Highly sensitive SERS detection of cancer proteins in low sample volume using hollow core photonic crystal fiber

Enzyme-linked immunosorbent assays (ELISA) are commonly used for detecting cancer proteins at concentration in the range of about ng-μg/mL. Hence it often fails to detect tumor markers at the early stages of cancer and other diseases where the amount of protein is extremely low. Herein, we report a novel photonic crystal fiber (PCF) based surface enhanced Raman scattering (SERS) sensing platform for the ultrasensitive detection of cancer proteins in an extremely low sample volume. As a proof of concept, epidermal growth factor receptors (EGFRs) in a lysate solution from human epithelial carcinoma cells were immobilized into the hollow core PCF. Highly sensitive detection of protein was achieved using anti-EGFR antibody conjugated SERS nanotag. This SERS nanotag probe was realized by anchoring highly active Raman molecules onto the gold nanoparticles followed by bioconjugation. The proposed sensing method can detect low amount of proteins at ～100 pg in a sample volume of ～10 nL. Our approach may lead to the highly sensitive protein sensing methodology for the early detection of diseases.     

Blood-Based Biomarkers of Aggressive Prostate Cancer

Purpose

Prostate cancer is a bimodal disease with aggressive and indolent forms. Current prostate-specific-antigen testing and digital rectal examination screening provide ambiguous results leading to both under-and over-treatment. Accurate, consistent diagnosis is crucial to risk-stratify patients and facilitate clinical decision making as to treatment versus active surveillance. Diagnosis is currently achieved by needle biopsy, a painful procedure. Thus, there is a clinical need for a minimally-invasive test to determine prostate cancer aggressiveness. A blood sample to predict Gleason score, which is known to reflect aggressiveness of the cancer, could serve as such a test.

Materials and Methods

Blood mRNA was isolated from North American and Malaysian prostate cancer patients/controls. Microarray analysis was conducted utilizing the Affymetrix U133 plus 2·0 platform. Expression profiles from 255 patients/controls generated 85 candidate biomarkers. Following quantitative real-time PCR (qRT-PCR) analysis, ten disease-associated biomarkers remained for paired statistical analysis and normalization.

Results

Microarray analysis was conducted to identify 85 genes differentially expressed between aggressive prostate cancer (Gleason score ≥8) and controls. Expression of these genes was qRT-PCR verified. Statistical analysis yielded a final seven-gene panel evaluated as six gene-ratio duplexes. This molecular signature predicted as aggressive (ie, Gleason score ≥8) 55% of G6 samples, 49% of G7(3+4), 79% of G7(4+3) and 83% of G8-10, while rejecting 98% of controls.

Conclusion

In this study, we have developed a novel, blood-based biomarker panel which can be used as the basis of a simple blood test to identify men with aggressive prostate cancer and thereby reduce the overdiagnosis and overtreatment that currently results from diagnosis using PSA alone. We discuss possible clinical uses of the panel to identify men more likely to benefit from biopsy and immediate therapy versus those more suited to an “active surveillance” strategy.     

Movements of Indian Flying Fox in Myanmar as a Guide to Human-Bat Interface Sites

EcoHealth - Frugivorous bats play a vital role in tropical ecosystems as pollinators and seed dispersers but are also important vectors of zoonotic diseases. Myanmar sits at the intersection of...     

The influence of preparative techniques on glycolytic metabolism in resting leucocytes

Literature values for ‘resting’ glycolytic and respiratory rates of guinea pig exudate polymorphonuclear leucocytes as reported by various authors were calculated to the same unit basis to determine what differences might exist. Comparison experiments investigated preparative techniques to determine the significance with respect to the variations discovered. Procedures using 12 or 0.1% casein in saline as peritoneal irritant, 0 or 37 °C collection temperatures, siliconed or non-siliconed glassware, and hemolysis treatment to remove red blood cells, did not affect glycolytic rate in leucocytes. Three different anticoagulants used, heparin, citrate, and ethylenediamine tetracetic acid (EDTA) did account for the variability in the reported glycolytic rates by increasing the lactate production as compared with cells not exposed to anticoagulant. Citrate and EDTA increased the lactate production approx. 65%, heparin 22%, with the increase apparently related to the calcium chelating ability. Calcium ions were found to depress the glycolytic lactate production proportionately as the concentration increased up to 0.0026 M in the incubation medium. The removal or absence of calcium ions from the medium increased the lactate formation.     

Molecular Parentage Analysis Is Essential in Breeding Asian Seabass

In aquaculture species, maintaining pedigree information and genetic variation in each generation is essential, but very difficult. In this study, we used nine microsatellites to genotype 2,520 offspring from four independent full-factorial crosses (10 males ×10 females) of Asian seabass to reconstruct pedigree and monitor the change of genetic variations. In all four crosses, over 96.8% of the offspring could be assigned to their parents, indicating the high power of the nine microsatellites for parentage assignment. This study revealed several interesting results: (1). In all four crosses, the contribution of parents to offspring was significantly uneven, and some dominant breeding fishes (i.e. brooders) were found; (2). In two mass crosses where the brooders were carefully checked for reproductive status, a majority (≥90%) of brooders contributed to offspring, whereas in another two crosses, where the brooders were randomly picked without checking reproductive status, only a few brooders (40.0–45.0%) produced offspring; (3). Females had more problems in successful spawning compared to males; and (4). In the two crosses where a few brooders produced offspring, there was a substantial loss in allelic (24.1–34.3%) and gene (20.5–25.7%) diversities in offspring, while in the other two crosses, the majority of allelic (96.8–97.0%) and gene diversities (94.8–97.1%) were maintained. These observations suggest that a routine molecular parentage analysis is required to maintain both allelic and gene diversity in breeding Asian seabass.     

Screening for microbial strains degrading glass fiber acrylic composite filters

In this study we have assessed the capacity of five fungal and two bacterial species to biodegrade glass fiber acrylic composite filters which are utilized in air conditioners. The strains used were Trichoderma harzianum (2 strains), Trichoderma koningii, Penicillium spp., Aspergillus niger, Pseudomonas aeruginosa and Pseudomonas fluorescens. Pre-sterilized filters were incubated in solid or liquid media at 30 °C for 21 days. Biodegradability was monitored by evaluating microbial colonization, increase in biomass and weight loss of filters coupons. Among the species under investigation, the two strains of T. harzianum (MYA198 and BCC5828) showed the best biodegradability performance and were used to analyse total carbon and esterase activity. Our results clearly indicate that cells grown in the presence of shredded filters display a hydrolytic activity and lead to a consistent removal of the organic portion of the tested filters. This study suggests that a solid state fermentation process in suitable bioreactors based on T. harzianum species could be a suitable approach to acrylic composite filter biodegradation.     

A role for focal adhesion kinase in hyluronan-dependent MMP-2 secretion in a human small-cell lung carcinoma cell line, QG90.

Hyluronan (HA), a nonsulfated high-molecular mass glycoaminoglycan, has been assigned as a clinical marker for the progression of various tumors. We found that HA stimulation of QG90, a cell line derived from human small-cell lung carcinoma, activates the secretion of matrix metalloproteinase-2 (MMP-2) in a focal adhesion kinase (FAK)-dependent manner. HA stimulation of QG90 cells activated MMP-2 secretion in a time-dependent manner. Larger sizes of HA seemed to have higher activities than smaller size one in MMP-2 secretion. Under HA stimulation, tyrosine phosphorylation of cellular proteins including FAK was activated. By use of antisense oligonucleotide to FAK, we found that FAK signaling was required for the activation of MMP-2 secretion and for the sustained activation of MAP kinase by HA treatment. These results strongly suggest that FAK-MAPK signaling is involved, at least in part, in HA-dependent activation of MMP-2 secretion in QG90 cells.     

Quantitative diagnosis of cervical neoplasia using fluorescence lifetime imaging on haematoxylin and eosin stained tissue sections

The use of conventional fluorescence microscopy for characterizing tissue pathological states is limited by overlapping spectra and the dependence on excitation power and fluorophore concentration. Fluorescence lifetime imaging microscopy (FLIM) can overcome these limitations due to its insensitivity to fluorophore concentration, excitation power and spectral similarity. This study investigates the diagnosis of early cervical cancer using FLIM and a neural network extreme learning machine classifier. A concurrently high sensitivity and specificity of 92.8% and 80.2%, respectively, were achieved. The results suggest that the proposed technique can be used to supplement the traditional histopathological examination of early cervical cancer. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Proteome analysis of gentisate-induced response in Pseudomonas alcaligenes NCIB 9867

Pseudomonas alcaligenes NCIB 9867 (P25X wild-type) is capable of degrading aromatic hydrocarbons via the gentisate pathway. Biochemical characterization of P25X mutants indicated that it has isofunctional enzymes for the mono- and dioxygenase-catalyzed reactions. One set of the enzymes is constitutive whereas the other is strictly inducible. To date, only the gene encoding the constitutively-expressed gentisate dioxygenase had been cloned and characterized. A mutant strain of P25X, designated G56, which had the constitutive copy of the gentisate 1,2-dioxygenase gene interrupted by a streptomycin/spectinomycin resistance gene cassette, was found to express gentisate dioxygenase, but only when the cells were induced by gentisate. The proteome profiles of P. alcaligenes P25X and mutant G56 cells grown in the presence and absence of gentisate were compared after two-dimensional polyacrylamide gel electrophoresis. Eight distinctive protein spots (designated M1-M8) which were observed only in induced cells of strain G56 but absent in noninduced cells were further analyzed by matrix-assisted laser desorption/ionization-time of flight, quadrupole-TOF and N-terminal sequencing. Of the 15 proteins (including seven up-regulated) examined, 13 showed sequence similarities to proteins with assigned functions in other microorganisms. The identification of protein M5 which showed high homology to a gentisate dioxygenase from Ralstonia sp. U2 indicated the putative function of this protein being consistent with the inducible gentisate 1,2-dioxygenase in P. alcaligenes. In addition, the induction of stress proteins and other adaptation phenomena were also observed.