Abstract: Objective　To analyze the treatment outcomes of elderly patients with pulmonary tuberculosis in Chongqing, so as to provide reference for the prevention and control strategies of the epidemic of elderly pulmonary tuberculosis. Methods　The data of tuberculosis cases aged ≥65 years in Chongqing from 2015 to 2020 were collected from the National Health Insurance Information Project Disease Prevention and Control Information system. Descriptive statistical methods were used to analyze the data. Results The registration rates of elderly active pulmonary tuberculosis patients and etiological positive patients were 110.95/10-5and 32.25/10-5 in 2015 and 84.06/10-5 and 57.29/10-5 in 2020. The annual decline rate of active tuberculosis registration was 5.40%, and the annual increase rate of pathogenic-positive tuberculosis registration was 12.18%. The registration rates of active tuberculosis patients and etiological positive patients in the whole population were 70.75/10-5 and 17.63/10-5 in 2015 and 50.34/10-5 and 29.14/10-5 in 2020. The annual decline rate of active tuberculosis registration was 6.58%, and the annual increase rate of pathogenic-positive tuberculosis registration was 10.57%. From 2015 to 2020, a total of 25 931 cases of elderly pulmonary tuberculosis were registered, of which 21 374 (82.43%) cases were successfully treated and 4 010 (15.80%) cases had unfavorable outcomes. The proportion of cured and death patients showed an increasing trend year by year (χ2trend=313.853, 100.502, P<0.01). From 2015 to 2020, the average annual successful treatment rate of elderly pulmonary tuberculosis in the whole city was 82.43%, with the lowest rate in southeast Chongqing (74.23%), followed by urban areas (81.99%). The success rate of elderly pulmonary tuberculosis treatment in the whole city, west Chongqing, northeast Chongqing and southeast Chongqing showed a downward trend year by year (χ2trend=230.199, 35.278, 108.076, 112.130, all P<0.01), with annual decline rates of 2.77%, 2.26%, 3.0% and 4.12%, respectively. Among the registered elderly patients, female, 65-<75 years old, Han nationality, newly diagnosed, no complications, and negative for etiology (χ2=15.234, 255.910, 146.842, 179.998, 25.575, 131.170, P<0.01) had higher success treatment rates. Conclusions The prevalence of pulmonary tuberculosis in the elderly population in Chongqing City is declining, but the positive registration rate of etiology is increasing annually, and the success rate of treatment is decreasing. Therefore, it is necessary to strengthen the systematic management, publicity and education of elderly patients (especially those in southeast Chongqing, male, positive patients and severe patients) to effectively control the epidemic of tuberculosis in the elderly.

Based on the transcriptome database of Salvia miltiorrhiza, specific primers were designed to clone a full-length cDNA of ent-kaurene oxidase synthase (SmKOL) using the RACE strategy. ORF Finder was used to find the open reading frame of SmKOL cDNA, and ClustalW has been performed to analysis the multiple amino acid sequence alignment. Phylogenetic tree has been constructed using MEGA 5.1. The transcription level of SmKOL from the hairy roots induced by elicitor methyl jasmonate (MeJA) was qualifiedby real-time quantitative PCR. The full length of SmKOL cDNA was of 1 884 bp nucleotides encoding 519 amino acids. The molecular weight of the SmKOL protein was about 58.88 kDa with isoelectric point (pI) of 7.62. Results of real-time quantitative PCR analyses indicated that the level of SmKOL mRNA expression in hairy roots was increased by elicitor oMeJA, and reached maximum in 36 h. The full-length cDNA of SmKOL was cloned from S. miltiorrhiza hairy root, which provides a target gene for further studies of its function, gibberellin biosynthesis and regulation of secondary metabolites.
Amino Acid Sequence ; Cloning, Molecular ; Computational Biology ; methods ; Cytochrome P-450 Enzyme System ; chemistry ; genetics ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Protein Structure, Tertiary ; Salvia miltiorrhiza ; enzymology

Objective:To observe the effect of acupuncture on the expression of mitochondrial proteome in hippocampus of senescence-accelerated mouse prone g (SAMPg) mice models with Alzheimer disease (AD),and to explore the possible protective mechanism of acupuncture on mitochondria.Methods:Sixty 6-month-old male SAMP8 mice were randomly divided into an acupuncture at acupoint group,an acupuncture at non-acupoint group and a model group,20 mice in each group.The 20 male senescence-accelerated mouse/resistance 1 (SAMR1) mice of the same age were used as a normal control group.Shenshu (BL 23),Baihui (GV 20),Xuehai (SP 10) and Geshu (BL 17) were selected for acupuncture intervention in acupuncture at acupoint group.After an 8-week intervention,mitochondrial tissues were extracted from the hippocampus.Differentially expressed proteins were identified by subcellular organelle proteomics.Western blot was used to verify the expressions of some related proteins in hippocampal mitochondria.Results:Compared with the model group,there were 13 differentially expressed protein spots in the acupuncture at acupoint group,of which,9 were up-regulated,including neurofilament light polypeptide (NFL),actin (cytoplasmic 1,database ID:ACTB),tubulin beta-2A chain (TBB2A),tropomodulin-2 (TMOD2),pyruvate dehydrogenase E1 component subunit beta (PDHE1-β),NADH-ubiquinone oxidoreductase 75 kDa subunit (database ID:NDUS1),heat shock cognate 71 kDa protein (HSC71),pyruvate dehydrogenase E1 component subunit alpha (PDHE1-α) and ATP synthase beta subunit (ATP-β);4 were down-regulated,including glial fibrillary acidic protein (GFAP),pyruvate dehydrogenase phosphatase 1 (PDP1),mitochondrial-processing peptidase subunit alpha (MMP-α) and adenosine kinase (ADK).According to the information provided in the protein database,most of the differentially expressed proteins involve the regulation of mitochondrial function and structure.The expression levels of NFL and TBB2A in the normal control group and the acupuncture at acupoint group were significantly higher than those in the acupuncture at non-acupoint group (P＜0.05).ATP-β and NDUS1 expression levels were significantly higher in the acupuncture at acupoint group than those in the acupuncture at non-acupoint group (P＜0.05);there was no significant difference between the acupuncture at non-acupoint group and the model group (P＞0.05).Conclusion:Acupuncture may achieve the potential therapeutic effect on AD by regulating the structure and functional proteins of hippocampal mitochondria.

Abstract?AIM: To investigate mechanism of bradykinin ( BK) on inflammations of retinal pigment epithelium ( RPE) cells.?METHODS: ARPE -19 cells were cultured in vitro, stimulated by 100nM BK for 24h. Cell morphology changes were observed by microscope, and BK receptor localization was detected through cell immunofluorescence. Changes of Ca2+in BK and BR antagonist stimuli were detected by laser scanning confocal microscopy.The expressions of COX-1, COX-2, eNOS and iNOS protein in control group and BK group were detected by Western Blot.?RESULTS: After the stimulation of BK, there was no significant changes of ARPE-19 cells in morphology.Kinin B1 receptors ( B1R ) and B2 receptors ( B2R ) could be detected in ARPE-19 cells.Compared with control group, Ca2+concentrations significantly increased in BK group; in B1R antagonist group and B2R antagonist group Ca2+concentrations increased less than BK group; B1R and B2R antagonist group showed no obvious changes in Ca2+concentrations.Compared with control group, COX-2 and iNOS protein concentrations were significantly increased in BK group (P<0.001).?CONCLUSION:BK induces the increasing expression of COX-2 and iNOS in the cultured ARPE cells through binding with either B1R or B2R.

Objective To investigate the expression and variation of MIP 1β,MIP-2,and IL-12p70 in mice with bloodstream infection caused by 4 kinds of bacteria.Methods CD-1 (ICR) mouse models of bloodstream infection with Staphylococcus aureus (S.aureus),Enterococcus f aecalis (E.f aecalis),Escherichia coli (E.coli),and K lebsiella pneumoniae (K.pneumoniae) were established.After mice in each trial group and PBS control group were infected by bacteria for 0.5h,1h,3h,6h,12h,24h,and 48h,concentrations of MIP-1β,MIP-2,and IL-12p70 were detected by Luminex liquid suspension chip system.Results Concentrations of MIP-1β increased significantly 1h after bacteria was in blood,S.aureus,E.faecalis,E.coli,K.pneumoniae,and control groups were (134.5 ± 18.3),(61.5 ± 15.4),(3 354.0 ±809.0),(6 888.4 ± 1 100.2),and (28.9 ± 4.6) pg/mL respectively;the peak values of IL-12p70 were (389.3 ± 118.1),(127.6 ± 10.0),(42.2 ± 3.5),(62.8 ± 8.4),and (4.8 ± 0.3) pg/mL respectively.Concentrations of MIP-1β and MIP-2 in E.coli and K.pneumoniae groups were significantly higher than other trial groups and control group (all P＜0.01),while concentrations of IL-12p70 in S.aureus and E.faecalis groups were both significantly higher than E.coli,K.pneumoniae,and control groups (all P＜0.01).Conclusion Concentrations of MIP-1β and MIP-2 in E.coli and K.pneumoniae groups were both significantly higher than those in S.aureus and E.faecalis groups,while concentrations of IL-12p70 in S.aureus and E.faecalis groups were both significantly higher than those in E.coli and K.pneumoniae groups.The combination detection of multiple cytokines or chemokines are valuable in predicting gram-positive or gram-negative bacterial infection,and can provide basis for treatment of early infection.

Objective To observe the effects of acupuncture on the behaviors and the expressions of Fis1 and OPA1, as well as mitochondrial ultrastructure in the hippocampus of mice with Alzheimer disease (AD); To explore the mechanism of action of acupuncture for AD.Methods Forty male SAMP8 mice were randomly divided into acupuncture group and model group, with 20 mice in each group. Another 20 male natural aging mice with the same age (SAMR1 mice) were set as the normal group. Acupuncture group chose Shenshu, Baihui, Xuehai and Geshu for intervention. 8 weeks later, Morris water maze was used to test the mice behaviors, and then hippocampus organization was taken. Western blot was used to detect the expressions of Fis1 and OPA1 and mitochondrial ultrastructure in hippocampal neurons of mice was observed by transmission electron microscopy.Results Compared with the model group, the escape latency of acupuncture group was significantly shortened (P<0.05), and the stay time in the former platform quadrant and former platform crossing times were significantly increased (P<0.05). The expression of Fis1 in the hippocampus of acupuncture group decreased significantly (P<0.05), while the expression of OPA1 increased significantly (P<0.05). The mitochondrial ultrastructure in hippocampus in the acupuncture group was effectively improved, and the mitochondrial surface density and body density were both increased in the acupuncture group compared with the model group (P<0.05).Conclusion Acupuncture may play a potential therapeutic role in AD by decreasing the expression of Fis1, increasing the expression of OPA1, recovering the injury of mitochondrial ultrastructure.

OBJECTIVETo construct a recombinant human adenovirus type 5 (Ad5) expressing luciferase and GFP reporter gene and detect neutralizing antibodies against adenovirus type 5 in common marmosets (Callithrix jacchus) to provide basic laboratory data for evaluating adenovirus vaccines.

METHODSLuciferase and GFP reporter genes from plasmid pHAGE-CMV-GFP were inserted into pDC315 to construct the recombinant adenovirus shutter plasmid pDC315-Luc-GFP. The shutter plasmid was co-transduced with pBHGlox(delta)E1,3Cre in 293A cell line to package the recombinant adenovirus rAd5/Luc/GFP. Three rounds of plaque formation experiment were performed to select the monoclonal adenovirus followed by purification with cesium chloride density gradient centrifugation and virus titration with TCID50 method. Chemiluminescence assay and flow cytometry were employed to detect the neutralizing antibody levels in 14 common marmosets.

RESULTSThe shuttle plasmid pDC315-Luc-GFP was successfully constructed and the recombinant adenovirus rAd5/Luc/GFP was packaged with a the titer reaching 6.9×10(11.5) PFU/mL. In the 14 marmosets, chemiluminescence assay identified 4 (28.6%) marmosets that were positive for Ad5-neutralizing antibodies, including 2 with a antibody titer of 1/16 and another 2 with a titer of 1/32; flow cytomery detected Ad5-neutralizing antibodies in 3 marmosets at the titer of 1/16.

CONCLUSIONChemiluminescence assay is a simple, sensitive, and accurate modality for detecting Ad5-neutralizing antibodies. Common marmosets have a very low positivity rate for Ad5-neutralizing antibodies and are therefore promising models for studying adenovirus-based vaccines and therapies.

Adenoviruses, Human ; immunology ; Animals ; Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Callithrix ; Cell Line ; Humans ; Immunity, Humoral ; Luciferases ; Plasmids

OBJECTIVETo study the clinicopathologic features, immunophenotype and prognosis of primary cutaneous anaplastic large cell lymphoma (C-ALCL).

METHODSEight cases of C-ALCL were enrolled into the study. The clinicopathologic features, immunohistochemical findings and results of in-situ hybridization for EBER 1/2 were analyzed.

RESULTSThree of the 8 patients were males and 5 were females. The median age was 49.5 years. C-ALCL often presented with solitary skin nodule, without systemic symptoms. Histologically, the lymphoma cells infiltrated the dermis and subcutis in a sheet-like pattern. They were of large size and showed conspicuous nuclear atypia. Immunohistochemical study showed that more than 75% of the lymphoma cells were positive for CD30. All cases expressed one to three T cell markers (CD3, CD5 or CD45RO) and cytotoxic granule-associated antigens (TIA-1, granzyme B or perforin). The staining for leukocyte common antigen was positive in all cases, while the expression of CD5, CD8, ALK-1 and epithelial membrane antigen was noted in 5, 1, 1 and 3 cases, respectively. The staining for CD15, CD20, CK and HMB45 was negative. In-situ hybridization for EBER 1/2 was also negative in all the cases studied. Follow-up information was available in 6 patients. Five of them were still alive and 1 died of unclear cause.

CONCLUSIONSC-ALCL has distinctive clinicopathologic and immunophenotypic features. It is not Epstein-Barr virus-related and often carries a favorable prognosis.

Adult ; Aged ; CD5 Antigens ; metabolism ; Child ; Combined Modality Therapy ; Female ; Follow-Up Studies ; Humans ; Immunophenotyping ; In Situ Hybridization ; Ki-1 Antigen ; metabolism ; Leukocyte Common Antigens ; metabolism ; Lymphoma, Primary Cutaneous Anaplastic Large Cell ; immunology ; metabolism ; pathology ; therapy ; Male ; Middle Aged ; Prognosis ; RNA, Viral ; metabolism ; Skin Neoplasms ; immunology ; metabolism ; pathology ; therapy ; Young Adult

Objective To investigate the effects of Bushen Huoxue acupuncture method on the behavioral changes and the proliferation of endogenous neural stem cells (NSCs) in hippocampus of Alzheimer disease (AD) model mice. Methods 18 male SAMP8 mice, seven months old, were randomly divided into acupuncture group, non-acupoint control group, and model control group. And another age-matched 6 male SAMR1 mice were prepared as normal control group. Mice in acupuncture group were intervened by acupuncture method in the acupoints of "Shenshu", "Baihui", "Xuehai", and "Geshu". Mice in non-acupoint control group were treated by stimulating the fixed non-point under the bilateral rib, while mice in model control group and normal control group were raised without special treatment but administered the stimulation of catching with the same time and the same stimulus intensity. All treatrment lasted for 8 weeks. After the intervention, the learning and memory abilitities and brain hippocampus Brd U positive cells of all groups were detected. Results Compared with the nomal control group, mice in the model control group had longer escape latency and less time spent in former platform quadrant (P＜0.05); the number of Brd U positive cells decreased significantly (P＜0.05). Compared with the model control group and the non-acupoint control group, mice in the acupuncture group had shorter escape latency and more time spent in former platform quadrant (P＜0.05); the number of Brd U positive cells increased significantly (P＜0.05). Conclusion Bushen Huoxue acupuncture method can improve the learning and memory abilities of SAMP8 mice AD model by inducing the proliferation of hippocampal endogenous NSCs.

OBJECTIVETo construct a cell model of 4.1R gene knockout in murine macrophage cell line RAW264.7 using CRISPR/Cas9 technique.

METHODSThree high?grade small?guide RNAs (sgRNAs) that could specifically identify 4.1R gene were synthesized and inserted into lentiCRISPRv2 plasmid. RAW264.7 cells were infected with sgRNA?Cas9 lentivirus from 293T cells transfected with the recombinant sgRNA?lentiCRISPRv2 plasmid, and the positive cells were screened using puromycin and the monoclonal cells were obtained. The expression of 4.1R protein in the monoclonal cells was measured by Western blotting, and the mutation site was confirmed by sequence analysis. Result A 4.1R gene knockout RAW264.7 cell line was obtained, which showed a 19?bp deletion mutation in the 4.1R gene sequence and obviously enhanced proliferation.

CONCLUSIONWe successfully constructed a 4.1R gene knockout macrophage cell line using CRISPR/Cas9 technique, which may facilitate further investigation of the function of 4.1R in macrophages.