scholarly journals Computational analysis of the effects of nitrogen source and sin1 knockout on biosilica morphology in the model diatom Thalassiosira pseudonana

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Szabolcs Horvát ◽  
Adeeba Fathima ◽  
Stefan Görlich ◽  
Michael Schlierf ◽  
Carl D. Modes ◽  
...  
Keyword(s):  
Nitrogen Source ◽  
Electron Micrographs ◽  
Self Assembly ◽  
Genetic Manipulation ◽  
Morphological Changes ◽  
Thalassiosira Pseudonana ◽  
Inorganic Materials ◽  
Automated Image Analysis ◽  
Loss Of Function ◽  
Knockout Mutant

AbstractMorphogenesis of the silica based cell walls of diatoms, a large group of microalgae, is a paradigm for the self-assembly of complex 3D nano- and microscale patterned inorganic materials. In recent years, loss-of-function studies using genetic manipulation were successfully applied for the identification of genes that guide silica morphogenesis in diatoms. These studies revealed that the loss of one gene can affect multiple morphological parameters, and the morphological changes can be rather subtle being blurred by natural variations in morphology even within the same clone. Both factors severely hamper the identification of morphological mutants using subjective by-eye inspection of electron micrographs. Here we have developed automated image analysis for objectively quantifying the morphology of ridge networks and pore densities from numerous electron micrographs of diatom biosilica. This study demonstrated differences in ridge network morphology and pore density in diatoms growing on ammonium rather than nitrate as the sole nitrogen source. Furthermore, it revealed shortcomings in previous by-eye evaluation of the biosilica phenotype of the silicanin-1 knockout mutant. We anticipate that the computational methods established in the present work will be invaluable for unraveling genotype–phenotype correlations in diatom biosilica morphogenesis.

Computational Analysis of The Effects of Nitrogen Source and Sin1 Knockout on Biosilica Morphology in The Model Diatom Thalassiosira Pseudonana

2020 ◽  
Author(s):  
Szabolcs Horvát ◽  
Adeeba Fathima ◽  
Stefan Görlich ◽  
Carl Modes ◽  
Michael Schlierf ◽  
...  
Keyword(s):  
Nitrogen Source ◽  
Electron Micrographs ◽  
Self Assembly ◽  
Genetic Manipulation ◽  
Morphological Changes ◽  
Thalassiosira Pseudonana ◽  
Inorganic Materials ◽  
Automated Image Analysis ◽  
Loss Of Function ◽  
Knockout Mutant

Abstract Morphogenesis of the silica based cell walls of diatoms, a large group of microalgae, is a paradigm for the self-assembly of complex 3D nano- and microscale patterned inorganic materials. In recent years, loss-of-function studies using genetic manipulation were successfully applied for the identification of genes that guide silica morphogenesis in diatoms. These studies revealed that the loss of one gene can affect multiple morphological parameters, and the morphological changes can be rather subtle being blurred by natural variations in morphology even within the same clone. Both factors severely hamper the identification of morphological mutants using subjective by-eye inspection of electron micrographs. Here we have developed automated image analysis for objectively quantifying the morphology of ridge networks and pore densities from numerous electron micrographs of diatom biosilica. This study demonstrated differences in ridge network morphology and pore density in diatoms growing on ammonium rather than nitrate as sole nitrogen source. Furthermore, it revealed shortcomings in previous by-eye evaluation of the biosilica phenotype of the silicanin-1 knockout mutant. We anticipate that the computational methods established in the present work, will be invaluable for unraveling genotype-phenotype correlations in diatom biosilica morphogenesis.

scholarly journals Tuba1a is uniquely important for axon guidance through midline commissural structures

2020 ◽  
Author(s):  
Georgia Buscaglia ◽  
Jayne Aiken ◽  
Katelyn J. Hoff ◽  
Kyle R. Northington ◽  
Emily A. Bates
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Intracellular Transport ◽  
Genetic Manipulation ◽  
Morphological Changes ◽  
Loss Of Function ◽  
Developmental Guidance ◽  
Genes Encoding ◽  
Developing Neurons

AbstractDeveloping neurons undergo dramatic morphological changes to appropriately migrate and extend axons to make synaptic connections. The microtubule cytoskeleton, made of α/β-tubulin dimers, drives neurite outgrowth, promotes neuronal growth cone responses, and facilitates intracellular transport of critical cargoes during neurodevelopment. TUBA1A constitutes the majority of α-tubulin in the developing brain and mutations to TUBA1A in humans cause severe brain malformations accompanied by varying neurological defects, collectively termed tubulinopathies. Studies of TUBA1A function in vivo have been limited by the presence of multiple genes encoding highly similar tubulin proteins, which prevents TUBA1A-specific antibody generation and makes genetic manipulation challenging. Here we present a novel tagging method for studying and manipulating TUBA1A in cells without impairing tubulin function. Using this tool, we show that a TUBA1A loss-of-function mutation TUBA1AN102D (TUBA1AND), reduced the amount of TUBA1A protein and prevented incorporation of TUBA1A into microtubule polymers. Reduced Tuba1a α-tubulin in heterozygous Tuba1aND/+ mice significantly impacted axon extension and impaired formation of forebrain commissures. Neurons with reduced Tuba1a caused by Tuba1aND had altered microtubule dynamics and slower neuron outgrowth compared to controls. Neurons deficient in Tuba1a failed to localize microtubule associated protein-1b (Map1b) to the developing growth cone, likely impacting reception of developmental guidance cues. Overall, we show that reduced Tuba1a is sufficient to support neuronal migration, but not axon guidance, and provide mechanistic insight as to how TUBA1A tunes microtubule function to support neurodevelopment.

scholarly journals Effect of captopril on post-infarction remodelling visualized by light sheet microscopy and echocardiography

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Urmas Roostalu ◽  
Louise Thisted ◽  
Jacob Lercke Skytte ◽  
Casper Gravesen Salinas ◽  
Philip Juhl Pedersen ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Light Sheet ◽  
Automated Image Analysis ◽  
Border Zone ◽  
Vascular Density ◽  
Mouse Heart ◽  
Imaging Method ◽  
Light Sheet Microscopy ◽  
Post Surgery ◽  
Captopril Treatment

AbstractAngiotensin converting enzyme inhibitors, among them captopril, improve survival following myocardial infarction (MI). The mechanisms of captopril action remain inadequately understood due to its diverse effects on multiple signalling pathways at different time periods following MI. Here we aimed to establish the role of captopril in late-stage post-MI remodelling. Left anterior descending artery (LAD) ligation or sham surgery was carried out in male C57BL/6J mice. Seven days post-surgery LAD ligated mice were allocated to daily vehicle or captopril treatment continued over four weeks. To provide comprehensive characterization of the changes in mouse heart following MI a 3D light sheet imaging method was established together with automated image analysis workflow. The combination of echocardiography and light sheet imaging enabled to assess cardiac function and the underlying morphological changes. We show that delayed captopril treatment does not affect infarct size but prevents left ventricle dilation and hypertrophy, resulting in improved ejection fraction. Quantification of lectin perfused blood vessels showed improved vascular density in the infarct border zone in captopril treated mice in comparison to vehicle dosed control mice. These results validate the applicability of combined echocardiographic and light sheet assessment of drug mode of action in preclinical cardiovascular research.

Pax-2 is required for mesenchyme-to-epithelium conversion during kidney development

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 711-720 ◽  
Author(s):  
U. W. Rothenpieler ◽  
G. R. Dressler
Keyword(s):  
Kidney Development ◽  
Morphological Changes ◽  
Cell Formation ◽  
Protein Accumulation ◽  
Metanephric Mesenchyme ◽  
Loss Of Function ◽  
Organ Cultures ◽  
Protein Levels ◽  
Differentiated Cells ◽  
Mesenchyme Cells

The conversion of mesenchyme to epithelium during the embryonic development of the mammalian kidney requires reciprocal inductive interactions between the ureter and the responding metanephric mesenchyme. The Pax-2 gene is activated in the mesenchyme in response to induction and is subsequently down-regulated in more differentiated cells derived from the mesenchyme. Pax-2 belongs to a family of genes, at least three of which encode morphogenetic regulatory transcription factors. In order to determine the role of Pax-2 during kidney development, we have generated a loss- of-function phenotype using antisense oligonucleotides in mouse kidney organ cultures. These oligonucleotides can specifically inhibit Pax-2 protein accumulation in kidney mesenchyme cells, where the intracellular concentrations are maximal. The kidney organ cultures were stained with uvomurulin and laminin antibodies as markers for epithelium formation. With significantly reduced Pax-2 protein levels, kidney mesenchyme cells fail to aggregate and do not undergo the sequential morphological changes characteristic of epithelial cell formation. The data demonstrate that Pax-2 function is required for the earliest phase of mesenchyme-to-epithelium conversion.

A Biological System Producing a Self-Assembling Cholesteric Protein Liquid Crystal

1971 ◽  
Vol 8 (1) ◽  
pp. 93-109
Author(s):  
A. C. NEVILLE ◽  
B. M. LUKE
Keyword(s):  
Liquid Crystal ◽  
Electron Density ◽  
Electron Micrographs ◽  
Self Assembly ◽  
Liquid Crystalline ◽  
Density Variation ◽  
Self Assembling ◽  
The Matrix ◽  
Electron Density Variation ◽  
Helicoidal Structure

The protein in the oothecal glands of praying mantids (Sphodromantis tenuidentata, Miomantis monacha) exists in the form of lamellar liquid crystalline spherulites, which coalesce as they flow out of a punctured gland tubule. Electron micrographs of sections of these spherulites after fixation show parabolic patterns of an electron-light component, set in a continuous matrix of protein. Such patterns arise in helicoidal systems (e.g. arthropod cuticle) and microdensitometric scans of the matrix show a rhythmical electron-density variation consistent with helicoidal structure. Double spiral patterns identical to those seen in liquid crystal spherulites are illustrated. These properties resemble those of cholesteric liquid crystals. The constructional units appear to be molecular rather than fibrillar as described by previous authors. The helicoidal architecture arises by self-assembly in the gland lumen. Lamellar surface structures self-assembled spontaneously on glass coverslips when the protein was left to stand for several days. When heated to 55 °C, the birefringent liquid crystalline protein abruptly changes to an isotropic gel, with associated loss of parabolic patterning in electron micrographs and of the rhythmical electron-density variation on microdensitometric scans. This behaviour is compared to the formation of gelatin from collagen, in terms of the randomization of an originally ordered secondary structure.

Automated image analysis applied to electron micrographs

1982 ◽  
Vol 6 (7) ◽  
pp. 656 ◽  
Author(s):  
C GRAVEKAMP ◽  
H KOERTEN ◽  
N VERWOERD ◽  
W DEBRUIJN ◽  
W DAEMS
Keyword(s):  
Image Analysis ◽  
Electron Micrographs ◽  
Automated Image Analysis

scholarly journals Genetic Manipulation of Transcriptional Regulators Alters Nicotine Biosynthesis in Tobacco

2020 ◽  
Vol 61 (6) ◽  
pp. 1041-1053 ◽  
Author(s):  
Shunya Hayashi ◽  
Mutsumi Watanabe ◽  
Makoto Kobayashi ◽  
Takayuki Tohge ◽  
Takashi Hashimoto ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Genetic Manipulation ◽  
Transcriptional Regulators ◽  
Knockout Mutant ◽  
Gene Encoding ◽  
Helix Loop Helix ◽  
Wide Range ◽  
Nicotine Biosynthesis ◽  
Transient Overexpression ◽  
The Impact

Abstract The toxic alkaloid nicotine is produced in the roots of Nicotiana species and primarily accumulates in leaves as a specialized metabolite. A series of metabolic and transport genes involved in the nicotine pathway are coordinately upregulated by a pair of jasmonate-responsive AP2/ERF-family transcription factors, NtERF189 and NtERF199, in the roots of Nicotiana tabacum (tobacco). In this study, we explored the potential of manipulating the expression of these transcriptional regulators to alter nicotine biosynthesis in tobacco. The transient overexpression of NtERF189 led to alkaloid production in the leaves of Nicotiana benthamiana and Nicotiana alata. This ectopic production was further enhanced by co-overexpressing a gene encoding a basic helix-loop-helix-family MYC2 transcription factor. Constitutive and leaf-specific overexpression of NtERF189 increased the accumulation of foliar alkaloids in transgenic tobacco plants but negatively affected plant growth. By contrast, in a knockout mutant of NtERF189 and NtERF199 obtained through CRISPR/Cas9-based genome editing, alkaloid levels were drastically reduced without causing major growth defects. Metabolite profiling revealed the impact of manipulating the nicotine pathway on a wide range of nitrogen- and carbon-containing metabolites. Our findings provide insights into the biotechnological applications of engineering metabolic pathways by targeting transcription factors.

Using Zebrafish to Test the Genetic Basis of Human Craniofacial Diseases

2017 ◽  
Vol 96 (11) ◽  
pp. 1192-1199 ◽  
Author(s):  
R. Grecco Machado ◽  
B. Frank Eames
Keyword(s):  
Candidate Genes ◽  
Genetic Manipulation ◽  
Association Studies ◽  
Underlying Disease ◽  
Genome Wide Association Studies ◽  
Loss Of Function ◽  
Genetic Loci ◽  
Large Numbers ◽  
Genetic Features ◽  
New Treatments

Genome-wide association studies (GWASs) opened an innovative and productive avenue to investigate the molecular basis of human craniofacial disease. However, GWASs identify candidate genes only; they do not prove that any particular one is the functional villain underlying disease or just an unlucky genomic bystander. Genetic manipulation of animal models is the best approach to reveal which genetic loci identified from human GWASs are functionally related to specific diseases. The purpose of this review is to discuss the potential of zebrafish to resolve which candidate genetic loci are mechanistic drivers of craniofacial diseases. Many anatomic, embryonic, and genetic features of craniofacial development are conserved among zebrafish and mammals, making zebrafish a good model of craniofacial diseases. Also, the ability to manipulate gene function in zebrafish was greatly expanded over the past 20 y, enabling systems such as Gateway Tol2 and CRISPR-Cas9 to test gain- and loss-of-function alleles identified from human GWASs in coding and noncoding regions of DNA. With the optimization of genetic editing methods, large numbers of candidate genes can be efficiently interrogated. Finding the functional villains that underlie diseases will permit new treatments and prevention strategies and will increase understanding of how gene pathways operate during normal development.

scholarly journals Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies

2016 ◽  
Vol 7 ◽  
pp. 613-629 ◽  
Author(s):  
Claudia Koch ◽  
Fabian J Eber ◽  
Carlos Azucena ◽  
Alexander Förste ◽  
Stefan Walheim ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Self Assembly ◽  
Building Blocks ◽  
Mosaic Disease ◽  
Inorganic Materials ◽  
High Yield ◽  
External Control ◽  
Immobilization Of Enzymes

The rod-shaped nanoparticles of the widespread plant pathogentobacco mosaic virus(TMV) have been a matter of intense debates and cutting-edge research for more than a hundred years. During the late 19th century, their behavior in filtration tests applied to the agent causing the 'plant mosaic disease' eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus–host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of 'smart' functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for monitoring blood sugar concentrations, might profit particularly from the presence of TMV rods: Their surfaces were recently shown to stabilize enzymatic activities upon repeated consecutive uses and over several weeks. This review gives the reader a ride through strikingly diverse achievements obtained with TMV-based particles, compares them to the progress with related viruses, and focuses on latest results revealing special advantages for enzyme-based biosensing formats, which might be of high interest for diagnostics employing 'systems-on-a-chip'.

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