Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis

Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)P3 (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42

Oxygen-sensing neurons reciprocally regulate peripheral lipid metabolism via neuropeptide signaling in <i>Caenorhabditis elegans</i>

<div><p>The mechanisms by which the sensory environment influences metabolic homeostasis remains poorly understood. In this report, we show that oxygen, a potent environmental signal, is an important regulator of whole body lipid metabolism. <i>C</i>. <i>elegans</i> oxygen-sensing neurons reciprocally regulate peripheral lipid metabolism under normoxia in the following way: under high oxygen and food absence, URX sensory neurons are activated, and stimulate fat loss in the intestine, the major metabolic organ for <i>C</i>. <i>elegans</i>. Under lower oxygen conditions or when food is present, the BAG sensory neurons respond by repressing the resting properties of the URX neurons. A genetic screen to identify modulators of this effect led to the identification of a BAG-neuron-specific neuropeptide called FLP-17, whose cognate receptor EGL-6 functions in URX neurons. Thus, BAG sensory neurons counterbalance the metabolic effect of tonically active URX neurons via neuropeptide communication. The combined regulatory actions of these neurons serve to precisely tune the rate and extent of fat loss to the availability of food and oxygen, and provides an interesting example of the myriad mechanisms underlying homeostatic control.</p></div

Group 4 ITI Consensus Report: Patient benefits following implant treatment in partially and fully edentulous patients

Objectives: The aim of Working Group 4 was to address patient benefits associated with implant dentistry. Focused questions on (a) dental patient-reported outcomes (dPROs), (b) improvement in orofacial function, and (c) preservation of orofacial tissues in partially and fully edentulous patients following provision of implant-retained/supported dental prostheses were addressed. Materials and methods: Three systematic reviews formed the basis for discussion. Participants developed statements and recommendations determined by group consensus based on the findings of the systematic reviews. These were then presented and accepted following further discussion and modifications as required by the plenary of the 7th ITI Consensus Conference, taking place in 2023 in Lisbon, Portugal. Results: Edentulous patients wearing complete dentures (CD) experience substantial improvements in overall dPROs and orofacial function following treatment with either complete implant-supported fixed dental prostheses (CIFDP) or implant overdentures (IODs). With respect to dPROs, mandibular IODs retained by two implants are superior to IODs retained by one implant. However, increasing the number of implants beyond two, does not further improve dPROs. In fully edentulous patients, rehabilitation with CIFDP or IOD is recommended to benefit the preservation of alveolar bone and masseter muscle thickness. Conclusions: Completely edentulous patients benefit substantially when at least the mandible is restored using an CIFDP or an IOD compared to CD. In fully edentulous patients, implant prostheses are the best option for tooth replacement. The availability of this treatment modality should be actively promoted in all edentulous communities, including those with limited access and means

Pheromone-sensing neurons regulate peripheral lipid metabolism in <i>Caenorhabditis elegans</i>

It is now established that the central nervous system plays an important role in regulating whole body metabolism and energy balance. However, the extent to which sensory systems relay environmental information to modulate metabolic events in peripheral tissues has remained poorly understood. In addition, it has been challenging to map the molecular mechanisms underlying discrete sensory modalities with respect to their role in lipid metabolism. In previous work our lab has identified instructive roles for serotonin signaling as a surrogate for food availability, as well as oxygen sensing, in the control of whole body metabolism. In this study, we now identify a role for a pair of pheromone-sensing neurons in regulating fat metabolism in C. elegans, which has emerged as a tractable and highly informative model to study the neurobiology of metabolism. A genetic screen revealed that GPA-3, a member of the Gα family of G proteins, regulates body fat content in the intestine, the major metabolic organ for C. elegans. Genetic and reconstitution studies revealed that the potent body fat phenotype of gpa-3 null mutants is controlled from a pair of neurons called ADL(L/R). We show that cAMP functions as the second messenger in the ADL neurons, and regulates body fat stores via the neurotransmitter acetylcholine, from downstream neurons. We find that the pheromone ascr#3, which is detected by the ADL neurons, regulates body fat stores in a GPA-3-dependent manner. We define here a third sensory modality, pheromone sensing, as a major regulator of body fat metabolism. The pheromone ascr#3 is an indicator of population density, thus we hypothesize that pheromone sensing provides a salient 'denominator' to evaluate the amount of food available within a population and to accordingly adjust metabolic rate and body fat levels

Engineering the Melanocortin-4 Receptor to Control Constitutive and Ligand-Mediated Gs Signaling In Vivo

The molecular and functional diversity of G protein–coupled receptors is essential to many physiological processes. However, this diversity presents a significant challenge to understanding the G protein–mediated signaling events that underlie a specific physiological response. To increase our understanding of these processes, we sought to gain control of the timing and specificity of Gs signaling in vivo. We used naturally occurring human mutations to develop two Gs-coupled engineered receptors that respond solely to a synthetic ligand (RASSLs). Our Gs-coupled RASSLs are based on the melanocortin-4 receptor, a centrally expressed receptor that plays an important role in the regulation of body weight. These RASSLs are not activated by the endogenous hormone α-melanocyte-stimulating hormone but respond potently to a selective synthetic ligand, tetrahydroisoquinoline. The RASSL variants reported here differ in their intrinsic basal activities, allowing the separation of the effects of basal signaling from ligand-mediated activation of the Gs pathway in vivo. These RASSLs can be used to activate Gs signaling in any tissue, but would be particularly useful for analyzing downstream events that mediate body weight regulation in mice. Our study also demonstrates the use of human genetic variation for protein engineering

Engineering GPCR signaling pathways with RASSLs

We are creating families of designer G-protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (Gs, Gi, Gq). These new advances are reviewed here to help facilitate the use of these powerful and diverse tools

Hyperactive Neuroendocrine Secretion Causes Size, Feeding, and Metabolic Defects of C. elegans Bardet-Biedl Syndrome Mutants

Bardet-Biedl syndrome, BBS, is a rare autosomal recessive disorder with clinical presentations including polydactyly, retinopathy, hyperphagia, obesity, short stature, cognitive impairment, and developmental delays. Disruptions of BBS proteins in a variety of organisms impair cilia formation and function and the multi-organ defects of BBS have been attributed to deficiencies in various cilia-associated signaling pathways. In C. elegans, bbs genes are expressed exclusively in the sixty ciliated sensory neurons of these animals and bbs mutants exhibit sensory defects as well as body size, feeding, and metabolic abnormalities. Here we show that in contrast to many other cilia-defective mutants, C. elegans bbs mutants exhibit increased release of dense-core vesicles and organism-wide phenotypes associated with enhanced activities of insulin, neuropeptide, and biogenic amine signaling pathways. We show that the altered body size, feeding, and metabolic abnormalities of bbs mutants can be corrected to wild-type levels by abrogating the enhanced secretion of dense-core vesicles without concomitant correction of ciliary defects. These findings expand the role of BBS proteins to the regulation of dense-core-vesicle exocytosis and suggest that some features of Bardet-Biedl Syndrome may be caused by excessive neuroendocrine secretion

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Measuring the burden of infodemics : summary of the methods and results of the fifth WHO infodemic management conference

Background: An infodemic is excess information, including false or misleading information, that spreads in digital and physical environments during a public health emergency. The COVID-19 pandemic has been accompanied by an unprecedented global infodemic that has led to confusion about the benefits of medical and public health interventions, with substantial impact on risk-taking and health-seeking behaviors, eroding trust in health authorities and compromising the effectiveness of public health responses and policies. Standardized measures are needed to quantify the harmful impacts of the infodemic in a systematic and methodologically robust manner, as well as harmonizing highly divergent approaches currently explored for this purpose. This can serve as a foundation for a systematic, evidence-based approach to monitoring, identifying, and mitigating future infodemic harms in emergency preparedness and prevention. Objective: In this paper, we summarize the Fifth World Health Organization (WHO) Infodemic Management Conference structure, proceedings, outcomes, and proposed actions seeking to identify the interdisciplinary approaches and frameworks needed to enable the measurement of the burden of infodemics. Methods: An iterative human-centered design (HCD) approach and concept mapping were used to facilitate focused discussions and allow for the generation of actionable outcomes and recommendations. The discussions included 86 participants representing diverse scientific disciplines and health authorities from 28 countries across all WHO regions, along with observers from civil society and global public health–implementing partners. A thematic map capturing the concepts matching the key contributing factors to the public health burden of infodemics was used throughout the conference to frame and contextualize discussions. Five key areas for immediate action were identified. Results: The 5 key areas for the development of metrics to assess the burden of infodemics and associated interventions included (1) developing standardized definitions and ensuring the adoption thereof; (2) improving the map of concepts influencing the burden of infodemics; (3) conducting a review of evidence, tools, and data sources; (4) setting up a technical working group; and (5) addressing immediate priorities for postpandemic recovery and resilience building. The summary report consolidated group input toward a common vocabulary with standardized terms, concepts, study designs, measures, and tools to estimate the burden of infodemics and the effectiveness of infodemic management interventions. Conclusions: Standardizing measurement is the basis for documenting the burden of infodemics on health systems and population health during emergencies. Investment is needed into the development of practical, affordable, evidence-based, and systematic methods that are legally and ethically balanced for monitoring infodemics; generating diagnostics, infodemic insights, and recommendations; and developing interventions, action-oriented guidance, policies, support options, mechanisms, and tools for infodemic managers and emergency program managers.peer-reviewe