Can acclimation of amphipods change their antioxidative response?

The aim of this study was to evaluate whether experimental conditions, particularly duration of acclimation in the laboratory with artificial water and diet prior to the actual experiment, can affect the antioxidant response of amphipods. This issue was evaluated using the Baikalian amphipod Eulimnogammarus cyaneus (Dyb.) exposed to two sources of natural organic matter (NOM). NOM elicits several oxidative stress symptoms and we used peroxidase activity as a representative response parameter. Pretest acclimation periods of the amphipods lasted from 3 days to 5 months. The exposure of E. cyaneus to NOM resulted in significant modulation of the peroxidase activity with a clear dependence on the duration of the acclimation period. Animals experiencing short-term acclimation showed reduced activity, whereas long-term acclimated animals exhibited an increase in peroxidase activity. We suggest that this difference was due to laboratory conditions particularly the artificial diet. This fact should be considered in future studies using field-collected animals kept in the laboratory for different periods of time.     

H1N1pdm influenza infection in hospitalized cancer patients: clinical evolution and viral analysis

Background

The novel influenza A pandemic virus (H1N1pdm) caused considerable morbidity and mortality worldwide in 2009. The aim of the present study was to evaluate the clinical course, duration of viral shedding, H1N1pdm evolution and emergence of antiviral resistance in hospitalized cancer patients with severe H1N1pdm infections during the winter of 2009 in Brazil.

Methods

We performed a prospective single-center cohort study in a cancer center in Rio de Janeiro, Brazil. Hospitalized patients with cancer and a confirmed diagnosis of influenza A H1N1pdm were evaluated. The main outcome measures in this study were in-hospital mortality, duration of viral shedding, viral persistence and both functional and molecular analyses of H1N1pdm susceptibility to oseltamivir.

Results

A total of 44 hospitalized patients with suspected influenza-like illness were screened. A total of 24 had diagnosed H1N1pdm infections. The overall hospital mortality in our cohort was 21%. Thirteen (54%) patients required intensive care. The median age of the studied cohort was 14.5 years (3–69 years). Eighteen (75%) patients had received chemotherapy in the previous month, and 14 were neutropenic at the onset of influenza. A total of 10 patients were evaluated for their duration of viral shedding, and 5 (50%) displayed prolonged viral shedding (median 23, range = 11–63 days); however, this was not associated with the emergence of a resistant H1N1pdm virus. Viral evolution was observed in sequentially collected samples.

Conclusions

Prolonged influenza A H1N1pdm shedding was observed in cancer patients. However, oseltamivir resistance was not detected. Taken together, our data suggest that severely ill cancer patients may constitute a pandemic virus reservoir with major implications for viral propagation.     

Endoplasmic reticulum stress in alveolar epithelial cells is prominent in IPF: association with altered surfactant protein processing and herpesvirus infection

Recent evidence suggests that dysfunctional type II alveolar epithelial cells (AECs) contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Based on the hypothesis that disease-causing mutations in surfactant protein C (SFTPC) provide an important paradigm for studying IPF, we investigated a potential mechanism of AEC dysfunction suggested to result from mutant SFTPC expression: induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We evaluated biopsies from 23 IPF patients (including 3 family members with L188Q SFTPC mutations, 10 individuals with familial interstitial pneumonia without SFTPC mutations, and 10 individuals with sporadic IPF) and sections from 10 control lungs. After demonstrating UPR activation in cultured A549 cells expressing mutant SFTPC, we identified prominent expression of UPR markers in AECs in the lungs of patients with SFTPC mutation-associated fibrosis. In individuals with familial interstitial pneumonia without SFTPC mutations and patients with sporadic IPF, we also found UPR activation selectively in AECs lining areas of fibrotic remodeling. Because herpesviruses are found frequently in IPF lungs and can induce ER stress, we investigated expression of viral proteins in lung biopsies. Herpesvirus protein expression was found in AECs from 15/23 IPF patients and colocalized with UPR markers in AECs from these patients. ER stress and UPR activation are found in the alveolar epithelium in patients with IPF and could contribute to disease progression. Activation of these pathways may result from altered surfactant protein processing or chronic herpesvirus infection.     

Starting to unravel the toxoglossan knot: molecular phylogeny of the "turrids" (Neogastropoda: Conoidea)

The superfamily Conoidea is one of the most speciose groups of marine mollusks, with estimates of about 340 recent valid genera and subgenera, and 4000 named living species. Previous classifications were based on shell and anatomical characters, and clades and phylogenetic relationships are far from well assessed. Based on a dataset of ca. 100 terminal taxa belonging to 57 genera, information provided by fragments of one mitochondrial (COI) and three nuclear (28S, 18S and H3) genes is used to infer the first molecular phylogeny of this group. Analyses are performed on each gene independently as well as for a data matrix where all genes are concatenated, using Maximum Likelihood, Maximum Parsimony and Bayesian approaches. Several well-supported clades are defined and are only partly identifiable to currently recognized families and subfamilies. The nested sampling used in our study allows a discussion of the classification at various taxonomical levels, and several genera, subfamilies and families are found polyphyletic.     

Dissecting the fidelity of bacteriophage RB69 DNA polymerase: site-specific modulation of fidelity by polymerase accessory proteins

Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3' exonuclease. Crystal structures have been determined for this enzyme with and without DNA substrates. We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol(+) Exo(+)) enzyme, an exonuclease-deficient mutator variant (Pol(+) Exo(-)), mutator variants with substitutions at Tyr(567) in the polymerase active site (Pol(M) Exo(+)), and the double mutator Pol(M) Exo(-). Comparing the mutational spectra of the Pol(+) Exo(-) and Pol(+) Exo(+) enzymes revealed the patterns and efficiencies of proofreading, while Tyr(567) was identified as an important determinant of base-selection fidelity. Here, we sought to determine how well the fidelities of the same enzymes are reflected in vitro. Compared to their behavior in vivo, the three mutator polymerases exhibited modestly higher mutation rates in vitro and their mutational predilections were also somewhat different. Although the RB69 gp43 accessory proteins exerted little or no effect on total mutation rates in vitro, they strongly affected mutation rates at many specific sites, increasing some rates and decreasing others.     

Venezuelan equine encephalitis virus replicon particles encoding respiratory syncytial virus surface glycoproteins induce protective mucosal responses in mice and cotton rats

Respiratory syncytial virus (RSV) is an important viral pathogen that causes severe lower respiratory tract infection in infants, the elderly, and immunocompromised individuals. There are no licensed RSV vaccines to date. To prevent RSV infection, immune responses in both the upper and lower respiratory tracts are required. Previously, immunization with Venezuelan equine encephalitis virus replicon particles (VRPs) demonstrated effectiveness in inducing mucosal protection against various pathogens. In this study, we developed VRPs encoding RSV fusion (F) or attachment (G) glycoproteins and evaluated the immunogenicity and efficacy of these vaccine candidates in mice and cotton rats. VRPs, when administered intranasally, induced surface glycoprotein-specific virus neutralizing antibodies in serum and immunoglobulin A (IgA) antibodies in secretions at the respiratory mucosa. In addition, fusion protein-encoding VRPs induced gamma interferon (IFN-γ)-secreting T cells in the lungs and spleen, as measured by reaction with an H-2K^d-restricted CD8⁺ T-cell epitope. In animals vaccinated with F protein VRPs, challenge virus replication was reduced below the level of detection in both the upper and lower respiratory tracts following intranasal RSV challenge, while in those vaccinated with G protein VRPs, challenge virus was detected in the upper but not the lower respiratory tract. Close examination of histopathology of the lungs of vaccinated animals following RSV challenge revealed no enhanced inflammation. Immunization with VRPs induced balanced Th1/Th2 immune responses, as measured by the cytokine profile in the lungs and antibody isotype of the humoral immune response. These results represent an important first step toward the use of VRPs encoding RSV proteins as a prophylactic vaccine for RSV.     

Overcoming MIII arrest from spontaneous activation in cultured rat oocytes

The rat oocyte spontaneously activates under a wide variety of conditions. This process progresses to MIII arrest that is not responsive to parthenogenetic activation and development. Insofar as activation involves extrusion of the second polar body (PBII), we set out to determine if preventing this step by inhibiting microfilaments would change the course of spontaneous activation (SA). In particular, how long does the effect of SA persist while retaining reversibility of PBII extrusion once inhibitors are removed? We wanted to determine if the eggs would be responsive to parthenogenetic activation and capable of resuming development once a permanent inhibition is achieved. We set out to determine whether SA would depend on the ovular age of oocytes. Inhibiting of PBII extrusion was achieved by affecting microtubules with demecolcine or nocodazole or actin filaments with cytochalasin B (CB) and cytochalasin D (CD). We found that all oocytes undergo SA and progression to MIII; however, the rapidity of spontaneous activation is a function of the ovular age of the oocyte. The resumption of the meiosis period changes dramatically from 20 to 180 min with decreasing ovular age. We established that suppression of PB formation can be effectively achieved in oocytes of younger ovular age, and that inhibition of PB extrusion became irreversible after 3.5 h of treatment. We established that drug-treated oocytes could undergo subsequent reactivation and in vitro development to blastocysts. The rate of in vitro development of cytochalasin-treated group was comparable to parthenogenetic controls, while nocodazole and demecolcine produced oocytes that developed at lower frequencies. Thus, the application of the microfilament inhibiting drugs helps to overcome the negative effect of SA that results in MIII arrest. Here we also show optimized parthenogenetic stimulation that resulted in development to the blastocyst stage at frequency comparable to development of fertilized embryos.