European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development

In situ hybridization (ISH) is used for the localization of specific nucleic acid sequences in cells or tissues by complementary binding of a nucleotide probe to a specific target nucleic acid sequence. In the last years, the specificity and sensitivity of ISH assays were improved by innovative techniques like synthetic nucleic acids and tandem oligonucleotide probes combined with signal amplification methods like branched DNA, hybridization chain reaction and tyramide signal amplification. These improvements increased the application spectrum for ISH on formalin-fixed paraffin-embedded tissues. ISH is a powerful tool to investigate DNA, mRNA transcripts, regulatory noncoding RNA, and therapeutic oligonucleotides. ISH can be used to obtain spatial information of a cell type, subcellular localization, or expression levels of targets. Since immunohistochemistry and ISH share similar workflows, their combination can address simultaneous transcriptomics and proteomics questions. The goal of this review paper is to revisit the current state of the scientific approaches in ISH and its application in drug research and development

Etude de l'Encéphalopathie Spongiforme Bovine Expérimentalement Transmise aux Ovins

To characterise in sheep the bovine spongiform encephalopathy agent (BSE) responsible for variant Creutzfeldt-Jakob disease in human beings, three ewes, presenting either a susceptible (2-ARQ/ARQ) or a resistant (1-ARR/ARR) genotype toward scrapie, were infected with a French BSE case. The research of abnormal prion protein (PrPsc) accumulating sites revealed traces of PrPsc in ARR/ARR sheep but did not distinguish BSE agent in ARQ/ARQ sheep from natural scrapie cases. The BSE agent was finally identified in these ewes by analysing protease cleavage sites of PrPsc and comparing, in mice infected with BSE, ovine-BSE and scrapie, the neuro-anatomical distribution of PrPsc deposits which was discriminant. Thus, as some properties of the BSE agent are maintained in sheep, it may be possible to identify this agent in sheep flocks using theses approaches.Dans le but de caractériser chez les ovins l'agent de l'encéphalopathie spongiforme bovine (ESB) responsable du variant de la maladie de Creutzfeldt-Jakob chez l'homme, trois brebis de génotype sensible (2-ARQ/ARQ) ou résistant (1-ARR/ARR) à la tremblante ont été infectées par un cas d'ESB français. La recherche des sites d'accumulation de la protéine prion anormale (PrPsc) a révélé des traces PrPsc chez le mouton ARR/ARR mais n'a pas permis de différencier l'ESB chez les moutons ARQ/ARQ de cas de tremblante naturelle. Chez ces brebis, l'agent de l'ESB a pu être en revanche identifié par l'analyse des sites de clivage de la PrPsc par les protéases comme par l'étude comparée, chez des souris infectées par l'ESB, l'ESB-ovine et la tremblante, de la distribution neuro-anatomique discriminante des dépôts de PrPsc. Ainsi, grâce au maintien de certaines de ses propriétés chez les ovins, il devrait être possible d'identifier l'agent de l'ESB dans les troupeaux par ces approches complémentaires

The Isolated Mouse Jejunal Afferent Nerve Assay as a Tool to Assess the Effect of Botulinum Neurotoxins in Visceral Nociception

For the past two decades, botulinum neurotoxin A (BoNT/A) has been described as a strong candidate in the treatment of pain. With the production of modified toxins and the potential new applications at the visceral level, there is a real need for tools allowing the assessment of these compounds. In this study, we evaluated the jejunal mesenteric afferent nerve assay to investigate BoNT/A effects on visceral nociception. This ex vivo model allowed the continuous recording of neuronal activity in response to various stimuli. BoNT/A was applied intraluminally during three successive distensions, and the jejunum was distended every 15 min for 3 h. Finally, samples were exposed to external capsaicin. BoNT/A intoxication was validated at the molecular level with the presence of cleaved synaptosomal-associated protein of 25 (SNAP25) in nerve terminals in the mucosa and musculosa layers 3 h after treatment. BoNT/A had a progressive inhibitory effect on multiunit discharge frequency induced by jejunal distension, with a significant decrease from 1 h after application without change in jejunal compliance. The capsaicin-induced discharge was also affected by the toxin. This assay allowed the description of an inhibitory effect of BoNT/A on afferent nerve activity in response to distension and capsaicin, suggesting BoNT/A could alleviate visceral nociception

A neonatal piglet model for investigating brain and cognitive development in small for gestational age human infants.

The piglet was investigated as a potential model for studying brain and cognitive deficits associated with being born small for gestational age (SGA). Naturally farrowed SGA (0.7-1.0 kg BW) and average for gestational age (AGA, 1.3-1.6 kg BW) piglets were obtained on postnatal day (PD) 2, placed in individual cages, and provided a nutritionally adequate milk replacer diet (285 ml/kg/d). Beginning at PD14, performance in a spatial T-maze task was assessed. At PD28, piglets were anesthetized for magnetic resonance (MR) imaging to assess brain structure (voxel-based morphometry), connectivity (diffusion-tensor imaging) and metabolites in the hippocampus and corpus callosum (proton MR spectroscopy). Piglets born SGA showed compensatory growth such that BW of SGA and AGA piglets was similar (P>0.05), by PD15. Birth weight affected maze performance, with SGA piglets taking longer to reach criterion than AGA piglets (p<0.01). Total brain volume of SGA and AGA piglets was similar (P<0.05), but overall, SGA piglets had less gray matter than AGA piglets (p<0.01) and tended to have a smaller internal capsule (p = 0.07). Group comparisons between SGA and AGA piglets defined 9 areas (≥ 20 clusters) where SGA piglets had less white matter (p<0.01); 2 areas where SGA piglets had more white matter (p<0.01); and 3 areas where SGA piglets had more gray matter (p<0.01). The impact of being born SGA on white matter was supported by a lower (p<0.04) fractional anisotropy value for SGA piglets, suggesting reduced white matter development and connectivity. None of the metabolites measured were different between groups. Collectively, the results show that SGA piglets have spatial learning deficits and abnormal development of white matter. As learning deficits and abnormalities in white matter are common in SGA human infants, the piglet is a tractable translational model that can be used to investigate SGA-associated cognitive deficits and potential interventions

The Expanding Therapeutic Utility of Botulinum Neurotoxins

Botulinum neurotoxin (BoNT) is a major therapeutic agent that is licensed in neurological indications, such as dystonia and spasticity. The BoNT family, which is produced in nature by clostridial bacteria, comprises several pharmacologically distinct proteins with distinct properties. In this review, we present an overview of the current therapeutic landscape and explore the diversity of BoNT proteins as future therapeutics. In recent years, novel indications have emerged in the fields of pain, migraine, overactive bladder, osteoarthritis, and wound healing. The study of biological effects distal to the injection site could provide future opportunities for disease-tailored BoNT therapies. However, there are some challenges in the pharmaceutical development of BoNTs, such as liquid and slow-release BoNT formulations; and, transdermal, transurothelial, and transepithelial delivery. Innovative approaches in the areas of formulation and delivery, together with highly sensitive analytical tools, will be key for the success of next generation BoNT clinical products

Additional file 1: Table S1. of Extract of Ginkgo biloba exacerbates liver metastasis in a mouse colon cancer Xenograft model

List of primers for PCR. Table S2 List of antibodies. (DOCX 17 kb

Fractional anisotropy (FA) values for AGA and SGA piglets.

<p>The FA value determined from Diffusion Tensor Imaging was lower in SGA piglets compared to AGA piglets (*p = 0.04). Data are shown as the mean ± SEM (n = 5).</p

Representative example of piglet Magnetic Resonance Spectroscopy Spectra.

<p>Representative example of piglet Magnetic Resonance Spectroscopy Spectra.</p

European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development.

In situ hybridization (ISH) is used for the localization of specific nucleic acid sequences in cells or tissues by complementary binding of a nucleotide probe to a specific target nucleic acid sequence. In the last years, the specificity and sensitivity of ISH assays were improved by innovative techniques like synthetic nucleic acids and tandem oligonucleotide probes combined with signal amplification methods like branched DNA, hybridization chain reaction and tyramide signal amplification. These improvements increased the application spectrum for ISH on formalin-fixed paraffin-embedded tissues. ISH is a powerful tool to investigate DNA, mRNA transcripts, regulatory noncoding RNA, and therapeutic oligonucleotides. ISH can be used to obtain spatial information of a cell type, subcellular localization, or expression levels of targets. Since immunohistochemistry and ISH share similar workflows, their combination can address simultaneous transcriptomics and proteomics questions. The goal of this review paper is to revisit the current state of the scientific approaches in ISH and its application in drug research and development

Voxel-based morphometry analysis using DARTEL showed increased gray matter volumes in the olfactory bulb and cortex of SGA piglets.

<p>The color bar indicates the pseudo-t statistic indicating level of significance.</p