Proteomics and genomics of urinary bladder cancer

Urinary bladder cancer is the fifth most common cancer in the United States. The National Cancer Institute estimates that the incidence rates will be 68 810 and the mortality rate will be 14 100 in the year 2008. Although the gold standards cytology and cystoscopy are specific for diagnosing bladder cancer, the former lacks the sensitivity to detect low‐grade tumors and the latter is very invasive and expensive. Therefore, scientists are interested in identifying reliable non‐invasive biomarkers that could be utilized in screening, leading to early detection and/or in predicting the progression of superficial tumors to invasive higher‐stage lesions with high specificity and sensitivity. Several biomarkers that indicate changes in the expression of proteins associated with increased risk have been identified. The purpose of this analysis is to provide an overview of the studies that have been conducted during the last decade that identify diagnostic and prognostic biomarkers via proteomic and genomic advancements.     

Proteomic analysis of BRCA1-depleted cell line reveals a putative role for replication protein A2 up-regulation in BRCA1 breast tumor development

Purpose : Germline mutations in BRCA1 result in a strong predisposition to breast cancer, with frequent loss of heterozygosity of the remaining wild‐type allele. The development of BRCA1 tumors is likely to depend on additional genetic alterations and gene expression changes which follow growth and DNA repair defects associated with BRCA1 deficiency. The identification of these modifications offers an opportunity to find surrogate markers of BRCA1 tumors. Here, we sought to identify differentially expressed proteins related to BRCA1 depletion. Experimental design : We used isogenic HeLa cells either stably knocked‐down or not for BRCA1 (BRCA1^KD) and compared protein profiles of these cells by DIGE. Results : We detected increased levels of Replication protein A2 (RPA2) in BRCA1^KD cells as compared to control cells. RPA2 is an essential protein required for DNA replication and repair. We further demonstrated that depletion of RPA2 subunit delays growth of BRCA1^KD respect to isogenic control cells. Strikingly, elevated levels of RPA2 were more frequently observed in BRCA1 tumors when triple‐negative tumors from BRCA1 mutation carriers (n=13) and non‐carriers (n=36) were stained in situ for RPA2. Conclusions and clinical relevance : RPA2 up‐regulation may thus be involved in the growth and/or survival of BRCA1 tumor cells and useful in immunohistochemical discrimination of triple‐negative BRCA1 tumors.     

Decreased annexin A3 expression correlates with tumor progression in papillary thyroid cancer

Purpose : The aim of this study is to identify the potential tumor markers that function in carcinogenesis and tumor progression, thus providing important diagnostic and prognostic information. Experimental design : We performed 2‐D gel electrophoresis and MALDI‐TOF MS to investigate the differentially expressed proteins in 25 papillary thyroid carcinoma tissues. For validation of candidate proteins and investigation of clinical significance, we performed Western, Northern blot analysis and immunohistochemical staining. Results : Our proteomic analyses revealed significantly decreased annexin A3 expression in papillary thyroid carcinoma at both the protein and mRNA levels, compared with normal thyroid tissue. ANXA3 immunoreactivity was not significantly correlated with lymph node metastasis, multifocality, capsular invasion or perithyroidal extension in thyroid cancer. However, the tumor subgroup with a lymph node metastasis score of >3 displayed significantly lower ANXA3 expression than did subgroups with negative and ≤3 scores (p=0.001). Moreover, ANXA3 expression was markedly lower in large tumors (>1 cm in diameter) than in microcarcinomas (p=0.001). Conclusion and clinical relevance : Decreased expression of ANXA3 in papillary thyroid cancer supports the idea that ANXA3 may be an effective marker of microcarcinoma, and a negative predictor of papillary thyroid cancer progression.     

Proteomic Helicobacter pylori biomarkers discriminative of low-grade gastric MALT lymphoma and duodenal ulcer

To date no reliable diagnostic method exists to predict, among the very large and clinically heterogeneous group of Helicobacter pylori‐infected patients, the extremely small group at risk for developing low‐grade gastric MALT lymphoma (LG‐MALT). Search of proteomic biomarkers holds promise for the classification of the H. pylori strains with regard to this severe clinical outcome. In the present study 69 H. pylori strains isolated from patients with two different H. pylori‐associated diseases, duodenal ulcer (DU, n=29) and LG‐MALT (n=40) were used. Protein expression patterns of the strains were analyzed by using the high‐throughput methodology SELDI. Selected proteins were purified by means of chromatographic and electrophoretic methods in view of further sequencing by LC‐MS/MS. Univariate analysis (Mann–Whitney test) of the protein expression patterns generated nine significant biomarkers that can discriminate between H. pylori strains from patients with DU and LG‐MALT. These biomarkers are of low molecular weight, ranging from 6 to 26.6 kDa. Among them, two are overexpressed in LG‐MALT strains and seven – in DU strains. Two biomarker proteins, one overexpressed in LG‐MALT strains (13.2 kDa) and another one – overexpressed in DU strains (26.6 kDa), were purified to homogeneity and identified by using LC‐MS/MS as a 50S ribosomal protein L7/L12 and a urease subunit, respectively. These biomarkers can be included in novel protein arrays for the differential diagnosis of H. pylori‐associated clinical outcomes.     

Neural network modeling of microwave FETs based on third‐order distortion characterization

A new method for characterization of HEMT distortion parameters, which extracts the coefficents of a Taylor series expansion of I_ds(V_gs, V_ds), including all cross‐terms, is developed from low‐frequency harmonic measurements. The extracted parameters will be used either in a Volterra series model around a fixed bias point for 3^rd‐order characterization of small‐signal I_ds nonlinearity, or in a large‐signal model of I_ds characteristic, where its partial derivatives are locally characterized up to the 3^rd order in the whole bias region, using a novel neural‐network representation. The two models are verified by one‐tone and two‐tone intermodulation distortion (IMD) tests on a PHEMT device. © 2006 Wiley Periodicals, Inc. Int J RF and Microwave CAE, 2006.     

A decomposition scheme for single stage scheduling problems

This paper introduces a general decomposition scheme for single stage scheduling problems with jobs that have arbitrary release dates. We assume that the objective function is monotone in the completion time of each job. The decomposition scheme has significant theoretical and practical relevance. When assuming equal processing times, we can reduce the number of steps required to solve several well-known nonpreemptive single machine scheduling problems by O(n³)\mathcal{O}(n^{3}), provided the processing time p is constant. Specifically, we develop new approaches that solve the problems 1|r _i ,p _i =p|∑f _i (C _i ) and 1|r _i ,p _i =p|∑w _i U _i in O(n⁴)\mathcal{O}(n^{4}) time; the algorithms that have been described in the literature for these problems operate in O(n⁷)\mathcal{O}(n^{7}). Moreover, solution approaches for NP\mathcal{NP}-hard problems with unequal processing times may also benefit from our decomposition rule. This is particularly true if p _max/p _min is close to 1. Using the decomposition rule, either the problem size is reduced or additional information about the maximal schedule length is obtained.     

A SOI-MEMS-based 3-DOF planar parallel-kinematics nanopositioning stage

This paper presents the design, kinematic and dynamic analysis, fabrication and characterization of a monolithic micro/nanopositioning three degrees-of-freedom (DOF) (XYθ) stage. The design of the proposed MEMS (micro-electro-mechanical system) stage is based on a parallel-kinematic mechanism (PKM) scheme that allows for translation in the XY plane and rotation about the Z axis, an increased motion range, and linear kinematics in the operating region (or work area) of the stage. The truss-like structure of the PKM results in higher modal frequencies by increasing the structural stiffness and reducing the moving mass of the stage. The stage is fabricated on a silicon-on-insulator (SOI) wafer using surface micromachining and deep reactive ion etching (DRIE) processes. Three sets of electrostatic linear comb drives jointly actuate the mechanism to produce motion in the X, Y and θ (rotation) directions. The fabricated stage provides a motion range of 18 μm and 1.72° at a driving voltage of 85 V. The resonant frequency of the stage under ambient conditions is 465 Hz. Additionally a high Q factor (66) is achieved from this parallel-kinematics mechanism design.     

Identification of Apolipoprotein AI as a serum biomarker of chronic kidney disease in liver transplant recipients, using proteomic techniques

Chronic kidney disease (CKD) is a common complication post‐orthotopic liver transplantation (OLT). Development of CKD is detected by monitoring serum urea and creatinine, however disease can occasionally be at an advanced stage before they become abnormal. Therefore, more accurate parameters are required. In order to identify novel biomarkers of CKD, serum was obtained from 47 OLT recipients with CKD (glomerular filtration rate <60 mL/min) and 23 with normal renal function (glomerular filtration rate >90 mL/min). Using the proteomic technique SELDI‐TOF‐MS, three protein biomarkers (55.6 kDa, 9.5 kDa and 11.4 kDa) were identified that, together, could stratify patients into cases or controls with a sensitivity and specificity of 93.6 and 91.3%, respectively. The area under the curve was 0.94. The primary splitter of the groups at 55.6 kDa was an alternative version of a molecule at 27.8 kDa, which was subsequently identified by 1‐D SDS‐PAGE and LC‐ESI‐MS/MS to be Apolipoprotein AI. Protein expression was shown to be reduced in CKD, by both ELISA (p = 0.057) and Western blot analysis (p = 0.003). Apolipoprotein AI is a novel, accurate marker of CKD post‐OLT. It does require further validation in a large, more diverse patient population but could potentially improve detection of CKD.     

Proteomic characterization of differentially expressed proteins in breast cancer: Expression of hnRNP H1, RKIP and GRP78 is strongly associated with HER-2/neu status

The human epidermal growth factor receptor type 2 (HER‐2/neu) oncoprotein is overexpressed in about 30% of breast cancers and associates with metastatic phenotypes of breast tumours. Dissecting the HER‐2/neu‐modulated molecules in cancer will be helpful in elucidating the underlying molecular mechanisms of HER‐2/neu‐driven tumourigenesis. We investigated the differential proteome profiles between microdissected HER‐2/neu‐positive and ‐negative tumours and unambiguously identified 21 proteins with diverse biological functions by peptide sequencing and NCBInr database interrogation. Six proteins were up‐regulated whereas 15 were down‐regulated in the HER‐2/neu‐positive tumours. Differential expressions of heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), 78 kDa glucose‐regulated protein (GRP78/Bip) and Raf‐1 kinase inhibitor protein (RKIP), which have not been previously reported as being linked to HER‐2/neu signalling, were further verified. Immunohistochemical staining on tissue microarray sections demonstrated a positive correlation of hnRNP H1 (p = 0.008) and negative correlations of GRP78 and RKIP (p = 0.018 and 0.013, respectively) with HER‐2/neu. Heregulin α1 enhanced hnRNP H1, but reduced GRP78 and RKIP expression in BT474 cells in a dose‐dependent manner, providing evidence of crosstalk between HER‐2/neu signalling and these modulators. Our studies have identified novel modulators that are likely to be intricately involved in HER‐2/neu‐driven tumour proliferation, invasion and metastasis.     

State of the art of 2D DIGE

Difference gel electrophoresis enables the accurate quantification of changes in the proteome including combinations of PTMs and protein isoform expression. Here, we review recent advances in study design, image acquisition, and statistical analysis. We also compare DIGE to established and emerging mass spectrometric analysis technologies. Despite these recent advances in MS and the still unsolved limitations of 2DE to map hydrophobic, high molecular weight proteins with extreme pIs, DIGE remains the most comprehensive top-down method to study changes in abundance of intact proteins.     

Impact of source/drain contacts formation of self‐aligned amorphous‐IGZO TFTs on their negative‐bias‐illumination‐stress stabilities

In this study, we have compared the performance of self‐aligned a‐IGZO thin‐film transistors (TFTs) whereby the source/drain (S/D) region's conductivity enhanced in three different ways, that is, using SiN_x interlayer plasma (hydrogen diffusion), using calcium (Ca as reducing metal) and using argon plasma (changing the atomic ratio). All these TFTs show comparable characteristics such as field‐effect mobility (μ_FE) of over 10.0 cm²/(V.s), sub‐threshold slope (SS^‐1) of 0.5 V/decade, and current ratio (I_ON/I_OFF) over 10⁸. However, under negative‐bias‐illumination‐stress (NBIS), all these TFTs showed strong degradation. We attributed this NBIS stability issue to the exposed S/D regions and changes in the conductivity of S/D contact regions. The hydrogen plasma‐treated TFTs showed the worst NBIS characteristics. This is linked to increased hydrogen diffusion from the S/D contact regions to the channel.     

Mass Spectrometric Analysis of Cerebrospinal Fluid Ubiquitin in Alzheimer's Disease and Parkinsonian Disorders

1 Purpose

Dysfunctional proteostasis, with decreased protein degradation and an accumulation of ubiquitin into aggregated protein inclusions, is a feature of neurodegenerative diseases. Identifying new potential biomarkers in cerebrospinal fluid (CSF) reflecting this process could contribute important information on pathophysiology.

2 Experimental design

A developed method combining SPE and PRM‐MS is employed to monitor the concentration of ubiquitin in CSF from subjects with Alzheimer's disease (AD), Parkinson's disease (PD), and progressive supranuclear palsy (PSP). Four independent cross‐sectional studies are conducted, studies 1–4, including controls (n = 86) and participants with AD (n = 60), PD (n = 15), and PSP (n = 11).

3 Results

The method shows a repeatability and intermediate precision not exceeding 6.1 and 7.9%, respectively. The determined LOD is 0.1 nm and the LOQ range between 0.625 and 80 nm . The CSF ubiquitin concentration is 1.2–1.5‐fold higher in AD patients compared with controls in the three independent AD‐control studies (Study 1, p < 0.001; Study 2, p < 0.001; and Study 3, p = 0.003). In the fourth study, there is no difference in PD or PSP, compared to controls.

4 Conclusion and clinical relevance

CSF ubiquitin may reflect dysfunctional proteostasis in AD. The described method can be used for further exploration of ubiquitin as a potential biomarker in neurodegenerative diseases.     

Influence of 8‐bit vs. 11‐bit digital displays on observer performance and visual search: A multi‐center evaluation

Abstract— Medical‐grade monochrome monitors typically display 8 bits of data. This study determined if 11‐bit displays could improve observer performance and decrease use of window/level. 8‐ and 11‐bit displays from three manufacturers were used at three sites. Six radiologists at each site viewed 100 DR chest images (half with a pulmonary nodule) on both displays. Decisions, confidence, nodule location, viewing time, and window/level use were recorded. There was no significant difference in ROC Az as a function of bit depth. The average Az with 8 bits was 0.8284 and with 11 bits was 0.8253. There was a significant difference in viewing time favoring the 11‐bit displays. Window/level use did not differ. Eye position was recorded on a subset of images at one site. Cumulative dwell times for each decision category were lower with the 11‐bit than with the 8‐bit display. When tested with t‐tests for paired observations, the TP (t = 1.452, p = 0.1507), FN (t = 0.050, p = 0.9609), and FP (t = 0.042, p = 0.9676) were not statistically significant. The difference in the TN decisions was statistically significant (t = 1.926, p = 0.05). 8‐bit displays will not impact negatively diagnostic accuracy, but using 11‐bit displays may improve workflow efficiency.     

On the connection between the exponential stability of C0-semigroups and the admissibility of a certain Sobolev space

Let T be a strongly continuous semigroup on a Banach space X and A its infinitesimal generator. We will prove that T is exponentially stable, if and only if, there exist p[1,∞) such that the space is admissible to the system Σ(A,I,I), defined below (i.e for all f belonging to the Sobolev space the convolution T*f lies in .     

A two‐stage hub location method for air transportation in Brazil

During the past decade, the Brazilian air passenger market has been growing at impressive rates suggesting the need for rationalization and the consistent use of the hub‐and‐spoke configuration. This research considers 135 airports, mostly extremely small, and the aim is to identify regional hubs that would accumulate traffic and feed a predefined restricted number of major hubs. The proposed approach is divided into two phases: the first phase considers all 135 existing commercial airports and using the p‐median problem identifies p airports that would play the role of regional hubs, and the second phase considers a problem with these p airports and solves the q‐hub location problem. Both phases are sequentially solved by the software AIMMS 3.9. The research has broad implications consistent with the publicized efforts to improve management practices, support a larger number of commercial airports, and face the expected peak demand in the upcoming international events. Given the growth in the air passenger market in Brazil, both phases may be seen as two independent objectives.     

Discovery of putative oocyte quality markers by comparative ExacTag proteomics

Purpose: Identification of the biomarkers of oocyte quality, and developmental and reprogramming potential is of importance to assisted reproductive technology in humans and animals. Experimental design: PerkinElmer ExacTag? Kit was used to label differentially proteins in pig oocyte extracts (oocyte proteome) and pig oocyte‐conditioned in vitro maturation media (oocyte secretome) obtained with high‐ and low‐quality oocytes. Results: We identified 16 major proteins in the oocyte proteome that were expressed differentially in high‐ versus low‐quality oocytes. More abundant proteins in the high‐quality oocyte proteome included kelch‐like ECH‐associated protein 1 (an adaptor for ubiquitin‐ligase CUL3), nuclear export factor CRM1 and ataxia‐telangiectasia mutated protein kinase. Dystrophin (DMD) was more abundant in low‐quality oocytes. In the secretome, we identified 110 proteins, including DMD and cystic fibrosis transmembrane conductance regulator, two proteins implicated in muscular dystrophy and cystic fibrosis, respectively. Monoubiquitin was identified in the low‐quality‐oocyte secretome. Conclusions and clinical implications: A direct, quantitative proteomic analysis of small oocyte protein samples can identify potential markers of oocyte quality without the need for a large amount of total protein. This approach will be applied to discovery of non‐invasive biomarkers of oocyte quality in assisted human reproduction and in large animal embryo transfer programs.     

Current‐mode ambient‐light sensing circuit using p‐type low‐temperature polycrystalline‐silicon TFTs and p‐i‐m diodes

Abstract— A current‐mode ambient‐light sensing circuit, which is composed of p‐intrinsic‐metal (p‐i‐m) diodes and p‐type low‐temperature polycrystalline‐silicon (LTPS) thin‐film transistors (TFTs) for autobrightness control of display panels. The proposed sensing circuit exhibits a wide dynamic range of 56 dB, while the output signal range is 1.8 times wider than that of a previously reported sensing circuit.     

Invited Paper: New technology for HTPS‐LCD panels for projection systems

Abstract— A new technology has been developed for high‐temperature‐polysilicon (HTPS) TFT liquid‐crystal panels employed in projection systems. It consists of vertically aligned nematic (VAN) liquid‐crystal, inorganic alignment layers, and a new driving technique. Full‐HD (1080p) resolution was realized in a 0.7‐in.‐diagonal device with high contrast and low power consumption.     

Human duodenal proteome modulations by glutamine and antioxidants

Purpose : Glutamine (Gln) has protective, anti‐inflammatory effects in animal models and humans. Antioxidant nutrients may exert synergistic effects on intestinal functions. Therefore, these combined nutrients may have a therapeutic potential during intestinal inflammation. This study was designed to investigate in humans the effects of a supplement composed of Gln and high‐dosed antioxidant micronutrients compared to isomolar Gln only, on duodenal proteome. Experimental design : Enteral perfusion of Gln (0.8 mmol . kg^?1. h^?1) or supplement was performed in two groups of six healthy volunteers during 5 h before taking endoscopic duodenal biopsies. Protein expression was analyzed by 2‐DE and the relevant proteins identified by MS/MS. Results : About 1500 protein spots were revealed in both supplement and Gln conditions. Comparative proteomics analysis indicated that 11 proteins were differentially and significantly (p≤0.05) expressed in response to the supplement. These proteins were essentially implicated in metabolism pathways, e.g. fatty acid binding protein‐1 and 40S ribosomal protein SA expressions were downregulated while manganese superoxide dismutase and retinal dehydrogenase‐1 expressions were upregulated. Conclusions and clinical relevance : This study provides new information on human duodenal proteome and its nutritional modulation, and supports further clinical investigations designed to evaluate the effects of Gln plus antioxidants during intestinal inflammation and cancer.