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ImportanceStroke is a leading cause of death and disability in the US. Accurate and updated measures of stroke burden are needed to guide public health policies.ObjectiveTo present burden estimates of ischemic and hemorrhagic stroke in the US in 2019 and describe trends from 1990 to 2019 by age, sex, and geographic location.Design, Setting, and ParticipantsAn in-depth cross-sectional analysis of the 2019 Global Burden of Disease study was conducted. The setting included the time period of 1990 to 2019 in the US. The study encompassed estimates for various types of strokes, including all strokes, ischemic strokes, intracerebral hemorrhages (ICHs), and subarachnoid hemorrhages (SAHs). The 2019 Global Burden of Disease results were released on October 20, 2020.ExposuresIn this study, no particular exposure was specifically targeted.Main Outcomes and MeasuresThe primary focus of this analysis centered on both overall and age-standardized estimates, stroke incidence, prevalence, mortality, and DALYs per 100 000 individuals.ResultsIn 2019, the US recorded 7.09 million prevalent strokes (4.07 million women [57.4%]; 3.02 million men [42.6%]), with 5.87 million being ischemic strokes (82.7%). Prevalence also included 0.66 million ICHs and 0.85 million SAHs. Although the absolute numbers of stroke cases, mortality, and DALYs surged from 1990 to 2019, the age-standardized rates either declined or remained steady. Notably, hemorrhagic strokes manifested a substantial increase, especially in mortality, compared with ischemic strokes (incidence of ischemic stroke increased by 13% [95% uncertainty interval (UI), 14.2%-11.9%]; incidence of ICH increased by 39.8% [95% UI, 38.9%-39.7%]; incidence of SAH increased by 50.9% [95% UI, 49.2%-52.6%]). The downturn in stroke mortality plateaued in the recent decade. There was a discernible heterogeneity in stroke burden trends, with older adults (50-74 years) experiencing a decrease in incidence in coastal areas (decreases up to 3.9% in Vermont), in contrast to an uptick observed in younger demographics (15-49 years) in the South and Midwest US (with increases up to 8.4% in Minnesota).Conclusions and RelevanceIn this cross-sectional study, the declining age-standardized stroke rates over the past 3 decades suggest progress in managing stroke-related outcomes. However, the increasing absolute burden of stroke, coupled with a notable rise in hemorrhagic stroke, suggests an evolving and substantial public health challenge in the US. Moreover, the significant disparities in stroke burden trends across different age groups and geographic locations underscore the necessity for region- and demography-specific interventions and policies to effectively mitigate the multifaceted and escalating burden of stroke in the country.

OBJECTIVE The objective of this study was to evaluate the 24-month durability of pain relief, function, quality of life, and safety outcomes for patients with nonsurgical refractory back pain (NSRBP) treated with high-frequency spinal cord stimulation (SCS) within a large, national, multicenter randomized controlled trial (RCT). METHODS Following the completion of an RCT comparing high-frequency SCS plus CMM with CMM alone for the treatment of NSRBP, patients gave additional consent for a follow-up extension to 24 months. Presented is the cohort analysis of all patients treated with high-frequency SCS following the optional crossover at 6 months. The outcomes assessed to 24 months included responder rate of ≥ 50% pain relief measured according to the visual analog scale [VAS]), disability (Oswestry Disability Index [ODI]), quality of life (EQ-5D 5-level [EQ-5D-5L]), opioid reduction. RESULTS Of the 125 patients who received a permanent implant, 121 completed the 12-month follow-up, 101 gave additional consent for extended follow-up, and 98 completed the 24-month follow-up. At 24 months after implantation, the mean back pain VAS score was reduced by 73% and the responder rate was 82%. ODI and EQ-5D-5L both improved by at least double the minimal clinically important difference for each measure. No unexpected adverse events were observed, and the rates of serious adverse events (3.4%) and device explantations (4.8%) were low. CONCLUSIONS The addition of high-frequency SCS to CMM in patients with NSRBP offers profound improvements at 24 months in pain, function, quality of life, and reduced opioid use. This study provides much-needed evidence to inform current clinical practice for managing patients with NSRBP.

Abstract Intraductal carcinoma of the prostate (IDC-P) is a lethal prostate cancer subtype that generally coexists with invasive high-grade prostate acinar adenocarcinoma (PAC) but exhibits distinct biological features compared with concomitant adenocarcinoma. In this study, we performed whole-exome, RNA, and DNA-methylation sequencing of IDC-P, concurrent invasive high-grade PAC lesions, and adjacent normal prostate tissues isolated from 22 radical prostatectomy specimens. Three evolutionary patterns of concurrent IDC-P and PAC were identified: early divergent, late divergent, and clonally distant. In contrast to those with a late divergent evolutionary pattern, tumors with clonally distant and early divergent evolutionary patterns showed higher genomic, epigenomic, transcriptional, and pathologic heterogeneity between IDC-P and PAC. Compared with coexisting PAC, IDC-P displayed increased expression of adverse prognosis–associated genes. Survival analysis based on an independent cohort of 505 patients with metastatic prostate cancer revealed that IDC-P carriers with lower risk International Society of Urological Pathology (ISUP) grade 1–4 adenocarcinoma displayed a castration-resistant free survival as poor as those with the highest risk ISUP grade 5 tumors that lacked concurrent IDC-P. Furthermore, IDC-P exhibited robust cell-cycle progression and androgen receptor activities, characterized by an enrichment of cellular proliferation–associated master regulators and genes involved in intratumoral androgen biosynthesis. Overall, this study provides a molecular groundwork for the aggressive behavior of IDC-P and could help identify potential strategies to improve treatment of IDC-P. Significance: The genomic, transcriptomic, and epigenomic characterization of concurrent intraductal carcinoma and adenocarcinoma of the prostate deepens the biological understanding of this lethal disease and provides a genetic basis for developing targeted therapies.

ImportanceLip, oral, and pharyngeal cancers are important contributors to cancer burden worldwide, and a comprehensive evaluation of their burden globally, regionally, and nationally is crucial for effective policy planning.ObjectiveTo analyze the total and risk-attributable burden of lip and oral cavity cancer (LOC) and other pharyngeal cancer (OPC) for 204 countries and territories and by Socio-demographic Index (SDI) using 2019 Global Burden of Diseases, Injuries, and Risk Factors (GBD) Study estimates.Evidence ReviewThe incidence, mortality, and disability-adjusted life years (DALYs) due to LOC and OPC from 1990 to 2019 were estimated using GBD 2019 methods. The GBD 2019 comparative risk assessment framework was used to estimate the proportion of deaths and DALYs for LOC and OPC attributable to smoking, tobacco, and alcohol consumption in 2019.FindingsIn 2019, 370 000 (95% uncertainty interval [UI], 338 000-401 000) cases and 199 000 (95% UI, 181 000-217 000) deaths for LOC and 167 000 (95% UI, 153 000-180 000) cases and 114 000 (95% UI, 103 000-126 000) deaths for OPC were estimated to occur globally, contributing 5.5 million (95% UI, 5.0-6.0 million) and 3.2 million (95% UI, 2.9-3.6 million) DALYs, respectively. From 1990 to 2019, low-middle and low SDI regions consistently showed the highest age-standardized mortality rates due to LOC and OPC, while the high SDI strata exhibited age-standardized incidence rates decreasing for LOC and increasing for OPC. Globally in 2019, smoking had the greatest contribution to risk-attributable OPC deaths for both sexes (55.8% [95% UI, 49.2%-62.0%] of all OPC deaths in male individuals and 17.4% [95% UI, 13.8%-21.2%] of all OPC deaths in female individuals). Smoking and alcohol both contributed to substantial LOC deaths globally among male individuals (42.3% [95% UI, 35.2%-48.6%] and 40.2% [95% UI, 33.3%-46.8%] of all risk-attributable cancer deaths, respectively), while chewing tobacco contributed to the greatest attributable LOC deaths among female individuals (27.6% [95% UI, 21.5%-33.8%]), driven by high risk-attributable burden in South and Southeast Asia.Conclusions and RelevanceIn this systematic analysis, disparities in LOC and OPC burden existed across the SDI spectrum, and a considerable percentage of burden was attributable to tobacco and alcohol use. These estimates can contribute to an understanding of the distribution and disparities in LOC and OPC burden globally and support cancer control planning efforts.

Abstract Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation Sayantani Sarkar Bhattacharya1, Taylor L. Witter1, Anh Q. T. Cong1, Elizabeth Bruinsma2, Swaathi Jayaraman2, Matthew P. Goetz2, John R. Hawse1, and Matthew J. Schellenberg1 1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN USA 55905 2Department of Oncology, Mayo Clinic, Rochester, MN USA 55905 Background: Z-Endoxifen (ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM), exhibited high antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancer. ENDX has also been shown to be a protein kinase C (PKC) inhibitor. PKCs participate in diverse cellular functions and their activity is often elevated in breast tumors. Guided by mechanistic insights from our recently determined crystal structure of PKCβI, we sought to determine the effects of ENDX on PKCβI in a breast cancer cell line model. Methods: To determine how ENDX regulates PKC activity, we used a Z’LYTE kinase activity assay. This Fluorescence Resonance Energy Transfer (FRET) based biochemical method can detect differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. We probed changes in activity for conventional and novel PKCs, as well as the purified catalytic domain of conventional PKCs in vitro. Alongside, as kinase activity of PKC relies on its spatial assembly, therefore we studied its intracellular localization using live cell confocal imaging. MCF7 cells expressing YFP-tagged PKCβI were grown in a glass bottom chamber and treated with ENDX and other modulators for relevant time and doses for this study. Images were taken using Zeiss LSM 780 confocal laser scanning microscope and analyzed in Zeiss-ZEN microscope software and GraphPad Prism 9. Results: Our data from an in vitro kinase assay indicates that ENDX inhibits the kinase activity of conventional (PKCβI) and novel (PKC𝛿) PKC isoforms with a similar IC50, however PKCβI catalytic domain is less sensitive to ENDX. We also identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory mechanism. Our live cell imaging study demonstrated that ENDX promotes the recruitment of PKCβI to the cell membrane in both a dose and time-dependent manner. Moreover, this translocation can also be mitigated by co-treatment with inhibitors of the PKC-regulator PHLPP phosphatases. Conclusion: Taken together, these results suggest the allosteric effects of ENDX trigger PKCβI recruitment to the cell membrane, yet since ENDX also inhibits kinase activity it suggests that ENDX triggers a non-productive interaction with the enzyme and "breaks" the well-known mechanism of PKC activation upon binding the cell membrane. Furthermore, ENDX is likely to trigger dephosphorylation and ultimately degradation, suggesting that ENDX represents a new mechanistic basis for targeting and downregulating PKC in cancer cells. Hence, the current study provides an integrated pattern of highly specific treatments regimen that can exploit PKCβI as a repurposing clinical target in breast cancer. Citation Format: Sayantani Sarkar Bhattacharya, Taylor L. Witter, Anh T. Cong, Elizabeth S. Bruinsma, Swaathi Jayaraman, Matthew P. Goetz, John Hawse, Matthew Schellenberg. Z-Endoxifen Allosterically Inhibits PKCβI and its Paradoxical Membrane Translocation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-03.

AbstractObjectiveThe aim of this study was to analyze a publicly available sample of rule-based phenotype definitions to characterize and evaluate the variability of logical constructs used.Materials and MethodsA sample of 33 preexisting phenotype definitions used in research that are represented using Fast Healthcare Interoperability Resources and Clinical Quality Language (CQL) was analyzed using automated analysis of the computable representation of the CQL libraries.ResultsMost of the phenotype definitions include narrative descriptions and flowcharts, while few provide pseudocode or executable artifacts. Most use 4 or fewer medical terminologies. The number of codes used ranges from 5 to 6865, and value sets from 1 to 19. We found that the most common expressions used were literal, data, and logical expressions. Aggregate and arithmetic expressions are the least common. Expression depth ranges from 4 to 27.DiscussionDespite the range of conditions, we found that all of the phenotype definitions consisted of logical criteria, representing both clinical and operational logic, and tabular data, consisting of codes from standard terminologies and keywords for natural language processing. The total number and variety of expressions are low, which may be to simplify implementation, or authors may limit complexity due to data availability constraints.ConclusionsThe phenotype definitions analyzed show significant variation in specific logical, arithmetic, and other operators but are all composed of the same high-level components, namely tabular data and logical expressions. A standard representation for phenotype definitions should support these formats and be modular to support localization and shared logic.

Abstract Mammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post‐translational modifications in the protein's native environment is desired. However, low or variable transfection efficiencies are challenges that must be overcome to use such an expression system. Expression of recombinant proteins as a fluorescent protein fusion enables real‐time monitoring of protein expression, and also provides an affinity handle for one‐step protein purification using a suitable affinity reagent. Here, we describe a panel of anti‐GFP and anti‐mCherry nanobody affinity matrices and their efficacy for purification of GFP/YFP or mCherry fusion proteins. We define the molecular basis by which they bind their target proteins using X‐ray crystallography. From these analyses, we define an optimal pair of nanobodies for purification of recombinant protein tagged with GFP/YFP or mCherry, and demonstrate these nanobody‐sepharose supports are stable to many rounds of cleaning and extended incubation in denaturing conditions. Finally, we demonstrate the utility of the mCherry‐tag system by using it to purify recombinant human topoisomerase 2α expressed in HEK293F cells. The mCherry‐tag and GFP/YFP‐tag expression systems can be utilized for recombinant protein expression individually or in tandem for mammalian protein expression systems where real‐time monitoring of protein expression levels and a high‐efficiency purification step is needed.

SARS-CoV-2 mRNA vaccination induces robust humoral and cellular immunity in the circulation; however, it is currently unknown whether it elicits effective immune responses in the respiratory tract, particularly against variants of concern (VOCs), including Omicron. We compared the SARS-CoV-2 S–specific total and neutralizing antibody responses, and B and T cell immunity, in the bronchoalveolar lavage fluid (BAL) and blood of COVID-19–vaccinated individuals and hospitalized patients. Vaccinated individuals had significantly lower levels of neutralizing antibody against D614G, Delta (B.1.617.2), and Omicron BA.1.1 in the BAL compared with COVID-19 convalescents despite robust S-specific antibody responses in the blood. Furthermore, mRNA vaccination induced circulating S-specific B and T cell immunity, but in contrast to COVID-19 convalescents, these responses were absent in the BAL of vaccinated individuals. Using a mouse immunization model, we demonstrated that systemic mRNA vaccination alone induced weak respiratory mucosal neutralizing antibody responses, especially against SARS-CoV-2 Omicron BA.1.1 in mice; however, a combination of systemic mRNA vaccination plus mucosal adenovirus-S immunization induced strong neutralizing antibody responses not only against the ancestral virus but also the Omicron BA.1.1 variant. Together, our study supports the contention that the current COVID-19 vaccines are highly effective against severe disease development, likely through recruiting circulating B and T cell responses during reinfection, but offer limited protection against breakthrough infection, especially by the Omicron sublineage. Hence, mucosal booster vaccination is needed to establish robust sterilizing immunity in the respiratory tract against SARS-CoV-2, including infection by the Omicron sublineage and future VOCs.