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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 66,973 bioRxiv papers from 294,811 authors.

Most downloaded bioRxiv papers, all time

in category bioengineering

1,325 results found. For more information, click each entry to expand.

1: Paperfuge: An ultra-low cost, hand-powered centrifuge inspired by the mechanics of a whirligig toy
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Posted to bioRxiv 30 Aug 2016

Paperfuge: An ultra-low cost, hand-powered centrifuge inspired by the mechanics of a whirligig toy
25,622 downloads bioengineering

M. Saad Bhamla, Brandon Benson, Chew Chai, Georgios Katsikis, Aanchal Johri, Manu Prakash

Sample preparation, including separation of plasma from whole blood or isolation of parasites, is an unmet challenge in many point of care (POC) diagnostics and requires centrifugation as the first key step. From the context of global health applications, commercial centrifuges are expensive, bulky and electricity-powered, leading to a critical bottle-neck in the development of decentralized, electricity-free POC diagnostic devices. By uncovering the fundamental mechanics of an ancient whirligig toy (3300 B.C.E), we design an ultra-low cost (20 cents), light-weight (2 g), human-powered centrifuge that is made out of paper ("paperfuge"). To push the operating limits of this unconventional centrifuge, we present an experimentally-validated theoretical model that describes the paperfuge as a non-linear, non-conservative oscillator system. We use this model to inform our design process, achieving speeds of 125,000 rpm and equivalent centrifugal forces of 30,000 g, with theoretical limits predicting one million rpm. We harness these speeds to separate pure plasma in less than 1.5 minutes and isolate malaria parasites in 15 minutes from whole human blood. By expanding the materials used, we implement centrifugal microfluidics using PDMS, plastic and 3D-printed devices, ultimately opening up new opportunities for electricity-free POC diagnostics, especially in resource-poor settings.

2: Directed evolution of TurboID for efficient proximity labeling in living cells and organisms
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Posted to bioRxiv 02 Oct 2017

Directed evolution of TurboID for efficient proximity labeling in living cells and organisms
14,438 downloads bioengineering

Tess C Branon, Justin A Bosch, Ariana D Sanchez, Namrata D Udeshi, Tanya Svinkina, Steven A. Carr, Jessica L Feldman, Norbert Perrimon, Alice Y. Ting

Protein interaction networks and protein compartmentation underlie every signaling process and regulatory mechanism in cells. Recently, proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, the two enzymes commonly used for PL come with tradeoffs: BioID is slow, requiring tagging times of 18-24 hours, while APEX peroxidase uses substrates that have limited cell permeability and high toxicity. To address these problems, we used yeast display-based directed evolution to engineer two mutants of biotin ligase, TurboID and miniTurbo, with much greater catalytic efficiency than BioID, and the ability to carry out PL in cells in much shorter time windows (as little as 10 minutes) with non-toxic and easily deliverable biotin. In addition to shortening PL time by 100-fold and increasing PL yield in cell culture, TurboID enabled biotin-based PL in new settings, including yeast, Drosophila, and C. elegans.

3: DNA microscopy: Optics-free spatio-genetic imaging by a stand-alone chemical reaction
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Posted to bioRxiv 19 Nov 2018

DNA microscopy: Optics-free spatio-genetic imaging by a stand-alone chemical reaction
8,339 downloads bioengineering

Joshua A. Weinstein, Aviv Regev, Feng Zhang

Analyzing the spatial organization of molecules in cells and tissues is a cornerstone of biological research and clinical practice. However, despite enormous progress in profiling the molecular constituents of cells, spatially mapping these constituents remains a disjointed and machinery-intensive process, relying on either light microscopy or direct physical registration and capture. Here, we demonstrate DNA microscopy, a new imaging modality for scalable, optics-free mapping of relative biomolecule positions. In DNA microscopy of transcripts, transcript molecules are tagged in situ with randomized nucleotides, labeling each molecule uniquely. A second in situ reaction then amplifies the tagged molecules, concatenates the resulting copies, and adds new randomized nucleotides to uniquely label each concatenation event. An algorithm decodes molecular proximities from these concatenated sequences, and infers physical images of the original transcripts at cellular resolution. Because its imaging power derives entirely from diffusive molecular dynamics, DNA microscopy constitutes a chemically encoded microscopy system.

4: Octopi: Open configurable high-throughput imaging platform for infectious disease diagnosis in the field
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Posted to bioRxiv 27 Jun 2019

Octopi: Open configurable high-throughput imaging platform for infectious disease diagnosis in the field
6,683 downloads bioengineering

Hongquan Li, Hazel Soto-Montoya, Maxime Voisin, Lucas Fuentes Valenzuela, Manu Prakash

Access to quantitative, robust, yet affordable diagnostic tools is necessary to reduce global infectious disease burden. Manual microscopy has served as a bedrock for diagnostics with wide adaptability, although at a cost of tedious labor and human errors. Automated robotic microscopes are poised to enable a new era of smart field microscopy but current platforms remain cost prohibitive and largely inflexible, especially for resource poor and field settings. Here we present Octopi, a low-cost ($250-$500) and reconfigurable autonomous microscopy platform capable of automated slide scanning and correlated bright-field and fluorescence imaging. Being highly modular, it also provides a framework for new disease-specific modules to be developed. We demonstrate the power of the platform by applying it to automated detection of malaria parasites in blood smears. Specifically, we discovered a spectral shift on the order of 10 nm for DAPI-stained Plasmodium falciparum malaria parasites. This shift allowed us to detect the parasites with a low magnification (equivalent to 10x) large field of view (2.56 mm^2) module. Combined with automated slide scanning, real time computer vision and machine learning-based classification, Octopi is able to screen more than 1.5 million red blood cells per minute for parasitemia quantification, with estimated diagnostic sensitivity and specificity exceeding 90% at parasitemia of 50/ul and 100% for parasitemia higher than 150/μl. With different modules, we further showed imaging of tissue slice and sputum sample on the platform. With roughly two orders of magnitude in cost reduction, Octopi opens up the possibility of a large robotic microscope network for improved disease diagnosis while providing an avenue for collective efforts for development of modular instruments.

5: Reading canonical and modified nucleotides in 16S ribosomal RNA using nanopore direct RNA sequencing
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Posted to bioRxiv 29 Apr 2017

Reading canonical and modified nucleotides in 16S ribosomal RNA using nanopore direct RNA sequencing
6,625 downloads bioengineering

Andrew M Smith, Miten Jain, Logan Mulroney, Daniel R Garalde, Mark Akeson

The ribosome small subunit is expressed in all living cells. It performs numerous essential functions during translation, including formation of the initiation complex and proofreading of base-pairs between mRNA codons and tRNA anticodons. The core constituent of the small ribosomal subunit is a ~1.5 kb RNA strand in prokaryotes (16S rRNA) and a homologous ~1.8 kb RNA strand in eukaryotes (18S rRNA). Traditional sequencing-by-synthesis (SBS) of rRNA genes or rRNA cDNA copies has achieved wide use as a "molecular chronometer" for phylogenetic studies [1], and as a tool for identifying infectious organisms in the clinic [2]. However, epigenetic modifications on rRNA are erased by SBS methods. Here we describe direct MinION nanopore sequencing of individual, full-length 16S rRNA absent reverse transcription or amplification. As little as 5 picograms (~10 attomole) of E. coli 16S rRNA was detected in 4.5 micrograms of total human RNA. Nanopore ionic current traces that deviated from canonical patterns revealed conserved 16S rRNA base modifications, and a 7-methylguanosine modification that confers aminoglycoside resistance to some pathological E. coli strains. This direct RNA sequencing technology has promise for rapid identification of microbes in the environment and in patient samples.

6: Exponential fluorescent amplification of individual RNAs using clampFISH probes
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Posted to bioRxiv 21 Nov 2017

Exponential fluorescent amplification of individual RNAs using clampFISH probes
5,791 downloads bioengineering

Sara H Rouhanifard, Ian A Mellis, Margaret Dunagin, Sareh Bayatpour, Orsolya Symmons, Allison Cote, Arjun Raj

Non-enzymatic, high-gain signal amplification methods with single-cell, single-molecule resolution are in great need. We present click-amplifying FISH (clampFISH) for the fluorescent detection of RNA that combines the specificity of oligonucleotides with bioorthogonal click chemistry in order to achieve high specificity and extremely high-gain (>400x) signal amplification. We show that clampFISH signal enables detection with low magnification microscopy and separation of cells by RNA levels via flow cytometry. Additionally, we show that the modular design of clampFISH probes enables multiplexing, that the locking mechanism prevents probe detachment in expansion microscopy, and that clampFISH works in tissue samples.

7: Corrigendum and follow-up: Whole genome sequencing of multiple CRISPR-edited mouse lines suggests no excess mutations.
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Posted to bioRxiv 23 Jun 2017

Corrigendum and follow-up: Whole genome sequencing of multiple CRISPR-edited mouse lines suggests no excess mutations.
5,455 downloads bioengineering

Kellie A. Schaefer, Benjamin W. Darbro, Diana F. Colgan, Stephen H. Tsang, Alexander G. Bassuk, Vinit B. Mahajan

Our previous publication suggested CRISPR-Cas9 editing at the zygotic stage might unexpectedly introduce a multitude of subtle but unintended mutations, an interpretation that not surprisingly raised numerous questions. The key issue is that since parental lines were not available, might the reported variants have been inherited? To expand upon the limited available whole genome data on whether CRISPR-edited mice show more genetic variation, whole-genome sequencing was performed on two other mouse lines that had undergone a CRISPR-editing procedure. Again, parents were not available for either the Capn5 nor Fblim1 CRISPR-edited mouse lines, so strain controls were examined. Additionally, we also include verification of variants detected in the initial mouse line. Taken together, these whole-genome-sequencing-level results support the idea that in specific cases, CRISPR-Cas9 editing can precisely edit the genome at the organismal level and may not introduce numerous, unintended, off-target mutations.

8: A rapid and tunable method to temporally control Cas9 expression enables the identification of essential genes and the interrogation of functional gene interactions in vitro and in vivo.
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Posted to bioRxiv 28 Jul 2015

A rapid and tunable method to temporally control Cas9 expression enables the identification of essential genes and the interrogation of functional gene interactions in vitro and in vivo.
5,364 downloads bioengineering

Serif Senturk, Nitin H Shirole, Dawid D. Nowak, Vincenzo Corbo, Alexander Vaughan, David A. Tuveson, Lloyd C. Trotman, Adam Kepecs, Frank Stegmeier, Raffaella Sordella

The Cas9/CRISPR system is a powerful tool for studying gene function. Here we describe a method that allows temporal control of Cas9/CRISPER activity based on conditional CAS9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 enables conditional rapid and reversible Cas9 expression in vitro and efficient gene-editing in the presence of a guide RNA. Further, we show that this strategy can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest, without the latter being co-modulated. In particular, when co-expressed with inducible Cre-ERT2, our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic identification of essential genes and the interrogation of genes functional interactions.

9: Scaling up DNA data storage and random access retrieval
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Posted to bioRxiv 07 Mar 2017

Scaling up DNA data storage and random access retrieval
4,541 downloads bioengineering

Lee Organick, Siena Dumas Ang, Yuan-Jyue Chen, Randolph Lopez, Sergey Yekhanin, Konstantin Makarychev, Miklos Z. Racz, Govinda Kamath, Parikshit Gopalan, Bichlien Nguyen, Christopher Takahashi, Sharon Newman, Hsing-Yeh Parker, Cyrus Rashtchian, Kendall Stewart, Gagan Gupta, Robert Carlson, John Mulligan, Douglas Carmean, Georg Seelig, Luis Ceze, Karin Strauss

Current storage technologies can no longer keep pace with exponentially growing amounts of data. Synthetic DNA offers an attractive alternative due to its potential information density of ~ 1018B/mm3, 107 times denser than magnetic tape, and potential durability of thousands of years. Recent advances in DNA data storage have highlighted technical challenges, in particular, coding and random access, but have stored only modest amounts of data in synthetic DNA. This paper demonstrates an end-to-end approach toward the viability of DNA data storage with large-scale random access. We encoded and stored 35 distinct files, totaling 200MB of data, in more than 13 million DNA oligonucleotides (about 2 billion nucleotides in total) and fully recovered the data with no bit errors, representing an advance of almost an order of magnitude compared to prior work. Our data curation focused on technologically advanced data types and historical relevance, including the Universal Declaration of Human Rights in over 100 languages, a high-definition music video of the band OK Go, and a CropTrust database of the seeds stored in the Svalbard Global Seed Vault. We developed a random access methodology based on selective amplification, for which we designed and validated a large library of primers, and successfully retrieved arbitrarily chosen items from a subset of our pool containing 10.3 million DNA sequences. Moreover, we developed a novel coding scheme that dramatically reduces the physical redundancy (sequencing read coverage) required for error-free decoding to a median of 5x, while maintaining levels of logical redundancy comparable to the best prior codes. We further stress-tested our coding approach by successfully decoding a file using the more error-prone nanopore-based sequencing. We provide a detailed analysis of errors in the process of writing, storing, and reading data from synthetic DNA at a large scale, which helps characterize DNA as a storage medium and justify our coding approach. Thus, we have demonstrated a significant improvement in data volume, random access, and encoding/decoding schemes that contribute to a whole-system vision for DNA data storage.

10: Deep learning achieves super-resolution in fluorescence microscopy
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Posted to bioRxiv 27 Apr 2018

Deep learning achieves super-resolution in fluorescence microscopy
4,343 downloads bioengineering

Hongda Wang, Yair Rivenson, Yiyin Jin, Zhensong Wei, Ronald Gao, Harun Günaydin, Laurent A. Bentolila, Aydogan Ozcan

We present a deep learning-based method for achieving super-resolution in fluorescence microscopy. This data-driven approach does not require any numerical models of the imaging process or the estimation of a point spread function, and is solely based on training a generative adversarial network, which statistically learns to transform low resolution input images into super-resolved ones. Using this method, we super-resolve wide-field images acquired with low numerical aperture objective lenses, matching the resolution that is acquired using high numerical aperture objectives. We also demonstrate that diffraction-limited confocal microscopy images can be transformed by the same framework into super-resolved fluorescence images, matching the image resolution acquired with a stimulated emission depletion (STED) microscope. The deep network rapidly outputs these super-resolution images, without any iterations or parameter search, and even works for types of samples that it was not trained for.

11: Multiplexed confocal and super-resolution fluorescence imaging of cytoskeletal and neuronal synapse proteins
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Posted to bioRxiv 25 Feb 2017

Multiplexed confocal and super-resolution fluorescence imaging of cytoskeletal and neuronal synapse proteins
4,227 downloads bioengineering

Syuan-Ming Guo, Remi Veneziano, Simon Gordonov, Li Li, Demian Park, Anthony B Kulesa, Paul C Blainey, Jeffrey R Cottrell, Edward S Boyden, Mark Bathe

Neuronal synapses contain dozens of protein species whose expression levels and localizations are key determinants of synaptic transmission and plasticity. The spectral properties of fluorophores used in conventional microscopy limit the number of measured proteins to four species within a given sample. The ability to perform high-throughput confocal or super-resolution imaging of many proteins simultaneously without limitation in target number imposed by this spectral limit would enable large-scale characterization of synaptic protein networks in situ. Here, we introduce PRISM: Probe-based Imaging for Sequential Multiplexing, a method that sequentially utilizes either high affinity Locked Nucleic Acid (LNA) or low affinity DNA probes to enable diffraction-limited confocal and PAINT-based super-resolution imaging. High-affinity LNA probes offer high-throughput, confocal-based imaging compared with PAINT, which uses low affinity probes to realize localization-based super-resolution imaging. Simultaneous immunostaining of all targets is performed prior to imaging, followed by sequential LNA/DNA probe exchange that requires only minutes under mild wash conditions. We apply PRISM to quantify the co-expression levels and nanometer-scale organization of one dozen cytoskeletal and synaptic proteins within individual neuronal synapses. Our approach is scalable to dozens of target proteins and is compatible with high-content screening platforms commonly used to interrogate phenotypic changes associated with genetic and drug perturbations in a variety of cell types.

12: BrainNet: A Multi-Person Brain-to-Brain Interface for Direct Collaboration Between Brains
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Posted to bioRxiv 26 Sep 2018

BrainNet: A Multi-Person Brain-to-Brain Interface for Direct Collaboration Between Brains
3,521 downloads bioengineering

Linxing Jiang, Andrea Stocco, Darby M. Losey, Justin A. Abernethy, Chantel S. Prat, Rajesh P. N. Rao

We present BrainNet which, to our knowledge, is the first multi-person non-invasive direct brain-to-brain interface for collaborative problem solving. The interface combines electroencephalography (EEG) to record brain signals and transcranial magnetic stimulation (TMS) to deliver information noninvasively to the brain. The interface allows three human subjects to collaborate using direct brain-to-brain communication. Two of the three subjects are designated as "Senders" whose brain signals are decoded using real-time EEG data analysis to extract decisions about whether to rotate a block in a Tetris-like game before it is dropped to fill a line. The Senders' decisions are transmitted via the Internet to the brain of a third subject, the "Receiver," who cannot see the game screen. The decisions are delivered to the Receiver's brain via magnetic stimulation of the occipital cortex. The Receiver integrates the information received and makes a decision using an EEG interface about either turning the block or keeping it in the same position. A second round of the game gives the Senders one more chance to validate and provide feedback to the Receiver's action. We evaluated the performance of BrainNet in terms of (1) Group-level performance during the game; (2) True/False positive rates of subjects' decisions; (3) Mutual information between subjects. Five groups of three subjects successfully used BrainNet to perform the Tetris task, with an average accuracy of 0.813. Furthermore, by varying the information reliability of the Senders by artificially injecting noise into one Sender's signal, we found that Receivers are able to learn which Sender is more reliable based solely on the information transmitted to their brains. Our results raise the possibility of future brain-to-brain interfaces that enable cooperative problem solving by humans using a "social network" of connected brains.

13: Genome Editing With Targeted Deaminases
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Posted to bioRxiv 28 Jul 2016

Genome Editing With Targeted Deaminases
3,433 downloads bioengineering

Luhan Yang, Adrian W Briggs, Wei Leong Chew, Prashant Mali, Marc Guell, John Aach, Daniel Bryan Goodman, David Cox, Yinan Kan, Emal Lesha, Venkataramanan Soundararajan, Feng Zhang, George Church

Precise genetic modifications are essential for biomedical research and gene therapy. Yet, traditional homology-directed genome editing is limited by the requirements for DNA cleavage, donor DNA template and the endogenous DNA break-repair machinery. Here we present programmable cytidine deaminases that enable site-specific cytidine to thymidine (C-to-T) genomic edits without the need for DNA cleavage. Our targeted deaminases are efficient and specific in Escherichia coli, converting a genomic C-to-T with 13% efficiency and 95% accuracy. Edited cells do not harbor unintended genomic abnormalities. These novel enzymes also function in human cells, leading to a site-specific C-to-T transition in 2.5% of cells with reduced toxicity compared with zinc-finger nucleases. Targeted deaminases therefore represent a platform for safer and effective genome editing in prokaryotes and eukaryotes, especially in systems where DSBs are toxic, such as human stem cells and repetitive elements targeting.

14: Design principles for open source bioinstrumentation: the poseidon syringe pump system as an example
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Posted to bioRxiv 17 Jan 2019

Design principles for open source bioinstrumentation: the poseidon syringe pump system as an example
3,076 downloads bioengineering

A. Sina Booeshaghi, Eduardo da Veiga Beltrame, Dylan Bannon, Jase Gehring, Lior Pachter

The poseidon syringe pump and microscope system is an open source alternative to commercial systems. It costs less than $400 and can be assembled in under an hour using the instructions and source files available at https://pachterlab.github.io/poseidon. We describe the poseidon system and use it to illustrate design principles that can facilitate the adoption and development of open source bioinstruments. The principles are functionality, robustness, simplicity, modularity, benchmarking, and documentation.

15: Rapid Sequential In Situ Multiplexing With DNA-Exchange-Imaging
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Posted to bioRxiv 20 Mar 2017

Rapid Sequential In Situ Multiplexing With DNA-Exchange-Imaging
3,013 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.

16: Shortwave Infrared Fluorescence Imaging with the Clinically Approved Near-Infrared Dye Indocyanine Green
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Posted to bioRxiv 28 Apr 2017

Shortwave Infrared Fluorescence Imaging with the Clinically Approved Near-Infrared Dye Indocyanine Green
2,909 downloads bioengineering

Jessica A. Carr, Daniel Franke, Justin R. Caram, Collin F. Perkinson, Vasileios Askoxylakis, Meenal Datta, Dai Fukumura, Rakesh K. Jain, Moungi G. Bawendi, Oliver T. Bruns

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.

17: An optimized toolkit for precision base editing
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Posted to bioRxiv 17 Apr 2018

An optimized toolkit for precision base editing
2,787 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.

18: High-Fidelity Nanopore Sequencing of Ultra-Short DNA Sequences
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Posted to bioRxiv 16 Feb 2019

High-Fidelity Nanopore Sequencing of Ultra-Short DNA Sequences
2,763 downloads bioengineering

Brandon D Wilson, Michael Eisenstein, Hyongsok Tom Soh

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.

19: An Analysis of Cochlear Implant Distortion from a User's Perspective
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Posted to bioRxiv 06 Mar 2014

An Analysis of Cochlear Implant Distortion from a User's Perspective
2,644 downloads bioengineering

Barry D. Jacobson

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.

20: Silk assembly integrates cells into a 3D fibrillar network that promotes cell spreading and proliferation
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Posted to bioRxiv 29 Aug 2018

Silk assembly integrates cells into a 3D fibrillar network that promotes cell spreading and proliferation
2,587 downloads bioengineering

Ulrika Johansson, Mona Widhe, Nancy Dekki Shalaly, Irene Linares Arregui, Linnea Nilebäck, Christos Panagiotis Tasiopoulos, Carolina Åstrand, Per-Olof Berggren, Christian Gasser, My Hedhammar

Tissues are built of cells integrated in an extracellular matrix (ECM) which provides a three-dimensional (3D) fibrillar network with specific sites for cell anchorage. By genetic engineering, motifs from the ECM can be functionally fused to recombinant silk proteins. Such a silk protein, FN-silk, which harbours a motif from fibronectin, has the ability to self-assemble into fibrillar networks under physiological-like conditions. Herein we describe a method by which mammalian cells are added to the silk solution before assembly, and thereby get uniformly integrated between the formed fibrils. In the resulting 3D scaffold, the cells proliferate and spread out with tissue-like morphology. Elongated cells containing filamentous actin and defined focal adhesion points confirm proper cell attachment to the FN-silk. The cells remain viable in culture for at least 90 days. The method is also scalable to macro-sized 3D cultures. Silk fibers with integrated cells are both strong and extendable, with mechanical properties similar to that of artery walls. The described method enables both differentiation of stem- or precursor cells in 3D and facile co-culture of several different cell types. We show that inclusion of endothelial cells leads to the formation of vessel-like structures throughout the tissue constructs. Hence, silk-assembly in presence of cells constitutes a viable option for 3D culture of cells integrated in a fibrillary ECM-like network, with potential as base for engineering of functional tissue.

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