Development and Validation of a UPLC-MS/MS Method to Monitor Cephapirin Excretion in Dairy Cows following Intramammary Infusion

Cephapirin, a cephalosporin antibiotic, is used by the majority of dairy farms in the US. Fecal and urinary excretion of cephapirin could introduce this compound into the environment when manure is land applied as fertilizer, and may cause development of bacterial resistance to antibiotics critical for human health. The environmental loading of cephapirin by the livestock industry remains un-assessed, largely due to a lack of appropriate analytical methods. Therefore, this study aimed to develop and validate a cephapirin quantification method to capture the temporal pattern of cephapirin excretion in dairy cows following intramammary infusion. The method includes an extraction with phosphate buffer and methanol, solid-phase extraction (SPE) clean-up, and quantification using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The LOQ values of the developed method were 4.02 µg kg⁻¹ and 0.96 µg L⁻¹ for feces and urine, respectively. This robust method recovered >60% and >80% cephapirin from spiked blank fecal and urine samples, respectively, with acceptable intra- and inter-day variation (<10%). Using this method, we detected trace amounts (µg kg⁻¹) of cephapirin in dairy cow feces, and cephapirin in urine was detected at very high concentrations (133 to 480 µg L⁻¹). Cephapirin was primarily excreted via urine and its urinary excretion was influenced by day (P = 0.03). Peak excretion (2.69 mg) was on day 1 following intramammary infusion and decreased sharply thereafter (0.19, 0.19, 0.08, and 0.17 mg on day 2, 3, 4, and 5, respectively) reflecting a quadratic pattern of excretion (Quadratic: P = 0.03). The described method for quantification of cephapirin in bovine feces and urine is sensitive, accurate, and robust and allowed to monitor the pattern of cephapirin excretion in dairy cows. This data will help develop manure segregation and treatment methods to minimize the risk of antibiotic loading to the environment from dairy farms.     

Incorporation of biological information in cancer risk assessment: Example — Vinyl chloride

Vinyl chloride (VC) is used as an example to demonstrate how biological information can be incorporated into quantitative risk assessment. The information included is the pharmacokinetics of VC in animals and humans and the data-generated hypothesis that VC primarily affects the initiation stage of the multistage carcinogenesis. The emphasis in this paper is on the improvement of risk assessment methodology rather than the risk assessment of VC per se.Sufficient data are available to construct physiologically-based pharmacokinetic models for both animals and humans. These models are used to calculate the metabolized dose corresponding to exposure scenarios in animals and in humans.On the basis of the data on liver angiosarcomas and carcinomas in rats, the cancer risk per unit of metabolized dose is comparable, irrespective of routes (oral or inhalation) of exposure. The tumor response from an intermittent/partial lifetime exposure is shown to be consistent with that from a lifetime exposure when VC is assumed to affect the first (initiation) stage of the multistage carcinogenic process. Furthermore, the risk estimates calculated on the basis of animal data are shown to be consistent with the human experience.     

Marine incursions,cryptic species and ecological diversification in Amazonia: the biogeographic history of the croaker genus Plagioscion (Sciaenidae)

Aim We propose a phylogenetic hypothesis for the marine‐derived sciaenid genus Plagioscion in the context of geomorphology and adaptation to freshwaters of South America, and assess the extent to which contemporary freshwater hydrochemical gradients influence diversification within a widely distributed Plagioscion species, Plagioscion squamosissimus. Location Amazon Basin and South America. Methods Using nuclear and mitochondrial DNA sequence data, phylogenetic analyses were conducted on the five nominal Plagioscion species, together with representatives from Pachyurus and Pachypops, using character and model‐based methods. Genealogical relationships and population genetic structure of 152 P. squamosissimus specimens sampled from the five major rivers and three hydrochemical settings/‘colours’ (i.e. white, black and clear water) of the Amazon Basin were assessed. Results Phylogenetic analyses support the monophyly of Plagioscion in South America and identify two putative cryptic species of Plagioscion. Divergence estimates suggest that the Plagioscion ancestor invaded South America via a northern route during the late Oligocene to early Miocene. Within P. squamosissimus a strong association of haplotype and water colour was observed, together with significant population structure detected between water colours. Main conclusions Our analyses of Plagioscion are consistent with a biogeographic scenario of early Miocene marine incursions into South America. Based on our phylogenetic results, the fossil record, geomorphological history and distributional data of extant Plagioscion species, we propose that marine incursions into western Venezuela between the late Oligocene and early Miocene were responsible for the adaptation to freshwaters in Plagioscion species. Following the termination of the marine incursions during the late Miocene and the establishment of the modern Amazon River, Plagioscion experienced a rapid diversification. Plagioscion squamosissimus arose during that time. The formation of the Amazon River probably facilitated population and range expansions for this species. Further, the large‐scale hydrochemical gradients within the Amazon Basin appear to be acting as ecological barriers maintaining population discontinuities in P. squamosissimus even in the face of gene flow. Our results highlight the importance of divergent natural selection through time in the generation and maintenance of sciaenid diversity in Amazonia.     

Tissue distribution and kininogen gene expression after acute-phase inflammation

A kinin-directed monoclonal antibody to kininogens has been developed by the fusion of murine myeloma cells with mouse splenocytes immunized with bradykinin-conjugated hemocyanin. The hybrid cells were screened by an enzyme-linked immunosorbent assay (ELISA) and a radioimmunoassay (RIA) for the secretion of antibodies to bradykinin. Ascitic fluids were produced and purified by a bradykinin-agarose affinity column. The monoclonal antibody (IgG1) bound to bradykinin, Lys-bradykinin, Met-Lys-bradykinin, and kininogens in ELISA. Further, this target-directed monoclonal antibody recognized purified low and high molecular weight bovine, human, or rat kininogens and T-kininogen in Western blotting. After turpentine-induced acute inflammation, rat kininogen levels increased dramatically in liver and serum as well as in the perfused pituitary, heart, lung, kidney, thymus, and other tissues, as identified by the kinin-directed kininogen antibody in Western blot analyses. The results were confirmed by measuring kinin equivalents of kininogens with a kinin RIA. During an induced inflammatory response, rat kininogens were localized immunohistochemically with the kinin-directed monoclonal antibody in parenchymal cells of liver, in acinar cells and some granular convoluted tubules of submandibular gland, and in the collecting tubules of kidney. Northern and cytoplasmic dot blot analyses using a kinin oligonucleotide probe showed that kininogen mRNA levels in liver but not in other tissues increase after turpentine-induced inflammation. The results indicated that rat kininogens are distributed in various tissues in addition to liver and only liver kininogen is induced by acute inflammation. The target-directed kininogen monoclonal antibody is a useful reagent for studying the structure, localization, and function of kininogens or any protein molecule containing the kinin moiety.     

Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype

The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2–4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.