Lectins As Effective Tools in the Study of the Biliary Network and the Parenchymal Architecture of the Zebrafish (Danio rerio) Liver.

Lectins are carbohydrate-binding proteins with specific affinity to glycoconjugates expressed in various tissues. Lectins are of substantial utility as research, histochemical, and diagnostic tools in mammalian systems. Reactivity of 12 commonly used plant-based lectins was studied in zebrafish liver. Four lectins, tomato lectin (TL), wheat germ agglutinin, concanavalin A, and Jacalin showed strong reactivity to hepatic parenchymal structures. Importantly, TL reacted to glycoconjugates within segments of the larval and adult intrahepatic biliary network, from canaliculi to bile ducts. We provide evidence that lectins can serve as important histochemical tools to investigate the structural and functional characteristics of the zebrafish liver.

Reprogramming of the LXRα Transcriptome Sustains Macrophage Secondary Inflammatory Responses.

Macrophages regulate essential aspects of innate immunity against pathogens. In response to microbial components, macrophages activate primary and secondary inflammatory gene programs crucial for host defense. The liver X receptors (LXRα, LXRß) are ligand-dependent nuclear receptors that direct gene expression important for cholesterol metabolism and inflammation, but little is known about the individual roles of LXRα and LXRß in antimicrobial responses. Here, the author demonstrate that induction of LXRα transcription by prolonged exposure to lipopolysaccharide (LPS) supports inflammatory gene expression in macrophages. LXRα transcription is induced by NF-κB and type-I interferon downstream of TLR4 activation. Moreover, LPS triggers a reprogramming of the LXRα cistrome that promotes cytokine and chemokine gene expression through direct LXRα binding to DNA consensus sequences within cis-regulatory regions including enhancers. LXRα-deficient macrophages present fewer binding of p65 NF-κB and reduced histone H3K27 acetylation at enhancers of secondary inflammatory response genes. Mice lacking LXRα in the hematopoietic compartment show impaired responses to bacterial endotoxin in peritonitis models, exhibiting reduced neutrophil infiltration and decreased expansion and inflammatory activation of recruited F4/80lo-MHC-IIhi peritoneal macrophages. Together, these results uncover a previously unrecognized function for LXRα-dependent transcriptional cis-activation of secondary inflammatory gene expression in macrophages and the host response to microbial ligands.

The role of microbiota on rheumatoid arthritis onset.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes inflammation and pain, which can lead to the loss of normal joint function. Although the exact cause of the disease is not yet fully understood, both environmental factors and genetics may play a role in its development. Moreover, research suggests microbiota contributes to the onset and progression of RA. People with RA show higher quantities of bacteria such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella copri, Proteus mirabilis, and Lactobacillus salivarius compared to healthy individuals. Conversely, studies propose that Lactobacillus casei, a probiotic bacterium with immunomodulatory properties, has beneficial effects for RA in murine and human models. Therefore, this work reviews the potential role of the gut microbiota in the development of RA and explores the feasibility of using probiotic bacteria as a supplementary treatment for this disease.

Water quality trends of streams in Puerto Rico: Evaluating 50 years of the Clean Water Act.

Water quality regulations entail a substantial commitment of resources from governments and private entities. It is important to continually evaluate the effectiveness of these regulations to ensure they are having the intended impact. In this paper, we evaluated nutrient data as indicators of primary productivity and dissolved oxygen (DO) concentrations and pH as response variables to assess historical water quality trends from 55 stations of Puerto Rico. The stations were divided into impaired versus non-impaired categories based on their historical total phosphorus (TP) mean concentration. Mean TP and total nitrogen (TN) concentrations were significantly higher in the impaired stations relative to the non-impaired stations. In contrast, DO mean concentrations and mean pH values were significantly lower in the impaired stations. A generalized additive mixed model was used to demonstrate temporal trends. A significant decrease in TP and TN concentrations was observed with time at the impaired stations. This was accompanied by significant increases in DO concentrations and pH. The non-impaired stations showed a marginal (statistically nonsignificant) decreasing trend with time. The large reductions in nutrient concentrations observed at the impaired stations seem to be related to the closure of several primary wastewater treatment plants (WWTPs) across the island. The conversion of abandoned crop agricultural lands into secondary forest in recent decades has resulted in small but significant decreases in TN (not TP) in receiving streams. We conclude that the Clean Water Act has promoted improvements in water quality in Puerto Rico by advancing upgrades in sanitary infrastructure and the regulation of point sources of pollution.

Transcriptomic and lipid profiling analysis reveals a functional interplay between testosterone and growth hormone in hypothyroid liver.

Preclinical and clinical studies suggest that hypothyroidism might cause hepatic endocrine and metabolic disturbances with features that mimic deficiencies of testosterone and/or GH. The absence of physiological interactions between testosterone and GH can be linked to male differentiated liver diseases. Testosterone plays relevant physiological effects on somatotropic-liver axis and liver composition and the liver is a primary organ of interactions between testosterone and GH. However, testosterone exerts many effects on liver through complex and poorly understood mechanisms. Testosterone impacts liver functions by binding to the Androgen Receptor, and, indirectly, through its conversion to estradiol, and cooperation with GH. However, the role of testosterone, and its interaction with GH, in the hypothyroid liver, remains unclear. In the present work, the effects of testosterone, and how they impact on GH-regulated whole transcriptome and lipid composition in the liver, were studied in the context of adult hypothyroid-orchiectomized rats. Testosterone replacement positively modulated somatotropic-liver axis and impacted liver transcriptome involved in lipid and glucose metabolism. In addition, testosterone enhanced the effects of GH on the transcriptome linked to lipid biosynthesis, oxidation-reduction, and metabolism of unsaturated and long-chain fatty acids (FA). However, testosterone decreased the hepatic content of cholesterol esters and triacylglycerols and increased fatty acids whereas GH increased neutral lipids and decreased polar lipids. Biological network analysis of the effects of testosterone on GH-regulated transcriptome confirmed a close connection with crucial proteins involved in steroid and fatty acid metabolism. Taken together, this comprehensive analysis of gene expression and lipid profiling in hypothyroid male liver reveals a functional interplay between testosterone and pulsed GH administration.

Zebrafish as a high throughput model for organ preservation and transplantation research.

Despite decades of effort, the preservation of complex organs for transplantation remains a significant barrier that exacerbates the organ shortage crisis. Progress in organ preservation research is significantly hindered by suboptimal research tools that force investigators to sacrifice translatability over throughput. For instance, simple model systems, such as single cell monolayers or co-cultures, lack native tissue structure and functional assessment, while mammalian whole organs are complex systems with confounding variables not compatible with high-throughput experimentation. In response, diverse fields and industries have bridged this experimental gap through the development of rich and robust resources for the use of zebrafish as a model organism. Through this study, we aim to demonstrate the value zebrafish pose for the fields of solid organ preservation and transplantation, especially with respect to experimental transplantation efforts. A wide array of methods were customized and validated for preservation-specific experimentation utilizing zebrafish, including the development of assays at multiple developmental stages (larvae and adult), methods for loading and unloading preservation agents, and the development of viability scores to quantify functional outcomes. Using this platform, the largest and most comprehensive screen of cryoprotectant agents (CPAs) was performed to determine their toxicity and efficiency at preserving complex organ systems using a high subzero approach called partial freezing (i.e., storage in the frozen state at -10°C). As a result, adult zebrafish cardiac function was successfully preserved after 5 days of partial freezing storage. In combination, the methods and techniques developed have the potential to drive and accelerate research in the fields of solid organ preservation and transplantation.

Dynamics of Collagen Oxidation and Cross Linking in Regenerating and Irreversibly Infarcted Myocardium.

In mammalian hearts myocardial infarction produces a permanent collagen-rich scar. Conversely, in zebrafish a collagen-rich scar forms but is completely resorbed as the myocardium regenerates. The formation of cross-links in collagen hinders its degradation but cross-linking has not been well characterized in zebrafish hearts. Here, a library of fluorescent probes to quantify collagen oxidation, the first step in collagen cross-link (CCL) formation, was developed. Myocardial injury in mice or zebrafish resulted in similar dynamics of collagen oxidation in the myocardium in the first month after injury. However, during this time, mature CCLs such as pyridinoline and deoxypyridinoline developed in the murine infarcts but not in the zebrafish hearts. High levels of newly oxidized collagen were still seen in murine scars with mature CCLs. These data suggest that fibrogenesis remains dynamic, even in mature scars, and that the absence of mature CCLs in zebrafish hearts may facilitate their ability to regenerate.

Qualitative Analysis of Men's Involvement in Family Planning in The Philippines: An Ecological Assessment.

While family planning (FP) programs have the capacity to empower women, support gender equality, and reduce poverty, male involvement is an influential factor for the uptake of FP that has been lacking. In the past decade, there have been more progressive FP policies and growing attention on male involvement in FP in the Philippines, providing an opportunity to develop evidence-based interventions to better integrate men into FP services by approaching care delivery from a family-focused perspective. This paper sought to understand the current role of men in FP services and explore how to strengthen facilitators and overcome barriers to optimize men's involvement in FP in the Philippines. Using the Ecological Model for Health Promotion, this qualitative study used convenience sampling to collect data through in-depth interviews and focus group discussions at all levels of the ecosystem. All data were collected in the Albay area, with the exception of some policy data collected in Manila. Qualitative analysis was guided by content analysis. The final sample included 66 participants across the ecosystem. Two primary themes emerged: (1) Resources and health care systems structure impact on male involvement in FP and (2) Education and training that support male involvement in FP. The findings of our qualitative study suggest that while men in the Philippines and their ecosystems support men's FP involvement, the inconsistent health care systems and protocols are not yet reaching men with information and education they need to help them make informed FP decisions with their female partners.

Pore-forming activity of S. pneumoniae pneumolysin disrupts the paracellular localization of the epithelial adherens junction protein E-cadherin.

Streptococcus pneumoniae, a common cause of community-acquired bacterial pneumonia, can cross the respiratory epithelial barrier to cause lethal septicemia and meningitis. S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers robust neutrophil (PMN) infiltration that promotes bacterial transepithelial migration in vitro and disseminated disease in mice. Apical infection of polarized respiratory epithelial monolayers by S. pneumoniae at a multiplicity of infection (MOI) of 20 resulted in recruitment of PMNs, loss of 50% of the monolayer, and PMN-dependent bacterial translocation. Reducing the MOI to 2 decreased PMN recruitment two-fold and preserved the monolayer, but apical-to-basolateral translocation of S. pneumoniae remained relatively efficient. At both MOI of 2 and 20, PLY was required for maximal PMN recruitment and bacterial translocation. Co-infection by wild-type S. pneumoniae restored translocation by a PLY-deficient mutant, indicating that PLY can act in trans. Investigating the contribution of S. pneumoniae infection on apical junction complexes in the absence of PMN transmigration, we found that S. pneumoniae infection triggered the cleavage and mislocalization of the adherens junction (AJ) protein E-cadherin. This disruption was PLY-dependent at MOI of 2 and was recapitulated by purified PLY, requiring its pore-forming activity. In contrast, at MOI of 20, E-cadherin disruption was independent of PLY, indicating that S. pneumoniae encodes multiple means to disrupt epithelial integrity. This disruption was insufficient to promote bacterial translocation in the absence of PMNs. Thus, S. pneumoniae triggers cleavage and mislocalization of E-cadherin through PLY-dependent and -independent mechanisms, but maximal bacterial translocation across epithelial monolayers requires PLY-dependent neutrophil transmigration.

Identification of Immunoglobulin Gene Rearrangement Biomarkers in Multiple Myeloma through cfDNA-Based Liquid Biopsy Using tchDNA-Seq.

Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of pathogenic CD138+ plasma cells (PPCs) in bone marrow (BM). Recent years have seen a significant increase in the treatment options for MM; however, most patients who achieve complete the response ultimately relapse. The earlier detection of tumor-related clonal DNA would thus be very beneficial for patients with MM and would enable timely therapeutic interventions to improve outcomes. Liquid biopsy of "cell-free DNA" (cfDNA) as a minimally invasive approach might be more effective than BM aspiration not only for the diagnosis but also for the detection of early recurrence. Most studies thus far have addressed the comparative quantification of patient-specific biomarkers in cfDNA with PPCs and BM samples, which have shown good correlations. However, there are limitations to this approach, such as the difficulty in obtaining enough circulating free tumor DNA to achieve sufficient sensitivity for the assessment of minimal residual disease. Herein, we summarize current data on methodologies to characterize MM, and we present evidence that targeted capture hybridization DNA sequencing (tchDNA-Seq) can provide robust biomarkers in cfDNA, including immunoglobulin (IG) rearrangements. We also show that detection can be improved by prior purification of the cfDNA. Overall, liquid biopsies of cfDNA to monitor IG rearrangements have the potential to provide important diagnostic, prognostic, and predictive information in patients with MM.

Neutralizing Antibodies against the SARS-CoV-2 Ancestral Strain and Omicron BA.1 Subvariant in Dogs and Cats in Mexico.

SARS-CoV-2 mainly affects humans; however, it is important to monitor the infection of companion and wild animals as possible reservoirs of this virus. In this sense, seroprevalence studies in companion animals, such as dogs and cats, provide important information about the epidemiology of SARS-CoV-2. This study aimed to evaluate the seroprevalence of neutralizing antibodies (nAbs) against the ancestral strain and the Omicron BA.1 subvariant in dogs and cats in Mexico. Six hundred and two samples were obtained from dogs (n = 574) and cats (n = 28). These samples were collected from the end of 2020 to December 2021 from different regions of Mexico. The presence of nAbs was evaluated using a plaque reduction neutralization test (PRNT) and microneutralization (MN) assays. The results showed that 14.2% of cats and 1.5% of dogs presented nAbs against the ancestral strain of SARS-CoV-2. The analysis of nAbs against Omicron BA.1 in cats showed the same percentage of positive animals but a reduced titer. In dogs, 1.2% showed nAbs against Omicron BA.1. These results indicate that nAbs were more frequent in cats than in dogs and that these nAbs have a lower capacity to neutralize the subvariant Omicron BA.1.

Real-life disease monitoring in follicular lymphoma patients using liquid biopsy ultra-deep sequencing and PET/CT.

In the present study, we screened 84 Follicular Lymphoma patients for somatic mutations suitable as liquid biopsy MRD biomarkers using a targeted next-generation sequencing (NGS) panel. We found trackable mutations in 95% of the lymph node samples and 80% of the liquid biopsy baseline samples. Then, we used an ultra-deep sequencing approach with 2 · 10-4 sensitivity (LiqBio-MRD) to track those mutations on 151 follow-up liquid biopsy samples from 54 treated patients. Positive LiqBio-MRD at first-line therapy correlated with a higher risk of progression both at the interim evaluation (HRINT 11.0, 95% CI 2.10-57.7, p = 0.005) and at the end of treatment (HREOT, HR 19.1, 95% CI 4.10-89.4, p < 0.001). Similar results were observed by PET/CT Deauville score, with a median PFS of 19 months vs. NR (p < 0.001) at the interim and 13 months vs. NR (p < 0.001) at EOT. LiqBio-MRD and PET/CT combined identified the patients that progressed in less than two years with 88% sensitivity and 100% specificity. Our results demonstrate that LiqBio-MRD is a robust and non-invasive approach, complementary to metabolic imaging, for identifying FL patients at high risk of failure during the treatment and should be considered in future response-adapted clinical trials.

Nematicidal, Acaricidal and Plant Growth-Promoting Activity of Enterobacter Endophytic Strains and Identification of Genes Associated with These Biological Activities in the Genomes.

In the present study, the nematicidal and acaricidal activity of three Enterobacter endophytic strains isolated from Mimosa pudica nodules was evaluated. The percentages of mortality of Enterobacter NOD4 against Panagrellus redivivus was 81.2%, and against Nacobbus aberrans 70.1%, Enterobacter NOD8 72.4% and 62.5%, and Enterobacter NOD10 64.8% and 58.7%, respectively. While against the Tyrophagus putrescentiae mite, the mortality percentages were 68.2% due to Enterobacter NOD4, 64.3% due to Enterobacter NOD8 and 77.8% due to Enterobacter NOD10. On the other hand, the ability of the three Enterobacter strains to produce indole acetic acid and phosphate solubilization, characteristics related to plant growth-promoting bacteria, was detected. Bioinformatic analysis of the genomes showed the presence of genes related to IAA production, phosphate solubilization, and nitrogen fixation. Phylogenetic analyzes of the recA gene, phylogenomics, and average nucleotide identity (ANI) allowed us to identify the strain Enterobacter NOD8 related to E. mori and Enterobacter NOD10 as E. asburiae, while Enterobacter NOD4 was identified as a possible new species of this species. The plant growth-promoting, acaricidal and nematicidal activity of the three Enterobacter strains makes them a potential agent to include in biocontrol alternatives and as growth-promoting bacteria in crops of agricultural interest.

NGS-Based Molecular Karyotyping of Multiple Myeloma: Results from the GEM12 Clinical Trial.

Next-generation sequencing (NGS) has greatly improved our ability to detect the genomic aberrations occurring in multiple myeloma (MM); however, its transfer to routine clinical labs and its validation in clinical trials remains to be established. We designed a capture-based NGS targeted panel to identify, in a single assay, known genetic alterations for the prognostic stratification of MM. The NGS panel was designed for the simultaneous study of single nucleotide and copy number variations, insertions and deletions, chromosomal translocations and V(D)J rearrangements. The panel was validated using a cohort of 149 MM patients enrolled in the GEM2012MENOS65 clinical trial. The results showed great global accuracy, with positive and negative predictive values close to 90% when compared with available data from fluorescence in situ hybridization and whole-exome sequencing. While the treatments used in the clinical trial showed high efficacy, patients defined as high-risk by the panel had shorter progression-free survival (p = 0.0015). As expected, the mutational status of TP53 was significant in predicting patient outcomes (p = 0.021). The NGS panel also efficiently detected clonal IGH rearrangements in 81% of patients. In conclusion, molecular karyotyping using a targeted NGS panel can identify relevant prognostic chromosomal abnormalities and translocations for the clinical management of MM patients.

Regulation of metabolic and transcriptional responses by the thyroid hormone in cellular models of murine macrophages.

Oncogene-immortalized bone marrow-derived macrophages are considered to be a good model for the study of immune cell functions, but the factors required for their survival and proliferation are still unknown. Although the effect of the thyroid hormones on global metabolic and transcriptional responses in macrophages has not yet been examined, there is increasing evidence that they could modulate macrophage functions. We show here that the thyroid hormone T3 is an absolute requirement for the growth of immortal macrophages. The hormone regulates the activity of the main signaling pathways required for proliferation and anabolic processes, including the phosphorylation of ERK and p38 MAPKs, AKT, ribosomal S6 protein, AMPK and Sirtuin-1. T3 also alters the levels of metabolites controlling transcriptional and post-transcriptional actions in macrophages, and causes widespread transcriptomic changes, up-regulating genes needed for protein synthesis and cell proliferation, while down-regulating genes involved in immune responses and endocytosis, among others. This is not observed in primary bone marrow-derived macrophages, where only p38 and AMPK activation is regulated by T3 and in which the metabolic and transcriptomic effects of the hormone are much weaker. However, the response to IFN-γ is reduced by T3 similarly in immortalized macrophages and in the primary cells, confirming previous results showing that the thyroid hormones can antagonize JAK/STAT-mediated signaling. These results provide new perspectives on the relevant pathways involved in proliferation and survival of macrophage cell culture models and on the crosstalk between the thyroid hormones and the immune system.

The Clonal Relationship Between the Ductal and Lobular Components of Mixed Ductal-Lobular Carcinomas Suggested a Ductal Origin in Most Tumors.

The relationship between the ductal and lobular components of invasive ductolobular carcinomas (IDLC) has not been fully elucidated. In this study, the molecular alterations of both components were analyzed in a series of 20 IDLC that were selected, not only by morphologic criteria, but also by the loss of E-cadherin expression in the lobular component. We found that 80% of tumors shared alterations of driver genes in both components, being PIK3CA the most common alteration. In addition, 45% of IDLC carried CDH1 mutations in their lobular component that were absent in the ductal component. Fluorescent in situ hybridization analysis of the CDH1 gene excluded homozygous CDH1 loss as a frequent cause of E-cadherin loss in tumors without CDH1 mutations. In addition, no pathogenic mutations of catenin genes were detected in this series of tumors. In 25% of tumors, actionable mutations in PIK3CA , AKT1 , and ERBB2 were found in only 1 component. Altogether, our results confirm that most IDLC derive from invasive carcinoma of no special type, in which a population of cells lose E-cadherin and acquire a lobular phenotype. The frequency of CDH1 mutations in IDLC appears to be lower than in conventional invasive lobular carcinomas, suggesting the implication of alternative mechanisms of E-cadherin loss. Moreover, molecular heterogeneity between ductal and lobular areas suggests the need for molecular characterization of both components to guide targeted therapies.

Improvement in the Thermostability of a Recombinant ß-Glucosidase Immobilized in Zeolite under Different Conditions.

ß-Glucosidase is part of the cellulases and is responsible for degrading cellobiose into glucose, a compound that can be used to produce biofuels. However, the use of the free enzyme makes the process more expensive. Enzyme immobilization improves catalytic characteristics and supports, such as zeolites, which have physical-chemical characteristics and ion exchange capacity that have a promising application in the biotechnological industry. This research aimed to immobilize by adsorption a recombinant ß-glucosidase from Trichoderma reesei, obtained in Escherichia coli BL21 (DE3), in a commercial zeolite. A Box Behnken statistical design was applied to find the optimal immobilization parameters, the stability against pH and temperature was determined, and the immobilized enzyme was characterized by SEM. The highest enzymatic activity was determined with 100 mg of zeolite at 35 °C and 175 min. Compared to the free enzyme, the immobilized recombinant ß-glucosidase presented greater activity from pH 2 to 4 and greater thermostability. The kinetic parameters were calculated, and a lower KM value was obtained for the immobilized enzyme compared to the free enzyme. The obtained immobilization parameters by a simple adsorption method and the significant operational stability indicate promising applications in different fields.

Detection of kinase domain mutations in BCR::ABL1 leukemia by ultra-deep sequencing of genomic DNA.

The screening of the BCR::ABL1 kinase domain (KD) mutation has become a routine analysis in case of warning/failure for chronic myeloid leukemia (CML) and B-cell precursor acute lymphoblastic leukemia (ALL) Philadelphia (Ph)-positive patients. In this study, we present a novel DNA-based next-generation sequencing (NGS) methodology for KD ABL1 mutation detection and monitoring with a 1.0E-4 sensitivity. This approach was validated with a well-stablished RNA-based nested NGS method. The correlation of both techniques for the quantification of ABL1 mutations was high (Pearson r = 0.858, p < 0.001), offering DNA-DeepNGS a sensitivity of 92% and specificity of 82%. The clinical impact was studied in a cohort of 129 patients (n = 67 for CML and n = 62 for B-ALL patients). A total of 162 samples (n = 86 CML and n = 76 B-ALL) were studied. Of them, 27 out of 86 harbored mutations (6 in warning and 21 in failure) for CML, and 13 out of 76 (2 diagnostic and 11 relapse samples) did in B-ALL patients. In addition, in four cases were detected mutation despite BCR::ABL1 < 1%. In conclusion, we were able to detect KD ABL1 mutations with a 1.0E-4 sensitivity by NGS using DNA as starting material even in patients with low levels of disease.

Dynamic Python-Based Method Provides Quantitative Analysis of Intercellular Junction Organization During S. pneumoniae Infection of the Respiratory Epithelium.

Many respiratory pathogens compromise epithelial barrier function during lung infection by disrupting intercellular junctions, such as adherens junctions and tight junctions, that maintain intercellular integrity. This includes Streptococcus pneumoniae, a leading cause of pneumonia, which can successfully breach the epithelial barrier and cause severe infections such as septicemia and meningitis. Fluorescence microscopy analysis on intercellular junction protein manipulation by respiratory pathogens has yielded major advances in our understanding of their pathogenesis. Unfortunately, a lack of automated image analysis tools that can tolerate variability in sample-sample staining has limited the accuracy in evaluating intercellular junction organization quantitatively. We have created an open source, automated Python computer script called "Intercellular Junction Organization Quantification" or IJOQ that can handle a high degree of sample-sample staining variability and robustly measure intercellular junction integrity. In silico validation of IJOQ was successful in analyzing computer generated images containing varying degrees of simulated intercellular junction disruption. Accurate IJOQ analysis was further confirmed using images generated from in vitro and in vivo bacterial infection models. When compared in parallel to a previously published, semi-automated script used to measure intercellular junction organization, IJOQ demonstrated superior analysis for all in vitro and in vivo experiments described herein. These data indicate that IJOQ is an unbiased, easy-to-use tool for fluorescence microscopy analysis and will serve as a valuable, automated resource to rapidly quantify intercellular junction disruption under diverse experimental conditions.