Chimeric Livers: Interspecies Blastocyst Complementation and Xenotransplantation for End-Stage Liver Disease.

Orthotopic liver transplantation (OLT) currently serves as the sole definitive treatment for thousands of patients suffering from end-stage liver disease; and the existing supply of donor livers for OLT is drastically outpaced by the increasing demand. To alleviate this significant gap in treatment, several experimental approaches have been devised with the aim of either offering interim support to patients waiting on the transplant list or bioengineering complete livers for OLT by infusing them with fresh hepatic cells. Recently, interspecies blastocyst complementation has emerged as a promising method for generating complete organs in utero over a short timeframe. When coupled with gene editing technology, it has brought about a potentially revolutionary transformation in regenerative medicine. Blastocyst complementation harbors notable potential for generating complete human livers in large animals, which could be used for xenotransplantation in humans, addressing the scarcity of livers for OLT. Nevertheless, substantial experimental and ethical challenges still need to be overcome to produce human livers in larger domestic animals like pigs. This review compiles the current understanding of interspecies blastocyst complementation and outlines future possibilities for liver xenotransplantation in humans.

Analysis of SARS-CoV-2 Variant-Specific Serum Antibody Post-Vaccination Utilizing Immortalized Human Hepatocyte-Like Cells (HLC) to Assess Development of Immunity.

Background: Our previous studies demonstrated that SARS-CoV-2 spike protein could bind to primary hepatocytes and immortalized Hepatocyte-like cells (HLC) via the asialoglycoprotein receptor-1 (ASGR-1). The binding of biotinylated spike protein could be inhibited by Spike-neutralizing monoclonal antibodies, anti-ASGR-1 antibodies and unlabeled spike protein. The cells were unable to bind Spike S1 and Spike S1 was incapable of blocking labeled Spike protein, suggesting that the Receptor Binding Domain (RBD) was not involved in the binding event. This study was done to investigate the utility of these cells and immortalized alveolar type 2-like (AT-2) cells in studying the development of variant-specific antibodies post-vaccination. Methods: Serum was collected from 10 individuals pre- and post-vaccination with the J&J, Moderna or Pfizer vaccines. The serum samples were quantified for variant-specific antibodies in a flow cytometry-based immunofluorescent assay utilizing beads coated with biotinylated variant spike proteins. Inhibition of spike protein binding to HLC and AT-2 cells by donor serum was analyzed by immunofluorescent confocal analysis. Results: All variant spike proteins bound to HLC and AT-2 cells. Post-vaccination serum samples demonstrated increases of SARS-CoV-2 antibody levels from 2 weeks to 2.5 months post-vaccination with associated increased spike-blocking capacity. It was also demonstrated that vaccination with all the available vaccines stimulated antibodies that inhibited binding of all the available variant spike proteins to both HLC and AT-2 cells. Conclusion: HLC, along with AT-2 cells, provides a useful platform to study the development of neutralizing antibodies post-vaccination. Vaccination with the 3 available vaccines all elicited neutralizing serum antibodies that inhibited binding of each of the variant spike proteins to both AT-2 and HLC cells. This study suggests that inhibition of spike binding to target cells may be a more useful technique to assess immunity than gross quantitation of antibody.

MicroRNA-206-3p suppresses hepatic lipogenesis and cholesterol synthesis while driving cholesterol efflux.

BACKGROUND AND AIMS: Hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia are interconnected metabolic disorders. This study is designed to characterize how microRNA-206-3p (miR-206) simultaneously prevents de novo lipogenesis (DNL), cholesterol synthesis, and VLDL production in hepatocytes while promoting cholesterol efflux in macrophages. APPROACH AND RESULTS: MiR-206 levels were reduced in hepatocytes and macrophages of mice subjected to a high-fat, high-cholesterol diet. A negative feedback between LXRα (liver X receptor alpha) and miR-206 is formed to maintain high LXRα and low miR-206 in hepatocytes. Systemic administration of miR-206 alleviated hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia in mice. A significant reduction in LDL cholesterol and VLDL cholesterol but unaltered HDL cholesterol was observed in miR-206-treated mice. Mirroring these findings, miR-206 reprogrammed the transcriptome of hepatocytes towards the inhibition of DNL, cholesterol synthesis, and assembly and secretion of VLDL. In macrophages, miR-206 activated the expression of genes regulating cholesterol efflux. Hepatocyte-specific expression of miR-206 reduced hepatic and circulating triglycerides and cholesterol, as well as VLDL production, while transplantation of macrophages bearing miR-206 facilitated cholesterol efflux. Mechanistically, miR-206 directly targeted Lxrα and Hmgcr in hepatocytes but facilitated expression of Lxrα in macrophages by targeting macrophage-specific tricho-rhino-phalangeal syndrome 1 (TRPS1), a transcription repressor of Lxrα . By targeting Hmgc r and Lxrα , miR-206 inhibited DNL, VLDL production, and cholesterol synthesis in hepatocytes, whereas it drove cholesterol efflux by activating the TRPS1-LXRα axis. CONCLUSIONS: MiR-206, through differentially modulating LXRα signaling in hepatocytes and macrophages, inhibits DNL, promotes cholesterol efflux, and concurrently hinders cholesterol synthesis and VLDL production. MiR-206 simulates the functions of lipid-lowering medications, statins, and LXRα agonists.

Treatment with tumor-treating fields (TTFields) suppresses intercellular tunneling nanotube formation in vitro and upregulates immuno-oncologic biomarkers in vivo in malignant mesothelioma.

Disruption of intercellular communication within tumors is emerging as a novel potential strategy for cancer-directed therapy. Tumor-Treating Fields (TTFields) therapy is a treatment modality that has itself emerged over the past decade in active clinical use for patients with glioblastoma and malignant mesothelioma, based on the principle of using low-intensity alternating electric fields to disrupt microtubules in cancer cells undergoing mitosis. There is a need to identify other cellular and molecular effects of this treatment approach that could explain reported increased overall survival when TTFields are added to standard systemic agents. Tunneling nanotube (TNTs) are cell-contact-dependent filamentous-actin-based cellular protrusions that can connect two or more cells at long-range. They are upregulated in cancer, facilitating cell growth, differentiation, and in the case of invasive cancer phenotypes, a more chemoresistant phenotype. To determine whether TNTs present a potential therapeutic target for TTFields, we applied TTFields to malignant pleural mesothelioma (MPM) cells forming TNTs in vitro. TTFields at 1.0 V/cm significantly suppressed TNT formation in biphasic subtype MPM, but not sarcomatoid MPM, independent of effects on cell number. TTFields did not significantly affect function of TNTs assessed by measuring intercellular transport of mitochondrial cargo via intact TNTs. We further leveraged a spatial transcriptomic approach to characterize TTFields-induced changes to molecular profiles in vivo using an animal model of MPM. We discovered TTFields induced 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. We visualized short TNTs in the dense stromatous tumor material selected as regions of interest for spatial genomic assessment. Superimposing these regions of interest from spatial genomics over the plane of TNT clusters imaged in intact tissue is a new method that we designate Spatial Profiling of Tunneling nanoTubes (SPOTT). In sum, these results position TNTs as potential therapeutic targets for TTFields-directed cancer treatment strategies. We also identified the ability of TTFields to remodel the tumor microenvironment landscape at the molecular level, thereby presenting a potential novel strategy for converting tumors at the cellular level from 'cold' to 'hot' for potential response to immunotherapeutic drugs.

A positive feedback between cholesterol synthesis and the pentose phosphate pathway rather than glycolysis promotes hepatocellular carcinoma.

Hepatic cholesterol accumulation and hypercholesterolemia are implicated in hepatocellular carcinoma (HCC). However, the therapeutic effects of cholesterol-lowering drugs on HCC are controversial, indicating that the relationship between cholesterol metabolism and HCC is more complex than anticipated. A positive feedback between cholesterol synthesis and the pentose phosphate pathway (PPP) rather than glycolysis was formed in tumors of c-Myc mice. Blocking the PPP prevented cholesterol synthesis and thereby HCC in c-Myc mice, while ablating glycolysis did not affect cholesterol synthesis and failed to prevent c-Myc-induced HCC. Unexpectedly, HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase) and G6PD (glucose-6-phosphate dehydrogenase), the rate-limiting enzymes of cholesterol synthesis and the PPP, were identified as direct targets of microRNA-206. By targeting Hmgcr and G6pd, microRNA-206 disrupted the positive feedback and fully prevented HCC in c-Myc mice, while 100% of control mice died of HCC. Disrupting the interaction of microRNA-206 with Hmgcr and G6pd restored cholesterol synthesis, the PPP and HCC growth that was inhibited by miR-206. This study identified a previously undescribed positive feedback loop between cholesterol synthesis and the PPP, which drives HCC, while microRNA-206 prevents HCC by disrupting this loop. Cholesterol synthesis as a process rather than cholesterol itself is the major contributor of HCC.

Cause of Death by Race and Ethnicity in Minnesota Before and During the COVID-19 Pandemic, 2019-2020.

OBJECTIVES: To measure changes in cause of death dynamics in 2019 and 2020 and the relationship between the concurrent occurrence of the COVID-19 pandemic and mortality outcome by race and ethnicity. PATIENTS AND METHODS: We used resident mortality data from the Minnesota Department of Health (MDH) to conduct a retrospective statistical analysis of deaths in Minnesota in 2019 relative to 2020 to assess changes in mortality in a pre-pandemic and pandemic period. RESULTS: COVID-19 strongly contributed to ethnicity-related mortality disparities in Minnesota. Not only was there a greater proportion of COVID-19 decedents within 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. CONCLUSIONS: This 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.

Liver Regeneration in Acute on Chronic Liver Failure.

Liver regeneration is a multifaceted process by which the organ regains its original size and histologic organization. In recent decades, substantial advances have been made in our understanding of the mechanisms underlying regeneration following loss of hepatic mass. Liver regeneration in acute liver failure possesses several classic pathways, while also exhibiting unique differences in key processes such as the roles of differentiated cells and stem cell analogs. Here we summarize these unique differences and new molecular mechanisms involving the gut-liver axis, immunomodulation, and microRNAs with an emphasis on applications to the patient population through stem cell therapies and prognostication.

In Silico Stage-Matching of Human, Marmoset, Mouse, and Pig Embryos to Enhance Organ Development Through Interspecies Chimerism.

Currently, there is a significant shortage of transplantable organs for patients in need. Interspecies chimerism and blastocyst complementation are alternatives for generating transplantable human organs in host animals such as pigs to meet this shortage. While successful interspecies chimerism and organ generation have been observed between evolutionarily close species such as rat and mouse, barriers still exist for more distant species pairs such as human-mouse, marmoset-mouse, human-pig, and others. One of the proposed barriers to chimerism is the difference in developmental stages between the donor cells and the host embryo at the time the cells are introduced into the host embryo. Hence, there is a logical effort to stage-match the donor cells with the host embryos for enhancing interspecies chimerism. In this study, we used an in silico approach to simultaneously stage-match the early developing embryos of four species, including human, marmoset, mouse, and pig based on transcriptome similarities. We used an unsupervised clustering algorithm to simultaneously stage-match all four species as well as Spearman's correlation analyses to stage-match pairs of donor-host species. From our stage-matching analyses, we found that the four stages that best matched with each other are the human blastocyst (E6/E7), the gastrulating mouse embryo (E6-E6.75), the marmoset late inner cell mass, and the pig late blastocyst. We further demonstrated that human pluripotent stem cells best matched with the mouse post-implantation stages. We also performed ontology analysis of the genes upregulated and commonly expressed between donor-host species pairs at their best matched stages. The stage-matching results predicted by this study will inform in vivo and in vitro interspecies chimerism and blastocyst complementation studies and can be used to match donor cells with host embryos between multiple species pairs to enhance chimerism for organogenesis.

Cause of Death by Race and Ethnicity in Minnesota Before and During the COVID-19 Pandemic, 2019-2020.

Objectives: To 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 Methods: We 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. Results: COVID-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. Conclusions: This 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.

MicroRNA-378a-3p prevents initiation and growth of colorectal cancer by fine tuning polyamine synthesis.

BACKGROUND: Inhibitors of ornithine decarboxylase (ODC) are effective at preventing colorectal cancer (CRC). However, their high toxicity limits their clinical application. This study was aimed to explore the potential of microRNAs (miRNAs) as an inhibitor of ODC. METHODS: miRNA array was used to identify dysregulated miRNAs in CRC tumors of mice and patients. Azoxymethane (AOM)/Dextran Sodium Sulfate (DSS) were used to induce CRC in mice. miRNA function in carcinogenesis was determined by soft-agar colony formation, flow cytometry, and wound healing of CRC cells. Mini-circle was used to deliver miRNA into colons. RESULTS: MiRNA profiling identified miR-378a-3p (miR-378a) as the most reduced miRNA in CRC tumors of patients and mice treated with AOM/DSS. Pathway array analysis revealed that miR-378a impaired c-MYC and ODC1 pathways. Further studies identified FOXQ1 (forkhead box Q1) and ODC1 as two direct targets of miR-378a. FOXQ1 activated transcription of c-MYC, a transcription activator of ODC1. In addition to directly targeting ODC1, miR-378a also inhibited expression of ODC1 via the FOXQ1-cMYC axis, thereby inhibiting polyamine synthesis in human CRC cells. Phenotypically, by reducing polyamine synthesis, miR-378a induced apoptosis and inhibited proliferation and migration of CRC cells, while disrupting the association of miR-378a with FOXQ1 and ODC1 offset the effects of miR-378a, suggesting that FOXQ1 and ODC1 were required for miR-378a to inhibit CRC cell growth. MiR-378a treatment robustly prevented growth of HCC by inhibiting polyamine synthesis in AOM/DSS mice. CONCLUSION: MiR-378a prevents CRC by inhibiting polyamine synthesis, suggesting its use as a novel ODC inhibitor against CRC.

Interspecies Chimeric Barriers for Generating Exogenic Organs and Cells for Transplantation.

A growing need for organs and novel cell-based therapies has provided a niche for approaches like interspecies chimeras. To generate organs from one donor species in another host species requires techniques such as blastocyst complementation and gene editing to successfully create an embryo that has cells from both the donor and the host. However, the task of developing highly efficacious and competent interspecies chimeras is met by many challenges. These interspecies chimeric barriers impede the formation of chimeras, often leading to lower levels of chimeric competency. The barriers that need to be addressed include the evolutionary distance between species, stage-matching, temporal and spatial synchronization of developmental timing, interspecies cell competition and the survival of pluripotent stem cells and embryos, compatibility of ligand-receptor signaling between species, and the ethical concerns of forming such models. By overcoming the interspecies chimera barriers and creating highly competent chimeras, the technology of organ and cellular generation can be honed and refined to develop fully functioning exogenic organs, tissues, and cells for transplantation.

Efficient silencing of hepatitis B virus S gene through CRISPR-mediated base editing.

Hepatitis B virus (HBV) infection is a major risk factor of liver cirrhosis and hepatocellular carcinoma. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been used to precisely edit the HBV genome and eliminate HBV through non-homologous end-joining repair of double-stranded break (DSB). However, the CRISPR/Cas9-mediated DSB triggers instability of host genome and exhibits low efficiency to edit genome, limiting its application. CRISPR cytidine base editors (CBEs) could silence genes by generating a premature stop codon. Here we developed a CRISPR base editor approach to precisely edit single nucleotide within the HBV genome to impair HBV gene expression. Specifically, a single-guide RNA (sgRNA) was designed to edit the 30th codon of HBV S gene, which encodes HBV surface antigen (HBsAg), from CAG (glutamine) to stop codon TAG. We next used human hepatoma PLC/PRF/5 cells carrying the HBV genome to establish a cell line that expresses a CBE (PLC/PRF/5-CBE). Lentivirus was used to introduce sgRNA into PLC/PRF/5-CBE cells. Phenotypically, 71% of PLC/PRF/5-CBE cells developed a premature stop codon within the S gene. Levels of HBs messenger RNA were significantly decreased. A 92% reduction of HBsAg secretion was observed in PLC/PRF/5-CBE cells. The intracellular HBsAg was also reduced by 84% after treatment of gRNA_S. Furthermore, no off-target effect was detected in predicted off-target loci within the HBV genome. Sequencing confirmed that 95%, 93%, 93%, 9%, and 72% S gene sequences of HBV genotypes B, C, F, G, and H had the binding site of sgRNA. Conclusion: Our findings indicate that CRISPR-mediated base editing is an efficient approach to silence the HBV S gene, suggesting its therapeutic potential to eliminate HBV.

MicroRNA-206 promotes the recruitment of CD8+ T cells by driving M1 polarisation of Kupffer cells.

OBJECTIVE: Kupffer cells (KCs) protect against hepatocellular carcinoma (HCC) by communicating with other immune cells. However, the underlying mechanism(s) of this process is incompletely understood. DESIGN: FVB/NJ mice were hydrodynamically injected with AKT/Ras and Sleeping Beauty transposon to induce HCC. Mini-circle and Sleeping Beauty were used to overexpress microRNA-206 in KCs of mice. Flow cytometry and immunostaining were used to evaluate the change in the immune system. RESULTS: Hydrodynamic injection of AKT/Ras into mice drove M2 polarisation of KCs and depletion of cytotoxic T cells (CTLs) and promoted HCC development. M1-to-M2 transition of KCs impaired microRNA-206 biogenesis. By targeting Klf4 (kruppel like factor 4) and, thereby, enhancing the production of M1 markers including C-C motif chemokine ligand 2 (CCL2), microRNA-206 promoted M1 polarisation of macrophages. Indeed, microRNA-206-mediated increase of CCL2 facilitated hepatic recruitment of CTLs via CCR2. Disrupting each component of the KLF4/CCL2/CCR2 axis impaired the ability of microRNA-206 to drive M1 polarisation of macrophages and recruit CTLs. In AKT/Ras mice, KC-specific expression of microRNA-206 drove M1 polarisation of KCs and hepatic recruitment of CTLs and fully prevented HCC, while 100% of control mice died from HCC. Disrupting the interaction between microRNA-206 and Klf4 in KCs and depletion of CD8+ T cells impaired the ability of miR-206 to prevent HCC. CONCLUSIONS: M2 polarisation of KCs is a major contributor of HCC in AKT/Ras mice. MicroRNA-206, by driving M1 polarisation of KCs, promoted the recruitment of CD8+ T cells and prevented HCC, suggesting its potential use as an immunotherapeutic approach.

MicroRNA-15a/16-1 Prevents Hepatocellular Carcinoma by Disrupting the Communication Between Kupffer Cells and Regulatory T Cells.

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 of AKT/Ras or c-Myc into mice resulted in hepatic enrichment of Tregs and 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. Hydrodynamic injection of microRNA-15a/16-1 fully prevented HCC in AKT/Ras and c-Myc mice, while 100% of control mice died of HCC. Therapeutically, microRNA-15a/16-1 promoted a regression of HCC in both mouse models, 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 nuclear factor-κB that activates transcription of Ccl22. By reducing CCL22 binding to C-C chemokine receptor type 4 on Tregs, microRNA-15a/16-1 impaired Treg chemotaxis. Disrupting the interaction between microRNA-15a/16-1 and nuclear factor-κB impaired the ability of microRNA-15a/16-1 to prevent hepatic Treg accumulation and HCC. Depletion of cluster of differentiation 8+ T cells and additional treatment of CCL22 recovered growth of HCC that was fully prevented by microRNA-15a/16. CONCLUSIONS: 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.

MicroRNA-206 enhances antitumor immunity by disrupting the communication between malignant hepatocytes and regulatory T cells in c-Myc mice.

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 TGFß1, which enhanced the suppressor function of Tregs and impaired the proliferation and cytotoxicity of CTLs. In addition to activating TGFß 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 TGFß1 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 TGFß1, which promoted HCC development. miR-206, by attenuating TGFß1 overproduction, disrupted the communication of malignant hepatocytes with CTLs and Tregs, which prevented HCC. miR-206 represents a potential immunotherapeutic agent against HCC.

The Proposal Preparation Program: A Group Mentoring, Faculty Development Model to Facilitate the Submission and Funding of NIH Grant Applications.

This article describes the University of Minnesota Medical School Proposal Preparation Program (P3). P3 is designed to develop grant-writing skills for assistant professors 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 providing structure, accountability, guidance, and support. Between sessions, 1 peer and 1 mentor are assigned (on a rotating basis) to critique each 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 1 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.

Differentiation of immortalized human multi-lineage progenitor to alveolar type 2-like cells: angiotensin-converting enzyme 2 expression and binding of severe acute respiratory syndrome coronavirus 2 spike and spike 1 proteins.

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 angiotensin-converting enzyme 2 (ACE-2) receptor to facilitate entry into the 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. AT2 cells express high levels of surface ACE-2 and thus are a leading target for primary infection by SARS-CoV-2. This study describes a method for directly differentiating telomerase reverse transcriptase-immortalized human cord blood-derived multi-lineage progenitor cells (MLPCs) 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, quantitative reverse transcription polymerase chain reaction and binding of biotinylated SARS-CoV-2 spike and spike 1 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. The authors defined the culture conditions that enabled AT2-like cells to be repeatedly passaged and cryopreserved without further differentiation to AT1. The authors' method provides a stable and renewable source of AT2 cells for respiratory viral binding, blocking and uptake studies.

Isolation of Mitochondria from Liver and Extraction of Total RNA and Protein: Analyses of miRNA and Protein Expression.

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.

Binding of the SARS-CoV-2 Spike Protein to the Asialoglycoprotein Receptor on Human Primary Hepatocytes and Immortalized Hepatocyte-Like Cells by Confocal Analysis.

BACKGROUND: 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.

Quantitative Assessment of Occipital Metabolic and Energetic Changes in Parkinson's Patients, Using In Vivo 31P MRS-Based Metabolic Imaging at 7T.

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.