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Daniel Edsgärd, Per Johnsson, Rickard Sandberg
As methods for measuring spatial gene expression at single-cell resolution become available, there is a need for computational analysis strategies. We present trendsceek, a method based on marked point processes that identifies genes with statistically significant spatial expression trends. trendsceek finds these genes in spatial transcriptomic and sequential fluorescence in situ hybridization data, and also reveals significant gene expression gradients and hot spots in low-dimensional projections of dissociated single-cell RNA-seq data...
March 19, 2018: Nature Methods
Lyndon da Cruz, Kate Fynes, Odysseas Georgiadis, Julie Kerby, Yvonne H Luo, Ahmad Ahmado, Amanda Vernon, Julie T Daniels, Britta Nommiste, Shazeen M Hasan, Sakina B Gooljar, Amanda-Jayne F Carr, Anthony Vugler, Conor M Ramsden, Magda Bictash, Mike Fenster, Juliette Steer, Tricia Harbinson, Anna Wilbrey, Adnan Tufail, Gang Feng, Mark Whitlock, Anthony G Robson, Graham E Holder, Mandeep S Sagoo, Peter T Loudon, Paul Whiting, Peter J Coffey
Age-related macular degeneration (AMD) remains a major cause of blindness, with dysfunction and loss of retinal pigment epithelium (RPE) central to disease progression. We engineered an RPE patch comprising a fully differentiated, human embryonic stem cell (hESC)-derived RPE monolayer on a coated, synthetic basement membrane. We delivered the patch, using a purpose-designed microsurgical tool, into the subretinal space of one eye in each of two patients with severe exudative AMD. Primary endpoints were incidence and severity of adverse events and proportion of subjects with improved best-corrected visual acuity of 15 letters or more...
March 19, 2018: Nature Biotechnology
Thomas H Segall-Shapiro, Eduardo D Sontag, Christopher A Voigt
The internal environment of growing cells is variable and dynamic, making it difficult to introduce reliable parts, such as promoters, for genetic engineering. Here, we applied control-theoretic ideas to design promoters that maintained constant levels of expression at any copy number. Theory predicts that independence to copy number can be achieved by using an incoherent feedforward loop (iFFL) if the negative regulation is perfectly non-cooperative. We engineered iFFLs into Escherichia coli promoters using transcription-activator-like effectors (TALEs)...
March 19, 2018: Nature Biotechnology
Xiaosa Li, Ying Wang, Yajing Liu, Bei Yang, Xiao Wang, Jia Wei, Zongyang Lu, Yuxi Zhang, Jing Wu, Xingxu Huang, Li Yang, Jia Chen
The targeting range of CRISPR-Cas9 base editors (BEs) is limited by their G/C-rich protospacer-adjacent motif (PAM) sequences. To overcome this limitation, we developed a CRISPR-Cpf1-based BE by fusing the rat cytosine deaminase APOBEC1 to a catalytically inactive version of Lachnospiraceae bacterium Cpf1. The base editor recognizes a T-rich PAM sequence and catalyzes C-to-T conversion in human cells, while inducing low levels of indels, non-C-to-T substitutions and off-target editing.
March 19, 2018: Nature Biotechnology
Jacqueline Cloos, Jeffrey R Harris, Jeroen J W M Janssen, Angele Kelder, F Huang, Gerrit Sijm, Maike Vonk, Alexander N Snel, Jennifer R Scheick, Willemijn J Scholten, Jannemieke Carbaat-Ham, Dennis Veldhuizen, Diana Hanekamp, Yvonne J M Oussoren-Brockhoff, Gertjan J L Kaspers, Gerrit J Schuurhuis, A Kate Sasser, Gert Ossenkoppele
Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival...
March 5, 2018: Journal of Visualized Experiments: JoVE
Zoran Marinović, Jelena Lujić, Eszter Kása, Zsolt Csenki, Béla Urbányi, Ákos Horváth
Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes...
March 4, 2018: Journal of Visualized Experiments: JoVE
Madeline Williams, Smrithi Prem, Xiaofeng Zhou, Paul Matteson, Percy Luk Yeung, Chi-Wei Lu, Zhiping Pang, Linda Brzustowicz, James H Millonig, Emanuel Dicicco-Bloom
Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons...
March 2, 2018: Journal of Visualized Experiments: JoVE
Kevin Isgrig, Wade W Chien
Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss. In this study, we describe the posterior semicircular canal approach as a viable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear...
March 2, 2018: Journal of Visualized Experiments: JoVE
Vid Leko, Smitha Sripathy, Robin L Adrianse, Taylor Loe, Angela Park, Uyen Lao, Eric J Foss, Marisa S Bartolomei, Antonio Bedalov
Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi)...
March 2, 2018: Journal of Visualized Experiments: JoVE
Diogo Castro Vaz, Edouard Lesne, Anke Sander, Hiroshi Naganuma, Eric Jacquet, Jacobo Santamaria, Agnès Barthélémy, Manuel Bibes
The quasi 2D electron system (q2DES) that forms at the interface between LaAlO3 (LAO) and SrTiO3 (STO) has attracted much attention from the oxide electronics community. One of its hallmark features is the existence of a critical LAO thickness of 4 unit-cells (uc) for interfacial conductivity to emerge. Although electrostatic mechanisms have been proposed in the past to describe the existence of this critical thickness, the importance of chemical defects has been recently accentuated. Here, we describe the growth of metal/LAO/STO heterostructures in an ultra-high vacuum (UHV) cluster system combining pulsed laser deposition (to grow the LAO), magnetron sputtering (to grow the metal) and X-ray photoelectron spectroscopy (XPS)...
February 8, 2018: Journal of Visualized Experiments: JoVE
Wyatt E Lanik, Lily Xu, Cliff J Luke, Elise Z Hu, Pranjal Agrawal, Victoria S Liu, Rajesh Kumar, Alexa M Bolock, Congrong Ma, Misty Good
Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine...
February 15, 2018: Journal of Visualized Experiments: JoVE
Fatima Cabral, Colton M Miller, Katrina M Kudrna, Blake E Hass, Jocelyn G Daubendiek, Brianna M Kellar, Edward N Harris
This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions...
February 12, 2018: Journal of Visualized Experiments: JoVE
Tara Nietzold, Bradley M West, Michael Stuckelberger, Barry Lai, Stefan Vogt, Mariana I Bertoni
The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical composition and elemental distribution within a material. Synchrotron-based XRF has become an integral characterization technique for a variety of research topics, particularly due to its non-destructive nature and its high sensitivity. Today, synchrotrons can acquire fluorescence data at spatial resolutions well below a micron, allowing for the evaluation of compositional variations at the nanoscale...
February 17, 2018: Journal of Visualized Experiments: JoVE
Mark J Bailey, Felix Broecker, Paul E Leon, Gene S Tan
Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-FcγR. Using this method, the contribution of specific Fc-FcγR interactions conferred by immunoglobulins can be determined using an in vitro assay...
February 23, 2018: Journal of Visualized Experiments: JoVE
Barun Das, Jashdeep Bhattacharjee, Preeti, Alaknanda Mishra, Kshama Jain, Srikanth Iyer, Ashwani Kesarwani, Parul Sahu, Prakriti Sinha, Perumal Nagarajan, Pramod Upadhyay
Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological conditions. Partial hepatectomy also facilitates the increased engraftment and proliferation of transplanted cells by accelerating neovascularization and cell migration towards the liver. Here, we describe a simple protocol for performing 30% hepatectomy and transplantation of cells in the spleen of a non-obese diabetic/severe combined immunodeficient NOD.SCID (NOD...
February 10, 2018: Journal of Visualized Experiments: JoVE
Jonathan T Ting, Brian R Lee, Peter Chong, Gilberto Soler-Llavina, Charles Cobbs, Christof Koch, Hongkui Zeng, Ed Lein
This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology...
February 26, 2018: Journal of Visualized Experiments: JoVE
Hao-Hsiang Hsu, John-Kevin Kracht, Laura Elisabeth Harder, Kerstin Rudnik, Gerd Lindner, Katharina Schimek, Uwe Marx, Ralf Pörtner
In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP))...
February 23, 2018: Journal of Visualized Experiments: JoVE
Hui Jiang, Yicun Wang, Zhantao Deng, Jiewen Jin, Jia Meng, Shuo Chen, Jun Wang, Yang Qiu, Ting Guo, Jianning Zhao
Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long follow-ups necessary for detection and sporadic occurrence, it is challenging to assess the pathogenesis ofparticle-induced osteolysis in clinical cases. Hence, optimal animal models are required for further studies. The murine model of calvarial osteolysis established by exposure to CoCrMo particles is an effective and valid tool for assessing the interactions between particles and various cells in aseptic loosening...
February 17, 2018: Journal of Visualized Experiments: JoVE
Paolo Giuseppe Limoli, Enzo Maria Vingolo, Celeste Limoli, Sergio Zaccaria Scalinci, Marcella Nebbioso
This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR)...
February 12, 2018: Journal of Visualized Experiments: JoVE
Grace H Hwang, Jessica L Hopkins, Philip W Jordan
Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries...
February 26, 2018: Journal of Visualized Experiments: JoVE
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