Laboratory Capabilities Research Articles

One Health surveillance-A cross-sectoral detection, characterization, and notification of foodborne pathogens.

Several Proficiency Test (PT) or External Quality Assessment (EQA) schemes are currently available for assessing the ability of laboratories to detect and characterize enteropathogenic bacteria, but they are usually targeting one sector, covering either public health, food safety or animal health. In addition to sector-specific PTs/EQAs for detection, cross-sectoral panels would be useful for assessment of the capacity to detect and characterize foodborne pathogens in a One Health (OH) perspective and further improving food safety and interpretation of cross-sectoral surveillance data. The aims of the study were to assess the cross-sectoral capability of European public health, animal health and food safety laboratories to detect, characterize and notify findings of the foodborne pathogens Campylobacter spp., Salmonella spp. and Yersinia enterocolitica, and to develop recommendations for future cross-sectoral PTs and EQAs within OH. The PT/EQA scheme developed within this study consisted of a test panel of five samples, designed to represent a theoretical outbreak scenario. A total of 15 laboratories from animal health, public health and food safety sectors were enrolled in eight countries: Denmark, France, Italy, the Netherlands, Poland, Spain, Sweden, and the United Kingdom. The laboratories analyzed the samples according to the methods used in the laboratory and reported the target organisms at species level, and if applicable, serovar for Salmonella and bioserotype for Yersinia. All 15 laboratories analyzed the samples for Salmonella, 13 for Campylobacter and 11 for Yersinia. Analytical errors were predominately false negative results. One sample (S. Stockholm and Y. enterocolitica O:3/BT4) with lower concentrations of target organisms was especially challenging, resulting in six out of seven false negative results. These findings were associated with laboratories using smaller sample sizes and not using enrichment methods. Detection of Salmonella was most commonly mandatory to notify within the three sectors in the eight countries participating in the pilot whereas findings of Campylobacter and Y. enterocolitica were notifiable from human samples, but less commonly from animal and food samples. The results of the pilot PT/EQA conducted in this study confirmed the possibility to apply a cross-sectoral approach for assessment of the joint OH capacity to detect and characterize foodborne pathogens.

Overview of the SARS-CoV-2 genotypes circulating in Latin America during 2021.

Latin America is one of the regions in which the COVID-19 pandemic has a stronger impact, with more than 72 million reported infections and 1.6 million deaths until June 2022. Since this region is ecologically diverse and is affected by enormous social inequalities, efforts to identify genomic patterns of the circulating SARS-CoV-2 genotypes are necessary for the suitable management of the pandemic. To contribute to the genomic surveillance of the SARS-CoV-2 in Latin America, we extended the number of SARS-CoV-2 genomes available from the region by sequencing and analyzing the viral genome from COVID-19 patients from seven countries (Argentina, Brazil, Costa Rica, Colombia, Mexico, Bolivia, and Peru). Subsequently, we analyzed the genomes circulating mainly during 2021 including records from GISAID database from Latin America. A total of 1,534 genome sequences were generated from seven countries, demonstrating the laboratory and bioinformatics capabilities for genomic surveillance of pathogens that have been developed locally. For Latin America, patterns regarding several variants associated with multiple re-introductions, a relatively low percentage of sequenced samples, as well as an increment in the mutation frequency since the beginning of the pandemic, are in line with worldwide data. Besides, some variants of concern (VOC) and variants of interest (VOI) such as Gamma, Mu and Lambda, and at least 83 other lineages have predominated locally with a country-specific enrichments. This work has contributed to the understanding of the dynamics of the pandemic in Latin America as part of the local and international efforts to achieve timely genomic surveillance of SARS-CoV-2.

Bridging the gap: strengthening testing capabilities of national medicines quality control laboratories in West Africa

Laboratory testing to assure the quality and safety of medicines is a critical component of public health. Across Africa, national regulatory authorities are challenged by a lack of sufficient capabilities within their national quality control laboratories (NQCLs) to perform analyses in a consistent, reliable, and accurate manner. The United States Agency for International Development founded-program, Promoting the Quality of Medicines and its successor Promoting the Quality of Medicines Plus, both implemented by United States Pharmacopeia, have worked to strengthen the testing capabilities of NQCLs in African countries. Their technical assistance aligned NQCLs’ operational practices and testing services with international standards and best practices and resulted in third-party recognition of capabilities and competence. This commentary provides a retrospective of key achievements of NQCLs in three West African countries, Ghana, Mali, and Nigeria, and illustrates success in addressing challenges through a complex multiyear venture. The commitment and dedication of NQCLs and their NRAs has resulted in international levels of compliance and competency.

Advancing biomechanics laboratories capabilities: A proposed framework for in-house technology development

BackgroundTechnological advancements have generated more opportunities to develop/distribute custom data analysis codes (e.g., automated events, biomechanical models, etc.). Industry standards for the code development process is regularly modeled to ensure product quality and usability. Procedural project management improves efficiency of the code development process by monitoring project planning, duration, analysis, success, and maintenance. The purpose of this study was to outline in the form of guidance to research labs, a framework that standardizes the development, management, testing, and documentation of various types of data analysis codes, utilized in the motion analysis laboratory setting. MethodsThis brief report outlines the workflow, briefly highlights its success a year after implementation, and provides a framework that can be adopted across laboratories of different sizes and those involved in multi-center collaborative studies. Specifically, the workflow outlined is initiated when a requestor has identified the need for a custom data analysis code. The workflow is complete and the code is released once the results of testing performed by a non-affiliated user, verifies that the code project workflow was followed appropriately, confirms a standard operating procedure has been finalized, and ensures the requestor and additional end-users are satisfied with the final product. FindingsGuidance documents and optimization of workflows are imperative for motion analysis laboratories managing numerous coding projects. InterpretationImplementation of the proposed framework is an effective approach to reduce workload, by minimizing redundancies, maximizing on the research team's expertise and promotes collaborative input which in turn allows for feedback along the process.

Fabrication, evaluation methodologies and models of slow-release fertilizers: A review

The development of slow-release fertilizers (SRFs) has attracted increasing attention, especially in the last two decades since the well-recognized environmental problem caused by overusing fertilizers. Different kinds of SRFs have been developed, including chemical or physical reactions, loading into the holders with porous and layer structures, coating, and holding by hydrogels, etc. Various techniques have been reviewed at different development stages and from different angles. Meanwhile, various methodologies have been developed and used to evaluate the release performances of different kinds of SRFs. However, the reported results are significantly different from the different evaluation methodologies. In this work, we have firstly briefly reviewed the development of SRFs according to the fabrication classification: (1) chemical synthesizing, (2) loading, (3) coating, (4) application of biodegradable polymers, and 5) hydrogel-based systems, then focused on reviewing different evaluation methodologies and their mechanisms. Various models used to describe and predicate release behaviors were also discussed. Generally, there are four kinds of evaluation methodologies: (1) immersing in water, (2) leaching by soil, (3) planting in greenhouse, and (4) yield comparison. It was found that most of the traditional synthesized products and coated fertilizers by conventional polymers are reasonably stable in water, so the method of immersing in water is suitable. Still, most of the water-sensitive coating or mixture-based products need to be evaluated through leaching by soil. Both greenhouse and yield comparison are simple and reliable methods and has been widely used for final evaluation and confirmation, but the weakness of time issue and unpredictable weather conditions limit their application during the initial developing step. It was found that there is no direct relationship between different methodologies since their releasing mechanisms are different even though the releasing trend is similar. Various evaluation methodologies need to be modified according to different products and adjusted according to laboratory capability (measurement techniques). The methods of element detection and environmental issues were also discussed. Various models used to describe and predicate releasing behaviors were reviewed in this paper. This paper also summarized the standards of SRFs from different countries. It was pointed out that the nutrient delivery to plants by actual controlled-release fertilizers should closely match the growing demand of the plants. A sustained supply of nutrients for the entire crop growth in a single dose (fertilizing) can save labor and handling costs. Furthermore, it is understandable that different plants grow favorably in different soil and conditions, so the fertilizers should also be evaluated in different environments and situations. The evaluation method should closely match plant growth requirements, such as rice in the water while corn is in solid soil.

APMP/TCRI key comparison report of measurement of air kerma for medium-energy x-rays (APMP.RI(I)-K3.2013)

Main textThe APMP/TCRI Dosimetry Working Group initiated the APMP.RI(I)-K3.2013 key comparison of measurement of air kerma for medium-energy x-rays (100 kV to 250 kV); measurements took place between 2015 and 2017. In total, 11 institutes took part in the comparison, among which 8 were APMP member laboratories, two from AFRIMETS and one from SIM. Three commercial cavity ionization chambers were used as transfer standards circulated among the participants. All the participants established 100, 135, 180 and 250 kV x-ray beam qualities equivalent to those of the BIPM. The ARPANSA and the NMIJ were linking laboratories and two participants subsequently made comparisons with the BIPM. The 7 other participants' degrees of equivalence fell within 11 parts in 103 of unity, well within the relevant expanded uncertainty (k = 2) in all cases. Excluding the BARC, the results were within 5.7 parts in 103, well within the relevant combined standard uncertainty. These results indicated that the calibration capabilities of most laboratories were in very good agreement and there were no serious discrepancies between them. The effect of implementing the recommendations of ICRU Report 90, which occurred on 1 January 2018 (after this comparison), is discussed and we deduce that the effect on the degrees of equivalence is negligible. The increase in the uncertainty in Wair has been added to all laboratories so that the degrees of equivalence and the uncertainties published here are applicable to post-ICRU 90 measurement results.To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database https://www.bipm.org/kcdb/.The final report has been peer-reviewed and approved for publication by the CCRI, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Centers for Disease Control and Prevention Participation in Cobalt Magnet National-Level Radiological Exercise.

Since September 11, 2001, the Centers for Disease Control and Prevention (CDC) has increased efforts to prepare the agency and public health partners for response to potential nuclear/radiological disasters. During the week of May 16-20, 2022, the CDC participated in a national-level radiological emergency exercise, Cobalt Magnet 22 (CM22). The exercise scenario consisted of a notional, failed search mission for a radiological dispersal device (RDD, "dirty bomb"), followed by its explosion during a public event in a large US city. Testing radioanalytical laboratory capabilities during a nuclear/radiological incident was an exercise objective, and developing clear messaging on low-dose exposure and long-term health concerns was a primary output of the exercise. The CDC practiced its activation protocols, exercised the establishment of its updated Incident Management System structure for radiation emergencies, and identified critical staffing needs for this type of response.

Antimicrobial stewardship program: Fighting antimicrobial resistance in India

Antimicrobial resistance (AMR) is a significant concern in India, contributing to increased medical costs, prolonged hospital stays, and mortality. Antimicrobial stewardship programs (AMSP) are organizational strategies in health-care systems designed to promote, monitor, and evaluate the rational use of antimicrobials to preserve their future effectiveness. The Indian council of medical research recognizes the need for AMS structures in healthcare institutions and has initiated actions to support this. These include developing an AMS curriculum, conducting workshops for increased awareness and education, and expanding surveillance of antimicrobial usage and resistance trends. However, many hospitals in India still lack structured processes for AMS, requiring a multidisciplinary approach involving various experts. AMSP contribute significantly to improving antibiotic use without compromising patient outcomes, such as optimal selection of agents, correct dosing, appropriate administration routes, and proper therapy duration. The aim is to enhance patient care and outcomes, reduce infection complications, and minimize the emergence of AMR. Key challenges faced in implementing AMS programs in India include lack of infrastructure and personnel, limited resources and funds, limited laboratory capability, absence of antibiotic prescription policy, irrational prescription and excessive use of broad-spectrum antibiotics, self-medication, inappropriate disposal of leftover antibiotics, lack of regulation, and treatment non-compliance. These challenges necessitate robust, collaborative, and multifaceted responses, including policy reform, investment in infrastructure development, expanded surveillance, improved professional practices, and public education.

MODERN ASPECTS OF DIAGNOSTICS OF AUTOIMMUNE DISEASES OF THE THYROID GLAND

Objective.This research ismeant to study the features of the clinical course and to developanoptimal algorithm for the diagnosis of autoimmune thyroiddiseases. Material and methods.The work is based on the examination and treatment data of 481 patients with autoimmune diseases of the thyroid gland, treated in the clinic.Diagnosis and treatment results of 481 patients were analyzed to develop an optimal algorithm for diagnosingautoimmune thyroiditis.The differential diagnostic capabilities of clinical, laboratory, and morphological examination methods of patients withautoimmune thyroiddiseases have been specified.The study of long-term results of treatment was carried out on 340patients, taking into account the various methods of treatment they underwent. Results.The analysis of existing diagnostic tools and methods allowed us to develop an optimal algorithm for diagnosingautoimmune thyroiddiseases, which is a complex of clinical, laboratory, and morphological methods that can reliably verify the diagnosis of autoimmune thyroiditis. Based on the examination results, it is possible to predict the likelihood of surgical treatment and to identify a group of patients in whom autoimmune processes can progress in the thyroid residue, contributing to the development of postoperative recurrence of the disease or causing its atrophy. Conclusion. Based on the study’s results, a rational algorithm for diagnostic search has been developed. The proposed algorithm allows, in the shortest possible time, to identify the presence of a form of autoimmune thyroiddisease and to determine the optimal tactics to treat patients withautoimmune thyroiditis based on clinical, laboratory, immunological tests and instrumental examinations.

Towards a robust test to detect gene doping for anabolic enhancement in human athletes.

Success in gene therapy in treating human disease makes this technology attractive to enhance athletic performance, creating the need for gene doping detection. In 2021, World Anti-Doping Agency (WADA) approved the first gene doping test. Here, we describe a new method to detect doping with four additional genes, follistatin, growth hormone 1, growth hormone-releasing hormone and insulin-like growth factor 1, that may improve performance by increasing muscle size and strength. The method utilises four hydrolysis probe-based polymerase chain reaction (PCR) assays that target the transgenes based on the coding sequence of the four endogenous genes. The assays are specific, reproducible and capable to detect five copies of transgene in the presence of very similar endogenous gene in 25,000 times excess. To underpin reliable and comparable routine method performance by doping testing laboratories, a synthetic reference material for the method was designed and generated following the ISO Guide 35. The complete method was validated in blood samples using plasma as extraction matrix and QIAamp DNA blood midi DNA extraction kit. All blood samples from different donors (n = 8) simulated to be negative or positive (1500 transgene copies spiked per millilitre of blood) for the transgenes were reported correctly. The new method that targets four additional genes will extend the capabilities of laboratories involved in doping control to protect athletes' health, fairness and equality.

Application of a Cholesterol-Based Stationary Phase for the Analysis of Brevetoxins.

A high-performance liquid chromatography protocol for the analysis of brevetoxins has been developed using a silica hydride-based cholesterol column. Brevetoxins are neurotoxins produced by harmful algae that have additional potential as drugs for a number of illnesses/diseases. To develop the optimum conditions, a number of different experimental approaches were tested. These include isocratic and gradient elution, different organic mobile phase components, and temperature variations. A separate protocol was developed for the compounds brevenal and brevenol, also produced by the same algae that make brevetoxins. Brevenal is a natural product under investigation as a therapy for chronic respiratory diseases, such as cystic fibrosis or asthma. The goal of this study was to provide a protocol for the analysis of these compounds that could be further developed into a validated method depending on a particular laboratory's capabilities and to highlight some of the unique features of the cholesterol stationary phase.

Comparing Data Generated Using Multiparametric Flow Cytometry, Capillary Electrophoresis, and Next-Generation Sequencing in a Cohort of B-Cell Lymphoproliferative Disorder Samples

Background: Determining whether suspected lymphoid proliferation disorder (LPD) samples are benign or malignant remains a significant challenge for the hematopathologist. For the vast majority of LPDs, the current gold standard includes assessment of clinical characteristics, cytomorphology, and immunophenotyping by flow cytometry; however, 10-15% of LPDs require further resolution by molecular methods, such as PCR-based capillary electrophoresis or next-generation sequencing, to rule out clonality based on V-D-J rearrangements in the immunoglobulin heavy-chain (IGH) gene. Invivoscribe (San Diego, CA) has developed multiple complementary methodologies to evaluate LPDs; including multiparametric flow cytometry (MFC), capillary electrophoresis (CE), and next-generation sequencing (NGS). To compare performance between these three methods, a cohort of 21 clinical samples were tested in a pilot study. Method: The CAP/CLIA Hematolymphoid Screening assay, a 10-color flow panel, was used to identify all major types of hematopoietic neoplasia according to the 2006 Bethesda Consensus and 2017 WHO Classification. For molecular testing, DNA was extracted from peripheral blood (PB), then PCR-based assays were used to determine presence or absence of clonality. The Research Use Only (RUO) molecular assays were designed for CE (ABI 3500xL) and NGS (MiSeqTM) detection, utilizing framework multiplex PCR master mixes targeting the conserved framework regions FR1, FR2, or FR3 within the VH and the JH regions of the IGH gene. Twenty-one (21) de-identified PB samples were collected from informed consensus donors (LabPMM, San Diego, CA) or from residual samples (Lahey Hospital, Burlington, MA) and tested under an IRB approved protocol. An aliquot of each PB sample was used for MFC evaluation, then the remaining DNA was extracted and tested using the CE and NGS methods. Test results were reported as "present abnormal" or "normal" for MFC and "clonal" or "non-clonal" for molecular assays based on pre-defined criteria. Results: The MFC assay identified 12 out of 21 (57%) samples as "present abnormal" and 9 out of 21 (43%) samples as "normal." Both molecular assays (CE and NGS) reported 12 out of 21 (57%) samples as "clonal" and 9 out of 21 (43%) samples as "non-clonal." The results between CE and NGS assays were 100% in agreement for clonality detection. Comparisons between the CE and MFC assays again resulted in 100% agreement; all 12 "present abnormal" samples by MFC were reported as "clonal" by CE, while all 9 "normal" samples by MFC were "non-clonal" by CE. In addition to presence of clonality, the NGS method was able to detect the prevalence of a specific clonal V-D-J rearrangement sequence (expressed as % reads), which ranged from 10% to 99% in the 12 clonal samples. This pilot study demonstrated excellent concordance (21/21) between these 3 methods (MFC, CE and NGS). Accordingly, MFC, CE and NGS are all suitable methods for the evaluation and stratification of B-cell LPD samples. The combined use of all 3 methods provides a comprehensive assessment of clonality: including surface markers identified by MFC, DNA rearrangement lengths from CE, and the specific DNA rearrangement sequences from NGS. In addition, the NGS method allows for tracking of these clonal sequences in relation to disease status. Conclusion: This pilot study demonstrated 100% concordance between 3 different methods (MFC, CE, and NGS) when evaluating samples from LPD subjects. Depending on laboratory capabilities and the type of information desired, any one of these technologies can provide appropriate results when studying clonality. The alignment between MFC and NGS allows the combined use of both methods to provide rapid diagnosis via the MFC service and identification of specific biomarkers for subsequent tracking of these clonal populations in clinical studies.

Pre-lab video demonstrations to enhance students' laboratory experience in a first-year chemical engineering class.

The limited capabilities of teaching laboratories, combined with an increasing number of students enrolled in university, require constant augmentation of instructional approaches. By enhancing laboratory demonstrations with digital technology, these structural issues can be addressed while at the same time enhancing student understanding and learning. Our case study focuses on the fermentation lab part of the Reaction Equilibria and Thermodynamics (RET) module, a first-year chemical engineering course at the University of Birmingham. Video demonstrations were used to introduce students to the laboratory set-ups and walk them through each step and technique. The video demonstrations allowed the students to attend the in-person lab sessions having established knowledge and understanding of the processes involved and the outcomes desired, which decreased the burden on the facilities and the staff. A knowledge-based quiz and a student survey conducted at the end of the module showed that the pre-lab videos encouraged more active participation in the laboratory sessions and reinforced learning. Approximately 70% of the students polled in the first survey conducted within this project felt more confident going into the laboratory sessions after watching the pre-lab videos and attempting the knowledge quiz, while 92% of the students polled in the second survey judged the pre-lab video sessions as beneficial to them. Overall, the teaching method has the potential to improve student participation and access, boost confidence and learning, and provided a more structured and flexible approach to laboratory learning outcomes.

A standardised low-cost membrane blood-feeder for Aedes aegypti made using common laboratory materials.

Blood feeding is a necessary part of laboratory studies involving mosquitoes and other hematophagous arthropods of interest in medical and ecological research. However, methods involving hosts may present serious risks, require ethics approvals and can be expensive. Here we describe an insect blood feeder made using common laboratory materials, which is low cost (<US$100) and can be constructed and operated with little technical expertise. We compared the blood feeder containing an artificial blood diet, Skitosnack, to direct human arm feeding for Aedes aegypti (Diptera: Culicidae), in terms of engorgement rate, fecundity and hatch rate. No significant difference in fecundity between the two approaches was found, (mean±SD); direct human arm: 56±26 eggs/female, artificial method: 47±25 eggs/female, P=0.569. Engorgement rates (direct human arm: 97.8±4%, artificial: 64.1±23%, P<0.05) and hatch rates (direct human arm: 75±12%, artificial: 59±14%, P<0.05) were lower in the artificially fed mosquitoes. Despite these differences, we maintained a healthy mosquito colony for 10 generations using the artificial feeding approach. Results from this comparison are within the range of other studies which compared direct host feeding with an artificial feeding method. We anticipate that the blood feeder presented here could substantially reduce costs usually required to establish a standardised and effective blood feeding method for maintaining mosquito colonies or conducting experiments, extending the capability of laboratories especially where research resources are limited, but vector-borne diseases common.

A calibration standard for low-frequency environmental magnetic field meters

In this paper, we present the establishment of a magnetic flux density standard in the national calibration laboratory in Croatia. A single-layer Helmholtz coil was constructed as a magnetic field source at frequencies from 25 Hz to 100 kHz to calibrate magnetic field meters up to field limits for occupational exposure. First, the calibration error margin was determined for probes up to 100 cm2 of the cross-sectional area with respect to the predefined calibration volume inside the coil. Next, the equivalent electrical model of the assembled standard was parameterized as a multiple resonance circuit to estimate the correction of the reference field with frequency. Finally, the calculated correction was experimentally verified using a single-turn induction loop with an amplifier. The resulting uncertainty budget of the standard confirms the laboratory's capability to calibrate environmental magnetometers with the best relative uncertainty of 0.8 %.

Global Antimicrobial Resistance and Use Surveillance System on the African continent: Early implementation 2017-2019.

BackgroundAntimicrobial resistance (AMR) is becoming a critical public health issue globally. The World Health Organization launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS) to support the strengthening of the AMR evidence base.ObjectiveThe article describes the evolution of national AMR surveillance systems and AMR data reporting of countries in the African continent between 2017 and 2019, and the constraints, perceived impact and value of the participation in GLASS.MethodsData on implementation of national surveillance systems and AMR rates were submitted to GLASS between 2017 and 2019 and summarised though descriptive statistics. The information on constraints and perceived impact and value in GLASS participation was collected though a set of questionnaires.ResultsBetween 2017 and 2019, Egypt, Ethiopia, Madagascar, Malawi, Mali, Mozambique, Nigeria, South Africa, Sudan, Tunisia, Uganda and Zambia submitted data to GLASS. The main constraints listed are linked to scarce laboratory capacity and capability, limited staffing, budget issues, and data management. Moreover, while the data are not yet nationally representative, high resistance rates were reported to commonly-used antibiotics, as the emerging resistance to last treatment options.ConclusionDespite the limitations, more and more countries in the African continent are working towards reaching a status that will enable them to report AMR data in a complete and systematic manner. Future improvements involve the expansion of routine surveillance capacity for several countries and the implementation of surveys that allow to effectively define the magnitude of AMR in the continent.

Calibration of image plate and back illuminated charge coupled device detectors at the thermal emission band of high Z target laser produced plasmas (80–800 eV)

We present a systematic method to absolutely calibrate detector efficiency vs photon energy using a laser produced plasma broadband x-ray source, a gold standard calibrated detector, and transmission gratings (TGs) as dispersive elements. Calibration uses one calibrated TG and a calibrated gold standard detector on one channel and a second calibrated TG and a detector to be calibrated on the other channel. Both channels simultaneously view the laser-produced plasma x-ray source from the same angle with respect to the laser beam and the planar target normal. Image plate detectors are calibrated for the first time at photon energies below 700 eV. Single shot simultaneous calibration of several detectors is possible, making this method an efficient and practical way to periodically calibrate detectors, using in-house capabilities of laser laboratories.

Seeds harvested during mowing from semi-natural grasslands as an ad hoc but effective solution for grassland restoration.

Species-rich grasslands formed by local ecotypes of native species provide numerous ecosystem services both in rural areas as well as urban grasslands. Nonetheless, their area is still too small, making grasslands one of the most frequently restored habitats. Successful restoration requires high-quality seed material, which is expensive and often not easy to acquire. In this study, we tested the potential of seeds accidentally collected during the mowing of a semi-natural grassland for grassland restoration. We tested seed purity, species composition, and germination capability in both laboratory and field conditions. Ninety percent of the collected material consisted of pure seeds of numerous species. Their germination capability was relatively low but still sufficient for successful grassland restoration under a typical seed density/mass per unit area seeding ratio. The germination capacity was the highest in the first two weeks after sowing and increased with overwintering seed storage. The results suggested that the seeds could be successfully used for species-rich grassland restoration. In terms of advantages, the seed mixture had a low cost and contained native species seeds representing local ecotypes. In terms of disadvantages, there was a relatively low amount of seed material and an inability to plan the time of seed harvesting. Thus, the use of the accidentally collected seeds can be considered an effective but rather ad hoc solution.

Gold nanomaterials and their potential use as cryo-electron tomography labels

Rapid advances in cryo-electron tomography (cryo-ET) are driving a revolution in cellular structural biology. However, unambiguous identification of specific biomolecules within cellular tomograms remains challenging. Overcoming this obstacle and reliably identifying targets in the crowded cellular environment is of major importance for the understanding of cellular function and is a pre-requisite for high-resolution structural analysis. The use of highly-specific, readily visualised and adjustable labels would help mitigate this issue, improving both data quality and sample throughput. While progress has been made in cryo-CLEM and in the development of cloneable high-density tags, technical issues persist and a robust ‘cryo-GFP’ remains elusive. Readily-synthesized gold nanomaterials conjugated to small ‘affinity modules’ may represent a solution. The synthesis of materials including gold nanoclusters (AuNCs) and gold nanoparticles (AuNPs) is increasingly well understood and is now within the capabilities of non-specialist laboratories. The remarkable chemical and photophysical properties of <3nm diameter nanomaterials and their emergence as tools with widespread biomedical application presents significant opportunities to the cryo-microscopy community. In this review, we will outline developments in the synthesis, functionalisation and labelling uses of both AuNPs and AuNCs in cryo-ET, while discussing their potential as multi-modal probes for cryo-CLEM.

Rapid Visual Detection of Plasmodium Using Recombinase-Aided Amplification With Lateral Flow Dipstick Assay.

BackgroundMalaria is a global public health problem. China has had no case of indigenous malaria since 2016. However, imported cases of malaria remain an issue among travelers, overseas workers, and foreign traders. Although these cases are always asymptomatic, if they donate blood, there is a great risk of transfusion transmitted-malaria (TTM). Therefore, blood banks need a rapid screening tool to detect Plasmodium species.MethodsWe designed an assay using recombinase-aided amplification (RAA) and a lateral-flow dipstick (LFD) (RAA-LFD) to detect the 18S ribosomal RNA gene of Plasmodium species. Sensitivity was evaluated using a recombinant plasmid and Plasmodium genomic DNA. Specificity was evaluated using DNA extracted from the blood of patients with malaria or other infectious parasites. For clinical assessment, blood samples from patients with malaria and blood donors were evaluated.ResultsThe RAA-LFD assay was performed in an incubator block at 37°C for 15 min, and the amplicons were visible to the naked eye on the flow dipsticks within 3 min. The sensitivity was 1 copy/μL of recombinant plasmid. For genomic DNA from whole blood of malaria patients infected with P. falciparum, P. vivax, P. ovale, and P. malariae, the sensitivity was 0.1 pg/μL, 10 pg/μL, 10-100 pg/μL, and 100pg/μL, respectively. The sensitivity of this assay was 100pg/μL. No cross-reaction with other transfusion-transmissible parasites was detected.ConclusionsThe results demonstrated that this RAA-LFD assay was suitable for reliable field detection of Plasmodium species in low-resource settings with limited laboratory capabilities.