Shewanella halifaxensis and Shewanella sediminis were among a few aquatic ?-proteobacteria that were psychrophiles and the first anaerobic bacteria that degraded hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Although many mesophilic or psychrophilic strains of Shewanella and ?-proteobacteria were sequenced for their genomes, the genomic evolution pathways for temperature adaptation were poorly understood. On the other hand, the genes responsible for anaerobic RDX mineralization pathways remain unknown. To determine the unique genomic properties of bacteria responsible for both cold-adaptation and RDX degradation, the genomes of S. halifaxensis and S. sediminis were sequenced and compared with 108 other ?-proteobacteria including Shewanella that differ in temperature and Na+ requirements, as well as RDX degradation capability. Results showed that for coping with marine environments their genomes had extensively exchanged with deep sea bacterial genomes. Many genes for Na+-dependent nutrient transporters were recruited to use the high Na+ content as an energy source. For coping with low temperatures, these two strains as well as other psychrophilic strains of Shewanella and ?-proteobacteria were found to decrease their genome G+C content and proteome alanine, proline and arginine content (p-value <0.01) to increase protein structural flexibility. Compared to poorer RDX-degrading strains, S. halifaxensis and S. sediminis have more number of genes for cytochromes and other enzymes related to RDX metabolic pathways. Experimentally, one cytochrome was found induced in S. halifaxensis by RDX when the chemical was the sole terminal electron acceptor. The isolated protein degraded RDX by mono-denitration and was identified as a multiheme 52 kDa cytochrome using a proteomic approach. The present analyses provided the first insight into divergent genomic evolution of bacterial strains for adaptation to the specific cold marine conditions and to the degradation of the pollutant RDX. The present study also provided the first evidence for the involvement of a specific c-type cytochrome in anaerobic RDX metabolism.
Ischemia-reperfusion (I/R) injury contributes to organ dysfunction in a variety of clinical disorders, including myocardial infarction, stroke, organ transplantation, and hemorrhagic shock. Recent investigations have demonstrated that apoptosis as an important mechanism of cell death leading to organ dysfunction following I/R. Intracellular danger-associated molecular patterns (DAMPs) released during cell death can activate cytoprotective responses by engaging receptors of the innate immune system.
Methodology/Principal FindingsIschemia was induced in the mouse hind limb by tourniquet or in the heart by coronary artery ligation. Reperfusion injury of skeletal or cardiac muscle was markedly reduced by intraperitoneal or subcutaneous injection of recombinant human (rh)BCL2 protein or rhBCL2-related protein A1 (BCL2A1) (50 ng/g) given prior to ischemia or at the time of reperfusion. The cytoprotective activity of extracellular rhBCL2 or rhBCL2A1 protein was mapped to the BH4 domain, as treatment with a mutant BCL2 protein lacking the BH4 domain was not protective, whereas peptides derived from the BH4 domain of BCL2 or the BH4-like domain of BCL2A1 were. Protection by extracellular rhBCL2 or rhBCL2A1 was associated with a reduction in apoptosis in skeletal and cardiac muscle following I/R, concomitant with increased expression of endogenous mouse BCL2 (mBCL2) protein. Notably, treatment with rhBCL2A1 protein did not protect mice deficient in toll-like receptor-2 (TLR2) or the adaptor protein, myeloid differentiation factor-88 (MyD88).
Conclusions/SignificanceTreatment with cytokine-like doses of rhBCL2 or rhBCL2A1 protein or BH4-domain peptides reduces apoptosis and tissue injury following I/R by a TLR2-MyD88-dependent mechanism. These findings establish a novel extracellular cytoprotective activity of BCL2 BH4-domain proteins as potent cytoprotective DAMPs.
Thymic epithelial cell (TEC) microenvironments are essential for the recruitment of T cell precursors from the bone marrow, as well as the subsequent expansion and selection of thymocytes resulting in a mature self-tolerant T cell repertoire. The molecular mechanisms, which control both the initial development and subsequent maintenance of these critical microenvironments, are poorly defined. Wnt signaling has been shown to be important to the development of several epithelial tissues and organs. Regulation of Wnt signaling has also been shown to impact both early thymocyte and thymic epithelial development. However, early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of canonical Wnt signaling in the maintenance of TECs in the postnatal thymus.
Methodology/Principal FindingsHere we demonstrate that tetracycline-regulated expression of the canonical Wnt inhibitor DKK1 in TECs localized in both the cortex and medulla of adult mice, results in rapid thymic degeneration characterized by a loss of ?NP63+ Foxn1+ and Aire+ TECs, loss of K5K8DP TECs thought to represent or contain an immature TEC progenitor, decreased TEC proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments.
Conclusions/SignificanceTaken together, the results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated with cancer therapy and bone marrow transplants.
The cellular form of the prion protein, PrPC, undergoes extensive proteolysis at the ? site (109K?H110). Expression of non-cleavable PrPC mutants in transgenic mice correlates with neurotoxicity, suggesting that ?-cleavage is important for PrPC physiology. To gain insights into the mechanisms of ?-cleavage, we generated a library of PrPC mutants with mutations in the region neighbouring the ?-cleavage site. The prevalence of C1, the carboxy adduct of ?-cleavage, was determined for each mutant. In cell lines of disparate origin, C1 prevalence was unaffected by variations in charge and hydrophobicity of the region neighbouring the ?-cleavage site, and by substitutions of the residues in the palindrome that flanks this site. Instead, ?-cleavage was size-dependently impaired by deletions within the domain 106–119. Almost no cleavage was observed upon full deletion of this domain. These results suggest that ?-cleavage is executed by an ?-PrPase whose activity, despite surprisingly limited sequence specificity, is dependent on the size of the central region of PrPC.
Alcoholism is associated with susceptibility to infectious disease, particularly bacterial pneumonia. In the present study we described characteristics in alcoholic patients with bacterial meningitis and delineate the differences with findings in non-alcoholic adults with bacterial meningitis.
Methods/Principal FindingsThis was a prospective nationwide observational cohort study including patients aged >16 years who had bacterial meningitis confirmed by culture of cerebrospinal fluid (696 episodes of bacterial meningitis occurring in 671 patients). Alcoholism was present in 27 of 686 recorded episodes of bacterial meningitis (4%) and alcoholics were more often male than non-alcoholics (82% vs 48%, P = 0.001). A higher proportion of alcoholics had underlying pneumonia (41% vs 11% P<0.001). Alcoholics were more likely to have meningitis due to infection with Streptococcus pneumoniae (70% vs 50%, P = 0.01) and Listeria monocytogenes (19% vs 4%, P = 0.005), whereas Neisseria meningitidis was more common in non-alcoholic patients (39% vs 4%, P = 0.01). A large proportion of alcoholics developed complications during clinical course (82% vs 62%, as compared with non-alcoholics; P = 0.04), often cardiorespiratory failure (52% vs 28%, as compared with non-alcoholics; P = 0.01). Alcoholic patients were at risk for unfavourable outcome (67% vs 33%, as compared with non-alcoholics; P<0.001).
Conclusions/SignificanceAlcoholic patients are at high risk for complications resulting in high morbidity and mortality. They are especially at risk for cardiorespiratory failure due to underlying pneumonia, and therefore, aggressive supportive care may be crucial in the treatment of these patients.
The urgent medical need for innovative approaches to control influenza is emphasized by the widespread resistance of circulating subtype H1N1 viruses to the leading antiviral drug oseltamivir, the pandemic threat posed by the occurrences of human infections with highly pathogenic avian H5N1 viruses, and indeed the evolving swine-origin H1N1 influenza pandemic. A recently discovered class of human monoclonal antibodies with the ability to neutralize a broad spectrum of influenza viruses (including H1, H2, H5, H6 and H9 subtypes) has the potential to prevent and treat influenza in humans. Here we report the latest efficacy data for a representative antibody of this novel class.
Methodology/Principal FindingsWe evaluated the prophylactic and therapeutic efficacy of the human monoclonal antibody CR6261 against lethal challenge with the highly pathogenic avian H5N1 virus in ferrets, the optimal model of human influenza infection. Survival rates, clinically relevant disease signs such as changes in body weight and temperature, virus replication in lungs and upper respiratory tract, as well as macro- and microscopic pathology were investigated. Prophylactic administration of 30 and 10 mg/kg CR6261 prior to viral challenge completely prevented mortality, weight loss and reduced the amount of infectious virus in the lungs by more than 99.9%, abolished shedding of virus in pharyngeal secretions and largely prevented H5N1-induced lung pathology. When administered therapeutically 1 day after challenge, 30 mg/kg CR6261 prevented death in all animals and blunted disease, as evidenced by decreased weight loss and temperature rise, reduced lung viral loads and shedding, and less lung damage.
Conclusions/SignificanceThese data demonstrate the prophylactic and therapeutic efficacy of this new class of human monoclonal antibodies in a highly stringent and clinically relevant animal model of influenza and justify clinical development of this approach as intervention for both seasonal and pandemic influenza.
Non-coding RNAs have critical functions in diverse biological processes, particularly in gene regulation. Viruses, like their host cells, employ such functional RNAs and the human cancer associated Epstein-Barr virus (EBV) is no exception. Nearly all EBV associated tumours express the EBV small, non-coding RNAs (EBERs) 1 and 2, however their role in viral pathogenesis remains largely obscure.
Methodology/Principal FindingsTo investigate the action of EBER1 in vivo, we produced ten transgenic mouse lines expressing EBER1 in the lymphoid compartment using the mouse immunoglobulin heavy chain intronic enhancer E?. Mice of several of these E?EBER1 lines developed lymphoid hyperplasia which in some cases proceeded to B cell malignancy. The hallmark of the transgenic phenotype is enlargement of the spleen and mesenteric lymph nodes and in some cases enlargement of the thymus, liver and peripheral lymph nodes. The tumours were found to be of B cell origin and showed clonal IgH rearrangements. In order to explore if EBER1 would cooperate with c-Myc (deregulated in Burkitt's lymphoma) to accelerate lymphomagenesis, a cross-breeding study was undertaken with E?EBER1 and E?Myc mice. While no significant reduction in latency to lymphoma onset was observed in bi-transgenic mice, c-Myc induction was detected in some E?EBER1 single transgenic tumours, indicative of a functional cooperation.
Conclusions/SignificanceThis study is the first to describe the in vivo expression of a polymerase III, non-coding viral gene and demonstrate its oncogenic potential. The data suggest that EBER1 plays an oncogenic role in EBV associated malignant disease.