Methods and Protocols最新文献

Heavy metal contamination of soil and water is a growing environmental concern, especially mercury, lead, and cadmium. Therefore, fast and reliable methodologies to assess contamination in the field are in demand. However, many methodologies require laborious, expensive, and cumbersome equipment that is not convenient for rapid field analysis. Mobile phone technology coupled with bioluminescent assays provides accessible hands-on alternatives that has already been shown to be feasible. Previously, we demonstrated that firefly luciferases can be harnessed as luminescence color-tuning sensors for toxic metals. An assay based on such a principle was already successfully applied for teaching biochemistry laboratory lessons, which demonstrates the effect of cadmium on enzyme function based on bioluminescence color change. For analytical detection of cadmium in water, here, we developed a novel bioluminescence assay using the cadmium-sensitive Amydetes vivianii firefly luciferase coupled with a cell phone provided with a program to quantify cadmium concentration based on luminescence color discrimination. The application has proven to be efficient with high precision between 0.10 and 2 mM of cadmium, being appliable to diluted water samples (0.1-2 µM) upon concentration and relying on reference cadmium standards values. The light emitted by the reference standards and samples in a dark box is captured by the smartphone's camera, which, using computer vision, automatically quantifies cadmium according to the RGB color. CadmiLume is a simple and easy luminescent enzymatic biosensor for cadmium contamination in water samples, which instantaneously can provide results with the convenience of a smartphone in the palm of one's hands.

Biomechanical parameters of plant tissues and organs are increasingly recognized as key factors in plant development and application, increasing the demand for convenient devices for their study. The paper presents an original device for performing a three-point bending test using the inverse method, which is a modification of the classical (straight) three-point test. The designed device was tested in experiments to determine the modulus of elasticity of flax plant stems, and the results were compared with data obtained using the vibration method and the straight three-point bending test on a commercial instrument. Due to the high sensitivity associated with its design features, the device for the inverse three-point bending test is characterized by being able to adequately measure elastic moduli in plant stems over a wide range of values, from tens of MPa to tens of GPa. It also allows checking the effect of humidity, temperature, and water content on the mechanical properties of samples and is equipped with an automation system. The proposed device is quite affordable and can be effectively used both for young stem parts, whose mechanical properties are based on a hydroskeleton, and for mature, poorly hydrated parts with cell walls highly developed in sclerenchymatous tissues.

Butterfly larvae display intricate cognitive capacities and behaviors, but relatively little is known about how those behaviors alter their brains at the molecular level. Here, we optimized a hybridization chain reaction 3.0 (HCR v3.0) protocol to visualize the expression of multiple RNA molecules in fixed larval brains of the African butterfly Bicyclus anynana. We optimized the polyacrylamide gel mounting, fixation, and sample permeabilization steps, and mapped the expression domains of ten genes in whole larval brain tissue at single-cell resolution. The genes included optomotor blind (omb), yellow-like, zinc finger protein SNAI2-like (SNAI2), weary (wry), extradenticle (exd), Synapsin, Distal-less (Dll), bric-à-brac 1 (bab1), dachshund (dac), and acetyl coenzyme A acetyltransferase B (AcatB). This method can be used alongside single-cell sequencing to visualize the spatial location of brain cells that change in gene expression or splicing patterns in response to specific behaviors or cognitive experiences.

Cholinesterase (ChE) is an essential enzyme for nervous system function, and its activity in the blood serves as a biomarker for pesticide exposure, necessitating accessible assessment methods in resource-limited areas. This study focused on developing and validating a micro-electrometric method to rapidly and reliably measure ChE activity in human blood to monitor pesticide exposure. A micro-electrometric method was designed to evaluate ChE activity in whole-blood samples, involving incubation with organophosphate and carbamate pesticides and subsequent pH measurement, and its performance was compared to that of the Ellman method. The optimized method was strongly correlated with the Ellman method (R² = 0.9147), effectively measuring dose-dependent ChE inhibition by mevinphos and carbofuran with stabilization after 10 min, demonstrating a high sensitivity suitable for field applications. The micro-electrometric method is easy, accurate, and inexpensive for measuring ChE activity. It has been proven to work by comparing it to other methods; therefore, it can be used in clinical and research settings with limited resources.

Volatile compounds (VCs) from fungi can promote plant growth, but their application methods are limited. Edible mushroom fungi beds (FBs) provide a readily available alternative source of fungal VCs, although their biostimulatory functions remain unvalidated. In this study, a novel, non-contact exposure method for applying VCs emitted from FBs to rice seedlings was developed. This marks the first evaluation of mushroom FBs as a direct source of bioactive VCs for plant growth promotion. Volatiles from two different edible mushroom FBs promoted shoot growth and increased biomass for rice seedlings. VCs from shiitake FBs significantly increased biomass by 67.4% while VCs from enokitake FBs by 39.5% compared to the control. The biomass-increasing effects were influenced by the quantity of shiitake FBs applied, with significant increases at 15 g, 30 g and 60 g applications. The VCs effects remained significant even when the FBs were covered with two types of gas-permeable polymer film. Chemical analysis of VCs from FBs identified several organic compounds and subsequent bioassays using synthetic VCs determined key bioactive VCs contributing to biomass increase at specific concentrations. This study presents a utilization method of waste mushroom FBs as sustainable, scalable, and cost-effective agricultural biostimulants.

In the context of wheelchair racing, research primarily focuses on studying wheelchair ergonomics and determining kinematic, kinetic, and rolling resistance variables. One factor identified as influencing athletes' performance is wheel skidding on the ground, a parameter complementary to rolling resistance. The objective of this study, therefore, is to identify, within a laboratory setting, the parameters that influence the risk of skidding in racing wheelchairs by measuring skidding torque. The ultimate goal is to enhance athletes' performance by optimizing the interaction between the athlete and their wheelchair, and the wheelchair and the environment. In this perspective, four parameters were examined: the type of tubular, the camber angle, the tire pressure, and the load applied to the wheel using a skidometer. This tool characterizes a tire's grip on a surface by measuring torques. The aim is to develop a system for classifying tire grip on dry athletics track at ambient temperature. The findings revealed that only the effects of load and tubular type had a significant impact on the torque values obtained. The tire that minimized the risk of skidding, among all tested combinations, is the Vittoria Pista Speed 23-28″. Furthermore, as the mass applied to the wheel increases, so do the resulting torques. This implies that a heavier athlete would require a greater force to be applied to the hand rim for the tire to skid. However, it was also demonstrated that the risk of skidding in a racing wheelchair is unlikely, as the torques obtained were over a range of 90 to 190 Nm. These values far exceed those typically exerted by para-athletes, which are a maximum of 60 Nm. The long-term goal would be to adjust the mode of torque application on the wheel using the skidometer for a more realistic field approach.

Genetic profiling and molecular biology methods have made it possible to study the etiology of the end-stage organ disease that led to transplantation, the genetic factors of compatibility and tolerance of the transplant, and the pharmacogenetics of immunosuppressive drugs and allowed for the development of monitoring methods for the early assessment of allograft rejection. This study aims to report the design and baseline characteristics of an integrated personalized genetic approach in solid organ transplantation, including whole-exome sequencing (WES) and the monitoring of dd-cfDNA by dPCR. Preliminary results reported female recipients with male donors undergoing two pediatric and five adult kidney and three heart transplantations. WES revealed a pathogenic mutation in RBM20 and VUS in TTN and PKP2 in heart recipients, while kidney donors presented mutations in UMOD and APOL1 associated with autosomal-dominant kidney diseases, highlighting the risks requiring the long-term monitoring of recipients, donors, and their family members. %dd-cfDNA levels were generally stable but elevated in cadaveric kidney recipient and one pediatric patient with infectious complications and genetic variants in the ABCB1 and ABCC2 genes. These findings highlight the potential of combining genetic and molecular biomarker-based approaches to improve donor-recipient matching, predict complications, and personalize post-transplant care, paving the way for precision medicine in transplantation.

Reporting guidelines are created with the intention to enhance the quality and transparency of reporting different research methods. Trochim's concept mapping (often referred to as group concept mapping) is a six-phase, participatory mixed-method approach to understanding complex constructs. Currently, there is no reporting guideline for concept mapping. Developing a reporting guideline typically follows a three-step process: 1. a systematic review to establish the need for a reporting guideline, 2. a Delphi study to identify candidate items, and 3. consolidation process to inform a draft guideline. We have previously reported our step 1, a systematic review of health-related concept mapping studies. In this paper, we report on steps 2 and 3, guideline development. In developing our reporting guideline, we opted to use concept mapping rather than the Delphi method. Stakeholders including researchers and experts in concept mapping were identified from papers included in our systematic review and invited to participate. Thirty-two stakeholders participated in the brainstorming phase of the concept mapping generating 96 discrete statements. The prioritisation and clustering phase involved 24 stakeholders. The final concept map included 11 clusters that represented key concepts for inclusion in the reporting guideline. The clusters were relatively small and positioned in a circle around the edge of the map, suggesting each was of equal importance and conceptually discreet. In phase 3, a guideline was drafted using the findings from both the phase 1 systematic review and phase 2 concept mapping study. The draft was reviewed by eight stakeholders (who had participated in our phase 2 concept mapping study) to check the completeness and clarity of expression of the items included in the guideline. The final reporting guideline (called the ConMapT) has 27-items organised under 14 headings. The guideline will be made freely available via the EQUATOR network. Registration: The study protocol was registered with the Open Science Framework (OSF) before recruiting the first study participant. The EQUATOR network has listed the study as a guideline under development.

Measuring the propulsion performance of sport wheelchairs in ecological conditions remains complex due to variations inherent in real-world practice. This study aims to develop a validation protocol for an instrumented wheel designed to measure propulsion power under ecological conditions. The wheel's precision was compared to that of the Lode Esseda roller ergometer, the gold standard for measuring the force exerted on both the left and right hands. Tests were conducted at three speeds (1, 2, and 3 m/s) on a multisport wheelchair. Results show a strong correlation between the two systems, confirmed by a repeated measures ANOVA test (p > 0.05) and a root mean square error (RMSE < 5%). Bland-Altman plots reveal good agreement despite discrepancies observed at high speeds, potentially due to mechanical limits. The proposed protocol validates the instrumented wheel and highlights the importance of multi-speed evaluation to ensure valid measurements in ecological conditions.

Background and aim: The use of mHealth, especially short-message text (SMS), has proven to be an effective intervention in promoting behavioral health outcomes in populations across different contexts and settings. While MomConnect, an mHealth technological device designed to enhance maternal and child health services in South Africa, offers various health-related contents aimed at improving maternal outcomes for pregnant and postpartum women, it currently lacks information on prenatal physical activity. However, physical activity and exercise during pregnancy is safe and beneficial for both the mother and the baby. This article outlines the protocol for designing and developing a prenatal physical activity and exercise text messaging content for the MomConnect device. To achieve this, the protocol aims to elucidate the preferences of prenatal physical activity and exercise text messages and ascertain the preferred amount of SMS messaging to inform the design of an intervention for the incorporation of prenatal physical activity and exercise text messages into the MomConnect device in South Africa.

Methods: We will apply a user-centred design approach conducted in three phases. First, a scoping literature review and interviews with pregnant women will be conducted to inform the formative stage for developing a desirable prototype SMS. Secondly, healthcare providers and pregnant women will be interviewed to collate data on the preferred SMS. Lastly, a cross-sectional survey will be conducted to determine the preferred quantity of SMS messaging to be incorporated in the MomConnect device.

Expected outcomes: A preferred or desirable prenatal physical activity and exercise SMS text message will inform the design of SMS text messages to be incorporated into the content of the MomConnect device to promote prenatal physical activity and exercise participation among women in the Eastern Cape Province. This study will develop a tailored mHealth intervention to improve prenatal physical activity participation and health behaviors among pregnant women in South Africa.