Machine intelligence-guided selection of optimized inhibitor for human immunodeficiency virus (HIV) from natural products.

The human immunodeficiency virus (HIV) connects to the cluster of differentiation (CD4) and any of the entry co-receptors (CCR5 and CXCR4); followed by unloading the viral genome, reverse transcriptase, and integrase enzymes within the host cell. The co-receptors facilitate the entry of virus and vital enzymes, leading to replication and pre-maturation of viral particles within the host. The protease enzyme transforms the immature viral vesicles into the mature virion. The pivotal role of co-receptors and enzymes in homeostasis and growth makes the crucial target for anti-HIV drug discovery, and the availability of X-ray crystal structures is an asset. Here, we used the machine intelligence-driven framework (A-HIOT) to identify and optimize target-based potential hit molecules for five significant protein targets from the ZINC15 database (natural products dataset). Following validation with dynamic motion behavior analysis and molecular dynamics simulation, the optimized hits were evaluated using in silico ADMET filtration. Furthermore, three molecules were screened, optimized, and validated: ZINC00005328058 for CCR5 and protease, ZINC000254014855 for CXCR4 and integrase, and ZINC000000538471 for reverse transcriptase. In clinical trials, the ZINC000254014855 and ZINC000254014855 were passed in primary screens for vif-HIV-1, and we reported the specific receptor as well as interactions. As a result, the validated molecules may be investigated further in experimental studies targeting specific receptors in order to design and synergize an anti-HIV regimen.

The paraventricular thalamus provides a polysynaptic brake on limbic CRF neurons to sex-dependently blunt binge alcohol drinking and avoidance behavior in mice.

Bed nucleus of the stria terminalis (BNST) neurons that synthesize corticotropin-releasing factor (CRF) drive binge alcohol drinking and anxiety. Here, we found that female C57BL/6J mice binge drink more than males and have greater basal BNSTCRF neuron excitability and synaptic excitation. We identified a dense VGLUT2 + synaptic input from the paraventricular thalamus (PVT) that releases glutamate directly onto BNSTCRF neurons but also engages a large BNST interneuron population to ultimately inhibit BNSTCRF neurons, and this polysynaptic PVTVGLUT2-BNSTCRF circuit is more robust in females than males. Chemogenetic inhibition of the PVTBNST projection promoted binge alcohol drinking only in female mice, while activation reduced avoidance behavior in both sexes. Lastly, repeated binge drinking produced a female-like phenotype in the male PVT-BNSTCRF excitatory synapse without altering the function of PVTBNST neurons per se. Our data describe a complex, feedforward inhibitory PVTVGLUT2-BNSTCRF circuit that is sex-dependent in its function, behavioral roles, and alcohol-induced plasticity.

Cre Recombinase Driver Mice Reveal Lineage-Dependent and -Independent Expression of Brs3 in the Mouse Brain.

Bombesin receptor subtype-3 (BRS3) is an orphan receptor that regulates energy homeostasis. We compared Brs3 driver mice with constitutive or inducible Cre recombinase activity. The constitutive BRS3-Cre mice show a reporter signal (Cre-dependent tdTomato) in the adult brain because of lineage tracing in the dentate gyrus, striatal patches, and indusium griseum, in addition to sites previously identified in the inducible BRS3-Cre mice (including hypothalamic and amygdala subregions, and parabrachial nucleus). We detected Brs3 reporter expression in the dentate gyrus at day 23 but not at postnatal day 1 or 5 months of age. Hypothalamic sites expressed reporter at all three time points, and striatal patches expressed Brs3 reporter at 1 day but not 5 months. Parabrachial nucleus Brs3 neurons project to the preoptic area, hypothalamus, amygdala, and thalamus. Both Cre recombinase insertions reduced Brs3 mRNA levels and BRS3 function, causing obesity phenotypes of different severity. These results demonstrate that driver mice should be characterized phenotypically and illustrate the need for knock-in strategies with less effect on the endogenous gene.

Non-viral strategies for delivering genome editing enzymes.

Genome-editing tools such as Cre recombinase (Cre), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein system have revolutionized biomedical research, agriculture, microbial engineering, and therapeutic development. Direct delivery of genome editing enzymes, as opposed to their corresponding DNA and mRNA precursors, is advantageous since they do not require transcription and/or translation. In addition, prolonged overexpression is a problem when delivering viral vector or plasmid DNA which is bypassed when delivering whole proteins. This lowers the risk of insertional mutagenesis and makes for relatively easier manufacturing. However, a major limitation of utilizing genome editing proteins in vivo is their low delivery efficiency, and currently the most successful strategy involves using potentially immunogenic viral vectors. This lack of safe and effective non-viral delivery systems is still a big hurdle for the clinical translation of such enzymes. This review discusses the challenges of non-viral delivery strategies of widely used genome editing enzymes, including Cre recombinase, ZFNs and TALENs, CRISPR/Cas9, and Cas12a (Cpf1) in their protein format and highlights recent innovations of non-viral delivery strategies which have the potential to overcome current delivery limitations and advance the clinical translation of genome editing.

An excitatory ventromedial hypothalamus to paraventricular thalamus circuit that suppresses food intake.

It is well recognized that ventromedial hypothalamus (VMH) serves as a satiety center in the brain. However, the feeding circuit for the VMH regulation of food intake remains to be defined. Here, we combine fiber photometry, chemo/optogenetics, virus-assisted retrograde tracing, ChR2-assisted circuit mapping and behavioral assays to show that selective activation of VMH neurons expressing steroidogenic factor 1 (SF1) rapidly inhibits food intake, VMH SF1 neurons project dense fibers to the paraventricular thalamus (PVT), selective chemo/optogenetic stimulation of the PVT-projecting SF1 neurons or their projections to the PVT inhibits food intake, and chemical genetic inactivation of PVT neurons diminishes SF1 neural inhibition of feeding. We also find that activation of SF1 neurons or their projections to the PVT elicits a flavor aversive effect, and selective optogenetic stimulation of ChR2-expressing SF1 projections to the PVT elicits direct excitatory postsynaptic currents. Together, our data reveal a neural circuit from VMH to PVT that inhibits food intake.

Conditional knockout of leptin receptor in neural stem cells leads to obesity in mice and affects neuronal differentiation in the hypothalamus early after birth.

Leptin, secreted by peripheral adipocytes, binds the leptin receptor (Lepr) in the hypothalamus, thereby contributing to the regulation of satiety and body weight. Lepr is expressed in the embryonic brain as early as embryonic day 12.5. However, the function of Lepr in neural precursor cells in the brain has not been resolved. To address this issue, we crossed the Leprflox/flox mice with each of Shh-Cre mice (Shh, sonic hedgehog) and Nestin (Nes)-Cre mice. We found that deletion of Lepr specifically in nestin-expressing cells led to extreme obesity, but the conditional null of Lepr in Shh-expressing cells had no obvious phenotype. Moreover, the level of leptin-activated pSTAT3 decreased in the anterior and central subregions of the arcuate hypothalamus of Shh-Cre; Leprflox/flox mice compared with the controls. By contrast, in Nes-Cre; Leprflox/flox mice, the level of leptin-activated pSTAT3 decreased in all subregions including the anterior, central, and posterior arcuate hypothalamus as well as the dorsomedial, ventromedial, and median eminence of the hypothalamus, revealing that the extensive lack of Lepr in the differentiated neurons of the hypothalamus in the conditional null mice. Notably, conditional deletion of Lepr in nestin-expressing cells enhanced the differentiation of neural precursor cells into neurons and oligodendroglia but inhibited differentiation into astrocytes early in postnatal development of hypothalamus. Our results suggest that Lepr expression in neural precursor cells is essential for maintaining normal body weight as well as the differentiation of neural precursor cells to the neural/glial fate in the hypothalamus shortly after birth.

[Involvement of GABAergic projection from central amygdaloid nucleus to paraventricular nucleus of hypothalamus in electroacupuncture induced regulation of gastric function in GAD2-Cre mice].

OBJECTIVE: To explore the effect of γ-aminobutyric acid (GABA)ergic neuronal circuit of the central amygdaloid nucleus (CeA) and the paraventricular nucleus of hypothalamus (PVN) on electroacupuncture (EA)-induced regulation of gastric function by way of CeA-PVN projection. METHODS: The present study included 3 parts: 1) C57BL/6 mice were randomly divided into control and EA groups (n=6 in each group). EA was applied to right "Weishu"(BL21, Back-shu point) and "Zhongwan"(CV12, Front-mu point) for 20 min, followed by detecting the expression of c-fos in the CeA and PVN by using immunofluorescence staining; 2) Microinjection of anterograde tracer (rAAV-EF1α-DIO-mcherry-WPRE-pA) into the CeA was conducted in GAD2-Cre mice for confirming the projection of GABAergic neurons from CeA to PVN; 3) GAD2-Cre mice were randomly divided into rAAV-DIO-mcherry (intra-CeA injection of rAAV-EF1α-DIO-mcherry-WPRE-pA), rAAV-DIO-hM3D（Gq）-mcherry(intra-CeA injection of rAAV-EF1α-DIO-hM3D(Gq)-mcherry-WPRE-pA) and rAAV-DIO-hM3D（Gq）-mcherry+EA groups(n=6 in each group). The food intake and gastric empty were detected, and the concentration of GABA in the PVN was assayed by using high performance liquid chromatography on the 28th day after intra-CeA injection. RESULTS: 1) The expression of c-fos in the CeA and PVN was significantly increased in the EA group relevant to the control group(P<0.01), suggesting an activation of neurons in both CeA and PVN after EA. 2) Following CeA injection of rAAV-EF1α-DIO-mcherry-WPRE-pA, the densely expressed virus GABAergic neurons were found in CeA and large number of projection fibers found in the PVN, suggesting a direct connection between CeA and PVN. 3) After activating the GABAergic neurons of CeA, the concentration of GABA in the PVN was obviously increased (P<0.01), the food intake and the gastric empty were considerably decreased relevant to the rAAV-DIO-mcherry group(P<0.01). Following EA intervention，the concentration of GABA in the PVN was obviously decreased(P<0.01), the food intake and the gastric empty were significantly increased relevant to the rAAV-DIO-hM3D（Gq）-mcherry group (P<0.01). CONCLUSION: EA of BL21 and CV12 (Back-shu and Front-mu acupoints) can increase food intake and gastric empty in GAD2-Cre mice, which may be achieved by suppressing the release of GABA in PVN through CeA-PVN GABAergic neural circuit.

Schisandrin B attenuates bleomycin-induced pulmonary fibrosis in mice through the wingless/integrase-1 signaling pathway.

Purpose/Aim: Pulmonary fibrosis (PF) is characterized by the progressive and ultimately fatal accumulation of fibroblasts and extracellular matrix in the lung that distorts its architecture and compromises its function.Objective: The present study investigated the potential protective effects of schisandrin B (Sch B) on the Wingless/Integrase-1 (Wnt) signaling pathway in attenuating inflammation and oxidative stress in ICR mice.Methods: Sixty healthy ICR mice were randomly divided into the following groups: control group, bleomycin (BLM) group, Sch B low dose (Sch B-L) group, Sch B medium dose (Sch B-M) group, Sch B high dose (Sch B-H) group, and dexamethasone (DXM) group. The expression of transforming growth factor (TGF)-ß1 was examined by ELISA. In addition, the levels of superoxide dismutase (SOD), hydroxyproline (HYP), and the total antioxidant capacity (T-AOC) were determined. The protein and mRNA levels of matrix metalloproteinase 7 (MMP7) and ß-catenin in mice were analyzed by western blot and quantitative real -quantitative time PCR (qRT-PCR), respectively.Results: Lung tissues from the BLM group exhibited significantly more inflammatory changes and a significantly greater number of collagen fibers than lung tissues from the control group. In addition, the lung tissues from these BLM-treated mice exhibited slightly increased MMP7 and ß-catenin protein expression. Lung tissues from the Sch B-H group exhibited fewer inflammatory changes and fewer collagen fibers than lung tissues from the BLM group. Furthermore, the lung tissues from the Sch B-H mice exhibited decreased HYP and TGF-ß1 levels, but increased SOD and T-AOC levels.Conclusions: The present study provided evidence that Sch B may be a potential therapeutic agent for the treatment of PF.

Fos-CreER-based genetic mapping of forebrain regions activated by acupuncture.

Acupuncture increasingly is accepted as a potential therapy for many diseases in the Western world. However, the mechanism of acupuncture is not well understood mechanistically. We have established that manual acupuncture (MA) at the Neiguan (P6) acupoint inhibits excitatory cardiovascular reflex responses through modulation of the autonomic nervous system in the brainstem. It is unclear whether P6 MA activates neurons in the brain regions beyond the brainstem. Thus, we mapped P6 specific neural activation by MA in the forebrain using the Fos-CreER; Ai9 mice model, which allows for enhanced sensitivity and efficiency compared to conventional immunohistochemical staining. Compared to sham-MA control without manual stimulation, we find that MA at P6 markedly increases c-Fos positive neurons in a number of the forebrain regions (n = 5 in each group). These activated regions include accumbens nucleus, caudate putamen, claustrum, bed nucleus of the stria terminalis, amygdaloid nucleus, ventral posterior division of the thalamic nucleus, paraventricular hypothalamic nucleus, arcuate hypothalamic nucleus, primary and secondary somatosensory cortex, ectorhinal cortex, and dorsolateral entorhinal cortex. As MA at P6 activates neurons in relatively broad brain networks beyond the brainstem, our data suggest that acupuncture at this acupoint has the potential to influence physiological functions associated with autonomic and non-autonomic nervous systems through its effects on multiple brain regions.

Enfermedad por VIH, Inhibidores de integrasa y el futuro de los controladores elite, revisión bibliográfica / VIH disease, Integrase Strand Transfer Inhibitors and the future of elite controllers: bibliographic review

La Guía Colombiana de práctica clínica para la atención de la infección por VIH / Sida en adolescentes y adultos incluye como primera línea de tratamiento el uso de Inhibidores de integrasa; sin embargo, no incluye recomendaciones que soporten la decisión de tratar a los pacientes controladores elite (CE). La definición de controladores elite es confusa pues varía de un estudio a otro y se desconoce si las recomendaciones de tratamiento, se pueden aplicar a los controladores de forma similar; tampoco existen mecanismos apropiados para el seguimiento sistemático de los controladores elite cuando se inicia en ellos una terapia antirretroviral. Este artículo es una revisión bibliográfica de la información disponible sobre la definición de los pacientes controladores, y los controladores elite, su evolución clinica e inmunológica, el tratamiento y las terapias disponibles en Colombia.

The Colombian Guide to Clinical Practice for HIV / AIDS Care in Adolescents and Adults, includes as first line of treatment the use of integrase inhibitors; however, there is no information to support the decision to treat elite control patients (EC). The definition of elite controller is confusing, because of the changes in definitions between studies, and it is unknown whether these recommendations apply to these patients in a similar way; and how should be systematic follow-up of elite controllers when antiretroviral therapy is initiated. Present paper is a bibliographic review of the available information on the definition of the controllers, and elite controllers its clinical and immunological evolution, the treatment and therapies available in Colombia.

Hierarchical organization of cortical and thalamic connectivity.

The mammalian cortex is a laminar structure containing many areas and cell types that are densely interconnected in complex ways, and for which generalizable principles of organization remain mostly unknown. Here we describe a major expansion of the Allen Mouse Brain Connectivity Atlas resource1, involving around a thousand new tracer experiments in the cortex and its main satellite structure, the thalamus. We used Cre driver lines (mice expressing Cre recombinase) to comprehensively and selectively label brain-wide connections by layer and class of projection neuron. Through observations of axon termination patterns, we have derived a set of generalized anatomical rules to describe corticocortical, thalamocortical and corticothalamic projections. We have built a model to assign connection patterns between areas as either feedforward or feedback, and generated testable predictions of hierarchical positions for individual cortical and thalamic areas and for cortical network modules. Our results show that cell-class-specific connections are organized in a shallow hierarchy within the mouse corticothalamic network.

The effect of acute or repeated stress on the corticotropin releasing factor system in the CRH-IRES-Cre mouse: A validation study.

Corticotropin releasing factor (CRF) is a key component of stress responsivity, modulating related behaviors including anxiety and reward. Difficulties identifying CRF neurons, using traditional approaches including immunohistochemistry, has led to the development of a number of transgenic CRF reporter mice. The Crh-IRES-Cre::Ai14 (tdTomato) reporter mouse is increasing in popularity as a useful tool to assess the localization, connectivity and function of CRF neurons in various stress-related behaviors. However, without proper characterization of reporter expression, the in vivo and in vitro manifestations resulting from the manipulation of these cells must be interpreted with caution. Here we mapped the distribution of tdTomato-expressing CRF cells throughout the rostro-caudal extent of the Crh-IRES-Cre::Ai14 mouse brain. To determine if reporter expression faithfully reproduced native CRF expression, we assessed the colocalization of CRF expression with tdTomato reporter expression across several brain regions. Good concordance was observed in the extended amygdala and paraventricular nucleus of the hypothalamus (PVN), while discrepancies were observed within the lateral hypothalamus and hippocampus. Finally, we examined the activation of CRF neurons in Crh-IRES-Cre::Ai14 mice in response to different types of stressors using Fos immunohistochemistry. Acute psychological (swim) and pharmacological (yohimbine) stress stimulated Fos-protein expression in PVN CRF neurons. Interestingly though, exposure to four daily restraint stress sessions followed by a novel acute stressor did not further recruit CRF neurons across any brain region examined. Our results highlight the importance of thoroughly characterizing reporter mice before use and suggest that acute versus repeated stress may differentially impact the CRF system. This article is part of the Special Issue entitled 'Hypothalamic Control of Homeostasis'.

A CreER mouse to study melanin concentrating hormone signaling in the developing brain.

The neuropeptide, melanin concentrating hormone (MCH), and its G protein-coupled receptor, melanin concentrating hormone receptor 1 (Mchr1), are expressed centrally in adult rodents. MCH signaling has been implicated in diverse behaviors such as feeding, sleep, anxiety, as well as addiction and reward. While a model utilizing the Mchr1 promoter to drive constitutive expression of Cre recombinase (Mchr1-Cre) exists, there is a need for an inducible Mchr1-Cre to determine the roles for this signaling pathway in neural development and adult neuronal function. Here, we generated a BAC transgenic mouse where the Mchr1 promotor drives expression of tamoxifen inducible CreER recombinase. Many aspects of the Mchr1-Cre expression pattern are recapitulated by the Mchr1-CreER model, though there are also notable differences. Most strikingly, compared to the constitutive model, the new Mchr1-CreER model shows strong expression in adult animals in hypothalamic brain regions involved in feeding behavior but diminished expression in regions involved in reward, such as the nucleus accumbens. The inducible Mchr1-CreER allele will help reveal the potential for Mchr1 signaling to impact neural development and subsequent behavioral phenotypes, as well as contribute to the understanding of the MCH signaling pathway in terminally differentiated adult neurons and the diverse behaviors that it influences.

In vivo clonal analysis reveals spatiotemporal regulation of thalamic nucleogenesis.

The thalamus, a crucial regulator of cortical functions, is composed of many nuclei arranged in a spatially complex pattern. Thalamic neurogenesis occurs over a short period during mammalian embryonic development. These features have hampered the effort to understand how regionalization, cell divisions, and fate specification are coordinated and produce a wide array of nuclei that exhibit distinct patterns of gene expression and functions. Here, we performed in vivo clonal analysis to track the divisions of individual progenitor cells and spatial allocation of their progeny in the developing mouse thalamus. Quantitative analysis of clone compositions revealed evidence for sequential generation of distinct sets of thalamic nuclei based on the location of the founder progenitor cells. Furthermore, we identified intermediate progenitor cells that produced neurons populating more than one thalamic nuclei, indicating a prolonged specification of nuclear fate. Our study reveals an organizational principle that governs the spatial and temporal progression of cell divisions and fate specification and provides a framework for studying cellular heterogeneity and connectivity in the mammalian thalamus.

Autocrine actions of prolactin contribute to the regulation of lactotroph function in vivo.

Prolactin (PRL), whose principal role is regulation of lactation, is mainly synthesized and secreted by lactotroph anterior pituitary cells. Its signaling is exerted via a transmembrane PRL receptor (PRLR) expressed in a wide variety of tissues, including the anterior pituitary. Dopamine, which is secreted by tuberoinfundibular hypothalamic neurons, is the major inhibitory regulator of prolactin secretion. Although PRL is well established to stimulate hypothalamic dopamine secretion, thereby exerting a negative feedback regulation on its own release, autocrine or paracrine actions of PRL on lactotroph cells have also been suggested. Within the pituitary, PRL may inhibit both lactotroph proliferation and secretion, but in vivo evaluation of these putative functions is limited. To determine whether the autocrine actions of prolactin have a significant role in the physiologic function of lactotrophs in vivo, we examined the consequences of conditional deletion of Prlr in lactotroph cells using a novel mouse line with loxP sites flanking the Prlr gene ( Prlrlox/lox) and Cre-recombinase (Cre) expressed under the control of the pituitary-specific Prl promoter. Prlrlox/lox/Prl-Cre mice have normal PRL levels and did not develop any pituitary lactotroph adenoma, even at 20 mo of age. Nevertheless, Prlrlox/lox/Prl-Cre mice displayed an increased dopaminergic inhibitory tone compared with control Prlrlox/lox mice. These results elegantly confirm an autocrine/paracrine feedback of PRL on lactotroph cells in vivo, which can be fully compensated by an intact hypothalamic feedback system.-Bernard, V., Lamothe, S., Beau, I., Guillou, A., Martin, A., Le Tissier, P., Grattan, D., Young, J., Binart, N. Autocrine actions of prolactin contribute to the regulation of lactotroph function in vivo.

Establishment of Cre-mediated HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral screening assays.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), existing in hepatocyte nuclei as a stable minichromosome, plays a central role in the life cycle of the virus and permits the persistence of infection. Despite being essential for HBV infection, little is known about the molecular mechanisms of cccDNA formation, regulation and degradation, and there is no therapeutic agents directly targeting cccDNA, fore mostly due to the lack of robust, reliable and quantifiable HBV cccDNA models. In this study, combined the Cre/loxP and sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2-60 copies of monomeric HBV genome flanked by loxP sites (HepG2-HBV/loxP). After Cre expression via adenoviral transduction, 3.3-kb recombinant cccDNA (rcccDNA) bearing a chimeric intron can be produced in the nuclei of these HepG2-HBV/loxP cells. The rcccDNA could be accurately quantified by quantitative PCR using specific primers and cccDNA pool generated in this model could be easily detected by Southern blotting using the digoxigenin probe system. We demonstrated that the rcccDNA was epigenetically organized as the natural minichromosome and served as the template supporting pgRNA transcription and viral replication. As the expression of HBV S antigen (HBsAg) is dependent on the newly generated cccDNA, HBsAg is the surrogate marker of cccDNA. Additionally, the efficacies of 3 classes of anti-HBV agents were evaluated in HepG2-HBV/loxP cells and antiviral activities with different mechanisms were confirmed. These data collectively suggested that HepG2-HBV/loxP cell system will be powerful platform for studying cccDNA related biological mechanisms and developing novel cccDNA targeting drugs.

Cre-expressing neurons in visual cortex of Ntsr1-Cre GN220 mice are corticothalamic and are depolarized by acetylcholine.

The Ntsr1-Cre GN220 mouse expresses Cre-recombinase in corticothalamic (CT) neurons in neocortical layer 6. It is not known if the other major types of pyramidal neurons in this layer also express this enzyme. By electrophysiological recordings in slices and histological analysis of the uptake of retrogradely transported beads we show that Cre-positive neurons are CT and not corticocortical or corticoclaustral types. Furthermore, we show that Ntsr1-Cre-positive cells are immuno-positive for the nuclear transcription factor Forkhead box protein P2 (FoxP2). We conclude that Cre-expression is limited to a specific type of pyramidal neuron: CT. However, it appears as not all CT neurons are Cre-expressing; there are indications that the penetrance of the gene is about 90%. We demonstrate the utility of assigning a specific identity to individual neurons by determining that the CT neurons are potently modulated by acetylcholine acting on both nicotinic and muscarinic acetylcholine receptors. These results corroborate the suggested function of these neurons in regulating the gain of thalamocortical transfer of sensory information depending on attentional demand and state of arousal.

A shortened tamoxifen induction scheme to induce CreER recombinase without side effects on the male mouse skeleton.

The selective estrogen receptor modulator tamoxifen exerts estrogen agonistic or antagonistic actions on several tissues, including bone. The off-target effects of tamoxifen are one of the most widely recognized pitfalls of tamoxifen-inducible Cre recombinases (CreERs), potentially confounding the phenotypic findings. Still, the validation of tamoxifen induction schemes that minimize the side effects of the drug has not been addressed. Here, we compared the side effects on the skeleton and other androgen-responsive targets of a shortened tamoxifen regimen (2 doses of 190 mg/kg body weight by oral gavage) to a standard protocol (4 doses) and determined their efficiency in inducing CreER-mediated gene deletion. In addition, both a vehicle- and a 10-dose group, which served as a positive control for tamoxifen side effects, were also included. For this purpose, we generated male mice with a floxed androgen receptor (AR) and a neuron-specifically expressed CreER. Treatment with two doses of tamoxifen was the only regimen that did not diminish androgenic bioactivity, as assessed by both seminal vesicles and levator ani/bulbocavernosus muscle weights and serum testosterone concentrations. Similarly, trabecular and cortical femoral bone structure were dramatically altered by both the standard and high-dose protocols but not by the shortened version. Serum osteocalcin and bone-gene expression analyses confirmed the absence of effects on bone by 2 doses of tamoxifen. This protocol decreased AR mRNA levels efficiently and specifically in the nervous system. Thus, we optimized a protocol for tamoxifen-induced CreER gene deletion in mice without off-target effects on bone and male reproductive organs.

Chemical, biological, and DNA markers for tracing slaughterhouse effluent.

Agricultural practices, if not managed correctly, can have a negative impact on receiving environments via waste disposal and discharge. In this study, a chicken slaughter facility on the rural outskirts of Sydney, Australia, has been identified as a possible source of persistent effluent discharge into a peri-urban catchment. Questions surrounding the facility's environmental management practices go back more than four decades. Despite there having never been a definitive determination of the facility's impact on local stream water quality, the New South Wales Environment Protection Authority (NSW EPA) has implemented numerous pollution reduction requirements to manage noise and water pollution at the slaughter facility. However, assessment of compliance remains complicated by potential additional sources of pollution in the catchment. To unravel this long-standing conundrum related to water pollution we apply a forensic, multiple lines of evidence approach to delineate the origin of the likely pollution source(s). Water samples collected between 2014 and 2016 from irrigation pipes and a watercourse exiting the slaughter facility had elevated concentrations of ammonia (max: 63,000µg/L), nitrogen (max: 67,000µg/L) and phosphorus (max: 39,000µg/L), which were significantly higher than samples from adjacent streams that did not receive direct runoff from the facility. Arsenic, sometimes utilised in growth promoting compounds, was detected in water discharging from the facility up to ~4 times (max 3.84µg/L) local background values (<0.5µg/L), with inorganic As(∑V+III) being the dominant species. The spatial association of elevated water pollution to the facility could not unequivocally distinguish a source and consequently DNA analysis of a suspected pollution discharge event was undertaken. Analysis of catchment runoff from several local streams showed that only water sampled at the downstream boundary of the facility tested positive for chicken DNA, with traces of duck DNA being absent, which was a potential confounder given that wild ducks are present in the area. Further, PCR analysis showed that only the discharge water emanating from the slaughter facility tested positive for a generalized marker of anthropogenic pollution, the clinical class 1 integron-integrase gene. The environmental data collected over a three-year period demonstrates that the slaughter facility is indisputably the primary source of water-borne pollution in the catchment. Moreover, application of DNA and PCR for confirming pollution sources demonstrates its potential for application by regulators in fingerprinting pollution sources.

Cholinergic neurons in the dorsomedial hypothalamus regulate food intake.

OBJECTIVE: Central cholinergic neural circuits play a role in the regulation of feeding behavior. The dorsomedial hypothalamus (DMH) is considered the appetite-stimulating center and contains cholinergic neurons. Here, we study the role of DMH cholinergic neurons in the control of food intake. METHODS: To selectively stimulate DMH cholinergic neurons, we expressed stimulatory designer receptors exclusively activated by designer drugs (DREADDs) and channelrhodopsins in DMH cholinergic neurons by injection of adeno-associated virus (AAV) vectors into the DMH of choline acetyltransferase (ChAT)-IRES-Cre mice. We also generated transgenic mice expressing channelrhodopsins in cholinergic neurons with the Cre-LoxP technique. To delete the Chat gene exclusively in the DMH, we injected an AAV carrying a Cre recombinase transgene into the DMH of floxed ChAT mice. Food intake was measured with and without selective stimulation of DMH cholinergic neurons. RESULTS: Mice lacking the Chat gene in the DMH show reduced body weight as compared to control. Chemogenetic activation of DMH cholinergic neurons promotes food intake. This orexigenic effect is further supported by experiments of optogenetic stimulation of DMH cholinergic neurons. DMH cholinergic neurons innervate pro-opiomelanocortin neurons in the arcuate nucleus of the hypothalamus (ARC). Treatment with acetylcholine (ACh) enhances GABAergic inhibitory transmission to ARC POMC neurons that is blocked by the muscarinic receptor antagonist. Direct activation of cholinergic fibers in the ARC readily stimulates food intake that is also abolished by the muscarinic receptor antagonist. CONCLUSION: ACh released from DMH cholinergic neurons regulates food intake and body weight. This effect is mediated in part through regulation of ARC POMC neurons. Activation of muscarinic receptors on GABAergic axon terminals enhances inhibitory tone to ARC POMC neurons. Hence, this novel DMHACh â†’ ARCPOMC pathway plays an important role in the control of food intake and body weight.