The Tec family of tyrosine kinases in T cells,amplifiers of T cell receptor signals

ITK and Rlk/Txk are the predominant Tec family of tyrosine kinases expressed in T cells, and are involved in T cell antigen receptor mediated activation of T cells. These kinases require prior activation of Lck, Zap-70 and PI3-kinase for efficient activation. They share major substrates with both Lck and Zap-70, however the pathways they regulate are unclear. Recent evidence suggests that these kinases may not activate unique pathways, but instead serve as amplifiers for the upstream kinases Lck and Zap-70. This review will discuss the evidence for this view.     

JNK Signalling Controls Remodelling of the Segment Boundary through Cell Reprogramming during Drosophila Morphogenesis

Segments are fundamental units in animal development which are made of distinct cell lineages separated by boundaries. Although boundaries show limited plasticity during their formation for sharpening, cell lineages make compartments that become tightly restricted as development goes on. Here, we characterize a unique case of breaking of the segment boundary in late drosophila embryos. During dorsal closure, specific cells from anterior compartments cross the segment boundary and enter the adjacent posterior compartments. This cell mixing behaviour is driven by an anterior-to-posterior reprogramming mechanism involving de novo expression of the homeodomain protein Engrailed. Mixing is accompanied by stereotyped local cell intercalation, converting the segment boundary into a relaxation compartment important for tension-release during morphogenesis. This process of lineage switching and cell remodelling is controlled by JNK signalling. Our results reveal plasticity of segment boundaries during late morphogenesis and a role for JNK-dependent developmental reprogramming in this process.     

Reduction of surgical site infection using a novel intervention (ROSSINI): study protocol for a randomised controlled trial

Background

Surgical site infection (SSI) is a common complication following abdominal surgery. It is associated with considerable morbidity and mortality, and its management results in significant cost to health services within both primary and secondary care. Some surgeons believe that the use of a wound-edge protection device may reduce the incidence of SSI. Whilst there is some encouraging evidence showing that such devices may lead to a reduction in SSI, there are no controlled trials of sufficient size or quality to support their routine use.

Methods/Design

750 patients will be recruited from around 20 surgical units within the United Kingdom. Patients undergoing laparotomy through any major abdominal incision for any indication, elective or emergency, are eligible. Patients under the age of 18, those undergoing a laparoscopic assisted procedure or who have undergone laparotomy within the previous 3 months, and those who are unable to give informed consent will be excluded. Patients will be randomised (1:1 ratio) to the use of a wound-edge protection device or no wound-edge protection device during surgery.Follow up will consist of blinded clinical wound reviews at 5-7 days and 30-33 days postoperatively with a self-completed questionnaire covering the intervening period. Quality of life questionnaires will be completed prior to surgery and at the subsequent wound review points and information on resource usage will also be captured.The primary outcome measure is SSI within 30 days of surgery. Secondary outcomes include the impact of the degree of wound contamination, patient comorbidity, and operative characteristics on the efficacy of a wound-edge protection device in reducing SSI and whether the use of a wound-edge protection device has an effect on health-related quality of life or length of hospital stay and is cost-effective.

Discussion

Rossini is the first multicentre observer-blinded randomised controlled trial of sufficient size and quality to establish whether the use of a wound-edge protection device in adult patients undergoing abdominal surgery leads to a lower rate of SSI. The results of this study will be used to inform current surgical practice and may potentially benefit patients undergoing surgery in the future.

Trial registration number

Current Controlled Trials ISRCTN: ISRCTN40402832

     

Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo

Sphingosine 1-phosphate (S1P, 1) regulates vascular barrier and lymphoid development, as well as lymphocyte egress from lymphoid organs, by activating high-affinity S1P1 receptors. We used reversible chemical probes (i) to gain mechanistic insights into S1P systems organization not accessible through genetic manipulations and (ii) to investigate their potential for therapeutic modulation. Vascular (but not airway) administration of the preferred R enantiomer of an in vivo-active chiral S1P1 receptor antagonist induced loss of capillary integrity in mouse skin and lung. In contrast, the antagonist did not affect the number of constitutive blood lymphocytes. Instead, alteration of lymphocyte trafficking and phenotype required supraphysiological elevation of S1P1 tone and was reversed by the antagonist. In vivo two-photon imaging of lymph nodes confirmed requirements for obligate agonism, and the data were consistent with the presence of a stromal barrier mechanism for gating lymphocyte egress. Thus, chemical modulation reveals differences in S1P-S1P1 'set points' among tissues and highlights both mechanistic advantages (lymphocyte sequestration) and risks (pulmonary edema) of therapeutic intervention.     

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

Background & Aims

Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen.

Methods

Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity.

Results

Cell preparation yielded the following cell counts per gram of liver tissue: 2.0±0.4×10⁷ hepatocytes, 1.8±0.5×10⁶ Kupffer cells, 4.3±1.9×10⁵ liver sinusoidal endothelial cells, and 3.2±0.5×10⁵ stellate cells. Hepatocytes were identified by albumin (95.5±1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5±1.2%) and exhibited phagocytic activity, as determined with 1μm latex beads. Endothelial cells were CD146⁺ (97.8±1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1±1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence.

Conclusions

Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.     

Shifts in archaeal communities associated with lithological and geochemical variations in subsurface Cretaceous rock

Subsurface microbial community structure in relation to geochemical gradients and lithology was investigated using a combination of molecular phylogenetic and geochemical analyses. Discreet groundwater and substratum samples were obtained from depths ranging from 182 to 190 m beneath the surface at approximately 10-cm intervals using a multilevel sampler (MLS) that straddled Cretaceous shale and sandstone formations at a site in the southern San Juan Basin in New Mexico. DNA and RNA were extracted directly from quartzite sand substratum loaded into individual cells of the MLS and colonized in situ. Polymerase chain reaction (PCR)-mediated T-RFLP analysis of archaeal rRNA genes (rDNA) in conjunction with partial sequencing analysis of archaeal rDNA libraries and quantitative RNA hybridization with oligonucleotide probes were used to probe community structure and function. Although total microbial populations remained relatively constant over the entire depth interval sampled, significant shifts in archaeal populations, predominantly methanogens, were observed. These shifts coincided with the geochemical transition from relatively high methane (26 mM), low sulphate (< 3 mg l(-1)) conditions in the region adjacent to the organic matter-rich shale to relatively low-methane (< 0.5 mM), high-sulphate (48 mg l(-1)) conditions in the organic-poor sandstone beneath the shale. These results indicated that active, phylogenetically diverse archaeal communities were present in the subsurface Cretaceous rock environment at this site and that major archaeal clades shifted dramatically over scales of tens of centimetres, corresponding to changes in the lithology and geochemical gradients.     

The nitric oxide (NO)-sensing repressor NsrR of Neisseria meningitidis has a compact regulon of genes involved in NO synthesis and detoxification

We have analyzed the extent of regulation by the nitric oxide (NO)-sensitive repressor NsrR from Neisseria meningitidis MC58, using microarray analysis. Target genes that appeared to be regulated by NsrR, based on a comparison between an nsrR mutant and a wild-type strain, were further investigated by quantitative real-time PCR, revealing a very compact set of genes, as follows: norB (encoding NO reductase), dnrN (encoding a protein putatively involved in the repair of nitrosative damage to iron-sulfur clusters), aniA (encoding nitrite reductase), nirV (a putative nitrite reductase assembly protein), and mobA (a gene associated with molybdenum metabolism in other species but with a frame shift in N. meningitidis). In all cases, NsrR acts as a repressor. The NO protection systems norB and dnrN are regulated by NO in an NsrR-dependent manner, whereas the NO protection system cytochrome c' (encoded by cycP) is not controlled by NO or NsrR, indicating that N. meningitidis expresses both constitutive and inducible NO protection systems. In addition, we present evidence to show that the anaerobic response regulator FNR is also sensitive to NO but less so than NsrR, resulting in complex regulation of promoters such as aniA, which is controlled by both FNR and NsrR: aniA was found to be maximally induced by intermediate NO concentrations, consistent with a regulatory system that allows expression during denitrification (in which NO accumulates) but is down-regulated as NO approaches toxic concentrations.     

Proteomic analysis of the eyespot of Chlamydomonas reinhardtii provides novel insights into its components and tactic movements

Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. To further understand the molecular organization of the eyespot apparatus and the phototactic movement that is controlled by light and the circadian clock, a detailed understanding of all components of the eyespot apparatus is needed. We developed a procedure to purify the eyespot apparatus from the green model alga Chlamydomonas reinhardtii. Its proteomic analysis resulted in the identification of 202 different proteins with at least two different peptides (984 in total). These data provide new insights into structural components of the eyespot apparatus, photoreceptors, retina(l)-related proteins, members of putative signaling pathways for phototaxis and chemotaxis, and metabolic pathways within an algal visual system. In addition, we have performed a functional analysis of one of the identified putative components of the phototactic signaling pathway, casein kinase 1 (CK1). CK1 is also present in the flagella and thus is a promising candidate for controlling behavioral responses to light. We demonstrate that silencing CK1 by RNA interference reduces its level in both flagella and eyespot. In addition, we show that silencing of CK1 results in severe disturbances in hatching, flagellum formation, and circadian control of phototaxis.     

Exploitation of archived marine nematodes — a hot lysis DNA extraction protocol for molecular studies

Museums and other research organizations around the world have large numbers of formalin-fixed marine invertebrates in their collections. These have the potential to be a valuable resource for molecular ecological studies, but the development of methodologies for the molecular analysis of formalin-fixed material has been slow. In this study, a hot lysis protocol accompanied by the use of a commercial DNA extraction kit has been employed for DNA recovery from archived marine nematodes, followed by PCR amplification and sequencing. In total, 25 specimens ranging from estuarine to deep sea environments were subjected to molecular analyses. Successful amplification and sequencing of the nuclear small subunit ribosomal RNA (18S rRNA) gene was achieved in all individuals. Additionally, some estuarine nematodes were tentatively identified to genus and species using a phylogenetic approach. In the future, this technique should prove to be profitable for the genetic study of a wide range of formalin-fixed marine invertebrates.