Comprehensive LC-MS/MS method combined with tandem hybrid hydrolysis for multiple exposure assessment of multiclass environmental pollutants.

Environmental pollutants (EPOLs), such as phthalates, volatile organic compounds, phenols, parabens, polycyclic aromatic hydrocarbons, pyrethroids, and environmental tobacco smoke, are highly heterogeneous compounds. Recently, attention has been drawn to the assessment of the combinatory effects of multiple EPs. To correlate multiple exposures with potential health implications, advanced comprehensive analytical methods covering multiclass EPOLs are essential. However, because of several technical problems associated with enzyme hydrolysis, simultaneous extraction, and multiresidue liquid chromatography-tandem mass spectrometry analysis, it is difficult to establish a comprehensive method covering a number of EPOLs in a single sample preparation and analytical run. We developed tandem hybrid hydrolysis, modified direct injection, and a comprehensive mobile phase to overcome these technical problems and established a comprehensive analytical method for simultaneous biomonitoring of multiclass EPOLs. Tandem hybrid hydrolysis using ß-glucuronidase and consecutive acid hydrolysis allowed selective hydrolysis of glucuronide- and sulfate-conjugated metabolites without phthalate degradation. The comprehensive mobile phase composed of 0.01% acetic acid and acetonitrile enabled us to simultaneously analyze 86 EPOLs, with good chromatographic behavior and ionization efficiency. Modified direct injection allowed a small amount of sample and simultaneous urinary extraction. The method was validated and applied to 39 urine samples from 19 mother-newborn pairs for multiple exposure assessment. Results showed that BP-3, a general component in sunblock products, and monoethyl phthalate, a metabolite of diethyl phthalate, exhibit a clear positive correlation between mothers and newborns. Therefore, the developed method has potential as a novel analytical tool for long-term, large-scale, and data-rich human biomonitoring of EPOLs.

Effects of a Δ-9-fatty acid desaturase and a cyclopropane-fatty acid synthase from the novel psychrophile Pseudomonas sp. B14-6 on bacterial membrane properties.

Psychrophilic bacteria, living at low and mild temperatures, can contribute significantly to our understanding of microbial responses to temperature, markedly occurring in the bacterial membrane. Here, a newly isolated strain, Pseudomonas sp. B14-6, was found to dynamically change its unsaturated fatty acid and cyclic fatty acid content depending on temperature which was revealed by phospholipid fatty acid (PLFA) analysis. Genome sequencing yielded the sequences of the genes Δ-9-fatty acid desaturase (desA) and cyclopropane-fatty acid-acyl-phospholipid synthase (cfa). Overexpression of desA in Escherichia coli led to an increase in the levels of unsaturated fatty acids, resulting in decreased membrane hydrophobicity and increased fluidity. Cfa proteins from different species were all found to promote bacterial growth, despite their sequence diversity. In conclusion, PLFA analysis and genome sequencing unraveled the temperature-related behavior of Pseudomonas sp. B14-6 and the functions of two membrane-related enzymes. Our results shed new light on temperature-dependent microbial behaviors and might allow to predict the consequences of global warming on microbial communities.

Rewiring FadR regulon for the selective production of ω-hydroxy palmitic acid from glucose in Escherichia coli.

ω-Hydroxy palmitic acid (ω-HPA) is a valuable compound for an ingredient of artificially synthesized ceramides and an additive for lubricants and adhesives. Production of such a fatty acid derivative is limited by chemical catalysis, but plausible by biocatalysis. However, its low productivity issue, including formations of unsaturated fatty acid (UFA) byproducts in host cells, remains as a hurdle toward industrial biological processes. In this study, to achieve selective and high-level production of ω-HPA from glucose in Escherichia coli, FadR, a native transcriptional regulator of fatty acid metabolism, and its regulon were engineered. First, FadR was co-expressed with a thioesterase with a specificity toward palmitic acid production to enhance palmitic acid production yield, but a considerable quantity of UFAs was also produced. In order to avoid the UFA production caused by fadR overexpression, FadR regulon was rewired by i) mutating FadR consensus binding sites of fabA or fabB, ii) integrating fabZ into fabI operon, and iii) enhancing the strength of fabI promoter. This approach led to dramatic increases in both proportion (48.3-83.0%) and titer (377.8 mg/L to 675.8 mg/L) of palmitic acid, mainly due to the decrease in UFA synthesis. Introducing a fatty acid ω-hydroxylase, CYP153A35, into the engineered strain resulted in a highly selective production of ω-HPA (83.5 mg/L) accounting for 87.5% of total ω-hydroxy fatty acids. Furthermore, strategies, such as i) enhancement in CYP153A35 activity, ii) expression of a fatty acid transporter, iii) supplementation of triton X-100, and iv) separation of the ω-HPA synthetic pathway into two strains for a co-culture system, were applied and resulted in 401.0 mg/L of ω-HPA production. For such selective productions of palmitic acid and ω-HPA, the rewiring of FadR regulation in E. coli is a promising strategy to develop an industrial process with economical downstream processing.

Cadaver skin allograft may improve mortality rate for burns involving over 30% of total body surface area: a propensity score analysis of data from four burn centers.

Cadaver skin is used for temporary wound covering, but there is insufficient evidence regarding its clinical usefulness in patients with major burns. We aimed to analyze the effect of cadaveric skin allograft on mortality rates in patients with burns involving > 30% of total body surface area (TBSA). Our study included 1282 patients with > 30% of TBSA burned admitted to four hospitals in Korea between June 1, 2008 and December 31, 2016. Of these, 698 patients underwent cadaver skin allograft (cadaver group), and 584 were treated with conventional treatment (non-cadaver group). We corrected the differences between the two groups using propensity score matching, and generated 474 propensity score-matched pairs. Overall 90-day in-hospital mortality rate among all patients was 35.3% (453/1282). There was a significant difference in 90-day in-hospital mortality between the two groups for both unmatched [cadaver vs. conventional, 31.7 vs. 39.7%; difference, 8.0; 95% confidence interval (CI) 2.8-13.3] and propensity-matched groups (37.8 vs. 47.3%; difference, 9.5; 95% CI 3.2-15.8). Logistic regression analyses showed a significant association between cadaver skin allograft and lower 90-day in-hospital mortality in the propensity-matched groups (odds ratio, 0.42; 95% CI 0.29-0.62). Patients with major burns who underwent cadaver skin allograft had a lower mortality rate compared to those who did not. Cadaver skin allograft may improve the survival of patients with major burns, especially in the early phase of injury.

Application of acetyl-CoA acetyltransferase (AtoAD) in Escherichia coli to increase 3-hydroxyvalerate fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

Polyhydroxyalkanoate (PHA) is a family of biodegradable polymers, and incorporation of different monomers can alter its physical properties. To produce the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) containing a high level of 3-hydroxyvalerate (3HV) by altering acetyl-CoA pool levels, we overexpressed an acetyl-CoA acetyltransferase (atoAD) in an engineered E. coli strain, YH090, carrying PHA synthetic genes bktB, phaB, and phaC. It was found that, with introduction of atoAD and with propionate as a co-substrate, 3HV fraction in PHA was increased up to 7.3-fold higher than a strain without atoAD expressed in trans (67.9 mol%). By the analysis of CoA pool concentrations in vivo and in vitro using HPLC and LC-MS, overexpression of AtoAD was shown to decrease the amount of acetyl-CoA and increase the propionyl-CoA/acetyl-CoA ratio, ultimately resulting in an increased 3HV fraction in PHA. Finally, synthesis of P(3HB-co-3HV) containing 57.9 mol% of 3HV was achieved by fed-batch fermentation of YJ101 with propionate.

MALDI-MS-based quantitative analysis for ketone containing homoserine lactones in Pseudomonas aeruginosa.

N-Acyl homoserine lactones (AHLs), quorum sensing molecules produced by Gram-negative bacteria, are used as important secondary metabolites for antibacterial drug development and cell-to-cell communication. Although various analytical techniques have been developed for detection and quantitation of AHLs from more complex bacterial culture media, only a few methods have been applied to AHL identification in physiological samples. Here, we developed a highly sensitive and reliable MALDI-based 3-oxo AHL quantitation method by employing Girard's reagent T (GT) to produce a permanent cationic charge state [M](+) at the ketone group of AHLs. After extracting AHLs from the supernatant of bacterial cultures using ethyl acetate, the extracts were subsequently derivatized with GT without any additional purification or desalting steps. The chemical derivatization of 3-oxo AHLs dramatically enhanced sensitivity (up to 60 000 times) by lowering the limit of detection (LOD, ∼0.5 fmol)/limit of quantitation (LOQ, ∼2.5 fmol). Additionally, the GT-derivatized 3-oxo AHLs allowed more accurate quantitative analysis from the Pseudomonas aeruginosa PAO1 culture supernatants. This method may be applied for developing high-throughput and sensitive detection methods of quorum sensing signal molecules in biofilm-related clinical applications such as virulence factor characterization and antibacterial drug development.

The production of ω-hydroxy palmitic acid using fatty acid metabolism and cofactor optimization in Escherichia coli.

Hydroxylated fatty acids (HFAs) are used as important precursors for bulk and fine chemicals in the chemical industry. Here, to overproduce long-chain (C16-C18) fatty acids and hydroxy fatty acid, their biosynthetic pathways including thioesterase (Lreu_0335) from Lactobacillus reuteri DSM20016, ß-hydroxyacyl-ACP dehydratase (fabZ) from Escherichia coli, and a P450 system (i.e., CYP153A from Marinobacter aquaeolei VT8 and camA/camB from Pseudomonas putida ATCC17453) were overexpressed. Acyl-CoA synthase (fadD) involved in fatty acid degradation by ß-oxidation was also deleted in E. coli BW25113. The engineered E. coli FFA4 strain without the P450 system could produce 503.0 mg/l of palmitic (C16) and 508.4 mg/l of stearic (C18) acids, of which the amounts are ca. 1.6- and 2.3-fold higher than those of the wild type. On the other hand, the E. coli HFA4 strain including the P450 system for ω-hydroxylation could produce 211.7 mg/l of ω-hydroxy palmitic acid, which was 42.1 ± 0.1 % of the generated palmitic acid, indicating that the hydroxylation reaction was the rate-determining step for the HFA production. For the maximum production of ω-hydroxy palmitic acid, NADH, i.e., an essential cofactor for P450 reaction, was overproduced by the integration of NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii into E. coli chromosome and the deletion of alcohol dehydrogenase (ADH). Finally, the NADH-level-optimized E. coli strain produced 610 mg/l of ω-hydroxy palmitic acid (ω-HPA), which was almost a threefold increase in its yield compared to the same strain without NADH overproduction.

CD39-mediated effect of human bone marrow-derived mesenchymal stem cells on the human Th17 cell function.

This study investigated the immune-modulatory effects of human bone marrow-derived mesenchymal stem cells (hBMSCs) on human Th17 cell function through the CD39-mediated adenosine-producing pathway. The suppressive effects of hBMSCs were evaluated by assessing their effects on the proliferation of Th17 cells and the secretion of interferon (IFN)-γ and interleukin (IL)-17A by Th17 cells with or without anti-CD39 treatment. Changes in CD39 and CD73 expression on the T cells with or without co-culture of hBMSCs were evaluated by flow cytometry. hBMSCs effectively suppressed the proliferation of Th17 cells and the secretion of both IL-17A and IFN-γ from Th17 cells using by both flow cytometry and ELISA, while anti-CD39 treatment significantly reduced the inhibitory effects of hBMSCs on the proliferation and secretion of the Th17 cells. The hBMSCs induced increased expression of the CD39 and CD73 on T cells correlated with the suppressive function of hBMSCs, which was accompanied by increased adenosine production. Our data suggests that hBMSCs can effectively suppress immune responses of the Th17 cells via the CD39-CD73-mediated adenosine-producing pathway.

One-step detection of procollagen type III N-terminal peptide as a fibrosis biomarker using fluorescent immunosensor (quenchbody).

BACKGROUND: Procollagen type III N-terminal peptide (P-III-NP) is a fibrosis biomarker associated with liver and cardiac fibrosis. Despite the value of P-III-NP as a biomarker, its analysis currently relies on enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), which require more than 3 h. To facilitate early diagnosis and treatment through rapid biomarker testing, we developed a one-step immunoassay for P-III-NP using a quenchbody, which is a fluorescence-labeled immunosensor for immediate signal generation. RESULTS: To create quenchbodies, the total mRNA of P-III-NP antibodies was extracted from early-developed hybridoma cells, and genes of variable regions were obtained through cDNA synthesis, inverse PCR, and sequencing. A single-chain variable fragment (scFv) with an N-terminal Cys-tag was expressed in E. coli Shuffle T7, resulting in a final yield of 9.8 mg L-1. The fluorescent dye was labeled on the Cys-tag of the anti-P-III-NP scFv using maleimide-thiol click chemistry, and the spacer arm lengths between the maleimide-fluorescent dyes were compared. Consequently, a TAMRA-C6-labeled quenchbody exhibited antigen-dependent fluorescence signals and demonstrated its ability to detect P-III-NP at concentrations as low as 0.46 ng mL-1 for buffer samples, 1.0 ng mL-1 for 2 % human serum samples. SIGNIFICANCE: This one-step P-III-NP detection method provides both qualitative and quantitative outcomes within a concise 5-min timeframe. Furthermore, its application can be expanded using a 96-well platform and human serum, making it a high-throughput and sensitive method for testing fibrotic biomarkers.

Biodegradation of oxidized low density polyethylene by Pelosinus fermentans lipase.

Polyethylene (PE) exhibits high resistance to degradation, contributing to plastic pollution. PE discarded into the environment is photo-oxidized by sunlight and oxygen. In this study, a key enzyme capable of degrading oxidized PE is reported for the first time. Twenty different enzymes from various lipase families were evaluated for hydrolytic activity using substrates mimicking oxidized PE. Among them, Pelosinus fermentans lipase 1 (PFL1) specifically cleaved the ester bonds within the oxidized carbon-carbon backbone. Moreover, PFL1 (6 µM) degraded oxidized PE film, reducing the weight average and number average molecular weights by 44.6 and 11.3 %, respectively, within five days. Finally, structural analysis and molecular docking simulations were performed to elucidate the degradation mechanism of PFL1. The oxidized PE-degrading enzyme reported here will provide the groundwork for advancing PE waste treatment technology and for engineering microbes to repurpose PE waste into valuable chemicals.

Activation of Aro80 transcription factor by heat-induced aromatic amino acid influx in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, transcription of ARO9 and ARO10 genes, involved in the catabolism of aromatic amino acids, is activated by Aro80 transcription factor in response to aromatic amino acids. Here we show that the transcription of ARO9 and ARO10 is also induced by heat shock in an Aro80-dependent manner. However, heat shock-related signaling pathways including PKA, PKC, and HOG pathways are not involved in the heat shock activation of Aro80. We elucidate that heat-induced increase in aromatic amino acid influx can lead to the inducer-dependent activation of Aro80 upon heat shock. Known aromatic amino acid permeases play an insignificant role in the heat-induced expression of ARO9 and ARO10, suggesting that an increase in plasma membrane fluidity might be responsible for the influx of aromatic amino acids during heat shock stress.

Phosphorylation of chloramphenicol by a recombinant protein Yhr2 from Streptomyces avermitilis MA4680.

Although phosphorylation of chloramphenicol has been shown to occur in the chloramphenicol producer, Streptomyces venezuelae, there are no reports on the existence of chloramphenicol phosphorylase in other Streptomyces species. In the present study, we report the modification of chloramphenicol by a recombinant protein, designated as Yhr2 (encoded by SAV_877), from Streptomyces avermitilis MA4680. Recombinant Yhr2 was expressed in Escherichia coli BL21 (DE3) and the cells expressing this recombinant protein were shown to phosphorylate chloramphenicol to a 3'-O-phosphoryl ester derivative, resulting in an inactivated form of the antibiotic. Expression of yhr2 conferred chloramphenicol resistance to E. coli cells up to 25 µg/mL and in an in vitro reaction, adenosine triphosphate (ATP), guanosine triphosphate (GTP), adenosine diphosphate (ADP) and guanosine diphosphate (GDP) were shown to be the phosphate donors for phosphorylation of chloramphenicol. This study highlights that antibiotic resistance conferring genes could be easily expressed and functionalized in other organisms that do not produce the respective antibiotic.

Simple visualization method for the c.577del of erythropoietin variant: CRISPR/dCas9-based single nucleotide polymorphism diagnosis.

One of the single nucleotide polymorphisms (SNPs) in human erythropoietin (hEPO), the c.577del variant, can produces 26 amino acids longer than the wild-type hEPO, posing a risk of misinterpretation in routine doping analysis. To prevent this, the World Anti-Doping Agency (WADA) included a procedure for reporting the sequencing results regarding the presence or absence of SNPs for suspected cases in the new version of the technical document for recombinant EPO in 2022. However, it is very expensive for anti-doping laboratories to purchase a gene sequencing analyzer, which costs hundreds of thousands of dollars, and only a few companies provide specific gene sequencing services with accredited certification. Therefore, in this study, we developed a simple visualization method for the c.577del of the EPO variant at the gene level. The gene fragment of the EPO gene, including c.577del, was amplified using a fast polymerase chain reaction (PCR), and the PCR products were incubated with the clustered regularly interspaced short palindromic repeats (CRISPR)/deadCas9 system using variant-specific single-guide RNA (sgRNA). This ribonucleoprotein complex binds specifically to the EPO variant gene fragment, causing a band shift on native-PAGE. We designed 4 sgRNAs that can bind only to the EPO variant or wild-type gene. In addition, an electrophoretic mobility shift assay on a gel demonstrated that one of the sgRNAs had a high level of specificity. Consequently, the c.577del variant was selectively detected by visualizing the target fragment of the gene on the gel within 3 h using only 3 µl of the whole blood.

CRISPR/deadCas9-based high-throughput gene doping analysis (HiGDA): A proof of concept for exogenous human erythropoietin gene doping detection.

A genetic approach targeted toward improving athletic performance is called gene doping and is prohibited by the World Anti-Doping Agency. Currently, the clustered regularly interspaced short palindromic repeats-associated protein (Cas)-related assays have been utilized to detect genetic deficiencies or mutations. Among the Cas proteins, deadCas9 (dCas9), a nuclease-deficient mutant of Cas9, acts as a DNA binding protein with a target-specific single guide RNA. On the basis of the principles, we developed a dCas9-based high-throughput gene doping analysis for exogenous gene detection. The assay comprises two distinctive dCas9s, a magnetic bead immobilized capture dCas9 for exogenous gene isolation and a biotinylated dCas9 with streptavidin-polyHRP that enables rapid signal amplification. For efficient biotin labeling via maleimide-thiol chemistry, two cysteine residues of dCas9 were structurally validated, and the Cys574 residue was identified as an essential labeling site. As a result, we succeeded in detecting the target gene in a concentration as low as 12.3 fM (7.41 × 105 copies) and up to 10 nM (6.07 × 1011 copies) in a whole blood sample within 1 h with HiGDA. Assuming an exogenous gene transfer scenario, we added a direct blood amplification step to establish a rapid analytical procedure while detecting target genes with high sensitivity. Finally, we detected the exogenous human erythropoietin gene at concentrations as low as 2.5 copies within 90 min in 5 µL of the blood sample. Herein, we propose that HiGDA is a very fast, highly sensitive, and practical detection method for actual doping field in the future.

Production and engineering of nanobody-based quenchbody sensors for detecting recombinant human growth hormone and its isoforms.

Due to athletes' misuse of recombinant human growth hormone (rhGH) for performance improvement, the World Anti-Doping Agency has designated rhGH as a prohibited substance. This study focuses on the development and improvement of a simple and fast rhGH detection method using a fluorescence-incorporated antibody sensor "Quenchbody (Q-body)" that activates upon antigen binding. Camelid-derived nanobodies were used to produce stable Q-bodies that withstand high temperatures and pH levels. Notably, pituitary human growth hormone (phGH) comprises two major isoforms, namely 22 and 20 kDa GH, which exist in a specific ratio, and the rhGH variant shares the same sequence as the 22 kDa GH isoform. Therefore, we aimed to discriminate rhGH abuse by analyzing its specific isoform ratio. Two nanobodies, NbPit (recognizing phGH) and NbRec (preferentially recognizing 22 kDa rhGH), were used to develop the Q-bodies. Nanobody production in Escherichia coli involved the utilization of a vector containing 6xHis-tag, and Q-bodies were obtained using a maleimide-thiol reaction between the N-terminal of the cysteine tag and a fluorescent dye. The addition of tryptophan residue through antibody engineering resulted in increased fluorescence intensity (FI) (from 2.58-fold to 3.04-fold). The limit of detection (LOD) was determined using a fluorescence response, with TAMRA-labeled NbRec successfully detecting 6.38 ng/ml of 22 kDa rhGH while unable to detect 20 kDa GH. However, ATTO520-labeled NbPit detected 7.00 ng/ml of 20 kDa GH and 2.20 ng/ml 22 kDa rhGH. Q-bodies successfully detected changes in the GH concentration ratio from 10 to 40 ng/ml in human serum within 10 min without requiring specialized equipment and kits. Overall, these findings have potential applications in the field of anti-doping measures and can contribute to improved monitoring and enforcement of rhGH misuse, ultimately enhancing fairness and integrity in competitive sports.

Engineering of daidzein 3'-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450.

A cytochrome P450 (CYP) enzyme, 3'-daidzein hydroxylase, CYP105D7 (3'-DH), responsible for daidzein hydroxylation at the 3'-position, was recently reported. CYP105D7 (3'-DH) is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product. Fusion-mediated construction with the reductase domain from self-sufficient CYP102D1 was done to increase electron transfer efficiency and coupling with the oxidative process. An artificial self-sufficient daidzein hydroxylase (3'-ASDH) displayed distinct spectral properties of both flavoprotein and CYP. The fusion enzyme catalyzed hydroxylation of daidzein more efficiently, with a k(cat)/K(m) value of 16.8 µM(-1) min(-1), which was about 24-fold higher than that of the 3'-DH-camA/B reconstituted enzyme. Finally, a recombinant Streptomyces avermitilis host for the expression of 3'-ASDH and production of the hydroxylated product was developed. The conversion that was attained (34.6%) was 5.2-fold higher than that of the wild-type.

Optimization, validation, and comparison of a rapid method for the quantification of insulin-like growth factor 1 in serum using liquid chromatography-high-resolution mass spectrometry.

Human insulin-like growth factor 1 (IGF-I) is the primary mediator of the effects of the growth hormone (GH). Therefore, it has been used as a biomarker to detect the abuse of GH in sports. The measurement of IGF-I relies on mass-based and immunological approaches to analysis. Among the mass-based analysis methods, liquid chromatography-mass spectrometry (LC-MS) has a number of functional advantages. LC-MS measurements based on the quantification of IGF-I, according to trypsin digestion, are used in the most common method of analyzing doping. However, this method is time-consuming and subject to experimental variability. In this study, we optimized a rapid method for detecting IGF-I without the trypsin digestion step. This method of analysis uses an ultra-centrifugal filter and an LC-HRMS through narrow-range mass scan method. To verify the validity of this method, eight categories of validation testing were applied with the following results: linearity, R2 > 0.99; limit of detection, 15 ng/ml; limit of quantification, 20 ng/ml; accuracy, >99%; recovery rate, >95%; carryover, <0.03; and inter- and intra-day precision values, %CV < 2% and %CV < 6%, respectively. Furthermore, we discussed the correlation of the quantified concentration from two other methods, immunoradiometric assay (IRMA) and parallel reaction monitoring method, using 209 serum samples. In conclusion, although both mass spectrometry-based methods worked equally well in terms of analytical performance and correlation with IRMA results, narrow-range mass scan method had several advantages, such as time and cost savings and reliable reproducibility, over the existing methods.

New application of the CRISPR-Cas9 system for site-specific exogenous gene doping analysis.

The increased potential for gene doping since the introduction of gene therapy presents the need to develop antidoping assays. We therefore aimed to develop a quick and simple method for the detection of specifically targeted exogenous doping genes utilizing an in vitro clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system. A human erythropoietin (hEPO) is a drug frequently used for doping in athletes, and gene doping using gene transfer techniques may be attempted. Therefore, we selected hEPO gene as a model of exogenous doping gene, and complemental single guide RNA (sgRNA) was designed to specifically bind to the four exon-exon junctions in the hEPO cDNA. For the rapid reaction of CRISPR-Cas9, further optimization was performed using an open-source program (CRISPOR) that avoids TT and GCC motifs before the protospacer adjacent motif (PAM) domain and predicts the efficiency of the sgRNA. We optimized the in vitro Cas9 assay and dual use of sgRNA for double cleavage and identified the limit of detection (LOD) of the 1.25 nM of the double cleavage method. We expect that the improved CRISPR-Cas9 method can be used for antidoping analysis of gene doping.

NaCl Concentration-Dependent Aminoglycoside Resistance of Halomonas socia CKY01 and Identification of Related Genes.

Among various species of marine bacteria, those belonging to the genus Halomonas have several promising applications and have been studied well. However, not much information has been available on their antibiotic resistance. In our efforts to learn about the antibiotic resistance of strain Halomonas socia CKY01, which showed production of various hydrolases and growth promotion by osmolytes in previous study, we found that it exhibited resistance to multiple antibiotics including kanamycin, ampicillin, oxacillin, carbenicillin, gentamicin, apramycin, tetracycline, and spectinomycin. However, the H. socia CKY01 resistance pattern to kanamycin, gentamicin, apramycin, tetracycline, and spectinomycin differed in the presence of 10% NaCl and 1% NaCl in the culture medium. To determine the mechanism underlying this NaCl concentration-dependent antibiotic resistance, we compared four aminoglycoside resistance genes under different salt conditions while also performing time-dependent reverse transcription PCR. We found that the aph2 gene encoding aminoglycoside phosphotransferase showed increased expression under the 10% rather than 1% NaCl conditions. When these genes were overexpressed in an Escherichia coli strain, pETDuet-1::aph2 showed a smaller inhibition zone in the presence of kanamycin, gentamicin, and apramycin than the respective control, suggesting aph2 was involved in aminoglycoside resistance. Our results demonstrated a more direct link between NaCl and aminoglycoside resistance exhibited by the H. socia CKY01 strain.

Characterization and structure identification of an antimicrobial peptide, hominicin, produced by Staphylococcus hominis MBBL 2-9.

Hominicin, antimicrobial peptide displaying potent activity against Staphylococcus aureus ATCC 25923, methicillin-resistant S. aureus (MRSA) ATCC 11435 and vancomycin-intermediate S. aureus (VISA) CCARM 3501, was purified by chloroform extraction, ion-exchange column chromatography and reverse-phase HPLC from culture supernatant of Staphylococcushominis MBBL 2-9. Hominicin exhibited heat stability up to 121 degrees C for 15min and activity under both acidic and basic conditions (from pH 2.0 to 10.0). Hominicin was cleaved into two fragments after treatment with proteinase K, resulting in the loss of its antibacterial activity, while it was resistant to trypsin, alpha-chymotrypsin, pepsin and lipase. The molecular mass of hominicin determined by mass spectrometry was 2038.4Da. LC-mass spectrometry and NMR spectroscopy analyses of the two fragments revealed the sequence of hominicin as DmIle-Dhb-Pro-Ala-Dhb-Pro-Phe-Dhb-Pro-Ala-Ile-Thr-Glu-Ile-Dhb-Ala-Ala-Val-Ile-Ala-Dmp, which had no similarity with other antimicrobial peptides previously reported. The present study is the first report of this novel antimicrobial peptide, which has uncommon amino acid residues like the ones in Class I group and shows potent activity against clinically relevant S. aureus, MRSA and VISA.