Comparison of MiSeq, MinION, and hybrid genome sequencing for analysis of Campylobacter jejuni.

The sequencing, assembly, and analysis of bacterial genomes is central to tracking and characterizing foodborne pathogens. The bulk of bacterial genome sequencing at the US Food and Drug Administration is performed using short-read Illumina MiSeq technology, resulting in highly accurate but fragmented genomic sequences. The MinION sequencer from Oxford Nanopore is an evolving technology that produces long-read sequencing data with low equipment cost. The goal of this study was to compare Campylobacter genome assemblies generated from MiSeq and MinION data independently, as well as hybrid genome assemblies combining both data types. Two reference strains and two field isolates of C. jejuni were sequenced using MiSeq and MinION, and the sequence data were assembled using the software programs SPAdes and Canu, respectively. Hybrid genome assembly was performed using the program Unicycler. Comparison of the C. jejuni 81-176 and RM1221 genome assemblies to the PacBio reference genomes revealed that the SPAdes assemblies had the most accurate nucleotide identity, while the hybrid assemblies were the most contiguous. Assemblies generated only from MinION data using Canu were the least accurate, containing many indels and substitutions that affected downstream analyses. The hybrid sequencing approach was the most useful for detecting plasmids, large genome rearrangements, and repetitive elements such as rRNA and tRNA genes. The full genomes of both C. jejuni field isolates were completed and circularized using hybrid sequencing, and a plasmid was detected in one isolate. Continued development of nanopore sequencing technologies will likely enhance the accuracy of hybrid genome assemblies and enable public health laboratories to routinely generate complete circularized bacterial genome sequences.

A Rapid Method for the Detection of Diarrhetic Shellfish Toxins and Azaspiracid Shellfish Toxins in Washington State Shellfish by Liquid Chromatography Tandem Mass Spectrometry.

BACKGROUND: Diarrhetic shellfish toxins (DSTs) in domestic shellfish and azaspiracids (AZAs) in imported products are emerging seafood safety issues in the United States. In addition to causing gastrointestinal illnesses, some of these toxins are also carcinogenic and genotoxic. Efficient analytical strategies are needed for their monitoring in U.S. domestic and imported shellfish. OBJECTIVE: In the US, DSTs and AZAs are the only lipophilic shellfish toxins addressed in regulations. Streamlining of existing methods for several classes of lipophilic toxins, based on liquid chromatography coupled with triple quadrupole mass spectrometry, was pursued. METHOD: The resulting simplified LC-MS/MS method is focused on the separation and detection of just the AZAs and total DSTs using a C18 Hypersil gold column. Filter vials are used to expedite and simplify sample handling. RESULTS: The method has a run time of 7.25 min. LOQs for the AZAs and DSTs in shellfish were 0.3-0.4 µg/kg. Recoveries (AZAs and total DSTs) for three spiking levels in three matrixes ranged from 68 to 129%. Trueness was established using certified reference materials. Method equivalence was established using shellfish provided blind by the Washington State Department of Health Public Health Laboratory (WA DOH PHL). Data obtained from these samples agreed well with data from another LC-MS/MS method used in harvest control by WA DOH PHL (R = 0.999; P < 0.0001). CONCLUSIONS: The LC-MS/MS method described offers more rapid sample handling and has excellent sensitivity, linearity, and repeatability.

Whole Genome Sequencing and Multiplex qPCR Methods to Identify Campylobacter jejuni Encoding cst-II or cst-III Sialyltransferase.

Campylobacter jejuni causes more than 2 million cases of gastroenteritis annually in the United States, and is also linked to the autoimmune sequelae Guillan-Barre syndrome (GBS). GBS often results in flaccid paralysis, as the myelin sheaths of nerve cells are degraded by the adaptive immune response. Certain strains of C. jejuni modify their lipooligosaccharide (LOS) with the addition of neuraminic acid, resulting in LOS moieties that are structurally similar to gangliosides present on nerve cells. This can trigger GBS in a susceptible host, as antibodies generated against C. jejuni can cross-react with gangliosides, leading to demyelination of nerves and a loss of signal transduction. The goal of this study was to develop a quantitative PCR (qPCR) method and use whole genome sequencing data to detect the Campylobacter sialyltransferase (cst) genes responsible for the addition of neuraminic acid to LOS. The qPCR method was used to screen a library of 89 C. jejuni field samples collected by the Food and Drug Administration Pacific Northwest Lab (PNL) as well as clinical isolates transferred to PNL. In silico analysis was used to screen 827 C. jejuni genomes in the FDA GenomeTrakr SRA database. The results indicate that a majority of C. jejuni strains could produce LOS with ganglioside mimicry, as 43.8% of PNL isolates and 46.9% of the GenomeTrakr isolates lacked the cst genes. The methods described in this study can be used by public health laboratories to rapidly determine whether a C. jejuni isolate has the potential to induce GBS. Based on these results, a majority of C. jejuni in the PNL collection and submitted to GenomeTrakr have the potential to produce LOS that mimics human gangliosides.

Chlorella diet alters mitochondrial cardiolipin contents differentially in organs of Danio rerio analyzed by a lipidomics approach.

The zebrafish (Danio rerio) is an important and widely used vertebrate model organism for the study of human diseases which include disorders caused by dysfunctional mitochondria. Mitochondria play an essential role in both energy metabolism and apoptosis, which are mediated through a mitochondrial phospholipid cardiolipin (CL). In order to examine the cardiolipin profile in the zebrafish model, we developed a CL analysis platform by using liquid chromatography-mass spectrometry (LC-MS). Meanwhile, we tested whether chlorella diet would alter the CL profile in the larval fish, and in various organs of the adult fish. The results showed that chlorella diet increased the chain length of CL in larval fish. In the adult zebrafish, the distribution patterns of CL species were similar between the adult brain and eye tissues, and between the heart and muscles. Interestingly, monolyso-cardiolipin (MLCL) was not detected in brain and eyes but found in other examined tissues, indicating a different remodeling mechanism to maintain the CL integrity. While the adult zebrafish were fed with chlorella for four weeks, the CL distribution showed an increase of the species of saturated acyl chains in the brain and eyes, but a decrease in the other organs. Moreover, chlorella diet led to a decrease of MLCL percentage in organs except the non-MLCL-containing brain and eyes. The CL analysis in the zebrafish provides an important tool for studying the mechanism of mitochondria diseases, and may also be useful for testing medical regimens targeting against the Barth Syndrome.

Breaking Through the Multi-Mesa-Channel Width Limited of Normally Off GaN HEMTs Through Modulation of the Via-Hole-Length.

We present new normally off GaN high-electron-mobility transistors (HEMTs) that overcome the typical limitations in multi-mesa-channel (MMC) width through modulation of the via-hole-length to regulate the charge neutrality screen effect. We have prepared enhancement-mode (E-mode) GaN HEMTs having widths of up to 300 nm, based on an enhanced surface pinning effect. E-mode GaN HEMTs having MMC structures and widths as well as via-hole-lengths of 100 nm/2 µm and 300 nm/6 µm, respectively, exhibited positive threshold voltages (V th) of 0.79 and 0.46 V, respectively. The on-resistances of the MMC and via-hole-length structures were lower than those of typical tri-gate nanoribbon GaN HEMTs. In addition, the devices not only achieved the E-mode but also improved the power performance of the GaN HEMTs and effectively mitigated the device thermal effect. We controlled the via-hole-length sidewall surface pinning effect to obtain the E-mode GaN HEMTs. Our findings suggest that via-hole-length normally off GaN HEMTs have great potential for use in next-generation power electronics.

Simultaneous Identification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari with SmartCycler-Based Multiplex Quantitative Polymerase Chain Reaction.

BACKGROUND: Consumption of Campylobacter contaminated food or water is a leading cause of human acute gastroenteritis. Campylobacter jejuni, Campylobacter coli, and Campylobacter lari account for over 95% of total Campylobacter infections. A multiplex quantitative polymerase chain reaction (qPCR) for simultaneous identification of C. jejuni, C. coli, and C. lari was developed for use with the SmartCycler II system. MATERIALS AND METHODS: We evaluated and combined previously described primers and probes for Campylobacter detection, designed a new internal amplification control, and optimized the multiplex qPCR for the detection of C. jejuni, C. coli, and C. lari. RESULTS: This method was 100% specific when tested against a panel of 32 target Campylobacter strains and 31 non-Campylobacter reference strains. Furthermore, there was no cross-reactivity with seven strains from four nontarget Campylobacter species. The amplification efficiency of each target in this multiplex qPCR was over 90%, and each coefficient of linearity was greater than 0.99. With artificially mixed genomic DNA, this method detected as few as two, three, and two genome copies of C. jejuni, C. coli, and C. lari, respectively. This method was also able to detect these three Campylobacter species in artificially contaminated milk with a sensitivity of five spiked cells of each target per reaction. CONCLUSION: The three Campylobacter targets were simultaneously identified using artificially mixed genomic DNA and spiked raw milk. This SmartCycler-based multiplex qPCR is a rapid, specific, and sensitive method to identify C. jejuni, C. coli, and C. lari.

Deferasirox-Iron Complex Formation Ratio as an Indicator of Long-term Chelation Efficacy in ß-Thalassemia Major.

BACKGROUND: ß-Thalassemia major patients with higher total drug levels [deferasirox (DEFR) plus its iron complex] do not yield better serum ferritin (SF) control. This study aimed to determine the concentrations of DEFR and its iron complex (Fe-[DEFR]2) in thalassemia patients to predict the chelation efficacy in terms of SF and cardiac T2* values. METHODS: Patients' steady-state drug levels at trough (Ctrough) and 2 hours postdose (C2h) were determined. Because iron deposition may cause changes in the hepatic metabolism of amino acids, the concentrations of 40 amino acids in plasma were also assayed at 2 hours postdose. RESULTS: A total of 28 patients either dosing daily or twice daily were recruited. After a 1-month DEFR maintenance therapy, 38.8% and 30% of patients from groups of once-daily and twice-daily, respectively, had a plasma DEFR-iron complex formation ratio higher than 0.05 [High Chelation Ratio, (HCR)]. After a 6-month follow-up, those patients who had a HCR (n = 10) at C2h showed more favorable median changes in SF and cardiac T2* values (-388.0, +10.1) than those with a low DEFR-iron complex formation ratio (Low Chelation Ratio; n = 18; +10.5; +4.5) compared with the baseline. The levels of plasma L-arginine, L-alanine, L-glycine, L-norleucine, and L-serine were significantly lower in patients with the low Chelation Ratio condition than the levels in HCR patients. CONCLUSIONS: This therapeutic drug monitoring study revealed that a DEFR-iron complex formation ratio at C2h might be an applicable indicator of the efficacy of long-term DEFR iron chelation therapy. A better iron-control response to DEFR was observed in the patients with HCRs. The trends for the ratio might have value in dose-setting and need to be validated in a larger cohort.

Genome sequencing and annotation of a Campylobacter coli strain isolated from milk with multidrug resistance.

As the most prevalent bacterial cause of human gastroenteritis, food-borne Campylobacter infections pose a serious threat to public health. Whole Genome Sequencing (WGS) is a tool providing quick and inexpensive approaches for analysis of food-borne pathogen epidemics. Here we report the WGS and annotation of a Campylobacter coli strain, FNW20G12, which was isolated from milk in the United States in 1997 and carries multidrug resistance. The draft genome of FNW20G12 (DDBJ/ENA/GenBank accession number LWIH00000000) contains 1, 855,435 bp (GC content 31.4%) with 1902 annotated coding regions, 48 RNAs and resistance to aminoglycoside, beta-lactams, tetracycline, as well as fluoroquinolones. There are very few genome reports of C. coli from dairy products with multidrug resistance. Here the draft genome of FNW20G12, a C. coli strain isolated from raw milk, is presented to aid in the epidemiology study of C. coli antimicrobial resistance and role in foodborne outbreak.

Simultaneous Determination of Plasma Deferasirox and Deferasirox-Iron Complex Using an HPLC-UV System and Pharmacokinetics of Deferasirox in Patients With ß-Thalassemia Major: Once-daily Versus Twice-daily Administration.

PURPOSE: Deferasirox (DEFR), when administered BID, improves iron overload and decreases DEFR-related adverse effects in patients with ß-thalassemia major. However, the pharmacokinetic (PK) disposition of DEFR and the iron-DEFR complex (Fe-[DEFR]2) in this dosing strategy is unclear. METHODS: Chromatographic analysis was performed using a solvent delivery system coupled to an HPLC-UV detector to determine the steady-state concentrations of DEFR (CDEFR) and Fe-(DEFR)2 (CFe-[DEFR]2) in ß-thalassemia major patients (n = 8) following either once-daily or BID dosing, during which the PK parameters of the 2 dosing schedules were compared. FINDINGS: An HPLC-UV system for the analysis of blood samples following solid-phase extraction was validated. Patients who received 40 mg/kg of DEFR had higher mean CDEFR and CFe-[DEFR]2 values at all sampling times. However, concentrations of iron-DEFR complex were similar in patients who received 30 or 40 mg/kg of DEFR in the once-daily group at the 6- to 24-hour sampling times. There was no significant difference in any of the PK parameters; however, DEFR administration BID increased the mean trough levels of DEFR (183.8 [157.5] µmol/L) compared with once daily (87.7 [56.8] µmol/L), whereas all the patients had increased peak levels per individual DEFR dose when they were switched from once daily to BID (139.0 [59.8] µmol/L vs 289.2 [145.8] µmol/L, respectively). IMPLICATIONS: Splitting the dose increased the peak levels of DEFR per unit dose in all patients and tends to increase drug exposures, but there were no significant differences in DEFR PK parameter estimates. Switching from once daily to BID may be considered for patients with an inadequate response to chelation therapy to achieve optimal drug levels. Further research is needed with a larger sample size to determine the clinical importance of the significant results due to the interindividual variability of DEFR.

Role of pirenoxine in the effects of catalin on in vitro ultraviolet-induced lens protein turbidity and selenite-induced cataractogenesis in vivo.

PURPOSE: In this study, we investigated the biochemical pharmacology of pirenoxine (PRX) and catalin under in vitro selenite/calcium- and ultraviolet (UV)-induced lens protein turbidity challenges. The systemic effects of catalin were determined using a selenite-induced cataractogenesis rat model. METHODS: In vitro cataractogenesis assay systems (including UVB/C photo-oxidation of lens crystallins, calpain-induced proteolysis, and selenite/calcium-induced turbidity of lens crystallin solutions) were used to screen the activity of PRX and catalin eye drop solutions. Turbidity was identified as the optical density measured using spectroscopy at 405 nm. We also determined the in vivo effects of catalin on cataract severity in a selenite-induced cataract rat model. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied to analyze the integrity of crystallin samples. RESULTS: PRX at 1,000 µM significantly delayed UVC-induced turbidity formation compared to controls after 4 h of UVC exposure (p<0.05), but not in groups incubated with PRX concentrations of <1,000 µM. Results were further confirmed by SDS-PAGE. The absolute γ-crystallin turbidity induced by 4 h of UVC exposure was ameliorated in the presence of catalin equivalent to 1~100 µM PRX in a concentration-dependent manner. Samples with catalin-formulated vehicle only (CataV) and those containing PRX equivalent to 100 µM had a similar protective effect after 4 h of UVC exposure compared to the controls (p<0.05). PRX at 0.03, 0.1, and 0.3 µM significantly delayed 10 mM selenite- and calcium-induced turbidity formation compared to controls on days 0~4 (p<0.05). Catalin (equivalent to 32, 80, and 100 µM PRX) had an initial protective effect against selenite-induced lens protein turbidity on day 1 (p<0.05). Subcutaneous pretreatment with catalin (5 mg/kg) also statistically decreased the mean cataract scores in selenite-induced cataract rats on post-induction day 3 compared to the controls (1.3±0.2 versus 2.4±0.4; p<0.05). However, catalin (equivalent to up to 100 µM PRX) did not inhibit calpain-induced proteolysis activated by calcium, and neither did 100 µM PRX. CONCLUSIONS: PRX at micromolar levels ameliorated selenite- and calcium-induced lens protein turbidity but required millimolar levels to protect against UVC irradiation. The observed inhibition of UVC-induced turbidity of lens crystallins by catalin at micromolar concentrations may have been a result of the catalin-formulated vehicle. Transient protection by catalin against selenite-induced turbidity of crystallin solutions in vitro was supported by the ameliorated cataract scores in the early stage of cataractogenesis in vivo by subcutaneously administered catalin. PRX could not inhibit calpain-induced proteolysis activated by calcium or catalin itself, and may be detrimental to crystallins under UVB exposure. Further studies on formulation modifications of catalin and recommended doses of PRX to optimize clinical efficacy by cataract type are warranted.

Simultaneous quantification of eight biogenic amine compounds in tuna by matrix solid-phase dispersion followed by HPLC-orbitrap mass spectrometry.

A method for the extraction of agmatine, cadaverine, histamine, phenyethylamine, putrescine, tryptamine, tyramine, and urocanic acid from canned tuna and frozen tuna loin matrices by matrix solid-phase dispersion, followed by separation and quantification of these compounds by ultrahigh-performance hydrophilic interaction chromatography (UHPLC-HILIC) with orbitrap mass spectrometric detection, is described. Tuna samples are dispersed in a CN-silica sorbent and eluted with a mixture of aqueous ammonium formate buffer and acetonitrile. Separation and detection are carried out on an Agilent 1200 high-performance liquid chromatograph coupled to a Thermo Exactive orbitrap mass spectrometer, and metformin is used as the internal standard. Spike recoveries are determined across a range of 20-100 ppm for each compound, and the method is validated with respect to linearity, reproducibility, accuracy, and limits of quantitation and detection. The method is demonstrated to be suitable for use in quantifying these target compounds in the studied matrices.

Analysis of carbon monoxide in commercially treated tuna (Thunnus spp.) and mahi-mahi (Coryphaena hippurus) by gas chromatography/mass spectrometry.

A simple and confirmative method for quantitative determination of carbon monoxide in tuna and mahi-mahi tissues using GC/MS, following chemical liberation of CO into headspace, is described. Carbon monoxide in recent years has been employed by the fishery industry to preserve fresh appearance in selected species of finfish during frozen storage, particularly in vacuum-packaged products. Indigenous CO contents of fresh Ahi tuna and mahi-mahi were examined using the method described in this study and found to be close to or less than 150 and 100 ng/g, respectively. Commercially CO-treated, vacuum-packaged tuna from multiple sources consistently showed CO level near or greater than 1 mug/g, while CO level in the only CO-treated frozen mahi-mahi sample was in the 500 ng/g range. The difference between untreated and treated specimens was in the range of 1 order of magnitude and thus suggested an easy quantitative and confirmative method of CO using widely available instrumentation that may be potentially useful for regulatory purpose in determining whether a commercially available product has been exposed to CO even if not labeled as such.

Selectivity and specificity of a chromogenic medium for detecting Vibrio parahaemolyticust.

The thiosulfate-citrate-bile salts-sucrose agar (TCBS) used in the most-probable-number method for detecting Vibrio parahaemolyticus cannot differentiate growth of V. parahaemolyticus from Vibrio vulnificus or Vibrio mimicus. This study examined the selectivity and specificity of Bio-Chrome Vibrio medium (BCVM), a chromogenic medium that detects V. parahaemolyticus on the basis of the formation of distinct purple colonies on the medium. A panel consisting of 221 strains of bacteria, including 179 Vibrio spp. and 42 non-Vibrio spp., were examined for their ability to grow and produce colored colonies on BCVM. Growth of Salmonella, Shigella, Escherichia coli, Enterobacter cloacae, Yersinia enterocolitica, and Aeromonas was inhibited by both BCVM and TCBS. All 148 strains of V. parahaemolyticus grew on BCVM, and 145 of them produced purple colonies. The remaining 31 Vibrio spp., except one strain of Vibrio fluvialis, were either unable to grow or produced blue-green or white colonies on BCVM. Bio-Chrome Vibrio medium was capable of differentiating V. parahaemolyticus from other species, including V. vulnificus and V. mimicus. Further studies are needed to evaluate the sensitivity and specificity of BCVM for detecting V. parahaemolyticus in foods.

Complexities in tetracycline analysis-chemistry, matrix extraction, cleanup, and liquid chromatography.

The extraction and cleanup of commonly used tetracyclines (oxytetracycline, tetracycline, chlortetracycline, and doxycycline) from sample matrix, and their subsequent determination via liquid chromatography can be problematic. Many manuscripts report on various challenges encountered when developing a method for tetracycline antibiotics determination. These complexities often result in less than perfect recoveries or chromatograms and are based on the underlying chemistry associated with tetracyclines. This review compiles, compares, and discusses the results and observations found in published methods, while focusing on chemical principles in order to increase the practicing chemist's understanding of TCs to aid him/her in developing useful analyses.