Targeted elimination of G proteins and arrestins defines their specific contributions to both intensity and duration of G protein-coupled receptor signalling

G protein-coupled receptors (GPCRs) can initiate intracellular signalling 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 signalling 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 signalling 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 signalling 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 signalling because the Gq/11-dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms whilst 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 signalling research and employ these cells to reveal a previously unappreciated interplay of signalling pathways where receptor phosphorylation can impact on ERK1/2 signalling through a mechanism that is likely independent of arrestins

Retrospective Analysis: Most Common Diagnoses Seen in a Primary Care Clinic and Corresponding Occupational Therapy Interventions

Background: The literature supports occupational therapy (OT) on primary care (PC) interprofessional teams; however, due to uncertainty regarding the role of, and reimbursement for, OT in PC, few occupational therapists practice in PC. This study addressed the first barrier by identifying the 15 most common diagnoses in a specific PC practice and determining how many of them have evidence-based OT interventions appropriate for their treatments. Method: A retrospective analysis of the ICD-10 codes used by one physician during a 12-month period was completed. These codes were reviewed and categorized using a functional classification system to determine the 15 most frequently occurring diagnostic categories. These diagnostic categories were compared to evidence-based industry standard OT interventions. Results: We reviewed 1,769 distinct ICD-10 codes and condensed them into 58 thematically grouped diagnostic categories. The 15 most frequent categories comprised 64% of the codes used. Evidence-based OT interventions to treat conditions directly, or address related underlying issues and common comorbidities, were identified for 100% of these categories. Discussion: Evidence-based OT interventions exist to treat aspects of 100% of the 15 most common conditions seen in PC. The findings support the growing body of literature that demonstrate the use of occupational therapists as interprofessional PC team members

Hospital Community Benefits After the ACA: The Emerging Federal Framework

Outlines the federal framework on requirements for hospitals to provide community benefit activities in exchange for tax-exempt status under the 2010 healthcare reform, including community health needs assessments; state policy options; and challenges

Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84

Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3′-diindolylmethane. 3,3′-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3′-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predicted and confirmed an integral role of arginine172, located in the 2nd extracellular loop, in the action of decanoic acid but not of 3,3′-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were able to block agonist actions of both decanoic acid and 3,3′-diindolylmethane at GPR84. However, although a radiolabelled form of a related antagonist, [3H]G9543, was able to bind with high affinity to GPR84, this was not competed for by increasing concentrations of either decanoic acid or 3,3′-diindolylmethane and was not affected adversely by mutation of arginine172. These studies identify three separable ligand binding sites within GPR84 and suggest that if medium chain fatty acids are true endogenous regulators then co-binding with a positive allosteric modulator would greatly enhance their function in physiological settings

Depletion of the yeast nuclear exosome subunit Rrp6 results in accumulation of polyadenylated RNAs in a discrete domain within the nucleolus

Copyright © 2007, American Society for Microbiology. All Rights ReservedRecent data reveal that a substantial fraction of transcripts generated by RNA polymerases I, II, and III are rapidly degraded in the nucleus by the combined action of the exosome and a noncanonical poly(A) polymerase activity. This work identifies a domain within the yeast nucleolus that is enriched in polyadenylated RNAs in the absence of the nuclear exosome RNase Rrp6 or the exosome cofactor Mtr4. In normal yeast cells, poly(A)(+) RNA was undetectable in the nucleolus but the depletion of either Rrp6 or Mtr4 led to the accumulation of polyadenylated RNAs in a discrete subnucleolar region. This nucleolar poly(A) domain is enriched for the U14 snoRNA and the snoRNP protein Nop1 but is distinct from the nucleolar body that functions in snoRNA maturation. In strains lacking both Rrp6 and the poly(A) polymerase Trf4, the accumulation of poly(A)(+) RNA was suppressed, suggesting the involvement of the Trf4-Air1/2-Mtr4 polyadenylation (TRAMP) complex. The accumulation of polyadenylated snoRNAs in a discrete nucleolar domain may promote their recognition as substrates for the exosome.T.C. was the recipient of a fellowship from Fundação para a Ciência e Tecnologia, Portugal. This work was supported by the European Commission QLG2-CT-2001-01554. D.T. and L.M. were supported by the Wellcome Trust.info:eu-repo/semantics/publishedVersio

Recommendations for Interpreting and Reporting Silent Carrier and Disease-Modifying Variants in SMA Testing Workflows

Carrier screening; Diagnosis; Spinal muscular atrophyCribado de portadores; Diagnóstico; Atrofia muscular espinalCribratge de portadors; Diagnòstic; Atròfia muscular espinalGenetic testing for SMA diagnosis, newborn screening, and carrier screening has become a significant public health interest worldwide, driven largely by the development of novel and effective molecular therapies for the treatment of spinal muscular atrophy (SMA) and the corresponding updates to testing guidelines. Concurrently, understanding of the underlying genetics of SMA and their correlation with a broad range of phenotypes and risk factors has also advanced, particularly with respect to variants that modulate disease severity or impact residual carrier risks. While testing guidelines are beginning to emphasize the importance of these variants, there are no clear guidelines on how to utilize them in a real-world setting. Given the need for clarity in practice, this review summarizes several clinically relevant variants in the SMN1 and SMN2 genes, including how they inform outcomes for spinal muscular atrophy carrier risk and disease prognosis.This work was partially supported by Grants from Biogen ESP-SMG-17-11256 (to E.F.T. supporting L.B.-P.), Roche and Spanish Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias and co-funded with ERDF funds (Grant No. FIS PI18/000687) (to E.F.T.)

Brief of Scholars of the History and Original Meaning of the Fourth Amendment as Amici Curiae in Support of Petitioner, Carpenter v. United States, No. 16-402 (U.S. Aug. 14, 2017)

Obtaining and examining cell site location records to find a person is a “search” in any normal sense of the word — a search of documents and a search for a person and her personal effects. It is therefore a “search” within the meaning of the Fourth Amendment in that it constitutes “examining,” “exploring,” “looking through,” “inquiring,” “seeking,” or “trying to find.” Nothing about the text of the Fourth Amendment, or the historical backdrop against which it was adopted, suggests that “search” should be construed more narrowly as, for example, intrusions upon subjectively manifested expectations of privacy that society is prepared to recognize as reasonable.Entrusting government agents with unfettered discretion to conduct searches using cell site location information undermines Fourth Amendment rights. The Amendment guarantees “[t]he right of the people to be secure in their persons, houses, papers, and effects, against unreasonable searches.” The Framers chose that language deliberately. It reflected the insecurity they suffered at the hands of “writs of assistance,” a form of general warrant that granted state agents broad discretion to search wherever they pleased. Such arbitrary power was “unreasonable” to the Framers, being “against the reason of the common law,” and it was intolerable because of its oppressive impact on “the people” as a whole. As emphasized in one of the seminal English cases that inspired the Amendment, this kind of general power to search was “totally subversive of the liberty of the subject.” James Otis’s famous speech denouncing a colonial writ of assistance similarly condemned those writs as “the worst instrument of arbitrary power,” placing “the liberty of every man in the hands of every petty officer.” Thus, although those who drafted and ratified the Fourth Amendment could not have anticipated cellphone technology, they would have recognized the dangers inherent in any state claim of unlimited authority to conduct searches for evidence of criminal activity. Cell site location information provides insight into where we go and what we do. Because this information is constantly generated and can be retrieved by the government long after the activities it memorializes have taken place, unfettered government access to cell site location information raises the specter of general searches and undermines the security of “the people.

Retrospective Analysis: Most Common Diagnoses Seen in a Primary Care Clinic and Corresponding Occupational Therapy Interventions

Background: The literature supports occupational therapy (OT) on primary care (PC) interprofessional teams; however, due to uncertainty regarding the role of, and reimbursement for, OT in PC, few occupational therapists practice in PC. This study addressed the first barrier by identifying the 15 most common diagnoses in a specific PC practice and determining how many of them have evidence-based OT interventions appropriate for their treatments. Method: A retrospective analysis of the ICD-10 codes used by one physician during a 12-month period was completed. These codes were reviewed and categorized using a functional classification system to determine the 15 most frequently occurring diagnostic categories. These diagnostic categories were compared to evidence-based industry standard OT interventions. Results: We reviewed 1,769 distinct ICD-10 codes and condensed them into 58 thematically grouped diagnostic categories. The 15 most frequent categories comprised 64% of the codes used. Evidence-based OT interventions to treat conditions directly, or address related underlying issues and common comorbidities, were identified for 100% of these categories. Discussion: Evidence-based OT interventions exist to treat aspects of 100% of the 15 most common conditions seen in PC. The findings support the growing body of literature that demonstrate the use of occupational therapists as interprofessional PC team members

Fatty acid 16:4(n-3) stimulates a GPR120-induced signaling cascade in splenic macrophages to promote chemotherapy resistance

Although chemotherapy is designed to eradicate tumor cells, it also has significant effects on normal tissues. The platinum-induced fatty acid 16:4(n-3) (hexadeca-4,7,10,13-tetraenoic acid) induces systemic resistance to a broad range of DNA-damaging chemotherapeutics. We show that 16:4(n-3) exerts its effect by activating splenic F4/80+/CD11blow macrophages, which results in production of chemoprotective lysophosphatidylcholines (LPCs). Pharmacologic studies, together with analysis of expression patterns, identified GPR120 on F4/80+/CD11blow macrophages as the relevant receptor for 16:4(n-3). Studies that used splenocytes from GPR120-deficient mice have confirmed this conclusion. Activation of the 16:4(n-3)-GPR120 axis led to enhanced cPLA2 activity in these splenic macrophages and secretion of the resistance-inducing lipid mediator, lysophosphatidylcholine(24:1). These studies identify a novel and unexpected function for GPR120 and suggest that antagonists of this receptor might be effective agents to limit development of chemotherapy resistance.—Houthuijzen, J. M., Oosterom, I., Hudson, B. D., Hirasawa, A., Daenen, L. G. M., McLean, C. M., Hansen, S. V. F., van Jaarsveld, M. T. M., Peeper, D. S., Jafari Sadatmand, S., Roodhart, J. M. L., van de Lest, C. H. A., Ulven, T., Ishihara, K., Milligan, G., Voest, E. E. Fatty acid 16:4(n-3) stimulates a GPR120-induced signaling cascade in splenic macrophages to promote chemotherapy resistance

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