Methods Protoc., Volume 6, Issue 5 (October 2023) – 31 articles

This protocol describes the methodology and methods for a collaborative project with eight Aboriginal and Torres Strait Islander primary health care (PHC) organisations, across three Australian states and one territory, to increase clinical service performance and access to preventive health and health promotion services for preventing, identifying, treating, and managing dementia risk in Aboriginal and Torres Strait Islander communities. Aboriginal participatory action research (APAR) methodology will be the framework for this project, incorporating continuous quality improvement (CQI), informed by research yarning with stakeholder groups, comprising community members and PHC staff and service providers and data collected from the auditing of client health records and the mapping of existing clinical processes and health services at each partnering PHC organisation. The qualitative and quantitative data will be summarised and discussed with stakeholder groups. Priorities will be identified and broken down into tangible PHC organisation deliverable strategies and programs, which will be co-developed with stakeholder groups and implemented cyclically over 24 months using the Plan, Do, Study, Act model of change. Key project outcome measures include increased clinical service performance and availability of preventive health and health promotion services for safeguarding against dementia. Project implementation will be evaluated for quality and transparency from an Indigenous perspective using an appropriate appraisal tool. The project processes, impact, and sustainability will be evaluated using the RE-AIM framework. A dementia safeguarding framework and accompanying tool kit will be developed from this work to support Aboriginal and Torres Strait Islander PHC organisations to identify, implement, and evaluate dementia safeguarding practice and service improvements on a broader scale. Full article

The sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA) is a membrane protein that is destabilized during purification in the absence of calcium ions. The disaccharide trehalose is a protein stabilizer that accumulates in the yeast cytoplasm when under stress. In the present work, SERCA was purified by including trehalose in the purification protocol. The purified SERCA showed high protein purity (~95%) and ATPase activity. ATP hydrolysis was dependent on the presence of Ca²⁺ and the enzyme kinetics showed a hyperbolic dependence on ATP (K_m = 12.16 ± 2.25 μM ATP). FITC labeling showed the integrity of the ATP-binding site and the identity of the isolated enzyme as a P-type ATPase. Circular dichroism (CD) spectral changes at a wavelength of 225 nm were observed upon titration with ATP, indicating α-helical rearrangements in the nucleotide-binding domain (N-domain), which correlated with ATP affinity (K_m). The presence of Ca²⁺ did not affect FITC labeling or the ATP-mediated structural changes at the N-domain. The use of trehalose in the SERCA purification protocol stabilized the enzyme. The isolated SERCA appears to be suitable for structural and ligand binding studies, e.g., for testing newly designed or natural inhibitors. The use of trehalose is recommended for the isolation of unstable enzymes. Full article

Concept mapping is a phased, mixed-method approach that is increasingly used in health research to develop an understanding of complex phenomena. The six phases of concept mapping are preparation, idea generation, structuring (clustering and prioritization), data analysis, interpretation, and utilization of the map. The reporting of concept mapping research requires the development of a specific reporting guideline. We conducted a systematic review to identify candidate reporting items for inclusion in a reporting guideline. Three databases (MEDLINE, CINAHL, and PsycInfo) were searched to identify studies that used concept mapping methodology. We included 75 concept mapping studies published since 2019 from which we extracted information about the quality of reporting. A third of the studies focused on public health. We identified 71 candidate items that relate to the quality of reporting concept mapping research. The rationale for the study, the focus prompt, procedures for brainstorming, and structuring statements were consistently reported across the included studies. The process for developing the focus prompt, the rationale for the size of the stakeholder groups, and the process for determining the final concept map were generally not reported. The findings from the review will be used to inform the development of our reporting guideline for concept mapping research. Full article

Hormonal changes and physiological alterations in pregnancy increase the susceptibility of the woman to oral diseases such as plaque-induced gingivitis. In individual oral prophylaxis, effective tooth brushing can reduce gingival inflammation. Therefore, it is necessary to update the scientific evidence to identify which type of toothbrush, manual or sonic-powered, is most effective in reducing the incidence of gingivitis in pregnant women. The aim of this clinical trial is to compare the biofilm control effectiveness of two manual and two sonic toothbrushes in pregnant women. This study is designed as a four-arm, parallel, randomized controlled trial with an allocation ratio of 1:1:1:1. The pregnant woman will be included at 15–18 weeks of amenorrhea and followed for 3 months. The primary outcome will be the change in the incidence of gingival bleeding from a baseline and various follow-up periods of the study. Secondary outcomes measures will be to compare clinical effects of the toothbrushes tested on (i) gingival inflammation, (ii) dental plaque, (iii) gingival attachment and (iv) periodontal pocket; and to assess toothbrush acceptability. Thus, identifying the best device for effective tooth brushing in pregnancy could be helpful in reducing and improving the incidence of gingival inflammation. Full article

Drosophila melanogaster is an excellent model for dissecting innate immune signaling and functions. Humoral and cellular immune mechanisms in the fly take place in the hemolymph, where host defense components are secreted and act in response to microbial invaders. Studying hemolymph factors is critical for understanding the regulation of the host’s antimicrobial immune system. Therefore, methods for extracting the fly hemolymph efficiently and in sufficient quantities are essential for isolating and characterizing immune proteins and peptides. Here, we describe a novel and simple hemolymph isolation protocol for single D. melanogaster male and female adults. This procedure substantially improves the already used technique and allows fly immunologists to explore innate immune hemolymph activity in D. melanogaster individuals. Full article

Limited epidemiological evidence suggests a link between antibiotic use and developing depression. This study seeks to investigate this association in depth, using a cohort of pregnant individuals. The primary aim is to explore any association between the use of antibiotics during pregnancy and the development of antenatal depressive symptoms up to the third trimester, as well as the use of antibiotics during pregnancy and within 12 months postpartum and the development of postpartum depressive symptoms. A national prospective, observational, longitudinal cohort study has been designed to examine these relationships. A sample size of 1500 pregnant individuals has been sought for this study, assuming 10 potential predictor variables (including antibiotic use) in the final multiple logistic regression model and allowing for a 30% drop-out rate. The development of depressive symptoms is considered either a diagnosis by a medical doctor and/or a scoring 13 or higher on the Edinburgh Postnatal Depression Scale. Data will be collected during the third trimester and at 6 weeks, 6 months, and 12 months postpartum. These surveys include variables previously identified as associated with antenatal and postpartum depression (e.g., level of social support, experience of intimate partner abuse, and obstetric complications), as well as antibiotic and probiotic use. This study will provide an update on the prevalence of the symptoms of depression during pregnancy and postpartum and its associated risk factors. It will also, for the first time, comprehensively explore the potential association between antibiotic use during pregnancy and up to 12 months postpartum and the development of depressive symptoms. Full article

In various drug delivery systems, solid lipid nanoparticles are dominantly lipid-based nanocarriers. Amiodarone hydrochloride is an antiarrhythmic agent used to treat severe rhythm disturbances. It has variable and hard-to-predict absorption in the gastrointestinal tract because of its low solubility and high permeability. The aims of this study were to improve its solubility by encapsulating amiodarone into solid lipid nanoparticles using two excipients—Compritol^® 888 ATO (pellets) (C888) as a lipid matrix and Transcutol^® (T) as a surfactant. Six types of amiodarone-loaded solid lipid nanoparticles (AMD-SLNs) were obtained using a hot homogenization technique followed by ultrasonication with varying sonication parameters. AMD-SLNs were characterized by their size distribution, polydispersity index, zeta potential, entrapment efficiency, and drug loading. Based on the initial evaluation of the entrapment efficiency, only three solid lipid nanoparticle formulations (P1, P3, and P5) were further tested. They were evaluated through scanning electron microscopy, Fourier-transform infrared spectrometry, near-infrared spectrometry, thermogravimetry, differential scanning calorimetry, and in vitro dissolution tests. The P5 formulation showed optimum pharmaco-technical properties, and it had the greatest potential to be used in oral pharmaceutical products for the controlled delivery of amiodarone. Full article

The rise of multidrug resistance of Pseudomonas aeruginosa highlights an increased need for selective and precise antimicrobial treatment. Drug efflux pumps are one of the major mechanisms of antimicrobial resistance found in many bacteria, including P. aeruginosa. Detection of efflux genes using a polymerase chain reaction (PCR)-based system would enable resistance detection and aid clinical decision making. Therefore, we aimed to develop and optimize a novel method herein referred to as “effluxR detection assay” using multiplex digital PCR (mdPCR) for detection of mex efflux pump genes in P. aeruginosa strains. The annealing/extension temperatures and gDNA concentrations were optimized to amplify mexB, mexD, and mexY using the multiplex quantitative PCR (mqPCR) system. We established the optimal mqPCR conditions for the assay (Ta of 59 °C with gDNA concentrations at or above 0.5 ng/µL). Using these conditions, we were able to successfully detect the presence of these genes in a quantity-dependent manner. The limit of detection for mex genes using the effluxR detection assay with mdPCR was 0.001 ng/µL (7.04–34.81 copies/µL). Moreover, using blind sample testing, we show that effluxR detection assay had 100% sensitivity and specificity for detecting mex genes in P. aeruginosa. In conclusion, the effluxR detection assay, using mdPCR, is able to identify the presence of multiple mex genes in P. aeruginosa that may aid clinical laboratory decisions and further epidemiological studies. Full article

Hydroalcoholic gels or hand sanitisers have become essential products to prevent and mitigate the transmission of COVID-19. Depending on their use, they can be classified as cosmetics (cleaning the skin) or biocides (with antimicrobial effects). The aim of this work was to determine sixty personal care products frequently found in cosmetic formulations, including fragrance allergens, synthetic musks, preservatives and plasticisers, in hydroalcoholic gels and evaluate their compliance with the current regulation. A simple and fast analytical methodology based on solid-phase microextraction followed by gas chromatography–tandem mass spectrometry (SPME-GC-MS/MS) was validated and applied to 67 real samples. Among the 60 target compounds, 47 of them were found in the analysed hand sanitisers, highlighting the high number of fragrance allergens (up to 23) at concentrations of up to 32,458 μg g⁻¹. Most of the samples did not comply with the labelling requirements of the EU Regulation No 1223/2009, and some of them even contained compounds banned in cosmetic products such as plasticisers. Method sustainability was also evaluated using the metric tool AGREEPrep, demonstrating its greenness. Full article

Although 2D in vitro cancer cell cultures have been used for decades as a first line-of-research tool to investigate antitumoral drugs and treatments, their use presents many drawbacks, including the poor resemblance of such cultures to the characteristics of in vivo tumors. To mitigate these drawbacks, 3D culture models have emerged as a more representative alternative. Cancer cells cultured as 3D structures have the advantage of resembling solid tumors in their architecture and in their resistance to chemotherapeutic drugs, in part because of restrained drug penetration. Additionally, these 3D structures create a more physiological environment for the study of immune cell invasion and migration, comparable to solid tumors. In this paper, we describe a fast and cost-effective step-by-step protocol for the generation of 3D spheres using ultra-low-attachment (ULA) multiwell plates, which can be incorporated into the normal workflow of any laboratory. Using this protocol, spheroids of different human cancer cell lines can be obtained and can then be characterized on the basis of their morphology, viability, and expression of specific markers. Full article

The aim of this research is to define optimal conditions to improve the stability of gold and silver nanoparticles’ anti-zearalenone antibody conjugates for their utilisation in lateral flow immunochromatographic assay (LFIA). The Turkevich–Frens method was used to synthesise gold nanoparticles (AuNPs), which were between 10 and 110 nm in diameter. Silver nanoparticles (AgNPs) with a size distribution of 2.5 to 100 nm were synthesised using sodium borohydride as a reducing agent. The onset of AuNP and AgNP aggregation occurred at 150 mM and 80 mM NaCl concentrations, respectively. Stable Au and Ag nanoparticle–antibody conjugates were achieved at 1.2 mM of K₂CO₃ concentration, which corresponds to the pH value of ≈7. Lastly, the highest degree of conjugation between Au and Ag nanoparticles and anti-zearalenone antibodies was at 4 and 6 µg/mL of antibody concentrations. The optimisation of the conjugation conditions can contribute to better stability of nanoparticles and their antibody conjugate and can improve the reproducibility of results of bioreporter molecules in biosensing lateral flow devices. Full article

Recent advances in phenotyping techniques have substantially improved the ability to mitigate type-II errors typically associated with high variance in phenotyping data sets. In particular, the implementation of automated techniques such as the High-Volume Instrument (HVI) and the Advanced Fiber Information System (AFIS) have significantly enhanced the reproducibility and standardization of various fiber quality measurements in cotton. However, micronaire is not a direct measure of either maturity or fineness, lending to limitations. AFIS only provides a calculated form of fiber diameter, not a direct measure, justifying the need for a visual-based reference method. Obtaining direct measurements of individual fibers through cross-sectional analysis and electron microscopy is a widely accepted standard but is time-consuming and requires the use of hazardous chemicals and specialized equipment. In this study, we present a simplified fiber histology and image acquisition technique that is both rapid and reproducible. We also introduce an automated image analysis program that utilizes machine learning to differentiate good fibers from bad and to subsequently collect critical phenotypic measurements. These methods have the potential to improve the efficiency of cotton fiber phenotyping, allowing for greater precision in unravelling the genetic architecture of critical traits such as fiber diameter, shape, areas of the secondary cell wall/lumen, and others, ultimately leading to larger genetic gains in fiber quality and improvements in cotton. Full article

The aim of the proposed scoping review is to describe and summarize studies assessing the associations between diet-related variables and depression in peri- and post-menopausal women. Studies examining the associations between diet-related variables and mental health indicators in women undergoing menopausal transition or in the post-menopausal period will be systematically retrieved via Medline, EMBASE, PsycINFO, Web of Science, and Scopus databases. All articles identified through the database searches will be imported into Covidence. Following the removal of duplicates, two authors will independently perform title and abstract screening, as well as full-text assessment against eligibility criteria. Data will be extracted using tables developed for observational and experimental studies. The methodological quality of randomized trials, cohort and cross-sectional studies, and case–control studies, will be assessed using the Cochrane risk-of-bias (RoB-2) tool, the NHLBI Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies, and the NHLBI Quality Assessment Tool for Case–Control studies, respectively. Data extraction tables will be used to produce two tables summarizing the main characteristics and findings of the studies included in the review. In the proposed review, we will systematically identify and summarize the currently available evidence on the association between diet-related variables and depression in peri- and post-menopausal women. To our knowledge, this is the first review focusing on this subgroup of the population. Protocol registration: osf.io/b89r6. Full article

Development of a rapid detection method for deoxycholic acid (DCA) is crucial for its diagnosis in the early stages of inflammation and cancer. In this study, we expressed a soluble recombinant anti-DCA single-chain variable fragment (scFv) in Escherichia coli. To convert scFv into a Quenchbody (Q-body), we labeled scFv using commercially available maleimide-linked fluorophores. The TAMRA-C5-maleimide-conjugated Q-body showed the highest response within a few minutes of DCA addition, indicating its applicability as a wash-free immunoassay probe for onsite DCA detection. Full article

Saccharomyces cerevisiae is a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield approximately 10⁶ cfu/µg of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 10⁸ cfu/µg plasmid DNA. The protocol generates sufficient transformants for 10–100× coverage of diverse genome libraries with small amounts of genomic libraries (0.1 µg of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries’ size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the method’s efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for downstream screening purposes while limiting the amount of the genomic library required. Full article

In pregnancies complicated by sickle cell disease (SCD), the maternal-fetal dyad is at high risk for mortality and morbidity. In healthy pregnancies, maternal nutritional status is a critical factor for the healthy growth and development of the fetus. However, there are no reviews of the current research on the nutritional status of pregnant women with SCD and pregnancy outcomes. First, we aim to assess the burden of malnutrition in pregnant women with SCD. Next, we aim to systematically evaluate if pregnant women with SCD who have poor nutritional status are at increased risk for adverse birth outcomes compared to pregnant women with sickle cell disease and normal nutritional status. We will systematically search multiple electronic databases. Our exposure is pregnant women with SCD and poor nutritional status. The primary outcomes of interest include low birth weight (categorical) and birth weight z-scores (continuous). We will also evaluate maternal and perinatal outcomes as secondary outcomes. We will evaluate the risk of bias and overall certainty of evidence with Risk of Bias in Non-randomized Studies—of Interventions (ROBINS-I), and the overall evidence will be assessed using Grading of Recommendation Assessment, Development, and Evaluation (GRADE) criteria. We will pool findings with a meta-analysis if sufficient homogeneity exists among studies. Findings will be published in a peer-reviewed journal and disseminated to SCD advocacy groups. PROSPERO registration number: CRD42023429412. Full article

The scratch assay is an in vitro assay that allows for high-throughput quantification of wound closure by keratinocytes and fibroblasts with relative ease. However, this assay is amenable to experimental variables, which can result in false-positive and false-negative data, making the interpretation of such data difficult. Also, data variability decreases the sensitivity of the scratch assay. Here, we identify important sources of data variation in the scratch assay and provide rational mitigation strategies that enable robust and highly reproducible quantification of scratch width and area, and ultimately the scratch closure rates. By eliminating these sources of variability, the sensitivity of the scratch assay is enhanced, thereby allowing for identification of dependent variables with wide-ranging impacts on wound closure in a robust and standardized manner. Full article

Background: Cardiac implantable electronic device (CIED) placement comes with certain complications. CIED infection is a severe adverse event related to CIED placement. In randomised controlled trials, the preoperative intravenous administration of antibiotics and the adjunctive use of an antibiotic mesh envelope resulted in significant reduction in infections related to cardiac implantable electronic devices. The adjunctive use of taurolidine for this purpose is relatively novel and not considered in the guidelines. The required evidence may consist of a set of clinical studies. Methods: The European TauroPace^TM registry (ETPR) prospectively evaluates every consecutive invasive procedure involving any CIED with adjunct TauroPace™ use in the contributing centres. As the estimation of the infection rate needs to be defensible, only interventions registered prior to the procedure will be followed-up. The endpoint is a major cardiac implantable electronic device infection according to the novel CIED infection criteria (1). Secondary endpoints comprise all-cause mortality, complications, adverse events of all grades, and major CIED infections during all follow-up examinations. The follow-up times are three months, twelve months, and eventually 36 months, as acute, subacute, and long-term CIED infections are of interest. Results: As the rate of CIED infections is expected to be very low, this registry is a multicentre, international project that will run for several years. Several reports are planned. The analyses will be included in the case number calculations for future randomised controlled trials. Conclusions: The ETPR will accumulate large case numbers to estimate small event rates more precisely; we intend to follow up on participants for years to reveal possible late effects. Full article

Tobacco use, particularly the initiation of smoking during adolescence and young adulthood, represents a significant public health concern in South Africa. The influence of socio-cultural factors, marketing strategies of the tobacco industry, and accessibility of tobacco products have all been implicated in this context. This systematic review and meta-analysis protocol aims to scrutinise the body of literature on this issue, providing a comprehensive understanding of the patterns and determinants of tobacco use among South African adolescents and young adults, with an eye towards informing more effective policy interventions. The available literature for studies on tobacco use will be systematically searched and reviewed. Five international scholarly databases, namely PubMed, MEDLINE, EMBASE, Global Health, and Scopus, will be searched. Peer-reviewed studies will be included if they are conducted in South Africa or South African provinces and if they include the prevalence of tobacco use among adolescents and young adults aged between 12 and 24 years. The results of such an analysis can guide future policy designs, enabling them to be more targeted and thus more effective. The findings can also have implications for shaping global tobacco control strategies, given the transferability of successful interventions across different populations and cultural contexts. This protocol has been registered in the PROSPERO database (ID: CRD42023428369). Full article

Introduction: Metabolic endotoxemia (ME) is the main cause of sub-clinical chronic inflammation, which subsequently triggers the onset of several chronic diseases. However, recent reports have indicated that dietary fiber (DF) contributes significantly to ameliorating ME and inflammation. This protocol aims to provide an outline of all procedures in synthesizing the available data on the effect of DF against ME. Methods: Following the PRISMA 2020 guidelines for preparing protocols, this protocol was registered in the International Prospective Registry of Systematic Reviews (PROSPERO) with registration number (CRD42023417833). In this review, we specifically focused on the inclusion of clinical trials that met the following criteria: they were published or available as preprints, employed random, quasi-random, or cross-over designs, and were exclusively documented in the English language. Clinical medical subject headings (MeSH) as search terms were used on prominent databases such as MEDLINE, COCHRANE library, PubMed, World Health Organization International Clinical Trials Registry Platforms, and US National Institutes of Health Ongoing Trials Register Clinicaltrials.gov. Results and discussion: This protocol will guide the exploration of articles that report changes in ME biomarkers in subjects supplemented with DF. The findings of this protocol will ensure a comprehensive evaluation of available evidence, provide a quantitative summary, identify patterns and trends, enhance statistical power, and address heterogeneity, which collectively will clarify the optimal types, doses, and duration of DF interventions for managing ME and low-grade inflammation. Ethics and dissemination: The quantitative data of clinical trials will be collected, and a meta-analysis will be performed using RevMan V.5.3 software. Therefore, no ethical approval is required. Full article

Poliomyelitis is a condition of great concern and is endemic in only two countries of the world: Pakistan and Afghanistan. Community mobilization plays a vital role in raising awareness and can help reduce polio vaccine refusals. The objective of this study will be to decrease polio vaccine refusals and zero-dose vaccines by motivating behavior change through the provision of conditional–collective–community-based incentives (C3Is) based on a reduction in polio vaccine refusals. The project will adopt a pretest/post-test quasi-experimental design with two intervention high-risk union councils (HRUCs) and two control union councils (UCs) of peri-urban (Karachi) and rural (Bannu) settings in Pakistan. A participatory community engagement and demand creation strategy with trust-building community mobilization with C3Is, to reduce vaccine refusals and improve polio immunization coverage in two HRUCs, will be used. These UCs will be divided into clusters based on the polio program framework and community groups will be formed in each cluster. These community groups will carry out awareness activities and will be given serial targets to reduce vaccine refusals and those who qualify will be provided C3Is. The project intends to create a replicable model that the government can integrate within health systems for long-term sustainability until the goal of eradication of poliovirus is achieved. The evaluation will be carried out by an independent data collection and analysis team at baseline and endline (after 12 months of intervention). The trial is registered with linicalTrials.gov with number NCT05721274. Full article

Used in solid-phase peptide synthesis (SPPS) for peptides with an acid termination, the 2-chlorotrityl chloride (2-CTC) resin is highly susceptible to moisture, leading to reduced resin loading and lower synthetic yields. It is therefore recommended that the resin be activated with thionyl chloride (SOCl₂) before peptide assembly. Here we present an optimized procedure for resin activation that minimizes the use of SOCl₂ as the activation reagent and reduces the activation time. Additionally, we demonstrate the feasibility of reusing the 2-CTC resin when following the activation protocol, achieving comparable results to the first usage of the resin. Moreover, we achieved different degrees of resin activation by varying the amount of SOCl₂. For instance, the use of 2% SOCl₂ in anhydrous dichloromethane (DCM) allowed up to 44% activation of the resin, thereby making it suitable for the synthesis of longer peptides. Alternatively, employing 25% SOCl₂ in anhydrous DCM resulted in up to 80% activation with a reaction time of only 5 min in both cases. Full article

The black soldier fly (BSF) is well known for its ability to biologically convert organic waste into insect biomass, including protein and oil, which can be utilised as animal feed. Since raw BSF products, such as BSF powder, are difficult to differentiate from other biological raw materials, therefore new analytical approaches are required. In this study, we have developed a new and fast method based on loop-mediated isothermal AMPlification (LAMP) reaction that can diagnose black soldier fly larvae and BSF byproducts with high accuracy, specificity and sensitivity. Species-specific primers for BSF were designed based on targeting the mitochondrial cytochrome C oxidase I (COI) gene. The assay was able to detect as low as 820 fg/L of BSF DNA in 60 min at 65 °C, which was a hundredfold higher than the detection limit of classical polymerase chain reaction and did not show cross-reactivity. In conclusion, the LAMP assay demonstrated excellent sensitivity and specificity to detect BSF and BSF byproducts, with a sampling-to-result identification time of 60 min. Full article

Advancements in single-cell-related technologies have opened new possibilities for analyzing rare cells, such as circulating tumor cells (CTCs) and rare immune cells. Among these techniques, single-cell proteomics, particularly single-cell mass spectrometric analysis (scMS), has gained significant attention due to its ability to directly measure transcripts without the need for specific reagents. However, the success of single-cell proteomics relies heavily on efficient sample preparation, as protein loss in low-concentration samples can profoundly impact the analysis. To address this challenge, an effective handling system for rare cells is essential for single-cell proteomic analysis. Herein, we propose a microfluidics-based method that offers highly efficient isolation, detection, and collection of rare cells (e.g., CTCs). The detailed fabrication process of the micropillar array-based microfluidic device is presented, along with its application for CTC isolation, identification, and collection for subsequent proteomic analysis. Full article

The recently discovered Jingmenvirus group includes viruses with a segmented genome, RNA of a positive polarity, and several proteins with distant homology to the proteins of the members of the genus Orthoflavivirus. Some Jingmenvirus group members, namely the Alongshan virus (ALSV) and Jingmen tick virus, are reported to be tick-borne human pathogens that can cause a wide variety of symptoms. The ALSV is widely distributed in Eurasia, yet no reliable assay that can detect it exists. We describe a qPCR system for ALSV detection. Our data showed that this system can detect as little as 10⁴ copies of the ALSV in a sample. The system showed no amplification of the common tick-borne viruses circulating in Eurasia, i.e., the Yanggou tick virus—which is another Jingmenvirus group member—or some known members of the genus Orthoflavivirus. The qPCR system was tested and had no nonspecific signal for the Ixodes ricinus, I. persulcatus, Dermacentor reticulatus, D. marginatus, Haemaphysalis concinna, and H. japonica ticks. The qPCR system had no nonspecific signal for human and sheep serum as well. Overall, the qPCR system described here can be used for reliable and quantitative ALSV detection. Full article

Our objectives are to perform (1) an umbrella review on diet and depression, (2) a systematic review update on dietary patterns and depression, and (3) updated meta-analyses using studies from the previous two objectives. Systematic reviews examining the relationships between diet and depression and primary studies on the relationship between dietary patterns and depression will be systematically retrieved via several databases. All articles identified through the database searches will be imported into Covidence. Following duplicates removal, two authors will independently perform title and abstract screening and full-text assessment against eligibility criteria. Data will be extracted using tables developed for both systematic reviews and primary studies. The methodological quality of systematic reviews will be assessed using the AMSTAR-2 tool. The risk of bias in randomized trials, cohort and cross-sectional studies, as well as case-control studies, will be assessed with the Cochrane risk-of-bias (RoB-2) tool, the NHLBI Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies, and the NHLBI Quality Assessment Tool for Case-Control studies, respectively. For each dietary variable, data extracted will be used to produce: (1) a summary of systematic reviews’ characteristics and results table, (2) a summary of the primary studies characteristics table, (3) a qualitative summary of results from the primary studies table, and (4) a quantitative summary of results in the form of forest plots. The certainty of evidence will be assessed using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) approach. Upon completion, this systematic review will be the most comprehensive and up-to-date synthesis of currently available evidence on the relationships between diet and depression. It will serve as a key reference to guide future research and as a resource for health professionals in the fields of nutrition and psychiatry. PROSPERO CRD42022343253. Full article

Fungi are well-known producers of chemically diverse and biologically active secondary metabolites. However, their production yields through fermentation may hamper structural analysis and biological activity downstream investigations. Herein, a systematic experimental design that varies multiple cultivation parameters, followed by chemometrics analysis on HPLC-UV-MS or UHPLC-HRMS/MS data, is presented to enhance the production yield of fungal natural products. The overall procedure typically requires 3–4 months of work when first developed, and up to 3 months as a routine procedure. Full article

Ulcerative colitis, characterized by its relapsing and remissive nature, negatively affects perception, body image, and overall quality of life. The associated financial burden underscores the need for alternative treatment approaches with fewer side effects, alongside pharmaceutical interventions. Montmorency tart cherries, rich in anthocyanins, have emerged as a potential natural anti-inflammatory agent for ulcerative colitis. This manuscript outlines the study protocol for a randomized placebo-controlled trial investigating the effects of Montmorency tart cherry in individuals with ulcerative colitis. The trial aims to recruit 40 participants with mild to moderate disease activity randomly assign them to either a Montmorency tart cherry or placebo group. The intervention will span 6 weeks, with baseline and 6-week assessments. The primary outcome measure is the Inflammatory Bowel Disease Quality of Life Questionnaire. Secondary outcomes include other health-related questionnaires and biological indices. Statistical analysis will adhere to an intention-to-treat approach using linear mixed effect models. Ethical approval has been obtained from the University of Hertfordshire (cLMS/SF/UH/05240), and the trial has been registered as a clinical trial (NCT05486507). The trial findings will be disseminated through a peer-reviewed publication in a scientific journal. Full article

Procuring high-grade RNA from mature coconut tissues is a tricky and labor-intensive process due to the intricate scaffold of polysaccharides, polyphenols, lipids, and proteins that form firm complexes with nucleic acids. However, we have effectively developed a novel method for the first time, letting the retrieval of high-grade RNA from the roots, endosperm, and mesocarp of mature coconut trees take place. In this method, we exploited dichloromethane as a replacement to phenol/chloroform for RNA recovery from mature coconut tissues. The amount of high-grade RNA acquired from the roots of mature coconut trees was 120.7 µg/g, with an A260/280 ratio of 1.95. Similarly, the mature coconut mesocarp yielded 134.6 µg/g FW of quality RNA with A260/280 ratio of 1.98, whereas the mature coconut endosperm produced 120.4 µg/g FW of quality RNA with A260/280 ratio of 2.01. Furthermore, the RNA isolation using the dichloromethane method exhibited excellent performance in downstream experiments, particularly in RT-PCR for cDNA production and amplification. On the contrary, the RNA plant kit, TRIZOL, and Cetyl Trimethyl Ammonium Bromide (CTAB) methods were unsuccessful in isolating substantial quantities of RNA with exceptional purities from the mentioned coconut tissues. In view of these findings, we conclude that the newly developed method will be pivotal in effectively extracting RNA with high purity from mature coconut tissues. Full article

Background: Childhood obesity has tripled, reaching critical levels of malnutrition. This factor is directly associated with a poorer health-related quality of life of the child and adolescent population. This article presents the study protocol of the project “Strong schoolchildren with a healthy lifestyle” (EF-Salud), which seeks to analyze the effects of a multicomponent program based on muscle strength exercises, sleep nutritional recommendations, and the use of screens in Chilean educational centers with extremely cold weather. Methods: The study protocol of a randomized controlled trial with a pre- and post-test conducted according to the CONSORT statement is reported. The total sample (n = 144) will be schoolchildren from six different school years, four of which will perform an intervention and two control. Intervention group 1 (from two different school years) will receive a muscular strength exercise program in the classroom once a day from Monday to Friday for six months and nutritional, sleep, and use of screens recommendations once a week. Intervention group 2 (from two different school years) will receive a program of nutritional, sleep, and use of screens recommendations once per week for six months. The control group (from two different school years) will carry out their usual school day in relation to physical education classes. Before and after the intervention, the investigators will evaluate the cardiovascular risk, physical condition, and lifestyle related to sleep and use of screens. Expected results: The schoolchildren in intervention group 1 will obtain significant results in increased strength, decreased cardiovascular risk, improved sleep habits, and fewer hours of screen use compared to the other two groups. Full article