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LoRa technology has gained popularity as one of the most widely used standards for device interconnection due to its ability to cover long distances and energy efficiency, making it a suitable choice for various Internet of Things (IoT) monitoring and control applications. In this sense, this work presents the development of a visual support tool for creating IoT devices with LoRa and LoRaWAN connectivity. This work significantly advances the state of the art in LoRa technology by introducing a novel visual support tool tailored for creating IoT devices with LoRa and LoRaWAN connectivity. By simplifying the development process and offering compatibility with multiple hardware solutions, this research not only facilitates the integration of LoRaWAN technology within educational settings but also paves the way for rapid prototyping of IoT nodes. The incorporation of block programming for LoRa and LoRaWAN using the Arduinoblocks framework as a graphical environment enhances the capabilities of the tool, positioning it as a comprehensive solution for efficient firmware generation. In addition to the visual tool for firmware generation, multiple compatible hardware solutions enable easy, economical, and stable development, offering a comprehensive hardware and software solution. The hardware proposal is based on an ESP32 microcontroller, known for its power and low cost, in conjunction with an RFM9x module that is based on SX127x LoRa transceivers. Finally, three successfully tested use cases and a discussion are presented.

The transfer from batch to flow chemistry is often based on commercial microfluidic equipment, such as costly complete reactor systems, which cannot be easily tailored to specific requirements of technologies such as DNA-encoded library technology (DELT), in particular for increasingly important photochemical reactions. Customized photoreactor concepts using rapid prototyping technology offer a modular, flexible, and affordable design that allows for adaptation to various applications. In order to validate the prototype reactors, a photochemical pinacol coupling reaction at 368 nm was conducted to demonstrate the transfer from batch to flow chemistry. The conversion rates were optimized by adapting the design parameters of the microfluidic flow photoreactor module. Subsequently, the photoreactor module has been extended to an application with DNA-tagged substrates by switching to LEDs with a wavelength of 454 nm. The successful recovery of DNA confirmed the feasibility of the modular-designed flow photo reactor. This collaborative approach holds enormous potential to drive the development of DELT and flow equipment design.
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
(Copyright © 2023 Dinter, Willems, Nissalk, Hastürk, Brunschweiger and Kockmann.)

Humans, Models, Anatomic, Computer Simulation, Hand, Anastomosis, Surgical education, Clinical Competence, Thoracic Surgical Procedures, and Simulation Training

Objective: The objective of this paper is to describe the techniques and process of developing and testing a take-home surgical anastomosis simulation model.
Design: Through an iterative process, a simulation model was customized and designed to target specific skill development and performance objectives that focused on anastomotic techniques in thoracic surgery and consist of 3D printed and silicone molded components. Various manufacturing techniques such as silicone dip spin coating and injection molding have been described in this paper and explored as part of the research and development process. The final prototype is a low-cost, take-home model with reusable and replaceable components.
Setting: The study took place at a single-center quaternary care university-affiliated hospital.
Participants: The participants included in the model testing were 10 senior thoracic surgery trainees who completed an in-person training session held during an annual hands- on thoracic surgery simulation course. Feedback was then collected in the form of an evaluation of the model from participants.
Results: All 10 participants had an opportunity to test the model and complete at least 1 pulmonary artery and bronchial anastomosis. The overall experience was rated highly, with minor feedback provided regarding the set- up and fidelity of the materials used for the anastomoses. Overall, the trainees agreed that the model was suitable for teaching advanced anastomotic techniques and expressed an interest in being able to use this model to practice skill development.
Conclusions: Developed simulation model can be easily reduced, with customized components that accurately simulate real-life vascular and bronchial components suitable for training of anastomoses technique amongst senior thoracic surgery trainees.
(Copyright © 2023. Published by Elsevier Inc.)

Models of urea kinetics facilitate a mechanistic understanding of urea transfer and provide a tool for optimizing dialysis efficacy. Dual-compartment models have largely replaced single-compartment models as they are able to accommodate the urea rebound on the cessation of dialysis. Modeling the kinetics of urea and other molecular species is frequently regarded as a rarefied academic exercise with little relevance at the bedside. We demonstrate the utility of System Dynamics in creating multi-compartment models of urea kinetics by developing a dual-compartment model that is efficient, intuitive, and widely accessible to a range of practitioners. Notwithstanding its simplicity, we show that the System Dynamics model compares favorably with the performance of a more complex volume-average model in terms of calibration to clinical data and parameter estimation. Its intuitive nature, ease of development/modification, and excellent performance with real-world data may make System Dynamics an invaluable tool in widening the accessibility of hemodialysis modeling.
(© 2023. The Author(s).)

Optical sensing offers several advantages owing to its non-invasiveness and high sensitivity. The miniaturization of optical sensors will mitigate spatial and weight constraints, expanding their applications and extending the principal advantages of optical sensing to different fields, such as healthcare, Internet of Things, artificial intelligence, and other aspects of society. In this study, we present the development of a miniature optical sensor for monitoring thrombi in extracorporeal membrane oxygenation (ECMO). The sensor, based on a complementary metal-oxide semiconductor integrated circuit (CMOS-IC), also serves as a photodiode, amplifier, and light-emitting diode (LED)-mounting substrate. It is sized 3.8 × 4.8 × 0.75 mm 3 and provides reflectance spectroscopy at three wavelengths. Based on semiconductor and microelectromechanical system (MEMS) processes, the design of the sensor achieves ultra-compact millimeter size, customizability, prototyping, and scalability for mass production, facilitating the development of miniature optical sensors for a variety of applications.

Since temperature and its spatial, and temporal variations affect a wide range of physical properties of material systems, they can be used to create reconfigurable spatial structures of various types in physical and biological objects. This paper presents an experimental optical setup for creating tunable two-dimensional temperature patterns on a micrometer scale. As an example of its practical application, we have produced temperature-induced magnetization landscapes in ferrimagnetic yttrium iron garnet films and investigated them using micro-focused Brillouin light scattering spectroscopy. It is shown that, due to the temperature dependence of the magnon spectrum, spatial temperature distributions can be visualized even for microscale thermal patterns.
(© 2023 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).)

Animals, Antivenins therapeutic use, Antibodies, Monoclonal therapeutic use, Snake Bites diagnosis, Snake Bites drug therapy, Crotalid Venoms therapeutic use, and Bothrops

Background: Brazil is home to a multitude of venomous snakes; perhaps the most medically relevant of which belong to the Bothrops genus. Bothrops spp. are responsible for roughly 70% of all snakebites in Brazil, and envenomings caused by their bites can be treated with three types of antivenom: bothropic antivenom, bothro-lachetic antivenom, and bothro-crotalic antivenom. The choice to administer antivenom depends on the severity of the envenoming, while the choice of antivenom depends on availability and on how certain the treating physician is that the patient was bitten by a bothropic snake. The diagnosis of a bothropic envenoming can be made based on expert identification of the dead snake or a photo thereof or based on a syndromic approach wherein the clinician examines the patient for characteristic manifestations of envenoming. This approach can be very effective but requires staff that has been trained in clinical snakebite management, which, unfortunately, far from all relevant staff has.
Results: In this article, we describe a prototype of the first lateral flow assay (LFA) capable of detecting venoms from Brazilian Bothrops spp. The monoclonal antibodies for the assay were generated using hybridoma technology and screened in sandwich enzyme-linked immunosorbent assays (ELISAs) to identify Bothrops spp.-specific antibody sandwich pairs. The prototype LFA is able to detect venom from several Bothrops spp. The LFA has a limit of detection (LoD) of 9.5 ng/mL in urine, when read with a commercial reader, and a visual LoD of approximately 25 ng/mL.
Significance: The work presented here serves as a proof of concept for a genus-specific venom detection kit that could support physicians in diagnosing Bothrops envenomings. Although further optimisation and testing is needed before the LFA can find clinical use, such a device could aid in decentralising antivenoms in the Brazilian Amazon and help ensure optimal snakebite management for even more victims of this highly neglected disease.
Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Cecilie Knudsen, Jonas A. Jürgensen, Søren H. Dam, Aleksander M. Haack, Rasmus U. W. Friis, and Andreas H. Laustsen are co-founders of VenomAid Diagnostics A/S. Jonas A. Jürgensen, Pelle D. Knudsen, and Georgina M. Ross are employed by VenomAid Diagnostics A/S. Cecilie Knudsen is an industrial PhD student at the Technical University of Denmark. Her PhD is co-sponsored by Innovation Fund Denmark and BioPorto Diagnostics A/S. Cecilie Knudsen, Jonas A. Jürgensen, Søren H. Dam, Aleksander M. Haack, Rasmus U. W. Friis, and Andreas H. Laustsen have been designated as inventors on a patent application related to the work presented here.
(Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Humans, Prospective Studies, Fatigue, Educational Status, Psychiatry, and Military Personnel

Background: Military service inherently includes frequent periods of high-stress training, operational tempo, and sustained deployments to austere far-forward environments. These occupational requirements can contribute to acute and chronic sleep disruption, fatigue, and behavioral health challenges related to acute and chronic stress and disruption of team dynamics. To date, there is no centralized mobile health platform that supports self- and supervised detection, monitoring, and management of sleep and behavioral health issues in garrison and during and after deployments.
Objective: The objective of this study was to adapt a clinical decision support platform for use outside clinical settings, in garrison, and during field exercises by medics and soldiers to monitor and manage sleep and behavioral health in operational settings.
Methods: To adapt an existing clinical decision support digital health platform, we first gathered system, content, and context-related requirements for a sleep and behavioral health management system from experts. Sleep and behavioral health assessments were then adapted for prospective digital data capture. Evidence-based and operationally relevant educational and interventional modules were formatted for digital delivery. These modules addressed the management and mitigation of sleep, circadian challenges, fatigue, stress responses, and team communication. Connectivity protocols were adapted to accommodate the absence of cellular or Wi-Fi access in deployed settings. The resulting apps were then tested in garrison and during 2 separate field exercises.
Results: Based on identified requirements, 2 Android smartphone apps were adapted for self-monitoring and management for soldiers (Soldier app) and team supervision and intervention by medics (Medic app). A total of 246 soldiers, including 28 medics, received training on how to use the apps. Both apps function as expected under conditions of limited connectivity during field exercises. Areas for future technology enhancement were also identified.
Conclusions: We demonstrated the feasibility of adapting a clinical decision support platform into Android smartphone-based apps to collect, save, and synthesize sleep and behavioral health data, as well as share data using adaptive data transfer protocols when Wi-Fi or cellular data are unavailable. The AIRE (Autonomous Connectivity Independent System for Remote Environments) prototype offers a novel self-management and supervised tool to augment capabilities for prospective monitoring, detection, and intervention for emerging sleep, fatigue, and behavioral health issues that are common in military and nonmilitary high-tempo occupations (eg, submarines, long-haul flights, space stations, and oil rigs) where medical expertise is limited.
(©Anne Germain, Megan Wolfson, I Wayan Pulantara, Meredith L Wallace, Katie Nugent, George Mesias, Kristina Clarke-Walper, Phillip J Quartana, Joshua Wilk. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 28.08.2023.)

Humans, Employment, and User-Centered Design

This paper presents elements of user-centered design and prototyping of a serious game. Produced within the framework of the SG-HANDI project, the serious game aims to raise awareness about integration, prevention of professional displacement and job retention of people with disabilities. This serious game is developed on an interactive RFID tabletop with tangible objects. It is intended to be used in a collective context involving one or more facilitators specialized in employment and disability, as well as the company's stakeholders to be made aware of the issue.

Lab-On-A-Chip Devices, Microfluidics, and Nucleic Acid Amplification Techniques methods

In this work, we demonstrate an inexpensive method of prototyping microfluidics using a desktop injection molding machine. A centrifugal microfluidic device with a novel central filling mechanism was developed to demonstrate the technique. We overcame the limitations of desktop machines in replicating microfluidic features by variotherm heating and cooling the mold between 50 °C and 110 °C within two minutes. Variotherm heating enabled good replication of microfeatures, with a coefficient of variation averaging only 3.6% attained for the measured widths of 100 μm wide molded channels. Using this methodology, we produced functional polystyrene centrifugal microfluidic chips, capable of aliquoting fluids into 5.0 μL reaction chambers with 97.5% accuracy. We performed allele-specific loop-mediated isothermal amplification (AS-LAMP) reactions for genotyping CYP2C19 alleles on these chips. Readouts were generated using optical pH sensors integrated onto chips, by drop-casting sensor precursor solutions into reaction chambers before final chip assembly. Positive reactions could be discerned by decreases in pH sensor fluorescence, thresholded against negative control reactions lacking the primers for nucleic acid amplification and with time-to-results averaging 38 minutes. Variotherm desktop injection molding can enable researchers to prototype microfluidic devices more cost-effectively, in an iterative fashion, due to reduced costs of smaller, in-house molds. Designs prototyped this way can be directly translated to mass production, enhancing their commercialization potential and positive impacts.

Three-dimensional printing technology has been used for more than three decades in many industries, including the automotive and aerospace industries. So far, the use of this technology in medicine has been limited only to 3D printing of anatomical models for educational and training purposes, which is due to the insufficient functional properties of the materials used in the process. Only recent advances in the development of innovative materials have resulted in the flourishing of the use of 3D printing in medicine and pharmacy. Currently, additive manufacturing technology is widely used in clinical fields. Rapid development can be observed in the design of implants and prostheses, the creation of biomedical models tailored to the needs of the patient and the bioprinting of tissues and living scaffolds for regenerative medicine. The purpose of this review is to characterize the most popular 3D printing techniques.

The increasing demand for seafood is responsible for many environmental impacts, especially caused by aquaculture. Shrimp accounts for a substantial part of seafood production and therefore also for negative effects associated with it. This work aimed to develop a mushroom-based shrimp analogue with a texture similar to shrimp using the fruiting bodies of pink oyster mushroom ( Pleurotus djamor ) and lion's mane ( Hericium erinaceus ). Three flushes of pink oyster mushrooms and a first flush of lion's mane mushroom were analysed regarding their nutritional composition and whether they are suitable shrimp alternatives. The two mushrooms are rich in proteins (∼32% and ∼26% w/w for the first flush of pink oyster and lion's mane, respectively). The protein content of pink oyster mushroom decreased and the dietary fibre content increased across the different flushes. The antioxidants in the mushrooms were extracted using different methods, whereby aqueous extracts mostly excelled in terms of antioxidant activity. Hydrolysis confirmed the presence of conjugated p -coumaric acid in both mushrooms and possibly conjugated caffeic acid in pink oyster. Texture analysis results of the prototypes were close to the values of fried shrimp. However, although the sensory qualities of the final prototypes were perceived as similar to shrimp, further improvements in the recipe are necessary to make the prototypes indistinguishable from shrimp.
Competing Interests: None.
(© 2023 The Author(s).)

This study proposes a design approach and the development of a low-power planar biped robot named YU-Bibot. The kinematic structure of the robot consists of six independently driven axes, and it weighs approximately 20 kg. Based on biomimetics, the robot dimensions were selected as the average anthropomorphic dimensions of the human lower extremities. The optimization of the mechanical design and actuator selection of the robot was based on the results of parametric simulations. The natural human walking gait was mimicked as a walking pattern in these simulations. As a result of the optimization, a low power-to-weight ratio of 30 W/kg was obtained. The drive system of the robot joints consists of servo-controlled brushless DC motors with reduction gears and additional bevel gears at the knee and ankle joints. The robot features spring-supported knee and ankle joints that counteract the robot's weight and compensate for the backlash present in these joints. The robot is constrained to move only in the sagittal plane by using a lateral support structure. The robot's feet are equipped with low-cost, force-sensitive resistor (FSR)-type sensors for monitoring ground contact and zero-moment point (ZMP) criterion. The experimental results indicate that the proposed robot mechanism can follow the posture commands accurately and demonstrate locomotion at moderate stability. The proposed parametric natural gait simulation-based design approach and the resulting biped robot design with a low power/weight ratio are the main contributions of this study.

The flapping-wing technology has emerged recently in the application of unmanned aerial robotics for autonomous flight, control, inspection, monitoring, and manipulation. Despite the advances in applications and outdoor manual flights (open-loop control), closed-loop control is yet to be investigated. This work presents a nonlinear optimal closed-loop control design via the state-dependent Riccati equation (SDRE) for a flapping-wing flying robot (FWFR). Considering that the dynamic modeling of the flapping-wing robot is complex, a proper model for the implementation of nonlinear control methods is demanded. This work proposes an alternative approach to deliver an equivalent dynamic for the translation of the system and a simplified model for orientation, to find equivalent dynamics for the whole system. The objective is to see the effect of flapping (periodic oscillation) on behavior through a simple model in simulation. Then the SDRE controller is applied to the derived model and implemented in simulations and experiments. The robot bird is a 1.6 m wingspan flapping-wing system (six-degree-of-freedom robot) with four actuators, three in the tail, and one as the flapping input. The underactuated system has been controlled successfully in position and orientation. The control loop is closed by the motion capture system in the indoor test bed where the experiments of flight have been successfully done.
Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest for this paper.
(Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Microfluidic devices have found extensive applications in mechanical, biomedical, chemical, and materials research. However, the high initial cost, low resolution, inferior feature fidelity, poor repeatability, rough surface finish, and long turn-around time of traditional prototyping methods limit their wider adoption. In this study, a strategic approach to a deterministic fabrication process based on in-situ image analysis and intermittent flow control called image-guided in-situ maskless lithography (IGIs-ML), has been proposed to overcome these challenges. By using dynamic image analysis and integrated flow control, IGIs-ML provides superior repeatability and fidelity of densely packed features across a large area and multiple devices. This general and robust approach enables the fabrication of a wide variety of microfluidic devices and resolves critical proximity effect and size limitations in rapid prototyping. The affordability and reliability of IGIs-ML make it a powerful tool for exploring the design space beyond the capabilities of traditional rapid prototyping.
(© 2023. The Author(s).)

In this work, we introduce a polymer version of a previously developed silicon MEMS drop deposition tool for surface functionalization that consists of a microcantilever integrating an open fluidic channel and a reservoir. The device is fabricated by laser stereolithography, which offers the advantages of low-cost and fast prototyping. Additionally, thanks to the ability to process multiple materials, a magnetic base is incorporated into the cantilever for convenient handling and attachment to the holder of a robotized stage used for spotting. Droplets with diameters ranging from ∼50 µm to ∼300 µm are printed upon direct contact of the cantilever tip with the surface to pattern. Liquid loading is achieved by fully immersing the cantilever into a reservoir drop, where a single load results in the deposition of more than 200 droplets. The influences of the size and shape of the cantilever tip and the reservoir on the printing outcome are studied. As a proof-of-concept of the biofunctionalization capability of this 3D printed droplet dispenser, microarrays of oligonucleotides and antibodies displaying high specificity and no cross-contamination are fabricated, and droplets are deposited at the tip of an optical fiber bundle.
Competing Interests: Conflict of interestThe authors declare no competing interests.
(© The Author(s) 2023.)

Animals, Bayes Theorem, Uncertainty, and Anthozoa

Emerging diseases can have devastating consequences for wildlife and require a rapid response. A critical first step towards developing appropriate management is identifying the etiology of the disease, which can be difficult to determine, particularly early in emergence. Gathering and synthesizing existing information about potential disease causes, by leveraging expert knowledge or relevant existing studies, provides a principled approach to quickly inform decision-making and management efforts. Additionally, updating the current state of knowledge as more information becomes available over time can reduce scientific uncertainty and lead to substantial improvement in the decision-making process and the application of management actions that incorporate and adapt to newly acquired scientific understanding. Here we present a rapid prototyping method for quantifying belief weights for competing hypotheses about the etiology of disease using a combination of formal expert elicitation and Bayesian hierarchical modeling. We illustrate the application of this approach for investigating the etiology of stony coral tissue loss disease (SCTLD) and discuss the opportunities and challenges of this approach for addressing emergent diseases. Lastly, we detail how our work may apply to other pressing management or conservation problems that require quick responses. We found the rapid prototyping methods to be an efficient and rapid means to narrow down the number of potential hypotheses, synthesize current understanding, and help prioritize future studies and experiments. This approach is rapid by providing a snapshot assessment of the current state of knowledge. It can also be updated periodically (e.g., annually) to assess changes in belief weights over time as scientific understanding increases. Synthesis and applications: The rapid prototyping approaches demonstrated here can be used to combine knowledge from multiple experts and/or studies to help with fast decision-making needed for urgent conservation issues including emerging diseases and other management problems that require rapid responses. These approaches can also be used to adjust belief weights over time as studies and expert knowledge accumulate and can be a helpful tool for adapting management decisions.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023 Elsevier Ltd. All rights reserved.)

Female, Humans, Pilot Projects, Endoscopes, Fallopian Tubes, Ovarian Neoplasms diagnostic imaging, and Ovarian Neoplasms pathology

Significance: High grade serous ovarian cancer is the most deadly gynecological cancer, and it is now believed that most cases originate in the fallopian tubes (FTs). Early detection of ovarian cancer could double the 5-year survival rate compared with late-stage diagnosis. Autofluorescence imaging can detect serous-origin precancerous and cancerous lesions in ex vivo FT and ovaries with good sensitivity and specificity. Multispectral fluorescence imaging (MFI) can differentiate healthy, benign, and malignant ovarian and FT tissues. Optical coherence tomography (OCT) reveals subsurface microstructural information and can distinguish normal and cancerous structure in ovaries and FTs.
Aim: We developed an FT endoscope, the falloposcope, as a method for detecting ovarian cancer with MFI and OCT. The falloposcope clinical prototype was tested in a pilot study with 12 volunteers to date to evaluate the safety and feasibility of FT imaging prior to standard of care salpingectomy in normal-risk volunteers. In this manuscript, we describe the multiple modifications made to the falloposcope to enhance robustness, usability, and image quality based on lessons learned in the clinical setting.
Approach: The ∼ 0.8    mm diameter falloposcope was introduced via a minimally invasive approach through a commercially available hysteroscope and introducing a catheter. A navigation video, MFI, and OCT of human FTs were obtained. Feedback from stakeholders on image quality and procedural difficulty was obtained.
Results: The falloposcope successfully obtained images in vivo . Considerable feedback was obtained, motivating iterative improvements, including accommodating the operating room environment, modifying the hysteroscope accessories, decreasing endoscope fragility and fiber breaks, optimizing software, improving fiber bundle images, decreasing gradient-index lens stray light, optimizing the proximal imaging system, and improving the illumination.
Conclusions: The initial clinical prototype falloposcope was able to image the FTs, and iterative prototyping has increased its robustness, functionality, and ease of use for future trials.
(© 2023 The Authors.)

Background: Design dynamics that evolve during a designer's prototyping process encapsulate important insights about the way the designer is using his or her knowledge, creativity, and reflective thinking. Nevertheless, the capturing of such dynamics is not always an easy task, as they are built through alternations between the self-first and self-third person views.
Objective: This study aimed at introducing a conceptual framework, namely 2D-ME, to provide an explainable domain that could express the dynamics across the design timeline during a prototyping process of serious games.
Methods: Within the 2D-ME framework, the Technological-Pedagogical-Content Knowledge (TPACK), its adaptation to the serious games (TPACK-Game), and the activity theory frameworks were combined to produce dynamic constructs that incorporate self-first and self-third person extension of the TPACK-Game to Games TPACK, rules, division of labor, and object. The dynamic interplay between such constructs was used as an adaptation engine within an optimization prototype process, so each sequential version of the latter could converge to the designer's initial idea of the serious game. Moreover, higher-order thinking is scaffolded with the internal Activity Interview Script proposed in this paper.
Results: An experimental case study of the application of the 2D-ME conceptual framework in the design of a light reflection game was showcased, revealing all the designer's dynamics, both from internal (via a diary) and external (via the prototype version) views. The findings of this case study exemplified the convergence of the prototyping process to an optimized output, by minimizing the mean square error between the conceptual (initial and updated) idea of the prototype, following explainable and tangible constructs within the 2D-ME framework.
Conclusions: The generic structure of the proposed 2D-ME framework allows its transferability to various levels of expertise in serious games mastering, and it is used both for the designer's process exploration and training of the novice ones.
(©Sofia Hadjileontiadou, Sofia B Dias, Leontios Hadjileontiadis. Originally published in JMIR Serious Games (https://games.jmir.org), 24.04.2023.)

Green Fluorescent Proteins chemistry, Green Fluorescent Proteins genetics, Biosynthetic Pathways, Metabolic Engineering methods, Nicotinamide Mononucleotide biosynthesis, Enzymes chemistry, and Enzymes genetics

Engineering biological systems to test new pathway variants containing different enzyme homologs is laborious and time-consuming. To tackle this challenge, a strategy was developed for rapidly prototyping enzyme homologs by combining cell-free protein synthesis (CFPS) with split green fluorescent protein (GFP). This strategy featured two main advantages: (1) dozens of enzyme homologs were parallelly produced by CFPS within hours, and (2) the expression level and activity of each homolog was determined simultaneously by using the split GFP assay. As a model, this strategy was applied to optimize a 3-step pathway for nicotinamide mononucleotide (NMN) synthesis. Ten enzyme homologs from different organisms were selected for each step. Here, the most productive homolog of each step was identified within 24 h rather than weeks or months. Finally, the titer of NMN was increased to 1213 mg/L by improving physiochemical conditions, tuning enzyme ratios and cofactor concentrations, and decreasing the feedback inhibition, which was a more than 12-fold improvement over the initial setup. This strategy would provide a promising way to accelerate design-build-test cycles for metabolic engineering to improve the production of desired products.
(© 2023 Wiley Periodicals LLC.)