GABI-Kat SimpleSearch: a flanking sequence tag (FST) database for the identification of T-DNA insertion mutants in Arabidopsis thaliana

SUMMARY: GABI-Kat SimpleSearch is a database of flanking sequence tags (FSTs) of T-DNA mutagenized Arabidopsis thaliana lines that were generated by the GABI-Kat project. Sequences flanking the T-DNA insertion sites were aligned to the A.thaliana genome sequence, annotated with information about the FST, the insertion site and the line from which the FST was derived. A web interface permits text-based as well as sequence-based searches for relevant insertions. GABI-Kat SimpleSearch aims to help biologists to quickly find T-DNA insertion mutants for their research. AVAILABILITY: http://www.mpiz-koeln.mpg.de/GABI-Kat/     

bHLH transcription factor Her5 links patterning to regional inhibition of neurogenesis at the midbrain-hindbrain boundary

The midbrain-hindbrain (MH) domain of the vertebrate embryonic neural plate displays a stereotypical profile of neuronal differentiation, organized around a neuron-free zone ('intervening zone', IZ) at the midbrain-hindbrain boundary (MHB). The mechanisms establishing this early pattern of neurogenesis are unknown. We demonstrate that the MHB is globally refractory to neurogenesis, and that forced neurogenesis in this area interferes with the continued expression of genes defining MHB identity. We further show that expression of the zebrafish bHLH Hairy/E(spl)-related factor Her5 prefigures and then precisely delineates the IZ throughout embryonic development. Using morpholino knock-down and conditional gain-of-function assays, we demonstrate that Her5 is essential to prevent neuronal differentiation and promote cell proliferation in a medial compartment of the IZ. We identify one probable target of this activity, the zebrafish Cdk inhibitor p27Xic1. Finally, although the her5 expression domain is determined by anteroposterior patterning cues, we show Her5 does not retroactively influence MH patterning. Together, our results highlight the existence of a mechanism that actively inhibits neurogenesis at the MHB, a process that shapes MH neurogenesis into a pattern of separate neuronal clusters and might ultimately be necessary to maintain MHB integrity. Her5 appears as a partially redundant component of this inhibitory process that helps translate early axial patterning information into a distinct spatiotemporal pattern of neurogenesis and cell proliferation within the MH domain.     

The cell wall of lactic acid bacteria: surface constituents and macromolecular conformations

A variety of strains of the genus Lactobacillus was investigated with respect to the structure, softness, and interactions of their outer surface layers in order to construct structure-property relations of the Gram-positive bacterial cell wall. The role of the conformational properties of the constituents of the outer cell-wall layers and their spatial distribution on the cell wall is emphasized. Atomic force microscopy was used to resolve the surface structure, interactions, and softness of the bacterial cell wall at nanometer-length scales and upwards. The pH-dependence of the electrophoretic mobility and a novel interfacial adhesion assay were used to analyze the average physicochemical properties of the bacterial strains. The bacterial surface is smooth when a compact layer of globular proteins constitutes the outer surface, e.g., the S-layer of L. crispatus DSM20584. In contrast, for two other S-layer containing strains (L. helveticus ATCC12046 and L. helveticus ATCC15009), the S-layer is covered by polymeric surface constituents which adopt a much more extended conformation and which confer a certain roughness to the surface. Consequently, the S-layer is important for the overall surface properties of L. crispatus, but not for the surface properties of L. helveticus. Both surface proteins (L. crispatus DSM20584) and (lipo)teichoic acids (L. johnsonii ATCC332) confer hydrophobic properties to the bacterial surface whereas polysaccharides (L. johnsonii DSM20533 and L. johnsonii ATCC 33200) render the bacterial surface hydrophilic. Using the interfacial adhesion assay, it was demonstrated that hydrophobic groups within the cell wall adsorb limited quantities of hydrophobic compounds. The present work demonstrates that the impressive variation in surface properties displayed by even a limited number of genetically-related bacterial strains can be understood in terms of established colloidal concepts, provided that sufficiently detailed structural, chemical, and conformational information on the surface constituents is available.     

Complete genome sequence and analysis of the multiresistant nosocomial pathogen Corynebacterium jeikeium K411, a lipid-requiring bacterium of the human skin flora

Corynebacterium jeikeium is a "lipophilic" and multidrug-resistant bacterial species of the human skin flora that has been recognized with increasing frequency as a serious nosocomial pathogen. Here we report the genome sequence of the clinical isolate C. jeikeium K411, which was initially recovered from the axilla of a bone marrow transplant patient. The genome of C. jeikeium K411 consists of a circular chromosome of 2,462,499 bp and the 14,323-bp bacteriocin-producing plasmid pKW4. The chromosome of C. jeikeium K411 contains 2,104 predicted coding sequences, 52% of which were considered to be orthologous with genes in the Corynebacterium glutamicum, Corynebacterium efficiens, and Corynebacterium diphtheriae genomes. These genes apparently represent the chromosomal backbone that is conserved between the four corynebacteria. Among the genes that lack an ortholog in the known corynebacterial genomes, many are located close to transposable elements or revealed an atypical G+C content, indicating that horizontal gene transfer played an important role in the acquisition of genes involved in iron and manganese homeostasis, in multidrug resistance, in bacterium-host interaction, and in virulence. Metabolic analyses of the genome sequence indicated that the "lipophilic" phenotype of C. jeikeium most likely originates from the absence of fatty acid synthase and thus represents a fatty acid auxotrophy. Accordingly, both the complete gene repertoire and the deduced lifestyle of C. jeikeium K411 largely reflect the strict dependence of growth on the presence of exogenous fatty acids. The predicted virulence factors of C. jeikeium K411 are apparently involved in ensuring the availability of exogenous fatty acids by damaging the host tissue.     

Hyperglycemia induces early upregulation of the calcium sensor KChIP3/DREAM/calsenilin in the rat retina

Hyperglycemia alters the tight control of intracellular calcium dynamics in retinal cells and may lead to the development of diabetic retinopathy. The potassium channel interacting protein 3 (KChIP3) also known as DREAM (Downstream Regulatory Element Antagonist Modulator) or calsenilin (KChIP3/DREAM/calsenilin), a member of the neuronal calcium sensor protein family, is expressed in Müller glial cells and upregulated under high glucose experimental culture conditions. Here, we analyzed the expression and function of KChIP3 in the retina of streptozotocin induced diabetic Long Evans rats by immunofluorescence confocal microscopy, western blot, co-immunoprecipitation, whole cell patch clamp recording on isolated cells and KChIP3 gene silencing by RNA interference. Three weeks after streptozotocin application, KChIP3 was increased throughout the different retinal layers and this process was not linked to augmented apoptosis. KChIP3 co-immunoprecipitated with voltage gated K(+) channels of the K(V)4.2-4.3 subtype in retinal extracts from control and hyperglycemic rats. Electrophysiological analysis showed that control cells did not express A type (K(V)4-mediated) K(+) currents but most of the cells from streptozotocin treated retinas displayed macroscopic currents with an inactivating component sensitive to 4-AP, suggesting the persistence of the A type currents at early times after treatment. siRNA analysis in Müller cells cultures grown under high glucose experimental conditions corroborated that, when the expression of KChIP3 is 50% reduced, the number of cells expressing A type currents decreases significantly. Together these data suggest an altered expression and function of KChIP3 after streptozotocin induced hyperglycemia that might help explain some pathological alterations in early diabetic retinopathy.     

Analysis of HIV Early Infant Diagnosis Data to Estimate Rates of Perinatal HIV Transmission in Zambia

Background

Mother-to-child transmission of HIV (MTCT) remains the most prevalent source of pediatric HIV infection. Most PMTCT (prevention of mother-to-child transmission of HIV) programs have concentrated monitoring and evaluation efforts on process rather than on outcome indicators. In this paper, we review service data from 28,320 children born to HIV-positive mothers to estimate MTCT rates.

Method

This study analyzed DNA PCR results and PMTCT data from perinatally exposed children zero to 12 months of age from five Zambian provinces between September 2007 and July 2010.

Results

The majority of children (58.6%) had a PCR test conducted between age six weeks and six months. Exclusive breastfeeding (56.8%) was the most frequent feeding method. An estimated 45.9% of mothers were below 30 years old and 93.3% had disclosed their HIV status. In terms of ARV regimen for PMTCT, 32.7% received AZT+single dose NVP (sdNVP), 30.9% received highly active antiretroviral treatment (HAART), 19.6% received sdNVP only and 12.9% received no ARVs. Transmission rates at six weeks when ARVs were received by both mother and baby, mother only, baby only, and none were 5.8%, 10.5%, 15.8% and 21.8% respectively. Transmission rates at six weeks where mother received HAART, AZT+sd NVP, sdNVP, and no intervention were 4.2%, 6.8%, 8.7% and 20.1% respectively. Based on adjusted analysis including ARV exposures and non ARV-related parameters, lower rates of positive PCR results were associated with 1) both mother and infant receiving prophylaxis, 2) children never breastfed and 3) mother being 30 years old or greater.Overall between September 2007 and July 2010, 12.2% of PCR results were HIV positive. Between September 2007 and January 2009, then between February 2009 and July 2010, proportions of positive PCR results were 15.1% and 11% respectively, a significant difference.

Conclusion

The use of ARV drugs reduces vertical transmission of HIV in a program setting. Non-chemoprophylactic factors also play a significant role in HIV transmission. The overall change in the proportions of positive PCR results over time is more likely an indication of better PMTCT implementation. Determination of the outcomes of PMTCT in program settings is feasible but requires accurate documentation and analysis.     

Genomic assessment of human cumulus cell marker genes as predictors of oocyte developmental competence: impact of various experimental factors

Background

Single embryo transfer (SET) is the most successful way to reduce the frequency of multiple pregnancies following in vitro fertilisation. However, selecting the embryo for SET with the highest chances of pregnancy remains a difficult challenge since morphological and kinetics criteria provide poor prediction of both developmental and implantation ability. Partly through the expression of specific genes, the oocyte-cumulus interaction helps the oocyte to acquire its developmental competence. Our aim was therefore to identify at the level of cumulus cells (CCs) genes related to oocyte developmental competence.

Methodology/Principal Findings

197 individual CCs were collected from 106 patients undergoing an intra-cytoplasmic sperm injection procedure. Gene expression of CCs was studied using microarray according to the nuclear maturity of the oocyte (immature vs. mature oocyte) and to the developmental competence of the oocyte (ability to reach the blastocyst stage after fertilisation). Microarray study was followed by a meta-analysis of the behaviour of these genes in other datasets available in Gene Expression Omnibus which showed the consistency of this list of genes. Finally, 8 genes were selected according to oocyte developmental competence from the 308 differentially expressed genes (p<0.0001) for further validation by quantitative PCR (qPCR). Three of these 8 selected genes were validated as potential biomarkers (PLIN2, RGS2 and ANG). Experimental factors such as inter-patient and qPCR series variability were then assessed using the Generalised Linear Mixed Model procedure, and only the expression level of RGS2 was confirmed to be related to oocyte developmental competence. The link between biomarkers and pregnancy was finally evaluated and level of RGS2 expression was also correlated with clinical pregnancy.

Conclusion/Significance

RGS2, known as a regulator of G protein signalling, was the only gene among our 8 selected candidates biomarkers of oocyte competence to cover many factors of variability, including inter-patient factors and experimental conditions.     

A generalized method for transfecting root epidermis uncovers endosomal dynamics in Arabidopsis root hairs

Progress in analysing the cellular functions of many structural proteins has accelerated through the use of confocal microscopy together with transient gene expression. Several methods for transient expression have been developed in the past few years, but their application has seen limited success beyond a few tractable species and tissues. We have developed a simple and efficient method to visualize fluorescent proteins in Arabidopsis root epidermis using co-cultivation of seedlings with Agrobacterium rhizogenes. The method is equally suitable for transient gene expression in other species, including Thellungiella, and can be combined with supporting molecular and biochemical analyses. The method promises significant advantages for study of membrane dynamics, cellular development and polar growth in root hairs without interference in the development of the plant. Since the method targets specifically the root epidermis, it also offers a powerful tool to approach issues of root-rhizosphere interactions, such as ion transport and nutrient acquisition. As a proof of principle, we carried out transfections with fluorescent markers for the plasma membrane (NpPMA2-GFP, Nicotiana plumbaginifolia L. Plasma Membrane H(+)-ATPase 2), the endoplasmic reticulum (YFP-HDEL), and the Golgi apparatus (sialyl transferase-GFP) to trace their distribution in growing Arabidopsis root hairs and epidermis. The results demonstrate that, in Arabidopsis root hairs, movement of the Golgi is faster than previously reported for tobacco leaf epidermal cells, consistent with the high secretory dynamics of the tip growing cell; they show a pattern to the endoplasmic reticulum within the cytoplasm that is more diffuse than found in tobacco leaf epidermis, and they confirm previous findings of a polarized distribution of the endoplasmic reticulum at the tip of growing root hairs.     

Genome sequence and description of Nesterenkonia massiliensis sp. nov. strain NP1T

Nesterenkonia massiliensis sp. nov., strain NP1^T, is the type strain of Nesterenkonia massiliensis sp. nov., a new species within the genus Nesterenkonia. This strain, whose genome is described here, was isolated from the feces of a 32-year-old French woman suffering from AIDS and living in Marseille. Nesterenkonia massiliensis is a Gram-positive aerobic coccus. Here, we describe the features of this bacterium, together with the complete genome sequencing and annotation. The 2,726,371 bp long genome (one chromosome but no plasmid) contains 2,663 protein-coding and 51 RNA genes, including 1 rRNA operon.