Propagation Characteristics of Fasting Duodeno-Jejunal Contractions in Healthy Controls Measured by Clustered Closely-spaced Manometric Sensors.

BACKGROUND/AIMS: High-resolution methods have advanced esophageal and anorectal manometry interpretation but are incompletely established for intestinal manometry. We characterized normal fasting duodeno-jejunal manometry parameters not measurable by standard techniques using clustered closely-spaced recordings. METHODS: Ten fasting recordings were performed in 8 healthy controls using catheters with 3-4 gastrointestinal manometry clusters with 1-2 cm channel spacing. Migrating motor complex phase III characteristics were quantified. Spatial-temporal contour plots measured propagation direction and velocity of individual contractions. Coupling was defined by pressure peak continuity within clusters. RESULTS: Twenty-three phase III complexes (11 antral, 12 intestinal origin) with 157 (95% CI, 104-211) minute periodicities, 6.99 (6.25-7.74) minute durations, 10.92 (10.68-11.16) cycle/minute frequencies, 73.6 (67.7-79.5) mmHg maximal amplitudes, and 4.20 (3.18-5.22) cm/minute propagation velocities were recorded. Coupling of individual contractions was 39.1% (32.1-46.1); 63.0% (54.4-71.6) of contractions were antegrade and 32.8% (24.1-41.5) were retrograde. Individual phase III contractions propagated > 35 fold faster (2.48 cm/sec; 95% CI, 2.25-2.71) than complexes themselves. Phase III complexes beyond the proximal jejunum were longer in duration (P = 0.025) and had poorer contractile coupling (P = 0.025) than proximal complexes. Coupling was greater with 1 cm channel spacing vs 2 cm (P ＜ 0.001). CONCLUSIONS: Intestinal manometry using clustered closely-spaced pressure ports characterizes novel antegrade and retrograde propagation and coupling properties which degrade in more distal jejunal segments. Coupling is greater with more closely-spaced recordings. Applying similar methods to dysmotility syndromes will define the relevance of these methods.

Propagation Characteristics of Fasting Duodeno-Jejunal Contractions in Healthy Controls Measured by Clustered Closely-spaced Manometric Sensors

BACKGROUND/AIMS: High-resolution methods have advanced esophageal and anorectal manometry interpretation but are incompletely established for intestinal manometry. We characterized normal fasting duodeno-jejunal manometry parameters not measurable by standard techniques using clustered closely-spaced recordings. METHODS: Ten fasting recordings were performed in 8 healthy controls using catheters with 3–4 gastrointestinal manometry clusters with 1–2 cm channel spacing. Migrating motor complex phase III characteristics were quantified. Spatial-temporal contour plots measured propagation direction and velocity of individual contractions. Coupling was defined by pressure peak continuity within clusters. RESULTS: Twenty-three phase III complexes (11 antral, 12 intestinal origin) with 157 (95% CI, 104–211) minute periodicities, 6.99 (6.25–7.74) minute durations, 10.92 (10.68–11.16) cycle/minute frequencies, 73.6 (67.7–79.5) mmHg maximal amplitudes, and 4.20 (3.18–5.22) cm/minute propagation velocities were recorded. Coupling of individual contractions was 39.1% (32.1–46.1); 63.0% (54.4–71.6) of contractions were antegrade and 32.8% (24.1–41.5) were retrograde. Individual phase III contractions propagated > 35 fold faster (2.48 cm/sec; 95% CI, 2.25–2.71) than complexes themselves. Phase III complexes beyond the proximal jejunum were longer in duration (P = 0.025) and had poorer contractile coupling (P = 0.025) than proximal complexes. Coupling was greater with 1 cm channel spacing vs 2 cm (P < 0.001). CONCLUSIONS: Intestinal manometry using clustered closely-spaced pressure ports characterizes novel antegrade and retrograde propagation and coupling properties which degrade in more distal jejunal segments. Coupling is greater with more closely-spaced recordings. Applying similar methods to dysmotility syndromes will define the relevance of these methods.

Effects of Specimen Collection Methodologies and Storage Conditions on the Short-Term Stability of Oral Microbiome Taxonomy.

UNLABELLED: Community profiling of the oral microbiome requires the recovery of quality sequences in order to accurately describe microbial community structure and composition. Our objective was to assess the effects of specimen collection method, storage medium, and storage conditions on the relative abundance of taxa in saliva and plaque identified using 16S rRNA genes. We also assessed short-term changes in taxon composition and relative abundance and compared the salivary and dental plaque communities in children and adults. Over a 2-week period, four successive saliva and dental plaque specimens were collected from four adults with no dental decay (108 samples), and two successive specimens were collected from six children with four or more erupted teeth (48 samples). There were minimal differences in community composition at the phylum and operational taxonomic unit levels between dental plaque collection using a scaler and collection using a CytoSoft brush. Plaque samples stored in OMNIgene medium showed higher within-sample Shannon diversity, were compositionally different, and were more similar to each other than plaque stored in liquid dental transport medium. Saliva samples stored in OMNIgene recovered similar communities for at least a week following storage at room temperature. However, the microbial communities recovered from plaque and saliva stored in OMNIgene were significantly different in composition from their counterparts stored in liquid dental transport medium. Dental plaque communities collected from the same tooth type over four successive visits from the same adult did not significantly differ in structure or composition. IMPORTANCE: Large-scale epidemiologic studies require collection over time and space, often with multiple teams collecting, storing, and processing data. Therefore, it is essential to understand how sensitive study results are to modest changes in collection and storage protocols that may occur with variation in personnel, resources available at a study site, and shipping requirements. The research presented in this paper measures the effects of multiple storage parameters and collection methodologies on the measured ecology of the oral microbiome from healthy adults and children. These results will potentially enable investigators to conduct oral microbiome studies at maximal efficiency by guiding informed administrative decisions pertaining to the necessary field or clinical work.

Mass-Selected Site-Specific Core-Fucosylation of Serum Proteins in Hepatocellular Carcinoma.

A mass spectrometry-based methodology has been developed to screen for changes in site-specific core-fucosylation (CF) of serum proteins in early stage HCC with different etiologies. The methods involve depletion of high-abundance proteins, trypsin digestion of medium-to-low-abundance proteins into peptides, iTRAQ labeling, and Lens culinaris Agglutinin (LCA) enrichment of CF peptides, followed by endoglycosidase F3 digestion before mass spectrometry analysis. 1300 CF peptides from 613 CF proteins were identified from patients sera, where 20 CF peptides were differentially expressed in alcohol (ALC)-related HCC samples compared with ALC-related cirrhosis samples and 26 CF peptides changed in hepatitis C virus (HCV)-related HCC samples compared with HCV-related cirrhosis samples. Among these, we found three CF peptides from fibronectin upregulated in ALC-related HCC samples compared with ALC-related cirrhosis samples with an AUC (area under the curve) value of 0.89 at site 1007 with a specificity of 85.7% at a sensitivity of 92.9% (generated with 10-fold cross-validation). When combined with the AFP index, the AUC value reached to 0.92 with a specificity of 92.9% at a sensitivity of 100%, significantly improved compared to that with AFP alone (LR test p < 0.001). In HCV-related samples, the CF level of cadherin-5 at site 61 showed the best AUC value of 0.75 but was not as promising as that of fibronectin site 1007 for ALC-related samples. The CF peptides of fibronectin may serve as potential biomarkers for early stage HCC screening in ALC-related cirrhosis patients.

Does Matching Quality Matter in Mode Comparison Studies?

This article provides a brief overview and comparison of three matching approaches in forming comparable groups for a study comparing test administration modes (i.e., computer-based tests [CBT] and paper-and-pencil tests [PPT]): (a) a propensity score matching approach proposed in this article, (b) the propensity score matching approach used by Lottridge, Nicewander, and Mitzel, and (c) a modified approach of matched samples comparability analyses (MSCA) mentioned by Way, Davis, and Fitzpatrick. Different matching approaches resulted in different matched data with differing degrees of matching quality, and matched data from each matching approach were then used in the mode comparison investigation. Construct equivalence was examined and the level of invariance was found to be consistent across modes for all three matching approaches. Raw-to-scale score conversion tables were created, and the impact on CBT students' proficiency classification was examined. The comparison of the number of CBT students whose proficiency classification would be affected and the equality of score distributions between modes on raw scores and scale scores across the three matching approaches indicate that the propensity score matching approach delineated in this article led to the most consistent evidence for the conclusion of the mode comparison.

Effect of hyperglycaemia on muscarinic M3 receptor expression and secretory sensitivity to cholinergic receptor activation in islets.

AIMS: Islets are innervated by parasympathetic nerves which release acetylcholine (ACh) to amplify glucose-induced insulin secretion, primarily via muscarinic M3 receptors (M3R). Here we investigate the consequence of chronic hyperglycaemia on islet M3R expression and secretory sensitivity of mouse islets to cholinergic receptor activation. METHODS: The impact of hyperglycaemia was studied in (i) islets isolated from ob/ob mice, (ii) alginate-encapsulated mouse islets transplanted intraperitoneally into streptozotocin-induced diabetic mice and (iii) mouse and human islets maintained in vitro at 5.5 or 16 mmol/l glucose. Blood glucose levels were assessed by a commercial glucose meter, insulin content by RIA and M3R expression by qPCR and immunohistochemistry. RESULTS: M3R mRNA expression was reduced in both ob/ob islets and islets maintained at 16 mmol/l glucose for 3 days (68 and 50% control, respectively). In all three models of hyperglycaemia the secretory sensitivity to the cholinergic receptor agonist, carbachol, was reduced by 60-70% compared to control islets. Treatment for 72 h with the irreversible PKC activator, PMA, or the PKC inhibitor, Gö6983, did not alter islet M3R mRNA expression nor did incubation with the PI3K-inhibitor, LY294002, although enhancement of glucose-induced insulin secretion by LY294002 was reduced in islets maintained at 16 mmol/l glucose, as was mRNA expression of the PI3K regulatory subunit, p85α. CONCLUSIONS: Cholinergic regulation of insulin release is impaired in three experimental islet models of hyperglycaemia consistent with reduced expression of M3 receptors. Our data suggest that the receptor downregulation is a PKC- and PI3K-independent consequence of the hyperglycaemic environment, and they imply that M3 receptors could be potential targets in the treatment of type 2 diabetes.

Glycoprotein biomarker panel for pancreatic cancer discovered by quantitative proteomics analysis.

Pancreatic cancer is a lethal disease where specific early detection biomarkers would be very valuable to improve outcomes in patients. Many previous studies have compared biosamples from pancreatic cancer patients with healthy controls to find potential biomarkers. However, a range of related disease conditions can influence the performance of these putative biomarkers, including pancreatitis and diabetes. In this study, quantitative proteomics methods were applied to discover potential serum glycoprotein biomarkers that distinguish pancreatic cancer from other pancreas related conditions (diabetes, cyst, chronic pancreatitis, obstructive jaundice) and healthy controls. Aleuria aurantia lectin (AAL) was used to extract fucosylated glycoproteins and then both TMT protein-level labeling and label-free quantitative analysis were performed to analyze glycoprotein differences from 179 serum samples across the six different conditions. A total of 243 and 354 serum proteins were identified and quantified by label-free and TMT protein-level quantitative strategies, respectively. Nineteen and 25 proteins were found to show significant differences in samples between the pancreatic cancer and other conditions using the label-free and TMT strategies, respectively, with 7 proteins considered significant in both methods. Significantly different glycoproteins were further validated by lectin-ELISA and ELISA assays. Four candidates were identified as potential markers with profiles found to be highly complementary with CA 19-9 (p < 0.001). Obstructive jaundice (OJ) was found to have a significant impact on the performance of every marker protein, including CA 19-9. The combination of α-1-antichymotrypsin (AACT), thrombospondin-1 (THBS1), and haptoglobin (HPT) outperformed CA 19-9 in distinguishing pancreatic cancer from normal controls (AUC = 0.95), diabetes (AUC = 0.89), cyst (AUC = 0.82), and chronic pancreatitis (AUC = 0.90). A marker panel of AACT, THBS1, HPT, and CA 19-9 showed a high diagnostic potential in distinguishing pancreatic cancer from other conditions with OJ (AUC = 0.92) or without OJ (AUC = 0.95).

Analysis of acquired genomic copy number aberrations and regions of loss of heterozygosity in acute myelogenous leukemia genomes using Affymetrix SNP 6.0 arrays and supporting software tools.

The application of SNP array technology to the analysis of cancer genomes has greatly advanced our knowledge of the incidence and functional consequences of acquired genomic copy number aberrations (aCNA) and LOH in various malignancies. The major challenges of using SNP arrays are accurately identifying acquired genomic DNA aberrations in the raw array data with very high sensitivity and specificity and meaningfully assessing the associations between these aberrations and biological characteristics or patient outcomes. Critical to the success and valid interpretation of data derived from SNP array profiling are (1) the purity of cells used as a source of template DNA; (2) the analysis of paired DNA samples (tumor and normal); (3) use of validated software tools for data analysis; (4) access to an acceptable gold standard for aCNA and LOH, including FISH data, cytogenetic results, and Q-PCR data; and (5) statistical support to employ or develop algorithmic approaches to SNP array data analysis. Overcalling of lesions including lack of validation and undercalling of lesions that display low fractional allelic representations are common problems. This guide should help the reader establish this powerful technology in the laboratory and aims to stimulate transition of SNP array profiling into clinical applications.

4α-phorbol 12,13-didecanoate activates cultured mouse dorsal root ganglia neurons independently of TRPV4.

BACKGROUND AND PURPOSE: The Ca(2+) -permeable cation channel TRPV4 is activated by mechanical disturbance of the cell membrane and is implicated in mechanical hyperalgesia. Nerve growth factor (NGF) is increased during inflammation and causes mechanical hyperalgesia. 4α-phorbol 12,13-didecanoate (4αPDD) has been described as a selective TRPV4 agonist. We investigated NGF-induced hyperalgesia in TRPV4 wild-type (+/+) and knockout (-/-) mice, and the increases in [Ca(2+) ](i) produced by 4αPDD in cultured mouse dorsal root ganglia neurons following exposure to NGF. EXPERIMENTAL APPROACH: Withdrawal thresholds to heat, von Frey hairs and pressure were measured in mice before and after systemic administration of NGF. Changes in intracellular Ca(2+) concentration were measured by ratiometric imaging with Fura-2 in cultured DRG and trigeminal ganglia (TG) neurons during perfusion of TRPV4 agonists. KEY RESULTS: Administration of NGF caused a significant sensitization to heat and von Frey stimuli in TRPV4 +/+ and -/- mice, but only TRPV4 +/+ mice showed sensitization to noxious pressure. 4αPDD stimulated a dose-dependent increase in [Ca(2+) ](i) in neurons from +/+ and -/- mice, with the proportion of responding neurons and magnitude of increase unaffected by the genotype. In contrast, the selective TRPV4 agonist GSK1016790A failed to stimulate an increase in intracellular Ca(2+) in cultured neurons. Responses to 4αPDD were unaffected by pretreatment with NGF. CONCLUSIONS AND IMPLICATIONS: TRPV4 contributes to mechanosensation in vivo, but there is little evidence for functional TRPV4 in cultured DRG and TG neurons. We conclude that 4αPDD activates these neurons independently of TRPV4, so it is not appropriate to refer to 4αPDD as a selective TRPV4 agonist.

Ontology-based combinatorial comparative analysis of adverse events associated with killed and live influenza vaccines.

Vaccine adverse events (VAEs) are adverse bodily changes occurring after vaccination. Understanding the adverse event (AE) profiles is a crucial step to identify serious AEs. Two different types of seasonal influenza vaccines have been used on the market: trivalent (killed) inactivated influenza vaccine (TIV) and trivalent live attenuated influenza vaccine (LAIV). Different adverse event profiles induced by these two groups of seasonal influenza vaccines were studied based on the data drawn from the CDC Vaccine Adverse Event Report System (VAERS). Extracted from VAERS were 37,621 AE reports for four TIVs (Afluria, Fluarix, Fluvirin, and Fluzone) and 3,707 AE reports for the only LAIV (FluMist). The AE report data were analyzed by a novel combinatorial, ontology-based detection of AE method (CODAE). CODAE detects AEs using Proportional Reporting Ratio (PRR), Chi-square significance test, and base level filtration, and groups identified AEs by ontology-based hierarchical classification. In total, 48 TIV-enriched and 68 LAIV-enriched AEs were identified (PRR>2, Chi-square score >4, and the number of cases >0.2% of total reports). These AE terms were classified using the Ontology of Adverse Events (OAE), MedDRA, and SNOMED-CT. The OAE method provided better classification results than the two other methods. Thirteen out of 48 TIV-enriched AEs were related to neurological and muscular processing such as paralysis, movement disorders, and muscular weakness. In contrast, 15 out of 68 LAIV-enriched AEs were associated with inflammatory response and respiratory system disorders. There were evidences of two severe adverse events (Guillain-Barre Syndrome and paralysis) present in TIV. Although these severe adverse events were at low incidence rate, they were found to be more significantly enriched in TIV-vaccinated patients than LAIV-vaccinated patients. Therefore, our novel combinatorial bioinformatics analysis discovered that LAIV had lower chance of inducing these two severe adverse events than TIV. In addition, our meta-analysis found that all previously reported positive correlation between GBS and influenza vaccine immunization were based on trivalent influenza vaccines instead of monovalent influenza vaccines.

Urine podocin:nephrin mRNA ratio (PNR) as a podocyte stress biomarker.

BACKGROUND: Proteinuria and/or albuminuria are widely used for noninvasive assessment of kidney diseases. However, proteinuria is a nonspecific marker of diverse forms of kidney injury, physiologic processes and filtration of small proteins of monoclonal and other pathologic processes. The opportunity to develop new glomerular disease biomarkers follows the realization that the degree of podocyte depletion determines the degree of glomerulosclerosis, and if persistent, determines the progression to end-stage kidney disease (ESKD). Podocyte cell lineage-specific mRNAs can be recovered in urine pellets of model systems and in humans. In model systems, progressive glomerular disease is associated with decreased nephrin mRNA steady-state levels compared with podocin mRNA. Thus, the urine podocin:nephrin mRNA ratio (PNR) could serve as a useful progression biomarker. The use of podocyte-specific transcript ratios also circumvents many problems inherent to urine assays. METHODS: To test this hypothesis, the human diphtheria toxin receptor (hDTR) rat model of progression was used to evaluate potentially useful urine mRNA biomarkers. We compared histologic progression parameters (glomerulosclerosis score, interstitial fibrosis score and percent of podocyte depletion) with clinical biomarkers [serum creatinine, systolic blood pressure (BP), 24-h urine volume, 24-h urine protein excretion and the urine protein:creatinine ratio(PCR)] and with the novel urine mRNA biomarkers. RESULTS: The PNR correlated with histologic outcome as well or better than routine clinical biomarkers and other urine mRNA biomarkers in the model system with high specificity and sensitivity, and a low coefficient of assay variation. CONCLUSIONS: We concluded that the PNR, used in combination with proteinuria, will be worth testing for its clinical diagnostic and decision-making utility.

Growth-dependent podocyte failure causes glomerulosclerosis.

Podocyte depletion leads to glomerulosclerosis, but whether an impaired capacity of podocytes to respond to hypertrophic stress also causes glomerulosclerosis is unknown. We generated transgenic Fischer 344 rats that express a dominant negative AA-4E-BP1 transgene driven by the podocin promoter; a member of the mammalian target of rapamycin complex 1 (mTORC1) pathway, 4E-BP1 modulates cap-dependent translation, which is a key determinant of a cell's hypertrophic response to nutrients and growth factors. AA-4E-BP1 rat podocytes expressed the transgene and had normal kidney histology and protein excretion at 100 g of body weight but developed ESRD by 12 months. Proteinuria and glomerulosclerosis were linearly related to both increasing body weight and transgene dose. Uni-nephrectomy reduced the body weight at which proteinuria first developed by 40%-50%. The initial histologic manifestation of disease was the appearance of bare areas of glomerular basement membrane from the pulling apart of podocyte foot processes, followed by adhesions to the Bowman capsule. Morphometric analysis confirmed the mismatch between glomerular tuft volume and total podocyte volume (number × size) per tuft in relation to weight gain and nephrectomy. Proteinuria and glomerulosclerosis did not develop if dietary calorie restriction prevented weight gain and glomerular enlargement. In summary, failure of podocytes to match glomerular tuft growth in response to growth signaling through the mTORC1 pathway can trigger proteinuria, glomerulosclerosis, and progression to ESRD. Reducing body weight and glomerular growth may be useful adjunctive therapies to slow or prevent progression to ESRD.

Nano-scale encapsulation enhances allograft survival and function of islets transplanted in a mouse model of diabetes.

AIMS/HYPOTHESIS: The success of islet transplantation as a treatment for type 1 diabetes is currently hampered by post-transplantation loss of functional islets through adverse immune and non-immune reactions. We aimed to test whether early islet loss can be limited and transplant survival improved by the application of conformal nano-coating layers to islets. METHODS: Our novel coating protocol used alternate layers of phosphorylcholine-derived polysaccharides (chitosan or chondroitin-4-sulphate) and alginate as coating materials, with the binding based on electrostatic complexation. The in vitro function of encapsulated mouse islets was studied by analysing islet secretory function and cell viability. The in vivo function was evaluated using syngeneic and allogeneic transplantation in the streptozotocin-induced mouse model of diabetes. RESULTS: Nano-scale encapsulated islets retained appropriate islet secretory function in vitro and were less susceptible to complement- and cytokine-induced apoptosis than non-encapsulated control islets. In in vivo experiments using a syngeneic mouse transplantation model, no deleterious responses to the coatings were observed in host animals, and the encapsulated islet grafts were effective in reversing hyperglycaemia. Allo-transplantation of the nano-coated islets resulted in preserved islet function post-implantation in five of seven mice throughout the 1 month monitoring period. CONCLUSIONS/INTERPRETATION: Nano-scale encapsulation offers localised immune protection for implanted islets, and may be able to limit early allograft loss and extend survival of transplanted islets. This versatile coating scheme has the potential to be integrated with tolerance induction mechanisms, thereby achieving long-term success in islet transplantation.

Angiotensin II-dependent persistent podocyte loss from destabilized glomeruli causes progression of end stage kidney disease.

Podocyte depletion is a major mechanism driving glomerulosclerosis. Progression is the process by which progressive glomerulosclerosis leads to end stage kidney disease (ESKD). In order to determine mechanisms contributing to persistent podocyte loss, we used a human diphtheria toxin transgenic rat model. After initial diphtheria toxin-induced podocyte injury (over 30% loss in 4 weeks), glomeruli became destabilized, resulting in continued autonomous podocyte loss causing global podocyte depletion (ESKD) by 13 weeks. This was monitored by urine mRNA analysis and by quantitating podocytes in glomeruli. Similar patterns of podocyte depletion were found in the puromycin aminonucleoside and 5/6 nephrectomy rat models of progressive end-stage disease. Angiotensin II blockade (combined enalapril and losartan) restabilized the glomeruli, and prevented continuous podocyte loss and progression to ESKD. Discontinuing angiotensin II blockade resulted in recurrent glomerular destabilization, podocyte loss, and progression to ESKD. Reduction in blood pressure alone did not reduce proteinuria or prevent podocyte loss from destabilized glomeruli. The protective effect of angiotensin II blockade was entirely accounted for by reduced podocyte loss. Thus, an initiating event resulting in a critical degree of podocyte depletion can destabilize glomeruli and initiate a superimposed angiotensin II-dependent podocyte loss process that accelerates progression resulting in eventual global podocyte depletion and ESKD. These events can be monitored noninvasively in real-time through urine mRNA assays.

Baseline plasma proteomic analysis to identify biomarkers that predict radiation-induced lung toxicity in patients receiving radiation for non-small cell lung cancer.

PURPOSE: To identify new plasma proteomic markers before radiotherapy start to predict later grade ≥2 radiation-induced lung toxicity (RILT2). METHODS: Fifty-seven patients with non-small cell lung cancer received radiotherapy (RT) were eligible. Forty-eight patients with minimum follow-up of 1 year, nine with RILT2 with tumor stage matched to 39 without RILT2, were enrolled for this analysis. Platelet-poor plasma was obtained within 2 weeks before radiotherapy. The plasma proteomes were compared using a multiplexed quantitative proteomics approach involving ExacTag labeling, reverse-phase high-performance liquid chromatography, and nano liquid chromatography electrospray ionization tandem mass spectrometry. Z scores and Bonferroni-adjusted p values for the two-sample mean comparison were used to identify the differential protein expression between patients with and without RILT2. RESULTS: More than 200 proteins were identified and quantified. After excluding proteins that were not detected in at least 40% of the 48 patient samples, C4b-binding protein alpha chain and vitronectin had significantly higher (p < 0.001 and p = 0.02) expression levels in patients with RILT2 compared with patients without RILT2. These two proteins were validated by Western blot. Ingenuity pathway analysis revealed that they both play important roles in the inflammatory response and are associated with the known pathways of radiation-induced lung damage. CONCLUSIONS: This proteomic approach demonstrates new plasma protein biomarkers before treatment for future studies on RILT2 prediction.

Mass spectrometric assay for analysis of haptoglobin fucosylation in pancreatic cancer.

A mass spectrometric method was developed to elucidate the N-glycan structures of serum glycoproteins and utilize fucosylated glycans as potential markers for pancreatic cancer. This assay was applied to haptoglobin in human serum where N-glycans derived from the serum of 16 pancreatic cancer patients were compared with those from 15 individuals with benign conditions (5 normals, 5 chronic pancreatitis, and 5 type II diabetes). This assay used only 10 µL of serum where haptoglobin was extracted using a monoclonal antibody and quantitative permethylation was performed on desialylated N-glycans followed by MALDI-QIT-TOF MS analysis. Eight desialylated N-glycan structures of haptoglobin were identified where a bifucosylated triantennary structure was reported for the first time in pancreatic cancer samples. Both core and antennary fucosylation were elevated in pancreatic cancer samples compared to samples from benign conditions. Fucosylation degree indices were calculated and show a significant difference between pancreatic cancer patients of all stages and the benign conditions analyzed. This study demonstrates that a serum assay based on haptoglobin fucosylation patterns using mass spectrometric analysis may serve as a novel method for the diagnosis of pancreatic cancer.

Identification of genes associated with complex traits by testing the genetic dissimilarity between individuals.

Using the exome sequencing data from 697 unrelated individuals and their simulated disease phenotypes from Genetic Analysis Workshop 17, we develop and apply a gene-based method to identify the relationship between a gene with multiple rare genetic variants and a phenotype. The method is based on the Mantel test, which assesses the correlation between two distance matrices using a permutation procedure. Using up to 100,000 permutations to estimate the statistical significance in 200 replicate data sets, we found that the method had 5.1% type I error at an α level of 0.05 and had various power to detect genes with simulated genetic associations. FLT1 and KDR had the most significant correlations with Q1 and were replicated 170 and 24 times, respectively, in 200 simulated data sets using a Bonferroni corrected p-value of 0.05 as a threshold. These results suggest that the distance correlation method can be used to identify genotype-phenotype association when multiple rare genetic variants in a gene are involved.

Quantitative proteomic profiling studies of pancreatic cancer stem cells.

Analyzing subpopulations of tumor cells in tissue is a challenging subject in proteomic studies. Pancreatic cancer stem cells (CSCs) are such a group of cells that only constitute 0.2-0.8% of the total tumor cells but have been found to be the origin of pancreatic cancer carcinogenesis and metastasis. Global proteome profiling of pancreatic CSCs from xenograft tumors in mice is a promising way to unveil the molecular machinery underlying the signaling pathways. However, the extremely low availability of pancreatic tissue CSCs (around 10,000 cells per xenograft tumor or patient sample) has limited the utilization of currently standard proteomic approaches which do not work effectively with such a small amount of material. Herein, we describe the profiling of the proteome of pancreatic CSCs using a capillary scale shotgun technique by coupling offline capillary isoelectric focusing(cIEF) with nano reversed phase liquid chromatography(RPLC) followed by spectral counting peptide quantification. A whole cell lysate from 10,000 cells which corresponds to approximately 1 microg of protein material is equally divided for three repeated cIEF separations where around 300 ng of peptide material is used in each run. In comparison with a nontumorigenic tumor cell sample, among 1159 distinct proteins identified with FDR less than 0.2%, 169 differentially expressed proteins are identified after multiple testing corrections where 24% of the proteins are upregulated in the CSCs group. Ingenuity Pathway analysis of these differential expression signatures further suggests significant involvement of signaling pathways related to apoptosis, cell proliferation, inflammation, and metastasis.

NFkappaB promotes inflammation, coagulation, and fibrosis in the aging glomerulus.

The peak prevalence of ESRD from glomerulosclerosis occurs at 70 to 79 years. To understand why old glomeruli are prone to failure, we analyzed the Fischer 344 rat model of aging under ad libitum-fed (rapid aging) and calorie-restricted (slowed aging) conditions. All glomerular cells contained genes whose expression changed "linearly" during adult life from 2 to 24 months: mesangial cells (e.g., MMP9), endothelial cells (e.g., ICAM and VCAM), parietal epithelial cells (e.g., ceruloplasmin), and podocytes (e.g., nephrin and prepronociceptin). Patterns of aging glomerular gene expression closely resembled atherosclerosis, including activation of endothelial cells, epithelial cells, and macrophages, as well as proinflammatory pathways related to cell adhesion, chemotaxis, blood coagulation, oxidoreductases, matrix metalloproteinases, and TGF-beta activation. We used a nonbiased data-mining approach to identify NFkappaB as the likely transcriptional regulator of these events. We confirmed NFkappaB activation by two independent methods: translocation of NFkappaB p50 to glomerular nuclei and ChIP assays demonstrating NFkappaB p50 binding to the kappaB motif of target genes in old versus young glomeruli. These data suggest that old glomeruli exhibit NFkappaB-associated up-regulation of a proinflammatory, procoagulable, and profibrotic phenotype compared with young glomeruli; these distinctions could explain their enhanced susceptibility to failure. Furthermore, these results provide a potential mechanistic explanation for the close relationship between ESRD and atherosclerotic organ failure as two parallel arms of age-associated NFkappaB-driven processes.

Identification of correlated genetic variants jointly associated with rheumatoid arthritis using ridge regression.

Using the North American Rheumatoid Arthritis Consortium genome-wide association dataset, we applied ridged, multiple least-squares regression to identify genetic variants with apparent unique contributions to variation of anti-cyclic citrullinated peptide (anti-CCP), a newly identified clinical risk factor for development of rheumatoid arthritis. Within a 2.7-Mbp region on chromosome 6 around the well studied HLA-DRB1 locus, ridge regression identified a single-nucleotide polymorphism that was associated with anti-CCP variation when including the additive effects of other single-nucleotide polymorphisms in a multivariable analysis, but that showed only a weak direct association with anti-CCP. This suggests that multivariable methods can be used to identify potentially relevant genetic variants in regions of interest that would be difficult to detect based on direct associations.