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Gene Library, Protein Biosynthesis, Synthetic Biology, High-Throughput Screening Assays, and Microfluidics methods

Engineering regulatory parts for improved performance in genetic programs has played a pivotal role in the development of the synthetic biology cell programming toolbox. Here, we report the development of a novel high-throughput platform for regulatory part prototyping and analysis that leverages the advantages of engineered DNA libraries, cell-free protein synthesis (CFPS), high-throughput emulsion droplet microfluidics, standard flow sorting adapted to screen droplet reactions, and next-generation sequencing (NGS). With this integrated platform, we screened the activity of millions of genetic parts within hours, followed by NGS retrieval of the improved designs. This in vitro platform is particularly valuable for engineering regulatory parts of nonmodel organisms, where in vivo high-throughput screening methods are not readily available. The platform can be extended to multipart screening of complete genetic programs to optimize yield and stability.

Purpose: Virtual reality (VR) can provide an added value for diagnosis and/or intervention planning. Several VR software implementations have been proposed but they are often application dependent. Previous attempts for a more generic solution incorporating VR in medical prototyping software (MeVisLab) were still lacking functionality precluding easy and flexible development.
Methods: We propose an alternative solution that uses rendering to a graphical processing unit (GPU) texture to enable rendering arbitrary Open Inventor scenes in a VR context. It facilitates flexible development of user interaction and rendering of more complex scenes involving multiple objects. We tested the platform in planning a transcatheter cardiac stent placement procedure.
Results: This approach proved to enable development of a particular implementation that facilitates planning of percutaneous treatment of a sinus venosus atrial septal defect. The implementation showed it is intuitive to plan and verify the procedure using VR.
Conclusion: An alternative implementation for linking OpenVR with MeVisLab is provided that offers more flexible development of VR prototypes which can facilitate further clinical validation of this technology in various medical disciplines.
(© 2022. CARS.)

Autotrophic Processes, Fermentation, Oxidation-Reduction, Carbon Cycle, and Escherichia coli metabolism

Carbon-negative synthesis of biochemical products has the potential to mitigate global CO 2 emissions. An attractive route to do this is the reverse β-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimize and implement r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screen 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways into Escherichia coli generates designer strains for the selective production of butanoic acid (4.9 ± 0.1 gL -1 ), as well as hexanoic acid (3.06 ± 0.03 gL -1 ) and 1-hexanol (1.0 ± 0.1 gL -1 ) at the best performance reported to date in this bacterium. We also generate Clostridium autoethanogenum strains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL -1 in a 1.5 L continuous fermentation. Our strategy enables optimization of r-BOX derived products for biomanufacturing and industrial biotechnology.
(© 2022. The Author(s).)

The use of personal protective equipment (PPE) has become essential to reduce the transmission of coronavirus disease 2019 (COVID-19) as it prevents the direct contact of body fluid aerosols expelled from carriers. However, many countries have reported critical supply shortages due to the spike in demand during the outbreak in 2020. One potential solution to ease pressure on conventional supply chains is the local fabrication of PPE, particularly face shields, due to their simplistic design. The purpose of this paper is to provide a research protocol and cost implications for the rapid development and manufacturing of face shields by individuals or companies with minimal equipment and materials. This article describes a best practice case study in which the establishment of a local manufacturing hub resulted in the swift production of 12,000 face shields over a seven-week period to meet PPE shortages in the North-West region of Ireland. Protocols and processes for the design, materials sourcing, prototyping, manufacturing, and distribution of face shields are described. Three types of face shields were designed and manufactured, including Flat, Laser-cut, and 3D-printed models. Of the models tested, the Flat model proved the most cost-effective (€0.51/unit), while the Laser-cut model was the most productive (245 units/day). The insights obtained from this study demonstrate the capacity for local voluntary workforces to be quickly mobilised in response to a healthcare emergency, such as the COVID-19 pandemic.
(© 2022 The Authors.)

Humans, Intelligence, and Software

Sensor networks have dynamically expanded our ability to monitor and study the world. Their presence and need keep increasing, and new hardware configurations expand the range of physical stimuli that can be accurately recorded. Sensors are also no longer simply recording the data, they process it and transform into something useful before uploading to the cloud. However, building sensor networks is costly and very time consuming. It is difficult to build upon other people's work and there are only a few open-source solutions for integrating different devices and sensing modalities. We introduce REIP, a Reconfigurable Environmental Intelligence Platform for fast sensor network prototyping. REIP's first and most central tool, implemented in this work, is an open-source software framework, an SDK, with a flexible modular API for data collection and analysis using multiple sensing modalities. REIP is developed with the aim of being user-friendly, device-agnostic, and easily extensible, allowing for fast prototyping of heterogeneous sensor networks. Furthermore, our software framework is implemented in Python to reduce the entrance barrier for future contributions. We demonstrate the potential and versatility of REIP in real world applications, along with performance studies and benchmark REIP SDK against similar systems.

Electrodes, Glucose, Gold chemistry, Hydrogen Peroxide, Silver, Electrochemical Techniques, and Microfluidics

We present a low-cost, accessible, and rapid fabrication process for electrochemical microfluidic sensors. This work leverages the accessibility of consumer-grade electronic craft cutters as the primary tool for patterning of sensor electrodes and microfluidic circuits, while commodity materials such as gold leaf, silver ink pen, double-sided tape, plastic transparency films, and fabric adhesives are used as its base structural materials. The device consists of three layers, the silver reference electrode layer at the top, the PET fluidic circuits in the middle and the gold sensing electrodes at the bottom. Separation of the silver reference electrode from the gold sensing electrodes reduces the possibility of cross-contamination during surface modification. A novel approach in mesoscale patterning of gold leaf electrodes can produce generic designs with dimensions as small as 250 μm. Silver electrodes with dimensions as small as 385 μm were drawn using a plotter and a silver ink pen, and fluid microchannels as small as 300 μm were fabricated using a sandwich of iron-on adhesives and PET. Device layers are then fused together using an office laminator. The integrated microfluidic electrochemical platform has electrode kinetics/performance of Δ Ep = 91.3 mV, Ipa / Ipc = 0.905, characterized by cyclic voltammetry using a standard ferrocyanide redox probe, and this was compared against a commercial screen-printed gold electrode (Δ Ep = 68.9 mV, Ipa / Ipc = 0.984). To validate the performance of the integrated microfluidic electrochemical platform, a catalytic hydrogen peroxide sensor and enzyme-coupled glucose biosensors were developed as demonstrators. Hydrogen peroxide quantitation achieves a limit of detection of 0.713 mM and sensitivity of 78.37 μA mM -1 cm -2 , while glucose has a limit of detection of 0.111 mM and sensitivity of 12.68 μA mM -1 cm -2 . This rapid process allows an iterative design-build-test cycle in under 2 hours. The upfront cost to set up the system is less than USD 520, with each device costing less than USD 0.12, making this manufacturing process suitable for low-resource laboratories or classroom settings.

Background: Augmented reality (AR) and brain-computer interface (BCI) are promising technologies that have a tremendous potential to revolutionize health care. While there has been a growing interest in these technologies for medical applications in the recent years, the combined use of AR and BCI remains a fairly unexplored area that offers significant opportunities for improving health care professional education and clinical practice. This paper describes a recent study to explore the integration of AR and BCI technologies for health care applications.
Objective: The described effort aims to advance an understanding of how AR and BCI technologies can effectively work together to transform modern health care practice by providing new mechanisms to improve patient and provider learning, communication, and shared decision-making.
Methods: The study methods included an environmental scan of AR and BCI technologies currently used in health care, a use case analysis for a combined AR-BCI capability, and development of an integrated AR-BCI prototype solution for health care applications.
Results: The study resulted in a novel interface technology solution that enables interoperability between consumer-grade wearable AR and BCI devices and provides the users with an ability to control digital objects in augmented reality using neural commands. The article discusses this novel solution within the context of practical digital health use cases developed during the course of the study where the combined AR and BCI technologies are anticipated to produce the most impact.
Conclusions: As one of the pioneering efforts in the area of AR and BCI integration, the study presents a practical implementation pathway for AR-BCI integration and provides directions for future research and innovation in this area.
(©Anya Andrews. Originally published in JMIR Formative Research (https://formative.jmir.org), 21.04.2022.)

Crown Lengthening, Humans, Printing, Three-Dimensional, Stereolithography, Computer-Aided Design, and Dental Implants

Progress with additive 3D printing is revolutionizing biomaterial manufacturing, including clinical dentistry and prosthodontics. Among the several 3D additive printing technologies, stereolithography is very popular as it utilizes light-activated resin for precise resolution. A simplified digital technique was used to fabricate two designs of a surgical guide for crown lengthening. Two cases are presented that utilized digital imaging and communications in medicine (DICOM) files obtained with computed tomography (CT) imaging and processed using four CAD software (Blue Sky Plan, Exocad, Meshmixer and 3D Slicer). The final models were converted to standard tessellation (STL) files and the guides were 3D printed with an additive stereolithography (SLA) printer. The first case was fabricated with a bone model from cone beam computed tomography (CBCT) data, and the second case was generated with intraoral and wax-up scans alone. Both methods appear to be equally effective compared to using a conventional method of guide frabication. However, proximal bone reduction was a concern with both designs. Digitally fabricated 3D printed surgical guide for crown lengthening has merit and a practical design is needed for future clinical validation.
(© 2021 by the American College of Prosthodontists.)

This paper demonstrates laser forming, localized heating with a laser to induce plastic deformation, can self-fold 2D printed circuit boards (PCBs) into 3D structures with electronic function. There are many methods for self-folding but few are compatible with electronic materials. We use a low-cost commercial laser writer to both cut and fold a commercial flexible PCB. Laser settings are tuned to select between cutting and folding with higher power resulting in cutting and lower power resulting in localized heating for folding into 3D shapes. Since the thin copper traces used in commercial PCBs are highly reflective and difficult to directly fold, two approaches are explored for enabling folding: plating with a nickel/gold coating or using a single, high-power laser exposure to oxidize the surface and improve laser absorption. We characterized the physical effect of the exposure on the sample as well as the fold angle as a function of laser passes and demonstrate the ability to lift weights comparable with circuit packages and passive components. This technique can form complex, multifold structures with integrated electronics; as a demonstrator, we fold a commercial board with a common timing circuit. Laser forming to add a third dimension to printed circuit boards is an important technology to enable the rapid prototyping of complex 3D electronics.

Exoskeletons and more in general wearable mechatronic devices represent a promising opportunity for rehabilitation and assistance to people presenting with temporary and/or permanent diseases. However, there are still some limits in the diffusion of robotic technologies for neuro-rehabilitation, notwithstanding their technological developments and evidence of clinical effectiveness. One of the main bottlenecks that constrain the complexity, weight, and costs of exoskeletons is represented by the actuators. This problem is particularly evident in devices designed for the upper limb, and in particular for the hand, in which dimension limits and kinematics complexity are particularly challenging. This study presents the design and prototyping of a hand finger exoskeleton. In particular, we focus on the design of a gear-based differential mechanism aimed at coupling the motion of two adjacent fingers and limiting the complexity and costs of the system. The exoskeleton is able to actuate the flexion/extension motion of the fingers and apply bidirectional forces, that is, it is able to both open and close the fingers. The kinematic structure of the finger actuation system has the peculiarity to present three DoFs when the exoskeleton is not worn and one DoF when it is worn, allowing better adaptability and higher wearability. The design of the gear-based differential is inspired by the mechanism widely used in the automotive field; it allows actuating two fingers with one actuator only, keeping their movements independent.
(Copyright © 2022 Dragusanu, Troisi, Villani, Prattichizzo and Malvezzi.)

Adult, Dental Pulp Cavity, Humans, Root Canal Therapy, Tooth Root, Transplantation, Autologous, Treatment Outcome, Surgery, Computer-Assisted, and Tooth

Background: Autotransplantation is a beneficial treatment with a high success rate for young patients. However, most adult patients require root canal treatment (RCT) of the donor teeth after the autotransplantation procedure, which causes a prolonged treatment time and additional expenses and increases the rate of future tooth fracture. Rapid prototyping (RP)-assisted autotransplantation shortens the extra-alveolar time and enables a superior clinical outcome. However, no cohort studies of the application of this method on adult populations have been reported.
Methods: This study is a retrospective cohort study. All patients underwent autotransplantation from 2012 to 2020 in the Kaohsiung and Chia-Yi branches of Chang Gung Memorial Hospital, and the procedure and clinical outcomes were analysed. Differences in clinical outcomes, age, sex, extra-alveolar time, fixation method, and RCT rate were compared between the two groups.
Results: We enrolled 21 patients, 13 treated using the conventional method and 8 treated using the RP-based technique. The RCT rates of the conventional group and RP group were 92.3% and 59%, respectively. The mean age of the two groups was significantly different (28.8 ± 10 vs. 21.6 ± 2.1); after performing subgroup analysis by excluding all of the patients aged > 40 years, we found that the RCT rates were still significantly different (91.0% vs. 50%). The mean extra-alveolar time was 43 s in the RP group, and the autotransplantation survival rate in both groups was 100%.
Conclusions: Rapid prototyping-assisted autotransplantation was successfully adopted for all patients in our study population. By shortening the extra-alveolar time, only 50% of the patients required a root canal treatment with a 100% autotransplantation survival rate.
Trial Registration: Retrospectively registered.
(© 2022. The Author(s).)

Electrodes, Ion-Selective Electrodes, Ions, Potassium, Potentiometry, Printing, Three-Dimensional, Sodium, Internet of Things, and Robotics

We report automated fabrication of solid-contact sodium-selective (Na + -ISEs) and potassium-selective electrodes (K + -ISEs) using a 3D printed liquid handling robot controlled with Internet of Things (IoT) technology. The printing system is affordable and can be customized for the use with micropipettes for applications such as drop-casting, biological assays, sample preparation, rinsing, cell culture, and online analyte monitoring using multi-well plates. The robot is more compact (25 × 30 × 35 cm) and user-friendly than commercially available systems and does not require mechatronic experience. For fabrication of ion-selective electrodes, a carbon black intermediate layer and ion-selective membrane were successively drop-cast on the surface of stencil-printed carbon electrode using the dispensing robot. The 3D-printed robot increased ISE robustness while decreasing the modification time by eliminating manual steps. The Na + -ISEs and K + -ISEs were characterized for their potentiometric responses using a custom-made, low-cost (<$25) multi-channel smartphone-based potentiometer capable of signal processing and wireless data transmission. The electrodes showed Nernstian responses of 58.2 ± 2.6 mV decade -1 and 56.1 ± 0.7 mV decade -1 for Na + and K + , respectively with an LOD of 1.0 × 10 -5  M. We successfully applied the ISEs for multiplexed detection of Na + and K + in urine and artificial sweat samples at clinically relevant concentration ranges. The 3D-printed pipetting robot cost $100 and will pave the way for more accessible mass production of ISEs for those who cannot afford the expensive commercial robots.
(Copyright © 2022 Elsevier B.V. All rights reserved.)

Traveling wave structures for lossless ion manipulation (TW-SLIM) has proven a valuable tool for the separation and study of gas-phase ions. Unfortunately, many of the traditional components of TW-SLIM experiments manifest practical and financial barriers to the technique's broad implementation. To this end, a series of technological innovations and methodologies are presented which enable for simplified SLIM experimentation and more rapid TW-SLIM prototyping. In addition to the use of multiple independent board sets that comprise the present SLIM system, we introduce a low-cost, multifunctional traveling wave generator to produce TW within the TW-SLIM. This square-wave producing unit proved effective in realizing TW-SLIM separations compared to traditional approaches. Maintaining a focus on lowering barriers to implementation, the present set of experiments explores the use of on-board injection (OBI) methods, which offer potential alternatives to ion funnel traps. These OBI techniques proved feasible and the ability of this simplified TW-SLIM platform to enhance ion accumulation was established. Further experimentation regarding ion accumulation revealed a complexity to ion accumulation within TW-SLIM that has yet to be expounded upon. Lastly, the ability of the presented TW-SLIM platform to store ions for extended periods (1 s) without significant loss (<10%) was demonstrated. The aforementioned experiments clearly establish the efficacy of a simplified TW-SLIM platform which promises to expand adoption and experimentation of the technique.
(Copyright © 2022 Elsevier B.V. All rights reserved.)

Transforming lab research into a sustainable business is becoming a trend in the microfluidic field. However, there are various challenges during the translation process due to the gaps between academia and industry, especially from laboratory prototyping to industrial scale-up production, which is critical for potential commercialization. In this Perspective, based on our experience in collaboration with stakeholders, e.g., biologists, microfluidic engineers, diagnostic specialists, and manufacturers, we aim to share our understanding of the manufacturing process chain of microfluidic cartridge from concept development and laboratory prototyping to scale-up production, where the scale-up production of commercial microfluidic cartridges is highlighted. Four suggestions from the aspect of cartridge design for manufacturing, professional involvement, material selection, and standardization are provided in order to help scientists from the laboratory to bring their innovations into pre-clinical, clinical, and mass production and improve the manufacturability of laboratory prototypes toward commercialization.
(© 2022 Author(s).)

Gait, Humans, Neural Networks, Computer, Recognition, Psychology, Apathy, and Wearable Electronic Devices

Human gait is a unique behavioral characteristic that can be used to recognize individuals. Collecting gait information widely by the means of wearable devices and recognizing people by the data has become a topic of research. While most prior studies collected gait information using inertial measurement units, we gather the data from 40 people using insoles, including pressure sensors, and precisely identify the gait phases from the long time series using the pressure data. In terms of recognizing people, there have been a few recent studies on neural network-based approaches for solving the open set gait recognition problem using wearable devices. Typically, these approaches determine decision boundaries in the latent space with a limited number of samples. Motivated by the fact that such methods are sensitive to the values of hyper-parameters, as our first contribution, we propose a new network model that is less sensitive to changes in the values using a new prototyping encoder-decoder network architecture. As our second contribution, to overcome the inherent limitations due to the lack of transparency and interpretability of neural networks, we propose a new module that enables us to analyze which part of the input is relevant to the overall recognition performance using explainable tools such as sensitivity analysis (SA) and layer-wise relevance propagation (LRP).

Capillary Action, Polymers, Porosity, Lab-On-A-Chip Devices, and Microfluidics

Paper-based microfluidics was initially developed for use in ultra-low-cost diagnostics powered passively by liquid wicking. However, there is significant untapped potential in using paper to internally guide porous microfluidic flows using externally applied pressure gradients. Here, we present a new technique for fabricating and utilizing low-cost polymer-laminated paper-based microfluidic devices using external pressure. Known as microfluidic pressure in paper (μPiP), devices fabricated by this technique are capable of sustaining a pressure gradient for use in precise liquid handling and manipulation applications similar to conventional microfluidic open-channel designs, but instead where fluid is driven directly through the porous paper structure. μPiP devices can be both rapidly prototyped or scalably manufactured and deployed at commercial scale with minimal time, equipment, and training requirements. We present an analysis of continuous pressure-driven flow in porous paper-based microfluidic channels and demonstrate broad applicability of this method in performing a variety of different liquid handling applications, including measuring red blood cell deformability and performing continuous free-flow DNA electrophoresis. This new platform offers a budget-friendly method for performing microfluidic operations for both academic prototyping and large-scale commercial device production.

Electrochemical Techniques, Electrodes, Microfluidics, Aptamers, Nucleotide, and Biosensing Techniques

We demonstrate the ability to rapidly prototype and fabricate an epoxy-embedded electrode platform and microfluidic device suitable for using electrochemical biosensors under flow conditions. We utilize three-dimensional (3-D) printing to rapidly prototype molds to fabricate epoxy-embedded electrodes in addition to molds for rapid prototyping of PDMS microfluidic components. We characterize the bare gold epoxy-embedded electrodes using ferricyanide as a redox indicator and then characterize the performance of an adenosine triphosphate (ATP) specific electrochemical, aptamer-based (E-AB) sensor. We then incorporate the ATP specific E-AB sensors into the microfluidic device to study and take advantage of the dynamic response this class of sensor offers. We were able to flow varying concentrations of target analyte and monitor the dynamic response of the sensors to the changing concentration. This work demonstrates the ability to rapidly prototype E-AB sensors under flow conditions using 3-D printing which can lead to rapid and affordable point-of-care or fieldable applications where dynamic measurements of concentration, specificity and sensitivity and multiplex detection are necessary.
(Copyright © 2021 Elsevier B.V. All rights reserved.)

Culture Media, Plastics, Workflow, Laboratories, and Printing, Three-Dimensional

Although the microbiology laboratory paradigm has increasingly changed from manual to automated procedures, and from functional to molecular methods, traditional culture methods remain vital. Using inexpensive desktop fused filament fabrication 3D printing, we designed, produced and tested rapid prototypes of customised labware for microbial culture namely frames to make dip slides, inoculation loops, multi-pin replicators, and multi-well culture plates for solid medium. These customised components were used to plate out samples onto solid media in various formats, and we illustrate how they can be suitable for many microbiological methods such as minimum inhibitory concentration tests, or for directly detecting pathogens from mastitis samples, illustrating the flexibility of rapid-prototyped culture consumable parts for streamlining microbiological methods. We describe the methodology needed for microbiologists to develop their own novel and unique tools, or to fabricate and customise existing consumables. A workflow is presented for designing and 3D printing labware and quickly producing easy-to-sterilise and re-useable plastic parts of great utility in the microbiology laboratory.
(© 2021 The Society for Applied Microbiology.)