scholarly journals Glyoxal Does Not Preserve Cellular Proteins as Accurately as PFA: A Microscopical Survey of Epitopes

2019 ◽  
Author(s):  
Ferda Topal Celikkan ◽  
Ceren Mungan ◽  
Merve Sucu ◽  
Fatma Uysal ◽  
Selda Kahveci ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Super Resolution ◽  
Cell Types ◽  
Cellular Proteins ◽  
Human Stem Cells ◽  
Cellular Targets ◽  
Molecular Alterations ◽  
Recent Developments ◽  
Spatio Temporal ◽  
Fixed Cell

AbstractChemical fixation is one of the most critical steps to retaining cellular targets as naturally as possible. Recent developments in microscopy allow sophisticated detection and measuring techniques with which spatio-temporal molecular alterations is conceivable. Here, we document the fixation competence of glyoxal (Gly), a less-toxic dialdehyde molecule, and paraformaldehyde (PFA) side-by-side (with or without Triton-X 100 permealization) in live- and fixed-cell preparations including human stem cells, spermatozoa, mouse oocytes/embryos using super-resolution microscopy. Although Gly seemed to act faster than PFA, catastrophic consequences were found not acceptable, especially in oocytes and embryos. Due to cell lysate and immunocytochemistry surveys, it was obvious that PFA is superior to Gly in retaining cellular proteins in situ with little/no background staining. In many samples, PFA revealed more reliable and consistent results regarding the protein quantity and cellular localization corresponding to previously defined patterns in the literature. Although the use of Gly is beneficial as indicated by previous reports, we concluded that it does not meet the requirement for proper fixation, at least for the tested cell types and proteins.

Gains achieved by molecular approaches in the area of lignification

2001 ◽  
Vol 73 (3) ◽  
pp. 561-566 ◽  
Author(s):  
Alain-M. Boudet ◽  
Matthieu Chabannes
Keyword(s):  
Molecular Biology ◽  
Lignin Content ◽  
Ectopic Expression ◽  
Lignin Biosynthesis ◽  
Cell Types ◽  
Molecular Approaches ◽  
Regulatory Circuits ◽  
Recent Developments ◽  
Spatio Temporal ◽  
Different Cell Types

In this article we highlight the contribution of molecular biology and lignin genetic engineering toward a better understanding of lignin biosynthesis and spatio-temporal deposition of lignin. Specific examples from the literature and from our laboratory will serve to underline the chemical flexibility of lignins, the complexity of the regulatory circuits involved in their synthesis, and the specific behavior of different cell types within the xylem. We will also focus on strategies aiming to reduce the lignin content or to modify the lignin composition of plants and present their impact on plant development. We will show that the ectopic expression of a specific transgene may have a different impact, depending on the genetic background, and that plants with a severe reduction in lignin content may undergo normal development. Lignification is currently benefiting enormously from recent developments in molecular biology and transgenesis, and the progress made opens the way for future developments to study how the walls of lignified plant cells are built and organized.

scholarly journals Revisiting the complexity of GLP-1 action-from sites of synthesis to receptor activation

2020 ◽  
Author(s):  
Brent A McLean ◽  
Chi Kin Wong ◽  
Jonathan E Campbell ◽  
David J Hodson ◽  
Stefan Trapp ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Hormone Secretion ◽  
Cell Types ◽  
Receptor Activation ◽  
Neural Pathways ◽  
Cellular Targets ◽  
Multiple Organs ◽  
Islet Hormone ◽  
Glucagon Like Peptide 1 ◽  
Gut Endocrine Cells

Abstract Glucagon-like peptide-1 (GLP-1) is produced in gut endocrine cells and in the brain, and acts through hormonal and neural pathways to regulate islet function, satiety, and gut motility, supporting development of GLP-1 receptor (GLP-1R) agonists for the treatment of diabetes and obesity. Classic notions of GLP-1 acting as a meal-stimulated hormone from the distal gut are challenged by data supporting production of GLP-1 in the endocrine pancreas, and by the importance of brain-derived GLP-1 in the control of neural activity. Moreover, attribution of direct vs. indirect actions of GLP-1 is difficult, as many tissue and cellular targets of GLP-1 action do not exhibit robust or detectable GLP-1R expression. Furthermore, reliable detection of the GLP-1R is technically challenging, highly method-dependent, and subject to misinterpretation. Here we revisit the actions of GLP-1, scrutinizing key concepts supporting gut vs. extra-intestinal GLP-1 synthesis and secretion. We discuss new insights refining cellular localization of GLP-1R expression and integrate recent data to refine our understanding of how and where GLP-1 acts to control inflammation, cardiovascular function, islet hormone secretion, gastric emptying, appetite, and body weight. These findings update our knowledge of cell types and mechanisms linking endogenous vs. pharmacological GLP-1 action to activation of the canonical GLP-1R, and the control of metabolic activity in multiple organs.

scholarly journals Astrocyte nanoscale morphology controls Ca2+ signals at tripartite synapses

2021 ◽  
Author(s):  
Audrey Denizot ◽  
Misa Arizono ◽  
U. Valentin Nägerl ◽  
Hugues Berry ◽  
De Schutter Erik
Keyword(s):  
Neuronal Activity ◽  
Diffusion Flux ◽  
Super Resolution ◽  
Signal Propagation ◽  
Computational Tools ◽  
Complex Morphology ◽  
Recent Developments ◽  
Spatio Temporal ◽  
Cellular Compartments ◽  
Super Resolution Microscopy

AbstractCa2+ signals in astrocytes can trigger the modulation of neuronal activity. Recent developments in Ca2+ imaging and super-resolution microscopy have allowed to characterize the complex morphology of astrocyte branchlets that communicate with neurons and the associated Ca2+ microdomains. Here, we use computational tools to investigate the causal relationship between branchlet morphology and spatio-temporal profile of Ca2+ signals. 3D reticular branchlet geometries were designed, alternating between large (nodes) and thinner cellular compartments (shafts). Simulations confirm experimental observations that a decreased shaft width is associated with a decreased diffusion flux from nodes, enhancing local Ca2+ activity. Upon successive neuronal stimuli, a decreased shaft width facilitates signal propagation in astrocyte branchlets. We further identify parameters that decrease local Ca2+ activity, such as a discontinuous ER geometry and an increased Ca2+ buffering. Overall, this study proposes key parameters that regulate Ca2+ activity locally, potentially favoring neuron-astrocyte communication at tripartite synapses.

Spatio-Temporal LED Driving for Subjective Super-Resolution of Grayscale Images

2019 ◽  
pp. 1243
Author(s):  
Kojiro Matsushita ◽  
Toyotaro Tokimoto ◽  
Kengo Fujii ◽  
Hirotsugu Yamamoto
Keyword(s):  
Super Resolution ◽  
Spatio Temporal ◽  
Grayscale Images

scholarly journals Hypoxia, Acidification and Inflammation: Partners in Crime in Parkinson’s Disease Pathogenesis?

Immuno ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 78-90
Author(s):  
Johannes Burtscher ◽  
Grégoire P. Millet
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Neurodegenerative Diseases ◽  
Current Knowledge ◽  
Cell Types ◽  
Brain Cell ◽  
Special Focus ◽  
Molecular Alterations ◽  
Research Fields

Like in other neurodegenerative diseases, protein aggregation, mitochondrial dysfunction, oxidative stress and neuroinflammation are hallmarks of Parkinson’s disease (PD). Differentiating characteristics of PD include the central role of α-synuclein in the aggregation pathology, a distinct vulnerability of the striato-nigral system with the related motor symptoms, as well as specific mitochondrial deficits. Which molecular alterations cause neurodegeneration and drive PD pathogenesis is poorly understood. Here, we summarize evidence of the involvement of three interdependent factors in PD and suggest that their interplay is likely a trigger and/or aggravator of PD-related neurodegeneration: hypoxia, acidification and inflammation. We aim to integrate the existing knowledge on the well-established role of inflammation and immunity, the emerging interest in the contribution of hypoxic insults and the rather neglected effects of brain acidification in PD pathogenesis. Their tight association as an important aspect of the disease merits detailed investigation. Consequences of related injuries are discussed in the context of aging and the interaction of different brain cell types, in particular with regard to potential consequences on the vulnerability of dopaminergic neurons in the substantia nigra. A special focus is put on the identification of current knowledge gaps and we emphasize the importance of related insights from other research fields, such as cancer research and immunometabolism, for neurodegeneration research. The highlighted interplay of hypoxia, acidification and inflammation is likely also of relevance for other neurodegenerative diseases, despite disease-specific biochemical and metabolic alterations.

scholarly journals Two-dimensional TIRF-SIM–traction force microscopy (2D TIRF-SIM-TFM)

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Liliana Barbieri ◽  
Huw Colin-York ◽  
Kseniya Korobchevskaya ◽  
Di Li ◽  
Deanna L. Wolfson ◽  
...  
Keyword(s):  
Resolution Enhancement ◽  
Traction Force ◽  
Super Resolution ◽  
Traction Force Microscopy ◽  
Two Dimensional ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Force Microscopy ◽  
Health And Disease ◽  
Spatio Temporal

AbstractQuantifying small, rapidly evolving forces generated by cells is a major challenge for the understanding of biomechanics and mechanobiology in health and disease. Traction force microscopy remains one of the most broadly applied force probing technologies but typically restricts itself to slow events over seconds and micron-scale displacements. Here, we improve >2-fold spatially and >10-fold temporally the resolution of planar cellular force probing compared to its related conventional modalities by combining fast two-dimensional total internal reflection fluorescence super-resolution structured illumination microscopy and traction force microscopy. This live-cell 2D TIRF-SIM-TFM methodology offers a combination of spatio-temporal resolution enhancement relevant to forces on the nano- and sub-second scales, opening up new aspects of mechanobiology to analysis.

scholarly journals mLoc-mRNA: predicting multiple sub-cellular localization of mRNAs using random forest algorithm coupled with feature selection via elastic net

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Prabina Kumar Meher ◽  
Anil Rai ◽  
Atmakuri Ramakrishna Rao
Keyword(s):  
Random Forest ◽  
Cellular Localization ◽  
Learning Algorithm ◽  
Elastic Net ◽  
Messenger Rnas ◽  
Computational Tool ◽  
Temporal Gene Expression ◽  
Single Location ◽  
Spatio Temporal ◽  
Localization Prediction

Abstract Background Localization of messenger RNAs (mRNAs) plays a crucial role in the growth and development of cells. Particularly, it plays a major role in regulating spatio-temporal gene expression. The in situ hybridization is a promising experimental technique used to determine the localization of mRNAs but it is costly and laborious. It is also a known fact that a single mRNA can be present in more than one location, whereas the existing computational tools are capable of predicting only a single location for such mRNAs. Thus, the development of high-end computational tool is required for reliable and timely prediction of multiple subcellular locations of mRNAs. Hence, we develop the present computational model to predict the multiple localizations of mRNAs. Results The mRNA sequences from 9 different localizations were considered. Each sequence was first transformed to a numeric feature vector of size 5460, based on the k-mer features of sizes 1–6. Out of 5460 k-mer features, 1812 important features were selected by the Elastic Net statistical model. The Random Forest supervised learning algorithm was then employed for predicting the localizations with the selected features. Five-fold cross-validation accuracies of 70.87, 68.32, 68.36, 68.79, 96.46, 73.44, 70.94, 97.42 and 71.77% were obtained for the cytoplasm, cytosol, endoplasmic reticulum, exosome, mitochondrion, nucleus, pseudopodium, posterior and ribosome respectively. With an independent test set, accuracies of 65.33, 73.37, 75.86, 72.99, 94.26, 70.91, 65.53, 93.60 and 73.45% were obtained for the respective localizations. The developed approach also achieved higher accuracies than the existing localization prediction tools. Conclusions This study presents a novel computational tool for predicting the multiple localization of mRNAs. Based on the proposed approach, an online prediction server “mLoc-mRNA” is accessible at http://cabgrid.res.in:8080/mlocmrna/. The developed approach is believed to supplement the existing tools and techniques for the localization prediction of mRNAs.

scholarly journals Molecular Alterations in Sporadic and SOD1-ALS Immortalized Lymphocytes: Towards a Personalized Therapy

2021 ◽  
Vol 22 (6) ◽  
pp. 3007
Author(s):  
Isabel Lastres-Becker ◽  
Gracia Porras ◽  
Marina Arribas-Blázquez ◽  
Inés Maestro ◽  
Daniel Borrego-Hernández ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Therapeutic Interventions ◽  
Autophagic Flux ◽  
Metabolic Disturbances ◽  
Molecular Alterations ◽  
Healthy Donors ◽  
Inflammatory Profile ◽  
Als Patients

Amyotrophic lateral sclerosis (ALS) is a fatal neurological condition where motor neurons (MNs) degenerate. Most of the ALS cases are sporadic (sALS), whereas 10% are hereditarily transmitted (fALS), among which mutations are found in the gene that codes for the enzyme superoxide dismutase 1 (SOD1). A central question in ALS field is whether causative mutations display selective alterations not found in sALS patients, or they converge on shared molecular pathways. To identify specific and common mechanisms for designing appropriate therapeutic interventions, we focused on the SOD1-mutated (SOD1-ALS) versus sALS patients. Since ALS pathology involves different cell types other than MNs, we generated lymphoblastoid cell lines (LCLs) from sALS and SOD1-ALS patients and healthy donors and investigated whether they show changes in oxidative stress, mitochondrial dysfunction, metabolic disturbances, the antioxidant NRF2 pathway, inflammatory profile, and autophagic flux. Both oxidative phosphorylation and glycolysis appear to be upregulated in lymphoblasts from sALS and SOD1-ALS. Our results indicate significant differences in NRF2/ARE pathway between sALS and SOD1-ALS lymphoblasts. Furthermore, levels of inflammatory cytokines and autophagic flux discriminate between sALS and SOD1-ALS lymphoblasts. Overall, different molecular mechanisms are involved in sALS and SOD1-ALS patients and thus, personalized medicine should be developed for each case.

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