AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragm...AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.展开更多
Hepatitis C virus (HCV) infection and associated liver diseases are still challenging and represent a significant health care burden around the world. Although, the treatment strategies have been improved by the devel...Hepatitis C virus (HCV) infection and associated liver diseases are still challenging and represent a significant health care burden around the world. Although, the treatment strategies have been improved by the development of novel direct-acting antivirals, but such therapeutic options are still expensive and beyond the financial range of the most infected individuals in developing or even in resource replete countries. It demands an urgent need to search novel and improved alternate treatment strategies to treat the infection. The present study was aimed to develop an in vitro stable cell culture system, persistently expressing HCV genotype 1a non-structural genes and to characterize the inhibitory effects of synthetic siRNAs (short interference RNA) directed against the most conserved regions of nonstructural genes in an in vitro cell culture model. The continuous expression of nonstructural genes for more than 30 days post transfection was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis in stable human hepatoma cell line (Huh-7). The gene expression studies revealed significantly reduced gene expression of HCV nonstructural genes (i.e., NS2, NS4A and NS5A) both at mRNA and protein levels when treated against genome specific synthetic siRNAs in stable cell lines (51%, 47% and 54% respectively, p < 0.05). Similarly, a vivid decrease in HCV viral titer was exhibited by synthetic siRNAs in an in vitro viral replicate cell culture model (58%, 48% and 50%, respectively, p < 0.05) determined by quantitative Real-Time PCR (qPCR). Our data indicate that siRNA mediated gene silencing may be considered a promising alternate treatment strategy against HCV in combination with other effective therapeutic regimens in future.展开更多
FMDV non-structural protein gene（NSPs） 3ABC, 3AB, 2C and 3D were amplified and cloned into expression plasmid pET-32a（+）. The recombinant NSPs were produced in E.coli and purified using Ni2+ affinity column. Weste...FMDV non-structural protein gene（NSPs） 3ABC, 3AB, 2C and 3D were amplified and cloned into expression plasmid pET-32a（+）. The recombinant NSPs were produced in E.coli and purified using Ni2+ affinity column. Western-blotting indicated that the NSPs were expressed correctly. Using the recombinant NSPs, indirect ELISAs have been set up to distinguish FMDV-infected pigs from vaccinated ones. Experimental results indicate that the immunogenesity of recombinant 3AB protein is strong and can be the ideal antigen for detection; the immunogenecity of 2C is weak and sensitivity of the assay is low; 3D is not an ideal antigen ,for the specificity of assay based on 3D is low.展开更多
The awn can contribute to photosynthesis and carbohydrates,enhancing grain yield in wheat.We mapped QAwn.sxau-5A,a major QTL for awn development in wheat(Triticum aestivum).This QTL was delimited to a 994-kb interval ...The awn can contribute to photosynthesis and carbohydrates,enhancing grain yield in wheat.We mapped QAwn.sxau-5A,a major QTL for awn development in wheat(Triticum aestivum).This QTL was delimited to a 994-kb interval at the B1 locus on chromosome 5A,which included the candidate gene encoding a zinc finger protein(TraesCS5A01G542800)as an awn length inhibitor(ALI).The Ali-A1 allele for the awnless trait showed abundant sequence differences in the promoter regions compared to the ali-A1 allele for the long-awn trait.The results of the swap experiment on the promoters from the two ALI-A1 alleles showed that the two promoters caused a difference in the protein level,indicating the gene was regulated at the transcript level.However,the ali-A1 allele contained an SNP that caused a premature stop codon in its coding region,resulting in a truncated protein compared to the functional Ali-A1 protein.The Ali-A1 protein contained two ethylene-responsive element binding factor-associated amphiphilic repression(EAR)motifs,one at the N terminus(EAR-N)and the other at the C terminus(EAR-C),and they were involved in interactions with the wheat co-repressor protein TOPLESS(TPL1).The ali-A1 protein retained the EAR-N motif but lost the EAR-C motif,resulting in the attenuated ability to interact with TPL1.The tpl1 mutant produced a longer awn compared to the wild type.Ali-A1 repressed the transcription of two downstream genes,TaLRP-A1 and TaARF-B1,involved in endogenous auxin concentrations and auxin responses in wheat.We concluded that the awn length is regulated not only by the ALI-A1 gene at transcript levels but also by Ali-A1 and TPL1 at the protein level in wheat.展开更多
BACKGROUND Ovarian cancer(OC)is the most lethal gynecological cancer among females,and its early diagnosis could help for better outcomes of the patients.AIM To investigate the utility of serum insulin-like growth fac...BACKGROUND Ovarian cancer(OC)is the most lethal gynecological cancer among females,and its early diagnosis could help for better outcomes of the patients.AIM To investigate the utility of serum insulin-like growth factors-binding proteins 2(IGFBP2),secreted phosphoprotein 1(SPP1),thrombospondin 1 protein(TSP1)and D-dimer levels in addition to currently used biomarkers[cancer antigen 125(CA125)and human epididymis protein 4(HE4)]in the diagnosis of epithelial OC(EOC).METHODS This is a case-control study that included fifty females diagnosed with EOC,10 females with benign ovarian masses recruited from the Egyptian National Cancer Institute,and 30 healthy females as a control group.All subjects were assessed for serum HE4,CA125,IGFBP2,TSP1 and SPP1 measurement by enzyme-linkedimmunosorbent assay.RESULTS There was a statistically significant difference in serum levels between EOC,benign ovarian masses,and healthy control groups regarding CA125 and SPP1(P<0.001 for both markers),while HE4 and IGFBP2 increased significantly in EOC compared to healthy control groups(P<0.001 for all markers)with no significant difference between EOC and benign ovarian masses groups.However,there was no statistically significant difference among EOC,benign ovarian masses,and healthy control groups regarding the TSP1 serum levels(P=0.051).Receiver operating characteristic analysis revealed that combined assessment of SPP1 with CA125 or TSP1 increased the diagnosis of EOC patients to a sensitivity,specificity,and area under curve of(93.3%,100%,0.968;respectively,P<0.001).CONCLUSION SPP1 may be a potential marker for the differentiation between benign and malignant ovarian masses,while IGFBP2 can differentiate between healthy females and females with ovarian masses.Combining SPP1 with CA125 or TSP1 provides high sensitivity and specificity for the detection of EOC patients.展开更多
Intrinsically disordered proteins(IDPs)and their regions(IDRs)play crucial roles in cellular func-tions despite their lack of stable three-dimensional structures.In this study,we investigate the interac-tions between ...Intrinsically disordered proteins(IDPs)and their regions(IDRs)play crucial roles in cellular func-tions despite their lack of stable three-dimensional structures.In this study,we investigate the interac-tions between the C-terminal do-main of protein 4.1G(4.1G CTD)and the nuclear mitotic apparatus protein(NuMA)under varying pH and salt ion conditions to under-stand the regulatory mechanisms affecting their binding.4.1G CTD and NuMA bind effec-tively under neutral and alkaline conditions,but their interaction is disrupted under acidic conditions(pH 3.6).The protonation of positively charged residues at the C-terminal of 4.1G CTD under acidic conditions leads to increased electrostatic repulsion,weakening the overall binding free energy.Secondary structure analysis shows that specific regions of 4.1G CTD re-main stable under both pH conditions,but the C-terminal region(aa 990−1000)and the N-terminal region of NuMA(aa 1800−1810)exhibit significant reductions in secondary struc-ture probability under acidic conditions.Contact map analysis and solvent-accessible surface area analysis further support these findings by showing a reduced contact probability be-tween these regions under pH 3.6.These results provide a comprehensive understanding of how pH and ionic strength regulate the binding dynamics of 4.1G CTD and NuMA,emphasiz-ing the regulatory role of electrostatic interactions.展开更多
Introduction:Diet intervention,especially supplementation with high-quality protein,is considered to be a critical strategy in sarcopenia.However,different sources and types of protein have different health impacts.Ob...Introduction:Diet intervention,especially supplementation with high-quality protein,is considered to be a critical strategy in sarcopenia.However,different sources and types of protein have different health impacts.Objectives:The aim of this study is to explore the differences in the ameliorative effects and mechanisms of different sources and types of proteins on sarcopenia,providing an optimal path for the prevention and treatment of sarcopenia.Methods:A sarcopenia model was established by intraperitoneal injection of dexamethasone(5 mg/kg).Sixty male C57BL/6 mice(8 months old)were randomly divided into the normal control,sarcopenia,goat whey protein,goat milk casein,bovine whey protein,and bovine milk casein groups.Animals were treated for 8 consecutive weeks.Organism-level and molecular phenotypes,16S rRNA gene sequencing,and untargeted metabolomics profiling based on GC-TOF/MS were employed to investigate the correlation between host metabolism,microbial metabolism,autophagy and inflammation and their influence on sarcopenia in C57BL/6 male mice.Results:All 4 proteins increased muscle mass,and goat whey protein improved muscle strength in sarcopenic mice.Goat and bovine milk proteins promoted muscle regeneration by increasing MyoD1 and MyoG expression,and the former had a more distinct effect in inducing autophagy and decreasing inflammation than the latter.In addition,goat whey protein and casein could modulate hostmicrobial arginine co-metabolism.Notably,goat milk proteins responded well to sarcopenia comorbidities,including sarcopenic obesity,osteosarcopenia,and osteoarthritis.Conclusion:The study confirmed that goat milk proteins were more effective than bovine milk proteins for the control of sarcopenia.Moreover,we found that whey protein and casein could modulate host-microbial arginine co-metabolism,which shows their potential as precision nutritional supplements for the management of sarcopenia.Our study provides theoretical support for the prevention and control of sarcopenia.展开更多
This article summarizes recent advances in the understanding of RNA-binding proteins(RBPs),with a focus on their roles in exercise-induced mRNA regulation and their implications for schizophrenia(SZ).RBPs are critical...This article summarizes recent advances in the understanding of RNA-binding proteins(RBPs),with a focus on their roles in exercise-induced mRNA regulation and their implications for schizophrenia(SZ).RBPs are critical regulators of mRNA stability,splicing,transport,translation,and degradation,directly influencing gene expression through sequence-and structure-specific binding.In the nervous system,RBPs sustain synaptic plasticity,neural development,and neuronal homeostasis.Emerging evidence shows that exercise modulates the expression and activity of RBPs,thereby influencing mRNA translation and neurotransmitter signaling,which may underlie its beneficial effects on brain function.Dysregulation of specific RBPs has been identified in SZ,implicating them in disrupted synaptic transmission,impaired plasticity,and neuroinflammation.RBPs involved in memory and emotional regulation show marked dysfunction in SZ patients.Some RBPs have been proposed as potential biomarkers for early diagnosis and treatment monitoring.Moreover,therapeutic modulation of RBPs,through pharmacological or behavioral interventions such as exercise,may restore neuronal function by targeting post-transcriptional gene regulation.Exercise,as a non-invasive modulator of RBP expression,holds promise as an adjunctive strategy in SZ treatment,particularly in early stages.Further research into RBP-mediated pathways may offer novel insights into SZ pathophysiology and inform the development of targeted interventions.展开更多
Bromodomain(BRD)-containing proteins are central mediators of gene regulation,serving as key components of chromatin remodeling complexes and histone recognition scaffolds.By specifically recognizing acetylated lysine...Bromodomain(BRD)-containing proteins are central mediators of gene regulation,serving as key components of chromatin remodeling complexes and histone recognition scaffolds.By specifically recognizing acetylated lysine residues on histones(Kac)via their conserved BRD,these proteins influence chromatin structure and gene expression.Although their overarching role is well-established,the precise molecular functions and mechanisms of individual BRD proteins remain incompletely characterized.The ciliate Tetrahymena thermophila,a unicellular eukaryote with a transcriptionally active macronucleus enriched in histone acetylation,is an excellent model for exploring the significance of BRD-containing proteins.In this comprehensive review,all BRD-containing proteins encoded in the T.thermophila genome are systematically examined,including their expression profiles,histone acetylation targets,interacting proteins,and potential roles.This review lays the groundwork for future investigations into the complex roles of BRD proteins in chromatin remodeling and transcription regulation,offering insights into basic eukaryotic biology and the molecular mechanisms underlying BRD-linked diseases.展开更多
Chemical modification of native peptides and proteins is a versatile strategy to facilitate late-stage diversification for functional studies.Among the proteogenic amino acids,lysine is extensively involved in posttra...Chemical modification of native peptides and proteins is a versatile strategy to facilitate late-stage diversification for functional studies.Among the proteogenic amino acids,lysine is extensively involved in posttranslational modifications and the binding of ligands to target proteins,making its selective modification attractive.However,lysine’s high natural abundance and solvent accessibility,as well as its relatively low reactivity to cysteine,necessitate addressing chemoselectivity and regioselectivity for the Lys modification of native proteins.Although Lys chemoselective modification methods have been well developed,achieving site-selective modification of a specific Lys residue remains a great challenge.In this review,we discussed the challenges of Lys selective modification,presented recent examples of Lys chemoselective modification,and summarized the currently known methods and strategies for Lys site-selective modification.We also included an outlook on potential solutions for Lys site-selective labeling and its potential applications in chemical biology and drug development.展开更多
Meiotic resumption in mammalian oocytes involves nuclear and organelle structural changes,notably the chromatin configuration transition from a non-surrounding nucleolus(NSN)to surrounding nucleolus(SN)in germinal ves...Meiotic resumption in mammalian oocytes involves nuclear and organelle structural changes,notably the chromatin configuration transition from a non-surrounding nucleolus(NSN)to surrounding nucleolus(SN)in germinal vesicle oocytes.In the current study,we found that nuclear speckles(NSs),a subnuclear structure mainly composed of serine-arginine(SR)proteins,changed from a diffuse spotted distribution in mouse NSN oocytes to an aggregated pattern in SN oocytes.We also found that the SR protein-specific kinase 1(SRPK1),an enzyme that phosphorylates SR proteins,co-localized with NSs at the SN stage,and that NSN oocytes failed to transition to SN oocytes after the inhibition of SRPK1 activity.Furthermore,the typical structure of the chromatin ring around the nucleolus in SN oocytes collapsed after treatment with an SRPK1 inhibitor.Mechanistically,phosphorylated SR proteins were found to be related to chromatin as shown by a salt extraction experiment,and in situ DNaseⅠassay showed that the accessibility of chromatin was enhanced in SN oocytes when SRPK1 was inhibited,accompanied by a decreased repressive modification on histone and the abnormal recurrence of a transcriptional signal.In conclusion,our results indicated that SRPK1-regulated phosphorylation of SR proteins was involved in the NSN-SN transition and played an important role in maintaining the condensed nucleus of SN oocytes via interacting with chromatin.展开更多
Vesicles of lipid bilayer can adopt a variety of shapes due to different coating proteins.The ability of proteins to reshape membrane is typically characterized by inducing spontaneous curvature of the membrane at the...Vesicles of lipid bilayer can adopt a variety of shapes due to different coating proteins.The ability of proteins to reshape membrane is typically characterized by inducing spontaneous curvature of the membrane at the coated area.BAR family proteins are known to have a crescent shape and can induce membrane curvature along their concaved body axis but not in the perpendicular direction.We model this type of proteins as a rod-shaped molecule with an orientation and induce normal curvature along its orientation in the tangential plane of the membrane surface.We show how a ring of these proteins reshapes an axisymmetric vesicle when the protein curvature or orientation is varied.A discontinuous shape transformation from a protrusion shape without a neck to a one with a neck is found.Increasing the rigidity of the protein ring is able to smooth out the transition.Furthermore,we show that varying the protein orientation is able to induce an hourglass-shaped neck,which is significantly narrower than the reciprocal of the protein curvature.Our results offer a new angle to rationalize the helical structure formed by many proteins that carry out membrane fission functions.展开更多
Biomolecular condensates,also known as membraneless organelles,play a crucial role in cellular organization by concentrating or sequestering biomolecules.Despite their importance,synthetically mimicking these organell...Biomolecular condensates,also known as membraneless organelles,play a crucial role in cellular organization by concentrating or sequestering biomolecules.Despite their importance,synthetically mimicking these organelles using non-peptidic small organic molecules has posed a significant challenge.The present study reports the discovery of D008,a self-assembling small molecule that sequesters a unique subset of RNA-binding proteins.Analysis and screening of a comprehensive collection of approximately 1 million compounds in the Chinese National Compound Library(Shanghai)identified 44 self-assembling small molecules in aqueous solutions.Subsequent screening of the focused library,coupled with proteome analysis,led to the discovery of D008 as a small organic molecule with the ability to condensate a specific subset of RNA-binding proteins.In vitro experiments demonstrated that the D008-induced sequestration of RNA-binding proteins impeded mRNA translation.D008 may offer a unique opportunity for studying the condensations of RNA-binding proteins and for developing an unprecedented class of small molecules that control gene expression.展开更多
HCLS1-associated protein X-1(HAX1)is a multifunctional mitochondrial protein involved in the regulation of apoptosis,a crucial process of programmed cell death,and mRNA processing.Despite its significance,limited stru...HCLS1-associated protein X-1(HAX1)is a multifunctional mitochondrial protein involved in the regulation of apoptosis,a crucial process of programmed cell death,and mRNA processing.Despite its significance,limited structural data is available for HAX1,hindering a comprehensive understanding of its biological function.Notably,the caseinolytic mitochondrial matrix peptidase chaperone subunit B(CLPB)has been identified as an interacting partner of HAX1,yet the biophysical properties and binding affinity governing their interaction remain poorly defined.In this study,we present a thorough biophysical characterization of full-length human HAX1 and CLPB,accomplished through recombinant expression and purification.By employing size exclusion chromatography,dynamic light scattering,and circular dichroism spectroscopy,we successfully established their biophysical properties,revealing contrasting structural features,with CLPB displaying a-helical content and HAX1 exhibiting a disordered nature.Moreover,we employed solutionstate nuclear magnetic resonance(NMR)spectroscopy to probe their binding affinity.Our findings demonstrate the formation of stable multimeric complexes between HAX1 and CLPB,and we quantified a dissociation constant in the low range of micro-molar for their high affinity interaction.These results lay the foundation for further in-depth investigations into the dynamics and energetics governing the HAX1-CLPB interaction,ultimately contributing to a comprehensive understanding of their functional mechanisms.展开更多
The soil-resident pathogen, Plasmodiophora brassicae, infects cruciferous crops, causing obligate parasitic clubroot disease and posing a significant threat to the Brassica vegetable industry in China. To learn more a...The soil-resident pathogen, Plasmodiophora brassicae, infects cruciferous crops, causing obligate parasitic clubroot disease and posing a significant threat to the Brassica vegetable industry in China. To learn more about its pathogenesis, we reported a Nanopore sequencing-derived25.3 Mb high-quality genome sequence of P. brassicae pathotype 4 strain(P.b 4). Comparing the P.b 4 genome with that of the published P.brassicae e3 genome(P.b e3) identified single nucleotide polymorphisms, structural variations, and small insertions and deletions. We then carried out RNA-sequencing of root samples from a clubroot-susceptible line at 5, 14, and 28 days after inoculation(DAI), and classified genes into five categories based on their expression patterns. Interestingly, 158 genes were highly expressed at 14 DAI, which were enriched in budding cell isotropic bud growth, ascospore wall assembly, spore wall assembly, spore wall biogenesis, and ascospore wall biogenesis.Subsequently, we bioinformatically predicted 555 secreted effector candidates, among which only 125 were expressed during infection and had amino acid lengths less than 400. The putative effector Pb010018, which was highly expressed at 14 DAI, was validated to have a signal peptide using a yeast secretion system. Luciferase activity and co-immunoprecipitation assays demonstrated that Pb010018 interacts with serine hydroxymethyltransferase BrSHMT1, and expression analysis showed that SHMT1 was upregulated in both Arabidopsis and B. rapa during infection. Furthermore, after infection, the Arabidopsis shmt1 mutant(atshmt1) showed reduced severity of clubroot disease, together with downregulated expression of Pb010018. Our results offer new insights into plant-pathogen interaction mechanisms, and provide the possibility for improving Brassica resistance to clubroot disease.展开更多
Objective Recent studies have overturned the traditional concept of the lung as a “sterile organ” revealing that pulmonary microbiota dysbiosis and abnormal surfactant proteins(SPs) expression are involved in the pr...Objective Recent studies have overturned the traditional concept of the lung as a “sterile organ” revealing that pulmonary microbiota dysbiosis and abnormal surfactant proteins(SPs) expression are involved in the progression of silicosis. This study aimed to investigate the relationship between abnormal SPs expression and dysbiosis of lung microbiota in silica-induced lung fibrosis, providing insights into mechanisms of silicosis.Methods Lung pathology, SPs expression, and microbiota composition were evaluated in silicaexposed mice. A mouse model of antibiotic-induced microbiota depletion was established, and alveolar structure and SPs expression were assessed. The roles of the lung microbiota and SPs in silicosis progression were further evaluated in mice with antibiotic-induced microbiota depletion, both with and without silica exposure.Results Silica exposure induced lung inflammation and fibrosis, along with increased expression of SPA expression. Antibiotics(Abx)-induced microbiota depletion elevated SP-A and SP-D expression.Furthermore, silica exposure altered lung microbiota composition, enriching potentially pathogenic taxa.However, antibiotic-induced microbiota depletion prior to silica exposure reduced silica-mediated lung fibrosis and inflammation.Conclusion Lung microbiota is associated with silica-induced lung injury. Overproduction of SP-A and SP-D, induced by Abx-induced microbiota depletion, may enhance the resistance of mouse lung tissue to silica-induced injury.展开更多
Essential proteins are crucial for biological processes and can be identified through both experimental and computational methods.While experimental approaches are highly accurate,they often demand extensive time and ...Essential proteins are crucial for biological processes and can be identified through both experimental and computational methods.While experimental approaches are highly accurate,they often demand extensive time and resources.To address these challenges,we present a computational ensemble learning framework designed to identify essential proteins more efficiently.Our method begins by using node2vec to transform proteins in the protein–protein interaction(PPI)network into continuous,low-dimensional vectors.We also extract a range of features from protein sequences,including graph-theory-based,information-based,compositional,and physiochemical attributes.Additionally,we leverage deep learning techniques to analyze high-dimensional position-specific scoring matrices(PSSMs)and capture evolutionary information.We then combine these features for classification using various machine learning algorithms.To enhance performance,we integrate the outputs of these algorithms through ensemble methods such as voting,weighted averaging,and stacking.This approach effectively addresses data imbalances and improves both robustness and accuracy.Our ensemble learning framework achieves an AUC of 0.960 and an accuracy of 0.9252,outperforming other computational methods.These results demonstrate the effectiveness of our approach in accurately identifying essential proteins and highlight its superior feature extraction capabilities.展开更多
Tyrosine residues in proteins can be nitrated to form 3-nitrotyrosine(3-NT)under the influence of ozone(O_(3))and nitrogen dioxide(NO_(2))in the air,which may introduce health impacts.A selective and sensitive enzyme-...Tyrosine residues in proteins can be nitrated to form 3-nitrotyrosine(3-NT)under the influence of ozone(O_(3))and nitrogen dioxide(NO_(2))in the air,which may introduce health impacts.A selective and sensitive enzyme-linked-immunoassay(ELISA)method was developed to determine 3-NT in modified model protein(bovine serum albumin,BSA)and ambient aerosol samples.The nitration degrees(NDs)of BSA in the exposure experiments with different durations were detected by both the ELISA and spectrophotometric methods(i.e.,ND_(ELISA) and ND_(SEC-PDA)),which show good coincidence.The kinetic investigation by both ΔND_(ELISA) and ΔND_(SEC-PDA) in the exposure experiments shows that the rate coefficients(k)of the pseudo-first-order kinetic rate reactions of protein nitration were comparable.These results indicate that direct detection of 3-NT by the ELISA method can be applied for laboratory exposure samples analysis for kinetic studies.Based on the selective detection of 3-NT,ND_(ELISA) provides a promising measure for the assessment of ND in model proteins.3-NT was alsomeasured in PM_(2.5) samples in summer in Guangzhou,southern China,ranging from 10.1 to 404 pg/m^(3),providing clear evidence of protein nitration in ambient aerosols.We further proposed that 3-NT/protein can be used as a proxy to evaluate protein nitration in ambient aerosols.A significant correlationwas observed between 3-NT/protein and O_(3),confirming the crucial role of O_(3) in protein nitration.Our results show that the direct detection of 3-NT by the ELISA method can be more widely applied in the laboratory and field-based studies for understanding the mechanisms of protein nitration.展开更多
The distance distributions between two site-specifically anchored spin labels in a protein,measured by pulsed electron-electron double resonance(PELDOR or DEER),provide rich sources of structural and conformational re...The distance distributions between two site-specifically anchored spin labels in a protein,measured by pulsed electron-electron double resonance(PELDOR or DEER),provide rich sources of structural and conformational restraints on the proteins or their complexes.The rigid connection of the nitroxide spin label to the protein improves the accuracy and precision of distance measurement.We report a new spin labelling approach by formation of thioester bond between nitroxide(NO)spin label,NOAI(NO spin labels activated by acetylimidazole),and a protein thiol,and this spin labeling method has demonstrated high performance in DEER distance measurement on proteins.The results showed that NOAI has shorter connection to the protein ligation site than 2,2,5,5-tetramethyl-pyrroline-1-oxyl methanethiosulfonate(MTSL)and 3-maleimido-proxyl(M-Prox)in the respective protein conjugate and produces narrower distance distributions for the tested proteins including ubiquitin(Ub),immunoglobulin-binding b1 domain of streptococcal protein G(GB1),and second mitochondria-derived activator of caspases(Smac).The NOAI protein conjugate connected by a thioester bond is resistant to reducing reagent and offers highfidelity DEER distance measurements in cell lysates.展开更多
DNA imaging and visualization techniques are crucial in biological experiments and have also emerged as a powerful method for single-molecule studies.Traditional intercalating dyes(e.g.,SYTOX,EtBr,GelRed)can stain DNA...DNA imaging and visualization techniques are crucial in biological experiments and have also emerged as a powerful method for single-molecule studies.Traditional intercalating dyes(e.g.,SYTOX,EtBr,GelRed)can stain DNA but may alter its structure and mechanical properties,and cause photocleavage.Recently,a novel fluorescent DNA-binding protein(FP-DBP)was introduced,which can stain DNA without sequence preference and without inducing photocleavage.In this study,using a custom-built magnetic tweezers system,we performed DNA stretching,twisting and unzipping experiments to compare the mechanical properties of DNA with and without two kinds of intercalating dyes(SYTOX Orange and GelRed)and mCherry FP-DBP.Our results show that mCherry FP-DBP does not affect DNA structure or mechanics,unlike SYTOX Orange and GelRed,making FP-DBP a promising tool for DNA visualization in single-molecule experiments.展开更多
基金Supported by National 863 Project,No.102-07-02-079th Five-Year Sci-Tech Plan,No.96-906A-03-08
文摘AIM: To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnostic reagents. METHODS: Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or) pGEX, and expressed in E.coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins. RESULTS: One clone with HGV fragment from core to E1 (G1), one from E2 (G31), three from NS3 (G6, G61, G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and showed obvious immunoreactivity by Western blotting. One clone with HGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins G1, G31, G61, G821 and G61-821 were detected in indirected ELISA as coating antigen respectively. Only recombinant G1 could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification. CONCLUSION: Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high-yield recombinant protein (G1) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.
文摘Hepatitis C virus (HCV) infection and associated liver diseases are still challenging and represent a significant health care burden around the world. Although, the treatment strategies have been improved by the development of novel direct-acting antivirals, but such therapeutic options are still expensive and beyond the financial range of the most infected individuals in developing or even in resource replete countries. It demands an urgent need to search novel and improved alternate treatment strategies to treat the infection. The present study was aimed to develop an in vitro stable cell culture system, persistently expressing HCV genotype 1a non-structural genes and to characterize the inhibitory effects of synthetic siRNAs (short interference RNA) directed against the most conserved regions of nonstructural genes in an in vitro cell culture model. The continuous expression of nonstructural genes for more than 30 days post transfection was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis in stable human hepatoma cell line (Huh-7). The gene expression studies revealed significantly reduced gene expression of HCV nonstructural genes (i.e., NS2, NS4A and NS5A) both at mRNA and protein levels when treated against genome specific synthetic siRNAs in stable cell lines (51%, 47% and 54% respectively, p < 0.05). Similarly, a vivid decrease in HCV viral titer was exhibited by synthetic siRNAs in an in vitro viral replicate cell culture model (58%, 48% and 50%, respectively, p < 0.05) determined by quantitative Real-Time PCR (qPCR). Our data indicate that siRNA mediated gene silencing may be considered a promising alternate treatment strategy against HCV in combination with other effective therapeutic regimens in future.
文摘FMDV non-structural protein gene（NSPs） 3ABC, 3AB, 2C and 3D were amplified and cloned into expression plasmid pET-32a（+）. The recombinant NSPs were produced in E.coli and purified using Ni2+ affinity column. Western-blotting indicated that the NSPs were expressed correctly. Using the recombinant NSPs, indirect ELISAs have been set up to distinguish FMDV-infected pigs from vaccinated ones. Experimental results indicate that the immunogenesity of recombinant 3AB protein is strong and can be the ideal antigen for detection; the immunogenecity of 2C is weak and sensitivity of the assay is low; 3D is not an ideal antigen ,for the specificity of assay based on 3D is low.
基金supported by the Grand Science and Technology Special Project in Shanxi Province(202201140601025-2)the National Natural Science Foundation of China(32201749)supported by the Agriculture and Food Research Initiative Competitive Grant 2022-68013-36439(WheatCAP)from the USDA National Institute of Food and Agriculture.
文摘The awn can contribute to photosynthesis and carbohydrates,enhancing grain yield in wheat.We mapped QAwn.sxau-5A,a major QTL for awn development in wheat(Triticum aestivum).This QTL was delimited to a 994-kb interval at the B1 locus on chromosome 5A,which included the candidate gene encoding a zinc finger protein(TraesCS5A01G542800)as an awn length inhibitor(ALI).The Ali-A1 allele for the awnless trait showed abundant sequence differences in the promoter regions compared to the ali-A1 allele for the long-awn trait.The results of the swap experiment on the promoters from the two ALI-A1 alleles showed that the two promoters caused a difference in the protein level,indicating the gene was regulated at the transcript level.However,the ali-A1 allele contained an SNP that caused a premature stop codon in its coding region,resulting in a truncated protein compared to the functional Ali-A1 protein.The Ali-A1 protein contained two ethylene-responsive element binding factor-associated amphiphilic repression(EAR)motifs,one at the N terminus(EAR-N)and the other at the C terminus(EAR-C),and they were involved in interactions with the wheat co-repressor protein TOPLESS(TPL1).The ali-A1 protein retained the EAR-N motif but lost the EAR-C motif,resulting in the attenuated ability to interact with TPL1.The tpl1 mutant produced a longer awn compared to the wild type.Ali-A1 repressed the transcription of two downstream genes,TaLRP-A1 and TaARF-B1,involved in endogenous auxin concentrations and auxin responses in wheat.We concluded that the awn length is regulated not only by the ALI-A1 gene at transcript levels but also by Ali-A1 and TPL1 at the protein level in wheat.
文摘BACKGROUND Ovarian cancer(OC)is the most lethal gynecological cancer among females,and its early diagnosis could help for better outcomes of the patients.AIM To investigate the utility of serum insulin-like growth factors-binding proteins 2(IGFBP2),secreted phosphoprotein 1(SPP1),thrombospondin 1 protein(TSP1)and D-dimer levels in addition to currently used biomarkers[cancer antigen 125(CA125)and human epididymis protein 4(HE4)]in the diagnosis of epithelial OC(EOC).METHODS This is a case-control study that included fifty females diagnosed with EOC,10 females with benign ovarian masses recruited from the Egyptian National Cancer Institute,and 30 healthy females as a control group.All subjects were assessed for serum HE4,CA125,IGFBP2,TSP1 and SPP1 measurement by enzyme-linkedimmunosorbent assay.RESULTS There was a statistically significant difference in serum levels between EOC,benign ovarian masses,and healthy control groups regarding CA125 and SPP1(P<0.001 for both markers),while HE4 and IGFBP2 increased significantly in EOC compared to healthy control groups(P<0.001 for all markers)with no significant difference between EOC and benign ovarian masses groups.However,there was no statistically significant difference among EOC,benign ovarian masses,and healthy control groups regarding the TSP1 serum levels(P=0.051).Receiver operating characteristic analysis revealed that combined assessment of SPP1 with CA125 or TSP1 increased the diagnosis of EOC patients to a sensitivity,specificity,and area under curve of(93.3%,100%,0.968;respectively,P<0.001).CONCLUSION SPP1 may be a potential marker for the differentiation between benign and malignant ovarian masses,while IGFBP2 can differentiate between healthy females and females with ovarian masses.Combining SPP1 with CA125 or TSP1 provides high sensitivity and specificity for the detection of EOC patients.
基金supported by the National Natural Science Foundation of China(No.22073018,No.22377015).
文摘Intrinsically disordered proteins(IDPs)and their regions(IDRs)play crucial roles in cellular func-tions despite their lack of stable three-dimensional structures.In this study,we investigate the interac-tions between the C-terminal do-main of protein 4.1G(4.1G CTD)and the nuclear mitotic apparatus protein(NuMA)under varying pH and salt ion conditions to under-stand the regulatory mechanisms affecting their binding.4.1G CTD and NuMA bind effec-tively under neutral and alkaline conditions,but their interaction is disrupted under acidic conditions(pH 3.6).The protonation of positively charged residues at the C-terminal of 4.1G CTD under acidic conditions leads to increased electrostatic repulsion,weakening the overall binding free energy.Secondary structure analysis shows that specific regions of 4.1G CTD re-main stable under both pH conditions,but the C-terminal region(aa 990−1000)and the N-terminal region of NuMA(aa 1800−1810)exhibit significant reductions in secondary struc-ture probability under acidic conditions.Contact map analysis and solvent-accessible surface area analysis further support these findings by showing a reduced contact probability be-tween these regions under pH 3.6.These results provide a comprehensive understanding of how pH and ionic strength regulate the binding dynamics of 4.1G CTD and NuMA,emphasiz-ing the regulatory role of electrostatic interactions.
基金supported by the Beijing Natural Science Foundation(7232236)the National Key R&D Program of China(2022YFF1100104)。
文摘Introduction:Diet intervention,especially supplementation with high-quality protein,is considered to be a critical strategy in sarcopenia.However,different sources and types of protein have different health impacts.Objectives:The aim of this study is to explore the differences in the ameliorative effects and mechanisms of different sources and types of proteins on sarcopenia,providing an optimal path for the prevention and treatment of sarcopenia.Methods:A sarcopenia model was established by intraperitoneal injection of dexamethasone(5 mg/kg).Sixty male C57BL/6 mice(8 months old)were randomly divided into the normal control,sarcopenia,goat whey protein,goat milk casein,bovine whey protein,and bovine milk casein groups.Animals were treated for 8 consecutive weeks.Organism-level and molecular phenotypes,16S rRNA gene sequencing,and untargeted metabolomics profiling based on GC-TOF/MS were employed to investigate the correlation between host metabolism,microbial metabolism,autophagy and inflammation and their influence on sarcopenia in C57BL/6 male mice.Results:All 4 proteins increased muscle mass,and goat whey protein improved muscle strength in sarcopenic mice.Goat and bovine milk proteins promoted muscle regeneration by increasing MyoD1 and MyoG expression,and the former had a more distinct effect in inducing autophagy and decreasing inflammation than the latter.In addition,goat whey protein and casein could modulate hostmicrobial arginine co-metabolism.Notably,goat milk proteins responded well to sarcopenia comorbidities,including sarcopenic obesity,osteosarcopenia,and osteoarthritis.Conclusion:The study confirmed that goat milk proteins were more effective than bovine milk proteins for the control of sarcopenia.Moreover,we found that whey protein and casein could modulate host-microbial arginine co-metabolism,which shows their potential as precision nutritional supplements for the management of sarcopenia.Our study provides theoretical support for the prevention and control of sarcopenia.
文摘This article summarizes recent advances in the understanding of RNA-binding proteins(RBPs),with a focus on their roles in exercise-induced mRNA regulation and their implications for schizophrenia(SZ).RBPs are critical regulators of mRNA stability,splicing,transport,translation,and degradation,directly influencing gene expression through sequence-and structure-specific binding.In the nervous system,RBPs sustain synaptic plasticity,neural development,and neuronal homeostasis.Emerging evidence shows that exercise modulates the expression and activity of RBPs,thereby influencing mRNA translation and neurotransmitter signaling,which may underlie its beneficial effects on brain function.Dysregulation of specific RBPs has been identified in SZ,implicating them in disrupted synaptic transmission,impaired plasticity,and neuroinflammation.RBPs involved in memory and emotional regulation show marked dysfunction in SZ patients.Some RBPs have been proposed as potential biomarkers for early diagnosis and treatment monitoring.Moreover,therapeutic modulation of RBPs,through pharmacological or behavioral interventions such as exercise,may restore neuronal function by targeting post-transcriptional gene regulation.Exercise,as a non-invasive modulator of RBP expression,holds promise as an adjunctive strategy in SZ treatment,particularly in early stages.Further research into RBP-mediated pathways may offer novel insights into SZ pathophysiology and inform the development of targeted interventions.
基金supported by the National Natural Science Foundation of China(32200399 to Y.W.,32125006 to S.G.)Natural Science Foundation of Shandong Province(ZR2024ZD40 to S.G.,ZR2024MC112 to Y.W.)+4 种基金Young Talent of Lifting Engineering for Science and Technology in Shandong,China(SDAST2024QTA008 to Y.W.)Fundamental Research Funds for the Central Universities(202441014 to Y.W.)Postdoctoral Fellowship Program of the China Postdoctoral Science Foundation(CPSF)(GZC20232503 to Y.L.)China Postdoctoral Science Foundation(2024M753050 to Y.L.)Laoshan Laboratory(LSKJ202203203 to S.G.)。
文摘Bromodomain(BRD)-containing proteins are central mediators of gene regulation,serving as key components of chromatin remodeling complexes and histone recognition scaffolds.By specifically recognizing acetylated lysine residues on histones(Kac)via their conserved BRD,these proteins influence chromatin structure and gene expression.Although their overarching role is well-established,the precise molecular functions and mechanisms of individual BRD proteins remain incompletely characterized.The ciliate Tetrahymena thermophila,a unicellular eukaryote with a transcriptionally active macronucleus enriched in histone acetylation,is an excellent model for exploring the significance of BRD-containing proteins.In this comprehensive review,all BRD-containing proteins encoded in the T.thermophila genome are systematically examined,including their expression profiles,histone acetylation targets,interacting proteins,and potential roles.This review lays the groundwork for future investigations into the complex roles of BRD proteins in chromatin remodeling and transcription regulation,offering insights into basic eukaryotic biology and the molecular mechanisms underlying BRD-linked diseases.
基金the National Natural Science Foundation of China(Nos.82373722,22077144)Hunan Provincial Natural Science Foundation of China(No.2023JJ30527)+2 种基金Guangdong Basic and Applied Basic Research Foundation(No.2023B1515040006)Guangdong Provincial Key Laboratory of Construction Foundation(No.2023B1212060022)Key Research and Development Program of Guangdong Province(No.2020B1111110003).
文摘Chemical modification of native peptides and proteins is a versatile strategy to facilitate late-stage diversification for functional studies.Among the proteogenic amino acids,lysine is extensively involved in posttranslational modifications and the binding of ligands to target proteins,making its selective modification attractive.However,lysine’s high natural abundance and solvent accessibility,as well as its relatively low reactivity to cysteine,necessitate addressing chemoselectivity and regioselectivity for the Lys modification of native proteins.Although Lys chemoselective modification methods have been well developed,achieving site-selective modification of a specific Lys residue remains a great challenge.In this review,we discussed the challenges of Lys selective modification,presented recent examples of Lys chemoselective modification,and summarized the currently known methods and strategies for Lys site-selective modification.We also included an outlook on potential solutions for Lys site-selective labeling and its potential applications in chemical biology and drug development.
基金National Natural Science Foundation of China(Grant Nos.32070838 and 82301874)Open Fund of State Key Laboratory of Reproductive Medicine,Nanjing Medical University(Grant No.SKLRM K202102)。
文摘Meiotic resumption in mammalian oocytes involves nuclear and organelle structural changes,notably the chromatin configuration transition from a non-surrounding nucleolus(NSN)to surrounding nucleolus(SN)in germinal vesicle oocytes.In the current study,we found that nuclear speckles(NSs),a subnuclear structure mainly composed of serine-arginine(SR)proteins,changed from a diffuse spotted distribution in mouse NSN oocytes to an aggregated pattern in SN oocytes.We also found that the SR protein-specific kinase 1(SRPK1),an enzyme that phosphorylates SR proteins,co-localized with NSs at the SN stage,and that NSN oocytes failed to transition to SN oocytes after the inhibition of SRPK1 activity.Furthermore,the typical structure of the chromatin ring around the nucleolus in SN oocytes collapsed after treatment with an SRPK1 inhibitor.Mechanistically,phosphorylated SR proteins were found to be related to chromatin as shown by a salt extraction experiment,and in situ DNaseⅠassay showed that the accessibility of chromatin was enhanced in SN oocytes when SRPK1 was inhibited,accompanied by a decreased repressive modification on histone and the abnormal recurrence of a transcriptional signal.In conclusion,our results indicated that SRPK1-regulated phosphorylation of SR proteins was involved in the NSN-SN transition and played an important role in maintaining the condensed nucleus of SN oocytes via interacting with chromatin.
基金support from the the National Natural Science Foundation of China(Grant Nos.12474199(RM)and 12374213(YC))Fundamental Research Funds for Central Universities of China(Grant No.20720240144(RM))111 Project(Grant No.B16029).
文摘Vesicles of lipid bilayer can adopt a variety of shapes due to different coating proteins.The ability of proteins to reshape membrane is typically characterized by inducing spontaneous curvature of the membrane at the coated area.BAR family proteins are known to have a crescent shape and can induce membrane curvature along their concaved body axis but not in the perpendicular direction.We model this type of proteins as a rod-shaped molecule with an orientation and induce normal curvature along its orientation in the tangential plane of the membrane surface.We show how a ring of these proteins reshapes an axisymmetric vesicle when the protein curvature or orientation is varied.A discontinuous shape transformation from a protrusion shape without a neck to a one with a neck is found.Increasing the rigidity of the protein ring is able to smooth out the transition.Furthermore,we show that varying the protein orientation is able to induce an hourglass-shaped neck,which is significantly narrower than the reciprocal of the protein curvature.Our results offer a new angle to rationalize the helical structure formed by many proteins that carry out membrane fission functions.
基金supported by JSPS(No.22H00350 to M.U.)Ministry of Health&Welfare,Republic of Korea(Korea Health Technology R&D Project through the Korea Health Industry Development Institute,No.HI19C1234 to H.K.)+3 种基金JST(the Establishment of University Fellowships towards the Creation of Science Technology Innovation,No.JPMJFS2123 to K.T.)supported and inspired by the International Collaborative Research Program of Institute for Chemical Research,Kyoto University(No.2024-84)Kyoto University On-Site Lab(Fudan-Kyoto Shanghai Lab)the international and interdisciplinary environments of JSPS CORE-to-CORE Program“Asian Chemical Biology Initiative”.
文摘Biomolecular condensates,also known as membraneless organelles,play a crucial role in cellular organization by concentrating or sequestering biomolecules.Despite their importance,synthetically mimicking these organelles using non-peptidic small organic molecules has posed a significant challenge.The present study reports the discovery of D008,a self-assembling small molecule that sequesters a unique subset of RNA-binding proteins.Analysis and screening of a comprehensive collection of approximately 1 million compounds in the Chinese National Compound Library(Shanghai)identified 44 self-assembling small molecules in aqueous solutions.Subsequent screening of the focused library,coupled with proteome analysis,led to the discovery of D008 as a small organic molecule with the ability to condensate a specific subset of RNA-binding proteins.In vitro experiments demonstrated that the D008-induced sequestration of RNA-binding proteins impeded mRNA translation.D008 may offer a unique opportunity for studying the condensations of RNA-binding proteins and for developing an unprecedented class of small molecules that control gene expression.
基金supported by grants from the Special Foundation of President of the Chinese Academy of Sciences(Grant No.,YZJJ2020QN27,YZJJ2021QN33)Anhui Provincial Natural Science Foundation(Grant No.,2108085MC79).
文摘HCLS1-associated protein X-1(HAX1)is a multifunctional mitochondrial protein involved in the regulation of apoptosis,a crucial process of programmed cell death,and mRNA processing.Despite its significance,limited structural data is available for HAX1,hindering a comprehensive understanding of its biological function.Notably,the caseinolytic mitochondrial matrix peptidase chaperone subunit B(CLPB)has been identified as an interacting partner of HAX1,yet the biophysical properties and binding affinity governing their interaction remain poorly defined.In this study,we present a thorough biophysical characterization of full-length human HAX1 and CLPB,accomplished through recombinant expression and purification.By employing size exclusion chromatography,dynamic light scattering,and circular dichroism spectroscopy,we successfully established their biophysical properties,revealing contrasting structural features,with CLPB displaying a-helical content and HAX1 exhibiting a disordered nature.Moreover,we employed solutionstate nuclear magnetic resonance(NMR)spectroscopy to probe their binding affinity.Our findings demonstrate the formation of stable multimeric complexes between HAX1 and CLPB,and we quantified a dissociation constant in the low range of micro-molar for their high affinity interaction.These results lay the foundation for further in-depth investigations into the dynamics and energetics governing the HAX1-CLPB interaction,ultimately contributing to a comprehensive understanding of their functional mechanisms.
基金supported by the Youth Foundation of Beijing Academy of Agriculture and Forestry Sciences[Grant No.QNJJ202242]the Excellent Young Scholars of Beijing Academy of Agriculture and Forestry Sciences[Grant No.YXQN202205]+3 种基金the Beijing Nova Program[Grant No.20220484052]the National Natural Science Foundation of China[Grant No.31801852]the Collaborative Innovation Center of Beijing Academy of Agriculture and Forestry Sciences[Grant No.KJCX201907-2]the Earmarked Fund for China Agriculture Research System[Grant No.CARS-23-A-05].
文摘The soil-resident pathogen, Plasmodiophora brassicae, infects cruciferous crops, causing obligate parasitic clubroot disease and posing a significant threat to the Brassica vegetable industry in China. To learn more about its pathogenesis, we reported a Nanopore sequencing-derived25.3 Mb high-quality genome sequence of P. brassicae pathotype 4 strain(P.b 4). Comparing the P.b 4 genome with that of the published P.brassicae e3 genome(P.b e3) identified single nucleotide polymorphisms, structural variations, and small insertions and deletions. We then carried out RNA-sequencing of root samples from a clubroot-susceptible line at 5, 14, and 28 days after inoculation(DAI), and classified genes into five categories based on their expression patterns. Interestingly, 158 genes were highly expressed at 14 DAI, which were enriched in budding cell isotropic bud growth, ascospore wall assembly, spore wall assembly, spore wall biogenesis, and ascospore wall biogenesis.Subsequently, we bioinformatically predicted 555 secreted effector candidates, among which only 125 were expressed during infection and had amino acid lengths less than 400. The putative effector Pb010018, which was highly expressed at 14 DAI, was validated to have a signal peptide using a yeast secretion system. Luciferase activity and co-immunoprecipitation assays demonstrated that Pb010018 interacts with serine hydroxymethyltransferase BrSHMT1, and expression analysis showed that SHMT1 was upregulated in both Arabidopsis and B. rapa during infection. Furthermore, after infection, the Arabidopsis shmt1 mutant(atshmt1) showed reduced severity of clubroot disease, together with downregulated expression of Pb010018. Our results offer new insights into plant-pathogen interaction mechanisms, and provide the possibility for improving Brassica resistance to clubroot disease.
基金supported by the National Natural Science Foundation of China Joint Fund for Regional Innovation and Development(Grant numbers [U21A20334])the Postgraduate Innovation Funding Project of Hebei Province(Grant numbers [CXZZBS2022116])。
文摘Objective Recent studies have overturned the traditional concept of the lung as a “sterile organ” revealing that pulmonary microbiota dysbiosis and abnormal surfactant proteins(SPs) expression are involved in the progression of silicosis. This study aimed to investigate the relationship between abnormal SPs expression and dysbiosis of lung microbiota in silica-induced lung fibrosis, providing insights into mechanisms of silicosis.Methods Lung pathology, SPs expression, and microbiota composition were evaluated in silicaexposed mice. A mouse model of antibiotic-induced microbiota depletion was established, and alveolar structure and SPs expression were assessed. The roles of the lung microbiota and SPs in silicosis progression were further evaluated in mice with antibiotic-induced microbiota depletion, both with and without silica exposure.Results Silica exposure induced lung inflammation and fibrosis, along with increased expression of SPA expression. Antibiotics(Abx)-induced microbiota depletion elevated SP-A and SP-D expression.Furthermore, silica exposure altered lung microbiota composition, enriching potentially pathogenic taxa.However, antibiotic-induced microbiota depletion prior to silica exposure reduced silica-mediated lung fibrosis and inflammation.Conclusion Lung microbiota is associated with silica-induced lung injury. Overproduction of SP-A and SP-D, induced by Abx-induced microbiota depletion, may enhance the resistance of mouse lung tissue to silica-induced injury.
基金financially supported by the National Key R&D Program of China(Grant No.2022YFF1202600)the National Natural Science Foundation of China(Grant No.82301158)+4 种基金Science and Technology Innovation Action Plan of Shanghai Science and Technology Committee(Grant No.22015820100)Two-hundred Talent Support(Grant No.20152224)Translational Medicine Innovation Project of Shanghai Jiao Tong University School of Medicine(Grant No.TM201915)Clinical Research Project of Multi-Disciplinary Team,Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine(Grant No.201914)China Postdoctoral Science Foundation(Grant No.2023M742332)。
文摘Essential proteins are crucial for biological processes and can be identified through both experimental and computational methods.While experimental approaches are highly accurate,they often demand extensive time and resources.To address these challenges,we present a computational ensemble learning framework designed to identify essential proteins more efficiently.Our method begins by using node2vec to transform proteins in the protein–protein interaction(PPI)network into continuous,low-dimensional vectors.We also extract a range of features from protein sequences,including graph-theory-based,information-based,compositional,and physiochemical attributes.Additionally,we leverage deep learning techniques to analyze high-dimensional position-specific scoring matrices(PSSMs)and capture evolutionary information.We then combine these features for classification using various machine learning algorithms.To enhance performance,we integrate the outputs of these algorithms through ensemble methods such as voting,weighted averaging,and stacking.This approach effectively addresses data imbalances and improves both robustness and accuracy.Our ensemble learning framework achieves an AUC of 0.960 and an accuracy of 0.9252,outperforming other computational methods.These results demonstrate the effectiveness of our approach in accurately identifying essential proteins and highlight its superior feature extraction capabilities.
基金supported by the National Natural Science Foundation of China(No.41975156).
文摘Tyrosine residues in proteins can be nitrated to form 3-nitrotyrosine(3-NT)under the influence of ozone(O_(3))and nitrogen dioxide(NO_(2))in the air,which may introduce health impacts.A selective and sensitive enzyme-linked-immunoassay(ELISA)method was developed to determine 3-NT in modified model protein(bovine serum albumin,BSA)and ambient aerosol samples.The nitration degrees(NDs)of BSA in the exposure experiments with different durations were detected by both the ELISA and spectrophotometric methods(i.e.,ND_(ELISA) and ND_(SEC-PDA)),which show good coincidence.The kinetic investigation by both ΔND_(ELISA) and ΔND_(SEC-PDA) in the exposure experiments shows that the rate coefficients(k)of the pseudo-first-order kinetic rate reactions of protein nitration were comparable.These results indicate that direct detection of 3-NT by the ELISA method can be applied for laboratory exposure samples analysis for kinetic studies.Based on the selective detection of 3-NT,ND_(ELISA) provides a promising measure for the assessment of ND in model proteins.3-NT was alsomeasured in PM_(2.5) samples in summer in Guangzhou,southern China,ranging from 10.1 to 404 pg/m^(3),providing clear evidence of protein nitration in ambient aerosols.We further proposed that 3-NT/protein can be used as a proxy to evaluate protein nitration in ambient aerosols.A significant correlationwas observed between 3-NT/protein and O_(3),confirming the crucial role of O_(3) in protein nitration.Our results show that the direct detection of 3-NT by the ELISA method can be more widely applied in the laboratory and field-based studies for understanding the mechanisms of protein nitration.
基金supported by National Natural Science Foundation of China(22161142018,21991081,22177056,and 22174074)the Ministry of Science and Technology of China(2021YFA1600304).
文摘The distance distributions between two site-specifically anchored spin labels in a protein,measured by pulsed electron-electron double resonance(PELDOR or DEER),provide rich sources of structural and conformational restraints on the proteins or their complexes.The rigid connection of the nitroxide spin label to the protein improves the accuracy and precision of distance measurement.We report a new spin labelling approach by formation of thioester bond between nitroxide(NO)spin label,NOAI(NO spin labels activated by acetylimidazole),and a protein thiol,and this spin labeling method has demonstrated high performance in DEER distance measurement on proteins.The results showed that NOAI has shorter connection to the protein ligation site than 2,2,5,5-tetramethyl-pyrroline-1-oxyl methanethiosulfonate(MTSL)and 3-maleimido-proxyl(M-Prox)in the respective protein conjugate and produces narrower distance distributions for the tested proteins including ubiquitin(Ub),immunoglobulin-binding b1 domain of streptococcal protein G(GB1),and second mitochondria-derived activator of caspases(Smac).The NOAI protein conjugate connected by a thioester bond is resistant to reducing reagent and offers highfidelity DEER distance measurements in cell lysates.
基金supported by the National Natural Science Foundation of China(Grant No.32371284)the Open Fund of the State Key Laboratory of Optoelectronic Materials and Technologies,Sun Yatsen University(Grant No.OEMT-2024-ZTS-04)support from the Physical Research Platform in the School of Physics,Sun Yatsen University(Grant No.PRPSP,SYSU).
文摘DNA imaging and visualization techniques are crucial in biological experiments and have also emerged as a powerful method for single-molecule studies.Traditional intercalating dyes(e.g.,SYTOX,EtBr,GelRed)can stain DNA but may alter its structure and mechanical properties,and cause photocleavage.Recently,a novel fluorescent DNA-binding protein(FP-DBP)was introduced,which can stain DNA without sequence preference and without inducing photocleavage.In this study,using a custom-built magnetic tweezers system,we performed DNA stretching,twisting and unzipping experiments to compare the mechanical properties of DNA with and without two kinds of intercalating dyes(SYTOX Orange and GelRed)and mCherry FP-DBP.Our results show that mCherry FP-DBP does not affect DNA structure or mechanics,unlike SYTOX Orange and GelRed,making FP-DBP a promising tool for DNA visualization in single-molecule experiments.
