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Bimolecular fluorescence complementation (BiFC) methodology uses split fluorescent proteins to detect interactions between proteins in living cells. To date, BiFC has been used to investigate receptor dimerization by splitting the fluorescent protein between the intracellular portions of different receptor components. We reasoned that attaching these split proteins to the extracellular N-terminus instead may improve the flexibility of this methodology and reduce the likelihood of impaired intracellular signal transduction. As a proof-of-concept, we used receptors for calcitonin gene-related peptide, which comprise heterodimers of either the calcitonin or calcitonin receptor-like receptor in complex with an accessory protein (receptor activity-modifying protein 1). We created fusion constructs in which split mVenus fragments were attached to either the C-termini or N-termini of receptor subunits. The resulting constructs were transfected into Cos7 and HEK293S cells, where we measured cAMP production in response to ligand stimulation, cell surface expression of receptor complexes, and BiFC fluorescence. Additionally, we investigated ligand-dependent internalization in HEK293S cells. We found N-terminal fusions were better tolerated with regards to cAMP signaling and receptor internalization. N-terminal fusions also allowed reconstitution of functional fluorescent mVenus proteins; however, fluorescence yields were lower than with C-terminal fusion. Our results suggest that BiFC methodologies can be applied to the receptor N-terminus, thereby increasing the flexibility of this approach, and enabling further insights into receptor dimerization., (© 2024 The Author(s).)

Type 2 diabetes (T2D) and obesity are prevalent metabolic disorders affecting millions of individuals worldwide. A new effective therapeutic drug called tirzepatide for the treatment of obesity and T2D is a dual agonist of the GIP receptor and GLP-1 receptor. Tirzepatide is clinically more effective than GLP-1 receptor agonists but the reasons why are not well understood. Tirzepatide reportedly stimulates the GIP receptor more potently than the GLP-1 receptor. However, tirzepatide signaling has not been thoroughly investigated at the E354 (wildtype) or Q354 (E354Q) GIP receptor variants. The E354Q variant is associated increased risk of T2D and lower body mass index. To better understand GIP receptor signaling we characterized the activity of endogenous agonists and tirzepatide at both GIP receptor variants. Using Cos7 cells we examined wildtype and E354Q GIP receptor signaling, analyzing cAMP and IP 1 accumulation as well as AKT, ERK1/2 and CREB phosphorylation. GIP(1-42) and GIP(1-30)NH 2 displayed equipotent effects on these pathways excluding CREB phosphorylation where GIP(1-30)NH 2 was more potent than GIP(1-42) at the E354Q GIP receptor. Tirzepatide favored cAMP signaling at both variants. These findings indicate that tirzepatide is a biased agonist towards Gα s signaling and suggests it equally activates the wildtype and E354Q GIP receptor variants. We also observed differences between the pharmacology of the GIP receptor variants with endogenous peptides, which may help to explain differences in phenotype. These findings contribute to a comprehensive understanding of GIP receptor signaling, and will aid development of therapies combating T2D and obesity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rees, Buttle, Tasma, Yang, Harris and Walker.)

Humans exhibit sex differences in the prevalence of many neurodevelopmental disorders and neurodegenerative diseases. Here, we generated one of the largest multi-brain-region bulk transcriptional datasets for the rhesus macaque and characterized sex-biased gene expression patterns to investigate the translatability of this species for sex-biased neurological conditions. We identify patterns similar to those in humans, which are associated with overlapping regulatory mechanisms, biological processes, and genes implicated in sex-biased human disorders, including autism. We also show that sex-biased genes exhibit greater genetic variance for expression and more tissue-specific expression patterns, which may facilitate rapid evolution of sex-biased genes. Our findings provide insights into the biological mechanisms underlying sex-biased disease and support the rhesus macaque model for the translational study of these conditions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The impact of COVID-19 has been far-reaching, and the field of neurology is no exception. Due to the long-hauler effect, a variety of chronic health consequences have occurred for some post-COVID patients. A subset of these long-hauler patients experienced symptoms of autonomic dysfunction and tested positive for postural orthostatic tachycardia syndrome (POTS) via autonomic testing., Methods: We conducted a chart review of a convenience sample from patients seen by neurologists at our tertiary care center for suspicion of post-COVID POTS. Patients included in our study had clearly defined POTS based on clinical criteria and positive tilt table test, were 81.25% female, and had an average age of approximately 36. Out of 16 patients, 12 had a confirmed positive COVID test result, with the remaining 4 having strong clinical suspicion for COVID infection. Our analysis examined the most bothersome 3 symptoms affecting each patient per the neurologist's note at their initial visit for post-COVID POTS, clinical presentation, comorbidities, neurological exam findings, autonomic testing results, and COMPASS-31 autonomic questionnaire and PROMIS fatigue survey results., Results: Palpitations (68.75%) and fatigue (62.5%) were the most common of the impactful symptoms reported by patients in their initial Cleveland Clinic neurology visit. The most frequent comorbidities in our sample were chronic migraines (37.5%), irritable bowel syndrome (IBS) (18.75%), and Raynaud's (18.75%). Neurological exam findings and autonomic testing results other than tilt table yielded variable findings without clear trends. Survey results showed substantial autonomic symptom burden (COMPASS-31 autonomic questionnaire average score 44.45) and high levels of fatigue (PROMIS fatigue survey average score 64.64) in post-COVID POTS patients., Conclusion: Our sample of post-COVID POTS patients are similar to the diagnosed POTS general population including in comorbidities and autonomic testing. Fatigue was identified by patients as a common and debilitating symptom. We hope that our study will be an early step toward further investigation of post-COVID POTS with focus on the trends identified in this chart review., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cantrell, Reid, Walker, Stallkamp Tidd, Zhang and Wilson.)

Background: The upper cervical dorsal root ganglia (DRG) are important for the transmission of sensory information associated with the back of the head and neck, contributing to head pain. Calcitonin receptor (CTR)-based receptors, such as the amylin 1 (AMY 1 ) receptor, and ligands, calcitonin gene-related peptide (CGRP) and amylin, have been linked to migraine and pain. However, the contribution of this system to nociception involving the cervical DRG is unclear. Therefore, this study aimed to determine the relative distribution of the CTR, CGRP, and amylin in upper cervical DRG., Methods: CTR, CGRP, and amylin immunofluorescence was examined relative to neural markers in C1/2 DRG from male and female mice, rats, and human cases. Immunofluorescence was supported by RNA-fluorescence in situ hybridization examining amylin mRNA distribution in rat DRG., Results: Amylin immunofluorescence was observed in neuronal soma and fibres. Amylin mRNA (Iapp) was also detected. Amylin and CGRP co-expression was observed in 19% (mouse), 17% (rat), and 36% (human) of DRG neurons in distinct vesicle-like neuronal puncta from one another. CTR immunoreactivity was present in DRG neurons, and both peptides produced receptor signalling in primary DRG cell cultures. CTR-positive neurons frequently co-expressed amylin and/or CGRP (66% rat; 84% human), with some sex differences., Conclusions: Amylin and CGRP could both be local peptide agonists for CTR-based receptors in upper cervical DRG, potentially acting through autocrine and/or paracrine signalling mechanisms to modulate neuron function. Amylin and its receptors could represent novel pain targets., (© 2024. The Author(s).)

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.16177. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

Inflammation is associated with the pathogenesis of myelodysplastic syndromes (MDS) and emerging evidence suggests that MDS hematopoietic stem and progenitor cells (HSPC) exhibit an altered response to inflammation. Deletion of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. Although this MDS subtype contains several haploinsufficient genes that impact innate immune signaling, the effects of inflammation on del(5q) MDS HSPC remains undefined. Utilizing a model of del(5q)-like MDS, inhibiting the IRAK1/4-TRAF6 axis improved cytopenias, suggesting that activation of innate immune pathways contributes to certain clinical features underlying the pathogenesis of low-risk MDS. However, low-grade inflammation in the del(5q)-like MDS model did not contribute to more severe disease but instead impaired the del(5q)-like HSPC as indicated by their diminished numbers, premature attrition and increased p53 expression. Del(5q)-like HSPC exposed to inflammation became less quiescent, but without affecting cell viability. Unexpectedly, the reduced cellular quiescence of del(5q) HSPC exposed to inflammation was restored by p53 deletion. These findings uncovered that inflammation confers a competitive advantage of functionally defective del(5q) HSPC upon loss of p53. Since TP53 mutations are enriched in del(5q) AML following an MDS diagnosis, increased p53 activation in del(5q) MDS HSPC due to inflammation may create a selective pressure for genetic inactivation of p53 or expansion of a pre-existing TP53-mutant clone.

End-stage kidney disease (ESKD) affects over 780,000 Americans and is associated with excess morbidity and premature death. Kidney disease health disparities are well-recognized, manifesting as ESKD overburden among racial and ethnic minority populations. Specifically, Black and Hispanic individuals have a 3.4-fold and 1.3-fold greater life risk of developing ESKD than their white counterparts. There is compelling evidence that communities of color have less opportunity to benefit from kidney-specific care throughout the course of their disease, from pre-ESKD, to ESKD home therapies and kidney transplantation. These healthcare inequities have the combined devastating impact of worse outcomes and quality of life for patients and families at a significant financial cost on the healthcare system. In the last three years, across two presidential administrations, bold, broad initiatives have been outlined that, together could lead to significant transformation in kidney health. The Advancing American Kidney Health (AAKH) initiative was established as a national framework to revolutionize kidney care but did not address health equity. More recently, the Advancing Racial Equity executive order was announced, outlining initiatives to promote equity for historically underserved communities. Building from these presidential directives, we outline strategies to address the complex issue of kidney health disparities, focusing on patient awareness, care delivery, scientific advancement, and workforce initiatives. An equity-focused framework will guide policy advancements to reduce the kidney disease burden in susceptible populations and positively impact the health and well-being of all Americans., Competing Interests: Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://cdt.amegroups.com/article/view/10.21037/cdt-22-566/coif). The series “Endovascular and Surgical Interventions in the End Stage Renal Disease Population” was commissioned by the editorial office without any funding or sponsorship. CAG is a council member of the American Society of Nephrology, speaker to COVID-19 for NIDDK and receives funding support from the National Institutes of Health and NIDDK. The authors have no other conflicts of interest to declare., (2023 Cardiovascular Diagnosis and Therapy. All rights reserved.)

We read with great interest the recent article by Yoo and colleagues [...].

Calcitonin gene-related peptide (CGRP) is a key component of migraine pathophysiology, yielding effective migraine therapeutics. CGRP receptors contain a core accessory protein subunit: receptor activity-modifying protein 1 (RAMP1). Understanding of RAMP1 expression is incomplete, partly due to the challenges in identifying specific and validated antibody tools. We profiled antibodies for immunodetection of RAMP1 using Western blotting, immunocytochemistry and immunohistochemistry, including using RAMP1 knockout mouse tissue. Most antibodies could detect RAMP1 in Western blotting and immunocytochemistry using transfected cells. Two antibodies (844, ab256575) could detect a RAMP1-like band in Western blots of rodent brain but not RAMP1 knockout mice. However, cross-reactivity with other proteins was evident for all antibodies. This cross-reactivity prevented clear conclusions about RAMP1 anatomical localization, as each antibody detected a distinct pattern of immunoreactivity in rodent brain. We cannot confidently attribute immunoreactivity produced by RAMP1 antibodies (including 844) to the presence of RAMP1 protein in immunohistochemical applications in brain tissue. RAMP1 expression in brain and other tissues therefore needs to be revisited using RAMP1 antibodies that have been comprehensively validated using multiple strategies to establish multiple lines of convincing evidence. As RAMP1 is important for other GPCR/ligand pairings, our results have broader significance beyond the CGRP field.

The related peptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) have diverse biological functions in peripheral tissues and the central nervous system. Therefore, these peptides and their three receptors represent potential drug targets for several conditions, including neurological and pain-related disorders. However, very little is known about how these peptides regulate their receptors through processes such as internalization. Therefore, we developed tools to study receptor regulation through the synthesis of fluorescently labeled analogues of PACAP-38, PACAP-27, and VIP using copper-mediated 1,3-dipolar cycloaddition of the Cy5 fluorophore. The functionality of Cy5-labeled peptides at their receptors was confirmed in cAMP accumulation assays. Internalization of the Cy5-labeled peptides was then examined and quantified at two distinct PAC 1 receptor splice variants, VPAC 1 and VPAC 2 receptors in transfected cells. All labeled peptides were functional, exhibiting comparable cAMP pharmacology to their unlabeled counterparts and underwent internalization in a time-dependent manner. Temporal differences in the internalization profiles were observed between Cy5-labeled peptides at the PAC 1n , PAC 1s , VPAC 1 , and VPAC 2 receptors. Interestingly, the pattern of Cy5-labeled peptide activity differed for cAMP accumulation and internalization, indicating that these peptides differentially stimulate cAMP accumulation and internalization and therefore display biased agonism. This novel insight into PACAP-responsive receptor signaling and internalization may provide a unique avenue for future therapeutic development. The fluorescently labeled PACAP and VIP peptides described herein, which we validated as tools to study receptor internalization, will have utility across a broad range of applications and provide greater insight into this receptor family., Competing Interests: The authors declare the following competing financial interest(s): D.L.H. is or has been a consultant or speaker for Lilly, Amgen, Teva, Intarcia, and Merck Sharp and Dohme and has received research funding from Living Cell Technologies and AbbVie in the past 3 years. C.S.W. has received research support from Living Cell Technologies and AbbVie., (© 2022 American Chemical Society.)

Introduction: Developmental and behavioral problems are prevalent in early childhood, whereas the workforce available to identify and address early problems is comparatively limited. Beyond workforce shortages, additional barriers to developing and training a highly skilled workforce in this area exist-particularly in rural, high-need, and underserved U.S. states. As the health care landscape emphasizes expertise in interdisciplinary care, training approaches that provide intensive learning opportunities for supporting a skilled early childhood developmental workforce necessitate novel training approaches. This Workforce Catalyst report summarizes the initial conceptualization, development, execution, and evaluation of a Child Health and Development Promotion (CHDP) postgraduate fellowship in a high need, underserved rural area., Method: Three cohorts totaling 15 trainees across fields including psychology, pediatric nursing, speech-language pathology, social work, and occupational therapy were recruited and cross-trained in an intensive postgraduate fellowship in early childhood development and behavior., Results: The CHDP fellowship led to experiences across the care continuum and resulted in multiple clinical, educational, and scholarly products. Outcomes revealed a training program aligned with Infant and Early Childhood Mental Health competencies, high in-state retention (71%) and employment (93%) following training, and graduates who report leadership positions and sharing of specialty developmental-behavioral knowledge in organizations focused on early childhood., Discussion: The CHDP Fellowship is a novel, immersive, and interdisciplinary training experience demonstrating positive initial training outcomes in Mississippi. The model and experience may serve as a roadmap for bolstering a skilled early childhood workforce in other underserved and high-need states. Aspects regarding scale of reach, funding, and accreditation are discussed as barriers. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide expressed in the trigeminal ganglia (TG). The TG conducts nociceptive signals in the head and may play roles in migraine. PACAP infusion provokes headaches in healthy individuals and migraine-like attacks in patients; however, it is not clear whether targeting this system could be therapeutically efficacious. To effectively target the PACAP system, an understanding of PACAP receptor distribution is required. Therefore, this study aimed to characterize commercially available antibodies and use these to detect PACAP-responsive receptors in the TG. Antibodies were initially validated in receptor transfected cell models and then used to explore receptor expression in rat and human TG. Antibodies were identified that could detect PACAP-responsive receptors, including the first antibody to differentiate between the PAC 1n and PAC 1s receptor splice variants. PAC 1 , VPAC 1 , and VPAC 2 receptor-like immunoreactivity were observed in subpopulations of both neuronal and glial-like cells in the TG. In this study, PAC 1 , VPAC 1 , and VPAC 2 receptors were detected in the TG, suggesting they are all potential targets to treat migraine. These antibodies may be useful tools to help elucidate PACAP-responsive receptor expression in tissues. However, most antibodies exhibited limitations, requiring the use of multiple methodologies and the careful inclusion of controls.

The neuropeptide calcitonin gene-related peptide (CGRP) is expressed in the trigeminal ganglia, a key site in craniofacial pain and migraine. CGRP potently activates two receptors: the CGRP receptor and the AMY 1 receptor. These receptors are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with either the calcitonin receptor-like receptor (CLR) to form the CGRP receptor or the calcitonin receptor (CTR) to form the AMY 1 receptor. The expression of the CGRP receptor in trigeminal ganglia has been described in several studies; however, there is comparatively limited data available describing AMY 1 receptor expression and in which cellular subtypes it is found. This research aimed to determine the relative distributions of the AMY 1 receptor subunit, CTR, and CGRP in neurons or glia in rat, mouse and human trigeminal ganglia. Antibodies against CTR, CGRP and neuronal/glial cell markers were applied to trigeminal ganglia sections to investigate their distribution. CTR-like and CGRP-like immunoreactivity were observed in both discrete and overlapping populations of neurons. In rats and mice, 30-40% of trigeminal ganglia neurons displayed CTR-like immunoreactivity in their cell bodies, with approximately 78-80% of these also containing CGRP-like immunoreactivity. Although human cases were more variable, a similar overall pattern of CTR-like immunoreactivity to rodents was observed in the human trigeminal ganglia. CTR and CGRP appeared to be primarily colocalized in small to medium sized neurons, suggesting that colocalization of CTR and CGRP may occur in C-fiber neurons. CGRP-like or CTR-like immunoreactivity were not typically observed in glial cells. Western blotting confirmed that CTR was expressed in the trigeminal ganglia of all three species. These results confirm that CTR is expressed in trigeminal ganglia neurons. The identification of populations of neurons that express both CGRP and CTR suggests that CGRP could act in an autocrine manner through a CTR-based receptor, such as the AMY 1 receptor. Overall, this suggests that a trigeminal ganglia CTR-based receptor may be activated during migraine and could therefore represent a potential target to develop treatments for craniofacial pain and migraine., Competing Interests: Authors D.L.H. has received research support from Living Cell Technologies and AbbVie and has acted as an advisor, speaker or consultant for Lilly, Teva, Merck and Amgen. C.S.W. has received research support from Living Cell Technologies and AbbVie. A.F.R has received research support from Lundbeck and has acted as an advisor or consultant for Lilly, Amgen, Novartis, Lundbeck, and Schedule One Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rees, Russo, O’Carroll, Hay and Walker.)

Reproduction and survival in most primate species reflects management of both competitive and cooperative relationships. Here, we investigated the links between neuroanatomy and sociality in free-ranging rhesus macaques. In adults, the number of social partners predicted the volume of the mid-superior temporal sulcus and ventral-dysgranular insula, implicated in social decision-making and empathy, respectively. We found no link between brain structure and other key social variables such as social status or indirect connectedness in adults, nor between maternal social networks or status and dependent infant brain structure. Our findings demonstrate that the size of specific brain structures varies with the number of direct affiliative social connections and suggest that this relationship may arise during development. These results reinforce proposed links between social network size, biological success, and the expansion of specific brain circuits.

Signaling through calcitonin gene-related peptide (CGRP) receptors is associated with pain, migraine, and energy expenditure. Small molecule and monoclonal antibody CGRP receptor antagonists that block endogenous CGRP action are in clinical use as anti-migraine therapies. By comparison, the potential utility of peptide antagonists has received less attention due to suboptimal pharmacokinetic properties. Lipidation is an established strategy to increase peptide half-life in vivo . This study aimed to explore the feasibility of developing lipidated CGRP peptide antagonists that retain receptor antagonist activity in vitro and attenuate endogenous CGRP action in vivo . CGRP peptide analogues based on the archetypal CGRP receptor antagonist, CGRP 8-37 , were palmitoylated at the N-terminus, position 24, and near the C-terminus at position 35. The antagonist activities of the lipidated peptide analogues were tested in vitro using transfected Cos-7 cells expressing either the human or mouse CGRP receptor, amylin subtype 1 (AMY 1 ) receptor, adrenomedullin (AM) receptors, or calcitonin receptor. Antagonist activities were also evaluated in SK-N-MC cells that endogenously express the human CGRP receptor. Lipidated peptides were then tested for their ability to antagonize endogenous CGRP action in vivo using a capsaicin-induced dermal vasodilation (CIDV) model in C57/BL6J mice. All lipidated peptides except for the C-terminally modified analogue retained potent antagonist activity compared to CGRP 8-37 towards the CGRP receptor. The lipidated peptides also retained, and sometimes gained, antagonist activities at AMY 1 , AM 1 and AM 2 receptors. Several lipidated peptides produced robust inhibition of CIDV in mice. This study demonstrates that selected lipidated peptide antagonists based on αCGRP 8-37 retain potent antagonist activity at the CGRP receptor and are capable of inhibition of endogenous CGRP action in vivo . These findings suggest that lipidation can be applied to peptide antagonists, such as αCGRP 8-37 and are a potential strategy for antagonizing CGRP action., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer DP declared a past co-authorship with several of the authors CSW, DLH to the handling editor., (Copyright © 2022 Jamaluddin, Chuang, Williams, Siow, Yang, Harris, Petersen, Bower, Chand, Brimble, Walker, Hay and Loomes.)

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of pre-leukemic cells that acquire specific mutations. Although individuals with CH are healthy, they are at an increased risk of developing myeloid malignancies, suggesting that additional alterations are needed for the transition from a pre-leukemia stage to frank leukemia. To identify signaling states that cooperate with pre-leukemic cells, we used an in vivo RNAi screening approach. One of the most prominent genes identified was the ubiquitin ligase TRAF6. Loss of TRAF6 in pre-leukemic cells results in overt myeloid leukemia and is associated with MYC-dependent stem cell signatures. TRAF6 is repressed in a subset of patients with myeloid malignancies, suggesting that subversion of TRAF6 signaling can lead to acute leukemia. Mechanistically, TRAF6 ubiquitinates MYC, an event that does not affect its protein stability but rather represses its functional activity by antagonizing an acetylation modification., Competing Interests: Declaration of interests D.T.S. serves on the scientific advisory board at Kurome Therapeutics and is a consultant for Kymera Therapeutics, Kurome Therapeutics, Captor Therapeutics, and Tolero Therapeutics. D.T.S. has equity in Kurome Therapeutics. The other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The development of several drugs that target the calcitonin gene-related peptide (CGRP) system has been a major breakthrough in the pharmacological management of migraine. These are divided into two major classes, antibodies which bind to the CGRP peptide, preventing it from activating CGRP receptors and receptor antagonists. Within the receptor antagonist class, there are two mechanisms of action, small molecule receptor antagonists and an antibody antagonist. This mini-review considers the pharmacology of these receptor targeted antagonist drugs at the CGRP receptor and closely related AMY 1 receptor, at which CGRP may also act. The antagonists are most potent at the CGRP receptor but can also show antagonism of the AMY 1 receptor. However, important data are missing and selectivity parameters cannot be provided for all antagonists. The clinical implications of AMY 1 receptor antagonism are unknown, but we urge consideration of this receptor as a potential contributing factor to CGRP and antagonist drug actions. LINKED ARTICLES: This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc., (© 2021 The British Pharmacological Society.)

The CGRP system has emerged as a key pharmacological target for the treatment of migraine. However, some individuals who suffer from migraine have low or no response to anti-CGRP or other treatments, suggesting the need for additional clinical targets. CGRP belongs to the calcitonin family of peptides, which includes calcitonin, amylin, adrenomedullin and adrenomedullin 2. These peptides display a range of pro-nociceptive and anti-nociceptive actions, in primary headache conditions such as migraine. Calcitonin family peptides also show expression at sites relevant to migraine and pain. This suggests that calcitonin family peptides and their receptors, beyond CGRP, may be therapeutically useful in the treatment of migraine and other pain disorders. This review considers the localisation of the calcitonin family in peripheral pain pathways and discusses how they may contribute to migraine and pain. LINKED ARTICLES: This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc., (© 2021 The British Pharmacological Society.)

Background and Purpose: The calcitonin (CT) receptor family is complex, comprising two receptors (the CT receptor [CTR] and the CTR-like receptor [CLR]), three accessory proteins (RAMPs) and multiple endogenous peptides. This family contains several important drug targets, including CGRP, which is targeted by migraine therapeutics. The pharmacology of this receptor family is poorly characterised in species other than rats and humans. To facilitate understanding of translational and preclinical data, we need to know the receptor pharmacology of this family in mice., Experimental Approach: Plasmids encoding mouse CLR/CTR and RAMPs were transiently transfected into Cos-7 cells. cAMP production was measured in response to agonists in the absence or presence of antagonists., Key Results: We report the first synthesis and characterisation of mouse adrenomedullin, adrenomedullin 2 and βCGRP and of mouse CTR without or with mouse RAMPs. Receptors containing m-CTR had subtly different pharmacology than human receptors; they were promiscuous in their pharmacology, both with and without RAMPs. Several peptides, including mouse αCGRP and mouse adrenomedullin 2, were potent agonists of the m-CTR:m-RAMP3 complex. Pharmacological profiles of receptors comprising m-CLR:m-RAMPs were generally similar to those of their human counterparts, albeit with reduced specificity., Conclusion and Implications: Mouse receptor pharmacology differed from that in humans, with mouse receptors displaying reduced discrimination between ligands. This creates challenges for interpreting which receptor may underlie an effect in preclinical models and thus translation of findings from mice to humans. It also highlights the need for new ligands to differentiate between these complexes., Linked Articles: This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary).. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc., (© 2021 The British Pharmacological Society.)

Background and Purpose: The pituitary adenylate cyclase-activating peptide (PACAP) family is of clinical interest for the treatment of migraine. These peptides activate three different PACAP-responsive class B G protein-coupled receptors: the PAC 1 , VPAC 1 and VPAC 2 receptors. The PAC 1 receptor may be alternatively spliced, generating variants that can differ in their pharmacological or signalling profiles. To inform drug discovery efforts targeting migraine, we need to better understand how the different PACAP-responsive receptors signal and how effectively these responses can be blocked by antagonists., Experimental Approach: The signalling profiles of the human PAC 1n , PAC 1s , VPAC 1 and VPAC 2 receptors were examined in transfected Cos7 cells for cAMP, IP 1 , pAkt, pERK and pCREB. Biased signalling was then quantified. The ability of antagonists to block PACAP-38, PACAP-27 or VIP stimulated cAMP accumulation at PACAP-responsive receptors was also determined., Key Results: PACAP-responsive receptors exhibited varied pharmacological profiles but activated signalling in a similar manner. The PAC 1n and PAC 1s receptors displayed distinct pharmacology. At the PAC 1s receptor, VIP and PHM were more potent than at the PAC 1n receptor. PACAP-responsive receptors displayed agonist-dependent antagonism where PACAP-38 was less effectively antagonised compared to PACAP-27 and VIP., Conclusions and Implications: The distinct pharmacological profile displayed by the PAC 1s receptor suggests that it can act as a dual receptor for VIP and PACAP. Furthermore, the effectiveness of blocking a signalling pathway can be influenced by which endogenous PACAP family agonist is present. These effects have potential implications for the development and effectiveness of drugs targeting the PACAP system., Linked Articles: This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc., (© 2021 The British Pharmacological Society.)

Cognitive fatigability is an objective performance decrement that occurs over time during a task requiring sustained cognitive effort. Although cognitive fatigability is a common and debilitating symptom in multiple sclerosis (MS), there is currently no standard for its quantification. The objective of this study was to validate the Paced Auditory Serial Addition Test (PASAT) discrete and regression-based normative data for quantifying performance and cognitive fatigability in an Ontario-based sample of individuals with MS. Healthy controls and individuals with MS completed the 3″ and 2″ versions of the PASAT. PASAT performance was measured with total correct, dyad, and percent dyad scores. Cognitive fatigability scores were calculated by comparing performance on the first half (or third) of the task to the last half (or third). The results revealed that the 3″ PASAT was sufficient to detect impaired performance and cognitive fatigability in individuals with MS given the increased difficulty of the 2″ version. In addition, using halves or thirds for calculating cognitive fatigability scores were equally effective methods for detecting impairment. Finally, both the discrete and regression-based norms classified a similar proportion of individuals with MS as having impaired performance and cognitive fatigability. These newly validated discrete and regression-based PASAT norms provide a new tool for clinicians to document statistically significant cognitive fatigability in their patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Walker, Berard and Walker.)

Inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC) has previously been shown to be a potential target for novel cancer therapeutics. One downstream consequence of PC-PLC activity is the activation of NF-κB, a nuclear transcription factor responsible for transcribing genes related to oncogenic traits, such as proliferation, angiogenesis, metastasis, and cancer cell survival. Another biological pathway linked to NF-κB is the exogenous delivery of nitric oxide (NO), which decreases NF-κB activity through an apparent negative-feedback loop. In this study, we designed and synthesised 13 novel NO-releasing derivatives of our previously reported class of PC-PLC inhibitors, 2-morpholinobenzoic acids. These molecules contained a secondary benzylamine group, which was readily nitrosylated and subsequently confirmed to release NO in vitro using a DAF-FM fluorescence-based assay. It was then discovered that these NO-releasing derivatives possessed significantly improved anti-proliferative activity in both MDA-MB-231 and HCT116 cancer cell lines compared to their non-nitrosylated parent compounds. These results confirmed that the inclusion of an exogenous NO-releasing functional group onto a known PC-PLC inhibitor enhances anti-proliferative activity and that this relationship can be exploited in order to further improve the anti-proliferative activity of current/future PC-PLC inhibitors.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

Amylin is a 37-amino acid endocrine hormone secreted from the pancreas in response to nutrient intake, acting centrally to promote meal-ending satiation. With many studies linking amylin action to the nervous system, determining the distribution or expression of amylin in the nervous system is critical. However, amylin shares sequence identity and structural homology to the related neuropeptide calcitonin gene-related peptide (CGRP). This creates challenges in identifying selective amylin antibodies that do not cross-react with CGRP, especially in neural tissues, where CGRP is densely packed into secretory vesicles. Here, we characterized eight amylin antibodies to determine their ability to detect amylin and cross-react with rat or human αCGRP, using immunoblots and preabsorption controls in rat pancreas. We observed that amylin antibodies frequently cross-reacted with αCGRP and are therefore not suitable for use in tissues that highly express CGRP. Earlier work using these antibodies should be revisited in light of our findings.

The "gepants" are a class of calcitonin gene-related peptide (CGRP) receptor antagonist molecules that have been developed for the prevention and treatment of migraine. Rimegepant is reported to act at the CGRP receptor, has good oral bioavailability, and has had positive clinical trial results. However, there is very little data available describing its receptor pharmacology. Importantly, rimegepant activity at the AMY 1 receptor, a second potent CGRP receptor that is known to be expressed in the trigeminovascular system, has not been reported. The ability of rimegepant to antagonize activation of human CGRP, AMY 1 , and related adrenomedullin receptors was determined in transfected in Cos7 cells. Rimegepant was an effective antagonist at both the CGRP and AMY 1 receptor. The antagonism of both CGRP and AMY 1 receptors may have implications for our understanding of the mechanism of action of rimegepant in the treatment of migraine., (Copyright © 2020 Pan, Siow, Hay and Walker.)

The corticotropin-releasing factor (CRF) receptors represent potential drug targets for the treatment of anxiety, stress, and other disorders. However, it is not known if endogenous CRF receptor agonists display biased signaling, how effective CRF receptor antagonists are at blocking different agonists and signaling pathways or how receptor activity-modifying proteins (RAMPs) effect these processes. This study aimed to address this by investigating agonist and antagonist action at CRF 1 and CRF 2 receptors. We used CRF 1 and CRF 2 receptor transfected Cos7 cells to assess the ability of CRF and urocortin (UCN) peptides to activate cAMP, inositol monophosphate (IP 1 ), and extracellular signal-regulated kinase 1/2 signaling and determined the ability of antagonists to block agonist-stimulated cAMP and IP 1 accumulation. The ability of RAMPs to interact with CRF receptors was also examined. At the CRF 1 receptor, CRF and UCN1 activated signaling in the same manner. However, at the CRF 2 receptor, UCN1 and UCN2 displayed similar signaling profiles, whereas CRF and UCN3 displayed bias away from IP 1 accumulation over cAMP. The antagonist potency was dependent on the receptor, agonist, and signaling pathway. CRF 1 and CRF 2 receptors had no effect on RAMP1 or RAMP2 surface expression. The presence of biased agonism and agonist-dependent antagonism at the CRF receptors offers new avenues for developing drugs tailored to activate a specific signaling pathway or block a specific agonist. Our findings suggest that the already complex CRF receptor pharmacology may be underappreciated and requires further investigation., (© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Class B G protein-coupled receptors are highly therapeutically relevant but challenges remain in identifying suitable small-molecule drugs. The calcitonin-like receptor (CLR) in particular is linked to conditions such as migraine, cardiovascular disease, and inflammatory bowel disease. The CLR cannot act as a cell-surface receptor alone but rather must couple to one of three receptor activity-modifying proteins (RAMPs), forming heterodimeric receptors for the peptides adrenomedullin and calcitonin gene-related peptide. These peptides have extended binding sites across their receptors. This is one reason why there are few small-molecule ligands that can modulate these receptors. Here we describe small molecules that are able to positively modulate the signaling of the CLR with all three RAMPs but are not active at the related calcitonin receptor. These compounds were selected from a β-arrestin recruitment screen, coupled with rounds of medicinal chemistry to improve their activity. Translational potential is shown as the compounds can positively modulate cAMP signaling in a vascular cell line model. Binding experiments do not support an extracellular domain binding site; however, molecular modeling reveals potential allosteric binding sites in multiple receptor regions. These are the first small-molecule positive modulators described for the CLR:RAMP complexes., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Calcitonin gene-related peptide (CGRP) is a neuropeptide that is involved in the transmission of pain. Drugs targeting CGRP or a CGRP receptor are efficacious in the treatment of migraine. The canonical CGRP receptor is a complex of a G protein-coupled receptor, the calcitonin-like receptor (CLR), with an accessory protein, receptor activity-modifying protein 1 (RAMP1). A second receptor, the AMY 1 receptor, a complex of the calcitonin receptor with RAMP1, is a dual high-affinity receptor for CGRP and amylin. Receptor regulatory processes, such as internalization, are crucial for controlling peptide and drug responsiveness. Given the importance of CGRP receptor activity in migraine we compared the internalization profiles of both receptors for CGRP using novel fluorescent probes and a combination of live cell imaging, fixed cell imaging, and ELISA. This revealed stark differences in the regulation of each receptor with the AMY 1 receptor unexpectedly showing little internalization., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Adrenomedullin (AM) is a 52 amino acid peptide that plays a regulatory role in the vasculature. Receptors for AM comprise the class B G protein-coupled receptor, the calcitonin-like receptor (CLR), in complex with one of three receptor activity-modifying proteins (RAMPs). The C-terminus of AM is involved in binding to the extracellular domain of the receptor, while the N-terminus is proposed to interact with the juxtamembranous portion of the receptor to activate signaling. There is currently limited information on the molecular determinants involved in AM signaling, thus we set out to define the importance of the AM N-terminus through five signaling pathways (cAMP production, ERK phosphorylation, CREB phosphorylation, Akt phosphorylation, and IP 1 production). We characterized the three CLR:RAMP complexes through the five pathways, finding that each had a distinct repertoire of intracellular signaling pathways that it is able to regulate. We then performed an alanine scan of AM from residues 15-31 and found that most residues could be substituted with only small effects on signaling, and that most substitutions affected signaling through all receptors and pathways in a similar manner. We identify F18, T20, L26, and I30 as being critical for AM function, while also identifying an analogue (AM 15-52 G19A) which has unique signaling properties relative to the unmodified AM. We interpret our findings in the context of new structural information, highlighting the complementary nature of structural biology and functional assays., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

The abundant femoral assemblage of Homo naledi found in the Dinaledi Chamber provides a unique opportunity to test hypotheses regarding the taxonomy, locomotion, and loading patterns of this species. Here we describe neck and shaft cross-sectional structure of all the femoral fossils recovered in the Dinaledi Chamber and compare them to a broad sample of fossil hominins, recent humans, and extant apes. Cross-sectional geometric (CSG) properties from the femoral neck (base of neck and midneck) and diaphysis (subtrochanteric region and midshaft) were obtained through CT scans for H. naledi and through CT scans or from the literature for the comparative sample. The comparison of CSG properties of H. naledi and the comparative samples shows that H. naledi femoral neck is quite derived with low superoinferior cortical thickness ratio and high relative cortical area. The neck appears superoinferiorly elongated because of two bony pilasters on its superior surface. Homo naledi femoral shaft shows a relatively thick cortex compared to the other hominins. The subtrochanteric region of the diaphysis is mediolaterally elongated resembling early hominins while the midshaft is anteroposteriorly elongated, indicating high mobility levels. In term of diaphyseal robusticity, the H. naledi femur is more gracile that other hominins and most apes. Homo naledi shows a unique combination of characteristics in its femur that undoubtedly indicate a species committed to terrestrial bipedalism but with a unique loading pattern of the femur possibly consequence of the unique postcranial anatomy of the species., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in a wide range of functions including vasodilatation, neuroprotection, nociception and neurogenic inflammation. PACAP activates three distinct receptors, the PAC 1 receptor, which responds to PACAP, and the VPAC 1 and VPAC 2 receptors, which respond to both PACAP and vasoactive intestinal polypeptide. The trigeminovascular system plays a key role in migraine and contains the trigeminal nerve, which is the major conduit of craniofacial pain. PACAP is expressed throughout the trigeminovascular system and in higher brain regions involved in processing pain. Evidence from human clinical studies suggests that PACAP may act outside the blood-brain barrier in the pathogenesis of migraine. However, the precise mechanisms involved remain unclear. PACAP potentially induces migraine attacks by activating different receptors in different cell types and tissues. This complexity prompted this review of PACAP receptor pharmacology, expression and function in the trigeminovascular system. Current evidence suggests that the PAC 1 receptor is the likely pathophysiological target of PACAP in migraine. However, multiple PACAP receptors are expressed in key parts of the trigeminovascular system and further work is required to determine their contribution to PACAP physiology and the pathology of migraine., Linked Articles: This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc., (© 2017 The British Pharmacological Society.)

The pancreatic peptide hormone, amylin, plays a critical role in the control of appetite, and synergizes with other key metabolic hormones such as glucagon-like peptide 1 (GLP-1). There is opportunity to develop potent and long-acting analogues of amylin or hybrids between these and GLP-1 mimetics for treating obesity. To achieve this, interrogation of how the 37 amino acid amylin peptide engages with its complex receptor system is required. We synthesized an extensive library of peptides to profile the human amylin sequence, determining the role of its disulfide loop, amidated C -terminus and receptor "capture" and "activation" regions in receptor signaling. We profiled four signaling pathways with different ligands at multiple receptor subtypes, in addition to exploring selectivity determinants between related receptors. Distinct roles for peptide subregions in receptor binding and activation were identified, resulting in peptides with greater activity than the native sequence. Enhanced peptide activity was preserved in the brainstem, the major biological target for amylin. Interpretation of our data using full-length active receptor models supported by molecular dynamics, metadynamics, and supervised molecular dynamics simulations guided the synthesis of a potent dual agonist of GLP-1 and amylin receptors. The data offer new insights into the function of peptide amidation, how allostery drives peptide-receptor interactions, and provide a valuable resource for the development of novel amylin agonists for treating diabetes and obesity., Competing Interests: The authors declare no competing financial interest., (Copyright © 2018 American Chemical Society.)

Body mass is an ecologically and biomechanically important variable in the study of hominin biology. Regression equations derived from recent human samples allow for the reasonable prediction of body mass of later, more human-like, and generally larger hominins from hip joint dimensions, but potential differences in hip biomechanics across hominin taxa render their use questionable with some earlier taxa (i.e., Australopithecus spp.). Morphometric prediction equations using stature and bi-iliac breadth avoid this problem, but their applicability to early hominins, some of which differ in both size and proportions from modern adult humans, has not been demonstrated. Here we use mean stature, bi-iliac breadth, and body mass from a global sample of human juveniles ranging in age from 6 to 12 years (n = 530 age- and sex-specific group annual means from 33 countries/regions) to evaluate the accuracy of several published morphometric prediction equations when applied to small humans. Though the body proportions of modern human juveniles likely differ from those of small-bodied early hominins, human juveniles (like fossil hominins) often differ in size and proportions from adult human reference samples and, accordingly, serve as a useful model for assessing the robustness of morphometric prediction equations. Morphometric equations based on adults systematically underpredict body mass in the youngest age groups and moderately overpredict body mass in the older groups, which fall in the body size range of adult Australopithecus (∼26-46 kg). Differences in body proportions, notably the ratio of lower limb length to stature, influence predictive accuracy. Ontogenetic changes in these body proportions likely influence the shift in prediction error (from under- to overprediction). However, because morphometric equations are reasonably accurate when applied to this juvenile test sample, we argue these equations may be used to predict body mass in small-bodied hominins, despite the potential for some error induced by differing body proportions and/or extrapolation beyond the original reference sample range., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Basal nuclear factor κB (NF-κB) activation is required for hematopoietic stem cell (HSC) homeostasis in the absence of inflammation; however, the upstream mediators of basal NF-κB signaling are less well understood. Here, we describe TRAF6 as an essential regulator of HSC homeostasis through basal activation of NF-κB. Hematopoietic-specific deletion of Traf6 resulted in impaired HSC self-renewal and fitness. Gene expression, RNA splicing, and molecular analyses of Traf6-deficient hematopoietic stem/progenitor cells (HSPCs) revealed changes in adaptive immune signaling, innate immune signaling, and NF-κB signaling, indicating that signaling via TRAF6 in the absence of cytokine stimulation and/or infection is required for HSC function. In addition, we established that loss of IκB kinase beta (IKKβ)-mediated NF-κB activation is responsible for the major hematopoietic defects observed in Traf6-deficient HSPC as deletion of IKKβ similarly resulted in impaired HSC self-renewal and fitness. Taken together, TRAF6 is required for HSC homeostasis by maintaining a minimal threshold level of IKKβ/NF-κB signaling., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chimpanzees are important referential models for the study of life history in hominin evolution. Age at sexual maturity and first reproduction are key life history milestones that mark the diversion of energy from growth to reproduction and are essential in comparing life history trajectories between chimpanzees and humans. Yet, accurate information on ages at these milestones in wild chimpanzees is difficult to obtain because most females transfer before breeding. Precise age at first birth is only known from a relatively small number of non-dispersing individuals. Moreover, due to small sample sizes, the degree to which age at maturation milestones varies is unknown. Here we report maturation milestones and explore sources of variance for 36 wild female chimpanzees of known age, including eight dispersing females born in Gombe National Park, Tanzania. Using Kaplan-Meier survival analysis, including censored intervals, we find an average age of 11.5 years (range 8.5-13.9) at sexual maturity and 14.9 years (range 11.1-22.1) at first birth. These values exceed previously published averages for wild chimpanzees by one or more years. Even in this larger sample, age at first birth is likely underestimated due to the disproportionate number of non-dispersing females, which, on average, give birth two years earlier than dispersing females. Model selection using Cox Proportional Hazards models shows that age at sexual maturity is delayed in females orphaned before age eight years and those born to low-ranking mothers. Age at first birth is most delayed in dispersing females and those orphaned before age eight years. These data provide improved estimates of maturation milestones in a population of wild female chimpanzees and indicate the importance of maternal factors in development., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The calcitonin/CGRP family of peptides includes calcitonin, α and β CGRP, amylin, adrenomedullin (AM) and adrenomedullin 2/intermedin (AM2/IMD). Their receptors consist of one of two GPCRs, the calcitonin receptor (CTR) or the calcitonin receptor-like receptor (CLR). Further diversity arises from heterodimerization of these GPCRs with one of three receptor activity-modifying proteins (RAMPs). This gives the CGRP receptor (CLR/RAMP1), the AM 1 and AM 2 receptors (CLR/RAMP2 or RAMP3) and the AMY 1, AMY 2 and AMY 3 receptors (CTR/RAMPs1-3 complexes, respectively). Apart from the CGRP receptor, there are only peptide antagonists widely available for these receptors, and these have limited selectivity, thus defining the function of each receptor in vivo remains challenging. Further challenges arise from the probable co-expression of CTR with the CTR/RAMP complexes and species-dependent splice variants of the CTR (CT (a) and CT (b) ). Furthermore, the AMY 1(a) receptor is activated equally well by both amylin and CGRP, and the preferred receptor for AM2/IMD has been unclear. However, there are clear therapeutic rationales for developing agents against the various receptors for these peptides. For example, many agents targeting the CGRP system are in clinical trials, and pramlintide, an amylin analogue, is an approved therapy for insulin-requiring diabetes. This review provides an update on the pharmacology of the calcitonin family of peptides by members of the corresponding subcommittee of the International Union of Basic and Clinical Pharmacology and colleagues., (© 2017 The British Pharmacological Society.)

Because of their ideal size and temperament, rabbits are commonly used in polyclonal antibody production. Immunostimulatory adjuvants-such as Freund complete and incomplete adjuvants as well as various proprietary products-trigger a robust immune response, which increases antibody concentrations. However, these adjuvants can cause excessive soft tissue reactions, prompting concerns regarding animal wellbeing. This study assessed the safety and efficacy of cationic liposome- oligonucleotide complexes (CLDC) as an alternative adjuvant to conventional adjuvants. On days 0 and 14, 15 female New Zealand white rabbits were vaccinated subcutaneously with 15 μg ovalbumin mixed with either CLDC, Freund adjuvant (day 0, complete; day 14, incomplete), or a proprietary adjuvant (n = 5 per group). Antibody titers were measured by direct ELISA on days 0, 14, and 28. Rabbits were palpated daily for lesion development, and all lesions were measured. Rabbits in all groups developed a significant antibody response to ovalbumin over 28 d. However, the differences between groups were not statistically significant. No rabbits in the CLDC group developed skin lesions, whereas 80% of rabbits that received Freund adjuvant and 100% of those that received the proprietary product developed skin lesions. This study demonstrates that CLDC may be a valuable and effective alternative adjuvant for polyclonal antibody production in rabbits-one that avoids the palpable injection-site lesions often seen with other adjuvants.

Humans' prolonged somatic development and life history are unique among primates, yet their evolutionary origins remain unclear. Dental development has been used as a proxy to reconstruct life history evolution in the hominin clade and indicates a recent emergence of the human developmental pattern. Here, we analyse tooth formation and eruption in two developing dentitions of Homo naledi , a late-surviving, morphologically mosaic hominin species. Deciduous dental development is more similar to humans than to chimpanzees, probably reflecting hominin symplesiomorphy rather than bearing life history significance. The later stages of permanent tooth development present a mix of human- and chimpanzee-like patterns. Surprisingly, the M 2 of H. naledi emerges late in the eruption sequence, a pattern previously unknown in fossil hominins and common in modern humans. This pattern has been argued to reflect a slow life history and is unexpected in a small-brained hominin. The geological age of H. naledi (approx. 300 kya), coupled with its small brain size and the dental development data presented here, raise questions about the relationship between dental development and other variables associated with life history., (© 2017 The Authors.)

Background and Purpose: CGRP is a potent vasodilator and nociceptive neuropeptide linked to migraine. CGRP receptors are heterodimers of receptor activity modifying protein 1 (RAMP1) and either calcitonin receptor-like receptor (CLR; forms canonical CGRP receptor) or calcitonin receptor (CT receptor; forms AMY 1 receptor). The goal of this study was to test whether transgenic mice globally expressing human RAMP1 have increased CGRP receptor activity and whether the receptors are sensitive to human selective antagonist telcagepant., Experimental Approach: cAMP production was measured in primary cultures of aortic smooth muscle and trigeminal ganglia neurons from global hRAMP1 mice and non-transgenic littermates. Functional activity and inhibition were compared with clonal cell lines expressing combinations of CLR or CT receptors with RAMP1., Key Results: Cultured smooth muscle from global hRAMP1 mice had a 10-fold greater CGRP-induced cAMP maximal response (Rmax) than non-transgenic littermates, with similar EC 50 s. In contrast, cultured trigeminal ganglia from global hRAMP1 mice had a 40-fold leftward shift of the EC 50 , with similar Rmax values as littermates. In both hRAMP1 cultures, telcagepant blocked CGRP-induced cAMP production, but was not effective in non-transgenic cultures. IC 50 values were closer to those observed for CT receptor/hRAMP1 than CLR/hRAMP1 in clonal cell lines., Conclusions and Implications: Overexpression of hRAMP1 increases CGRP signalling by changing the maximal response or ligand sensitivity, depending on tissue type. Furthermore, telcagepant inhibited transgenic hRAMP1 CGRP receptors, but the degree of inhibition suggests that the transgenic mice are only partially humanized or both canonical CGRP and AMY 1 receptors are functional in trigeminal ganglia neurons and vascular smooth muscle., (© 2017 The British Pharmacological Society.)

The Rising Star cave system has produced abundant fossil hominin remains within the Dinaledi Chamber, representing a minimum of 15 individuals attributed to Homo naledi . Further exploration led to the discovery of hominin material, now comprising 131 hominin specimens, within a second chamber, the Lesedi Chamber. The Lesedi Chamber is far separated from the Dinaledi Chamber within the Rising Star cave system, and represents a second depositional context for hominin remains. In each of three collection areas within the Lesedi Chamber, diagnostic skeletal material allows a clear attribution to H. naledi . Both adult and immature material is present. The hominin remains represent at least three individuals based upon duplication of elements, but more individuals are likely present based upon the spatial context. The most significant specimen is the near-complete cranium of a large individual, designated LES1, with an endocranial volume of approximately 610 ml and associated postcranial remains. The Lesedi Chamber skeletal sample extends our knowledge of the morphology and variation of H. naledi , and evidence of H. naledi from both recovery localities shows a consistent pattern of differentiation from other hominin species.

This paper describes the 108 femoral, patellar, tibial, and fibular elements of a new species of Homo (Homo naledi) discovered in the Dinaledi chamber of the Rising Star cave system in South Africa. Homo naledi possesses a mosaic of primitive, derived, and unique traits functionally indicative of a bipedal hominin adapted for long distance walking and possibly running. Traits shared with australopiths include an anteroposteriorly compressed femoral neck, a mediolaterally compressed tibia, and a relatively circular fibular neck. Traits shared with Homo include a well-marked linea aspera, anteroposteriorly thick patellae, relatively long tibiae, and gracile fibulae with laterally oriented lateral malleoli. Unique features include the presence of two pillars on the superior aspect of the femoral neck and a tubercular distal insertion of the pes anserinus on the tibia. The mosaic morphology of the H. naledi thigh and leg appears most consistent with a species intermediate between Australopithecus spp. and Homo erectus and, accordingly, may offer insight into the nature of the earliest members of genus Homo. These fossils also expand the morphological diversity of the Homo lower limb, perhaps indicative of locomotor diversity in our genus., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The evolutionary transition from an ape-like to human-like upper extremity occurred in the context of a behavioral shift from an upper limb predominantly involved in locomotion to one adapted for manipulation. Selection for overarm throwing and endurance running is thought to have further shaped modern human shoulder girdle morphology and its position about the thorax. Homo naledi (Dinaledi Chamber, Rising Star Cave, Cradle of Humankind, South Africa) combines an australopith-like cranial capacity with dental characteristics akin to early Homo. Although the hand, foot, and lower limb display many derived morphologies, the upper limb retains many primitive traits. Here, we describe the H. naledi upper extremity (excluding the hand) in detail and in a comparative context to evaluate the diversity of clavicular, scapular, humeral, radial, and ulnar morphology among early hominins and later Homo. Homo naledi had a scapula with a markedly cranially-oriented glenoid, a humerus with extremely low torsion, and an australopith-like clavicle. These traits indicate that the H. naledi scapula was situated superiorly and laterally on the thorax. This shoulder girdle configuration is more similar to that of Australopithecus and distinct from that of modern humans, whose scapulae are positioned low and dorsally about the thorax. Although early Homo erectus maintains many primitive clavicular and humeral features, its derived scapular morphology suggests a loss of climbing adaptations. In contrast, the H. naledi upper limb is markedly primitive, retaining morphology conducive to climbing while lacking many of the derived features related to effective throwing or running purported to characterize other members of early Homo., (Published by Elsevier Ltd.)

The calcitonin receptor (CTR) is relevant to three hormonal systems: amylin, calcitonin, and calcitonin gene-related peptide (CGRP). Receptors for amylin and calcitonin are targets for treating obesity, diabetes, and bone disorders. CGRP receptors represent a target for pain and migraine. Amylin receptors (AMY) are a heterodimer formed by the coexpression of CTR with receptor activity-modifying proteins (RAMPs). CTR with RAMP1 responds potently to both amylin and CGRP. The brain stem is a major site of action for circulating amylin and is a rich site of CGRP binding. This study aimed to enhance our understanding of these hormone systems by mapping CTR expression in the human brain stem, specifically the medulla oblongata. Widespread CTR-like immunoreactivity was observed throughout the medulla. Dense CTR staining was noted in several discrete nuclei, including the nucleus of the solitary tract, the hypoglossal nucleus, the cuneate nucleus, spinal trigeminal nucleus, the gracile nucleus, and the inferior olivary nucleus. CTR staining was also observed in the area postrema, the lateral reticular nucleus, and the pyramidal tract. The extensive expression of CTR in the medulla suggests that CTR may be involved in a wider range of functions than currently appreciated., (Copyright © 2016 the American Physiological Society.)

Renal depletion of myo-inositol (MI) is associated with the pathogenesis of diabetic nephropathy in animal models, but the underlying mechanisms involved are unclear. We hypothesized that MI depletion was due to changes in inositol metabolism and therefore examined the expression of genes regulating de novo biosynthesis, reabsorption, and catabolism of MI. We also extended the analyses from diabetes mellitus to animal models of dietary-induced obesity and hypertension. We found that renal MI depletion was pervasive across these three distinct disease states in the relative order: hypertension (-51%)>diabetes mellitus (-35%)>dietary-induced obesity (-19%). In 4-wk diabetic kidneys and in kidneys derived from insulin-resistant and hypertensive rats, MI depletion was correlated with activity of the MI-degrading enzyme myo-inositol oxygenase (MIOX). By contrast, there was decreased MIOX expression in 8-wk diabetic kidneys. Immunohistochemistry localized the MI-degrading pathway comprising MIOX and the glucuronate-xylulose (GX) pathway to the proximal tubules within the renal cortex. These findings indicate that MI depletion could reflect increased catabolism through MIOX and the GX pathway and implicate a common pathological mechanism contributing to renal oxidative stress in metabolic disease., (Copyright © 2015 the American Physiological Society.)