Lindsey E. Romick-Rosendale

NMR-based metabonomics analysis of mouse urine and fecal extracts following oral treatment with the broad-spectrum antibiotic enrofloxacin (Baytril).

The human gastrointestinal tract is home to hundreds of species of bacteria and the balance between beneficial and pathogenic bacteria plays a critical role in human health and disease. The human infant, however, is born with a sterile gut and the complex gastrointestinal host/bacterial ecosystem is only established after birth by rapid bacterial colonization. Composition of newborn gut flora depends on several factors including type of birth (Ceasarian or natural), manner of early feeding (breast milk or formula), and exposure to local, physical environment. Imbalance in normal, healthy gut flora contributes to several adult human diseases including inflammatory bowel (ulcerative colitis and Crohns disease) and Clostridium difficile associated disease, and early childhood diseases such as necrotizing enterocolitis. As a first step towards characterization of the role of gut bacteria in human health and disease, we conducted an 850 MHz 1H nuclear magnetic resonance spectroscopy study to monitor changes in metabolic profiles of urine and fecal extracts of 15 mice following gut sterilization by the broad‐spectrum antibiotic enrofloxacin (also known as Baytril). Ten metabolites changed in urine following enrofloxacin treatment including decreased acetate due to loss of microbial catabolism of sugars and polysaccharides, decreased trimethylamine‐N‐oxide due to loss of microbial catabolism of choline, and increased creatine and creatinine due to loss of microbial enzyme degradation. Eight metabolites changed in fecal extracts of mice treated with enrofloxacin including depletion of amino acids produced by microbial proteases, reduction in metabolites generated by lactate‐utilizing bacteria, and increased urea caused by loss of microbial ureases. Copyright

Statistical significance analysis of nuclear magnetic resonance-based metabonomics data.

Use of nuclear magnetic resonance (NMR)-based metabonomics to search for human disease biomarkers is becoming increasingly common. For many researchers, the ultimate goal is translation from biomarker discovery to clinical application. Studies typically involve investigators from diverse educational and training backgrounds, including physicians, academic researchers, and clinical staff. In evaluating potential biomarkers, clinicians routinely use statistical significance testing language, whereas academicians typically use multivariate statistical analysis techniques that do not perform statistical significance evaluation. In this article, we outline an approach to integrate statistical significance testing with conventional principal components analysis data representation. A decision tree algorithm is introduced to select and apply appropriate statistical tests to loadings plot data, which are then heat map color-coded according to P score, enabling direct visual assessment of statistical significance. A multiple comparisons correction must be applied to determine P scores from which reliable inferences can be made. Knowledge of means and standard deviations of statistically significant buckets enabled computation of effect sizes and study sizes for a given statistical power. Methods were demonstrated using data from a previous study. Integrated metabonomics data assessment methodology should facilitate translation of NMR-based metabonomics discovery of human disease biomarkers to clinical use.

The Fanconi anemia pathway: repairing the link between DNA damage and squamous cell carcinoma.

Fanconi anemia (FA) is a rare inherited recessive disease caused by mutations in one of fifteen genes known to encode FA pathway components. In response to DNA damage, nuclear FA proteins associate into high molecular weight complexes through a cascade of post-translational modifications and physical interactions, followed by the repair of damaged DNA. Hematopoietic cells are particularly sensitive to the loss of these interactions, and bone marrow failure occurs almost universally in FA patients. FA as a disease is further characterized by cancer susceptibility, which highlights the importance of the FA pathway in tumor suppression, and will be the focus of this review. Acute myeloid leukemia is the most common cancer type, often subsequent to bone marrow failure. However, FA patients are also at an extreme risk of squamous cell carcinoma (SCC) of the head and neck and gynecological tract, with an even greater incidence in those individuals who have received a bone marrow transplant and recovered from hematopoietic disease. FA tumor suppression in hematopoietic versus epithelial compartments could be mechanistically similar or distinct. Definition of compartment specific FA activities is now critical to assess the effects of todays bone marrow failure treatments on tomorrows solid tumor development. It is our hope that current therapies can then be optimized to decrease the risk of malignant transformation in both hematopoietic and epithelial cells. Here we review our current understanding of the mechanisms of action of the Fanconi anemia pathway as it contributes to stress responses, DNA repair and squamous cell carcinoma susceptibility.

Identification of urinary metabolites that distinguish membranous lupus nephritis from proliferative lupus nephritis and focal segmental glomerulosclerosis.

IntroductionSystemic lupus erythematosus (SLE or lupus) is a chronic autoimmune disease, and kidney involvement with SLE, a.k.a. lupus nephritis (LN), is a frequent and severe complication of SLE that increases patient morbidity and mortality. About 50% of patients with SLE encounter renal abnormalities which, if left untreated, can lead to end-stage renal disease. Kidney biopsy is considered the criterion standard for diagnosis and staging of LN using the International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification, which was developed to help predict renal outcomes and assist with medical decision-making. However, kidney biopsy-based classification of LN is highly invasive and impractical for real-time monitoring of LN status. Here, nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling was used to identify urinary metabolites that discriminated between proliferative and pure membranous LN as defined by the ISN/RPS classification, and between LN and primary focal segmental glomerulosclerosis (FSGS).MethodsMetabolic profiling was conducted using urine samples of patients with proliferative LN without membranous features (Class III/IV; n = 7) or pure membranous LN (Class V; n = 7). Patients with primary FSGS and proteinuria (n = 10) served as disease controls. For each patient, demographic information and clinical data was obtained and a random urine sample collected to measure NMR spectra. Data and sample collection for patients with LN occurred around the time of kidney biopsy. Metabolic profiling analysis was done by visual inspection and principal component analysis.ResultsUrinary citrate levels were 8-fold lower in Class V LN compared to Class III/IV patients, who had normal levels of urinary citrate (P < 0.05). Class III/IV LN patients had > 10-fold lower levels of urinary taurine compared to Class V patients, who had mostly normal levels (P < 0.01). Class V LN patients had normal urinary hippurate levels compared to FSGS patients, who completely lacked urinary hippurate (P < 0.001).ConclusionsThis pilot study indicated differences in urinary metabolites between proliferative LN and pure membranous LN patients, and between LN and FSGS patients. If confirmed in larger studies, these urine metabolites may serve as biomarkers to help discriminate between different classes of LN, and between LN and FSGS.

Mechanistic underpinnings of dehydration stress in the American dog tick revealed through RNA-Seq and metabolomics.

ABSTRACT Ticks are obligate blood feeders but spend the majority of their lifetime off-host where they must contend with a multitude of environmental stresses. Survival under desiccating conditions is a determinant for habitats where ticks can become established, and water-balance characteristics of ticks have been extensively studied. However, little is known about the underlying aspects associated with dehydration stress in ticks. In this study, we examined the response of male American dog ticks, Dermacentor variabilis, to dehydration using a combined transcriptomics and metabolomics approach. During dehydration, 497 genes were differentially expressed, including an up-regulation of stress-response and protein-catabolism genes and concurrent down-regulation of several energetically expensive biological processes. Accumulation of several metabolites, including specific amino acids, glycerol and gamma aminobutyric acid (GABA), and transcript shifts in the associated pathways for generating these metabolites indicated congruence between changes in the metabolome and gene expression. Ticks treated with exogenous glycerol and GABA demonstrated altered water-balance characteristics; specifically, increased water absorption at high relative humidity. Finally, we observed changes in locomotor activity in response to dehydration, but this change was not influenced by the accumulation of GABA. Overall, the responses to dehydration by these ticks were similar to those observed in other dehydration-tolerant arthropods, but several molecular and behavioral responses are distinct from those associated with other taxa. Highlighted Article: Ticks employ a suite of molecular and biochemical mechanisms to survive dehydration stress; strategies include limiting damage and maximizing recovery when conditions allow.

Defects in the Fanconi anemia pathway in head and neck cancer cells stimulate tumor cell invasion through DNA-PK and Rac1 signaling

Purpose: Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and Fanconi anemia (FA) gene mutations and transcriptional repression are common. Invasive tumor behavior is associated with poor outcome, but relevant pathways triggering invasion are poorly understood. There is a significant need to improve our understanding of genetic pathways and molecular mechanisms driving advanced tumor phenotypes, to develop tailored therapies. Here we sought to investigate the phenotypic and molecular consequences of FA pathway loss in HNSCC cells. Experimental Design: Using sporadic HNSCC cell lines with and without FA gene knockdown, we sought to characterize the phenotypic and molecular consequences of FA deficiency. FA pathway inactivation was confirmed by the detection of classic hallmarks of FA following exposure to DNA cross-linkers. Cells were subjected to RNA sequencing with qRT-PCR validation, followed by cellular adhesion and invasion assays in the presence and absence of DNA-dependent protein kinase (DNA-PK) and Rac1 inhibitors. Results: We demonstrate that FA loss in HNSCC cells leads to cytoskeletal reorganization and invasive tumor cell behavior in the absence of proliferative gains. We further demonstrate that cellular invasion following FA loss is mediated, at least in part, through NHEJ-associated DNA-PK and downstream Rac1 GTPase activity. Conclusions: These findings demonstrate that FA loss stimulates HNSCC cell motility and invasion, and implicate a targetable DNA-PK/Rac1 signaling axis in advanced tumor phenotypes. Clin Cancer Res; 22(8); 2062–73. ©2015 AACR.

A Potential Biomarker for Acute Kidney Injury in Preterm Infants from Metabolic Profiling.

Background Currently used biomarkers for acute kidney injury (AKI), namely Ngal, SCr, and BUN, are inadequate for timely detection of AKI in preterm infants. Methods Nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling was conducted on urines from 20 preterm infants to determine if novel metabolic biomarkers could be identified for early detection of AKI. Urines were collected from every patient each day for the first 14 days of life. NMR spectra were measured for all urines and metabolic profiling analysis conducted. Results One metabolite, carnitine, increased significantly in urines of three extremely low birth weight (ELBW) infants starting on day five of life. The three affected infants either received prolonged antibiotic treatment, extended treatment with indomethacin, or both. One ELBW patient who received both treatments and reached the highest urinary carnitine level died on day 10 of life due to localized gut perforation complicated by suspected AKI. Conclusions It was concluded that carnitine increased in the three neonates in part due to antibiotic- and/or indomethacin-induced AKI. It is hypothesized that combined antibiotic and indomethacin treatment promoted AKI resulting in reduced proximal renal tubule reabsorption of carnitine and that β-lactam antibiotics blocked renal carnitine uptake by human organic cation transporter, hOCTN2.

A review of metabolomics approaches and their application in identifying causal pathways of childhood asthma

Because asthma is a disease that results from host-environment interactions, an approach that allows assessment of the effect of the environment on the host is needed to understand the disease. Metabolomics has appealing potential as an application to study pathways to childhood asthma development. The objective of this review is to provide an overview of metabolomics methods and their application to understanding host-environment pathways in asthma development. We reviewed recent literature on advances in metabolomics and their application to study pathways to childhood asthma development. We highlight the (1) potential of metabolomics in understanding the pathogenesis of disease and the discovery of biomarkers; (2) choice of metabolomics techniques, biospecimen handling, and data analysis; (3) application to studying the role of the environment on asthma development; (4) review of metabolomics applied to the outcome of asthma; (5) recommendations for application of metabolomics-based -omics data integration in understanding disease pathogenesis; and (6) limitations. In conclusion, metabolomics allows use of biospecimens to identify useful biomarkers and pathways involved in disease development and subsequently to inform a greater understanding of disease pathogenesis and endotypes and prediction of the clinical course of childhood asthma phenotypes.

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis

The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth.

Feasibility Study of NMR Based Serum Metabolomic Profiling to Animal Health Monitoring: A Case Study on Iron Storage Disease in Captive Sumatran Rhinoceros (Dicerorhinus sumatrensis)

A variety of wildlife species maintained in captivity are susceptible to iron storage disease (ISD), or hemochromatosis, a disease resulting from the deposition of excess iron into insoluble iron clusters in soft tissue. Sumatran rhinoceros (Dicerorhinus sumatrensis) is one of the rhinoceros species that has evolutionarily adapted to a low-iron diet and is susceptible to iron overload. Hemosiderosis is reported at necropsy in many African black and Sumatran rhinoceroses but only a small number of animals reportedly die from hemochromatosis. The underlying cause and reasons for differences in susceptibility to hemochromatosis within the taxon remains unclear. Although serum ferritin concentrations have been useful in monitoring the progression of ISD in many species, there is some question regarding their value in diagnosing hemochromatosis in the Sumatran rhino. To investigate the metabolic changes during the development of hemochromatosis and possibly increase our understanding of its progression and individual susceptibility differences, the serum metabolome from a Sumatran rhinoceros was investigated by nuclear magnetic resonance (NMR)-based metabolomics. The study involved samples from female rhinoceros at the Cincinnati Zoo (n = 3), including two animals that died from liver failure caused by ISD, and the Sungai Dusun Rhinoceros Conservation Centre in Peninsular Malaysia (n = 4). Principal component analysis was performed to visually and statistically compare the metabolic profiles of the healthy animals. The results indicated that significant differences were present between the animals at the zoo and the animals in the conservation center. A comparison of the 43 serum metabolomes of three zoo rhinoceros showed two distinct groupings, healthy (n = 30) and unhealthy (n = 13). A total of eighteen altered metabolites were identified in healthy versus unhealthy samples. Results strongly suggest that NMR-based metabolomics is a valuable tool for animal health monitoring and may provide insight into the progression of this and other insidious diseases.