The high content of sucrose and raffinose reduces the prebiotic value of soybean oligosaccharides. Fructan sucrases can catalyze the conversion of sucrose and raffinose to high-value products such as fructooligosaccharides and melibiose. To obtain a fructan sucrase that can efficiently convert soybean oligosaccharides, we first mined the fructan sucrase gene from microorganisms in the coastal areas of Xisha Islands and Bohai Bay and then characterized the enzymatic and catalytic properties of the enzyme. Finally, recombinant extracellular expression of this gene was carried out in Bacillus subtilis. The results showed that a novel fructan sucrase, BhLS 39, was mined from Bacillus halotolerans. With sucrose and raffinose as substrates, BhLS 39 showed the optimal temperatures of 50 ℃ and 55 ℃, optimal pH 5.5 for both, and K_cat/K_m ratio of 3.4 and 6.6 L/(mmol·s), respectively. When 400 g/L raffinose was used as the substrate, the melibiose conversion rate was 84.6% after 30 min treatment with 5 U BhLS 39. Furthermore, BhLS 39 catalyzed the conversion of sucrose to produce levan-type-fructooligosaccharide and levan. Then, the recombinant extracellular expression of BhLS 39 in B. subtilis was achieved. The co-expression of the intracellular chaperone DnaK and the extracellular chaperone PrsA increased the extracellular activity of the recombinant BhLS 39 by 5.2 folds to 17 U/mL compared with that of the control strain. BhLS 39 obtained in this study is conducive to improving the quality and economic benefits of soybean oligosaccharides. At the same time, the strategy used here to enhance the extracellular expression of BhLS 39 will also promote the efficient recombinant expression of other proteins in B. subtilis.
Oligosaccharides/metabolism* ; Glycine max/metabolism* ; Bacillus subtilis/metabolism* ; Sucrase/biosynthesis* ; Raffinose/metabolism* ; Fructans/metabolism* ; Sucrose/metabolism* ; Bacillus/genetics* ; Recombinant Proteins/biosynthesis* ; Bacterial Proteins/biosynthesis*

Bone metabolism includes the processes of bone formation and bone resorption, both of which maintain bone metabolic homeostasis in the body. Adipose browning is a biological process that converts energy-storing white fat into thermogenic brown fat, which is induced by environment, exercise, nutrients, and signaling molecules. Adipose browning regulates bone metabolism in vivo and affects osteogenic and osteoblastic capacity through the secretion of adipokines such as fibroblast growth factor-21, adiponectin, insulin-like growth factor-1, and bone morphogenetic proteins. Adipose browning can also affect bone metabolism through immune pathways mediated by the gut microbiota. The browning ability of fat decreases in the aging body, which is related to the imbalance of bone metabolism in the state of osteoporosis. In children and adolescents, fat browning is active and bone metabolism is maintained in a healthy state. Exercise and supplementation of nutrients (capsaicin, resveratrol, quercetin, etc.) can promote the browning of adipose tissue and maintain brown adipose tissue, which plays a positive role in bone metabolism. In the future, it is of great significance to clarify the specific regulatory mode between adipose browning and bone metabolism for the treatment of bone metabolism-related diseases by intervening adipose browning.

BACKGROUND:There is controversy regarding the need for marrow reaming in intertrochanteric fractures of the femur.Some believe that unreaming shortens operative time,reduces bleeding,and decreases intraoperative risk in elderly patients,but there is no basis for whether this move reduces the effectiveness of intramedullary nail support.Others believe that reaming allows for the selection of thicker diameter intramedullary nails for better mechanical support,but basic studies have shown that this approach carries risks such as fat embolism and destruction of bone(especially in elderly patients with osteoporosis). OBJECTIVE:To analyze the mechanical distribution characteristics of reamed and unreamed proximal femoral nail antirotation-Ⅱ in the treatment of type 31-A3 intertrochanteric fractures by finite element analysis. METHODS:A healthy volunteer was included,and CT scans of his femur were obtained in DICOM format,and the files were sequentially imported into Mimics,Geomagic Wrap,SolidWorks,Hypermesh,and Ansys software for processing.The A3.1,A3.2,and A3.3 intertrochanteric fracture models were obtained and assembled with 9 mm,11 mm diameter,and 170 mm length intramedullary nails,respectively,followed by assigning material properties,setting the interaction relationship of each contact surface and defining the load and boundary conditions,and then solved.The femoral stress distribution,internal fixation stress distribution,femoral displacement,and internal fixation displacement were observed in different models. RESULTS AND CONCLUSION:(1)The femoral stress was less than that of unreamed intramedullary nail fixation for each type of fracture,and the maximum stress value of the femur for A3.3 fracture was greater than that of A3.1 and A3.2.(2)The internal fixation stress was greater than that of unreamed intramedullary nail fixation for each type of fracture,and the maximum stress value of internal fixation for A3.3 fracture was greater than that of A3.1.(3)Reamed versus unreamed intramedullary nailing has less effect on femoral and internal fixation displacement and more effect on stress.(4)It is indicated that the use of reamed intramedullary nail fixation results in a reduction in femoral stress,an increase in the stress borne by the internal fixation as a whole,and a reduction in the stress borne by the distal locking nail.The use of reamed intramedullary nail fixation may provide better treatment results compared to unreamed intramedullary nail fixation.

Objective : To investigate the clinical and molecular epidemiological characteristics of 10 patients with enterovirus (EV)-D68 infections in the respiratory tract in Shaoxing City, Zhejiang Province, so as to provide insights into formulation of control interventions for EV-D68 infections. Methods: Clinical specimens were sampled from patients with acute respiratory tract infections (ARTIs) admitted to sentinel hospitals in Shaoxing City from 2021 to 2022, and EV-D68 was detected using real-time fluorescent PCR assay and sequencing of the VP1 region of enterovirus. The epidemiological and etiological characteristics of EV-D68 infected cases were descriptively analyzed. Results: A total of 3 009 specimens were sampled from patients with ARTIs from 2021 to 2022, and the detection of EV-D68 was 0.33%. Of all EV-D68-infected patients, there were 6 men and 4 women, and 5 cases under 18 years of age, 2 cases at ages of 18 to 60 years and 3 cases at ages of over 60 years. EV-D68 infection predominantly occurred in summer (5 cases detected between May and July) and autumn (5 cases detected between September and October). The main clinical symptoms included fever (10 cases), sore throat (9 cases) and cough (8 cases), and all 10 cases recovered well, with no deaths reported. Sequencing identified D3 subtype in all 10 specimens positive for EV-D68. Conclusions The ARTIs caused by EV-D68 occurred predominantly among children under 18 years of age in Shaoxing City, and was highly prevalent in summer and autumn. D3 was the predominant enterovirus subtype.

Objective To explore the relationship between serum Visfatin,pentraxin 3(PTX3),and carbo-hydrate antigen 19-9(CA19-9)levels and prognosis in patients with acute cholecystitis.Methods A total of 228 patients with acute cholecystitis admitted to a hospital from September 2020 to September 2023 were se-lected as the study objects.According to the severity of the patients,228 patients were divided into mild group(n=81),moderate group(n=102)and severe group(n=45).The patients were divided into good prognosis group(n=186)and bad prognosis group(n=42).General data of patients were collected,serum Visfatin and PTX3 levels were detected by enzyme linked immunosorbent assay,and serum CA19-9 levels were determined by chemiluminescence immunoassay.Univariate and multivariate analysis of prognostic factors in patients with acute cholecystitis was performed,and the diagnostic value of serum Visfatin,PTX3 and CA19-9 levels in patients with acute cholecystitis was analyzed by drawing receiver operating characteristic curve.Results Ser-um Visfatin,PTX3 and CA19-9 in moderate and severe groups were significantly different from those in mild group(P＜0.05).There were significant differences in serum Visfatin,PTX3 and CA19-9 between severe group and moderate group(P＜0.05).Gallbladder thickness,long diameter and short diameter of gallbladder,serum Visfatin,PTX3 and CA19-9 levels in the poor prognosis group were significantly different from those in the good prognosis group(P＜0.05).Multivariate Logistic regression analysis showed that serum Visfatin,PTX3 and CA19-9 levels were the influential factors for poor prognosis of patients(P＜0.05).The area under the curve of serum Visfatin,PTX3 and CA19-9 combined diagnosis was significantly larger than that of Visfa-tin alone diagnosis(Z=4.577,P＜0.001)and PTX3 alone diagnosis(Z=3.132,P=0.002)and the area un-der the curve of CA19-9 diagnosis alone(Z=2.766,P=0.006).Conclusion Serum Visfatin,PTX3 and CA19-9 are elevated in patients with acute cholecystitis,which is related to the patient's condition.The combi-nation of the three is of better value in the diagnosis of poor prognosis.

Objectives:To analyze the safety and clinical effect of airway diagnosis in multifactor suspicious difficult airway patients with complex mouth restriction after entering the operating room.Methods:From March 2018 to May 2023, a total of 21 patients with suspicious difficult airway with complex mouth restriction were collected from the Northwest Women′s and Children′s Hospital and the Third Affiliated Hospital of Air Force Military Medical University, who were evaluated according to LEMON airway evaluation rule. Ventilation capacity score, airway airway score with soft vision mirror and comprehensive airway score of difficult airway were performed after admission to the operating room. Patients diagnosed with non-difficult airway were divided into group N and patients diagnosed with difficult airway were divided into group D. The number and proportion of patients in the two groups were counted, and the process of airway diagnosis, the way of airway establishment, the results, the frequency of hypoventilation during airway establishment, the number of intubation, and the time of airway establishment were analyzed.Results:A total of 15 patients in group N, accounting for 71.4%, underwent rapid intravenous induction nasal intubation after diagnosis of non-difficult airway, and the intubation was successful all the time. No hypoventilation occurred in all patients during intubation. A total of 6 patients in the group D, accounting for 28.6%, were assisted by pure oxygen mask ventilation after diagnosis of difficult airway, and underwent awake nasotracheal intubation after recovery. 4 patients were successfully intubated once, 2 patients were successfully intubated twice, and 1 patient experienced one hypopnea during the evaluation of ventilation capacity. The airway establishment time of the group N was (9.3±0.8)min, and that of the group D was (20.0±2.0)min, the difference was statistically significant ( P<0.05). The airway establishment time of all patients was (12.0±5.0)min, and the awake intubation time was (12.0±0.7.0)min. The proportion of patients satisfied with anesthesia in the group N was higher than that in the group D, and the difference was statistically significant ( P<0.05). No agitation during sevoflurane induction or recovery was observed in both groups. Conclusions:The diagnosis procedure of difficult airway can be safely applied to patients with multi-factor suspicious difficult airway with complex mouth restriction. Most of these patients do not have truly difficult airways; The application of difficult airway diagnosis procedures in these patients can significantly reduce unnecessary awake endotracheal intubation; Compared with the direct choice of awake intubation, following the difficult airway diagnosis procedure does not increase airway establishment time in such patients.

Objective:To investigate the effect of long non-coding RNA (lncRNA) H19 regulating miRNA-675 (miR-675) and phosphatase and tensin homologue-deleted chromosome ten gene (PTEN) on the cell proliferation of glioma.Methods:Glioma cell lines U87-MG and U251 were chosen. The siRNA online design tool wad used to design small interfering RNA (siRNA) targeting H19. U87-MG and U251 cell lines with the stable knockdown of H19 were constructed (the stable knockdown of H19 group), and the cells randomly transfected with siRNA plasmid were taken as the control group, and normal cultured cells were treated as the blank group. Additionally, miR-675 and control microRNA were transfected into U87-MG and U251 with the stable knockdown of H19 (the overexpressing miR-675 group and the corresponding control group). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative expression levels of miR-675 and H19 in each group; the methyl thiazolyl tetrazolium (MTT) assay was used to detect the cell proliferation ability; the dual luciferase reporter gene assay was used to verify the targeting relationship between miR-675 and PTEN; Western blot was used to detect the relative expression level of PTEN protein.Results:The MTT assay results showed that the proliferation ability of U87-MG and U251 cells in the stable knockdown of H19 group was lower than that of the corresponding control group; and the differences in cell proliferation ability of all the groups after 48 h of culture were statistically significant (all P < 0.05). qRT-PCR detection results showed that the relative expression level of miR-675 in U251 cells in the stable knockdown of H19 group and the corresponding control group was 0.329±0.009 and 1.043±0.087, respectively, and the difference was statistically significant ( t = 14.15, P < 0.001); the relative expression level of miR-675 in U87-MG cells in the stable knockdown of H19 group and the corresponding control group was 0.299±0.009 and 1.027±0.106, respectively, and the difference was statistically significant ( t = 11.85, P < 0.001); the relative expression level of miR-675 in U87-MG and U251 cells in the stable knockdown of H19 group was lower than that of the corresponding control group. The dual luciferase reporter gene assay verified that miR-675 could bind to the 3'-UTR of PTEN. Western blot detection results showed that the relative expression level of PTEN protein in U87-MG and U251 cells in the stable knockdown of H19 group was higher than that of the corresponding control group and the blank group; in the U87-MG and U251 cells in the stable knockdown of H19 group, the relative expression level of PTEN in the overexpressing miR-675 group was lower than that of the corresponding blank group and the control group. In the U87-MG and U251 cells in the stable knockdown of H19 group, the cell proliferation ability of the overexpressing miR-675 group was higher than that of the corresponding blank group and the control group; the differences in cell proliferation ability of all the groups after 48 h of culture were statistically significant (all P < 0.05). Conclusions:lncRNA H19 may regulate the cell proliferation of glioma cells through the miR-675-PTEN signaling pathway.

Objective: To explore the possible mechanism of moxibustion in myocardial protection of rats undergoing long-term fatigue exercise based on observing the classical pyroptosis pathway mediated by nuclear factor kappa-B (NF-κB)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/cysteinyl aspartate specific proteinase 1 (Caspase-1).Methods: A total of 50 specific-pathogen-free male Sprague-Dawley rats were bought. Ten unqualified rats were excluded, and the remaining 40 rats were divided into a normal group, a normal + Shenque (CV8) group, a model group, a model + non-meridian non-point group, and a model + Shenque (CV8) group according to the random number table method, with 8 rats in each group. Except for rats in the normal group and the normal + Shenque (CV8) group, rats in the other three groups were trained with an incline running table exercise protocol to create a long-term fatigue exercise model, 1 h/time, once a day for 5 d with 2 d off, for a total of 8 weeks. Rats in the normal group received no modeling or intervention. Rats in the normal + Shenque (CV8) group were not modeled but received mild moxibustion at Shenque (CV8); those in the model group were modeled only without intervention; those in the model + non-meridian non-point group received moxibustion at non-meridian and non-point spots after the modeling; those in the model + Shenque (CV8) group received moxibustion at Shenque (CV8) after modeling. The above moxibustion interventions were performed for 15 min/time once daily, for 5 d with 2 d off per week and a total of 8 weeks. Blood was collected from the femoral artery 4 h after the last exercise, and the serum interleukin (IL)-1β and IL-18 levels were measured. The NF-κB, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), Caspase-1, and gasdermin D (GSDMD) expression levels were detected by Western blotting. Myocardial morphology and pyroptosis were observed by hematoxylin-eosin (HE) staining and electron microscopy. Results: The HE staining results showed that the myocardial cells in the model group and the model + non-meridian non-point group were disorganized with blurred transverse lines, widened interstitial spaces, interstitial edema, and inflammatory cell infiltration. The structure of myocardial cells in the model + Shenque (CV8) group was clearly visible, with slightly widened interstitial spaces and occasional infiltration of inflammatory cells in the interstitium. Compared with the normal group, the serum IL-1β and IL-18 levels were increased, and myocardial NF-κB, NLRP3, ASC, Caspase-1, and GSDMD expression levels were elevated in the model group and the model + non-meridian non-point group (P<0.01). Compared with the model group, the above indicators did not change significantly in the model + non-meridian non-point group, while all the above indicators were decreased in the model + Shenque (CV8) group (P<0.01). Compared with the model + non-meridian non-point group, all the above biochemical indicators were decreased in the model + Shenque (CV8) group (P<0.01). Transmission electron microscopy showed that the mitochondria number was increased in the model group and the model + non-meridian non-point group, some of the mitochondrial lumen was irregularly enlarged, the cell membrane structure was unclear, and chromatin was aggregated. The mitochondria number was increased, the swelling was reduced, and the nuclear membrane structure was more intact in the model + Shenque (CV8) group. Conclusion: Moxibustion at Shenque (CV8) regulates the NF-κB/NLRP3/Caspase-1 pathway and reduces the pyroptosisin the myocardium of rats with long-term fatigue exercise, thus reducing the myocardial injury caused by long-term fatigue exercise.

ObjectiveTo investigate the effect of microemulsion on the distribution of index components in different phases of Zexietang extract based on high performance liquid chromatography（HPLC） and phase separation process. MethodParticle size meter and transmission electron microscope were used to characterize the colloidal particles in blank microemulsion， aqueous extract of Zexietang and microemulsion extract of Zexietang. The phase separation process was established by high-speed centrifugation and dialysis， and based on this process， the aqueous extract and microemulsion extract of Zexietang were separated into the true solution phase， the colloidal phase and the precipitation phase， respectively. The contents of six components， including atractylenolide Ⅲ， atractylenolide Ⅱ， 23-acetyl alisol C， alisol A， alisol B and alisol B 23-acetate， were determined by HPLC with the mobile phase of water（A）-acetonitrile（B） for gradient elution（0-5 min， 40%-43%B； 5-20 min， 43%-45%B； 20-45 min. 45%-60%B； 45-75 min， 60%-80%B）. The solubility of the index components in water and microemulsion was determined by saturation solubility method. ResultThe colloidal particles in the aqueous extract， microemulsion extract and blank microemulsion were all spherical， and the particle size， polydispersity index（PDI） and Zeta potential of the colloidal particles were in the order of aqueous extract >microemulsion extract >blank microemulsion. The results of phase separation showed that the colloidal phase and the true solution phase could be completely separated by dialysis for 2.5 h， and the phase separation process was tested to be stable and feasible. Compared with the aqueous extract of Zexietang， the use of microemulsion as an extraction solvent could increase the contents of atractylenolide Ⅲ， 23-acetyl alisol C， atractylenolide Ⅱ ， alisol A， alisol B and alisol B 23-acetate by 3.75， 6.82， 35.47， 10.66， 35.41， 27.75-fold， and could increase the extraction efficiencies of the latter five constituents by 2.03， 1.15， 1.70， 6.43， 5.53 times. The solubility test showed that the microemulsion could significantly improve the solubility of atractylenolide Ⅱ， alisol A， alisol B and alisol B 23-acetate， but it had less effect on the solubility of atractylenolide Ⅲ and 23-acetyl alisol C. ConclusionMicroemulsion can improve the extraction efficiency and increase the distribution of the index components in the colloidal phase state of Zexietang to different degrees， providing a reference for the feasibility of microemulsion as an extraction solvent for traditional Chinese medicine.

Objective : To explore the nucleotide variation and protein amino acid changes of E4 and L2 genes of Human Papillomavirus 16 (HPV16) , and to analyze the evolutionary characteristics of HPV16 virus. Methods : 40 HPV16 infection⁃positive cervical exfoliated cells samples and tissue cell samples were collected from hospital , viral DNA was extracted , Sanger sequencing perform in cervical exfoliated cells DNA and high⁃throughput sequencing technology sequenced in cervical tissues DNA for E4 and L2 genes of HPV16 , HPV16 E4 and L2 gene phylogenetic evolution trees were constructed , and variation of HPV16 E4 and L2 genes were analyzed. Results : There were 72 HPV16 E4 variant samples with nucleotide variants (4 missense mutations and 7 synonymous mutations) at 10 sites , HPV16 L2 gene variants in 74 samples , and nucleotide variants (23 missense mutations and 18 synonymous mutations) at 40 sites. The variation frequency of T4177C , A4288C and A4654C in cervical cancer was significantly higher than that in non⁃cervical cancer, and the difference was statistically significant (P < 0. 05) . Conclusion ① The main HPV16 virus strains in Xinjiang are European strains , and a few are Asian strains. ② The mutation frequency of T4177C , A4288C and A4654C in HPV16 L2 gene is higher than that in non⁃cervical cancer, and G4181A is related to the Asian strain.