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 fields of Life Sciences, Chemistry, and Biomedical 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 24.9 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 2024).
- 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:
2.3 (2023);
5-Year Impact Factor:
2.2 (2023)
Latest Articles
Full Validation and Application to Clinical Research of a High-Performance Liquid Chromatography Method for the Assessment of Urinary 3-Indoxyl Sulfate in Pediatric Patients with Hematopoietic Stem Cell Transplant
Methods Protoc. 2024, 7(4), 64; https://doi.org/10.3390/mps7040064 - 19 Aug 2024
Abstract
3-indoxyl sulfate (3-IS) results from a hepatic transformation of indole, a tryptophan degradation product produced by commensal gut bacteria. The metabolite has shown promise as a biomarker of dysbiosis and clinical outcomes following hematopoietic stem cell transplant (HSCT) in adults. Nonetheless, there is
[...] Read more.
3-indoxyl sulfate (3-IS) results from a hepatic transformation of indole, a tryptophan degradation product produced by commensal gut bacteria. The metabolite has shown promise as a biomarker of dysbiosis and clinical outcomes following hematopoietic stem cell transplant (HSCT) in adults. Nonetheless, there is a paucity of data regarding microbiome health and outcomes in the pediatric HSCT setting. We developed and thoroughly validated an affordable high-performance liquid chromatography/fluorescence detector (HPLC-FLD) method to quantify 3-IS in urine for use in the pediatric setting. Chromatographic separation was achieved on a C18 column (250 × 4.6 mm × 5 μm) with a mobile phase consisting of pH 4.0 acetic acid-triethylamine buffer and acetonitrile (88:12, v/v), eluted isocratically at 1 mL/min. 3-IS fluorescence detection was set at excitation/emission of 280 and 375, respectively. The method was fully validated according to FDA-specified limits including selectivity, linearity (0.10 to 10.00 mg/L, r2 > 0.997), intra- and inter-day accuracy, and precision. 3-IS stability was confirmed after three freeze–thaw cycles, for short- and medium-term on a benchtop and at 4 °C and for long-term up to 60 days at −20 °C. The validated method was used to quantify 3-IS in urine samples from HSCT pediatric patients.
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(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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Open AccessProtocol
Evaluation of an Italian Population-Based Programme for Risk Assessment and Genetic Counselling and Testing for BRCA1/2-Related Hereditary Breast and Ovarian Cancer after 10 Years of Operation: An Observational Study Protocol
by
Stefano Ferretti, Priscilla Sassoli de Bianchi, Debora Canuti, Cinzia Campari, Laura Cortesi, Valentina Arcangeli, Elena Barbieri, Cecilia D’Aloia, Rita Danesi, Pierandrea De Iaco, Margherita De Lillo, Laura Lombardo, Gabriella Moretti, Antonino Musolino, Dante Palli, Caterina Palmonari, Mila Ravegnani, Alfredo Tafà, Alessandra Tononi, Daniela Turchetti, Claudio Zamagni, Valentina Zampiga, Lauro Bucchi and the HBOC Study Groupadd
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Methods Protoc. 2024, 7(4), 63; https://doi.org/10.3390/mps7040063 - 13 Aug 2024
Abstract
Hereditary breast/ovarian cancer (HBOC) syndrome is caused by the inheritance of monoallelic germline BRCA1/2 gene mutations. If BRCA1/2 mutation carriers are identified before the disease develops, effective actions against HBOC can be taken, including intensive screening, risk-reducing mastectomy and salpingo-oophorectomy, and risk-reducing medications.
[...] Read more.
Hereditary breast/ovarian cancer (HBOC) syndrome is caused by the inheritance of monoallelic germline BRCA1/2 gene mutations. If BRCA1/2 mutation carriers are identified before the disease develops, effective actions against HBOC can be taken, including intensive screening, risk-reducing mastectomy and salpingo-oophorectomy, and risk-reducing medications. The Italian National Prevention Plan mandates the creation of regional BRCA genetic testing programmes. So far, however, only informal data have been reported on their implementation. We have designed a study aimed at evaluating the results of a population-based programme for risk assessment and genetic counselling and testing for BRCA1/2-related HBOC that is underway in the Emilia-Romagna region (northern Italy). The programme—which is entirely free—includes basic screening with an estimate of the likelihood of carrying a BRCA1/2 mutation using a familial risk assessment tool, a closer examination of women with suspected risk increase, an assessment of the need for further genetic counselling and, if needed, genetic testing and risk-reducing interventions. In this paper, the design of the programme and the protocol of the study are presented. The study has an observational, historical cohort design. Eligible are the women found to be at an increased risk of HBOC (profile 3 women). The main objectives are (i) to determine the precision of the programme in measuring the level of risk of HBOC for profile 3 women; (ii) to determine the characteristics of profile 3 women and their association with the risk management strategy chosen; (iii) to compare the age at onset, histologic type, tumour stage, molecular subtype, and prognosis of breast/ovarian cancers observed in the cohort of profile 3 women with the features of sporadic cancers observed in the general female population; (iv) to determine the level and the determinants of adherence to recommendations; and (v) to determine the appropriateness and timing of risk-reducing surgery and medications. Investigating the quality and results of the programme is necessary because the best practices in risk assessment and genetic counselling and testing for BRCA1/2-related cancer and the challenges they encounter should be identified and shared. The study has the potential to provide sound empirical evidence for the factors affecting the effectiveness of this type of service.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessArticle
A Mathematical Model of Pressure Ulcer Formation to Facilitate Prevention and Management
by
Ioannis G. Violaris, Konstantinos Kalafatakis, Nikolaos Giannakeas, Alexandros T. Tzallas and Markos Tsipouras
Methods Protoc. 2024, 7(4), 62; https://doi.org/10.3390/mps7040062 - 13 Aug 2024
Abstract
Pressure ulcers are a frequent issue involving localized damage to the skin and underlying tissues, commonly arising from prolonged hospitalization and immobilization. This paper introduces a mathematical model designed to elucidate the mechanics behind pressure ulcer formation, aiming to predict its occurrence and
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Pressure ulcers are a frequent issue involving localized damage to the skin and underlying tissues, commonly arising from prolonged hospitalization and immobilization. This paper introduces a mathematical model designed to elucidate the mechanics behind pressure ulcer formation, aiming to predict its occurrence and assist in its prevention. Utilizing differential geometry and elasticity theory, the model represents human skin and simulates its deformation under pressure. Additionally, a system of ordinary differential equations is employed to predict the outcomes of these deformations, estimating the cellular death rate in skin tissues and underlying layers. The model also incorporates changes in blood flow resulting from alterations in skin geometry. This comprehensive approach provides new insights into the optimal bed surfaces required to prevent pressure ulcers and offers a general predictive method to aid healthcare personnel in making informed decisions for at-risk patients. Compared to existing models in the literature, our model delivers a more thorough prediction method that aligns well with current data. It can forecast the time required for an immobilized individual to develop an ulcer in various body parts, considering different initial health conditions and treatment strategies.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessProtocol
The Sri Lanka Mother and Newborn Growth (S-MaNGro) Cohort: Protocol of a Nationwide Prospective Study
by
Malshani Lakshika Pathirathna, Megumi Haruna, Satoshi Sasaki, Kaori Yonezawa, Yuriko Usui and Yasuhiro Hagiwara
Methods Protoc. 2024, 7(4), 61; https://doi.org/10.3390/mps7040061 - 6 Aug 2024
Abstract
Perinatal cohort studies with a prospective longitudinal design are critical for determining the effects of early-life exposures on offspring’s health outcomes. The Sri Lanka Mother and Newborn Growth cohort study aims to investigate the impact of maternal nutritional and psychosocial factors on newborns’
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Perinatal cohort studies with a prospective longitudinal design are critical for determining the effects of early-life exposures on offspring’s health outcomes. The Sri Lanka Mother and Newborn Growth cohort study aims to investigate the impact of maternal nutritional and psychosocial factors on newborns’ birth weight in the Sri Lankan context. This paper presents the methodology of participant recruitment, follow-ups, an overview of measurements, and planned data analyses. This study included a nationally representative sample of Sri Lankan pregnant women recruited in their first trimester of pregnancy. Follow-up assessments were conducted once during the second and third trimesters of pregnancy and after the baby’s birth, prospectively tracking the women’s dietary intake, mental health, hemoglobin concentrations, and gestational weight gain data. Once the participants delivered their babies, the data on gestational age, sex of the newborn, birth weight, length and occipitofrontal circumference at birth, and mode of delivery were collected. Between August 2022 and August 2023, we recruited 2000 first-trimester pregnant women to the cohort and continued to follow up with them until the baby’s birth. The response rates were 90.4%, 81.4%, and 75.2% in the first, second, and third follow-ups. We plan to analyze the data in July 2024. We expect this study to provide valuable insights into various early-life exposures affecting neonatal birth weight. The study’s findings will serve as a valuable information resource for a broader scientific community, enabling the development of effective policies to prevent low-birth-weight deliveries in low-resource settings.
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(This article belongs to the Section Public Health Research)
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Open AccessArticle
Phytochemical Composition and Antimicrobial and Antibiofilm Effect of Myrciaria cauliflora Hydroethanolic Extract against Staphylococcus aureus and Acinetobacter baumannii
by
Luciane Dias de Oliveira, Ana Luisa Monteiro Ribeiro, Sthéfani de Oliveira Dias, Geovani Moreira da Cruz, Raquel Teles de Menezes, Lara Steffany de Carvalho, Mariana Gadelho Gimenez Diamantino, Thaís Cristine Pereira, Maria Cristina Marcucci and Amjad Abu Hasna
Methods Protoc. 2024, 7(4), 60; https://doi.org/10.3390/mps7040060 - 4 Aug 2024
Abstract
Staphylococcus aureus and Acinetobacter baumannii are opportunistic pathogens, and both are involved in different oral infections. This work aimed to analyze the phytochemical composition of Myrciaria cauliflora hydroethanolic extract and to evaluate its antimicrobial and antibiofilm action against Staphylococcus aureus (ATCC 6538) and
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Staphylococcus aureus and Acinetobacter baumannii are opportunistic pathogens, and both are involved in different oral infections. This work aimed to analyze the phytochemical composition of Myrciaria cauliflora hydroethanolic extract and to evaluate its antimicrobial and antibiofilm action against Staphylococcus aureus (ATCC 6538) and Acinetobacter baumannii (ATCC 19606; multi-resistant clinical strains 58004, 50098, 566006, and H557). Myrciaria cauliflora hydroethanolic extract was prepared, and the content of soluble solids, flavonoids, and phenols was quantified. High-performance liquid chromatography (HPLC) was performed later. The minimum inhibitory concentration was determined using the broth microdilution method according to the Clinical and Laboratory Standards Institute, standard M7-A6, and subsequently, its minimum bactericidal concentration was determined. Then, the most effective concentrations were analyzed against biofilms. Statistical analysis was performed using the ANOVA method with Tukey’s test. The soluble solids content in the prepared hydroethanolic extract of M. cauliflora was 2.22%. Additionally, the total flavonoid content, measured using the quercetin standard curve, was 0.040 mg/mL. Furthermore, the total phenol content, determined using the gallic acid standard curve, was 0.729 mg/mL. HPLC analysis presented peaks of gallic acid (11.80 m), p-coumaric acid (12.09 m), cinnamic acid derivative (19.02 m), and ellagic acid (29.83 m). The extract demonstrated antimicrobial and antibiofilm action against all tested strains. However, the most effective antibacterial concentration against all the tested bacteria was 5.55 mg/mL. Therefore, these chemical components justify that M. cauliflora hydroethanolic extract is effective in reducing biofilm formation in S. aureus (standard strain) and A. baumannii (standard and clinical strains).
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
Research on the Design and Performance of Plant Volatile Organic Compounds Water Removal Device Based on Optimized Filler Ratio
by
Yali Yuan, Huasen Wang, Zhihong Sun and Chao Yu
Methods Protoc. 2024, 7(4), 59; https://doi.org/10.3390/mps7040059 - 31 Jul 2024
Abstract
This study focuses on the development and optimization of a water removal device for biogenic volatile organic compounds (BVOCs) from plant emissions. BVOCs play a crucial role in various ecological processes and have potential therapeutic effects on human health. However, it is challenging
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This study focuses on the development and optimization of a water removal device for biogenic volatile organic compounds (BVOCs) from plant emissions. BVOCs play a crucial role in various ecological processes and have potential therapeutic effects on human health. However, it is challenging to accurately detect and analyze BVOCs due to their very low concentrations and interference by water vapor. This study systematically evaluates different filler materials and ratios to alleviate water vapor interference while maintaining BVOCs’ integrity. The experimental results demonstrate that the combination of MgSO4 + Na2SO4 mixed filling and CuSO4 layered filling in a 3:3:1 ratio can effectively improve the collection efficiency and detection accuracy of BVOCs. Meanwhile, the effectiveness of the device in improving the detection of volatile compounds in plant samples is also confirmed by the VOC verification experiments on Michelia maudiae and Cinnamomum camphora tree species after mechanical damage. The experimental results show that the device is effective in improving the detection of volatile compounds in plant samples. The findings provide a powerful technical means for exploring the role of BVOCs in environmental monitoring and scientific research.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessStudy Protocol
A Multicenter, Single-Arm, Prospective Trial to Evaluate Efficacy and Safety of Dose-Dense Methotrexate, Vinblastine, Doxorubicin, and Carboplatin (DD-MVACarbo) Chemotherapy for Cisplatin-Ineligible Patients with Advanced Urothelial Cancer: Study Protocol of the CARBUNCLE Trial
by
Makito Miyake, Satoshi Anai, Yusuke Iemura, Kazuki Ichikawa, Tatsuki Miyamoto, Atsushi Tomioka, Masaomi Kuwada, Yoshitaka Itami, Yukinari Hosokawa, Yoshiaki Matsumura, Eijiro Okajima, Kazumasa Torimoto, Nobutaka Nishimura, Mitsuru Tomizawa, Takuto Shimizu, Shunta Hori, Yosuke Morizawa, Daisuke Gotoh, Yasushi Nakai and Kiyohide Fujimoto
Methods Protoc. 2024, 7(4), 58; https://doi.org/10.3390/mps7040058 - 29 Jul 2024
Abstract
Unresectable, metastatic, advanced urothelial carcinoma (aUC) is an aggressive disease and is treated with platinum-containing first-line chemotherapy, followed by immune checkpoint inhibitors and antibody–drug conjugates. Response to first-line chemotherapy is a vital priority in sequential treatment strategies because a better response to first-line
[...] Read more.
Unresectable, metastatic, advanced urothelial carcinoma (aUC) is an aggressive disease and is treated with platinum-containing first-line chemotherapy, followed by immune checkpoint inhibitors and antibody–drug conjugates. Response to first-line chemotherapy is a vital priority in sequential treatment strategies because a better response to first-line chemotherapy is associated with a better response to subsequent therapies. Gemcitabine plus carboplatin chemotherapy is conventionally recommended for cisplatin-ineligible patients. This multicenter, single-arm prospective trial will investigate whether dose-dense methotrexate, vinblastine, doxorubicin, and carboplatin (DD-MVACarbo) chemotherapy is superior to gemcitabine plus carboplatin chemotherapy in terms of efficacy in platinum-naïve, cisplatin-ineligible patients with aUC. After screening and registration, a total of 46 patients will be treated with this novel chemotherapy regimen. The primary endpoint is the objective response rate. The secondary endpoints include disease control rate, patient-reported outcomes, and adverse events. No evidence of this novel intervention is available as of July 2024. The results are expected to change the standard of care and improve the management of patients with aUC.
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(This article belongs to the Section Public Health Research)
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Open AccessProtocol
A Protocol to Extract a Specific Genomic Region from a Public Whole-Genome Database and Modify Analytical Bin Length for Population Genetic Studies
by
Muhammad Shoaib Akhtar and Shoji Kawamura
Methods Protoc. 2024, 7(4), 57; https://doi.org/10.3390/mps7040057 - 27 Jul 2024
Abstract
With the advent of “next-generation” sequencing and the continuous reduction in sequencing costs, an increasing amount of genomic data has emerged, such as whole-genome, whole-exome, and targeted sequencing data. These applications are popular not only in mega sequencing projects, such as the 1000
[...] Read more.
With the advent of “next-generation” sequencing and the continuous reduction in sequencing costs, an increasing amount of genomic data has emerged, such as whole-genome, whole-exome, and targeted sequencing data. These applications are popular not only in mega sequencing projects, such as the 1000 Genomes Project and UK BioBank, but also among individual researchers. Evolutionary genetic analyses, such as the dN/dS ratio and Tajima’s D, are demanded more and more for whole-genome-level population data. These analyses are often carried out under a uniform custom bin size across the genome. However, these analyses require subdivision of a genomic region into functional units, such as protein-coding regions, introns, and untranslated regions, and computing these genetic measures for large-scale data remains challenging. In a recent investigation, we successfully devised a method to address this issue. This method requires a multi-sample VCF file containing population data, a reference genome, target regions in the BED file, and a list of samples to be included in the analysis. Given that the targeted regions are extracted in a new VCF file, targeted population genetic analysis can be performed. We conducted Tajima’s D analysis using this approach on intact and pseudogenes, as well as non-coding regions.
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(This article belongs to the Section Omics and High Throughput)
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Open AccessArticle
Image Processing Analysis of Plant Morphometry with Examples from the Genus Sedum (Crassulaceae)
by
Mădălina Borcă, Alexandru Borcă, Alin Ciobica, Gabriela Halitchi and Andrei Stoie
Methods Protoc. 2024, 7(4), 56; https://doi.org/10.3390/mps7040056 - 24 Jul 2024
Abstract
The complex systematics of the genus Sedum, the difficulties of its classification and the ambiguity of the concrete identification of the taxa brought about the need to implement a measurement system adaptable to field conditions, so as to facilitate the accuracy of
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The complex systematics of the genus Sedum, the difficulties of its classification and the ambiguity of the concrete identification of the taxa brought about the need to implement a measurement system adaptable to field conditions, so as to facilitate the accuracy of data collection, avoiding the etiolation of samples and, therefore, the deterioration of the morphological structures subject to analysis. Thus, our study describes a digitization of the classic method of making measurements using millimeter paper, thus facilitating the subsequent statistical processing of quantifiable values. Depending on the number of pixels in the photos taken and the pixel/millimeter ratio, a variable measurement scale can be created depending on the size of the analyzed taxomes. The method used adds to the classic taxonomy, which is based on the analysis of morphological characteristics to determine the species of these succulent plants. The applicability of our method is shown by means of the example of an analysis performed on the flowers of the native species of the genus Sedum in the territory of Romania.
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(This article belongs to the Special Issue Plant Tissue Culture for Crop Improvement)
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Open AccessArticle
Odours in Asphalt: Analysis of the Release of H2S from Bitumen by a Mass Spectrometric Residual Gas Analyser
by
Marcello Monteleone, Giuseppe Di Luca, Marcello Filomia, Alessio Fuoco, Alberto Figoli and Johannes Carolus Jansen
Methods Protoc. 2024, 7(4), 55; https://doi.org/10.3390/mps7040055 - 15 Jul 2024
Abstract
During the production and laying phases of hot-mixing asphalt (HMA), various volatile organic compounds (VOCs) and noxious gases such as H2S are released into the atmosphere. These emissions are a serious environmental problem, a risk to human health, and expose workers
[...] Read more.
During the production and laying phases of hot-mixing asphalt (HMA), various volatile organic compounds (VOCs) and noxious gases such as H2S are released into the atmosphere. These emissions are a serious environmental problem, a risk to human health, and expose workers and residents to unfriendly odours. The aim of this study was the development of a fast and sensitive analytical method to detect the H2S emitted from hot bituminous binder that is generally used in the various stages of asphalt production, processing, handling and during road construction. The method consisted in the analysis of evolved H2S from a flask with molten bitumen, using nitrogen as a carrier gas to lead the volatile compounds into a residual gas analyser equipped with a quadrupole mass spectrometer. The analysis was performed following the H2S-specific signals at m/z 33 (HS+) and at m/z 34 (H2S+) in real time, directly on the sample without laborious and expensive pre-treatments and with short response times (<6 s). Calibration with a standard mixture of 1000 ppm of H2S in nitrogen allows semi-quantitative H2S detection. The sensitivity and rapidity of the method were evaluated by quenching the release of sulphur compounds with commercial odour-suppressing agents. Upon addition of 0.1% of additive in two minutes, the H2S signal drops about 80% in two minutes, confirming the good response of the method, even with a very complex matrix.
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(This article belongs to the Special Issue Feature Papers in Methods and Protocols 2024)
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Open AccessStudy Protocol
Protocol for the Development and Initial Validation of the COG-IMPACT Tool: A Purpose-Built Unmet Needs Assessment for Cancer-Related Cognitive Impairment
by
Darren Haywood, Moira O’Connor, Frank D. Baughman, Alexandre Chan, Raymond J. Chan, Evan Dauer, Haryana M. Dhillon, Ashley M. Henneghan, Blake J. Lawrence, Maryam Lustberg, Janette L. Vardy, Susan L. Rossell and Nicolas H. Hart
Methods Protoc. 2024, 7(4), 54; https://doi.org/10.3390/mps7040054 - 10 Jul 2024
Abstract
(1) Background: A significant proportion of cancer survivors report experiencing a cognitive ‘fog’ that affects their ability to think coherently and quickly, and reason with clarity. This has been referred to as cancer-related cognitive impairment (CRCI). CRCI has extensive impacts on the
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(1) Background: A significant proportion of cancer survivors report experiencing a cognitive ‘fog’ that affects their ability to think coherently and quickly, and reason with clarity. This has been referred to as cancer-related cognitive impairment (CRCI). CRCI has extensive impacts on the daily lives of people living with or beyond cancer, including occupational, social, and psychological functioning. Oncology health professionals report feeling under-resourced to effectively assess the needs of an individual with CRCI and then provide optimal care and referral. (2) Methods: The objective of this project is to develop and provide an initial validation of the first purpose-built unmet needs assessment for CRCI: the Unmet Needs Assessment of Cancer-Related Cognitive Impairment Impact (COG-IMPACT). We will use a multiple-stage, co-design, mixed-methods approach to develop and provide an initial validation of the COG-IMPACT. (3) Results: The primary anticipated result of this research is the production of the COG-IMPACT, the first purpose-built unmet needs assessment for CRCI. The assessment could be used by health professionals to understand the unmet needs and facilitate optimal care and referral for cancer survivors, by survivors to elucidate their supportive needs and advocate for their care, and by researchers to examine the correlates of unmet needs relating to CRCI, as well as how best to support people with CRCI.
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(This article belongs to the Section Public Health Research)
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Open AccessCommunication
Suitability of XRF for Routine Analysis of Multi-Elemental Composition: A Multi-Standard Verification
by
Riccardo Fedeli, Luigi Antonello Di Lella and Stefano Loppi
Methods Protoc. 2024, 7(4), 53; https://doi.org/10.3390/mps7040053 - 5 Jul 2024
Abstract
This study investigated the suitability of X-ray fluorescence (XRF) analysis for routine multi-elemental composition analysis, checking its analytical capabilities by measuring a wide array of certified reference materials of soil and plant origin. A portable XRF analyzer was used to evaluate 32 soil
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This study investigated the suitability of X-ray fluorescence (XRF) analysis for routine multi-elemental composition analysis, checking its analytical capabilities by measuring a wide array of certified reference materials of soil and plant origin. A portable XRF analyzer was used to evaluate 32 soil and 12 plant standard materials, using both the Soil and Geochem mode, with sequential beams, allowing the detection of a wide range of elements. Recovery rates were calculated by comparing XRF measurements with certified values, and their correlations were verified through the Spearman coefficient. The results demonstrated the reliability of XRF measurements for soil samples, with a large number of elements showing a good or very good recovery and strong correlations with certified values. For plant samples, XRF largely overestimated the certified values, but the strong statistically significant correlations for almost all tested elements allowed us to correct this systematic bias, using the reported median value for dividing the value obtained via XRF. The Geochem mode emerged as more reliable for a larger number of elements. It was concluded that XRF may be a suitable alternative to ICP-MS in routine multi-elemental composition analysis.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessReview
Methods to Investigate the Secretome of Senescent Cells
by
Afshin Samiminemati, Domenico Aprile, Dario Siniscalco and Giovanni Di Bernardo
Methods Protoc. 2024, 7(4), 52; https://doi.org/10.3390/mps7040052 - 2 Jul 2024
Abstract
The word “secretome” was first used to describe the proteins that cells secrete under different circumstances; however, recent studies have proven the existence of other molecules such as RNA and chemical compounds in the secretome. The study of secretome has significance for the
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The word “secretome” was first used to describe the proteins that cells secrete under different circumstances; however, recent studies have proven the existence of other molecules such as RNA and chemical compounds in the secretome. The study of secretome has significance for the diagnosis and treatment of disease as it provides insight into cellular functions, including immune responses, development, and homeostasis. By halting cell division, cellular senescence plays a role in both cancer defense and aging by secreting substances known as senescence-associated secretory phenotypes (SASP). A variety of techniques could be used to analyze the secretome: protein-based approaches like mass spectrometry and protein microarrays, nucleic acid-based methods like RNA sequencing, microarrays, and in silico prediction. Each method offers unique advantages and limitations in characterizing secreted molecules. Top-down and bottom-up strategies for thorough secretome analysis are became possible by mass spectrometry. Understanding cellular function, disease causes, and proper treatment targets is aided by these methodologies. Their approaches, benefits, and drawbacks will all be discussed in this review.
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(This article belongs to the Section Molecular and Cellular Biology)
Open AccessReview
A Review of Histological Techniques for Differentiating Human Bone from Animal Bone
by
Emanuela Stan, Camelia-Oana Muresan, Ecaterina Daescu, Raluca Dumache, Veronica Ciocan, Stefania Ungureanu, Dan Costachescu and Alexandra Enache
Methods Protoc. 2024, 7(4), 51; https://doi.org/10.3390/mps7040051 - 30 Jun 2024
Abstract
The first step in anthropological study is the positive identification of human remains, which can be a challenging undertaking when bones are broken. When bone pieces from different species are mixed together, it can be crucial to distinguish between them in forensic and
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The first step in anthropological study is the positive identification of human remains, which can be a challenging undertaking when bones are broken. When bone pieces from different species are mixed together, it can be crucial to distinguish between them in forensic and archaeological contexts. For years, anthropology and archaeology have employed the histomorphological analysis of bones to evaluate species-specific variations. Based on variations in the dimensions and configuration of Haversian systems between the two groups, these techniques have been devised to distinguish between non-human and human bones. All of those techniques concentrate on a very particular kind of bone, zone, and segment. Histomorphometric techniques make the assumption that there are size, form, and quantity variations between non-humans and humans. The structural components of Haversian bones are significant enough to use discriminant function analysis to separate one from the other. This review proposes a comprehensive literature analysis of the various strategies or techniques available for distinguishing human from non-human bones to demonstrate that histomorphological analysis is the most effective method to be used in the case of inadequate or compromised samples.
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(This article belongs to the Section Tissue Engineering and Organoids)
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Open AccessProtocol
In Vitro Methods for Assessing the Antibacterial and Antibiofilm Properties of Essential Oils as Potential Root Canal Irrigants—A Simplified Description of the Technical Steps
by
Jihad Diouchi, Jelena Marinković, Milica Nemoda, Lhoussaine El Rhaffari, Babacar Toure and Sonia Ghoul
Methods Protoc. 2024, 7(4), 50; https://doi.org/10.3390/mps7040050 - 25 Jun 2024
Abstract
Background: Essential oils have gained in significance due to their various biological activities, and there is a growing demand for them in many industries. The present article focuses on the technical steps for an in vitro evaluation of the antibacterial and antibiofilm activities
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Background: Essential oils have gained in significance due to their various biological activities, and there is a growing demand for them in many industries. The present article focuses on the technical steps for an in vitro evaluation of the antibacterial and antibiofilm activities of essential oils for potential use as root canal irrigant in dentistry. Methods: The bioactivities of the essential oil were investigated through in vitro assays. The gram-positive bacterium Enterococcus faecalis was used as a micro-organism model. The antibacterial activity of the essential oil was assessed using the microdilution method, and resazurin staining to determine the minimal inhibition concentrations (MICs) and the minimal bactericidal concentrations (MBCs). The antibiofilm effect was evaluated spectrophotometrically at 570 nm using the microplate cultivation technique and crystal violet staining. Conclusions: This article features a detailed in vitro protocol to facilitate the preparation of the essential oil samples, the bacterial suspension, and the methods used for assessment of the antibiofilm and antibacterial activities of the essential oil. The advantages of these approaches are presented in relation to the limits linked to the choice of the bacteria and the essential oil.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays
by
Janina Fischer, Jan Ole Kaufmann and Michael G. Weller
Methods Protoc. 2024, 7(3), 49; https://doi.org/10.3390/mps7030049 - 13 Jun 2024
Abstract
The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody–antigen pair. Many methods based on
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The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody–antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise and effort that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC50 approaches the Kd value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to Kd. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols.
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(This article belongs to the Section Biochemical and Chemical Analysis & Synthesis)
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Open AccessProtocol
A Comprehensive Methodology for Microbial Strain Typing Using Fourier-Transform Infrared Spectroscopy
by
Francis Muchaamba and Roger Stephan
Methods Protoc. 2024, 7(3), 48; https://doi.org/10.3390/mps7030048 - 11 Jun 2024
Abstract
Timely and accurate detection and characterization of microbial threats is crucial for effective infection and outbreak management. Additionally, in food production, rapid microbe identification is indispensable for maintaining quality control and hygiene standards. Current methods for typing microbial strains often rely on labor-intensive,
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Timely and accurate detection and characterization of microbial threats is crucial for effective infection and outbreak management. Additionally, in food production, rapid microbe identification is indispensable for maintaining quality control and hygiene standards. Current methods for typing microbial strains often rely on labor-intensive, time-consuming, and expensive DNA- and sera-serotyping techniques, limiting their applicability in rapid-response scenarios. In this context, the IR Biotyper®, utilizing Fourier-transform infrared (FTIR) spectroscopy, offers a novel approach, providing specific spectra for fast strain typing within 3 h. This methodology article serves as a comprehensive resource for researchers and technicians aiming to utilize FTIR spectroscopy for microbial strain typing. It encompasses detailed guidelines on sample preparation, data acquisition, and analysis techniques, ensuring the generation of reliable and reproducible results. We highlight the IR Biotyper®’s rapid and accurate discrimination capabilities, showcasing its potential for real-time pathogen monitoring and source-tracking to enhance public health and food safety. We propose its integration as an early screening method, followed by more detailed analysis with whole-genome sequencing, to optimize detection accuracy and response efficiency in microbial surveillance systems.
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(This article belongs to the Section Molecular and Cellular Biology)
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Open AccessStudy Protocol
Evaluation of the Sexually Transmitted Infections Programme among Key and Priority Populations in Primary Healthcare Facilities to Inform a Targeted Response: A Protocol Paper
by
Mohlago Ablonia Seloka, Edith Phalane and Refilwe Nancy Phaswana-Mafuya
Methods Protoc. 2024, 7(3), 47; https://doi.org/10.3390/mps7030047 - 5 Jun 2024
Abstract
Primary healthcare facilities lack routine diagnostic screening due to resource limitations and dependence on syndromic management, resulting in an unprecedented prevalence and incidence of sexually transmitted infections (STIs), particularly among key and priority populations. Specific focuses are essential to strengthen current STI control
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Primary healthcare facilities lack routine diagnostic screening due to resource limitations and dependence on syndromic management, resulting in an unprecedented prevalence and incidence of sexually transmitted infections (STIs), particularly among key and priority populations. Specific focuses are essential to strengthen current STI control measures. Therefore, this article describes the protocol for evaluating STI programme among key and priority populations in selected primary healthcare facilities in South Africa. We will employ an exploratory, descriptive research design to assess the STI programme in terms of its facility operations, functions, scope, gaps, delivery services, STI surveillance methods, and indicators in the selected primary healthcare facilities. A purposive sample of 15–20 STI programme stakeholders will be selected from five primary healthcare facilities in Limpopo Province, South Africa. The programme evaluation will use the World Health Organization assessment checklist tool, a globally recognised and validated instrument comprising open- and closed-ended questions to assess the STI programme. This tool, known for its credibility and reliability, ensures the study’s validity. Quantitative data will be captured on STATA software (College Station, TX, USA) version 18 for descriptive analysis and presented as the mean and standard deviation for continuous variables, proportions and percentages for categorical variables. A p ≤ 0.05 will demonstrate a statistically significant level. Thematic content analysis will be conducted for the qualitative data using Atlas. ti software (Technical University, Berlin, Germany) version 23.1. The study’s results will inform new approaches to strengthen STI coverage, service delivery, and linkage to care.
Full article
(This article belongs to the Section Public Health Research)
Open AccessReview
Advances in 3D Inner Ear Reconstruction Software for Cochlear Implants: A Comprehensive Review
by
Michail Athanasopoulos, Pinelopi Samara and Ioannis Athanasopoulos
Methods Protoc. 2024, 7(3), 46; https://doi.org/10.3390/mps7030046 - 23 May 2024
Abstract
Auditory impairment stands as a pervasive global issue, exerting significant effects on individuals’ daily functioning and interpersonal engagements. Cochlear implants (CIs) have risen as a cutting-edge solution for severe to profound hearing loss, directly stimulating the auditory nerve with electrical signals. The success
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Auditory impairment stands as a pervasive global issue, exerting significant effects on individuals’ daily functioning and interpersonal engagements. Cochlear implants (CIs) have risen as a cutting-edge solution for severe to profound hearing loss, directly stimulating the auditory nerve with electrical signals. The success of CI procedures hinges on precise pre-operative planning and post-operative evaluation, highlighting the significance of advanced three-dimensional (3D) inner ear reconstruction software. Accurate pre-operative imaging is vital for identifying anatomical landmarks and assessing cochlear deformities. Tools like 3D Slicer, Amira and OTOPLAN provide detailed depictions of cochlear anatomy, aiding surgeons in simulating implantation scenarios and refining surgical approaches. Post-operative scans play a crucial role in detecting complications and ensuring CI longevity. Despite technological advancements, challenges such as standardization and optimization persist. This review explores the role of 3D inner ear reconstruction software in patient selection, surgical planning, and post-operative assessment, tracing its evolution and emphasizing features like image segmentation and virtual simulation. It addresses software limitations and proposes solutions, advocating for their integration into clinical practice. Ultimately, this review underscores the impact of 3D inner ear reconstruction software on cochlear implantation, connecting innovation with precision medicine.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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Open AccessStudy Protocol
MUltiparametric Score for Ventilation Discontinuation in Intensive Care Patients: A Protocol for an Observational Study
by
Iacopo Cappellini, Andrea Cardoni, Lorenzo Campagnola and Guglielmo Consales
Methods Protoc. 2024, 7(3), 45; https://doi.org/10.3390/mps7030045 - 20 May 2024
Abstract
Background: Mechanical ventilation significantly improves patient survival but is associated with complications, increasing healthcare costs and morbidity. Identifying optimal weaning times is paramount to minimize these risks, yet current methods rely heavily on clinical judgment, lacking specificity. Methods: This study introduces a novel
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Background: Mechanical ventilation significantly improves patient survival but is associated with complications, increasing healthcare costs and morbidity. Identifying optimal weaning times is paramount to minimize these risks, yet current methods rely heavily on clinical judgment, lacking specificity. Methods: This study introduces a novel multiparametric predictive score, the MUSVIP (MUltiparametric Score for Ventilation discontinuation in Intensive care Patients), aimed at accurately predicting successful extubation. Conducted at Santo Stefano Hospital’s ICU, this single-center, observational, prospective cohort study will span over 12 months, enrolling adult patients undergoing invasive mechanical ventilation. The MUSVIP integrates variables measured before and during a spontaneous breathing trial (SBT) to formulate a predictive score. Results: Preliminary analyses suggest an Area Under the Curve (AUC) of 0.815 for the MUSVIP, indicating high predictive capacity. By systematically applying this score, we anticipate identifying patients likely to succeed in weaning earlier, potentially reducing ICU length of stay and associated healthcare costs. Conclusion: This study’s findings could significantly influence clinical practices, offering a robust, easy-to-use tool for optimizing weaning processes in ICUs.
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(This article belongs to the Section Biomedical Sciences and Physiology)
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