (p < 0.05 in both HomoA and HomoB). This protective effect was associated with an increase in IFN\u3b3 and CD69 mRNA expression and in the percentage of perforin-expressing CD107+CD8+ cells. RhERAP2 addition also resulted in an increase in CD8+ activated lymphocyte (CD25+HLA-DRII+) and Effector Memory/Terminally differentiated CD8+ T cells ratio. Conclusions: This is the first report providing evidence for the release of ERAP2 in the secretome of immunocompetent cells. Data herein also indicate that exogenous ERAP2-FL exerts its protective function against HIV-1 infection, even in HomoB subjects who do not genetically produce it. Presumably, this defensive extracellular feature is only partially dependent on immune system modulation

Biomarker testing in oncology - Requirements for organizing external quality assessment programs to improve the performance of laboratory testing:revision of an expert opinion paper on behalf of IQNPath ABSL

In personalized medicine, predictive biomarker testing is the basis for an appropriate choice of therapy for patients with cancer. An important tool for laboratories to ensure accurate results is participation in external quality assurance (EQA) programs. Several providers offer predictive EQA programs for different cancer types, test methods, and sample types. In 2013, a guideline was published on the requirements for organizing high-quality EQA programs in molecular pathology. Now, after six years, steps were taken to further harmonize these EQA programs as an initiative by IQNPath ABSL, an umbrella organization founded by various EQA providers. This revision is based on current knowledge, adds recommendations for programs developed for predictive biomarkers by in situ methodologies (immunohistochemistry and in situ hybridization), and emphasized transparency and an evidence-based approach. In addition, this updated version also has the aim to give an overview of current practices from various EQA providers

IQN path ASBL report from the first European cfDNA consensus meeting:expert opinion on the minimal requirements for clinical ctDNA testing

Liquid biopsy testing is a new laboratory-based method that detects tumour mutations in circulating free DNA (cfDNA) derived from minimally invasive blood sampling techniques. Recognising the significance for clinical testing, in 2017, IQN Path provided external quality assessment for liquid biopsy testing. Representatives of those participating laboratories were invited to attend a workshop to discuss the findings and how to achieve quality implementation of cfDNA testing in the clinical setting, the discussion and outcomes of this consensus meeting are described below. Predictive molecular profiling using tumour tissue in order to select cancer patients eligible for targeted therapy is now routine in diagnostic pathology. If insufficient tumour tissue material is available, in some circumstances, recent European Medicines Agency (EMA) guidance recommends mutation testing with plasma cfDNA. Clinical applications of cfDNA include treatment selection based on clinically relevant mutations derived from pre-treatment samples and the detection of resistant mutations upon progression of the disease. In order to identify tumour-related mutations in amongst other nucleic acid material found in plasma samples, highly sensitive laboratory methods are needed. In the workshop, we discussed the variable approaches taken with regard to cfDNA extraction methods, the tests, and considered the impact of false-negative test results. We explored the lack of standardisation of complex testing procedures ranging from plasma collection, transport, processing and storage, cfDNA extraction, and mutation analysis, to interpretation and reporting of results. We will also address the current status of clinical validation and clinical utility, and its use in current diagnosis. This workshop revealed a need for guidelines on with standardised procedures for clinical cfDNA testing and reporting, and a requirement for cfDNA-based external quality assessment programs

SMYD3 promotes the epithelial-mesenchymal transition in breast cancer

SMYD3 is a methylase previously linked to cancer cell invasion and migration. Here we show that SMYD3 favors TGF\u3b2-induced epithelial-mesenchymal transition (EMT) in mammary epithelial cells, promoting mesenchymal and EMT transcription factors expression. SMYD3 directly interacts with SMAD3 but it is unnecessary for SMAD2/3 phosphorylation and nuclear translocation. Conversely, SMYD3 is indispensable for SMAD3 direct association to EMT genes regulatory regions. Accordingly, SMYD3 knockdown or its pharmacological blockade with the BCI121 inhibitor dramatically reduce TGF\u3b2-induced SMAD3 association to the chromatin. Remarkably, BCI121 treatment attenuates mesenchymal genes transcription in the mesenchymal-like MDA-MB-231 cell line and reduces their invasive ability in vivo, in a zebrafish xenograft model. In addition, clinical datasets analysis revealed that higher SMYD3 levels are linked to a less favorable prognosis in claudin-low breast cancers and to a reduced metastasis free survival in breast cancer patients. Overall, our data point at SMYD3 as a pivotal SMAD3 cofactor that promotes TGF\u3b2-dependent mesenchymal gene expression and cell migration in breast cancer, and support SMYD3 as a promising pharmacological target for anti-cancer therapy

International pilot external quality assessment scheme for analysis and reporting of circulating tumour DNA

Background Molecular analysis of circulating tumour DNA (ctDNA) is becoming increasingly important in clinical treatment decisions. A pilot External Quality Assessment (EQA) scheme for ctDNA analysis was organized by four European EQA providers under the umbrella organization IQN Path, in order to investigate the feasibility of delivering an EQA to assess the detection of clinically relevant variants in plasma circulating cell-free DNA (cfDNA) and to analyze reporting formats. Methods Thirty-two experienced laboratories received 5 samples for EGFR mutation analysis and/or 5 samples for KRAS and NRAS mutation analysis. Samples were artificially manufactured to contain 3 mL of human plasma with 20 ng/mL of fragmented ctDNA and variants at allelic frequencies of 1 and 5%. Results The scheme error rate was 20.1%. Higher error rates were observed for RAS testing when compared to EGFR analysis, for allelic frequencies of 1% compared to 5%, and for cases including 2 different variants. The reports over-interpreted wild-type results and frequently failed to comment on the amount of cfDNA extracted. Conclusions The pilot scheme demonstrated the feasibility of delivering a ctDNA EQA scheme and the need for such a scheme due to high error rates in detecting low frequency clinically relevant variants. Recommendations to improve reporting of cfDNA are provided