Development of a nanozyme-based electrochemical catalyst for real-time biomarker sensing of superoxide and nitric oxide anions released from living cells and exogenous donors.

Developing quantitative biosensors of superoxide (O2•-) and nitric oxide (NO) anion is crucial for pathological research. As of today, the main challenge for electrochemical detection is to develop high-selectivity nano-mimetic materials to replace natural enzymes. In this study, the dendritic-like morphological structure of silver organic framework (Ag-MOF) was successfully synthesized via a solvothermal strategy. Owing to the introduction of polymeric composites results in improved electrical conductivity and catalytic activity, which promotes mass transfer and leads to faster electron efficiency. For monitoring the electrochemical signals of O2•- and NO, the Ag-MOF electrode substrate was produced by drop-coating, and composites were designed by cyclic voltammetric potential cycles. The designed electrode substrates demonstrate high sensitivity, wide linear concentrations of 1 nM-1000 µM and 1 nM-850 µM, and low detection limits of 0.27 nM and 0.34 nM (S/N = 3) against O2•- and NO. Aside from that, the sensor successfully monitored the cellular release of O2•-, and NO from HepG2 and RAW 264.7 living cells and has the potential to monitor exogenous NO release from donors of Diethylamine (DEA)-NONOate and sodium nitroprusside (SNP). Additionally, the developed system was applied to the analysis of O2•- and NO in real biological fluid samples, and the results were good satisfactory (94.10-99.57 ± 1.23%). The designed system provides a novel approach to obtaining a good electrochemical biosensor platform that is highly selective, stable, and flexible. Finally, the proposed method provides a quantitative way to follow the dynamic changes in O2•- and NO in biological systems.

Persistence and dynamic structures of diverse cephalosporinase genes in nontyphoidal Salmonella in cross-sectional surveillance in Taiwan.

OBJECTIVES: Nontyphoidal Salmonella (NTS) is a major foodborne pathogen causing from acute gastroenteritis to bacteraemia, particularly in paediatric and elderly patients. Antimicrobial resistance of NTS, especially resistance to extended-spectrum cephalosporins, has emerged over the past decades. METHODS: Thirteen NTS isolates resistant to ceftriaxone or cefotaxime were collected from a teaching hospital in Taipei, and another three from a tertiary hospital, in New Taipei City, Taiwan, from September 2018 to December 2019. Ten other archived isolates from 2000 to 2017 were also obtained. Complete genomes of the 26 isolates were obtained. Serovars, sequence types, resistomes, genetic relatedness, and sequence comparison of plasmids were analyzed. RESULTS: Serogroups B, C2 and E were significantly associated with ampicillin resistance. Over 90% of these 26 isolates are susceptible to carbapenems and colistin. Genomic epidemiology of these isolates shows that blaCMY-2-harbouring isolates in different serovars were prevalent over two decades, presumably resulting from highly mobile IncI1 plasmid harbouring blaCMY-2. One type of the IncI1 plasmids contained a mobile element, IS26, which might be involved in the acquisition of antimicrobial resistance genes. Two emerging serovars, S. Goldcoast ST358 harbouring blaCTX-M-55 on IncHI2 plasmids and S. Anatum ST64 harbouring blaDHA-1 on IncA/C2 plasmids persisted in Taiwan, possibly through the clonal spread. Integration of complete or partial plasmid sequences into host chromosomes or multiplications of the antimicrobial resistance genes also appears to be mediated by IS26, in the two emerging clones. CONCLUSION: The dynamic movement of cephalosporinase genes mediated by IS26 in NTS is of great concern.

Electrochemical sensing of dual biomolecules in live cells and whole blood samples: A flexible gold wire-modified copper-organic framework-based hybrid composite.

For clinical research, the precise measurement of hydrogen peroxide (H2O2) and glucose (Glu) is of paramount importance, due to their imbalanced concentrations in blood glucose, and reactive oxygen species (ROS) play a huge role in COVID-19 viral disease. It is critical to construct and develop a simple, rapid, flexible, long-term, and sensitive detection of H2O2 and glucose. In this paper, we have developed a unique morphological structure of MOF(Cu) on a single-walled carbon nanotube-modified gold wire (swnt@gw). Highly designed frameworks with nanotube composites enhance electron rate-transfer behavior while extending conductance and electroactive surface area.The composite sensing system delivers wide linear-range concentrations, low detection limit, and interference-free performance in co-existence with other biomolecules and metal ions. Endogenous quantitative tracking of H2O2 was performed in macrophage live-cells with the help of a strong stimulator lipopolysaccharide.The composite device was effectively utilized for the measurement of H2O2 and glucose in turbid samples of whole blood and milk samples without a pretreatment process. The practical results of biofluids showed favorable voltammetric results and acceptance recovery percentage levels between 97.49 and 98.88%. Finally, a flexible MOF-based hybrid system may provide a suitable detection platform in the construction of electro-biosensors and hold potential promise for clinical-sensory applications.

Tailoring of peroxidase mimetics bifunctional nanocomposite: Dual mode electro-spectroscopic screening of cholesterol and hydrogen peroxide in real food samples and live cells.

A simple and rapid screening of biomarkers in clinical and food matrices is urgently needed to diagnose cardiovascular diseases. The cholesterol (Chol) and hydrogen peroxide (H2O2) are critical bio-indicators, which require more inventive detection techniques to be applied to real food, and bio-samples. In this study, a robust dual sensor was developed for Chol and H2O2 using hybrid catalyst. Bovine serum albumin (BSA)-capped nanocatalyst was potentially catalyzed 3,3',5,5'-tetramethylbenzidine (TMB), and H2O2. The enzymatic nanoelectrocatalyst delivered a wide range of signaling concentrations from 250 nM to 3.0 mM and 100 nM to 10 mM, limit of detection (LOD) of 53.2 nM and 18.4 nM for Chol and H2O2. The cholesterol oxidase-BSA-AuNPs-metal-free organic framework (ChOx-BSA-AuNPs-MFOF) based electrode surface effectively operated in live-cells and real-food samples. The enzymatic sensor exhibits adequate recovery of real-food samples (96.96-99.44%). Finally, the proposed system is a suitable choice for the potential applications of Chol and H2O2 in clinical and food chemistry.

Intelligent image analysis recognizes important orchid viral diseases.

Phalaenopsis orchids are one of the most important exporting commodities for Taiwan. Most orchids are planted and grown in greenhouses. Early detection of orchid diseases is crucially valuable to orchid farmers during orchid cultivation. At present, orchid viral diseases are generally identified with manual observation and the judgment of the grower's experience. The most commonly used assays for virus identification are nucleic acid amplification and serology. However, it is neither time nor cost efficient. Therefore, this study aimed to create a system for automatically identifying the common viral diseases in orchids using the orchid image. Our methods include the following steps: the image preprocessing by color space transformation and gamma correction, detection of leaves by a U-net model, removal of non-leaf fragment areas by connected component labeling, feature acquisition of leaf texture, and disease identification by the two-stage model with the integration of a random forest model and an inception network (deep learning) model. Thereby, the proposed system achieved the excellent accuracy of 0.9707 and 0.9180 for the image segmentation of orchid leaves and disease identification, respectively. Furthermore, this system outperformed the naked-eye identification for the easily misidentified categories [cymbidium mosaic virus (CymMV) and odontoglossum ringspot virus (ORSV)] with the accuracy of 0.842 using two-stage model and 0.667 by naked-eye identification. This system would benefit the orchid disease recognition for Phalaenopsis cultivation.

Effects of colonization-associated gene yqiC on global transcriptome, cellular respiration, and oxidative stress in Salmonella Typhimurium.

BACKGROUND: yqiC is required for colonizing the Salmonella enterica serovar Typhimurium (S. Typhimurium) in human cells; however, how yqiC regulates nontyphoidal Salmonella (NTS) genes to influence bacteria-host interactions remains unclear. METHODS: The global transcriptomes of S. Typhimurium yqiC-deleted mutant (ΔyqiC) and its wild-type strain SL1344 after 2 h of in vitro infection with Caco-2 cells were obtained through RNA sequencing to conduct comparisons and identify major yqiC-regulated genes, particularly those involved in Salmonella pathogenicity islands (SPIs), ubiquinone and menaquinone biosynthesis, electron transportation chains (ETCs), and carbohydrate/energy metabolism. A Seahorse XFp Analyzer and assays of NADH/NAD+ and H2O2 were used to compare oxygen consumption and extracellular acidification, glycolysis parameters, adenosine triphosphate (ATP) generation, NADH/NAD+ ratios, and H2O2 production between ΔyqiC and SL1344. RESULTS: After S. Typhimurium interacts with Caco-2 cells, yqiC represses gene upregulation in aspartate carbamoyl transferase, type 1 fimbriae, and iron-sulfur assembly, and it is required for expressing ilvB operon, flagellin, tdcABCD, and dmsAB. Furthermore, yqiC is required for expressing mainly SPI-1 genes and specific SPI-4, SPI-5, and SPI-6 genes; however, it diversely regulates SPI-2 and SPI-3 gene expression. yqiC significantly contributes to menD expression in menaquinone biosynthesis. A Kyoto Encyclopedia of Genes and Genomes analysis revealed the extensive association of yqiC with carbohydrate and energy metabolism. yqiC contributes to ATP generation, and the analyzer results demonstrate that yqiC is required for maintaining cellular respiration and metabolic potential under energy stress and for achieving glycolysis, glycolytic capacity, and glycolytic reserve. yqiC is also required for expressing ndh, cydA, nuoE, and sdhB but suppresses cyoC upregulation in the ETC of aerobically and anaerobically grown S. Typhimurium; priming with Caco-2 cells caused a reversed regulation of yiqC toward upregulation in these ETC complex genes. Furthermore, yqiC is required for maintaining NADH/NAD+ redox status and H2O2 production. CONCLUSIONS: Specific unreported genes that were considerably regulated by the colonization-associated gene yqiC in NTS were identified, and the key role and tentative mechanisms of yqiC in the extensive modulation of virulence factors, SPIs, ubiquinone and menaquinone biosynthesis, ETCs, glycolysis, and oxidative stress were discovered.

Particulate Cell Wall Materials of Lactobacillus acidophilus as Vaccine Adjuvant.

We evaluated Lactobacillus acidophilus (LA) for adjuvant application in animal vaccines. LA particles (LAPs) are made by treating LA with purification processes and high-pressure homogenization (HPH). We found that LAPs treated with HPH with trehalose and emulsifiers had an average particle size of 179 nm, considerably smaller than LAPs without additives. First, we evaluated the adjuvanticity of LAPs using a murine model with ovalbumin antigens, revealing that LAPs, especially in a five-fold concentration, could induce a considerable antibody response compared with other current adjuvants. In poultry vaccination tests using inactivated Newcastle disease virus, LAPs alone could induce a similar antibody response compared to commercial water-in-oil (W/O) adjuvant ISA70, a commercial adjuvant, at weeks 4 and 6; however, they declined faster than ISA70 at weeks 8 and 10. LAPs added to conventional adjuvant materials, such as mineral oil-based O/W emulsions, showed similar adjuvanticity to ISA70. LA-H5-C, composed of carbomer, emulsifiers and trehalose showed no significant body weight change in acute toxicity compared to other adjuvants including ISA70, making formulated LAPs a potential candidate for use as a veterinary vaccine adjuvant.

Correlation between Sperm Micro Ribonucleic Acid-34b and -34c Levels and Clinical Outcomes of Intracytoplasmic Sperm Injection in Men with Male Factor Infertility.

Few studies have examined the correlation between sperm miRNA levels and clinical outcomes of intracytoplasmic sperm injection (ICSI). In this study, we aimed to assess the correlation of sperm miR-34b, miR-34c, miR-122, and miR-429 levels with ICSI outcomes in men with teratozoospermia and asthenozoospermia. TaqMan microRNA quantitative polymerase chain reaction was used to evaluate the relative expression of miRNAs in sperm. The relative miRNA levels quantified using a comparative method found that the four miRNAs were not associated with fertilization rate and early embryo development. However, revels of miR-34b and miR-34c in teratozoospermia sperm of the live birth group were significantly higher than those in the non-live birth group. Receiver operating characteristic curve analysis revealed that the optimal cut-off delta cycle threshold values of miR-34b and miR-34c were 8.630 and 7.883, respectively. Statistical analysis found that the levels of miR-34b and the miR-34c in teratozoospermic and asthenozoospermic sperm above the thresholds were not associated with the fertilization rate and the high-quality embryo rate above 50%; however, they were more likely to exhibit higher implantation, pregnancy, and live birth rates. miR-34b and miR-34c were significantly associated with ICSI clinical outcomes in male factor infertility, especially teratozoospermia. Further validation is required before it becomes a clinically valid reference indicator.

A Soil-Isolated Streptomyces spororaveus Species Produces a High-Molecular-Weight Antibiotic AF1 against Fungi and Gram-Positive Bacteria.

The overuse of antibiotics has resulted in the emergence of antibiotic resistance, not only in bacteria but also in fungi. Streptomyces are known to produce numerous secondary metabolites including clinically useful antibiotics. In this study, we screened for antibiotic-producing actinobacteria from soils in Taipei and discovered a Streptomyces strain SC26 that displayed antimicrobial activities against Gram-positive bacteria and fungi, but the compounds are heat-labile. Upon UV mutagenesis, a late-sporulation mutant SC263 was isolated with the same antibiotic spectrum but increased in thermostability. The nature of the antibiotic is not clear, but its activity was resistant to proteolytic, nucleolytic and pancreatic digestions, and was retained by the 100 kDa membrane during filtration. To gather more information on SC263, the genome was sequenced, which produced three contigs with a total of 8.2 Mb and was assigned to the species of Streptomyces spororaveus based on the average nucleotide identity to the reference species S. spororaveus NBRC 15456.

First report of youcai mosaic virus in firecracker flower (Crossandra infundibuliformis) in Taiwan.

Firecracker flower or crossandra (Crossandra infundibuliformis), an ornamental native to southern Asia, is commonly grown as bedding plants in the garden. In January 2021, crossandra plants showing mosaic, chlorotic ringspot, and leaf deformation (Suppl. Fig. 1) were observed at a recreational farm in Zhuolan Township (Miauli County, Taiwan) (E120°82'62'', N24°33'30'') . Transmission electron microscope (JEM-1400, JOEL, Japan) examination by negative staining of 10 affected plants indicated the presence of particles resembling a tobamovirus in all examined affected samples. However, no tobamovirus-like particles were observed in the crude sap prepared from healthy crossandra leaf tissues. Total RNA was extracted from affected leaves and used for RT-PCR amplification using the tobamovirus group-specific primer pair Tob Uni1 (5'-ATTTAAGTGGAGGGAAAACCACT-3') and Tob Uni2 (5'-GTYGTTGATGAGTTCGTGGA-3') (Letschert et al., 2002). A cDNA fragment of about 650-bp was amplified and Sanger sequenced (ABI PRISM 3730 DNA Sequencer, Biotechnology Center at National Chung Hsing University, Taichung, Taiwan ), revealing 98% sequence identity to that of a Brassica isolate of youcai mosaic virus (YoMV, AY318866). The virus was isolated through mechanical inoculation onto Chenopodium quinoa to yield two pure isolates, designated FC-1 and FC-2. Mechanical inoculation of FC-1 and FC-2 back to virus-free Crossandra infundibuliformis plants (5 for each isolate) resulted in systemic mosaic, chlorotic ringspot, and leaf deformation. All mock and healthy controls were symptomless and failed to obtain any RT-PCR products with YoMV-specific primers CPF1 (5'- ATGGTTTACAACATCACGAG-3') and CPR1 (5'-CTATGTAGCTGGCGCAGTAG-3'). Systemic symptoms of mild mosaic, ringspot, leafroll, and necrosis appeared on some of the tobaccos (Nicotiana benthamiana, N. tabacum, N. rustica), and cruciferous vegetables (Brassica rapa subsp. chinensis cv. Known-You No.2), B. rapa subsp. pekinensis cv. Autumn Sun), B. oleracea var. italica cv. Ching Hua), B. oleracea var. capitata cv. Green Peak), and Raphanus sativus cv. Snow Lady). However, inoculation of FC-1 and FC-2 on pepper (Capsicum annuum cv. Blue Star) resulted in severe necrosis on leaves and necrotic sunken spots on petioles and stems causing acute wilting and quick death. In stark contrast, FC-1 and FC-2 only induced local lesions on inoculated leaves of Chenopodium quinoa, Gomphrena globose, and Carica papaya. The infectivity of FC isolates to all plants used in host range tests were further confirmed by RT-PCR as mentioned. Oligonucleotide primers (Suppl. Table 1) specifically complementary to YoMV sequence were designed and used to amplify full-length genomic sequences of FC-1 and FC-2 isolates by RT-PCR and Sanger sequencing. Sequence analysis revealed that the genome of both FC-1 and FC-2 isolates consists of 6302 nucleotides. The viral genome has four open reading frames encoding a small replicase subunit, a RNA-dependent RNA polymerase , a movement protein, and a coat protein (CP), respectively. Both sequences, which shared 99.6% identity with each other, have been deposited in the NCBI database (Genbank Accession Numbers LC701592 and LC701593). FC-1 and FC-2 and the deduced amino acid sequences of CP shared 91.2% - 98.9% and 93.4 - 99.4% similarities, respectively, to those of published YoMV strains, confirming the identity of FC-1 and FC-2. RT-PCR analyses detected YoMV in all (100%) crossandra samples collected from the field (Suppl. Fig. 2). YoMV, formerly named Chinese rape mosaic virus (CRMV) or oilseed rape mosaic virus (ORMV) (Zhu et al., 2001), has been reported to infect cruciferous, solanaceous, and ornamental crops in Asia and Europe (Ju et al., 2019). The firecracker flower is a common and popular ornamental in Taiwan, even though its economic values are not so important. However, finding YoMV in firecracker flower may have epidemiological impacts as YoMV can infect economically important cruciferous and solanaceous crops. To our knowledge, this is the first report of YoMV infecting firecracker flowers in Taiwan.

Enhancement of Swimming Endurance by Herbal Supplement M3P.

OBJECTIVE: To investigate the effect of M3P (containing Deer antler, Cordyceps sinensis, Rhodiola rosea, and Panax ginseng); an herbal remedy with the function of tonifying Kidney (Shen) and invigorating Spleen (Pi), replenishing qi and nourishing blood; on fatigue alleviation, endurance capacity and toxicity. METHODS: Swimming with weight-loading of 24 male ICR mice was used to evaluate the endurance capacity, and fatigue-related plasma biomarkers were determined. Mice were randomly assigned to control or M3P treatment groups with 6 mice for each group and were orally administered with M3P everyday for 8 weeks at doses 0, 10, 33 or 100 mg/kg. Swimming time to exhaustion was measured in a specialized water tank. Lliver and kidney functions, body weight, and hematological profile were determined to evaluate the safety and toxicity after long-term M3P administration. RESULTS: M3P supplementation 100 mg/kg significantly increased swimming endurance time up to approximate 2.4 folds of controls (P<0.05). The plasma concentrations of cortisol and hepatic glycogen content were significantly increased in mice received M3P (P<0.05, P<0.01 respectively). The lactic acid level and blood glucose were not changed after M3P treatment (P>0.05). The liver and kidney functions muscle damage biomarker creatine, body weight, and hemograms were not altered in M3P supplementation (P>0.05). CONCLUSION: M3P supplementation may improve swimming endurance accompanied by increasing hepatic glycogen content and serum cortisol level without major toxicity.

Convenient and ultrasensitive detection of live Salmonella using ratiometric electrochemical molecular substrates.

Salmonella contamination is a major concern in food and public health safety, and carrying out episodic monitoring of Salmonella contamination in food and water bodies is essential for safeguarding public health and the economy. Therefore, there is an urgent need to develop an easy-to-operate Salmonella-targeting point-of-care detection platform. To this end, we designed two activity-based latent ratiometric electrochemical molecular substrates, denoted as Sal-CAF and Sal-NBAF, specifically for achieving easy, rapid, and selective profiling of Salmonella esterase (a Salmonella biomarker) under physiological conditions. The octyl esters of the substrates were cleaved by the esterase and triggered the trimethyl lock to eject the electron-rich aminoferrocene derivatives (CAF and NBAF), and the corresponding electrochemical signals were tracked at the negative region (-0.08 V vs Ag/AgCl) of the voltammetric spectrum. The Sal-CAF substrate was used to determine the concentration of Salmonella in a wide dynamic range (1.03 × 105-1.1 × 1010 CFU mL-1) with a low detection limit of 39.27 × 103 CFU mL-1. The developed probes were tested against various bacteria but were only activated by live Salmonella. Furthermore, the Sal-CAF probe was used directly in quantifying spiked live Salmonella spiked in milk samples and also used to effectively monitor and quantify Salmonella production in real-time. These achievements indicated the Sal-CAF probe to be a promising platform for point-of-care Salmonella analysis.

Genotypic Diversity of Ciprofloxacin Nonsusceptibility and Its Relationship with Minimum Inhibitory Concentrations in Nontyphoidal Salmonella Clinical Isolates in Taiwan.

This study analyzed the genetic diversity of ciprofloxacin (CIP) nonsusceptibility and the relationship between two major mechanisms and minimum inhibitory concentrations (MICs) of CIP in nontyphoidal Salmonella (NTS). Chromosomal mutations in quinolone resistance-determining regions (QRDRs) and plasmid-mediated quinolone resistance (PMQR) genes were searched from ResFinder, ARG-ANNOT, and PubMed for designing the sequencing regions in gyrA, gyrB, parC, and parE, and the 13 polymerase chain reactions for PMQR genes. We found that QRDR mutations were detected in gyrA (82.1%), parC (59.0%), and parE (20.5%) but not in gyrB among the 39 isolates. Five of the 13 PMQR genes were identified, including oqxA (28.2%), oqxB (28.2%), qnrS (18.0%), aac(6')-Ib-cr (10.3%), and qnrB (5.1%), which correlated with the MICs of CIP within 0.25-2 µg/mL, and it was found that oxqAB contributed more than qnr genes to increase the MICs. All the isolates contained either QRDR mutations (53.8%), PMQR genes (15.4%), or both (30.8%). QRDR mutations (84.6%) were more commonly detected than PMQR genes (46.2%). QRDR mutation numbers were significantly associated with MICs (p < 0.001). Double mutations in gyrA and parC determined high CIP resistance (MICs ≥ 4 µg/mL). PMQR genes contributed to intermediate to low CIP resistance (MICs 0.25-2 µg/mL), thus providing insights into mechanisms underlying CIP resistance.

An Actinobacterial Isolate, Streptomyces sp. YX44, Produces Broad-Spectrum Antibiotics That Strongly Inhibit Staphylococcus aureus.

The need for new antibiotics is increasing due to their overuse, and antibiotic resistance has become one of the major threats worldwide to public health, food safety, and clinical treatment. In this study, we describe an actinobacterial isolate, YX44, which belongs to the genus Streptomyces. This Streptomyces was isolated from a drinking pipe located in Osaka, Japan, and has the ability to inhibit Gram-positive bacteria, Gram-negative bacteria, and various fungi. YX44 fermentation broth shows strong activity against Escherichia coli and Staphylococcus aureus, as well as also inhibiting clinical isolates of multidrug-resistant Staphylococcus aureus. The YX44 antibacterial substances in the broth are relatively heat-stable, show high stability from the pH range 1 to 11, and have good solubility in both organic and non-organic solvents. Size-exclusion chromatography revealed that the YX44 antibacterial compounds are less than 1000 Da in size. LC-MS was able to identify three possible candidate molecules with molecular weights of 308, 365, 460, and 653 g/mol; none of these sizes correspond to any well-known antibiotics. Our results show that Streptomyces sp. YX44 seems to produce a number of novel antibiotics with high pH stability and good solubility that have significant activity against S. aureus, including multidrug-resistant strains.

Molecular Epidemiology and Characterization of Carbapenem-Resistant Klebsiella pneumoniae Isolated from Urine at a Teaching Hospital in Taiwan.

Urinary tract infections (UTIs) are common in clinics and hospitals and are associated with a high economic burden. Enterobacterium Klebsiella pneumoniae is a prevalent agent causing UTIs. A high prevalence of carbapenem-resistant K. pneumoniae (CRKP) has emerged recently and is continuing to increase. Seventeen urinary CRKP isolates collected at a teaching hospital in Taiwan from December 2016 to September 2017 were analyzed to elucidate their drug resistance mechanisms. Two-thirds of the isolates were obtained from outpatients. Antimicrobial susceptibility tests demonstrated multidrug resistance in all the isolates. Multilocus sequence typing analysis showed high diversity among the isolates. PCR analysis demonstrated the presence of carbapenemases in three isolates. All isolates carried at least one other extended-spectrum ß-lactamase, including TEM, DHA, and CTX-M. Fifteen isolates contained mutations in one of the outer membrane porins that were assessed. The expression levels of the acrB and/or oqxB efflux pump genes, as determined by qRT-PCR, were upregulated in 11 isolates. Six isolates might have utilized other efflux pumps or antimicrobial resistance mechanisms. These analyses demonstrated a highly diverse population and the presence of complex resistance mechanisms in urinary isolates of K. pneumoniae.

Role of wzxE in Salmonella Typhimurium lipopolysaccharide biosynthesis and interleukin-8 secretion regulation in human intestinal epithelial cells.

In Salmonella Typhimurium (S. Typhimurium), lipopolysaccharide (LPS) anchored on the bacterial outer membrane is a major immune stimulus that can broadly activate immune cells and induce innate immune responses. wzxE is involved in bacterial LPS biosynthesis but has rarely been reported in Salmonella; wzxE encodes a flipase that can flip the precursor of LPS across the membrane into the periplasm space. Our preliminary data showed that the wzxE transposon mutant of S. Typhimurium could not significantly adhere to and invade into HEp-2 cells, but the mechanism remains unknown. In this study, we infected human LS174T, Caco-2, HeLa, and THP-1 cells with the wild-type S. Typhimurium strain SL1344, its wzxE mutant, and its complemented strain. wzxE depletion significantly attenuated bacterial adhesion and internalization in the four cell types. In addition, the postinfectious production of interleukin-8 (IL-8) was significantly decreased in the Caco-2 cells infected with the wzxE mutant. Bacterial LPS stained with polymyxin B probe also exhibited a reduced signal in the wzxE mutant. The silver staining of purified LPS demonstrated a significant reduction of the O-antigen (OAg) chain in the wzxE mutant. To confirm the role of OAg in the wzxE mutant during infection, we treated the HT-29 cells with the S. Typhimurium strain SL1344, its wzxE mutant, and their purified LPS, which revealed significantly decreased IL-8 secretion in the HT-29 cells treated with purified LPS from the wzxE mutant and with the wzxE mutant. In conclusion, wzxE mediates LPS biosynthesis and plays a major role in bacterial pathogenesis by regulating OAg flipping.

Characterization of a New Monopartite Begomovirus with a Betasatellite Associated with Leaf Curl, Yellow Vein, and Vein Enation in Hibiscus rosa-sinensis.

A new begomovirus, tentatively named hibiscus yellow vein leaf curl virus (HYVLCV), was identified in Hibiscus rosa-sinensis plants showing symptoms of leaf curl, yellow vein, and vein enation on the undersides of the leaf in Taiwan. Sequence analysis of the full-length HYVLCV genome from the rolling cycle amplicon revealed a genome of 2,740 nucleotides that contains six open reading frames and a conserved sequence (5'-TAATATTAC-3') commonly found in geminiviral genomes. HYVLCV shares the highest nucleotide identity (88.8%) with cotton leaf curl Multan virus (CLCuMuV) genome, which is lower than the criteria (91%) set for species demarcation in the genus Begomovirus. No begomoviral DNA-B was detected; however, a begomovirus-associated DNA betasatellite (DNA-ß) was detected. The DNA-ß (1,355 nucleotides) shares the highest nucleotide identity (78.6%) with malvastrum yellow vein betasatellite (MaYVB). Because the identity is slightly higher than the criteria (78%) set for the species demarcation threshold for a distinct DNA-ß species, the DNA-ß of HYVLCV reported in this study is considered the same species of MaYVB and tentatively named MaYVB-Hib. An expected 1,498-bp fragment was amplified with two HYVLCV-specific primers from 10 of 11 field-collected samples. Four independent amplicons were sequenced, revealing 100% nucleotide identity with the HYVLCV genome. Agroinoculation of a dimer of the infectious monopartite genome alone to Nicotiana benthamiana resulted in mild symptoms at 28 days postinoculation (dpi); coagroinoculation with the DNA-ß satellite resulted in severe symptoms at 12 dpi. HYVLCV could be transmitted to healthy H. rosa-sinensis by grafting, resulting in yellow vein symptoms at 30 dpi.

Biological, Pathological, and Molecular Characteristics of a New Potyvirus, Dendrobium Chlorotic Mosaic Virus, Infecting Dendrobium Orchid.

Dendrobium smillieae is one of the popular orchids in Taiwan. This report describes a new potyvirus tentatively named Dendrobium chlorotic mosaic virus (DeCMV) causing chlorotic and mosaic symptoms in D. smillieae. Enzyme-linked immunosorbent assay (ELISA) tests using six antisera against orchid-infecting viruses revealed that only a monoclonal antibody against the potyvirus group reacted positively with crude saps prepared from a symptomatic dendrobium orchid. Potyvirus-like, flexuous, filamentous particles were observed under an electron microscope, measuring approximately 700 to 800 nm in length and 11 to 12 nm in diameter. Sequence analyses revealed that DeCMV coat protein gene shared 59.6 to 66.0% nucleotide sequence identity and 57.6 to 66.0% amino acid sequence identity, whereas the DeCMV complete genome shared 54.1 to 57.3% nucleotide sequence identity and 43.7 to 49.5% amino acid sequence identity with those other known potyviruses. These similarity levels were much lower than the criteria set for species demarcation in potyviruses. Thus, DeCMV can be considered a new potyvirus. The whole DeCMV genome contains 10,041 nucleotides (GenBank accession no. MK241979) and encodes a polyprotein that is predicted to produce 10 proteins by proteolytic cleavage. In a pathogenicity test, results of inoculation assays demonstrated that DeCMV can be transmitted to dendrobium orchids by grafting and mechanical inoculation, as verified by ELISA and western blot analyses using the DeCMV polyclonal antiserum and by reverse transcription polymerase chain reaction using the coat protein gene-specific primers. The inoculated orchids developed similar chlorotic and mosaic symptoms. In conclusion, DeCMV is a novel orchid-infecting potyvirus, and this is the first report of a new potyvirus that infects dendrobium orchids in Taiwan.

Electrochemical Molecular Switch for the Selective Profiling of Cysteine in Live Cells and Whole Blood and for the Quantification of Aminoacylase-1.

A first-of-a-kind latent electrochemical redox probe, ferrocene carbamate phenyl acrylate (FCPA), was developed for the selective detection of cysteine (Cys) and aminoacylase (ACY-1). The electrochemical signal generated by this probe was shown to be highly specific to Cys and insensitive to other amino acids and biological redox reactants. The FCPA-incorporated electrochemical sensor exhibited a broad dynamic range of 0.25-100 µM toward Cys. This probe also proficiently monitored the ACY-1-catalyzed biochemical transformation of N-acetylcysteine (NAC) into Cys, and this proficiency was used to develop an electrochemical assay for quantifying active ACY-1, which it did so in a dynamic range of 10-200 pM (0.1-2 mU/cm3) with a detection limit of 1 pM (0.01 mU/cm3). Furthermore, the probe was utilized in real-time tracking and quantification of cellular Cys production, specifically in Escherichia coli W3110, along with a whole blood assay to determine levels of Cys and spiked ACY-1 in blood with a reliable analytical performance.

A highly efficient targeted recombination system for engineering linear chromosomes of industrial bacteria Streptomyces.

Soil bacteria Streptomyces are the most important producers of secondary metabolites, including most known antibiotics. These bacteria and their close relatives are unique in possessing linear chromosomes, which typically harbor 20 to 30 biosynthetic gene clusters of tens to hundreds of kb in length. Many Streptomyces chromosomes are accompanied by linear plasmids with sizes ranging from several to several hundred kb. The large linear plasmids also often contain biosynthetic gene clusters. We have developed a targeted recombination procedure for arm exchanges between a linear plasmid and a linear chromosome. A chromosomal segment inserted in an artificially constructed plasmid allows homologous recombination between the two replicons at the homology. Depending on the design, the recombination may result in two recombinant replicons or a single recombinant chromosome with the loss of the recombinant plasmid that lacks a replication origin. The efficiency of such targeted recombination ranges from 9 to 83% depending on the locations of the homology (and thus the size of the chromosomal arm exchanged), essentially eliminating the necessity of selection. The targeted recombination is useful for the efficient engineering of the Streptomyces genome for large-scale deletion, addition, and shuffling.