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Coronary artery calcification (CAC), a measure of subclinical atherosclerosis, predicts future symptomatic coronary artery disease (CAD). Identifying genetic risk factors for CAC may point to new therapeutic avenues for prevention. Currently, there are only four known risk loci for CAC identified from genome-wide association studies (GWAS) in the general population. Here we conducted the largest multi-ancestry GWAS meta-analysis of CAC to date, which comprised 26,909 individuals of European ancestry and 8,867 individuals of African ancestry. We identified 11 independent risk loci, of which eight were new for CAC and five had not been reported for CAD. These new CAC loci are related to bone mineralization, phosphate catabolism and hormone metabolic pathways. Several new loci harbor candidate causal genes supported by multiple lines of functional evidence and are regulators of smooth muscle cell-mediated calcification ex vivo and in vitro. Together, these findings help refine the genetic architecture of CAC and extend our understanding of the biological and potential druggable pathways underlying CAC.

Background: Exposure to Δ9-tetrahydrocannabinol (THC) is an established risk factor for later-life neuropsychiatric vulnerability, including mood- and anxiety-related symptoms. The psychotropic effects of THC on affect and anxiogenic behavioral phenomena are known to target the striatal network, particularly the nucleus accumbens, a neural region linked to mood and anxiety disorder pathophysiology. THC may increase neuroinflammatory responses via the redox system and dysregulate inhibitory and excitatory neural balance in various brain circuits, including the striatum. Thus, interventions that can induce antioxidant effects may counteract the neurodevelopmental impacts of THC exposure. Methods: In the current study, we used an established preclinical adolescent rat model to examine the impacts of adolescent THC exposure on various behavioral, molecular, and neuronal biomarkers associated with increased mood and anxiety disorder vulnerability. Moreover, we investigated the protective properties of the antioxidant N-acetylcysteine against THC-related pathology. Results: We demonstrated that adolescent THC exposure induced long-lasting anxiety- and depressive-like phenotypes concomitant with differential neuronal and molecular abnormalities in the two subregions of the nucleus accumbens, the shell and the core. In addition, we report for the first time that N-acetylcysteine can prevent THC-induced accumbal pathophysiology and associated behavioral abnormalities. Conclusions: The preventive effects of this antioxidant intervention highlight the critical role of redox mechanisms underlying cannabinoid-induced neurodevelopmental pathology and identify a potential intervention strategy for the prevention and/or reversal of these pathophysiological sequelae.

Background/Objective: Immune checkpoint inhibitors (ICI), including Programmed Cell Death 1, Programmed Cell Death Ligand 1, and Cytotoxic T-lymphocyte Associated Antigen 4 inhibitors, upregulate T-cell responses against tumor cells and are becoming a cornerstone in the treatment of various advanced solid and hematological cancers. Mulvihill-Smith Syndrome (MSS) is a rare genetic syndrome that has been associated with metabolic abnormalities and early-onset tumors, including malignancies. We report the first known case of ICI-induced hyponatremia attributable to syndrome of inappropriate antidiuretic hormone ADH release (SIADH) in a patient with MSS. Case Report: A 23-year-old female patient with MSS and hepatocellular carcinoma presented with recurrent hyponatremia. Assessment of fluid status and electrolytes revealed a euvolemic, hypotonic process consistent with SIADH shortly after initiating adjuvant therapy with atezolizumab, a Programmed Cell Death Ligand 1 inhibitor. Discussion: Endocrine etiologies for euvolemic hypotonic hyponatremia, including adrenal insufficiency and hypothyroidism, were excluded. The diagnosis of SIADH was confirmed based on electrolyte and osmolality studies. Sodium levels normalized with fluid restriction. Given the onset of hyponatremia 30 days after atezolizumab initiation, we posit that atezolizumab triggered severe hyponatremia due to SIADH. Conclusion: With the expanding utilization of ICIs, including in patients predisposed to malignancies such as MSS, vigilant monitoring for ICI-mediated electrolyte imbalances is crucial. Monitoring for hyponatremia and SIADH in the setting of ICI therapy is recommended.

Abstract Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods.

Abstract Analysis of single cancer cells is critical to obtain accurate patient diagnosis and prognosis. In this work, we report the selective dielectrophoretic capture and electrochemical analysis of single melanoma cells at an array of interlocked spiral bipolar electrodes (iBPEs). Following dielectrophoretic capture, individual melanoma cells are hydrodynamically transferred into picoliter‐scale chambers for subsequent analysis. The interlocked spiral end of the iBPE (the sensing pole) is utilized to read out an electrochemical enzyme‐linked immunosorbent assay (eELISA), which quantifies the expression of a cell surface antigen, melanoma cell adhesion marker (MCAM). The opposite pole of each BPE is located in a fluidically isolated compartment containing reagents for electrogenerated chemiluminescence (ECL), such that luminescence reports iBPE current. In a preliminary device design, the ECL intensity was insufficient to detect MCAM expression on single cells. To achieve single‐cell analysis, we decreased the gap size between the interlocked spirals tenfold (5.0 μm to 0.5 μm), thereby creating a more sensitive biosensor by enhanced redox cycling of the product of eELISA. This work is significant because it allows for the selective isolation and sensitive analysis of individual melanoma cells in a device amenable to point‐of‐care (POC) application by combining dielectrophoresis (DEP) with interdigitated bipolar electrodes (IDBPEs).

BackgroundClinical decision support (CDS) tools that incorporate machine learning–derived content have the potential to transform clinical care by augmenting clinicians’ expertise. To realize this potential, such tools must be designed to fit the dynamic work systems of the clinicians who use them. We propose the use of academic detailing—personal visits to clinicians by an expert in a specific health IT tool—as a method for both ensuring the correct understanding of that tool and its evidence base and identifying factors influencing the tool’s implementation. ObjectiveThis study aimed to assess academic detailing as a method for simultaneously ensuring the correct understanding of an emergency department–based CDS tool to prevent future falls and identifying factors impacting clinicians’ use of the tool through an analysis of the resultant qualitative data. MethodsPreviously, our team designed a CDS tool to identify patients aged 65 years and older who are at the highest risk of future falls and prompt an interruptive alert to clinicians, suggesting the patient be referred to a mobility and falls clinic for an evidence-based preventative intervention. We conducted 10-minute academic detailing interviews (n=16) with resident emergency medicine physicians and advanced practice providers who had encountered our CDS tool in practice. We conducted an inductive, team-based content analysis to identify factors that influenced clinicians’ use of the CDS tool. ResultsThe following categories of factors that impacted clinicians’ use of the CDS were identified: (1) aspects of the CDS tool’s design (2) clinicians’ understanding (or misunderstanding) of the CDS or referral process, (3) the busy nature of the emergency department environment, (4) clinicians’ perceptions of the patient and their associated fall risk, and (5) the opacity of the referral process. Additionally, clinician education was done to address any misconceptions about the CDS tool or referral process, for example, demonstrating how simple it is to place a referral via the CDS and clarifying which clinic the referral goes to. ConclusionsOur study demonstrates the use of academic detailing for supporting the implementation of health information technologies, allowing us to identify factors that impacted clinicians’ use of the CDS while concurrently educating clinicians to ensure the correct understanding of the CDS tool and intervention. Thus, academic detailing can inform both real-time adjustments of a tool’s implementation, for example, refinement of the language used to introduce the tool, and larger scale redesign of the CDS tool to better fit the dynamic work environment of clinicians.

ABSTRACT Although genetic manipulation is one of the hallmarks of model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the black-legged tick Ixodes scapularis, an arthropod that serves as a vector for several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats [(CRISPR)/CRISPR-associated protein 9 (Cas9)] genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology-dependent recombination. Targeting the E3 ubiquitin ligase x-linked inhibitor of apoptosis (xiap) and its substrate p47 led to an alteration in molecular signaling within the immune deficiency network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for the genetic engineering of I. scapularis ticks, which currently limit efficient and scalable molecular genetic screens in vivo.IMPORTANCEGenetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology-dependent recombination. We ablated the expression of xiap and p47, two signaling molecules present in the immune deficiency (IMD) pathway of Ixodes scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agent Anaplasma phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the black-legged tick I. scapularis.

Surface enhanced Raman scattering (SERS) is a powerful tool for vibrational spectroscopy, providing orders of magnitude increase in chemical sensitivity compared to spontaneous Raman scattering. Yet it remains a challenge to synthesize robust, uniform SERS substrates quickly and easily. Lithographic approaches to produce substrates can achieve high, uniform sensitivity but are expensive and complex, thus difficult to scale. Facile solution-phase chemical approaches often result in unreliable SERS substrates due to heterogeneous arrangement of "hot spots" throughout the material. Here we demonstrate the synthesis and characterization of a homogeneous gold nanofoam (AuNF) substrate produced by a rapid, one-pot, four-ingredient synthetic approach. AuNFs are rapidly nucleated with macroscale porosity and then chemically roughened to produce nanoscale features that confer homogeneous and high signal enhancement (~109) across large areas, a comparable performance to lithographically produced substrates.

As a rapid, label-free, non-destructive analytical measurement requiring little to no sample preparation, Raman spectroscopy shows great promise for liquid biopsy cancer detection and diagnosis. We carried out Raman analysis and mass spectrometry of plasma and saliva from more than 50 subjects in a cohort of head and neck cancer patients and benign controls (e.g., patients with benign oral masses). Unsupervised data models were built to assess diagnostic performance. Raman spectra collected from either biofluid provided moderate performance to discriminate cancer samples. However, by fusing together the Raman spectra of plasma and saliva for each patient, subsequent analytical models delivered an impressive sensitivity, specificity, and accuracy of 96.3%, 85.7%, and 91.7%, respectively. We further confirmed that the metabolites driving the differences in Raman spectra for our models are among the same ones that drive mass spectrometry models, unifying the two techniques and validating the underlying ability of Raman to assess metabolite composition. This study bolsters the relevance of Raman to provide additive value by probing the unique chemical compositions across biofluid sources. Ultimately, we show that a simple data augmentation routine of fusing plasma and saliva spectra provided significantly higher clinical value than either biofluid alone, pushing forward the potential of clinical translation of Raman spectroscopy for liquid biopsy cancer diagnostics.

BackgroundTetraspanin expression of extracellular vesicles (EVs) is often used as a surrogate for their detection and classification, a practice that typically assumes their consistent expression across EV sources.ResultsHere we demonstrate that there are distinct patterns in colocalization of tetraspanin expression of EVs enriched from a variety of in vitro and in vivo sources. We report an optimized method for the use of single particle antibody-capture and fluorescence detection to identify subpopulations according to tetraspanin expression and compare our findings with nanoscale flow cytometry. We found that tetraspanin profile is consistent from a given EV source regardless of isolation method, but that tetraspanin profiles are distinct across various sources. Tetraspanin profiles measured by flow cytometry do not totally agree, suggesting that limitations in subpopulation detection significantly impact apparent protein expression. We further analyzed tetraspanin expression of single EVs captured non-specifically, revealing that tetraspanin capture can bias the apparent multiplexed tetraspanin profile. Finally, we demonstrate that this bias can have significant impact on diagnostic sensitivity for tumor-associated EV surface markers.ConclusionOur findings may reveal key insights into protein expression heterogeneity of EVs that better inform EV capture and detection platforms for diagnostic or other downstream use.

Tetraspanin expression of extracellular vesicles (EVs) is often used as a surrogate for their general detection and classification from background contaminants. This common practice typically assumes a consistent expression of tetraspanins across EV sources, thus obscuring subpopulations of variable or limited tetraspanin expression. While some recent studies indicate differential expression of tetraspanins across bulk isolated EVs, here we present analysis of single EVs isolated using various field-standard methods from a variety of in vitro and in vivo sources to identify distinct patterns in colocalization of tetraspanin expression. We report an optimized method for the use of antibodycapture single particle interferometric reflectance imaging sensing (SP-IRIS) and fluorescence detection to identify subpopulations according to tetraspanin expression and compare our findings with nanoscale flow cytometry. Using SP-IRIS and immunofluorescence, we report that tetraspanin profile is consistent from a given EV source regardless of isolation method, but that tetraspanin profiles are distinct across various sources. Tetraspanin profiles as measured by flow cytometry do not share similar trends, suggesting that limitations in subpopulation detection significantly impact apparent protein expression. We further analyzed tetraspanin expression of single EVs captured non-specifically, revealing that tetraspanin capture can bias the apparent multiplexed tetraspanin profile. Finally, we demonstrate that this bias can have significant impact on diagnostic sensitivity for tumor-associated EV surface markers. Our findings may reveal key insights into the complexities of the EV biogenesis and signaling pathways and better inform EV capture and detection platforms for diagnostic or other downstream use.