PhaseFIT: live-organoid phase-fluorescent image transformation via generative AI.

Organoid models have provided a powerful platform for mechanistic investigations into fundamental biological processes involved in the development and function of organs. Despite the potential for image-based phenotypic quantification of organoids, their complex 3D structure, and the time-consuming and labor-intensive nature of immunofluorescent staining present significant challenges. In this work, we developed a virtual painting system, PhaseFIT (phase-fluorescent image transformation) utilizing customized and morphologically rich 2.5D intestinal organoids, which generate virtual fluorescent images for phenotypic quantification via accessible and low-cost organoid phase images. This system is driven by a novel segmentation-informed deep generative model that specializes in segmenting overlap and proximity between objects. The model enables an annotation-free digital transformation from phase-contrast to multi-channel fluorescent images. The virtual painting results of nuclei, secretory cell markers, and stem cells demonstrate that PhaseFIT outperforms the existing deep learning-based stain transformation models by generating fine-grained visual content. We further validated the efficiency and accuracy of PhaseFIT to quantify the impacts of three compounds on crypt formation, cell population, and cell stemness. PhaseFIT is the first deep learning-enabled virtual painting system focused on live organoids, enabling large-scale, informative, and efficient organoid phenotypic quantification. PhaseFIT would enable the use of organoids in high-throughput drug screening applications.

Physical properties of food or bile redirection do not contribute to the intestinal adaptations after Roux-en-Y Gastric Bypass in rats.

Objective: Metabolic and morphological adaptations of the intestine have been suggested to play a role in the various therapeutic benefits of Roux-en-Y Gastric Bypass (RYGB) surgery. However, the precise underlying mechanisms remain unclear. In this study, the effects of physical properties of ingested food and redirection of biliopancreatic secretions on intestinal remodeling were investigated in RYGB operated rats. Methods: RYGB employing two different Roux Limb (RL) lengths was performed on high fat diet induced obese rats. Post-operatively, rats were fed either Solid or isocaloric Liquid diets. Metabolic and morphological remodeling of intestine was compared across both diet forms (Solid and Liquid diets) and surgical models (Short RL and Long RL). Results: RYGB surgery in rats induced weight loss and improved glucose tolerance which was independent of physical properties of ingested food and biliopancreatic secretions. Intestinal glucose utilization after RYGB was not determined by either food form or biliopancreatic secretions. The GLUT-1 expression in RL was not influenced by physical properties of food. Furthermore, both physical properties of food and biliopancreatic secretions showed no effects on intestinal morphological adaptations after RYGB. Conclusion: Results of this study demonstrate that physical properties of food and bile redirection are not major determinants of intestinal remodeling after RYGB in rats.

Stiffness Restricts the Stemness of the Intestinal Stem Cells and Skews Their Differentiation Toward Goblet Cells.

BACKGROUND & AIMS: Fibrosis and tissue stiffening are hallmarks of inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). METHODS: We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs in vivo. RESULTS: We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+-proliferating cells. Conversely, cells expressing the stem cell marker, olfactomedin-4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of olfactomedin-4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs toward goblet cells. Furthermore, analysis of colon samples from murine colitis models and patients with IBD demonstrated cellular and molecular remodeling reminiscent of those observed in vitro. CONCLUSIONS: Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.

Interplay between nematic and cholesteric interactions in self-consistent field theory.

Chirality is a design feature of a number of biomolecules (e.g., collagen). In these molecules, cholesteric (chiral-nematic) behavior emerges from a combination of the tendency for the biopolymers to align (nematic interactions) and for the alignment direction to change with position, rotating around an axis normal to the alignment direction. This paper presents self-consistent field theory (SCFT) of chiral-nematic polymers, which takes into account polymer flexibility and the orientational degrees of freedom of polymer segments. Using the resulting SCFT, we construct a phase diagram showing regions of stability for isotropic, nematic, and cholesteric phases. Furthermore, we find that nematic interactions can stabilize the cholesteric phase, pushing the isotropic-cholesteric phase transition to lower cholesteric interaction strength, until the isotropic-nematic-cholesteric triple point is reached.

Rewiring of the Liver Transcriptome across Multiple Time-Scales Is Associated with the Weight Loss-Independent Resolution of NAFLD Following RYGB.

Roux-en-Y gastric bypass (RYGB) surgery potently improves obesity and a myriad of obesity-associated co-morbidities including type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). Time-series omics data are increasingly being utilized to provide insight into the mechanistic underpinnings that correspond to metabolic adaptations in RYGB. However, the conventional computational biology methods used to interpret these temporal multi-dimensional datasets have been generally limited to pathway enrichment analysis (PEA) of isolated pair-wise comparisons based on either experimental condition or time point, neither of which adequately capture responses to perturbations that span multiple time scales. To address this, we have developed a novel graph network-based analysis workflow designed to identify modules enriched with biomolecules that share common dynamic profiles, where the network is constructed from all known biological interactions available through the Kyoto Encyclopedia of Genes and Genomes (KEGG) resource. This methodology was applied to time-series RNAseq transcriptomics data collected on rodent liver samples following RYGB, and those of sham-operated and weight-matched control groups, to elucidate the molecular pathways involved in the improvement of as NAFLD. We report several network modules exhibiting a statistically significant enrichment of genes whose expression trends capture acute-phase as well as long term physiological responses to RYGB in a single analysis. Of note, we found the HIF1 and P53 signaling cascades to be associated with the immediate and the long-term response to RYGB, respectively. The discovery of less intuitive network modules that may have gone overlooked with conventional PEA techniques provides a framework for identifying novel drug targets for NAFLD and other metabolic syndrome co-morbidities.

Mechanoscopy: A Novel Device and Procedure for in vivo Detection of Chronic Colitis in Mice.

BACKGROUND: Gut stiffening caused by fibrosis plays a critical role in the progression of inflammatory bowel disease (IBD) and colon cancer. Previous studies have characterized the biomechanical response of healthy and pathological gut, with most measurements obtained ex vivo. METHODS: Here, we developed a device and accompanying procedure for in vivo quantification of gut stiffness, termed mechanoscopy. Mechanoscopy includes a flexible balloon catheter, pressure sensor, syringe pump, and control system. The control system activates the balloon catheter and performs automated measurements of the gut stress-strain biomechanical response. RESULTS: A gut stiffness index (GSI) is identified based on the slope of the obtained stress-strain response. Using a colitis mouse model, we demonstrated that GSI positively correlates with the extent of gut fibrosis, the severity of mucosal damage, and the infiltration of immune cells. Furthermore, a critical strain value is suggested, and GSI efficiently detects pathological gut fibrotic stiffening when the strain exceeds this value. CONCLUSIONS: Based on these results, we envision that mechanoscopy and GSI will facilitate the clinical diagnosis of IBD.

Here, we present a novel procedure/device, termed mechanoscopy, which we have demonstrated to accurately detect and differentiate between fibrosis and inflammation in rodent models of colitis. Thus, mechanoscopy offers a translationally relevant approach for ultrasensitive and minimally invasive IBD diagnosis.

Self-consistent field theory of chiral nematic worm-like chains.

Many macromolecules of biological and technological interest are both chiral and semi-flexible. DNA and collagen are good examples. Such molecules often form chiral nematic (or cholesteric) phases, as is well-documented in collagen and chitin. This work presents a method for studying cholesteric phases in the highly successful self-consistent field theory of worm-like chains, offering a new way of studying many biologically relevant molecules. The method involves an effective Hamiltonian with a chiral term inspired by the Oseen-Frank (OF) model of liquid crystals. This method is then used to examine the formation of cholesteric phases in chiral-nematic worm-like chains as a function of polymer flexibility, as well as the optimal cholesteric pitch and distribution of polymer segment orientations. Our approach not only allows for the determination of the isotropic-cholesteric transition and segment distributions, beyond what the OF model promises, but also explicitly incorporates polymer flexibility into the study of the cholesteric phase, offering a more complete understanding of the behavior of semiflexible chiral-nematic polymers.

Nematic ordering of worm-like polymers near an interface.

The phase behavior of semi-flexible polymers is integral to various contexts, from materials science to biophysics, many of which utilize or require specific confinement geometries as well as the orientational behavior of the polymers. Inspired by collagen assembly, we study the orientational ordering of semi-flexible polymers, modeled as Maier-Saupe worm-like chains, using self-consistent field theory. We first examine the bulk behavior of these polymers, locating the isotropic-nematic transition and delineating the limit of stability of the isotropic and nematic phases. This effort explains how nematic ordering emerges from the isotropic phase and offers insight into how different (e.g., mono-domain vs multi-domain) nematic phases form. We then clarify the influence of planar confinement on the nematic ordering of the polymers. We find that while the presence of a single confining wall does not shift the location of nematic transition, it aligns the polymers in parallel to the wall; above the onset of nematic transition, this preference tends to propagate into the bulk phase. Introducing a second, perpendicular, wall leads to a nematic phase that is parallel to both walls, allowing the ordering direction to be uniquely set by the geometry of the experimental setup. The advantage of wall-confinement is that it can be used to propagate mono-domain nematic phases into the bulk phase.

A theoretical model of collective cell polarization and alignment.

Collective cell polarization and alignment play important roles in tissue morphogenesis, wound healing and cancer metastasis. How cells sense the direction and position in these processes, however, has not been fully understood. Here we construct a theoretical model based on describing cell layer as a nemato-elastic medium, by which the cell polarization, cell alignment and cell active contraction are explicitly expressed as functions of components of the nematic order parameter. To determine the order parameter we derive two sets of governing equations, one for the force equilibrium of the system, and the other for the minimization of the system's free energy including the energy of cell polarization and alignment. By solving these coupled governing equations, we can predict the effects of substrate stiffness, geometries of cell layers, external forces and myosin activity on the direction- and position-dependent cell aspect ratio and cell orientation. Moreover, the axisymmetric problem with cells on a ring-like pattern is solved analytically, and the analytical solution for cell aspect ratio are governed by parameter groups which include the stiffness of the cell and the substrate, the strength of myosin activity and the external forces. Our predictions of the cell aspect ratio and orientation are generally comparable to experimental observations. These results show that the pattern of cell polarization is determined by the anisotropic degree of active contractile stress, and suggest a stress-driven polarization mechanism that enables cells to sense their spatial positions to develop direction- and position-dependent behavior. This, in turn, sheds light on the ways to control pattern formation in tissue engineering for potential biomedical applications.

Rejuvenation of aged rat skin with pulsed electric fields.

The demand for skin rejuvenation procedures has progressively increased in the past decade. Additionally, clinical trials have shown that current therapies might cause downtime and side effects in patients including prolonged erythema, scarring, and dyspigmentation. The goal of this study was to explore the effect of partial irreversible electroporation (pIRE) with pulsed electric fields in aged skin rejuvenation as a novel, non-invasive skin resurfacing technique. In this study, we used an experimental model of aged rats. We showed that treatment with pIRE promoted keratinocyte proliferation and blood flow in aged rat skin. We also found significant evidence indicating that pIRE reformed the dermal extracellular matrix (ECM). Both the collagen protein and fibre density in aged skin increased after pIRE administration. Furthermore, using an image-processing algorithm, we found that the collagen fibre orientation in the histological sections did not change, indicating a lack of scar formation in the treated areas. The results showed that pIRE approach could effectively stimulate keratinocyte proliferation, ECM synthesis, and angiogenesis in an aged rat model.

Nonalcoholic fatty liver disease and gastric bypass surgery regulate serum and hepatic levels of pyruvate kinase isoenzyme M2.

Treatment of nonalcoholic fatty liver disease (NAFLD) focuses on the underlying metabolic syndrome, and Roux-en-Y gastric bypass surgery (RYGB) remains one of the most effective options. In rodents and human patients, RYGB induces an increase in the gene and protein expression levels of the M2 isoenzyme of pyruvate kinase (PKM2) in the jejunum. Since PKM2 can be secreted in the circulation, our hypothesis was that the circulating levels of PKM2 increase after RYGB. Our data, however, revealed an unexpected finding and a potential new role of PKM2 for the natural history of metabolic syndrome and NAFLD. Contrary to our initial hypothesis, RYGB-treated patients had decreased PKM2 blood levels compared with a well-matched group of patients with severe obesity before RYGB. Interestingly, PKM2 serum concentration correlated with body mass index before but not after the surgery. This prompted us to evaluate other potential mechanisms and sites of PKM2 regulation by the metabolic syndrome and RYGB. We found that in patients with NAFLD and nonalcoholic steatohepatitis (NASH), the liver had increased PKM2 expression levels, and the enzyme appears to be specifically localized in Kupffer cells. The study of murine models of metabolic syndrome and NASH replicated this pattern of expression, further suggesting a metabolic link between hepatic PKM2 and NAFLD. Therefore, we conclude that PKM2 serum and hepatic levels increase in both metabolic syndrome and NAFLD and decrease after RYGB. Thus, PKM2 may represent a new target for monitoring and treatment of NAFLD.

Metabolomic Modularity Analysis (MMA) to Quantify Human Liver Perfusion Dynamics.

Large-scale -omics data are now ubiquitously utilized to capture and interpret global responses to perturbations in biological systems, such as the impact of disease states on cells, tissues, and whole organs. Metabolomics data, in particular, are difficult to interpret for providing physiological insight because predefined biochemical pathways used for analysis are inherently biased and fail to capture more complex network interactions that span multiple canonical pathways. In this study, we introduce a nov-el approach coined Metabolomic Modularity Analysis (MMA) as a graph-based algorithm to systematically identify metabolic modules of reactions enriched with metabolites flagged to be statistically significant. A defining feature of the algorithm is its ability to determine modularity that highlights interactions between reactions mediated by the production and consumption of cofactors and other hub metabolites. As a case study, we evaluated the metabolic dynamics of discarded human livers using time-course metabolomics data and MMA to identify modules that explain the observed physiological changes leading to liver recovery during subnormothermic machine perfusion (SNMP). MMA was performed on a large scale liver-specific human metabolic network that was weighted based on metabolomics data and identified cofactor-mediated modules that would not have been discovered by traditional metabolic pathway analyses.

Regulation of Energy Homeostasis After Gastric Bypass Surgery.

The obesity epidemic continues to escalate each year in the United States more than anywhere else in the world. The existing pharmaceutical and other nonsurgical treatments for morbid obesity produce suboptimal physiologic outcomes compared with those of Roux-en-Y gastric bypass (RYGB) surgery. RYGB has been the gold standard of bariatric surgery because the beneficial long-term outcomes, which include sustainable weight loss and type 2 diabetes mellitus (T2DM) resolution, are far superior to those obtained with other bariatric surgeries. However, the current understanding of RYGB's mechanisms of actions remains limited and incomplete. There is an urgent need to understand these mechanisms as gaining this knowledge may lead to the development of innovative and less invasive procedures and/or medical devices, which can mirror the favorable outcomes of RYGB surgery. In this review, we highlight current observations of the metabolic and physiologic events following RYGB, with a particular focus on the role of the anatomical reconfiguration of the gastrointestinal tract after RYGB.

Multi-omic network-based interrogation of rat liver metabolism following gastric bypass surgery featuring SWATH proteomics.

Morbidly obese patients often elect for Roux-en-Y gastric bypass (RYGB), a form of bariatric surgery that triggers a remarkable 30% reduction in excess body weight and reversal of insulin resistance for those who are type II diabetic. A more complete understanding of the underlying molecular mechanisms that drive the complex metabolic reprogramming post-RYGB could lead to innovative non-invasive therapeutics that mimic the beneficial effects of the surgery, namely weight loss, achievement of glycemic control, or reversal of non-alcoholic steatohepatitis (NASH). To facilitate these discoveries, we hereby demonstrate the first multi-omic interrogation of a rodent RYGB model to reveal tissue-specific pathway modules implicated in the control of body weight regulation and energy homeostasis. In this study, we focus on and evaluate liver metabolism three months following RYGB in rats using both SWATH proteomics, a burgeoning label free approach using high resolution mass spectrometry to quantify protein levels in biological samples, as well as MRM metabolomics. The SWATH analysis enabled the quantification of 1378 proteins in liver tissue extracts, of which we report the significant down-regulation of Thrsp and Acot13 in RYGB as putative targets of lipid metabolism for weight loss. Furthermore, we develop a computational graph-based metabolic network module detection algorithm for the discovery of non-canonical pathways, or sub-networks, enriched with significantly elevated or depleted metabolites and proteins in RYGB-treated rat livers. The analysis revealed a network connection between the depleted protein Baat and the depleted metabolite taurine, corroborating the clinical observation that taurine-conjugated bile acid levels are perturbed post-RYGB.

Human Corneal Fibroblast Pattern Evolution and Matrix Synthesis on Mechanically Biased Substrates.

In a fibroblast colony model of corneal stromal development, we asked how physiological tension influences the patterning dynamics of fibroblasts and the orientation of deposited extracellular matrix (ECM). Using long-term live-cell microscopy, enabled by an optically accessible mechanobioreactor, a primary human corneal fibroblast colony was cultured on three types of substrates: a mechanically biased, loaded, dense, disorganized collagen substrate (LDDCS), a glass coverslip, and an unloaded, dense, disorganized collagen substrate (UDDCS). On LDDCS, fibroblast orientation and migration along a preferred angle developed early, cell orientation was correlated over long distances, and the colony pattern was stable. On glass, fibroblast orientation was poorly correlated, developed more slowly, and colony patterns were metastable. On UDDCS, cell orientation was correlated over shorter distances compared with LDDCS specimens. On all substrates, the ECM pattern reflected the cell pattern. In summary, mechanically biasing the collagen substrate altered the early migration behavior of individual cells, leading to stable emergent cell patterning, which set the template for newly synthesized ECM.

Metabolic profiling during ex vivo machine perfusion of the human liver.

As donor organ shortages persist, functional machine perfusion is under investigation to improve preservation of the donor liver. The transplantation of donation after circulatory death (DCD) livers is limited by poor outcomes, but its application may be expanded by ex vivo repair and assessment of the organ before transplantation. Here we employed subnormothermic (21 °C) machine perfusion of discarded human livers combined with metabolomics to gain insight into metabolic recovery during machine perfusion. Improvements in energetic cofactors and redox shifts were observed, as well as reversal of ischemia-induced alterations in selected pathways, including lactate metabolism and increased TCA cycle intermediates. We next evaluated whether DCD livers with steatotic and severe ischemic injury could be discriminated from 'transplantable' DCD livers. Metabolomic profiling was able to cluster livers with similar metabolic patterns based on the degree of injury. Moreover, perfusion parameters combined with differences in metabolic factors suggest variable mechanisms that result in poor energy recovery in injured livers. We conclude that machine perfusion combined with metabolomics has significant potential as a clinical instrument for the assessment of preserved livers.

Surgical models of Roux-en-Y gastric bypass surgery and sleeve gastrectomy in rats and mice.

Bariatric surgery is the only definitive solution currently available for the present obesity pandemic. These operations typically involve reconfiguration of gastrointestinal tract anatomy and impose profound metabolic and physiological benefits, such as substantially reducing body weight and ameliorating type II diabetes. Therefore, animal models of these surgeries offer unique and exciting opportunities to delineate the underlying mechanisms that contribute to the resolution of obesity and diabetes. Here we describe a standardized procedure for mouse and rat models of Roux-en-Y gastric bypass (80-90 min operative time) and sleeve gastrectomy (30-45 min operative time), which, to a high degree, resembles operations in humans. We also provide detailed protocols for both pre- and postoperative techniques that ensure a high success rate in the operations. These protocols provide the opportunity to mechanistically investigate the systemic effects of the surgical interventions, such as regulation of body weight, glucose homeostasis and gut microbiome.

Lymph node fibroblastic reticular cell transplants show robust therapeutic efficacy in high-mortality murine sepsis.

Sepsis is an aggressive inflammatory syndrome and a global health burden estimated to kill 7.3 million people annually. Single-target molecular therapies have not addressed the multiple disease pathways triggered by septic injury. Cell therapies might offer a broader set of mechanisms of action that benefit complex, multifocal disease processes. We describe a population of immune-specialized myofibroblasts derived from lymph node tissue, termed fibroblastic reticular cells (FRCs). Because FRCs have an immunoregulatory function in lymph nodes, we hypothesized that ex vivo-expanded FRCs would control inflammation when administered therapeutically. Indeed, a single injection of ex vivo-expanded allogeneic FRCs reduced mortality in mouse models of sepsis when administered at early or late time points after septic onset. Mice treated with FRCs exhibited lower local and systemic concentrations of proinflammatory cytokines and reduced bacteremia. When administered 4 hours after induction of lipopolysaccharide endotoxemia, or cecal ligation and puncture (CLP) sepsis in mice, FRCs reduced deaths by at least 70%. When administered late in disease (16 hours after CLP), FRCs still conveyed a robust survival advantage (44% survival compared to 0% for controls). FRC therapy was dependent on the metabolic activity of nitric oxide synthase 2 (NOS2) as the primary molecular mechanism of drug action in the mice. Together, these data describe a new anti-inflammatory cell type and provide preclinical evidence for therapeutic efficacy in severe sepsis that warrants further translational study.