Journal Description
Methods and Protocols
Methods and Protocols
is an international, peer-reviewed, open access journal aiming to establish and describe new experimental techniques in the biological and medical sciences, published bimonthly online by MDPI.
- Open Access— free for readers, with article processing charges (APC) paid by authors or their institutions.
- High Visibility: indexed within Scopus, ESCI (Web of Science), PubMed, PMC, CAPlus / SciFinder, and other databases.
- Journal Rank: CiteScore - Q2 (Biochemistry, Genetics and Molecular Biology (miscellaneous))
- Rapid Publication: manuscripts are peer-reviewed and a first decision is provided to authors approximately 27.2 days after submission; acceptance to publication is undertaken in 3.7 days (median values for papers published in this journal in the first half of 2023).
- Recognition of Reviewers: reviewers who provide timely, thorough peer-review reports receive vouchers entitling them to a discount on the APC of their next publication in any MDPI journal, in appreciation of the work done.
Impact Factor:
2.4 (2022);
5-Year Impact Factor:
2.4 (2022)
Latest Articles
Comparative Analysis of Primers Used for 16S rRNA Gene Sequencing in Oral Microbiome Studies
Methods Protoc. 2023, 6(4), 71; https://doi.org/10.3390/mps6040071 - 06 Aug 2023
Abstract
Recent advances in genomic technologies have enabled more in-depth study of the oral microbiome. In this study, we compared the amplicons generated by primers targeting different sites of the 16S rRNA gene found in the Human Oral Microbiome Database (HOMD). Six sets of
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Recent advances in genomic technologies have enabled more in-depth study of the oral microbiome. In this study, we compared the amplicons generated by primers targeting different sites of the 16S rRNA gene found in the Human Oral Microbiome Database (HOMD). Six sets of primer targeting V1–V2, V1–V3, V3–V4, V4–V5, V5–V7 and V6–V8 regions of 16S rRNA were tested via in silico simulation. Primers targeting the V1–V2, V3–V4, and V4–V5 regions generated more than 90% of the original input sequences. Primers targeting the V1–V2 and V1–V3 regions exhibited a low number of mismatches and unclassified sequences at the taxonomic level, but there were notable discrepancies at the species level. Phylogenetic tree comparisons showed primers targeting the V1–V2 and V3–V4 regions showed performances similar to primers targeting the whole 16s RNA region in terms of separating total oral microbiomes and periodontopathogens. In an analysis of clinical oral samples, V1–V2 primers showed superior performance for identifying more taxa and had better resolution sensitivity for Streptococcus than V3–V4 primers. In conclusion, primers targeting the V1–V2 region of 16S rRNA showed the best performance for oral microbiome studies. In addition, the study demonstrates the need for careful PCR primer selections.
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(This article belongs to the Section Omics and High Throughput)
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Open AccessArticle
Application of a Customised Franz-Type Cell Coupled with HPTLC to Monitor the Timed Release of Bioactive Components in Complex Honey Matrices
by
, , , , , and
Methods Protoc. 2023, 6(4), 70; https://doi.org/10.3390/mps6040070 - 03 Aug 2023
Abstract
The aim of this study was to assess the release profile of components in five different honeys (a New Zealand Manuka and two Western Australian honeys, a Jarrah honey and a Coastal Peppermint honey) and their corresponding honey-loaded gel formulations using a custom-designed
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The aim of this study was to assess the release profile of components in five different honeys (a New Zealand Manuka and two Western Australian honeys, a Jarrah honey and a Coastal Peppermint honey) and their corresponding honey-loaded gel formulations using a custom-designed Franz-type diffusion cell in combination with High-Performance Thin-Layer Chromatography (HPTLC). To validate the suitability of the customised setup, release data using this new approach were compared with data obtained using a commercial Franz cell apparatus, which is an established analytical tool to monitor the release of active ingredients from topical semisolid products. The release profiles of active compounds from pure honey and honey-loaded formulations were found to be comparable in both types of Franz cells. For example, when released either from pure honey or its corresponding pre-gel formulation, the percentage release of two Jarrah honey constituents, represented by distinct bands at RF 0.21 and 0.53 and as analysed by HPTLC, was not significantly different (p = 0.9986) at 12 h with over 99% of these honey constituents being released in both apparatus. Compared to the commercial Franz diffusion cell, the customised Franz cell offers several advantages, including easy and convenient sample application, the requirement of only small sample quantities, a large diffusion surface area, an ability to analyse 20 samples in a single experiment, and lower cost compared to purchasing a commercial Franz cell. Thus, the newly developed approach coupled with HPTLC is conducive to monitor the release profile of minor honey constituents from pure honeys and honey-loaded semisolid formulations and might also be applicable to other complex natural-product-based products.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessArticle
Ultrasound-Assisted Anthocyanins Extraction from Pigmented Corn: Optimization Using Response Surface Methodology
Methods Protoc. 2023, 6(4), 69; https://doi.org/10.3390/mps6040069 - 30 Jul 2023
Abstract
This study aimed to determine the optimal UAE conditions for extracting anthocyanins from pigmented corn using the Box–Behnken design (BBD). Six anthocyanins were identified in the samples and were used as response variables to evaluate the effects of the following working variables: extraction
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This study aimed to determine the optimal UAE conditions for extracting anthocyanins from pigmented corn using the Box–Behnken design (BBD). Six anthocyanins were identified in the samples and were used as response variables to evaluate the effects of the following working variables: extraction solvent pH (2–7), temperature (10–70 °C), solvent composition (0–50% methanol in water), and ultrasound power (20–80%). The extraction time (5–25 min) was evaluated for complete recovery. Response surface methodology suggested optimal conditions, specifically 36% methanol in water with pH 7 at 70 °C using 73% ultrasound power for 10 min. The method was validated with a high level of accuracy (>90% of recovery) and high precision (CV < 5% for both repeatability and intermediate precision). Finally, the proposed analytical extraction method was successfully applied to determine anthocyanins that covered a wide concentration range (36.47–551.92 mg kg−1) in several pigmented corn samples revealing potential varieties providing more health benefits.
Full article
(This article belongs to the Special Issue Women’s Special Issue Series: Analytical Methods)
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Open AccessProtocol
Geriatric Oncology in the Instagram Era: Feasibility and Acceptability Randomised Controlled Trial on Adopting PhotoVoice to Enable Empowerment, Patient-Centred Care, and Shared Decision Making—Study Protocol
by
, , , , , , and
Methods Protoc. 2023, 6(4), 68; https://doi.org/10.3390/mps6040068 - 26 Jul 2023
Abstract
Geriatric assessment (GA) is fundamental to optimising cancer care in older adults, yet implementing comprehensive GA tools in real-world clinical settings remains a challenge. This study aims to assess the feasibility and acceptability of integrating information from patient-derived photographs (PhotoVoice) into enhanced supportive
[...] Read more.
Geriatric assessment (GA) is fundamental to optimising cancer care in older adults, yet implementing comprehensive GA tools in real-world clinical settings remains a challenge. This study aims to assess the feasibility and acceptability of integrating information from patient-derived photographs (PhotoVoice) into enhanced supportive care (ESC) for older adults with cancer. A feasibility randomised controlled trial will be conducted at a regional cancer care centre in Australia. Participants aged 70 and above will be randomised into two groups: PhotoVoice plus ESC or usual care (ESC) alone. In the PhotoVoice group, participants will provide four photographs for deduction of representations of different aspects of their lives using photo-elicitation techniques. ESC will be conducted for both groups, incorporating PhotoVoice analysis in the intervention group. PhotoVoice may improve patient-centred care outcomes, including enhanced communication, shared decision making, and identification of patient priorities and barriers. Findings will provide insights into implementing PhotoVoice in geriatric assessment and guide future trials in cancer among older adults.
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(This article belongs to the Section Public Health Research)
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Open AccessProtocol
Variations in Microcirculatory and Hemodynamic Parameters during Oncological Demolitive–Reconstructive Head and Neck Surgery: A Protocol for an Observational Study
Methods Protoc. 2023, 6(4), 67; https://doi.org/10.3390/mps6040067 - 21 Jul 2023
Abstract
(1) Background: Oncological demolitive–reconstructive surgeries in the head and neck region cause significant stress on patients’ biohumoural, cardiac, and vascular systems, leading to disturbances in macrocirculatory and microcirculatory parameters. Traditional monitoring addresses the symptoms, but not the underlying cause. Microcirculatory assessments complement macrocirculatory
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(1) Background: Oncological demolitive–reconstructive surgeries in the head and neck region cause significant stress on patients’ biohumoural, cardiac, and vascular systems, leading to disturbances in macrocirculatory and microcirculatory parameters. Traditional monitoring addresses the symptoms, but not the underlying cause. Microcirculatory assessments complement macrocirculatory monitoring, and bladder-catheter-based technology offers a better representation of central microcirculation. Flap reconstruction surgeries involve demolitive and reconstructive phases, requiring optimal tissue perfusion. The literature lacks a consensus on macro–microcirculation coupling, and there is no agreement on the use of vasopressors during head and neck surgeries. Evidence-based guidelines are lacking, resulting in variations in vasopressor administration. (2) Methods: This is a 12-month observational, prospective study conducted in a single center. It aims to evaluate the impact of macro–microcirculation coupling on clinical complications in head and neck surgery. All consecutive patients undergoing oncologic surgery requiring flap reconstruction and meeting the inclusion criteria will be enrolled. The study will utilize standard hemodynamic monitoring and bladder catheterization for measuring urine output and temperature. (3) Conclusions: The study aims to evaluate the coupling of macro- and microcirculation in head and neck surgeries, assess hemodynamic parameters and microcirculatory changes, and investigate their association with postoperative complications. The results can enhance patient care and surgical outcomes.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessProtocol
Ex Vivo Perfusion of Porcine Pancreas and Liver Sourced from Commercial Abattoirs after Circulatory Death as a Research Resource: A Methodological Study
by
, , , , , , , and
Methods Protoc. 2023, 6(4), 66; https://doi.org/10.3390/mps6040066 - 12 Jul 2023
Abstract
Background: Machine perfusion (MP) is increasingly used for human transplant organ preservation. The use of MP for research purposes is another opportunity for this technology. The porcine pancreas and liver are similar in anatomical size and function to their human counterparts, making them
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Background: Machine perfusion (MP) is increasingly used for human transplant organ preservation. The use of MP for research purposes is another opportunity for this technology. The porcine pancreas and liver are similar in anatomical size and function to their human counterparts, making them an excellent resource for research, but they have some important differences from human organs which can influence their research use. In this paper, we describe a technique developed and tested for the retrieval of porcine organs for use in research on perfused viable organs. Methods: Whole-organ porcine pancreata and livers were harvested at a commercial abattoir, following standard slaughterhouse processes. The standard slaughterhouse process involved a thoracotomy and mid-line laparotomy, and all the thoracoabdominal organs were removed. The pancreas, fixed in the retroperitoneum, was carefully dissected from its attachments to the surrounding structures, and tissue planes between the pancreas, spleen, duodenum, and colon were meticulously identified and dissected. Vessel exposure and division: The aorta, portal vein (PV), hepatic vein (HV), and hepatic artery (HA) were dissected and isolated, preserving the input and output channels for the liver and pancreas. A distal 3 cm of the aorta was preserved and divided and served as the input for the pancreas perfusions. The liver, PV, HV, and HA were preserved and divided to preserve the physiological channels of the input (PV and HA) and output (HV) for the liver perfusions. The porcine hepatic and pancreas anatomy shares significant resemblance with the gross anatomy found in humans, and this was taken into consideration when designing the perfusion circuitry. The porcine pancreas and spleen shared a common blood supply, with branches arising from the splenic artery. The organs were flushed with cold, heparinised normal saline and transported in a temperature-regulated receptacle maintained at a core temperature between 4 and 8 °C, in line with the standards of static cold storage (SCS), to a dedicated perfusion lab and perfused using our novel perfusion machine with autologous, heparinised porcine blood, also collected at the abattoir.
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(This article belongs to the Section Synthetic and Systems Biology)
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Open AccessArticle
Assessment of SARS-CoV-2 (COVID-19) Clinical Mouthwash Protocol and Prevalence of the Oral Pathogen Scardovia wiggsiae: A Pilot Study of Antibacterial Effects
by
, , , , , , , and
Methods Protoc. 2023, 6(4), 65; https://doi.org/10.3390/mps6040065 - 06 Jul 2023
Abstract
One protocol in healthcare facilities and dental offices due to the COVID-19 pandemic for reducing the amount of detectable oral SARS-CoV-2 has been gargling with mouthwash for 60 s. This protocol lasts longer than the daily routine for most patients and may have
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One protocol in healthcare facilities and dental offices due to the COVID-19 pandemic for reducing the amount of detectable oral SARS-CoV-2 has been gargling with mouthwash for 60 s. This protocol lasts longer than the daily routine for most patients and may have unexpected benefits in reducing oral microbes as a result. This project evaluated the prevalence of the newly identified oral pathogen Scardovia wiggsiae before and after this procedure to determine any measurable effects. Using an approved protocol, n = 36 pre-mouthwash patient samples, n = 36 matched post-mouthwash samples, and n = 36 matched recall samples were identified (total sample number n = 108). DNA was isolated from each sample (pre-, post-mouthwash, and recall). Screening using qPCR and validated primers revealed n = 10/36 or 27.8% tested positive for Scardovia among the pre-mouthwash (Sample A) isolates with n = 3/36 or 8.3% testing positive among the post-mouthwash (Sample B) isolates. Screening of the recall (Sample C) samples has revealed n = 10/36, or 27.8% once again tested positive for Scardovia, demonstrating that this pathogen was found among a significant proportion of pediatric patient samples. Moreover, the COVID-19-related procedure of requiring sustained mouth washing prior to clinical treatment appears to reduce the levels of detectable Scardovia, at least initially. However, this study found no long-term effects using this isolated protocol.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessArticle
Development of a Wound-Healing Protocol for In Vitro Evaluation of Urothelial Cell Growth
Methods Protoc. 2023, 6(4), 64; https://doi.org/10.3390/mps6040064 - 05 Jul 2023
Abstract
Urethral healing is plagued by strictures, impacting quality of life and medical costs. Various growth factors (GFs) have shown promise as therapeutic approaches to improve healing, but there is no protocol for in vitro comparison between GFs. This study focuses the development of
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Urethral healing is plagued by strictures, impacting quality of life and medical costs. Various growth factors (GFs) have shown promise as therapeutic approaches to improve healing, but there is no protocol for in vitro comparison between GFs. This study focuses the development of a biomimetic in vitro urothelial healing assay designed to mimic early in vivo healing, followed by an evaluation of urothelial cell growth in response to GFs. Methods: Wound-healing assays were developed with human urothelial cells and used to compared six GFs (EGF, FGF-2, IGF-1, PDGF, TGF-β1, and VEGF) at three concentrations (1 ng/mL, 10 ng/mL, and 100 ng/mL) over a 48 h period. A commercial GF-containing medium (EGF, TGF-α, KGF, and Extract P) and a GF-free medium were used as controls. Results: There was a statistically significant increase in cell growth for IGF-1 at 10 and 100 ng/mL compared to both controls (p < 0.05). There was a statistically significant increase in cell growth for EGF at all concentrations compared to the GF-free medium control (p < 0.05). Conclusion: This study shows the development of a clinically relevant wound-healing assay to evaluate urothelial cell growth. It is the first to compare GFs for future use in reconstructive techniques to improve urethral healing.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessBrief Report
Precise Analysis of Nanoparticle Size Distribution in TEM Image
by
and
Methods Protoc. 2023, 6(4), 63; https://doi.org/10.3390/mps6040063 - 03 Jul 2023
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As an essential characterization, size distribution is an important indicator for the synthesis, optimization, and application of nanoparticles. Electron microscopes such as transmission electron microscopes (TEMs) are commonly utilized to collect size information on nanoparticles. However, the current popular statistical method of manually
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As an essential characterization, size distribution is an important indicator for the synthesis, optimization, and application of nanoparticles. Electron microscopes such as transmission electron microscopes (TEMs) are commonly utilized to collect size information on nanoparticles. However, the current popular statistical method of manually measuring large particles one by one, using a ruler tool in the corresponding image analysis software is time-consuming and can introduce manual errors. Moreover, it is difficult to determine the measurement interval for irregularly shaped nanoparticles. Therefore, it is necessary to use an efficient and standard method to perform size distribution analysis of nanoparticles. In this work, we use basic ImageJ software (1.53 t) to analyze the size of typical silica nanoparticles in a TEM image and use Origin software to process the data, to obtain its accurate distribution quickly. Using it as a template, we believe that this work can provide a paradigm for the standardized analysis of nanoparticle size.
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Open AccessProtocol
A New and Profitable Protocol to DNA Extraction in Limnospira maxima
by
, , , and
Methods Protoc. 2023, 6(4), 62; https://doi.org/10.3390/mps6040062 - 01 Jul 2023
Abstract
Limnospira maxima is a remarkable organism showing great potential as a versatile and sustainable food source, offering a powerful solution to address the pressing issues of malnutrition and undernourishment worldwide. L. maxima contains high amounts of proteins, vitamins, minerals, and essential fatty acids.
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Limnospira maxima is a remarkable organism showing great potential as a versatile and sustainable food source, offering a powerful solution to address the pressing issues of malnutrition and undernourishment worldwide. L. maxima contains high amounts of proteins, vitamins, minerals, and essential fatty acids. It can be grown in both bioreactors and open systems; however, before considering industrial production, optimization studies of the cultivation must be conducted to obtain knowledge about the ideal environmental conditions. Additionally, for the molecular typing of L. maxima strains and their industrial scaling, high-quality and large quantity DNA extraction is required. Notwithstanding, DNA extraction from L. maxima can be challenging due to the low amount of DNA in cells and the presence of difficult-to-remove substances such as polysaccharides and polyphenols. In this study, the quality and quantity of DNA extracted from two types of L. maxima samples (Limnospira maxima strain SISCA accession GenBank: OR195505.1) were evaluated using three commercially available DNA extraction kits and two types of input biological material. The results showed that Pbact-P kit had the highest quantity and quality of DNA, while CTAB-P allowed for a higher quantity and quality of RNA, making them optimal protocols for nucleic acid extraction to improve PCR, rt-PCR, and genome sequencing of L. maxima compared with other extraction methods.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessProtocol
Proposed Protocol for Field Testing of Endurance Fitness of Young Labrador Retrievers
Methods Protoc. 2023, 6(4), 61; https://doi.org/10.3390/mps6040061 - 28 Jun 2023
Abstract
The number of dogs and, with it, dog sports are growing in popularity, and the training of dogs begins at an early age. Although fitness testing is an imperative part of purposeful training and sports, to our knowledge, no objective field tests are
[...] Read more.
The number of dogs and, with it, dog sports are growing in popularity, and the training of dogs begins at an early age. Although fitness testing is an imperative part of purposeful training and sports, to our knowledge, no objective field tests are available for measuring young dogs’ endurance fitness. The aim of this study is to describe a simple, easy-to-repeat, and inexpensive way to test training intervention effects on endurance fitness in young Labrador Retrievers. Healthy client-owned 16-week-old Labrador Retrievers will be recruited and divided into test and control groups. The test group will have an eight-week training program followed by a four-week detraining period, while the control group will live a normal puppy life. All dogs will be tested for endurance fitness four times at four-week intervals: at baseline, one month later, two months later at the end of the training period, and one month after ending the training program. Each of the four testing sessions will be identical and will consist of four measurements of heart rate (HR) and blood lactate (BL): at baseline, after trotting 1000 m, after sprinting 200 m, and at recovery 5–8 min after the sprint. The training-induced changes in endurance fitness are evaluated by changes in HR and heart rate recovery times (HRR), BL, and running times.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessReview
New Prospects in the Electroanalysis of Heavy Metal Ions (Cd, Pb, Zn, Cu): Development and Application of Novel Electrode Surfaces
Methods Protoc. 2023, 6(4), 60; https://doi.org/10.3390/mps6040060 - 26 Jun 2023
Abstract
The detection of toxic heavy metal ions, especially cadmium (Cd), lead (Pb), zinc (Zn), and copper (Cu), is a global problem due to ongoing pollution incidents and continuous anthropogenic and industrial activities. Therefore, it is important to develop effective detection techniques to determine
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The detection of toxic heavy metal ions, especially cadmium (Cd), lead (Pb), zinc (Zn), and copper (Cu), is a global problem due to ongoing pollution incidents and continuous anthropogenic and industrial activities. Therefore, it is important to develop effective detection techniques to determine the levels of pollution from heavy metal ions in various media. Electrochemical techniques, more specifically voltammetry, due to its properties, is a promising method for the simultaneous detection of heavy metal ions. This review examines the current trends related to electrode formation and analysis techniques used. In addition, there is a reference to advanced detection methods based on the nanoparticles that have been developed so far, as well as formation with bismuth and the emerging technique of screen-printed electrodes. Finally, the advantages of using these methods are highlighted, while a discussion is presented on the benefits arising from nanotechnology, as it gives researchers new ideas for integrating these technologies into devices that can be used anywhere at any time. Reference is also made to the speciation of metals and how it affects their toxicity, as it is an important subject of research.
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(This article belongs to the Special Issue Women’s Special Issue Series: Analytical Methods)
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Open AccessTechnical Note
Optimized Protocol for Preservation of Human Platelet Samples for Fluorometric Polyphosphate Quantification
Methods Protoc. 2023, 6(4), 59; https://doi.org/10.3390/mps6040059 - 22 Jun 2023
Abstract
Platelet polyphosphate (polyP) can be conveniently quantified by exploiting a recent methodological breakthrough using 4′,6-diamidino-2-phenylindole (DAPI). However, the preservation of these biological samples has not yet been standardized. In a preliminary study, potential protocols were screened, while accepted protocols were further tested in
[...] Read more.
Platelet polyphosphate (polyP) can be conveniently quantified by exploiting a recent methodological breakthrough using 4′,6-diamidino-2-phenylindole (DAPI). However, the preservation of these biological samples has not yet been standardized. In a preliminary study, potential protocols were screened, while accepted protocols were further tested in this study. Pure-platelet-rich plasma (P-PRP) samples and washed platelet suspensions were prepared using blood obtained from non-smoking healthy male donors and were fixed with ThromboFix for 20–24 h at 4 °C. Mass polyP levels were determined using a fluorometer at wavelengths of 425 and 525 nm. Platelet polyP levels were normalized to platelet counts. Statistical analyses were performed using non-parametric tests. Platelet polyP levels significantly decreased by 20% after 7 days in the platelet suspension maintained under fixed conditions at 4 °C (control). In contrast, the platelet polyP levels in both the P-PRP and washed platelet suspensions were maintained without a significant reduction for up to 6 weeks by removing ThromboFix after fixation and subsequent freezing in pure water at −80 °C. Fluorometric polyP quantification often interferes with the low specificity of DAPI binding and the wavelength used. Our validated protocols will enable long-term preservation and high-throughput polyP quantification and can be applied to relatively large cohort studies.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
Non-Invasive Mapping of Cerebral Autoregulation Using Near-Infrared Spectroscopy: A Study Protocol
by
, , , , and
Methods Protoc. 2023, 6(3), 58; https://doi.org/10.3390/mps6030058 - 09 Jun 2023
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The ability of cerebral vessels to maintain a fairly constant cerebral blood flow is referred to as cerebral autoregulation (CA). Using near-infrared spectroscopy (NIRS) paired with arterial blood pressure (ABP) monitoring, continuous CA can be assessed non-invasively. Recent advances in NIRS technology can
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The ability of cerebral vessels to maintain a fairly constant cerebral blood flow is referred to as cerebral autoregulation (CA). Using near-infrared spectroscopy (NIRS) paired with arterial blood pressure (ABP) monitoring, continuous CA can be assessed non-invasively. Recent advances in NIRS technology can help improve the understanding of continuously assessed CA in humans with high spatial and temporal resolutions. We describe a study protocol for creating a new wearable and portable imaging system that derives CA maps of the entire brain with high sampling rates at each point. The first objective is to evaluate the CA mapping system’s performance during various perturbations using a block-trial design in 50 healthy volunteers. The second objective is to explore the impact of age and sex on regional disparities in CA using static recording and perturbation testing in 200 healthy volunteers. Using entirely non-invasive NIRS and ABP systems, we hope to prove the feasibility of deriving CA maps of the entire brain with high spatial and temporal resolutions. The development of this imaging system could potentially revolutionize the way we monitor brain physiology in humans since it would allow for an entirely non-invasive continuous assessment of regional differences in CA and improve our understanding of the impact of the aging process on cerebral vessel function.
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Open AccessArticle
A Custom Solution for Acoustic Startle Response Setup with Spike2-Based Data Acquisition Interface
Methods Protoc. 2023, 6(3), 57; https://doi.org/10.3390/mps6030057 - 08 Jun 2023
Abstract
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
[...] Read more.
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.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessArticle
Practical Method to Evaluate the Stiffness of Fractured Radius
Methods Protoc. 2023, 6(3), 56; https://doi.org/10.3390/mps6030056 - 05 Jun 2023
Abstract
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
[...] Read more.
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.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessTechnical Note
On-Slide Lambda Protein Phosphatase-Mediated Dephosphorylation of Fixed Samples
Methods Protoc. 2023, 6(3), 55; https://doi.org/10.3390/mps6030055 - 27 May 2023
Abstract
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
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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.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessProtocol
A Modified Protocol for the Isolation, Culture, and Characterization of Human Smooth Muscle Cells from the Umbilical Cord
Methods Protoc. 2023, 6(3), 54; https://doi.org/10.3390/mps6030054 - 26 May 2023
Abstract
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
[...] Read more.
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.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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An Improved Technique for Genotyping the ABCB1 Gene Variant of Exon 21
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, , , , and
Methods Protoc. 2023, 6(3), 53; https://doi.org/10.3390/mps6030053 - 26 May 2023
Abstract
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
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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.
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(This article belongs to the Special Issue Methods and Protocols 2023)
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Non-Antibiotic Prophylaxis for Recurrent UTIs in Neurogenic Lower Urinary Tract Dysfunction (NAPRUN): Study Protocol for a Prospective, Longitudinal Multi-Arm Observational Study
by
, , , , , , , , and
Methods Protoc. 2023, 6(3), 52; https://doi.org/10.3390/mps6030052 - 24 May 2023
Abstract
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,
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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.
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