Err-Cheng Chan

Urinary 8-hydroxydeoxyguanosine and its analogs as DNA marker of oxidative stress: development of an ELISA and measurement in both bladder and prostate cancers

BACKGROUND 8-hydroxydeoxyguanosine (8-OHdG) is the most frequently detected and studied DNA lesion. Upon DNA repair, 8-OHdG is excreted in the urine. Urinary 8-OHdG is now considered as a biomarker of generalized, cellular oxidative stress and is linked to degenerative diseases including cancer. METHODS We developed a competitive enzyme-linked immunosorbent assay (ELISA) for urinary 8-OHdG by coating BSA conjugated 8-hydroxyguanine (8-OHG) on a microplate. Urine specimens containing 8-OHdG and monoclonal anti-8-OHdG antibody were incubated together in the microwell. Final quantification of bound anti-8-OHdG antibody was estimated by the addition of HRP-conjugated sheep-anti-mouse antibody. RESULTS The concentration range of the calibration curve was 0-60 ng/ml. The sensitivity of the assay was 0.5 ng/ml. The within-day precision and day-to-day precision were <10%. The ELISA correlated well with a commercial kit (r=0.9). Our assay measured not only 8-OHdG but also 8-OHG and 8-hyroxyguanine in urine. Increased urinary concentration of 8-OHdG and its analogs were detected in both patients with bladder cancer (70.5+/-38.2 ng/mg creatinine) and prostate cancer (58.8+/-43.4 ng/mg creatinine) as compared to the healthy control (36.1+/-24.5 ng/mg creatinine). CONCLUSION Our preliminary data suggest that the competitive ELISA for 8-OHdG and its analogs appears to be a simple method for quantifying the extent of oxidative stress and may have potential for identifying cancer risk.

Detection of rare mutant K-ras DNA in a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe

The major problem of using somatic mutations as markers of malignancy is that the clinical samples are frequently containing a trace amounts of mutant allele in a large excess of wild-type DNA. Most methods developed thus far for the purpose of tickling this difficult problem require multiple procedural steps that are laborious. We report herein the development of a rapid and simple protocol for detecting a trace amounts of mutant K-ras in a single tube, one-step format. In a capillary PCR, a 17mer peptide nucleic acid (PNA) complementary to the wild-type sequence and spanning codons 12 and 13 of the K-ras oncogene was used to clamp-PCR for wild-type, but not mutant alleles. The designated PNA was labeled with a fluorescent dye for use as a sensor probe, which differentiated all 12 possible mutations from the wild-type by a melting temperature (Tm) shift in a range of 9 to 16°C. An extension temperature of 60°C and an opposite primer 97 nt away from the PNA were required to obtain full suppression of wild-type PCR. After optimization, the reaction detected mutant templates in a ratio of 1:10 000 wild-type alleles. Using this newly devised protocol, we have been able to detect 19 mutants in a group of 24 serum samples obtained from patients with pancreatic cancer. Taken together, our data suggest that this newly devised protocol can serve as an useful tool for cancer screening as well as in the detection of rare mutation in many diseases.

Novel biodegradable sandwich-structured nanofibrous drug-eluting membranes for repair of infected wounds: an in vitro and in vivo study

Background The purpose of this study was to develop novel sandwich-structured nanofibrous membranes to provide sustained-release delivery of vancomycin, gentamicin, and lidocaine for repair of infected wounds. Methods To prepare the biodegradable membranes, poly(D, L)-lactide-co-glycolide (PLGA), collagen, and various pharmaceuticals, including vancomycin, gentamicin, and lidocaine, were first dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol. They were electrospun into sandwich-structured membranes with PLGA/collagen as the surface layers and PLGA/drugs as the core. An elution method and a high-pressure liquid chromatography assay were used to characterize in vivo and in vitro drug release from the membranes. In addition, repair of infected wounds in rats was studied. Histological examination of epithelialization and granulation at the wound site was also performed. Results The biodegradable nanofibrous membranes released large amounts of vancomycin and gentamicin (well above the minimum inhibition concentration) and lidocaine in vivo for more than 3 weeks. A bacterial inhibition test was carried out to determine the relative activity of the antibiotics released. The bioactivity ranged from 40% to 100%. The nanofibrous membranes were functionally active in treating infected wounds, and were very effective as accelerators in early-stage wound healing. Conclusion Using the electrospinning technique, we will be able to manufacture biodegradable, biomimetic, nanofibrous, extracellular membranes for long-term delivery of various drugs.

An Informatics-assisted Label-free Approach for Personalized Tissue Membrane Proteomics: Case Study on Colorectal Cancer

We developed a multiplexed label-free quantification strategy, which integrates an efficient gel-assisted digestion protocol, high-performance liquid chromatography tandem MS analysis, and a bioinformatics alignment method to determine personalized proteomic profiles for membrane proteins in human tissues. This strategy provided accurate (6% error) and reproducible (34% relative S.D.) quantification of three independently purified membrane fractions from the same human colorectal cancer (CRC) tissue. Using CRC as a model, we constructed the personalized membrane protein atlas of paired tumor and adjacent normal tissues from 28 patients with different stages of CRC. Without fractionation, this strategy confidently quantified 856 proteins (≥2 unique peptides) across different patients, including the first and robust detection (Mascot score: 22,074) of the well-documented CRC marker, carcinoembryonic antigen 5 by a discovery-type proteomics approach. Further validation of a panel of proteins, annexin A4, neutrophils defensin A1, and claudin 3, confirmed differential expression levels and high occurrences (48–70%) in 60 CRC patients. The most significant discovery is the overexpression of stomatin-like 2 (STOML2) for early diagnostic and prognostic potential. Increased expression of STOML2 was associated with decreased CRC-related survival; the mean survival period was 34.77 ± 2.03 months in patients with high STOML2 expression, whereas 53.67 ± 3.46 months was obtained for patients with low STOML2 expression. Further analysis by ELISA verified that plasma concentrations of STOML2 in early-stage CRC patients were elevated as compared with those of healthy individuals (p < 0.001), suggesting that STOML2 may be a noninvasive serological biomarker for early CRC diagnosis. The overall sensitivity of STOML2 for CRC detection was 71%, which increased to 87% when combined with CEA measurements. This study demonstrated a sensitive, label-free strategy for differential analysis of tissue membrane proteome, which may provide a roadmap for the subsequent identification of molecular target candidates of multiple cancer types.

Comparison of membrane fraction proteomic profiles of normal and cancerous human colorectal tissues with gel-assisted digestion and iTRAQ labeling mass spectrometry

The aim of this study was to uncover the membrane protein profile differences between colorectal carcinoma and neighboring normal mucosa from colorectal cancer patients. Information from cellular membrane proteomes can be used not only to study the roles of membrane proteins in fundamental biological processes, but also to discover novel targets for improving the management of colorectal cancer patients. We used solvent extraction and a gel‐assisted digestion method, together with isobaric tags with related and absolute quantitation (iTRAQ) reagents to label tumoral and adjacent normal tissues in a pairwise manner (n = 8). For high‐throughput quantification, these digested labeled peptides were combined and simultaneously analyzed using LC‐MS/MS. Using the shotgun approach, we identified a total of 438 distinct proteins from membrane fractions of all eight patients. After comparing protein expression between cancerous and corresponding normal tissue, we identified 34 upregulated and eight downregulated proteins with expression changes greater than twofold (Student’s t‐test, P < 0.05). Among these, the overexpression of well‐established biomarkers such as carcinoembryonic antigens (CEACAM5, CEACAM6), as well as claudin‐3, HLA class I histocompatibility antigen A‐1, tapasin and mitochondrial solute carrier family 25A4 were confirmed by western blotting. We conclude that gel‐assisted digestion and iTRAQ labeling MS is a potential approach for uncovering and comparing membrane protein profiles of tissue samples that has the potential to identify novel biomarkers.

Development of a biodegradable antibiotic delivery system.

Antibiotic beads have been used as a drug delivery system for the treatment of various surgical infections. In this study, the copolymer 50:50 poly(DL-lactide):co-glycolide was mixed with vancomycin powder and hot compressing molded at 55 degrees C to form five types of biodegradable antibiotic beads. The beads were placed in 1 mL of phosphate buffered saline and incubated at 37 degrees C. The phosphate buffered saline was changed daily, and the removed buffer solutions were stored at -70 degrees C until the antibiotic concentration in each sample was determined by high performance liquid chromatography system assay. The concentration of vancomycin in each sample was well above the breakpoint sensitivity concentration (the antibiotic concentration at the transition point between bacterial killing and resistance to the antibiotic) for more than 32 days. The release was most marked during the first 48 hours. All copolymer 50:50 poly(DI lactide):co-glycolide biodegradable beads released high concentrations of the antibiotics in vitro for the time needed to treat bone infections (4 to 6 weeks). The diameter of the sample inhibition zone ranged from 6.5 mm to 10 mm, and the relative activity of vancomycin ranged from 12.5% to 100%. Copolymers with low heat of formation temperatures are required for making a controlled release system to prevent antibiotic decomposition, which occurs when using the hot compressing molded method. The rate and duration of release from the antibiotic beads can be adjusted by varying the diameter of the beads. This offers a convenient method to adjust the release rate to meet the specific antibiotic requirements for different patients.

Identifying Differentially Expressed Genes Associated with Metastasis of Follicular Thyroid Cancer by cDNA Expression Array

Patients with follicular thyroid carcinoma have a higher incidence of metastasis than papillary thyroid carcinoma when thyroid cancer is diagnosed. The cDNA expression array technology is utilized herein to profile differentially expressed genes from metastatic human follicular thyroid carcinoma and reveal new tumor markers as well as target genes for therapeutic intervention. Tissue samples were obtained during surgical resection of the thyroid follicular carcinoma and metastatic tissue in the brain of the same patient. Two identical Atlas human cDNA expression arrays were hybridized with 32P-labeled cDNA probes derived from RNA of either primary thyroid cancer or metastatic tissue. Parallel analysis of the hybridized signals allowed us to identify the alteration of gene expression in the metastasis process. Eighteen genes significantly overexpressed and 40 genes significantly underexpressed were identified in the metastatic thyroid cancer. Genes that displayed an altered expression were associated with the processes of cell cycle regulation, apoptosis, DNA damage response, angiogenesis, cell adhesion and mobility, invasion, and immune response. An expression profile of genes that are associated with metastasis process of follicular thyroid cancer was also discussed. Further investigation is required to understand the precise relationship between the altered expression of these genes and the metastasis process of follicular thyroid cancer.

In Vitro Elution of Antibiotic from Antibiotic-impregnated Biodegradable Calcium Alginate Wound Dressing

OBJECTIVE The authors investigated the calcium alginate dressing as a drug-delivery system for the treatment of various surgical infections. METHODS Cytotoxicity of the calcium alginate dressing to fibroblasts and HeLa cells was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MITT) colorimetric assay. The calcium alginate dressing was mixed with vancomycin, and lyophilized or not lyophilized to form two types of antibiotic dressings. The antibiotic dressings were placed in 2 mL of phosphate buffered saline (PBS) or in PBS containing 0.01% calcium ions, and incubated at 37 degrees C. The PBS was changed daily, and the removed solutions were stored at -70 degrees C until the antibiotic concentration in each sample was determined by high performance liquid chromatography assay. RESULTS The results suggested that the antibiotic dressings present no obvious toxic risk to their use as a drug-delivery system. The concentration of vancomycin in each sample was well above the breakpoint sensitivity concentration (the antibiotic concentration at the transition point between bacterial kill. ing and resistance to the antibiotic) for more than 14 days. The release was most marked during the first 48 hours. The concentration of calcium ions in PBS and the lyophilization of the manufacture process of antibiotic dressings prolonged the antibiotic diffusion duration. The diameter of the sample inhibition zone ranged from 10 to 11 mm, and the relative activity of vancomycin ranged from 62.88% to 92.18%. CONCLUSION All antibiotic dressings released bactericidal concentrations of the antibiotics in vitro for the period of time needed to treat surgical infections. This study offers a convenient method to meet the specific antibiotic requirement for different patients.

Comparison of KRAS mutation analysis of primary tumors and matched circulating cell-free DNA in plasmas of patients with colorectal cancer

Colorectal cancer (CRC) patients with KRAS mutations do not benefit from epidermal growth factor receptor (EGFR) targeted therapy. In clinical practice, identifying patients with KRAS mutations is critical prior to EGFR targeting therapy, and gene testing is generally performed using the DNA extracted from tumor tissue. The aim of this study was to compare the presence of KRAS mutations in circulating cell-free DNA (cfDNA) and primary tumor tissue using a peptide nucleic acid mediated polymerase chain reaction. We extracted and analyzed the DNA from plasmas and corresponding primary tumor samples from 52 patients with CRC. The results demonstrated that the detection rate of KRAS sequence variations was 50% (26 of 52) in plasma samples and 28.8% (15 of 52) in resected primary tumor tissue samples. The majority of KRAS mutations detected in tumors were also found in matched plasma specimens with an agreement rate of 78.8%. Eleven plasma cfDNA were found positive for KRAS mutation but not in their corresponding tissue. In conclusion, our results suggest that circulating cfDNA provides a better representation of the malignant disease as a whole and could be a reliable source of diagnostic DNA to replace the tumor tissue in a diagnostic setting.

Expression of caspid protein VP1 for use as antigen for the diagnosis of enterovirus 71 infection

To produce enterovirus 71 antigen for diagnostic purposes, the gene encoding the entire capsid protein VP1 was amplified by reverse transcription‐polymerase chain reaction (RT‐PCR), cloned and expressed in Escherichia coli as a poly‐histidine fusion protein. Western blotting experiments with sera from patients with enterovirus 71 infection indicated that immunoglobulin G (IgG) and IgM antibodies bound to a single polypeptide VP1. According to these results, IgM anti‐VP1 appeared in sera of patients with a symptomatic enterovirus 71 acute infection, whereas IgG anti‐VP1 was present in sera of past infection. This finding suggests that detecting IgG and IgM immune responses against linear epitopes of recombinant VP1 is an effective means of determining the different phases of enterovirus 71 infection. In addition, sera containing coxsackie virus 16 (CA16) antibodies did not cross‐react with the recombinant VP1 of enterovirus 71, despite the homology between VP1 proteins of both viruses. Comparison with reference PCR and neutralization assays showed these antibody tests to be appropriate for the serodiagnosis of enterovirus 71 infection. J. Med. Virol. 61:228–234, 2000.