ACLY alternative splicing correlates with cancer phenotypes.

ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA for protein acetylation. ACLY has two major splice isoforms: the full-length canonical "long" isoform and an uncharacterized "short" isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within an intrinsically disordered region and includes at least one dynamically phosphorylated residue. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the percent spliced in (PSI) of exon 14 is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types. This prompted us to explore potential biochemical and functional differences between ACLY isoforms. Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology nor did it attenuate tumor burden in a genetic model of intestinal cancer. Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive.

Acetylcarnitine shuttling links mitochondrial metabolism to histone acetylation and lipogenesis.

The metabolite acetyl-CoA is necessary for both lipid synthesis in the cytosol and histone acetylation in the nucleus. The two canonical precursors to acetyl-CoA in the nuclear-cytoplasmic compartment are citrate and acetate, which are processed to acetyl-CoA by ATP-citrate lyase (ACLY) and acyl-CoA synthetase short-chain 2 (ACSS2), respectively. It is unclear whether other substantial routes to nuclear-cytosolic acetyl-CoA exist. To investigate this, we generated cancer cell lines lacking both ACLY and ACSS2 [double knockout (DKO) cells]. Using stable isotope tracing, we show that both glucose and fatty acids contribute to acetyl-CoA pools and histone acetylation in DKO cells and that acetylcarnitine shuttling can transfer two-carbon units from mitochondria to cytosol. Further, in the absence of ACLY, glucose can feed fatty acid synthesis in a carnitine responsive and carnitine acetyltransferase (CrAT)-dependent manner. The data define acetylcarnitine as an ACLY- and ACSS2-independent precursor to nuclear-cytosolic acetyl-CoA that can support acetylation, fatty acid synthesis, and cell growth.

Acly Deficiency Enhances Myelopoiesis through Acetyl Coenzyme A and Metabolic-Epigenetic Cross-Talk.

Hematopoiesis integrates cytokine signaling, metabolism, and epigenetic modifications to regulate blood cell generation. These processes are linked, as metabolites provide essential substrates for epigenetic marks. In this study, we demonstrate that ATP citrate lyase (Acly), which metabolizes citrate to generate cytosolic acetyl-CoA and is of clinical interest, can regulate chromatin accessibility to limit myeloid differentiation. Acly was tested for a role in murine hematopoiesis by small-molecule inhibition or genetic deletion in lineage-depleted, c-Kit-enriched hematopoietic stem and progenitor cells from Mus musculus. Treatments increased the abundance of cell populations that expressed the myeloid integrin CD11b and other markers of myeloid differentiation. When single-cell RNA sequencing was performed, we found that Acly inhibitor-treated hematopoietic stem and progenitor cells exhibited greater gene expression signatures for macrophages and enrichment of these populations. Similarly, the single-cell assay for transposase-accessible chromatin sequencing showed increased chromatin accessibility at genes associated with myeloid differentiation, including CD11b, CD11c, and IRF8. Mechanistically, Acly deficiency altered chromatin accessibility and expression of multiple C/EBP family transcription factors known to regulate myeloid differentiation and cell metabolism, with increased Cebpe and decreased Cebpa and Cebpb. This effect of Acly deficiency was accompanied by altered mitochondrial metabolism with decreased mitochondrial polarization but increased mitochondrial content and production of reactive oxygen species. The bias to myeloid differentiation appeared due to insufficient generation of acetyl-CoA, as exogenous acetate to support alternate compensatory pathways to produce acetyl-CoA reversed this phenotype. Acly inhibition thus can promote myelopoiesis through deprivation of acetyl-CoA and altered histone acetylome to regulate C/EBP transcription factor family activity for myeloid differentiation.

Intrinsic antiviral immunity of barrier cells revealed by an iPSC-derived blood-brain barrier cellular model.

Physiological blood-tissue barriers play a critical role in separating the circulation from immune-privileged sites and denying access to blood-borne viruses. The mechanism of virus restriction by these barriers is poorly understood. We utilize induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (iBMECs) to study virus-blood-brain barrier (BBB) interactions. These iPSC-derived cells faithfully recapitulate a striking difference in in vivo neuroinvasion by two alphavirus isolates and are selectively permissive to neurotropic flaviviruses. A model of cocultured iBMECs and astrocytes exhibits high transendothelial electrical resistance and blocks non-neurotropic flaviviruses from getting across the barrier. We find that iBMECs constitutively express an interferon-induced gene, IFITM1, which preferentially restricts the replication of non-neurotropic flaviviruses. Barrier cells from blood-testis and blood-retinal barriers also constitutively express IFITMs that contribute to the viral resistance. Our application of a renewable human iPSC-based model for studying virus-BBB interactions reveals that intrinsic immunity at the barriers contributes to virus exclusion.

Generation of hypothalamic arcuate organoids from human induced pluripotent stem cells.

Human brain organoids represent remarkable platforms for recapitulating features of human brain development and diseases. Existing organoid models do not resolve fine brain subregions, such as different nuclei in the hypothalamus. We report the generation of arcuate organoids (ARCOs) from human induced pluripotent stem cells (iPSCs) to model the development of the human hypothalamic arcuate nucleus. Single-cell RNA sequencing of ARCOs revealed significant molecular heterogeneity underlying different arcuate cell types, and machine learning-aided analysis based on the neonatal human hypothalamus single-nucleus transcriptome further showed a human arcuate nucleus molecular signature. We also explored ARCOs generated from Prader-Willi syndrome (PWS) patient iPSCs. These organoids exhibit aberrant differentiation and transcriptomic dysregulation similar to postnatal hypothalamus of PWS patients, indicative of cellular differentiation deficits and exacerbated inflammatory responses. Thus, patient iPSC-derived ARCOs represent a promising experimental model for investigating nucleus-specific features and disease-relevant mechanisms during early human arcuate development.

A tutorial review of mathematical techniques for quantifying tumor heterogeneity.

Intra-tumor and inter-patient heterogeneity are two challenges in developing mathematical models for precision medicine diagnostics. Here we review several techniques that can be used to aid the mathematical modeller in inferring and quantifying both sources of heterogeneity from patient data. These techniques include virtual populations, nonlinear mixed effects modeling, non-parametric estimation, Bayesian techniques, and machine learning. We create simulated virtual populations in this study and then apply the four remaining methods to these datasets to highlight the strengths and weak-nesses of each technique. We provide all code used in this review at https://github.com/jtnardin/Tumor-Heterogeneity/ so that this study may serve as a tutorial for the mathematical modelling community. This review article was a product of a Tumor Heterogeneity Working Group as part of the 2018-2019 Program on Statistical, Mathematical, and Computational Methods for Precision Medicine which took place at the Statistical and Applied Mathematical Sciences Institute.

Sliced Human Cortical Organoids for Modeling Distinct Cortical Layer Formation.

Human brain organoids provide unique platforms for modeling development and diseases by recapitulating the architecture of the embryonic brain. However, current organoid methods are limited by interior hypoxia and cell death due to insufficient surface diffusion, preventing generation of architecture resembling late developmental stages. Here, we report the sliced neocortical organoid (SNO) system, which bypasses the diffusion limit to prevent cell death over long-term cultures. This method leads to sustained neurogenesis and formation of an expanded cortical plate that establishes distinct upper and deep cortical layers for neurons and astrocytes, resembling the third trimester embryonic human neocortex. Using the SNO system, we further identify a critical role of WNT/ß-catenin signaling in regulating human cortical neuron subtype fate specification, which is disrupted by a psychiatric-disorder-associated genetic mutation in patient induced pluripotent stem cell (iPSC)-derived SNOs. These results demonstrate the utility of SNOs for investigating previously inaccessible human-specific, late-stage cortical development and disease-relevant mechanisms.

A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter- and Intra-tumoral Heterogeneity.

Glioblastomas exhibit vast inter- and intra-tumoral heterogeneity, complicating the development of effective therapeutic strategies. Current in vitro models are limited in preserving the cellular and mutational diversity of parental tumors and require a prolonged generation time. Here, we report methods for generating and biobanking patient-derived glioblastoma organoids (GBOs) that recapitulate the histological features, cellular diversity, gene expression, and mutational profiles of their corresponding parental tumors. GBOs can be generated quickly with high reliability and exhibit rapid, aggressive infiltration when transplanted into adult rodent brains. We further demonstrate the utility of GBOs to test personalized therapies by correlating GBO mutational profiles with responses to specific drugs and by modeling chimeric antigen receptor T cell immunotherapy. Our studies show that GBOs maintain many key features of glioblastomas and can be rapidly deployed to investigate patient-specific treatment strategies. Additionally, our live biobank establishes a rich resource for basic and translational glioblastoma research.

Bilateral Symmetric and Asymmetric Superior Rectus Recession for Patients with Dissociated Vertical Deviation.

PURPOSE: To evaluate the outcome of bilateral symmetric and asymmetric superior rectus (SR) recessions in patients with bilateral dissociated vertical deviation (DVD) without oblique dysfunction and determine factors that might influence the surgical outcome. DESIGN: Retrospective study. METHODS: All patients who underwent bilateral SR recession for bilateral DVD from January 2012 to December 2016 at an eye hospital in New Delhi, India were included. Indication for surgery was decompensated DVD in 1 or both eyes. Symmetric SR recession was performed for symmetric DVD and asymmetric SR recession was performed for asymmetric DVD of 10 prism diopters (PD) or more. Patients with a minimum follow-up of 6 months were included. Success was defined as absence of manifest DVD in both eyes at the final postoperative visit. RESULTS: Medical records of 27 patients were analyzed. Their median age at surgery was 8 years [interquartile range (IQR), 6-10 years] and mean follow-up was 16.3 months (range, 6-48 months). Symmetric and asymmetric surgeries were performed in 19 and 8 patients, respectively. The amount of SR recession performed ranged from 5 to 10 mm. The median DVD reduced from 9 PD (IQR, 6-14 PD) to 5 PD (IQR, 3-8 PD) in the right eye (P = 0.015) and 9 PD (IQR, 7-12 PD) to 6 PD (IQR, 3-10 PD) in the left eye (P = 0.016) after surgery. Successful outcome was seen in 63% of patients. There was no difference in successful outcome with respect to age, sex, preoperative horizontal deviation, preoperative vertical deviation, postoperative residual horizontal deviation, surgical technique, asymmetry of SR recession, and magnitude of preoperative DVD. Patients with a preference for fixation were more likely to have a successful outcome. CONCLUSIONS: Bilateral SR recession resulted in a success rate of 63% after single operation for bilateral DVD without oblique dysfunction. We found that the probability of a successful outcome did not depend on age at surgery, sex, preoperative horizontal or vertical deviation, magnitude of preoperative DVD, symmetry of SR recession, or surgical technique.

Epigenetic crosstalk: Pharmacological inhibition of HDACs can rescue defective synaptic morphology and neurotransmission phenotypes associated with loss of the chromatin reader Kismet.

We are beginning to appreciate the complex mechanisms by which epigenetic proteins control chromatin dynamics to tightly regulate normal development. However, the interaction between these proteins, particularly in the context of neuronal function, remains poorly understood. Here, we demonstrate that the activity of histone deacetylases (HDACs) opposes that of a chromatin remodeling enzyme at the Drosophila neuromuscular junction (NMJ). Pharmacological inhibition of HDAC function reverses loss of function phenotypes associated with Kismet, a chromodomain helicase DNA-binding (CHD) protein. Inhibition of HDACs suppresses motor deficits, overgrowth of the NMJ, and defective neurotransmission associated with loss of Kismet. We hypothesize that Kismet and HDACs may converge on a similar set of target genes in the nervous system. Our results provide further understanding into the complex interactions between epigenetic protein function in vivo.

Development of a formula for estimating plasma free cortisol concentration from a measured total cortisol concentration when elastase-cleaved and intact corticosteroid binding globulin coexist.

Cortisol bound to corticosteroid binding globulin (CBG) contributes up to 90% of the total cortisol concentration in circulation. Therefore, changes in the binding kinetics of cortisol to CBG can potentially impact on the concentration of free cortisol, the only form that is responsible for the physiological function of the hormone. When CBG is cleaved into elastase-cleaved CBG (eCBG) by the activity of neutrophil elastase, its affinity for cortisol is reduced. Therefore, when eCBG coexists with intact CBG (iCBG) in plasma, the calculation of free cortisol concentration based on the formulae that considers only one CBG pool with the same affinity for cortisol may be inappropriate. In this study, we developed in vivo and in vitro models of cortisol partitioning which considers two CBG pools, iCBG and eCBG, with different affinities for cortisol, and deduce a new formula for calculating plasma free cortisol concentration. The formula provides better estimates of free cortisol concentration than previously used formulae when measurements of the concentrations of the two CBG forms are available. The model can also be used to estimate the affinity of CBG and albumin for cortisol in different clinical groups. We found no significant difference in the estimated affinity of CBG and albumin for cortisol in normal, sepsis and septic shock groups, although free cortisol was higher in sepsis and septic shock groups. The in vivo model also demonstrated that the concentration of interstitial free cortisol is increased locally at a site of inflammation where iCBG is cleaved to form eCBG by the activity of elastase released by neutrophils. This supports the argument that the cleavage of iCBG at sites of inflammation leads to more lower-affinity eCBG and may be a mechanism that permits the local concentration of free cortisol to increase at these sites, while allowing basal free cortisol concentrations at other sites to remain unaffected.

The role of enzyme compartmentalization on the regulation of steroid synthesis.

Steroidogenic enzymes can be compartmentalized at different levels, some by virtue of being membrane bound in specific intra-cellular compartments. Although both 3ß-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3ß-HSD) and 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) are expressed in the endoplasmic reticulum (ER) membrane, these proteins may still be spatially separated within this membrane system. Side chain cleavage cytochrome P450 (P450scc) is anchored to the inner mitochondrial membrane and this organelle is the major source of pregnenolone (P5) feeding steroidogenesis. Furthermore, steroidogenic enzymes can also be partitioned in different cells. Although well recognized, the effect of enzyme compartmentalization on the rate of steroid production and the balance of different steroids is unclear. This study uses mathematical modeling to investigate the effect of enzyme compartmentalization on steroid synthesis in a human-ovine-bovine model of steroid synthesis. The study shows that the spatial separation of steroidogenic enzymes within the ER has a minimal effect on the rate of steroid synthesis. The compartmentalization of the enzymes into different organelles of a cell creates cellular steroid gradients and can affect the balance of the different steroid products. The partitioning of steroidogenic enzymes in different cells reduces the rate of steroid synthesis. The greater is the distance between the cells that contain different enzymes, the more the rate of steroid synthesis is reduced. Additionally, when 3ß-HSD is not in the same cell with P450scc (the P5 source) and P450c17, the ratio of the Δ(5)-pathway products' concentrations to the Δ(4)-pathway products' concentrations is increased. However, none of these levels of compartmentalization of steroidogenic enzymes alter the qualitative behaviors of steroid synthesis in response to variation in an enzyme activity or P5 supply.

Multilevel regulation of steroid synthesis and metabolism in the bovine placenta.

Steroid hormones play critical roles in almost all physiological processes in male and female reproduction. In a normal pregnancy, the concentrations of steroid hormones in maternal and foetal blood vary with gestation in response to changing needs. The placenta plays a central role in producing the appropriate steroids to support the pregnancy by coordinating its own steroidogenic activity with that of the corpus luteum and responding to foetal signals. Although much is known about the steroidogenic potential of the bovine placenta, far less is known about how the placenta integrates the synthesis of steroids with their subsequent metabolism and clearance to achieve appropriate local and peripheral concentrations of steroids in maternal and foetal blood at each stage of gestation. This review focuses on the current knowledge of the temporal and spatial regulation and compartmentalization of the biochemical pathways by which potent steroid hormones are synthesized and metabolized in the bovine placenta. The aim is to increase our understanding of how the balance of synthesis and metabolism determines placental steroid output as it changes with development and differentiation, and how this is regulated in response to the variations in the foetal signals and luteal secretory activity. The review highlights knowledge gaps and suggests that mathematical modelling can help understand the effect of different levels of regulation on the steroidogenic output of an organ, such as the bovine placenta.

Variation in 3ß-hydroxysteroid dehydrogenase activity and in pregnenolone supply rate can paradoxically alter androstenedione synthesis.

The 3ß-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3ß-HSD) and 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) enzymes are important in determining the balance of the synthesis of different steroids such as progesterone (P4), glucocorticoids, androgens, and estrogens. How this is achieved is not a simple matter because each of the two enzymes utilizes more than one substrate and some substrates are shared in common between the two enzymes. The two synthetic pathways, Δ(4) and Δ(5), are interlinked such that it is difficult to predict how the synthesis of each steroid changes when any of the enzyme activities is varied. In addition, the P450c17 enzyme exhibits different substrate specificities among species, particularly with respect to the 17,20-lyase activity. The mathematical model developed in this study simulates the network of reactions catalyzed by 3ß-HSD and P450c17 that characterizes steroid synthesis in human, non-human primate, ovine, and bovine species. In these species, P450c17 has negligible 17,20-lyase activity with the Δ(4)-steroid 17α-hydroxy-progesterone (17OH-P4); therefore androstenedione (A4) is synthesized efficiently only from dehydroepiandrosterone (DHEA) through the Δ(5) pathway. The model helps to understand the interplay between fluxes through the Δ(4) and Δ(5) pathways in this network, and how this determines the response of steroid synthesis to the variation in 3ß-HSD activity or in the supply of the precursor substrate, pregnenolone (P5). The model simulations show that A4 synthesis can change paradoxically when 3ß-HSD activity is varied. A decrease in 3ß-HSD activity to a certain point can increase A4 synthesis by favouring metabolism through the Δ(5) pathway, though further decrease in 3ß-HSD activity beyond that point eventually limits A4 synthesis. The model also showed that due to the competitive inhibition of the enzymes' activities by substrates and products, increasing the rate of P5 supply above a certain point can suppress the synthesis of A4, DHEA, and 17OH-P4, and consequently drive more P5 towards P4 synthesis.