Cyclin-dependent kinases 7 and 9 specifically regulate neutrophil transcription and their inhibition drives apoptosis to promote resolution of inflammation

Terminally differentiated neutrophils are short-lived but the key effector cells of the innate immune response, and have a prominent role in the pathogenesis and propagation of many inflammatory diseases. Delayed apoptosis, which is responsible for their extended longevity, is critically dependent on a balance of intracellular survival versus pro-apoptotic proteins. Here, we elucidate the mechanism by which the cyclin-dependent kinase (CDK) inhibitor drugs such as R-roscovitine and DRB (5,6-dichloro-1-beta--ribofuranosylbenzimidazole) mediate neutrophil apoptosis. We demonstrate (by a combination of microarray, confocal microscopy, apoptosis assays and western blotting) that the phosphorylation of RNA polymerase II by CDKs 7 and 9 is inhibited by R-roscovitine and that specific effects on neutrophil transcriptional capacity are responsible for neutrophil apoptosis. Finally, we show that specific CDK7 and 9 inhibition with DRB drives resolution of neutrophil-dominant inflammation. Thus, we highlight a novel mechanism that controls both primary human neutrophil transcription and apoptosis that could be targeted by selective CDK inhibitor drugs to resolve established inflammation

A cardinal role for cathepsin D in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci

The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP) and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D-/- hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function

Training evaluation: a case study of training Iranian health managers

<p>Abstract</p> <p>Background</p> <p>The Ministry of Health and Medical Education in the Islamic Republic of Iran has undertaken a reform of its health system, in which-lower level managers are given new roles and responsibilities in a decentralized system. To support these efforts, a United Kingdom-based university was contracted by the World Health Organization to design a series of courses for health managers and trainers. This process was also intended to develop the capacity of the National Public Health Management Centre in Tabriz, Iran, to enable it to organize relevant short courses in health management on a continuing basis. A total of seven short training courses were implemented, three in the United Kingdom and four in Tabriz, with 35 participants. A detailed evaluation of the courses was undertaken to guide future development of the training programmes.</p> <p>Methods</p> <p>The Kirkpatrick framework for evaluation of training was used to measure participants' reactions, learning, application to the job, and to a lesser extent, organizational impact. Particular emphasis was put on application of learning to the participants' job. A structured questionnaire was administered to 23 participants, out of 35, between one and 13 months after they had attended the courses. Respondents, like the training course participants, were predominantly from provincial universities, with both health system and academic responsibilities. Interviews with key informants and ex-trainees provided supplemental information, especially on organizational impact.</p> <p>Results</p> <p>Participants' preferred interactive methods for learning about health planning and management. They found the course content to be relevant, but with an overemphasis on theory compared to practical, locally-specific information. In terms of application of learning to their jobs, participants found specific information and skills to be most useful, such as health systems research and group work/problem solving. The least useful areas were those that dealt with training and leadership. Participants reported little difficulty in applying learning deemed "useful", and had applied it often. In general, a learning area was used less when it was found difficult to apply, with a few exceptions, such as problem-solving. Four fifths of respondents claimed they could perform their jobs better because of new skills and more in-depth understanding of health systems, and one third had been asked to train their colleagues, indicating a potential for impact on their organization. Interviews with key informants indicated that job performance of trainees had improved.</p> <p>Conclusion</p> <p>The health management training programmes in Iran, and the external university involved in capacity building, benefited from following basic principles of good training practice, which incorporated needs assessment, selection of participants and definition of appropriate learning outcomes, course content and methods, along with focused evaluation. Contracts for external assistance should include specific mention of capacity building, and allow for the collaborative development of courses and of evaluation plans, in order to build capacity of local partners throughout the training cycle. This would also help to develop training content that uses material from local health management situations to demonstrate key theories and develop locally required skills. Training evaluations should as a minimum assess participants' reactions and learning for every course. Communication of evaluation results should be designed to ensure that data informs training activities, as well as the health and human resources managers who are investing in the development of their staff.</p

Regional variations in and correlates of disability-free life expectancy among older adults in China

<p>Abstract</p> <p>Background</p> <p>Considerable socioeconomic and health inequalities have been reported in China. However, because of a lack of appropriate data, limited research has been conducted on variations in disability-free life expectancy (DFLE) among older adults. This study aimed to use the most up-to-date disability survey data to explore geographical variations in DFLE at age 60 in China and to identify the socioeconomic and health care factors that partially account for these variations.</p> <p>Methods</p> <p>This study used 2006 mortality data extrapolated from the 1990 and 2000 Census and disability data from a national disability survey conducted in 2006. Disability was performance based and was diagnosed by trained physicians. DFLE was calculated by region using the Sullivan method. Multiple linear regression models by gender were conducted to explore correlates of DFLE.</p> <p>Results</p> <p>DFLE at age 60 varied widely by region, from 11.2 to 20.8 years in 2006. Per capita gross domestic product, proportion of urban residents, and access to health care were the primary factors associated with geographical variations in DFLE.</p> <p>Conclusion</p> <p>The pattern of differences in DFLE by region mirrors the pattern of regional economic development in China. Countermeasures to decrease regional differences in DFLE include accelerating regional economic development and improving health care distribution.</p

The Evolution of Compact Binary Star Systems

We review the formation and evolution of compact binary stars consisting of white dwarfs (WDs), neutron stars (NSs), and black holes (BHs). Binary NSs and BHs are thought to be the primary astrophysical sources of gravitational waves (GWs) within the frequency band of ground-based detectors, while compact binaries of WDs are important sources of GWs at lower frequencies to be covered by space interferometers (LISA). Major uncertainties in the current understanding of properties of NSs and BHs most relevant to the GW studies are discussed, including the treatment of the natal kicks which compact stellar remnants acquire during the core collapse of massive stars and the common envelope phase of binary evolution. We discuss the coalescence rates of binary NSs and BHs and prospects for their detections, the formation and evolution of binary WDs and their observational manifestations. Special attention is given to AM CVn-stars -- compact binaries in which the Roche lobe is filled by another WD or a low-mass partially degenerate helium-star, as these stars are thought to be the best LISA verification binary GW sources.Comment: 105 pages, 18 figure

Evolutionary History of the Vertebrate Mitogen Activated Protein Kinases Family

Background: The mitogen activated protein kinases (MAPK) family pathway is implicated in diverse cellular processes and pathways essential to most organisms. Its evolution is conserved throughout the eukaryotic kingdoms. However, the detailed evolutionary history of the vertebrate MAPK family is largely unclear. Methodology/Principal Findings: The MAPK family members were collected from literatures or by searching the genomes of several vertebrates and invertebrates with the known MAPK sequences as queries. We found that vertebrates had significantly more MAPK family members than invertebrates, and the vertebrate MAPK family originated from 3 progenitors, suggesting that a burst of gene duplication events had occurred after the divergence of vertebrates from invertebrates. Conservation of evolutionary synteny was observed in the vertebrate MAPK subfamilies 4, 6, 7, and 11 to 14. Based on synteny and phylogenetic relationships, MAPK12 appeared to have arisen from a tandem duplication of MAPK11 and the MAPK13-MAPK14 gene unit was from a segmental duplication of the MAPK11-MAPK12 gene unit. Adaptive evolution analyses reveal that purifying selection drove the evolution of MAPK family, implying strong functional constraints of MAPK genes. Intriguingly, however, intron losses were specifically observed in the MAPK4 and MAPK7 genes, but not in their flanking genes, during the evolution from teleosts to amphibians and mammals. The specific occurrence of intron losses in the MAPK4 and MAPK7 subfamilies might be associated with adaptive evolution of the vertebrates by enhancing the gen

Association of FcγRIIa R131H polymorphism with idiopathic pulmonary fibrosis severity and progression

<p>Abstract</p> <p>Background</p> <p>A significant genetic component has been described for idiopathic pulmonary fibrosis (IPF). The R131H (rs1801274) polymorphism of the IgG receptor FcγRIIa determines receptor affinity for IgG subclasses and is associated with several chronic inflammatory diseases. We investigated whether this polymorphism is associated with IPF susceptibility or progression.</p> <p>Methods</p> <p>In a case-control study, we compared the distribution of FcγRIIa R131H genotypes in 142 patients with IPF and in 218 controls using allele-specific PCR amplification.</p> <p>Results</p> <p>No differences in the frequency of FcγRIIa genotypes were evident between IPF patients and control subjects. However, significantly impaired pulmonary function at diagnosis was observed in HH compared to RR homozygotes, with evidence of more severe restriction (reduced forced vital capacity (FVC)) and lower diffusing capacity for carbon monoxide (D<smcaps>L</smcaps>_CO). Similarly, increased frequency of the H131 allele was observed in patients with severe disease (D<smcaps>L</smcaps>_CO< 40% predicted) (0.53 vs. 0.38; p = 0.03). Furthermore, the H131 allele was associated with progressive pulmonary fibrosis as determined by > 10% drop in FVC and/or > 15% fall in D<smcaps>L</smcaps>_COat 12 months after baseline (0.48 vs. 0.33; p = 0.023).</p> <p>Conclusions</p> <p>These findings support an association between the FcγRIIa R131H polymorphism and IPF severity and progression, supporting the involvement of immunological mechanisms in IPF pathogenesis.</p

Characterization of Spontaneous Bone Marrow Recovery after Sublethal Total Body Irradiation: Importance of the Osteoblastic/Adipocytic Balance

Many studies have already examined the hematopoietic recovery after irradiation but paid with very little attention to the bone marrow microenvironment. Nonetheless previous studies in a murine model of reversible radio-induced bone marrow aplasia have shown a significant increase in alkaline phosphatase activity (ALP) prior to hematopoietic regeneration. This increase in ALP activity was not due to cell proliferation but could be attributed to modifications of the properties of mesenchymal stem cells (MSC). We thus undertook a study to assess the kinetics of the evolution of MSC correlated to their hematopoietic supportive capacities in mice treated with sub lethal total body irradiation. In our study, colony-forming units – fibroblasts (CFU-Fs) assay showed a significant MSC rate increase in irradiated bone marrows. CFU-Fs colonies still possessed differentiation capacities of MSC but colonies from mice sacrificed 3 days after irradiation displayed high rates of ALP activity and a transient increase in osteoblastic markers expression while pparγ and neuropilin-1 decreased. Hematopoietic supportive capacities of CFU-Fs were also modified: as compared to controls, irradiated CFU-Fs significantly increased the proliferation rate of hematopoietic precursors and accelerated the differentiation toward the granulocytic lineage. Our data provide the first evidence of the key role exerted by the balance between osteoblasts and adipocytes in spontaneous bone marrow regeneration. First, (pre)osteoblast differentiation from MSC stimulated hematopoietic precursor's proliferation and granulopoietic regeneration. Then, in a second time (pre)osteoblasts progressively disappeared in favour of adipocytic cells which down regulated the proliferation and granulocytic differentiation and then contributed to a return to pre-irradiation conditions

Loss of estrogen receptor β decreases mitochondrial energetic potential and increases thrombogenicity of platelets in aged female mice

Platelets derived from aged (reproductively senescent) female mice with genetic deletion of estrogen receptor beta (βER) are more thrombogenic than those from age-matched wild-type (WT) mice. Intracellular processes contributing to this increased thrombogenicity are not known. Experiments were designed to identify subcellular localization of estrogen receptors and evaluate both glycolytic and mitochondrial energetic processes which might affect platelet activation. Platelets and blood from aged (22–24 months) WT and estrogen receptor β knockout (βERKO) female mice were used in this study. Body, spleen weight, and serum concentrations of follicle-stimulating hormone and 17β-estradiol were comparable between WT and βERKO mice. Number of spontaneous deaths was greater in the βERKO colony (50% compared to 30% in WT) over the course of 24 months. In resting (nonactivated) platelets, estrogen receptors did not appear to colocalize with mitochondria by immunostaining. Lactate production and mitochondrial membrane potential of intact platelets were similar in both groups of mice. However, activities of NADH dehydrogenase, cytochrome bc1 complex, and cytochrome c oxidase of the electron transport chain were reduced in mitochondria isolated from platelets from βERKO compared to WT mice. There were a significantly higher number of phosphatidylserine-expressing platelet-derived microvesicles in the plasma and a greater thrombin-generating capacity in βERKO compared to WT mice. These results suggest that deficiencies in βER affect energy metabolism of platelets resulting in greater production of circulating thrombogenic microvesicles and could potentially explain increased predisposition to thromboembolism in some elderly females

Prenylation Inhibition-Induced Cell Death in Melanoma: Reduced Sensitivity in BRAF Mutant/PTEN Wild-Type Melanoma Cells.

While targeted therapy brought a new era in the treatment of BRAF mutant melanoma, therapeutic options for non-BRAF mutant cases are still limited. In order to explore the antitumor activity of prenylation inhibition we investigated the response to zoledronic acid treatment in thirteen human melanoma cell lines with known BRAF, NRAS and PTEN mutational status. Effect of zoledronic acid on proliferation, clonogenic potential, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were investigated in vitro with SRB, TUNEL and PARP cleavage assays and videomicroscopy and immunoblot measurements, respectively. Subcutaneous and spleen-to-liver colonization xenograft mouse models were used to evaluate the influence of zoledronic acid treatment on primary and disseminated tumor growth of melanoma cells in vivo. Zoledronic acid more efficiently decreased short-term in vitro viability in NRAS mutant cells when compared to BRAF mutant and BRAF/NRAS wild-type cells. In line with this finding, following treatment decreased activation of ribosomal protein S6 was found in NRAS mutant cells. Zoledronic acid demonstrated no significant synergism in cell viability inhibition or apoptosis induction with cisplatin or DTIC treatment in vitro. Importantly, zoledronic acid could inhibit clonogenic growth in the majority of melanoma cell lines except in the three BRAF mutant but PTEN wild-type melanoma lines. A similar pattern was observed in apoptosis induction experiments. In vivo zoledronic acid did not inhibit the subcutaneous growth or spleen-to-liver colonization of melanoma cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. Nevertheless, we also demonstrated that therapeutic sensitivity might be influenced by the PTEN status of BRAF mutant melanoma cells. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target prenylation in melanoma cells