Functional Screening of Candidate Causal Genes for Insulin Resistance in Human Preadipocytes and Adipocytes.

Rationale: Genome-wide association studies have identified genetic loci associated with insulin resistance (IR) but pinpointing the causal genes of a risk locus has been challenging. Objective: To identify candidate causal genes for IR, we screened regional and biologically plausible genes (16 in total) near the top 10 IR-loci in risk-relevant cell types, namely preadipocytes and adipocytes. Methods and Results: We generated 16 human Simpson-Golabi-Behmel syndrome preadipocyte knockout lines each with a single IR-gene knocked out by lentivirus-mediated CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. We evaluated each gene knockout by screening IR-relevant phenotypes in the 3 insulin-sensitizing mechanisms, including adipogenesis, lipid metabolism, and insulin signaling. We performed genetic analyses using data on the genotype-tissue expression portal expression quantitative trait loci database and accelerating medicines partnership type 2 diabetes mellitus Knowledge Portal to evaluate whether candidate genes prioritized by our in vitro studies were expression quantitative trait loci genes in human subcutaneous adipose tissue, and whether expression of these genes is associated with risk of IR, type 2 diabetes mellitus, and cardiovascular diseases. We further validated the functions of 3 new adipose IR genes by overexpression-based phenotypic rescue in the Simpson-Golabi-Behmel syndrome preadipocyte knockout lines. Twelve genes, PPARG, IRS-1, FST, PEPD, PDGFC, MAP3K1, GRB14, ARL15, ANKRD55, RSPO3, COBLL1, and LYPLAL1, showed diverse phenotypes in the 3 insulin-sensitizing mechanisms, and the first 7 of these genes could affect all the 3 mechanisms. Five out of 6 expression quantitative trait loci genes are among the top candidate causal genes and the abnormal expression levels of these genes (IRS-1, GRB14, FST, PEPD, and PDGFC) in human subcutaneous adipose tissue could be associated with increased risk of IR, type 2 diabetes mellitus, and cardiovascular disease. Phenotypic rescue by overexpression of the candidate causal genes (FST, PEPD, and PDGFC) in the Simpson-Golabi-Behmel syndrome preadipocyte knockout lines confirmed their function in adipose IR. Conclusions: Twelve genes showed diverse phenotypes indicating differential roles in insulin sensitization, suggesting mechanisms bridging the association of their genomic loci with IR. We prioritized PPARG, IRS-1, GRB14, MAP3K1, FST, PEPD, and PDGFC as top candidate genes. Our work points to novel roles for FST, PEPD, and PDGFC in adipose tissue, with consequences for cardiometabolic diseases.

Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells.

Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal, we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation.

Adipocyte insulin receptor activity maintains adipose tissue mass and lifespan.

Type 2 diabetes follows a well-defined progressive pathogenesis, beginning with insulin resistance in metabolic tissues such as the adipose. Intracellular signaling downstream of insulin receptor activation regulates critical metabolic functions of adipose tissue, including glucose uptake, lipogenesis, lipolysis and adipokine secretion. Previous studies have used the aP2 promoter to drive Cre recombinase expression in adipose tissue. Insulin receptor (IR) knockout mice created using this aP2-Cre strategy (FIRKO mice) were protected from obesity and glucose intolerance. Later studies demonstrated the promiscuity of the aP2 promoter, casting doubts upon the tissue specificity of aP2-Cre models. It is our goal to use the increased precision of the Adipoq promoter to investigate adipocyte-specific IR function. Towards this end we generated an adipocyte-specific IR knockout (AIRKO) mouse using an Adipoq-driven Cre recombinase. Here we report AIRKO mice are less insulin sensitive throughout life, and less glucose tolerant than wild-type (WT) littermates at the age of 16 weeks. In contrast to WT littermates, the insulin sensitivity of AIRKO mice is unaffected by age or dietary regimen. At any age, AIRKO mice are comparably insulin resistant to old or obese WT mice and have a significantly reduced lifespan. Similar results were obtained when these phenotypes were re-examined in FIRKO mice. We also found that the AIRKO mouse is protected from high-fat diet-induced weight gain, corresponding with a 90% reduction in tissue weight of major adipose depots compared to WT littermates. Adipose tissue mass reduction is accompanied by hepatomegaly and increased hepatic steatosis. These data indicate that adipocyte IR function is crucial to systemic energy metabolism and has profound effects on adiposity, hepatic homeostasis and lifespan.

Transcriptional activation by NFκB increases perlecan/HSPG2 expression in the desmoplastic prostate tumor microenvironment.

Perlecan/HSPG2, a heparan sulfate proteoglycan typically found at tissue borders including those separating epithelia and connective tissue, increases near sites of invasion of primary prostatic tumors as previously shown for other proteins involved in desmoplastic tissue reaction. Studies of prostate cancer cells and stromal cells from both prostate and bone, the major site for prostate cancer metastasis, showed that cancer cells and a subset of stromal cells increased production of perlecan in response to cytokines present in the tumor microenvironment. In silico analysis of the HSPG2 promoter revealed two conserved NFκB binding sites, in addition to the previously reported SMAD3 binding sites. By systematically transfecting cells with a variety of reporter constructs including sequences up to 2.6 kb from the start site of transcription, we identified an active cis element in the distal region of the HSPG2 promoter, and showed that it functions in regulating transcription of HSPG2. Treatment with TNF-α and/or TGFß1 identified TNF-α as a major cytokine regulator of perlecan production. TNF-α treatment also triggered p65 nuclear translocation and binding to the HSPG2 regulatory region in stromal cells and cancer cells. In addition to stromal induction of perlecan production in the prostate, we identified a matrix-secreting bone marrow stromal cell type that may represent the source for increases in perlecan in the metastatic bone marrow environment. These studies implicate perlecan in cytokine-mediated, innate tissue responses to cancer cell invasion, a process we suggest reflects a modified wound healing tissue response co-opted by prostate cancer cells.

p62/SQSTM1 is required for cell survival of apoptosis-resistant bone metastatic prostate cancer cell lines.

BACKGROUND: Bone marrow stromal cell (BMSC) paracrine factor(s) can induce apoptosis in bone metastatic prostate cancer (PCa) cell lines. However, the PCa cells that escape BMSC-induced apoptosis can upregulate cytoprotective autophagy. METHODS: C4-2, C4-2B, MDA PCa 2a, MDA PCa 2b, VCaP, PC3, or DU145 PCa cell lines were grown in BMSC conditioned medium and analyzed for mRNA and/or protein accumulation of p62 (also known as sequestome-1/SQSTM1), Microtubule-associated protein 1 light chain 3B (LC3B), or lysosomal-associated membrane protein 1 (LAMP1) using quantitative polymerase chain reaction (QPCR), Western blot, or immunofluorescence. Small interfering RNA (siRNA) was used to determine if p62 is necessary PCa cell survival. RESULTS: BMSC paracrine signaling upregulated p62 mRNA and protein in a subset of the PCa cell lines. The PCa cell lines that were insensitive to BMSC-induced apoptosis and autophagy induction had elevated basal p62 mRNA and protein. In the BMSC-insensitive PCa cell lines, siRNA knockdown of p62 was cytotoxic and immunostaining showed peri-nuclear clustering of autolysosomes. However, in the BMSC-sensitive PCa cell lines, p62 siRNA knockdown was not appreciably cytotoxic and did not affect autolysosome subcellular localization. CONCLUSIONS: A pattern emerges wherein the BMSC-sensitive PCa cell lines are known to be osteoblastic and express the androgen receptor, while the BMSC-insensitive PCa cell lines are characteristically osteolytic and do not express the androgen receptor. Furthermore, BMSC-insensitive PCa may have evolved a dependency on p62 for cell survival that could be exploited to target and kill these apoptosis-resistant PCa cells in the bone.

Benchmark Outcomes in Deceased Donor Kidney Transplantation: A Multicenter Analysis of 80 996 Transplants From 126 Centers.

Background: We defined clinically relevant benchmark values in deceased donor kidney transplantation (KT), to assess the best achievable results in low-risk patient cohorts from experienced centers. Methods: We identified the "ideal" cases from the United Network for Organ Sharing Standard Transplant Analysis and Research files from centers performing ≥50 KT per year between 2010 and 2018. Cases have been selected based on the kidney donor profile index values (<35%), a cold ischemia time (CIT) ≤18 h, a HLA mismatch ≤4, and excluding blood group (ABO) incompatible, dual and combined transplants. The outcomes of the benchmark cohort have been compared with a group of patients excluded from the benchmark cohort because but not meeting 1 or more of the abovementioned criteria. Results: The 171 424 KT patients in the United Network for Organ Sharing Standard Transplant Analysis and Research files were screened and 8694 benchmark cases of a total of 80 996 KT (10.7%) from 126 centers meeting the selection criteria were identified. The benchmarks for 1-, 3-, and 5-y patient survival are ≥97%, ≥92.5%, and ≥86.7%, and ≥95.4%, ≥87.8%, and ≥79.6% for graft survival. Benchmark cutoff for hospital length of stay is ≤5 d, ≤23.6% for delayed graft function, and ≤7.5% and ≤9.1% for 6-mo and 1-y incidence of acute rejection. Overall 1-, 3-, and 5-y actuarial graft survivals were 96.6%, 91.1%, and 84.2% versus 93.5%, 85.4%, and 75.5% in the benchmark and comparison groups, respectively (P < 0.001). Overall 1-, 3-, and 5-y actuarial patient survivals were 98.1%, 94.8%, and 90.0% versus 96.6%, 91.1%, and 83.0% in the benchmark and comparison groups, respectively (P < 0.001). Conclusions: For the first time, we quantified the best achievable postoperative results in an ideal scenario in deceased donor KT, aimed at improving the clinical practice guided by the comparison of center performances with the ideal outcomes defined.

mARC1 Is the Main Contributor to Metabolic Reduction of N-Hydroxyurea.

N-Hydroxyurea has been known since the 1960s as an antiproliferative drug and is used both in oncology and for treatment of hematological disorders such as sickle cell anemia where very high daily doses are administered. It is assumed that the cellular effect of N-hydroxyurea is caused by inhibition of ribonucleotide reductase, while alternative mechanisms, e.g., generation of nitric oxide, have also been proposed. Despite its many therapeutic applications, the metabolism of hydroxyurea is largely unexplored. The major elimination pathway of N-hydroxyurea is the reduction to urea. Since the mitochondrial amidoxime reducing component (mARC) is known for its N-reductive activity, we investigated the reduction of NHU by this enzyme system. This study presents in vitro and in vivo evidence that this reductive biotransformation is specifically mediated by the mARC1. Inactivation by mARC1 is a possible explanation for the high doses of NHU required for treatment.

mARC1 in MASLD: Modulation of lipid accumulation in human hepatocytes and adipocytes.

BACKGROUND: Mutations in the gene MTARC1 (mitochondrial amidoxime-reducing component 1) protect carriers from metabolic dysfunction-associated steatohepatitis (MASH) and cirrhosis. MTARC1 encodes the mARC1 enzyme, which is localized to the mitochondria and has no known MASH-relevant molecular function. Our studies aimed to expand on the published human genetic mARC1 data and to observe the molecular effects of mARC1 modulation in preclinical MASH models. METHODS AND RESULTS: We identified a novel human structural variant deletion in MTARC1, which is associated with various biomarkers of liver health, including alanine aminotransferase levels. Phenome-wide Mendelian Randomization analyses additionally identified novel putatively causal associations between MTARC1 expression, and esophageal varices and cardiorespiratory traits. We observed that protective MTARC1 variants decreased protein accumulation in in vitro overexpression systems and used genetic tools to study mARC1 depletion in relevant human and mouse systems. Hepatocyte mARC1 knockdown in murine MASH models reduced body weight, liver steatosis, oxidative stress, cell death, and fibrogenesis markers. mARC1 siRNA treatment and overexpression modulated lipid accumulation and cell death consistently in primary human hepatocytes, hepatocyte cell lines, and primary human adipocytes. mARC1 depletion affected the accumulation of distinct lipid species and the expression of inflammatory and mitochondrial pathway genes/proteins in both in vitro and in vivo models. CONCLUSIONS: Depleting hepatocyte mARC1 improved metabolic dysfunction-associated steatotic liver disease-related outcomes. Given the functional role of mARC1 in human adipocyte lipid accumulation, systemic targeting of mARC1 should be considered when designing mARC1 therapies. Our data point to plasma lipid biomarkers predictive of mARC1 abundance, such as Ceramide 22:1. We propose future areas of study to describe the precise molecular function of mARC1, including lipid trafficking and subcellular location within or around the mitochondria and endoplasmic reticulum.

Low vs Standard-Dose Indocyanine Green in the Identification of Biliary Anatomy Using Near-Infrared Fluorescence Imaging: A Multicenter Randomized Controlled Trial.

BACKGROUND: Near-infrared fluorescence imaging using intravenous indocyanine green (ICG) facilitates intraoperative identification of biliary anatomy. We hypothesize that a much lower dose of ICG than the standard decreases hepatic and background fluorescence and improves bile duct visualization. STUDY DESIGN: In this multicenter randomized controlled trial, 55 adult patients undergoing laparoscopic cholecystectomy were randomized to low-dose (0.05 mg) or standard-dose (2.5 mg) ICG preoperatively on the day of surgery. A quantitative assessment was performed on recorded videos from the operation using ImageJ software to quantify the fluorescence intensity of the bile duct, liver, and surrounding/background fat. Operating surgeons blinded to ICG dose provided a qualitative assessment of various aspects of the visualization of the extrahepatic biliary tree comparing near-infrared fluorescence to standard visible light imaging using a scale of 1 to 5 (1, unsatisfactory; 5, excellent). Quantitative and qualitative scores were compared between the groups to determine any significant differences between the doses. RESULTS: The bile duct-to-liver and bile duct-to-background fat fluorescence intensity ratios were significantly higher for the low-dose group compared with the standard-dose group (3.6 vs 0.68, p < 0.0001; and 7.5 vs 3.3, p < 0.0001, respectively). Low-dose ICG had a slightly higher (ie better) mean score on the qualitative assessment compared to the standard dose, although the differences were not statistically significant. CONCLUSIONS: Low-dose ICG leads to quantitative improvement of biliary visualization using near-infrared fluorescence imaging by minimizing liver fluorescence; this further facilitates routine use during hepatobiliary operations.

Pre- and Post-Portosystemic Shunt Placement Metabolomics Reveal Molecular Signatures for the Development of Hepatic Encephalopathy.

Hepatic encephalopathy (HE) is a common complication of advanced liver disease causing brain dysfunction. This is likely due to the accumulation of unfiltered toxins within the bloodstream. A known risk factor for developing or worsening HE is the placement of a transjugular intrahepatic portosystemic shunt (TIPS), which connects the pre-hepatic and post-hepatic circulation allowing some blood to bypass the dysfunctional liver and decreases portal hypertension. To better understand the pathophysiology of post-TIPS HE, we conducted a multi-center prospective cohort study employing metabolomic analyses on hepatic vein and peripheral vein blood samples from participants with cirrhosis undergoing elective TIPS placement, measuring chemical modifications and changes in concentrations of metabolites resulting from TIPS placement. In doing so, we identified numerous alterations in metabolites, including bile acids, glycerophosphocholines, and bilirubins possibly implicated in the development and severity of HE.

Portosystemic shunt placement reveals blood signatures for the development of hepatic encephalopathy through mass spectrometry.

Elective transjugular intrahepatic portosystemic shunt (TIPS) placement can worsen cognitive dysfunction in hepatic encephalopathy (HE) patients due to toxins, including possible microbial metabolites, entering the systemic circulation. We conducted untargeted metabolomics on a prospective cohort of 22 patients with cirrhosis undergoing elective TIPS placement and followed them up to one year post TIPS for HE development. Here we suggest that pre-existing intrahepatic shunting predicts HE severity post-TIPS. Bile acid levels decrease in the peripheral vein post-TIPS, and the abundances of three specific conjugated di- and tri-hydroxylated bile acids are inversely correlated with HE grade. Bilirubins and glycerophosphocholines undergo chemical modifications pre- to post-TIPS and based on HE grade. Our results suggest that TIPS-induced metabolome changes can impact HE development, and that pre-existing intrahepatic shunting could be used to predict HE severity post-TIPS.

Personalized Tacrolimus Dosing After Liver Transplantation: A Randomized Clinical Trial.

Background: Inter- and intra-individual variability in tacrolimus dose requirements mandates empirical clinician-titrated dosing that frequently results in deviation from a narrow target range. Improved methods to individually dose tacrolimus are needed. Our objective was to determine whether a quantitative, dynamically-customized, phenotypic-outcome-guided dosing method termed Phenotypic Personalized Medicine (PPM) would improve target drug trough maintenance. Methods: In a single-center, randomized, pragmatic clinical trial ( NCT03527238 ), 62 adults were screened, enrolled, and randomized prior to liver transplantation 1:1 to standard-of-care (SOC) clinician-determined or PPM-guided dosing of tacrolimus. The primary outcome measure was percent days with large (>2 ng/mL) deviation from target range from transplant to discharge. Secondary outcomes included percent days outside-of-target-range and mean area-under-the-curve (AUC) outside-of-target-range per day. Safety measures included rejection, graft failure, death, infection, nephrotoxicity, or neurotoxicity. Results: 56 (29 SOC, 27 PPM) patients completed the study. The primary outcome measure was found to be significantly different between the two groups. Patients in the SOC group had a mean of 38.4% of post-transplant days with large deviations from target range; the PPM group had 24.3% of post-transplant days with large deviations; (difference -14.1%, 95% CI: -26.7 to -1.5 %, P=0.029). No significant differences were found in the secondary outcomes. In post-hoc analysis, the SOC group had a 50% longer median length-of-stay than the PPM group [15 days (Q1-Q3: 11-20) versus 10 days (Q1-Q3: 8.5-12); difference 5 days, 95% CI: 2-8 days, P=0.0026]. Conclusions: PPM guided tacrolimus dosing leads to better drug level maintenance than SOC. The PPM approach leads to actionable dosing recommendations on a day-to-day basis. Lay Summary: In a study on 62 adults who underwent liver transplantation, researchers investigated whether a new dosing method called Phenotypic Personalized Medicine (PPM) would improve daily dosing of the immunosuppression drug tacrolimus. They found that PPM guided tacrolimus dosing leads to better drug level maintenance than the standard-of-care clinician-determined dosing. This means that the PPM approach leads to actionable dosing recommendations on a day-to-day basis and can help improve patient outcomes.

A new assay for functional screening of BRCA2 linker region mutations identifies variants that alter chemoresistance to cisplatin.

Variants of unknown significance (VUS) complicate the assignment of risk to new DNA sequence variants found in at-risk populations. This study focused on the poorly studied linker region of the cancer-associated BRCA2 protein encoded by exons twelve through fourteen of BRCA2. To develop a new method to characterize VUS in this region of BRCA2, we first chose to study 4 reported VUS occurring on evolutionarily conserved residues within the linker region. To determine if these VUS represent neutral changes or if they impact the function of the BRCA2 protein, we stably transfected expression plasmids encoding wild-type or each mutant peptide into T47D breast cancer cells, which are wild-type for BRCA2. Four mutant peptide expressing cell lines and a wild-type linker region expressing cell line next were studied by challenging transfected cell lines with the DNA crosslinking compound cisplatin (10µM) for 5days. Expression of the wild-type linker region and certain mutant linker peptides (N2452D and I2285V) decreased apoptosis (as demonstrated by cell death detection assay) in transfected cell lines, indicating that the linker region peptide directly or indirectly affects the DNA damage repair pathway. By determining the cell survival and assaying the apoptotic index of treated cell lines, one could potentially use this screen to determine that a particular VUS has a functional impact on BRCA2 function, and hence is of functional significance. We conclude that this method is useful for screening the effect of linker region VUS on BRCA2 function, and to identify mutations for further testing. We also conclude that mutations in the linker region may have heretofore unappreciated roles in BRCA2 function.

Clinical Utility and Cost of Postoperative Hemoglobin Level Testing Following Robotic-assisted Hysterectomy for Endometrial Cancer.

AIM: Our aim was to assess the clinical utility of postoperative hemoglobin testing following hysterectomy. PATIENTS AND METHODS: We carried out a retrospective cohort study of patients who underwent robotic surgery at an academic center during a 44-month study period. Data included demographics and perioperative outcomes. The mean postoperative decrease in hemoglobin level was evaluated using numerical and categorical variables. RESULTS: A total of 201 women were included. A total of 45 (22.4%) developed symptoms suggestive of hemodynamic compromise. When compared to asymptomatic patients, these patients were no different in operative time, estimated blood loss, pre- or post-operative hemoglobin levels, or the change in hemoglobin levels. Symptomatic patients did receive less fluid intraoperatively (1.2 vs. 1.5 l; p<0.0001). Perioperative outcomes were not associated with a greater postoperative decrease in hemoglobin (Hb). Postoperative anemia was associated with preoperative anemia (0% vs. 45%; p<0.0001). Patients with postoperative anemia were also more likely to be re-admitted within 30 days after surgery (7% vs. 23%; p=0.025). Of the three patients who received blood transfusions postoperatively, all three had preoperative Hb<9.5 g/dl, compared to 2.5% of those who were not transfused (p<0.0001). Using Institutional charges and Medicare reimbursement rates for blood hemoglobin testing, savings were estimated to be $3,629 and $1,236, respectively, during the study period. CONCLUSION: Postoperative Hb testing may be safely avoided unless starting Hb is less <10 g/dl. Clinical practice change can reduce healthcare costs without hindering patient care.

Implementation of an external female urinary catheter as an alternative to an indwelling urinary catheter.

BACKGROUND: Until recently, there has been a lack of viable alternative to an indwelling urinary catheter for female patients that require precise urine output measurements. With the introduction of external female urinary catheters, we can now substitute this type of device for an indwelling urinary catheter in many patients, decreasing their risk of catheter-associated urinary tract infections. METHODS: In this retrospective study, we analyzed the impact of a hospital-wide implementation of an external female urinary catheter at a large academic medical center. The study included female patients, greater than 18 years of age. We compared a 12-month period before and after device implementation to assess the impact on indwelling urinary catheter utilization and catheter-associated urinary tract infections rate. RESULTS: Data included over 220,000 patient days, over 10,000 external urinary catheter days and 33,000 indwelling urinary catheter days. We found a statistically significant decrease in indwelling urinary catheter utilization following the implementation of the external female urinary catheter, but only in intensive care units. CONCLUSIONS: It is our recommendation that facilities first implement the device in ICUs as this level of care was where we observed the most significant impact.

Racial Disparity in Liver Transplantation Listing.

BACKGROUND: Previous studies have demonstrated disparities in transplantation for women, non-Caucasians, the uninsured or publicly insured, and rural populations. We sought to correlate transplant center characteristics with patient access to the waiting list and liver transplantation. We hypothesized that liver transplant centers vary greatly in providing equitable access to the waiting list and liver transplantation. STUDY DESIGN: Center-specific, adult, deceased-donor liver transplant and waitlist data for the years 2013 to 2018 were obtained from the United Network for Organ Sharing. Waitlist race/ethnicity distributions from liver transplant centers performing ≥ 250 transplants over this period (n = 109) were compared with those of their donor service area, as calculated from 5-year US Census Bureau estimates of 2017. Center-specific characteristics correlating with disparities were analyzed using a linear regression model with a log transformed outcome. RESULTS: Non-Hispanic Blacks (NHBs) are under-represented in liver transplant listing compared with center donation service area (88/109, 81%), whereas, non-Hispanic Whites are over-represented (65/109, 58%) (p < 0.0001). Hispanics were also under-represented on the waitlist at the majority of transplant centers (68/109, 62%) (p = 0.02). Although the racial/ethnic distribution of transplantation is more reflective of the waitlist, there is a higher than expected rate of transplantation for NHBs compared to the waitlist. Predictors of disparity in listing include percentage of transplant recipients at the center who had private insurance, racial composition of the donation service area, and the distance recipients had to travel for transplant. CONCLUSIONS: Non-Hispanic Blacks are listed for liver transplantation less than would be expected. Once listed, however, racial disparities in transplantation are greatly diminished. Improvements in access to adequate health insurance appear to be essential to diminishing disparities in access to this life-saving care.

Cell-specific transcriptional control of mitochondrial metabolism by TIF1γ drives erythropoiesis.

Transcription and metabolism both influence cell function, but dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. We discovered, using a chemical suppressor screen, that inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) rescues erythroid differentiation in bloodless zebrafish moonshine (mon) mutant embryos defective for transcriptional intermediary factor 1 gamma (tif1γ). This rescue depends on the functional link of DHODH to mitochondrial respiration. The transcription elongation factor TIF1γ directly controls coenzyme Q (CoQ) synthesis gene expression. Upon tif1γ loss, CoQ levels are reduced, and a high succinate/α-ketoglutarate ratio leads to increased histone methylation. A CoQ analog rescues mon's bloodless phenotype. These results demonstrate that mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage.

Targeted Protein Degradation Phenotypic Studies Using HaloTag CRISPR/Cas9 Endogenous Tagging Coupled with HaloPROTAC3.

To assess the role of a protein, protein loss phenotypic studies can be used, most commonly through mutagenesis RNAi or CRISPR knockout. Such studies have been critical for the understanding of protein function and the identification of putative therapeutic targets for numerous human disease states. However, these methodological approaches present challenges because they are not easily reversible, and if an essential gene is targeted, an associated loss of cell viability can potentially hinder further studies. Here we present a reversible and conditional live-cell knockout strategy that is applicable to numerous proteins. This modular protein-tagging approach regulates target loss at the protein, rather than the genomic, level through the use of HaloPROTAC3, which specifically degrades HaloTag fusion proteins via recruitment of the VHL E3 ligase component. To enable HaloTag-mediated degradation of endogenous proteins, we provide protocols for HaloTag genomic insertion at the protein N or C terminus via CRISPR/Cas9 and use of HaloTag fluorescent ligands to enrich edited cells via Fluorescence-Activated Cell Sorting (FACS). Using these approaches, endogenous HaloTag fusion proteins present in various subcellular locations can be degraded by HaloPROTAC3. As detecting the degradation of endogenous targets is challenging, the 11-amino-acid peptide tag HiBiT is added to the HaloTag fusion to allows the sensitive luminescence detection of HaloTag fusion levels without the use of antibodies. Lastly, we demonstrate, through comparison of HaloPROTAC3 degradation with that of another fusion tag PROTAC, dTAG-13, that HaloPROTAC3 has a faster degradation rate and similar extent of degradation. © 2020 The Authors. Basic Protocol 1: CRISPR/Cas9 insertion of HaloTag or HiBiT-HaloTag Basic Protocol 2: HaloPROTAC3 degradation of endogenous HaloTag fusions.

Mitoregulin Controls ß-Oxidation in Human and Mouse Adipocytes.

We previously discovered in mouse adipocytes an lncRNA (the homolog of human LINC00116) regulating adipogenesis that contains a highly conserved coding region. Here, we show human protein expression of a peptide within LINC00116, and demonstrate that this peptide modulates triglyceride clearance in human adipocytes by regulating lipolysis and mitochondrial ß-oxidation. This gene has previously been identified as mitoregulin (MTLN). We conclude that MTLN has a regulatory role in adipocyte metabolism as demonstrated by systemic lipid phenotypes in knockout mice. We also assert its adipocyte-autonomous phenotypes in both isolated murine adipocytes as well as human stem cell-derived adipocytes. MTLN directly interacts with the ß subunit of the mitochondrial trifunctional protein, an enzyme critical in the ß-oxidation of long-chain fatty acids. Our human and murine models contend that MTLN could be an avenue for further therapeutic research, albeit not without caveats, for example, by promoting white adipocyte triglyceride clearance in obese subjects.

Fattening chips: hypertrophy, feeding, and fasting of human white adipocytes in vitro.

Adipose is a distributed organ that performs vital endocrine and energy homeostatic functions. Hypertrophy of white adipocytes is a primary mode of both adaptive and maladaptive weight gain in animals and predicts metabolic syndrome independent of obesity. Due to the failure of conventional culture to recapitulate adipocyte hypertrophy, technology for production of adult-size adipocytes would enable applications such as in vitro testing of weight loss therapeutics. To model adaptive adipocyte hypertrophy in vitro, we designed and built fat-on-a-chip using fiber networks inspired by extracellular matrix in adipose tissue. Fiber networks extended the lifespan of differentiated adipocytes, enabling growth to adult sizes. By micropatterning preadipocytes in a native cytoarchitecture and by adjusting cell-to-cell spacing, rates of hypertrophy were controlled independent of culture time or differentiation efficiency. In vitro hypertrophy followed a nonlinear, nonexponential growth model similar to human development and elicited transcriptomic changes that increased overall similarity with primary tissue. Cells on the chip responded to simulated meals and starvation, which potentiated some adipocyte endocrine and metabolic functions. To test the utility of the platform for therapeutic development, transcriptional network analysis was performed, and retinoic acid receptors were identified as candidate drug targets. Regulation by retinoid signaling was suggested further by pharmacological modulation, where activation accelerated and inhibition slowed hypertrophy. Altogether, this work presents technology for mature adipocyte engineering, addresses the regulation of cell growth, and informs broader applications for synthetic adipose in pharmaceutical development, regenerative medicine, and cellular agriculture.