Understanding Racial/Ethinic Disparities in COVID Mortality Using a Novel Metric: COVID Excess Mortality Percentage.

Prior research on racial/ethnic disparities in COVID-19 mortality has often not considered to what extent they reflect COVID-19-specific factors, versus preexisting health differences. This study examines how racial/ethnic disparities in COVID-19 mortality vary with age, gender, and time period over April - December 2020 in the US, using mortality from other natural causes to proxy for underlying health. We study a novel measure, the COVID Excess Mortality Percentage (CEMP), defined as the COVID-19 mortality rate, divided by the non-COVID natural mortality rate, converted to a percentage, where the CEMP denominator, controls, albeit imperfectly, for differences in population health. Disparities measured using CEMP deviate substantially from those in prior research. In particular, we find very high disparities (up to 12:1) in CEMP rates for Hispanics versus Whites, particularly for non-elderly men. Asians also have elevated CEMP rates versus Whites, which were obscured in prior work by lower overall Asian mortality. Native Americans and Blacks have significant disparities compared to Whites populations, but CEMP ratios to Whites are lower than ratios reported in other work. This is because the higher COVID-19 mortality for Blacks and Native Americans comes partly from higher general mortality risk, and partly from COVID-specific risk.

Minimal requirements for the epigenetic inheritance of engineered silent chromatin domains.

Mechanisms enabling genetically identical cells to differentially regulate gene expression are complex and central to organismal development and evolution. While gene silencing pathways involving DNA sequence-specific recruitment of histone-modifying enzymes are prevalent in nature, examples of sequence-independent heritable gene silencing are scarce. Studies of the fission yeast Schizosaccharomyces pombe indicate that sequence-independent propagation of heterochromatin can occur but requires numerous multisubunit protein complexes and their diverse activities. Such complexity has so far precluded a coherent articulation of the minimal requirements for heritable gene silencing by conventional in vitro reconstitution approaches. Here, we take an unconventional approach to defining these requirements by engineering sequence-independent silent chromatin inheritance in budding yeast Saccharomyces cerevisiae cells. The mechanism conferring memory upon these cells is remarkably simple and requires only two proteins, one that recognizes histone H3 lysine 9 methylation (H3K9me) and catalyzes the deacetylation of histone H4 lysine 16 (H4K16), and another that recognizes deacetylated H4K16 and catalyzes H3K9me. Together, these bilingual "read-write" proteins form an interdependent positive feedback loop that is sufficient for the transmission of DNA sequence-independent silent information over multiple generations.

Evidence on COVID-19 Mortality and Disparities Using a Novel Measure, COVID excess mortality percentage: Evidence from Indiana, Wisconsin, and Illinois.

COVID-19 mortality rates increase rapidly with age, are higher among men than women, and vary across racial/ethnic groups, but this is also true for other natural causes of death. Prior research on COVID-19 mortality rates and racial/ethnic disparities in those rates has not considered to what extent disparities reflect COVID-19-specific factors, versus preexisting health differences. This study examines both questions. We study the COVID-19-related increase in mortality risk and racial/ethnic disparities in COVID-19 mortality, and how both vary with age, gender, and time period. We use a novel measure validated in prior work, the COVID Excess Mortality Percentage (CEMP), defined as the COVID-19 mortality rate (Covid-MR), divided by the non-COVID natural mortality rate during the same time period (non-Covid NMR), converted to a percentage. The CEMP denominator uses Non-COVID NMR to adjust COVID-19 mortality risk for underlying population health. The CEMP measure generates insights which differ from those using two common measures-the COVID-MR and the all-cause excess mortality rate. By studying both CEMP and COVID-MRMR, we can separate the effects of background health from Covid-specific factors affecting COVID-19 mortality. We study how CEMP and COVID-MR vary by age, gender, race/ethnicity, and time period, using data on all adult decedents from natural causes in Indiana and Wisconsin over April 2020-June 2022 and Illinois over April 2020-December 2021. CEMP levels for racial and ethnic minority groups can be very high relative to White levels, especially for Hispanics in 2020 and the first-half of 2021. For example, during 2020, CEMP for Hispanics aged 18-59 was 68.9% versus 7.2% for non-Hispanic Whites; a ratio of 9.57:1. CEMP disparities are substantial but less extreme for other demographic groups. Disparities were generally lower after age 60 and declined over our sample period. Differences in socio-economic status and education explain only a small part of these disparities.

Measuring prion propagation in single bacteria elucidates a mechanism of loss.

Prions are self-propagating protein aggregates formed by specific proteins that can adopt alternative folds. Prions were discovered as the cause of the fatal transmissible spongiform encephalopathies in mammals, but prions can also constitute nontoxic protein-based elements of inheritance in fungi and other species. Prion propagation has recently been shown to occur in bacteria for more than a hundred cell divisions, yet a fraction of cells in these lineages lost the prion through an unknown mechanism. Here, we investigate prion propagation in single bacterial cells as they divide using microfluidics and fluorescence microscopy. We show that the propagation occurs in two distinct modes. In a fraction of the population, cells had multiple small visible aggregates and lost the prion through random partitioning of aggregates to one of the two daughter cells at division. In the other subpopulation, cells had a stable large aggregate localized to the pole; upon division the mother cell retained this polar aggregate and a daughter cell was generated that contained small aggregates. Extending our findings to prion domains from two orthologous proteins, we observe similar propagation and loss properties. Our findings also provide support for the suggestion that bacterial prions can form more than one self-propagating state. We implement a stochastic version of the molecular model of prion propagation from yeast and mammals that recapitulates all the observed single-cell properties. This model highlights challenges for prion propagation that are unique to prokaryotes and illustrates the conservation of fundamental characteristics of prion propagation.

Selection Effects and COVID-19 Mortality Risk after Pfizer vs. Moderna Vaccination: Evidence from Linked Mortality and Vaccination Records.

Prior research generally finds that the Pfizer-BioNTech (BNT162b2) and Moderna (mRNA1273) COVID-19 vaccines provide similar protection against mortality, sometimes with a Moderna advantage due to slower waning. However, most comparisons do not address selection effects for those who are vaccinated and with which vaccine. We report evidence on large selection effects, and use a novel method to control for these effects. Instead of directly studying COVID-19 mortality, we study the COVID-19 excess mortality percentage (CEMP), defined as the COVID-19 deaths divided by non-COVID-19 natural deaths for the same population, converted to a percentage. The CEMP measure uses non-COVID-19 natural deaths to proxy for population health and control for selection effects. We report the relative mortality risk (RMR) for each vaccine relative to the unvaccinated population and to the other vaccine, using linked mortality and vaccination records for all adults in Milwaukee County, Wisconsin, from 1 April 2021 through 30 June 2022. For two-dose vaccinees aged 60+, RMRs for Pfizer vaccinees were consistently over twice those for Moderna, and averaged 248% of Moderna (95% CI = 175%,353%). In the Omicron period, Pfizer RMR was 57% versus 23% for Moderna. Both vaccines demonstrated waning of two-dose effectiveness over time, especially for ages 60+. For booster recipients, the Pfizer-Moderna gap is much smaller and statistically insignificant. A possible explanation for the Moderna advantage for older persons is the higher Moderna dose of 100 µg, versus 30 µg for Pfizer. Younger persons (aged 18-59) were well-protected against death by two doses of either vaccine, and highly protected by three doses (no deaths among over 100,000 vaccinees). These results support the importance of a booster dose for ages 60+, especially for Pfizer recipients. They suggest, but do not prove, that a larger vaccine dose may be appropriate for older persons than for younger persons.

Using a deep learning approach for implanted seed detection on fluoroscopy images in prostate brachytherapy.

Purpose: To apply a deep learning approach to automatically detect implanted seeds on a fluoroscopy image in prostate brachytherapy. Material and methods: Forty-eight fluoroscopy images of patients, who underwent permanent seed implant (PSI) were used for this study after our Institutional Review Boards approval. Pre-processing procedures that were used to prepare for the training data, included encapsulating each seed in a bounding box, re-normalizing seed dimension, cropping to a region of prostate, and converting fluoroscopy image to PNG format. We employed a pre-trained faster region convolutional neural network (R-CNN) from PyTorch library for automatic seed detection, and leave-one-out cross-validation (LOOCV) procedure was applied to evaluate the performance of the model. Results: Almost all cases had mean average precision (mAP) greater than 0.91, with most cases (83.3%) having a mean average recall (mAR) above 0.9. All cases achieved F1-scores exceeding 0.91. The averaged results for all the cases were 0.979, 0.937, and 0.957 for mAP, mAR, and F1-score, respectively. Conclusions: Although there are limitations shown in interpreting overlapping seeds, our model is reasonably accurate and shows potential for further applications.

Understanding COVID-19 Vaccine Effectiveness against Death Using a Novel Measure: COVID Excess Mortality Percentage.

COVID-19 vaccines have saved millions of lives; however, understanding the long-term effectiveness of these vaccines is imperative to developing recommendations for booster doses and other precautions. Comparisons of mortality rates between more and less vaccinated groups may be misleading due to selection bias, as these groups may differ in underlying health status. We studied all adult deaths during the period of 1 April 2021-30 June 2022 in Milwaukee County, Wisconsin, linked to vaccination records, and we used mortality from other natural causes to proxy for underlying health. We report relative COVID-19 mortality risk (RMR) for those vaccinated with two and three doses versus the unvaccinated, using a novel outcome measure that controls for selection effects. This measure, COVID Excess Mortality Percentage (CEMP), uses the non-COVID natural mortality rate (Non-COVID-NMR) as a measure of population risk of COVID mortality without vaccination. We validate this measure during the pre-vaccine period (Pearson correlation coefficient = 0.97) and demonstrate that selection effects are large, with non-COVID-NMRs for two-dose vaccinees often less than half those for the unvaccinated, and non-COVID NMRs often still lower for three-dose (booster) recipients. Progressive waning of two-dose effectiveness is observed, with an RMR of 10.6% for two-dose vaccinees aged 60+ versus the unvaccinated during April-June 2021, rising steadily to 36.2% during the Omicron period (January-June, 2022). A booster dose reduced RMR to 9.5% and 10.8% for ages 60+ during the two periods when boosters were available (October-December, 2021; January-June, 2022). Boosters thus provide important additional protection against mortality.

Understanding COVID-19 Vaccine Effectiveness Against Death Using a Novel Measure: COVID Excess Mortality Percentage.

COVID-19 vaccines have saved millions of lives and prevented countless adverse patient disease outcomes. Understanding the long-term effectiveness of these vaccines is imperative to developing recommendations for precautions and booster doses. Comparisons between more and less vaccinated groups may be misleading due to selection bias, as these groups may differ in underlying health status and thus risk of adverse COVID-19 outcomes. We study all adult deaths over April 1, 2021-June 30, 2022 in Milwaukee County, Wisconsin, linked to vaccination records, use mortality from other natural causes to proxy for underlying health, and report relative COVID-19 mortality risk (RMR) for vaccinees versus the unvaccinated, using a novel outcome measure that controls for selection effects. This measure, COVID Excess Mortality Percentage (CEMP) uses the non-COVID natural mortality rate (Non-Covid-NMR) as a measure of population risk of COVID mortality without vaccination. We validate this measure during the pre-vaccine period (r = 0.97) and demonstrate that selection effects are large, with Non-Covid-NMRs for two-dose vaccinees less than half those for the unvaccinated, and Non-COVID NMRs still lower for three dose (booster) recipients. Progressive waning of two-dose effectiveness is observed, with relative mortality risk (RMR) for two-dose vaccinees aged 60 + versus the unvaccinated of 11% during April-June 2021, rising steadily to 36% during the Omicron period (January-June, 2022). Notably, a booster dose reduced RMR to 10-11% for ages 60+. Boosters thus provide important additional protection against mortality.

Bispecific antibody targeting TROP2xCD3 suppresses tumor growth of triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is a subtype of breast cancers with poor prognosis and targeted drug therapies are limited. To develop novel and efficacious therapies for TNBC, we developed a bispecific antibody F7AK3 that recognizes both trophoblast cell surface antigen 2 (TROP2) and CD3 and evaluated its antitumor activities both in vitro and in vivo. METHODS: The binding affinities of F7AK3 to the two targets, TROP2 and CD3, were evaluated by surface plasmon resonance. Binding of F7AK3 to TNBC cells and T cells were evaluated by flow cytometry. Immunofluorescent staining was performed to demonstrate the interactions between T cells with TNBC cells. The cytotoxicity of T cells against TNBC cell lines and primary tumor cells mediated by F7AK3 were determined in vitro. In vivo antitumor activity of F7AK3 was investigated in a xenograft TNBC tumor model, using immunodeficient mice that were reconstituted with human peripheral blood mononuclear cells. RESULTS: We demonstrated that F7AK3 binds specifically to human TROP2 and CD3 antigens, as well as TNBC cell lines and primary tumor cells. Human T cells can only be activated by F7AK3 in the presence of target tumor cells. F7AK3 recruits T cells to TROP2+ tumor cells in vitro and into tumor tissues in vivo. Antitumor growth activity of F7AK3 is observed in a xenograft TNBC tumor model. CONCLUSION: This study showed the antitumor potential of an anti-TROP2xCD3 bispecific antibody F7AK3 to TNBC tumor cells both in vitro and in vivo. These data demonstrate that F7AK3 has the potential to treat TNBC patients, which warrants further preclinical and clinical evaluation of the F7AK3 in advanced or metastatic TNBC patients.

The state of the art of bispecific antibodies for treating human malignancies.

Bispecific antibodies (bsAb) that target two independent epitopes or antigens have been extensively explored in translational and clinical studies since they were first developed in the 1960s. Many bsAbs are being tested in clinical trials for treating a variety of diseases, mostly cancer. Here, we provide an overview of various types of bsAbs in clinical studies and discuss their targets, safety profiles, and efficacy. We also highlight the current challenges, potential solutions, and future directions of bsAb development for cancer treatment.

Distinguishing moral hazard from access for high-cost healthcare under insurance.

CONTEXT: Health policy has long been preoccupied with the problem that health insurance stimulates spending ("moral hazard"). However, much health spending is costly healthcare that uninsured individuals could not otherwise access. Field studies comparing those with more or less insurance cannot disaggregate moral hazard versus access. Moreover, studies of patients consuming routine low-dollar healthcare are not informative for the high-dollar healthcare that drives most of aggregate healthcare spending in the United States. METHODS: We test indemnities as an alternative theory-driven counterfactual. Such conditional cash transfers would maintain an opportunity cost for patients, unlike standard insurance, but also guarantee access to the care. Since indemnities do not exist in U.S. healthcare, we fielded two blinded vignette-based survey experiments with 3,000 respondents, randomized to eight clinical vignettes and three insurance types. Our replication uses a population that is weighted to national demographics on three dimensions. FINDINGS: Most or all of the spending due to insurance would occur even under an indemnity. The waste attributable to moral hazard is undetectable. CONCLUSIONS: For high-cost care, policymakers should be more concerned about the foregone efficient spending for those lacking full insurance, rather than the wasteful spending that occurs with full insurance.

Isolation of Antibody Binders to MISIIR from a Phage Display Library by Sorting.

Cell surface antigens represent the most common targets for antibody-based cancer therapy. Isolation of lead antibodies to these membrane targets from antibody repertoires, such as immunized or naïve phage display libraries, has been a challenging task, which is an outstanding issue when soluble portion of the target(s) is not available, and/or a naïve phage display library is used. Common cell-based panning methods often encounter numerous difficulties, including high background and loss of cells during repeated washes. Here we described a novel FACS sorter-based protocol to isolate single-chain Fv molecules specific for defined antigen MSIIR expressed on stably transformed mammalian cells, and screening of unique binders to the tumor target.

A bacteria-based genetic assay detects prion formation.

Prions are infectious, self-propagating protein aggregates that are notorious for causing devastating neurodegenerative diseases in mammals. Recent evidence supports the existence of prions in bacteria. However, the evaluation of candidate bacterial prion-forming proteins has been hampered by the lack of genetic assays for detecting their conversion to an aggregated prion conformation. Here we describe a bacteria-based genetic assay that distinguishes cells carrying a model yeast prion protein in its nonprion and prion forms. We then use this assay to investigate the prion-forming potential of single-stranded DNA-binding protein (SSB) of Campylobacter hominis Our findings indicate that SSB possesses a prion-forming domain that can transition between nonprion and prion conformations. Furthermore, we show that bacterial cells can propagate the prion form over 100 generations in a manner that depends on the disaggregase ClpB. The bacteria-based genetic tool we present may facilitate the investigation of prion-like phenomena in all domains of life.

A highly potent extended half-life antibody as a potential RSV vaccine surrogate for all infants.

Prevention of respiratory syncytial virus (RSV) illness in all infants is a major public health priority. However, no vaccine is currently available to protect this vulnerable population. Palivizumab, the only approved agent for RSV prophylaxis, is limited to high-risk infants, and the cost associated with the requirement for dosing throughout the RSV season makes its use impractical for all infants. We describe the development of a monoclonal antibody as potential RSV prophylaxis for all infants with a single intramuscular dose. MEDI8897*, a highly potent human antibody, was optimized from antibody D25, which targets the prefusion conformation of the RSV fusion (F) protein. Crystallographic analysis of Fab in complex with RSV F from subtypes A and B reveals that MEDI8897* binds a highly conserved epitope. MEDI8897* neutralizes a diverse panel of RSV A and B strains with >50-fold higher activity than palivizumab. At similar serum concentrations, prophylactic administration of MEDI8897* was ninefold more potent than palivizumab at reducing pulmonary viral loads by >3 logs in cotton rats infected with either RSV A or B subtypes. MEDI8897 was generated by the introduction of triple amino acid substitutions (YTE) into the Fc domain of MEDI8897*, which led to more than threefold increased half-life in cynomolgus monkeys compared to non-YTE antibody. Considering the pharmacokinetics of palivizumab in infants, which necessitates five monthly doses for protection during an RSV season, the high potency and extended half-life of MEDI8897 support its development as a cost-effective option to protect all infants from RSV disease with once-per-RSV-season dosing in the clinic.

A bacterial global regulator forms a prion.

Prions are self-propagating protein aggregates that act as protein-based elements of inheritance in fungi. Although prevalent in eukaryotes, prions have not been identified in bacteria. Here we found that a bacterial protein, transcription terminator Rho of Clostridium botulinum (Cb-Rho), could form a prion. We identified a candidate prion-forming domain (cPrD) in Cb-Rho and showed that it conferred amyloidogenicity on Cb-Rho and could functionally replace the PrD of a yeast prion-forming protein. Furthermore, its cPrD enabled Cb-Rho to access alternative conformations in Escherichia coli-a soluble form that terminated transcription efficiently and an aggregated, self-propagating prion form that was functionally compromised. The prion form caused genome-wide changes in the transcriptome. Thus, Cb-Rho functions as a protein-based element of inheritance in bacteria, suggesting that the emergence of prions predates the evolutionary split between eukaryotes and bacteria.

Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes.

Influenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.

A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2-Targeted Therapy.

Antibody-drug conjugate (ADC) which delivers cytotoxic drugs specifically into targeted cells through internalization and lysosomal trafficking has emerged as an effective cancer therapy. We show that a bivalent biparatopic antibody targeting two non-overlapping epitopes on HER2 can induce HER2 receptor clustering, which in turn promotes robust internalization, lysosomal trafficking, and degradation. When conjugated with a tubulysin-based microtubule inhibitor, the biparatopic ADC demonstrates superior anti-tumor activity over ado-trastuzumab emtansine (T-DM1) in tumor models representing various patient subpopulations, including T-DM1 eligible, T-DM1 ineligible, and T-DM1 relapsed/refractory. Our findings indicate that this biparatopic ADC has promising potential as an effective therapy for metastatic breast cancer and a broader patient population may benefit from this unique HER2-targeting ADC.

The bacterial cell cycle regulator GcrA is a σ70 cofactor that drives gene expression from a subset of methylated promoters.

Cell cycle progression in most organisms requires tightly regulated programs of gene expression. The transcription factors involved typically stimulate gene expression by binding specific DNA sequences in promoters and recruiting RNA polymerase. Here, we found that the essential cell cycle regulator GcrA in Caulobacter crescentus activates the transcription of target genes in a fundamentally different manner. GcrA forms a stable complex with RNA polymerase and localizes to almost all active σ(70)-dependent promoters in vivo but activates transcription primarily at promoters harboring certain DNA methylation sites. Whereas most transcription factors that contact σ(70) interact with domain 4, GcrA interfaces with domain 2, the region that binds the -10 element during strand separation. Using kinetic analyses and a reconstituted in vitro transcription assay, we demonstrated that GcrA can stabilize RNA polymerase binding and directly stimulate open complex formation to activate transcription. Guided by these studies, we identified a regulon of ∼ 200 genes, providing new insight into the essential functions of GcrA. Collectively, our work reveals a new mechanism for transcriptional regulation, and we discuss the potential benefits of activating transcription by promoting RNA polymerase isomerization rather than recruitment exclusively.