Sleep fragmentation increases blood pressure and is associated with alterations in the gut microbiome and fecal metabolome in rats.

The gut microbiota, via the production of metabolites entering the circulation, plays a role in blood pressure regulation. Blood pressure is also affected by the characteristics of sleep. To date, no studies have examined relationships among the gut microbiota/metabolites, blood pressure, and sleep. We hypothesized that fragmented sleep is associated with elevated mean arterial pressure, an altered and dysbiotic gut microbial community, and changes in fecal metabolites. In our model system, rats were randomized to 8 h of sleep fragmentation during the rest phase (light phase) or were undisturbed (controls) for 28 consecutive days. Rats underwent sleep and blood pressure recordings, and fecal samples were analyzed during: baseline (days -4 to -1), early sleep fragmentation (days 0-3), midsleep fragmentation (days 6-13), late sleep fragmentation (days 20-27), and recovery/rest (days 28-34). Less sleep per hour during the sleep fragmentation period was associated with increased mean arterial pressure. Analyses of gut microbial communities and metabolites revealed that putative short chain fatty acid-producing bacteria were differentially abundant between control and intervention animals during mid-/late sleep fragmentation and recovery. Midsleep fragmentation was also characterized by lower alpha diversity, lower Firmicutes:Bacteroidetes ratio, and higher Proteobacteria in intervention rats. Elevated putative succinate-producing bacteria and acetate-producing bacteria were associated with lower and higher mean arterial pressure, respectively, and untargeted metabolomics analysis demonstrates that certain fecal metabolites are significantly correlated with blood pressure. These data reveal associations between sleep fragmentation, mean arterial pressure, and the gut microbiome/fecal metabolome and provide insight to links between disrupted sleep and cardiovascular pathology.

Antibiotic Exposure and Reduced Short Chain Fatty Acid Production after Hematopoietic Stem Cell Transplant.

Human studies have shown loss of diversity of the gut microbiome following hematopoietic stem cell transplantation (HSCT) in association with significant gut injury caused by the preparative regimen. Prolonged antibiotic use worsens loss of microbiome diversity and increases risk of complications such as graft-versus-host disease (GVHD). Our data support the hypothesis that loss of intestinal commensals that produce short-chain fatty acids (SCFAs) may increase dysbiosis. Here, we report an extensive longitudinal examination of changes in the luminal SCFAs in children undergoing allogeneic HSCT, and the relationship of those changes to the microbiota and antibiotic exposure. We found significant and progressive alterations in butyrate, and in additional SCFAs in stool in the first 14 days after transplant, a finding not observed in published mouse studies. SCFA levels were lower in children receiving antibiotics with activity against anaerobic organisms. Moreover, day 14 post-HSCT butyrate and propionate levels are lower in children who went on to develop GVHD, although our disease population was small. These data provide insight into the mechanism of prior observations that loss of diversity and increased antibiotic use are associated with GVHD following HSCT. Our findings offer potential modifiable targets to reduce risk of GVHD and improve survival after HSCT.

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

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.

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 today's bone marrow failure treatments on tomorrow's 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.

Head and Neck Cancer Susceptibility and Metabolism in Fanconi Anemia.

Fanconi anemia (FA) is a rare inherited, generally autosomal recessive syndrome, but it displays X-linked or dominant negative inheritance for certain genes. FA is characterized by a deficiency in DNA damage repair that results in bone marrow failure, and in an increased risk for various epithelial tumors, most commonly squamous cell carcinomas of the head and neck (HNSCC) and of the esophagus, anogenital tract and skin. Individuals with FA exhibit increased human papilloma virus (HPV) prevalence. Furthermore, a subset of anogenital squamous cell carcinomas (SCCs) in FA harbor HPV sequences and FA-deficient laboratory models reveal molecular crosstalk between HPV and FA proteins. However, a definitive role for HPV in HNSCC development in the FA patient population is unproven. Cellular metabolism plays an integral role in tissue homeostasis, and metabolic deregulation is a known hallmark of cancer progression that supports uncontrolled proliferation, tumor development and metastatic dissemination. The metabolic consequences of FA deficiency in keratinocytes and associated impact on the development of SCC in the FA population is poorly understood. Herein, we review the current literature on the metabolic consequences of FA deficiency and potential effects of resulting metabolic reprogramming on FA cancer phenotypes.

Maternal regulation of biliary disease in neonates via gut microbial metabolites.

Maternal seeding of the microbiome in neonates promotes a long-lasting biological footprint, but how it impacts disease susceptibility in early life remains unknown. We hypothesized that feeding butyrate to pregnant mice influences the newborn's susceptibility to biliary atresia, a severe cholangiopathy of neonates. Here, we show that butyrate administration to mothers renders newborn mice resistant to inflammation and injury of bile ducts and improves survival. The prevention of hepatic immune cell activation and survival trait is linked to fecal signatures of Bacteroidetes and Clostridia and increases glutamate/glutamine and hypoxanthine in stool metabolites of newborn mice. In human neonates with biliary atresia, the fecal microbiome signature of these bacteria is under-represented, with suppression of glutamate/glutamine and increased hypoxanthine pathways. The direct administration of butyrate or glutamine to newborn mice attenuates the disease phenotype, but only glutamine renders bile duct epithelial cells resistant to cytotoxicity by natural killer cells. Thus, maternal intake of butyrate influences the fecal microbial population and metabolites in newborn mice and the phenotypic expression of experimental biliary atresia, with glutamine promoting survival of bile duct epithelial cells.

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.

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 Crohn's 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 (1)H 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.

Chronic Dysregulation of Cortical and Subcortical Metabolism After Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) is a leading cause of death and long-term disability worldwide. Although chronic disability is common after TBI, effective treatments remain elusive and chronic TBI pathophysiology is not well understood. Early after TBI, brain metabolism is disrupted due to unregulated ion release, mitochondrial damage, and interruption of molecular trafficking. This metabolic disruption causes at least part of the TBI pathology. However, it is not clear how persistent or pervasive metabolic injury is at later stages of injury. Using untargeted 1H-NMR metabolomics, we examined ex vivo hippocampus, striatum, thalamus, frontal cortex, and brainstem tissue in a rat lateral fluid percussion model of chronic brain injury. We found altered tissue concentrations of metabolites in the hippocampus and thalamus consistent with dysregulation of energy metabolism and excitatory neurotransmission. Furthermore, differential correlation analysis provided additional evidence of metabolic dysregulation, most notably in brainstem and frontal cortex, suggesting that metabolic consequences of injury are persistent and widespread. Interestingly, the patterns of network changes were region-specific. The individual metabolic signatures after injury in different structures of the brain at rest may reflect different compensatory mechanisms engaged to meet variable metabolic demands across brain regions.

Reduced Gut Microbiome Diversity and Metabolome Differences in Rhinoceros Species at Risk for Iron Overload Disorder.

Iron overload disorder (IOD) affects many wildlife species cared for ex situ. Two of the four rhinoceros species in human care, Sumatran rhinoceros (Dicerorhinus sumatrensis) and black rhinoceros (Diceros bicornis), are susceptible, whereas the other two, white rhinoceros (Ceratotherium simum) and greater one-horned (GOH) rhinoceros (Rhinoceros unicornis), are relatively resistant to IOD. Complex interrelationships exist between mammalian hosts, their indigenous gut microbiota, metabolome, physical condition, and iron availability. The goal of this study was to gain insight into these relationships within the family Rhinocerotidae. Specific objectives were to (1) characterize the gut microbiome and metabolome of four rhinoceros species; (2) compare the microbiome and metabolome of IOD-susceptible and IOD-resistant rhinoceros species; and (3) identify variation in the microbiome and metabolome associated with compromised health or disease in IOD-susceptible rhinoceroses. Fecal samples were collected from 31 rhinoceroses (Sumatran rhinoceros, n = 3; black rhinoceros, n = 6; GOH rhinoceros, n = 9; white rhinoceros, n = 13) located at five facilities, and matched fecal aliquots were processed for microbiome and metabolome analyses using 16S rRNA gene sequencing and nuclear magnetic resonance spectroscopy, respectively. Despite the phylogenetic disparity and dissimilar zoo diets of the hosts, the structure of the fecal microbiota of the two IOD-susceptible rhinoceros species were more closely related to each other than to those of the two IOD-resistant species (Bray-Curtis dissimilarity; IOD-susceptible vs. IOD-resistant p-value < 0.001). In addition, IOD-susceptible rhinoceroses exhibited less microbial diversity than their IOD-resistant relatives (Shannon diversity; p-value < 0.001) which could have health implications. Of note, the black rhinoceros was distinct among the four rhinoceros species with the most divergent fecal metabolome; interestingly, it contained higher concentrations of short chain fatty acids. Neither age nor sex were associated with differences in microbial community composition (p = 0.253 and 0.488, respectively) or fecal metabolomic profile (p = 0.634 and 0.332, respectively). Differences in the distal gut microbiomes between IOD-resistant and IOD-susceptible rhinoceroses support hypotheses that gut microbes play a role in host iron acquisition, and further studies and experiments to test these hypotheses are warranted.

Lipidomic Profiling Links the Fanconi Anemia Pathway to Glycosphingolipid Metabolism in Head and Neck Cancer Cells.

Purpose: Mutations in Fanconi anemia (FA) genes are common in sporadic squamous cell carcinoma of the head and neck (HNSCC), and we have previously demonstrated that FA pathway depletion in HNSCC cell lines stimulates invasion. The goal of our studies was to use a systems approach in order to define FA pathway-dependent lipid metabolism and to extract lipid-based signatures and effectors of invasion in FA-deficient cells.Experimental Design: We subjected FA-isogenic HNSCC keratinocyte cell lines to untargeted and targeted lipidomics analyses to discover novel biomarkers and candidate therapeutic targets in FA-deficient cells. Cellular invasion assays were carried out in the presence and absence of N-butyldeoxynojirimycin (NB-DNJ), a biosynthetic inhibitor of the newly identified class of gangliosides, to investigate the requirement of ganglioside upregulation in FA-deficient HNSCC cells.Results: The most notable element of the lipid profiling results was a consistent elevation of glycosphingolipids, and particularly the accumulation of gangliosides. Conversely, repression of this same class of lipids was observed upon genetic correction of FA patient-derived HNSCC cells. Functional studies demonstrate that ganglioside upregulation is required for HNSCC cell invasion driven by FA pathway loss. The motility of nontransformed keratinocytes in response to FA loss displayed a similar dependence, thus supporting early and late roles for the FA pathway in controlling keratinocyte invasion through lipid regulation.Conclusions: Elevation of glycosphingolipids including the ganglioside GM3 in response to FA loss stimulates invasive characteristics of immortalized and transformed keratinocytes. An inhibitor of glycosphingolipid biosynthesis NB-DNJ attenuates invasive characteristics of FA-deficient HNSCC cells. Clin Cancer Res; 24(11); 2700-9. ©2018 AACR.

Dehydration prompts increased activity and blood feeding by mosquitoes.

Current insights into the mosquito dehydration response rely on studies that examine specific responses but ultimately fail to provide an encompassing view of mosquito biology. Here, we examined underlying changes in the biology of mosquitoes associated with dehydration. Specifically, we show that dehydration increases blood feeding in the northern house mosquito, Culex pipiens, which was the result of both higher activity and a greater tendency to land on a host. Similar observations were noted for Aedes aegypti and Anopheles quadrimaculatus. RNA-seq and metabolome analyses in C. pipiens following dehydration revealed that factors associated with carbohydrate metabolism are altered, specifically the breakdown of trehalose. Suppression of trehalose breakdown in C. pipiens by RNA interference reduced phenotypes associated with lower hydration levels. Lastly, mesocosm studies for C. pipiens confirmed that dehydrated mosquitoes were more likely to host feed under ecologically relevant conditions. Disease modeling indicates dehydration bouts will likely enhance viral transmission. This dehydration-induced increase in blood feeding is therefore likely to occur regularly and intensify during periods when availability of water is low.

Pathobiological Pseudohypoxia as a Putative Mechanism Underlying Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic disorders that are incurable with conventional therapy. Their incidence is increasing with global population aging. Although many genetic, epigenetic, splicing, and metabolic aberrations have been identified in patients with MDS, their clinical features are quite similar. Here, we show that hypoxia-independent activation of hypoxia-inducible factor 1α (HIF1A) signaling is both necessary and sufficient to induce dysplastic and cytopenic MDS phenotypes. The HIF1A transcriptional signature is generally activated in MDS patient bone marrow stem/progenitors. Major MDS-associated mutations (Dnmt3a, Tet2, Asxl1, Runx1, and Mll1) activate the HIF1A signature. Although inducible activation of HIF1A signaling in hematopoietic cells is sufficient to induce MDS phenotypes, both genetic and chemical inhibition of HIF1A signaling rescues MDS phenotypes in a mouse model of MDS. These findings reveal HIF1A as a central pathobiologic mediator of MDS and as an effective therapeutic target for a broad spectrum of patients with MDS.Significance: We showed that dysregulation of HIF1A signaling could generate the clinically relevant diversity of MDS phenotypes by functioning as a signaling funnel for MDS driver mutations. This could resolve the disconnection between genotypes and phenotypes and provide a new clue as to how a variety of driver mutations cause common MDS phenotypes. Cancer Discov; 8(11); 1438-57. ©2018 AACR. See related commentary by Chen and Steidl, p. 1355 This article is highlighted in the In This Issue feature, p. 1333.

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.

PLK1 inhibition enhances temozolomide efficacy in IDH1 mutant gliomas.

Despite multimodal therapy with radiation and the DNA alkylating agent temozolomide (TMZ), malignant gliomas remain incurable. Up to 90% of grades II-III gliomas contain a single mutant isocitrate dehydrogenase 1 (IDH1) allele. IDH1 mutant-mediated transformation is associated with TMZ resistance; however, there is no clinically available means of sensitizing IDH1 mutant tumors to TMZ. In this study we sought to identify a targetable mechanism of TMZ resistance in IDH1 mutant tumors to enhance TMZ efficacy. IDH1 mutant astrocytes rapidly bypassed the G2 checkpoint with unrepaired DNA damage following TMZ treatment. Checkpoint adaptation was accompanied by PLK1 activation and IDH1 mutant astrocytes were more sensitive to treatment with BI2536 and TMZ in combination (<20% clonogenic survival) than either TMZ (~60%) or BI2536 (~75%) as single agents. In vivo, TMZ or BI2536 alone had little effect on tumor size. Combination treatment caused marked tumor shrinkage in all mice and complete tumor regression in 5 of 8 mice. Mutant IDH1 promotes checkpoint adaptation which can be exploited therapeutically with the combination of TMZ and a PLK1 inhibitor, indicating PLK1 inhibitors may be clinically valuable in the treatment of IDH1 mutant gliomas.

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.

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.

Prolonged antibiotic use induces intestinal injury in mice that is repaired after removing antibiotic pressure: implications for empiric antibiotic therapy.

Metabolic profiling of urine and fecal extracts, histological investigation of intestinal ilea, and fecal metagenomics analyses were used to investigate effects of prolonged antibiotic use in mice. The study provides insight into the effects of extended empiric antibiotic therapy in humans. Mice were administered a broad-spectrum antibiotic for four consecutive days followed by oral gavage with Clostridium butyricum, an opportunistic gram-positive pathogenic bacteria commonly isolated in fecal and blood cultures of necrotizing enterocolitis patients. Metagenomics data indicated loss of bacterial diversity after 4 days on antibiotics that was restored after removing antibiotic pressure. Histological analyses indicated damage to ileal villi after antibiotic treatment that underwent repair after lifting antibiotic pressure. Metabolic profiling confirmed intestinal injury in antibiotic-treated mice indicated by increased urinary trans-4-hydroxy-l-proline, a breakdown product of collagen present in connective tissue of ileal villi that may serve as a biomarker for antibiotic-induced injury in at risk populations.

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 ß-lactam antibiotics blocked renal carnitine uptake by human organic cation transporter, hOCTN2.

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

INTRODUCTION: Systemic 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). METHODS: Metabolic 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. RESULTS: Urinary 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). CONCLUSIONS: This 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.