Molecular cloning and characterization of a novel alpha 1,2-fucosyltransferase (CE2FT-1) from Caenorhabditis elegans

Here we report the discovery of a unique fucosyltransferase (FT) in Caenorhabditis elegans. In studying the activities of FTs in extracts of adult C. elegans, we detected activity toward the unusual disaccharide acceptors Galbeta1-4Xyl-R and Galbeta1-6GlcNAc-R to generate products with the general structure Fucalpha1-2Galbeta1-R. We identified a gene encoding a unique alpha1,2FT (designated CE2FT-1), which contains an open reading frame encoding a predicted protein of 355 amino acids with the type 2 topology and domain structure typical of other glycosyltransferases. The predicted cDNA for CE2FT-1 has very low identity (5-10%) at the amino acid level to alpha1,2FT sequences in humans, rabbits, and mice. Recombinant CE2FT-1 expressed in human 293T cells has high alpha1,2FT activity toward the simple acceptor Galbeta-O-phenyl acceptor to generate Fucalpha1-2Galbeta-R, which in this respect resembles mammalian alpha1,2FTs. However, CE2FT-1 is otherwise completely different from known alpha1,2FTs in its acceptor specificity, since it is unable to fucosylate either Galbeta1-4Glcbeta-R or free lactose and prefers the unusual acceptors Galbeta1-4Xylbeta-R and Galbeta1-6GlcNAc-R. Promoter analysis of the CE2FT-1 gene using green fluorescent protein reporter constructs demonstrates that CE2FT-1 is expressed in single cells of early stage embryos and exclusively in the 20 intestinal cells of L(1)-L(4) and adult worms. These and other results suggest that multiple fucosyltransferase genes in C. elegans may encode enzymes with unique activities, expression, and developmental roles.     

The Effect of Protease Inhibitors on the Induction of Osteoarthritis-Related Biomarkers in Bovine Full-Depth Cartilage Explants

Objective

The specific degradation of type II collagen and aggrecan by matrix metalloproteinase (MMP)-9, -13 and ADAMTS-4 and -5 (aggrecanase-1 and -2) in the cartilage matrix is a critical step in pathology of osteoarthritis (OA). The aims of this study were: i) To investigate the relative contribution of ADAMTS-4 and ADAMTS-5 to cartilage degradation upon catabolic stimulation; ii) To investigate the effect of regulating the activities of key enzymes by mean of broad-spectrum inhibitors.

Methods

Bovine full-depth cartilage explants stimulated with tumor necrosis factor alpha (TNF-α) and Oncostatin M (OSM) were cultured for 21 days with or without a number of inhibitors targeting different types of proteases. Monoclonal antibodies were raised against the active sites of ADAMTS-4, -5, MMP-9 and -13, and 4 ELISAs were developed and technically validated. In addition, the established AGNxI (ADAMTS-degraded aggrecan), AGNxII (MMP-degraded aggrecan), and CTX-II (MMP-derived type II collagen) were quantified in the explants-conditioned media.

Results

We found that: i) Active ADAMTS-4, MMP-9, -13 were released in the late stage of TNF-α/ OSM stimulation, whereas no significant active ADAMTS-5 was detected in either extracts or supernatants; ii) Active ADAMTS-4 was primarily responsible for E³⁷³-³⁷⁴A bond cleavage in aggrecan in this setting; and iii) The compensatory mechanism could be triggered following the blockage of the enzyme caused by inhibitors.

Conclusions

ADAMTS-4 appeared to be the major protease for the generation of ³⁷⁴ARGS aggrecan fragment in the TNF-α/OSM stimulated bovine cartilage explants. This study addresses the need to determine the roles of ADAMTS-4 and ADAMTS-5 in human articular degradation in OA and hence identify the attractive target for slowing down human cartilage breakdown.     

Suppression of Graft Regeneration,Not Ischemia/Reperfusion Injury,Is the Primary Cause of Small-for-Size Syndrome after Partial Liver Transplantation in Mice

Background

Ischemia/reperfusion injury (IRI) is commonly considered to play a crucial role in the pathogenesis of small-for-size syndrome (SFSS) after liver transplantation. Rapid regeneration is also considered essential for the survival of SFS grafts.

Methods

Mouse models of full-size orthotopic liver transplantation, 50% partial liver transplantation and 30% partial liver transplantation were established. Survival rate and serum alanine aminotransferase were observed. IRI was assessed by hepatic pathologic alterations, apoptosis and necrosis. Regeneration response was detected by mitotic index, BrdU incorporation and PCNA, Cyclin D1 and Cyclin E expression. The expression of mTOR, AKT, ERK, JNK2 and p70S6K, also involved in regeneration signaling pathways, were analyzed as well.

Results

30% partial liver graft resulted in a significantly low 7-day survival rate (P = 0.002) with no marked difference in tissue injury compared with the 50% partial graft group. Serum alanine aminotransferase levels were not significantly different between partial transplantation and full-size transplantation. Western blot analysis of caspase-3 and TUNEL staining also indicated no significant difference in apoptosis response between 30% partial transplantation and half-size or full-size transplantation (P = 0.436, P = 0.113, respectively). However, liver regeneration response indicators, mitotic index (P<0.0001) and BrdU (P = 0.0022), were markedly lower in 30% LTx compared with 50% LTx. Suppressed expression of PCNA, cyclin D1, cyclin E, mTOR, JNK2, AKT, ERK and p70S6K was also detected by western blot.

Conclusions

Liver regeneration is markedly suppressed in SFSS, and is more likely the primary cause of SFSS, rather than ischemia/reperfusion injury. Therapy for recovering graft regeneration could be a potentially important strategy to reduce the incidence of SFSS.     

Inhibiting of circ-TLK1 inhibits the progression of glioma through down-regulating PANX1 via targeting miR-17-5p

Glioma remains the most common malignant tumors in the central nervous system and often has poor prognosis. In recent years, it has been gradually revealed that non-coding RNA effects glioma progression. In this study, we aimed to investigate the significance of circular RNA TLK1 (Circ-TLK1) in predicting the survival of glioma patients as well as its role in glioma development via both in-vitro and in-vivo experiments. We found that Circ-TLK1 was conspicuously up-regulated in glioma tissues compared with adjacent normal tissues, and the up-regulated Circ-TLK1 was significantly correlated with glioma patients’ larger tumor volume and higher grades. Functionally, Circ-TLK1 over-expression facilitated glioma growth, migration and invasion, inhibited cell apoptosis, and accelerated PANX1/MAPK/ERK expression, while Circ-TLK1 low expression had the opposite effects. In addition, bioinformatics analysis showed that miR-17-5p was a potential target of Circ-TLK1 and targeted at PANX1. Furthermore, through dual luciferase viability assay, Circ-TLK1 acted as a competing endogenous RNA by sponging miR-17-5p, which targeted and inhibited PANX1/MAPK/ERK expression. MiR-17-5p overexpression mitigated glioma progression, which was significantly inhibited with Circ-TLK1 upregulation. In conclusion, this study confirmed a novel axis of Circ-TLK1-miR-17-5p-PANX1 in modulating glioma development, providing more references for glioma diagnosis and targeted therapy.

     

Acute Myocardial Infarction and Pulmonary Diseases Result in Two Different Degradation Profiles of Elastin as Quantified by Two Novel ELISAs

Background

Elastin is a signature protein of the arteries and lungs, thus it was hypothesized that elastin is subject to enzymatic degradation during cardiovascular and pulmonary diseases. The aim was to investigate if different fragments of the same protein entail different information associated to two different diseases and if these fragments have the potential of being diagnostic biomarkers.

Methods

Monoclonal antibodies were raised against an identified fragment (the ELM-2 neoepitope) generated at the amino acid position ‘552 in elastin by matrix metalloproteinase (MMP) −9/−12. A newly identified ELM neoepitope was generated by the same proteases but at amino acid position ‘441. The distribution of ELM-2 and ELM, in human arterial plaques and fibrotic lung tissues were investigated by immunohistochemistry. A competitive ELISA for ELM-2 was developed. The clinical relevance of the ELM and ELM-2 ELISAs was evaluated in patients with acute myocardial infarction (AMI), no AMI, high coronary calcium, or low coronary calcium. The serological release of ELM-2 in patients with chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF) was compared to controls.

Results

ELM and ELM-2 neoepitopes were both localized in diseased carotid arteries and fibrotic lungs. In the cardiovascular cohort, ELM-2 levels were 66% higher in serum from AMI patients compared to patients with no AMI (p<0.01). Levels of ELM were not significantly increased in these patients and no correlation was observed between ELM-2 and ELM. ELM-2 was not elevated in the COPD and IPF patients and was not correlated to ELM. ELM was shown to be correlated with smoking habits (p<0.01).

Conclusions

The ELM-2 neoepitope was related to AMI whereas the ELM neoepitope was related to pulmonary diseases. These results indicate that elastin neoepitopes generated by the same proteases but at different amino acid sites provide different tissue-related information depending on the disease in question.     

MMEJ-KO: a web tool for designing paired CRISPR guide RNAs for microhomology-mediated end joining fragment deletion

正Dear Editor,Clustered regularly interspaced short palindromic repeats associated protein (CRISPR/Cas) systems are convenient and versatile tools for genome editing. Directed by a guide RNA (gRNA), the Cas nuclease produces double-strand break (DSB) at the target site, generally resulting in small base insertion/deletion mutations by the non-homologous end joining (NHEJ) DNA repair.     

Developing a Time Series Predictive Model for Dengue in Zhongshan,China Based on Weather and Guangzhou Dengue Surveillance Data

Background

Dengue is a re-emerging infectious disease of humans, rapidly growing from endemic areas to dengue-free regions due to favorable conditions. In recent decades, Guangzhou has again suffered from several big outbreaks of dengue; as have its neighboring cities. This study aims to examine the impact of dengue epidemics in Guangzhou, China, and to develop a predictive model for Zhongshan based on local weather conditions and Guangzhou dengue surveillance information.

Methods

We obtained weekly dengue case data from 1^st January, 2005 to 31^st December, 2014 for Guangzhou and Zhongshan city from the Chinese National Disease Surveillance Reporting System. Meteorological data was collected from the Zhongshan Weather Bureau and demographic data was collected from the Zhongshan Statistical Bureau. A negative binomial regression model with a log link function was used to analyze the relationship between weekly dengue cases in Guangzhou and Zhongshan, controlling for meteorological factors. Cross-correlation functions were applied to identify the time lags of the effect of each weather factor on weekly dengue cases. Models were validated using receiver operating characteristic (ROC) curves and k-fold cross-validation.

Results

Our results showed that weekly dengue cases in Zhongshan were significantly associated with dengue cases in Guangzhou after the treatment of a 5 weeks prior moving average (Relative Risk (RR) = 2.016, 95% Confidence Interval (CI): 1.845–2.203), controlling for weather factors including minimum temperature, relative humidity, and rainfall. ROC curve analysis indicated our forecasting model performed well at different prediction thresholds, with 0.969 area under the receiver operating characteristic curve (AUC) for a threshold of 3 cases per week, 0.957 AUC for a threshold of 2 cases per week, and 0.938 AUC for a threshold of 1 case per week. Models established during k-fold cross-validation also had considerable AUC (average 0.938–0.967). The sensitivity and specificity obtained from k-fold cross-validation was 78.83% and 92.48% respectively, with a forecasting threshold of 3 cases per week; 91.17% and 91.39%, with a threshold of 2 cases; and 85.16% and 87.25% with a threshold of 1 case. The out-of-sample prediction for the epidemics in 2014 also showed satisfactory performance.

Conclusion

Our study findings suggest that the occurrence of dengue outbreaks in Guangzhou could impact dengue outbreaks in Zhongshan under suitable weather conditions. Future studies should focus on developing integrated early warning systems for dengue transmission including local weather and human movement.     

Climate and the Timing of Imported Cases as Determinants of the Dengue Outbreak in Guangzhou, 2014: Evidence from a Mathematical Model

As the world’s fastest spreading vector-borne disease, dengue was estimated to infect more than 390 million people in 2010, a 30-fold increase in the past half century. Although considered to be a non-endemic country, mainland China had 55,114 reported dengue cases from 2005 to 2014, of which 47,056 occurred in 2014. Furthermore, 94% of the indigenous cases in this time period were reported in Guangdong Province, 83% of which were in Guangzhou City. In order to determine the possible determinants of the unprecedented outbreak in 2014, a population-based deterministic model was developed to describe dengue transmission dynamics in Guangzhou. Regional sensitivity analysis (RSA) was adopted to calibrate the model and entomological surveillance data was used to validate the mosquito submodel. Different scenarios were created to investigate the roles of the timing of an imported case, climate, vertical transmission from mosquitoes to their offspring, and intervention. The results suggested that an early imported case was the most important factor in determining the 2014 outbreak characteristics. Precipitation and temperature can also change the transmission dynamics. Extraordinary high precipitation in May and August, 2014 appears to have increased vector abundance. Considering the relatively small number of cases in 2013, the effect of vertical transmission was less important. The earlier and more frequent intervention in 2014 also appeared to be effective. If the intervention in 2014 was the same as that in 2013, the outbreak size may have been over an order of magnitude higher than the observed number of new cases in 2014.The early date of the first imported and locally transmitted case was largely responsible for the outbreak in 2014, but it was influenced by intervention, climate and vertical transmission. Early detection and response to imported cases in the spring and early summer is crucial to avoid large outbreaks in the future.