Content In Biological Samples Research Articles

Automated phase reconstruction and super-resolution with deep learning in digital holography

Digital holography can provide quantitative phase images that are related to the shape and content of biological samples. In particular, high-resolution phase images contain more important details and information about the sample. However, to obtain a high-resolution phase image, various phase reconstruction processes must be performed, while the finite pixel size of the sensor needs to be overcome. We propose a deep learning model that can obtain high-resolution phase images from low-resolution holograms. The proposed model consists of image translation and super-resolution parts, and performs phase reconstruction and the super-resolution process at the same time. We successfully generated sophisticated phase values that closely resembled real images for three scaling factors of (×2, ×3, and ×4). Comparative evaluations with various deep learning models demonstrated the favorable performance of our proposed model. Multi-scale training was also possible, so it was shown that high-resolution phase images could be generated regardless of the scale factor. The proposed model can automatically generate accurate high-resolution phase images from low-resolution holograms, reducing the cost of digital holography, and providing great benefits to biological sample measurement processes.

Biomarkers of arsenic exposure in biogeochemical provinces

Introduction. One of the direct methods of assessing the impact of contaminants on public health is the use of biological markers that allow determining the presence of a substance or its metabolite in human biological tissues and the dose received from all sources of this substance. 
 The purpose of this study was to determine the dependence of arsenic content in biological samples (hair) 
 of exposed residents on the level of its content in drinking water and to substantiate the effectiveness of the use of this biomarker in the conditions of a hydrogeochemical province in the Republic of Dagestan.
 Material and methods. During this research the arsenic content in the hair of the exposed residents was measured with further comparison of the data obtained with its content in drinking groundwater. To determine the arsenic content in the hair of the exposed residents, an atomic absorption method was used with preliminary preparation of biosubstrate samples. Socio-demographic data were obtained from a questionnaire. Statistical data processing was carried out using Microsoft Excel.
 Results. In the course of this study, a correlation was revealed between the arsenic content in the hair of the exposed residents and its content in the drinking water consumed, and a direct proportional relationship between these values was also revealed. Thus, with an increase in the concentration of arsenic in water by 
 0.1 mg/l, the arsenic content in the hair increases by 0.14 mkg/g. So, using of the hair of exposed residents as a biomarker of long-term exposure is recommended to small doses of arsenic in a biogeochemical province.
 limitation of the study is the small sample size. However, this does not prevent the assessment of the effectiveness of using hair as a biomarker of arsenic exposure and preliminary conclusions for subsequent in-depth analysis of biomonitoring in the conditions of biogeochemical provinces.
 Conclusion. As a rule, the content of natural toxic elements is characterized by trace amounts in the conditions of biogeochemical provinces. Biomonitoring will reveal the level of stress on the body and timely apply preventive methods before the clinical signs of intoxication are revealed.

Development of Methyl Ester Antibody-Based Competitive Indirect ELISA for Quantitative Detection of Mitragynine in Human Urine.

Mitragynine is the main psychoactive compound of Mitragyna speciosa Korth. (kratom). This alkaloid could render psychotropic effects and is often misused as a substitute for commercial drugs. Nowadays, the increasing popularity of kratom has led to the development of a rapid and effective detection method. The detection of mitragynine in a biological sample such as urine requires a highly sensitive and specific method due to the complex nature of mitragynine in urine. Enzyme-linked immunosorbent assay (ELISA) is well known as a rapid screening method for biological samples. In this study, a competitive indirect ELISA was successfully developed using MG-22-OCH3 IgG as a detection antibody for mitragynine in human urine. The mitragynine immunoassay showed a limit of detection and a limit of quantification of 0.412 and 1.25 μg/mL, respectively. The measurement range was between 0.01 and 100.0 μg/mL, with a minimal inhibition (IC50) value of 0.152 μg/mL. The developed ELISA was validated using a gold method such as high-performance liquid chromatography-mass spectrometry (HPLC-MS). The percentage of recovery and the coefficient of variation (CV) for the ELISA and LCMS/MS analyses were 84.0-95.70%, 99.20-112.0%, 7.69-9.78%, and 2.86-6.62%, respectively. This indicates that the developed ELISA is a reliable method that can be used as a rapid approach for quantifying mitragynine content in biological samples.

Fast automated quantitative phase reconstruction in digital holography with unsupervised deep learning

Digital holography can provide quantitative phase images related to the morphology and content of biological samples. To reconstruct an accurate phase image, several processes, such as phase unwrapping, focusing, and calculation of digital reference wave and numerical propagation, are essential. However, this process is time-consuming. We propose a model that performs phase reconstruction in one-step and two-step using an unsupervised image-to-image translation structure. The two-step reconstruction model translates the phase image, which is obtained by performing numerical propagation on the hologram, into an accurate phase image, whereas the one-step reconstruction model directly translates the hologram into an accurate phase image. The proposed model shows similar high-performance reconstruction to the supervised learning model used in many previous studies. However, since supervised learning is trained in strict pairs, many target domain data (accurate phase imagery) is required. Since the proposed model is trained by unsupervised learning, phase reconstruction can be performed with a small amount of target domain data. The proposed method can help to observe the morphology and movement of biological cells in real-time applications.

Exosomal lipid PI4P regulates small extracellular vesicle secretion by modulating intraluminal vesicle formation.

Membrane lipids play vital roles in small extracellular vesicle (sEV) biogenesis. However, the function of various lipids in the biogenesis of sEVs is still poorly understood. Phosphoinositolphosphates (PIPs), a group of the most critical lipids in vesicle transport, can undergo rapid conversion in response to a variety of cell signals, which in turn influence the generation of vesicles. Due to the challenge in detecting the low amount of PIP content in biological samples, the function of PIPs in sEVs has been insufficiently investigated. Here, we employed an LC-MS/MS method to detect the levels of PIPs in sEVs. We revealed phosphatidylinositol-4-phosphate (PI4P) was the main PI-monophosphate in macrophage-derived sEVs. The release of sEVs was regulated in a time-dependent manner and correlated with the PI4P level during the lipopolysaccharide (LPS) stimulation. In terms of mechanism, within 10h of LPS treatment, the LPS-induced production of type I interferon inhibited the expression of PIP-5-kinase-1-gamma, which increased the PI4P content on multivesicular bodies (MVBs) and recruited RAB10, member RAS oncogene family, to promote sEV generation. When LPS stimulation was extended to 24h, the heat shock protein family A member 5 (HSPA5) expression level was elevated. PI4P interacted with HSPA5 on the Golgi or endoplasmic reticulum away from MVBs, which disrupted the continuous fast sEV release. In conclusion, the present study demonstrated an inducible sEV release model response to LPS treatment. The inducible release may be due to PI4P regulating the generation of intraluminal vesicles secreted as sEVs.

Assessment of uranium content in biological samples of the population living in the vicinity of uranium production

Pollution of the environment by industrial waste is one of the urgent problems today. Therefore, for the population living near the storage of radioactive waste, it is important to study its impact on the human body for many years and determine ways to solve this problem. The article presents an overview of the issues of ensuring radiation safety in the areas of radioactive waste warehouses, data on the current state of radioactive waste storage sites in the country. It also provides the characteristic of the ways of movement of radionuclides, mainly in the objects of the uranium series environment, as well as an overview of the main methods for assessing the content of radionuclides in the human body and calculating radiation doses. The main attention was paid to the determination of uranium in biological samples (hair, nails, urine).

Highly sensitive cholesterol biosensor based on electron mediator thionine and cubic-shaped Cu2O nanomaterials

The preparation of an electrochemical biosensor applicable for the highly sensitive detection of cholesterol content in biological samples in clinical practice is studied in this paper. Commonly used in biosensors, nano-cuprous oxide (Cu2O) contains active electron-hole pairs and a large specific surface area, while thionine (TH) improves the efficiency of electron transfer between the enzyme and the electrode. In this study, a cholesterol biosensor, cholesterol oxidase (ChOx)/thionine/nano-cuprous oxide/ glassy carbon electrode (GCE) (ChOx/TH/Cu2O/GCE), is prepared using cubic-shaped Cu2O as the nanomaterial, TH as an electron mediator, and chitosan (CS) as a cross-linking agent to immobilize cholesterol oxidase. The results show that Cu2O nanoparticles (NPs) are successfully synthesized, and the cholesterol biosensor prepared from synthesized Cu2O NPs exhibits excellent electrochemical characteristics, good linearity (R2 = 0.997) over a substrate concentration range of 10 to 1000 µM, a sensitivity of 70.22 µA µM−1 cm−2, and a low detection limit of 0.0018 µM for the substrate. The ChOx, which immobilizes on the composite electrode, shows a strong affinity for cholesterol due to its low Michaelis–Menten constant (Km) value (25.359 µM). Moreover, with strong anti-interference, good reproducibility, and stability, the cholesterol biosensor can achieve a recovery rate of 99.71 % to 107.75 % according to a recovery test of actual spiked samples. Thus, the prepared nanomaterial-based cholesterol biosensor can be applied to the highly sensitive detection of cholesterol in actual biological samples.

Determination of mitochondrial complex I content in biological samples

Determination of mitochondrial complex I content in biological samples

Automated phase unwrapping in digital holography with deep learning.

Digital holography can provide quantitative phase images related to the morphology and content of biological samples. After the numerical image reconstruction, the phase values are limited between -π and π; thus, discontinuity may occur due to the modulo 2π operation. We propose a new deep learning model that can automatically reconstruct unwrapped focused-phase images by combining digital holography and a Pix2Pix generative adversarial network (GAN) for image-to-image translation. Compared with numerical phase unwrapping methods, the proposed GAN model overcomes the difficulty of accurate phase unwrapping due to abrupt phase changes and can perform phase unwrapping at a twice faster rate. We show that the proposed model can generalize well to different types of cell images and has high performance compared to recent U-net models. The proposed method can be useful in observing the morphology and movement of biological cells in real-time applications.

Improved methods to characterize the length and quantity of highly unstable PolySialic acids subject category: (Carbohydrates, chromatographic techniques)

Polysialic acid (polySia) is a linear homopolymer of α2-8-linked sialic acids that is highly expressed during early stages of mammalian brain development and modulates a multitude of cellular functions. While degree of polymerization (DP) can affect such functions, currently available methods do not accurately characterize this parameter, because of the instability of the polymer. We developed two improved methods to characterize the DP and total polySia content in biological samples. PolySia chains with exposed reducing termini can be derivatized with DMB for subsequent HPLC analysis. However, application to biological samples of polySia-glycoproteins requires release of polySia chains from the underlying glycan, which is difficult to achieve without concurrent partial hydrolysis of the α2-8-linkages of the polySia chain, affecting its accurate characterization. We report an approach to protect internal α2-8sia linkages of long polySia chains, using previously known esterification conditions that generate stable polylactone structures. Such polylactonized molecules are more stable during acid hydrolysis release and acidic DMB derivatization. Additionally, we used the highly specific Endoneuraminidase-NF enzyme to discriminate polysialic acid and other sialic acid and developed an approach to precisely measure the total content of polySia in a biological sample. These two methods provide improved quantification and characterization of polySia.

Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples

Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 104 min−1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 103 complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.

Elemental plasma content and urinary excretion in vineyard farmers occupationally exposed to pesticides in southern Brazil.

This is a cross-sectional study with data and biological material collection from vineyard farmers in southern Brazil. An interview was carried out through a questionnaire developed according to the reference guide of the state government. Plasma and urine samples were screened for Aluminum, Chromium, Manganese, Copper, Nickel, Cobalt, Zinc, Arsenic, Selenium, Cadmium, Antimony, Barium, Mercury, Lead and Uranium, with a technique for fast determination of these elemental contents in biological material utilizing dynamic reaction cell inductively coupled mass spectrometry. Principal component analysis was used to identify associations between these elemental contents in biological samples and the information obtained from the interviews. The farmers showed some trace elements in plasma and urine at a higher concentration than unexposed populations from other studies. This study highlights recent findings of trace elements in biological material and their association with characteristics of pesticide use. In addition, it also contributes to the gap in the literature regarding trace elements content in plasma and urine of workers exposed to pesticides.

PSV-4 Determination of Deoxynivalenol (DON) Content in Biological Samples as an Indicator of DON Intake in Grower-finisher Pigs

Abstract Due to difficulties in obtaining consistent and/or reliable measures of deoxynivalenol (DON) in complete swine diets, we investigated whether measuring DON in biological samples could be used as an indicator of DON ingestion in pigs. In this study, graded levels of DON (1, 3, or 5 ppm) were fed to grower-finisher pigs for a period of 77-d. On d 35 and 77 of the study, urine samples were quantitatively collected over a 24-h period and blood samples were collected between 3 – 4 h after the morning meal on each of those days for serum DON analysis. For direct quantification of DON in urine, high-performance liquid chromatography with tandem mass spectrometry was performed. For serum samples, indirect quantification of DON was performed via enzymatic hydrolysis. We observed that DON content in urine increased linearly as intake of DON increased (Fig.1A; P < 0.05). Analysis of DON in serum follow a similar trend, where serum DON content was increased as DON intake increased (Fig.1B; P < 0.05). An average of 30% of DON ingested was recovered as DON in urine over a 24-h period. In summary, there was a linear relationship between DON intake and DON content in both urine and blood serum, therefore, analyzing DON concentration in serum and urine could be used as a tool to estimate for DON exposure in pigs under controlled conditions.

A simple and reliable refractometric method to determine the total solids concentration of the cervico-vaginal bovine mucus samples

Cervico-vaginal mucus (CVM) is a viscoelastic substance continuously produced by secretory cells of the endocervix and the vagina of cows. Its physicochemical composition varies depending on the hormonal status of the estrous cycle. In veterinary medicine refractometry is a widely diffused technique to determine total solids (TS) content of biological samples, but there are not published data of CVM total solids from refractometric measures. Refractometric TS determination contributes to the qualitative constituents analysis of CVM, additionally it is an easier and more inexpensive technique than gravimetric TS determination. The main goal of the present paper was to validate a refractometric method to estimate TS concentration of the soluble fraction of CVM samples. Samples were collected from seventy-three Holando Argentino cows of Santa Fe province farms in Argentina. Cows were classified in three experimental groups: healthy, subclinical (SE) and clinical endometritis (CE) group. To achieve a solubilisation protocol for CVM samples, four Triton™ X-100 concentrations were tested. Refractive index (RI) and gravimetric total solid (gTS) concentration of solubilised samples were determined for the three experimental groups. A mathematical equation was determined with the experimental data from the healthy group, in order to obtain calculated total solid concentration (cTS) from refractivity (R) values. To validate the RI method for CVM samples, cTS concentrations were compared with gTS concentrations from endometritis group samples. Triton™ X-100 0.01% (V/V) improved CVM samples handling and did not change physicochemical parameters (gTS, Na+ and K+ concentration, and RI values). The linear regression equation obtained was: cTS (g/dL) = (R – 0.67)/16.2, r2 = 0.91. Correlation between gTS and cTS concentration was: r = 0.97 for SE group and r = 0.97 for CE group. The homogenization protocol allowed the measurement of physicochemical parameters without altering their values. A high correlation coefficient between cTS and gTS postulates refractometry as an accurate method to determine TS concentration for solubilised CVM samples.

Synthesis of copper oxides-graphene composites for glucose sensing

A multi-gram preparation of graphene nanoplatelets decorated with CuO leaf-like structures and Cu2O nanocubes can be accomplished with ease from graphite using an environmentally friendly method. The composites' synthesis is conveniently achieved within an hour through a high shear mixing pre-treatment of the graphite in water, followed by the addition of copper salts to the resulting slurry and their subsequent conversion to cupric or cuprous oxide-few layer graphite composites of varying weight ratios. The materials, characterized by XRD, Raman, SEM, TEM, and TGA exhibit good electrochemical glucose oxidation sensing capacity. Cyclic voltammetry of modified glassy carbon electrodes prepared with a 1:0.2 weight ratio of graphene nanoplatelets to CuO or Cu2O composites shows low detection limits of 0.25 μM (S/N = 3) for glucose and high sensitivities of 845 and 1,108 μA cm–2 mM–1, respectively. The sensors also show exceptionally low current response to the commonly found interferents such as uric acid, dopamine, and ascorbic acid. The materials' ease of manufacturing and good sensing capabilities suggests their potential use in determining glucose contents in biological samples and industrial applications.

Quantitative Determination of Rutin in Biological Samples of Rats Upon Administration of Primula veris L. Herbal Dense Extract

Rutin contents in biological samples of blood plasma, cardiomyocytes, and mitochondria of cardiomyocytes in rats after administration of Primula veris L. dense herbal extract were determined using a quantitative HPLC method. The optimum conditions for quantitative determination of rutin were KH2PO4 buffer (50 mM, pH 4.6), 82:18% (v/v) aqueous to organic phase ratio, 30°C, detection at 205 nm, flow rate 1 mL/min, 12 MPa pressure in chromatographic system, and retention time 6 – 6.5 min. Rutin was found to enter the blood and distribute into the heart, in which its concentration was 5.34 times higher than in blood, and to penetrate mitochondria, in which its concentration was 1.7 times higher than in plasma and 3.18 times lower than in the heart.

Effect of long-term feeding of graded levels of deoxynivalenol (DON) on growth performance, nutrient utilization, and organ health in finishing pigs and DON content in biological samples.

The prevalence of deoxynivalenol (DON) is a concern for swine producers, and although there has been extensive research into the effects of DON in pigs, focus has been in young pigs and/or in short-term studies. The objective of the study was to determine the effect of long-term exposure to DON-contaminated diets in finisher pigs. A total of 200 pigs (76.6 ± 3.9 kg initial weight) were group housed (five pigs per pen; n = 10 pens/treatment) in a 6-wk trial. Pigs were fed a wheat-barley-soybean meal-based control (CONT) diet with no DON or the basal diet in which clean wheat was replaced by DON-contaminated wheat and wheat screenings to provide DON content of 1, 3, or 5 ppm (DON1, DON3, and DON5, respectively). Individual BW and pen feed intake were recorded weekly to calculate average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F). Blood was collected on days 0, 14, and 43 and analyzed for indicators of liver and kidney health. Nitrogen (N)-balance was conducted immediately following the growth performance period to determine the effect of DON on nutrient utilization. Blood and urine samples collected during N balance were analyzed for DON content. Feeding DON reduced (P < 0.05) ADFI and ADG from days 0 to 28 compared with CONT, after which there was no effect of diet on ADFI and ADG. The G:F was lower (P < 0.05) in DON5 fed pigs compared with all treatments during days 0 to 7; however, no treatment effects on G:F was observed from days 8 to 42. Nitrogen retention was lower (P < 0.05) in DON3 and DON5 compared with DON1-fed pigs. Nitrogen retention efficiency was higher (P < 0.05) in DON1 compared with DON3 and DON5 and protein deposition for DON1 pigs was higher (P < 0.05) than all treatments. There were no treatment effects on indicators of liver and kidney health. As dietary DON intake increased, concentration of DON in blood and urine increased. Overall, although there was an initial decrease in ADG and ADFI in pigs receiving diets containing >1 ppm DON, pig performance recovered after a period of time, whereas nutrient utilization continued to be affected after recovery of performance. Moreover, the lack of DON on G:F indicates that the negative effects of DON on growth performance are largely due to reduced feed intake. Overall, although pigs maybe capable of adapting to intake of DON-contaminated diets, their final body weight will be reduced when fed diets containing >1 ppm DON.

Improved HPLC Conditions to Determine Eumelanin and Pheomelanin Contents in Biological Samples Using an Ion Pair Reagent.

Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin and pheomelanin, two major classes of melanin pigments, affords pyrrole-2,3,5-tricarboxylic acid (PTCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) from eumelanin and thiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA) from pheomelanin. Quantification of these five markers by HPLC provides useful information on the quantity and structural diversity of melanins in various biological samples. HPLC analysis of these markers using the original method of 0.1 M potassium phosphate buffer (pH 2.1):methanol = 99:1 (85:15 for PTeCA) on a reversed-phase column had some problems, including the short lifetime of the column and, except for the major eumelanin marker PTCA, other markers were occasionally overlapped by interfering peaks in samples containing only trace levels of these markers. These problems can be overcome by the addition of an ion pair reagent for anions, such as tetra-n-butylammonium bromide (1 mM), to retard the elution of di-, tri- and tetra-carboxylic acids. The methanol concentration was increased to 17% (30% for PTeCA) and the linearity, reproducibility, and recovery of the markers with this improved method is good to excellent. This improved HPLC method was compared to the original method using synthetic melanins, mouse hair, human hair, and human epidermal samples. In addition to PTCA, TTCA, a major marker for pheomelanin, showed excellent correlations between both HPLC methods. The other markers showed an attenuation of the interfering peaks with the improved method. We recommend this improved HPLC method for the quantitative analysis of melanin markers following AHPO because of its simplicity, accuracy, and reproducibility.

Two-dimensional titanium carbide (MXene) nanosheets as an efficient electrocatalyst for 4-nitroquinoline N-oxide detection

Two-dimensional (2D) MXene-based electrode materials have recently gained attention in the field of electrochemical sensors. Herein, we report the first titanium carbide (TiC)-based electrochemical sensor for the detection of 4-nitroquinoline N-oxide (4-NO). X-ray diffraction, field emission scanning electron microscopy, elemental mapping, and energy-dispersive X-ray spectroscopy were used to examine the structural and compositional properties of TiC. With the 2D MXene structure, the TiC-modified electrode provides excellent conductivity, an electron transfer boost, large surface area, and nanoscale effects, which improve the electrochemical activity for 4-NO detection. The fabricated electrochemical sensor shows a strong voltammetry performance at −0.32 V with a wide linear range (0.01–114 μM and 133–650 μM) and a low limit of detection (2 nM) for 4-NO detection. The real-time analysis of 4-NO content in biological samples was successfully conducted and afforded good recoveries. Our method of design produces an efficient electrode material for 4-NO detection with outstanding electrochemical performance.

A review of applications of Raman spectroscopy in immunology

Vibrational spectroscopic techniques have recently gained increasing clinical importance as non-invasive, rapid and inexpensive methods to obtain information on the content of biological samples. For some time Raman spectroscopy has been involved in preclinical applications, mainly in the cancer space, with evolving applications towards new horizons in the dermatology and companion diagnostics arena. It is attractive as an analytical technique due to its exquisite sensitivity, label-free operation and low water detectivity such that in-vivo applications are possible. In cytometry, Raman spectroscopy has been applied to the analysis of single cells providing a label-free alternative to cell classification approaches in the laboratory. In this review we collate in-vitro, ex-vivo and in-vivo examples of research using Raman spectroscopy for the detection, quantification and analysis of immune signaling at the cellular level. While cancer biology has recently focussed on the role of immunological signals in the development of the disease, it is timely to examine these applications as research in this space evolves.