We constructed plasmid pMTac to overexpress 3-ketosteroid-Δ1-dehydrogenase (KSDD) in Mycobacterium neoaurum JC-12 for improving androst-1,4-diene-3,17-dione (ADD) production. To construct pMTac, pACE promoter on pMF41 was replaced by tac promoter, and then four recombinants were constructed, which were M. neoaurum JC-12/pMF41-gfp, M. neoaurum JC-12/pMTac-gfp, M. neoaurum JC-12/pMF41-ksdd and M. neoaurum JC-12/pMTac-ksdd. Fluorescence detection results show that much more green fluorescent protein (GFP) was expressed in M. neoaurum JC-12/pMTac-ksdd than M. neoaurum JC-12/pMF41-ksdd. The activity of KSDD was 2.41 U/mg in M. neoaurum JC-12/pMTac-ksdd, 6.53-fold as that of M. neoaurum JC-12 and 4.36-fold as that of M. neoaurum JC-12/pMF41-ksdd. In shake flask fermentation, ADD production of M. neoaurum JC-12/pMTac-ksdd was 5.94 g/L, increased about 22.2% compared to the original strain M. neoaurum JC-12 and 12.7% to M. neoaurum JC-12/pMF41-ksdd. AD (4-androstene-3,17-dione) production of JC-12/pMTac-ksdd was 0.17 g/L, decreased 81.5% compared to M. neoaurum JC-12 and 71.2% to M neoaurum JC-12/pMF41-ksdd. In the 5 L fermenter, 20 g/L phytosterols was used as substrate, ADD production of M. neoaurum JC-12/pMTac-ksdd was improved to 10.28 g/L. pMTac is favorable for expressing KSDD in M. neoaurum JC-12, and overexpression of KSDD has beneficial effect on ADD producing, and it is the highest level ever reported using fermentation method in M. neoaurum.
Androstadienes ; metabolism ; Fermentation ; Industrial Microbiology ; Mycobacterium ; Oxidoreductases ; genetics ; metabolism ; Phytosterols ; metabolism ; Plasmids

Objective:To detect the expression of BTLA on Treg cells of HIV-infected patients and investigate the role of BTLA in HIV infection.Methods: Forty-four HIV-1-infected patients (twenty-four early HIV infection,fourteen chronic HIV-infected patients with CD4+ T counts> 200 cells/μl,AIDS patients with CD4+T counts<200 cells/μl) and nine healthy people served as normal controls were selected to detect the expression of BTLA on Treg cells by flow cytometry.The correlations between BTLA expression on Treg cells and disease progression or immune activation were studied.Results: There was a higher percentage of BTLA on Treg cells in chronic HIV patients and AIDS patients than that in early HIV infected patients(P<0.05,P<0.01),and the expression of BTLA on Treg cells in AIDS patients was higher than that in normal controls(P<0.05).The expression of BTLA on Treg cells was negatively correlated with CD4+T lymphocyte counts and positively correlated with viral load (P<0.001,P<0.01).The percentage of BTLA on Treg cells was positively correlated with CD4+CD38+T lymphocytes and CD4+HLA-DR+T lymphocytes(P<0.001,P<0.001).Conclusion: Increased BTLA expression on HIV-infected Treg cells is associated with disease progression,suggesting that it may accelerate disease progression by enhancing Treg cells inhibitory function and may provide intervention information for HIV infection in the future.

Objective:To study the changing characteristics and impact factors of helical tomotherapy (HT)for longitudinal dose fall-off outside the target, in order to guide the plan junction or pretreatment target and implementation efficiency in clinical.Methods:Eight patients with head and neck tumors admitted to the Department of Oncology Radiotherapy of the First Affiliated Hospital of Zhengzhou University in December 2019 were retrospectively selected as the research objects. The planning target area and dose drop structure were outlined in the head and neck images with a thickness of 1 mm obtained by Siemens SOMATOM Definition AS positioning computerized tomography (CT). Different field widths (FW, 5.0 cm/2.5 cm/1.0 cm) and pitches (0.430/0.287/0.215) were assembled for planning with the same modulation factor (1.8), finest does calculation grid (0.195 cm ×0.195 cm) and other planning parameters were consistent. The plans were designed by different parameters, and the result was analyzed by univariate analysis.Results:The that different pitch curves coincided under the same field width by comparative analyzing, so pitchs had no effect on dose drop. The different field width curves were independent of each other, indicating that the field width had an effect on dose drop in the head and foot direction. The relationship between the longitudinal dose drop speed outside the target and the change of the field width was inversely correlated: the larger field widths meant the slower dose fall-off and the larger penumbra, while the smaller field widths meant the faster fall-off and the smaller penumbra. When the dose fall-off to 50% of the prescribed dose, the distance from the target was approximately equal to half the field widths, and the pitchs had not affect the rate of dose-drop, while the dose at different distances from the target boundary could be calculated by the fitting formulas. The field widths and pitchs had little effect on the CI and HI index of the target, relatively, the target area was best when the field width was 2.5 cm. The total beam-on time gradually decreased with the increase of the field widths and pitches.Conclusions:When segment target therapy needs to consider planning junction, execution efficiency, and controlling longitudinal dose fall-off and considered the execution, the optimal planned parameters such as field widths and pitches could be selected or the target at the junction regions could be adducted according to the longitudinal dose drop formula, so as to achieve the ideal dose distribution.

Objective To discover more novel bat viruses and molecularly characterize bat-borne bocavirus diversity in Yunnan.Methods Twenty-six Aselliscus stoliczkanus were sampled in Jinghong , Yunnan, and subjected to viral metagenomic analysis.Specific PCR was used to detect any bocavirus in these samples based on the metagenomic result , while full genome was amplified and compared with other bocaviruses .Results and Conclusion Totally, 3 of the 26 (11.5%) bats were positive for bocavirus, the full genome of which contained 5203 nucleotides and could encode NS1, NP and VP1/VP2 proteins.Phylogenetic analysis showed that this virus shared up to 58.7% and 53.3% amino acid identities with canine bocavirus 1 and canine minute virus .According to ICTV criteria (85%amino acid sequence identity ) on a new species of bocavirus , this virus could be a novel species within genus Bocaparvovirus .This study provides important data to better understand viral diversity in bats and to uncover the relationship between bocavirus and its hosts .

Objective To assess the influences of early implementation of patient-controlled epidural analgesia (PCEA) in labor on uterine myoelectrical activity and delivery outcomes. Methods A prospective study was conducted on 240 singleton cephalic primiparae with spontaneous labor at Guangzhou Women and Children's Medical Center from January 2015 to October 2018. Those women, who were ready to accept PCEA, were randomly assigned to early- or late-PCEA group based on cervical dilation of 0-3 cm or 3-6 cm at the time of commencing PCEA, while those who refused PCEA in labor were classified as non-PCEA group. Uterine electromyographic activity and visual analogue score (VAS)were recorded before and 1 h and 2 h after PCEA. Patient satisfaction with labor, duration of the first stage of labor, volume of postpartum bleeding within 2 h after delivery and neonatal Apgar score were compared between different groups using multivariate analysis of variance, repeated measures analysis of variance, LSD-t test or Chi-square test. Results The VAS values 1 h after PCEA in the early- and late-PCEA group were both lower than that in the non-PCEA group (2.08±1.34 and 2.00±1.28 vs 7.65±1.04, LSD-t were － 27.713 and － 27.663, P<0.001) and those before PCEA (7.65±0.91 and 7.62±0.86, LSD-t were －32.879 and －33.349, P<0.001). The VAS values 2 h after PCEA in the early- and late-PCEA group were both lower than that in the non-PCEA group (1.63±1.53 and 1.41±1.56 vs 7.66±0.87, LSD-t were －27.018 and －27.823, P<0.001) and those before PCEA (LSD-t were －31.379 and －32.718, P<0.001).The patient satisfaction rate with labor was higher in the early-PCEA group comparing to the late-PCEA group [80.0% (72/90) vs 61.1% (55/90), P<0.001], and the two figures above were both higher than that of the non-PCEA group [20.0% (12/60), both P<0.001]. There was no significant difference in the duration of the first stage of labor, the volume of postpartum blood loss 2 h after delivery, oxytocin usage rate, the rate of convertion to cesarean section, neonatal birth weight or Apgar score at 1 or 5 min among the three groups (all P>0.05). There was also no significant difference in uterine electromyographic parameters among the three groups before or 2 h after PCEA (all P>0.05). The number and duration of burst, power density spectrum peak frequency, root mean square and total power 1 h after PCEA in the early- and later-PCEA group were all lower than those in the non-PCEA group [4.80±2.49 and 5.54±3.04 vs 9.67±2.44; (34.41±1.21) and (36.94±1.18) vs (41.68±1.53) s; (0.36±0.08) and (0.36±0.07) vs (0.48±0.05) Hz ; (0.05±0.04) and (0.05±0.05) vs (0.07±0.05) mV; (4.33±0.51) and (5.36 ±0.59) vs (9.90±1.43) pV2; all P<0.05]. Conclusions The effect of PCEA on uterine myoelectrical activity has no association with the commencing time. While early PCEA could alleviate the labor pain as soon as possible, which enable us to improve the efficacy of labor analgesia, patient satisfaction and maternal and neonatal safety without increasing cesarean section rate.

Objective: To achieve quantitative analysis of image quality parameters and establish warning and action thresholds for the on-board imaging (OBI) system of linear accelerator. Methods: The Catphan604 phantom was repeatedly scanned in the Full-Fan and Half-Fan CBCT scanning modes on a Varian EDGE linear accelerator, and the software based on Python language development in-house was utilized to analyze image quality parameters, such as CT number linearity, geometric consistency, slice thickness, spatial resolution, uniformity and low-contrast resolution. The quantitative analysis results of each image quality parameter obtained from 16 times of scanning within 16 months were normalized to the mean and the standard deviations were recorded. A run chart analysis was created to determine the warnings and action thresholds. Results: The software built in-house can quantitatively analyze the image parameters of the two scanning modes of OBI system. The low-contrast resolution of Half-Fan was better than that of Full-Fan, whereas the spatial resolution of Full-Fan was superior to that of Half-Fan. One standard deviation (1σ) was set as the warning threshold and 2 standard deviations (2σ) as the action threshold, respectively. The tolerance level of Half-Fan was smaller than that of Full-Fan. Conclusion Self-developed software enables quantitative analysis of accelerator image quality parameters, establishes warning and action tolerance of quality assurance and provides guidance for image quality assurance under image-guided radiotherapy specification.

Objective To analyze the clinical value of the second generation hybrid capture nucleic acid detection kit (DH3) of human papillomavirus (HPV) in screening cervical cancer and precancerous lesions.Methods The cervical exfoliated cell samples of 480 women in gynecologic clinic were collected and detected by DH3,TCT and HPV-PCR.Colposcopic pathological examination was performed in women with TCT ≥ ASC-US.With the pathological results as the golden standard,the diagnostic value of DH3 was analyzed.Results The pathological results showed that 370 cases were normal (77.08％),59 cases had benign lesions (≤CIN Ⅰ) (12.29％) and 51 were high-risk cases (≥CIN Ⅱ) (10.63％).The positive diagnostic rates of TCT,HPV-PCR and DH3 were 26.04％,32.08％ and 27.08％,respectively.The concordancerate of DH3 and TCT was 94.79％ (P ＜ 0.01),and the concordance rate with HPV-PCR was 93.13％ (P ＜ 0.01).The sensitivity,specificity,positive predictive value,negative predictive value and accuracy rate of DH3 were 98.18％,87.57％,70.13％,99.39％ and 90％,respectively.The ROC area of detection of high-risk (CIN Ⅱ) was Z =0.887 (95 ％ CI 0.785 ～ 0.918,P ＜ 0.01).Conclusion DH3 kit has a high degree of consistency with TCT and HPV-PCR in the detection of HPV cervical cancer and precancerous lesions.The sensitivity,specificity and accuracy are high,and it is easy to operate.

Objective To investigate the relationships of serum angiopoietin-like protein 4(ANGPTL4)and fibroblast growth factor-23(FGF-23)levels with the severity and prognosis of patients with diabet-ic nephropathy.Methods A total of 120 patients(diabetic nephropathy group)with diabetic nephrop-athy were selected from July 2018 to July 2020 and divided into mild group(n=62)and severe group(n=58)according to the staging criteria of diabetic nephropathy;based on the prognosis within 3 years,they were also divided into good prognosis group(n=94)and poor prognosis group(n=26).Addition-ally,102 diabetic patients were enrolled as diabetic group,and 81 healthy volunteers were selected as control group.The serum levels of ANGPTL4 and FGF-23 in diabetic nephropathy patients were detected by enzyme-linked immunosorbent assay(ELISA).Logistic regression analysis was used to analyze the influencing factors of prognosis,and receiver operating characteristic(ROC)curves were drawn to ana-lyze the values of ANGPTL4,FGF-23 and their combination in assessing the severity and prognosis of diabetic nephropathy.Results The levels of ANGPTL4 and FGF-23 in the diabetic nephropathy group and simple diabetic group were significantly higher than those in the control group(P＜0.05);compared with the simple diabetic group,the levels of ANGPTL4 and FGF-23 in the diabetic ne-phropathy group were significantly increased(P＜0.05);the levels of ANGPTL4 and FGF-23 in the severe group were significantly higher than those in the mild group(P＜0.05);the ROC curve analysis revealed that the areas under the curve(AUCs)for assessing disease severity of ANGPTL4,FGF-23 and their combination were 0.748,0.781 and 0.858 respectively,and the combined diagnosis was significantly better than FGF-23(Z=2.149,P=0.032)and ANGPTL4(Z=2.886,P=0.004)alone;the levels of ANGPTL4 and FGF-23 in the good prognosis group were significantly lower than those in the poor prognosis group(P＜0.05);significant differences were observed in diabetes dura-tion,blood urea nitrogen(BUN),serum creatinine(Scr),fasting blood glucose(FBG),glycated hemoglobin(HbA1C),hypertension,ANGPTL4 and FGF-23 levels between diabetic nephropathy pa-tients with different prognoses(P＜0.05);the Scr,ANGPTL4 and FGF-23 levels were all risk factors affecting the prognosis of diabetic nephropathy(P＜0.05);the ROC curve analysis for prognosis as-sessment showed that the AUCs of ANGPTL4,FGF-23 and their combination were 0.774,0.795 and 0.874 respectively;the combined diagnosis was significantly better than FGF-23(Z=2.385,P=0.171)and ANGPTL4(Z=2.317,P=0.021)alone.Conclusion The serum levels of ANGPTL4 and FGF-23 are significantly increased in diabetic nephropathy patients,and they have certain refer-ence value for the clinical diagnosis and prognosis assessment of diabetic nephropathy patients.

Objective To analyze the clinical value of the second generation hybrid capture nucleic acid detection kit (DH3) of human papillomavirus (HPV) in screening cervical cancer and precancerous lesions.Methods The cervical exfoliated cell samples of 480 women in gynecologic clinic were collected and detected by DH3,TCT and HPV-PCR.Colposcopic pathological examination was performed in women with TCT ≥ ASC-US.With the pathological results as the golden standard,the diagnostic value of DH3 was analyzed.Results The pathological results showed that 370 cases were normal (77.08％),59 cases had benign lesions (≤CIN Ⅰ) (12.29％) and 51 were high-risk cases (≥CIN Ⅱ) (10.63％).The positive diagnostic rates of TCT,HPV-PCR and DH3 were 26.04％,32.08％ and 27.08％,respectively.The concordancerate of DH3 and TCT was 94.79％ (P ＜ 0.01),and the concordance rate with HPV-PCR was 93.13％ (P ＜ 0.01).The sensitivity,specificity,positive predictive value,negative predictive value and accuracy rate of DH3 were 98.18％,87.57％,70.13％,99.39％ and 90％,respectively.The ROC area of detection of high-risk (CIN Ⅱ) was Z =0.887 (95 ％ CI 0.785 ～ 0.918,P ＜ 0.01).Conclusion DH3 kit has a high degree of consistency with TCT and HPV-PCR in the detection of HPV cervical cancer and precancerous lesions.The sensitivity,specificity and accuracy are high,and it is easy to operate.

Objective To investigate the relationships of serum angiopoietin-like protein 4(ANGPTL4)and fibroblast growth factor-23(FGF-23)levels with the severity and prognosis of patients with diabet-ic nephropathy.Methods A total of 120 patients(diabetic nephropathy group)with diabetic nephrop-athy were selected from July 2018 to July 2020 and divided into mild group(n=62)and severe group(n=58)according to the staging criteria of diabetic nephropathy;based on the prognosis within 3 years,they were also divided into good prognosis group(n=94)and poor prognosis group(n=26).Addition-ally,102 diabetic patients were enrolled as diabetic group,and 81 healthy volunteers were selected as control group.The serum levels of ANGPTL4 and FGF-23 in diabetic nephropathy patients were detected by enzyme-linked immunosorbent assay(ELISA).Logistic regression analysis was used to analyze the influencing factors of prognosis,and receiver operating characteristic(ROC)curves were drawn to ana-lyze the values of ANGPTL4,FGF-23 and their combination in assessing the severity and prognosis of diabetic nephropathy.Results The levels of ANGPTL4 and FGF-23 in the diabetic nephropathy group and simple diabetic group were significantly higher than those in the control group(P＜0.05);compared with the simple diabetic group,the levels of ANGPTL4 and FGF-23 in the diabetic ne-phropathy group were significantly increased(P＜0.05);the levels of ANGPTL4 and FGF-23 in the severe group were significantly higher than those in the mild group(P＜0.05);the ROC curve analysis revealed that the areas under the curve(AUCs)for assessing disease severity of ANGPTL4,FGF-23 and their combination were 0.748,0.781 and 0.858 respectively,and the combined diagnosis was significantly better than FGF-23(Z=2.149,P=0.032)and ANGPTL4(Z=2.886,P=0.004)alone;the levels of ANGPTL4 and FGF-23 in the good prognosis group were significantly lower than those in the poor prognosis group(P＜0.05);significant differences were observed in diabetes dura-tion,blood urea nitrogen(BUN),serum creatinine(Scr),fasting blood glucose(FBG),glycated hemoglobin(HbA1C),hypertension,ANGPTL4 and FGF-23 levels between diabetic nephropathy pa-tients with different prognoses(P＜0.05);the Scr,ANGPTL4 and FGF-23 levels were all risk factors affecting the prognosis of diabetic nephropathy(P＜0.05);the ROC curve analysis for prognosis as-sessment showed that the AUCs of ANGPTL4,FGF-23 and their combination were 0.774,0.795 and 0.874 respectively;the combined diagnosis was significantly better than FGF-23(Z=2.385,P=0.171)and ANGPTL4(Z=2.317,P=0.021)alone.Conclusion The serum levels of ANGPTL4 and FGF-23 are significantly increased in diabetic nephropathy patients,and they have certain refer-ence value for the clinical diagnosis and prognosis assessment of diabetic nephropathy patients.