Liver-specific Mettl14 deletion induces nuclear heterotypia and dysregulates RNA export machinery.

Modification of RNA with N6-methyladenosine (m6A) has gained attention in recent years as a general mechanism of gene regulation. In the liver, m6A, along with its associated machinery, has been studied as a potential biomarker of disease and cancer, with impacts on metabolism, cell cycle regulation, and pro-cancer state signaling. However these observational data have yet to be causally examined in vivo. For example, neither perturbation of the key m6A writers Mettl3 and Mettl14, nor the m6A readers Ythdf1 and Ythdf2 have been thoroughly mechanistically characterized in vivo as they have been in vitro. To understand the functions of these machineries, we developed mouse models and found that deleting Mettl14 led to progressive liver injury characterized by nuclear heterotypia, with changes in mRNA splicing, processing and export leading to increases in mRNA surveillance and recycling.

A nucleosome switch primes Hepatitis B Virus infection.

Chronic hepatitis B virus (HBV) infection is an incurable global health threat responsible for causing liver disease and hepatocellular carcinoma. During the genesis of infection, HBV establishes an independent minichromosome consisting of the viral covalently closed circular DNA (cccDNA) genome and host histones. The viral X gene must be expressed immediately upon infection to induce degradation of the host silencing factor, Smc5/6. However, the relationship between cccDNA chromatinization and X gene transcription remains poorly understood. Establishing a reconstituted viral minichromosome platform, we found that nucleosome occupancy in cccDNA drives X transcription. We corroborated these findings in cells and further showed that the chromatin destabilizing molecule CBL137 inhibits X transcription and HBV infection in hepatocytes. Our results shed light on a long-standing paradox and represent a potential new therapeutic avenue for the treatment of chronic HBV infection.

Space radiation damage rescued by inhibition of key spaceflight associated miRNAs.

Our previous research revealed a key microRNA signature that is associated with spaceflight that can be used as a biomarker and to develop countermeasure treatments to mitigate the damage caused by space radiation. Here, we expand on this work to determine the biological factors rescued by the countermeasure treatment. We performed RNA-sequencing and transcriptomic analysis on 3D microvessel cell cultures exposed to simulated deep space radiation (0.5 Gy of Galactic Cosmic Radiation) with and without the antagonists to three microRNAs: miR-16-5p, miR-125b-5p, and let-7a-5p (i.e., antagomirs). Significant reduction of inflammation and DNA double strand breaks (DSBs) activity and rescue of mitochondria functions are observed after antagomir treatment. Using data from astronaut participants in the NASA Twin Study, Inspiration4, and JAXA missions, we reveal the genes and pathways implicated in the action of these antagomirs are altered in humans. Our findings indicate a countermeasure strategy that can potentially be utilized by astronauts in spaceflight missions to mitigate space radiation damage.

SCARPET: site-specific quantification of methylated and nonmethylated adenosines reveals m6A stoichiometry.

m6A has different stoichiometry at different positions in different mRNAs. However, the exact stoichiometry of m6A is difficult to measure. Here, we describe SCARPET (site-specific cleavage and radioactive-labeling followed by purification, exonuclease digestion, and thin-layer chromatography), a simple and streamlined biochemical assay for quantifying m6A at any specific site in any mRNA. SCARPET involves a site-specific cleavage of mRNA immediately 5' of an adenosine site in an mRNA. This site is radiolabeled with 32P, and after a series of steps to purify the RNA and to remove nonspecific signals, the nucleotide is resolved by TLC to visualize A and m6A at this site. Quantification of these spots reveals the m6A stoichiometry at the site of interest. SCARPET can be applied to poly(A)-enriched RNA, or preferably purified mRNA, which produces more accurate m6A stoichiometry measurements. We show that sample processing steps of SCARPET can be performed in a single day, and results in a specific and accurate measurement of m6A stoichiometry at specific sites in mRNA. Using SCARPET, we measure exact m6A stoichiometries in specific mRNAs and show that Zika genomic RNA lacks m6A at previously mapped sites. SCARPET will be useful for testing specific sites for their m6A stoichiometry and to assess how m6A stoichiometry changes in different conditions and cellular contexts.

SARS-CoV-2 infection causes dopaminergic neuron senescence.

COVID-19 patients commonly present with signs of central nervous system and/or peripheral nervous system dysfunction. Here, we show that midbrain dopamine (DA) neurons derived from human pluripotent stem cells (hPSCs) are selectively susceptible and permissive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. SARS-CoV-2 infection of DA neurons triggers an inflammatory and cellular senescence response. High-throughput screening in hPSC-derived DA neurons identified several FDA-approved drugs that can rescue the cellular senescence phenotype by preventing SARS-CoV-2 infection. We also identified the inflammatory and cellular senescence signature and low levels of SARS-CoV-2 transcripts in human substantia nigra tissue of COVID-19 patients. Furthermore, we observed reduced numbers of neuromelanin+ and tyrosine-hydroxylase (TH)+ DA neurons and fibers in a cohort of severe COVID-19 patients. Our findings demonstrate that hPSC-derived DA neurons are susceptible to SARS-CoV-2, identify candidate neuroprotective drugs for COVID-19 patients, and suggest the need for careful, long-term monitoring of neurological problems in COVID-19 patients.

The impact of epitranscriptomic modifications on liver disease.

RNA modifications have emerged as important mechanisms of gene regulation. Developmental, metabolic, and cell cycle regulatory processes are all affected by epitranscriptomic modifications, which control gene expression in a dynamic manner. The hepatic tissue is highly metabolically active and has an impressive ability to regenerate after injury. Cell proliferation, differentiation, and metabolism, which are all essential to the liver response to injury and regeneration, are regulated via RNA modification. Two such modifications, N6-methyladenosine (m6A)and 5-methylcytosine (m5C), have been identified as prognostic disease markers and potential therapeutic targets for liver diseases. Here, we describe progress in understanding the role of RNA modifications in liver biology and disease and discuss specific areas where unexpected results could lead to improved future understanding.

Mesenteric Parametrial Fat Pad Surgery for in vivo Implantation of Hepatocytes in Nude Mice.

Cell-based liver therapies utilizing functionally stabilized engineered hepatic tissue hold promise in improving host liver functions and are emerging as a potential alternative to whole-organ transplantation. Owing to the ability to accommodate a large ex vivo engineered hepatocyte mass and dense vascularization, the mesenteric parametrial fat pad in female nude mice forms an ideal anatomic microenvironment for ectopic hepatocyte transplantation. However, the lack of any reported protocol detailing the presurgical preparation and construction of the engineered hepatic hydrogel, fat pad surgery, and postsurgical care and bioluminescence imaging to confirm in vivo hepatocyte implantation makes it challenging to reliably perform and test engraftment and integration with the host. In this report, we provide a step-by-step protocol for in vivo hepatocyte implantation, including preparation of hepatic tissue for implantation, the surgery process, and bioluminescence imaging to assess survival of functional hepatocytes. This will be a valuable protocol for researchers in the fields of tissue engineering, transplantation, and regenerative medicine. Key features • Primary human hepatocytes transduced ex vivo with a lentiviral vector carrying firefly luciferase are surgically implanted onto the fat pad. • Bioluminescence helps monitor survival of transplanted hepatic tissue over time. • Applicable for assessment of graft survival, graft-host integration, and liver regeneration.

Organelle stress and alterations in interorganelle crosstalk during liver fibrosis.

The synchronous functioning and quality control of organelles ensure cell survival and function and are essential for maintaining homeostasis. Prolonged exposure to stressors (viruses, bacteria, parasitic infections, alcohol, drugs) or genetic mutations often disrupt the functional integrity of organelles which plays a critical role in the initiation and progression of several diseases including chronic liver diseases. One of the most important pathologic consequences of chronic liver diseases is liver fibrosis, characterized by tissue scarring due to the progressive accumulation of extracellular matrix components. Left untreated, fibrosis may advance to life-threatening complications such as cirrhosis, hepatic decompensation, and HCC, which collectively accounts for ∼1 million deaths per year worldwide. Owing to the lack of treatment options that can regress or reverse cirrhosis, liver transplantation is currently the only available treatment for end-stage liver disease. However, the limited supply of usable donor organs, adverse effects of lifelong immunosuppressive regimes, and financial considerations pose major challenges and limit its application. Hence, effective therapeutic strategies are urgently needed. An improved understanding of the organelle-level regulation of fibrosis can help devise effective antifibrotic therapies focused on reducing organelle stress, limiting organelle damage, improving interorganelle crosstalk, and restoring organelle homeostasis; and could be a potential clinical option to avoid transplantation. This review provides a timely update on the recent findings and mechanisms covering organelle-specific dysfunctions in liver fibrosis, highlights how correction of organelle functions opens new treatment avenues and discusses the potential challenges to clinical application.

A comprehensive SARS-CoV-2 and COVID-19 review, Part 2: host extracellular to systemic effects of SARS-CoV-2 infection.

COVID-19, the disease caused by SARS-CoV-2, has caused significant morbidity and mortality worldwide. The betacoronavirus continues to evolve with global health implications as we race to learn more to curb its transmission, evolution, and sequelae. The focus of this review, the second of a three-part series, is on the biological effects of the SARS-CoV-2 virus on post-acute disease in the context of tissue and organ adaptations and damage. We highlight the current knowledge and describe how virological, animal, and clinical studies have shed light on the mechanisms driving the varied clinical diagnoses and observations of COVID-19 patients. Moreover, we describe how investigations into SARS-CoV-2 effects have informed the understanding of viral pathogenesis and provide innovative pathways for future research on the mechanisms of viral diseases.

Epinephrine inhibits PI3Kα via the Hippo kinases.

The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.

Epigenetic memory of coronavirus infection in innate immune cells and their progenitors.

Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.

Core mitochondrial genes are down-regulated during SARS-CoV-2 infection of rodent and human hosts.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins bind to host mitochondrial proteins, likely inhibiting oxidative phosphorylation (OXPHOS) and stimulating glycolysis. We analyzed mitochondrial gene expression in nasopharyngeal and autopsy tissues from patients with coronavirus disease 2019 (COVID-19). In nasopharyngeal samples with declining viral titers, the virus blocked the transcription of a subset of nuclear DNA (nDNA)-encoded mitochondrial OXPHOS genes, induced the expression of microRNA 2392, activated HIF-1α to induce glycolysis, and activated host immune defenses including the integrated stress response. In autopsy tissues from patients with COVID-19, SARS-CoV-2 was no longer present, and mitochondrial gene transcription had recovered in the lungs. However, nDNA mitochondrial gene expression remained suppressed in autopsy tissue from the heart and, to a lesser extent, kidney, and liver, whereas mitochondrial DNA transcription was induced and host-immune defense pathways were activated. During early SARS-CoV-2 infection of hamsters with peak lung viral load, mitochondrial gene expression in the lung was minimally perturbed but was down-regulated in the cerebellum and up-regulated in the striatum even though no SARS-CoV-2 was detected in the brain. During the mid-phase SARS-CoV-2 infection of mice, mitochondrial gene expression was starting to recover in mouse lungs. These data suggest that when the viral titer first peaks, there is a systemic host response followed by viral suppression of mitochondrial gene transcription and induction of glycolysis leading to the deployment of antiviral immune defenses. Even when the virus was cleared and lung mitochondrial function had recovered, mitochondrial function in the heart, kidney, liver, and lymph nodes remained impaired, potentially leading to severe COVID-19 pathology.

Targeted viral adaptation generates a simian-tropic hepatitis B virus that infects marmoset cells.

Hepatitis B virus (HBV) only infects humans and chimpanzees, posing major challenges for modeling HBV infection and chronic viral hepatitis. The major barrier in establishing HBV infection in non-human primates lies at incompatibilities between HBV and simian orthologues of the HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Through mutagenesis analysis and screening among NTCP orthologues from Old World monkeys, New World monkeys and prosimians, we determined key residues responsible for viral binding and internalization, respectively and identified marmosets as a suitable candidate for HBV infection. Primary marmoset hepatocytes and induced pluripotent stem cell-derived hepatocyte-like cells support HBV and more efficient woolly monkey HBV (WMHBV) infection. Adapted chimeric HBV genome harboring residues 1-48 of WMHBV preS1 generated here led to a more efficient infection than wild-type HBV in primary and stem cell derived marmoset hepatocytes. Collectively, our data demonstrate that minimal targeted simianization of HBV can break the species barrier in small NHPs, paving the path for an HBV primate model.

Tumour extracellular vesicles and particles induce liver metabolic dysfunction.

Cancer alters the function of multiple organs beyond those targeted by metastasis1,2. Here we show that inflammation, fatty liver and dysregulated metabolism are hallmarks of systemically affected livers in mouse models and in patients with extrahepatic metastasis. We identified tumour-derived extracellular vesicles and particles (EVPs) as crucial mediators of cancer-induced hepatic reprogramming, which could be reversed by reducing tumour EVP secretion via depletion of Rab27a. All EVP subpopulations, exosomes and principally exomeres, could dysregulate hepatic function. The fatty acid cargo of tumour EVPs-particularly palmitic acid-induced secretion of tumour necrosis factor (TNF) by Kupffer cells, generating a pro-inflammatory microenvironment, suppressing fatty acid metabolism and oxidative phosphorylation, and promoting fatty liver formation. Notably, Kupffer cell ablation or TNF blockade markedly decreased tumour-induced fatty liver generation. Tumour implantation or pre-treatment with tumour EVPs diminished cytochrome P450 gene expression and attenuated drug metabolism in a TNF-dependent manner. We also observed fatty liver and decreased cytochrome P450 expression at diagnosis in tumour-free livers of patients with pancreatic cancer who later developed extrahepatic metastasis, highlighting the clinical relevance of our findings. Notably, tumour EVP education enhanced side effects of chemotherapy, including bone marrow suppression and cardiotoxicity, suggesting that metabolic reprogramming of the liver by tumour-derived EVPs may limit chemotherapy tolerance in patients with cancer. Our results reveal how tumour-derived EVPs dysregulate hepatic function and their targetable potential, alongside TNF inhibition, for preventing fatty liver formation and enhancing the efficacy of chemotherapy.

Astrocytic TDP-43 dysregulation impairs memory by modulating antiviral pathways and interferon-inducible chemokines.

Transactivating response region DNA binding protein 43 (TDP-43) pathology is prevalent in dementia, but the cell type-specific effects of TDP-43 pathology are not clear, and therapeutic strategies to alleviate TDP-43-linked cognitive decline are lacking. We found that patients with Alzheimer's disease or frontotemporal dementia have aberrant TDP-43 accumulation in hippocampal astrocytes. In mouse models, induction of widespread or hippocampus-targeted accumulation in astrocytic TDP-43 caused progressive memory loss and localized changes in antiviral gene expression. These changes were cell-autonomous and correlated with impaired astrocytic defense against infectious viruses. Among the changes, astrocytes had elevated levels of interferon-inducible chemokines, and neurons had elevated levels of the corresponding chemokine receptor CXCR3 in presynaptic terminals. CXCR3 stimulation altered presynaptic function and promoted neuronal hyperexcitability, akin to the effects of astrocytic TDP-43 dysregulation, and blockade of CXCR3 reduced this activity. Ablation of CXCR3 also prevented TDP-43-linked memory loss. Thus, astrocytic TDP-43 dysfunction contributes to cognitive impairment through aberrant chemokine-mediated astrocytic-neuronal interactions.

A multi-organoid platform identifies CIART as a key factor for SARS-CoV-2 infection.

COVID-19 is a systemic disease involving multiple organs. We previously established a platform to derive organoids and cells from human pluripotent stem cells to model SARS-CoV-2 infection and perform drug screens1,2. This provided insight into cellular tropism and the host response, yet the molecular mechanisms regulating SARS-CoV-2 infection remain poorly defined. Here we systematically examined changes in transcript profiles caused by SARS-CoV-2 infection at different multiplicities of infection for lung airway organoids, lung alveolar organoids and cardiomyocytes, and identified several genes that are generally implicated in controlling SARS-CoV-2 infection, including CIART, the circadian-associated repressor of transcription. Lung airway organoids, lung alveolar organoids and cardiomyocytes derived from isogenic CIART-/- human pluripotent stem cells were significantly resistant to SARS-CoV-2 infection, independently of viral entry. Single-cell RNA-sequencing analysis further validated the decreased levels of SARS-CoV-2 infection in ciliated-like cells of lung airway organoids. CUT&RUN, ATAC-seq and RNA-sequencing analyses showed that CIART controls SARS-CoV-2 infection at least in part through the regulation of NR4A1, a gene also identified from the multi-organoid analysis. Finally, transcriptional profiling and pharmacological inhibition led to the discovery that the Retinoid X Receptor pathway regulates SARS-CoV-2 infection downstream of CIART and NR4A1. The multi-organoid platform identified the role of circadian-clock regulation in SARS-CoV-2 infection, which provides potential therapeutic targets for protection against COVID-19 across organ systems.

SARS CoV-2 detected in neonatal stool remote from maternal COVID-19 during pregnancy.

BACKGROUND: In utero transmission of SARS coronavirus 2 (SARS-CoV-2) has not been fully investigated. We investigated whether newborns of mothers with COVID-19 during pregnancy might harbor SARS-CoV-2 in the gastrointestinal tract. METHODS: This cohort study investigated stool from 14 newborns born at 25-41 weeks admitted at delivery to our urban academic hospital whose mothers had COVID-19 during pregnancy. Eleven mothers had COVID-19 resolved more than 10 weeks before delivery. Newborn stool was evaluated for SARS-CoV-2 RNA, Spike protein, and induction of inflammatory cytokines interleukin-6 (IL-6) and interferon-γ (IFN-γ) in macrophages. RESULTS: Despite negative SARS CoV-2 nasal PCRs from all newborns, viral RNAs and Spike protein were detected in the stool of 11 out of 14 newborns as early as the first day of life and increased over time in 6. Stool homogenates from all 14 newborns elicited elevated inflammatory IL-6 and IFN-γ from macrophages. Most newborns were clinically well except for one death from gestational autoimmune liver disease and another who developed necrotizing enterocolitis. CONCLUSIONS: These findings suggest in utero transmission of SARS-CoV-2 and possible persistent intestinal viral reservoirs in the newborns. Further investigation is required to understand the mechanisms and their clinical implications. IMPACT: SARS-CoV-2 RNAs or Spike protein was detected in the stool of 11 out of 14 preterm newborns born to mothers with resolved COVID-19 weeks prior to delivery despite negative newborn nasal PCR swabs. These novel findings suggest risk of in utero SARS-CoV-2 transmission to the fetal intestine during gestation. The presence of SARS-CoV-2 RNAs and Spike protein in the intestines of newborns may potentially impact the development of the gut microbiome and the immune system; the long-term health impact on the preterm infants should be further investigated.

Multiple Facets of Cellular Homeostasis and Regeneration of the Mammalian Liver.

Liver regeneration occurs in response to diverse injuries and is capable of functionally reestablishing the lost parenchyma. This phenomenon has been known since antiquity, encapsulated in the Greek myth where Prometheus was to be punished by Zeus for sharing the gift of fire with humanity by having an eagle eat his liver daily, only to have the liver regrow back, thus ensuring eternal suffering and punishment. Today, this process is actively leveraged clinically during living donor liver transplantation whereby up to a two-thirds hepatectomy (resection or removal of part of the liver) on a donor is used for transplant to a recipient. The donor liver rapidly regenerates to recover the lost parenchymal mass to form a functional tissue. This astonishing regenerative process and unique capacity of the liver are examined in further detail in this review.