Metabolomics analysis reveals novel serum metabolite alterations in cancer cachexia.

Background: Cachexia is a body wasting syndrome that significantly affects well-being and prognosis of cancer patients, without effective treatment. Serum metabolites take part in pathophysiological processes of cancer cachexia, but apart from altered levels of select serum metabolites, little is known on the global changes of the overall serum metabolome, which represents a functional readout of the whole-body metabolic state. Here, we aimed to comprehensively characterize serum metabolite alterations and analyze associated pathways in cachectic cancer patients to gain new insights that could help instruct strategies for novel interventions of greater clinical benefit. Methods: Serum was sampled from 120 metastatic cancer patients (stage UICC IV). Patients were grouped as cachectic or non-cachectic according to the criteria for cancer cachexia agreed upon international consensus (main criterium: weight loss adjusted to body mass index). Samples were pooled by cachexia phenotype and assayed using non-targeted gas chromatography-mass spectrometry (GC-MS). Normalized metabolite levels were compared using t-test (p < 0.05, adjusted for false discovery rate) and partial least squares discriminant analysis (PLS-DA). Machine-learning models were applied to identify metabolite signatures for separating cachexia states. Significant metabolites underwent MetaboAnalyst 5.0 pathway analysis. Results: Comparative analyses included 78 cachectic and 42 non-cachectic patients. Cachectic patients exhibited 19 annotable, significantly elevated (including glucose and fructose) or decreased (mostly amino acids) metabolites associating with aminoacyl-tRNA, glutathione and amino acid metabolism pathways. PLS-DA showed distinct clusters (accuracy: 85.6%), and machine-learning models identified metabolic signatures for separating cachectic states (accuracy: 83.2%; area under ROC: 88.0%). We newly identified altered blood levels of erythronic acid and glucuronic acid in human cancer cachexia, potentially linked to pentose-phosphate and detoxification pathways. Conclusion: We found both known and yet unknown serum metabolite and metabolic pathway alterations in cachectic cancer patients that collectively support a whole-body metabolic state with impaired detoxification capability, altered glucose and fructose metabolism, and substrate supply for increased and/or distinct metabolic needs of cachexia-associated tumors. These findings together imply vulnerabilities, dependencies and targets for novel interventions that have potential to make a significant impact on future research in an important field of cancer patient care.

Pyruvate dehydrogenase fuels a critical citrate pool that is essential for Th17 cell effector functions.

Pyruvate dehydrogenase (PDH) is the central enzyme connecting glycolysis and the tricarboxylic acid (TCA) cycle. The importance of PDH function in T helper 17 (Th17) cells still remains to be studied. Here, we show that PDH is essential for the generation of a glucose-derived citrate pool needed for Th17 cell proliferation, survival, and effector function. In vivo, mice harboring a T cell-specific deletion of PDH are less susceptible to developing experimental autoimmune encephalomyelitis. Mechanistically, the absence of PDH in Th17 cells increases glutaminolysis, glycolysis, and lipid uptake in a mammalian target of rapamycin (mTOR)-dependent manner. However, cellular citrate remains critically low in mutant Th17 cells, which interferes with oxidative phosphorylation (OXPHOS), lipid synthesis, and histone acetylation, crucial for transcription of Th17 signature genes. Increasing cellular citrate in PDH-deficient Th17 cells restores their metabolism and function, identifying a metabolic feedback loop within the central carbon metabolism that may offer possibilities for therapeutically targeting Th17 cell-driven autoimmunity.

Plasma Metabolome Alterations Discriminate between COVID-19 and Non-COVID-19 Pneumonia.

Pneumonia is a common cause of morbidity and mortality and is most often caused by bacterial pathogens. COVID-19 is characterized by lung infection with potential progressive organ failure. The systemic consequences of both disease on the systemic blood metabolome are not fully understood. The aim of this study was to compare the blood metabolome of both diseases and we hypothesize that plasma metabolomics may help to identify the systemic effects of these diseases. Therefore, we profiled the plasma metabolome of 43 cases of COVID-19 pneumonia, 23 cases of non-COVID-19 pneumonia, and 26 controls using a non-targeted approach. Metabolic alterations differentiating the three groups were detected, with specific metabolic changes distinguishing the two types of pneumonia groups. A comparison of venous and arterial blood plasma samples from the same subjects revealed the distinct metabolic effects of pulmonary pneumonia. In addition, a machine learning signature of four metabolites was predictive of the disease outcome of COVID-19 subjects with an area under the curve (AUC) of 86 ± 10 %. Overall, the results of this study uncover systemic metabolic changes that could be linked to the etiology of COVID-19 pneumonia and non-COVID-19 pneumonia.

BCAT1 redox function maintains mitotic fidelity.

The metabolic enzyme branched-chain amino acid transaminase 1 (BCAT1) drives cell proliferation in aggressive cancers such as glioblastoma. Here, we show that BCAT1 localizes to mitotic structures and has a non-metabolic function as a mitotic regulator. Furthermore, BCAT1 is required for chromosome segregation in cancer and induced pluripotent stem cells and tumor growth in human cerebral organoid and mouse syngraft models. Applying gene knockout and rescue strategies, we show that the BCAT1 CXXC redox motif is crucial for controlling cysteine sulfenylation specifically in mitotic cells, promoting Aurora kinase B localization to centromeres, and securing accurate chromosome segregation. These findings offer an explanation for the well-established role of BCAT1 in promoting cancer cell proliferation. In summary, our data establish BCAT1 as a component of the mitotic apparatus that safeguards mitotic fidelity through a moonlighting redox functionality.

Complexity of subcellular metabolism: strategies for compartment-specific profiling.

Subcellular compartmentalization provides cells with tremendous advantages for the operation of cellular metabolism. Spatial separation of metabolism generates microenvironments with distinct concentrations of metabolites and cofactors allowing the cell to execute otherwise thermodynamically exclusive reactions simultaneously. Moreover, compartmentalization is also involved in the fine-tuned regulation of gene expression. Thus, the elucidation of compartment-specific metabolic processes has become essential for a thorough understanding of cellular metabolism. The limited availability of reliable methods to inspect metabolic activities in subcellular compartments has been a major impediment in this field. During the last decade, progress has been greatly extended through methodological advances in subcellular metabolic profiling. This review summarizes recent strategies applied for the direct and indirect assessment of compartment-specific metabolism. We review contemporary techniques, recent advances, limitations, and future trends in the field of subcellular metabolomics.

SiMeEx, a simplified method for metabolite extraction of adherent mammalian cells.

A reliable method for metabolite extraction is central to mass spectrometry-based metabolomics. However, existing methods are lengthy, mostly due to the step of scraping cells from cell culture vessels, which restricts metabolomics in broader application such as lower cell numbers and high-throughput studies. Here, we present a simplified metabolite extraction (SiMeEx) method, to efficiently and quickly extract metabolites from adherent mammalian cells. Our method excludes the cell scraping step and therefore allows for a more efficient extraction of polar metabolites in less than 30 min per 12-well plate. We demonstrate that SiMeEx achieves the same metabolite recovery as using a standard method containing a scraping step, in various immortalized and primary cells. Omitting cell scraping does not compromise the performance of non-targeted and targeted GC-MS analysis, but enables metabolome analysis of cell culture on smaller well sizes down to 96-well plates. Therefore, SiMeEx demonstrates advantages not only on time and resources, but also on the applicability in high-throughput studies.

Lipidomics investigations into the tissue phospholipidomic landscape of invasive ductal carcinoma of the breast.

The need of identifying alternative therapeutic targets for invasive ductal carcinoma (IDC) of the breast with high specificity and sensitivity for effective therapeutic intervention is crucial for lowering the risk of fatality. Lipidomics has emerged as a key area for the discovery of potential candidates owing to its several shared pathways between cancer cell proliferation and survival. In the current study, we performed comparative phospholipidomic analysis of IDC, benign and control tissue samples of the breast to identify the significant lipid alterations associated with malignant transformation. A total of 33 each age-matched tissue samples from malignant, benign and control were analyzed to identify the altered phospholipids by using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM/MS). A combination of univariate and multivariate statistical approaches was used to select the phospholipid species with the highest contribution in group segregation. Furthermore, these altered phospholipids were structurally confirmed by tandem mass spectrometry. A total of 244 phospholipids were detected consistently at quantifiable levels, out of which 32 were significantly altered in IDC of the breast. Moreover, in pairwise comparison of IDC against benign and control samples, 11 phospholipids were found to be significantly differentially expressed. Particularly, LPI 20:3, PE (22:1/22:2), LPE 20:0 and PC (20:4/22:4) were observed to be most significantly associated with IDC tissue samples. Apart from that, we also identified that long-chain unsaturated fatty acids were enriched in the IDC tissue samples as compared to benign and control samples, indicating its possible association with the invasive phenotype.

Rewiring of Metabolic Network in Mycobacterium tuberculosis During Adaptation to Different Stresses.

Metabolic adaptation of Mycobacterium tuberculosis (M. tuberculosis) to microbicidal intracellular environment of host macrophages is fundamental to its pathogenicity. However, an in-depth understanding of metabolic adjustments through key reaction pathways and networks is limited. To understand how such changes occur, we measured the cellular metabolome of M. tuberculosis subjected to four microbicidal stresses using liquid chromatography-mass spectrometric multiple reactions monitoring (LC-MRM/MS). Overall, 87 metabolites were identified. The metabolites best describing the separation between stresses were identified through multivariate analysis. The coupling of the metabolite measurements with existing genome-scale metabolic model, and using constraint-based simulation led to several new concepts and unreported observations in M. tuberculosis; such as (i) the high levels of released ammonia as an adaptive response to acidic stress was due to increased flux through L-asparaginase rather than urease activity; (ii) nutrient starvation-induced anaplerotic pathway for generation of TCA intermediates from phosphoenolpyruvate using phosphoenolpyruvate kinase; (iii) quenching of protons through GABA shunt pathway or sugar alcohols as possible mechanisms of early adaptation to acidic and oxidative stresses; and (iv) usage of alternate cofactors by the same enzyme as a possible mechanism of rewiring metabolic pathways to overcome stresses. Besides providing new leads and important nodes that can be used for designing intervention strategies, the study advocates the strength of applying flux balance analyses coupled with metabolomics to get a global picture of complex metabolic adjustments.

Serum metabolomic alterations in multiple myeloma revealed by targeted and untargeted metabolomics approaches: a pilot study.

Multiple myeloma (MM) is the second most prevalent hematological malignancy characterized by rapid proliferation of plasma cells, which leads to overproduction of antibodies. MM affects around 15% of all hemato-oncology cases across the world. The present study involves identification of metabolomic alterations in the serum of an MM cohort compared to healthy controls using both LC-MRM/MS based targeted and GC-MS based untargeted approaches. Several MM specific serum metabolomic signatures were observed in this study. A total of 54 metabolites were identified as being significantly altered in MM cohort, out of which, 26 metabolites were identified from LC-MRM/MS based targeted analysis, whereas 28 metabolites were identified from the GC-MS based untargeted analysis. Receiver operating characteristic (ROC) curve analysis demonstrated that six metabolites each from both the datasets can be projected as marker metabolites to discriminate MM subjects with higher specificity and sensitivity. Moreover, pathway analysis deciphered that several metabolic pathways were altered in MM including pyrimidine metabolism, purine metabolism, amino acid metabolism, nitrogen metabolism, sulfur metabolism, and the citrate cycle. Comprehensively, this study contributes valuable information regarding MM induced serum metabolite alterations and their pathways, which could offer further insights into this cancer.

Current Understanding of the Potential of Proteomics and Metabolomics Approaches in Cancer Chemoresistance: A Focus on Multiple Myeloma.

Chemoresistance is one of the major hurdles in cancer treatment leading to recurrence of cancer and affects the overall survival of patients. Cancer chemoresistance can be associated with various phenomena including modulation of vital cellular pathways. Unrevealing these alterations could provide a better understanding of chemoresistance and assist in the identification of new targets to overcome it. Recent advances in the field of proteomics and metabolomics have substantially helped in the identification of potential targets for chemoresistance in various cancers. This review highlights the potential of proteomics and metabolomics research to explore the putative targets associated with cancer chemoresistance with a special focus on Multiple Myeloma (MM). MM is a type of hematological malignancy which constitutes about 13% of all blood cell cancers. The therapeutic advancements for MM have increased the median overall survival rate to over 3-fold in the last one and half decade. Although in recent times, significant improvements in the overall survival rate of MM are achieved, MM remains an incurable disease with unpredictable refractory mechanisms. In spite of therapeutic advances, chemoresistance thrives to be a major hurdle in the treatment of multiple myeloma which demands a better understanding of chemoresistance. In this review, we have attempted to highlight the potential applications of proteomics and metabolomics research in the understanding of chemoresistance in MM.

Metabolomic alterations in invasive ductal carcinoma of breast: A comprehensive metabolomic study using tissue and serum samples.

Invasive ductal carcinoma (IDC) is the most common type of breast cancer and the leading cause of breast cancer related mortality. In the present study, metabolomic profiles of 72 tissue samples and 146 serum samples were analysed using targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM/MS) and untargeted gas chromatography mass spectrometry (GC-MS) approaches. Combination of univariate and multivariate statistical treatment identified significant alterations of 42 and 32 metabolites in tissue and serum samples of IDC, respectively when compared to control. Some of the metabolite changes from tissue were also reflected in serum, indicating a bi-directional interaction of metabolites in IDC. Additionally, 8 tissue metabolites and 9 serum metabolites showed progressive change from control to benign to IDC suggesting their possible role in malignant transformation. We have identified a panel of three metabolites viz. tryptophan, tyrosine, and creatine in tissue and serum, which could be useful in screening of IDC subjects from both control and benign. The metabolomic alterations in IDC showed perturbations in purine and pyrimidine metabolism, amino sugar metabolism, amino acid metabolism, fatty acid biosynthesis etc. Comprehensively, this study provides valuable insights into metabolic adaptations of IDC, which can help to identify diagnostic markers as well as potential therapeutic targets.

A non-invasive approach to explore the discriminatory potential of the urinary volatilome of invasive ductal carcinoma of the breast.

Worldwide, breast invasive ductal carcinoma (IDC) accounts for the majority of the reported cases of this form of cancer. IDC effective management, as for any form of cancer, would greatly benefit from early diagnosis. This, however, due to various socio-economic reasons, is very far for the reality in developing countries like India, where cancer diagnosis is often carried out at late stages when disease management is troublesome. With the present work, we aim to evaluate a simple analytical methodology to identify a set of volatile organic compounds (VOCs) in urine samples, as a biosignature for IDC. Using solid-phase microextraction followed by gas chromatography/mass spectrometry, a panel of 14 urinary VOCs was found to discriminate IDC (n = 65) from a healthy control (HC) group (n = 70) through multivariate statistical treatments. Furthermore, metabolic pathway analysis revealed various dysregulated pathways involved in IDC patients hinting that their detailed investigations could lead to novel mechanistic insights into the disease pathophysiology. In addition, we validated the expression pattern of five of these VOCs namely 2-ethyl-1-hexanol, isolongifolenone, furan, dodecanoic acid, 2-methoxy-phenol in another external cohort of 59 urinary samples (IDC = 32 and HC = 27) and found their expression pattern to be consistent with the primary sample set. To our knowledge, this is the first study exploring breast IDC volatome alterations in Indian patients.

Investigation of altered urinary metabolomic profiles of invasive ductal carcinoma of breast using targeted and untargeted approaches.

INTRODUCTION: Invasive ductal carcinoma (IDC) is a type of breast cancer, usually detected in advanced stages due to its asymptomatic nature which ultimately leads to low survival rate. Identification of urinary metabolic adaptations induced by IDC to understand the disease pathophysiology and monitor therapy response would be a helpful approach in clinical settings. Moreover, its non-invasive and cost effective strategy better suited to minimize apprehension among high risk population. OBJECTIVE: This study aims toward investigating the urinary metabolic alterations of IDC by targeted (LC-MRM/MS) and untargeted (GC-MS) approaches for the better understanding of the disease pathophysiology and monitoring therapy response. METHODS: Urinary metabolic alterations of IDC subjects (63) and control subjects (63) were explored by targeted (LC-MRM/MS) and untargeted (GC-MS) approaches. IDC specific urinary metabolomics signature was extracted by applying both univariate and multivariate statistical tools. RESULTS: Statistical analysis identified 39 urinary metabolites with the highest contribution to metabolomic alterations specific to IDC. Out of which, 19 metabolites were identified from targeted LC-MRM/MS analysis, while 20 were identified from the untargeted GC-MS analysis. Receiver operator characteristic (ROC) curve analysis evidenced 6 most discriminatory metabolites from each type of approach that could differentiate between IDC subjects and controls with higher sensitivity and specificity. Furthermore, metabolic pathway analysis depicted several dysregulated pathways in IDC including sugar, amino acid, nucleotide metabolism, TCA cycle etc. CONCLUSIONS: Overall, this study provides valuable inputs regarding altered urinary metabolites which improved our knowledge on urinary metabolomic alterations induced by IDC. Moreover, this study identified several dysregulated metabolic pathways which offer further insight into the disease pathophysiology.

The expression of proteins involved in digestion and detoxification are regulated in Helicoverpa armigera to cope up with chlorpyrifos insecticide.

Helicoverpa armigera is a key pest in many vital crops, which is mainly controlled by chemical strategies. To manage this pest is becoming challenging due to its ability and evolution of resistance against insecticides. Further, its subsequent spread on nonhost plant is remarkable in recent times. Hence, decoding resistance mechanism against phytochemicals and synthetic insecticides is a major challenge. The present work describes that the digestion, defense and immunity related enzymes are associated with chlorpyrifos resistance in H. armigera. Proteomic analysis of H. armigera gut tissue upon feeding on chlorpyrifos containing diet (CH) and artificial diet (AD) using nano-liquid chromatography-mass spectrometry identified upregulated 23-proteins in CH fed larvae. Database searches combined with gene ontology analysis revealed that the identified gut proteins engrossed in digestion, proteins crucial for immunity, adaptive responses to stress, and detoxification. Biochemical and quantitative real-time polymerase chain reaction analysis of candidate proteins indicated that insects were struggling to get nutrients and energy in presence of CH, while at the same time endeavoring to metabolize chlorpyrifos. Moreover, we proposed a potential processing pathway of chlorpyrifos in H. armigera gut by examining the metabolites using gas chromatography-mass spectrometry. H. armigera exhibit a range of intriguing behavioral, morphological adaptations and resistance to insecticides by regulating expression of proteins involved in digestion and detoxification mechanisms to cope up with chlorpyrifos. In these contexts, as gut is a rich repository of biological information; profound analysis of gut tissues can give clues of detoxification and resistance mechanism in insects.