Perilla (Perilla frutescens L.) is an important edible-medicinal oil crop, with its seed containing 46%-58% oil. Of perilla seed oil, α-linolenic acid (C18:3) accounts for more than 60%. Lysophosphatidic acid acyltransferase (LPAT) is one of the key enzymes responsible for triacylglycerol assembly in plant seeds, controlling the metabolic flow from lysophosphatidic acid to phosphatidic acid. In this study, the LPAT2 gene from the developing seeds of perilla was cloned and designated as PfLPAT2. The expression profile of PfLPAT2 gene was examined in various tissues and different seed development stages of perilla (10, 20, 30, and 40 days after flowering, DAF) by quantitative real-time PCR (qRT-PCR). In order to detect the subcellular localization of PfLPAT2 protein, a fusion expression vector containing PfLPAT2 and GFP was constructed and transformed into Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration. In order to explore the enzymatic activity and biological function of PfLPAT2 protein, an E. coli expression vector, a yeast expression vector and a constitutive plant overexpression vector were constructed and transformed into an E. coli mutant SM2-1, a wild-type Saccharomyces cerevisiae strain INVSc1, and a common tobacco (Nicotiana tabacum, variety: Sumsun NN, SNN), respectively. The results showed that the PfLPAT2 open reading frame (ORF) sequence was 1 155 bp in length, encoding 384 amino acid residues. Functional structure domain prediction showed that PfLPAT2 protein has a typical conserved domain of lysophosphatidic acid acyltransferase. qRT-PCR analysis indicated that PfLPAT2 gene was expressed in all tissues tested, with the peak level in seed of 20 DAF of perilla. Subcellular localization prediction showed that PfLPAT2 protein is localized in cytoplasm. Functional complementation assay of PfLPAT2 in E. coli LPAAT mutant (SM2-1) showed that PfLPAT2 could restore the lipid biosynthesis of SM2-1 cell membrane and possess LPAT enzyme activity. The total oil content in the PfLPAT2 transgenic yeast was significantly increased, and the content of each fatty acid component changed compared with that of the non-transgenic control strain. Particularly, oleic acid (C18:1) in the transgenic yeast significantly increased, indicating that PfLPAT2 has a higher substrate preference for C18:1. Importantly, total fatty acid content in the transgenic tobacco leaves increased by about 0.42 times compared to that of the controls, with the C18:1 content doubled. The increased total oil content and the altered fatty acid composition in transgenic tobacco lines demonstrated that the heterologous expression of PfLPAT2 could promote host oil biosynthesis and the accumulation of health-promoting fatty acids (C18:1 and C18:3). This study will provide a theoretical basis and genetic elements for in-depth analysis of the molecular regulation mechanism of perilla oil, especially the synthesis of unsaturated fatty acids, which is beneficial to the genetic improvement of oil quality of oil crops.
Acyltransferases ; Cloning, Molecular ; Escherichia coli/metabolism* ; Fatty Acids ; Perilla frutescens/metabolism* ; Plant Oils ; Plant Proteins/metabolism* ; Saccharomyces cerevisiae/metabolism* ; Seeds/chemistry* ; Tobacco/genetics*

Production of biofuels such as ethanol from non-grain crops may contribute to alleviating the global energy crisis and reducing the potential threat to food security. Tobacco (Nicotiana tabacum) is a commercial crop with high biomass yield. Breeding of starch-rich tobacco plants may provide alternative raw materials for the production of fuel ethanol. We cloned the small subunit gene NtSSU of ADP-glucose pyrophosphorylase (NtAGPase), which controls starch biosynthesis in tobacco, and constructed a plant expression vector pCAMBIA1303-NtSSU. The NtSSU gene was overexpressed in tobacco upon Agrobacterium-mediated leaf disc transformation. Phenotypic analysis showed that overexpression of NtSSU gene promoted the accumulation of starch in tobacco leaves, and the content of starch in tobacco leaves increased from 17.5% to 41.7%. The growth rate and biomass yield of the transgenic tobacco with NtSSU gene were also significantly increased. The results revealed that overexpression of NtSSU gene could effectively redirect more photosynthesis carbon flux into starch biosynthesis pathway, which led to an increased biomass yield but did not generate negative effects on other agronomic traits. Therefore, NtSSU gene can be used as an excellent target gene in plant breeding to enrich starch accumulation in vegetative organs to develop new germplasm dedicated to fuel ethanol production.
Biomass ; Gene Expression Regulation, Plant ; Plant Breeding ; Plant Leaves/genetics* ; Plants, Genetically Modified/metabolism* ; Starch ; Tobacco/metabolism*

Objective To investigate the related factors of the prognosis of arterial embolectomy of acute cardiogenic cerebral embolism caused by atrial fibrillation.Methods The clinical data of using vein thrombolysis bridging artery embolectomy or arterial embolectomy alone for the treatment of patients with acute cardiogenic cerebral embolism of cerebral large artery occlusion due to atrial fibrillation were analyzed retrospectively.From January 2015 to December 2016,22 consecutive inpatients with cardioembolic cerebral embolism caused by paroxysmal or persistent atrial fibrillation were enrolled,including 10 patients with the 90-day modified Rankin Scale (mRS) score 0-2 (good recovery group) and 12 patients with mRS scores 3-6 (poor recovery group).The clinical features,imaging data,and treatment of the patients in both groups were compared.The factors such as age,gender,preoperative international standardization ratio (INR),embolism position,whether bridging vein thrombolysis before thrombectomy,National Institutes of Health Stroke Scale (NIHSS) score at the onset,time of onset to reperfusion (TOR),whether using tirofiban,times of thrombectomy,modified Thrombolysis In Cerebral Infarction (mTICI) blood flow grade,and postoperative intracranial symptomatic intracerebral hemorrhage were analyzed.Results There were no significant differences in age,gender,preoperative INR,embolism position,the number of intravenous thrombolysis before thrombectomy,the number of using tirofiban in surgery,the proportion of the above mTICI 2b grade,and the proportion of symptomatic cerebral hemorrhage after surgery of the patients between the two groups (P>0.05).The NIHSS score 15.2±2.0 at the onset in the good recovery group was lower than 22.9±8.4 in the poor recovery group.There was significant difference between the two groups (P<0.05).The TOR time (307±86 min) in the good recovery group was less than that of the poor recovery group (426±145 min).There was significant difference between the two groups (P<0.05).Embolectomy was performed 1.5 (0.5,3.0) times in the good recovery group,which was less than the poor recovery group (4.0 [2.0,7.0] times).There was significant difference between the two groups (P<0.05).Conclusions Shortening the time of reperfusion and reducing the number of embolectomy during operation are the important factors for improving the prognosis of patients when atrial fibrillation causes arterial embolectomy in patients with acute cerebral embolism.However,a study of larger sample is needed for further exploration.

OBJECTIVETo investigate the causes and strategy of difficult intraarterial mechanical thrombectomy (≥3 times) in patients with acute ischemic stroke (AIS).

METHODSThe clinical data of 8 cases of AIS with thrombectomy ≥3 times admitted in Department of Neurology, the 306(th) Hospital of People's Liberation Army from June to October in 2015 was analyzed retrospectively. There were 7 male and 1 female patients, aged from 38 to 86 years with an average age of (70±15) years, in which 5 cases were cardiogenic cerebral embolism and 3 cases were large artery atherosclerotic infarction. The National Institute of Health stroke scale (NIHSS) score (M (QR)) was 16 (12) before procedure and modified thrombolysis in cerebral infarction (mTICI)score were 0 in all the patients. Solitaire AB was used in thrombectomy in the occlusion of the arteries.

RESULTSThe causes of difficult intraarterial thrombectomy included multiple thrombus, tortuosity in vascular paths, guiding catheter being placed below the internal carotid artery siphon leading to weak strength of suction and support of stent, embolus dropping in the thrombectomy and inadequate anesthesia. After successful thrombectomy 3 cases had mTICI score of 2a, 4 cases of 2b, 1 case of 3. The NIHSS score was 5 (24) at 7(th) day after treatment. At the 90-day follow-up 5 patients had good prognosis (modified Rankin score 0 to 2) and 3 had disability (modified Rankin score 3 to 4).

CONCLUSIONCases of AIS with difficult intraarterial thrombectomy can be treated by improving thrombectomy materials and technique, reasonable anesthesia and perioperative medication in decision-making strategy.

Adult ; Aged ; Aged, 80 and over ; Carotid Artery, Internal ; pathology ; Female ; Humans ; Intracranial Embolism ; surgery ; Male ; Middle Aged ; Retrospective Studies ; Stents ; Stroke ; surgery ; Thrombectomy ; Treatment Outcome

OBJECTIVE:To establish a method for the simultaneous determination of(R,S)-goitrin,chlorogenic acid,luteolo-side and isochlorogenic acid A in Xiaoer ganmao granule. METHODS:HPLC was performed on the column of Hedera C18 with mo-bile phase of acetonitrile-0.1% formic acid aqueous at a flow rate of 1.0 ml/min,the detection wavelength was 254 nm,330 nm and 370 nm,column temperature was 40 ℃,and the injection volume was 5 μl,RESULTS:The linear range was 6.6-105 μg/ml for (R,S)-goitrin (r=0.999 9),9-140 μg/ml for chlorogenic acid (r=0.999 9),9-144 μg/ml for luteoloside (r=0.999 8) and 9-138 μg/ml for isochlorogenic acid A(r=0.999 6);the limits of quantitation were 330 ng,450 ng,450 ng and 450 ng,limits of detection were 66 ng,90 ng,90 ng and 90 ng,respectively;RSDs of precision,stability and reproducibility tests were lower than 3%;recoveries were 95.01%-98.77%(RSD=1.48%,n=6),95.14%-98.91%(RSD=1.52%,n=6),95.11%-97.54%(RSD=0.93%,n=6) and 95.58%-99.63%(RSD=1.73%,n=6). CONCLUSIONS:The method is simple and accurate,and suitable for the simultaneous determination of(R,S)-goitrin,chlorogenic acid,luteoloside and isochlorogenic acid A in Xiaoer ganmao granule.

The lack of effective in vitro infection model for hepatitis E virus (HEV) has greatly hindered the quantitative analysis of neutralizing titers of anti-HEV antibodies and human sera, thus impeding further studies of HEV-stimulated antibody responses and the immunological mechanisms. In order to improve this situation, the infection of HepG2 cells that are inefficient for HEV replication was continuously monitored until the viral load reached the limit of detection on day 13, the results of which confirmed the feasibility of using this cell line to establish the infection model. Then, neutralization assays of five anti-HEV murine monoclonal antibodies and serum samples collected from four HEV vaccine recipients (collected before and after vaccination) were performed by 96 multi-channel parallel infections, nucleic acid extraction, and qPCR. The results showed that the cell model can be applied for quantitative evaluation of the neutralizing capacity of different antibodies and antiserum samples from HEV vaccine recipients. In this study, we have successfully established a high-throughput in vitro HEV replication model, which will prove to be useful for the evaluation of HEV vaccines and studies of HEV epitopes.
Animals ; Antibodies, Viral ; analysis ; immunology ; Hepatitis Antibodies ; analysis ; immunology ; Hepatitis E ; immunology ; virology ; Hepatitis E virus ; chemistry ; immunology ; physiology ; High-Throughput Screening Assays ; methods ; Humans ; Mice ; Mice, Inbred BALB C ; Neutralization Tests ; methods ; Virus Replication

Objective To investigate the relationship between the evaluation of cerebral perfusion with CT perfusion (CTP)imaging and cognitive impairment in patients with asymptomatic severe internal carotid stenosis. Methods A total of 104 patients with asymptomatic severe unilateral internal carotid artery origin stenosis (the unilateral stenosis rate ≥70% and the contralateral stenosis rate < 30%)were enrolled respectively. After conducting Montreal Cognitive Assessment (MoCA)scores,they were divided into a non-cognitive impairment group (n = 24;MoCA ≥26)and a cognitive impairment group (n = 80;MoCA <26). All patients were performed digital subtraction angiography (DSA)and / or CT angiography (CTA)examinations. Their unilateral severe stenosis was confirmed,and they underwent brain CTP examinations. The relative cerebral blood flow (rCBF),relative cerebral blood volume (rCBV),relative mean transit time (rMTT),and relative time to peak (rTTP)were calculate by CTP. The presence rate of collateral circulation in 96 patients was calculated by DSA. The presence rate of collateral circulation,and relative perfusion parameters of the 2 groups were compared. Results (1)The MoCA score in patients of the non-cognitive impairment group was 27. 8 ± 1. 7,and the MoCA score in patients of the cognitive impairment group was 21. 4 ± 3. 1. There was significant difference between the 2 groups (t = 17. 959, P <0. 05). (2)The rate of 96 patients having collateral circulation was 68. 4% (52 / 76)in the cognitive impairment group,and in the non-cognitive impairment group was 60. 0% (12/ 20). There was no significant difference (P >0. 05). The CTP parameters rMTT,rTTP,rCBV,and rCBF in the non-cognitive impairment group were 1. 074 ± 0. 066,1. 103 ± 0. 032,1. 045 ± 0. 021 and 1. 066 ± 0. 040,respectively;the CTP parameters rMTT,rTTP,rCBV,and rCBF in the cognitive impairment group were 1. 241 ± 0. 169, 1. 328 ± 0. 248,1. 046 ± 0. 030,and 1. 093 ± 0. 058,respectively. The rTTP and rMTT of the cognitive impairment were longer than those of the non-cognitive impairment group. There were significant differences in rTTP and rMTT between the 2 groups (P < 0. 05),but there were no significant differences in rCBF and rCBV between the 2 groups (P >0.05). Conclusion Most of the patients with asymptomatic severe internal carotid stenosis has cognitive impairment,and cerebral perfusion caused by stenosis is significantly slower in patients with cognitive impairment than in those with noncognitive impairment.

Objective To investigate the clinical characteristics of cognitive impairment in the patients with asymptomatic unilateral severe internal carotid artery stenosis (ICAS). Methods A total of 80 patients with unilateral severe carotid stenosis (stenosis rate ≥70%)and 40 patients without carotid stenosis (control group)diagnosed by digital substract angiography (DSA)were analyzed retrospectively. According to the stenotic sides,the patients with severe ICAS were divided into a left stenosis group and a right stenosis group (n = 40 in each group). The North American Symptomatic Carotid Endarterectomy Trial (NASCET)criteria were used to grade the degree of stenosis. Montreal cognitive assessment (MoCA)was used to evaluate the cognitive function of the patients,and then the cognitive function of the patients in 3 groups was assessed. Results The MoCA total scores,visuospatial and executive functions,and language ability,and delayed memory scores of the patients in both left and right groups were lower than those of the control group. There were significant differences (21. 8 ± 3. 1,3. 4 ± 1. 3,1. 8 ± 0. 6,and 1. 6 ± 1. 3,respec-tively in the left stenosis group;22. 6 ± 2. 5,3. 5 ± 1. 1,1. 9 ± 0. 6,and 1. 7 ± 1. 4,respectively in the right stenosis group;and 26. 4 ± 1. 8,4. 2 ± 0. 9,2. 7 ± 0. 6,and 3. 8 ± 1. 0,respectively in the control group;all P < 0. 01). There were no significant differences in naming,attention,abstract ability,orientation ability scores compared with the control group (all P > 0. 05). There were no significant differences in the MoCA total scores and each single test score of the patients between the left stenosis and the right stenosis groups (P > 0. 05). Conclusion The patients with asymptomatic unilateral severe ICAS have cognitive impairment generally;it is characterized by delay memory,visuospatial and executive functions,and language dysfunction.

Objective To study the changes of the putamen metabolites with magnetic resonance spectrum(MRS),and to explore possible underlying unrecongnised aetiological factor and pathophysiology mechanism in the central nervous system of the patients with Tourette' s syndrome.Methods Twenty-two cases of Tourette' s syndrome,and twenty-two gender and age-matched subjects ( the control subjects ) were performed on a clinical 3.0 T MRI system.Proton prob-voxel spectroscopy imaging (1H-MRS) was obtained from two sides of the putamen.The metabolites included N-acetylaspartate (NAA),creatine and phosphocreatine ( Cr),choline-containing compounds (Cho),and myoinositol ( MI ).The value of the NAA,Cr,Cho,and MI were calculated by integration of their peaks.The ratios of NAA/Cr,Cho/Cr,ML/Cr were calculated respectively.Repeated measures analysis of variance ( ANOVA ) was used to test both the value of NAA/Cr,Cho/Cr,MI/Cr of the putamen for group difference,with group as between-subjects factor and side as within-subjects factor.Results The NAA/Cr ratio in patients ( left:1.29 ± 0.13 ; right:1.34 + 0.15 ) was significantly lower than that in the control subjects ( left:1.50 ± 0.08,T =1.962,P ＜0.05 ; right:1.52 ± 0.11,T =1.865,P ＜ 0.05 ).There was no significant difference in the Cho/Cr and MI/Cr ratio between both groups.Conclusion The abnormalities of the structure and(or) function in the putamens of patients may be the one of the underlying anaetiological factors and pathophysiology mechanisms of the Tourette' s syndrome.

ObjectiveTo investigate the central nervous system mechanisms of active of movement switch in the patients with Tourette' s syndrome( TS),and to explore the possible underlying distinct neural networks for tic trigger and generation. Methods Participants were 14 patients with TS and 14 age- and gender-matched healthy volunteers with no history of physical,psychiatric or neurological disease: All patients were assessed with functional magnetic resonances imaging (fMRI)of the brain during the intermittent performance of finger-tapping switch tasks,Blood-oxygen-level dependent-fMRI was performed using a 3.0 Tesla MR.The area over which the activation was distributed was calculated,and the activation volumes were also compared between the patients with TS and the control subjects.ResultsThe regions activated in the patients with TS and in the volunteers were similar in several brain regions,including contralateral precentral and postcentral gyrus,contralateral mesia pre-front gyrus,contralateral cingulate gyrus,contralateral insula and ipsilataral cerebellum. There were also many different activation areas between the patients and the control subjects. The patients with TS demonstrated more significant and extended activation in the contralateral pre- and postcentral gyrus than the healthy volunteers.The volume of the left pre- and postcentral gyrus of the TS patients was (8.024 ±0.071 ) cm3,while the volume of the left pre-and postcentral gyrus of the control subjects was ( 6.480 ± 0.026) cm3 ( t =3.026,P ＜ 0.01 ) ; The volume of the right pre- and postcentral gyrus was (6.192 ± 0.019) cm3 in the TS cases,while there was (5.608 ±0.037) cm3 in the control subjects (t =2.752,P ＜0.05).There were significant differences in the volumes of bilateral pre- and postcetral gyrus between the TS and control subjects. The activations of conralateral thalamus without contralateral insula were found in the patients with TS. Conversely, the contralateral insula activation without thalamus activation could be found in the healthy volunteers.ConclusionThe thalamus might play an important role in the aetiological and physiopathologic mechanisms of the TS. The thalamus along with the parietal cortex,cingulate cortex and insular cortex appear to constitute a distinct neural network for tic trigger and generation.