The objective of this study was to develop a rapid and precise detection technique for por-cine Senecavirus A(SVA)employing reverse transcription recombinase polymerase amplification(RT-RAA)in conjunction with CRISPR Cas13a technology.Additionally,the study aimed to opti-mize the assay's reaction conditions to enhance amplification efficiency.Eight RT-RAA primer sets were designed based on the conserved gene sequence of porcine SVA,and a series of reaction condi-tions were evaluated to refine the RT-RAA reaction system.Subsequently,CRISPR-derived RNA(crRNA)sequences were developed and selected to construct the RT-RAA-CRISPR reaction sys-tem.The method's specificity was determined by examining six prevalent porcine pathogenic nucleic acids,while its sensitivity was assessed using SVA cRNA standards quantified by digital PCR.The method's stability and the consistency of clinical sample analysis were also evaluated.The findings revealed that the optimized RT-RAA and CRISPR reaction systems exhibited the highest amplifi-cation efficiency at a reaction temperature of 37 ℃.Among the eight crRNAs,five were identified as exhibiting the strongest detection signals.The formulated RT-RAA-CRISPR Cas13a method demonstrated exceptional specificity,showing no cross-reactivity with other common porcine disea-ses,including ASFV,PRRSV,PEDV,PCV2,CSFV,and PRV.The method achieved high sensitivi-ty,detecting as low as 0.86 copies/μL of SVA,and exhibited stable fluorescence output,robust re-producibility,and the ability to complete clinical sample analysis within 50 minutes.Consistency e-valuation with six positive and 58 negative samples indicated 100％agreement in outcomes.These results substantiate that the study successfully established a rapid and specific RT-RAA-CRISPR Cas13a detection method for the on-site identification of porcine Senecavirus A,demonstrating high specificity and sensitivity,and holds promise for application in SVA monitoring and control initia-tives.

The objective of this study was to develop a rapid and precise detection technique for por-cine Senecavirus A(SVA)employing reverse transcription recombinase polymerase amplification(RT-RAA)in conjunction with CRISPR Cas13a technology.Additionally,the study aimed to opti-mize the assay's reaction conditions to enhance amplification efficiency.Eight RT-RAA primer sets were designed based on the conserved gene sequence of porcine SVA,and a series of reaction condi-tions were evaluated to refine the RT-RAA reaction system.Subsequently,CRISPR-derived RNA(crRNA)sequences were developed and selected to construct the RT-RAA-CRISPR reaction sys-tem.The method's specificity was determined by examining six prevalent porcine pathogenic nucleic acids,while its sensitivity was assessed using SVA cRNA standards quantified by digital PCR.The method's stability and the consistency of clinical sample analysis were also evaluated.The findings revealed that the optimized RT-RAA and CRISPR reaction systems exhibited the highest amplifi-cation efficiency at a reaction temperature of 37 ℃.Among the eight crRNAs,five were identified as exhibiting the strongest detection signals.The formulated RT-RAA-CRISPR Cas13a method demonstrated exceptional specificity,showing no cross-reactivity with other common porcine disea-ses,including ASFV,PRRSV,PEDV,PCV2,CSFV,and PRV.The method achieved high sensitivi-ty,detecting as low as 0.86 copies/μL of SVA,and exhibited stable fluorescence output,robust re-producibility,and the ability to complete clinical sample analysis within 50 minutes.Consistency e-valuation with six positive and 58 negative samples indicated 100％agreement in outcomes.These results substantiate that the study successfully established a rapid and specific RT-RAA-CRISPR Cas13a detection method for the on-site identification of porcine Senecavirus A,demonstrating high specificity and sensitivity,and holds promise for application in SVA monitoring and control initia-tives.

Tourette syndrome(TS)is a chronic neurodevelopmental disorder.Acupuncture can effectively improve the clinical symptoms of TS patients.This article systematically summarized the clinical research status of acupuncture for the treatment of TS in recent years from the aspects of characteristic acupuncture methods,characteristic needles and comprehensive therapies,and put forward suggestions and prospects for systematically elaborating the peripheral-central mechanism of acupuncture for TS around the intestinal immunity and brain network mechanism in the future,so as to provide reference for optimizing clinical research and treatment.

Objective To explore the effect of drug injection under bronchoscopy on the retreatment of smear positive cavitary pulmonary tuberculosis. Methods From June 2016 to December 2017,164 cases of pul-monary tuberculosis with smear Yang cavity type were selected,which were divided into 2 groups according to the random digital table method,each groups has 82 cases.The control group received routine treatment,the observa-tion group underwent bronchoscopy with Kangfuxin Liquid combined with drug injection therapy.The clinical effect of the two groups,the changes of lung function before and after treatment and the improvement of clinical symp-toms were compared.Results The clinical curative effect of the treatment group was better than the control group, which difference was statistically significant(P < 0.05). FEV1,FEV1/FVC,PEF index in the observation group were better than the control group,which difference was statistically significant(P<0.05).The complication rate of observation group was lower than the control group,which difference was statistically significant(P < 0.05). Conclusion Drug injection under bronchoscopy can significantly improve the clinical efficacy and lung function and promote clinical symptoms in patients with retreated smear positive pulmonary tuberculosis.It is worthy of popu-larization and application.

It is not fully clear why there is a higher contribution of pluripotent stem cells (PSCs) to the chimera produced by injection of PSCs into 4-cell or 8-cell stage embryos compared with blastocyst injection. Here, we show that not only embryonic stem cells (ESCs) but also induced pluripotent stem cells (iPSCs) can generate F0 nearly 100% donor cell-derived mice by 4-cell stage embryo injection, and the approach has a "dose effect". Through an analysis of the PSC-secreted proteins, Activin A was found to impede epiblast (EPI) lineage development while promoting trophectoderm (TE) differentiation, resulting in replacement of the EPI lineage of host embryos with PSCs. Interestingly, the injection of ESCs into blastocysts cultured with Activin A (cultured from 4-cell stage to early blastocyst at E3.5) could increase the contribution of ESCs to the chimera. The results indicated that PSCs secrete protein Activin A to improve their EPI competency after injection into recipient embryos through influencing the development of mouse early embryos. This result is useful for optimizing the chimera production system and for a deep understanding of PSCs effects on early embryo development.
Activins ; metabolism ; Animals ; Cells, Cultured ; Embryonic Development ; Germ Layers ; metabolism ; Mice ; Pluripotent Stem Cells ; cytology ; metabolism

Objective To investigate the impact of AG490 on the blood-brain barrier (BBB ) permeability and the expression of interleukin-6 (IL-6 )and tumor necrosis factor-α(TNF-α)after traumatic brain injury (TBI)in rats. Methods A total of 144 healthy male SD rats were randomly divided into a control group,a trauma group,and an AG490 intervention group (n=48 in each group). The rats in each group were redivided into four subgroups (4 h,1 d,3 d,and 7 d subgroups)according to the time points after cerebral injury (n=12 in each subgroup). A brain trauma models were induced by hydraulic shock method. Evans blue was used to determine the changes of the BBB permeability after cerebral injury in each group. Real-time fluorescence quantitative PCR was to detect the expression levels of TNF-αand IL-6 mRNA in rat brain tissue. Immunohistochemistry was used to detect the expression of human phospho tyrosine kinase (P-JAK2). Results (1)The permeability of BBB:The permeability of BBB increased at 4 h,1 d,3 d and 7 d after brain injury in the trauma group (Evans blue permeation:10. 4 ± 1. 2,16. 0 ± 1. 4,22. 3 ± 2. 0,and 8. 4 ± 0. 9μg/g,respectively). Compared with the control group, there were significant differences (all P<0. 01). The Evans blue permeation of the AG490 intervention group were 9. 1 ± 1. 0,12. 8 ± 1. 1,17. 5 ± 1. 4 and 7. 1 ± 0. 8μg/g,respectively at each time point,and they were all significantly lower than those of the trauma group (all P<0. 01). (2)The expression of IL-6 and TNF-α mRNA:The expression levels of IL-6 mRNA and TNF-α mRNA at 4 h,1 d,3 d and 7 d after traumatic brain injury in the trauma group were 2. 31 ± 0. 35,2. 73 ± 0. 35,3. 32 ± 0. 29,2. 14 ± 0. 24 and 7. 46 ± 1. 18,9. 42 ± 1. 54,13. 76 ± 1. 89,and 6. 28 ± 1. 00,respectively,they were all significantly higher than those of the control group (all P<0. 01). The expression levels of IL-6 mRNA and TNF-α mRNA of the AG490 intervention group were 1. 14 ± 0. 22,1. 54 ± 0. 23,1. 94 ± 0. 32,1. 26 ± 0. 21 and 5. 57 ± 0. 88, 7. 78 ± 1. 02,11. 51 ± 1. 29,and 5. 05 ± 0. 97,respectively,they were all lower than those of the trauma group,but they still higher than the control group. There were significant differences (all P<0. 01). (3 )The expression of P-JAK2:The expression levels of P-JAK2-positive cells at each time point after traumatic brain injury in the trauma group were significantly higher than the control group (all P<0. 01),they were 17. 4 ± 2. 7,56. 2 ± 6. 7,26. 1 ± 5. 4,and 15. 3 ± 2. 5,respectively;those of the AG490 intervention group were 12. 2 ± 1. 4,41. 5 ± 4. 6,19. 4 ± 4. 1,and 9. 6 ± 2. 0,respectively,they were all lower than those of the trauma group,but still higher than the control group. There were significant differences (all P<0. 01). Conclusion During the acute phase after TBI,AG490 may activate the factor signaling pathways by inhibiting the non-receptor tyrosine kinase/signal transduction and transcription,significantly inhibit the expression of brain tissue inflammatory cytokines IL-6 IL-6 and TNF-α,reduce the BBB damage,and help to reduce secondary brain injury.

P2X7 is the most important subtype of the ATP receptors known so far. Recent investigations showed that the downstream signaling pathway of P2X7 is coupled with several key inflammatory molecules including IL-1beta and IL-18, this suggests P2X7 might have roles in the inflammatory diseases. Moreover, attenuation of P2X7 by selective antagonists in vitro and knockout mice in vivo reducing the inflammatory response indicated that P2X7 is a potential therapeutic target for inflammatory diseases. However, most previous studies on P2X7 were focused on nerve system diseases most, while its effects in inflammatory respiratory diseases, especially in asthma, chronic obstructive pulmonary disease (COPD) and lung cancer have been poorly investigated. In this paper, we reviewed the research progress on the structure, distribution, biological activities of P2X7 and its relationship with inflammatory respiratory diseases including asthma, COPD and lung cancer, along with the development of P2X7 antagonist as therapeutics.

We established a cell based high throughput screening model by calcium assay on fluorometric imaging plate reader for finding modulators of TRPV3. The TRPV3 expression vector was transfected into HEK-293 and stable cell line expressing TRPV3 was selected with antibiotics. Upon TRPV3 specific modulators stimulated, pharmacological characteristics of TRPV3 over expression cell line were detected by calcium assay on fluorometric imaging plate reader. Assay conditions were optimized and stability of the model was observed. The reliability and accuracy of application to 96 and 384 well format high throughput screening were also evaluated. A stable HEK-293 cell line highly expressing TRPV3 was established. TRPV3 specific modulators could modulate calcium signal through TRPV3 in dose dependent manner. Optimized screening condition was established by assay development. This model is stable and sensitive, and meets the requirement of high throughput screening by Z'factor validation and Spiking test. This cell model can be applied to screening TRPV3 modulators by calcium assay.
Calcium ; metabolism ; Cell Line ; Humans ; Ion Transport ; Kidney ; cytology ; Membrane Transport Modulators ; metabolism ; Models, Biological ; TRPV Cation Channels ; biosynthesis ; genetics ; Transfection

Transient receptor potential A1 (TRPAl) is a cold sensitive cation channel, which could also be activated by various pungent compounds. As a transduction channel in a number of sensory modalities, TRPAl expressing in heterogonous systems serves to provide great convenience in pharmacological analysis and functional investigation. Due to cellular toxicity, establishment of stable TRPAl cell line has always been challenging. Nevertheless, the first stable human embryonic kidney (HEK-293) cell line with un-controlled expression of TRPAl was successfully established. It was also confirmed that this stable cell line retained TRPAl expression for more than 25 passages in culture. The functional analysis of the cell response verified the stability and specificity of this novel recombinant TRPAl cell line. Altogether, the data indicated this TRPAl-HEK cell line would be a useful tool for functional analysis of TRPAl and for the development of high throughput screening (HTS) compatible assay in the effort to identify TRPAl modulators.

AIM:To study the blocking effect of shRNA on the expression of PSMA gene in LNCaP cell line by using shRNA eukaryotic expression vector.METHODS:Three pairs of DNA templates coding shRNA,synthesized against PSMA and cloned into the vector pSilencer 2.1-U6-neo,which was named pSilencer 2.1-U6-neo-shRNA,were identified by restriction endonuclease digestion analysis and DNA sequencing.LNCaP cells were then transfected with these three pSilencer 2.1-U6-neo-shRNAs and the negative control pSilencer 2.1-U6-neo-NC.After G418 selection,the cells were selected and the interfering effect was detected by RT-PCR and Western blotting.The biological behaviours of the transfected LNCaP cells were also tested.RESULTS:Restriction endonuclease digestion analysis and DNA sequencing results all showed that the 3 target segments were cloned into pSilencer 2.1-U6-neo vector respectively.After transfected into LNCaP cells,the inhibitory ratio of PSMA mRNA was 33.15%,9.26% and 41.97% respectively,and that of PSMA protein was 26.26%,6.47%,40.69% respectively.The p-shRNA3 was chosen to test the cell growth and its invasive power in vitro.The results showed that after interfering,the invasiveness of LNCaP cells were enhanced.CONCLUSION:The vector-based shRNA on PSMA gene effectively knocks down the PSMA gene expression.The successful construction of PSMA shRNA makes it possible for further study of the interaction between PSMA and prostate cancer.