Constitutive and inducible aspects of nitrate-nitrogen uptake by Chlamydomonas reinhardtii

Similarly to higher plant root systems, Chlamydomonas reinhardtii Dangeard (UTEX 90) cells exhibited biphasic NO₃^? uptake kinetics. The uptake pattern was similar in cells cultured in 10 mM NO₃^? (NO₃^?-grown), 0.25 mM NO₃^? (N-limited) or 10 mM NO₃^? followed by an 18-h period of N-deprivation (N-starved). In all cell types there was an apparent phase transition in uptake at 1.1 mM NO₃^?, although there were variations in the uptake V_max of both isotherms. The rate of uptake via isotherm 0 ([NO₃^?]<1.1 mM) in N-limited cells was higher than that of either NO₃^?-grown or N-starved cells. In contrast, NO₃^?-grown and N-limited cells exhibited comparable V_max values when supplied with 1.1 to 1.8 mM NO₃^? (isotherm 1). When supplied with 1.6 mM NO₃^?, both N-limited and N-starved cells exhibited enhanced linear uptake after 60 min of incubation. We ascribed this to an induction phenomenon. This trend was not observed when NO₃^?-grown cells were supplied with 1.6 mM NO₃^?, or when N-limited and N-starved cells were supplied with 0.6 mM NO₃^?. The ‘inducible’ aspect of uptake by N-limited cells was blocked by cycloheximide (10 mg l^?1), but not by actinomycin D (5 mg l^?1), thus indicating the involvement of a translational or post-translational event. To investigate this phenomenon further, we analysed the cell proteins of N-limited cells supplied with either 0.6 or 1.6 mM NO₃^? for 90 min, using two-dimensional gel electrophoresis. Comparison of protein profiles enabled the identification of a single cell membrane-associated polypeptide (21 kDa, pI ca 5.5) and ten soluble fraction polypeptides (17–73 kDa, pI ca 5.0 to 7.1) unique to the high NO₃^? treatment. We propose that the ‘inducible’ portion of NO₃^? uptake may provide the means by which C. reinhardtii cells regulate uptake in accordance with assimilatory capacity.     

Molecular variation at the SLC6A3 locus predicts lifetime risk of PTSD in the Detroit Neighborhood Health Study

Recent work suggests that the 9-repeat (9R) allele located in the 3'UTR VNTR of the SLC6A3 gene increases risk of posttraumatic stress disorder (PTSD). However, no study reporting this association to date has been based on population-based samples. Furthermore, no study of which we are aware has assessed the joint action of genetic and DNA methylation variation at SLC6A3 on risk of PTSD. In this study, we assessed whether molecular variation at SLC6A3 locus influences risk of PTSD. Participants (n?=?320; 62 cases/258 controls) were drawn from an urban, community-based sample of predominantly African American Detroit adult residents, and included those who had completed a baseline telephone survey, had provided blood specimens, and had a homozygous genotype for either the 9R or 10R allele or a heterozygous 9R/10R genotype. The influence of DNA methylation variation in the SLC6A3 promoter locus was also assessed in a subset of participants with available methylation data (n?=?83; 16 cases/67 controls). In the full analytic sample, 9R allele carriers had almost double the risk of lifetime PTSD compared to 10R/10R genotype carriers (OR?=?1.98, 95% CI?=?1.02-3.86), controlling for age, sex, race, socioeconomic status, number of traumas, smoking, and lifetime depression. In the subsample of participants with available methylation data, a significant (p?=?0.008) interaction was observed whereby 9R allele carriers showed an increased risk of lifetime PTSD only in conjunction with high methylation in the SLC6A3 promoter locus, controlling for the same covariates. Our results confirm previous reports supporting a role for the 9R allele in increasing susceptibility to PTSD. They further extend these findings by providing preliminary evidence that a "double hit" model, including both a putatively reduced-function allele and high methylation in the promoter region, may more accurately capture molecular risk of PTSD at the SLC6A3 locus.     

Evidence for the Neurotrophic Regulation of Collagen-Tailed Acetylcholinesterase in Immature Skeletal Muscle by β-Endorphin

Abstract: Acetylcholinesterase (AChE) was extracted in a high-saline medium from gastrocnemius muscles of rat embryos and young rats aged 14 days'gestation to 40 days post partum. The molecular forms of the enzyme were separated by low-salt precipitation, followed by velocity sedimentation. During gestation, all molecular forms increased in activity, particularly the 16 S (A₁₂) form. During the first 2 weeks of life, there was a large increase in the activity of soluble AChE (G forms), whilst the activity of insoluble AChE (A forms) was reduced. Denervation of the muscle reversed the change in the relative proportions of the molecular forms. The embryonic pattern of activities of AChE forms persisted in cultures of myotubes obtained at 20 days'gestation and maintained in the absence of spinal cord. When myotubes were maintained in medium previously conditioned by developing spinal cord explants, 16 S AChE declined while the soluble (4 and 6 S) forms increased in activity in a manner resembling that seen in early postnatal muscles in vivo . β-Endorphin (β-EP) immunoreactivity was detected in the spinal cord-conditioned medium and was identified by HPLC and ion-exchange chromatography as β-EP-(l–31) plus its shortened and N -acetylated forms. Cultivation of myotubes in the presence of synthetic camel β-EP resulted in a reversible change in the pattern of AChE forms which was similar to that seen with spinal cord-conditioned medium. These studies provide evidence for the neuroregulation of AChE A and G forms in immature skeletal muscle. A major candidate for this role is β-EP, produced and released by developing spinal cord.     

The HopF family of Pseudomonas syringae type III secreted effectors

Pseudomonas syringae is a bacterial phytopathogen that utilizes the type III secretion system to inject effector proteins into plant host cells. Pseudomonas syringae can infect a wide range of plant hosts, including agronomically important crops such as tomatoes and beans. The ability of P. syringae to infect such numerous hosts is caused, in part, by the diversity of effectors employed by this phytopathogen. Over 60 different effector families exist in P. syringae; one such family is HopF, which contains over 100 distinct alleles. Despite this diversity, research has focused on only two members of this family: HopF1 from P. syringae pathovar phaseolicola 1449B and HopF2 from P. syringae pathovar tomato DC3000. In this study, we review the research on HopF family members, including their host targets and molecular mechanisms of immunity suppression, and their enzymatic function. We also provide a phylogenetic analysis of this expanding effector family which provides a basis for a proposed nomenclature to guide future research. The extensive genetic diversity that exists within the HopF family presents a great opportunity to study how functional diversification on an effector family contributes to host specialization.     

Portal motor velocity and internal force resisting viral DNA packaging in bacteriophage phi29

During the assembly of many viruses, a powerful molecular motor compacts the genome into a preassembled capsid. Here, we present measurements of viral DNA packaging in bacteriophage phi29 using an improved optical tweezers method that allows DNA translocation to be measured from initiation to completion. This method allowed us to study the previously uncharacterized early stages of packaging and facilitated more accurate measurement of the length of DNA packaged. We measured the motor velocity versus load at near-zero filling and developed a ramped DNA stretching technique that allowed us to measure the velocity versus capsid filling at near-zero load. These measurements reveal that the motor can generate significantly higher velocities and forces than detected previously. Toward the end of packaging, the internal force resisting DNA confinement rises steeply, consistent with the trend predicted by many theoretical models. However, the force rises to a higher magnitude, particularly during the early stages of packaging, than predicted by models that assume coaxial inverse spooling of the DNA. This finding suggests that the DNA is not arranged in that conformation during the early stages of packaging and indicates that internal force is available to drive complete genome ejection in vitro. The maximum force exceeds 100 pN, which is about one-half that predicted to rupture the capsid shell.     

Interleukin-6 is a potential biomarker for severe pandemic H1N1 influenza A infection

Pandemic H1N1 influenza A (H1N1pdm) is currently a dominant circulating influenza strain worldwide. Severe cases of H1N1pdm infection are characterized by prolonged activation of the immune response, yet the specific role of inflammatory mediators in disease is poorly understood. The inflammatory cytokine IL-6 has been implicated in both seasonal and severe pandemic H1N1 influenza A (H1N1pdm) infection. Here, we investigated the role of IL-6 in severe H1N1pdm infection. We found IL-6 to be an important feature of the host response in both humans and mice infected with H1N1pdm. Elevated levels of IL-6 were associated with severe disease in patients hospitalized with H1N1pdm infection. Notably, serum IL-6 levels associated strongly with the requirement of critical care admission and were predictive of fatal outcome. In C57BL/6J, BALB/cJ, and B6129SF2/J mice, infection with A/Mexico/4108/2009 (H1N1pdm) consistently triggered severe disease and increased IL-6 levels in both lung and serum. Furthermore, in our lethal C57BL/6J mouse model of H1N1pdm infection, global gene expression analysis indicated a pronounced IL-6 associated inflammatory response. Subsequently, we examined disease and outcome in IL-6 deficient mice infected with H1N1pdm. No significant differences in survival, weight loss, viral load, or pathology were observed between IL-6 deficient and wild-type mice following infection. Taken together, our findings suggest IL-6 may be a potential disease severity biomarker, but may not be a suitable therapeutic target in cases of severe H1N1pdm infection due to our mouse data.     

Solid state fermentation and fractionation of oat straw by Basidiomycetes

Summary Seventee white-rot and brown-rot fungi were screened for their ability to fractionate the lignocellulose structure of oat straw through the preferential attack of lignin or cellulose. Fermentations were carried out under solid-state conditions with 25 g quantities of straw. The fermented straw was analyzed for weight loss, Klason lignin loss and cellulase digestion. All the fungi attacked both lignin and carbohydrate fractions causing 3–28% weight losses and 26–34 g/100 g enzymatic digestibility. Polyporus tulipiferae, Phanerochaete chrysosporium and Polyporus sp. were tested for the effects of various nitrogen, phosphate and carbon levels, incubation temperatures and incubation time. The three fungi had different responses to these factors.     

Patient-Reported Outcome (PRO) Assessment in Clinical Trials: A Systematic Review of Guidance for Trial Protocol Writers

Background

Evidence suggests there are inconsistencies in patient-reported outcome (PRO) assessment and reporting in clinical trials, which may limit the use of these data to inform patient care. For trials with a PRO endpoint, routine inclusion of key PRO information in the protocol may help improve trial conduct and the reporting and appraisal of PRO results; however, it is currently unclear exactly what PRO-specific information should be included. The aim of this review was to summarize the current PRO-specific guidance for clinical trial protocol developers.

Methods and Findings

We searched the MEDLINE, EMBASE, CINHAL and Cochrane Library databases (inception to February 2013) for PRO-specific guidance regarding trial protocol development. Further guidance documents were identified via Google, Google scholar, requests to members of the UK Clinical Research Collaboration registered clinical trials units and international experts. Two independent investigators undertook title/abstract screening, full text review and data extraction, with a third involved in the event of disagreement. 21,175 citations were screened and 54 met the inclusion criteria. Guidance documents were difficult to access: electronic database searches identified just 8 documents, with the remaining 46 sourced elsewhere (5 from citation tracking, 27 from hand searching, 7 from the grey literature review and 7 from experts). 162 unique PRO-specific protocol recommendations were extracted from included documents. A further 10 PRO recommendations were identified relating to supporting trial documentation. Only 5/162 (3%) recommendations appeared in ≥50% of guidance documents reviewed, indicating a lack of consistency.

Conclusions

PRO-specific protocol guidelines were difficult to access, lacked consistency and may be challenging to implement in practice. There is a need to develop easily accessible consensus-driven PRO protocol guidance. Guidance should be aimed at ensuring key PRO information is routinely included in appropriate trial protocols, in order to facilitate rigorous collection/reporting of PRO data, to effectively inform patient care.