Methods and Protocols

Replicating in vivo conditions is essential for understanding immune responses and measuring immune biomarkers in blood. Sampling immune biomarkers in plasma or serum often fails to detect disease-relevant signals, possibly because these markers are sequestered in immune cells or extracellular vesicles. Furthermore, traditional whole blood cultures using external media may not accurately mimic the physiological environment of blood cells. To address these limitations, we developed a strategy using whole blood cell lysates and supernatants to optimize biomarker detection. Additionally, we employed neat whole blood culture methods, preserving the natural cellular and biochemical environment to assess sensitivity to immune modulators, such as lipopolysaccharide (LPS). This cost-effective approach minimizes variability and contamination risks. By utilizing Luminex multiplex immunoassays, we profiled immune biomarkers with higher sensitivity and efficiency than traditional ELISAs. Blood samples from individuals with high alcohol consumption validated our method by assessing biomarker levels before and after LPS stimulation, providing insights into intracellular responses and inflammatory pathways. This method enhances our understanding of inflammatory processes in blood cells, demonstrating the advantages of cell lysates, supernatants, and advanced multiplex assays in immunological research. Full article

Early initiation of palliative care in patients with advanced chronic conditions significantly improves their quality of care; however, variability in disease trajectories complicates such interventions’ timing. The NECPAL CCOMS-ICO prognostic tool was developed as a straightforward instrument to help healthcare providers in all clinical settings promptly identify patients with advanced chronic conditions who require palliative care, thereby enhancing service planning and delivery. Its latest version, 4.0, 2021, for the first time, incorporates a patient survival estimation. Nevertheless, validation is necessary. This study aims to validate the NECPAL version 4.0 tool in an independent cohort. It is an observational, prospective study involving outpatients and hospitalized non-randomized patients at Hospital Universitario Infanta Leonor–Virgen de la Torre in Madrid, Spain, all of whom have at least one advanced chronic condition. The study is scheduled to last 6 years, including a recruitment period of 30 months starting 1 February 2024, followed by a 12-month follow-up period for each patient. This is the first prospective study designed to validate the NECPAL version 4.0 instrument. Implementing this tool would allow the identification of patients with advanced chronic conditions and unmet palliative care needs and determine the more appropriate care pathway at the proper moment. Full article

Myocardial infarction with non-obstructive coronary arteries (MINOCA) is a working diagnosis encompassing several pathophysiological mechanisms with specific treatments and different prognoses. Despite the absence of obstructive coronary artery disease, MINOCA has proven to be associated with a significant risk of mortality, angina burden, and socioeconomic costs. However, due to the heterogeneous nature of this clinical condition and the absence of randomized clinical trials, evidence supporting a standardized diagnostic algorithm and the clinical management of these patients is lacking. The PROMISE trial is the first randomized clinical trial evaluating the effectiveness of a precision medicine approach strategy in improving the outcomes and quality of life of patients with MINOCA, offering new insights into personalized treatment strategies. This review article discusses the promise of a precision medicine approach in patients with MINOCA, highlighting the potential innovations and challenges of a personalized medicine strategy in MINOCA. Full article

Antigen presentation on major histocompatibility complex (MHC) molecules is central to the initiation of immune responses, and a lot of our understanding about the antigen processing and presentation pathway has been gained through studies in mice. MHC molecules are the most genetically diverse genes; consequently, mouse strains differ substantially in their MHC make up and resulting antigen presentation. Swiss mice are commonly used in pharmacological research, yet our understanding of antigen presentation in this strain is surprisingly limited. Here, we have tested a range of anti-MHC antibodies and present a range of clones suitable to analyse MHC class I and class II molecules in Swiss mice who have the H2-q MHC haplotype. Moreover, we demonstrate using immunopeptidomics that clones 28-12-8, 34-1-2, MKD6, and N22 are also suited to isolate MHC class I and class II ligands in this mouse strain. Thus, this work also establishes a first experimental account of the H2-q-derived thymus and spleen immunopeptidome in Swiss mice which bears strong resemblance with ligands isolated from the H2-d MHC haplotype of Balb/C mice. The analysis of source proteins shows common but also organ- and function-specific antigen presentation in line with the involvement of the thymus in tolerance induction and the function of the spleen as a site of immune responses. Full article

Type 2 diabetes mellitus (T2DM) significantly impairs fracture healing, with neutrophils playing a crucial role in this process. In T2DM, these immune cells are over-activated, leading to the excessive release of neutrophil extracellular traps (NETs), increasing inflammation and hindering recovery. Thus, a need for markers to assess patients in the risk group arises. This study demonstrates that circulating cell-free DNA (cfDNA) can be efficiently quantified from serum samples by a single-step qPCR and be used as a marker for NETosis. Our results revealed that trauma patients with T2DM have the highest cfDNA levels, followed by trauma patients, and the healthy group has the lowest. The method shows strong correlations between cfDNA and neutrophil-specific markers such as MPO, citH3, AZU1, and α-defensin, highlighting its potential as a rapid indicator of NETosis. This approach could allow the timely interference for high-risk patients, ultimately improving healing outcomes and reducing complications such as chronic inflammation, non-union fractures, and diabetic foot ulcers. Full article

Background: Evidence supporting the validity of photogrammetry for assessing body segment alignment remains limited, with most studies focusing on spinal evaluation. Thus, there is a lack of robust research examining its use for other body segments such as the lower limbs. Objective: This study aimed to evaluate the concurrent validity of three photogrammetric methods for measuring knee alignment in the sagittal plane with and without corrections for potential rotational deviations in the participant’s thigh and leg. Methods: A total of 21 adults underwent sequential evaluations involving panoramic radiography of the lower limbs and photogrammetry at a private radiology clinic. Photogrammetric analysis involved identifying the following anatomical landmarks: the greater trochanter of the femur (GTF), the lateral condyle of the femur (LCF), the head of the fibula (HF), and lateral malleolus (LM). Three photogrammetric methods were employed: (1) the condylar angle (CA) defined by the GTF, LCF, and LM points; (2) the fibula head angle (FHA) defined by the GTF, HF, and LM points; and (3) the four-point angle (4PA) incorporating the GTF, LCF, HF, and LM. Concurrent validity was assessed using correlation analysis, agreement with radiographic measurements, and the root mean square error (RMSE). Each photogrammetric method was tested using raw (CA, FHA, and 4PA) and corrected (CAcorr, FHAcorr, and 4PAcorr) values, accounting for thigh and/or leg rotational deviations. Results: Correcting for thigh and leg rotations significantly improved the validity metrics for all methods. The best performance was observed with the corrected condylar angle (CAcorr: r = 0.746; adjusted r² = 0.533; RMSE = 2.9°) and the corrected four-point angle (4PAcorr: r = 0.733; adjusted r² = 0.513; RMSE = 3.0°); however, the measurements presented proportional errors, possible due the method of assessment of rotations. Conclusions: The findings validate the evaluated photogrammetric methods for assessing sagittal knee alignment. Accounting for thigh and leg rotational deviations is critical for achieving accurate measurements, raising the need of accurate tools for measuring rotational changes in the lower limbs to avoid errors. Full article

Strength development through physical exercise enhances neuromodulator production, neural connectivity, and motor unit efficiency. Beyond physical benefits, understanding individuals’ perceptions, opinions, and knowledge can optimize engagement in exercise. However, existing literature lacks studies examining these factors alongside strength development. This study aimed to investigate whether the effectiveness of strength training protocols is associated with individuals’ perceptions, opinions, and knowledge, thereby establishing a link between performance enhancement and awareness of the physiological demands of exercise. The findings seek to highlight the educational potential of physical exercise in promoting psychophysical well-being. A total of 24 participants (14 males, 10 females), aged 35–55 years with varying occupational backgrounds and sedentary levels, were recruited. A strength development protocol was administered, and the participants completed perception-based questionnaires at three time points. Statistical analyses, including repeated-measures ANOVA, Friedman’s test, and post hoc comparisons, were conducted. Significant strength improvements were observed, specifically in the Hand Grip Test (p < 0.01). An increase, but non-significant, emerged in the Isometric Mid-Thigh Pull from 1850 ± 210 N to 2270 ± 190 N. The participants also reported a 35% increase in motivation to engage in exercise and a 42% reduction in sedentary behavior. Additionally, 78% of the participants demonstrated greater awareness of exercise benefits, correlating positively with physical improvements. The findings indicate that strength development is associated with increased awareness of the benefits of physical exercise, supporting its use as an educational tool to enhance engagement and adherence to exercise protocols. Full article

Steam-assisted gravity drainage (SAGD) is a widely used thermal enhanced oil recovery (EOR) technique in North America, particularly in high-permeability oil sand reservoirs. While effective, its economic viability has declined due to low oil prices and high greenhouse gas (GHG) emissions from the steam generation. To improve cost-effectiveness and reduce emissions, solvent-assisted SAGD techniques have been explored. Expanding Solvent-SAGD (ES-SAGD) involves co-injecting light hydrocarbons like propane or butane with steam to enhance oil viscosity reduction. This approach lowers the steam–oil ratio by combining solvent dissolution effects with thermal effects. However, the high cost of solvents, particularly butane, challenges its commercial feasibility. Propane is cheaper but less effective, while butane improves performance but remains expensive. This research aims to optimize ES-SAGD by using a propane–butane mixture to achieve efficient performance at a lower cost than pure butane. A linear sand pack is used to evaluate different propane/butane compositions, maintaining constant operational conditions and a solvent concentration of 15 vol.%. Temperature monitoring provides insights into steam chamber growth. Results show that solvent injection significantly enhances ES-SAGD performance compared to conventional SAGD. Performance improves with increasing butane concentration, up to 80% butane in the C₃–C₄ mixture at the test pressure and ambient temperature. Propane alone results in the lowest system temperature, while conventional SAGD reaches the highest temperature. These findings highlight the potential of optimized solvent mixtures to improve ES-SAGD efficiency while reducing costs and GHG emissions. Full article

Short hairpin RNAs (shRNAs) are potent tools for gene silencing, offering therapeutic potential for gene and cell therapy applications. However, their efficacy and safety depend on precise processing by the RNA interference machinery and the generation of minimal by-products. In this protocol, we describe how to systematically analyze the processing of therapeutic small RNAs by DROSHA and DICER1 and their incorporation into functional AGO complexes. Using standard small RNA sequencing and tailored bioinformatic analysis (QuagmiR), we evaluate the different steps of shRNA maturation that influence processing efficiency and specificity. We provide guidelines for troubleshooting common design pitfalls and off-target effects in transcriptome-wide profiling to identify unintended mRNA targeting via the miRNA-like effect. We provide examples of the bioinformatic analysis that can be performed to characterize therapeutic shRNA. Finally, we provide guidelines for troubleshooting shRNA designs that result in suboptimal processing or undesired off-target effects. This protocol underscores the importance of rational shRNA design to enhance specificity and reduce biogenesis by-products that can lead to off-target effects, providing a framework for optimizing the use of small RNAs in gene and cell therapies. Full article

We aimed to develop a species-specific ELISA for qualitatively and quantitatively determining serum amyloid A (SAA) in horses. Current methods for measuring SAA in horses utilize ELISA or immunoturbidimetric tests designed for human SAA, which are not specific to horses. Mice and rabbits were used to generate polyclonal antibodies against equine SAA. The study examined serum samples from 32 horses with acute inflammatory disease (SG) and 25 clinically healthy horses. Furthermore, the SAAeq kinetics were observed in three horses from the SG group at three different timepoints. The SAA-ELISA established a cut-off at 0.06 OD₄₉₂nm, where values equal to or higher than this were deemed positive, while values below it was considered negative. The test exhibited a sensitivity of 94% and specificity of 92%, resulting in an overall accuracy of 93%. The positive and negative predictive values were 94% and 92%, respectively. Coefficients of variation for inter- and intra-assay were 6.1% and 7.46% for SG and 9.6% and 9.63% for the control group (CG). The detection limit was determined to be 0.067. The SAA-ELISA proved its worth by demonstrating satisfactory performance, paving the way for the development of automated quantitative tests and species-specific semi-quantitative tests. This paves the way for their application in practical field settings. Full article

Background: Synthetic DNA aptamers are a class of molecules with potential applications in medicine, serving as molecular sensors or ligands for targeted drug delivery. Systematic evolution of ligands by exponential enrichment (SELEX) is a technology for selecting functional aptamers that was first reported three decades ago and has been actively developed since. SELEX involves multiple iterations of two fundamental steps: (i) target affinity-based partitioning of aptamers from a random library and (ii) amplification of selected aptamers by PCR, followed by isolation of single-stranded DNA (ssDNA). SELEX protocols have diversified considerably, with numerous variations possible for each step. This heterogeneity makes it challenging to identify optimal methods. Comparative analysis of different approaches for the major stages of SELEX is therefore of considerable practical importance. Methods: Four widely used methods for ssDNA generation were performed in parallel: (a) PCR followed by digestion of the antisense strand with exonuclease lambda, (b) PCR with an extended primer followed by size-dependent strand separation using denaturing PAGE, (c) asymmetric PCR, and (d) asymmetric PCR with a primer-blocker. Results: The specificity, efficiency, reproducibility, and duration of each method were compared. Conclusions: Asymmetric PCR with a primer-blocker yielded the most favorable results. Full article

An accurate histopathological evaluation of radical cystectomy (RC) specimens is crucial for optimal tumor staging, prognosis, and therapeutic decision making. The increasing demand for precision medicine and multidisciplinary oncological management emphasizes the necessity for standardized protocols in the handling and sampling of bladder cancer specimens. The effective processing of RC specimens begins with the integration of clinical and anamnestic data, along with appropriate formalin fixation methods to meet diagnostic needs. The pathologist must meticulously document the macroscopic characteristics and dimensions of the surgical specimen, especially in post-neoadjuvant chemotherapy (post-NAC) cases where the primary tumor may not be macroscopically visible. Sampling strategies should ensure a comprehensive assessment of the primary tumor and any extra-organ or metastatic involvement. Despite international guidelines, variability in pathology practices persists, particularly concerning prostate sampling in RC and the use of frozen sections for margin assessment. Addressing these challenges necessitates a consensus-driven, standardized approach to improve the reproducibility and quality of histopathological data. By addressing gaps in current pathology practices, this review advocates for uniform protocols that enhance diagnostic accuracy, ultimately improving patient care and clinical decision making. Full article

(1) Background: Research has consistently demonstrated that regular physical activity (PA) is associated with several benefits among adolescents. However, PA levels among adolescents are low worldwide and tend to decrease with age. Consequently, researchers aim to identify psychological antecedents of PA to inform effective interventions, including in physical education (PE) settings. PE lessons provide an ideal environment for conveying health-related messages to adolescents. (2) Methods: This project aims to develop a three-month face-to-face and web-based intervention program for PE teachers to increase autonomy-, competence-, and relatedness-supportive behavior toward their students (main trial phase 1: min n = 78; main trial phase 2: min n = 116) and to avoid the respective need-thwarting behaviors toward their students. The effectiveness of the intervention program is examined at multiple time points during the three-month period. (3) Results: After the intervention program, it is expected that the experimental group students demonstrate significantly higher intrinsic motivation toward PA and are significantly more physically active, as measured by accelerometers, compared to control group students. Additionally, the unique effects of autonomy, competence, and relatedness support interventions on students’ intrinsic motivation and PA will be investigated. (4) Conclusions: This project provides highly valuable insights for PE teacher training to increase students’ intrinsic motivation and their overall PA. Full article

Heavy metal contamination of soil and water is a growing environmental concern, especially mercury, lead, and cadmium. Therefore, fast and reliable methodologies to assess contamination in the field are in demand. However, many methodologies require laborious, expensive, and cumbersome equipment that is not convenient for rapid field analysis. Mobile phone technology coupled with bioluminescent assays provides accessible hands-on alternatives that has already been shown to be feasible. Previously, we demonstrated that firefly luciferases can be harnessed as luminescence color-tuning sensors for toxic metals. An assay based on such a principle was already successfully applied for teaching biochemistry laboratory lessons, which demonstrates the effect of cadmium on enzyme function based on bioluminescence color change. For analytical detection of cadmium in water, here, we developed a novel bioluminescence assay using the cadmium-sensitive Amydetes vivianii firefly luciferase coupled with a cell phone provided with a program to quantify cadmium concentration based on luminescence color discrimination. The application has proven to be efficient with high precision between 0.10 and 2 mM of cadmium, being appliable to diluted water samples (0.1–2 µM) upon concentration and relying on reference cadmium standards values. The light emitted by the reference standards and samples in a dark box is captured by the smartphone’s camera, which, using computer vision, automatically quantifies cadmium according to the RGB color. CadmiLume is a simple and easy luminescent enzymatic biosensor for cadmium contamination in water samples, which instantaneously can provide results with the convenience of a smartphone in the palm of one’s hands. Full article

Biomechanical parameters of plant tissues and organs are increasingly recognized as key factors in plant development and application, increasing the demand for convenient devices for their study. The paper presents an original device for performing a three-point bending test using the inverse method, which is a modification of the classical (straight) three-point test. The designed device was tested in experiments to determine the modulus of elasticity of flax plant stems, and the results were compared with data obtained using the vibration method and the straight three-point bending test on a commercial instrument. Due to the high sensitivity associated with its design features, the device for the inverse three-point bending test is characterized by being able to adequately measure elastic moduli in plant stems over a wide range of values, from tens of MPa to tens of GPa. It also allows checking the effect of humidity, temperature, and water content on the mechanical properties of samples and is equipped with an automation system. The proposed device is quite affordable and can be effectively used both for young stem parts, whose mechanical properties are based on a hydroskeleton, and for mature, poorly hydrated parts with cell walls highly developed in sclerenchymatous tissues. Full article

Butterfly larvae display intricate cognitive capacities and behaviors, but relatively little is known about how those behaviors alter their brains at the molecular level. Here, we optimized a hybridization chain reaction 3.0 (HCR v3.0) protocol to visualize the expression of multiple RNA molecules in fixed larval brains of the African butterfly Bicyclus anynana. We optimized the polyacrylamide gel mounting, fixation, and sample permeabilization steps, and mapped the expression domains of ten genes in whole larval brain tissue at single-cell resolution. The genes included optomotor blind (omb), yellow-like, zinc finger protein SNAI2-like (SNAI2), weary (wry), extradenticle (exd), Synapsin, Distal-less (Dll), bric-à-brac 1 (bab1), dachshund (dac), and acetyl coenzyme A acetyltransferase B (AcatB). This method can be used alongside single-cell sequencing to visualize the spatial location of brain cells that change in gene expression or splicing patterns in response to specific behaviors or cognitive experiences. Full article

Cholinesterase (ChE) is an essential enzyme for nervous system function, and its activity in the blood serves as a biomarker for pesticide exposure, necessitating accessible assessment methods in resource-limited areas. This study focused on developing and validating a micro-electrometric method to rapidly and reliably measure ChE activity in human blood to monitor pesticide exposure. A micro-electrometric method was designed to evaluate ChE activity in whole-blood samples, involving incubation with organophosphate and carbamate pesticides and subsequent pH measurement, and its performance was compared to that of the Ellman method. The optimized method was strongly correlated with the Ellman method (R² = 0.9147), effectively measuring dose-dependent ChE inhibition by mevinphos and carbofuran with stabilization after 10 min, demonstrating a high sensitivity suitable for field applications. The micro-electrometric method is easy, accurate, and inexpensive for measuring ChE activity. It has been proven to work by comparing it to other methods; therefore, it can be used in clinical and research settings with limited resources. Full article

Volatile compounds (VCs) from fungi can promote plant growth, but their application methods are limited. Edible mushroom fungi beds (FBs) provide a readily available alternative source of fungal VCs, although their biostimulatory functions remain unvalidated. In this study, a novel, non-contact exposure method for applying VCs emitted from FBs to rice seedlings was developed. This marks the first evaluation of mushroom FBs as a direct source of bioactive VCs for plant growth promotion. Volatiles from two different edible mushroom FBs promoted shoot growth and increased biomass for rice seedlings. VCs from shiitake FBs significantly increased biomass by 67.4% while VCs from enokitake FBs by 39.5% compared to the control. The biomass-increasing effects were influenced by the quantity of shiitake FBs applied, with significant increases at 15 g, 30 g and 60 g applications. The VCs effects remained significant even when the FBs were covered with two types of gas-permeable polymer film. Chemical analysis of VCs from FBs identified several organic compounds and subsequent bioassays using synthetic VCs determined key bioactive VCs contributing to biomass increase at specific concentrations. This study presents a utilization method of waste mushroom FBs as sustainable, scalable, and cost-effective agricultural biostimulants. Full article

In the context of wheelchair racing, research primarily focuses on studying wheelchair ergonomics and determining kinematic, kinetic, and rolling resistance variables. One factor identified as influencing athletes’ performance is wheel skidding on the ground, a parameter complementary to rolling resistance. The objective of this study, therefore, is to identify, within a laboratory setting, the parameters that influence the risk of skidding in racing wheelchairs by measuring skidding torque. The ultimate goal is to enhance athletes’ performance by optimizing the interaction between the athlete and their wheelchair, and the wheelchair and the environment. In this perspective, four parameters were examined: the type of tubular, the camber angle, the tire pressure, and the load applied to the wheel using a skidometer. This tool characterizes a tire’s grip on a surface by measuring torques. The aim is to develop a system for classifying tire grip on dry athletics track at ambient temperature. The findings revealed that only the effects of load and tubular type had a significant impact on the torque values obtained. The tire that minimized the risk of skidding, among all tested combinations, is the Vittoria Pista Speed 23–28″. Furthermore, as the mass applied to the wheel increases, so do the resulting torques. This implies that a heavier athlete would require a greater force to be applied to the hand rim for the tire to skid. However, it was also demonstrated that the risk of skidding in a racing wheelchair is unlikely, as the torques obtained were over a range of 90 to 190 Nm. These values far exceed those typically exerted by para-athletes, which are a maximum of 60 Nm. The long-term goal would be to adjust the mode of torque application on the wheel using the skidometer for a more realistic field approach. Full article