A Simple Method for Creating a High-Content Microscope for Imaging Multiplexed Tissue Microarrays.

High-throughput, high-content imaging technologies and multiplex slide scanning have become widely used. Advantages of these approaches include the ability to archive digital copies of slides, review slides as teams using virtual microscopy software, and standardize analytical approaches. The cost and hardware and software inflexibility of dedicated slide scanning devices can, however, complicate implementation. Here, we describe a simple method that allows any microscope to be used for slide scanning. The only requirements are that the microscope be equipped with a motorized filter turret or wheels (for multi-channel fluorescence) and a motorized xyz stage. This example uses MetaMorph software, but the same principles can be used with any microscope control software that includes a few standard functions and allows programming of simple command routines, or journals. The series of journals that implement the method perform key functions, including assistance in defining an unlimited number of regions of interest (ROI) and imaging parameters. Fully automated acquisition is rapid, taking less than 3 hr to image fifty 2.5-mm ROIs in four channels. Following acquisition, images can be easily stitched and displayed using open-source or commercial image-processing and virtual microscope applications. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Hardware and software configuration Basic Protocol 2: Create a preliminary scan Basic Protocol 3: Select, save, and position ROIs Basic Protocol 4: Determine and set autofocus parameters Basic Protocol 5: Acquire tiled images Basic Protocol 6: Review the scans Basic Protocol 7: Reimage ROIs as needed Basic Protocol 8: Stitch, stack, and assemble images Basic Protocol 9: Repeat scanning for multiplex immunostaining or background subtraction.

Type I IFNs and CD8 T cells increase intestinal barrier permeability after chronic viral infection.

Intestinal barrier leakage constitutes a potential therapeutic target for many inflammatory diseases and represents a disease progression marker during chronic viral infections. However, the causes of altered gut barrier remain mostly unknown. Using murine infection with lymphocytic choriomeningitis virus, we demonstrate that, in contrast to an acute viral strain, a persistent viral isolate leads to long-term viral replication in hematopoietic and mesenchymal cells, but not epithelial cells (IECs), in the intestine. Viral persistence drove sustained intestinal epithelial barrier leakage, which was characterized by increased paracellular flux of small molecules and was associated with enhanced colitis susceptibility. Type I IFN signaling caused tight junction dysregulation in IECs, promoted gut microbiome shifts and enhanced intestinal CD8 T cell responses. Notably, both type I IFN receptor blockade and CD8 T cell depletion prevented infection-induced barrier leakage. Our study demonstrates that infection with a virus that persistently replicates in the intestinal mucosa increases epithelial barrier permeability and reveals type I IFNs and CD8 T cells as causative factors of intestinal leakage during chronic infections.

Differential regulation of claudin-2 and claudin-15 expression in children and adults with malabsorptive disease.

Intestinal Na+-nutrient cotransport depends on claudin-2 and claudin-15 mediated Na+ recycling. Expression of these proteins is coordinately regulated during postnatal development. While expression of claudin-2 and claudin-15 has been studied in inflammatory bowel disease (IBD) and celiac disease (CD), it has not been assessed in other malabsorptive diseases, and no reports have compared expression in children and adults. We used quantitative immunofluorescence microscopy to assess claudin-2 and claudin-15 expression in duodenal biopsies from children and adults with malabsorptive disease and healthy controls. Consistent with previous work in rodents, claudin-2 expression in healthy children was markedly greater, and claudin-15 expression was less, than that in adults. Claudin-2 expression was increased in adults with CD and downregulated in children with graft-versus-host disease (GVHD). In contrast, claudin-15 expression was reduced in adults with GVHD and common variable immunodeficiency (CVID). These data show that one of the two Na+/water pore-forming claudins is upregulated in CD and downregulated in GVHD and CVID. The specific claudin whose expression changes, however, reflects the age of the patient (child or adult). We conclude that contributions of claudin-2 and claudin-15 to pathophysiology of and responses to diarrhea in children and adults with GVHD and CVID differ from those in CD and IBD.

Inflammation-induced Occludin Downregulation Limits Epithelial Apoptosis by Suppressing Caspase-3 Expression.

BACKGROUND & AIMS: Epithelial tight junctions are compromised in gastrointestinal disease. Processes that contribute to the resulting barrier loss include endocytic occludin removal from the tight junction and reduced occludin expression. Nevertheless, the relatively-normal basal phenotype of occludin knockout (KO) mice has been taken as evidence that occludin does not contribute to gastrointestinal barrier function. We asked whether stress could unmask occludin functions within intestinal epithelia. METHODS: Wildtype (WT), universal and intestinal epithelial-specific occludin KO, and villin-EGFP-occludin transgenic mice as well as WT and occludin knockdown (KD) Caco-2BBe cell monolayers were challenged with DSS, TNBS, staurosporine, 5-FU, or TNF. Occludin and caspase-3 expression were assessed in patient biopsies. RESULTS: Intestinal epithelial occludin loss limited severity of DSS- and TNBS-induced colitis due to epithelial resistance to apoptosis; activation of both intrinsic and extrinsic apoptotic pathways was blocked in occludin KO epithelia. Promoter analysis revealed that occludin enhances CASP3 transcription and, conversely, that occludin downregulation reduces caspase-3 expression. Analysis of biopsies from Crohn's disease and ulcerative colitis patients and normal controls demonstrated that disease-associated occludin downregulation was accompanied by and correlated with reduced caspase-3 expression. In vitro, cytokine-induced occludin downregulation resulted in reduced caspase-3 expression and resistance to intrinsic and extrinsic pathway apoptosis, demonstrating an overall protective effect of inflammation-induced occludin loss. CONCLUSIONS: The tight junction protein occludin regulates apoptosis by enhancing caspase-3 transcription. These data suggest that reduced epithelial caspase-3 expression downstream of occludin downregulation is a previously-unappreciated anti-apoptotic process that contributes to mucosal homeostasis in inflammatory conditions.

Slowed gastric emptying and improved oral glucose tolerance produced by a nanomolar-potency inhibitor of calcium-activated chloride channel TMEM16A.

Interstitial cells of Cajal, which express the calcium-activated chloride channel transmembrane member 16A (TMEM16A), are an important determinant of gastrointestinal (GI) motility. We previously identified the acylaminocycloalkylthiophene class of TMEM16A inhibitors, which, following medicinal chemistry, gave analog 2-bromodifluoroacetylamino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide (TMinh-23) with 30 nM half-maximal inhibitory concentration. Here, we tested the efficacy of TMinh-23 for inhibition of GI motility in mice. In isolated murine gastric antrum, TMinh-23 strongly inhibited spontaneous and carbachol-stimulated rhythmic contractions. Pharmacokinetic analysis showed predicted therapeutic concentrations of TMinh-23 for at least 4 h following a single oral or intraperitoneal dose at 10 mg/kg. Gastric emptying, as assessed following an oral bolus of phenol red or independently by [99mTc]-diethylenetriamine pentaacetic acid scintigraphy, was reduced by TMinh-23 by ∼60% at 20 min. Interestingly, there was little effect of TMinh-23 on baseline whole-gut transit time or time to diarrhea induced by castor oil. Consequent to the delay in gastric emptying, TMinh-23 administration significantly reduced the elevation in blood sugar in mice following an oral but not intraperitoneal glucose load. These results provide pharmacological evidence for involvement of TMEM16A in gastric emptying and suggest the utility of TMEM16A inhibition in disorders of accelerated gastric emptying, such as dumping syndrome, and potentially for improving glucose tolerance in diabetes mellitus/metabolic syndrome and enhancing satiety in obesity.-Cil, O., Anderson, M. O., Yen, R., Kelleher, B., Huynh, T. L., Seo, Y., Nilsen, S. P., Turner, J. R., Verkman, A. S. Slowed gastric emptying and improved oral glucose tolerance produced by a nanomolar-potency inhibitor of calcium-activated chloride channel TMEM16A.

fruitless regulates aggression and dominance in Drosophila.

When competing for resources, two Drosophila melanogaster flies of the same sex fight each other. Males and females fight with distinctly different styles, and males but not females establish dominance relationships. Here we show that sex-specific splicing of the fruitless gene plays a critical role in determining who and how a fly fights, and whether a dominance relationship forms.

Gender-selective patterns of aggressive behavior in Drosophila melanogaster.

Complex behaviors, such as aggression, are comprised of distinct stereospecific behavioral patterns (modules). How such patterns get wired into nervous systems remains unknown. Recently, we reported on a quantitative analysis of fighting behavior in male flies of the common Canton-S strain of Drosophila melanogaster. Here, we report a similar analysis of fighting behavior in females of the same species. Fights were carried out between pairs of virgin and pairs of mated females in competition for a yeast resource. Each fight was videotaped and analyzed by using transition matrices and Markov chain analyses. We observe only small difference in fighting intensity between virgin and mated females. In contrast to what is seen in male fights, however, no clear hierarchical relationship is formed in the female fights. A further comparison of the behavioral patterns making up male and female fights reveals that some modules are shared by both sexes, whereas others are highly selective. Within the shared components, transitions between the modules also show gender-selective differences. By using the powerful genetic methods available for examining behavior in fruit flies, it should be possible to use the gender-selective differences in fighting behavior to address the question of how these behavioral patterns get established in the brains of fruit flies.