Methods and Protocols

This article presents a low-cost and flexible software solution for acoustic startle response (ASR) test that can be used with a Spike2-based interface. ASR is a reflexive response to an unexpected, loud acoustic stimulus, and prepulse inhibition (PPI) is a phenomenon in which the startle response is reduced when preceded by a weak prestimulus of the same modality. Measuring PPI is important because changes in PPI have been observed in patients with various psychiatric and neurological disorders. Commercial ASR testing systems are expensive, and their closed source code affects their transparency and result reproducibility. The proposed software is easy to install and use. The Spike2 script is customizable and supports a wide range of PPI protocols. As an example of PPI recording, the article presents data obtained in female rats, both wild-type (WT) and dopamine transporter knockout (DAT-KO), showing the same tendency as the data obtained in males, with ASR on a single pulse higher than ASR on prepulse+pulse, and PPI reduced in DAT-KO rats compared to WT. Full article

Distal radius fractures (DRFs) are one of the most common fractures of the upper extremity system. To evaluate the performance of DRF treatments, the construct (i.e., a DRF fixed by an implant) was compressed at the distal radius in the axial direction to evaluate the compressive stiffness. In previous studies, various constructs of both cadaveric and synthetic radii have been proposed for biomechanical testing for DRF. Unfortunately, high deviations of the measured stiffness have been reported across the literature, which may relate to the inconsistency of applied mechanical actions (i.e., the tested radii may under various combinations including compression, bending, and shear). In the present study, a biomechanical apparatus and an experimental procedure were proposed for the biomechanical testing of radii under pure compression. After the biomechanical tests of synthetic radii, it was found that the standard deviation of stiffness was significantly lower than that in previous studies. Thus, the biomechanical apparatus and the experimental procedure were proven to be a practical method for the evaluation of radii stiffness. Full article

Protein phosphorylation is a ubiquitous post-translational modification that regulates a plethora of intracellular processes, making its analysis crucial for understanding intracellular dynamics. The commonly used methods, such as radioactive labeling and gel electrophoresis, do not provide information about subcellular localization. Immunofluorescence using phospho-specific antibodies and subsequent analysis via microscopy allows researchers to assess subcellular localization, but it typically lacks validation whether the observed fluorescent signal is phosphorylation specific. In this study, an on-slide dephosphorylation assay coupled with immunofluorescence staining using phospho-specific antibodies on fixed samples is proposed as a fast and simple approach to validate phosphorylated proteins in their native subcellular context. The assay was validated using antibodies against two different phosphorylated target proteins, connexin 43 phosphorylated at serine 373, and phosphorylated substrates of protein kinase A, with a dramatic reduction in the signal upon dephosphorylation. The proposed approach provides a convenient way to validate phosphorylated proteins without the need for additional sample preparation steps, reducing the time and effort required for analysis, while minimizing the risk of protein loss or alteration. Full article

Background: Vascular smooth muscle cells (VSMCs) and vascular endothelial cells are key participants in the pathogenesis of atherosclerosis. Human umbilical vein endothelial cells (HUVECs) and VSMCs are useful models to design therapeutic strategies for many cardiovascular diseases (CVDs). However, procuring a VSMC cell line by researchers, to model atherosclerosis, for example, is impeded by time and cost limitations, as well as by many other logistic problems in many countries. Results: This article describes a protocol for the quick and cheap isolation of VSMCs from human umbilical cords using a mechanical and enzymatic method. This VSMC protocol yields a confluent primary culture that could be obtained within 10 days and sub-cultured for 8–10 passages. The isolated cells are characterized by their morphology and the expression of mRNA of marker proteins analyzed by reverse transcription polymerase chain reaction (RT-qPCR). Conclusion: The protocol described herein for the isolation of VSMCs from human umbilical cords is easy and is time- and cost-efficient. Isolated cells are useful models for understanding the mechanisms underlying many pathophysiological conditions. Full article

The Multidrug Resistance protein (ABCB1, MDR1) is involved in the transport of xenobiotics and antiretroviral drugs. Some variants of the ABCB1 gene are of clinical importance; among them, exon 12 (c.1236C>T, rs1128503), 21 (c.2677G>T/A, rs2032582), and 26 (c.3435C>T, rs1045642) have a high incidence in Caucasians. Several protocols have been used for genotyping the exon 21 variants, such as allele-specific PCR-RFLP using adapted primer to generate a digestion site for several enzymes and automatic sequencing to detect the SNVs, TaqMan Allele Discrimination assay and High-Resolution Melter analysis (HRMA). The aim was to describe a new approach to genotype the three variants c.2677G>T/A for the exon 21 doing only one PCR with the corresponding primers and the digestion of the PCR product with two restriction enzymes: BrsI to identify A allele and BseYI to differentiate between G or T. An improvement of this methodology was also described. The proposal technique here described is demonstrated to be very efficient, easy, fast, reproducible, and cost-effective. Full article

Introduction: Patients with neurogenic lower urinary tract dysfunction (NLUTD) reliant on intermittent self-catheterization for bladder emptying are at an increased risk of recurrent urinary tract infections (rUTI). So far, the most common practice in the prevention of rUTIs is long-term low-dose antibiotic prophylaxis, phytotherapy, and immunomodulation, whereby antibiotic prophylaxis inevitably leads to the emergence of drug-resistant pathogens and difficulty in treating infections. Therefore, non-antibiotic alternatives in the prevention of rUTIs are urgently required. We aim to identify the comparative clinical effectiveness of a non-antibiotic prophylaxis regimen in the prevention of recurrent urinary tract infections in patients with neurogenic bladder dysfunction who practice intermittent self-catheterization. Methods and analysis: In this multi-centre, prospective longitudinal multi-arm observational study, a total of 785 patients practising intermittent self-catheterisation due to NLUTD will be included. After inclusion, non-antibiotic prophylaxis regimens will be instilled with either UroVaxom^® (OM-89) standard regimen, StroVac^® (bacterial lysate vaccine) standard regimen, Angocin^®, D-mannose (oral dose 2 g), bladder irrigation with saline (once per day). The management protocols will be pre-defined, but the selection of the protocol will be at the clinicians’ discretion. Patients will be followed for 12 months from the onset of the prophylaxis protocol. The primary outcome is to identify the incidence of breakthrough infections. The secondary outcomes are adverse events associated with the prophylaxis regimens and the severity of breakthrough infections. Other outcomes include the exploration of change in susceptibility pattern via the optional rectal and perineal swab, as well as health-related quality of life over time (HRQoL), which will be measured in a random subgroup of 30 patients. Ethics and dissemination: Ethical approval for this study has been granted by the ethical review board of the University Medical Centre Rostock (A 2021-0238 from 28 October 2021). The results will be published in a peer-reviewed journal and presented at relevant meetings. Study registration number: German Clinical Trials Register: Number DRKS00029142. Full article

Background: Despite the known benefits of physical activity in cancer survivors, adherence to exercise guidelines remains low. Known barriers to adhering to guidelines include a lack of time and an unwillingness to return to treatment facilities. Virtual exercise programming could assist in mitigating these barriers. This protocol presents a single arm pilot study exploring the feasibility of personalized Zoom-delivered exercise training for breast and prostate cancer survivors. A secondary objective is to determine the preliminary efficacy of participation on body composition, estimated VO_2max, hand grip, one repetition maximum leg press, resting heart rate, resting blood pressure, exercise self-efficacy, and intentions to remain active. Methods: Breast (n = 10) and prostate (n = 10) cancer survivors will participate in a 24-week feasibility study, including (1) 12 weeks of one-on-one virtual personal training with an exercise physiologist (EP) via Zoom, and (2) individual exercise for a 12-week follow-up period using recordings of Zoom sessions for guidance. Physical assessments and surveys will be implemented at baseline, 12 weeks, and at the end of the study (24 weeks from baseline). Conclusions: While virtual exercise programming became popularized during the pandemic, evidence is still required to understand whether it can successfully address barriers and promote participation. Full article

In ophthalmic research, there is a strong need for in vitro corneal cell models. Here, we describe different protocols for the cultivation of primary corneal cells that were isolated from porcine eyes. This primary cell culture can be used to test new therapeutic options for corneal diseases, such as dry eye disease, traumatic injuries, or corneal infections, and to study limbal epithelial stem cell (LESC) expansion. Two different isolation methods were performed: the outgrowth and the collagenase method. To perform the outgrowth protocol, small explants of the corneal limbus were generated and incubated in culture flasks in an incubator for 4–5 weeks. Regarding the collagenase method, to extract corneal cells, porcine corneas were removed, cut into small pieces, and incubated with collagenase. After incubation and centrifugation, the cells were seeded in 6- or 12-well plates and incubated in an incubator for 2–3 weeks. The differences between corneal cell cultivation with fetal bovine serum (FBS) and without it are also discussed. Therefore, the main advantages of the outgrowth method are that it requires fewer porcine eyes, and it takes less time to be performed compared to the collagenase method. On the other hand, with the collagenase method, mature cells are obtained earlier, at about 2 to 3 weeks. Full article

In the past decades, we have witnessed tremendous developments in endovascular surgery. Nowadays, highly complex procedures are performed by minimally invasive means. A key point is equipment improvement. Modern C-arms provide advanced imaging capabilities, facilitating endovascular navigation with an adequate open surgical environment. Nevertheless, radiation exposure remains an issue of concern. This study aims to analyze radiation used during endovascular procedures according to complexity, comparing a mobile X-ray system with a hybrid room (fixed X-ray system). This is an observational and prospective study based on a cohort of non-randomized patients treated by endovascular procedures in a Vascular Surgery department using two imaging systems. The study is planned for a 3-year duration with a recruitment period of 30 months (beginning 20 July 2021) and a 1-month follow-up period for each patient. This is the first prospective study designed to describe the radiation dose according to the complexity of the procedure. Another strength of this study is that radiologic variables are obtained directly from the C-arm and no additional measurements are required for feasibility benefit. The results from this study will help us determine the level of radiation in different endovascular procedures, in view of their complexity. Full article

Introduction: Midwives have the potential to significantly contribute to health-delivery systems by providing sexual, reproductive, maternal, newborn, and adolescent health (SRMNAH) care. However, scant research finds barriers to understanding what midwives need to realize their full potential. There are gaps in the definition of a midwife and an understanding of effective means to support the implementation of midwifery care. Mentorship has been found to support systems and healthcare providers to improve care availability and quality. Objectives: We describe the methodology of an integrative review that aims to generate evidence of the impact of introducing midwives and also on-site facility mentoring to better understand facilitators and barriers to implementation of the quality and availability of SRMNAH services in low- and middle-income countries (LMICs). Methods: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines will be used to carry out the integrative review. Four electronic bibliographic databases, PubMed MEDLINE, EMBASE, Scopus, and CINAHL, will be used to identify eligible studies. All types of qualitative or quantitative studies will be considered. Eligible studies will be screened according to Population, Intervention, Comparison, and Outcome (PICO) inclusion criteria, and data will be extracted against a predetermined format. The aspects of health system strengthening in providing improved SRMNCH care will be examined in this review to generate evidence on how midwives and mentorship can improve routine care and health outcomes using the World Health Organization’s Six Building Blocks approach. The quality of the articles will be thematically analyzed in four areas: coherence and integrity, appropriateness for answering the question, relevance and focus, and overall assessment using the Gough weight-of-evidence framework. Expected results: The literature review will consider assessing both upstream health systems regulators and downstream effectors for implementing midwifery interventions. Within this building block framework, this research will report on the outcomes and experiences of introducing midwives and the effectiveness of mentoring midwives and other staff in midwives’ roles in improving care quality and health outcomes. Full article

The effect of arbitrary stimulus selection is a persistent concern when employing implicit measures. The current study tests a data-driven multi-step procedure to create stimulus items using a combination of free-recall and survey data. Six sets of stimulus items were created, representing healthy food and high sugar items in children, adolescents, and adults. Selected items were highly representative of the target concepts, in frequent use, and of near equal length. Tests of the piloted items in two samples showed slightly higher implicit measure–behavior relations compared to a previously used measure, providing preliminary support for the value in empirically based stimulus selection. Further, the items reported as being the most associated with their target concepts differed notably from what one may expect from the guidelines or population consumption patterns, highlighting the importance of informed stimulus selection. Full article

The longitudinal monitoring of patient circulating tumor DNA (ctDNA) provides a powerful method for tracking the progression, remission, and recurrence of several types of cancer. Often, clinical and research approaches involve the manual review of individual liquid biopsy reports after sampling and genomic testing. Here, we describe a process developed to integrate techniques utilized in data science within a cancer research framework. Using data collection, an analysis that classifies genetic cancer mutations as pathogenic, and a patient matching methodology that identifies the same donor within all liquid biopsy reports, the manual work for research personnel is drastically reduced. Automated dashboards provide longitudinal views of patient data for research studies to investigate tumor progression and treatment efficacy via the identification of ctDNA variant allele frequencies over time. Full article

The last 18 years have brought an increasing interest in the therapeutic use of perinatal derivatives (PnD). Preclinical studies used to assess the potential of PnD therapy include a broad range of study designs. The COST SPRINT Action (CA17116) aims to provide systematic and comprehensive reviews of preclinical studies for the understanding of the therapeutic potential and mechanisms of PnD in diseases and injuries that benefit from PnD therapy. Here we describe the publication search and data mining, extraction, and synthesis strategies employed to collect and prepare the published data selected for meta-analyses and reviews of the efficacy of PnD therapies for different diseases and injuries. A coordinated effort was made to prepare the data suitable to make statements for the treatment efficacy of the different types of PnD, routes, time points, and frequencies of administration, and the dosage based on clinically relevant effects resulting in clear increase, recovery or amelioration of the specific tissue or organ function. According to recently proposed guidelines, the harmonization of the nomenclature of PnD types will allow for the assessment of the most efficient treatments in various disease models. Experts within the COST SPRINT Action (CA17116), together with external collaborators, are doing the meta-analyses and reviews using the data prepared with the strategies presented here in the relevant disease or research fields. Our final aim is to provide standards to assess the safety and clinical benefit of PnD and to minimize redundancy in the use of animal models following the 3R principles for animal experimentation. Full article

The detection and quantification of protein–protein interactions (PPIs) is a crucial technique that often involves the use of recombinant proteins with fusion protein tags, such as maltose-binding protein (MBP) and glutathione-S-transferase (GST). In this study, we improved the cohesive and sticky properties of gelatinized starch by supplementing it with agarose, resulting in a harder gel that could coat the bottom of a microtiter plate. The resulting gelatinized starch/agarose mixture allowed for the efficient immobilization of MBP-tagged proteins on the coated plates, enabling the use of indirect ELISA-like PPI assays. By using the enzymatic activity of GST as an indicator, we succeeded in determining the dissociation constants between MBP-tagged and GST-tagged proteins on 96-well microtiter plates and a microplate reader without any expensive specialized equipment. Full article

Traditional Western blotting is one of the most used analytical techniques in biological research. However, it can be time-consuming and suffer from a lack of reproducibility. Consequently, devices with different degrees of automation have been developed. These include semi-automated techniques and fully automated devices that replicate all stages downstream of the sample preparation, including sample size separation, immunoblotting, imaging, and analysis. We directly compared traditional Western blotting with two different automated systems, iBind™ Flex, which is a semi-automated system designed to perform the immunoblotting, and JESS Simple Western™, a fully automated and capillary-based system performing all steps downstream of sample preparation and loading, including imaging and image analysis. We found that a fully automated system can save time and importantly offer valuable sensitivity. This is particularly beneficial for limited sample amounts. The downside of automation is the cost of devices and reagents. Nevertheless, automation can be a good option to increase output and facilitate sensitive protein analyses. Full article

Outer membrane vesicles (OMVs) are lipid structures containing various biomolecules in their native environment and are spontaneously shed by gram-negative bacteria. OMVs perform several biological functions critical to both bacterial physiology and pathogenicity. Scientific research on OMV function and biogenesis requires a standardized and robust method of isolating these vesicles from bacterial cultures that reliably provide high-purity OMVs. Herein, we describe an optimized protocol to isolate OMVs from overnight cultures of three different strains of nontypeable Haemophilus influenzae (NTHi) for use in different downstream applications. Involving mainly differential centrifugation of the culture supernatant, the procedure described is relatively simple, efficient, and generates high-quality OMV preparations from each strain tested with sufficient yields, while preserving the native outer membrane composition. Full article

While the general reliability of the Y balance test has been previously found to be excellent, earlier reviews highlighted a need for a more consistent methodology between studies. The purpose of this test–retest intrarater reliability study was to assess the intrarater reliability of the YBT using different methodologies regarding normalisation for leg length, number of repetitions, and score calculation. Sixteen healthy adult novice recreational runners aged 18–55 years, both women and men, were reviewed in a laboratory environment. Mean calculated scores, intraclass correlation coefficient, standard error of measurement, and minimal detectable change were calculated and analysed between different leg length normalisation and score calculation methods. The number of repetitions needed to reach a plateauing of results was analysed from the mean proportion of maximal reach per successful repetition. The intrarater reliability of the YBT was found to be good to excellent, and it was not affected by the method of score calculation or leg length measurement. The test results plateaued after the sixth successful repetition. Based on this study, it is suggested to use anterior superior iliac spine–medial malleolus length for leg length normalisation because this method was proposed in the original YBT protocol. At least seven successful repetitions should be performed to reach a result plateau. The average of the best three repetitions should be used to mitigate possible outliers and account for the learning effects seen in this study. Full article

Medicinal and herbal plants are abundant sources of phytochemicals, which are biologically active compounds with potential health benefits. The characterisation of phytochemicals has been the subject of many studies, but there is a lack of comprehensive assays to accurately assess the main phytochemical categories and their antioxidant properties. To address this, the present study has developed a multiparametric protocol comprising eight biochemical assays, which quantify the major categories of phytochemicals, including polyphenols, tannins and flavonoids, as well as their antioxidant and scavenging potential. The presented protocol offers several advantages over other methods, including higher sensitivity and significantly lower cost, making it a simpler and more affordable approach compared to commercial kits. The protocol was tested on two datasets with seventeen distinct herbal and medicinal plants, and the results demonstrated its effectiveness in accurately characterising the phytochemical composition of plant samples. The modular design of the protocol allows its adaptation to any spectrophotometric instrumentation, while all assays are simple to follow and require a minimum number of analytical steps. Full article

The application of the CRISPR/Cas9-based genome editing technique to the yeast Saccharomyces cerevisiae has made it possible to simultaneously modify several sites, particularly to integrate several expression cassettes. The existing methods provide high efficiency for such modifications; however, common protocols include several preparatory steps, namely, the construction of an intermediate Cas9-expressing strain, the assembly of a plasmid bearing several single guide RNA (sgRNA) expression cassettes, and the surrounding integrated DNA fragments with long flanks for recombination with target loci. Since these preparatory steps are time consuming and may not be desirable in some types of experiments, we explored the possibility of multiple integration without these steps. We have demonstrated that it is possible to skip them simultaneously and integrate up to three expression cassettes into separate sites by transforming the recipient strain with the Cas9 expression plasmid, three differently marked sgRNA plasmids, and three donor DNAs flanked with short (70 bp) arms for recombination. This finding increases the flexibility of choosing the optimal experimental design for multiple editing of the genome of S. cerevisiae and can significantly accelerate such experiments. Full article