Parallel Samples Research Articles

Profiling DNA Methylation in Human Naïve Pluripotent Stem Cells.

DNA methylation represents one of the best characterized epigenetic modifications. Inparticular, global demethylation is a common feature of epigenetic reprogramming to naïve pluripotency in human and mouse pluripotent stem cells. In parallel to the global changes, several locus-specific changes to the DNA methylation landscape occur and also loss of imprinting has been observed in naïve human pluripotent stem cells. The current gold standard to assess and quantitively map DNA methylation is bisulfite sequencing. Various protocols are available for genome-wide bisulfite sequencing and here I describe an optimized method based on Post Bisulfite Adapter Tagging (PBAT) for low amounts of DNA or cells, with as little as 50 cells as minimum requirement, and with the possibility to process a large number of samples in parallel. I outline the basic bioinformatic steps needed to process rawIllumina sequencing data and then describe the inital steps ofthe analysis of DNA methylation datasets, including an assessment of imprint control regions.

Vapour source and spatiotemporal variation of precipitation isotopes in Southwest Spain

AbstractThe δ2H and δ18O composition of 77 precipitation samples collected between January 2014 and April 2019 from two sites across the Guadalquivir Basin, SW Spain, were analysed. The first site is located in an urban area of Seville at 100 km distance to the Atlantic coast and the second site is located in a dune area of the Doñana National Park a few kilometres from the coast. Sampling was performed within intervals of at least 14 days if rain occurred but frequently intervals were longer according to the rainfall incidence. Samples from both sites are available for the period February 2016 to June 2018 with six samples containing identical rain events at both locations. Precipitation weighted averages and local meteoric water lines produced by weighting and non‐weighting regression methods are presented for its use in hydrological applications. Results show a remarkably high variability in δ2H and δ18O values and precipitation weighted average d‐excess values of 11.8‰ and 13‰ at the sites Plaza de España and Doñana, respectively. Temperature and amount effects were found to be weak. A significant influence of secondary evaporation for single rainfall events during summer was identified by enriched isotopic signatures with reduced d‐excess values plotting close or below the global meteoric water line. Backward trajectory analysis of 115 days with daily rainfall above 3 mm yield a predominant Atlantic Ocean vapour source with negligible Mediterranean influence and therefore, d‐excess variability is attributed to the different ocean surface conditions of relative humidity and sea surface temperature. Parallel sampling indicate very similar isotopic signatures at both sites and point to the existence of thermal effects of the Plaza de España site in Seville city during the summer season.

Data Mining-Based Stability and Prescription Analysis of Neonatal Parenteral Nutrition Solution.

In order to evaluate the stability of neonatal parenteral nutrition solution, in this paper, the prescription of neonatal parenteral nutrition solution was investigated and analyzed. The formula of neonatal parenteral nutrition solution used, particularly the one utilized in this study, is commonly used in clinical practice. All the neonatal parenteral nutrition solution required for the test was prepared on the purification workbench in a sterile environment. The time points of stability of parenteral nutrient solution were 0, 12, and 24 hours, respectively, and three parallel samples were taken at each time point. Likewise, to investigate the stability of two kinds of fat milk injection in parenteral nutrition solution of neonates and provide a reference for subsequent experiments and to investigate the influence of electrolyte, amino acid, temperature, pH value, mixing sequence, and the final concentration of glucose on the stability of neonatal parenteral nutrition solution, the stability indexes of neonatal parenteral nutrition liquid mainly include appearance, pH, insoluble particles, fat milk particle size, and particle size distribution. Neonatal parenteral nutrition solution prescriptions from the First Affiliated Hospital of Jinan University, specifically from January to June 2019, were collected and statistically processed. The experimental data were processed by SPSS 19.0 software and data mining technology. The results were expressed as mean ± standard deviation and statistically processed by ANOVA. P < 0.05 was considered statistically significant. The results showed that the stability of neonatal parenteral nutrient solution was influenced by many factors. The formula of neonatal parenteral nutrition solution is generally reasonable, but there are unreasonable phenomena which are needed to be improved further if feasible.

Coagulation parameters in the newborn and infant- the Copenhagen Baby Heart and COMPARE studies.

The coagulation system is not fully developed at birth and matures during the first months of infancy, complicating clinical decision making within hemostasis. This study evaluates coagulation parameters at birth and two months after birth, and tests whether cord blood can be used as a proxy for neonatal venous blood measurements. The Copenhagen Baby Heart Study (CBHS) and the COMPARE study comprise 13,237 cord blood samples and 444 parallel neonatal venous blood samples, with a two month follow-up in 362 children. Because coagulation parameters differed according to gestational age (GA), all analyses were stratified by GA. For neonatal venous blood, reference intervals for activated partial thromboplastin time (APTT) and prothrombin time (PT) were 28-43s and 33-61% for GA 37-39 and 24-38s and 30-65% for GA 40-42. Reference intervals for international normalized ratio (INR) and thrombocyte count were 1.1-1.7 and 194-409×109/L for GA 37-39 and 1.2-1.8 and 188-433×109/L for GA 40-42. Correlation coefficients between umbilical cord and neonatal venous blood for APTT, PT, INR, and thrombocyte count were 0.68, 0.72, 0.69, and 0.77 respectively, and the distributions of the parameters did not differ between the two types of blood (all p-values>0.05). This study describes new GA dependent reference intervals for common coagulation parameters in newborns and suggests that cord blood may serve as a proxy for neonatal venous blood for these traits. Such data will likely improve clinical decision making within hemostasis among newborn and infant children.

A 10‐MHz to 50‐GHz low‐jitter multiphase clock generator for high‐speed oscilloscope in 0.15‐μm GaAs technology

AbstractThe data acquisition system is widely used in electronic measuring instruments, for example, the oscilloscope. The development direction of the oscilloscope reflects the research status and development trend of the data acquisition system. Currently, the index of oscilloscopes is progressing toward a high sampling rate, and the performance of the analog‐to‐digital converter (ADC) has become the main bottleneck to improve the sampling rate of the acquisition system. The alternate sampling technology entails multichannel low sampling rate ADCs to achieve high sampling rate data acquisition through parallel sampling and realize data reconstruction through software. The sampling clock is the most important and difficult part of the system. For ADCs with the 100‐GS/s sampling rate and 10‐bit resolution, the jitter of the sampling clock needs to be in sub‐100 fs along with multiple phases. Based on the principle of injection locking, a 150‐GS/s sampling clock is proposed in this paper, which is implemented in a 0.15‐μm GaAs process, and the 50‐GHz locking range is achieved. The measurement results show that the sampling frequency is 150 GS/s, the root mean square (RMS) jitter is 63 fs with the integrated range from 1 kHz to 10 MHz, and the phase noise can meet the sampling requirements of the 10‐bit oscilloscope, which can provide a clocking scheme for small and low‐power ultrahigh‐speed oscilloscope.

A new trauma frontier: Exploratory pilot study of platelet transcriptomics in trauma patients.

The earliest measurable changes to postinjury platelet biology may be in the platelet transcriptome, as platelets are known to carry messenger ribonucleic acids (RNAs), and there is evidence in other inflammatory and infectious disease states of differential and alternative platelet RNA splicing in response to changing physiology. Thus, the aim of this exploratory pilot study was to examine the platelet transcriptome and platelet RNA splicing signatures in trauma patients compared with healthy donors. Preresuscitation platelets purified from trauma patients (n = 9) and healthy donors (n = 5) were assayed using deep RNA sequencing. Differential gene expression analysis, weighted gene coexpression network analysis, and differential alternative splicing analyses were performed. In parallel samples, platelet function was measured with platelet aggregometry, and clot formation was measured with thromboelastography. Differential gene expression analysis identified 49 platelet RNAs to have differing abundance between trauma patients and healthy donors. Weighted gene coexpression network analysis identified coexpressed platelet RNAs that correlated with platelet aggregation. Differential alternative splicing analyses revealed 1,188 splicing events across 462 platelet RNAs that were highly statistically significant (false discovery rate <0.001) in trauma patients compared with healthy donors. Unsupervised principal component analysis of these platelet RNA splicing signatures segregated trauma patients in two main clusters separate from healthy controls. Our findings provide evidence of finetuning of the platelet transcriptome through differential alternative splicing of platelet RNA in trauma patients and that this finetuning may have relevance to downstream platelet signaling. Additional investigations of the trauma platelet transcriptome should be pursued to improve our understanding of the platelet functional responses to trauma on a molecular level.

Microscale characterisation of the time-dependent mechanical behaviour of brain white matter

Brain mechanics is a topic of deep interest because of the significant role of mechanical cues in both brain function and form. Specifically, capturing the heterogeneous and anisotropic behaviour of cerebral white matter (WM) is extremely challenging and yet the data on WM at a spatial resolution relevant to tissue components are sparse. To investigate the time-dependent mechanical behaviour of WM, and its dependence on local microstructural features when subjected to small deformations, we conducted atomic force microscopy (AFM) stress relaxation experiments on corpus callosum (CC), corona radiata (CR) and fornix (FO) of fresh ovine brain. Our experimental results show a dependency of the tissue mechanical response on axons orientation, with e.g. the stiffness of perpendicular and parallel samples is different in all three regions of WM whereas the relaxation behaviour is different for the CC and FO regions.An inverse modelling approach was adopted to extract Prony series parameters of the tissue components, i.e. axons and extra cellular matrix with its accessory cells, from experimental data. Using a bottom-up approach, we developed analytical and FEA estimates that are in good agreement with our experimental results.Our systematic characterisation of sheep brain WM using a combination of AFM experiments and micromechanical models provide a significant contribution for predicting localised time-dependent mechanics of brain tissue. This information can lead to more accurate computational simulations, therefore aiding the development of surgical robotic solutions for drug delivery and accurate tissue mimics, as well as the determination of criteria for tissue injury and predict brain development and disease progression.

Strategies for sample labelling and library preparation in DNA metabarcoding studies.

Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample‐specific nucleotide identifiers are added to the amplicons prior to sequencing. The latter enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and during “library preparation”, that is, when amplicons are prepared for sequencing. Different strategies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using metabarcoding, ensuring that data generation is robust and fit for the chosen purpose is critically important for practitioners seeking to employ metabarcoding for biodiversity assessments. Here, we present an overview of the three main workflows for sample‐specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms; one‐step PCR, two‐step PCR, and tagged PCR. Further, we distill the key considerations for researchers seeking to select an appropriate metabarcoding strategy for their specific study. Ultimately, by gaining insights into the consequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications.

A Parallelized Nanofluidic Device for High-Throughput Optical DNA Mapping of Bacterial Plasmids.

Optical DNA mapping (ODM) has developed into an important technique for DNA analysis, where single DNA molecules are sequence-specifically labeled and stretched, for example, in nanofluidic channels. We have developed an ODM assay to analyze bacterial plasmids—circular extrachromosomal DNA that often carry genes that make bacteria resistant to antibiotics. As for most techniques, the next important step is to increase throughput and automation. In this work, we designed and fabricated a nanofluidic device that, together with a simple automation routine, allows parallel analysis of up to 10 samples at the same time. Using plasmids encoding extended-spectrum beta-lactamases (ESBL), isolated from Escherichia coli and Klebsiella pneumoniae, we demonstrate the multiplexing capabilities of the device when it comes to both many samples in parallel and different resistance genes. As a final example, we combined the device with a novel protocol for rapid cultivation and extraction of plasmids from fecal samples collected from patients. This combined protocol will make it possible to analyze many patient samples in one device already on the day the sample is collected, which is an important step forward for the ODM analysis of plasmids in clinical diagnostics.

A cell-free high throughput assay for assessment of SARS-CoV-2 neutralizing antibodies

Highly accurate serological tests are key to assessing the prevalence of SARS-CoV-2 antibodies and the level of immunity in the population. This is important to predict the current and future status of the pandemic. With the recent emergence of new and more infectious SARS-CoV-2 variants, assays allowing for high throughput analysis of antibodies able to neutralize SARS-CoV-2 become even more important. Here, we report the development and validation of a robust, high throughput method, which enables the assessment of antibodies inhibiting the binding between the SARS-CoV-2 spike protein and angiotensin converting enzyme 2 (ACE2). The assay uses recombinantly produced spike-f and ACE2 and is performed in a bead array format, which allows analysis of up to 384 samples in parallel per instrument over seven hours, demanding only one hour of manual handling. The method is compared to a microneutralization assay utilising live SARS-CoV-2 and is shown to deliver highly correlating data. Further, a comparison with a serological method that measures all antibodies recognizing the spike protein shows that this type of assessment provides important insights into the neutralizing efficiency of the antibodies, especially for individuals with low antibody levels. This method can be an important and valuable tool for large-scale assessment of antibody-based neutralization, including neutralization of new spike variants that might emerge.

Efficient EM Optimization Exploiting Parallel Local Sampling Strategy and Bayesian Optimization for Microwave Applications

In electromagnetic (EM) optimization of microwave design, a computationally bad starting point usually leads the local optimization process to be stuck into local optimum, which does not satisfy the design specifications. In this situation, global optimization methods can be an alternative to achieve the final optimal solution. However, global optimization methods always suffer from a relatively low convergence rate. To address this problem, we propose an efficient EM optimization technique with a novel parallel local sampling strategy and Bayesian optimization (BO) for microwave applications in this article. We develop a new parallel local sampling strategy to increase the exploitation ability near the potential optimal solution in each optimization iteration and improve the convergence of the entire optimization process. The local sampling range in each iteration is determined based on the derivative information of the current potential optimal solution. While conventional BO only uses the information of potential optimal solutions in each iteration during optimization, we propose to exploit both the generated local samples and the potential optimal solutions together to build a surrogate model and guide the optimization. Therefore, the exploration and exploitation during the proposed EM optimization are well balanced, and the entire EM optimization process is effectively accelerated in comparison to other existing global methods. Examples of EM optimizations of microwave components are used to demonstrate the proposed technique.

Influence of Large-scale Field Structures on the Scaling Anisotropy in 3D MHD Turbulence

Abstract Magnetohydrodynamic (MHD) turbulence is revealed to have scaling anisotropy based on structure function calculations. Recent studies on solar wind turbulence found that the scaling anisotropy disappears when removing large-scale field structures. This finding raises questions as to whether numerical MHD turbulences have large-scale field structures. How do these structures affect the scaling anisotropy therein? Here we investigate these questions with a driven compressible three-dimensional MHD turbulence. We introduce a new method to check how the random stationarity condition is satisfied. We find for the first time in the numerical MHD turbulence that the large-scale field structures destroy the random stationarity of the local fields and make samplings nonparallel to the instantaneous fields be calculated as apparent parallel samplings. This mixture makes statistical calculations show anisotropic scaling of the turbulence. When we select only the random stationary data intervals, the statistical results show an isotropic nature. We also find that among the large-scale field structures, one-third are tangential discontinuities (TDs), one-third are rotational discontinuities (RDs), and the rest are EDs (either TD or RD). These results show that the large-scale structures in the numerical MHD turbulence have important influence on the structure function analysis.

Fast data-driven learning of parallel MRI sampling patterns for large scale problems

In this study, a fast data-driven optimization approach, named bias-accelerated subset selection (BASS), is proposed for learning efficacious sampling patterns (SPs) with the purpose of reducing scan time in large-dimensional parallel MRI. BASS is applicable when Cartesian fully-sampled k-space measurements of specific anatomy are available for training and the reconstruction method for undersampled measurements is specified; such information is used to define the efficacy of any SP for recovering the values at the non-sampled k-space points. BASS produces a sequence of SPs with the aim of finding one of a specified size with (near) optimal efficacy. BASS was tested with five reconstruction methods for parallel MRI based on low-rankness and sparsity that allow a free choice of the SP. Three datasets were used for testing, two of high-resolution brain images (text {T}_{2}-weighted images and, respectively, text {T}_{1rho }-weighted images) and another of knee images for quantitative mapping of the cartilage. The proposed approach has low computational cost and fast convergence; in the tested cases it obtained SPs up to 50 times faster than the currently best greedy approach. Reconstruction quality increased by up to 45% over that provided by variable density and Poisson disk SPs, for the same scan time. Optionally, the scan time can be nearly halved without loss of reconstruction quality. Quantitative MRI and prospective accelerated MRI results show improvements. Compared with greedy approaches, BASS rapidly learns effective SPs for various reconstruction methods, using larger SPs and larger datasets; enabling better selection of sampling-reconstruction pairs for specific MRI problems.

Characterization of Asbestos in Ambient Air During Refractory Material Production from Magnesite Ore

Asbestos is a general term employed for crystallized silicate minerals in fibril form. It can be commonly found in nature and from which fibre uses in the industry through processing are obtained. It is dielectric and fibrous, having an elastic feature. However, there is limited information about the air quality in terms of asbestos for the companies that produce refractory materials from magnesite ore. This deficiency in the literature can be eliminated with this study for a company with high quality and durable refractory materials. Among the most commonly used asbestos analysis are scanning electron microscope (SEM), transmission electron microscope (TEM). Asbestos analyses have been performed in two different institutions on the parallel samples whose preliminary processes have been completed. According to the results of the examinations conducted by SEM, 23 of the fibrils observed in a total area are bigger than five µmin lengths and less than 3 µm in width. Three of these fibrils were observed to have the characteristics of chrysotile fibres morphemically and elementally. In addition, airborne samples were analyzed using a TEM instrument. According to the results obtained, 13 of the fibrils observed in total were bigger than 5 µm in length and less than 3 µm in width and based on ED analysis and elemental composition (EDXA) results of these fibrils, and they were not observed to have the characteristics of chrysotile fibres. The majority of the fibrils observed were noted to be other inorganic minerals (lizardite) fibrils. Additionally, the obtained results show that the asbestos concentration in the air is below the limit specified by the provision (0,1 fiber/cm3).

Determination of Hydrogen Peroxide Migration Amount from Bamboo and Wooden Chopsticks to Food Simulants and Its Migration Law

Objective: Established a measurement method of hydrogen peroxide in different food simulants, studied the migration amount of hydrogen peroxide from bamboo and wooden chopsticks to different food simulants and its migration laws. Method: Used spectrophotometer to determine hydrogen peroxide, established a standard curve of hydrogen peroxide mass in different food simulants (water-based non-acid food simulators, water-based acid food simulators, alcohol food simulators, oil-based food simulators) and absorbance, used correlation coefficient, accuracy and precision as inspection indicators to verify the methodology, taking the type of the food simulants, time, frequency and temperature as investigating factors, the migration rule of hydrogen peroxide from bamboo and wooden chopsticks to the food simulants was initially explored. Result: The correlation coefficient of the obtained standard curve was greater than 0.999, the recovery rate of standard addition was 84%~113%, the maximum relative deviation (RSD) between spiked parallel samples were all lessed than 10%. The migration amount of hydrogen peroxide from bamboo and wood chopsticks to the oil-based simulant was smallest under the same conditions, the migration of hydrogen peroxide from bamboo and wooden chopsticks had a short rapid release process, and then converted to a long-term slow migration release, the migration was a long time repeated process, and there was still migration detected at the 8th time, as the temperature increased, the amount of migration increased significantly. Conclusion: Spectrophotometric determination of hydrogen peroxide amount in different food simulants was accurate and reliable, hydrogen peroxide migration were detected in different food simulants of some bamboo and wood chopsticks by this method, its migration law was a long time and repeated process, the migration rate could be significantly increased by increased the temperature, the remigration amount of hydrogen peroxide in bamboo and wood chopsticks could effectively lowered by boiled in water at 100 °C.

Desiccant drying prior to hermetic storage extends viability and reduces bruchid (Callosobruchus chinensis L.) infestation of mung bean (Vigna radiata (L.) R. Wilczek) seeds

Seeds of mung bean (Vigna radiata (L.) R. Wilczek) are subject to loss of viability due to aging and damage from pulse beetles (or bruchids; Callosobruchus spp.) infestation during storage. We investigated whether seed drying using desiccants and hermetic packaging would prevent or ameliorate these consequences of storage. Sun-dried mung bean seeds at a moisture content of 10% were subjected to further drying for 72 h using five different desiccants: Drying Beads® (a zeolite-based desiccant), silica gel, sodium aluminum silicate, activated alumina, and cow-dung ash (a traditional desiccant). Seeds were subsequently stored in hermetic plastic containers in the presence of these desiccants under ambient conditions along with sun-dried seeds stored in cloth bags or in hermetic containers. In addition, parallel samples of each treatment were inoculated with one pair of bruchid beetles (C. chinensis L.) and stored under the same conditions. The seed drying treatments did not affect initial seed quality (germination percentage and seedling vigor) significantly. After storage for 9 months at ambient temperatures, seeds dried using Drying Beads, silica gel, sodium aluminum silicate and activated alumina had higher germination percentages, seedling vigor indices and soil emergence, and lower electrical conductivity (leakage upon imbibition) and fungal infestation compared to other conditions. In addition, the mung bean seeds inoculated with bruchids and stored with these effective desiccants had less damage, oviposition, and insect respiratory activity in the hermetic containers and maintained higher seed germination and seedling vigor after six months of storage compared to other treatments and controls. The results demonstrate the superior ability of desiccants to quickly and safely dry seeds prior to and during storage and the benefits of such drying and hermetic storage conditions for preventing seed deterioration and insect damage during storage.

P13.22 Menglia: an innovative approach to liquid biopsies in glioma patients

Abstract BACKGROUND Gliomas are the second most frequent primary brain tumor. Since 2016 gliomas are classified according to histological, and molecular features such as IDH status and 1p19q codeletion. Recently oncological research has focused on liquid biopsies to reduce the need for invasive diagnostic tests. Cerebrospinal fluid (CSF) is the principal source of brain tumor biomarkers. Modern technologies currently allow us to analyze proteomic and metabolomic tumor profiles, in order to find new biomarkers without being constrained by a priori hypothesis. Several data from the literature also suggests that inflammatory cells and cytokines in tumors contribute to tumor growth, progression and immunosuppression. The possibility of simultaneously testing different inflammatory molecules on the biological fluids could lead to the identification of new biological markers with prognostic and predictive value for treatment response. STUDY OBJECTIVES - Identification of protein and/or lipid biomarkers specific to the glioma subgroup among those under analysis. - Identification of tumor group- and subgroup-specific CSF and/or serum inflammatory biomarkers among those tested. - Correlation of biomarkers with Progression Free Survival (PFS), Overall Survival (OS). ELIGIBILITY CRITERIA - Patients with a radiological suspected diagnosis of glioma eligible for surgery. - Age ≥ 18 years. - Availability of histological samples, CSF, blood and urine. - Patients with unconfirmed histological diagnosis of glioma will be excluded. - Patients who have never received any systemic or local treatment for CNS diseases. STUDY DESIGN This is a prospective monocentric study involving mass spectrometry analysis for characterization of proteins and metabolites and ELISA analysis for the characterization of different cytokines in parallel anonymized biological samples consisting of tumor tissue, CSF, blood and urine from glioma patients, divided into three different molecular subtypes: 1p19q codelet/IDH mutated, 1p19q non-codelet/IDH mutated and 1p19q non-codelet/IDH wt. Biological samples will be taken at surgery, at 24–48 h after surgery and every 3 months up to 1 year. The control group will include patients with subarachnoid hemorrhage or hydrocephalus, either undergoing external ventricular shunting or undergoing ventricular catheter placement/revision surgery. STUDY ANALYSIS Data interpretation will be based on a statistical analysis of the data, applying tools such as Principal Component Analysis (PCA) Weighted Correlation Network Analysis (WGNCA), Multiple Venn Diagram, T-Test, ANOVA, Clustering and Gene Ontology Enrichment, non-parametric Mann-Whitney test. For the assessment of clinical outcome, survival curves will be compared between different groups of patients who share a similar protein/metabolomic/inflammatory profile.

Comparison of parallel infill sampling criteria for optimization problems

The efficient global optimization (EGO) algorithm based on the Kriging surrogate model is popular. One of the main problems is to determine the infill sampling criteria. An adaptive distance function is proposed and applied to the EI, PI, and MP criteria. The criteria base on a fix distance is also investigated. Seven test problems were used to evaluate these criteria. The results show that the MMP criterion has poor global search ability. PEI, KB and DMP criteria perform better and have better convergence speed and stability.

Application of approach for development of hptlc identification and quantification methods for determination of phenolic compounds and terpenoids of several Thymus L. species

The aim of this work was to improve the approaches for standardization of Thymus L. species by development of HPTLC identification of phenolic compounds (flavonoids and phenylpropanoids) and terpenoids (essential oils and triterpenoids) and quantitative determination of rosmarinic acid and flavonoids for six Thymus L. species of Ukraine flora, as well as determination of chromatographic profiles of Thymus L. species’ extracts obtained using different extraction methods.&#x0D; Materials and methods. Objects were: samples of dry herb of Th.serpyllum, Th.vulgaris, Th.Marschallianus, Th.Pallasianus, Th.calcareus, and Th.moldavicus of Ukraine origin collected in 2019-2021 years or acquired commercially on Ukraine market. Instruments: CAMAG HPTLC Herbal System, visionCats 2.5. The analytical grade reagents were used. Reference substances were purchased from Extrasynthese, Sigma Aldrich. Chromatography was performed on HPTLC plates Si 60 F254, Merck according to the developed methods.&#x0D; Results. A new approach for quality control of different Thymus L. species of Ukraine flora included the development of HPTLC methods for identification of main groups of bioactive substances of these species, such as flavonoids and phenylpropanoids, essential oils, triterpenoids; development of quantification method of rosmarinic acid and assay of total flavonoids, expressed as luteolin-7-O-glucoside. The characteristic HPTLC fingerprints of six Thymus L. species in three mobile phases of different polarities that cover a wide range of bioactive substances were established. The content of rosmarinic acid in different Thymus L. species samples was in the range of 0,11-0,72 %: Th.moldavicus – 0,11 %; Th.Marschallianus – 0,19-0,27 %; Th.serpyllum – 0,38 %; Th.vulgaris – 0,51 %; Th.calcareus – 0,56 %; T.Pallasianus – 0,72 %. The total flavonoids content, expressed as luteolin-7-O-glucoside, was in the range of 0,8-2,72 %: Th.moldavicus – 0,8 %; Th.serpyllum – 0,87 %; Th.vulgaris – 1,06 %; Th.Pallasianus – 1,28 %; Th.Marschallianus – 1,89 %; Th.calcareus – 2,72 %.&#x0D; Conclusions. The proposed scientific approach for quality evaluation of Thymus L. species using HPTLC allows to determine comprehensive information of chemical composition and content of active substances of multiple samples in parallel, in a cost and time-efficient manner

Standardized high-throughput biomonitoring using DNA metabarcoding: Strategies for the adoption of automated liquid handlers

Reliable and comprehensive monitoring data are required to trace and counteract biodiversity loss. High-throughput metabarcoding using DNA extracted from community samples (bulk) or from water or sediment (environmental DNA) has revolutionized biomonitoring, given the capability to assess biodiversity across the tree of life rapidly with feasible effort and at a modest price. DNA metabarcoding can be upscaled to process hundreds of samples in parallel. However, while automated high-throughput analysis workflows are well-established in the medical sector, manual sample processing still predominates in biomonitoring laboratory workflows limiting the upscaling and standardization for routine monitoring applications. Here we present an automated, scalable, and reproducible metabarcoding workflow to extract DNA from bulk samples, perform PCR and library preparation on a liquid handler. Key features are the independent sample replication throughout the workflow and the use of many negative controls for quality assurance and quality control. We generated two datasets: i) a validation dataset consisting of 42 individual arthropod specimens of different species, and ii) a routine monitoring dataset consisting of 60 stream macroinvertebrate bulk samples. As a marker, we used the mitochondrial COI gene. Our results show that the developed single-deck workflow is free of laboratory-derived contamination and produces highly consistent results. Minor deviations between replicates are mostly due to stochastic differences for low abundant OTUs. Thus, we successfully demonstrated that robotic liquid handling can be used reliably from DNA extraction to final library preparation on a single deck, thereby substantially increasing throughput, reducing costs, and increasing data robustness for biodiversity assessments and monitoring.