Cell-free protein expression system is a new method to express target protein in vitro and has been widely applied to the study of protein structure, protein function and other related fields. Preparation of cell extract is one of the key factors that affect the efficiency of the cell-free system. To improve the efficiency and economical feasibility of cell-free protein synthesis, we discussed the parameters during the preparation of the cell extract. These parameters include centrifugation speed, pre-incubation, and dialysis. We used the green fluorescent protein as the reporter protein, and obtained a simple procedure for the preparation of Escherichia coli cell extract. A simple centrifugation step (12 000 x g, 10 min) followed by a brief incubation was sufficient for the preparation of an active cell extract to support protein expression with higher productivity (209 microg/mL). Compared to the traditional E. coli S30 procedure, the processing time was reduced by 62%, and the productivity was increased by 2.6 times. The new procedure will make the advantage of cell-free technology more obvious, and promote its wider application.
Cell Fractionation ; methods ; Cell-Free System ; Escherichia coli ; cytology ; genetics ; metabolism ; Escherichia coli Proteins ; biosynthesis ; chemistry ; isolation & purification ; Green Fluorescent Proteins ; metabolism

Objective To investigate the total examinations of conventional X-ray diagnosis and CT diagnosis of radiation diagnosis and treatment institutions,in order to explore the distribution characteristic of radiological diagnosis frequency and diagnostic patients,and to estimate the application frequency of medical X-ray diagnosis in Heilongjiang province.Methods The questionnaire was sent to all radiation diagnosis and treatment institutions in forms of public document issued by the administrative department of health and family planning of Heilongjiang province.Basic situations and patient numbers of conventional X-ray diagnosis (interventional diagnosis not included) and CT diagnosis in 2016 were collected and summarized combined with sampled on-site verification.The application frequency of medical X-ray diagnosis was obtained using permanent resident population in Heilongjiang province in 2016.Results Totally 1 645 medical radiation institutions were investigated,including 81 tertiary hospitals,359 secondary hospitals,808 primary hospitals or unrated medical institutions,and 397 private dental clinics.The examinations of conventional X-ray diagnosis and CT diagnosis were 7 706 050 and 7 063 734,respectively.The application frequency of conventional X-ray diagnosis and CT diagnosis was 200.9 and 184.1 examinations per 1 000 population,respectively,and varied from 98.0 to 274.7 in different areas.The ratio of examinations in public medical institutions to that in private medical institutions was 7.47 ∶ 1.Conclusions 50％ of the total radiological diagnosis were made in tertiary hospitals,and only 13％ were made in medical institutions below primary in Heilongjiang.Meanwhile,although the number of public and private medical institutions was at the same scale,the total examination of radiological diagnosis in public medical institutions was 7 times of that in private medical institutions.The application frequency of conventional X-ray diagnosis and CT diagnosis in Heilongjiang province in 2016 was similar to that of Jiangsu province in 2015.

Objective:To prepare the whole bladder acellular matrix (BAM) using the self-designed perfusion decellularization system, and evaluate the feasibility of constructing the tissue engineering bladder with the adipose-derived stem cells (ADSCs).Methods:This study was conducted from October 2020 to April 2021. The self-designed perfusion decellularization system was used, and four different decellularization protocols (group A, group B, group C and group D) were formulated, according to the flow direction of the perfusate and the action time of different decellularization solutions. Among them, the urethral orifice of the bladder tissue was used as the outflow tract of the perfusion fluid in groups A and B. The top of the bladder was cut off and used as the outflow tract of the perfusion fluid in groups C and D. In groups A and C, 1% Triton X-100 was treated for 6 h, and 1% sodium dodecyl sulfate (SDS) was treated for 2 h. In groups B and D, 1% Triton X-100 was treated for 7 h, and 1% sodium dodecyl sulfate (SDS) was treated for 1 h. In addition, the tissue in the normal bladder group was directly obtained from the natural bladder tissue, which did not require perfusion, cryopreservation and thawing. The fast and efficient decellularization protocol was screened out through HE, DAPI, Masson trichrome and Alcian Blue staining and quantitative analyses to prepare the whole bladder scaffold. The prepared BAM was used as the scaffold material, and the ADSCs were used as the seeding cells to construct the tissue engineering bladder. HE and DAPI staining were used to observe the distribution of ADSCs on the BAM.Results:HE and DAPI staining showed that there was no obvious nuclear residue in the group C. Masson trichrome and Alcian Blue staining showed that the collagen structure and glycosaminoglycan were well preserved in the group C. There was no significant difference in bladder wall thickness between the group C and the normal bladder group [(975.44±158.62)μm vs.(1 064.49±168.52)μm, P > 0.05]. The DNA content in the group C [(43.59 ±4.59) ng/mg] was lower than that in the normal bladder group, group A, group B and group D [(532.50±26.69), (135.17±6.99), (182.49±13.69) and(84.00±4.38)ng/mg], and the difference was statistically significant ( P<0.05). The collagen content [(10.98 ± 0.29)μg/mg] and glycosaminoglycan content [(2.30±0.18)μg/mg] in group C were not significantly different with those in the normal bladder group [(11.69±0.49) and (2.36±0.09)μg/mg, P>0.05]. Scanning electron microscopy showed that a large number of pore structures could be observed on the surface of the prepared BAM in groups A-D and were facilitated to cell adhesion. The isolated and cultured ADSCs were identified by flow cytometry to confirm the positive expression of CD90 and CD29, and the negative expression of CD45 and CD106. Live/dead staining and CCK-8 detection confirmed that the prepared BAM in the group C had no cytotoxicity. HE and DAPI staining showed that a large number of ADSCs were distributed on the surface and inside of the tissue engineering bladder. Conclusions:The whole bladder shape BAM prepared by the self-designed perfusion decellularization system could be used as the scaffold material for bladder tissue engineering, and the constructed tissue engineering bladder could be used for bladder repair and reconstruction.

Objective:This study aimed to investigate the physical properties, biocompatibility, and 3D printing performance of a novel hybrid bioink composed of gelatin methacrylated (GelMA) and chitin nanocrystal (ChiNC).Methods:The study was conducted from May 2021 to December 2022, four different bioinks were prepared by adding varying amounts of ChiNC to GelMA bioink. The GelMA concentration in all four bioinks was 100 mg/ml, while the ChiNC concentrations were 0 mg/ml (no ChiNC added), 5 mg/ml, 10 mg/ml, and 20 mg/ml, respectively, named as GC0, GC5, GC10, and GC20 bioinks. The cross-sectional morphology of the hydrogels formed after photocuring the four bioinks was observed using scanning electron microscopy, and the porosity was calculated. Weighing the hydrogels before and after swelling, and then calculate the equilibrium swelling rate. HUVECs were seeded on the surfaces of the hydrogels prepared from the four bioinks and cultured in medium. Cell proliferation was assessed using CCK-8 assays at 1d, 3d, and 7d to compare the proliferation rates of cells on the four hydrogels. HUVECs were added to the four bioinks, and grid-like scaffolds were printed and cultured in medium. Live-Dead staining was performed at 1d and 7d to observe cell viability. Compare the printing effect of bioinks by observing its forming continuous threads properties during extrusion. Finally, tissue-engineered bladder patches simulating the mucosal layer, submucosal layer, and muscular layer anatomical structures of the bladder wall were 3D bioprinted using the optimized bioink composition, and the stability and fidelity of the printed structures were observed to further validate the feasibility of printing multi-layered complex structures with the bioink.Results:Scanning electron microscopy revealed that the porosity of the GC0, GC5, GC10, and GC20 hydrogels were (51.43±6.23)%, (51.85±6.47)%, (50.55±4.59)%, and (42.49±2.20)%, respectively. The differences in porosity between the GC0 group and the other three groups were not statistically significant ( P=0.9994, P=0.9948, P=0.1200). The equilibrium swelling ratio of the other three groups [(8.81±0.41), (7.95±0.19), (7.71±0.14)] was significantly lower than that of the GC0 group (9.37 ± 0.49), and the differences were statistically significant ( P=0.0457, P<0.01, P<0.01). CCK-8 assay showed no significant difference in absorbance value between the GC10 group (0.360±0.009) and the GC0 group (0.357±0.007), GC5 group (0.350±0.012), and GC20 group (0.345±0.018) on the first day ( P=0.9332, P=0.5464, P=0.4937). However, on the third day, the absorbance value of the GC10 group (0.755±0.012) was significantly higher than that of the GC0 group (0.634±0.010), GC5 group (0.704±0.009), and GC20 group (0.653±0.015) ( P<0.01, P=0.0033, P=0.0002). On the seventh day, the absorbance value of the GC10 group (1.001±0.031) was significantly higher than that of the GC0 group (0.846±0.026), GC5 group (0.930±0.043), and GC20 group (0.841±0.024)( P=0.0012, P=0.1390, P=0.0010). The addition of human umbilical vein endothelial cells (HUVECs) into the four groups of hydrogels enabled the printing of grid-like scaffolds, and Live-Dead staining was performed on day 1 and day 7. The cell viability of HUVECs in the four groups on day 1 was (90.13±1.63)%, (90.6±2.45)%, (92.58±2.15)%, and (91.40±3.17)%, respectively. There were no statistically significant differences between the GC0 group and the other three groups ( P=0.9869, P=0.3093, P=0.8008). On day 7, the cell viability was (89.97±3.10)%, (92.18±2.21)%, (92.05±2.25)%, and (90.12±1.97)% for the four groups, respectively. There were no statistically significant differences between the GC0 group and the other three groups ( P=0.3965, P=0.4511, P=0.9995). Bioink extrusion test showed that the GC0 hydrogel could be extruded continuously and form threads at temperatures between 24℃ and 25℃, while the GC10 hydrogel could be extruded continuously and form threads at temperatures between 24℃ and 27℃. Printing tissue engineered bladder patches simulating the anatomical structure of the bladder mucosal layer, submucosal layer, and muscular layer using GC10 bioink, and the printed patches were stable, without collapse, and had high fidelity. Conclusions:Adding ChiNC to GelMA promotes cell adhesion, proliferation, and expands the printing window of GelMA bioink. The biocompatibility of the mixed bioink prepared by adding 10 mg/ml ChiNC in GelMA is good, capable of printing tissue-engineered bladder patches that mimic the anatomical structure of natural bladder walls.

Objective:To investigate the effect of tissue engineered bladder patch constructed by double-layer silk scaffold and adipose-derived stem cells (ADSCs) in the repair and reconstruction of bladder.Methods:This study was conducted from May 2020 to March 2021. The silk fibroin (SF) aqueous solution was obtained from silkworm cocoons, and a double-layer silk scaffold composed of silk fibroin film and silk fibroin sponge was further prepared. The rat ADSCs were isolated, cultured, and the ADSCs surface markers (CD29, CD90, CD45, CD106) were identified by flow cytometry. The ADSCs were planted on a double-layer silk scaffold to construct a tissue-engineered bladder patch. Thirty-six male SD rats were randomly divided into three groups: tissue engineered bladder patch group (SF-ADSCs group, n=15), double-layer silk scaffold group (SF group, n=15), control group ( n=6). The tissue engineered bladder patch (SF-ADSCs group) and double-layer silk scaffold (SF group) were wrapped on the omentum to promote vascularization. The vascularization was evaluated by HE and immunofluorescence staining. The wrapped tissue engineered bladder patch and double-layer silk scaffold were used to repair the defective bladder. In the control group (six rats), the incision was closed immediately after the bladder tissue fully exposed. At 4 weeks and 12 weeks after operation, the general morphology of bladder tissue and cystography were performed to evaluate the recovery of bladder morphology. After the graft was harvested, HE and Masson's trichrome staining and immunofluorescence staining were used to observe the regeneration of bladder wall tissue. Urodynamics was used to assess the recovery of bladder function at 12 weeks after operation. Results:The flow cytometry results confirmed that the isolated cells positively expressed CD29 and CD90, and there was no significant expression of CD45 and CD106. Gross observation and scanning electron microscope confirmed that the preparation of double-layer silk scaffold not only had a pore structure that was conducive to cell planting, but also had good toughness and was facilitated to surgical suture. The number (43.50±2.66) and area (0.73±0.03)% of vascular-like structures in the SF-ADSCs group after the omentum encapsulation was significantly higher than that in the SF group [(24.50±3.51), (0.55±0.05)%], and the difference was statistically significant ( P<0.05). At 4 weeks after bladder repair, the histological staining of the grafts in the SF-ADSCs and SF groups showed a large number of degraded fragments of double-layer silk scaffold. At 12 weeks, the morphology of the graft in the SF-ADSCs group showed uniform bladder morphology, which was similar to that of normal bladder tissue. Immunofluorescence staining showed that the continuous urothelial layer, abundant smooth muscle tissue, vascular structure and regenerated neurons could be observed in the SF-ADSCs group. Urodynamic test showed that the bladder maximum volume (0.74±0.03)ml and compliance (16.68±0.44)μl/cm H 2O in the SF-ADSCs group, which were better than that in the SF group [(0.47±0.05)ml, (14.89±0.37)μl/cm H 2O], but lower than that in the control group [(1.12±0.08)ml, (19.34±0.45)μl/cm H 2O], and the difference was statistically significant ( P<0.05). Conclusions:The tissue engineered bladder patch constructed with double-layer silk scaffolds and ADSCs could promote the morphological repair of bladder tissue, the regeneration of bladder wall structure and the recovery of bladder physiological function.

Objective To observe the clinical effect of "palace bone-setting" Dieda-Wanying cream combined with Yuan Shu Zhi Pai Zi fixation in the treatment of acute ankle sprain.Methods A total of 60 patients with acute ankle sprain were collected and randomly divided into observation group and control group according to the random number table method,with 30 cases in each group.The observation group was treated with "Palace Bone-setting" Dieda-Wanying cream combined with Yuan Shu Zhi Pai Zi for fixation.The control group was treated with external application of diclofenac diethylamine emulsion and fixation of elastic bandage.Two groups were treated for 14 days.The patients pain disappeared time and the swelling subsided,the VAS scale for assessment of ankle pain observed.The American Orthopedic Foot and Ankle Society (AOFAS) Ankle-after scoring system was used to assess the Ankle function,evaluation of clinical curative effect.Results The pain disappear time (9.7 ± 1.6 d vs.13.4 ± 3.5 d,t=5.638),regression and time (3.5 ± 1.3 d vs.6.7 ± 1.1 d,t=10.292) in the observation group were significantly earlier than those in the control group (P＜0.01).After treatment,VAS score (1.9 ± 1.1 vs.3.3 ± 1.3,t=4.503) of the observation group significantly lower than that of the control group (P＜0.01);AOFAS score (93.6 ± 3.9 vs.84.2 ± 5.1,t=8.019) significantly higher than the control group (P＜0.01).Total effective rate in the observation group was 96.7％ (29/30),and the control group was 80.0％ (24/30),which the difference was statistically significant (x2=4.043,P=0.044).Conclusions The "Palace Bone-setting" Dieda-Wanying cream combined with Yuan Shu Zhi Pai Zi fixation has definite curative effect on acute ankle sprain,relieve pain,swelling and promote the ankle function.

ObjectiveTo observe the clinical efficacy of herb-partitioned moxibustion on the navel combined with Yishen Tongluo prescription in the treatment of infertility induced by idiopathic asthenozoospermia （iAZS） with kidney-Yang deficiency and collateral obstruction syndrome and its effect on sperm DNA damage and superoxide dismutase （SOD） in the seminal plasma. MethodsA total of 112 eligible patients who met the inclusion criteria were randomly divided into an observation group （56 cases） and a control group （56 cases）. The patients in the observation group were treated with herb-partitioned moxibustion on the navel combined with Yishen Tongluo prescription，while those in the control group received levocarnitine oral liquid. The primary observation indicators included spouse pregnancy rate，progressive motility （PR），and total sperm motility，and the secondary observation indicators included sperm DNA fragmentation index （DFI），SOD in the seminal plasma， and improvement of TCM syndromes. The treatment cycle was 12 weeks. Before and after treatment，the PR，total sperm motility，sperm DFI，SOD in the seminal plasma， and TCM syndrome scores were recorded. The patients were followed up for 12 weeks and the pregnancy status of spouses within 24 weeks （half a year） was recorded. The clinical efficacy of the two groups was evaluated. ResultThe pregnancy rate of spouses in the observation group was 15.69% （8/51）， higher than 3.85% （2/52） in the control group （χ²=4.118，P<0.05）. The total effective rate of the observation group was 88.24%（45/51）， superior to 69.23% （36/52）in the control group （Z=-3.402，P<0.01）. After treatment， PR， total sperm motility，sperm DFI， SOD in the seminal plasma， and TCM syndromes of the two groups were improved compared with those before treatment （P<0.05）， and the observation group was superior to the control group （P<0.05）. ConclusionHerb-partitioned moxibustion on the navel combined with Yishen Tongluo prescription in the treatment of iAZS-induced infertility patients with kidney-Yang deficiency and collateral obstruction syndrome can increase PR，total sperm motility， and SOD level in the seminal plasma， reduce sperm DFI，improve the TCM symptoms of patients， and improve the pregnancy rate of spouses. The mechanism may be attributed to the fact that this treatment can increase the SOD level in the seminal plasma of patients，enhance the body's antioxidant function，protect sperm from oxidative stress damage，and reduce sperm DFI.

Objective:To investigate the prognostic value and related factors of heart-type fatty acid binding protein (H-FABP) in patients with heart failure.Methods:A total of 877 consecutive patients who were admitted to heart failure care unit of Fuwai hospital and diagnosed as heart failure from July 2015 to July 2017 were enrolled in this study. Baseline serum H-FABP concentration was measured by fluorescence lateral flow immunoassay. According to serum H-FABP levels, patients were divided into three groups: low H-FABP group (H-FABP≤4.04 ng/ml, n=292), middle H-FABP group (H-FABP 4.04-7.02 ng/ml, n=292) and high H-FABP group (H-FABP≥7.02 ng/ml, n=293). The general clinical characteristics were collected and compared among the three groups. According to whether heart failure was caused by coronary artery disease or not, patients with heart failure were divided into ischemic heart failure and non-ischemic heart failure. Multivariate linear regression analysis was performed to explore the independent risk factors of H-FABP. The primary endpoint events were the composite of all-cause death or heart transplantation. Multivariate Cox regression analyses, receiver operating characteristic (ROC) curves, risk prediction tests with multivariate Cox regression model and Kaplan-Meier analyses were conducted to investigate the relationship between H-FABP and the prognosis of heart failure. Results:Multivariate linear regression analysis showed that age, coronary artery disease, alanine aminotransferase, uric acid and N-terminal pro-B type natriuretic peptide (NT-proBNP) were positively associated with H-FABP (β=0.012, 0.238, 0.001, 0.345 and 0.063 respectively,all P<0.05), while female, hemoglobin, albumin, sodium, and estimated glomerular filtration rate (eGFR) were negatively associated with H-FABP (β=-0.184, -0.006, -0.016, -0.034 and -0.006 respectively, all P<0.05). One hundred and nineteen patients (13.6%) lost to follow-up, and 246 patients (32.5%) suffered from all-cause death or heart transplantation during the median follow-up duration of 931 (412-1 185) days. Multivariate Cox regression analysis showed that baseline H-FABP (log 2H-FABP) level was the independent predictor of all-cause death or heart transplantation in patients with heart failure ( HR=1.39, P<0.001). ROC curves showed that baseline H-FABP was a predictor of all-cause death or heart transplantation in patients with heart failure within 3 months, 1 year and 2 years (areas under the curves were 0.69, 0.69 and 0.71 respectively), and the best cut-off values were 5.85 ng/ml, 6.54 ng/ml and 6.54 ng/ml respectively. Risk prediction test with multivariate Cox regression model showed that baseline H-FABP could provide additional prognostic value in predicting all-cause death or heart transplantation for patients with heart failure on top of basic model and baseline NT-proBNP ( P<0.001). Taking 6.54 ng/ml and trisected levels of H-FABP as cut-off values respectively, Kaplan-Meier analyses showed that the survival rates were significantly different among the two or three groups ( P<0.001). Subgroup analyses showed that baseline H-FABP (log 2H-FABP) level was an independent predictor of all-cause death or heart transplantation in patients with ischemic heart failure ( HR=1.74, P<0.001), as well as in patients with non-ischemic heart failure ( HR=1.28, P=0.027). Conclusions:Age, sex, coronary artery disease, hemoglobin, albumin, alanine aminotransferase, sodium, eGFR, uric acid and NT-proBNP are associated with H-FABP level. Baseline H-FABP level is an independent predictor of all-cause death or heart transplantation in patients with heart failure. On top of basic model and baseline NT-proBNP, baseline H-FABP could provide additional prognostic value in predicting adverse events for patients with heart failure.

Cyclic lipopeptide has extensive application prospect in the field of medicine due to its unique chemical structure and biological activity. This study aims to obtain high purity of cyclic lipopeptide monomer from Bacillus amyloliquefaciems strain Q-426, and illuminate preliminary antitumor mechanism of C-15 Bacillomycin D and C-16 Bacillomycin D. Firstly, crude cyclic lipopeptide solution was prepared by two-steps purification of acid precipitation and double-resins chromatography. In order to obtain purer product preparative HPLC was utilized to separate and purify cyclic lipopeptide. Component 1 and component 2 were detected as C-15 Bacillomycin D and C-16 Bacillomycin D by HPLC-MS and ESI-MS/MS. Secondly, the effect of C-15 Bacillomycin D, C-16 Bacillomycin D and their mixture (1:1, mol:mol) on cell proliferation was measured using human cancer cells (Hela, MG, Hep-G2 and HT-29). The cyclic peptide showed a dose dependent manner on the cell proliferation inhibition of Hela and MG cells. Finally, the results of the scratch wound healing assay and FACS analysis revealed that C-16 Bacillomycin D can effectively influence the cells migration and the cells treated with C-16 Bacillomycin D showed typical apoptotic morphology with the increase of drug concentration in the early apoptosis, late apoptosis percentage increased, and G₀G₁ arrest was induced significantly.

The purpose of this study is to provide a simple and reliable genetic typing approach for molecular drug susceptibility test of Mycobacterium tuberculosis, through the developing of fluorescence molecular marker of rifampicin resistance gene rpoB. Eleven fluorescent molecular markers of the rpoB gene were established by using the sequence difference between the amino acid positions 531, 526, 516, 511 and 513 of rpoB gene of rifampicin-resistant strains and the alleles of rifampicin-sensitive strains, combined with the PARMS technique (Penta-primer amplification refractory mutation system). We used 104 clinical isolates of Mycobacterium tuberculosis to validate this marker and it was verified by sequencing as 100% correct. These samples were also tested with proportional drug sensitivity test. The coincidence rate was 94.23%. The molecular markers had high reliability for genotyping of rpoB gene. It can also detect low-concentration drug-resistant samples (511/533 unit point mutations) whose phenotypic susceptibility cannot be detected. The eleven sets of fluorescent molecular markers could cover 92%-96% of rpoB gene mutation types of rifampicin-resistant strains, and provide new idea for rapid detection of rifampin-resistant Mycobacterium tuberculosis.
Bacterial Proteins/genetics* ; DNA-Directed RNA Polymerases/genetics* ; Drug Resistance, Bacterial/genetics* ; Microbial Sensitivity Tests ; Mutation ; Mycobacterium tuberculosis/genetics* ; Reproducibility of Results ; Rifampin/pharmacology* ; Technology