An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus

In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively drop out a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. miRNA-219 and miR-196a KOs both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.

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Segregation of neural crest specific lineage trajectories from a heterogeneous neural plate border territory only emerges at neurulation

10.1101/2021.08.08.455573 ◽

2021 ◽

Author(s):

Ruth Williams ◽

Martyna Lukoseviciute ◽

Tatjana Sauka-Spengler ◽

Marianne E Bronner

Keyword(s):

Neural Crest ◽

Neural Plate ◽

Developmental Trajectory ◽

Rna Seq ◽

Transcriptional Signature ◽

Transcriptional Changes ◽

Neural Plate Border ◽

Neural Ectoderm ◽

Vertebrate Embryos

The epiblast of vertebrate embryos is comprised of neural and non-neural ectoderm, with the border territory at their intersection harbouring neural crest and cranial placode progenitors. Here we profile avian epiblast cells as a function of time using single-cell RNA-seq to define transcriptional changes in the emerging ‘neural plate border’. The results reveal gradual establishment of heterogeneous neural plate border signatures, including novel genes that we validate by fluorescent in situ hybridisation. Developmental trajectory analysis shows that segregation of neural plate border lineages only commences at early neurulation, rather than at gastrulation as previously predicted. We find that cells expressing the prospective neural crest marker Pax7 contribute to multiple lineages, and a subset of premigratory neural crest cells shares a transcriptional signature with their border precursors. Together, our results suggest that cells at the neural plate border remain heterogeneous until early neurulation, at which time progenitors become progressively allocated toward defined lineages.

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A novel approach for preparing in-situ nitrogen doped carbon via pyrolysis of bean pulp for supercapacitors

Energy ◽

10.1016/j.energy.2020.119227 ◽

2021 ◽

Vol 216 ◽

pp. 119227

Author(s):

Yan Ding ◽

Yunchao Li ◽

Yujie Dai ◽

Xinhong Han ◽

Bo Xing ◽

...

Keyword(s):

Nitrogen Doped ◽

Novel Approach ◽

Nitrogen Doped Carbon

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Phosphorylation of GAPVD1 Is Regulated by the PER Complex and Linked to GAPVD1 Degradation

International Journal of Molecular Sciences ◽

10.3390/ijms22073787 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3787

Author(s):

Hussam Ibrahim ◽

Philipp Reus ◽

Anna Katharina Mundorf ◽

Anna-Lena Grothoff ◽

Valerie Rudenko ◽

...

Keyword(s):

Circadian Clock ◽

Transcriptional Repression ◽

Small Gtpase ◽

Main Role ◽

Interacting Proteins ◽

Casein Kinase 1 ◽

Bona Fide ◽

Kinetics Of

Repressor protein period (PER) complexes play a central role in the molecular oscillator mechanism of the mammalian circadian clock. While the main role of nuclear PER complexes is transcriptional repression, much less is known about the functions of cytoplasmic PER complexes. We found with a biochemical screen for PER2-interacting proteins that the small GTPase regulator GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), which has been identified previously as a component of cytoplasmic PER complexes in mice, is also a bona fide component of human PER complexes. We show that in situ GAPVD1 is closely associated with casein kinase 1 delta (CSNK1D), a kinase that regulates PER2 levels through a phosphoswitch mechanism, and that CSNK1D regulates the phosphorylation of GAPVD1. Moreover, phosphorylation determines the kinetics of GAPVD1 degradation and is controlled by PER2 and a C-terminal autoinhibitory domain in CSNK1D, indicating that the regulation of GAPVD1 phosphorylation is a novel function of cytoplasmic PER complexes and might be part of the oscillator mechanism or an output function of the circadian clock.

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Novel Approach of In-situ Nickel Capture Technology to Recycle Silver and Palladium from Waste Nickel-rich Multilayer Ceramic Capacitors

Journal of Cleaner Production ◽

10.1016/j.jclepro.2020.125650 ◽

2020 ◽

pp. 125650

Author(s):

Ya Liu ◽

Qingming Song ◽

Lingen Zhang ◽

Zhenming Xu

Keyword(s):

Multilayer Ceramic Capacitors ◽

Novel Approach ◽

Ceramic Capacitors ◽

Multilayer Ceramic

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In-situ constructing uniform polymer network for iron oxide microspheres: A novel approach to improve the cycling stability of the conversion electrodes through chemical interaction

Journal of Power Sources ◽

10.1016/j.jpowsour.2021.229510 ◽

2021 ◽

Vol 489 ◽

pp. 229510

Author(s):

Ting Li ◽

Hao Li ◽

Aiqiong Qin ◽

Daohong Zhang ◽

Fei Xu

Keyword(s):

Iron Oxide ◽

Chemical Interaction ◽

Polymer Network ◽

Cycling Stability ◽

Novel Approach

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In Situ Generation of Green Hybrid Nanofibrillar Polymer-Polymer Composites—A Novel Approach to the Triple Shape Memory Polymer Formation

Polymers ◽

10.3390/polym13121900 ◽

2021 ◽

Vol 13 (12) ◽

pp. 1900

Author(s):

Ramin Hosseinnezhad ◽

Iurii Vozniak ◽

Fahmi Zaïri

Keyword(s):

Polymer Blends ◽

Shape Memory ◽

Shape Memory Polymer ◽

Cellulose Nanofibers ◽

Polymeric Materials ◽

Novel Approach ◽

Phase Transition Temperatures ◽

Triple Shape Memory

The paper discusses the possibility of using in situ generated hybrid polymer-polymer nanocomposites as polymeric materials with triple shape memory, which, unlike conventional polymer blends with triple shape memory, are characterized by fully separated phase transition temperatures and strongest bonding between the polymer blends phase interfaces which are critical to the shape fixing and recovery. This was demonstrated using the three-component system polylactide/polybutylene adipateterephthalate/cellulose nanofibers (PLA/PBAT/CNFs). The role of in situ generated PBAT nanofibers and CNFs in the formation of efficient physical crosslinks at PLA-PBAT, PLA-CNF and PBAT-CNF interfaces and the effect of CNFs on the PBAT fibrillation and crystallization processes were elucidated. The in situ generated composites showed drastically higher values of strain recovery ratios, strain fixity ratios, faster recovery rate and better mechanical properties compared to the blend.

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A Novel Cobalt Metallopolymer with Redox-Matched Conjugated Organic Backbone via Electropolymerization of a Readily Available N4 Cobalt Complex

Polymers ◽

10.3390/polym13101667 ◽

2021 ◽

Vol 13 (10) ◽

pp. 1667

Author(s):

Mikhail Karushev

Keyword(s):

Redox Reactions ◽

Repeat Unit ◽

Cobalt Complex ◽

Radical Cations ◽

Electrochemical Quartz Crystal Microbalance ◽

Practical Applications ◽

Sensing Applications ◽

Novel Approach ◽

Redox Switching

Fast and reversible cobalt-centered redox reactions in metallopolymers are the key to using these materials in energy storage, electrocatalytic, and sensing applications. Metal-centered electrochemical activity can be enhanced via redox matching of the conjugated organic backbone and cobalt centers. In this study, we present a novel approach to redox matching via modification of the cobalt coordination site: a conductive electrochemically active polymer was electro-synthesized from [Co(Amben)] complex (Amben = N,N′-bis(o-aminobenzylidene)ethylenediamine) for the first time. The poly-[Co(Amben)] films were investigated by cyclic voltammetry, electrochemical quartz crystal microbalance (EQCM), in situ UV‑vis-NIR spectroelectrochemistry, and in situ conductance measurements between −0.9 and 1.3 V vs. Ag/Ag+. The polymer displayed multistep redox processes involving reversible transfer of the total of 1.25 electrons per repeat unit. The findings indicate consecutive formation of three redox states during reversible electrochemical oxidation of the polymer film, which were identified as benzidine radical cations, Co(III) ions, and benzidine di-cations. The Co(II)/Co(III) redox switching is retained in the thick polymer films because it occurs at potentials of high polymer conductivity due to the optimum redox matching of the Co(II)/Co(III) redox pair with the organic conjugated backbone. It makes poly-[Co(Amben)] suitable for various practical applications based on cobalt-mediated redox reactions.

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Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining

Scientific Reports ◽

10.1038/s41598-020-78640-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Robert J. Francis ◽

Gillian Robb ◽

Lee McCann ◽

Bhagwati Khatri ◽

James Keeble ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Three Dimensional ◽

Large Field ◽

Staining Procedure ◽

3D Analysis ◽

Mycobacterium Tuberculosis Infection ◽

In Vivo Models ◽

Novel Approach

AbstractTuberculosis (TB) preclinical testing relies on in vivo models including the mouse aerosol challenge model. The only method of determining colony morphometrics of TB infection in a tissue in situ is two-dimensional (2D) histopathology. 2D measurements consider heterogeneity within a single observable section but not above and below, which could contain critical information. Here we describe a novel approach, using optical clearing and a novel staining procedure with confocal microscopy and mesoscopy, for three-dimensional (3D) measurement of TB infection within lesions at sub-cellular resolution over a large field of view. We show TB morphometrics can be determined within lesion pathology, and differences in infection with different strains of Mycobacterium tuberculosis. Mesoscopy combined with the novel CUBIC Acid-Fast (CAF) staining procedure enables a quantitative approach to measure TB infection and allows 3D analysis of infection, providing a framework which could be used in the analysis of TB infection in situ.

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In Situ Hyperspectral Raman Imaging: A New Method to Investigate Sintering Processes of Ceramic Material at High-temperature

Applied Sciences ◽

10.3390/app9071310 ◽

2019 ◽

Vol 9 (7) ◽

pp. 1310 ◽

Cited By ~ 6

Author(s):

Kerstin Hauke ◽

Johannes Kehren ◽

Nadine Böhme ◽

Sinje Zimmer ◽

Thorsten Geisler

Keyword(s):

High Temperature ◽

Raman Imaging ◽

In Situ Studies ◽

Kinetic Information ◽

Novel Approach ◽

In Situ Investigation ◽

Dependent Growth ◽

Micrometer Scale ◽

Sintering Processes

In the last decades, Raman spectroscopy has become an important tool to identify and investigate minerals, gases, glasses, and organic material at room temperature. In combination with high-temperature and high-pressure devices, however, the in situ investigation of mineral transformation reactions and their kinetics is nowadays also possible. Here, we present a novel approach to in situ studies for the sintering process of silicate ceramics by hyperspectral Raman imaging. This imaging technique allows studying high-temperature solid-solid and/or solid-melt reactions spatially and temporally resolved, and opens up new avenues to study and visualize high-temperature sintering processes in multi-component systems. After describing in detail the methodology, the results of three application examples are presented and discussed. These experiments demonstrate the power of hyperspectral Raman imaging for in situ studies of the mechanism(s) of solid-solid or solid-melt reactions at high-temperature with a micrometer-scale resolution as well as to gain kinetic information from the temperature- and time-dependent growth and breakdown of minerals during isothermal or isochronal sintering.

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