Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity.

The adenosine A1 receptor (A1-AR) is a G-protein-coupled receptor that plays a vital role in cardiac, renal, and neuronal processes but remains poorly targeted by current drugs. We determined a 3.2 Å crystal structure of the A1-AR bound to the selective covalent antagonist, DU172, and identified striking differences to the previously solved adenosine A2A receptor (A2A-AR) structure. Mutational and computational analysis of A1-AR revealed a distinct conformation of the second extracellular loop and a wider extracellular cavity with a secondary binding pocket that can accommodate orthosteric and allosteric ligands. We propose that conformational differences in these regions, rather than amino-acid divergence, underlie drug selectivity between these adenosine receptor subtypes. Our findings provide a molecular basis for AR subtype selectivity with implications for understanding the mechanisms governing allosteric modulation of these receptors, allowing the design of more selective agents for the treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain.

Positive allosteric mechanisms of adenosine A1 receptor-mediated analgesia.

The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.

Elemental Sulfur Promoted Cyclization of Aryl Hydrazones and Aryl Isothiocyanates Yielding 2-Imino-1,3,4-thiadiazoles.

We report a method for using elemental sulfur to facilitate the cyclization of aryl hydrazones and aryl isothiocyanates, affording biorelated 2-imino-1,3,4-thiadiazoles. Reactions progressed in the presence of elemental sulfur, N-methylmorpholine base, and DMSO solvent, while were tolerant of a wide range of functionalities including halogen, nitro, cyano, methylsulfonyl, and heterocyclic groups. The method appears to offer a general pathway for using simple, cheap, and stable reagents to afford triaryl-substituted 2-imino-1,3,4-thiadiazoles under relatively mild conditions.

Impact of the COVID-19 pandemic on mental health and viral suppression among persons living with HIV in western Washington.

The COVID-19 pandemic and social distancing measures elevated stress levels globally, exacerbating mental health challenges for people with HIV (PWH). We examined the effect of COVID-19-related stress on mental health among PWH in western Washington, exploring whether social support and coping self-efficacy were protective. Data on COVID-19-related stress, mental health, social support, and coping self-efficacy were collected using online surveys during the pandemic. Pre-COVID-19 mental health data were available for a subset of participants and were linked with the survey data. In the total sample (N = 373), COVID-19-stress was associated with elevated depression (PHQ-8, ß = 0.21, 95%CI [0.10, 0.32]) and anxiety (GAD-7, ß = 0.28, 95%CI [0.17, 0.39]). Among the subset of respondents with pre-pandemic mental health data (N = 103), COVID-19-related stress was associated with elevated PHQ-8 scores (ß = 0.35, 95%CI [0.15, 0.56]) and GAD-7 scores (ß = 0.35, 95%CI [0.16, 0.54]), adjusted for baseline mental health and other confounders. Coping self-efficacy was negatively associated with GAD-7 scores (ß = -0.01, 95%CI [-0.01, 0.00]), while social support was negatively associated with PHQ-8 scores (ß = -0.06, 95%CI [-0.12, -0.01]). Viral suppression before and during the pandemic did not differ among participants with available data. While COVID-19-related stress predicted elevated depression and anxiety symptoms among PWH, social support and coping self-efficacy were protective.

Cobalt ferrite nanoparticles for annulation of C3-substituted nitroarenes and aryl isothiocyanates.

The synthesis of medicinally relevant N-aryl-substituted 2-aminobenzothiazoles often uses 2-aminothiophenol derivatives, which are not commercially abundant, as starting materials. Herein, we report a method for the annulation of C3-substituted nitroarenes and aryl isothiocyanates towards the synthesis of 2-aminobenzothiazoles. Reactions proceeded in the presence of cobalt ferrite nanoparticles as a catalyst, DABCO as a base, and DMF as a promoter. The cobalt ferrite nanoparticles could be recovered after each run and reused up to 3 times while the product yield was not diminished. Our method appears to be the first example of the direct use of substituted nitroarenes for yielding 2-aminobenzothiazoles.

Synthesis of 2-benzyl benzoxazoles and benzothiazoles via elemental sulfur promoted cyclization of styrenes with 2-nitrophenols and N,N-dialkyl-3-nitroanilines.

Herein we report a method for affording 2-benzyl benzoxazoles from substituted styrenes and 2-nitrophenols. The success of this method relies on the use of simple reagents, namely elemental sulfur and DABCO. A combination of identical reagents was utilized for the annulation of styrenes with N,N-dialkyl-3-nitroanilines to afford 2-benzyl benzothiazoles. Overall, benzoxazoles and benzothiazoles bearing useful functionalities such as halogens, amines, and heterocyclic groups were isolated in moderate to good yields. Our methods are a rare example of divergent transformations of substituted nitroarenes towards 2-benzyl benzoxazoles and benzothiazoles.

Development and validation of multiplex real-time PCR for simultaneous detection of six bacterial pathogens causing lower respiratory tract infections and antimicrobial resistance genes.

BACKGROUND: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae and Staphylococcus aureus are major bacterial causes of lower respiratory tract infections (LRTIs) globally, leading to substantial morbidity and mortality. The rapid increase of antimicrobial resistance (AMR) in these pathogens poses significant challenges for their effective antibiotic therapy. In low-resourced settings, patients with LRTIs are prescribed antibiotics empirically while awaiting several days for culture results. Rapid pathogen and AMR gene detection could prompt optimal antibiotic use and improve outcomes. METHODS: Here, we developed multiplex quantitative real-time PCR using EvaGreen dye and melting curve analysis to rapidly identify six major pathogens and fourteen AMR genes directly from respiratory samples. The reproducibility, linearity, limit of detection (LOD) of real-time PCR assays for pathogen detection were evaluated using DNA control mixes and spiked tracheal aspirate. The performance of RT-PCR assays was subsequently compared with the gold standard, conventional culture on 50 tracheal aspirate and sputum specimens of ICU patients. RESULTS: The sensitivity of RT-PCR assays was 100% for K. pneumoniae, A. baumannii, P. aeruginosa, E. coli and 63.6% for S. aureus and the specificity ranged from 87.5% to 97.6%. The kappa correlation values of all pathogens between the two methods varied from 0.63 to 0.95. The limit of detection of target bacteria was 1600 CFU/ml. The quantitative results from the PCR assays demonstrated 100% concordance with quantitative culture of tracheal aspirates. Compared to culture, PCR assays exhibited higher sensitivity in detecting mixed infections and S. pneumoniae. There was a high level of concordance between the detection of AMR gene and AMR phenotype in single infections. CONCLUSIONS: Our multiplex quantitative RT-PCR assays are fast and simple, but sensitive and specific in detecting six bacterial pathogens of LRTIs and their antimicrobial resistance genes and should be further evaluated for clinical utility.

Structure of the adenosine-bound human adenosine A1 receptor-Gi complex.

The class A adenosine A1 receptor (A1R) is a G-protein-coupled receptor that preferentially couples to inhibitory Gi/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A1R in complex with adenosine and heterotrimeric Gi2 protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive A1R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A1R primarily via amino acids in the C terminus of the Gαi α5-helix, concomitant with a 10.5 Å outward movement of the A1R transmembrane domain 6. Comparison with the agonist-bound ß2 adrenergic receptor-Gs-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A1R structure provides molecular insights into receptor and G-protein selectivity.

Metabolic Engineering of Corynebacterium glutamicum for the Production of Flavonoids and Stilbenoids.

Flavonoids and stilbenoids, crucial secondary metabolites abundant in plants and fungi, display diverse biological and pharmaceutical activities, including potent antioxidant, anti-inflammatory, and antimicrobial effects. However, conventional production methods, such as chemical synthesis and plant extraction, face challenges in sustainability and yield. Hence, there is a notable shift towards biological production using microorganisms like Escherichia coli and yeast. Yet, the drawbacks of using E. coli and yeast as hosts for these compounds persist. For instance, yeast's complex glycosylation profile can lead to intricate protein production scenarios, including hyperglycosylation issues. Consequently, Corynebacterium glutamicum emerges as a promising alternative, given its adaptability and recent advances in metabolic engineering. Although extensively used in biotechnological applications, the potential production of flavonoid and stilbenoid in engineered C. glutamicum remains largely untapped compared to E. coli. This review explores the potential of metabolic engineering in C. glutamicum for biosynthesis, highlighting its versatility as a cell factory and assessing optimization strategies for these pathways. Additionally, various metabolic engineering methods, including genomic editing and biosensors, and cofactor regeneration are evaluated, with a focus on C. glutamicum. Through comprehensive discussion, the review offers insights into future perspectives in production, aiding researchers and industry professionals in the field.

Advancing Urology Resident Surgical Autonomy.

PURPOSE OF REVIEW: This paper aims to survey current literature on urologic graduate medical education focusing on surgical autonomy. RECENT FINDINGS: Affording appropriate levels of surgical autonomy has a key role in the education of urologic trainees and perceived preparedness for independent practice. Recent studies in surgical resident autonomy have demonstrated a reduction in autonomy for trainees in recent years. Efforts to advance the state of modern surgical training include creation of targeted curricula, enhanced with use of surgical simulation, and structured feedback. Decline in surgical autonomy for urology residents may influence confidence after completion of their residency. Further study is needed into the declining levels of urology resident autonomy, how it affects urologists entering independent practice, and what interventions can advance autonomy in modern urologic training.

Swept under the carpet: a qualitative study of patient perspectives on Long COVID, treatments, services, and mental health.

BACKGROUND: A constellation of often disabling long-term physical symptoms enduring after an acute SARS-COV-2 infection is commonly referred to as Long COVID. Since Long COVID is a new clinical entity, research is required to clarify treatment needs and experiences of individuals affected. This qualitative descriptive study aimed to provide insight into Long COVID treatment and service experiences and preferences of individuals experiencing Long COVID and the intersections with mental health. METHODS: The study was conducted out of a tertiary care mental health hospital, with online recruitment from the community across Canada. A total of 47 individuals (average age = 44.9) participated in one of 11 focus groups between June and December 2022. Five focus groups were conducted with participants who had pre-existing mental health concerns prior to contracting SARS-CoV-2, and six were with people with Long COVID but without pre-existing mental health concerns. A semi-structured interview guide asked about service experiences and service preferences, including mental health and well-being services. Discussions were recorded, transcribed, and analyzed using codebook thematic analysis. RESULTS: When accessing services for Long COVID, patients experienced: (1) systemic barriers to accessing care, and (2) challenges navigating the unknowns of Long COVID, leading to (3) negative impacts on patient emotional well-being and recovery. Participants called for improvements in Long COVID care, with a focus on: (1) developing Long COVID-specific knowledge and services, (2) enhancing support for financial well-being, daily living, and building a Long COVID community, and (3) improving awareness and the public representation of Long COVID. CONCLUSIONS: Substantial treatment barriers generate considerable burden for individuals living with Long COVID. There is a pressing need to improve treatment, social supports, and the social representation of Long COVID to create integrated, accessible, responsive, and ongoing support systems.

Interventions for mental health, cognition, and psychological wellbeing in long COVID: a systematic review of registered trials.

BACKGROUND: Among patients diagnosed with COVID-19, a substantial proportion are experiencing ongoing symptoms for months after infection, known as 'long COVID'. Long COVID is associated with a wide range of physical and neuropsychological symptoms, including impacts on mental health, cognition, and psychological wellbeing. However, intervention research is only beginning to emerge. This systematic review synthesizes currently registered trials examining interventions for mental health, cognition, and psychological wellbeing in patients with long COVID. METHODS: Standard systematic review guidelines were followed. Trials registered in two large trial registries in 2020 to May 2022 were reviewed. Included studies were narratively synthesized by type of intervention and a risk-of-bias assessment was conducted. RESULTS: Forty-two registered trials were included, with a total target sample size of 5814 participants. These include 11 psychological interventions, five pharmacological and other medical interventions, and five evaluating herbal, nutritional, or natural supplement interventions. An additional nine trials are examining cognitive and neurorehabilitation interventions and 12 are examining physiotherapy or physical rehabilitation. Most trials are randomized, but many are feasibility trials; trials are evaluating a wide spectrum of outcomes. CONCLUSIONS: While there is a newly emerging body of research testing interventions for mental health, cognition, and psychological wellbeing in long COVID, the breadth and scope of the research remains limited. It is urgently incumbent on researchers to expand upon the intervention research currently under way, in order to generate high-quality evidence on a wide range of candidate interventions for diverse long COVID patient populations.

Resources for Enhancing Alzheimer's Caregiver Health in Vietnam (REACH VN): Exploratory Analyses of Outcomes of a Cluster Randomized Controlled Trial to Test the Feasibility and Preliminary Efficacy of a Family Dementia Caregiver Intervention in Vietnam.

OBJECTIVE: Few support services and caregiving interventions exist to support family caregivers in low- and middle-income countries (LMIC). This paper presents exploratory analyses of outcomes of Resources for Enhancing Alzheimer's Caregiver Health in Vietnam (REACH VN). METHODS: A cluster randomized controlled trial (RCT) was conducted in Soc Son, a semi-rural area in Hanoi. Nine clusters with 60 caregivers were randomized to either an enhanced control group or REACH VN, an in-home, multicomponent, family caregiver support intervention delivered over two to three months. Outcomes were assessed at baseline and three months. RESULTS: Caregivers in the intervention group experienced a significantly greater reduction in frustration levels compared to those in the control group. There were differences in other outcomes (e.g., care recipient problem behaviors and associated caregiver bother) favoring the intervention condition that did not reach statistical significance. CONCLUSION: We found additional evidence that REACH VN is a promising intervention to improve family caregiver outcomes in Vietnam.

KLF4 (Kruppel-Like Factor 4)-Dependent Perivascular Plasticity Contributes to Adipose Tissue inflammation.

OBJECTIVE: Smooth muscle cells and pericytes display remarkable plasticity during injury and disease progression. Here, we tested the hypothesis that perivascular cells give rise to Klf4-dependent macrophage-like cells that augment adipose tissue (AT) inflammation and metabolic dysfunction associated with diet-induced obesity (DIO). Approach and Results: Using Myh11-CreERT2 eYFP (enhanced yellow fluorescent protein) mice and flow cytometry of the stromovascular fraction of epididymal AT, we observed a large fraction of smooth muscle cells and pericytes lineage traced eYFP+ cells expressing macrophage markers. Subsequent single-cell RNA sequencing, however, showed that the majority of these cells had no detectable eYFP transcript. Further exploration revealed that intraperitoneal injection of tamoxifen in peanut oil, used for generating conditional knockout or reporter mice in thousands of previous studies, resulted in large increase in the autofluorescence and false identification of macrophages within epididymal AT as being eYFP+; and unintended proinflammatory consequences. Using newly generated Myh11-DreERT2tdTomato mice given oral tamoxifen, we virtually eliminated the problem with autofluorescence and identified 8 perivascular cell dominated clusters, half of which were altered upon DIO. Given that perivascular cell KLF4 (kruppel-like factor 4) can have beneficial or detrimental effects, we tested its role in obesity-associated AT inflammation. While smooth muscle cells and pericytes-specific Klf4 knockout (smooth muscle cells and pericytes Klf4Δ/Δ) mice were not protected from DIO, they displayed improved glucose tolerance upon DIO, and showed marked decreases in proinflammatory macrophages and increases in LYVE1+ lymphatic endothelial cells in the epididymal AT. CONCLUSIONS: Perivascular cells within the AT microvasculature dynamically respond to DIO and modulate tissue inflammation and metabolism in a KLF4-dependent manner.

Thermal induced changes of rice straw phytolith in relation to arsenic release: A perspective of rice straw arsenic under open burning.

On-site open burning is a common practice for handling rice straw, but its negative impacts, e.g., biomass loss and air pollution, are largely debated worldwide. To address the negative effects of open burning, many efforts have been made to 'ignite' worldwide bans. However, these bans are likely based on a singular view in which some positive aspects of open burning are overlooked. In this study, we aimed to determine the thermal-induced changes of straw and straw arsenic (As) under open burning and heat-treatments (in the temperature range from 300 to 900 °C). It was found that silica phase in rice straw (so-called phytolith) can encapsulate As in its structure. Open burning or heat-treatment of straw resulted in a tighter association of As and phytolith, thereby reducing dissolution of As. We proposed an opinion that open burning causes air pollution, but it can increase the activity of phytolith in sequestrating As, enabling delayed As cycle in rice ecosystems. The combat of on-site open burning of rice straw to reduce air pollution will alter straw handling routines, thereby changing the cycle of straw phytolith and the route of straw As.

Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis.

BACKGROUND: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. METHODS: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. RESULTS: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11-, Lgals3+ population with a chondrocyte-like gene signature that was markedly reduced with SMC-Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. CONCLUSIONS: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3+ osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.

Fast and Sensitive Real-Time PCR Detection of Major Antiviral-Drug Resistance Mutations in Chronic Hepatitis B Patients by Use of a Predesigned Panel of Locked-Nucleic-Acid TaqMan Probes.

We developed a novel real-time PCR assay that simultaneously evaluates 11 major nucleos(t)ide antiviral (NA) drug resistance mutations (mt) in chronic hepatitis B patients (CHB), including L180M, M204I/V, and V207M (lamivudine [LMV] resistance), N/H238A/T (adefovir [ADF] resistance), which are circulating in Vietnam; and T184G/L, S202I, and M250V (entecavir [ETV] resistance) and A194T (tenofovir resistance), which have been recently reported in several studies across the globe. We detected drug-resistant mt in hepatitis B virus (HBV) samples using our predesigned panel of allele-specific locked-nucleic acid (LNA) probes. Our assay had a high sensitivity of 5% in a low-HBV DNA population of ≥5 × 103 IU/ml and was validated in a cohort of 130 treatment-naive children and 98 NA-experienced adults with CHB. Single-point mt for LMV and ADF resistance were detected in 57.7% and 54.1% of the child and adult samples, respectively, with rtV207M (children, 42.3%; adults, 36.7%) and rtN238T/A (children, 15.4%; adults, 16.3%) being the most frequent mt in these populations. Multiple-point mt, including rtL180M-rtM204V- rtN238A and rtL180M-rtM204I, were identified in only two children, resulting in LMV-ADF resistance and reduced ETV susceptibility. In conclusion, this assay accurately identified the mt profile of children (98.4%) and adults (91.2%) with CHB, which is comparable to established methods. This fast and sensitive screening method can be used for the detection of major NA-resistant mt circulating in developing countries, as well as providing a model for the development of similar mt-detection assays, especially for use in nonhospitalized patients who need their results within half a day, before starting treatment.

The zebrafish as a model for gastrointestinal tract-microbe interactions.

The zebrafish (Danio rerio) has become a widely used vertebrate model for bacterial, fungal, viral, and protozoan infections. Due to its genetic tractability, large clutch sizes, ease of manipulation, and optical transparency during early life stages, it is a particularly useful model to address questions about the cellular microbiology of host-microbe interactions. Although its use as a model for systemic infections, as well as infections localised to the hindbrain and swimbladder having been thoroughly reviewed, studies focusing on host-microbe interactions in the zebrafish gastrointestinal tract have been neglected. Here, we summarise recent findings regarding the developmental and immune biology of the gastrointestinal tract, drawing parallels to mammalian systems. We discuss the use of adult and larval zebrafish as models for gastrointestinal infections, and more generally, for studies of host-microbe interactions in the gut.

Allogeneic administration of human umbilical cord-derived mesenchymal stem/stromal cells for bronchopulmonary dysplasia: preliminary outcomes in four Vietnamese infants.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a severe condition in premature infants that compromises lung function and necessitates oxygen support. Despite major improvements in perinatal care minimizing the devastating effects, BPD remains the most frequent complication of extreme preterm birth. Our study reports the safety of the allogeneic administration of umbilical cord-derived mesenchymal stem/stromal cells (allo-UC-MSCs) and the progression of lung development in four infants with established BPD. METHODS: UC tissue was collected from a healthy donor, followed by propagation at the Stem Cell Core Facility at Vinmec Research Institute of Stem Cell and Gene Technology. UC-MSC culture was conducted under xeno- and serum-free conditions. Four patients with established BPD were enrolled in this study between May 25, 2018, and December 31, 2018. All four patients received two intravenous doses of allo-UC-MSCs (1 million cells/kg patient body weight (PBW) per dose) with an intervening interval of 7 days. Safety and patient conditions were evaluated during hospitalization and at 7 days and 1, 6 and 12 months postdischarge. RESULTS: No intervention-associated severe adverse events or prespecified adverse events were observed in the four patients throughout the study period. At the time of this report, all patients had recovered from BPD and were weaned off of oxygen support. Chest X-rays and CT scans confirmed the progressive reductions in fibrosis. CONCLUSIONS: Allo-UC-MSC administration is safe in preterm infants with established BPD. Trial registration This preliminary study was approved by the Vinmec International Hospital Ethics Board (approval number: 88/2019/QD-VMEC; retrospectively registered March 12, 2019).

Human Monocyte Heterogeneity as Revealed by High-Dimensional Mass Cytometry.

Objective- Three distinct human monocyte subsets have been identified based on the surface marker expression of CD14 and CD16. We hypothesized that monocytes were likely more heterogeneous in composition. Approach and Results- We used the high dimensionality of mass cytometry together with the FlowSOM clustering algorithm to accurately identify and define monocyte subsets in blood of healthy human subjects and those with coronary artery disease (CAD). To study the behavior and functionality of the newly defined monocyte subsets, we performed RNA sequencing, transwell migration, and efferocytosis assays. Here, we identify 8 human monocyte subsets based on their surface marker phenotype. We found that 3 of these subsets fall within the CD16+ nonclassical monocyte population and 4 subsets belong to the CD14+ classical monocytes, illustrating significant monocyte heterogeneity in humans. As nonclassical monocytes are important in modulating atherosclerosis in mice, we studied the functions of our 3 newly identified nonclassical monocytes in subjects with CAD. We found a marked expansion of a Slan+CXCR6+ nonclassical monocyte subset in CAD subjects, which was positively correlated with CAD severity. This nonclassical subset can migrate towards CXCL16 and shows an increased efferocytosis capacity, indicating it may play an atheroprotective role. Conclusions- Our data demonstrate that human nonclassical monocytes are a heterogeneous population, existing of several subsets with functional differences. These subsets have changed frequencies in the setting of severe CAD. Understanding how these newly identified subsets modulate CAD will be important for CAD-based therapies that target myeloid cells.