Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 84,908 bioRxiv papers from 365,213 authors.
Most downloaded bioRxiv papers, all time
in category bioengineering
1,793 results found. For more information, click each entry to expand.
3,247 downloads bioengineering
Nanopore sequencing offers a portable and affordable alternative to sequencing-by-synthesis methods but suffers from lower accuracy and cannot sequence ultra-short DNA. This puts applications such as molecular diagnostics based on the analysis of cell-free DNA or single-nucleotide variants (SNV) out of reach. To overcome these limitations, we report a nanopore-based sequencing strategy in which short target sequences are first circularized and then amplified via rolling-circle amplification to produce long stretches of concatemeric repeats. These can be sequenced on the MinION platform from Oxford Nanopore Technologies (ONT), and the resulting repeat sequences aligned to produce a highly-accurate consensus that reduces the high error-rate present in the individual repeats. Using this approach, we demonstrate for the first time the ability to obtain unbiased and accurate nanopore data for target DNA sequences of < 100 bp. Critically, this approach is sensitive enough to achieve SNV discrimination in mixtures of sequences and even enables quantitative detection of specific variants present at ratios of < 10%. Our method is simple, cost-effective, and only requires well-established processes. It therefore expands the utility of nanopore sequencing for molecular diagnostics and other applications, especially in resource-limited settings.
3,111 downloads bioengineering
Fluorescence imaging is a method of real-time molecular tracking in vivo that has enabled many clinical technologies. Imaging in the shortwave infrared region (SWIR, 1-2 μm) promises higher contrast, sensitivity, and penetration depths compared to conventional visible and near-infrared (NIR) fluorescence imaging. However, adoption of SWIR imaging in clinical settings has been limited, due in part to the absence of FDA-approved fluorophores with peak emission in the SWIR. Here, we show that commercially available NIR dyes, including the FDA-approved contrast agent indocyanine green (ICG), exhibit optical properties suitable for in vivo SWIR fluorescence imaging. Despite the fact that their emission reaches a maximum in the NIR, these dyes can be imaged non-invasively in vivo in the SWIR spectral region, even beyond 1500 nm. We demonstrate real-time fluorescence angiography at wavelengths beyond 1300 nm using ICG at clinically relevant doses. Furthermore, we show tumor-targeted SWIR imaging with trastuzumab labeled with IRDye 800CW, a NIR dye currently being tested in multiple phase II clinical trials. Our findings suggest that high-contrast SWIR fluorescence imaging can be implemented alongside existing imaging modalities by switching the detection of conventional NIR fluorescence systems from silicon-based NIR cameras to emerging indium gallium arsenide (InGaAs) SWIR cameras. Using ICG in particular opens the possibility of translating SWIR fluorescence imaging to human clinical applications.
3,098 downloads bioengineering
Yu Wang, Johannes B Woehrstein, Noah Donoghue, Mingjie Dai, Maier S. Avendaño, Ron C.J. Schackmann, Jason J. Zoeller, Shan Shan H. Wang, Paul W Tillberg, Demian Park, Sylvain W Lapan, Edward S Boyden, Joan S Brugge, Pascal S Kaeser, George M. Church, Sarit S Agasti, Ralf Jungmann, Peng Yin
To decipher the molecular mechanism of biological function, it is critical to map the molecular composition of individual cells in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (hours to overnight in practice) immunostaining. To overcome this speed restriction and develop a practical platform for rapid in situ multiplexing, we describe here DNA-Exchange-Imaging, which allows single-step immunostaining with DNA-barcoded antibodies, followed by rapid (minutes) buffer exchange of fluorophore-bearing DNA imager strands. By eliminating the need for multiple rounds of immunostaining, DEI enables rapid spectrally unlimited sequential imaging. The programmability of DNA-Exchange-Imaging further allows us to adapt it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here), achieving highly multiplexed in situ protein visualization in diverse samples (including neuronal and tumor cells as well as fresh-frozen or paraffin-embedded tissue sections) and at multiple desired resolution scales (from ~300 nm down to sub-20-nm). Validation highlights include 8-target imaging using single-channel Exchange-Confocal in tens of micron thick retina tissue sections in 2-3 hours (as compared to days required in principle by previous methods using comparable equipment), and 8-target super-resolution imaging with ~20 nm resolution using Exchange-PAINT in primary neurons. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.
2,930 downloads bioengineering
Maria Paz Zafra, Emma M Schatoff, Alyna Katti, Miguel Foronda, Marco Breinig, Anabel Y Schweitzer, Amber Simon, Teng Han, Sukanya Goswami, Emma Montgomery, Jordana Thibado, Francisco J. Sánchez-Rivera, Junwei Shi, Christopher R. Vakoc, Scott W Lowe, Darjus F Tschaharganeh, Lukas E Dow
CRISPR base editing is a potentially powerful technology that enables the creation of genetic mutations with single base pair resolution. By re-engineering both DNA and protein sequences, we developed a collection of constitutive and inducible base editing vector systems that dramatically improve the ease and efficiency by which single nucleotide variants can be created. This new toolkit is effective in a wide range of model systems and provides a means for efficient in vivo somatic base editing.
2,924 downloads bioengineering
We describe our first-hand experience with a cochlear implant (CI), being both a recent recipient and a hearing researcher. We note the promising loudness, but very unpleasant distortion, which makes understanding speech difficult in many environments, including in noise, on the phone or through the radio. We also discuss the extreme unpleasantness of music, which makes recognizing familiar melodies very difficult. We investigate the causes of the above problems through mathematical analysis and computer simulations of sound mixtures, and find that surprisingly, the culprit appears to be non-biological in origin, but primarily due to the envelope-based signal processing algorithms currently used. This distortion is generated before the signal even enters the cochlea. Hence, the long-held belief that inter-electrode interference or current spreading is the cause, appears incorrect. We explain that envelope processing may have been originally instituted based on an inaccurate understanding of the role of place coding vs. temporal coding, or alternatively, because of an incorrect analogy to radio modulation theory. On the basis of our analysis, we suggest immediate concrete steps, some possibly in firmware alone, that may lead to a much improved experience.
2,854 downloads bioengineering
The remarkable mechanical performance of biological materials is based on intricate structure-function relationships. Nanoindentation has become the primary tool for characterising biological materials, as it allows to relate structural changes to variations in mechanical properties on small scales. However, the respective theoretical background and associated interpretation of the parameters measured via indentation derives largely from research on "traditional" engineering materials such as metals or ceramics. Here, we discuss the functional relevance of indentation hardness in biological materials by presenting a meta-analysis of its relationship with indentation modulus. Across seven orders of magnitude, indentation hardness was directly proportional to indentation modulus, illustrating that hardness is not an independent material property. Using a lumped parameter model to deconvolute indentation hardness into components arising from reversible and irreversible deformation, we establish criteria which allow to interpret differences in indentation hardness across or within biological materials. The ratio between hardness and modulus arises as a key parameter, which is a proxy for the ratio between irreversible and reversible deformation during indentation, and the material's yield strength. Indentation hardness generally increases upon material dehydration, however to a larger extend than expected from accompanying changes in indentation modulus, indicating that water acts as a "plasticiser". A detailed discussion of the role of indentation hardness, modulus and toughness in damage control during sharp or blunt indentation yields comprehensive guidelines for a performance-based ranking of biological materials, and suggests that quasi-plastic deformation is a frequent yet poorly understood damage mode, highlighting an important area of future research.
2,813 downloads bioengineering
We have engineered light-gated channelrhodopsins (ChRs) whose current strength and light sensitivity enable minimally-invasive neuronal circuit interrogation. Current ChR tools applied to the mammalian brain require intracranial surgery for transgene delivery and implantation of invasive fiber-optic cables to produce light-dependent activation of a small volume of brain tissue [~1 mm3]. To enable optogenetics for large brain volumes and without the need for invasive implants, our ChR engineering approach leverages the significant literature of ChR variants to train statistical models for the design of new, high-performance ChRs. With Gaussian Process models trained on a limited experimental set of 102 functionally characterized ChR variants, we designed high-photocurrent ChRs with unprecedented light sensitivity; three of these, ChRger1, ChRger2, and ChRger3, enable optogenetic activation of the nervous system via minimally-invasive systemic transgene delivery with rAAV-PHP.eB, which was not possible previously due to low per-cell transgene copy produced by systemic delivery. These engineered ChRs enable light-induced neuronal excitation without invasive intracranial surgery for virus delivery or fiber optic implantation, i.e. they enable minimally-invasive optogenetics.
2,779 downloads bioengineering
The term biosensors encompasses devices that have the potential to quantify physiological, immunological and behavioural responses of livestock and multiple animal species. Novel biosensing methodologies offer highly specialized monitoring devices for the specific measurement of individual and multiple parameters covering an animal physiology as well as monitoring of an animals environment. These devices are not only highly specific and sensitive for the parameters being analysed, but they are also reliable and easy to use, and can accelerate the monitoring process. Novel biosensors in livestock management provide significant benefits and applications in disease detection and isolation, health monitoring and detection of reproductive cycles, as well as monitoring physiological well-being of the animal via analysis of the animals environment. With the development of integrated systems and the Internet of Things, the continuously monitoring devices are expected to become affordable. The data generated from integrated livestock monitoring is anticipated to assist farmers and the agricultural industry to improve animal productivity in the future. The data is expected to reduce the impact of the livestock industry on the environment, while at the same time driving the new wave towards the improvements of viable farming techniques. This review focuses on the emerging technological advancements in monitoring of livestock health for detailed, precise information on productivity, as well as physiology and well-being. Biosensors will contribute to the 4th revolution in agriculture by incorporating innovative technologies into cost-effective diagnostic methods that can mitigate the potentially catastrophic effects of infectious outbreaks in farmed animals.
2,763 downloads bioengineering
David N. Nguyen, Theodore L. Roth, Jonathan Li, Peixin Amy Chen, Murad R. Mamedov, Linda T Vo, Victoria Tobin, Ryan Apathy, Daniel Goodman, Eric Shifrut, Jeffrey A. Bluestone, Jennifer M Puck, Francis C Szoka, Alexander Marson
Virus-modified T cells are approved for cancer immunotherapy, but more versatile and precise genome modifications are needed for a wider range of adoptive cellular therapies. We recently developed a non-viral CRISPR-Cas9 system for genomic site-specific integration of large DNA sequences in primary human T cells. Here, we report two key improvements for efficiency and viability in an expanded variety of clinically-relevant primary cell types. We discovered that addition of truncated Cas9 target sequences (tCTS) at the ends of the homology directed repair (HDR) templates can interact with Cas9 ribonucleoproteins (RNPs) to 'shuttle' the template and enhance targeting efficiency. Further, stabilizing the Cas9 RNPs into nanoparticles with poly(glutamic acid) improved editing, reduced toxicity, and enabled lyophilized storage without loss of activity. Combining the tCTS HDR template modifications with polymer-stabilized nanoparticles increased gene targeting efficiency and viable cell yield across multiple genomic loci in diverse cell types. This system is an inexpensive, user-friendly delivery platform for non-viral genome reprogramming that we successfully applied in regulatory T cells (Tregs), γδ-T cells, B cells, NK cells, and primary and iPS-derived hematopoietic stem progenitor cells (HSPCs).
2,755 downloads bioengineering
A summary is presented of five mechanical parameters from human lower limb skeletal muscles critical for Hill-based muscle modeling: the optimal fiber length, the fiber pennation angle, the physiological cross-sectional area (PCSA), the unloaded tendon length, and the fast-twitch fiber fraction. The data presented are drawn from a total of 29 publications including human cadaver studies, in vivo imaging studies of live humans, musculoskeletal modeling studies, and combinations of these methods. Where possible, parameter values were adjusted from the referenced data to present them with consistent definitions (normalization of measured fiber lengths to optimal sarcomere length, and calculation of PCSA as the ratio of fiber volume to fiber length). It is seen that within a specific muscle, optimal fiber lengths are fairly consistent between studies, pennation angles and PCSAs vary widely between studies, and data for unloaded tendon length are comparatively sparse. Few studies have reported fiber type fractions for a large number of muscles. Guidelines for implementing these parameter values in muscle modeling and musculoskeletal modeling are suggested.
2,727 downloads bioengineering
We present critical improvements to recently invented Expansion Microscopy (ExM), which resolve the incompatibility between ExM and single molecule super-resolution microscopy STORM. Specifically, the improved ExM circumvents the massive, 50-100%, bleaching of fluorophores during sample preparations, and preserve the efficiency of enzymatic oxygen scavenging systems in expanded samples. These improvements open up new avenues for Ex-STORM - expanding a sample mechanically, and then visualizing the sample with STORM.
2,667 downloads bioengineering
Selective-plane illumination microscopy (SPIM) provides unparalleled advantages for long-term volumetric imaging of living organisms. In order to achieve high-resolution imaging in common biological sample holders, we designed a high numerical aperture (NA) epi-illumination SPIM (eSPIM) system, which utilizes a single objective and has an identical sample interface as an inverted fluorescence microscope with no additional reflection elements. This system has an effective detection NA of > 1.06. We demonstrated multicolor and fast volumetric imaging of live cells and single-molecule super-resolution microscopy using our system.
2,627 downloads bioengineering
Single-cell RNA-sequencing (scRNA-seq) has become an essential tool for characterizing multi-celled eukaryotic systems but current methods are not compatible with bacteria. Here, we introduce microSPLiT, a low cost and high-throughput scRNA-seq method that works for gram-negative and gram-positive bacteria and can resolve transcriptional states that remain hidden at a population level. We applied microSPLiT to >25,000 Bacillus subtilis cells sampled from different growth stages, creating a detailed atlas of changes in metabolism and lifestyle. We not only retrieve detailed gene expression profiles associated with known but rare states such as competence and PBSX prophage induction, but also identify novel and unexpected gene expression states including heterogeneous activation of a niche metabolic pathway in a subpopulation of cells. microSPLiT empowers high-throughput analysis of gene expression in complex bacterial communities.
2,598 downloads bioengineering
APEX is an engineered peroxidase that catalyzes the oxidation of a wide range of substrates, facilitating its use in a variety of applications, from subcellular staining for electron microscopy to proximity biotinylation for spatial proteomics and transcriptomics. To further advance the capabilities of APEX, we used directed evolution to engineer a split APEX tool (sAPEX). Twenty rounds of FACS-based selections from yeast-displayed fragment libraries, using three different yeast display configurations, produced a 200-amino acid N-terminal fragment (with 9 mutations relative to APEX2) called AP and a 50-amino acid C-terminal fragment called EX. AP and EX fragments were each inactive on their own but reconstituted to give peroxidase activity when driven together by a molecular interaction. We demonstrate sAPEX reconstitution in the mammalian cytosol, on engineered RNA motifs within telomerase noncoding RNA, and at mitochondria-endoplasmic reticulum contact sites.
2,556 downloads bioengineering
CRISPR-Cas technologies have transformed genome-editing of experimental organisms and have immense therapeutic potential. Despite significant advances in our understanding of the CRISPR-Cas9 system, concerns remain over the potential for off-target effects. Recent studies have addressed these concerns using whole-genome sequencing (WGS) of gene-edited embryos or animals to search for de novo mutations (DNMs), which may represent candidate changes induced by poor editing fidelity. Critically, these studies used strain-matched but not pedigree-matched controls and thus were unable to reliably distinguish generational or colony-related differences from true DNMs. Here we used a trio design and whole genome sequenced 8 parents and 19 embryos, where 10 of the embryos were mutagenised with well-characterised gRNAs targeting the coat colour Tyrosinase (Tyr) locus. Detailed analyses of these whole genome data allowed us to conclude that if CRISPR mutagenesis were causing SNV or indel off-target mutations in treated embryos, then the number of these mutations is not statistically distinguishable from the background rate of DNMs occurring due to other processes.
2,540 downloads bioengineering
DeepImageJ is a user-friendly plugin that enables the generic use in FIJI/ImageJ of pre-trained deep learning (DL) models provided by their developers. The plugin acts as a software layer between TensorFlow and FIJI/ImageJ, runs on a standard CPU-based computer and can be used without any DL expertise. Beyond its direct use, we expect DeepImageJ to contribute to the spread and assessment of DL models in life-sciences applications and bioimage informatics.
2,518 downloads bioengineering
Scientific communities are drawn to the open source model as an increasingly utilitarian method to produce and share work. Initially used as a means to develop freely available soft- ware, open source projects have been applied to hardware including scientific tools. Increasing convenience of 3D printing has fueled the proliferation of open labware projects aiming to develop and share designs for scientific tools that can be produced in-house as cheap alter- natives to commercial products. We present our design of a micropipette that is assembled from 3D-printable parts and some hardware that works by actuating a disposable syringe to a user adjustable limit. Graduations on the syringe are used to accurately adjust the set point to the desired volume. Our open design printed micropipette is assessed in comparison to a commercial pipette and meets ISO 8655 standards.
2,505 downloads bioengineering
Potassium ion (K+) homeostasis and dynamics play critical roles in regulating various biological activities, and the ability to monitor K+ spatial-temporal dynamics is critical to understanding these biological functions. Here we report the design and characterization of a Förster resonance energy transfer (FRET)-based genetically encoded K+ indicator, KIRIN1, constructed by inserting a bacterial cytosolic K+ binding protein (Kbp) between a fluorescent protein (FP) FRET pair, mCerulean3 and cp173Venus. Binding of K+ induces a conformational change in Kbp, resulting in an increase in FRET efficiency. KIRIN1 was able to detect K+ at physiologically relevant concentrations in vitro and is highly selective toward K+ over Na+. We further demonstrated that KIRIN1 allowed real-time imaging of pharmacologically induced depletion of cytosolic K+ in live cells, and KIRIN1 also enabled optical tracing of K+ efflux and reuptake in neurons upon glutamate stimulation in cultured primary neurons. These results demonstrate that KIRIN1 is a valuable tool to detect K+ in vitro and in live cells.
2,500 downloads bioengineering
The study of cellular processes occurring inside intact organisms and the development of cell-based diagnostic and therapeutic agents requires methods to visualize cellular functions such as gene expression in deep tissues. Ultrasound is a widely used biomedical technology enabling deep-tissue imaging with high spatial and temporal resolution. However, no genetically encoded molecular reporters are available to connect ultrasound contrast to gene expression in mammalian cells. To address this limitation, we introduce the first mammalian acoustic reporter genes. Starting with an eleven-gene polycistronic gene cluster derived from bacteria, we engineered a eukaryotic genetic program whose introduction into mammalian cells results in the expression of a unique class of intracellular air-filled protein nanostructures called gas vesicles. The scattering of ultrasound by these nanostructures allows mammalian cells to be visualized at volumetric densities below 0.5%, enables the monitoring of dynamic circuit-driven gene expression, and permits high-resolution imaging of gene expression in living animals. These mammalian acoustic reporter genes enable previously impossible approaches to monitoring the location, viability and function of mammalian cells in vivo.
2,487 downloads bioengineering
Objective. Recent research has shown that auditory closed-loop stimulations can enhance sleep slow oscillations (SO) to improve N3 sleep quality and cognition. Previous studies have been conducted in a lab environment and on a small sample size. The present study aimed at validating and assessing the performance of a novel ambulatory wireless dry-EEG device (WDD), for auditory closed-loop stimulations of SO during N3 sleep at home. Material and Methods. The performance of the WDD to detect N3 sleep automatically and to send auditory closed-loop stimulations on SO were tested on 20 young healthy subjects who slept with both the WDD and a miniaturized polysomnography (part 1) in both stimulated and sham nights within a double blind, randomized and crossover design. The electrophysiological effects of auditory closed-loop stimulation on delta power increase were assessed after 1 and 10 nights of stimulations on an observational pilot study in the home environment including 90 middle-aged subjects (part 2). Results. The sensitivity and specificity of the WDD to automatically detect N3 sleep in real-time were 0.70 and 0.90, respectively. The stimulation accuracy of the SO ascending-phase targeting was 45+/-52 degrees. The stimulation protocol induced an increase of 39.5 % of delta power after the stimulations. The increase of SO response to auditory stimulations remained at the same level after 10 consecutive nights. . Conclusion. The WDD shows good performances to automatically detect in real-time N3 sleep and to send auditory closed-loop stimulations on SO accurately. These stimulations increased the SO amplitude during N3 sleep without any adaptation effect after 10 consecutive nights. This tool provides new perspectives to figure out novel sleep EEG biomarkers in longitudinal studies and can be interesting to conduct broad studies on the effects of auditory stimulations during sleep.
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