Understanding the role of dietary sulforaphane in regulating metabolic pathways using genomic approaches

Introduction: Sulforaphane (4-methyl sulfinylbutyl isothiocyanate), a phytochemical derived from broccoli, has been linked to many health benefits in model systems, primarily through the activation of NRF2 (Nuclear factor erythroid-2 related factor), which regulates cellular antioxidant response. Recent evidence suggests that SF may improve glucose regulation in diabetic patients, but the molecular pathways or the role of NRF2 are yet unclear. This work set out to assess the molecular mechanisms by which SF regulates energy metabolism in the liver under conditions that represent different cellular metabolic states. Methods: Established liver hepatocellular carcinoma cells (HepG2) were treated with physiological concentrations of SF (10 µM) under varying glucose concentrations; no (0 mM), basal (5 mM), and high glucose (25 mM). Metabolic phenotyping was undertaken using the Seahorse Extracellular Flux Analyser, untargeted metabolomics, and subsequent experiments with stable isotope tracers of glucose and glutamine, coupled with Gas-Chromatography and Mass Spectrometry (GC-MS). Whole transcriptome was obtained through Illumina RNA sequencing. Finally, the genome-editing technique CRISPR-Cas 9 was applied to assess whether NRF2 mediates the metabolic changes. Results: Real time energy production assessed using the Seahorse Extracellular Flux Analyser demonstrated that SF reduced both mitochondrial respiration and glycolysis in HepG2 cells in a high glucose environment. At the same time, the expression of GSH biosynthetic genes and levels of reduced glutathione (GSH) were significantly increased. To support GSH synthesis, SF altered levels of the three amino acids that are the biosynthetic building blocks; namely, increased intracellular utilization of glycine and glutamate, by redirecting the latter away from the TCA cycle, as well as increased the import of cysteine from the media. To support the cellular antioxidant enzyme response, SF also altered pathways generating NADPH, the necessary cofactor for these oxidoreductase reactions, namely pentose phosphate pathway (PPP) and 1Cmetabolism. Firstly, SF increased genes in the PPP pathway, including glucose-6- phosphate dehydrogenase, the rate limiting enzyme, and increased the PPP metabolite ribulose-5-phosphate, suggesting that excess glucose is likely redirected towards PPP, away from glycolysis. Secondly, SF upregulated genes in the folate cycle, namely 10- formyltetrahydrofolate dehydrogenase (ALDH1L1) and monofunctional C1- 3 tetrahydrofolate synthase (MTHFD1L) and utilized serine as a methyl donor for THF to support the 1C metabolism. Finally, SF downregulated the biosynthesis of the unsaturated fatty acids gene set, which is an NADPH consuming pathway. Finally, transcriptomic and targeted metabolomics LC-MS analysis of NRF2KD HepG2 cells generated using CRISPR-Cas 9 genome editing revealed that the above metabolic effects are mediated through NRF2. Conclusions: The results suggest that SF rewires central metabolism to suppress the metabolic dysregulation induced by excessive glucose and identify glucose biosynthesis and 1Cmetabolism as key mechanistic pathways

Numerical Relativity Simulations of the Neutron Star Merger GW170817: Long-Term Remnant Evolutions, Winds, Remnant Disks, and Nucleosynthesis

We present a systematic numerical-relativity study of the dynamical ejecta, winds and nucleosynthesis in neutron star merger remnants. Binaries with the chirp mass compatible with GW170817, different mass ratios, and five microphysical equations of state (EOS) are simulated with an approximate neutrino transport and a subgrid model for magnetohydrodynamics turbulence up to 100 milliseconds postmerger. Spiral density waves propagating from the neutron star remnant to the disk trigger a wind with mass flux ${\sim}0.1{-}0.5\,{\rm M_\odot/s}$ persisting for the entire simulation as long as the remnant does not collapse to black hole. This wind has average electron fraction $\gtrsim 0.3$ and average velocity ${\sim}0.1-0.17\,$c and thus is a site for the production of weak $r$-process elements (mass number $A<195$). Disks around long-lived remnants have masses ${\sim}0.1{-}0.2\,{\rm M_\odot}$, temperatures peaking at $\lesssim10\,$MeV near the inner edge, and a characteristic double-peak distribution in entropy resulting from shocks propagating through the disk. The dynamical and spiral-wave ejecta computed in our targeted simulations are not compatible with those inferred from AT2017gfo using two-components kilonova models. Rather, they indicate that multi-component kilonova models including disk winds are necessary to interpret AT2017gfo. The nucleosynthesis in the combined dynamical ejecta and spiral-wave wind in the comparable-mass long-lived mergers robustly accounts for all the $r$-process peaks, from mass number ${\sim}75$ to actinides in terms of solar abundances. Total abundandes are weakly dependent on the EOS, while the mass ratio affect the production of first peak elements.Comment: 20 pages, 13 figures, 3 table

The early life microbiota protects neonatal mice from pathological small intestinal epithelial cell shedding

The early life gut microbiota plays a crucial role in regulating and maintaining the intestinal barrier, with disturbances in these communities linked to dysregulated renewal and replenishment of intestinal epithelial cells. Here we sought to determine pathological cell shedding outcomes throughout the postnatal developmental period, and which host and microbial factors mediate these responses. Surprisingly, neonatal mice (Day 14 and 21) were highly refractory to induction of cell shedding after intraperitoneal administration of liposaccharide (LPS), with Day 29 mice showing strong pathological responses, more similar to those observed in adult mice. These differential responses were not linked to defects in the cellular mechanisms and pathways known to regulate cell shedding responses. When we profiled microbiota and metabolites, we observed significant alterations. Neonatal mice had high relative abundances of Streptococcus, Escherichia, and Enterococcus and increased primary bile acids. In contrast, older mice were dominated by Candidatus Arthromitus, Alistipes, and Lachnoclostridium, and had increased concentrations of SCFAs and methyamines. Antibiotic treatment of neonates restored LPS-induced small intestinal cell shedding, whereas adult fecal microbiota transplant alone had no effect. Our findings further support the importance of the early life window for microbiota-epithelial interactions in the presence of inflammatory stimuli and highlights areas for further investigation

GSH Levels Serve As a Biological Redox Switch Regulating Sulforaphane-Induced Cell Fate in Human Lens Cells

Purpose: Sulforaphane (SFN) is a therapeutic phytochemical agent for many health conditions. SFN-induced cytotoxicity is shown to have promise in preventing posterior capsule opacification (PCO). In the current study, we aimed to elucidate key processes and mechanisms linking SFN treatment to lens cell death. Methods: The human lens epithelial cell line FHL124 and central anterior epithelium were used as experimental models. Cell death was assessed by microscopic observation and cell damage/viability assays. Gene or protein levels were assessed by TaqMan RT-PCR or immunoblotting. Mitochondrial networks and DNA damage were assessed by immunofluorescence. Mitochondrial membrane potential, activating transcription factor 6 (ATF6) activity, ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), and glutathione reductase (GR) activity were measured using different light reporter assays. SFN metabolites were analyzed by LC-MS/MS. Results: Treatment with N-acetylcysteine (NAC), a reactive oxygen species scavenger, prevented SFN-induced cell death in both models. NAC also significantly protected FHL124 cells from SFN-induced mitochondrial dysfunctions, endoplasmic reticulum stress (ERS), DNA damage and autophagy. SFN significantly depleted GSH, the major antioxidant in the eye, and reduced GR activity, despite doubling its protein levels. The most abundant SFN conjugate detected in lens cells following SFN application was SFN–GSH. The addition of GSH protected lens cells from all SFN-induced cellular events. Conclusions: SFN depletes GSH levels in lens cells through conjugation and inhibition of GR activity. This leads to increased reactive oxygen species and oxidative stress that trigger mitochondrial dysfunction, ERS, autophagy, and DNA damage, leading to cell death. In summary, the work presented provides a mechanistic understanding to support the therapeutic application of SFN for PCO and other disorders

Accretion-induced prompt black hole formation in asymmetric neutron star mergers, dynamical ejecta and kilonova signals

We present new numerical relativity results of neutron star mergers with chirp mass $1.188M_\odot$ and mass ratios $q=1.67$ and $q=1.8$ using finite-temperature equations of state (EOS), approximate neutrino transport and a subgrid model for magnetohydrodynamics-induced turbulent viscosity. The EOS are compatible with nuclear and astrophysical constraints and include a new microphysical model derived from ab-initio calculations based on the Brueckner-Hartree-Fock approach. We report for the first time evidence for accretion-induced prompt collapse in high-mass-ratio mergers, in which the tidal disruption of the companion and its accretion onto the primary star determine prompt black hole formation. As a result of the tidal disruption, an accretion disc of neutron-rich and cold matter forms with baryon masses ${\sim}0.15M_\odot$, and it is significantly heavier than the remnant discs in equal-masses prompt collapse mergers. Massive dynamical ejecta of order ${\sim}0.01M_\odot$ also originate from the tidal disruption. They are neutron rich and expand from the orbital plane with a crescent-like geometry. Consequently, bright, red and temporally extended kilonova emission is predicted from these mergers. Our results show that prompt black hole mergers can power bright electromagnetic counterparts for high-mass-ratio binaries, and that the binary mass ratio can be in principle constrained from multimessenger observations.Comment: 20 pages, 21 figures, 4 table

Metabolic regulation of macrophages by SIRT1 determines activation during cholestatic liver disease in mice

Background & Aims: Inflammation is the hallmark of chronic liver disease. Metabolism is a key determinant to regulate the activation of immune cells. Here, we define the role of sirtuin 1 (SIRT1), a main metabolic regulator, in controlling the activation of macrophages during cholestatic liver disease and in response to endotoxin. Methods: We have used mice overexpressing SIRT1, which we treated with intraperitoneal lipopolysaccharides or induced cholestasis by bile duct ligation. Bone marrow–derived macrophages were used for mechanistic in vitro studies. Finally, PEPC-Boy mice were used for adoptive transfer experiments to elucidate the impact of SIRT1-overexpressing macrophages in contributing to cholestatic liver disease. Results: We found that SIRT1 overexpression promotes increased liver inflammation and liver injury after lipopolysaccharide/GalN and bile duct ligation; this was associated with an increased activation of the inflammasome in macrophages. Mechanistically, SIRT1 overexpression associated with the activation of the mammalian target of rapamycin (mTOR) pathway that led to increased activation of macrophages, which showed metabolic rewiring with increased glycolysis and broken tricarboxylic acid cycle in response to endotoxin in vitro. Activation of the SIRT1/mTOR axis in macrophages associated with the activation of the inflammasome and the attenuation of autophagy. Ultimately, in an in vivo model of cholestatic disease, the transplantation of SIRT1-overexpressing myeloid cells contributed to liver injury and fibrosis. Conclusions: Our study provides novel mechanistic insights into the regulation of macrophages during cholestatic disease and the response to endotoxin, in which the SIRT1/mTOR crosstalk regulates macrophage activation controlling the inflammasome, autophagy and metabolic rewiring

Transcriptional changes in prostate of men on active surveillance after a 12-mo glucoraphanin-rich broccoli intervention—results from the Effect of Sulforaphane on prostate CAncer PrEvention (ESCAPE) randomized controlled trial

Background Epidemiological evidence suggests that consumption of cruciferous vegetables is associated with reduced risk of prostate cancer progression, largely attributed to the biological activity of glucosinolate degradation products, such as sulforaphane derived from glucoraphanin. Because there are few therapeutic interventions for men on active surveillance for prostate cancer to reduce the risk of cancer progression, dietary approaches are an appealing option for patients. Objective We evaluated whether consumption of a glucoraphanin-rich broccoli soup for 1 y leads to changes in gene expression in prostate tissue of men with localized prostate cancer. Methods Forty-nine men on active surveillance completed a 3-arm parallel randomized double-blinded intervention study for 12 mo and underwent transperineal template biopsy procedures and dietary assessment at the start and end of the study. Patients received a weekly 300 mL portion of soup made from a standard broccoli (control) or from 1 of 2 experimental broccoli genotypes with enhanced concentrations of glucoraphanin, delivering 3 and 7 times that of the control, respectively. Gene expression in tissues from each patient obtained before and after the dietary intervention was quantified by RNA sequencing followed by gene set enrichment analyses. Results In the control arm, there were several hundred changes in gene expression in nonneoplastic tissue during the 12 mo. These were associated with an increase in expression of potentially oncogenic pathways including inflammation processes and epithelial–mesenchymal transition. Changes in gene expression and associated oncogenic pathways were attenuated in men on the glucoraphanin-rich broccoli soup in a dose-dependent manner. Although the study was not powered to assess clinical progression, an inverse association between consumption of cruciferous vegetables and cancer progression was observed. Conclusion Consuming glucoraphanin-rich broccoli soup affected gene expression in the prostate of men on active surveillance, consistent with a reduction in the risk of cancer progression. This trial was registered at clinicaltrials.gov as NCT01950143

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M&gt;70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0&lt;e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level