National Center for Complementary and Integrative Health

PIEZO channels are mechanosensitive ion channels conserved from plants to humans, yet structures exist for only a few mammalian orthologs. We define the structural and functional diversity of Caenorhabditis elegans PEZO-1, a single gene with extensive alternative splicing, by determining cryo-electron microscopy structures of three representative isoforms: G (full length), K (lacking the pore-distal N-terminal blade), and L (missing most of the blade). PEZO-1G displays mechanically evoked currents yet adopts a compact, semi-flattened conformation that significantly differs from the mammalian domes. The blades exhibit a three-step slope architecture stabilized by inter-blade latching among transmembrane helical units, yielding a circular, steering-wheel-like arrangement. A wider cap enables distinct blade-cap contacts that stabilize a "toggle-down" conformation. Isoform K also exhibits mechanically evoked currents, indicating that the pore-distal N-terminal blade is dispensable for mechanoactivation. Computational membrane-deformation modeling indicates that the isoforms impose distinct curvatures on the bilayer. Our findings indicate an evolutionarily distinct architecture for PEZO-1.

Abstract Recent studies determined that in glioblastoma, tumor cells integrate into neural networks, forming synapses with neurons, enhancing neuronal firing facilitating tumor growth. We sought to understand how neuronal activity alters the function of immune cells in glioblastoma using both murine models and freshly acquired human glioblastoma tissue from surgical specimens. We first used chemogenetic stimulation (AAV5-SYN1-hM3Dq) of an RCAS-tva mouse glioblastoma model (Ntv-a Ink4a-Arf-/-) to identify which tumor immune populations are impacted by enhanced neuronal firing in vivo. Using high-dimensional spectral flow cytometry, we found an increase in microglia following stimulation. We then used human glioblastoma tissue to study a possible neuron-myeloid interaction. Immunofluorescent staining of these tumors showed that approximately 50% of the Iba1+ myeloid cells colocalized with the presynaptic neuronal marker synapsin 1, confirming the neuron-myeloid interaction. We then sorted CD11b+ myeloid cells from these patient samples. Bulk RNA-seq revealed high expression of neurotransmitter receptors, predominantly the Gi protein-coupled P2Y12 receptor for ADP. Calcium fluorescence imaging, calcium flow cytometry, and a phagocytic assay showed that the cells were primarily responsive to ADP among the neurotransmitters examined, including glutamate, norepinephrine, GABA, and acetylcholine. Analysis of bulk RNA-seq from 286 brain tumor patients found that P2Y12 had a higher positive correlation with M1-like markers and a negative correlation with M2-like markers than all other neurotransmitter receptor genes and others such as ADRB2, CX3CR1, TMEM119, TREM2, and CSF1R. To understand how the ADP-P2Y12 axis affects myeloid phenotypic function in vivo, we conducted flow cytometry on our SB28 mouse brain tumor tissues. We found that P2Y12-positive microglia had an increased M1-like and decreased M2-like phenotype compared to P2Y12-negative microglia, based on functional marker expression. Overall, we conclude that neuronal excitation affects microglia in the glioblastoma microenvironment, primarily through an ADP-P2Y12 axis, which could be an important target for enhancing response to immunotherapy.

Pain is an inherently salient multidimensional experience that signals potential bodily threats and promotes nocifensive behaviours. Any stimulus can be salient depending on its features and context. This poses a challenge in delineating pain-specific processes in the brain, rather than salience-driven activity. It is thus essential to salience match control (innocuous) stimuli and noxious stimuli, to remove salience effects, when aiming to delineate pain-specific mechanisms. Previous studies have salience-matched either through subjective salience ratings or the skin conductance response (SCR). The construct of salience is not intuitive, and thus, matching through self-report poses challenges. SCR is used as a proxy measure that captures physiological arousal, which overcomes the nebulous construct of salience. However, SCR cannot be used to salience match in real time (ie, during an experiment) and assumes an association between salience and physiological arousal elicited by painful and non-painful stimuli, but this has not been explicitly tested. To determine whether salience and physiological arousal are associated, 41 healthy adults experienced 30 heat pain and 30 non-painful electric stimuli of varying intensities. Stimuli were subjectively matched for salience, and SCR was measured to each presentation. A linear mixed model found no differences in SCR between salience-matched heat and electric stimuli. A mediation analysis showed that salience fully mediated the relationship between stimulus intensity and SCR. In conclusion, salience and physiological arousal are associated, and subjective salience ratings are suitable for salience matching pain with non-painful stimuli. Future work can thus use subjective salience ratings to delineate pain-specific processes.

Although acupuncture points (acupoints) have been used for thousands of years, their exact biological basis remains uncertain. This knowledge gap has hindered both acupuncture research and the integration of acupuncture into clinical care. In response to a consensus in the scientific community about the need for an open-access database where researchers can deposit, share, and compare anatomical and physiological data associated with acupoints, the National Center for Complementary and Integrative Health has funded a project to develop such a database, the Topological Atlas and Repository for Acupoint Research (TARA). TARA will enable researchers to synthesize different types of data to develop a comprehensive understanding of acupoints and help elucidate the mechanisms of acupuncture. For example, one area of research that may benefit from this resource is the developing concept that stimulating points on the surface of the body may have physiological effects through the interstitium-the connective tissue between fascial layers-in addition to effects through pathways within the nervous system.

Objective: Federally Qualified Health Centers (FQHCs) provide comprehensive primary care to underserved populations. While the presence of chiropractic services in these multidisciplinary systems is of growing interest, little is known. The purpose of this study is to identify and map where Doctors of Chiropractic (DCs) are employed or providing care within FQHCs and describe their employment characteristics. Methods: Websites for FQHC clinics identified by the Health Resources and Services Administration were reviewed to determine whether chiropractic services are offered and/or a DC is employed at that clinic. Identified DCs were invited to participate in a cross-sectional survey, which included questions about their employment. Analysis of survey data utilized descriptive statistics and content analysis of open-ended questions. Results: We identified 233 DCs working full- or part-time in 146 of 1537 (9.5%) FQHC systems, with two thirds of those employing more than one DC. Chiropractic services are delivered at FQHCs in 28/50 U.S. states, 75.0% of which offer a chiropractic benefit in their state Medicaid program. California had the largest concentration of FQHCs offering chiropractic services (65/146, 44.5%). Of surveys sent to 206 viable DC e-mail addresses, 101 were completed (49.0% response rate). Most DCs responded to personal (39.0%) or advertised (26.0%) solicitation for employment by the FQHC, while some DCs (14.0%) initiated the opportunity. Average employment at the FQHC was 5.1 years. Credentialing levels and compensation structures were inconsistent. DCs predominantly spend their time on clinical care (mean 88.0%) compared with administrative tasks (mean 10.4%). Conclusion: This study provides important baseline information about the presence of chiropractic within FQHCs and DCs employment characteristics. Future research should include exploring the roles DCs fulfill within FQHCs, skills necessary for successful collaboration, and barriers to incorporating chiropractic services within these systems.