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.     

MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25 years, and lack of auto-antibodies. It accounts for 2–5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil.     

Free-living amoebae promote Candida auris survival and proliferation in water

Candida auris is an emerging species responsible for life-threatening infections. Its ability to be resistant to most systemic antifungal classes and its capacity to persist in a hospital environment have led to health concerns. Currently, data about environmental reservoirs are limited but remain essential in control of C. auris spread. The aim of our study was to explore the interactions between C. auris and two free-living amoeba (FLA) species, Vermamoeba vermiformis and Acanthamoeba castellanii, potentially found in the same water environment. Candida auris was incubated with FLA trophozoites or their culture supernatants. The number of FLA and yeasts was determined at different times and transmission electron microscopy (TEM) was performed. Supernatants of FLAs promoted yeast survival and proliferation. Internalization of viable C. auris within both FLA species was also evidenced by TEM. A water environmental reservoir of C. auris can therefore be considered through FLAs and contamination of the hospital water networks would consequently be possible.     

The effects of androgens on the β-glands of the tokay (Gekko gecko): Modification of an hypothesis

Study of the posterior abdominal epidermis in hypophysectomized/thyroidectomized male and female tokays following surgery, and subsequent androgen therapy, indicates that, contrary to a previous model, all aspects of β-gland differentiation are under direct androgenic control. On the other hand, another epidermal specialization, the digital foot-pad, shows a pattern of histogenesis directly comparable to that of β-glands, but is unaffected by androgens. These data are discussed with respect to the evolution of glandular epidermal specializations in gekkonid lizards and the possible role of androgens in modifying the control of cell differentiation in lizard epidermis.     

Chromatographic separation of extruded iron-sulfur cores from the apoproteins of Clostridium pasteurianum and spinach ferredoxins in aqueous Triton X-100/urea

Iron-sulfur core extrusions from spinach [( 2Fe-2S]) and Clostridium pasteurianum (2[4Fe-4S]) ferredoxins in aqueous Triton X-100/urea containing excess benzenethiol yield quantitatively [FenSn(SPh)4]2- with n = 2 and n = 4, respectively. The iron-sulfur cluster can be separated from the corresponding apoprotein by rapid passage of the extrusion mixture over a small anaerobic column of Whatman DE-52 anion-exchange cellulose. Essentially quantitative recovery of [FenSn (SPh)4]2- is achieved in the eluate. The apoprotein remaining on the column can be eluted with 0.5 M NaCl. Most of the residual Triton X-100 and benzenethiol can be removed by passage of the apoprotein eluate over a small column of Bio-Beads SM-2, a hydrophobic polystyrene adsorbent. Apoprotein recovery is comparable to that obtained by other chromatographic methods. At least with spinach ferredoxin, the apoprotein prepared in this fashion can be reconstituted. The procedures developed in this work are potentially most applicable to selective removal of [2Fe-2S] and [4Fe-4S] centers from a multicenter enzyme without irreversible denaturation.     

Chromosome transfer and R-prime formation by an RP4::mini-Mu derivative in Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Proteus mirabilis

We have introduced into the wide host range conjugative plasmid RP4, a mini-Mu derivative which was known to be able to transpose spontaneously in E. coli K-12, and to induce in such a host several kinds of chromosomal rearrangements including replicon fusions. Unlike RP4, RP4::mini-Mu can mediate the transfer of the host chromosome to a recipient bacterium and generate R primes at high frequencies (10^?4 for the transfer of a given marker, 10^?5 for the formation of R primes carrying a given marker). Two such RP4::mini-Mu plasmids were introduced into one Salmonella typhimurium strain, one Klebsiella pneumoniae strain, and one Proteus mirabilis strain. Each of these three strains were mated with an E. coli K-12 recipient and transconjugants carrying R primes were recovered in all three cases at frequencies ranging from 5 × 10^?6 to 10^?7.     

In vitro anti-HIV activity of phosphorothioate alpha-anomeric oligodeoxynucleotides

Oligonucleotide analogs consisting exclusively of alpha-anomeric deoxynucleoside units bridged with phosphorothioate linkages have been synthesized and tested in vitro as antiviral agents against human immunodeficiency virus (HIV) in human T cells. Two 28-mers, an homopolymer alpha-S-dC28 and an oligomer alpha-S-anti-rev complementary to the initiation site of the regulatory viral gene rev exhibited antiviral activities comparable to those reported for the corresponding beta-anomeric phosphorothioate analogs. In contrast, a nuclease-resistant homopolymer, alpha-dC28 was inactive. Their preliminary results would indicate that the origin of oligonucleotide phosphorothioate anti-HIV activity is not exclusively correlated with their higher nuclease resistance.     

Kinetics of phenylalanine absorption by the rat intestine in vivo after distal resection

The kinetics of L-phenylalanine absorption across rat small intestine in sham and 50% distal resected animals, in vivo, have been studied by perfusing jejunal loops and monitoring the disappearance of the substrate from the perfusate. After 5 months postresection the total phenylalanine absorption was increased. The relationship between total absorption of substrate and its concentration in the bulk phase shows a non-saturable component and a saturable one that can be inhibited by methionine, both in control and remnant jejunum. The slope of the line that represents the non-saturable component is greater in remnant jejunum, indicating that the apparent mass-transfer coefficient, K'D, was increased by distal resection. The kinetic analysis of the saturable component shows that Jmax was unaltered and the apparent semisaturation constant, K'M, was slightly decreased by distal small intestine resection. Correction of the kinetic constant for the unstirred water layer effects shows that the differences between 'real' KD values of the two experimental groups increase whereas 'real' KM values do not change significantly. This indicates that the observed increase in total intestinal absorption in resected animals appears to result from an increase in the intestinal passive permeability.