How clot composition influences fibrinolysis in the acute phase of stroke: a proteomic study of cerebral thrombi.

Background: The dramatic clinical improvement offered by mechanical thrombectomy (MT) raised questions about the relevance of prior intravenous thrombolysis (IVT) in large vessel occlusion strokes. Hence, studying IVT susceptibility and its dependence on thrombus composition is crucial. We used observational proteomic study of whole thrombi retrieved by MT to identify factors associated with fibrin content and fibrinolytic activity (FA). Methods: In 104 stroke patients, the thrombi proteome was established by mass spectrometry coupled to liquid chromatography. FA were estimated in clots both outside (FAout) by measuring D-dimer levels at the blood-thrombus interface and inside (FAin) by evaluating the ratio of fibrinogen ⍺ to its plasmin-cleaved forms using proteomics coupled with protein electrophoresis. The factors associated with fibrin content, FAin and FAout were determined by IVT-adjusted linear regression. Results: FAout (p<0.0001) and FAin (p=0.0147) were driven by rt-PA administration (47/104) and thrombus composition. Indeed, FAout was greater with fibrin-rich than erythrocyte-rich thrombi, presumably because of more (r)t-PA substrates. Thus, FAout was increased with cardioembolic thrombi (72/104), which are rich in fibrin (p=0.0300). Opposite results were found inside thrombus, suggesting that (r)t-PA penetrability was hampered by the density of the fibrinous cap. Moreover, blood cells had a strong impact on thrombus structure and susceptibility to (r)t-PA. Indeed, fibrin content was negatively associated with erythrocyte-specific proteins in the thrombus, admission hematocrit (p=0.0139) and hemoglobin level (p=0.0080), which underlines the key role of erythrocytes in thrombus composition. Also, an increased number of neutrophils impaired FAout (p=0.0225), which suggests that their aggregation around the thrombus prevented the (r)t-PA attack. Only FAout was significantly associated with reduced thrombus weight (p=0.0310), increased recanalization rate (p=0.0150), good clinical outcome (p=0.0480) and reduced mortality (p=0.0080). Conclusions: Proteomics can offer new insights into the close relation between thrombus composition and susceptibility to fibrinolysis, paving the way for new adjuvant therapies.

Identification of Metabolomic Markers in Frozen or Formalin-Fixed and Paraffin-Embedded Samples of Diffuse Glioma from Adults.

The aim of this study was to identify metabolomic signatures associated with the gliomagenesis pathway (IDH-mutant or IDH-wt) and tumor grade of diffuse gliomas (DGs) according to the 2021 WHO classification on frozen samples and to evaluate the diagnostic performances of these signatures in tumor samples that are formalin-fixed and paraffin-embedded (FFPE). An untargeted metabolomic study was performed using liquid chromatography/mass spectrometry on a cohort of 213 DG samples. Logistic regression with LASSO penalization was used on the frozen samples to build classification models in order to identify IDH-mutant vs. IDH-wildtype DG and high-grade vs low-grade DG samples. 2-Hydroxyglutarate (2HG) was a metabolite of interest to predict IDH mutational status and aminoadipic acid (AAA) and guanidinoacetic acid (GAA) were significantly associated with grade. The diagnostic performances of the models were 82.6% AUC, 70.6% sensitivity and 80.4% specificity for 2HG to predict IDH status and 84.7% AUC, 78.1% sensitivity and 73.4% specificity for AAA and GAA to predict grade from FFPE samples. Thus, this study showed that AAA and GAA are two novel metabolites of interest in DG and that metabolomic data can be useful in the classification of DG, both in frozen and FFPE samples.

Survival analysis of patient groups defined by unsupervised machine learning clustering methods based on patient metabolomic data.

Purpose: Meta-analyses failed to accurately identify patients with non-metastatic breast cancer who are likely to benefit from chemotherapy, and metabolomics could provide new answers. In our previous published work, patients were clustered using five different unsupervised machine learning (ML) methods resulting in the identification of three clusters with distinct clinical and simulated survival data. The objective of this study was to evaluate the survival outcomes, with extended follow-up, using the same 5 different methods of unsupervised machine learning. Experimental design: Forty-nine patients, diagnosed between 2013 and 2016, with non-metastatic BC were included retrospectively. Median follow-up was extended to 85.8 months. 449 metabolites were extracted from tumor resection samples by combined Liquid chromatography-mass spectrometry (LC-MS). Survival analyses were reported grouping together Cluster 1 and 2 versus cluster 3. Bootstrap optimization was applied. Results: PCA k-means, K-sparse and Spectral clustering were the most effective methods to predict 2-year progression-free survival with bootstrap optimization (PFSb); as bootstrap example, with PCA k-means method, PFSb were 94% for cluster 1&2 versus 82% for cluster 3 (p = 0.01). PCA k-means method performed best, with higher reproducibility (mean HR=2 (95%CI [1.4-2.7]); probability of p ≤ 0.05 85%). Cancer-specific survival (CSS) and overall survival (OS) analyses highlighted a discrepancy between the 5 ML unsupervised methods. Conclusion: Our study is a proof-of-principle that it is possible to use unsupervised ML methods on metabolomic data to predict PFS survival outcomes, with the best performance for PCA k-means. A larger population study is needed to draw conclusions from CSS and OS analyses.

Pilot Study on the Use of Untargeted Metabolomic Fingerprinting of Liquid-Cytology Fluids as a Diagnostic Tool of Malignancy for Thyroid Nodules.

Although it is the gold standard for assessing the malignancy of thyroid nodules (TNs) preoperatively, the cytological analysis of fine-needle aspiration cytology (FNAC) samples results in 20-30% of cases in indeterminate lesions (ITNs). As two-thirds of these lesions will appear benign after diagnostic surgery, improved preoperative diagnostic methods need to be developed. In this pilot study, we evaluate if the metabolomic profiles of liquid-based (CytoRich®) FNAC samples of benign and malignant nodules can allow the molecular diagnosis of TNs. We performed untargeted metabolomic analyses with CytoRich® FNAC in a monocentric retrospective study. The cohort was composed of cytologically benign TNs, histologically benign or papillary thyroid carcinomas (PTCs) cytologically ITNs, and suspicious/malignant TNs histologically confirmed as PTCs. The diagnostic performance of the identified metabolomic signature was assessed using several supervised classification methods. Seventy-eight patients were enrolled in the study. We identified 7690 peaks, of which 2697 ions were included for further analysis. We selected a metabolomic signature composed of the top 15 metabolites. Among all the supervised classification methods, the supervised autoencoder deep neural network exhibited the best performance, with an accuracy of 0.957 (0.842-1), an AUC of 0.945 (0.833-1), and an F1 score of 0.947 (0.842-1). Here, we report a promising new ancillary molecular technique to differentiate PTCs from benign TNs (including among ITNs) based on the metabolomic signature of FNAC sample fluids. Further studies with larger cohorts are now needed to identify a larger number of biomarkers and obtain more robust signatures.

Metabolomics Profiling of Tunisian Sonchus oleraceus L. Extracts and Their Antioxidant Activities.

Sonchus oleraceus (L.) L. (Asteraceae) is an edible wild plant, known for its uses in traditional medicine. The aim of this study is to explore the phytochemical composition of the aerial parts (AP) and roots (R) of aqueous extracts of Sonchus oleraceus L. growing in Tunisia, using liquid chromatography-tandem mass spectrometry(LC/MS/MS), and determine the content of polyphenols and antioxidant activities. Results showed that aqueous extracts of AP and R contained, respectively, 195.25±33 µg/g and 118.66±14 µg/g gallic acid equivalent (GAE), and 52.58±7 µg/g and 3.2±0.3µg/g quercetin equivalent. AP and R extracts also contained tannins, 581.78±33 µg/g and 948.44±19 µg/g GAE. The AP extract in the 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activities, hydroxyl radical scavenging (OH-) and in cupric reducing antioxidant activity (CUPRAC) assays were respectively 0.325±0.036 mg/mL, 0.053±0.018 mg/mL, 0.696±0.031 mg/mL and 60.94±0.004 µMTE/g, while the R extract using the same assays showed, 0.209±0.052 mg/mL, 0.034±0.002 mg/mL, 0.444±0.014 mg/mL and 50.63±0.006 µM Trolox equivalent/g, respectively. A total of 68 compounds were tentatively identified by LC/MS/MS in both extracts in which quinic acid, pyrogallol, osthrutin, piperine, gentisic acid, fisetin, luteolin, caffeic acid, gingerol, were the most abundant in the LC/MS/MS spectrum. Many of these metabolites were found for the first time in Tunisian Sonchus oleraceus L. which may take account for the antioxidant activities exhibited by the plant.

Gut-specific telomerase expression counteracts systemic aging in telomerase-deficient zebrafish.

Telomere shortening is a hallmark of aging and is counteracted by telomerase. As in humans, the zebrafish gut is one of the organs with the fastest rate of telomere decline, triggering early tissue dysfunction during normal zebrafish aging and in prematurely aged telomerase mutants. However, whether telomere-dependent aging of an individual organ, the gut, causes systemic aging is unknown. Here we show that tissue-specific telomerase expression in the gut can prevent telomere shortening and rescues premature aging of tert-/-. Induction of telomerase rescues gut senescence and low cell proliferation, while restoring tissue integrity, inflammation and age-dependent microbiota dysbiosis. Averting gut aging causes systemic beneficial impacts, rescuing aging of distant organs such as reproductive and hematopoietic systems. Conclusively, we show that gut-specific telomerase expression extends the lifespan of tert-/- by 40%, while ameliorating natural aging. Our work demonstrates that gut-specific rescue of telomerase expression leading to telomere elongation is sufficient to systemically counteract aging in zebrafish.

Metabolomic Signatures of Scarff-Bloom-Richardson (SBR) Grade in Non-Metastatic Breast Cancer.

PURPOSE: Identification of metabolomic biomarkers of high SBR grade in non-metastatic breast cancer. METHODS: This retrospective bicentric metabolomic analysis included a training set (n = 51) and a validation set (n = 49) of breast cancer tumors, all classified as high-grade (grade III) or low-grade (grade I-II). Metabolomes of tissue samples were studied by liquid chromatography coupled with mass spectrometry. RESULTS: A molecular signature of the top 12 metabolites was identified from a database of 602 frequently predicted metabolites. Partial least squares discriminant analyses showed that accuracies were 0.81 and 0.82, the R2 scores were 0.57 and 0.55, and the Q2 scores were 0.44431 and 0.40147 for the training set and validation set, respectively; areas under the curve for the Receiver Operating Characteristic Curve were 0.882 and 0.886. The most relevant metabolite was diacetylspermine. Metabolite set enrichment analyses and metabolic pathway analyses highlighted the tryptophan metabolism pathway, but the concentration of individual metabolites varied between tumor samples. CONCLUSIONS: This study indicates that high-grade invasive tumors are related to diacetylspermine and tryptophan metabolism, both involved in the inhibition of the immune response. Targeting these pathways could restore anti-tumor immunity and have a synergistic effect with immunotherapy. Recent studies could not demonstrate the effectiveness of this strategy, but the use of theragnostic metabolomic signatures should allow better selection of patients.

Plasma Rhenium and Selenium Concentrations After Repeated Daily Oral Administration of Rhenium(I)-diselenoether in 4T1 Breast Tumor-bearing Mice.

BACKGROUND/AIM: Rhenium(I)-diselenoether (Re-diSe) is a compound combining a rhenium tricarbonyl(I) core with a diselenide ligand. A high dose of 60 mg/kg had a pro-tumor effect in a previous study, in non-immune deficient 4T1 tumor-bearing mice, while doses of 1 and 10 mg/kg did not affect tumor growth, after repeated oral administrations. This study aimed to examine the tumor effects of a lower dose of 0.1 mg/kg with the same experimental design and to assay plasma Re and Se concentrations. MATERIALS AND METHODS: Syngenic BALB/cByJ (JAX) mice were orthotopically inoculated with 4T1 mammary breast cancer cells. Re-diSe was daily administered orally for 23 days at doses of 0.1, 1, and 10 mg/kg, whereas controls received no treatment. Tumor and mice weights were measured at the end of the experiment. Plasma Re and Se concentrations were assayed by an inductively coupled plasma sector field mass spectrometry instrument (ICP-sf-MS). RESULTS: The weight of the tumors did not vary in treated versus non-treated mice. The limit of detection (LOD) of Re was 0.34 nmol/l. Plasma Re concentrations were 14±20 nmol/l at doses of 0.1 mg/kg, and increased at higher doses, up to 792±167 nmol/l at doses of 10 mg/kg. Plasma Se concentrations were significantly increased in mice treated with the dose of 0.1 mg/kg (4,262±1,511 nmol/l) versus controls (1,262±888 nmol/l), but not from 0.1 to 1 mg/kg, nor from 1 to 10 mg/kg. CONCLUSION: The 0.1 mg/kg dose of Re-diSe resulted in detectable plasma Re concentrations and significantly increased plasma Se concentrations. In the future, doses as low as 0.1 mg/kg of Re-diSe will be tested, exploring its potential immune interest as a metronomic schedule of treatment, but in mouse models that readily develop extensive metastatic disease.

Untargeted plasma metabolomic fingerprinting highlights several biomarkers for the diagnosis and prognosis of coronavirus disease 19.

Objectives: The COVID-19 pandemic has been a serious worldwide public health crisis since 2020 and is still challenging healthcare systems. New tools for the prognosis and diagnosis of COVID-19 patients remain important issues. Design: Here, we studied the metabolome of plasma samples of COVID-19 patients for the identification of prognosis biomarkers. Patients: Plasma samples of eighty-six SARS-CoV-2-infected subjects and 24 healthy controls were collected during the first peak of the COVID-19 pandemic in France in 2020. Main results: Plasma metabolome fingerprinting allowed the successful discrimination of healthy controls, mild SARS-CoV-2 subjects, and moderate and severe COVID-19 patients at hospital admission. We found a strong effect of SARS-CoV-2 infection on the plasma metabolome in mild cases. Our results revealed that plasma lipids and alterations in their saturation level are important biomarkers for the detection of the infection. We also identified deoxy-fructosyl-amino acids as new putative plasma biomarkers for SARS-CoV-2 infection and COVID-19 severity. Finally, our results highlight a key role for plasma levels of tryptophan and kynurenine in the symptoms of COVID-19 patients. Conclusion: Our results showed that plasma metabolome profiling is an efficient tool for the diagnosis and prognosis of SARS-CoV-2 infection.

Relationship between the oxidative status and the tumor growth in transplanted triple-negative 4T1 breast tumor mice after oral administration of rhenium(I)-diselenoether.

BACKGROUND: Selective inhibitory effects of rhenium(I)-diselenoether (Re-diSe) were observed in cultured breast malignant cells. They were attributed to a decrease in Reactive Oxygen Species (ROS) production. A concomitant decrease in the production of Transforming Growth Factor-beta (TGFß1), Insulin Growth Factor 1 (IGF1), and Vascular Endothelial Growth Factor A (VEGFA) by the malignant cells was also observed. AIM: The study aimed to investigate the anti-tumor effects of Re-diSe on mice bearing 4T1 breast tumors, an experimental model of triple-negative breast cancer, and correlate them with several biomarkers. MATERIAL AND METHODS: 4T1 mammary breast cancer cells were orthotopically inoculated into syngenic BALB/c Jack mice. Different doses of Re-diSe (1, 10, and 60 mg/kg) were administered orally for 23 consecutive days to assess the efficacy and toxicity. The oxidative status was evaluated by assaying Advanced Oxidative Protein Products (AOPP), and by the dinitrophenylhydrazone (DNPH) test in plasma of healthy mice, non-treated tumor-bearing mice (controls), treated tumor-bearing mice, and tumors in all tumor-bearing mice. Tumor necrosis factor (TNFα), VEGFA, VEGFB, TGFß1, Interferon, and selenoprotein P (selenoP) were selected as biomarkers. RESULTS: Doses of 1 and 10 mg/kg did not affect the tumor weights. There was a significant increase in the tumor weights in mice treated with the maximum dose of 60 mg/kg, concomitantly with a significant decrease in AOPP, TNFα, and TGFß1 in the tumors. SelenoP concentrations increased in the plasma but not in the tumors. CONCLUSION: We did not confirm the anti-tumor activity of the Re-diSe compound in this experiment. However, the transplantation of the tumor cells did not induce an expected pro-oxidative status without any increase of the oxidative biomarkers in the plasma of controls compared to healthy mice. This condition could be essential to evaluate the effect of an antioxidant drug. The choice of the experimental model will be primordial to assess the effects of the Re-diSe compound in further studies.

Cross Pharmacological, Biochemical and Computational Studies of a Human Kv3.1b Inhibitor from Androctonus australis Venom.

The voltage-gated K+ channels Kv3.1 display fast activation and deactivation kinetics and are known to have a crucial contribution to the fast-spiking phenotype of certain neurons. AahG50, as a natural product extracted from Androctonus australis hector venom, inhibits selectively Kv3.1 channels. In the present study, we focused on the biochemical and pharmacological characterization of the component in AahG50 scorpion venom that potently and selectively blocks the Kv3.1 channels. We used a combined optimization through advanced biochemical purification and patch-clamp screening steps to characterize the peptide in AahG50 active on Kv3.1 channels. We described the inhibitory effect of a toxin on Kv3.1 unitary current in black lipid bilayers. In silico, docking experiments are used to study the molecular details of the binding. We identified the first scorpion venom peptide inhibiting Kv3.1 current at 170 nM. This toxin is the alpha-KTx 15.1, which occludes the Kv3.1 channel pore by means of the lysine 27 lateral chain. This study highlights, for the first time, the modulation of the Kv3.1 by alpha-KTx 15.1, which could be an interesting starting compound for developing therapeutic biomolecules against Kv3.1-associated diseases.

The Carcinogen Cadmium Activates Lysine 63 (K63)-Linked Ubiquitin-Dependent Signaling and Inhibits Selective Autophagy.

Signaling, proliferation, and inflammation are dependent on K63-linked ubiquitination-conjugation of a chain of ubiquitin molecules linked via lysine 63. However, very little information is currently available about how K63-linked ubiquitination is subverted in cancer. The present study provides, for the first time, evidence that cadmium (Cd), a widespread environmental carcinogen, is a potent activator of K63-linked ubiquitination, independently of oxidative damage, activation of ubiquitin ligase, or proteasome impairment. We show that Cd induces the formation of protein aggregates that sequester and inactivate cylindromatosis (CYLD) and selective autophagy, two tumor suppressors that deubiquitinate and degrade K63-ubiquitinated proteins, respectively. The aggregates are constituted of substrates of selective autophagy-SQSTM1, K63-ubiquitinated proteins, and mitochondria. These protein aggregates also cluster double-membrane remnants, which suggests an impairment in autophagosome maturation. However, failure to eliminate these selective cargos is not due to alterations in the general autophagy process, as degradation of long-lived proteins occurs normally. We propose that the simultaneous disruption of CYLD and selective autophagy by Cd feeds a vicious cycle that further amplifies K63-linked ubiquitination and downstream activation of the NF-κB pathway, processes that support cancer progression. These novel findings link together impairment of selective autophagy, K63-linked ubiquitination, and carcinogenesis.

Combined Omic Analyzes of Cerebral Thrombi: A New Molecular Approach to Identify Cardioembolic Stroke Origin.

Background and Purpose: The diagnosis of cardioembolic stroke can be challenging for patient management in secondary stroke prevention, particularly in the case of covert paroxysmal atrial fibrillation. The molecular composition of a cerebral thrombus is related to its origin. Therefore, proteomic and metabolomic analyses of the retrieved thrombotic material should allow the identification of biomarkers or signatures to improve the etiological diagnosis of stroke. Methods: In this pilot study, the proteome and metabolome of cerebral thrombi from atherothrombotic and cardioembolic stroke patients were studied according to ASCOD phenotyping (A: atherosclerosis; S: small-vessel disease; C: cardiac pathology; O: other causes; D: dissection), with the highest causality grade, from the ThrombiOMIC cohort (consecutive patients with stroke recanalized by mechanical thrombectomy in an acute phase). Proteomic and metabolomic results were used separately or combined, and the obtained omic signatures were compared with classical cardioembolic stroke predictors using pairwise comparisons of the area under receiver operating characteristics. Results: Among 59 patients of the ThrombiOMIC cohort, 34 patients with stroke showed a cardioembolic phenotype and 7 had an atherothrombotic phenotype. Two thousand four hundred fifty-six proteins and 5019 molecular features of the cerebral thrombi were identified using untargeted proteomic and metabolomic approaches, respectively. Area under receiver operating characteristics to predict the cardioembolic origin of stroke were calculated using the proteomic results (0.945 [95% CI, 0.871­1]), the metabolomic results (0.836 [95% CI, 0.714­0.958]), and combined signatures (0.996 [95% CI, 0.984­1]). The diagnostic performance of the combined signatures was significantly higher than that of classical predictors such as the plasmatic BNP (B-type natriuretic peptide) level (area under receiver operating characteristics, 0.803 [95% CI, 0.629­0.976]). Conclusions: The combined proteomic and metabolomic analyses of retrieved cerebral thrombi is a very promising molecular approach to predict the cardioembolic cause of stroke and to improve secondary stroke prevention strategies.

Proteomic analysis identified LBP and CD14 as key proteins in blood/biphasic calcium phosphate microparticle interactions.

Immediately upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to the biomaterial surface and the protein layer affects both blood cell functions and biomaterial bioactivity. Previously, we reported that 80-200 µm biphasic calcium phosphate (BCP) microparticles embedded in a blood clot, induce ectopic woven bone formation in mice, when 200-500 µm BCP particles induce mainly fibrous tissue. Here, in a LC-MS/MS proteomic study we compared the differentially expressed blood proteins (plasma and blood cell proteins) and the deregulated signaling pathways of these osteogenic and fibrogenic blood composites. We showed that blood/BCP-induced osteogenesis is associated with a higher expression of fibrinogen (FGN) and an upregulation of the Myd88- and NF-κB-dependent TLR4 signaling cascade. We also highlighted the key role of the LBP/CD14 proteins in the TLR4 activation of blood cells by BCP particles. As FGN is an endogenous ligand of TLR4, able to modulate blood composite stiffness, we propose that different FGN concentrations modify the blood clot mechanical properties, which in turn modulate BCP/blood composite osteoactivity through TLR4 signaling. The present findings provide an insight at the protein level, into the mechanisms leading to an efficient bone reconstruction by blood/BCP composites. STATEMENT OF SIGNIFICANCE: Upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to bone substitute surface and this protein layer affects both biomaterial bioactivity and bone healing. Therefore, for the best outcome for patients, it is crucial to understand the molecular interactions between blood and bone scaffolds. Biphasic calcium phosphate (BCP) ceramics are considered as the gold standard in bone reconstruction surgery. Here, using proteomic analyses we showed that the osteogenic properties of 80-200 µm BCP particles embedded in a blood clot is associated with a higher expression of fibrinogen. Fibrinogen upregulates the Myd88- and NF-κB-dependent TLR4 pathway in blood cells and, BCP-induced TLR4 activation is mediated by the LBP and CD14 proteins.

Ingested Ketone Ester Leads to a Rapid Rise of Acetyl-CoA and Competes with Glucose Metabolism in the Brain of Non-Fasted Mice.

The role of ketone bodies in the cerebral energy homeostasis of neurological diseases has begun to attract recent attention particularly in acute neurological diseases. In ketogenic therapies, ketosis is achieved by either a ketogenic diet or by the administration of exogenous ketone bodies. The oral ingestion of the ketone ester (KE), (R)-3-hydroxybutyl (R)-3-hydroxybutyrate, is a new method to generate rapid and significant ketosis (i.e., above 6 mmol/L) in humans. KE is hydrolyzed into ß-hydroxybutyrate (ßHB) and its precursor 1,3-butanediol. Here, we investigate the effect of oral KE administration (3 mg KE/g of body weight) on brain metabolism of non-fasted mice using liquid chromatography in tandem with mass spectrometry. Ketosis (Cmax = 6.83 ± 0.19 mmol/L) was obtained at Tmax = 30 min after oral KE-gavage. We found that ßHB uptake into the brain strongly correlated with the plasma ßHB concentration and was preferentially distributed in the neocortex. We showed for the first time that oral KE led to an increase of acetyl-CoA and citric cycle intermediates in the brain of non-fasted mice. Furthermore, we found that the increased level of acetyl-CoA inhibited glycolysis by a feedback mechanism and thus competed with glucose under physiological conditions. The brain pharmacodynamics of this oral KE strongly suggest that this agent should be considered for acute neurological diseases.

Tumor microenvironment affects exogenous sodium/iodide symporter expression.

For decades, sodium/iodide symporter NIS-mediated iodide uptake has played a crucial role in the radioactive ablation of thyroid cancer cells. NIS-based gene therapy has also become a promising tool for the treatment of tumors of extrathyroidal origin. But its applicability has been hampered by reduced expression of NIS, resulting in a moderated capacity to accumulate 131I and in inefficient ablation. Despite numerous preclinical enhancement strategies, the understanding of NIS expression within tumors remains limited. This study aims at a better understanding of the functional behavior of exogenous NIS expression in the context of malignant solid tumors that are characterized by rapid growth with an insufficient vasculature, leading to hypoxia and quiescence. Using subcutaneous HT29NIS and K7M2NIS tumors, we show that NIS-mediated uptake and NIS expression at the plasma membrane of cancer cells are impaired in the intratumoral regions. For a better understanding of the underlying molecular mechanisms induced by hypoxia and quiescence (separately and in combination), we performed experiments on HT29NIS cancer cells. Hypoxia and quiescence were both found to impair NIS-mediated uptake through mechanisms including NIS mis-localization. Modifications in the expression of proteins and metabolites involved in plasma membrane localization and in energy metabolism were found using untargeted proteomics and metabolomics approaches. In conclusion, our results provide evidence that hypoxia and quiescence impair NIS expression at the plasma membrane, and iodide uptake. Our study also shows that the tumor microenvironment is an important parameter for successful NIS-based cancer treatment.

Metabolome of Cerebral Thrombi Reveals an Association between High Glycemia at Stroke Onset and Good Clinical Outcome.

Despite the fact that glucose is the main fuel of the brain, hyperglycemia at hospital admission is generally associated with a poor functional outcome in stroke patients. This paradox may be explained by the lack of information about the blood glucose level at stroke onset. Here, we analyzed the metabolome of blood cells entrapped in cerebral thrombi to gain insight into their metabolism at stroke onset. Fourty-one consecutive stroke patients completely recanalized by mechanical thrombectomy within 6 h were included. The metabolome of retrieved thrombi was analyzed by liquid chromatography tandem with mass spectrometry. Discriminant Analysis (sparse Partial Least Squares Discriminant Analysis (sPLS-DA)) was performed to identify classification models and significant associated features of favorable clinical outcome at 3 months (modified Rankin Scale (mRS) < 2). sPLS-DA of the metabolomes of cerebral thrombi discriminated between stroke patients with a favorable or poor clinical outcome (Area Under the Curve (AUC) = 0.992 (0.931-1)). In addition, our results revealed that high sorbitol and glucose levels in the thrombi positively correlated with favorable clinical outcomes. Sorbitol, a short-term glycemic index reflecting a high blood glucose level at stroke onset, was found to be an independent predictor of good outcome (AUC = 0.908 (0.807-0.995)). This study demonstrates that a high blood glucose level at stroke onset is beneficial to the clinical outcome of the patient.

Comparison of unsupervised machine-learning methods to identify metabolomic signatures in patients with localized breast cancer.

Genomics and transcriptomics have led to the widely-used molecular classification of breast cancer (BC). However, heterogeneous biological behaviors persist within breast cancer subtypes. Metabolomics is a rapidly-expanding field of study dedicated to cellular metabolisms affected by the environment. The aim of this study was to compare metabolomic signatures of BC obtained by 5 different unsupervised machine learning (ML) methods. Fifty-two consecutive patients with BC with an indication for adjuvant chemotherapy between 2013 and 2016 were retrospectively included. We performed metabolomic profiling of tumor resection samples using liquid chromatography-mass spectrometry. Here, four hundred and forty-nine identified metabolites were selected for further analysis. Clusters obtained using 5 unsupervised ML methods (PCA k-means, sparse k-means, spectral clustering, SIMLR and k-sparse) were compared in terms of clinical and biological characteristics. With an optimal partitioning parameter k = 3, the five methods identified three prognosis groups of patients (favorable, intermediate, unfavorable) with different clinical and biological profiles. SIMLR and K-sparse methods were the most effective techniques in terms of clustering. In-silico survival analysis revealed a significant difference for 5-year predicted OS between the 3 clusters. Further pathway analysis using the 449 selected metabolites showed significant differences in amino acid and glucose metabolism between BC histologic subtypes. Our results provide proof-of-concept for the use of unsupervised ML metabolomics enabling stratification and personalized management of BC patients. The design of novel computational methods incorporating ML and bioinformatics techniques should make available tools particularly suited to improving the outcome of cancer treatment and reducing cancer-related mortalities.

Retinoic acid synthesis by ALDH1A proteins is dispensable for meiosis initiation in the mouse fetal ovary.

In mammals, the timing of meiosis entry is regulated by signals from the gonadal environment. All-trans retinoic acid (ATRA) signaling is considered the key pathway that promotes Stra8 (stimulated by retinoic acid 8) expression and, in turn, meiosis entry. This model, however, is debated because it is based on analyzing the effects of exogenous ATRA on ex vivo gonadal cultures, which not accurately reflects the role of endogenous ATRA. Aldh1a1 and Aldh1a2, two retinaldehyde dehydrogenases synthesizing ATRA, are expressed in the mouse ovaries when meiosis initiates. Contrary to the present view, here, we demonstrate that ATRA-responsive cells are scarce in the ovary. Using three distinct gene deletion models for Aldh1a1;Aldh1a2;Aldh1a3, we show that Stra8 expression is independent of ATRA production by ALDH1A proteins and that germ cells progress through meiosis. Together, these data demonstrate that ATRA signaling is dispensable for instructing meiosis initiation in female germ cells.

Proteomic Analysis of Iodinated Contrast Agent-Induced Perturbation of Thyroid Iodide Uptake.

(1) Background: We recently showed that iodinated contrast media (ICM) reduced thyroid uptake of iodide independently of free iodide through a mechanism different from that of NaI and involving a dramatic and long-lasting decrease in Na/I symporter expression. The present study aimed at comparing the response of the thyroid to ICM and NaI using a quantitative proteomic approach. (2) Methods: Scintiscans were performed on ICM-treated patients. Micro Single-Photon Emission Computed Tomography (microSPECT/CT) imaging was used to assess thyroid uptakes in ICM- or NaI-treated mice and their response to recombinant human thyroid-stimulating hormone. Total thyroid iodide content and proteome was determined in control, NaI-, or ICM-treated animals. (3) Results: The inhibitory effect of ICM in patients was selectively observed on thyroids but not on salivary glands for up to two months after a systemic administration. An elevated level of iodide was observed in thyroids from NaI-treated mice but not in those from ICM animals. Exposure of the thyroid to NaI modulates 15 cellular pathways, most of which are also affected by ICM treatment (including the elF4 and P706SK cell signaling pathway and INSR identified as an upstream activator in both treatments). In addition, ICM modulates 16 distinct pathways and failed to affect thyroid iodide content. Finally, administration of ICM reduces thyroid-stimulating hormone (TSH) receptor expression which results in a loss of TSH-induced iodide uptake by the thyroid. (4) Conclusions: Common intracellular mechanisms are involved in the ICM- and NaI-induced reduction of iodide uptake. However, ICM fails to affect thyroid iodide content which suggests that the modulation of these common pathways is triggered by separate effectors. ICM also modulates numerous distinct pathways which may account for its long-lasting effect on thyroid uptake. These observations may have implications in the management of patients affected by differentiated thyroid carcinomas who have been exposed to ICM. They also provide the basis for the utilization of ICM-based compounds in radioprotection of the thyroid.