Protocol standardization and sharing are crucial for reproducibility in life sciences. In spite of numerous efforts for standardized protocol description, adherence to these standards in literature ...remains largely inconsistent. Curation of protocols are especially challenging due to the labor intensive process, requiring expert domain knowledge of each experimental procedure. Recent advancements in Large Language Models (LLMs) offer a promising solution to interpret and curate knowledge from complex scientific literature. In this work, we develop ProtoCode, a tool leveraging fine-tune LLMs to curate protocols into intermediate representation formats which can be interpretable by both human and machine interfaces. Our proof-of-concept, focused on polymerase chain reaction (PCR) protocols, retrieves information from PCR protocols at an accuracy ranging 69–100 % depending on the information content. In all tested protocols, we demonstrate that ProtoCode successfully converts literature-based protocols into correct operational files for multiple thermal cycler systems. In conclusion, ProtoCode can alleviate labor intensive curation and standardization of life science protocols to enhance research reproducibility by providing a reliable, automated means to process and standardize protocols. ProtoCode is freely available as a web server at https://curation.taxila.io/ProtoCode/.
The research in extracellular vesicles (EVs) has been rising during the last decade. However, there is no clear consensus on the most accurate protocol to isolate and analyze them. Besides, most of ...the current protocols are difficult to implement in a hospital setting due to being very time-consuming or to requirements of specific infrastructure. Thus, our aim is to compare five different protocols (comprising two different medium-speed differential centrifugation protocols; commercially polymeric precipitation - exoquick - acid precipitation; and ultracentrifugation) for blood and urine samples to determine the most suitable one for the isolation of EVs. Nanoparticle tracking analysis, flow cytometry, western blot (WB), electronic microscopy, and spectrophotometry were used to characterize basic aspects of EVs such as concentration, size distribution, cell-origin and transmembrane markers, and RNA concentration. The highest EV concentrations were obtained using the exoquick protocol, followed by both differential centrifugation protocols, while the ultracentrifugation and acid-precipitation protocols yielded considerably lower EV concentrations. The five protocols isolated EVs of similar characteristics regarding markers and RNA concentration; however, standard protocol recovered only small EVs. EV isolated with exoquick presented difficult to be analyzed with WB. The RNA concentrations obtained from urine-derived EVs were similar to those obtained from blood-derived ones, despite the urine EV concentration being 10-20 times lower. We consider that a medium-speed differential centrifugation could be suitable to be applied in a hospital setting as it requires the simplest infrastructure and recovers higher concentration of EV than standard protocol. A workflow from sampling to characterization of EVs is proposed.
The species composition of fish burgers - declared as composed by European seabass (Dicentrarchus labrax) - collected in the context of a seafood company self-control was assessed using ...metabarcoding. Positive/negative controls, replicates, samples processed in dirty laboratory environment were also included as quality measures. A ≈ 200 bp of the 16S rRNA gene was selected as molecular target. The sequencing was performed on Illumina platform, and the data were analysed using DADA2 R package. The species taxonomic assignment was performed using Blastn against GenBank (identity value ≥ 99 %). The sequences assigned to D. labrax were highly predominant in all the products, with percentages ≥ 99.34 %, except for one, where also a high number of sequences assigned to Atlantic salmon (Salmo salar) were found (12.41 %). Sequences identified with other species (seafood, mammals, insects) were in percentage ≤ 0.57 %, and in the 14 % of the cases they even did not achieve the 0.001 %. A threshold value of 3.3 % to remove false positives was fixed based on the results of the positive controls. Overall, metabarcoding was proved as effective technique to assess the ingredients contained in complex seafood products. However, further investigation including a higher sample number and inter-laboratory validation should be performed to validate the procedure.
•The species composition of fish burgers was assessed using metabarcoding.•A number of experimental controls was included to assess the process quality.•A threshold value to adjust the data respect to DNA contamination was established.•All the products except for one were compliant respect to the declared ingredient.•Standard Operating Procedures should be defined to harmonize protocols.
Hyperpolarized (HP) 129Xe MRI uniquely images pulmonary ventilation, gas exchange, and terminal airway morphology rapidly and safely, providing novel information not possible using conventional ...imaging modalities or pulmonary function tests. As such, there is mounting interest in expanding the use of biomarkers derived from HP 129Xe MRI as outcome measures in multi‐site clinical trials across a range of pulmonary disorders. Until recently, HP 129Xe MRI techniques have been developed largely independently at a limited number of academic centers, without harmonizing acquisition strategies. To promote uniformity and adoption of HP 129Xe MRI more widely in translational research, multi‐site trials, and ultimately clinical practice, this position paper from the 129Xe MRI Clinical Trials Consortium (https://cpir.cchmc.org/XeMRICTC) recommends standard protocols to harmonize methods for image acquisition in HP 129Xe MRI. Recommendations are described for the most common HP gas MRI techniques—calibration, ventilation, alveolar‐airspace size, and gas exchange—across MRI scanner manufacturers most used for this application. Moreover, recommendations are described for 129Xe dose volumes and breath‐hold standardization to further foster consistency of imaging studies. The intention is that sites with HP 129Xe MRI capabilities can readily implement these methods to obtain consistent high‐quality images that provide regional insight into lung structure and function. While this document represents consensus at a snapshot in time, a roadmap for technical developments is provided that will further increase image quality and efficiency. These standardized dosing and imaging protocols will facilitate the wider adoption of HP 129Xe MRI for multi‐site pulmonary research.
Human umbilical cord–derived mesenchymal stromal cells (hUC-MSCs) are increasingly used in research and therapy. To obtain hUC-MSCs, a diversity of isolation and expansion methods are applied. Here, ...we report on a robust and standardized method for hUC-MSC isolation and expansion.
Using 90 hUC donors, we compared and optimized critical variables during each phase of the multi-step procedure involving UC collection, processing, MSC isolation, expansion and characterization. Furthermore, we assessed the effect of donor-to-donor variability regarding UC morphology and donor attributes on hUC-MSC characteristics.
We demonstrated robustness of our method across 90 UC donors at each step of the procedure. With our method, UCs can be collected up to 6 h after birth, and UC-processing can be initiated up to 48 h after collection without impacting on hUC-MSC characteristics. The removal of blood vessels before explant cultures improved hUC-MSC purity. Expansion in Minimum essential medium α supplemented with human platelet lysate increased reproducibility of the expansion rate and MSC characteristics as compared with Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum. The isolated hUC-MSCs showed a purity of ∼98.9%, a viability of >97% and a high proliferative capacity. Trilineage differentiation capacity of hUC-MSCs was reduced as compared with bone marrow-derived MSCs. Functional assays indicated that the hUC-MSCs were able to inhibit T-cell proliferation demonstrating their immune-modulatory capacity.
We present a robust and standardized method to isolate and expand hUC-MSCs, minimizing technical variability and thereby lay a foundation to advance reliability and comparability of results obtained from different donors and different studies.
Display omitted
To avoid loss of genetic information in environmental DNA (eDNA) field samples, the preservation of nucleic acids during field sampling is a critical step. In the development of standard operating ...procedures (SOPs) for eDNA-based compliance monitoring, the effect of different routinely used sediment preservations on biological community structures serving as bioindicators has gone untested. We compared eDNA metabarcoding results of marine bacterial communities from sample aliquots that were treated with a nucleic acid preservation solution (treated samples) and aliquots that were frozen without further treatment (non-treated samples). Sediment samples were obtained from coastal locations subjected to different stressors (aquaculture, urbanization, industry). DNA extraction efficiency, bacterial community profiles, and measures of alpha- and beta-diversity were highly congruent between treated and non-treated samples. As both preservation methods provide the same relevant information to environmental managers and regulators, we recommend the inclusion of both methods into SOPs for biomonitoring in marine coastal environments.
•We assessed effects of sample preservation for eDNA-based biomonitoring.•We compared freezing versus preservation-solution treatment.•DNA extraction efficiency was highly congruent among treatments.•Bacterial community profiles as bioindicators were highly similar among treatments.•Standard Operating Procedures (SOPs) for biomonitoring can use both treatments.
In the review of total phenolic contents (TPCs) of acacia, lime, and chestnut honey samples from several literature sources, large differences were noticed, which cannot be attributed only to ...seasonal or geographical variations. The dependence of TPC on the process of construction of the calibration line is illustrated in the measurement of acacia, lime, and chestnut honey types from Croatia and neighbouring countries (Serbia, Italy, and Hungary). TPCs are determined for 39 uni-floral honey samples by four calibration lines and four TPC values are obtained for each honey sample. Obtained results are compared mutually, as well as with the literature results for honey samples of the same type. For each honey type, the average of all determined TPCs determined in this study is in the middle of literature values. The average TPC values for chestnut honey samples were found to be 1.5 and 3 times higher than those for lime and acacia, respectively. The effects of two factors regularly considered in the determination of calibration lines are analyzed: (1) the concentration range of the standard chemical and (2) whether the calibration line is drawn through the origin, or not. The final results strongly depend on these two factors that should be considered in future TPC estimations.
This work is licensed under a Creative Commons Attribution 4.0 International License .
In this study we examined 6080 data gathered by our research group during more than 20 years of research on the moss biomonitoring technique, in order to quantify the variability generated by ...different aspects of the protocol and to calculate the overall measurement uncertainty associated with the technique. The median variance of the concentrations of different pollutants measured in moss tissues attributed to the different methodological aspects was high, reaching values of 2851 (ng·g−1)2 for Cd (sample treatment), 35.1 (μg·g−1)2 for Cu (sample treatment), 861.7 (ng·g−1)2 and for Hg (material selection). These variances correspond to standard deviations that constitute 67, 126 and 59% the regional background levels of these elements in the study region. The overall measurement uncertainty associated with the worst experimental protocol (5 subsamples, refrigerated, washed, 5 × 5 m size of the sampling area and once a year sampling) was between 2 and 6 times higher than that associated with the optimal protocol (30 subsamples, dried, unwashed, 20 × 20 m size of the sampling area and once a week sampling), and between 1.5 and 7 times higher than that associated with the standardized protocol (30 subsamples and once a year sampling). The overall measurement uncertainty associated with the standardized protocol could generate variations of between 14 and 47% in the regional background levels of Cd, Cu, Hg, Pb and Zn in the study area and much higher levels of variation in polluted sampling sites. We demonstrated that although the overall measurement uncertainty of the technique is still high, it can be reduced by using already well defined aspects of the protocol. Further standardization of the protocol together with application of the information on the overall measurement uncertainty would improve the reliability and comparability of the results of different biomonitoring studies, thus extending use of the technique beyond the context of scientific research.
Display omitted
•The overall measurement uncertainty associated with the moss technique was calculated.•Uncertainty decreased when using the worst vs. standardized vs. optimal experimental set up.•Although the overall uncertainty of the technique is still high, it can be reduced.•Standardization of the protocol improves the reliability and comparability of the results.
Measurement uncertainty associated to the results of the moss technique is significantly reduced when applying a standardized protocol.
PDF
