Cross-Platform Synaptic Network Analysis of Human Entorhinal Cortex Identifies TWF2 as a Modulator of Dendritic Spine Length.

Proteomic studies using postmortem human brain tissue samples have yielded robust assessments of the aging and neurodegenerative disease(s) proteomes. While these analyses provide lists of molecular alterations in human conditions, like Alzheimer's disease (AD), identifying individual proteins that affect biological processes remains a challenge. To complicate matters, protein targets may be highly understudied and have limited information on their function. To address these hurdles, we sought to establish a blueprint to aid selection and functional validation of targets from proteomic datasets. A cross-platform pipeline was engineered to focus on synaptic processes in the entorhinal cortex (EC) of human patients, including controls, preclinical AD, and AD cases. Label-free quantification mass spectrometry (MS) data (n = 2260 proteins) was generated on synaptosome fractionated tissue from Brodmann area 28 (BA28; n = 58 samples). In parallel, dendritic spine density and morphology was measured in the same individuals. Weighted gene co-expression network analysis was used to construct a network of protein co-expression modules that were correlated with dendritic spine metrics. Module-trait correlations were used to guide unbiased selection of Twinfilin-2 (TWF2), which was the top hub protein of a module that positively correlated with thin spine length. Using CRISPR-dCas9 activation strategies, we demonstrated that boosting endogenous TWF2 protein levels in primary hippocampal neurons increased thin spine length, thus providing experimental validation for the human network analysis. Collectively, this study describes alterations in dendritic spine density and morphology as well as synaptic proteins and phosphorylated tau from the entorhinal cortex of preclinical and advanced stage AD patients.SIGNIFICANCE STATEMENT Proteomic studies can yield vast lists of molecules that are altered under various experimental or disease conditions. Here, we provide a blueprint to facilitate mechanistic validation of protein targets from human brain proteomic datasets. We conducted a proteomic analysis of human entorhinal cortex (EC) samples spanning cognitively normal and Alzheimer's disease (AD) cases with a comparison of dendritic spine morphology in the same samples. Network integration of proteomics with dendritic spine measurements allowed for unbiased discovery of Twinfilin-2 (TWF2) as a regulator of dendritic spine length. A proof-of-concept experiment in cultured neurons demonstrated that altering Twinfilin-2 protein level induced corresponding changes in dendritic spine length, thus providing experimental validation for the computational framework.

Characteristics of rehabilitation duration in patients with intracerebral hemorrhage.

Background and Purpose: No consensus exists on the ideal duration of rehabilitation in patients with intracerebral hemorrhage (ICH). The aim of this study is to identify demographic and clinical characteristics associated with rehabilitation duration (RD) in patients with ICH during hospitalization. Methods: This retrospective study followed consecutive patients admitted to a tertiary care center between 2016 and 2019 with primary diagnosis of ICH. The primary outcome, RD was calculated by adding the total number of therapy (speech, occupational, and physical) minutes received during admission, divided by the hospital length of stay. Variables abstracted included demographic and clinical characteristics (ICH score, neurosurgical procedure). Multiple linear regression models were used to measure adjusted association between select predictors and mean RD. Results: A total of 316 ICH patients (62.5 ± 15.9 years, 49 % White) were included in the final analysis. Compared to Whites, other racial minorities (ß= -9.017, p = 0.002) received rehabilitation therapy for a significantly shorter duration in the adjusted model. Age was significantly associated with having higher RD (ß=0.320, p < 0.001). Patients with lobar ICH (ß= -7.486, p = 0.014) had significantly shorter RD compared to deep. ICH score was significantly associated with having a lower RD (ß= -8.624, p < 0.001). Conclusion: Age, race, ICH score, and location were significantly associated with RD. Non-White patients had significantly shorter RD, indicating a potential racial disparity in the rehabilitation of patients with ICH.

Palliative Care Clinicians' Views on Metrics for Successful Specialist Palliative Care Delivery in the ICU.

CONTEXT: A quarter of palliative care (PC) clinicians' consultations are now requested from the intensive care unit (ICU). Despite this high usage, a standardized set of quality metrics for PC delivery in the ICU does not exist. OBJECTIVES: To explore PC clinicians' views on how to best measure quality of care delivery in their role as a consultant in the ICU setting. METHODS: Secondary analysis of a parent dataset consisting of qualitative data from semi-structured interviews exploring ways to optimize PC clinicians' role in the ICU. Nineteen participants were recruited across five academic medical centers in the US. Participants included PC physicians (n = 14), nurse practitioners (n = 2), and social workers (n = 3). Thematic analysis with an inductive approach was used to generate themes. RESULTS: We identified two central themes: difficulties in measuring PC quality in the ICU (theme 1) and tension between the role of PC and metrics (theme 2). Theme 1 had two subthemes related to logistical challenges in measuring outcomes and PC clinicians' preference for metrics that incorporate subjective feedback from patients, family members, and the primary ICU team. Theme 2 described how PC clinicians often felt a disconnect between the goal of meeting a metric and their goals in delivering high-quality clinical care. CONCLUSION: Our findings provide insight into PC clinician perspectives on quality metrics and identify major barriers that need to be addressed to successfully implement quality measurement in the ICU setting.

Disparities in Race and Ethnicity Reporting and Representation for Clinical Trials in Stroke: 2010 to 2020.

BACKGROUND: Racial and ethnic minority groups are at a higher stroke risk and have poor poststroke outcomes. The aim of this study was to assess the frequency of race reporting and proportions of race and ethnicity representation in stroke-related clinical trials. METHODS AND RESULTS: This is a descriptive study of stroke-related clinical trials completed between January 1, 2010 and December 31, 2020, and registered on ClinicalTrials.gov. Trials conducted in the United States, related to stroke and enrolling participants ≥18 years, were considered eligible. Trials were reviewed for availability of published results, data on race and ethnicity distribution, and trial characteristics. Overall, 60.1% of published trials reported race or ethnicity of participants, with a 2.6-fold increase in reporting between 2010 and 2020. White patients represented 65.0% of the participants, followed by 24.8% Black, 2.4% Asian or Pacific Islander, and <1% Native American and multiracial participants; 9.0% were of Hispanic ethnicity. These trends remained consistent throughout the study period, except in 2018, when a higher proportion of Black participants (53.1%) was enrolled compared with White participants (35.8%). Trials with the National Institutes of Health/federal funding had higher enrollment of Black (28.1%) and Hispanic (13.8%) participants compared with other funding sources. Behavioral intervention trials had the most diverse enrollment with equal enrollment of Black and White participants (41.1%) and 14.5% Hispanic participants. CONCLUSIONS: Despite the increase in race and ethnicity reporting between 2010 and 2020, the representation of racial and ethnic minority groups remains low in stroke trials. Funding initiatives may influence diversity efforts in trial enrollment.

Hierarchical assembly and modeling of DNA nanotube networks using Y-shaped DNA origami seeds.

DNA nanotechnology offers many means to synthesize custom nanostructured materials from the ground up in a hierarchical fashion. While the assembly of DNA nanostructures from small (nanometer-scale) monomeric components has been studied extensively, how the hierarchical assembly of rigid or semi-flexible units produces multi-micron scale structures is less understood. Here we demonstrate a mechanism for assembling micron-scale semi-flexible DNA nanotubes into extended structures. These nanotubes assemble from nanometer-scale tile monomers into materials via heterogeneous nucleation from rigid, Y-shaped DNA origami seeds to form Y-seeded nanotube architectures. These structures then assemble into networks via nanotube end-to-end joining. We measure the kinetics of network growth and find that the assembly of networks can be approximated by a model of hierarchical assembly that assumes a single joining rate between DNA nanotube ends. Because the number of nucleation sites on Y-seeds and their spatial arrangement can be systematically varied by design, this hierarchical assembly process could be used to form a wide variety of networks and to understand the assembly mechanisms that lead to different types of material architectures at length scales of tens to hundreds of microns.

Design and pilot test of an implicit bias mitigation curriculum for clinicians.

Introduction: Clinician implicit racial bias (IB) may lead to lower quality care and adverse health outcomes for Black patients. Educational efforts to train clinicians to mitigate IB vary widely and have insufficient evidence of impact. We developed and pilot-tested an evidence-based clinician IB curriculum, "REACHing Equity." Methods: To assess acceptability and feasibility, we conducted an uncontrolled one-arm pilot trial with post-intervention assessments. REACHing Equity is designed for clinicians to: (1) acquire knowledge about IB and its impact on healthcare, (2) increase awareness of one's own capacity for IB, and (3) develop skills to mitigate IB in the clinical encounter. We delivered REACHing Equity virtually in three facilitated, interactive sessions over 7-9 weeks. Participants were health care providers who completed baseline and end-of-study evaluation surveys. Results: Of approximately 1,592 clinicians invited, 37 participated, of whom 29 self-identified as women and 24 as non-Hispanic White. Attendance averaged 90% per session; 78% attended all 3 sessions. Response rate for evaluation surveys was 67%. Most respondents agreed or strongly agreed that the curriculum objectives were met, and that REACHing Equity equipped them to mitigate the impact of implicit bias in clinical care. Participants consistently reported higher self-efficacy for mitigating IB after compared to before completing the curriculum. Conclusions: Despite apparent barriers to clinician participation, we demonstrated feasibility and acceptability of the REACHing Equity intervention. Further research is needed to develop objective measures of uptake and clinician skill, test the impact of REACHing Equity on clinically relevant outcomes, and refine the curriculum for uptake and dissemination.ClinicalTrials.gov ID: NCT03415308.

Analysis of cellularity in H&E-stained rat bone marrow tissue via deep learning.

Our objective was to develop an automated deep-learning-based method to evaluate cellularity in rat bone marrow hematoxylin and eosin whole slide images for preclinical safety assessment. We trained a shallow CNN for segmenting marrow, 2 Mask R-CNN models for segmenting megakaryocytes (MKCs), and small hematopoietic cells (SHCs), and a SegNet model for segmenting red blood cells. We incorporated the models into a pipeline that identifies and counts MKCs and SHCs in rat bone marrow. We compared cell segmentation and counts that our method generated to those that pathologists generated on 10 slides with a range of cell depletion levels from 10 studies. For SHCs, we compared cell counts that our method generated to counts generated by Cellpose and Stardist. The median Dice and object Dice scores for MKCs using our method vs pathologist consensus and the inter- and intra-pathologist variation were comparable, with overlapping first-third quartile ranges. For SHCs, the median scores were close, with first-third quartile ranges partially overlapping intra-pathologist variation. For SHCs, in comparison to Cellpose and Stardist, counts from our method were closer to pathologist counts, with a smaller 95% limits of agreement range. The performance of the bone marrow analysis pipeline supports its incorporation into routine use as an aid for hematotoxicity assessment by pathologists. The pipeline could help expedite hematotoxicity assessment in preclinical studies and consequently could expedite drug development. The method may enable meta-analysis of rat bone marrow characteristics from future and historical whole slide images and may generate new biological insights from cross-study comparisons.

Comparison of Golgi-Cox and Intracellular Loading of Lucifer Yellow for Dendritic Spine Density and Morphology Analysis in the Mouse Brain.

Dendritic spines are small protrusions on dendrites that serve as the postsynaptic site of the majority of excitatory synapses. These structures are important for normal synaptic transmission, and alterations in their density and morphology have been documented in various disease states. Over 130 years ago, Ramón y Cajal used Golgi-stained tissue sections to study dendritic morphology. Despite the array of technological advances, including iontophoretic microinjection of Lucifer yellow (LY) fluorescent dye, Golgi staining continues to be one of the most popular approaches to visualize dendritic spines. Here, we compared dendritic spine density and morphology among pyramidal neurons in layers 2/3 of the mouse medial prefrontal cortex (mPFC) and pyramidal neurons in hippocampal CA1 using three-dimensional digital reconstructions of (1) brightfield microscopy z-stacks of Golgi-impregnated dendrites and (2) confocal microscopy z-stacks of LY-filled dendrites. Analysis of spine density revealed that the LY microinjection approach enabled detection of approximately three times as many spines as the Golgi staining approach in both brain regions. Spine volume measurements were larger using Golgi staining compared to LY microinjection in both mPFC and CA1. Spine length was mostly comparable between techniques in both regions. In the mPFC, head diameter was similar for Golgi staining and LY microinjection. However, in CA1, head diameter was approximately 50% smaller on LY-filled dendrites compared to Golgi staining. These results indicate that Golgi staining and LY microinjection yield different spine density and morphology measurements, with Golgi staining failing to detect dendritic spines and overestimating spine size.

Evaluating a couple communication skills training (CCST) intervention for advanced cancer: study protocol for a randomized controlled trial.

BACKGROUND: For patients and their intimate partners, advanced cancer poses significant challenges that can negatively impact both individuals and their relationship. Prior studies have found evidence that couple-based communication skills interventions can to be beneficial for patients and partners. However, these studies have been limited by reliance on in-person treatment delivery and have not targeted couples at high risk for poor outcomes. This study tests the efficacy of a Couples Communication Skills Training (CCST) intervention delivered via videoconference for couples reporting high levels of holding back from discussing cancer-related concerns, a variable associated with poorer psychological and relationship functioning. METHODS: This RCT is designed to evaluate the efficacy of CCST in improving patient and partner relationship functioning (primary outcome). Secondary outcomes include patient and partner psychological functioning and patient symptoms and health care use. We also examine the role of objective and self-reported communication behaviors as mediators of treatment effects. Two hundred thirty patients with advanced lung, gastrointestinal, genitourinary, and breast cancer and their partners will be randomized to CCST or an education control intervention. Participants in both conditions complete self-reported outcome measures at baseline, mid-treatment, post-treatment, and 3 months post-treatment. Objective measures of communication are derived from video-recorded couple conversations collected at baseline and post-treatment. An implementation-related process evaluation (assessing implementation outcomes and potential barriers to/facilitators of implementation) will be conducted to inform future efforts to implement CCST in real-world settings. DISCUSSION: This trial can yield important new knowledge about effective ways to improve patient and partner adjustment to advanced cancer. TRIAL REGISTRATION: This study trial is registered at clinicaltrials.gov (Trial # NCT04590885); registration date: October 19, 2020.

Tumid Lupus Erythematosus: A Rare and Distinctive Variant of Cutaneous Lupus Erythematosus Masquerading as Urticarial Vasculitis.

Tumid lupus erythematosus (TLE) is a rare form of chronic cutaneous lupus that has triggered much debate regarding its clinical and histopathological features. It has been classically defined as annular erythematous, succulent, plaques involving the face and trunk that typically are devoid of any papulosquamous features such as scale and follicular plugging. These lesions are a clinical mimicker of other urticarial lesions such as urticarial vasculitis and lymphocytic infiltrate of Jessner. We report a case of TLE presenting in a 49-year-old Caucasian female whose initial clinical presentation was concerning for urticarial vasculitis due to presence of urticarial-like lesions present for approximately three months. Laboratory studies and histopathological correlations confirmed the diagnosis of TLE and the patient was successfully treated with topical corticosteroids.

Immune checkpoints and cancer in the immunogenomics era.

Immune checkpoints have been the subject of a wave of new studies. Among these checkpoints are tytotoxic T-lymphocyte-associated antigen 4, checkpoints programmed death-1 and programmed death-ligand 1; their blockades have been approved by the Food and Drug Administration for therapy of melanoma and other types of cancers. Immunogenomics, which combines the latest nucleic acid sequencing strategy with immunotherapy, provides precise information about genomic alterations (e.g. mutations) and enables a paradigm shift of immune checkpoint therapy from tumor types to molecular signatures. Studying these critical checkpoints in relation to genomic mutations and neoantigens has produced groundbreaking results. This article examines these studies and delves into the relationships between immune checkpoint blockade and tumors harboring certain genomic mutations. Moreover, this article reviews recent studies on resistance to immune checkpoint therapy.

Development of three quantitative real-time PCR assays for the detection of Rickettsia raoultii, Rickettsia slovaca, and Rickettsia aeschlimannii and their validation with ticks from the country of Georgia and the Republic of Azerbaijan.

A previous surveillance study of human pathogens within ticks collected in the country of Georgia showed a relatively high infection rate for Rickettsia raoultii, R. slovaca, and R. aeschlimannii. These 3 spotted fever group rickettsiae are human pathogens: R. raoultii and R. slovaca cause tick-borne lymphadenopathy (TIBOLA), and R. aeschlimannii causes an infection characterized by fever and maculopapular rash. Three quantitative real-time polymerase chain reaction (qPCR) assays, Rraoul, Rslov, and Raesch were developed and optimized to detect R. raoultii, R. slovaca, and R. aeschlimannii, respectively, by targeting fragments of the outer membrane protein B gene (ompB) using species-specific molecular beacon or TaqMan probes. The 3 qPCR assays showed 100% specificity when tested against a rickettsiae DNA panel (n=20) and a bacteria DNA panel (n=12). The limit of detection was found to be at least 3 copies per reaction for all assays. Validation of the assays using previously investigated tick nucleic acid preparations, which included Rickettsia-free tick samples, tick samples that contain R. raoultii, R. slovaca, R. aeschlimannii, and other Rickettsia spp., gave 100% sensitivity for all 3 qPCR assays. In addition, a total of 65 tick nucleic acid preparations (representing 259 individual ticks) collected from the country of Georgia and the Republic of Azerbaijan in 2009 was tested using the 3 qPCR assays. R. raoultii, R. slovaca, and R. aeschlimannii were not detected in any ticks (n=31) from the Republic of Azerbaijan, but in the ticks from the country of Georgia (n=228) the minimal infection rate for R. raoultii and R. slovaca in Dermacentor marginatus was 10% and 4%, respectively, and for R. aeschlimannii in Haemaphysalis sulcata and Hyalomma spp. it was 1.9% and 20%, respectively.