Project description:Mammals display wide range of variation in their lifespan. Investigating the molecular networks that distinguish long- from short-lived species has proven useful to identify determinants of longevity. Here, we compared the liver of long-lived naked mole-rats (NMRs) and the phylogenetically closely related, shorter-lived, guinea pigs using an integrated omic approach. We found that NMRs livers display a unique expression pattern of mitochondrial proteins that result in distinct metabolic features of their mitochondria. For instance, we observed a generally reduced respiration rate associated with lower protein levels of respiratory chain components, particularly complex I, and increased capacity to utilize fatty acids. Interestingly, we show that the same molecular networks are affected during aging in both NMR and humans, supporting a direct link to the extraordinary longevity of both species. Finally, we identified a novel longevity pathway and validated it experimentally in the nematode C. elegans.
Project description:IntroductionThe aims of this study were to evaluate urine flow cytometry (UFC) as a tool to screen urine samples of urological patients for bacteriuria and to compare UFC and dipstick analysis with urine culture in a patient cohort at a urological department of a university hospital.Methods and materialWe screened 662 urine samples from urological patients (75.2% male; 80.7% inpatients; mean age 58 years). UFC results were compared to microbiological urine culture.ResultsThe accuracy in using the UFC-based parameters for detecting cultural bacteriuria was 91.99% and 88.97% for ≥105 colony-forming units (CFU)/mL and ≥104 CFU/mL, respectively. UFC and leukocyte dipstick analysis measured leukocyturia similarly (Pearson correlation coefficient 0.87, p value <0.01%), but dipstick analysis scored less accurately on bacteriuria (accuracy 59.37% and 62.69%). UFC remained effective in subgroup analysis of patients of both sexes and with different urological conditions with its overall use only slightly impaired when assessing gross hematuria (NPV 84.62% for ≥104 CFU/mL). UFC also reliably removed those urine samples below cutoffs with negative predictive values of 99.28% for ≥105 CFU/mL and 95.86% for ≥104 CFU/mL.ConclusionCounting bacteria with UFC is an accurate and rapid method to determine significant bacteriuria in urological patients and is superior to dipstick analysis or indirect surrogate parameters such as leukocyturia. When UFC is available, we recommend it to be used for the diagnosis of bacteriuria over findings obtained by dipstick analysis.
Project description:Urine is an equally attractive biofluid for metabolomics analysis, as it is a challenging matrix analytically. Accurate urine metabolite concentration estimates by Nuclear Magnetic Resonance (NMR) are hampered by pH and ionic strength differences between samples, resulting in large peak shift variability. Here we show that calculating the spectra of original samples from mixtures of samples using linear algebra reduces the shift problems and makes various error estimates possible. Since the use of two-dimensional (2D) NMR to confirm metabolite annotations is effectively impossible to employ on every sample of large sample sets, stabilization of metabolite peak positions increases the confidence in identifying metabolites, avoiding the pitfall of oranges-to-apples comparisons.
Project description:IntroductionSpectroscopic analysis of urine samples from laboratory animals can be used to predict the efficacy and side effects of drugs. This employs methods combining (1)H NMR spectroscopy with quantification of biomarkers or with multivariate data analysis. The most critical steps in data evaluation are analytical reproducibility of NMR data (collection, storage, and processing) and the health status of the animals, which may influence urine pH and osmolarity.MethodsWe treated rats with a solvent, a diuretic, or a nephrotoxicant and collected urine samples. Samples were titrated to pH 3 to 9, or salt concentrations increased up to 20-fold. The effects of storage conditions and freeze-thaw cycles were monitored. Selected metabolites and multivariate data analysis were evaluated after (1)H NMR spectroscopy.ResultsWe showed that variation of pH from 3 to 9 and increases in osmolarity up to 6-fold had no effect on the quantification of the metabolites or on multivariate data analysis. Storage led to changes after 14 days at 4°C or after 12 months at -20°C, independent of sample composition. Multiple freeze-thaw cycles did not affect data analysis.ConclusionReproducibility of NMR measurements is not dependent on sample composition under physiological or pathological conditions.
Project description:Platinum-based cytostatic drugs are one of the most widely used cancer treatments. They are excreted via the urinary tract and can reach the environment through wastewater, posing a risk to human health due to their side effects. Four identification and quantification techniques, including liquid chromatography (LC) separation coupled to (i) a diode array ultraviolet (UV(DAD)) (ii), mass spectrometer in single ion monitoring mode (LC-MS) and (iii) multiple reaction monitoring mode (LC-MS/MS) and (iv) derivatization with diethyldithiocarbamate prior to LC-MS/MS analysis, have been optimized and compared for the multiresidue determination of main platinized cytostatic drugs (cisplatin, carboplatin, and oxaliplatin) in urine samples. Parameters that affect the efficiency of the chromatographic separation and analytical determination of different methods (column, mobile phase, wavelength, precursor ions, fragmentor, and product ions) were optimized. Analytical features, such as matrix effect, sensitivity, precision, selectivity, and linearity, were calculated. In terms of selectivity, the derivatization technique was discarded since it was only applicable to the platinated sum. A high dilution of the sample with LC-UV(DAD) was needed to reduce the matrix effect. Overall, the LC-MS/MS method presented the best analytical features (% RSD ≤ 12.8%, R2 ≥ 0.991, or method-detection limits between 0.01-1 µg mL-1). The selected method was applied to the quantification of platinized cytostatic drugs in hospital urine samples from oncologic patients.
Project description:IntroductionAbsolute quantification of individual metabolites in complex biological samples is crucial in targeted metabolomic profiling.ObjectivesAn inter-laboratory test was performed to evaluate the impact of the NMR software, peak-area determination method (integration vs. deconvolution) and operator on quantification trueness and precision.MethodsA synthetic urine containing 32 compounds was prepared. One site prepared the urine and calibration samples, and performed NMR acquisition. NMR spectra were acquired with two pulse sequences including water suppression used in routine analyses. The pre-processed spectra were sent to the other sites where each operator quantified the metabolites using internal referencing or external calibration, and his/her favourite in-house, open-access or commercial NMR tool.ResultsFor 1D NMR measurements with solvent presaturation during the recovery delay (zgpr), 20 metabolites were successfully quantified by all processing strategies. Some metabolites could not be quantified by some methods. For internal referencing with TSP, only one half of the metabolites were quantified with a trueness below 5%. With peak integration and external calibration, about 90% of the metabolites were quantified with a trueness below 5%. The NMRProcFlow integration module allowed the quantification of several additional metabolites. The number of quantified metabolites and quantification trueness improved for some metabolites with deconvolution tools. Trueness and precision were not significantly different between zgpr- and NOESYpr-based spectra for about 70% of the variables.ConclusionExternal calibration performed better than TSP internal referencing. Inter-laboratory tests are useful when choosing to better rationalize the choice of quantification tools for NMR-based metabolomic profiling and confirm the value of spectra deconvolution tools.
Project description:Several estimating equations for predicting 24-h urinary sodium (24-hUNa) excretion using spot urine (SU) samples have been developed, but have not been readily available to Chinese populations. We aimed to compare and validate the six existing methods at population level and individual level. We extracted 1671 adults eligible for both 24-h urine and SU sample collection. Mean biases (95% CI) of predicting 24-hUNa excretion using six formulas were 58.6 (54.7, 62.5) mmol for Kawasaki, -2.7 (-6.2, 0.9) mmol for Tanaka, -24.5 (-28.0, -21.0) mmol for the International Cooperative Study on Salt, Other Factors, and Blood Pressure (INTERSALT) with potassium, -26.8 (-30.1, -23.3) mmol for INTERSALT without potassium, 5.9 (2.3, 9.6) mmol for Toft, and -24.2 (-27.7, -20.6) mmol for Whitton. The proportions of relative difference >40% with the six methods were nearly a third, and the proportions of absolute difference >51.3 mmol/24-h (3 g/day salt) were more than 40%. The misclassification rate were all >55% for the six methods at the individual level. Although the Tanaka method could offer a plausible estimation for surveillance of the population sodium excretion in Shandong province, caution remains when using the Tanaka formula for other provincial populations in China. However, these predictive methods were inadequate to evaluate individual sodium excretion.
Project description:While urine-based sampling for human papillomavirus (HPV) is being explored as a simple and noninvasive approach for cervical cancer screening, data comparing HPV genotyping in urine and those in cellular sampling of the cervix and vulva, and their correlation with rigorously confirmed cervical disease status, are sparse. We performed HPV genotyping on voided-urine and clinician-collected vulvar and cervical samples from 72 women undergoing colposcopy. Although urine-based HPV carcinogenic HPV detection was lower (58.3%) than cervical (73.6%) and vulvar (72.1%) detection (P = 0.05 and 0.07, respectively), the agreement of urine HPV with cervical and vulvar HPV was moderate (kappa = 0.55) and substantial (kappa = 0.62), respectively. Urine-based carcinogenic HPV detection had a clinical sensitivity of 80.8% (95% confidence interval [CI] = 60.7 to 93.5) and a specificity of 53.3% (95% CI = 37.9 to 68.3) for diagnosing cervical intraepithelial neoplasia grades 2/3 (CIN2/3) on histology; 90.0% of CIN3 was positive for urine HPV. The corresponding sensitivity and specificity values for vulvar sampling were 92% (95% CI = 74 to 99) and 40.5% (95% CI = 25.6 to 56.7), and those for cervical sampling were 96.2% (95% CI = 80.4 to 99.9) and 40% (95% CI = 25.7 to 55.7), respectively. HPV16 was the most common carcinogenic genotype detectable in 25% of urine, 33.8% of vulvar, and 31.9% of cervical samples overall, with prevalence increasing with cervical disease grade, regardless of the sampling method. Stronger cervical HPV PCR signal strengths were associated with increased frequency of urine HPV detection. In summary, the relatively lower detection rates but comparable clinical performance of urine-based HPV sampling underscore the need for larger studies to evaluate urine-based sampling for cervical cancer screening, epidemiologic studies, and postvaccination HPV disease surveillance.
Project description:Human papillomavirus (HPV) is the leading cause of cervical cancer. Urine-based HPV testing offers a simple and non-invasive method because of its increasing acceptance. A total of 164 pairs of cervical swab and urine samples from Thai women who underwent cervical cancer screening were used for HPV testing with HPV GenoArray Diagnostic Kits. The overall concordance percentage for HPV detection in the cervical swab and urine samples was 65.2%. The HPV genotypes most commonly detected were HPV16 and HPV18. An analysis of the urine samples and a second analysis of the cervical swab samples showed that the differences in the overall HPV detection rate between women with normal and abnormal cytology were not significant (p > 0.05). Urine samples processed with the GenoArray assay is an alternative for women who decline to undergo Pap smear even though it is not ideal as the first-line screening option.