A fasciclin-like arabinogalactan-protein (FLA) mutant of Arabidopsis thaliana, fla1, shows defects in shoot regeneration

BACKGROUND: The fasciclin-like arabinogalactan-proteins (FLAs) are an enigmatic class of 21 members within the larger family of arabinogalactan-proteins (AGPs) in Arabidopsis thaliana. Located at the cell surface, in the cell wall/plasma membrane, they are implicated in many developmental roles yet their function remains largely undefined. Fasciclin (FAS) domains are putative cell-adhesion domains found in extracellular matrix proteins of organisms from all kingdoms, but the juxtaposition of FAS domains with highly glycosylated AGP domains is unique to plants. Recent studies have started to elucidate the role of FLAs in Arabidopsis development. FLAs containing a single FAS domain are important for the integrity and elasticity of the plant cell wall matrix (FLA11 and FLA12) and FLA3 is involved in microspore development. FLA4/SOS5 with two FAS domains and two AGP domains has a role in maintaining proper cell expansion under salt stressed conditions. The role of other FLAs remains to be uncovered. METHOD/PRINCIPAL FINDINGS: Here we describe the characterisation of a T-DNA insertion mutant in the FLA1 gene (At5g55730). Under standard growth conditions fla1-1 mutants have no obvious phenotype. Based on gene expression studies, a putative role for FLA1 in callus induction was investigated and revealed that fla1-1 has a reduced ability to regenerate shoots in an in vitro shoot-induction assay. Analysis of FLA1p:GUS reporter lines show that FLA1 is expressed in several tissues including stomata, trichomes, the vasculature of leaves, the primary root tip and in lateral roots near the junction of the primary root. CONCLUSION: The results of the developmental expression of FLA1 and characterisation of the fla1 mutant support a role for FLA1 in the early events of lateral root development and shoot development in tissue culture, prior to cell-type specification.     

Incidence and risk factors of serious adverse events during antituberculous treatment in Rwanda: a prospective cohort study

Background

Tuberculosis (TB) and TB-human immunodeficiency virus infection (HIV) coinfection is a major public health concern in resource-limited settings. Although TB treatment is challenging in HIV-infected patients because of treatment interactions, immunopathological reactions, and concurrent infections, few prospective studies have addressed this in sub-Saharan Africa.In this study we aimed to determine incidence, causes of, and risk factors for serious adverse events among patients on first-line antituberculous treatment, as well as its impact on antituberculous treatment outcome.

Methods and findings

Prospective observational cohort study of adults treated for TB at the Internal Medicine department of the Kigali University Hospital from May 2008 through August 2009.Of 263 patients enrolled, 253 were retained for analysis: median age 35 (Interquartile range, IQR 28–40), 55% male, 66% HIV-positive with a median CD4 count 104 cells/mm³ (IQR 44–248 cells/mm³). Forty percent had pulmonary TB, 43% extrapulmonary TB and 17% a mixed form. Sixty-four (26%) developed a serious adverse event; 58/167 (35%) HIV-infected vs. 6/86 (7%) HIV-uninfected individuals. Commonest events were concurrent infection (n = 32), drug-induced hepatitis (n = 24) and paradoxical reactions/TB-IRIS (n = 23).HIV-infection (adjusted Hazard Ratio, aHR 3.4, 95% Confidence Interval, CI 1.4–8.7) and extrapulmonary TB (aHR 2, 95%CI 1.1–3.7) were associated with an increased risk of serious adverse events. For TB/HIV co-infected patients, extrapulmonary TB (aHR 2.0, 95%CI 1.1–3.9) and CD4 count <100 cells/mm3 at TB diagnosis (aHR 1.7, 95%CI 1.0–2.9) were independent predictors. Adverse events were associated with an almost two-fold higher risk of unsuccessful treatment outcome at 6 months (HR 1.89, 95%CI 1.3–3.0).

Conclusion

Adverse events frequently complicate the course of antituberculous treatment and worsen treatment outcome, particularly in patients with extrapulmonary TB and advanced immunodeficiency. Concurrent infection accounts for most events. Our data suggest that deterioration in a patient already receiving antituberculous treatment should prompt an aggressive search for additional infections.     

EscE and EscG Are Cochaperones for the Type III Needle Protein EscF of Enteropathogenic Escherichia coli

Type III secretion systems (T3SSs) are central virulence mechanisms used by a variety of Gram-negative bacteria to inject effector proteins into host cells. The needle polymer is an essential part of the T3SS that provides the effector proteins a continuous channel into the host cytoplasm. It has been shown for a few T3SSs that two chaperones stabilize the needle protein within the bacterial cytosol to prevent its premature polymerization. In this study, we characterized the chaperones of the enteropathogenic Escherichia coli (EPEC) needle protein EscF. We found that Orf2 and Orf29, two poorly characterized proteins encoded within the EPEC locus of enterocyte effacement (LEE), function as the needle protein cochaperones. Our finding demonstrated that both Orf2 and Orf29 are essential for type III secretion (T3S). In addition, we found that Orf2 and Orf29 associate with the bacterial membrane and form a complex with EscF. Orf2 and Orf29 were also shown to disrupt the polymerization of EscF in vitro. Prediction of the tertiary structures of Orf2 and Orf29 showed high structural homology to chaperones of other T3SS needle proteins. Overall, our data suggest that Orf2 and Orf29 function as the chaperones of the needle protein, and therefore, they have been renamed EscE and EscG.     

Testing Bergmann’s rule and the Rosenzweig hypothesis with craniometric studies of the South American sea lion

We tested the validity of Bergmann’s rule and Rosenzweig’s hypothesis through an analysis of the geographical variation of the skull size of Otaria flavescens along the entire distribution range of the species (except Brazil). We quantified the sizes of 606 adult South American sea lion skulls measured in seven localities of Peru, Chile, Uruguay, Argentina, and the Falkland/Malvinas Islands. Geographical and environmental variables included latitude, longitude, and monthly minimum, maximum, and mean air and ocean temperatures. We also included information on fish landings as a proxy for productivity. Males showed a positive relationship between condylobasal length (CBL) and latitude, and between CBL and the six temperature variables. By contrast, females showed a negative relationship between CBL and the same variables. Finally, female skull size showed a significant and positive correlation with fish landings, while males did not show any relationship with this variable. The body size of males conformed to Bergmann’s rule, with larger individuals found in southern localities of South America. Females followed the converse of Bergmann’s rule at the intraspecific level, but showed a positive relationship with the proxy for productivity, thus supporting Rosenzweig’s hypothesis. Differences in the factors that drive body size in females and males may be explained by their different life-history strategies. Our analyses demonstrate that latitude and temperature are not the only factors that explain spatial variation in body size: others such as food availability are also important for explaining the ecogeographical patterns found in O. flavescens.     

Bacterial Oxidation of Molybdenum in Ore Deposits

Some microorganisms can use reduced compounds of molybdenum as energy sources for autotrophic growth. A culture of molybdenum‐oxidizing bacteria was obtained by enrichment in a mineral‐salts medium by using samples of oxidized molybdenum ore from a molybdenum deposit as inoculum. In laboratory experiments, powellite (CaMoO₄) was obtained. The role of microorganisms in the geochemistry of molybdenum is discussed.     

One antitoxin—two functions: SR4 controls toxin mRNA decay and translation

Type I toxin–antitoxin systems encoded on bacterial chromosomes became the focus of research during the past years. However, little is known in terms of structural requirements, kinetics of interaction with their targets and regulatory mechanisms of the antitoxin RNAs. Here, we present a combined in vitro and in vivo analysis of the bsrG/SR4 type I toxin–antitoxin system from Bacillus subtilis. The secondary structures of SR4 and bsrG mRNA and of the SR4/bsrG RNA complex were determined, apparent binding rate constants calculated and functional segments required for complex formation narrowed down. The initial contact between SR4 and its target was shown to involve the SR4 terminator loop and loop 3 of bsrG mRNA. Additionally, a contribution of the stem of SR4 stem-loop 3 to target binding was found. On SR4/bsrG complex formation, a 4 bp double-stranded region sequestering the bsrG Shine Dalgarno (SD) sequence was extended to 8 bp. Experimental evidence was obtained that this extended region caused translation inhibition of bsrG mRNA. Therefore, we conclude that SR4 does not only promote degradation of the toxin mRNA but also additionally inhibit its translation. This is the first case of a dual-acting antitoxin RNA.     

T cell profiling reveals high CD4+CTLA-4+ T cell frequency as dominant predictor for survival after Prostate GVAX/ipilimumab treatment

Immune checkpoint blockade enhances antitumor responses, but can also lead to severe immune-related adverse events (IRAE). To avoid unnecessary exposure to these potentially hazardous agents, it is important to identify biomarkers that correlate with clinical activity and can be used to select patients that will benefit from immune checkpoint blockade. To understand the consequences of CTLA-4 blockade and identify biomarkers for clinical efficacy and/or survival, an exploratory T cell monitoring study was performed in a phase I/II dose escalation/expansion trial (n = 28) of combined Prostate GVAX/ipilimumab immunotherapy. Phenotypic T cell monitoring in peripheral blood before and after Prostate GVAX/ipilimumab treatment revealed striking differences between patients who benefited from therapy and patients that did not. Treatment-induced rises in absolute lymphocyte counts, CD4⁺ T cell differentiation, and CD4⁺ and CD8⁺ T cell activation were all associated with clinical benefit. Moreover, significantly prolonged overall survival (OS) was observed for patients with high pre-treatment frequencies of CD4⁺CTLA-4⁺, CD4⁺PD-1⁺, or differentiated (i.e., non-naive) CD8⁺ T cells or low pre-treatment frequencies of differentiated CD4⁺ or regulatory T cells. Unsupervised clustering of these immune biomarkers revealed cancer-related expression of CTLA-4⁺ in CD4⁺ T cells to be a dominant predictor for survival after Prostate GVAX/ipilimumab therapy and to thus provide a putative and much-needed biomarker for patient selection prior to therapeutic CTLA4 blockade.     

24 Evolutionary dynamics of inteins in bacteria

Inteins are protein sequences that autocatalytically splice themselves out of protein precursors – analogous to introns – and ligate the flanking regions into a functional protein. Inteins are present in all three kingdoms of life, but have a sporadic distribution. They are found predominantly in proteins involved in DNA replication and repair such as helicases. The distribution of inteins suggests an adaptive function. The evolutionary forces which shaped the observed distribution of inteins are generally unknown. Some authors view inteins only as the selfish elements and argue that frequent horizontal transfer is behind inteins sporadic dissemination (Gogarten et al., 2002). On the other hand, the ancient nature of the inteins and the process of gain/loss could lead to the scattered distribution of inteins among species (Pietrokovski, 2001). It is necessary to note that the exclusively selfish nature of inteins is questionable; recent findings support the hypothesis of possible functional roles of inteins in protein regulation (Callahan et al., 2011). Moreover, both hypotheses were built on a limited number of the intein representatives. The amount of genomic data available for bacteria is enormous and in silico analysis for diverse inteins is warranted. We decided to take advantage of these microbial genomic data and performed comprehensive mining for the inteins using a bioinformatic pipeline. Altogether, 1757 species were analysed from 19 major phyla yielding more than 4500 intein-like sequences. The majority of these bacterial inteins were not described previously. Approximately 55% of the inteins were found in polymerases, helicases, or recombinases (Figure 1). Phylogenetic analysis indicated the complex evolutionary dynamics of inteins which includes horizontal transfers, high evolutionary rates coupled with recurrent gains, and losses. The preponderance of inteins in helicases and reductases is being investigated in terms of functional relevance.     

Surrendering Pets To Shelters: The Relinquisher's Perspective

ABSTRACT

The intention of this research was not to justify the relinquishment of pets to shelters, but to provide a detailed and impartial view of the relinquisher's perspective. This perspective, as revealed in 38 interviews, was contrary to the view of relinquishment that commonly prevails in shelter cultures; namely, that their decisions are trivial or casual. These interviews exposed a processes that began long before releases were signed and animals were turned over to shelter staff. All of the individuals and families interviewed struggled with the decision to give up their pets. This struggle often manifested as procrastination, as attachment issues and negative perceptions of shelters were balanced against the circumstances threatening the pet's position in the family. Attempts at resolution made by poorly informed pet owners were frequently inefficient and consequently unsuccessful. Owners commonly tolerated circumstances until the reasons for relinquishment overcame attachment and negative perceptions of shelters.