Approaches for Mapping and Analysis of R-loops.

R-loops are nucleic acid structures composed of a DNA:RNA hybrid with a displaced non-template single-stranded DNA. Current approaches to identify and map R-loop formation across the genome employ either an antibody targeted against R-loops (S9.6) or a catalytically inactivated form of RNase H1 (dRNH1), a nuclease that can bind and resolve DNA:RNA hybrids via RNA exonuclease activity. This overview article outlines several ways to map R-loops using either methodology, explaining the differences and similarities among the approaches. Bioinformatic analysis of R-loops involves several layers of quality control and processing before visualizing the data. This article provides resources and tools that can be used to accurately process R-loop mapping data and explains the advantages and disadvantages of the resources as compared to one another. © 2024 Wiley Periodicals LLC.

The ratting of North America: A 350-year retrospective on Rattus species compositions and competition.

While the impacts of black (Rattus rattus) and brown (Rattus norvegicus) rats on human society are well documented-including the spread of disease, broad-scale environmental destruction, and billions spent annually on animal control-little is known about their ecology and behavior in urban areas due to the challenges of studying animals in city environments. We use isotopic and ZooMS analysis of archaeological (1550s-1900 CE) rat remains from eastern North America to provide a large-scale framework for species arrival, interspecific competition, and dietary ecology. Brown rats arrived earlier than expected and rapidly outcompeted black rats in coastal urban areas. This replacement happened despite evidence that the two species occupy different trophic positions. Findings include the earliest molecularly confirmed brown rat in the Americas and show a deep ecological structure to how rats exploit human-structured areas, with implications for understanding urban zoonosis, rat management, and ecosystem planning as well as broader themes of rat dispersal, phylogeny, evolutionary ecology, and climate impacts.

The long shadow of woody encroachment: An integrated approach to modeling grassland songbird habitat.

Animals must track resources over relatively fine spatial and temporal scales, particularly in disturbance-mediated systems like grasslands. Grassland birds respond to habitat heterogeneity by dispersing among sites within and between years, yet we know little about how they make post-dispersal settlement decisions. Many methods exist to quantify the resource selection of mobile taxa, but the habitat data used in these models are frequently not collected at the same location or time that individuals were present. This spatiotemporal misalignment may lead to incorrect interpretations and adverse conservation outcomes, particularly in dynamic systems. To investigate the extent to which spatially and temporally dynamic vegetation conditions and topography drive grassland bird settlement decisions, we integrated multiple data sources from our study site to predict slope, vegetation height, and multiple metrics of vegetation cover at any point in space and time within the temporal and spatial scope of our study. We paired these predictions with avian mark-resight data for 8 years at the Konza Prairie Biological Station in NE Kansas to evaluate territory selection for Grasshopper Sparrows (Ammodramus savannarum), Dickcissels (Spiza americana), and Eastern Meadowlarks (Sturnella magna). Each species selected different types and amounts of herbaceous vegetation cover, but all three species preferred relatively flat areas with less than 6% shrub cover and less than 1% tree cover. We evaluated several scenarios of woody vegetation removal and found that, with a targeted approach, the simulated removal of just one isolated tree in the uplands created up to 14 ha of grassland bird habitat. This study supports growing evidence that small amounts of woody encroachment can fragment landscapes, augmenting conservation threats to grassland systems. Conversely, these results demonstrate that drastic increases in bird habitat area could be achieved through relatively efficient management interventions. The results and approaches reported pave the way for more efficient conservation efforts in grasslands and other systems through spatiotemporal alignment of habitat with animal behaviors and simulated impacts of management interventions.

Multi-omics analyses reveal novel effects of PLCγ2 deficiency in the mouse brain.

Phospholipase C gamma-2 (PLCγ2) catalyzes the hydrolysis of the membrane phosphatidylinositol-4,5-bisphosphate (PIP2) to form diacylglycerol (DAG) and inositol trisphosphate (IP3), which subsequently feed into numerous downstream signaling pathways. PLCG2 polymorphisms are associated with both reduced and increased risk of Alzheimer's disease (AD) and with longevity. In the brain, PLCG2 is highly expressed in microglia, where it is proposed to regulate phagocytosis, secretion of cytokines/chemokines, cell survival and proliferation. We analyzed the brains of three-month-old PLCγ2 knockout (KO), heterozygous (HET), and wild-type (WT) mice using multiomics approaches, including shotgun lipidomics, proteomics, and gene expression profiling, and immunofluorescence. Lipidomic analyses revealed sex-specific losses of total cerebrum PIP2 and decreasing trends of DAG content in KOs. In addition, PLCγ2 depletion led to significant losses of myelin-specific lipids and decreasing trends of myelin-enriched lipids. Consistent with our lipidomics results, RNA profiling revealed sex-specific changes in the expression levels of several myelin-related genes. Further, consistent with the available literature, gene expression profiling revealed subtle changes on microglia phenotype in mature adult KOs under baseline conditions, suggestive of reduced microglia reactivity. Immunohistochemistry confirmed subtle differences in density of microglia and oligodendrocytes in KOs. Exploratory proteomic pathway analyses revealed changes in KO and HET females compared to WTs, with over-abundant proteins pointing to mTOR signaling, and under-abundant proteins to oligodendrocytes. Overall, our data indicate that loss of PLCγ2 has subtle effects on brain homeostasis that may underlie enhanced vulnerability to AD pathology and aging via novel mechanisms in addition to regulation of microglia function.

Systematic analysis and characterization of long non-coding RNA genes in inflammatory bowel disease.

The cases of inflammatory bowel disease (IBD) are increasing rapidly around the world. Due to the multifactorial causes of IBD, there is an urgent need to understand the pathogenesis of IBD. As such, the usage of high-throughput techniques to profile genetic mutations, microbiome environments, transcriptome and proteome (e.g. lipidome) is increasing to understand the molecular changes associated with IBD, including two major etiologies of IBD: Crohn disease (CD) and ulcerative colitis (UC). In the case of transcriptome data, RNA sequencing (RNA-seq) technique is used frequently. However, only protein-coding genes are analyzed, leaving behind all other RNAs, including non-coding RNAs (ncRNAs) to be unexplored. Among these ncRNAs, long non-coding RNAs (lncRNAs) may hold keys to understand the pathogenesis of IBD as lncRNAs are expressed in a cell/tissue-specific manner and dysregulated in a disease, such as IBD. However, it is rare that RNA-seq data are analyzed for lncRNAs. To fill this gap in knowledge, we re-analyzed RNA-seq data of CD and UC patients compared with the healthy donors to dissect the expression profiles of lncRNA genes. As inflammation plays key roles in the pathogenesis of IBD, we conducted loss-of-function experiments to provide functional data of IBD-specific lncRNA, lung cancer associated transcript 1 (LUCAT1), in an in vitro model of macrophage polarization. To further facilitate the lncRNA research in IBD, we built a web database, IBDB (https://ibd-db.shinyapps.io/IBDB/), to provide a one-stop-shop for expression profiling of protein-coding and lncRNA genes in IBD patients compared with healthy donors.

Promoter hypermethylation of RARB and GSTP1 genes in plasma cell-free DNA as breast cancer biomarkers in Peruvian women.

BACKGROUND: Promoter hypermethylation is one of the enabling mechanisms of hallmarks of cancer. Tumor suppressor genes like RARB and GSTP1 have been reported as hypermethylated in breast cancer tumors compared with normal tissues in several populations. This case-control study aimed to determine the association between the promoter methylation ratio (PMR) of RARB and GSTP1 genes (separately and as a group) with breast cancer and its clinical-pathological variables in Peruvian patients, using a liquid biopsy approach. METHODS: A total of 58 breast cancer patients and 58 healthy controls, matched by age, participated in the study. We exacted cell-free DNA (cfDNA) from blood plasma and converted it by bisulfite salts. Methylight PCR was performed to obtain the PMR value of the studied genes. We determined the association between PMR and breast cancer, in addition to other clinicopathological variables. The sensitivity and specificity of the PMR of these genes were obtained. RESULTS: A significant association was not found between breast cancer and the RARB PMR (OR = 1.90; 95% CI [0.62-6.18]; p = 0.210) or the GSTP1 PMR (OR = 6.57; 95% CI [0.75-307.66]; p = 0.114). The combination of the RARB + GSTP1 PMR was associated with breast cancer (OR = 2.81; 95% CI [1.02-8.22]; p = 0.026), controls under 50 years old (p = 0.048), patients older than 50 (p = 0.007), and postmenopausal (p = 0.034). The PMR of both genes showed a specificity of 86.21% and a sensitivity of 31.03%. CONCLUSION: Promoter hypermethylation of RARB + GSTP1 genes is associated with breast cancer, older age, and postmenopausal Peruvian patients. The methylated promoter of the RARB + GSTP1 genes needs further validation to be used as a biomarker for liquid biopsy and as a recommendation criterion for additional tests in asymptomatic women younger than 50 years.

Oxygen insufflation through the bronchoscope channel for sedation-induced hypoxia: safe and effective.

OBJECTIVES: To evaluate both efficacy and safety parameters for insufflation through the bronchoscope as a method of recovery from sedation-induced hypoxia. To explore parameters applicable to use in human beings using an animal model. MATERIALS AND METHODS: Two adult pigs were sedated enough to depress respiratory drive. The effects of insufflation at 15 l/min (the upper limits of flow that might be used clinically) were then evaluated. Pressure and volume responses to bronchoscopy during intubation and without an endotracheal tube in place were recorded. Several assays were performed for each scenario, with each animal acting as its own control. Recovery from hypoxemia using insufflation was compared with recovery using mechanical ventilation. RESULTS: Insufflation was effective, with rapid increases in fraction of inspired oxygen (FIO2), saturation, and partial pressure of arterial oxygen (PaO2). The rate of recovery using insufflation was faster than that from institution of mechanical ventilation. Insufflation in an intubated animal with cuff inflated led to a rapid and dangerous rise in pressure. With balloon deflated, there were no adverse pressure consequences from insufflation via the endotracheal tube at a rate of 15 l/min. CONCLUSION: Insufflation through the bronchoscope for episodes of sedation-induced hypoxia should be safe and effective as long as not delivered within a closed system.

Exploration and analysis of R-loop mapping data with RLBase.

R-loops are three-stranded nucleic acid structures formed from the hybridization of RNA and DNA. In 2012, Ginno et al. introduced the first R-loop mapping method. Since that time, dozens of R-loop mapping studies have been conducted, yielding hundreds of publicly available datasets. Current R-loop databases provide only limited access to these data. Moreover, no web tools for analyzing user-supplied R-loop datasets have yet been described. In our recent work, we reprocessed 810 R-loop mapping samples, building the largest R-loop data resource to date. We also defined R-loop consensus regions and developed a framework for R-loop data analysis. Now, we introduce RLBase, a user-friendly database that provides the capability to (i) explore hundreds of public R-loop mapping datasets, (ii) explore R-loop consensus regions, (iii) analyze user-supplied data and (iv) download standardized and reprocessed datasets. RLBase is directly accessible via the following URL: https://gccri.bishop-lab.uthscsa.edu/shiny/rlbase/.

A Spectrum of Metastases to the Breast: Radiologic-Pathologic Correlation.

Metastases to the breast from non-mammary origin are rare. The majority of these lesions appear as secondary manifestations of melanoma and lymphoma, followed by lung carcinomas, gynecological carcinomas, and sarcomas. There has been a steady trend of an increase in diagnosis of intramammary metastases owing to the current advances in imaging technology. Imaging features depend on the type of primary neoplasm and route of dissemination, some of which resemble primary breast cancer and benign breast entities. There are certain imaging features that raise the level of suspicion for metastases in the correct clinical context. However, imaging manifestations of intramammary metastases do not always comply with the known classic patterns. The aim of this review is to clarify these features, emphasizing radiologic-pathologic correlation and a multidisciplinary approach, since most cases are found in patients with advanced disease.

Chip-based digital Polymerase Chain Reaction as quantitative technique for the detection of PIK3CA mutations in breast cancer patients.

Background: PIK3CA is a gene frequently mutated in breast cancer. With the FDA approval of alpelisib, the evaluation of PIK3CA for activating mutations is becoming routinely. Novel platforms for gene analysis as digital PCR (dPCR) are emerging as a potential replacement for the traditional Sanger sequencing. However, there are still few studies on chip-based dPCR to detect mutations in tumor samples. Thus, this cross-sectional study aimed to assess the sensibility of a chip-based dPCR to detect and quantify PIK3CA mutations and compare its performance with Sanger sequencing. Materials and Methods: Tumor samples from 57 breast cancer patients (22 pre-treatment samples, 32 tumors after neoadjuvant chemotherapy, and three lymph nodes) were collected and analyzed by Sanger sequencing and dPCR for the three PIK3CA most relevant mutations (p.E545K, p. H1047R, and p. H1047L). Digital PCR sensitivity, specificity, and overall performance were estimated by contingency tables, receptor operator characteristic (ROC), and area under the curve (AUC). Association of PIK3CA mutations with clinicopathological variables was conducted. Results: Sanger sequencing identified PIK3CA mutations in six patients (10.5%), two with p. H1047R, and four with p. E545K. Digital PCR confirmed those mutations and identified 19 additional patients with at least one mutation. Comparison between dPCR and Sanger sequencing showed a sensitivity of 100% (95% CI 53-100%), and a specificity of 84.2% (95% CI 83-84.2%). Besides, p. H1047R mutation detected by dPCR showed a significant association with breast cancer phenotype (p = 0.019) and lymphatic nodes infiltration (p = 0.046). Conclusions: Digital PCR showed a high sensitivity to detect mutations in tumor samples and it might be capable to detect low-rate mutations and tumor subpopulations not detected by Sanger sequencing.

Systematic Analysis of Long Non-Coding RNA Genes in Nonalcoholic Fatty Liver Disease.

The largest solid organ in humans, the liver, performs a variety of functions to sustain life. When damaged, cells in the liver can regenerate themselves to maintain normal liver physiology. However, some damage is beyond repair, which necessitates liver transplantation. Increasing rates of obesity, Western diets (i.e., rich in processed carbohydrates and saturated fats), and cardiometabolic diseases are interlinked to liver diseases, including non-alcoholic fatty liver disease (NAFLD), which is a collective term to describe the excess accumulation of fat in the liver of people who drink little to no alcohol. Alarmingly, the prevalence of NAFLD extends to 25% of the world population, which calls for the urgent need to understand the disease mechanism of NAFLD. Here, we performed secondary analyses of published RNA sequencing (RNA-seq) data of NAFLD patients compared to healthy and obese individuals to identify long non-coding RNAs (lncRNAs) that may underly the disease mechanism of NAFLD. Similar to protein-coding genes, many lncRNAs are dysregulated in NAFLD patients compared to healthy and obese individuals, suggesting that understanding the functions of dysregulated lncRNAs may shed light on the pathology of NAFLD. To demonstrate the functional importance of lncRNAs in the liver, loss-of-function experiments were performed for one NAFLD-related lncRNA, LINC01639, which showed that it is involved in the regulation of genes related to apoptosis, TNF/TGF, cytokine signaling, and growth factors as well as genes upregulated in NAFLD. Since there is no lncRNA database focused on the liver, especially NAFLD, we built a web database, LiverDB, to further facilitate functional and mechanistic studies of hepatic lncRNAs.

Development and characterization of a mass cytometry panel for detecting the effect of acute doxorubicin exposure on murine cardiac nonmyocytes.

Childhood cancer survivors (CCSs) face lifelong side effects related to their treatment with chemotherapy. Anthracycline agents, such as doxorubicin (DOX), are important in the treatment of childhood cancers but are associated with cardiotoxicity. Cardiac toxicities represent a significant source of chronic disability that cancer survivors face; despite this, the chronic cardiotoxicity phenotype and how it relates to acute toxicity remains poorly defined. To address this critical knowledge gap, we studied the acute effect of DOX on murine cardiac nonmyocytes in vivo. Determination of the acute cellular effects of DOX on nonmyocytes, a cell pool with finite replicative capacity, provides a basis for understanding the pathogenesis of the chronic heart disease that CCSs face. To investigate the acute cellular effects of DOX, we present single-cell RNA sequencing (scRNAseq) data from homeostatic cardiac nonmyocytes and compare it with preexisting datasets, as well as a novel CyTOF datasets. SCANPY, a python-based single-cell analysis, was used to assess the heterogeneity of cells detected in scRNAseq and CyTOF. To further assist in CyTOF data annotation, joint analyses of scRNAseq and CyTOF data using an artificial neural network known as sparse autoencoder for clustering, imputation, and embedding (SAUCIE) are performed. Lastly, the panel is tested on a mouse model of acute DOX exposure at two time points (24 and 72 h) after the last dose of doxorubicin and examined with joint clustering. In sum, we report the first ever CyTOF study of cardiac nonmyocytes and characterize the effect of acute DOX exposure with scRNAseq and CyTOF.NEW & NOTEWORTHY We describe the first mass cytometry studies of murine cardiac nonmyocytes. The mass cytometry panel is compared with single-cell RNA sequencing data. Homeostatic cardiac nonmyocytes are characterized by mass cytometry to identify and quantify four major cell populations: endothelial cells, fibroblasts, leukocytes, and pericytes. The single-cell acute nonmyocyte response to doxorubicin is studied at 24 and 72 h after doxorubicin exposure given daily for 5 days at a dose of 4 mg/kg/day.

Quality-controlled R-loop meta-analysis reveals the characteristics of R-loop consensus regions.

R-loops are three-stranded nucleic acid structures formed from the hybridization of RNA and DNA. While the pathological consequences of R-loops have been well-studied to date, the locations, classes, and dynamics of physiological R-loops remain poorly understood. R-loop mapping studies provide insight into R-loop dynamics, but their findings are challenging to generalize. This is due to the narrow biological scope of individual studies, the limitations of each mapping modality, and, in some cases, poor data quality. In this study, we reprocessed 810 R-loop mapping datasets from a wide array of biological conditions and mapping modalities. From this data resource, we developed an accurate R-loop data quality control method, and we reveal the extent of poor-quality data within previously published studies. We then identified a set of high-confidence R-loop mapping samples and used them to define consensus R-loop sites called 'R-loop regions' (RL regions). In the process, we identified a stark divergence between RL regions detected by S9.6 and dRNH-based mapping methods, particularly with respect to R-loop size, location, and colocalization with RNA binding factors. Taken together, this work provides a much-needed method to assess R-loop data quality and offers novel context regarding the differences between dRNH- and S9.6-based R-loop mapping approaches.

Current Status of Epitranscriptomic Marks Affecting lncRNA Structures and Functions.

Long non-coding RNAs (lncRNAs) belong to a class of non-protein-coding RNAs with their lengths longer than 200 nucleotides. Most of the mammalian genome is transcribed as RNA, yet only a small percent of the transcribed RNA corresponds to exons of protein-coding genes. Thus, the number of lncRNAs is predicted to be several times higher than that of protein-coding genes. Because of sheer number of lncRNAs, it is often difficult to elucidate the functions of all lncRNAs, especially those arising from their relationship to their binding partners, such as DNA, RNA, and proteins. Due to their binding to other macromolecules, it has become evident that the structures of lncRNAs influence their functions. In this regard, the recent development of epitranscriptomics (the field of study to investigate RNA modifications) has become important to further elucidate the structures and functions of lncRNAs. In this review, the current status of lncRNA structures and functions influenced by epitranscriptomic marks is discussed.

FibroDB: Expression Analysis of Protein-Coding and Long Non-Coding RNA Genes in Fibrosis.

Most long non-coding RNAs (lncRNAs) are expressed at lower levels than protein-coding genes and their expression is often restricted to specific cell types, certain time points during development, and various stress and disease conditions, respectively. To revisit this long-held concept, we focused on fibroblasts, a common cell type in various organs and tissues. Using fibroblasts and changes in their expression profiles during fibrosis as a model system, we show that the overall expression level of lncRNA genes is significantly lower than that of protein-coding genes. Furthermore, we identified lncRNA genes whose expression is upregulated during fibrosis. Using dermal fibroblasts as a model, we performed loss-of-function experiments and show that the knockdown of the lncRNAs LINC00622 and LINC01711 result in gene expression changes associated with cellular and inflammatory responses, respectively. Since there are no lncRNA databases focused on fibroblasts and fibrosis, we built a web application, FibroDB, to further promote functional and mechanistic studies of fibrotic lncRNAs.

Cost-Benefit Analysis of Robotic vs. Laparoscopic Hepatectomy: A Propensity-Matched Retrospective Cohort Study of American College of Surgeons National Surgical Quality Improvement Program Database.

BACKGROUND: Robotic and laparoscopic hepatectomies having increased utilization as minimally invasive techniques are explored for hepatobiliary malignancies. Although the data on outcomes from these 2 approaches are emerging, the cost-benefit analysis of these approaches remains sparse. This study compares the costs associated with robotic vs. laparoscopic liver resections, taking into account 30-day complications. METHODS: Using the American College of Surgeons National Surgical Quality Improvement Program database, a propensity-matched cohort of patients with laparoscopic or robotic liver resections between 2014 and 2017 was identified. Costs were assigned to perioperative variables, including operating room (OR) time, length of stay, blood transfusions, and 30-day complications. Cost estimates were obtained from the Centers for Medicare and Medicaid Services billing data (2017), American Hospital Association data (2017), relevant literature, and local institutional cost data. RESULTS: In our matched cohort of 454 patients (227 per group), total costs associated with laparoscopic liver resections were estimated at $5.5 M ($24 K per patient) vs. $6.8 M ($29.8 K per patient) for robotic liver resections (21.3% difference, P < .001). The higher cost associated with robotic hepatectomies was related to blood transfusions ($22.0 K vs. $12.1 K, P = .02), length of stay ($2.05 M vs. $1.76 M, P = .046), and OR time ($4.01 M vs. $3.24 M, P < .0001). DISCUSSION: Robotic hepatectomies were associated with higher costs compared to laparoscopic hepatectomies. The 2 major contributors to the cost disparity were increased OR time and increased length of stay. Future studies are warranted to analyze high-volume Minimally Invasive Surgery surgeons' impact in specialty centers on potentially mitigating this current cost disparity.

Effect of preoperative radiation on free flap outcomes for head and neck reconstruction: An updated systematic review and meta-analysis.

BACKGROUND: There is an ongoing debate about whether neoadjuvant radiation therapy is associated with higher rates of postoperative complications after head and neck reconstruction. Herle et al. conducted a systematic review in 2014 of 24 studies, finding higher complication rates in irradiated fields. We sought to perform an exhaustive updated systematic review and meta-analysis. METHODS: We conducted an updated systematic review of the literature, as outlined in our protocol, which was registered on PROSPERO. Databases included Medline, Embase, Cochrane Central, and Web of Science. There were no limits placed on the date range, place of publication, or origin. Exclusion criteria included patients less than 18 years of age, studies with less than 20 participants (n < 20), case studies, skull base reconstructions, and local tissue rearrangements. The combined results of the studies and relative risks (RR) were calculated. RESULTS: 53 studies were included for analysis, including 5,086 free flaps in an irradiated field, and 9,110 free flaps in a non-irradiated field. Of the 53 studies, 21 studies overlapped with those discussed in Herle et al.'s study, with a total of 32 additional studies. Neoadjuvant radiation was found to be a statistically significant risk factor for postoperative complications (RR 1.579, P < 0.001), total flap failure (RR, 1.565; P < 0.001), and fistula (RR, 1.810; P < 0.001). Our work reaffirmed the findings of the Herle et al. CONCLUSION: Preoperative radiation was associated with a statistically significant increase in the risk of total flap failure, fistula, and total complications but not partial flap failure. These high-morbidity complications must be taken into consideration when determining which patients should receive neoadjuvant radiation therapy.

Highlights from the 7th Oncological Pathology Conference 'Pathological Anatomy in the context of the National Cancer Law: An overview of the Latin American experience', 15, 22 and 23 July 2022, Trujillo, Peru.

The seventh session of the Oncological Pathology Conference (JoPaO) entitled 'Pathological Anatomy in the context of the National Cancer Law: An overview of the Latin American experience', was held virtually on July 15, 22 and 23. Peru was the headquarters for this event, where 17 national and international professors of high academic standing participated. They interacted in a multidisciplinary context through talks with national panellists and the general public. The recent promulgation of the 'National Cancer Law' fosters the development of discussion forums to analyse the national realities and uphold continuous learning about experiences in other Latin American countries with successful cancer programmes, in which pathology holds a principal role. The topics addressed during this JoPaO included the exchange of Latin American cancer management experiences, an emphasis on investments in and the development of strategic plans to improve care, the use of new technologies, laboratory quality control, and the need to advance scientific research.

Development of a Model for Abdominal Aortic Aneurysms in Swine.

INTRODUCTION: Producing a reliable large-animal model of AAA has proven challenging. We sought to create a reproducible swine model of AAA using enzymatic degradation of the aortic wall. METHODS: Twelve male Yorkshire swine received periadventitial injections of type 1 collagenase and porcine pancreatic elastase into a 4 cm segment of infrarenal aorta. Nine survived until postoperative day (POD) 21. Aortic growth was monitored at 7 and 14 days using ultrasound. The animals were euthanized on POD 21, and the suprarenal (control) and infrarenal aorta were harvested for analysis, after gross measurement of aortic diameter (AD). Tensile strength was measured and additional segments were collected for histopathological analysis. PCR of matrix metalloproteinases (MMP9) was conducted. Groups were compared with paired t-tests, or ANOVA, where appropriate. RESULTS: Average percent growth of AD at POD 21 for treated segments was 27% versus 4.5% for control tissue. The average difference in AD by subject, was 26.7% (P<0.001). Aortic medial thickness was decreased in treated tissue; 235 µm versus 645 µm (P<0.0001). Quantities of both medial elastin fibers, and smooth muscles cells were decreased in treated tissue; 1.8% compared to 9.9% (P<0.0001), and 24% versus 37.4%, respectively. Tensile strength was also decreased in treated tissue; 16.7 MPa versus 29.5 MPa (P=0.0002). A 12-fold increase in expression of MMP9 mRNA was also demonstrated in aneurysmal tissue (P=0.002) CONCLUSION: A reproducible, large-animal model of AAA, with anatomical, histopathological, and biomechanical properties that are clinically translatable, can be achieved with extraluminal enzymatic degradation.