USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling

Protein kinase ALK3/BMPR1A mediates bone morphogenetic protein (BMP) signalling through phosphorylation and activation of SMADs 1/5/8. SMAD6, a transcriptional target of BMP, negatively regulates the BMP pathway by recruiting E3 ubiquitin ligases and targeting ALK3 for ubiquitin-mediated degradation. Here, we identify a deubiquitylating enzyme USP15 as an interactor of SMAD6 and ALK3. We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3. RNAi-mediated depletion of USP15 increases ALK3 K48-linked polyubiquitylation, and reduces both BMP-induced SMAD1 phosphorylation and transcription of BMP target genes. We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation. Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis

Probing the unfolded protein response in long-lived naked mole-rats.

The long-living naked mole-rat (NMR) shows negligible senescence and resistance to age-associated diseases. Recent evidence, based on protein-level assays, suggests that enhanced protein homeostasis machinery contributes to NMR stress-resistance and longevity. Here, we develop NMR-specific, transcriptional assays for measuring the unfolded protein response (UPR), a component of ER proteostasis. By varying doses and response times of pharmacological ER stressors applied to NMR kidney fibroblasts, we probe the NMR UPR in detail, demonstrating that NMR fibroblasts have a higher UPR activation threshold compared to mouse fibroblasts under mild ER-stress induction; whereas temporal analysis reveals that severe ER-stress induction results in no comparative differences. Probing NMR UPR activation with our robust assays may lead to insights into the proteostasis and ageing relationship.Herchel Smith PhD Research Scholarship Rosetrees Trust Gates Cambridge Trust scholarshi

Acid and inflammatory sensitisation of naked mole-rat colonic afferent nerves.

Acid sensing in the gastrointestinal tract is required for gut homeostasis and the detection of tissue acidosis caused by ischaemia, inflammation and infection. In the colorectum, activation of colonic afferents by low pH contributes to visceral hypersensitivity and abdominal pain in human disease including during inflammatory bowel disease. The naked mole-rat (Heterocephalus glaber) shows no pain-related behaviour to subcutaneous acid injection and cutaneous afferents are insensitive to acid, an adaptation thought to be a consequence of the subterranean, likely hypercapnic, environment in which it lives. As such we sought to investigate naked mole-rat interoception within the gastrointestinal tract and how this differed from the mouse (Mus Musculus). Here, we show the presence of calcitonin gene-related peptide expressing extrinsic nerve fibres innervating both mesenteric blood vessels and the myenteric plexi of the smooth muscle layers of the naked mole-rat colorectum. Using ex vivo colonic-nerve electrophysiological recordings, we show differential sensitivity of naked mole-rat, compared to mouse, colonic afferents to acid and the prototypic inflammatory mediator bradykinin, but not direct mechanical stimuli. In naked mole-rat, but not mouse, we observed mechanical hypersensitivity to acid, whilst both species sensitised to bradykinin. Collectively, these findings suggest that naked mole-rat colonic afferents are capable of detecting acidic stimuli; however, their intracellular coupling to downstream molecular effectors of neuronal excitability and mechanotransduction likely differs between species.The authors declare no competing financial interests. This work was supported by Rosetrees Postdoctoral Grant (A1296; JRFH and EStJS), BBSRC grant (BB/R006210/1; JRFH and EStJS), Versus Arthritis Pain Challenge Grant (RG21973; GC and EStJS), AstraZeneca PhD studentshipt (KHB), EMBO Long-Term Fellowship (ALTF1565-2015; ZH) and University of Cambridge Vice Chancellor’s Award (TST)

Single-cell RNAseq reveals seven classes of colonic sensory neuron.

OBJECTIVE: Integration of nutritional, microbial and inflammatory events along the gut-brain axis can alter bowel physiology and organism behaviour. Colonic sensory neurons activate reflex pathways and give rise to conscious sensation, but the diversity and division of function within these neurons is poorly understood. The identification of signalling pathways contributing to visceral sensation is constrained by a paucity of molecular markers. Here we address this by comprehensive transcriptomic profiling and unsupervised clustering of individual mouse colonic sensory neurons. DESIGN: Unbiased single-cell RNA-sequencing was performed on retrogradely traced mouse colonic sensory neurons isolated from both thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglia associated with lumbar splanchnic and pelvic spinal pathways, respectively. Identified neuronal subtypes were validated by single-cell qRT-PCR, immunohistochemistry (IHC) and Ca2+-imaging. RESULTS: Transcriptomic profiling and unsupervised clustering of 314 colonic sensory neurons revealed seven neuronal subtypes. Of these, five neuronal subtypes accounted for 99% of TL neurons, with LS neurons almost exclusively populating the remaining two subtypes. We identify and classify neurons based on novel subtype-specific marker genes using single-cell qRT-PCR and IHC to validate subtypes derived from RNA-sequencing. Lastly, functional Ca2+-imaging was conducted on colonic sensory neurons to demonstrate subtype-selective differential agonist activation. CONCLUSIONS: We identify seven subtypes of colonic sensory neurons using unbiased single-cell RNA-sequencing and confirm translation of patterning to protein expression, describing sensory diversity encompassing all modalities of colonic neuronal sensitivity. These results provide a pathway to molecular interrogation of colonic sensory innervation in health and disease, together with identifying novel targets for drug development

Functional and Molecular Characterization of Mechanoinsensitive "Silent" Nociceptors.

Mechanical and thermal hyperalgesia (pain hypersensitivity) are cardinal signs of inflammation. Although the mechanism underlying thermal hyperalgesia is well understood, the cellular and molecular basis of mechanical hyperalgesia is poorly described. Here, we have identified a subset of peptidergic C-fiber nociceptors that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli when exposed to the inflammatory mediator nerve growth factor (NGF). Strikingly, NGF did not affect mechanosensitivity of other nociceptors. We show that these mechanoinsensitive "silent" nociceptors are characterized by the expression of the nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) and that the mechanically gated ion channel PIEZO2 mediates NGF-induced mechanosensitivity in these neurons. Retrograde tracing revealed that CHRNA3+ nociceptors account for ∼50% of all peptidergic nociceptive afferents innervating visceral organs and deep somatic tissues. Hence, our data suggest that NGF-induced "un-silencing" of CHRNA3+ nociceptors significantly contributes to the development of mechanical hyperalgesia during inflammation

Estrogen receptor-α and progesterone receptor are expressed in label-retaining mammary epithelial cells that divide asymmetrically and retain their template DNA strands

INTRODUCTION: Stem cells of somatic tissues are hypothesized to protect themselves from mutation and cancer risk through a process of selective segregation of their template DNA strands during asymmetric division. Mouse mammary epithelium contains label-retaining epithelial cells that divide asymmetrically and retain their template DNA. METHOD: Immunohistochemistry was used in murine mammary glands that had been labeled with [(3)H]thymidine during allometric growth to investigate the co-expression of DNA label retention and estrogen receptor (ER)-α or progesterone receptor (PR). Using the same methods, we investigated the co-localization of [(3)H]thymidine and ER-α or PR in mammary tissue from mice that had received treatment with estrogen, progesterone, and prolactin subsequent to a long chase period to identify label-retaining cells. RESULTS: Label-retaining epithelial cells (LRECs) comprised approximately 2.0% of the entire mammary epithelium. ER-α-positive and PR-positive cells represented about 30–40% of the LREC subpopulation. Administration of estrogen, progesterone, and prolactin altered the percentage of LRECs expressing ER-α. CONCLUSION: The results presented here support the premise that there is a subpopulation of LRECs in the murine mammary gland that is positive for ER-α and/or PR. This suggests that certain mammary LRECs (potentially stem cells) remain stably positive for these receptors, raising the possibility that LRECs comprise a hierarchy of asymmetrically cycling mammary stem/progenitor cells that are distinguished by the presence or absence of nuclear steroid receptor expression

Edible crabs “Go West”: migrations and incubation cycle of Cancer pagurus revealed by electronic tags

Crustaceans are key components of marine ecosystems which, like other exploited marine taxa, show seasonable patterns of distribution and activity, with consequences for their availability to capture by targeted fisheries. Despite concerns over the sustainability of crab fisheries worldwide, difficulties in observing crabs’ behaviour over their annual cycles, and the timings and durations of reproduction, remain poorly understood. From the release of 128 mature female edible crabs tagged with electronic data storage tags (DSTs), we demonstrate predominantly westward migration in the English Channel. Eastern Channel crabs migrated further than western Channel crabs, while crabs released outside the Channel showed little or no migration. Individual migrations were punctuated by a 7-month hiatus, when crabs remained stationary, coincident with the main period of crab spawning and egg incubation. Incubation commenced earlier in the west, from late October onwards, and brooding locations, determined using tidal geolocation, occurred throughout the species range. With an overall return rate of 34%, our results demonstrate that previous reluctance to tag crabs with relatively high-cost DSTs for fear of loss following moulting is unfounded, and that DSTs can generate precise information with regards life-history metrics that would be unachievable using other conventional means

The material properties of naked mole-rat hyaluronan.

Hyaluronan (HA) is a key component of the extracellular matrix. Given the fundamental role of HA in the cancer resistance of the naked mole-rat (NMR), we undertook to explore the structural and soft matter properties of this species-specific variant, a necessary step for its development as a biomaterial. We examined HA extracted from NMR brain, lung, and skin, as well as that isolated from the medium of immortalised cells. In common with mouse HA, NMR HA forms a range of assemblies corresponding to a wide distribution of molecular weights. However, unique to the NMR, are highly folded structures, whose characteristic morphology is dependent on the tissue type. Skin HA forms tightly packed assemblies that have spring-like mechanical properties in addition to a strong affinity for water. Brain HA forms three dimensional folded structures similar to the macroscopic appearance of the gyri and sulci of the human brain. Lung HA forms an impenetrable mesh of interwoven folds in a morphology that can only be described as resembling a snowman. Unlike HA that is commercially available, NMR HA readily forms robust gels without the need for chemical cross-linking. NMR HA gels sharply transition from viscoelastic to elastic like properties upon dehydration or repeated loading. In addition, NMR HA can form ordered thin films with an underlying semi-crystalline structure. Given the role of HA in maintaining hydration in the skin it is plausible that the folded structures contribute to both the elasticity and youthfulness of NMR skin. It is also possible that such densely folded materials could present a considerable barrier to cell invasion throughout the tissues, a useful characteristic for a biomaterial.This work was supported by a Cancer Research UK/RCUK Multidisciplinary Project Award (C56829/A22053) to K.R., E.S. and D.F. and a Cancer Research UK Career Establishment Award (C47525/A17348) to W.T.K. F.H. and S.C. were supported by Gates Cambridge Trust scholarships

The Space Coronagraph Optical Bench (SCoOB): 1. Design and Assembly of a Vacuum-compatible Coronagraph Testbed for Spaceborne High-Contrast Imaging Technology

The development of spaceborne coronagraphic technology is of paramount importance to the detection of habitable exoplanets in visible light. In space, coronagraphs are able to bypass the limitations imposed by the atmosphere to reach deeper contrasts and detect faint companions close to their host star. To effectively test this technology in a flight-like environment, a high-contrast imaging testbed must be designed for operation in a thermal vacuum (TVAC) chamber. A TVAC-compatible high-contrast imaging testbed is undergoing development at the University of Arizona inspired by a previous mission concept: The Coronagraphic Debris and Exoplanet Exploring Payload (CDEEP). The testbed currently operates at visible wavelengths and features a Boston Micromachines Kilo-C DM for wavefront control. Both a vector vortex coronagraph and a knife-edge Lyot coronagraph operating mode are under test. The optics will be mounted to a 1 x 2 meter pneumatically isolated optical bench designed to operate at 10^-8 torr and achieve raw contrasts of 10^-8 or better. The validation of our optical surface quality, alignment procedure, and first light results are presented. We also report on the status of the testbed's integration in the vaccum chamber.Comment: 14 pages, 9 figure