Computational design of non-porous pH-responsive antibody nanoparticles.

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and is important for targeted delivery of biologics. Here we describe the design of octahedral non-porous nanoparticles with a targeting antibody on the two-fold symmetry axis, a designed trimer programmed to disassemble below a tunable pH transition point on the three-fold axis, and a designed tetramer on the four-fold symmetry axis. Designed non-covalent interfaces guide cooperative nanoparticle assembly from independently purified components, and a cryo-EM density map closely matches the computational design model. The designed nanoparticles can package protein and nucleic acid payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between 5.9 and 6.7. The ability to incorporate almost any antibody into a non-porous pH-dependent nanoparticle opens up new routes to antibody-directed targeted delivery.

The natural history of celiac artery aneurysms.

OBJECTIVES: The goal of this study was to document the natural history of celiac artery aneurysms (CAAs). BACKGROUND: Celiac artery aneurysms are rare. Existing literature is skewed towards outcomes after intervention of large, symptomatic aneurysms but the behavior of untreated CAAs is poorly understood. METHODS: This is a single institution, retrospective analysis of patients with CAA diagnosed by CT imaging (2015-2019) identified through an institutional radiology database. Radiologic, demographic, and follow-up data were analyzed. The primary endpoint was the mean growth rate of CAAs. RESULTS: Of the 76 patients included, 86.8% were men with a mean age at presentation of 69.8 years. The mean CAA diameter on index imaging was 15.4 +/- 3.8 mm (range, 7-30 mm). All were classified as true aneurysms and 76.3% were saccular. All patients had clinical follow-up with mean follow-up 31.2 months +/- 21.6 months. No patient developed symptoms or rupture. The mean radiological follow-up among 45 patients was 25.2 +/- 16.8 months. Over this period, 16 CAAs (35.6%) enlarged, while 29 (64.4%) remained stable. One patient (1.3%) underwent intervention for increasing size in the setting of a chronic dissection. On multivariate analysis, age <70 was significantly associated with increased risk of aneurysm growth. CONCLUSIONS: In this institutional review of patients with CAAs, the majority of aneurysms remained stable in size, with no patients developing symptoms or rupture over clinical follow-up. Given the observed benign behavior of these aneurysms, guidelines that suggest conservative management of CAAs less than 2 cm seems appropriate.

Strategies for improving the performance of prediction models for response to immune checkpoint blockade therapy in cancer.

Immune checkpoint blockade (ICB) therapy holds promise for bringing long-lasting clinical gains for the treatment of cancer. However, studies show that only a fraction of patients respond to the treatment. In this regard, it is valuable to develop gene expression signatures based on RNA sequencing (RNAseq) data and machine learning methods to predict a patient's response to the ICB therapy, which contributes to more personalized treatment strategy and better management of cancer patients. However, due to the limited sample size of ICB trials with RNAseq data available and the vast number of candidate gene expression features, it is challenging to develop well-performed gene expression signatures. In this study, we used several published melanoma datasets and investigated approaches that can improve the construction of gene expression-based prediction models. We found that merging datasets from multiple studies and incorporating prior biological knowledge yielded prediction models with higher predictive accuracies. Our finding suggests that these two strategies are of high value to identify ICB response biomarkers in future studies.

The substantial burden of iatrogenic vascular injury on the vascular surgery workforce at an academic medical center.

OBJECTIVE: Vascular surgeons are often called upon to provide emergent surgical assistance to other specialties for iatrogenic complications, both intraoperatively and in the inpatient setting. The management of iatrogenic vascular injury remains a critical role of the vascular surgeon, especially in the context of the increasing adoption of percutaneous procedures by other specialties. This study aims to characterize consultation timing, management, and outcomes for iatrogenic vascular injuries. METHODS: This study identified patients for whom vascular surgery was consulted for iatrogenic vascular complications from February 1, 2022, to May 12, 2023. Patient information, including demographic information, injury details, and details of any operative intervention, was retrospectively collected from February 1, 2022, to October 13, 2022, and prospectively collected for the remainder of the study period. Analyses were performed with R (version 2022.02.03). RESULTS: There were 87 patients with consultations related to iatrogenic vascular injury. Of these, 42 (46%) were female and the mean age was 59 years (±18 years). The most common consulting services were cardiology (32%), cardiothoracic surgery (26%), general surgery (8%), and neurointerventional radiology (10%). Reasons for consultation included hemorrhage (36%), limb ischemia (36%), and treatment of pseudoaneurysm (23%). A total of 24% of consults were intraoperative, 20% of consults related to extracorporeal membrane oxygenation cannulation, and 16% of consults related to ventricular assist devices including left ventricular assist device and intra-aortic balloon pump. The majority of these consult requests (60%) occurred during evening and night hours (5 PM to 7 AM). Emergent intervention was required in 62% of cases and consisted of primary open surgical repair of arterial injury (54%), endovascular intervention (21%), and open thromboembolectomy (15%). Overall, in-hospital mortality for the patient cohort was 20% and the reintervention rate was 23%, reflecting the underlying complexity of the illness and nature of the vascular injury in this patient group. CONCLUSIONS: Vascular surgeons play an essential role in managing emergent life-threatening hemorrhagic and ischemic iatrogenic vascular complications in the hospitalized setting. The complications require immediate bedside or intraoperative consult and often emergent open surgical or endovascular intervention. Furthermore, many of these require urgent management in the evening or overnight hours, and therefore the high frequency of these events represents a potential significant resource utilization and workforce issue to the vascular surgery workforce.

Antidepressant Dispensing to US Adolescents and Young Adults: 2016-2022.

BACKGROUND: Mental health worsened in adolescents and young adults after the coronavirus disease 2019 (COVID-19) outbreak in March 2020, but whether antidepressant dispensing to this population changed is unknown. METHODS: We identified antidepressant prescriptions dispensed to US individuals aged 12 to 25 years from 2016 to 2022 using the IQVIA Longitudinal Prescription Database, an all-payer national database. The outcome was the monthly antidepressant dispensing rate, defined as the monthly number of individuals with ≥1 dispensed antidepressant prescription per 100 000 people. We fitted linear segmented regression models assessing for level or slope changes during March 2020 and conducted subgroup analyses by sex and age group. RESULTS: Between January 2016 and December 2022, the monthly antidepressant dispensing rate increased 66.3%, from 2575.9 to 4284.8. Before March 2020, this rate increased by 17.0 per month (95% confidence interval: 15.2 to 18.8). The COVID-19 outbreak was not associated with a level change but was associated with a slope increase of 10.8 per month (95% confidence interval: 4.9 to 16.7). The monthly antidepressant dispensing rate increased 63.5% faster from March 2020 onwards compared with beforehand. In subgroup analyses, this rate increased 129.6% and 56.5% faster from March 2020 onwards compared with beforehand among females aged 12 to 17 years and 18 to 25 years, respectively. In contrast, the outbreak was associated with a level decrease among males aged 12 to 17 years and was not associated with a level or slope change among males aged 18 to 25 years. CONCLUSIONS: Antidepressant dispensing to adolescents and young adults was rising before the COVID-19 outbreak and rose 63.5% faster afterward. This change was driven by increased antidepressant dispensing to females and occurred despite decreased dispensing to male adolescents.

Computationally designed sensors detect endogenous Ras activity and signaling effectors at subcellular resolution.

The utility of genetically encoded biosensors for sensing the activity of signaling proteins has been hampered by a lack of strategies for matching sensor sensitivity to the physiological concentration range of the target. Here we used computational protein design to generate intracellular sensors of Ras activity (LOCKR-based Sensor for Ras activity (Ras-LOCKR-S)) and proximity labelers of the Ras signaling environment (LOCKR-based, Ras activity-dependent Proximity Labeler (Ras-LOCKR-PL)). These tools allow the detection of endogenous Ras activity and labeling of the surrounding environment at subcellular resolution. Using these sensors in human cancer cell lines, we identified Ras-interacting proteins in oncogenic EML4-Alk granules and found that Src-Associated in Mitosis 68-kDa (SAM68) protein specifically enhances Ras activity in the granules. The ability to subcellularly localize endogenous Ras activity should deepen our understanding of Ras function in health and disease and may suggest potential therapeutic strategies.

ASSOCIATION OF METFORMIN USE WITH RISK OF NEWLY ONSET NEOVASCULAR AGE-RELATED MACULAR DEGENERATION DEVELOPMENT.

PURPOSE: To investigate if metformin use reduces the odds of developing new neovascular AMD (nAMD). METHODS: This is a case-control study of 86,930 subjects with new diagnoses of nAMD and 86,918 matched control subjects using the Merative Marketscan Research Databases. Subjects were analyzed using multivariable conditional logistic regression to identify the risks of various exposures on developing nAMD. A subgroup analysis of 22,117 diabetic cases and 21,616 diabetic control subjects was also performed. RESULTS: Metformin use was associated with reduced odds ratio of developing nAMD (odds ratio 0.95, 95% confidence interval 0.91-0.98) in full sample and diabetic cohort particularly in patients without any diabetic retinopathy-an effect that persisted after Bonferroni correction. In the diabetic cohort without diabetic retinopathy, reduced odds ratio was observed at 24-month cumulative doses of 1 to 300 g, 301 to 630 g, and 631 to 1,080 g. CONCLUSION: Metformin use was associated with reduced odds ratio of nAMD, particularly in patients without diabetic retinopathy. The protective effect was noted for 24-month cumulative doses below 1,080 g. Metformin may be a novel preventive strategy for nAMD.

Association of Contrast Sensitivity With Eye Disease and Vision-Related Quality of Life.

PURPOSE: To investigate contrast sensitivity (CS) as a screening tool to detect eye disease and assess its association with both eye disease and vision-related quality of life. DESIGN: Cross-sectional study. METHODS: Setting and population: Adults receiving care from a free clinic and a Federally Qualified Health Center in Michigan. MAIN OUTCOME MEASURES: Screening positive for eye disease and Visual Function Questionnaire (VFQ) score. OBSERVATION: Participants received a vision exam reviewed via telemedicine for disease, completed a demographic survey, and the 9-item VFQ. The ability of CS to predict eye disease was explored and area under the curve (AUC) is reported. Logistic and linear regression were used to investigate the continuous effect of CS on the probability of screening positive for eye disease and VFQ score, respectively, adjusting for age and visual acuity. RESULTS: 1159 included participants were, on average, 54.9 ± 14.5 years old, 62% identified as female, 34% as White, 54% as Black, 10% as Hispanic/Latino, and reported mean VFQ score of 79.7 ± 15.3. CS ranged from 0.00 to 1.95 log units (mean = 1.54 ± 0.24), 21% of eyes had glaucoma, 19% cataract, 6% DR, and 2% AMD. AUCs were 0.53 to 0.73. A 0.3 log unit decrease in better eye CS was associated with increased odds of glaucoma (odds ratio [OR] = 1.35, confidence interval [CI] = 1.09-1.67), cataract (OR = 1.35, CI = 1.05-1.72), DR (OR = 2.05, CI = 1.51-2.77), and AMD (OR = 2.08, CI = 1.10-3.91). A 0.3 log unit increase in better eye CS was associated with a 5.9 unit increase in VFQ. CONCLUSION: While CS alone is not sufficient to identify people with eye disease, it is an important measure of visual function that can add value to comprehensive eye screening.

The natural history and long-term follow-up of splenic artery aneurysms.

OBJECTIVE: Although splenic artery aneurysms (SAAs) are the most common visceral aneurysm, there is a paucity of literature on the behavior of these entities. The objective of this study was to review the natural history of patients with SAA. METHODS: This single-institution, retrospective analysis studied patients with SAA diagnosed by computed tomography imaging between 2015 and 2019, identified by our institutional radiology database. Imaging, demographic, and clinical data were obtained via the electronic medical record. The growth rate was calculated for patients with radiologic follow-up. RESULTS: The cohort consisted of 853 patients with 890 SAAs, of whom 692 were female (81.2%). There were 37 women (5.3%) of childbearing age (15-50 years). The mean age at diagnosis was 70.9 years (range: 28-100 years). Frequently observed medical comorbidities included hypertension (70.2%), hypercholesterolemia (54.7%), and prior smoking (32.2%). Imaging indications included abdominal pain (37.3%), unrelated follow-up (28.0%), and follow-up of a previously noted visceral artery aneurysm (8.6%). The mean diameter at diagnosis was 13.3 ± 6.3 mm. Anatomic locations included the splenic hilum (36.0%), distal splenic artery (30.3%), midsplenic artery (23.9%), and proximal splenic artery (9.7%). Radiographically, the majority were saccular aneurysms (72.4%) with calcifications (88.5%). One patient (38-year-old woman) was initially diagnosed at the time of rupture of a 25 mm aneurysm; this patient underwent immediate endovascular intervention with no complications. The mean clinical follow-up among 812 patients was 4.1 ± 4.0 years, and the mean radiological follow-up among 514 patients was 3.8 ± 6.8 years. Of the latter, 122 patients (23.7%) experienced growth. Aneurysm growth rates for initial sizes <10 mm (n = 123), 10 to 19 mm (n = 353), 20 to 29 mm (n = 34), and >30 mm (n = 4) were 0.166 mm/y, 0.172 mm/y, 0.383 mm/y, and 0.246 mm/y, respectively. Of the entire cohort, 27 patients (3.2%) eventually underwent intervention (81.5% endovascular), with the most common indications including size/growth criteria (70.4%) and symptom development (18.5%). On multivariate analysis, only prior tobacco use was significantly associated with aneurysm growth (P = .028). CONCLUSIONS: The majority of SAAs in this cohort remained stable in size, with few patients requiring intervention over a mean follow-up of 4 years. Current guidelines recommending treatment of asymptomatic aneurysms >30 mm appear appropriate given their slow progression. Despite societal recommendations for intervention for all SAAs among women of childbearing age, only a minority underwent vascular surgical consultation and intervention in this series, indicating that these recommendations are likely not well known in the general medical community.

Visual Impairment from Uncorrected Refractive Error among Participants in a Novel Program to Improve Eye Care Access among Low-Income Adults in Michigan.

PURPOSE: To assess the rate of visual impairment (VI) from uncorrected refractive error (URE) and associations with demographic and socioeconomic factors among low-income patients presenting to the Michigan Screening and Intervention for Glaucoma and Eye Health through Telemedicine (MI-SIGHT) program. DESIGN: Cross-sectional study. PARTICIPANTS: Adults ≥ 18 years without acute ocular symptoms. METHODS: MI-SIGHT program participants received a telemedicine-based eye disease screening and ordered glasses through an online optical store. Participants were categorized based on refractive error (RE) status: VI from URE (presenting visual acuity [PVA], ≤ 20/50; best-corrected visual acuity [BCVA], ≥ 20/40), URE without VI (PVA, ≥ 20/40; ≥ 2-line improvement to BCVA), and no or adequately corrected RE (PVA, ≥ 20/40; < 2-line improvement to BCVA). Patient demographics, self-reported visual function, and satisfaction with glasses obtained through the program were compared among groups using analysis of variance, Kruskal-Wallis, chi-square, and Fisher exact testing. MAIN OUTCOME MEASURES: PVA, BCVA, and presence of VI (defined as PVA ≤ 20/50). RESULTS: Of 1171 participants enrolled in the MI-SIGHT program during the first year, average age was 55.1 years (SD = 14.5), 37.7% were male, 54.1% identified as Black, and 1166 (99.6%) had both PVA and BCVA measured. VI was observed in 120 participants (10.3%); 96 had VI from URE (8.2%), 168 participants (14.4%) had URE without VI, and 878 (75.3%) had no or adequately corrected RE. A smaller percentage of participants with VI resulting from URE reported having a college degree, and a larger percentage reported income < $10 000 compared with participants with no or adequately corrected RE (3.2% vs. 14.2% [P = 0.02]; 45.5% vs. 21.6% [P < 0.0001], respectively). Visual function was lowest among participants with VI from URE, followed by those with URE without VI, and then those with no or adequately corrected RE (9-item National Eye Institute Visual Function Questionnaire composite score, 67.3 ± 19.6 vs. 77.0 ± 14.4 vs. 82.2 ± 13.3, respectively; P < 0.0001). In total, 71.2% (n = 830) ordered glasses for an average cost of $36.80 ± $32.60; 97.7% were satisfied with their glasses. CONCLUSIONS: URE was the main cause of VI at 2 clinics serving low-income communities and was associated with reduced vision-related quality of life. An online optical store with lower prices made eyeglasses accessible to low-income patients. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Techniques and Preferences for Nonvalved Aqueous Shunts: A Survey of American Glaucoma Society Members.

PURPOSE: To assess practice patterns and opinions of glaucoma specialists regarding indications, surgical technique, and postoperative management for nonvalved aqueous shunts. DESIGN: Anonymous online survey study. PARTICIPANTS: American Glaucoma Society (AGS) members. METHODS: An anonymous online survey was distributed to glaucoma specialists via the AGS forum from June to August 2022. MAIN OUTCOME MEASURES: Survey questions and responses were assessed in 4 sections: (1) general demographics and practice patterns; (2) nonvalved tubes vs. trabeculectomy; (3) nonvalved tubes vs. valved tubes; and (4) nonvalved tube techniques. RESULTS: There were 132 respondents; nonvalved tubes were reported to be performed more often than trabeculectomy by 61% of respondents within 5 years of completing training and 23% of respondents with more than 15 years since completing training. The most frequently preferred types of nonvalved tubes were Baerveldt-350 (41%), Baerveldt-250 (27%), and ClearPath-250 (18%). In patients with lower target intraocular pressure (IOP), 92% of respondents preferred trabeculectomy over nonvalved tube; 33% cited a cutoff of < 12 mmHg, and 31% cited a cutoff of < 15 mmHg. In patients with higher preoperative IOP, 50% of respondents preferred valved over nonvalved tubes; 29% cited a cutoff of > 40 mmHg, and 38% cited a cutoff of > 30 mmHg. The most frequently used ligature was 7-0 Vicryl (69%). The most frequently used strategies for early IOP lowering were fenestrations without wicks (70%) and with wicks (22%), with one 10-0 Nylon being the most used wick technique (22%). Overall, 37% of respondents use a ripcord; among ripcord users, 55% use it for hypotony prevention (3-0 Prolene most common for this purpose at 35%), and 40% use it for optional early IOP lowering (4-0 Nylon most common for this purpose at 21%). If IOP is too high at postoperative week 4, 38% of respondents do not open the tube early. CONCLUSIONS: We demonstrate significant heterogeneity regarding specific indications, surgical technique, and postoperative management for nonvalved tubes. Future work is needed to identify and develop standardized guidelines alongside best practices. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

A large-scale retrospective study enabled deep-learning based pathological assessment of frozen procurement kidney biopsies to predict graft loss and guide organ utilization.

Lesion scores on procurement donor biopsies are commonly used to guide organ utilization for deceased-donor kidneys. However, frozen sections present challenges for histological scoring, leading to inter- and intra-observer variability and inappropriate discard. Therefore, we constructed deep-learning based models to recognize kidney tissue compartments in hematoxylin & eosin-stained sections from procurement needle biopsies performed nationwide in years 2011-2020. To do this, we extracted whole-slide abnormality features from 2431 kidneys and correlated with pathologists' scores and transplant outcomes. A Kidney Donor Quality Score (KDQS) was derived and used in combination with recipient demographic and peri-transplant characteristics to predict graft loss or assist organ utilization. The performance on wedge biopsies was additionally evaluated. Our model identified 96% and 91% of normal/sclerotic glomeruli respectively; 94% of arteries/arterial intimal fibrosis; 90% of tubules. Whole-slide features of Sclerotic Glomeruli (GS)%, Arterial Intimal Fibrosis (AIF)%, and Interstitial Space Abnormality (ISA)% demonstrated strong correlations with corresponding pathologists' scores of all 2431 kidneys, but had superior associations with post-transplant estimated glomerular filtration rates in 2033 and graft loss in 1560 kidneys. The combination of KDQS and other factors predicted one- and four-year graft loss in a discovery set of 520 kidneys and a validation set of 1040 kidneys. By using the composite KDQS of 398 discarded kidneys due to "biopsy findings", we suggest that if transplanted, 110 discarded kidneys could have had similar survival to that of other transplanted kidneys. Thus, our composite KDQS and survival prediction models may facilitate risk stratification and organ utilization while potentially reducing unnecessary organ discard.

Opioid Prescribing by US Surgeons, 2016-2022.

This cross-sectional study investigates the rate and dosing of opioid prescriptions among US surgeons from 2016 to 2022.

Oral Metformin Inhibits Choroidal Neovascularization by Modulating the Gut-Retina Axis.

Purpose: Emerging data indicate that metformin may prevent the development of age-related macular degeneration (AMD). Whereas the underlying mechanisms of metformin's anti-aging properties remain undetermined, one proposed avenue is the gut microbiome. Using the laser-induced choroidal neovascularization (CNV) model, we investigate the effects of oral metformin on CNV, retinal pigment epithelium (RPE)/choroid transcriptome, and gut microbiota. Methods: Specific pathogen free (SPF) male mice were treated via daily oral gavage of metformin 300 mg/kg or vehicle. Male mice were selected to minimize sex-specific differences to laser induction and response to metformin. Laser-induced CNV size and macrophage/microglial infiltration were assessed by isolectin and Iba1 immunostaining. High-throughput RNA-seq of the RPE/choroid was performed using Illumina. Fecal pellets were analyzed for gut microbiota composition/pathways with 16S rRNA sequencing/shotgun metagenomics, as well as microbial-derived metabolites, including small-chain fatty acids and bile acids. Investigation was repeated in metformin-treated germ-free (GF) mice and antibiotic-treated/GF mice receiving fecal microbiota transplantation (FMT) from metformin-treated SPF mice. Results: Metformin treatment reduced CNV size (P < 0.01) and decreased Iba1+ macrophage/microglial infiltration (P < 0.005). One hundred forty-five differentially expressed genes were identified in the metformin-treated group (P < 0.05) with a downregulation in pro-angiogenic genes Tie1, Pgf, and Gata2. Furthermore, metformin altered the gut microbiome in favor of Bifidobacterium and Akkermansia, with a significant increase in fecal levels of butyrate, succinate, and cholic acid. Metformin did not suppress CNV in GF mice but colonization of microbiome-depleted mice with metformin-derived FMT suppressed CNV. Conclusions: These data suggest that oral metformin suppresses CNV, the hallmark lesion of advanced neovascular AMD, via gut microbiome modulation.

A comparative study of clustering methods on gene expression data for lung cancer prognosis.

Lung cancer subtyping based on gene expression data is important for identifying patient subgroups with differing survival prognosis to facilitate customized treatment strategies for each subtype of patients. Unsupervised clustering methods are the traditional approach for clustering patients into subtypes. However, since those methods cluster patients based only on gene expression data, the resulting clusters may not always be relevant to the survival outcome of interest. In recent years, semi-supervised and supervised methods have been proposed, which leverage the survival outcome data to identify clusters more relevant to survival prognosis. This paper aims to compare the performance of different clustering methods for identifying clinically prognostic lung cancer subtypes based on two lung adenocarcinoma datasets. For each method, we clustered patients into two clusters and assessed the difference in patient survival time between clusters. Unsupervised methods were found to have large logrank p-values and no significant results in most cases. Semi-supervised and supervised methods had improved performance over unsupervised methods and very significant p-values. These results indicate that unsupervised methods are not capable of identifying clusters with significant differences in survival prognosis in most cases, while supervised and semi-supervised methods can better cluster patients into clinically useful subtypes.

Association between the COVID-19 outbreak and opioid prescribing by U.S. dentists.

BACKGROUND: U.S. data on opioid prescribing by dentists are limited to 2019. More recent data are needed to understand the effect of the COVID-19 outbreak on dental opioid prescribing, characterize current practices, and determine if dental opioid stewardship initiatives are still warranted. OBJECTIVE: To evaluate the association between the COVID-19 outbreak and the rate of opioid prescribing by U.S. dentists. METHODS: During February-April 2023, the authors conducted a cross-sectional analysis of the IQVIA Longitudinal Prescription Database, which reports 92% of prescriptions dispensed in U.S. retail pharmacies. The authors calculated the monthly dental opioid dispensing rate, defined as the monthly number of dispensed opioid prescriptions from dentists per 100,000 U.S. individuals, during January 2016-February 2020 and June 2020-December 2022. To prevent distortions in trends, data from March-May 2020, when dental opioid dispensing declined sharply, were excluded. Using linear segmented regression models, the authors assessed for level and slope changes in the dental opioid dispensing rate during June 2020. RESULTS: Analyses included 81,189,605 dental opioid prescriptions. The annual number of prescriptions declined from 16,105,634 in 2016 to 8,910,437 in 2022 (-44.7%). During January 2016-February 2020, the dental opioid dispensing rate declined -3.9 (95% CI: -4.3, -3.6) per month. In June 2020, this rate abruptly increased by 31.4 (95% CI: 19.3, 43.5) and the monthly decline in the dental opioid dispensing rate slowed to -2.1 (95% CI: -2.6, -1.6) per month. As a result, 6.1 million more dental opioid prescriptions were dispensed during June 2020-December 2022 than would be predicted had trends during January 2016-February 2020 continued. DISCUSSION: U.S. dental opioid prescribing is declining, but the rate of this decline slowed after the COVID-19 outbreak. Findings highlight the continued importance of dental opioid stewardship initiatives.

Multiomic screening of invasive GBM cells reveals targetable transsulfuration pathway alterations.

While the poor prognosis of glioblastoma arises from the invasion of a subset of tumor cells, little is known of the metabolic alterations within these cells that fuel invasion. We integrated spatially addressable hydrogel biomaterial platforms, patient site-directed biopsies, and multiomics analyses to define metabolic drivers of invasive glioblastoma cells. Metabolomics and lipidomics revealed elevations in the redox buffers cystathionine, hexosylceramides, and glucosyl ceramides in the invasive front of both hydrogel-cultured tumors and patient site-directed biopsies, with immunofluorescence indicating elevated reactive oxygen species (ROS) markers in invasive cells. Transcriptomics confirmed upregulation of ROS-producing and response genes at the invasive front in both hydrogel models and patient tumors. Among oncologic ROS, H2O2 specifically promoted glioblastoma invasion in 3D hydrogel spheroid cultures. A CRISPR metabolic gene screen revealed cystathionine γ-lyase (CTH), which converts cystathionine to the nonessential amino acid cysteine in the transsulfuration pathway, to be essential for glioblastoma invasion. Correspondingly, supplementing CTH knockdown cells with exogenous cysteine rescued invasion. Pharmacologic CTH inhibition suppressed glioblastoma invasion, while CTH knockdown slowed glioblastoma invasion in vivo. Our studies highlight the importance of ROS metabolism in invasive glioblastoma cells and support further exploration of the transsulfuration pathway as a mechanistic and therapeutic target.

De novo designed Hsp70 activator dissolves intracellular condensates.

Protein quality control (PQC) is carried out in part by the chaperone Hsp70, in concert with adapters of the J-domain protein (JDP) family. The JDPs, also called Hsp40s, are thought to recruit Hsp70 into complexes with specific client proteins. However, the molecular principles regulating this process are not well understood. We describe the de novo design of a set of Hsp70 binding proteins that either inhibited or stimulated Hsp70's ATPase activity; a stimulating design promoted the refolding of denatured luciferase in vitro, similar to native JDPs. Targeting of this design to intracellular condensates resulted in their nearly complete dissolution. The designs inform our understanding of chaperone structure-function relationships and provide a general and modular way to target PQC systems to condensates and other cellular targets.

Designed Endocytosis-Triggering Proteins mediate Targeted Degradation.

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC1,2 and KineTAC3, have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.

Single-cell signaling analysis reveals that Major Vault Protein facilitates RasG12C inhibitor resistance.

Recently developed covalent inhibitors for RasG12C provide the first pharmacological tools to target mutant Ras-driven cancers. However, the rapid development of resistance to current clinical Ras G12C inhibitors is common. Presumably, a subpopulation of RasG12C-expressing cells adapt their signaling to evade these inhibitors and the mechanisms for this phenomenon are unclear due to the lack of tools that can measure signaling with single-cell resolution. Here, we utilized recently developed Ras sensors to profile the environment of active Ras and to measure the activity of endogenous Ras in order to pair structure (Ras signalosome) to function (Ras activity), respectively, at a single-cell level. With this approach, we identified a subpopulation of KRasG12C cells treated with RasG12C-GDP inhibitors underwent oncogenic signaling and metabolic changes driven by WT Ras at the golgi and mutant Ras at the mitochondria, respectively. Our Ras sensors identified Major Vault Protein (MVP) as a mediator of Ras activation at both compartments by scaffolding Ras signaling pathway components and metabolite channels. We found that recently developed RasG12C-GTP inhibitors also led to MVP-mediated WT Ras signaling at the golgi, demonstrating that this a general mechanism RasG12C inhibitor resistance. Overall, single-cell analysis of structure-function relationships enabled the discovery of a RasG12C inhibitor-resistant subpopulation driven by MVP, providing insight into the complex and heterogenous rewiring occurring during drug resistance in cancer.