Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System.

CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression-all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses.

MassDash: A Web-Based Dashboard for Data-Independent Acquisition Mass Spectrometry Visualization.

With the increased usage and diversity of methods and instruments being applied to analyze Data-Independent Acquisition (DIA) data, visualization is becoming increasingly important to validate automated software results. Here we present MassDash, a cross-platform DIA mass spectrometry visualization and validation software for comparing features and results across popular tools. MassDash provides a web-based interface and Python package for interactive feature visualizations and summary report plots across multiple automated DIA feature detection tools, including OpenSwath, DIA-NN, and dreamDIA. Furthermore, MassDash processes peptides on the fly, enabling interactive visualization of peptides across dozens of runs simultaneously on a personal computer. MassDash supports various multidimensional visualizations across retention time, ion mobility, m/z, and intensity, providing additional insights into the data. The modular framework is easily extendable, enabling rapid algorithm development of novel peak-picker techniques, such as deep-learning-based approaches and refinement of existing tools. MassDash is open-source under a BSD 3-Clause license and freely available at https://github.com/Roestlab/massdash, and a demo version can be accessed at https://massdash.streamlit.app.

Exploring the frontier of rapid prototyping technologies for plant synthetic biology and what could lie beyond.

Realizing the full potential of plant synthetic biology both to elucidate the relationship between genotype and phenotype and to apply these insights to engineer traits requires rapidly iterating through design-build-test cycles. However, the months-long process of transgenesis, the long generation times, and the size-based limitations on experimentation have stymied progress by limiting the speed and scale of these cycles. Herein, we review a representative sample of recent studies that demonstrate a variety of rapid prototyping technologies that overcome some of these bottlenecks and accelerate progress. However, each of them has caveats that limit their broad utility. Their complementary strengths and weaknesses point to the intriguing possibility that these strategies could be combined in the future to enable rapid and scalable deployment of synthetic biology in plants.

Inertially enhanced mass transport using 3D-printed porous flow-through electrodes with periodic lattice structures.

Electrochemical reactors utilizing flow-through electrodes (FTEs) provide an attractive path toward the efficient utilization of electrical energy, but their commercial viability and ultimate adoption hinge on attaining high currents to drive productivity and cost competitiveness. Conventional FTEs composed of random, porous media provide limited opportunity for architectural control and engineering of microscale transport. Alternatively, the design freedom engendered by additively manufacturing FTEs yields additional opportunities to further drive performance via flow engineering. Through experiment and validated continuum computation we analyze the mass transfer in three-dimensional (3D)-printed porous FTEs with periodic lattice structures and show that, in contrast to conventional electrodes, the mesoscopic length scales in 3D-printed electrodes lead to an increase in the mass correlation exponent as inertial flow effects dominate. The inertially enhanced mass transport yields mass transfer coefficients that exceed previously reported 3D-printed FTEs by 10 to 100 times, bringing 3D-printed FTE performance on par with conventional materials.

Advantages of stereolithographic 3D printing in the fabrication of the Affiblot device for dot-blot assays.

In stereolithographic (SLA) 3D printing, objects are constructed by exposing layers of photocurable resin to UV light. It is a highly user-friendly fabrication method that opens a possibility for technology sharing through CAD file online libraries. Here, we present a prototyping procedure of a microfluidics-enhanced dot-blot device (Affiblot) designed for simple and inexpensive screening of affinity molecule characteristics (antibodies, oligonucleotides, cell receptors, etc.). The incorporation of microfluidic features makes sample processing user-friendly, less time-consuming, and less laborious, all performed completely on-device, distinguishing it from other dot-blot devices. Initially, the Affiblot device was fabricated using CNC machining, which required significant investment in manual post-processing and resulted in low reproducibility. Utilization of SLA 3D printing reduced the amount of manual post-processing, which significantly streamlined the prototyping process. Moreover, it enabled the fabrication of previously impossible features, including internal fluidic channels. While 3D printing of sub-millimeter microchannels usually requires custom-built printers, we were able to fabricate microfluidic features on a readily available commercial printer. Open microchannels in the size range 200-300 µm could be fabricated with reliable repeatability and sealed with a replaceable foil. Economic aspects of device fabrication are also discussed.

Demonstration of a Transparent and Adhesive Sealing Top for Microfluidic Lab-Chip Applications.

A transparent and adhesive film-based enclosing and sealing method is here presented for out-of-cleanroom-based open-form microfluidic devices. The commercially available polyester flexible film known as Microseal 'B' is presented in this paper as a cover seal for open-form microfluidic devices. This film is adaptable to high working temperatures and is biocompatible. The quality of the sealing film was investigated by leak tests, fluorescence tests, and contact angle measurements. The investigations revealed its sealing strength, fluorescence detection compatibility, and surface wettability. It was found that the proposed sealing polyester film on the 3D-printed device could sustain a gauge pressure of 2.7 atm at a flow rate of 4 mL/min without any leaks. It also provided fluorescence detection compatibility and an intensity-to-background ratio in the range of 2.3 to 4.5 for particle sizes of 5 µm and 15 µm, respectively, which is comparable with the performances of other sealing materials. The film's hydrophobicity is comparable to other polymers used in microfluidics. This paper concludes by showcasing some applications of such transparent tops in classical microfluidic devices used for droplet generation and fluid mixing, in order to demonstrate the prospects of this fabrication technique in lab-on-a-chip devices.

Wearable Robot Design Optimization Using Closed-Form Human-Robot Dynamic Interaction Model.

Wearable robots are emerging as a viable and effective solution for assisting and enabling people who suffer from balance and mobility disorders. Virtual prototyping is a powerful tool to design robots, preventing the costly iterative physical prototyping and testing. Design of wearable robots through modelling, however, often involves computationally expensive and error-prone multi-body simulations wrapped in an optimization framework to simulate human-robot-environment interactions. This paper proposes a framework to make the human-robot link segment system statically determinate, allowing for the closed-form inverse dynamics formulation of the link-segment model to be solved directly in order to simulate human-robot dynamic interactions. The paper also uses a technique developed by the authors to estimate the walking ground reactions from reference kinematic data, avoiding the need to measure them. The proposed framework is (a) computationally efficient and (b) transparent and easy to interpret, and (c) eliminates the need for optimization, detailed musculoskeletal modelling and measuring ground reaction forces for normal walking simulations. It is used to optimise the position of hip and ankle joints and the actuator torque-velocity requirements for a seven segments of a lower-limb wearable robot that is attached to the user at the shoes and pelvis. Gait measurements were carried out on six healthy subjects, and the data were used for design optimization and validation. The new technique promises to offer a significant advance in the way in which wearable robots can be designed.

Cell-Free Synthesis: Expediting Biomanufacturing of Chemical and Biological Molecules.

The increasing demand for sustainable alternatives underscores the critical need for a shift away from traditional hydrocarbon-dependent processes. In this landscape, biomanufacturing emerges as a compelling solution, offering a pathway to produce essential chemical materials with significantly reduced environmental impacts. By utilizing engineered microorganisms and biomass as raw materials, biomanufacturing seeks to achieve a carbon-neutral footprint, effectively counteracting the carbon dioxide emissions associated with fossil fuel use. The efficiency and specificity of biocatalysts further contribute to lowering energy consumption and enhancing the sustainability of the production process. Within this context, cell-free synthesis emerges as a promising approach to accelerate the shift towards biomanufacturing. Operating with cellular machinery in a controlled environment, cell-free synthesis offers multiple advantages: it enables the rapid evaluation of biosynthetic pathways and optimization of the conditions for the synthesis of specific chemicals. It also holds potential as an on-demand platform for the production of personalized and specialized products. This review explores recent progress in cell-free synthesis, highlighting its potential to expedite the transformation of chemical processes into more sustainable biomanufacturing practices. We discuss how cell-free techniques not only accelerate the development of new bioproducts but also broaden the horizons for sustainable chemical production. Additionally, we address the challenges of scaling these technologies for commercial use and ensuring their affordability, which are critical for cell-free systems to meet the future demands of industries and fully realize their potential.

Modular training model for education of students in restorative and prosthodontic dentistry.

INTRODUCTION: At the moment, no commercial model solution is available for the individualisation of the dentition depending on the clinical case scenario. Furthermore, the realistic training of most restorative and prosthodontic procedures on a single dental study model is not possible. The aim of this study was the creation of a new training model to fill this gap. MATERIALS AND METHODS: Complete upper and lower jaw models were created based on existing scans and radiological data from a patient. All components for 100 complete models and 1128 teeth for the training were produced with a SLA-printer. Overall, 94 voluntary students attending the first and second preclinical course in prosthodontics tested the functionality of the model with three different tooth types against a standard dental study model and real teeth. After the training, the model was rated in a questionnaire. RESULTS: The production of the models and teeth was feasible. The overall rating of the different teeth was worse for type I (Ø 3.6 ± 1.1), significantly better for type II (Ø 2.5 ± 1.0) and type III (Ø 2.4 ± 1.0) than a standard typodont tooth (Ø 2.7 ± 1.1). The new model was rated significantly better overall (Ø 2.6 ± 1.0) than the standard training model (Ø 3.0 ± 1.1). CONCLUSIONS: The aim of this study was fulfilled. A superior training model was created with equivalent and better tooth types. The new teeth were outstanding in terms of cost-efficiency, appearance and feeling during preparation.

The 3D-printed shell complete denture technique: Simplifying prosthodontic diagnosis prior to implant planning.

Traditionally, artificial teeth arrangements or the definitive complete dentures are used to establish important prosthodontic parameters such as the occlusal plane orientation, vertical dimension, and the incisal edge position. The relationship of these elements with the underlying bony structures is commonly evaluated using advanced planning protocols such as the dual scan technique. This technique article presents an uncomplicated alternative approach to establish these parameters intraorally using a 3D-printed shell complete denture generated from a 3D scan of the patient's existing complete denture.

Zirconia Implants Produced by Additive Manufacturing - A Scoping Review.

INTRODUCTION: Additive manufacturing is a tool with potential use in medicine and dentistry. The manufacture of metals and composites is already advanced, however, concerns about titanium hypersensitivity, tissue staining, and corrosion caused by gradual material degradation encourage research into more biocompatible alternatives. OBJECTIVE: This systematic scoping review aimed to gather studies that evaluated zirconia implants produced by additive manufacturing to describe the current stage of the printing technique and the final product. METHODS: Searches in Embase, PubMed, SCOPUS, Web of Science, and Google Scholar databases were enriched with manual searches between February and March 2021 and updated in June 2022 using keywords: zirconium implants, zirconium oxide, additive manufacturing, rapid prototyping, 3D printing, selective laser melting, and electron beam melting. The criteria included studies that evaluated or described zirconia implants obtained by 3D printing, with a direct relationship to dentistry or orthopedics. RESULTS: The database search resulted in 671 articles. Eight articles were selected for full reading and remained in this systematic review. CONCLUSION: The printing technique for zirconia implants is promising. However, further studies are required before implants produced by the printing technique can be tested clinically. The literature with results regarding the impression product is still limited.

Three-dimensional printed models can reduce costs and surgical time for complex proximal humeral fractures: preoperative planning, patient satisfaction, and improved resident skills.

BACKGROUND: Proximal humeral fractures (PHFs) are still controversial with regards to treatment and are difficult to classify. The study's objective is to show that preoperative planning performed while handling a three-dimensional (3D) printed anatomical model of the fracture can ensure a better understanding of trauma for both surgeons and patients. MATERIALS AND METHODS: Twenty patients (group A, cases) with complex PHF were evaluated preoperatively by reproducing life-size, full-touch 3D anatomical models. Intraoperative blood loss, radiographic controls, duration of surgery, and clinical outcomes of patients in group A were compared with 20 patients (group B, controls) who underwent standard preoperative evaluation. Additionally, senior surgeons and residents, as well as group A patients, answered a questionnaire to evaluate innovative preoperative planning and patient compliance. Cost analysis was evaluated. RESULTS: Intraoperative radiography controls and length of operation were significantly shorter in group A. There were no differences in clinical outcomes or blood loss. Patients claim a better understanding of the trauma suffered and the proposed treatment. Surgeons assert that the planning of the definitive operation with 3D models has had a good impact. The development of this tool has been well received by the residents. The surgery was reduced in length by 15%, resulting in savings of about EUR 400 for each intervention. CONCLUSIONS: Fewer intraoperative radiography checks, shorter surgeries, and better patient compliance reduce radiation exposure for patients and healthcare staff, enhance surgical outcomes while reducing expenses, and lower the risk of medicolegal claims. LEVEL OF EVIDENCE: Level I, prospective randomized case-control study.

Establishing a versatile toolkit of flux enhanced strains and cell extracts for pathway prototyping.

Building and optimizing biosynthetic pathways in engineered cells holds promise to address societal needs in energy, materials, and medicine, but it is often time-consuming. Cell-free synthetic biology has emerged as a powerful tool to accelerate design-build-test-learn cycles for pathway engineering with increased tolerance to toxic compounds. However, most cell-free pathway prototyping to date has been performed in extracts from wildtype cells which often do not have sufficient flux towards the pathways of interest, which can be enhanced by engineering. Here, to address this gap, we create a set of engineered Escherichia coli and Saccharomyces cerevisiae strains rewired via CRISPR-dCas9 to achieve high-flux toward key metabolic precursors; namely, acetyl-CoA, shikimate, triose-phosphate, oxaloacetate, α-ketoglutarate, and glucose-6-phosphate. Cell-free extracts generated from these strains are used for targeted enzyme screening in vitro. As model systems, we assess in vivo and in vitro production of triacetic acid lactone from acetyl-CoA and muconic acid from the shikimate pathway. The need for these platforms is exemplified by the fact that muconic acid cannot be detected in wildtype extracts provided with the same biosynthetic enzymes. We also perform metabolomic comparison to understand biochemical differences between the cellular and cell-free muconic acid synthesis systems (E. coli and S. cerevisiae cells and cell extracts with and without metabolic rewiring). While any given pathway has different interfaces with metabolism, we anticipate that this set of pre-optimized, flux enhanced cell extracts will enable prototyping efforts for new biosynthetic pathways and the discovery of biochemical functions of enzymes.

Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell-free protein synthesis with split GFP.

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.

Research relating to three-dimensional (3D) printing in spine surgery: a bibliometric analysis.

PURPOSE: Although numerous publications on three-dimensional printing (3DP) in spine surgery have been published, bibliometric analysis studies are scarce. Thus, this study aimed to present a bibliometric analysis of the status, hot spots, and frontiers of 3DP in spine surgery and associated research disciplines. METHODS: All publications relating to the utilization of 3DP in spine surgery from 1999 to May 9, 2022, were retrieved from the Web of Science. The bibliometric analysis was performed using CiteSpace software, and information on the country, institution, author, journal, and keywords for each publication was collected. RESULTS: A total of 270 articles were identified. From 2016 onward, a significant increase in publications on spinal surgery was observed. China was the most productive and influential country (98 publications) and H-index (22), followed by the USA and Australia. The most productive institution was Capital Medical University (9 publications). P. S. D'urso (8 publications, 46 citations) and R. J. Mobbs (8 publications, 39 citations) were the most prolific authors. European Spine Journal contributed the highest number of publications. The eight main clusters were: "rapid prototyping" #0, "3D printed" #1, "spine fusion" #2, "scoliosis" #3, "spine surgery" #4, "patient-specific" #5, "nervous system" #6, and "neuronavigation" #7. The strongest keyword bursts in 3DP in spine surgery were "fixation," "drill template," "instrumentation," "fusion," "complication," and "atlantoaxial instability." CONCLUSION: This analysis provides information on research trends and frontiers in the application of 3DP in spine surgery, as well as research and collaboration partners, institutions, and countries.

Comparisons of 3D printed materials for biomedical imaging applications.

In biomedical imaging, it is desirable that custom-made accessories for restraint, anesthesia, and monitoring can be easily cleaned and not interfere with the imaging quality or analyses. With the rise of 3D printing as a form of rapid prototyping or manufacturing for imaging tools and accessories, it is important to understand which printable materials are durable and not likely to interfere with imaging applications. Here, 15 3D printable materials were evaluated for radiodensity, optical properties, simulated wear, and capacity for repeated cleaning and disinfection. Materials that were durable, easily cleaned, and not expected to interfere with CT, PET, or optical imaging applications were identified.

A guide for selecting 3D printed materials for custom research tools through characterization of their merits and limitations in biomedical imaging.

Rapid IoT Prototyping: A Visual Programming Tool and Hardware Solutions for LoRa-Based Devices.

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.

SENS+: A Co-Existing Fabrication System for a Smart DFA Environment Based on Energy Fusion Information.

In factories, energy conservation is a crucial issue. The co-fabrication space is a modern-day equivalent of a new factory type, and it makes use of Internet of Things (IoT) devices, such as sensors, software, and online connectivity, to keep track of various building features, analyze data, and produce reports on usage patterns and trends that can be used to improve building operations and the environment. The co-fabrication user requires dynamic and flexible space, which is different from the conventional user's usage. Because the user composition in a co-fabrication space is dynamic and unstable, we cannot use the conventional approach to assess their usage and rentals. Prototyping necessitates a specifically designed energy-saving strategy. The research uses a "seeing-moving-seeing" design thinking framework, which enables designers to more easily convey their ideas to others through direct observation of the outcomes of their intuitive designs and the representation of their works through design media. The three components of human behavior, physical manufacture, and digital interaction are primarily the focus of this work. The computing system that connects the physical machine is created through communication between the designer and the digital interface, giving the designer control over the physical machine. It is an interactive fabrication process formed by behavior. The Sensible Energy System+ is an interactive fabrication process of virtual and real coexistence created by combining the already-existing technology, the prototype fabrication machine, and SENS. This process analyzes each step of the fabrication process and energy, fits it into the computing system mode to control the prototype fabrication machine, and reduces the problem between virtual and physical fabrication and energy consumption.

Characterizing user needs for Beescape: A spatial decision support tool focused on pollinator health.

Interactive maps can serve as powerful environmental decision-support tools. However, designing an interactive map that meets the needs of diverse constituencies is a challenge. In this article, we evaluate and characterize user needs for an interactive map and spatial decision-support tool called Beescape. Beescape is designed to visualize resources and environmental risks to bees and other pollinators (such as availability of nutritional resources from flowering plants and exposure to pesticides) in order to help users make informed decisions about managing bee populations and associated landscapes. We conducted a needs assessment workshop with twenty stakeholders from four user groups including beekeepers, growers, conservationists, and pollinator scientists to elicit their knowledge to guide future Beescape development. The results of the workshop identify current analytical gaps with the existing Beescape prototype, including the need for predictive and historical tools, more actionable data layers, finer-grain spatial data, and better explanations on what data represent and how they were created. Our findings on user's analytical, informational, and interface needs can be utilized to guide the future development of spatial decision support tools like Beescape, and our methodological approach may apply to other environmental informatics tools where it is important to design for multiple constituent user groups.

Recent Advances in the Applications of Additive Manufacturing (3D Printing) in Drug Delivery: A Comprehensive Review.

There has been a tremendous increase in the investigations of three-dimensional (3D) printing for biomedical and pharmaceutical applications, and drug delivery in particular, ever since the US FDA approved the first 3D printed medicine, SPRITAM® (levetiracetam) in 2015. Three-dimensional printing, also known as additive manufacturing, involves various manufacturing techniques like fused-deposition modeling, 3D inkjet, stereolithography, direct powder extrusion, and selective laser sintering, among other 3D printing techniques, which are based on the digitally controlled layer-by-layer deposition of materials to form various geometries of printlets. In contrast to conventional manufacturing methods, 3D printing technologies provide the unique and important opportunity for the fabrication of personalized dosage forms, which is an important aspect in addressing diverse patient medical needs. There is however the need to speed up the use of 3D printing in the biopharmaceutical industry and clinical settings, and this can be made possible through the integration of modern technologies like artificial intelligence, machine learning, and Internet of Things, into additive manufacturing. This will lead to less human involvement and expertise, independent, streamlined, and intelligent production of personalized medicines. Four-dimensional (4D) printing is another important additive manufacturing technique similar to 3D printing, but adds a 4th dimension defined as time, to the printing. This paper aims to give a detailed review of the applications and principles of operation of various 3D printing technologies in drug delivery, and the materials used in 3D printing, and highlight the challenges and opportunities of additive manufacturing, while introducing the concept of 4D printing and its pharmaceutical applications.