Gamma-Glutamylcysteine Production Using Phytochelatin Synthase-Like Enzyme Derived from Nostoc sp. Covalently Immobilized on a Cellulose Carrier.

Gamma-glutamylcysteine (γ-EC) is an intermediate generated in the de novo synthesis of glutathione (GSH). Recent studies have revealed that the administration of γ-EC shows neuroprotective effects against oxidative stress in age-related disorders and chronic diseases like Alzhiemer's disease in model animals, which is not expected function in GSH. A phytochelatin synthase-like enzyme derived from Nostoc sp. (NsPCS) mediates γ-EC synthesis from GSH. To achieve low-cost and stable commercial level supply, the availability of immobilized NsPCS for γ-EC production was investigated in this study. Among the tested immobilization techniques, covalent binding to the cellulose carrier was most effective, and could convert GSH completely to γ-EC without decreasing the yield. The stable conversion of γ-EC from 100 mM GSH was achieved by both batch repeated and continuous reactions using the immobilized NsPCS on cellulose sheet and column shape monolith, respectively. The immobilization of NsPCS on those carriers is promising alternative technique for high-yielding and cost-effective production of γ-EC on its commercial applications.

[Ideal Pharmaceutical Education Viewed from Quality Assurance on Third-party Accreditation].

The first-phase third-party accreditation conducted by the Japan Accreditation Board for Pharmaceutical Education between FY 2013 and 2019 revealed various issues regarding current pharmacy education programs. In addition, the report of the Ministry of Health, Labour and Welfare's Study Group on the Training and Quality Improvement of Pharmacists, which was published in 2021, identified a broad range of issues in pharmaceutical education related to pharmacist training and quality improvement. Many of these issues are concerned with university curricula; thus, in order to ensure and enhance the quality of pharmaceutical education and develop pharmaceutical human resources that society demands, it is extremely important for each university to improve its curricula. Since revision of the model core curriculum for pharmaceutical education is currently underway, in this symposium, we held a discussion to identify points that need to be improved regarding the current model core curriculum to ensure and enhance the quality of pharmaceutical education in light of the above-mentioned issues, and incorporate the corresponding measures into the basic policy for revision of the model core curriculum.

Physicians' and Nurses' Perceptions of Pharmacists' Competencies, and Their Needs of Pharmacists during COVID-19.

Objective: To understand how physicians and nurses evaluate Japanese pharmacists' observed competencies and to explore potential new roles for pharmacists during COVID-19. Methods: A web-based Japanese survey with 25 items assessing physicians' and nurses' workplaces and the degree of their relationship with pharmacists in their daily work was conducted (Intage, Inc., Tokyo, Japan) in Japan in June 2021 (for one week beginning on 22 June). The survey asked physicians and nurses whether pharmacists had the required professional competencies and whether the needs of physicians and nurses were met by pharmacists in their workplaces. The scored questionnaire data, which used a Likert scale, were calculated as the mean and standard deviation (S.D.). The perception assessment scale used four levels (1, Agree; 2, Slightly agree; 3, Slightly disagree; and 4, Disagree). Results: This perception study ultimately obtained responses from 304 physicians and 336 nurses. Most pharmacists' competencies were evaluated as "Agree" or "Slightly agree" by the physicians and nurses. However, the competencies for "Fundamental basic science" and "Prescription analytical skill or case analytical skill" were evaluated significantly lower by physicians than by nurses (Mann−Whitney U test, p < 0.01). Regarding physicians' and nurses' needs from pharmacists, nurses hoped that pharmacists could play a greater role as healthcare professionals in response to all items; in contrast, physicians hoped that pharmacists could play a greater role as healthcare professionals in response to five items. The common items were related to the role of healthcare professionals in the community. Conclusion: Our research is necessary for facilitating interprofessional collaboration and reflecting these results in pharmacy education by allowing physicians and nurses to assess the competencies of pharmacists and to understand their needs; however, these data are from only one country.

Reactivity of γ-glutamyl-cysteine with intracellular and extracellular glutathione metabolic enzymes.

Gamma-glutamyl-cysteine (γ-EC) is a precursor of glutathione (GSH) biosynthesis. We investigated whether it functions as a substrate for three intracellular and one extracellular GSH metabolic enzymes, which mediate the antioxidant defence function of GSH. Among them, glutathione peroxidase, glutathione S-transferase and γ-glutamyl transferase (GGT) exhibited substrate specificity for γ-EC, whereas glutathione reductase did not. The specificities of γ-EC and its disulphide form to GGT were comparable to GSH and its oxidized form, GSSG respectively. These results indicate that they can supply GSH constituent amino acids, glutamate, cysteine and cystine through degradation by GGT. γ-EC may contribute valuable antioxidant defence properties as a food and cosmetic additive.

Optimization of Reaction Conditions for γ-Glutamylcysteine Production from Glutathione Using a Phytochelatin Synthase-Like Enzyme from Nostoc sp. Pasteur Culture Collection 7120.

γ-Glutamylcysteine (γ-EC) has antioxidant properties similar to those of glutathione (GSH) and acts as its precursor in mammals. There are a few procedures for the production of γ-EC, such as chemical synthesis or enzymatic synthesis from glutamate and cysteine; however, they are very costly and not suitable for industrial production. A phytochelatin synthase-like enzyme derived from Nostoc sp. Pasteur Culture Collection 7120 (NsPCS) catalyzes the hydrolysis of GSH to γ-EC and glycine in the absence of ATP or other additives. Our research aims to establish an alternative γ-EC production procedure with low cost and high productivity. To this end, we optimized the reaction conditions of NsPCS and characterized its properties in this study. We found that 200 mM potassium phosphate buffer, pH 8.0, at 37 °C, had the highest NsPCS activity among the conditions we tested. Under these conditions, NsPCS had a Km of 385 µM and a Vmax of 26 mol/min/mg-protein. In addition, NsPCS converted 100 mM GSH into γ-EC with high yields. These results suggest that the NsPCS reaction has great potential for the low-cost, industrial-scale production of γ-EC.

In Vitro Model for a Drug Assessment of Cytochrome P450 Family 3 Subfamily A Member 4 Substrates Using Human Induced Pluripotent Stem Cells and Genome Editing Technology.

In drug development, a system for predicting drug metabolism and drug-induced toxicity is necessary to ensure drug safety. Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) is an important drug-metabolizing enzyme expressed in the liver and small intestine, and predicting CYP3A4-mediated drug metabolism and drug-induced toxicity is essential. We previously developed procedures to differentiate human induced pluripotent stem (iPS) cells into hepatocyte-like cells (HLCs) or intestinal epithelial-like cells (IECs) with a fetal phenotype as well as a highly efficient genome editing technology that could enhance the homologous recombination efficiency at any locus, including CYP3A4. By using human iPS cells and our genome editing technology, we generated CYP3A4-knockout (KO) iPS cell-derived HLCs and IECs for the evaluation of CYP3A4-mediated drug metabolism and drug-induced toxicity. CYP3A4 deficiency did not affect pluripotency and hepatic and intestinal differentiation capacities, and CYP3A4 activity was entirely eradicated by CYP3A4 KO. Off-target effects (e.g., inhibition of bile acid excretion) were hardly observed in CYP3A4-KO cells but were observed in CYP3A4 inhibitor-treated (e.g., ketoconazole) cells. To evaluate whether drug-induced hepatotoxicity and enterotoxicity could be predicted using our model, we exposed CYP3A4-KO HLCs and IECs to acetaminophen, amiodarone, desipramine, leflunomide, tacrine, and tolcapone and confirmed that these cells could predict CYP3A4-mediated toxicity. Finally, we examined whether the therapeutic effects of an anti-hepatitis C virus (HCV) drug metabolized by CYP3A4 would be predicted using our model. CYP3A4-KO HLCs were treated with asunaprevir (antiviral drug metabolized by CYP3A4) after HCV infection, and the anti-viral effect was indeed strengthened by CYP3A4 KO. Conclusion: We succeeded in generating a novel evaluation system for prediction of CYP3A4-mediated drug metabolism and drug-induced toxicity.

Development and Evaluation of a System for the Semi-Quantitative Determination of the Physical Properties of Skin After Exposure to Silver Nanoparticles.

In order to ensure the safe usage of silver nanoparticles (nAgs) in cosmetics, it is necessary to reveal the physical properties of nAgs inside the skin, as these properties may change during the process of percutaneous absorption. In this study, we aimed to establish an analytical system based on single particle inductively coupled plasma mass spectrometry (sp-ICP-MS) to determine the physical properties of nAgs in the skin. First, we optimized a pretreatment method for solubilizing the skin samples and then showed that most of the nAgs were recovered by sodium hydroxide treatment while remaining in particle form. For separating the skin into the epidermis and dermis, we screened several conditions of microwave irradiation. The sp-ICP-MS analysis indicated that the application of 200 W for 30 s was optimal, as this condition ensured complete separation of skin layers without changing the physical properties of the majority of nAgs. Finally, we evaluated the in vivo application by analyzing the quantity as well as the physical properties of Ag in the epidermis, dermis, and peripheral blood of mice after exposing the skin to nAgs or Ag+. Subsequent sp-ICP-MS analysis indicated that nAgs could be absorbed and distributed into the deeper layers in the ionized form, whereas Ag+ was absorbed and distributed without a change in physical properties. This study indicates that in order to obtain a comprehensive understanding of the response of skin following exposure to nAgs, it is essential to consider the distribution and particle size of not only nAgs but also Ag+ released from nAgs into the skin.

Comparison of commercially available media for hepatic differentiation and hepatocyte maintenance.

Human hepatocytes are essential materials in pharmaceutical researches. Not only primary human hepatocytes (PHH) but also human iPS cell-derived hepatocyte-like cells (human iPS-HLCs) are expected to be applied as materials for pharmaceutical researches. To date, several culture media have been developed for culturing human hepatocytes. However, there have been no reports comparing these media to determine which is most suitable for culturing human hepatocytes. In this study, we compared five commercial media (Hepatocyte Culture Medium (HCM), HepatoZYME-SFM, Cellartis Power Primary HEP Medium, DMEM/F12, and William's E Medium (WEM)) to determine which is most suitable for culturing PHH and human iPS-HLCs. In hepatic differentiation of human iPS cells (day 14-25 of differentiation), albumin (ALB) and urea secretion abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM or WEM. During maintenance of human iPS-HLCs, ALB and urea producing abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM. Importantly, we found that human iPS-HLCs cultured in HCM were maintained for 3 weeks or more without impairment of their hepatic functions. These results suggest that it is necessary to select an optimal medium for hepatic differentiation and maintenance of human iPS-HLCs. In the case of PHH culture, there was little difference in hepatic functions among the five media. However, the CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM and WEM. In conclusion, it is important to select the optimal medium for specific application when carrying out pharmaceutical researches using human hepatocytes.

Establishment of SLC15A1/PEPT1-Knockout Human-Induced Pluripotent Stem Cell Line for Intestinal Drug Absorption Studies.

Because many peptide and peptide-mimetic drugs are substrates of peptide transporter 1, it is important to evaluate the peptide transporter 1-mediated intestinal absorption of drug candidates in the early phase of drug development. Although intestinal cell lines treated with inhibitors of peptide transporter 1 are widely used to examine whether drug candidates are substrates for peptide transporter 1, these inhibitors are not sufficiently specific for peptide transporter 1. In this study, to generate a more precise evaluation model, we established peptide transporter 1-knockout induced pluripotent stem cells (iPSCs) by using a CRISPR-Cas9 system and differentiated the cells into intestinal epithelial-like cells. The permeability value and uptake capacity of glycylsarcosine (substrate of peptide transporter 1) in peptide transporter 1-knockout intestinal epithelial-like cells were significantly lower than those in wild-type intestinal epithelial-like cells, suggesting that peptide transporter 1 was successfully depleted in the epithelial cells. Taken together, our model can be useful in the development of peptide and peptide-mimetic drugs.

Mycobacterium smegmatis alters the production of secondary metabolites by marine-derived Aspergillus niger.

It is generally accepted that fungi have a number of dormant gene clusters for the synthesis of secondary metabolites, and the activation of these gene clusters can expand the diversity of secondary metabolites in culture. Recent studies have revealed that the mycolic acid-containing bacterium Tsukamurella pulmonis activates dormant gene clusters in the bacterial genus Streptomyces. However, it is not clear whether the mycolic acid-containing bacteria activate dormant gene clusters of fungi. We performed co-culture experiments using marine-derived Aspergillus niger with Mycobacterium smegmatis, a mycolic acid-containing bacteria. The co-cultivation resulted in the production of a pigment by A. niger and increased cytotoxic activity of the extract against human prostate cancer DU145 cells. An analysis of secondary metabolites in the extract of the co-culture broth revealed that the increase in cytotoxic activity was caused by the production of malformin C (1), and that TMC-256A1 (2), desmethylkotanin (3), and aurasperone C (4) were selectively produced under co-culture conditions. In addition, further study suggested that direct interaction between the two microorganisms was necessary for the production of the pigment and the cytotoxic compound malformin C (1) from A. niger. Given the biological activities of malformin C, including cytotoxic activity, our approach for increasing the production of bioactive secondary metabolites has important practical applications and may facilitate structural analyses of novel bioactive compounds.

Development and evaluation of a simultaneous and efficient quantification strategy for final prostanoid metabolites in urine.

Prostanoids (PNs) play critical roles in various physiological and pathological processes. Therefore, it is important to understand the alternation of PN expression profiles. However, a simultaneous and efficient quantification system for final PN metabolites in urine has not yet been established. Here, we developed and evaluated a novel method to quantify all final PN metabolites. By purification using a reverse phase solid phase extraction (SPE) column, the matrix effects against the final PGD2, PGE2, and PGF2α metabolites were low, and their accuracies were nearly 100%. The matrix effects against the final PGI2 and TXA2 metabolites were high using reverse phase SPE column purification alone. By applying a tandem SPE method that combined reverse phase and ion exchange SPE columns, the matrix effects decreased so that the accuracy was nearly 100%. To validate the reliability of the method, each final metabolite was quantified from mouse urine to which the PNs (PGD2, PGE2, and PGI2) were intravenously administered. As a result, the amounts of PN metabolites were correlated with those of the PNs administered to the blood in a dose-dependent manner. To validate the method using human samples, the urinary metabolites of Crohn's disease (CD, a PN-related disease) patients and healthy individuals were quantified. All five metabolites were successfully quantified. Only final PGE2 metabolite levels were significantly higher in CD patients than those in healthy individuals, so that the urinary metabolite profiles of CD patients is determined. In conclusion, we developed a novel method to quantify all final PN metabolites simultaneously and efficiently and demonstrated the practicality of the method using human CD patient samples.

Generation of Human iPSC-Derived Intestinal Epithelial Cell Monolayers by CDX2 Transduction.

BACKGROUND & AIMS: To develop an effective and safe orally administered drug, it is important to predict its intestinal absorption rate, intestinal first-pass effect, and drug-drug interactions of orally administered drugs. However, there is no existing model to comprehensively predict the intestinal pharmacokinetics and drug-response of orally administered drugs. In this study, we attempted to generate homogenous and functional intestinal epithelial cells from human induced pluripotent stem (iPS) cells for pharmaceutical research. METHODS: We generated almost-homogenous Villin- and zonula occludens-1 (ZO1)-positive intestinal epithelial cells by caudal-related homeobox transcription factor 2 (CDX2) transduction into human iPS cell-derived intestinal progenitor cells. RESULTS: The drug absorption rates in human iPS cell-derived intestinal epithelial cell monolayers (iPS-IECM) were highly correlated with those in humans (R2=0.91). The expression levels of cytochrome P450 (CYP) 3A4, a dominant drug-metabolizing enzyme in the small intestine, in human iPS-IECM were similar to those in human small intestine in vivo. In addition, intestinal availability in human iPS-IECM (the fraction passing the gut wall: Fg=0.73) was more similar to that in the human small intestine in vivo (Fg=0.57) than to that in Caco-2 cells (Fg=0.99), a human colorectal adenocarcinoma cell line. Moreover, the drug-drug interaction and drug-food interaction could be observed by using our human iPS-IECM in the presence of an inducer and inhibitor of CYP3A4, i.e., rifampicin and grape fruit juice, respectively. CONCLUSION: Taking these results together, we succeeded in generating the human iPS-IECM that can be applied to various intestinal pharmacokinetics and drug-response tests of orally administered drugs.

Optimization and Evaluation of Pretreatment Method for sp-ICP-MS to Reveal the Distribution of Silver Nanoparticles in the Body.

The prevalent use of engineered nanoparticles (ENPs) has increased our exposure to these particles. The current available analytical techniques fail to simultaneously quantify and analyze the physical properties of ENPs in biological tissues. Therefore, new methods are required to evaluate the exposure conditions to ENPs. Single particle inductively coupled plasma-mass spectrometry (sp-ICP-MS) is an attractive approach that can perform quantitative and qualitative analyses of ENPs. However, the application of this approach for biological samples is limited because of the lack of pretreatment methods for effectively recovering ENPs from biological tissues. In this study, we evaluated various pretreatment methods and identified the optimal pretreatment conditions for sp-ICP-MS analyses of ENPs in biological tissues using silver nanoparticles (nAg) as a model. We screened five reagents as pretreatment solvents (sodium hydroxide, tetramethylammonium hydroxide, nitric acid, hydrochloric acid, and proteinase K). Our results showed that treatment with sodium hydroxide was optimal for detecting nAg in the mouse liver. Moreover, this pretreatment method can be applied to other organs, such as the heart, lung, spleen, and kidney. Finally, we evaluated the applicability of this method by analyzing the quantity and physical properties of silver in the mouse blood and liver, after intravenous administration of nAg or silver ion. Our sp-ICP-MS method revealed that nAg administered into the blood was partially ionized and tended to be distributed in the particle form (approximately 80%) in the liver and in ionic form (approximately 95%) in the blood. In conclusion, we optimized pretreatment strategies for sp-ICP-MS evaluation of ENPs in biological tissues and demonstrated its applicability by evaluating the changes in the physical properties of nAg in the liver and blood. We also showed that partial changes from the particle form to the ionic form of nAg influences their kinetics and distribution when administered to mice.

Development of a Paper-Based Luminescence Bioassay for Therapeutic Monitoring of Aminoglycosides: a Proof-of-Concept Study.

Aminoglycosides are widely used antibiotics that bind to the bacterial 30S ribosomal subunit to inhibit translation. Owing to their adverse side effects and narrow therapeutic index, monitoring blood levels of aminoglycosides is important to maximize their effectiveness and minimize their toxicity. Current monitoring techniques require a well-equipped diagnostic laboratory. The present study aimed to present a proof-of-concept for a simple, low-cost biochemical assay utilizing a paper platform for the detection of serum/whole blood aminoglycosides. A paper-based bioassay chip for the assay was developed by spotting and freeze-drying cell-free transcription/translation reaction machinery for a luminescent reporter protein (NanoLuc) within an array of wax circles printed on filter paper. The paper-based chip could be used to quantify serum/whole blood aminoglycosides within clinically relevant concentrations in 30-60 min by spotting minimal volumes of samples, followed by the NanoLuc substrate, in the wax circles and detecting the associated changes in luminescence signals, using a simple digital camera. Furthermore, a one-pot assay in which cell-free transcription/translation reaction machinery and NanoLuc substrate are mixed in advance and embedded in paper could be used to detect an aminoglycoside in serum. Overall, our paper-based bioassay can potentially provide a basic platform for the simple and low-cost therapeutic monitoring of aminoglycosides, especially in resource-limited regions.

Modeling of Hepatic Drug Metabolism and Responses in CYP2C19 Poor Metabolizer Using Genetically Manipulated Human iPS cells.

Cytochrome P450 family 2 subfamily C member 19 (CYP2C19), in liver, plays important roles in terms of drug metabolism. It is known that CYP2C19 poor metabolizers (PMs) lack CYP2C19 metabolic capacity. Thus, unexpected drug-induced liver injury or decrease of drug efficacy would be caused in CYP2C19 substrate-treated CYP2C19 PMs. However, it is difficult to evaluate the safety and effectiveness of drugs and candidate compounds for CYP2C19 PMs because there is currently no model for this phenotype. Here, using human induced pluripotent stem cells (human iPS cells) and our highly efficient genome-editing and hepatocyte differentiation technologies, we generated CYP2C19-knockout human iPS cell-derived hepatocyte-like cells (CYP2C19-KO HLCs) as a novel CYP2C19 PM model for drug development and research. The gene expression levels of hepatocyte markers were similar between wild-type iPS cell-derived hepatocyte-like cells (WT HLCs) and CYP2C19-KO HLCs, suggesting that CYP2C19 deficiency did not affect the hepatic differentiation potency. We also examined CYP2C19 metabolic activity by measuring S-mephenytoin metabolites using ultra-performance liquid chromatography-tandem mass spectrometry. The CYP2C19 metabolic activity was almost eliminated by CYP2C19 knockout. Additionally, we evaluated whether clopidogrel (CYP2C19 substrate)-induced liver toxicity could be predicted using our model. Unexpectedly, there was no significant difference in cell viability between clopidogrel-treated WT HLCs and CYP2C19-KO HLCs. However, the cell viability in clopidogrel- and ketoconazole (CYP3A4 inhibitor)-treated CYP2C19-KO HLCs was significantly enhanced as compared with that in clopidogrel- and DMSO-treated CYP2C19-KO HLCs. This result suggests that CYP2C19-KO HLCs can predict clopidogrel-induced liver toxicity. We succeeded in generating CYP2C19 PM model cells using human iPS cells and genome-editing technologies for pharmaceutical research. SIGNIFICANCE STATEMENT: Although unexpected drug-induced liver injury or decrease of drug efficacy would be caused in CYP2C19 substrate-treated CYP2C19 poor metabolizers, it is difficult to evaluate the safety and effectiveness of drugs and candidate compounds for CYP2C19 poor metabolizers because there is currently no model for this phenotype. Using human iPS cells and our highly efficient genome editing and hepatocyte differentiation technologies, we generated CYP2C19-knockout human iPS cell-derived hepatocyte-like cells as a novel CYP2C19 poor metabolizer model for drug development and research.

Efficient Generation of Small Intestinal Epithelial-like Cells from Human iPSCs for Drug Absorption and Metabolism Studies.

The small intestine plays an important role in the absorption and metabolism of oral drugs. In the current evaluation system, it is difficult to predict the precise absorption and metabolism of oral drugs. In this study, we generated small intestinal epithelial-like cells from human induced pluripotent stem cells (hiPS-SIECs), which could be applied to drug absorption and metabolism studies. The small intestinal epithelial-like cells were efficiently generated from human induced pluripotent stem cell by treatment with WNT3A, R-spondin 3, Noggin, EGF, IGF-1, SB202190, and dexamethasone. The gene expression levels of small intestinal epithelial cell (SIEC) markers were similar between the hiPS-SIECs and human adult small intestine. Importantly, the gene expression levels of colonic epithelial cell markers in the hiPS-SIECs were much lower than those in human adult colon. The hiPS-SIECs generated by our protocol exerted various SIEC functions. In conclusion, the hiPS-SIECs can be utilized for evaluation of drug absorption and metabolism.

Paper-based luminescence bioassay method embedding a sequence of enzymatic reactions to detect sulfonamide groups.

Sulfonamide residue in foodstuffs and the environment is a serious global concern for their contribution to the occurrence of antibiotic-resistant bacteria, especially in developing countries. Here, we describe a novel, simple, and low-cost bioassay for sulfonamides, which has high potential versatility for use in low-resource settings. The bioassay method is based on a purpose-built luminescent assay reaction that detects sulfonamide groups. The luminescent assay reaction comprises dihydropteroate synthase, a target enzyme of sulfonamides, and luminescent pyrophosphate detection reagent, which triggers a sequence of biomolecular reactions that convert sulfonamides to emit luminescence. The novel assay detected at least six different sulfonamides with an estimated limit of detection of <25 ng ml-1 in a solution-phase assay using a microplate reader. More importantly, the luminescent assay reaction functioned even after spotting and freeze-drying on a wax pattern-printed paper platform. The paper-embedded luminescent assay reaction showed response signals to sulfadiazine within 30 min at a limit of detection similar to that of the solution-phase assay using a microplate reader. The signal could be recorded using a digital camera in the dark and required no other laboratory infrastructure, freeing the assay from the constraints of a well-fitted laboratory.

The presence of colistin resistance gene mcr-1 and -3 in ESBL producing Escherichia coli isolated from food in Ho Chi Minh City, Vietnam.

Colistin is indicated for the treatment of multidrug-resistant gram-negative bacterial infections. However, the spread of colistin-resistant bacteria harbouring an mcr gene has become a serious concern. This study investigated local foods in Vietnam for contamination with colistin-resistant bacteria. A total of 261 extended-spectrum ß-lactamase (ESBL)- and AmpC-producing Escherichia coli isolates from 330 meat and seafood products were analysed for colistin susceptibility and the presence of mcr genes. Approximately, 24% (62/261) of ESBL- or AmpC-producing E. coli isolates showed colistin resistance; 97% (60/62) of colistin-resistant isolates harboured mcr-1, whereas 3% (2/62) harboured mcr-3. As the result of plasmid analysis of two strains, both plasmids harbouring mcr-3 revealed that plasmid replicon type was IncFII. Sequencing analysis indicated that an insertion sequence was present near mcr-3, suggesting that IncFII plasmids harbouring mcr-3 could be transferred to other bacterial species by horizontal transfer of the plasmid or transfer with some insertion sequence. In conclusion, ESBL-producing E. coli and AmpC-producing E. coli have acquired colistin resistance because 24% of such isolates show colistin resistance and 3% of the colistin-resistant strains harbour mcr-3. We reported the present of the mcr-3-carrying ESBL-producing E. coli isolated from pork in Vietnam.

Isolation and characterization of a stress-responsive gene encoding a CHRD domain-containing protein from a halotolerant green alga.

The genetic basis of stress resistance in extremophilic microalgae is not well studied. In this study, a gene of unknown function, the cluster58 or CL58 gene, was identified from the halotolerant green alga Chlamydomonas W80 and characterized. The CL58 gene encodes a protein containing a domain of unknown function, the CHRD domain, and a putative secretory signaling sequence at its N-terminus. The levels of CL58 mRNA increased in response to high copper levels and low temperatures. When the CL58 gene was heterologously expressed as a fusion gene with the NanoLuc luciferase gene in Chlamydomonas reinhardtii, a majority of the NanoLuc activity was detected in the culture medium compared with that in the intracellular fraction. A mutagenic analysis revealed that the putative secretory signaling sequence was sufficient for the secretion of the CL58-NanoLuc fusion protein. In addition, we expressed the protein encoded by the CL58 gene in Escherichia coli; the recombinant, soluble protein was then purified. In summary, we identified a novel gene from C. W80 that appears to encode a stress-responsive, CHRD domain-containing secreted protein.