Methods Protoc., Volume 7, Issue 6 (December 2024) – 13 articles

Cyclic peptides have higher stability and better properties as therapeutic agents than their linear peptide analogues. Consequently, intramolecular click chemistry is becoming an increasingly popular method for the synthesis of cyclic peptides from their isomeric linear peptides. However, assessing the purity of these cyclic peptides by mass spectrometry is a significant challenge, as the linear and cyclic peptides have identical masses. In this paper, we have evaluated the analytical capabilities of energy-resolved mass spectrometry (ER MS) and mid-infrared microscopy (IR) to address this challenge. On the one hand, mixtures of both peptides were subjected to collision-induced dissociation tandem mass spectrometry (CID MS/MS) experiments in an ion trap mass spectrometer at several excitation energies. Two different calibration models were used: a univariate model (at a single excitation voltage) and a multivariate model (using multiple excitation voltages). The multivariate model demonstrated slightly enhanced analytical performance, which can be attributed to more effective signal averaging when multiple excitation voltages are considered. On the other hand, IR microscopy was used for the quantification of the relative amount of linear peptide. This was achieved through univariate calibration, based on the absorbance of an alkyne band specific to the linear peptide, and through Partial Least Squares (PLS) multivariate calibration. The PLS calibration model demonstrated superior performance in comparison to univariate calibration, indicating that consideration of the full IR spectrum is preferable to focusing on the specific peak of the linear peptide. The advantage of IR microscopy is that it is linear across the entire working interval, from linear peptide molar ratios of 0 (equivalent to pure cyclic peptide) up to 1 (pure linear peptide). In contrast, the ER MS calibration models exhibited linearity only up to 0.3 linear peptide molar ratio. However, ER MS showed better performances in terms of the limit of detection, intermediate precision and the root-mean-square-error of calibration. Therefore, ER MS is the optimal choice for the detection and quantification of the lowest relative amounts of linear peptides. Full article

Immunohistochemical (IHC) studies of formalin-fixed paraffin-embedded (FFPE) samples are a gold standard in oncology for tumor characterization, and the identification of prognostic and predictive markers. However, despite the abundance of archived FFPE samples, their research use is limited due to the labor-intensive nature of IHC on large cohorts. This study aimed to create a high-throughput workflow using modern technologies to facilitate IHC biomarker studies on large patient groups. Semiautomatic constructed tissue microarrays (TMAs) were created for two tumor patient cohorts and IHC stained for seven antibodies (ABs). AB expression in the tumor and surrounding stroma was quantified using the AI-supported image analysis software QuPath. The data were correlated with clinicopathological information using an R-script, all results were automatically compiled into formatted reports. By minimizing labor time to 7.7%—compared to whole-slide studies—the established workflow significantly reduced human and material resource consumption. It successfully correlated AB expression with overall patient survival and additional clinicopathological data, providing publication-ready figures and tables. The AI-assisted high-throughput TMA workflow, validated on two patient cohorts, streamlines modern histopathological research by offering cost and time efficiency compared to traditional whole-slide studies. It maintains research quality and preserves patient tissue while significantly reducing material and human resources, making it ideal for high-throughput research centers and collaborations. Full article

This study critically investigates the aluminium chloride–based colorimetric determination of the total flavonoid content (TFC) of honey. Following a comprehensive review of the recent literature reporting the use of the assay in the determination of TFC in honey, 10 honeys of different botanical origins were investigated using the colorimetric method alongside an artificial honey that was used as a control. Using spiking experiments, this study demonstrates that the flavonoid concentrations commonly found in honey are too low for a direct measurement and thus some of the TFC data reported in the literature might more likely be a reflection of the honey’s inherent colour rather than a product of the coordination complex formed specifically between flavonoids and Al³⁺ ions. This paper highlights the importance of correct blanking and suggests alternative approaches to the traditional TFC assay for honey to ensure analysis results that are truly reflective of honey’s TFC. Full article

Escherichia coli O157:H7, a Shiga-toxin-producing E. coli (STEC), is an important pathogen related to foodborne disease that is responsible for a growing number of outbreaks worldwide and has been detected in processed meats, dairy, and fresh vegetables. Although culturing is the gold standard method for detection of this bacterium, molecular methods based on nucleic acid amplification techniques such as PCR are becoming more common because of their rapidity, sensitivity, and specificity. However, to ensure reliable results among the several alternative PCR protocols (e.g., commercial kits and reference methods), different measurement assurance tools, including validated methods, reference materials, and proficiency tests, among others, are required. Herein, we present a digital PCR method validation for E. coli O157:H7 detection and quantification using seven specific gene sequences; this method quantified nucleic acids from different E. coli serotypes, with a detection range of 6.6 to 7900 copies/µL and a repeatability standard deviation over the concentration range of 1% to 13.6%. The relative standard uncertainty was 3.5–14.6%, and the detection limit was 0.27 copies/µL. Subsequently, two batches of a candidate reference material based on E. coli O157:H7 genomic DNA were then produced and characterized for evaluation of copy number concentration with the validated ddPCR method, with assigned values of 164,770 ± 9251 and 172 ± 9 copies/μL. Thus, this study demonstrated the development of a validated method and reference material for dPCR and qPCR detection of E. coli O157:H7, a key STEC responsible for food poisoning. Full article

Using the internet to recruit participants into research trials is effective but can attract high numbers of fraudulent attempts, particularly via social media. We drew upon the previous literature to rigorously identify and remove fraudulent attempts when recruiting rural residents into a community-based health improvement intervention trial. Our objectives herein were to describe our dynamic process for identifying fraudulent attempts, quantify the fraudulent attempts identified by each action, and make recommendations for minimizing fraudulent responses. The analysis was descriptive. Validation methods occurred in four phases: (1) recruitment and screening for eligibility and validation; (2) investigative periods requiring greater scrutiny; (3) baseline data cleaning; and (4) validation during the first annual follow-up survey. A total of 19,665 attempts to enroll were recorded, 74.4% of which were considered fraudulent. Automated checks for IP addresses outside study areas (22.1%) and reCAPTCHA screening (10.1%) efficiently identified many fraudulent attempts. Active investigative procedures identified the most fraudulent cases (33.7%) but required time-consuming interaction between researchers and individuals attempting to enroll. Some automated validation was overly zealous: 32.1% of all consented individuals who provided an invalid birthdate at follow-up were actively contacted by researchers and could verify or correct their birthdate. We anticipate fraudulent responses will grow increasingly nuanced and adaptive given recent advances in generative artificial intelligence. Researchers will need to balance automated and active validation techniques adapted to the topic of interest, population being recruited, and acceptable participant burden. Full article

Over the last few decades, malaria-derived extracellular vesicles (EVs) have gained increasing interest due to their role in disease pathophysiology and parasite biology. Unlike other EV research fields, the isolation of malaria EVs is not standardized, hampering inter-study comparisons. Most malaria EV studies isolate vesicles by the “gold-standard” technique of differential (ultra)centrifugation (DC). Here, we describe in detail an optimized and reproducible protocol for the isolation of malaria-derived EVs by DC. The protocol begins with a description of cultivating high-parasitemia, synchronous P. falciparum cultures that are the source of EV-containing conditioned culture media. The isolation protocol generates two EV subtypes, and we provide details of characterizing these distinct subtypes by analyzing human and parasite proteins by Western blot analysis. We identify some of these proteins as suitable markers for malaria EV subpopulations and subtypes. Full article

The present study proposes the use of Near-Infrared (NIR) spectroscopy as a rapid method for estimating the growth of Spirulina platensis cultures, avoiding any sample manipulation or pretreatment. NIR spectroscopy in diffuse reflectance mode was used on culture volumes as received, with Principal Component Analysis (PCA) and Partial Least Squares (PLS) linear regression, for developing the calibration model in the wavelength range of 1000–2500 nm, in order to choose the appropriate wavelength to estimate the growth of the microalga. The local reflectance maximum at 1062.6 nm, connected with reduced water absorption and scattering effects by the microalga, was identified from PCA as the positive peak in the first loading plot, correlating diffuse reflectance with dilution levels. The calibration curve of diffuse reflectance at 1062.6 nm in response to dilution presented strong linearity, supported by a coefficient of determination (R²) of 0.995. Cross-validation of NIR spectra with a S. platensis culture confirmed the method’s reliability, showing that the growth follows an exponential pattern. The study shows that diffuse reflectance NIR spectroscopy can be used for the rapid monitoring of Spirulina platensis growth. Full article

Background: The aim of this study is to analyze the reproducibility of sample thickness measurements taken by a non-experienced user by comparing a standard digital vernier caliper, with four different protocols, to a specialized thickness gauge. Methods: The current study is a methodological study where we examined the thickness of the porcine arterial wall in the thoracic aorta of six pigs. Two adjacent samples of 10 × 10 mm from each aorta were excised longitudinally from the anterior wall, resulting in twelve specimens. Five protocols were employed to measure the thickness of each sample. In four of these protocols, digital vernier calipers (Multicomp PRO MP012475) were utilized, while the fifth protocol utilized a specialized digital thickness gauge (Mitutoyo 547-500S, Mitutoyo Corp., Kawasaki, Japan). Results: We observed a higher average thickness of the samples during the initial measurement compared to the second measurement (1.11 ± 0.16 vs. 0.94 ± 0.17, p = 0.0319) with the first protocol and smaller values than those determined at the last measurement (0.93 ± 0.15 vs. 1.10 ± 0.15, p = 0.0135) for the third protocol. Further, with the digital vernier calipers, we recorded lower values for all four protocols than for the digital thickness gauge determinations. In addition, we computed the ratio of the thicknesses measured during the first, second, and third measurements to analyze how consistent the values were across the three consecutive measurements, with no difference regarding the third, fourth, and control protocols. Conclusions: The digital thickness gauge offers dependable measurements, regardless of the user’s expertise in assessing tissue thickness, and demonstrates a substantially higher reproducibility when compared to the digital vernier. We also found that taking an average of the thickness measurements from four specific points on each half of the sides or on each diagonal of each corner yielded consistently reliable results over time when using a standard digital vernier caliper instead of a specialized one. Full article

South Africa developed the differentiated service delivery (DSD) model to improve access to healthcare for people living with HIV (PLHIV), especially key populations (KPs) including female sex workers (FSWs) who often face barriers in accessing HIV services. The DSD model, aims to reduce the burden on healthcare users, healthcare workers, and the healthcare system, can significantly benefit this group. However, the success of the DSD model in achieving the desired HIV treatment outcomes for FSWs has been barely evaluated. This paper describes the protocol for evaluation of the DSD model in improving HIV treatment outcomes among FSWs in Gauteng Province of South Africa. Both qualitative and quantitative methods will be utilized to address three study objectives: stakeholder analysis, mapping, and in-depth interviews (objective 1); programme evaluation of the DSD model in selected sites (objective 2); and development of a framework for optimizing the DSD model in improving HIV treatment outcomes (objective 3). Quantitative statistical analysis will be performed using STATA version 17 (College Station, TX, USA). Qualitative analysis will be performed using ATLAS.ti. This study will provide new insights into the utilization of the DSD model among FSWs in South Africa. It will also inform new strategies for the DSD model’s implementation in the country. This study will contribute towards the development of a framework for strengthening the DSD model in improving HIV treatment outcomes among FSWs in Gauteng Province. Full article

Kappaphycus alvarezii is a red seaweed used globally in various biotechnological processes. To ensure the content and stability of its bioactive compounds postharvest, suitable drying protocols must be adopted to provide high-quality raw materials for industrial use. This study aimed to analyze the influence of freeze-drying and oven-drying on the total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity (FRAP and DPPH assays), total carotenoid content (TC), and lipase (LA) and protease activity (PA) of K. alvarezii samples collected over the seasons in sea farms in southern Brazil. The freeze-drying technique was found to be more effective regarding superior contents of TPC (39.23 to 127.74 mg GAE/100 g) and TC (10.27 to 75.33 μg/g), as well as DPPH (6.12 to 8.91 mg/100 g). In turn, oven-drying proved to be the best method regarding the TFC (4.99 to 12.29 mg QE/100 g) and PA (119.50 to 1485.09 U/g), with better performance in the FRAP (0.28 to 0.70 mmol/100 g). In this way, it appears that the drying process of the algal biomass can be selected depending on the required traits of the biomass for the intended industrial application. In terms of cost-effectiveness, drying the biomass using oven-drying can be considered appropriate. Full article

The valorization of coffee cherry waste through hydrothermal carbonization (HTC) was investigated using various organic and inorganic acid catalysts to produce platform chemicals. This study aimed to evaluate the effectiveness of these catalysts for enhancing reaction rates, improving yields, and promoting selectivity. The results showed that sulfuric acid and adipic acid were the most effective, each resulting in a 20% increase in the total yield, demonstrating the potential of organic acids as efficient catalysts in HTC. Other catalysts, such as benzoic acid and phenylacetic acid, also showed promising results, while butyric acid significantly decreased the total yield. The most abundantly produced platform chemicals were sugars, followed by formic acid, levulinic acid, HMF, and furfural. These findings highlight the potential of coffee cherry waste as a valuable resource for producing key chemicals, and the feasibility of hydrothermal carbonization as a sustainable approach for biomass valorization. This study emphasizes the importance of selecting the appropriate catalysts to optimize the conversion process and maximize the extraction of valuable chemicals. The environmental and economic implications of these findings are significant, as they can contribute to the development of sustainable and efficient biomass utilization technologies that could transform agricultural waste into high-value products while reducing waste and promoting a circular economy. Full article

Continuous advances in neurological research techniques are enabling researchers to further understand the neural mechanisms that regulate energy balance. In this review, we specifically highlight key tools and techniques and explore how they have been applied to study the role of the hypothalamic arcuate nucleus in feeding behaviors. Additionally, we provide a detailed discussion of the advantages and limitations associated with each methodology. Full article

The monitoring of species in a habitat is important to ensure biological diversity. Environmental DNA (eDNA) can infer the presence–absence of species and enable rapid action to avoid threatening factors in ecosystems in the case of non-indigenous species. Loop-mediated isothermal amplification (LAMP) assays for molecular amplification are rapidly gaining popularity in species detection, but LAMP remains an underutilized method for eDNA-based monitoring practices. The most effective combination for successful species monitoring may be the collection of eDNA or biological traces collected by nanofiltration followed by LAMP-based species detection initiatives. Here, we used LAMP analysis to detect the eDNA of Esox lucius (Northern Pike), Anguilla anguilla (European Eel), and Salmo salar (Atlantic Salmon) in Borre Lake and in the Drammen River. The selection of species is based on the categories of regionally invasive species, endangered species, and species of least concern. Two target genes were considered for each species and LAMP primers were designed. Our study showed that LAMP is an effective tool for discovering specific fish eDNA (analysis) to maintain aquatic ecosystems. Full article