Beneficial effects of MGL-3196 and BAM15 combination in a mouse model of fatty liver disease.

BACKGROUND AND AIM: Metabolic dysfunction-associated steatohepatitis (MASH) is a metabolic disorder with limited treatment options. The thyroid hormone receptor (THR)-ß agonist resmetirom/MGL-3196 (MGL) increases liver fat oxidation and has been approved for treating adult MASH. However, over 60% of patients receiving MGL treatment do not achieve MASH resolution. Therefore, we investigated the potential for combination therapy of MGL with the mitochondrial uncoupler BAM15 to improve fatty liver disease outcomes in the GAN mouse model of MASH. METHODS: C57BL/6J male mice were fed GAN diet for 38 weeks before stratification and randomization to treatments including MGL, BAM15, MGL + BAM15, or no drug control for 8 weeks. Treatments were admixed in diet and mice were pair-fed to control for drug intake. Treatment effectiveness was assessed by body weight, body composition, energy expenditure, glucose tolerance, tissue lipid content, and histological analyses. RESULTS: MGL + BAM15 treatment resulted in better efficacy versus GAN control mice than either monotherapy in the context of energy expenditure, liver fat loss, glucose control, and fatty liver disease activity score. Improvements in ALT, liver mass, and plasma cholesterol were primarily driven by MGL, while improvements in body fat were primarily driven by BAM15. No treatments altered liver fibrosis. CONCLUSIONS: MGL + BAM15 treatment had overall better efficacy to improve metabolic outcomes in mice fed GAN diet than either monotherapy alone. These data warrant further investigation into combination therapies of THR-ß agonists and mitochondrial uncouplers for the potential treatment of disorders related to fatty liver, obesity, and insulin resistance.

Calcium tunneling through the ER and transfer to other organelles for optimal signaling in Toxoplasma gondii.

Ca2+ signaling in cells begins with the opening of Ca2+ channels in either the plasma membrane (PM) or the endoplasmic reticulum (ER) and results in a dramatic increase in the physiologically low (<100 nM) cytosolic Ca2+ level. The temporal and spatial Ca2+ levels are well regulated to enable precise and specific activation of critical biological processes. Ca2+ signaling regulates pathogenic features of apicomplexan parasites like Toxoplasma gondii which infects approximately one-third of the world's population. T. gondii relies on Ca2+ signals to stimulate traits of its infection cycle and several Ca2+ signaling elements play essential roles in its parasitic cycle. Active egress, an essential step for the infection cycle of T. gondii is preceded by a large increase in cytosolic Ca2+ most likely by release from intracellular stores. Intracellular parasites take up Ca2+ from the host cell during host Ca2+ signaling events to replenish intracellular stores. In this work, we investigated the mechanism by which intracellular stores are replenished with Ca2+ and demonstrated a central role for the SERCA-Ca2+-ATPase to keep not only the ER filled with Ca2+ but also acidic stores. We also show mitochondrial Ca2+ uptake, by transfer of Ca2+ from the ER most likely through membrane contact sites. We propose a central role for the ER in tunneling of calcium from the extracellular milieu through the ER to other organelles.

Fertility protection during chemotherapy treatment by boosting the NAD(P)+ metabolome.

Chemotherapy induced ovarian failure and infertility is an important concern in female cancer patients of reproductive age or younger, and non-invasive, pharmacological approaches to maintain ovarian function are urgently needed. Given the role of reduced nicotinamide adenine dinucleotide phosphate (NADPH) as an essential cofactor for drug detoxification, we sought to test whether boosting the NAD(P)+ metabolome could protect ovarian function. We show that pharmacological or transgenic strategies to replenish the NAD+ metabolome ameliorates chemotherapy induced female infertility in mice, as measured by oocyte yield, follicle health, and functional breeding trials. Importantly, treatment of a triple-negative breast cancer mouse model with the NAD+ precursor nicotinamide mononucleotide (NMN) reduced tumour growth and did not impair the efficacy of chemotherapy drugs in vivo or in diverse cancer cell lines. Overall, these findings raise the possibility that NAD+ precursors could be a non-invasive strategy for maintaining ovarian function in cancer patients, with potential benefits in cancer therapy.

Web-Based Tool Designed to Encourage Supplemental Nutrition Assistance Program Use in Urban College Students: Usability Testing Study.

BACKGROUND: Food insecurity continues to be a risk for college students in the United States. It is associated with numerous problems, such as chronic health conditions, increased stress and anxiety, and a lower grade point average. After COVID-19, the Supplemental Nutrition Assistance Program (SNAP) benefits were extended to college-aged students; however, there were some barriers to participation, which persisted such as lack of perceived food insecurity risk, lack of knowledge regarding the SNAP application process, the complexity of determining eligibility, and stigma associated with needing social assistance. A technology-enhanced tool was developed to address these barriers to SNAP enrollment and encourage at-risk college students to apply for SNAP. OBJECTIVE: The purpose of this study was to test the usability and acceptability of a web-based SNAP screening tool designed for college-aged students. METHODS: College students aged 18-25 years were recruited to participate in 2 rounds of usability testing during fall 2022. Participants tested the prototype of a web-based SNAP screener tool using a standardized think-aloud method. The usability and acceptability of the tool were assessed using a semistructured interview and a 10-item validated System Usability Scale questionnaire. Audio recordings and field notes were systematically reviewed by extracting and sorting feedback as positive or negative comments. System Usability Scale questionnaire data were analyzed using the Wilcoxon signed rank test and sign test. RESULTS: A total of 12 students (mean age 21.8, SD 2.8 years; n=6, 50% undergraduate; n=11, 92% female; n=7, 58% Hispanic or Black or African American; n=9, 78% low or very low food security) participated in both rounds of user testing. Round 1 testing highlighted overall positive experiences with the tool, with most participants (10/12) stating that the website fulfills its primary objective as a support tool to encourage college students to apply for SNAP. However, issues related to user interface design, navigation, and wording of some questions in the screening tool were noted. Key changes after round 1 reflected these concerns, including improved design of response buttons and tool logo and improved clarity of screening questions. The overall system usability showed slight, but not statistically significant, improvement between round 1 and round 2 (91.25 vs 92.50; P=.10, respectively). CONCLUSIONS: Overall usability findings suggest that this web-based tool was highly usable and acceptable to urban college students and could be an effective and appealing approach as a support tool to introduce college students to the SNAP application process. The findings from this study will inform further development of the tool, which could eventually be disseminated publicly among various college campuses.

Invited Commentary: Mixing multiple imputation and bootstrapping for variance estimation.

In 2023, Martinez et al. examined trends in the inclusion, conceptualization, operationalization and analysis of race and ethnicity among studies published in US epidemiology journals. Based on a random sample of papers (N=1,050) published from 1995-2018, the authors describe the treatment of race, ethnicity, and ethnorace in the analytic sample (N=414, 39% of baseline sample) over time. Between 32% and 19% of studies in each time stratum lacked race data; 61% to 34% lacked ethnicity data. The review supplies stark evidence of the routine omission and variability of measures of race and ethnicity in epidemiologic research. Informed by public health critical race praxis (PHCRP), this commentary discusses the implications of four problems the findings suggest pervade epidemiology: 1) a general lack of clarity about what race and ethnicity are; 2) the limited use of critical race or other theory; 3) an ironic lack of rigor in measuring race and ethnicity; and, 4) the ordinariness of racism and white supremacy in epidemiology. The identified practices reflect neither current publication guidelines nor the state of the knowledge on race, ethnicity and racism; therefore, we conclude by offering recommendations to move epidemiology toward more rigorous research in an increasingly diverse society.

Beneficial effects of simultaneously targeting calorie intake and calorie efficiency in diet-induced obese mice.

Semaglutide is an anti-diabetes and weight loss drug that decreases food intake, slows gastric emptying, and increases insulin secretion. Patients begin treatment with low-dose semaglutide and increase dosage over time as efficacy plateaus. With increasing dosage, there is also greater incidence of gastrointestinal side effects. One reason for the plateau in semaglutide efficacy despite continued low food intake is due to compensatory actions whereby the body becomes more metabolically efficient to defend against further weight loss. Mitochondrial uncoupler drugs decrease metabolic efficiency, therefore we sought to investigate the combination therapy of semaglutide with the mitochondrial uncoupler BAM15 in diet-induced obese mice. Mice were fed high-fat western diet (WD) and stratified into six treatment groups including WD control, BAM15, low-dose semaglutide without or with BAM15, and high-dose semaglutide without or with BAM15. Combining BAM15 with either semaglutide dose decreased body fat and liver triglycerides, which was not achieved by any monotherapy, while high-dose semaglutide with BAM15 had the greatest effect on glucose homeostasis. This study demonstrates a novel approach to improve weight loss without loss of lean mass and improve glucose control by simultaneously targeting energy intake and energy efficiency. Such a combination may decrease the need for semaglutide dose escalation and hence minimize potential gastrointestinal side effects.

Contrasting MEG effects of anodal and cathodal high-definition TDCS on sensorimotor activity during voluntary finger movements.

Introduction: Protocols for noninvasive brain stimulation (NIBS) are generally categorized as "excitatory" or "inhibitory" based on their ability to produce short-term modulation of motor-evoked potentials (MEPs) in peripheral muscles, when applied to motor cortex. Anodal and cathodal stimulation are widely considered excitatory and inhibitory, respectively, on this basis. However, it is poorly understood whether such polarity-dependent changes apply for neural signals generated during task performance, at rest, or in response to sensory stimulation. Methods: To characterize such changes, we measured spontaneous and movement-related neural activity with magnetoencephalography (MEG) before and after high-definition transcranial direct-current stimulation (HD-TDCS) of the left motor cortex (M1), while participants performed simple finger movements with the left and right hands. Results: Anodal HD-TDCS (excitatory) decreased the movement-related cortical fields (MRCF) localized to left M1 during contralateral right finger movements while cathodal HD-TDCS (inhibitory), increased them. In contrast, oscillatory signatures of voluntary motor output were not differentially affected by the two stimulation protocols, and tended to decrease in magnitude over the course of the experiment regardless. Spontaneous resting state oscillations were not affected either. Discussion: MRCFs are thought to reflect reafferent proprioceptive input to motor cortex following movements. Thus, these results suggest that processing of incoming sensory information may be affected by TDCS in a polarity-dependent manner that is opposite that seen for MEPs-increases in cortical excitability as defined by MEPs may correspond to reduced responses to afferent input, and vice-versa.

Liver-Selective Imidazolopyrazine Mitochondrial Uncoupler SHD865 Reverses Adiposity and Glucose Intolerance in Mice.

Excess body fat is a risk factor for metabolic diseases and is a leading preventable cause of morbidity and mortality worldwide. There is a strong need to find new treatments that decrease the burden of obesity and lower the risk of obesity-related comorbidities, including cardiovascular disease and type 2 diabetes. Pharmacologic mitochondrial uncouplers represent a potential treatment for obesity through their ability to increase nutrient oxidation. Herein, we report the in vitro and in vivo characterization of compound SHD865, the first compound to be studied in vivo in a newly discovered class of imidazolopyrazine mitochondrial uncouplers. SHD865 is a derivative of the furazanopyrazine uncoupler BAM15. SHD865 is a milder mitochondrial uncoupler than BAM15 that results in a lower maximal respiration rate. In a mouse model of diet-induced adiposity, 6-week treatment with SHD865 completely restored normal body composition and glucose tolerance to levels like those of chow-fed controls, without altering food intake. SHD865 treatment also corrected liver steatosis and plasma hyperlipidemia to normal levels comparable with chow-fed controls. SHD865 has maximal oral bioavailability in rats and slow clearance in human microsomes and hepatocytes. Collectively, these data identify the potential of imidazolopyrazine mitochondrial uncouplers as drug candidates for the treatment of obesity-related disorders.

SIRT2 transgenic over-expression does not impact lifespan in mice.

The NAD+ -dependent deacylase family of sirtuin enzymes have been implicated in biological ageing, late-life health and overall lifespan, though of these members, a role for sirtuin-2 (SIRT2) is less clear. Transgenic overexpression of SIRT2 in the BubR1 hypomorph model of progeria can rescue many aspects of health and increase overall lifespan, due to a specific interaction between SIRT2 and BubR1 that improves the stability of this protein. It is less clear whether SIRT2 is relevant to biological ageing outside of a model where BubR1 is under-expressed. Here, we sought to test whether SIRT2 over-expression would impact the overall health and lifespan of mice on a nonprogeroid, wild-type background. While we previously found that SIRT2 transgenic overexpression prolonged female fertility, here, we did not observe any additional impact on health or lifespan, which was measured in both male and female mice on standard chow diets, and in males challenged with a high-fat diet. At the biochemical level, NMR studies revealed an increase in total levels of a number of metabolites in the brain of SIRT2-Tg animals, pointing to a potential impact in cell composition; however, this did not translate into functional differences. Overall, we conclude that strategies to enhance SIRT2 protein levels may not lead to increased longevity.

Nuclear speckles regulate HIF-2α programs and correlate with patient survival in kidney cancer.

Nuclear speckles are membrane-less bodies within the cell nucleus enriched in RNA biogenesis, processing, and export factors. In this study we investigated speckle phenotype variation in human cancer, finding a reproducible speckle signature, based on RNA expression of speckle-resident proteins, across >20 cancer types. Of these, clear cell renal cell carcinoma (ccRCC) exhibited a clear correlation between the presence of this speckle expression signature, imaging-based speckle phenotype, and clinical outcomes. ccRCC is typified by hyperactivation of the HIF-2α transcription factor, and we demonstrate here that HIF-2α drives physical association of a select subset of its target genes with nuclear speckles. Disruption of HIF-2α-driven speckle association via deletion of its speckle targeting motifs (STMs)-defined in this study-led to defective induction of speckle-associating HIF-2α target genes without impacting non-speckle-associating HIF-2α target genes. We further identify the RNA export complex, TREX, as being specifically altered in speckle signature, and knockdown of key TREX component, ALYREF, also compromises speckle-associated gene expression. By integrating tissue culture functional studies with tumor genomic and imaging analysis, we show that HIF-2α gene regulatory programs are impacted by specific manipulation of speckle phenotype and by abrogation of speckle targeting abilities of HIF-2α. These findings suggest that, in ccRCC, a key biological function of nuclear speckles is to modulate expression of a specific subset of HIF-2α-regulated target genes that, in turn, influence patient outcomes. We also identify STMs in other transcription factors, suggesting that DNA-speckle targeting may be a general mechanism of gene regulation.

Host-microbiome interactions in nicotinamide mononucleotide (NMN) deamidation.

The nicotinamide adenine dinucleotide (NAD+ ) precursor nicotinamide mononucleotide (NMN) is a proposed therapy for age-related disease, whereby it is assumed that NMN is incorporated into NAD+ through the canonical recycling pathway. During oral delivery, NMN is exposed to the gut microbiome, which could modify the NAD+ metabolome through enzyme activities not present in the mammalian host. We show that orally delivered NMN can undergo deamidation and incorporation in mammalian tissue via the de novo pathway, which is reduced in animals treated with antibiotics to ablate the gut microbiome. Antibiotics increased the availability of NAD+ metabolites, suggesting the microbiome could be in competition with the host for dietary NAD+ precursors. These findings highlight new interactions between NMN and the gut microbiome.

Spurious intragenic transcription is a feature of mammalian cellular senescence and tissue aging.

Mammalian aging is characterized by the progressive loss of tissue function and increased risk for disease. Accumulation of senescent cells in aging tissues partly contributes to this decline, and targeted depletion of senescent cells in vivo ameliorates many age-related phenotypes. The fundamental molecular mechanisms responsible for the decline of cellular health and fitness during senescence and aging are largely unknown. In this study, we investigated whether chromatin-mediated loss of transcriptional fidelity, known to contribute to fitness and survival in yeast and worms, also occurs during human cellular senescence and mouse aging. Our findings reveal aberrant transcription initiation inside genes during senescence and aging that co-occurs with changes in the chromatin landscape. Interventions that alter these spurious transcripts have profound consequences on cellular health, primarily affecting intracellular signal transduction pathways. We propose that age-related spurious transcription promotes a noisy transcriptome and degradation of coherent transcriptional networks.

Oxadiazolopyridine Derivatives as Efficacious Mitochondrial Uncouplers in the Prevention of Diet-Induced Obesity.

Small-molecule mitochondrial uncouplers are gaining recognition as potential therapeutics for metabolic diseases such as obesity, diabetes, and nonalcoholic steatohepatitis (NASH). Specifically, heterocycles derived from BAM15, a potent and mitochondria-selective uncoupler, have yielded promising preclinical candidates that are efficacious in animal models of obesity and NASH. In this study, we report the structure-activity relationship studies of 6-amino-[1,2,5]oxadiazolo[3,4-b]pyridin-5-ol derivatives. Using oxygen consumption rate as a readout of mitochondrial uncoupling, we established 5-hydroxyoxadiazolopyridines as mild uncouplers. In particular, SHM115, which contains a pentafluoro aniline, had an EC50 value of 17 µM and exhibited 75% oral bioavailability. SHM115 treatment increased the energy expenditure and lowered the body fat mass in two diet-induced obesity mouse models, including an obesity prevention model and an obesity reversal model. Taken together, our findings demonstrate the therapeutic potential of mild mitochondrial uncouplers for the prevention of diet-induced obesity.

The Heptaprenyl Diphosphate Synthase (Coq1) Is the Target of a Lipophilic Bisphosphonate That Protects Mice against Toxoplasma gondii Infection.

Prenyldiphosphate synthases catalyze the reaction of allylic diphosphates with one or more isopentenyl diphosphate molecules to form compounds such as farnesyl diphosphate, used in, e.g., sterol biosynthesis and protein prenylation, as well as longer "polyprenyl" diphosphates, used in ubiquinone and menaquinone biosynthesis. Quinones play an essential role in electron transport and are associated with the inner mitochondrial membrane due to the presence of the polyprenyl group. In this work, we investigated the synthesis of the polyprenyl diphosphate that alkylates the ubiquinone ring precursor in Toxoplasma gondii, an opportunistic pathogen that causes serious disease in immunocompromised patients and the unborn fetus. The enzyme that catalyzes this early step of the ubiquinone synthesis is Coq1 (TgCoq1), and we show that it produces the C35 species heptaprenyl diphosphate. TgCoq1 localizes to the mitochondrion and is essential for in vitro T. gondii growth. We demonstrate that the growth defect of a T. gondii TgCoq1 mutant is rescued by complementation with a homologous TgCoq1 gene or with a (C45) solanesyl diphosphate synthase from Trypanosoma cruzi (TcSPPS). We find that a lipophilic bisphosphonate (BPH-1218) inhibits T. gondii growth at low-nanomolar concentrations, while overexpression of the TgCoq1 enzyme dramatically reduced growth inhibition by the bisphosphonate. Both the severe growth defect of the mutant and the inhibition by BPH-1218 were rescued by supplementation with a long-chain (C30) ubiquinone (UQ6). Importantly, BPH-1218 also protected mice against a lethal T. gondii infection. TgCoq1 thus represents a potential drug target that could be exploited for improved chemotherapy of toxoplasmosis. IMPORTANCE Millions of people are infected with Toxoplasma gondii, and the available treatment for toxoplasmosis is not ideal. Most of the drugs currently used are only effective for the acute infection, and treatment can trigger serious side effects requiring changes in the therapeutic approach. There is, therefore, a compelling need for safe and effective treatments for toxoplasmosis. In this work, we characterize an enzyme of the mitochondrion of T. gondii that can be inhibited by an isoprenoid pathway inhibitor. We present evidence that demonstrates that inhibition of the enzyme is linked to parasite death. In addition, the inhibitor can protect mice against a lethal dose of T. gondii. Our results thus reveal a promising chemotherapeutic target for the development of new medicines for toxoplasmosis.

Genome-wide association mapping and genomic prediction for kernel color traits in intermediate wheatgrass (Thinopyrum intermedium).

BACKGROUND: Intermediate wheatgrass (IWG) is a novel perennial grain crop currently undergoing domestication. It offers important ecosystem benefits while producing grain suitable for human consumption. Several aspects of plant biology and genetic control are yet to be studied in this new crop. To understand trait behavior and genetic characterization of kernel color in IWG breeding germplasm from the University of Minnesota was evaluated for the CIELAB components (L*, a*, b*) and visual differences. Trait values were used in a genome-wide association scan to reveal genomic regions controlling IWG's kernel color. The usability of genomic prediction in predicting kernel color traits was also evaluated using a four-fold cross validation method. RESULTS: A wide phenotypic variation was observed for all four kernel color traits with pairwise trait correlations ranging from - 0.85 to 0.27. Medium to high estimates of broad sense trait heritabilities were observed and ranged from 0.41 to 0.78. A genome-wide association scan with single SNP markers detected 20 significant marker-trait associations in 9 chromosomes and 23 associations in 10 chromosomes using multi-allelic haplotype blocks. Four of the 20 significant SNP markers and six of the 23 significant haplotype blocks were common between two or more traits. Evaluation of genomic prediction of kernel color traits revealed the visual score to have highest mean predictive ability (r2 = 0.53); r2 for the CIELAB traits ranged from 0.29-0.33. A search for candidate genes led to detection of seven IWG genes in strong alignment with MYB36 transcription factors from other cereal crops of the Triticeae tribe. Three of these seven IWG genes had moderate similarities with R-A1, R-B1, and R-D1, the three genes that control grain color in wheat. CONCLUSIONS: We characterized the distribution of kernel color in IWG for the first time, which revealed a broad phenotypic diversity in an elite breeding germplasm. Identification of genetic loci controlling the trait and a proof-of-concept that genomic selection might be useful in selecting genotypes of interest could help accelerate the breeding of this novel crop towards specific end-use.

Endothelial Cell-Specific Molecule-1 Inhibits Albuminuria in Diabetic Mice.

Background: Diabetic kidney disease (DKD) is the most common cause of kidney failure in the world, and novel predictive biomarkers and molecular mechanisms of disease are needed. Endothelial cell-specific molecule-1 (Esm-1) is a secreted proteoglycan that attenuates inflammation. We previously identified that a glomerular deficiency of Esm-1 associates with more pronounced albuminuria and glomerular inflammation in DKD-susceptible relative to DKD-resistant mice, but its contribution to DKD remains unexplored. Methods: Using hydrodynamic tail-vein injection, we overexpress Esm-1 in DKD-susceptible DBA/2 mice and delete Esm-1 in DKD-resistant C57BL/6 mice to study the contribution of Esm-1 to DKD. We analyze clinical indices of DKD, leukocyte infiltration, podocytopenia, and extracellular matrix production. We also study transcriptomic changes to assess potential mechanisms of Esm-1 in glomeruli. Results: In DKD-susceptible mice, Esm-1 inversely correlates with albuminuria and glomerular leukocyte infiltration. We show that overexpression of Esm-1 reduces albuminuria and diabetes-induced podocyte injury, independent of changes in leukocyte infiltration. Using a complementary approach, we find that constitutive deletion of Esm-1 in DKD-resistant mice modestly increases the degree of diabetes-induced albuminuria versus wild-type controls. By glomerular RNAseq, we identify that Esm-1 attenuates expression of kidney disease-promoting and interferon (IFN)-related genes, including Ackr2 and Cxcl11. Conclusions: We demonstrate that, in DKD-susceptible mice, Esm-1 protects against diabetes-induced albuminuria and podocytopathy, possibly through select IFN signaling. Companion studies in patients with diabetes suggest a role of Esm-1 in human DKD.

Emerging Epigenetic Therapies for Brain Tumors.

Malignant brain tumors are among the most intractable cancers, including malignancies such as glioblastoma, diffuse midline glioma, medulloblastoma, and ependymoma. Unfortunately, treatment options for these brain tumors have been inadequate and complex, leading to poor prognoses and creating a need for new treatment modalities. Aberrant epigenetics define these types of tumors, with underlying changes in DNA methylation, histone modifications, chromatin structure and noncoding RNAs. Epigenetic-targeted therapies are an alternative that have the potential to reverse the epigenetic deregulation underpinning brain malignancies. Various drugs targeting epigenetic regulators have shown promise in preclinical and clinical testing. In this review, we highlight some of the recent emerging epigenetic targeted therapies for brain tumors being evaluated in the discovery phase and in clinical trials.