Phosphorylation of Rab5a Protein by Protein Kinase C? Is Crucial for T-cell Migration

Rab GTPases control membrane traffic and receptor-mediated endocytosis. Within this context, Rab5a plays an important role in the spatial regulation of intracellular transport and signal transduction processes. Here, we report a previously uncharacterized role for Rab5a in the regulation of T-cell motility. We show that Rab5a physically associates with protein kinase Cϵ (PKCϵ) in migrating T-cells. After stimulation of T-cells through the integrin LFA-1 or the chemokine receptor CXCR4, Rab5a is phosphorylated on an N-terminal Thr-7 site by PKCϵ. Both Rab5a and PKCϵ dynamically interact at the centrosomal region of migrating cells, and PKCϵ-mediated phosphorylation on Thr-7 regulates Rab5a trafficking to the cell leading edge. Furthermore, we demonstrate that Rab5a Thr-7 phosphorylation is functionally necessary for Rac1 activation, actin rearrangement, and T-cell motility. We present a novel mechanism by which a PKCϵ-Rab5a-Rac1 axis regulates cytoskeleton remodeling and T-cell migration, both of which are central for the adaptive immune response.     

Emergence of airway smooth muscle functions related to structural malleability

The function of a complex system such as a smooth muscle cell is the result of the active interaction among molecules and molecular aggregates. Emergent macroscopic manifestations of these molecular interactions, such as the length-force relationship and its associated length adaptation, are well documented, but the molecular constituents and organization that give rise to these emergent muscle behaviors remain largely unknown. In this minireview, we describe emergent properties of airway smooth muscle that seem to have originated from inherent fragility of the cellular structures, which has been increasingly recognized as a unique and important smooth muscle attribute. We also describe molecular interactions (based on direct and indirect evidence) that may confer malleability on fragile structural elements that in turn may allow the muscle to adapt to large and frequent changes in cell dimensions. Understanding how smooth muscle works may hinge on how well we can relate molecular events to its emergent macroscopic functions.     

Characterization of the human gastric fluid proteome reveals distinct pH-dependent protein profiles: implications for biomarker studies

Gastric fluid is a source of gastric cancer biomarkers. However, very little is known about the normal gastric fluid proteome and its biological variations. In this study, we performed a comprehensive analysis of the human gastric fluid proteome using samples obtained from individuals with benign gastric conditions. Gastric fluid proteins were prefractionated using ultracentrifuge filters (3 kDa cutoff) and analyzed by two-dimensional gel electrophoresis (2-DE) and multidimensional LC-MS/MS. Our 2-DE analysis of 170 gastric fluid samples revealed distinct protein profiles for acidic and neutral samples, highlighting pH effects on protein composition. By 2D LC-MS/MS analysis of pooled samples, we identified 284 and 347 proteins in acidic and neutral samples respectively (FDR ≤1%), of which 265 proteins (72.4%) overlapped. However, unlike neutral samples, most proteins in acidic samples were identified from peptides in the filtrate (i.e., <3 kDa). Consistent with this finding, immunoblot analysis of six potential gastric cancer biomarkers rarely detected full-length proteins in acidic samples. These findings have important implications for biomarker studies because a majority of gastric cancer patients have neutral gastric fluid compared to noncancer controls. Consequently, sample stratification, choice of proteomic approaches, and validation strategy can profoundly affect the interpretation of biomarker findings. These observations should help to refine gastric fluid biomarker studies.     

A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.     

Mutations in enterobacterial common antigen biosynthesis restore outer membrane barrier function in Escherichia coli tol-pal mutants

The outer membrane (OM) is an essential component of the Gram-negative bacterial envelope that protects the cells against external threats. To maintain a functional OM, cells require distinct mechanisms to ensure balance of proteins and lipids in the membrane. Mutations in OM biogenesis and/or homeostasis pathways often result in permeability defects, but how molecular changes in the OM affect barrier function is unclear. Here, we seek potential mechanism(s) that can alleviate permeability defects in Escherichia coli cells lacking the Tol-Pal complex, which accumulate excess PLs in the OM. We identify mutations in enterobacterial common antigen (ECA) biosynthesis that re-establish OM barrier function against large hydrophilic molecules, yet did not restore lipid homeostasis. Furthermore, we demonstrate that build-up of biosynthetic intermediates, but not loss of ECA itself, contributes to the rescue. This suppression of OM phenotypes is unrelated to known effects that accumulation of ECA intermediates have on the cell wall. Finally, we reveal that an unusual diacylglycerol pyrophosphoryl-linked lipid species also accumulates in ECA mutants, and might play a role in the rescue phenotype. Our work provides insights into how OM barrier function can be restored independent of lipid homeostasis, and highlights previously unappreciated effects of ECA-related species in OM biology.     

Investigations into the nature of growth-related proteolysis in human fibroblasts

Previous experiments have shown that cultured human fibroblasts possess a cell-surface proteinase (the growth-related proteinase; GRP) which is essential to cell proliferation. In the present work, proteinase inhibition in defined and complex serum-free media and in pre-conditioned normal medium, still resulted in a corresponding inhibition of cell proliferation. Proteinase inhibition also blocked the action of a range of peptide growth factors and of a phorbol ester. Elevated extracellular calcium concentrations were still mitogenic in the presence of proteinase inhibitors. Proteinase inhibition did not affect the mobilisation of intracellular calcium, nor the metabolism of inositol phosphate derivatives in response to a mitogenic stimulus.     

Influence of calcium on myosin thick filament formation in intact airway smooth muscle

Myosin thick filaments have been shown tobe structurally labile in intact smooth muscles. Although the mechanismof thick filament assembly/disassembly for purified myosins in solution has been well described, regulation of thick filament formation inintact muscle is still poorly understood. The present study investigates the effect of resting calcium level on thick filament maintenance in intact airway smooth muscle and on thick filament formation during activation. Cross-sectional density of the thick filaments measured electron microscopically showed that the density increased substantially (144%) when the muscle was activated. Theabundance of filamentous myosins in relaxed muscle was calcium sensitive; in the absence of calcium (with EGTA), the filament densitydeceased by 35%. Length oscillation imposed on the muscle underzero-calcium conditions produced no further reduction in the density.Isometric force and filament density recovered fully after reincubationof the muscle in normal physiological saline. The results suggest thatin airway smooth muscle, filamentous myosins exist in equilibrium withmonomeric myosins; muscle activation favors filament formation, and theresting calcium level is crucial for preservation of the filaments inthe relaxed state.

     

Improving the Clinical Diagnosis of Influenza—a Comparative Analysis of New Influenza A (H1N1) Cases

Background

The presentation of new influenza A(H1N1) is broad and evolving as it continues to affect different geographic locations and populations. To improve the accuracy of predicting influenza infection in an outpatient setting, we undertook a comparative analysis of H1N1(2009), seasonal influenza, and persons with acute respiratory illness (ARI) in an outpatient setting.

Methodology/Principal Findings

Comparative analyses of one hundred non-matched cases each of PCR confirmed H1N1(2009), seasonal influenza, and ARI cases. Multivariate analysis was performed to look for predictors of influenza infection. Receiver operating characteristic curves were constructed for various combinations of clinical and laboratory case definitions. The initial clinical and laboratory features of H1N1(2009) and seasonal influenza were similar. Among ARI cases, fever, cough, headache, rhinorrhea, the absence of leukocytosis, and a normal chest radiograph positively predict for both PCR-confirmed H1N1-2009 and seasonal influenza infection. The sensitivity and specificity of current WHO and CDC influenza-like illness (ILI) criteria were modest in predicting influenza infection. However, the combination of WHO ILI criteria with the absence of leukocytosis greatly improved the accuracy of diagnosing H1N1(2009) and seasonal influenza (positive LR of 7.8 (95%CI 3.5–17.5) and 9.2 (95%CI 4.1–20.3) respectively).

Conclusions/Significance

The clinical presentation of H1N1(2009) infection is largely indistinguishable from that of seasonal influenza. Among patients with acute respiratory illness, features such as a temperature greater than 38°C, rhinorrhea, a normal chest radiograph, and the absence of leukocytosis or significant gastrointestinal symptoms were all positively associated with H1N1(2009) and seasonal influenza infection. An enhanced ILI criteria that combines both a symptom complex with the absence of leukocytosis on testing can improve the accuracy of predicting both seasonal and H1N1-2009 influenza infection.     

Engineering application of anaerobic ammonium oxidation process in wastewater treatment

Anaerobic ammonium oxidation (Anammox), a promising biological nitrogen removal process, has been verified as an efficient, sustainable and cost-effective alternative to conventional nitrification and denitrification processes. To date, more than 110 full-scale anammox plants have been installed and are in operation, treating industrial NH₄ ⁺-rich wastewater worldwide, and anammox-based technologies are flourishing. This review the current state of the art for engineering applications of the anammox process, including various anammox-based technologies, reactor selection and attempts to apply it at different wastewater plants. Process control and implementation for stable performance are discussed as well as some remaining issues concerning engineering application are exposed, including the start-up period, process disturbances, greenhouse gas emissions and especially mainstream anammox applications. Finally, further development of the anammox engineering application is proposed in this review.