Development Of Isotags For Nmr Based Metabolite Profiling And Applications

NMR spectroscopy is a powerful analytical tool for both qualitative and quantitative metabolite profiling analysis. However, accurate quantitative analysis of biological systems especially using one dimensional NMR has been challenging due to signal overlap. In contrast, the enhanced resolution and sensitivity offered by chemoselective isotope tags have enabled new and enhanced methods for detecting hundreds of quantifiable metabolites in biofluids using NMR spectroscopy or mass spectrometry. In this thesis we show improved sensitivity and resolution of NMR experiments imparted by 15N and 13C isotope tagging which enables the accurate analysis of plasma metabolites. To date, isotope tagging has been used in conjunction with a single analytical platform. The inability to detect the same metabolites using the complementary analytical techniques of NMR and mass spectrometry has hindered the correlation of data derived from the two powerful platforms for applications such as biomarker discovery or the identification of unknown metabolites. To address this problem, we describe a smart isotope tag, 15N-cholamine, which possesses two important properties: an NMR sensitive isotope, and a permanent charge for MS sensitivity. Finally, we present a study on metabolite profiling using intact breast cancer tissue samples in which we exploit the combined strength of NMR and multivariate statistical methods for metabolite profiling

Antibiotic Resistance of Helicobacter pylori Strains in the United Arab Emirates and its Relation to the Gene Associated to Gastric Cancer “Cag A Gene”

Helicobacter pylori is a flagellated corkscrew, slow growing neutrophilic gram negative ureolytic organism. It has an extraordinary ability to establish infection in human stomach that can last for years or decades. Its eradication remains an important public health challenge especially in light of broadening indications and increasing antimicrobial resistance. AIM: i) Identification of H. pylori in UAE patients ii) Determination of the prevalence of antibiotic resistance genes (mutation in 23S rRNA gene in clarithromycin, and deletion in RdxA gene in metronidazole) among H. pylori strains isolated from U.A.E patients by using molecular methods, iii) Ascertain whether Cag A- positive H. pylori strains correlates with the antibiotic resistance strains or not and iv) Screening for new H. pylori strains in UAE through the phylogenetic analysis of the 23S rRNA middle region of the gene. METHODS: The identification of H. pylori in UAE patients was carried on by primary screening for H. pylori using CLO test while the confirmation test were done using PCR technique. The prevalence of antibiotic resistance genes (mutation in 23S rRNA gene in clarithromycin, and deletion in RdxA gene in metronidazole) among H. pylori strains isolated from U.A.E patients and Cag A gene relation between this gene and antibiotic resistant genes were studied by PCR and sequencing technique. Phylogenetic analysis of the 23S rRNA gene was analyzed by using the software ClustalX, version 2. Reference sequences used in the alignment was obtained from NCBI data base for all 23s rRNA from different H. pylori strains. RESULTS: 26 out of 90 biopsy samples were positive for H. pylori using PCR whereas only 22 were positive when tested by CLO test. Resistance to clarithromycin and metronidazole was detected in 9 and 3 of strains, respectively. Of the clarithromycin resistant strains, 2 strains had the A2142G mutation in the 23S rRNA gene, 5 strains in A2143G, 1 strain in A2143C and 1 strain of highly changed in sequence. Of the metronidazole resistant strains, deletion in rdxA gene was detected in 3 strains which were negative for CLO test. DNA sequence phylogenetic analysis of the 23S rRNA middle region of the gene indicates that the strains from UAE harbor a unique 23S rRNA sequences that is common among isolates from the UAE patients and different than other strains published in the NCBI database. CONCLUSION: This study is the first time done in the UAE where a significant proportion of gastric mucosal biopsies obtained in the UAE are positive for Genes associated with Clarithromycin and Metronidazole resistance (mainly in Clarithromycin). A2143G remains the most prevalent point mutation involved, thus suggesting that new therapeutic strategies are neede

Development of Electroanalytical Methods for the Determination of Sialic Acid as a Biomarker for Cancer

Sialic acid (SA) is a general term for a family composed of 43 derivatives of neuraminic acid. Whereas N-acetylneuraminic acid (NANA) is the most commonly occurring sialic acid in human. There has been a great interest in the determination of SA in humans because variations in SA level was linked to different medical conditions and diseases. In particular, serum SA are elevated in several types of cancers. SA also exists in Erythropoietin (EPO), a hormone, which induces the production of red blood cells and hence used in the treatment of anemia. Although of the great physiological significance of SA and the attractive merits offered by the electrochemical techniques, there was a notable absence of literature describing electrochemical methods for the determination of SA. This surprising observation triggered this project to develop and to evaluate the first flow injection analysis (FIA) system as well as the first biosensor - based on amperometric transduction - for simple, fast, direct, and reliable determination of SA for clinical applications. The principle of the present work is based on a sequence of two enzyme,s i.e., Nacetylneuraminic acid aldolase (NANA-aldolase) and pyruvate oxidase (PO) which catalyze a two-step conversion of SA into H2O2, which could be detected by anodic amperometry using platinum electrode polarized 0.6 V vs Ag/AgCI. The first phase of the current project was to investigate the effect of different experimental variables on the generation of hydrogen peroxide by the sequence action of the two enzymes. This initial study is carried out using the two enzymes in the soluble form (i.e., homogenous enzyme catalysis). The obtained optimum experimental conditions for hydrogen peroxide generation and detection are 0.1 M phosphate buffer pH 6.3 at 37°C, using NANA-aldolase/PO activity ratio of 1.5 and a thiamine pyrophosphate (TPP) cofactor concentration of 0.5-2 mM. The second phase aimed to construct an FIA system based on an immobilized enzyme reactor (IER) and an amperometric detector for the generated hydrogen peroxide. The IER is prepared by co-immobilization of the two enzymes on controlled pore glass beads activated with glutaraldehyde and packed in a glass tube (3-5 cm in length). A tubular platinum detector of large surface area is suggested in this work and proved efficient to enhance the sensitivity of SA determination by the proposed FIA system. The entire FIA system is evaluated under the optimum conditions obtained from the initial investigation. The obtained linear range, analysis time, and sensitivity could be easily tuned to meet the required performance characteristics by controlling the carrier buffer flow rate, and the injected sample volume. The determination of SA in real samples using the proposed FIA system is presented as well. The third phase is devoted to the most challenging task in this project, i.e., to construct a prototype SA biosensor which necessitated co-immobilization of the two enzymes as well as their integration in a close proximity of platinum electrode surface. Although, three methods are tested for enzyme immobilization, the method based on glutaraldehyde crosslinking with BSA proved the most efficient. A novel microporous polyester membrane is used as a substrate for the enzyme layer, which provided high adhesion and reproducible fabrication of the enzyme layer. The optimum pH of the crosslinked enzyme system is ~ 1 pH higher (~ pH 7.3) than that obtained with homogenous catalysis. Careful optimization of enzyme layer composition and thickness allowed stable and fast response with detection limit of less than 10 µM SA .. Protection of the platinum surface with an inner electropolymeric layer enhanced the selectivity of the SA biosensor in the presence of interfering oxidizable species such as ascorbic and uric acids and acetaminophen. The favorable performance characteristics of the developed SA biosensor allowed its successful application in the determination of SA in simulated serum sample and real biological samples. The obtained performance characteristics of the newly developed electrochemical methods suggest their wide use in the numerous clinical applications and in particular as a non-specific tumor marker and to monitor tumor therapy

The Relationship between Retinal Vascular Reactivity and Arteriolar Diameter

ABSTRACT Purpose: The primary aim of the study (i.e. Chapter 3) was to compare the magnitude of retinal vascular reactivity in arterioles of varying diameter in healthy, young subjects. The secondary aims were to determine: a) if there are any order effects in terms of provoking vasoconstriction or vasodilation first; and b) the repeatability of the vascular reactivity measurements. An additional aim (i.e. Chapter 4) was to determine the effect of healthy aging on the relationship between retinal vascular reactivity and vessel diameter. Method: The sample comprised 10 healthy, young subjects (mean age 26.5 years, SD 4.04) and 7 healthy, older subjects (mean age 55.43 years, SD 5.41). Each subject from the young age group attended for three sessions. The first session was used to determine eligibility and select hemodynamic measurement sites. At sessions 2 and 3, O2 and CO2 were sequentially administered to the subjects using a face mask and sequential re-breathing circuit (to maintain standardized hyperoxia and hypercapnia). The order of vasoconstriction and vasodilation was varied across sessions 2 and 3. The design of the protocol was simplified for the subjects from the older age group. Each subject from the older group attended for one visit. O2 and CO2 were administered to the subjects using a face mask and sequential re-breathing circuit. The order of gas provocation was varied among the subjects (i.e. hyperoxia or hypercapnia first). For both groups, measurements of vessel diameter, centerline blood velocity and derived blood flow were acquired at each condition (i.e. baseline, during stabilized vasoconstriction, vasodilation, and recovery) at two discrete measurement sites along the supero-temporal arteriole. Results: The results of the repeated measures ANOVA showed a significant difference between the narrow and wide measurement sites for the younger group for flow (p≤ 0.0003) and a significant influence of inspired gas provocation on flow for both protocols (p<0.0001). In addition, the interaction of measurement site and inspired gas provocation was significant (p<0.0001). The magnitude of retinal vascular reactivity showed a significantly greater blood flow response for the wide measurement site (p<0.0001). O2 provocation resulted in vasoconstriction that was still present up to 10 minutes after cessation of the stimulus (order effect of O2; p≤0.046). No such order effect was apparent for CO2 provocation (order effect of CO2; p=0.352). The group mean blood flow Coefficient of Repeatability (COR) for the narrow measurement site was 0.74 µl/min (relative to group mean flow of 4.85 µl/min ± SD 1.31) and for the wide measurement site was 1.49 µl/min (relative to group mean flow of 11.29 µl/min ± SD 3.55). There was no difference between the young and the older age groups in retinal vascular reactivity for both the narrow (two-tailed Student t-test, p=0.8692) and wide (two-tailed Student t-test, p=0.2795) measurement sites. Conclusion: This study demonstrated that the magnitude of retinal vascular reactivity was greater for arteriolar measurement sites with wider baseline vessel diameters. In addition, it demonstrated that hyperoxic provocation resulted in a persistent vasoconstriction up to 10 minutes after cessation of the stimulus. The study demonstrated that the repeatability of retinal blood flow measurements in absolute terms is lower for smaller diameter vessels. Finally, this study also suggests that age does not affect the relationship between retinal vascular reactivity and vessel diameter

Retinal Blood Oxygen Saturation and Angiogenic and Inflammatory Biomarkers in Type 2 Diabetes

Introduction: Diabetic retinopathy (DR) is a major source of visual loss in the world, including North America. A number of hyperglycemia-related pathways have been associated with DR onset and progression. Disturbances in the retinal vasculature appear to play a vital role in DR, resulting in biochemical and functional vascular changes. Therefore, this study investigated retinal blood oxygen saturation and angiogenic and inflammatory biomarkers in DR. Methods: Chapter 3 and 5: FD-OCT Doppler blood flow was non-invasively measured using a prototype system based on the RTVue (Optovue Inc., USA). A minimum of six separate FD-OCT Doppler measurements was acquired. Chapter 4, 5, 7: Non-invasive hyperspectral retinal (HR) imaging was acquired in participants with mild-to-moderate NPDR and age-matched controls. For each subject, six repeated HRC images were acquired at wavelengths of 586 and 605nm. Results: Chapter3: The individual COV medians for retinal blood flow were 7.5% and 9.2% for young and elderly subjects, respectively. The group mean CORs for retinal blood flow for young participants were 6.4µl/min and for elderly subjects were 10.5µl/min. Chapter 4: Retinal blood oxygen saturation in the arterioles of healthy controls was 92.97±1.6%, and in the venules was 55.90±4.8%. Retinal blood oxygen saturation for diabetic subjects with NPDR was significantly higher at 94.65±2.2% (p=0.015) in the arterioles and 64.13±4.3% (p<0.001) in the venules. Chapter 5: Total retinal blood flow was significantly lower in NPDR when compared to controls (42.66 vs 32.97; p=0.004). There was no relationship between total retinal blood flow and venular oxygen saturation (r=0.2). Chapter 6: Angiopoietin 2, IL-8, HGF was significantly higher in NPDR patients than in control patients (p=0.005, p=0.034, p=0.018, respectively) and EGF was significantly lower in NPDR patients when compared to controls (p=0.025). Chapter 7: The study demonstrated a correlation between retinal blood oxygen saturation and Ang 2, HGF and EGF but did not find any correlation for IL-8, TGF-β even though these biomarkers were significantly higher in the diabetic group. Conclusions: Chapter 3: Doppler OCT gave consistent and repeatable blood flow measurements within retinal venules in normal subjects. Chapter 4: A higher blood oxygen saturation could be the result of less oxygen consumtion due to cell death. Chapter 5: There is no correlation between retinal blood flow and retinal blood oxygen saturation. Chapter 6: Further investigation of Ang 2, HGF, IL-8, EGF, TGF-β could be used to better understand the pathophysiology of DR. Chapter 7: The result of this study revealed a relationship between the biomarkers that might result in cell death and higher retinal blood oxygen saturation.1 yea

Variability and repeatability of quantitative, fourier-domain optical coherence tomography doppler blood flow in young and elderly healthy subjects

Purpose. The purpose of this study was to determine the within-session variability and between-session repeatability of spectral Fourier-domain optical coherence tomography (Doppler FD-OCT) Doppler retinal blood flow measurements in young and elderly subjects. Methods. Doppler FD-OCT blood flow was measured using the RTVue system. One eye of each of 20 healthy young (24.7 ± 2.7 years) and 16 healthy elderly (64.6 ±5.1 years) subjects was randomly selected, and the pupil was dilated. The double circular scanning pattern of the RTVue was employed. Six Doppler FD-OCT measurements (i.e., each separate measurement comprising an upper and a lower nasal pupil scan) were acquired at each session. Measurements were repeated approximately 2 weeks later. Total retinal blood flow was calculated by summing flow from all detectable venules surrounding the optic nerve head. The coefficient of variation (COV) and coefficient of repeatability (COR) were calculated for each individual. Results. The individual COVs for retinal blood flow for young subjects ranged from 0.4% to 20.4% (median 7.5%) and for the elderly subjects ranged from 0.6% to 34.6% (median 9.2%). The group mean CORs for retinal blood flow for young participants were 6.4 μL/min (median 5.91 μL/min, relative to a mean effect 39.8 μL/min) and for elderly subjects were 10.5 μL/min (median 9.2 μL/min, relative to a mean effect 46.4 μL/min). Conclusions. Doppler FD-OCT gave consistent and repeatable blood flow measurements within retinal venules in normal subjects. Considering the individual variation in blood flow measurements, confidence limits for retinal hemodynamics need to be determined on an individual basis

Cost-Effectiveness Analysis of Stockholm 3 Testing Compared to PSA as the Primary Blood Test in the Prostate Cancer Diagnostic Pathway:A Decision Tree Approach

OBJECTIVE: This study evaluated the cost effectiveness of using Stockholm 3 (STHLM3) testing compared to the prostate-specific antigen (PSA) test in the diagnostic pathway for prostate cancer. METHODS: We created a decision tree model for PSA (current standard) and STHLM3 (new alternative). Cost effectiveness was evaluated in a hypothetical cohort of male individuals aged 50–69 years. The study applied a Danish hospital perspective with a time frame restricted to the prostate cancer diagnostic pathway, beginning with the initial PSA/STHLM3 test, and ending with biopsy and histopathological diagnosis. Estimated values from the decision-analytical model were used to calculate the incremental cost-effectiveness ratio. Deterministic and probabilistic sensitivity analyses were conducted to test the robustness of the base-case analysis. RESULTS: The model-based analysis revealed that STHLM3 testing was more effective than the PSA, but also more costly, with an incremental cost-effectiveness ratio of €511.7 (95% credible interval, 359.9–674.3) for each additional correctly classified individual. In the deterministic sensitivity analysis, variations in the cost of STHLM3 had the greatest influence on the incremental cost-effectiveness ratio. In the probabilistic sensitivity analysis, all iterations were positioned in the north-east quadrant of the incremental cost-effectiveness scatterplot. At a willingness to pay of €700 for an additional correctly classified individual, STHLM3 had a 100% probability of being cost effective. CONCLUSIONS: Compared to the PSA test as the initial testing modality in the prostate cancer diagnostic workup, STHLM3 testing showed improved incremental effectiveness, however, at additional costs. The results were sensitive to the cost of the STHLM3 test; therefore, a lower cost of the STHLM3 test would improve its cost effectiveness compared with PSA tests. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40258-022-00741-0

LXRs regulate features of age-related macular degeneration and may be a potential therapeutic target

Effective treatments and animal models for the most prevalent neurodegenerative form of blindness in elderly people, called age-related macular degeneration (AMD), are lacking. Genome-wide association studies have identified lipid metabolism and inflammation as AMD-associated pathogenic pathways. Given liver X receptors (LXRs), encoded by the nuclear receptor subfamily 1 group H members 2 and 3 (NR1H3 and NR1H2), are master regulators of these pathways, herein we investigated the role of LXR in human and mouse eyes as a function of age and disease and tested the therapeutic potential of targeting LXR. We identified immunopositive LXR fragments in human extracellular early dry AMD lesions and a decrease in LXR expression within the retinal pigment epithelium (RPE) as a function of age. Aged mice lacking LXR presented with isoform-dependent ocular pathologies. Specifically, loss of the Nr1h3 isoform resulted in pathobiologies aligned with AMD, supported by compromised visual function, accumulation of native and oxidized lipids in the outer retina, and upregulation of ocular inflammatory cytokines, while absence of Nr1h2 was associated with ocular lipoidal degeneration. LXR activation not only ameliorated lipid accumulation and oxidant-induced injury in RPE cells but also decreased ocular inflammatory markers and lipid deposition in a mouse model, thereby providing translational support for pursuing LXR-active pharmaceuticals as potential therapies for dry AMD