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Introduction: Nitrogen dioxide (NO 2 ) is known to be a trigger for asthma exacerbation. However, little is known about the role of seasonal variation in indoor and outdoor NO 2 levels in childhood asthma in a mixed rural-urban setting of North America. Methods: This prospective cohort study, as a feasibility study, included 62 families with children (5-17 years) that had diagnosed persistent asthma residing in Olmsted County, Minnesota. Indoor and outdoor NO 2 concentrations were measured using passive air samples over 2 weeks in winter and 2 weeks in summer. We assessed seasonal variation in NO 2 levels in urban and rural residential areas and the association with asthma control status collected from participants’ asthma diaries during the study period. Results: Outdoor NO 2 levels were lower (median: 2.4 parts per billion (ppb) in summer, 3.9 ppb in winter) than the Environmental Protection Agency (EPA) annual standard (53 ppb). In winter, a higher level of outdoor NO 2 was significantly associated with urban residential living area ( P = .014) and lower socioeconomic status (SES) ( P = .027). For both seasons, indoor NO 2 was significantly higher ( P

Summary: Presynaptic boutons in the mammalian brain are typically small and difficult to manipulate and study. Here, we present a protocol applying HaloTag self-labeling technology to detect de novo local protein synthesis in intact presynaptic mossy fiber boutons from acute mouse hippocampal slices. We describe stereotaxic injection of HaloTag-expressing virus into the brain region of interest, followed by brain slice preparation. We then detail the labeling of HaloTag-fused protein and image acquisition to visualize the labeled protein in an intact circuit.For complete details on the use and execution of this protocol, please refer to Monday et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The approval of radiation oncology care by insurance companies is burdensome for providers. In this topic discussion, we attempt to provide practical recommendations for how to deal with peer-to-peer phone calls as well as how to improve the timeliness and quality of subsequent letters of appeal. [ABSTRACT FROM AUTHOR]

Rural populations are more vulnerable to the impacts of COVID-19 compared to their urban counterparts as they are more likely to be older, uninsured, to have more underlying medical conditions, and live further from medical care facilities. We engaged the Southeastern MN (SEMN) community (N = 7,781, 51% rural) to conduct a survey of motivators and barriers to masking to prevent COVID-19. We also assessed preferences for types of and modalities to receive education/intervention, exploring both individual and environmental factors primarily consistent with Social Cognitive Theory. Our results indicated rural compared to urban residents performed fewer COVID-19 prevention behaviors (e.g. 62% rural vs. 77% urban residents reported wearing a mask all of the time in public, p

Biosynthesis of the complex diterpenoid antibiotic pleuromutilin relies on a bifunctional (di)terpene synthase, and here site-directed mutagenesis was used to knockout either of the two active sites. This enabled characterization of the novel ring contracted intermediate produced by the initiating class II diterpene cyclase active site. Quantum chemical calculations further indicate the importance of reactant configuration for this intriguing ring rearrangement.

Sesterterpenoids are a relatively rare class of plant terpenes. Sesterterpene synthase (STS)-mediated cyclization of the linear C25 isoprenoid precursor geranylfarnesyl diphosphate (GFPP) defines sesterterpene scaffolds. So far only a very limited number of STSs have been characterized. The discovery of three new plant STSs is reported that produce a suite of sesterterpenes with unprecedented 6/11/5 and 6/6/7/5 fused ring systems when transiently co-expressed with a GFPP synthase in Nicotiana benthamiana. Structural elucidation, feeding experiments, and quantum chemical calculations suggest that these STSs catalyze an unusual cyclization path involving reprotonation, intramolecular 1,6 proton transfer, and concerted but asynchronous bicyclization events. The cyclization is diverted from those catalyzed by the characterized plant STSs by forming unified 15/5 bicyclic sesterterpene intermediates. Mutagenesis further revealed a conserved amino acid residue implicated in reprotonation.

In this paper, as a purpose to apply the supersaturated solid-solutionized Al-9Mg alloy to the structural sheet parts of automotive, tensile tests were conducted under the various conditions and a constitutive equation was derived from the tensile test results. Al-9Mg alloy was produced using a special Mg master alloy containing Al2Ca during the casting process and extruded into the sheet. In order to study the deformation behavior of Al-9Mg alloy in warm temperature forming environments, tensile tests were conducted under the temperature of 373 K-573 K and the strain rate of 0.001/s~0.1/s. In addition, by using the raw data obtained from tensile tests, a constitutive equation of the Al-9Mg alloy was derived for predicting the optimized condition of the hot stamping process. Al-9Mg alloy showed uncommon deformation behavior at the 373 K and 473 K temperature conditions. The calculated curves from the constitutive equation well-matched with the measured curves from the experiments particularly under the low temperature and high strain rate conditions.

The first chemical synthesis of pentacyclic onocerane triterpenoids has been achieved. A putative biomimetic tricyclization cascade is employed to forge a fused decalin-/oxepane ring system. The synthetic route proceeds to (+)-cupacinoxepin in seven steps and to (+)-onoceranoxide in eight steps in the longest linear sequence, when starting from geranyl chloride and (+)-sclareolide. The bioinspired epoxypolyene cyclization is supported by computational and enzymatic studies.

Abstract Background A subgroup of patients with asthma has been reported to have an increased risk for asthma-associated infectious and inflammatory multimorbidities (AIMs). To systematically investigate the association of asthma with AIMs using a large patient cohort, it is desired to leverage a broad range of electronic health record (EHR) data sources to automatically identify AIMs accurately and efficiently. Methods We established an expert consensus for an operational definition for each AIM from EHR through a modified Delphi technique. A series of questions about the operational definition of 19 AIMS (11 infectious diseases and 8 inflammatory diseases) was generated by a core team of experts who considered feasibility, balance between sensitivity and specificity, and generalizability. Eight internal and 5 external expert panelists were invited to individually complete a series of online questionnaires and provide judgement and feedback throughout three sequential internal rounds and two external rounds. Panelists’ responses were collected, descriptive statistics tabulated, and results reported back to the entire group. Following each round the core team of experts made iterative edits to the operational definitions until a moderate (≥ 60%) or strong (≥ 80%) level of consensus among the panel was achieved. Results Response rates for each Delphi round were 100% in all 5 rounds with the achievement of the following consensus levels: (1) Internal panel consensus: 100% for 8 definitions, 88% for 10 definitions, and 75% for 1 definition, (2) External panel consensus: 100% for 12 definitions and 80% for 7 definitions. Conclusions The final operational definitions of AIMs established through a modified Delphi technique can serve as a foundation for developing computational algorithms to automatically identify AIMs from EHRs to enable large scale research studies on patient’s multimorbidities associated with asthma.

Objective: To perform a geospatial and temporal trend analysis for coronavirus disease 2019 (COVID-19) in a Midwest community to identify and characterize hot spots for COVID-19. Participants and Methods: We conducted a population-based longitudinal surveillance assessing the semimonthly geospatial trends of the prevalence of test confirmed COVID-19 cases in Olmsted County, Minnesota, from March 11, 2020, through October 31, 2020. As urban areas accounted for 84% of the population and 86% of all COVID-19 cases in Olmsted County, MN, we determined hot spots for COVID-19 in urban areas (Rochester and other small cities) of Olmsted County, MN, during the study period by using kernel density analysis with a half-mile bandwidth. Results: As of October 31, 2020, a total of 37,141 individuals (30%) were tested at least once, of whom 2433 (7%) tested positive. Testing rates among race groups were similar: 29% (black), 30% (Hispanic), 25% (Asian), and 31% (white). Ten urban hot spots accounted for 590 cases at 220 addresses (2.68 cases per address) as compared with 1843 cases at 1292 addresses in areas outside hot spots (1.43 cases per address). Overall, 12% of the population residing in hot spots accounted for 24% of all COVID-19 cases. Hot spots were concentrated in neighborhoods with low-income apartments and mobile home communities. People living in hot spots tended to be minorities and from a lower socioeconomic background. Conclusion: Geographic and residential risk factors might considerably account for the overall burden of COVID-19 and its associated racial/ethnic and socioeconomic disparities. Results could geospatially guide community outreach efforts (eg, testing/tracing and vaccine rollout) for populations at risk for COVID-19.

Caspase-1 signaling in myeloid suppressor cells can promote T-cell independent cancer progression, but the regulation of inflammasome signaling within the highly heterogeneous myeloid population in the tumor milieu remains elusive. To resolve this complexity, single cell transcriptomic profile of Head and Neck Squamous Cell Carcinoma (HNSCC) identified distinct inflammasome-associated genes within specific clusters of tumor-infiltrating myeloid cells. Among these myeloid cells, the sensor protein, NLRP3, and downstream effector IL-1β transcripts were enriched in discreet monocytic and macrophage subtypes in the TME. We showed that deletion of NLRP3, but not AIM2, phenocopied caspase-1/IL-1β dependent tumor progression in vivo. Paradoxically, we found myeloid-intrinsic caspase-1 signaling increased myeloid survival contrary to what would be predicted from the canonical pyroptotic function of caspase-1. This myeloid NLRP3/IL-1β signaling axis promotion of tumor growth was found to be gasdermin D independent. Mechanistically, we found that phagocyte-mediated efferocytosis of dying tumor cells in the TME directly activated NLRP3-dependent inflammasome signaling to drive IL-1β secretion. Subsequently we showed that NLRP3-mediated IL-1β production drives tumor growth in vivo. Dynamic RNA velocity analysis showed a robust directional flow from efferocytosis gene-set high macrophages to an inflammasome gene-set high macrophage population. We provide a novel efferocytosis-dependent inflammasome signaling pathway which mediates homeostatic tumor cell apoptosis that characterizes chronic inflammation-induced malignancy.

Intramolecular chirality transfer [2 + 2] cycloaddition of enantiomerically enriched allenoates and alkenes is presented. The use of a chiral catalyst was found to be critical to achieve high levels of diastereoselectivity compared to use of an achiral catalyst. The method developed leads to highly substituted cyclobutanes that would be difficult to prepare by alternative methods.

Sesterterpenoids are a rare terpene class harboring untapped chemodiversity and bioactivities. Their structural diversity originates primarily from the scaffold-generating sesterterpene synthases (STSs). In fungi, all six known STSs are bifunctional, containing C-terminal trans-prenyltransferase (PT) and N-terminal terpene synthase (TPS) domains. In plants, two colocalized PT and TPS gene pairs from Arabidopsis thaliana were recently reported to synthesize sesterterpenes. However, the landscape of PT and TPS genes in plant genomes is unclear. Here, using a customized algorithm for systematically searching plant genomes, we reveal a suite of physically colocalized pairs of PT and TPS genes for the biosynthesis of a large sesterterpene repertoire in the wider Brassicaceae. Transient expression of seven TPSs from A. thaliana, Capsella rubella, and Brassica oleracea in Nicotiana benthamiana yielded fungal-type sesterterpenes with tri-, tetra-, and pentacyclic scaffolds, and notably (-)-ent-quiannulatene, an enantiomer of the fungal metabolite (+)-quiannulatene. Protein and structural modeling analysis identified an amino acid site implicated in structural diversification. Mutation of this site in one STS (AtTPS19) resulted in premature termination of carbocation intermediates and accumulation of bi-, tri-, and tetracyclic sesterterpenes, revealing the cyclization path for the pentacyclic sesterterpene (-)-retigeranin B. These structural and mechanistic insights, together with phylogenetic analysis, suggest convergent evolution of plant and fungal STSs, and also indicate that the colocalized PT-TPS gene pairs in the Brassicaceae may have originated from a common ancestral gene pair present before speciation. Our findings further provide opportunities for rapid discovery and production of sesterterpenes through metabolic and protein engineering.

ObjectivesThis study assessed the effect of 2 types of hydroxyethyl starches (HES) on renal integrity and blood transfusion in cardiac surgery patients.DesignRetrospective investigation.SettingPatients from a single tertiary medical center.ParticipantsInclusion criteria included coronary artery bypass graft (CABG) and/or valve surgery that included cardiopulmonary bypass with aortic cross-clamping.InterventionsIntraoperative HES and blood product administration.Measurements and main resultsThe study comprised 1,265 patients who met inclusion criteria. Of these patients, 70% received HES, and of these, 47% received

Hypertension is a leading cause of global disease, mortality, and disability. While individuals of African descent suffer a disproportionate burden of hypertension and its complications, they have been underrepresented in genetic studies. To identify novel susceptibility loci for blood pressure and hypertension in people of African ancestry, we performed both single and multiple-trait genome-wide association analyses. We analyzed 21 genome-wide association studies comprised of 31,968 individuals of African ancestry, and validated our results with additional 54,395 individuals from multi-ethnic studies. These analyses identified nine loci with eleven independent variants which reached genome-wide significance (P < 1.25×10-8) for either systolic and diastolic blood pressure, hypertension, or for combined traits. Single-trait analyses identified two loci (TARID/TCF21 and LLPH/TMBIM4) and multiple-trait analyses identified one novel locus (FRMD3) for blood pressure. At these three loci, as well as at GRP20/CDH17, associated variants had alleles common only in African-ancestry populations. Functional annotation showed enrichment for genes expressed in immune and kidney cells, as well as in heart and vascular cells/tissues. Experiments driven by these findings and using angiotensin-II induced hypertension in mice showed altered kidney mRNA expression of six genes, suggesting their potential role in hypertension. Our study provides new evidence for genes related to hypertension susceptibility, and the need to study African-ancestry populations in order to identify biologic factors contributing to hypertension.

BackgroundRigid sternal fixation (RSF) has been shown to reduce sternal wound complications in high-risk patients. However, the higher initial cost continues to deter its use. This study evaluates the cost of caring for high-risk sternotomy patients who underwent RSF compared with those who underwent sternal closure with a modified wire technique (MWT).MethodsA retrospective single institution review of high-risk patients who underwent MWT (n = 45) and RSF (n = 30) for primary sternal closure from 2006 to 2009 was conducted. Total hospital cost, revenue, and net cost associated with surgery and subsequent care were analyzed.ResultsOverall rates of wound dehiscence and wound infections (superficial and deep) were higher in MWT patients (n = 14, 13, and 7, respectively) than RSF patients (n = 3, 2, and 0, respectively; P < 0.05). Modified wire technique patients also required more operations (mean ± SEM: 0.4 ± 0.1 vs 0.1 ± 0.1; P = 0.045), and had longer follow-up time (55.0 ± 9.1 vs 13.4 ± 10.5 days; P = 0.004). Overall, the hospital suffered a greater loss caring for MWT patients (US $18,903 ± 2,160) than RSF patients (US $8,935 ± 2,647). Modified wire technique patients who developed a complication had higher costs associated with their operative hospitalization, outpatient care, and home health than RSF patients (total net loss: US $41,436 ± 7327 vs US $10,612 ± 4,258; P = 0.034).ConclusionsIn high-risk patients, RSF is associated with lower rates of infections, including the "never event" mediastinitis, compared with MWT. Moreover, despite the initial higher cost, RSF affords an overall lower cost of care compared with MWT in patients at high-risk for developing sternal complications.

With the clinical approval of T-cell–dependent immune checkpoint inhibitors for many cancers, therapeutic cancer vaccines have re-emerged as a promising immunotherapy. Cancer vaccines require the addition of immunostimulatory adjuvants to increase vaccine immunogenicity, and increasingly multiple adjuvants are used in combination to bolster further and shape cellular immunity to tumor antigens. However, rigorous quantification of adjuvants’ synergistic interactions is challenging due to partial redundancy in costimulatory molecules and cytokine production, leading to the common assumption that combining both adjuvants at the maximum tolerated dose results in optimal efficacy. Herein, we examine this maximum dose assumption and find combinations of these doses are suboptimal. Instead, we optimized dendritic cell activation by extending the Multidimensional Synergy of Combinations (MuSyC) framework that measures the synergy of efficacy and potency between two vaccine adjuvants. Initially, we performed a preliminary in vitro screening of clinically translatable adjuvant receptor targets (TLR, STING, NLL, and RIG-I). We determined that STING agonist (CDN) plus TLR4 agonist (MPL-A) or TLR7/8 agonist (R848) as the best pairwise combinations for dendritic cell activation. In addition, we found that the combination of R848 and CDN is synergistically efficacious and potent in activating both murine and human antigen-presenting cells (APCs) in vitro. These two selected adjuvants were then used to estimate a MuSyC-dose optimized for in vivo T-cell priming using ovalbumin-based peptide vaccines. Finally, using B16 melanoma and MOC1 head and neck cancer models, MuSyC-dose–based adjuvating of cancer vaccines improved the antitumor response, increased tumor-infiltrating lymphocytes, and induced novel myeloid tumor infiltration changes. Further, the MuSyC-dose–based adjuvants approach did not cause additional weight changes or increased plasma cytokine levels compared to CDN alone. Collectively, our findings offer a proof of principle that our MuSyC-extended approach can be used to optimize cancer vaccine formulations for immunotherapy.

BackgroundAtrial fibrillation represents the most common arrhythmia leading to increased morbidity and mortality, yet, current treatment strategies have proven inadequate. Conventional treatment with antiarrhythmic drugs carries a high risk for proarrhythmias. The soluble epoxide hydrolase enzyme catalyzes the hydrolysis of anti-inflammatory epoxy fatty acids, including epoxyeicosatrienoic acids from arachidonic acid to the corresponding proinflammatory diols. Therefore, the goal of the study is to directly test the hypotheses that inhibition of the soluble epoxide hydrolase enzyme can result in an increase in the levels of epoxyeicosatrienoic acids, leading to the attenuation of atrial structural and electric remodeling and the prevention of atrial fibrillation.Methods and resultsFor the first time, we report findings that inhibition of soluble epoxide hydrolase reduces inflammation, oxidative stress, atrial structural, and electric remodeling. Treatment with soluble epoxide hydrolase inhibitor significantly reduces the activation of key inflammatory signaling molecules, including the transcription factor nuclear factor κ-light-chain-enhancer, mitogen-activated protein kinase, and transforming growth factor-β.ConclusionsThis study provides insights into the underlying molecular mechanisms leading to atrial fibrillation by inflammation and represents a paradigm shift from conventional antiarrhythmic drugs, which block downstream events to a novel upstream therapeutic target by counteracting the inflammatory processes in atrial fibrillation.

Through site-directed mutagenesis targeted at identification of the catalytic base in the rice (Oryza sativa) syn-copalyl diphosphate synthase OsCPS4, changes to a single residue (H501) were found to induce rearrangement rather than immediate deprotonation of the initially formed bicycle, leading to production of the novel compound syn-halimadienyl diphosphate. These mutational results are combined with quantum chemical calculations to provide insight into the underlying reaction mechanism.

Substitution of a histidine, comprising part of the catalytic base group in the ent-copalyl diphosphate synthases found in all seed plants for gibberellin phytohormone metabolism, by a larger aromatic residue leads to rearrangements. Through a series of 1,2-hydride and methyl shifts of the initially formed bicycle predominant formation of (-)-kolavenyl diphosphate is observed. Further mutational analysis and quantum chemical calculations provide mechanistic insight into the basis for this profound effect on product outcome.

To date, few scholars in counselor education have attended to the processes and impacts of introducing business related concepts within counseling curricula. The authors describe the development, implementation and evaluation of a graduate-level course titled Entrepreneurship in Clinical Settings wherein students were tasked with producing a business plan for their ideal clinical practice. Implications and recommendations are explored.

Abstract Introduction: Prior studies indicate greater disease burden for obesity among rural compared with urban residents but no differences for mood disorder based on geographic location. Recent attention has focused on the need to examine regional rural–urban disparities in disease burden. We focused on mood disorders and obesity prevalence within three southeastern Minnesota counties served by the Mayo Clinic Center for Translational Science Award, in Rochester, Minnesota, as these were top priorities identified in community health needs assessments. Methods: Cross-sectional study to assess the association of rural–urban locality on 5-year (2009–2014) prevalence of mood disorder and obesity obtained using the Rochester Epidemiological Project medical records linkage system, among subjects residing in three mixed rural–urban counties on April 1, 2014. Multivariable analyses adjusted for demographics, socioeconomic status using an individual housing-based measure, and counties. Results: The study cohort (percent rural location) included 91,202 (15%) for Olmsted, 10,197 (51%) in Dodge, and 10,184 (57%) in Wabasha counties. On multivariate analysis, 5-year prevalence of mood disorders and obesity was significantly greater for urban compared with rural residents, after adjusting for confounders; odds ratios (95% confidence intervals): 1.21 (1.17–1.26), P < 0.001, and 1.05 (1.01–1.10), P = 0.016, respectively. Observed effects were not modified in additional models adjusted for health care utilization (HCU; ≥1 general medical examination visit and flu vaccination). Conclusions: Rural–urban health disparities for burden of mood disorders and obesity are independent of socioeconomic status and HCU in a Midwestern community. It is important to assess potential regional heterogeneity of rural–urban disparities on health outcomes.

Abstract Background: Given the significant health effects, we assessed geospatial patterns of adverse events (AEs), defined as physical or sexual abuse and accidents or poisonings at home, among children in a mixed rural–urban community. Methods: We conducted a population-based cohort study of children (

Peter Windsor,1 Syseng Khounsy,2 Francesca Earp,1 Isabel MacPhillamy,1 James Young,1 Russell Bush1 1The University of Sydney, Sydney School of Veterinary Science, Camden, NSW, 2570, Australia; 2Department of Livestock and Fisheries, Ministry of Livestock and Fisheries, Vientiane, Lao People’s Democratic RepublicCorrespondence: Peter WindsorThe University of Sydney, Sydney School of Veterinary Science, Camden, NSW 2570, AustraliaTel +61 204-3898-3367Email peter.windsor@sydney.edu.auIntroduction: Foot-and-mouth disease (FMD) causes negative impacts on global food security, the livestock trade, national economies, and farming-family livelihoods, particularly in resource-poor developing countries with inadequate biosecurity and low levels of vaccination from inadequate veterinary services. As smallholder farmers have limited understanding of disease-risk management, their focus in FMD outbreaks is on accessing clinically effective therapies. However, most are provided inappropriate traditional treatments and/or topical or parenteral antibiotics, often delivered by paraveterinarians inadequately trained in antimicrobial custodianship. This results in negative financial impacts on livelihoods plus risks of food safety and development of antimicrobial resistance. We report the use of a novel pain-relief therapy for FMD.Methods: Clinical examinations in an outbreak of suspected FMD in April 2019 in Muang Khay village in Luang Prabang province, Laos confirmed signs and lesions of severe, subacute, ulcerative glossitis and interdigital dermatitis, typical of FMD. All affected buffalo (n=99) and cattle (n=37) presented for treatment in a population of 238 large ruminants, from 15 of 136 households, were administered a topical anesthetic pain-relief product (PRP) wound gel by spray-on (10– 30 mL per animal) formulation developed for aversive husbandry procedures (Tri-Solfen, Animal Ethics, Australia).Results: Treatment with PRP resulted in immediate improvement in demeanor and locomotion, and no adverse events were observed. On follow-up interview, all owners confirmed that their animals were eating within 2 days and lesions had healed within 5 days. Having experienced the positive clinical impacts of PRP on affected animals, these and surrounding farmers were keen to purchase the PRP for future use. The veterinary authorities rapidly registered the PRP for FMD therapy in Laos due to the observed efficacy.Discussion: These findings suggest a potential paradigm shift from treating FMD with expensive antimicrobials, which risks antimicrobial resistance, to a new, less expensive therapeutic approach that reduces animal suffering and may motivate farmers to report disease to access treatment. Use of the PRP is suggested as an innovation that may improve future FMD management, particularly in developing countries.Keywords: Laos, cattle, buffalo, therapy, topical anesthesia, antisepsis

Summary: Many steps of the MHC class I antigen processing pathway can be predicted using computational methods. Here we show that epitope predictions can be further improved by considering abundance levels of peptides' source proteins. We utilized biophysical principles and existing MHC binding prediction tools in concert with abundance estimates of source proteins to derive a function that estimates the likelihood of a peptide to be an MHC class I ligand. We found that this combination improved predictions for both naturally eluted ligands and cancer neoantigen epitopes. We compared the use of different measures of antigen abundance, including mRNA expression by RNA-Seq, gene translation by Ribo-Seq, and protein abundance by proteomics on a dataset of SARS-CoV-2 epitopes. Epitope predictions were improved above binding predictions alone in all cases and gave the highest performance when using proteomic data. Our results highlight the value of incorporating antigen abundance levels to improve epitope predictions.

The RNA binding protein FMRP regulates the synthesis of synaptic and nuclear proteins within different compartments of a neuron.

Abstract Background: Studies examining the role of geographic factors in coronavirus disease-2019 (COVID-19) epidemiology among rural populations are lacking. Methods: Our study is a population-based longitudinal study based on rural residents in four southeast Minnesota counties from March through October 2020. We used a kernel density estimation approach to identify hotspots for COVID-19 cases. Temporal trends of cases and testing were examined by generating a series of hotspot maps during the study period. Household/individual-level socioeconomic status (SES) was measured using the HOUSES index and examined for association between identified hotspots and SES. Results: During the study period, 24,243 of 90,975 residents (26.6%) were tested for COVID-19 at least once; 1498 (6.2%) of these tested positive. Compared to other rural residents, hotspot residents were overall younger (median age: 40.5 vs 43.2), more likely to be minorities (10.7% vs 9.7%), and of higher SES (lowest HOUSES [SES] quadrant: 14.6% vs 18.7%). Hotspots accounted for 30.1% of cases (14.5% of population) for rural cities and 60.8% of cases (27.1% of population) for townships. Lower SES and minority households were primarily affected early in the pandemic and higher SES and non-minority households affected later. Conclusion: In rural areas of these four counties in Minnesota, geographic factors (hotspots) play a significant role in the overall burden of COVID-19 with associated racial/ethnic and SES disparities, of which pattern differed by the timing of the pandemic (earlier in pandemic vs later). The study results could more precisely guide community outreach efforts (e.g., public health education, testing/tracing, and vaccine roll out) to those residing in hotspots.