Matrix stiffness increases energy efficiency of endothelial cells.

To form blood vessels, endothelial cells rearrange their cytoskeleton, generate traction stresses, migrate, and proliferate, all of which require energy. Despite these energetic costs, stiffening of the extracellular matrix promotes tumor angiogenesis and increases cell contractility. However, the interplay between extracellular matrix, cell contractility, and cellular energetics remains mechanistically unclear. Here, we utilized polyacrylamide substrates with various stiffnesses, a real-time biosensor of ATP, and traction force microscopy to show that endothelial cells exhibit increasing traction forces and energy usage trend as substrate stiffness increases. Inhibition of cytoskeleton reorganization via ROCK inhibition resulted in decreased cellular energy efficiency, and an opposite trend was found when cells were treated with manganese to promote integrin affinity. Altogether, our data reveal a link between matrix stiffness, cell contractility, and cell energetics, suggesting that endothelial cells on stiffer substrates can better convert intracellular energy into cellular traction forces. Given the critical role of cellular metabolism in cell function, our study also suggests that not only energy production but also the efficiency of its use plays a vital role in regulating cell behaviors and may help explain how increased matrix stiffness promotes angiogenesis.

Immunological isolation and characterization of neuronal progenitors from human dental pulp: A laboratory-based investigation.

AIMS: Dental pulp stem cells (DPSCs) contain a population of stem cells with a broad range of differentiation potentials, as well as more lineage-committed progenitors. Such heterogeneity is a significant obstacle to experimental and clinical applications. The aim of this study is to isolate and characterize a homogenous neuronal progenitor cell population from human DPSCs. METHODOLOGY: Polysialylated-neural cell adhesion molecule (PSA-NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic-activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non-neural markers was used to characterize the magnetically isolated PSA-NCAM+ fraction. PSA-NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic-AMP, neurotrophin-3, B27 and N2 supplements to induce neuronal differentiation. Both PSA-NCAM+ and differentiated PSA-NCAM+ cells were used in Ca2+ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization. RESULTS: PSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic-activated cell sorting and anti-PSA-NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA-NCAM+ cells. Immunofluorescent staining revealed expression of pan-neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP-induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast-voltage-responding fluorescent molecule, FluoVolt™ in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations. CONCLUSIONS: In summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural-like properties that can be effectively isolated with magnetic-activated cell sorting (MACS).

Synergy between Laminin-Derived Elastin-like Polypeptides (LELPs) Optimizes Cell Spreading.

A major component of the extracellular matrix (ECM), laminins, modulates cells via diverse receptors. Their fragments have emerging utility as components of "ECM-mimetics" optimized to promote cell-based therapies. Recently, we reported that a bioactive laminin peptide known as A99 enhanced cell binding and spreading via fusion to an elastin-like polypeptide (ELP). The ELP "handle" serves as a rapid, noncovalent strategy to concentrate bioactive peptide mixtures onto a surface. We now report that this strategy can be further generalized across an expanded panel of additional laminin-derived elastin-like polypeptides (LELPs). A99 (AGTFALRGDNPQG), A2G80 (VQLRNGFPYFSY), AG73 (RKRLQVQLSIRT), and EF1m (LQLQEGRLHFMFD) all promote cell spreading while showing morphologically distinct F-actin formation. Equimolar mixtures of A99:A2G80-LELPs have synergistic effects on adhesion and spreading. Finally, three of these ECM-mimetics promote the neurite outgrowth of PC-12 cells. The evidence presented here demonstrates the potential of ELPs to deposit ECM-mimetics with applications in regenerative medicine, cell therapy, and tissue engineering.

Diagnostic delay, comorbid hidradenitis suppurativa and the prognostic value of bacterial culture in folliculitis decalvans: A cohort study.

BACKGROUND: Folliculitis decalvans (FD) is a type of primary neutrophilic cicatricial alopecia often leading to irreversible hair loss. Data on its epidemiology, clinical features, outcomes, and prognostic factors are limited. OBJECTIVE: To evaluate a cohort of patients with FD and identify characteristics of severe disease and prognostic factors which impede remission. PATIENTS AND METHODS: This retrospective cohort study included 192 patients diagnosed with FD and followed for at least six months at a tertiary center between 2010 and 2020. RESULTS: There was a diagnostic delay averaging 22.2 (± 29.7) months. Comorbid follicular occlusion disorders were common. Bacterial cultures were positive in 45.6% of the cases, with Staphylococcus (S.) aureus being the most common pathogen. Severe disease was associated with comorbid hidradenitis suppurativa and a positive bacterial culture, particularly S. aureus. 50.7% of patients experienced complete remission: 32% within the first six months of treatment and 18.7% later during follow-up. Relapses were frequent. Negative prognostic factors for achieving remission included younger age and a positive bacterial culture. CONCLUSIONS: There is a need for the education of dermatologists to reduce the diagnostic delay. Screening FD patients for comorbid hidradenitis suppurativa and obtaining bacterial cultures is important for treatment planning.

ADEPT: Autoencoder with differentially expressed genes and imputation for robust spatial transcriptomics clustering.

Advancements in spatial transcriptomics (ST) have enabled an in-depth understanding of complex tissues by quantifying gene expression at spatially localized spots. Several notable clustering methods have been introduced to utilize both spatial and transcriptional information in the analysis of ST datasets. However, data quality across different ST sequencing techniques and types of datasets influence the performance of different methods and benchmarks. To harness spatial context and transcriptional profile in ST data, we developed a graph-based, multi-stage framework for robust clustering, called ADEPT. To control and stabilize data quality, ADEPT relies on a graph autoencoder backbone and performs an iterative clustering on imputed, differentially expressed genes-based matrices to minimize the variance of clustering results. ADEPT outperformed other popular methods on ST data generated by different platforms across analyses such as spatial domain identification, visualization, spatial trajectory inference, and data denoising.

Matrix stiffness regulates tumor cell intravasation through expression and ESRP1-mediated alternative splicing of MENA.

During intravasation, cancer cells cross the endothelial barrier and enter the circulation. Extracellular matrix stiffening has been correlated with tumor metastatic potential; however, little is known about the effects of matrix stiffness on intravasation. Here, we utilize in vitro systems, a mouse model, specimens from patients with breast cancer, and RNA expression profiles from The Cancer Genome Atlas Program (TCGA) to investigate the molecular mechanism by which matrix stiffening promotes tumor cell intravasation. Our data show that heightened matrix stiffness increases MENA expression, which promotes contractility and intravasation through focal adhesion kinase activity. Further, matrix stiffening decreases epithelial splicing regulatory protein 1 (ESRP1) expression, which triggers alternative splicing of MENA, decreases the expression of MENA11a, and enhances contractility and intravasation. Altogether, our data indicate that matrix stiffness regulates tumor cell intravasation through enhanced expression and ESRP1-mediated alternative splicing of MENA, providing a mechanism by which matrix stiffness regulates tumor cell intravasation.

The miRNA Landscape of Lacrimal Glands in a Murine Model of Autoimmune Dacryoadenitis.

Purpose: To analyze the changes in the lacrimal gland (LG) miRNAome from male nonobese diabetic (NOD) mice with autoimmune dacryoadenitis compared with LG from healthy male BALB/c and dacryoadenitis-free female NOD mice. Methods: LG from these mice were collected for small RNA sequencing to identify dysregulated miRNAs; hits were validated by RT-qPCR in male NOD and BALB/c LG. Dysregulation of validated species within immune cell-enriched cell fractions and epithelial-enriched cell fractions from LG was probed by RT-qPCR. Ingenuity pathway analysis identified putative miRNA targets, which were examined in publicly available mRNA-seq datasets. Western blotting and confocal imaging of immunofluorescence enabled validation of some molecular changes at the protein level. Results: Male NOD LG exhibited 15 and 13 significantly up- and downregulated miRNAs, respectively. Dysregulated expression of 14 of these miRNAs (9 upregulated, 5 downregulated) was validated in male NOD versus BALB/c LG by RT-qPCR. Seven of the upregulated miRNAs were increased owing to their abundance in immune cell-enriched cell fractions, whereas four downregulated miRNAs were largely expressed in epithelial-enriched cell fractions. Ingenuity pathway analysis predicted the upregulation of IL-6 and IL-6-like pathways as an outcome of miRNA dysregulation. Increased expression of several genes in these pathways was confirmed by mRNA-seq analysis, whereas immunoblotting and immunofluorescence confirmed Ingenuity pathway analysis-predicted changes for IL-6Rα and gp130/IL-6st. Conclusions: Male NOD mouse LG exhibit multiple dysregulated miRNAs owing to the presence of infiltrating immune cells, and decreased acinar cell content. The observed dysregulation may increase IL-6Rα and gp130/IL-6st on acini and IL-6Rα on specific lymphocytes, enhancing IL-6 and IL-6-like cytokine signaling.

Tear miRNAs Identified in a Murine Model of Sjögren's Syndrome as Potential Diagnostic Biomarkers and Indicators of Disease Mechanism.

Objective: The tear miRNAome of the male NOD mouse, a model of ocular symptoms of Sjögren's syndrome (SS), was analyzed to identify unique miRNAs. Methods: Male NOD mice, aged 12-14 weeks, were used to identify tear miRNAs associated with development of autoimmune dacryoadenitis. Age- and sex-matched male BALB/c mice served as healthy controls while age-matched female NOD mice that do not develop the autoimmune dacryoadenitis characteristic of SS were used as additional controls. Total RNA was isolated from stimulated tears pooled from 5 mice per sample and tear miRNAs were sequenced and analyzed. Putative miRNA hits were validated in additional mouse cohorts as well as in tears of SS patients versus patients with another form of dry eye disease, meibomian gland disease (MGD) using qRT-PCR. The pathways influenced by the validated hits were identified using Ingenuity Pathway Analysis. Results: In comparison to tears from both healthy (male BALB/c) and additional control (female NOD) mice, initial analy1sis identified 7 upregulated and 7 downregulated miRNAs in male NOD mouse tears. Of these, 8 were validated by RT-qPCR in tears from additional mouse cohorts. miRNAs previously implicated in SS pathology included mmu-miR-146a/b-5p, which were significantly downregulated, as well as mmu-miR-150-5p and mmu-miR-181a-5p, which were upregulated in male NOD mouse tears. All other validated hits including the upregulated miR-181b-5p and mmu-miR-203-3p, as well as the downregulated mmu-miR-322-5p and mmu-miR-503-5p, represent novel putative indicators of autoimmune dacryoadenitis in SS. When compared to tears from patients with MGD, miRNAs hsa-miR-203a-3p, hsa-miR-181a-5p and hsa-miR-181b-5p were also significantly increased in tears of SS patients. Conclusions: A panel of differentially expressed miRNAs were identified in tears of male NOD mice, with some preliminary validation in SS patients, including some never previously linked to SS. These may have potential utility as indicators of ocular symptoms of SS; evaluation of the pathways influenced by these dysregulated miRNAs may also provide further insights into SS pathogenesis.

Quality and Safety Considerations in Stereotactic Radiosurgery and Stereotactic Body Radiation Therapy: An ASTRO Safety White Paper Update.

PURPOSE: This updated report on stereotactic radiosurgery (SRS) and stereotactic body radiation therapy (SBRT) is part of a series of consensus-based white papers previously published addressing patient safety. Since the first white papers were published, SRS and SBRT technology and procedures have progressed significantly such that these procedures are now more commonly used. The complexity and submillimeter accuracy, and delivery of a higher dose per fraction requires an emphasis on best practices for technical, dosimetric, and quality assurance. Therefore, quality and patient safety considerations for these techniques remain an important area of focus. METHOD: The American Society for Radiation Oncology convened a task force to assess the original SRS/SBRT white paper and update content where appropriate. Recommendations were created using a consensus-building methodology and task force members indicated their level of agreement based on a 5-point Likert scale, from "strongly agree" to "strongly disagree." A prespecified threshold of ≥75% of raters who select "strongly agree" or "agree" indicated consensus. SUMMARY: This white paper builds on the previous version and uses of other guidance documents to broadly address SRS and SBRT delivery, primarily focusing on processes related to quality and safety. SRS and SBRT require a team-based approach, staffed by appropriately trained and credentialed specialists as well as significant personnel resources, specialized technology, and implementation time. A thorough feasibility analysis of resources is required to achieve the clinical and technical goals and thoroughly discussed with all personnel before undertaking new disease sites. A comprehensive quality assurance program must be developed, using established treatment guidelines, to ensure SRS and SBRT are performed in a safe and effective manner. Patient safety in SRS/SBRT is everyone's responsibility and professional organizations, regulators, vendors, and end-users must demonstrate a clear commitment to working together to ensure the highest levels of safety.

Improved Sobriety Rates After Brain-Computer Interface-Based Cognitive Remediation Training.

Up to 80% of individuals seeking treatment fail in their attempts at sobriety. This study investigated whether 1) a cognitive remediation therapy (CRT) program augmented with a brain-computer interface (BCI) to influence brain performance metrics would increase participants' self-agency by restoring cognitive control performance; and 2) that ability increase would produce increased sobriety rates, greater than published treatment rates. The study employed a retrospective chart review structured to replicate a switching replication methodology (i.e., waitlist group) using a pre-test and post-test profile analysis quasi-experimental design. Participants' records were organized into treatment and non-treatment groups. Adult poly-substance users were recruited from alcohol and other drugs (AOD) use outpatient programs and AOD use treatment centers in the United States. Participants volunteered for pre- and post-testing without treatment (n = 121) or chose to enter the treatment program (n = 200). The treatment group engaged in a 48-session BCI/CRT augmented treatment program. Pre- and post-treatment measures comprised 14 areas from the Woodcock-Johnson Cognitive Abilities III Assessment Battery. An 18-month follow-up assessment measured maintenance of sobriety. After testing the difference for all variables across time between test groups, a significant multivariate effect was found. In addition, at 18 months post-treatment, 89% of the treatment group maintained sobriety, compared to 31% of the non-treatment group. Consistent with addiction neurobehavioral imbalance models, traditional treatment programs augmented with BCI/CRT training, focused on improving cognitive control abilities, may strengthen self-control and improve sobriety rates.

Treatment of Acute Graft-versus-Host Disease in Liver Transplant Recipients.

Acute graft-versus-host disease (aGvHD) is a rare complication of liver transplantation associated with high morbidity and mortality. Death typically occurs due to complications related to severe infection, shock, and multiorgan failure. The clinical presentation involves dysfunction of multiple organ systems with overlapping symptoms that often results in a diagnostic delay. As there are a limited number of cases reported in the literature, there are no clear guidelines for treatment. Many different therapeutic measures have been utilized that target various immune system pathways, but steroids remain the first line of therapy. We report on two patients who developed aGvHD after liver transplantation who were treated with ruxolitinib, a novel Janus kinase 1/2 (JAK) inhibitor that has been shown to improve outcomes in steroid refractory cases of aGvHD after allogenic hematopoietic stem cell transplantation. We reviewed the literature to discuss various therapeutic options currently available for aGvHD after liver transplantation.

Assessment of a Diagnostic Classification System for Management of Lesions to Exclude Melanoma.

Importance: The proposed MOLEM (Management of Lesion to Exclude Melanoma) schema is more clinically relevant than Melanocytic Pathology Assessment Tool and Hierarchy for Diagnosis (MATH-Dx) for the management classification of melanocytic and nonmelanocytic lesions excised to exclude melanoma. A more standardized way of establishing diagnostic criteria will be crucial in the training of artificial intelligence (AI) algorithms. Objective: To examine pathologists' variability, reliability, and confidence in reporting melanocytic and nonmelanocytic lesions excised to exclude melanoma using the MOLEM schema in a population of higher-risk patients. Design, Setting, and Participants: This cohort study enrolled higher-risk patients referred to a primary care skin clinic in New South Wales, Australia, between April 2019 and December 2019. Baseline demographic characteristics including age, sex, and related clinical details (eg, history of melanoma) were collected. Patients with lesions suspicious for melanoma assessed by a primary care physician underwent clinical evaluation, dermoscopy imaging, and subsequent excision biopsy of the suspected lesion(s). A total of 217 lesions removed and prepared by conventional histologic method and stained with hematoxylin-eosin were reviewed by up to 9 independent pathologists for diagnosis using the MOLEM reporting schema. Pathologists evaluating for MOLEM schema were masked to the original histopathologic diagnosis. Main Outcomes and Measures: Characteristics of the lesions were described and the concordance of cases per MOLEM class was assessed. Interrater agreement and the agreement between pathologists' ratings and the majority MOLEM diagnosis were calculated by Gwet AC1 with quadratic weighting applied. The diagnostic confidence of pathologists was then assessed. Results: A total of 197 patients were included in the study (102 [51.8%] male; 95 [48.2%] female); mean (SD) age was 64.2 (15.8) years (range, 24-93 years). Overall, 217 index lesions were assessed with a total of 1516 histological diagnoses. Of 1516 diagnoses, 677 (44.7%) were classified as MOLEM class I; 120 (7.9%) as MOLEM class II; 564 (37.2%) as MOLEM class III; 114 (7.5%) as MOLEM class IV; and 55 (3.6%) as MOLEM class V. Concordance rates per MOLEM class were 88.6% (class I), 50.8% (class II), 76.2% (class III), 77.2% (class IV), and 74.2% (class V). The quadratic weighted interrater agreement was 91.3%, with a Gwet AC1 coefficient of 0.76 (95% CI, 0.72-0.81). The quadratic weighted agreement between pathologists' ratings and majority MOLEM was 94.7%, with a Gwet AC1 coefficient of 0.86 (95% CI, 0.84-0.88). The confidence in diagnosis data showed a relatively high level of confidence (between 1.0 and 1.5) when diagnosing classes I (mean [SD], 1.3 [0.3]), IV (1.3 [0.3]) and V (1.1 [0.1]); while classes II (1.8 [0.2]) and III (1.5 [0.4]) were diagnosed with a lower level of pathologist confidence (≥1.5). The quadratic weighted interrater confidence rating agreement was 95.2%, with a Gwet AC1 coefficient of 0.92 (95% CI, 0.90-0.94) for the 1314 confidence ratings collected. The confidence agreement for each MOLEM class was 95.0% (class I), 93.5% (class II), 95.3% (class III), 96.5% (class IV), and 97.5% (class V). Conclusions and Relevance: The proposed MOLEM schema better reflects clinical practice than the MPATH-Dx schema in lesions excised to exclude melanoma by combining diagnoses with similar prognostic outcomes for melanocytic and nonmelanocytic lesions into standardized classification categories. Pathologists' level of confidence appeared to follow the MOLEM schema diagnostic concordance trend, ie, atypical naevi and melanoma in situ diagnoses were the least agreed upon and the most challenging for pathologists to confidently diagnose.

New Extensibility and Scripting Tools in the ImageJ Ecosystem.

ImageJ provides a framework for image processing across scientific domains while being fully open source. Over the years ImageJ has been substantially extended to support novel applications in scientific imaging as they emerge, particularly in the area of biological microscopy, with functionality made more accessible via the Fiji distribution of ImageJ. Within this software ecosystem, work has been done to extend the accessibility of ImageJ to utilize scripting, macros, and plugins in a variety of programming scenarios, e.g., from Groovy and Python and in Jupyter notebooks and cloud computing. We provide five protocols that demonstrate the extensibility of ImageJ for various workflows in image processing. We focus first on Fluorescence Lifetime Imaging Microscopy (FLIM) data, since this requires significant processing to provide quantitative insights into the microenvironments of cells. Second, we show how ImageJ can now be utilized for common image processing techniques, specifically image deconvolution and inversion, while highlighting the new, built-in features of ImageJ-particularly its capacity to run completely headless and the Ops matching feature that selects the optimal algorithm for a given function and data input, thereby enabling processing speedup. Collectively, these protocols can be used as a basis for automating biological image processing workflows. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Using PyImageJ for FLIM data processing Alternate Protocol: Groovy FLIMJ in Jupyter Notebooks Basic Protocol 2: Using ImageJ Ops for image deconvolution Support Protocol 1: Using ImageJ Ops matching feature for image inversion Support Protocol 2: Headless ImageJ deconvolution.

Histologic Patterns and Clues to Autoinflammatory Diseases in Children: What a Cutaneous Biopsy Can Tell Us.

Autoinflammation is defined by aberrant, antigen-independent activation of the innate immune signaling pathways. This leads to increased, pro-inflammatory cytokine expression and subsequent inflammation. In contrast, autoimmune and allergic diseases are antigen-directed immune responses from activation of the adaptive immune system. The innate and adaptive immune signaling pathways are closely interconnected. The group of 'complex multigenic diseases' are a result of mutual dysregulation of both the autoinflammatory and autoimmune physiologic components. In contrast, monogenic autoinflammatory syndromes (MAIS) result from single mutations and are exclusively autoinflammatory in their pathogenesis. Studying the clinical and histopathological findings for the various MAIS explains the phenotypical correlates of their specific mutations. This review aims to group the histopathologic clues for autoinflammation into three recognizable patterns. The presence of these histologic patterns in a pediatric patient with recurrent fevers and systemic inflammation should raise suspicion of an autoinflammatory component in MAIS, or, more frequently, in a complex multigenic disease. The three major histopathological patterns seen in autoinflammation are as follows: (i) the 'neutrophilic' pattern, seen in urticarial neutrophilic dermatosis, pustular psoriasis, aseptic neutrophilic folliculitis, and Sweet's syndrome; (ii) the 'vasculitic' pattern seen in small vessel-vasculitis (including hypersensitivity/leukocytoclastic vasculitis, thrombosing microangiopathy and lymphocytic vasculitis), and intermediate-sized vessel vasculitis, mimicking polyarteritis nodosa; and (iii) the 'granulomatous' pattern. Beyond these three patterns, there are additional histopathologic clues, which are detailed below. It is important for a dermatopathologist to recognize the patterns of autoinflammation, so that a diagnosis of MAIS or complex multigenic diseases may be obtained. Finally, careful histopathologic analyses could contribute to a better understanding of the various clinical manifestations of autoinflammation.