A Structural View of Negative Regulation of the Toll-like Receptor-Mediated Inflammatory Pathway

Even though the Toll-like receptor (TLR) pathway is integral to inflammatory defense mechanisms, its excessive signaling may be devastating. Cells have acquired a cascade of strategies to regulate TLR signaling by targeting protein-protein interactions, or ubiquitin chains, but the details of the inhibition mechanisms are still unclear. Here, we provide the structural basis for the regulation of TLR signaling by constructing architectures of protein-protein interactions. Structural data suggest that 1) Toll/IL-1R (TIR) domain-containing regulators (BCAP, SIGIRR, and ST2) interfere with TIR domain signalosome formation; 2) major deubiquitinases such as A20, CYLD, and DUBA prevent association of TRAF6 and TRAF3 with their partners, in addition to removing K63-linked ubiquitin chains that serve as a docking platform for downstream effectors; 3) alternative downstream pathways of TLRs also restrict signaling by competing to bind common partners through shared binding sites. We also performed in silico mutagenesis analysis to characterize the effects of oncogenic mutations on the negative regulators and to observe the cellular outcome (whether there is/is not inflammation). Missense mutations that fall on interfaces and nonsense/frameshift mutations that result in truncated negative regulators disrupt the interactions with the targets, thereby enabling constitutive activation of the nuclear factor-kappa B, and contributing to chronic inflammation, autoimmune diseases, and oncogenesis.     

Increasing proteome coverage with offline RP HPLC coupled to online RP nanoLC-MS

Fractionation prior to mass spectrometry is an indispensable step in proteomics. In this paper we report the success of performing offline reversed phase high pressure liquid chromatography (HPLC) fractionation on a C18 2.0 mm×150 mm column at the peptide level with microliter per minute flow rates prior to online nano-flow reversed phase liquid chromatography mass spectrometry (nanoLC-MS) using the well-studied fungus Saccharomyces cerevisiae. A C18 75 μm×150 mm column was used online and the online elution gradients for each fraction were adjusted in order to obtain well resolved separation. Comparing this method directly to only performing nanoLC-MS we observed a 61.6% increase in the number of identified proteins. At a 1% false discovery rate 1028 proteins were identified using two dimensions of RPLC versus 636 proteins identified in a single nano-flow separation. The majority of proteins identified by one dimension of nano-LC were present in the proteins identified in our two dimensional strategy. Although increasing analysis time, this non-orthogonal and facile pre-fractionation method affords a more comprehensive examination of the proteome.     

Influence of pH and temperature on soluble substrate generation with primary sludge fermentation

The study was undertaken to evaluate the effect of pH and temperature control on the generation of soluble fermentation products from primary sludge. The effect was tested by running parallel experiments under pH and temperature controlled and uncontrolled conditions. In fermentation experiments conducted at 20 degrees C without pH control, the average soluble COD release was 14 mg per liter of wastewater treated, representing a potential increase of 5% in the biodegradable COD content of the primary sedimentation effluent. The corresponding average VFA generation was 9.2mg COD l(-1). The nutrient release was practically negligible and stayed at 0.4 mg l(-1) for nitrogen and 0.1mg l(-1) for phosphorus. Acetic acid accounted more than 45% of the generated VFA in all experimental runs. The acetic acid content of the VFA decreased with increasing initial VSS concentrations and higher pH levels. VFA generation by fermentation was significantly affected with temperature and pH control. Temperature change between 10 and 24 degrees C induced a five-fold increase in VFA generation, from 610 mg l(-1) at 10 degrees C to 2950 mg l(-1) at 24 degrees C.     

A novel cry2Ab gene from the indigenous isolate Bacillus thuringiensis subsp. kurstaki

A novel cry2Ab gene was cloned and sequenced from the indigenous isolate of Bacillus thuringiensis subsp. kurstaki. This gene was designated as cry2Ab25 and its sequence revealed an open reading frame of 1,902 bp encoding a 633 aa protein with calculated molecular mass of 70 kDa and pI value of 8.98. The amino acid sequence of the Cry2Ab25 protein was compared with previously known Cry2Ab toxins, and the phylogenetic relationships among them were determined. The deduced amino acid sequence of the Cry2Ab25 protein showed 99% homology to the known Cry2Ab proteins, except for Cry2Ab10 and Cry2Ab12 with 97% homology, and a variation in one amino acid residue in comparison with all known Cry2Ab proteins. The cry2Ab25 gene was expressed in Escherichia coli BL21(DE3) cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Cry2Ab25 protein is about 70 kDa. The toxin expressed in BL21(DE3) exhibited high toxicity against Malacosoma neustria and Rhagoletis cerasi with 73% and 75% mortality after 5 days of treatment, respectively.     

Immobilization of apricot pectinesterase (Prunus armeniaca L.) on porous glass beads and its characterization

Pectinesterase isolated from Malatya apricot pulp was covalently immobilized onto glutaraldehyde-containing amino group functionalized porous glass beads surface by chemical immobilization at pH 8.0. The amount of covalently bound apricot PE was found 1.721 mg/g glass support. The properties of immobilized enzyme were investigated and compared to those of free enzyme. The effect of various parameters such as pH, temperature, activation energy, heat and storage stability on immobilized enzyme were investigated. Optimum pH and temperature were determined to be 8.0 and 50 °C, respectively. The immobilized PE exhibited better thermostability than the free one. Kinetic parameters of the immobilized enzyme (K_m and V_max values) were also evaluated. The K_m was 0.71 mM and the V_max was 0.64 μmol min^?1 mg^?1. No drastic change was observed in the K_m and V_max values. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. Thermal and storage stability experiments were also carried out. It was observed that the immobilized enzyme had longer storage stability and retained 50% of its initial activity during 30 days.     

Incidence, risk factors and mortality of nosocomial pneumonia in Intensive Care Units: A prospective study

Background

Increasing antimicrobial resistance among the key pathogens responsible for community-acquired respiratory tract infections has the potential to limit the effectiveness of antibiotics available to treat these infections. Since there are regional differences in the susceptibility patterns observed and treatment is frequently empirical, the selection of antibiotic therapy may be challenging. PROTEKT, a global, longitudinal multicentre surveillance study, tracks the activity of telithromycin and comparator antibacterial agents against key respiratory tract pathogens.

Methods

In this analysis, we examine the prevalence of antibacterial resistance in 1,336 bacterial pathogens, isolated from adult and paediatric patients clinically diagnosed with acute bacterial sinusitis (ABS).

Results and discussion

In total, 58.0%, 66.1%, and 55.8% of S. pneumoniae isolates were susceptible to penicillin, cefuroxime, and clarithromycin respectively. Combined macrolide resistance and reduced susceptibility to penicillin was present in 200/640 (31.3 %) of S. pneumoniae isolates (128 isolates were resistant to penicillin [MIC >= 2 mg/L], 72 intermediate [MIC 0.12–1 mg/L]) while 99.5% and 95.5% of isolates were susceptible to telithromycin and amoxicillin-clavulanate, respectively. In total, 88.2%, 87.5%, 99.4%, 100%, and 100% of H. influenzae isolates were susceptible to ampicillin, clarithromycin, cefuroxime, telithromycin, and amoxicillin-clavulanate, respectively. In vitro, telithromycin demonstrated the highest activity against M. catarrhalis (MIC₅₀ = 0.06 mg/L, MIC₉₀ = 0.12 mg/L).

Conclusion

The high in vitro activity of against pathogens commonly isolated in ABS, together with a once daily dosing regimen and clinical efficacy with 5-day course of therapy, suggest that telithromycin may play a role in the empiric treatment of ABS.     

Enrichment of lignocellulose-degrading microbial communities from natural and engineered methanogenic environments

The aim of this study was to develop an effective bioaugmentation concept for anaerobic digesters treating lignocellulosic biomass such as straw. For that purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from cow and goat rumen fluid as well as from a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate). The bacterial communities of the enriched cultures and the different inocula were examined by 454 amplicon sequencing of 16S rRNA genes while the methanogenic archaeal communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the mcrA gene. Bacteroidetes was the most abundant phylum in all samples. Within the Bacteroidetes phylum, Bacteroidaceae was the most abundant family in the rumen-derived enrichment cultures, whereas Porphyromonadaceae was the predominant one in the reactor-derived culture. Additionally, the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure, but still the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures. The bioaugmentation potential of the enriched methanogenic cultures will be evaluated in further studies.

     

Volatile Oil Yield and Composition,Total Phenolic Content,Antioxidant Activity and Secondary Metabolite Content of Collected Thymus praecox Species in Rize

In this study, the volatile oil yield (Clevenger), volatile oil (VO) composition (Gas Chromatography), phenolic contents (UV-VIS Spectrophotometer), antioxidant activities (UV-VIS Spectrophotometer) and secondary metabolite content (High Pressure Liquid Chromatography) of 11 Thymus praecox subspecies were evaluated. The most detected chemical class were oxygenated monoterpenes (55.18–86.1 %) in investigated samples. In the present study rosmarinic acid, isoquercitrin, gallocatechin and thymol could be detected in high amounts. The min. and max. content values of Flora/Field Samples were 1543.241 and 890.3-1425.3 for rosmarinic acid, 139.44-287.894 and 129.9-312.2 for thymol, 38.619-121.424 and 26.3-112.9 for gallocatechin as mg/g DW. Principal Component Analysis was used to differentiate Thymus praecox species regarding volatile oil composition and secondary metabolite content. The results demonstrated that T. praecox collected from the Rize flora and cultivated afterwards showed variability based on investigated characteristics. Finally, the Thymus praecox samples displaying high bioactive compounds present useful information for further investigations and applications.