Strategies to rescue steatotic livers before transplantation in clinical and experimental studies

The shortage of donor livers has led to an increased use of organs from expanded criteria donors. Included are livers with steatosis, a metabolic abnormality that increases the likelihood of graft complications posttransplantation. After a brief introduction on the etiology, pathophysiology, categories and experimental models of hepatic steatosis, we herein review the methods to rescue steatotic donor livers before transplantation applied in clinical and experimental studies. The methods span the spectrum of encouraging donor weight loss, employing drug therapy, heat shock preconditioning, ischemia preconditioning and selective anesthesia on donors, and the treatment on isolated grafts during preservation. These methods work at different stages of transplantation process, although share similar molecular mechanisms including lipid metabolism stimulation through enzymes or nuclear receptor e.g. , peroxisomal proliferator-activated receptor, or anti-inflammation through suppressing cytokines e.g. , tumor necrosis factor-α, or antioxidant therapies to alleviate oxidative stress. This similarity of molecular mechanisms implies possible future attempts to reinforce each approach by repeating the same treatment approach at several stages of procurement and preservation, as well as utilizing these alternative approaches in tandem.

Mechanical force drives the polarization and orientation of cells

Collective cells are organized to form specific patterns which play important roles in various physiological and pathological processes, such as tissue morphogenesis, wound healing, and cancer invasion. Compared to single cell behaviors, which has been intensively studied from many aspects (cell migration, adhesion, polarization, proliferation, etc.) and at various scales (molecular, subcellular, and cellular), the multiple cell behaviors are relatively less understood, particularly in a quantitative manner. In this paper, we will present our recent studies of collective polarization and orientation of multiple cells through both experimental measurement and theoretical modeling, including those cell behaviors on/in 2D and 3D substrate/tissue. We find that the collective cell behaviors, including polarization, alignment and migration are closely related to local stress states in cell layer or tissue, which demonstrate the crucial roles of mechanical forces in the living organisms. Specifically, the cells prefer to polarize and align along the maximum principal stress in the cell layer, and the aspect ratio of cell increases with the in-plane maximum shear stress, suggesting that the maximum shear stress is the underlying driving force of cell polarization and orientation. This theory of stress-driven cell behaviors of polarization and orientation provides a new perspective for understanding cell behaviors in living organisms and the guideline for tissue engineering in biomedical applications.

Supramolecular hybrid hydrogels as rapidly on-demand dissoluble,self-healing,and biocompatible burn dressings

Despite decades of efforts,state-of-the-art synthetic burn dressings to treat partial-thickness burns are still far from ideal.Current dressings adhere to the wound and necessitate debridement.This work describes the first“supramolecular hybrid hydrogel(SHH)”burn dressing that is biocompatible,self-healable,and on-demand dissoluble for easy and trauma-free removal,prepared by a simple,fast,and scalable method.These SHHs leverage the interactions of a custom-designed cationic copolymer via host-guest chemistry with cucurbit[7]uril and electrostatic interactions with clay nanosheets coated with an anionic polymer to achieve enhanced mechanical properties and fast on-demand dissolution.The SHHs show high mechanical strength(>50 kPa),self-heal rapidly in~1 min,and dissolve quickly(4-6 min)using an amantadine hydrochloride(AH)solution that breaks the supramolecular interactions in the SHHs.Neither the SHHs nor the AH solution has any adverse effects on human dermal fibroblasts or epidermal keratinocytes in vitro.The SHHs also do not elicit any significant cytokine response in vitro.Furthermore,in vivo murine experiments show no immune or inflammatory cell infiltration in the subcutaneous tissue and no change in circulatory cytokines compared to sham controls.Thus,these SHHs present excellent burn dressing candidates to reduce the time of pain and time associated with dressing changes.

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.

Layer-by-layer functionalized nanotube arrays:A versatile microfluidic platform for biodetection

We demonstrate the layer-by-layer(LbL)assembly of polyelectrolyte multilayers(PEM)on three-dimensional nanofiber scaffolds.High porosity(99%)aligned carbon nanotube(CNT)arrays are photolithographically patterned into elements that act as textured scaffolds for the creation of functionally coated(nano)porous materials.Nanometer-scale bilayers of poly(allylamine hydrochloride)/poly(styrene sulfonate)(PAH/SPS)are formed conformally on the individual nanotubes by repeated deposition from aqueous solution in microfluidic channels.Computational and experimental results show that the LbL deposition is dominated by the diffusive transport of the polymeric constituents,and we use this understanding to demonstrate spatial tailoring on the patterned nanoporous elements.A proof-of-principle application,microfluidic bioparticle capture using N-hydroxysuccinimide-biotin binding for the isolation of prostate-specific antigen(PSA),is demonstrated.

Regulation of Torpor in the Gray Mouse Lemur:Transcriptional and Translational Controls and Role of AMPK Signaling

The gray mouse lemur（Microcebus murinus） is one of few primate species that is able to enter daily torpor or prolonged hibernation in response to environmental stresses. With an emerging significance to human health research, lemurs present an optimal model for exploring molecular adaptations that regulate primate hypometabolism. A fundamental challenge is how to effectively regulate energy expensive cellular processes（e.g., transcription and translation） during transitionsto/from torpor without disrupting cellular homeostasis. One such regulatory mechanism is reversible posttranslational modification of selected protein targets that offers fine cellular control without the energetic burden. This study investigates the role of phosphorylation and/or acetylation in regulating key factors involved in energy homeostasis（AMP-activated protein kinase, or AMPK, signaling pathway）, m RNA translation（eukaryotic initiation factor 2a or e IF2 a, eukaryotic initiation factor 4E or e IF4 E, and initiation factor 4E binding protein or 4EBP）, and gene transcription（histone H3） in six tissues of torpid and aroused gray mouse lemurs. Our results indicated selective tissue-specific changes of these regulatory proteins. The relative level of Thr172-phosphorylated AMPKa was significantly elevated in the heart but reduced in brown adipose tissue during daily torpor, as compared to the aroused lemurs, implicating the regulation of AMPK activity during daily torpor in these tissues. Interestingly, the levels of the phosphorylated e IFs were largely unaltered between aroused and torpid animals. Phosphorylation and acetylation of histone H3 were examined as a marker for transcriptional regulation. Compared to the aroused lemurs, level of Ser10-phosphorylated histone H3 decreased significantly in white adipose tissue during torpor, suggesting global suppression of gene transcription. However, a significant increase in acetyl-histone H3 in the heart of torpid lemurs indicated a possible stimulation of transcriptional activity of this tissue. Overall, our study demonstrates that AMPK signaling and posttranslational regulation of selected proteins may play crucial roles in the control of transcription/translation during daily torpor in mouse lemurs.

Induction of Antioxidant and Heat Shock Protein Responses During Torpor in the Gray Mouse Lemur, Microcebus murinus

A natural tolerance of various environmental stresses is typically supported by various cytoprotective mechanisms that protect macromolecules and promote extended viability. Among these are antioxidant defenses that help to limit damage from reactive oxygen species and chaperones that help to minimize protein misfolding or unfolding under stress conditions. To understand the molecular mechanisms that act to protect cells during primate torpor, the present study characterizes antioxidant and heat shock protein（HSP） responses in various organs of control（aroused）and torpid gray mouse lemurs, Microcebus murinus. Protein expression of HSP70 and HSP90 a was elevated to 1.26 and 1.49 fold, respectively, in brown adipose tissue during torpor as compared with control animals, whereas HSP60 in liver of torpid animals was 1.15 fold of that in control（P 〈 0.05）. Among antioxidant enzymes, protein levels of thioredoxin 1 were elevated to 2.19 fold in white adipose tissue during torpor, whereas Cu–Zn superoxide dismutase 1 levels rose to 1.1 fold in skeletal muscle（P 〈 0.05）. Additionally, total antioxidant capacity was increased to 1.6 fold in liver during torpor（P 〈 0.05）, while remaining unchanged in the five other tissues. Overall, our data suggest that antioxidant and HSP responses are modified in a tissue-specific manner during daily torpor in gray mouse lemurs. Furthermore, our data also show that cytoprotective strategies employed during primate torpor are distinct from the strategies in rodent hibernation as reported in previous studies.

Primate Torpor: Regulation of Stress-activated Protein Kinases During Daily Torpor in the Gray Mouse Lemur, Microcebus murinus

Very few selected species of primates are known to be capable of entering torpor. This exciting discovery means that the ability to enter a natural state of dormancy is an ancestral trait among primates and, in phylogenetic terms, is very close to the human lineage. To explore the regulatory mechanisms that underlie primate torpor, we analyzed signal transduction cascades to discover those involved in coordinating tissue responses during torpor. The responses of mitogen-activated protein kinase（MAPK） family members to primate torpor were compared in six organs of control（aroused） versus torpid gray mouse lemurs, Microcebus murinus. The proteins examined include extracellular signal-regulated kinases（ERKs）, c-jun NH2-terminal kinases（JNKs）, MAPK kinase（MEK）, and p38, in addition to stress-related proteins p53 and heat shock protein 27（HSP27）. The activation of specific MAPK signal transduction pathways may provide a mechanism to regulate the expression of torpor-responsive genes or the regulation of selected downstream cellular processes. In response to torpor, each MAPK subfamily responded differently during torpor and each showed organ-specific patterns of response. For example, skeletal muscle displayed elevated relative phosphorylation of ERK1/2 during torpor. Interestingly, adipose tissues showed the highest degree of MAPK activation. Brown adipose tissue displayed an activation of ERK1/2 and p38, whereas white adipose tissue showed activation of ERK1/2, p38, MEK, and JNK during torpor. Importantly, both adipose tissues possess specialized functions that are critical for torpor, with brown adipose required for non-shivering thermogenesis and white adipose utilized as the primary source of lipid fuel for torpor. Overall, these data indicate crucial roles of MAPKs in the regulation of primate organs during torpor.

Potassium channels as potential drug targets for limb wound repair and regeneration

Background:Ion channels are a large family of transmembrane proteins,accessible by soluble membraneimpermeable molecules,and thus are targets for development of therapeutic drugs.Ion channels are the second most common target for existing drugs,after G protein-coupled receptors,and are expected to make a big impact on precision medicine in many different diseases includingwound repair and regeneration.Research has shown that endogenous bioelectric signaling mediated by ion channels is critical in non-mammalian limb regeneration.However,the role of ion channels in regeneration of limbs in mammalian systems is not yet defined.Methods:To explore the role of potassium channels in limb wound repair and regeneration,the hindlimbs of mouse embryos were amputated at E12.5 when the wound is expected to regenerate and E15.5 when the wound is not expected to regenerate,and gene expression of potassium channels was studied.Results:Most of the potassium channels were downregulated,except for the potassium channel kcnj8(Kir6.1)which was upregulated in E12.5 embryos after amputation.Conclusion:This study provides a new mouse limb regeneration model and demonstrates that potassium channels are potential drug targets for limb wound healing and regeneration.

Cytokine and Antioxidant Regulation in the Intestine of the Gray Mouse Lemur (Microcebus murinus) During Torpor

During food shortages, the gray mouse lemur（Microcebus murinus） of Madagascar experiences daily torpor thereby reducing energy expenditures. The present study aimed to understand the impacts of torpor on the immune system and antioxidant response in the gut of these animals.This interaction may be of critical importance given the trade-off between the energetically costly immune response and the need to defend against pathogen entry during hypometabolism. The protein levels of cytokines and antioxidants were measured in the small intestine（duodenum, jejunum,and ileum） and large intestine of aroused and torpid lemurs. While there was a significant decrease of some pro-inflammatory cytokines（IL-6 and TNF-a） in the duodenum and jejunum during torpor as compared to aroused animals, there was no change in anti-inflammatory cytokines. We observed decreased levels of cytokines（IL-12p70 and M-CSF）, and several chemokines（MCP-1and MIP-2） but an increase in MIP-1a in the jejunum of the torpid animals. In addition, we evaluated antioxidant response by examining the protein levels of antioxidant enzymes and total antioxidant capacity provided by metabolites such as glutathione（and others）. Our results indicatedthat levels of antioxidant enzymes did not change between torpor and aroused states, although antioxidant capacity was significantly higher in the ileum during torpor. These data suggest a suppression of the immune response, likely as an energy conservation measure, and a limited role of antioxidant defenses in supporting torpor in lemur intestine.

Regulation of the PI3K/AKT Pathway and Fuel Utilization During Primate Torpor in the Gray Mouse Lemur, Microcebus murinus

Gray mouse lemurs（Microcebus murinus） from Madagascar present an excellent model for studies of torpor regulation in a primate species. In the present study, we analyzed the response of the insulin signaling pathway as well as controls on carbohydrate sparing in six different tissues of torpid versus aroused gray mouse lemurs. We found that the relative level of phospho-insulin receptor substrate（IRS-1） was significantly increased in muscle, whereas the level of phospho-insulin receptor（IR） was decreased in white adipose tissue（WAT） of torpid animals, both suggesting an inhibition of insulin/insulin-like growth factor-1（IGF-1） signaling during torpor in these tissues. By contrast, the level of phospho-IR was increased in the liver. Interestingly, muscle,WAT, and liver occupy central roles in whole body homeostasis and each displays regulatory controls operating at the plasma membrane. Changes in other tissues included an increase in phosphoglycogen synthase kinase 3a（GSK3a） and decrease in phospho-ribosomal protein S6（RPS6） in the heart, and a decrease in phospho-mammalian target of rapamycin（m TOR） in the kidney. Pyruvate dehydrogenase（PDH） that gates carbohydrate entry into mitochondria is inhibited via phosphorylation by pyruvate dehydrogenase kinase（e.g., PDK4）. In the skeletal muscle, the protein expression of PDK4 and phosphorylated PDH at Ser 300 was increased, suggesting inhibition during torpor. In contrast, there were no changes in levels of PDH expression and phosphorylation in other tissues comparing torpid and aroused animals. Information gained from these studies highlight the molecular controls that help to regulate metabolic rate depression and balance energetics during primate torpor.

Modulation of Gene Expression in Key Survival Pathways During Daily Torpor in the Gray Mouse Lemur, Microcebus murinus

A variety of mammals employ torpor as an energy-saving strategy in environments of marginal or severe stress either on a daily basis during their inactive period or on a seasonal basis during prolonged multi-day hibernation. Recently, a few Madagascar lemur species have been identified as the only primates that exhibit torpor; one of these is the gray mouse lemur（Microcebus murinus）. To explore the regulatory mechanisms that underlie daily torpor in a primate, we analyzed the expression of 28 selected genes that represent crucial survival pathways known to be involved in squirrel and bat hibernation. Array-based real-time PCR was used to compare gene expression in control（aroused） versus torpid lemurs in five tissues including the liver, kidney,skeletal muscle, heart, and brown adipose tissue. Significant differences in gene expression during torpor were revealed among genes involved in glycolysis, fatty acid metabolism, antioxidant defense, apoptosis, hypoxia signaling, and protein protection. The results showed upregulation of select genes primarily in liver and brown adipose tissue. For instance, both tissues showed elevated gene expression of peroxisome proliferator activated receptor gamma（ppargc）, ferritin（fth1）, and protein chaperones during torpor. Overall, the data show that the expression of only a few genes changed during lemur daily torpor, as compared with the broader expression changes reported for hibernation in ground squirrels. These results provide an indication that the alterations in gene expression required for torpor in lemurs are not as extensive as those needed for winter hibernation in squirrel models. However, identification of crucial genes with altered expression that support lemur torpor provides key targets to be explored and manipulated toward a goal of translational applications of inducible torpor as a treatment option in human biomedicine.