Barley Protein LFBEP-C1 from Lactiplantibacillus plantarum dy-1 Fermented Barley Extracts by Inhibiting Lipid Accumulation in a Caenorhabditis elegans Model

Objective This study aimed to investigate the lipid-lowering activity of LFBEP-C1 in high glucose-fed Caenorhabditis elegans(C.elegans).Methods In this study,the fermented barley protein LFBEP-C1 was prepared and tested for its potential anti-obesity effects on C.elegans.The worms were fed Escherichia coli OP50(E.coli OP50),glucose,and different concentrations of LFBEP-C1.Body size,lifespan,movement,triglyceride content,and gene expression were analyzed.The results were analyzed using ANOVA and Tukey's multiple comparison test.Results Compared with the model group,the head-swing frequency of C.elegans in the group of LFBEP-C1 at 20μg/mL increased by 33.88%,and the body-bending frequency increased by 27.09%.This indicated that LFBEP-C1 improved the locomotive ability of C.elegans.The average lifespan of C.elegans reached 13.55 days,and the body length and width of the C.elegans decreased after LFBEP-C1 intake.Additionally,LFBEP-C1 reduced the content of lipid accumulation and triglyceride levels.The expression levels of sbp-1,daf-2,and mdt-15 significantly decreased,while those of daf-16,tph-1,mod-1,and ser-4 significantly increased after LFBEP-C1 intake.Changes in these genes explain the signaling pathways that regulate lipid metabolism.Conclusion LFBEP-C1 significantly reduced lipid deposition in C.elegans fed a high-glucose diet and alleviated the adverse effects of a high-glucose diet on the development,lifespan,and exercise behavior of C.elegans.In addition,LFBEP-C1 regulated lipid metabolism mainly by mediating the expression of genes in the sterol regulatory element-binding protein,insulin,and 5-hydroxytryptamine signaling pathways.

In situ direct reprogramming of astrocytes to neurons via polypyrimidine tract-binding protein 1 knockdown in a mouse model of ischemic stroke

In situ direct reprogramming technology can directly convert endogenous glial cells into functional neurons in vivo for central nervous system repair. Polypyrimidine tract-binding protein 1(PTB) knockdown has been shown to reprogram astrocytes to functional neurons in situ. In this study, we used AAV-PHP.e B-GFAP-sh PTB to knockdown PTB in a mouse model of ischemic stroke induced by endothelin-1, and investigated the effects of GFAP-sh PTB-mediated direct reprogramming to neurons. Our results showed that in the mouse model of ischemic stroke, PTB knockdown effectively reprogrammed GFAP-positive cells to neurons in ischemic foci, restored neural tissue structure, reduced inflammatory response, and improved behavioral function. These findings validate the effectiveness of in situ transdifferentiation of astrocytes, and suggest that the approach may be a promising strategy for stroke treatment.

Low Selenium and Low Protein Exacerbate Myocardial Damage in Keshan Disease by Affecting the PINK1/Parkin-mediated Mitochondrial Autophagy Pathway

Objective Keshan disease(KD)is a myocardial mitochondrial disease closely related to insufficient selenium(Se)and protein intake.PTEN induced putative kinase 1(PINK1)/Parkin mediated mitochondrial autophagy regulates various physiological and pathological processes in the body.This study aimed to elucidate the relationship between PINK1/Parkin-regulated mitochondrial autophagy and KD-related myocardial injury.Methods A low Se and low protein animal model was established.One hundred Wistar rats were randomly divided into 5 groups(control group,low Se group,low protein group,low Se+low protein group,and corn from KD area group).The JC-1 method was used to detect the mitochondrial membrane potential(MMP).ELISA was used to detect serum creatine kinase MB(CK-MB),cardiac troponin I(cTnI),and mitochondrial-glutamicoxalacetic transaminase(M-GOT)levels.RT-PCR and Western blot analysis were used to detect the expression of PINK1,Parkin,sequestome 1(P62),and microtubule-associated proteins1A/1B light chain 3B(MAP1LC3B).Results The MMP was significantly decreased and the activity of CK-MB,cTnI,and M-GOT significantly increased in each experimental group(low Se group,low protein group,low Se+low protein group and corn from KD area group)compared with the control group(P<0.05 for all).The mRNA and protein expression levels of PINK1,Parkin and MAP1LC3B were profoundly increased,and those of P62 markedly decreased in the experimental groups compared with the control group(P<0.05 for all).Conclusion Low Se and low protein levels exacerbate myocardial damage in KD by affecting the PINK1/Parkin-mediated mitochondrial autophagy pathway.

C-reactive protein to albumin ratio predict responses to programmed cell death-1 inhibitors in hepatocellular carcinoma patients

BACKGROUND Over the years,programmed cell death-1(PD-1)inhibitors have been routinely used for hepatocellular carcinoma(HCC)treatment and yielded improved survival outcomes.Nonetheless,significant heterogeneity surrounds the outcomes of most studies.Therefore,it is critical to search for biomarkers that predict the efficacy of PD-1 inhibitors in patients with HCC.AIM To investigate the role of the C-reactive protein to albumin ratio(CAR)in evaluating the efficacy of PD-1 inhibitors for HCC.METHODS The clinical data of 160 patients with HCC treated with PD-1 inhibitors from January 2018 to November 2022 at the First Affiliated Hospital of Guangxi Medical University were retrospectively analyzed.RESULTS The optimal cut-off value for CAR based on progression-free survival(PFS)was determined to be 1.20 using x-tile software.Cox proportional risk model was used to determine the factors affecting prognosis.Eastern Cooperative Oncology Group performance status[hazard ratio(HR)=1.754,95%confidence interval(95%CI)=1.045-2.944,P=0.033],CAR(HR=2.118,95%CI=1.057-4.243,P=0.034)and tumor number(HR=2.932,95%CI=1.246-6.897,P=0.014)were independent prognostic factors for overall survival.CAR(HR=2.730,95%CI=1.502-4.961,P=0.001),tumor number(HR=1.584,95%CI=1.003-2.500,P=0.048)and neutrophil to lymphocyte ratio(HR=1.120,95%CI=1.022-1.228,P=0.015)were independent prognostic factors for PFS.Two nomograms were constructed based on independent prognostic factors.The C-index index and calibration plots confirmed that the nomogram is a reliable risk prediction tool.The ROC curve and decision curve analysis confirmed that the nomogram has a good predictive effect as well as a net clinical benefit.CONCLUSION Overall,we reveal that the CAR is a potential predictor of short-and long-term prognosis in patients with HCC treated with PD-1 inhibitors.If further verified,CAR-based nomogram may increase the number of markers that predict individualized prognosis.

Polycytosine RNA-binding protein 1 regulates osteoblast function via a ferroptosis pathway in type 2 diabetic osteoporosis

BACKGROUND Recently,type 2 diabetic osteoporosis(T2DOP)has become a research hotspot for the complications of diabetes,but the specific mechanism of its occurrence and development remains unknown.Ferroptosis caused by iron overload is con-sidered an important cause of T2DOP.Polycytosine RNA-binding protein 1(PCBP1),an iron ion chaperone,is considered a protector of ferroptosis.AIM To investigate the existence of ferroptosis and specific role of PCBP1 in the development of type 2 diabetes.METHODS A cell counting kit-8 assay was used to detect changes in osteoblast viability under high glucose(HG)and/or ferroptosis inhibitors at different concentrations and times.Transmission electron microscopy was used to examine the morpho-logical changes in the mitochondria of osteoblasts under HG,and western blotting was used to detect the expression levels of PCBP1,ferritin,and the ferroptosis-related protein glutathione peroxidase 4(GPX4).A lentivirus silenced and overex-pressed PCBP1.Western blotting was used to detect the expression levels of the osteoblast functional proteins osteoprotegerin(OPG)and osteocalcin(OCN),whereas flow cytometry was used to detect changes in reactive oxygen species(ROS)levels in each group.RESULTS Under HG,the viability of osteoblasts was considerably decreased,the number of mitochondria undergoing atrophy was considerably increased,PCBP1 and ferritin expression levels were increased,and GPX4 expression was decreased.Western blotting results demonstrated that infection with lentivirus overexpressing PCBP1,increased the expression levels of ferritin,GPX4,OPG,and OCN,compared with the HG group.Flow cytometry results showed a reduction in ROS,and an opposite result was obtained after silencing PCBP1.CONCLUSION PCBP1 may protect osteoblasts and reduce the harm caused by ferroptosis by promoting ferritin expression under a HG environment.Moreover,PCBP1 may be a potential therapeutic target for T2DOP.

AAV mediated carboxyl terminus of Hsp70 interacting protein overexpression mitigates the cognitive and pathological phenotypes of APP/PS1 mice

The E3 ubiquitin ligase,carboxyl terminus of heat shock protein 70(Hsp70)interacting protein(CHIP),also functions as a co-chaperone and plays a crucial role in the protein quality control system.In this study,we aimed to investigate the neuroprotective effect of overexpressed CHIP on Alzheimer’s disease.We used an adeno-associated virus vector that can cross the blood-brain barrier to mediate CHIP overexpression in APP/PS1 mouse brain.CHIP overexpression significantly ameliorated the performance of APP/PS1 mice in the Morris water maze and nest building tests,reduced amyloid-βplaques,and decreased the expression of both amyloid-βand phosphorylated tau.CHIP also alleviated the concentration of microglia and astrocytes around plaques.In APP/PS1 mice of a younger age,CHIP overexpression promoted an increase in ADAM10 expression and inhibitedβ-site APP cleaving enzyme 1,insulin degrading enzyme,and neprilysin expression.Levels of HSP70 and HSP40,which have functional relevance to CHIP,were also increased.Single nuclei transcriptome sequencing in the hippocampus of CHIP overexpressed mice showed that the lysosomal pathway and oligodendrocyte-related biological processes were up-regulated,which may also reflect a potential mechanism for the neuroprotective effect of CHIP.Our research shows that CHIP effectively reduces the behavior and pathological manifestations of APP/PS1 mice.Indeed,overexpression of CHIP could be a beneficial approach for the treatment of Alzheimer’s disease.

Protein-protein Interaction Between Domains of PDZ and BAR from PICK1

Two DNA fragments encoding PDZ domain （21-110 residues） and BAR domain （ 150-360 residues） from PICK1 （1-416 residues） were amplified by PCR and then introduced into vectors, pET-32M and pMAL-e2X respectively to generate recombinant plasmids, pE-pdz and pM-bar. Having been separately transferred into the hosts E. coli BL21 and E. coli JM109, these two strains can express fusion proteins： His-tagged PDZ（PDZ domain） and maltose binding protein-BAR（ MBP-BAR domain） respectively, as confirmed by both SDS-PAGE and Wostem blotting. The interaction between these two domains is dose-dependence, as identified by a pull-down test. Moreover, it has been shown from the ELISA analysis that the actual amount of PDZ bound to MBP-BAR-amylose beads reaches （ 16 ± 0. 5）%, as calculated by the molar ratio of PDZ to MBP-BAR. In addition, the interaction between BAR（bait） and PDZ（prey） in vivo was also examined with a yeast two-hybrid system.

MMP15、PICK1基因在山羊睾丸中的表达研究

MMP15、PICK1基因均是哺乳动物性腺组织中影响性腺生理功能正常发挥的重要影响因子,但它们在黔北麻羊睾丸组织中的表达规律尚不明晰。为研究黔北麻羊不同月龄的睾丸中MMP15、PICK1基因的表达情况,本试验以黔北麻羊为研究对象,通过qRT-PCR技术分别检测MMP15与PICK1基因在1、3、6、9、12月龄的黔北麻羊睾丸中的表达水平并建立基因表达的标准曲线。结果显示:MMP15、PICK1基因在黔北麻羊不同月龄睾丸组织中均有表达;MMP15基因在黔北麻羊不同月龄睾丸中的表达量从高到低排列为:12月龄>1月龄>9月龄>3月龄>6月龄;12月龄与1月龄的表达差异不显著(P>0.05),二者表达量均极显著高于3月龄、6月龄、9月龄(P<0.01);9月龄表达显著高于3月龄(P<0.05),极显著高于6月龄(P<0.01);3月龄表达极显著高于6月龄(P<0.01)。PICK1基因在黔北麻羊不同月龄睾丸中的表达量从高到低排列分别是:3月龄>6月龄>12月龄>9月龄>1月龄;3、6月龄其表达量极显著高于9、12月龄(P<0.01);1月龄与其他几个月龄相比,其表达量均极显著低于其他月龄(P<0.01)。结合黔北麻羊的生理机能发育情况,黔北麻羊在6月龄时已经基本达到性成熟,所以此时睾丸内细胞活动增强,睾丸机能活跃。本次试验结果表明,黔北麻羊睾丸生精功能的正常发挥或与MMP15及PICK1基因的表达量有关。

Calcitriol attenuates liver fibrosis through hepatitis C virus nonstructural protein 3-transactivated protein 1-mediated TGF β1/Smad3 and NF-κB signaling pathways

BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.

Truncating PICK1 Variant Identified in Azoospermia Affected Mitochondrial Dysfunction in Knockout Mice

Objective The protein interacting with C kinase 1(PICK1)plays a critical role in vesicle trafficking,and its deficiency in sperm cells results in abnormal vesicle trafficking from Golgi to acrosome,which eventually disrupts acrosome formation and leads to male infertility.Methods An azoospermia sample was filtered,and the laboratory detection and clinical phenotype indicated typical azoospermia in the patient.We sequenced all of the exons in the PICK1 gene and found that there was a novel homozygous variant in the PICK1 gene,c.364delA(p.Lys122SerfsX8),and this protein structure truncating variant seriously affected the biological function.Then we constructed a PICK1 knockout mouse model using clustered regularly interspaced short palindromic repeat cutting technology(CRISPRc).Results The sperm from PICK1 knockout mice showed acrosome and nucleus abnormalities,as well as dysfunctional mitochondrial sheath formation.Both the total sperm and motility sperm counts were decreased in the PICK1 knockout mice compared to wild-type mice.Moreover,the mitochondrial dysfunction was verified in the mice.These defects in the male PICK1 knockout mice may have eventually led to complete infertility.Conclusion The c.364delA novel variant in the PICK1 gene associated with clinical infertility,and pathogenic variants in the PICK1 may cause azoospermia or asthenospermia by impairing mitochondrial function in both mice and humans.

Transcription factor glucocorticoid modulatory element-binding protein 1 promotes hepatocellular carcinoma progression by activating Yes-associate protein 1

BACKGROUND Glucocorticoid modulatory element-binding protein 1(GMEB1),which has been identified as a transcription factor,is a protein widely expressed in various tissues.Reportedly,the dysregulation of GMEB1 is linked to the genesis and development of multiple cancers.AIM To explore GMEB1’s biological functions in hepatocellular carcinoma(HCC)and figuring out the molecular mechanism.METHODS GMEB1 expression in HCC tissues was analyzed employing the StarBase database.Immunohistochemical staining,Western blotting and quantitative realtime PCR were conducted to examine GMEB1 and Yes-associate protein 1(YAP1)expression in HCC cells and tissues.Cell counting kit-8 assay,Transwell assay and flow cytometry were utilized to examine HCC cell proliferation,migration,invasion and apoptosis,respectively.The JASPAR database was employed for predicting the binding site of GMEB1 with YAP1 promoter.Dual-luciferase reporter gene assay and chromatin immunoprecipitation-qPCR were conducted to verify the binding relationship of GMEB1 with YAP1 promoter region.RESULTS GMEB1 was up-regulated in HCC cells and tissues,and GMEB1 expression was correlated to the tumor size and TNM stage of HCC patients.GMEB1 overexpression facilitated HCC cell multiplication,migration,and invasion,and suppressed the apoptosis,whereas GMEB1 knockdown had the opposite effects.GMEB1 bound to YAP1 promoter region and positively regulated YAP1 expression in HCC cells.CONCLUSION GMEB1 facilitates HCC malignant proliferation and metastasis by promoting the transcription of the YAP1 promoter region.

Death-associated protein kinase 1 is associated with cognitive dysfunction in major depressive disorder

We previously showed that death-associated protein kinase 1(DAPK1)expression is increased in hippocampal tissue in a mouse model of major depressive disorde and is related to cognitive dysfunction in Alzheimer's disease.In addition,depression is a risk factor for developing Alzheimer's disease,as well as an early clinical manifestation of Alzheimer's disease.Meanwhile,cognitive dysfunction is a distinctive feature of major depressive disorder.Therefore,DAPK1 may be related to cognitive dysfunction in major depressive disorder.In this study,we established a mouse model of major depressive disorder by housing mice individually and exposing them to chronic,mild,unpredictable stressors.We found that DAPK1 and tau protein levels were increased in the hippocampal CA3 area,and tau was hyperphosphorylated at Thr231,Ser262,and Ser396 in these mice.Furthermore,DAPK1 shifted from axonal expression to overexpression on the cell membrane.Exercise and treatment with the antidepressant drug citalopram decreased DAPK1 expression and tau protein phosphorylation in hippocampal tissue and improved both depressive symptoms and cognitive dysfunction.These results indicate that DAPK1 may be a potential reason and therapeutic target of cognitive dysfunction in major depressive disorder.

Calcium/calmodulin modulates salt responses by binding a novel interacting protein SAMS1 in peanut(Arachis hypogaea L.)

The Ca^(2+)/CaM signal transduction pathway helps plants adapt to environmental stress. However, our knowledge on the functional proteins of C^(2+)/CaM pathway in peanut(Arachis hypogeae L.) remains limited. In the present study, a novel calmodulin 4(CaM4)-binding protein S-adenosyl-methionine synthetase 1(SAMS1) in peanut was identified using a yeast two-hybrid assay. Expression of AhSAMS1was induced by Ca^(2+), ABA, and salt stress. To elucidate the function of AhSAMS1, physiological and phenotypic analyses were performed with wild-type and transgenic materials. Overexpression of AhSAMS1increased spermidine and spermidine synthesis while decreased the contents of ethylene, thereby eliminating excessive reactive oxygen species(ROS) in transgenic lines under salt stress. AhSAMS1 reduced uptake of Na+and leakage of K+from mesophyll cells, and was less sensitive to salt stress during early seedling growth, in agreement with the induction of SOS and NHX genes Transcriptomics combined with epigenetic regulation uncovered relationships between differentially expressed genes and differentially methylated regions, which raised the salt tolerance and plants growth. Our findings support a model in which the role of AhSAMS1 in the ROS-dependent regulation of ion homeostasis was enhanced by Ca^(2+)/CaM while AhSAMS1-induced methylation was regulated by CaM, thus providing a new strategy for increasing the tolerance of plants to salt stress.

A pilot study of the relative number of circulating tumor cells and leukocytes containing actin-binding proteins in head and neck cancer patients

Circulating tumor cells(CTCs)play an important role in tumor metastases,which is positively correlated with an increased risk of death.Actin-binding proteins,including cofilin(CFL1),profilin 1(PFN1),and adenylate cyclase-associated protein 1(CAP1),are thought to be involved in tumor cell motility and metastasis,specifically in head and neck squamous cell carcinoma(HNSCC).However,currently,there are no published studies on CFL1,PFN1,and CAP1 in CTCs and leukocytes in HNSCC patients.We assessed serum levels of CFL1,PFN1,and CAP1 and the number of CTCs and leukocytes containing these proteins in blood from 31 HNSCC patients(T1-4N0-2M0).The analysis used flow cytometry and an enzyme-linked immunosorbent assay kit.We found that CAP1+CTCs and CAP1+leukocyte subpopulations were prevalent in these HNSCC patient samples,while the prevalence rates of CFL1+and PFN1+CTCs were relatively low.Patients with stage T2-4N1-2M0 had CFL1+and PFN1+CTCs with an elevated PFN1 serum level,compared with the T1-3N0M0 group.In summary,the PFN1 serum level and the relative number of PFN1+CD326+CTCs could be valuable prognostic markers for HNSCC metastases.The current study is the first to obtain data regarding the contents of actin-binding proteins(ABPs)in CTCs,and leukocytes in blood from HNSCC patients.This is also the first to assess the relationship between the number of CTCs subgroups and disease characteristics.

Photoprotective Effects of D1 Protein Turnover and the Lutein Cycle on Three Ephemeral Plants under Heat Stress

To clarify the characteristics of photoinhibition and the primary defense mechanisms of ephemeral plant leaves against photodestruction under high temperature stress,inhibitors and the technology to determine chlorophyll fluorescence were used to explore the protective effects of D1 protein turnover and the lutein cycle in the high temperature stress of the leaves of three ephemeral plants.The results showed that the maximum light conversion efficiency(Fv/Fm)of the ephemeral plant leaves decreased,and the initial fluorescence(Fo)increased under 35℃±1℃ heat stress for 1-4 h or on sunny days in the summer.Both Fv/Fm and Fo could be recovered after 8 h of darkness or afternoon weakening of the external temperature.Streptomycin sulfate(SM)or dithiothreitol(DTT)accelerated the decrease of Fv/Fm and the photochemical quenching coefficient(qP)in the leaves of three ephemeral plants at high temperature,and the decrease was greater in the SM than in the DTT treatment.When the high temperature stress was prolonged,the Y(II)values of light energy distribution parameters of PSII decreased,and the Y(NPQ)and Y(NO)values increased gradually in all the treatment groups of the three ephemeral plants.The results showed that the leaves of the three ephemeral plants had their own highly advanced mechanisms to protect against photodamage,which inhibited the turnover of D1 protein and xanthophyll cycle.This can damage the PSII reaction center in the leaves of the three ephemeral plants under high temperature.The protective effect of D1 protein turnover on heat stress in Erodium oxyrrhynchum and Senecio subdentatus was greater than that of the lutein cycle,while the protective effect of lutein cycle was greater than that of D1 protein turnover in Heliotropium acutiflorum subjected to heat damage.

Protein Profiles of Pod Borer Maruca Resistant Transgenic Cowpea

The grain legume cowpea Vigna unguiculata (L.) Walp. is a major protein source used for food and feed in Sub-Saharan Africa. The crop is affected by the pod borer Maruca vitrata against which transgenic lines were developed as part of the genetic control approach. This study aimed to assess the protein profiles in seeds and leaves of transgenic cowpea lines and their non-transgenic near-isogenic counterparts. Crude protein content was determined by the Kjeldahl method, and soluble proteins were quantified using Bradford dye binding assay. The average crude protein content ranged between 21.61% and 26.58% in the seeds and between 10.86% and 17.90% in the leaves. Total solubility varied between 13.03% and 20.64%. Osborne’s protein fractions contents in the seeds were 52.41% - 69.52% (albumin), 4.62% - 7.19% (globulin), 7.95% - 11.40% (glutelin) and 3% - 4% (prolamin). In any case, protein content differed significantly between cowpea genotypes but not between pairs of transgenic/non-transgenic lines. Insecticidal Cry1Ab protein expressed by transgenic lines was only detected in the albumin and globulin fractions. Altogether, these findings enhance our understanding of the effects of genetic modification on cowpea protein content and composition, with potential implications for nutritional and safety assessments.

Deleted in liver cancer 1 suppresses the growth of prostate cancer cells through inhibiting Rho-associated protein kinase pathway

Objective:Deleted in liver cancer 1(DLC1)is a GTPase-activating protein that is reported as a suppressor in certain human cancers.However,the detailed biological function of DLC1 is still unclear in human prostate cancer(PCa).In the present study,we aimed to explore the function of DLC1 in PCa cells.Methods:Silencing and overexpression of DLC1 were induced in an androgen-sensitive PCa cell line(LNCaP)using RNA interference and lentiviral vector transduction.The Cell Counting Kit-8 assay was performed to determine cell proliferation.The cell cycle was examined by performing a propidium iodide staining assay.Results:Our results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of LNCaP cells.Moreover,DLC1 expression was negatively correlated with Rho-associated protein kinase(ROCK)expression in LNCaP cells.Importantly,this study showed that the ROCK inhibitor Y27632 restored the function of DLC1 in LNCaP cells and reduced the tumorigenicity of LNCaP cells in vivo.Conclusion:Our results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of PCa cells and negatively correlated with ROCK expression in PCa cells and tissue.

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.

Effectiveness of conjunctival bleb scarring by knockdown of heat shock protein 47 in rat model

AIM:To evaluate the effectiveness of knock-down of heat shock protein 47(HSP47)on conjunctival bleb scarring in a rat model and its possible mechanism.METHODS:Male Sprague–Dawley rats were used for glaucoma filtration surgery(GFS)and were treated with either phosphate buffered solution,shControl,mitomycin C,or sh-HSP47 using a microsyringe immediately after GFS.The morphology of filtering blebs was observed postoperatively.The levels of HSP47 were analyzed at 2,5,8,and 11d after GFS via real‑time quantitative polymerase chain reaction(PCR)and Western blot.The silencing effect of HSP47,the expression of collagen I and III,and the potential signaling pathways of HSP47 during scarification were explored 11d post GFS.The protein levels of transforming growth factor-β1(TGF-β1),phospho-Smad2(pSmad2),phospho-Smad3(p-Smad3),and phospho-p38(p-p38)were also analyzed using Western blot.RESULTS:Sh-HSP47 treatment significantly prolonged the functional filtration bleb retention.The levels of HSP47 were increased significantly at 5,8,and 11d postoperatively compared to the control group(P<0.05,P<0.01,and P<0.001).The levels of HSP47 protein at day 11 postoperatively were significantly down-regulated after HSP47 silencing using sh-HSP47 adenovirus transfection(P<0.01).Expression levels of collagen I and III within the blebs were significantly reduced in the absence of HSP47(P<0.01).Moreover,the protein levels of TGF-β1,p-Smad2/3,and p-p38 were dramatically inhibited after treatment with sh-HSP47(P<0.01).CONCLUSION:The inhibitory effects of HSP47 knockdown on scarring after GFS have the potential to be an efficacious therapeutic option for the treatment of conjunctival bleb scarring.

Changes and significance of serum ubiquitin carboxyl-terminal hydrolase L1 and glial fibrillary acidic protein in patients with glioma

BACKGROUND Brain gliomas are malignant tumors with high postoperative recurrence rates.Early prediction of prognosis using specific indicators is of great significance.AIM To assess changes in ubiquitin carboxy-terminal hydrolase L1(UCH-L1)and glial fibrillary acidic protein(GFAP)levels in patients with glioma pre-and postoperatively.METHODS Between June 2018 and June 2021,91 patients with gliomas who underwent surgery at our hospital were enrolled in the glioma group.Sixty healthy volunteers were included in the control group.Serum UCH-L1 and GFAP levels were measured in peripheral blood collected from patients with glioma before and 3 d after surgery.UCH-L1 and GFAP levels in patients with glioma with different clinicopathological characteristics were compared before and after surgery.The patients were followed-up until February 2022.Postoperative glioma recurrence was recorded to determine the serum UCH-L1 and GFAP levels,which could assist in predicting postoperative glioma recurrence.RESULTS UCH-L1 and GFAP levels in patients with glioma decreased significantly 3 d after surgery compared to those before therapy(P<0.05).However,UCH-L1 and GFAP levels in the glioma group were significantly higher than those in the control group before and after surgery(P<0.05).There were no statistically significant differences in preoperative serum UCH-L1 and GFAP levels among patients with glioma according to sex,age,pathological type,tumor location,or number of lesions(P>0.05).Serum UCH-L1 and GFAP levels were significantly lower in the patients with WHO grade I-II tumors than in those with gradeⅢ-IV tumors(P<0.05).Serum UCH-L1 and GFAP levels were lower in the patients with tumor diameter≤5 cm than in those with diameter>5 cm,in which the differences were statistically significant(P<0.05).Glioma recurred in 22 patients.The preoperative and 3-d postoperative serum UCH-L1 and GFAP levels were significantly higher in the recurrence group than these in the non-recurrence group(P<0.05).Receiver operating characteristic curves were plotted.The areas under the curves of preoperative serum UCH-L1 and GFAP levels for predicting postoperative glioma recurrence were 0.785 and 0.775,respectively.However,the efficacy of serum UCH-L1 and GFAP levels 3 d after surgery in predicting postoperative glioma recurrence was slightly lower compared with their preoperative levels.CONCLUSION UCH-L1 and GFAP efficiently reflected the development and recurrence of gliomas and could be used as potential indicators for the recurrence and prognosis of glioma.