Non-canonical transcriptional regulation of the poor prognostic factor UGT2B17 in chronic lymphocytic leukemic and normal B cells.

BACKGROUND: High expression of the glycosyltransferase UGT2B17 represents an independent adverse prognostic marker in chronic lymphocytic leukemia (CLL). It also constitutes a predictive marker for therapeutic response and a drug resistance mechanism. The key determinants driving expression of the UGT2B17 gene in normal and leukemic B-cells remain undefined. The UGT2B17 transcriptome is complex and is comprised of at least 10 alternative transcripts, identified by previous RNA-sequencing of liver and intestine. We hypothesized that the transcriptional program regulating UGT2B17 in B-lymphocytes is distinct from the canonical expression previously characterized in the liver. RESULTS: RNA-sequencing and genomics data revealed a specific genomic landscape at the UGT2B17 locus in normal and leukemic B-cells. RNA-sequencing and quantitative PCR data indicated that the UGT2B17 enzyme is solely encoded by alternative transcripts expressed in CLL patient cells and not by the canonical transcript widely expressed in the liver and intestine. Chromatin accessible regions (ATAC-Seq) in CLL cells mapped with alternative promoters and non-coding exons, which may be derived from endogenous retrotransposon elements. By luciferase reporter assays, we identified key cis-regulatory STAT3, RELA and interferon regulatory factor (IRF) binding sequences driving the expression of UGT2B17 in lymphoblastoid and leukemic B-cells. Electrophoretic mobility shift assays and pharmacological inhibition demonstrated key roles for the CLL prosurvival transcription factors STAT3 and NF-κB in the leukemic expression of UGT2B17. CONCLUSIONS: UGT2B17 expression in B-CLL is driven by key regulators of CLL progression. Our data suggest that a NF-κB/STAT3/IRF/UGT2B17 axis may represent a novel B-cell pathway promoting disease progression and drug resistance.

Supercritical Carbon Dioxide Decellularization of Porcine Nerve Matrix for Regenerative Medicine.

Tissue engineering scaffolds are often made from the decellularization of tissues. The decellularization of tissues caused by prolonged contact with aqueous detergents might harm the microstructure and leave cytotoxic residues. In this research, we developed a new technique to use supercritical carbon dioxide (Sc-CO2)-based decellularization for porcine nerve tissue. The effect of decellularization was analyzed by histological examination, including Hematoxylin and Eosin, Masson's Trichrome staining, and 4',6-diamidino-2-phenylindole staining. Moreover, biochemical analysis of the decellularized tissues was also performed by measuring DNA content, amount of collagen, and glycosaminoglycans (GAGs) after decellularization. The results showed that the tissue structure was preserved, cells were removed, and the essential components of extracellular matrix, such as collagen fibers, elastin fibers, and GAG fibers, remained after decellularization. In addition, the DNA content was decreased compared with native tissue, and the concentration of collagen and GAGs in the decellularized nerve tissue was the same as in native tissue. The in vivo experiment in the rat model showed that after 6 months of decellularized nerve implantation, the sciatic function index was confirmed to recover in decellularized nerve. Morphological analysis displayed a range of infiltrated cells in the decellularized nerve, similar to that in native tissue, and the number of Schwann cells that play essential for motor function and sensory in the decellularized nerve was confirmed. These findings indicate that tissue decellularization using Sc-CO2 has been successfully used in tissue engineering.

Untargeted metabolomics identifies metabolic dysregulation of sphingolipids associated with aggressive chronic lymphocytic leukaemia and poor survival.

BACKGROUND: Metabolic dependencies of chronic lymphocytic leukaemia (CLL) cells may represent new personalized treatment approaches in patients harbouring unfavourable features. METHODS: Here, we used untargeted metabolomics and lipidomics analyses to isolate metabolomic features associated with aggressive CLL and poor survival outcomes. We initially focused on profiles associated with overexpression of the adverse metabolic marker glycosyltransferase (UGT2B17) associated with poor survival and drug resistance. RESULTS: Leukaemic B-cell metabolomes indicated a significant perturbation in lipids, predominantly bio-active sphingolipids. Expression of numerous enzyme-encoding genes of sphingolipid biosynthesis pathways was significantly associated with shorter patient survival. Targeted metabolomics further exposed higher circulating levels of glucosylceramides (C16:0 GluCer) in CLL patients relative to healthy donors and an aggressive cancer biology. In multivariate analyses, C16:0 GluCer and sphinganine were independent prognostic markers and were inversely linked to treatment-free survival. These two sphingolipid species function as antagonistic mediators, with sphinganine being pro-apoptotic and GluCer being pro-proliferative, tested in leukemic B-CLL cell models. Blocking GluCer synthesis using ceramide glucosyltransferase inhibitors induced cell death and reduced the proliferative phenotype, which further sensitized a leukaemic B-cell model to the anti-leukaemics fludarabine and ibrutinib in vitro. CONCLUSIONS: Specific sphingolipids may serve as prognostic markers in CLL, and inhibiting enzymatic pathways involved in their biosynthesis has potential as a therapaeutic approach.

Self-Organized Phase-Composite Nanocrystal Solids with Superior Charge Transport.

Interparticle electronic coupling is essential for self-assembled colloidal nanocrystal (NC) solid semiconductors to fulfill their wide-tunable electrical and optoelectrical properties, but it has been limited by disorders. Here, a disorder-tolerant coupling approach is presented by synthesizing self-organized NC solids based on amorphous/nanocrystalline phase-composites. The ZnO amorphous matrix, which infills the space between the less regularly ordered ZnO NCs, enables robust electronic coupling between neighboring NCs via the resonant wave function overlap, leading to a disorder-tolerant resonant conducting state. Field-effect transistors based on phase-composite semiconductors show delocalized band-like transport with superior field-effect mobility values (∼75 cm2 V-1 s-1), compared to amorphous or polycrystalline ZnO semiconductors. Furthermore, the broad amorphous matrix can mitigate interfacial defects between crystalline regions through atomic relaxation, in contrast to narrow grain boundaries in polycrystalline films, resulting in a significantly low interface trap density for phase-composite NC solids. Density function theory calculations and quantum transport simulations using the nonequilibrium Green's function formalism elucidate the origins of superior and highly disorder-tolerant electron transport in phase-composite NC solids. Our report introduces a new class of NC solids complementary to the colloidal counterpart and will be applicable to CMOS-compatible emerging device technologies.

Impact of sodium chloride and carbon dioxide on conidial germination and radial growth of Penicillium camemberti.

Penicillium camemberti is a domesticated species adapted to the dairy environment, which is used as adjunct cultures to ripen soft cheeses. A recent population genomics analysis on P. camemberti revealed that P. camemberti is a clonal lineage with two varieties almost identical genetically but with contrasting phenotypes in terms of growth, color, mycotoxin production and inhibition of contaminants. P. camemberti variety camemberti is found on Camembert and Brie cheeses, and P. camemberti variety caseifulvum is mainly found on other cheeses like Saint-Marcellin and Rigotte de Condrieu. This study aimed to evaluate the impact of water activity (aw) reduced by sodium chloride (NaCl) and the increase of carbon dioxide (CO2) partial pressure, on conidial germination and growth of two varieties of P. camemberti: var. Camemberti and var. Caseifulvum. Mathematical models were used to describe the responses of P. camemberti strains to both abiotic factors. The results showed that these genetically distant strains had similar responses to increase in NaCl and CO2 partial pressure. The estimated cardinal values were very close between the strains although all estimated cardinal values were significantly different (Likelihood ratio tests, pvalue = 0.05%). These results suggest that intraspecific variability could be more exacerbated during fungal growth compared with conidial germination, especially in terms of macroscopic morphology. Indeed, var. Caseifulvum seemed to be more sensitive to an increase of CO2 partial pressure, as shown by the fungal morphology, with the occurrence of irregular outgrowths, while the morphology of var. Camemberti remains circular. These data could make it possible to improve the control of fungal development as a function of salt and carbon dioxide partial pressure. These abiotic factors could serve as technological barriers to prevent spoilage and increase the shelf life of cheeses. The present data will allow more precise predictions of fungal proliferation as a function of salt and carbon dioxide partial pressure, which are significant technological hurdles in cheese production.

Impact of carbon dioxide on the radial growth of fungi isolated from dairy environment.

In dairy industry, filamentous fungi are used as adjunct cultures in fermented products for their technological properties but they could also be responsible for food spoilage and mycotoxin production. The consumer demands about free-preservative products has increased in recent years and lead to develop alternative methods for food preservation. Modified Atmosphere Packaging (MAP) can inhibit fungal growth and therefore increase the food product shelf-life. This study aimed to evaluate radial growth as a function of CO2 and more particularly carbonic acid for fourteen adjuncts and/or fungal spoiler isolated from dairy products or dairy environment by using predictive mycology tools. The impact of the different chemical species linked to CO2 (notably carbonic acid) were study because it was reported previously that undissociated carbonic acid impacted bacterial growth and bicarbonates ions were involved in modifications of physiological process of fungal cells. A significant diversity in the responses of selected strains was observed. Mucor circinelloides had the fastest growth rates (µ > 11 mm. day-1) while Bisifusarium domesticum, Cladosporium herbarum and Penicillium bialowiezense had the slowest growth rates (µ < 1 mm. day-1). Independently of the medium pH, the majority of strains were sensitive to total carbonic acid. In this case, it was not possible to conclude if CO2 active form was gaseous or aqueous so modeling were performed as a function of CO2 percentage. Only Geotrichum candidum and M. circinelloides strains were sensitive to undissociated carbonic acid. Among the fourteen strains, P. bialowiezense was the less sensitive strain to CO2, no growth was observed at 50% of CO2 only for this strain. M. lanceolatus was the less sensitive strain to CO2, the CO250 which reduce the growth rates by 50% was estimated at 138% of CO2. Low CO2 percentage improved the growth of Penicillium expansum, Penicillium roqueforti and Paecilomyces niveus. Mathematical models (without and with optimum) were suggested to describe the impact of CO2 percentage or undissociated carbonic acid concentration on fungal growth rate.

A liquid chromatography tandem mass spectrometry method for plasma glucagon.

Glucagon is a peptide involved in controlling the body's blood glucose levels. The majority of analytical methods used for its quantitation are based on immunoassays that suffer from cross-reactivity with other peptides. For accurate routine analysis a liquid chromatography tandem mass spectrometry (LC-MSMS) was developed. Glucagon was extracted from plasma samples by a combination of protein precipitation using ethanol and mixed anion exchange solid phase extraction. Linearity for glucagon was above 0.99 (r2) up to a concentration range of 771 ng/L with a lower limit of quantification established at 19 ng/L. Precision of the method was below 9% (coefficient of variation). Recovery was 93%. Correlations with the existing immunoassay displayed a significant negative bias.

A Non-Canonical Role for the Glycosyltransferase Enzyme UGT2B17 as a Novel Constituent of the B Cell Receptor Signalosome.

In chronic lymphocytic leukemia (CLL), an elevated glycosyltransferase UGT2B17 expression (UGT2B17HI) identifies a subgroup of patients with shorter survival and poor drug response. We uncovered a mechanism, possibly independent of its enzymatic function, characterized by an enhanced expression and signaling of the proximal effectors of the pro-survival B cell receptor (BCR) pathway and elevated Bruton tyrosine kinase (BTK) phosphorylation in B-CLL cells from UGT2B17HI patients. A prominent feature of B-CLL cells is the strong correlation of UGT2B17 expression with the adverse marker ZAP70 encoding a tyrosine kinase that promotes B-CLL cell survival. Their combined high expression levels in the treatment of naïve patients further defined a prognostic group with the highest risk of poor survival. In leukemic cells, UGT2B17 knockout and repression of ZAP70 reduced proliferation, suggesting that the function of UGT2B17 might involve ZAP70. Mechanistically, UGT2B17 interacted with several kinases of the BCR pathway, including ZAP70, SYK, and BTK, revealing a potential therapeutic vulnerability. The dual SYK and JAK/STAT6 inhibitor cerdulatinib most effectively compromised the proliferative advantage conferred by UGT2B17 compared to the selective BTK inhibitor ibrutinib. Findings point to an oncogenic role for UGT2B17 as a novel constituent of BCR signalosome also connected with microenvironmental signaling.

Informing One Health Anthrax Surveillance and Vaccination Strategy from Spatial Analysis of Anthrax in Humans and Livestock in Ha Giang Province, Vietnam (1999-2020).

Anthrax, caused by Bacillus anthracis, has a nearly global distribution but is understudied in Southeast Asia, including Vietnam. Here, we used historical data from 1999 to 2020 in Ha Giang, a province in northern Vietnam. The objectives were to describe the spatiotemporal patterns and epidemiology of human and livestock anthrax in the province and compare livestock vaccine coverage with human and livestock anthrax incidence. Annual incidence rates (per 10,000) for humans, buffalo/cattle, and goats were used to explore anthrax patterns and for comparison with livestock annual vaccine variations. A data subset describes anthrax epidemiology in humans by gender, age, source of infection, type of anthrax, admission site, and season. Zonal statistics and SaTScan were used to identify spatial and space-time clusters of human anthrax. SaTScan revealed space-time clusters in 1999, 2004, and 2007-2008 in the province, including in the northeastern, eastern, and western areas. Most human anthrax was reported between July and October. Most patients were male, aged 15-59 years, who had handled sick animals and/or consumed contaminated meat. High case-fatality rates were reported with gastrointestinal or respiratory cases. Our data suggest that vaccination in buffalo and cattle reduces the disease burden in humans and vaccinated animals but does not reduce the incidence in unvaccinated animals (goats). This study identified spatial areas of high risk for anthrax and can inform One Health surveillance and livestock vaccination planning in contextual settings similar to Ha Giang province.

Extensive metabolic consequences of human glycosyltransferase gene knockouts in prostate cancer.

BACKGROUND: Naturally occurring germline gene deletions (KO) represent a unique setting to interrogate gene functions. Complete deletions and differential expression of the human glycosyltransferase UGT2B17 and UGT2B28 genes are linked to prostate cancer (PCa) risk and progression, leukaemia, autoimmune and other diseases. METHODS: The systemic metabolic consequences of UGT deficiencies were examined using untargeted and targeted mass spectrometry-based metabolomics profiling of carefully matched, treatment-naive PCa cases. RESULTS: Each UGT KO differentially affected over 5% of the 1545 measured metabolites, with divergent metabolic perturbations influencing the same pathways. Several of the perturbed metabolites are known to promote PCa growth, invasion and metastasis, including steroids, ceramides and kynurenine. In UGT2B17 KO, reduced levels of inactive steroid-glucuronides were compensated by sulfated derivatives that constitute circulating steroid reservoirs. UGT2B28 KO presented remarkably lower levels of oxylipins paralleled by reduced inflammatory mediators, but higher ceramides unveiled as substrates of the enzyme in PCa cells. CONCLUSION: The distinctive and broad metabolic rewiring caused by UGT KO reinforces the need to examine their unique and divergent functions in PCa biology.

A liquid chromatography-mass spectrometry assay for the quantification of nucleotide sugars in human plasma and urine specimens and its clinical application.

Nucleotide sugars and more specifically UDP-sugars, represent a major source of energy, key components of extracellular matrix, glycosylation and glucuronidation reactions, and emerge as important signaling molecules through P2Y14 purinergic receptor. Despite their pivotal role in a variety of physiological and pathological processes and their potential as biomarkers, UDP-sugar composition of biological fluids remains poorly studied. We developed a liquid chromatography electrospray ionization tandem mass spectrometry in multiple reaction monitoring mode for the simultaneous quantification of UDP-glucose, UDP-galactose, UDP-glucuronic acid, UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine in human blood and urine. Relative to existing methods, UDP-sugar recovery was enhanced with perchloric acid and ammonium formate during sample preparation that also significantly improved chromatographic stability. Performance of the assay was validated and allowed the absolute quantification of UDP-sugars with a wide dynamic range (0.1 to 200 ng/mL) using stable deuterated isotopes as internal standards. We report a fast (13 min run) and sensitive method (limit of detection: 10-30 pg/mL; lower limit of quantification ≤ 0.2 ng/ml) to simultaneously quantify five UDP-sugars in a low volume (100 µL) of plasma and urine. Findings identified sex-specific profiles in both plasma and urine of healthy subjects. Applicability was also successfully demonstrated in specimens collected from individuals displaying a variety of medical conditions. This validated method was optimized for a high-throughput assessment of UDP-sugars in specimens of clinical importance and enabled an accurate and reliable absolute quantification of important UDP-sugars in diverse clinical contexts.

Impact of water activity on the radial growth of fungi in a dairy environment.

Filamentous fungi are used in the dairy industry as adjunct cultures in fermented products, but can also lead to food spoilage, waste and economic losses. The control of filamentous fungi with abiotic factors contributes to longer food shelf life and prevention of fungal spoilage. One of the main abiotic factors for controlling fungal growth in foods is water activity (aw). This study aimed to evaluate radial growth as a function of aw for sixteen fungal adjuncts and/or spoilers isolated from dairy products or a dairy environment. Glycerol (a non-ionic compound) and sodium chloride (NaCl, an ionic compound) were used to adjust the aw of culture media. This study showed significant diversity in the responses of the tested fungal strains as a function of medium aw. The growth response of Penicillium bialowiezense and Sporendonema casei was binary, with no clear decrease of growth rate until the growth limit, when the aw was reduced. For the strains of Bisifusarium domesticum, Mucor circinelloides and Penicillium camemberti, a decrease of aw had the same impact on radial growth rate regardless of the aw belonging to their growth range. For the strains of Aspergillus flavus, Cladosporium herbarum, Geotrichum candidum, Mucor lanceolatus, Penicillium expansum, Penicillium fuscoglaucum, Penicillium nalgiovense, Paecilomyces niveus, Penicillium roqueforti, Penicillium solitum and Scopulariopsis asperula, the impact of a decrease in aw was more pronounced at high aw than at low aw. A mathematical model was suggested to describe this impact on the radial growth rate. For all tested species, radial growth was more sensitive to NaCl than glycerol. The ionic strength of NaCl mainly explains the difference in the effects of the two solutes.

Low level of Fibrillarin, a ribosome biogenesis factor, is a new independent marker of poor outcome in breast cancer.

BACKGROUND: A current critical need remains in the identification of prognostic and predictive markers in early breast cancer. It appears that a distinctive trait of cancer cells is their addiction to hyperactivation of ribosome biogenesis. Thus, ribosome biogenesis might be an innovative source of biomarkers that remains to be evaluated. METHODS: Here, fibrillarin (FBL) was used as a surrogate marker of ribosome biogenesis due to its essential role in the early steps of ribosome biogenesis and its association with poor prognosis in breast cancer when overexpressed. Using 3,275 non-metastatic primary breast tumors, we analysed FBL mRNA expression levels and protein nucleolar organisation. Usage of TCGA dataset allowed transcriptomic comparison between the different FBL expression levels-related breast tumours. RESULTS: We unexpectedly discovered that in addition to breast tumours expressing high level of FBL, about 10% of the breast tumors express low level of FBL. A correlation between low FBL mRNA level and lack of FBL detection at protein level using immunohistochemistry was observed. Interestingly, multivariate analyses revealed that these low FBL tumors displayed poor outcome compared to current clinical gold standards. Transcriptomic data revealed that FBL expression is proportionally associated with distinct amount of ribosomes, low FBL level being associated with low amount of ribosomes. Moreover, the molecular programs supported by low and high FBL expressing tumors were distinct. CONCLUSION: Altogether, we identified FBL as a powerful ribosome biogenesis-related independent marker of breast cancer outcome. Surprisingly we unveil a dual association of the ribosome biogenesis FBL factor with prognosis. These data suggest that hyper- but also hypo-activation of ribosome biogenesis are molecular traits of distinct tumors.

Design and validation of an LC-MS/MS method for simultaneous quantification of asymmetric dimethylguanidino valeric acid, asymmetric dimethylarginine and symmetric dimethylarginine in human plasma.

Asymmetric dimethylguanidino valeric acid (ADGV), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are three arginine metabolites which have utility in the assessment of cardiovascular disease, renal disease and non-alcoholic fatty liver disease (NAFLD). Translation of these research metabolomic markers into routine clinical use requires the development of robust assays with appropriately assessed preanalytical variables and traceable clinical reference intervals. A hydrophilic interaction liquid chromatography (HILIC) tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of ADGV, ADMA and SDMA was developed. Sample stability and collection conditions were scrutinised to determine any preanalytical factors that could affect quantification under routine laboratory conditions. Patient samples from 120 males and 120 females were used to derive preliminary reference intervals. All three analytes were quantifiable in human plasma using unique MS/MS transitions. The analytes were stable for up to a week once separated from red cells, though reduced stability was observed upon extraction of the analytes from plasma. The assay was linear for concentration of ADGV between 1.6 nmol/L and 200 nmol/L and for ADMA and SDMA between 0.1 µmol/L and 4.0 µmol/L. The accuracy for all analytes was 97-103% and interday and intraday imprecisions (coefficients of variation) were less than 10%. ADGV concentrations were noted to be lower in the female reference population when compared to males. The analytical method shows excellent performance and is sufficiently robust to be used in the clinical investigation of cardiovascular disease and NAFLD.

Streamlined three step total vitamin C analysis by HILIC-UV for laboratory testing.

OBJECTIVES: In the clinical setting, the analysis and quantification of vitamin C (ascorbic acid) poses several challenges including analyte instability and poor retention by reverse phase HPLC systems. In this article we describe a rapid hydrophilic interaction chromatography ultraviolet method for the measurement of total vitamin C in plasma which overcomes these issues. METHODS: Ascorbic acid and the internal standard were separated under isocratic conditions using a Waters BEH-Amide column and a mobile phase containing 0.005 M potassium phosphate in 80% acetonitrile. RESULTS: The proposed method was validated and showed good precision (coefficient of variation <5%), accuracy (>99%), and analyte stability after extraction (>24 h). CONCLUSIONS: The simple sample preparation allows full automation and rapid analytical run times of the assay and is therefore suitable for a high-throughput clinical chromatography laboratory.

Intraspecific variability in cardinal growth temperatures and water activities within a large diversity of Penicillium roqueforti strains.

Different strains of a given fungal species may display heterogeneous growth behavior in response to environmental factors. In predictive mycology, the consideration of such variability during data collection could improve the robustness of predictive models. Among food-borne fungi, Penicillium roqueforti is a major food spoiler species which is also used as a ripening culture for blue cheese manufacturing. In the present study, we investigated the intraspecific variability of cardinal temperatures and water activities (aw), namely, minimal (Tmin and awmin), optimal (Topt and awopt) and maximal (Tmax) temperatures and/or aw estimated with the cardinal model for radial growth, of 29 Penicillium roqueforti strains belonging to 3 genetically distinct populations. The mean values of cardinal temperatures and aw for radial growth varied significantly across the tested strains, except for Tmax which was constant. In addition, the relationship between the intraspecific variability of the biological response to temperature and aw and putative genetic populations (based on microsatellite markers) within the selected P. roqueforti strains was investigated. Even though no clear relationship was identified between growth parameters and ecological characteristics, PCA confirmed that certain strains had marginal growth response to temperature or aw. Overall, the present data support the idea that a better knowledge of the response to abiotic factors such as temperature and aw at an intraspecific level would be useful to model fungal growth in predictive mycology approaches.

Dual Analysis of N-methyl-2-pyridone-5-carboxamide and N-1-methylnicotinamide in Urine by HPLC with UV Detection.

N-methyl-2-pyridone-5-carboxamide (2PYr) and N-1-methylnicotinamide (NMN) are metabolites of the water soluble Vitamin B3 (Nicotinamide). Limited methodologies exist for their dual chromatographic analysis in urine samples. In this study, we developed a method for analysis of both 2PYr and NMN by ultraviolet detection. Urine samples were treated to a salting-out assisted liquid/liquid extraction for the extraction of 2PYr and cation exchange for NMN. Both analytes were separated on a Biphenyl 100 × 2.1 mm, 2.6-µm column. The new assay's performance (specifically 2PYr) was compared against the existing testing protocol (based on a previously published method). Linearity for both analytes was above 0.99 (r2) up to a concentration range of: 1500 µmol/L (2PYr) and 150 µmol/L (NMN). Intra-assay and inter-assay precision of the method was below 8% (coefficient of variation) except at the lower limit of quantification where it was below 20%. Recovery of 2PYr was above 80% and NMN above 90%. A significant positive bias was observed with 2PYr against the existing method. This new method allows for both 2PYr and NMN to be chromatographed and overcomes sample preparation issues in urine 2PYr analysis.

Coronavirus testing indicates transmission risk increases along wildlife supply chains for human consumption in Viet Nam, 2013-2014

Outbreaks of emerging coronaviruses in the past two decades and the current pandemic of a novel coronavirus (SARS-CoV-2) that emerged in China highlight the importance of this viral family as a zoonotic public health threat. To gain a better understanding of coronavirus presence and diversity in wildlife at wildlife-human interfaces in three southern provinces in Viet Nam 2013-2014, we used consensus Polymerase Chain Reactions to detect coronavirus sequences. In comparison to previous studies, we observed high proportions of positive samples among field rats (34.0%, 239/702) destined for human consumption and insectivorous bats in guano farms (74.8%, 234/313) adjacent to human dwellings. Most notably among field rats, the odds of coronavirus RNA detection significantly increased along the supply chain from field rats sold by traders (reference group; 20.7% positivity, 39/188) by a factor of 2.2 for field rats sold in large markets (32.0%, 116/363) and 10.0 for field rats sold and served in restaurants (55.6%, 84/151). Coronaviruses were detected in the majority of wildlife farms (60.7%, 17/28) and in the Malayan porcupines (6.0%, 20/331) and bamboo rats (6.3%, 6/96) that are farmed. We identified six known coronaviruses in bats and rodents, clustered in three Coronaviridae genera, including the Alpha-, Beta-, and Gammacoronaviruses. Our analysis also suggested either mixing of animal excreta in the environment or interspecies transmission of coronaviruses, as both bat and avian coronaviruses were detected in rodent feces in the trade. The mixing of multiple coronaviruses, and their apparent amplification along the wildlife supply chain into restaurants, suggests maximal risk for end consumers and likely underpins the mechanisms of zoonotic spillover to people.

A high performance liquid chromatography fluorescence method for the analysis of both pyridoxal-5-phosphate and thiamine pyrophosphate in whole blood.

BACKGROUND AND AIMS: The active coenzymes of the water soluble vitamins B1 and B6 (thiamine pyrophosphate (TPP) and pyridoxal-5-phosphate (P5P) respectively) play an important role in numerous bodily functions. The simultaneous analysis of both these analytes is limited to either mass spectrometry based methods or commercial kit suppliers. In this study we developed a novel method for analysis of both TPP and P5P by fluorescence detection. METHODS: Briefly, whole blood samples are precipitated by trichloroacetic acid, and P5P and TPP are both derivatised before separation on a C18-PFP column. The new assay's performance was compared against a recent cycle from an external quality assurance program (RCPAQAP) and the current only existing commercial kit (n = 76). RESULTS: Linearity for both analytes was above 0.99 (r2) up to a concentration range of: 4000 nmol/L (P5P) and 2000 nmol/L (TPP). Precision of the method (intra-day and inter-day) compared against commercial quality control material was below 6% (coefficient of variation). Recovery of both compounds exceeded 90%. Accuracy of the protocol displayed satisfactory results in proficiency testing and had an acceptable level of agreement with the existing current kit method. CONCLUSIONS: Overall, this method provides an economical alternative in routine clinical diagnostic laboratories wishing to perform P5P and TPP analysis.