Small molecule bio-signature in childhood intra-thoracic tuberculosis identified by metabolomics.

The diagnosis of pediatric tuberculosis (TB) remains a major challenge, hence the evaluation of new tools for improved diagnostics is urgently required. We investigated the serum metabolic profile of children with culture-confirmed intra-thoracic TB (ITTB) (n = 23) and compared it with those of non-TB controls (NTCs) (n = 13) using proton NMR spectroscopy-based targeted and untargeted metabolomics approaches. In targeted metabolic profiling, five metabolites (histidine, glycerophosphocholine, creatine/phosphocreatine, acetate, and choline) differentiated TB children from NTCs. Additionally, seven discriminatory metabolites (N-α-acetyl-lysine, polyunsaturated fatty acids, phenylalanine, lysine, lipids, glutamate + glutamine, and dimethylglycine) were identified in untargeted metabolic profiling. The pathway analysis revealed alterations in six metabolic pathways. The altered metabolites were associated with impaired protein synthesis, hindered anti-inflammatory and cytoprotective mechanisms, abnormalities in energy generation processes and membrane metabolism, and deregulated fatty acid and lipid metabolisms in children with ITTB. The diagnostic significance of the classification models obtained from significantly distinguishing metabolites showed sensitivity, specificity, and area under the curve of 78.2%, 84.6%, and 0.86, respectively, in the targeted profiling and 92.3%, 100%, and 0.99, respectively, in the untargeted profiling. Our findings highlight detectable metabolic changes in childhood ITTB; however, further validation is warranted in a large cohort of the pediatric population.

Multiparametric Magnetic Resonance Imaging and Metabolic Characterization of Patient-Derived Xenograft Models of Clear Cell Renal Cell Carcinoma.

Patient-derived xenografts (PDX) are high-fidelity cancer models typically credentialled by genomics, transcriptomics and proteomics. Characterization of metabolic reprogramming, a hallmark of cancer, is less frequent. Dysregulated metabolism is a key feature of clear cell renal cell carcinoma (ccRCC) and authentic preclinical models are needed to evaluate novel imaging and therapeutic approaches targeting metabolism. We characterized 5 PDX from high-grade or metastatic ccRCC by multiparametric magnetic resonance imaging (MRI) and steady state metabolic profiling and flux analysis. Similar to MRI of clinical ccRCC, T2-weighted images of orthotopic tumors of most PDX were homogeneous. The increased hyperintense (cystic) areas observed in one PDX mimicked the cystic phenotype typical of some RCC. The negligible hypointense (necrotic) areas of PDX grown under the highly vascularized renal capsule are beneficial for preclinical studies. Mean apparent diffusion coefficient (ADC) values were equivalent to those of ccRCC in human patients. Hyperpolarized (HP) [1-13C]pyruvate MRI of PDX showed high glycolytic activity typical of high-grade primary and metastatic ccRCC with considerable intra- and inter-tumoral variability, as has been observed in clinical HP MRI of ccRCC. Comparison of steady state metabolite concentrations and metabolic flux in [U-13C]glucose-labeled tumors highlighted the distinctive phenotypes of two PDX with elevated levels of numerous metabolites and increased fractional enrichment of lactate and/or glutamate, capturing the metabolic heterogeneity of glycolysis and the TCA cycle in clinical ccRCC. Culturing PDX cells and reimplanting to generate xenografts (XEN), or passaging PDX in vivo, altered some imaging and metabolic characteristics while transcription remained like that of the original PDX. These findings show that PDX are realistic models of ccRCC for imaging and metabolic studies but that the plasticity of metabolism must be considered when manipulating PDX for preclinical studies.

Tc-99m-tamoxifen: A novel diagnostic imaging agent for estrogen receptor-expressing breast cancer patients.

PURPOSE The aim of the study was to radiolabel, characterize, and perform in vitro and in vivo assessment of Technetium-99m (Tc-99m) tamoxifen for screening ER expressing lesions in breast cancer patients. METHODS In this study, tamoxifen has been radiolabeled with Tc-99m via Tc-99m-tricarbonyl core. The characterization and quality control tests of Tc-99m-tamoxifen were performed. In vitro recep- tor binding and blocking studies were performed in both positive control (MCF-7) and negative control cell lines (MDA-MB-231). Normal biodistribution studies were performed in female Wistar albino rats. The pilot clinical studies were performed in 4 ER-expressing breast cancer patients. Of the 4 patients, 1 was on tamoxifen therapy. All 4 patients had also undergone Fluorine-18 fluorodeoxyglucose (F-18-FDG) positron emission tomography/computed tomography. RESULTS Tamoxifen was radiolabeled with Tc-99m via Tc-99m-tricarbonyl core with more than 95% radio- chemical yield. Mass spectra showed a peak corresponding to the molecular weight of Tc-99m- tricarbonyl and Tc-99m-tamoxifen. The site of binding of Tc-99m-tricarbonyl with tamoxifen was determined by proton nuclear magnetic resonance. The Tc-99m-tamoxifen showed 30% binding with MCF-7 and only 1%-2% receptor binding with MDA-MB-231 cell lines. Also, the percentage of receptor binding was drastically reduced (up to 72%) when ER was saturated with 50 times the excess molar ratio of unlabeled tamoxifen. In a pilot patient study, Tc-99m-tamoxifen uptake was observed in primary and metastatic lesions. However, no uptake was observed in a patient who was on tamoxifen therapy. The uptake of F-18-FDG was noted in all the patients. CONCLUSION Tamoxifen was radiolabeled with an in-house-synthesized Tc-99m-tricarbonyl core. The radio- labeled complex has been characterized and evaluated for receptor specificity in in vitro and in vivo studies. Also, this is the first clinical study using Tc-99m-tamoxifen for imaging ER. More patients need to be evaluated to further explore the role of Tc-99m-tamoxifen in ER-expressing lesions.

NMR based metabolic profiling of patients with potential celiac disease elucidating early biochemical changes of gluten-sensitivity: A pilot study.

BACKGROUND: The patients with positive celiac disease (CeD) specific serology, but no evidence of intestinal inflammation are defined as potential celiac disease (PCeD) patients. About one-third of PCeD patients develop intestinal inflammation over time. The present study investigated the metabolome of small intestinal biopsies, blood plasma, and urine of patients with PCeD to understand the biochemical changes underlying the CeD. METHODS: The metabolic profiles of small intestinal biopsies, blood plasma, and urine of patients with PCeD (n = 7) were compared with CeD (n = 64) and controls (n = 15) [disease controls (DC) and healthy controls (HC)] using 1H NMR spectroscopy. RESULTS: The intestinal mucosa of PCeD showed lower levels of histidine, glycine, tyrosine, and tryptophan compared to DC. Altered levels of 6 metabolites (glucose, acetate, acetoacetate, ß-hydroxybutyrate, pyruvate, arginine) in blood plasma and two metabolites (succinate and aminohippurate) in urine were observed in PCeD compared to HC. The PLS-DA model built on the concentration of blood plasma showed separate clustering for PCeD and CeD patients. CONCLUSION: Altered metabolic profile of PCeD suggested that gluten intolerance was evident at the metabolic level before the intestinal damage. Altered energy metabolism and lower cytoprotective activity (histidine, glycine, arginine) indicated vulnerability to develop intestinal inflammation in PCeD over time. Our study may provide an insight into early biochemical processes of the progression of PCeD to CeD.

Dihydromyricetin alleviates cerebral ischemia-reperfusion injury by attenuating apoptosis and astrogliosis in peri-infarct cortex.

OBJECTIVES: In ischemic stroke, reperfusion after thrombolysis is associated with secondary brain damage. Dihydromyricetin (DHM), a flavonoid, has shown neuroprotective effects through anti-oxidant, anti-inflammatory and anti-apoptotic properties. This study investigates the potential of DHM, given postreperfusion in middle cerebral artery occlusion (MCAo) model of stroke in rats. METHODS: MCAo surgery was performed in male Wistar rats. Reperfusion was performed after 90 min of ischemia. DHM (50 and 100 mg/kg) was administered 10-15 min and 2 h postreperfusion followed by daily dosing for 2 more days. Neurobehavioral parameters and infarct size (TTC staining) were assessed after 72 h. The effective dose (100 mg/kg) was then used to study reduction in infarct size (measured by MRI) and effect on apoptosis (evaluated by protein expression of Bax, Bcl-2 and cleaved caspase-3 and TUNEL assay) in peri-infarct cortex. Furthermore, effects of DHM on neuronal damage and activation of astrocytes were studied by immunofluorescence. RESULTS: Poststroke DHM (100 mg/kg) administered for 3 days showed significant improvements in motor-coordination and infarct damage (TTC staining and MRI). MCAo-induced altered apoptotic proteins were normalized to a significant extent in peri-infarct cortex with DHM treatment. Data from TUNEL assay were complementary to the effects on apoptotic proteins. Additionally, DHM caused a significant reduction in the number of reactive astrocytes when compared with the MCAo group. DISCUSSION: This study demonstrated the efficacy of subacute DHM treatment in ischemia/reperfusion injury by modulating apoptosis and astrogliosis in the peri-infarct cortex. This suggests the potential of DHM in attenuating disease progression.

Urine metabolite profiling of Indian Antarctic Expedition members: NMR spectroscopy-based metabolomic investigation.

The southernmost region of earth, Antarctica, has world's most challenging environments. Those who live for long time and work in Antarctic stations are subjected to environmental stresses such as cold weather, photoperiod variations leading to disrupted sleep cycles, constrained living spaces, dry air, non-availability of fresh food items, and high electromagnetic radiations, psychological factors, such as geographical and social isolation, etc. All these factors have a significant impact on the human body. The present study investigated the impact of Antarctica harsh environment on human physiology and its metabolic processes by evaluating urine metabolome, using 1H NMR spectroscopy and analyzing certain physiological and clinical parameters for correlation with physiological expression data and metabolite results. Two study groups - before Antarctic exposure (B) and after Antarctic exposure (E), consisting of 11 subjects, exposed to one-month summer expedition, were compared. 35 metabolites in urine samples were identified from the 700 MHz 1H NMR spectra from where integral intensity of 22 important metabolites was determined. Univariate analysis indicated significant decrease in the levels of citrate and creatinine in samples collected post-expedition. Multivariate analysis was also performed using 1H NMR spectroscopy, because independent metabolite abundances may complement each other in predicting the dependent variables. 10 metabolites were identified among the groups; the OPLS-DA and VIP score indicated variation in appearance of metabolites over different time periods with insignificant change in the intensities. Metabolite results illustrate the impact of environmental stress or altered life style including the diet with absence of fresh fruits and vegetables, on the pathophysiology of the human health. Metabolic adaptation to Antarctic environmental stressors may help to highlight the effect of short-term physiological status and provide important information during Antarctic expeditions to formulate management programmes.

Re-188-tricarbonyl tamoxifen as a theranostic radiopharmaceutical for estrogen receptor expressing breast cancers: radiolabeling, characterization and in-vitro cytotoxic assessment.

PURPOSE: Development of a novel theranostic radiopharmaceutical for estrogen receptor, expressing unresectable primary and metastatic breast cancers. METHODS: Tamoxifen was radiolabeled with Rhenium-188 (Re-188) through tricarbonyl core. Radiolabeled complex was characterized by 1proton nuclear magnetic resonance spectroscopy and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). Various quality control tests such as sterility, apyrogenicity, and radiochemical purity (RCP) were performed to assess the suitability of the radiopharmaceutical for intravenous administration. In-vitro cell culture studies were performed for cytotoxic assessment. In addition to this, exposure due to different doses of Re-188-tricarbonyl tamoxifen was also calculated. RESULTS: Re-188-tricarbonyl and Re-188-tricarbonyl tamoxifen showed more than 99% RCP. Sample was found to be sterile and pyrogens levels were within the permissible limit. Re-188-tricarbonyl tamoxifen was successfully characterized by MALDI-TOF and 1H-NMR spectroscopy. Re-188 (1.480 MBq) and tamoxifen (0.027 or 0.054 µM) individually showed 36 and 70% cell death, respectively. However, radiolabeled complex (Re-188-tricarbonyl tamoxifen) with the same amount of radioactivity (1.480 MBq) increased the cell death to more than 90% with one-fifth to one-tenth molar concentration of tamoxifen (0.0054 µM). CONCLUSION: Re-188-tricarbonyl tamoxifen can be synthesized in-house in radiopharmacy lab. Radionuclide therapy with Re-188-tricarbonyl tamoxifen can be given using 10 times less amount of tamoxifen as compared to cold tamoxifen.

Resistance to Androgen Deprivation Leads to Altered Metabolism in Human and Murine Prostate Cancer Cell and Tumor Models.

Currently, no clinical methods reliably predict the development of castration-resistant prostate cancer (CRPC) that occurs almost universally in men undergoing androgen deprivation therapy. Hyperpolarized (HP) 13C magnetic resonance imaging (MRI) could potentially detect the incipient emergence of CRPC based on early metabolic changes. To characterize metabolic shifts occurring upon the transition from androgen-dependent to castration-resistant prostate cancer (PCa), the metabolism of [U-13C]glucose and [U-13C]glutamine was analyzed by nuclear magnetic resonance spectroscopy. Comparison of steady-state metabolite concentrations and fractional enrichment in androgen-dependent LNCaP cells and transgenic adenocarcinoma of the murine prostate (TRAMP) murine tumors versus castration-resistant PC-3 cells and treatment-driven CRPC TRAMP tumors demonstrated that CRPC was associated with upregulation of glycolysis, tricarboxylic acid metabolism of pyruvate; and glutamine, glutaminolysis, and glutathione synthesis. These findings were supported by 13C isotopomer modeling showing increased flux through pyruvate dehydrogenase (PDH) and anaplerosis; enzymatic assays showing increased lactate dehydrogenase, PDH and glutaminase activity; and oxygen consumption measurements demonstrating increased dependence on anaplerotic fuel sources for mitochondrial respiration in CRPC. Consistent with ex vivo metabolomic studies, HP [1-13C]pyruvate distinguished androgen-dependent PCa from CRPC in cell and tumor models based on significantly increased HP [1-13C]lactate.

Abnormalities in metabolic pathways in celiac disease investigated by the metabolic profiling of small intestinal mucosa, blood plasma and urine by NMR spectroscopy.

Celiac disease (CeD) is an autoimmune enteropathy caused by gluten intake in genetically predisposed individuals. We investigated the metabolism of CeD by metabolic profiling of intestinal mucosa, blood plasma and urine using NMR spectroscopy and multivariate analysis. The metabolic profile of the small intestinal mucosa was compared between patients with CeD (n = 64) and disease controls (DCs, n = 30). The blood plasma and urinary metabolomes of CeD patients were compared with healthy controls (HCs, n = 39). Twelve metabolites (proline (Pro), arginine (Arg), glycine (Gly), histidine (His), glutamate (Glu), aspartate, tryptophan (Trp), fumarate, formate, succinate (Succ), glycerophosphocholine (GPC) and allantoin (Alln)) of intestinal mucosa differentiated CeD from controls. The metabolome of blood plasma with 18 metabolites (Pro, Arg, Gly, alanine, Glu, glutamine, glucose (Glc), lactate (Lac), acetate (Ace), acetoacetate (AcAc), ß-hydroxybutyrate (ß-OHB), pyruvate (Pyr), Succ, citrate (Cit), choline (Cho), creatine (Cr), phosphocreatine (PCr) and creatinine) and 9 metabolites of urine (Pro, Trp, ß-OHB, Pyr, Succ, N-methylnicotinamide (NMN), aminohippurate (AHA), indoxyl sulfate (IS) and Alln) distinguished CeD from HCs. Our data demonstrated changes in nine metabolic pathways. The altered metabolites were associated with increased oxidative stress (Alln), impaired healing and repair mechanisms (Pro, Arg), compromised anti-inflammatory and cytoprotective processes (Gly, His, NMN), altered energy metabolism (Glc, Lac, ß-OHB, Ace, AcAc, Pyr, Succ, Cit, Cho, Cr and PCr), impaired membrane metabolism (GPC and Cho) and intestinal dysbiosis (AHA and IS). An orthogonal partial least square discriminant analysis model provided clear differentiation between patients with CeD and controls in all three specimens. A classification model built by combining the distinguishing metabolites of blood plasma and urine samples gave an AUC of 0.99 with 97.7% sensitivity, 93.3% specificity and a predictive accuracy of 95.1%, which was higher than for the models built separately using small intestinal mucosa, blood plasma and urine. In conclusion, a panel of metabolic biomarkers in intestinal biopsies, plasma and urine samples has potential to differentiate CeD from controls and may complement traditional tests to improve the diagnosis of CeD.

Disinfection of dentinal tubules with 2% Chlorhexidine gel, Calcium hydroxide and herbal intracanal medicaments against Enterococcus faecalis: An in-vitro study.

AIM: This in vitro study was conducted to evaluate the disinfection of dentinal tubules using 2% Chlorhexidine gel, Honey, Aloe vera gel, Curcuma longa, Propolis gel and Calcium hydroxide against Enterococcus faecalis. MATERIALS AND METHOD: Two hundred and ten human mandibular first premolars were infected with Enterococcus faecalis for 21 days. Samples were divided into 7 groups. Group I- Saline (negative control), Group II- 2% Chlorhexidine gel(CHX), Group III- honey, Group IV- Aloe vera gel, Group V- 20% Curcuma longa gel, Group VI- Propolis gel and Group VII -Calcium hydroxide (CH). At the end of 1, 3 and 5 days, the antimicrobial efficacy of medicaments against E.faecalis was assessed at the depths of 200µm and 400µm. RESULTS: 2% Chlorhexidine gel was most effective followed by Propolis and Curcuma longa. CONCLUSION: 2% Chlorhexidine gel gave the best results. Among the herbal extracts Propolis and Curcuma longa hold a promising future but to implement their use as sole intracanal medicaments clinically, further in vivo and long term studies are warranted.

Altered breast milk components in preeclampsia; An in-vitro proton NMR spectroscopy study.

OBJECTIVE: To investigate the metabolic profile of milk on day 3 and at the 6th month of lactation in mothers with preeclampsia (PE) and normotensive mothers. STUDY DESIGN: Women with PE (n=29) and control women (n=31) were recruited for this study. Milk was collected on day 3 and at the 6th month of lactation. Proton NMR spectroscopy was used to identify 25 milk metabolites (alpha-lactose, beta-lactose, oligosaccharides, myo-inositol, alanine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, tyrosine, valine, acetone, citrate, creatine, phosphocreatine, acetate, choline, lactate, lipid, phosphocholine and glycerophosphocholine). Principle component analysis (PCA) and Partial Least Square Discriminant Analysis (PLS-DA) were carried out to identify differences in milk metabolite composition between both the groups. RESULTS: The levels of milk metabolites varied between the control and PE groups. Alpha and beta-lactose, glycine, glycerophosphocholine (p<0.01 for all); glutamate, glutamine and phosphocholine levels (p<0.05 for all) were increased at the 6th month as compared to day 3 of lactation in the control group. However, in the PE group, only glycerophosphocholine level showed an increase (p<0.01) at the 6th month. The levels of acetate, acetone (p<0.05 for both) and creatine (p<0.01) decreased at the 6th month as compared to day 3 of lactation in both groups. However, the levels of oligosaccharides were similar between groups and also similar at day 3 and at the 6th month of lactation. CONCLUSION: Our data indicates differential levels of metabolites in the milk of women with PE. Future studies are required to investigate the associations between milk components and infant growth and development.

Metabolic abnormalities of gastrointestinal mucosa in celiac disease: An in vitro proton nuclear magnetic resonance spectroscopy study.

BACKGROUND AND AIM: Celiac disease (CeD) is a common autoimmune disorder in which ingestion of gluten and related proteins leads to inflammation in the small intestine. Although the histological findings in CeD are characteristic, they are not specific. In this study, proton nuclear magnetic resonance (NMR) spectroscopy was used to investigate the differences in metabolic profile of duodenal mucosal biopsies of patients with CeD and controls to find out the biomarker/s of villous atrophy. METHODS: Duodenal mucosal biopsies were collected from 29 CeD patients (mean age 26.2 ± 10.8 years) and 17 controls (mean age 34.1 ± 11.1 years) and were subjected to proton NMR spectroscopy following perchloric acid extraction. Assignment of metabolite resonances was carried out and their concentrations were determined. For comparison between the groups unpaired t-test/Wilcoxon rank sum test was used. Partial least squares-discriminant analysis was performed to study the clustering behavior of the samples from CeD patients and controls using the Unscrambler 10.2 software. RESULTS: Partial least squares-discriminant analysis clearly differentiated CeD patients from controls. Significantly higher concentrations of isoleucine, leucine, aspartate, succinate, and pyruvate, and lower concentration of glycerophosphocholine, were observed in the duodenal mucosa of CeD patients compared with controls. The results suggest abnormalities in glycolysis, Krebs cycle (energy deficiency), and amino acid metabolism, which may affect the biosynthetic pathways and consequently contribute to villous atrophy. CONCLUSIONS: NMR spectroscopy with multivariate analysis of duodenal mucosal biopsies revealed a characteristic metabolic profile in CeD patients. The work provided an insight in determining biomarker/s for villous atrophy and diagnosis of CeD patients.