New Short Tandem Repeat-Based Molecular Typing Method for Pneumocystis jirovecii Reveals Intrahospital Transmission between Patients from Different Wards

Pneumocystis pneumonia is a severe opportunistic infection in immunocompromised patients caused by the unusual fungus Pneumocystis jirovecii. Transmission is airborne, with both immunocompromised and immunocompetent individuals acting as a reservoir for the fungus. Numerous reports of outbreaks in renal transplant units demonstrate the need for valid genotyping methods to detect transmission of a given genotype. Here, we developed a short tandem repeat (STR)-based molecular typing method for P. jirovecii. We analyzed the P. jirovecii genome and selected six genomic STR markers located on different contigs of the genome. We then tested these markers in 106 P. jirovecii PCR-positive respiratory samples collected between October 2010 and November 2013 from 91 patients with various underlying medical conditions. Unique (one allele per marker) and multiple (more than one allele per marker) genotypes were observed in 34 (32%) and 72 (68%) samples, respectively. A genotype could be assigned to 55 samples (54 patients) and 61 different genotypes were identified in total with a discriminatory power of 0.992. Analysis of the allelic distribution of the six markers and minimum spanning tree analysis of the 61 genotypes identified a specific genotype (Gt21) in our hospital, which may have been transmitted between 10 patients including six renal transplant recipients. Our STR-based molecular typing method is a quick, cheap and reliable approach to genotype Pneumocystis jirovecii in hospital settings and is sensitive enough to detect minor genotypes, thus enabling the study of the transmission and pathophysiology of Pneumocystis pneumonia.     

Using budding yeast to screen for anti-prion drugs

Prions are misfolded proteins capable of propagating their altered conformation which are commonly considered as the causative agent of transmissible spongiform encephalopathies, a class of fatal neurodegenerative diseases. Currently, no treatment for prion-based diseases is available. Recently we have developed a rapid, yeast-based, two-step assay to screen for anti-prion drugs [1]. This new method allowed us to identify several compounds that are effective in vivo against budding yeast [PSI+] and [URE3] prions but also able to promote mammalian prion clearance in three different cell culture-based assays. Taken together, these results validate our method as an economic and efficient high-throughput screening approach to identify novel prion inhibitors or to carry on comprehensive structure-activity studies for already isolated anti-mammalian prion drugs. These results suggest furthermore that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to human and thus amenable to pharmacological and genetic analysis. Finally, it would be very interesting to test active drugs isolated using the yeast-based assay in models for other diseases (neurodegenerative or not) involving amyloid fibers like Huntington's, Parkinson's or Alzheimer's diseases.     

Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2’-O-Methylation Mutant

Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.     

Species and endosymbiont diversity of Bemisia tabaci (Homoptera: Aleyrodidae) on vegetable crops in Senegal

Bemisia tabaci‐transmitted geminiviruses are one of the major threats on cassava and vegetable crops in Africa. However, to date, few studies are available on the diversity of B. tabaci and their associated endosymbionts in Africa. More than 28 species have been described in the complex of B. tabaci cryptic species; among them, 2 are invasive pests worldwide: MED and MEAM1. In order to assess the species diversity of B. tabaci in vegetable crops in Senegal, several samplings in different localities, hosts and seasons were collected and analyzed with nuclear (microsatellite) and mitochondrial (COI) markers. The bacterial endosymbiont community was also studied for each sample. Two species were detected: MED Q1 and MEAM1 B. Patterns of MED Q1 (dominance on most of the samples and sites, highest nuclear and mitochondrial diversity and broader secondary endosymbiont community: Hamiltonella, Cardinium, Wolbachia and Rickettsia), point toward a predominant resident begomovirus vector group for MED Q1 on market gardening crops. Furthermore, the lower prevalence of the second species MEAM1 B, its lower nuclear and mitochondrial diversity and a narrower secondary endosymbiont community (Hamiltonella/Rickettsia), indicate that this genetic group is exotic and results from a recent invasion in this area.     

Inflorescences vs leaves: a distinct modulation of carbon metabolism process during Botrytis infection

Plant growth and survival depends critically on photo assimilates. Pathogen infection leads to changes in carbohydrate metabolism of plants. In this study, we monitored changes in the carbohydrate metabolism in the grapevine inflorescence and leaves using Botrytis cinerea and Botrytis pseudo cinerea. Fluctuations in gas exchange were correlated with variations in chlorophyll a fluorescence. During infection, the inflorescences showed an increase in net photosynthesis (Pn) with a stomatal limitation. In leaves, photosynthesis decreased, with a non‐stomatal limitation. A decrease in the effective photosystem II (PSII) quantum yield (ΦPSII) was accompanied by an increase in photochemical quenching (qP) and non‐photochemical quenching (qN). The enhancement of qP and ΦPSII could explain the observed increase in Pn. In leaves, the significant decline in ΦPSII and qP, and increase in qN suggest that energy was mostly oriented toward heat dissipation instead of CO₂ fixation. The accumulation of glucose and sucrose in inflorescences and glucose and fructose in the leaves during infection indicate that the plant's carbon metabolism is differently regulated in these two organs. While a strong accumulation of starch was observed at 24 and 48 hours post‐inoculation (hpi) with both species of Botrytis in the inflorescences, a significant decrease with B. cinerea at 24 hpi and a significant increase with B. pseudo cinerea at 48 hpi were observed in the leaves. On the basis of these results, it can be said that during pathogen attack, the metabolism of grapevine inflorescence and leaf is modified suggesting distinct mechanisms modifying gas exchange, PSII activity and sugar contents in these two organs.     

Indirect Hemagglutinating Antibody Response to Herpesvirus hominis Types 1 and 2 in Immunized Laboratory Animals and in Natural Infections of Man

Indirect hemagglutinating (IHA) antibody responses to Herpesvirus hominis types 1 and 2 (HVH-1 and HVH-2) were compared to complement-fixing and neutralizing antibody responses in immunized laboratory animals (rabbits, guinea pigs, and hamsters) and in natural infections of man. With the immunized animals, type specificity was seen only in the IHA test and only with antisera produced in hamsters and in the rabbits immunized with HVH-2. In human nongenital infections (considered to be caused predominately by HVH-1), IHA and neutralizing antibodies developed at about the same rate and reached approximately the same levels for HVH-1 and HVH-2. IHA titers tended to be higher than neutralizing antibody titers for both virus types. In genital infections (considered to be caused predominately by HVH-2), there was a rapid IHA antibody response to HVH-2, and the early HVH-2 antibody demonstrable by IHA, but not by neutralization tests, was found to be immunoglobulin M in nature. In genital infections, IHA titers for HVH-2 were markedly higher than neutralization titers, but there was no pronounced difference in neutralizing the IHA antibody titers for HVH-1. Several patients with genital infections fialed to develop IHA antibody for HVH-1. The IHA test possessed no greater sensitivity than did complement fixation or neutralization tests for serodiagnosis of HVH infections. Despite the fact that a number of patients with genital infections produced IHA antibody only for HVH-2, the test was no more effective than the neutralization test in providing a type-specific serodiagnosis of infection, due largely to the fact that the rapid IHA antibody response to HVH-2 prevented demonstration of a further, significant antibody titer increase in a number of cases.     

Dereplication of natural products from complex extracts by regression analysis and molecular networking: case study of redox-active compounds from <Emphasis Type="Italic">Viola alba</Emphasis> subsp. <Emphasis Type="Italic">dehnhardtii</Emphasis>

Introduction

In natural product research, bioassay-guided fractionation was previously widely employed but is now judged to be inadequate in terms of time and cost, particularly if only known compounds are ultimately isolated. The development of metabolomics, along with improvements in analytical tools, allows comprehensive metabolite profiling. This enables dereplication to target unknown active compounds early in the purification workflow.

Objectives

Starting from an ethanolic extract of violet leaves, this study aims to predict redox active compounds within a complex matrix through an untargeted metabolomics approach and correlation analysis.

Methods

Rapid fractionation of crude extracts was carried out followed by multivariate data analysis (MVA) of liquid chromatography–high resolution mass spectrometry (LC–HRMS) profiles. In parallel, redox active properties were evaluated by the capacity of the molecules to reduce 2,2-diphenyl-1-picrylhydrazyl (DPPH^·) and superoxide (O₂ ^·?) radicals using UV–Vis and electron spin resonance spectroscopies (ESR), respectively. A spectral similarity network (molecular networking) was used to highlight clusters involved in the observed redox activities.

Results

Dereplication on Viola alba subsp. dehnhardtii highlighted a reproducible pool of redox active molecules. Polyphenols, particularly O-glycosylated coumarins and C-glycosylated flavonoids, were identified and de novo dereplicated through molecular networking. Confirmatory analyses were undertaken by thin layer chromatography (TLC)–DPPH–MS assays and nuclear magnetic resonance (NMR) spectra of the most active compounds.

Conclusion

Our dereplication strategy allowed the screening of leaf extracts to highlight new biologically active metabolites in few steps with a limited amount of crude material and reduced time-consuming manipulations. This approach could be applied to any kind of natural extract for the study of various biological activities.

     

The production of fluorescent transgenic trout to study <Emphasis Type="Italic">in vitro</Emphasis> myogenic cell differentiation

Background  

Fish skeletal muscle growth involves the activation of a resident myogenic stem cell population, referred to as satellite cells, that can fuse with pre-existing muscle fibers or among themselves to generate a new fiber. In order to monitor the regulation of myogenic cell differentiation and fusion by various extrinsic factors, we generated transgenic trout (Oncorhynchus mykiss) carrying a construct containing the green fluorescent protein reporter gene driven by a fast myosin light chain 2 (MlC2f) promoter, and cultivated genetically modified myogenic cells derived from these fish.     

Lipase-catalyzed synthesis of chlorogenate fatty esters in solvent-free medium

Chlorogenic acid (5-caffeoylquinic acid or 5-CQA) is an hydrophilic phenolic compound with antioxidant properties. Because of its high polarity, its antioxidant properties may be altered when formulated in oil based food or cosmetic preparations. Therefore, there is an interest in trying to enhance its hydrophobicity by grafting of an aliphatic chain. Such lipophilization reactions can be generally achieved through enzymatic catalysis. Our study consisted in synthesizing fatty cholorogenate esters in a two steps reaction. Firstly, 5-CQA was chemically esterified by methanol using an Amberlite IR120 H resin to obtain methyl chlorogenate that is more soluble in the fatty alcohols than 5-CQA. Secondly, this chlorogenate intermediate was transesterified with fatty alcohols of various chain lengths (C₄, C₈, C₁₂, or C₁₆) in the presence of Candida antarctica B lipase. Under optimal reaction conditions (a_w = 0.05; 5% (w/w) of biocatalyst), the transesterification rates were until two-fold higher than in the direct lipase-catalyzed esterification of chlorogenic acid by the same alcohols. The two-step reaction overall yield was between 61 and 93% depending on the alcohol chain length, whereas it was 40–60% for the direct esterification with the same alcohols.     

TGF‐β induces SOX2 expression in a time‐dependent manner in human melanoma cells

The sry‐related high‐mobility box (SOX)‐2 protein has recently been proven to play a significant role in progression, metastasis, and clinical prognosis spanning several cancer types. Research on the role of SOX2 in melanoma is limited and currently little is known about the mechanistic function of this gene in this context. Here, we observed high expression of SOX2 in both human melanoma cell lines and primary melanomas in contrast to melanocytic nevi. This overexpression in melanoma can, in part, be explained by extra gene copy numbers of SOX2 in primary samples. Interestingly, we were able to induce SOX2 expression, mediated by SOX4, via TGF‐β1 stimulation in a time‐dependent manner. Moreover, the knockdown of SOX2 impaired TGF‐β‐induced invasiveness. This phenotype switch can be explained by SOX2‐mediated cross talk between TGF‐β and non‐canonical Wnt signaling. Thus, we propose that SOX2 is involved in the critical TGF‐β signaling pathway, which has been shown to correlate with melanoma aggressiveness and metastasis. In conclusion, we have identified a novel downstream factor of TGF‐β signaling in melanoma, which may have further implications in the clinic.