UCH-L1(ubiquitin carboxy-terminal hydrolases L1)is a member of the carboxyl-terminal ubiquitin hydrolase family,naturally,that there is hydrolase activity,however,and that there is ligase acitivity,which is different from the other members of UCHs.UCH-L1 hydrolase activity could keep the pool of free ubiquitin and compromise the ubiquitin-dependent degradation pathway,while UCH-L1 dimerizational-dependent,ubiquityl ligase activity could produce undegradable,K63-linked polyubiquitin chains that could inhibit proteasomal activity.Therefore,UCH-L1 is involved in the more diverse physiological activities,including neuron formation,gonadal development and fertilization.Mutation of UCH-L1 is linked to the neurodegenerative diseases,such as Parkinson's disease.Moreover,abnormal expression of UCH-L1 is response to carcinogenesis in many tissue such as thyroid lung.

OBJECTIVETo develop a hydroponic Nicotiana cultivation system for rapid and high-yield transient expression of recombinant proteins under laboratory conditions.

METHODSTo establish the hydroponic cultivation system, several parameters were examined to define the optimal conditions for the expression of recombinant proteins in plants. We used the green fluorescent protein (GFP) and the geminiviral plant transient expression vector as the model protein/expression vector. We examined the impact of Nicotiana species, the density and time of Agrobacterium infiltration, and the post-infiltration growth period on the accumulation of GFP. The expression levels of GFP in Nicotiana leaves were then examined by Western blotting and ELISA.

RESULTSOur data indicated that a hydroponic Nicotiana cultivation system with a light intensity of 9000 LX/layer, a light cycle of 16 h day/8 h night, a temperature regime of 28 degrees celsius; day/21 degrees celsius; night, and a relative humidity of 80% could support the optimal plant growth and protein expression. After agroinfiltration with pBYGFPDsRed.R/LBA4404, high levels of GFP expression were observed in both N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants cultured with this hydroponic cultivation system. An optimal GFP expression was achieved in both Nicotiana species leaves 4 days after infiltration by Agrobacterium with an OD(600) of 0.8. At a given time point, the average biomass of N. tobaccum (cv. Yuyan No.5) was significantly higher than that of N. benthamiana. The leaves from 6-week-old N. benthamiana plants and 5-week-old N. tobaccum (cv. Yuyan No.5) plants could be the optimal material for agroinfiltration.

CONCLUSIONWe have established a hydroponic cultivation system that allows robust growth of N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants and the optimal GFP expression in the artificial climate box.

Gene Expression Regulation, Plant ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hydroponics ; methods ; Plants, Genetically Modified ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Tobacco ; genetics ; growth & development

OBJECTIVETo study the effect of RNA interference (RNAi) on small heat shock protein (sHSP) Sjp40 of Schistosoma japonicum and its synergistic effect on the expression of SjHSP60, SjHSP70, and SjHSP90 mRNA, and observe the mRNA expression levels of Sjp40, SjHSP60, SjHSP70, and SjHSP90 in different stages of S.japonicum.

METHODSDouble-stranded RNA (dsRNA) of Sjp40 (dsSjp40) and a control dsRNA of green fluorescent protein (dsGFP) were generated by in vitro transcription and transfected into adult worm by immersing the worm in dsRNA solution. The total RNA and proteins were isolated simultaneously from the adult worms using TRIzol reagent 7 days after transfection. The expression levels of Sjp40, SjHSP60, SjHSP70, and SjHSP90 mRNA and the expression level of Sjp40 protein were determined by quantitative real-time PCR (qPCR) and Western blotting, respectively. The mRNA expression of HSPs of S. japonicum in different stages was evaluated by qPCR.

RESULTSCompared with those in the control worms transfected with dsGFP, Sjp40 mRNA level was decreased by 80% in the worms transfected with dsSjp40, and the level of Sjp40 protein showed also a significant decrease. The mRNA expression levels of SjHSP60, SjHSP70, and SjHSP90 did not show an obvious synergism after Sjp40 RNAi. The expression profiles of Sjp40, SjHSP60, SjHSP70, and SjHSP90 showed significant differences in different stages of S. japonicum, and the expression level of Sjp40 mRNA in the egg stage was much higher than that of other HSP genes.

CONCLUSIONdsSjp40-RNAi can induce effective suppression of Sjp40 gene expression at both mRNA and protein levels, but no obvious synergism occurs in the mRNA expressions of SjHSP60, SjHSP70, and SjHSP90.

Animals ; Gene Expression Profiling ; Heat-Shock Proteins, Small ; genetics ; Helminth Proteins ; genetics ; RNA Interference ; RNA, Double-Stranded ; RNA, Messenger ; genetics ; Schistosoma japonicum ; genetics

Objective:To investigate the clinical value of hematoma volume estimated by 3D-Slicer in predicting symptomatic cerebral vasospasm (sCVS) after aneurysmal subarachnoid hemorrhage (aSAH).Methods:CT images on admission of 84 aSAH patients, admitted to our hospital from January 2018 to June 2021, were collected. The hematoma volume of these patients was measured by 3D-Slicer (3D-Slicer hematoma volume). Patients were divided into sCVS group and non-sCVS group according to the occurrence of postoperative sCVS. The differences of 3D-Slicer hematoma volume, Fisher grading scale and modified Fisher grading scale between the two groups were compared by statistical methods, and the independent risk factors for sCVS after aSAH were screened. The differences of 3D-Slicer hematoma volume among patients with different Fisher grading scale or modified Fisher grading scale were compared.Results:There was no significant difference in Fisher grading scale and modified Fisher grading scale between sCVS group and non-SCVS group ( P>0.05), but the 3D-Slicer hematoma volume in sCVS group was significantly larger than that in non-SCVS group ( P<0.05). 3D-Slicer hematoma volume ( OR=1.061, 95%CI: 1.004-1.120, P=0.034) was independent risk factors for sCVS after aSAH. The comparison of 3D-Slicer hematoma volume among patients with different Fisher grading scale or modified Fisher grading scale showed statistical difference ( P<0.05). Conclusion:As compared with Fisher grading scale and modified Fisher grading scale, the hematoma volume measured by 3D-Slicer has more advantage in predicting sCVS after aSAH.

Objective To study the protective effect of helium preconditioning (HPC) on rats with acute transient focal cerebral ischemia/reperfusion (I/R) injury and its possible mechanism.Methods Healthy adult SD rats were divided into 3 groups:sham-operated group,I/R group and HPC group; the rat models of focal cerebral I/R in the later two groups were established by middle cerebral artery occlusion (MCAO,2 h) and reperfusion (24 h).Rats in the HPC group were exposed to the helium for 5 min×3 times (5 min interval) 24 h before MCAO.Neurological deficit scale was performed; the degrees of cerebral injury (infarct volume) were evaluated; BCA protein quantification was used to measure the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content.The morphological changes of the brain were observed by Nissl staining.Results As compared with those in the I/P group,the neurological deficit scale scores were deceased,infracted volume and content of MDA were decreased,and SOD activity was increased in HPC group,with significant differences (P＜ 0.05); the number of surviving neurons in the brain cortex and hippocampus of HPC group was increased as compared with that in the I/R group.Conclusion HPC has a protective effect against acute focal cerebral I/R injury,whose mechanism may be related to scavenging of flee radicals by spermine.

Objective To investigate the effect ofcranioplasty on prognosis of patients accepted intracranial aneurysm clipping combined with simultaneous decompressive craniectomy.Methods One hundred and forty-four patients accepted intracranial aneurysm clipping combined with simultaneous decompressive craniectomy (first-stage operation) in our hospital from January 2013 to June 2017 were chosen;there were 56 patients without cranioplasty in the control group and 88 patients with cranioplasty (second-stage operation) in the observation group.The degrees of coma before first-stage operation were assessed by Glasgow coma scale (GCS).The general state three months after first-stage operation was assessed by GCS and activity of daily living (ADL) scale.The prognoses of these patients 9 and 15 months after first-stage operation were assessed by Glasgow outcome scale (GOS) and ADL scale.The clinical data,prognoses and incidence of hydrocephalus of patients from the two groups were compared.Related factors associated with hydrocephalus were analyzed by multivariate Logistic regression analysis.Results GCS,GOS and ADL scale scores in the observation group 9 and 15 months after first-stage operation were all significantly higher than those in the control group (P＜0.05);incidence of hydrocephalus in the observation group after first-stage operation (31.82％) was significantly lower than that in the control group (62.5％,P＜0.05).Logistic regression model revealed that cranioplasty,Hunt-Hess grading and Fisher grading were independent related factors for incidence of hydrocephalus (P＜0.05);cranioplasty was the protective factor of hydrocephalus (OR=0.126),and Hunt-Hess grading and Fisher grading were the risk factors of hydrocephalus (OR=5.311 and 5.073).Conclusion Cranioplasty can reduce the incidence of hydrocephalus and improve the prognosis of patients accepted intracranial aneurysm clipping combined with simultaneous decompressive craniectomy.

OBJECTIVETo examine the expression profile and immunofluorescence localization of the major egg antigen p40 of Schistosoma japonicum (Sjp40) during granuloma formation in the liver of infected New Zealand white rabbits.

METHODSNew Zealand white rabbits were infected with S. japonicum cercariae, and the livers were harvested at 29 and 45 days post-infection (dpi). The total RNA of the liver tissues was extracted for expression profiling of Sjp40 by quantitative reverse transcription-PCR (qRT-PCR) with GAPDH of S. japonicum as the endogenous reference gene. The expression of Sjp40 in the liver were detected by Western blotting using anti-Sjp40 monoclonal antibody (mAb) 9G7 or anti-Toxoplasma gondii tSAG1 mAb Y3A8 (control) as the primary antibody. Paraffin sections of the liver were prepared for observing egg granuloma formation using HE staining and for indirect immunofluorescence assay of Sjp40 location in the trapped eggs and egg granulomas.

RESULTSThe level of Sjp40 mRNA in the eggs trapped in rabbit livers was significantly higher at 45 dpi than that at 29 dpi (P<0.05), and Western blotting confirmed the presence of Sjp40 protein in the rabbit livers at both 29 and 45 dpi. Immunofluorescence assay demonstrated localized expression of Sjp40 in the immature eggs in the rabbit liver at 29 dpi, but at 45 dpi fluorescence was detected in clusters of mature eggs containing miracidium and in the surrounding egg granulomas.

CONCLUSIONSThe transcriptional levels of Sjp40 significantly increased with the maturation of eggs trapped in the rabbit livers. Sjp40 protein spread from the eggs to the surrounding egg granuloma at 45 dpi when acute liver granulomatous lesions occur, suggesting that Sjp40 plays a key role in egg granulomas formation in the livers of infected New Zealand white rabbits.

Animals ; Antibodies, Monoclonal ; Antigens, Helminth ; metabolism ; Fluorescent Antibody Technique ; Gene Expression Profiling ; Granuloma ; parasitology ; Helminth Proteins ; metabolism ; Liver ; parasitology ; RNA, Messenger ; Rabbits ; Schistosoma japonicum ; Schistosomiasis japonica

Folliculogenesis is essential for production of female gametes in vertebrates. However, the molecular mechanisms underlying follicle development, particularly apoptosis regulation in ovary, remain elusive. Here, we generated sox3 knockout zebrafish lines using CRISPR/Cas9. sox3 knockout led to follicle development retardation and a reduced fecundity in females. Comparative analysis of transcriptome between sox3 and wild-type ovaries revealed that Sox3 was involved in pathways of ovarian steroidogenesis and apoptosis. Knockout of sox3 promoted follicle apoptosis and obvious apoptosis signals were detected in somatic cells of stages III and IV follicles of sox3 ovaries. Moreover, Sox3 can bind to and activate the promoter of cyp19a1a. Up-regulation of Cyp19a1a expression promoted 17β-estradiol synthesis, which inhibited apoptosis in follicle development. Thus, Sox3 functions as a regulator of Cyp19a1a expression, via 17β-E2 linking apoptosis suppression, which is implicated in improving female fecundity.

Background: Cardiopulmonary resuscitation (CPR) strategies in COVID-19 patients differ from those in patients suffering from cardiogenic cardiac arrest. During CPR, both healthcare and non-healthcare workers who provide resuscitation are at risk of infection. The Working Group for Expert Consensus on Prevention and Cardiopulmonary Resuscitation for Cardiac Arrest in COVID-19 has developed this Chinese Expert Consensus to guide clinical practice of CPR in COVID-19 patients. Main recommendations: 1) A medical team should be assigned to evaluate severe and critical COVID-19 for early monitoring of cardiac-arrest warning signs. 2) Psychological counseling and treatment are highly recommended, since sympathetic and vagal abnormalities induced by psychological stress from the COVID-19 pandemic can induce cardiac arrest. 3) Healthcare workers should wear personal protective equipment (PPE). 4) Mouth-to-mouth ventilation should be avoided on patients suspected of having or diagnosed with COVID-19. 5) Hands-only chest compression and mechanical chest compression are recommended. 6) Tracheal-intubation procedures should be optimized and tracheal-intubation strategies should be implemented early. 7) CPR should be provided for 20-30 min. 8) Various factors should be taken into consideration such as the interests of patients and family members, ethics, transmission risks, and laws and regulations governing infectious disease control. Changes in management: The following changes or modifications to CPR strategy in COVID-19 patients are proposed: 1) Healthcare workers should wear PPE. 2) Hands-only chest compression and mechanical chest compression can be implemented to reduce or avoid the spread of viruses by aerosols. 3) Both the benefits to patients and the risk of infection should be considered. 4) Hhealthcare workers should be fully aware of and trained in CPR strategies and procedures specifically for patients with COVID-19.