Phagocytosis and phagosome maturation are regulated by calcium in J774 macrophages interacting with unopsonized prey

Phagocytosis by neutrophils, macrophages, and other professional phagocytes requires rapid remodeling of actin. Early phagosomes are surrounded by a rim of F-actin that is disassembled during phagosomoal maturation. Breakdown of periphagosomal F-actin and phagolysosome fusion are calcium dependent processes in neutrophils interacting with serum-opsonized prey, but appears to be calcium independent in macrophages interacting with serum- or IgG-opsonized prey. In the present study, we found that calcium was necessary for phagocytosis, breakdown of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey. We also observed that lipophosphoglycan (LPG) from Leishmania donovani promastigotes required calcium to exert its inhibitory effect on macrophage phagocytosis and periphagosomal F-actin breakdown. We conclude that calcium is essential for phagocytosis, depolymerization of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey, as well as for proper functioning of LPG.     

MSCs—cells with many sides

The field of mesenchymal stromal cell (MSC) biology and clinical cellular therapy has grown exponentially over the last few decades. With discovery of multiple tissue specific sources of stromal cells, invariably being termed MSCs, and their increasing clinical application, there is a need to further delineate the true definition of a mesenchymal stromal cell and to recognise the inherit differences between cell sources; both their potential and limitations.In this review, we discuss the importance of considering every stromal cell source as an independent entity and the need to critically evaluate and appreciate the true phenotype of these cells and their safety when considering their use in novel cell therapies.     

Prevalence and risk factors of vertebral compression fractures in female SLE patients

Introduction

The present study objective was to evaluate the incidence of methotrexate (MTX)-specific liver lesions from the analysis of a liver biopsy of inflammatory arthritis patients with elevated liver enzymes.

Methods

A case-control study was performed with 1,571 arthritis patients on long-term low-dose MTX therapy. Results of liver biopsy were analyzed in 41 patients with elevated liver enzymes. The expression of autoimmune markers was also assessed. This population was compared with 41 disease control subjects obtained from the same database, also on MTX but without elevated liver enzymes, matched for age, sex and rheumatic disease.

Results

Compared with the disease controls, patients with liver biopsy showed lower disease duration and lower MTX exposure, weekly and cumulative doses, reflecting shorter treatment duration due to liver abnormalities. Liver biopsies showed 17 autoimmune hepatitis-like (AIH-like) lesions, 13 nonalcoholic steatohepatitis-like lesions, seven limited liver lesions, and two primary biliary cirrhoses. However, MTX-specific lesions with dystrophic nuclei in hepatocytes were seen in only two cases. Liver biopsy lesions were associated with autoimmune markers (P = 0.007); notably, AIH-like lesions were associated with rheumatoid arthritis and with the presence of the HLA-DR shared epitope.

Conclusions

MTX-specific liver lesions are rarely observed in arthritis patients under long-term MTX therapy and elevated liver enzymes.     

Bme585 I [5'-CCCGC(4/6)-3'], a new isoschizomer of restriction endonuclease Fau I, isolated from a strain of Bacillus mesentericus

Bme585 I is a new member of the restriction endonuclease type IIS family. It was partially purified from the heterothrophic, mesophilic bacterial strain Bacillus mesentericus 585 by ammonium sulphate precipitation and phosphocellulose column chromatography. Bme585 I is a monomeric protein with a molecular mass of 62 kD. The enzyme is active over a broad pH range from 7.0 to 8.8, has a temperature optimum of 37 degrees C and tolerance of NaCl in reaction buffer from 0 to 400 mM. Bme585 I recognizes the asymmetric sequence 5'-CCCGC(4/6)-3' and is therefore an isoschizomer of restriction endonuclease Fau I.     

Novel Systems for Dynamically Assessing Insulin Action in Live Cells Reveals Heterogeneity in the Insulin Response

Regulated GLUT4 trafficking is a key action of insulin. Quantitative stepwise analysis of this process provides a powerful tool for pinpointing regulatory nodes that contribute to insulin regulation and insulin resistance. We describe a novel GLUT4 construct and workflow for the streamlined dissection of GLUT4 trafficking; from simple high throughput screens to high resolution analyses of individual vesicles. We reveal single cell heterogeneity in insulin action highlighting the utility of this approach – each cell displayed a unique and highly reproducible insulin response, implying that each cell is hard‐wired to produce a specific output in response to a given stimulus. These data highlight that the response of a cell population to insulin is underpinned by extensive heterogeneity at the single cell level. This heterogeneity is pre‐programmed within each cell and is not the result of intracellular stochastic events.     

Rapid genotyping of the osteoporosis-associated polymorphic transcription factor Sp1 binding site in the COL1A1 gene by pyrosequencing

We describe a novel polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) sequencingbased assay for rapid genotyping of the polymorphic Sp1 binding site in the COL1A1 gene (1). A single nucleotide G-->T substitution polymorphism at this GC-rich site has recently been reported to be a predictive genetic marker for low bone mineral density (BMD). To simplify screening for this marker, we optimized PCR conditions and subjected the amplicons to pyrosequencing, which is a convenient high-throughput sequence analysis technique, readily amenable to automation. The analysis of 200 deidentified convenience DNA samples extracted from blood revealed genotype frequences in Hardy-Weinberg equilibrium (SS 68.0%, Ss 28.5%, and ss 3.5%) in agreement with other studies of European populations. This study demonstrates for the first time that pyrosequencing can be used for rapid identification of the osteoporosis-associated single nucleotide polymorphism (SNP) in the COL1A1 gene.     

Distribution and antifungal susceptibility of yeasts isolates from intensive care unit patients

Yeasts frequently colonize non-sterile sites in the body. The aim of the study was to determine distribution in clinical samples and antifungal susceptibility to five antifungals. From January 2013 through June 2015, 800 isolates were obtained from intensive care unit patients. Candida albicans (58.9%), Candida glabrata (20.4%), Candida krusei (8.6%), and Candida parapsilosis (3.6%) were the leading species. Majority of the C. albicans isolates were susceptible to the fluconazole. Elevated voriconazole minimal inhibitory concentrations (MICs) were observed in isolates exhibiting high fluconazole MICs, most frequently in C. glabrata. Isolates with echinocandins MICs suggesting reduced susceptibility were only sporadic cases with the exception of Trichosporon spp. The amphotericin B MICs were slightly higher for some C. krusei.     

Synthesis of novel potent hepatitis C virus NS3 protease inhibitors: discovery of 4-hydroxy-cyclopent-2-ene-1,2-dicarboxylic acid as a N-acyl-L-hydroxyproline bioisostere

Potent tetrapeptidic inhibitors of the HCV NS3 protease have been developed incorporating 4-hydroxy-cyclopent-2-ene-1,2-dicarboxylic acid as a new N-acyl-l-hydroxyproline mimic. The hydroxycyclopentene template was synthesized in eight steps from commercially available (syn)-tetrahydrophthalic anhydride. Three different amino acids were explored in the P1-position and in the P2-position the hydroxyl group of the cyclopentene template was substituted with 7-methoxy-2-phenyl-quinolin-4-ol. The P3/P4-positions were then optimized from a set of six amino acid derivatives. All inhibitors were evaluated in an in vitro assay using the full-length NS3 protease. Several potent inhibitors were identified, the most promising exhibiting a K(i) value of 1.1nM.     

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene.