A Fast-Tracking Sample Preparation Protocol for Proteomics of Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Archived tumor specimens are routinely preserved by formalin fixation and paraffin embedding. Despite the conventional wisdom that proteomics might be ineffective due to the cross-linking and pre-analytical variables, these samples have utility for both discovery and targeted proteomics. Building on this capability, proteomics approaches can be used to maximize our understanding of cancer biology and clinical relevance by studying preserved tumor tissues annotated with the patients' medical histories. Proteomics of formalin-fixed paraffin-embedded (FFPE) tissues also integrates with histological evaluation and molecular pathology strategies, so that additional collection of research biopsies or resected tumor aliquots is not needed. The acquisition of data from the same tumor sample also overcomes concerns about biological variation between samples due to intratumoral heterogeneity. However, the protein extraction and proteomics sample preparation from FFPE samples can be onerous, particularly for small (i.e., limited or precious) samples. Therefore, we provide a protocol for a recently introduced kit-based EasyPep method with benchmarking against a modified version of the well-established filter-aided sample preparation strategy using laser-capture microdissected lung adenocarcinoma tissues from a genetically engineered mouse model. This model system allows control over the tumor preparation and pre-analytical variables while also supporting the development of methods for spatial proteomics to examine intratumoral heterogeneity. Data are posted in ProteomeXchange (PXD045879).

Informed development of a multi-species biofilm in chronic obstructive pulmonary disease.

Recent evidence indicates that microbial biofilm aggregates inhabit the lungs of COPD patients and actively contribute towards chronic colonization and repeat infections. However, there are no contextually relevant complex biofilm models for COPD research. In this study, a meta-analysis of the lung microbiome in COPD was used to inform development of an optimized biofilm model composed of genera highly associated with COPD. Bioinformatic analysis showed that although diversity matrices of COPD microbiomes were similar to healthy controls, and internal compositions made it possible to accurately differentiate between these cohorts (AUC = 0.939). Genera that best defined these patients included Haemophilus, Moraxella and Streptococcus. Many studies fail to account for fungi; therefore, Candida albicans was included in the creation of an interkingdom biofilm model. These organisms formed a biofilm capable of tolerating high concentrations of antimicrobial therapies with no significant reductions in viability. However, combined therapies of antibiotics and an antifungal resulted in significant reductions in viable cells throughout the biofilm (p < 0.05). This biofilm model is representative of the COPD lung microbiome and results from in vitro antimicrobial challenge experiments indicate that targeting both bacteria and fungi in these interkingdom communities will be required for more positive clinical outcomes.

Grading of lung adenocarcinomas with simultaneous segmentation by artificial intelligence (GLASS-AI).

Preclinical genetically engineered mouse models (GEMMs) of lung adenocarcinoma are invaluable for investigating molecular drivers of tumor formation, progression, and therapeutic resistance. However, histological analysis of these GEMMs requires significant time and training to ensure accuracy and consistency. To achieve a more objective and standardized analysis, we used machine learning to create GLASS-AI, a histological image analysis tool that the broader cancer research community can utilize to grade, segment, and analyze tumors in preclinical models of lung adenocarcinoma. GLASS-AI demonstrates strong agreement with expert human raters while uncovering a significant degree of unreported intratumor heterogeneity. Integrating immunohistochemical staining with high-resolution grade analysis by GLASS-AI identified dysregulation of Mapk/Erk signaling in high-grade lung adenocarcinomas and locally advanced tumor regions. Our work demonstrates the benefit of employing GLASS-AI in preclinical lung adenocarcinoma models and the power of integrating machine learning and molecular biology techniques for studying the molecular pathways that underlie cancer progression.

SINE RNA of the imprinted miRNA clusters mediates constitutive type III interferon expression and antiviral protection in hemochorial placentas.

Hemochorial placentas have evolved defense mechanisms to prevent the vertical transmission of viruses to the immunologically underdeveloped fetus. Unlike somatic cells that require pathogen-associated molecular patterns to stimulate interferon production, placental trophoblasts constitutively produce type III interferons (IFNL) through an unknown mechanism. We demonstrate that transcripts of short interspersed nuclear elements (SINEs) embedded in miRNA clusters within the placenta trigger a viral mimicry response that induces IFNL and confers antiviral protection. Alu SINEs within primate-specific chromosome 19 (C19MC) and B1 SINEs within rodent-specific microRNA cluster on chromosome 2 (C2MC) produce dsRNAs that activate RIG-I-like receptors (RLRs) and downstream IFNL production. Homozygous C2MC knockout mouse trophoblast stem (mTS) cells and placentas lose intrinsic IFN expression and antiviral protection, whereas B1 RNA overexpression restores C2MCΔ/Δ mTS cell viral resistance. Our results uncover a convergently evolved mechanism whereby SINE RNAs drive antiviral resistance in hemochorial placentas, placing SINEs as integral players in innate immunity.

Managing Scapular Dyskinesis.

Scapular dyskinesis, the impairment of optimal scapular position and motion, is common in association with shoulder injury. A comprehensive evaluation process can show the causative factors and lead to effective treatment protocols. The complexity of scapular motion and the integrated relationship between the scapula, humerus, trunk, and legs suggest a need to develop rehabilitation programs that involve all segments working as a unit rather than isolated components. This is best accomplished with an integrated rehabilitation approach that includes rectifying deficits in mobility, strength, and motor control but not overtly focusing on any one area.

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis.

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

Abbreviated Outpatient Upper Extremity Fracture Care to Avoid Clinic and Hospital Environmental Encounters During the COVID-19 Pandemic: A New Approach to Fracture Care?

BACKGROUND: To minimize in-person visits during the COVID-19 pandemic, a new fracture care protocol for children with complete and stable, nondisplaced or minimally displaced upper extremity (UE) fractures has been implemented. This protocol involves immobilization with a bivalved cast, which allows for home cast removal during a telemedicine visit, and no follow-up radiographs, thus eliminating the requirement for a return to clinic. The purpose of this study is to evaluate the outcomes and parent satisfaction of this new abbreviated fracture care protocol. METHODS: Between May 2020 and April 2021, during the COVID-19 pandemic, children with complete and stable, nondisplaced or minimally displaced UE fractures were treated with a bivalved cast and 1 follow-up telemedicine visit for home cast removal. A prospective longitudinal study of these patients was performed. The PROMIS Upper Extremity questionnaire was administered at enrollment and 3 months follow-up. Parents completed a satisfaction survey after home cast removal. Demographic data and information regarding complications were collected. A historical cohort of controls treated with standard cast in 2019 was used for comparison. RESULTS: A total of 56 patients with a mean age of 8±3 years (range 2 to 15) were prospectively enrolled in this study. Parent-reported PROMIS Upper Extremity scores showed a significant increase from 24.9 (95% confidence interval=20.8-29.1) at enrollment to 51.6 (95% confidence interval=50.8-52.5) at 3 months follow-up (P<0.001). Results of the satisfaction survey (n=39) showed all parents were either very satisfied (85%) or satisfied (15%). In addition, 10% of parents would have initially preferred to come into clinic for cast removal and 90% of parents would prefer this new treatment plan in the future. Patients in the abbreviated care cohort returned to clinic for a median 1 in-person visits, compared with 2 for historical controls (n=183, P<0.001). Abbreviated care patients received fewer (1.0) radiographs than controls (2.0, P<0.001). Complication rate did not differ between the groups (P=0.77). CONCLUSIONS: Complete and stable, nonminimally or minimally displaced UE fractures can be cared for safely and effectively in a single in-person visit, with a telemedicine cast removal visit. Parents are satisfied with this abbreviated protocol and prefer it to additional in-person visits. LEVEL OF EVIDENCE: Level III.

Deep Learning of Histopathology Images at the Single Cell Level.

The tumor immune microenvironment (TIME) encompasses many heterogeneous cell types that engage in extensive crosstalk among the cancer, immune, and stromal components. The spatial organization of these different cell types in TIME could be used as biomarkers for predicting drug responses, prognosis and metastasis. Recently, deep learning approaches have been widely used for digital histopathology images for cancer diagnoses and prognoses. Furthermore, some recent approaches have attempted to integrate spatial and molecular omics data to better characterize the TIME. In this review we focus on machine learning-based digital histopathology image analysis methods for characterizing tumor ecosystem. In this review, we will consider three different scales of histopathological analyses that machine learning can operate within: whole slide image (WSI)-level, region of interest (ROI)-level, and cell-level. We will systematically review the various machine learning methods in these three scales with a focus on cell-level analysis. We will provide a perspective of workflow on generating cell-level training data sets using immunohistochemistry markers to "weakly-label" the cell types. We will describe some common steps in the workflow of preparing the data, as well as some limitations of this approach. Finally, we will discuss future opportunities of integrating molecular omics data with digital histopathology images for characterizing tumor ecosystem.

The Serpin Superfamily and Their Role in the Regulation and Dysfunction of Serine Protease Activity in COPD and Other Chronic Lung Diseases.

Chronic obstructive pulmonary disease (COPD) is a debilitating heterogeneous disease characterised by unregulated proteolytic destruction of lung tissue mediated via a protease-antiprotease imbalance. In COPD, the relationship between the neutrophil serine protease, neutrophil elastase, and its endogenous inhibitor, alpha-1-antitrypsin (AAT) is the best characterised. AAT belongs to a superfamily of serine protease inhibitors known as serpins. Advances in screening technologies have, however, resulted in many members of the serpin superfamily being identified as having differential expression across a multitude of chronic lung diseases compared to healthy individuals. Serpins exhibit a unique suicide-substrate mechanism of inhibition during which they undergo a dramatic conformational change to a more stable form. A limitation is that this also renders them susceptible to disease-causing mutations. Identification of the extent of their physiological/pathological role in the airways would allow further expansion of knowledge regarding the complexity of protease regulation in the lung and may provide wider opportunity for their use as therapeutics to aid the management of COPD and other chronic airways diseases.

Trypsin-Like Proteases and Their Role in Muco-Obstructive Lung Diseases.

Trypsin-like proteases (TLPs) belong to a family of serine enzymes with primary substrate specificities for the basic residues, lysine and arginine, in the P1 position. Whilst initially perceived as soluble enzymes that are extracellularly secreted, a number of novel TLPs that are anchored in the cell membrane have since been discovered. Muco-obstructive lung diseases (MucOLDs) are characterised by the accumulation of hyper-concentrated mucus in the small airways, leading to persistent inflammation, infection and dysregulated protease activity. Although neutrophilic serine proteases, particularly neutrophil elastase, have been implicated in the propagation of inflammation and local tissue destruction, it is likely that the serine TLPs also contribute to various disease-relevant processes given the roles that a number of these enzymes play in the activation of both the epithelial sodium channel (ENaC) and protease-activated receptor 2 (PAR2). More recently, significant attention has focused on the activation of viruses such as SARS-CoV-2 by host TLPs. The purpose of this review was to highlight key TLPs linked to the activation of ENaC and PAR2 and their association with airway dehydration and inflammatory signalling pathways, respectively. The role of TLPs in viral infectivity will also be discussed in the context of the inhibition of TLP activities and the potential of these proteases as therapeutic targets.

Losartan protects endothelium-dependent relaxation in vivo in a murine model of rheumatoid arthritis.

Angiotensin II-type 1 receptor stimulation is recognised to promote inflammation, a state central to the development and maintenance of rheumatoid arthritis. Herein we examined the use of losartan, an angiotensin II-type 1 receptor antagonist, on vascular reactivity, knee joint diameter and behavioural assessment of pain in a Freund's complete adjuvant (FCA) mouse model of joint inflammation. Monoarthritis was induced via FCA in the presence or absence of losartan with naive mice serving as controls. Knee joint swelling, joint pain (assessed by dynamic weight bearing of limb use), knee joint artery reactivity (assessed ex vivo) and blood perfusion of the knee joint (assessed in vivo) were determined. FCA mediated a significant increase in knee joint diameter and reduced weight-bearing (a surrogate for pain sensation) of the affected limb. Notably, these phenomena were substantially reduced when mice were prophylactically treated with losartan. Assessment of arterial relaxation and blood perfusion with acetylcholine stimulation revealed that FCA resulted in significant vascular dysfunction, which was resolved to naïve levels with losartan treatment. Through the actions of losartan, these findings indicate that the angiotensin II-type 1 receptor is a likely therapeutic target of importance in the development of the physical changes, pain sensation and vascular dysfunction found in inflammatory arthritis.

TIMEx: tumor-immune microenvironment deconvolution web-portal for bulk transcriptomics using pan-cancer scRNA-seq signatures.

SUMMARY: The heterogeneous cell types of the tumor-immune microenvironment (TIME) play key roles in determining cancer progression, metastasis and response to treatment. We report the development of TIMEx, a novel TIME deconvolution method emphasizing on estimating infiltrating immune cells for bulk transcriptomics using pan-cancer single-cell RNA-seq signatures. We also implemented a comprehensive, user-friendly web-portal for users to evaluate TIMEx and other deconvolution methods with bulk transcriptomic profiles. AVAILABILITY AND IMPLEMENTATION: TIMEx web-portal is freely accessible at http://timex.moffitt.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Self-assembled miRNA-switch nanoparticles target denuded regions and prevent restenosis.

Cardiovascular disease is the leading cause of death and disability worldwide. Effective delivery of cell-selective therapies that target atherosclerotic plaques and neointimal growth while sparing the endothelium remains the Achilles heel of percutaneous interventions. The current study utilizes synthetic microRNA switch therapy that self-assembles to form a compacted, nuclease-resistant nanoparticle <200 nM in size when mixed with cationic amphipathic cell-penetrating peptide (p5RHH). These nanoparticles possess intrinsic endosomolytic activity that requires endosomal acidification. When administered in a femoral artery wire injury mouse model in vivo, the mRNA-p5RHH nanoparticles deliver their payload specifically to the regions of endothelial denudation and not to the lungs, liver, kidney, or spleen. Moreover, repeated administration of nanoparticles containing a microRNA switch, consisting of synthetically modified mRNA encoding for the cyclin-dependent kinase inhibitor p27Kip1 that contains one complementary target sequence of the endothelial cell-specific miR-126 at its 5' UTR, drastically reduced neointima formation after wire injury and allowed for vessel reendothelialization. This cell-selective nanotherapy is a valuable tool that has the potential to advance the fight against neointimal hyperplasia and atherosclerosis.

Protease activated receptor 2 and matriptase expression in the joints of cats with and without osteoarthritis.

OBJECTIVES: The aim of this study was to determine the presence of protease-activated receptor 2 (PAR2) and matriptase proteins and quantify PAR2 and matriptase mRNA expression in the articular cartilage and synovial membrane of cats with and without osteoarthritis (OA). METHODS: A total of 28 articular cartilage samples from adult cats (14 OA and 14 normal), 10 synovial membranes from adult cats (five OA and five normal) and three cartilage samples from 9-week-old fetal cats were used. The presence of PAR2 and matriptase in the cartilage and synovial membrane of the adult samples was detected by immunohistochemical (IHC) staining, while real-time PCR was used for mRNA expression analyses in all samples. RESULTS: PAR2 was detected in all OA and normal articular cartilage and synovial membrane samples but confined to only a few superficial chondrocytes in the normal samples. Matriptase was only detected in OA articular cartilage and synovial membrane samples. PAR2 and matriptase mRNA expression were, however, detected in all cartilage and synovial membrane samples. PAR2 and matriptase mRNA expression levels in OA articular cartilage were five (P <0.001) and 3.3 (P <0.001) times higher than that of the healthy group, respectively. There was no significant difference (P = 0.05) in the OA synovial membrane PAR2 and matriptase mRNA expression compared with the normal samples. CONCLUSIONS AND RELEVANCE: Detection of PAR2 and matriptase proteins and gene expression in feline articular tissues is a novel and important finding, and supports the hypothesis that serine proteases are involved in the pathogenesis of feline OA. The consistent presence of PAR2 and matriptase protein in the cytoplasm of OA chondrocytes suggests a possible involvement of proteases in cartilage degradation. Further investigations into the PAR2 and matriptase pathobiology could enhance our understanding of the proteolytic cascades in feline OA, which might lead to the development of novel therapeutic strategies.

Left-sided congenitally unguarded tricuspid valve with congenitally corrected transposition of the great arteries: A rare diagnosis confirmed by three-dimensional echocardiography.

Left-sided unguarded tricuspid valve disease with congenitally corrected transposition of the great arteries (ccTGA) is a rare cardiac malformation, only reported a few times in the literature. Two-dimensional echocardiography (2DE) uses standard views to diagnose tricuspid valve disease. Advanced imaging techniques, such as three-dimensional echocardiography, allow for simultaneous visualization of the tricuspid valve annulus and all leaflets. Three-dimensional echocardiography (3DE) may be useful in distinguishing unguarded tricuspid valve orifice from other forms of tricuspid valve disease.

Chromosome 19 microRNA cluster enhances cell reprogramming by inhibiting epithelial-to-mesenchymal transition.

During implantation, cytotrophoblasts undergo epithelial-to-mesenchymal transition (EMT) as they differentiate into invasive extravillous trophoblasts (EVTs). The primate-specific microRNA cluster on chromosome 19 (C19MC) is exclusively expressed in the placenta, embryonic stem cells and certain cancers however, its role in EMT gene regulation is unknown. In situ hybridization for miR-517a/c, a C19MC cistron microRNA, in first trimester human placentas displayed strong expression in villous trophoblasts and a gradual decrease from proximal to distal cell columns as cytotrophoblasts differentiate into invasive EVTs. To investigate the role of C19MC in the regulation of EMT genes, we employed the CRISPR/dCas9 Synergistic Activation Mediator (SAM) system, which induced robust transcriptional activation of the entire C19MC cistron and resulted in suppression of EMT associated genes. Exposure of human iPSCs to hypoxia or differentiation of iPSCs into either cytotrophoblast-stem-like cells or EVT-like cells under hypoxia reduced C19MC expression and increased EMT genes. Furthermore, transcriptional activation of the C19MC cistron induced the expression of OCT4 and FGF4 and accelerated cellular reprogramming. This study establishes the CRISPR/dCas9 SAM as a powerful tool that enables activation of the entire C19MC cistron and uncovers its novel role in suppressing EMT genes critical for maintaining the epithelial cytotrophoblasts stem cell phenotype.

Exosomes in intercellular communication and implications for osteoarthritis.

Osteoarthritis (OA) is the most prevalent of the musculoskeletal conditions and represents a significant public health burden. While degeneration of articular cartilage is a key feature, it is now increasingly recognized as a complex condition affecting the whole joint, with synovial inflammation present in a significant proportion of patients. As a secretory tissue, the OA synovium is a rich source of both soluble inflammatory mediators and extracellular vesicles, including exosomes, which have been implicated in cell-cell communication. Exosome cargo has been found to include proteins, lipids and various RNA subtypes such as mRNA and miRNA, potentially capable of regulating gene expression in target cells and tissues. Profiling of exosome cargo and understanding effects on cartilage could elucidate novel regulatory mechanisms within the joint, providing insight for targeted treatment. The aim of this article is to review current literature on exosome biology, highlighting the relevance and application for OA pathogenesis.

The Transcriptomic Toolbox: Resources for Interpreting Large Gene Expression Data within a Precision Medicine Context for Metabolic Disease Atherosclerosis.

As one of the most widespread metabolic diseases, atherosclerosis affects nearly everyone as they age; arteries gradually narrow from plaque accumulation over time reducing oxygenated blood flow to central and periphery causing heart disease, stroke, kidney problems, and even pulmonary disease. Personalized medicine promises to bring treatments based on individual genome sequencing that precisely target the molecular pathways underlying atherosclerosis and its symptoms, but to date only a few genotypes have been identified. A promising alternative to this genetic approach is the identification of pathways altered in atherosclerosis by transcriptome analysis of atherosclerotic tissues to target specific aspects of disease. Transcriptomics is a potentially useful tool for both diagnostics and discovery science, exposing novel cellular and molecular mechanisms in clinical and translational models, and depending on experimental design to identify and test novel therapeutics. The cost and time required for transcriptome analysis has been greatly reduced by the development of next generation sequencing. The goal of this resource article is to provide background and a guide to appropriate technologies and downstream analyses in transcriptomics experiments generating ever-increasing amounts of gene expression data.

Nucleotide Modification Alters MicroRNA-Dependent Silencing of MicroRNA Switches.

mRNA therapeutics hold great promise for the treatment of human diseases. While incorporating naturally occurring modified nucleotides during synthesis has greatly increased their potency and safety, challenges in selective expression have hindered clinical applications. MicroRNA (miRNA)-regulated in vitro-transcribed mRNAs, called miRNA switches, have been used to control the expression of exogenous mRNA in a cell-selective manner. However, the effect of nucleotide modifications on miRNA-dependent silencing has not been examined. Here we show that the incorporation of pseudouridine, N1-methylpseudourdine, or pseudouridine and 5-methylcytidine, which increases translation, tends to decrease the regulation of miRNA switches. Moreover, pseudouridine and 5-methylcytidine modification enables one miRNA target site at the 3' UTR to be as effective as four target sites. We also demonstrate that the effects of pseudouridine, pseudouridine and 5-methylcytidine, and N1-methylpseudourdine modification are miRNA switch specific and do not correlate with the proportion of modified nucleotides in the miRNA target site. Furthermore, modified miRNA switches containing seed-complementary target sites are poorly regulated by miRNA. We also show that placing the miRNA target site in the 5' UTR of the miRNA switch abolishes the effect of nucleotide modification on miRNA-dependent silencing. This work provides insights into the influence of nucleotide modifications on miRNA-dependent silencing and informs the design of optimal miRNA switches.