Daily consumption of ketone ester, bis-octanoyl (R)-1,3-butanediol, is safe and tolerable in healthy older adults, a randomized, parallel arm, double-blind, placebo-controlled, pilot study.

Objectives: Ketone bodies are endogenous metabolites produced during fasting or a ketogenic diet that have pleiotropic effects on aging pathways. Ketone esters (KEs) are compounds that induce ketosis without dietary changes, but KEs have not been studied in an older adult population. The primary objective of this trial was to determine tolerability and safety of KE ingestion in older adults. Design: Randomized, placebo-controlled, double-blinded, parallel-arm trial, with a 12-week intervention period ( NCT05585762 ). Setting: General community, Northern California, USA. Participants: Community-dwelling older adults, independent in activities of daily living, with no unstable acute medical conditions (n=30) were randomized and n=23 (M= 14, F=9) completed the protocol. Intervention: Participants were randomly allocated to consume either KE (bis-octanoyl (R)-1,3-butanediol) or a taste, appearance, and calorie-matched placebo (PLA) containing canola oil. Measurements: Tolerability was assessed using a composite score from a daily log for 2-weeks, and then via a bi-weekly phone interview. Safety was assessed by vital signs and lab tests at screening and weeks 0, 4 and 12, along with tabulation of adverse events. Results: There was no difference in the prespecified primary outcome of proportion of participants reporting moderate or severe nausea, headache, or dizziness on more than one day in a two-week reporting period (KE n =2 (14.3% [90% CI = 2.6 - 38.5]); PLA n=1 (7.1% [90% CI = 0.4 - 29.7]). Dropouts numbered four in the PLA group and two in the KE group. A greater number of symptoms were reported in both groups during the first two weeks; symptoms were reported less frequently between 2 - 12 weeks. There were no clinically relevant changes in safety labs or vital signs in either group. Conclusions: This KE was safe and well-tolerated in healthy older adults. These results provide a foundation for use of KEs in aging research. Highlights: Ketones esters induce ketosis without dietary changes and may target aging biologyStudies of ketone esters were limited in duration and focused on younger adultsWe found ketone esters were safe and tolerable for 12 weeks in healthy older adults.

A ketogenic diet reduces age-induced chronic neuroinflammation in mice Running title: ketogenic diet and brain inflammaging.

Beta-hydroxybutyrate (BHB) is a ketone body synthesized during fasting or strenuous exercise. Our previous study demonstrated that a cyclic ketogenic diet (KD), which induces BHB levels similar to fasting every other week, reduces midlife mortality and improves memory in aging mice. BHB actively regulates gene expression and inflammatory activation through non-energetic signaling pathways. Neither of these activities has been well-characterized in the brain and they may represent mechanisms by which BHB affects brain function during aging. First, we analyzed hepatic gene expression in an aging KD-treated mouse cohort using bulk RNA-seq. In addition to the downregulation of TOR pathway activity, cyclic KD reduces inflammatory gene expression in the liver. We observed via flow cytometry that KD also modulates age-related systemic T cell functions. Next, we investigated whether BHB affects brain cells transcriptionally in vitro. Gene expression analysis in primary human brain cells (microglia, astrocytes, neurons) using RNA-seq shows that BHB causes a mild level of inflammation in all three cell types. However, BHB inhibits the more pronounced LPS-induced inflammatory gene activation in microglia. Furthermore, we confirmed that BHB similarly reduces LPS-induced inflammation in primary mouse microglia and bone marrow-derived macrophages (BMDMs). BHB is recognized as an inhibitor of histone deacetylase (HDAC), an inhibitor of NLRP3 inflammasome, and an agonist of the GPCR Hcar2. Nevertheless, in microglia, BHB's anti-inflammatory effects are independent of these known mechanisms. Finally, we examined the brain gene expression of 12-month-old male mice fed with one-week and one-year cyclic KD. While a one-week KD increases inflammatory signaling, a one-year cyclic KD reduces neuroinflammation induced by aging. In summary, our findings demonstrate that BHB mitigates the microglial response to inflammatory stimuli, like LPS, possibly leading to decreased chronic inflammation in the brain after long-term KD treatment in aging mice.

Proteome and Secretome Dynamics of Human Retinal Pigment Epithelium in Response to Reactive Oxygen Species.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries, and is characterized by slow retinal degeneration linked to chronic reactive oxygen species (ROS) in the retinal pigmented epithelium (RPE). The molecular mechanisms leading to RPE dysfunction in response to ROS are unclear. Here, human stem cell-derived RPE samples were stressed with ROS for 1 or 3 weeks, and both intracellular and secreted proteomes were quantified by mass spectrometry. ROS increased glycolytic proteins but decreased mitochondrial complex I subunits, as well as membrane proteins required for endocytosis. RPE secreted over 1,000 proteins, many of which changed significantly due to ROS. Notably, secreted APOE is decreased 4-fold, and urotensin-II, the strongest known vasoconstrictor, doubled. Furthermore, secreted TGF-beta is increased, and its cognate signaler BMP1 decreased in the secretome. Together, our results paint a detailed molecular picture of the retinal stress response in space and time.

Illustrating the potency of current Good Manufacturing Practice-compliant induced pluripotent stem cell lines as a source of multiple cell lineages using standardized protocols.

BACKGROUND AIMS: We have previously reported the generation of a current Good Manufacture Practice (cGMP)-compliant induced pluripotent stem cell (iPSC) line for clinical applications. Here we show that multiple cellular products currently being considered for therapy can be generated from a single master cell bank of this or any other clinically compliant iPSC line METHODS: Using a stock at passage 20 prepared from the cGMP-compliant working cell bank (WCB), we tested differentiation into therapeutically relevant cell types of the three germ layers using standardized but generic protocols. Cells that we generated include (i) neural stem cells, dopaminergic neurons and astrocytes; (ii) retinal cells (retinal pigment epithelium and photoreceptors); and (iii) hepatocyte, endothelial and mesenchymal cells. To confirm that these generic protocols can also be used for other iPSC lines, we tested the reproducibility of our methodology with a second clinically compliant line RESULTS: Our results confirmed that well-characterized iPSC lines have broad potency, and, despite allelic variability, the same protocols could be used with minimal modifications with multiple qualified lines. In addition, we introduced a constitutively expressed GFP cassette in Chr13 safe harbor site using a standardized previously described method and observed no significant difference in growth and differentiation between the engineered line and the control line indicating that engineered products can be made using a standardized methodology CONCLUSIONS: We believe that our demonstration that multiple products can be made from the same WCB and that the same protocols can be used with multiple lines offers a path to a cost-effective strategy for developing cellular products from iPSC lines.

A novel AhR ligand, 2AI, protects the retina from environmental stress.

Various retinal degenerative diseases including dry and neovascular age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy are associated with the degeneration of the retinal pigmented epithelial (RPE) layer of the retina. This consequently results in the death of rod and cone photoreceptors that they support, structurally and functionally leading to legal or complete blindness. Therefore, developing therapeutic strategies to preserve cellular homeostasis in the RPE would be a favorable asset in the clinic. The aryl hydrocarbon receptor (AhR) is a conserved, environmental ligand-dependent, per ARNT-sim (PAS) domain containing bHLH transcription factor that mediates adaptive response to stress via its downstream transcriptional targets. Using in silico, in vitro and in vivo assays, we identified 2,2'-aminophenyl indole (2AI) as a potent synthetic ligand of AhR that protects RPE cells in vitro from lipid peroxidation cytotoxicity mediated by 4-hydroxynonenal (4HNE) as well as the retina in vivo from light-damage. Additionally, metabolic characterization of this molecule by LC-MS suggests that 2AI alters the lipid metabolism of RPE cells, enhancing the intracellular levels of palmitoleic acid. Finally, we show that, as a downstream effector of 2AI-mediated AhR activation, palmitoleic acid protects RPE cells from 4HNE-mediated stress, and light mediated retinal degeneration in mice.

Modeling the Dynamic AMD-Associated Chronic Oxidative Stress Changes in Human ESC and iPSC-Derived RPE Cells.

PURPOSE: Here we use human embryonic stem cells (hESCs) and human-induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells to model chronic oxidative stress in vitro. This model allows us to understand the evolution of chronic stress response in RPE in vivo, as well as to monitor microRNAs changes. Finally, we use this in vitro model to identify a partial agonist of NRF2 that is protective against reactive oxygen species (ROS)-induced cytotoxicity. METHODS: The hESCs and hiPSCs were differentiated toward an RPE fate. Upon maturation, RPE cells were subjected to chronic oxidative stress using Paraquat (PQ). The cells were then analyzed using immunocytochemistry and quantitative RT-PCR to look for changes in gene expression and microRNA changes. Small molecules targeting NRF2 pathways were utilized to look for protection against oxidative stress-induced apoptosis. RESULTS: We show that 160 µM PQ can be used to generate a model of chronic oxidative stress in RPE cells derived from hESCs and hiPSCs. Using this model, we characterize the NRF2 pathway effectors during the early and late stages of chronic oxidative stress and identify microRNAs changes during oxidative stress. We find that hsa-miR144 modulates NRF2 activity during ROS stress. Lastly, we found a small molecule modulator of NRF2 that plays a protective role against oxidative stress-induced RPE apoptosis. CONCLUSIONS: In summary, pluripotent stem cell-derived retinal cells can be used to model retinal diseases in a dish. This can provide an unprecedented opportunity to understand the evolution of disease processes and allow us to identify novel therapeutics.

Splendid symmetry: crystallization of an unbridged isomer of Co2(CO)8 in Co2(CO)8.C60.

Cocrystallization of Co2(CO)8 with C60 traps the cobalt carbonyl molecule as the D(3d) isomer that otherwise is not available for crystallographic analysis.

Cleavage of the indium(III) octaethyloxophlorin dimer, {In(III)(OEPO)}2, with Lewis bases. Importance of outer-sphere hydrogen bonding in adduct structures.

Splitting of the oxygen-bridged dimer {In(III)(OEPO)}(2) [where (OEPO)(3-) is the trianion of octaethyloxophlorin] by potential axial ligands has been examined and compared to results obtained previously for the cleavage of {Fe(III)(OEPO)}(2). Treatment of {In(III)(OEPO)}(2) with an excess of imidazole (im) produced the crystalline complex {(im)(2)In(III)(OEPO...im)}(2).(im)(2)In(III)(OEPO).2Cl(2)C(6)H(4). This solid contains two different (im)(2)In(III)(OEPO) units that are bridged through hydrogen bonding by an uncoordinated imidazole. Treatment of {In(III)(OEPO)}(2) with an excess of pyridine (py) produced (py)(2)In(III)(OEPO), which is isostructural with (py)(2)Fe(III)(OEPO). Although {Fe(III)(OEPO)}(2) reacted with xylyl isocyanide (xylylNC) to form the novel free-radical complex (2,6-xylylNC)(2)Fe(II)(OEPO(*)) [where (OEPO(*))(2-) is the radical dianion of octaethyloxophlorin], {In(III)(OEPO)}(2) was unreactive toward xylyl isocyanide.

Isolation of a novel chromium(III) binding protein from bovine liver tissue after chromium(VI) exposure.

In the ongoing investigation into the biological importance and toxicity issues surrounding the bioinorganic chemistry of chromium, the accepted literature procedure for the isolation of the biological form of chromium, low molecular weight chromium binding protein (LMWCr) or chromodulin, was investigated for its specificity. When chromium(VI) is added to bovine liver homogenate, results presented here indicate at least four chromium(III) binding peptides and proteins are produced and that the process is non-specific for the isolation of LMWCr. A novel trivalent chromium containing protein (1) has been isolated to purity and initial characterization is reported here. Chromium(III) identification was determined by optical spectroscopy and diphenylcarbazide testing. This chromium binding protein has a molecular weight of 15.6kDa, which was determined from both gel-electrophoresis and mass spectrometry. The protein is comprised primarily of Asx, Glx, His, Gly/Thr, Ala, and Lys in a 1.00:2.51:0.37:2.09:0.39:1.17 ratio and is anionic at pH 7.4. In addition, the protein binds approximately 2.5 chromium(III) ions per molecule.