Richer than previously probed: An application of 1H NMR reveals one hundred metabolites using only fifty microliter serum.

The classical approach restricts the detection of metabolites in serum samples by using nuclear magnetic resonance (NMR) spectroscopy; however, the presence of copious proteins and lipoproteins emphasize and necessitate the development of a contemporary, high-throughput tactic. To eliminate the lipoproteins and proteins from sera to engender filtered sera (FS), the study was executed with 50 µl serum obtained from five healthy individuals with 5 years of age difference from 25 to 45 years old and the application of a unique mechanical filter with molecular weight cut-off value of 2KDa. The 10 µl FS from each individual was pooled to make 50 µl final volume filled in a co-axial tube for acquisition of a battery of 1D/2D investigations at 800 MHz NMR spectrometer and the assigned metabolites was confirmed through mass spectrometry as well as by comparing 1H NMR spectra of individual metabolites. This innovative tactic is commissioning to reveal more than 100 metabolites. In contrast to the protein precipitation method, 24 new metabolites were recognized in the present study. The present innovative approach characterizes nucleosides, nitrogenous bases, and volatile metabolites that possibly produce a landmark for the delineation of a comprehensive metabolic profile applicable for detection of the molecular cause of pathogenicity, early-stage disease detection and prognosis, inborn error of metabolism, and forensic investigations exerting the least volume of FS and NMR spectroscopy. The assignment of 100 metabolites using 1H NMR-based FS is described for the first time in the present report.

Evaluation of prostate cancer tissue metabolomics: would clinics utilise it for diagnosis?

The difficulty of diagnosing prostate cancer (PC) with the available biomarkers frequently leads to over-diagnosis and overtreatment of PC, underscoring the need for novel molecular signatures. The purpose of this review is to provide a summary of the currently available cellular metabolomics for PC molecular signatures. A comprehensive search on PubMed was conducted to find studies published between January 2004 and August 2022 that reported biomarkers for PC detection, development, aggressiveness, recurrence and treatment response. Although potential studies have reported the presence of distinguishing molecules that can distinguish between benign and cancerous prostate tissue. However, there are few studies looking into signature molecules linked to disease development, therapy response or tumour recurrence. The majority of these studies use high-dimensional datasets, and the number of potential metabolites investigated frequently exceeds the size of the available samples. In light of this, pre-analytical, statistical, methodological and confounding factors such as antiandrogen therapy (NAT) may also be linked to the identified chemometric multivariate differences between PC and relevant control samples in the datasets. Despite the methodological and procedural challenges, a range of methodological groups and processes have consistently identified a number of signature metabolites and pathways that appear to imply a substantial involvement in the cellular metabolomics of PC for tumour formation and recurrence.

Relevance of emerging metabolomics-based biomarkers of prostate cancer: a systematic review.

Prostate cancer (PC) presents great challenges in early diagnosis and often leads to unnecessary invasive procedures as well as over diagnosis and treatment, thus highlighting the need for promising early diagnostic biomarkers. The aim of this review is to provide an up-to-date summary of chronologically existing metabolomics PC biomarkers, their potential to improve clinical PC diagnosis and to reduce the proliferation and monitoring of PC. The systematic research was conducted on PubMed in accordance with PRISMA guidelines to report PC biomarkers. The majority of the studies distinguished malignant from benign prostate and few explored the biomarkers associated with the progression of PC. The present review summarises the primary outcomes of most significant studies to extend our knowledge of PC metabolomics biomarkers. We observed divergent inter-laboratory technical procedures employing different statistical approaches produced abundant information regarding PC metabolites perturbation. Since PC metabolomics is still in its early phase, it is vital that we dig out the most specific, sensitive and accurate metabolic signatures and conduct more studies with milestone findings with comparable sample sizes to validate and corroborate the findings.

Metabolomics of prostate cancer: Knock-in versus knock-out prostate.

Several metabolomics-derived biomarkers of prostate cancer (PC) have been reported with pre-radical prostatectomy (RP) (knock-in PC) conditions; however, uncontested PC biomarkers panel appraisal and investigation of correlative evidence of these measures is lacking through post-RP (knock-out PC). We sought to explore patients' filtered serum-based metabolomics derived signature measures in knock-in PC (n = 90) using nuclear magnetic resonance spectroscopy and multiple rigorous statistical analyses, and to develop the correlative evidence of these measures through knock-out PC (n = 90) follow-up on the 15th and 30th days. The glutamate, citrate and glycine were observed as hallmarks of PC. Observed trends revealed; augmented glutamate level in knock-in PC following a sudden drop and subsequently upside of glutamate at 15th and 30th days of knock-out PC, reduction of citrate in knock-in PC subsequently gradual increase of citrate in knock-out PC, and glycine lessening in knock-in PC following augmentation on 30th day of knock-out PC. This study-based evidence clears the doubts regarding the discovery of metabolomics-derived PC biomarkers.

NMR-derived targeted serum metabolic biomarkers appraisal of bladder cancer: A pre- and post-operative evaluation.

With high morbidity and mortality, urinary bladder cancer (BC) ranks fifth among common cancers globally. The inherent limitations of urine cytology and cystoscopy, and marginal enhancements in the rate of survival promt us to develop surrogate serum based metabolic biomarkers of screening, identification, and follow-up protocols of management for BC patients. Earlier, we exhibited that abnormal expression levels of dimethylamine (DMA), malonate, lactate, glutamine, histidine, and valine in serum may be used as signature metabolites to differentiate BC from healthy controls (HC) (J. Proteome Res. 2013; 12(12):5839-50). Here we further gauge and validate these observations by comparing pre-operative to post-operative follow-up BC patients. This study was conducted on 160 sera samples involving HC (n = 52), pre-operative (n = 55) and post-operative (n = 53) BC cases. 1H nuclear magnetic resonance (NMR) spectroscopy was used to generate serum metabolic profiles and to gauge aberrantly expressed metabolites. The targeted metabolomic approach revealed that the expression levels of these signature metabolites were progressively and significantly decreased in post-operative follow-up at the interval of 30, 60, and 90 days compared to pre-operative BC sera samples and were maintained at HC levels. Serum metabolic biomarkers appear to be an inspiring and least-invasive tactic for detection and prognosticating BC patient follow-up.

Role of metabolomics-derived biomarkers to identify renal cell carcinoma: a comprehensive perspective of the past ten years and advancements.

Introduction: Renal cell carcinoma (RCC) is one of the most prevalent metabolic diseases and a leading cause of utmost mortality among men globally. In spite of extensive development in technology for biomarker discovery during the last 10 years, the currently used clinical biomarkers are still unable to detect RCC at early and progression stages. Hence, the development of new and precise biomarkers is most important to improve the clinical management of RCC and reduce the level of mortality.Area covered: For the detection of RCC at an early stage; a new branch of omics technology - metabolomics - has been introduced. Mainly two techniques (mass spectrometry and nuclear magnetic resonance spectroscopy) have been exploited to execute metabolomics. Precisely, metabolomics showed promising and powerful approach to identify novel RCC biomarker. The present review discussed and the literature search to narrate the outcomes of the past 10 years of studies related to RCC pathophysiology, metabolomics, advancements, and shortcomings.Expert opinion: Although, compared to mass spectrometric tactic, nuclear magnetic resonance is moving fast to achieve the aim and in vivo application for diagnosis and management of RCC, metabolomics-based research in RCC is still in its infancy stage.

Relevance of MIC-1 in the Era of PSA as a Serum Based Predictor of Prostate Cancer: A Critical Evaluation.

To reduce the ambiguity of contradictory observations in different studies regarding the expression level of Macrophage Inhibitory Cytokine-1 (MIC-1) in serum in prostate cancer (PC), benign prostatic hyperplasia (BPH) and healthy controls (HC), we designed this double-blind study. The study comprises 240 sera from PC, BPH and HC subjects. The expression level of MIC-1 in PC, BPH and HC were appraised using Western blot (WB) and ELISA based approach. WB and ELISA appraisal reveals that the expression level of MIC-1 is significantly higher in PC than in HC or BPH subjects. Regression analysis revealed a significant correlation between MIC-1 vs. PSA (r = 0.09; p < 0.001) and MIC-1 vs. GS (r = 0.7; p < 0.001). ROC analysis using discriminant predicted probability revealed that the MIC-1 was better than PSA. Moreover, the combination of MIC-1 and PSA was allowing 99.1% AUC for the differentiation of BPH + PC from HC, 97.9% AUC for differentiation of BPH from HC, 98.6% AUC for differentiation of PC from HC, and 96.7% AUC for the differentiation of PC from BPH. The augmented expression of MIC-1 in PC compared to BPH and HC subjects is in concurrent of the over-expression of MIC-1 in PC reports and confiscates the contradictory findings of other studies.

Serum-based protein biomarkers of bladder cancer: A pre- and post-operative evaluation.

Urinary bladder cancer (BC) is the fifth most common cancer worldwide with alarming mortality. Shortcomings of urine cytology and cystoscopy and sparse improvements in the survival rate prompt us to evolve surrogate serum based protein biomarkers to identify BC at an early stage. Previously, we showed that aberrant expression of S100A4, S100A8, S100A9, carbonic anhydrase I (CA I) and Annexin V proteins in pre-operative BC serum compared to healthy controls (HC) (Clin Chim Acta, 2014; 36: 97-103). Here we further evaluate and validate these findings with follow-up post-operative BC patients. This study was conducted on 160 sera samples comprising healthy controls (HC, n=52), pre-operative (n=55) and post-operative (n=53) BC patients. Enzyme-linked immunosorbent assay (ELISA) was used to appraise the aberrantly expressed proteins. ELISA results revealed that the expression levels of S100A8, S100A9, S100A4, and CA I were gradually and significantly reduced; concomitantly, Annexin V was progressively and significantly increased in post-operative compared to pre-operative BC sera samples. Serum protein biomarkers appear to be an encouraging and least-invasive approach for BC identification and prognosticating patient outcomes.

Proteometabolomics of bladder cancer: current and future prospects.

Urinary bladder cancer (BC) is fifth most common cancer worldwide; the diagnostic methods are mostly instrumental approaches including cystoscopy and cytology. Since BC recurrence rate is high, consequently requires long-term follow-up. The molecular assays that can precisely identify BC at an early stage are obligatory. Although several noninvasive urine and blood samples based biomarkers have been proposed in the last decade but only few have been approved by Food and drug administration (FDA) for clinical purpose. Hence the search for more suitable biomarker is still on. In this review, we summarize the urine and blood based metabolic and protein tests not only for determination but also BC patient surveillance.

Low- and high-grade bladder cancer appraisal via serum-based proteomics approach.

OBJECTIVE: To address the shortcomings of urine cytology and cystoscopy for screening and grading of urinary bladder cancer (BC) we applied a serum-based proteomics approach as a surrogate tactic for rapid BC probing. METHODS: This study was performed on 90 sera samples comprising of low-grade (LG, n=33) and high-grade (HG, n=32) BC, and healthy controls (HC, n=25). Two-dimensional gel electrophoresis (2DE) tactic was executed to describe serum proteome. MALDI-TOF-MS (MS) was used to identify the characteristics of aberrantly expressed proteins in 2DE and validated using Western blot (WB) and ELISA approach. Receiver operating characteristics (ROC) curve analysis was also performed to determine the clinical usefulness of these proteins to discriminate among LG, HG and HC cohorts. RESULTS: This comprehensive approach of 2DE, MS, WB and ELISA reveals five differentially expressed proteins. Among them two biomarkers (S100A8 and S100A9) were able to accurately (ROC, 0.946) distinguish 81% of BC (LG+HG) cases compared to HC with highest sensitivity and specificity. With a comparable tactic, two biomarkers (S100A8 and S100A4) were able to precisely (ROC, 0.941) discriminate 92% of LG cases from HG with utmost sensitivity and specificity. CONCLUSIONS: Serum proteomics probing appears to be an encouraging and least-invasive tactic for screening and grading of BC.

Low- and high-grade bladder cancer determination via human serum-based metabolomics approach.

To address the shortcomings of urine cytology and cystoscopy for probing and grading urinary bladder cancer (BC), we applied (1)H nuclear magnetic resonance (NMR) spectroscopy as a surrogate method for the identification of BC. This study includes 99 serum samples comprising low-grade (LG; n = 36) and high-grade (HG; n = 31) BC as well as healthy controls (HC; n = 32). (1)H NMR-derived serum data were analyzed using orthogonal partial least-squares discriminant analysis (OPLS-DA). OPLS-DA-derived model validity was confirmed using an internal and external cross-validation. Internal validation was performed using the initial samples (n = 99) data set. External validation was performed on a new batch of suspected BC patients (n = 106) through a double-blind study. Receiver operating characteristic (ROC) curve analysis was also performed. OPLS-DA-derived serum metabolomics (six biomarkers, ROC; 0.99) were able to discriminate 95% of BC cases with 96% sensitivity and 94% specificity when compared to HC. Likewise (three biomarkers, ROC; 0.99), 98% of cases of LG were able to differentiate from HG with 97% sensitivity and 99% specificity. External validation reveals comparable results to the internal validation. (1)H NMR-based serum metabolic screening appears to be a promising and less invasive approach for probing and grading BC in contrast to the highly invasive and painful cystoscopic approach for BC detection.

Efficacy of Withania somnifera on seminal plasma metabolites of infertile males: a proton NMR study at 800 MHz.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Indian systems of medicine use roots of Withania somnifera for impotence, infertility treatment, stress, and the aging process. Although Withania somnifera improves semen quality by regulating reproductive hormone levels and oxidative stress, the molecular mechanism is not clear. AIM OF THE STUDY: Our study uses high-resolution Nuclear Magnetic Resonance (NMR) spectroscopy to explore the scientific basis to reveal the pre- and post-treatment efficacy of Withania somnifera on seminal plasma of infertile men-which remains unexplored to date. MATERIALS AND METHODS: A total of 180 infertile male patients were administered Withania somnifera root powder at the rate of 5 g/d for a 3-month period. The study included age-matched, healthy men as a control (n=50) group. Proton NMR spectroscopy was used to measure lactate, alanine, glutamate, glutamine, citrate, lysine, choline, glycerophosphocholine (GPC), glycine, tyrosine, histidine, phenylalanine, and uridine in all seminal plasma samples. To appraise infertility levels, we also measured sperm concentration, motility, lipid peroxide, and hormonal perturbation. RESULTS: Withania somnifera therapy repairs the disturbed concentrations of lactate, alanine, citrate, GPC, histidine, and phenylalanine in seminal plasma and recovers the quality of semen of post-treated compared to pre-treated infertile men. Serum biochemistry was also improved over post-therapy in infertile men. Our findings reveal that Withania somnifera not only reboots enzymatic activity of metabolic pathways and energy metabolism but also invigorates the harmonic balance of seminal plasma metabolites and reproductive hormones in infertile men. CONCLUSION: The results suggest that Withania somnifera may be used as an empirical therapy for clinical management and treatment of infertility.

Augmentation of therapeutic responses in melanoma by inhibition of IRAK-1,-4.

Toll-like receptors (TLR) are expressed by a variety of cancers, including melanoma, but their functional contributions in cancer cells are uncertain. To approach this question, we evaluated the effects of stimulating or inhibiting the TLR/IL-1 receptor-associated kinases IRAK-1 and IRAK-4 in melanoma cells where their functions are largely unexplored. TLRs and TLR-related proteins were variably expressed in melanoma cell lines, with 42% expressing activated phospho-IRAK-1 constitutively and 85% expressing high levels of phospho-IRAK-4 in the absence of TLR stimulation. Immunohistochemical evaluation of melanoma tumor biopsies (n = 242) revealed two distinct patient populations, one that expressed p-IRAK-4 levels similar to normal skin (55%) and one with significantly higher levels than normal skin (45%). Levels of p-IRAK-4 levels did not correlate with clinical stage, gender, or age, but attenuated IRAK-1,-4 signaling with pharmacologic inhibitors or siRNA-enhanced cell death in vitro in combination with vinblastine. Moreover, in a xenograft mouse model of melanoma, the combined pharmacologic treatment delayed tumor growth and prolonged survival compared with subjects receiving single agent therapy. We propose p-IRAK-4 as a novel inflammation and prosurvival marker in melanoma with the potential to serve as a therapeutic target to enhance chemotherapeutic responses.

Redirecting gene-modified T cells toward various cancer types using tagged antibodies.

PURPOSE: To develop an adaptable gene-based vector that will confer immune cell specificity to various cancer types. EXPERIMENTAL DESIGN: Human and mouse T cells were genetically engineered to express a chimeric antigen receptor (CAR) that binds a fluorescein isothiocyanate (FITC) molecule, termed anti-FITC CAR T cells. Various antibodies (Ab) currently in clinical use including cetuximab (Ctx), trastuzumab (Her2), and rituximab (Rtx) were conjugated with FITC and tested for their ability to bind tumor cells, activate T cells, and induce antitumor effects in vitro and in vivo. RESULTS: Anti-FITC CAR T cells recognize various cancer types when bound with FITC-labeled Abs resulting in efficient target lysis, T-cell proliferation, and cytokine/chemokine production. The treatment of immunocompromised mice with human anti-FITC CAR T cells plus FITC-labeled cetuximab (FITC-Ctx) delayed the growth of colon cancer but unexpectedly led to the outgrowth of EGF receptor (EGFR)-negative tumor cells. On the other hand, in a human pancreatic cancer cell line with uniform EGFR expression, anti-FITC CAR T cells plus FITC-Ctx eradicated preestablished late-stage tumors. In immunocompetent mice, anti-FITC CAR T cells exhibited potent antitumor activity against syngeneic mouse breast cancer expressing Her2 and B-cell lymphoma expressing CD20 by combining with FITC-Her2 and FITC-Rtx, respectively. In addition, the activity of anti-FITC CAR T cells could be attenuated by subsequent injections of nonspecific FITC-IgG. CONCLUSION: These studies highlight an applicability of anti-tag CAR technology to treat patients with different types of cancers and a possibility to regulate CAR T-cell functions with competing FITC molecules.

Metabolomics of urinary tract infection: a new uroscope in town.

Urinary tract infection (UTI) is a potentially life-threatening infectious disease. For rapid directed therapy of UTIs, it is essential to determine the causative microorganism. To date, there is no single test that has been proven to reliably, rapidly and accurately identify the etiologic organism in UTI. The molecular methods for diagnosing the cause of UTI and prognostic development of clinically important metabolomic evaluations and their limitations for use in the diagnosis and monitoring of infections are discussed in this review article. The application of the emerging investigative device NMR spectroscopy as a surrogate method for the diagnosis of UTI is also addressed.

A proton NMR study of the effect of Mucuna pruriens on seminal plasma metabolites of infertile males.

The objective of this study was to employ proton nuclear magnetic resonance ((1)H NMR) spectroscopy to evaluate the impact of Mucuna pruriens seeds on the metabolic profile of seminal plasma of infertile patients. A total of 180 infertile patients were administered M. pruriens seed powder for a period of three months. Age-matched healthy men comprised the control (n=50) group in the study. Lactate, alanine, choline, citrate, glycerophosphocholine (GPC), glutamine, tyrosine, histidine, phenylalanine, and uridine were measured in seminal plasma by (1)H NMR spectroscopy. To evaluate the degree of infertility and extent of hormonal imbalance induced by this milieu, separate sperm concentration, motility, lipid peroxide in seminal plasma and LH, FSH, T, and PRL hormone concentration in serum were measured using standard laboratory methods and RIA, respectively, in the same subjects. M. pruriens therapy rectifies the perturbed alanine, citrate, GPC, histidine and phenylalanine content in seminal plasma and improves the semen quality of post-treated infertile men with compared to pre-treated. Concomitantly, clinical variables in seminal plasma and blood serum were also improved over post therapy in infertile men. On the basis of these observations, it may be proposed that M. pruriens seed powder not only reactivates the enzymatic activity of metabolic pathways and energy metabolism but also rejuvenates the harmonic balance of male reproductive hormones in infertile men. These findings open more opportunities for infertility treatment and management by improving semen quality.