The Evaluation of a Lateral Flow Strip Based on the Covalently Fixed "End-On" Orientation of an Antibody for Listeria monocytogenes Detection.

In this study, the covalently fixed "end-on" orientation of a monoclonal Listeria monocytogenes antibody (mAb-Lis) to amino terminated oligo (ethylene glycol)-capped gold nanoparticles (NH2-TEG-AuNPs) was used to fabricate an in-house lateral flow strip (LFS), namely, the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS. The aim was to evaluate the performance of the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS in detecting L. monocytogenes. The proposed LFS enabled the sensitive detection of L. monocytogenes in 15 min with a visual limit of detection of 102 CFU/mL. Quantitative analysis indicated an LOD at 10 CFU/mL. The fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS showed no cross-reactivity with other pathogenic bacteria and practical performance across different food matrices, including human blood, milk, and mushroom samples. Furthermore, the clinical performance of the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS for detecting L. monocytogenes was evaluated by using 12 clinical samples validated by the hemoculture method. It demonstrated excellent concordance with the reference methods, with no false-positive or false-negative results observed. Therefore, the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS serves as a promising candidate for a point-of-care test (POCT), enabling the rapid, precise, and highly sensitive detection of L. monocytogenes in clinical samples and contaminated food.

Serum CCDC25 Levels as a Potential Marker for Metabolic Syndrome.

BACKGROUND/AIM: Metabolic syndrome (MetS) stands as a significant risk for developing various severe health problems. Therefore, the discovery of biomarkers capable of predicting the progression of metabolic conditions is crucial for improving overall health outcomes. Recently, we reported that coiled-coil domain containing 25 (CCDC25) might be associated with key proteins involved in metabolic pathways, by bioinformatics analysis. Thus, we assumed that serum CCDC25 levels might have an association with MetS status. PATIENTS AND METHODS: In this study, based on the modified National Cholesterol Education Program-Adult Treatment Panel III (modified NCEP-ATP III) criteria, the participants who had three or more of abnormal criteria were defined as MetS, and those who had 1 or 2 abnormal criteria as pre-MetS groups; those who had no abnormal criteria were classified as the healthy control (HC) group. Serum CCDC25 levels were measured using the dot blot assay. RESULTS: The results showed that serum CCDC25 levels of the MetS group (0.072±0.026 ng/µl) were significantly higher (p<0.001) than that of pre-MetS (0.031±0.011 ng/µl) or HC groups (0.018±0.007 ng/µl). We can discern a consistent trend indicating that serum CCDC25 level is well correlated with the number of abnormal criteria of MetS of each participant. Although serum CCDC25 levels correlated with the distribution of all 5 MetS criteria, the highest correlation was seen in serum CCDC25 levels and triglyceride (TG) levels, with r=0.563, followed by systolic blood pressure (SBP) levels (r=0.557) and high-density lipoprotein-cholesterol (HDL-C) levels (r=-0.545). CONCLUSION: CCDC25 showed correlations with all MetS parameters, particularly with TG, SBP, and HDL-C. This prompts speculation that heightened CCDC25 levels may indicate the development and/or progression of those MetS-associated diseases.

Anticancer and anti-angiogenic activities of mannooligosaccharides extracted from coconut meal on colorectal carcinoma cells in vitro.

Colorectal carcinoma (CRC) is one of the most common malignancies, though there are no effective therapeutic regimens at present. This study aimed to investigate the inhibitory effects of mannooligosaccharides extracted from coconut meal (CMOSs) on the proliferation and migration of human colorectal cancer HCT116 cells in vitro. The results showed that CMOSs exhibited significant inhibitory activity against HCT116 cell proliferation in a concentration-dependent manner with less cytotoxic effects on the Vero normal cells. CMOSs displayed the ability to increase the activation of caspase-8, -9, and -3/7, as well as the generation of reactive oxygen species (ROS). Moreover, CMOSs suppressed HCT116 cell migration in vitro. Interestingly, treatment of human microvascular endothelial cells (HMVECs) with CMOSs resulted in the inhibition of cell proliferation, cell migration, and capillary-like tube formation, suggesting its anti-vascular angiogenesis. In summary, the results of this study indicate that CMOSs could be a valuable therapeutic candidate for CRC treatment.

Identification of Glucose-6 Phosphate Dehydrogenase Deficient Patients Using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy Using Partial Least Squares Discriminant Analysis in Aqueous Blood Samples.

Glucose-6 phosphate dehydrogenase (G6PD) deficiency is an X-linked blood disease that affects 400 million people globally and is especially prevalent in malaria-endemic regions. A significant portion of carriers are asymptomatic and undiagnosed posing complications in the eradication of malaria as it restricts the types of drugs used for malaria treatment. A simple and accurate diagnosis of the deficiency is vital in the eradication of malaria. In this study, we investigate the potential of attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) as a diagnostic technique for G6PD deficiency. Venous blood samples were collected in lithium heparin anticoagulant tubes from G6PD partial and fully deficient volunteers, n = 17, and normal volunteers, n = 59, in Khon Kaen, Thailand. Spectra of aqueous and dry samples were acquired of whole blood, plasma, and red blood cells, and modeled using partial least squares discriminant analysis (PLS-DA). PLS-DA modeling resulted in a sensitivity of 0.800 and specificity of 0.800 correctly classifying fully deficient participants as well as a majority of partially deficient females who are often misdiagnosed as normal by current screening methods. The viability of utilizing aqueous samples has always been hindered by the variability of hydration in the sample, but by employing multicurve curve resolution-alternating least squares to subtract water from each sample we are able to produce high-quality spectra with minimized water contributions. The approach shows proof of principle that ATR FT-IR combined with multivariate data analysis could become a frontline screening tool for G6PD deficiency by improving tailored drug treatments and ultimately saving lives.

Development of a Prognostic Score for Cholangiocarcinoma Patients Using a Combination of Biochemical Parameters.

BACKGROUND/AIM: Prognosis of cholangiocarcinoma (CCA), especially of intrahepatic CCA (iCCA), is poor primarily due to difficulties in earlier diagnosis. Since the majority of iCCA patients are elders, their prognosis cannot be correctly predicted by pathological features and/or resection status alone. Consideration for comorbidity and/or risks of subclinical diseases at diagnosis is critically necessary for the prediction of prognosis of iCCA patients. This study aimed to develop a simple but reliable scoring system for prognosis of iCCA patients at the time of diagnosis. PATIENTS AND METHODS: Serum samples from 152 iCCA patients were collected, and four commonly used biochemical markers, serum aspartate aminotransferase, alkaline phosphatase, cystatin C and creatinine-based estimated glomerular filtration rate were measured. Then, the values of individual patients were scored as 0, 1, and 2 (low, medium, and high) by tertiles or clinically relevant cut-off points and summed to construct a prognostic score with a range between 0 to 8. RESULTS: Patients with high scores of 2-4 and 5-8 exhibited significantly shorter survival times compared to those with low scores of 0-1 (Chi-square: 15.75, p<0.001). Cox regression analysis suggested that the score could be an independent predictor for the survival of iCCA patients. The odds of advanced tumor stage in high score iCCA patients (2-4 and 5-8) were 12.310 (95%CI=2.241-67.605) and 23.964 (95%CI=3.296-174.216), respectively. This scoring system allowed further stratification of death rates per 100 person-years of iCCA patients. CONCLUSION: The ability of such a simple scoring system to discriminate risk might be helpful for iCCA patients to determine therapeutic programs at the time of diagnosis.

High Level of Serum Coiled-coil Domain Containing 25 (CCDC25) as a Diagnostic Marker for Cholangiocarcinoma But Not for Other Cancers.

BACKGROUND/AIM: Recently, we reported that coiled-coil domain containing 25 (CCDC25) protein is elevated in the sera of patients with cholangiocarcinoma (CCA) and is suggested to be a diagnostic biomarker for CCA. This study aimed to examine whether serum CCDC25 level can be a unique biomarker for CCA. Bioinformatic analyses using Human Protein Atlas (HPA) database and Gene Expression Profiling Interactive Analysis 2 (GEPIA2) indicated that CCDC25 protein and mRNA are expressed not only in CCA but also in other cancers, such as colorectal cancer (CRC), breast cancer (BC), and hepatocellular carcinoma (HCC), all of which are the top 5 cancers highly prevalent in Thailand. MATERIALS AND METHODS: Using a quantitative dot blot assay, serum CCDC25 levels were measured for 30 healthy controls (HC), 34 CRC, 42 BC, 43 HCC, and 83 CCA. RESULTS: The serum CCDC25 levels of CCA patients (0.193±0.039 ng/µl) were significantly higher than those of CRC (0.019±0.006 ng/µl), BC (0.036±0.015 ng/µl), HCC (0.035±0.016 ng/µl), and higher than those of HC (0.012±0.003 ng/µl). The serum CCDC25 level can discriminate CCA from the HC, CRC, BC, and HCC with a sensitivity of 100, 99, 94, and 94%, respectively, and specificity of 100, 100, 98, and 95%, respectively. CONCLUSION: CCDC25 is a candidate diagnostic biomarker for CCA.

Green synthesized apigenin conjugated gold nanoparticles inhibit cholangiocarcinoma cell activity and endothelial cell angiogenesis in vitro.

Cholangiocarcinoma (CCA) is a rare malignancy of the biliary tract with extremely poor clinical outcomes due to a lack of effective therapies to improve disease management. The emerging green synthesis of gold nanoparticles (AuNPs) has extensively provided their use in biomedical applications. In this study, we developed AuNPs via reducing gold salts with apigenin (4',5,7-trihydroxyflavone). The synthesized apigenin-conjugated AuNPs (api-AuNPs) were physicochemically characterized by various techniques before evaluation their biological and functional inhibition in a CCA cell line, KKU-M055. The mean size of api-AuNPs was 90.34 ± 22.82 nm with zeta potential of -36 ± 0.55. The half-maximal inhibitory concentration (IC50, 0.8 mg/mL) of api-AuNPs on cell proliferation of KKU-M055 was 1.9-fold less than that of an immortalized human cholangiocyte cell line, MMNK1 (IC50, 1.5 mg/mL). Moreover, api-AuNPs induced cell apoptosis via the up-regulation of Bax, Bid, and Caspase 3, and down-regulation of Bcl2, leading to elevated caspase 3/7, 8, 9 activities and reactive oxygen species (ROS) production. The api-AuNPs significantly inhibited the migration of KKU-M055 cells and suppressed the proliferation, migration, and in vitro tube formation of vascular endothelial cells. Collectively, our findings indicate the dual abilities of api-AuNPs that potentially inhibit cancer cell growth and motility as well as endothelial cell-mediated angiogenesis, which may offer a novel therapeutic avenue to treat CCA patients effectively.

Prediction of CIAPIN1 (Cytokine-Induced Apoptosis Inhibitor 1) Signaling Pathway and Its Role in Cholangiocarcinoma Metastasis.

Cholangiocarcinoma (CCA), a malignancy of the biliary epithelium, can arise at any point in the biliary system. We previously reported that CIAPIN1 is detectable in the sera and that its overexpression was associated with poor prognosis and metastasis of CCA patients. In this study, we investigated further its expression in CCA tissues, biological functions, and related signaling pathways in CCA cells. First, we examined CIAPIN1 expression in CCA tissues of 39 CCA patients using immunohistochemistry (IHC). Then, CIAPIN1-related proteins expressed in CCA cells were identified using RNA interference (siRNA) and liquid chromatography-mass spectrometry (LC-MS/MS). To predict the functions and signaling pathways of CIAPIN1 in CCA cells, the identified proteins were analyzed using bioinformatics tools. Then, to validate the biological functions of CIAPIN1 in the CCA cell line, transwell migration/invasion assays were used. CIAPIN1 was overexpressed in CCA tissues compared with adjacent noncancerous tissues. Its overexpression was correlated with lymph node metastasis. Bioinformatic analyses predicted that CIAPIN1 is connected to the TGF-ß/SMADs signaling pathway via nitric oxide synthase 1 (NOS1) and is involved in the metastasis of CCA cells. In fact, cell migration and invasion activities of the KKU-100 CCA cell line were significantly suppressed by CIAPIN1 gene silencing. Our results unravel its novel function and potential signaling pathway in metastasis of CCA cells. CIAPIN1 can be a poor prognostic factor and can be a promising target molecule for CCA chemotherapy.

3D Silk Fibroin-Gelatin/Hyaluronic Acid/Heparan Sulfate Scaffold Enhances Expression of Stemness and EMT Markers in Cholangiocarcinoma.

BACKGROUND/AIM: Cholangiocarcinoma (CCA) is a stem cell-based cancer. The in vivo tumor microenvironment is not present in two-dimensional (2D) cultures, which is one of the limitations in cancer stem cell (CSC) research. Thus, we aimed to establish three-dimensional (3D) culture mimicking extracellular matrix (ECM) that could serve as a niche for CSC enrichment in CCA. MATERIALS AND METHODS: Silk fibroin-gelatin/hyaluronic acid/heparan sulfate (SF-GHHs) scaffolds were fabricated by lyophilization in various ratios and compared to silk fibroin (SF) scaffold. The physical and biological characteristics of the scaffolds were investigated. RESULTS: The SF-GHHs 1:2 scaffold with pore size of 350±102 µm harbored optimal porosity, good water uptake, and stable beta-sheet that supported the increase in KKU-213A cell proliferation and aggregation. The CSC and the epithelial-mesenchymal transition (EMT) markers were significantly upregulated in this scaffold compared to 2D. Moreover, drug sensitivity against cisplatin and gemcitabine in 3D culture was significantly higher than that in 2D culture. CONCLUSION: The SF-GHHs 1:2 scaffold could simulate ECM that may serve as a CSC niche of CCA, and reinforce stemness and EMT properties, suggesting its suitability for 3D CCA model, which supports CSC and new targeting drug research in CCA.

Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy Discriminates the Elderly with a Low and High Percentage of Pathogenic CD4+ T Cells.

In the aging process, the presence of interleukin (IL)-17-producing CD4+CD28-NKG2D+T cells (called pathogenic CD4+ T cells) is strongly associated with inflammation and the development of various diseases. Thus, their presence needs to be monitored. The emergence of attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy empowered with machine learning is a breakthrough in the field of medical diagnostics. This study aimed to discriminate between the elderly with a low percentage (LP; ≤3%) and a high percentage (HP; ≥6%) of pathogenic CD4+CD28-NKG2D+IL17+ T cells by utilizing ATR-FTIR coupled with machine learning algorithms. ATR spectra of serum, exosome, and HDL from both groups were explored in this study. Only exosome spectra in the 1700-1500 cm-1 region exhibited possible discrimination for the LP and HP groups based on principal component analysis (PCA). Furthermore, partial least square-discriminant analysis (PLS-DA) could differentiate both groups using the 1700-1500 cm-1 region of exosome ATR spectra with 64% accuracy, 69% sensitivity, and 61% specificity. To obtain better classification performance, several spectral models were then established using advanced machine learning algorithms, including J48 decision tree, support vector machine (SVM), random forest (RF), and neural network (NN). Herein, NN was considered to be the best model with an accuracy of 100%, sensitivity of 100%, and specificity of 100% using serum spectra in the region of 1800-900 cm-1. Exosome spectra in the 1700-1500 and combined 3000-2800 and 1800-900 cm-1 regions using the NN algorithm gave the same accuracy performance of 95% with a variation in sensitivity and specificity. HDL spectra with the NN algorithm also showed excellent test performance in the 1800-900 cm-1 region with 97% accuracy, 100% sensitivity, and 95% specificity. This study demonstrates that ATR-FTIR coupled with machine learning algorithms can be used to study immunosenescence. Furthermore, this approach can possibly be applied to monitor the presence of pathogenic CD4+ T cells in the elderly. Due to the limited number of samples used in this study, it is necessary to conduct a large-scale study to obtain more robust classification models and to assess the true clinical diagnostic performance.

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.

Serum Levels of Cytokine-Induced Apoptosis Inhibitor 1 (CIAPIN1) as a Potential Prognostic Biomarker of Cholangiocarcinoma.

The mortality rate of cholangiocarcinoma (CCA) is high since there is a lack of a non-invasive technique to accurately detect tumors at the early stage. CCA biomarkers are consistently needed for various purposes including screening, early diagnosis, prognosis and follow-up. Herein, using bioinformatic analysis of our mitochondrial proteome database of CCA tissues, we identified cytokine-induced apoptosis inhibitor 1 (CIAPIN1) as a potential prognostic biomarker for CCA. CIAPIN1 levels in the sera of 159 CCA patients and 93 healthy controls (HC) were measured using a dot blot assay. The median level ± quartile deviation of CIAPIN1 level in the sera of CCA patient group was 0.5144 ± 0.34 µg/µL, which was significantly higher than 0.2427 ± 0.09 µg/µL of the HC group (p < 0.0001). In CCA patients, higher serum CIAPIN1 level was significantly associated with lymph node metastasis (p = 0.024) and shorter overall survival time (p = 0.001, Kaplan-Meier test). Cox regression analysis showed that the serum CIAPIN1 level can be an independent prognostic indicator for the survival of CCA patients. Moreover, for the prediction of CCA prognosis, CIAPIN1 is superior to CEA, CA19-9 and ALP. In conclusion, CIAPIN1 can be a serum biomarker candidate for the poor prognosis of CCA.

Kallikrein-11, in Association with Coiled-Coil Domain Containing 25, as a Potential Prognostic Marker for Cholangiocarcinoma with Lymph Node Metastasis.

Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes. Currently, the treatment and prognosis for CCA are mostly poor. Recently, we have reported that coiled-coil domain containing 25 (CCDC25) protein level in the sera may be a diagnostic marker for CCA. Subsequently, we identified three binding proteins of CCDC25 and found that kallikrein-11 (KLK11) expression was highest among those binding proteins. In this study, we investigated CCDC25 and KLK11 expression in CCA and adjacent normal tissues (n = 18) using immunohistochemistry. The results demonstrated that the expressions of CCDC25 and KLK11 in CCA tissues were both significantly higher than the adjacent tissues (p < 0.001 and p = 0.001, respectively). Then, using GEPIA bioinformatics analysis, KLK11 mRNA was significantly overexpressed in CCA tumor tissues compared with normal tissues (p < 0.05). Moreover, CCDC25 expression was positively correlated with KLK11 expression in CCA with lymph node metastasis (p = 0.028, r = 0.593). An analysis for the interaction of KLK11 with CCDC25 and other proteins, using STRING version 11.0, revealed that CCDC25 and KLK11 correlated with metastasis-related proteins. In addition, Kaplan-Meier survival curve analysis revealed that a high expression of KLK11 was associated with the poor prognosis of CCA. In conclusion, KLK11 is, as a binding protein for CCDC25, possibly involved in the metastatic process of CCA. KLK11 may be used as a prognostic marker for CCA.

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.

Synchrotron FTIR microspectroscopy revealed apoptosis-induced biomolecular changes of cholangiocarcinoma cells treated with ursolic acid.

BACKGROUND: Ursolic acid (UA) is a natural triterpenoid which possesses anti-cancer activity. However, little is known regarding the activity and molecular mechanism of UA in cholangiocarcinoma (CCA). Thus, we investigated the effects of UA on growth inhibition and apoptosis induction through biomolecular changes in KKU-213 and KKU-055 CCA cell lines. METHODS: The anti-proliferative effect of UA against CCA cells was evaluated using SRB assay. Changes in biomolecules were assessed by SR-FTIR microspectroscopy combined with PCA and conventional methods (i.e., Annexin V-FITC/PI staining for lipid alteration and apoptosis induction; Western blot analysis and caspase-3/7 activity assay for apoptotic protein detection). RESULTS: UA suppressed the proliferation of CCA cells in a dose- and time-dependent manner. SR-FTIR data revealed a significant alteration in lipids attributable to changes in apoptotic cell membranes, confirmed by Annexin V-FITC/PI staining. SR-FTIR data showed that UA promoted changes in the protein secondary structure. Elevated expression of Bax and decreased expression of Bcl-2 and survivin/BIRC5 along with augmented caspase-3/7 activity supported alterations in apoptosis-related proteins. CONCLUSIONS: SR-FTIR microspectroscopy was successfully used as a label-free technique to monitor apoptosis-induced biomolecular changes in UA-treated CCA cells. UA exerted the cytotoxic and apoptotic activities in CCA cells through alterations in membrane lipids and apoptotic proteins. UA could be a potential anti-CCA candidate and a chemical starting point for the discovery of novel anti-cancer agents. SIGNIFICANCE: Our present study showed the first evidence that UA exhibited the anti-proliferative and pro-apoptotic activities toward CCA cells through changes in biomolecules, notably lipids and proteins.

Attenuated total reflection: Fourier transform infrared spectroscopy for detection of heterogeneous vancomycin-intermediate Staphylococcus aureus.

Staphylococcus aureus strains resistant to the last line antibiotic, vancomycin, have been of clinical concern. These include heterogeneous vancomycin-intermediate S. aureus (hVISA) and VISA. The hVISA phenotype cannot be detected by routine laboratory methods. Characterization of hVISA/VISA by new technologies is necessary to differentiate them rapidly from the vancomycin-susceptible isolates (VSSA). In this study, we developed a model for discrimination of hVISA from VSSA by using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy combined with multivariate data analysis, displaying a phenotypic signature of the bacteria. ATR-FTIR spectra were acquired from a total of 59 clinical methicillin-resistant S. aureus (MRSA) isolates comprising 28 hVISA and 31 VSSA strains. Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) were used to analyze 351 spectra of 39 isolates and develop a discrimination model for identifying hVISA and VSSA. The classification model, which was used for blind testing of 90 spectra from each of 10 hVISA, and 10 VSSA isolates, provided 100% sensitivity and specificity. The modeling revealed that the major discrimination between hVISA and VSSA phenotypes involved bands related to cell wall content (1087 and 1057 cm-1). This study showed that ATR-FTIR technique may be an alternative method for rapid detection of low-level vancomycin-resistant S. aureus.

Infrared spectroscopy coupled to cloud-based data management as a tool to diagnose malaria: a pilot study in a malaria-endemic country.

BACKGROUND: Widespread elimination of malaria requires an ultra-sensitive detection method that can detect low parasitaemia levels seen in asymptomatic carriers who act as reservoirs for further transmission of the disease, but is inexpensive and easy to deploy in the field in low income settings. It was hypothesized that a new method of malaria detection based on infrared spectroscopy, shown in the laboratory to have similar sensitivity to PCR based detection, could prove effective in detecting malaria in a field setting using cheap portable units with data management systems allowing them to be used by users inexpert in spectroscopy. This study was designed to determine whether the methodology developed in the laboratory could be translated to the field to diagnose the presence of Plasmodium in the blood of patients presenting at hospital with symptoms of malaria, as a precursor to trials testing the sensitivity of to detect asymptomatic carriers. METHODS: The field study tested 318 patients presenting with suspected malaria at four regional clinics in Thailand. Two portable infrared spectrometers were employed, operated from a laptop computer or a mobile telephone with in-built software that guided the user through the simple measurement steps. Diagnostic modelling and validation testing using linear and machine learning approaches was performed against the gold standard qPCR. Sample spectra from 318 patients were used for building calibration models (112 positive and 110 negative samples according to PCR testing) and independent validation testing (39 positive and 57 negatives samples by PCR). RESULTS: The machine learning classification (support vector machines; SVM) performed with 92% sensitivity (3 false negatives) and 97% specificity (2 false positives). The Area Under the Receiver Operation Curve (AUROC) for the SVM classification was 0.98. These results may be better than as stated as one of the spectroscopy false positives was infected by a Plasmodium species other than Plasmodium falciparum or Plasmodium vivax, not detected by the PCR primers employed. CONCLUSIONS: In conclusion, it was demonstrated that ATR-FTIR spectroscopy could be used as an efficient and reliable malaria diagnostic tool and has the potential to be developed for use at point of care under tropical field conditions with spectra able to be analysed via a Cloud-based system, and the diagnostic results returned to the user's mobile telephone or computer. The combination of accessibility to mass screening, high sensitivity and selectivity, low logistics requirements and portability, makes this new approach a potentially outstanding tool in the context of malaria elimination programmes. The next step in the experimental programme now underway is to reduce the sample requirements to fingerprick volumes.

The development of simultaneous measurement of viral load and physical status for human papillomavirus 16 and 18 co-infection using multiplex quantitative polymerase chain reaction.

Persistent infection with human papillomavirus (HPV) type 16 and 18 is known to be a major risk factor for cervical cancer. Increased prevalence of co-infection with these high-risk types has been observed in pre-cancerous and cancerous tissues. The determination of physical status and copy numbers of viruses is therefore useful in clinical settings. A simple multiplex quantitative polymerase chain reaction (qPCR) for HPV16/HPV18 co-infection in one tube reaction was established in the present study using TaqMan®-based PCR for E2 and E6 viral DNA. The detection range was up to 106 copies with 100% specificity and high precision (CV of cycle time <0.5%). The analytical accuracy and robustness were verified by competitive assay using an unequal mixture of HPV16/HPV18 DNA. No significant effect was demonstrated when compared with the simplex qPCR. The detection of physical status was evaluated in cervical samples, including 5 pre-cancerous and 15 cancerous samples. No significant difference was observed between simplex and multiplex qPCR (P=0.372). In conclusion, the developed multiplex qPCR method successfully demonstrated the viral status of the common HPV types in one tube. This assay will facilitate viral assessment and monitoring of cervical cancer associated with HPV16 and HPV18 co-infection.

Classification of Gemcitabine resistant Cholangiocarcinoma cell lines using synchrotron FTIR microspectroscopy.

Cholangiocarcinoma (CCA), a cancer of bile duct epithelium, is a major health problem in Thailand especially in the northeast. Overall treatment outcomes have not shown much improvement because the disease is usually detected at an advanced stage and often shows chemotherapeutic resistance. High-throughput Fourier Transform Infrared (FTIR) microspectroscopy can be used for cell classification and has the potential to diagnose cancer and possibly predict chemo-response. This study was aimed to differentiate gemcitabine-sensitive and gemcitabine-resistant induction in two CCA cell lines (KKU-M139 and KKU-M214) and xenograft tissues using synchrotron-FTIR microspectroscopy. Partial Least Squares Discriminant Analysis (PLS-DA) could discriminate between chemo-sensitive and chemo-resistant cells in the FTIR fingerprint spectral region (1800-1000 cm-1 ) with more than 90% sensitivity and specificity. The chemo-resistant and chemo-sensitive phenotypes were different in protein (amide I, amide II), lipids (carbonyl group and CH3 deformation) and phosphodiester from nucleic acids. Additionally, spectra from xenograft tissues showed similar results to the cell line study with marked differences between chemo-resistant and chemo-sensitive CCA tissues, and PLS-DA could discriminate the chemotherapeutic response with 98% sensitivity and specificity. This is the first study to demonstrate the use of FTIR microspectroscopy to assess chemo-response both in vitro and in vivo.

Gold Nanoparticles Enhance the Anticancer Activity of Gallic Acid against Cholangiocarcinoma Cell Lines.

Gold nanoparticles (GNPs) were conjugated with gallic acid (GA) at various concentrations between 30 and 150 µM and characterized using transmission electron microscopy (TEM) and UV-Vis spectroscopy (UV-VIS). The anticancer activities of the gallic acid-stabilized gold nanoparticles against well-differentiated (M213) and moderately differentiated (M214) adenocarcinomas were then determined using a neutral red assay. The GA mechanism of action was evaluated using Fourier transform infrared (FTIR) microspectroscopy. Distinctive features of the FTIR spectra between the control and GA-treated cells were confirmed by principal component analysis (PCA). The surface plasmon resonance spectra of the GNPs had a maximum absorption at 520 nm, whereas GNPs-GA shifted the maximum absorption values. In an in vitro study, the complexed GNPs-GA had an increased ability to inhibit the proliferation of cancer cells that was statistically significant (P<0.0001) in both M213 and M214 cells compared to GA alone, indicating that the anticancer activity of GA can be improved by conjugation with GNPs. Moreover, PCA revealed that exposure of the tested cells to GA resulted in significant changes in their cell membrane lipids and fatty acids, which may enhance the efficacy of this anticancer activity regarding apoptosis pathways.