Objective To explore the regulation of the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)-mediated inflammatory response in chronic obstructive pulmonary disease(COPD)by modified Shengxian Decoction through the lung-gut axis.Methods Thirty rats were divided into three groups:Control group,COPD group,and COPD+modified Shengxian Decoction(SXT)group,with 10 rats in each.The COPD model was established using passive smoking combined with intratracheal instillation of lipopolysaccharide(LPS).General symptoms and signs of the rats were monitored during the modeling and intervention periods.Hematoxylin and eosin(HE)staining and immunohistochemistry(IHC)were used to observe lung tissue structure.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory cytokines interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α)in lung tissues.Flow cytometry was used to detect the number of type Ⅱ innate lymphoid cells(nILC2)and type 2 innate lymphoid cells(iILC2)in lung and intestinal tissues.Illumina MiSeq sequencing technology was used to perform 16S rRNA gene sequencing on rat feces to analyze the gut microbiota structure.Gas chromatography-mass spectrometry(GC-MS)was used to determine the content of short-chain fatty acids(SCFAs)in rat feces.Western blotting was used to detect the expressions of related proteins in the PI3K/AKT pathway.Results Compared with the Control group,the COPD group showed significantly reduced lung function indicators,increased heart rate and decreased body mass,while the SXT group showed significant improvement in lung function and general signs(P＜0.05).HE staining showed that the COPD group had lung tissue damage filled with inflammatory cells,while the SXT group had significantly fewer inflammatory cells.IHC results showed that the SXT group had significantly reduced expression of caspase-3 protein(P＜0.05).ELISA results showed that the levels of IL-6 and TNF-α were significantly increased in the COPD group,while the SXT group showed significant improvement in inflammatory damage.The ratio of nILC2 to iILC2 in lung and intestinal tissues was significantly reduced in the COPD group,indicating a significant inflammatory response,while the SXT group showed significant improvement(P＜0.05).The levels of ILC2 cytokines IL-13 and IL-4 were significantly increased in the COPD group,while the SXT group had significantly reduced IL-13 and IL-4 levels.The relative abundance of lung and gut microbiota in the SXT group was significantly higher than that in the Control and COPD groups(P＜0.05).Beta diversity index analysis showed significant differences in species diversity among the three groups(P＜0.05).GC-MS detected six types of SCFAs in rat feces:acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid,and valeric acid.Their levels were lower in the COPD group than in the Control group,but the levels in the SXT group were higher than those in the COPD group.Western blotting results showed that the expressions of p-PI3K,PI3K,p-AKT,AKT,p-NF-κB,and NF-κB proteins were significantly reduced in the SXT group compared to the COPD group(P＜0.05).ELISA results showed that the SXT group had significantly downregulated expression levels of IL-1β and IL-10 compared to the COPD group(P＜0.05).Conclusion Modified Shengxian Decoction can alleviate COPD inflammation.It may mediate the inflammatory response in COPD by inhibiting iILC2 cell activity and expressions of related proteins in the PI3K/AKT signaling pathway through gut microbiota metabolism.

BACKGROUND:Apoptosis and autophagy imbalance in the hippocampal region of perimenopausal depressed rats are closely related to cognitive decline.Whether aerobic exercise can reduce apoptosis by promoting hippocampal autophagy and thus improve the learning and memory abilities of perimenopausal depressed rats is not clear.OBJECTIVE:To investigate the possible mechanism by which 4-week moderate-intensity treadmill exercise improves learning memory ability in ovariectomized stressed rats.METHODS:Forty Sprague-Dawely rats were randomly divided into four groups,namely,sham operation group(n=10),ovariectomized group(n=10),ovariectomized stress group(n=10)and ovariectomized stress exercise group(n=10).Except for the sham operation group,the ovaries were removed in the other three groups to establish a perimenopausal rat model,and then a depressed rat model was established by chronic unpredictable stress in the latter two groups.The rats in the ovariectomized stress exercise group underwent a 4-week moderate-intensity treadmill exercise.Tail suspension test and sucrose preference test were performed to text depression-like behaviors in rats after exercise and stress.The eight-arm maze experiment was used to test the learning and memory behaviors of rats after exercise and stress.Western blot was used to detect the protein levels of AMP-activated protein kinase/UNC-51 like autophagy activating kinase 1/mammalian target of rapamycin(AMPK/mTOR/ULK1),hippocampus apoptotic factor Caspase-3 and the protein expression of autophagy markers LC-3II/Beclin-1 in the hippocampus.RESULTS AND CONCLUSION:(1)Compared with the sham operation group,rats in the ovariectomized and ovariectomized stress groups had prolonged resting time in the tail suspension test and decreased sugar-water intake and sugar-water preference in the sucrose preference test.(2)Ovary removal reduced the learning memory capacity of rats,as evidenced behaviorally by a significant increase in the number of working memory errors,the number of reference memory errors,and the completion time,and an even more pronounced increase in the above measures in the ovariectomized stress group.(3)Compared with the ovariectomized group,there was a significant reduction in the number of working memory errors,the number of reference memory errors,and the completion time in the ovariectomized stress group.(4)Compared with the sham operation group,in the ovariectomized and ovariectomized stress groups,the expression of hippocampal apoptotic factor Caspase 3 protein was significantly elevated,the expression of autophagy-related factors proteins Beclin-1 and LC3II,as well as the protein expression of AMPK and ULK1,was decreased,whereas the expression of mTOR protein was elevated.Changes in the above indicators were more significant in the ovariectomized stress group.(5)Compared with the ovariectomized stress group,in the ovariectomized stress exercise group,the protein expression of Caspase 3 was significantly decreased,the protein expression of Beclin-1 and LC3II was significantly increased,the protein expression of AMPK and ULK1 was significantly increased,and the protein expression of mTOR was significantly reduced.To conclude,4-week moderate-intensity treadmill exercise may promote cellular autophagy and reduce apoptosis through the AMPK/mTOR/ULK1 autophagy signaling pathway,thereby enhancing the learning and memory capacity of rats with ovariectomized depression

Objective To explore the regulation of the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)-mediated inflammatory response in chronic obstructive pulmonary disease(COPD)by modified Shengxian Decoction through the lung-gut axis.Methods Thirty rats were divided into three groups:Control group,COPD group,and COPD+modified Shengxian Decoction(SXT)group,with 10 rats in each.The COPD model was established using passive smoking combined with intratracheal instillation of lipopolysaccharide(LPS).General symptoms and signs of the rats were monitored during the modeling and intervention periods.Hematoxylin and eosin(HE)staining and immunohistochemistry(IHC)were used to observe lung tissue structure.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory cytokines interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α)in lung tissues.Flow cytometry was used to detect the number of type Ⅱ innate lymphoid cells(nILC2)and type 2 innate lymphoid cells(iILC2)in lung and intestinal tissues.Illumina MiSeq sequencing technology was used to perform 16S rRNA gene sequencing on rat feces to analyze the gut microbiota structure.Gas chromatography-mass spectrometry(GC-MS)was used to determine the content of short-chain fatty acids(SCFAs)in rat feces.Western blotting was used to detect the expressions of related proteins in the PI3K/AKT pathway.Results Compared with the Control group,the COPD group showed significantly reduced lung function indicators,increased heart rate and decreased body mass,while the SXT group showed significant improvement in lung function and general signs(P＜0.05).HE staining showed that the COPD group had lung tissue damage filled with inflammatory cells,while the SXT group had significantly fewer inflammatory cells.IHC results showed that the SXT group had significantly reduced expression of caspase-3 protein(P＜0.05).ELISA results showed that the levels of IL-6 and TNF-α were significantly increased in the COPD group,while the SXT group showed significant improvement in inflammatory damage.The ratio of nILC2 to iILC2 in lung and intestinal tissues was significantly reduced in the COPD group,indicating a significant inflammatory response,while the SXT group showed significant improvement(P＜0.05).The levels of ILC2 cytokines IL-13 and IL-4 were significantly increased in the COPD group,while the SXT group had significantly reduced IL-13 and IL-4 levels.The relative abundance of lung and gut microbiota in the SXT group was significantly higher than that in the Control and COPD groups(P＜0.05).Beta diversity index analysis showed significant differences in species diversity among the three groups(P＜0.05).GC-MS detected six types of SCFAs in rat feces:acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid,and valeric acid.Their levels were lower in the COPD group than in the Control group,but the levels in the SXT group were higher than those in the COPD group.Western blotting results showed that the expressions of p-PI3K,PI3K,p-AKT,AKT,p-NF-κB,and NF-κB proteins were significantly reduced in the SXT group compared to the COPD group(P＜0.05).ELISA results showed that the SXT group had significantly downregulated expression levels of IL-1β and IL-10 compared to the COPD group(P＜0.05).Conclusion Modified Shengxian Decoction can alleviate COPD inflammation.It may mediate the inflammatory response in COPD by inhibiting iILC2 cell activity and expressions of related proteins in the PI3K/AKT signaling pathway through gut microbiota metabolism.

BACKGROUND:Apoptosis and autophagy imbalance in the hippocampal region of perimenopausal depressed rats are closely related to cognitive decline.Whether aerobic exercise can reduce apoptosis by promoting hippocampal autophagy and thus improve the learning and memory abilities of perimenopausal depressed rats is not clear.OBJECTIVE:To investigate the possible mechanism by which 4-week moderate-intensity treadmill exercise improves learning memory ability in ovariectomized stressed rats.METHODS:Forty Sprague-Dawely rats were randomly divided into four groups,namely,sham operation group(n=10),ovariectomized group(n=10),ovariectomized stress group(n=10)and ovariectomized stress exercise group(n=10).Except for the sham operation group,the ovaries were removed in the other three groups to establish a perimenopausal rat model,and then a depressed rat model was established by chronic unpredictable stress in the latter two groups.The rats in the ovariectomized stress exercise group underwent a 4-week moderate-intensity treadmill exercise.Tail suspension test and sucrose preference test were performed to text depression-like behaviors in rats after exercise and stress.The eight-arm maze experiment was used to test the learning and memory behaviors of rats after exercise and stress.Western blot was used to detect the protein levels of AMP-activated protein kinase/UNC-51 like autophagy activating kinase 1/mammalian target of rapamycin(AMPK/mTOR/ULK1),hippocampus apoptotic factor Caspase-3 and the protein expression of autophagy markers LC-3II/Beclin-1 in the hippocampus.RESULTS AND CONCLUSION:(1)Compared with the sham operation group,rats in the ovariectomized and ovariectomized stress groups had prolonged resting time in the tail suspension test and decreased sugar-water intake and sugar-water preference in the sucrose preference test.(2)Ovary removal reduced the learning memory capacity of rats,as evidenced behaviorally by a significant increase in the number of working memory errors,the number of reference memory errors,and the completion time,and an even more pronounced increase in the above measures in the ovariectomized stress group.(3)Compared with the ovariectomized group,there was a significant reduction in the number of working memory errors,the number of reference memory errors,and the completion time in the ovariectomized stress group.(4)Compared with the sham operation group,in the ovariectomized and ovariectomized stress groups,the expression of hippocampal apoptotic factor Caspase 3 protein was significantly elevated,the expression of autophagy-related factors proteins Beclin-1 and LC3II,as well as the protein expression of AMPK and ULK1,was decreased,whereas the expression of mTOR protein was elevated.Changes in the above indicators were more significant in the ovariectomized stress group.(5)Compared with the ovariectomized stress group,in the ovariectomized stress exercise group,the protein expression of Caspase 3 was significantly decreased,the protein expression of Beclin-1 and LC3II was significantly increased,the protein expression of AMPK and ULK1 was significantly increased,and the protein expression of mTOR was significantly reduced.To conclude,4-week moderate-intensity treadmill exercise may promote cellular autophagy and reduce apoptosis through the AMPK/mTOR/ULK1 autophagy signaling pathway,thereby enhancing the learning and memory capacity of rats with ovariectomized depression

Objective:To study the clinical value of serum microRNA-34a (miR-34a) and aryl hydrocarbon receptor nuclear transcript-like protein 1 (BMAL1) in cervical cancer and their relationship with high-risk human papillomavirus (HR-HPV) infection.Methods:The clinical data of 76 patients with cervical cancer and 50 patients with benign cervical diseases in the Affiliated Hospital of Jining Medical University from January to December 2022 were retrospectively analyzed. The expression levels of serum miR-34a and BMAL1 were detected by real-time fluorescence quantitative polymerase chain reaction, and HR-HPV infection was detected by flow fluorescent hybridization. The patients were followed up until December 2023, and the death and poor prognosis (death, tumor recurrence and progression and severe complications at 1-year of follow-up) were recorded. The receiver operating characteristic (ROC) curve was used to analyze the efficacy of miR-34a, BMAL1 and related indexes in evaluating the poor prognosis in patients with cervical cancer at 1-year. Multivariate Cox regression analysis was used to analyze the independent risk factors for death in patients with cervical cancer. The Kaplan-Meier survival curve was used to analyze the relationship between miR-34a, BMAL1 expression and survival period, and the log-rank test was used for comparison.Results:The expression level of serum miR-34a in patients with cervical cancer was significantly lower than that in patients with benign cervical lesions (0.46 ± 0.08 vs. 0.67 ± 0.11), the expression level of serum BMAL1 was significantly higher than that in patients with benign cervical lesions (0.58 ± 0.07 vs. 0.41 ± 0.07), and there were statistical differences ( t= 12.40 and 13.34, P<0.01). The expression levels of serum miR-34a and BMAL1 in patients with cervical cancer were associated with tumor differentiation, myometrial invasion depth, lymph node metastasis, distant metastasis and International Federation of Gynecology and Obstetrics (FIGO) stage, and there were statistical differences ( P<0.05 or<0.01); they were not associated with age, menopause and pathological type, and there were no statistical differences ( P>0.05). In patients with cervical cancer, the expression level of miR-34a in patients with HR-HPV positive infection (60 cases) was significantly lower than that in patients with HR-HPV negative infection (16 cases): 0.41 ± 0.07 vs. 0.49 ± 0.08, the expression level of BMAL1 was significantly higher than that in patients with HR-HPV negative infection: 0.65 ± 0.09 vs. 0.53 ± 0.06, and there were statistical differences ( t = 3.68 and 4.24, P<0.05 or<0.01). In patients with benign cervical diseases, there were no statistical differences in the expression levels of miR-34a and BMAL1 between patients with HR-HPV positive infection (7 cases) and patients with HR-HPV negative infection (43 cases) ( P>0.05). ROC curve analysis result showed that miR-34a combined with BMAL1 had the highest sensitivity (90.4%), specificity (89.9%) and area under curve (0.911) in assessing the 1-year poor prognosis in patients with cervical cancer ( P<0.01), and the optimal cutoff values of miR-34a and BMAL1 expression level were ≤0.39 and ≥0.64. Multivariate Cox regression analysis result showed that poor differentiation, myometrial invasion depth ≥1/2, lymph node metastasis, distant metastasis, FIGO stage Ⅲ+Ⅳ, miR-34a expression level ≤0.46 and BMAL1 expression level ≥0.58 were independent risk factors for death in patients with cervical cancer ( OR = 1.857, 2.125, 2.337, 2.751, 2.457, 3.885 and 3.666; 95% CI 0.845 to 5.788, 0.726 to 5.924, 0.709 to 5.631, 0.693 to 5.727, 0.801 to 5.936, 1.244 to 6.423 and 1.031 to 5.612; P<0.01). Kaplan-Meier survival curve analysis result showed that the median survival time in cervical cancer patients with miR-34a expression level ≤0.39 and BMAL1 expression level ≥0.64 (21 cases) was significantly lower than that in the other cervical cancer patients (miR-34a expression level>0.39 or BMAL1 expression level<0.64, 55 cases): (26.4 ± 4.2) months vs. (34.2 ± 5.6) months, log-rank χ2 = 17.12, P<0.05. Conclusions:The expression level of serum miR-34a in patients with cervical cancer is significantly reduced and the expression level of BMAL1 is significantly increased, which is related to the condition, prognosis and HR-HPV infection. It can be used as a marker for the assessment of the condition and prognosis of cervical cancer. The combined detection of the two can significantly improve the sensitivity and specificity in predicting poor prognosis of cervical cancer.

Currently, there is still no effective curative treatment for the development of late-stage liver fibrosis. Here, we have illustrated that TB001, a dual glucagon-like peptide-1 receptor/glucagon receptor (GLP-1R/GCGR) agonist with higher affinity towards GCGR, could retard the progression of liver fibrosis in various rodent models, with remarkable potency, selectivity, extended half-life and low toxicity. Four types of liver fibrosis animal models which were induced by CCl₄, α-naphthyl-isothiocyanate (ANIT), bile duct ligation (BDL) and Schistosoma japonicum were used in our study. We found that TB001 treatment dose-dependently significantly attenuated liver injury and collagen accumulation in these animal models. In addition to decreased levels of extracellular matrix (ECM) accumulation during hepatic injury, activation of hepatic stellate cells was also inhibited via suppression of TGF-β expression as well as downstream Smad signaling pathways particularly in CCl₄-and S. japonicum-induced liver fibrosis. Moreover, TB001 attenuated liver fibrosis through blocking downstream activation of pro-inflammatory nuclear factor kappa B/NF-kappa-B inhibitor alpha (NFκB/IKBα) pathways as well as c-Jun N-terminal kinase (JNK)-dependent induction of hepatocyte apoptosis. Furthermore, GLP-1R and/or GCGR knock-down results represented GCGR played an important role in ameliorating CCl₄-induced hepatic fibrosis. Therefore, TB001 can be used as a promising therapeutic candidate for the treatment of multiple causes of hepatic fibrosis demonstrated by our extensive pre-clinical evaluation of TB001.

Objective:To investigate the present situation of unintended pregnancy within two years postpartum and its influencing factors in China.Methods:Participants who delivered a live birth at 60 hospitals in 15 provinces in the eastern, central and western regions of China during July 2015 to June 2016 were interviewed by using structured questionnaire. Information on occurrence of unintended pregnancy within 2 years after delivery, postpartum contraceptive use, sexual resumption, breastfeeding, and women′s socio-demographic characteristics, and so on, were collected. Life-table analysis, cluster log-rank tests and a 2-level Cox regression model were used for data analysis.Results:A total of 18 045 postpartum women were investigated. The cumulative 1- and 2-year unintended pregnancy rates after delivery were 5.3% (95% CI: 4.5%－6.1%) and 13.1% (95% CI: 11.3%－14.8%), respectively. Cox regression model analysis showed that the risk of unintended pregnancy within 2 years postpartum were increased in younger women, ethnic minorities, women with abortion history, and those who had a vaginal delivery with short lactation time and late postpartum contraceptive initiation (all P<0.01). The risk of postpartum unintended pregnancy was not associated with geographic regions and hospitals where women gave a birth (all P>0.05). Conclusions:In China, the risk of unintended pregnancy within 2 years after delivery is relatively high. Service institutions and service providers should improve the quality of postpartum family planning services, promote the use of high effect contraceptive methods, and educate women to use a method at the time of their sexual resumption or even before.

OBJECTIVE: To evaluate the accuracy and diagnostic value of bronchoalveolar lavage fluid galactomannan test (BALF-GM) combined with serum GM test on invasive pulmonary aspergillosis (IPA). METHODS: 190 cases of BALF-GM and 4 787 cases of serum GM specimens suspected of fungal infection in patients admitted to Affiliated Hospital of Jining Medical University from January 2016 to June 2018 were enrolled and analyzed. All patients were classified into clinically confirmed IPA, clinically diagnosed IPA, suspected IPA and excluded IPA according to the classification standard of Expert consensus on diagnosis and treatment of pulmonary mycosis. The coincidence rate of BALF and serum GM test results with clinical diagnosis was analyzed. Receiver operating characteristic (ROC) curve was performed, and the diagnostic value of BALF and serum GM test alone or in combination for IPA was evaluated. Subgroup analysis was performed in patients with normal or abnormal immune function, and the sensitivity and specificity of BALF and serum GM test were compared separately or jointly. RESULTS: The positive rate of BALF-GM was 46.8% (89/190), and 10.4% (497/4 787) on serum GM. Among them, 156 patients were both tested on BALF and serum GM. There were 44 cases with both positive in BALF and serum GM, the coincidence rate of clinical definite was 93.2% (41/44). There were 34 cases with positive BALF-GM and negative GM test in serum, and the coincidence rate of clinical definite was 64.7% (22/34). There were 56 cases positive in serum GM and negative in BALF-GM, and the coincidence rate of clinical definite was 48.2% (27/56). BALF and serum GM tests were both negative in 22 cases, and the coincidence rate of exclusion diagnosis was 90.9% (20/22). ROC curve analysis showed that the diagnostic value of BALF-GM test combined with serum GM test for IPA was better than that of BALF-GM test or serum GM test alone [area under ROC curve (AUC): 0.992 vs. 0.983, 0.976]. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 95.3%, 87.0%, 93.2% and 90.9%, respectively. Subgroup analysis showed that among 89 patients with positive BALF-GM test, 85 cases (95.5%) had normal immune function and 4 cases (4.5%) had unknown condition. Among 497 patients with positive serum GM test, 12 cases (2.4%) had normal immune function, 372 cases (74.9%) had abnormal immune function and 113 cases (22.7%) were uncertain. It was shown by ROC curve analysis that the sensitivity of positive BALF-GM test in diagnosis of IPA in patients with normal immune function was higher than that of positive serum GM test (95.6% vs. 88.9%), while the sensitivity of positive serum GM test in patients with abnormal immune function was higher than that of positive BALF-GM test (91.8% vs. 89.9%). CONCLUSIONS The results of BALF and serum GM tests are in good agreement with clinical diagnosis, and the combined detection of BALF and serum GM is more valuable for IPA diagnosis than single detection, especially for patients with unknown immune function.
Bronchoalveolar Lavage Fluid/chemistry* ; Galactose/analogs & derivatives* ; Humans ; Invasive Pulmonary Aspergillosis/diagnosis* ; Mannans/blood* ; Sensitivity and Specificity

TCM variety derived from classical or proved prescriptions and was improved through the innovation and development of clinical practices,controlling of medicinal raw material and producing process.The current protection system of TCM variety does not match the developing rule of TCM,in that there is no particular article to regulate traditional classical or proved prescriptions,self-made TCM preparations in hospitals,the genuineness of medicinal raw material and manufacturing techniques,and also lacks the relevant investment return mechanism.It was recommended of creating TCM protection category,protecting self-made TCM preparations in hospitals,regulating genuineness of medicinal raw material and establishing know-how protecting mechanisms,while protecting test data were suggested to be introduced for perfecting the protection system of TCM variety.

OBJECTIVETo understand the prevalence of high risk behaviors and influencing factors among HIV infected persons aged ≥50 years.

METHODSFace to face questionnaire interview was conducted among the HIV infected persons selected in Jianshui, Gejiu and Mengzi counties in Yunnan province through random sampling in June 2015. The sample size was 450.

RESULTSAmong the HIV infected persons surveyed, 41.2% (122/296) had sexual behaviors with their spouses during past year, and the consistent condom use rate was 66.4% (81/122). Among the HIV infected males, 8.9% (28/313) had commercial sexual behaviors during past year, and the consistent condom use rate was 17.9% (5/28). Among the HIV infected females, 0.7% were still engaged in commercial sex service during past year. Among the 450 HIV infected persons, 32 (7.1%) reported having casual sex behaviors during past years, and the consistent condom use rate was 18.7% (6/32). The rate of commercial sexual behavior in urban residents (13.4%, 19/115) was higher than that in rural residents (4.5%, 9/198), the difference was statistically significant (χ(2)=11.715, P=0.001). The risk factors for commercial sex behaviors included lack of family and social support, aged 50-59 years, living in urban area, higher income and being male. The risk factors for using no condom included living in rural area, lower education level, lack of family and social support and higher income.

CONCLUSIONSRisk sex behaviors are still prevalent in HIV infected people aged >50 years, which exacerbated HIV transmission. Further efforts should be focused on the education about AIDS prevention and control and promoting protected sexual behaviors. Additional effort should be done to improve the family and social support for HIV infected people aged >50 years. Moreover, comprehensive intervention for low-paid female sex workers also needs to be strengthened.

China ; epidemiology ; Condoms ; utilization ; Female ; HIV Infections ; epidemiology ; Humans ; Male ; Middle Aged ; Prevalence ; Risk Factors ; Risk-Taking ; Sex Work ; psychology ; statistics & numerical data ; Sexual Behavior ; psychology ; statistics & numerical data ; Surveys and Questionnaires