Background Arsenic exposure has been demonstrated to induce apoptosis. The fibronectin type III structural domain 3B protein (FNDC3B) has been shown to promote cancer cell proliferation; however, its role in arsenic-induced apoptosis remains to be elucidated. Objective To investigate the effects of sodium arsenite (NaAsO₂) and its metabolites on the expression of FNDC3B gene in A549 cells and to understand the function of FNDC3B gene in A549 cells. Methods (1) A549 cells were exposed to varying final concentrations of NaAsO₂ and their optical density at 450 nm values were measured by Cell Counting Kit-8 (CCK-8) after 48 h. Survival curves were plotted, and a final exposure dose was selected according to the survival rate. Total protein and RNA were extracted by exposing A549 cells to high (30 µmol·L⁻¹), medium (20 µmol·L⁻¹), and low (10 µmol·L⁻¹) NaAsO₂ concentrations, high (30 µmol·L⁻¹) monomethylarsinic acid (MMA), and high (30 µmol·L⁻¹) dimethylarsinic acid for a period of 48 h. mRNA expression and the protein expression of the FNDC3B gene was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB), while the protein ubiquitination expression of the FNDC3B gene was detected by co-immunoprecipitation (CO-IP) and WB assay. (2) Knockdown of FNDC3B gene expression was achieved in A549 cells by siRNA interference. The si-FNDC3B fragment was transfected in A549 cells for 48 h. The mRNA and protein expression of FNDC3B gene was then detected by qRT-PCR and WB assay. Cell viability was determined through CCK-8 assay. Hoechst 33342/propidium iodide (PI) double staining and JC-1 mitochondrial membrane potential assay were employed to detect both early and late apoptosis, while cleaved caspase3 protein and P53 signalling pathway related protein expressions were evaluated by WB. Results (1) The CCK-8 results demonstrated a decline in the viability of A549 cells with an increase in NaAsO₂ concentration, with an inhibitory concentration at 50% of 38.12 µmol·L⁻¹. The qRT-PCR results demonstrated that compared to the control group, varying concentrations of NaAsO₂ (10, 20, and 30 µmol·L⁻¹) significantly upregulated the mRNA expression of FNDC3B gene (P<0.01). In contrast, MMA and DMA showed no significant effect on FNDC3B mRNA expression (P>0.05). The WB analysis revealed that the protein expression of FNDC3B was reduced in the NaAsO₂-treated group compared to the control, accompanied by elevated ubiquitination levels of FNDC3B protein, particularly at the K48 ubiquitination site. MMA and DMA exhibited no impact on FNDC3B protein expression. (2) Following the specific knockdown of FNDC3B expression in A549 cells, the CCK-8 assay demonstrated a significant reduction in cell viability in the silenced FNDC3B group (si-FNDC3B) compared to the control group. The JC-1 assay demonstrated that the mitochondrial membrane potential was diminished in the si-FNDC3B group relative to the control group. The Hoechst 33342/PI staining assay revealed that the si-FNDC3B group exhibited a notable degree of apoptosis. The si-FNDC3B group also displayed substantial apoptosis. The WB analysis indicated that the relative expressions of cleaved caspase3, P53, MDM2, Bad, and Bax proteins were elevated in the si-FNDC3B group in comparison to the control group. Conclusion The presence of NaAsO₂ is observed to promote the ubiquitination expression of the FNDC3B protein, which in turn reduces the expression of FNDC3B protein. However, the main metabolites DMA and MMA have no effect on the expression of FNDC3B. Furthermore, the silencing of FNDC3B is observed to inhibit the viability of A549 cells and promote apoptosis, a phenomenon related to the activation of P53 signaling pathway.

To screen new antigens for novel tuberculosis(TB)vaccine research,we used bioinformatics to predict the B and T cell epitopes of Rv2318,and evaluated the immunogenicity of Rv2318 and its T/B epitope peptides(Rv2318p).The recombinant plas-mids pET32a-Rv2318 and pET32a-Rv2318p were constructed through gene synthesis methods.The recombinant proteins were ex-pressed in a prokaryotic system and purified with nickel affinity chromatography.Proteins were identified with SDS-PAGE and western blotting.BALB/c mice were immunized subcutaneously with the recombinant proteins to evaluate immunogenicity.Sera were collected,and antigen specific antibody titers were evaluated with ELISA.Splenocytes were isolated,and cytokines and T cell proliferation were analyzed with ELISA and flow cytometry,respectively.Rv2318 included two epitope fragments,aa10-130 and 350-410.SDS-PAGE and western blotting indicated that the target proteins were expressed and purified correctly,and their relative molecular weights were-approximately 68 kD and 42 kD,respectively.Rv2318 and Rv2318p induced stronger humoral immune responses than observed in the control groups(P<0.000 1,n=6).Compared with Rv2318,Rv2318p showed significantly greater enhancement of specific IgG and IgG subclass antibodies(P<0.000 1,n=6).In addition,Rv2318p increased the ratio of IgG2a/IgG1,thus indicating that it primarily induced a cellular immune response biased toward the Th1 type.Cytokine experiments revealed that IFN-γ,IL-2,IL-6,and IL-4 significantly increased after immunization with Rv2318p(P all<0.01,n=6),particularly Th1 type cytokines(IFN-γ and IL-2).Furthermore,Rv2318 increased the expression of only IL-2 and IL-6,particularly IL-6(P all<0.01,n=6).Although Rv2318 in-duced more IFN-γ,we observed no significant difference between Rv2318 and PBS immunized mice.Importantly,Rv2318p stimu-lated mice to express IFN-γ at 842 pg/mL,approximately 3 times the level elicited by Rv2318.Whereas both proteins increased the proportions of CD4+and CD8+T cells,Rv2318p promoted greater proliferation of T lymphocytes.These data indicated that both Rv2318 and its epitope peptides enhanced humoral and cellular immune responses,whereas the epitope peptides notably triggered a stronger Th1 type cellular response.In conclusion,the recombinant protein Rv2318 and its epitope peptides showed favorable immunogenicity,and the immunogenicity of Rv2318p was superior to that of Rv2318.This study provides a theoretical basis for TB vaccine development.

This study was aimed at analyzing the epidemiological and drug resistance characteristics of tuberculosis at a desig-nated tuberculosis hospital in Hunan Province in 2024.Patients diagnosed with TB at the hospital between April and October 2024 were included in the study.Demographic data,clinical information,and drug sensitivity test results were collected from the hospital′s electronic medical record system.Descriptive statistics,the chi-square test,and logistic regression were used to analyze the epidemic characteristics,drug resistance characteristics,and factors influencing tuberculosis.Whole genome sequencing of isolates was per-formed,and lineage classification and drug resistance gene mutations were detected with TB-Profiler.The male-to-female ratio was 2.72∶1,and the median age was 56(IQR:43-66)years.Among the 391 patients,most were farmers(46.8%,183/391)and were pri-marily from Changsha(41.1%,162/391).Significant differences were observed in sex and occupation between pulmonary tuberculosis(PTB)and extrapulmonary tuberculosis(EPTB).The overall prevalence of any type of drug resistance of tuberculosis was 33.25%,and the multidrug resistance TB(MDR-TB)and poly-drug resistance(PR-TB)rates were 14.23%and 4.35%,respectively.The re-sistance rates to rifampicin(RIF),isoniazid(INH),ethambutol(EMB),and streptomycin(SM)were 17.90%,22.25%,6.39%,and 20.20%,respectively.Multivariable logistic regression analysis indicated that both diabetes(OR:2.295,95%CI:1.082-4.866)and retreatment(OR:17.822,95%CI:8.343-38.072)were risk factors for developing MDR-TB.Lineage 2(L2)strains accounted for 64.40%(136/191),whereas lineage 4(L4)accounted for 28.80%(55/191).The most common drug resistance mutations were katG Ser315Thr(62.50%,20/32)for INH,rpoB Ser450Leu(50.00%,12/24)for RIF,embB Met306Val(55.56%,5/9)for EMB,and rpsL Lys43Arg(80.95%,34/42)for SM.In conclusion,TB drug resistance was found to be a serious problem at a designated tu-berculosis hospital in Hunan in 2024.Strengthening the treatment and management of patients infected with L2 strains,those with co-morbid diabetes,and retreatment cases is crucial for preventing and controlling the emergence of drug-resistant TB.

To screen new antigens for novel tuberculosis(TB)vaccine research,we used bioinformatics to predict the B and T cell epitopes of Rv2318,and evaluated the immunogenicity of Rv2318 and its T/B epitope peptides(Rv2318p).The recombinant plas-mids pET32a-Rv2318 and pET32a-Rv2318p were constructed through gene synthesis methods.The recombinant proteins were ex-pressed in a prokaryotic system and purified with nickel affinity chromatography.Proteins were identified with SDS-PAGE and western blotting.BALB/c mice were immunized subcutaneously with the recombinant proteins to evaluate immunogenicity.Sera were collected,and antigen specific antibody titers were evaluated with ELISA.Splenocytes were isolated,and cytokines and T cell proliferation were analyzed with ELISA and flow cytometry,respectively.Rv2318 included two epitope fragments,aa10-130 and 350-410.SDS-PAGE and western blotting indicated that the target proteins were expressed and purified correctly,and their relative molecular weights were-approximately 68 kD and 42 kD,respectively.Rv2318 and Rv2318p induced stronger humoral immune responses than observed in the control groups(P<0.000 1,n=6).Compared with Rv2318,Rv2318p showed significantly greater enhancement of specific IgG and IgG subclass antibodies(P<0.000 1,n=6).In addition,Rv2318p increased the ratio of IgG2a/IgG1,thus indicating that it primarily induced a cellular immune response biased toward the Th1 type.Cytokine experiments revealed that IFN-γ,IL-2,IL-6,and IL-4 significantly increased after immunization with Rv2318p(P all<0.01,n=6),particularly Th1 type cytokines(IFN-γ and IL-2).Furthermore,Rv2318 increased the expression of only IL-2 and IL-6,particularly IL-6(P all<0.01,n=6).Although Rv2318 in-duced more IFN-γ,we observed no significant difference between Rv2318 and PBS immunized mice.Importantly,Rv2318p stimu-lated mice to express IFN-γ at 842 pg/mL,approximately 3 times the level elicited by Rv2318.Whereas both proteins increased the proportions of CD4+and CD8+T cells,Rv2318p promoted greater proliferation of T lymphocytes.These data indicated that both Rv2318 and its epitope peptides enhanced humoral and cellular immune responses,whereas the epitope peptides notably triggered a stronger Th1 type cellular response.In conclusion,the recombinant protein Rv2318 and its epitope peptides showed favorable immunogenicity,and the immunogenicity of Rv2318p was superior to that of Rv2318.This study provides a theoretical basis for TB vaccine development.

This study was aimed at analyzing the epidemiological and drug resistance characteristics of tuberculosis at a desig-nated tuberculosis hospital in Hunan Province in 2024.Patients diagnosed with TB at the hospital between April and October 2024 were included in the study.Demographic data,clinical information,and drug sensitivity test results were collected from the hospital′s electronic medical record system.Descriptive statistics,the chi-square test,and logistic regression were used to analyze the epidemic characteristics,drug resistance characteristics,and factors influencing tuberculosis.Whole genome sequencing of isolates was per-formed,and lineage classification and drug resistance gene mutations were detected with TB-Profiler.The male-to-female ratio was 2.72∶1,and the median age was 56(IQR:43-66)years.Among the 391 patients,most were farmers(46.8%,183/391)and were pri-marily from Changsha(41.1%,162/391).Significant differences were observed in sex and occupation between pulmonary tuberculosis(PTB)and extrapulmonary tuberculosis(EPTB).The overall prevalence of any type of drug resistance of tuberculosis was 33.25%,and the multidrug resistance TB(MDR-TB)and poly-drug resistance(PR-TB)rates were 14.23%and 4.35%,respectively.The re-sistance rates to rifampicin(RIF),isoniazid(INH),ethambutol(EMB),and streptomycin(SM)were 17.90%,22.25%,6.39%,and 20.20%,respectively.Multivariable logistic regression analysis indicated that both diabetes(OR:2.295,95%CI:1.082-4.866)and retreatment(OR:17.822,95%CI:8.343-38.072)were risk factors for developing MDR-TB.Lineage 2(L2)strains accounted for 64.40%(136/191),whereas lineage 4(L4)accounted for 28.80%(55/191).The most common drug resistance mutations were katG Ser315Thr(62.50%,20/32)for INH,rpoB Ser450Leu(50.00%,12/24)for RIF,embB Met306Val(55.56%,5/9)for EMB,and rpsL Lys43Arg(80.95%,34/42)for SM.In conclusion,TB drug resistance was found to be a serious problem at a designated tu-berculosis hospital in Hunan in 2024.Strengthening the treatment and management of patients infected with L2 strains,those with co-morbid diabetes,and retreatment cases is crucial for preventing and controlling the emergence of drug-resistant TB.

Background Arsenic is a human carcinogen. Arsenic and its metabolites affect the expression of p53, but whether there are any changes of p53 phosphorylation and ubiquitination levels in human bronchial epithelium cells (BEAS-2B) are not clear after exposure to arsenic and its metabolites. Objective To study the effects of arsenic and its metabolites monomethylarsic acid (MMA) and dimethylarsinic acid (DMA) on the expression of tumor suppressor gene p53 in BEAS-2B cells. Methods Different concentrations of sodium arsenite (NaAsO₂) were used to infect BEAS-2B cells, and the cell viability was detected with CCK-8 reagent to determine the dose and time of NaAsO₂ used for the following study. Based on the results of cell viability, the cells were divided into two panels: a sodium arsenide panel and an arsenic methylation metabolite penal. The doses of sodium arsenite were 0, 2, 4, and 6 μmol·L⁻¹ NaAsO₂; the arsenic methylation metabolite panel consisted of 0 μmol·L⁻¹ NaAsO₂ group (control), 6 μmol· L⁻¹ MMA group, 6 μmol· L⁻¹ DMA group， and 6 μmol· L⁻¹ NaAsO₂ group. The cells were collected after 48 h treatment, and the total protein and total RNA were extracted. The relative levels of p53 mRNA expression were determined by quantitative real-time polymerase chain reaction (qRT-PCR), the relative expression levels of p53 protein, p53 Ser9 and Ser15 phosphorylated proteins were determined by Western blot, and the level of p53 ubiquitination was detected by co-immunoprecipitation (CO-IP). Results Compared with the control group, the cell viability rates in all BEAS-2B cells treated by NaAsO₂ were significantly reduced (P<0.05), and the 50% cell viability was observed at 6 μmol·L⁻¹. Compared with the control group, the relative expression level of p53 mRNA gradually decreased after NaAsO₂ (2, 4, 6 μmol·L⁻¹) treatment (P<0.05), the relative expression levels of p53 protein and Ser9 phosphorylated protein induced by NaAsO₂ also decreased gradually (P<0.05), and the relative expression level of p53 Ser15 phosphorylated protein induced by NaAsO₂ followed the same pattern, but it was only lower than that of the control group in the 6 μmol·L⁻¹ NaAsO₂ group (P<0.05). Compared with the control group, there were no significant effects on the relative expression levels of p53 mRNA, p53 protein, Ser9 and Ser15 phosphorylated proteins in the MMA group and the DMA group. Compared with the control group, the expression level of p53 ubiquitination was significantly decreased and the expression of K48 ubiquitination decreased significantly after NaAsO₂ infection. Conclusion Arsenic causes a decrease in the expression of the p53 protein in BEAS-2B cells, largely due to inhibition of the phosphorylated pathway and a decrease in mRNA expression, and protein changes caused by a decrease in p53 ubiquitination do not play a dominant role. MMA and DMA do not affect p53 gene expression.

Objective:To preliminarily evaluate the immunogenicity and efficacy of two novel tuberculosis vaccine candidates (a fusion multicomponent protein EPDPA015f and a mixed multicomponent protein EPDPA015m) and to provide a new antigen combination for the development of tuberculosis vaccines.Methods:Recombinant plasmids for the expression of EPDPA015f and EPDPA015m proteins were constructed. Six-week-old BALB/c mice were immunized with EPDPA015f or EPDPA015m in combination with aluminium adjuvant (50 μg/mouse) for three times with an interval of 10 d. The mice were sacrificed 10 d after the last immunization to collect blood and spleen samples. Serum antibody titers and cytokine levels were measured by ELISA, Luminex technique and enzyme-linked immunospot assay (ELISPOT). Mycobacterial growth inhibition assay (MGIA) was used to detect the ability of mouse splenocytes to inhibit the growth of Mtb in vitro. One-way analysis of variance and t-test were used for statistical analysis. Results:Both EPDPA015f and EPDPA015m could induce the production of various cytokines and IgG antibodies at a high level. The levels of cytokines related to Th1 (IL-2, TNF-α, IFN-γ), Th2 (IL-4, IL-6, IL-10) and Th17 (IL-17) as well as other proinflammatory cytokines (GM-CSF, IL-12) were higher in the EPDPA015f group than in the adjuvant group ( P<0.05). The titer of IgG antibody induced by EPDPA015f was as high as 1∶4×10 6. The results of MGIA showed that the numbers of Mtb (lgCFU) in the PBS, adjuvant, EPDPA015f and EPDPA015m groups were 3.46±0.11, 3.51±0.06, 2.98±0.09 and 3.19±0.08, respectively. The number of colonies in the EPDPA015f group was the least as compared with that in the other three groups ( P<0.001, P<0.001, P<0.01). Conclusions:The vaccine candidate EPDPA015f could elicit more comprehensive and high-level cellular and humoral immune responses, and exhibited superior in vitro inhibitory activity against the growth of Mtb. EPDPA015f had the potential to be used as a preventive vaccine or a booster vaccine

Objective:To evaluate the immunogenicity and protective effect of a multicomponent recombinant protein vaccine EPRHP014 constructed independently and provide a scientific basis for developing new tuberculosis (TB) vaccine and effective prevention and control of TB.Methods:Three full-length Mycobacterium ( M.) tuberculosis protein antigens (EsxH, Rv2628, and HspX) and two epitope-predicted and optimized epitope-dominant protein antigens (nPPE18 and nPstS1) were selected, from which five protein antigens were used to construct a protein antigen composition EPRHP014, including a fusion expression multi-component protein antigen (EPRHP014f) and a multi-component mixed protein antigen (EPRHP014m) formed with the five single protein using clone, purification, and purification respectively. Multicomponent protein vaccines EPRHP014f and EPRHP014m were prepared with aluminum adjuvant, and the BCG vaccine was used as a control. ELISA detected the titer of serum-specific antibodies, the secretion of various cytokines was detected by ELISpot and Luminex, and immune protection was observed by the M.tuberculosis growth inhibition test in vitro. The results were statistically analyzed by t-test or rank sum test, and P<0.05 was considered a statistically significant difference. Results:Mice Immunized with EPRHP014m and EPRHP014f could produce highly effective IgG antibodies and their subtypes IgG1 and IgG2a, and the antibody titers were similar to those of mice immunized with BCG, with no statistical significance ( P>0.05). The number of spot-forming cells (SFC) secreting IFN-γ and IL-4 induced by EPRHP014f group was significantly higher than those by EPRHP014m group and BCG group ( P<0.05), but there was no significant difference in the number of SFC for IFN-γ and IL-4 induced between EPRHP014m group and BCG group ( P>0.05). The secretion levels of GM-CSF and IL-12p70 induced by the EPRHP014m group were higher than those of the BCG group ( P<0.05), but there was no significant difference in the levels of IL-6 and IL-10 induced between EPRHP014m group and BCG group ( P>0.05). There was no significant difference in the secretions of IL-6, IL-10, IL-12, and GM-CSF between the EPRHP014f and BCG groups ( P>0.05). EPRHP014m group, EPRHP014f group, and BCG group had obvious antibacterial effects in vitro, and the difference was insignificant ( P>0.05). Conclusion:Both EPRHP014f and EPRHP014m can induce strong humoral and cellular immune responses in mice after immunization, and have a strong ability to inhibit the growth of M. tuberculosis in vitro, indicating that the antigen composition EPRHP014 has good potential in the development and application of TB vaccine.

Objective To investigate the changes of erythrocyte parameters and the value of differential diagnosis in pregnant women with β-mediterranean anemia.Methods A total of 300 pregnancy women from July 2014 to December 2015 in Center Hospital of Longgang were recruited in this study,100 pregnant women with β-mediterranean anemia in β-mediterranean anemia pregnancy group,100 healthy pregnant women in normal pregnancy group,100 pregnant women with iron deficiency anemia in iron deficiency anemia pregnancy group.Mean red cell volume(MCV),mean erythrocyte hemoglobin(MCH),reticulocyte percentage(Ret%) were detected and compared in the three groups.Results Compared with the normal pregnancy group and iron deficiency anemia pregnancy group,the MCV,MCH significantly reduced,and Ret% significantly rised in the β-mediterranean anemia pregnant group,the differences were significant(P<0.05).The best cut-off value of Ret% was 1.7% in differential diagnosis of β-mediterranean anemia pregnancy and iron deficiency anemia pregnancy,the sensitivity was 63.00%,the specificity was 74.00%,the area under of receiver operating characteristic curve was 0.841.The sensitivity of joint detection including MCV,MCH and Ret% in differential diagnosis of β-mediterranean anemia pregnancy and iron deficiency anemia pregnancy was 84.00%,the specificity was 90.00%.Conclusion MCV,MCH and Ret% in pregnancy women with β-mediterranean anemia changes significant compared with normal pregnancy group and iron deficiency anemia pregnancy group,the joint detection including MCV,MCH and Ret% could significantly improve the differential diagnosis of β-mediterranean anemia and iron deficiency anemia in pregnancy women.

Objective To determine the contents of four effective constituents in Euphorbiae Semen decoction;To provide evidence for Euphorbiae Semen decoction into clinical application. Methods Established quantitative analysis multi-components by single marker method was used to determine the contents of diterpenoids constituents, such as euphorbia storoid, euphorbia factor L2, and euphorbia factor L3. HPLC method was used to determine the contents of aesculetin. Results Contents of euphorbia storoid, euphorbia factor L2, and euphorbia factor L3 in smashed Euphorbiae Semen decoction were 0.015 9%, 0.005 9% and 0.024 1%, respectively. However, the contents of the above three constituents could not be detected in whole Euphorbiae Semen decoction. The content of aesculetin (0.693 6%) in smashed Euphorbiae Semen decoction was more than that in whole Euphorbiae Semen decoction (0.288 2%). Conclusion Decoction digestion effect of diterpenoids constituents in Euphorbiae Semen decoction is not good. Decocting with water is not suitable for the clinical application of Euphorbiae Semen for purgation and diuresis. Aesculetin in smashed Euphorbiae Semen decoction has good decoction digestion effect, in which clinical use for antisepsis and anti-inflammation is effective.