An Automated Micro-Total Immunoassay System for Measuring Cancer-Associated α2,3-linked Sialyl N-Glycan-Carrying Prostate-Specific Antigen May Improve the Accuracy of Prostate Cancer Diagnosis.

The low specificity of the prostate-specific antigen (PSA) for early detection of prostate cancer (PCa) is a major issue worldwide. The aim of this study to examine whether the serum PCa-associated α2,3-linked sialyl N-glycan-carrying PSA (S2,3PSA) ratio measured by automated micro-total immunoassay systems (µTAS system) can be applied as a diagnostic marker of PCa. The µTAS system can utilize affinity-based separation involving noncovalent interaction between the immunocomplex of S2,3PSA and Maackia amurensis lectin to simultaneously determine concentrations of free PSA and S2,3PSA. To validate quantitative performance, both recombinant S2,3PSA and benign-associated α2,6-linked sialyl N-glycan-carrying PSA (S2,6PSA) purified from culture supernatant of PSA cDNA transiently-transfected Chinese hamster ovary (CHO)-K1 cells were used as standard protein. Between 2007 and 2016, fifty patients with biopsy-proven PCa were pair-matched for age and PSA levels, with the same number of benign prostatic hyperplasia (BPH) patients used to validate the diagnostic performance of serum S2,3PSA ratio. A recombinant S2,3PSA- and S2,6PSA-spiked sample was clearly discriminated by µTAS system. Limit of detection of S2,3PSA was 0.05 ng/mL and coefficient variation was less than 3.1%. The area under the curve (AUC) for detection of PCa for the S2,3PSA ratio (%S2,3PSA) with cutoff value 43.85% (AUC; 0.8340) was much superior to total PSA (AUC; 0.5062) using validation sample set. Although the present results are preliminary, the newly developed µTAS platform for measuring %S2,3PSA can achieve the required assay performance specifications for use in the practical and clinical setting and may improve the accuracy of PCa diagnosis. Additional validation studies are warranted.

[Development and Clinical Application of Glycan-Targeted Biomarkers for Prostate Cancer].

Prostate cancer (PCa) is now the most common male malignant tumor in both Japan and Western countries. Prostate-specific antigen (PSA) has been widely used for the early detection of PCa; however, it is not an ideal biomarker due to its low specificity. Aberrant glycosylation is closely associated with malignant transformation and cancer progression. Recent advances in glycobiology techniques can be applied to the development of novel biomarkers for PCa. We previously identified PCa-associated aberrant glycosylation on PSA, that is, α2,3-linked sialylation as an additional terminal N-glycan on free PSA(S2,3PSA). We then developed a new assay system for the measurement of S2,3PSA utilizing the µTAS method. The area under the curve (AUC) for the detection of PCa with the %S2,3PSA ratio was significantly better than that with total PSA. Another urgent issue in clinical practice for PCa is the over-treatment of patients with a low malignant potential, as aggressive treatment is not necessary. To overcome this problem, it is essential to develop a useful tool for the measurement of the malignant potential. Core2 ,ß-1,6-N-acetylglucosaminyltransferase-1 (GCNT1, C2GnT) is a key enzyme that forms core 2-branched 0-glycans. Its expression is associated with the progression of several cancers. We established a mouse IgG monoclonal antibody (mAb) against GCNT1 and examined the relationship of GCNT1 expression with the clinicopathological status of PCa. GCNT1- negative patients were associated with significantly better PSA-free survival compared with GCNT1-positive patients. Furthermore, we established new methods for GCNT1 detection using urine samples of PCa patients. Immunoblotting was used to examine post-digital rectal examination (DRE) urine from PCa patients. Over 90% of GCNT1-positive PCa patients with high concentrations of serum PSA showed extracapsular extension in prostatectomy specimens. In conclusion, the clinical application of glycobiology techniques is a promising approach to develop novel biomarkers for PCa.

ZnO nanopowder induced light scattering for improved visualization of emission sites in carbon nanotube films and arrays.

We report on ZnO nanopowder induced light scattering for improved visualization of emission sites in carbon nanotube films and arrays. We observed a significant reduction of the internal multiple light scattering phenomena, which are characteristic for ZnO micropowders. The microsized grains of the commercially available ZnO:Zn (P 15) were reduced to the nanometre scale by pulsed laser ablation at an oxygen ambient pressure of 10 kPa. Our investigations show no crystalline change and no shift of the broad green emission peak at 500 nm for the ZnO nanopowder. For the application in field emission displays, we demonstrate the possibility of achieving cathodoluminescence with a fine pitch size of 100 microm of the patterned pixels without requiring additional electron beam focusing and without a black matrix. Moreover, the presented results show the feasibility of employing ZnO nanopowder as a detection material for the phosphorus screen method, which is able to localize emission sites of carbon nanotube films and arrays with an accuracy comparable to scanning anode field emission microscopy.

Differential detection of KRAS mutations in codons 12 and 13 with a modified loop-hybrid (LH) mobility shift assay using an insert-type LH-generator.

BACKGROUND: The loop-hybrid mobility shift assay (LH-MSA) was previously developed for the rapid detection of the EGFR mutation L858R for predicting clinical responses to gefitinib in lung cancer. Recently, clinical importance of determining KRAS mutations has been demonstrated in colorectal tumors as tumors harboring mutated KRAS genes were not responsive to therapy with EGFR-targeted antibodies such as cetuximab. METHODS: We developed a new version of the LH-MSA using an insert-type LH generator that was capable of detecting all 12 KRAS mutations in codons 12 and 13. RESULTS: Feasibility evaluation was performed with this new LH-MSA on 215 colorectal cancer specimens. KRAS codon 12 mutations were detected in 23% specimens and codon 13 mutations in 6.5% specimens by LH-MSA at a rate better than by direct sequencing. CONCLUSIONS: Using the new method, the G13D mutation was readily distinguishable from other KRAS mutations in codon 12 and, therefore, would be advantageous for clinical applications.

Simple and precise detection of UGT1A1 polymorphisms with a modified loop-hybrid mobility shift assay using Cy5-labeled loop probes.

BACKGROUND: Irinotecan, an inhibitor of topoisomerase I, has been widely used as an important anti-cancer therapeutic drug. Deleterious effects of the drug in hypersensitive patients are known to be associated with genetic polymorphisms of the UGT1A1 gene, namely the polymorphic variants, *28 and *6. METHODS: A modified form of loop-hybrid mobility shift assay using a Cy5-tagged loop-hybrid probe was proposed as a precise and easy method of determining TA repeat polymorphisms at the *28 locus. RESULTS: In this modified method, only loop-hybrid bands were detected by a Cy5-fluorescent signal, despite several irregular electrophoretic bands due to TA repeats in the PCR product. CONCLUSIONS: When a loop-hybrid using a Cy5-tagged probe for the *28 locus and *6 locus were combined and used for mobility shift assay, simultaneous typing of the *28 and *6 variants was achieved in a single lane.

Use of transcriptional sequencing in difficult to read areas of the genome.

In genome and cDNA sequencing projects, current cycle sequencing often encounters difficult-to-sequence templates which have unique secondary structures due to GC-rich composition or repeated regions. Due to the formation of stable secondary structures, remarkable decreases in fluorescent signals are observed in cycle sequencing reactions. It is not easy to determine the nucleotide sequences of these regions. Although several modifications of sequencing reactions have been tried to overcome these problems, some unreadable regions remain as gaps in genome sequencing projects. Here, we further developed transcriptional sequencing technology and evaluated the sequencing accuracy in these regions. The method was successively applied to artificial GC cluster templates and putative secondary structure-forming templates from genomic and cDNA clones. Our results indicate that transcriptional sequencing is a powerful and accurate method for GC-rich regions, simple sequence repeats, hairpins (inverted repeats), tandem repeat DNA templates, and gap-closing in draft sequencing data.