AtSYP71 Is Important for Plant Cell Wall Biosynthesis and Stress Adaptation.

Background: SYP71, the plant-specific Qc-SNARE protein, is reported to regulate vesicle trafficking. SYP71 is localized on the ER, endosome, plasma membrane and cell plate, suggesting its multiple functions. Lotus SYP71 is essential for symbiotic nitrogen fixation in nodules. AtSYP71, GmSYP71 and OsSYP71 are implicated in plant resistance to pathogenesis. To date, SYP71 regulatory role on plant development remain unclear.Results: AtSYP71-knockout mutant atsyp71-4 was lethal at early development stage. Early development of AtSYP71-knockdown mutant atsyp71-2 was delayed, and stress response was also affected. Confocal images revealed that protein secretion was blocked in atsyp71-2. Transcriptomic analysis indicated that metabolism, response to environmental stimuli pathways and apoplast components were influenced in atsyp71-2. Moreover, the contents of lignin, cellulose and flavonoids as well as cell wall structures were also altered.Conclusion: Our findings suggested that AtSYP71 is essential for plant development. AtSYP71 probably regulates plant development, metabolism and environmental adaptation by affecting cell wall homeostasis via mediating secretion of materials and regulators required for cell wall biosynthesis and dynamics.

Post hoc Correction of Chromatic Aberrations in Large-Scale Volumetric Images in Confocal Microscopy

Over the last decade, tissue-clearing techniques have expanded the scale of volumetric fluorescence imaging of the brain, allowing for the comprehensive analysis of neuronal circuits at a millimeter scale. Multicolor imaging is particularly powerful for circuit tracing with fluorescence microscopy. However, multicolor imaging of large samples often suffers from chromatic aberration, where different excitation wavelengths of light have different focal points. In this study, we evaluated chromatic aberrations for representative objective lenses and a clearing agent with confocal microscopy and found that axial aberration is particularly problematic. Moreover, the axial chromatic aberrations were often depth-dependent. Therefore, we developed a program that is able to align depths for different fluorescence channels based on reference samples with fluorescent beads or data from guide stars within biological samples. We showed that this correction program can successfully correct chromatic aberrations found in confocal images of multicolor-labeled brain tissues. Our simple post hoc correction strategy is useful to obtain large-scale multicolor images of cleared tissues with minimal chromatic aberrations.

Macros to Quantify Exosome Release and Autophagy at the Neuromuscular Junction of Drosophila Melanogaster

Automatic quantification of image parameters is a powerful and necessary tool to explore and analyze crucial cell biological processes. This article describes two ImageJ/Fiji automated macros to approach the analysis of synaptic autophagy and exosome release from 2D confocal images. Emerging studies point out that exosome biogenesis and autophagy share molecular and organelle components. Indeed, the crosstalk between these two processes may be relevant for brain physiology, neuronal development, and the onset/progression of neurodegenerative disorders. In this context, we describe here the macros “Autophagoquant” and “Exoquant” to assess the quantification of autophagosomes and exosomes at the neuronal presynapse of the Neuromuscular Junction (NMJ) in Drosophila melanogaster using confocal microscopy images. The Drosophila NMJ is a valuable model for the study of synapse biology, autophagy, and exosome release. By use of Autophagoquant and Exoquant, researchers can have an unbiased, standardized, and rapid tool to analyze autophagy and exosomal release in Drosophila NMJ.Code available at: https://github.com/IreneSaMi/Exoquant-Autophagoquant

Gossypol Treatment Restores Insufficient Apoptotic Function of DFF40/CAD in Human Glioblastoma Cells

Glioblastoma (GBM) is a highly aggressive brain tumor and almost all patients die because of relapses. GBM-derived cells undergo cell death without nuclear fragmentation upon treatment with different apoptotic agents. Nuclear dismantling determines the point-of-no-return in the apoptotic process. DFF40/CAD is the main endonuclease implicated in apoptotic nuclear disassembly. To be properly activated, DFF40/CAD should reside in the cytosol. However, the endonuclease is poorly expressed in the cytosol and remains cumulated in the nucleus of GBM cells. Here, by employing commercial and non-commercial patient-derived GBM cells, we demonstrate that the natural terpenoid aldehyde gossypol prompts DFF40/CAD-dependent nuclear fragmentation. A comparative analysis between gossypol- and staurosporine-treated cells evidenced that levels of neither caspase activation nor DNA damage were correlated with the ability of each compound to induce nuclear fragmentation. Deconvoluted confocal images revealed that DFF40/CAD was almost completely excluded from the nucleus early after the staurosporine challenge. However, gossypol-treated cells maintained DFF40/CAD in the nucleus for longer times, shaping a ribbon-like structure piercing the nuclear fragments and building a network of bridged masses of compacted chromatin. Therefore, GBM cells can fragment their nuclei if treated with the adequate insult, making the cell death process irreversible.

In Vitro Biofilm Formation on Aryl Ketone Polymer (AKP), A New Denture Material, Compared with That on Three Traditional Dental Denture Materials

Control of denture plaque biofilms is a practical approach to preventing persistent oral infections such as denture stomatitis. Objectives. This study compared in vitro biofilm attachment and growth on a new denture material, Ultaire® AKP, with that on traditional denture materials including cobalt chrome (CoCr), polymethyl methacrylate (PMMA), and polyoxymethylene (POM). Methods. Microbial biofilms were grown with cultures of Candida albicans, Streptococcus mutans UA159, or a mixed Streptococcus spp. (S. mutans 700610/Streptococcus sanguinis BAA-1455) for 6 hours in a static protocol or 24 hours in a dynamic protocol for each material. Adherent biofilm cells were removed, and viable colony-forming units (CFUs) were enumerated. Confocal microscopy of the 24-hour Streptococcus spp. biofilms was used to determine biofilm mass and roughness coefficients. Results. The rank order of C. albicans attachment after 6 hours was CoCr > PMMA ∗ > Ultaire® AKP ∗ ( ∗ vs CoCr, p ≤ 0.05 ), and that for 24-hour biofilm growth was CoCr > Ultaire® AKP ∗ > PMMA ∗ ( ∗ vs CoCr, p ≤ 0.05 ). The rank order of S. mutans biofilm attachment was CoCr > POM > Ultaire® AKP ∗ > PMMA ∗ ( ∗ vs CoCr, p ≤ 0.05 ), and that for the 24-hour Streptococcus spp. biofilm growth was POM > Ultaire® AKP > PMMA > CoCr ∗ ( ∗ vs POM, p ≤ 0.05 ). Confocal images revealed structural differences in Streptococcus spp. biofilms on CoCr compared with the other test materials. Significantly lower roughness coefficients of Streptococcus spp. biofilms on Ultaire® AKP were noted, suggesting that these biofilms were less differentiated. Ultaire® AKP promoted significantly less C. albicans and S. mutans biofilm attachment than CoCr at 6 hours and C. albicans growth at 24 hours. Streptococcus spp. biofilms on Ultaire® AKP were less differentiated than those on other test materials. Conclusion. In addition to its material strength, Ultaire® AKP represents an attractive option for denture material in removable partial dentures.

CaMKII activation in early diabetic hearts induces altered sarcoplasmic reticulum-mitochondria signaling

Prediabetic myocardium, induced by fructose-rich diet (FRD), is prone to increased sarcoplasmic reticulum (SR)-Ca2+ leak and arrhythmias due to increased activity of the Ca2+/calmodulin protein kinase II (CaMKII). However, little is known about the role of SR-mitochondria microdomains, mitochondrial structure, and mitochondrial metabolisms. To address this knowledge gap we measured SR-mitochondrial proximity, intracellular Ca2+, and mitochondrial metabolism in wild type (WT) and AC3-I transgenic mice, with myocardial-targeted CaMKII inhibition, fed with control diet (CD) or with FRD. Confocal images showed significantly increased spontaneous Ca2+ release events in FRD vs. CD WT cardiomyocytes. [3H]-Ryanodine binding assay revealed higher [3H]Ry binding in FRD than CD WT hearts. O2 consumption at State 4 and hydrogen peroxide (H2O2) production rate were increased, while respiratory control rate (RCR) and Ca2+ retention capacity (CRC) were decreased in FRD vs. CD WT isolated mitochondria. Transmission Electron Microscopy (TEM) images showed increased proximity at the SR-mitochondria microdomains, associated with increased tethering proteins, Mfn2, Grp75, and VDAC in FRD vs. CD WT. Mitochondria diameter was decrease and roundness and density were increased in FRD vs. CD WT specimens. The fission protein, Drp1 was significantly increased while the fusion protein, Opa1 was unchanged in FRD vs. CD WT hearts. These differences were prevented in AC3-I mice. We conclude that SR-mitochondria microdomains are subject to CaMKII-dependent remodeling, involving SR-Ca2+ leak and mitochondria fission, in prediabetic mice induced by FRD. We speculate that CaMKII hyperactivity induces SR-Ca2+ leak by RyR2 activation which in turn increases mitochondria Ca2+ content due to the enhanced SR-mitochondria tethering, decreasing CRC.

Fluorescent Staining of Silicone Micro-and Nano-patterns for Their Optical Imaging

Performance of engineered surfaces can be enhanced by making them hydrophobic or superhydrophobic via coating them with low-surface-energy micro-and nano-patterns. However, the wetting phenomena of particularly irregular shape and spacing (super)hydrophobic patterns such as polysiloxane coatings are not yet fully understood from a microscopic perspective. Here, we show a new method to collect 3D confocal images from irregular polysiloxane micro-and nanorods from a single rod resolution to discuss their wetting response over long liquid/solid interaction times and quantify the length and diameter of these rods. To collect such 3D confocal images, fluorescent dye containing water droplets were left on our superhydrophobic and hydrophobic polysiloxane coated surfaces. Then their liquid/solid interfaces were imaged at different staining scenarios: (i) using different fluorescent dyes, (ii) when the droplets were in contact with surfaces, or (iii) after the droplets were taken away from the surface at the end of staining. Using such staining strategies, we could resolve the micro-and nanorods from root to top and determine their length and diameter, which were then found to be in good agreement with those obtained from their electron microscopy images. 3D confocal images in this paper, for the first time, present the long-time existence of more than one wetting state under the same droplet in contact with surfaces, as well as external and internal three-phase contact lines shifting and pinning. In the end, these findings were used to explain the time-dependent wetting kinetics of our surfaces. We believe that the proposed imaging strategy here will, in the future, be used to study many other irregular patterned (super)antiwetting surfaces to describe their wetting theory, which is today impossible due to the complicated surface geometry of these irregular patterns.

Differential Labeling of Chemically Modified Peptides and Lipids among Cyanobacteria Planktothrix and Microcystis

The cyanoHAB forming cyanobacteria Microcystis and Planktothrix frequently produce high intracellular amounts of microcystins (MCs) or anabaenopeptins (APs). In this study, chemically modified MCs and APs have been localized on a subcellular level in Microcystis and Planktothrix applying copper-catalyzed alkyne-azide cycloaddition (CuACC). For this purpose, three different non-natural amino acids carrying alkyne or azide moieties were fed to individual P. agardhii strains No371/1 and CYA126/8 as well as to M. aeruginosa strain Hofbauer showing promiscuous incorporation of various amino acid substrates during non-ribosomal peptide synthesis (NRPS). Moreover, CYA126/8 peptide knock-out mutants and non-toxic strain Synechocystis PCC6803 were processed under identical conditions. Simultaneous labeling of modified peptides with ALEXA405 and ALEXA488 and lipid staining with BODIPY 505/515 were performed to investigate the intracellular location of the modified peptides. Pearson correlation coefficients (PCC) obtained from confocal images were calculated between the different fluorophores and the natural autofluorescence (AF), and between labeled modified peptides and dyed lipids to investigate the spatial overlap between peptides and the photosynthetic complex, and between peptides and lipids. Overall, labeling of modified MCs (M. aeruginosa) and APs (P. agardhii) using both fluorophores revealed increased intensity in MC/AP producing strains. For Synechocystis lacking NRPS, no labeling using either ALEXA405 or ALEXA488 was observed. Lipid staining in M. aeruginosa and Synechocystis was intense while in Planktothrix it was more variable. When compared with AF, both modified peptides and lipids showed a heterologous distribution. In comparison, the correlation between stained lipids and labeled peptides was not increased suggesting a reduced spatial overlap.

Evolutionary 3D Image Segmentation of Curve Epithelial Tissues of Drosophila melanogaster

Analysing biological images coming from the microscope is challenging; not only is it complex to acquire the images, but also the three-dimensional shapes found on them. Thus, using automatic approaches that could learn and embrace that variance would be highly interesting for the field. Here, we use an evolutionary algorithm to obtain the 3D cell shape of curve epithelial tissues. Our approach is based on the application of a 3D segmentation algorithm called LimeSeg, which is a segmentation software that uses a particle-based active contour method. This program needs the fine-tuning of some hyperparameters that could present a long number of combinations, with the selection of the best parametrisation being highly time-consuming. Our evolutionary algorithm automatically selects the best possible parametrisation with which it can perform an accurate and non-supervised segmentation of 3D curved epithelial tissues. This way, we combine the segmentation potential of LimeSeg and optimise the parameters selection by adding automatisation. This methodology has been applied to three datasets of confocal images from Drosophila melanogaster, where a good convergence has been observed in the evaluation of the solutions. Our experimental results confirm the proper performing of the algorithm, whose segmented images have been compared to those manually obtained for the same tissues.