Artificial Samples Research Articles

A novel way to evaluate autoantibody interference in samples with mixed antinuclear antibody patterns in the HEp-2 cell based indirect immunofluorescence assay and comparison of conventional microscopic and computer-aided pattern recognition

BackgroundA major challenge of the HEp-2 cell-based indirect immunofluorescence (IIF) assays is the correct identification of the individual anti-nuclear antibodies (ANAs) if more than one is present in a sample. We created artificial mixes by pooling two different samples with a single autoantibody in different titers. Comparison of the expected and observed patterns and titers clarifies the interference between the two tested ANAs. MethodsSerum samples with a single homogeneous or speckled ANA pattern were serially diluted and mixed in 16 combinations, providing end-point titers of 1:5,120 to 1:80 for both patterns. These mixes were tested by a HEp-2 IIF assay and were evaluated by conventional evaluation, the EUROPattern (EPa) system and on-screen analysis. ResultsHomogeneous pattern can alter the identification of the speckled pattern much more than vice versa, but both has an interfering effect on the other. The effect of the interfering on the tested pattern was higher if the titer of the former one was higher. The pattern recognition efficacy of conventional and the on-screen evaluation was similar and superior compared to the EPa analysis. ConclusionsThe application of artificial mixed samples can help the evaluation of the efficacy of manual and computer-aided ANA HEp-2 pattern recognition.

Adsorption and detection tramadol in aqueous solution by fluorescent- magnetic molecularly imprinting polymers

BackgroundThe usage of tramadol (TR) as one of the most common analgesics is expansively growing in the world. Therefore, there is an urgent requirement for a rapid and reliable method for the on-site detection of tramadol in different samples. The elimination half-life of tramadol is about 6 h, and its removal from body is relatively slow. So, it is very critical providing a method for rapid detection of tramadol. MethodsIn this study, three magnetic nanocomposites (Fe3O4, Fe3O4/SiO2, and Fe3O4/SiO2/CMC) and molecularly imprinted polymers (MIPs) based on those in the presence of graphene quantum dots (GQDs) were synthesized and investigated for selective adsorption and detection of TR in aqueous solution. For the characterization of synthesized materials, XRD, FT-IR, SEM, VSM, and the response surface method (RSM) were also employed to optimize and estimate the effective parameters of TR adsorption by these materials. Significant findingsThe results of investigating the absorption ability of nanocomposites showed that the Fe3O4/SiO2/CMC composite has higher absorption ability than the other two nanocomposites. Moreover, Fe3O4/SiO2/CMC has an excellent ability to adsorb of TR with a removal percentage of 97.46 % at pH = 7, initial TR concentration = 15 mg/L, composite dosage = 1.5 g/L, and contact time = 6 min. According to the experimental results, the imprinting factors (IF) of MIP-Fe3O4, MIP-Fe3O4/SiO2, and MIP-Fe3O4/SiO2/CMC are 1.02, 1.72, and 2.19, respectively. Therefore, the MIP-Fe3O4/SiO2/CMC was chosen as a selective sensor, and the sensitivity of its response was investigated. The detection of TR was performed by fluorescence spectroscopy at a concentration range of TR of 0 to 250 µM, and a sensitivity of 3.13 a.u./µM and limit of detection of 2.08 µM were determined. Also, the river water and artificial urine samples (with TR of 10 µM) was detected using MIP-Fe3O4/SiO2/CMC, and the results of the sensor showed a TR concentration of river water is 9.97 µM and artificial urine is 10.456 µM and which is very good and acceptable precision.

Analysis of influencing factors of aircraft fuel tank flammability

PurposeIt is an indispensable part of airworthiness certification to evaluate the fuel tank flammability exposure time for transport aircraft. There are many factors and complex coupling relationships affecting the fuel tank flammability exposure time. The current work not only lacks a comprehensive analysis of these factors but also lacks the significance of each factor, the interaction relationship and the prediction method of flammability exposure time. The lack of research in these aspects seriously restricts the smooth development of the airworthiness forensics work of domestic large aircraft. This paper aims to clarify the internal relationship between user input parameters and predict the flammability exposure time of fuel tanks for transport aircraft.Design/methodology/approachBased on the requirements of airworthiness certification for large aircraft, an in-depth analysis of the Monte Carlo flammability evaluation source procedures specified in China Civil Aviation Regulation/FAR25 airworthiness regulations was made, the internal relationship between factors affecting the fuel tank flammability exposure time was clarified and the significant effects and interactions of input parameters in the Monte Carlo evaluation model were studied using the response surface method. And the BP artificial neural network training samples with high significance factors were used to establish the prediction model of flammability exposure time.FindingsThe input parameters in the Monte Carlo program directly or indirectly affect the fuel tank flammability exposure time by means of the influence on the flammability limit or fuel temperature. Among the factors affecting flammability exposure time, the cruising Mach number, balance temperature difference and maximum range are the most significant, and they are all positively correlated with flammability exposure time. Although there are interactions among all factors, the degree of influence on flammability exposure time is not the same. The interaction between maximum range and equilibrium temperature difference is more significant than other factors. The prediction model of flammability exposure time based on multifactor interaction and BP neural network has good accuracy and can be applied to the prediction of fuel tank flammability exposure time.Originality/valueThe flammability exposure time prediction model was established based on multifactor interaction and BP neural network. The limited test results were combined with intelligent algorithm to achieve rapid prediction, which saved the test cost and time.

Chemical Nose Strategy with Metabolic Labeling and "Antibiotic-Responsive Spectrum" Enables Accurate and Rapid Pathogen Identification.

The worldwide antimicrobial resistance (AMR) dilemma urgently requires rapid and accurate pathogen phenotype discrimination and antibiotic resistance identification. The conventional protocols are either time-consuming or depend on expensive instrumentations. Herein, we demonstrate a metabolic-labeling-assisted chemical nose strategy for phenotyping classification and antibiotic resistance identification of pathogens based on the "antibiotic-responsive spectrum" of different pathogens. d-Amino acids with click handles were metabolically incorporated into the cell wall of pathogens for further clicking with dibenzocyclooctyne-functionalized upconversion nanoparticles (DBCO-UCNPs) in the presence/absence of six types of antibiotics, which generates seven-channel sensing responses. With the assistance of machine learning algorithms, eight types of pathogens, including three types of antibiotic-resistant bacteria, can be well classified and discriminated in terms of microbial taxonomies, Gram phenotypes, and antibiotic resistance. The present metabolic-labeling-assisted strategy exhibits good anti-interference capability and improved discrimination ability rooted in the unique sensing mechanism. Sensitive identification of pathogens with 100% accuracy from artificial urinary tract infection samples at a concentration as low as 105 CFU/mL was achieved. Pathogens outside of the training set can also be discriminated well. This clearly demonstrated the potential of the present strategy in the identification of unknown pathogens in clinical samples.

Capillary electrophoresis with capacitively coupled contactless conductivity detection (C4 D) for rapid and simple determination of lactate in sweat.

An analytical method based on capillary electrophoresis (CE) using capacitively coupled contactless conductivity detection (C4 D) was developed and validated for fast, straightforward, and reliable determination of lactate in artificial and human sweat samples. The background electrolyte was composed of equimolar concentrations (10mmol/L) of 2-(N-morpholino)ethanesulfonic acid and histidine, with 0.2mmol/L of cetyltrimethylammonium bromide as electroosmotic flow inverter. The limit of detection and quantification were 3.1 and 10.3µmol/L, respectively. Recoveries in the 97 to 118% range were obtained using sweat samples spiked with lactate at three concentration levels, indicating an acceptable accuracy. The intraday and interday precisions were 1.49 and 7.08%, respectively. The proposed CE-C4 D method can be a starting point for monitoring lactate concentrations in sweat samples for diagnostics, physiological studies, and sports performance assessment applications.

Label-Free, Impedance-Based Biosensor for Kidney Disease Biomarker Uromodulin.

We demonstrate the development of a label-free, impedance-based biosensor by using a passivation layer of 50-nm tantalum pentoxide (Ta2O5) on interdigitated electrodes (IDE). This layer was fabricated by atomic layer deposition (ALD) and has a high dielectric constant (high-κ), which improves the capacitive property of the IDE. We validate the biosensor's performance by measuring uromodulin, a urine biomarker for kidney tubular damage, from artificial urine samples. The passivation layer is functionalized with uromodulin antibodies for selective binding. The passivated IDE enables the non-faradaic impedance measurement of uromodulin concentrations with a measurement range from 0.5 ng/mL to 8 ng/mL and with a relative change in impedance of 15 % per ng/mL at a frequency of 150 Hz (log scale). This work presents a concept for point-of-care biosensing applications for disease biomarkers.

Microplastic Removal from Road Stormwater Runoff using Lab-scale Bioretention Cell

Microplastic removal from stormwater runoff from roads is necessary to reduce the effect of microplastic pollution in water bodies. Bioretention is a potential technology to remove microplastics in stormwater runoff from roads. A lab-scale experiment was conducted to determine the efficiency, effect on vegetation and discharge variation, and the kinetics of microplastic removal from stormwater runoff from roads using a bioretention cell. The experiment was done using an artificial sample based on visual characterization of stormwater runoff from highways, commercial, and residential roads. The vegetations that were examined were Vetivera sp. and Hibiscus sp. The operational discharge was varied based on rainfall intensity categories. The result showed that the removal efficiency was in the range of 92.4 to 99.3% with a mean of 97.2%. Statistical analysis (α = 5%) showed that variation in vegetation and discharge had no significant effect on microplastic removal using bioretention. The first-order kinetic analysis showed that the kinetic removal constant of the bioretention with Vetivera sp., bioretention with Hibiscus sp., and bioretention without vegetation was 0.0356, 0.034, and 0.0327, respectively. These results indicate that bioretention with Hibiscus sp. removed more microplastics at greater depths than with Vetivera sp.

Reconstruction of private genomes through reference-based genotype imputation

BackgroundGenotype imputation is an essential step in genetic studies to improve data quality and statistical power. Public imputation servers are widely used by researchers to impute their data using otherwise access-controlled reference panels of high-fidelity genomes held by these servers.ResultsWe report evidence against the prevailing assumption that providing access to panels only indirectly via imputation servers poses a negligible privacy risk to individuals in the panels. To this end, we present algorithmic strategies for adaptively constructing artificial input samples and interpreting their imputation results that lead to the accurate reconstruction of reference panel haplotypes. We illustrate this possibility on three reference panels of real genomes for a range of imputation tools and output settings. Moreover, we demonstrate that reconstructed haplotypes from the same individual could be linked via their genetic relatives using our Bayesian linking algorithm, which allows a substantial portion of the individual’s diploid genome to be reassembled. We also provide population genetic estimates of the proportion of a panel that could be linked when an adversary holds a varying number of genomes from the same population.ConclusionsOur results show that genomes in imputation server reference panels can be vulnerable to reconstruction, implying that additional safeguards may need to be considered. We suggest possible mitigation measures based on our findings. Our work illustrates the value of adversarial algorithms in uncovering new privacy risks to help inform the genomics community towards secure data sharing practices.

A Study on Behaviour of Clayey and Silty Soils with Granite Dust

A massive amount of granite dust is produced as a byproduct of processing granite rock. Disposing of granite dust is a fatal to human health and is a environmental toxic for land disposal. An attempt was made to use such a material in construction, decrease the disposing problems. Soil, an important material bearing the load of the resisting structure can be available in various layers beneath the earth. Clayey soil and mixture of silt in soil layers exhibits inadequate load bearing ability and shows significant changes in volume with respect to changes in moisture levels. Stabilization of soil is the process that improves the behavior of soil properties. The study investigates the percentage of granite dust that can be a best stabilizer to silty and clayey soil. A series of laboratory tests were conducted to investigate the effects of granite dust. Artificial soil sample were prepared with 0%, 12%, 16%, 20%and 24% with additive as granite dust on silty and clayey soils. Chemical composition of the granite dust was studied for the application of granite dust as a stabilizer. Comprehensive soil tests like liquid limit, plastic limit, Maximum dry density, swell index, shear strength, CBR were performed. A significant decrease was observed in liquid and plastic limit varying with granite dust for both the soils. The dry density increases with an increase in % of granite dust. Swell index shows a decrease rate with increase in granite dust. The shear strength and CBR values gave appropriate increase conclude that granite dust can acts good soil stabilizer for clayey and silty soils.

Methods of applied utilization of generative adversarial networks in graphic data processing

The paper explores an important area of artificial intelligence — Generative Adversarial Networks (GANs), which are used to create high-quality artificial data samples. GANs have undergone significant development and application in various sectors, including the processing of graphical data. The report focuses on the practical use of GANs and their architecture. It discusses the fundamental principles of GAN operation, highlights the advantages and disadvantages, including issues with training, vanishing gradients, and convergence oscillations, and describes measures to overcome these problems. It also examines current research in the field of GANs and their applications in various domains, including cybersecurity, medicine, forensics, and computer vision. Practical results from the report's authors regarding their own GAN experiments, optimization, and architecture improvements are presented. The research aims to analyze the architectural features of GANs to enhance their training process

Exploiting second-order advantage for simultaneous biosensing of romidepsin and vorinostat in the presence of belinostat as uncalibrated interference in human serum samples

In this work, a novel, ultra-sensitive, selective, and multi-electrochemical techniques biosensor was fabricated for simultaneous determination of romidepsin (RD) and vorinostat (VN) in the presence of belinostat (BS) as uncalibrated interference in human serum samples. A glassy carbon electrode (GCE) was modified with chitosan-ionic liquid (CS-IL) which was used as a platform to immobilize histone deacetylase (HDAC) by the use of glutaraldehyde. The RD and VN as inhibitors of the HDAC were trapped by the HDAC onto the biosensor surface which didn’t show any response at the surface of the CS-IL/GCE. In order to simultaneous biosensing of RD and VN, the biosensor was immersed into a probe solution to get a response from the biosensor. By individual immersion of the biosensor into RD and VN solutions different steric hindrances were occurred at the biosensor surface which caused generation of two different responses from the biosensor. The biosensor responses were individually calibrated and used to develop second-order calibration models to support the biosensor for simultaneous determination of RD and VN. Second-order hydrodynamic square wave voltammetric (HSWV) data were generated and modeled by PARAFAC2, MCR-ALS, PARASIAS, and U-PCA/RBL to find the best method to couple with outputs of the biosensor. The results confirmed the best performance for the biosensor- U-PCA/RBL for simultaneous determination of RD and VN in the presence of BS in artificial human serum samples. The biosensor-U-PCA/RBL combinatorial method was also successful in simultaneous determination of RD and VN in the presence of BS in real human serum samples with a comparable performance with HPLC as the reference method.

Electrocatalytic aptasensor for bacterial detection exploiting ferricyanide reduction by methylene blue on mixed PEG/aptamer monolayers

Pathogen-triggered infections are the most severe global threat to human health, and to provide their timely treatment and prevention, robust methods for rapid and reliable identification of pathogenic microorganisms are required. Here, we have developed a fast and inexpensive electrocatalytic aptamer assay enabling specific and ultrasensitive detection of E. coli. E. coli, a biomarker of environmental contamination and infections, was captured on the mixed aptamer/thiolated PEG self-assembled monolayers formed on electrochemically pre-treated gold screen-printed electrodes (SPE). Signals from aptamer - E. coli binding were amplified by electrocatalytic reduction of ferricyanide mediated by methylene blue (MB) adsorbed on bacterial and aptamer surfaces. PEG operated as an antifouling agent and inhibited direct (not MB-mediated) discharge of ferricyanide. The assay allowed from 10 to 1000 CFU mL−1E. coli detection in 30 min, with no interference from B. subtilis, in buffer and artificial urine samples. This electrocatalytic approach is fast, specific, sensitive, and can be used directly in in-field and point-of-care applications for analysis of bacteria in human environment.

Ultrahighly Sensitive and Selective Glutathione Sensor Based on Carbon Dot-Functionalized Solution-Gate Graphene Transistor.

A new type of carbon dot (CD)-functionalized solution-gated graphene transistor (SGGT) sensor was designed and fabricated for the highly sensitive and highly selective detection of glutathione (GSH). The CDs were synthesized via a one-step hydrothermal method using DL-thioctic acid and triethylenetetramine (TETA) as sources of S, N, and C. The CDs have abundant amino and carboxyl groups and were used to modify the surface of the gate electrode of SGGT as probes for detecting GSH. Remarkably, the CDs-SGGT sensor exhibited excellent selectivity and ultrahigh sensitivity to GSH, with an ultralow limit of detection (LOD) of up to 10-19 M. To the best of our knowledge, the sensor outperforms previously reported systems. Moreover, the CDs-SGGT sensor shows rapid detection and good stability. More importantly, the detection of GSH in artificial serum samples was successfully demonstrated.

Wrinkled MXene-modified screen-printed electrodes for highly efficient sarcosine detection

MXenes are gaining immense attention owing to their robust conductivity, presence of favorable surface-functional groups and excellent hydrophilicity. Herein, a simple solution-based approach was adopted to functionalize few-layer thick MXene-Ti3C2Tx with chitosan (Chi) molecules, creating a stable electro-active screen-printed electrode (SPE) platform for the detection of sarcosine (SAR), a new biomarker for prostate cancer. In comparison to pristine MXene, the chitosan-functionalized Ti3C2Tx (MX-Chi/SPE) offered favorable surface functionalization for easy immobilization of sarcosine oxidase (SOx) enzymes, which led to the improved enzymatic oxidation of SAR. The detection mechanism relied upon the efficient reduction of enzymatically produced H2O2 over MX-Chi/SPE, enabling selective detection of SAR both in low (0.1–0.3 µM) and high (1.5–3.5 µM) concentration ranges, with an estimated limit of detection (LOD) values of 0.012 µM and 0.14 µM (S/N = 3), respectively. Due to its robust enzymatic configuration, the biosensor enabled selective detection of SAR even in the presence of interfering species like ascorbic acid, uric acid, and glucose. Furthermore, the biosensor demonstrated excellent signal repeatability and stability and effectively recovered spiked SAR levels (98–106%) from artificial urine samples. This approach demonstrated the successful integration of MXenes as redox-active materials in clinical biosensors with a simple electrode preparation procedure, which has the potential to be extended to other MXene compositions.

Development and performance of a next generation sequencing (NGS) assay for monitoring of dd-cfDNA post solid organ transplantation

The aim of this study was to evaluate the analytical performance of a novel NGS assay, intended for monitoring of donor-derived cell-free DNA (dd-cfDNA), and describe its validity in clinical plasma samples from kidney transplanted patients. Artificial and clinical samples with increasing amounts of patient DNA were evaluated using NGS analysis of indel markers. Monitoring of dd-cfDNA with the NGS assay presented herein demonstrated a sensitivity of ≥0.1% dd-cfDNA and excellent accuracy (R2 0.99) throughout an extensive range of dd-cfDNA (0.1–30%). The precision of the test was determined for two levels (0.1% (LoD) and 1%) of dd-cfDNA. The between run precision (CV%) for the respective level was 16% and 9% and the corresponding result for the within run precision was 19% and 7%. To evaluate performance of the assay in clinical samples, 507 retrospective monitoring samples from 21 patients transplanted either with kidneys from living or deceased donors were analyzed. Monitoring samples were sampled at multiple time points from 24 h up to 90 days post-transplantation. We show that in one patient, increase of dd-cfDNA preceded increase of creatinine caused by acute cellular rejection by several days. In conclusion, the NGS assay displayed a combination of high sensitivity with good accuracy and precision in both artificial and clinical dd-cfDNA samples.

Visualization and characterization of experimental hydraulic fractures interacting with karst fracture-cavity distributions

Karst fracture-cavity carbonate reservoirs, in which natural cavities are connected by natural fractures to form cavity clusters in many circumstances, have become significant fields of oil and gas exploration and exploitation. Proppant fracturing is considered as the best method for exploiting carbonate reservoirs; however, previous studies primarily focused on the effects of individual types of geological formations, such as natural fractures or cavities, on fracture propagation. In this study, true-triaxial physical simulation experiments were systematically performed under four types of stress difference conditions after the accurate prefabrication of four types of different fracture-cavity distributions in artificial samples. Subsequently, the interaction mechanism between the hydraulic fractures and fracture-cavity structures was systematically analyzed in combination with the stress distribution, cross-sectional morphology of the main propagation path, and three-dimensional visualization of the overall fracture network. It was found that the propagation of hydraulic fractures near the cavity was inhibited by the stress concentration surrounding the cavity. In contrast, a natural fracture with a smaller approach angle (0° and 30°) around the cavity can alleviate the stress concentration and significantly facilitate the connection with the cavity. In addition, the hydraulic fracture crossed the natural fracture at the 45° approach angle and bypassed the cavity under higher stress difference conditions. A new stimulation effectiveness evaluation index was established based on the stimulated reservoir area (SRA), tortuosity of the hydraulic fractures (T), and connectivity index (CI) of the cavities. These findings provide new insights into the fracturing design of carbonate reservoirs.

Simultaneous preconcentration of 9Be and cosmogenic 10Be for determination of the 10Be/9Be ratio in (coastal) seawater

AbstractBeryllium isotopes have emerged as a quantitative tracer of continental weathering, but accurate and precise determination of the cosmogenic 10Be and stable 9Be in seawater is challenging, because seawater contains high concentrations of matrix elements but extremely low concentrations of 9Be and 10Be. In this study, we develop a new, time‐efficient procedure for the simultaneous preconcentration of 9Be and 10Be from (coastal) seawater based on the iron co‐precipitation method. The concentrations of 9Be, 10Be, and the resulting 10Be/9Be ratio for Changjiang Estuary water derived from the new procedure agree well with those obtained from the conventional procedure requiring separate preconcentration for 9Be and 10Be determinations. By avoiding the separate preconcentration, our newly developed procedure contributes toward more time‐efficient handling of samples, less sample cross‐contamination, and a more reliable 10Be/9Be ratio. Prior to this, we validated the iron co‐precipitation method using artificial seawater and natural water samples from the Amazon Estuary regarding: (1) the “matrix effect” for Be analysis, (2) its extraction efficiency for pg g−1 levels Be in the presence and absence of organic matter, and (3) the data comparability with another preconcentration method. We calculated that for the determination of 9Be and 10Be in most open ocean seawater with typical 10Be concentrations of > 500 atoms g−1, good precisions (< 5%) can be achieved using less than 3 liters of seawater compared to more than 20 liters routinely used previously. Even for coastal seawater with extremely low 10Be concentration (e.g., 100 atoms g−1), we estimate a maximum amount of 10 liters to be adequate.

The Effect of Ficin Immobilized on Carboxymethyl Chitosan on Biofilms of Oral Pathogens.

In the last decade, Ficin, a proteolytic enzyme extracted from the latex sap of the wild fig tree, has been widely investigated as a promising tool for the treatment of microbial biofilms, wound healing, and oral care. Here we report the antibiofilm properties of the enzyme immobilized on soluble carboxymethyl chitosan (CMCh) and CMCh itself. Ficin was immobilized on CMCh with molecular weights of either 200, 350 or 600 kDa. Among them, the carrier with a molecular weight of 200 kDa bound the maximum amount of enzyme, binding up to 49% of the total protein compared to 19-32% of the total protein bound to other CMChs. Treatment with pure CMCh led to the destruction of biofilms formed by Streptococcus salivarius, Streptococcus gordonii, Streptococcus mutans, and Candida albicans, while no apparent effect on Staphylococcus aureus was observed. A soluble Ficin was less efficient in the destruction of the biofilms formed by Streptococcus sobrinus and S. gordonii. By contrast, treatment with CMCh200-immobilized Ficin led to a significant reduction of the biofilms of the primary colonizers S. gordonii and S. mutans. In model biofilms obtained by the inoculation of swabs from teeth of healthy volunteers, the destruction of the biofilm by both soluble and immobilized Ficin was observed, although the degree of the destruction varied between artificial plaque samples. Nevertheless, combined treatment of oral Streptococci biofilm by enzyme and chlorhexidine for 3 h led to a significant decrease in the viability of biofilm-embedded cells, compared to solely chlorhexidine application. This suggests that the use of either soluble or immobilized Ficin would allow decreasing the amount and/or concentration of the antiseptics required for oral care or improving the efficiency of oral cavity sanitization.

The Applicability of Chromatographic Retention Modeling on Chiral Stationary Phases in Reverse-Phase Mode: A Case Study for Ezetimibe and Its Impurities.

Mechanistic modeling is useful for predicting and modulating selectivity even in early chromatographic method development. This approach is also in accordance with current analytical quality using design principles and is highly welcomed by the authorities. The aim of this study was to investigate the separation behavior of two different types of chiral stationary phases (CSPs) for the separation of ezetimibe and its related substances using the mechanistic retention modeling approach offered by the Drylab software (version 4.5) package. Based on the obtained results, both CSPs presented with chemoselectivity towards the impurities of ezetimibe. The cyclodextrin-based CSP displayed a higher separation capacity and was able to separate seven related substances from the active pharmaceutical ingredient, while the cellulose-based column enabled the baseline resolution of six impurities from ezetimibe. Generally, the accuracy of predicted retention times was lower for the polysaccharide CSP, which could indicate the presence of additional secondary interactions between the analytes and the CSP. It was also demonstrated that the combination of mechanistic modeling and an experimental design approach can be applied to method development on CSPs in reverse-phase mode. The applicability of the methods was tested on spiked artificial placebo samples, while intraday and long-term (2 years) method repeatability was also challenged through comparing the obtained retention times and resolution values. The results indicated the excellent robustness of the selected setpoints. Overall, our findings indicate that the chiral columns could offer orthogonal selectivity to traditional reverse-phase columns for the separation of structurally similar compounds.

Diversity and distribution of benthic dinoflagellates in Tonga include the potentially harmful genera Gambierdiscus and Fukuyoa

Benthic dinoflagellates that can cause illness, such as ciguatera poisoning (CP), are prevalent around the Pacific but are poorly described in many locations. This study represents the first ecological assessment of benthic harmful algae species in the Kingdom of Tonga, a country where CP occurs regularly. Surveys were conducted in June 2016 in the Tongatapu island group, and in June 2017 across three island groups: Ha'apai, Vava'u, and Tongatapu. Shallow subtidal coastal habitats were investigated by measuring water quality parameters and conducting quadrat surveys. Microalgae samples were collected using either macrophyte collection or the artificial substrate method. Benthic dinoflagellates (Gambierdiscus and/or Fukuyoa, Ostreopsis, and Prorocentrum) were counted using light microscopy, followed by molecular analyses (real-time PCR in 2016 and high throughput sequencing (metabarcoding) in 2017) to identify Gambierdiscus and Fukuyoa to species level. Six species were detected from the Tongatapu island group in 2016 (G. australes, G. carpenteri, G. honu, G. pacificus, F. paulensis, and F. ruetzleri) using real-time PCR. Using the metabarcoding approach in 2017, a total of eight species (G. australes, G. carpenteri, G. honu, G. pacificus, G. cheloniae, G. lewisii, G. polynesiensis, and F. yasumotoi) were detected. Species were detected in mixed assemblages of up to six species, with G. pacificus and G. carpenteri being the most frequently observed. Ha'apai had the highest diversity with eight species detected, which identifies this area as a Gambierdiscus diversity ‘hotspot’. Vava'u and Tongatapu had three and six species found respectively. Gambierdiscus polynesiensis, a described ciguatoxin producer and proposed causative agent of CP was found only in Ha'apai and Vava'u in 2017, but not in Tongatapu in either year. Ostreopsis spp. and Prorocentrum spp. were also frequently observed, with Prorocentrum most abundant at the majority of sites. In 2016, the highest number of Gambierdiscus and/or Fukuyoa cells were observed on seagrass (Halodule uninervis) from Sopu, Tongatapu. In 2017, the highest numbers of Gambierdiscus and/or Fukuyoa from artificial substrate samples were recorded in the Halimeda dominant habitat at Neiafu Tahi, Vava'u, a low energy site. This raised the question of the effect of wave motion or currents on abundance measurements from artificial substrates. Differences in detection were noticed between macrophytes and artificial substrates, with higher numbers of species found on artificial substrates. This study provides a baseline of benthic dinoflagellate distributions and diversity for Tonga that may be used for future studies and the development of monitoring programmes.