Human keratinocyte growth factor-1 (hKGF-1), a member of the fibroblast growth factor (FGF) family, plays crucial roles in organ development, cell proliferation, wound healing, and tissue repair, representing one of the most effective and specific growth factors for skin repair. However, obtaining recombinant hKGF-1 remains challenging due to its universally low expression efficiency in vitro. This study employs the Bombyx mori baculovirus expression system to establish a technological platform that utilizes the economically important insect Bombyx mori as a bioreactor for high-efficiency and low-cost expression and production of recombinant human keratinocyte growth factor 1 (hKGF-1) protein, ultimately achieving high-level expression of hKGF-1 in Bombyx mori ovary cell line (BmN). In this study, we optimized the hKGF-1 sequence based on the codon preference of baculovirus. By fusing hKGF-1 with polyhedrin (highly expressed in this system) and adding extra promoters and enhancers, we significantly improved the expreesion level of hKGF-1 in Bombyx mori cells. The results demonstrated that the aforementioned strategies significantly enhanced the expression level of hKGF-1 in Bombyx mori cells. SDS-PAGE and Western blotting results revealed that the highest hKGF-1 expression (accounting for 8.7% of total cellular protein) was achieved when the Polh promoter was combined in tandem with the P6.9 promoter and hKGF-1 was fused with a 15-residue polyhedrin fragment for co-expression. The optimal harvest time was determined to be 120 h post transfection. This study achieved the efficient expression of hKGF-1 in Bombyx mori cells, establishing an ideal technological platform for the industrial utilization of recombinant hKGF-1. The developed methodology not only provides valuable technical references for the production of other growth factors and complex proteins, but also demonstrates significant implications for employing silkworms as bioreactors for recombinant human protein expression.
Bombyx/metabolism* ; Animals ; Baculoviridae/metabolism* ; Humans ; Fibroblast Growth Factor 7/biosynthesis* ; Recombinant Proteins/genetics* ; Cell Line ; Genetic Vectors/genetics*

Bombyx mori nucleopolyhedrovirus (BmNPV) is extremely harmful to the silk industry. The polyhedrin, which encodes the polyhedrin (Polh), can be expressed at ultra-high levels and form occlusion bodies in the nucleus, embedding the progeny virus within it. However, the detailed mechanism by which polyhedrin is transported into the host cell nucleus remains unknown. Clarifying the nuclear import mechanisms of viral proteins can help us develop better prevention and treatment measures against baculoviruses. This study employed molecular cloning, co-immunoprecipitation, and immunofluorescence to analyze in detail the expression pattern of the highly expressed polyhedrin in the very late stage of the virus, and further revealed that the host protein importin α participates in the nuclear import of polyhedrin through protein interactions. This study provides a reference for further elucidating the nuclear import mechanisms of the baculovirus proteins including polyhedrin that can enter the nucleus.
Nucleopolyhedroviruses/metabolism* ; Active Transport, Cell Nucleus ; Animals ; Bombyx/virology* ; alpha Karyopherins/metabolism* ; Cell Nucleus/metabolism* ; Viral Structural Proteins/metabolism* ; Occlusion Body Matrix Proteins

Objective To explore the influencing factors of neonatal pneumonia complicated with myocardial damage and Th1/Th2 cytokines,and construct a line chart model.Methods A total of 390 neonates with pneumonia who were treated in Mianyang People's Hospital were selected as the study subjects and randomly divided into a modeling cohort(n=273)and a validation cohort(n=117)according to a 7∶3 ratio.They were further divided into myocardial damage group and non-myocardial damage group according to whether they had concurrent myocardial damage.Enzyme linked immunosorbent assay(ELISA)was used to measure Th1/Th2 cytokines(IFN-γ,IL-6,TNF-α,IL-4,IL-2 and IL-10),and the Mindray BS-280 automatic biochemical analyzer was used to measure hs-cTn I,CK-MB,LDH and CK.Logistic regression equation was used to screen the influencing factors of neonatal pneumonia complicated with myocardial damage.R software was used to construct a line chart model,and the receiver operating characteristic curve(ROC)and area under the ROC curve(AUC)were used to analyze the predictive ability of the model.The Hosmer-Lemeshow goodness-of-fit test was used,and a calibration curve was drawn to evaluate the calibration of the model.The decision curve analysis(DCA)was used to evaluate the clinical effectiveness.Results The incidence of myocardial damage in 390 neonates with pneumonia was 28.21%.Modeling cohort and validetion cohort,the 1min Apgar score in the myocardial damage group was lower than that in the non-myocardial damage group(t=3.314,2.619),and CK-MB,LDH,CK,hs-cTnI,IL-2,IFN-γ,TNF-α,IL-6,IL-10 and IL-4 were higher than those in the non-myocardial damage group(t=5.805～18.914),and the proportions of severe pneumonia,low birthweight infant,premature infants were higher than those in the non-myocardial damage group(χ2=4.464～41.497),and the differences were statistically significant(all P<0.05),respectively.The Logistic regression equation showed that low birth weight,1-minute Apgar score,premature birth,hs-cTnI,IL-2,IFN-γ,IL-6 and IL-4 were factors affecting neonatal pneumonia complicated with myocardial damage(Wald χ2=10.330～14.328,all P<0.05).The AUC(95%CI)of the nomogram model constructed based on the factors affecting neonatal pneumonia complicated with myocardial damage was 0.880(0.839～0.921)in the modeling cohort and 0.910(0.856～0.964)in the validation cohort,with slopes of the calibration curves close to 1,and the clinical net benefit rate was the highest in the ranges of 0.1～0.8 and 0.0～0.7.Conclusion The nomogram model,which includes Th1/Th2 cytokines,hs-cTnI,1-minute Apgar score,premature infants and low-birth-weight infants has high predictive value for neonatal pneumonia complicated with myocardial damage.It can help clinicians identify high-risk populations,take reasonable diagnostic and treatment measures,and reduce the risk of myocardial damage.

Objective To explore the influencing factors of neonatal pneumonia complicated with myocardial damage and Th1/Th2 cytokines,and construct a line chart model.Methods A total of 390 neonates with pneumonia who were treated in Mianyang People's Hospital were selected as the study subjects and randomly divided into a modeling cohort(n=273)and a validation cohort(n=117)according to a 7∶3 ratio.They were further divided into myocardial damage group and non-myocardial damage group according to whether they had concurrent myocardial damage.Enzyme linked immunosorbent assay(ELISA)was used to measure Th1/Th2 cytokines(IFN-γ,IL-6,TNF-α,IL-4,IL-2 and IL-10),and the Mindray BS-280 automatic biochemical analyzer was used to measure hs-cTn I,CK-MB,LDH and CK.Logistic regression equation was used to screen the influencing factors of neonatal pneumonia complicated with myocardial damage.R software was used to construct a line chart model,and the receiver operating characteristic curve(ROC)and area under the ROC curve(AUC)were used to analyze the predictive ability of the model.The Hosmer-Lemeshow goodness-of-fit test was used,and a calibration curve was drawn to evaluate the calibration of the model.The decision curve analysis(DCA)was used to evaluate the clinical effectiveness.Results The incidence of myocardial damage in 390 neonates with pneumonia was 28.21%.Modeling cohort and validetion cohort,the 1min Apgar score in the myocardial damage group was lower than that in the non-myocardial damage group(t=3.314,2.619),and CK-MB,LDH,CK,hs-cTnI,IL-2,IFN-γ,TNF-α,IL-6,IL-10 and IL-4 were higher than those in the non-myocardial damage group(t=5.805～18.914),and the proportions of severe pneumonia,low birthweight infant,premature infants were higher than those in the non-myocardial damage group(χ2=4.464～41.497),and the differences were statistically significant(all P<0.05),respectively.The Logistic regression equation showed that low birth weight,1-minute Apgar score,premature birth,hs-cTnI,IL-2,IFN-γ,IL-6 and IL-4 were factors affecting neonatal pneumonia complicated with myocardial damage(Wald χ2=10.330～14.328,all P<0.05).The AUC(95%CI)of the nomogram model constructed based on the factors affecting neonatal pneumonia complicated with myocardial damage was 0.880(0.839～0.921)in the modeling cohort and 0.910(0.856～0.964)in the validation cohort,with slopes of the calibration curves close to 1,and the clinical net benefit rate was the highest in the ranges of 0.1～0.8 and 0.0～0.7.Conclusion The nomogram model,which includes Th1/Th2 cytokines,hs-cTnI,1-minute Apgar score,premature infants and low-birth-weight infants has high predictive value for neonatal pneumonia complicated with myocardial damage.It can help clinicians identify high-risk populations,take reasonable diagnostic and treatment measures,and reduce the risk of myocardial damage.

Objective:To investigate the perioperative impact of closure of mesenteric fissure in laparoscopic right hemicolectomy.Methods:The prospective randomized controlled trial was conducted. The clinical data of 320 patients who underwent laparoscopic right hemicolectomy in 11 medical centers, including The First Affiliated Hospital of China Medical University et al, from November 2022 to August 2023 were selected. Based on block randomization, patients were alloca-ted into the mesenteric fissure non-closure group and the mesenteric fissure closure group. Observa-tion indicators: (1) grouping of the enrolled patients; (2) intraoperative conditions; (3) postopera-tive conditions. Measurement data with skewed distribution were represented as M( Q1, Q3) and com-parison between groups was conducted using the Mann-Whitney U test. Count data were represen-ted as absolute numbers or percentages, and comparison between groups was conducted using the chi-square test or Fisher's exact probability. Comparison of ordinal data was conducted using the rank sum test. Comparison of visual analog scores was analyzed using generalized estimating equations. Results:(1) Grouping of the enrolled patients. A total of 320 patients with colon cancer were screened for eligibility, including 156 males and 164 females, aged 68(59,73)years. All the 320 patients were allocated into the mesenteric fissure non-closure group with 164 cases and the mesenteric fissure closure group with 156 cases. There was no significant difference in the age, body mass index, American Society of Anesthesiologist score, maximum tumor diameter, anastomosis location, anastomosis method, surgical approach, range of lymph node dissection, tumor staging between the two groups ( P>0.05) and there was a significant difference in the sex between them ( P<0.05). (2) Intraoperative conditions. There was no significant difference between the mesenteric fissure closure group and the mesenteric fissure non-closure group in the volume of intraoperative blood loss, operation time, conversion to laparotomy, intraoperative complication ( P>0.05). Three patients in the mesenteric fissure non-closure group were converted to laparotomy. One patient in the mesenteric fissure closure group was converted to laparotomy, and 2 cases with intraoperative complication were mesenteric hematoma. (3) Postoperative conditions. There was no significant difference between the mesenteric fissure non-closure group and the mesenteric fissure closure group in the overall postoperative complications ( χ2=0.28, P>0.05). There was no significant difference in the occurrence of postoperative intestinal obstruction, abdominal distension, ascites, pleural effusion, gastric paralysis, anastomotic bleeding, anastomotic leakage, or surgical wound infection between the two groups ( P>0.05). There was no significant difference between the two groups in the reoperation, postoperative gastric tube replacement. There was no significant differ-ence in time to postoperative first flatus, time to postoperative initial liquid food intake, time to post-operative resumption of bowel movements, duration of postoperative hospital stay, total hospital expenses between the two groups ( Z=-0.01, 0.43, 1.04, -0.54, -0.36, P>0.05). One patient in the mesenteric fissure non-closure group received reoperation. No perioperative internal hernia or death occurred in either group. The visual analog score decreased with time in both groups. There was no significant difference in the visual analog score between the mesenteric fissure closure group and the mesenteric fissure non-closure group [ β=-0.20(-0.53,0.13), P>0.05]. Conclusion:Compared with closure of mesenteric fissure, non-closure of mesenteric fissure during laparoscopic right hemi-colectomy dose not increase perioperative complications or postoperative management risk.

Background::Bladder cancer, characterized by a high potential of tumor recurrence, has high lifelong monitoring and treatment costs. To date, tumor cells with intrinsic softness have been identified to function as cancer stem cells in several cancer types. Nonetheless, the existence of soft tumor cells in bladder tumors remains elusive. Thus, our study aimed to develop a microbarrier microfluidic chip to efficiently isolate deformable tumor cells from distinct types of bladder cancer cells.Methods::The stiffness of bladder cancer cells was determined by atomic force microscopy (AFM). The modified microfluidic chip was utilized to separate soft cells, and the 3D Matrigel culture system was to maintain the softness of tumor cells. Expression patterns of integrin β8 (ITGB8), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were determined by Western blotting. Double immunostaining was conducted to examine the interaction between F-actin and tripartite motif containing 59 (TRIM59). The stem-cell-like characteristics of soft cells were explored by colony formation assay and in vivo studies upon xenografted tumor models. Results::Using our newly designed microfluidic approach, we identified a small fraction of soft tumor cells in bladder cancer cells. More importantly, the existence of soft tumor cells was confirmed in clinical human bladder cancer specimens, in which the number of soft tumor cells was associated with tumor relapse. Furthermore, we demonstrated that the biomechanical stimuli arising from 3D Matrigel activated the F-actin/ITGB8/TRIM59/AKT/mTOR/glycolysis pathways to enhance the softness and tumorigenic capacity of tumor cells. Simultaneously, we detected a remarkable up-regulation in ITGB8, TRIM59, and phospho-AKT in clinical bladder recurrent tumors compared with their non-recurrent counterparts.Conclusions::The ITGB8/TRIM59/AKT/mTOR/glycolysis axis plays a crucial role in modulating tumor softness and stemness. Meanwhile, the soft tumor cells become more sensitive to chemotherapy after stiffening, that offers new insights for hampering tumor progression and recurrence.

OBJECTIVE To study the protective effect and possible mechanism of soybean isoflavones against threatened miscarriage rats. METHODS Female mice were selected to promote estrus and mate with male mice. After pregnancy， they were randomly divided into normal group （purified water， i.g.， n＝10）， model group （purified water， i.g.， n＝9）， positive control drug group （progesterone 4 mg/kg， i.m.， n＝9）， low-， medium- and high-dose soybean isoflavone groups （25， 50 and 100 mg/kg， i.g.， n＝10）. Except for the normal group， the rest were given mifepristone+misoprostol on the 8th day of pregnancy to establish threatened miscarriage model， and then given purified water or drugs， once a day， on days 1-7 and 9-12 of pregnancy， respectively. At 14 days of pregnancy， the rates of fetal protection were counted. Serum levels of β-human chorionic gonadotrophin （β-HCG） and progesterone （P） in rats were detected. Pathological and morphological changes in rat placenta and decidua tissues were observed， and the apoptosis indexes of cells were detected； mRNA and protein expressions of factor of apoptosis related （Fas）， factor of apoptosis related ligand （FasL）， proliferating cell nuclear antigen （PCNA） and heparin binding epidermal growth factor （HB-EGF） were determined in placenta tissues， and mRNA and protein expressions of Fas， PCNA and HB-EGF in decidua tissues were detected. RESULTS In the model group， the placental tissues of rats were hyperemia and dilatation， with fewer and irregular blood vessels； severe stromal edema，inflammatory cell infiltration and iron-choledrin depositionwere observed. Compared with model group， the fetal survival rates， serum levels of β-HCG and P， the expressions of PCNA and HB-EGF mRNA and proteins in the placenta and decidua tissue of soybean isoflavone groups increased significantly （P＜ 0.05）， while pathological changes were improved significantly； cell apoptosis index in the placenta and decidua tissue， the expressions of Fas， FasL mRNA and proteins in the placenta and Fas mRNA and protein in the decidua tissue decreased significantly （P＜0.05）. The effect of soybean isoflavones was dose-dependent （P＜0.05）. CONCLUSIONS Soybean isoflavone has protective effect on threatened miscarriage， the mechanism of which is related to down-regulating the expressions of Fas and FasL mRNA and protein at the maternal-fetal interface， and up-regulating the expressions of mRNA and protein of PCNA and HB- EGF.

Human epidermal growth factor (hEGF) can be applied in the treatment of surgical trauma (burns, scalds), tissue repair, skin moisturizing, beauty, skincare, etc. However, the low expression and high cost limit the application of hEGF. In order to improve the expression level of hEGF and reduce the production cost, considering the high expression of polyhedrin, this study fused a partial sequence of polyhedrin with hEGF and expressed the fused sequence by using a silkworm baculovirus expression vector system. In view of the small molecular weight of hEGF, we connected hEGF genes in series and optimized the codons to construct multiple fusion expression vectors by fusing different partial sequences of polyhedrin at the N-terminus. The results showed that through the above strategy, the protein expression level of hEGF was significantly increased. The expression vector containing three concatenated hEGF genes with optimized codons and fused with the sequence encoding 25 or 35 residues at the N-terminus of polyhedrin showed the highest expression level.
Humans ; Epidermal Growth Factor/biosynthesis* ; Genetic Vectors/genetics* ; Recombinant Fusion Proteins/biosynthesis* ; Animals ; Bombyx/metabolism* ; Occlusion Body Matrix Proteins/genetics* ; Nucleopolyhedroviruses/genetics* ; Amino Acid Sequence

Male ; Humans ; Aged ; Sleep Duration ; Aging ; Testosterone ; China ; Sleep

Based on single-cell sequencing of the hippocampi of 5x familiar Alzheimer's disease(5x FAD)and wild type mice at 2-,12-,and 24-month of age,we found an increased percentage of microglia in aging and Alzheimer's disease(AD)mice.Blood brain barrier injury may also have contributed to this increase.Immune regulation by microglia plays a major role in the progression of aging and AD,according to the functions of 41 intersecting differentially expressed genes in microglia.Signaling crosstalk between C-C motif chemokine ligand(CCL)and major histocompatibility complex-1 bridges intercellular communi-cation in the hippocampus during aging and AD.The amyloid precursor protein(APP)and colony stimulating factor(CSF)signals drive 5x FAD to deviate from aging track to AD occurrence among intercellular communication in hippocampus.Microglia are involved in the progression of aging and AD can be divided into 10 functional types.The strength of the interaction among microglial subtypes weakened with aging,and the CCL and CSF signaling pathways were the fundamental bridge of communication among microglial subtypes.