Status epilepticus (SE) induces neuroinflammation and epileptogenesis, but the mechanisms are not yet fully delineated. The Fyn, a non-receptor Src family tyrosine kinase (SFK), and its immediate downstream target, PKCδ are emerging as potential mediators of neuroinflammation. In order to first determine the role of Fyn kinase signaling in SE, we tested the efficacy of a SFK inhibitor, saracatinib (25mg/kg, oral) in C57BL/6J mouse kainate model of acute seizures. Saracatinib pretreatment dampened SE severity and completely prevented mortality. We further utilized fyn and fyn mice (wildtype control for the fyn mice on same genetic background), and the rat kainate model, treated with saracatinib post-SE, to validate the role of Fyn/SFK in SE and epileptogenesis. We observed significant reduction in SE severity, epileptiform spikes, and electrographic non-convulsive seizures in fyn mice when compared to fyn mice. Interestingly, significant reductions in phosphorylated pSrc-416 and PKCδ (pPKCδ-507) and naive PKCδ were observed in fyn mice as compared to fyn mice suggesting that PKCδ signaling is a downstream mediator of Fyn in SE and epileptogenesis. Notably, fyn mice also showed a reduction in key proinflammatory mediators TNF-α, IL-1β, and iNOS mRNA expression; serum IL-6 and IL-12 levels; and nitro-oxidative stress markers such as 4-HNE, gp91, and 3-NT in the hippocampus. Immunohistochemistry revealed a significant increase in reactive microgliosis and neurodegeneration in the hippocampus and hilus of dentate gyrus in fyn mice in contrast to fyn mice. Interestingly, we did not observe upregulation of Fyn in pyramidal neurons of the hippocampus during post-SE in fyn mice, but it was upregulated in hilar neurons of the dentate gyrus when compared to naïve control. In reactive microglia, both Fyn and PKCδ were persistently upregulated during post-SE suggesting that Fyn-PKCδ may drive neuroinflammation during epileptogenesis. Since disabling the Fyn kinase prior to SE, either by treating with saracatinib or fyn gene knockout, suppressed seizures and the subsequent epileptogenic events, we further tested whether Fyn/SFK inhibition during post-SE modifies epileptogenesis. Telemetry-implanted, SE-induced, rats were treated with saracatinib and continuously monitored for a month. At 2h post-diazepam, the saracatinib (25mg/kg) or the vehicle was administered orally and repeated twice daily for first three days followed by a single dose/day for the next four days. The saracatinib post-treatment prevented epileptogenesis in >50% of the rats and significantly reduced spontaneous seizures and epileptiform spikes in the rest (one animal did not respond) when compared to the vehicle treated group, which had >24 seizures in a month. Collectively, the findings suggest that Fyn/SFK is a potential mediator of epileptogenesis and a therapeutic target to prevent/treat seizures and epileptogenesis.
Purpose: Compare the precision of doxycycline quantification in tear fluid collected with either Schirmer strips or polyvinyl acetal (PVA) sponges following oral drug administration.Methods: Three dogs and 3 cats were administered doxycycline orally at a dose of 4.2–5 mg/kg every 12 h for 6 consecutive days. At day 5 and 6, blood and tear fluid were sampled to capture doxycycline trough and maximal concentrations. Tear fluid was collected 3 times (spaced 10 min apart) at each session with the absorbent material placed in the lower conjunctival fornix until the 20-mm mark was reached (Schirmer strip, one eye) or for 1 min (PVA sponge, other eye). Tear extraction was performed with either centrifugation or elution in methanol. Doxycycline concentrations were measured with liquid chromatography–mass spectrometry. Low (100 ng/mL) and high (1,000 ng/mL) tear concentrations measured in vivo were spiked into each absorbent material in vitro to evaluate percentage drug recovery.Results: After oral administration of doxycycline, the drug reached the tear compartment at concentrations of 45.1–900.7 ng/mL in cats and 45.4–632.0 ng/mL in dogs, representing a tear-to-serum ratio of 12% and 16%, respectively. Doxycycline tear concentrations were significantly more precise when tear collection was performed with Schirmer strips rather than PVA sponges (P = 0.007), but were not correlated with tear flow rate. In vitro doxycycline recovery was poor to moderate (<75%).Conclusions: Schirmer strips represent a good option for lacrimal doxycycline quantification, although the collection and subsequent extraction have to be optimized to improve drug recovery.
Sow mortality attributable to pelvic organ prolapse (POP) has increased in the U.S. swine industry and continues to worsen. Two main objectives of this study were, (1) to develop a perineal scoring system that can be correlated with POP risk, and (2) identify POP risk-associated biological factors. To assess POP risk during late gestation, sows (n = 213) were scored using a newly developed perineal scoring (PS) system. Sows scored as PS1 (low), PS2 (moderate), or PS3 (high) based on POP risk. Subsequently, 1.5, 0.8, and 23.1% of sows scored PS1, PS2, or PS3, respectively, experienced POP. To identify biomarkers, serum and vaginal swabs were collected from late gestation sows differing in PS. Using GC–MS, 82 serum metabolite differences between PS1 and PS3 animals (P < 0.05) were identified. Vaginal swabs were utilized for 16S rRNA gene sequencing and differences in vaginal microbiomes between PS1 and PS3 animals were detected on a community level (P < 0.01) along with differences in abundances of 89 operational taxonomic units (P < 0.05). Collectively, these data demonstrate that sows with greater POP risk have differential serum metabolites and vaginal microflora. Additionally, an initial and novel characterization of the sow vaginal microbiome was determined.
BackgroundCorrect annotation of function is essential if one is to take full advantage of the vast amounts of genomic sequence data. The accuracy of sequence-based functional annotations is often variable, particularly if the sequence homology to a known function is low. Indeed recent work has shown that even proteins with very high sequence identity can have different folds and functions, and therefore caution is needed in assigning functions by sequence homology in the absence of experimental validation. Experimental methods are therefore needed to efficiently evaluate annotations in a way that complements current high throughput technologies. Here, we describe the use of nuclear magnetic resonance (NMR)-based ligand screening as a tool for testing functional assignments of putative enzymes that may be of variable reliability.ResultsThe target genes for this study are putative enzymes from the methanogenic archaeon Methanosarcina acetivorans (MA) that have been selected after manual genome re-annotation and demonstrate detectable in vivo expression at the level of the transcriptome. The experimental approach begins with heterologous E. coli expression and purification of individual MA gene products. An NMR-based ligand screen of the purified protein then identifies possible substrates or products from a library of candidate compounds chosen from the putative pathway and other related pathways. These data are used to determine if the current sequence-based annotation is likely to be correct. For a number of case studies, additional experiments (such as in vivo genetic complementation) were performed to determine function so that the reliability of the NMR screen could be independently assessed.ConclusionsIn all examples studied, the NMR screen was indicative of whether the functional annotation was correct. Thus, the case studies described demonstrate that NMR-based ligand screening is an effective and rapid tool for confirming or negating the annotated gene function of putative enzymes. In particular, no protein-specific assay needs to be developed, which makes the approach broadly applicable for validating putative functions using an automated pipeline strategy.
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