Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection

Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation.     

Implications of Cardiovascular Disease Risk Assessment Using the WHO/ISH Risk Prediction Charts in Rural India

Cardiovascular disease (CVD) risk in India is currently assessed using the World Health Organization/International Society for Hypertension (WHO/ISH) risk prediction charts since no population-specific models exist. The WHO/ISH risk prediction charts have two versions—one with total cholesterol as a predictor (the high information (HI) model) and the other without (the low information (LI) model). However, information on the WHO/ISH risk prediction charts including guidance on which version to use and when, as well as relative performance of the LI and HI models, is limited. This article aims to, firstly, quantify the relative performance of the LI and HI WHO/ISH risk prediction (for WHO-South East Asian Region D) using data from rural India. Secondly, we propose a pre-screening (simplified) point-of-care (POC) test to identify patients who are likely to benefit from a total cholesterol (TC) test, and subsequently when the LI model is preferential to HI model. Analysis was performed using cross-sectional data from rural Andhra Pradesh collected in 2005 with recorded blood cholesterol measurements (N = 1066). CVD risk was computed using both LI and HI models, and high risk individuals who needed treatment(T _HR) were subsequently identified based on clinical guidelines. Model development for the POC assessment of a TC test was performed through three machine learning techniques: Support Vector Machine (SVM), Regularised Logistic Regression (RLR), and Random Forests (RF) along with a feature selection process. Disagreement in CVD risk predicted by LI and HI WHO/ISH models was 14.5% (n = 155; p<0.01) overall and comprised 36 clinically relevant T _HR patients (31% of patients identified as T _HR by using either model). Using two patient-specific parameters (age, systolic blood pressure), our POC assessment can pre-determine the benefit of TC testing and choose the appropriate risk model (out-of-sample AUCs:RF-0.85,SVM-0.84,RLR:0.82 and maximum sensitivity-98%). The identification of patients benefitting from a TC test for CVD risk stratification can aid planning for resource-allocation and save costs for large-scale screening programmes.     

Sequencing two Tyr::CreERT2 transgenic mouse lines

The Cre/loxP system is a powerful tool that has allowed the study of the effects of specific genes of interest in various biological settings. The Tyr::CreER^T2 system allows for the targeted expression and activity of the Cre enzyme in the melanocyte lineage following treatment with tamoxifen, thus providing spatial and temporal control of the expression of specific target genes. Two independent transgenic mouse models, each containing a Tyr::CreER^T2 transgene, have been generated and are widely used to study melanocyte transformation. In this study, we performed whole genome sequencing (WGS) on genomic DNA from the two Tyr::CreER^T2 mouse models and identified their sites of integration in the C57BL/6 genome. Based on these results, we designed PCR primers to accurately, and efficiently, genotype transgenic mice. Finally, we discussed some of the advantages of each transgenic mouse model.     

First partial cranium of Togocetus from Kpogamé (Togo) and the protocetid diversity in the Togolese phosphate basin

Earliest cetaceans (whales) originated from the early Eocene of Indo-Pakistan, but the group dispersed through most of the oceans of the planet by the late middle to late Eocene. This late Eocene global distribution indicates that important dispersal events took place during the middle Eocene (Lutetian), a globally undersampled time interval that is well documented in the Togolese phosphate series. We report here the first discovery of a partial cetacean cranium from middle Eocene deposits of Togo (West Africa). A 3D model of the cranium and teeth was reconstructed in order to reveal hidden anatomical features. The dental and cranial characteristics of the Togolese specimen recall those of protocetid taxa described in Africa, Asia, and North America, but also display significant differences. In particular, we show that the new specimen shares a number of morphological features with the Togolese taxon Togocetus. Such a hypothesis is further supported by a cladistic analysis including 45 taxa and 167 morphological characters, which recovers the new specimen close to Togocetus as the first offshoot of protocetids. Phylogenetic analysis including all the protocetids remains of Kpogamé confirms the singular diversity of the Togolese phosphate basin, and enables to examine potential connections with faunas from contemporaneous localities in Africa.     

Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid–specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.     

Does placenta position modify the risk of placental abruption in car crashes?

The goal of this paper is to study the influence of placenta position on the risk of placenta abruption following a car crash involving a full term pregnant woman in the driver's seat. We developed an anatomically precise finite element numerical model of a pregnant woman. This numerical model was validated using experimental data from crash tests using cadavers given to science. For a frontal impact at a speed of 20 km/h, the mean value of peak strain levels at the utero-placenta interface were found to be close to 20%, independent of placenta position, representing an adverse foetal outcome risk of approximately 15%. This study found no significant effect of placenta position on the risk of placenta abruption. Our anatomically precise approach does however confirm the interest of using a numerical model when studying injury mechanisms in pregnant women involved in car crashes.     

Increased Expression of a Phloem Membrane Protein Encoded by NHL26 Alters Phloem Export and Sugar Partitioning in Arabidopsis

The complex process of phloem sugar transport involves symplasmic and apoplasmic events. We characterized Arabidopsis thaliana lines ectopically expressing a phloem-specific gene encoding NDR1/HIN1-like26 (NHL26), a putative membrane protein. NHL26 overexpressor plants grew more slowly than wild-type plants, accumulated high levels of carbohydrates in mature leaves, and had a higher shoot biomass, contrasting with slower root growth and a lower seed yield. Similar effects were observed when NHL26 was overexpressed in companion cells, under the control of a companion cell–specific promoter. The soluble sugar content of the phloem sap and sink organs was lower than that in the wild type, providing evidence of a sugar export defect. This was confirmed in a phloem-export assay with the symplastic tracer carboxyfluorescein diacetate. Leaf sugar accumulation was accompanied by higher organic acid, amino acid, and protein contents, whereas analysis of the metabolite profile of phloem sap exudate revealed no change in amino acid or organic acid content, indicating a specific effect on sugar export. NHL26 was found to be located in the phloem plasmodesmata and the endoplasmic reticulum. These findings reveal that NHL26 accumulation affects either the permeability of plasmodesmata or sugar signaling in companion cells, with a specific effect on sugar export.     

Symptomatic Versus Inapparent Outcome in Repeat Dengue Virus Infections Is Influenced by the Time Interval between Infections and Study Year

Four dengue virus serotypes (DENV1-4) circulate globally, causing more human illness than any other arthropod-borne virus. Dengue can present as a range of clinical manifestations from undifferentiated fever to Dengue Fever to severe, life-threatening syndromes. However, most DENV infections are inapparent. Yet, little is known about determinants of inapparent versus symptomatic DENV infection outcome. Here, we analyzed over 2,000 DENV infections from 2004 to 2011 in a prospective pediatric cohort study in Managua, Nicaragua. Symptomatic cases were captured at the study health center, and paired healthy annual samples were examined on a yearly basis using serological methods to identify inapparent DENV infections. Overall, inapparent and symptomatic DENV infections were equally distributed by sex. The mean age of infection was 1.2 years higher for symptomatic DENV infections as compared to inapparent infections. Although inapparent versus symptomatic outcome did not differ by infection number (first, second or third/post-second DENV infections), substantial variation in the proportion of symptomatic DENV infections among all DENV infections was observed across study years. In participants with repeat DENV infections, the time interval between a first inapparent DENV infection and a second inapparent infection was significantly shorter than the interval between a first inapparent and a second symptomatic infection. This difference was not observed in subsequent infections. This result was confirmed using two different serological techniques that measure total anti-DENV antibodies and serotype-specific neutralizing antibodies, respectively. Taken together, these findings show that, in this study, age, study year and time interval between consecutive DENV infections influence inapparent versus symptomatic infection outcome, while sex and infection number had no significant effect. Moreover, these results suggest that the window of cross-protection induced by a first infection with DENV against a second symptomatic infection is approximately 2 years. These findings are important for modeling dengue epidemics and development of vaccines.     

Bcl-xL Silencing Induces Alterations in hsa-miR-608 Expression and Subsequent Cell Death in A549 and SK-LU1 Human Lung Adenocarcinoma Cells

Bcl-xL is an anti-apoptotic protein that is frequently found to be overexpressed in non-small cell lung cancer leading to an inhibition of apoptosis and poor prognosis. Recently, the role of miRNAs in regulating apoptosis and cell survival during tumorigenesis has become evident, with cancer cells showing perturbed expression of various miRNAs. In this study, we utilized miRNA microarrays to determine if miRNA dysregulation in bcl-xL silenced lung adenocarcinoma cells could be involved in regulating cell death. Short interfering RNA-based transfection of A549 and SK-LU1 lung adenocarcinoma cells was successful in inducing a reduction in bcl-xL expression levels, resulting in a decrease in cell viability. A total of 10 miRNAs were found to be significantly differentially expressed when compared between siRNA-transfected and non-transfected cells including hsa-miR-181a, hsa-miR-769-5p, hsa-miR-361-5p, hsa-miR-1304 and hsa-miR-608. When overexpression studies on hsa-miR-608 was performed via transfection of miRNA mimics, cell death was found to be induced in A549 and SK-LU1 cells in comparison to untreated cells. This effect was reversed when knockdown studies involving anti-sense inhibitors were introduced. Combination of siRNA based silencing of bcl-xL (siBcl-xL) followed by anti-sense inhibitor transfection led to a decrease in the apoptotic population of A549 and SK-LU1 cells in comparison to cells only treated with siBcl-xL, illustrating the connection between bcl-xL, hsa-miR-608 and cell death. Gene target prediction analysis implicated the PI3K/AKT, WNT, TGF-β, and ERK signaling pathways as targets of bcl-xL induced miRNA alterations. We have demonstrated that bcl-xL silencing in A549 and SK-LU1 cells leads to the occurrence of cell death through the dysregulation of specific miRNAs. This study also provides a platform for anti-sense gene therapy whereby miRNA expression can be exploited to increase the apoptotic properties in lung adenocarcinoma cells.