A novel cell response triggered by interphase centromere structural instability

Interphase centromeres are crucial domains for the proper assembly of kinetochores at the onset of mitosis. However, it is not known whether the centromere structure is under tight control during interphase. This study uses the peculiar property of the infected cell protein 0 of herpes simplex virus type 1 to induce centromeric structural damage, revealing a novel cell response triggered by centromere destabilization. It involves centromeric accumulation of the Cajal body-associated coilin and fibrillarin as well as the survival motor neuron proteins. The response, which we have termed interphase centromere damage response (iCDR), was observed in all tested human and mouse cells, indicative of a conserved mechanism. Knockdown cells for several constitutive centromere proteins have shown that the loss of centromeric protein B provokes the centromeric accumulation of coilin. We propose that the iCDR is part of a novel safeguard mechanism that is dedicated to maintaining interphase centromeres compatible with the correct assembly of kinetochores, microtubule binding, and completion of mitosis.     

Progesterone-Based Therapy Protects Against Influenza by Promoting Lung Repair and Recovery in Females

Over 100 million women use progesterone therapies worldwide. Despite having immunomodulatory and repair properties, their effects on the outcome of viral diseases outside of the reproductive tract have not been evaluated. Administration of exogenous progesterone (at concentrations that mimic the luteal phase) to progesterone-depleted adult female mice conferred protection from both lethal and sublethal influenza A virus (IAV) infection. Progesterone treatment altered the inflammatory environment of the lungs, but had no effects on viral load. Progesterone treatment promoted faster recovery by increasing TGF-β, IL-6, IL-22, numbers of regulatory Th17 cells expressing CD39, and cellular proliferation, reducing protein leakage into the airway, improving pulmonary function, and upregulating the epidermal growth factor amphiregulin (AREG) in the lungs. Administration of rAREG to progesterone-depleted females promoted pulmonary repair and improved the outcome of IAV infection. Progesterone-treatment of AREG-deficient females could not restore protection, indicating that progesterone-mediated induction of AREG caused repair in the lungs and accelerated recovery from IAV infection. Repair and production of AREG by damaged respiratory epithelial cell cultures in vitro was increased by progesterone. Our results illustrate that progesterone is a critical host factor mediating production of AREG by epithelial cells and pulmonary tissue repair following infection, which has important implications for women’s health.     

Association of acylated cationic decapeptides with dipalmitoylphosphatidylserine-dipalmitoylphosphatidylcholine lipid membranes.

The interaction of three acylated and cationic decapeptides with lipid membranes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) has been studied by means of fluorescence spectroscopy and differential scanning calorimetry (DSC). The synthetic model decapeptides that are N-terminally linked with C(2), C(8), and C(14) acyl chains contain four basic histidine residues in their identical amino acid sequence. A binding model, based on changes in the intrinsic fluorescent properties of the peptides upon association with the DPPC-DPPS membranes, is used to estimate the peptide-membrane dissociation constants. The results clearly show that all three peptides have a higher affinity to liposomes containing DPPS lipids due to non-specific electrostatic interactions between the cationic peptides and the anionic DPPS lipids. Furthermore, it is found that the acyl chain length of the peptides plays a crucial role for the binding. A preference for fluid phase membranes as compared to gel phase membranes is generally observed for all three peptides. DSC is used to characterise the influence of the three peptides on the thermodynamic phase behaviour of the binary DPPC-DPPS lipid mixture. The extent of peptide association deduced from the heat capacity measurements suggests a strong binding and membrane insertion of the C(14) acylated peptide in accordance with the fluorescence measurements.     

Palmitoylation of the Influenza A Virus M2 Protein Is Not Required for Virus Replication In Vitro but Contributes to Virus Virulence

The influenza A virus M2 protein has important roles during virus entry and in the assembly of infectious virus particles. The cytoplasmic tail of the protein can be palmitoylated at a cysteine residue, but this residue is not conserved in a number of human influenza A virus isolates. Recombinant viruses encoding M2 proteins with a serine substituted for the cysteine at position 50 were generated in the A/WSN/33 (H1N1) and A/Udorn/72 (H3N2) genetic backgrounds. The recombinant viruses were not attenuated for replication in MDCK cells, Calu-3 cells, or in primary differentiated murine trachea epithelial cell cultures, indicating there was no significant contribution of M2 palmitoylation to virus replication in vitro. The A/WSN/33 M2C50S virus displayed a slightly reduced virulence after infection of mice, suggesting that there may be novel functions for M2 palmitoylation during in vivo infection.Influenza A virus is a member of the Orthomyxoviridae and contains a segmented, negative-sense RNA genome that codes for 10 or 11 proteins, depending upon the virus strain (11). The integral membrane protein M2 is the viral ion channel protein that is required during virus entry (29) and for the production of infectious virus particles (4, 10, 12, 13). The sequences responsible for the latter map to the cytoplasmic tail of the protein and overlap with a number of sites for posttranslational modification, which include palmitoylation and phosphorylation (7, 26, 31). Palmitoylation occurs on the cysteine present at amino acid 50 and is not required for ion channel activity of the M2 protein from A/Udorn/72 (H3N2) (7). Palmitoylation of M2 appeared to be dispensable for the production of infectious virus particles using a reassortant virus consisting of seven segments from an H3N8 subtype virus (A/Equine/Miami/63) and the M segment from an H1N1 subtype virus (A/Puerto Rico/8/34) (2). No studies examining the role of M2 palmitoylation in the context of a naturally occurring influenza A virus strain have been published to date.The significance of palmitoylation of the influenza A virus hemagglutinin (HA) protein can vary among virus strains. Palmitoylation of HA from an H7 and an H1 but not an H3 subtype is required for efficient membrane fusion (5, 24, 32), whereas palmitoylation of HA from an H3 but not an H1 subtype is required for virus assembly (5). An analysis of 3,532 sequences of influenza isolates from humans revealed that the M2 residue C50 is conserved in a strain-specific manner. A total of 2,602 of 2,610 H3N2 sequences code for a cysteine at this position; the cysteine, however, is conserved in only 330 of 1,051 H1N1 sequences (data not shown). A serine residue is substituted for cysteine in the majority of the H1N1 viruses that do not have a cytoplasmic palmitoylation site; the newly emerged 2009 H1N1 influenza A viruses, however, do have a cysteine at this position (3). The sequence alignment data are consistent with a strain-specific selective pressure to maintain the palmitoylation site on the M2 protein. Interestingly, other M2 cytoplasmic tail sequences display differential effects on infectious virus production, depending on the strain used (12).To investigate the role of M2 palmitoylation in influenza A virus replication, we substituted a serine for the cysteine residue at position 50 (C50S) of the M2 protein in two influenza A virus strains, A/Udorn/72 (H3N2) (rUdorn) and A/WSN/33 (H1N1) (rWSN). The resultant viruses were tested for their ability to replicate in tissue culture cells, and the mouse-adapted virus was tested for virulence in a mouse model of infection. Neither mutant virus showed any defect in virus replication in tissue culture cells, in differentiated murine primary trachea epithelial cells (mTEC), or in the lungs of infected mice. The viruses lacking a palmitoylation site, however, did have a modest reduction in virulence, suggesting that M2 palmitoylation is dispensable for in vitro replication but contributes to virus virulence in vivo.     

17beta-estradiol alters the activity of conventional and IFN-producing killer dendritic cells

Estrogens increase aspects of innate immunity and contribute to sex differences in the prevalence of autoimmune diseases and in response to infection. The goal of the present study was to assess whether exposure to 17beta-estradiol (E2) affects the development and function of bone marrow-derived dendritic cells and to determine whether similar changes are observed in CD11c(+) splenocytes exposed to E2 in vivo. E2 facilitated the differentiation of BM precursor cells into functional CD11c(+)CD11b(+)MHC class II(+) dendritic cells (DCs) with increased expression of the costimulatory molecules CD40 and CD86. Exposure of bone marrow-derived dendritic cells to E2 also enhanced production of IL-12 in response to the TLR ligands, CpG and LPS. In contrast, CD11c(+) cells isolated from the spleens of female C57BL/6 mice that were intact, ovariectomized, or ovariectomized with E2 replacement exhibited no differences in the number or activity of CD11c(+)CD11b(+)MHC class II(+) DCs. The presence of E2 in vivo, however, increased the number of CD11c(+)CD49b(+)NK1.1(low) cells and reduced numbers of CD11c(+)CD49b(+)NK1.1(high) cells, a surface phenotype for IFN-producing killer DCs (IKDCs). Ultrastructural analysis demonstrated that CD11c(+)NK1.1(+) populations were comprised of cells that had the appearance of both DCs and IKDCs. CD11c(+) splenocytes isolated from animals with supplemental E2 produced more IFN-gamma in response to IL-12 and IL-18. These data illustrate that E2 has differential effects on the development and function of DCs and IKDCs and provide evidence that E2 may strengthen innate immunity by enhancing IFN-gamma production by CD11c(+) cells.     

Myocardial performance index in female athletes

Background

Long-term intensive training leads to morphological and mechanical changes in the heart generally known as “athlete’s heart”. Previous studies have suggested that the diastolic and systolic function of the ventricles is unaltered in athletes compared to sedentary.The purpose of this study was to investigate myocardial performance index (MPI) by pulsed wave Doppler (PWD) and by tissue Doppler imaging (TDI) in female elite athletes compared to sedentary controls.

Methods

The study consisted of 32 athletes (mean age 20 ± 2 years) and 34 sedentary controls (mean age 23 ± 2 years). MPI by PWD and TDI were measured in the left (LV) and right ventricle (RV) in both groups. Moreover, comparisons of MPI by the two methods and between the LV and RV within the two groups were made.

Results

There were no significant differences in MPI between athletes and controls (p > 0.05), whereas the LV had significantly higher MPI compared to RV (p < 0.001, in athletes and controls). The agreement and the correlation between the two methods measuring MPI showed low agreement and no correlation (athletes RV r = ?0.027, LV r = 0.12; controls RV r = 0.20, LV r = 0.30).

Conclusion

The global function of the LV and RV measured by MPI with PWD and TDI is similar in female athletes compared to sedentary controls. Conversely, both MPI by PWD and by TDI shows a significant difference between the LV and RV. However, the agreement and correlation between conventional methods of measuring MPI by PWD compared to MPI by TDI is very poor in both these populations.

     

Age-related adaptation of bone-PDL-tooth complex: Rattus-Norvegicus as a model system

Functional loads on an organ induce tissue adaptations by converting mechanical energy into chemical energy at a cell-level. The transducing capacity of cells alters physico-chemical properties of tissues, developing a positive feedback commonly recognized as the form-function relationship. In this study, organ and tissue adaptations were mapped in the bone-tooth complex by identifying and correlating biomolecular expressions to physico-chemical properties in rats from 1.5 to 15 months. However, future research using hard and soft chow over relevant age groups would decouple the function related effects from aging affects. Progressive curvature in the distal root with increased root resorption was observed using micro X-ray computed tomography. Resorption was correlated to the increased activity of multinucleated osteoclasts on the distal side of the molars until 6 months using tartrate resistant acid phosphatase (TRAP). Interestingly, mononucleated TRAP positive cells within PDL vasculature were observed in older rats. Higher levels of glycosaminoglycans were identified at PDL-bone and PDL-cementum entheses using alcian blue stain. Decreasing biochemical gradients from coronal to apical zones, specifically biomolecules that can induce osteogenic (biglycan) and fibrogenic (fibromodulin, decorin) phenotypes, and PDL-specific negative regulator of mineralization (asporin) were observed using immunohistochemistry. Heterogeneous distribution of Ca and P in alveolar bone, and relatively lower contents at the entheses, were observed using energy dispersive X-ray analysis. No correlation between age and microhardness of alveolar bone (0.7 ± 0.1 to 0.9 ± 0.2 GPa) and cementum (0.6 ± 0.1 to 0.8 ± 0.3 GPa) was observed using a microindenter. However, hardness of cementum and alveolar bone at any given age were significantly different (P<0.05). These observations should be taken into account as baseline parameters, during development (1.5 to 4 months), growth (4 to 10 months), followed by a senescent phase (10 to 15 months), from which deviations due to experimentally induced perturbations can be effectively investigated.     

Expression of extracellular polysaccharides and proteins by clinical isolates of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in response to environmental conditions

The opportunistic pathogen Pseudomonas aeruginosa causes chronic respiratory infections in patients with cystic fibrosis (CF). Persistence of this bacterium is attributed to its ability to form biofilms which rely on an extracellular polymeric substance matrix. Extracellular polysaccharides (EPS) and secreted proteins are key matrix components of P. aeruginosa biofilms. Recently, nebulized magnesium sulfate has been reported as a significant bronchodilator for asthmatic patients including CF. However, the impact of magnesium sulfate on the virulence effect of P. aeruginosa is lacking. In this report, we investigated the influence of magnesium sulfate and other environmental factors on the synthesis of alginate and secretion of proteins by a mucoid and a non-mucoid strain of P. aeruginosa, respectively. By applying the Plackett-Burman and Box-Behnken experimental designs, we found that phosphates (6.0 g/l), ammonium sulfate (4.0 g/l), and trace elements (0.6 mg/l) markedly supported alginate production by the mucoid strain. However, ferrous sulfate (0.3 mg/l), magnesium sulfate (0.02 g/l), and phosphates (6.0 g/l) reinforced the secretion of proteins by the non-mucoid strain.     

Wetting and capillary condensation as means of protein organization in membranes.

Wetting and capillary condensation are thermodynamic phenomena in which the special affinity of interfaces to a thermodynamic phase, relative to the stable bulk phase, leads to the stabilization of a wetting phase at the interfaces. Wetting and capillary condensation are here proposed as mechanisms that in membranes may serve to induce special lipid phases in between integral membrane proteins leading to long-range lipid-mediated joining forces acting between the proteins and hence providing a means of protein organization. The consequences of wetting in terms of protein aggregation and protein clustering are derived both within a simple phenomenological theory as well as within a concrete calculation on a microscopic model of lipid-protein interactions that accounts for the lipid bilayer phase equilibria and direct lipid-protein interactions governed by hydrophobic matching between the lipid bilayer hydrophobic thickness and the length of the hydrophobic membrane domain. The theoretical results are expected to be relevant for optimizing the experimental conditions required for forming protein aggregates and regular protein arrays in membranes.