This study was aimed at preliminarily investigating the molecular biological functions of the PPE65 protein from Myco-bacterium tuberculosis,and providing foundational data for tuberculosis prevention and control.The basic biological properties of the PPE65 gene-encoded protein were predicted with bioinformatics tools.Sequence information for the Mycobacterium tuberculosis Rv3621c gene and PPE65 protein was retrieved from the NCBI database.The Rv3621c gene was amplified through PCR with the H37Rv genome as a template,then cloned into the pET22b(+)expression vector.The recombinant pET22b(+)-PPE65 plasmid was transformed into Escherichia coli BL21(DE3)competent cells for IPTG-induced expression.Solubility analysis,purification,and identification of the recombinant PPE65 protein were performed.BEAS-2B cells were treated with various concentrations of PPE65 protein for 24 h,and cell proliferation was assessed with CCK-8 assays.PPE65 was found to be composed of 413 amino acids and to have a molecular formula of C????H????N???O???S??,a relative molecular mass of 40 679.88,a theoretical isoelectric point of 4.60,an ali-phatic index of 81.94,and an average hydrophilicity value of 0.319,thus indicating a stable hydrophobic protein lacking signal pep-tides or transmembrane domains.Secondary structure analysis revealed 53.03%α-helix(Hh),2.66%β-sheet(Ee),and 44.31%ran-dom coil(Cc).Bioinformatics predictions identified 38 B-cell epitopes and 22 CTL/Th-cell epitopes.The full-length PPE65 gene(1 308 bp)was confirmed through double restriction enzyme digestion and sequencing,thereby validating the correct construction of the pET22b(+)-PPE65 recombinant plasmid.SDS-PAGE analysis demonstrated that the recombinant protein was found in inclusion bodies,and a single band at 43.7 kDa was observed after purification.Western blotting revealed specific binding to mouse-derived His monoclonal antibodies,thereby confirming successful expression of the PPE65 protein.BEAS-2B cells treated with a PPE65 protein concentration gradient(2.5-20 μg/mL)exhibited a dose-dependent increase in cell number.Compared with those in the PBS control group,TGF-β relative expression levels were significantly higher in all treatment groups(t2.5=4.893,P<0.001,t5.0=4.640,P<0.05,t10=7.535,P<0.05,t20=16.44,P<0.000 1).This study elucidated the structural characteristics of the PPE65 protein,successfully obtained the recombinant protein through prokaryotic expression and purification,and demonstrated its ability to promote BEAS-2B cell proliferation.The underlying mechanism might involve suppression of TGF-β/S mad signaling pathway activation.These findings provide a theoretical basis for understanding the role and regulatory mechanisms of PPE65 during M.tuberculosis infection.

This study was aimed at preliminarily investigating the molecular biological functions of the PPE65 protein from Myco-bacterium tuberculosis,and providing foundational data for tuberculosis prevention and control.The basic biological properties of the PPE65 gene-encoded protein were predicted with bioinformatics tools.Sequence information for the Mycobacterium tuberculosis Rv3621c gene and PPE65 protein was retrieved from the NCBI database.The Rv3621c gene was amplified through PCR with the H37Rv genome as a template,then cloned into the pET22b(+)expression vector.The recombinant pET22b(+)-PPE65 plasmid was transformed into Escherichia coli BL21(DE3)competent cells for IPTG-induced expression.Solubility analysis,purification,and identification of the recombinant PPE65 protein were performed.BEAS-2B cells were treated with various concentrations of PPE65 protein for 24 h,and cell proliferation was assessed with CCK-8 assays.PPE65 was found to be composed of 413 amino acids and to have a molecular formula of C????H????N???O???S??,a relative molecular mass of 40 679.88,a theoretical isoelectric point of 4.60,an ali-phatic index of 81.94,and an average hydrophilicity value of 0.319,thus indicating a stable hydrophobic protein lacking signal pep-tides or transmembrane domains.Secondary structure analysis revealed 53.03%α-helix(Hh),2.66%β-sheet(Ee),and 44.31%ran-dom coil(Cc).Bioinformatics predictions identified 38 B-cell epitopes and 22 CTL/Th-cell epitopes.The full-length PPE65 gene(1 308 bp)was confirmed through double restriction enzyme digestion and sequencing,thereby validating the correct construction of the pET22b(+)-PPE65 recombinant plasmid.SDS-PAGE analysis demonstrated that the recombinant protein was found in inclusion bodies,and a single band at 43.7 kDa was observed after purification.Western blotting revealed specific binding to mouse-derived His monoclonal antibodies,thereby confirming successful expression of the PPE65 protein.BEAS-2B cells treated with a PPE65 protein concentration gradient(2.5-20 μg/mL)exhibited a dose-dependent increase in cell number.Compared with those in the PBS control group,TGF-β relative expression levels were significantly higher in all treatment groups(t2.5=4.893,P<0.001,t5.0=4.640,P<0.05,t10=7.535,P<0.05,t20=16.44,P<0.000 1).This study elucidated the structural characteristics of the PPE65 protein,successfully obtained the recombinant protein through prokaryotic expression and purification,and demonstrated its ability to promote BEAS-2B cell proliferation.The underlying mechanism might involve suppression of TGF-β/S mad signaling pathway activation.These findings provide a theoretical basis for understanding the role and regulatory mechanisms of PPE65 during M.tuberculosis infection.

Objective:To evaluate the safety and efficacy of Toumai ? endoscopic robotic system in radical prostatectomy. Methods:This study was a single-center phase Ⅲ randomized controlled study. From June 2020 to January 2021, patients with prostate cancer who met the inclusion criteria in Changhai Hospital Affiliated to Naval Military Medical University were divided into the experimental group and the control group by random table method. Inclusion criteria included aged 18 to 80 years, pathologically diagnosed as prostate cancer, clinical stage ≤T 2N 0M 0. Exclusion criteria included patients requiring emergency surgery, having serious cardiovascular diseases and cannot tolerate surgery, having participated in other investigational drug or device clinical trials within the last 3 months. The experimental group used Toumai ? laparoscopic robotic system, and the continence group used the Da Vinci robotic system. The patients in both groups underwent radical prostatectomy via a transabdominal approach, which was performed by two surgeons. The clinical characteristics between the two groups were compared, related adverse events were recorded, and PSA and urinary continence were followed up one month after the operation. Results:A total of 44 patients were enrolled in this study, including 22 cases in the experimental group and 22 cases in the control group. The mean age of patients in the trial group and the control group was (67.7±7.5) years and (66.4±6.3) years, respectively. The median PSA at diagnosis was 10.5 (7.7, 23.7) ng/ ml and 13.5 (8.9, 24.7) ng/ ml, respectively. Biopsy Gleason score of 6, 7, 8 and 9 in experimental group were 13.6% (3/22), 68.2% (15/22), 4.5% (1/22) and 13.6% (3/22), respectively, and in the control group were 4.5% (1/22), 59.1% (13/22), 22.7% (5/22) and 13.6% (3/22) respectively. The middle risk and high risk group in the experimental group was 50.0% (11/22), 50.0% (11/22), and the control group was 36.4% (8/22), 63.6% (14/22). There was no statistical difference between the two groups.The operations in both groups were successfully performed. There were no conversions to open or laparoscopic surgeries, and no Clavien-Dindo grade Ⅲcomplications. There was no significant difference in the estimated blood loss during the operation [(109.1±51.6)ml vs.(94.5±51.6)ml] and the blood transfusion rate [9.1%(2/22)vs. 4.5%(1/22)] in both groups. The operation time was significantly higher in the experimental group than that in the control group [164.5(130.5, 214.3) min vs. 88.0(65.3, 110.5)min, P<0.001]. The positive rate of surgical margin was 13.6% (3/22) in the experimental group and 36.4% (8/22) in the control group, respectively, showing no significant difference. The pathologic stages of pT 2, pT 3a and pT 3bin experimental group were 63.6% (14/22), 13.6% (3/22) and 22.7% (5/22), respectively, while those in control group were 36.3% (8/22), 40.9% (9/22) and 22.7% (5/22), respectively, showing no significant difference. The recovery rates of urine control in the experimental group and the control group were 22.7% (5/22) and 22.7% (5/22), respectively. The median PSA in the experimental group and the control group were 0.055 (0.021, 0.103) ng/ ml and 0.032 (0.010, 0.089) ng/ ml, respectively, with no statistical difference. Conclusions:The Toumai ? endoscopic robotic system can successfully perform radical prostatectomy, based on insignificant difference from Da Vinci robotic system in safety and efficacy. The short-term follow-up showed that tumor control and urinary continence have recovered well in the test group. The long-term effect of the new system on tumor control and functional recovery after radical prostatectomy needs further multi-center studies.

Objective To evaluate the efficacy and safety of intravitreal ranibizumab injection combined with triamcinolone acetonide (TA) for macular edema secondary to type Ⅱ optic disc vasculitis.Methods Totally 19 cases(19 eyes) with macular edema secondary to type Ⅱ optic disc vasculitis from January 2013 to December 2015 were injected ranibizumab intravitreally and triamcinolone acetonide 2 weeks later.The visual acuity,intraocular pressure,ocular fundus,OCT,FFA and related complications were observed during the 6 months follow-up period.If the macuiar edema returns during the follow-up period,the eyes would be given ranibizumab and TA again until the macular edema subsided.Results The visual acuity was 0.16 ± 0.09 and the central retinal thickness was (694.88 ± 79.06) μm before treatment.Compared with pretreatment,the visual acuity of all cases were improved in different degree except for one at 1 month,3 months and 6 months (all P ＜ 0.05).Also central retinal thickness were decreased and macular edema were subsided,there were statistical differences (all P ＜0.05).Compared with results at 1 month,the visual acuity and central retinal thickness was no significant different at 3 months and 6 months (all P ＞ 0.05).The intraocular pressure of all cases were normal throughout the follow-up period but one was increased transitorily in the first day after intravitreal rauibizumab injection.FFA examination at 1 month showed retinal hemorrhage and effusion absorbed obviously,besides,the optic disc and macular fluorescein leakage reduced significantly.The macular edema of 1 eye returned at 3 months and subsided after giving ranibizumab and TA again.The average number of injected eyes was 2.11 times.No complications associated with intravitreal injection and drugs were observed during the follow-up period.Conclusion It is effective and safe that intravitreal ranibizumab junction combined with TA for macular edema secondary to type Ⅱ optic disc vasculitis,which not only shorten the period of treatment but also reduce the times of drug injection,surgical trauma and complications.

Objective To investigate the safety and effectiveness of open radical prostatectomy (ORP) for locally advanced prostate cancer (LAPC).Methods From January 2012 to April 2017,132 cases underwent ORP were included.The mean age was 65.1 years old (ranged 41 to 83 years old),median PSA was 28.9 ng/ml (ranged 1.2 to 319.7 ng/ml) and mean Glcason score was 8.0(ranged 6.0 to 10.0).The number of clinical stage T3aN0,T3bN0,T4N0 and T1 ～4N1 were 92 cases(69.7％),20 cases (15.2％),8 cases (6.1％) and 12 cases (9.0％),respectively.Results The median length of hospital day,mean operative time and median blood loss were 9 d,180 min and 350 ml respectively.The intraoperative complication rate was 3.0％ (4/132),including 2 rectum injury and 2 iliac vessel injury.Pathological tumor stage revealed that ≤ pT2 N0 7 cases (5.3％),pT3a N0 61 cases (46.2％),pT3b N0 38 cases (28.8％),pT4N0 12 cases (9.1％) and pT1～4N1 14 cases (10.6％).The mean Gleason score was 8.0 (ranged 6 tol0).The numbers of patients with perineural invasion,seminal vesicle invasion and positive surgical margin were 81 cases (61.4％),49 cases (37.1％) and 41 cases (31.1％) respectively.The median follow-up duration was 24.1 (ranged 1.8 to 62.2) months.The rate of postoperative complications was 3.0％ (4/132) including 1 urethral stricture,1 wound infection,1 intestinal fistula and 1 lymphatic fistula.The rates of patients with urinary continence 1,3,6 and 12 months after surgery were 30.4％ (38/125)、63.9％ (76/119)、72.6％ (82/112)、89.1％ (90/101).The rates of adjuvant hormonal therapy and radiotherapy were 34.1％ (45/132) and 38.6％ (51/132).One patient (0.8％) died of lung cancer.The rate of biochemical recurrence(BCR) was 25.8％ (34/132).The 5-year BCRfree survival rate was 57.2％ (95％ CI 41.9％ ～ 70.6％).Conclusion The oncological control and functional recovery outcomes of ORP for locally advanced prostate cancer were reliable.

Objective To explore the effect of continuing nursing mode in women with stress urinary incontinence who accept the pelvic floor functional exercise at home.Methods By convenient sampling method, 60 patients with incontinence were selected, who received four weeks pelvic floor electrophysiological treatment and biofeedback treatment in Peking Union Medical College Hospital, and then received 12 weeks of pelvic floor rehabilitation therapy at home . All patients were divided into control group and observation group on average. The patients of the control group underwent routine health education, while the patients of the observation group were given pelvic floor muscle tissue by electrical stimulation and the continuation of nursing mode, then we compared two groups of maternal pelvic floor functional exercise adherence, 1 hours urine pad test for lateral leakage urine volume, ICI-Q-SF score for subjective improvement.Results After interventions, the excise compliance of pelvic floor muscle was better in the observation group than that of the control group (P<0.05); 3 and 6 months after intervention, the lateral leakage urine volumes were both decreased in two groups (P<0.05); both scores of ICI-Q-SF in two groups were improved, but that of the observation group was more obvious (P<0.05).Conclusions Planned continuing nursing mode can maintain and strengthen the clinical treatment of urinary incontinence, and increase the compliance of pelvic floor muscle function exercise in patients with urinary incontinence.

Typodont models of bilateral canines gingival displaced vertically for 3 mm and bilateral lateral incisors palatal displaced horizontally for 3 mm are made and every kind of the two kind models was divided into four groups: MDD, MBT, self-ligating and Tip-Edge. Each group of the two kinds of models had 5 models, so a total of 40 models for the two kinds of models were set up. The experimental models were aligned with a 0.30 mm of nickel titanium round wire in a water-bath with constant temperature 45 degrees C for 30 minutes. The three-dimensional digital images of the experimental models before and after the water bath were obtained by scanning with the three dimensional optical scanner ATOS. Geomagic software was used to overlap the digital images taken before and after the water bath treatment. The vertical changes of sign points of canines, the movements of sign points of lateral incisors in the sagittal plane and the horizontal plane were quantified by using the Color map. The data was then processed by a two-factor analysis of variance by using SAS 9.2 software package. Analysis of the results shows that the vertical changes of sign points of canines , the movements of sign points of lateral incisors in the sagittal plane and the horizontal plane of MDD group are all less than those in the other three groups, a statistically significant difference (P < 0.0001). And the size of the teeth displacement is directly related to the size of orthodontic force on the typodont models. Thus the preliminary results suggest that MDD appliance might have advantages of light force in the alignment stage, and that the possible relevant factors are the improvement of its sub-slot and the single ligation wing design.
Cuspid ; Dental Models ; Humans ; Imaging, Three-Dimensional ; methods ; Incisor ; anatomy & histology ; Malocclusion ; therapy ; Orthodontic Appliance Design ; Tooth Movement Techniques ; instrumentation ; methods

Naturally occurring mutations in surface proteins of Hepatitis B virus(HBV)usually result in altered hepatitis B surface antigen(HBsAg)secretion efficiency.In the present study,we reported two conserved residues,M75 and M103 with respect to HBsAg,mutations of which not only attenuated HBsAg secretion(M75 only),but also suppressed HBV genome replication without compromising the overlapping p-gene product.We also found M75 and M103 can initiate truncated surface protein(TSPs)synthesis upon over-expression of full-length surface proteins,which may possibly contribute to HBV genome replication.However,attempts to rescue replicationdefective HBV mutant by co-expression of TSPs initiated from M75 or M103 were unsuccessful,which indicated surface proteins rather than the putative TSPs were involved in regulation of HBV genome replication.

The female sheep reproductive tracts were freshly collected from a local meat processing plant and used as experimental materials. Two antibacterial peptides were isolated and characterized from female sheep reproductive tracts by two consecutive chromatographic steps. The peptide isolation procedures included acetic acid extraction, dialyzed, gel filtration chromatography on Sephadex G-50, and reverse phase high-performance liquid chromatography (RP-HPLC). Their molecular mass were 4 820.47 u and 4 012.5 u, respectively, analyzed by MALDI-TOF-MS. The partial N-terminal amino acid sequences of two peptides were determined as AYVLDEPKP and YDSGA, respectively, by Edman degradation. The antimicrobial activity was tested during each purification step by the radial diffusion plate assay and broth microdilution method. These two peptides showed good antimicrobial activities against reference strains of G~+(S. Aureus ATCC2592 and Streptococcu ATCC55121), G~-(E. Coli ATCC25922) and fungi(C. Albicans ATCC2002). The peptides did not show active hemolytic activity against rabbit blood red cells and had no significant effects on human blood coagulation system. The discovery of antibacterial peptides in sheep reproductive system reveals that antibacterial peptides may play a role in innate immunity against microorganisms in a wide range of animal species.