Digital microfluidic assay for protein detection.

Global studies of the human proteome have revealed a plethora of putative protein biomarkers. However, their application for early disease detection remains at a standstill without suitable methods to realize their utility in the clinical setting. There thus continues to be tremendous interest in developing new technology for sensitive protein detection that is both low in cost and carries a small footprint to be able to be used at the point of care. The current gold standard method for protein biomarker detection is the ELISA, which measures protein abundance using bulky fluorescent scanners that lack portability. Here, we present a digital microfluidic platform for protein biomarker detection that is low in cost compared with standard optical detection methods, without any compromise in sensitivity. This platform furthermore makes use of simple electronics, enabling its translation into a portable handheld device, and has been developed in a manner that can easily be adapted to assay different types of proteomic biomarkers. We demonstrate its utility in quantifying not only protein abundance, but also activity. Interleukin-6 abundance could be assayed from concentrations as low as 50 pM (an order of magnitude lower than that detectable by a comparable laboratory designed ELISA) using less than 5 µL of sample, and Abelson tyrosine kinase activity was detectable in samples containing 100 pM of kinase.

Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations.

We present a simple molecular indexing method for quantitative targeted RNA sequencing, in which mRNAs of interest are selectively captured from complex cDNA libraries and sequenced to determine their absolute concentrations. cDNA fragments are individually labeled so that each molecule can be tracked from the original sample through the library preparation and sequencing process. Multiple copies of cDNA fragments of identical sequence become distinct through labeling, and replicate clones created during PCR amplification steps can be identified and assigned to their distinct parent molecules. Selective capture enables efficient use of sequencing for deep sampling and for the absolute quantitation of rare or transient transcripts that would otherwise escape detection by standard sequencing methods. We have also constructed a set of synthetic barcoded RNA molecules, which can be introduced as controls into the sample preparation mix and used to monitor the efficiency of library construction. The quantitative targeted sequencing revealed extremely low efficiency in standard library preparations, which were further confirmed by using synthetic barcoded RNA molecules. This finding shows that standard library preparation methods result in the loss of rare transcripts and highlights the need for monitoring library efficiency and for developing more efficient sample preparation methods.

Genomic responses in mouse models poorly mimic human inflammatory diseases.

A cornerstone of modern biomedical research is the use of mouse models to explore basic pathophysiological mechanisms, evaluate new therapeutic approaches, and make go or no-go decisions to carry new drug candidates forward into clinical trials. Systematic studies evaluating how well murine models mimic human inflammatory diseases are nonexistent. Here, we show that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another. Among genes changed significantly in humans, the murine orthologs are close to random in matching their human counterparts (e.g., R(2) between 0.0 and 0.1). In addition to improvements in the current animal model systems, our study supports higher priority for translational medical research to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory diseases.

Benchmarking outcomes in the critically injured burn patient.

OBJECTIVE: To determine and compare outcomes with accepted benchmarks in burn care at 6 academic burn centers. BACKGROUND: Since the 1960s, US morbidity and mortality rates have declined tremendously for burn patients, likely related to improvements in surgical and critical care treatment. We describe the baseline patient characteristics and well-defined outcomes for major burn injuries. METHODS: We followed 300 adults and 241 children from 2003 to 2009 through hospitalization, using standard operating procedures developed at study onset. We created an extensive database on patient and injury characteristics, anatomic and physiological derangement, clinical treatment, and outcomes. These data were compared with existing benchmarks in burn care. RESULTS: Study patients were critically injured, as demonstrated by mean % total body surface area (TBSA) (41.2 ± 18.3 for adults and 57.8 ± 18.2 for children) and presence of inhalation injury in 38% of the adults and 54.8% of the children. Mortality in adults was 14.1% for those younger than 55 years and 38.5% for those aged 55 years and older. Mortality in patients younger than 17 years was 7.9%. Overall, the multiple organ failure rate was 27%. When controlling for age and % TBSA, presence of inhalation injury continues to be significant. CONCLUSIONS: This study provides the current benchmark for major burn patients. Mortality rates, notwithstanding significant % TBSA and presence of inhalation injury, have significantly declined compared with previous benchmarks. Modern day surgical and medically intensive management has markedly improved to the point where we can expect patients younger than 55 years with severe burn injuries and inhalation injury to survive these devastating conditions.

Single cell mutational analysis of PIK3CA in circulating tumor cells and metastases in breast cancer reveals heterogeneity, discordance, and mutation persistence in cultured disseminated tumor cells from bone marrow.

BACKGROUND: Therapeutic decisions in cancer are generally guided by molecular biomarkers or, for some newer therapeutics, primary tumor genotype. However, because biomarkers or genotypes may change as new metastases emerge, circulating tumor cells (CTCs) from blood are being investigated for a role in guiding real-time drug selection during disease progression, expecting that CTCs will comprehensively represent the full spectrum of genomic changes in metastases. However, information is limited regarding mutational heterogeneity among CTCs and metastases in breast cancer as discerned by single cell analysis. The presence of disseminated tumor cells (DTCs) in bone marrow also carry prognostic significance in breast cancer, but with variability between CTC and DTC detection. Here we analyze a series of single tumor cells, CTCs, and DTCs for PIK3CA mutations and report CTC and corresponding metastatic genotypes. METHODS: We used the MagSweeper, an immunomagnetic separation device, to capture live single tumor cells from breast cancer patients' primary and metastatic tissues, blood, and bone marrow. Single cells were screened for mutations in exons 9 and 20 of the PIK3CA gene. Captured DTCs grown in cell culture were also sequenced for PIK3CA mutations. RESULTS: Among 242 individual tumor cells isolated from 17 patients and tested for mutations, 48 mutated tumor cells were identified in three patients. Single cell analyses revealed mutational heterogeneity among CTCs and tumor cells in tissues. In a patient followed serially, there was mutational discordance between CTCs, DTCs, and metastases, and among CTCs isolated at different time points. DTCs from this patient propagated in vitro contained a PIK3CA mutation, which was maintained despite morphological changes during 21 days of cell culture. CONCLUSIONS: Single cell analysis of CTCs can demonstrate genotypic heterogeneity, changes over time, and discordance from DTCs and distant metastases. We present a cautionary case showing that CTCs from any single blood draw do not always reflect metastatic genotype, and that CTC and DTC analyses may provide independent clinical information. Isolated DTCs remain viable and can be propagated in culture while maintaining their original mutational status, potentially serving as a future resource for investigating new drug therapies.

High-quality DNA sequence capture of 524 disease candidate genes.

The accurate and complete selection of candidate genomic regions from a DNA sample before sequencing is critical in molecular diagnostics. Several recently developed technologies await substantial improvements in performance, cost, and multiplex sample processing. Here we present the utility of long padlock probes (LPPs) for targeted exon capture followed by array-based sequencing. We found that on average 92% of 5,471 exons from 524 nuclear-encoded mitochondrial genes were successfully amplified from genomic DNA from 63 individuals. Only 144 exons did not amplify in any sample due to high GC content. One LPP was sufficient to capture sequences from <100-500 bp in length and only a single-tube capture reaction and one microarray was required per sample. Our approach was highly reproducible and quick (<8 h) and detected DNA variants at high accuracy (false discovery rate 1%, false negative rate 3%) on the basis of known sample SNPs and Sanger sequence verification. In a patient with clinical and biochemical presentation of ornithine transcarbamylase (OTC) deficiency, we identified copy-number differences in the OTC gene at exon-level resolution. This shows the ability of LPPs to accurately preserve a sample's genome information and provides a cost-effective strategy to identify both single nucleotide changes and structural variants in targeted resequencing.

Human transcriptome array for high-throughput clinical studies.

A 6.9 million-feature oligonucleotide array of the human transcriptome [Glue Grant human transcriptome (GG-H array)] has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing as well as detection of coding SNPs and noncoding transcripts. The performance of the array was examined and compared with mRNA sequencing (RNA-Seq) results over multiple independent replicates of liver and muscle samples. Compared with RNA-Seq of 46 million uniquely mappable reads per replicate, the GG-H array is highly reproducible in estimating gene and exon abundance. Although both platforms detect similar expression changes at the gene level, the GG-H array is more sensitive at the exon level. Deeper sequencing is required to adequately cover low-abundance transcripts. The array has been implemented in a multicenter clinical program and has generated high-quality, reproducible data. Considering the clinical trial requirements of cost, sample availability, and throughput, the GG-H array has a wide range of applications. An emerging approach for large-scale clinical genomic studies is to first use RNA-Seq to the sufficient depth for the discovery of transcriptome elements relevant to the disease process followed by high-throughput and reliable screening of these elements on thousands of patient samples using custom-designed arrays.

Development of a genomic metric that can be rapidly used to predict clinical outcome in severely injured trauma patients.

OBJECTIVE: Many patients have complicated recoveries following severe trauma due to the development of organ injury. Physiological and anatomical prognosticators have had limited success in predicting clinical trajectories. We report on the development and retrospective validation of a simple genomic composite score that can be rapidly used to predict clinical outcomes. DESIGN: Retrospective cohort study. SETTING: Multi-institutional level 1 trauma centers. PATIENTS: Data were collected from 167 severely traumatized (injury severity score >15) adult (18-55 yr) patients. METHODS: Microarray-derived genomic data obtained from 167 severely traumatized patients over 28 days were assessed for differences in messenger RNA abundance among individuals with different clinical trajectories. Once a set of genes was identified based on differences in expression over the entire study period, messenger RNA abundance from these subjects obtained in the first 24 hours was analyzed in a blinded fashion using a rapid multiplex platform, and genomic data reduced to a single metric. RESULTS: From the existing genomic dataset, we identified 63 genes whose leukocyte expression differed between an uncomplicated and complicated clinical outcome over 28 days. Using a multiplex approach that can quantitate messenger RNA abundance in less than 12 hours, we reassessed total messenger RNA abundance from the first 24 hours after trauma and reduced the genomic data to a single composite score using the difference from reference. This composite score showed good discriminatory capacity to distinguish patients with a complicated outcome (area under a receiver-operator curve, 0.811; p <0.001). This was significantly better than the predictive power of either Acute Physiology and Chronic Health Evaluation II or new injury severity score scoring systems. CONCLUSIONS: A rapid genomic composite score obtained in the first 24 hours after trauma can retrospectively identify trauma patients who are likely to develop complicated clinical trajectories. A novel platform is described in which this genomic score can be obtained within 12 hours of blood collection, making it available for clinical decision making.

Benchmarking outcomes in the critically injured trauma patient and the effect of implementing standard operating procedures.

OBJECTIVE: To determine and compare outcomes with accepted benchmarks in trauma care at 7 academic level I trauma centers in which patients were treated on the basis of a series of standard operating procedures (SOPs). BACKGROUND: Injury remains the leading cause of death for those younger than 45 years. This study describes the baseline patient characteristics and well-defined outcomes of persons hospitalized in the United States for severe blunt trauma. METHODS: We followed 1637 trauma patients from 2003 to 2009 up to 28 hospital days using SOPs developed at the onset of the study. An extensive database on patient and injury characteristics, clinical treatment, and outcomes was created. These data were compared with existing trauma benchmarks. RESULTS: The study patients were critically injured and were in shock. SOP compliance improved 10% to 40% during the study period. Multiple organ failure and mortality rates were 34.8% and 16.7%, respectively. Time to recovery, defined as the time until the patient was free of organ failure for at least 2 consecutive days, was developed as a new outcome measure. There was a reduction in mortality rate in the cohort during the study that cannot be explained by changes in the patient population. CONCLUSIONS: This study provides the current benchmark and the overall positive effect of implementing SOPs for severely injured patients. Over the course of the study, there were improvements in morbidity and mortality rates and increasing compliance with SOPs. Mortality was surprisingly low, given the degree of injury, and improved over the duration of the study, which correlated with improved SOP compliance.

Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device.

The enumeration of rare circulating epithelial cells (CEpCs) in the peripheral blood of metastatic cancer patients has shown promise for improved cancer prognosis. Moving beyond enumeration, molecular analysis of CEpCs may provide candidate surrogate endpoints to diagnose, treat, and monitor malignancy directly from the blood samples. Thorough molecular analysis of CEpCs requires the development of new sample preparation methods that yield easily accessible and purified CEpCs for downstream biochemical assays. Here, we describe a new immunomagnetic cell separator, the MagSweeper, which gently enriches target cells and eliminates cells that are not bound to magnetic particles. The isolated cells are easily accessible and can be extracted individually based on their physical characteristics to deplete any cells nonspecifically bound to beads. We have shown that our device can process 9 mL of blood per hour and captures >50% of CEpCs as measured in spiking experiments. We have shown that the separation process does not perturb the gene expression of rare cells. To determine the efficiency of our platform in isolating CEpCs from patients, we have isolated CEpCs from all 47 tubes of 9-mL blood samples collected from 17 women with metastatic breast cancer. In contrast, we could not find any circulating epithelial cells in samples from 5 healthy donors. The isolated CEpCs are all stored individually for further molecular analysis.

A network-based analysis of systemic inflammation in humans.

Oligonucleotide and complementary DNA microarrays are being used to subclassify histologically similar tumours, monitor disease progress, and individualize treatment regimens. However, extracting new biological insight from high-throughput genomic studies of human diseases is a challenge, limited by difficulties in recognizing and evaluating relevant biological processes from huge quantities of experimental data. Here we present a structured network knowledge-base approach to analyse genome-wide transcriptional responses in the context of known functional interrelationships among proteins, small molecules and phenotypes. This approach was used to analyse changes in blood leukocyte gene expression patterns in human subjects receiving an inflammatory stimulus (bacterial endotoxin). We explore the known genome-wide interaction network to identify significant functional modules perturbed in response to this stimulus. Our analysis reveals that the human blood leukocyte response to acute systemic inflammation includes the transient dysregulation of leukocyte bioenergetics and modulation of translational machinery. These findings provide insight into the regulation of global leukocyte activities as they relate to innate immune system tolerance and increased susceptibility to infection in humans.

Variation in the TLR4 gene influences the risk of organ failure and shock posttrauma: a cohort study.

BACKGROUND: Genetic variation contributes to risk and outcomes of sepsis. We sought to determine whether variation in inflammation related genes is associated with severity of sepsis in trauma patients. METHODS: A cohort of severely injured Caucasian patients was studied and genotyped for candidate single nucleotide polymorphisms (SNPs). These were toll-like receptor 4 (TLR4) A896G, tumor necrosis factor-alpha G-308A, interleukin-6 G-174C, interleukin-1beta C-31T, and cluster of differentiation marker 14C-159T. SNP genotypes among patients with sepsis and complicated sepsis were analyzed by chi2 and logistic regression. Six haplotype-tagging SNPs in the TLR4 gene were subsequently examined to analyze their influence on TLR4 A896G SNPs relationship to sepsis severity. RESULTS: We enrolled 598 patients. Complicated sepsis developed in 147 (25%). Adjusting for independent risk factors, carriage of the variant TLR4 896 G allele was associated with decreased risk of complicated sepsis (odds ratio = 0.3, 95% confidence interval, 0.1-0.7, p = 0.008). Furthermore, two haplotypes seemed to better characterize this risk than the variant TLR4 896G allele. The variant TLR4 896G allele is linked to one common haplotype, which seems to confer a considerably reduced risk of complicated sepsis. (aOR = 0.2 95% confidence interval, 0.05-0.7, p = 0.01). CONCLUSIONS: Variation within TLR4 gene is associated with severity of posttraumatic sepsis. This risk may not be solely related to TLR4 A896G SNP. It is likely that other, uncharacterized variations in the TLR4 gene contribute to sepsis severity. A thorough evaluation of variability within the TLR4 gene is needed to characterize sepsis risk.

Murine intramyocellular lipids quantified by NMR act as metabolic biomarkers in burn trauma.

It has been suggested that intramyocellular lipids (IMCLs) may serve as biomarkers of insulin resistance and mitochondrial dysfunction. Using a hind-limb mouse model of burn trauma, we tested the hypothesis that severe localized burn trauma involving 5% of the total body surface area causes a local increase in IMCLs in the leg skeletal muscle. We quantified IMCLs from ex vivo intact tissue specimens using High-Resolution Magic Angle Spinning (HRMAS) 1H NMR and characterized the accompanying gene expression patterns in burned versus control skeletal muscle specimens. We also quantified plasma-free fatty acids (FFAs) in burn versus control mice. Our results from HRMAS 1H NMR measurements indicated that IMCL levels were significantly increased in mice exposed to burn trauma. Furthermore, plasma FFA levels were also significantly increased, and gene expression of Glut4, insulin receptor substrate 1 (IRS1), glycolytic genes, and PGC-1beta was downregulated in these mice. Backward stepwise multiple linear regression analysis demonstrated that IMCL levels correlated significantly with FFA levels, which were a significant predictor of IRS1 and PGC-1beta gene expression. We conclude from these findings that IMCLs can serve as metabolic biomarkers in burn trauma and that FFAs and IMCLs may signal altered metabolic gene expression. This signaling may result in the observed burn-induced insulin resistance and skeletal muscle mitochondrial dysfunction. We believe that IMCLs may therefore be useful biomarkers in predicting the therapeutic effectiveness of hypolipidemic agents for patients with severe burns.

Reduced rate of adenosine triphosphate synthesis by in vivo 31P nuclear magnetic resonance spectroscopy and downregulation of PGC-1beta in distal skeletal muscle following burn.

Using a mouse model of burn trauma, we tested the hypothesis that severe burn trauma corresponding to 30% of total body surface area (TBSA) causes reduction in adenosine triphosphate (ATP) synthesis in distal skeletal muscle. We employed in vivo 31P nuclear magnetic resonance (NMR) in intact mice to assess the rate of ATP synthesis, and characterized the concomitant gene expression patterns in skeletal muscle in burned (30% TBSA) versus control mice. Our NMR results showed a significantly reduced rate of ATP synthesis and were complemented by genomic results showing downregulation of the ATP synthase mitochondrial F1 F0 complex and PGC-1beta gene expression. Our findings suggest that inflammation and muscle atrophy in burns are due to a reduced ATP synthesis rate that may be regulated upstream by PGC-1beta. These findings implicate mitochondrial dysfunction in distal skeletal muscle following burn injury. That PGC-1beta is a highly inducible factor in most tissues and responds to common calcium and cyclic adenosine monophosphate (cAMP) signaling pathways strongly suggests that it may be possible to develop drugs that can induce PGC-1beta.

Proton NMR spectroscopy shows lipids accumulate in skeletal muscle in response to burn trauma-induced apoptosis.

Burn trauma triggers hypermetabolism and muscle wasting via increased cellular protein degradation and apoptosis. Proton nuclear magnetic resonance (1H NMR) spectroscopy can detect mobile lipids in vivo. To examine the local effects of burn in skeletal muscle, we performed in vivo 1H NMR on mice 3 days after burn trauma; and ex vivo, high-resolution, magic angle spinning (1)H NMR on intact excised mouse muscle samples before and 1 and 3 days after burn. These samples were then analyzed for apoptotic nuclei using a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. To confirm our NMR and cell biology results, we used transcriptome analysis to demonstrate that burn trauma alters the expression of genes involved in lipid metabolism and apoptosis. Our results demonstrate that burn injury results in a localized intramyocellular lipid accumulation, which in turn is accompanied by burn-induced apoptosis and mitochondrial dysfunction, as seen by the up-regulation of apoptotic genes and down-regulation of genes that encode lipid oxidation and the peroxisomal proliferator activator receptor gamma coactivator PGC-1beta. Moreover, the increased levels of bisallylic methylene fatty acyl protons (2.8 ppm) and vinyl protons (5.4 ppm), in conjunction with the TUNEL assay results, further suggest that burn trauma results in apoptosis. Together, our results provide new insight into the local physiological changes that occur in skeletal muscle after severe burn trauma.

Uncoupling protein 3 expression and intramyocellular lipid accumulation by NMR following local burn trauma.

Burn trauma is a clinical condition accompanied by muscle wasting that severely impedes rehabilitation in burn survivors. Mitochondrial uncoupling protein 3 (UCP3) is uniformly expressed in myoskeletal mitochondria and its expression has been found to increase in other clinical syndromes that, like burn trauma, are associated with muscle wasting (e.g., starvation, fasting, cancer, sepsis). The aim of this study was to explore the effects of burn trauma on UCP3 expression, intramyocellular lipids, and plasma-free fatty acids. Mice were studied at 6 h, 1 d and 3 d after nonlethal hindlimb burn trauma. Intramyocellular lipids in hindlimb skeletal muscle samples collected from burned and normal mice were measured using 1H NMR spectroscopy on a Bruker 14.1 Tesla spectrometer at 4 degrees C. UCP3 mRNA and protein levels were also measured in these samples. Plasma-free fatty acids were measured in burned and normal mice. Local burn trauma was found to result in: 1) upregulation of UCP3 mRNA and protein expression in hindlimb myoskeletal mitochondria by 6 h postburn; 2) increased intramyocellular lipids; and 3) increased plasma-free fatty acids. Our findings show that the increase in UCP3 after burn trauma may be linked to burn-induced alterations in lipid metabolism. Such a link could reveal novel insights into how processes related to energy metabolism are controlled in burn and suggest that induction of UCP3 by burn in skeletal muscle is protective by either activating cellular redox signaling and/or mitochondrial uncoupling.

In vivo high-resolution magic angle spinning magnetic and electron paramagnetic resonance spectroscopic analysis of mitochondria-targeted peptide in Drosophila melanogaster with trauma-induced thoracic injury.

Trauma is the most common cause of mortality among individuals aged between 1 and 44 years and the third leading cause of mortality overall in the US. In this study, we examined the effects of trauma on the expression of genes in Drosophila melanogaster, a useful model for investigating genetics and physiology. After trauma was induced by a non-lethal needle puncture of the thorax, we observed the differential expression of genes encoding for mitochondrial uncoupling proteins, as well as those encoding for apoptosis-related and insulin signaling-related proteins, thus indicating muscle functional dysregulation. These results prompted us to examine the link between insulin signaling and mitochondrial dysfunction using in vivo nuclear magnetic resonance (NMR) with complementary electron paramagnetic resonance (EPR) spectroscopy. Trauma significantly increased insulin resistance biomarkers, and the NMR spectral profile of the aged flies with trauma-induced thoracic injury resembled that of insulin-resistant chico mutant flies. In addition, the mitochondrial redox status, as measured by EPR, was significantly altered following trauma, indicating mitochondrial uncoupling. A mitochondria-targeted compound, Szeto-Schiller (SS)-31 that promotes adenosine triphosphate (ATP) synthesis normalized the NMR spectral profile, as well as the mitochondrial redox status of the flies with trauma-induced thoracic injury, as assessed by EPR. Based on these findings, we propose a molecular mechanism responsible for trauma-related mortality and also propose that trauma sequelae in aging are linked to insulin signaling and mitochondrial dysfunction. Our findings further suggest that SS-31 attenuates trauma-associated pathological changes.