SHORT COMMUNICATION: Double pronuclei injection of DNA into zygotes increases yields of transgenic mouse lines

Transgenic mice are increasingly used for gene function and regulation studies of mammalian genes. A major limitation is the necessity to produce a large number of founder animals to obtain one line with the desired expression pattern. We developed a method, the 'double pronuclei injection', that doubles the yield of transgenic mouse lines obtained from each injection session, thereby reducing the time, effort and costs of generating transgenic mice. Three transgenic vectors were microinjected into the male and female pronuclei of zygotes. Approximately half of the resulting born mice were transgenic. This represented a 60% increase in the yield of founders per injected zygote, and a 100% increase in the yield of transgenic mice per born animal, when compared to yields obtained using single pronucleus injection. This method should prove useful for generating large numbers of transgenic mice for gene regulation studies and for conditional gene ablation     

Reconstruction of a beech population bottleneck using archival demographic information and Bayesian analysis of genetic data

Range expansion and contraction has occurred in the history of most species and can seriously impact patterns of genetic diversity. Historical data about range change are rare and generally appropriate for studies at large scales, whereas the individual pollen and seed dispersal events that form the basis of geneflow and colonization generally occur at a local scale. In this study, we investigated range change in Fagus sylvatica on Mont Ventoux, France, using historical data from 1838 to the present and approximate Bayesian computation (ABC) analyses of genetic data. From the historical data, we identified a population minimum in 1845 and located remnant populations at least 200 years old. The ABC analysis selected a demographic scenario with three populations, corresponding to two remnant populations and one area of recent expansion. It also identified expansion from a smaller ancestral population but did not find that this expansion followed a population bottleneck, as suggested by the historical data. Despite a strong support to the selected scenario for our data set, the ABC approach showed a low power to discriminate among scenarios on average and a low ability to accurately estimate effective population sizes and divergence dates, probably due to the temporal scale of the study. This study provides an unusual opportunity to test ABC analysis in a system with a well-documented demographic history and identify discrepancies between the results of historical, classical population genetic and ABC analyses. The results also provide valuable insights into genetic processes at work at a fine spatial and temporal scale in range change and colonization.     

Protein interactions between surface annexin A2 and S100A10 mediate adhesion of breast cancer cells to microvascular endothelial cells

Annexin A2 (AnxA2) and S100A10 are known to form a molecular complex. Using fluorescence-based binding assays, we show that both proteins are localised on the cell surface, in a molecular form that allows mutual interaction. We hypothesized that binding between these proteins could facilitate cell–cell interactions. For cells that express surface S100A10 and surface annexin A2, cell–cell interactions can be blocked by competing with the interaction between these proteins. Thus an annexin A2-S100A10 molecular bridge participates in cell–cell interactions, revealing a hitherto unexplored function of this protein interaction.     

A Novel Mouse Model for Stable Engraftment of a Human Immune System and Human Hepatocytes

Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodium parasites leading to acute or chronic diseases constitute a global health challenge. The species tropism of these hepatotropic pathogens is restricted to chimpanzees and humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathology is intimately related to the degree and quality of the immune response. As a first step to decipher the immune response to infected hepatocytes, we developed an animal model harboring both a human immune system (HIS) and human hepatocytes (HUHEP) in BALB/c Rag2^-/- IL-2Rγc^-/- NOD.sirpa uPA^tg/tg mice. The extent and kinetics of human hepatocyte engraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocytes were polarized and mature in vivo, resulting in 20–50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepatic compartments were humanized with mature B cells, NK cells and naïve T cells, as well as monocytes and dendritic cells. Taken together, these results demonstrate that HIS-HUHEP mice can be stably (> 5 months) and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens and to test novel drug strategies or vaccine candidates.     

Systematic Literature Review and Meta-Analysis of Renal Function in Human Immunodeficiency Virus (HIV)-Infected Patients Treated with Atazanavir (ATV)-Based Regimens

Some HIV antiretroviral therapies (ART) have been associated with renal toxicities, which become of increasing concern as HIV-infected patients age and develop comorbidities. The objective of this study was to evaluate the relative impact of atazanavir (ATV)-based regimens on the renal function of adult patients with HIV. We conducted a systematic literature review by searching PubMed, EMBASE, Cochrane library, and the CRD from 2000 until March 2013. Major HIV-related conferences occurring in the past two years were also searched. All randomized clinical trials and large cohort studies assessing renal function in treatment-naïve and/or treatment-experienced HIV patients on ATV-based regimens were included. Fixed-effect mixed-treatment network analyses were carried out on the most frequently reported renal outcomes. 23 studies met the inclusion criteria, and change in estimated glomerular filtration rate (eGFR) from baseline to 48 weeks was identified as the main outcome. Two networks including, respectively, six studies (using the Cockcroft-Gault method) and four studies (using MDRD and CKD-EPI) were analysed. With CG network, ATV/r + TDF/FTC was associated with lower impact on the decline of eGFR than ATV/cobicistat + TDF/FTC but with higher decrease in eGFR than ATV/r + ABC/3TC (difference in mean change from baseline in eGFR repectively +3.67 and –3.89). The use of ATV/cobicistat + TDF/FTC led to a similar decline in eGFR as EVG/cobicistat/TDF/FTC. With respect to third agents combined with TDF/FTC, ATV/r had a lower increase in eGFR in comparison to EFV, and no difference was shown when compared to SQV/r and DRV/r. The effect of ATV-based regimens on renal function at 48 weeks appears similar to other ART regimens and appears to be modest regardless of boosting agent or backbone, although TDF containing backbones consistently leads to greater decline in eGFR.     

Atherosclerotic Plaque Destabilization in Mice: A Comparative Study

Atherosclerosis-associated diseases are the main cause of mortality and morbidity in western societies. The progression of atherosclerosis is a dynamic process evolving from early to advanced lesions that may become rupture-prone vulnerable plaques. Acute coronary syndromes are the clinical manifestation of life-threatening thrombotic events associated with high-risk vulnerable plaques. Hyperlipidemic mouse models have been extensively used in studying the mechanisms controlling initiation and progression of atherosclerosis. However, the understanding of mechanisms leading to atherosclerotic plaque destabilization has been hampered by the lack of proper animal models mimicking this process. Although various mouse models generate atherosclerotic plaques with histological features of human advanced lesions, a consensus model to study atherosclerotic plaque destabilization is still lacking. Hence, we studied the degree and features of plaque vulnerability in different mouse models of atherosclerotic plaque destabilization and find that the model based on the placement of a shear stress modifier in combination with hypercholesterolemia represent with high incidence the most human like lesions compared to the other models.     

Tissue stretch induces nuclear remodeling in connective tissue fibroblasts

Studies in cultured cells have shown that nuclear shape is an important factor influencing nuclear function, and that mechanical forces applied to the cell can directly affect nuclear shape. In a previous study, we demonstrated that stretching of whole mouse subcutaneous tissue causes dynamic cytoskeletal remodeling with perinuclear redistribution of α-actin in fibroblasts within the tissue. We have further shown that the nuclei of these fibroblasts have deep invaginations containing α-actin. In the current study, we hypothesized that tissue stretch would cause nuclear remodeling with a reduced amount of nuclear invagination, measurable as a change in nuclear concavity. Subcutaneous areolar connective tissue samples were excised from 28 mice and randomized to either tissue stretch or no stretch for 30 min, then examined with histochemistry and confocal microscopy. In stretched tissue (vs. non-stretched), fibroblast nuclei had a larger cross-sectional area (P < 0.001), smaller thickness (P < 0.03) in the plane of the tissue, and smaller relative concavity (P < 0.005) indicating an increase in nuclear convexity. The stretch-induced loss of invaginations may have important influences on gene expression, RNA trafficking and/or cell differentiation.     

A cluster of Ankyrin and Ankyrin-TPR repeat genes is associated with panicle branching diversity in rice

The number of grains per panicle is an important yield-related trait in cereals which depends in part on panicle branching complexity. One component of this complexity is the number of secondary branches per panicle. Previously, a GWAS site associated with secondary branch and spikelet numbers per panicle in rice was identified. Here we combined gene capture, bi-parental genetic population analysis, expression profiling and transgenic approaches in order to investigate the functional significance of a cluster of 6 ANK and ANK-TPR genes within the QTL. Four of the ANK and ANK-TPR genes present a differential expression associated with panicle secondary branch number in contrasted accessions. These differential expression patterns correlate in the different alleles of these genes with specific deletions of potential cis-regulatory sequences in their promoters. Two of these genes were confirmed through functional analysis as playing a role in the control of panicle architecture. Our findings indicate that secondary branching diversity in the rice panicle is governed in part by differentially expressed genes within this cluster encoding ANK and ANK-TPR domain proteins that may act as positive or negative regulators of panicle meristem’s identity transition from indeterminate to determinate state.