Dual Checkpoint Aptamer Immunotherapy: Unveiling Tailored Cancer Treatment Targeting CTLA-4 and NKG2A.

Recent strides in immunotherapy have illuminated the crucial role of CTLA-4 and PD-1/PD-L1 pathways in contemporary oncology, presenting both promises and challenges in response rates and adverse effects. This study employs a computational biology tool (in silico approach) to craft aptamers capable of binding to dual receptors, namely, inhibitory CTLA4 and NKG2A, thereby unleashing both T and NK cells and enhancing CD8+ T and NK cell functions for tumor cell lysis. Computational analysis highlighted AYA22T-R2-13 with HADDOCK scores of -78.2 ± 10.2 (with CTLA4), -60.0 ± 4.2 (with NKG2A), and -77.5 ± 5.6 (with CD94/NKG2A). Confirmation of aptamer binding to targeted proteins was attained via ELISA and flow cytometry methods. In vitro biological functionality was assessed using lactate dehydrogenase (LDH) cytotoxicity assay. Direct and competitive assays using ELISA and flow cytometry demonstrated the selective binding of AYA22T-R2-13 to CTLA4 and NKG2A proteins, as well as to the cell surface receptors of IL-2-stimulated T cells and NK cells. This binding was inhibited in the presence of competition from CTLA4 or NKG2A proteins. Remarkably, the blockade of CTLA4 or NKG2A by AYA22T-R2-13 augmented human CD8 T cell- and NK cell-mediated tumor cell lysis in vitro. Our findings highlight the precise binding specificity of AYA22T-R2-13 for CTLA4-B7-1/B7-2 (CD80/CD86) or CD94/NKG2A-HLA-E interactions, positioning it as a valuable tool for immune checkpoint blockade aptamer research in murine tumor models. These in vitro studies establish a promising foundation for further enhancing binding capacity and establishing efficacy and safety in animal models. Consequently, our results underscore the potential of AYA22T-R2-13 in cancer immunotherapy, offering high specificity, low toxicity, and the potential for cost-effective production.

Photodynamic bone stabilization for traumatic and pathologic fractures: a systematic review of utilization, complications, and patient-reported outcomes.

INTRODUCTION: The photodynamic bone stabilization system (PBSS) was was developed in 2010, and in 2018 gained FDA approval in the United States. Given its relative novelty, our analysis sought to analyze the available literature exploring the indications, outcomes, and complications of the PBSS. METHODS: We performed a systematic review (PROSPERO registration of study protocol: CRD42022363065, October 8th, 2022). PubMed, EBSCOHost, and Google Scholar electronic databases were queried to identify articles evaluating PBSS in the treatment of pathologic or traumatic fractures between January 1 2010 and 15 October 2022. The quality of the included studies was assessed using the Methodological Index for Nonrandomized Studies tool. RESULTS: Our initial search yielded 326 publications, which were then screened for appropriate studies that aligned with the purpose of our review. A total of thirteen studies, comprising seven case series, four case reports, and two cohort studies. The total sample size of the included studies consisted of 345 patients, with 242 females (70%) and 103 males (30%). The implants were most commonly utilized in the humerus (41%), radius (12%), and metacarpal (12%). The most common complications were related to broken implants (5%) and dislocation (1%). Most studies reported complete fracture healing and return of full strength and range of motion. CONCLUSION: Despite being a relatively novel technology, PBSS appears to be a viable option for fracture stabilization. Most studies included in our analysis reported complete fracture healing and return of function with minimal complications.

Metagenomic evaluation of the performance of passive Moore swabs for sewage monitoring relative to composite sampling over time resolved deployments.

Moore swabs have re-emerged as a versatile tool in the field of wastewater-based epidemiology during the COVID-19 pandemic and offer unique advantages for monitoring pathogens in sewer systems, especially at the neighborhood-level. However, whether Moore swabs provide comparable results to more commonly used composite samples remains to be rigorously tested including the optimal duration of Moore swab deployment. This study provides new insights into these issues by comparing the results from Moore swab samples to those of paired composite samples collected from the same sewer lines continuously over six to seventy-two hours post-deployment, during low COVID-19 prevalence periods. Our results show that Moore swabs accumulated approximately 10-fold higher PMMoV concentrations (on a basis of mL of Moore swab squeezed filtrate to mL of composite sewage) and showed comparable trends in terms of bacterial species abundance when compared to composite samples. Moore swabs also generally captured higher SARS-CoV-2 N1/N2 RNA concentrations than composite samples. Moore swabs showed comparable trends in terms of abundance dynamics of the sewage microbiome to composite samples and variable signs of saturation over time that were site and/or microbial population-specific. Based on our dual ddRT-PCR and shotgun metagenomic approach, we find that Moore swabs at our sites were optimally deployed for 6 h at a time at two sites.

Risk of Fracture and Complications After Fixation in Patients With Pre-injury Psychiatric Illness: A Propensity-Matched Cohort Study.

OBJECTIVES: The purpose was to describe the frequency of orthopaedic trauma and postsurgical complications associated with psychiatric diagnoses. DESIGN: Query of TriNetx Analytics Network. SETTING: Participating hospitals. PATIENT SELECTION CRITERIA: Those ≥18 years old with psychiatric illness and orthopaedic trauma. OUTCOME MEASURES AND COMPARISONS: Fractures and postoperative complications were described. A 1:1 propensity score matching function was used. Odds ratios compared intercohort complications. RESULTS: A total of 11,266,415 patients were identified with a psychiatric diagnosis, including bipolar disorder (8.9%), schizophrenia (3.3%), major depression (12.4%), stress-related disorder (9.6%), anxiety disorder (64.5%), borderline personality disorder (1.1%), or antisocial personality (0.2%). Prevalence of 30.2% was found for a fracture and at least 1 psychiatric diagnosis. Antisocial personality disorder had the highest risk ratio relative to people without that mental disorder (relative risk [RR] = 5.09) of having 1 or more associated fracture, followed by depression (RR = 3.03), stress-related disorders (RR = 3.00), anxiety disorders (RR = 2.97), borderline personality disorder (RR = 2.92), bipolar disorder (RR = 2.80), and schizophrenia (RR = 2.69). Patients with at least 1 psychiatric comorbidity had greater risk of pulmonary embolism, superficial and deep surgical site infections, pneumonia, urinary tract infection, deep venous thrombosis, osteonecrosis, and complex regional pain syndrome by 1 month after fixation, when compared with patients without psychiatric disorder. By 1 year, they were also at an increased risk for stroke and myocardial infarction. CONCLUSIONS: All psychiatric comorbidities were associated with increased RR of fracture and higher odds of complications compared with patients without psychiatric comorbidities. Providers should be aware of preexisting psychiatric diagnoses during treatment of acute injuries because of these risks. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Impact of external beam radiation on total shoulder arthroplasty outcomes: a propensity-matched cohort study.

BACKGROUND: External beam radiation therapy has a number of deleterious effects on the body, and a number of post-operative complications have been reported for several surgeries including total knee arthroplasty. However, few studies have investigated the impact of external beam radiation therapy for total shoulder arthroplasty (TSA). Our study aimed to assess the systemic and joint complications associated with TSA in patients with prior radiation exposures, as well as evaluate the surgical outcomes of radiation patients compared to non-radiation TSA patients. MATERIALS AND METHODS: A retrospective cohort analysis was conducted using the TriNetX Analytics Network. A 1:1 propensity score matching function was utilized to create two cohorts with matched baseline characteristics within the TriNetX network. Comparisons of the primary and secondary outcomes between the two cohorts were made using odds ratios. A p value of < 0.05 was determined to be significant. RESULTS: A total of 75,510 patients that received TSA were identified with 1505 having a history of radiation therapy (RT) and 73,605 with no radiation therapy (non-RT). After propensity matching, both groups contained 1484 patients. RT patients were at higher risk for developing prosthetic joint infection, acute renal failure, altered mental state, cerebrovascular event, DVT, PE, pneumonia, respiratory failure, and UTI compared to non-RT patients at different time points (p < 0.5). CONCLUSION: Patients with prior history of external beam radiation undergoing TSA had a higher risk of systemic complications and prosthetic joint infection compared to patients without a prior history. These complications suggest a more complicated post-operative management course for these patients.

Recovery Services for Interpersonal Violence Victims on Healthcare Use at a Trauma Center.

INTRODUCTION: Treatment of interpersonal violence (IPV) patients is often complicated by social and mental health comorbidities. New American College of Surgeons (ACS) requirements include provision of psychosocial support services for recovery after injury. We aim to describe utilization and patient outcomes after provision of Trauma Recovery Services (TRS) at our institution for the IPV population. These services include assistance with food, housing, criminal justice, and advocacy. METHODS: IPV patients were identified between September 6, 2018 and December 20, 2020. Demographic information was collected. TRS utilization and specific services rendered were identified. Primary outcome measures included initial length of stay (LOS), number of subsequent emergency department (ED) visits, and outpatient visits within 1 y after the initial injury. Statistical analyses included t-tests, Chi-squared tests, and multivariate regression analyses. RESULTS: A total of 502 patients were included in the final cohort, and 394 patients (78.5%) accepted the utilization of TRS services after initial interaction. Patients were on average 33.4 y old, and 59.4% were females. Patients who were older (P < 0.001) and homeless (P = 0.004) were more likely to use TRS, while victims of sexual assault (P < 0.001) and single patients (P = 0.041) were less likely. Patients who utilized TRS had longer initial LOS (P < 0.001), more ED visits (P < 0.001), and more outpatient visits (P = 0.01) related to the initial complaint, independent of potential confounders on multivariate linear regression. Food and housing service utilization associated with LOS (P = 0.01), ED visits (P < 0.001), and outpatient visits (P < 0.001). Additionally, transportation services were associated with longer LOS (P = 0.01) while patient advocacy services were associated with more ED visits (P = 0.03). CONCLUSIONS: TRS was extensively utilized by IPV patients, and associated with more follow-up appointments, ED visits, and longer LOS. Emphasis on injury mechanisms, baseline demographics, and social features may further characterize patients in need who tend toward utilization.

T helper (Th) cell profiles and cytokines/chemokines in characterization, treatment, and monitoring of autoimmune diseases.

Autoimmune diseases (AD) consist of a spectrum of disease entities whose etiologies are very complex and still not well understood. Every individual has the potential for developing AD under appropriate conditions because the body contains lymphocytes that are potentially reactive with self-antigens. The aims of this study are to (1) explore the flow cytometry method to identify the frequency of various circulating CD4+ T helper (Th) cell-subsets, including Th1, Th2, Th9, Th17, Th17.1, and Th22; (2) In parallel, to examine multiplex ELISA method for pathogenic inflammatory cytokines/chemokines, and (3) To assess the correlation of expression of T cell-subsets with serum cytokines/chemokines and understand its clinical importance with available AD treatments. We analyzed Th17, Th17.1, Th22, Th2, Th1, and Th9 Th cell populations and compared the concentrations of 67 cytokines/chemokines in healthy as well as AD-diagnosed patients. We observed that patients with autoimmune markers had significantly elevated percentages of naïve (Th17, Th22, and Th9) as well as memory (Th17 and Th22) Th cell-subsets, along with increased concentrations of cytokines/chemokines (Eotaxin, TNFß, and FABP4). The percentage of Th cell-subsets correlated positively or negatively with the production of cytokines/chemokines of patients diagnosed with AD. Our study demonstrates that the naïve and memory Th cell-subsets with positive correlations to cytokines/chemokines show new diagnostic markers to predict the patients' outcome, while the negative correlation of cytokines/chemokines shows the response to autoimmune therapies. Our findings of Th cell-subsets by flow cytometry and cytokines/chemokines by multiplex ELISA suggest that CCR6+ Th cell-subsets (Th17, Th17.1, Th22, and Th9) contribute to our understanding of the pathogenesis of AD and identify the new onset of AD from the autoimmune spectrum. Our findings highlight the importance of CCR6+ as a possible marker in the characterization, treatment, and monitoring of AD.

New High-Affinity Thrombin Aptamers for Advancing Coagulation Therapy: Balancing Thrombin Inhibition for Clot Prevention and Effective Bleeding Management with Antidote.

Thrombin is a key enzyme involved in blood clotting, and its dysregulation can lead to thrombotic diseases such as stroke, myocardial infarction, and deep vein thrombosis. Thrombin aptamers have the potential to be used as therapeutic agents to prevent or treat thrombotic diseases. Thrombin DNA aptamers developed in our laboratory exhibit high affinity and specificity to thrombin. In vitro assays have demonstrated their efficacy by significantly decreasing Factor II activity and increasing PT and APTT times in both plasma and whole blood. Aptamers AYA1809002 and AYA1809004, the two most potent aptamers, exhibit high affinity for their target, with affinity constants (Kd) of 10 nM and 13 nM, respectively. Furthermore, the in vitro activity of these aptamers displays dose-dependent behavior, highlighting their efficacy in a concentration-dependent manner. In vitro stability assessments reveal that the aptamers remain stable in plasma and whole blood for up to 24 h. This finding is crucial for their potential application in clinical settings. Importantly, the thrombin inhibitory activity of the aptamers can be reversed by employing reverse complement sequences, providing a mechanism to counteract their anticoagulant effects when necessary to avoid excessive bleeding. These thrombin aptamers have been determined to be safe, with no observed mutagenic or immunogenic effects. Overall, these findings highlight the promising characteristics of these newly developed thrombin DNA aptamers, emphasizing their potential for therapeutic applications in the field of anticoagulation therapy. Moreover, the inclusion of an antidote in the coagulation therapy regimen can improve patient safety, ensure greater therapeutic efficacy, and minimize risk during emergency situations.

Target-Captured mRNA from Murine Olfactory Bulb.

The external world is perceived via sensory receptors arranged in highly organized systems according to functional strategies, which in turn reflect features of critical importance to both the sense and the animal. Thus describing the receptor organization and functional strategies of olfaction, the sense of smell, is crucial for a concrete understanding of the fundamental principles of the system's architecture. Sensory processing in olfactory systems is organized across olfactory bulb (OB) glomeruli, wherein axons of peripheral sensory neurons expressing the same olfactory receptor (OR) co-terminate to transmit receptor-specific activity to mitral/tufted cells, projection neurons in the olfactory bulb. Understanding how receptors map to glomeruli is therefore critical to understanding olfaction.Here we describe a method to enrich low-abundant OR mRNAs from the mouse OB for spatial transcriptomics to generate high-throughput mapping of receptors to glomeruli [2]. Our method combines sequential sectioning along the anteroposterior, dorsoventral, and mediolateral axes with target capture enrichment sequencing to overcome low-abundance target expression. This strategy spatially mapped 86% of olfactory receptors across the olfactory bulb and uncovered a relationship between olfactory receptor sequence and glomerular position.

Press Releases of Drug-Related Randomized Trial Results Prior to Publication in High-Impact Journals: an Observational Study.

IMPORTANCE: Results from high-profile randomized controlled trials (RCTs) are routinely reported through press release months prior to peer-reviewed publication. There are potential benefits to press releases (e.g., knowledge dissemination, ensuring regulatory compliance), but also potential drawbacks (e.g., selective reporting, positive "spin"). OBJECTIVE: To characterize the practice of press release predating the publication of a drug-related RCT in a peer-reviewed journal ("preemptive press release"), including factors associated with this practice. DESIGN, SETTING, AND PARTICIPANTS: We systematically reviewed all RCTs of medications published between 2015 and 2019 in the New England Journal of Medicine (NEJM), Journal of the American Medical Association (JAMA), and Lancet. Press releases were identified using a systematic search of the grey literature (e.g., press release databases, study sponsor websites). An RCT was considered to have a preemptive press release if the press release was published at least three months (90 days) prior to the date of publication in a peer-reviewed journal. MAIN OUTCOMES AND MEASURES: Presence of preemptive press release, defined as a press-release at least 90 days prior to the date of publication in a peer-reviewed journal. As secondary measures for dissemination, we also assessed citation count and Altmetric score. RESULTS: We identified 988 RCTs, of which 172 (17%) had a press release published at least 90 days before the date of peer-reviewed publication. Press releases were published a median of 246 days (interquartile range [IQR] 169-366 days) before publication in a peer-reviewed journal. In the multivariable logistic regression model, the strongest predictor of having a preemptive press release was funding by a pharmaceutical company (odds ratio 13, 95% CI 7, 25). Approximately 85% of RCTs with preemptive press releases had a positive primary outcome and, concordantly, 81% of the corresponding press releases had a positive headline. Multivariable regression models identified studies with a preemptive press release had a similar Altmetric score (median - 15, 95% CI - 33, 12) and higher median citation count (median 22 [95% CI 10 to 33] compared to studies without a preemptive press release. CONCLUSIONS AND RELEVANCE: Preemptive press releases were common, most often issued for trials funded by a pharmaceutical company, and typically preceded publication in a peer-reviewed journal by approximately eight months.

Simultaneous detection and quantification of multiple pathogen targets in wastewater.

Wastewater-based epidemiology has emerged as a critical tool for public health surveillance, building on decades of environmental surveillance work for pathogens such as poliovirus. Work to date has been limited to monitoring a single pathogen or small numbers of pathogens in targeted studies; however, few studies consider simultaneous quantitative analysis of a wide variety of pathogens, which could greatly increase the utility of wastewater surveillance. We developed a novel quantitative multi-pathogen surveillance approach (35 pathogen targets including bacteria, viruses, protozoa, and helminths) using TaqMan Array Cards (TAC) and applied the method on concentrated wastewater samples collected at four wastewater treatment plants in Atlanta, GA from February to October of 2020. From sewersheds serving approximately 2 million people, we detected a wide range of targets including many we expected to find in wastewater (e.g., enterotoxigenic E. coli and Giardia in 97% of 29 samples at stable concentrations) as well as unexpected targets including Strongyloides stercoralis (a human threadworm rarely observed in the USA). Other notable detections included SARS-CoV-2, but also several pathogen targets that are not commonly included in wastewater surveillance like Acanthamoeba spp., Balantidium coli, Entamoeba histolytica, astrovirus, norovirus, and sapovirus. Our data suggest broad utility in expanding the scope of enteric pathogen surveillance in wastewaters, with potential for application in a variety of settings where pathogen quantification in fecal waste streams can inform public health surveillance and selection of control measures to limit infections.

Complications in Osteonecrosis Patients Following Total Knee Arthroplasty: A Propensity-Matched Cohort Study.

BACKGROUND: Several studies have demonstrated high complication rates in osteonecrosis (ON) patients undergoing total hip arthroplasty. However, there is a paucity of literature regarding outcomes of total knee arthroplasty (TKA) in ON patients. Our study aimed to assess preoperative risk factors associated with the development of ON and determine the incidence of postoperative complications up to one year following TKA. METHODS: A retrospective cohort study was conducted using a large national database. Patients who had a primary TKA and ON were isolated using Current Procedural Terminology code 27447 and ICD-10-CM code M87, respectively. A total of 185,045 patients were identified, including 181,151 patients who had a TKA and 3,894 patients who had a TKA and ON. After propensity matching, both groups each contained 3,758 patients. Intercohort comparisons of primary and secondary outcomes after propensity score matching were made using the odds ratio. A P value of < .01 was determined to be significant. RESULTS: The ON patients were found to have an increased risk for prosthetic joint infection, urinary tract infection, deep vein thrombosis, pulmonary embolism, wound dehiscence pneumonia, and the development of heterotopic ossification at different time points. Osteonecrosis patients had an increased risk of revision at the 1-year time point (odds ratio = 2.068, P < .0001). CONCLUSION: The ON patients had a higher risk of systemic and joint complications than non-ON patients. These complications suggest a more complicated management course for patients who have ON prior to and after TKA.

Zika virus RNA persistence and recovery in water and wastewater: An approach for Zika virus surveillance in resource-constrained settings.

During the 2015-2016 Zika virus (ZIKV) epidemic in the Americas, serological cross-reactivity with other flaviviruses and relatively high costs of nucleic acid testing in the region hindered the capacity for widespread diagnostic testing. In such cases where individual testing is not feasible, wastewater monitoring approaches may offer a means of community-level public health surveillance. To inform such approaches, we characterized the persistence and recovery of ZIKV RNA in experiments where we spiked cultured ZIKV into surface water, wastewater, and a combination of both to examine the potential for detection in open sewers serving communities most affected by the ZIKV outbreak, such as those in Salvador, Bahia, Brazil. We used reverse transcription droplet digital PCR to quantify ZIKV RNA. In our persistence experiments, we found that the persistence of ZIKV RNA decreased with increasing temperature, significantly decreased in surface water versus wastewater, and significantly decreased when the initial concentration of virus was lowered by one order of magnitude. In our recovery experiments, we found higher percent recovery of ZIKV RNA in pellets versus supernatants from the same sample, higher recoveries in pellets using skimmed milk flocculation, lower recoveries of ZIKV RNA in surface water versus wastewater, and lower recoveries from a freeze thaw. We also analyzed samples collected from Salvador, Brazil during the ZIKV outbreak (2015-2016) that consisted of archived samples obtained from open sewers or environmental waters thought to be contaminated by sewage. Although we did not detect any ZIKV RNA in the archived Brazil samples, results from these persistence and recovery experiments serve to inform future wastewater monitoring efforts in open sewers, an understudied and important application of wastewater monitoring.

The Surgical Management of Migraines and Chronic Headaches: A Cross-sectional Review of American Insurance Coverage.

BACKGROUND: Migraine headache can be an extremely debilitating condition, with pharmacotherapy for prophylaxis or treatment of acute symptoms being unsuccessful in a large proportion of patients. Surgical management of migraine has recently gained popularity as an alternative to pharmacotherapy for severe disease. However, the novel nature of these procedures may lead to variable insurance coverage, limiting access to care. METHODS: A cross-sectional analysis of 101 US insurance companies was conducted. Companies were chosen based on greatest market share and enrollment per state. A Web-based search or phone call identified whether each company had a publicly available policy on nonsurgical or surgical management of migraine or headache. For companies with an available policy, coverage was categorized into covered, covered on a case-by-case basis, or never covered, with criteria required for coverage collected and categorized. RESULTS: Of the 101 evaluated insurers, significantly fewer companies had a policy on surgical treatment for migraine or headache (n = 52 [52%]) compared with nonsurgical treatment (n = 78 [78%]) (P < 0.001). For companies with a policy, the most frequently covered nonsurgical treatments were biofeedback (n = 23 [92%]) and botulism toxin injections (n = 61 [88%]). Headaches were an approved indication for occipital nerve stimulation in 4% (n = 2) of company policies and nerve decompression in 2% (n = 1) of policies. Migraines were never offered preauthorized coverage for surgical procedures. CONCLUSION: Approximately half of US insurance companies have a publicly available policy on surgical management of migraine or headache. Surgical treatment was seldom covered for the indication of headache and would never receive preauthorized coverage for migraine. Lack of coverage may create challenges in accessing surgical treatment. Additional prospective, controlled studies are necessary to further support the efficacy of surgical treatment.

Impact of presentation at conference with timed release of academic publication.

Fast-tracking publication of original research to coincide with a conference presentation ("coordinated publication") is a mechanism of rapidly disseminating new data. How often this occurs, whether its frequency is changing, and the impact of this approach on information dissemination, is unknown. Our objective was to describe the characteristics of coordinated publications, how the practice has changed over time, and evaluate its potential impact on dissemination of study results. We conducted a cross-sectional study of randomized controlled trials published in NEJM, Lancet, and JAMA between January 1, 2015, and December 31, 2019. Among the 1533 included randomized controlled trials, 502 (33%) had coordinated publications. Coordinated publications increased from 30% [n = 94] in 2015 to 37% [n = 136] in 2019. Coordinated publications were more likely to be unblinded (61% [n = 305] vs. 52% [n = 532]) and more likely to be funded by industry (50% [n = 249] vs. 30% [n = 311]). The strongest predictor of a coordinated publication was cardiovascular disease subspecialty (OR = 3.96, 95% CI [2.95, 5.36]). The median number of citations (188 vs. 98) and the median Altmetric score (318 vs. 182) were higher for coordinated publications than non-coordinated publications. These differences persisted in a multivariable regression model. Coordinated publication is increasingly common. While coordinated publications may generate greater attention, they were observed to be more likely to be unblinded and more likely to be funded by industry, raising questions about the value and intentions of such promotion.

Phenotypic screen identifies the natural product silymarin as a novel anti-inflammatory analgesic.

Sensory neuron hyperexcitability is a critical driver of pathological pain and can result from axon damage, inflammation, or neuronal stress. G-protein coupled receptor signaling can induce pain amplification by modulating the activation of Trp-family ionotropic receptors and voltage-gated ion channels. Here, we sought to use calcium imaging to identify novel inhibitors of the intracellular pathways that mediate sensory neuron sensitization and lead to hyperexcitability. We identified a novel stimulus cocktail, consisting of the SSTR2 agonist L-054,264 and the S1PR3 agonist CYM5541, that elicits calcium responses in mouse primary sensory neurons in vitro as well as pain and thermal hypersensitivity in mice in vivo. We screened a library of 906 bioactive compounds and identified 24 hits that reduced calcium flux elicited by L-054,264/CYM5541. Among these hits, silymarin, a natural product derived from milk thistle, strongly reduced activation by the stimulation cocktail, as well as by a distinct inflammatory cocktail containing bradykinin and prostaglandin E2. Silymarin had no effect on sensory neuron excitability at baseline, but reduced calcium flux via Orai channels and downstream mediators of phospholipase C signaling. In vivo, silymarin pretreatment blocked development of adjuvant-mediated thermal hypersensitivity, indicating potential use as an anti-inflammatory analgesic.

Recurrent repeat expansions in human cancer genomes.

Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases1,2. However, repeat expansions are often not explored beyond neurological and neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs, a phenomenon termed microsatellite instability; however, larger repeat expansions have not been systematically analysed in cancer3-8. Here we identified TR expansions in 2,622 cancer genomes spanning 29 cancer types. In seven cancer types, we found 160 recurrent repeat expansions (rREs), most of which (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with enrichment near candidate cis-regulatory elements, suggesting a potential role in gene regulation. One rRE, a GAAA-repeat expansion, located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, in preliminary experiments, treating cells that harbour this rRE with a GAAA-targeting molecule led to a dose-dependent decrease in cell proliferation. Overall, our results suggest that rREs may be an important but unexplored source of genetic variation in human cancer, and we provide a comprehensive catalogue for further study.

Parallel deployment of passive and composite samplers for surveillance and variant profiling of SARS-CoV-2 in sewage.

Wastewater-based epidemiology during the COVID-19 pandemic has proven useful for public health decision-making but is often hampered by sampling methodology constraints, particularly at the building- or neighborhood-level. Time-weighted composite samples are commonly used; however, autosamplers are expensive and can be affected by intermittent flows in sub-sewershed contexts. In this study, we compared time-weighted composite, grab, and passive sampling via Moore swabs, at four locations across a college campus to understand the utility of passive sampling. After optimizing the methods for sample handling and processing for viral RNA extraction, we quantified SARS-CoV-2 N1 and N2, as well as a fecal strength indicator, PMMoV, by ddRT-PCR and applied tiled amplicon sequencing of the SARS-CoV-2 genome. Passive samples compared favorably with composite samples in our study area: for samples collected concurrently, 42 % of the samples agreed between Moore swab and composite samples and 58 % of the samples were positive for SARS-CoV-2 using Moore swabs while composite samples were below the limit of detection. Variant profiles from Moore swabs showed a shift from variant BA.1 to BA.2, consistent with in-person saliva samples. These data have implications for the broader implementation of sewage surveillance without advanced sampling technologies and for the utilization of passive sampling approaches for other emerging pathogens.

The Markup on Orthopaedic Services: An Analysis of 2014-2019 Medicare Data and the Potential for Surprise Billing.

BACKGROUND: Markups on charges for medical services have the potential to result in "surprise billing," especially for out-of-network and uninsured patients. Although previously analyzed in other surgical subspecialties, the distribution and level of cost-to-charge ratios (CCRs) for orthopaedic services have yet to be studied. Therefore, our analysis sought to evaluate the CCRs for orthopaedic surgery services provided to Medicare beneficiaries throughout the United States. METHODS: Orthopaedic services provided to Medicare Part B beneficiaries between 2014 and 2019 were identified in the Physician & Other Practitioners database of the Centers for Medicare & Medicaid Services (CMS). CCRs, representing the ratio between the actual payment provided by CMS and the charge submitted by the provider, were calculated for each service. Descriptive statistics were calculated for CCRs at the national, state, and service-code levels. The coefficient of variation (CoV) was utilized to evaluate variability in CCRs across services and states. Additionally, Mann-Kendall tests were performed to evaluate trends in CCRs for included services over the time frame. RESULTS: Our analysis included an annual mean of 47,247,928 services provided by a mean of 23,185 orthopaedic surgeons over the study period. In the non-facility setting, there was a decrease in median CCRs for orthopaedic surgery services (0.29 to 0.27; p = 0.024). No changes were demonstrated for facility-based services. Service codes related to trigger finger procedures (0.18 to 0.17; p = 0.004), physical therapy (0.40 to 0.36; p = 0.035), and new patient visits (0.52 to 0.46; p = 0.035) demonstrated significant decreases in median CCRs. Only shoulder arthroscopy demonstrated a significant increase in median CCR (0.09 to 0.10; p = 0.003). High dispersion in CCRs was demonstrated for 16 (80%) of the 20 evaluated services. Wide variations in CCRs were demonstrated across individual states (median, 0.57; interquartile range width, 0.53). CONCLUSIONS: Our analysis demonstrated low and variable CCRs for commonly performed orthopaedic services in the U.S. These findings serve to inform and help improve related price transparency policies. Additionally, our analysis encourages increased efforts at preventing these low CCRs from limiting care in vulnerable populations.

Highly efficacious and safe neutralizing DNA aptamer of SARS-CoV-2 as an emerging therapy for COVID-19 disease.

BACKGROUND: The paucity of SARS-CoV-2-specific virulence factors has greatly hampered the therapeutic management of patients with COVID-19 disease. Although available vaccines and approved therapies have shown tremendous benefits, the continuous emergence of new variants of SARS-CoV-2 and side effects of existing treatments continue to challenge therapy, necessitating the development of a novel effective therapy. We have previously shown that our developed novel single-stranded DNA aptamers not only target the trimer S protein of SARS-CoV-2, but also block the interaction between ACE2 receptors and trimer S protein of Wuhan origin, Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants of SARS-CoV-2. We herein performed in vivo experiments that administer the aptamer to the lungs by intubation as well as in vitro studies utilizing PBMCs to prove the efficacy and safety of our most effective aptamer, AYA2012004_L. METHODS: In vivo studies were conducted in transgenic mice expressing human ACE2 (K18hACE2), C57BL/6J, and Balb/cJ. Flow cytometry was used to check S-protein expressing pseudo-virus-like particles (VLP) uptake by the lung cells and test the immuogenicity of AYA2012004_L. Ames test was used to assess mutagenicity of AYA2012004_L. RT-PCR and histopathology were used to determine the biodistribution and toxicity of AYA2012004_L in vital organs of mice. RESULTS: We measured the in vivo uptake of VLPs by lung cells by detecting GFP signal using flow cytometry. AYA2012004_L specifically neutralized VLP uptake and also showed no inflammatory response in mice lungs. In addition, AYA2012004_L did not induce inflammatory response in the lungs of Th1 and Th2 mouse models as well as human PBMCs. AYA2012004_L was detectable in mice lungs and noticeable in insignificant amounts in other vital organs. Accumulation of AYA2012004_L in organs decreased over time. AYA2012004_L did not induce degenerative signs in tissues as seen by histopathology and did not cause changes in the body weight of mice. Ames test also certified that AYA2012004_L is non-mutagenic and proved it to be safe for in vivo studies. CONCLUSIONS: Our aptamer is safe, effective, and can neutralize the uptake of VLPs by lung cells when administered locally suggesting that it can be used as a potential therapeutic agent for COVID-19 management.