Assessment of the Genetic Structure and Diversity of Soybean (Glycine max L.) Germplasm Using Diversity Array Technology and Single Nucleotide Polymorphism Markers

Knowledge of the genetic structure and diversity of germplasm collections is crucial for sustainable genetic improvement through hybridization programs and rapid adaptation to changing breeding objectives. The objective of this study was to determine the genetic diversity and population structure of 281 International Institute of Tropical Agriculture (IITA) soybean accessions using diversity array technology (DArT) and single nucleotide polymorphism (SNP) markers for the efficient utilization of these accessions. From the results, the SNP and DArT markers were well distributed across the 20 soybean chromosomes. The cluster and principal component analyses revealed the genetic diversity among the 281 accessions by grouping them into two stratifications, a grouping that was also evident from the population structure analysis, which divided the 281 accessions into two distinct groups. The analysis of molecular variance revealed that 97% and 98% of the genetic variances using SNP and DArT markers, respectively, were within the population. Genetic diversity indices such as Shannon’s diversity index, diversity and unbiased diversity revealed the diversity among the different populations of the soybean accessions. The SNP and DArT markers used provided similar information on the structure, diversity and polymorphism of the accessions, which indicates the applicability of the DArT marker in genetic diversity studies. Our study provides information about the genetic structure and diversity of the IITA soybean accessions that will allow for the efficient utilization of these accessions in soybean improvement programs, especially in Africa.

CSF-Neurofilament Light Chain Levels in NMDAR and LGI1 Encephalitis: A National Cohort Study

Background and ObjectivesThe two most common autoimmune encephalitides (AE), N-methyl-D-Aspartate receptor (NMDAR) and Leucine-rich Glioma-Inactivated 1 (LGI1) encephalitis, have been known for more than a decade. Nevertheless, no well-established biomarkers to guide treatment or estimate prognosis exist. Neurofilament light chain (NfL) has become an unspecific screening marker of axonal damage in CNS diseases, and has proven useful as a diagnostic and disease activity marker in neuroinflammatory diseases. Only limited reports on NfL in AE exist. We investigated NfL levels at diagnosis and follow-up in NMDAR and LGI1-AE patients, and evaluated the utility of CSF-NfL as a biomarker in AE.MethodsPatients were included from the National Danish AE cohort (2009-present) and diagnosed based upon autoantibody positivity and diagnostic consensus criteria. CSF-NfL was analyzed by single molecule array technology. Clinical and diagnostic information was retrospectively evaluated and related to NfL levels at baseline and follow-up. NMDAR-AE patients were subdivided into: idiopathic/teratoma associated or secondary NMDAR-AE (post-viral or concomitant with malignancies/demyelinating disease).ResultsA total of 74 CSF samples from 53 AE patients (37 NMDAR and 16 LGI1 positive) were included in the study. Longitudinal CSF-NfL levels was measured in 21 patients. Median follow-up time was 23.8 and 43.9 months for NMDAR and LGI1-AE respectively. Major findings of this study are: i) CSF-NfL levels were higher in LGI1-AE than in idiopathic/teratoma associated NMDAR-AE at diagnosis; ii) CSF-NfL levels in NMDAR-AE patients distinguished idiopathic/teratoma cases from cases with other underlying etiologies (post-viral or malignancies/demyelinating diseases) and iii) Elevated CSF-NfL at diagnosis seems to be associated with worse long-term disease outcomes in both NMDAR and LGI1-AE.DiscussionCSF-NfL measurement may be beneficial as a prognostic biomarker in NMDAR and LGI1-AE, and high CSF-NfL could foster search for underlying etiologies in NMDAR-AE. Further studies on larger cohorts, using standardized methods, are warranted.

Species monitoring through space and time — combining microfluidic SNP genotyping and environmental niche modelling on herbarium specimens of the rare Northern dragonhead, Dracocephalum ruyschiana (Lamiaceae)

Aim: We have studied population genetic change through time in the Northern dragonhead, Dracocephalum ruyschiana (Lamiaceae); a plant species that has experienced a drastic population decline and habitat loss in Europe. We aimed at adding a historic level to the monitoring of dragonhead by testing a microfluidic SNP array approach on herbarium specimens up to 200 years old and comparing the genomic results with that of modern populations in Norway. We also aimed to gain a more holistic species knowledge to guide monitoring efforts by combining herbarium genomics with ecological niche modelling (ENM). Location: Europe (mainly Norway) Methods: We have applied a microfluidic array consisting of 96 SNP markers on 130 herbarium specimens collected from 1820 to 2008. Obtained genotype data were compared with SNP data from modern samples using various population genetic analyses. We used sample metadata and observational records to model the species’ environmental niche. Results: The SNP array successfully genotyped all included herbarium specimens but was less capable of capturing diversity outside of Norway, which was genetically highly divergent from the Norwegian dragonheads. The historic-modern comparison revealed similar genetic structure in space and limited change through time in Norway. The ENM suggests that dragonhead has not fully achieved its potential distribution in Norway, which is anchored in warmer and drier regions, including areas where it does not occur today. Main conclusions: With the appropriate design procedures, the SNP array technology is promising for genotyping old herbarium specimens; an invaluable source of information from the past. We found no signs of the severe reduction in population size in our temporal genomic data of Norwegian dragonhead. Regardless, the regional populations in Norway are genetically divergent, both from each other and more so from populations outside of Norway, rendering continued protection of all existing populations of the species relevant.

Probing Multivalent Carbohydrate-Protein Interactions With On-Chip Synthesized Glycopeptides Using Different Functionalized Surfaces

Multivalent ligand–protein interactions are a commonly employed approach by nature in many biological processes. Single glycan–protein interactions are often weak, but their affinity and specificity can be drastically enhanced by engaging multiple binding sites. Microarray technology allows for quick, parallel screening of such interactions. Yet, current glycan microarray methodologies usually neglect defined multivalent presentation. Our laser-based array technology allows for a flexible, cost-efficient, and rapid in situ chemical synthesis of peptide scaffolds directly on functionalized glass slides. Using copper(I)-catalyzed azide–alkyne cycloaddition, different monomer sugar azides were attached to the scaffolds, resulting in spatially defined multivalent glycopeptides on the solid support. Studying their interaction with several different lectins showed that not only the spatially defined sugar presentation, but also the surface functionalization and wettability, as well as accessibility and flexibility, play an essential role in such interactions. Therefore, different commercially available functionalized glass slides were equipped with a polyethylene glycol (PEG) linker to demonstrate its effect on glycan–lectin interactions. Moreover, different monomer sugar azides with and without an additional PEG-spacer were attached to the peptide scaffold to increase flexibility and thereby improve binding affinity. A variety of fluorescently labeled lectins were probed, indicating that different lectin–glycan pairs require different surface functionalization and spacers for enhanced binding. This approach allows for rapid screening and evaluation of spacing-, density-, ligand and surface-dependent parameters, to find optimal lectin binders.

Interlaboratory collaboration to determine the performance of the Randox food diagnostics biochip array technology for the simultaneous quantitative detection of seven mycotoxins in feed

An inter-laboratory collaborative study was performed to evaluate the performance of the Biochip Array Technology (BAT) Myco 7 method. The Myco 7 Array is a method which simultaneously and quantitatively detects 20 mycotoxins including aflatoxins B1, B2, G1 and G2, ochratoxin A, deoxynivalenol, zearalenone, fumonisin B1, B2 and B3 and T-2 and HT-2 toxin. The BAT Myco 7 method was collaboratively evaluated by nine government and private Association of American Feed Control Officials (AAFCO) laboratories. Samples were analysed in a proficiency testing round format. Seventeen blind samples were analysed on the same equipment using Myco 7 kits. 99% of the results fell within an acceptable Z-score range of -2|<Z<|+2. Deoxynivalenol had a 100% Z-score pass rate, while a 99% pass was recorded for aflatoxins, zearalenone, ochratoxin A and fumonisins. T-2 toxin had a 97% Z-score pass rate. HorRat analysis for reproducibility used a range of 0.3<|HorRat|≤2. The target was met for deoxynivalenol, zearalenone, T-2 and HT-2 toxin, and aflatoxins B1, B2, G1 and G2 assays. Fumonisins and ochratoxin A assays had a 93% and 94% pass, respectively. The reproducibility co-efficiency of variation was between 16 and 20% meeting set criterion of <40% and is, therefore, fit-for-purpose for use in the AAFCO control programs for mycotoxins.

Presentation of cytokine profile in relation to oxidative stress parameters in patients with severe COVID-19: a case-control pilot study

Introduction: COVID-19 can be worsened by hyper-production of cytokines accompanied by increased level of oxidative stress. The aim of this study was to investigate the correlation between a set of cytokines and the markers of the oxidative stress. Methods: The levels of cytokines IL-2, IL-4, IL-6, IL8, IL-10, VEGF, IFN-γ, TNF-α, IL-1α, MCP-1 and EGF were determined by using High Sensitivity Evidence Investigator™ Biochip Array technology. The oxidative stress parameters (d-ROM, PAT, OS index) were measured in serum on FRAS5 analytical photometric system. Results: IL-6, IL-8, IL-10, VEGF, MCP-1 and EGF were significantly higher (p<0.05) in the patients with severe COVID-19 with increased levels of IL-2, IFN-y, TNF-α and IL-1α. The d-ROM, OS index, and PAT were significantly higher (p<0.05) in severe COVID-19 patients. IL-6 demonstrated the strongest correlation with all of the markers of the oxidative stress, d-ROM (r=0.9725, p=0.0001), PAT (r=0.5000, p=0.0001) and OS index (r=0.9593, p=0.012). Similar behavior was evidenced between IFN-y and d-ROM (r=0.4006, p=0.0001), PAT (r=0.6030, p=0.0001) and OS index (r=0.4298, p=0.012). Conclusion: The oxidative stress markers show good correlation with the tested cytokines which can be measured at the beginning of the disease in a primary care setting to predict the course of COVID-19.

Quantifying SARS-CoV-2 nucleocapsid antigen in oropharyngeal swabs using single molecule array technology

This study aimed to develop a highly sensitive SARS-CoV-2 nucleocapsid antigen assay using the single molecule array (Simoa) technology and compare it with real time RT-PCR as used in routine clinical practice with the ambition to achieve a comparative technical and clinical sensitivity. Samples were available from 148 SARS-CoV-2 real time RT-PCR positive and 73 SARS-CoV-2 real time RT-PCR negative oropharyngeal swabs. For determination of technical sensitivity SARS-CoV-2 virus culture material was used. The samples were treated with lysis buffer and analyzed using both an in-house and a pre-commercial SARS-CoV-2 nucleocapsid antigen assay on Simoa. Both nucleocapsid antigen assays have a technical sensitivity corresponding to around 100 SARS-CoV-2 RNA molecules/mL. Using a cut-off at 0.1 pg/mL the pre-commercial SARS-CoV-2 nucleocapsid antigen assay had a sensitivity of 96% (95% CI 91.4–98.5%) and specificity of 100% (95% CI 95.1–100%). In comparison the in-house nucleocapsid antigen assay had sensitivity of 95% (95% CI 89.3–98.1%) and a specificity of 100% (95% CI 95.1–100%) using a cut-off at 0.01 pg/mL. The two SARS-CoV-2 nucleocapsid antigen assays correlated with r = 0.91 (P < 0.0001). The in-house and the pre-commercial SARS-CoV-2 nucleocapsid antigen assay demonstrated technical and clinical sensitivity comparable to real-time RT-PCR methods for identifying SARS-CoV-2 infected patients and thus can be used clinically as well as serve as a reference method for antigen Point of Care Testing.