Post-translational modifications of prostaglandin-endoperoxide synthase 2 in colorectal cancer:An update

The biosynthesis of prostanoids is involved in both physiological and pathological processes. The expression of prostaglandin-endoperoxide synthase 2(PTGS2; also known as COX-2) has been traditionally associated to the onset of several pathologies, from inflammation to cardiovascular, gastrointestinal and oncologic events. For this reason, the search of selective PTGS2 inhibitors has been a focus for therapeutic interventions. In addition to the classic non-steroidal anti-inflammatory drugs, selective and specific PTGS2 inhibitors, termed coxibs, have been generated and widely used. PTGS2 activity is less restrictive in terms of substrate specificity than the homeostatic counterpart PTGS1, and it accounts for the elevated prostanoid synthesis that accompanies several pathologies. The main regulation of PTGS2 occurs at the transcription level. In addition to this, the stability of the mRNA is finely regulated through the interaction with several cytoplasmic elements, ranging from specificmicroR NAs to proteins that control mR NA degradation. Moreover, the protein has been recognized to be the substrate for several post-translational modifications that affect both the enzyme activity and the targeting for degradation via proteasomal and non-proteasomal mechanisms. Among these modifications, phosphorylation, glycosylation and covalent modifications by reactive lipidic intermediates and by free radicals associated to the proinflammatory condition appear to be the main changes. Identification of these post-translational modifications is relevant to better understand the role of PTGS2 in several pathologies and to establish a correct analysis of the potential function of this protein in diseases progress. Finally, these modifications can be used as biomarkers to establish correlations with other parameters, including the immunomodulation dependent on molecular pathological epidemiology determinants, which may provide a better frame for potential therapeutic interventions.

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.

cDNA Cloning and Expression Analysis of the Chalcone Synthases (CHS) in <i>Osmanthus fragrans</i>

In the flavonoid biosynthesis pathway, Chalcone synthase (CHS) is involved in the formation of the pigment and has been shown to be a rate-limiting enzyme for the synthesis of flavonoids. In this study, a PCR approach was used to clone a Chalcone synthases cDNA from flower of sweet osmanthus “Chenghong Dangui” and it was designated as OfCHS (O. fragrans, CHS). The cDNA was 1383 bp long and a coding sequence (CDS) of 1173 bp encoding a polypeptide of 391 amino acids with an estimated molecular mass of 39.9 kDa. The theoretical isoelectric point was 6.23. Phylogenetic analysis demonstrated that OfCHS clustered with Olea europaea, Solenostemon scutellarioides, Perilla frutescens, Antirrhinum majus and Digitalis lanata. We also detected the expression of OfCHS in different tissues in “Dangui” and in two cultivars with varied coloration, “Zi Yingui” and “Chenghong Dangui” at different floral stages using quantitative real-time PCR. We observed that OfCHS transcript was higher in leaves than in petals in “Dangui”. The transcripts of OfCHS in “Zi Yingui” petals were higher than those in “Dangui” at three stages especially at xianyan stage and there was no significant difference between the two cultivars in the full flowering stage. “Chenghong Dangui” has a relatively high anthocyanin content compared to “Zi Yingui”. The relative amount of anthocyanin of “Chenghong Dangui” initially increases, and then decreases during the bloom period. However, the expression of CHS is the highest at the initial flowering stage. These data suggest that the OfCHS does not play a key role in the accumulation of total flavonoid in this cultivar. These data could contribute to explain the different accumulation of flavonoids in petals of the two cultivars.

Role of Nitric Oxide and Nitric Oxide Synthases in Ischemia-reperfusion Injury in Rat Organotypic Hippocampus Slice

To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthasc isoforms （nNOS and iNOS）, rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia by oxygen glucose deprivation （OGD） for 30 min and then placed in the normal culture condition. The ischemia-reperfusion produced a time-dependent increase in nitrite levels in the culture medium. Reverse transcriptional-polymerase chain reaction showed augmented levels of mRNA for both nNOS and iNOS when compared with control at 12 h and remained increase at 36 h after OGD （P〈0.05）. The protein levels of both nitric oxide synthase isoforms increased significantly as determined by Western Blot. OGD also caused neurotoxicity in this model as revealed by the elevated lactate dehydrogenase （LDH） efflux into the incubation solution. The resuits suggest that organotypic hippocampus slice is a useful model in studying ischemia-reperfusion brain injury. NO and NOS may play a critical role in the ischemia-reperfusion brain damage in vitro.

In vitro demonstration of interactions among zinc-binding domains of cellulose synthases in Arabidopsis and aspen

Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible interactions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were detected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant species could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.

Two farnesyl pyrophosphate synthases,GhFPS1–2,in Gossypium hirsutum are involved in the biosynthesis of farnesol to attract parasitoid wasps

Sesquiterpenoids play an import role in the direct or indirect defense of plants.Farnesyl pyrophosphate synthases(FPSs)catalyze the biosynthesis of farnesyl pyrophosphate,which is a key precursor of farnesol and(E)-β-farnesene.In the current study,two FPS genes in Gossypium hirsutum,GhFPS1 and GhFPS2,were heterologously cloned and functionally characterized in a greenhouse setting.The open reading frames for full-length GhFPS1 and GhFPS2 were each 1029 nucleotides,and encoded two proteins of 342 amino acids with molecular weights of 39.4 kDa.The deduced amino acid sequences of GhFPS1–2 showed high identity to FPSs of other plants.Quantitative real-time PCR analysis revealed that GhFPS1 and GhFPS2 were highly expressed in G.hirsutum leaves,and were upregulated in methyl jasmonate(MeJA)-,methyl salicylate(MeSA)-and aphid infestation-treated cotton plants.The recombinant proteins of either GhFPS1 or GhFPS2 plus calf intestinal alkaline phosphatase could convert geranyl diphosphate(GPP)or isopentenyl diphosphate(IPP)to one major product,farnesol.Moreover,in electrophysiological response and Y-tube olfactometer assays,farnesol showed obvious attractiveness to female Aphidius gifuensis,which is an important parasitic wasp of aphids.Our findings suggest that two GhFPSs are involved in farnesol biosynthesis and they play a crucial role in indirect defense of cotton against aphid infestation.

Differential expression of apoptosis related proteins and nitric oxide synthases in Epstein Barr associated gastric carcinomas

瞄准：为了决定 Epstein Barr 的发生，病毒在巴西联系了胃的癌(GC ) 并且比较 apoptosis 的表情在 EBV 之间的相关蛋白质和氮的氧化物 synthases 积极、否定的胃的癌。方法：编码 EBV 的小 RNA-1 (EBER-1 ) 和 PCR 的原位杂交被执行在 GC 识别 EBV 的存在。Immunohistochemistry 被用来在 25 EBV 积极 GC 并且在 103 EBV 否定 GCS 识别 bcl-2， bcl-xl， bak， bax， p53， NOS-1， NOS-2，和 NOS-3 蛋白质的表情。结果：GC (25/208 ) 的 12% 盒子显示出 EBER-1 和 EBNA-1 表示。盒子是优先地 diffuse 打字与强烈淋巴在基质渗入。EBV 联系了 GC bcl-2 蛋白质的显示出的更高的表示和 bak 蛋白质的更低的表示与比在 EBV 否定 GC。确实， NOS-1 和 NOS-3 的表情经常在 EBV 被观察联系 GC。结论：我们的数据建议 EBV 感染可以保护肿瘤房间免受 apoptosis 的伤害，为永久房间骑车和增长给他们能力。另外， EBV 积极 GC 显示出能影响肿瘤前进和攻击性的组成的 NOS 的高表示。

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。