An Albumin-Holliday Junction Biomolecular Modular Design for Programmable Multifunctionality and Prolonged Circulation.

Combinatorial properties such as long-circulation and site- and cell-specific engagement need to be built into the design of advanced drug delivery systems to maximize drug payload efficacy. This work introduces a four-stranded oligonucleotide Holliday Junction (HJ) motif bearing functional moieties covalently conjugated to recombinant human albumin (rHA) to give a "plug-and-play" rHA-HJ multifunctional biomolecular assembly with extended circulation. Electrophoretic gel-shift assays show successful functionalization and purity of the individual high-performance liquid chromatography-purified modules as well as efficient assembly of the rHA-HJ construct. Inclusion of an epidermal growth factor receptor (EGFR)-targeting nanobody module facilitates specific binding to EGFR-expressing cells resulting in approximately 150-fold increased fluorescence intensity determined by flow cytometric analysis compared to assemblies absent of nanobody inclusion. A cellular recycling assay demonstrated retained albumin-neonatal Fc receptor (FcRn) binding affinity and accompanying FcRn-driven cellular recycling. This translated to a 4-fold circulatory half-life extension (2.2 and 0.55 h, for the rHA-HJ and HJ, respectively) in a double transgenic humanized FcRn/albumin mouse. This work introduces a novel biomolecular albumin-nucleic acid construct with extended circulatory half-life and programmable multifunctionality due to its modular design.

Associated adverse health outcomes of polypharmacy and potentially inappropriate medications in community-dwelling older adults with diabetes.

Aim: This study aimed to identify the association of chronic polypharmacy and potentially inappropriate medications (PIMs) with adverse health outcomes (AHOs) in community-dwelling older adults with diabetes in China. Methods: A 2-year retrospective cohort study was conducted using 11,829 community-followed older adults with diabetes and medical records from 83 hospitals and 702 primary care centers in Shenzhen, China. Chronic polypharmacy and PIMs were identified from prescription records using Beers' criteria, and their associated AHO was analyzed using multivariable logistic regression analysis. Results: The prevalence of chronic polypharmacy and at least one PIM exposure was 46.37% and 55.09%, respectively. The top five PIMs were diuretics, benzodiazepines, first-generation antihistamines, sulfonylureas, and insulin (sliding scale). Chronic polypharmacy was positively associated with all-cause hospital admission, admission for coronary heart disease, admission for stroke, admission for dementia, and emergency department visits. Exposure to PIMs was positively associated with all-cause hospital admission, admission for heart failure (PIMs ≥2), admission for stroke (PIMs ≥3), emergency department visits, bone fracture, constipation, and diarrhea. Conclusion: Chronic polypharmacy and PIMs were prevalent in older adults with diabetes in Chinese communities. Iatrogenic exposure to chronic polypharmacy and PIMs is associated with a higher incidence of different AHOs. This observational evidence highlights the necessity of patient-centered medication reviews for chronic polypharmacy and PIMs use in older patients with diabetes in primary care facilities in China and draws attention to the caution of polypharmacy, especially PIM use in older adults with diabetes in clinical practice.

Prevalence of potentially inappropriate medications and association with comorbidities in older adults with diabetes in an outpatient visitation setting.

Aims: Potentially inappropriate medications had been found associated with adverse drug events such as falls, emergency department admissions and hospital readmissions. There is lack of information about the prevalence of potentially inappropriate medications and associated chronic conditions in older patients with diabetes in China. This study aimed to assess the prevalence of potentially inappropriate medications in older adults with diabetes in an outpatient visitation setting and the association with polypharmacy due to comorbidities. Materials and methods: This was a 3-year repeated cross-sectional study which conducted in outpatient setting of 52 hospitals in Shenzhen, China, using 2019 Beers criteria. The prevalence of potentially inappropriate medications, polypharmacy and comorbidities in older adults with diabetes in an outpatient setting was expressed as percentages. Logistic models were used to investigate the association between potentially inappropriate medication exposure and age, sex, polypharmacy and comorbidities. Results: Among the 28,484 older adults with diabetes in 2015, 31,757 in 2016 and 24,675 in 2017, the prevalence of potentially inappropriate medications was 43.2%, 44.88% and 42.40%, respectively. The top five potentially inappropriate medications were diuretics (20.56%), benzodiazepines (13.85%), androgens (13.18%), non-steroidal anti-inflammatory drugs (12.94%) and sulfonylureas (6.23%). After adjustment for age and polypharmacy, the probability of potentially inappropriate medication exposure was associated with chronic gastrointestinal diseases, followed by osteoarthritis and rheumatoid arthritis, chronic pulmonary disease, chronic kidney disease, tumor, dementia, chronic liver disease, hypertension, cardiovascular disease, cerebrovascular disease and hyperlipemia. Conclusion: Potentially inappropriate medications were common in older patients with diabetes in an outpatient visitation setting. Higher probability of potentially inappropriate medication exposure was associated with the comorbidity chronic gastrointestinal diseases as well as osteoarthritis and rheumatoid arthritis. To ensure that iatrogenic risks remain minimal for older adults with diabetes, the clinical comorbidities should be considered.

Risk stratification of cutaneous melanoma reveals carcinogen metabolism enrichment and immune inhibition in high-risk patients.

Cutaneous melanoma (CM) is the most lethal form of skin cancer. Risk assessment should facilitate stratified surveillance and guide treatment selection. Here, based on the mRNA-seq data from 419 CM patients in the Cancer Genome Atlas (TCGA), we developed a prognostic 21-gene signature to distinguish the outcomes of high- and low-risk patients, which was further validated in two external cohorts. The signature achieved a higher C-index as compared with other known biomarkers and clinical characteristics in both the TCGA and validation cohorts. Notably, in high-risk patients the expression levels of three driver genes, BRAF, NRAS, and NF1 in the MAPK pathway, were lower but exhibited a stronger positive correlation as compared with low-risk patients. Moreover, the genes involved in nicotinamide adenine dinucleotide metabolism were negatively correlated with the expression of BRAF in the high-risk group. Function analysis revealed that the upregulated genes in the high-risk group were enriched in the cytochrome P450-mediated metabolism of chemical carcinogens. Furthermore, the low-risk group had high levels of gamma delta T cells infiltration, while regulatory T cells were accumulated in the high-risk group. The present study offers a promising new prognostic signature for CM, and provides insight into the mechanisms of melanoma progression.

Better prognostic determination and feature characterization of cutaneous melanoma through integrative genomic analysis.

Melanoma is the most dangerous type of skin cancer and has highly heterogeneous features. Despite progress in melanoma classification, interpatient heterogeneity remains difficult to predict, especially in terms of long-term survival. Here, based on mRNA-seq, miRNA-seq and DNA methylation data from 447 cutaneous melanoma patients in the Cancer Genome Atlas, we performed integrative and single-dataset clustering analyses. A novel group of patients was identified, including 301 with better, 55 with poorer and 91 with intermediate prognoses. Immune genes were upregulated in the better prognostic group, and higher immune scores (representing a greater extent of immune cell infiltration into tumor tissues) were associated with better prognoses. Higher expression of 115 genes was determined to predict better outcomes. The better prognostic group also exhibited DNA hypomethylation, and immune pathways were enriched among the hypomethylated genes. Using exome-seq data from the same patients, we observed that the better prognostic group harbored the highest number of mutations. The mutational signature in the better prognostic group was associated with ultraviolet light exposure. These integrated investigations have potential therapeutic significance, as they clarify the molecular heterogeneity of cutaneous melanoma and enhance its classification.

Independent Validation of a Diagnostic Noninvasive 3-MicroRNA Ratio Model (uCaP) for Prostate Cancer in Cell-Free Urine.

BACKGROUND: Detection of prostate cancer (PC) based on serum prostate-specific antigen (PSA) testing leads to many unnecessary prostate biopsies, overdiagnosis, and overtreatment of clinically insignificant tumors. Thus, novel and more accurate molecular biomarkers are required. METHODS: Using reverse transcription quantitative PCR, we measured the concentrations of 45 preselected microRNAs (miRNAs) in extracellular vesicle-enriched cell-free urine samples from 4 independent patient cohorts from Spain and Denmark, including 758 patients with clinically localized PC, 289 noncancer controls with benign prostatic hyperplasia (BPH), and 233 patients undergoing initial transrectal ultrasound (TRUS)-guided prostate biopsy owing to PC suspicion (101 with benign and 132 with malignant outcome). Diagnostic potential was assessed by ROC and decision curve analysis. RESULTS: We identified and successfully validated 8 upregulated and 21 downregulated miRNAs in urine from PC patients. Furthermore, we validated a previously identified 3-miRNA diagnostic ratio model, uCaP (miR-222-3p*miR-24-3p/miR-30c-5p). High uCaP scores were distinctive of PC in urine samples from BPH vs PC patients in 3 independent cohorts [area under the curve (AUC) = 0.84, 0.71, 0.72]. Additionally, uCaP predicted TRUS biopsy results with greater accuracy than PSA (AUC uCaP = 0.644; AUC PSA = 0.527) for patients within the diagnostic gray zone (PSA ≤ 10 ng/mL). CONCLUSIONS: We successfully validated a urine-based diagnostic 3-miRNA signature for PC (uCaP) in 3 independent patient cohorts from 2 countries. In the future, the simple and noninvasive uCaP test may be used to help more accurately select patients for prostate biopsy. Prospective clinical validation is warranted.

Methylation-Based Classification of Cervical Squamous Cell Carcinoma into Two New Subclasses Differing in Immune-Related Gene Expression.

Cervical cancer is traditionally classified into two major histological subtypes, cervical squamous cell carcinoma (CSCC) and cervical adenocarcinoma (CA). However, heterogeneity exists among patients, comprising possible subpopulations with distinct molecular profiles. We applied consensus clustering to 307 methylation samples with cervical cancer from The Cancer Genome Atlas (TCGA). Fisher's exact test was used to perform transcription factors (TFs) and genomic region enrichment. Gene expression profiles were downloaded from TCGA to assess expression differences. Immune cell fraction was calculated to quantify the immune cells infiltration. Putative neo-epitopes were predicted from somatic mutations. Three subclasses were identified: Class 1 correlating with the CA subtype and Classes 2 and 3 dividing the CSCC subtype into two subclasses. We found the hypomethylated probes in Class 3 exhibited strong enrichment in promoter region as compared with Class 2. Five TFs significantly enriched in the hypomethylated promoters and their highly expressed target genes in Class 3 functionally involved in the immune pathway. Gene function analysis revealed that immune-related genes were significantly increased in Class 3, and a higher level of immune cell infiltration was estimated. High expression of 24 immune genes exhibited a better overall survival and correlated with neo-epitope burden. Additionally, we found only two immune-related driver genes, CARD11 and JAK3, to be significantly increased in Class 3. Our analyses provide a classification of the largest CSCC subtype into two new subclasses, revealing they harbored differences in immune-related gene expression.

Palmitoylated phosphodiester gapmer designs with albumin binding capacity and maintained in vitro gene silencing activity.

BACKGROUND: Antisense gapmer oligonucleotide drugs require delivery and biodistribution enabling technologies to increase in vivo efficacy. An attractive approach is their binding and consequent transport by the endogenous human serum albumin pool as mediated by fatty acid incorporation into the gapmer design. METHODS: The present study investigated the effect of palmitoyl modification and position on albumin-binding, cellular uptake and in vitro gene silencing of gapmers with either a phosphorothioate (PS) or phosphodiester (PO) backbone. RESULTS: Two palmitoyls positioned exclusively at the 5' end, or a single palmitoyl at both the 3' and 5' positions, showed similar binding to human serum albumin as demonstrated by a gel-shift assay. Decreased cellular uptake determined by flow cytometry (27% compared to nonpalmitoyl gapmers) was observed for palmitoylated Cy5.5 labelled gapmers. However, HER3 (human epidermal growth factor receptor 3) gene silencing was exhibited by the palmitoylated gapmers with transfection agent in PC-3 and Caco-2 cells (68% and 62%, respectively), which was comparable to nonpalmitoyl gapmers (68% and 82%, respectively). Importantly, PO gapmers with a single palmitoyl positioned at both the 3' and 5' positions showed high silencing efficiencies (68% and 66% in PC-3 and Caco-2 cells, respectively) similar to those of PS nonpalmitoylated gapmers (67% and 66% in PC-3 and Caco-2 cells, respectively) in the absence of a transfection agent. CONCLUSIONS: The present study defines phosphodiester gapmer design criteria exhibiting high gene silencing activity and albumin binding that may be utilized with potentially less in vivo toxicity that can be associated with phosphorothioate gapmer designs.

A benchmark study of sequence alignment methods for protein clustering.

BACKGROUND: Protein sequence alignment analyses have become a crucial step for many bioinformatics studies during the past decades. Multiple sequence alignment (MSA) and pair-wise sequence alignment (PSA) are two major approaches in sequence alignment. Former benchmark studies revealed drawbacks of MSA methods on nucleotide sequence alignments. To test whether similar drawbacks also influence protein sequence alignment analyses, we propose a new benchmark framework for protein clustering based on cluster validity. This new framework directly reflects the biological ground truth of the application scenarios that adopt sequence alignments, and evaluates the alignment quality according to the achievement of the biological goal, rather than the comparison on sequence level only, which averts the biases introduced by alignment scores or manual alignment templates. Compared with former studies, we calculate the cluster validity score based on sequence distances instead of clustering results. This strategy could avoid the influence brought by different clustering methods thus make results more dependable. RESULTS: Results showed that PSA methods performed better than MSA methods on most of the BAliBASE benchmark datasets. Analyses on the 80 re-sampled benchmark datasets constructed by randomly choosing 90% of each dataset 10 times showed similar results. CONCLUSIONS: These results validated that the drawbacks of MSA methods revealed in nucleotide level also existed in protein sequence alignment analyses and affect the accuracy of results.

A hyaluronic acid-based hydrogel enabling CD44-mediated chondrocyte binding and gapmer oligonucleotide release for modulation of gene expression in osteoarthritis.

Hyaluronic acid (HA) is an attractive biomaterial for osteoarthritis (OA) treatment due to inherent functional and compatibility properties as an endogenous knee joint component. In this work, we describe a HA-based hydrogel with the dual functionality of increased CD44-dependent chondrocyte binding and controlled release of gapmer antisense oligonucleotides for unassisted cellular entry and subsequent gene silencing activity. A Schiff base-mediated gelation method was used to produce a panel of hydrogels varying in the aldehyde-modified HA (900kDa) to chitosan ratios (3:7, 5:5 and 7:3) for identifying designs displaying optimal engagement of OA patient-derived CD44-expressing chondrocytes. Correlation was found between cell binding and CD44 expression, with maximal binding exhibited at a HA/chitosan ratio of 7:3, that was 181% higher than CD44-negative MCF-7 cell control cells. Transfection agent-free uptake into OA chondrocytes of fluorescent 13-mer DNA oligonucleotides with a flanked locked nucleic acid (LNA) gapmer design, in contrast to naked siRNA, was demonstrated by confocal and flow cytometric analysis. A sustained and complete release over 5days was found with the 7:3 hydrogel, in contrast, the 5:5 and 3:7 hydrogel released 60% and 43% of loaded gapmers, respectively over the same period. A COX-2-specific gapmer designed with maximal chondrocyte gene silencing (~70% silencing efficiency at 500nM compared with a mismatch gapmer sequence) resulted in effective COX-2 silencing over 14days in hydrogels seeded with OA chondrocytes, with significant difference exhibited between day 3 and 10. This work introduces a novel HA-based CD44-mediated cellular binding and gapmer controlled release platform to modulate cellular gene expression.

Evolving-Pattern Analysis of Transient and Long-Term Biomarkers for Cancers: Hepatocellular Carcinoma as a Case.

Cancer is a complex disease arises from combinations of changes that occur over a period of time. With the development of bioinformatics, more and more biomarkers representing changes in cancers had been identified using gene expression profiles. However, biomarkers alone are quite limited in explaining the molecular processes occurred in the due process. In this paper, we develop an evolving-pattern analysis pipeline for in-depth studies of gene expression changes during different disease stages, choosing hepatocellular carcinoma (HCC) as a case. Enrichment analyses were performed on three levels: functional terms, validated genes, and regulation factors for all the biomarkers to find out their biological characters. Our results show that biomarkers with distinct evolving patterns exhibit quite different characteristics on functional and regulation levels. For the case of HCC, transient biomarkers are mostly annotated to metabolic processes, while long-term biomarkers are mostly annotated to regulation processes, with a larger number of enriched regulation factors. Furthermore, our pipeline reveals the important roles of microRNAs in various evolving patterns, which are known to be closely related to HCC. These results confirm that evolving-pattern analysis may provide a new sight for in-depth studies of biomarkers and diseases.

Evolving-pattern analysis of transient and long-term biomarkers for cancers: Hepatocellular carcinoma as a case.

Cancer is a complex disease arises from combinations of changes that occur over a period of time. With the development of bioinformatics, more and more biomarkers representing changes in cancers had been identified using gene expression profiles. However, biomarkers alone are quite limited in explaining the molecular processed occurred in the due process. In this paper, we develop an evolving-pattern analysis pipeline for in-depth studies of gene expression changes during different disease stages, choosing hepatocellular carcinoma (HCC) as a case. Enrichment analyses were performed on three levels: functional terms, validated genes, and regulation factors for all the biomarkers to find out their biological characters. Our results show that biomarkers with distinct evolving patterns exhibit quite different characteristics on functional and regulation levels. For the case of HCC, transient biomarkers are mostly annotated to metabolic processes, while long-term biomarkers are mostly annotated to regulation processes, with a larger number of enriched regulation factors. Furthermore, our pipeline reveals the important roles of microRNAs in various evolving patterns, which are known to be closely related to HCC. These results confirm that evolving-pattern analysis may provide a new sight for in-depth studies of biomarkers and diseases.

Development of a 5-fluorouracil-loaded PLGA microsphere delivery system by a solid-in-oil-in-hydrophilic oil (S/O/hO) novel method for the treatment of tumors.

Tumor treatment requires a long-term regimen of chemotherapy, and both surgical tumor resection and radiation therapy are also used. The present study aimed to develop a novel method for 5-fluorouracil (5-FU)-loaded microspheres which enhance the therapeutic effects of chemotherapy, the quality of life of patients and reduce chemotherapy systemic side-effects. The preparation of a 5-FU microsphere delivery system by a solid-in-oil-in-hydrophilic oil (S/O/hO) novel method was carried out and then in vitro and in vivo evaluation of the 5-FU-microsphere delivery system was conducted. The 5-FU microsphere delivery system prepared had sustained-release function and achieved local treatment efficacy for tumors. The encapsulation efficiency of the 5-FU microsphere delivery system was >90% [better than the fabrication method using water-in-oil-in-water (W/O/W)]. The drug release profile from the 5-FU-loaded sustained-release microsphere delivery system matched the pseudo zero-order equation for 30 days in vitro. The plasma concentration of 5-FU was higher than the water solution by subcutaneous injection. The tumor growth rate of rabbits using the 5-FU microsphere delivery system was much lower than the rate in rabbit using a subcutaneous injection of 5-FU water solution. The 5-FU-loaded sustained-release microspheres using the novel method (S/O/hO) is a potential and effective method with which to inhibit tumor growth.

The relationship between the 24 h blood pressure variability and carotid intima-media thickness: a compared study.

Large blood pressure variability (BPV) will not only harm the target organ but also increase the possibility of the cardiovascular events. Since the damage of vascular system always leads to the alteration of the carotid wall, the structure and function of the carotid artery have been extensively examined in previous studies. In this work we conduct a study (60 subjects, aged 33-79) to evaluate the relationship between BPV and carotid intima-media thickness (IMT) in Shenzhen, which is one large city in the southern area of China. In our study, the blood pressure (BP) was collected using the 24 h ambulatory BP monitoring, and the BPV was evaluated using standard deviation (SD), coefficient of variation (CV), and average real variability (ARV) during 24 h, daytime and nighttime. All the IMT measurements are collected by ultrasound. The results show that both the daytime, and 24 h systolic BPV evaluated by three indices are positively associated with IMT. Among them, daytime systolic BPV evaluated with ARV is the best variable to represent the increasing of carotid IMT. In addition, after adjusting by age, sex, smoking, hypertension, and mean BP and PP values, 24 h diastolic BPV evaluated with SD also presents the favorable performance.

PEG-PCL-DEX polymersome-protamine vector as an efficient gene delivery system via PEG-guided self-assembly.

AIM: The nonviral carrier system based on the triblock copolymer PEG-PCL-DEX (PPD) and protamine was developed for nucleic acid delivery. MATERIALS & METHODS: Self-assembly occurred in the PEG continuous phase to form 'dextran-interior' polymersomes. siRNA can be condensed by protamine and encapsulated into PPD polymersomes in order to form the PPD-protamine siRNA nanoparticles by thermodynamically preferential partition between the PEG continuous phase and the dextran cavity. RESULTS: This system can package siRNA into PPD polymersomes to form 145.2 ± 8.02-nm (± standard deviation) nanoparticles, and the ζ-potential can be reduced to approximately 0 mV. PPD-protamine siRNA nanoparticles achieved cellular uptake of siRNA in SMMC-7721 cells with negligible cytotoxicity, and the GL3 gene expression can be reduced to 61.73 ± 6.25%. A biodistribution study of nanoparticles suggested that the PPD-protamine siRNA nanoparticles mainly accumulated in liver. CONCLUSION: All of these results suggest that PPD-protamine carriers may offer a promising gene delivery strategy for the treatment of liver-related disease.

Long-acting preparations of exenatide.

Exenatide has been widely used for the treatment of type 2 diabetes mellitus. However, its short plasma half-life of 2.4 hours has limited its clinical application. The exenatide products on the market, twice-daily Byetta™ and once-weekly Bydureon™ (both Amylin Pharmaceuticals, San Diego, CA, USA), are still not perfect. Many researchers have attempted to prolong the acting time of exenatide by preparing sustained-release dosage forms, modifying its structure, gene therapies, and other means. This review summarizes recent advances in long-acting exenatide preparations.

Local antitumor effects of intratumoral delivery of rlL-2 loaded sustained-release dextran/PLGA-PLA core/shell microspheres.

In this study, we formulated a rIL-2 loaded sustained-release dextran/PLGA-PLA core/shell microsphere, mimicking the paracrine mechanisms of cytokine action, to investigate its local antitumor efficacy. The presented microspheres were formed in two steps: rIL-2 was firstly loaded into dextran particles to keep its bioactivity by a unique method of stabilizing aqueous-aqueous "emulsion"; subsequently, the particles were encapsulated into poly(dl-lactide-co-glycolide)/polylactic acid (PLGA/PLA). A stable sustained release behavior in vitro was achieved for a period of about 25 days. In the subcutaneous colon carcinoma BALB/c mice models, a single dose of microspheres was introtumorally administrated and compared with multiple doses of rIL-2 solution to investigate the long acting effect of microspheres on tumor. The animal experiments showed the local efficacy at tumor site mediated by rIL-2 from a single dose of microspheres was better than that of multiple rIL-2 solution injections. Based on the experimental results, we conclude that rlL-2 loaded sustained-release dextran/PLGA-PLA core/shell microspheres represent a promising approach for local cancer treatment in animals.

A novel preparation method for silicone oil nanoemulsions and its application for coating hair with silicone.

BACKGROUND: Silicone oil, as a major component in conditioner, is beneficial in the moisture preservation and lubrication of hair. However, it is difficult for silicone oil to directly absorb on the hair surface because of its hydrophobicity. Stable nanoemulsions containing silicone oil may present as a potential solution to this problem. METHODS: Silicone oil nanoemulsions were prepared using the oil-in-water method with nonionic surfactants. Emulsion particle size and distribution were characterized by scanning electron microscopy. The kinetic stability of this nanoemulsion system was investigated under accelerated stability tests and long-term storage. The effect of silicone oil deposition on hair was examined by analyzing the element of hair after treatment of silicone oil nanoemulsions. RESULTS: Nonionic surfactants such as Span 80 and Tween 80 are suitable emulsifiers to prepare oil-in-water nanoemulsions that are both thermodynamically stable and can enhance the absorption of silicone oil on hair surface. CONCLUSION: The silicone oil-in-water nanoemulsions containing nonionic surfactants present as a promising solution to improve the silicone oil deposition on the hair surface for hair care applications.

A candidate molecular biomarker panel for the detection of bladder cancer.

BACKGROUND: Bladder cancer is among the five most common malignancies worldwide, and due to high rates of recurrence, one of the most prevalent. Improvements in noninvasive urine-based assays to detect bladder cancer would benefit both patients and health care systems. In this study, the goal was to identify urothelial cell transcriptomic signatures associated with bladder cancer. METHODS: Gene expression profiling (Affymetrix U133 Plus 2.0 arrays) was applied to exfoliated urothelia obtained from a cohort of 92 subjects with known bladder disease status. Computational analyses identified candidate biomarkers of bladder cancer and an optimal predictive model was derived. Selected targets from the profiling analyses were monitored in an independent cohort of 81 subjects using quantitative real-time PCR (RT-PCR). RESULTS: Transcriptome profiling data analysis identified 52 genes associated with bladder cancer (P ≤ 0.001) and gene models that optimally predicted class label were derived. RT-PCR analysis of 48 selected targets in an independent cohort identified a 14-gene diagnostic signature that predicted the presence of bladder cancer with high accuracy. CONCLUSIONS: Exfoliated urothelia sampling provides a robust analyte for the evaluation of patients with suspected bladder cancer. The refinement and validation of the multigene urothelial cell signatures identified in this preliminary study may lead to accurate, noninvasive assays for the detection of bladder cancer. IMPACT: The development of an accurate, noninvasive bladder cancer detection assay would benefit both the patient and health care systems through better detection, monitoring, and control of disease.