Antibody–drug conjugates (ADCs) are effective targeted therapeutics but are limited in their ability to incorporate less-potent payloads, varied drug mechanisms of action, different drug release mechanisms and tunable drug-to-antibody ratios. Here we introduce a technology to overcome these limitations called ‘antibody–bottlebrush prodrug conjugates’ (ABCs). An ABC consists of an IgG1 monoclonal antibody covalently conjugated to the terminus of a compact bivalent bottlebrush prodrug that has payloads bound through cleavable linkers and polyethylene glycol branches. This design enables the synthesis of ABCs with tunable average drug-to-antibody ratios up to two orders of magnitude greater than those of traditional ADCs. We demonstrate the functional flexibility and manufacturing efficiency of this technology by synthesizing more than 10 different ABCs targeting either HER2 or MUC1 using drugs with potencies spanning several orders of magnitude as well as imaging agents for ABC visualization and photocatalysts for proximity-based labeling of the ABC interactome. ABCs display high target engagement, high cell uptake and improved efficacy in tumor models compared to conventional HER2-targeted ADCs, suggesting promise for clinical translation. Antibody–bottlebrush conjugates expand the options for drug cargos compared to antibody–drug conjugates.

Mitochondrial dysfunction is a characteristic trait of human and rodent obesity, insulin resistance and fatty liver disease. Here we show that high-fat diet (HFD) feeding causes mitochondrial fragmentation in inguinal white adipocytes from male mice, leading to reduced oxidative capacity by a process dependent on the small GTPase RalA. RalA expression and activity are increased in white adipocytes after HFD. Targeted deletion of RalA in white adipocytes prevents fragmentation of mitochondria and diminishes HFD-induced weight gain by increasing fatty acid oxidation. Mechanistically, RalA increases fission in adipocytes by reversing the inhibitory Ser637 phosphorylation of the fission protein Drp1, leading to more mitochondrial fragmentation. Adipose tissue expression of the human homolog of Drp1, DNM1L , is positively correlated with obesity and insulin resistance. Thus, chronic activation of RalA plays a key role in repressing energy expenditure in obese adipose tissue by shifting the balance of mitochondrial dynamics toward excessive fission, contributing to weight gain and metabolic dysfunction.

Despite numerous female contraceptive options, nearly half of all pregnancies are unintended. Family planning choices for men are currently limited to unreliable condoms and invasive vasectomies with questionable reversibility. Here, we report the development of an oral contraceptive approach based on transcriptional disruption of cyclical gene expression patterns during spermatogenesis. Spermatogenesis involves a continuous series of self-renewal and differentiation programs of spermatogonial stem cells (SSCs) that is regulated by retinoic acid (RA)-dependent activation of receptors (RARs), which control target gene expression through association with corepressor proteins. We have found that the interaction between RAR and the corepressor silencing mediator of retinoid and thyroid hormone receptors (SMRT) is essential for spermatogenesis. In a genetically engineered mouse model that negates SMRT-RAR binding (SMRTmRID mice), the synchronized, cyclic expression of RAR-dependent genes along the seminiferous tubules is disrupted. Notably, the presence of an RA-resistant SSC population that survives RAR de-repression suggests that the infertility attributed to the loss of SMRT-mediated repression is reversible. Supporting this notion, we show that inhibiting the action of the SMRT complex with chronic, low-dose oral administration of a histone deacetylase inhibitor reversibly blocks spermatogenesis and fertility without affecting libido. This demonstration validates pharmacologic targeting of the SMRT repressor complex for non-hormonal male contraception.

The Association of Women's Health, Obstetric and Neonatal Nurses recently recommended continued use of supplemental oxygen (O2) for intrauterine resuscitation despite ACOG recommendations to the contrary. There is a need to address the safety of this practice given neonatal and adult literature that demonstrate a link between excess O2 exposure and free radical-mediated oxidative cell damage. We tested the hypothesis that O2, compared with room air (RA), for Category II fetal heart tracings (FHT) is associated with an increase in umbilical artery (UA) and maternal measures of oxidative stress. This was a single-center randomized controlled trial. Singleton pregnancies ³ 37 weeks with Category II FHT necessitating intrauterine resuscitation in active labor were randomized to RA or 10L/min O2 by face mask until delivery. The primary outcomes were two UA markers of oxidative stress via free radical-mediated lipid peroxidation: malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). They were measured using commercially available thiobarbituric acid and competitive ELISA reactive substances’ assays. Secondary outcomes were maternal plasma MDA and 4-HNE at time of delivery, UA cord gases, and mode of delivery.185 patients were needed for 80% power to detect a 10% difference in UA MDA levels between groups. We increased the sample size to 212 to accommodate 15% loss to unattainable UA samples. Analysis was by intention-to-treat. Of the 212 patients randomized, 153 had UA samples that were adequate for oxidative stress analysis; 74 O2 and 79 RA. Baseline characteristics and alternate intrauterine resuscitation techniques were similar between groups. There was no difference in median UA MDA or 4-HNE between O2 and RA groups (Figure). There was no difference in maternal MDA or 4-HNE, UA gases, or mode of delivery between groups. Among patients with Category II FHT in active labor, intrauterine resuscitation with supplemental O2 is not associated with an increase in fetal or maternal lipid peroxidation markers. Clinical outcomes were similar between O2 and RA groups.

Somatic mutations and homozygous deletions targeting chromatin regulators in ASCP genomes. IGV views of A) TP53Y205S and MSL2C44W in 3.8N population sorted from primary tumor 9R. B) KRASG12D and KANSL1Q396X in flow sorted tumor fraction in PAX217. Sorted 2.0N fraction was used as a patient matched normal for 9R while patient matched lymphocytes were used as a source of normal DNA for PAX217. Homozygous deletions (log2 ratio <-3.0) targeting ASXL2 and ARID2 in C) PENN040 and D) PAX208.

Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) hijack the epigenetic machinery in stromal components to establish a desmoplastic and therapeutic resistant tumor microenvironment (TME). Here we identify Class I histone deacetylases (HDACs) as key epigenetic factors facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in pancreatic stromal fibroblasts. Mechanistically, HDAC-mediated changes in chromatin architecture enable the activation of pro-desmoplastic programs directed by serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also coordinate fibroblast pro-inflammatory programs inducing leukemia inhibitory factor (LIF) expression, supporting paracrine pro-tumorigenic crosstalk. HDAC depletion in cancer-associated fibroblasts (CAFs) and treatment with the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a lipogenic fibroblast subpopulation, a potential precursor for myofibroblasts in the PDAC stroma. Overall, our study reveals the stromal targeting potential of HDACi, highlighting the utility of this epigenetic modulating approach in PDAC therapeutics.

Tibial malunion and nonunion are complications that may follow the repair of traumatic tibial shaft fractures. Management may sometime require osteotomy and bone transport. In recent years, there has been a paradigm shift in the management of intercalary tibial defects away from bone transport via external fixation towards intramedullary lengthening nails. This shift necessitates a re-evaluation of the approach to infection and soft tissue defects.We describe the case of a young man with a two-year history of infected tibial nonunion who underwent osteotomy and debridement before requiring free tissue transfer and eventual bone transport. Second, we present the case of a middle-aged man with a 30-year history of infected tibial malunion. Prior to placement of the intramedullary nail, debridement and osteotomy were performed, followed by two free anterolateral thigh flaps and tissue rearrangement.The use of free tissue transfers and emphasis on achieving an anatomically correct lower extremity prior to the initiation of bone transport resulted in minimal external fixation time, treatment compliance, and satisfactory clinical outcomes. Although multiple treatments can produce successful bone transport, no cohesive treatment algorithm exists that addresses infection, external fixation time, healing, and psychological burden.The paradigm shift towards intramedullary bone transport devices requires the use of antibiotic implants and prioritization of the soft tissue envelope prior to device implantation to prevent hardware infection and reoperation. An algorithmic management approach by an orthoplastic surgical team that includes an orthopedic surgeon and microsurgeon is recommended.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy in need of new therapeutic options. Using unbiased analyses of super-enhancers (SEs) as sentinels of core genes involved in cell-specific function, here we uncover a druggable SE-mediated RNA-binding protein (RBP) cascade that supports PDAC growth through enhanced mRNA translation. This cascade is driven by a SE associated with the RBP heterogeneous nuclear ribonucleoprotein F, which stabilizes protein arginine methyltransferase 1 (PRMT1) to, in turn, control the translational mediator ubiquitin-associated protein 2-like. All three of these genes and the regulatory SE are essential for PDAC growth and coordinately regulated by the Myc oncogene. In line with this, modulation of the RBP network by PRMT1 inhibition reveals a unique vulnerability in Myc-high PDAC patient organoids and markedly reduces tumor growth in male mice. Our study highlights a functional link between epigenetic regulation and mRNA translation and identifies components that comprise unexpected therapeutic targets for PDAC.

BACKGROUND: Cardiac metabolic dysfunction is a hallmark of heart failure (HF). Estrogen-related receptors ERRα and ERRγ are essential regulators of cardiac metabolism. Therefore, activation of ERR could be a potential therapeutic intervention for HF. However, in vivo studies demonstrating the potential usefulness of ERR agonist for HF treatment are lacking, because compounds with pharmacokinetics appropriate for in vivo use have not been available. METHODS: Using a structure-based design approach, we designed and synthesized 2 structurally distinct pan-ERR agonists, SLU-PP-332 and SLU-PP-915. We investigated the effect of ERR agonist on cardiac function in a pressure overload–induced HF model in vivo. We conducted comprehensive functional, multi-omics (RNA sequencing and metabolomics studies), and genetic dependency studies both in vivo and in vitro to dissect the molecular mechanism, ERR isoform dependency, and target specificity. RESULTS: Both SLU-PP-332 and SLU-PP-915 significantly improved ejection fraction, ameliorated fibrosis, and increased survival associated with pressure overload–induced HF without affecting cardiac hypertrophy. A broad spectrum of metabolic genes was transcriptionally activated by ERR agonists, particularly genes involved in fatty acid metabolism and mitochondrial function. Metabolomics analysis showed substantial normalization of metabolic profiles in fatty acid/lipid and tricarboxylic acid/oxidative phosphorylation metabolites in the mouse heart with 6-week pressure overload. ERR agonists increase mitochondria oxidative capacity and fatty acid use in vitro and in vivo. Using both in vitro and in vivo genetic dependency experiments, we show that ERRγ is the main mediator of ERR agonism-induced transcriptional regulation and cardioprotection and definitively demonstrated target specificity. ERR agonism also led to downregulation of cell cycle and development pathways, which was partially mediated by E2F1 in cardiomyocytes. CONCLUSIONS: ERR agonists maintain oxidative metabolism, which confers cardiac protection against pressure overload–induced HF in vivo. Our results provide direct pharmacologic evidence supporting the further development of ERR agonists as novel HF therapeutics.

Focal amplicons in ASCP genomes. The CNV profiles of A) A129 and B) PAX232 included focal high level (log2 ratio >2) amplicons that targeted the TLK2 and the ASH2L loci respectively. The X and Y axes in the CGH plots represent chromosome and log2 ratios for each genome. Red shaded areas denote homozygous deletions.

Mitochondrial dysfunction is a characteristic trait of human and rodent obesity, insulin resistance, and fatty liver disease. Here we report that mitochondria undergo fragmentation and reduced oxidative capacity specifically in inguinal white adipose tissue after feeding mice high fat diet (HFD) by a process dependent on the small GTPase RalA. RalA expression and activity are increased in white adipocytes from mice fed HFD. Targeted deletion of Rala in white adipocytes prevents the obesity-induced fragmentation of mitochondria and produces mice resistant to HFD-induced weight gain via increased fatty acid oxidation. As a result, these mice also exhibit improved glucose tolerance and liver function. In vitro mechanistic studies revealed that RalA suppresses mitochondrial oxidative function in adipocytes by increasing fission through reversing the protein kinase A-catalyzed inhibitory Ser637phosphorylation of the mitochondrial fission protein Drp1. Active RalA recruits protein phosphatase 2A (PP2Aa) to specifically dephosphorylate this inhibitory site on Drp1, activating the protein, thus increasing mitochondrial fission. Adipose tissue expression of the human homolog of Drp1, DNML1, is positively correlated with obesity and insulin resistance in patients. Thus, chronic activation of RalA plays a key role in repressing energy expenditure in obese adipose tissue by shifting the balance of mitochondrial dynamics towards excessive fission, contributing to weight gain and related metabolic dysfunction.

ABSTRACT Objectives Most human in‐vivo placental imaging techniques are unable to distinguish and characterize various placental compartments, such as the intervillous space (IVS), placental vessels (PV) and placental tissue (PT), limiting their specificity. We describe a method that employs T2* and diffusion‐weighted magnetic resonance imaging (MRI) data to differentiate automatically placental compartments, quantify their oxygenation properties and identify placental lesions (PL) in vivo . We also investigate the association between placental oxygenation patterns and fetal brain oxygenation. Methods This was a prospective study conducted between 2018 and 2021 in which dual‐contrast clinical MRI data (T2* and diffusion‐weighted MRI) were acquired from patients between 20 and 38 weeks' gestation. We trained a fuzzy clustering method to analyze T2* and diffusion‐weighted MRI data and assign placental voxels to one of four clusters, based on their distinct imaging domain features. The new method divided automatically the placenta into IVS, PV, PT and PL compartments and characterized their oxygenation changes throughout pregnancy. Results A total of 27 patients were recruited, of whom five developed pregnancy complications. Total placental oxygenation level and T2* did not demonstrate a statistically significant temporal correlation with gestational age (GA) ( R 2 = 0.060, P = 0.27). In contrast, the oxygenation level reflected by T2* values in the placental IVS ( R 2 = 0.51, P = 0.0002) and PV ( R 2 = 0.76, P = 1.1 × 10 −7 ) decreased significantly with advancing GA. Oxygenation levels in the PT did not show any temporal change during pregnancy ( R 2 = 0.00044, P = 0.93). A strong spatial‐dependent correlation between PV oxygenation level and GA was observed. The strongest negative correlation between PV oxygenation and GA ( R 2 = 0.73, P = 4.5 × 10 −7 ) was found at the fetal‐vessel‐dominated region close to the chorionic plate. The location and extent of the placental abnormality were automatically delineated and quantified in the five women with clinically confirmed placental pathology. Compared to the averaged total placental oxygenation, placental IVS oxygenation level best reflected fetal brain oxygenation level during fetal development. Conclusion Based on clinically feasible dual‐MRI, our method enables accurate spatiotemporal quantification of placental compartment and fetal brain oxygenation across different GAs. This information should improve our knowledge of human placenta development and its relationship with normal and abnormal pregnancy. © 2022 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Over the last decades, epidemiology and functional studies have started to reveal a pivotal role of vitamin D in both type 1 and type 2 diabetes pathogenesis. Acting through the vitamin D receptor (VDR), vitamin D regulates insulin secretion in pancreatic islets and insulin sensitivity in multiple peripheral metabolic organs. In vitro studies and both T1D and T2D animal models showed that vitamin D can improve glucose homeostasis by enhancing insulin secretion, reducing inflammation, reducing autoimmunity, preserving beta cell mass, and sensitizing insulin action. Conversely, vitamin D deficiency has been shown relevant in increasing T1D and T2D incidence. While clinical trials testing the hypothesis that vitamin D improves glycemia in T2D have shown conflicting results, subgroup and meta-analyses support the idea that raising serum vitamin D levels may reduce the progression from prediabetes to T2D. In this review, we summarize current knowledge on the molecular mechanisms of vitamin D in insulin secretion, insulin sensitivity, and immunity, as well as the observational and interventional human studies investigating the use of vitamin D as a treatment for diabetes.

Repetitive physical exercise induces physiological adaptations in skeletal muscle that improves exercise performance and is effective for the prevention and treatment of several diseases. Genetic evidence indicates that the orphan nuclear receptors estrogen receptor-related receptors (ERRs) play an important role in skeletal muscle exercise capacity. Three ERR subtypes exist (ERRα, β, and γ), and although ERRβ/γ agonists have been designed, there have been significant difficulties in designing compounds with ERRα agonist activity. Additionally, there are limited synthetic agonists that can be used to target ERRs in vivo. Here, we report the identification of a synthetic ERR pan agonist, SLU-PP-332, that targets all three ERRs but has the highest potency for ERRα. Additionally, SLU-PP-332 has sufficient pharmacokinetic properties to be used as an in vivo chemical tool. SLU-PP-332 increases mitochondrial function and cellular respiration in a skeletal muscle cell line. When administered to mice, SLU-PP-332 increased the type IIa oxidative skeletal muscle fibers and enhanced exercise endurance. We also observed that SLU-PP-332 induced an ERRα-specific acute aerobic exercise genetic program, and the ERRα activation was critical for enhancing exercise endurance in mice. These data indicate the feasibility of targeting ERRα for the development of compounds that act as exercise mimetics that may be effective in the treatment of numerous metabolic disorders and to improve muscle function in the aging.

The dysregulation of energy homeostasis in obesity involves multihormone resistance. Although leptin and insulin resistance have been well characterized, catecholamine resistance remains largely unexplored. Murine β3-adrenergic receptor expression in adipocytes is orders of magnitude higher compared with that of other isoforms. While resistant to classical desensitization pathways, its mRNA (Adrb3) and protein expression are dramatically downregulated after ligand exposure (homologous desensitization). β3-Adrenergic receptor downregulation also occurs after high-fat diet feeding, concurrent with catecholamine resistance and elevated inflammation. This downregulation is recapitulated in vitro by TNF-α treatment (heterologous desensitization). Both homologous and heterologous desensitization of Adrb3 were triggered by induction of the pseudokinase TRIB1 downstream of the EPAC/RAP2A/PI-PLC pathway. TRIB1 in turn degraded the primary transcriptional activator of Adrb3, CEBPα. EPAC/RAP inhibition enhanced catecholamine-stimulated lipolysis and energy expenditure in obese mice. Moreover, adipose tissue expression of genes in this pathway correlated with body weight extremes in a cohort of genetically diverse mice and with BMI in 2 independent cohorts of humans. These data implicate a signaling axis that may explain reduced hormone-stimulated lipolysis in obesity and resistance to therapeutic interventions with β3-adrenergic receptor agonists.

Inexorable increases in insulin resistance, lipolysis, and hepatic glucose production (HGP) are hallmarks of type 2 diabetes. Previously, we showed that peripheral delivery of exogenous fibroblast growth factor 1 (FGF1) has robust anti-diabetic effects mediated by the adipose FGF receptor (FGFR) 1. However, its mechanism of action is not known. Here, we report that FGF1 acutely lowers HGP by suppressing adipose lipolysis. On a molecular level, FGF1 inhibits the cAMP-protein kinase A axis by activating phosphodiesterase 4D (PDE4D), which separates it mechanistically from the inhibitory actions of insulin via PDE3B. We identify Ser44 as an FGF1-induced regulatory phosphorylation site in PDE4D that is modulated by the feed-fast cycle. These findings establish the FGF1/PDE4 pathway as an alternate regulator of the adipose-HGP axis and identify FGF1 as an unrecognized regulator of fatty acid homeostasis.

The burden of nonalcoholic fatty liver disease (NAFLD) is rising globally. Cardiovascular disease is the leading cause of death in patients with NAFLD. Nearly half of individuals with NAFLD have coronary heart disease, and more than a third have carotid artery atherosclerosis. Individuals with NAFLD are at a substantially higher risk of fatal and nonfatal cardiovascular events. NAFLD and cardiovascular disease share multiple common disease mechanisms, such as systemic inflammation, insulin resistance, genetic risk variants, and gut microbial dysbiosis. In this review, we discuss the epidemiology of cardiovascular disease in NAFLD, and highlight common risk factors. In addition, we examine recent advances evaluating the shared disease mechanisms between NAFLD and cardiovascular disease. In conclusion, multidisciplinary collaborations are required to further our understanding of the complex relationship between NAFLD and cardiovascular disease and potentially identify therapeutic targets.