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.

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.

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.

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.

Telomere associated primase Tap repairs truncated telomeres of Streptomyces.

Replication of the linear chromosomes of soil bacteria Streptomyces proceeds from an internal origin towards the telomeres, followed by patching of the resulting terminal single-strand overhangs by DNA synthesis using terminal proteins as the primer, which remains covalently bound to the 5΄ ends of the DNA. In most Streptomyces chromosomes, the end patching requires the single-strand overhangs, terminal protein Tpg, and terminal associated protein Tap. The telomere overhangs contain several palindromic sequences capable of forming stable hairpins. Previous in vitro deoxynucleotidylation studies indicated that Tap adds the Palindrome I sequence to Tpg, which is extended by a polymerase to fill the gap. In this study, the stringency of Palindrome I sequence was examined by an in vitro deoxynucleotidylation system and in vivo replication. Several nt in Palindrome I were identified to be critical for priming. While the first 3 G on the template were required for deoxynucleotidylation in vitro, deletions of them could be suppressed by the presence of dGTP. In vivo, deletions of these G were also tolerated, and the telomere sequence was restored in the linear plasmid DNA. Our results indicated that the truncated telomeres were repaired by extension synthesis by Tap on the foldback Palindrome I sequence.

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.

Indoor Scene Point Cloud Registration Algorithm Based on RGB-D Camera Calibration.

With the increasing popularity of RGB-depth (RGB-D) sensor, research on the use of RGB-D sensors to reconstruct three-dimensional (3D) indoor scenes has gained more and more attention. In this paper, an automatic point cloud registration algorithm is proposed to efficiently handle the task of 3D indoor scene reconstruction using pan-tilt platforms on a fixed position. The proposed algorithm aims to align multiple point clouds using extrinsic parameters of the RGB-D camera obtained from every preset pan-tilt control point. A computationally efficient global registration method is proposed based on transformation matrices formed by the offline calibrated extrinsic parameters. Then, a local registration method, which is an optional operation in the proposed algorithm, is employed to refine the preliminary alignment result. Experimental results validate the quality and computational efficiency of the proposed point cloud alignment algorithm by comparing it with two state-of-the-art methods.

A new electrochemical substrate for rapid and sensitive in vivo monitoring of ß-galactosidase gene expressions.

A 4-Methoxyphenyl-ß-galactopyranoside (4-MPGal) substrate incorporating 4-methoxy phenol (4-MP) as an electrochemical reporter is described for the monitoring of ß-Galactosidase (ß-Gal) gene expressions. ß-Gal derived from Escherichia coli (E. coli) and Aspergillus oryzae (A. oryzae) were investigated, while a graphene oxide film modified electrode was employed as the transducer. The electrochemical signal of 4-MPG within 4-MPGal was masked by protecting their hydroxyl group with galactose. The externally added ß-Gal triggered the deprotection through specific enzymatic hydrolysis with concomitant release of 4-MP. The apparent Km and Vmax values of 4-MPGal are determined to be 0.21 mM and 0.51 µM min(-1) mg of ß-Gal(-1) (E. coli), which is consistent with the previous reports. To detect ß-Gal derived from E. coli, cyclic voltammetry (CV) provides linear ranges of 12-1200 ng mL(-1) and 1.2-12 µg mL(-1) with a limit of detection (LOD) of 5 ng mL(-1), while differential pulse voltammetry (DPV) shows a linear range of 1.2-120 ng mL(-1) and LOD of 1 ng mL(-1). To detect ß-Gal derived from A. oryzae, CV provides linear ranges of 0.1-100 ng mL(-1) and 0.1-1 µg mL(-1) with a LOD of 0.06 ng mL(-1), while DPV shows a linear range of 10 pg mL(-1)-10 ng mL(-1) with a LOD of 8 pg mL(-1). Moreover, we set up a platform for the real-time in vivo monitoring of ß-Gal gene expressions in E. coli cultivated through microbiological culture. The developed sensing platform using 4-MPGal as a substrate is simple, rapid, sensitive, specific and advantageous over its laborious optical analogues.

Live and heat-killed Lactobacillus rhamnosus GG upregulate gene expression of pro-inflammatory cytokines in 5-fluorouracil-pretreated Caco-2 cells.

PURPOSE: This study investigates whether post-chemotherapeutic use of live and heat-killed Lactobacillus rhamnosus GG can modulate the expression of three pro-inflammatory cytokines in 5-fluorouracil (5-FU)-induced intestinal mucositis in vitro. METHODS: Live L. rhamnosus GG and heat-killed L. rhamnosus GG were observed using scanning electron microscopy. To establish the duration required for optimal expression of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interleukin-12 (IL-12), 5 µM of 5-FU was selected to treat 10-day-old Caco-2 cells for 4, 6, 8, and 24 h. Caco-2 cells were treated with 5-FU (5 µM) for 4 h, followed by the administration of live L. rhamnosus GG (multiplicity of infection = 25), and heat-killed L. rhamnosus GG for 2 and 4 h. Finally, total cellular RNA was isolated to quantify mRNA expression of TNF-α, MCP-1, and IL-12 using real-time PCR. RESULTS: The results demonstrated that heat-killed L. rhamnosus GG remained structurally intact with elongation. A biphasic upregulated expression of TNF-α, MCP-1, and IL-12 was observed in 5-FU-treated Caco-2 cells at 4 and 24 h. Compared to non-L. rhamnosus GG controls in 5-FU-pretreated Caco-2 cells, a 2-h treatment of heat-killed L. rhamnosus GG significantly upregulated the MCP-1 expression (p < 0.05), and both live and heat-killed L. rhamnosus GG treatments lasting 4 h upregulated the TNF-α and MCP-1 expression (p < 0.05). Only live L. rhamnosus GG upregulated the IL-12 expression (p < 0.05). CONCLUSIONS: Post-chemotherapeutic use of live or heat-killed L. rhamnosus GG can upregulate the gene expression of 5-FU-induced pro-inflammatory cytokines in Caco-2 cells. Human intestinal epithelium may be vulnerable to the post-chemotherapeutic use of L. rhamnosus GG in 5-FU-induced mucositis that requires further in vivo studies for clarification.

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.

Linear Streptomyces plasmids form superhelical circles through interactions between their terminal proteins.

Linear chromosomes and linear plasmids of Streptomyces possess covalently bound terminal proteins (TPs) at the 5' ends of their telomeres. These TPs are proposed to act as primers for DNA synthesis that patches the single-stranded gaps at the 3' ends during replication. Most ('archetypal') Streptomyces TPs (designated Tpg) are highly conserved in size and sequence. In addition, there are a number of atypical TPs with heterologous sequences and sizes, one of which is Tpc that caps SCP1 plasmid of Streptomyces coelicolor. Interactions between the TPs on the linear Streptomyces replicons have been suggested by electrophoretic behaviors of TP-capped DNA and circular genetic maps of Streptomyces chromosomes. Using chemical cross-linking, we demonstrated intramolecular and intermolecular interactions in vivo between Tpgs, between Tpcs and between Tpg and Tpc. Interactions between the chromosomal and plasmid telomeres were also detected in vivo. The intramolecular telomere interactions produced negative superhelicity in the linear DNA, which was relaxed by topoisomerase I. Such intramolecular association between the TPs poses a post-replicational complication in the formation of a pseudo-dimeric structure that requires resolution by exchanging TPs or DNA.

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.

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.

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.

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.

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.

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.