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R Verboket, M Leiblein, C Seebach, C Nau, M Janko, M Bellen, H Bönig, D Henrich, I Marzi
OBJECTIVES: Reconstruction of long segmental bone defects is demanding for patients and surgeons, and associated with long-term treatment periods and substantial complication rates in addition to high costs. While defects up to 4-5 cm length might be filled up with autologous bone graft, heterologous bone from cadavers, or artificial bone graft substitutes, current options to reconstruct bone defects greater than 5 cm consist of either vascularized free bone transfers, the Masquelet technique or the Ilizarov distraction osteogenesis...
January 19, 2018: European Journal of Trauma and Emergency Surgery: Official Publication of the European Trauma Society
Micaela Tamara Vitor, Sébastien Sart, Antoine Barizien, Lucimara Gaziola De La Torre, Charles N Baroud
Transient gene expression (TGE) technology enables the rapid production of large amount of recombinant proteins, without the need of fastidious screening of the producing cells required for stable transfection (ST). However, several barriers must be overcome before reaching the production yields using ST. For optimizing the production yields from suspended cells using TGE, a better understanding of the transfection conditions at the single cell level are required. In this study, a universal droplet microfluidic platform was used to assess the heterogeneities of CHO-S population transiently transfected with cationic liposomes (CL) (lipoplexes) complexed with GFP-coding plasmid DNA (pDNA)...
January 19, 2018: Scientific Reports
Lieselot Deleye, Ann-Sophie Vander Plaetsen, Jana Weymaere, Dieter Deforce, Filip Van Nieuwerburgh
To allow multiple genetic analyses on a single cell, whole genome amplification (WGA) is required. Unfortunately, studies comparing different WGA methods for downstream human identification Short Tandem Repeat (STR) analysis remain absent. Therefore, the aim of this work was to assess the performance of four commercially available WGA kits for downstream human identification STR profiling on a B-lymphoblastoid cell line. The performance was assessed using an input of one or three micromanipulated cells. REPLI-g showed a very low dropout rate, as it was the only WGA method in this study that could provide a complete STR profile in some of its samples...
January 19, 2018: Scientific Reports
Seong Jun Park, Sanggeun Song, Gil-Suk Yang, Philip M Kim, Sangwoon Yoon, Ji-Hyun Kim, Jaeyoung Sung
Gene expression is a complex stochastic process composed of numerous enzymatic reactions with rates coupled to hidden cell-state variables. Despite advances in single-cell technologies, the lack of a theory accurately describing the gene expression process has restricted a robust, quantitative understanding of gene expression variability among cells. Here we present the Chemical Fluctuation Theorem (CFT), providing an accurate relationship between the environment-coupled chemical dynamics of gene expression and gene expression variability...
January 19, 2018: Nature Communications
Veena S Patil, Ariel Madrigal, Benjamin J Schmiedel, James Clarke, Patrick O'Rourke, Aruna D de Silva, Eva Harris, Bjoern Peters, Gregory Seumois, Daniela Weiskopf, Alessandro Sette, Pandurangan Vijayanand
CD4+ cytotoxic T lymphocytes (CD4-CTLs) have been reported to play a protective role in several viral infections. However, little is known in humans about the biology of CD4-CTL generation, their functional properties, and heterogeneity, especially in relation to other well-described CD4+ memory T cell subsets. We performed single-cell RNA sequencing in more than 9000 cells to unravel CD4-CTL heterogeneity, transcriptional profile, and clonality in humans. Single-cell differential gene expression analysis revealed a spectrum of known transcripts, including several linked to cytotoxic and costimulatory function that are expressed at higher levels in the TEMRA (effector memory T cells expressing CD45RA) subset, which is highly enriched for CD4-CTLs, compared with CD4+ T cells in the central memory (TCM) and effector memory (TEM) subsets...
January 19, 2018: Science Immunology
Qian-Hao Zhu, Yuman Yuan, Warwick Stiller, Yinhua Jia, Pengpeng Wang, Zhaoe Pan, Xiongming Du, Danny Llewellyn, Iain Wilson
Cotton fibres are single-celled trichomes arising from the epidermal cells of the seed coat and may be either long (lint) or very short (fuzz). The dominant fuzzless N1 of Gossypium hirsutum is a defective allele of the At-subgenome homoeolog of MYB25-like, but the genetic components underlying the recessive fuzzless trait from G. barbadense (Gb) are unknown. We have identified five genetic loci, including a major contributing locus containing MYB25-like_Dt, associated with Gb fuzzless seeds based on genotyping of fuzzy and fuzzless near isogenic lines (NILs) from an interspecies cross (G...
January 17, 2018: Journal of Experimental Botany
Makusu Tsutsui, Masayoshi Tanaka, Takahiro Marui, Kazumichi Yokota, Takeshi Yoshida, Akihide Arima, Wataru Tonomura, Masateru Taniguchi, Takashi Washio, Mina Okochi, Tomoji Kawai
Bioinspired pore sensing for selective detection of flagellated bacteria was investigated. The Au micropore wall surface was modified with a synthetic peptide designed from toll-like receptor 5 (TLR5) to mimic the pathogen-recognition capability. We found that intermolecular interactions between the TLR5-derived recognition peptides and flagella induce ligand-specific perturbations in the translocation dynamics of Escherichia coli, which facilitated the discrimination between the wild-type and flagellin-deletion mutant (ΔfliC) by the resistive pulse patterns thereby demonstrating the sensing of bacteria at a single-cell level...
January 19, 2018: Analytical Chemistry
Aaron J Wilk, Catherine A Blish
NK cells are innate lymphocytes with important roles in immunoregulation, immunosurveillance, and cytokine production. Originally defined on the functional basis of their "natural" ability to lyse tumor targets and thought to be a relatively homogeneous group of lymphocytes, NK cells possess a remarkable degree of phenotypic and functional diversity due to the combinatorial expression of an array of activating and inhibitory receptors. Diversification of NK cells is multifaceted: mechanisms of NK cell education that promote self-tolerance result in a heterogeneous repertoire that further diversifies upon encounters with viral pathogens...
January 19, 2018: Journal of Leukocyte Biology
Xingrui Li, Dongfeng Zhang, Huimin Zhang, Zhichao Guan, Yanling Song, Ruochen Liu, Zhi Zhu, Chaoyong James Yang
Compartmentalization of aqueous samples in uniform emulsion droplets has proven to be a useful tool for many chemical, biological, and biomedical applications. Herein, we introduce an array-based emulsification method for rapid and easy generation of monodisperse agarose-in-oil droplets in a PDMS microwell array. The microwells are filled with agarose solution, and subsequent addition of hot oil results in immediate formation of agarose droplets due to the surface-tension of the liquid solution. Because droplet size is determined solely by the array unit dimensions, uniform droplets with pre-selectable diameters ranging from 20 µm to 100 µm can be produced with relative standard deviations less than 3...
January 19, 2018: Analytical Chemistry
Verónica Urdaneta, Josep Casadesús
Adaptation to bile is the ability to endure the lethal effects of bile salts after growth on sublethal concentrations. Surveys of adaptation to bile in Salmonella enterica ser. Tyhimurium reveal that active efflux is essential for adaptation while other bacterial functions involved in bile resistance are not. Among S. enterica mutants lacking one or more efflux systems, only strains lacking AcrAB are unable to adapt, thus revealing an essential role for AcrAB. Transcription of the acrAB operon is upregulated in the presence of a sublethal concentration of sodium deoxycholate (DOC) while other efflux loci are either weakly upregulated or irresponsive...
January 19, 2018: Environmental Microbiology
K S Jayaprakash, A K Sen
We report the continuous splitting of aqueous droplets at the interface between two co-flowing immiscible oil streams in a microchannel. The aqueous droplets initially present in a primary continuous stream (CP1) migrate into a secondary continuous stream (CP2) when the ratio of the non-inertial lift force to the interfacial tension force exceeds a critical value (K. S. Jayaprakash, U. Banerjee and A. K. Sen, Langmuir, 2016, 32, 2136-2143). Here, experiments were performed to understand the droplet splitting phenomenon and demonstrate the splitting of droplets encapsulating microbeads and cells...
January 19, 2018: Soft Matter
Xiuling Fu, Fangfang He, Yuhao Li, Allahverdi Shahveranov, Andrew Paul Hutchins
Organisms are made of a limited number of cell types that combine to form higher order tissues and organs. Cell types have traditionally been defined by their morphologies or biological activity, yet the underlying molecular controls of cell type remain unclear. The onset of single cell technologies, and more recently genomics (particularly single cell genomics), has substantially increased the understanding of the concept of cell type, but has also increased the complexity of this understanding. These new technologies have added a new genome wide molecular dimension to the description of cell type, with genome-wide expression and epigenetic data acting as a cell type 'fingerprint' to describe the cell state...
December 2017: Cell Regeneration
Etmar Bulk, Nadzeya Kramko, Ivan Liashkovich, Felix Glaser, Hermann Schillers, Hans-Joachim Schnittler, Hans Oberleithner, Albrecht Schwab
Early metastasis leads to poor prognosis of lung cancer patients, whose 5-year survival rate is only 15%. We could recently show that the Ca2+ sensitive K+ channel KCa3.1 promotes aggressive behavior of non-small cell lung cancer (NSCLC) cells and that it can serve as a prognostic marker in NSCLC. Since NSCLC patients die of metastases, we investigated whether KCa3.1 channels contribute to poor patient prognosis by regulating distinct steps of the metastatic cascade. We investigated the extravasation of NSCLC cells and focused on their adhesion to endothelial cells and on transendothelial migration...
December 22, 2017: Oncotarget
Niek van Wietmarschen, Sarra Merzouk, Nancy Halsema, Diana C J Spierings, Victor Guryev, Peter M Lansdorp
Bloom syndrome is a cancer predisposition disorder caused by mutations in the BLM helicase gene. Cells from persons with Bloom syndrome exhibit striking genomic instability characterized by excessive sister chromatid exchange events (SCEs). We applied single-cell DNA template strand sequencing (Strand-seq) to map the genomic locations of SCEs. Our results show that in the absence of BLM, SCEs in human and murine cells do not occur randomly throughout the genome but are strikingly enriched at coding regions, specifically at sites of guanine quadruplex (G4) motifs in transcribed genes...
January 18, 2018: Nature Communications
Eric Libby, William W Driscoll, William C Ratcliff
Programmed cell death (PCD) occurs in both unicellular and multicellular organisms. While PCD plays a key role in the development and maintenance of multicellular organisms, explaining why single-celled organisms would evolve to actively commit suicide has been far more challenging. Here, we explore the potential for PCD to act as an accessory to microbial bet-hedging strategies that utilize stochastic phenotype switching. We consider organisms that face unpredictable and recurring disasters, in which fitness depends on effective phenotypic diversification...
January 18, 2018: Scientific Reports
Davide Risso, Fanny Perraudeau, Svetlana Gribkova, Sandrine Dudoit, Jean-Philippe Vert
Single-cell RNA-sequencing (scRNA-seq) is a powerful high-throughput technique that enables researchers to measure genome-wide transcription levels at the resolution of single cells. Because of the low amount of RNA present in a single cell, some genes may fail to be detected even though they are expressed; these genes are usually referred to as dropouts. Here, we present a general and flexible zero-inflated negative binomial model (ZINB-WaVE), which leads to low-dimensional representations of the data that account for zero inflation (dropouts), over-dispersion, and the count nature of the data...
January 18, 2018: Nature Communications
Chen Jia, Michael Q Zhang, Hong Qian
Single-cell gene expression is inherently stochastic; its emergent behavior can be defined in terms of the chemical master equation describing the evolution of the mRNA and protein copy numbers as the latter tends to infinity. We establish two types of "macroscopic limits": the Kurtz limit is consistent with the classical chemical kinetics, while the Lévy limit provides a theoretical foundation for an empirical equation proposed in N. Friedman et al., Phys. Rev. Lett. 97, 168302 (2006)PRLTAO0031-900710.1103/PhysRevLett...
October 2017: Physical Review. E
David B Saakian
We investigate the collective stationary sensing using N communicative cells, which involves surface receptors, diffusive signaling molecules, and cell-cell communication messengers. We restrict the scenarios to the signal-to-noise ratios (SNRs) for both strong communication and extrinsic noise only. We modified a previous model [Bialek and Setayeshgar, Proc. Natl. Acad. Sci. USA 102, 10040 (2005)PNASA60027-842410.1073/pnas.0504321102] to eliminate the singularities in the fluctuation correlations by considering a uniform receptor distribution over the surface of each cell with a finite radius a...
October 2017: Physical Review. E
Jens Doose, Benjamin Lindner
Single cell stimulation in vivo is a powerful tool to investigate the properties of single neurons and their functionality in neural networks. We present a method to determine a cell-specific stimulus that reliably evokes a prescribed spike train with high temporal precision of action potentials. We test the performance of this stimulus in simulations for two different stochastic neuron models. For a broad range of parameters and a neuron firing with intermediate firing rates (20-40 Hz) the reliability in evoking the prescribed spike train is close to its theoretical maximum that is mainly determined by the level of intrinsic noise...
September 2017: Physical Review. E
Chen Jia
Here we develop an effective approach to simplify two-time-scale Markov chains with infinite state spaces by removal of states with fast leaving rates, which improves the simplification method of finite Markov chains. We introduce the concept of fast transition paths and show that the effective transitions of the reduced chain can be represented as the superposition of the direct transitions and the indirect transitions via all the fast transition paths. Furthermore, we apply our simplification approach to the standard Markov model of single-cell stochastic gene expression and provide a mathematical theory of random gene expression bursts...
September 2017: Physical Review. E
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