Draft genome sequence data of heavy metal-resistant Morganella morganii WA01/MUTU, a silver nanoparticle (AgNP) synthesising bacterium.

Morganella morganii WA01/MUTU is a heavy metal tolerant strain capable of producing silver nanoparticles (AgNPs) from AgNO3. Here we present the draft genome sequence of M. morganii WA01/MUTU isolated from a water sample collected in Nakhon Pathom province, Thailand. The draft genome was sequenced on the Illumina NextSeq 550 sequencer. The genome consisted of 34 contigs with a total size of 3,991,804 bp, an N50 value of 364,423 bp and a GC content of 50.93%. The digital DNA-DNA hybridisation (dDDH) between WA01/MUTU and Morganella morganii (NBRC 3848) was 83.9%, identifying the strain as Morganella morganii. The data presented here can be used in comparative genomics to identify gene clusters involved in AgNP biosynthesis and secondary metabolite production. The draft genome sequence data was deposited at NCBI under Bioproject accession number PRJNA493966.

Annexin A2 Improves the Osteogenic Differentiation of Mesenchymal Stem Cells Exposed to High-Glucose Conditions through Lessening the Senescence.

Osteoporosis is frequently found in chronic diabetic patients, and it results in an increased risk of bone fractures occurring. The underlying mechanism of osteoporosis in diabetic patients is still largely unknown. Annexin A2 (ANXA2), a family of calcium-binding proteins, has been reported to be involved in many biological process including bone remodeling. This study aimed to investigate the role of ANXA2 in mesenchymal stem cells (MSCs) during in vitro osteoinduction under high-glucose concentrations. Osteogenic gene expression, calcium deposition, and cellular senescence were determined. The high-glucose conditions reduced the osteogenic differentiation potential of the MSCs along with the lower expression of ANXA2. Moreover, the high-glucose conditions increased the cellular senescence of the MSCs as determined by senescence-associated ß-galactosidase staining and the expression of p16, p21, and p53 genes. The addition of recombinant ANXA2 could recover the glucose-induced deterioration of the osteogenic differentiation of the MSCs and ameliorate the glucose-induced cellular senescence of the MSCs. A Western blot analysis revealed an increase in p53 and phosphorylated p53 (Ser 15), which was decreased by recombinant ANXA2 in MSC osteoblastic differentiation under high-glucose conditions. Our study suggested that the alteration of ANXA2 in high-glucose conditions may be one of the plausible factors in the deterioration of bones in diabetic patients by triggering cellular senescence.

Boiling, Blanching, and Stir-Frying Markedly Reduce Pesticide Residues in Vegetables.

Nowadays, a lot of produce (fruits and vegetables) sold in many countries are contaminated with pesticide residues, which cause severe effects on consumer health, such as cancer and neurological disorders. Therefore, this study aims to determine whether cooking processes can reduce the pesticide residues in commonly consumed vegetables (Chinese kale and yard long beans) in Thailand. For cooking experiments, the two vegetables were cooked using three different processes: boiling, blanching, and stir-frying. After the treatments, all cooked and control samples were subjected to extraction and GC-MS/MS analysis for 88 pesticides. The results demonstrated that pesticide residues were reduced by 18-71% after boiling, 36-100% after blanching, and 25-60% after stir-frying for Chinese kale. For yard long beans, pesticide residues were reduced by 38-100% after boiling, 27-28% after blanching, and 35-63% after stir-frying. Therefore, cooking vegetables are proven to protect consumers from ingesting pesticide residues.

Protective Efficacy of Spilanthes acmella Murr. Extracts and Bioactive Constituents in Neuronal Cell Death.

Spilanthes acmella Murr., a well-known Thai traditional medicine, has been used for treatment of toothache, rheumatism, and fever. Diverse pharmacological activities of S. acmella Murr. have been reported. In this study, antioxidative and neuroprotective effects of S. acmella Murr. extracts as well as bioactive scopoletin, vanillic acid, and trans-ferulic acid found in the aerial parts of this plant species have been described. Protective effect of S. acmella Murr. extracts and bioactive compounds on dexamethasone-induced neuronal cell death was investigated. Different plant crude ethyl acetate (EtOAc) and methanol (MeOH) extracts including pure compounds of S. acmella Murr. were evaluated in human neuroblastoma SH-SY5Y cells. Cytotoxic effects were performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Mechanisms involved in the antioxidant effects of S. acmella Murr. regarding the activation of antioxidant marker proteins such as superoxide dismutase 2 (SOD2) and sirtuin 3 (SIRT3) were determined using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay, Western blot analysis, and immunocytochemistry. Dexamethasone significantly caused the decrease of SH-SY5Y cell viability. Conversely, the increases in reactive oxygen species (ROS), autophagy, and apoptosis were observed in dexamethasone-treated cells. S. acmella Murr. MeOH and EtOAc extracts, as well as the bioactive compounds, reversed the toxic effect of dexamethasone by increasing the cell viability, SIRT3 protein expression but reducing the ROS, autophagy, and apoptosis. This study demonstrated that S. acmella Murr. may exert its protective effects against ROS through SOD2 and SIRT3 signaling pathways in dexamethasone-induced neurotoxicity. S. acmella Murr. may be a candidate therapy for neuroprotection.

Emergence of plasmid-mediated colistin resistance mcr-3.5 gene in Citrobacter amalonaticus and Citrobacter sedlakii isolated from healthy individual in Thailand.

Citrobacter spp. are Gram-negative bacteria commonly found in environments and intestinal tracts of humans and animals. They are generally susceptible to third-generation cephalosporins, carbapenems and colistin. However, several antibiotic resistant genes have been increasingly reported in Citrobacter spp., which leads to the postulation that Citrobacter spp. could potentially be a reservoir for spreading of antimicrobial resistant genes. In this study, we characterized two colistin-resistant Citrobacter spp. isolated from the feces of a healthy individual in Thailand. Based on MALDI-TOF and ribosomal multilocus sequence typing, both strains were identified as Citrobacter sedlakii and Citrobacter amalonaticus. Genomic analysis and S1-nuclease pulsed field gel electrophoresis/DNA hybridization revealed that Citrobacter sedlakii and Citrobacter amalonaticus harbored mcr-3.5 gene on pSY_CS01 and pSY_CA01 plasmids, respectively. Both plasmids belonged to IncFII(pCoo) replicon type, contained the same genetic context (Tn3-IS1-ΔTnAs2-mcr-3.5-dgkA-IS91) and exhibited high transferring frequencies ranging from 1.03×10-4 - 4.6×10-4 CFU/recipient cell Escherichia coli J53. Colistin-MICs of transconjugants increased ≥ 16-fold suggesting that mcr-3.5 on these plasmids can be expressed in other species. However, beside mcr, other major antimicrobial resistant determinants in multidrug resistant Enterobacterales were not found in these two isolates. These findings indicate that mcr gene continued to evolve in the absence of antibiotics selective pressure. Our results also support the hypothesis that Citrobacter could be a reservoir for spreading of antimicrobial resistant genes. To the best of our knowledge, this is the first report that discovered human-derived Citrobacter spp. that harbored mcr but no other major antimicrobial resistant determinants. Also, this is the first report that described the presence of mcr gene in C. sedlakii and mcr-3 in C. amalonaticus.

Proteomic study of in vitro osteogenic differentiation of mesenchymal stem cells in high glucose condition.

Patients with diabetes have been widely reported to be at an increased risk of secondary osteoporosis. Osteoporosis is caused by an imbalance in bone remodeling due to increased bone resorption and/or decreased osteoblast-dependent bone formation. In this study, mesenchymal stem cells (MSCs) were used as a disease model to determine the effects of high glucose levels on MSC-osteoblast development. The results indicated that under high glucose conditions, MSCs had reduced cell viability and increased number of ß-galactosidase-positive cells. Furthermore, in vitro osteogenesis was shown to be reduced in MSCs cultured in osteogenic differentiation medium at 10, 25, and 40 mM glucose as demonstrated by Alizarin red S staining and alkaline phosphatase activity assay. Moreover, a proteomic study was performed in MSCs cultured with 25 and 40 mM glucose. The proteomic results demonstrated that 12 proteins were up- and downregulated in bone marrow-derived mesenchymal stem cells cultured with high glucose in a dose-dependent manner. The findings presented here contribute to our understanding of the mechanism of diabetes mellitus responsible for bone loss. However, the exact mechanism of action of hyperglycemia on bone deformability requires additional studies.

Roles of kininogen-1, basement membrane specific heparan sulfate proteoglycan core protein, and roundabout homolog 4 as potential urinary protein biomarkers in diabetic nephropathy.

Diabetic nephropathy, a major complication of diabetes mellitus (DM), is increasing worldwide and the large majority of patients have type 2 DM. Microalbuminuria has been used as a diagnostic marker of diabetic nephropathy. But owing to its insufficient sensitivity and specificity, other biomarkers are being sought. In addition, the pathophysiology of diabetic nephropathy is not fully understood and declines in renal function occur even without microalbuminuria. In this study, we investigated urinary proteins from three study groups (controls, and type 2 diabetic subjects with or without microalbuminuria). Non-targeted label-free Nano-LC QTOF analysis was conducted to discover underlying mechanisms and protein networks, and targeted label-free Nano-LC QTOF with SWATH was performed to qualify discovered protein candidates. Twenty-eight proteins were identified as candidates and functionally analyzed via String DB, gene ontology and pathway analysis. Four predictive mechanisms were analyzed: i) response to stimulus, ii) platelet activation, signaling and aggregation, iii) ECM-receptor interaction, and iv) angiogenesis. These mechanisms can provoke kidney dysfunction in type 2 diabetic patients via endothelial cell damage and glomerulus structural alteration. Based on these analyses, three proteins (kininogen-1, basement membrane-specific heparan sulfate proteoglycan core protein, and roundabout homolog 4) were proposed for further study as potential biomarkers. Our findings provide insights that may improve methods for both prevention and diagnosis of diabetic nephropathy.

HCVpred: A web server for predicting the bioactivity of hepatitis C virus NS5B inhibitors.

Hepatitis C virus (HCV) is one of the major causes of liver disease affecting an estimated 170 million people culminating in 300,000 deaths from cirrhosis or liver cancer. NS5B is one of three potential therapeutic targets against HCV (i.e., the other two being NS3/4A and NS5A) that is central to viral replication. In this study, we developed a classification structure-activity relationship (CSAR) model for identifying substructures giving rise to anti-HCV activities among a set of 578 non-redundant compounds. NS5B inhibitors were described by a set of 12 fingerprint descriptors and predictive models were constructed from 100 independent data splits using the random forest algorithm. The modelability (MODI index) of the data set was determined to be robust with a value of 0.88 exceeding established threshold of 0.65. The predictive performance was deduced by the accuracy, sensitivity, specificity, and Matthews correlation coefficient, which was found to be statistically robust (i.e., the former three parameters afforded values in excess of 0.8 while the latter statistical parameter provided a value >0.7). An in-depth analysis of the top 20 important descriptors revealed that aromatic ring and alkyl side chains are important for NS5B inhibition. Finally, the predictive model is deployed as a publicly accessible HCVpred web server (available at http://codes.bio/hcvpred/) that would allow users to predict the biological activity as being active or inactive against HCV NS5B. Thus, the knowledge and web server presented herein can be used in the design of more potent and specific drugs against the HCV NS5B.

Proteomics and bioinformatics analysis reveal potential roles of cadmium-binding proteins in cadmium tolerance and accumulation of Enterobacter cloacae.

BACKGROUND: Enterobacter cloacae (EC) is a Gram-negative bacterium that has been utilized extensively in biotechnological and environmental science applications, possibly because of its high capability for adapting itself and surviving in hazardous conditions. A search for the EC from agricultural and industrial areas that possesses high capability to tolerate and/or accumulate cadmium ions has been conducted in this study. Plausible mechanisms of cellular adaptations in the presence of toxic cadmium have also been proposed. METHODS: Nine strains of EC were isolated and subsequently identified by biochemical characterization and MALDI-Biotyper. Minimum inhibitory concentrations (MICs) against cadmium, zinc and copper ions were determined by agar dilution method. Growth tolerance against cadmium ions was spectrophotometrically monitored at 600 nm. Cadmium accumulation at both cellular and protein levels was investigated using atomic absorption spectrophotometer. Proteomics analysis by 2D-DIGE in conjunction with protein identification by QTOF-LC-MS/MS was used to study differentially expressed proteins between the tolerant and intolerant strains as consequences of cadmium exposure. Expression of such proteins was confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Bioinformatics tools were applied to propose the functional roles of cadmium-binding protein and its association in cadmium tolerance mechanisms. RESULTS: The cadmium-tolerant strain (EC01) and intolerant strain (EC07) with the MICs of 1.6 and 0.4 mM, respectively, were isolated. The whole cell lysate of EC01 exhibited approximately two-fold higher in cadmium binding capability than those of the EC07 and ATCC 13047, possibly by the expression of Cd-binding proteins. Our proteomics analysis revealed the higher expression of DUF326-like domain (a high cysteine-rich protein) of up to 220 fold in the EC01 than that of the EC07. Confirmation of the transcription level of this gene by qRT-PCR revealed a 14-fold induction in the EC01. Regulation of the DUF326-like domain in EC01 was more pronounced to mediate rapid cadmium accumulation (in 6 h) and tolerance than the other resistance mechanisms found in the ATCC 13047 and the EC07 strains. The only one major responsive protein against toxic cadmium found in these three strains belonged to an antioxidative enzyme, namely catalase. The unique proteins found in the ATCC 13047 and EC07 were identified as two groups: (i) ATP synthase subunit alpha, putative hydrolase and superoxide dismutase and (ii) OmpX, protein YciF, OmpC porin, DNA protection during starvation protein, and TrpR binding protein WrbA, respectively. CONCLUSION: All these findings gain insights not only into the molecular mechanisms of cadmium tolerance in EC but also open up a high feasibility to apply the newly discovered DUF326-like domain as cadmium biosorbents for environmental remediation in the future.

Production and Characterization of Recombinant Wild Type Uricase from Indonesian Coelacanth (L. menadoensis) and Improvement of Its Thermostability by In Silico Rational Design and Disulphide Bridges Engineering.

The ideal therapeutic uricase (UOX) is expected to have the following properties; high expression level, high activity, high thermostability, high solubility and low immunogenicity. The latter property is believed to depend largely on sequence identity to the deduced human UOX (dH-UOX). Herein, we explored L. menadoensis uricase (LM-UOX) and found that it has 65% sequence identity to dH-UOX, 68% to the therapeutic chimeric porcine-baboon UOX (PBC) and 70% to the resurrected ancient mammal UOX. To study its biochemical properties, recombinant LM-UOX was produced in E. coli and purified to more than 95% homogeneity. The enzyme had specific activity up to 10.45 unit/mg, which was about 2-fold higher than that of the PBC. One-litre culture yielded purified protein up to 132 mg. Based on homology modelling, we successfully engineered I27C/N289C mutant, which was proven to contain inter-subunit disulphide bridges. The mutant had similar specific activity and production yield to that of wild type (WT) but its thermostability was dramatically improved. Up on storage at -20 °C and 4 °C, the mutant retained ~100% activity for at least 60 days. By keeping at 37 °C, the mutant retained ~100% activity for 15 days, which was 120-fold longer than that of the wild type. Thus, the I27C/N289C mutant has potential to be developed for treatment of hyperuricemia.

Repositioning of 8-hydroxyquinoline derivatives as a new promising candidate for combating multidrug resistant Neisseria gonorrhoeae.

The multidrug resistance of Neisseria gonorrhoeae becomes a public health problem worldwide, especially the strain H041 that showed the decrease susceptibility to ceftriaxone which is the last resort for gonorrhea treatment. Therefore, the simultaneous discovery and development of a new compound to fight this pathogen is urgently required. In this study, 8-hydroxyquinoline (8HQ) and derivatives were evaluated for their antimicrobial activities against the gonococcal pathogen using spectinomycin as the reference drug. The results showed that 8HQ derivatives gave an excellent antimicrobial potency. Particularly, the dihalogenated 8HQ (iodoquinol, clioquinol and 5,7-diCl-8HQ) exerted the high activity with MIC range of 0.08-0.15 µM, 0.10-0.20 µM and 0.28-0.56 µM, respectively, compared with the reference drug (MIC = 16 µg/mL or 48.14 µM). Moreover, these compounds were also shown to be non-cytotoxic/very high safety index. The findings reveal that these three compounds could be further developed as a new antimicrobial agent for fighting the gonorrheal disease.

Metal complexation by histidine-rich peptides confers protective roles against cadmium stress in Escherichia coli as revealed by proteomics analysis.

The underlying mechanism and cellular responses of bacteria against toxic cadmium ions is still not fully understood. Herein, Escherichia coli TG1 expressing hexahistidine-green fluorescent protein (His6GFP) and cells expressing polyhistidine-fused to the outer membrane protein A (His-OmpA) were applied as models to investigate roles of cytoplasmic metal complexation and metal chelation at the surface membrane, respectively, upon exposure to cadmium stress. Two-dimensional gel electrophoresis (2-DE) and two-dimensional difference in gel electrophoresis (2D-DIGE) in conjunction with mass spectrometry-based protein identification had successfully revealed the low level expression of antioxidative enzymes and stress-responsive proteins such as manganese-superoxide dismutase (MnSOD; +1.65 fold), alkyl hydroperoxide reductase subunit C (AhpC; +1.03 fold) and DNA starvation/stationary phase protection protein (Dps; -1.02 fold) in cells expressing His6GFP in the presence of 0.2 mM cadmium ions. By contrarily, cadmium exposure led to the up-regulation of MnSOD of up to +7.20 and +3.08 fold in TG1-carrying pUC19 control plasmid and TG1 expressing native GFP, respectively, for defensive purposes against Cd-induced oxidative cell damage. Our findings strongly support the idea that complex formation between cadmium ions and His6GFP could prevent reactive oxygen species (ROS) caused by interaction between Cd2+ and electron transport chain. This coincided with the evidence that cells expressing His6GFP could maintain their growth pattern in a similar fashion as that of the control cells even in the presence of harmful cadmium. Interestingly, overexpression of either OmpA or His-OmpA in E. coli cells has also been proven to confer protection against cadmium toxicity as comparable to that observed in cells expressing His6GFP. Blockage of metal uptake as a consequence of anchored polyhistidine residues on surface membrane limited certain amount of cadmium ions in which some portion could pass through and exert their toxic effects to cells as observed by the increased expression of MnSOD of up to +9.91 and +3.31 fold in case of TG1 expressing only OmpA and His-OmpA, respectively. Plausible mechanisms of cellular responses and protein mapping in the presence of cadmium ions were discussed. Taken together, we propose that the intracellular complexation of cadmium ions by metal-binding regions provides more efficiency to cope with cadmium stress than the blockage of metal uptake at the surface membrane. Such findings provide insights into the molecular mechanism and cellular adaptation against cadmium toxicity in bacteria.

Oxidative responses and defense mechanism of hyperpigmented P. aeruginosa as characterized by proteomics and metabolomics.

Pseudomonas aeruginosa is known to produce multiple types of pigment which are involved in its pathogenicity and survival in certain environments. Herein, we reported the identification of P. aeruginosa dark-brown hyperpigmented (HP) strains which have been isolated from clinical samples. In order to study the role of these dark-brown containing secretions, alterations of metabolic processes and cellular responses under microenvironment of this bacterial pathogen, two-dimensional gel electrophoresis (2-DE) in conjunction with peptide mass fingerprinting (PMF) were performed. Protein spots showing the most significant differences and high spot optical density values were selected for further characterization. Fold difference of protein expression levels among those spots were calculated. Three major groups of proteins including anti-oxidant enzyme such as catalase, alkyl hydroperoxide reductase and also iron-superoxide dismutase (Fe-SOD), transmembrane proteins as well as proteins involved in energy metabolism such as ATP synthase and pyruvate/2-oxoglutarate dehydrogenase were significantly decreased in P. aeruginosa HP. Whereas, malate syntase and isocitrate lyase, the key enzyme in glyoxylate cycle as well as alcohol dehydrogenase were significantly increased in P. aeruginosa HP, as compared to the reference strain ATCC 27853. Moreover, the HP exerted SOD-like activity with its IC50 equal to 0.26 mg/ml as measured by NBT assay. Corresponding to secretomic metabolome identification, elevated amounts of anti-oxidant compounds are detected in P. aeruginosa HP than those observed in ATCC 27853. Our findings indicated successful use of proteomics and metabolomics for understanding cell responses and defense mechanisms of P. aeruginosa dark-brown hyperpigmented strains upon surviving in its microenvironment.

Multiplex PCR scheme for variant plasmid mediated class C ß-lactamase typing.

BACKGROUND: An increasing of prevalence and diversification of plasmid-mediated AmpC (pAmpC) has been emerged worldwide. The incidence of pAmpC resulted in increasing ß-lactamase production and conferred resistance to almost all ß-lactam antibiotics excluding carbapenems. The lack of standard method for pAmpC identification and classification exert a challenge in epidemiological surveillance and infection control practices. METHODS: A robust, single tube multiplex PCR has been developed to classify six different pAmpC groups including CIT (CMY-2 like, LAT and CFE), ECB (ACT, MIR), MOX & CMY-1 like, DHA, ACC, and FOX. The developed method was optimized and validated by testing of sensitivity and specificity. RESULTS: Developed method can detect crude extracted DNA template at nano-scale (2.5 ηg) and has high discriminatory power as compared to phenotypic and commercial genotypic method. CONCLUSION: The developed method can be utilized for tracking the changes of clinically important resistance patterns and further investigation of occurrence and distribution of plasmid-mediated AmpC types.

Comparative proteomics analysis of Neisseria gonorrhoeae strains in response to extended-spectrum cephalosporins.

Neisseria gonorrhoeae strains displaying reduced susceptibility and resistance to extended-spectrum cephalosporins (ESCs) are major public health concerns. Although resistance mechanisms of ESCs have extensively been studied, the proteome-wide investigation on the biological response to the antibiotic stress is still limited. Herein, a proteomics approach based on two-dimensional gel electrophoresis and MALDI-TOF/TOF-MS analysis was applied to investigate the global protein expression under ESC stresses of ESC-susceptible and ESC-reduced susceptible N. gonorrhoeae strains. Upon exposure to ceftriaxone, 14 and 21 proteins of ESC-susceptible and ESC-reduced susceptible strains, respectively, were shown to be differentially expressed. In the meanwhile, differential expressions of 13 and 17 proteins were detected under cefixime stress for ESC-susceptible and ESC-reduced susceptible strains, respectively. ESC antibiotics have been proven to trigger the expression of several proteins implicated in a variety of biological functions including transport system, energy metabolism, stress response and pathogenic virulence factors. Interestingly, macrophage infectivity potentiators (Ng-MIP) showed increased expression for ESC-reduced susceptible strain under ESC stress. The altered expression of Ng-MIP was found to be a unique response to ESC stresses. Our finding proposes a broad view on proteomic changes in N. gonorrhoeae in response to ESC antibiotics that provides further insights into the gonococcal antimicrobial resistance and physiological adaptation mechanism.

Prevalence of Thalassemia Traits and Iron Deficiency Anemia in Sindh, Pakistan.

Among microcytic hypochromic anemias, the most common disorders are iron deficiency anemia and co-pathological conditions such as α- or ß-thalassemia (α- or ß-thal) traits. The aim of the present study was to determine the frequency and prevalence of iron deficiency anemia and α- or ß-thal traits based on clinical laboratory data across different ethnic groups in five districts of Sindh Province, Pakistan. The present retrospective study analyzed 3 years (2012-2015) of encoded and unlinked clinical laboratory data, and identified 3030 microcytic hypochromic anemia cases. The data contained complete blood counts (CBCs) with smear morphology examinations, serum ferritin levels, and hemoglobin (Hb) electrophoreses. After reviewing the data, 994 confirmed subjects (iron deficiency anemia and α- and ß-thal traits) were then selected for the present study. The prevalence of α- and ß-thal traits was highest in Badin district (35.27%), while the prevalence of iron deficiency anemia was highest in Larkana district (30.73%). According to the ethnic-wise distribution, higher numbers of α- and ß-thal trait cases were seen in the Sindhi ethnic group [375 (64.21%) and 283 (69.02%), respectively] than in the other ethnic groups. In addition, a higher distribution of ß-thal trait cases was observed in the Sindhi ethnic group [n = 327 (56%)] in α- and ß-thal cases overall. Findings from the present study strongly suggested that screening is important not only for ß-thal trait but also other traits as well. However, careful monitoring of CBC parameters, including red blood cell (RBC) indices and morphology, along with clinical findings are essential to diagnose carrier cases, especially in high prevalence areas.

High-fat diet-induced plasma protein and liver changes in obese rats can be attenuated by melatonin supplementation.

Obesity triggers changes in protein expression in various organs that might participate in the pathogenesis of obesity. Melatonin has been reported to prevent or attenuate such pathological protein changes in several chronic diseases. However, such melatonin effects on plasma proteins have not yet been studied in an obesity model. Using a proteomic approach, we investigated the effect of melatonin on plasma protein profiles after rats were fed a high-fat diet (HFD) to induce obesity. We hypothesized that melatonin would attenuate abnormal protein expression in obese rats. After 10weeks of the HFD, animals displayed increased body weight and fat accumulation as well as increased glucose levels, indicating an obesity-induced prediabetes mellitus-like state. Two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry revealed 12 proteins whose expression was altered in response to the HFD and the melatonin treatment. The altered proteins are related to the development of liver pathology, such as cirrhosis (α1-antiproteinase), thrombosis (fibrinogen, plasminogen), and inflammation (mannose-binding protein A, complement C4, complement factor B), contributing to liver steatosis or hepatic cell death. Melatonin treatment most probably reduced the severity of the HFD-induced obesity by reducing the amplitude of HFD-induced plasma protein changes. In conclusion, we identified several potential biomarkers associated with the progression of obesity and its complications, such as liver damage. Furthermore, our findings reveal melatonin's beneficial effect of attenuating plasma protein changes and liver pathogenesis in obese rats.

Improving enzymatic activities and thermostability of a tri-functional enzyme with SOD, catalase and cell-permeable activities.

Synergistic action of major antioxidant enzymes, e.g., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) is known to be more effective than the action of any single enzyme. Recently, we have engineered a tri-functional enzyme, 6His-MnSOD-TAT/CAT-MnSOD (M-TAT/CM), with SOD, CAT and cell-permeable activities. The protein actively internalized into the cells and showed superior protection against oxidative stress-induced cell death over native enzymes fused with TAT. To improve its molecular size, enzymatic activity and stability, in this study, MnSOD portions of the engineered protein were replaced by CuZnSOD, which is the smallest and the most heat resistant SOD isoform. The newly engineered protein, CAT-CuZnSOD/6His-CuZnSOD-TAT (CS/S-TAT), had a 42% reduction in molecular size and an increase in SOD and CAT activities by 22% and 99%, respectively. After incubation at 70°C for 10min, the CS/S-TAT retained residual SOD activity up to 54% while SOD activity of the M-TAT/CM was completely abolished. Moreover, the protein exhibited a 5-fold improvement in half-life at 70°C. Thus, this work provides insights into the design and synthesis of a smaller but much more stable multifunctional antioxidant enzyme with ability to enter mammalian cells for further application as protective/therapeutic agent against oxidative stress-related conditions.

Paper-based acetylcholinesterase inhibition assay combining a wet system for organophosphate and carbamate pesticides detection.

A dramatic increase in pesticide usage in agriculture highlights the need for on-site monitoring for public health and safety. Here, a paper-based sensor combined with a wet system was developed for the simple and rapid screening of organophosphate (OP) and carbamate (CM) pesticides based on the inhibition of acetylcholinesterase (AChE). The paper-based sensor was designed as a foldable device consisting of a cover and detection sheets pre-prepared with indoxyl acetate and AChE, respectively. The paper-based sensor requires only the incubation of a sample on the test zone for 10 minutes, followed by closing of the foldable sheet to initiate the enzymatic reaction. Importantly, the buffer loading hole was additionally designed on the cover sheet to facilitate the interaction of the coated substrate and the immobilized enzyme. This subsequently facilitates the mixing of indoxyl acetate with AChE, resulting in the improved analytical performance of the sensor. The absence or decrease in blue color produced by the AChE hydrolysis of indoxyl acetate can be observed in the presence of OPs and CMs. Under optimized conditions and using image analysis, the limit of detection (LOD) of carbofuran, dichlorvos, carbaryl, paraoxon, and pirimicarb are 0.003, 0.3, 0.5, 0.6, and 0.6 ppm, respectively. The assay could be applied to determine OP and CM residues in spiked food samples. Visual interpretation of the color signal was clearly observed at the concentration of 5 mg/kg. Furthermore, a self-contained sample pre-concentration approach greatly enhanced the detection sensitivity. The paper-based device developed here is low-cost, requires minimal reagents and is easy to handle. As such, it would be practically useful for pesticide screening by non-professional end-users.