Discovery of proqodine A derivatives with antitumor activity targeting NAD(P)H: quinone oxidoreductase 1 and nicotinamide phosphoribosyltransferase / 中国天然药物

NAD(P)H: quinone oxidoreductase 1 (NQO1) is a flavin protease highly expressed in various cancer cells. NQO1 catalyzes a futile redox cycle in substrates, leading to substantial reactive oxygen species (ROS) production. This ROS generation results in extensive DNA damage and elevated poly (ADP-ribose) polymerase 1 (PARP1)-mediated consumption of nicotinamide adenine dinucleotide (NAD+), ultimately causing cell death. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage synthesis pathway, emerges as a critical target in cancer therapy. The concurrent inhibition of NQO1 and NAMPT triggers hyperactivation of PARP1 and intensive NAD+ depletion. In this study, we designed, synthesized, and assessed a novel series of proqodine A derivatives targeting both NQO1 and NAMPT. Among these, compound T8 demonstrated potent antitumor properties. Specifically, T8 selectively inhibited the proliferation of MCF-7 cells and induced apoptosis through mechanisms dependent on both NQO1 and NAMPT. This discovery offers a promising new molecular entity for advancing anticancer research.

Modification of C20 oxidase in tanshinone biosynthesis pathway / 中国中药杂志

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.

Genotype-phenotype correlations, surgical selections, and postoperative complications of 5α-reductase 2 deficiency in 69 children with hypospadias / 亚洲男科学杂志(英文版)

5α-reductase 2 deficiency prevents testosterone from being converted to dihydrotestosterone, which causes abnormal urogenital sinus development. The aim of this study was to analyze the relationship between genotype-phenotype, surgical selections, and postoperative complications of 5α-reductase 2-deficient patients with hypospadias. We retrospectively evaluated the medical records of patients who were diagnosed with 5α-reductase 2 deficiency after genetic testing in the Department of Endocrinology and underwent initial hypospadias surgery in the Department of Urology in Beijing Children's Hospital, Capital Medical University (Beijing, China), from April 2007 to December 2021. A total of 69 patients were included in this study; the mean age at surgery was 34.1 months, and the average follow-up time was 54.1 months. Sixty children were treated with preoperative hormone stimulation (PHS) to promote penile growth. The average penis length and glans width were increased by 1.46 cm and 0.62 cm, respectively. The most frequent mutations were p.R227Q (39.1%, 54/138), p.Q6* (15.2%, 21/138), p.G203S (12.3%, 17/138), and p.R246Q (11.6%, 16/138). In 64 patients who were followed up, 43 had a one-stage operation and 21 had a staged operation, and there were significant differences in external masculinization score (EMS) ( P = 0.008) and the average number of operation required to cure ( P < 0.001) between one-stage and staged operations. PHS had a positive effect ( P < 0.001) on penile development. The p.R227Q mutation was associated with higher EMS and less severe hypospadias. One-stage surgery can be selected if conditions permit. The growth and development of children are acceptable in the long term, but penis growth remains unsatisfactory. Long-term complications of hypospadias should be considered during puberty.

Analysis of clinical phenotypes and MMACHC gene variants in 65 children with Methylmalonic acidemia and homocysteinemia / 中华医学遗传学杂志

OBJECTIVE@#To carry out Sanger sequencing for MMACHC gene variants among 65 Chinese pedigrees affected with combined methylmalonic aciduria and homocysteinemia, and summarize their genetic and clinical characteristics and prognosis.@*METHODS@#Clinical characteristics of the 65 children identified with Methylmalonic acidemia and homocysteinemia at the Children's Hospital Affiliated to Zhengzhou University (Zhengzhou Children's Hospital) from April 2017 to April 2022 were selected as the study subjects. Potential variants of the MMACHC gene were detected by direct sequencing of the PCR products.@*RESULTS@#The median age of the 65 children was 3 months (14 days to 17 years old). These included 28 cases (43.08%) from neonatal screening, 11 cases (16.92%) with a history of jaundice, and 9 cases (13.85%) with various degrees of anemia. The main clinical symptoms included development delay, slow growth, epilepsy, hydrocephalus, lethargy, feeding difficulty, regression or decline in motor ability, recurrent respiratory infections, anemia, jaundice, respiratory and heart failures, hydrocephalus, limb weakness, and hypertension. Blood and urine tandem mass spectrometry screening has revealed increase of methylmalonic acid, propionyl carnitine, propionyl carnitine/acetylcarnitine ratio, and propionyl carnitine/free carnitine ratio to various extents, and blood homocysteine was increased in all patients. The detection rate of genetic variants was 98.46% (128/130), and in total 22 types of MMACHC gene variants were detected. The most common ones have included c.609G>A (W203X) (58/128), c.658-660del (K220del) (19/128), and c.80A>G (Q27A) (16/128). Two novel variants have been identified, namely c.565C>T (p.R189C) and c.624_ 625delTG (p.A208Afs), which were respectively predicted as likely pathogenic (PM2_Supporting+PM3+PP2+PP3) and pathogenic (PVS1+PM2_Supporting+PM3+PP2) based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). Exon 4 had the highest frequency for the detection.@*CONCLUSION@#Identification of MMACHC gene variants has confirmed the diagnosis in the children, among which the c.609G>A variant has the highest frequency. Discovery of the new variants has enriched the mutational spectrum of the MMACHC gene.

Transcriptomic analysis of the ΔPaLoc mutant of Clostridioides difficile and verification of its toxicity / 中华预防医学杂志

Objective: Comparative analyses of wild-type Clostridioides difficile 630 (Cd630) strain and pathogenicity locus (PaLoc) knockout mutant (ΔPaLoc) by using RNA-seq technology. Analysis of differential expression of Cd630 wild-type strain and ΔPaLoc mutant strain and measurement of its cellular virulence changes. Lay the foundation for the construction of an toxin-attenuated vaccine strain against Clostridioides difficile. Methods: Analysis of Cd630 and ΔPaLoc mutant strains using high-throughput sequencing (RNA-seq). Clustering differentially expressed genes and screening differentially expressed genes by DESeq software. Further analysis of differential genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, cytotoxicity assays of ΔPaLoc and Cd630 strains were performed in the African monkey kidney epithelial cell (Vero) and the human colonic cell (Caco-2) lines. Results: The transcriptome data showed that the ΔPaLoc mutant toxin genes tcdA and tcdB were not transcribed. Compared to the wild-type strain, CD630_36010, CD630_020910,CD630_02080 and cel genes upregulated 17.92,11.40,8.93 and 7.55 fold, respectively. Whereas the hom2 (high serine dehydrogenase), the CD630_15810 (spore-forming protein), CD630_23230 (zinc-binding dehydrogenase) and CD630_23240 (galactitol 1-phosphate 5-dehydrogenase) genes were down-regulated by 0.06, 0.075, 0.133 and 0.183 fold, respectively. The GO and KEGG enrichment analyses showed that the differentially transcribed genes in ΔPaLoc were enriched in the density-sensing system, ABC transport system, two-component system, phosphotransferase (PTS) system, and sugar metabolism pathway, as well as vancomycin resistance-related pathways. Cytotoxicity assays showed that the ΔPaLoc mutant strain lost its virulence to Vero and Caco-2 cells compared to the wild-type Cd630 strain. Conclusion: Transcriptional sequencing analysis of the Cd630 and ΔPaLoc mutant strains showed that the toxin genes were not transcribed. Those other differential genes could provide a reference for further studies on the physiological and biochemical properties of the ΔPaLoc mutant strain. Cytotoxicity assays confirmed that the ΔPaLoc mutant lost virulence to Vero and Caco-2 cells, thus laying the foundation for constructing an toxin-attenuated vaccine strain against C. difficile.

Pre-conception carrier screening for 21 inherited metabolic diseases in a Chinese population / 中华医学遗传学杂志

OBJECTIVE@#To determine the carrier rate for 21 inherited metabolic diseases among a Chinese population of childbearing age.@*METHODS@#A total of 897 unrelated healthy individuals (including 143 couples) were recruited, and DNA was extracted from their peripheral blood samples. Whole exome sequencing (WES) was carried out to screen potential variants among 54 genes associated with 21 inherited metabolic diseases. Pathogenic and likely pathogenic variants and unreported loss-of-function variants were analyzed.@*RESULTS@#One hundred fourty types of pathogenic/likely pathogenic variants (with an overall number of 183) and unreported loss-of-function variants were detected, which yield a frequency of 0.20 per capita. A husband and wife were both found to carry pathogenic variants of the SLC25A13 gene and have given birth to a healthy baby with the aid of preimplantation genetic diagnosis. The detected variants have involved 40 genes, with the most common ones including ATP7B, SLC25A13, PAH, CBS and MMACHC. Based on the Hardy-Weinberg equilibrium, the incidence of the 21 inherited metabolic diseases in the population was approximately 1/1100, with the five diseases with higher incidence including citrullinemia, methylmalonic acidemia, Wilson disease, glycogen storage disease, and phenylketonuria.@*CONCLUSION@#This study has preliminarily determined the carrier rate and incidence of 21 inherited metabolic diseases among a Chinese population of childbearing age, which has provided valuable information for the design of neonatal screening program for inherited metabolic diseases. Pre-conception carrier screening can provide an important measure for the prevention of transmission of Mendelian disorders in the population.

Genetic analysis of 21 cases of methylmalonic acidemia / 中华医学遗传学杂志

OBJECTIVE@#To carry out genetic analysis for 21 patients with methylmalonic acidemia (MMA) and provide genetic counseling for their families.@*METHODS@#Next generation sequencing (panel) was used to detect the pathogenic variants underlying the disease.@*RESULTS@#In total 29 variant sites of MMUT, MMAA, MMUT were identified in the 21 patients, with common variants including c.323G>A (10%), c.917C>T (10%), c.984delC (10%) of MMUT gene, and c.609G>A (45%), c.80A>G (10%) , c.567dupT (10%) of MMACHC gene. Among these, c.2000A>G of MMUT, c.298G>T of MMACHC and c.734-7A>G of MMAA gene were unreported previously.@*CONCLUSION@#Genetic testing for MMA patients can clarify the cause of the disease and provide a basis for the clinical diagnosis. Discovery of novel variants has enriched the mutational spectrum of MMA.

Current status of whitening agents and enzymes in Dentistry

Abstract This study reviews the knowledge on the use of conventional dental whitening and the use of enzymes as a new approach in bleaching. A review of the literature was based on academic articles and on patents related to the use of enzymes in dental bleaching. Tooth whitening techniques used nowadays are well reported in the literature, and its mechanism of action consists of an oxidoreduction reaction with the release of free radicals. The great instability of radicals, when in contact with the tissues, promotes oxidation and reduction in the size of the pigment chains incorporated into them. These pigments are eventually broken down into smaller and smaller molecular chains and end up being diffused from the dental structure. In turn, the use of enzymes aimed at tooth whitening can be a less harmful alternative to the tooth because their specificity regarding the substrate makes them of great interest to perform specific reactions, reducing collateral effects. The use of proteolytic enzymes and oxidoreductases paired with the application of peroxides, can be a promising alternative for obtaining even better results in the dental bleaching process.

Downregulation of ceramide synthase 1 promotes oral cancer through endoplasmic reticulum stress / 国际口腔科学杂志·英文版

C18 ceramide plays an important role in the occurrence and development of oral squamous cell carcinoma. However, the function of ceramide synthase 1, a key enzyme in C18 ceramide synthesis, in oral squamous cell carcinoma is still unclear. The aim of our study was to investigate the relationship between ceramide synthase 1 and oral cancer. In this study, we found that the expression of ceramide synthase 1 was downregulated in oral cancer tissues and cell lines. In a mouse oral squamous cell carcinoma model induced by 4-nitroquinolin-1-oxide, ceramide synthase 1 knockout was associated with the severity of oral malignant transformation. Immunohistochemical studies showed significant upregulation of PCNA, MMP2, MMP9, and BCL2 expression and downregulation of BAX expression in the pathological hyperplastic area. In addition, ceramide synthase 1 knockdown promoted cell proliferation, migration, and invasion in vitro. Overexpression of CERS1 obtained the opposite effect. Ceramide synthase 1 knockdown caused endoplasmic reticulum stress and induced the VEGFA upregulation. Activating transcription factor 4 is responsible for ceramide synthase 1 knockdown caused VEGFA transcriptional upregulation. In addition, mild endoplasmic reticulum stress caused by ceramide synthase 1 knockdown could induce cisplatin resistance. Taken together, our study suggests that ceramide synthase 1 is downregulated in oral cancer and promotes the aggressiveness of oral squamous cell carcinoma and chemotherapeutic drug resistance.

Evaluation of Multidrug Resistant Loop-mediated Isothermal Amplification Assay for Detecting the Drug Resistance of / 生物医学与环境科学（英文）

Objective@#To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the @*Methods@#MDR-LAMP assay was evaluated using 100 @*Results@#The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of @*Conclusion@#MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of

Progress in vitamin C biosynthesis related dehydrogenases / 生物工程学报

Vitamin C is an essential vitamin for human beings. It has a huge market in the fields of food and pharmaceuticals. 2-keto-L-gulonic acid is an important precursor to produce vitamin C by microbial fermentation in industrial. In microbial fermentations, the L-sorbose pathway and the D-gluconate pathway have been the focus of research because of high yield. This article aims at stating recent research progress in dehydrogenases related to biosynthesis of vitamin C in the L-sorbose pathway and the D-gluconate pathway. The properties of dehydrogenase in terms of localization, substrate specificity, cofactors, and electron transport carrier are elaborated. And then, the main problems and strategies are reviewed in the L-sorbose pathway and in the D-gluconate pathway. Finally, future research on the dehydrogenases in the biosynthesis of vitamin C through L-sorbose pathway and D-gluconate pathway is discussed.

Preparation of anti EGFR scFv::FTH1/FTH1 nanoparticles for asthma treatment / 生物工程学报

Asthma is a common respiratory disease that affects 300 million of people worldwide, posing a serious health risk and medical burden. Development of new anti-asthmatic drugs and alternative treatment regimens is therefore encouraged. Recent studies have shown that Epidermal Growth Factor Receptor (EGFR) is involved in asthma development. In order to construct nanoparticles targeting EGFR for asthma treatment, a single chain antibody fragment (scFv) against EGFR was genetically engineered and modified at the N-terminal end of the human ferritin H-chain (FTH1) to construct Anti EGFR scFv::FTH1/FTH1 nanoparticles. Transmission electron microscopy showed that the nanoparticles were self-assembled into hollow cage-like structures with the particle size of about 12 nm. Semi-quantitative analysis of the purified nanoparticles by SDS-PAGE revealed the mass ratio of FTH1 to Anti EGFR scFv::FTH1 was 7:3. In House Dust Mite (HDM) driven models, Anti EGFR scFv::FTH1/FTH1 nanoparticles efficiently attenuated several key features of asthma, including goblet cell hyperplasia, mucous metaplasia and subepithelial fibrosis, showing the potential of using ferritin based nanoparticle for asthma treatment.

Advances of structure, function, and catalytic mechanism of methyl-coenzyme M reductase / 生物工程学报

Methanogens are unique microorganisms for methane production and the main contributor of the biogenic methane in atmosphere. Methyl-coenzyme M reductase (Mcr) catalyzes the last step of methane production in methanogenesis and the first step of methane activation in anaerobic oxidation of methane. The genes encoding this enzyme are highly conserved and are widely used as a marker in the identification and phylogenetic study of archaea. There has been a longstanding interest in its unique cofactor F430 and the underpinning mechanisms of enzymatic cleavage of alkane C-H bond. The recent breakthroughs of high-resolution protein and catalytic-transition-state structures further advanced the structure-function study of Mcr. In particular, the recent discovery of methyl-coenzyme M reductase-like (Mcr-like) enzymes that activates the anaerobic degradation of non-methane alkanes has attracted much interest in the molecular mechanisms of C-H activation without oxygen. This review summarized the advances on function-structure-mechanism study of Mcr/Mcr-like enzymes. Additionally, future directions in anaerobic oxidation of alkanes and greenhouse-gas control using Mcr/Mcr-like enzymes were proposed.

Expression and characterization of recombinant wheat quiescin sulfhydryl oxidase and its effect on bread quality / 生物工程学报

Wheat quiescin sulfhydryl oxidase was expressed in Escherichia coli for developing a new biological flour improver. The synthesized wqsox gene was constructed into the vector pMAL-c5x and expressed in E. coli, then the expression conditions of recombinant protein was optimized. The MBP fusion label in recombinant protein was removed by protease digestion after affinity purification. Moreover, enzymatic properties of the purified wQSOX and its effect on bread quality were investigated. The synthesized wqsox gene contained 1 359 bp and encoded 453 amino acids with a deduced molecular weight of 51 kDa. The constructed recombinant vector pMAL-c5x-wqsox could successfully express soluble recombinant protein MBP-wQSOX in E. coli Rosetta gamiB(DE3), and the optimal induced expression conditions for recombinant protein were 25 °C, 0.3 mmol/L IPTG and 6 h. MBP fusion tag was cut out by factor Xa protease and wQSOX was prepared after affinity purification. wQSOX could catalyze the oxidation of DTT, GSH and Cys, accompanying the production of H2O2, and exhibited the highest substrate specificity for DTT. Furthermore, enzymatic properties results demonstrated that the optimal temperature and pH for wQSOX catalyzing oxidation of DTT was 50 °C and 10.0, respectively, and wQSOX presented a good stability under high temperature and alkaline environment. The addition of wQSOX with 1.1 U/g flour significantly (P<0.05) increased 26.4% specific volume of the bread, and reduced 20.5% hardness and 24.8% chewiness of bread crumb compared to the control, indicating a remarkable ability to improve the quality of bread.

Application and the limitation of next generation sequencing for the diagnosis of methylmalonic acidemia / 中华医学遗传学杂志

OBJECTIVE@#To identify genetic variants among patients with methylmalonic acidemia and provide genetic evidence for prenatal diagnosis.@*METHODS@#Thirty-one probands and their parents were subjected to next generation sequencing (NGS). Suspected variants were verified by Sanger sequencing.@*RESULTS@#25 probands or their parents were found to harbor previously known pathogenic or likely pathogenic variants, and three probands were found to carry heterozygous MMACHC exonic deletion. The overall diagnostic yield was 90.32%.@*CONCLUSION@#NGS can improve the detection rate for methylmalonic acidemia for its accuracy and efficiency, yet the detection of exonic deletion is required to further improve the diagnostic yield. The identification of specific variants provided evidence for prenatal diagnosis.

P-I metalloproteinases and L-amino acid oxidases from Bothrops species inhibit angiogenesis

Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia. In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. Methods: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. Results: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially β chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. Conclusion: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo.(AU)

Cardioprotective effect of eicosapentaenoic acid during ischemia and reperfusion / Efeito cardioprotetor do ácido eicosapentaenóico durante a isquemia e reperfusão

Objective: In this work, rats isolated hearts were infused EPA before the ischemia period and during reperfusion for available get well in parameter relatives to redox reactions. Methods: The effect of EPA was tested on isolated hearts induced to ischemia and reperfusion, treatment occurred at different times (ischemia or reperfusion). Antioxidant capacity against peroxyl radicals, glutathione cysteine ligase activity, glutathione concentration, lactate dehydrogenase, and creatine kinase concentration was analyzed. Results: Hearts treated with eicosapentaenoic acid had the minor generation of species reactive oxygen and lipid damage after reperfusion. The GSH concentration was higher when the hearts were treated with eicosapentaenoic acid in the period of reperfusion. Conclusion: In conclusion, this study demonstrates that the dose of EPA (20µM) used before ischemia can act as a cardioprotective antioxidant molecule, prevented damage heart from ischemic and reperfusion injury

Objetivo: Neste trabalho, corações isolados de ratos foram infundidos com EPA antes do período de isquemia e durante a reperfusão para obtenção de melhora em parâmetros relativos às reações redox. Métodos: O efeito do EPA foi testado em corações isolados induzidos a isquemia e reperfusão, o tratamento ocorreu em diferentes momentos (isquemia ou reperfusão). A capacidade antioxidante contra os radicais peroxil, atividade da glutationa cisteína ligase, concentração de glutationa, lactato desidrogenase e concentração de creatina quinase foi analisada. Resultados: Corações tratados com ácido eicosapentaenóico tiveram a menor geração de espécies reativas de oxigênio e danos lipídicos após a reperfusão. A concentração de GSH foi maior quando os corações foram tratados com ácido eicosapentaenóico no período de reperfusão. Conclusão: Em conclusão, este estudo demonstra que a dose de EPA (20µM) utilizada antes da isquemia pode atuar como uma molécula antioxidante cardioprotetora, prevenindo danos ao coração por isquemia e lesão de reperfusão.

Exploring the five-paced viper (Deinagkistrodon acutus) venom proteome by integrating a combinatorial peptide ligand library approach with shotgun LC-MS/MS

Snake venoms are complex mixtures of toxic proteins or peptides encoded by various gene families that function synergistically to incapacitate prey. In the present study, in order to unravel the proteomic repertoire of Deinagkistrodon acutus venom, some trace abundance components were analyzed. Methods Shotgun proteomic approach combined with shotgun nano-LC-ESI-MS/MS were employed to characterize the medically important D. acutus venom, after collected samples were enriched with the combinatorial peptide ligand library (CPLL). Results This avenue helped us find some trace components, undetected before, in D. acutus venom. The results indicated that D. acutus venom comprised 84 distinct proteins from 10 toxin families and 12 other proteins. These results are more than twice the number of venom components obtained from previous studies, which were only 29 distinct proteins obtained through RP-HPLC for the venom of the same species. The present results indicated that in D. acutus venom, the most abundant components (66.9%) included metalloproteinases, serine proteinases, and C-type lectin proteins; the medium abundant components (13%) comprised phospholipases A2 (PLA2) and 5'-nucleotidases and nucleases; whereas least abundant components (6%) were aminopeptidases, L-amino acid oxidases (LAAO), neurotoxins and disintegrins; and the trace components. The last were undetected before the use of conventional shotgun proteomics combined with shotgun nano-LC-ESI-MS/MS, such as cysteine-rich secretory proteins Da-CRPa, phospholipases B-like 1, phospholipases B (PLB), nerve growth factors (NGF), glutaminyl-peptide cyclortransferases (QC), and vascular non-inflammatory molecules 2 (VNN2). Conclusion These findings demonstrated that the CPLL enrichment method worked well in finding the trace toxin proteins in D. acutus venom, in contrast with the previous venomic characterization of D. acutus by conventional LC-MS/MS. In conclusion, this approach combined with the CPLL enrichment was effective for allowing us to explore the hidden D. acutus venomic profile and extended the list of potential venom toxins.(AU)

Reusable Enzymatic Strip for Detection of Arsenic

HIGHLIGHTS Arsenic is considered as one of the highly hazardous elements in the environment and a serious carcinogen for the human health. An enzymatic method has been described by using arsenite oxidase for arsenic detection. Residual activity of the immobilized enzyme was 43% of the initial activity after being recycled 10 times.

Abstract Arsenic is considered as one of the highly hazardous elements in the environment and a serious carcinogen for the human health. More attention has taken towards the arsenic due to its presence in ground water in India, China, Bangladesh, Inner Mongolia and several other regions of the world. It's been a challenge to remove arsenic due to the lack of its efficient detection approach in the complicated environmental matrix. The proposed method describes an enzymatic method for arsenic determination using arsenite oxidase, which catalyzes the oxidation of arsenite to arsenate. Hence, a colorimetric PVC strip with immobilized arsenite oxidase has been developed to detect the arsenic concentration and also having potential for the field-testing. The influence of the optimal conditions i.e. pH, temperature, storage stability, and reusability of free and immobilized enzyme were evaluated and compared. The results have shown that the stabilities were significantly enhanced compared with free counterpart. Residual activity of the immobilized enzyme was 43% of the initial activity after being recycled 10 times. We approve that this novel low cost immobilized carrier presents a new approach in large scale applications and expected to act as a model for establishment of indigenous arsenic sensor in miniature form.