A comparative study of CARE 2D and N2V 2D for tissue-specific denoising in second harmonic generation imaging.

This study explored the application of deep learning in second harmonic generation (SHG) microscopy, a rapidly growing area. This study focuses on the impact of glycerol concentration on image noise in SHG microscopy and compares two image restoration techniques: Noise-to-Void 2D (N2V 2D, no reference image restoration) and content-aware image restoration (CARE 2D, full reference image restoration). We demonstrated that N2V 2D effectively restored the images affected by high glycerol concentrations. To reduce sample exposure and damage, this study further addresses low-power SHG imaging by reducing the laser power by 70% using deep learning techniques. CARE 2D excels in preserving detailed structures, whereas N2V 2D maintains natural muscle structure. This study highlights the strengths and limitations of these models in specific SHG microscopy applications, offering valuable insights and potential advancements in the field .

Excess natural-cause mortality in US counties and its association with reported COVID-19 deaths.

In the United States, estimates of excess deaths attributable to the COVID-19 pandemic have consistently surpassed reported COVID-19 death counts. Excess deaths reported to non-COVID-19 natural causes may represent unrecognized COVID-19 deaths, deaths caused by pandemic health care interruptions, and/or deaths from the pandemic's socioeconomic impacts. The geographic and temporal distribution of these deaths may help to evaluate which explanation is most plausible. We developed a Bayesian hierarchical model to produce monthly estimates of excess natural-cause mortality for US counties over the first 30 mo of the pandemic. From March 2020 through August 2022, 1,194,610 excess natural-cause deaths occurred nationally [90% PI (Posterior Interval): 1,046,000 to 1,340,204]. A total of 162,886 of these excess natural-cause deaths (90% PI: 14,276 to 308,480) were not reported to COVID-19. Overall, 15.8 excess deaths were reported to non-COVID-19 natural causes for every 100 reported COVID-19 deaths. This number was greater in nonmetropolitan counties (36.0 deaths), the West (Rocky Mountain states: 31.6 deaths; Pacific states: 25.5 deaths), and the South (East South Central states: 26.0 deaths; South Atlantic states: 25.0 deaths; West South Central states: 24.2 deaths). In contrast, reported COVID-19 death counts surpassed estimates of excess natural-cause deaths in metropolitan counties in the New England and Middle Atlantic states. Increases in reported COVID-19 deaths correlated temporally with increases in excess deaths reported to non-COVID-19 natural causes in the same and/or prior month. This suggests that many excess deaths reported to non-COVID-19 natural causes during the first 30 mo of the pandemic in the United States were unrecognized COVID-19 deaths.

Tunable PhenoCycler imaging of the murine pre-clinical tumour microenvironments.

BACKGROUND: The tumour microenvironment (TME) consists of tumour-supportive immune cells, endothelial cells, and fibroblasts. PhenoCycler, a high-plex single cell spatial biology imaging platform, is used to characterize the complexity of the TME. Researchers worldwide harvest and bank tissues from mouse models which are employed to model a plethora of human disease. With the explosion of interest in spatial biology, these panoplies of archival tissues provide a valuable resource to answer new questions. Here, we describe our protocols for developing tunable PhenoCycler multiplexed imaging panels and describe our open-source data analysis pipeline. Using these protocols, we used PhenoCycler to spatially resolve the TME of 8 routinely employed pre-clinical models of lymphoma, breast cancer, and melanoma preserved as FFPE. RESULTS: Our data reveal distinct TMEs in the different cancer models that were imaged and show that cell-cell contacts differ depending on the tumour type examined. For instance, we found that the immune infiltration in a murine model of melanoma is altered in cellular organization in melanomas that become resistant to αPD-1 therapy, with depletions in a number of cell-cell interactions. CONCLUSIONS: This work presents a valuable resource study seamlessly adaptable to any field of research involving murine models. The methodology described allows researchers to address newly formed hypotheses using archival materials, bypassing the new to perform new mouse studies.

COVID-19 and All-Cause Mortality by Race, Ethnicity, and Age Across Five Periods of the Pandemic in the United States.

Racial/ethnic and age disparities in COVID-19 and all-cause mortality during 2020 are well documented, but less is known about their evolution over time. We examine changes in age-specific mortality across five pandemic periods in the United States from March 2020 to December 2022 among four racial/ethnic groups (non-Hispanic White, non-Hispanic Black, Hispanic, and non-Hispanic Asian) for ages 35+. We fit Gompertz models to all-cause and COVID-19 death rates by 5-year age groups and construct age-specific racial/ethnic mortality ratios across an Initial peak (Mar-Aug 2020), Winter peak (Nov 2020-Feb 2021), Delta peak (Aug-Oct 2021), Omicron peak (Nov 2021-Feb 2022), and Endemic period (Mar-Dec 2022). We then compare to all-cause patterns observed in 2019. The steep age gradients in COVID-19 mortality in the Initial and Winter peak shifted during the Delta peak, with substantial increases in mortality at working ages, before gradually returning to an older age pattern in the subsequent periods. We find a disproportionate COVID-19 mortality burden on racial and ethnic minority populations early in the pandemic, which led to an increase in all-cause mortality disparities and a temporary elimination of the Hispanic mortality advantage at certain age groups. Mortality disparities narrowed over time, with racial/ethnic all-cause inequalities during the Endemic period generally returning to pre-pandemic levels. Black and Hispanic populations, however, faced a younger age gradient in all-cause mortality in the Endemic period relative to 2019, with younger Hispanic and Black adults in a slightly disadvantageous position and older Black adults in a slightly advantageous position, relative to before the pandemic.

Monthly excess mortality across counties in the United States during the COVID-19 pandemic, March 2020 to February 2022.

Excess mortality is the difference between expected and observed mortality in a given period and has emerged as a leading measure of the COVID-19 pandemic's mortality impact. Spatially and temporally granular estimates of excess mortality are needed to understand which areas have been most impacted by the pandemic, evaluate exacerbating factors, and inform response efforts. We estimated all-cause excess mortality for the United States from March 2020 through February 2022 by county and month using a Bayesian hierarchical model trained on data from 2015 to 2019. An estimated 1,179,024 excess deaths occurred during the first 2 years of the pandemic (first: 634,830; second: 544,194). Overall, excess mortality decreased in large metropolitan counties but increased in nonmetropolitan counties. Despite the initial concentration of mortality in large metropolitan Northeastern counties, nonmetropolitan Southern counties had the highest cumulative relative excess mortality by July 2021. These results highlight the need for investments in rural health as the pandemic's rural impact grows.

COVID-19 Mortality by Race and Ethnicity in US Metropolitan and Nonmetropolitan Areas, March 2020 to February 2022.

Importance: Prior research has established that Hispanic and non-Hispanic Black residents in the US experienced substantially higher COVID-19 mortality rates in 2020 than non-Hispanic White residents owing to structural racism. In 2021, these disparities decreased. Objective: To assess to what extent national decreases in racial and ethnic disparities in COVID-19 mortality between the initial pandemic wave and subsequent Omicron wave reflect reductions in mortality vs other factors, such as the pandemic's changing geography. Design, Setting, and Participants: This cross-sectional study was conducted using data from the US Centers for Disease Control and Prevention for COVID-19 deaths from March 1, 2020, through February 28, 2022, among adults aged 25 years and older residing in the US. Deaths were examined by race and ethnicity across metropolitan and nonmetropolitan areas, and the national decrease in racial and ethnic disparities between initial and Omicron waves was decomposed. Data were analyzed from June 2021 through March 2023. Exposures: Metropolitan vs nonmetropolitan areas and race and ethnicity. Main Outcomes and Measures: Age-standardized death rates. Results: There were death certificates for 977 018 US adults aged 25 years and older (mean [SD] age, 73.6 [14.6] years; 435 943 female [44.6%]; 156 948 Hispanic [16.1%], 140 513 non-Hispanic Black [14.4%], and 629 578 non-Hispanic White [64.4%]) that included a mention of COVID-19. The proportion of COVID-19 deaths among adults residing in nonmetropolitan areas increased from 5944 of 110 526 deaths (5.4%) during the initial wave to a peak of 40 360 of 172 515 deaths (23.4%) during the Delta wave; the proportion was 45 183 of 210 554 deaths (21.5%) during the Omicron wave. The national disparity in age-standardized COVID-19 death rates per 100 000 person-years for non-Hispanic Black compared with non-Hispanic White adults decreased from 339 to 45 deaths from the initial to Omicron wave, or by 293 deaths. After standardizing for age and racial and ethnic differences by metropolitan vs nonmetropolitan residence, increases in death rates among non-Hispanic White adults explained 120 deaths/100 000 person-years of the decrease (40.7%); 58 deaths/100 000 person-years in the decrease (19.6%) were explained by shifts in mortality to nonmetropolitan areas, where a disproportionate share of non-Hispanic White adults reside. The remaining 116 deaths/100 000 person-years in the decrease (39.6%) were explained by decreases in death rates in non-Hispanic Black adults. Conclusions and Relevance: This study found that most of the national decrease in racial and ethnic disparities in COVID-19 mortality between the initial and Omicron waves was explained by increased mortality among non-Hispanic White adults and changes in the geographic spread of the pandemic. These findings suggest that despite media reports of a decline in disparities, there is a continued need to prioritize racial health equity in the pandemic response.

Nonlinear microscopy and deep learning classification for mammary gland microenvironment studies.

Tumors, their microenvironment, and the mechanisms by which collagen morphology changes throughout cancer progression have recently been a topic of interest. Second harmonic generation (SHG) and polarization second harmonic (P-SHG) microscopy are label-free, hallmark methods that can highlight this alteration in the extracellular matrix (ECM). This article uses automated sample scanning SHG and P-SHG microscopy to investigate ECM deposition associated with tumors residing in the mammary gland. We show two different analysis approaches using the acquired images to distinguish collagen fibrillar orientation changes in the ECM. Lastly, we apply a supervised deep-learning model to classify naïve and tumor-bearing mammary gland SHG images. We benchmark the trained model using transfer learning with the well-known MobileNetV2 architecture. By fine-tuning the different parameters of these models, we show a trained deep-learning model that suits such a small dataset with 73% accuracy.

Associations between mortality from COVID-19 and other causes: A state-level analysis.

BACKGROUND: During the COVID-19 pandemic, the high death toll from COVID-19 was accompanied by a rise in mortality from other causes of death. The objective of this study was to identify the relationship between mortality from COVID-19 and changes in mortality from specific causes of death by exploiting spatial variation in these relationships across US states. METHODS: We use cause-specific mortality data from CDC Wonder and population estimates from the US Census Bureau to examine relationships at the state level between mortality from COVID-19 and changes in mortality from other causes of death. We calculate age-standardized death rates (ASDR) for three age groups, nine underlying causes of death, and all 50 states and the District of Columbia between the first full year of the pandemic (March 2020-February 2021) and the year prior (March 2019-February 2020). We then estimate the relationship between changes in cause-specific ASDR and COVID-19 ASDR using linear regression analysis weighted by the size of the state's population. RESULTS: We estimate that causes of death other than COVID-19 represent 19.6% of the total mortality burden associated with COVID-19 during the first year of the COVID-19 pandemic. At ages 25+, circulatory disease accounted for 51.3% of this burden while dementia (16.4%), other respiratory diseases (12.4%), influenza/pneumonia (8.7%) and diabetes (8.6%) also contribute. In contrast, there was an inverse association across states between COVID-19 death rates and changes in death rates from cancer. We found no state-level association between COVID-19 mortality and rising mortality from external causes. CONCLUSIONS: States with unusually high death rates from COVID-19 experienced an even larger mortality burden than implied by those rates alone. Circulatory disease served as the most important route through which COVID-19 mortality affected death rates from other causes of death. Dementia and other respiratory diseases made the second and third largest contributions. In contrast, mortality from neoplasms tended to decline in states with the highest death rates from COVID-19. Such information may help to inform state-level responses aimed at easing the full mortality burden of the COVID-19 pandemic.

The use of opioids in the management of chronic pain: Synopsis of the 2022 Updated U.S. Department of Veterans Affairs and U.S. Department of Defense Clinical Practice Guideline

In May 2022, leadership within the U.S. Department of Veterans Affairs (VA) and U.S. Department of Defense (DoD) approved a joint clinical practice guideline for the use of opioids when managing chronic pain. This synopsis summarizes the recommendations that the authors believe are the most important to highlight. In December 2020, the VA/DoD Evidence-Based Practice Work Group assembled a team to update the 2017 VA/DoD Clinical Practice Guideline for Opioid Therapy for Chronic Pain. The guideline development team included clinical stakeholders and conformed to the National Academy of Medicine's tenets for trustworthy clinical practice guidelines. The guideline team developed key questions to guide a systematic evidence review that was done by an independent third party and distilled 20 recommendations for care using the GRADE (Grading of Recommendations Assessment, Development and Evaluation) system. The guideline team also created 3 one-page algorithms to help guide clinical decision making. This synopsis presents the recommendations and highlights selected recommendations on the basis of clinical relevance. This guideline is intended for clinicians who may be considering opioid therapy to manage patients with chronic pain. This synopsis reviews updated recommendations for the initiation and continuation of opioid therapy; dose, duration, and taper of opioids; screening, assessment, and evaluation; and risk mitigation. New additions are highlighted, including recommendations about the use of buprenorphine instead of full agonist opioids; assessing for behavioral health conditions and factors associated with higher risk for harm, such as pain catastrophizing; and the use of pain and opioid education to reduce the risk for prolonged opioid use for postsurgical pain.

Differences Between Reported COVID-19 Deaths and Estimated Excess Deaths in Counties Across the United States, March 2020 to February 2022.

Accurate and timely tracking of COVID-19 deaths is essential to a well-functioning public health surveillance system. The extent to which official COVID-19 death tallies have captured the true toll of the pandemic in the United States is unknown. In the current study, we develop a Bayesian hierarchical model to estimate monthly excess mortality in each county over the first two years of the pandemic and compare these estimates to the number of deaths officially attributed to Covid-19 on death certificates. Overall, we estimated that 268,176 excess deaths were not reported as Covid-19 deaths during the first two years of the Covid-19 pandemic, which represented 23.7% of all excess deaths that occurred. Differences between excess deaths and reported COVID-19 deaths were substantial in both the first and second year of the pandemic. Excess deaths were less likely to be reported as COVID-19 deaths in the Mountain division, in the South, and in nonmetro counties. The number of excess deaths exceeded COVID-19 deaths in all Census divisions except for the New England and Middle Atlantic divisions where there were more COVID-19 deaths than excess deaths in large metro areas and medium or small metro areas. Increases in excess deaths not assigned to COVID-19 followed similar patterns over time to increases in reported COVID-19 deaths and typically preceded or occurred concurrently with increases in reported COVID-19 deaths. Estimates from this study can be used to inform targeting of resources to areas in which the true toll of the COVID-19 pandemic has been underestimated.

Proteomic Assessment of the Murine Mammary Gland Extracellular Matrix.

The extracellular matrix (ECM) is a molecular scaffold mainly comprising fibrous proteins, glycoproteins, proteoglycans, and polysaccharides. Aside from acting as a structural support, the ECM's composition dictates cell-matrix interactions at the biochemical and biophysical level. In the context of cancer, the ECM is a critical component of the tumor microenvironment (TME) and dysregulation of its deposition and remodelling has been shown to promote tumor onset, progression, and metastasis. Here, we describe a robust protocol for the isolation and subsequent proteomic analysis of the ECM of murine mammary glands, for downstream assays studying the role of the ECM in breast cancer. The protocol yields sufficient protein amounts to enable not only the global quantitation of protein expression but furthermore the enrichment and quantitative analysis of post-translationally modified peptides to study aberrant signalling.

Phosphorylation of eIF4E in the stroma drives the production and spatial organisation of collagen type I in the mammary gland.

The extracellular matrix (ECM) plays critical roles in breast cancer development. Whether ECM composition is regulated by the phosphorylation of eIF4E on serine 209, an event required for tumorigenesis, has not been explored. Herein, we used proteomics and mouse modeling to investigate the impact of mutating serine 209 to alanine on eIF4E (i.e., S209A) on mammary gland (MG) ECM. The proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD028953. We discovered that S209A knock-in mice, expressing a non-phosphorylatable form of eIF4E, have less collagen-I deposition in native and tumor-bearing MGs, leading to altered tumor cell invasion. Additionally, phospho-eIF4E deficiency impacts collagen topology; fibers at the tumor-stroma boundary in phospho-eIF4E-deficient mice run parallel to the tumor edge but radiate outwards in wild-type mice. Finally, a phospho-eIF4E-deficient tumor microenvironment resists anti-PD-1 therapy-induced collagen deposition, correlating with an increased anti-tumor response to immunotherapy. Clinically, we showed that collagen-I and phospho-eIF4E are positively correlated in human breast cancer samples, and that stromal phospho-eIF4E expression is influenced by tumor proximity. Together, our work defines the importance of phosphorylation of eIF4E on S209 as a regulator of MG collagen architecture in the tumor microenvironment, thereby positioning phospho-eIF4E as a therapeutic target to augment response to therapy.

Monthly excess mortality across counties in the United States during the Covid-19 pandemic, March 2020 to February 2022.

Excess mortality is the difference between expected and observed mortality in a given period and has emerged as a leading measure of the overall impact of the Covid-19 pandemic that is not biased by differences in testing or cause-of-death assignment. Spatially and temporally granular estimates of excess mortality are needed to understand which areas have been most impacted by the pandemic, evaluate exacerbating and mitigating factors, and inform response efforts, including allocating resources to affected communities. We estimated all-cause excess mortality for the United States from March 2020 through February 2022 by county and month using a Bayesian hierarchical model trained on data from 2015 to 2019. An estimated 1,159,580 excess deaths occurred during the first two years of the pandemic (first: 620,872; second: 538,708). Overall, excess mortality decreased in large metropolitan counties, but increased in nonmetro counties, between the first and second years of the pandemic. Despite the initial concentration of mortality in large metropolitan Northeast counties, beginning in February 2021, nonmetro South counties had the highest cumulative relative excess mortality. These results highlight the need for investments in rural health as the pandemic's disproportionate impact on rural areas continues to grow.

A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens.

Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a 'green' xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.

Self-care and Wellness Checks in Emergency Field Hospitals During COVID-19 Pandemic: A New Self-check Tool for Military Personnel and Civilians.

The authors explore the impact of cumulative stress on United States (US) military service members (SM), including soldiers and medical personnel, deployed to serve in New York City (NYC) communities. Their mission was to assist in establishing emergency field hospitals during the COVID-19 pandemic. Causative biopsychosocial factors are presented, as well as the impact of wellness checks, which were utilized to monitor the mood and morale of frontline healthcare providers, military personnel, and infected patients in a 2,500-bed emergency field hospital and a 1,000-bed Naval hospital ship operating in the metropolitan NYC area. The authors introduce a self-care and wellness tool, which assesses five core domains (physical, mental, emotional, social, and spiritual) for the purpose of assessing and improving individual overall well-being during periods of heightened stress. This instrument could aid attending medical personnel in identifying patients at risk of suicide. Likewise, the utility of this self-care tool is applicable to both military SM and civilians, and includes soldiers and medical personnel.

PRMT7 ablation stimulates anti-tumor immunity and sensitizes melanoma to immune checkpoint blockade.

Despite the success of immune checkpoint inhibitor (ICI) therapy for cancer, resistance and relapse are frequent. Combination therapies are expected to enhance response rates and overcome this resistance. Herein, we report that combining PRMT7 inhibition with ICI therapy induces a strong anti-tumor T cell immunity and restrains tumor growth in vivo by increasing immune cell infiltration. PRMT7-deficient B16.F10 melanoma exhibits increased expression of genes in the interferon pathway, antigen presentation, and chemokine signaling. PRMT7 deficiency or inhibition with SGC3027 in B16.F10 melanoma results in reduced DNMT expression, loss of DNA methylation in the regulatory regions of endogenous retroviral elements (ERVs) causing their increased expression. PRMT7-deficient cells increase RIG-I and MDA5 expression with a reduction in the H4R3me2s repressive histone mark at their gene promoters. Our findings identify PRMT7 as a regulatory checkpoint for RIG-I, MDA5, and their ERV-double-stranded RNA (dsRNA) ligands, facilitating immune escape and anti-tumor T cell immunity to restrain tumor growth.

Acquired Resistance to EZH2 Inhibitor GSK343 Promotes the Differentiation of Human DLBCL Cell Lines toward an ABC-Like Phenotype.

Diffuse large B-cell lymphoma (DLBCL) accounts for 40% of non-Hodgkin lymphoma, and 30% to 40% of patients will succumb to relapsed/refractory disease (rrDLBCL). Patients with rrDLBCL generally have low long-term survival rates due to a lack of efficient salvage therapies. Small-molecule inhibitors targeting the histone methyltransferase EZH2 represent an emerging group of novel therapeutics that show promising clinical efficacy in patients with rrDLBCL. The mechanisms that control acquired resistance to this class of targeted therapies, however, remain poorly understood. Here, we develop a model of resistance to the EZH2 inhibitor (EZH2i) GSK343 and use RNA-seq data and in vitro investigation to show that GCB (germinal center B-cell)-DLBCL cell lines with acquired drug resistance differentiate toward an ABC (activated B-cell)-DLBCL phenotype. We further observe that the development of resistance to GSK343 is sufficient to induce cross-resistance to other EZH2i. Notably, we identify the immune receptor SLAMF7 as upregulated in EZH2i-resistant cells, using chromatin immunoprecipitation profiling to uncover the changes in chromatin landscape remodeling that permit this altered gene expression. Collectively, our data reveal a previously unreported response to the development of EZH2i resistance in DLBCL, while providing strong rationale for pursuing investigation of dual-targeting of EZH2 and SLAMF7 in rrDLBCL.

County-level estimates of excess mortality associated with COVID-19 in the United States.

The COVID-19 pandemic in the U.S. has been largely monitored using death certificates containing reference to COVID-19. However, prior analyses reveal that a significant percentage of excess deaths associated with the pandemic were not directly assigned to COVID-19. In this study, we estimated a generalized linear model of expected mortality based on historical trends in deaths by county of residence between 2011 and 2019. We used the results of the model to generate estimates of excess mortality and excess deaths not assigned to COVID-19 in 2020 for 1470 county sets in the U.S. representing 3138 counties. Across the country, we estimated that 438,386 excess deaths occurred in 2020, among which 87.5% were assigned to COVID-19. Some regions (Mideast, Great Lakes, New England, and Far West) reported the most excess deaths in large central metros, whereas other regions (Southwest, Southeast, Plains, and Rocky Mountains) reported the highest excess mortality in nonmetro areas. The proportion assigned to COVID-19 was lowest in large central metro areas (79.3%). Regionally, the proportion of excess deaths assigned to COVID-19 was lowest in the Southeast (81.6%), Southwest (82.6%), Far West (83.7%), and Rocky Mountains (86.7%). Across the regions, the number of excess deaths exceeded the number of directly assigned COVID-19 deaths in most counties. The exception to this pattern occurred in New England, which reported more directly assigned COVID-19 deaths than excess deaths in metro and nonmetro areas. Many county sets had substantial numbers of excess deaths that were not accounted for in direct COVID-19 death counts. Estimates of excess mortality at the local level can inform the allocation of resources to areas most impacted by the pandemic and contribute to positive behavior feedback loops, such as increases in mask-wearing and vaccine uptake.