TLR2 signaling is critical for Mycoplasma pneumoniae-induced airway mucin expression

Excessive airway mucin production contributes to airway obstruction in lung diseases such as asthma and chronic obstructive pulmonary disease. Respiratory infections, such as atypical bacterium Mycoplasma pneumoniae (Mp), have been proposed to worsen asthma and chronic obstructive pulmonary disease in part through increasing mucin. However, the molecular mechanisms involved in infection-induced airway mucin overexpression remain to be determined. TLRs have been recently shown to be a critical component in host innate immune response to infections. TLR2 signaling has been proposed to be involved in inflammatory cell activation by mycoplasma-derived lipoproteins. In this study, we show that TLR2 signaling is critical in Mp-induced airway mucin expression in mice and human lung epithelial cells. Respiratory Mp infection in BALB/c mice activated TLR2 signaling and increased airway mucin. A TLR2-neutralizing Ab significantly reduced mucin expression in Mp-infected BALB/c mice. Furthermore, Mp-induced airway mucin was abolished in TLR2 gene-deficient C57BL/6 mice. Additionally, Mp was shown to increase human lung A549 epithelial cell mucin expression, which was inhibited by the overexpression of a human TLR2 dominant-negative mutant. These results clearly demonstrate that respiratory Mp infection increases airway mucin expression, which is dependent on the activation of TLR2 signaling.     

A wide host-range metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase

Using DNA obtained from the metagenome of an anaerobic digestor in a waste water treatment plant, we constructed a gene library cloned in the wide host-range cosmid pLAFR3. One cosmid enabled Rhizobium leguminosarum to grow on ethanol as sole carbon and energy source, this being due to the presence of a gene, termed adhEMeta. The AdhEMeta protein most closely resembles the AdhE alcohol dehydrogenase of Clostridium acetobutylicum, where it catalyses the formation of ethanol and butanol in a two-step reductive process. However, cloned adhEMeta did not confer ethanol utilization ability to Escherichia coli or to Pseudomonas aeruginosa, even though it was transcribed in both these hosts. Further, cell-free extracts of E. coli and R. leguminosarum containing cloned adhEMeta had butanol and ethanol dehydrogenase activities when assayed in vitro. In contrast to the well-studied AdhE proteins of C. acetobutylicum and E. coli, the enzyme specified by adhEMeta is not inactivated by oxygen and it enables alcohol to be catabolized. Cloned adhEMeta did, however, confer one phenotype to E. coli. AdhE- mutants of E. coli fail to ferment glucose and introduction of adhEMeta restored the growth of such mutants when grown under fermentative conditions. These observations show that the use of wide host-range vectors enhances the efficacy with which metagenomic libraries can be screened for genes that confer novel functions.     

A high-throughput turbidometric assay for screening inhibitors of protein disulfide isomerase activity

Protein disulfide isomerase (PDI) plays a key role in protein folding by catalyzing rearrangements of disulfide bonds in substrate proteins following their synthesis in eukaryotic cells. Besides its major role in the processing and maturation of secretory proteins in the endoplasmic reticulum, this enzyme and its homologs have been implicated in multiple important cellular processes; however, they have not served as targets for the development of therapeutic agents. The authors developed a high-throughput screening assay for PDI and its homologous enzymes in 384-well microplates. The method is based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol and measures the aggregation of reduced insulin chains at 650 nm. This kinetic assay was converted to an end-point assay by using hydrogen peroxide as a stop reagent. The feasibility of this high-throughput assay for screening chemical libraries was demonstrated in a pilot screen. The authors show that this homogenous turbidometric assay is robust and cost-effective and can be applied to identify PDI inhibitors from chemical libraries, opening this class of enzymes for therapeutic exploration.     

Comparison of techniques for the measurement of skin temperature during exercise in a hot,humid environment

Exercising or working in a hot, humid environment can results in the onset of heat-related illness when an individual''s temperature is not carefully monitored. The purpose of the present study was to compare three techniques (data loggers, thermal imaging, and wired electrodes) for the measurement of peripheral (bicep) and central (abdominal) skin temperature. Young men and women (N = 30) were recruited to complete the present study. The three skin temperature measurements were made at 0 and every 10-min during 40-min (60% VO₂max) of cycling in a hot (39±2°C), humid (45±5% RH) environment. Data was statistically analyzed using the Bland-Altman method and correlation analysis. For abdominal skin temperature, the Bland-Altman limits of agreement indicated that data loggers (1.5) were a better index of wired than was thermal imaging (3.5), For the bicep skin temperature the limits of agreement was similar between data loggers (1.9) and thermal (1.9), suggesting the both were suitable measurements. We also found that when skin temperature exceeded 35°C, we observed progressively better prediction between data loggers, thermal imaging, and wired skin sensors. This report describes the potential for the use of data loggers and thermal imaging to be used as alternative measures of skin temperature in exercising, human subjects.     

Anatomical Abnormalities in Gray and White Matter of the Cortical Surface in Persons with Schizophrenia

Background

Although schizophrenia has been associated with abnormalities in brain anatomy, imaging studies have not fully determined the nature and relative contributions of gray matter (GM) and white matter (WM) disturbances underlying these findings. We sought to determine the pattern and distribution of these GM and WM abnormalities. Furthermore, we aimed to clarify the contribution of abnormalities in cortical thickness and cortical surface area to the reduced GM volumes reported in schizophrenia.

Methods

We recruited 76 persons with schizophrenia and 57 healthy controls from the community and obtained measures of cortical and WM surface areas, of local volumes along the brain and WM surfaces, and of cortical thickness.

Results

We detected reduced local volumes in patients along corresponding locations of the brain and WM surfaces in addition to bilateral greater thickness of perisylvian cortices and thinner cortex in the superior frontal and cingulate gyri. Total cortical and WM surface areas were reduced. Patients with worse performance on the serial-position task, a measure of working memory, had a higher burden of WM abnormalities.

Conclusions

Reduced local volumes along the surface of the brain mirrored the locations of abnormalities along the surface of the underlying WM, rather than of abnormalities of cortical thickness. Moreover, anatomical features of white matter, but not cortical thickness, correlated with measures of working memory. We propose that reductions in WM and smaller total cortical surface area could be central anatomical abnormalities in schizophrenia, driving, at least partially, the reduced regional GM volumes often observed in this illness.     

The Association of Maximum Body Weight on the Development of Type 2 Diabetes and Microvascular Complications: MAXWEL Study

Background

Obesity precedes the development of type 2 diabetes (T2D). However, the relationship between the magnitude and rate of weight gain to T2D development and complications, especially in non-White populations, has received less attention.

Methods and Findings

We determined the association of rate and magnitude of weight gain to age at T2D diagnosis (Age_T2D), HbA1c at T2D diagnosis (HbA1c_T2D), microalbuminuria, and diabetic retinopathy after adjusting for sex, BMI at age 20 years, lifestyles, family history of T2D and/or blood pressure and lipids in 2164 Korean subjects aged ≥30 years and newly diagnosed with diabetes. Body weight at age 20 years (Wt_20y) was obtained by recall or from participants’ medical, school, or military records. Participants recalled their maximum weight (Wt_max) prior to T2D diagnosis and age at maximum weight (Age_{max_wt}). The rate of weight gain (Rate_{max_wt}) was calculated from magnitude of weight gain (ΔWt = Wt_max–Wt_20y) divided by ΔTime (Age_{max_wt} –20 years). The mean Age_{max_wt} and Age_T2D were 41.5±10.9 years and 50.1±10.5 years, respectively. The Wt_20y and Wt_max were 59.9±10.5 kg and 72.9±11.4 kg, respectively. The Rate_{max_wt} was 0.56±0.50 kg/year. After adjusting for risk factors, greater ΔWt and higher Rate_{max_wt} were significantly associated with earlier Age_T2D, higher HbA1c_T2D after additional adjusting for Age_T2D, and microalbuminuria after further adjusting for HbA1c_T2D and lipid profiles. Greater ΔWt and higher Rate_{max_wt} were also significantly associated with diabetic retinopathy.

Conclusions

This finding supports public health recommendations to reduce the risk of T2D and its complications by preventing weight gain from early adulthood.     

New azole antagonists with high affinity for the P2Y1 receptor

Five-membered-ring heterocyclic urea mimics have been found to be potent and selective antagonists of the P2Y₁ receptor. SAR of the various heterocyclic replacements is presented, as well as side-chain SAR of the more potent thiadiazole ring system which leads to thiadiazole 4c as a new antiplatelet agent.     

2-Aminothiazole based P2Y1 antagonists as novel antiplatelet agents

ADP receptors, P2Y₁ and P2Y₁₂ have been recognized as potential targets for antithrombotic drugs. A series of P2Y₁ antagonists that contain 2-aminothiazoles as urea surrogates were discovered. Extensive SAR of the thiazole ring is described. The most potent compound 7j showed good P2Y₁ binding (K_i = 12 nM), moderate antagonism of platelet aggregation (PA IC₅₀ = 5.2 μM) and acceptable PK in rats.     

Triazolo and imidazo dihydropyrazolopyrimidine potassium channel antagonists

Previously disclosed C6 amido and benzimidazole dihydropyrazolopyrimidines were potent and selective blockers of I_Kur current. Syntheses and SAR for C6 triazolo and imidazo dihydropyrazolopyrimidines series are described. Trifluoromethylcyclohexyl N(1) triazole, compound 51, was identified as a potent and selective K_v1.5 inhibitor with an acceptable PK and liability profile.     

Radiolabelled proteomics to determine differential functioning of Accumulibacter during the anaerobic and aerobic phases of a bioreactor operating for enhanced biological phosphorus removal

Proteins synthesized by the mixed microbial community of two sequencing batch reactors run for enhanced biological phosphorus removal (EBPR) during aerobic and anaerobic reactor phases were compared, using mass spectrometry‐based proteomics and radiolabelling. Both sludges were dominated by polyphosphate‐accumulating organisms belonging to Candidatis Accumulibacter and the majority of proteins identified matched closest to these bacteria. Enzymes from the Embden–Meyerhof–Parnas pathway were identified, suggesting this is the major glycolytic pathway for these Accumulibacter populations. Enhanced aerobic synthesis of glyoxylate cycle enzymes suggests this cycle is important during the aerobic phase of EBPR. In one sludge, several TCA cycle enzymes showed enhanced aerobic synthesis, suggesting this cycle is unimportant anaerobically. The second sludge showed enhanced synthesis of TCA cycle enzymes under anaerobic conditions, suggesting full or partial TCA cycle operation anaerobically. A phylogenetic analysis of Accumulibacter polyphosphate kinase genes from each sludge demonstrated different Accumulibacter populations dominated the two sludges. Thus, TCA cycle activity differences may be due to Accumulibacter strain differences. The major fatty acids present in Accumulibacter‐dominated sludge include palmitic, hexadecenoic and cis‐vaccenic acid and fatty acid content increased by approximately 20% during the anaerobic phase. We hypothesize that this is associated with increased anaerobic phospholipid membrane biosynthesis, to accommodate intracellular polyhydroxyalkanoate granules.