Objective To establish models for real-time dynamic monitoring of intracellular cytoplasmic adenosine triphosphate(ATP)and mitochondrial ATP levels in cells in order to study the changes in metabolic processes.Methods The lentiviral plasmids of the cytoplasmic chemogenetic green fluorescent protein(GFP)ATP probe(Chemo-G)and those of the mitochondrial-localized chemogenetic GFP ATP probe(mito-Chemo-G)were constructed before being transfected into HEK293T together with the helper plasmids,respectively.Chemo-G and mito-Chemo-G lentiviruses were obtained.HeLa cells were infected with the lentivirus.Puromycin resistance selection and flow cytometry cell sorting were employed to identify and isolate the infected HeLa cells.The growth and GFP expressions of HeLa cells were observed.A live-cell imaging system was used for continuous imaging of the cells,with stimuli added at specific time points to alter intracellular ATP levels to observe changes in the fluorescence intensity of the ATP probe.Results Lentiviral plasmids containing Chemo-G and mito-Chemo-G sequences were constructed.Two cell lines which could stably express Chemo-G and mito-Chemo-G were established that exhibited strong growth and accurate intracellular fluorescence localization.Live-cell imaging revealed that after the addition of 2-deoxy-D-glucose(2-DG)into HeLa-Chemo-G,the fluorescence resonance energy transfer(FRET)/GFP ratio showed a decrease that was partially reversed by the addition of glucose.The FRET/GFP ratio increased after histamine stimulation,but rapidly decreased after the addition of oligomycin.Conclusion The Chemo-G and mito-Chemo-G lentiviral vectors and stably transfected cell lines HeLa-Chemo-G and HeLa-mito-Chemo-G are constructed,which provides reliable experimental models for studying cellular metabolism and changes in intracellular ATP levels.

Objective To establish a stable in vitro model of CD8+T cell exhaustion.Methods CD8+T cells were isolated and purified from the spleens of ovalbumin-specific CD8+T cell receptor(OT-I)transgenic mice and subjected to chronic antigen stimulation to induce exhaustion in vitro.Flow cytometry was employed to evaluate the expressions of exhaustion markers,secretion of effector cytokines,and transcription factor profiles in CD8+T cells.Exhausted and effector(non-exhausted)CD8+T cells were co-cultured with tumor cells before tumor cell viability was measured to assess the cytotoxic potential of CD8+T cells.Additionally,N-acetyl-L-cysteine(N-AC)was used as a positive control during exhaustion induction to validate the model.Results Chronic stimulation resulted in a significant upregulation of inhibitory receptors,including programmed cell death protein 1(PD-1),T cell immunoglobulin and mucin domain-containing protein 3(TIM-3),T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motifdomain(TIGIT),and lymphocyte activation gene 3(LAG-3).Concurrently,the secretion of key effector cytokines such as tumor necrosis factor-α(TNF-α)and interferon-γ(IFN-γ)was markedly reduced.Exhausted CD8+T cells exhibited diminished cytotoxicity against tumor cells compared to effector CD8+T cells.Notably,treatment with N-AC effectively restored the function of exhausted CD8+T cells and enhanced their anti-tumor activity.Conclusion This study has established an effective in vitro model for CD8+T cell exhaustion.The use of N-AC demonstrates its potential to restore functionality in exhausted CD8+T cells,underscoring the reliability and utility of this model for investigating the anti-tumor potential of exhausted T cells.

Objective To construct an NZB/W(F1)mouse model of systemic lupus erythematosus and evaluate the effects of nicotinamide on each index of lupus nephritis pathogenesis of NZB/W(F1)mice in order to provide data for research on the role of nicotinamide in the treatment of lupus nephritis.Methods Female NZB/W(F1)mice were obtained by crossing male NZW mice with female NZB ones.Urine samples were collected using metabolic cages and proteinuria test strips were used to detect proteinuria.Blood samples were collected through the orbital venous plexus in mice.Enzyme-linked immunosorbent assay(ELISA)was used to detect the level of anti-dsDNA antibody.Levels of serum creatinine and urea nitrogen and liver function indexes were detected using an automatic blood analyzer.Hematoxylin-eosin staining and immunofluorescence technique were used to detect the pathological state of the kidney.Results The levels of proteinuria,double-stranded DNA antibodies,serum creatinine,and urea nitrogen were gradually increased during the natural course of the disease in female NZB/W(F1)mice,indicating that the lupus nephritis disease model was constructed in female NZB/W(F1)mice.Compared to the control group,nicotinamide feeding could obviously decrease the level of proteinuria(P=0.0070),inhibit the production of double-stranded DNA antibodies(P=0.0325),and retard the progression of serum creatinine(P=0.0067)and urea nitrogen indexes(P=0.0166)in serum.In addition,the pathological state of the kidney in the nicotinamide feeding group was significantly alleviated compared with the control group.Conclusion A lupus nephritis disease model is constructed in NZB/W(F1)mice.Nicotinamide feeding can obviously alleviate the disease state of lupus nephritis in NZB/W(F1)mice.

Objective:To understand the current situation of nurses in 52 hospitals in China on mastery of knowledge about skin injury in the elderly based on the background of mixed-mode homogenization training.Methods:Using the convenient sampling method, a total of 1 067 nurses from 52 hospitals in China were selected as the research objects in January 2021. A self-designed questionnaire on knowledge of skin injury in the elderly was used to investigate the nurses through the questionnaire star and univariate analysis was used to analyze the influencing factors. A total of 1 067 questionnaires were distributed and 1 067 valid questionnaires were recovered, and the effective recovery rate was 100%.Results:The knowledge scores of pressure injury, incontinence-associated dermatitis, skin tear and xerosis cutis among 1067 nurses were (95.66±7.37) , (95.65±9.15) , (91.37±15.45) and (87.67±15.91) , respectively. The results of univariate analysis showed that hospital grade was the influencing factor of nurses' knowledge score of pressure injury, skin tear and incontinence-associated dermatitis ( P<0.05) , educational background was the influencing factor of nurses' knowledge score of skin tear ( P<0.05) , professional title was the influencing factor of nurses' knowledge scores of pressure injury, incontinence-associated dermatitis and xerosis cutis ( P<0.05) . Conclusions:Hospitals at all levels need to strengthen the theoretical and practical knowledge training for nurses on skin xerosis and skin tear in the elderly, especially for nurses with primary titles and lower education in grassroots hospitals.

In order to obtain the lead compound for treatment of rheumatoid arthritis (RA), in this study, therapeutic efficacy of three bispecific antibodies (BsAB-1, BsAB-2 and BsAB-3) against both hIL-1beta and hIL-17 were compared on CIA model mice. First, by ELISA method we compared the binding capacity of the three bispecific antibodies to the two antigens. The results showed that all three antibodies could simultaneously bind both antigens, among these antibodies, BsAB-1 was superior over BsAB-2 and BsAB-3. CIA model was established with chicken type II collagen (CII) and developed RA-like symptoms such as ankle swelling, skin tight, hind foot skin hyperemia. The CIA mice were treated with three antibodies once every two days for total of 29 days. Compared with the CIA model mice, the RA-like symptoms of the antibody treated-mice significantly relieved, while the BsAB-1 treated-mice were almost recovered. CII antibody level in the serum and cytokines (IL-2, IL-1beta, IL-17A and TNF-alpha) expression in the spleen were examined. Compared with the CIA model mice, all three antibodies could significantly reduce CII antibody and cytokine expression levels. BsAB-1 antibody was more potent than BsAB-2 and BsAB-3. In summary, BsAB-1 is superior over BsAB-2 and BsAB-3 in amelioration of RA symptoms and regulation of CII antibody production and pro-inflammatory cytokine expression, therefore, BsAB-1 can be chosen as a lead compound for further development of drug candidate for treatment of RA.

To investigate the effect of hSCGF-alpha on human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs), we obtained hSCGF-alpha using genetic engineering, hSCGF-alpha gene was amplified from hUCMSCs cDNA using two-step PCR and was inserted into pET-28a(+) plasmid vector. Induced by IPTG at 20 degrees Celsius for 24 h, the fusion protein expressed in E. coli BL21 (DE3) was mainly existing in soluble form. The recombinant hSCGF-a was purified using NI-NTA affinity chromatography and the purity was up to 90%. The colony forming test revealed that combined use hSCGF-alpha and rmGM-CSF (recombinant murine GM-colony stimulating factor, rmGM-CSF) had granulocyte/macrophage (GM) promoting effects on murine bone marrow GM progenitor. In addition, the results indicated that hSCGF-alpha and rhGM-CSF had stimulatory effect on hUCMSCs and their synergetic effect was the strongest.
Cell Proliferation ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Drug Synergism ; Escherichia coli ; genetics ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Stem Cell Factor ; biosynthesis ; genetics ; Umbilical Cord ; cytology

Cyanovirin-N (CVN) is an 11 kDa anti-HIV protein originally isolated from extracts of a cyanobacterium, Nostoc ellipsosporum. The protein binds with high affinity to the viral envelope glycoprotein gp120 and irreversibly inactivates diverse HIV strains. A fusion gene consisting of cvn, sumo and 6xHis tag was synthesized by PCR according to the codon bias of Escherichia coli. The fusion protein is expressed in the cytoplasm of E. coli in a soluble form and up to 28% of the total protein. The recombinant CVN was purified to homogeneity by 2 rounds of Ni-NTA affinity chromatography and one round of SUMO protease cleavage. Bioactivity assay demonstrated that SUMO-CVN and CVN bound to gp120 with nanomolar concentration. In addition, CVN showed potent anti-HSV-1 and anti-HIV-1 activities in in vitro cellular assays. Therefore, the 6xHis SUMO fusion expression and purification system provides a better approach for large scale production of CVN for further microbicide development.
Antiviral Agents ; isolation & purification ; metabolism ; pharmacology ; Bacterial Proteins ; biosynthesis ; genetics ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; pharmacology ; Escherichia coli ; genetics ; metabolism ; HIV-1 ; drug effects ; Herpesvirus 1, Human ; drug effects ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; pharmacology

Adolescent ; Adult ; Endoscopy ; Ethmoid Sinus ; surgery ; Eyelids ; surgery ; Female ; Humans ; Male ; Middle Aged ; Orbital Fractures ; surgery ; Otorhinolaryngologic Surgical Procedures ; methods ; Young Adult