The M1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes.

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M1 muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)-linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M1 and anti-green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M1-mEGFP was expressed at levels equal to the M1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M1-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M1-mEGFP-expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M1-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

G protein-receptor kinases 5/6 are the key regulators of G protein-coupled receptor 35-arrestin interactions.

Human G protein-coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phospho-acceptor amino acids within its C-terminal tail. Alteration of both Ser300 and Ser303 to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome edited to lack expression of combinations of G protein receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser300 and Ser303 (orce; the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues. Extensions of these studies demonstrated the importance of the GRK5/6-mediated phosphorylation of these amino acids for agonist-induced internalization of the receptor. Homology and predictive modeling of the interaction of human GPR35 with GRKs showed that the N terminus of GRK5 is likely to dock in the same methionine pocket on the intracellular face of GPR35 as the C terminus of the α5 helix of Gα13 and, that while this is also the case for GRK6, GRK2 and GRK3 are unable to do so effectively. These studies provide unique and wide-ranging insights into modes of regulation of GPR35, a receptor that is currently attracting considerable interest as a novel therapeutic target in diseases including ulcerative colitis.

EHF is essential for epidermal and colonic epithelial homeostasis, and suppresses Apc-initiated colonic tumorigenesis.

Ets homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) family of transcription factors. To investigate its role in development and epithelial homeostasis, we generated a series of novel mouse strains in which the Ets DNA-binding domain of Ehf was deleted in all tissues (Ehf-/-) or specifically in the gut epithelium. Ehf-/- mice were born at the expected Mendelian ratio, but showed reduced body weight gain, and developed a series of pathologies requiring most Ehf-/- mice to reach an ethical endpoint before reaching 1 year of age. These included papillomas in the facial skin, abscesses in the preputial glands (males) or vulvae (females), and corneal ulcers. Ehf-/-mice also displayed increased susceptibility to experimentally induced colitis, which was confirmed in intestinal-specific Ehf knockout mice. Gut-specific Ehf deletion also impaired goblet cell differentiation, induced extensive transcriptional reprogramming in the colonic epithelium and enhanced Apc-initiated adenoma development. The Ets DNA-binding domain of EHF is therefore essential for postnatal homeostasis of the epidermis and colonic epithelium, and its loss promotes colonic tumour development.

Biased M1 muscarinic receptor mutant mice show accelerated progression of prion neurodegenerative disease.

There are currently no treatments that can slow the progression of neurodegenerative diseases, such as Alzheimer's disease (AD). There is, however, a growing body of evidence that activation of the M1 muscarinic acetylcholine receptor (M1-receptor) can not only restore memory loss in AD patients but in preclinical animal models can also slow neurodegenerative disease progression. The generation of an effective medicine targeting the M1-receptor has however been severely hampered by associated cholinergic adverse responses. By using genetically engineered mouse models that express a G protein-biased M1-receptor, we recently established that M1-receptor mediated adverse responses can be minimized by ensuring activating ligands maintain receptor phosphorylation/arrestin-dependent signaling. Here, we use these same genetic models in concert with murine prion disease, a terminal neurodegenerative disease showing key hallmarks of AD, to establish that phosphorylation/arrestin-dependent signaling delivers neuroprotection that both extends normal animal behavior and prolongs the life span of prion-diseased mice. Our data point to an important neuroprotective property inherent to the M1-receptor and indicate that next generation M1-receptor ligands designed to drive receptor phosphorylation/arrestin-dependent signaling would potentially show low adverse responses while delivering neuroprotection that will slow disease progression.

Inviting the Patient to Talk About a Conversation They Had with Another Healthcare Practitioner: A Way of Promoting Discussion About Disease Progression and End of Life in Palliative Care Interactions.

Discussing disease progression is a core task in palliative care. This is especially important when there are indications that a patient considers their death as less imminent than the clinical team does. This article examines a communicative action that palliative medicine doctors use to address such discrepancies in knowledge and understanding of the patient's prognosis: inviting the patient to talk about the contents of a conversation they had with another healthcare practitioner. The study used conversation analysis to examine five consultations in which this action was identified. These were part of a larger data set of 37 consultations recorded in a large UK hospice and involving patients with palliative care needs, sometimes accompanied by family or friends, and palliative medicine doctors. Findings are that the action of inviting the patient to talk about a previous conversation creates an opportunity for patients to articulate what they know and understand about their disease progression - but without requiring them to do so. Discussing such sensitive matters is thus made a matter of 'opting in' (rather than 'opting out'). Doctors thereby avoid being interactionally accountable for directly initiating a potentially distressing topic. The article shows how the task of discussing disease progression and end of life is intertwined with the delicate management of patients' displayed states of awareness regarding their disease progression. The study thus has practical implications by documenting ways in which clinicians can help patients realign their expectations about such delicate matters.

Brief cognitive behavioural therapy for eating disorders symptomatology among a mixed sample of adolescents and young adults in primary care: A non-randomised feasibility and pilot study.

OBJECTIVE: Brief and accessible therapies for people with an eating disorder is an important health target. Ten-session cognitive behavioural therapy (CBT-T) is a brief treatment evaluated in people with a non-underweight eating disorder. This study aimed to evaluate the feasibility and preliminary effectiveness of CBT-T for young people in primary care. METHOD: This cohort pilot study used group (adolescents vs. young adults) by time (over four time points) Generalised Linear Mixed Model analysis. Participants included 13-25-year-olds attending an early intervention mental health service, receiving 10 sessions of CBT-T. Feasibility was assessed using recruitment, retention and satisfaction. Eating and other pathology measures were administered at baseline, weeks four and 10, and 12-week follow-up. RESULTS: Of the 63 commencing treatment, 38 completed 10 CBT-T sessions (60%). Most (94%) reported high treatment satisfaction. Significant reductions in eating pathology, depression and stress were found. Age group did not yield differences in CBT-T outcome, with large to very large effect sizes across outcome variables. Anxiety was associated with attrition. CONCLUSION: This study provides preliminary support for the use of CBT-T in primary care, across adolescence and early adulthood. Findings require replication in other clinical settings and comparison to other clinical approaches and control populations.

Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor.

G protein-coupled receptor 35 (GPR35) is poorly characterized but nevertheless has been revealed to have diverse roles in areas including lower gut inflammation and pain. The development of novel reagents and tools will greatly enhance analysis of GPR35 functions in health and disease. Here, we used mass spectrometry, mutagenesis, and [32P] orthophosphate labeling to identify that all five hydroxy-amino acids in the C-terminal tail of human GPR35a became phosphorylated in response to agonist occupancy of the receptor and that, apart from Ser294, each of these contributed to interactions with arretin-3, which inhibits further G protein-coupled receptor signaling. We found that Ser303 was key to such interactions; the serine corresponding to human GPR35a residue 303 also played a dominant role in arrestin-3 interactions for both mouse and rat GPR35. We also demonstrated that fully phospho-site-deficient mutants of human GPR35a and mouse GPR35 failed to interact effectively with arrestin-3, and the human phospho-deficient variant was not internalized from the surface of cells in response to agonist treatment. Even in cells stably expressing species orthologues of GPR35, a substantial proportion of the expressed protein(s) was determined to be immature. Finally, phospho-site-specific antisera targeting the region encompassing Ser303 in human (Ser301 in mouse) GPR35a identified only the mature forms of GPR35 and provided effective sensors of the activation status of the receptors both in immunoblotting and immunocytochemical studies. Such antisera may be useful tools to evaluate target engagement in drug discovery and target validation programs.

Selective phosphorylation of threonine residues defines GPR84-arrestin interactions of biased ligands.

GPR84 is an immune cell-expressed, proinflammatory receptor currently being assessed as a therapeutic target in conditions including fibrosis and inflammatory bowel disease. Although it was previously shown that the orthosteric GPR84 activators 2-HTP and 6-OAU promoted its interactions with arrestin-3, a G protein-biased agonist DL-175 did not. Here, we show that replacement of all 21 serine and threonine residues within i-loop 3 of GPR84, but not the two serines in the C-terminal tail, eliminated the incorporation of [32P] and greatly reduced receptor-arrestin-3 interactions promoted by 2-HTP. GPR84 was phosphorylated constitutively on residues Ser221 and Ser224, while various other amino acids are phosphorylated in response to 2-HTP. Consistent with this, an antiserum able to identify pSer221/pSer224 recognized GPR84 from cells treated with and without activators, whereas an antiserum able to identify pThr263/pThr264 only recognized GPR84 after exposure to 2-HTP and not DL-175. Two distinct GPR84 antagonists as well as inhibition of G protein-coupled receptor kinase 2/3 prevented phosphorylation of pThr263/pThr264, but neither strategy affected constitutive phosphorylation of Ser221/Ser224. Furthermore, mutation of residues Thr263 and Thr264 to alanine generated a variant of GPR84 also limited in 2-HTP-induced interactions with arrestin-2 and -3. By contrast, this mutant was unaffected in its capacity to reduce cAMP levels. Taken together, these results define a key pair of threonine residues, regulated only by subsets of GPR84 small molecule activators and by GRK2/3 that define effective interactions with arrestins and provide novel tools to monitor the phosphorylation and functional status of GPR84.

Intestinal-specific Hdac3 deletion increases susceptibility to colitis and small intestinal tumor development in mice fed a high-fat diet.

High-fat (HF) diets (HFDs) and inflammation are risk factors for colon cancer; however, the underlying mechanisms remain to be fully elucidated. The transcriptional corepressor HDAC3 has recently emerged as a key regulator of intestinal epithelial responses to diet and inflammation with intestinal-specific Hdac3 deletion (Hdac3IKO) in mice increasing fatty acid oxidation genes and the rate of fatty acid oxidation in enterocytes. Hdac3IKO mice are also predisposed to experimentally induced colitis; however, whether this is driven by the intestinal metabolic reprogramming and whether this predisposes these mice to intestinal tumorigenesis is unknown. Herein, we examined the effects of intestinal-specific Hdac3 deletion on colitis-associated intestinal tumorigenesis in mice fed a standard (STD) or HFD. Hdac3IKO mice were highly prone to experimentally induced colitis, which was further enhanced by an HFD. Hdac3 deletion also accelerated intestinal tumor development, specifically when fed an HFD and most notably in the small intestine where lipid absorption is maximal. Expression of proteins involved in fatty acid metabolism and oxidation (SCD1, EHHADH) were elevated in the small intestine of Hdac3IKO mice fed an HFD, and these mice displayed increased levels of lipid peroxidation, DNA damage, and apoptosis in their villi, as well as extensive expansion of the stem cell and progenitor cell compartment. These findings reveal a novel role for Hdac3 in suppressing colitis and intestinal tumorigenesis, particularly in the context of consumption of an HFD, and reveal a potential mechanism by which HFDs may increase intestinal tumorigenesis by increasing fatty acid oxidation, DNA damage, and intestinal epithelial cell turnover.NEW & NOTEWORTHY We reveal a novel role for the transcriptional corepressor Hdac3 in suppressing colitis and intestinal tumorigenesis, particularly in the context of consumption of an HFD, and reveal a potential mechanism by which HFDs may increase intestinal tumorigenesis by increasing fatty acid oxidation, DNA damage, and intestinal epithelial cell turnover. We also identify a unique mouse model for investigating the complex interplay between diet, metabolic reprogramming, and tumor predisposition in the intestinal epithelium.

Feeding after spawning and energy balance at spawning are associated with repeat spawning interval in steelhead trout.

Consecutive and skip repeat spawning (1- or ≥2-year spawning interval) life histories commonly occur in seasonally breeding iteroparous fishes. Spawning interval variation is driven by energetic status and impacts fisheries management. In salmonids, energetic status (either absolute level of energy reserves or the rate of change of energy reserves, i.e., energy balance) is thought to determine reproductive trajectory during a critical period ∼1 year prior to initial spawning. However, information on repeat spawners is lacking. To examine the timing and the aspects of energetic status that regulate repeat spawning interval, female steelhead trout (Oncorhynchus mykiss) were fasted for 10 weeks after spawning and then fed ad libitum and compared to ad libitum fed controls. Plasma growth hormone (GH) and insulin-like growth factor-I (IGF-I) levels were measured to assess long-term energy balance. Plasma estradiol levels showed that some fish in both groups initiated a consecutive spawning cycle. In fasted fish, GH was lower at spawning in consecutive versus skip spawners. In consecutive spawners, GH was higher at spawning in fed versus fasted fish. These results suggest that fish with a less negative energy balance at spawning initiated reproductive development in the absence of feeding, but that feeding during the post-spawning period enabled initiation of reproduction in some fish with a more negative energy balance at spawning. Thus, both energy balance at spawning and feeding after spawning regulated reproductive schedules. These results show that the critical period model of salmonid maturation applies to regulation of repeat spawning, and that the reproductive decision window extends into the first 10 weeks after spawning.

Effects of post-spawning ration restriction on reproductive development and the growth hormone/insulin-like growth factor-1 axis in female rainbow trout (Oncorhynchus mykiss).

In iteroparous female salmonids, the growth and reproductive endocrine axes interact during the period after spawning. Energy depletion due to pre-spawn fasting, migration, and ovarian development must be restored, and the next reproductive cycle is initiated in consecutively maturing fish. In the natural environment, food availability is often limited during the post-spawn period. To investigate the growth and reproductive endocrinology of the post-spawn period, we sampled female rainbow trout over the 30 weeks following their first spawning. Fish were fasted for 2 months prior to spawning, then fed a standard or a restricted ration. Analysis was confined to reproductive fish. Plasma estradiol-17ß decreased during the 8 weeks following spawning and then began increasing in both ration groups and was lower in feed-restricted versus standard ration fish from 8 weeks onward. Plasma insulin-like growth factor-1 increased over the same period and then remained constant in both ration groups and was lower in feed-restricted versus standard ration fish from week 8 to week 30. Plasma growth hormone decreased following spawning in standard ration fish and became elevated in feed-restricted versus standard ration fish at 20- and 30-weeks post-spawn. Growth rates, condition factor, and muscle lipid levels were higher in standard ration versus feed-restricted fish within 2-4 weeks after spawning. These results suggest that two phases occurred during the post-spawn period: recovery from spawning and restoration of energy reserves over weeks 0 to 8, followed by adjustment of the growth and reproductive endocrine axes to ration level over weeks 8 to 30.

Dual targeting of FGFR3 and ERBB3 enhances the efficacy of FGFR inhibitors in FGFR3 fusion-driven bladder cancer.

BACKGROUND: Mutations and fusions in Fibroblast Growth Factor Receptor 3 (FGFR3) occur in 10-20% of metastatic urothelial carcinomas and confer sensitivity to FGFR inhibitors. However, responses to these agents are often short-lived due to the development of acquired resistance. The objective of this study was to identify mechanisms of resistance to FGFR inhibitors in two previously uncharacterised bladder cancer cell lines harbouring FGFR3 fusions and assess rational combination therapies to enhance sensitivity to these agents. METHODS: Acquired resistance to FGFR inhibitors was generated in two FGFR3 fusion harbouring cell lines, SW780 (FGFR3-BAIAP2L1 fusion) and RT4 (FGFR3-TACC3 fusion), by long-term exposure to the FGFR inhibitor BGJ398. Changes in levels of receptor tyrosine kinases were assessed by phospho-RTK arrays and immunoblotting. Changes in cell viability and proliferation were assessed by the Cell-Titre Glo assay and by propidium iodide staining and FACS analysis. RESULTS: Long term treatment of FGFR3-fusion harbouring SW780 and RT4 bladder cancer cell lines with the FGFR inhibitor BGJ398 resulted in the establishment of resistant clones. These clones were cross-resistant to the clinically approved FGFR inhibitor erdafitinib and the covalently binding irreversible FGFR inhibitor TAS-120, but remained sensitive to the MEK inhibitor trametinib, indicating resistance is mediated by alternate activation of MAPK signalling. The FGFR inhibitor-resistant SW780 and RT4 lines displayed increased expression of pERBB3, and strikingly, combination treatment with an FGFR inhibitor and the ATP-competitive pan-ERBB inhibitor AZD8931 overcame this resistance. Notably, rapid induction of pERBB3 and reactivation of pERK also occurred in parental FGFR3 fusion-driven lines within 24 h of FGFR inhibitor treatment, and combination treatment with an FGFR inhibitor and AZD8931 delayed the reactivation of pERBB3 and pERK and synergistically inhibited cell proliferation. CONCLUSIONS: We demonstrate that increased expression of pERBB3 is a key mechanism of adaptive resistance to FGFR inhibitors in FGFR3-fusion driven bladder cancers, and that this also occurs rapidly following FGFR inhibitor treatment. Our findings demonstrate that resistance can be overcome by combination treatment with a pan-ERBB inhibitor and suggest that upfront combination treatment with FGFR and pan-ERBB inhibitors warrants further investigation for FGFR3-fusion harbouring bladder cancers.

RealTalk evidence-based communication training resources: development of conversation analysis-based materials to support training in end-of-life-related health and social care conversations.

Training to enhance healthcare practitioners' capabilities in engaging people in sensitive and end-of life-related conversations is in demand. However, evaluations have either not measured, or found very limited impact on actual practice and patient experience. Training effectiveness is improved when it is based on in-depth evidence, reflects the complexity of real-life interactions, and instils principles adaptable to everyday practice. A relatively new source of in-depth evidence and practice-relevant insights on healthcare interactions is conversation analytic research, a form of observational analysis of real-life interactions. However, conversation analytic research findings have largely been disseminated by and for scientists, rather than clinicians and trainers. We used conversation analytic evidence to develop resources for use by healthcare trainers. The aim was to increase training's evidence-base and authenticity. We further aimed to develop resources applicable to working with learners ranging from novices to advanced practitioners. METHODS:  Using an intervention development approach, we created online video-clips and supplementary written materials for professionals who deliver training, supervision, and support in healthcare communication for staff and students. The materials were reviewed by an advisory group comprising clinicians, lay consultees, educators, and researchers, and piloted by trainers in UK universities, NHS organisations and independent hospices. We refined materials based on their feedback. RESULTS:  The resulting 'RealTalk' resources focus on practices for communicating with patients and their companions about end-of-life and prognosis. Two core training modules were developed, each comprising several patient case studies featuring video-clips from real-life healthcare consultations. The clips featured practices that patients and experienced practitioners use in approaching end-of-life matters. The case studies also included evidence-based descriptions of observable practices and the principles underlying these, alongside transcripts and case synopses. CONCLUSIONS:  RealTalk training resources aim to facilitate evidence-based, experiential and reflective learning, focusing on communication challenges, practices and principles for end-of-life-related interactions. The resources are designed for use by trainers for delivering all levels of training, from introductory to advanced, in both formal and informal training settings. Our development process may serve as a blueprint for the production of future evidence-based training resources based on conversation analytic research.

Communicating with patients and families about illness progression and end of life: a review of studies using direct observation of clinical practice.

BACKGROUND: There is growing recognition that a diverse range of healthcare professionals need competence in palliative approaches to care. Effective communication is a core component of such practice. This article informs evidence-based communication about illness progression and end of life through a rapid review of studies that directly observe how experienced clinicians manage such discussions. METHODS: The current rapid review updates findings of a 2014 systematic review, focussing more specifically on evidence related to illness progression and end-of-life conversations. Literature searches were conducted in nine bibliographic databases. Studies using conversation analysis or discourse analysis to examine recordings of actual conversations about illness progression or end of life were eligible for inclusion in the review. An aggregative approach was used to synthesise the findings of included studies. RESULTS: Following screening, 26 sources were deemed to meet eligibility criteria. Synthesis of study findings identified the structure and functioning of ten communication practices used in discussions about illness progression and end-of-life. CONCLUSION: The ten practices identified underpin five evidence-based recommendations for communicating with patients or family members about illness progression and end of life.

Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation.

Despite recent advances in structural definition of GPCR-G protein complexes, the basis of receptor selectivity between G proteins remains unclear. The Gα12 and Gα13 subtypes together form the least studied group of heterotrimeric G proteins. G protein-coupled receptor 35 (GPR35) has been suggested to couple efficiently to Gα13 but weakly to Gα12. Using combinations of cells genome-edited to not express G proteins and bioluminescence resonance energy transfer-based sensors, we confirmed marked selectivity of GPR35 for Gα13. Incorporating Gα12/Gα13 chimeras and individual residue swap mutations into these sensors defined that selectivity between Gα13 and Gα12 was imbued largely by a single leucine-to-isoleucine variation at position G.H5.23. Indeed, leucine could not be substituted by other amino acids in Gα13 without almost complete loss of GPR35 coupling. The critical importance of leucine at G.H5.23 for GPR35-G protein interaction was further demonstrated by introduction of this leucine into Gαq, resulting in the gain of coupling to GPR35. These studies demonstrate that Gα13 is markedly the most effective G protein for interaction with GPR35 and that selection between Gα13 and Gα12 is dictated largely by a single conservative amino acid variation.-Mackenzie, A. E., Quon, T., Lin, L.-C., Hauser, A. S., Jenkins, L., Inoue, A., Tobin, A. B., Gloriam, D. E., Hudson, B. D., Milligan, G. Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation.

Evidence for the Existence of a CXCL17 Receptor Distinct from GPR35.

The chemokine CXCL17 is associated with the innate response in mucosal tissues but is poorly characterized. Similarly, the G protein-coupled receptor GPR35, expressed by monocytes and mast cells, has been implicated in the immune response, although its precise role is ill-defined. A recent manuscript reported that GPR35 was able to signal in response to CXCL17, which we set out to confirm in this study. GPR35 was readily expressed using transfection systems but failed to signal in response to CXCL17 in assays of ß-arrestin recruitment, inositol phosphate production, calcium flux, and receptor endocytosis. Similarly, in chemotaxis assays, GPR35 did not confirm sensitivity to a range of CXCL17 concentrations above that observed in the parental cell line. We subsequently employed a real time chemotaxis assay (TAXIScan) to investigate the migratory responses of human monocytes and the monocytic cell line THP-1 to a gradient of CXCL17. Freshly isolated human monocytes displayed no obvious migration to CXCL17. Resting THP-1 cells showed a trend toward directional migration along a CXCL17 gradient, which was significantly enhanced by overnight incubation with PGE2 However, pretreatment of PGE2-treated THP-1 cells with the well-characterized GPR35 antagonist ML145 did not significantly impair their migratory responses to CXCL17 gradient. CXCL17 was susceptible to cleavage with chymase, although this had little effect its ability to recruit THP-1 cells. We therefore conclude that GPR35 is unlikely to be a bona fide receptor for CXCL17 and that THP-1 cells express an as yet unidentified receptor for CXCL17.

How to perform and interpret the sweat test.

Cystic fibrosis (CF) is the most common life-threatening autosomal-recessive disease affecting Caucasians in the western world. The sweat test is the main diagnostic test for CF. It is indicated as part of the clinical assessment for infants that have picked up on the national neonatal screening programme. It may also be requested where clinical suspicion of a diagnosis of CF exists despite normal screening results. This article outlines the physiological basis behind sweat testing and the technical aspects of performing the test. Indications for performing the test are also considered. The article aims to provide clinicians with a guide to interpretation of results.

Clinical exome sequencing reveals locus heterogeneity and phenotypic variability of cohesinopathies.

PURPOSE: Defects in the cohesin pathway are associated with cohesinopathies, notably Cornelia de Lange syndrome (CdLS). We aimed to delineate pathogenic variants in known and candidate cohesinopathy genes from a clinical exome perspective. METHODS: We retrospectively studied patients referred for clinical exome sequencing (CES, N = 10,698). Patients with causative variants in novel or recently described cohesinopathy genes were enrolled for phenotypic characterization. RESULTS: Pathogenic or likely pathogenic single-nucleotide and insertion/deletion variants (SNVs/indels) were identified in established disease genes including NIPBL (N = 5), SMC1A (N = 14), SMC3 (N = 4), RAD21 (N = 2), and HDAC8 (N = 8). The phenotypes in this genetically defined cohort skew towards the mild end of CdLS spectrum as compared with phenotype-driven cohorts. Candidate or recently reported cohesinopathy genes were supported by de novo SNVs/indels in STAG1 (N = 3), STAG2 (N = 5), PDS5A (N = 1), and WAPL (N = 1), and one inherited SNV in PDS5A. We also identified copy-number deletions affecting STAG1 (two de novo, one of unknown inheritance) and STAG2 (one of unknown inheritance). Patients with STAG1 and STAG2 variants presented with overlapping features yet without characteristic facial features of CdLS. CONCLUSION: CES effectively identified disease-causing alleles at the mild end of the cohensinopathy spectrum and enabled characterization of candidate disease genes.

Altering the Proteoglycan State of Transforming Growth Factor ß Type III Receptor (TßRIII)/Betaglycan Modulates Canonical Wnt/ß-Catenin Signaling.

Hyperactive Wnt/ß-catenin signaling is linked to cancer progression and developmental abnormalities, making identification of mechanisms controlling Wnt/ß-catenin signaling vital. Transforming growth factor ß type III receptor (TßRIII/betaglycan) is a transmembrane proteoglycan co-receptor that exists with or without heparan and/or chondroitin sulfate glycosaminoglycan (GAG) modifications in cells and has established roles in development and cancer. Our studies here demonstrate that TßRIII, independent of its TGFß co-receptor function, regulates canonical Wnt3a signaling by controlling Wnt3a availability through its sulfated GAG chains. Our findings revealed, for the first time, opposing functions for the different GAG modifications on TßRIII suggesting that Wnt interactions with the TßRIII heparan sulfate chains result in inhibition of Wnt signaling, likely via Wnt sequestration, whereas the chondroitin sulfate GAG chains on TßRIII promote Wnt3a signaling. These studies identify a novel, dual role for TßRIII/betaglycan and define a key requirement for the balance between chondroitin sulfate and heparan sulfate chains in dictating ligand responses with implications for both development and cancer.

Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling.

G protein-coupled receptors (GPCRs) can initiate intracellular signaling cascades by coupling to an array of heterotrimeric G proteins and arrestin adaptor proteins. Understanding the contribution of each of these coupling options to GPCR signaling has been hampered by a paucity of tools to selectively perturb receptor function. Here we employ CRISPR/Cas9 genome editing to eliminate selected G proteins (Gαq and Gα11) or arrestin2 and arrestin3 from HEK293 cells together with the elimination of receptor phosphorylation sites to define the relative contribution of G proteins, arrestins, and receptor phosphorylation to the signaling outcomes of the free fatty acid receptor 4 (FFA4). A lack of FFA4-mediated elevation of intracellular Ca2+ in Gαq/Gα11-null cells and agonist-mediated receptor internalization in arrestin2/3-null cells confirmed previously reported canonical signaling features of this receptor, thereby validating the genome-edited HEK293 cells. FFA4-mediated ERK1/2 activation was totally dependent on Gq/11 but intriguingly was substantially enhanced for FFA4 receptors lacking sites of regulated phosphorylation. This was not due to a simple lack of desensitization of Gq/11 signaling because the Gq/11-dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms, whereas a substantially enhanced ERK1/2 response was only observed for receptors lacking phosphorylation sites and not in arrestin2/3-null cells. In conclusion, we validate CRISPR/Cas9 engineered HEK293 cells lacking Gq/11 or arrestin2/3 as systems for GPCR signaling research and employ these cells to reveal a previously unappreciated interplay of signaling pathways where receptor phosphorylation can impact on ERK1/2 signaling through a mechanism that is likely independent of arrestins.