K2P18.1 translates T cell receptor signals into thymic regulatory T cell development

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that driv...     

Mycobacterium tuberculosis Bacteremia in a Cohort of HIV-Infected Patients Hospitalized with Severe Sepsis in Uganda–High Frequency,Low Clinical Sand Derivation of a Clinical Prediction Score

Background

When manifested as Mycobacterium tuberculosis (MTB) bacteremia, disseminated MTB infection clinically mimics other serious blood stream infections often hindering early diagnosis and initiation of potentially life-saving anti-tuberculosis therapy. In a cohort of hospitalized HIV-infected Ugandan patients with severe sepsis, we report the frequency, management and outcomes of patients with MTB bacteremia and propose a risk score based on clinical predictors of MTB bacteremia.

Methods

We prospectively enrolled adult patients with severe sepsis at two Ugandan hospitals and obtained blood cultures for MTB identification. Multivariable logistic regression modeling was used to determine predictors of MTB bacteremia and to inform the stratification of patients into MTB bacteremia risk categories based on relevant patient characteristics.

Results

Among 368 HIV-infected patients with a syndrome of severe sepsis, eighty-six (23%) had MTB bacteremia. Patients with MTB bacteremia had a significantly lower median CD4 count (17 vs 64 lymphocytes/mm³, p<0.001) and a higher 30-day mortality (53% vs 32%, p = 0.001) than patients without MTB bacteremia. A minority of patients with MTB bacteremia underwent standard MTB diagnostic testing (24%) or received empiric anti-tuberculosis therapy (15%). Independent factors associated with MTB bacteremia included male sex, increased heart rate, low CD4 count, absence of highly active anti-retroviral therapy, chief complaint of fever, low serum sodium and low hemoglobin. A risk score derived from a model containing these independent predictors had good predictive accuracy [area under the curve = 0.85, 95% CI 0.80–0.89].

Conclusions

Nearly 1 in 4 adult HIV-infected patients hospitalized with severe sepsis in 2 Ugandan hospitals had MTB bacteremia. Among patients in whom MTB was suspected, standard tests for diagnosing pulmonary MTB were inaccurate for correctly classifying patients with or without bloodstream MTB infection. A MTB bacteremia risk score can improve early diagnosis of MTB bacteremia particularly in settings with increased HIV and MTB co-infection.     

Heme Amplifies the Innate Immune Response to Microbial Molecules through Spleen Tyrosine Kinase (Syk)-dependent Reactive Oxygen Species Generation

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor κB (NF-κB) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.     

The sterol-sensing domain: multiple families, a unique role?

The "sterol-sensing domain" (SSD) is conserved across phyla and is present in several membrane proteins, such as Patched (a Hedgehog receptor) and NPC-1 (the protein defective in Niemann-Pick type C1 disease). The role of the SSD is perhaps best understood from the standpoint of its involvement in cholesterol homeostasis. This article discusses how the SSD appears to function as a regulatory domain involved in linking vesicle trafficking and protein localization with such varied processes as cholesterol homeostasis, cell signalling and cytokinesis.     

Selective breeding for high endurance running increases hindlimb symmetry

Comparative studies provide correlational evidence of morphological adaptations for high locomotor performance, such as the classical indicators of cursoriality in mammals, long limbs and high metatarsal/femur ratios. More recently, enlarged femoral condyles have been suggested as an adaptation for high endurance running in the genus Homo. Asymmetry of locomotor appendages should adversely affect locomotor abilities, but this has not been studied in a rigorous evolutionary context. We used experimental evolution to test for morphological adaptations associated with high voluntary wheel running in selectively bred lines of mice. Surprisingly, the classical indicators of cursoriality had not evolved in concert with high activity levels. Instead, high runners had larger femoral condyles and reduced directional asymmetry of hindlimb bones. We hypothesize that greater limb symmetry and larger femoral heads are general adaptations associated with sustained, high-speed locomotion.     

Recombinant expression of Ole e 6, a Cys-enriched pollen allergen, in Pichia pastoris yeast: detection of partial oxidation of methionine by NMR

Olive pollen is one of the main causes of allergy in Mediterranean countries. Ole e 6, an olive pollen allergen, is a small (5.8 kDa) and acidic protein (pI 4.2) and no homologous proteins have been isolated or characterized so far. Ole e 6 has been efficiently expressed in the methylotrophic yeast Pichia pastoris. The cDNA encoding Ole e 6 was inserted into the plasmid vector pPIC9 and overexpressed in GS115 yeast cells. The recombinant product was purified by size-exclusion chromatography followed by reverse-phase HPLC. N-terminal sequencing, amino acid composition analysis, CD, NMR, and IgG-binding experiments were employed to characterize the purified protein. NMR data revealed the oxidation of the methionine at position 28 in approximately 50% of the recombinant protein but, although this alters its electrophoretic behavior, it did not affect folding or IgG-binding properties of rOle e 6. The recombinant form of Ole e 6 expressed in P. pastoris can be employed for structural and biochemical studies.     

Contact guidance mediated three-dimensional cell migration is regulated by Rho/ROCK-dependent matrix reorganization

Cells generate mechanical force to organize the extracellular matrix (ECM) and drive important developmental and reparative processes. Likewise, tumor cells invading into three-dimensional (3D) matrices remodel the ECM microenvironment. Importantly, we previously reported a distinct radial reorganization of the collagen matrix surrounding tumors that facilitates local invasion. Here we describe a mechanism by which cells utilize contractility events to reorganize the ECM to provide contact guidance that facilitates 3D migration. Using novel assays to differentially organize the collagen matrix we show that alignment of collagen perpendicular to the tumor-explant boundary promotes local invasion of both human and mouse mammary epithelial cells. In contrast, organizing the collagen matrix to mimic the ECM organization associated with noninvading regions of tumors suppresses 3D migration/invasion. Moreover, we demonstrate that matrix reorganization is contractility-dependent and that the Rho/Rho kinase pathway is necessary for collagen alignment to provide contact guidance. Yet, if matrices are prealigned, inhibiting neither Rho nor Rho kinase inhibits 3D migration, which supports our conclusion that Rho-mediated matrix alignment is an early step in the invasion process, preceding and subsequently facilitating 3D migration.     

Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding

Human telomeres are composed of duplex TTAGGG repeats and a 3' single-stranded DNA tail. The telomeric DNA is protected and regulated by the shelterin proteins, including the protection of telomeres 1 (POT1) protein that binds telomeric single-stranded DNA. The single-stranded tail can fold into G-quadruplex (G4) DNA. Both POT1 and G4 DNA play important roles in regulating telomere length homeostasis. To date, most studies have focused on individual quadruplexes formed by four TTAGGG repeats. Telomeric tails in human cells have on average six times as many repeats, and no structural studies have examined POT1 binding in competition with G4 DNA folding. Using single molecule atomic force microscopy imaging, we observed that the majority of the telomeric tails of 16 repeats formed two quadruplexes even though four were possible. The result that physiological telomeric tails rarely form the maximum potential number of G4 units provides a structural basis for the coexistence of G4 and POT1 on the same DNA molecule, which is observed directly in the captured atomic force microscopy images. We further observed that POT1 is significantly more effective in disrupting quadruplex DNA on long telomeric tails than an antisense oligonucleotide, indicating a novel POT1 activity beyond simply preventing quadruplex folding.     

Identification of a hydrogen peroxide signalling pathway in the control of light-dependent germination in Arabidopsis

Germination is controlled by external factors, such as temperature, water, light and by hormone balance. Recently, reactive oxygen species (ROS) have been shown to act as messengers during plant development, stress responses and programmed cell death. We analyzed the role of ROS during germination and demonstrated that ROS in addition to their role as cell wall loosening factor are essential signalling molecules in this process. Indeed, we showed that ROS are released prior to endosperm rupture, that their production is required for germination, and that class III peroxidases, as ROS level regulators, colocalized with ROS production. Among ROS, H₂O₂ modifies, during germination early steps, the expression of genes encoding for enzymes regulating ROS levels. This pointing out a regulatory feedback loop for ROS production. Measurements of endogenous levels of ROS following application of GA and ABA suggested that ABA inhibits germination by repressing ROS accumulation, and that, conversely, GA triggers germination by promoting an increase of ROS levels. We followed the early visible steps of germination (testa and endosperm rupture) in Arabidopsis seeds treated by specific ROS scavengers and as the light quality perception is necessary for a regular germination, we examined the germination in presence of exogenous H₂O₂ in different light qualities. H₂O₂ either promoted germination or repressed germination depending on the light wavelengths, showing that H₂O₂ acts as a signal molecule regulating germination in a light-dependent manner. Using photoreceptors null-mutants and GA-deficient mutants, we showed that H₂O₂-dependent promotion of germination relies on phytochrome signalling, but not on cryptochrome signalling, and that ROS signalling requires GA signalling.