A multisite study to develop and validate first trimester, circulating microparticle biomarkers for tiered risk stratification of spontaneous preterm birth in nulliparas.

BACKGROUND: Despite much research, advances in early prediction of spontaneous preterm birth (sPTB) has been slow. The evolving field of circulating microparticle (CMP) biology may identify novel blood-based, and clinically useful, biomarkers. OBJECTIVE: To test the ability of a previously identified, 7-marker set of CMP-derived proteins from the first trimester of pregnancy, in the form of an in vitro diagnostic multivariate index assay (IVDMIA), to stratify pregnant patients according to their risk for sPTB. STUDY DESIGN: We employed a previously validated set of CMP protein biomarkers, utilizing mass spectrometry assays and a nested case-control design in a subset of participants from the Nulliparous Pregnancy Outcomes Study: monitoring mothers-to-be (nuMoM2b). We evaluated these biomarkers in the form of an IVDMIA to predict risk for sPTB at different gestational ages. Plasma samples collected at 9- to 13-weeks' gestation were analyzed. The IVDMIA assigned subjects to 1 of 3 sPTB risk categories: low risk (LR), moderate risk (MR), or high risk (HR). Independent validation on a set-aside set confirmed the IVDMIA's performance in risk stratification. RESULTS: Samples from 400 participants from the nuMoM2b cohort were used for the study; of these, 160 delivered<37 weeks and 240 delivered at term. Through Monte Carlo simulation in which the validation results were adjusted based on actual weekly sPTB incidence rates in the nuMoM2b cohort, the IVDMIA stratifications demonstrated statistically significant differences among the risk groups in time-to-event (birth) analysis (P<.0001). The incidence-rate adjusted cumulative risks of sPTB at ≤32 weeks' gestation were 0.4%, 1.6%, and 7.5%, respectively for the LR, MR, and HR groups, respectively. Compared to the LR group, the corresponding risk ratios of the IVDMIA assigned MR and HR group were 4.25 (95% confidence interval [CI] 2.2-7.9) and 19.92 (95% CI 10.4-37.4), respectively. CONCLUSION: A first trimester CMP protein biomarker panel can be used to stratify risk for sPTB at different gestational ages. Such a multitiered stratification tool could be used to assess risk early in pregnancy to enable timely clinical management and interventions, and, ultimately, to enable the development of tailored care pathways for sPTB prevention.

Circulating microparticle proteins obtained in the late first trimester predict spontaneous preterm birth at less than 35 weeks' gestation: a panel validation with specific characterization by parity.

BACKGROUND: We have previously shown that protein biomarkers associated with circulating microparticles proteins (CMPs) obtained at the end of the first trimester may detect physiologic changes in maternal-fetal interaction such that the risk of spontaneous preterm delivery ≤35 weeks can be stratified. OBJECTIVES: We present here a study extension and validation of the CMP protein multiplex concept using a larger sample set from a multicenter population that allows for model derivation in a training set and characterization in a separate testing set. MATERIALS AND METHODS: Ethylenediaminetetraacetic acid (EDTA) plasma was obtained from 3 established biobanks (Seattle, Boston, and Pittsburgh). Samples were from patients at a median of 10-12 weeks' gestation, and the CMPs were isolated via size-exclusion chromatography followed by protein identification via targeted protein analysis using liquid chromatography-multiple reaction monitoring-mass (LC-MRM) spectrometry. A total of 87 women delivered at ≤35 weeks, and 174 women who delivered at term were matched by maternal age (±2 years) and gestational age at sample draw (±2 weeks). From our prior work, the CMP protein multiplex comprising F13A, FBLN1, IC1, ITIH2, and LCAT was selected for validation. RESULTS: For delivery at ≤35 weeks, the receiver operating characteristic (ROC) curve for a panel of CMP proteins (F13A, FBLN1, IC1, ITIH2, and LCAT) revealed an associated area under the ROC curve (AUC) of 0.74 (95% CI, 0.63-0.81). A separate panel of markers (IC1, LCAT, TRFE, and ITIH4), which stratified risk among mothers with a parity of 0, showed an AUC of 0.77 (95% CI, 0.61-0.90). CONCLUSION: We have identified a set of CMP proteins that provide, at 10-12 weeks gestation, a clinically useful AUC in an independent test population. Furthermore, we determined that parity is pertinent to the diagnostic testing performance of the biomarkers for risk stratification.

Evaluation of proteomic biomarkers associated with circulating microparticles as an effective means to stratify the risk of spontaneous preterm birth.

BACKGROUND: The analysis of circulating microparticles in pregnancy is of revolutionary potential because it represents an in vivo biopsy of active gestational tissues. OBJECTIVE: We hypothesized that circulating microparticle signaling will differ in pregnancies that experience spontaneous preterm birth from those delivering at term and that these differences will be evident many weeks in advance of clinical presentation. STUDY DESIGN: Utilizing plasma specimens obtained between 10 and 12 weeks' gestation as part of a prospectively collected birth cohort in which pregnancy outcomes are independently validated by 2 board-certified maternal-fetal medicine physicians, 25 singleton cases of spontaneous preterm birth ≤ 34 weeks were matched by maternal age, race, and gestational age of sampling (±2 weeks) with 50 uncomplicated term deliveries. Circulating microparticles from these first-trimester specimens were isolated and analyzed by multiple reaction monitoring mass spectrometry for potential protein biomarkers following previous studies. Markers with robust univariate performance in correlating spontaneous preterm birth were further evaluated for their biological relevance via a combined functional profiling/pathway analysis and for multivariate performance. RESULTS: Among the 132 proteins evaluated, 62 demonstrated robust power of detecting spontaneous preterm birth in a bootstrap receiver-operating characteristic curve analysis at a false discovery rate of < 20% estimated via label permutation. Differential dependency network analysis identified spontaneous preterm birth-associated coexpression patterns linked to biological processes of inflammation, wound healing, and the coagulation cascade. Linear modeling of spontaneous preterm birth using a multiplex of the candidate biomarkers with a fixed sensitivity of 80% exhibited a specificity of 83% with median area under the curve of 0.89. These results indicate a strong potential of multivariate model development for informative risk stratification. CONCLUSION: This project has identified functional proteomic factors with associated biological processes that are already unique in their expression profiles at 10-12 weeks among women who go on to deliver spontaneously ≤ 34 weeks. These changes, with further validation, will allow the stratification of patients at risk of spontaneous preterm birth before clinical presentation.

Performance evaluation of a SARS-CoV-2 and influenza A/B combo rapid antigen test.

Introduction: The global COVID-19 pandemic and seasonal influenza outbreaks have drawn attention to the critical need for accurate and efficient diagnostic tools. Methods: The performance of the InstaView COVID-19/Flu Ag Combo Test, which was designed to simultaneously detect the SARS-CoV-2, influenza A, and influenza B viruses, was analytically and clinically evaluated. Results: The InstaView COVID-19/Flu Ag Combo Test exhibited robust detection capabilities, accurately identifying SARS-CoV-2, influenza A, and influenza B viruses over a wide concentration range (1.41 × 103 to 7.05 × 104 TCID50/mL). Extensive testing against potential cross-reactants and interferences yielded no false-positive results, indicating the high specificity of the test. Clinical evaluation further confirmed the kit's reliability, with sensitivity ranging from 95.1% to 98.2% for SARS-CoV-2, 88.9%-95.2% for influenza A, and 91.7%-100% for influenza B depending on the sample type. The specificity was consistently 100% for all of the targeted viruses. Discussion: The InstaView COVID-19/Flu Ag Combo Test thus demonstrated high performance, ease of use, rapid results, and the ability to precisely detect SARS-CoV-2 and influenza A/B infections, making it an effective tool in streamlining diagnostic workflows, optimizing resource allocation, and improving patient outcomes.

Klotho ameliorates chemically induced endoplasmic reticulum (ER) stress signaling.

BACKGROUND: Both endoplasmic reticulum (ER) stress, a fundamental cell response associated with stress-initiated unfolded protein response (UPR), and loss of Klotho, an anti-aging hormone linked to NF-κB-induced inflammation, occur in chronic metabolic diseases such as obesity and type 2 diabetes. We investigated if the loss of Klotho is causally linked to increased ER stress. METHODS: We treated human renal epithelial HK-2, alveolar epithelial A549, HEK293, and SH-SH-SY5Y neuroblastoma cells with ER stress-inducing agents, thapsigargin and/or tunicamycin. Effects of overexpression or siRNA-mediated knockdown of Klotho on UPR signaling was investigated by immunoblotting and Real-time PCR. RESULTS: Elevated Klotho levels in HK-2 cells decreased expression of ER stress markers phospho--IRE1, XBP-1s, BiP, CHOP, pJNK, and phospho-p38, all of which were elevated in response to tunicamycin and/or thapsigargin. Similar results were observed using A549 cells for XBP-1s, BiP, and CHOP in response to thapsigargin. Conversely, knockdown of Klotho in HEK 293 cells using siRNA caused further thapsigargin-induced increases in pIRE-1, XBP-1s, and BiP. Klotho overexpression in A549 cells blocked thapsigargin-induced caspase and PARP cleavage and improved cell viability. CONCLUSION: Our data indicate that Klotho has an important role in regulating ER stress and that loss of Klotho is causally linked to ER stress-induced apoptosis.

Long-term alterations in maternal plasma proteome after sFlt1-induced preeclampsia in mice.

OBJECTIVE: Preeclampsia is associated with long-term adverse maternal health, such as cardiovascular and metabolic diseases. The objective of this study was to determine whether preeclampsia in a well-characterized animal model that was induced by overexpression of soluble fms-like tyrosine kinase-1 (sFlt1) results in alterations in the maternal circulating proteome that persist long after delivery. STUDY DESIGN: CD-1 mice at day 8 of gestation were injected with adenovirus that carried sFlt1 or the murine immunoglobulin G2α Fc fragment as control. Depleted maternal plasma was analyzed 6 months after delivery by label-free liquid chromatography-mass spectrometry assay. The tandem mass spectrometry data were searched against a mouse database, and the resultant intensity data were used to compare abundance of proteins across disease/control plasma pool. Results were analyzed with ingenuity pathways analysis. Right-tailed Fisher exact test was used to calculate a probability value. RESULTS: Of 150 proteins that are common for both groups, ingenuity pathways analysis determined 105 proteins that were ready for analysis. Diseases and disorders analysis showed significant enrichment of proteins that are associated with cardiovascular disease. Within this cluster, the most abundant proteins were associated with vascular disease, atherosclerosis, and atherosclerotic lesions. Other top disease clusters were inflammatory response, organismal injury and abnormalities, and hematologic and metabolic disease. CONCLUSION: Exposure to sFlt1-induced preeclampsia alters multiple biologic functions in mothers that persist later in life. Our results suggest that some of the long-term adverse outcomes that are associated with preeclampsia actually may be a consequence rather than a mere unmasking of an underlying predisposition. If similar results are found in humans, the development of preventive strategies for preeclampsia should also improve long-term maternal health.

Circulating microparticle proteins predict pregnancies complicated by placenta accreta spectrum.

Placenta accreta spectrum (PAS) is characterized by abnormal attachment of the placenta to the uterus, and attempts at placental delivery can lead to catastrophic maternal hemorrhage and death. Multidisciplinary delivery planning can significantly improve outcomes; however, current diagnostics are lacking as approximately half of pregnancies with PAS are undiagnosed prior to delivery. This is a nested case-control study of 35 cases and 70 controls with the primary objective of identifying circulating microparticle (CMP) protein panels that identify pregnancies complicated by PAS. Size exclusion chromatography and liquid chromatography with tandem mass spectrometry were used for CMP protein isolation and identification, respectively. A two-step iterative workflow was used to establish putative panels. Using plasma sampled at a median of 26 weeks' gestation, five CMP proteins distinguished PAS from controls with a mean area under the curve (AUC) of 0.83. For a separate sample taken at a median of 35 weeks' gestation, the mean AUC was 0.78. In the second trimester, canonical pathway analyses demonstrate over-representation of processes related to iron homeostasis and erythropoietin signaling. In the third trimester, these analyses revealed abnormal immune function. CMP proteins classify PAS well prior to delivery and have potential to significantly reduce maternal morbidity and mortality.

High throughput short interfering RNA (siRNA) screening of the human kinome identifies novel kinases controlling the canonical nuclear factor-κB (NF-κB) activation pathway.

Nuclear factor-κB (NF-κB) is an inducible cytoplasmic transcription factor that plays a role as a master regulator of airway mucosal inflammation. The prototypical ("canonical") NF-κB pathway controls cytoplasmic to nuclear translocation in response to stimulation by the mononuclear cytokine, TNF. Despite intensive investigation, the spectrum of kinases involved in the canonical NF-κB pathway has not yet been systematically determined. Here we have applied a high throughput siRNA-mediated loss-of-function screening assay to identify novel kinases important in TNF-induced NF-κB signaling. Type II A549 epithelial cells stably expressing an IL-8/luciferase reporter gene optimized for high throughput siRNA format (Z' score of 0.65) and siRNAs for 636 human kinases were reverse-transfected and screened in the assay. 36 candidate genes were identified that inhibited TNF signaling with a Z score deviation of <-1.3 in replicate plates. From this group, 11 kinases were selected for independent validation, of which eight were successfully silenced. Six kinases were validated, including ATM, CDK2, -5, and -7, CALM3, MAPAKP5, and MAP3K/MEKK3. The surprising function of ATM in TNF signaling was confirmed where reduced NF-κB/RelA translocation and Ser-276 phosphorylation were seen in ATM(-/-) mouse embryo fibroblasts. These data indicate that ATM is a key regulatory kinase that may control global NF-κB activation in the TNF-induced canonical pathway.

Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma.

Gene expression profiling identified human melanoma cells demonstrating increased cell motility and invasiveness. The gene WNT5A best determined in vitro invasive behavior. Melanoma cells were transfected with vectors constitutively overexpressing Wnt5a. Consistent changes included actin reorganization and increased cell adhesion. No increase in beta-catenin expression or nuclear translocation was observed. There was, however, a dramatic increase in activated PKC. In direct correlation with Wnt5a expression and PKC activation, there was an increase in melanoma cell invasion. Blocking this pathway using antibodies to Frizzled-5, the receptor for Wnt5a, inhibited PKC activity and cellular invasion. Furthermore, Wnt5a expression in human melanoma biopsies directly correlated to increasing tumor grade. These observations support a role for Wnt5a in human melanoma progression.

Protein microarrays: meeting analytical challenges for clinical applications.

Protein microarrays, one emerging class of proteomic technologies, have broad applications for discovery and quantitative analysis. A rapidly expanding use of this technology is the acquisition of information about the posttranslational modifications of proteins reflecting the activity state of signal pathways and networks, and is now employed for the analysis of biopsy samples in clinical trial research.

Comprehensive mapping of the human kinome to epidermal growth factor receptor signaling.

Disregulation of epidermal growth factor receptor (EGFR) signaling directly promotes bypass of proliferation and survival restraints in a high frequency of epithelia-derived cancer. As such, much effort is currently focused on decoding the molecular architecture supporting EGFR activation and function. Here, we have leveraged high throughput reverse phase protein lysate arrays, with a sensitive fluorescent nanocrystal-based phosphoprotein detection assay, together with large scale siRNA-mediated loss of function to execute a quantitative interrogation of all elements of the human kinome supporting EGF-dependent signaling. This screening platform has captured multiple novel contributions of diverse protein kinases to modulation of EGFR signal generation, signal amplitude, and signal duration. As examples, the prometastatic SNF1/AMPK-related kinase hormonally upregulated Neu kinase was found to support EGFR activation in response to ligand binding, whereas the enigmatic kinase MGC16169 selectively supports coupling of active EGFR to ERK1/2 regulation. Of note, the receptor tyrosine kinase MERTK and the pyrimidine kinase UCK1 were both found to be required for surface accumulation of EGFR and subsequent pathway activation in multiple cancer cell backgrounds and may represent new targets for therapeutic intervention.

Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule.

Klotho has profound effects on phosphate metabolism, but the mechanisms of how Klotho affects phosphate homeostasis is unknown. We detected Klotho in the proximal tubule cell, brush border, and urinary lumen, where phosphate homeostasis resides. Increasing Klotho in the kidney and urine chronically by transgenic overexpression or acutely by intravenous infusion caused hypophosphatemia, phosphaturia from decreased proximal phosphate reabsorption, and decreased activity and protein of the principal renal phosphate transporter NaPi-2a. The phosphaturic effect was present in FGF23-null mice, indicating a direct action distinct from Klotho's known role as a coreceptor for FGF23. Direct inhibition of NaPi-2a by Klotho was confirmed in cultured cells and in cell-free membrane vesicles characterized by acute inhibition of transport activity followed by decreased cell surface protein. Transport inhibition can be mimicked by recombinant beta-glucuronidase and is associated with proteolytic degradation and reduced surface NaPi-2a. The inhibitory effect of Klotho on NaPi-2a was blocked by beta-glucuronidase inhibitor but not by protease inhibitor. Klotho is a novel phosphaturic substance that acts as an enzyme in the proximal tubule urinary lumen by modifying glycans, which cause decreased transporter activity, followed by proteolytic degradation and possibly internalization of NaPi-2a from the apical membrane.

Removal of sialic acid involving Klotho causes cell-surface retention of TRPV5 channel via binding to galectin-1.

Klotho is a mammalian senescence-suppression protein that has homology with glycosidases. The extracellular domain of Klotho is secreted into urine and blood and may function as a humoral factor. Klotho-deficient mice have accelerated aging and imbalance of ion homeostasis. Klotho treatment increases cell-surface abundance of the renal epithelial Ca(2+) channel TRPV5 by modifying its N-linked glycans. However, the precise sugar substrate and mechanism for regulation by Klotho is not known. Here, we report that the extracellular domain of Klotho activates plasma-membrane resident TRPV5 through removing terminal sialic acids from their glycan chains. Removal of sialic acids exposes underlying disaccharide galactose-N-acetylglucosamine, a ligand for a ubiquitous galactoside-binding lectin galectin-1. Binding to galectin-1 lattice at the extracellular surface leads to accumulation of functional TRPV5 on the plasma membrane. Knockdown of beta-galactoside alpha2,6-sialyltransferase (ST6Gal-1) by RNA interference, but not other sialyltransferases, in a human cell line prevents the regulation by Klotho. Moreover, the regulation by Klotho is absent in a hamster cell line that lacks endogenous ST6Gal-1, but is restored by forced expression of recombinant ST6Gal-1. Thus, Klotho participates in specific removal of alpha2,6-linked sialic acids and regulates cell surface retention of TRPV5 through this activity. This action of Klotho represents a novel mechanism for regulation of the activity of cell-surface glycoproteins and likely contributes to maintenance of calcium balance by Klotho.

Vitamin D Assessment Over 48 Weeks in Treatment-Naive HIV Individuals Starting Lopinavir/Ritonavir Monotherapy.

BACKGROUND: Vitamin D deficiency is common in HIV population and has been associated with increased comorbidity risk and poor immunologic status. OBJECTIVE: To evaluate the effect of protease inhibitor lopinavir/ritonavir monotherapy on changes in serum 25-hydroxyvitamin D [25(OH)D] over 48 weeks. METHODS: Thirty-four treatment-naïve HIV individuals initiating lopinavir/ritonavir monotherapy and receiving clinical care from private practice in Houston, Texas, were included. Serum 25-hydroxyvitamin D levels from stored plasma samples collected from IMANI-2 pilot study at both baseline and 48 weeks were analyzed using LC-MS assays. Mean 25(OH)D at baseline and 48 weeks were compared using paired t-tests. Linear regression analysis was used to evaluate factors associated with changes in 25(OH)D. Logistic regression analyses were used to determine the effect of vitamin D status and covariates on CD4 cell count recovery. RESULTS: Mean 25(OH)D was significantly higher at 48 weeks (26.3 ng/mL (SD + 14.9); p=0.0003) compared to baseline (19.8 ng/mL (SD +12.1), with fewer individuals having vitamin D deficiency (41.2%) and severe deficiency (11.8%). Both body mass index and baseline CD4 cell count were significant independent covariates associated with 25(OH)D changes over 48 weeks. Baseline vitamin D status did not affect CD4 cell count recovery. However, in a 24-week multivariate analysis, current tobacco use was significantly associated with a decreased odds of CD4 cell count recovery (AOR 0.106, 95% CI 0.018-0.606; p=0.012). CONCLUSION: Individuals treated with lopinavir/ritonavir monotherapy had significantly higher 25(OH)D after 48 weeks. Current tobacco users had significantly diminished CD4 cell count recovery after starting treatment, warranting further clinical investigation.

Hyperaldosteronism in Klotho-deficient mice.

Klotho is a membrane protein participating in the inhibitory effect of FGF23 on the formation of 1,25-dihydroxyvitamin-D(3) [1,25(OH)(2)D(3)]. It participates in the regulation of renal tubular phosphate reabsorption and stimulates renal tubular Ca(2+) reabsorption. Klotho hypomorphic mice (klotho(hm)) suffer from severe growth deficit, rapid aging, and early death, events largely reversed by a vitamin D-deficient diet. The present study explored the role of Klotho deficiency in mineral and electrolyte metabolism. To this end, klotho(hm) mice and wild-type mice (klotho(+/+)) were subjected to a normal (D(+)) or vitamin D-deficient (D(-)) diet or to a vitamin D-deficient diet for 4 wk and then to a normal diet (D(-/+)). At the age of 8 wk, body weight was significantly lower in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice, klotho(hm)D(-) mice, and klotho(hm)D(-/+) mice. Plasma concentrations of 1,25(OH)(2)D(3,) adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH), and aldosterone were significantly higher in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice. Plasma volume was significantly smaller in klotho(hm)D(-/+) mice, and plasma urea, Ca(2+), phosphate and Na(+), but not K(+) concentrations were significantly higher in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice. The differences were partially abrogated by a vitamin D-deficient diet. Moreover, the hyperaldosteronism was partially reversed by Ca(2+)-deficient diet. Ussing chamber experiments revealed a marked increase in amiloride-sensitive current across the colonic epithelium, pointing to enhanced epithelial sodium channel (ENaC) activity. A salt-deficient diet tended to decrease and a salt-rich diet significantly increased the life span of klotho(hm)D(+) mice. In conclusion, the present observation disclose that the excessive formation of 1,25(OH)(2)D(3) in Klotho-deficient mice results in extracellular volume depletion, which significantly contributes to the shortening of life span.

Late first trimester circulating microparticle proteins predict the risk of preeclampsia < 35 weeks and suggest phenotypic differences among affected cases.

We hypothesize that first trimester circulating micro particle (CMP) proteins will define preeclampsia risk while identifying clusters of disease subtypes among cases. We performed a nested case-control analysis among women with and without preeclampsia. Cases diagnosed < 34 weeks' gestation were matched to controls. Plasma CMPs were isolated via size exclusion chromatography and analyzed using global proteome profiling based on HRAM mass spectrometry. Logistic models then determined feature selection with best performing models determined by cross-validation. K-means clustering examined cases for phenotypic subtypes and biological pathway enrichment was examined. Our results indicated that the proteins distinguishing cases from controls were enriched in biological pathways involved in blood coagulation, hemostasis and tissue repair. A panel consisting of C1RL, GP1BA, VTNC, and ZA2G demonstrated the best distinguishing performance (AUC of 0.79). Among the cases of preeclampsia, two phenotypic sub clusters distinguished cases; one enriched for platelet degranulation and blood coagulation pathways and the other for complement and immune response-associated pathways (corrected p < 0.001). Significantly, the second of the two clusters demonstrated lower gestational age at delivery (p = 0.049), increased protein excretion (p = 0.01), more extreme laboratory derangement (p < 0.0001) and marginally increased diastolic pressure (p = 0.09). We conclude that CMP-associated proteins at 12 weeks' gestation predict the overall risk of developing early preeclampsia and indicate distinct subtypes of pathophysiology and clinical morbidity.

Application of multiple statistical tests to enhance mass spectrometry-based biomarker discovery.

BACKGROUND: Mass spectrometry-based biomarker discovery has long been hampered by the difficulty in reconciling lists of discriminatory peaks identified by different laboratories for the same diseases studied. We describe a multi-statistical analysis procedure that combines several independent computational methods. This approach capitalizes on the strengths of each to analyze the same high-resolution mass spectral data set to discover consensus differential mass peaks that should be robust biomarkers for distinguishing between disease states. RESULTS: The proposed methodology was applied to a pilot narcolepsy study using logistic regression, hierarchical clustering, t-test, and CART. Consensus, differential mass peaks with high predictive power were identified across three of the four statistical platforms. Based on the diagnostic accuracy measures investigated, the performance of the consensus-peak model was a compromise between logistic regression and CART, which produced better models than hierarchical clustering and t-test. However, consensus peaks confer a higher level of confidence in their ability to distinguish between disease states since they do not represent peaks that are a result of biases to a particular statistical algorithm. Instead, they were selected as differential across differing data distribution assumptions, demonstrating their true discriminatory potential. CONCLUSION: The methodology described here is applicable to any high-resolution MALDI mass spectrometry-derived data set with minimal mass drift which is essential for peak-to-peak comparison studies. Four statistical approaches with differing data distribution assumptions were applied to the same raw data set to obtain consensus peaks that were found to be statistically differential between the two groups compared. These consensus peaks demonstrated high diagnostic accuracy when used to form a predictive model as evaluated by receiver operating characteristics curve analysis. They should demonstrate a higher discriminatory ability as they are not biased to a particular algorithm. Thus, they are prime candidates for downstream identification and validation efforts.

Accelerated suicidal erythrocyte death in Klotho-deficient mice.

Klotho, a membrane protein mainly expressed in parathyroid glands, kidney, and choroid plexus, counteracts aging and increases the life span. Accordingly, life span is significantly shorter in Klotho-deficient mice (klotho(-/-)) than in their wild-type littermates (klotho(+/+)). The pleotropic effects of Klotho include inhibition of 1,25-dihydroxyvitamin D(3)(1,25(OH)(2)D(3)) formation. Vitamin D-deficient diet reverses the shortening of life span in klotho(-/-) mice. In a variety of cells, 1,25(OH)(2)D(3) stimulates Ca(2+) entry. In erythrocytes, increased Ca(2+) entry stimulates suicidal erythrocyte death, which is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. The present study explored the putative impact of Klotho on eryptosis. According to Fluo3 fluorescence, cytosolic Ca(2+) concentration was significantly larger in klotho(-/-) erythrocytes as compared to klotho(+/+) erythrocytes. According to annexin V-binding, phosphatidylserine exposure was significantly enhanced, and according to forward scatter, cell volume significantly decreased in klotho(-/-) erythrocytes as compared to klotho(+/+) erythrocytes. Energy depletion (13 h glucose depletion) and oxidative stress (35 min 1 mM tert-butyl-hydroxyl-peroxide [tert-BOOH]) increased phosphatidylserine exposure to values again significantly larger in klotho(-/-) erythrocytes as compared to klotho(+/+) erythrocytes. Reticulocyte number was significantly increased in klotho (-/-) mice, pointing to enhanced erythrocyte turnover. Vitamin D-deficient diet reversed the enhanced Ca(2+) entry and annexin V-binding of klotho(-/-) erythrocytes. The present observations reveal a novel function of Klotho, i.e., the at least partially vitamin D-dependent regulation of cytosolic Ca(2+) activity in and suicidal death of erythrocytes.

Biomarker selection and sample prediction for multi-category disease on MALDI-TOF data.

MOTIVATION: Diseases normally progress through several stages. Therefore, biomarkers corresponding to each stage may exist. To deal with such a multi-category problem, including sample stage prediction and biomarker selection, we propose methods for classification and feature selection. The proposed classification method is based on two schemes: error-correcting output coding (ECOC) and pairwise coupling (PWC). The final decision for a test sample prediction is an integration of these two schemes. The biomarker pattern for distinguishing each disease category from another one is achieved by the development of an extended Markov blanket (EMB) feature selection method. RESULTS: In this study, a liver cancer matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) dataset was used, which comprises hepatocellular carcinoma (HCC), cirrhosis, and healthy spectra. Peak patterns were discovered for distinguishing pairwise categories among the three classes. Importance and reliability of individual peaks were presented by the measurements of certain weight values and frequencies. The classification capability of the proposed approach was compared with classical ECOC, random forest, Naive Bayes, and J48 methods. AVAILABILITY: Supplementary materials are available at http://visionlab.uta.edu/biomarker/bioinfo.htm.

Validation and Clinical Applications of a Comprehensive Next Generation Sequencing System for Molecular Characterization of Solid Cancer Tissues.

Identification of somatic molecular alterations in primary and metastatic solid tumor specimens can provide critical information regarding tumor biology and its heterogeneity, and enables the detection of molecular markers for clinical personalized treatment assignment. However, the optimal methods and target genes for clinical use are still being in development. Toward this end, we validated a targeted amplification-based NGS panel (Oncomine comprehensive assay v1) on a personal genome machine sequencer for molecular profiling of solid tumors. This panel covers 143 genes, and requires low amounts of DNA (20 ng) and RNA (10 ng). We used 27 FFPE tissue specimens, 10 cell lines, and 24 commercial reference materials to evaluate the performance characteristics of this assay. We also evaluated the performance of the assay on 26 OCT-embedded fresh frozen specimens (OEFF). The assay was found to be highly specific (>99%) and sensitive (>99%), with low false-positive and false-negative rates for single-nucleotide variants, indels, copy number alterations, and gene fusions. Our results indicate that this is a reliable method to determine molecular alterations in both fixed and fresh frozen solid tumor samples, including core needle biopsies.