Objective To investigate the computer-assisted cognitive training (CACT) on cognitive function and functional independence of stroke patients. Methods Sixty patients were randomly divided into 2 groups: the CACT group and the control group. All patients received routine training. Patients in the CACT group received computer-assisted cognitive training additionally. Before and after the CACT, the cognitive function and functional independence of patients were assessed by MMSE, MoCA and FIM assays. Results Before the CACT, no significant differences in the scores of MMSE, MoCA and FIM were found between the two groups (P > 0.05). After the CACT, scores of MMSE, MoCA and FIM were improved in both groups, especially in the CACT group, with statistical differences (P < 0.05). Conclusions CACT can help improve the cognitive function and functional independence in stroke patients.

Objective To explore the central mechanism of acupuncture at Baihui (GV20) for memory impairment after stroke. Methods 32 stroke patients were randomized to receive acupuncture at Baihui (GV20) (as observation group) and acupuncture at Yanglingquan (GB34) (as control group) for 8 weeks. At the meantime, all participants received routine treatment including physical and occupational therapies. They were scanned with resting-state functional magnetic resonance imaging (fMRI) to find functional connectivity and assessed with Wechsler Memory Scale (WMS) before and after treatment. The results of WMS and functional connectivity were analyzed with Pearson's correlation. Results The memory scores and memory quotient improved significantly after treatment in the observation group (P<0.05). The functional connectivity significantly increased occurred in the left hippocampus to right inferior frontal gyrus, right middle frontal gyrus and left inferior frontal gyrus; and right hippocampus to left middle frontal gyrus, left superior frontal gyrus and left parietal lobe. Significant correlations were found between memory quotient and functional connectivity of hippocampus to frontal lobe and left parietal lobe in the observation group. There was no statistical significance in memory scores and memory quotient in the control group. The functional connectivity significantly decreased in left hippocampus to right middle occipital gyrus, and right hippocampus to right superior temporal gyrus and right posterior lobe of cerebellum. There was no statistical correlation between functional connectivity and results of WMS. Conclusion The acupuncture at Baihui can improves memory ability of stroke patients, which may associate with the increase of functional connectivity of hippocampus with frontal lobes and parietal lobes.

Objective To investigate the cross reaction characteristics of recombinant human papillomavirus 16 type L2 full-length fusion protein in HPV types of 6, 11, 18.Methods The serum samples of 108 condyloma acuminatum patients, 156 cervix cancer patients and 100 healthy control subjects were collected.The gene of full-length HPV16 L2 was amplificated from the tissue DNA of cervical cancer patient and inserted into the prokaryotic expression vector PGEX-4T-1 to construct the recombinant plasmid PGEX-4T-1-HPV16 L2.After sequencing identification, the recombinant plasmid was tranformed into E.coli BL21 (DE3).After induction by IPTG, the fusion protein containing HPV16 L2 was expressed and analyzed by both SDS-PAGE and WB.Furthermore, the specific binding capacity of the fusion protein to the HPV 6,11, 16 and 18 DNA positive patient's sera were analyzed by WB.The fusion protein was purified with NiNTA Agarose Kit and coated with ELISA reaction plates.The specific serum IgG were detected by indirect ELISA.Results The recombinant plasmid PGEX-4T-1-HPV16 L2 was constructed successfully. Highly expressed HPV16 L2 full-length fusion protein was obtained and the expression level was 27.2 %.The relative molecular mass(Mr) of the fusion protein is about 82 × 103, which matches up to the expected Mr.Meanwhile, the sera of HPV 6,11,16,18 DNA positive patients were used as the primary antibody and the Mr of the specific band was detected to be about 82 × 103 by WB.The results of indirect ELISA showed that the average levels of specific IgG in condyloma acuminatum group, cervical cancer group and healthy control subjects were 0.848 ±0.257, 0.822 ±0.247 and 0.173 ±0.143 with the positive rate of 92.6%, 94.2%and 8.0% respectively.There was no significance of the specific IgG levels between condyloma acuminatum group and cervical cancer group ( F = 0, P ＞ 0.05 ), but there was significant difference of specific IgG levels and positivity among the three groups ( F = 305.201 ,x2 = 253.178, P ＜ 0.01 ).Conclusions The HPV16 L2 full-length fusion protein has better antigenicity.However cross reactions with HPV6, 11 and 18 were found.It can be applied in serological screening reagents for HPV infection and associated cancer.

Objective To explore the HLA-A2 restriction and immunogenicity of 5 previously identified HCV-speeific CTL epitopes. Methods Based on T2 cell, to explore the HLA-A2 restriction of previously identified HCV-specific CTL epitopes by MHC-peptide complex stabilization assay;To detect pep-tide-specific CTL in HLA-A2~+ PBMC stimulated by HLA-A2-restricted peptides by intracellular cytokine staining(ICS) and ELISPOT; To explore the cytotoxicity of peptide-specific CTL to same peptide-loaded T2 cells (target cells) by CTL cytotoxicity test. Results Among 5 previously identified CTL epitopes NS4b_78 (SMMAFSAAL) and NS5a_367 (TVSSALAEL) have high-affinity for HLA-A2 molecules(FI 1) ;ELISPOT results shown that NS4b_78(SMMAFSAAL) and NSSa_367(TVSSALAEL) induced high levels of IFN-γ-se-creting cells [(60±6) SFC/10~4 PBMC vs (4±1 ) SFC/10~4 PBMC, P < 0.01 ; (10 ± 3 ) SFC/10~4 PBMC vs (2±1 ) SFC/10~4 PBMC, P <0.01, respectively] ;ICS results indicated that there were high percentages of CD8~+ IFN-γ~+ T cells in total CD8~+T cells stimulated by these peptides [(2.33 ±0.22 ) % vs (0.05±0.01)%, P <0.001 ; (0.36±0.06)% vs (0.03±0.01)%, P <0.001, respectively]. Furthermore,peptide-specific CTL could effectively kill same peptide-loadcd T2 cells. Conclusion NS4b_78 (SMMAF-SAAL) and NSSa_367 (TVSSALAEL) were identified as HLA-A2-restricted CTL epitopes which could in-duce immune response in vitro.

Objective To express in prokaryotic system and to analyze the antigenic specificity of EB virus(EBV) latent membrane protein 2(LMP2) multi-epitopes gene rich of T cell and B cell epitopes.Methods Using on-line prediction service, T cell epitopes and B cell epitopes of EB virus latent membrane protein 2 were predicted. The genes rich of CTL and th cell epitopes were selected as the candidate gene sequences, while B cell epitopes around them were taken into account. The finial selected multi-epitope gene was synthesized after being optimized according to prokaryotic codon bias and inserted into prokaryotic expression vector pET32a( + ) to get the recombinant plasmid: pET32a( + )/EBV-LMP2 multi-epitopes. After transformed into E. coli BL21 (DE3) and induced by IPTG, the target multi-epitopes gene can be expressed as Trx-His fusion protein. The expression products can be identified by SDS-PAGE and Western blot. Moreover, rabbit serum antibody to EBV membrane protein and nasopharyngeal carcinoma(NPC) patient serum were used respectively to detect the antigenic specificity of the multi-epitopes. Meanwhile, 6-8 weeks female BALB/c mice were immunized with EBV-LMP2 multi-epitope at 2 week intervals, three times in all, Trx-His protein and PBS were set as the control groups. At the second week after the last immunization, the mice were sacrificed. LDH and indirect ELISA were taken to detect the specific spleen CTL activityand specific IgG in serum, which reflected the immunogenicity of the EBV-LMP2 multi-epitope. Results Two amino acid sequences which locate at the LMP2 (aa195 -232 ) and LMP2 (aa419-436 ) were selected and connected in series to be the target gene. The recombinant plasmid containing EBV-LMP2 multi-epitope gene successfully constructed and the target protein was expressed in E. coli BL21 ( DE3 ). The relative molecular mass(Mr) of The expression products is about 27 × 103 , which matches up to the expected Mr. The antigenic specificity of the multi-epitopes protein was identified by Western blot and the multi-epitopes protein also can be detected by rabbit serum antibody to EBV membrane protein and NPC patient serum respectively. In the result of the animal experiment, EBV-LMP2 multi-epitope was able to induce the specific CTL activity in BALB/c mice. With the increasing of the effector: target ( E: T) 1: 5,1: 10, 1: 25, the CTL activity was also increased wih( 12.52% + 2.59% ), (21.80% + 1.08% ), (23.68% + 3.74% ) respectively; EBV-LMP2 multi-epitope was able to induce LMP2-specific antibody response(A490 =0.258 +0.040) as compared with Trx-His protein(A490 =0.095 +0.011) and PBS(A490 =0.068 +0.014,P＜0.05＝. Conclusion The EBV-LMP2 multi-epitopes gene was designed successfully and expressed precisely in prokaryotic expression system with good antigenicity and immunogenicity.

Objective To observe the functional connectivity (FC) pattern linking the hippocampus with the rest of the brain in ischemic stroke patients with cognitive dysfunction,especially the default mode network (DMN).Methods Resting-state functional connectivity magnetic resonance imaging (fMRI) was performed on 15 ischemic stroke patients with cognitive dysfunction (the patient group) and 10 normal elderly controls.The bilateral hippocampus was the region of interest.Correlation analyses yielded a mapping of cerebral functional connectivity activation for both groups.Results Compared with the healthy controls,the patient group showed weakened functional connectivity between the hippocampus and other regions including the cingulate gyrus,the superior,middle and inferior frontal lobes,the inferior parietal lobule and the superior temporal gyrus.But there was enhanced functional connectivity with the cerebellar posterior lobe,the occipital lobe,the medial temporal lobe,the precuneus and the calcarine.The results were significantly different between the two groups.Conclusion The functional connectivity pattern of the hippocampus is impaired in stroke patients with cognitive dysfunction.Reduced functional connectivity between brain regions may be one cause of cognitive dysfunction after stroke,and enhanced functional connectivity may be an appropriate compensatory mechanism.

UL48 plays essential role in replication of MDV genome and interacts with UL36 as well as other MDV tegument proteins.To investigate the interaction between UL48 and UL36 during MDV oncogenisis,antibody against UL48 was prepared and characterized in current study.UL48 gene was amplified from MDV-Ⅰ genome and then subcloned into pTYB1 and pGEX-4T3 vectors for UL48 expression with induction of IPTG in BL21(DE3) E..coli cells.Chitin-sepharose and Glutathion-sepharose were,respectively,used to purify fusion protein intein-UL48 and GST-UL48.Four subcutaneous injections of intein-UL48 fusion protein were done on the lower back and the thigh of rabbit and then other three injections with an interval 10 days.The titer of antibody was measured by the sandwich ELISA with UL48 protein isolated from GST-UL48 after cleavage of thrombin.Western blot was carried out for specificity analysis of antibody against UL48 protein.The results suggested that UL48 antibody was succesfully prepared,and its titer was 1 ∶ 512 000.

Ischemic stroke is a major health crisis causing high mortality and morbidity. The key treatment relies on the rapid intervention to dissolve thrombus, to reduce bleeding side effect and re-canalize clotted blood vessels using clot lysis drugs. Tissue plasminogen activator (tPA) is the only FDA-approved drug for ischemic stroke, but it has many limitations in clinical use. In recent years, the development of thrombolytic drugs and treatment strategies based on tPA has been progressed rapidly. Here we review the recent progress in this field, including the contributions from us and others, to promote the future development of novel thrombolytic drugs.
Brain Ischemia/drug therapy* ; Fibrinolytic Agents/therapeutic use* ; Humans ; Research/trends* ; Stroke/drug therapy* ; Thrombolytic Therapy/trends* ; Tissue Plasminogen Activator/therapeutic use*

Objective: To elucidate the anti-inflammatory mechanism of Reduning Injection (RDN) by analyzing the potential biomarkers and metabolic pathways of the carrageenan-induced inflammatory model from the overall metabolic level. Methods: Rat inflammatory model was established by carrageenan. UPLC-Q-TOF/MS was used to detect and analyze changes of endogenous metabolites in the serum and urine of carrageenan-induced inflammatory rats. Combined with multivariate analysis and databases analysis, inflammatory-related potential biomarkers were screened and identified to analyze possible metabolic pathways. The reliability and biological significance of these biomarkers was verified by metabolic network analysis and correlation analysis with pharmacodynamic indicators. Results: A total of 16 potential biomarkers were screened and identified by multivariate analysis and metabolite databases, among which 13 species could be adjusted by RDN. The metabolism pathway analysis revealed that histidine metabolism, sphingolipid metabolism, and tyrosine metabolism were greatly disturbed. Their biomarkers involved urocanic acid, sphingosine, and norepinephrine, all of which showed a callback trend after RDN treatment. The three biomarkers had a certain correlation with some known inflammatory-related small molecules (histamine, arachidonic acid, Leukotriene B4, and PGE