Maternal hypothyroidism in mice influences glucose metabolism in adult offspring.

AIMS/HYPOTHESIS: During pregnancy, maternal metabolic disease and hormonal imbalance may alter fetal beta cell development and/or proliferation, thus leading to an increased risk for developing type 2 diabetes in adulthood. Although thyroid hormones play an important role in fetal endocrine pancreas development, the impact of maternal hypothyroidism on glucose homeostasis in adult offspring remains poorly understood. METHODS: We investigated this using a mouse model of hypothyroidism, induced by administration of an iodine-deficient diet supplemented with propylthiouracil during gestation. RESULTS: Here, we show that, when fed normal chow, adult mice born to hypothyroid mothers were more glucose-tolerant due to beta cell hyperproliferation (two- to threefold increase in Ki67-positive beta cells) and increased insulin sensitivity. However, following 8 weeks of high-fat feeding, these offspring gained 20% more body weight, became profoundly hyperinsulinaemic (with a 50% increase in fasting insulin concentration), insulin-resistant and glucose-intolerant compared with controls from euthyroid mothers. Furthermore, altered glucose metabolism was maintained in a second generation of animals. CONCLUSIONS/INTERPRETATION: Therefore, gestational hypothyroidism induces long-term alterations in endocrine pancreas function, which may have implications for type 2 diabetes prevention in affected individuals.

Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum.

The parasite Toxoplasma gondii replicates in a specialized intracellular vacuole and causes disease in many species. Protection from toxoplasmosis is mediated by CD8(+) T cells, but the T. gondii antigens and host genes required for eliciting protective immunity are poorly defined. Here we identified GRA6, a polymorphic protein secreted in the parasitophorous vacuole, as the source of the immunodominant and protective decapeptide HF10 presented by the H-2L(d) major histocompatibility complex class I molecule. Presentation of the HF10-H-2L(d) ligand required proteolysis by ERAAP, the endoplasmic reticulum aminopeptidase associated with antigen processing. Consequently, expansion of protective CD8(+) T cell populations was impaired in T. gondii-infected ERAAP-deficient mice, which were more susceptible to toxoplasmosis. Thus, endoplasmic reticulum proteolysis is critical for eliciting protective immunity to a vacuolar parasite.

Dynamics of T cell, antigen-presenting cell, and pathogen interactions during recall responses in the lymph node.

Memory T cells circulate through lymph nodes where they are poised to respond rapidly upon re-exposure to a pathogen; however, the dynamics of memory T cell, antigen-presenting cell, and pathogen interactions during recall responses are largely unknown. We used a mouse model of infection with the intracellular protozoan parasite, Toxoplasma gondii, in conjunction with two-photon microscopy, to address this question. After challenge, memory T cells migrated more rapidly than naive T cells, relocalized toward the subcapsular sinus (SCS) near invaded macrophages, and engaged in prolonged interactions with infected cells. Parasite invasion of T cells occurred by direct transfer of the parasite from the target cell into the T cell and corresponded to an antigen-specific increase in the rate of T cell invasion. Our results provide insight into cellular interactions during recall responses and suggest a mechanism of pathogen subversion of the immune response.

Dynamics of neutrophil migration in lymph nodes during infection.

Although the signals that control neutrophil migration from the blood to sites of infection have been well characterized, little is known about their migration patterns within lymph nodes or the strategies that neutrophils use to find their local sites of action. To address these questions, we used two-photon scanning-laser microscopy to examine neutrophil migration in intact lymph nodes during infection with an intracellular parasite, Toxoplasma gondii. We found that neutrophils formed both small, transient and large, persistent swarms via a coordinated migration pattern. We provided evidence that cooperative action of neutrophils and parasite egress from host cells could trigger swarm formation. Neutrophil swarm formation coincided in space and time with the removal of macrophages that line the subcapsular sinus of the lymph node. Our data provide insights into the cellular mechanisms underlying neutrophil swarming and suggest new roles for neutrophils in shaping immune responses.

Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons.

To maintain homeostasis, hypothalamic neurons in the arcuate nucleus must dynamically sense and integrate a multitude of peripheral signals. Blood-borne molecules must therefore be able to circumvent the tightly sealed vasculature of the blood-brain barrier to rapidly access their target neurons. However, how information encoded by circulating appetite-modifying hormones is conveyed to central hypothalamic neurons remains largely unexplored. Using in vivo multiphoton microscopy together with fluorescently labeled ligands, we demonstrate that circulating ghrelin, a versatile regulator of energy expenditure and feeding behavior, rapidly binds neurons in the vicinity of fenestrated capillaries, and that the number of labeled cell bodies varies with feeding status. Thus, by virtue of its vascular connections, the hypothalamus is able to directly sense peripheral signals, modifying energy status accordingly.

Redistribution of PDGFRß cells and NG2DsRed pericytes at the cerebrovasculature after status epilepticus.

PURPOSE: The role of cerebrovascular dysfunction in seizure disorders is recognized. Blood-brain barrier (BBB) damage in epilepsy has been linked to endothelial and glial pathophysiological changes. Little is known about the involvement of pericytes, a cell type that contributes to BBB function. METHODS: NG2DsRed mice were used to visualize cerebrovascular pericytes. The pattern of vascular and parenchymal distributions of platelet-derived growth factor receptor beta (PDGFRß) cells was evaluated by immunohistochemistry. Status epilepticus was induced in NG2DsRed or C57BL/6J mice by intraperitoneal kainic acid (KA). Animals were perfused intracardially using FITC-Dextran or FITC-Albumin to visualize the cerebrovasculature. Colocalization was performed between NG2DsRed, PDGFRß and microglia IBA-1. Confocal 3D vessel reconstruction was used to visualize changes in cell morphology and position. PDGFRß expression was also evaluated in vitro using organotypic hippocampal cultures (OHC) treated with kainic acid to induce seizure-like activity. Co-localization of PDGFRß with the vascular marker RECA-1 and NG2 was performed. Finally, we assessed the expression of PDGFRß in brain specimens obtained from a cohort of patients affected by drug resistant epilepsy compared to available autoptic brain. RESULTS: In vivo, severe status epilepticus (SE) altered NG2DsRed vascular coverage. We found dishomogenous NG2DsRed perivascular ramifications after SE and compared to control. Concomitantly, PDGFRß(+) cells re-distributed towards the cerebrovasculature after severe SE. Cerebrovascular NG2DsRed partially colocalized with PDGFRß(+) while parenchymal PDGFRß(+) cells did not colocalize with IBA-1(+) microglia. Using in vitro OHC we found decreased NG2 vascular staining and increased PDGFRß(+) ramifications associated with RECA-1(+) microvessels after seizure-like activity. Cellular PDGFRß and NG2(+) colocalization was observed in the parenchyma. Finally, analysis of human TLE brains revealed perivascular and parenchymal PDGFRß(+) cell distributions resembling the murine in vivo and in vitro results. PDGFRß(+) cells at the cerebrovasculature were more frequent in TLE brain tissues as compared to the autoptic control. CONCLUSIONS: The rearrangement of PDGFRß(+) and vascular NG2DsRed cells after SE suggests a possible involvement of pericytes in the cerebrovascular modifications observed in epilepsy. The functional role of vascular-parenchymal PDGFRß(+) cell redistribution and the relevance of a pericyte response to SE remain to be fully elucidated.

Time-division multiplexing for myoelectric closed-loop control using electrotactile feedback.

BACKGROUND: Restoring sensory feedback in myoelectric prostheses is still an open challenge. Closing the loop might lead to a more effective utilization and better integration of these systems into the body scheme of the user. Electrotactile stimulation can be employed to transmit the feedback information to the user, but it represents a strong interference to the recording of the myoelectric signals that are used for control. Time-division multiplexing (TDM) can be applied to avoid this interference by performing the stimulation and recording in dedicated, non-overlapping time windows. METHODS: A closed-loop compensatory tracking task with myocontrol and electrotactile stimulation was used to investigate how the duration of the feedback window (FW) influences the ability to perceive the feedback information and react with an appropriate control action. Nine subjects performed eight trials with continuous recording and contralateral feedback (CONT-CLT) and TDM with ispilateral stimulation and recording using the FW of 40 ms (TDM40), 100 ms (TDM100) and 300 ms (TDM300). The tracking quality was evaluated by comparing the reference and generated trajectories using cross-correlation coefficient (CCCOEF), time delay, root mean square tracking error, and the amount of overshoot. RESULTS: The control performance in CONT-CLT was the best in all the outcome measures. The overall worst performance was obtained using TDM with the shortest FW (TDM40). There was no significant difference between TDM100 and TDM300, and the quality of tracking in these two conditions was high (CCCOEF ~ 0.95). The results demonstrated that FW duration is indeed an important parameter in TDM, which appears to have an optimal value. Among the tested cases, the FW duration of 100 ms seems to be the best trade-off between the quality of perception and a limited command update rate. CONCLUSIONS: This study represents the first systematic evaluation of a TDM-based approach for closing the loop using electrotactile feedback in myoelectric systems. The overall conclusion is that TDM is a feasible and attractive method for closed-loop myocontrol, since it is easy to implement (software-only solution), has limited impact on the performance when using proper FW duration, and might decrease habituation due to burst-like stimulation delivery.

Hi-M: A Multiplex Oligopaint FISH Method to Capture Chromatin Conformations In Situ and Accompanying Open-Source Acquisition Software.

The simultaneous observation of three-dimensional (3D) chromatin structure and transcription in single cells is critical to understand how DNA is organized inside cells and how this organization influences or is affected by other processes, such as transcription. We have recently introduced an innovative technology known as Hi-M, which enables the sequential tagging, 3D visualization, and precise localization of multiple genomic DNA regions alongside RNA expression within individual cells. In this chapter, we present a comprehensive guide outlining the creation of probes, as well as sample preparation and labeling. Finally, we provide a step-by-step guide to conduct a complete Hi-M acquisition using our open-source software package, Qudi-HiM, which controls the robotic microscope handling the entire acquisition procedure.

pyHiM: a new open-source, multi-platform software package for spatial genomics based on multiplexed DNA-FISH imaging.

Genome-wide ensemble sequencing methods improved our understanding of chromatin organization in eukaryotes but lack the ability to capture single-cell heterogeneity and spatial organization. To overcome these limitations, new imaging-based methods have emerged, giving rise to the field of spatial genomics. Here, we present pyHiM, a user-friendly python toolbox specifically designed for the analysis of multiplexed DNA-FISH data and the reconstruction of chromatin traces in individual cells. pyHiM employs a modular architecture, allowing independent execution of analysis steps and customization according to sample specificity and computing resources. pyHiM aims to facilitate the democratization and standardization of spatial genomics analysis.

Multiplexed chromatin imaging reveals predominantly pairwise long-range coordination between Drosophila Polycomb genes.

Polycomb (Pc) group proteins are transcriptional regulators with key roles in development, cell identity, and differentiation. Pc-bound chromatin regions form repressive domains that interact in 3D to assemble repressive nuclear compartments. Here, we use multiplexed chromatin imaging to investigate whether Pc compartments involve the clustering of multiple Pc domains during Drosophila development. Notably, 3D proximity between Pc targets is rare and involves predominantly pairwise interactions. These 3D proximities are particularly enhanced in segments where Pc genes are co-repressed. In addition, segment-specific expression of Hox Pc targets leads to their spatial segregation from Pc-repressed genes. Finally, non-Hox Pc targets are more proximal in regions where they are co-expressed. These results indicate that long-range Pc interactions are temporally and spatially regulated during differentiation and development but do not induce frequent clustering of multiple distant Pc genes.

Actions of a picomolar short-acting S1P1 agonist in S1P1-eGFP knock-in mice.

Sphingosine 1-phosphate receptor 1 (S1P(1)) is critical for lymphocyte recirculation and is a clinical target for treatment of multiple sclerosis. By generating a short-duration S1P(1) agonist and mice in which fluorescently tagged S1P(1) replaces wild-type receptor, we elucidate physiological and agonist-perturbed changes in expression of S1P(1) at a subcellular level in vivo. We demonstrate differential downregulation of S1P(1) on lymphocytes and endothelia after agonist treatment.

SAR analysis of novel non-peptidic NPBWR1 (GPR7) antagonists.

In this Letter we report on the advances in our NPBWR1 antagonist program aimed at optimizing the 5-chloro-2-(3,5-dimethylphenyl)-4-(4-methoxyphenoxy)pyridazin-3(2H)-one lead molecule previously obtained from a high-throughput screening (HTS)-derived hit. Synthesis and structure-activity relationships (SAR) studies around the 3,5-dimethylphenyl and 4-methoxyphenyl regions resulted in the identification of a novel series of non-peptidic submicromolar NPBWR1 antagonists based on a 5-chloro-4-(4-alkoxyphenoxy)-2-(benzyl)pyridazin-3(2H)-one chemotype. Amongst them, 5-chloro-2-(9H-fluoren-9-yl)-4-(4-methoxyphenoxy)pyridazin-3(2H)-one 9h (CYM50769) inhibited NPW activation of NPBWR1 with a submicromolar IC(50), and displayed high selectivity against a broad array of off-targets with pharmaceutical relevance. Our medicinal chemistry study provides innovative non-peptidic selective NPBWR1 antagonists that may enable to clarify the biological role and therapeutic utility of the target receptor in the regulation of feeding behavior, pain, stress, and neuroendocrine function.

Discovery, design and synthesis of a selective S1P(3) receptor allosteric agonist.

Potent and selective S1P3 receptor (S1P3-R) agonists may represent important proof-of-principle tools used to clarify the receptor biological function and assess the therapeutic potential of the S1P3-R in cardiovascular, inflammatory and pulmonary diseases. N,N-Dicyclohexyl-5-propylisoxazole-3-carboxamide was identified by a high-throughput screening of MLSMR library as a promising S1P3-R agonist. Rational chemical modifications of the hit allowed the identification of N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide, a S1P3-R agonist endowed with submicromolar activity and exquisite selectivity over the remaining S1P1,2,4,5-R family members. A combination of ligand competition, site-directed mutagenesis and molecular modeling studies showed that the N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide is an allosteric agonist and binds to the S1P3-R in a manner that does not disrupt the S1P3-R-S1P binding. The lead molecule herein disclosed constitutes a valuable pharmacological tool to explore the molecular basis of the receptor function, and provides the bases for further rational design of more potent and drug-like S1P3-R allosteric agonists.

A sphingosine 1-phosphate receptor 2 selective allosteric agonist.

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.

Pituitary growth hormone network responses are sexually dimorphic and regulated by gonadal steroids in adulthood.

There are well-recognized sex differences in many pituitary endocrine axes, usually thought to be generated by gonadal steroid imprinting of the neuroendocrine hypothalamus. However, the recognition that growth hormone (GH) cells are arranged in functionally organized networks raises the possibility that the responses of the network are different in males and females. We studied this by directly monitoring the calcium responses to an identical GH-releasing hormone (GHRH) stimulus in populations of individual GH cells in slices taken from male and female murine GH-eGFP pituitary glands. We found that the GH cell network responses are sexually dimorphic, with a higher proportion of responding cells in males than in females, correlated with greater GH release from male slices. Repetitive waves of calcium spiking activity were triggered by GHRH in some males, but were never observed in females. This was not due to a permanent difference in the network architecture between male and female mice; rather, the sex difference in the proportions of GH cells responding to GHRH were switched by postpubertal gonadectomy and reversed with hormone replacements, suggesting that the network responses are dynamically regulated in adulthood by gonadal steroids. Thus, the pituitary gland contributes to the sexually dimorphic patterns of GH secretion that play an important role in differences in growth and metabolism between the sexes.

Contributions of 3D chromatin structure to cell-type-specific gene regulation.

Eukaryotic genomes are organized in 3D in a multiscale manner, and different mechanisms acting at each of these scales can contribute to transcriptional regulation. However, the large single-cell variability in 3D chromatin structures represents a challenge to understand how transcription may be differentially regulated between cell types in a robust and efficient manner. Here, we describe the different mechanisms by which 3D chromatin structure was shown to contribute to cell-type-specific transcriptional regulation. Excitingly, several novel methodologies able to measure 3D chromatin conformation and transcription in single cells in their native tissue context, or to detect the dynamics of cis-regulatory interactions, are starting to allow quantitative dissection of chromatin structure noise and relate it to how transcription may be regulated between different cell types and cell states.

Median eminence blood flow influences food intake by regulating ghrelin access to the metabolic brain.

Central integration of peripheral appetite-regulating signals ensures maintenance of energy homeostasis. Thus, plasticity of circulating molecule access to neuronal circuits involved in feeding behavior plays a key role in the adaptive response to metabolic changes. However, the mechanisms involved remain poorly understood despite their relevance for therapeutic development. Here, we investigated the role of median eminence mural cells, including smooth muscle cells and pericytes, in modulating gut hormone effects on orexigenic/anorexigenic circuits. We found that conditional activation of median eminence vascular cells impinged on local blood flow velocity and altered ghrelin-stimulated food intake by delaying ghrelin access to target neurons. Thus, activation of median eminence vascular cells modulates food intake in response to peripheral ghrelin by reducing local blood flow velocity and access to the metabolic brain.

Remote Monitoring of Positive Airway Pressure Data: Challenges, Pitfalls, and Strategies to Consider for Optimal Data Science Applications.

Over recent years, positive airway pressure (PAP) remote monitoring has transformed the management of OSA and produced a large amount of data. Accumulated PAP data provide valuable and objective information regarding patient treatment adherence and efficiency. However, the majority of studies that have analyzed longitudinal PAP remote monitoring have summarized data trajectories in static and simplistic metrics for PAP adherence and the residual apnea-hypopnea index by the use of mean or median values. The aims of this article are to suggest directions for improving data cleaning and processing and to address major concerns for the following data science applications: (1) conditions for residual apnea-hypopnea index reliability, (2) lack of standardization of indicators provided by different PAP models, (3) missing values, and (4) consideration of treatment interruptions. To allow fair comparison among studies and to avoid biases in computation, PAP data processing and management should be conducted rigorously with these points in mind. PAP remote monitoring data contain a wealth of information that currently is underused in the field of sleep research. Improving the quality and standardizing data handling could facilitate data sharing among specialists worldwide and enable artificial intelligence strategies to be applied in the field of sleep apnea.

Identification of lead compounds targeting the cathepsin B-like enzyme of Eimeria tenella.

Cysteine peptidases have been implicated in the development and pathogenesis of Eimeria. We have identified a single-copy cathepsin B-like cysteine peptidase gene in the genome database of Eimeria tenella (EtCatB). Molecular modeling of the predicted protein suggested that it differs significantly from host enzymes and could be a good drug target. EtCatB was expressed and secreted as a soluble, active, glycosylated mature enzyme from Pichia pastoris. Biochemical characterization of the recombinant enzyme confirmed that it is cathepsin B-like. Screening of a focused library against the enzyme identified three inhibitors (a nitrile, a thiosemicarbazone, and an oxazolone) that can be used as leads for novel drug discovery against Eimeria. The oxazolone scaffold is a novel cysteine peptidase inhibitor; it may thus find widespread use.

Discovery, design and synthesis of novel potent and selective sphingosine-1-phosphate 4 receptor (S1P4-R) agonists.

High affinity and selective small molecule agonists of the S1P(4) receptor (S1P(4)-R) may have significant therapeutic utility in diverse disease areas including autoimmune diseases, viral infections and thrombocytopenia. A high-throughput screening (HTS) of the Molecular Libraries-Small Molecule Repository library identified 3-(2-(2,4-dichlorophenoxy)ethoxy)-6-methyl-2-nitropyridine as a moderately potent and selective S1P(4)-R hit agonist. Design, synthesis and systematic structure-activity relationships study of the HTS-derived hit led to the development of novel potent S1P(4)-R agonists exquisitely selective over the remaining S1P(1-3,5)-Rs family members. Remarkably, the molecules herein reported provide novel pharmacological tools to decipher the biological function and assess the therapeutic utility of the S1P(4)-R.