Methods and Protocols最新文献

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.

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.

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.

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.

Isolation of African swine fever virus (ASFV) is a critical step towards the identification, titration, characterization, and even modification of the virus. Therefore, it is important to identify a suitable cell line that supports the efficient replication of ASFV for these purposes. This should be achieved even when starting with a low virus load, as in the case of isolating the virus from field samples. This article presents a detailed protocol on the preparation of porcine bone marrow primary (PBMP) cell culture, which has a high sensitivity towards ASFV, resulting in high viral yields with a minimal risk of bacterial contamination.

Recent advances in genomic technologies have enabled more in-depth study of the oral microbiome. In this study, we compared the amplicons generated by primers targeting different sites of the 16S rRNA gene found in the Human Oral Microbiome Database (HOMD). Six sets of primer targeting V1-V2, V1-V3, V3-V4, V4-V5, V5-V7 and V6-V8 regions of 16S rRNA were tested via in silico simulation. Primers targeting the V1-V2, V3-V4, and V4-V5 regions generated more than 90% of the original input sequences. Primers targeting the V1-V2 and V1-V3 regions exhibited a low number of mismatches and unclassified sequences at the taxonomic level, but there were notable discrepancies at the species level. Phylogenetic tree comparisons showed primers targeting the V1-V2 and V3-V4 regions showed performances similar to primers targeting the whole 16s RNA region in terms of separating total oral microbiomes and periodontopathogens. In an analysis of clinical oral samples, V1-V2 primers showed superior performance for identifying more taxa and had better resolution sensitivity for Streptococcus than V3-V4 primers. In conclusion, primers targeting the V1-V2 region of 16S rRNA showed the best performance for oral microbiome studies. In addition, the study demonstrates the need for careful PCR primer selections.

The aim of this study was to assess the release profile of components in five different honeys (a New Zealand Manuka and two Western Australian honeys, a Jarrah honey and a Coastal Peppermint honey) and their corresponding honey-loaded gel formulations using a custom-designed Franz-type diffusion cell in combination with High-Performance Thin-Layer Chromatography (HPTLC). To validate the suitability of the customised setup, release data using this new approach were compared with data obtained using a commercial Franz cell apparatus, which is an established analytical tool to monitor the release of active ingredients from topical semisolid products. The release profiles of active compounds from pure honey and honey-loaded formulations were found to be comparable in both types of Franz cells. For example, when released either from pure honey or its corresponding pre-gel formulation, the percentage release of two Jarrah honey constituents, represented by distinct bands at R_F 0.21 and 0.53 and as analysed by HPTLC, was not significantly different (p = 0.9986) at 12 h with over 99% of these honey constituents being released in both apparatus. Compared to the commercial Franz diffusion cell, the customised Franz cell offers several advantages, including easy and convenient sample application, the requirement of only small sample quantities, a large diffusion surface area, an ability to analyse 20 samples in a single experiment, and lower cost compared to purchasing a commercial Franz cell. Thus, the newly developed approach coupled with HPTLC is conducive to monitor the release profile of minor honey constituents from pure honeys and honey-loaded semisolid formulations and might also be applicable to other complex natural-product-based products.

This study aimed to determine the optimal UAE conditions for extracting anthocyanins from pigmented corn using the Box-Behnken design (BBD). Six anthocyanins were identified in the samples and were used as response variables to evaluate the effects of the following working variables: extraction solvent pH (2-7), temperature (10-70 °C), solvent composition (0-50% methanol in water), and ultrasound power (20-80%). The extraction time (5-25 min) was evaluated for complete recovery. Response surface methodology suggested optimal conditions, specifically 36% methanol in water with pH 7 at 70 °C using 73% ultrasound power for 10 min. The method was validated with a high level of accuracy (>90% of recovery) and high precision (CV < 5% for both repeatability and intermediate precision). Finally, the proposed analytical extraction method was successfully applied to determine anthocyanins that covered a wide concentration range (36.47-551.92 mg kg^-1) in several pigmented corn samples revealing potential varieties providing more health benefits.

Geriatric assessment (GA) is fundamental to optimising cancer care in older adults, yet implementing comprehensive GA tools in real-world clinical settings remains a challenge. This study aims to assess the feasibility and acceptability of integrating information from patient-derived photographs (PhotoVoice) into enhanced supportive care (ESC) for older adults with cancer. A feasibility randomised controlled trial will be conducted at a regional cancer care centre in Australia. Participants aged 70 and above will be randomised into two groups: PhotoVoice plus ESC or usual care (ESC) alone. In the PhotoVoice group, participants will provide four photographs for deduction of representations of different aspects of their lives using photo-elicitation techniques. ESC will be conducted for both groups, incorporating PhotoVoice analysis in the intervention group. PhotoVoice may improve patient-centred care outcomes, including enhanced communication, shared decision making, and identification of patient priorities and barriers. Findings will provide insights into implementing PhotoVoice in geriatric assessment and guide future trials in cancer among older adults.