Novel Pan-ERR Agonists Ameliorate Heart Failure Through Enhancing Cardiac Fatty Acid Metabolism and Mitochondrial Function.

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.

Effect of Protonation on the Molecular Structure of Adenosine 5'-Triphosphate: A Combined Theoretical and Near Edge X-ray Absorption Fine Structure Study.

The present work combines the near edge X-ray absorption mass spectrometry of a protonated adenosine 5'-triphosphate (ATP) molecule isolated in an ion trap with (time-dependent) density functional theory calculations. Our study unravels the effect of protonation on the ATP structure and its spectral properties, providing structure-property relationships at atomistic resolution for protonated ATP (ATPH) isolated in the gas-phase conditions. On the other hand, the present C and N K-edge X-ray absorption spectra of isolated ATPH appear closely like those previously reported for solvated ATP at low pH. Therefore, the present work should be relevant for further investigation and modeling of structure-function properties of protonated adenine and ATP in complex biological environments.

Deconvolution of cancer cell states by the XDec-SM method.

Proper characterization of cancer cell states within the tumor microenvironment is a key to accurately identifying matching experimental models and the development of precision therapies. To reconstruct this information from bulk RNA-seq profiles, we developed the XDec Simplex Mapping (XDec-SM) reference-optional deconvolution method that maps tumors and the states of constituent cells onto a biologically interpretable low-dimensional space. The method identifies gene sets informative for deconvolution from relevant single-cell profiling data when such profiles are available. When applied to breast tumors in The Cancer Genome Atlas (TCGA), XDec-SM infers the identity of constituent cell types and their proportions. XDec-SM also infers cancer cells states within individual tumors that associate with DNA methylation patterns, driver somatic mutations, pathway activation and metabolic coupling between stromal and breast cancer cells. By projecting tumors, cancer cell lines, and PDX models onto the same map, we identify in vitro and in vivo models with matching cancer cell states. Map position is also predictive of therapy response, thus opening the prospects for precision therapy informed by experiments in model systems matched to tumors in vivo by cancer cell state.

Clipless skeletonized internal mammary artery harvesting with harmonic technology.

Since the introduction of the mammary artery harvesting procedure, electrocautery has been used as a standard method of care. However, mammary artery spasm, subadventitial haematoma and mammary artery damage due to clips dispositioning or high thermal energy injury have been recorded. To achieve a perfect mammary artery graft, we propose the usage of a high-frequency ultrasound device, usually recognized as a harmonic scalpel. It reduces thermal-related injuries, the use of clips and the risk of mammary artery spasm and/or dissection.

Identifying biomarkers of differential chemotherapy response in TNBC patient-derived xenografts with a CTD/WGCNA approach.

Although systemic chemotherapy remains the standard of care for TNBC, even combination chemotherapy is often ineffective. The identification of biomarkers for differential chemotherapy response would allow for the selection of responsive patients, thus maximizing efficacy and minimizing toxicities. Here, we leverage TNBC PDXs to identify biomarkers of response. To demonstrate their ability to function as a preclinical cohort, PDXs were characterized using DNA sequencing, transcriptomics, and proteomics to show consistency with clinical samples. We then developed a network-based approach (CTD/WGCNA) to identify biomarkers of response to carboplatin (MSI1, TMSB15A, ARHGDIB, GGT1, SV2A, SEC14L2, SERPINI1, ADAMTS20, DGKQ) and docetaxel (c, MAGED4, CERS1, ST8SIA2, KIF24, PARPBP). CTD/WGCNA multigene biomarkers are predictive in PDX datasets (RNAseq and Affymetrix) for both taxane- (docetaxel or paclitaxel) and platinum-based (carboplatin or cisplatin) response, thereby demonstrating cross-expression platform and cross-drug class robustness. These biomarkers were also predictive in clinical datasets, thus demonstrating translational potential.

XDec-CHI reveals immunosuppressive interactions in pancreatic ductal adenocarcinoma.

Most cancers harbor a diverse collection of cell types including a typically heterogeneous cancer cell fraction. To reconstruct cell-intrinsic and heterotypic interactions driving tumor progression, we combine the XDec deconvolution method with cell-type-specific gene expression correlation analysis into the XDec-CHI method. XDec-CHI identifies intra- and inter-cellular pathways using correlation and places them in the context of specific tumor subtypes, as defined by the state of constituent cancer cells. We make the method web-accessible for analysis of publicly accessible pancreatic ductal adenocarcinoma, breast, head and neck, glioblastoma, and glioma tumors. We apply the method to TCGA and ICGC datasets to identify immune-suppressive interactions within PDAC tumors that are relevant for immunotherapies targeting PD-L1. Subtype-specific interactions derived from correlative analyses validated in co-culture experiments suggest PDAC subtypes have distinct therapeutic weaknesses, with Basal-like and MSLN-high Classical B tumors most likely to respond to therapies targeting PD-L1.

Immunologically "cold" triple negative breast cancers engraft at a higher rate in patient derived xenografts.

TNBC is a heterogeneous subtype of breast cancer, and only a subset of TNBC can be established as PDXs. Here, we show that there is an engraftment bias toward TNBC with low levels of immune cell infiltration. Additionally, TNBC that failed to engraft show gene expression consistent with a cancer-promoting immunological state, leading us to hypothesize that the immunological state of the tumor and possibly the state of the immune system of the host may be essential for engraftment.

MPAPASS software enables stitched multiplex, multidimensional EV repertoire analysis and a standard framework for reporting bead-based assays.

Extracellular vesicles (EVs) of various types are released or shed from all cells. EVs carry proteins and contain additional protein and nucleic acid cargo that relates to their biogenesis and cell of origin. EV cargo in liquid biopsies is of widespread interest owing to its ability to provide a retrospective snapshot of cell state at the time of EV release. For the purposes of EV cargo analysis and repertoire profiling, multiplex assays are an essential tool in multiparametric analyte studies but are still being developed for high-parameter EV protein detection. Although bead-based EV multiplex analyses offer EV profiling capabilities with conventional flow cytometers, the utilization of EV multiplex assays has been limited by the lack of software analysis tools for such assays. To facilitate robust EV repertoire studies, we developed multiplex analysis post-acquisition analysis (MPAPASS) open-source software for stitched multiplex analysis, EV database-compatible reporting, and visualization of EV repertoires.

Consistent characterization of the electronic ground state of iron(II) phthalocyanine from valence and core-shell electron spectroscopy.

We studied the iron(II) phthalocyanine molecule in the gas-phase. It is a complex transition organometallic compound, for which, the characterization of its electronic ground state is still debated more than 50 years after the first published study. Here, we show that to determine its electronic ground state, one needs a large corpus of data sets and a consistent theoretical methodology to simulate them. By simulating valence and core-shell electron spectra, we determined that the ground state is a 3Eg and that the ligand-to-metal charge transfer has a large influence on the spectra.

Epigenetic and senescence markers indicate an accelerated ageing-like state in women with preeclamptic pregnancies.

BACKGROUND: Preeclampsia is a pregnancy-specific hypertensive disorder characterized by proteinuria and/or multisystem involvement. Disease-specific therapy has yet to be developed due to the lack of understanding of underlying mechanism(s). We postulate that accelerated ageing in general, and particularly cellular senescence, play a role in its pathophysiology. METHODS: We compared women with preeclampsia vs. normotensive pregnancies with respect to epigenetic markers of ageing and markers of senescence in tissues/organs affected by preeclampsia (blood, urine, adipose tissue, and kidney). FINDINGS: We demonstrate that preeclamptic compared to normotensive pregnant women: (i) undergo accelerated epigenetic ageing during pregnancy, as demonstrated by an "epigenetic clock"; (ii) exhibit higher levels/expression of senescence-associated secretory phenotype factors in blood and adipose tissue; (iii) display increased expression of p16INK4A in adipose tissue and renal sections, and (iv) demonstrate decreased levels of urinary α-Klotho (an anti-ageing protein) at the time of delivery. Finally, we provide data indicating that pre-treatment with dasatinib, a senolytic agent, rescues the angiogenic potential of mesenchymal stem cells (MSC) obtained from preeclamptic pregnancies, and promotes angiogenesis, even under pro-inflammatory conditions. INTERPRETATION: Taken together, our results identify senescence as one of the mechanisms underpinning the pathophysiology of preeclampsia. Therapeutic strategies that target senescent cells may offer novel mechanism-based treatments for preeclampsia. FUNDING: This work was supported by NIH grants, R01 HL136348, R37 AG013925, P01 AG062413, R01 DK11916, generous gifts from the Connor Fund, Robert J. and Theresa W. Ryan and from The George G. Beasley family, the Noaber Foundation, and the Henry and Emma Meyer Professorship in Molecular Genetics.

Testicular germ cell tumors arise in the absence of sex-specific differentiation.

In response to signals from the embryonic testis, the germ cell intrinsic factor NANOS2 coordinates a transcriptional program necessary for the differentiation of pluripotent-like primordial germ cells toward a unipotent spermatogonial stem cell fate. Emerging evidence indicates that genetic risk factors contribute to testicular germ cell tumor initiation by disrupting sex-specific differentiation. Here, using the 129.MOLF-Chr19 mouse model of testicular teratomas and a NANOS2 reporter allele, we report that the developmental phenotypes required for tumorigenesis, including failure to enter mitotic arrest, retention of pluripotency and delayed sex-specific differentiation, were exclusive to a subpopulation of germ cells failing to express NANOS2. Single-cell RNA sequencing revealed that embryonic day 15.5 NANOS2-deficient germ cells and embryonal carcinoma cells developed a transcriptional profile enriched for MYC signaling, NODAL signaling and primed pluripotency. Moreover, lineage-tracing experiments demonstrated that embryonal carcinoma cells arose exclusively from germ cells failing to express NANOS2. Our results indicate that NANOS2 is the nexus through which several genetic risk factors influence tumor susceptibility. We propose that, in the absence of sex specification, signals native to the developing testis drive germ cell transformation.

CTD: An information-theoretic algorithm to interpret sets of metabolomic and transcriptomic perturbations in the context of graphical models.

We consider the following general family of algorithmic problems that arises in transcriptomics, metabolomics and other fields: given a weighted graph G and a subset of its nodes S, find subsets of S that show significant connectedness within G. A specific solution to this problem may be defined by devising a scoring function, the Maximum Clique problem being a classic example, where S includes all nodes in G and where the score is defined by the size of the largest subset of S fully connected within G. Major practical obstacles for the plethora of algorithms addressing this type of problem include computational efficiency and, particularly for more complex scores which take edge weights into account, the computational cost of permutation testing, a statistical procedure required to obtain a bound on the p-value for a connectedness score. To address these problems, we developed CTD, "Connect the Dots", a fast algorithm based on data compression that detects highly connected subsets within S. CTD provides information-theoretic upper bounds on p-values when S contains a small fraction of nodes in G without requiring computationally costly permutation testing. We apply the CTD algorithm to interpret multi-metabolite perturbations due to inborn errors of metabolism and multi-transcript perturbations associated with breast cancer in the context of disease-specific Gaussian Markov Random Field networks learned directly from respective molecular profiling data.

Histoepigenetic analysis of the mesothelin network within pancreatic ductal adenocarcinoma cells reveals regulation of retinoic acid receptor gamma and AKT by mesothelin.

To enable computational analysis of regulatory networks within the cancer cell in its natural tumor microenvironment, we develop a two-stage histoepigenetic analysis method. The first stage involves iterative computational deconvolution to estimate sample-specific cancer-cell intrinsic expression of a gene of interest. The second stage places the gene within a network module. We validate the method in simulation experiments, show improved performance relative to differential expression analysis from bulk samples, and apply it to illuminate the role of the mesothelin (MSLN) network in pancreatic ductal adenocarcinoma (PDAC). The network analysis and subsequent experimental validation in a panel of PDAC cell lines suggests AKT activation by MSLN through two known activators, retinoic acid receptor gamma (RARG) and tyrosine kinase non receptor 2 (TNK2). Taken together, these results demonstrate the potential of histoepigenetic analysis to reveal cancer-cell specific molecular interactions directly from patient tumor profiles.

Oxygen K-shell spectroscopy of isolated progressively solvated peptide.

Gas-phase near-edge X-ray-absorption fine structure (NEXAFS) action spectroscopy around the oxygen K-edge and mass spectrometry were employed to probe isolated substance P (SP) molecular ions, both bare and progressively solvated with 4 and 11 water molecules. Detailed mass spectra of bare and hydrated precursors are presented for the resonant photon energy of 532 eV that corresponds to O1s →π(amide)* core excitation, triggering resonant Auger decay and fragmentation from the ionized radical molecular system. The fragmentation pattern of doubly protonated SP hydrated with 4 water molecules clearly shows a series of abundant doubly charged backbone fragments, as well as triply charged precursor with small neutral losses, all preserving full water cluster. This is drastically different from the collisional induced dissociation of the hydrated peptide where the water loss is a dominant relaxation process. Moreover, the action NEXAFS obtained from several resolved small backbone fragments revealed increased fragmentation of hydrated SP relative to the bare one, due to a resonant O1s excitation of the attached water molecules. Such unexpected result inspires further experimental developments to investigate possible nonlocal energy transfer from the solvent to the biomolecules within the first solvation shell. The experiment is supported by molecular dynamics and DFT calculations to estimate the intensity of the resonant X-ray absorption of bare and hydrated SP around peptide and water O1s excitation region.

Polymorphic presentation of an acute aortic syndrome.

We describe a case of a patient with acute aortic syndrome referred to an emergent surgery. Aortic dissection presented with all-in-one imaging and pathologic entities: aortic dissection with intimal flap and true/false lumen, intramural hematoma and penetrating aortic ulcer. Usually, only one of these entities is present in a single patient, but it is possible that one entity evolve into or coexist with another. Extended hemiarch replacement was performed and the patient was discharged in a good condition.

Glioma-Derived miRNA-Containing Extracellular Vesicles Induce Angiogenesis by Reprogramming Brain Endothelial Cells.

Glioblastoma (GBM) is characterized by aberrant vascularization and a complex tumor microenvironment. The failure of anti-angiogenic therapies suggests pathways of GBM neovascularization, possibly attributable to glioblastoma stem cells (GSCs) and their interplay with the tumor microenvironment. It has been established that GSC-derived extracellular vesicles (GSC-EVs) and their cargoes are proangiogenic in vitro. To further elucidate EV-mediated mechanisms of neovascularization in vitro, we perform RNA-seq and DNA methylation profiling of human brain endothelial cells exposed to GSC-EVs. To correlate these results to tumors in vivo, we perform histoepigenetic analysis of GBM molecular profiles in the TCGA collection. Remarkably, GSC-EVs and normal vascular growth factors stimulate highly distinct gene regulatory responses that converge on angiogenesis. The response to GSC-EVs shows a footprint of post-transcriptional gene silencing by EV-derived miRNAs. Our results provide insights into targetable angiogenesis pathways in GBM and miRNA candidates for liquid biopsy biomarkers.

Guidelines for cell-type heterogeneity quantification based on a comparative analysis of reference-free DNA methylation deconvolution software.

BACKGROUND: Cell-type heterogeneity of tumors is a key factor in tumor progression and response to chemotherapy. Tumor cell-type heterogeneity, defined as the proportion of the various cell-types in a tumor, can be inferred from DNA methylation of surgical specimens. However, confounding factors known to associate with methylation values, such as age and sex, complicate accurate inference of cell-type proportions. While reference-free algorithms have been developed to infer cell-type proportions from DNA methylation, a comparative evaluation of the performance of these methods is still lacking. RESULTS: Here we use simulations to evaluate several computational pipelines based on the software packages MeDeCom, EDec, and RefFreeEWAS. We identify that accounting for confounders, feature selection, and the choice of the number of estimated cell types are critical steps for inferring cell-type proportions. We find that removal of methylation probes which are correlated with confounder variables reduces the error of inference by 30-35%, and that selection of cell-type informative probes has similar effect. We show that Cattell's rule based on the scree plot is a powerful tool to determine the number of cell-types. Once the pre-processing steps are achieved, the three deconvolution methods provide comparable results. We observe that all the algorithms' performance improves when inter-sample variation of cell-type proportions is large or when the number of available samples is large. We find that under specific circumstances the methods are sensitive to the initialization method, suggesting that averaging different solutions or optimizing initialization is an avenue for future research. CONCLUSION: Based on the lessons learned, to facilitate pipeline validation and catalyze further pipeline improvement by the community, we develop a benchmark pipeline for inference of cell-type proportions and implement it in the R package medepir.

Anthocyanins Protect Hepatocytes against CCl4-Induced Acute Liver Injury in Rats by Inhibiting Pro-inflammatory mediators, Polyamine Catabolism, Lipocalin-2, and Excessive Proliferation of Kupffer Cells.

: This study examined the hepatoprotective and anti-inflammatory effects of anthocyanins from Vaccinim myrtillus (bilberry) fruit extract on the acute liver failure caused by carbon tetrachloride-CCl4 (3 mL/kg, i.p.). The preventive treatment of the bilberry extract (200 mg anthocyanins/kg, orally, 7 days) prior to the exposure to the CCl4 resulted in an evident decrease in markers of liver damage (glutamate dehydrogenase, sorbitol dehydrogenase, malate dehydrogenase), and reduced pro-oxidative (conjugated dienes, lipid hydroperoxide, thiobarbituric acid reactive substances, advanced oxidation protein products, NADPH oxidase, hydrogen peroxide, oxidized glutathione), and pro-inflammatory markers (tumor necrosis factor-alpha, interleukin-6, nitrite, myeloperoxidase, inducible nitric oxide synthase, cyclooxygenase-2, CD68, lipocalin-2), and also caused a significant decrease in the dissipation of the liver antioxidative defence capacities (reduced glutathione, glutathione S-transferase, and quinone reductase) in comparison to the results detected in the animals treated with CCl4 exclusively. The administration of the anthocyanins prevented the arginine metabolism's diversion towards the citrulline, decreased the catabolism of polyamines (the activity of putrescine oxidase and spermine oxidase), and significantly reduced the excessive activation and hyperplasia of the Kupffer cells. There was also an absence of necrosis, in regard to the toxic effect of CCl4 alone. The hepatoprotective mechanisms of bilberry extract are based on the inhibition of pro-oxidative mediators, strong anti-inflammatory properties, inducing of hepatic phase II antioxidant enzymes (glutathione S-transferase, quinone reductase) and reduced glutathione, hypoplasia of Kupffer cells, and a decrease in the catabolism of polyamines.