A(1) adenosine receptors in human neutrophils: direct binding and electron microscope visualization.

By occupying specific surface receptors, adenosine and adenosine analogues modulate neutrophil functions; in particular, functional and biochemical studies have shown that A(1) adenosine receptors modulate chemotaxis in response to chemotactic peptides. Until now, the characteristics of the specific agonist binding and the visualization of A(1) receptors in human neutrophils have not been investigated. In the present study, we used the agonist [(3)H] CHA for radioligand binding studies and a CHA-biotin XX probe in order to visualize the A(1) binding sites in human neutrophils, ultrastructurally, by conjugation with colloidal gold-streptavidin. [(3)H] CHA bound A(1) adenosine receptors with selectivity and specificity, although with a low binding capacity. Scatchard analysis showed a Kd value of 1.4 +/- 0.08 nM and a maximum density of binding sites of 7.1 +/- 0.37 fmol/mg of proteins. The good affinity and selectivity of the CHA-biotin XX probe for A(1) adenosine receptors allowed us to visualize them, after conjugation with colloidal gold-streptavidin, as electron-dense gold particles on the neutrophil surface and inside the cell. The internalization of the ligand-receptor complex was followed in a controlled temperature system, and occurred through a receptor-mediated pathway. The kinetics of the intracellular trafficking was fast, taking less than 5 min. These data suggest that the CHA-biotin XX-streptavidin-gold complex is a useful marker for the specific labelling of A(1) binding sites and to follow the intracellular trafficking of these receptors.     

Serum Level of Zinc and Copper in Sudanese Women with Polycystic Ovarian Syndrome

The paper’s objective was to estimate weekly Hg intake from fish meals based on intervention research. Total Hg (THg) concentrations in blood and hair samples collected from men (n = 67) from an intervention study as well as muscular tissues of fresh and after heat-treating fish were determined using the thermal decomposition amalgamation atomic absorption spectrometry method (TDA-AAS) using direct mercury analyzer (DMA-80). The mean of the estimated weekly intake (EWI) was estimated at 0.62 μg/kg bw/week in the range 0.36–0.96 μg/kg body weight (bw) /week through the consumption of 4 edible marine fish species every day (for 10 days) by the participants from the intervention research in Lodz, Poland. The Hg intake in the volunteers in our intervention study accounted for 38.6% of the provisional tolerable weekly intake (PTWI) (1.6 μg/kg bw, weekly) value. The average Hg concentration in the analyzed fish ranged from 0.018 ± 0.006 mg/kg wet weight (Gadus chalcogrammus) to 0.105 ± 0.015 mg/kg wet weight (Macruronus magellanicus). The results for the average consumers were within PTWI of methylmercury (MeHg). Moreover, the average concentration of Hg in the selected fish after heat treatment did not exceed the maximum permitted concentrations for MeHg (MPCs = 0.5 mg/kg wet weight) in food set by the European Commission Regulation (EC/1881/2006). Hence, the risk of adverse effects of MeHg for the participants is substantially low.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

In vitro methods for antifungal susceptibility testing of Trichophyton spp.

In general, methods to test the susceptibility of fungi to antifungal drugs require standardized techniques, but so far there is no methodology that is widely applicable to dermatophytes. Here we introduced modifications to the protocols from documents of the National Committee for Clinical Laboratory Standards (CLSI) M38-A and the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) that are usually applied to moulds and fermentative yeasts, in order to adjust the conditions for the growth of dermatophytes. The modifications included: growth on potato dextrose agar supplemented with 2 % in-house rice flour to encourage sporulation, the addition of 2 % glucose to the culture media (RPMI-1640), and an incubation temperature of 28 °C. In addition, the incubation period was 7 d, the minimum inhibitory concentration (MIC) was defined as 80 % growth inhibition endpoints for azole agents, and the inocula only contained microconidia. Results obtained by both tested methodologies were very similar to the ones reported by other researchers. MIC₉₀ (MIC at which 90% of isolates tested were inhibited) values were identical for four out of five antifungal drugs tested and there was only a difference of one or two dilutions when MIC₅₀ values were compared. Although the modifications introduced did not interfere with the results, more studies are necessary to establish a standard technique to test susceptibility of dermatophytes to antifungal drugs.     

Lifestyle factors and quality of life among primary health care physicians in Madinah,Saudi Arabia

BackgroundPhysicians are considered to be a high-risk population for a poor quality of life (QoL), but few studies of lifestyle factors include the QoL among them.ObjectivesThis study aimed to investigate the relationship between lifestyle factors and a positive QoL among primary health care (PHC) physicians.MethodsA cross-sectional study was conducted at 20 primary healthcare centers in Madinah, Saudi Arabia. A self-administered questionnaire was used, including sociodemographic characteristics, lifestyle data, and the short version of the World Health Organization Quality of Life questionnaire. Appropriate statistical analyses were used, including multivariate logistic regression models.ResultsThe response rate was 85.7% (72/84) physicians. The mean score of the total QoL and its four studied domains (physical, psychological, social, and environmental) was relatively high, with no statistically significant difference between the consultants and general practitioners. The positive total QoL in this study was significantly lower among physicians with obesity (OR = 0.55, 95%CI = 0.25–0.97), those using butter and animal fat for cooking (OR = 0.10, 95%CI = 0.02–0.81), and those eating meals out > 3 times per week (OR = 0.30, 95%CI = 0.10–0.90). Although non-significant, vegetable consumption and a high level of physical activity were associated with a positive QoL, with adjusted ORs of 2.5 (95%CI = 0.82–7.58) and 1.5 (95%CI = 0.33–6.65), respectively.ConclusionThe findings indicate a relatively good QoL among the participating physicians; however, a lower QoL was associated with unhealthy lifestyle factors. QoL was significantly associated with obesity, cooking practices, and eating meals from restaurants.     

Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability

Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.     

CC and CXC chemokines induce airway smooth muscle proliferation and survival

The increase in airway smooth muscle (ASM) mass is a major structural change in asthma. This increase has been attributed to ASM cell (ASMC) hyperplasia and hypertrophy. The distance between ASMC and the epithelium is reduced, suggesting migration of smooth muscle cells toward the epithelium. Recent studies have suggested a role of chemokines in ASMC migration toward the epithelium; however, chemokines have other biological effects. The objective of the current study is to test the hypothesis that chemokines (eotaxin, RANTES, IL-8, and MIP-1α) can directly influence ASMC mass by increasing the rate of proliferation or enhancing the survival of these cells. Human ASMCs were exposed to different concentrations of eotaxin, RANTES, IL-8, or MIP-1α. To test for proliferation, matched control and stimulated ASMC were pulsed with [(3)H]thymidine, or ASMCs were stained with BrdU and then analyzed with flow cytometry. Apoptosis was measured using Annexin V staining and flow cytometry. Expression of phosphorylated p42/p44 and MAPKs was assessed by Western blot. In a concentration-dependent manner, chemokines including eotaxin, RANTES, IL-8, and MIP-1α increased ASMC's [(3)H]thymidine incorporation and DNA synthesis. IL-8, eotaxin, and MIP-1α decreased the rate of apoptosis of ASMCs compared with the matched controls. A significant increase in phosphorylated p42/p44 MAPKs was seen after treating ASMCs with RANTES and eotaxin. Moreover, inhibition of p42/p44 MAPK phosphorylation reduced the level of chemokine-induced ASM proliferation. We conclude that chemokines might contribute to airway remodeling seen in asthma by enhancing the number and survival of ASMCs.     

Integrated metagenomic and metaproteomic analyses of marine biofilm communities

Metagenomic and metaproteomic analyses were utilized to determine the composition and function of complex air–water interface biofilms sampled from the hulls of two US Navy destroyers. Prokaryotic community analyses using PhyloChip-based 16S rDNA profiling revealed two significantly different and taxonomically rich biofilm communities (6,942 taxa) in which the majority of unique taxa were ascribed to members of the Gammaproteobacteria, Alphaproteobacteria and Clostridia. Although metagenomic sequencing indicated that both biofilms were dominated by prokaryotic sequence reads (> 91%) with the majority of the bacterial reads belonging to the Alphaproteobacteria, the Ship-1 metagenome harbored greater organismal and functional diversity and was comparatively enriched for sequences from Cyanobacteria, Bacteroidetes and macroscopic eukaryotes, whereas the Ship-2 metagenome was enriched for sequences from Proteobacteria and microscopic photosynthetic eukaryotes. Qualitative liquid chromatography-tandem mass spectrometry metaproteome analyses identified 678 unique proteins, revealed little overlap in species and protein composition between the ships and contrasted with the metagenomic data in that ~80% of classified and annotated proteins were of eukaryotic origin and dominated by members of the Bacillariophyta, Cnidaria, Chordata and Arthropoda (data deposited to the ProteomeXchange, identifier PXD000961). Within the shared metaproteome, quantitative ¹⁸O and iTRAQ analyses demonstrated a significantly greater abundance of structural proteins from macroscopic eukaryotes on Ship-1 and diatom photosynthesis proteins on Ship-2. Photosynthetic pigment composition and elemental analyses confirmed that both biofilms were dominated by phototrophic processes. These data begin to provide a better understanding of the complex organismal and biomolecular composition of marine biofilms while highlighting caveats in the interpretation of stand-alone environmental ‘-omics’ datasets.