Investigation of Limitations in the Detection of Antibody + Antigen Complexes Using the Silicon-on-Insulator Field-Effect Transistor Biosensor.

The SOI-FET biosensor (silicon-on-insulator field-effect transistor) for virus detection is a promising device in the fields of medicine, virology, biotechnology, and the environment. However, the applications of modern biosensors face numerous problems and require improvement. Some of these problems can be attributed to sensor design, while others can be attributed to technological limitations. The aim of this work is to conduct a theoretical investigation of the "antibody + antigen" complex (AB + AG) detection processes of a SOI-FET biosensor, which may also solve some of the aforementioned problems. Our investigation concentrates on the analysis of the probability of AB + AG complex detection and evaluation. Poisson probability density distribution was used to estimate the probability of the adsorption of the target molecules on the biosensor's surface and, consequently, to obtain correct detection results. Many implicit and unexpected causes of error detection have been identified for AB + AG complexes using SOI-FET biosensors. We showed that accuracy and time of detection depend on the number of SOI-FET biosensors on a crystal.

Nanoribbon Biosensor-Based Detection of microRNA Markers of Prostate Cancer.

Prostate cancer (PC) is one of the major causes of death among elderly men. PC is often diagnosed later in progression due to asymptomatic early stages. Early detection of PC is thus crucial for effective PC treatment. The aim of this study is the simultaneous highly sensitive detection of a palette of PC-associated microRNAs (miRNAs) in human plasma samples. With this aim, a nanoribbon biosensor system based on "silicon-on-insulator" structures (SOI-NR biosensor) has been employed. In order to provide biospecific detection of the target miRNAs, the surface of individual nanoribbons has been sensitized with DNA oligonucleotide probes (oDNA probes) complementary to the target miRNAs. The lowest concentration of nucleic acids, detectable with our biosensor, has been found to be 1.1 × 10-17 M. The successful detection of target miRNAs, isolated from real plasma samples of PC patients, has also been demonstrated. We believe that the development of highly sensitive nanotechnology-based biosensors for the detection of PC markers is a step towards personalized medicine.

Omics Untargeted Key Script: R-Based Software Toolbox for Untargeted Metabolomics with Bladder Cancer Biomarkers Discovery Case Study.

Large-scale untargeted LC-MS-based metabolomic profiling is a valuable source for systems biology and biomarker discovery. Data analysis and processing are major tasks due to the high complexity of generated signals and the presence of unwanted variations. In the present study, we introduce an R-based open-source collection of scripts called OUKS (Omics Untargeted Key Script), which provides comprehensive data processing. OUKS is developed by integrating various R packages and metabolomics software tools and can be easily set up and prepared to create a custom pipeline. Novel computational features are related to quality control samples-based signal processing and are implemented by gradient boosting, tree-based, and other nonlinear regression algorithms. Bladder cancer biomarkers discovery study which is based on untargeted LC-MS profiling of urine samples is performed to demonstrate exhaustive functionality of the developed software tool. Unique examination among dozens of metabolomics-specific data curation methods was carried out at each processing step. As a result, potential biomarkers were identified, statistically validated, and described by metabolism disorders. Our study demonstrates that OUKS helps to make untargeted LC-MS metabolomic profiling with the latest computational features readily accessible in a ready-to-use unified manner to a research community.

"Silicon-On-Insulator"-Based Biosensor for the Detection of MicroRNA Markers of Ovarian Cancer.

Ovarian cancer is a gynecological cancer characterized by a high mortality rate and tumor heterogeneity. Its early detection and primary prophylaxis are difficult to perform. Detecting biomarkers for ovarian cancer plays a pivotal role in therapy effectiveness and affects patients' survival. This study demonstrates the detection of microRNAs (miRNAs), which were reported to be associated with ovarian cancer tumorigenesis, with a nanowire biosensor based on silicon-on-insulator structures (SOI-NW biosensor). The advantages of the method proposed for miRNA detection using the SOI-NW biosensor are as follows: (1) no need for additional labeling or amplification reaction during sample preparation, and (2) real-time detection of target biomolecules. The detecting component of the biosensor is a chip with an array of 3 µm wide, 10 µm long silicon nanowires on its surface. The SOI-NW chip was fabricated using the "top-down" method, which is compatible with large-scale CMOS technology. Oligonucleotide probes (oDNA probes) carrying sequences complementary to the target miRNAs were covalently immobilized on the nanowire surface to ensure high-sensitivity biospecific sensing of the target biomolecules. The study involved two experimental series. Detection of model DNA oligonucleotides being synthetic analogs of the target miRNAs was carried out to assess the method's sensitivity. The lowest concentration of the target oligonucleotides detectable in buffer solution was 1.1 × 10-16 M. In the second experimental series, detection of miRNAs (miRNA-21, miRNA-141, and miRNA-200a) isolated from blood plasma samples collected from patients having a verified diagnosis of ovarian cancer was performed. The results of our present study represent a step towards the development of novel highly sensitive diagnostic systems for the early revelation of ovarian cancer in women.

Micro-Raman Characterization of Structural Features of High-k Stack Layer of SOI Nanowire Chip, Designed to Detect Circular RNA Associated with the Development of Glioma.

The application of micro-Raman spectroscopy was used for characterization of structural features of the high-k stack (h-k) layer of "silicon-on-insulator" (SOI) nanowire (NW) chip (h-k-SOI-NW chip), including Al2O3 and HfO2 in various combinations after heat treatment from 425 to 1000 °C. After that, the NW structures h-k-SOI-NW chip was created using gas plasma etching optical lithography. The stability of the signals from the monocrine phase of HfO2 was shown. Significant differences were found in the elastic stresses of the silicon layers for very thick (>200 nm) Al2O3 layers. In the UV spectra of SOI layers of a silicon substrate with HfO2, shoulders in the Raman spectrum were observed at 480-490 cm-1 of single-phonon scattering. The h-k-SOI-NW chip created in this way has been used for the detection of DNA-oligonucleotide sequences (oDNA), that became a synthetic analog of circular RNA-circ-SHKBP1 associated with the development of glioma at a concentration of 1.1 × 10-16 M. The possibility of using such h-k-SOI NW chips for the detection of circ-SHKBP1 in blood plasma of patients diagnosed with neoplasm of uncertain nature of the brain and central nervous system was shown.

Detection of Urothelial Bladder Cancer Based on Urine and Tissue Telomerase Activity Measured by Novel RT-TRAP-2PCR Method.

PURPOSE: To assess the diagnostic performance of urine telomerase activity (TA) in detecting bladder cancer (BCa) using the modified Telomeric Repeat Amplification Protocol (TRAP) and the Real Time Telomeric Repeat Amplification Protocol with double Polymerase Chain Reaction (RT-TRAP-2PCR). METHODS: In this case-control study, matching urine (in the pre- and post-surgical period) and tissue samples from 68 patients with BCa were assessed for TA. As a control, 45 urine samples were examined from non-BCa patients. TA levels were measured using TRAP and RT-TRAP-2 PCR methods. RESULTS: Preoperative urinary TA was elevated in 64 (94.1%) of the 68 BCa patients. Urine TA was undetectable in 44 control patients, while TA was detected in one patient with histologically verified cystitis. Sensitivity for BCa detection of 94.1% and specificity of 97.8% were observed for urinary TA, while tissue TA had 100% sensitivity and 97.8% specificity. Both urine and tissue TA levels were not significantly higher in patients with muscle-invasive disease compared to those with non-muscle invasive BCa (p > 0.05). Urine and tissue TA levels were not associated with higher tumor grade, stage, and number of tumors (p > 0.05). However, the association was found between higher urinary and tissue TA levels with tumor size ≥ 3 cm (p = 0.02 and p = 0.01, respectively). During the first postoperative year, 17 BCa patients experienced disease recurrence, and urinary TA was present in 14 (82.4%) of these patients. The sensitivity and specificity of urinary TA levels for BCa recurrence in patients with non-muscle invasive bladder cancer (NMIBC) during follow-up were 82% and 94.4%, respectively. CONCLUSIONS: This pilot study demonstrates a high diagnostic performance of urinary and tissue TA levels measured by a new RT-TRAP-2PCR method for detecting and monitoring BCa. Additionally, the association was found between higher urinary and tissue TA levels with tumor size ≥ 3 cm; however, higher TA levels failed for significant correlation with advanced tumor stage and grade. Our study could serve as a benchmark for the evaluation of novel biomarkers using the RT-TRAP-2PCR method.

Detection of Influenza Virus Using a SOI-Nanoribbon Chip, Based on an N-Type Field-Effect Transistor.

The detection of influenza A virions with a nanoribbon detector (NR detector) has been demonstrated. Chips for the detector have been fabricated based on silicon-on-insulator nanoribbon structures (SOI nanoribbon chip), using a complementary metal-oxide-semiconductor (CMOS)-compatible technology-by means of gas-phase etching and standard optical photolithography. The surface of the SOI nanoribbon chip contains a matrix of 10 nanoribbon (NR) sensor elements. SOI nanoribbon chips of n-type conductance have been used for this study. For biospecific detection of target particles, antibodies against influenza virus have been covalently immobilized onto NRs. Influenza A virus detection was performed by real-time registration of the source-drain current through the NRs. The detection of the target viral particles was carried out in buffer solutions at the target particles concentration within the range from 107 to 103 viral particles per milliliter (VP/mL). The lowest detectable concentration of the target viral particles was 6 × 10-16 M (corresponding to 104 VP/mL). The use of solutions containing ~109 to 1010 VP/mL resulted in saturation of the sensor surface with the target virions. In the saturation mode, detection was impossible.

hTERT, hTR and TERT promoter mutations as markers for urological cancers detection: A systematic review.

The clinical relevance of telomerase subunits (human reverse transcriptase - hTERT, and human telomerase RNA - hTR) and TERT promotor mutations as biomarkers in genitourinary cancers was reviewed through the systematic analysis of the current literature. We performed a systematic literature search using 2 databases (Medline and Scopus) over the past 20 years. Primary outcomes were sensitivity and specificity of hTR, hTERT and TERT promoter mutations. Secondary outcomes were the biomarkers predictive values for tumor characteristics. Regarding bladder cancer, hTERT in urine showed high sensitivity (mean values: 55%-96%), and specificity (69%-100%): it correlated with bladder cancer grade and/or stage. hTR sensitivity ranged from 77% to 92%. With adapted cut-off, it demonstrated 72% to 89% specificity. TERT promoter mutation rate was up to 80% both in tissue and urine, resulting in 62%-92% sensitivity for primary tumors and 42% for relapse. Specificity ranged from 73% to 96%, no correlations with stage were observed. In prostate cancer, hTERT in tissue, prostate secretion and serum showed high sensitivity (97.9%, 36%, and 79.2%-97.5%, respectively) and specificity values (70%, 66%, 60%-100%). hTR showed very high sensitivity (88% in serum and 100% in tissue) although specificity values were highly variable depending on the series and techniques (0%-96.5%). In RCC, hTERT sensitivity on tissue ranged from 90 to 97%, specificity from 25 to 58%. There was an association of hTERT expression with tumor stage and grade. hTERT showed high accuracy in genitourinary cancers, while the value of hTR was more controversial. hTERT and TERT promotor mutations may have predictive value for bladder cancer and RCC staging and grading, while no such relationship was observed in CaP. Although telomerase subunits showed clinically relevant values in genitourinary cancers, developing fast and cost-effective methods is required before contemplating routine use.

Highly Sensitive Detection of CA 125 Protein with the Use of an n-Type Nanowire Biosensor.

The detection of CA 125 protein in a solution using a silicon-on-insulator (SOI)-nanowire biosensor with n-type chip has been experimentally demonstrated. The surface of nanowires was modified by covalent immobilization of antibodies against CA 125 in order to provide the biospecificity of the target protein detection. We have demonstrated that the biosensor signal, which results from the biospecific interaction between CA 125 and the covalently immobilized antibodies, increases with the increase in the protein concentration. At that, the minimum concentration, at which the target protein was detectable with the SOI-nanowire biosensor, amounted to 1.5 × 10-16 M.

Telomeres and Telomerase in Heart Ontogenesis, Aging and Regeneration.

The main purpose of the review article is to assess the contributions of telomere length and telomerase activity to the cardiac function at different stages of development and clarify their role in cardiac disorders. It has been shown that the telomerase complex and telomeres are of great importance in many periods of ontogenesis due to the regulation of the proliferative capacity of heart cells. The review article also discusses the problems of heart regeneration and the identification of possible causes of dysfunction of telomeres and telomerase.

Detection of Marker miRNAs, Associated with Prostate Cancer, in Plasma Using SOI-NW Biosensor in Direct and Inversion Modes.

Information about the characteristics of measuring chips according to their storage conditions is of great importance for clinical diagnosis. In our present work, we have studied the capability of chips to detect nanowire biosensors when they are either freshly prepared or have been stored for either one or two years in a clean room. Potential to detect DNA oligonucleotides (oDNAs)-synthetic analogues of microRNAs (miRNAs) 198 and 429 that are associated with the development of prostate cancer (PCa)-in buffer solution was demonstrated using a nanowire biosensor based on silicon-on-insulator structures (SOI-NW biosensor). To provide biospecific detection, nanowire surfaces were sensitized with oligonucleotide probes (oDNA probes) complimentary to the known sequences of miRNA 183 and 484. In this study it is demonstrated that freshly prepared SOI-NW biosensor chips with n-type conductance and immobilized oDNA probes exhibit responses to the addition of complimentary oDNAs in buffer, leading to decreases in chips' conductance at a concentration of 3.3 × 10-16 M. The influence of storage time on the characteristics of SOI-NW biosensor chips is also studied herein. It is shown that a two-year storage of the chips leads to significant changes in their characteristics, resulting in "inverse" sensitivity toward negatively charged oDNA probes (i.e., through an increase in chips' conductance). It is concluded that the surface layer makes the main contribution to conductance of the biosensor chip. Our results indicate that the detection of target nucleic acid molecules can be carried out with high sensitivity using sensor chips after long-term storage, but that changes in their surface properties, which lead to inversed detection signals, must be taken into account. Examples of the applications of such chips for the detection of cancer-associated microRNAs in plasma samples of patients with diagnosed prostate cancer are given. The results obtained herein are useful for the development of highly sensitive nanowire-based diagnostic systems for the revelation of (prostate) cancer-associated microRNAs in human plasma.

Micro-Raman Spectroscopy for Monitoring of Deposition Quality of High-k Stack Protective Layer onto Nanowire FET Chips for Highly Sensitive miRNA Detection.

Application of micro-Raman spectroscopy for the monitoring of quality of high-k (h-k) dielectric protective layer deposition onto the surface of a nanowire (NW) chip has been demonstrated. A NW chip based on silicon-on-insulator (SOI) structures, protected with a layer of high-k dielectric ((h-k)-SOI-NW chip), has been employed for highly sensitive detection of microRNA (miRNA) associated with oncological diseases. The protective dielectric included a 2-nm-thick Al2O3 surface layer and a 8-nm-thick HfO2 layer, deposited onto a silicon SOI-NW chip. Such a chip had increased time stability upon operation in solution, as compared with an unprotected SOI-NW chip with native oxide. The (h-k)-SOI-NW biosensor has been employed for the detection of DNA oligonucleotide (oDNA), which is a synthetic analogue of miRNA-21 associated with oncological diseases. To provide biospecificity of the detection, the surface of (h-k)-SOI-NW chip was modified with oligonucleotide probe molecules (oDVA probes) complementary to the sequence of the target biomolecule. Concentration sensitivity of the (h-k)-SOI-NW biosensor at the level of DL~10-16 M has been demonstrated.

The Effects of PPAR Stimulation on Cardiac Metabolic Pathways in Barth Syndrome Mice.

Aim: Tafazzin knockdown (TazKD) in mice is widely used to create an experimental model of Barth syndrome (BTHS) that exhibits dilated cardiomyopathy and impaired exercise capacity. Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that play essential roles as transcription factors in the regulation of carbohydrate, lipid, and protein metabolism. We hypothesized that the activation of PPAR signaling with PPAR agonist bezafibrate (BF) may ameliorate impaired cardiac and skeletal muscle function in TazKD mice. This study examined the effects of BF on cardiac function, exercise capacity, and metabolic status in the heart of TazKD mice. Additionally, we elucidated the impact of PPAR activation on molecular pathways in TazKD hearts. Methods: BF (0.05% w/w) was given to TazKD mice with rodent chow. Cardiac function in wild type-, TazKD-, and BF-treated TazKD mice was evaluated by echocardiography. Exercise capacity was evaluated by exercising mice on the treadmill until exhaustion. The impact of BF on metabolic pathways was evaluated by analyzing the total transcriptome of the heart by RNA sequencing. Results: The uptake of BF during a 4-month period at a clinically relevant dose effectively protected the cardiac left ventricular systolic function in TazKD mice. BF alone did not improve the exercise capacity however, in combination with everyday voluntary running on the running wheel BF significantly ameliorated the impaired exercise capacity in TazKD mice. Analysis of cardiac transcriptome revealed that BF upregulated PPAR downstream target genes involved in a wide spectrum of metabolic (energy and protein) pathways as well as chromatin modification and RNA processing. In addition, the Ostn gene, which encodes the metabolic hormone musclin, is highly induced in TazKD myocardium and human failing hearts, likely as a compensatory response to diminished bioenergetic homeostasis in cardiomyocytes. Conclusion: The PPAR agonist BF at a clinically relevant dose has the therapeutic potential to attenuate cardiac dysfunction, and possibly exercise intolerance in BTHS. The role of musclin in the failing heart should be further investigated.

Implication of Gastric Cancer Molecular Genetic Markers in Surgical Practice.

INTRODUCTION: We have investigated aberrant methylation of genes CDH1, RASSF1A, MLH1, N33, DAPK, expression of genes hTERT, MMP7, MMP9, BIRC5 (survivin), PTGS2, and activity of telomerase of 106 gastric tumor samples obtained intra-operatively and 53 gastric tumor samples from the same group of patients obtained endoscopically before surgery. Biopsy specimens obtained from 50 patients with chronic calculous cholecystitis were used as a control group. Together with tissue samples obtained from different sites remote to tumors, a total of 727 samples have been studied. The selected parameters comprise a system of molecular markers that can be used in both diagnostics of gastric cancer and in dynamic monitoring of patients after surgery. Special attention was paid to the use of molecular markers for the diagnostics of malignant process in the material obtained endoscopically since the efficacy of morphological diagnostics in biopsies is compromised by intratumoral heterogeneity, which may prevent reliable identification of tumor cells in the sampling. Our data indicated that certain molecular genetic events provided more sensitive yet specific markers of the tumor. CONCLUSION: We demonstrated that molecular profiles detected in preoperative biopsies were confirmed by the material obtained intra-operatively. The use of endoscopic material facilitates gastric tumors pre-operative diagnostics, improving early detection of gastric cancer and potential effective treatment strategies.

The PPAR pan-agonist bezafibrate ameliorates cardiomyopathy in a mouse model of Barth syndrome.

BACKGROUND: The PGC-1α/PPAR axis has been proposed as a potential therapeutic target for several metabolic disorders. The aim was to evaluate the efficacy of the pan-PPAR agonist, bezafibrate, in tafazzin knockdown mice (TazKD), a mouse model of Barth syndrome that exhibits age-dependent dilated cardiomyopathy with left ventricular (LV) dysfunction. RESULTS: The effect of bezafibrate on cardiac function was evaluated by echocardiography in TazKD mice with or without beta-adrenergic stress. Adrenergic stress by chronic isoproterenol infusion exacerbates the cardiac phenotype in TazKD mice, significantly depressing LV systolic function by 4.5 months of age. Bezafibrate intake over 2 months substantially ameliorates the development of LV systolic dysfunction in isoproterenol-stressed TazKD mice. Without beta-adrenergic stress, TazKD mice develop dilated cardiomyopathy by 7 months of age. Prolonged treatment with suprapharmacological dose of bezafibrate (0.5% in rodent diet) over a 4-month period effectively prevented LV dilation in mice isoproterenol treatment. Bezafibrate increased mitochondrial biogenesis, however also promoted oxidative stress in cardiomyocytes. Surprisingly, improvement of systolic function in bezafibrate-treated mice was accompanied with simultaneous reduction of cardiolipin content and increase of monolysocardiolipin levels in cardiac muscle. CONCLUSIONS: Thus, we demonstrate that bezafibrate has a potent therapeutic effect on preventing cardiac dysfunction in a mouse model of Barth syndrome with obvious implications for treating the human disease. Additional studies are needed to assess the potential benefits of PPAR agonists in humans with Barth syndrome.

Identification of Novel Epigenetic Markers of Prostate Cancer by NotI-Microarray Analysis.

A significant need for reliable and accurate cancer diagnostics and prognosis compels the search for novel biomarkers that would be able to discriminate between indolent and aggressive tumors at the early stages of disease. The aim of this work was identification of potential diagnostic biomarkers for characterization of different types of prostate tumors. NotI-microarrays with 180 clones associated with chromosome 3 genes/loci were applied to determine genetic and epigenetic alterations in 33 prostate tumors. For 88 clones, aberrations were detected in more than 10% of tumors. The major types of alterations were DNA methylation and/or deletions. Frequent methylation of the discovered loci was confirmed by bisulfite sequencing on selective sampling of genes: FGF12, GATA2, and LMCD1. Three genes (BHLHE40, BCL6, and ITGA9) were tested for expression level alterations using qPCR, and downregulation associated with hypermethylation was shown in the majority of tumors. Based on these data, we proposed the set of potential biomarkers for detection of prostate cancer and discrimination between prostate tumors with different malignancy and aggressiveness: BHLHE40, FOXP1, LOC285205, ITGA9, CTDSPL, FGF12, LOC440944/SETD5, VHL, CLCN2, OSBPL10/ZNF860, LMCD1, FAM19A4, CAND2, MAP4, KY, and LRRC58. Moreover, we probabilistically estimated putative functional relations between the genes within each set using the network enrichment analysis.