Prediction of occupational coal workers′ pneumoconiosis incidence by a multi-layer perceptron neural network model / 中国职业医学

OBJECTIVE: To construct and verify the incidence prediction model of occupational coal workers′ pneumoconiosis(CWP) in coal mine workers exposed to dust(hereinafter referred to as ″dust exposure″) based on a multi-layer perceptron(MLP) neural network, and explore its application value in predicting CWP incidence. METHODS: A total of 17 023 dust exposed workers in a coal mining group in Hebei Province from 1970 to 2017 were selected as the research subjects by a typical sampling method. Among them, 839 patients were confirmed as CWP and 16 185 workers did not suffered from CWP. The MLP neural network model was established with the incidence of CWP as the target output variable, and the type of work, age, beginning year of dust exposure, observation year(i.e. incubation period) and cumulative dust exposure as the input variable. The receiver operating characteristic(ROC) curve was used to evaluate the predictive ability of the built model. The established model was used to predict the high-risk group and key monitoring group population of CWP in dust-exposed workers in the following 10 years. RESULTS: There were 44 synapses in the hidden layer of the established MLP neural network model. The area under ROC curve was 0.91. The accuracy, sensitivity and specificity of the model were 92.7%, 74.8% and 93.6%, respectively. In the validation samples, the accuracy, sensitivity and specificity were 92.1%, 70.5% and 93.2%, respectively. The MLP neural network model was used to predict 1 534 workers with high risk of CWP in the following 10 years, and the individuals were located. The number of workers in need of actively monitored was 7 599. Among them, it is predicted that the incidence of CWP in different types of dust exposed workers in the following 10 years from high to low is tunneling worker, coal miner, mixing worker and auxiliary worker(P<0.01). The earlier the dust exposure began, the higher the risk of CWP(P<0.01). CONCLUSION: The MLP neural network model based on the type of work, age, beginning year of dust exposure, incubation period and cumulative dust exposure has a good performance in predicting the incidence of CWP in coal mine dust exposure workers, and can provide a reference for early preventive management measures to prevent and cure CWP.

Exploration of dominant diseases and clinical application characteristics of wrist-ankle acupuncture therapy based on data mining technology / 中国针灸

OBJECTIVE@#To analyze the literature regarding wrist-ankle acupuncture therapy by data mining technology, and the dominant diseases of wrist-ankle acupuncture therapy were summarized to provide reference for evaluation of specificity effect of wrist-ankle acupuncture therapy.@*METHODS@#The journal articles regarding wrist-ankle acupuncture therapy from the CNKI, WANFANG, and VIP since January 1, 1975 to December 31, 2017, and the medical cases regarding wrist-ankle acupuncture therapy in - and were retrieved. Based on the disease types of wrist-ankle acupuncture therapy in the journal articles and medical cases, the association rules method of data mining technology was applied to calculate frequent itemsets. The self-developed database platform for wrist-ankle acupuncture therapy was used to extract and summarize the information to explore the rules of clinical application.@*RESULTS@#The scope of disease involved a total of 7 disease entities in the literature regarding wrist-ankle acupuncture therapy. In the journal articles, there were 83 disease types and the most common surgical diseases were soft tissue injury and periarthritis of shoulder, the frequency were 34 and 24 times respectively. The total effective rates of wrist-ankle acupuncture therapy were 92.74% in internal medicine, 91.39% in surgery, 91.51% in gynecology, 90.88% in dermatology, 96.20% in pediatric, 89.05% in ophthalmology and otorhinolaryngology and 88.78% in psychiatry. In the medical cases of wrist-ankle acupuncture therapy, there were 118 disease types and the most common diseases were pain and syndrome, herpes zoster was also a common disease. Psychiatric diseases used more wrist-ankle acupuncture therapy, and the common diseases were anxiety and mania.@*CONCLUSION@#Wrist-ankle acupuncture therapy is widely used in treatment of clinical diseases and has significant therapeutic effects. It is mostly used in the treatment of surgery, internal medicine (neurology particularly), dermatology (herpes zoster particularly). Besides, the wrist-ankle acupuncture therapy is also applicable in the treatment of diseases of ophthalmology and otorhinolaryngology, gynecology, pediatric and psychiatry.

Effect of Human Umbilical Cord Mesenchymal Stem Cells on Etoposide-induced Nalm-6 Cell Apoptosis / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSC) on VP16-induced apoptosis of Nalm-6 cells.</p><p><b>METHODS</b>hUC-MSC were isolated and identified using morphological observation and flow cytometry, then Nalm-6 cells were treated with hUC-MSC with or without VP16. Apoptosis and cell cycle were assayed by FACS. The mRNA levels of apoptosis-related genes BCL-2, BAX and caspase-3 were detected by quantitative RT-PCR, and the protein levels of BCL-2, BAX and caspase-3 were examined by Western blot.</p><p><b>RESULTS</b>FACS showed that hUC-MSC inhibited the proliferation and decreased apoptosis of Nalm-6 cells resulted from VP16. The quantitative RT-PCR showed that hUC-MSC increased the mRNA expression level of BCL-2 and decreased the expression level of BAX and caspase-3 (P < 0.05). Western blot showed that the protein expression level of BCL-2 increased, and expression level of BAX and caspase-3 decreased in Nalm-6 cells after co-culture with hUC-MSC (P < 0.05).</p><p><b>CONCLUSION</b>hUC-MSC may protect Nalm-6 cells from apoptosis induced by VP16 through regulation of BCL-2, BAX and caspase-3.</p>

The Expression of MicroRNA-155 in Plasma and Tissue Is Matched in Human Laryngeal Squamous Cell Carcinoma

PURPOSE: Tumor-associated microRNAs have been detected in cancer, though whether plasma microRNA-155 (miR-155) could be a potential biomarker for laryngeal squamous cell carcinoma (LSCC) prognosis is unclear. We aimed to determine how miR-155 can be used to predict the clinical characteristics of patients with LSCC and correctly diagnose them. MATERIALS AND METHODS: We collected tissue samples and peripheral blood samples before and after treatment from 280 LSCC cases and 560 controls. Real-time quantitative reverse transcription PCR was employed in this study to compare the relative expression of miR-155. RESULTS: A total of 280 LSCC patients and 560 age- and sex-matched controls were included in the study. The miR-155 level was more up-regulated in LSCC tissue than in the non-tumor tissues (13.6+/-2.4 vs. 3.1+/-0.80, p<0.001). Additionally, a significantly higher miR-155 level in plasma samples from LSCC patients than in those of the controls (8.9+/-1.25 vs. 1.8+/-0.8, p<0.001) was reported. Tissue miR-155 showed an area under the curve (AUC) of 0.933, with a sensitivity of 82.6% and a specificity of 89.2%. The AUC for plasma miR-155 was 0.757, with a sensitivity of 58.4% and a specificity of 69.5%. When early LSCC in TNM I stage was considered, tissue miR-155 showed an area under the curve of 0.804, with a sensitivity of 85.2% and a specificity of 87.3%. CONCLUSION: The expression of tissue and plasma miR-155 were significantly up-regulated in patients with LSCC. Our work will serve as a basis for further investigation, preferably large-scale validation in clinical trials.

Research of bleeding volume and method in blood-letting acupuncture therapy based on data mining / 中国针灸

Through computer-based technology and data mining method, with treatment in cases of bloodletting acupuncture therapy in collected literature as sample data, the association rule in data mining was applied. According to self-built database platform, the data was input, arranged and summarized, and eventually required data was acquired to perform the data mining of bleeding volume and method in blood-letting acupuncture therapy, which summarized its application rules and clinical values to provide better guide for clinical practice. There were 9 kinds of blood-letting tools in the literature, in which the frequency of three-edge needle was the highest, accounting for 84.4% (1239/1468). The bleeding volume was classified into six levels, in which less volume (less than 0.1 mL) had the highest frequency (401 times). According to the results of the data mining, blood-letting acupuncture therapy was widely applied in clinical practice of acupuncture, in which use of three-edge needle and less volume (less than 0.1 mL) of blood were the most common, however, there was no central tendency in general.

Comparison of different moxibustion techniques in clinical treatment of diseases based on data mining / 中国针灸

<p><b>OBJECTIVE</b>To explore the specific efficacy of different moxibustion techniques in treatment of common diseases and clinical indications, and compare the specificity in clinical indications and efficacy among different moxibustion techniques so as to guide clinical practice better.</p><p><b>METHODS</b>The modern computerization and data mining technology were adopted to set up moxibustion literature database. The relevant literature of moxibustion techniques in recent 60 years were collected, screened, examined, extracted and analyzed statistically so as to explore the advantages of different moxibustion techniques in clinical treatment.</p><p><b>RESULTS</b>(1) Of 2,516 literature, moxa stick, moxe cone and moxa device were used in the highest frequency in internal medicine department, for 730 times, 278 times and 102 times respectively. The warm needling technique was used in the highest frequency, for 70 times in the surgical department. (2) In the dermatology department, the curative rate with moxa cone was the highest, 75%. In the ear-nose-throat department, the warm needing technique and moxa device achieved the highest curative rate, 49% for both of them. In the internal medicine department and surgical department, the curative rate of warm needling technique was 53% and 58% respectively. In the gynecology department, the curative rate of moxa device was the highest, 59%. In the pediatrics department, the curative rate of moxa cone was the highest, 80%. (3) The numbers of priority disorders, frequency ≥20 times: 24 kinds of disease for moxa stick, five kinds of disease for moxa cone, 2 kinds of disease for warm needling technqiue and one disorder for moxa device. Facial paralysis, diarrhea, lumbar and leg pain and elbow and knee swelling pain were of the highest priority, treated with these 4 moxibustion techniques, with a certain of literature research values. (4) The warm needling technique achieved the better efficacy on elbow and knee swelling pain, lumbar and leg pain and diarrhea compared with the other three techniques and the curative rate was higher. The moxa device tecnique achieved the higher curative rate for facial paralysis compared with the other three techniques.</p><p><b>CONCLUSION</b>Through the comparison of application frequency, curative rate, clinical application frequency in disorders and the efficacy of priority disorders in the treatment with different moxibustion techniques, it is found that moxa stick, moxa cone and moxa device are simple in manipulation, safe and effective. Hence, they can be extensively used in the treatment of common disorders in every department in clinic. The warm needling technique acts on the body by the co-work of needling and warming stimulation of mugwort. It achieves the particular effect on the disorders with complicated etiologies compared with the other three techniques. It can be chosen in priority for the disorders caused by blockage in meridian and collateral and stagnation of qi and blood.</p>

Diseases treated by moxibustion and fire needling in clinical practice based on data mining: a comparison study / 中国针灸

<p><b>OBJECTIVE</b>To discuss the specificity of diseases treated by moxibustion and fire needling in clinical practice, so as to provide references for clinical treatment.</p><p><b>METHODS</b>With data mining of modern computer technique, journal and literature databases regarding moxibustion and fire needling were established, respectively. Literature regarding moxibustion and fire needling for the past 60 years has been collected, screened, included, reviewed and abstracted. The utility rate of moxibustion and fire needling in each department was calculated, frequency of diseases in clinical practice was summarized, and diseases which had differences in clinical practice in each department were screened; also the advantages of disease categories and clinical practice between two kinds of therapies in each department were compared.</p><p><b>RESULTS</b>(1) The utility rate of moxibustion was highest in department of internal medicine and surgery, which were 43.6% and 28.1%, respectively; the utility rate of fire needling was highest in surgery and dermatological department, which were 53.7% and 23.8%, respectively. (2) According to the comparison and analysis on diseases treated by two therapies in clinic, among 26 kinds of gynecology diseases that were treated by moxibustion, 20 kinds were not involved with fire needling; among 22 kinds of pediatrics diseases that were treated by moxibustion, 20 kinds were not involved with fire needling. It was certain that the difference of the two therapies in clinical application was more significant in gynecology and pediatrics than that in the rest four departments. (3) Among the diseases which had differences in clinical practice in each department, the ones involved with moxibustion alone were insomnia, distention and fullness, consumptive fatigue in the department of internal medicine, blood-vessel Bi, stiff neck and hernia in surgery department, urticarial, skin Bi and skin cancer in dermatological department, malposition, infertility and amenorrhea in gynecology department, diarrhea, indigestion and stomachache in pediatrics department, blepharoptosis, blurred vision and dryness syndrome in ENT department; the ones involved with fire needling alone were numbness, coldness syndrome and acute renal colic in the department of internal medicine, lipoma, soft tissue injury and papilloma in surgery department, bromhidrosis, freckle and erysipelas in dermatological department, uterine fibroid in gynecology department, umbilical polyp in pediatrics department, auricle pseudocyst, starred nebula and phlegmatic mass in ENT department.</p><p><b>CONCLUSION</b>Moxibustion is frequently applied in department of internal medicine and surgery, while fire needling is frequently used in surgery and dermatological department; the application of moxibustion is broader than that of fire needling in gynecology and pediatrics department. Among the diseases which have differences in clinical practice in each department, moxibustion is commonly seen for clinical symptoms featured with deficiency syndrome, while fire needling is commonly applied for the diseases that have obvious local symptoms.</p>

Exploration on the application characteristics of bloodletting therapy for the diseases relevant with soft tissue infection by data mining technology / 中国针灸

<p><b>OBJECTIVE</b>Using the data mining technology to analyze the application law of bloodletting therapy for the diseases relevant with soft tissue infection and explore the effect specificity of bloodletting therapy.</p><p><b>METHODS</b>The variety of diseases treated with bloodletting therapy was taken as the sample data by collecting the reports from the periodical literatures. The network database of bloodletting literature was set up. The association rule of data mining technology was adopted to extract the information on the diseases relevant with soft tissue infection in the periodical literatures. The frequency item of the superiority disease category was calculated. And the deep mining was done in terms of the needle device, point selection and the bleeding amount separately. The application law of bloodletting therapy was summarized in the treatment of the diseases relevant with soft tissue infection.</p><p><b>RESULTS</b>Fourteen kinds of relevant diseases of soft tissue infection treated with bloodletting therapy were included in the contemporary periodical literatures, of which, the reported case number and the reporting frequency were the highest for hordeolum and breast abscess. The three-edged needle was used the most frequently in the manipulation. The acupoints selected were the local Ashi points, combined with the symptomatic acupoints. The bleeding amount was not normalized by the ideas of different physicians.</p><p><b>CONCLUSION</b>The bloodletting therapy achieves the better curative effect on some diseases relevant with soft tissue infection and this therapy deserves to be further promoted in clinical practice.</p>

Acupoint catgut-embedding therapy: superiorities and principles of application / 中国针灸

<p><b>OBJECTIVE</b>To analyze the superiorities of acupoint catgut-embedding therapy, discuss its law of clinical application and provide scientific decision-making for clinical treatment.</p><p><b>METHODS</b>Literatures on acupoint catgut-embedding therapy in the recent 40 years were selected, input, examined and verified, picked up and analyzed by establishing database with the modern computer technology.</p><p><b>RESULTS</b>(1) One thousand and seventy-five literatures were input. It shows that the acupoint catgut-embedding therapy has an extensive application in all departments, especially in the internal department, accounting for 48.54% (50/103) of the total disease category. It has the most extensive application on treatment of epigastric pain, with the frequency of 102 times, and obesity of 74 times. The next is surgery, accounting for 14.56% (15/103). The major application is on low back pain and leg pain with the frequency of 79 times. Psoriasis, with the frequency of 30 times, holds the major application in dermatological department. And blepharoplasty, with the frequency of 30 times, gains the most application in department of ophthalmology and otorhinolaryngology. (2) In the included literatures, selection of adjacent acupoints and distal acupoints are held as the major method of acupoint selection. The adjusted lumbar puncture needle is taken as the major tool for the acupoint catgut-embedding therapy. And catguts of different sizes are adopted for the operation. (3) Analysis of the therapeutic effect shows that acupoint catgut-embedding therapy has obvious effect in all departments, especially in surgery and dermatology, with the total effective rate over 90%.</p><p><b>CONCLUSION</b>Epigastric pain, obesity, epilepsy, asthma, abdominal pain, facial paralysis and constipation of the internal medicine, low back pain and leg pain of the surgical department, psoriasis of the dermatological department and blepharoplasty of the department of ophthalmology and otorhinolaryngology are considered as the dominant diseases for acupoint catgut-embedding therapy.</p>

Specificity of acupuncture-moxibustion effect and its research value / 中国针灸

Acupuncture-moxibustion is the major method and process in acupuncture treatment. And obvious therapeutic effect can be found toward different phases of syndromes or various diseases. The concept of specificity of acupuncture-moxibustion effect is firstly put forward by this article, which refers to the corresponding relationship between different acupuncture and moxibustion techniques and the best indications or phases of syndromes. Meanwhile, through analysis of literatures concerning comparison on effects of different acupuncture-moxibustion techniques, different manipulations of the same acupuncture technique and different parameters of the same technique, the subjective existence of the specificity of acupuncture-moxibustion effect is approved. The recognition on specificity of acupuncture-moxibustion effect by domestic experts is also expounded. It is demonstrated that the study of specificity of acupuncture-moxibustion effect can be advanced through revealing of the law and features of its specificity, which is considered to be significant for clinical decision, educational implement and scientific research designation of acupuncture and moxibustion.

The effect of inhibitory and activating natural killer cell immunoglobulin receptor on haplo-identical hematopoietic stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of inhibitory and activating KIRs on a cohort of Chinese leukemia patients who received haplo-identical hematopoietic stem cell transplantation (HSCT).</p><p><b>METHODS</b>Donor's inhibitory and activating KIRs and recipient's HLA-C from 47 cases who received haplo-identical HSCT were tested by PCR-SSP. 2 year overall survival (OS), incidence of severe (grade III to IV) acute GVHD (aGVHD) and relapse rate (RR) were analyzed.</p><p><b>RESULTS</b>(1) According to Matched (M) vs Mis-Matched (MM) between donor's inhibitory KIR and recipient's HLA-C1/C2 subgroup, 2 year OS rate in M group [(87.5 +/- 8.3)%] was significantly higher than that in MM group (54.5 +/- 9.0)%, (P = 0.03). Lower incidence of relapse rate was seen in M group than in M/MM groups [0 vs (25.4 +/- 9.5)%, P = 0.05]. In 30 cases of myeloid leukemia patients, there was lower RR in M group than in MM groups [0 vs (35.0 +/- 14.4)%, P = 0.04]. (2) According to the 3 activating KIR genes: KIR2DS1/ KIR2DS2/ KIR2DS3, lower incidence of grade III-IV aGVHD was seen in KIR2DS1 (+) group than in KIR2DS1 (-) group (13% vs 28%, respectively, P > 0.05); and so was done in KIR2DS3 (+) group (11% vs 26%, respectively, P > 0.05). The RR was lower in KIR2DS2 (+) group [0% vs (17.3 +/- 7.1)%, respectively, P > 0.05].</p><p><b>CONCLUSIONS</b>In our haplo-identical HSCT setting, match between donor's inhibitory KIR and recipient's HLA-C can significantly reduce the incidence of relapse rate and improve OS. Although lower incidences of severe aGVHD are noted in the donors with KIR2DS1 (+) or KIR2DS3 (+), and lower relapse rate is noted in the donors with KIR2DS2 (+) but without statistic difference, no remarkable effects of activating KIRs on OS have been found in our relatively small clinical series.</p>

Influence of HLA-A/B/C/DRB1/DQB1 on unrelated donor hematopoietic stem cell transplantation / 中国实验血液学杂志

This study was purposed to explore the influence of number and locus of HLA allele mismatch on unrelated donor hematopoietic stem cell transplantation (URHSCT) in Chinese Han population. Total 10 alleles within the HLA-A/B/C/DRB1/DQB1 loci were analyzed by PCR-SSP for 101 pairs of donor and recipients who received URHSCT. 101 cases of URHSCT were divided into four groups: HLA-allele 10/10 match (n = 30), 9/10 (n = 32), 8/10 (n = 31) and 7/10 match (n = 8). The correlation of the HLA with overall survival (OS ≥ 1 year), incidence of acute GVHD (aGVHD) of grade II to IV and relapse rate of primary diseases were evaluated. The results showed that (1) The OS rates in HLA-10/10 and 9/10 groups were higher than that in HLA-8/10 match group (78% and 82% vs 50%, p = 0.39); incidence of aGVHD in the HLA-10/10 were lower than that in HLA-9/10 and HLA-8/10 group (0% vs 10% and 10%; p = 0.28); relapse rates among the 3 groups were close (16%, 18% and 20%, respectively). Although there were only 8 cases in HLA-7/10 match URHSCT, the data indicated that they were safe and effective; (2) Compared to the HLA-10/10 match URHSCT (n = 30), the HLA-C mismatch URHSCT (n = 12) harbored higher incidence of severe aGVHD (0% vs 25%, p = 0.006), longer OS (77% vs 85%, p = 0.30), and tendency to low relapse rate (8% vs17%, p = 0.47); (3) According to HLA-C1/C2, the ligands of inhibitory KIR, the 42 cases of HLA-10/10 match URHSCT and HLA-C mismatch URHSCT were grouped into donor/recipient HLA-C1/C2 match and mis-match subgroups. There was no difference between the two subgroups for OS, incidence of aGVHD and relapse rate (78% vs 80%, 14% vs 20%, and 5% vs 20%). It is concluded that for 0 to 2 locus of HLA allele mismatch in URHSCT, the fewer mismatch numbers, the longer OS, but with similar aGVHD incidence and the relapse rate; triple HLA allele mismatch (HLA-7/10 match) is safe in URHSCT. The HLA-C mismatch may be related to higher incidence of aGVHD and lower relapse rate and prolonged OS, remaining to be further studied.

Role of HLA protein three-dimensional conformation difference in unrelated hematopoietic stem cell transplantation / 中国实验血液学杂志

This study was purposed to investigate the value of Histocheck and HLA-Matchmaker softwares in evaluating influence of HLA protein three dimensional conformation among individuals on outcome of unrelated donor hematopoietic stem cell transplantation (URHSCT). Data of the HLA-A/B/C/DRB1/DQB1 genotypes from 62 cases of URHSCT (HLA-allele 10/10 match 30 cases, 9/10 match 32 cases) were input into Histocheck and HLA-Matchmaker softwares respectively. The relationship between the software dissimilar scores and the 1 year overall survival (OS), incidence of aGVHD of III-IV grade and relapse rate was analyzed. The results showed that (1) with increase of the Histocheck scores, incidence of aGVHD of III-IV increased from 0% to 20% (p = 0.25), while no or mild aGVHD occurred in 70% cases with the high scores. For the relapsed cases, there was no significant difference between the cases with low scores and with highest scores (relapse rate 20%) except that 9 cases had no relapse in the group with higher score (11 - 20). (2) the analysis using HLA matchmaker software showed that incidence of aGVHD of III-IV grade increased with the increase of numbers of mismatch Eplets, arranging from 0% to 30%, the incidence of moderate aGVHD reduced (p = 0.019), whereas 60% cases in highest scores group had moderate aGVHD. No relapse occurred in the group with higher scores (≥ 3) (n = 10), whereas high relapse rate appeared in the lower score group (20%, p = 0.54). It is concluded that the value of Histocheck and HLA-Mtchmaker software for analysing the outcome of URHSCT may be similar despite of different calculating methods; for the certain pair of recipient and donor, correlation of the two score systems with incidence of aGVHD and relapse rate is similar, but with less accuracy; The HLA Matchmaker software appears better than Histocheck software in terms of correlation.

Cloning of the Phosphatidylserine Synthase Gene and Its Secretive Expression in Bacillus subtilis / 中国生物工程杂志

The gene encoding the phosphatidylserine synthase in Escherichia coli K12 Sgal-(ExPASy P23830) was amplified by PCR. After DNA sequence analysis, it was inserted into the inducible expressive shuttle vector pBES of Bacillus subtilis, which was constructed in the lab, and the recombinant plasmid pBES-pss was transformed into competent cells of the Bacillus subtilis strain DB104. The positive transformant DB104 (pBES-pss) was grown on Bacillus subtilis common fermentation medium, which contained 30?g/ml kanamycin. After 2 hours cultivation, sucrose was added and increased to the final concentration of 2% for induction and this phosphatidylserine synthase was secreted into the medium. The result of SDS-PAGE showed that the molecular weight of the protein was 52kDa and the result of enzyme coupling colorimetric method showed that the enzyme activity was 1.50U/ml. The recombinant Bacillus subtilis has increased the yield of phosphatidylserine synthase which will be used for industrial biosynthesis of phosphatidylserine.

Purification and Properties of Neutral Protease from Bacillus Subtilis ZC-7 / 中国生物工程杂志

Bacillus subtilis ZC-7 was obtained by implantation with N+ ions beam to B.subtilis AS1.398,and compared with the AS1.398 neutral protease,the enzyme activity of ZC-7 neutral protease was about 1 timeshigher in previous research.A neutral protease was purified from the culture of B.Subtilis ZC-7 by the procedures including amoninium sulfate precipitation,ultrafiltration,DEAE-Sepharose Fast Flow chromatography and Sephadex G-75 chromatography.By multi-step purification,the ZC-7 neutral protease was purified to 78.5 folds and its yield was 27.7%,at last,the specific activity of ZC-7 neutral protease was up to 4.1?105U/mg.Analysed by SDS-PAGE,the purified protease has shown a molecular mass of about 42kDa.The Km for casein hydrolysis was 3.67?10-3?g/ml and the Vmax was 12.21?g/min.The optimum pH and temperature forhydrolysis of casein were 7.0 and 55℃,respectively.This protease was stable up to 40℃ within the pH range of 6.5 and 8.0.EDTA,isopropanol and alcohol nearly inhibited its activity while some ions such as Ca2+,Mg2+,Fe3+ can improve its activity.In addition,it could resist 1 mol/L H2O2.

Studies on Gene Knocking Out of 3-Ketosteriod-1-Dehydrogenase in Mycobacterium neoaurum / 中国生物工程杂志

The selective side-chain cleavage of phytosterol to 4-androstene-3,17-dione(4-AD)and 1,4-androstadiene-3,17-dione(ADD)by Mycobacterium sp.was described.Because of the similarity in chemical structure between 4-AD and ADD,it is difficult to separate them from the fermentation broth.So far,it has been verified that the ADD can be produced by dehydrogenation of 4-AD.In this reaction,3-Ketosteriod-1-Dehydrogenase(ksdD)plays an important role.The gene knocking out method was used to solve the problem.Partial sequence of ksdD was obtained by PCR which was 631bp in length.Then,a targeting vector pUC19-MK was constructed,which was electroporate into the original strain Mycobacterium neoauru.The method of homologous recombination was used to knock out ksdD gene located in the chromosome of Mycobacterium neoauru.In this way,ksdD would lose its enzyme activity.In the result,5 transformants were screened.The experiments of steroid transformation by the transformants were carried out.The productivity of 4-AD reached 17.52% after 144h,which is 192% higher than the original strain.Meanwhile,the productivity of ADD reached 6.12%,which is 89.9% lower than the original strain.