The effect of pH on the production of cellulases in Aspergillus terreus

Summary The optimal pH for the production of extracellular cellulolytic enzymes in the wild strain of Aspergillus terreus was shown to be pH 5.0. After 160 h of cultivation, carboxymethyl cellulase reached 9.0 IU/ml, filterpaper, cellulase 0.5 IU/ml and -glucosidase 0.9 IU/ml. The rate of synthesis of CM- and FP-cellulases decreased after 90 h of cultivation but -glucosidase was produced linearly for 160 h. Some of the enzymes produced were released into the medium during the fungal growth while others remained bound. The binding of enzymes to cells and residual crystalline cellulose was strongly affected by the pH of the medium. FP-cellulase and particularly -glucosidase were bound more effectively, at lower pHs. Cold shock treatment of the cell suspension increased the activities of FP- and CM-cellulases but -glucosidase activity was not affected.     

Phospholipase Dδ assists to cortical microtubule recovery after salt stress

The dynamic microtubule cytoskeleton plays fundamental roles in the growth and development of plants including regulation of their responses to environmental stress. Plants exposed to hyper-osmotic stress commonly acclimate, acquiring tolerance to variable stress levels. The underlying cellular mechanisms are largely unknown. Here, we show, for the first time, by in vivo imaging approach that linear patterns of phospholipase Dδ match the localization of microtubules in various biological systems, validating previously predicted connection between phospholipase Dδ and microtubules. Both the microtubule and linear phospholipase Dδ structures were disintegrated in a few minutes after treatment with oryzalin or salt. Moreover, by using immunofluorescence confocal microscopy of the cells in the root elongation zone of Arabidopsis, we have shown that the cortical microtubules rapidly depolymerized within 30 min of treatment with 150 or 200 mM NaCl. Within 5 h of treatment, the density of microtubule arrays was partially restored. A T-DNA insertional mutant lacking phospholipase Dδ showed poor recovery of microtubule arrays following salt exposition. The restoration of microtubules was significantly retarded as well as the rate of root growth, but roots of overexpressor GFP-PLDδ prepared in our lab, have grown slightly better compared to wild-type plants. Our results indicate that phospholipase Dδ is involved in salt stress tolerance, possibly by direct anchoring and stabilization of de novo emerging microtubules to the plasma membrane, providing novel insight into common molecular mechanism during various stress events.     

Hyperbaric Oxygen Therapy Can Diminish Fibromyalgia Syndrome – Prospective Clinical Trial

BackgroundFibromyalgia Syndrome (FMS) is a persistent and debilitating disorder estimated to impair the quality of life of 2–4% of the population, with 9:1 female-to-male incidence ratio. FMS is an important representative example of central nervous system sensitization and is associated with abnormal brain activity. Key symptoms include chronic widespread pain, allodynia and diffuse tenderness, along with fatigue and sleep disturbance. The syndrome is still elusive and refractory. The goal of this study was to evaluate the effect of hyperbaric oxygen therapy (HBOT) on symptoms and brain activity in FMS.ConclusionsThe study provides evidence that HBOT can improve the symptoms and life quality of FMS patients. Moreover, it shows that HBOT can induce neuroplasticity and significantly rectify abnormal brain activity in pain related areas of FMS patients.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01827683","term_id":"NCT01827683"}}NCT01827683     

Serum calprotectin (S100A8/9): an independent predictor of ultrasound synovitis in patients with rheumatoid arthritis

IntroductionCalprotectin, a heterodimeric complex of S100A8/9 (MRP8/14), has been proposed as an important serum biomarker that reflects disease activity and structural joint damage in rheumatoid arthritis (RA). The objective of this cross-sectional study was to test the hypothesis that calprotectin is associated with clinical and ultrasound-determined disease activity in patients with RA.MethodsA total of 37 patients with RA (including 24 females, a mean disease duration of 20 months) underwent a clinical examination and 7-joint ultrasound score (German US-7) of the clinically dominant hand and foot to assess synovitis by grey-scale (GS) and synovial vascularity by power Doppler (PD) ultrasound using semiquantitative 0–3 grading. The levels of serum calprotectin and C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were determined at the time of the ultrasound assessment. We analysed the relationship between serum calprotectin level, traditional inflammatory markers, and ultrasound-determined synovitis.ResultsThe levels of serum calprotectin were significantly correlated with swollen joint count (r = 0.465, p < 0.005), DAS28-ESR (r = 0.430, p < 0.01), ESR (r = 0.370, p < 0.05) and, in particular, CRP (r = 0.629, p < 0.001). Calprotectin was significantly associated with GS (r = 0.359, p < 0.05) and PD synovitis scores (r = 0.497, p < 0.005). Using multivariate regression analysis, calprotectin, adjusted for age and sex, was a better predictor of PD synovitis score (R² = 0.765, p < 0.001) than CRP (R² = 0.496, p < 0.001).ConclusionsThe serum levels of calprotectin are significantly associated with clinical, laboratory and ultrasound assessments of RA disease activity. These results suggest that calprotectin might be superior to CRP for monitoring ultrasound-determined synovial inflammation in RA patients.     

The American brine shrimp as an exotic invasive species in the western Mediterranean

The hypersaline environments and salterns present in the western Mediterranean region (including Italy, southern France, the Iberian Peninsula and Morocco) contain autochthonous forms of the brine shrimp Artemia, with parthenogenetic diploid and tetraploid strains coexisting with the bisexual species A. salina. Introduced populations of the American brine shrimp A. franciscana have also been recorded in these Mediterranean environments since the 1980s. Based on brine shrimp cyst samples collected in these countries from 1980 until 2002, we were able to establish the present distribution of autochthonous brine shrimps and of A. franciscana, which is shown to be an expanding invasive species. The results obtained show that A. franciscana is now the dominant Artemia species in Portuguese salterns, along the French Mediterranean coast and in Cadiz bay (Spain). Co-occurrence of autochthonous (parthenogenetic) and American brine shrimp populations was observed in Morocco (Mar Chica) and France (Aigues Mortes), whereas A. franciscana was not found in Italian cyst samples. The results suggest these exotic A. franciscana populations originate as intentional or non-intentional inoculations through aquacultural (hatchery effluents) or pet market activities, and suggest that the native species can be rapidly replaced by the exotic species. This revised version was published online in July 2006 with corrections to the Cover Date.     

New triphosphate conjugates bearing reporter groups: labeling of DNA fragments for microarray analysis

A simple and convenient method for incorporation of fluorescent or ligand groups into 3'-termini of DNA fragments is proposed. A set of triphosphoric acid monoesters bearing fluorescent groups or biotin attached to the triphosphate fragment through linkers of different lengths and structures was synthesized. All the compounds were substrates for calf thymus terminal deoxynucleotidyltransferase and were used for incorporation of marker groups into 3'-termini of DNA fragments. The compounds were successfully applied for DNA labeling during post-PCR target preparation for microarray analysis.     

Preparation of a monolithic capillary column with immobilized alpha-mannose for affinity chromatography of lectins

A simple method for the preparation of an affinity monolithic (also called continuous bed) capillary column for alpha-mannose-specific lectins is described. 2-Hydroxyethyl methacrylate in combination with (+)-N,N -diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) as crosslinkers, were used as monomers for the monolith. After oxidation of DATD with periodate, alpha-mannose with spacer was bound to the aldehyde groups of the polymeric skeleton via reductive amination to form an affinity column for the separation, enrichment or binding studies of mannose-specific lectins. The permeability of the column was excellent. The porosity of the monolith was investigated by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC). The affinity of the monolith was evaluated by frontal analysis (FA) and fluorescence microscopy (FM) using fluorescently labeled concanavalin (Con A). Frontal affinity chromatography showed a specific interaction of two different lectins with the alpha-mannose-modified monolith. According to FM the affinity sites were evenly distributed over the monolithic bed.     

H2AX: functional roles and potential applications

Upon DNA double-strand break (DSB) induction in mammals, the histone H2A variant, H2AX, becomes rapidly phosphorylated at serine 139. This modified form, termed γ-H2AX, is easily identified with antibodies and serves as a sensitive indicator of DNA DSB formation. This review focuses on the potential clinical applications of γ-H2AX detection in cancer and in response to other cellular stresses. In addition, the role of H2AX in homeostasis and disease will be discussed. Recent work indicates that γ-H2AX detection may become a powerful tool for monitoring genotoxic events associated with cancer development and tumor progression.     

Comparative Structure-Function Analysis of Mannose-Specific FimH Adhesins from Klebsiella pneumoniae and Escherichia coli

FimH, the adhesive subunit of type 1 fimbriae expressed by many enterobacteria, mediates mannose-sensitive binding to target host cells. At the same time, fine receptor-structural specificities of FimH from different species can be substantially different, affecting bacterial tissue tropism and, as a result, the role of the particular fimbriae in pathogenesis. In this study, we compared functional properties of the FimH proteins from Escherichia coli and Klebsiella pneumoniae, which are both 279 amino acids in length but differ by some ∼15% of residues. We show that K. pneumoniae FimH is unable to mediate adhesion in a monomannose-specific manner via terminally exposed Manα(1-2) residues in N-linked oligosaccharides, which are the structural basis of the tropism of E. coli FimH for uroepithelial cells. However, K. pneumoniae FimH can bind to the terminally exposed Manα(1-3)Manβ(1-4)GlcNAcβ1 trisaccharide, though only in a shear-dependent manner, wherein the binding is marginal at low shear force but enhanced sevenfold under increased shear. A single mutation in the K. pneumoniae FimH, S62A, converts the mode of binding from shear dependent to shear independent. This mutation has occurred naturally in the course of endemic circulation of a nosocomial uropathogenic clone and is identical to a pathogenicity-adaptive mutation found in highly virulent uropathogenic strains of E. coli, in which it also eliminates the dependence of E. coli binding on shear. The shear-dependent binding properties of the K. pneumoniae and E. coli FimH proteins are mediated via an allosteric catch bond mechanism. Thus, despite differences in FimH structure and fine receptor specificity, the shear-dependent nature of FimH-mediated adhesion is highly conserved between bacterial species, supporting its remarkable physiological significance.The most common type of adhesive organelle in the Enterobacteriaceae is the type 1 fimbria, which has been most extensively studied in Escherichia coli. The corresponding structures of Klebsiella pneumoniae are similar to those of E. coli with regard to genetic composition and regulation (15). Type 1 fimbriae are composed primarily of the structural subunit FimA, with minor amounts of three ancillary subunits, FimF, FimG, and the mannose-specific adhesin FimH. The FimH adhesin is an allosteric protein that mediates the catch bond mechanism of adhesion where the binding is increased under increased shear stress (48).It has been demonstrated in E. coli that FimH has two domains, the mannose-binding lectin domain (from amino acid [aa] 1 through 156) and the fimbria-incorporating pilin domain (from aa 160 through 279), connected via a 3-aa-long linker chain (6). A mannose-binding site is located at the top of the lectin domain, at the opposite end from the interdomain linker (17).Several studies have demonstrated that type 1 fimbriae play an important role in E. coli urinary tract infection (UTI) (7, 21, 23, 35). In addition, in urinary E. coli isolates, the FimH adhesin accumulates amino acid replacements which increase tropism for the uroepithelium and various components of basement membranes (21, 30, 35, 37, 49). Most of the replacements increase the monomannose binding capability of FimH under low shear, by altering allosteric catch bond properties of the protein (48). The mutated FimH variants were shown to provide an advantage in colonization of the urinary tract in the mouse model (35) and correlate with the overall extraintestinal virulence of E. coli (16). Thus, FimH mutations are pathoadaptive in nature.Klebsiella pneumoniae is recognized as an important opportunistic pathogen frequently causing UTIs, septicemia, or pneumonia in immunocompromised individuals (29). It is responsible for up to 10% of all nosocomial bacterial infections (18, 41). K. pneumoniae is ubiquitous in nature, and it has been shown that environmental isolates are phenotypically indistinguishable from clinical isolates (22, 26, 27, 29, 33). Furthermore, it has been demonstrated that environmental isolates of K. pneumoniae are as virulent as clinical isolates (28, 45).K. pneumoniae possesses a number of known virulence factors, including a pronounced capsule, type 3 fimbriae, and type 1 fimbriae (29, 44). Type 1 fimbriae produced by K. pneumoniae are described as functionally and structurally similar to type 1 fimbriae from E. coli (25) and have been shown to play a significant role in K. pneumoniae UTI (32, 43).We have previously shown that mature FimH from 54 isolates of K. pneumoniae (isolated from urine, blood, liver, and the environment) is represented by seven protein variants due to point amino acid replacements. (42) When K. pneumoniae FimH was aligned with the FimH of E. coli, they showed ∼85% similarity at the amino acid level. Furthermore, a majority (14 out of 21 isolates) of the K. pneumoniae strains isolated from patients with UTI grouped into a single clonal group based on multilocus sequence typing, but fimH in one isolate in the group differed from the others by a single nucleotide mutation resulting in an amino acid change, serine to alanine, in position 62 (42). The same mutation has been found in FimH of a highly uropathogenic clone of E. coli and significantly increases the adhesin''s ability to adhere to monomannose under low or no shear (19, 39, 50).In this study, we describe the extent and pattern of structural variability of the FimH protein from K. pneumoniae and perform comparative analyses of the functional properties of FimH from both K. pneumonae and E. coli.