At low levels, inorganic mercury interference with antigen signaling is associated with modifications to a panel of novel phosphoserine sites in B cell receptor pathway proteins.

Epidemiological studies indicate that human and animal exposure to environmental mercury (Hg) disrupts normal immune system function, but the molecular mechanism responsible for this is still unresolved. We have previously utilized phospho-proteomic mass spectrometry to demonstrate that in the absence of B Cell Receptor (BCR) stimulation, exposure of B cells to Hg induces significant changes to a great many elements of the BCR signaling pathway in a concentration dependent manner. In this report, we have extended those initial findings by utilizing mass spectrometry to evaluate in detail the effect of low-level Hg exposure on BCR induced phospho-proteomic changes. Specifically, murine WEHI-231 B lymphoma cells were exposed to environmentally relevant levels of Hg with or without concomitant BCR stimulation. The cellular phospho-proteomes were then profiled by LC-MS/MS. We found that for low-level exposures, Hg interference with signal transduction across the BCR pathway was predominantly associated with modification of phosphorylation of 12 phosphosites located on seven different proteins. Nine sites were serine, two sites tyrosine and one site threonine. Most of these sites are novel, in the sense that only the two tyrosine and one of the serine sites have previously been reported to be associated with BCR signaling.

Depletion of Mdig Changes Proteomic Profiling in Triple Negative Breast Cancer Cells.

Triple-negative breast cancers are highly aggressive with an overall poor prognosis and limited therapeutic options. We had previously investigated the role of mdig, an oncogenic gene induced by some environmental risk factors, on the pathogenesis of breast cancer. However, a comprehensive analysis of the proteomic profile affected by mdig in triple-negative breast cancer has not been determined yet. Using label-free bottom-up quantitative proteomics, we compared wildtype control and mdig knockout MDA-MB-231 cells and identified the proteins and pathways that are significantly altered with mdig deletion. A total of 904 differentially expressed (p < 0.005) proteins were identified in the KO cells. Approximately 30 pathways and networks linked to the pathogenicity of breast cancer were either up- or downregulated, such as EIF2 signaling, the unfolded protein response, and isoleucine degradation I. Ingenuity Pathway Analysis established that the differentially expressed proteins have relevant biological actions in cell growth, motility, and malignancy. These data provide the first insight into protein expression patterns in breast cancer associated with a complete disruption of the mdig gene and yielded substantial information on the key proteins, biological processes, and pathways modulated by mdig that contribute to breast cancer tumorigenicity and invasiveness.

Revisiting the trapping of noble gases (He-Kr) by the triatomic H3+ and Li3+ species: a density functional reactivity theory study.

Small atomic clusters with exotic stability, bonding, aromaticity, and reactivity properties can be made use of for various purposes. In this work, we revisit the trapping of noble gas atoms (He-Kr) by the triatomic H3+ and Li3+ species by using some analytical tools from density functional theory, conceptual density functional theory, and the information-theoretic approach. Our results showcase that though similar in geometry, H3+ and Li3+ exhibit markedly different behavior in bonding, aromaticity, and reactivity properties after the addition of noble gas atoms. Moreover, the exchange-correlation interaction and steric effect are key energy components in stabilizing the clusters. This study also finds that the origin of the molecular stability of these species is due to the spatial delocalization of the electron density distribution. Our work provides an additional arsenal towards a better understanding of small atomic clusters capturing noble gases.

Density functional theory studies of boron clusters with exotic properties in bonding, aromaticity and reactivity.

Atomic clusters are unique in many perspectives because of their size and structure features and are continuously being applied for different purposes. To unveil their unconventional properties, in this work, using neutral tetraboron clusters as illustrative examples, we study their exotic behaviors in bonding, aromaticity, and reactivity. We show that both double and triple bonds can be formed, ring current patterns can be totally different, and both electrophilic and nucleophilic reactivities can coexist simultaneously. These features are often in contrast with our conventional chemical wisdom and could enrich the possibility for their potential applications. The methodologies employed in this work can be readily applied to other systems. Our studies should help us better appreciate atomic clusters with many atypical properties and henceforth yield novel applications.

Emergence of Klebsiella pneumoniae ST307 Co-Producing CTX-M with SHV and KPC from Paediatric Patients at Shenzhen Children's Hospital, China.

AIM: We investigated the clonal diversity of carbapenemase-producing Klebsiella pneumoniae isolates from the Shenzhen Children's Hospital, China, and drew conclusions on the clinical and public health impact of these isolates as multidrug-resistant. METHODS: From January 2014 to December 2018, a total number of 36 unique carbapenemase-producing clinical isolates of Klebsiella pneumoniae were collected out of 900 clinical isolates in paediatric patients from the Shenzhen Children's Hospital, China. After carbapenemase production confirmation, antimicrobial susceptibility, resistance determinants and phylogenetic relationship were determined. RESULTS: The isolates showed resistance to ceftazidime, ertapenem, ampicillin, cefazolin, ceftriaxone, cefotetan, ticarcillin, cefaclor, cefpodoxime, azlocillin, cefcapene, mezlocillin and ampicillin-sulbactam. Of the 36 Klebsiella pneumoniae carbapenemase genes coding isolates, bla NDM was the mostly detected 50% (n=18) followed by bla KPC and bla IMP 19% (n=7), bla VIM 17% (n=6), bla OXA-48-like 8% (n=3) and bla SME 5% (n=2), whereas extended-spectrum ß-lactamase (bla SHV) was predominantly detected 92% (n=33) followed by bla CTX-M 53% (n=19) and bla CMY 28% (n=10). Pulsed-field gel electrophoresis typing showed eight different patterns, and twenty-five distinct sequences types were observed with ST307 being predominantly identified 11% (n=4), followed by ST2407 8% (n=3). Plasmid replicon typing results indicated that IncFIS, IncHI2, IncFIC and IncFIA plasmids carry bla CTX-M, bla SHV and bla NDM genes. CONCLUSION: This study reports on the occurrence and spread of carbapenemase and extended-spectrum ß-lactamase encoding genes co-existence in sporadic Klebsiella pneumoniae ST307 in paediatric patients from the Shenzhen Children's Hospital, China.

Local Temperature as a Chemical Reactivity Descriptor.

Using the electron density and its associated quantities in a molecular system to quantify chemical reactivity in density functional theory is of considerable recent interest. Local temperature based on the kinetic energy density is an intrinsic property of a molecular system, which can be employed for this purpose. In this work, we explore such a possibility. To this end, we examine the local behavior of local temperature with a few choices of the kinetic energy density, apply it to determine regioselectivity of nucleophilic and electrophilic compounds, and then investigate its performance in appreciating reactions along the intrinsic reaction pathway for exothermic, endothermic, and thermoneutral transformations. Our results confirm that local temperature can be used as an effective descriptor of molecular reactivity.

Pharmacokinetics of a novel triazine ethanamizuril in rats and broiler chickens.

Ethanamizuril is a new triazine compound that displays potent anticoccidial activity against chicken coccidiosis caused by Eimeria tenella. We studied the pharmacokinetics of ethanamizuril in rats and chickens after single oral doses of one, two and three times the minimum effective dose Ethanamizuril was readily absorbed at all three doses and the plasma concentration reached was maximal within 5h and progressively declined over time. The mean peak plasma concentrations (Cmax) and the area under the concentration-time curve (AUC) were both dose-dependent. These results provide pharmacokinetic profiles of ethanamizuril for future preclinical studies and clinical use.

Development and validation of an UPLC-UV method for determination of a novel triazine coccidiostat ethanamizuril and its metabolite M3 in chicken tissues.

Ethanamizuril (EZL) is a novel anti-coccidial triazine synthesized by the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. As a novel coccidiostat, it is essential to study its residue in food animal tissues. In this study, a reliable UPLC method for the determination of ethanamizuril (EZL) and one of its metabolites (M3) in chicken muscle, skin and fat, liver, and kidney samples was developed. Analytes were extracted using acetonitrile and 10% sodium carbonate solution, defatted by n-hexane, and further purified by using an Oasis MCX cartridge. The calibration curves for both test compounds were linear in all tissue matrices we tested: muscle, skin and fat, liver and kidney. The mean relative recoveries of EZL and M3 were from 87.3% to 103.2% between all tissue samples. The inter-day and intra-day relative standard deviations were within 15% and 20%, respectively. This method is accurate, reproducible and is ready to be field-tested.

Development of a modified QuEChERS method for the determination of veterinary antibiotics in swine manure by liquid chromatography tandem mass spectrometry.

A QuEChERS (quick, easy, cheap, effective, rugged and safe) based methodology was developed for the rapid, simultaneous quantification and identification of 26 veterinary drugs in swine manure by liquid chromatography tandem mass spectrometry. The selected antibiotics included tetracyclines, sulfonamides, macrolides, fluoroquinolones, lincosamides and pleuromutilins. This is the first study to determine pleuromutilin levels in manure. The QuEChERS process involved two simple steps. First, sample extraction with methanol: acetonitrile: 0.1M EDTA-McIlvaine buffer followed by phase separation with MgSO4: NaCl addition. The supernatant was then extracted and cleaned by dispersive solid-phase extraction using a primary-secondary amine (PSA) and octadecylsilane (C18) support. The proposed method provides a linearity in the range of 1-500ngmL-1 and linear regression coefficients (r) were greater than 0.996. MDL and MQL ranged between 0.01-1.86µgkg(-1) and 0.05-5.91µgkg(-1), respectively. Recoveries ranged from 61.39 to 105.65% with the exception of sulfaquinoxaline (55.7-56.8%) and valnemulin (33.7-37.7%). This method resulted in good precision (repeatability and reproducibility) and relative standard deviations less than 17% within the same day, and lower than 20% between days. The method was then applied to study the swine manure samples collected from Guangdong, China. Chlortetracycline, tetracycline, doxycycline, sulfadimidine and tilmicosin were detected in all samples indicating high residuals in manure. In fact tilmicosin was detected at 14400µgkg(-1) suggesting that prudent treatment of manure should be conducted to prevent environmental contamination. In conclusion, this workflow can provide a simpler and more cost-effective alternative to conventional methods and is compatible with processing large sample numbers over a short time period.

In Vivo Pharmacodynamics of Cefquinome in a Neutropenic Mouse Thigh Model of Streptococcus suis Serotype 2 at Varied Initial Inoculum Sizes.

Streptococcus suis serotype 2 is an emerging zoonotic pathogen and causes severe disease in both pigs and human beings. Cefquinome (CEQ), a fourth-generation cephalosporin, exhibits broad-spectrum activity against Gram-positive bacteria such as S. suis. This study evaluated the in vitro and in vivo antimicrobial activities of CEQ against four strains of S. suis serotype 2 in a murine neutropenic thigh infection model. We investigated the effect of varied inoculum sizes (10(6) to 10(8) CFU/thigh) on the pharmacokinetic (PK)/pharmacodynamic (PD) indices and magnitudes of a particular PK/PD index or dose required for efficacy. Dose fractionation studies included total CEQ doses ranging from 0.625 to 640 mg/kg/24 h. Data were analyzed via a maximum effect (Emax) model using nonlinear regression. The PK/PD studies demonstrated that the percentage of time that serum drug levels were above the MIC of free drug (%ƒT>MIC) in a 24-h dosing interval was the primary index driving the efficacy of both inoculum sizes (R(2) = 91% and R(2) = 63%). CEQ doses of 2.5 and 40 mg/kg body weight produced prolonged postantibiotic effects (PAEs) of 2.45 to 8.55 h. Inoculum sizes had a significant influence on CEQ efficacy. Compared to the CEQ exposure and dosages in tests using standard inocula, a 4-fold dose (P = 0.006) and a 2-fold exposure time (P = 0.01) were required for a 1-log kill using large inocula of 10(8) CFU/thigh.

Metabolism of nitazoxanide in rats, pigs, and chickens: Application of liquid chromatography coupled to hybrid linear ion trap/Orbitrap mass spectrometer.

Nitazoxanide (NTZ) is a nitrothiazole benzamide compound with a broad activity spectrum against parasites, Gram-positive and Gram-negative anaerobic bacteria, and viruses. In this study, hybrid linear ion trap/Orbitrap mass spectrometer providing a high mass resolution and accuracy was used to investigate the metabolism of NTZ in rats, pigs, and chickens. The results revealed that acetylation and glucuronidation were the main metabolic pathways in rats and pigs, whereas acetylation and sulfation were the major metabolic pathways in chickens, which indicated interspecies variations in drug metabolism and elimination. With the accurate mass data and the characteristic MS(n) product ions, we identified six metabolites in which tizoxanide and hydroxylated tizoxanide were phase I metabolites and tizoxanide glucuronide, tizoxanide glucose, tizoxanide sulfate and hydroxyl tizoxanide sulfate were phase II metabolites. Hydroxylated tizoxanide and tizoxanide glucose were identified for the first time. All the comprehensive data were provided to make out the metabolism of NTZ in rats, pigs and chickens more clearly.

[Determination of cefalonium residue in milk by high performance liquid chromatography-tandem mass spectrometry].

An analytical method was developed for the determination of cefalonium in milk by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). A total of 1 g milk was deproteinized by acetonitrile. The supernatant was transferred into a test tube to be blown dry with N2 at 37 degrees C. Then the residue was dissolved with methanol-0.1% formic acid in water (3:7, v/v). The sample was determined by HPLC-MS/MS after the purification. The chromatographic separation was achieved on a C18 column using 0.1% formic acid in water and acetonitrile as mobile phases with gradient elution. Qualitative and quantitative analyses were achieved by HPLC-MS/MS under positive ionization and multiple reaction monitoring (MRM) mode. Matrix-matched calibration curve was used for the quantification. Good correlation coefficients were obtained (r > 0. 999) in the mass concentration range of 2-200 microg/L. The limit of detection (LOD, S/N > or = 3) was 0.5 microg/kg in milk, and the limit of quantification (LOQ, S/N 10) was 2 microg/kg. The mean recoveries at the four levels of LOQ, 1/2MRL (maximum residue level), MRL, 2MRL were between 78.5% and 86.2%, with the intra-day relative standard deviations (RSDs) of 1.5% to 6.2% and inter-day RSDs of 2.9% to 5.6%. In conclusion, the established method can be applied for the determination of cefalonium residue in milk.

Identification and elucidation of the structure of in vivo metabolites of diaveridine in chicken.

Diaveridine (DVD) is a popular antibacterial synergist that is widely used in combination with sulfonamide. It has been reported to be genotoxic to mammalian cells, but more studies are required to clarify this. Moreover, there is very little information on its pharmacokinetics, metabolic elimination and mechanism of toxicity. Therefore, in order to gain a better understanding of the metabolism of DVD, we performed high-performance liquid chromatography linear ion trapped orbitrap mass spectrometer (LC-LTQ-Orbitrap). With this approach, we identified 15 metabolites of DVD in chicken after a single oral administration of DVD; 10 of these metabolites have been identified in vivo for the first time. Nine phase I and five phase II metabolites were detected in the plasma, and eight phase I and six phase II metabolites were found in feces. The major phase I metabolites were formed via the O-demethylation and N-oxidation pathways, and the major phase II metabolites were glucuronide conjugates. These results are essential for understanding this compound more clearly and lay the basis for further studies about the metabolism of DVD. Therefore, using this approach, we were able to identify and characterize metabolites of DVD with high sensitivity and resolution. We were able to detect a broad range of metabolites, even some trace ones and some so far unknown metabolites.

Simultaneous determination of major type-B trichothecenes and the de-epoxy metabolite of deoxynivalenol in chicken tissues by HPLC-MS/MS.

In this study, a specific and sensitive LC-MS/MS method for the simultaneous analysis of type-B trichothecenes (deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol) and the de-epoxy metabolite of deoxynivalenol (de-epoxy-deoxynivalenol) in chicken muscle, liver, kidney, and fat tissues was developed and validated. The method involved an extraction step using ethyl acetate, followed by the evaporation of the supernatant, which was further purified by an Oasis HLB SPE cartridge (Waters, Milford, MA, USA). Chromatographic separation was performed on a C18 column by detection with MS in multiple-reaction monitoring mode and using a gradient elution program with 0.1% formic acid in water and methanol. The correlation coefficients (r) for each calibration curve were >0.99 within the experimental concentration range. The extraction recoveries ranged from 73.7 to 106.4%, with intraday and interday RSD < 11.6% at three levels of concentrations of 2, 10, and 100 µg/kg. The decision limits and the detection capabilities of the analytes in the chicken tissues ranged from 0.16 to 0.92 and 0.68 to 2.07 µg/kg, respectively. The results demonstrated the applicability of this sensitive procedure to the determination of trichothecenes in chicken tissue samples.

[Determination of clavulanic acid residue in milk by high performance liquid chromatography-tandem mass spectrometry].

An analytical method was developed for the determination of clavulanic acid (CLAV) in milk by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). A 2 g milk sample was deproteinized by ethanol. The supernatant was transferred into a pear-shaped bottle to be evaporated to about 0.5 mL, and the residue was dissolved with ammonium acetate solution. The sample was determined by HPLC-MS/MS after the purification. The chromatographic separation was achieved on a Luna 5u C8 column using 0.1% formic acid in water and acetonitrile as mobile phases with gradient elution. The identification of CLAV was carried out by MS/MS equipped with electrospray ionization in negative scanning and multiple reaction monitoring (MRM) modes. Matrix-matched calibration standard was used for the quantification. The calibration curve showed perfect linear in the range of 10 - 400 microg/kg with the correlation coefficient of 0.999. The limit of detection (LOD, S/N > or = 3) was 10 microg/kg in milk, and the limit of quantification (LOQ, S/N > or = 10) was 20 microg/kg. The mean recoveries varied from 80.00% to 91.25% at the four spiked levels of LOQ, 1/2MRL (the maximum residue limit), MRL, and 2MRL with the relative standard deviations of 5.60% -8.77%. In conclusion, the established method can be applied for the determination of CLAV residues in milk.