scholarly journals One cell, one drop, one click: hybrid microfluidic mammalian single-cell isolation

2020 ◽  
Author(s):  
Kenza Samlali ◽  
Fatemeh Ahmadi ◽  
Angela B.V. Quach ◽  
Guy Soffer ◽  
Steve C.C. Shih
Keyword(s):  
User Interface ◽  
Single Cells ◽  
Electrode System ◽  
Loss Of Function ◽  
Precision Control ◽  
Knock Out ◽  
On Demand ◽  
Complex Process ◽  
Single Cell Isolation ◽  
Hybrid Microfluidics

AbstractThe process of generating a stable knockout cell line is a complex process that can take several months to complete. In this work, we introduce a microfluidic method that is capable of isolating single cells, selecting successful edited clones, and expansion of these isoclones. Using a hybrid microfluidics method, droplets in channels can be individually addressed using a co-planar electrode system. In our hybrid microfluidic device, we show that we can trap single cells and subsequently encapsulate them on demand into pL-sized droplets. Furthermore, individual cells inside the droplet can be released from the traps or merged with other droplets by simply applying an electric potential to the electrodes that is actuated through a user interface. We use this high precision control to sort and to recover single isoclones to establish monoclonal cell lines, which is demonstrated with a heterozygous NCI-H1299 lung squamous cell population resulting from loss-of-function eGFP and RAF1 gene knock-out transfections.

A microfluidic device enabling deterministic single cell trapping and release

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Huichao Chai ◽  
Yongxiang Feng ◽  
Fei Liang ◽  
Wenhui Wang
Keyword(s):  
Chemical Analysis ◽  
Single Cell ◽  
Microfluidic Device ◽  
Single Cells ◽  
Cell Isolation ◽  
Cell Trapping ◽  
Analysis Techniques ◽  
Single Cell Trapping ◽  
Single Cell Isolation ◽  
Made In

Successful single-cell isolation is a pivotal technique for subsequent biological and chemical analysis of single cells. Although significant advances have been made in single-cell isolation and analysis techniques, most passive...

scholarly journals Mbov_0503 Encodes a Novel Cytoadhesin that Facilitates Mycoplasma bovis Interaction with Tight Junctions

2020 ◽  
Vol 8 (2) ◽  
pp. 164 ◽  
Author(s):  
Xifang Zhu ◽  
Yaqi Dong ◽  
Eric Baranowski ◽  
Xixi Li ◽  
Gang Zhao ◽  
...  
Keyword(s):  
Host Cell ◽  
Tight Junctions ◽  
Binding Capacity ◽  
Host Cells ◽  
Mycoplasma Bovis ◽  
Knock Out ◽  
Cell Monolayers ◽  
Protein Levels ◽  
Complex Process ◽  
Host Cell Surface

Molecules contributing to microbial cytoadhesion are important virulence factors. In Mycoplasma bovis, a minimal bacterium but an important cattle pathogen, binding to host cells is emerging as a complex process involving a broad range of surface-exposed structures. Here, a new cytoadhesin of M. bovis was identified by producing a collection of individual knock-out mutants and evaluating their binding to embryonic bovine lung cells. The cytoadhesive-properties of this surface-exposed protein, which is encoded by Mbov_0503 in strain HB0801, were demonstrated at both the mycoplasma cell and protein levels using confocal microscopy and ELISA. Although Mbov_0503 disruption was only associated in M. bovis with a partial reduction of its binding capacity, this moderate effect was sufficient to affect M. bovis interaction with the host-cell tight junctions, and to reduce the translocation of this mycoplasma across epithelial cell monolayers. Besides demonstrating the capacity of M. bovis to disrupt tight junctions, these results identified novel properties associated with cytoadhesin that might contribute to virulence and host colonization. These findings provide new insights into the complex interplay taking place between wall-less mycoplasmas and the host-cell surface.

scholarly journals Notch Signaling in Skeletal Development, Homeostasis and Pathogenesis

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 332 ◽  
Author(s):  
Jennifer T. Zieba ◽  
Yi-Ting Chen ◽  
Brendan H. Lee ◽  
Yangjin Bae
Keyword(s):  
Notch Signaling ◽  
Cell Fate ◽  
Human Genetics ◽  
Skeletal Development ◽  
Gene Activation ◽  
Fracture Repair ◽  
Loss Of Function ◽  
Age Related ◽  
Complex Process ◽  
And Cartilage

Skeletal development is a complex process which requires the tight regulation of gene activation and suppression in response to local signaling pathways. Among these pathways, Notch signaling is implicated in governing cell fate determination, proliferation, differentiation and apoptosis of skeletal cells-osteoblasts, osteoclasts, osteocytes and chondrocytes. Moreover, human genetic mutations in Notch components emphasize the critical roles of Notch signaling in skeletal development and homeostasis. In this review, we focus on the physiological roles of Notch signaling in skeletogenesis, postnatal bone and cartilage homeostasis and fracture repair. We also discuss the pathological gain- and loss-of-function of Notch signaling in bone and cartilage, resulting in osteosarcoma and age-related degenerative diseases, such as osteoporosis and osteoarthritis. Understanding the physiological and pathological function of Notch signaling in skeletal tissues using animal models and human genetics will provide new insights into disease pathogenesis and offer novel approaches for the treatment of bone/cartilage diseases.

scholarly journals On-Demand Intracellular Delivery of Single Particles in Single Cells by 3D Hollow Nanoelectrodes

Nano Letters ◽  
2019 ◽  
Vol 19 (2) ◽  
pp. 722-731 ◽  
Author(s):  
Jian-An Huang ◽  
Valeria Caprettini ◽  
Yingqi Zhao ◽  
Giovanni Melle ◽  
Nicolò Maccaferri ◽  
...  
Keyword(s):  
Single Cells ◽  
Intracellular Delivery ◽  
Single Particles ◽  
On Demand

C/EBPα-Induced Microrna-30c Directly Targets Notch1 During Granulopoiesis and Is Repressed in Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3514-3514
Author(s):  
Christiane Katzerke ◽  
Vikas Madan ◽  
Dennis Gerloff ◽  
Daniela Braeuer-Hartmann ◽  
Jens-Uwe Hartmann ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Protein Expression ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Locked Nucleic Acid ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Granulocytic Differentiation ◽  
Knock Out ◽  
Acute Myeloid

Abstract Abstract 3514 The transcription factor CCAAT Enhancer Binding Protein alpha (C/EBPα) is crucial for normal granulopoiesis and frequently disrupted in acute myeloid leukemia (AML). Mutations in the CEBPA gene are reported for about 10% of all AML. Loss of function of C/EBPα leads to a block of myeloid differentiation. MicroRNAs (miR) inhibiting translation of mRNA into protein were identified as critical players in granulocytic differentiation. We and others have already shown that C/EBPα exerts its effects by regulating miRNAs such as miR-223 and miR-34a. In a global microRNA-array we found miR-30c as a novel most important target of C/EBPα during granulopoiesis. Wild-type C/EBPα-p42 up regulates miR-30c expression, whereas the C/EBPα-p30 mutant, found in AML, does not. Furthermore, miR-30c expression is up regulated by G-CSF during granulocytic differentiation of primary human CD34-positive progenitor cells and down regulated in various subtypes of AML. Among these, miR-30c is significantly down regulated in samples from AML with normal karyotype and CEBPA mutations compared to AML patients with CEBPA wildtype. In mice, miR-30c shows a high expression in GMP and granulocytes. An induced tissue specific knock-out of C/EBPα in mice leads to a significant suppression of miR-30c expression in bone marrow cells. A luciferase reporter assay identifies NOTCH1 as a direct target of miR-30c as evident by its binding to the 3'UTR. Recent studies show that Notch1 blocks protein expression of C/EBPα-p42 by activation of Tribbles homolog 2 (Trib2). On the other hand, Proteins of Notch1 and Trib2 are down regulated by C/EBPα-p42. A block of miR-30c by locked nucleic acid (LNA) oligonucleotides prevents C/EBPα–induced downregulation of Notch1 protein expression, thus, miR-30c is necessary for C/EBPα to block NOTCH1. In this direction, the network leads to a block of granulopoiesis and further to leukemogenesis. Our study indicates that C/EBPα-induced miR-30c inactivates Notch1 during granulopoiesis and is down regulated in AML. This data stress the important role of deregulated miRNA expression in leukemia and may provide novel biomarkers and therapeutic targets in AML. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership.

scholarly journals The function of the transmembrane and cytoplasmic domains of pneumococcal penicillin-binding proteins 2x and 2b extends beyond that of simple anchoring devices

Microbiology ◽  
2014 ◽  
Vol 160 (8) ◽  
pp. 1585-1598 ◽  
Author(s):  
Kari Helene Berg ◽  
Daniel Straume ◽  
Leiv Sigve Håvarstein
Keyword(s):  
Binding Proteins ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Cytoplasmic Domain ◽  
Loss Of Function ◽  
Penicillin Binding Proteins ◽  
Catalytic Domains ◽  
Complex Process ◽  
Cytoplasmic Tails ◽  
Cross Links

The biosynthesis of cell-wall peptidoglycan is a complex process that involves six different penicillin-binding proteins (PBPs) in Streptococcus pneumoniae. Two of these, PBP2x and PBP2b, are monofunctional transpeptidases that catalyse the formation of peptide cross-links between adjacent glycan strands. Both of them are bitopic membrane proteins with a small cytoplasmic and a large extracellular domain. PBP2x and PBP2b are essential for septal and peripheral peptidoglycan synthesis, respectively. Although several studies have investigated the properties of their extracellular catalytic domains, it is not known whether the role of their N-terminal non-catalytic domains extends beyond that of being simple anchoring devices. We therefore decided to use reciprocal domain swapping and mutational analysis to gain more information about the biological function of the membrane anchors and cytoplasmic tails of PBP2x and PBP2b. In the case of PBP2x both domains are essential, but neither the membrane anchor nor the cytoplasmic domain of PBP2x appear to serve as major localization signals. Instead, our results suggest that they are involved in interactions with other components of the divisome. Mutations of conserved amino acids in the cytoplasmic domain of PBP2x resulted in loss of function, underlining the importance of this region. The cytoplasmic domain of PBP2b could be swapped with the corresponding domain from PBP2x, whereas replacement of the PBP2b transmembrane domain with the corresponding PBP2x domain gave rise to slow-growing cells with grossly abnormal morphology. When both domains were exchanged simultaneously the cells were no longer viable.

scholarly journals Efficient single-cell poration by microsecond laser pulses

Lab on a Chip ◽  
2015 ◽  
Vol 15 (2) ◽  
pp. 581-588 ◽  
Author(s):  
Qihui Fan ◽  
Wenqi Hu ◽  
Aaron T. Ohta
Keyword(s):  
Laser Pulse ◽  
Single Cell ◽  
Single Cells ◽  
Laser Pulses ◽  
On Demand

Microsecond-laser-pulse-induced microbubbles can porate specific single cells on demand at up to 95% efficiency, including the delivery of plasmids and multiple types of payloads.

scholarly journals High-Throughput Single-Cell Cultivation on Microfluidic Streak Plates

2016 ◽  
Vol 82 (7) ◽  
pp. 2210-2218 ◽  
Author(s):  
Cheng-Ying Jiang ◽  
Libing Dong ◽  
Jian-Kang Zhao ◽  
Xiaofang Hu ◽  
Chaohua Shen ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Response Analysis ◽  
Bacterial Strains ◽  
Content Type ◽  
Chemical Gradients ◽  
Droplet Array ◽  
Polycyclic Aromatic ◽  
Single Cell Isolation

ABSTRACTThis paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for comprehensive dose-response analysis. The MSP method was validated by using single-cell isolation ofEscherichia coliand antimicrobial susceptibility testing ofPseudomonas aeruginosaPAO1. The robustness of the MSP work flow was demonstrated by cultivating a soil community that degrades polycyclic aromatic hydrocarbons. Cultivation in droplets enabled detection of the richest species diversity with better coverage of rare species. Moreover, isolation and cultivation of bacterial strains by MSP led to the discovery of several species with high degradation efficiency, including fourMycobacteriumisolates and a previously unknown fluoranthene-degradingBlastococcusspecies.

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