A potential role for the Src-like adapter protein SLAP-2 in signaling by the colony stimulating factor-1 receptor

The development of macrophages from myeloid progenitor cells is primarily controlled by the growth factor colony stimulating factor-1 (CSF-1) and its cognate receptor, a transmembrane tyrosine kinase encoded by the c-Fms proto-oncogene. The CSF-1 receptor exerts its biological effects on cells via a range of signaling proteins including Erk1/2 and Akt. Here we have investigated the potential involvement of the Src-like adapter protein (SLAP-2) in signaling by the CSF-1 receptor in mouse bone marrow-derived macrophages. RT-PCR analysis revealed constitutive expression of the SLAP-2 gene in bone marrow macrophages. Surprisingly, co-immunoprecipitation and GST binding experiments demonstrated that the CSF-1 receptor could bind to SLAP-2 in a ligand-independent manner. Furthermore, the binding of SLAP-2 to the CSF-1 receptor involved multiple domains of SLAP-2. SLAP-2 also bound c-Cbl, with the interaction being mediated, at least in part, by the unique C-terminal domain of SLAP-2. Overexpression of SLAP-2 in bone marrow macrophages partially suppressed the CSF-1-induced tyrosine phosphorylation and/or expression level of a approximately 80 kDa protein without affecting CSF-1-induced global tyrosine phosphorylation, or activation of Akt or Erk1/2. Significantly, CSF-1 stimulation induced serine phosphorylation of SLAP-2. Pharmacologic inhibition of specific protein kinases revealed that CSF-1-induced phosphorylation of SLAP-2 was dependent on JNK activity. Taken together, our results suggest that SLAP-2 could potentially be involved in signaling by the CSF-1 receptor.     

Porphyromonas gingivalis-derived RgpA-Kgp Complex Activates the Macrophage Urokinase Plasminogen Activator System: IMPLICATIONS FOR PERIODONTITIS*

Urokinase plasminogen activator (uPA) converts plasminogen to plasmin, resulting in a proteolytic cascade that has been implicated in tissue destruction during inflammation. Periodontitis is a highly prevalent chronic inflammatory disease characterized by destruction of the tissue and bone that support the teeth. We demonstrate that stimulation of macrophages with the arginine- and lysine-specific cysteine protease complex (RgpA-Kgp complex), produced by the keystone pathogen Porphyromonas gingivalis, dramatically increased their ability to degrade matrix in a uPA-dependent manner. We show that the RgpA-Kgp complex cleaves the inactive zymogens, pro-uPA (at consensus sites Lys¹⁵⁸-Ile¹⁵⁹ and Lys¹³⁵-Lys¹³⁶) and plasminogen, yielding active uPA and plasmin, respectively. These findings are consistent with activation of the uPA proteolytic cascade by P. gingivalis being required for the pathogen to induce alveolar bone loss in a model of periodontitis and reveal a new host-pathogen interaction in which P. gingivalis activates a critical host proteolytic pathway to promote tissue destruction and pathogen virulence.     

Genetic association analysis highlights new loci that modulate hematological trait variation in Caucasians and African Americans

Red blood cell, white blood cell, and platelet measures, including their count, sub-type and volume, are important diagnostic and prognostic clinical parameters for several human diseases. To identify novel loci associated with hematological traits, and compare the architecture of these phenotypes between ethnic groups, the CARe Project genotyped 49,094 single nucleotide polymorphisms (SNPs) that capture variation in ~2,100 candidate genes in DNA of 23,439 Caucasians and 7,112 African Americans from five population-based cohorts. We found strong novel associations between erythrocyte phenotypes and the glucose-6 phosphate dehydrogenase (G6PD) A-allele in African Americans (rs1050828, P<2.0×10(-13), T-allele associated with lower red blood cell count, hemoglobin, and hematocrit, and higher mean corpuscular volume), and between platelet count and a SNP at the tropomyosin-4 (TPM4) locus (rs8109288, P=3.0×10(-7) in Caucasians; P=3.0×10(-7) in African Americans, T-allele associated with lower platelet count). We strongly replicated many genetic associations to blood cell phenotypes previously established in Caucasians. A common variant of the α-globin (HBA2-HBA1) locus was associated with red blood cell traits in African Americans, but not in Caucasians (rs1211375, P<7×10(-8), A-allele associated with lower hemoglobin, mean corpuscular hemoglobin, and mean corpuscular volume). Our results show similarities but also differences in the genetic regulation of hematological traits in European- and African-derived populations, and highlight the role of natural selection in shaping these differences.     

Synaptic and intrinsic determinants of the phase resetting curve for weak coupling

A phase resetting curve (PRC) keeps track of the extent to which a perturbation at a given phase advances or delays the next spike, and can be used to predict phase locking in networks of oscillators. The PRC can be estimated by convolving the waveform of the perturbation with the infinitesimal PRC (iPRC) under the assumption of weak coupling. The iPRC is often defined with respect to an infinitesimal current as z_i(ϕ), where ϕ is phase, but can also be defined with respect to an infinitesimal conductance change as z_g(ϕ). In this paper, we first show that the two approaches are equivalent. Coupling waveforms corresponding to synapses with different time courses sample z_g(ϕ) in predictably different ways. We show that for oscillators with Type I excitability, an anomalous region in z_g(ϕ) with opposite sign to that seen otherwise is often observed during an action potential. If the duration of the synaptic perturbation is such that it effectively samples this region, PRCs with both advances and delays can be observed despite Type I excitability. We also show that changing the duration of a perturbation so that it preferentially samples regions of stable or unstable slopes in z_g(ϕ) can stabilize or destabilize synchrony in a network with the corresponding dynamics.     

Genome-wide association study of white blood cell count in 16,388 African Americans: the continental origins and genetic epidemiology network (COGENT)

Total white blood cell (WBC) and neutrophil counts are lower among individuals of African descent due to the common African-derived "null" variant of the Duffy Antigen Receptor for Chemokines (DARC) gene. Additional common genetic polymorphisms were recently associated with total WBC and WBC sub-type levels in European and Japanese populations. No additional loci that account for WBC variability have been identified in African Americans. In order to address this, we performed a large genome-wide association study (GWAS) of total WBC and cell subtype counts in 16,388 African-American participants from 7 population-based cohorts available in the Continental Origins and Genetic Epidemiology Network. In addition to the DARC locus on chromosome 1q23, we identified two other regions (chromosomes 4q13 and 16q22) associated with WBC in African Americans (P<2.5×10(-8)). The lead SNP (rs9131) on chromosome 4q13 is located in the CXCL2 gene, which encodes a chemotactic cytokine for polymorphonuclear leukocytes. Independent evidence of the novel CXCL2 association with WBC was present in 3,551 Hispanic Americans, 14,767 Japanese, and 19,509 European Americans. The index SNP (rs12149261) on chromosome 16q22 associated with WBC count is located in a large inter-chromosomal segmental duplication encompassing part of the hydrocephalus inducing homolog (HYDIN) gene. We demonstrate that the chromosome 16q22 association finding is most likely due to a genotyping artifact as a consequence of sequence similarity between duplicated regions on chromosomes 16q22 and 1q21. Among the WBC loci recently identified in European or Japanese populations, replication was observed in our African-American meta-analysis for rs445 of CDK6 on chromosome 7q21 and rs4065321 of PSMD3-CSF3 region on chromosome 17q21. In summary, the CXCL2, CDK6, and PSMD3-CSF3 regions are associated with WBC count in African American and other populations. We also demonstrate that large inter-chromosomal duplications can result in false positive associations in GWAS.     

Molecular docking and dynamics simulations of A.niger RNase from Aspergillus niger ATCC26550: for potential prevention of human cancer

The aim of the present research was to study the anticancer effects of Aspergillus niger (A.niger) RNase. We found that RNase (A.niger RNase) significantly and dose dependently inhibited invasiveness of breast cancer cell line MDA MB 231 by 55 % (P?<?0.01) at 1 μM concentration. At a concentration of 2 μM, the anti invasive effect of the enzyme increased to 90 % (P?<?0.002). Keeping the aim to determine molecular level interactions (molecular simulations and protein docking) of human actin with A.niger RNase we extended our work in-vitro to in-silico studies. To gain better relaxation and accurate arrangement of atoms, refinement was done on the human actin and A.niger RNase by energy minimization (EM) and molecular dynamics (MD) simulations using 43A² force field of Gromacs96 implemented in the Gromacs 4.0.5 package, finally the interaction energies were calculated by protein-protein docking using the HEX. These in vitro and in-silico structural studies prove the effective inhibition of actin activity by A.niger RNase in neoplastic cells and thereby provide new insights for the development of novel anti cancer drugs.     

Insights from the molecular docking of withanolide derivatives to the target protein PknG from Mycobacterium tuberculosis

A crucial virulence factor for intracellular Mycobacterium tuberculosis survival is Protein kinase G (PknG), a eukaryotic-like serinethreonine protein kinase expressed by pathogenic mycobacteria that blocks the intracellular degradation of mycobacteria in lysosomes. Inhibition of PknG results in mycobacterial transfer to lysosomes. Withania somnifera, a reputed herb in ayurvedic medicine, comprises a large number of steroidal lactones known as withanolides which show various pharmacological activities. We describe the docking of 26 withanferin and 14 withanolides from Withania somnifera into the three dimensional structure of PknG of M. tuberculosis using GLIDE. The inhibitor binding positions and affinity were evaluated using scoring functions- Glidescore. The withanolide E, F and D and Withaferin - diacetate 2 phenoxy ethyl carbonate were identified as potential inhibitors of PknG. The available drug molecules and the ligand AX20017 showed hydrogen bond interaction with the aminoacid residues Glu233 and Val235. In addition to Val235 the other amino acids, Gly237, Gln238 and Ser239 are important for withanolide inhibitor recognition via hydrogen bonding mechanisms.     

Micro-cloning of an economic rattan palm Calamus thwaitesii for eco-restoration programme

Axillary shoots were induced from shoot tip of Calamus thwaitesii suckers on Murashige and Skoog medium supplemented with 0.4 mg/L N6-benzylaminopurine and 0.1 mg/L each of thidiazuron and α-naphthaleneacetic acid (NAA). The shoots initiated were subcultured to fresh media of the same composition for shoot multiplication and multiplied shoots were transferred to half strength MS hormone-free media for shoot elongation. The elongated shoots (～5cm) were then re-cultured to the media supplemented with 3.0 mg/L indole-3-butyric acid/4.0 mg/L NAA to raise plantlets which were subsequently analysed for genetic fidelity using inter simple sequence repeat markers. Out of 183 bands scored, 178 bands were monomorphic indicating 97.2% similarity. The observed low level of polymorphism between genotypes supports genetic consistency of these micro-clones that are likely to be genetically true to their parental origin. The clones thus obtained were hardened in the specially fabricated mist house at 29 ±2°C and 80±5% relative humidity for 3 months followed by shifting to green house for another 3 months of nursery establishment. The established plants when reintroduced to the selected forest segments of the Western Ghats, Kerala (India) showed 79.3% survival rate after 2 years of field transfer. The viable and highly reproducible in vitro cloning protocol demonstrated here for the first time can be used for the production of elite female clones for aforestation activities and sustained delivery of high quality raw materials to cane processing units for strengthening cane industry.     

Use of Xpert MTB/RIF in Decentralized Public Health Settings and Its Effect on Pulmonary TB and DR-TB Case Finding in India

BackgroundXpert MTB/RIF, the first automated molecular test for tuberculosis, is transforming the diagnostic landscape in high-burden settings. This study assessed the impact of up-front Xpert MTB/RIF testing on detection of pulmonary tuberculosis (PTB) and rifampicin-resistant PTB (DR-TB) cases in India.MethodsThis demonstration study was implemented in 18 sub-district level TB programme units (TUs) in India in diverse geographic and demographic settings covering a population of 8.8 million. A baseline phase in 14 TUs captured programmatic baseline data, and an intervention phase in 18 TUs had Xpert MTB/RIF offered to all presumptive TB patients. We estimated changes in detection of TB and DR-TB, the former using binomial regression models to adjust for clustering and covariates.ResultsIn the 14 study TUs, which participated in both phases, 10,675 and 70,556 presumptive TB patients were enrolled in the baseline and intervention phase, respectively, and 1,532 (14.4%) and 14,299 (20.3%) bacteriologically confirmed PTB cases were detected. The implementation of Xpert MTB/RIF was associated with increases in both notification rates of bacteriologically confirmed TB cases (adjusted incidence rate ratio [aIRR] 1.39; CI 1.18-1.64), and proportion of bacteriological confirmed TB cases among presumptive TB cases (adjusted risk ratio (aRR) 1.33; CI 1.6-1.52). Compared with the baseline strategy of selective drug-susceptibility testing only for PTB cases at high risk of drug-resistant TB, Xpert MTB/RIF implementation increased rifampicin resistant TB case detection by over fivefold. Among, 2765 rifampicin resistance cases detected, 1055 were retested with conventional drug susceptibility testing (DST). Positive predictive value (PPV) of rifampicin resistance detected by Xpert MTB/RIF was 94.7% (CI 91.3-98.1), in comparison to conventional DST.ConclusionIntroduction of Xpert MTB/RIF as initial diagnostic test for TB in public health facilities significantly increased case-notification rates of all bacteriologically confirmed TB by 39% and rifampicin-resistant TB case notification by fivefold.