Objective To investigate the effect of a nucleic acid purifier combined with a vacuum concentrator on DNA recovery rate.Methods Using 39 tubes of 007 standard samples,two 30 μL samples were taken from each tube.Samples were purified using the PrepFiler ExpressTM BTA Purification Kit on the AutoMate ExpressTM purifier,with elution volumes of 30 μL and 200 μL.Samples with 200 μL eluent were concentrated to 30 μL using a trace DNA sample vacuum concentrator.DNA solution concentrations were measured,and recovery rates were calculated and compared.Results The measured DNA concentration of the 007 standard samples was 0.08～0.15(0.11±0.02)ng/μL,showing a significant difference from the kit-labeled concentration of 0.1 ng/μL(t=3.88,P<0.01).The DNA solution concentration in the purified and concentrated group(200 μL eluent concentrated to 30 μL)[0.05～0.13(0.08±0.02)ng/μL]was significantly higher than the purified group(30 μL eluent)[0.02～0.08(0.05±0.01)ng/μL](t=21.20,P<0.01).The DNA recovery rate of the purified and concentrated group[54.55%～92.86%(75.41±10.12)%]was substantially higher than the purified group[25.00%～54.55%(39.79±7.13)%](t=19.79,P<0.01).Conclusion Utilizing a large-volume elution system in nucleic acid purification can significantly enhance DNA recovery rate.Combining this method with a vacuum concentrator effectively increases DNA solution concentration,thereby improving DNA testing success rates.

The pathogenesis of chronic fatigue syndrome(CFS)remains poorly defined,and while the tryptophan metabolic pathway is closely associated with the development of CFS,the underlying mechanisms are not yet fully understood.This review examines the alterations in tryptophan metabolites and related enzymes in both peripheral and central systems of CFS patients,with a particular focus on the involvement of the tryptophan pathway in the gut-brain axis(GBA).Furthermore,it provides a comprehensive analysis of the mechanistic roles of tryptophan metabolites in modulating the progression of CFS,aiming to elucidate the current evidence and potential driving mechanisms linking the tryptophan metabolic pathway to CFS,thereby promoting new insights and advancements in this research domain.

Aim To examine the pathogenesis of disul-fide death gene in vascular dementia(VD)by bioin-formatics analysis of disulfide death differentially ex-pressed genes(DEGs)combined with experimental verification.Methods The death DEGs of disulfide were screened and their correlation was analyzed.The VD patients data in the data set were analyzed by clus-tering and typing and gene set variation.The clustering risk of DEGs was tested with a nomogram model,and the optimal learning model was predicted.After the es-tablishment of VD rat model,water maze test,HE stai-ning and RT-qPCR detection were performed to verify the results of health information.Results Four DEGs including SLC7A11 were obtained,which had antago-nistic or synergistic interaction with each other.The genetic data could be divided into two subtypes with significant differences.After typing,VD disulfide DEGs were mainly concentrated in GnRH signaling pathways.The accuracy of the nomogram prediction model was high.Generalized linear was the best ma-chine learning model.Compared with the sham opera-tion group,the escape latency of rats in the model group was prolonged,the number of crossing platforms decreased,the relative mRNA expression levels of Slc3a2 and Slc7a11 decreased,and LRPPRC in-creased.Conclusions SLC7A11 and other disulfide death DEGs and its related GnRH signaling pathway may be an important part of the pathogenesis of VD di-sulfide death.SLC3A2,LRPPRC and SLC7A11 can be used as characteristic genes in the regulation of VD by disulfide death,which may affect VD progression through the regulation of disulfide death.

To construct a safe and effective multi-epitope vaccine against the S1 protein of chicken infectious bronchitis virus(IBV).In this study,homologous and non-homologous dominant epitopes of IBV M41,T,QX and H120 virulent strain S1 proteins were screened by various online bioprediction software,respectively,and a new peptide W with high immunogenicity was construc-ted by connecting the screened B-cell and T-cell epitopes with a linker peptide.W was ligated to the truncated sequence of the four viral strains by T2A yietding to the eukaryotic expression vector pEGFP-N1,and it was identified by PCR and double digestion,the obtained recombinant plasmid was transfected into HEK293A cells and target protein expression was measured by Western blot.The constructed plasmid was injected intramuscularly twice to detect the antibody level,cytokine level,and peripheral blood T cell subsets were detected after two immunizations.The epitope pro-tein W was successfully constructed,which was structurally stable,antigenic,and soluble;the re-combinant plasmid pEGFP-WMQtH,pEGFP-W,and pEGFP-MQtH matched the expected size;anti-IBV IgG antibody levels in pEGFP-N1 was increased greatly compared to the PBS group.cyto-kines IL-2,and γ interferon(IFN-γ)were increased greatly(P＜0.05);peripheral blood CD4+/CD8a value(P＜0.05)was increased greatly.The W epitope protein was successfully constructed,which can effectively activate the humoral immunity and cellular immunity against four infectious bronchitis viruses(IBV),laying a foundation for the development of an effective vaccine against IB.

Aim To explore the effects of nuciferine on cognitive behavior and the underlying mechanisms,white matter injury(WMI),neuroinflammation,and ferroptosis in mice with chronic ischemic WMI.Meth-ods Sixty C57BL/6 mice were divided into a control group,a bilateral common carotid artery stenosis(BCAS)model group,and low/high-dose nuciferine groups(20/40 mg·kg-1).A chronic ischemic WMI model was established using BCAS surgery.Following eight weeks of treatment,cognitive behavior(Y-maze,novel object recognition,Morris water maze),white matter integrity(LFB/MBP staining),microglial acti-vation(Iba-1 immunofluorescence),inflammatory cy-tokines(ELISA for TNF-α,IL-1β,IL-6),ferroptosis markers(Fe2+,ROS,MDA,GSH),mitochondrial ultrastructure(electron microscopy),and protein ex-pression of the PI3K/Akt and NRF2/xCT/GPX4 signa-ling pathways(Western blot)were evaluated.Results Compared with the control group,the BCAS group showed significant cognitive decline(P＜0.05),re-duced myelin density,elevated inflammatory cytokines and ferroptosis markers(Fe2+,ROS,MDA),shrunk-en mitochondria,and downregulated PI3K/Akt and NRF2/xCT/GPX4 pathway proteins(P＜0.05).Nu-ciferine intervention significantly ameliorated these in-juries in BCAS mice,with the high-dose group exhibi-ting superior effects(P＜0.05).Conclusions Nu-ciferine exerts protective effects against chronic ische-mic WMI and cognitive impairment by activating the PI3K/Akt and NRF2/xCT/GPX4 signaling pathways,thereby suppressing neuroinflammation and ferroptosis.

Hepatocellular carcinoma(HCC),which is essentially primary liver cancer,is closely related to CD8+T cell immune infiltration and immune suppression.We constructed a CD8+T cells related risk score model to pre-dict the prognosis of HCC patients and provided therapeutic guidance based on the risk score.Using integrated bulk RNA sequencing(RNA-seq)and single-cell RNA sequencing(scRNA-seq)datasets,we identified stable CD8+T cell signatures.Based on these signatures,a 3-gene risk score model,comprised of KLRB1,RGS2,and TN-FRSF1B was constructed.The risk score model was well validated through an independent external validation co-hort.We divided patients into high-risk and low-risk groups according to the risk score and compared the differ-ences in immune microenvironment between these two groups.Compared with low-risk patients,high-risk patients have higher M2-type macrophage content(P＜0.0001)and lower CD8+T cells infiltration(P＜0.0001).High-risk patients predict worse response to immunotherapy treatment than low-risk patients(P＜0.01).Drug sensitivity a-nalysis shows that PI3K-β inhibitor AZD6482 and TGFβRII inhibitor SB505124 may be suitable therapies for high-risk patients,while the IGF-1R inhibitor BMS-754807 or the novel pyrimidine-based anti-tumor metabolic drug Gemcitabine could be potential therapeutic choices for low-risk patients.Moreover,expression of these 3-gene mod-el was verified by immunohistochemistry.In summary,the establishment and validation of a CD8+T cell-derived risk model can more accurately predict the prognosis of HCC patients and guide the construction of personalized treatment plans.

Hepatocellular carcinoma(HCC),which is essentially primary liver cancer,is closely related to CD8+T cell immune infiltration and immune suppression.We constructed a CD8+T cells related risk score model to pre-dict the prognosis of HCC patients and provided therapeutic guidance based on the risk score.Using integrated bulk RNA sequencing(RNA-seq)and single-cell RNA sequencing(scRNA-seq)datasets,we identified stable CD8+T cell signatures.Based on these signatures,a 3-gene risk score model,comprised of KLRB1,RGS2,and TN-FRSF1B was constructed.The risk score model was well validated through an independent external validation co-hort.We divided patients into high-risk and low-risk groups according to the risk score and compared the differ-ences in immune microenvironment between these two groups.Compared with low-risk patients,high-risk patients have higher M2-type macrophage content(P＜0.0001)and lower CD8+T cells infiltration(P＜0.0001).High-risk patients predict worse response to immunotherapy treatment than low-risk patients(P＜0.01).Drug sensitivity a-nalysis shows that PI3K-β inhibitor AZD6482 and TGFβRII inhibitor SB505124 may be suitable therapies for high-risk patients,while the IGF-1R inhibitor BMS-754807 or the novel pyrimidine-based anti-tumor metabolic drug Gemcitabine could be potential therapeutic choices for low-risk patients.Moreover,expression of these 3-gene mod-el was verified by immunohistochemistry.In summary,the establishment and validation of a CD8+T cell-derived risk model can more accurately predict the prognosis of HCC patients and guide the construction of personalized treatment plans.

AIM To study the chemical constituents from ethyl acetate fraction of Balanophora harlandii Hook.f.and their tyrosinase inhibitory activity.METHODS Separation and purification were performed using silica gel,MCI,ODS,Sephadex LH-20 and semi-preparative HPLC,then the structures of obtained compounds were identified by physicochemical properties and spectral data.The monophenolase inhibitory activity was determined by the tyrosinase-catalyzed oxidation of L-tyrosine.RESULTS Twenty-four compounds were isolated and identified as sesamin(1),methyl caffeate(2),quercetin(3),5,7-dihydroxychromanone(4),methyl 3,4-dihydroxybenzoate(5),esculetin(6),kaempferol(7),naringenin(8),pyrogallic acid(9),pinosylvin(10),methyl propionate(11),caffeic acid(12),saccharinol(13),ferulic acid(14),trans-p-hydroxycinnamic acid(15),cinnamic acid(16),vanillic acid(17),vanillin(18),4-hydroxyacetophenone(19),4-hydroxybenzaldehyde(20),apigenin(21),(-)-isolariciresinol(22),(-)-secoisolariciresinol(23)and meso-2,3-di(3′,4′-methylenedioxybenzyl)butane-1,4-diol(24).The IC50 values of compounds 3,5,7,8,19,and 20 ranged from(0.246 5±0.028 3)to(1.278 2±0.021 3)mmol/L.CONCLUSION Compounds 1-9、11、15、17-21、24 are isolated from this plant for the first time,and 1,6,9,17-19,24 are first isolated from genus Balanophora.Compounds 3、5、7、8、19 and 20 have tyrosinase inhibitory activity.

To construct a safe and effective multi-epitope vaccine against the S1 protein of chicken infectious bronchitis virus(IBV).In this study,homologous and non-homologous dominant epitopes of IBV M41,T,QX and H120 virulent strain S1 proteins were screened by various online bioprediction software,respectively,and a new peptide W with high immunogenicity was construc-ted by connecting the screened B-cell and T-cell epitopes with a linker peptide.W was ligated to the truncated sequence of the four viral strains by T2A yietding to the eukaryotic expression vector pEGFP-N1,and it was identified by PCR and double digestion,the obtained recombinant plasmid was transfected into HEK293A cells and target protein expression was measured by Western blot.The constructed plasmid was injected intramuscularly twice to detect the antibody level,cytokine level,and peripheral blood T cell subsets were detected after two immunizations.The epitope pro-tein W was successfully constructed,which was structurally stable,antigenic,and soluble;the re-combinant plasmid pEGFP-WMQtH,pEGFP-W,and pEGFP-MQtH matched the expected size;anti-IBV IgG antibody levels in pEGFP-N1 was increased greatly compared to the PBS group.cyto-kines IL-2,and γ interferon(IFN-γ)were increased greatly(P＜0.05);peripheral blood CD4+/CD8a value(P＜0.05)was increased greatly.The W epitope protein was successfully constructed,which can effectively activate the humoral immunity and cellular immunity against four infectious bronchitis viruses(IBV),laying a foundation for the development of an effective vaccine against IB.

High-performance liquid chromatography(HPLC) was used to determine the content of three major components in Citri Reticulatae Semen(CRS), including limonin, nomilin, and obacunone. The chromaticity of the CRS sample during salt processing and stir-frying was measured using a color difference meter. Next, the relationship between the color and content of the salt-processed CRS sample was investigated through correlation analysis. By integrating the oil bath technique for processing simulation with HPLC, the changes in the relative content of nomilin and its transformation products were analyzed, with its structural transformation pattern during processing identified. Additionally, RAW264.7 cells were induced with lipopolysaccharides(LPSs) to establish an inflammatory model, and the anti-inflammatory activity of nomilin and its transformation product, namely obacunone was evaluated. The results indicated that as processing progressed, E~*ab and L~* values showed a downward trend; a~* values exhibited a slow increase over a certain period, followed by no significant changes, and b~* values remained stable with no significant changes over a certain period and then started to decrease. The limonin content remained barely unchanged; the nomilin content decreased, and the obacunone increased significantly. The changing trends in content and color parameters during salt-processing and stir-frying were basically consistent. The content of nomilin and obacunone was significantly correlated with the colorimetric values(L~*, a~*, b~*, and E~*ab), while limonin content showed no significant correlation with these values. By analyzing HPLC patterns of nomylin at different heating temperatures and time, it was found that under conditions of 200-250 ℃ for heating of 5-60 min, the content of nomilin significantly decreased, while the obacunone content increased pronouncedly. The in vitro anti-inflammatory activity results indicated that compared to the model group, the group with a high concentration of nomilin and the groups with varying concentrations of obacunone showed significantly reduced release of nitric oxide(NO)(P<0.01). When both were at the same concentration, obacunone showed better performance in inhibiting NO release. In this study, the obvious correlation between the color and content of major components during the processing of CRS samples was identified, and the dynamic patterns of quality change in CRS samples during processing were revealed. Additionally, the study revealed and confirmed the transformation of nomilin into obacunone during processing, with the in vitro anti-inflammatory activity of obacunone significantly greater than that of nomilin. These findings provided a scientific basis for CRS processing optimization, tablet quality control, and its clinical application.
Mice ; Animals ; Drugs, Chinese Herbal/pharmacology* ; RAW 264.7 Cells ; Limonins/chemistry* ; Chromatography, High Pressure Liquid ; Citrus/chemistry* ; Color ; Benzoxepins/chemistry* ; Anti-Inflammatory Agents/chemistry*