Modes of Calreticulin Recruitment to the Major Histocompatibility Complex Class I Assembly Pathway

Major histocompatibility complex (MHC) class I molecules are ligands for T-cell receptors of CD8⁺ T cells and inhibitory receptors of natural killer cells. Assembly of the heavy chain, light chain, and peptide components of MHC class I molecules occurs in the endoplasmic reticulum (ER). Specific assembly factors and generic ER chaperones, collectively called the MHC class I peptide loading complex (PLC), are required for MHC class I assembly. Calreticulin has an important role within the PLC and induces MHC class I cell surface expression, but the interactions and mechanisms involved are incompletely understood. We show that interactions with the thiol oxidoreductase ERp57 and substrate glycans are important for the recruitment of calreticulin into the PLC and for its functional activities in MHC class I assembly. The glycan and ERp57 binding sites of calreticulin contribute directly or indirectly to complexes between calreticulin and the MHC class I assembly factor tapasin and are important for maintaining steady-state levels of both tapasin and MHC class I heavy chains. A number of destabilizing conditions and mutations induce generic polypeptide binding sites on calreticulin and contribute to calreticulin-mediated suppression of misfolded protein aggregation in vitro. We show that generic polypeptide binding sites per se are insufficient for stable recruitment of calreticulin to PLC substrates in cells. However, such binding sites could contribute to substrate stabilization in a step that follows the glycan and ERp57-dependent recruitment of calreticulin to the PLC.     

Chronic Parasitic Infection Maintains High Frequencies of Short-Lived Ly6C+CD4+ Effector T Cells That Are Required for Protection against Re-infection

In contrast to the ability of long-lived CD8⁺ memory T cells to mediate protection against systemic viral infections, the relationship between CD4⁺ T cell memory and acquired resistance against infectious pathogens remains poorly defined. This is especially true for T helper 1 (Th1) concomitant immunity, in which protection against reinfection coincides with a persisting primary infection. In these situations, pre-existing effector CD4 T cells generated by ongoing chronic infection, not memory cells, may be essential for protection against reinfection. We present a systematic study of the tissue homing properties, functionality, and life span of subsets of memory and effector CD4 T cells activated in the setting of chronic Leishmania major infection in resistant C57Bl/6 mice. We found that pre-existing, CD44⁺CD62L⁻T-bet⁺Ly6C⁺ effector (T_EFF) cells that are short-lived in the absence of infection and are not derived from memory cells reactivated by secondary challenge, mediate concomitant immunity. Upon adoptive transfer and challenge, non-dividing Ly6C⁺ T_EFF cells preferentially homed to the skin, released IFN-γ, and conferred protection as compared to CD44⁺CD62L⁻Ly6C⁻ effector memory or CD44⁺CD62L⁺Ly6C⁻ central memory cells. During chronic infection, Ly6C⁺ T_EFF cells were maintained at high frequencies via reactivation of T_CM and the T_EFF themselves. The lack of effective vaccines for many chronic diseases may be because protection against infectious challenge requires the maintenance of pre-existing T_EFF cells, and is therefore not amenable to conventional, memory inducing, vaccination strategies.     

The Genome Sequence DataBase (GSDB): improving data quality and data access.

In 1997 the primary focus of the Genome Sequence DataBase (GSDB; www. ncgr.org/gsdb ) located at the National Center for Genome Resources was to improve data quality and accessibility. Efforts to increase the quality of data within the database included two major projects; one to identify and remove all vector contamination from sequences in the database and one to create premier sequence sets (including both alignments and discontiguous sequences). Data accessibility was improved during the course of the last year in several ways. First, a graphical database sequence viewer was made available to researchers. Second, an update process was implemented for the web-based query tool, Maestro. Third, a web-based tool, Excerpt, was developed to retrieve selected regions of any sequence in the database. And lastly, a GSDB flatfile that contains annotation unique to GSDB (e.g., sequence analysis and alignment data) was developed. Additionally, the GSDB web site provides a tool for the detection of matrix attachment regions (MARs), which can be used to identify regions of high coding potential. The ultimate goal of this work is to make GSDB a more useful resource for genomic comparison studies and gene level studies by improving data quality and by providing data access capabilities that are consistent with the needs of both types of studies.     

Effect of cholesterol depletion on the pore dilation of TRPV1

The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.     

Ring-cleaving cyanuric acid amidohydrolase activity in the atrazine-mineralizing Ralstonia basilensis M91-3

The purpose of this study was to characterize the cyanuric acid amidohydrolase reaction in Ralstonia basilensis M91-3, an atrazine-mineralizing soil bacterium. This ring fission reaction is the last aromatic step in the degradative pathway of atrazine and other s-triazines. The products and molar stoichiometry of the cyanuric acid amidohydrolase reaction were one mol biuret (H₂N·CO·NH·CO·NH₂) and one mol CO₂ per mol cyanuric acid hydrolyzed, as confirmed by ¹³C-NMR and gas chromatography. The optimum pH and temperature, substrate specificity, and kinetic parameters were also characterized for the purified enzyme. The native enzyme had two forms of different sizes, 204?kDa and 160?kDa. Each was a tetramer or pentamer of 44?kDa and 33?kDa, respectively.     

Cytosolic isocitrate dehydrogenase in humans, mice, and voles and phylogenetic analysis of the enzyme family

In this study, we report cDNA sequences of the cytosolic NADP-dependent isocitrate dehydrogenase for humans, mice, and two species of voles (Microtus mexicanus and Microtus ochrogaster). Inferred amino acid sequences from these taxa display a high level of amino acid sequence conservation, comparable to that of myosin beta heavy chain, and share known structural features. A Caenorhabditis elegans enzyme that was previously identified as a protein similar to isocitrate dehydrogenase is most likely the NADP-dependent cytosolic isocitrate dehydrogenase enzyme equivalent, based on amino acid similarity to mammalian enzymes and phylogenetic analysis. We also suggest that NADP-dependent isocitrate dehydrogenases characterized from alfalfa, soybean, and eucalyptus are most likely cytosolic enzymes. The phylogenetic tree of various isocitrate dehydrogenases from eukaryotic sources revealed that independent gene duplications may have given rise to the cytosolic and mitochondrial forms of NADP-dependent isocitrate dehydrogenase in animals and fungi. There appears to be no statistical support for a hypothesis that the mitochondrial and cytosolic forms of the enzyme are orthologous in these groups. A possible scenario of the evolution of NADP-dependent isocitrate dehydrogenases is proposed.      

CoMEt: a statistical approach to identify combinations of mutually exclusive alterations in cancer

Cancer is a heterogeneous disease with different combinations of genetic alterations driving its development in different individuals. We introduce CoMEt, an algorithm to identify combinations of alterations that exhibit a pattern of mutual exclusivity across individuals, often observed for alterations in the same pathway. CoMEt includes an exact statistical test for mutual exclusivity and techniques to perform simultaneous analysis of multiple sets of mutually exclusive and subtype-specific alterations. We demonstrate that CoMEt outperforms existing approaches on simulated and real data. We apply CoMEt to five different cancer types, identifying both known cancer genes and pathways, and novel putative cancer genes.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0700-7) contains supplementary material, which is available to authorized users.     

Biological treatment and toxicity of low concentrations of oily wastewater (bilgewater)

The biodegradability and toxicity of low concentrations of oily wastewater (bilgewater) were tested under simulated sanitary wastewater treatment conditions. This was done to establish the feasibility of a combined shipboard oily and nonoily wastewater treatment system. The biodegradability of oily wastewater was determined by proxy; 14C-labeled dodecane, toluene, and phenanthrene (representing alkane, aromatic, and polyaromatic compounds, respectively) were mineralized in petroleum fuels and lubricants. We found that low concentrations of oily wastewater components were mineralized, even in the presence of more abundant substrates (such as synthetic graywater, containing vegetable oil, detergent, gelatin, and starch). The toxic effects of diesel fuel and several other components of oily wastewater (such as surfactants and a synthetic lubricant) on a na?ve wastewater assemblage was also tested. In concentrations much higher than would be expected under normal shipboard conditions, we found no evidence of toxic effects of the bilgewater compounds tested. Thus, a combined shipboard bilgewater and sanitary wastewater system might be feasible.     

Short‐term effects of herbicides and a prescribed fire on restoration of a shrub‐encroached pine savanna

Shrub encroachment occurring worldwide in savannas and grasslands has commonly been hypothesized to result from anthropogenically altered environments. Two disturbance‐based approaches to restoration have involved: (1) application of selective herbicides to reduce density/cover of shrubs; (2) reinstatement of natural fire regimes to generate environmental conditions favoring herbaceous species. We studied short‐term responses of native shrubs, vines, and grasses in a Louisiana pine savanna to herbicides followed by a prescribed fire and fire alone. Plots established in the summer, 2013, were hand‐sprayed in the fall with Imazapyr and Triclopyr, Triclopyr alone, or no herbicide, then prescribed burned the following spring. Numbers of species of shrubs and vines at scales of 1 and 100 m², numbers of stems and regrowth of stems produced by six common species of shrubs, and the number of flowering culms of perennial C₄ grasses were assessed postfire in 2014. Compared with fire alone, herbicides followed by fire resulted in (1) small reductions in species richness of shrubs and no effects on vines, (2) fewer stems comprising shrub genets, but similar postfire regrowth of resprouting shrub stems, and (3) fewer flowering culms of C₄ grasses. Underground storage organs of savanna shrubs and vines survived both aboveground disturbances. Thus, single applications of herbicides followed by fires reduced, but did not reverse shrub encroachment and negatively affected grasses. Because effects of herbicides overrode those of prescribed fires, these disturbances did not act synergistically, suggesting that reinstating natural fire regimes should be a priority in restoration of savannas and grasslands.