Objective To establish a stable,reliable,and clinically relevant porcine model of endotoxin-induced acute respiratory distress syndrome(ARDS).Methods Ten 8-month-old male Bama minipigs were deeply sedated,followed by invasive mechanical ventilation and electrocardiographic monitoring.Lipopolysaccharide(LPS)was intravenously pumped at 600 μg/(kg·h)for 3 hours,then maintained at 15 μg/(kg·h)thereafter.Dynamic monitoring was performed at five time points after LPS injection(LPS 0,1,3,5,and 8 h),including arterial blood gas analysis and chest computed tomography(CT)scans.Pathological examination of lung tissues obtained via bronchoscopic biopsy(HE staining and transmission electron microscopy)was conducted.These indicators were comprehensively used to evaluate the success of the animal model.Results At 5 hours after LPS administration,8 minipigs developed symptoms such as skin cyanosis,elevated body temperature,and respiratory distress.The oxygenation index decreased to<300 mmHg.Chest CT scans showed diffuse pulmonary infiltrates.Histopathology revealed alveolar edema and hyaline membrane formation.Transmission electron microscopy demonstrated disruption of pulmonary blood-air barrier,depletion of lamellar bodies in type Ⅱ pneumocytes,inflammatory cell infiltration,and exudation of plasma proteins and fibrin.Compared with LPS 0 h,at LPS 8 h,the oxygenation index and arterial blood pH were significantly decreased(P<0.001),while blood lactic acid and serum potassium were significantly increased(P<0.05);serum calcium and base excess were significantly decreased(P<0.05),and the lung injury score based on HE-stained lung sections was significantly increased(P<0.01).Conclusion The porcine ARDS model established by continuous LPS injection can dynamically simulate the pathophysiological characteristics and typical pathological manifestations of clinical septic ARDS,making it an effective tool to study the pathogenesis,prevention,and treatment strategies of septic ARDS.

Acquired resistance to the proteasome inhibitor bortezomib(BTZ)poses a significant chal-lenge in the treatment of multiple myeloma(MM).During the acquisition of BTZ resistance,metabolic reprogramming is actively engaged in MM cells.However,the key regulatory genes and molecular mecha-nisms mediating bortezomib resistance through this metabolic rewiring have not been fully elucidated.This study aims to investigate the regulatory role of pyrimidine metabolites in drug resistance of MM and their underlying molecular mechanisms.Screening via CCK-8 assays demonstrated that the pyrimidine metabolite uridine is associated with BTZ resistance in MM(P＜0.05).In vitro experiments,including CCK-8 assays,Western blotting,and flow cytometry,demonstrated that uridine partially suppresses bort-ezomib-induced apoptosis in MM cells(P＜0.05).In vivo,experiments utilizing Vk*MYC mouse mod-els,subcutaneous tumor models,and intramedullary bone marrow transplantation models showed that the combination of BTZ and uridine significantly accelerated tumor growth,exacerbated bone destruction,and increased tumor cell infiltration in tumor-bearing mice(P＜0.05).RNA sequencing analysis re-vealed that uridine primarily affects mitochondrial translation in MM at the transcriptional level.Seahorse energy metabolism assays demonstrated that uridine enhances mitochondrial oxidative phosphorylation without significantly altering glycolysis.Transcriptomic analysis further identified a significant upregula-tion of cytochrome c oxidase subunit 5B(COX5B)transcription in uridine-treated groups(P＜0.05).Functional studies confirmed that COX5B is a key molecule mediating uridine's effects on mitochondrial function in MM cells.In conclusion,uridine promotes BTZ resistance in MM by upregulating COX5B transcription and protein expression,thereby enhancing cytochrome c oxidase activity and regulating mito-chondrial oxidative phosphorylation.This study delineates the role of uridine in the development of borte-zomib resistance in MM and elucidates its COX5B-mediated metabolic reprogramming mechanism,provi-ding a theoretical foundation for developing targeted therapies against relapsed/refractory MM.

Acquired resistance to the proteasome inhibitor bortezomib(BTZ)poses a significant chal-lenge in the treatment of multiple myeloma(MM).During the acquisition of BTZ resistance,metabolic reprogramming is actively engaged in MM cells.However,the key regulatory genes and molecular mecha-nisms mediating bortezomib resistance through this metabolic rewiring have not been fully elucidated.This study aims to investigate the regulatory role of pyrimidine metabolites in drug resistance of MM and their underlying molecular mechanisms.Screening via CCK-8 assays demonstrated that the pyrimidine metabolite uridine is associated with BTZ resistance in MM(P＜0.05).In vitro experiments,including CCK-8 assays,Western blotting,and flow cytometry,demonstrated that uridine partially suppresses bort-ezomib-induced apoptosis in MM cells(P＜0.05).In vivo,experiments utilizing Vk*MYC mouse mod-els,subcutaneous tumor models,and intramedullary bone marrow transplantation models showed that the combination of BTZ and uridine significantly accelerated tumor growth,exacerbated bone destruction,and increased tumor cell infiltration in tumor-bearing mice(P＜0.05).RNA sequencing analysis re-vealed that uridine primarily affects mitochondrial translation in MM at the transcriptional level.Seahorse energy metabolism assays demonstrated that uridine enhances mitochondrial oxidative phosphorylation without significantly altering glycolysis.Transcriptomic analysis further identified a significant upregula-tion of cytochrome c oxidase subunit 5B(COX5B)transcription in uridine-treated groups(P＜0.05).Functional studies confirmed that COX5B is a key molecule mediating uridine's effects on mitochondrial function in MM cells.In conclusion,uridine promotes BTZ resistance in MM by upregulating COX5B transcription and protein expression,thereby enhancing cytochrome c oxidase activity and regulating mito-chondrial oxidative phosphorylation.This study delineates the role of uridine in the development of borte-zomib resistance in MM and elucidates its COX5B-mediated metabolic reprogramming mechanism,provi-ding a theoretical foundation for developing targeted therapies against relapsed/refractory MM.

Objective:To compare and analyze the changes of aggregation rate and routine parameters of platelets stored in temperature cycle,cold storage at 4 ℃ and oscillating storage at 22 ℃,so as to provide more experimental data for platelet preservation methods.Methods:Blood samples were collected at 5 time points on the 1st,2nd,3rd,4th and 6th day after platelet cycling preservation at temperature,cold storage at 4 ℃,and oscillating storage at 22 ℃.Platelet maximum aggregation rate(MAR)and routine parameters including platelet count(PLT),mean platelet volume(MPV),platelet distribution width(PDW)and platelet-larger cell ratio(P-LCR)were detected.Results:The platelet MAR of three groups showed a significant decrease trend with the preservation time,the fastest decrease was in the 22 ℃ group,the slowest was in the 4 ℃ group,and the temperature cycle group was between the two groups.On the 3rd day of preservation,the platelet MAR in 4 ℃ group was still in the normal range(MAR＞60％),while in temperature cycle group was about 50％,and in 22 ℃ group was the lowest.On the 4th day of preservation,platelet MAR in all the three groups was lower than 50％,and that in temperature cycle group was significantly lower than in 4 ℃ group but higher than in 22 ℃ group(both P＜0.05).On the 6th day of preservation,platelet MAR in the temperature cycle group was significantly lower than that in the 4 ℃ group(P＜0.05),but there was no statistically significant difference compared to 22 ℃ group(P＞0.05).PLT values in the three groups were all significantly decreased with the preservation time extension,and were significantly lower than those in the early stage of preservation within 6 days(all P＜0.05).PDW in temperature cycle group had no significant change within 6 days of preservation,but MPV and P-LCR were significantly increased.MPV,PDW and P-LCR all decreased significantly in 4 ℃ group within 6 days of preservation but increased in 22 ℃ group.Under the same storage days,PLT value of temperature cycle group had no significant difference with that of 4 ℃ group and 22 ℃ group,while MPV,PDW and P-LCR values were significantly higher than 4 ℃ group but lower than 22 ℃ group(all P＜0.05).Conclusion:The aggregation function and routine parameters changes of temperature circulating preserved platelets are between 4 and 22 ℃.

This work was aimed at predicting and verifying B-cell epitopes of SARS-CoV-2 E protein through bioinformatics methods,and clarifying the dominant B cell epitopes with mouse polyclonal antibody serum prepared through SARS-CoV-2 re-combinant E protein immunization and human positive serum vaccinated with inactivated SARS-CoV-2 vaccine.The structural and B-cell linear epitopes of SARS-CoV-2 E protein were predicted with SOPMA,Expasy,SWISS-MODEL,IEDB database,and Bepid-2.0 software.Candidate epitopes were expressed as GST-tagged recombinant protein fragments in an E.coli sys-tem,and their immunoreactivity with mouse and human poly-clonal positive serum against SARS-CoV-2 E protein was de-tected by western blotting and indirect ELISA,respectively.The epitope prediction results showed that E protein contained linear B cell epitopes Ser6-Val14 and Tyr57-Pro71,and the conformational epitopes of Glu8-Val12,Leu39-Tyr59,and Ser60-Leu65.The GST tagged recombinant E protein fragments of E1 and E3,containing Ser6-Val14 and Tyr57-Pro71 epitopes,respectively,as well as E2 without an epitope sequence as a control,were expressed in an E.coli expression system and successfully purified with an Ni-NTA column.Western blotting and indirect ELISA analysis indicated that all mouse and human SARS-CoV-2 positive sera positively reacted with only E1 and E3 proteins,but negatively reacted with E2 protein,thus indicating that the corresponding epitope prediction with Ser6-Val14 and Tyr57-Pro71 was correct.This study successfully predicted and preliminarily identified two linear B cell epitopes of SARS-CoV-2 E protein,thus providing a reference for the preparation of new coronavirus vaccines and the analysis of immune response characteristics.

Ion concentration polarization (ICP) is an electrical transport phenomenon that occurs at the micro-nano interface under the action of an applied electric field, and the ICP phenomenon can be used to enrich charged particles with high efficiency. The microfluidic chip has the advantages of high precision, high efficiency, easy integration and miniaturization in biochemical analysis, which provides a new solution and technical way for biochemical analysis. In response to the demand for the detection of trace charged target analytes in sample solution, the advantages of high enrichment multiplicity, convenient operation and easy integration of ICP are utilized to provide an effective way for microfluidic biochemical detection. The combination of ICP phenomenon and microfluidic analysis technology has been widely used in the fields of pre-enrichment of charged particles, separation of targets, and detection of target analytes in biochemical analysis. In this paper, the principle of ICP and the microfluidic ICP chip are briefly introduced. Under the action of external electric field, the co-ions pass through the ion-selective nanochannel, the counterions are rejected at the boundary of nanochannel to form a depletion zone, and the charged samples will be enriched at the boundary of the depletion zone. Then the preparation techniques and methods of ICP chips are summarized. Among them, the design of microfluidic channel structure and the preparation and design of nanostructures are emphasized. The basic single-channel structure is analyzed, and the parallel-channel structure as well as the integrated multi-functional microfluidic ICP chip are sorted out and summarized. The preparation methods of nanostructures in ICP chips and their respective advantages and disadvantages are listed, and it is summarized that the current mainstream means are the embedding method and the self-assembly method, and attention is paid to the design of nanostructures preparation methods by both of them. In addition, this paper also discusses how to optimize the enrichment efficiency of ICP chip, through the introduction of multi-field coupling, valve control and other means to achieve the optimization of the enrichment efficiency of target substances. Meanwhile, this paper provides a classified overview of the progress of application of ICP chips in biochemical analysis and detection. ICP chips have been widely used in the research and development of biosensors, which can be used for the enrichment and separation of a variety of analytes including small molecules, nucleic acids, proteins, and cells, etc. By changing the design of microfluidic structures, integrating detection methods and modifying specific antibodies, ICP chips have shown great potential in the fields of rapid enrichment and pre-processing of targets, separation of targets and highly sensitive detection. Finally, it is pointed out that ICP chips are facing challenges in improving enrichment efficiency and selectivity, and solving the problems of fluid control, mixing and transport to match the biological properties of target assay, and that microfluidic ICP chips have been continuously promoting the development of ICP chips through the improvement of materials, chip design and integration of multifunctional units, opening up new possibilities in the field of biochemical analysis methods and applications. It can be seen that microfluidic ICP chips have the advantages of low sample flow rate, good separation and enrichment, high detection efficiency, and easy integration and miniaturization, which have shown good research significance and practical prospects in the field of biochemical detection.

Objective:To investigate the effect of transconjunctival lower blepharoplasty combined with stromal vascular fraction-gel (SVF-gel) transplantation in suborbital rejuvenation.Methods:The clinical data of patients with suborbital rejuvenation who were admitted to the Department of Plastic Surgery, Wuhan No.1 Hospital from April 2019 to April 2021 were retrospectively analyzed. Hirmand classification of tear trough deformity was performed based on preoperative photographs. The expanded orbital septum fat was removed through the conjunctival approach, and the SVF-gel was extracted from the fat which was harvested from abdomen or thigh. The tear trough or suborbital depression was corrected by SVF-gel injection. The patients were followed up and the complications related to the operation were recorded. According to tear trough rating scale (TTRS), the depth of tear trough depression, the degree of pigmentation, the sagging degree of zygomatic fat pad and the degree of lower eyelid skin fold were scored before and after operation. The higher the total score indicates the greater severity of the lacrimal groove deformity. The morphology of the orbital-buccal junction was rated (0 to 3) according to the Barton aesthetic rating criteria. The aesthetic effects of lower eyelid bag and tear trough correction were comprehensively analyzed based on the satisfaction questionnaire (very satisfied, satisfied, dissatisfied 3 grades). SPSS 22.0 software was used for statistical analysis, and the normally distributed measurement data were represented by Mean±SD. Paired sample t-test was used to compare preoperative and postoperative TTRS scores, and P<0.05 was considered statistically significant. Results:A total of 92 patients with bilateral lower eyelid bag accompanied by tear trough or middle facial depression were included. There were 15 males and 77 females, aged (38.8±9.6) years (21-55 years). Preoperative Hirmand tear trough classification was performed as type Ⅱ in 37 cases, as type Ⅲ in 55 cases. All patients had conjunctival edema and lower eyelid swelling of different degrees 3 days after surgery, among which 35 patients had skin ecchymosis of different degrees, which recovered spontaneously 1 to 2 weeks after surgery, and no complications such as hematoma, diplopia, fat liquefaction, fat nodule and fat embolism occurred. Postoperative follow-up was conducted for 6 to 12 months. Comparison of preoperative and postoperative TTRS scores: compared with preoperative results, the TTRS scores of Hirmand type Ⅱ patients [(2.37±0.78) points vs. (4.83±0.98) points, t=36.54, P<0.001] and type Ⅲ patients [(2.61±0.60) points vs. (5.38±0.93) points, t=55.20, P<0.001] were significantly decreased after surgery, and the differences were statistically significant. Comparison of Barton aesthetic rating before and after surgery: for Hirmand type Ⅱ patients, preoperative rating was grade 1 in 7 cases, grade 2 in 19 cases, grade 3 in 11 cases, and postoperative improvement was grade 0 in 36 cases and grade 1 in 1 case; for Hirmand type Ⅲ patients, preoperative ratings were grade 2 in 20 cases, grade 3 in 35 cases, and postoperative improvement was grade 0 in 50 cases, grade 1 in 5 cases. Postoperative patient satisfaction survey: 85 cases (92.4%) were very satisfied, 5 cases (5.4%) were satisfied, 2 cases (2.2%) were dissatisfied. For the 2 patients who were not satisfied, one patient had no obvious improvement due to tear trough deformity, and was filled with SVF-gel for the second time 3 months later, achieving satisfactory results. One patient still had baggy eye on the outer side of the lower eyelid after surgery, which was corrected again 3 months later and achieved satisfactory results. Conclusion:For patients with lower eyelid bag with tear trough or suborbital depression without obvious skin relaxation, lower eyelid bag correction through conjunctival approach combined with SVF-gel transplantation can achieve the effect of rejuvenation of the suborbital region.

This study aimed to investigate the effect and molecular mechanism of sinomenine on proliferation, apoptosis, metastasis, and combination with inhibitors in human hepatocellular carcinoma HepG2 cells and SK-HEP-1 cells. The effect of sinomenine on the growth ability of HepG2 and SK-HEP-1 cells were investigated by CCK-8 assay, colony formation assay, and BeyoClick~(TM) EdU-488 staining. The effect of sinomenine on DNA damage was detected by immunofluorescence assay, and the effect of sinomenine on apoptosis of human hepatocellular carcinoma cells was clarified by Hoechst 33258 staining and CellEvent~(TM) Cystein-3/7Green ReadyProbes~(TM) reagent assay. Cell invasion assay and 3D tumor cell spheroid invasion assay were performed to investigate the effect of sinomenine on the invasion ability of human hepatocellular carcinoma cells in vitro. The effect of sinomenine on the regulation of protein expression related to the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription 3(STAT3) signaling pathway in HepG2 and SK-HEP-1 cells was examined by Western blot. Molecular docking was used to evaluate the strength of affinity of sinomenine to the target cysteinyl aspartate specific proteinase-3(caspase-3) and STAT3, and combined with CCK-8 assay to detect the changes in cell viability after combination with STAT3 inhibitor JSI-124 in combination with CCK-8 assay. The results showed that sinomenine could significantly reduce the cell viability of human hepatocellular carcinoma cells in a concentration-and time-dependent manner, significantly inhibit the clonogenic ability of human hepatocellular carcinoma cells, and weaken the invasive ability of human hepatocellular carcinoma cells in vitro. In addition, sinomenine could up-regulate the cleaved level of poly ADP-ribose polymerase(PARP), a marker of apoptosis, and down-regulate the protein levels of p-Akt, p-mTOR, and p-STAT3 in human hepatocellular carcinoma cells. Molecular docking results showed that sinomenine had good affinity with the targets caspase-3 and STAT3, and the sensitivity of sinomenine to hepatocellular carcinoma cells was diminished after STAT3 was inhibited. Therefore, sinomenine can inhibit the proliferation and invasion of human hepatocellular carcinoma cells and induce apoptosis, and the mechanism may be attributed to the activation of caspase-3 signaling and inhibition of the Akt/mTOR/STAT3 pathway. This study can provide a new reference for the in-depth research and clinical application of sinomenine and is of great significance to further promote the scientific development and utilization of sinomenine.
Humans ; Carcinoma, Hepatocellular/genetics* ; Proto-Oncogene Proteins c-akt/metabolism* ; Caspase 3/metabolism* ; Liver Neoplasms/genetics* ; Molecular Docking Simulation ; Sincalide/pharmacology* ; Cell Line, Tumor ; Cell Proliferation ; Hep G2 Cells ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis

This study investigated the effect and mechanism of morin in inducing autophagy and apoptosis in hepatocellular carcinoma cells through the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription protein 3(STAT3) pathway. Human hepatocellular carcinoma SK-HEP-1 cells were stimulated with different concentrations of morin(0, 50, 100, 125, 200, and 250 μmol·L~(-1)). The effect of morin on the viability of SK-HEP-1 cells was detected by Cell Counting Kit-8(CCK-8). The effect of morin on the proliferation and apoptosis of SK-HEP-1 cells was investigated using colony formation assay, flow cytometry, and BeyoClick~(TM) EdU-488 with different concentrations of morin(0, 125, and 250 μmol·L~(-1)). The changes in the autophagy level of cells treated with morin were examined by transmission electron microscopy and autophagy inhibitors. The impact of morin on the expression levels of proteins related to the Akt/mTOR/STAT3 pathway was verified by Western blot. Compared with the control group, the morin groups showed decreased viability of SK-HEP-1 cells in a time-and concentration-dependent manner, increased number of apoptotic cells, up-regulated expression level of apoptosis marker PARP, up-regulated phosphorylation level of apoptosis-regulating protein H2AX, decreased number of positive cells and the colony formation rate, an upward trend of expression levels of autophagy-related proteins LC3-Ⅱ, Atg5, and Atg7, and decreased phosphorylation levels of Akt, mTOR, and STAT3. These results suggest that morin can promote apoptosis, inhibit proliferation, and induce autophagy in hepatocellular carcinoma cells, and its mechanism of action may be related to the Akt/mTOR/STAT3 pathway.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis ; Autophagy ; Cell Proliferation ; Cell Line, Tumor ; STAT3 Transcription Factor/metabolism*

Objective:To investigate the effect of self-made medical ventilation chair in patients with acute respiratory distress syndrome (ARDS) ventilated in prone position, with the aim of reducing the occurrence of complications in patients with ARDS ventilated in prone position.Methods:This study was a quasi experimental research method. In this study, 78 patients admitted to the respiratory intensive care unit of Changsha Central Hospital affiliated to South China University from October 2019 to September 2021 were selected for the study by convenience sampling method, and were divided into a control group and a experimental group according to the order of admission, with 39 cases in each group. The experimental group was ventilated in the prone position using a self-made medical ventilation chair, and the control group was ventilated in the prone position using the conventional turning method, comparing the facial skin injury, tracheal displacement, tracheal tube obstruction, and RICU hospitalization time in the two groups.Results:At the end of prone position ventilation, the incidence of facial skin intact and redness and swelling was 56.41% (22/39) and 43.59% (17/39) in the experimental group and 25.64% (10/39) and 69.23% (27/39) in the control group, respectively, with statistically significant differences ( χ2=7.63, 5.21, both P<0.05); there was no statistically significant difference in the incidence of facial skin breakdown between the two groups ( P>0.05); the incidence of complications was 5.13% (2/39) in the experimental group and 20.51% (8/39) in the control group, with statistically significant differences ( χ2=4.13, P<0.05); the duration of RICU stay was (13.34 ± 3.85) days in the experimental group and ( 15.80 ± 5.55) days, with a statistically significant difference ( t=2.25, P<0.05). Conclusions:The use of self-made medical ventilation chair can reduce the facial skin damage of patients, reduce the occurrence of related complications, and shorten the hospitalization time of RICU. It is worth popularizing and applying in ICU.