Target Deletion of the Cytoskeleton-Associated Protein Palladin Does Not Impair Neurite Outgrowth in Mice

Palladin is an actin cytoskeleton–associated protein which is crucial for cell morphogenesis and motility. Previous studies have shown that palladin is localized to the axonal growth cone in neurons and may play an important role in axonal extension. Previously, we have generated palladin knockout mice which display cranial neural tube closure defect and embryonic lethality before embryonic day 15.5 (E15.5). To further study the role of palladin in the developing nervous system, we examined the innervation of palladin-deficient mouse embryos since the 200 kd, 140 kd, 90–92 kd and 50 kd palladin isoforms were undetectable in the mutant mouse embryo brain. Contrary to the results of previous studies, we found no inhibition of the axonal extension in palladin-deficient mouse embryos. The cortical neurons derived from palladin-deficient mice also showed no significant difference in neurite outgrowth as compared with those from wild-type mice. Moreover, no difference was found in neurite outgrowth of neural stem cell derived-neurons between palladin-deficient mice and wild-type mice. In conclusion, these results suggest that palladin is dispensable for normal neurite outgrowth in mice

Understanding the consumption of folic acid during preconception, among Pakistani, Bangladeshi and white British mothers in Luton, UK: a qualitative study

Background To review the similarities and differences in Pakistani, Bangladeshi and White British mothers health beliefs (attitudes, knowledge and perceptions) and health behaviour regarding their consumption of folic acid pre-conception, to reduce the risk of neural tube defects. Methods Our study used a descriptive qualitative research approach, implementing face-to-face focus group discussions with Pakistani, Bangladeshi or White British mothers (normal birth outcomes and mothers with poor birth outcomes) and semi-structured interviews or focus groups with service providers using semi-structured topic guides. This method is well suited for under researched areas where in-depth information is sought. There were three sample groups: 1. Pakistani, Bangladeshi and White British mothers with normal birth outcomes (delivery after 37 weeks of gestation, in the preceding 6 to 24 months, weighing 2500 g and living within a specified postcode area in Luton, UK). 2. Pakistani Bangladeshi and white British bereaved mothers who had suffered a perinatal mortality (preceding 6 to 24 months, residing within a specificied postcode area). 3. Healthcare professionals working on the local maternity care pathway (i.e. services providing preconception, antenatal, antepartum and postpartum care). Transcribed discussions were analysed using the Framework Analysis approach. Results The majority of mothers in this sample did not understand the benefits or optimal time to take folic acid pre-conception. Conversely, healthcare professionals believed the majority of women did consume folic acid, prior to conception. Conclusions There is a need to increase public health awareness of the optimal time and subsequent benefits for taking folic acid, to prevent neural tube defects.</p

Chondrocyte Deformations as a Function of Tibiofemoral Joint Loading Predicted by a Generalized High-Throughput Pipeline of Multi-Scale Simulations

Cells of the musculoskeletal system are known to respond to mechanical loading and chondrocytes within the cartilage are not an exception. However, understanding how joint level loads relate to cell level deformations, e.g. in the cartilage, is not a straightforward task. In this study, a multi-scale analysis pipeline was implemented to post-process the results of a macro-scale finite element (FE) tibiofemoral joint model to provide joint mechanics based displacement boundary conditions to micro-scale cellular FE models of the cartilage, for the purpose of characterizing chondrocyte deformations in relation to tibiofemoral joint loading. It was possible to identify the load distribution within the knee among its tissue structures and ultimately within the cartilage among its extracellular matrix, pericellular environment and resident chondrocytes. Various cellular deformation metrics (aspect ratio change, volumetric strain, cellular effective strain and maximum shear strain) were calculated. To illustrate further utility of this multi-scale modeling pipeline, two micro-scale cartilage constructs were considered: an idealized single cell at the centroid of a 100×100×100 μm block commonly used in past research studies, and an anatomically based (11 cell model of the same volume) representation of the middle zone of tibiofemoral cartilage. In both cases, chondrocytes experienced amplified deformations compared to those at the macro-scale, predicted by simulating one body weight compressive loading on the tibiofemoral joint. In the 11 cell case, all cells experienced less deformation than the single cell case, and also exhibited a larger variance in deformation compared to other cells residing in the same block. The coupling method proved to be highly scalable due to micro-scale model independence that allowed for exploitation of distributed memory computing architecture. The method’s generalized nature also allows for substitution of any macro-scale and/or micro-scale model providing application for other multi-scale continuum mechanics problems

A High-Throughput Platform for Lentiviral Overexpression Screening of the Human ORFeome

In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2′-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes

Sequestration of Highly Expressed mRNAs in Cytoplasmic Granules, P-Bodies, and Stress Granules Enhances Cell Viability

Transcriptome analyses indicate that a core 10%–15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress

Genetic signatures of variation in population size in a native fungal pathogen after the recent intensive plantation of its host tree

Historical fluctuations in forests’ distribution driven by past climate changes and anthropogenic activities can have large impacts on the demographic history of pathogens that have a long co-evolution history with these host trees. Using a population genetic approach, we investigated that hypothesis by reconstructing the demographic history of Armillaria ostoyae, one of the major pathogens of the maritime pine (Pinus pinaster), in the largest monospecific pine planted forest in Europe (south-western France). Genetic structure analyses and approximate Bayesian computation approaches revealed that a single pathogen population underwent a severe reduction in effective size (12 times lower) 1080–2080 generations ago, followed by an expansion (4 times higher) during the last 4 generations. These results are consistent with the history of the maritime pine forest in the region characterized by a strong recession during the last glaciation (~19 000 years ago) and massive plantations during the second half of the nineteenth century. Results suggest that recent and intensive plantations of a host tree population have offered the opportunity for a rapid spread and adaptation of their pathogens

Interplay between SIN3A and STAT3 Mediates Chromatin Conformational Changes and GFAP Expression during Cellular Differentiation

BACKGROUND: Neurons and astrocytes are generated from common neural precursors, yet neurogenesis precedes astrocyte formation during embryogenesis. The mechanisms of neural development underlying suppression and de-suppression of differentiation-related genes for cell fate specifications are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: By using an in vitro system in which NTera-2 cells were induced to differentiate into an astrocyte-like lineage, we revealed a novel role for Sin3A in maintaining the suppression of GFAP in NTera-2 cells. Sin3A coupled with MeCP2 bound to the GFAP promoter and their occupancies were correlated with repression of GFAP transcription. The repression by Sin3A and MeCP2 may be an essential mechanism underlying the inhibition of cell differentiation. Upon commitment toward an astrocyte-like lineage, Sin3A- MeCP2 departed from the promoter and activated STAT3 simultaneously bound to the promoter and exon 1 of GFAP; meanwhile, olig2 was exported from nuclei to the cytoplasm. This suggested that a three-dimensional or higher-order structure was provoked by STAT3 binding between the promoter and proximal coding regions. STAT3 then recruited CBP/p300 to exon 1 and targeted the promoter for histone H3K9 and H3K14 acetylation. The CBP/p300-mediated histone modification further facilitates chromatin remodeling, thereby enhancing H3K4 trimethylation and recruitment of RNA polymerase II to activate GFAP gene transcription. CONCLUSIONS/SIGNIFICANCE: These results provide evidence that exchange of repressor and activator complexes and epigenetic modifications are critical strategies for cellular differentiation and lineage-specific gene expression

Tension stimulation drives tissue formation in scaffold-free systems

Scaffold-free systems have emerged as viable approaches for engineering load-bearing tissues. However, the tensile properties of engineered tissues have remained far below the values for native tissue. Here, by using self-assembled articular cartilage as a model to examine the effects of intermittent and continuous tension stimulation on tissue formation, we show that the application of tension alone, or in combination with matrix remodelling and synthesis agents, leads to neocartilage with tensile properties approaching those of native tissue. Implantation of tension-stimulated tissues results in neotissues that are morphologically reminiscent of native cartilage. We also show that tension stimulation can be translated to a human cell source to generate anisotropic human neocartilage with enhanced tensile properties. Tension stimulation, which results in nearly sixfold improvements in tensile properties over unstimulated controls, may allow the engineering of mechanically robust biological replacements of native tissue