Global market demand for chicken breast muscle with high yield and quality, together with the high incidence rate of breast muscle abnormalities in recent years highlights the need for tools that can ...provide a rapid and precise evaluation of breast muscle development and morphology. In this study, we used a novel deep learning-based automated image analysis workflow combining Fiji (ImageJ) with Cellpose and MorphoLibJ plugins to generate an automated diameter and cross-sectional area quantification for broiler breast muscle. We compared data of myofiber diameter from 14-day-old broiler chicks, generated either by manual analysis or by automated analysis. Comparison between manual and automated analysis methods exhibited a striking accuracy rate of up to 99.91%. Moreover, the automated analysis method was much faster. When the automated analysis method was implemented on 84 breast muscle cross-section images it characterized 59,128 myofibers within 4.2 h, while manual analysis of 27 breast muscle cross-section images enabled analysis of 17,333 myofibers in 54 h. The automated image analysis method was also more productive, producing data sets of both diameter and cross-sectional area at an 80-fold higher rate than the manual analysis (26,279 vs. 321 data sets per hour, respectively). In order to demonstrate the ability of this automated image analysis tool to detect differences in breast muscle histomorphology, we applied it on cross sections from chicks of control and in ovo feeding group, injected with a methionine source 2-hydroxy-4-(methylthio) butanoic calcium salt (HMTBa), known to effect skeletal muscle histomorphology. Analysis was performed on 19,807 myofibers from the control group and 21,755 myofibers from the HMTBa group and was completed in less than 1 h. The clear advantages of this automated image analysis workflow characterized by high precision, high speed, and high productiveness demonstrate its potential to be implemented as a reproducible and readily adaptable research or diagnostic tool for chicken breast muscle development and morphology.
In many taxa, females store sperm in specialized storage organs. Most insect sperm storage organs have a tubular structure, typically consisting of a central lumen surrounded by epithelial cells. ...These specialized tubules perform the essential tasks of transporting sperm through the female reproductive tract and supporting long-term sperm survival and function. Little is known about the way in which female sperm storage organs provide an environment conducive to sperm survival. We address this using a combined light microscopy, micro computed tomography (microCT), and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) approach for high-resolution correlative three-dimensional imaging to advance our understanding of sperm-female interactions in Drosophila melanogaster. Using this multimodal approach, we were able to scan the lower female reproductive tract and distal portion of the seminal receptacle at low magnification, and to subsequently zoom in for further analysis on an ultrastructural level. Our findings highlight aspects of the way in which the seminal receptacle keeps sperm viable in the lumen, and set the stage for further studies. The methods developed are suitable not only for Drosophila but also for other organisms with soft, delicate tissues.
Plant pathogenic fungi are a major threat to food security and impose a severe economic burden, thus there is a continuous need to develop new strategies to manage them. NAD
is a co-factor in ...numerous enzymatic activities and determines the metabolic fate of the cell. Therefore, maintenance of NAD
concentration is important for cellular viability. Consequently, the NAD
biosynthetic pathway and redox homeostasis was suggested as a target for antifungal development. We aimed to study how
senses and responds to nicotinaldehyde (NA), an inhibitor of Pnc1, a key enzyme in the salvage pathway of NAD
biosynthesis. We were able to show that NA was inhibitory in high concentrations to several fungal plant pathogens, with much milder effects on tomato growth. Under low nutrient conditions NA reduced the total amounts of NAD
in the fungal cell, a trend that was also observed in rich media, although without statistical significance. In low and high nutrient availability NA dramatically reduced the NAD
/NADH ratio. After exposure to NA, NADH levels were increased and NAD
levels and the biomass were greatly reduced. Cells responded to NA by up-regulation of oxidoreductases, with hardly any up-regulation of the classic response to oxidative stress. Direct measurement of oxidative stress response showed that unlike formaldehyde and hydrogen peroxide, NA caused reductive rather than oxidative stress. Surprisingly, alcohol dehydrogenases were significantly up-regulated more than any other dehydrogenases, including aldehyde dehydrogenases. We propose that conidia of
efficiently detoxified the aldehyde group of NA by reducing NAD
to NADH; the high concentrations of the latter provoked the expression of alcohol dehydrogenases that in yeast can act to reduce NADH and increase NAD
amounts, respectively. Overall, the results suggest that targeting NAD
biosynthesis pathway and redox homeostasis can be a potential approach to manage fungal plant pathogens. Many of the natural antifungal compounds produced by bio-control agents or even the natural biome are aldehydes, and thus the results presented here predict the possible response of
to wide sources of toxicity in the environment.
We explored the structural features of recombinant ostreolysin A (rOlyA), a protein produced by Pleurotus ostreatus and responsible for binding to α/β-tubulin. We found that rOlyA cell ...internalization is essential for the induction of adipocyte-associated activity, which is mediated by the interaction of rOlyA and microtubule proteins. We created different point mutations at conserved tryptophan (W) sites in rOlyA and analyzed their biological activity in HIB-1B preadipocytes. We demonstrated that the protein’s cell-internalization ability and the differentiated phenotype induced, such as small lipid-droplet formation and gene expression of mitogenesis activity, were impaired in point-mutated proteins W96A and W28A, where W was converted to alanine (A). We also showed that an rOlyA homologue, OlyA6 complexed with mCherry, cannot bind to β-tubulin and does not induce mitochondrial biosynthesis-associated markers, suggesting that the OlyA6 region masked by mCherry is involved in β-tubulin binding. Protein–protein docking simulations were carried out to investigate the binding mode of rOlyA with β-tubulin. Taken together, we identified functional sites in rOlyA that are essential for its binding to β-tubulin and its adipocyte-associated biological activity.
Environmental stresses are among the major factors that limit crop productivity and plant growth. Various nondestructive approaches for monitoring plant stress states have been developed. However, ...early sensing of the initial biochemical events during stress responses remains a significant challenge. In this work, we established whole-plant redox imaging using potato (Solanum tuberosum) plants expressing a chloroplast-targeted redox-sensitive green fluorescence protein 2 (roGFP2), which reports the glutathione redox potential (EGSH). Ratiometric imaging analysis demonstrated the probe response to redox perturbations induced by H2O2, DTT, or a GSH biosynthesis inhibitor. We mapped alterations in the chloroplast EGSH under several stress conditions including, high-light (HL), cold, and drought. An extremely high increase in chloroplast EGSH was observed under the combination of HL and low temperatures, conditions that specifically induce PSI photoinhibition. Intriguingly, we noted a higher reduced state in newly developed compared with mature leaves under steady-state and stress conditions, suggesting a graded stress sensitivity as part of the plant strategies for coping with stress. The presented observations suggest that whole-plant redox imaging can serve as a powerful tool for the basic understanding of plant stress responses and applied agricultural research, such as toward improving phenotyping capabilities in breeding programs and early detection of stress responses in the field.
Abstract
Background
HIV persists in a long-lived infected CD4 T cell reservoir, which harbors an integrated transcriptionally latent virus. We reported that cytotoxic CD8 T cells conjugate with, and ...kill, autologous HIV-infected CD4 T cells isolated from all stages of HIV infection including reservoir cells of patients under antiretroviral therapy (ART) (image 1). This killing is attenuated by HIV Nef protein. We also previously reported that FasL presented by the cytotoxic T cells interacts with Fas on the surface of the target cells, resulting in the apoptosis of the target cells. Wajant demonstrated that Fas/FasL interaction can result in the target cell activation through NFkB, whereas NFkB binding site is present in the Long Terminal Repeat of HIV. Therefore, we hypothesize that the interaction of FasL and autologous CD8 T cells with CD4 T cells can result in the reactivation of latent HIV.
Conjugation and apoptosis of CD4 T cells by autologous CD8 T cells procured from patients on ART and analyzed by ImageStream.
The CD4 T cells in apoptosis are positive for activated caspase 3 (yellow) and DNA fragmentation/ TUNEL assay (orange). CD4 T cells (green), CD8 T cells (red), DAPI (blue).
Methods
CD4 T cells and CD8 T cells were procured from the PBMC of 20 HIV-infected patients on ART with undetectable viral load and 10 healthy volunteers. The cells were isolated using magnetic beads. Resting memory CD4 T cells (CD25-, CD69- and HLA-DR-) were isolated using a two-step bead depletion purification procedure. These cells were then incubated with soluble FasL (sFasL) and autologous CD8 T cells, for 2 and 16 hours. P24 was looked for in the supernatant by ELISA, and the expression of viral proteins was looked for inside CD4 T cells using immunohistochemistry and confocal microscopy
Results
CD4 T cells and resting memory CD4 T cells, procured from PBMC of HIV-infected patients on ART, showed HIV reactivation after incubation with sFasL and autologous CD8 T cells. This reactivation was demonstrated by the appearance of P24 in the supernatant (figure 1). HIV proteins p24, gp120, and Nef appeared inside the CD4 T cells (Image 2). HIV reactivation was not demonstrated in the CD4 T cells procured from HIV-infected patients that were incubated without sFasL or autologous CD8 T cells for 18 hours (figure 1).
Immunohistochemistry and confocal microscopy of CD4 T cells incubated with sFasL for 18 hours.
The cells were then incubated with a primary fluorescent antibody against HIV proteins (P24, Nef, GP120) and a secondary antibody ALEXA594 (red). The cells were also marked with DAPI (blue). We can see the expression of HIV proteins inside the CD4 T cells (red arrows).
ELISA for the expression of P24 in the supernatant of CD4 T cells procured from HIV-infected patients, following different manipulations
On the left, we note positive ELISA for P24 in the supernatant of the CD4 T cells of patients 11 to 16 (pg/ml). On the right, we note positive ELISA for P24 in the supernatant of the latent CD4 T cells of patients 17-20 (pg/ml).
Conclusion
HIV manipulates the cellular immune system in two ways; First, HIV attenuates CD8 T cells killing of HIV-infected CD4 T cells by the HIV-Nef protein. Second, reactivation of latent HIV by CD8 T cells and sFasL, that results in further infection of additional CD4 T cells.
Disclosures
All Authors: No reported disclosures
Apicomplexan parasites are well-known to modulate their host cells at diverse functional levels. As such, apicomplexan-induced alteration of host cellular cell cycle was described and appeared ...dependent on both, parasite species and host cell type. As a striking evidence of species-specific reactions, we here show that Eimeria bovis drives primary bovine umbilical vein endothelial cells (BUVECs) into a senescence-like phenotype during merogony I. In line with senescence characteristics, E. bovis induces a phenotypic change in host cell nuclei being characterized by nucleolar fusion and heterochromatin-enriched peripheries. By fibrillarin staining we confirm nucleoli sizes to be increased and their number per nucleus to be reduced in E. bovis-infected BUVECs. Additionally, nuclei of E. bovis-infected BUVECs showed enhanced signals for HH3K9me2 as heterochromatin marker thereby indicating an infection-induced change in heterochromatin transition. Furthermore, E. bovis-infected BUVECs show an enhanced β-galactosidase activity, which is a well-known marker of senescence. Referring to cell cycle progression, protein abundance profiles in E. bovis-infected endothelial cells revealed an up-regulation of cyclin E1 thereby indicating a cell cycle arrest at G1/S transition, signifying a senescence key feature. Similarly, abundance of G2 phase-specific cyclin B1 was found to be downregulated at the late phase of macromeront formation. Overall, these data indicate that the slow proliferative intracellular parasite E. bovis drives its host endothelial cells in a senescence-like status. So far, it remains to be elucidated whether this phenomenon indeed reflects an intentionally induced mechanism to profit from host cell-derived energy and metabolites present in a non-dividing cellular status.
Apicomplexan parasites are well-known to modulate their host cells at diverse functional levels. As such, apicomplexan-induced alteration of host cellular cell cycle was described and appeared ...dependent on both, parasite species and host cell type. As a striking evidence of species-specific reactions, we here show that Eimeria bovis drives primary bovine umbilical vein endothelial cells (BUVECs) into a senescence-like phenotype during merogony I. In line with senescence characteristics, E. bovis induces a phenotypic change in host cell nuclei being characterized by nucleolar fusion and heterochromatin-enriched peripheries. By fibrillarin staining we confirm nucleoli sizes to be increased and their number per nucleus to be reduced in E. bovis-infected BUVECs. Additionally, nuclei of E. bovis-infected BUVECs showed enhanced signals for HH3K9me2 as heterochromatin marker thereby indicating an infection-induced change in heterochromatin transition. Furthermore, E. bovis-infected BUVECs show an enhanced β-galactosidase activity, which is a well-known marker of senescence. Referring to cell cycle progression, protein abundance profiles in E. bovis-infected endothelial cells revealed an up-regulation of cyclin E1 thereby indicating a cell cycle arrest at G1/S transition, signifying a senescence key feature. Similarly, abundance of G2 phase-specific cyclin B1 was found to be downregulated at the late phase of macromeront formation. Overall, these data indicate that the slow proliferative intracellular parasite E. bovis drives its host endothelial cells in a senescence-like status. So far, it remains to be elucidated whether this phenomenon indeed reflects an intentionally induced mechanism to profit from host cell-derived energy and metabolites present in a non-dividing cellular status.
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