Rat Counterpart Research Articles

Tabernanthalog and ibogainalog inhibit the α7 and α9α10 nicotinic acetylcholine receptors via different mechanisms and with higher potency than the GABAA receptor and CaV2.2 channel

In this study, we have investigated the pharmacological activity and structural interaction of two novel psychoplastogens, tabernanthalog (TBG) and ibogainalog (IBG) at heterologously-expressed rat (r) and human (h) nicotinic acetylcholine receptors (nAChRs), the rα1β2γ2L γ-aminobutyric acid type A receptor (GABAAR), and the human voltage-gated N-type calcium channel (CaV2.2 channel). Both compounds inhibited the nAChRs with the following receptor selectivity: α9α10 > α7 > α3β2 ≅ α3β4, indicating that β2/β4 subunits are relatively less important for their activity. The potencies of TBG and IBG were comparable at hα7 and hα9α10 subtypes, and comparable to their rat counterparts. TBG- and IBG-induced inhibition of rα7 was ACh concentration-independent and voltage-dependent, whereas rα9α10 inhibition was ACh concentration-dependent and voltage-independent, suggesting that they interact with the α7 ion channel pore and α9α10 orthosteric ligand binding site, respectively. These results were supported by molecular docking studies showing that at the α7 model TBG forms stable interactions with luminal rings at 9′, 13′, and 16′, whereas IBG mostly interacts with the extracellular-transmembrane junction. In the α9α10 model, however, these compounds interacted with several residues from the principal (+) and complementary (–) sides in the transmitter binding site. Ibogaminalog (DM506) also interacted with a non-luminal site at α7, and one α9α10 orthosteric site. TBG and IBG inhibited the GABAAR and CaV2.2 channels with 10 to 30-fold lower potencies. In sum, we show that TBG and IBG inhibit the α7 and α9α10 nAChRs by noncompetitive and competitive mechanisms, respectively, and with higher potency than the GABAAR and CaV2.2 channel.

372 Differences in Oligodendrogenesis Between Human and Rodent Spinal Cord Stem Cells

INTRODUCTION: Research in pre-clinical rodent models suggests that targeting the endogenous neural stem/progenitor cells (NSPCs) in the spinal cord may hold the key to stimulating oligodendrocyte regeneration. However, it remains to be seen whether human spinal cord NSPCs possess the same regenerative potential as their rat counterparts. METHODS: To assess the potential for oligodendrogenesis between human and rat spinal cord NSPCs, primary tissue from adult donors and female rats was harvested and cultured using the Neurosphere assay. To induce spontaneous differentiation, primary-derived NSPCs were treated with 1% fetal bovine serum (n = 15 humans, n = 10 rats). To further promote oligodendrocyte differentiation, pdNSPCs were treated with 40 ng/µL or 100 ng/µL of platelet-derived growth factor AA (PDGF-AA) (n = 3 humans, n = 3 rats). Immunocytochemistry and RNA sequencing were then employed to compare the transcriptomes, with the differentially expressed genes and gene sets analyzed via DESeq and GSEA. RESULTS: Human NSPCs showed a reduced potential for oligodendrocyte generation compared to rat NSPCs (0.013 ± 0.01% and 0.029 ± 0.01% O4+ after one and two weeks in humans; 4.9 ± 0.4% and 6.3 ± 0.6% O4+ after one and two weeks in rats). PDGF-AA treatment at 40 ng/µL for one week was able to effectively promote oligodendrocyte differentiation in rat NSPCs, but had a minimal effect on human NSPCs (8.5 ± 1.4 fold increase in O4+). OLIG1/2, SOX10, and CNP gene transcripts were enriched in rat NSPCs. CONCLUSIONS: We compared the potential of oligodendrogenesis between primary human and rat spinal cord NSPCs and found a significantly lower capacity in the human NSPCs both functionally and transcriptionally, which could be an obstacle to the successful application of myelinating techniques.

Age-dependent assessment of selenium nanoparticles: biodistribution and toxicity study in young and adult rats.

Aim: To study the biodistribution and toxicology of selenium nanoparticles (SeNPs) versus their bulk counterpart in young and adult male rats in a 28-day study. Methods: SeNPs were synthesized and conjugated with indocyanine green to assess comparative biodistribution by in vivo imaging and further characterized by transmission electron microscopy, Fourier transform infrared, scanning electron microscopy/energy dispersive x-ray spectroscopy, UV and ζ-analysis. The toxicity of bulk selenium was evaluated relative to its nano form by hematology indices, redox, inflammatory markers and histopathology. Results: Indocyanine green-conjugated nanoparticles showed preferential accumulation in the liver, followed by testis and kidney. The protective effect of SeNPs was more significantly observed in young livers than in adults compared with the bulk counterpart. Conclusion: Age-dependentmonitoring and diagnosis of toxicity may need different biomarkers of selenium and may also provide better understanding of SeNPs as therapeutics.

Increased depressive-like behaviour of postpartum Flinders sensitive and resistant line rats is reversed by a predictable postpartum stressor

During the peripartum period, women are at an increased risk to develop perinatal distress, presenting as symptoms of depression and/or anxiety. Yet, due to practical and ethical restrictions, our understanding of this condition remains limited. Animal studies that focus on the neuropsychiatric mechanisms associated with the postpartum period, often ignore the genetical predisposition factor. We therefore investigated whether pregnancy could alter the bio-behavioural profile of the Flinders sensitive and resistant line rats, and whether these effects are exacerbated by a postpartum stressor. Postpartum dams were compared to nulliparous controls in behavioural tests, analysing depressive- and anxiety-like behaviours. Next, postpartum dams were subjected to a maternal separation and early weaning (MSEW) regimen, with their behaviour and serotonergic and noradrenergic concentrations compared to rats not separated from their pups. Regardless of strain, pregnancy decreased time spent in the open arms of the elevated plus maze and hippocampal serotonin concentrations. Time spent immobile in the forced swim test was also increased, with a significant effect in the FRL and a strong trend in the FSL rats. MSEW reversed these behaviours in both strains and increased social interaction with a male counterpart in the FSL rats, without influencing hippocampal or cortical serotonin or norepinephrine. Taken together, these results suggest that pregnancy influences postpartum behaviour, in a strain-dependent manner. Contrary to what we expected, MSEW overall decreased depressive- and anxiety-like behaviours, with strain specific differences, indicating that a chronic, predictable stressor may not necessarily adversely affect postpartum behaviour.

Examining mGlu5 Receptor Availability as a Predictor of Vulnerability to PTSD: An [18F]FPEB and PET Study in Male and Female Rats

Background Females are twice as likely to experience post-traumatic stress disorder (PTSD) than males, yet specific factors contributing to this greater risk are not fully understood. Our clinical and recent preclinical findings suggest a role for the metabotropic glutamate receptor 5 (mGlu5) in PTSD and differential involvement between males and females. Methods Here, we further investigate whether mGlu5 receptor availability may contribute to individual and sex differences in PTSD susceptibility by quantifying receptor availability using the mGlu5 receptor-specific radiotracer, [18F]FPEB, and positron emission tomography in male (n = 16) and female (n = 16) rats before and after traumatic footshock exposure (FE) and assessment of stress-enhanced fear learning (SEFL) susceptibility, as compared with no-shock controls (CON; n = 7 male; n = 8 female). Results Overall, FE rats displayed greater fear generalization as compared with CON (p < .001). Further, greater mGlu5 receptor availability at baseline (p = .003) and post-test (p = .005) was significantly associated with expression of the SEFL phenotype. Notably, FE female rats displayed a shift to more passive coping (ie, freezing), and displayed greater SEFL susceptibility (p = .01), and had lower baseline mGlu5 availability (p = .03) relative to their FE male rat counterparts. Conclusion Results are consistent with clinical findings of higher mGlu5 receptor availability in PTSD, and add to growing evidence implicating these receptors in the pathophysiology of PTSD and sex-differences in susceptibility for this disorder.

Homology of neocortical areas in rats and primates based on cortical type analysis: an update of the Hypothesis on the Dual Origin of the Neocortex

Sixty years ago, Friedrich Sanides traced the origin of the tangential expansion of the primate neocortex to two ancestral anlagen in the allocortex of reptiles and mammals, and proposed the Hypothesis on the Dual Origin of the Neocortex. According to Sanides, paraolfactory and parahippocampal gradients of laminar elaboration expanded in evolution by addition of successive concentric rings of gradually different cortical types inside the allocortical ring. Rodents had fewer rings and primates had more rings in the inner part of the cortex. In the present article, we perform cortical type analysis of the neocortex of adult rats, Rhesus macaques, and humans to propose hypotheses on homology of cortical areas applying the principles of the Hypothesis on the Dual Origin of the Neocortex. We show that areas in the outer rings of the neocortex have comparable laminar elaboration in rats and primates, while most 6-layer eulaminate areas in the innermost rings of primate neocortex lack homologous counterparts in rats. We also represent the topological distribution of cortical types in simplified flat maps of the cerebral cortex of monotremes, rats, and primates. Finally, we propose an elaboration of the Hypothesis on the Dual Origin of the Neocortex in the context of modern studies of pallial patterning that integrates the specification of pallial sectors in development of vertebrate embryos. The updated version of the hypothesis of Sanides provides explanation for the emergence of cortical hierarchies in mammals and will guide future research in the phylogenetic origin of neocortical areas.

Innate lymphoid cell characterization in the rat and their correlation to gut commensal microbes.

Innate lymphoid cells (ILCs) are important for tissue immune homeostasis, and are thoroughly characterized in mice and humans. Here, we have performed in-depth characterization of rat ILCs. Rat ILCs were identified based on differential expression of transcription factors and lack of lineage markers. ILC3s represented the major ILC population of the small intestine, while ILC2s were infrequent but most prominent in liver and adipose tissue. Two major subsets of group 1 ILCs were defined. Lineage- T-bet+ Eomes+ cells were identified as conventional NK cells, while lineage- T-bet+ Eomes- cells were identified as the probable rat counterpart of ILC1s based on their selective expression of the ILC marker CD200R. Rat ILC1s were particularly abundant in liver and intestinal tissues, and were functionally similar to NK cells. Single-cell transcriptomics of spleen and liver cells confirmed the main division of NK cells and ILC1-like cells, and demonstrated Granzyme A as an additional ILC1 marker. We further report differential distributions of NK cells and ILCs along the small and large intestines, and the association of certain bacterial taxa to frequencies of ILCs. In conclusion, we provide a framework for future studies of ILCs in diverse rat experimental models, and novel data on the potential interplay between commensals and intestinal ILCs.

Connections of the mouse subfornical region of the lateral hypothalamus (LHsf).

The lateral hypothalamus is a major integrative hub with a complex architecture characterized by intricate and overlapping cellular populations expressing a large variety of neuro-mediators. In rats, the subfornical lateral hypothalamus (LHsf) was identified as a discrete area with very specific outputs, receiving a strong input from the nucleus incertus, and involved in defensive and foraging behaviors. We identified in the mouse lateral hypothalamus a discrete subfornical region where a conspicuous cluster of neurons express the mu opioid receptor. We thus examined the inputs and outputs of this LHsf region in mice using retrograde tracing with the cholera toxin B subunit and anterograde tracing with biotin dextran amine, respectively. We identified a connectivity profile largely similar, although not identical, to what has been described in rats. Indeed, the mouse LHsf has strong reciprocal connections with the lateral septum, the ventromedial hypothalamic nucleus and the dorsal pre-mammillary nucleus, in addition to a dense output to the lateral habenula. However, the light input from the nucleus incertus and the moderate bidirectional connectivity with nucleus accumbens are specific to the mouse LHsf. A preliminary neurochemical study showed that LHsf neurons expressing mu opioid receptors also co-express calcitonin gene-related peptide or somatostatin and that the reciprocal connection between the LHsf and the lateral septum may be functionally modulated by enkephalins acting on mu opioid receptors. These results suggest that the mouse LHsf may be hodologically and functionally comparable to its rat counterpart, but more atypical connections also suggest a role in consummatory behaviors.

Wild Rats, Laboratory Rats, Pet Rats: Global Seoul Hantavirus Disease Revisited.

Recent reports from Europe and the USA described Seoul orthohantavirus infection in pet rats and their breeders/owners, suggesting the potential emergence of a “new” public health problem. Wild and laboratory rat-induced Seoul infections have, however, been described since the early eighties, due to the omnipresence of the rodent reservoir, the brown rat Rattus norvegicus. Recent studies showed no fundamental differences between the pathogenicity and phylogeny of pet rat-induced Seoul orthohantaviruses and their formerly described wild or laboratory rat counterparts. The paucity of diagnosed Seoul virus-induced disease in the West is in striking contrast to the thousands of cases recorded since the 1980s in the Far East, particularly in China. This review of four continents (Asia, Europe, America, and Africa) puts this “emerging infection” into a historical perspective, concluding there is an urgent need for greater medical awareness of Seoul virus-induced human pathology in many parts of the world. Given the mostly milder and atypical clinical presentation, sometimes even with preserved normal kidney function, the importance of simple but repeated urine examination is stressed, since initial but transient proteinuria and microhematuria are rarely lacking.

Bioavailability of docosahexaenoic acid 22:6(n-3) from enantiopure triacylglycerols and their regioisomeric counterpart in rats

Lack of synthetic enantiospecific triacylglycerols (TAGs) has hindered our understanding of the impact of TAG structure on the absorption and metabolic fate of fatty acids (FAs). In a five-day feeding trial with mildly (n-3) deficient rats, the bioavailability of docosahexaenoic acid [22:6(n-3), DHA] and stearic acid (18:0) from the two different enantiomers of TAG: sn-22:6(n-3)-18:0-18:0 and sn-18:0-18:0-22:6(n-3), and their regioisomeric TAG: sn-18:0-22:6(n-3)-18:0 was compared. Less secretion of fecal DHA was detected from the sn-2 position compared with the sn-1 and sn-3 positions, but no difference was found in DHA content of the fasting plasma or in the weight of the body or organs. 18:0 was lost to feces mainly as cleaved from the primary positions but also as glycerol-bound. The 5-day intervention in rats was long enough to modify the fatty acid profile of plasma phospholipids.

Comparative neuropharmacology of N-(2-methoxybenzyl)-2,5-dimethoxyphenethylamine (NBOMe) hallucinogens and their 2C counterparts in male rats

2,5-Dimethoxyphenethylamines (2C compounds) are 5-HT2A/2C receptor agonists that induce hallucinogenic effects. N-methoxybenzylation of 2C compounds markedly increases their affinity for 5-HT2A receptors, and two such analogs, 2-(4-chloro-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25C-NBOMe) and 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe), have emerged in recreational drug markets. Here, we investigated the neuropharmacology of 25C-NBOMe and 25I-NBOMe in rats, as compared to their 2C analogs and the prototypical 5-HT2A/2C agonist 1-(4-iodo-2,5-dimethoxyphenyl)propan-2-amine (DOI). Compounds were tested in vitro using 5-HT2A receptor binding and calcium mobilization assays. For in vivo experiments, 25C-NBOMe (0.01–0.3 mg/kg), 25I-NBOMe (0.01–0.3 mg/kg), 2-(4-chloro-2,5-dimethoxyphenyl)ethanamine (2C-C) (0.1–3.0 mg/kg), 2-(4-iodo-2,5-dimethoxyphenyl)ethanamine (2C-I) (0.1–3.0 mg/kg) and DOI (0.03–1.0 mg/kg) were administered subcutaneously (sc) to male rats, and 5-HT2A-mediated behaviors were assessed. NBOMes displayed higher affinity for 5-HT2A receptors than their 2C counterparts but were substantially weaker in functional assays. 25C-NBOMe and 25I-NBOMe were much more potent at inducing wet dog shakes (WDS) and back muscle contractions (BMC) when compared to 2C-C and 2C-I. Pretreatment with the selective 5-HT2A antagonist (R)-(2,3-dimethoxyphenyl){1-[2-(4-fluorophenyl)ethyl]-4-piperidinyl}methanol (M100907) reversed behaviors produced by all agonists. Interestingly, binding affinities at the 5-HT2A receptor were significantly correlated with potencies to induce BMC but not WDS. Our findings show that NBOMes are highly potent 5-HT2A agonists in rats, similar to effects in mice, and consistent with the reported hallucinogenic effects in human users.This article is part of the Special Issue entitled ‘Psychedelics: New Doors, Altered Perceptions’.

Acute oral toxicity study of magnesium oxide nanoparticles and microparticles in female albino Wistar rats

Advancements in nanotechnology have led to the development of the nanomedicine, which involves nanodevices for diagnostic and therapeutic purposes. A key requirement for the successful use of the nanoparticles (NPs) in biomedical applications is their good dispensability, colloidal stability in biological media, internalization efficiency, and low toxicity. Therefore, toxicological profiling is necessary to understand the mechanism of NPs and microparticles (MPs). MgO NPs have attracted wide scientific interest due to ease of synthesis, chemical stability and unique properties. However, their toxic effects on humans should also be of concern with the increased applications of nano MgO. The present study was aimed to assess the toxicological potential of MgO NPs in comparison to their micron counterparts in female Wistar rats. Toxicity was evaluated using genotoxicity, histological, biochemical, antioxidant and biodistribution parameters post administration of MgO particles to rats through oral route. The results obtained from the investigation revealed that the acute exposure to the high doses of MgO NPs produced significant (p < 0.01) DNA damage and biochemical alterations. Antioxidant assays revealed prominent oxidative stress at the high dose level for both the particles. Toxicokinetic analysis showed significant levels of Mg accumulation in the liver and kidney tissues apart from urine and feces. Further, mechanistic investigational reports are warranted to document safe exposure levels and health implications post exposure to high levels of NPs.

Anatomical Organization of Urocortin 3-Synthesizing Neurons and Immunoreactive Terminals in the Central Nervous System of Non-Human Primates [Sapajus spp.

Urocortin 3 (UCN3) is a neuropeptide member of the corticotropin-releasing factor (CRF) peptide family that acts as a selective endogenous ligand for the CRF, subtype 2 (CRF2) receptor. Immunohistochemistry and in situ hybridization data from rodents revealed UCN3-containing neurons in discrete regions of the central nervous system (CNS), such as the medial preoptic nucleus, the rostral perifornical area (PFA), the medial nucleus of the amygdala and the superior paraolivary nucleus. UCN3-immunoreactive (UCN3-ir) terminals are distributed throughout regions that mostly overlap with regions of CRF2 messenger RNA (mRNA) expression. Currently, no similar mapping exists for non-human primates. To better understand the role of this neuropeptide, we aimed to study the UCN3 distribution in the brains of New World monkeys of the Sapajus genus. To this end, we analyzed the gene and peptide sequences in these animals and performed immunohistochemistry and in situ hybridization to identify UCN3 synthesis sites and to determine the distribution of UCN3-ir terminals. The sequencing of the Sapajus spp. UCN3-coding gene revealed 88% and 65% identity to the human and rat counterparts, respectively. Additionally, using a probe generated from monkey cDNA and an antiserum raised against human UCN3, we found that labeled cells are mainly located in the hypothalamic and limbic regions. UCN3-ir axons and terminals are primarily distributed in the ventromedial hypothalamic nucleus (VMH) and the lateral septal nucleus (LS). Our results demonstrate that UCN3-producing neurons in the CNS of monkeys are phylogenetically conserved compared to those of the rodent brain, that the distribution of fibers agrees with the distribution of CRF2 in other primates and that there is anatomical evidence for the participation of UCN3 in neuroendocrine control in primates.

Rat astrocytes are more supportive for mouse OPC self-renewal than mouse astrocytes in culture.

Mouse primary oligodendrocyte precursor cells (OPCs) are increasingly used to study the molecular mechanisms underlying the phenotype changes in oligodendrocyte differentiation and axonal myelination observed in transgenic or mutant mouse models. However, mouse OPCs are much more difficult to be isolated by the simple dissociation culture of brain tissues than their rat counterparts. To date, the mechanisms underlying the species difference in OPC preparation remain obscure. In this study, we showed that astrocytes from rats have a stronger effect than those from mouse in promoting OPC proliferation and survival in vitro. Mouse astrocytes displayed significantly weaker viability in culture and reduced potential in maintaining OPC self-renewal, as confirmed by culturing OPCs with conditioned media from rat or mouse astrocytes. These results explained the reason for why stratified cultures of OPCs and astrocytes are difficult to be achieved in mouse CNS tissues. Based on these findings, we adopted inactivated rat astrocytes as feeder cells to support the self-renewal of mouse cortical OPCs and preparation of high-purity mouse OPCs. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 907-916, 2017.

Degradation of Serotonin N-Acetyltransferase, a Circadian Regulator, by the N-end Rule Pathway

Serotonin N-acetyltransferase (AANAT) converts serotonin to N-acetylserotonin (NAS), a distinct biological regulator and the immediate precursor of melatonin, a circulating hormone that influences circadian processes, including sleep. N-terminal sequences of AANAT enzymes vary among vertebrates. Mechanisms that regulate the levels of AANAT are incompletely understood. Previous findings were consistent with the possibility that AANAT may be controlled through its degradation by the N-end rule pathway. By expressing the rat and human AANATs and their mutants not only in mammalian cells but also in the yeast Saccharomyces cerevisiae, and by taking advantage of yeast genetics, we show here that two "complementary" forms of rat AANAT are targeted for degradation by two "complementary" branches of the N-end rule pathway. Specifically, the N(α)-terminally acetylated (Nt-acetylated) Ac-AANAT is destroyed through the recognition of its Nt-acetylated N-terminal Met residue by the Ac/N-end rule pathway, whereas the non-Nt-acetylated AANAT is targeted by the Arg/N-end rule pathway, which recognizes the unacetylated N-terminal Met-Leu sequence of rat AANAT. We also show, by constructing lysine-to-arginine mutants of rat AANAT, that its degradation is mediated by polyubiquitylation of its Lys residue(s). Human AANAT, whose N-terminal sequence differs from that of rodent AANATs, is longer-lived than its rat counterpart and appears to be refractory to degradation by the N-end rule pathway. Together, these and related results indicate both a major involvement of the N-end rule pathway in the control of rodent AANATs and substantial differences in the regulation of rodent and human AANATs that stem from differences in their N-terminal sequences.

Garlic decreases liver and kidney receptor for advanced glycation end products expression in experimental diabetes

The up-regulation of the receptor for advanced glycation end products (RAGE) has been implicated as a major mediator in the development and progression of diabetic nephropathy and hepatic fibrogenesis. The present study was designed to investigate the potential of garlic (Allium sativum L.) to modulate the level of expression of RAGE in renal and hepatic tissues of diabetic rats. Three groups of rats were studied after 8 weeks following diabetes induction: normal, streptozotocin-induced diabetic (control diabetic), and garlic-treated diabetic rats. A polyclonal antibody of proven specificity to RAGE indicated in immunohistochemical assays that RAGE labeling was significantly increased in renal and hepatic tissues of control diabetic rats compared to the normal group. The increased RAGE labeling involved mesangial cells in glomeruli exhibiting signs of mesangial expansion, mesangial nodule formation and glomerulosclerosis. In the liver, a significant up-regulation of RAGE was observed in hepatocytes and bile ducts and vessels in portal tracts. In 2-dimensional Western blots, RAGE expression in both tissues was dominated by heterogeneous charge variants, represented by 46–50kDa isoforms with more basic pIs compared to their counterparts in normal rats. Compared to control diabetic rats, RAGE labeling in the garlic-treated diabetic group was significantly reduced throughout renal and hepatic regions and was marked by the expression of 43–50kDa acidic charge variants comparable to those observed in normal rats. The capacity of garlic to modulate diabetes-induced up-regulation of selective RAGE polymorphic variants may be implicated in attenuating the detrimental consequences of excessive RAGE signaling manifested by diabetes-associated disorders.

Molecular engineering of high affinity single-chain antibody fragment for endothelial targeting of proteins and nanocarriers in rodents and humans

Endothelial cells (EC) represent an important target for pharmacologic intervention, given their central role in a wide variety of human pathophysiologic processes. Studies in lab animal species have established that conjugation of drugs and carriers with antibodies directed to surface targets like the Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, a highly expressed endothelial transmembrane protein) help to achieve specific therapeutic interventions in ECs. To translate such “vascular immunotargeting” to clinical practice, it is necessary to replace antibodies by advanced ligands that are more amenable to use in humans. We report the molecular design of a single chain variable antibody fragment (scFv) that binds with high affinity to human PECAM-1 and cross-reacts with its counterpart in rats and other animal species, allowing parallel testing in vivo and in human endothelial cells in microfluidic model. Site-specific modification of the scFv allows conjugation of protein cargo and liposomes, enabling their endothelial targeting in these models. This study provides a template for molecular engineering of ligands, enabling studies of drug targeting in animal species and subsequent use in humans.

Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies.

The conventional approach of double immunostaining to visualize more than one protein in tissues or cells using antibodies from two different host species is not always feasible due to limitations with antibody availability. Previously reported methodologies for performing multiple immunostains on the same tissue or cells with antibodies originating from the same species are varied in their complexity, sensitivity, and approach to prevent unwanted interactions between antibodies. In the ever-expanding field of macrophage biology, much more is known about mouse and human macrophages than their rat counterparts. The limited availability of validated and well-characterized monoclonal antibodies from different species is one factor responsible for preventing advances in rat macrophage biology. Here we describe an immunostaining method for identifying and examining rat macrophages that is sufficiently sensitive for use in formalin-fixed paraffin-embedded tissue and that uses only commercially available reagents and antibodies. This method can be used to help characterize both physiological and pathophysiological processes in rat macrophages and can be adapted for use with any two antibodies from the same species of origin as long as one of the antibodies is biotinylated.

Tissue-specific expression of histone H3 variants diversified after species separation.

BackgroundThe selective incorporation of appropriate histone variants into chromatin is critical for the regulation of genome function. Although many histone variants have been identified, a complete list has not been compiled.ResultsWe screened mouse, rat and human genomes by in silico hybridization using canonical histone sequences. In the mouse genome, we identified 14 uncharacterized H3 genes, among which 13 are similar to H3.3 and do not have human or rat counterparts, and one is similar to human testis-specific H3 variant, H3T/H3.4, and had a rat paralog. Although some of these genes were previously annotated as pseudogenes, their tissue-specific expression was confirmed by sequencing the 3′-UTR regions of the transcripts. Certain new variants were also detected at the protein level by mass spectrometry. When expressed as GFP-tagged versions in mouse C2C12 cells, some variants were stably incorporated into chromatin and the genome-wide distributions of most variants were similar to that of H3.3. Moreover, forced expression of H3 variants in chromatin resulted in alternate gene expression patterns after cell differentiation.ConclusionsWe comprehensively identified and characterized novel mouse H3 variant genes that encoded highly conserved amino acid sequences compared to known histone H3. We speculated that the diversity of H3 variants acquired after species separation played a role in regulating tissue-specific gene expression in individual species. Their biological relevance and evolutionary aspect involving pseudogene diversification will be addressed by further functional analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0027-3) contains supplementary material, which is available to authorized users.

An invertebrate model for CNS drug discovery: Transcriptomic and functional analysis of a mammalian P-glycoprotein ortholog

BackgroundABC efflux transporters at the blood brain barrier (BBB), namely the P-glycoprotein (P-gp), restrain the development of central nervous system (CNS) drugs. Consequently, early screening of CNS drug candidates is pivotal to identify those affected by efflux activity. Therefore, simple, high-throughput and predictive screening models are required. The grasshopper (locust) has been developed as an invertebrate in situ model for BBB permeability assessment, as it has shown similarities to vertebrate models. MethodsTranscriptome profiling of ABC efflux transporters in the locust brain was performed. Subsequently, identified transcripts were matched with their counterparts in human, rat, mouse and Drosophila melanogaster, based on amino acid sequence similarity, and phylogenetic trees were constructed to reveal the most likely evolutionary history of the proteins. Further, functional characterization of a P-gp ortholog was achieved through transport studies, using a selective P-gp substrate and locust brain in situ, followed by kinetic analyses. ResultsA protein with high sequence similarity to the ABCB1 gene of vertebrates was found in the locust brain, which encodes P-gp in human and is considered the most vital efflux pump. Functionally, this model showed transport kinetic behaviors comparable to those obtained from in vitro models. Particularly, substrate affinity of the putative P-gp was observed as in P-gp expressing cells lines, used for predicting drug penetration across biological barriers. ConclusionFindings suggest a conserved mechanism of brain efflux activity between insects and vertebrates, confirming that this model holds promise for inexpensive and high-throughput screening relative to in vivo models, for CNS drug discovery.