Content In Biological Samples Research Articles

Determination of Chitin Content in Insects: An Alternate Method Based on Calcofluor Staining.

Chitin is an aminopolysaccharide present in yeast cells and arthropod cuticle and is one of the most abundant biopolymers. The conventional methods for the quantitation of chitin content in biological samples are based on its hydrolysis (acid or enzymatic), and the assessment of the byproduct, glucosamine. However, previously described methodologies are time-consuming, laborious, low throughput, and not applicable to insect samples in many cases. Here we describe a new approach to chitin content quantitation based on calcofluor fluorescent brightener staining of samples, followed by microplate fluorescence readings. Calcofluor is a specific chitin stain commonly used for topological localization of the polymer. The protocol was tested in three important disease vector species, namely Lutzomyia longipalpis, Aedes aegypti, and Rhodnius prolixus, and then compared to a classic colorimetric chitin assessment method. Results show that chitin content in the tested insects can vary largely in a range of 8–4600 micrograms of chitin per insect, depending on species, sex, and instar. Comparisons between measurements from the previous protocol and calcofluor method showed statistically significant differences in some samples. However, the difference might be due to interference in the classic method from non-chitin sources of glucosamine and reducing agents. Furthermore, chitinase hydrolysis reduces the total chitin mass estimated between 36 and 74%, consolidating the fluorescent measurements as actual stained chitin in the same extent that was observed with the standard protocol. Therefore, the calcofluor staining method revealed to be a fast and reliable technique for chitin quantitation in homogenized insect samples.

New approach to investigate Common Variable Immunodeficiency patients using spectrochemical analysis of blood

Common variable immune deficiency (CVID) is a primary immunodeficiency disease, characterized by hypogammaglobulinemia, recurrent infections and various complications. The clinical heterogeneity of CVID has hindered identification of an underlying immune defect; diagnosis relies on clinical judgement, alongside evidence-based criteria. The lack of pathognomonic clinical or laboratory features leads to average diagnostic delays of 5 years or more from the onset. Vibrational spectroscopic techniques such as Fourier-transform infrared (FTIR) spectroscopy have recently gained increasing clinical importance, being rapid-, non-invasive and inexpensive methods to obtain information on the content of biological samples. This has led us to apply FTIR spectroscopy to the investigation of blood samples from a cohort of CVID patients; revealing spectral features capable of stratifying CVID patients from healthy controls with sensitivities and specificities of 97% and 93%, respectively for serum, and 94% and 95%, respectively for plasma. Furthermore we identified several discriminating spectral biomarkers; wavenumbers in regions indicative of nucleic acids (984 cm−1, 1053 cm−1, 1084 cm−1, 1115 cm−1, 1528 cm−1, 1639 cm−1), and a collagen-associated biomarker (1528 cm−1), which may represent future candidate biomarkers and provide new knowledge on the aetiology of CVID. This proof-of-concept study provides a basis for developing a novel diagnostic tool for CVID.

Novel Applications of Lead Acetate and Flow Cytometry Methods for Detection of Sulfur-Containing Molecules

Hydrogen sulfide (H2S) is the most recently established gaseous vasodilator, enzymatically produced from cysteine metabolism, involved in a number of pathophysiological processes. However, its accurate detection in vivo is critical due to its volatility and tendency to form sulfane sulfur derivatives, thus limiting the data interpretation of its biological roles. We developed new applications of the simple and rapid method to measure H2S release in cell culture systems, based on the lead acetate strip test. This test, previously prevalently used in microbiology, was compared with the agar trap method, applied, in parallel, on both cell cultures and cell-free samples. Sulfane sulfur represents the major species derived from intracellular H2S. Various fluorescent probes are available for quantitation of H2S derivatives intracellularly. We present here an alternative to the classic imaging method for sulfane sulfur evaluation, running on a flow cytometer, based on SSP4 probe labeling. Flow cytometry turned out to be more direct, fully quantitative and less time-consuming compared to microscopy and more precise with respect to the fluorescence multi-plate reader assay. The new application methods for H2S determination appear to be fully suitable for the analysis of H2S release and sulfane sulfur content in biological samples.

Diagnosis of plant diseases using the Nanopore sequencing platform

Reliable detection and identification of plant pathogens are essential for disease control strategies. Diagnostic methods commonly used to detect plant pathogens have limitations such as requirement of prior knowledge of the genome sequence, low sensitivity and a restricted ability to detect several pathogens simultaneously. The development of advanced DNA sequencing technologies has enabled determination of total nucleic acid content in biological samples. The possibility of using the single‐molecule sequencing platform of Oxford Nanopore as a general method for diagnosis of plant diseases was examined. It was tested by sequencing DNA or RNA isolated from tissues with symptoms from plants of several families inoculated with known pathogens (e.g. bacteria, viruses, fungi, phytoplasma). Additionally, samples of groups of 200 seeds containing one infected seed of each of two or three pathogens, as well as samples with symptoms but unidentified pathogens were tested. Sequencing results were analysed with Nanopore data analysis tools. In all the inoculated plants, pathogens were identified in real time within 1–2 h of running the Nanopore sequencer and were classified to the species or genus level. DNA sequencing or direct RNA sequencing of samples with unidentified disease agents were validated by conventional diagnostic procedures (e.g. PCR, ELISA, Koch test), which supported the results obtained by Nanopore sequencing. The advantages of this technology include: long read lengths, fast run times, portability, low cost and the possibility of use in every laboratory. This study indicates that adoption of the Nanopore platform will be greatly advantageous for routine laboratory diagnosis.

Terahertz time-domain spectroscopy for non-invasive assessment of water content in biological samples.

We apply terahertz time-domain spectroscopy for the quantitative non-invasive assessment of the water content in biological samples, such as Carpinus caroliniana tree leaves and pork muscles. The developed experimental terahertz time-domain spectroscopy system operates both in transmission and reflection modes. The Landau-Looyenga-Lifshitz-based model is used for the calculation of the water concentration within the samples. The results of the water concentration measurements are compared with the results of the gravimetric measurements. The obtained results show that the water content in biological samples can be measured non-invasively, with a high accuracy, utilizing terahertz waves in transmission and reflection modes.

Robust Analysis of Time Series in Virome Metagenomics.

Metagenomics is a powerful tool for assessing the functional and taxonomic contents in biological samples as it makes feasible to study, simultaneously, the whole living community related to a host organism or medium: all the microbes, including virus, bacteria, archaea, fungi, and protists. New DNA and RNA sequencing technologies are dramatically decreasing the cost per sequenced base, so metagenomic sequencing is becoming more and more widespread in biomedical and environmental research. This is opening the possibility of complete longitudinal metagenomic studies, which could unravel the dynamics of microbial communities including intra-microbiome and host-microbiome interactions through in-depth analysis of time series. For viruses, this is particularly interesting because it allows broad interaction studies of viruses and hosts in different time scales, as in bacteria-phages coevolution studies.This chapter presents computational methods for an automatic and robust analysis of metagenomic time series in virome metagenomics (RATSVM). The same theoretical frame and computational protocol is also suitable for longitudinal studies of spatial series to uncover the dynamics of a microbial community with viruses along a selected dimension in the space. In order to conveniently illustrate the procedure, real data from a published virome study is used. The computational protocol presented here requires only basic computational knowledge. Several scripts have been prepared to ease and automate the most complicate steps, they are available in the RATSVM public repository. For some of the methods a mid-range computing server is advisable, and for some others, it is required. A fat-node with large memory and fast I/O would be the best choice for optimum results.

A method for determining polysaccharide content in biological samples

Difficulties in the determination of polysaccharide content in biological samples has been one of the bottlenecks limiting the development of polysaccharides pharmacokinetics, greatly due to the complicated chemical structure of these compounds. In this study, we established a simple, reproducible and reliable method for the determination of polysaccharide content in biological samples. Polysaccharides were replaced by fluorescein isothiocyanate (FITC) and iodine to generate a complex. The iodine content in the complex was measured by inductively coupled plasma-mass spectrometry (ICP-MS) to indirectly reflect polysaccharide content. We investigated the stability of the complex, carried on methodological validation and proceeded to determine the polysaccharide content in rat blood and organs after oral administration of iodine substituted polysaccharides using ICP-MS. The results showed that the iodine complex was stable in vivo and in vitro. In the bioavailability of polysaccharides, after 1h, the absorption rate of polysaccharides in blood was the highest, reaching 5.84%. After 3h, the bioaccessibility was differently distributed in various organs, reaching 1% on average across organs.

Relationship between arsenic and selenium in workers occupationally exposed to inorganic arsenic

The interaction between arsenic (As) and selenium (Se) has been one of the most extensively studied. The antagonism between As and Se suggests that low Se status plays an important role in aggravating arsenic toxicity in diseases development. The objective of this study was to assess the Se contents in biological samples of inorganic As exposed workers (n=61) and in non-exposed subjects (n=52). Median (Me) total arsenic concentration in urine of exposed workers was 21.83μg/g creat. (interquartile range (IQR) 15.49–39.77) and was significantly higher than in the control group – (Me 3.75μg/g creat. (IQR 2.52–9.26), p<0.0001). The median serum Se concentrations in the study group and the control were: 54.20μg/l (IQR 44.2–73.10μg/l) and 55.45μg/l (IQR 38.5–69.60μg/l) respectively and did not differ significantly between the groups. In the exposed group we observed significantly higher urine concentrations of selenosugar 1 (SeSug 1) and selenosugar 3 (SeSug3) than in the control group Me: 1.68μg/g creat. (IQR 1.25–2.97 vs Me: 1.07μg/g creat. (IQR 0.86–1.29μg/g), p<0.0001 for SeSug1; Me: 0.45μg/g creat. (IQR 0.26–0.69) vs Me: 0.28μg/g creat. (IQR 0.17–0.45μg/g), p=0.0021). In the multivariate model, after adjusting to cofounders (age, BMI, job seniority time, consumption of fish and seafood and smoking habits) the high rate of arsenic urine wash out (measured as a sum of iAs+MMA+DMA) was significantly associated with the high total selenium urine excretion (B=0.14 (95%CI (confidence interval) 0.05–0.23)). Combination of both arsenic and selenium status to assess the risk of arsenic-induced diseases requires more studies with regard to both the analysis of speciation, genetics and the influence of factors such as nutritional status.

Determination of the selenium content in biological samples by gamma - gamma coincidence method

At Dalat nuclear research reactor, the gamma - gamma coincidence method is eficiently used in data nuclear research direction. By this method, some laboratories in the world test neutron activation analysis of geological, biological and environmental samples. In this paper, we present the determination of the selenium content in biological sample by gamma – gamma coincidence method. The results showed that the compton scattering was suppressed. The ratio of peak counts/back was improved up to 64.2 times. The detection limit for selenium in biological sample was enhanced to 8.9 times to compare to the method one detector system.

A comprehensive profiling of sulfatides in myelin from mouse brain using liquid chromatography coupled to high-resolution accurate tandem mass spectrometry

Sulfatides are sulfoglycolipids found in the myelin sheath. The composition ratio of sulfatide molecular species changes with age, and it has also been associated with the pathogenesis of various human central nervous system diseases. However, profiling sulfatides in biological samples is difficult, due to the great variety of molecular species. In this work, a new, easy and reliable liquid chromatography-electrospray tandem mass spectrometry (LC-ESI(+)-MS/MS) method has been developed to profile sulfatide content in biological samples of myelin. The ‘wrong-way-round’ ionization effect has been described for this type of molecules for the first time, making it possible to correctly identify as many as 37 different sulfatides in mouse brain myelin samples, including molecules with different fatty acid chain lengths and varying degrees of unsaturation and hydroxylation. A chemometric analysis of their relative abundances showed that the main difference among individuals of different ages was the content of sulfatides with odd-numbered fatty acid chains, in addition to hydroxylated species.

Highly specific and sensitive immunoassay for the measurement of prostaglandin E2 in biological fluids.

Lack of specificity of anti-PGE2 antibodies is a long-standing problem. Given quite a few analogs and low PGE2 content in biological fluids, it is quite important to simultaneously meet the demands of high specificity and sensitivity. Highly specific anti-PGE2 antibodies were obtained by combined use of cationic carrier protein and Mannich reaction. The cross-reactivity values of the resultant polyclonal and monoclonal antibodies against eight analogs were <14 and <5%, respectively. Furthermore, we established a highly sensitive ELISA, which could be applied to direct analysis of PGE2 at the pg/ml level (LOQ = 15.6 pg/ml). We provide an appropriate strategy to develop a highly-specific and sensitive immunoassay for measuring low PGE2 content in biological samples.

Nanotherapeutics With Anthracyclines: Methods of Determination and Quantification of Anthracyclines in Biological Samples

Anthracyclines, e.g. doxorubicin, pirarubicin, are widely used as cytostatic agents in the polymer nanotherapeutics designed for the highly effective antitumor therapy with reduced side effects. However, their precise dosage scheme needs to be optimized, which requires an accurate method for their quantification on the cellular level in vitro during nanocarrier development and in body fluids and tissues during testing in vivo. Various methods detecting the anthracycline content in biological samples have already been designed. Most of them are highly demanding and they differ in exactness and reproducibility. The cellular uptake and localization is predominantly observed and determined by microscopy techniques, the anthracycline content is usually quantified by chromatographic analysis using fluorescence detection. We reviewed and compared published methods concerning the detection of anthracycline nanocarriers.

Accelerator mass spectrometry analysis of 14C-oxaliplatin concentrations in biological samples and 14C contents in biological samples and antineoplastic agents

Accelerator mass spectrometry (AMS) is expected to play an important role in microdose trials. In this study, we measured the 14C concentration in 14C-oxaliplatin-spiked serum, urine and supernatant of fecal homogenate samples in our Yamagata University (YU) – AMS system. The calibration curves of 14C concentration in serum, urine and supernatant of fecal homogenate were linear (the correlation coefficients were ⩾0.9893), and the precision and accuracy was within the acceptance criteria.To examine a 14C content of water in three vacuum blood collection tubes and a syringe were measured. 14C was not detected from water in these devices. The mean 14C content in urine samples of 6 healthy Japanese volunteers was 0.144dpm/mL, and the intra-day fluctuation of 14C content in urine from a volunteer was little. The antineoplastic agents are administered to the patients in combination. Then, 14C contents of the antineoplastic agents were quantitated. 14C contents were different among 10 antineoplastic agents; 14C contents of paclitaxel injection and docetaxel hydrate injection were higher than those of the other injections.These results indicate that our quantitation method using YU-AMS system is suited for microdosing studies and that measurement of baseline and co-administered drugs might be necessary for the studies in low concentrations.

Quantitative Analysis of Structural Isomers of Linear Pentaamines by GC–MS

Structural isomers of long linear polyamines are often highly difficult to differentially quantify due to their structural similarities. In this study, we focused on pentaamines, aminopropylcanavalmine [H2N(CH2)3NH(CH2)4NH(CH2)3NH(CH2)4NH2, (3-4-3-4)] and N1-aminopropyl-N8-aminobutylspermidine [H2N(CH2)3NH(CH2)3NH(CH2)4NH(CH2)4NH2, (3-3-4-4)], which have never been separated. We analyzed a mixture of the heptafluorobutyryl derivatives of these pentaamines by gas chromatography–mass spectrometry (GC–MS) and found that the polyamine content and intensity of the unique fragment ions detected in MS spectra exhibited a strong correlation. The calibration curves of appropriate combinations of the peak intensities versus pentaamine mixing ratios showed high linearity (r > 0.99) in the mixing ratio range of 0–2.33. These data indicated that the pentaamine content in the pentaamine mixed solutions could be determined using appropriate calibration curves. Therefore, this method can potentially be applied to measure the pentaamine content of biological samples.

Determination of Ti from TiO2 Nanoparticles in Biological Materials by Different ICP-MS Instruments: Method Validation and Applications

The accurate determination of nanoparticles (NPs) content in biological samples is of great interest for studying its potential impacts on health. This paper describes a method validation process for Ti determination from TiO2 NPs in biological matrixes using a closed-vessel microwave digestion with a mixture of 3 mL of HNO3 and 0.4 mL of HF and a detection by quadrupole inductively coupled plasma-mass spectrometry (Q-ICP-MS), in both standard and collision/ reaction cell (CCT) modes. As no suitable certified reference materials are available, several internal reference materials (IRMs) were prepared precisely and they were used for the optimisation and the analytical quality assurance of the method. Several criteria such as linearity, limits of quantification (LOQ), precision under repeatability conditions and intermediate precision reproducibility were evaluated. Furthermore, the method was compared on different Q-ICP-MS devices and on a high resolution (HR)-ICP-MS using the prepared IRMs and various samples of rat tissues exposed to two different sizes of TiO2 NPs. The results demonstrated that the Q-ICP-MS method evaluated permitted an accurate determination of Ti present as TiO2 NPs in both standard and CCT modes, with a greater accuracy in CCT mode at low concentration levels near the LOQ of 0.10 mg kg-1.

Pressurized Flow Solubilization System Using Electromagnetic Induction Heating Technique for Simultaneous Determination of Inorganic Elements (Ba, Ca, Cd, Cu, Fe, Mg, Mn, Na, Pb, Sr, Zn) in Sonicate Slurries of Biological Materials by Microwave Induced Plasma Optical Emission Spectrometry (MIP-OES)

A pressurized continuous flow system using electromagnetic induction heating technique has been developed for the determination of trace and minor element content in biological samples by microwave induced plasma-optical emission spectrometry (MIP-OES). The system allows the continuous solubilization of samples at an optimized temperature of 120 °C and pressure of 3.5 bar in 40 min. A system was used to perform off-line solubilization of slurried samples of biological tissues, non-fat milk powder and lichen (3% m/v). Recovery of trace and minor elements averaged 99.2 ± 0.1% was performed by MIP-OES using external calibration technique. The accuracy of the method was proved using certified reference materials from the National Research Council Canada (NRCC, Dogfish Liver, DOLT-2), National Institute of Standards and Technology (NIST, (Non-Fat Milk Powder, NIST 1549) and International Atomic Energy Agency (IAEA, Lichen, IAEA-336). Satisfied analytical results were also obtained in real sample analysis of milk powder, barley and cinnamon.

Development of an analytical method for assessment of silver nanoparticle content in biological matrices by inductively coupled plasma mass spectrometry.

Silver nanoparticles (AgNPs) are a broad class of synthetic nanoparticles that are utilized in a wide variety of consumer products as antimicrobial agents. Despite their widespread use, a detailed understanding of their toxicological characteristics and biological and environmental hazards is not available. To support research into the biodistribution and toxicology of AgNPs, it is necessary to develop a suitable method for the assessment of AgNP content in biological samples. Two methods were developed and validated to analyze citrate-coated AgNP content that utilize acid digestion of rodent feces and liver tissue samples, and a third method was developed for the dilution and direct analysis of rodent urine samples. Following sample preparation, the silver content of each sample was determined by inductively coupled plasma mass spectrometry (ICP-MS) to quantify the silver and AgNP levels present. Analysis of rat feces matrix yielded analytical recoveries ranging from 82 to 93%. Liver tissue spiked with a formulation of AgNPs over a range of concentrations yielded analytical recoveries between 88 and 90%, providing acceptable accuracy results. The analysis of silver in urine samples exhibited recovery values ranging from 80 to 85% for AgNP formulations and 62-84% for standard silver ion solutions. All determinations exhibited a high degree of analytical precision. The results obtained here suggest that matrix interference plays a minimal role in AgNP recovery in feces and liver tissue, while the urine matrix can exhibit a significant effect on the determination of silver content.

A new simple fluorimetric method to assay cytosolic ATP content: application to durum wheat seedlings to assess modulation of mitochondrial potassium channel and uncoupling protein activity under hyperosmotic stress

AbstractThe assays commonly used to determine ATP content in biological samples generally measure total cellular ATP content, but not the different subcellular pools. In this study a new simple method for measuring ATP content in a cytosol-enriched fraction (CEF) was developed, based on a rapid cytosolic ATP extraction (by an isotonic grinding medium that preserves organelle integrity) and its detection monitoring the NADPH fluorescence generated via hexokinase/glucose-6-phosphate dehydrogenase coupled reactions. Four protocols, differing for timing of NADPH generation and for either the presence or absence of some inhibitors of ATP and NADPH metabolism, were compared by determining CEF-ATP, as well as total ATP, in durum wheat (Triticum durum Desf.) etiolated seedlings. The best protocol was the one adopting both simultaneous NADPH generation and use of inhibitors during tissue homogenization. This protocol also showed higher performance than the classical trichloroacetic acid extraction. Using the new method, CEF-ATP content was assessed in control, salt- and osmotic-stressed seedlings, resulting 2.68 ± 0.04, 1.69 ± 0.12 (−40%) and 1.35 ± 0.16 (−50%) μmol/g dry weight, respectively. Finally, the effects of this stress-dependent decrease of cytosolic ATP were evaluated with respect to a possible modulation of two mitochondrial energy-dissipating systems, the uncoupling protein (PUCP) and the K+ channel (PmitoKATP), both inhibited by cytosolic ATP. Experiments carried out at different physiological ATP concentrations suggest that the decreased cytosolic ATP content occurring under hyperosmotic stress may contribute to attenuate inhibition of PmitoKATP, thus promoting its activity (up to about 90%), but not of PUCP, that appears to lose ATP sensitivity under stress condition.

True Color Molecular Imaging with METRiCS OCT

Being able to assess, structure and quantify the molecular content of biological samples in vivo is paramount to improving our understanding of many diseases. To this end, we have developed molecular imaging true-color spectroscopic optical coherence tomography (METRiCS OCT), which derives functional information from the spatially resolved spectral content of scattered and absorbed light.

The human urinary proteome: combinational approaches to comprehensive mapping

The development methodologies for the assessment of the protein content of biological samples have been in the ‘eye of the storm’ in proteomics for almost two decades. The work of Zerefos et al. is a continuation of this trend, focusing on analysis of urinary proteins using a combination of separation methodologies. In this work, the authors employ a previously analyzed control urine sample. Three different methodologies are presented, involving the combination of classical separation approaches, such as SDS-PAGE, preparative electrophoresis and liquid chromatography and standard mass spectrometer instrumentation. Several hundred proteins were reported following the application of a typical proteomics workflow and the use of a data meta-analysis platform to enhance the credibility of the final output. This is also established through cross-method (within this study) as well as cross-study (comparison of this with other main studies in the field) data comparisons. Emphasis is placed on the presentation of experimental identifiers as well as information provided at the peptide level.