Translocator protein (TSPO), also known as the peripheral benzodiazepine receptor, is a highly conserved outer mitochondrial membrane protein present in specific subpopulations of cells within ...different tissues. In recent studies, the presumptive model depicting mammalian TSPO as a critical cholesterol transporter for steroidogenesis has been refuted by studies examining effects of Tspo gene deletion in vivo and in vitro, biochemical testing of TSPO cholesterol transport function, and specificity of TSPO-mediated pharmacological responses. Nevertheless, high TSPO expression in steroid-producing cells seemed to indicate an alternate function for this protein in steroidogenic mitochondria. To seek an explanation, we used CRISPR/Cas9-mediated TSPO knockout steroidogenic MA-10 Leydig cell (MA-10:TspoΔ/Δ) clones to examine changes to core mitochondrial functions resulting from TSPO deficiency. We observed that 1) MA-10:TspoΔ/Δ cells had a shift in substrate utilization for energy production from glucose to fatty acids with significantly higher mitochondrial fatty acid oxidation (FAO), and increased reactive oxygen species production; and 2) oxygen consumption rate, mitochondrial membrane potential, and proton leak were not different between MA-10:TspoΔ/Δ and MA-10:Tspo+/+ control cells. Consistent with this finding, TSPO-deficient adrenal glands from global TSPO knockout (Tspo−/−) mice also showed up-regulation of genes involved in FAO compared with the TSPO floxed (Tspofl/fl) controls. These results demonstrate the first experimental evidence that TSPO can affect mitochondrial energy homeostasis through modulation of FAO, a function that appears to be consistent with high levels of TSPO expression observed in cell types active in lipid storage/metabolism.
The synthesis of steroid hormones occurs in specific cells and tissues in the body in response to trophic hormones and other signals. In order to synthesize steroids de novo, cholesterol, the ...precursor of all steroid hormones, must be mobilized from cellular stores to the inner mitochondrial membrane (IMM) to be converted into the first steroid formed, pregnenolone. This delivery of cholesterol to the IMM is the rate-limiting step in this process, and has long been known to require the rapid synthesis of a new protein(s) in response to stimulation. Although several possibilities for this protein have arisen over the past few decades, most of the recent attention to fill this role has centered on the candidacies of the proteins the Translocator Protein (TSPO) and the Steroidogenic Acute Regulatory Protein (StAR). In this review, the process of regulating steroidogenesis is briefly described, the characteristics of the candidate proteins and the data supporting their candidacies summarized, and some recent findings that propose a serious challenge for the role of TSPO in this process are discussed.
•Steroid biosynthesis is controlled by the transfer of cholesterol to the inner mitochondrial membrane.•Both the Steroidogenic Acute Regulatory (StAR) and Translocator (TSPO) proteins have been implicated in this process.•Knockout of StAR results in a complete inability to synthesize steroids in both humans and mice.•Knockout of TSPO has been shown to have no effect on steroid synthesis in mice in two separate laboratories.•Previous in vitro studies that demonstrated an essential function for TSPO in steroidogenesis have not been reproducible.
MA-10 cells, established 4 decades ago from a murine Leydig cell tumor, has served as a key model system for studying steroidogenesis. Despite a precipitous loss in their innate ability to respond to ...luteinizing hormone (LH), the use of a cell-permeable cAMP analog for induction ensured their continued use. In parallel, a paradigm that serum-free conditions are essential for trophic steroidogenic stimulation was rationalized. Through the selection of LH-responsive single-cell MA-10Slip clones, we uncovered that Leydig cells remain responsive in the presence of serum in vitro and that exogenous cholesterol delivery by lipoproteins provided a significantly elevated steroid biosynthetic response (>2-fold). In scrutinizing the underlying regulation, systems biology of the MA-10 cell proteome identified multiple Rho-GTPase signaling pathways as highly enriched. Testing Rho function in steroidogenesis revealed that its modulation can negate the specific elevation in steroid biosynthesis observed in the presence of lipoproteins/serum. This signaling modality primarily linked to the regulation of endocytic traffic is evident only in the presence of exogenous cholesterol. Inhibiting Rho function in vivo also decreased hCG-induced testosterone production in mice. Collectively, our findings dispel a long-held view that the use of serum could confound or interfere with trophic stimulation and underscore the need for exogenous lipoproteins when dissecting physiological signaling and cholesterol trafficking for steroid biosynthesis in vitro. The LH-responsive MA-10Slip clones derived in this study present a reformed platform enabling biomimicry to study the cellular and molecular basis of mammalian steroidogenesis.
Translocator protein (TSPO) is a mitochondrial outer membrane protein of unknown function with high physiological expression in steroidogenic cells. Using TSPO gene–deleted mice, we recently ...demonstrated that TSPO function is not essential for steroidogenesis. The first link between TSPO and steroidogenesis was established in studies showing modest increases in progesterone production by adrenocortical and Leydig tumor cell lines after treatment with PK11195. To reconcile discrepancies between physiological and pharmacological interpretations of TSPO function, we generated TSPO-knockout MA-10 mouse Leydig tumor cells (MA-10:TspoΔ/Δ) and examined their steroidogenic potential after exposure to either dibutyryl-cAMP or PK11195. Progesterone production in MA-10:TspoΔ/Δ after dibutyryl-cAMP was not different from control MA-10:Tspo+/+ cells, confirming that TSPO function is not essential for steroidogenesis. Interestingly, when treated with increasing concentrations of PK11195, both control MA-10:Tspo+/+ cells and MA-10:TspoΔ/Δ cells responded in a similar dose-dependent manner showing increases in progesterone production. These results show that the pharmacological effect of PK11195 on steroidogenesis is not mediated through TSPO.
AB-423 is a member of the sulfamoylbenzamide (SBA) class of hepatitis B virus (HBV) capsid inhibitors in phase 1 clinical trials. In cell culture models, AB-423 showed potent inhibition of HBV ...replication (50% effective concentration EC
= 0.08 to 0.27 μM; EC
= 0.33 to 1.32 μM) with no significant cytotoxicity (50% cytotoxic concentration > 10 μM). Addition of 40% human serum resulted in a 5-fold increase in the EC
s. AB-423 inhibited HBV genotypes A through D and nucleos(t)ide-resistant variants
Treatment of HepDES19 cells with AB-423 resulted in capsid particles devoid of encapsidated pregenomic RNA and relaxed circular DNA (rcDNA), indicating that it is a class II capsid inhibitor. In a
infection model, AB-423 prevented the conversion of encapsidated rcDNA to covalently closed circular DNA, presumably by interfering with the capsid uncoating process. Molecular docking of AB-423 into crystal structures of heteroaryldihydropyrimidines and an SBA and biochemical studies suggest that AB-423 likely also binds to the dimer-dimer interface of core protein.
dual combination studies with AB-423 and anti-HBV agents, such as nucleos(t)ide analogs, RNA interference agents, or interferon alpha, resulted in additive to synergistic antiviral activity. Pharmacokinetic studies with AB-423 in CD-1 mice showed significant systemic exposures and higher levels of accumulation in the liver. A 7-day twice-daily administration of AB-423 in a hydrodynamic injection mouse model of HBV infection resulted in a dose-dependent reduction in serum HBV DNA levels, and combination with entecavir or ARB-1467 resulted in a trend toward antiviral activity greater than that of either agent alone, consistent with the results of the
combination studies. The overall preclinical profile of AB-423 supports its further evaluation for safety, pharmacokinetics, and antiviral activity in patients with chronic hepatitis B.
Objective
Although individual-level social determinants of health (SDH) are known to influence 30-day readmission risk, contextual-level associations with readmission are poorly understood among ...children. This study explores associations between neighborhood disadvantage measured by Area Deprivation Index (ADI) and pediatric 30-day readmissions.
Methods
This retrospective cohort study included discharges of patients aged < 20 years from Maryland’s 2013–2016 all-payer dataset. The ADI, which quantifies 17 indicators of neighborhood socioeconomic disadvantage within census block groups, is used as a proxy for contextual-level SDH. Readmissions were identified with the 30-day Pediatric All-Condition Readmissions measure. Associations between ADI and readmission were identified with generalized estimating equations adjusted for patient demographics and clinical severity (Chronic Condition Indicator CCI, Pediatric Medical Complexity Algorithm PMCA, Index Hospital All Patients Refined Diagnosis Related Groups APR-DRG), and hospital discharge volume.
Results
Discharges (n = 138,998) were mostly female (52.7%), publicly insured (55.1%), urban-dwelling (93.0%), with low clinical severity levels (0–1 CCIs 82.3%, minor APR-DRG severity 48.4%). Overall readmission rate was 4.0%. Compared to the least disadvantaged ADI quartile, readmissions for the most disadvantaged quartile were significantly more likely (aOR 1.19, 95% CI 1.09–1.30). After adjustment, readmissions were associated with public insurance and indicators of medical complexity (higher number of CCIs, complex-chronic disease PMCA, and APR-DRG severity).
Conclusion
In this all-payer, statewide sample, living in the most socioeconomically disadvantaged neighborhoods independently predicted pediatric readmission. While the relative magnitude of neighborhood disadvantage was modest compared to medical complexity, disadvantage is modifiable and thus represents an important consideration for prevention and risk stratification efforts.
Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial ...permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo−/−) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.
Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a ...cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.
Airborne particular matter (PM) pollution is an increasing global issue and alternative sources of filter fibers are now an area of significant focus. Compared with relatively mature hazardous gas ...treatments, state of the art high-efficiency PM filters still lack thermal decomposition ability for organic PM pollutants, such as soot from coal-fired power plants and waste-combustion incinerators, resulting in frequent replacement, high cost, and second-hand pollution. In this manuscript, we propose a bottom-up synthesis method to make the first all-thermal-catalyst air filter (ATCAF). Self-assembled from ∼50 nm diameter TiO2 fibers, ATCAF could not only capture the combustion-generated PM pollutants with >99.999% efficiency but also catalyze the complete decomposition of the as-captured hydrocarbon pollutants at high temperature. It has the potential of in situ eliminating the PM pollutants from burning of hydrocarbon materials leveraging the burning heat.
Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies ...generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitosis and kinetochore function; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.
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