Strengthening the US Public Health Lab System: Summary of Coronavirus Disease (COVID-19) After Action Review Workshop.

Context: The COVID-19 pandemic highlighted the significance of public health laboratories across the United States, while also revealing weaknesses in the laboratory system.

Objective: To identify actionable recommendations for building a more resilient public health laboratory system based on previously published lessons learned from COVID-19.

Design, Setting, And Participants: In April 2023, the Association of Public Health Laboratories, in cooperation with RAND , convened a 1.

Does it matter that standard preparedness indices did not predict COVID-19 outcomes?

A number of scientific publications and commentaries have suggested that standard preparedness indices such as the Global Health Security Index (GHSI) and Joint External Evaluation (JEE) scores did not predict COVID-19 outcomes. To some, the failure of these metrics to be predictive demonstrates the need for a fundamental reassessment which better aligns preparedness measurement with operational capacities in real-world stress situations, including the points at which coordination structures and decision-making may fail. There are, however, several reasons why these instruments should not be so easily rejected as preparedness measures.

A peer assessment approach for learning from public health emergencies.

As an alternative to standard quality improvement approaches and to commonly used after action report/improvement plans, we developed and tested a peer assessment approach for learning from singular public health emergencies. In this approach, health departments engage peers to analyze critical incidents, with the goal of aiding organizational learning within and across public health emergency preparedness systems. We systematically reviewed the literature in this area, formed a practitioner advisory panel to help translate these methods into a protocol, applied it retrospectively to case studies, and later field-tested the protocol in two locations.

Building performance-based accountability with limited empirical evidence: performance measurement for public health preparedness.

Efforts to respond to performance-based accountability mandates for public health emergency preparedness have been hindered by a weak evidence base linking preparedness activities with response outcomes. We describe an approach to measure development that was successfully implemented in the Centers for Disease Control and Prevention Public Health Emergency Preparedness Cooperative Agreement. The approach leverages insights from process mapping and experts to guide measure selection, and provides mechanisms for reducing performance-irrelevant variation in measurement data.

Gpr3 stimulates Aβ production via interactions with APP and β-arrestin2.

The orphan G protein-coupled receptor (GPCR) GPR3 enhances the processing of Amyloid Precursor Protein (APP) to the neurotoxic beta-amyloid (Aβ) peptide via incompletely understood mechanisms. Through overexpression and shRNA knockdown experiments in HEK293 cells, we show that β-arrestin2 (βarr2), a GPCR-interacting scaffold protein reported to bind γ-secretase, is an essential factor for GPR3-stimulated Aβ production. For a panel of GPR3 receptor mutants, the degree of stimulation of Aβ production correlates with receptor-β-arrestin binding and receptor trafficking to endocytic vesicles.

Phosphorylation of threonine-19 of PSD-95 by GSK-3β is required for PSD-95 mobilization and long-term depression.

Activity of glycogen synthase kinase-3β (GSK-3β) is required for long-term depression (LTD) via molecular mechanisms that are incompletely understood. Here, we report that PSD-95, a major scaffold protein of the postsynaptic density (PSD) that promotes synaptic strength, is phosphorylated on threonine-19 (T19) by GSK-3β. In cultured rat hippocampal neurons, phosphorylation of T19 increases rapidly with chemical LTD and is attenuated by pharmacologic or genetic suppression of GSK-3β.

In vivo monitoring of recovery from neurodegeneration in conditional transgenic SCA1 mice.

Reliable and objective markers of neuronal function and pathology that can directly assess the effects of neuroprotective treatments in the brain are urgently needed for clinical trials in neurodegenerative diseases. Here we assessed the sensitivity of high field proton magnetic resonance spectroscopy ((1)H MRS) to monitor reversal of neurodegeneration by taking advantage of a well characterized conditional mouse model of spinocerebellar ataxia type 1 (SCA1), where the cerebellar pathology and ataxic phenotype are reversible by doxycycline administration. Transgene expression was suppressed by feeding the mice with chow that contains doxycycline from 6 to 12 weeks of age in an early stage group and from 12 to 24 weeks in a mid-stage group.

PSD-95 is required to sustain the molecular organization of the postsynaptic density.

PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNA interference knockdown of PSD-95 leads to loss of entire patches of PSD material, and electron microscopy tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA receptor-type, but not NMDA receptor-type, structures.

Federal initiative increases community preparedness for public health emergencies.

Policy makers need to know whether federal programs can improve community-level readiness for large-scale public health emergencies, and how to design such programs to increase their impact. This article describes an evaluation of the Cities Readiness Initiative, a federal program to improve communities' ability to dispense medications rapidly during emergencies. The program helped increase staffing, improve planning and partnerships, and streamline medication-dispensing procedures.

TULP3 bridges the IFT-A complex and membrane phosphoinositides to promote trafficking of G protein-coupled receptors into primary cilia.

Primary cilia function as a sensory signaling compartment in processes ranging from mammalian Hedgehog signaling to neuronal control of obesity. Intraflagellar transport (IFT) is an ancient, conserved mechanism required to assemble cilia and for trafficking within cilia. The link between IFT, sensory signaling, and obesity is not clearly defined, but some novel monogenic obesity disorders may be linked to ciliary defects.

Noninvasive detection of presymptomatic and progressive neurodegeneration in a mouse model of spinocerebellar ataxia type 1.

Recent studies with a conditional mouse model of spinocerebellar ataxia type 1 (SCA1) suggest that neuronal dysfunction is reversible and neurodegeneration preventable with early interventions. Success of such interventions will depend on early detection of neuronal and glial abnormalities before cell loss and availability of objective methods to monitor progressive neurodegeneration. Cerebellar concentrations of N-acetylaspartate (NAA), myo-inositol, and glutamate as measured by magnetic resonance spectroscopy (MRS) correlate with ataxia scores of patients with SCA1, indicating their potential as reliable biomarkers of neurodegeneration.

Neurochemical changes in the rat prefrontal cortex following acute phencyclidine treatment: an in vivo localized (1)H MRS study.

Acute phencyclidine (PCP) administration mimics some aspects of schizophrenia in rats, such as behavioral alterations, increased dopaminergic activity and prefrontal cortex dysfunction. In this study, we used single-voxel (1)H-MRS to investigate neurochemical changes in rat prefrontal cortex in vivo before and after an acute injection of PCP. A short-echo time sequence (STEAM) was used to acquire spectra in a 32-microL voxel positioned in the prefrontal cortex area of 12 rats anesthetized with isoflurane.

How can we strengthen the evidence base in public health preparedness?

The lack of frequent real-world opportunities to study preparedness for large-scale public health emergencies has hindered the development of an evidence base to support best practices, performance measures, standards, and other tools needed to assess and improve the nation's multibillion dollar investment in public health preparedness. In this article, we argue that initial funding priorities for public health systems research on preparedness should focus on using engineering-style methods to identify core preparedness processes, developing novel data sources and measures based on smaller-scale proxy events, and developing performance improvement approaches to support the translation of research into practice within the wide variety of public health systems found in the nation.

S-nitrosylation of beta-arrestin regulates beta-adrenergic receptor trafficking.

Signal transduction through G protein-coupled receptors (GPCRs) is regulated by receptor desensitization and internalization that follow agonist stimulation. Nitric oxide (NO) can influence these processes, but the cellular source of NO bioactivity and the effects of NO on GPCR-mediated signal transduction are incompletely understood. Here, we show in cells and mice that beta-arrestin 2, a central element in GPCR trafficking, interacts with and is S-nitrosylated at a single cysteine by endothelial NO synthase (eNOS), and that S-nitrosylation of beta-arrestin 2 is promoted by endogenous S-nitrosogluthathione.

Beta-arrestin scaffolding of phosphatidylinositol 4-phosphate 5-kinase Ialpha promotes agonist-stimulated sequestration of the beta2-adrenergic receptor.

Members of the seven-transmembrane receptor (7TMR) superfamily are sequestered from the plasma membrane following stimulation both to limit cellular responses as well as to initiate novel G protein-independent signaling pathways. The best studied mechanism for 7TMR internalization is via clathrin-coated pits, where clathrin and adaptor protein complex 2 nucleate and polymerize upon encountering the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) to form the outer layer of the clathrin-coated vesicle. Activated receptors are recruited to clathrin-coated pits by beta-arrestins, scaffolding proteins that interact with agonist-occupied 7TMRs as well as adaptor protein complex 2 and clathrin.

Targeting of diacylglycerol degradation to M1 muscarinic receptors by beta-arrestins.

Seven-transmembrane receptor (7TMR) signaling is transduced by second messengers such as diacylglycerol (DAG) generated in response to the heterotrimeric guanine nucleotide-binding protein Gq and is terminated by receptor desensitization and degradation of the second messengers. We show that beta-arrestins coordinate both processes for the Gq-coupled M1 muscarinic receptor. beta-Arrestins physically interact with diacylglycerol kinases (DGKs), enzymes that degrade DAG.

Public health preparedness: evolution or revolution?

The recent emphasis on preparedness has created heightened expectations and has raised questions about the extent to which U.S. public health systems have evolved in recent years.

Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation.

Parathyroid hormone (PTH) regulates calcium homeostasis via the type I PTH/PTH-related peptide (PTH/PTHrP) receptor (PTH1R). The purpose of the present study was to identify the contributions of distinct signaling mechanisms to PTH-stimulated activation of the mitogen-activated protein kinases (MAPK) ERK1/2. In Human embryonic kidney 293 (HEK293) cells transiently transfected with hPTH1R, PTH stimulated a robust increase in ERK activity.

beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor.

Physiological effects of beta adrenergic receptor (beta2AR) stimulation have been classically shown to result from G(s)-dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein beta-arrestins mediate beta2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the beta2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on G(s)-G(i)/protein kinase A (PKA) or beta-arrestins.

Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2.

Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times. This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes. We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo.

Beta-arrestin 2 mediates endocytosis of type III TGF-beta receptor and down-regulation of its signaling.

beta-Arrestins bind to activated seven transmembrane-spanning (7TMS) receptors (G protein-coupled receptors) after the receptors are phosphorylated by G protein-coupled receptor kinases (GRKs), thereby regulating their signaling and internalization. Here, we demonstrate an unexpected and analogous role of beta-arrestin 2 (betaarr2) for the single transmembrane-spanning type III transforming growth factor-beta (TGF-beta) receptor (TbetaRIII, also referred to as betaglycan). Binding of betaarr2 to TbetaRIII was also triggered by phosphorylation of the receptor on its cytoplasmic domain (likely at threonine 841).

The adaptor protein beta-arrestin2 enhances endocytosis of the low density lipoprotein receptor.

Endocytosis of the low density lipoprotein (LDL) receptor (LDLR) in coated pits employs the clathrin adaptor protein ARH. Similarly, agonist-dependent endocytosis of heptahelical receptors in coated pits employs the clathrin adaptor beta-arrestin proteins. In mice fed a high fat diet, we found that homozygous deficiency of beta-arrestin2 increased total and LDL plus intermediate-density lipoprotein cholesterol levels by 23 and 53%, respectively (p < 0.

G-protein-coupled receptor (GPCR) kinase phosphorylation and beta-arrestin recruitment regulate the constitutive signaling activity of the human cytomegalovirus US28 GPCR.

Phosphorylation of G-protein-coupled receptors (GPCRs) by GRKs and subsequent recruitment of beta-arrestins to agonist-occupied receptors serves to terminate or attenuate signaling by blocking G-proteins from further interaction with the receptors. Human cytomegalovirus encodes a GPCR termed US28 that is homologous to the human chemokine family of GPCRs but differs from the cellular receptors in that it maintains high constitutive activity in the absence of agonist. Although US28 is constitutively active, mechanisms that regulate this activity are unknown.

Desensitization, internalization, and signaling functions of beta-arrestins demonstrated by RNA interference.

Beta-arrestins bind to activated G protein-coupled receptor kinase-phosphorylated receptors, which leads to their desensitization with respect to G proteins, internalization via clathrin-coated pits, and signaling via a growing list of "scaffolded" pathways. To facilitate the discovery of novel adaptor and signaling roles of beta-arrestins, we have developed and validated a generally applicable interfering RNA approach for selectively suppressing beta-arrestins 1 or 2 expression by up to 95%. Beta-arrestin depletion in HEK293 cells leads to enhanced cAMP generation in response to beta(2)-adrenergic receptor stimulation, markedly reduced beta(2)-adrenergic receptor and angiotensin II receptor internalization and impaired activation of the MAP kinases ERK 1 and 2 by angiotensin II.