Incorporation of Three Extracyclic Arginine Residues into a Melanocortin Macrocyclic Agonist (c[Pro-His-DPhe-Arg-Trp-Dap-Lys(Arg-Arg-Arg-Ac)-DPro]) Decreases Food Intake When Administered Intrathecally or Subcutaneously Compared to a Macrocyclic Ligand Lacking Extracyclic Arginine Residues (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro)].

Of the three Food and Drug Administration-approved melanocortin peptide drugs, two possess a cyclic scaffold, demonstrating that cyclized melanocortin peptides have therapeutic relevance. An extracyclic Arg residue, critical for pharmacological activity in the approved melanocortin cyclic drug setmelanotide, has also been demonstrated to increase the signal when fluorescently labeled cell-penetrating cyclic peptides are incubated with HeLa cells, with the maximal signal observed with three extracyclic Arg amino acids. Herein, a branching Lys residue was substituted into two macrocyclic melanocortin peptide agonists to incorporate 0-3 extracyclic Arg amino acids. Incorporation of the Arg residues resulted in equipotent or increased agonist potency at the mouse melanocortin receptors in vitro, suggesting that these substitutions were tolerated in the macrocyclic scaffolds. Further in vivo evaluation of one parent ligand (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-Pro]) and the three Arg derivative (c[Pro-His-DPhe-Arg-Trp-Dap-Lys(Ac-Arg-Arg-Arg)-Pro)] demonstrated that the three Arg derivative further decreased food intake compared to the parent macrocycle when the compounds were administered either via intrathecal injection or subcutaneous dosing. This suggests that three extracyclic Arg amino acids may be beneficial in the design of cyclic melanocortin ligands and that in vitro pharmacological profiling may not predict the in vivo efficacy of melanocortin ligands.

Human long noncoding RNA, VILMIR, is induced by major respiratory viral infections and modulates the host interferon response.

Long noncoding RNAs (lncRNAs) are a newer class of noncoding transcripts identified as key regulators of biological processes. Here we aimed to identify novel lncRNA targets that play critical roles in major human respiratory viral infections by systematically mining large-scale transcriptomic datasets. Using bulk RNA-sequencing (RNA-seq) analysis, we identified a previously uncharacterized lncRNA, named virus inducible lncRNA modulator of interferon response (VILMIR), that was consistently upregulated after in vitro influenza infection across multiple human epithelial cell lines and influenza A virus subtypes. VILMIR was also upregulated after SARS-CoV-2 and RSV infections in vitro. We experimentally confirmed the response of VILMIR to influenza infection and interferon-beta (IFN-ß) treatment in the A549 human epithelial cell line and found the expression of VILMIR was robustly induced by IFN-ß treatment in a dose and time-specific manner. Single cell RNA-seq analysis of bronchoalveolar lavage fluid (BALF) samples from COVID-19 patients uncovered that VILMIR was upregulated across various cell types including at least five immune cells. The upregulation of VILMIR in immune cells was further confirmed in the human T cell and monocyte cell lines, SUP-T1 and THP-1, after IFN-ß treatment. Finally, we found that knockdown of VILMIR expression reduced the magnitude of host transcriptional responses to IFN-ß treatment in A549 cells. Together, our results show that VILMIR is a novel interferon-stimulated gene (ISG) that regulates the host interferon response and may be a potential therapeutic target for human respiratory viral infections upon further mechanistic investigation.

Buprenorphine prescribing and treatment accessibility in response to regulation changes due to the COVID-19 public health emergency.

BACKGROUND: In 2021, over 80,000 fatal overdoses occurred in the United States. Since 2020, the federal government has enacted multiple regulatory changes around buprenorphine prescribing for opioid use disorder (OUD) to increase access to buprenorphine. This study aims to explore trends in buprenorphine treatment initiation pre- and post-public health emergency to evaluate changes in the context of X-waiver relaxations and telehealth allowances. METHODS: In a cross-sectional study, all RI residents who filled a buprenorphine prescription at a pharmacy in Rhode Island (RI), Massachusetts, and Connecticut between January 2017 and December 2023 were obtained from the RI Prescription Drug Monitoring Program (PDMP). The study excluded buprenorphine products not approved for OUD treatment from the analysis. Identified individuals had initiated buprenorphine for OUD during the study period if they did not have a prior prescription or if they had >30 days without buprenorphine exposure between their prescriptions. Spearman's rank correlation tests were used to identify significant associations between outcomes and regulation changes. RESULTS: The average number of patients dispensed buprenorphine did not significantly change over the study period, however the average number of initiates significantly decreased (ρ = -0.38255, p = .0003). The average number of providers prescribing CII-CV substances in RI has increased 3.4 % over the study period. The average percentage of prescribers in the PDMP prescribing buprenorphine for OUD doubled (ρ = 0.96075, p < .0001). CONCLUSION: Though efforts have been made to increase buprenorphine initiation, buprenorphine initiates remain well below pre-PHE levels. Efforts must continue to eliminate existing barriers to treatment and improve access to individuals seeking treatment.

Accuracy of Video-Based Gait Analysis Using Pose Estimation During Treadmill Walking Versus Overground Walking in Persons After Stroke.

OBJECTIVE: Video-based pose estimation is an emerging technology that shows significant promise for improving clinical gait analysis by enabling quantitative movement analysis with little costs of money, time, or effort. The objective of this study is to determine the accuracy of pose estimation-based gait analysis when video recordings are constrained to 3 common clinical or in-home settings (ie, frontal and sagittal views of overground walking and sagittal views of treadmill walking). METHODS: Simultaneous video and motion capture recordings were collected from 30 persons after stroke during overground and treadmill walking. Spatiotemporal and kinematic gait parameters were calculated from videos using an open-source human pose estimation algorithm and from motion capture data using traditional gait analysis. Repeated-measures analyses of variance were then used to assess the accuracy of the pose estimation-based gait analysis across the different settings, and the authors examined Pearson and intraclass correlations with ground-truth motion capture data. RESULTS: Sagittal videos of overground and treadmill walking led to more accurate measurements of spatiotemporal gait parameters versus frontal videos of overground walking. Sagittal videos of overground walking resulted in the strongest correlations between video-based and motion capture measurements of lower extremity joint kinematics. Video-based measurements of hip and knee kinematics showed stronger correlations with motion capture versus ankle kinematics for both overground and treadmill walking. CONCLUSION: Video-based gait analysis using pose estimation provides accurate measurements of step length, step time, and hip and knee kinematics during overground and treadmill walking in persons after stroke. Generally, sagittal videos of overground gait provide the most accurate results. IMPACT: Many clinicians lack access to expensive gait analysis tools that can help identify patient-specific gait deviations and guide therapy decisions. These findings show that video-based methods that require only common household devices provide accurate measurements of a variety of gait parameters in persons after stroke and could make quantitative gait analysis significantly more accessible.

Differences in Substance Use and Harm Reduction Practices by Race and Ethnicity: Rhode Island Harm Reduction Surveillance System, 2021-2022.

CONTEXT: In the United States, minority populations are disproportionately affected by the overdose epidemic, have higher mortality rates, and unequal access to harm reduction and treatment services. OBJECTIVE: This analysis aims to better understand harm reduction utilization and substance use patterns among minority populations to improve overdose outreach and prevention initiatives in Rhode Island. DESIGN: The present analysis used data from the Harm Reduction Surveillance System from January 2021 to December 2022 (N = 393). Chi-square tests and multivariable regression models were used to investigate differences in substance use behaviors by race and ethnicity. SETTING: Rhode Island. PARTICIPANTS: Participants include individuals who self-reported the use of illicit drugs, currently reside in Rhode Island, and were older than 18 years. MAIN OUTCOME MEASURES: Methods of drug use and uptake of harm reduction practices. RESULTS: Among survey participants, 41% were non-Hispanic White, 57% were aged 25 to 44 years, 62% identified as male, and 95% had health insurance coverage. Most participants reported smoking as their method of drug use (90%) and harm reduction practices were underutilized by all race and ethnicity groups. Fewer non-Hispanic Black participants reported carrying naloxone compared to the other race and ethnicity groups. Non-Hispanic Black and Hispanic participants were significantly less likely to inject drugs compared with non-Hispanic White participants (adjusted odds ratio [AOR] = 0.14; 95% confidence interval [CI], 0.04-0.45) (AOR = 0.40; 95% CI, 0.18-0.90). CONCLUSIONS: Smoking was the most common self-reported method of substance administration for all participants, whereas injection was more prevalent among non-Hispanic White participants. There is a continued need for minority-led and culturally informed harm reduction and treatment services for minority populations.

Discovery of a Pan-Melanocortin Receptor Antagonist [Ac-DPhe(pI)-Arg-Nal(2')-Orn-NH2] at the MC1R, MC3R, MC4R, and MC5R that Mediates an Increased Feeding Response in Mice and a 40-Fold Selective MC1R Antagonist [Ac-DPhe(pI)-DArg-Nal(2')-Arg-NH2].

Discovery of pan-antagonist ligands for the melanocortin receptors will help identify the physiological activities controlled by these receptors. The previously reported MC3R/MC4R antagonist Ac-DPhe(pI)-Arg-Nal(2')-Arg-NH2 was identified herein, for the first time, to possess MC1R and MC5R antagonist activity. Further structure-activity relationship studies probing the second and fourth positions were performed toward the goal of identifying potent melanocortin antagonists. Of the 21 tetrapeptides synthesized, 13 possessed MC1R, MC3R, MC4R, and MC5R antagonist activity. Three tetrapeptides were more than 10-fold selective for the mMC1R, including 8 (LTT1-44, Ac-DPhe(pI)-DArg-Nal(2')-Arg-NH2) that possessed 80 nM mMC1R antagonist potency and was at least 40-fold selective over the mMC3R, mMC4R, and mMC5R. Nine tetrapeptides were selective for the mMC4R, including 14 [SSM1-8, Ac-DPhe(pI)-Arg-Nal(2')-Orn-NH2] with an mMC4R antagonist potency of 1.6 nM. This compound was administered IT into mice, resulting in a dose-dependent increase in the food intake and demonstrating the in vivo utility of this compound series.

Development of allosteric and selective CDK2 inhibitors for contraception with negative cooperativity to cyclin binding.

Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception. However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs. In this paper, we describe the development and mechanism of action of type III inhibitors that bind CDK2 with nanomolar affinity. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as contraceptive agents is seen by incubation with spermatocyte chromosome spreads from mouse testicular explants, where they recapitulate Cdk2-/- and Spdya-/- phenotypes.

Screening through Lead Optimization of High Affinity, Allosteric Cyclin-Dependent Kinase 2 (CDK2) Inhibitors as Male Contraceptives That Reduce Sperm Counts in Mice.

Although cyclin-dependent kinase 2 (CDK2) is a validated target for both cancer and contraception, developing a CDK2 inhibitor with exquisite selectivity has been challenging due to the structural similarity of the ATP-binding site, where most kinase inhibitors bind. We previously discovered an allosteric pocket in CDK2 with the potential to bind a selective compound and then discovered and structurally confirmed an anthranilic acid scaffold that binds this pocket with high affinity. These allosteric inhibitors are selective for CDK2 over structurally similar CDK1 and show contraceptive potential. Herein, we describe the screening and optimization that led to compounds like EF-4-177 with nanomolar affinity for CDK2. EF-4-177 is metabolically stable, orally bioavailable, and significantly disrupts spermatogenesis, demonstrating this series' therapeutic potential. This work details the discovery of the highest affinity allosteric CDK inhibitors reported and shows promise for this series to yield an efficacious and selective allosteric CDK2 inhibitor.

Prevalence of individual brain and eye defects potentially related to Zika virus in pregnancy in 22 U.S. states and territories, January 2016 to June 2017.

During the Centers for Disease Control and Prevention's Zika Virus Response, birth defects surveillance programs adapted to monitor birth defects potentially related to Zika virus (ZIKV) infection during pregnancy. Pregnancy outcomes occurring during January 2016 to June 2017 in 22 U.S. states and territories were used to estimate the prevalence of those brain and eye defects potentially related to ZIKV. Jurisdictions were divided into three groups: areas with widespread ZIKV transmission, areas with limited local ZIKV transmission, and areas without local ZIKV transmission. Prevalence estimates for selected brain and eye defects and microcephaly per 10,000 live births were estimated. Prevalence ratios (PRs) and 95% confidence intervals (CIs) were estimated using Poisson regression for areas with widespread and limited ZIKV transmission compared with areas without local ZIKV transmission. Defects with significantly higher prevalence in areas of widespread transmission were pooled, and PRs were calculated by quarter, comparing subsequent quarters to the first quarter (January-March 2016). Nine defects had significantly higher prevalence in areas of widespread transmission. The highest PRs were seen in intracranial calcifications (PR = 12.6, 95% CI [7.4, 21.3]), chorioretinal abnormalities (12.5 [7.1, 22.3]), brainstem abnormalities (9.3 [4.7, 18.4]), and cerebral/cortical atrophy (6.7 [4.2, 10.8]). The PR of the nine pooled defects was significantly higher in three quarters in areas with widespread transmission. The largest difference in prevalence was observed for defects consistently reported in infants with congenital ZIKV infection. Birth defects surveillance programs could consider monitoring a subset of birth defects potentially related to ZIKV in pregnancy.

NCOA4-Mediated Ferritinophagy Is a Pancreatic Cancer Dependency via Maintenance of Iron Bioavailability for Iron-Sulfur Cluster Proteins.

Pancreatic ductal adenocarcinomas (PDAC) depend on autophagy for survival; however, the metabolic substrates that autophagy provides to drive PDAC progression are unclear. Ferritin, the cellular iron storage complex, is targeted for lysosomal degradation (ferritinophagy) by the selective autophagy adaptor NCOA4, resulting in release of iron for cellular utilization. Using patient-derived and murine models of PDAC, we demonstrate that ferritinophagy is upregulated in PDAC to sustain iron availability, thereby promoting tumor progression. Quantitative proteomics reveals that ferritinophagy fuels iron-sulfur cluster protein synthesis to support mitochondrial homeostasis. Targeting NCOA4 leads to tumor growth delay and prolonged survival but with the development of compensatory iron acquisition pathways. Finally, enhanced ferritinophagy accelerates PDAC tumorigenesis, and an elevated ferritinophagy expression signature predicts for poor prognosis in patients with PDAC. Together, our data reveal that the maintenance of iron homeostasis is a critical function of PDAC autophagy, and we define NCOA4-mediated ferritinophagy as a therapeutic target in PDAC. SIGNIFICANCE: Autophagy and iron metabolism are metabolic dependencies in PDAC. However, targeted therapies for these pathways are lacking. We identify NCOA4-mediated selective autophagy of ferritin ("ferritinophagy") as upregulated in PDAC. Ferritinophagy supports PDAC iron metabolism and thereby tumor progression and represents a new therapeutic target in PDAC. See related commentary by Jain and Amaravadi, p. 2023. See related article by Ravichandran et al., p. 2198. This article is highlighted in the In This Issue feature, p. 2007.

Insights from biosurveillance: non-fatal opioid overdoses in Rhode Island 2019-21.

BACKGROUND AND AIMS: Opioids biosurveillance is a new approach to public health surveillance of non-fatal overdoses that relies upon laboratory analysis of residual biospecimens from hospitals treating opioids overdoses. In Rhode Island (RI), USA, hospitals report suspected opioid overdoses to the Department of Health. Residual specimens associated with these overdoses are submitted to the State Health Laboratories for further characterization. This surveillance project aimed to characterize non-fatal overdoses through toxicological testing of urine specimens associated with non-fatal overdoses during the initial 2-year period of biosurveillance implementation in RI to assess the feasibility and public health utility of this approach. METHODS: This study included individuals who presented for treatment for a suspected opioid overdose in 10 RI hospitals between July 2019 and June 2021. Urine samples were received for 1354 unique overdose encounters corresponding to reported overdoses. Some individuals experienced multiple overdoses during this time. Urine samples were extracted and then analyzed by liquid chromatography tandem mass spectrometry with a panel consisting of 1033 opiates, synthetic opioids, fentanyl analogs and select metabolites. Temporal and spatial trends were evaluated for the studied population. RESULTS: A total of 1354 samples were tested for the presence of opioids in urine collected from individuals who experienced a suspected overdose. Fentanyl (and/or norfentanyl) was present in 79% of all samples in which opioids were found (n = 1033). Fentanyl analogs varied in their contribution to these totals, with observations ranging as high as 35% of all opioid-containing samples in August 2019 and May 2021. CONCLUSIONS: Laboratory identification of opioids involved in suspected overdoses shows that fentanyl and its analogs are the main drivers of the opioids overdose epidemic in Rhode Island, USA. The biosurveillance approach is unique in its ability to quantify contributions of novel fentanyl analogs to the burden of overdoses.