Biodistribution of cerium dioxide and titanium dioxide nanomaterials in rats after single and repeated inhalation exposures.

BACKGROUND: Physiologically based kinetic models facilitate the safety assessment of inhaled engineered nanomaterials (ENMs). To develop these models, high quality datasets on well-characterized ENMs are needed. However, there are at present, several data gaps in the systemic availability of poorly soluble particles after inhalation. The aim of the present study was therefore to acquire two comparable datasets to parametrize a physiologically-based kinetic model. METHOD: Rats were exposed to cerium dioxide (CeO2, 28.4 ± 10.4 nm) and titanium dioxide (TiO2, 21.6 ± 1.5 nm) ENMs in a single nose-only exposure to 20 mg/m3 or a repeated exposure of 2 × 5 days to 5 mg/m3. Different dose levels were obtained by varying the exposure time for 30 min, 2 or 6 h per day. The content of cerium or titanium in three compartments of the lung (tissue, epithelial lining fluid and freely moving cells), mediastinal lymph nodes, liver, spleen, kidney, blood and excreta was measured by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) at various time points post-exposure. As biodistribution is best studied at sub-toxic dose levels, lactate dehydrogenase (LDH), total protein, total cell numbers and differential cell counts were determined in bronchoalveolar lavage fluid (BALF). RESULTS: Although similar lung deposited doses were obtained for both materials, exposure to CeO2 induced persistent inflammation indicated by neutrophil granulocytes influx and exhibited an increased lung elimination half-time, while exposure to TiO2 did not. The lavaged lung tissue contained the highest metal concentration compared to the lavage fluid and cells in the lavage fluid for both materials. Increased cerium concentrations above control levels in secondary organs such as lymph nodes, liver, spleen, kidney, urine and faeces were detected, while for titanium this was found in lymph nodes and liver after repeated exposure and in blood and faeces after a single exposure. CONCLUSION: We have provided insight in the distribution kinetics of these two ENMs based on experimental data and modelling. The study design allows extrapolation at different dose-levels and study durations. Despite equal dose levels of both ENMs, we observed different distribution patterns, that, in part may be explained by subtle differences in biological responses in the lung.

Harmonizing the symphony of chimeric antigen receptor T cell immunotherapy with the elegance of biomaterials.

Chimeric antigen receptor T cell (CAR-T) immunotherapy has become a heated field of cancer research, demonstrating revolutionary efficacy in refractory and relapsed hematologic malignancies. However, CAR-T therapy has still encountered tough challenges, including complicated and lengthy manufacturing procedures, mediocre targeted delivery, limited therapeutic effect against solid tumors and difficulties in real-time in vivo monitoring. To overcome these limitations, various versatile biomaterials have been used in the above aspects and have improved CAR-T therapy impressively. This review mainly summarizes the latest research progress of biomaterials promoting CAR-T therapy in manufacturing, enhancing targeted delivery and tumor infiltration, and dramatic in vivo tracking to provide new insights and inspiration for clinical treatment.

Design, Fabrication, and Implantation of Invasive Microelectrode Arrays as in vivo Brain Machine Interfaces: A Comprehensive Review.

Invasive Microelectrode Arrays (MEAs) have been a significant and useful tool for us to gain a fundamental understanding of how the brain works through high spatiotemporal resolution neuron-level recordings and/or stimulations. Through decades of research, various types of microwire, silicon, and flexible substrate-based MEAs have been developed using the evolving new materials, novel design concepts, and cutting-edge advanced manufacturing capabilities. Surgical implantation of the latest minimal damaging flexible MEAs through the hard-to-penetrate brain membranes introduces new challenges and thus the development of implantation strategies and instruments for the latest MEAs. In this paper, studies on the design considerations and enabling manufacturing processes of various invasive MEAs as in vivo brain-machine interfaces have been reviewed to facilitate the development as well as the state-of-art of such brain-machine interfaces from an engineering perspective. The challenges and solution strategies developed for surgically implanting such interfaces into the brain have also been evaluated and summarized. Finally, the research gaps have been identified in the design, manufacturing, and implantation perspectives, and future research prospects in invasive MEA development have been proposed.

The mouse metabolic phenotyping center (MMPC) live consortium: an NIH resource for in vivo characterization of mouse models of diabetes and obesity.

The Mouse Metabolic Phenotyping Center (MMPC)Live Program was established in 2023 by the National Institute for Diabetes, Digestive and Kidney Diseases (NIDDK) at the National Institutes of Health (NIH) to advance biomedical research by providing the scientific community with standardized, high quality phenotyping services for mouse models of diabetes and obesity. Emerging as the next iteration of the MMPC Program which served the biomedical research community for 20 years (2001-2021), MMPCLive is designed as an outwardly-facing consortium of service cores that collaborate to provide reduced-cost consultation and metabolic, physiologic, and behavioral phenotyping tests on live mice for U.S. biomedical researchers. Four MMPCLive Centers located at universities around the country perform complex and often unique procedures in vivo on a fee for service basis, typically on mice shipped from the client or directly from a repository or vendor. Current areas of expertise include energy balance and body composition, insulin action and secretion, whole body carbohydrate and lipid metabolism, cardiovascular and renal function, food intake and behavior, microbiome and xenometabolism, and metabolic pathway kinetics. Additionally, an opportunity arose to reduce barriers to access and expand the diversity of the biomedical research workforce by establishing the VIBRANT Program. Directed at researchers historically underrepresented in the biomedical sciences, VIBRANT-eligible investigators have access to testing services, travel and career development awards, expert advice and experimental design consultation, and short internships to learn test technologies. Data derived from experiments run by the Centers belongs to the researchers submitting mice for testing which can be made publicly available and accessible from the MMPCLive database following publication. In addition to services, MMPCLive staff provide expertise and advice to researchers, develop and refine test protocols, engage in outreach activities, publish scientific and technical papers, and conduct educational workshops and training sessions to aid researchers in unraveling the heterogeneity of diabetes and obesity.

Mechanical and Thermal Stimulation for Studying the Somatosensory System: a Review on Devices and Methods.

The somatosensory system is widely studied to understand its functioning mechanisms. Multiple tests, based on different devices and methods, have been performed not only on humans but also on animals and ex-vivo models. Depending on the nature of the sample under analysis and on the scientific aims of interest, several solutions for experimental stimulation and for investigations on sensation or pain have been adopted. In this review paper, an overview of the available devices and methods has been reported, also analyzing the representative values adopted during literature experiments. Among the various physical stimulations used to study the somatosensory system, we focused only on mechanical and thermal ones. Based on the analysis of their main features and on literature studies, we pointed out the most suitable solution for humans, rodents, and ex-vivo models and investigation aims (sensation and pain).

Albumin-Mediated Drug Uptake by Organic Anion Transporter 1/3 Is Real: Implications for the Prediction of Active Renal Secretion Clearance.

Modulation of the transport-mediated active uptake by human serum albumin (HSA) for highly protein-bound substrates has been reported and improved the in vitro-to-in vivo extrapolation (IVIVE) of hepatic clearance. However, evidence for the relevance of such a phenomenon in the case of renal transporters is sparse. In this study, transport of renal organic anion transporter 1 or 3 (OAT1/3) substrates into conditionally immortalized proximal tubular epithelial cells transduced with OAT1/3 was measured in the presence and absence of 1 and 4% HSA while keeping the unbound substrate concentration constant (based on measured fraction unbound, fu,inc). In the presence of 4% HSA, the unbound intrinsic active uptake clearance (CLint,u,active) of six highly protein-bound substrates increased substantially relative to the HSA-free control (3.5- to 122-fold for the OAT1 CLint,u,active, and up to 28-fold for the OAT3 CLint,u,active). The albumin-mediated uptake effect (fold increase in CLint,u,active) was more pronounced with highly bound substrates compared to no effect seen for weakly protein-bound substrates adefovir (OAT1-specific) and oseltamivir carboxylate (OAT3-specific). The relationship between OAT1/3 CLint,u,active and fu,inc agreed with the facilitated-dissociation model; a relationship was established between the albumin-mediated fold change in CLint,u,active and fu,inc for both the OAT1 and OAT3, with implications for IVIVE modeling. The relative activity factor and the relative expression factor based on global proteomic quantification of in vitro OAT1/3 expression were applied for IVIVE of renal clearance. The inclusion of HSA improved the bottom-up prediction of the level of OAT1/3-mediated secretion and renal clearance (CLsec and CLr), in contrast to the underprediction observed with the control (HSA-free) scenario. For the first time, this study confirmed the presence of the albumin-mediated uptake effect with renal OAT1/3 transporters; the extent of the effect was more pronounced for highly protein-bound substrates. We recommend the inclusion of HSA in routine in vitro OAT1/3 assays due to considerable improvements in the IVIVE of CLsec and CLr.

Large-Scale Formation and Long-Term Culture of Hepatocyte Organoids From Streamlined In Vivo Genome-Edited GGTA1-/- Pigs for Bioartificial Liver Applications.

Hepatocyte transplantation and bioartificial liver (BAL) systems hold significant promise as less invasive alternatives to traditional transplantation, providing crucial temporary support for patients with acute and chronic liver failure. Although human hepatocytes are ideal, their use is limited by ethical concerns and donor availability, leading to the use of porcine hepatocytes in BAL systems due to their functional similarities. Recent advancements in gene-editing technology have improved porcine organ xenotransplantation clinical trials by addressing immune rejection issues. Gene-edited pigs, such as alpha-1,3-galactosyltransferase (GGTA1) knockout pigs, offer a secure source of primary cells for BAL systems. Our research focuses on optimizing the safety and functionality of porcine primary hepatocytes during large-scale cultivation. We achieved this by creating GGTA1 knockout pigs through one-step delivery of CRISPR/Cas9 to pig zygotes via oviduct injection of rAAV, and enhancing hepatocyte viability and function by co-culturing hepatocytes with Roof plate-specific spondin 1 overexpressing HUVECs (R-HUVECs). Using a Rocker culture system, approximately 1010 primary porcine hepatocytes and R-HUVECs rapidly formed organoids with a diameter of 92.1 ± 28.1 µm within 24 h. These organoids not only maintained excellent functionality but also supported partial hepatocyte self-renewal during long-term culture over 28 days. Gene-edited primary porcine hepatocyte organoids will significantly advance the applications of hepatocyte transplantation and BAL systems.

Perovskites as Multiphoton Fluorescence Contrast Agents for In Vivo Imaging.

Multiphoton fluorescence microscopy is a powerful tool for imaging and exploring biological tissue at subcellular spatial resolution while minimizing photobleaching and autofluorescence. For optimal performance in multiphoton microscopy, materials exhibiting a large multiphoton absorption cross section (σn) and fluorescence quantum yield are desired. Notably, perovskite nanocrystals (CsPbX3, PNCs) exhibit exceptionally large two-, three-, up to five photon absorption cross section (σ2 ∼ 106 GM, σ3 ∼ 10-73 cm6s2 photon-2, σ5 ∼ 10-136 cm10s4 photon-4), along with near unity fluorescence quantum yield, making them desirable for deep tissue applications. Here, we employed PNCs as contrast agents to image mesenchymal stromal cells in a living mouse. The PNCs were stabilized by encapsulating them in a SiO2 matrix (∼60-70 nm in diameter), offering versatility for subsequent surface modification to target specific biological entities for both diagnostic and therapeutic applications. Multiphoton imaging of PNCs offers substantial benefits for dynamic tracking of cells in deep tissue, such as in understanding immune cell migration and other biological processes in both healthy and diseased tissues.

Dietary incorporation of magnetic bentonite nanocomposite: impacts on in vitro fermentation pattern, nutrient digestibility, and growth performance of Baluchi male lambs.

Background: Incorporation of bentonite into the diets of ruminants can be helpful to maximize their performance. Modifying the structure of bentonite to nano and nanocomposite has improved their chemical stability and physicochemical properties, enhancing adsorption, absorption, and cation exchange capacity. Aims: This study aimed to assess the effect of magnetic bentonite nanocomposite (MBNC) on in vivo and in vitro fermentation process patterns, nutrient digestibility, and growth performance of Baluchi male lambs. Methods: Effects of control (basal diet), natural bentonite (NB) (10 g/kg dry matter (DM)), processed bentonite (PB) (5 and 10 g/kg DM basal diet), and MBNC (5 and 10 g/kg DM basal diet) on gas production (GP), and the fermentation process were determined using in vitro GP technique. For the in vivo experiment, 20 Baluchi male lambs were used with 4 experimental treatments: control, NB (5 g/kg DM), PB (5 g/kg DM), and MBNC (5 g/kg DM) and 5 replications in a completely randomized design for 60 consecutive days. Results: The potential for GP and its fractional rates were significantly decreased and increased in MBNC, respectively (P<0.01). The lowest cumulative GP, and CH4 yield were observed in MBNC (P<0.05). In vitro, DM and organic matter (OM) digestibility and all fermentation parameters increased with the addition of two levels of MBNC to the culture medium (P<0.01). Except for feed conversion ratio (FCR), other growth performance parameters increased with the addition of MBNC to the diet (P<0.01). The ruminal pH, total volatile fatty acids (TVFA), acetate, and propionate significantly increased when MBNC incorporated to the diet (P<0.01). The NH3-N (P<0.001) was significantly decreased in MBNC. The bentonite supplementation decreased acetate to propionate (P=0.001) compared to the control. Conclusion: Adding MBNC at the 5 g/kg diet DM level can be used as a useful supplement to optimize rumen fermentation pattern, reduce methane production, and increase lamb performance.

Effects of Latilactobacillus sakei LZ217 on Gastric Mucosal Colonization, Metabolic Interference, and Urease Expression in Helicobacter pylori Infection.

Emerging evidence suggests differential antagonism of lactic acid-producing bacteria (LAB) to Helicobacter pylori, posing challenges to human health and food safety due to unclear mechanisms. This study assessed 21 LAB strains from various sources on H. pylori growth, urease activity, and coaggregation. Composite scoring revealed that Latilactobacillus sakei LZ217, derived from fresh milk, demonstrates strong inhibitory effects on both H. pylori growth and urease activity. L. sakei LZ217 significantly reduced H. pylori adherence of gastric cells in vitro, with inhibition ratios of 47.62%. Furthermore, in vivo results showed that L. sakei LZ217 alleviated H. pylori-induced gastric mucosa damage and inflammation in mice. Metabolomic exploration revealed metabolic perturbations in H. pylori induced by L. sakei LZ217, including reduced amino acid levels (e.g., isoleucine, leucine, glutamate, aspartate, and phenylalanine) and impaired carbohydrate and nucleotide synthesis, contributing to the suppression of ureA (28.30%), ureE (84.88%), and ureF (59.59%) expressions in H. pylori. This study underscores the efficacy of LAB against H. pylori and highlights metabolic pathways as promising targets for future interventions against H. pylori growth and colonization.

Preclinical evidence of the anti-inflammatory effect and toxicological safety of aryl-cyclohexanone in vivo.

BACKGROUND: Respiratory distress syndrome is a complex inflammatory condition defined by the presence of acute hypoxemia and cellular infiltration with diffuse alveolar injury following a tissue injury, such as acute lung injury. The inflammatory process involved in this pathology is a defense mechanism of the body against infectious agents and/or tissue injuries. However, when the condition is not reversed, it becomes a significant cause of tissue damage, sometimes leading to loss of function of the affected organ. Therefore, it is essential to understand the mechanisms underlying inflammation, as well as the development of new therapeutic agents that reduce inflammatory damage in these cases. Aryl-cyclohexanone derivatives have previously shown significant anti-inflammatory activity linked to an immunomodulatory capacity in vitro and may be good candidates for therapies in which inflammation plays a central role. METHODS: Was evaluated the anti-inflammatory capacity of a synthesized molecule aryl-cyclohexanone in the murine model of lipopolysaccharide (LPS)-induced acute lung injury. The assessment of acute oral toxicity follows the Organization for Economic Co-operation and Development (OECD) guideline 423. RESULTS: The results demonstrated that the studied molecule protects against LPS-induced inflammation. We observed a decrease in the migration of total and differential leukocytes to the bronchoalveolar lavage fluid (BALF), in addition to a reduction in exudation, myeloperoxidase (MPO) activity, nitric oxide metabolites, and the secretion of pro-inflammatory cytokines (alpha tumor necrosis factors [TNF-α], interleukin-6 [IL-6], interferon-gamma [IFN-γ], and monocyte chemoattractant protein-1 [MCP-1]). Finally, aryl cyclohexanone did not show signs of acute oral toxicity (OECD 423). CONCLUSIONS: The results prove our hypothesis that aryl-cyclohexanone is a promising molecule for developing a new, safe anti-inflammatory drug.

Contribution of Continued Dermal Exposure of PFAS-Containing Sunscreens to Internal Exposure: Extrapolation from In Vitro and In Vivo Tests to Physiologically Based Toxicokinetic Models.

Per- and polyfluoroalkyl substances (PFASs) are widely present in sunscreen products as either active ingredients or impurities. They may penetrate the human skin barrier and then pose potential health risks. Herein, we aimed to develop a physiologically based toxicokinetic (PBTK) model capable of predicting the body loading of PFASs after repeated, long-term dermal application of commercial sunscreens. Ten laboratory-prepared sunscreens, generally falling into two categories of water-in-oil (W/O) and oil-in-water (O/W) sunscreens, were subject to in vitro percutaneous penetration test to assess the impacts of four sunscreen ingredients on PFAS penetration. According to the results, two sunscreen formulas representing W/O and O/W types that mostly enhanced PFAS dermal absorption were then selected for a subsequent 30 day in vivo exposure experiment in mice. PBTK models were successfully established based on the time-dependent PFAS concentrations in mouse tissues (R2 = 0.885-0.947) and validated through another 30 day repeated exposure experiment in mice using two commercially available sunscreens containing PFASs (R2 = 0.809-0.835). The PBTK model results suggest that applying sunscreen of the same amount on a larger skin area is more conducive to PFAS permeation, thus enhancing the exposure risk. This emphasizes the need for caution in practical sunscreen application scenarios, particularly during the summer months.

Refining urine collection in mice: Development of an innovative urine collection device.

Urine collection can be challenging in studies involving small rodents like mice, as the actual methods of collection are anxiogenic and constrain animal welfare while having high variability in the volume of urine collected. To improve the current methods and eventually reduce the impact on the well-being of mice, we developed an innovative 3D-printed urine collection device (UCD). This two-compartment UCD is shaped to fit in classical husbandry cages and allows urine collection by spontaneous urination from two mice housed in their own cage without cross-contamination while enabling potential social interactions. We used our UCD to study the evolution of urinary parameters related to renal functions in a model of antibody-mediated chronic kidney disease. Overall, we report here a time-saving and affordable method for urine collection providing a large amount of uncontaminated urine and which we believe may improve animal welfare in comparison with other methods.

Harmony in nature's elixir: a comprehensive exploration of ethanol and nano-formulated extracts from Passiflora incarnata leaves: unveiling in vitro cytotoxicity, acute and sub-acute toxicity profiles in Swiss albino mice.

We analyzed the toxic effect of the ethanolic extract of Passiflora incarnata (EEP) and its nanoformulation (N-EEP) in the in vitro and in vivo models (zebrafish embryos and Swiss albino mice). The EEP composition was verified by phytochemical and GC-MS analysis. The synthesized N-EEP was characterized using UV-visible spectroscopy and scanning electron microscopy. In vitro results showed both EEP and N-EEP have a dose-dependent effect in L132 cells (normal embryonic lung cells). In zebrafish embryos, no developmental changes were observed for both EEP and N-EEP at 200 µg/ml. The acute and sub-acute toxicity of EEP and N-EEP was identified by oral administration in Swiss albino mice. A single-day oral dose of EEP and N-EEP at different concentrations was administered for acute toxicity, and changes in body weight, food, water intake, temperature, respiration rate, skin color changes, and eye color till 72 h was observed. In a sub-acute toxicity study, 28 days oral administration of different concentrations of EEP and N-EEP was done. Hematological analysis, serum hepatic biochemical parameter analysis, and histopathological analysis for the liver, kidney, spleen, intestine, and heart were performed. The results indicated that lower than 600 mg/kg of EEP and N-EEP can safely be used for the remediation of a spectrum of diseases.

Deep learning method with integrated invertible wavelet scattering for improving the quality of in vivo cardiac DTI.

Objective Respiratory motion, cardiac motion, and inherently low signal-to-noise ratio (SNR) are major limitations of in vivo cardiac diffusion tensor imaging (DTI). We propose a novel enhancement method that uses unsupervised learning based invertible wavelet scattering (IWS) to improve the quality of in vivo cardiac DTI. Approach Our method starts by extracting nearly transformation-invariant features from multiple cardiac diffusion-weighted (DW) image acquisitions using multi-scale wavelet scattering (WS). The relationship between the WS coefficients and DW images is learned through a multiscale encoder and a decoder network. Using the trained encoder, the deep features of WS coefficients of multiple DW image acquisitions are further extracted and then fused using an average rule. Finally, using the fused WS features and trained decoder, the enhanced DW images are derived. Main Results We evaluated the performance of the proposed method by comparing it with several methods on three in vivo cardiac DTI datasets in terms of SNR, contrast to noise ratio (CNR), fractional anisotropy (FA), mean diffusivity (MD), and helix angle (HA). Compared to the best comparison method, SNR/CNR of diastolic, gastric peristalsis influenced, and end systolic DW images were improved by 1%/16%, 5%/6%, and 56%/30%, respectively. The approach also yielded consistent FA and MD values and more coherent helical fiber structures than the comparison methods used in this work. Significance The ablation results verify that using the transformation-invariant and noise-robust wavelet scattering features enables effective exploration of useful information from limited data. This provides a potential means to alleviate the dependence of the fusion results on the number of repeated acquisitions, which is beneficial for dealing with noise and residual motion issues simultaneously, thereby improving the quality of in vivo cardiac DTI.

Recent Advances in DNA-Based Nanoprobes for In vivo MiRNA Imaging.

As a post transcriptional regulator of gene expression, miRNA is closely related to many major human diseases, especially cancer. Therefore, its precise detection is very important for disease diagnosis and treatment. With the advancement of fluorescent dye and imaging technology, the focus has shifted from in vitro microRNAs (miRNA) detection to in vivo miRNA imaging. This concept review summarizes signal amplification strategies including DNAzyme catalytic reaction, hybrid chain reaction (HCR), catalytic hairpin assembly (CHA) to enhance detection signal of lowly expressed miRNAs; external stimuli of ultraviolet (UV) light or near-infrared region (NIR) light, and internal stimuli such as adenosine triphosphate (ATP), glutathione (GSH), protease and cell membrane protein to prevent nonspecific activation for the avoidance of false positive signal; and the development of fluorescent probes with emission in NIR for in vivo miRNA imaging; as well as rare earth nanoparticle based the second near-infrared window (NIR-II) nanoprobes with excellent tissue penetration and depth for in vivo miRNA imaging. The concept review also indicated current challenges for in vivo miRNA imaging including the dynamic monitoring of miRNA expression change and simultaneous in vivo imaging of multiple miRNAs.

Improvements in precision and accuracy of complex- relative to real-domain linear combination model spectral fitting not necessarily recovered by zero filling.

Although the information obtained from in vivo proton magnetic resonance spectroscopy (1H MRS) presents a complex-valued spectrum, spectral quantification generally employs linear combination model (LCM) fitting using the real spectrum alone. There is currently no known investigation comparing fit results obtained from LCM fitting over the full complex data versus the real data and how these results might be affected by common spectral preprocessing procedure zero filling. Here, we employ linear combination modeling of simulated and measured spectral data to examine two major ideas: first, whether use of the full complex rather than real-only data can provide improvements in quantification by linear combination modeling and, second, to what extent zero filling might influence these improvements. We examine these questions by evaluating the errors of linear combination model fits in the complex versus real domains against three classes of synthetic data: simulated Lorentzian singlets, simulated metabolite spectra excluding the baseline, and simulated metabolite spectra including measured in vivo baselines. We observed that complex fitting provides consistent improvements in fit accuracy and precision across all three data types. While zero filling obviates the accuracy and precision benefit of complex fitting for Lorentzian singlets and metabolite spectra lacking baselines, it does not necessarily do so for complex spectra including measured in vivo baselines. Overall, performing linear combination modeling in the complex domain can improve metabolite quantification accuracy relative to real fits alone. While this benefit can be similarly achieved via zero filling for some spectra with flat baselines, this is not invariably the case for all baseline types exhibited by measured in vivo data.

In vivo fitness of sul gene-dependent sulfonamide-resistant Escherichia coli in the mammalian gut.

The widespread sulfonamide resistance genes sul1, sul2, and sul3 in food and gut bacteria have attracted considerable attention. In this study, we assessed the in vivo fitness of sul gene-dependent sulfonamide-resistant Escherichia coli, using a murine model. High fitness costs were incurred for sul1 and sul3 gene-dependent E. coli strains in vivo. A fitness advantage was found in three of the eight mice after intragastric administration of sul2 gene-dependent E. coli strains. We isolated three compensatory mutant strains (CMSs) independently from three mice that outcompeted the parent strain P2 in vivo. Whole-genome sequencing revealed seven identical single nucleotide polymorphism (SNP) mutations in the three CMSs compared with strain P2, an additional SNP mutation in strain S2-2, and two additional SNP mutations in strain S2-3. Furthermore, tandem mass tag-based quantitative proteomic analysis revealed abundant differentially expressed proteins (DEPs) in the CMSs compared with P2. Of these, seven key fitness-related DEPs distributed in two-component systems, galactose and tryptophan metabolism pathways, were verified using parallel reaction monitoring analysis. The DEPs in the CMSs influenced bacterial motility, environmental stress tolerance, colonization ability, carbohydrate utilization, cell morphology maintenance, and chemotaxis to restore fitness costs and adapt to the mammalian gut environment.IMPORTANCESulfonamides are traditional synthetic antimicrobial agents used in clinical and veterinary medical settings. Their long-term excessive overuse has resulted in widespread microbial resistance, limiting their application for medical interventions. Resistance to sulfonamides is primarily conferred by the alternative genes sul1, sul2, and sul3 encoding dihydropteroate synthase in bacteria. Studying the potential fitness cost of these sul genes is crucial for understanding the evolution and transmission of sulfonamide-resistant bacteria. In vitro studies have been conducted on the fitness cost of sul genes in bacteria. In this study, we provide critical insights into bacterial adaptation and transmission using an in vivo approach.

ChEC-Seq: A Comprehensive Guide for Scalable and Cost-Efficient Genome-Wide Profiling in Saccharomyces cerevisiae.

Chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq) is a profiling method for protein-DNA interactions that can detect binding locations in vivo, does not require antibodies or fixation, and provides genome-wide coverage at near nucleotide resolution.The core of this method is an MNase fusion of the target protein, which allows it, when triggered by calcium exposure, to cut DNA at its binding sites and to generate small DNA fragments that can be readily separated from the rest of the genome and sequenced.Improvements since the original protocol have increased the ease, lowered the costs, and multiplied the throughput of this method to enable a scale and resolution of experiments not available with traditional methods such as ChIP-seq. This method describes each step from the initial creation and verification of the MNase-tagged yeast strains, over the ChEC MNase activation and small fragment purification procedure to the sequencing library preparation. It also briefly touches on the bioinformatic steps necessary to create meaningful genome-wide binding profiles.

Assessing In Vivo Bacterial Extracellular Vesicle (BEV) Biodistribution Using Fluorescent Lipophilic Membrane Stains.

Bacterial extracellular vesicles (BEVs) are nano-size vesicles containing a cargo of bioactive molecules that can play key roles in microbe-microbe and microbe-host interactions. In tracking their biodistribution in vivo, BEVs can cross several physical host barriers including the intestinal epithelium, vascular endothelium, and blood-brain-barrier (BBB) to ultimately accumulate in tissues such as the liver, lungs, spleen, and the brain. This tissue-specific dissemination has been exploited for the delivery of biomolecules such as vaccines for mucosal delivery. Although numerous strategies for labeling and tracking BEVs have been described, most have constraints that impact on interpreting in vivo bioimaging patterns. Here, we describe a general method for labeling BEVs using lipophilic fluorescent membrane stains which can be adopted by non-expert users. We also describe how the procedure can be used to overcome potential limitations. Furthermore, we outline methods of quantitative ex vivo tissue imaging that can be used to evaluate BEV organ trafficking.