Methods Protoc., Volume 6, Issue 6 (December 2023) – 16 articles

Perturbation of angiogenesis is associated with a variety of diseases and pro- as well as antiangiogenic therapies are being actively explored. Additionally, unintended adverse drug effects on angiogenesis might lead to promotion of tumor progression and cardiovascular complications. Several tri-dimensional microfluidic vessel-on-chip systems have been described that allow a more accurate investigation of vascular physiology and pathology, compared to the two-dimensional static culture of endothelial cells. The OrganoPlate^® angiogenesis-on-chip system has been demonstrated to be amenable to high-throughput screening for the antiangiogenic properties of molecules. We set out to adapt this system for high-throughput screening of molecules with proangiogenic properties. Our technical advancement of the OrganoPlate^® angiogenesis-on-chip assay expands its applicability in the early screening of both anti- as well as proangiogenic properties of compounds for therapeutic modulation of angiogenesis as well as the identification of angiogenesis-associated drug-induced vascular toxicities. Full article

Bone metastases cause morbidity and mortality in several human cancer forms. Experimental models are used to unravel the mechanisms and identify possible treatment targets. The location inside the skeleton complicates accurate assessment. This study evaluates the performance of magnetic resonance imaging (MRI) of prostate cancer tumors growing intratibially in mice. MRI detected intratibial tumor lesions with a sensitivity and specificity of 100% and 89%, respectively, compared to histological evaluation. Location and some phenotypical features could also be readily detected with MRI. Regarding volume estimation, the correlation between MRI and histological assessment was high (p < 0.001, r = 0.936). In conclusion, this study finds MRI to be a reliable tool for in vivo, non-invasive, non-ionizing, real-time monitoring of intratibial tumor growth. Full article

Background: The global COVID-19 pandemic has profoundly affected public health. Directly, the pandemic resulted in over 6.6 million deaths, numerous hospitalizations, and widespread illness. The pandemic has also affected health indirectly through government-imposed protective measures, causing decline in mental well-being and increasing social isolation. Unlike previous disasters or crises, the pandemic’s worldwide and enduring impact necessitates a unique research approach. The Network for Health Research in Disasters in the Netherlands responded by initiating a longitudinal, extensive research project called the Integrated Health Monitor COVID-19. The Integrated Health Monitor COVID-19 explores both the direct and indirect health effects of the pandemic at the population level. Methods: The Integrated Health Monitor COVID-19 employs a dual-pronged monitoring strategy alongside an annual literature review. This strategy comprises short-cycle monitoring (conducted quarterly) and long-cycle monitoring (conducted once every one or two years). This comprehensive approach enables the evaluation of health trends during the pandemic, facilitating comparisons with pre-pandemic levels and identification of risk and protective factors. Both monitoring methods incorporate data from surveys and general practice registries. The integration of annual literature reviews with these measurements enables iterative research, while dialogues on policy and practice improvements enhance the knowledge-to-action process. Discussion: Much of the existing knowledge about the potential impact of the COVID-19 pandemic is derived from research on sudden-onset disasters limited to specific geographical areas. This study is anticipated to provide valuable fresh insights into the evolving dynamics of population health and specific vulnerabilities within the ongoing pandemic context. Full article

Quantifying axonal branching is crucial for understanding neural circuit function, developmental and regeneration processes and disease mechanisms. Factors that regulate patterns of axonal arborization and tune neuronal circuits are investigated for their implication in various disorders in brain connectivity. The lack of a reliable and user-friendly method makes the quantitative analysis of axon morphology difficult. Specifically, methods to visualize and quantify the complex axon arborization are challenging to implement and apply practically. Our study was aimed at developing a robust but simple method of quantification that used ImageJ 2D analysis and compared it with Imaris visualization and analysis of 3D images. We used zebrafish fluorescent transgenic lines to perform in vivo imaging of developing motor neuron axons that adequately reflected the complexity of axonal networks. Our new method, developed on ImageJ, is easy and fast, giving access to new information such as collateral distribution along the axonal shaft. This study describes step-by-step procedures that can be easily applied to a variety of organisms and in vitro systems. Our study provides a basis for further exploration of neural circuits to gain new insights into neuronal disorders and potential therapeutic interventions. Full article

Background: The health of children’s lower extremities and feet is a focus area for caregivers and healthcare professionals such as doctors, school nurses, and podiatrists. Our study aims to investigate the general health status of Danish children’s lower extremities and feet to identify anthropometric parameters that might be preconditions for pain and evaluate for foot diseases and whether they are associated with pain intensity and location, three-dimensional foot dimensions and foot pressure mapping, shoe dimensions, types and intensity of sports activity, quality of life, and foot health. The aim is that we will be able to identify parameters pre-dispositioning for pain, thus providing recommendations for sports activities in relation to the anthropometric conditions of a child as a potential preventive measure for pain. This analysis will be stratified by socioeconomic status on a group level, and this perspective will be able to provide preventative recommendations to prevent pain. Methods: This study is a cross-sectional examination of a thousand children in the first, fifth, and ninth grades in randomized selected Danish primary schools. We will perform a clinical examination of the lower extremities and feet for misalignments, deformities, and diseases as well as rotational status and range of motion. Moreover, we will evaluate their pain levels, sports activities, three-dimensional foot dimensions, plantar pressure, footwear, and patient-related outcome measures (PROMs) for foot health and quality of life. Results: We aim to provide an anthropometrical overview of the lower extremities and feet in children. The obtained basic understanding of healthy normal material in children will be analyzed for its relationships with pain level, sports activities, and socioeconomic status on a group level. This could potentially provide us with an understanding of the factors that impact lower extremity and foot diseases in children. In conclusion, examining children’s lower extremities and feet in Danish primary schools is a step toward identifying areas of improvement in self-care and shoe fitting, mapping podiatry-related needs of care in children’s feet, and providing parental recommendations for preventive actions on shoe fitting and the choice and intensity of sports activity concerning pain. Conclusions: The tenet of this study is a long-term follow-up to evaluate the long-term socioeconomic course on a group level, foot status, and sports activity, using patient-related outcome measures evaluating quality of life and other lifestyle factors such as emotional functioning, social functioning and interaction, and school functioning. Potentially, this will improve children’s quality of life and prevent future diseases. Full article

Background. The aim of this study was to compare shoulder movement measurements between a Kinect-based markerless ROM assessment device (POM-Checker) and a 3D motion capture analysis system (BTS SMART DX-400). Methods. This was a single-visit clinical trial designed to evaluate the validity and reliability of the POM-Checker. The primary outcome was to assess the equivalence between two measurement devices within the same set of participants, aiming to evaluate the validity of the POM-Checker compared to the gold standard device (3D Motion Analysis System). As this was a pilot study, six participants were included. Results. The intraclass correlation coefficient (ICC) and the corresponding 95% confidence intervals (CIs) were used to assess the reproducibility of the measurements. Among the 18 movements analyzed, 16 exhibited ICC values of >0.75, indicating excellent reproducibility. Conclusion. The results showed that the POM-checker is reliable and validated to measure the range of motion of the shoulder joint. Full article

Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for indica cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega indica rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of Agrobacterium culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of indica rice cultivars for functional genomics studies and crop improvement. Full article

Cell-free RNAs (cfRNAs) are promising analytes as non-invasive biomarkers and have even greater potential if tied in with metabolomics. Plasma is an optimal source for cfRNAs but is often derived from a variety of anticoagulants. Plasma obtained in heparin is suitable for metabolomics but is difficult to utilize for qPCR-based downstream analysis. In the present study, we aimed to develop a simple, time-efficient, and cost-effective heparinase protocol, followed by library preparation and sequencing of human plasma cfRNAs drawn and stored in heparin at −80 °C for several years. Blood was collected in CPT™ sodium heparin tubes from patients with chronic HCV infection (NCT02400216) at the National Institutes of Health (NIH) Clinical Center. Plasma cfRNAs were treated with heparinase I and used for library preparation and next-generation sequencing (NGS). Heparinase treatment maintained RNA integrity and allowed for successful library preparation for all the study subjects even with 7 ng of cfRNAs as starting material. The classification report derived from Pavian R package v1.2.0 showed no artificial reads. The abundance of chordate over microbial reads suggests no addition of experimental error through heparinase I treatment. We report a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years. This is an effective demonstration of utilizing heparin plasma for NGS and downstream transcriptomic research, which could then be integrated with metabolomics from the same samples, maximizing efficiency and minimizing blood draws. Full article

As our readers know, Methods and Protocols is a multidisciplinary peer-reviewed scientific journal that provides a forum to the publication of novel approaches in the fields of Life Sciences, Chemistry, and Biomedical Sciences and their intersection with other related scientific fields such as Physics, Earth Sciences, and Environmental Research [...] Full article

One approach to enhance the bioavailability and half-life of peptides in vivo is through N-methylation of one or more of the amino acids within the peptide sequence. However, commercially available Fmoc-N-Me-AA-OHs are limited and often expensive. In this study, a solid-phase synthesis method for Fmoc-N-Me-AA-OH was developed using a 2-chlorotrityl chloride (2-CTC) resin as a temporary protective group for the carboxylic acid strategy. Two strategies for the alkylation step were compared, employing either dimethyl sulfate or methyl iodide in the Biron−Kessler method. In this work we tested the protocol with two amino acids: Fmoc-Thr(tBu)-OH and Fmoc-βAla-OH. The first one is an alpha amino acid, very hindered and with the amine group directly influenced by the electronic effects of the carboxy group, whereas in Fmoc-βAla-OH, the presence of a methylene group weakens this influence due to the intervening carbon atoms. The desired amino acids, Fmoc-N-Me-Thr(tBu)-OH and Fmoc-N-Me-βAla-OH, were synthesized by both strategies with high yield and purity. Full article

Bio-SELEX is a revolutionary method for the discovery of novel biomarkers within biological samples, offering profound insights into diagnosing both infectious and non-infectious diseases. This innovative strategy involves three crucial steps: Traditional SELEX, Pull Down, and mass spectrometry. Firstly, Traditional SELEX involves the systematic selection of specific nucleic acid sequences (aptamers) that bind to the target molecules of interest. These aptamers are generated through iterative rounds of selection, amplification, and enrichment, ultimately yielding highly selective ligands. Secondly, the Pull-Down phase employs these aptamers to capture and isolate the target biomarkers from complex biological samples. This step ensures the specificity of the selected aptamers in binding to their intended targets. Lastly, mass spectrometry is utilized to identify and quantify the captured biomarkers, providing precise information about their presence and concentration in the sample. These quantitative data are invaluable in disease diagnosis and monitoring. Bio-SELEX’s significance lies in its ability to discover biomarkers for a wide range of diseases, spanning infectious and non-infectious conditions. This approach holds great promise for early disease detection, personalized medicine, and the development of targeted therapies. By harnessing the power of aptamers and mass spectrometry, Bio-SELEX advances our understanding of disease biology and opens new avenues for improved healthcare. Full article

Whole-brain imaging is important for understanding brain functions through deciphering tissue structures, neuronal circuits, and single-neuron tracing. Thus, many clearing methods have been developed to acquire whole-brain images or images of three-dimensional thick tissues. However, there are several limitations to imaging whole-brain volumes, including long image acquisition times, large volumes of data, and a long post-image process. Based on these limitations, many researchers are unsure about which light microscopy is most suitable for imaging thick tissues. Here, we compared fast-confocal microscopy with light-sheet fluorescence microscopy for whole-brain three-dimensional imaging, which can acquire images the fastest. To compare the two types of microscopies for large-volume imaging, we performed tissue clearing of a whole mouse brain, and changed the sample chamber and low- magnification objective lens and modified the sample holder of a light-sheet fluorescence microscope. We found out that light-sheet fluorescence microscopy using a 2.5× objective lens possesses several advantages, including saving time, large-volume image acquisitions, and high Z-resolution, over fast-confocal microscopy, which uses a 4× objective lens. Therefore, we suggest that light-sheet fluorescence microscopy is suitable for whole mouse brain imaging and for obtaining high-resolution three-dimensional images. Full article

Alcohol-associated liver disease (ALD) is a major global health issue, contributing significantly to morbidity and mortality worldwide. Among the ALD subtypes, alcohol-associated hepatitis poses a severe and urgent medical challenge with high short-term mortality rates. Despite extensive research, the current therapeutic approaches for alcohol-associated hepatitis have limited efficacy, necessitating novel interventions. Recent studies have highlighted the crucial role of the gut microbiota in ALD pathogenesis, particularly Enterococcus faecalis (E. faecalis) and its cytolysin exotoxin. This study presents the development of a standardized real-time quantitative polymerase chain reaction (RT-qPCR) assay to detect and quantify cytolysin in fecal samples from patients with alcohol-associated hepatitis. The diagnostic assay allows for an association analysis between cytolysin-positive E. faecalis and disease severity as well as mortality. This assay was developed to standardize the identification of cytolysin-positive patients who can be selected for clinical trials. Full article

The horizontal flatbed electrophoresis method is employed to separate protein samples, providing greater flexibility for various electrophoretic applications and easier sample loading compared to its vertical counterpart. In the currently available equipment setup, cathode and anode electrodes are positioned on top of a gel at each end. Since an electric field enters the gel from the top, its strength gradually weakens from the top to the bottom of the gel. When examining the interior of gels following electrophoretic separation, the uneven electric field causes the protein bands to lie down forward in the direction of migration, leading to an increase in bandwidth. This issue has remained unaddressed for several decades. To address this problem, new clamp-shaped and double-deck electrodes were developed to apply an electric field simultaneously from both the top and bottom of the gel. Both of these new electrodes facilitated the formation of perpendicular protein band shapes and enhanced resolution at a comparable level. Due to their ease of use, double-deck electrodes are recommended. By combining these new electrodes with the field inversion gel electrophoresis (FIGE) technique, the protein bands could be focused and aligned nearly vertically, resulting in the highest level of electrophoretic resolution. Our electrodes are compatible with polyacrylamide gels of varying sizes, buffer systems, and sample well formats. They can be easily manufactured and seamlessly integrated into existing laboratory instruments for practical use. Full article

This research evaluates extracts from the bark of Heliocarpus popayanensis and Triumfetta bogotensis as coagulating agents for removing turbidity in domestic wastewater, considering the coagulant dosage and pH of the wastewater. ANOVA was conducted to assess differences between the coagulants, dosages, and pH, with three pH levels (5, 8, and 9) and six dosages (7, 9, 11, 13, 15, and 17 mL per 1000 mL of wastewater) at a significance level of α = 0.05, and both the p-value and effect size were evaluated. This study found that the mucilaginous compound from the bark of Triumfetta bogotensis performed better in reducing turbidity levels, with an average reduction of 30.2 NTU (Nephelometric Turbidity Unit) (CI [25.9 NTU; 34.5 NTU], α = 0.05) at a pH of 5, and an average initial NTU of 102.2. This represents an average reduction of 70.45%. The dosage factor did not show significant effects on turbidity reduction, which opens the possibility for further study to determine the optimal dosage of the best coagulant. Full article

We describe the development and validation of a new high performance liquid chromatography (HPLC) method for analysis of a combination of the first-line anti-tubercular drugs isoniazid, pyrazinamide, and rifampicin together with clofazimine. This is a unique challenge since clofazimine and rifampicin are relatively highly lipophilic drugs, whereas isoniazid and pyrazinamide are considerably more hydrophilic. Thus, clear separation of peaks and quantification of four individual drugs can present difficulties during the development of an analytical method. Detection was established at two wavelengths—254 nm for isoniazid and pyrazinamide and 320 nm for clofazimine and rifampicin. Gradient elution was employed using 0.1% aqueous formic acid (A) and acetonitrile (B); clear separation of the four drugs was achieved within 10 min. A linear relationship was indicated by a correlation coefficient (r²) of 0.9999 for each anti-tubercular drug, respectively. The limit of detection (LOD) for the individual drugs was 0.70 µg/mL (isoniazid), 0.30 µg/mL (pyrazinamide), 0.20 µg/mL (rifampicin) and 0.20 µg/mL (clofazimine). Precision experiments rendered a mean recovery percentage of 101.25% (isoniazid), 98.70% (pyrazinamide), 99.68% (rifampicin) and 97.14% (clofazimine). This HPLC method was validated and is reliable, repeatable, and accurate for the purpose of conducting simultaneous HPLC analyses of the four anti-tubercular drugs. Full article