Reproducible cancer biomarker discovery in SELDI-TOF MS using different pre-processing algorithms

Background

There has been much interest in differentiating diseased and normal samples using biomarkers derived from mass spectrometry (MS) studies. However, biomarker identification for specific diseases has been hindered by irreproducibility. Specifically, a peak profile extracted from a dataset for biomarker identification depends on a data pre-processing algorithm. Until now, no widely accepted agreement has been reached.

Results

In this paper, we investigated the consistency of biomarker identification using differentially expressed (DE) peaks from peak profiles produced by three widely used average spectrum-dependent pre-processing algorithms based on SELDI-TOF MS data for prostate and breast cancers. Our results revealed two important factors that affect the consistency of DE peak identification using different algorithms. One factor is that some DE peaks selected from one peak profile were not detected as peaks in other profiles, and the second factor is that the statistical power of identifying DE peaks in large peak profiles with many peaks may be low due to the large scale of the tests and small number of samples. Furthermore, we demonstrated that the DE peak detection power in large profiles could be improved by the stratified false discovery rate (FDR) control approach and that the reproducibility of DE peak detection could thereby be increased.

Conclusions

Comparing and evaluating pre-processing algorithms in terms of reproducibility can elucidate the relationship among different algorithms and also help in selecting a pre-processing algorithm. The DE peaks selected from small peak profiles with few peaks for a dataset tend to be reproducibly detected in large peak profiles, which suggests that a suitable pre-processing algorithm should be able to produce peaks sufficient for identifying useful and reproducible biomarkers.     

Reduction of plasma gelsolin levels correlates with development of multiple organ dysfunction syndrome and fatal outcome in burn patients

Background

Depletion of the circulating actin-binding protein, plasma gelsolin (pGSN) has been described in critically ill surgical patients. We hypothesized that the extent of pGSN reduction might correlate with different outcome of burn patients. The study was performed to evaluate the prognostic implications of pGSN levels on the development of multiple organ dysfunction syndrome (MODS) and fatal outcome in a group of severely burn patients.

Methods and Findings

95 patients were included, and they were divided into three groups with different burn area: group I (n = 33), group II (n = 32) and group III (n = 30). According to whether there was development of MODS or not, patients were divided into MODS group (n = 28) and none-MODS group (n = 67); then the patients with MODS were further divided into non-survivor group (n = 17) and survivor group (n = 11). The peripheral blood samples were collected on postburn days (PBD) 1, 3, 7, 14, and 21. The levels of pGSN were determined and T cells were procured from the blood. The contents of cytokines (IL-2, IL-4 and IFN-γ) released by T cells were also measured. The related factors of prognosis were analyzed by using multivariate logistic regression analysis. The results showed that pGSN concentrations, as well as the levels of IL-2 and IFN-γ, decreased markedly on PBD 1–21, whereas, the levels of IL-4 increased markedly in all burn groups as compared with normal controls (P<0.05 or P<0.01), and there were obviously differences between group I and group III (P<0.05 or P<0.01). The similar results were found in MODS patients and the non-survivor group as compared with those without MODS and the survival group on days 3–21 postburn (P<0.05 or P<0.01). Moreover, as the pGSN levels decreased, the incidence of septic complication as well as MODS remarkably increased.

Conclusions

pGSN levels appear to be an early prognostic marker in patients suffering from major burns.     

Early elevation of matrix metalloproteinase-8 and -9 in pediatric ARDS is associated with an increased risk of prolonged mechanical ventilation

Background

Matrix metalloproteinases (MMP) -8 and -9 may play key roles in the modulation of neutrophilic lung inflammation seen in pediatric Acute Respiratory Distress Syndrome (ARDS). We aimed to perform a comprehensive analysis of MMP-8 and MMP-9 activity in tracheal aspirates of pediatric ARDS patients compared with non-ARDS controls, testing whether increased MMP-8 and -9 activities were associated with clinical outcomes.

Methods

Tracheal aspirates were collected from 33 pediatric ARDS patients and 21 non-ARDS controls at 48 hours of intubation, and serially for those who remained intubated greater than five days. MMPs, tissue inhibitor of metalloproteinases (TIMPs), human neutrophil elastase (HNE) and myeloperoxidase (MPO) activity were measured by ELISA, and correlated with clinical indicators of disease severity such as PRISM (Pediatric Risk of Mortality) scores, oxygen index (OI), multi-organ system failure (MOSF) and clinical outcome measures including length of intubation, ventilator-free days (VFDs) and mortality in the Pediatric Intensive Care Unit (PICU).

Results

Active MMP-9 was elevated early in pediatric ARDS subjects compared to non-ARDS controls. Higher MMP-8 and active MMP-9 levels at 48 hours correlated with a longer course of mechanical ventilation (r = 0.41, p = 0.018 and r = 0.75, p<0.001; respectively) and fewer number of VFDs (r = −0.43, p = 0.013 and r = −0.76, p<0.001; respectively), independent of age, gender and severity of illness. Patients with the highest number of ventilator days had the highest levels of active MMP-9. MMP-9 and to a lesser extent MMP-8 activities in tracheal aspirates from ARDS subjects were sensitive to blockade by small molecule inhibitors.

Conclusions

Higher MMP-8 and active MMP-9 levels at 48 hours of disease onset are associated with a longer duration of mechanical ventilation and fewer ventilator-free days among pediatric patients with ARDS. Together, these results identify early biomarkers predictive of disease course and potential therapeutic targets for this life threatening disease.     

A conserved cysteine motif is critical for rice ceramide kinase activity and function

Background

Ceramide kinase (CERK) is a key regulator of cell survival in dicotyledonous plants and animals. Much less is known about the roles of CERK and ceramides in mediating cellular processes in monocot plants. Here, we report the characterization of a ceramide kinase, OsCERK, from rice (Oryza sativa spp. Japonica cv. Nipponbare) and investigate the effects of ceramides on rice cell viability.

Principal Findings

OsCERK can complement the Arabidopsis CERK mutant acd5. Recombinant OsCERK has ceramide kinase activity with Michaelis-Menten kinetics and optimal activity at 7.0 pH and 40°C. Mg²⁺ activates OsCERK in a concentration-dependent manner. Importantly, a CXXXCXXC motif, conserved in all ceramide kinases and important for the activity of the human enzyme, is critical for OsCERK enzyme activity and in planta function. In a rice protoplast system, inhibition of CERK leads to cell death and the ratio of added ceramide and ceramide-1-phosphate, CERK''s substrate and product, respectively, influences cell survival. Ceramide-induced rice cell death has apoptotic features and is an active process that requires both de novo protein synthesis and phosphorylation, respectively. Finally, mitochondria membrane potential loss previously associated with ceramide-induced cell death in Arabidopsis was also found in rice, but it occurred with different timing.

Conclusions

OsCERK is a bona fide ceramide kinase with a functionally and evolutionarily conserved Cys-rich motif that plays an important role in modulating cell fate in plants. The vital function of the conserved motif in both human and rice CERKs suggests that the biochemical mechanism of CERKs is similar in animals and plants. Furthermore, ceramides induce cell death with similar features in monocot and dicot plants.     

Altered expression of insulin receptor isoforms in breast cancer

Purpose

Insulin-like growth factor (IGF) signaling through human insulin receptor isoform A (IR-A) contributes to tumorigenesis and intrinsic resistance to anti-IGF1R therapy. In the present study, we (a) developed quantitative TaqMan real time-PCR-based assays (qRT-PCR) to measure human insulin receptor isoforms with high specificity, (b) evaluated isoform expression levels in molecularly-defined breast cancer subtypes, and (c) identified the IR-A:IR-B mRNA ratio as a potential biomarker guiding patient stratification for anti-IGF therapies.

Experimental Design

mRNA expression levels of IR-A and IR-B were measured in 42 primary breast cancers and 19 matched adjacent normal tissues with TaqMan qRT-PCR assays. The results were further confirmed in 165 breast cancers. The tumor samples were profiled using whole genome microarrays and subsequently subtyped using the PAM50 breast cancer gene signature. The relationship between the IR-A:IR-B ratio and cancer subtype, as well as markers of proliferation were characterized.

Results

The mRNA expression levels of IR-A in the breast tumors were similar to those observed in the adjacent normal tissues, while the mRNA levels of IR-B were significantly decreased in tumors. The IR-A:IR-B ratio was significantly higher in luminal B breast cancer than in luminal A. Strong concordance between the IR-A:IR-B ratio and the composite Oncotype DX proliferation score was observed for stratifying the latter two breast cancer subtypes.

Conclusions

The reduction in IR-B expression is the key to the altered IR-A:IR-B ratio observed in breast cancer. The IR-A:IR-B ratio may have biomarker utility in guiding a patient stratification strategy for an anti-IGF therapeutic.     

Altered connectivity pattern of hubs in default-mode network with Alzheimer's disease: an Granger causality modeling approach

Background

Evidences from normal subjects suggest that the default-mode network (DMN) has posterior cingulate cortex (PCC), medial prefrontal cortex (MPFC) and inferior parietal cortex (IPC) as its hubs; meanwhile, these DMN nodes are often found to be abnormally recruited in Alzheimer''s disease (AD) patients. The issues on how these hubs interact to each other, with the rest nodes of the DMN and the altered pattern of hubs with respect to AD, are still on going discussion for eventual final clarification.

Principal Findings

To address these issues, we investigated the causal influences between any pair of nodes within the DMN using Granger causality analysis and graph-theoretic methods on resting-state fMRI data of 12 young subjects, 16 old normal controls and 15 AD patients respectively. We found that: (1) PCC/MPFC/IPC, especially the PCC, showed the widest and distinctive causal effects on the DMN dynamics in young group; (2) the pattern of DMN hubs was abnormal in AD patients compared to old control: MPFC and IPC had obvious causal interaction disruption with other nodes; the PCC showed outstanding performance for it was the only region having causal relation with all other nodes significantly; (3) the altered relation between hubs and other DMN nodes held potential as a noninvasive biomarker of AD.

Conclusions

Our study, to the best of our knowledge, is the first to support the hub configuration of the DMN from the perspective of causal relationship, and reveal abnormal pattern of the DMN hubs in AD. Findings from young subjects provide additional evidence for the role of PCC/MPFC/IPC acting as hubs in the DMN. Compared to old control, MPFC and IPC lost their roles as hubs owing to the obvious causal interaction disruption, and PCC was preserved as the only hub showing significant causal relations with all other nodes.     

Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells

Background

Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined.

Methodology/Principal Findings

Distinct cytoplasmic rods (∼3–10 µm in length) and rings (∼2–5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin.

Conclusions/Significance

RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.     

Association of adiponectin SNP+45 and SNP+276 with type 2 diabetes in Han Chinese populations: a meta-analysis of 26 case-control studies

Recently, many studies have reported that the SNP+45(T>G) and SNP+276(G>T) polymorphisms in the adiponectin gene are associated with type 2 diabetes (T2DM) in the Chinese Han population. However, the previous studies yielded many conflicting results. Thus, a meta-analysis of the association of the adiponectin gene with T2DM in the Chinese Han population is required. In the current study, we first determined the distribution of the adiponectin SNP+276 polymorphism in T2DM and nondiabetes (NDM) control groups. Our results suggested that the genotype and allele frequencies for SNP+276 did not differ significantly between the T2DM and NDM groups. Then, a meta-analysis of 23 case-control studies of SNP+45, with a total of 4161 T2DM patients and 3709 controls, and 11 case-control studies of SNP+276, with 2533 T2DM patients and 2212 controls, was performed. All subjects were Han Chinese. The fixed-effects model and random-effects model were applied for dichotomous outcomes to combine the results of the included studies. The results revealed a trend towards an increased risk of T2DM for the SNP+45G allele as compared with the SNP+45T allele (OR = 1.34; 95% CI, 1.11–1.62; P<0.01) in the Chinese Han population. However, there was no association between SNP+276 and T2DM (OR = 0.90; 95% CI, 0.73–1.10; P = 0.31). The results of our association study showed there was no association between the adiponectin SNP+276 polymorphism and T2DM in the Yunnan Han population. The meta-analysis results suggested that the SNP+45G allele might be a susceptibility allele for T2DM in the Chinese Han population. However, we did not observe an association between SNP+276 and T2DM.     

Genome dynamics and diversity of Shigella species, the etiologic agents of bacillary dysentery

The Shigella bacteria cause bacillary dysentery, which remains a significant threat to public health. The genus status and species classification appear no longer valid, as compelling evidence indicates that Shigella, as well as enteroinvasive Escherichia coli, are derived from multiple origins of E.coli and form a single pathovar. Nevertheless, Shigella dysenteriae serotype 1 causes deadly epidemics but Shigella boydii is restricted to the Indian subcontinent, while Shigella flexneri and Shigella sonnei are prevalent in developing and developed countries respectively. To begin to explain these distinctive epidemiological and pathological features at the genome level, we have carried out comparative genomics on four representative strains. Each of the Shigella genomes includes a virulence plasmid that encodes conserved primary virulence determinants. The Shigella chromosomes share most of their genes with that of E.coli K12 strain MG1655, but each has over 200 pseudogenes, 300 approximately 700 copies of insertion sequence (IS) elements, and numerous deletions, insertions, translocations and inversions. There is extensive diversity of putative virulence genes, mostly acquired via bacteriophage-mediated lateral gene transfer. Hence, via convergent evolution involving gain and loss of functions, through bacteriophage-mediated gene acquisition, IS-mediated DNA rearrangements and formation of pseudogenes, the Shigella spp. became highly specific human pathogens with variable epidemiological and pathological features.     

Interactions between 11beta-hydroxysteroid dehydrogenase and COX-2 in kidney

The syndrome of apparent mineralocorticoid excess (SAME) is an autosomal recessive form of salt-sensitive hypertension caused by deficiency of the kidney type 2 11beta-hydroxysteroid dehydrogenase (11betaHSD2). In this disorder, cortisol is not inactivated by 11betaHSD2, occupies mineralocorticoid receptors (MRs), and causes excessive sodium retention and hypertension. In renal medulla, prostaglandins derived from cyclooxygenase-2 (COX-2) stimulate sodium and water excretion, and renal medullary COX-2 expression increases after mineralocorticoid administration. We investigated whether medullary COX-2 also increases in rats with 11betaHSD2 inhibition and examined its possible role in the development of hypertension. 11betaHSD2 inhibition increased medullary and decreased cortical COX-2 expression in adult rats and induced high blood pressure in high-salt-treated rats. COX-2 inhibition had no effect on blood pressure in control animals but further increased blood pressure in high-salt-treated rats with 11betaHSD2 inhibition. COX-1 inhibition had no effect on blood pressure in either control or experimental animals. 11betaHSD2 inhibition also led to medullary COX-2 increase and cortical COX-2 decrease in weaning rats, primarily through activation of MRs. In the suckling rats, medullary COX-2 expression was very low, consistent with a urinary concentrating defect. 11betaHSD2 inhibition had no effect on either cortical or medullary COX-2 expression in the suckling rats, consistent with low levels of circulating corticosterone in these animals. These data indicate that COX-2 plays a modulating role in the development of hypertension due to 11betaHSD2 deficiency and that 11betaHSD2 regulates renal COX-2 expression by preventing glucocorticoid access to MRs during postnatal development.