A spatiotemporal atlas of mouse liver homeostasis and regeneration.

The mechanism by which mammalian liver cell responses are coordinated during tissue homeostasis and perturbation is poorly understood, representing a major obstacle in our understanding of many diseases. This knowledge gap is caused by the difficulty involved with studying multiple cell types in different states and locations, particularly when these are transient. We have combined Stereo-seq (spatiotemporal enhanced resolution omics-sequencing) with single-cell transcriptomic profiling of 473,290 cells to generate a high-definition spatiotemporal atlas of mouse liver homeostasis and regeneration at the whole-lobe scale. Our integrative study dissects in detail the molecular gradients controlling liver cell function, systematically defining how gene networks are dynamically modulated through intercellular communication to promote regeneration. Among other important regulators, we identified the transcriptional cofactor TBL1XR1 as a rheostat linking inflammation to Wnt/ß-catenin signaling for facilitating hepatocyte proliferation. Our data and analytical pipelines lay the foundation for future high-definition tissue-scale atlases of organ physiology and malfunction.

"The Good, the Bad, and the Ugly" - About Diverse Phenotypes of Hepatic Stellate Cells in the Liver.

Hepatic stellate cells (HSCs) and their activated derivatives, often referred to as myofibroblasts (MFs), play a key role in progression of chronic liver injuries leading to fibrosis, cirrhosis, and hepatocellular carcinoma. Until recently, MFs were considered a homogenous cell type majorly due to lack of techniques that allow complex molecular studies at a single-cell resolution. Recent technical advancements in genetic lineage-tracing models as well as the exponential growth of studies with single-cell transcriptome and proteome analyses have uncovered hidden heterogeneities among the HSC and MF populations in healthy states as well as chronic liver injuries at the various stages of tissue deformation. The identification of different phenotypes along the HSC/MF axis, which either maintain essential liver functions ("good" HSCs), emerge during fibrosis ("bad" HSCs), or even promote hepatocellular carcinoma ("ugly" HSCs), may lay the foundation for targeting a particular MF phenotype as potential treatment for chronic liver injuries.

Bioartificial liver with reprogrammed hepatocytes ready for prime time.

In this issue, Wang et al.1 provide evidence of the pre-clinical as well as the clinical utility of in vitro-generated directly reprogrammed human hepatocytes in bioartificial liver. This approach will help offer patients a more curative surgical therapy for liver cancer and improve survival rates.

Cell atlas of CCl 4-induced progressive liver fibrosis reveals stage-specific responses.

Chronic liver injury leads to progressive liver fibrosis and ultimately cirrhosis, a major cause of morbidity and mortality worldwide. However, there are currently no effective anti-fibrotic therapies available, especially for late-stage patients, which is partly attributed to the major knowledge gap regarding liver cell heterogeneity and cell-specific responses in different fibrosis stages. To reveal the multicellular networks regulating mammalian liver fibrosis from mild to severe phenotypes, we generated a single-nucleus transcriptomic atlas encompassing 49 919 nuclei corresponding to all main liver cell types at different stages of murine carbon tetrachloride (CCl 4)-induced progressive liver fibrosis. Integrative analysis distinguished the sequential responses to injury of hepatocytes, hepatic stellate cells and endothelial cells. Moreover, we reconstructed cell-cell interactions and gene regulatory networks implicated in these processes. These integrative analyses uncovered previously overlooked aspects of hepatocyte proliferation exhaustion and disrupted pericentral metabolic functions, dysfunction for clearance by apoptosis of activated hepatic stellate cells, accumulation of pro-fibrotic signals, and the switch from an anti-angiogenic to a pro-angiogenic program during CCl 4-induced progressive liver fibrosis. Our dataset thus constitutes a useful resource for understanding the molecular basis of progressive liver fibrosis using a relevant animal model.

Synergistic Combinations of FDA-Approved Drugs with Ceftobiprole against Methicillin-Resistant Staphylococcus aureus.

New strategies are urgently needed to address the public health threat of antimicrobial resistance. Synergistic agent combinations provide one possible pathway toward addressing this need and are also of fundamental mechanistic interest. Effective methods for comprehensively identifying synergistic agent combinations are required for such efforts. In this study, an FDA-approved drug library was screened against methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300) in the absence and presence of sub-MIC levels of ceftobiprole, a PBP2a-targeted anti-MRSA ß-lactam. This screening identified numerous potential synergistic agent combinations, which were then confirmed and characterized for synergy using checkerboard analyses. The initial group of synergistic agents (sum of the minimum fractional inhibitory concentration ∑FICmin ≤0.5) were all ß-lactamase-resistant ß-lactams (cloxacillin, dicloxacillin, flucloxacillin, oxacillin, nafcillin, and cefotaxime). Cloxacillin-the agent with the greatest synergy with ceftobiprole-is also highly synergistic with ceftaroline, another PBP2a-targeted ß-lactam. Further follow-up studies revealed a range of ceftobiprole synergies with other ß-lactams, including with imipenem, meropenem, piperacillin, tazobactam, and cefoxitin. Interestingly, given that essentially all other ceftobiprole-ß-lactam combinations showed synergy, ceftaroline and ceftobiprole showed no synergy. Modest to no synergy (0.5 < ∑FICmin ≤ 1.0) was observed for several non-ß-lactam agents, including vancomycin, daptomycin, balofloxacin, and floxuridine. Mupirocin had antagonistic activity with ceftobiprole. Flucloxacillin appeared particularly promising, with both a low intrinsic MIC and good synergy with ceftobiprole. That so many ß-lactam combinations with ceftobiprole show synergy suggests that ß-lactam combinations can generally increase ß-lactam effectiveness and may also be useful in reducing resistance emergence and spread in MRSA. IMPORTANCE Antimicrobial resistance represents a serious threat to public health. Antibacterial agent combinations provide a potential approach to combating this problem, and synergistic agent combinations-in which each agent enhances the antimicrobial activity of the other-are particularly valuable in this regard. Ceftobiprole is a late-generation ß-lactam antibiotic developed for MRSA infections. Resistance has emerged to ceftobiprole, jeopardizing this agent's effectiveness. To identify synergistic agent combinations with ceftobiprole, an FDA-approved drug library was screened for potential synergistic combinations with ceftobiprole. This screening and follow-up studies identified numerous ß-lactams with ceftobiprole synergy.

Deconvolution of multichannel LC-MS/MS chromatograms of glucosamine-phosphates: Evidence of a GlmS regulatory difference between Staphylococcus aureus and Enterococcus faecium.

Resolving isomeric analytes is challenging given their physical similarity - making chromatographic resolution difficult, and their identical masses - making simple mass resolution impossible. MS/MS data provides a means to resolve isomeric analytes if their MS/MS intensity profiles are sufficiently different. Glucosamine-6-phosphate (GlcN-6P) and glucosamine-1-phosphate (GlcN-1P) are early bacterial cell wall intermediates. These and other isomeric hexosamine-phosphates are highly polar and unretained on reverse-phase chromatography media. Three commercially available hexosamine-phosphate standards (GlcN-6P, GlcN-1P, and GalN-1P) were derivatized with octanoic anhydride, and chromatographic conditions were established to resolve these analytes on C18 columns. GlcN-1P and GalN-1P overlapped chromatographically under all tested chromatography conditions. Three MS/MS fragments (79, 97, and 199 m/z) were common to all three commercially available hexosamine-phosphates. Intensity ratios of the three MS/MS fragments from these three hexosamine-phosphate standards were used to deconvolute mixture chromatograms of these standards by non-negative linear regression. This approach allowed the complete resolution of these analytes. The chromatographically overlapping GlcN-1P and GalN-1P, which shared similar but modestly different MS/MS intensity profiles, were fully resolved with this non-negative deconvolution approach. This approach was then applied to MRSA, VSE, and VRE bacterial extracts before and after exposure to vancomycin. This demonstrated a substantial (3-fold) increase in GlcN-6P in vancomycin-treated MRSA samples but not in vancomycin-treated VSE or VRE samples. These observations appear to localize previously observed differences between MRSA and VRE/VSE peptidoglycan biosynthesis regulation to GlmS, which synthesizes GlcN-6P and is the product of a regulatory ribozyme sensitive to the levels of GlcN-6P.

Therapeutic HNF4A mRNA attenuates liver fibrosis in a preclinical model.

BACKGROUND & AIMS: Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive approach that, until recently, has remained poorly explored. In this study, we examined the therapeutic utility of mRNA delivery for liver fibrosis and cirrhosis. Specifically, we aimed to demonstrate the therapeutic efficacy of human hepatocyte nuclear factor alpha (HNF4A) mRNA in mouse models of fibrosis and cirrhosis. METHODS: We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNPs) encapsulating HNF4A mRNA. To identify potential mechanisms of action, we performed microarray-based gene expression profiling, single-cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation. RESULTS: Expression of HNF4A mRNA led to restoration of the metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of LNP-encapsulated HNF4A mRNA induced a robust inhibition of fibrogenesis in 4 independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells. CONCLUSION: Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver. LAY SUMMARY: Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality worldwide. Herein, we take advantage of a promising therapeutic approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides proof-of-concept that mRNA therapy is a promising strategy for reversing liver fibrosis and cirrhosis.

Functional Genomic Screening During Somatic Cell Reprogramming Identifies DKK3 as a Roadblock of Organ Regeneration.

Somatic cell reprogramming and tissue repair share relevant factors and molecular programs. Here, Dickkopf-3 (DKK3) is identified as novel factor for organ regeneration using combined transcription-factor-induced reprogramming and RNA-interference techniques. Loss of Dkk3 enhances the generation of induced pluripotent stem cells but does not affect de novo derivation of embryonic stem cells, three-germ-layer differentiation or colony formation capacity of liver and pancreatic organoids. However, DKK3 expression levels in wildtype animals and serum levels in human patients are elevated upon injury. Accordingly, Dkk3-null mice display less liver damage upon acute and chronic failure mediated by increased proliferation in hepatocytes and LGR5+ liver progenitor cell population, respectively. Similarly, recovery from experimental pancreatitis is accelerated. Regeneration onset occurs in the acinar compartment accompanied by virtually abolished canonical-Wnt-signaling in Dkk3-null animals. This results in reduced expression of the Hedgehog repressor Gli3 and increased Hedgehog-signaling activity upon Dkk3 loss. Collectively, these data reveal Dkk3 as a key regulator of organ regeneration via a direct, previously unacknowledged link between DKK3, canonical-Wnt-, and Hedgehog-signaling.

Hyaluronic Acid-Targeted Stimuli-Sensitive Nanomicelles Co-Encapsulating Paclitaxel and Ritonavir to Overcome Multi-Drug Resistance in Metastatic Breast Cancer and Triple-Negative Breast Cancer Cells.

Active targeting and overcoming multi-drug resistance (MDR) can be some of the important attributes of targeted therapy for metastatic breast cancer (MBC) and triple-negative breast cancer (TNBC) treatment. In this study, we constructed a hyaluronic acid (HA)-decorated mixed nanomicelles-encapsulating chemotherapeutic agent paclitaxel (PTX) and P-glycoprotein inhibitor ritonavir (RTV). HA was conjugated to poly (lactide) co-(glycolide) (PLGA) polymer by disulfide bonds (HA-ss-PLGA). HA is a natural ligand for CD44 receptors overexpressed in breast cancer cells. Disulfide bonds undergo rapid reduction in the presence of glutathione, present in breast cancer cells. The addition of RTV can inhibit the P-gp and CYP3A4-mediated metabolism of PTX, thus aiding in reversing MDR and sensitizing the cells toward PTX. An in vitro uptake and cytotoxicity study in MBC MCF-7 and TNBC MDA-MB-231 cell lines demonstrated the effective uptake of the nanomicelles and drug PTX compared to non-neoplastic breast epithelium MCF-12A cells. Interestingly, in vitro potency determination showed a reduction in mitochondrial membrane potential and reactive oxygen species in breast cancer cell lines, indicating effective apoptosis of cancer cells. Thus, stimuli-sensitive nanomicelles along with HA targeting and RTV addition can effectively serve as a chemotherapeutic drug delivery agent for MBC and TNBC.

p53-Independent Induction of p21 Fails to Control Regeneration and Hepatocarcinogenesis in a Murine Liver Injury Model.

BACKGROUND & AIMS: A coordinated stress and regenerative response is important after hepatocyte damage. Here, we investigate the phenotypes that result from genetic abrogation of individual components of the checkpoint kinase 2/transformation-related protein 53 (p53)/cyclin-dependent kinase inhibitor 1A (p21) pathway in a murine model of metabolic liver injury. METHODS: Nitisinone was reduced or withdrawn in Fah-/- mice lacking Chk2, p53, or p21, and survival, tumor development, liver injury, and regeneration were analyzed. Partial hepatectomies were performed and mice were challenged with the Fas antibody Jo2. RESULTS: In a model of metabolic liver injury, loss of p53, but not Chk2, impairs the oxidative stress response and aggravates liver damage, indicative of a direct p53-dependent protective effect on hepatocytes. Cell-cycle control during chronic liver injury critically depends on the presence of both p53 and its downstream effector p21. In p53-deficient hepatocytes, unchecked proliferation occurs despite a strong induction of p21, showing a complex interdependency between p21 and p53. The increased regenerative potential in the absence of p53 cannot fully compensate the surplus injury and is not sufficient to promote survival. Despite the distinct phenotypes associated with the loss of individual components of the DNA damage response, gene expression patterns are dominated by the severity of liver injury, but reflect distinct effects of p53 on proliferation and the anti-oxidative stress response. CONCLUSIONS: Characteristic phenotypes result from the genetic abrogation of individual components of the DNA damage-response cascade in a liver injury model. The extent to which loss of gene function can be compensated, or affects injury and proliferation, is related to the level at which the cascade is interrupted. Accession numbers of repository for expression microarray data: GSE156983, GSE156263, GSE156852, and GSE156252.

MicroRNA-342-3p is a potent tumour suppressor in hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a cancer with multiple aetiologies and widespread prevalence. Largely refractory to current treatments, HCC is the fourth leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) are important regulators in HCCs. We aimed to identify tumour suppressor miRNAs during tumour regression in a conditional c-MYC-driven mouse model (LT2/MYC) of HCC, and to evaluate their therapeutic potential for HCC treatment. METHODS: We performed miRNA expression profiling of developed and regressing LT2/MYC tumours and in-depth in vitro gain- and loss-of-function analyses. The effect of adeno-associated virus (AAV) vector-mediated miR-342-3p treatment was evaluated in 3 HCC mouse models. RESULTS: We identified miR-342-3p as a tumour suppressor miRNA in HCC, with increased expression in regressing tumours. Forced miR-342-3p expression in hepatoma cells showed significantly decreased cell proliferation, migration, and colony formation. In vivo administration of AAV-miR-342-3p led to significant attenuation of tumour development and increased overall survival. We identified monocarboxylic acid transporter 1 (MCT1) as a bona fide target of miR-342-3p in HCC. We show that the tumour suppressor role of miR-342-3p is executed partly by modulating the lactate transport function of MCT1. Importantly, we find miR-342-3p downregulated in tumours from patients with HCC compared with matched non-tumour tissues, inversely correlating with MCT1 expression. We observed similar findings in TCGA-LIHC data. CONCLUSIONS: In our study, we identified and validated miR-342-3p as a tumour suppressor miRNA in HCC. We demonstrated its therapeutic efficacy in significantly attenuating tumour development, and prolonging survival, in different HCC mouse models. Identification of miR-342-3p as an effective tumour suppressor opens a therapeutic avenue for miRNA-mediated attenuation of HCC development. LAY SUMMARY: Hepatocellular carcinoma (HCC), the most common type of liver cancer, affects diverse populations and has a global impact, being the fourth leading cause of cancer deaths worldwide. There are currently no systemic therapies for HCC that can significantly prolong long-term survival. Thus, novel effective treatment options are urgently required. To understand the molecular basis of tumour regression, we compared tumours and regressing liver tumours in mice. We show that a small non-coding miRNA, miR-342-3p, is a tumour suppressor in HCC. Expression of miR-342-3p is low in tumours and high in regressing tumours. When miR-342-3p is delivered to mouse livers with HCC, it can significantly slow down liver tumour development and improve survival. Our study highlights the promising therapeutic potential of miR-342-3p intervention in HCC.

MicroRNA-125b-5p Regulates Hepatocyte Proliferation During the Termination Phase of Liver Regeneration.

The ability of the liver to regenerate and restore mass limits the increasing mortality rate due to life-threatening liver diseases. Successful liver regeneration is accomplished in multiple stages, of which the priming and proliferation phases are well studied. However, the regulatory pathways, specifically microRNA (miRNA)-mediated posttranscriptional regulation, which prevent uncontrolled proliferation and mediate the termination of liver regeneration, are not well understood. We identified differentially regulated miRNAs during the termination phase after 2/3 partial hepatectomy (PH) in mice, which is a well-established mouse model of liver regeneration. We further evaluated the function of differentially regulated miRNAs in primary mouse hepatocytes by using mimics and inhibitors and in vivo by using adeno-associated virus (AAV) serotype 8. A candidate miRNA target was identified by messenger RNA array in silico analyses and validated in primary mouse and human hepatocytes. Using miRNA profiling, we discovered miR-125b-5p as a novel regulator of hepatocyte proliferation in the late phase of liver regeneration. AAV-mediated miR-125b-5p delivery in mice enhanced the endogenous regenerative capacity and resulted in improved restoration of liver mass after 2/3 PH. Further, we found that ankyrin repeat and BTB/POZ domain containing protein 1 (Abtb1) is a direct target of miR-125b-5p in primary mouse and human hepatocytes and contributes to the pro-proliferative activity of miR-125b-5p by forkhead box G1 (FOXG1) and the cyclin-dependent kinase inhibitor 1A (p21) pathway. Conclusion: miR-125b-5p has an important role in regulating hepatocyte proliferation in the termination phase of liver regeneration and may serve as a potential therapeutic target in various liver diseases that often exhibit deregulated hepatocyte proliferation.

c-di-AMP Accumulation Impairs Muropeptide Synthesis in Listeria monocytogenes.

Cyclic di-AMP (c-di-AMP) is an essential and ubiquitous second messenger among bacteria. c-di-AMP regulates many cellular pathways through direct binding to several molecular targets in bacterial cells. c-di-AMP depletion is well known to destabilize the bacterial cell wall, resulting in increased bacteriolysis and enhanced susceptibility to cell wall targeting antibiotics. Using the human pathogen Listeria monocytogenes as a model, we found that c-di-AMP accumulation also impaired cell envelope integrity. An L. monocytogenes mutant deleted for c-di-AMP phosphodiesterases (pdeA pgpH mutant) exhibited a 4-fold increase in c-di-AMP levels and several cell wall defects. For instance, the pdeA pgpH mutant was defective for the synthesis of peptidoglycan muropeptides and was susceptible to cell wall-targeting antimicrobials. Among different muropeptide precursors, we found that the pdeA pgpH strain was particularly impaired in the synthesis of d-Ala-d-Ala, which is required to complete the pentapeptide stem associated with UDP-N-acetylmuramic acid (MurNAc). This was consistent with an increased sensitivity to d-cycloserine, which inhibits the d-alanine branch of peptidoglycan synthesis. Finally, upon examining d-Ala:d-Ala ligase (Ddl), which catalyzes the conversion of d-Ala to d-Ala-d-Ala, we found that its activity was activated by K+ Based on previous reports that c-di-AMP inhibits K+ uptake, we propose that c-di-AMP accumulation impairs peptidoglycan synthesis, partially through the deprivation of cytoplasmic K+ levels, which are required for cell wall-synthetic enzymes.IMPORTANCE The bacterial second messenger c-di-AMP is produced by a large number of bacteria and conditionally essential to many species. Conversely, c-di-AMP accumulation is also toxic to bacterial physiology and pathogenesis, but its mechanisms are largely undefined. We found that in Listeria monocytogenes, elevated c-di-AMP levels diminished muropeptide synthesis and increased susceptibility to cell wall-targeting antimicrobials. Cell wall defects might be an important mechanism for attenuated virulence in bacteria with high c-di-AMP levels.

MicroRNA-221: A Fine Tuner and Potential Biomarker of Chronic Liver Injury.

The last decade has witnessed significant advancements in our understanding of how small noncoding RNAs, such as microRNAs (miRNAs), regulate disease progression. One such miRNA, miR-221, has been shown to play a key role in the progression of liver fibrosis, a common feature of most liver diseases. Many reports have demonstrated the upregulation of miR-221 in liver fibrosis caused by multiple etiologies such as viral infections and nonalcoholic steatohepatitis. Inhibition of miR-221 via different strategies has shown promising results in terms of the suppression of fibrogenic gene signatures in vitro, as well as in vivo, in independent mouse models of liver fibrosis. In addition, miR-221 has also been suggested as a noninvasive serum biomarker for liver fibrosis and cirrhosis. In this review, we discuss the biology of miR-221, its significance and use as a biomarker during progression of liver fibrosis, and finally, potential and robust approaches that can be utilized to suppress liver fibrosis via inhibition of miR-221.

Innovations, challenges, and minimal information for standardization of humanized mice.

Mice xenotransplanted with human cells and/or expressing human gene products (also known as "humanized mice") recapitulate the human evolutionary specialization and diversity of genotypic and phenotypic traits. These models can provide a relevant in vivo context for understanding of human-specific physiology and pathologies. Humanized mice have advanced toward mainstream preclinical models and are now at the forefront of biomedical research. Here, we considered innovations and challenges regarding the reconstitution of human immunity and human tissues, modeling of human infections and cancer, and the use of humanized mice for testing drugs or regenerative therapy products. As the number of publications exploring different facets of humanized mouse models has steadily increased in past years, it is becoming evident that standardized reporting is needed in the field. Therefore, an international community-driven resource called "Minimal Information for Standardization of Humanized Mice" (MISHUM) has been created for the purpose of enhancing rigor and reproducibility of studies in the field. Within MISHUM, we propose comprehensive guidelines for reporting critical information generated using humanized mice.

Ex Vivo/In vivo Gene Editing in Hepatocytes Using "All-in-One" CRISPR-Adeno-Associated Virus Vectors with a Self-Linearizing Repair Template.

Adeno-associated virus (AAV)-based vectors are considered efficient and safe gene delivery systems in gene therapy. We combined two guide RNA genes, Cas9, and a self-linearizing repair template in one vector (AIO-SL) to correct fumarylacetoacetate hydrolase (FAH) deficiency in mice. The vector genome of 5.73 kb was packaged into VP2-depleted AAV particles (AAV2/8ΔVP2), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2ΔVP2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8ΔVP2 likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an ∼6-fold higher degree of template integration than vectors without template self-linearization. Subsequent analysis revealed that AAV8 particles, in contrast to AAV2, incorporate oversized genomes distinctly greater than 5.2 kb. Finally, our AAV8-based vector represents a promising tool for gene editing strategies to correct monogenic liver diseases requiring (large) fragment removal and/or simultaneous sequence replacement.