Eugenol targeting CrtM inhibits the biosynthesis of staphyloxanthin in Staphylococcus aureus

Staphylococcus aureus is a serious foodborne pathogen threatening food safety and public health.Especially the emergence of methicillin-resistant Staphylococcus aureus(MRSA)increased the difficulty of S.aureus treatment.Staphyloxanthin is a crucial virulence factor of S.aureus.Blocking staphyloxanthin production could help the host immune system counteract the invading S.aureus cells.In this study,we first screened for staphyloxanthin inhibitors using a virtual screening method.The outcome of the virtual screening method resulted in the identification of eugenol(300μg/mL),which significantly inhibits the staphyloxanthin production in S.aureus ATCC 29213,S.aureus Newman,MRSA ATCC 43300 and MRSA ATCC BAA1717by 84.2%,63.5%,68.1%,and 79.5%,respectively.The outcome of the growth curve assay,field-emission scanning electron,and confocal laser scanning microscopy analyses confirmed that eugenol at the test concentration did not affect the morphology and growth of S.aureus.Moreover,the survival rate of S.aureus ATCC 29213 and MRSA ATCC 43300 under H_(2)O_(2) pressure decreased to 51.9%and 45.5%in the presence of eugenol,respectively.The quantitative RT-PCR and molecular simulation studies revealed that eugenol targets staphyloxanthin biosynthesis by downregulating the transcription of the crtM gene and inhibiting the activity of the CrtM enzyme.Taken together,we first determined that eugenol was a prominent compound for staphyloxanthin inhibitor to combat S.aureus especially MRSA infections.

Effect of mutations on acetohydroxyacid synthase(AHAS)function in Cyperus difformis L.

Cyperus difformis L.is a troublesome weed in paddy fields and has attracted attention due to its resistance to acetohydroxyacid synthase(AHAS)inhibitors.It was found that the amino acid mutation in AHAS was the primary cause for the resistance of Cyperus difformis.However,the effect of different mutations on AHAS function is not clear in Cyperus difformis.To confirm the effect of mutations on AHAS function,six biotypes were collected,including Pro197Arg,Pro197Ser,Pro197Leu,Asp376Glu,Trp574Leu and wild type,from Hunan,Anhui,Jiangxi and Jiangsu provinces,China and the function of AHAS was characterized.The AHAS in vitro inhibition assay results indicated that the mutations decreased the sensitivity of AHAS to pyrazosulfuron-ethyl,in which the I_(50)(the half maximal inhibitory concentration)of wild type AHAS was 0.04μmol L^(-1)and Asp376Glu,Pro197Leu,Pro197Arg,Pro197Ser and Trp574Leu mutations were 3.98,11.50,40.38,38.19 and 311.43μmol L^(-1),respectively.In the determination of enzyme kinetics parameters,the Km and the maximum reaction velocity(Vmax)of the wild type were 5.18 mmol L^(-1)and 0.12 nmol mg^(-1)min^(-1),respectively,and the Km values of AHAS with Asp376Glu,Trp574Leu,Pro197Leu and Pro197Ser mutations were 0.38-0.93 times of the wild type.The Km value of the Pro197Arg mutation was 1.14times of the wild type,and the Vmax values of the five mutations were 1.17-3.33-fold compared to the wild type.It was found that the mutations increased the affinity of AHAS to the substrate,except for the Pro197Arg mutation.At a concentration of 0.0032-100 mmol L^(-1)branched-chain amino acids(BCAAs),the sensitivity of the other four mutant AHAS biotypes to feedback inhibition decreased,except for the Pro197Arg mutation.This study elucidated the effect of different mutations on AHAS function in Cyperus difformis and provided ideas for further study of resistance development.

The gene encoding flavonol synthase contributes to lesion mimic in wheat

Lesion mimic often exhibits leaf disease-like symptoms even in the absence of pathogen infection,and is characterized by a hypersensitive-response(HR)that closely linked to plant disease resistance.Despite this,only a few lesion mimic genes have been identified in wheat.In this investigation,a lesion mimic wheat mutant named je0297 was discovered,showing no alteration in yield components when compared to the wild type(WT).Segregation ratio analysis of the F_(2)individuals resulting from the cross between the WT and the mutant revealed that the lesion mimic was governed by a single recessive gene in je0297.Using Bulked segregant analysis(BSA)and exome capture sequencing,we mapped the lesion mimic gene designated as lm6 to chromosome 6BL.Further gene fine mapping using 3315 F_(2)individuals delimited the lm6 within a 1.18 Mb region.Within this region,we identified 16 high-confidence genes,with only two displaying mutations in je0297.Notably,one of the two genes,responsible for encoding flavonol synthase,exhibited altered expression levels.Subsequent phenotype analysis of TILLING mutants confirmed that the gene encoding flavonol synthase was indeed the causal gene for lm6.Transcriptome sequencing analysis revealed that the DEGs between the WT and mutant were significantly enriched in KEGG pathways related to flavonoid biosynthesis,including flavone and flavonol biosynthesis,isoflavonoid biosynthesis,and flavonoid biosynthesis pathways.Furthermore,more than 30 pathogen infection-related(PR)genes exhibited upregulation in the mutant.Corresponding to this expression pattern,the flavonoid content in je0297 showed a significant decrease in the 4^(th)leaf,accompanied by a notable accumulation of reactive oxygen,which likely contributed to the development of lesion mimic in the mutant.This investigation enhances our comprehension of cell death signaling pathways and provides a valuable gene resource for the breeding of disease-resistant wheat.

Genome-wide identification and function analysis of the sucrose phosphate synthase MdSPS gene family in apple

Sucrose phosphate synthase(SPS)is a rate-limiting enzyme that works in conjunction with sucrose-6-phosphate phosphatase(SPP)for sucrose synthesis,and it plays an essential role in energy provisioning during growth and development in plants as well as improving fruit quality.However,studies on the systematic analysis and evolutionary pattern of the SPS gene family in apple are still lacking.In the present study,a total of seven MdSPS and four MdSPP genes were identified from the Malus domestica genome GDDH13 v1.1.The gene structures and their promoter cis-elements,protein conserved motifs,subcellular localizations,physiological functions and biochemical properties were analyzed.A chromosomal location and gene-duplication analysis demonstrated that whole-genome duplication(WGD)and segmental duplication played vital roles in MdSPS gene family expansion.The Ka/Ks ratio of pairwise MdSPS genes indicated that the members of this family have undergone strong purifying selection during domestication.Furthermore,three SPS gene subfamilies were classified based on phylogenetic relationships,and old gene duplications and significantly divergent evolutionary rates were observed among the SPS gene subfamilies.In addition,a major gene related to sucrose accumulation(MdSPSA2.3)was identified according to the highly consistent trends in the changes of its expression in four apple varieties(‘Golden Delicious’,‘Fuji’,‘Qinguan’and‘Honeycrisp’)and the correlation between gene expression and soluble sugar content during fruit development.Furthermore,the virus-induced silencing of MdSPSA2.3 confirmed its function in sucrose accumulation in apple fruit.The present study lays a theoretical foundation for better clarifying the biological functions of the MdSPS genes during apple fruit development.

Glycogen Synthase Kinase-3β,NLRP3 Inflammasome,and Alzheimer's Disease

Alzheimer’s disease (AD) is the most prevalent cause of dementia worldwide. Because of the progressive neurodegeneration, individual cognitive and behavioral functions are impaired, affecting the quality of life of millions of people. Although the exact pathogenesis of AD has not been fully elucidated, amyloid plaques, neurofibrillary tangles (NFTs), and sustaining neuroinflammation dominate its characteristics. As one of the major tau kinases leading to hyperphosphorylation and aggregation of tau, glycogen synthase kinase-3β (GSK-3β) has been drawing great attention in various AD studies. Another research focus of AD in recent years is the inflammasome, a multiprotein complex acting as a regulator in immunological reactions to exogenous and endogenous danger signals, of which the Nod-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome has been studied mostly in AD and proven to play a significant role in AD development by its activation and downstream effects such as caspase-1 maturation and interleukin (IL)-1β release. Studies have shown that the NLRP3 inflammasome is activated in a GSK-3β-dependent way and that inhibition of the NLRP3 inflammasome downregulates GSK-3β, suggesting that these two important proteins are closely related. This article reviews the respective roles of GSK-3β and the NLRP3 inflammasome in AD as well as their relationship and interaction.

Metformin promotes angiogenesis and functional recovery in aged mice after spinal cord injury by adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury.However,its effect on spinal cord injury in aged mice remains unclear.Considering the essential role of angiogenesis during the regeneration process,we hypothesized that metformin activates the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway in endothelial cells,thereby promoting microvascular regeneration in aged mice after spinal cord injury.In this study,we established young and aged mouse models of contusive spinal cord injury using a modified Allen method.We found that aging hindered the recovery of neurological function and the formation of blood vessels in the spinal cord.Treatment with metformin promoted spinal cord microvascular endothelial cell migration and blood vessel formation in vitro.Furthermore,intraperitoneal injection of metformin in an in vivo model promoted endothelial cell proliferation and increased the density of new blood vessels in the spinal cord,thereby improving neurological function.The role of metformin was reversed by compound C,an adenosine monophosphate-activated protein kinase inhibitor,both in vivo and in vitro,suggesting that the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway likely regulates metformin-mediated angiogenesis after spinal cord injury.These findings suggest that metformin promotes vascular regeneration in the injured spinal cord by activating the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway,thereby improving the neurological function of aged mice after spinal cord injury.

Neuronal nitric oxide synthase/reactive oxygen species pathway is involved in apoptosis and pyroptosis in epilepsy

Dysfunction of neuronal nitric oxide synthase contributes to neurotoxicity,which triggers cell death in various neuropathological diseases,including epilepsy.Studies have shown that inhibition of neuronal nitric oxide synthase activity increases the epilepsy threshold,that is,has an anticonvulsant effect.However,the exact role and potential mechanism of neuronal nitric oxide synthase in seizures are still unclear.In this study,we performed RNA sequencing,functional enrichment analysis,and weighted gene coexpression network analysis of the hippocampus of tremor rats,a rat model of genetic epilepsy.We found damaged hippocampal mitochondria and abnormal succinate dehydrogenase level and Na+-K+-ATPase activity.In addition,we used a pilocarpine-induced N2a cell model to mimic epileptic injury.After application of neuronal nitric oxide synthase inhibitor 7-nitroindazole,changes in malondialdehyde,lactate dehydrogenase and superoxide dismutase,which are associated with oxidative stress,were reversed,and the increase in reactive oxygen species level was reversed by 7-nitroindazole or reactive oxygen species inhibitor N-acetylcysteine.Application of 7-nitroindazole or N-acetylcysteine downregulated the expression of caspase-3 and cytochrome c and reversed the apoptosis of epileptic cells.Furthermore,7-nitroindazole or N-acetylcysteine downregulated the abnormally high expression of NLRP3,gasdermin-D,interleukin-1βand interleukin-18.This indicated that 7-nitroindazole and N-acetylcysteine each reversed epileptic cell death.Taken together,our findings suggest that the neuronal nitric oxide synthase/reactive oxygen species pathway is involved in pyroptosis of epileptic cells,and inhibiting neuronal nitric oxide synthase activity or its induced oxidative stress may play a neuroprotective role in epilepsy.

Identifi cation of sesquiterpene synthase genes in the genome of Aquilaria sinensis and characterization of anα-humulene synthase

Sesquiterpenes are the major pharmacodynamic components of agarwood,a precious traditional Chinese medicine obtained from the resinous portions of Aquilaria sinensis trees that form in response to environmental stressors.To characterize the sesquiterpene synthases responsible for sesquiterpene production in A.sinensis,a bioinformatics analysis of the genome of A.sinensis identifi ed six new terpene synthase genes,and 16 sesquiterpene synthase genes were identifi ed as type TPS-a in a phylogenetic analysis.The expression patterns for eight of the sesquiterpene synthase genes after treatment with various hormones or hydrogen peroxide were analyzed by real-time quantitative PCR.The results suggest that 100μM methyl jasmonate,ethephon,(±)-abscisic acid or hydrogen peroxide could be eff ective short-term eff ectors to increase the expression of sesquiterpene synthase genes,while 1 mM methyl salicylate may have long-term eff ects on increasing the expression of specifi c sesquiterpene synthase genes(e.g.,As-SesTPS,AsVS,AsTPS12 and AsTPS29).The expression changes in these genes under various conditions refl ected their specifi c roles during abiotic or biotic stresses.Heterologous expression of a novel A.sinensis sesquiterpene synthase gene,AsTPS2,in Escherichia coli produced a major humulene product,so AsTPS2 is renamed AsHS1.AsHS1 is diff erent from ASS1,AsSesTPS,and AsVS,for mainly producingα-humulene.Based on the predicted space conformation of the AsHS1 model,the small ligand molecule may bind to the free amino acid by hydrogen bonding for the catalytic function of the enzyme,while the substrate farnesyl diphosphate(FPP)probably binds to the free amino acid on one side of the RxR motif.Arg450,Asp453,Asp454,Thr457,and Glu461 from the NSE/DTE motif and D307 and D311 from the DDxxD motif were found to form a polar interaction with two Mg^(2+)clusters by docking.The Mg^(2+)-bound DDxxD and NSE/DTE motifs and the free RXR motif are jointly directed into the catalytic pocket of AsHS1.Comparison of the tertiary structural models of AsHS1 with ASS1 showed that they diff ered in structures in several positions,such as surrounding the secondary catalytic pocket,which may lead to diff erences in catalytic products.Based on the results,biosynthetic pathways for specifi c sesquiterpenes such asα-humulene in A.sinensis are proposed.This study provides novel insights into the functions of the sesquiterpene synthases of A.sinensis and enriches knowledge on agarwood formation.

Mutations in Plasmodium knowlesi Kelch protein 13 and the dihydropteroate synthase gene in clinical samples

Objective:To determine the genetic diversity,natural selection and mutations in Plasmodium(P.)knowlesi drug resistant molecular markers Kelch 13 and dhps gene in clinical samples of Malaysia.Methods:P.knowlesi full-length gene sequences Kelch 13 gene(PkK13)from 40 samples and dhps gene from 30 samples originating from Malaysian Borneo were retrieved from public databases.Genetic diversity,natural selection,and phylogenetic analysis of gene sequences were analysed using DNAsp v5.10 and MEGA v5.2.Results:Seventy-two single nucleotide polymorphic sites(SNPs)across the full-length PkK13 gene(63 synonymous substitutions and 9 non-synonymous substitutions)with nucleotide diversity ofπ~0.005 was observed.Analysis of the full-length Pkdhps gene revealed 73 SNPs andπ~0.006(44 synonymous substitutions and 29 non-synonymous substitutions).A high number of haplotypes(PkK13;H=37 and Pkdhps;H=29)with haplotype diversity of Hd~0.99 were found in both genes,indicating population expansion.Nine mutant alleles were identified in PkK13 amino acid alignment of which,7(Asp3Glu,Lys50Gln,Lys53Glu,Ser123Thr,Ser127Pro,Ser149Thr and Ala169Thr)were within the Plasmodium specific domain,2(Val372Ile and Lys424Asn)were in the BTB/POZ domain and no mutation was observed within the kelch propeller domain.The 29 non-synonymous mutations in the Pkdhps gene were novel and only presented in exon 1 and 2.Conclusions:Monitoring the mutations from clinical samples collected from all states of Malaysia along with clinical efficacy studies will be necessary to determine the drug resistance in P.knowlesi.

A nonsynonymous mutation in an acetolactate synthase gene (Gh_D10G1253) is required for tolerance to imidazolinone herbicides in cotton

Background Herbicide tolerance in crops enables them to survive when lethal doses of herbicides are applied to surrounding weeds.Herbicide-tolerant crops can be developed through transgenic approaches or traditional mutagenesis approaches.At present,no transgenic herbicide tolerant cotton have been commercialized in China due to the genetically-modified organism(GMO)regulation law.We aim to develop a non-transgenic herbicide-tolerant cotton through ethyl methanesulfonate(EMS)mutagenesis,offering an alternative choice for weed management.Results Seeds of an elite cotton cultivar Lumianyan 37(Lu37)were treated with EMS,and a mutant Lu37-1 showed strong tolerance to imidazolinone(IMI)herbicides was identified.A novel nonsynonymous substitution mutation Ser642Asn at acetolactate synthase(ALS)(Gh_D10G1253)in Lu37-1 mutant line was found to be the potential cause to the IMI herbicides tolerance in cotton.The Ser642Asn mutation in ALS did not present among the genomes of natural Gossypium species.Cleaved amplified polymorphic sequence(CAPS)markers were developed to identify the ALS mutant allele.The Arabidopsis overexpressing the mutanted ALS also showed high tolerance to IMI herbicides.Conclusion The nonsynonymous substitution mutation Ser642Asn of the ALS gene Gh_D10G1253 is a novel identi-fied mutation in cotton.This substitution mutation has also been identified in the orthologous ALS genes in other crops.This mutant ALS allele can be used to develop IMI herbicide-tolerant crops via a non-transgenic or transgenic approach.

Identification and Pathogen Stimulation Patterns of Neuronal Nitric Oxide Synthase(nNOS)in Black Rockfish(Sebastes schlegelii)

Neuronal nitric oxide synthase(nNOS)was the producer of nitric oxide(NO)which played important gas messenger molecules in biological process.It also can take effect as immune regulation molecule in organism.Black rockfish(Sebastes schlegelii)is an important economic fish which were widely farmed in East Asia countries.Meanwhile,the pathogenic bacteria such as the Edwardsiella tarda and Vibrio anguillarum in seawater always brought serious obstacles to their healthy growth.In order to explore the expression pattern of n NOS gene under the pathogen stimulation and predict its immune function,the n NOS gene in black rockfish named Ssn NOS was identified.It was 3780 bp in length,located on chromosome 6,and contained 27 coding domain sequence(CDs).According to the phylogenetic analysis,the Ssn NOS showed closest relative to the counterpart gene of swamp eel(Monopterus albus).Meanwhile,analysis of Ssn NOS expression in various healthy tissues showed that Ssn NOS expression level was highest in healthy brain tissues,followed by intestinal tissues.In addition,Ssn NOS showed significant expression changes in response to stimulation by two pathogens.Particular in gill,the expression of Ssn NOS after pathogenic stimulation increased significantly.The Elisa analysis showed the Ssn NOS content in gills was much higher than that in other tissues at all time points.Moreover,the expression patterns of Ssn NOS in brain,intestine and kidney after stimulation by pathogens showed a distinct expression pattern which first down-regulated and then up-regulated.Therefore,the Ssn NOS may be an important signaling molecule for fish to respond rapidly in immune stimulation.

ZmMS39 encodes a callose synthase essential for male fertility in maize(Zea mays L.)

Callose contributes to many biological processes of higher plants including pollen development,cell plate and vascular tissue formation,as well as regulating the transport function of plasmodesmata.The functions of callose synthase genes in maize have been little studied.We describe a maize male-sterile mutant 39(ms39)characterized by reduced plant height.In this study,we confirmed using CRISPR/Cas9 technology that a mutation in Zm00001d043909(ZmCals12),encoding a callose synthase,is responsible for the male sterility of the ms39 mutant.Compared with male-fertile plants,callose deposition around the dyads and tetrads in ms39 anthers was significantly reduced.Increased cell autophagy observed in ms39 anthers may have been due to the premature programmed cell death of tapetal cells,leading to collapse of the anther wall structure.Disordered glucose metabolism in ms39 may have intensified autophagy in anthers.Evaluation of the ms39 gene on maize heterosis by paired-crossed experiment with 11 maize inbred lines indicated that ms39 can be used for maize hybrid seed production.

Prostaglandin F_(2α)synthase promotes oxaliplatin resistance in colorectal cancer through prostaglandin F_(2α)-dependent and F_(2α)-independent mechanism

BACKGROUND Oxaliplatin(Oxa)is the first-line chemotherapy drug for colorectal cancer(CRC),and Oxa resistance is crucial for treatment failure.Prostaglandin F_(2α)synthase(PGF 2α)(PGFS),an enzyme that catalyzes the production of PGF_(2α),is involved in the proliferation and growth of a variety of tumors.However,the role of PGFS in Oxa resistance in CRC remains unclear.AIM To explore the role and related mechanisms of PGFS in mediating Oxa resistance in CRC.METHODS The PGFS expression level was examined in 37 pairs of CRC tissues and paracancerous tissues at both the mRNA and protein levels.Overexpression or knockdown of PGFS was performed in CRC cell lines with acquired Oxa resistance(HCT116-OxR and HCT8-OxR)and their parental cell lines(HCT116 and HCT8)to assess its influence on cell proliferation,chemoresistance,apoptosis,and DNA damage.For determination of the underlying mechanisms,CRC cells were examined for platinum-DNA adducts and reactive oxygen species(ROS)levels in the presence of a PGFS inhibitor or its products.RESULTS Both the protein and mRNA levels of PGFS were increased in the 37 examined CRC tissues compared to the adjacent normal tissues.Oxa induced PGFS expression in the parental HCT116 and HCT8 cells in a dosedependent manner.Furthermore,overexpression of PGFS in parental CRC cells significantly attenuated Oxainduced proliferative suppression,apoptosis,and DNA damage.In contrast,knockdown of PGFS in Oxa-resistant HCT116 and HCT8 cells(HCT116-OxR and HCT8-OxR)accentuated the effect of Oxa treatment in vitro and in vivo.The addition of the PGFS inhibitor indomethacin enhanced the cytotoxicity caused by Oxa.Treatment with the PGFS-catalyzed product PGF_(2α)reversed the effect of PGFS knockdown on Oxa sensitivity.Interestingly,PGFS inhibited the formation of platinum-DNA adducts in a PGF_(2α)-independent manner.PGF_(2α)exerts its protective effect against DNA damage by reducing ROS levels.CONCLUSION PGFS promotes resistance to Oxa in CRC via both PGF_(2α)-dependent and PGF_(2α)-independent mechanisms.

Correlation between the expressions of metastasis-associated factor-1 in colon cancer and vacuolar ATP synthase

BACKGROUND Clinical prognosis often worsens due to high recurrence rates following radical surgery for colon cancer.The examination of high-risk recurrence factors post-surgery provides critical insights for disease evaluation and treatment planning.AIM To explore the relationship between metastasis-associated factor-1 in colon cancer(MACC1)and vacuolar ATP synthase(V-ATPase)expression in colon cancer tissues,and recurrence rate in patients undergoing radical colon cancer surgery.METHODS We selected 104 patients treated with radical colon cancer surgery at our hospital from January 2018 to June 2021.Immunohistochemical staining was utilized to assess the expression levels of MACC1 and V-ATPase in these patients.RESULTS The rates of MACC1 and V-ATPase positivity were 64.42%and 67.31%,respe-ctively,in colon cancer tissues,which were significantly higher than in paracan-cerous tissues(P<0.05).Among patients with TNM stage III,medium to low differentiation,and lymph node metastasis,the positive rates of MACC1 and V-ATPase were significantly elevated in comparison to patients with TNM stage I-II,high differentiation,and no lymph node metastasis(P<0.05).The rate of MACC1 positivity was 76.67%in patients with tumor diameters>5 cm,notably higher than in patients with tumor diameters≤5 cm(P<0.05).We observed a positive correlation between MACC1 and V-ATPase expression(rs=0.797,P<0.05).The positive rates of MACC1 and V-ATPase were significantly higher in patients with recurrence compared to those without(P<0.05).Logistic regression analysis revealed TNM stage,lymph node metastasis,MACC1 expression,and V-ATPase expression as risk factors for postoperative colon cancer recurrence(OR=6.322,3.435,2.683,and 2.421;P<0.05).CONCLUSION The upregulated expression of MACC1 and V-ATPase in colon cancer patients appears to correlate with clinicopathological features and post-radical surgery recurrence.

内皮型一氧化氮合酶在运动预适应改善心肌缺血-再灌注损伤中的作用

背景:运动是防治各种心血管疾病并保护心脏免受缺血-再灌注损伤的有效策略,其作用机制有待深入研究。目的:观察有氧运动预适应对心肌缺血-再灌注损伤的影响,并探讨内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)激活(包括偶联和磷酸化)在其间的作用。方法:取80只成年Wistar大鼠,采用随机数字表法分为安静组(n=40)和运动组(n=40),运动组进行8周有氧运动,安静组在鼠笼内安静饲养。8周后进行3项实验:①实验1:末次训练后,检测大鼠心功能、心脏NO代谢物含量及心脏eNOS、磷酸化eNOS-S1177、eNOS二聚体、eNOS单体的蛋白表达量;②实验2:将大鼠分为安静对照组、运动对照组、安静+eNOS抑制剂组、运动+eNOS抑制剂组,均进行体外心肌缺血-再灌注损伤实验,安静+eNOS抑制剂组、运动+eNOS抑制剂组再灌注前10 min持续灌注eNOS抑制剂,再灌注3 h后检测心功能与心肌梗死面积;③实验3:将大鼠分为安静对照组、运动对照组、安静+eNOS偶联剂组和运动+eNOS偶联剂组,均进行体外心肌缺血-再灌注损伤实验,安静+eNOS偶联剂组和运动+eNOS偶联剂组再灌注前10 min持续灌注eNOS偶联剂,再灌注3 h后检测心肌梗死面积、心脏NO代谢物含量及心脏eNOS、磷酸化eNOS-S1177、eNOS二聚体、eNOS单体和3-硝基酪氨酸的蛋白表达量(其中,磷酸化eNOS-S1177/eNOS比值反映eNOS磷酸化/去磷酸化水平,eNOS二聚体/单体比值反映eNOS偶联/解偶联水平)。结果与结论:①实验1:与安静组比较,运动组大鼠心输出量、左心室射血分数升高(P<0.05),亚硝酸盐和S-亚硝基硫醇含量升高(P<0.05),磷酸化eNOS-S1177、eNOS蛋白表达和磷酸化eNOS-S1177/eNOS比值上调(P<0.05),eNOS二聚体蛋白表达和eNOS二聚体/单体比值升高(P<0.05);②实验2:与安静对照组比较,运动对照组左心室发展压升高(P<0.05),心肌梗死面积下降(P<0.05);与运动对照组比较,运动+eNOS抑制剂组左心室发展压降低(P<0.05),心肌梗死面积增加(P<0.05);③实验3:与安静对照组比较,运动对照组左心室发展压升高(P<0.05),心肌梗死面积下降(P<0.05),磷酸化eNOS-S1177/eNOS比值下降(P<0.05),eNOS二聚体/单体比值下降(P<0.05),S-亚硝基硫醇含量增加(P<0.05),3-硝基酪氨酸蛋白表达量下调(P<0.05);与运动对照组比较,运动+eNOS偶联剂组左心室发展压降低(P<0.05),心肌梗死面积增加(P<0.05),磷酸化eNOS-S1177/eNOS比值升高(P<0.05),eNOS二聚体/单体比值升高(P<0.05),3-硝基酪氨酸蛋白表达升高(P<0.05);④结果表明:有氧运动预适应可诱导心脏保护效应,其机制与心脏缺血-再灌注期间eNOS解偶联以及去磷酸化进而抑制NO过度产生并降低硝基-氧化应激有关。

基于Fas/FasL信号通路对桂药生精胶囊治疗少精子症的临床研究

目的探讨桂药生精胶囊对少精子症(肾精亏虚型)患者的精子浓度(SC)、前向运动精子比例(PR)、PR+非前向运动精子比例(NP)、精子总数、精子脂肪酸合成酶(Fas)信使RNA(mRNA)、脂肪酸合成酶配体(FasL)mRNA表达水平及精浆Fas蛋白、FasL蛋白表达水平的影响。方法选取2019年10月至2020年3月就诊于广西中医药大学附属瑞康医院男性科门诊的少精子症(肾精亏虚型)患者60例,随机分为对照组和治疗组,每组30例。对照组采用左卡尼汀治疗,治疗组在对照组的基础上联合桂药生精胶囊治疗,12周为1个疗程。观察治疗前后两组患者的SC、PR、PR+NP、精子总数、精子Fas mRNA、FasL mRNA和精浆Fas蛋白、FasL蛋白表达水平。结果治疗组总有效率比对照组高(P<0.05)。治疗前两组患者SC、PR、PR+NP和精子总数比较,差异均无统计学意义(P>0.05)。治疗后两组患者精子SC、PR、PR+NP和精子总数均比治疗前高(P<0.05),且治疗组患者精子SC、PR和精子总数高于对照组(P<0.05)。治疗前两组患者精浆Fas和FasL蛋白表达水平比较,差异均无统计学意义(P>0.05)。治疗后两组精浆Fas蛋白表达水平低于治疗前(P<0.05),且治疗组Fas蛋白表达水平低于对照组(P<0.05)。治疗前两组患者精子Fas mRNA和FasL mRNA表达水平比较,差异均无统计学意义(P>0.05)。治疗后两组患者精子Fas mRNA和FasL mRNA表达水平低于治疗前(P<0.05),且治疗组Fas mRNA表达水平低于对照组(P<0.05)。结论桂药生精胶囊能明显提高少精子症(肾精亏虚型)患者的SC、PR、精子总数,降低精子Fas mRNA及精浆Fas蛋白的表达水平。

海洋链霉菌酮合成酶结构域基因的克隆及分析

海洋链霉菌通过聚酮合酶(PKS)合成许多结构和功能多样且具有药用价值的聚酮化合物(PKs),酮合成酶结构域(KS)作为PKS的核心结构域,可催化底物与伸长的聚酮之间的脱羧缩合,在聚酮化合物生物合成中起着重要作用。本文通过对从海洋链霉菌Streptomyces sp.X66基因组DNA克隆获得的ks基因的生物信息学分析表明,该ks基因序列长945 bp,BLAST序列比对显示其具有典型的酮合酶结构域的功能区域。理化分析显示其拟编码309个氨基酸,理论等电点为6.60,原子组成为C1401H2239N425O419S8,不稳定指数为42.11,平均亲水系数为0.112,编码产物为酸性疏水不稳定蛋白,且不含信号肽和跨膜结构,二级结构以无规则卷曲和α-螺旋为主,SDS-PAGE显示其分子量约为55 kDa。通过对ks基因的研究,为进一步解析聚酮化合物合成代谢中的调控机制及组合生物学和体外酶系合成聚酮化合物提供参考。

青蒿琥酯调节cGAS-STING信号通路对抑郁症模型小鼠神经炎性反应的影响

目的探讨青蒿琥酯(ART)调节环磷酸鸟苷-腺苷酸合成酶(cGAS)-干扰素基因刺激因子(STING)通路对抑郁症小鼠神经炎性反应的影响。方法小鼠分为模型组、对照组、ART低剂量组、ART高剂量组、氟西汀组、ART高剂量+RocA(cGAS-STING通路激活剂)组。糖水消耗实验、强迫游泳实验评估小鼠的抑郁行为;HE染色检测海马组织病理变化;ELISA检测白细胞介素(IL)-6、肿瘤坏死因子-α(TNF-α)、五羟色胺(5-HT)、多巴胺(DA)水平;TUNEL染色检测神经元凋亡;Western blot检测Bcl-2相关X蛋白(Bax)、p53、cGAS、STING蛋白。结果与对照组相比,模型组小鼠表现出神经元性脓疱变性,糖水消耗率、5-HT、DA水平降低,强迫游泳静止时间延长,IL-6、TNF-α水平、神经元凋亡率及Bax、p53、cGAS、STING蛋白表达升高(P<0.05);与模型组相比,ART低剂量组、ART高剂量组、氟西汀组小鼠海马神经元损伤有所缓解,糖水消耗率、5-HT、DA水平升高,强迫游泳静止时间缩短,IL-6、TNF-α水平、神经元凋亡率及Bax、p53、cGAS、STING蛋白表达降低(P<0.05);RocA逆转了高剂量ART对小鼠抑郁症的改善作用。结论ART抑制抑郁症小鼠神经炎性反应及神经元凋亡,上调胺类神经递质水平的机制可能与阻断cGAS-STING通路有关。

基于Fas/FasL信号通路探索舒芬太尼对急性心肌梗死大鼠心功能和心肌细胞凋亡的影响

目的基于脂肪酸合成酶(Fas)/脂肪酸合成酶配体(FasL)信号通路探索舒芬太尼对急性心肌梗死(AMI)大鼠心功能和心肌细胞凋亡的影响。方法选择健康雄性SD大鼠108只,适应性饲养7天,随机分为对照组、模型组、舒芬太尼低剂量组、舒芬太尼高剂量组、舒芬太尼高剂量+Fas阴性对照组、舒芬太尼高剂量+Fas慢病毒组,每组18只。模型组、舒芬太尼低剂量组、舒芬太尼高剂量组、舒芬太尼高剂量+Fas阴性对照组、舒芬太尼高剂量+Fas慢病毒组通过冠状动脉左前降支结扎法制作AMI模型;对照组除不结扎冠状动脉左前降支外,其余步骤与AMI模型相同。舒芬太尼低剂量组和舒芬太尼高剂量组分别于AMI模型制作成功后腹腔注射0.1、1µg/kg舒芬太尼。舒芬太尼高剂量+Fas阴性对照组和舒芬太尼高剂量+Fas慢病毒组分别于AMI模型制作成功后腹腔注射1µg/kg舒芬太尼,尾静脉注射200 nmol/kg NC shRNA慢病毒或Fas shRNA慢病毒。对照组和模型组腹腔注射等量生理盐水。术后72 h,采集大鼠尾静脉血,采用ELISA法检测血清肌钙蛋白T;采用超声心动图检测左心室射血分数(LVEF)、左心室缩短分数(LVFS)、左心室舒张末期内径(LVEDd)和左心室收缩末期内径(LVESd)。待大鼠心功能检测完成后,腹主动脉取血,采用ELISA法检测血清TNF-α、IL-6。所有大鼠断头处死,留取心脏,随机取6只大鼠的心脏组织,TTC染色,计算心肌梗死面积;随机取6只大鼠的心脏组织,HE染色,观察心肌组织病理形态变化,采用TUNEL法检测细胞凋亡情况;取剩余6只大鼠的心脏组织,采用Western blotting法检测细胞凋亡相关蛋白Bcl-2、Bax、Caspase-3及Fas/FasL信号通路相关蛋白表达。结果与对照组比较,模型组血清肌钙蛋白T水平升高,LVEDd、LVESd升高,LVEF、LVFS降低,血清TNF-α、IL-6水平升高,心肌梗死面积增大,细胞凋亡率及Bax、Caspase-3、Fas、FasL蛋白表达升高,Bcl-2蛋白表达下降(P均<0.05);与对照组比较,模型组心肌细胞形态模糊、纹理消失、排列紊乱、心肌间小血管扩张、细胞数量减少,可见大量炎性细胞浸润。与模型组比较,舒芬太尼低剂量组和舒芬太尼高剂量组血清肌钙蛋白T水平降低,LVEDd、LVESd降低,LVEF、LVFS升高,血清TNF-α、IL-6水平降低,心肌梗死面积减小,细胞凋亡率及Bax、Caspase-3、Fas、FasL蛋白表达降低,Bcl-2蛋白表达升高(P均<0.05);与模型组比较,舒芬太尼低剂量组和舒芬太尼高剂量组心肌细胞形态、纹理、排列、血管扩张、细胞数量及炎性细胞浸润显著改善。以舒芬太尼高剂量组上述效果改善较为明显。与舒芬太尼高剂量+Fas阴性对照组比较,舒芬太尼高剂量+Fas慢病毒组血清肌钙蛋白T水平升高,LVEDd、LVESd升高,LVEF、LVFS降低,血清TNF-α、IL-6水平升高,心肌梗死面积增大,细胞凋亡率及Bax、Caspase-3、Fas、FasL蛋白表达升高,Bcl-2蛋白表达降低(P均<0.05);与舒芬太尼高剂量+Fas阴性对照组比较,舒芬太尼高剂量+Fas慢病毒组心肌细胞形态模糊、纹理消失、排列紊乱、心肌间小血管扩张、细胞数量减少,炎性细胞浸润明显。结论舒芬太尼可改善AMI大鼠心功能,抑制心肌细胞凋亡,并且1µg/kg舒芬太尼的作用效果要优于0.1µg/kg舒芬太尼;抑制Fas/FasL信号通路激活可能是舒芬太尼改善AMI大鼠心功能的作用机制之一。

肺腺癌组织中前列腺素内过氧化物合酶2表达水平与表皮生长因子受体基因突变的相关性分析

目的 分析肺腺癌组织中前列腺素内过氧化物合酶2(PTGS2)表达水平与表皮生长因子受体(EGFR)基因突变的相关性。方法 选取57例肺腺癌患者为研究对象,收集肺腺癌组织及其相应癌旁组织。采用实时荧光定量PCR(RT-PCR)法检测癌组织及癌旁组织中PTGS2 mRNA表达水平;采用免疫组织化学法分析PTGS2蛋白表达;采用RT-PCR法检测癌组织及癌旁组织EGFR基因突变情况。分析肺腺癌患者临床病理参数与PTGS2 mRNA水平、EGFR基因突变情况的关系。采用Spearman秩相关分析探讨肺腺癌患者EGFR基因突变情况与PTGS2 mRNA水平的相关性。结果 57例肺腺癌患者中,5例癌旁组织EGFR基因突变型患者,其对应癌组织也均发生突变,且为同一突变类型。26例(45.61%)癌组织EGFR基因突变型患者,未发现双重突变,其中19外显子突变17例(29.82%),均为缺失突变;21外显子突变9例(15.79%),均为L858R点突变。癌组织中PTGS2mRNA表达水平、PTGS2蛋白阳性率及EGFR基因突变型比例高于癌旁组织,EGFR基因野生型比例低于癌旁组织(P<0.01)。肺腺癌患者性别、TNM分期、吸烟与PTGS2 mRNA表达水平、EGFR基因突变情况有关(P<0.05或0.01)。EGFR基因突变型PTGS2 mRNA表达水平高于EGFR基因野生型(P<0.01)。肺腺癌患者EGFR基因突变与PTGS2 mRNA表达水平呈正相关(r=0.512,P<0.01)。结论 肺腺癌患者癌组织中PTGS2mRNA表达水平及PTGS2蛋白阳性率升高,且与EGFR基因突变关系密切,二者可能共同影响疾病进程。