Methods and Protocols

Recommendations released by the CDC in 2023 address the need to demonstrate that the RNA genome of positive-strand RNA viruses is inactivated in addition to viral particles. This recommendation is in response to the similarities between host mRNA and the viral genome that allow the viral RNA to be used as a template by host replication mechanisms to produce infectious viruses; therefore, there is concern that through artificial introduction into host cells, active positive-strand RNA genomes can be utilized to produce infectious viruses out of a containment facility. Utilizing 10% formalin for 7 days or 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M cacodylate buffer (glut/PFA) for 2 days to fix eastern equine encephalitis virus (EEEV)-infected non-human primate (NHP) brain tissue was found to effectively inactivate EEEV particles and genomic RNA. The methods assessed in this paper outline an effective means to validate both genomic RNA and viral particle inactivation. Full article

High-grade serous ovarian cancer (HGSOC) remains the most lethal gynecological malignancy, and there is still an unmet medical need to deepen basic research on its origins and mechanisms of progression. Patient-derived organoids of high-grade serous ovarian cancer (HGSOC-PDO) are a powerful model to study the complexity of ovarian cancer as they maintain, in vitro, the mutational profile and cellular architecture of the cancer tissue. Genetic modifications by lentiviral transduction allow novel insights into signaling pathways and the potential identification of biomarkers regarding the evolution of drug resistance. Here, we provide an in-depth and detailed protocol to successfully modify the gene expression of HGSOC-PDOs by lentiviral transduction. As an example, we validate our protocol and create a stable knockdown of the MACC1 oncogene with an efficacy of ≥72% in two HGSOC-PDO lines, which remained stable for >3 months in culture. Moreover, we explain step-by-step the sample preparation for the validation procedures on transcriptional (qPCR) and protein (Western Blot) levels. Sustained downregulation of specific genes by lentiviral transduction enables the analysis of the resulting phenotypic and morphological changes. It serves as a valuable in-vitro model to study the mechanisms of ovarian cancer pathogenesis and allows for the evaluation of therapeutic approaches. Full article

The accurate measurement of spatiotemporal parameters, such as step length and step frequency, is crucial for analyzing running and sprinting performance. Traditional methods like video analysis and force platforms are either time consuming or limited in scope, prompting the need for more efficient technologies. This study evaluates the effectiveness of a commercial Global Positioning System (GPS) unit integrated with an Inertial Measurement Unit (IMU) in capturing these parameters during sprints at varying velocities. Five experienced male runners performed six 40 m sprints at three velocity conditions (S: Slow, M: Medium, F: Fast) while equipped with a GPS-IMU system and an optical system as the gold standard reference. A total of 398 steps were analyzed for this study. Step frequency, step length and step velocity were extracted and compared using statistical methods, including the coefficient of determination (r²) and root mean square error (RMSE). Results indicated a very large agreement between the embedded system and the reference system, for the step frequency (r² = 0.92, RMSE = 0.14 Hz), for the step length (r² = 0.91, RMSE = 0.07 m) and the step velocity (r² = 0.99, RMSE = 0.17 m/s). The GPS-IMU system accurately measured spatiotemporal parameters across different running velocities, demonstrating low relative errors and high precision. This study demonstrates that GPS-IMU systems can provide comprehensive spatiotemporal data, making them valuable for both training and competition. The integration of these technologies offers practical benefits, helping coaches better understand and enhance running performance. Future improvements in sample rate acquisition GPS-IMU technology could further increase measurement accuracy and expand its application in elite sports. Full article

An electrochemical investigation of 1,2- and 1,4-dihydroxybenzenes was carried out with platinum macro- and microelectrodes using square wave and cyclic voltammetry techniques. Furthermore, the effect of the two solvents—acetic acid and ethyl acetate—was compared. When using square wave voltammetry, signals only appeared at lower frequencies and only when the supporting electrolyte was in excess, as expected due to the relatively low permittivity of the used solvents. The behavior of hydroquinone and catechol did not differ significantly from that of their derivatives (dihydroxybenzaldehydes, dihydroxybenzoic acids and 2′,5′-dihydroxyacetophenone). When the cyclic voltammetric experiments using a microelectrode were extended to higher anodic potentials, electrode fouling was very significant in ethyl acetate after the potential region where steady-state oxidation to the corresponding quinone occurs. The substituent effect was not significant here either, which was proven by using different functional groups in different positions. In contrast, the position had a dramatic influence on the susceptibility to electropolymerization, as 1,2-dihydroxybenzenes—independent of the nature of the substituent on the benzene ring—deactivated the electrode, while 1,4-dihydroxybenzenes did not, possibly due to the different solubilities of the polymers formed from the primary oxidation product (quinones). A user-friendly analytical procedure is also proposed that uses an electropolymerization reaction and does not require frequent cleaning of the electrode via polishing, which is required usually especially with a microelectrode. Full article

High-quality RNA is crucial in clinical diagnostics and precision medicine. Formalin-fixed and paraffin-embedded (FFPE) tissues pose a challenge due to nucleic acid fragmentation and crosslinking. In this pilot study, various commercially available techniques for extracting RNA from small FFPE samples were compared. We evaluated the KingFisher Duo automated system or the manual MagMAX FFPE DNA/RNA Ultra Kit as an RNA extraction method combined with either a xylene, d-limonene, or AutoLys M tubes deparaffinization method. Additionally, the automated Maxwell RSC RNA FFPE kit and the High Pure FFPET RNA Isolation Kit were examined using FFPE samples from inflammatory bowel disease (IBD) patients, as well as samples from ovarian, kidney, and breast cancer and the skin. The KingFisher Duo system gave a higher yield and more consistent RNA quantities, especially from small volumes of IBD samples, compared to manual extraction. The deparaffinization method also impacted results, with AutoLys M tubes proving effective in combination with the KingFisher Duo system. Conversely, the High Pure kit exhibited higher yields for larger FFPE samples. While RNA integrity is a critical factor, particularly for messenger RNA (mRNA) expression studies, its role is less prominent in microRNA (miRNA) analyses. Recognizing this, our study focused on RNA yield and purity (A260/A230) to evaluate RNA extraction methods for various sample types. These findings emphasize the importance of selecting appropriate RNA extraction methods based on sample characteristics and research goals, highlighting the performance of automated methods and the impact of deparaffinization choices. The findings contribute to refining RNA extraction for molecular biology analyses, suggesting avenues for further exploration, including cost-effectiveness under specific experimental conditions. Full article

The multifunctional catalytic hemoglobin from the terebellid polychaete Amphitrite ornata, also named dehaloperoxidase (AoDHP), utilizes the typical oxygen transport function in addition to four observed activities involved in substrate oxidation. The multifunctional ability of AoDHP is presently a rare observation, and there exists a limitation for how novel dehaloperoxidases can be identified from macrobenthic infauna. In order to discover more infaunal DHP-bearing candidates, we have devised a facilitated method for an accurate taxonomic identification that places visual and molecular taxonomic approaches in parallel. Traditional visual taxonomic species identification by the non-specialist, at least for A. ornata or even for other marine worms, is a very difficult and time-consuming task since a large diversity is present and the method is restricted to adult worm specimens. The work herein aimed to describe a method that simplifies the taxonomic identification of A. ornata in particular through the assessment of its mitochondrial cytochrome c oxidase subunit I gene by employing the DNA barcoding technique. Furthermore, whole-worm specimens of A. ornata were used to extract and purify AoDHP followed by an H₂O₂-dependent peroxidase activity assay evaluation against substrate 2,4,6-trichlorophenol. AoDHP isoenzyme A was also overexpressed as the recombinant protein in Escherichia coli, and its peroxidase activity parameters were compared to AoDHP from the natural source. The activity assay assessment indicated a tight correlation for all Michaelis–Menten parameters evaluated. We conclude that the method described herein exhibits a streamlined approach to identify the polychaete A. ornata, which can be adopted by the non-specialist, and the full procedure is predicted to facilitate the discovery of novel dehaloperoxidases from other marine invertebrates. Full article

Both the prevalence and mortality of liver cancers continue to rise. Early surgical interventions, including liver transplantation or resection, remain the only curative treatment. Nerves in the periphery influence tumor growth within visceral organs. Emerging cancer neuroscience efforts linked parasympathetic vagus nerves with tumor pathology, underscoring the value of vagal nerve denervation methods within cancer mouse models. Here, we describe a selective hepatic vagotomy that largely maintains non-liver parasympathetic innervation in mice. To address vagal interactions in hepatic tumor pathology, we provide an adapted methodology utilizing an established liver metastatic model. We anticipate that this methodology will expand the burgeoning field of cancer neuroscience, enabling the study of the neuroimmune, neurometabolic, and/or nerve–microbiota interactions shaping liver cancer progression and treatment. Full article

Background: About 287,000 women died globally during their pregnancy journey in 2020, yet most of these deaths could have been prevented. In Uganda, studies show that using Community Health Worker (CHW) visits to households with a pregnant woman can support the prevention of adverse maternal and neonatal outcomes. One such intervention is through the timed and targeted counselling (ttC) approach, where CHWs deliver tailored messages to mothers and their male caregivers at key stages of pregnancy. This study aims to evaluate the impact of the ttC approach on maternal health in Northern Uganda. The main outcomes include antenatal care attendance, advised place of delivery, and postnatal care visit. Methods: We will employ a cross-sectional quasi-experimental design, with retrospective data to compare an intervention group (where ttC is implemented) to a control group (without intervention) using the propensity score matching (PSM) technique applying a 1:1 ratio with a caliper width of 20% of the standard deviation to estimate the average treatment effects. Adjusted odds ratios after generating matched pairs will be reported with 95% confidence intervals with Rosenbaum sensitivity analysis carried out for robustness. Discussion: These findings can be used to modify the implementation of the ttC approach, thereby enhancing its efficiency and effectiveness. Full article

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