Search

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with limited treatment options and a poor prognosis. The critical role of epigenetic alterations such as changes in DNA methylation, histones modifications, and chromatin remodeling, in pancreatic tumors progression is becoming increasingly recognized. Moreover, in PDAC these aberrant epigenetic mechanisms can also limit therapy efficacy. This study aimed to investigate the expression and prognostic significance of a key epigenetic complex encompassing DNA methyltransferase-1 (DNMT1), the histone methyltransferase G9a, and the scaffold protein UHRF1 in PDAC. We also evaluated the therapeutic potential of an innovative inhibitor targeting these epigenetic effectors. Methods Immunohistochemical analysis of DNMT1, G9a, and UHRF1 expression was conducted in human PDAC tissue samples. Staining was semi-quantitatively scored, and overexpression was defined as moderate to strong positivity. The prognostic impact was assessed by correlating protein expression with patient survival. The antitumoral effects of the dual DNMT1-G9a inhibitor CM272 were tested in PDAC cell lines, followed by transcriptomic analyses to identify underlying mechanisms. The in vivo antitumoral efficacy of CM272 was evaluated in PDAC xenograft and syngeneic mouse models, both alone and in combination with anti-PD1 immunotherapy. Results DNMT1, G9a, and UHRF1 were significantly overexpressed in PDAC cells and stroma compared to normal pancreatic tissues. Simultaneous overexpression of the three proteins was associated with significantly reduced survival in resected PDAC patients. CM272 exhibited potent antiproliferative activity in PDAC cell lines, inducing apoptosis and altering key metabolic and cell cycle-related genes. CM272 also enhanced chemotherapy sensitivity and significantly inhibited tumor growth in vivo without detectable toxicity. Combination of CM272 with anti-PD1 therapy further improved antitumor responses and immune cell infiltration, particularly CD4 + and CD8 + T cells. Conclusions The combined overexpression of DNMT1, G9a, and UHRF1 in PDAC is a strong predictor of poor prognosis. CM272, by targeting this epigenetic complex, shows promising therapeutic potential by inducing apoptosis, reprogramming metabolic pathways, and enhancing immune responses. The combination of CM272 with immunotherapy offers a novel, effective treatment strategy for PDAC.

Abstract The treatment of non-small cell lung cancer (NSCLC) patients has significantly improved with recent therapeutic strategies; however, many patients still do not benefit from them. As a result, new treatment approaches are urgently needed. In this study, we evaluated the antitumor efficacy of co-targeting G9a and DNMT1 enzymes and its potential as a cancer drug sensitizer. We observed co-expression and overexpression of G9a and DNMT1 in NSCLC, which were associated with poor prognosis. Co-targeting G9a/DNMT1 with the drug CM-272 reduced proliferation and induced cell death in a panel of human and murine NSCLC cell lines. Additionally, the transcriptomes of these cells were reprogrammed to become highly responsive to chemotherapy (cisplatin), targeted therapy (trametinib), and epigenetic therapy (vorinostat). In vivo, CM-272 reduced tumor volume in human and murine cell-derived cancer models, and this effect was synergistically enhanced by cisplatin. The expression of SCARA5 and AOX1 was induced by CM-272, and both proteins were found to be essential for the antiproliferative response, as gene silencing decreased cytotoxicity. Furthermore, the expression of SCARA5 and AOX1 was positively correlated with each other and inversely correlated with G9a and DNMT1 expression in NSCLC patients. SCARA5 and AOX1 DNA promoters were hypermethylated in NSCLC, and SCARA5 methylation was identified as an epigenetic biomarker in tumors and liquid biopsies from NSCLC patients. Thus, we demonstrate that co-targeting G9a/DNMT1 is a promising strategy to enhance the efficacy of cancer drugs, and SCARA5 methylation could serve as a non-invasive biomarker to monitor tumor progression.

Abstract The development of computational tools for the systematic prediction of metabolic vulnerabilities of cancer cells constitutes a central question in systems biology. Here, we present gmctool, a freely accessible online tool that allows us to accomplish this task in a simple, efficient and intuitive environment. gmctool exploits the concept of genetic Minimal Cut Sets (gMCSs), a theoretical approach to synthetic lethality based on genome-scale metabolic networks, including a unique database of synthetic lethals computed from Human1, the most recent metabolic reconstruction of human cells. gmctool introduces qualitative and quantitative improvements over our previously developed algorithms to predict, visualize and analyze metabolic vulnerabilities in cancer, demonstrating a superior performance than competing algorithms. A detailed illustration of gmctool is presented for multiple myeloma (MM), an incurable hematological malignancy. We provide in vitro experimental evidence for the essentiality of CTPS1 (CTPS synthase) and UAP1 (UDP-N-Acetylglucosamine Pyrophosphorylase 1) in specific MM patient subgroups.

Abstract Despite the development of novel therapies for acute myeloid leukemia, outcomes remain poor for most patients, and therapeutic improvements are an urgent unmet need. Although treatment regimens promoting differentiation have succeeded in the treatment of acute promyelocytic leukemia, their role in other acute myeloid leukemia subtypes needs to be explored. Here we identify and characterize two lysine deacetylase inhibitors, CM-444 and CM-1758, exhibiting the capacity to promote myeloid differentiation in all acute myeloid leukemia subtypes at low non-cytotoxic doses, unlike other commercial histone deacetylase inhibitors. Analyzing the acetylome after CM-444 and CM-1758 treatment reveals modulation of non-histone proteins involved in the enhancer–promoter chromatin regulatory complex, including bromodomain proteins. This acetylation is essential for enhancing the expression of key transcription factors directly involved in the differentiation therapy induced by CM-444/CM-1758 in acute myeloid leukemia. In summary, these compounds may represent effective differentiation-based therapeutic agents across acute myeloid leukemia subtypes with a potential mechanism for the treatment of acute myeloid leukemia.

Aims: Rotator cuff (RC) injuries are characterized by tendon rupture, muscle atrophy, retraction, and fatty infiltration, which increase injury severity and jeopardize adequate tendon repair. Epigenetic drugs, such as histone deacetylase inhibitors (HDACis), possess the capacity to redefine the molecular signature of cells, and they may have the potential to inhibit the transformation of the fibro-adipogenic progenitors (FAPs) within the skeletal muscle into adipocyte-like cells, concurrently enhancing the myogenic potential of the satellite cells. Methods: HDACis were added to FAPs and satellite cell cultures isolated from mice. The HDACi vorinostat was additionally administered into a RC injury animal model. Histological analysis was carried out on the isolated supra- and infraspinatus muscles to assess vorinostat anti-muscle degeneration potential. Results: Vorinostat, a HDACi compound, blocked the adipogenic transformation of muscle-associated FAPs in culture, promoting myogenic progression of the satellite cells. Furthermore, it protected muscle from degeneration after acute RC in mice in the earlier muscle degenerative stage after tenotomy. Conclusion: The HDACi vorinostat may be a candidate to prevent early muscular degeneration after RC injury. Cite this article: Bone Joint Res 2024;13(4):169–183.

Abstract Alterations in the epigenetic machinery in both tumor and immune cells contribute to bladder cancer (BC) development, constituting a promising target as an alternative therapeutic option. Here, we have explored the effects of a novel histone deacetylase (HDAC) inhibitor CM-1758, alone or in combination with immune checkpoint inhibitors (ICI) in BC. We determined the antitumor effects of CM-1758 in various BC cell lines together with the induction of broad transcriptional changes, with focus on the epigenetic regulation of PD-L1. Using an immunocompetent syngeneic mouse model of metastatic BC, we studied the effects of CM-1758 alone or in combination with anti-PD-L1 not only on tumor cells, but also in the tumor microenvironment. In vitro, we found that CM-1758 has cytotoxic and cytostatic effects either by inducing apoptosis or cell cycle arrest in BC cells at low micromolar levels. PD-L1 is epigenetically regulated by histone acetylation marks and is induced after treatment with CM-1758. We also observed that treatment with CM-1758 led to an important delay in tumor growth and a higher CD8 + T cell tumor infiltration. Moreover, anti-PD-L1 alone or in combination with CM-1758 reprogramed macrophage differentiation towards a M1-like polarization state and increased of pro-inflammatory cytokines systemically, yielding potential further antitumor effects. Our results suggest the possibility of combining HDAC inhibitors with immunotherapies for the management of advanced metastatic BC.

Abstract Drug combinations are key to circumvent resistance mechanisms compromising response to single anti-cancer targeted therapies. The implementation of combinatorial approaches involving MEK1/2 or KRASG12C inhibitors in the context of KRAS-mutated lung cancers focuses fundamentally on targeting KRAS proximal activators or effectors. However, the antitumor effect is highly determined by compensatory mechanisms arising in defined cell types or tumor subgroups. A potential strategy to find drug combinations targeting a larger fraction of KRAS-mutated lung cancers may capitalize on the common, distal gene expression output elicited by oncogenic KRAS. By integrating a signature-driven drug repurposing approach with a pairwise pharmacological screen, here we show synergistic drug combinations consisting of multi-tyrosine kinase PKC inhibitors together with MEK1/2 or KRASG12C inhibitors. Such combinations elicit a cytotoxic response in both in vitro and in vivo models, which in part involves inhibition of the PKC inhibitor target AURKB. Proteome profiling links dysregulation of MYC expression to the effect of both PKC inhibitor-based drug combinations. Furthermore, MYC overexpression appears as a resistance mechanism to MEK1/2 and KRASG12C inhibitors. Our study provides a rational framework for selecting drugs entering combinatorial strategies and unveils MEK1/2- and KRASG12C-based therapies for lung cancer.

Vancomycin-resistant Enterococcus (VRE) represents a major threat for patients’ health. Here, using a mouse model, the authors show that specific commensals restrict VRE gut colonization through depletion of fructose, a nutrient source that boosts VRE growth in vivo.

Multidrug-resistant Enterobacteriaceae (MRE) represent a major threat for patients’ health. Here, the authors describe how cooperation between gut commensal bacteria (Lactobacillus spp. And Clostridiales) restrict MRE colonization in mice and patients

Abstract Background Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA. Methods Cholangiocarcinogenesis was induced in rats (TAA) and mice (Jnk Δhepa + CCl4 + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRASG12D cells. Cell signaling, growth, gene regulation and [U-13C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model. Results Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRASG12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRASG12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRASG12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression. Conclusions In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA.

BackgroundThe main objective was to evaluate the efficacy of intranasal photodynamic therapy (PDT) in SARS-CoV-2 mildly symptomatic carriers on decreasing the infectivity period. SARS-CoV-2-specific immune-stimulating effects and safety were also analysed.MethodsWe performed a randomized, placebo-controlled, clinical trial in a tertiary hospital (NCT05184205). Patients with a positive SARS-CoV-2 PCR in the last 48 hours were recruited and aleatorily assigned to PDT or placebo. Patients with pneumonia were excluded. Participants and investigators were masked to group assignment. The primary outcome was the reduction in in vitro infectivity of nasopharyngeal samples at days 3 and 7. Additional outcomes included safety assessment and quantification of humoral and T-cell immune-responses.FindingsPatients were recruited between December 2021 and February 2022. Most were previously healthy adults vaccinated against COVID-19 and most carried Omicron variant. 38 patients were assigned to placebo and 37 to PDT. Intranasal PDT reduced infectivity at day 3 post-treatment when compared to placebo with a β-coefficient of -812.2 (CI95%= -478660 – -1.3, p34) at day 7 was higher on the PDT-group, with an OR of 0.15 (CI95%=0.04-0.58). There was a decay in anti-Spike titre and specific SARS-CoV-2 T cell immunity in the placebo group 10 and 20 weeks after infection, but not in the PDT-group. No serious adverse events were reported.InterpretationIntranasal-PDT is safe in pauci-symptomatic COVID-19 patients, it reduces SARS-CoV-2 infectivity and decelerates the decline SARS-CoV-2 specific immune-responses.

Lysosomal storage disorders (LSDs) constitute a large group of rare, multisystemic, inherited disorders of metabolism, characterized by defects in lysosomal enzymes, accessory proteins, membrane transporters or trafficking proteins. Pompe disease (PD) is produced by mutations in the acid alpha-glucosidase (GAA) lysosomal enzyme. This enzymatic deficiency leads to the aberrant accumulation of glycogen in the lysosome. The onset of symptoms, including a variety of neurological and multiple-organ pathologies, can range from birth to adulthood, and disease severity can vary between individuals. Although very significant advances related to the development of new treatments, and also to the improvement of newborn screening programs and tools for a more accurate diagnosis and follow-up of patients, have occurred over recent years, there exists an unmet need for further understanding the molecular mechanisms underlying the progression of the disease. Also, the reason why currently available treatments lose effectiveness over time in some patients is not completely understood. In this scenario, characterization of the metabolic phenotype is a valuable approach to gain insights into the global impact of lysosomal dysfunction, and its potential correlation with clinical progression and response to therapies. These approaches represent a discovery tool for investigating disease-induced modifications in the complete metabolic profile, including large numbers of metabolites that are simultaneously analyzed, enabling the identification of novel potential biomarkers associated with these conditions. This review aims to highlight the most relevant findings of recently published omics-based studies with a particular focus on describing the clinical potential of the specific metabolic phenotypes associated to different subgroups of PD patients.

Abstract Incidence of pneumococcal disease has increased worldwide in recent years. Response to pneumococcal vaccine is usually measured using the multiserotype enzyme-linked immunosorbent assay (ELISA) pneumococcal test. However, this approach presents several limitations. Therefore, the introduction of new and more robust analytical approaches able to provide information on the efficacy of the pneumococcal vaccine would be very beneficial for the clinical management of patients. Surface plasmon resonance (SPR) has been shown to offer a valuable understanding of vaccines’ properties over the last years. The aim of this study is to evaluate the reliability of SPR for the anti-pneumococcal capsular polysaccharides (anti-PnPs) IgGs quantification in vaccinated. Fast protein liquid chromatography (FPLC) was used for the isolation of total IgGs from serum samples of vaccinated patients. Binding-SPR assays were performed to study the interaction between anti-PnPs IgGs and PCV13. A robust correlation was found between serum levels of anti-PnPs IgGs, measured by ELISA, and the SPR signal. Moreover, it was possible to correctly classify patients into “non-responder”, “responder” and “high-responder” groups according to their specific SPR PCV13 response profiles. SPR technology provides a valuable tool for reliably characterize the interaction between anti-PnPs IgGs and PCV13 in a very short experimental time.

Summary: Cell reprogramming is thought to be associated with a full metabolic switch from an oxidative- to a glycolytic-based metabolism. However, neither the dynamics nor the factors controlling this metabolic switch are fully understood. By using cellular, biochemical, protein array, metabolomic, and respirometry analyses, we found that c-MYC establishes a robust bivalent energetics program early in cell reprogramming. Cells prone to undergo reprogramming exhibit high mitochondrial membrane potential and display a hybrid metabolism. We conclude that MYC proteins orchestrate a rewiring of somatic cell metabolism early in cell reprogramming, whereby somatic cells acquire the phenotypic plasticity necessary for their transition to pluripotency in response to either intrinsic or external cues. : Torres and colleagues describe an MYC-dependent remodeling of mitochondrial dynamics and metabolism during the first stage of cell reprogramming. Endogenous MYC activity was found to be necessary for cell reprogramming, likely by establishing a hybrid metabolic state characterized by elevated glycolytic flux and a somatic oxidative metabolic rate. MYC polarized mitochondria by increasing ATPIF1 and labeled cells prone to cell reprogramming. Keywords: cell reprogramming, metabolism, mitochondrial dynamics, cell signaling

Parkinson’s disease (PD) is the second-most common neurodegenerative disorder, whose physiopathology is still unclear. Moreover, there is an urgent need to discover new biomarkers and therapeutic targets to facilitate its diagnosis and treatment. Previous studies performed in PD models and samples from PD patients already demonstrated that metabolic alterations are associated with this disease. In this context, the aim of this study is to provide a better understanding of metabolic disturbances underlying PD pathogenesis. To achieve this goal, we used a Drosophila PD model based on inactivation of the DJ-1β gene (ortholog of human DJ-1). Metabolomic analyses were performed in 1-day-old and 15-day-old DJ-1β mutants and control flies using 1H nuclear magnetic resonance spectroscopy, combined with expression and enzymatic activity assays of proteins implicated in altered pathways. Our results showed that the PD model flies exhibited protein metabolism alterations, a shift fromthe tricarboxylic acid cycle to glycolytic pathway to obtain ATP, together with an increase in the expression of some urea cycle enzymes. Thus, these metabolic changes could contribute to PD pathogenesis and might constitute possible therapeutic targets and/or biomarkers for this disease.

Pharmacometabolomics (PMx) studies aim to predict individual differences in treatment response and in the development of adverse effects associated with specific drug treatments. Overall, these studies inform us about how individuals will respond to a drug treatment based on their metabolic profiles obtained before, during, or after the therapeutic intervention. In the era of precision medicine, metabolic profiles hold great potential to guide patient selection and stratification in clinical trials, with a focus on improving drug efficacy and safety. Metabolomics is closely related to the phenotype as alterations in metabolism reflect changes in the preceding cascade of genomics, transcriptomics, and proteomics changes, thus providing a significant advance over other omics approaches. Nuclear Magnetic Resonance (NMR) is one of the most widely used analytical platforms in metabolomics studies. In fact, since the introduction of PMx studies in 2006, the number of NMR-based PMx studies has been continuously growing and has provided novel insights into the specific metabolic changes associated with different mechanisms of action and/or toxic effects. This review presents an up-to-date summary of NMR-based PMx studies performed over the last 10 years. Our main objective is to discuss the experimental approaches used for the characterization of the metabolic changes associated with specific therapeutic interventions, the most relevant results obtained so far, and some of the remaining challenges in this area.

(1) Background: The ability of cancer cells to evade the immune system is due in part to their capacity to induce and recruit T regulatory cells (Tregs) to the tumor microenvironment. Strategies proposed to improve antitumor immunity by depleting Tregs generally lack specificity and raise the possibility of autoimmunity. Therefore, we propose to control Tregs by their functional inactivation rather than depletion. Tregs are characterized by the expression of the Forkhead box protein 3 (FOXP3) transcription factor, which is considered their “master regulator”. Its interaction with DNA is assisted primarily by its interaction with other proteins in the so-called “Foxp3 interactome”, which elicits much of the characteristic Treg cell transcriptional signature. We speculated that the disruption of such a protein complex by using synthetic peptides able to bind Foxp3 might have an impact on the functionality of Treg cells and thus have a therapeutic potential in cancer treatment. (2) Methods: By using a phage-displayed peptide library, or short synthetic peptides encompassing Foxp3 fragments, or by studying the crystal structure of the Foxp3:NFAT complex, we have identified a series of peptides that are able to bind Foxp3 and inhibit Treg activity. (3) Results: We identified some peptides encompassing fragments of the leuzin zipper or the C terminal domain of Foxp3 with the capacity to inhibit Treg activity in vitro. The acetylation/amidation of linear peptides, head-to-tail cyclization, the incorporation of non-natural aminoacids, or the incorporation of cell-penetrating peptide motifs increased in some cases the Foxp3 binding capacity and Treg inhibitory activity of the identified peptides. Some of them have shown antitumoral activity in vivo. (4) Conclusions: Synthetic peptides constitute an alternative to inhibit Foxp3 protein–protein interactions intracellularly and impair Treg immunosuppressive activity. These peptides might be considered as potential hit compounds on the design of new immunotherapeutic approaches against cancer.

The present study was aimed at determining the metabolic profile of PMNs in obese subjects, and to explore its potential relationship with insulin resistance (IR). To achieve this goal, a pilot clinical study was performed using PMNs from 17 patients with obesity and IR, and 17 lean controls without IR, which was validated in an additional smaller cohort (consisting of 10 patients and 10 controls). PMNs were isolated from peripheral blood and nuclear magnetic resonance was used to perform the metabolomic analysis. A total of 48 metabolites were quantified. The main metabolic change found in PMNs was a significant increase in 2-aminoisobutyric acid with a direct correlation with HOMA-IR (p

Prostate cancer (PCa) is one of the most frequently diagnosed cancers and a leading cause of death among men worldwide. Despite extensive efforts in biomarker discovery during the last years, currently used clinical biomarkers are still lacking enough specificity and sensitivity for PCa early detection, patient prognosis, and monitoring. Therefore, more precise biomarkers are required to improve the clinical management of PCa patients. In this context, metabolomics has shown to be a promising and powerful tool to identify novel PCa biomarkers in biofluids. Thus, changes in polyamines, tricarboxylic acid (TCA) cycle, amino acids, and fatty acids metabolism have been reported in different studies analyzing PCa patients’ biofluids. The review provides an up-to-date summary of the main metabolic alterations that have been described in biofluid-based studies of PCa patients, as well as a discussion regarding their potential to improve clinical PCa diagnosis and prognosis. Furthermore, a summary of the most significant findings reported in these studies and the connections and interactions between the different metabolic changes described has also been included, aiming to better describe the specific metabolic signature associated to PCa.

[This corrects the article DOI: 10.1371/journal.pone.0182985.].

Human peripheral blood cells are relevant ex vivo models for characterizing diseases and evaluating the pharmacological effects of therapeutic interventions, as they provide a close reflection of an individual pathophysiological state. In this work, a new approach to evaluate the impact of nanoparticles on the three main fractions of human peripheral blood cells by nuclear magnetic resonance spectroscopy is shown. Thus, a comprehensive protocol has been set-up including the separation of blood cells, their in vitro treatment with nanoparticles and the extraction and characterization of metabolites by nuclear magnetic resonance. This method was applied to assess the effect of gold nanoparticles, either coated with chitosan or supported on ceria, on peripheral blood cells from healthy individuals. A clear antioxidant effect was observed for chitosan-coated gold nanoparticles by a significant increase in reduced glutathione, that was much less pronounced for gold-cerium nanoparticles. In addition, the analysis revealed significant alterations of several other pathways, which were stronger for gold-cerium nanoparticles. These results are in accordance with the toxicological data previously reported for these materials, confirming the value of the current methodology.

Intrauterine growth restriction (IUGR) is a risk factor for abnormal neurodevelopment. We studied a rabbit model of IUGR by magnetic resonance imaging (MRI) and spectroscopy (MRS), to assess in vivo brain structural and metabolic consequences, and identify potential metabolic biomarkers for clinical translation.IUGR was induced in 3 pregnant rabbits at gestational day 25, by 40-50% uteroplacental vessel ligation in one horn; the contralateral horn was used as control. Fetuses were delivered at day 30 and weighted. A total of 6 controls and 5 IUGR pups underwent T2-w MRI and localized proton MRS within the first 8 hours of life, at 7T. Changes in brain tissue volumes and respective contributions to each MRS voxel were estimated by semi-automated registration of MRI images with a digital atlas of the rabbit brain. MRS data were used for: (i) absolute metabolite quantifications, using linear fitting; (ii) local temperature estimations, based on the water chemical shift; and (iii) classification, using spectral pattern analysis.Lower birth weight was associated with (i) smaller brain sizes, (ii) slightly lower brain temperatures, and (iii) differential metabolite profile changes in specific regions of the brain parenchyma. Specifically, we found estimated lower levels of aspartate and N-acetylaspartate (NAA) in the cerebral cortex and hippocampus (suggesting neuronal impairment), and higher glycine levels in the striatum (possible marker of brain injury). Our results also suggest that the metabolic changes in cortical regions are more prevalent than those detected in hippocampus and striatum.IUGR was associated with brain metabolic changes in vivo, which correlate well with the neurostructural changes and neurodevelopment problems described in IUGR. Metabolic parameters could constitute non invasive biomarkers for the diagnosis and abnormal neurodevelopment of perinatal origin.

Excessive apoptosis induces unwanted cell death and promotes pathological conditions. Drug discovery efforts aimed at decreasing apoptotic damage initially targeted the inhibition of effector caspases. Although such inhibitors were effective, safety problems led to slow pharmacological development. Therefore, apoptosis inhibition is still considered an unmet medical need.The interaction between Apaf-1 and the inhibitors was confirmed by NMR. Target specificity was evaluated in cellular models by siRNa based approaches. Cell recovery was confirmed by MTT, clonogenicity and flow cytometry assays. The efficiency of the compounds as antiapoptotic agents was tested in cellular and in vivo models of protection upon cisplatin induced ototoxicity in a zebrafish model and from hypoxia and reperfusion kidney damage in a rat model of hot ischemia.Apaf-1 inhibitors decreased Cytc release and apoptosome-mediated activation of procaspase-9 preventing cell and tissue damage in ex vivo experiments and in vivo animal models of apoptotic damage. Our results provide evidence that Apaf-1 pharmacological inhibition has therapeutic potential for the treatment of apoptosis-related diseases.

ObjectivesTo characterize via NMR spectroscopy the full spectrum of metabolic changes in umbilical vein blood plasma of newborns diagnosed with different clinical forms of intrauterine growth restriction (IUGR).Methods23 early IUGR cases and matched 23 adequate-for-gestational-age (AGA) controls and 56 late IUGR cases with 56 matched AGAs were included in this study. Early IUGR was defined as a birth weight 35 weeks. This group was subdivided in 18 vasodilated (VD) and 38 non-VD late IUGR fetuses. All AGA patients had a birth weight >10(th) centile. (1)H nuclear magnetic resonance (NMR) metabolomics of the blood samples collected from the umbilical vein at delivery was obtained. Multivariate statistical analysis identified several metabolites that allowed the discrimination between the different IUGR subgroups, and their comparative levels were quantified from the NMR data.ResultsThe NMR-based analysis showed increased unsaturated lipids and VLDL levels in both early and late IUGR samples, decreased glucose and increased acetone levels in early IUGR. Non-significant trends for decreased glucose and increased acetone levels were present in late IUGR, which followed a severity gradient when the VD and non-VD subgroups were considered. Regarding amino acids and derivatives, early IUGR showed significantly increased glutamine and creatine levels, whereas the amounts of phenylalanine and tyrosine were decreased in early and late-VD IUGR samples. Valine and leucine were decreased in late IUGR samples. Choline levels were decreased in all clinical subforms of IUGR.ConclusionsIUGR is not associated with a unique metabolic profile, but important changes are present in different clinical subsets used in research and clinical practice. These results may help in characterizing comprehensively specific alterations underlying different IUGR subsets.

BACKGROUND: Cell culture media conditioned by human foreskin fibroblasts (HFFs) provide a complex supplement of protein and metabolic factors that support in vitro proliferation of human embryonic stem cells (hESCs). However, the conditioning process is variable with different media batches often exhibiting differing capacities to maintain hESCs in culture. While recent studies have examined the protein complement of conditioned culture media, detailed information regarding the metabolic component of this media is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Using a (1)H-Nuclear Magnetic Resonance ((1)H-NMR) metabonomics approach, 32 metabolites and small compounds were identified and quantified in media conditioned by passage 11 HFFs (CMp11). A number of metabolites were secreted by HFFs with significantly higher concentration of lactate, alanine, and formate detected in CMp11 compared to non-conditioned media. In contrast, levels of tryptophan, folate and niacinamide were depleted in CMp11 indicating the utilisation of these metabolites by HFFs. Multivariate statistical analysis of the (1)H-NMR data revealed marked age-related differences in the metabolic profile of CMp11 collected from HFFs every 24 h over 72 h. Additionally, the metabolic profile of CMp11 was altered following freezing at -20°C for 2 weeks. CM derived from passage 18 HFFs (CMp18) was found to be ineffective at supporting hESCs in an undifferentiated state beyond 5 days culture. Multivariate statistical comparison of CMp11 and CMp18 metabolic profiles enabled rapid and clear discrimination between the two media with CMp18 containing lower concentrations of lactate and alanine as well as higher concentrations of glucose and glutamine. CONCLUSIONS/SIGNIFICANCE: (1)H-NMR-based metabonomics offers a rapid and accurate method of characterising hESC conditioning media and is a valuable tool for monitoring, controlling and optimising hESC culture media preparation.

Conventional patent-based drug development incentives work badly for the developing world, where commercial markets are usually small to non-existent. For this reason, the past decade has seen extensive experimentation with alternative R&D institutions ranging from private-public partnerships to development prizes. Despite extensive discussion, however, one of the most promising avenues-open source drug discovery-has remained elusive. We argue that the stumbling block has been the absence of a critical mass of preexisting work that volunteers can improve through a series of granular contributions. Historically, open source software collaborations have almost never succeeded without such "kernels".HERE, WE USE A COMPUTATIONAL PIPELINE FOR: (i) comparative structure modeling of target proteins, (ii) predicting the localization of ligand binding sites on their surfaces, and (iii) assessing the similarity of the predicted ligands to known drugs. Our kernel currently contains 143 and 297 protein targets from ten pathogen genomes that are predicted to bind a known drug or a molecule similar to a known drug, respectively. The kernel provides a source of potential drug targets and drug candidates around which an online open source community can nucleate. Using NMR spectroscopy, we have experimentally tested our predictions for two of these targets, confirming one and invalidating the other.The TDI kernel, which is being offered under the Creative Commons attribution share-alike license for free and unrestricted use, can be accessed on the World Wide Web at http://www.tropicaldisease.org. We hope that the kernel will facilitate collaborative efforts towards the discovery of new drugs against parasites that cause tropical diseases.