Structure of the N-terminal ankyrin repeat domain of the TRPV2 ion channel

The TRPV ion channels mediate responses to many sensory stimuli including heat, low pH, neuropeptides, and chemical ligands. All TRPV subfamily members contain an intracellular N-terminal ankyrin repeat domain (ARD), a prevalent protein interaction motif. The 1.6-A crystal structure of the TRPV2-ARD, with six ankyrin repeats, reveals several atypical structural features. Repeats one through three display unusually long and flexible fingers with a large number of exposed aromatic residues, whereas repeats five and six have unusually long outer helices. Furthermore, a large counterclockwise twist observed in the stacking of repeats four and five breaks the regularity of the domain, altering the shape of surfaces available for interactions with proteins or other cellular ligands. Both solution studies and crystal packing interactions indicate that the TRPV2-ARD does not form homo-oligomers, suggesting that the ARD of TRPV ion channels may be used for interactions with regulatory factors rather than in promoting tetrameric assembly of the ion channels.     

Human macrophage migration inhibitory factor: a proven immunomodulatory cytokine?

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory mediator with the ability to induce various immunomodulatory responses and override glucocorticoid-driven immunosuppression. Some of these functions have been linked to the unusual enzymatic properties of the protein, namely tautomerase and oxidoreductase activities. However, there are conflicting reports regarding the functional role of these enzymatic properties in normal physiological homeostasis and disease progression. Therefore, we have produced a highly pure, virtually endotoxin-free recombinant MIF preparation and fully characterized this using a variety of biochemical and biophysical approaches. The recombinant protein, with demonstrable enzymatic activity, was then used to systematically examine the biological activity of MIF. Surprisingly, treatment with MIF alone failed to induce cytokine expression, with the exception of IL-8. However, co-treatment of lipopolysaccharide (LPS) in conjunction with MIF produced synergistic secretion of tumor necrosis factor-alpha, interleukin (IL)-1, and IL-8 compared with LPS alone. The potentiating effect of MIF was seen at physiologically relevant concentrations. These data suggest that MIF has no conventional cytokine activity but, rather, acts to modulate and amplify the response to LPS.     

Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis

Apoptosis is essential for clearance of potentially injurious inflammatory cells and subsequent efficient resolution of inflammation. Here we report that human neutrophils contain functionally active cyclin-dependent kinases (CDKs), and that structurally diverse CDK inhibitors induce caspase-dependent apoptosis and override powerful anti-apoptosis signals from survival factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that the CDK inhibitor R-roscovitine (Seliciclib or CYC202) markedly enhances resolution of established neutrophil-dependent inflammation in carrageenan-elicited acute pleurisy, bleomycin-induced lung injury, and passively induced arthritis in mice. In the pleurisy model, the caspase inhibitor zVAD-fmk prevents R-roscovitine-enhanced resolution of inflammation, indicating that this CDK inhibitor augments inflammatory cell apoptosis. We also provide evidence that R-roscovitine promotes apoptosis by reducing concentrations of the anti-apoptotic protein Mcl-1. Thus, CDK inhibitors enhance the resolution of established inflammation by promoting apoptosis of inflammatory cells, thereby demonstrating a hitherto unrecognized potential for the treatment of inflammatory disorders.     

Basic Biology and Mechanisms of Neural Ciliogenesis and the B9 Family

Although the discovery of cilia is one of the earliest in cell biology, the past two decades have witnessed an explosion of new insight into these enigmatic organelles. While long believed to be vestigial, cilia have recently moved into the spotlight as key players in multiple cellular processes, including brain development and homeostasis. This review focuses on the rapidly expanding basic biology of neural cilia, with special emphasis on the newly emerging B9 family of proteins. In particular, recent findings have identified a critical role for the B9 complex in a network of protein interactions that take place at the ciliary transition zone (TZ). We describe the essential role of these protein complexes in signaling cascades that require primary (nonmotile) cilia, including the sonic hedgehog pathway. Loss or dysfunction of ciliary trafficking and TZ function are linked to a number of neurologic diseases, which we propose to classify as neural ciliopathies. When taken together, the studies reviewed herein point to critical roles played by neural cilia, both in normal physiology and in disease.     

Biophysical and mechanistic insights into novel allosteric inhibitor of spleen tyrosine kinase

Extracellular stimulation of the B cell receptor or mast cell FcεRI receptor activates a cascade of protein kinases, ultimately leading to antigenic or inflammation immune responses, respectively. Syk is a soluble kinase responsible for transmission of the receptor activation signal from the membrane to cytosolic targets. Control of Syk function is, therefore, critical to the human antigenic and inflammation immune response, and an inhibitor of Syk could provide therapy for autoimmune or inflammation diseases. We report here a novel allosteric Syk inhibitor, X1, that is noncompetitive against ATP (K(i) 4 ± 1 μM) and substrate peptide (K(i) 5 ± 1 μM), and competitive against activation of Syk by its upstream regulatory kinase LynB (K(i) 4 ± 1 μM). The inhibition mechanism was interrogated using a combination of structural, biophysical, and kinetic methods, which suggest the compound inhibits Syk by reinforcing the natural regulatory interactions between the SH2 and kinase domains. This novel mode of inhibition provides a new opportunity to improve the selectivity profile of Syk inhibitors for the development of safer drug candidates.     

Hydra, a versatile model to study the homeostatic and developmental functions of cell death

In the freshwater cnidarian polyp Hydra, cell death takes place in multiple contexts. Indeed apoptosis occurs during oogenesis and spermatogenesis, during starvation, and in early head regenerating tips, promoting local compensatory proliferation at the boundary between heterografts. Apoptosis can also be induced upon exposure to pro-apoptotic agents (colchicine, wortmannin), upon heat-shock in the thermosensitive sf-1 mutant, and upon wounding. In all these contexts, the cells that undergo cell death belong predominantly to the interstitial cell lineage, whereas the epithelial cells, which are rather resistant to pro-apoptotic signals, engulf the apoptotic bodies. Beside this clear difference between the interstitial and the epithelial cell lineages, the different interstitial cell derivatives also show noticeable variations in their respective apoptotic sensitivity, with the precursor cells appearing as the most sensitive to pro-apoptotic signals. The apoptotic machinery has been well conserved across evolution. However, its specific role and regulation in each context are not known yet. Tools that help characterize apoptotic activity in Hydra have recently been developed. Among them, the aposensor Apoliner initially designed in Drosophila reliably measures wortmannin-induced apoptotic activity in a biochemical assay. Also, flow cytometry and TUNEL analyses help identify distinctive features between wortmannin-induced and heat-shock induced apoptosis in the sf-1 strain. Thanks to the live imaging tools already available, Hydra now offers a model system with which the functions of the apoptotic machinery to maintain long-term homeostasis, stem cell renewal, germ cell production, active developmental processes and non-self response can be deciphered.     

A systems approach to improving rural care in Ethiopia

Background

Multiple interventions have been launched to improve the quality, access, and utilization of primary health care in rural, low-income settings; however, the success of these interventions varies substantially, even within single studies where the measured impact of interventions differs across sites, centers, and regions. Accordingly, we sought to examine the variation in impact of a health systems strengthening intervention and understand factors that might explain the variation in impact across primary health care units.

Methodology/Principal Findings

We conducted a mixed methods positive deviance study of 20 Primary Health Care Units (PHCUs) in rural Ethiopia. Using longitudinal data from the Ethiopia Millennium Rural Initiative (EMRI), we identified PHCUs with consistently higher performance (n = 2), most improved performance (n = 3), or consistently lower performance (n = 2) in the provision of antenatal care, HIV testing in antenatal care, and skilled birth attendance rates. Using data from site visits and in-depth interviews (n = 51), we applied the constant comparative method of qualitative data analysis to identify key themes that distinguished PHCUs with different performance trajectories. Key themes that distinguished PHCUs were 1) managerial problem solving capacity, 2) relationship with the woreda (district) health office, and 3) community engagement. In higher performing PHCUs and those with the greatest improvement after the EMRI intervention, health center and health post staff were more able to solve day-to-day problems, staff had better relationships with the woreda health official, and PHCU communities'' leadership, particularly religious leadership, were strongly engaged with the health improvement effort. Distance from the nearest city, quality of roads and transportation, and cultural norms did not differ substantially among PHCUs.

Conclusions/Significance

Effective health strengthening efforts may require intensive development of managerial problem solving skills, strong relationships with government offices that oversee front-line providers, and committed community leadership to succeed.     

A genome-wide study of cytogenetic changes in colorectal cancer using SNP microarrays: opportunities for future personalized treatment

In colorectal cancer (CRC), chromosomal instability (CIN) is typically studied using comparative-genomic hybridization (CGH) arrays. We studied paired (tumor and surrounding healthy) fresh frozen tissue from 86 CRC patients using Illumina's Infinium-based SNP array. This method allowed us to study CIN in CRC, with simultaneous analysis of copy number (CN) and B-allele frequency (BAF)--a representation of allelic composition. These data helped us to detect mono-allelic and bi-allelic amplifications/deletion, copy neutral loss of heterozygosity, and levels of mosaicism for mixed cell populations, some of which can not be assessed with other methods that do not measure BAF. We identified associations between CN abnormalities and different CRC phenotypes (histological diagnosis, location, tumor grade, stage, MSI and presence of lymph node metastasis). We showed commonalities between regions of CN change observed in CRC and the regions reported in previous studies of other solid cancers (e.g. amplifications of 20q, 13q, 8q, 5p and deletions of 18q, 17p and 8p). From Therapeutic Target Database, we identified relevant drugs, targeted to the genes located in these regions with CN changes, approved or in trials for other cancers and common diseases. These drugs may be considered for future therapeutic trials in CRC, based on personalized cytogenetic diagnosis. We also found many regions, harboring genes, which are not currently targeted by any relevant drugs that may be considered for future drug discovery studies. Our study shows the application of high density SNP arrays for cytogenetic study in CRC and its potential utility for personalized treatment.     

DNA damage response by single-strand breaks in terminally differentiated muscle cells and the control of muscle integrity

DNA single-strand breaks (SSB) formation coordinates the myogenic program, and defects in SSB repair in post-mitotic cells have been associated with human diseases. However, the DNA damage response by SSB in terminally differentiated cells has not been explored yet. Here we show that mouse post-mitotic muscle cells accumulate SSB after alkylation damage, but they are extraordinarily resistant to the killing effects of a variety of SSB-inducers. We demonstrate that, upon SSB induction, phosphorylation of H2AX occurs in myotubes and is largely ataxia telangiectasia mutated (ATM)-dependent. However, the DNA damage signaling cascade downstream of ATM is defective as shown by lack of p53 increase and phosphorylation at serine 18 (human serine 15). The stabilization of p53 by nutlin-3 was ineffective in activating the cell death pathway, indicating that the resistance to SSB inducers is due to defective p53 downstream signaling. The induction of specific types of damage is required to activate the cell death program in myotubes. Besides the topoisomerase inhibitor doxorubicin known for its cardiotoxicity, we show that the mitochondria-specific inhibitor menadione is able to activate p53 and to kill effectively myotubes. Cell killing is p53-dependent as demonstrated by full protection of myotubes lacking p53, but there is a restriction of p53-activated genes. This new information may have important therapeutic implications in the prevention of muscle cell toxicity.     

Low Dose Antigen Exposure for a Finite Period in Newborn Rats Prevents Induction of Mucosal Tolerance

Background

In adult rats, initial exposure to antigens by a mucosal route triggers tolerance such that any subsequent re-exposure, even by a systemic route, results in suppression of immunity. The newborn’s gut is semi-permeable for a finite period to allow maternal antibodies to enter the newborn’s circulation. We propose that antigens introduced in extreme early life can readily traverse the gut wall and therefore circumvent induction of mucosal tolerance.

Methodology/Principle Findings

Rat pups were gavaged with low-doses of ovalbumin (OVA; oral exposure group) or saline (parenteral control group) every second day for several weeks followed by an intraperitoneal (i.p.) injection at 1 month of age. When gavage was initiated the day after birth, newborn oral exposure pups responded with significantly higher anti-OVA IgA, IgM, IgG2a, and IgG1 titres in their serum and anti-OVA IgA, IgG2a and IgG1 titres in their lungs compared to negative control pups. Oral exposure alone failed to induce immunity. Pups exposed to the same treatment regimen starting at 14 days of age showed induction of mucosal tolerance after i.p. immunization. Newborn oral exposure groups subjected to secondary i.p. immunization responded with significantly increased humoral immunity in lung and sera suggesting that once antigen-specific mucosal tolerance if circumvented, it persists. Lymphocytes derived from mesenteric lymph node cells re-simulated with OVA ex vivo, from newborn oral exposure pups exposed to secondary immunization produced significantly higher IFN-γ expression and lymphocyte proliferation relative to control pups indicating prevention of tolerance in the cell-mediated immune system.

Conclusions/Significance

This work demonstrates that newborns may be uniquely qualified to prevent induction of mucosal tolerance to oral antigens. These results should be further explored to establish whether prevention of tolerance by early life oral vaccination can be exploited to prime for mucosal as well as systemic immunity and thus protect this susceptible population against infectious diseases.