To address the role of Pr1 gene in the process of Hirsutella sinensis infecting Hepialus gonggaensis, differential expression of subtilisin-like protease gene was detected. In the present study, Pr1 gene analogues from H. sinensis were obtained by PCR strategy using specific primers designed from conserved regions of Pr1 gene reported in the GenBank. Then we detected the changes in the expression of Pr1 gene before and after infecting H. gonggaensis using real-time quantitative PCR. We obtained the partial sequence of Pr1 gene with the length of 535 bp (GenBank accession: KC009680). Real-time PCR results showed that the expression level of Pr1 gene was significantly different among 8 samples (P < 0.01). Pr1 gene showed the obvious higher expression level (2-3 folds) after infecting the H. gonggaensis, suggesting that the Pr1 gene may play an important role in the process of H. sinensis infecting H. gonggaensis. The present study paves a way for further identification on infectivity assessment of H. sinensis.

Hirudo nipponica Whitman,a Chinese medicine which has a strong effect on platelet aggregation and antithrombin activity,is involved in the studies.The current situation is adverse to the cultivation,development and utilization of H.nipponica.Prior to summarizing the current research status and the unresolved problems and the prospective of H.nipponica,domestic and foreign literatures over the H.nipponica was discussed and judged,aiming at providing a reference for the comprehensive development and utilization of H.nipponica.

Fertilization is a crucial step for origin of life.During Assisted Reproductive Technologies (ART),total fertilization failure is complex and unpredictable.Total fertilization failure may related to some abnormal cellular mechanistic events,such as:any stage of sperm and cumulus-oocyte-complexes penetration,sperm-zona pellucida binding / penetration,sperm-oocyte membrane binding,oocyte activation,sperm discondensation or pronuclear formation.Most of total fertilization failure could be solved by intracytoplasmic sperm injection.But oocytes of some patient still can't fertilize successfully,even though assisted oocyte activation be used.As for total fertilization failure patients in ART,combining the mature of oocyte,sperm quality and some trail to improve clinical protocol in later cycle may prevent failure to happen again.

Objective To analyze the clinical features of patients with open injuries combined with infections in Chinese Wenchuan earthquake and summarize the therapeutic experience. Methods An analysis was done on 54 patients with open injuries combined with infection transferred to our depart-ment from May 13, 2008 to June 10, 2008. Thorough debridement was performed based on general sup-porting management and anti-bacterial thempies done on the basis of results of bacteria culture and sus-ceptibility test. In the meantime, other managements including high-pressure oxygen therapy and external fixation were done for open fractures. Results Infection could be found in all open injuries, primarily combined infections with G+ coccus and G- bacillus or anaerobic. Moreover, most of the patients were infected by anaerobia. Of all, 1 patient with amputation received multiple debridement and repair because of severe infection of the amputation site and no death occurred. All patients recovered uneventfully, with sound wound healing, except for 2 patients. External fixation was stable and in good position. Conclu-sions The repeated and thorough debridement and the timely closure of wounds on the basis of energeti-cally anti-infection measures are key to diminishing the mortality rate and mutilation rate. Appropriate ad-junctive therapy can dramatically improve the curative effect.

OBJECTIVETo conduct a clinical trial of ketogenic diet (KD) in patients with acute spinal cord injury (SCI) and evaluate its safety and feasibility by measuring blood ketone bodies and blood glucose levels.

METHODTen patients with acute SCI were recruited in the trial during the period from May, 2012 to October, 2013. The patients received a standard KD after fasting for 48 h. The levels of blood ketone, blood glucose and uric ketone were tested daily, and routine blood examination, electrolytes, liver and kidney function, body mass index (BMI), sensory and motor function, and adverse reactions were monitored weekly to assess the safety and feasibility of KD.

RESULTSKD treatment lasted for a mean of 12.9 days (4 to 29 days) in these patients. In all the patients, blood ketone level increased during the fasting and maintained a level above 2.0 mmol/L after taking KD, while the uric ketone level ranged from +++ to ++++. The blood glucose level was in the normal range during KD. Except for blood chloride level and BMI, routine blood test results, electrolytes, liver and kidney function showed no significant changes after KD. No significant changes were observed in the sensation of light touch and pinprick. The average motor ASIA score increased from 33.3 to 35.1 after KD. Gastrointestinal dysfunction (diarrhea, nausea, poor appetite, gastric pain, and abdominal distension) was recorded in 5 patients, hypoglycemia occurred in one patient early after KD, and one patient experienced urticaria during KD. All the adverse reactions were relieved after symptomatic treatments.

CONCLUSIONThis preliminary clinical trial demonstrated that KD could increase ketone bodies level and maintain a normal blood glucose level, suggesting its safety and feasibility in patients with acute SCI.

Adolescent ; Adult ; Aged ; Diet, Ketogenic ; adverse effects ; methods ; Feasibility Studies ; Female ; Humans ; Male ; Middle Aged ; Spinal Cord Injuries ; diet therapy ; Treatment Outcome ; Young Adult

Objective To conduct a clinical trial of ketogenic diet (KD) in patients with acute spinal cord injury (SCI) and evaluate its safety and feasibility by measuring blood ketone bodies and blood glucose levels. Method Ten patients with acute SCI were recruited in the trial during the period from May, 2012 to October, 2013. The patients received a standard KD after fasting for 48 h. The levels of blood ketone, blood glucose and uric ketone were tested daily, and routine blood examination, electrolytes, liver and kidney function, body mass index (BMI), sensory and motor function, and adverse reactions were monitored weekly to assess the safety and feasibility of KD. Results KD treatment lasted for a mean of 12.9 days (4 to 29 days) in these patients. In all the patients, blood ketone level increased during the fasting and maintained a level above 2.0 mmol/L after taking KD, while the uric ketone level ranged from+++to++++. The blood glucose level was in the normal range during KD. Except for blood chloride level and BMI, routine blood test results, electrolytes, liver and kidney function showed no significant changes after KD. No significant changes were observed in the sensation of light touch and pinprick. The average motor ASIA score increased from 33.3 to 35.1 after KD. Gastrointestinal dysfunction (diarrhea, nausea, poor appetite, gastric pain, and abdominal distension) was recorded in 5 patients, hypoglycemia occurred in one patient early after KD, and one patient experienced urticaria during KD. All the adverse reactions were relieved after symptomatic treatments. Conclusion This preliminary clinical trial demonstrated that KD could increase ketone bodies level and maintain a normal blood glucose level, suggesting its safety and feasibility in patients with acute SCI.

Objective To conduct a clinical trial of ketogenic diet (KD) in patients with acute spinal cord injury (SCI) and evaluate its safety and feasibility by measuring blood ketone bodies and blood glucose levels. Method Ten patients with acute SCI were recruited in the trial during the period from May, 2012 to October, 2013. The patients received a standard KD after fasting for 48 h. The levels of blood ketone, blood glucose and uric ketone were tested daily, and routine blood examination, electrolytes, liver and kidney function, body mass index (BMI), sensory and motor function, and adverse reactions were monitored weekly to assess the safety and feasibility of KD. Results KD treatment lasted for a mean of 12.9 days (4 to 29 days) in these patients. In all the patients, blood ketone level increased during the fasting and maintained a level above 2.0 mmol/L after taking KD, while the uric ketone level ranged from+++to++++. The blood glucose level was in the normal range during KD. Except for blood chloride level and BMI, routine blood test results, electrolytes, liver and kidney function showed no significant changes after KD. No significant changes were observed in the sensation of light touch and pinprick. The average motor ASIA score increased from 33.3 to 35.1 after KD. Gastrointestinal dysfunction (diarrhea, nausea, poor appetite, gastric pain, and abdominal distension) was recorded in 5 patients, hypoglycemia occurred in one patient early after KD, and one patient experienced urticaria during KD. All the adverse reactions were relieved after symptomatic treatments. Conclusion This preliminary clinical trial demonstrated that KD could increase ketone bodies level and maintain a normal blood glucose level, suggesting its safety and feasibility in patients with acute SCI.

Objective:To establish a feasible mouse model of post-stroke dysphagia(PSD).Methods:Thirty C57BL/6 male mice were randomly divided into a sham-operated group(Sham)and a model group(PSD),and the PSD mouse model was made by the photothrombosis method(PT)method,and the sham-operated group was only injec-ted with rose bengal staining solution in the tail vein.The cerebral blood flow of the mice was measured by laser scatter imaging,the ratio of cerebral infarct area was detected by TTC staining,the electromyographic area of the in vivo pha-ryngeal muscle group of mice swallowing was recorded by a multi-conductor physiological recorder MP160,the drinking function of the mice was measured by the 4-min water drinking experiment,and the weight changes were recorded,respectively,at 1,3,and 7 d.Results:Cerebral blood flow decreased at all time points,with a sharp drop in cerebral blood flow at 1 d,gradual recovery of cerebral blood flowat 3 and 7 d,establishment of collateral circulation,and gradu-al reduction of cerebral infarction area;compared with the Sham group,the myoelectric area of the PSD group was reduced at 1 and 3 d(P＜0.05),but with a trend of gradual recovery,and there was no significant difference between the PSD group and the Sham group at 7 d,and water consumption and weight decreased at 4 min at 1,3,and 7 d(P＜0.05).Conclusion:The mice showed some degree of dysphagia symptoms and are expected to be a translational model for PSD.

Objective To clone the HnGUA gene from Hirudo nipponia and conduct bioinformatics analysis,protein prokaryotic expression analysis and gene differential expression analysis.Methods Based on the transcriptome data of H.nipponia in the previous study,the full-length cDNA of HnGUA was cloned by rapid amplification of cDNA ends(RACE),and bioinformatics analysis was performed.The prokaryotic expression vector was constructed,transformed into Escherichia coli BL21(DE3)competent cells and the expression of recombinant protein was induced by IPTG.The qPCR was used to further analyze the tissue-specific expression of HnGUA.Results The size of HnGUA gene was 504 bp,containing an open reading frame(ORF)of 231 bp and encoding 76 amino acids.Its protein molecular weight and isoelectric point are 8.17 kDa and 4.44,respectively.Multiple sequence alignment analysis showed that HnGUA was highly homologous to genes in other leech species that encode inhibitory proteins.The results of the prokaryotic expression analysis showed that the constructed pET32a-HnGUA vector could be successfully expressed in E.coli BL21(DE3),and the SDS-PAGE results showed that the induced recombinantly expressed HnGUA protein was around 6 kDa,which was basically consistent with the predicted protein size.The results of the Real-time PCR revealed spatial and temporal differences in the expression profiles of HnGUA,with high levels of expression detected in the skin and crop tissues.Conclusion This study represents the first successful cloning of the HnGUA gene from H.nipponia and the expression of the corresponding recombinant protein in E.coli.It provides a foundation for future exploration of the biosynthetic pathways and molecular regulatory mechanisms of active small anticoagulant molecules in leeches.