Preparation of Tc99m-Labeled Pseudomonas Bacteriophage without Adversely Impacting Infectivity or Biodistribution.

Bacteriophages (phages) are ubiquitous viruses which have adapted to infect and replicate within target bacteria, their only known hosts, in a strain specific fashion with minimal cross infectivity. The recent steep rise in antibiotic resistance throughout the world has renewed interest in adapting phages for the imaging and treatment of bacterial infection in humans. In this article, we describe the current limitations surrounding the radiolabeling of phage for the imaging and treatment of bacterial infection and methods to overcome these difficulties. Specifically, we examined the effects of hydrazinonicotinamide conjugation and removal of bacterial DNA on the infectivity, biodistribution, and radionuclide imaging of a phage lytic for a clinically relevant strain of Pseudomonas aeruginosa, a common Gram-negative bacterial pathogen often resistant to multiple antibiotics. We found that all but the briefest reaction of concentrated phage with hydrazinonicotinamide (≤3 min) resulted in nearly complete loss of infectivity. Furthermore, we determined that digestion and removal of bacterial DNA was needed to avoid high nonspecific uptake of hydrazinonicotinamide-labeled phage within the liver and spleen as well as prolonged circulation in the blood. We also demonstrate the surprisingly wide soft tissue and organ biodistribution and rapid pharmacokinetics of 99mTc-hydrazinonicotinamide-labeled phage in normal mice as well as its imaging characteristics and efficacy in wounded mice infected with bioluminescent Pseudomonas aeruginosa. In conclusion, the preservation of phage infectivity and removal of all bacterial containments including DNA are critical methodologic considerations in the labeling of phages for imaging and therapy.

Automated Spatial Omics Landscape Analysis Approach Reveals Novel Tissue Architectures in Ulcerative Colitis.

The utility of spatial omics in leveraging cellular interactions in normal and diseased states for precision medicine is hampered by a lack of strategies for matching disease states with spatial heterogeneity-guided cellular annotations. Here we use a spatial context-dependent approach that matches spatial pattern detection to cell annotation. Using this approach in existing datasets from ulcerative colitis patient colonic biopsies, we identified architectural complexities and associated difficult-to-detect rare cell types in ulcerative colitis germinal-center B cell follicles. Our approach deepens our understanding of health and disease pathogenesis, illustrates a strategy for automating nested architecture detection for highly multiplexed spatial biology data, and informs precision diagnosis and therapeutic strategies.

Highlighting the Undetectable - Fluorescence Molecular Imaging in Gastrointestinal Endoscopy.

Flexible high-definition white-light endoscopy is the current gold standard in screening for cancer and its precursor lesions in the gastrointestinal tract. However, miss rates are high, especially in populations at high risk for developing gastrointestinal cancer (e.g., inflammatory bowel disease, Lynch syndrome, or Barrett's esophagus) where lesions tend to be flat and subtle. Fluorescence molecular endoscopy (FME) enables intraluminal visualization of (pre)malignant lesions based on specific biomolecular features rather than morphology by using fluorescently labeled molecular probes that bind to specific molecular targets. This strategy has the potential to serve as a valuable tool for the clinician to improve endoscopic lesion detection and real-time clinical decision-making. This narrative review presents an overview of recent advances in FME, focusing on probe development, techniques, and clinical evidence. Future perspectives will also be addressed, such as the use of FME in patient stratification for targeted therapies and potential alliances with artificial intelligence. KEY MESSAGES: • Fluorescence molecular endoscopy is a relatively new technology that enables safe and real-time endoscopic lesion visualization based on specific molecular features rather than on morphology, thereby adding a layer of information to endoscopy, like in PET-CT imaging. • Recently the transition from preclinical to clinical studies has been made, with promising results regarding enhancing detection of flat and subtle lesions in the colon and esophagus. However, clinical evidence needs to be strengthened by larger patient studies with stratified study designs. • In the future fluorescence molecular endoscopy could serve as a valuable tool in clinical workflows to improve detection in high-risk populations like patients with Barrett's esophagus, Lynch syndrome, and inflammatory bowel syndrome, where flat and subtle lesions tend to be malignant up to five times more often. • Fluorescence molecular endoscopy has the potential to assess therapy responsiveness in vivo for targeted therapies, thereby playing a role in personalizing medicine. • To further reduce high miss rates due to human and technical factors, joint application of artificial intelligence and fluorescence molecular endoscopy are likely to generate added value.

A tissue atlas of ulcerative colitis revealing evidence of sex-dependent differences in disease-driving inflammatory cell types and resistance to TNF inhibitor therapy.

Although literature suggests that resistance to TNF inhibitor (TNFi) therapy in patients with ulcerative colitis (UC) is partially linked to immune cell populations in the inflamed region, there is still substantial uncertainty underlying the relevant spatial context. Here, we used the highly multiplexed immunofluorescence imaging technology CODEX to create a publicly browsable tissue atlas of inflammation in 42 tissue regions from 29 patients with UC and 5 healthy individuals. We analyzed 52 biomarkers on 1,710,973 spatially resolved single cells to determine cell types, cell-cell contacts, and cellular neighborhoods. We observed that cellular functional states are associated with cellular neighborhoods. We further observed that a subset of inflammatory cell types and cellular neighborhoods are present in patients with UC with TNFi treatment, potentially indicating resistant niches. Last, we explored applying convolutional neural networks (CNNs) to our dataset with respect to patient clinical variables. We note concerns and offer guidelines for reporting CNN-based predictions in similar datasets.

Rapid assessment of changes in phage bioactivity using dynamic light scattering.

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. Here, we use Dynamic Light Scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-year-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web-application (Phage-ELF) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and non-destructive tool for quality control of phage preparations in academic and commercial settings.

Rapid assessment of changes in phage bioactivity using dynamic light scattering.

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. In this study, we use dynamic light scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-y-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web application (Phage-Estimator of Lytic Function) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and nondestructive tool for quality control of phage preparations in academic and commercial settings.

Infection with Listeria monocytogenes alters the placental transcriptome and eicosanome.

INTRODUCTION: Placental infection and inflammation are risk factors for adverse pregnancy outcomes, including preterm labor. However, the mechanisms underlying these outcomes are poorly understood. METHODS: To study this response, we have employed a pregnant mouse model of placental infection caused by the bacterial pathogen Listeria monocyogenes, which infects the human placenta. Through in vivo bioluminescence imaging, we confirm the presence of placental infection and quantify relative infection levels. Infected and control placentas were collected on embryonic day 18 for RNA sequencing to evaluate gene expression signatures associated with infection by Listeria. RESULTS: We identified an enrichment of genes associated with eicosanoid biosynthesis, suggesting an increase in eicosanoid production in infected tissues. Because of the known importance of eicosanoids in inflammation and timing of labor, we quantified eicosanoid levels in infected and uninfected placentas using semi-targeted mass spectrometry. We found a significant increase in the concentrations of several key eicosanoids: leukotriene B4, lipoxin A4, prostaglandin A2, prostaglandin D2, and eicosatrienoic acid. DISCUSSION: Our study provides a likely explanation for dysregulation of the timing of labor following placental infection. Further, our results suggest potential biomarkers of placental pathology and targets for clinical intervention.

Gut Microbiome in Inflammatory Bowel Disease: Role in Pathogenesis, Dietary Modulation, and Colitis-Associated Colon Cancer.

The gut microbiome has increasingly been recognized as a critical and central factor in inflammatory bowel disease (IBD). Here, we review specific microorganisms that have been suggested to play a role in the pathogenesis of IBD and the current state of fecal microbial transplants as a therapeutic strategy in IBD. We discuss specific nutritional and dietary interventions in IBD and their effects on gut microbiota composition. Finally, we examine the role and mechanisms of the gut microbiome in mediating colitis-associated colon cancer.

Abnormal microRNA expression in Ts65Dn hippocampus and whole blood: contributions to Down syndrome phenotypes.

Down syndrome (DS; trisomy 21) is one of the most common genetic causes of intellectual disability, which is attributed to triplication of genes located on chromosome 21. Elevated levels of several microRNAs (miRNAs) located on chromosome 21 have been reported in human DS heart and brain tissues. The Ts65Dn mouse model is the most investigated DS model with a triplicated segment of mouse chromosome 16 harboring genes orthologous to those on human chromosome 21. Using ABI TaqMan miRNA arrays, we found a set of miRNAs that were significantly up- or downregulated in the Ts65Dn hippocampus compared to euploid controls. Furthermore, miR-155 and miR-802 showed significant overexpression in the Ts65Dn hippocampus, thereby confirming results of previous studies. Interestingly, miR-155 and miR-802 were also overexpressed in the Ts65Dn whole blood but not in lung tissue. We also found overexpression of the miR-155 precursors, pri- and pre-miR-155 derived from the miR-155 host gene, known as B cell integration cluster, suggesting enhanced biogenesis of miR-155. Bioinformatic analysis revealed that neurodevelopment, differentiation of neuroglia, apoptosis, cell cycle, and signaling pathways including ERK/MAPK, protein kinase C, phosphatidylinositol 3-kinase, m-TOR and calcium signaling are likely targets of these miRNAs. We selected some of these potential gene targets and found downregulation of mRNA encoding Ship1, Mecp2 and Ezh2 in Ts65Dn hippocampus. Interestingly, the miR-155 target gene Ship1 (inositol phosphatase) was also downregulated in Ts65Dn whole blood but not in lung tissue. Our findings provide insights into miRNA-mediated gene regulation in Ts65Dn mice and their potential contribution to impaired hippocampal synaptic plasticity and neurogenesis, as well as hemopoietic abnormalities observed in DS.

Antimicrobial peptides and the gut microbiome in inflammatory bowel disease.

Antimicrobial peptides (AMP) are highly diverse and dynamic molecules that are expressed by specific intestinal epithelial cells, Paneth cells, as well as immune cells in the gastrointestinal (GI) tract. They play critical roles in maintaining tolerance to gut microbiota and protecting against enteric infections. Given that disruptions in tolerance to commensal microbiota and loss of barrier function play major roles in the pathogenesis of inflammatory bowel disease (IBD) and converge on the function of AMP, the significance of AMP as potential biomarkers and novel therapeutic targets in IBD have been increasingly recognized in recent years. In this frontier article, we discuss the function and mechanisms of AMP in the GI tract, examine the interaction of AMP with the gut microbiome, explore the role of AMP in the pathogenesis of IBD, and review translational applications of AMP in patients with IBD.

Neuronal and vascular deficits following chronic adaptation to high altitude.

We sought to understand the mechanisms underlying cognitive deficits that are reported to affect non-native subjects following their prolonged stay and/or work at high altitude (HA). We found that mice exposed to a simulated environment of 5000 m exhibit deficits in hippocampal learning and memory accompanied by abnormalities in brain MR imaging. Exposure (1-8 months) to HA led to an increase in brain ventricular volume, a reduction in relative cerebral blood flow and changes in diffusion tensor imaging (DTI) derived parameters within the hippocampus and corpus callosum. Furthermore, neuropathological examination revealed significant expansion of the neurovascular network, microglia activation and demyelination within the corpus callosum. Electrophysiological recordings from the corpus callosum indicated that axonal excitabilities are increased while refractory periods are longer despite a lack of change in action potential conduction velocities of both myelinated and unmyelinated fibers. Next generation RNA-sequencing identified alterations in hippocampal and amygdala transcriptome signaling pathways linked to angiogenesis, neuroinflammation and myelination. Our findings reveal that exposure to hypobaric-hypoxia triggers maladaptive responses inducing cognitive deficits and suggest potential mechanisms underlying the adverse impacts of staying or traveling at high altitude.

Identification of recently handled materials by analysis of latent human fingerprints using infrared spectromicroscopy.

Analysis of fingerprints has predominantly focused on matching the pattern of ridges to a specific person as a form of identification. The present work focuses on identifying extrinsic materials that are left within a person's fingerprint after recent handling of such materials. Specifically, we employed infrared spectromicroscopy to locate and positively identify microscopic particles from a mixture of common materials in the latent human fingerprints of volunteer subjects. We were able to find and correctly identify all test substances based on their unique infrared spectral signatures. Spectral imaging is demonstrated as a method for automating recognition of specific substances in a fingerprint. We also demonstrate the use of attenuated total reflectance (ATR) and synchrotron-based infrared spectromicroscopy for obtaining high-quality spectra from particles that were too thick or too small, respectively, for reflection/absorption measurements. We believe the application of this rapid, nondestructive analytical technique to the forensic study of latent human fingerprints has the potential to add a new layer of information available to investigators. Using fingerprints to not only identify who was present at a crime scene, but also to link who was handling key materials, will be a powerful investigative tool.