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Background & Aims Hepatocellular carcinoma (HCC) is characterized by intratumoral accumulation of regulatory T-cells (Tregs), which suppresses antitumor immunity. This study was designed to investigate how microRNAs regulate immunosuppression in HCC. Methods FVB/NJ mice were hydrodynamically injected with AKT/Ras or c-Myc and Sleeping Beauty transposon to induce HCC. The Sleeping Beauty system was used to deliver microRNA-15a/16-1 into livers of mice. Flow cytometry and immunostaining were used to determine changes in the immune system. Results Hydrodynamic injection (HDI) of AKT/Ras or c-Myc into mice resulted in hepatic enrichment of Tregs, reduced cytotoxic T cells (CTLs) and HCC development. HCC impaired microRNA-15a/16-1 biogenesis in Kupffer cells (KCs) of AKT/Ras and c-Myc mice. HDI of microRNA-15a/16-1 fully prevented HCC in AKT/Ras and c-Myc mice, while 100% of control mice died from HCC. Therapeutically, microRNA-15a/16-1 promoted a regression of HCC in both mouse models. MicroRNA-15a/16-1 impaired hepatic enrichment of Tregs and increased hepatic CTLs. Mechanistically, a significant increase was observed in serum C-C motif chemokine 22 (CCL22) and transcription of Ccl22 in KCs of AKT/Ras and c-Myc mice. MicroRNA-15a/16-1 prevented KCs from overproducing CCL22 by inhibiting NF-κB that activates transcription of Ccl22. By reducing CCL22 binding to CCR4 on Tregs, microRNA-15a/16-1 impaired Treg chemotaxis. Disrupting the interaction between microRNA-15a/16-1 and NF-κB impaired the ability of microRNA-15a/16-1 to prevent hepatic Treg accumulation and HCC. Depletion of CD8+ T cells and additional treatment of CCL22 recovered growth of HCC that was fully prevented by miR-15a/16. Conclusion MicroRNA-15a/16-1 attenuates immunosuppression by disrupting CCL22-mediated communication between KCs and Tregs. MicroRNA-15a/16-1 represents a potential immunotherapy against HCC.

Supplementary Table 2 from Gene Networks and microRNAs Implicated in Aggressive Prostate Cancer

Free Article from Gene Networks and microRNAs Implicated in Aggressive Prostate Cancer

Prostate cancer, a complex disease, can be relatively harmless or extremely aggressive. To identify candidate genes involved in causal pathways of aggressive prostate cancer, we implemented a systems biology approach by combining differential expression analysis and coexpression network analysis to evaluate transcriptional profiles using lymphoblastoid cell lines from 62 prostate cancer patients with aggressive phenotype (Gleason grade ≥ 8) and 63 prostate cancer patients with nonaggressive phenotype (Gleason grade ≤ 5). From 13,935 mRNA genes and 273 microRNAs (miRNA) tested, we identified significant differences in 1,100 mRNAs and 7 miRNAs with a false discovery rate (FDR) of P = 3.67 × 10−11). The module of interest was characterized by overrepresentation of cell cycle–related genes (FDR = 3.50 × 10−50). From this module, we further defined 20 hub genes that were highly connected to other genes. Interestingly, 5 of the 7 differentially expressed miRNAs have been implicated in cell cycle regulation and 2 (miR-145 and miR-331-3p) are predicted to target 3 of the 20 hub genes. Ectopic expression of these two miRNAs reduced expression of target hub genes and subsequently resulted in cell growth inhibition and apoptosis. These results suggest that cell cycle is likely to be a molecular pathway causing aggressive phenotype of prostate cancer. Further characterization of cell cycle–related genes (particularly, the hub genes) and miRNAs that regulate these hub genes could facilitate identification of candidate genes responsible for the aggressive phenotype and lead to a better understanding of prostate cancer etiology and progression. [Cancer Res 2009;69(24):9490–7]

Supplementary Table 3 from Gene Networks and microRNAs Implicated in Aggressive Prostate Cancer

Supplementary Table 1 from Gene Networks and microRNAs Implicated in Aggressive Prostate Cancer

ObjectivesTo measure changes in cause of death dynamics in 2019 and 2020 and the relationship between concurrent occurrence of the COVID-19 pandemic and mortality outcome by race and ethnicity.Patients and MethodsWe used resident mortality data from the Minnesota Department of Health (MDH) to conduct retrospective statistical analysis of deaths in Minnesota in 2019 relative to 2020 to assess changes in mortality in a pre-pandemic and pandemic period.ResultsCOVID-19 strongly contributed to ethnicity-related mortality disparities in Minnesota. Not only was there a greater proportion of COVID-19 decedents within the Black and Hispanic populations, but their average decedent age was markedly lower relative to the White population. The Black population experienced a disproportionate increase in decedents with a 34% increase during 2020 compared to 2019.ConclusionsThis retrospective analysis of death dynamics and mortality outcomes in Minnesota from 2019 to 2020 demonstrated an increase in adverse mortality outcomes relative to the pre-pandemic period that disproportionately impacted Black and Hispanic minority populations. Access to non-pharmaceutical interventions combating COVID-19 infection in Black and Hispanic communities should be expanded in Minnesota.

Along with the nasal epithelium, the lung epithelium is a portal of entry for sudden acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and many other respiratory viruses. In the case of SARS-CoV-2, the virus surface spike proteins bind to the ACE-2 receptor to facilitate entry into respiratory epithelium. Alveolar type 2 (AT2) cells are committed respiratory progenitor cells responsible for the integrity and regeneration of the respiratory epithelium and production of respiratory surfactant proteins. They express high levels of surface ACE-2 and thus are a leading target for primary infection by the SARS-CoV-2. This study describes a method to directly differentiate TERT-immortalized human cord blood-derived multilineage progenitor cells (MLPC) to AT2-like cells for the purpose of generating an in vitro cellular platform for viral studies. Differentiation was confirmed with the acquisition of AT2 and absence of alveolar type 1 (AT1) specific markers by confocal microscopy. Expression of the ACE-2 receptor was confirmed by immunofluorescence antibody staining, qRT-PCR, and binding of biotinylated SARS-CoV-2 spike (S and S1) proteins. The binding of biotinylated spike proteins was specifically blocked by unlabeled spike proteins and neutralizing antibodies. Additionally, it was demonstrated that the spike protein was internalized after binding to the surface membrane of the cells. We defined the culture conditions that enabled AT2-like cells to be repeatedly passaged and cryopreserved without further differentiation to AT1. Our method provides a stable and renewable source of AT2 cells for respiratory viral binding, blocking and uptake studies., Graphical Abstract AT2-like cells differentiated from TERT-immortalized human MLPC expressed ACE-2, bound SARS-CoV-2 spike proteins and differentiated between RBD-specific and non-RBD-specific neutralizing antibodies. This long-lived cell line could provide a reliable tool to study spike protein binding and inhibition by antibodies or other potential therapeutics designed to inhibit infection by the SARS-CoV-2 virus.Image, graphical abstract

BACKGROUND AND AIMS Intertumoral accumulation of regulatory T cells (Tregs) has been implicated in the pathogenesis of HCC. Because of poor understanding of the immunosuppression mechanism(s) in HCC, immunotherapy is largely unsuccessful for the treatment of HCC. APPROACH AND RESULTS Hydrodynamic injection (HDI) of c-Myc into mice resulted in enlarged spleens and lethal HCC associated with an increase in hepatic Tregs and depletion of CTLs (cytotoxic T lymphocytes). Malignant hepatocytes in c-Myc mice overproduced TGFβ1, which enhanced the suppressor function of Tregs and impaired the proliferation and cytotoxicity of CTLs. In addition to activating TGFβ signaling, c-Myc synergized with Yin Yang 1 to impair microRNA-206 (miR-206) biogenesis. HDI of miR-206 fully prevented HCC and the associated enlargement of the spleen, whereas 100% of control mice died from HCC within 5-9 weeks postinjection. Mechanistically, by directly targeting errant kirsten ras oncogene (KRAS) signaling, miR-206 impeded the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) axis that drives expression of Tgfb1. By blocking the KRAS/MEK/ERK axis, miR-206 prevented TGFβ1 overproduction, thereby impairing the suppressor function and expansion of Tregs, but enhancing the expansion and cytotoxic program of CTLs. Disrupting the interaction between miR-206 and Kras offset the roles of miR-206 in inhibiting immunosuppression and HCC. Depletion of CD8+ T cells impaired the ability of miR-206 to inhibit HCC. CONCLUSIONS c-Myc-educated hepatocytes promoted immunosuppression by overproducing TGFβ1, which promoted HCC development. miR-206, by attenuating TGFβ1 overproduction, disrupted the communication of malignant hepatocytes with CTLs and Tregs, which prevented HCC. miR-206 represents a potential immunotherapeutic agent against HCC.

Intercellular communication is critical for the development of invasive cancers. Multiple forms of intercellular communication have been well characterized, involving diffusible soluble factors or contact-dependent channels for immediately adjacent cells. Over the past 1-2 decades, the emergence of a unique form of F-actin-based cellular protrusion known as tunneling nanotubes (TNTs) has filled the niche of long-range cell-contact dependent intercellular communication that facilitates cell growth, differentiation, and in the case of invasive cancer phenotypes, a more chemoresistant phenotype. The cellular machinery of TNT-mediated transport is an area of active investigation, and microtubules have been implicated in this process as they are in other membranous protrusions. Tumor-Treating Fields (TTFields) therapy is a novel therapeutic strategy in clinical use for patients with advanced cancers, based on the principle of using low-intensity alternating electric fields to disrupt microtubules in cancer cells undergoing mitosis. Other mechanisms of action have also been demonstrated. In this study, we investigated the effects of TTFields on TNTs in malignant pleural mesothelioma (MPM)in vitroand also on the spatial transcriptomic landscapein vivo. We found that applying TTFields at 1.0 V/cm significantly suppressed TNT formation in a biphasic MPM cell line (MSTO-211H), but not in sarcomatoid MPM (VAMT). At these parameters, TTFields significantly reduced cell count in MSTO-211H, but did not significantly alter intercellular transport of mitochondria via intact TNTs. To understand how TTFields may impact expression of genes with known involvement to TNT formation and overall tumor growth, we performed spatial genomic assessment of TTFields-treated tumors from anin vivoanimal model of MPM, and detected upregulation of immuno-oncologic biomarkers with simultaneous downregulation of pathways associated with cell hyperproliferation, invasion, and other critical regulators of oncogenic growth. Several molecular classes and pathways coincide with markers that we and others have found to be differentially expressed in cancer cell TNTs, including MPM specifically. In this study, we report novel cellular and molecular effects of TTFields in relation to tumor communication networks enabled by TNTs and related molecular pathways. These results position TNTs as potential therapeutic targets for TTFields-directed cancer treatment strategies; and also identify the ability of TTFields to potentially remodel the tumor microenvironment, thus enhancing response to immunotherapeutic drugs.

Daniel P Collins,1 Clifford J Steer2,3 1CMDG, LLC, Saint Paul, MN, USA; 2Department of Medicine, University of Minnesota Medical School, Minneapolis, MN, USA; 3Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN, USACorrespondence: Daniel P Collins Email dc@cmdgllc.comBackground: The SARS-CoV-2 virus may have direct or indirect effects on other human organs beyond the respiratory system and including the liver, via binding of the spike protein. This study investigated the potential direct interactions with the liver by comparing the binding of SARS-CoV-2 spike proteins to human AT2-like cells, primary human hepatocytes and immortalized hepatocyte-like hybrid cells. Receptors with binding specificity for SARS-CoV-2 spike protein on AT2 cells and hepatocytes were identified.Methods: The specific binding of biotinylated spike and spike 1 proteins to undifferentiated human E12 MLPC (E12), E12 differentiated alveolar type 2 (AT2) cells, primary human hepatocytes (PHH) and E12 human hepatocyte-like hybrid cells (HLC) was studied by confocal microscopy. We investigated the expression of ACE-2, binding of biotinylated spike protein, biotinylated spike 1 and inhibition of binding by unlabeled spike protein, two neutralizing antibodies and an antibody directed against the hepatocyte asialoglycoprotein receptor 1 (ASGr1).Results: E12 MLPC did not express ACE-2 and did not bind either of spike or spike 1 proteins. AT2-like cells expressed ACE-2 and bound both spike and spike 1. Both PHH and HLC did not express ACE-2 and did not bind spike 1 protein. However, both PHH and HLC actively bound the spike protein. Biotinylated spike protein binding was inhibited by unlabeled spike but not spike 1 protein on PHH and HLC. Two commercial neutralizing antibodies blocked the binding of the spike to PHH and HLC but only one blocked binding to AT2. An antibody to the hepatocyte ASGr1 blocked the binding of the spike protein to PHH and HLC.Conclusion: The absence of ACE-2 receptors and inhibition of spike binding by an antibody to the ASGr1 on both PHH and HLC suggested that the spike protein interacts with the ASGr1. The differential antibody blocking of spike binding to AT2, PHH and HLC indicated that neutralizing activity of SARS-CoV-2 binding might involve additional mechanisms beyond RBD binding to ACE-2.Keywords: asialoglycoprotein receptor, E12 MLPC, SARS-CoV-2, AT2, human hepatocytes, spike proteins

Background Research directed towards drug development, metabolism, and liver functions often utilize primary hepatocytes (PH) for preliminary in vitro studies. Variability in the in vitro functionality of PH and the unsuitability of hepatocarcinoma cells for these studies have driven researchers to look to ESC, iPS, and other stem cell types using differentiation protocols to provide more reliable and available cells. This study describes the development of hepatocyte-like cells through the in vitro differentiation of human TERT-immortalized cord blood-derived multi-lineage progenitor cells (MLPC). The E12 clonal cell line derived from polyclonal TERT-transfected cells was used throughout the study. Methods E12 MLPC were subjected to a three-step differentiation protocol using alternating combinations of growth factors, cytokines, and maturational factors. Cells at various stages of differentiation were analyzed for consistency with PH by morphology, immunohistochemistry, urea production, and gene expression. Results E12 MLPC were shown to significantly change morphology with each stage of differentiation. Coincidental with the morphological changes in the cells, immunohistochemistry data documented the differentiation to committed endoderm by the expression of SOX-17 and GATA-4; the progression to committed hepatocyte-like cells by the expression of a large number of markers including α-fetoprotein and albumin; and the final differentiation by the expression of nuclear and cytoplasmic HNF4. Fully differentiated cells demonstrated gene expression, urea production, and immunohistochemistry consistent with PH. A methodology and medium formulation to continuously expand the E12-derived hepatocyte-like cells is described. Conclusion The availability of immortalized hepatocyte-like cell lines could provide a consistent tool for the study of hepatic diseases, drug discovery, and the development of cellular therapies for liver disorders. Utilization of these techniques could provide a basis for the development of bridge therapies for liver failure patients awaiting transplant.

Mitochondrial dysfunction is implicated in the pathogenesis of Parkinson's disease. Preliminary data have shown lower brain adenosine triphosphate (ATP) levels in Parkinson's disease versus age-matched healthy controls. Ursodeoxycholic acid (UDCA) may improve impaired mitochondrial function. Our objective was to evaluate UDCA tolerability, pharmacokinetics, and its effect on brain bioenergetics in individuals with Parkinson's disease. An open-label, prospective, multiple-ascending-dose study of oral UDCA in 5 individuals with Parkinson's disease was completed. A blood safety panel, plasma concentrations of UDCA and UDCA conjugates, and brain ATP levels were measured before and after therapy (week 1: 15 mg/kg/day; week 2: 30 mg/kg/day; and weeks 3-6: 50 mg/kg/day). UDCA and conjugates were measured using liquid chromatography-mass spectrometry. ATP levels and ATPase activity were measured using 7-Tesla 31 P magnetic resonance spectroscopy. Secondary measures included the Unified Parkinson's Disease Rating Scale and Montreal Cognitive Assessment. UDCA was generally well tolerated. The most frequent adverse event was gastrointestinal discomfort, rated by subjects as mild to moderate. Noncompartmental pharmacokinetic analysis resulted in (mean ± standard deviation) a maximum concentration of 8749 ± 2840 ng/mL and half-life of 2.1 ± 0.71 hr. Magnetic resonance spectroscopy data were obtained in 3 individuals with Parkinson's disease and showed modest increases in ATP and decreases in ATPase activity. Changes in Unified Parkinson's Disease Rating Scale (parts I-IV) and Montreal Cognitive Assessment scores (mean ± standard deviation) were -4.6 ± 6.4 and 2 ± 1.7, respectively. This is the first report of UDCA use in individuals with Parkinson's disease. Its pharmacokinetics are variable, and at high doses it appears reasonably well tolerated. Our findings warrant additional studies of its effect on brain bioenergetics.

Background Primary human hepatocytes (PHHs) are the ideal candidates for studying critical liver functions such as drug metabolism and toxicity. However, as they are isolated from discarded livers that are unsuitable for transplantation, they possess limited expansion ability in vitro and their enzymatic functions deteriorate rapidly because they are often of poor quality. Therefore, there is a compelling reason to find reliable alternative sources of hepatocytes. Methods In this study, we report on efficient and robust differentiation of embryonic stem cells (ESC) from the common marmoset Callithrix jacchus into functional hepatocyte-like cells (HLC) using a simple, and reproducible three-step procedure. ESC-derived HLCs were examined by morphological analysis and tested for their expression of hepatocyte-specific markers using a combination of immunohistochemistry, RT-PCR, and biochemical assays. Primary human hepatocytes were used as controls. Results ESC-derived HLCs expressed each of the hepatocyte-specific markers tested, including albumin; α-fetoprotein; asialoglycoprotein receptor 1; α-1 antitrypsin; hepatocyte nuclear factors 1α and 4; cytokeratin 18; hepatocyte growth factor receptor; transferrin; tyrosine aminotransferase; alkaline phosphatase; c-reactive protein; cytochrome P450 enzymes CYP1A2, CYP2E1 and CYP3A4; and coagulation factors FVII and FIX. They were functionally competent as demonstrated by biochemical assays in addition to producing urea. Conclusion Our data strongly suggest that marmoset HLCs possess characteristics similar to those of PHHs. They could, therefore, be invaluable for studies on drug metabolism and cell transplantation therapy for a variety of liver disorders. Because of the similarities in the anatomical and physiological features of the common marmoset to that of humans, Callithrix jacchus is an appropriate animal model to study human disease conditions and cellular functions.

The large number of monogenic metabolic disorders originating in the liver poses a unique opportunity for development of gene therapy modalities to pursue curative approaches. Various disorders have been successfully treated via liver-directed gene therapy, though most of the advances have been in animal models, with only limited success in clinical trials. Pre-clinical data in animals using non-viral approaches, including the Sleeping Beauty transposon system, are discussed. The various advances with viral vectors for liver-directed gene therapy are also a focus of this review, including retroviral, adenoviral, recombinant adeno-associated viral, and SV40 vectors. Genome editing techniques, including zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats (CRISPR), are also described. Further, the various controversies in the field with regards to somatic vs. germline editing using CRISPR in humans are explored, while also highlighting the myriad of preclinical advances. Lastly, newer technologies are reviewed, including base editing and prime editing, which use CRISPR with exciting adjunctive properties to avoid double-stranded breaks and thus the recruitment of endogenous repair mechanisms. While encouraging results have been achieved recently, there are still significant challenges to overcome prior to the broad use of vector-based and genome editing techniques in the clinical arena. As these technologies mature, the promise of a cure for many disabling inherited metabolic disorders is within reach, and urgently needed.

The secondary bile acid ursodeoxycholic acid (UDCA) has long been known to have medicinal properties. As the therapeutically active component of bear bile, it has been used for centuries in traditional Chinese medicine to treat a range of conditions, while manufactured UDCA has been used for decades in Western medicine to treat cholestatic liver diseases. The beneficial qualities of UDCA are thought to be due to its well-established cytoprotective and anti-inflammatory actions. In addition to its established role in treating liver diseases, UDCA is now under investigation for numerous conditions associated with inflammation and apoptosis, including neurological, ocular, metabolic, and cardiovascular diseases. Here, we review the growing evidence base from in vitro and in vivo models to suggest that UDCA may also have a role to play in the therapy of inflammatory bowel diseases.

Cirrhosis is characterized by extensive hepatic fibrosis, and it is the 14th leading cause of death worldwide. Numerous contributing conditions have been implicated in its development, including infectious etiologies, medication overdose or adverse effects, ingestible toxins, autoimmunity, hemochromatosis, Wilson's disease and primary biliary cholangitis to list a few. It is associated with portal hypertension and its stigmata (varices, ascites, hepatic encephalopathy, combined coagulopathy and thrombophilia), and it is a major risk factor for hepatocellular carcinoma. Currently, orthotopic liver transplantation has been the only curative modality to treat cirrhosis, and the scarcity of donors results in many people waiting years for a transplant. Identification of novel targets for pharmacologic therapy through elucidation of key mechanistic components to induce fibrosis reversal is the subject of intense research. Development of robust models of hepatic fibrosis to faithfully characterize the interplay between activated hepatic stellate cells (the principal fibrogenic contributor to fibrosis initiation and perpetuation), hepatocytes and extracellular matrix components has the potential to identify critical components and mechanisms that can be exploited for targeted treatment. In this review, we will highlight key cellular pathways involved in the pathophysiology of fibrosis from extracellular ligands, effectors and receptors, to nuclear receptors, epigenetic mechanisms, energy homeostasis and cytokines. Further, molecular pathways of hepatic stellate cell deactivation are discussed, including apoptosis, senescence and reversal or transdifferentiation to an inactivated state resembling quiescence. Lastly, clinical evidence of fibrosis reversal induced by biologics and small molecules is summarized, current compounds under clinical trials are described and efforts for treatment of hepatic fibrosis with mesenchymal stem cells are highlighted. An enhanced understanding of the rich tapestry of cellular processes identified in the initiation, perpetuation and resolution of hepatic fibrosis, driven principally through phenotypic switching of hepatic stellate cells, should lead to a breakthrough in potential therapeutic modalities.

Blastocyst complementation combined with gene editing is an emerging approach in the field of regenerative medicine that could potentially solve the worldwide problem of organ shortages for transplantation. In theory, blastocyst complementation can generate fully functional human organs or tissues, grown within genetically engineered livestock animals. Targeted deletion of a specific gene(s) using gene editing to cause deficiencies in organ development can open a niche for human stem cells to occupy, thus generating human tissues. Within this review, we will focus on the pancreas, liver, heart, kidney, lung, and skeletal muscle, as well as cells of the immune and nervous systems. Within each of these organ systems, we identify and discuss (i) the common causes of organ failure; (ii) the current state of regenerative therapies; and (iii) the candidate genes to knockout and enable specific exogenous organ development via the use of blastocyst complementation. We also highlight some of the current barriers limiting the success of blastocyst complementation.

This article describes the University of Minnesota Medical School Proposal Preparation Program (P3). P3 is designed to develop grant-writing skills for assistant professors who are preparing their first K- or R-series application to the National Institutes of Health (NIH). Three 4-month P3 cycles are conducted annually. For each cycle, a cohort of around 10 assistant professor participants and 5 regular faculty mentors meet for ten ~2-hour group sessions. Participants receive iterative oral and written feedback on their proposals-in-development within a small, interdisciplinary, group mentoring setting that provides structure, accountability, guidance, and support. Between sessions, one peer and one mentor are assigned (on a rotating basis) to critique a participant's developing application. The sessions include a brief mentor-led presentation on a particular grant section followed by discussion of each participant's application conducted by the assigned reviewers. The cycle concludes with a mock NIH review session, in which each participant is matched with a University of Minnesota faculty content expert who critiques their completed application using NIH guidelines. In a survey sent to all past P3 participants as of 2018 (n = 194), 88% of respondents reported having submitted their P3-developed NIH grant, and 35% of these submitters reported funding success. A separate analysis of institutional data for all past P3 participants as of 2016 (n = 165) showed that 73% submitted at least one NIH proposal since completing P3 and that 43% of these had acquired NIH funding, for a combined total of $193 million in funding awarded. The estimated rate at which participants obtained funding for their P3-developed grant application (~35%) exceeds the national annual NIH grant funding rates (~20%) by approximately 50%. This article provides the practical information needed for other institutions to implement a P3-like program and presents a cost-benefit analysis showing the advantages of doing so.

Several studies have indicated the presence of microRNAs (miRNAs) within mitochondria although the origin, as well as the biological function, of these mitochondrially located miRNAs is largely unknown. The identification and significance of this subcellular localization is gaining increasing relevance to the pathogenesis of certain disease states. Here, we describe the isolation of highly purified mitochondria from rat liver by differential centrifugation, followed by RNAse A treatment to eliminate contaminating RNA. The coupled extraction of total RNA and protein is a more efficient design for allowing the downstream evaluation of miRNA and protein expression in mitochondria.

Background There are over 17,000 patients in the US waiting to receive liver transplants, and these numbers are increasing dramatically. Significant effort is being made to obtain functional hepatocytes and liver tissue that can for therapeutic use in patients. Blastocyst complementation is a challenging, innovative technology that could fundamentally change the future of organ transplantation. It requires the knockout (KO) of genes essential for cell or organ development in early stage host embryos followed by injection of donor pluripotent stem cells (PSCs) into host blastocysts to generate chimeric offspring in which progeny of the donor cells populate the open niche to develop functional tissues and organs. Methods The HHEX gene is necessary for proper liver development. We engineered loss of HHEX gene expression in early mouse and pig embryos and performed intraspecies blastocyst complementation of HHEX KO embryos with eGFP-labeled PSCs in order to rescue the loss of liver development. Results Loss of HHEX gene expression resulted in embryonic lethality at day 10.5 in mice and produced characteristics of lethality at day 18 in pigs, with absence of liver tissue in both species. Analyses of mouse and pig HHEX KO fetuses confirmed significant loss of liver-specific gene and protein expression. Intraspecies blastocyst complementation restored liver formation and liver-specific proteins in both mouse and pig. Livers in complemented chimeric fetuses in both species were comprised of eGFP-labeled donor-derived cells and survived beyond the previously observed time of HHEX KO embryonic lethality. Conclusions This work demonstrates that loss of liver development in the HHEX KO can be rescued via blastocyst complementation in both mice and pigs. This complementation strategy is the first step towards generating interspecies chimeras for the goal of producing human liver cells, tissues, and potentially complete organs for clinical transplantation.

Abnormal energy metabolism associated with mitochondrial dysfunction is thought to be a major contributor to the progression of neurodegenerative diseases such as Parkinson’s disease (PD). Recent advancements in the field of magnetic resonance (MR) based metabolic imaging provide state-of-the-art technologies for non-invasively probing cerebral energy metabolism under various brain conditions. In this proof-of-principle clinical study, we employed quantitative 31P MR spectroscopy (MRS) imaging techniques to determine a constellation of metabolic and bioenergetic parameters, including cerebral adenosine triphosphate (ATP) and other phosphorous metabolite concentrations, intracellular pH and nicotinamide adenine dinucleotide (NAD) redox ratio, and ATP production rates in the occipital lobe of cognitive-normal PD patients, and then we compared them with age-sex matched healthy controls. Small but statistically significant differences in intracellular pH, NAD and ATP contents and ATPase enzyme activity between the two groups were detected, suggesting that subtle defects in energy metabolism and mitochondrial function are quantifiable before regional neurological deficits or pathogenesis begin to occur in these patients. Pilot data aiming to evaluate the bioenergetic effect of mitochondrial-protective bile acid, ursodeoxycholic acid (UDCA) were also obtained. These results collectively demonstrated that in vivo 31P MRS-based neuroimaging can non-invasively and quantitatively assess key metabolic-energetic metrics in the human brain. This provides an exciting opportunity to better understand neurodegenerative diseases, their progression and response to treatment.

Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) proteins can be designed to bind specified DNA and RNA sequences and hold great promise for the accurate detection of nucleic acids for diagnostics. We integrated commercially available reagents into a CRISPR/Cas9-based lateral flow assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences with single-base specificity. This approach requires minimal equipment and represents a simplified platform for field-based deployment. We also developed a rapid, multiplex fluorescence CRISPR/Cas9 nuclease cleavage assay capable of detecting and differentiating SARS-CoV-2, influenza A and B, and respiratory syncytial virus in a single reaction. Our findings provide proof-of-principle for CRISPR/Cas9 point-of-care diagnosis as well as a scalable fluorescent platform for identifying respiratory viral pathogens with overlapping symptomology.

Monogenic metabolic disorders of hepatic origin number in the hundreds, and for many, liver transplantation remains the only cure. Liver-targeted gene therapy is an attractive treatment modality for many of these conditions, and there have been significant advances at both the preclinical and clinical stages. Viral vectors, including retroviruses, lentiviruses, adenovirus-based vectors, adeno-associated viruses and simian virus 40, have differing safety, efficacy and immunogenic profiles, and several of these have been used in clinical trials with variable success. In this review, we profile viral vectors and non-viral vectors, together with various payloads, including emerging therapies based on RNA, that are entering clinical trials. Genome editing technologies are explored, from earlier to more recent novel approaches that are more efficient, specific and safe in reaching their target sites. The various curative approaches for the multitude of monogenic hepatic metabolic disorders currently at the clinical development stage portend a favorable outlook for this class of genetic disorders.

The incidence of liver disease is increasing significantly worldwide and, as a result, there is a pressing need to develop new technologies and applications for end-stage liver diseases. For many of them, orthotopic liver transplantation is the only viable therapeutic option. Stem cells that are capable of differentiating into all liver cell types and could closely mimic human liver disease are extremely valuable for disease modeling, tissue regeneration and repair, and for drug metabolism studies to develop novel therapeutic treatments. Despite the extensive research efforts, positive results from rodent models have not translated meaningfully into realistic preclinical models and therapies. The common marmoset Callithrix jacchus has emerged as a viable non-human primate model to study various human diseases because of its distinct features and close physiologic, genetic and metabolic similarities to humans. C. jacchus embryonic stem cells (cjESC) and recently generated cjESC-derived hepatocyte-like cells (cjESC-HLCs) could fill the gaps in disease modeling, liver regeneration and metabolic studies. They are extremely useful for cell therapy to regenerate and repair damaged liver tissues in vivo as they could efficiently engraft into the liver parenchyma. For in vitro studies, they would be advantageous for drug design and metabolism in developing novel drugs and cell-based therapies. Specifically, they express both phase I and II metabolic enzymes that share similar substrate specificities, inhibition and induction characteristics, and drug metabolism as their human counterparts. In addition, cjESCs and cjESC-HLCs are advantageous for investigations on emerging research areas, including blastocyst complementation to generate entire livers, and bioengineering of discarded livers to regenerate whole livers for transplantation.

The incidence of liver disease is increasing significantly worldwide and, as a result, there is a pressing need to develop new technologies and applications for end-stage liver diseases. For many of them, orthotopic liver transplantation is the only viable therapeutic option. Stem cells that are capable of differentiating into all liver cell types and could closely mimic human liver disease are extremely valuable for disease modeling, tissue regeneration and repair, and for drug metabolism studies to develop novel therapeutic treatments. Despite the extensive research efforts, positive results from rodent models have not translated meaningfully into realistic preclinical models and therapies. The common marmoset Callithrix jacchus has emerged as a viable non-human primate model to study various human diseases because of its distinct features and close physiologic, genetic and metabolic similarities to humans. C. jacchus embryonic stem cells (cjESC) and recently generated cjESC-derived hepatocyte-like cells (cjESC-HLCs) could fill the gaps in disease modeling, liver regeneration and metabolic studies. They are extremely useful for cell therapy to regenerate and repair damaged liver tissues in vivo as they could efficiently engraft into the liver parenchyma. For in vitro studies, they would be advantageous for drug design and metabolism in developing novel drugs and cell-based therapies. Specifically, they express both phase I and II metabolic enzymes that share similar substrate specificities, inhibition and induction characteristics, and drug metabolism as their human counterparts. In addition, cjESCs and cjESC-HLCs are advantageous for investigations on emerging research areas, including blastocyst complementation to generate entire livers, and bioengineering of discarded livers to regenerate whole livers for transplantation.

Corona Virus Disease 2019 (COVID-19) has affected over 8 million people worldwide. We underscore the potential benefits of conducting a randomized open-label unblinded clinical trial to evaluate the role of ursodeoxycholic acid (UDCA) in the treatment of COVID-19. Some COVID-19 patients are characterized with cytokine storm syndrome that can cause severe and irreversible damage to organs leading to multi-organ failure and death. Therefore, it is critical to control both programmed cell death (apoptosis) and the hyper-immune inflammatory response in COVID-19 patients to reduce the rising morbidity and mortality. UDCA is an existing drug with proven safety profiles that can reduce inflammation and prevent cell death. National Geographic reported that, “China Promotes Bear Bile as Coronavirus Treatment”. Bear bile is rich in UDCA, comprising up to 40–50% of the total bile acid. UDCA is a logical and attainable replacement for bear bile that is available in pill form and merits clinical trial consideration.

Nonalcoholic fatty liver disease (NAFLD) is a major risk factor of many end‐stage liver diseases. Alterations in microRNA expression have been reported in patients with NAFLD. However, the transcriptional mechanism(s) of dysregulated microRNAs under the state of NAFLD is poorly described, and microRNAs that regulate the pathogenesis of NAFLD synergistically with their regulators remain unknown. Here we report that microRNA‐378 expression is significantly increased in fatty livers of mice and patients with NAFLD. Although microRNA‐378 locates within the intron of Ppargc1β (peroxisome proliferator‐activated receptor γ coactivator 1‐beta), there was a significant uncoupling of Ppargc1β mRNA and microRNA‐378 levels in both sources of fatty livers. Further studies identified a full‐length primary transcript of microRNA‐378. LXRα (liver X receptor alpha) functioned as a transcription activator of microRNA‐378 and a repressor of Ppargc1β transcription. It is known that miR‐378 is an inhibitor of fatty acid oxidation (FAO) and the function of Ppargc1β is opposite to that of miR‐378. GW3965 treatment (LXRα agonist) of murine hepatocytes and mice increased microRNA‐378 and reduced Ppargc1β, which subsequently impaired FAO and aggravated hepatosteatosis. In contrast, additional treatment of miR‐378 inhibitor or Ppargc1β, which knocked down increased miR‐378 or recovered expression of Ppargc1β, offset the effects of GW3965. Liver‐specific ablation of Lxrα led to decreased miR‐378 and increased Ppargc1β, which subsequently improved FAO and reduced hepatosteatosis. Conclusion: Our findings indicated that miR‐378 possesses its own transcription machinery, which challenges the well‐established dogma that miR‐378 transcription is controlled by the promoter of Ppargc1β. LXRα selectively activates transcription of miR‐378 and inhibits expression of Ppargc1β, which synergistically impairs FAO. In addition to lipogenesis, impaired FAO by miR‐378 in part contributes to LXRα‐induced hepatosteatosis.

Background: Application of alternating-electric fields as cancer-directed therapy known as Tumor-Treating Fields (TTFields) has been shown to be effective by exerting dipole alignment forces on polar microtubule subunits and dielectrophoretic forces in the cytokinetic furrow. This effect results in disruption of mitosis at the cellular level, and at the clinical level with significantly prolonged overall survival of patients with glioblastoma and malignant pleural mesothelioma (MPM). The molecular alterations that occur at the genomic and transcriptomic levels following TTFields treatment are unknown. We applied a spatial omics approach to elucidate spatial intratumoral effects of TTFields at the molecular level in regions of interest (ROI) in a mouse model of MPM. Methods: Eight Balb/C mice were injected with AB1 MPM cells until sizable tumors were observed (2-3 weeks). TTFields were applied, with heat sham used as a negative control. After 14 days (6 days of treatment, 2 days of rest, then 6 more days of treatment) tumors were resected, fixed and paraffin embedded. Following the Nanostring GeoMx protocol, thin sections of all eight tumors were placed on a slide, and incubated with both Ki-67 antibodies, and a GeoMx Mouse Cancer Transcriptome Atlas RNA probe set. Using Ki-67 staining as a guide, 12 ROIs were selected across each tumor to capture intratumoral heterogeneity, including the core and periphery of each tumor The DSP barcoded RNA probes were cleaved, sequenced and analyzed. We compared differential expression of subset gene classes of TTFields vs heat sham treated tumors; results were further stratified into Ki-67 high and low subsets for each sample. Results were assessed by Gene Set Enrichment. Results: Sham-treated tumors grew to over 300 mm3; TTFields-treated tumors averaged 100 mm3, confirming anti-tumor effect. Gene Set Enrichment Analysis uncovered upregulation of genes associated with interferon-alpha and -gamma responses in TTFields-treated tumors, which also displayed downregulation of pathway components associated with glycolysis, mTOR signaling, oxidative phosphorylation, cell invasion, hypoxia, and TNF-alpha signaling. Spatial analysis detected a heterogeneous response and differential expression between different portions of tumors. Conclusions: TTFields application induces clear patterns of differential expression in the transcriptome of treated tumors, including an increasingly immune-stimulated tumor microenvironment that also affects molecular pathways critical to cellular proliferation and invasion. These findings point the way toward improved understanding of timing and sequences of TTFields in relation to systemic cytotoxic, targeted, and immune modulation forms of therapeutic strategies. Our finding of upregulated immune response implicates TTFields as a potential synergistic tactic when coupled with immunotherapeutic approaches. Citation Format: Emil Lou, Katherine Ladner, Kerem Wainer-Katsir, Karina Deniz, Yaara Porat, Boris Brant, Shiri Davidi, Yuki Padmanabhan, Phillip Wong, Amrinder Nain, Clifford J. Steer, Moshe Giladi. Spatial omic changes of malignant pleural mesothelioma following treatment using tumor-treating fields [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2037.

Technology for precise and efficient genetic editing is constantly evolving and is now capable of human clinical applications. Autoimmune and inflammatory diseases are chronic, disabling, sometimes life-threatening, conditions that feature heritable components. Both primary genetic lesions and the inflammatory pathobiology underlying these diseases represent fertile soil for new therapies based on the capabilities of gene editing. The ability to orchestrate precise targeted modifications to the genome will likely enable cell-based therapies for inflammatory diseases such as monogenic autoinflammatory disease, acquired autoimmune disease and for regenerative medicine in the setting of an inflammatory environment. Here, we discuss recent advances in genome editing and their evolving applications in immunoinflammatory diseases. Strengths and limitations of older genetic modification tools are compared with CRISPR/Cas9, base editing, RNA editing, targeted activators and repressors of transcription and targeted epigenetic modifiers. Commonly employed delivery vehicles to target cells or tissues of interest with genetic modification machinery, including viral, non-viral and cellular vectors, are described. Finally, applications in animal and human models of inflammatory diseases are discussed. Use of chimeric autoantigen receptor T cells, correction of monogenic diseases with genetically edited haematopoietic stem and progenitor cells, engineering of induced pluripotent stem cells and ex vivo expansion and modification of regulatory T cells for a range of chronic inflammatory diseases are reviewed.

Backgrounds The incidence of nonalcoholic fatty liver disease (NAFLD) is rapidly increasing due to the prevalence of obesity. NAFLD is a major risk factor of hepatocellular carcinoma (HCC). Even with successful surgical removal, the presence of NAFLD is associated with an increased recurrence of HCC. Despite the extensive study of NAFLD, its underlying mechanism(s) remains essentially unknown and there are no FDA-approved drugs for its treatment. Alterations in microRNA (miR) expression have been observed in human fatty livers. However, regulatory mechanism(s) of miRNA biogenesis and their role in regulating the development of NAFLD is poorly described. Methods We used immunohistochemistry, luciferase assays and immunoblotting to study the regulatory mechanism of miR-378 biogenesis. Wild-type mice kept on a high fat diet (HFD) were injected with miR-378 inhibitors or a mini-circle expression system containing miR-378 to study loss and gain-of functions of miR-378. Results miR-378 was significantly increased in fatty livers of dietary obese mice and human hepatoma HepG2 cells with accumulated lipid. Further studies identified NRF1 (Nuclear receptor factor 1), a key regulator of fatty acid oxidation (FAO), as a direct target of miR-378. Overexpression of miR-378 impaired FAO and promoted lipid accumulation in murine hepatoma Hepa1-6 cells. In contrast, knockdown of miR-378 using its ASO (anti-sense oligo) improved FAO and reduced intracellular lipid content in Hepa1-6 cells. Liver-specific expression of miR-378 impaired FAO, which subsequently promoted the development of hepatosteatosis. Antagonizing miR-378 via injecting miR-378-ASO into HFD-treated mice led to increased expression of Nrf1, improved FAO and decreased hepatosteatosis. Additional knockdown of up-regulated Nrf1 offset the effects of miR-378-ASO, suggesting that Nrf1 mediated the inhibitory effect of miR-378-ASO on hepatosteatosis. Furthermore, Nrf1 was identified as a transcriptional repressor of miR-378. Ablation of Nrf1 using its shRNA in livers led to increased miR-378, which subsequently resulted in reduced FAO and elevated hepatic lipid content. Conclusions These findings identified a negative feedback loop between miR-378 and Nrf1 that promotes the pathogenesis of hepatosteatosis, and suggests the use of miR-378 as a potential therapeutic target for NAFLD.