Molecular interactions between the Wilms\u27 tumor transcription factor and the aryl hydrocarbon receptor during nephrogenesis and its impact on fetal programming of renal disease.

Embryonic development requires the orchestration of temporally precise genetic events that culminate in the formation of a complete organism. The molecular mechanisms responsible for ontogenesis are regulated by environmental and somatic factors in utero that activate or repress the expression of numerous genetic elements resulting in fetal programming of adult diseases. The aryl hydrocarbon receptor (AHR) is an important nuclear transcription factor both during embryogenesis and throughout maturity in multiple organisms. Building upon interesting studies establishing a direct, novel link between AHR and post-transcriptional regulation of the Wilms\u27 tumor suppressor ( WT1 ) gene, the overall goal of the project was to elucidate the role of AHR in the regulation of WT1 during nephrogenesis. The evidence shows that in utero exposure to environmentally relevant exposures of benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) and AHR ligand, results in AHR allele-specific deficits in renal development manifested as decreased numbers of glomeruli, induction in urinary albumin, and podocytopenia. These findings were consistent with metanephric and organ culture data where PAHs caused AHR allele-specific reduction in renal cell differentiation markers, dysregulation of WT1 mRNA splice variants, and decreases in direct WT1 transcriptional targets. Observed deficits were linked to disruption of constitutive AHR signaling, as siRNA-mediated AHR degradation reproduced similar effects. Collectively, this evidence defined a novel role for the AHR in renal development and in fetal programming of PAH-induced environmental disease

Albumin-Like Proteins Are Critical Regulators of Vascular Redox Signaling

This laboratory previously identified an albumin-like protein (denoted as p70) as a component of the macromolecular complex assembled within the 5 -regulatory region of redox-sensitive genes in vascular smooth muscle cells (vSMCs). Here we show that p70 is present in the cytosolic and nuclear compartments of vSMCs and dynamically responsive to redox status. Intense cytoplasmic and perinuclear staining, coupled with enhanced nuclear localization, was observed in vSMCs, but not HepG2 cells, treated with benzo(a)pyrene (BaP), H 2 O 2 , or N-acetylcysteine, agents known to modulate redox status. 3 RACE indicated that p70 is not generated as a product of endogenous gene expression, but rather taken up from the extracellular compartment. While p70 was undetectable in cells grown for 24 hours under serum-free conditions, cell-associated, acid-resistant albumin was detected 30 min after the addition of exogenous albumin. vSMCs incubated at 4 ∘ C with 100 g/mL unlabeled BSA and 10 g/mL FITC-BSA for 60 minutes and switched to 37 ∘ C to examine temperature-sensitive label uptake showed punctate structures throughout the cell consistent with albumin internalization at the higher temperature. Albumin was found to influence redox-signaling, as evidenced by modulation of cyp1a1 gsta1 and Ha-ras gene inducibility. Together, these results implicate albumin and albumin-like proteins as critical regulators of vascular redox signaling

A Mutant Ahr Allele Protects the Embryonic Kidney from Hydrocarbon-Induced Deficits in Fetal Programming

Background: The use of experimental model systems has expedited the elucidation of pathogenetic mechanisms of renal developmental disease in humans and the identification of genes that orchestrate developmental programming during nephrogenesis

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Molecular interactions between the Wilms' tumor transcription factor and the aryl hydrocarbon receptor during nephrogenesis and its impact on fetal programming of renal disease

Embryonic development requires the orchestration of temporally precise genetic events that culminate in the formation of a complete organism. The molecular mechanisms responsible for ontogenesis are regulated by environmental and somatic factors in utero that activate or repress the expression of numerous genetic elements resulting in fetal programming of adult diseases. The aryl hydrocarbon receptor (AHR) is an important nuclear transcription factor both during embryogenesis and throughout maturity in multiple organisms. Building upon interesting studies establishing a direct, novel link between AHR and post-transcriptional regulation of the Wilms' tumor suppressor (WT1 ) gene, the overall goal of the project was to elucidate the role of AHR in the regulation of WT1 during nephrogenesis. The evidence shows that in utero exposure to environmentally relevant exposures of benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) and AHR ligand, results in AHR allele-specific deficits in renal development manifested as decreased numbers of glomeruli, induction in urinary albumin, and podocytopenia. These findings were consistent with metanephric and organ culture data where PAHs caused AHR allele-specific reduction in renal cell differentiation markers, dysregulation of WT1 mRNA splice variants, and decreases in direct WT1 transcriptional targets. Observed deficits were linked to disruption of constitutive AHR signaling, as siRNA-mediated AHR degradation reproduced similar effects. Collectively, this evidence defined a novel role for the AHR in renal development and in fetal programming of PAH-induced environmental disease

Albumin-Like Proteins Are Critical Regulators of Vascular Redox Signaling

This laboratory previously identified an albumin-like protein (denoted as p70) as a component of the macromolecular complex assembled within the 5′-regulatory region of redox-sensitive genes in vascular smooth muscle cells (vSMCs). Here we show that p70 is present in the cytosolic and nuclear compartments of vSMCs and dynamically responsive to redox status. Intense cytoplasmic and perinuclear staining, coupled with enhanced nuclear localization, was observed in vSMCs, but not HepG2 cells, treated with benzo(a)pyrene (BaP), H2O2, or N-acetylcysteine, agents known to modulate redox status. 3′ RACE indicated that p70 is not generated as a product of endogenous gene expression, but rather taken up from the extracellular compartment. While p70 was undetectable in cells grown for 24 hours under serum-free conditions, cell-associated, acid-resistant albumin was detected 30 min after the addition of exogenous albumin. vSMCs incubated at 4°C with 100 μg/mL unlabeled BSA and 10 μg/mL FITC-BSA for 60 minutes and switched to 37°C to examine temperature-sensitive label uptake showed punctate structures throughout the cell consistent with albumin internalization at the higher temperature. Albumin was found to influence redox-signaling, as evidenced by modulation of cyp1a1 gsta1 and Ha-ras gene inducibility. Together, these results implicate albumin and albumin-like proteins as critical regulators of vascular redox signaling

Discovery of AM-6494: A Potent and Orally Efficacious β-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitor with in Vivo Selectivity over BACE2

β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is an aspartyl protease that plays a key role in the production of amyloid β (Aβ) in the brain and has been extensively pursued as a target for the treatment of Alzheimer’s disease (AD). BACE2, an aspartyl protease that is structurally related to BACE1, has been recently reported to be involved in melanosome maturation and pigmentation. Herein, we describe the development of a series of cyclopropylthiazines as potent and orally efficacious BACE1 inhibitors. Lead optimization led to the identification of 20, a molecule with biochemical IC50 BACE2/BACE1 ratio of 47. Administration of 20 resulted in no skin/fur color change in a 13-day mouse hypopigmentation study and demonstrated robust and sustained reduction of CSF and brain Aβ40 levels in rat and monkey pharmacodynamic models. On the basis of a compelling data package, 20 (AM-6494) was advanced to preclinical development