Correlative montage parallel array cryo-tomography for in situ structural cell biology.

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here we present robust tools for montage parallel array cryo-tomography (MPACT) tailored for vitrified specimens. The combination of correlative cryo-fluorescence microscopy, focused-ion-beam milling, substrate micropatterning, and MPACT supports studies that contextually define the three-dimensional architecture of cells. To further extend the flexibility of MPACT, tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

Neural network for autonomous segmentation and volumetric assessment of clot and edema in acute and subacute intracerebral hemorrhages.

INTRODUCTION: Minimally-invasive surgical techniques for intracerebral hemorrhage (ICH) evacuation use imaging to guide the suction, lysing and/or drainage from the hemorrhage site via various designs. A previous international surgical study has shown that reduction of hematoma volume below 15 ml is indicative of improved long term patient outcomes. The study noted a need for tools to periodically visualize remaining clot during intervention to increase the likelihood of evacuating sufficient clot volumes without endangering rebleeds. Robust segmentation of MRI could guide surgeons and radiologists regarding remaining regions and approaches for prudent evacuation. We thus propose a Convolutional Neural Network (CNN) to identify and autonomously segment clot and peripheral edema in MR images of the brain and generate an estimate of the remaining clot volume. MATERIALS AND METHODS: We used a retrospective, locally-acquired dataset of ICH patient scans taken on 3 T MRI scanners. Three sets of ground truth manual segmentations were independently generated by two imaging scientists and one radiology fellow. Evaluation of clot age was determined based on relative contrast of hemorrhage components and reviewed by a neurosurgeon. Model accuracy was determined by pixel-wise Dice coefficient (DC) calculations between each ground truth manual segmentation and the machine-derived autonomous segmentations. RESULTS: The model produced autonomous segmentations of clot core with an average DC of 0.75 ± 0.21 relative to manual segmentations of the same scans. For edema, it produced segmentations with an average DC of 0.68 ± 0.16 relative to manual. From these pixel-wise segmentations, clot volume can be calculated. Model-produced segmentations underestimated clot volumes by an average of 17% relative to ground-truth. CONCLUSION: The machine learning models were able to identify and segment volumes of ICH components swiftly and accurately.

Integrated Fluorescence Microscopy (iFLM) for Cryo-FIB-milling and In-situ Cryo-ET.

Correlative cryo-FLM-FIB milling is a powerful sample preparation technique for in situ cryo-ET. However, correlative workflows that incorporate precise targeting remain challenging. Here, we demonstrate the development and use of an integrated Fluorescence Light Microscope (iFLM) module within a cryo-FIB-SEM to enable a coordinate-based two-point 3D correlative workflow. The iFLM guided targeting of regions of interest coupled with an automated milling process of the cryo-FIB-SEM instrument allows for the efficient preparation of 9-12 ∼200 nm thick lamellae within 24 hours. Using regular and montage-cryo-ET data collection schemes, we acquired data from FIB-milled lamellae of HeLa cells to examine cellular ultrastructure. Overall, this workflow facilitates on-the-fly targeting and automated FIB-milling of cryo-preserved cells, bacteria, and possibly high pressure frozen tissue, to produce lamellae for downstream cryo-ET data collection.

Evaluation of sarcopenia in patients receiving intravesical Bacillus Calmette-Guérin for non-muscle invasive bladder cancer.

BACKGROUND: Sarcopenia is associated with adverse outcomes for patients with muscle invasive bladder cancer (MIBC), but less is known about its impact in the setting of non-muscle invasive bladder cancer (NMIBC). Sarcopenia, skeletal muscle density, and adipose tissue area have been studied as markers of malnutrition and can be determined radiographically. The purpose of this study is to characterize the prevalence of sarcopenia in patients with NMIBC receiving intravesical Bacillus Calmette-Guérin (BCG). METHODS: Following institutional review board approval, patients with NMIBC having received intravesical BCG were identified using institutional pharmacy records. Patients having undergone computed tomography (CT) of the abdomen and pelvis within 90 days of treatment were included in the analysis. Using sliceOmatic 5.0 software, skeletal muscle area (cm2) was measured at the L3 level to calculate skeletal muscle index (SMI), a marker of sarcopenia. Subcutaneous, visceral, and intramuscular adipose tissue areas in addition to skeletal muscle density were also measured. Frailty was evaluated as a secondary aim using the 5-Item Modified Frailty Index (mFI-5). Using predefined cutoffs, the prevalence of sarcopenia was determined. Descriptive statistics were used to characterize frailty and secondary body composition characteristics. Statistical analysis was performed to evaluate the impact of sarcopenia on recurrence rate and progression. RESULTS: A total of 308 patients having received BCG between 2015 and 2020 were identified, of which 90 met criteria for analysis. Nearly all (94%) patients completed at least 5 out of 6 BCG induction instillations. Median body mass index (kg/m2) was 27.64 (IQR 24.9, 30.5) for females and 27.7 (IQR 24.9, 30.66) for males. Median SMI (cm2/m2) was 49.44 (IQR 39.39, 55.17) for females and 49.58 (IQR 40.25, 55.58) for males. A majority (61%) of patients were found to be sarcopenic. High-risk frailty was identified 36% of patients. There was no association between sarcopenia and recurrence rate or progression. CONCLUSIONS: Sarcopenia and frailty are highly prevalent amongst patients with NMIBC. A diagnosis of NMIBC represents a window of opportunity to identify and intervene on modifiable risk factors such as sarcopenia and frailty, which are associated with adverse outcomes in more advanced disease states.

A multisystem, dimensional interplay of assets versus adversities: Revised benevolent childhood experiences (BCEs) in the context of childhood maltreatment, threat, and deprivation.

This study expanded the Benevolent Childhood Experiences scale (termed the "BCEs-Original" scale) with 10 new multisystem items and identified a subset of items (termed the "BCEs-Revised" scale) that are systematically less commonly reported across samples. Total BCEs-Revised scores were tested against total BCEs-Original scores and three dimensions of childhood adversity (maltreatment, threat, and deprivation) as predictors of young adulthood mental health problems (depression, anxiety, and PTSD symptoms). Hypotheses expected stronger inverse associations of BCEs-Revised scores than BCEs-Original scores with all mental health problems. Participants were 1,746 U.S. young adults (M = 26.6 years, SD = 4.7, range = 19-35 years; 55.3% female, 42.4% male, 2.3% gender non-conforming; 67.0% White, 10.3% Asian, 8.6% Black, 8.4% Latine, 5.7% other) who completed a 20-item BCEs scale and well-validated instruments on childhood adversities and mental health problems. Compared to BCEs-Original scores, BCEs-Revised scores were significantly more strongly inversely associated with all mental health outcomes. Compared to childhood threat and deprivation, maltreatment was significantly more strongly associated with PTSD symptoms. After controlling for current depression symptoms, BCEs-Revised scores interacted with maltreatment to predict PTSD symptoms. Maltreatment and BCEs-Revised scores also influenced PTSD symptoms in person-oriented analyses. The BCEs-Revised scale has strong psychometric properties and unique strengths in research and practice. Implications for multisystem resilience are discussed.

PET/MRI in Gynecologic Malignancy.

Patients with gynecologic malignancies often require a multimodality imaging approach for initial staging, treatment response assessment, and surveillance. MRI imaging and PET are two well-established and widely accepted modalities in this setting. Although PET and MRI imaging are often acquired separately on two platforms (a PET/computed tomography [CT] and an MRI imaging scanner), hybrid PET/MRI scanners offer the potential for comprehensive disease assessment in one visit. Gynecologic malignancies have been one of the most successful areas for implementation of PET/MRI. This article provides an overview of the role of this platform in the care of patients with gynecologic malignancies.

Childhood Centeredness is a Broader Predictor of Young Adulthood Mental Health than Childhood Adversity, Attachment, and Other Positive Childhood Experiences.

This study introduced the novel concept of Centeredness, a measure of the emotional atmosphere of the family of origin and a target adult individual's perception of feeling safe, accepted, and supported from childhood primary caregivers and other family members. This study developed a Centeredness scale for adult respondents and tested hypotheses that higher levels of overall Centeredness would predict lower levels of depression and anxiety symptoms; suicidal thoughts and behaviors (STBs); and aggressive behavior; and higher levels of life satisfaction. Predictive effects of Centeredness were compared against attachment-related anxiety and avoidance, and adverse and benevolent childhood experiences (ACEs and BCEs). Participants were recruited via the Prolific-Academic (Pro-A) survey panel into two large independent samples of US young adults aged 19-35 years [Sample 1 (test sample), N = 548, 53.5% female, 2.2% gender non-conforming, 68.3% White, recruited before the pandemic; Sample 2 (replication sample), N = 1,198, 56.2% female, 2.3% gender non-conforming, 66.4% White; recruited during the pandemic]. Participants completed the novel Centeredness scale, which showed strong psychometric properties, and standardized, publicly available assessments of childhood experiences and mental health outcomes. Centeredness was the only variable that significantly predicted each mental health outcome across both samples. BCEs predicted all outcomes except aggressive behavior in the test sample. Centeredness and BCEs were also the only two variables that significantly predicted a dimensional mental health composite in both samples. Neither attachment-related anxiety and avoidance nor ACEs were as broadly predictive. The Centeredness scale assesses emotional aspects of childhood family relationships with individuals of diverse backgrounds and family compositions. Clinical and cultural implications are discussed. Supplementary Information: The online version contains supplementary material available at 10.1007/s42844-023-00089-x.

Federated learning enables big data for rare cancer boundary detection.

Although machine learning (ML) has shown promise across disciplines, out-of-sample generalizability is concerning. This is currently addressed by sharing multi-site data, but such centralization is challenging/infeasible to scale due to various limitations. Federated ML (FL) provides an alternative paradigm for accurate and generalizable ML, by only sharing numerical model updates. Here we present the largest FL study to-date, involving data from 71 sites across 6 continents, to generate an automatic tumor boundary detector for the rare disease of glioblastoma, reporting the largest such dataset in the literature (n = 6, 314). We demonstrate a 33% delineation improvement for the surgically targetable tumor, and 23% for the complete tumor extent, over a publicly trained model. We anticipate our study to: 1) enable more healthcare studies informed by large diverse data, ensuring meaningful results for rare diseases and underrepresented populations, 2) facilitate further analyses for glioblastoma by releasing our consensus model, and 3) demonstrate the FL effectiveness at such scale and task-complexity as a paradigm shift for multi-site collaborations, alleviating the need for data-sharing.

Escherichia coli O127 group 4 capsule proteins assemble at the outer membrane.

Enteropathogenic Escherichia coli O127 is encapsulated by a protective layer of polysaccharide made of the same strain specific O-antigen as the serotype lipopolysaccharide. Seven genes encoding capsule export functions comprise the group 4 capsule (gfc) operon. Genes gfcE, etk and etp encode homologs of the group 1 capsule secretion system but the upstream gfcABCD genes encode unknown functions specific to group 4 capsule export. We have developed an expression system for the large-scale production of the outer membrane protein GfcD. Contrary to annotations, we find that GfcD is a non-acylated integral membrane protein. Circular dichroism spectroscopy, light-scattering data, and the HHomp server suggested that GfcD is a monomeric ß-barrel with 26 ß-strands and an internal globular domain. We identified a set of novel protein-protein interactions between GfcB, GfcC, and GfcD, both in vivo and in vitro, and quantified the binding properties with isothermal calorimetry and biolayer interferometry. GfcC and GfcB form a high-affinity heterodimer with a KD near 100 nM. This heterodimer binds to GfcD (KD = 28 µM) significantly better than either GfcB or GfcC alone. These gfc proteins may form a complex at the outer membrane for group 4 capsule secretion or for a yet unknown function.

A comprehensive characterization of the cell-free transcriptome reveals tissue- and subtype-specific biomarkers for cancer detection.

Cell-free RNA (cfRNA) is a promising analyte for cancer detection. However, a comprehensive assessment of cfRNA in individuals with and without cancer has not been conducted. We perform the first transcriptome-wide characterization of cfRNA in cancer (stage III breast [n = 46], lung [n = 30]) and non-cancer (n = 89) participants from the Circulating Cell-free Genome Atlas (NCT02889978). Of 57,820 annotated genes, 39,564 (68%) are not detected in cfRNA from non-cancer individuals. Within these low-noise regions, we identify tissue- and cancer-specific genes, defined as "dark channel biomarker" (DCB) genes, that are recurrently detected in individuals with cancer. DCB levels in plasma correlate with tumor shedding rate and RNA expression in matched tissue, suggesting that DCBs with high expression in tumor tissue could enhance cancer detection in patients with low levels of circulating tumor DNA. Overall, cfRNA provides a unique opportunity to detect cancer, predict the tumor tissue of origin, and determine the cancer subtype.

CorRelator: Interactive software for real-time high precision cryo-correlative light and electron microscopy.

Cryo-correlative light and electron microscopy (CLEM) is a technique that uses the spatiotemporal cues from fluorescence light microscopy (FLM) to investigate the high-resolution ultrastructure of biological samples by cryo-electron microscopy (cryo-EM). Cryo-CLEM provides advantages for identifying and distinguishing fluorescently labeled proteins, macromolecular complexes, and organelles from the cellular environment. Challenges remain on how correlation workflows and software tools are implemented on different microscope platforms to support automated cryo-EM data acquisition. Here, we present CorRelator: an open-source desktop application that bridges between cryo-FLM and real-time cryo-EM/ET automated data collection. CorRelator implements a pixel-coordinate-to-stage-position transformation for flexible, high accuracy on-the-fly and post-acquisition correlation. CorRelator can be integrated into cryo-CLEM workflows and easily adapted to standard fluorescence and transmission electron microscope (TEM) system configurations. CorRelator was benchmarked under live-cell and cryogenic conditions using several FLM and TEM instruments, demonstrating that CorRelator reliably supports real-time, automated correlative cryo-EM/ET acquisition, through a combination of software-aided and interactive alignment. CorRelator is a cross-platform software package featuring an intuitive Graphical User Interface (GUI) that guides the user through the correlation process. CorRelator source code is available at: https://github.com/wright-cemrc-projects/corr.

Catastrophic Antiphospholipid Syndrome Presenting with Genitourinary Manifestations.

Catastrophic antiphospholipid syndrome (CAPS) is a rare disorder characterized by acute, multi-system dysfunc- tion due to small-vessel thrombosis related to anti-phospholipid antibodies. Here we present an unusual case of CAPS presenting with genitourinary manifestations. A 72-year-old male developed a series of symptoms over the course of two weeks. His symptoms included testicular inflammation, scrotal edema, priapism, hematuria, penile eschar, elbow eschars, and acute kidney injury. He was found to have anti-phospholipid antibodies and treated with anticoagulation, high-dose steroids and plasma exchange. His symptoms resolved with minimal lasting effects. This case is unique to the literature because of the extensive genitourinary involvement.