PAC1 is the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) preferring receptor, which belongs to class B G protein-coupled receptors (GPCR) family. PAC1 mediates the most effects of PACAP as neurotransmitter, neuroregulator and neuroprotectant, while its high expression has close relationship with some physiological and pathological processes such as nerve-injury and tumor. To further understand the function of PAC1, a cell line that expressed inducible PAC1 was constructed to achieve Doxycycline (Dox) dependent expression of PAC1 in CHO (Chinese hamster ovary) cell using the improved Tet (tetracycline)-on Advanced System. First, the PAC1-EYFP fusion gene composed of PAC1 gene and gene encoding EYFP (enhanced yellow fluorescent protein) was sub-cloned to the tetracycline response element pTRE-Tight vector to construct the recombinant vector pEYFP-PAC1-EYFP by double enzyme digestion. Second, the tetracycline regulation components pTet-On advanced vector and the response element pTRE-PAC1-EYFP vector were both introduced into CHO cells successively and the positive clones were screened with G418 and hygromycin respectively. Third, the controlled expression of PAC1-EYFP in CHO was induced by tetracycline analogues Dox in different concentrations and the different levels of receptor PAC1-EYFP were detected. The results of fluorescence analysis and western blotting show that the cell strain with Dox dependent expression of PAC1-EYFP named PAC1-Tet-CHO was obtained. Moreover, in PAC1-Tet-CHO cells the expression of PAC1-EYFP was induced by Dox in a dose-dependent manner. The inducible expression of PAC1 still was stable after sub-culturing for more than 10 passages. It was also found by MTT assay that the higher expression level of PAC1 endowed the cells with higher proliferative viabilities. The construction of controlled expression system of PAC1 will lay a foundation for the further research on PAC1 profiles.
Animals ; Blotting, Western ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Genetic Vectors ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I ; biosynthesis

Objective To evaluate the effect of traditional Chinese medicine nursing technology among patients with ulcerative colitis.Methods Totally 170 patients with ulcerative colitis were recruited.All patients received routine treatment and received transitional care in inpatient department including diet guidance,illness response and anal guidance on contraction exercise,etc.What's more,the observation group implemented traditional Chinese medicine nursing techniques,including auricular pressure paste,acupuncture point application,acupressure,moxibustion,etc.After 7 and 14 days,we compared the patients' disease activity,sleep quality by Grading scale for Traditional Chinese Medicine symptoms and signs of ulcerative colitis,improved Mayo question,Pittsburgh Quality Sleep Index(PQSI).Results On the 7th and 14th day after intervention,the scores of symptoms and signs in the observation group were (9.73 ± 3.49),(4.54 ± 2.75) points,lower than those in the control group (10.72 ± 4.00),(8.22 ±3.06) points,and the difference was statistically significant (t =1.973,7.878,P =0.042,0.000).On the 14th day after intervention,the Mayo score of observation group was (3.33 ± 1.82) points,lower than that of control group (4.56 ± 1.77) points.The difference was statistically significant (t =4.278,P =0.000).The PSQI score of observation group was (5.67 ± 2.87) points,lower than that of control group (8.53 ± 3.13)points.The difference was statistically significant (t =5.934,P =0.000).Conclusions Traditional Chinese medicine transitional care could effectively improve the symptom of disease and sleep quality among patients with ulcerative colitis.

Objective:To grasp the distribution of fine antigenic epitope profiles of nucleoprotein (NP) and glycoprotein (GP) fragments of Crimean-Congo hemorrhagic fever virus (CCHFV) and to clarify the value of dominant antigenic epitopes in laboratory testing of Crimean-Congo hemorrhagic fever (CCHF).Methods:In a minimal synthetic short peptide consisting of 8 amino acids was segmentally expressed by CCHFV YL04057 strain using a modified bio-peptide synthesis method from 2014 to 2021 in the laboratory of Xinjiang University, College of Life Sciences. Using CCHFV polyclonal antibody or monoclonal antibody 14B7 (IgM) or CCHFV-positive sheep serum as antibodies, the minimal antigenic epitopes (BCEs) with antigenic activity on NP and GP fragments were identified by immunoblotting, and the obtained BCEs with sequence polymorphism were spatially clustered with CCHFV from different regions using the neighbor-joining method to determine the combination mode of BCEs with geographical correlation of regional distribution, to explore its application in establishing serological diagnosis. A prokaryotic expression plasmid (pET-32a), an E. coli expression plasmid (pGEX-KG) and a prokaryotic expression plasmid with an incomplete glutathione (GST188) tag (pXXGST-ST-1) were used to construct and express six dominant antigenic epitopes of different peptide lengths on NP fragments, and an indirect Enzyme-linked immunosorbent assay (ELISA) was established. CCHF sheep serum identified by immunofluorescence assay (IFA) was used as a control, and the specificity, sensitivity and overall compliance of the recombinant proteins with different peptide lengths of antigenic epitopes with IFA assay results were statistically analyzed. Results:CCHFV, NP and GP fragments had a total of 30 antigenically active BCEs, among which the core intermediate fragment NP2 (aa 170 th-305 th), which had a concentration of antigenic epitopes in the NP fragment, has 6 BCEs, and the NP1 (aa 1 st-200 th) and NP3 (aa 286 th-482 nd) at both ends have 9 BCEs; the Gc (aa 1 st-558 th) and Gn (aa 533 th-708 th) fragments of the GP fragment have 14 BCEs and a long antigenic peptide (AP) containing 15 amino acids, and the amino acid sequence homology of the NP fragment BCEs was 97.1% and that of the GP fragment BCEs was 89.1%. There was a significant difference ( P=0.0281, P<0.05). Among the 9 BCEs with sequence polymorphism in the GP fragment, 6 combined BCEs from GnEc1, GnE2, GnE4, GcE3, GcE6 and GcAP-4 (Ap) could cluster 15 CCHFV strains from different regions of the world into 5 geographical taxa, AsiaⅠ, AsiaⅡ, AficaⅠ, AficaⅡ and Europe. The constructs expressing PET-32a-NP (full length), PGEX-KG-NP2 (aa 170 th-305 th), pGEX-KG-NP2-1 (aa 235 th-275 th), PGEX-KG-NP2-1-1 (aa 237 th-256 th), pXXGST-1-NP2-1-2 (aa 250 th-265 th) and PGEX KG-NP2-1-3 (aa 260 th-276 th), six recombinant proteins CCHFV NP rabbit polyclonal antiserum (pAb) Western Blotting reaction positive, 33 sheep sera tested by IFA XHF as a reference, the sensitivity of the assay established by indirect ELISA using the recombinant proteins constructed from two fragments of NP2 and NP2-1 as antigens. The sensitivity, specificity and overall compliance were the best, with 73.4% (11/15) and 66.7% (10/15) for sensitivity, 100% (18/18) and 94.4% (17/18) for specificity, and 87.9% (29/33) and 81.8% (27/33) for overall compliance. Conclusion:CCHFV NP and GP are distributed with a high number of BCEs with antigenic immunoreactivity, among which the dominant antigenic epitopes are of high value in the laboratory serological diagnosis of CCHF.

OBJECTIVETo explore the biological characteristics and the immuno-suppression function of tolerogenic dendritic cells (tDC) induced by tacrolimus.

METHODSHuman monocytes derived from peripheral blood were cultured in the cGMP-compliant CellGro DC medium supplemented with GM-CSF and IL-4 to obtain dendritic cells (DCs), and 0.1 μmol/L immunosuppressive drug tacrolimus was added to the culture medium at the third and fifth day to obtain tDCs. The molecular markers of them and the livability were assayed by flow cytometry. Then the tolerance functionality of tDCs induced by many agents and these tDCs modulated allogeneic CD4 T cells was determined via CFSE proliferation assay. And the research also analyzed the biological characters and immunosuppression function of tDCs induced by tacrolimus after storing.

RESULTStDCs induced by tacrolimus exhibit a typical tolerogenic phenotype, whose level of costimulatory molecules CD80, CD83, CD86 and HLA-DR is (2.95 ± 1.32)%, (2.33 ± 1.60)%, (90.02 ± 7.42)% and (91.80 ± 6.18)%, respectively. It's survival rate was (85.2 ± 4.72)%. And immunosuppressive drugs didn't influence the differentiation of tDCs from monocytes. tDCs induced by immunosuppressive drugs dexamethasone, cyclosporin A and tacrolimus had lower immunogenic than control DCs as CD4+ T proliferation rate of tDCs induced by tacrolimus is 0.42% and could not primed allogeneic CD4+ T cells proliferation. Functional analyses showed that tDCs induced by tacrolimus can more effectively suppressed mature DC-induced T cell proliferation than other tDCs, whose inhibition rate can reach (67.01 ± 19.73)%. Importantly, tDCs induced by tacrolimus had phenotypical and functional stability after storing.

CONCLUSIONtDCs induced by tacrolimus with tolerance functionality are a promising cellular therapeutic for immunomodulation.

Cell Proliferation ; drug effects ; Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; Humans ; Immune Tolerance ; drug effects ; Lymphocyte Culture Test, Mixed ; Tacrolimus ; pharmacology

ObjectiveTo investigate the dietary preference and nutritional knowledge needs of the elderly people who dined at meal service sites. MethodsUsing the form of stratified and convenience sampling method with self-designed questionnaire was used， in November 2021， to select 700 elderly people who dine at meal service sites in 7 jurisdictions in Shanghai were selected， and a self-designed questionnaire was used to investigate the basic information. Results91.64% of the elderly surveyed would eat at relatively fixed meal service sites， and the total Dietary Diversity Score （DDS9） was 3.56±1.46. 41.45% of the elderly with diseases preferred unhealthy cooking methods. Only 8.03% of the surveyed seniors said they were unwilling to accept targeted and personalized nutrition tips and reminders. Multivariate logistic regression analysis showed that the probability reaching the “understanding” level of “Food Guide Pagoda for Chinese Residents” and “Four Principles Recommended by the Core Dietary Guidelines for the Elderly” was different in the elderly with different education levels. The willingness of the elderly to expect to receive different nutrition tips and reminders was related to whether they cared about the corresponding contents. There was a statistically significant difference （P<0.05） among the elderly who were concerned about different health problems in terms of the willingness to receive different nutritional tips. There were significant differences in the proportion of elderly people with different health status for intervention （χ²=5.402， P<0.05）. ConclusionThe elderly who dine at meal service sites are highly dependent on the sites， have a low level of dietary diversification， and do not have a high degree of understanding of nutrition-related knowledge， and have a high demand for targeted nutritional interventions. Nutritional interventions for the sick elderly should be piloted through multiple channels.