Acute Downregulation but Not Genetic Ablation of Murine MCU Impairs Suppressive Capacity of Regulatory CD4 T Cells.

By virtue of mitochondrial control of energy production, reactive oxygen species (ROS) generation, and maintenance of Ca2+ homeostasis, mitochondria play an essential role in modulating T cell function. The mitochondrial Ca2+ uniporter (MCU) is the pore-forming unit in the main protein complex mediating mitochondrial Ca2+ uptake. Recently, MCU has been shown to modulate Ca2+ signals at subcellular organellar interfaces, thus fine-tuning NFAT translocation and T cell activation. The mechanisms underlying this modulation and whether MCU has additional T cell subpopulation-specific effects remain elusive. However, mice with germline or tissue-specific ablation of Mcu did not show impaired T cell responses in vitro or in vivo, indicating that 'chronic' loss of MCU can be functionally compensated in lymphocytes. The current work aimed to specifically investigate whether and how MCU influences the suppressive potential of regulatory CD4 T cells (Treg). We show that, in contrast to genetic ablation, acute siRNA-mediated downregulation of Mcu in murine Tregs results in a significant reduction both in mitochondrial Ca2+ uptake and in the suppressive capacity of Tregs, while the ratios of Treg subpopulations and the expression of hallmark transcription factors were not affected. These findings suggest that permanent genetic inactivation of MCU may result in compensatory adaptive mechanisms, masking the effects on the suppressive capacity of Tregs.

Ovulation is triggered by a cyclical modulation of gonadotropes into a hyperexcitable state.

Gonadotropes in the anterior pituitary gland are essential for fertility and provide a functional link between the brain and the gonads. To trigger ovulation, gonadotrope cells release massive amounts of luteinizing hormone (LH). The mechanism underlying this remains unclear. Here, we utilize a mouse model expressing a genetically encoded Ca2+ indicator exclusively in gonadotropes to dissect this mechanism in intact pituitaries. We demonstrate that female gonadotropes exclusively exhibit a state of hyperexcitability during the LH surge, resulting in spontaneous [Ca2+]i transients in these cells, which persist in the absence of any in vivo hormonal signals. L-type Ca2+ channels and transient receptor potential channel A1 (TRPA1) together with intracellular reactive oxygen species (ROS) levels ensure this state of hyperexcitability. Consistent with this, virus-assisted triple knockout of Trpa1 and L-type Ca2+ subunits in gonadotropes leads to vaginal closure in cycling females. Our data provide insight into molecular mechanisms required for ovulation and reproductive success in mammals.

SMS reminders increase on-time vaccination in Aboriginal and Torres Strait Islander infants.

Abstract: Timely immunisation is important to protect children from communicable diseases. However, immunisation uptake in Aboriginal and Torres Strait Islander children under the age of two years is often lower than in non-Indigenous children. This contributes to the gap in health outcomes between Aboriginal and Torres Strait Islander children and non-Indigenous children. We have tested the effectiveness of short message service (SMS) reminders in improving timeliness of childhood immunisation in Aboriginal and Torres Strait Islander infants in regional Queensland, Australia. Reminders were sent to parents of Aboriginal and Torres Strait Islander children, at five immunisation age milestones: six weeks, four months, six months, 12 months, and 18 months. There was a significant improvement in the proportion of children vaccinated on-time (within 30 days of the due date), compared to an earlier age cohort, at all milestones except 12 months. The absolute risk difference (ARD) of on-time vaccination between the two cohorts ranged between 4.7% (95% confidence interval [95% CI]: 1.1-8.2%, at six weeks) and 12.9% (95% CI: 7.4-18.5%, at six months). The likelihood of on-time vaccination (rate ratio, RR) in the intervention group compared to the control group ranged from 1.05 (95% CI: 1.01-1.10, at six weeks) to 1.31 (95% CI: 1.14-1.50, at 18 months). SMS reminders were associated with an improvement in immunisation timeliness in Aboriginal and Torres Strait Islander infants at all age milestones measured except 12 months.

Bitter taste cells in the ventricular walls of the murine brain regulate glucose homeostasis.

The median eminence (ME) is a circumventricular organ at the base of the brain that controls body homeostasis. Tanycytes are its specialized glial cells that constitute the ventricular walls and regulate different physiological states, however individual signaling pathways in these cells are incompletely understood. Here, we identify a functional tanycyte subpopulation that expresses key taste transduction genes including bitter taste receptors, the G protein gustducin and the gustatory ion channel TRPM5 (M5). M5 tanycytes have access to blood-borne cues via processes extended towards diaphragmed endothelial fenestrations in the ME and mediate bidirectional communication between the cerebrospinal fluid and blood. This subpopulation responds to metabolic signals including leptin and other hormonal cues and is transcriptionally reprogrammed upon fasting. Acute M5 tanycyte activation induces insulin secretion and acute diphtheria toxin-mediated M5 tanycyte depletion results in impaired glucose tolerance in diet-induced obese mice. We provide a cellular and molecular framework that defines how bitter taste cells in the ME integrate chemosensation with metabolism.

Intra-pituitary follicle-stimulating hormone signaling regulates hepatic lipid metabolism in mice.

Inter-organ communication is a major hallmark of health and is often orchestrated by hormones released by the anterior pituitary gland. Pituitary gonadotropes secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to regulate gonadal function and control fertility. Whether FSH and LH also act on organs other than the gonads is debated. Here, we find that gonadotrope depletion in adult female mice triggers profound hypogonadism, obesity, glucose intolerance, fatty liver, and bone loss. The absence of sex steroids precipitates these phenotypes, with the notable exception of fatty liver, which results from ovary-independent actions of FSH. We uncover paracrine FSH action on pituitary corticotropes as a mechanism to restrain the production of corticosterone and prevent hepatic steatosis. Our data demonstrate that functional communication of two distinct hormone-secreting cell populations in the pituitary regulates hepatic lipid metabolism.

Using Outcome Trajectory Evaluation to Assess HarvestPlus' Contribution to the Development of National Biofortification Breeding Programs.

Improving policies-broadly defined-is at the heart of the structural transformation agenda. This paper describes the use of a new evaluation method-outcome trajectory evaluation (OTE), based on both evaluation and policy process theory-to explore the influence of HarvestPlus, a large and complex research for development program focused on improving nutrition, on a specific policy outcome, namely the establishment of biofortification crop breeding programs in national agricultural research institutes in Bangladesh, India, and Rwanda. The findings support claims of significant HarvestPlus contributions while also raising issues that need to be monitored to ensure sustainability. The paper also discusses the pros and cons of the OTE approach in terms of methodological rigor and the accumulation of learning from one evaluation to the next.

L'amélioration des politiques, au sens large, est au cœur du programme de transformation structurelle. Cet article décrit l'utilisation d'une nouvelle méthode d'évaluation - l'évaluation de la trajectoire des résultats (outcome trajectory evaluation ou OTE en anglais), basée à la fois sur la théorie de l'évaluation et du processus politique - pour explorer l'influence de HarvestPlus, un vaste et complexe programme de recherche pour le développement axé sur l'amélioration de la nutrition, et orienté vers un résultat politique, à savoir la mise en place de programmes de biofortification et de sélection de cultures dans les instituts nationaux de recherche agricole au Bangladesh, en Inde et au Rwanda. Les résultats appuient les allégations selon lesquelles HarvestPlus permet des contributions importantes, tout en soulevant des problèmes qui doivent être surveillés pour assurer la durabilité. L'article discute également des avantages et des inconvénients de l'approche OTE en termes de rigueur méthodologique et d'accumulation d'apprentissage d'une évaluation à l'autre.

Combining mass spectrometry and genetic labeling in mice to report TRP channel expression.

Transient receptor potential (TRP) ion channels play important roles in fundamental biological processes throughout the body of humans and mice. TRP channel dysfunction manifests in different disease states, therefore, these channels may represent promising therapeutic targets in treating these conditions. Many TRP channels are expressed in several organs suggesting multiple functions and making it challenging to untangle the systemic pathophysiology of TRP dysfunction. Detailed characterization of the expression pattern of the individual TRP channels throughout the organism is thus essential to interpret data such as those derived from systemic phenotyping of global TRP knockout mice. Murine TRP channel reporter strains enable reliable labeling of TRP expression with a fluorescent marker. Here we present an optimized method to visualize primary TRP-expressing cells with single cell resolution throughout the entire organism. In parallel, we methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest. The TRP protein expression data are then correlated with the GFP reporter expression data. The combined methodological approach presented here can be adopted to generate expression data for other genes of interest and reporter mice.•We present an optimized method to systemically characterize gene expression in fluorescent reporter mouse strains with a single cell resolution.•We methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest in mice.

Whole-body analysis of TRPML3 (MCOLN3) expression using a GFP-reporter mouse model reveals widespread expression in secretory cells and endocrine glands.

TRPML3 (mucolipin 3, MCOLN3) is an endolysosomal cation channel belonging to the TRPML subfamily of transient receptor potential channels. Gain-of-function mutations in the Trpml3 gene cause deafness, circling behavior and coat color dilution in mice due to cell death of TRPML3-expressing hair cells of the inner ear or skin melanocytes, respectively. Furthermore, TRPML3 was found to play a role in the long term survival of cochlear hair cells (its absence contributing to presbycusis), in specialized giant lysosomes that neonatal (birth to weaning) enterocytes used for the uptake and digestion of maternal milk nutrients, and in the expulsion of exosome-encased bacteria such as uropathogenic E. coli, infecting bladder epithelial cells. Recently, TRPML3 was found to be expressed at high levels in alveolar macrophages and loss of TRPML3 results in a lung emphysema phenotype, confirmed in two independently engineered Trpml3 knockout lines. TRPML3 is not ubiquitously expressed like its relative TRPML1 and thus cellular expression of TRPML3 on a whole-tissue level remains, with the exceptions mentioned above, largely elusive. To overcome this problem, we generated a τGFP reporter mouse model for TRPML3 and compared expression data obtained from this model by immunofluorescence on tissue sections with immunohistochemistry using TRPML3 antibodies and in situ hybridization. We thus uncovered expression in several organs and distinct cell types. We confirmed TRPML3 expression in both neonatal and adult alveolar macrophages, in melanocytes of hair follicles and glabrous skin, in principle cells of the collecting duct of the neonatal and adult kidney, and in olfactory sensory neurons of the olfactory epithelium, including its fibres protruding to the glomeruli of the olfactory bulb. Additionally, we localized TRPML3 in several glands including parathyroid, thyroid, salivary, adrenal, and pituitary gland, testes and ovaries, suggestive of potential roles for the channel in secretion or uptake of different hormones.

Subunit composition, molecular environment, and activation of native TRPC channels encoded by their interactomes.

In the mammalian brain TRPC channels, a family of Ca2+-permeable cation channels, are involved in a variety of processes from neuronal growth and synapse formation to transmitter release, synaptic transmission and plasticity. The molecular appearance and operation of native TRPC channels, however, remained poorly understood. Here, we used high-resolution proteomics to show that TRPC channels in the rodent brain are macro-molecular complexes of more than 1 MDa in size that result from the co-assembly of the tetrameric channel core with an ensemble of interacting proteins (interactome). The core(s) of TRPC1-, C4-, and C5-containing channels are mostly heteromers with defined stoichiometries for each subtype, whereas TRPC3, C6, and C7 preferentially form homomers. In addition, TRPC1/C4/C5 channels may co-assemble with the metabotropic glutamate receptor mGluR1, thus guaranteeing both specificity and reliability of channel activation via the phospholipase-Ca2+ pathway. Our results unveil the subunit composition of native TRPC channels and resolve the molecular details underlying their activation.

TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann-Pick type C1, and Batten disease.

Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. LSDs manifest often with neurodegenerative symptoms, typically starting in early childhood, and going along with a strongly reduced life expectancy and quality of life. We show here that small molecule activation of the Ca2+ -permeable endolysosomal two-pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with LSDs such as cholesterol or lipofuscin accumulation, or the formation of abnormal vacuoles seen by electron microscopy. Rescue effects by TPC2 activation, which promotes lysosomal exocytosis and autophagy, were assessed in mucolipidosis type IV (MLIV), Niemann-Pick type C1, and Batten disease patient fibroblasts, and in neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. For in vivo proof of concept, we tested TPC2 activation in the MLIV mouse model. In sum, our data suggest that TPC2 is a promising target for the treatment of different types of LSDs, both in vitro and in-vivo.

Bitter taste signaling in tracheal epithelial brush cells elicits innate immune responses to bacterial infection.

Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into first-line defense mechanisms against bacterial infections of the lung.

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice.

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.

Prolactin-sensitive olfactory sensory neurons regulate male preference in female mice by modulating responses to chemosensory cues.

Chemosensory cues detected in the nose need to be integrated with the hormonal status to trigger appropriate behaviors, but the neural circuits linking the olfactory and the endocrine system are insufficiently understood. Here, we characterize olfactory sensory neurons in the murine nose that respond to the pituitary hormone prolactin. Deletion of prolactin receptor in these cells results in impaired detection of social odors and blunts male preference in females. The prolactin-responsive olfactory sensory neurons exhibit a distinctive projection pattern to the brain that is similar across different individuals and express a limited subset of chemosensory receptors. Prolactin modulates the responses within these neurons to discrete chemosensory cues contained in male urine, providing a mechanism by which the hormonal status can be directly linked with distinct olfactory cues to generate appropriate behavioral responses.

A community-based sleep educational intervention for children with autism spectrum disorder.

BACKGROUND: Sleep problems are common in children with autism spectrum disorder (ASD). Sleep education, effective in improving sleep in ASD, may be difficult to access. We determined if community-based pediatric therapists could successfully deliver sleep educational interventions to caregivers of children with ASD. METHODS: A seven-week feasibility study was conducted consisting of 10 children and caregivers. This feasibility study informed the development of a 16-week preliminary effectiveness study, which consisted of 33 children and caregivers. Children, ages 2-12 years, with a clinical diagnosis of autism and caregiver-reported sleep onset delay of 30 min were included. Community therapists underwent comprehensive training in sleep education and then met with caregiver participants to provide sleep education to each family. Semi-structured qualitative interviews were conducted with all families who completed study procedures.In the feasibility and preliminary effectiveness studies, child participants wore an actigraphy watch (at baseline and after sleep education) and caregivers completed the Child Sleep Habits Questionnaire and Family Inventory of Sleep Habits at baseline and after sleep education; the Child Behavior Checklist was also completed by caregivers in the preliminary effectiveness study. RESULTS: Educator fidelity to the manualized curriculum was maintained. Caregivers showed appropriate understanding, comfort, and implementation of the curriculum. Qualitative and quantitative measures, including caregiver surveys and actigraphy, showed improvements in child sleep and behavior. CONCLUSIONS: Community-based therapists can successfully deliver sleep education to families of children with ASD, which has favorable implications for improving access to care in this population.

Comments on the evolution of TRPV6.

It is well known that not all biological findings derived from animals can be directly applied to humans. The TRPV6 protein may serve as an example which highlights these inter-species differences as an example of parallel evolutionary pathways. TRPV6 (and TRPV5) belong to a family of ion channels from the transient receptor potential group but are selectively permeable for Ca2+, in contrast to other members of the family. Sequences with recognizable similarity to TRPV6 can already be found in archaebacteria. These ancient sequences show clear similarity to the ion-conducting pore of TRPV6. Over the course of evolution, the duplication of the TRPV6 gene gave rise to TRPV5. Duplications of the complete genome as well as subsequent loss of genetic material have led to a variety of different TRPV5/6 combinations. In addition, there is an N-terminal extension of the protein in placental animals. This extension causes translation of TRPV6 to be initiated from an ACG codon. Inactivation of one TRPV6 allele can be correlated with alcohol-independent pancreatitis in humans while inactivation of both alleles leads to skeletal dysplasia of newborn babies. The latter effect is not observed in mice, implying that the effects due to perturbations in TRPV6 levels are much more pronounced in humans.

Odontoblast TRPC5 channels signal cold pain in teeth.

Teeth are composed of many tissues, covered by an inflexible and obdurate enamel. Unlike most other tissues, teeth become extremely cold sensitive when inflamed. The mechanisms of this cold sensation are not understood. Here, we clarify the molecular and cellular components of the dental cold sensing system and show that sensory transduction of cold stimuli in teeth requires odontoblasts. TRPC5 is a cold sensor in healthy teeth and, with TRPA1, is sufficient for cold sensing. The odontoblast appears as the direct site of TRPC5 cold transduction and provides a mechanism for prolonged cold sensing via TRPC5's relative sensitivity to intracellular calcium and lack of desensitization. Our data provide concrete functional evidence that equipping odontoblasts with the cold-sensor TRPC5 expands traditional odontoblast functions and renders it a previously unknown integral cellular component of the dental cold sensing system.

Human TRPV6-pathies caused by gene mutations.

The TRP-family of ion channels consists of 27 members in humans. Most TRP channels are non- selective cation channels with the exception of TRPV5 and TRPV6 which exhibit a high permeability for Ca2+ ions. A functional channel is formed by 4 identical subunits [1]. A growing number of mutations are present in human TRPV6 genes which alter channel function and can lead to elevated blood levels of the parathyroid hormone accompanied by transient hyperparathyroidism. Recent publications suggest that TRPV6 mutations could also trigger non-alcoholic chronic pancreatitis. This review summarises the consequences of these mutations within the TRPV6 gene.

TRPC channels regulate Ca2+-signaling and short-term plasticity of fast glutamatergic synapses.

Transient receptor potential (TRP) proteins form Ca2+-permeable, nonselective cation channels, but their role in neuronal Ca2+ homeostasis is elusive. In the present paper, we show that TRPC channels potently regulate synaptic plasticity by changing the presynaptic Ca2+-homeostasis of hippocampal neurons. Specifically, loss of TRPC1/C4/C5 channels decreases basal-evoked secretion, reduces the pool size of readily releasable vesicles, and accelerates synaptic depression during high-frequency stimulation (HFS). In contrast, primary TRPC5 channel-expressing neurons, identified by a novel TRPC5-τ-green fluorescent protein (τGFP) knockin mouse line, show strong short-term enhancement (STE) of synaptic signaling during HFS, indicating a key role of TRPC5 in short-term plasticity. Lentiviral expression of either TRPC1 or TRPC5 turns classic synaptic depression of wild-type neurons into STE, demonstrating that TRPCs are instrumental in regulating synaptic plasticity. Presynaptic Ca2+ imaging shows that TRPC activity strongly boosts synaptic Ca2+ dynamics, showing that TRPC channels provide an additional presynaptic Ca2+ entry pathway, which efficiently regulates synaptic strength and plasticity.

Widespread Cell-Specific Prolactin Receptor Expression in Multiple Murine Organs.

The prolactin receptor (Prlr) mediates not only the multiple effects of prolactin, but also those of the placental lactogens and, in humans, some actions of growth hormone. Although Prlr expression has been reported to be widespread in the body, specific cellular expression patterns within tissues are undefined for many organs. One persisting problem in investigating Prlr function is that the protein is difficult to detect using conventional methods. To allow investigation of Prlr expression with a single cell resolution, we have recently developed a knock-in mouse strain in which Cre recombinase is expressed together with the long isoform of the Prlr using an internal ribosome entry site. When crossed to a Cre-dependent reporter mouse strain, Cre-mediated recombination will genetically label cells that acutely express the Prlr as well as cells that have transiently expressed the Prlr during development. We report here the anatomical distribution of cells which express the fluorescent reporter τ green fluorescent protein in a total of 38 organs prepared from young adult male and female Prlr reporter mice. Our results establish a resource for dissecting the functional role of Prlr in multiple murine tissues.

Do vaccination interventions have effects? A study on how poultry vaccination interventions change smallholder farmer knowledge, attitudes, and practice in villages in Kenya and Tanzania.

Poultry are important for many poor households in developing countries, but there are many constraints to poultry production, including disease. One of the most important diseases of chickens is Newcastle disease (ND). Even though there are effective vaccines against this disease available in most countries, uptake by small-scale poultry keepers is often low. In this study, two areas in Kenya and Tanzania were studied, where some villages had received additional support to get vaccination and other villages had not. In Kenya, 320 households from 10 villages were interviewed, of which half of the villages had active promotion of vaccination through village-based advisors. In Tanzania, 457 households were interviewed, of which 241 came from villages that have had active support through either a project or government extension services. Knowledge about vaccines and the attitudes towards vaccinating against ND was evaluated using mixed multivariable logistic models. Results indicate that in Kenya, the most important determinants for understanding the function of a vaccine were having had support in the village and to have knowledge about ND signs, while in Tanzania gender and previous vaccine use were important in addition to having had support. Attitudes towards vaccination were mainly determined by knowledge, where more knowledge about how vaccines work in general or about ND contributed to more positive attitudes. Among Kenyan farmers that had never used the vaccine before, the amount of birds they lost to disease and predators also influenced attitudes. In conclusion, this study supports the notion that knowledge is a very important component of extension support and that simply making vaccines available may not be sufficient for high levels of uptake.