Monitoring the Progression of Liver Fluke-Induced Cholangiocarcinoma in a Hamster Model Using Synchrotron FTIR Microspectroscopy and Focal Plane Array Infrared Imaging.

Cholangiocarcinoma (CCA) is a bile duct cancer that originates in the bile duct epithelium. Northeastern Thailand has the highest incidence of CCA, and there is a direct correlation with liver fluke (Opisthorchis viverrini) infection. The high mortality rate of CCA is a consequence of delayed diagnosis. Fourier transform infrared (FTIR) spectroscopy is a powerful technique that detects the absorbance of molecular vibrations and is perfectly suited for the interrogation of biological samples. In this study, we applied synchrotron radiation-FTIR (SR-FTIR) microspectroscopy and focal plane array (FPA-FTIR) microspectroscopy to characterize periductal fibrosis and bile duct cells progressing to CCA induced by inoculating O. viverrini metacercariae into hamsters. SR-FTIR and FPA-FTIR measurements were performed in liver sections harvested from 1-, 2-, 3-, and 6-month post-infected hamsters compared to uninfected liver tissues. Principal component analysis (PCA) of the tissue samples showed a clear discrimination among uninfected and early-stage (1 and 2 months) and cancerous-stage (3 and 6 months) tissues. The discrimination is based on intensity changes in the phosphodiester band (1081 cm-1), amino acid residue (∼1396 cm-1), and C═O stretching carboxylic esters (1745 cm-1). Infected tissues also show definitive bands at ∼1280, 1234, and 1201 cm-1 characteristic of the collagen triplet and indicative of fibrosis. Hierarchical cluster analysis (HCA) was performed on the FPA data and showed a classification into specific cell types. Hepatocyte, fibrotic lesion, and bile duct (cancer) were classified and HCA mapping showed similar cellular distribution pattern compared to Sirius red staining. This study was also extended to less invasive sample analysis using attenuated total reflectance-FTIR (ATR-FTIR) spectroscopy. Sera from O. viverrini-infected and uninfected hamsters were analyzed using multivariate analysis, including principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA). PCA was able to classify spectra of normal, early-stage CCA, and CCA, while the PLS-DA gave 100% accuracy for the validation. The model was established from 17 samples (11 normal, 6 cancer) in the calibration set and 9 samples in the validation set (4 normal, 2 cancer, 3 precancerous). These results indicate that FTIR-based technology is a potential tool to detect the progression of CCA, especially in the early stages of the disease.

DNA aptamer-functionalized PDA nanoparticles: from colloidal chemistry to biosensor applications.

Polydopamine nanoparticles (PDA NPs) are widely utilized in the field of biomedical science for surface functionalization because of their unique characteristics, such as simple and low-cost preparation methods, good adhesive properties, and ability to incorporate amine and oxygen-rich chemical groups. However, challenges in the application of PDA NPs as surface coatings on electrode surfaces and in conjugation with biomolecules for electrochemical sensors still exist. In this work, we aimed to develop an electrochemical interface based on PDA NPs conjugated with a DNA aptamer for the detection of glycated albumin (GA) and to study DNA aptamers on the surfaces of PDA NPs to understand the aptamer-PDA surface interactions using molecular dynamics (MD) simulation. PDA NPs were synthesized by the oxidation of dopamine in Tris buffer at pH 10.5, conjugated with DNA aptamers specific to GA at different concentrations (0.05, 0.5, and 5 µM), and deposited on screen-printed carbon electrodes (SPCEs). The charge transfer resistance of the PDA NP-coated SPCEs decreased, indicating that the PDA NP composite is a conductive bioorganic material. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) confirmed that the PDA NPs were spherical, and dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy data indicated the successful conjugation of the aptamers on the PDA NPs. The as-prepared electrochemical interface was employed for the detection of GA. The detection limit was 0.17 µg/mL. For MD simulation, anti-GA aptamer through the 5'terminal end in a single-stranded DNA-aptamer structure and NH2 linker showed a stable structure with its axis perpendicular to the PDA surface. These findings provide insights into improved biosensor design and have demonstrated the potential for employing electrochemical PDA NP interfaces in point-of-care applications.

Characterisation of chondrogenic differentiation of human mesenchymal stem cells using synchrotron FTIR microspectroscopy.

A major limiting factor in stem cell therapy is the accurate identification of the differentiation state of cells destined for transplantation. This study aimed to evaluate the potential of synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy as a novel technique to probe the differentiation state of human mesenchymal stem cells (hMSCs) to chondrocytes over a period of 7, 14 and 21 days of induction. The chondrogenic markers were determined using reverse transcription polymerase chain reaction, histology and immunohistochemistry. The changes of average spectra located near 1338-1230 and 1175-960 cm(-1) indicated increased levels of collagen and aggrecan, respectively, in chondrocyte-induced hMSCs compared with control cells. Classification of independent test spectra using partial least squares discriminant analysis (PLS-DA) could distinguish control and chondrocyte-induced cells with 100% accuracy. We conclude that the SR-FTIR microspectroscopy technique is sensitive for monitoring the differentiation state of stem cells under chondrogenic induction particularly at an early stage. It provides biochemical information that is complimentary to that obtained from conventional techniques, and may give more unambiguous results particularly at the very early stage of cellular differentiation. In addition, the spectroscopic approach is more straightforward, non-destructive and requires less sample preparation compared with the conventional methodologies.

Detection of Human Cholangiocarcinoma Markers in Serum Using Infrared Spectroscopy.

Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium. Opisthorchis viverrini infection is a known high-risk factor for CCA and in found, predominantly, in Northeast Thailand. The silent disease development and ineffective diagnosis have led to late-stage detection and reduction in the survival rate. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) is currently being explored as a diagnostic tool in medicine. In this study, we apply ATR-FTIR to discriminate CCA sera from hepatocellular carcinoma (HCC), biliary disease (BD) and healthy donors using a multivariate analysis. Spectral markers differing from healthy ones are observed in the collagen band at 1284, 1339 and 1035 cm-1, the phosphate band (vsPO2-) at 1073 cm-1, the polysaccharides band at 1152 cm-1 and 1747 cm-1 of lipid ester carbonyl. A Principal Component Analysis (PCA) shows discrimination between CCA and healthy sera using the 1400-1000 cm-1 region and the combined 1800-1700 + 1400-1000 cm-1 region. Partial Least Square-Discriminant Analysis (PLS-DA) scores plots in four of five regions investigated, namely, the 1400-1000 cm-1, 1800-1000 cm-1, 3000-2800 + 1800-1000 cm-1 and 1800-1700 + 1400-1000 cm-1 regions, show discrimination between sera from CCA and healthy volunteers. It was not possible to separate CCA from HCC and BD by PCA and PLS-DA. CCA spectral modelling is established using the PLS-DA, Support Vector Machine (SVM), Random Forest (RF) and Neural Network (NN). The best model is the NN, which achieved a sensitivity of 80-100% and a specificity between 83 and 100% for CCA, depending on the spectral window used to model the spectra. This study demonstrates the potential of ATR-FTIR spectroscopy and spectral modelling as an additional tool to discriminate CCA from other conditions.

Simultaneous manganese adsorption and biotransformation by Streptomyces violarus strain SBP1 cell-immobilized biochar.

Consumption of water containing high proportions of manganese could cause Parkinson's like symptoms and damage the central nervous systems. This study aims to investigate the potential of manganese removal through the development of microbial cell-immobilized biochar. The wood vinegar industry generates a large volume of carbonized wood waste (natural biochar) from the pyrolytic process. This is the first investigation utilizing this low value waste combined with biological treatment for water purification. Raw and hydrogen peroxide-modified biochars were used to immobilize an effective manganese-oxidizing bacterium, Streptomyces violarus strain SBP1 (SBP1). The results demonstrated that the modified biochar had a higher proportion of oxygen-containing functional groups leading to better manganese removal. Manganese adsorption by the modified biochar fitted pseudo-second-order and Langmuir models with the maximum adsorption capacity of 1.15 mg g-1. The modified biochar with SBP1 provided the highest removal efficiency at 78%. The advanced synchrotron analyses demonstrated that manganese removal by the biochar with SBP1 is due to the synergistic combination of manganese adsorption by biochars and biological oxidation by SBP1.

Discrimination of micromass-induced chondrocytes from human mesenchymal stem cells by focal plane array-Fourier transform infrared microspectroscopy.

Rapid and sensitive methods for identifying stem cell differentiation state are required for facilitating future stem cell therapies. We aimed to evaluate the capability of focal plane array-Fourier transform infrared (FPA-FTIR) microspectroscopy for characterising the differentiation of chondrocytes from human mesenchymal stem cells (hMSCs). Successful induction was validated by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis for collagen and aggrecan expression as chondrocyte markers in parallel with the spectroscopy. Spectra derived from chondrocyte-induced cells revealed strong IR absorbance bands attributed to collagen near 1338 and 1234 cm(-1) and proteoglycan at 1245 and 1175-960 cm(-1) compared to the non-induced cells. In addition, spectra from control and induced cells are segregated into separate clusters in partial least squares discriminant analysis score plots at the very early stages of induction and discrimination of an independent set of validation spectra with 100% accuracy. The predominant bands responsible for this discrimination were associated with collagen and aggrecan protein concordant with those obtained from RT-PCR and Western blot techniques. Our findings support the capability of FPA-FTIR microspectroscopy as a label-free tool for stem cell characterization allowing rapid and sensitive detection of macromolecular changes during chondrogenic differentiation.