9α-hydroxy-4-androstene-3,17-dione (9-OH-AD) is an important intermediate in the steroidal drugs production. 3-ketosteroid-9α-hydroxylase (KSH), a two protein system of KshA and KshB, is a key-enzyme in the microbial steroid ring B-opening pathway. KSH catalyzes the transformation of 4-androstene-3,17-dione (AD) into 9-OH-AD specifically. In the present study, the putative KshA and KshB genes were cloned from Mycobacterium smegmatis mc(2)155 and Gordonia neofelifaecis NRRL B-59395 respectively, and were inserted into the expression vector pNIT, the co-expression plasmids of kshA-kshB were obtained and electroporated into Mycobacterium sp. NRRL B-3805 cells. The recombinants were used to transform steroids, the main product was characterized as 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), showing that kshA and kshB were expressed successfully. Different from the original strain Mycobacterium sp. NRRL B-3805 that accumulates 4-androstene-3,17-dione, the recombinants accumulates 9α-hydroxy-4-androstene-3,17-dione as the main product. This results indicates that the putative genes kshA, kshB encode active KshA and KshB, respectively. The process of biotransformation was investigated and the results show that phytosterol is the most suitable substrate for biotransformation, kshA and kshB from M. smegmatis mc(2)155 seemed to exhibit high activity, because the resultant recombinant of them catalyzed the biotransformation of phytosterol to 9-OH-AD in a percent conversion of 90%, which was much higher than that of G. neofelifaecis NRRL B-59395. This study on the manipulation of the ksh genes in Mycobacterium sp. NRRL B-3805 provides a new pathway for producing steroid medicines.
Androstenedione ; analogs & derivatives ; biosynthesis ; Bacterial Proteins ; genetics ; metabolism ; Biotransformation ; Ketosteroids ; Mixed Function Oxygenases ; genetics ; metabolism ; Mycobacterium ; metabolism ; Mycobacterium smegmatis ; enzymology ; Plasmids

Objective:To construct an evaluation system for clinical thinking ability of general practitioners in the treatment of multimorbidity.Methods:This was a cross-sectional study. The draft of evaluation indexes for clinical thinking ability of general practitioners in treatment of multimorbidity was preliminary developed through literature review, collation, analysis and discussion. Nineteen clinical and teaching experts of general practice were selected for consultation via anonymous convenient sampling. From January to June 2022, 2 rounds of expert consultation were conducted using the Delphi method. During the first round of consultation, according to the survey feedback, we modified and improved the evaluation system of general practitioners′ clinical thinking ability for multi-disease co-treatment. During the second round, experts were asked to assess the importance of each index, and to calculate the weight of each index accordingly. Questionnaires were sent to experts via letters. The content of the questionnaires encompasses the basic information of experts, evaluation for various indexes and relevant opinions. The mean value of importance assignment ≥3.5, coefficient of variation ≤0.25 and the full score frequency ≥30% were taken as the criteria. Indexes unsatisfying the criteria were removed, so that the final index system could be constructed.Results:The average age of 19 experts was 50.2 years old, 9 of them were male. A total of 2 rounds of expert consultation were conducted, 19 questionnaires were issued in each round, and 19 effective questionnaires were received afterwards. In the first round of consultation, 10 experts put forward revised opinions, and some indexes were adjusted according to the definition criteria and the discussion of the research group. In the second round of consultation, 3 experts put forward suggestions for modification. According to the definition criteria, no need to delete the indexes. After discussion by the research group, some indexes were adjusted, and finally an evaluation system of clinical thinking ability for multi-disease co-treatment of general practitioners was established, including 4 first-level indexes and 30 second-level indexes. The weights of the 4 first-level indexes in descending order were "overall thinking ability" (38.01%), "diagnostic thinking ability" (33.96%), "evidence-based thinking ability" (14.75%), and "critical thinking ability" (13.28%). Among the 30 secondary indexes, the top 5 were "ability to identify and handle priority emergency incidents" (5.04%), "risk assessment and critical illness identification ability" (4.63%), "emergency referral ability" (4.61%), "communication and expression ability" (4.57%), and "standardized diagnosis and treatment ability" (4.23%).Conclusion:This study successfully constructed an evaluation system for clinical thinking ability of general practitioners in the treatment of multimorbidity.

Objective: To observe the efficacy and safety of CTD (cyclophosphamide, thalidomide, dexamethasone) and PCD (bortezomib, cyclophosphamide, dexamethasone) regimens in treatment of patients with newly diagnosed multiple myeloma (NDMM) . Methods: A retrospective analysis was carried out on 88 cases of NDMM patients admitted to our hospital from July 2013 to January 2016, including 49 cases in CTD group and 39 cases in PCD group. The outcomes of two different regimens were analyzed, including response, prognosis, and adverse events. Results: The total overall remission rates (ORR, better than PR) of CTD and PCD were 65.3% (32/49) and 84.6% (33/39) , while very good partial response (VGPR) were 30.6% (15/49) and 53.8% (21/39) , and differences were statistically significant (P=0.041, P=0.028) . The median follow-up was 11.5 (3-33) months. The median progression-free survival (PFS) was (23.0±4.5) months in CTD groups, but it was not achieved in PCD group, with statistically significant differences (P=0.050) . Medial overall survival was not achieved in both two groups, without statistically significant difference (P=0.257) . There were statistical differences between patients with minor response (MR) and patients without MR in medium OS in CTD group (P=0.005) , and there were statistical difference between patients with VGPR and without VGPR in medium OS in CTD group (P=0.042) . Infection was a common adverse event in two groups. The incidences of peripheral neuropathy and herpes zoster were markedly higher in PCD group than CTD group, and the incidences of thrombus, palpation and rash, etc., were higher in CTD group. Conclusion Both CTD and PCD regimens were effective first-line induction chemotherapy choice for NDMM. PCD regimen is better than CTD in treatment power and deep remission.

Duodenal cancer is a clinically rare gastrointestinal malignant tumor. Its early symptoms lack specificity, making it difficult to diagnose clinically and easy to be misdiagnosed and missed. The treatment of duodenal cancer is mainly surgery, supplemented by radiotherapy and chemotherapy. Most patients have already experienced local invasion or distant metastasis at the time of first diagnosis, and lack of effective treatment, the prognosis is extremely poor. In recent years, a series of studies have pointed out that targeted therapy and immunotherapy can improve the prognosis of patients with duodenal cancer. This article reviews the progress of diagnosis and treatment of duodenal cancer, hoping to help guide clinical diagnosis and treatment and develop new treatment options.