Identification of halohydrin dehalogenase mutants that resist COBE inhibition

The biocatalytic cascade conversion of ethyl 4-chloroacetoacetate (COBE) to ethyl (R)-4-cyano-3-hydroxybutyrate ((R)-HN) for the preparation of atorvastatin represents significant economic and environmental benefits, and is catalyzed by alcohol dehydrogenase and halohydrin dehalogenase (HHDH). However, as the activity of HHDH is inhibited by COBE, the cascade reaction is an inefficient one-pot reaction. In this study, substrate inhibition kinetics analysis was performed and the inhibition by COBE was found to be competitive reversible inhibition. Molecular simulation analysis was used to determine the inhibition mechanism by COBE. The results showed that COBE bound to the active center of HHDH via the formation of hydrogen bonds with the OH groups of S132 and Y145. Site saturation mutagenesis of residues around the active site and at the entrance of the access tunnel was performed, and two target mutant residues were identified, F136 and W249. Small focused mutagenesis on these two residues was performed and the F136V/W249F mutant was successfully found to relieve the activity inhibition of HHDH by COBE. The half inhibiting concentration of mutant F136V/W249F was found to be 20-fold higher than wild-type HHDH. The efficiency of the multi-enzymatic one-pot system for the synthesis of (R)-HN from COBE using mutant F136V/W249F was improved significantly.     

饥饿对南方鲇幼鱼游泳能力个体变异和重复性的影响

为考察肉食性鱼类有氧和无氧运动能力的种内个体变异、重复性及其对饥饿的响应, 研究以南方鲇（Silurus meridionalis Chen）幼鱼为实验对象, 在（250.5）℃条件下测定对照组（n=28）和饥饿组（n=29）的临界游泳速度（Critical swimming speed, Ucrit）、暴发游泳速度（Constant acceleration speed, Ucat）和固定流速耐受时间（Endurance）, 分析游泳能力的个体变异、稳定性及饥饿的影响。结果显示: （1）饥饿组的体重、体长和肥满度均分别显著下降了（15.100.86）%、（2.570.40）%和（7.941.59）%（P0.05）, 而对照组无明显变化; （2）对照组的Ucat和耐受时间无明显变化（P0.05）, 但Ucrit下降（6.632.25）%（P=0.031）, 而除耐受时间外饥饿组其Ucrit和Ucat分别显著下降了（26.002.76）%和（13.681.86）%（P0.001）, 并饥饿组Ucrit的下降比例显著大于其Ucat（P0.001）; （3）并且对照组三个指标的变异系数（Coefficient of variation, CV）变化方向和程度不尽相同, 饥饿组的Ucrit、Ucat和耐受时间的CV全部增加; （4）南方鲇幼鱼Ucrit和Ucat呈正相关且2周的饥饿并未改变此正相关。饥饿明显降低南方鲇幼鱼两种游泳能力并导致游泳能力的个体变异变大, 但没有改变该种鱼的有氧运动能力和无氧运动能力之间的内在正相关关系。研究表明无氧运动能力在环境改变后显得更为保守, 种内个体变异的变动可能有利于在相同自然选择压力下种内个体采取不同的捕食和避敌对策。       

A phylo-functional core of gut microbiota in healthy young Chinese cohorts across lifestyles,geography and ethnicities

Structural profiling of healthy human gut microbiota across heterogeneous populations is necessary for benchmarking and characterizing the potential ecosystem services provided by particular gut symbionts for maintaining the health of their hosts. Here we performed a large structural survey of fecal microbiota in 314 healthy young adults, covering 20 rural and urban cohorts from 7 ethnic groups living in 9 provinces throughout China. Canonical analysis of unweighted UniFrac principal coordinates clustered the subjects mainly by their ethnicities/geography and less so by lifestyles. Nine predominant genera, all of which are known to contain short-chain fatty acid producers, co-occurred in all individuals and collectively represented nearly half of the total sequences. Interestingly, species-level compositional profiles within these nine genera still discriminated the subjects according to their ethnicities/geography and lifestyles. Therefore, a phylogenetically diverse core of gut microbiota at the genus level may be commonly shared by distinctive healthy populations as functionally indispensable ecosystem service providers for the hosts.     

Proteomic Analysis of Fetal Ovary Reveals That Ovarian Developmental Potential Is Greater in Meishan Pigs than in Yorkshire Pigs

Time-dependent expression of functional proteins in fetal ovaries is important to understand the developmental process of the ovary. This study was carried out to enhance our understanding of the developmental process of porcine fetal ovaries and to better address the differences in fetal ovary development of local and foreign pigs. The objective of the present study is to test the expression of key proteins that regulate the growth and development of fetal ovaries in Meishan and Yorkshire porcine breeds by using proteomics technology. Six Meishan and 6 Yorkshire pregnant gilts were used in this experiment. Fetal ovaries were obtained from Yorkshire and Meishan gilts on days 55 and 90 of the gestation period. Using 2D-DIGE (two dimensional-difference in gel electrophoresis) analysis, the results showed that there are about 1551 and 1400 proteins in gilt fetal ovaries on days 55 and 90, respectively of the gestation. Using MALDI TOF-TOF MS analysis, 27 differentially expressed proteins were identified in the fetal ovaries of the 2 breeds on day 55 of gestation, and a total of 18 proteins were identified on day 90 of gestation. These differentially expressed proteins were involved in the regulation of biological processes (cell death, stress response, cytoskeletal proteins) and molecular functions (enzyme regulator activity). We also found that alpha-1-antitrypsin, actin, vimentin, and PP2A proteins promote the formation of primordial follicles in the ovaries of Yorkshire pigs on day 55 of gestation while low expression heat shock proteins and high expression alpha-fetoproteins (AFP) may promote Meishan fetal ovarian follicular development on day 90 of gestation. These findings provide a deeper understanding of how reduced expression of heat shock proteins and increased expression of AFP can significantly reduce the risk of reproductive disease in obese Meishan sows. Our study also shows how these proteins can increase the ovulation rate and may be responsible for the low reproductive efficiency reported in other obese breeds. The ovarian developmental potential was found to be greater in Meishan pigs than in Yorkshire pigs.     

NGF Modulates trkANGFR/p75NTR in αSMA-Expressing Conjunctival Fibroblasts from Human Ocular Cicatricial Pemphigoid (OCP)

Objective

In a previous study, we reported the upregulation of Nerve Growth Factor (NGF) and trkA^NGFR expression in Ocular Cicatricial Pemphigoid (OCP), an inflammatory and remodeling eye disease. Herein, we hypothesize a potential NGF-driven mechanism on fibroblasts (FBs) during OCP remodeling events. To verify, human derived OCP-FBs were isolated and characterized either at baseline or after NGF exposure.

Materials and Methods

Conjunctival biopsies were obtained from 7 patients having OCP and 6 control subjects (cataract surgery). Both conjunctivas and primary FB cultures were characterised for αSMA, NGF and trkA^NGFR/p75^NTR expression. Subcultures were exposed to NGF and evaluated for αSMA, NGF, trkA^NGFR/p75^NTR expression as well as TGFβ1/IL4 release. For analysis, early and advanced subgroups were defined according to clinical parameters.

Results

OCP-conjunctivas showed αSMA-expressing FBs and high NGF levels. Advanced OCP-FBs showed higher αSMA expression associated with higher p75^NTR and lower trkA^NGFR expression, as compared to early counterparts. αSMA expression was in keeping with disease severity and correlated to p75^NTR. NGF exposure did not affect trkA^NGFR levels in early OCP-FBs while decreased both αSMA/p75^NTR expression and TGFβ1/IL4 release. These effects were not observed in advanced OCP-FBs.

Conclusions

Taken together, these data are suggestive for a NGF/p75^NTR task in the potential modulation of OCP fibrosis and encourages further studies to fully understand the underlying mechanism occurring in fibrosis. NGF/p75^NTR might be viewed as a potential therapeutic target.     

Internalization of the TGF-β type I receptor into caveolin-1 and EEA1 double-positive early endosomes

Endocytosis and intracellular sorting of transforming growth factor-β (TGF-β) receptors play an important regulatory role in TGF-β signaling. Two major endocytic pathways, clathrin- and caveolae-mediated endocytosis, have been reported to independently mediate the internalization of TGF-β receptors. In this study, we demonstrate that the clathrin- and caveolae-mediated endocytic pathways can converge during TGF-β receptor endocytic trafficking. By tracking the intracellular dynamics of fluorescently-labeled TGF-β type I receptor (TβRI), we found that after mediating TβRI internalization, certain clathrin-coated vesicles and caveolar vesicles are fused underneath the plasma membrane, forming a novel type of caveolin-1 and clathrin double-positive vesicles. Under the regulation of Rab5, the fused vesicles are targeted to early endosomes and thus deliver the internalized TβRI to the caveolin-1 and EEA1 double-positive early endosomes (caveolin-1-positive early endosomes). We further showed that the caveolin-1-positive early endosomes are positive for Smad3/SARA, Rab11 and Smad7/Smurf2, and may act as a multifunctional device for TGF-β signaling and TGF-β receptor recycling and degradation. Therefore, these findings uncover a novel scenario of endocytosis, the direct fusion of clathrin-coated and caveolae vesicles during TGF-β receptor endocytic trafficking, which leads to the formation of the multifunctional sorting device, caveolin-1-positive early endosomes, for TGF-β receptors.     

Inorganic and Organic Nitrogen Acquisition by a Fern Dicranopteris dichotoma in a Subtropical Forest in South China

The fern Dicranopteris dichotoma is an important pioneer species of the understory in Masson pine (Pinus massoniana) forests growing on acidic soils in the subtropical and tropical China. To improve our understanding of the role of D. dichotoma in nitrogen (N) uptake of these forests, a short-term ¹⁵N experiment was conducted at mountain ridge (MR, with low N level) and mountain foot (MF, with high N level). We injected ¹⁵N tracers as ¹⁵NH₄, ¹⁵NO₃ or ¹⁵N-glycine into the soil surrounding each plant at both MR and MF sites. Three hours after tracer injection, the fern D. dichotoma took up ¹⁵NH₄ ⁺ significantly faster at MF than at MR, but it showed significantly slower uptake of ¹⁵NO₃ ⁻ at MF than at MR. Consequently, ¹⁵NO₃ ⁻ made greater contribution to the total N uptake (50% to the total N uptake) at MR than at MF, but ¹⁵N-glycine only contributed around 11% at both sites. Twenty-four hours after tracer injection, D. dichotoma preferred ¹⁵NH₄ ⁺ (63%) at MR, whereas it preferred ¹⁵NO₃ ⁻ (47%) at MF. We concluded that the D. dichotoma responds distinctly in its uptake pattern for three available N species over temporal and spatial scales, but mainly relies on inorganic N species in the subtropical forest. This suggests that the fern employs different strategies to acquire available N which depends on N levels and time.     

Dominance of CTX-M-Type Extended-Spectrum β-Lactamase (ESBL)-Producing Escherichia coli Isolated from Patients with Community-Onset and Hospital-Onset Infection in China

Objective

To investigate CTX-M genotypes among extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) isolated from patients with community-onset and hospital-onset infections in China, their clonality and the distribution of CTX-M variants in different specimens of community-onset and hospital-onset infections.

Methods

ESBL-EC isolates were collected from general hospitals from 2011 to 2012 in China. Broth microdilution method antimicrobial susceptibility testing of 16 antibiotics was performed. Clinical data from community-onset and hospital-onset infections due to ESBL-EC were analyzed. ESBL-encoding genes were amplified by PCR and sequenced, and multilocus sequence typing (MLST) was performed for a random selection of predominant CTX-M type strains identified.

Results

A total of 1,168 ESBL-EC isolates were obtained from various clinical specimens, 41.7% of which were responsible for causing community-onset infections. The presence of urinary calculi was higher in community-onset infections, whereas malignancy, cardiovascular and cerebrovascular diseases, dementia, chronic renal disease, diabetes mellitus and surgical treatment were found to have higher proportions in hospital-onset infections. There was no significant difference in trauma between community-onset and hospital-onset infections. 96.2% of the isolates were detected to harbor bla _CTX-M genes. bla _CTX-M-1 group and bla _CTX-M-9 group were detected at 40.7% and 48.7% respectively, and both positive group accounted for 10.6%. bla _CTX-M-55 (24.8%) and bla _CTX-M-15 (18.2%) were the major genotypes in bla _CTX-M-1 group while bla _CTX-M-14 (46.8%) was predominant in bla _CTX-M-9 group. A comparison of bla _CTX-M distribution in different specimens between ESBL-EC causing community-onset and hospital-onset infection showed no significant difference. A total of 229 isolates were tested for MLST. ST131 (14%) was the predominant type. ST648, ST405 and ST1193 were also detected.

Conclusions

Community-onset ESBL-EC has emerged as a common pathogen in China. CTX-M-14 is the most commonly encountered, CTX-M-55 and CTX-M-15 have spread rapidly. ST131 is the predominant clonal group, and the great diversity of CTX-M-producing isolates of E. coli has emerged in China.