Similar Localization Pattern Research Articles

Ftsz-Mediated Fission of a Cuboid Bacterial Symbiont

Less than a handful of cuboid and squared cells have been described in nature which makes them a rarity. Here, we show how Candidatus Thiosymbion cuboideus, a cube-like gammaproteobacterium, reproduces on the surface of marine free-living nematodes. Morphometric analysis showed that most non-dividing Ca . T. cuboideus cells are squared and 1.3 μm-thick on average. Immunostaining of symbiont cells with an anti-fimbriae antibody revealed that they are host-polarized, as these appendages exclusively localized at the host-proximal (animal-attached) pole. Moreover, by applying a fluorescently labelled metabolic probe to track new cell wall insertion in vivo , we observed that the host-attached pole started to septate before the distal one. Proximal-to-distal insertion of new cell wall material at the septum displayed a similar localization pattern as the tubulin homolog FtsZ. Although this has been shown to arrange into squares in synthetically remodelled cuboid cells, here we show that FtsZ may also mediate the division of naturally occurring ones. This implies that, even in natural settings, membrane roundness is not required for FtsZ function.

MreB and MreC act as the geometric moderators of the cell wall synthetic machinery in Thermus thermophiles

How cell morphology is maintained in thermophilic bacteria is unknown. In this study, the functions and mechanisms of the potential cell shape determinants (e.g. MreB, MreC, MreD and RodA homologues) of the model extremely thermophilic bacterium Thermus thermophilus were initially analyzed. Deletion of mreC, mreD or rodA only resulted in heterozygous mutants indicating that these genes are all essential. In the MreB-inhibited (by A22) strain and the heterozygous mreC, mreD or rodA mutant, cell morphologies were drastically changed, and enlarged spherical cells were eventually dead indicating that they are vital for cell shape maintenance. When fused to sGFP, MreB, MreC, MreD, RodA, and the enzymes involved in peptidoglycan synthesis (e.g. PBP2 and MurG) exhibited similar subcellular localization pattern, appearing as patches, or bands slightly angled to the cell length. The localizations and functions of all the 6 proteins required a natural peptidoglycan synthesis pattern, additionally those of MreD, RodA and MurG were dependent on MreB polymerization. Consistently, through comprehensive bacterial two-hybrid analyses, it was revealed that MreB could interact with itself, MreC, MreD, RodA and MurG, and MreC could associate with PBP2. In conclusion, in T. thermophilus, MreB, MreC, MreD, RodA and the peptidoglycan synthesis enzymes probably form a network of interactions centered with MreB and bridged with MreC, thereby maintaining cell morphology.

Color and direction-invariant nonlocal self-similarity prior and its application to color image denoising

Nonlocal self-similarity (NSS) is one of the most commonly used priors in computer vision and image processing. It aims to make use of the fact that a natural image often possesses many repetitive local patterns, and thus a local image patch always has many similar patches across the image. Through compensatively integrating these similar image patches, their insightful patterns hiding under corrupted noises can be intrinsically extracted. However, for using this prior knowledge, current methods search the similar patches by using simple block matching strategy with Euclidean distance, which largely ignores those patches containing similar local patterns but with different texture-directions and colors. To more sufficiently explore similar patches over an image, in this paper, we propose two new representations for image patches, which facilitate an easy NSS prior for measuring direction-invariant and color-invariant nonlocal self-similarity possessed by image patches. Specifically, based on this prior term, we formulate the color image denoising problem as a concise Bayesian posterior estimation framework, and design an efficient expectation-maximization (EM) algorithm to solve it. A series of experiments implemented on simulated and real noisy color images demonstrate the superiority of the proposed method as compared with the state-of-the-arts both visually and quantitatively, verifying the potential usefulness of this new NSS prior.

Trafficking of a Resistance to Thyroid Hormone Syndrome Mutant: A Role of Calcium Signaling?

Thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily that serve as transcription factors for thyroid hormone‐responsive genes. TRs undergo nucleocytoplasmic shuttling mediated by nuclear localization signals (NLS) and nuclear export signals (NES) that interact with importins and exportins, respectively. Mutations in TRs offset the balance between import and export, and TR mislocalization has been hypothesized to contribute to disease pathogenesis such as in cancer and Resistance to Thyroid Hormone (RTH) syndrome (1). Severe bone malformations and delayed linear growth often characterize the phenotype of patients with RTH syndrome. A patient with a specific, atypical RTH point mutation in TRα1 (N359Y) also exhibit hypercalcemia (2). The purpose of this study was to quantify the intracellular localization pattern of RTH(N359Y) and to test the hypothesis that calcium plays a role in regulating trafficking of this RTH mutant.First, we performed in silico protein prediction modelling for both wild‐type TRα1 and RTH(N359Y). Both TRs have similar structure overall with perturbations in the DNA‐binding‐domain and Hinge domain. Transient transfection of HeLa (human) cells with GFP‐tagged wild‐type TRα1 and RTH(N359Y) were utilized to quantify the distribution patterns through the nuclear‐to‐cytoplasmic fluorescence ratio. RTH(N359Y) had similar localization patterns compared to wild‐type (p>0.05), with both receptors primarily localized to the nucleus. In contrast, when exposed to calcium‐enriched media, RTH(N359Y) showed a significantly increased nuclear localization compared to the wild‐type (p<0.05).To further test our hypothesis, we examined the distribution patterns of a membrane‐associated, N‐terminal truncated TRα1, namely p30, which has been reported to associate with Caveolin‐1 in the lipid‐microdomain with other proteins involved with the cGMP‐NO signaling cascade (3). Despite lacking the DNA‐binding‐domain, p30 reconstituted in MC3T3 lead to T3‐dependent proliferation. Therefore, if p30 localizes at the plasma membrane with Caveolin‐1, thyroid hormone stimulation could mediate import of calcium from the extracellular media and serve as an in vivo approach to test the impact of calcium on receptor trafficking for wild‐type TRα1 and RTH(N359Y). Confocal analysis, however, showed that GFP‐tagged p30 does not localize at the plasma membrane, up to 36 hours post‐transfection. Therefore, other approaches will need to be utilized to ascertain the role of calcium in the trafficking of this RTH mutant.An understanding of how calcium regulates the trafficking of RTH(N359Y) could provide insight into the connection between RTH syndrome and hypercalcemia and more generally the synergistic role of calcium signaling and nuclear trafficking.Support or Funding InformationNIH Grant #2R15DK058028 to L.A.A.

Monitoring Glaucomatous Functional Loss Using an Artificial Intelligence–Enabled Dashboard

To develop an artificial intelligence (AI) dashboard for monitoring glaucomatous functional loss. Retrospective, cross-sectional, longitudinal cohort study. Of 31 591 visual fields (VFs) on 8077 subjects, 13 231 VFs from the most recent visit of each patient were included to develop the AI dashboard. Longitudinal VFs from 287 eyes with glaucoma were used to validate the models. We entered VF data from the most recent visit of glaucomatous and nonglaucomatous patients into a "pipeline" that included principal component analysis (PCA), manifold learning, and unsupervised clustering to identify eyes with similar global, hemifield, and local patterns of VF loss. We visualized the results on a map, which we refer to as an "AI-enabled glaucoma dashboard." We used density-based clustering and the VF decomposition method called "archetypal analysis" to annotate the dashboard. Finally, we used 2 separate benchmark datasets-one representing "likely nonprogression" and the other representing "likely progression"-to validate the dashboard and assess its ability to portray functional change over time in glaucoma. The severity and extent of functional loss and characteristic patterns of VF loss in patients with glaucoma. After building the dashboard, we identified 32 nonoverlapping clusters. Each cluster on the dashboard corresponded to a particular global functional severity, an extent of VF loss into different hemifields, and characteristic local patterns of VF loss. By using 2 independent benchmark datasets and a definition of stability as trajectories not passing through over 2 clusters in a left or downward direction, the specificity for detecting "likely nonprogression" was 94% and the sensitivity for detecting "likely progression" was 77%. The AI-enabled glaucoma dashboard, developed using a large VF dataset containing a broad spectrum of visual deficit types, has the potential to provide clinicians with a user-friendly tool for determination of the severity of glaucomatous vision deficit, the spatial extent of the damage, and a means for monitoring the disease progression.

Identification of the cleavage sites leading to the shed forms of human and mouse anti-aging and cognition-enhancing protein Klotho.

Klotho is an age-extending, cognition-enhancing protein found to be down-regulated in aged mammals when age-related diseases start to appear. Low levels of Klotho occur in neurodegenerative diseases, kidney disease and many cancers. Many normal and pathologic processes involve the proteolytic shedding of membrane proteins. Transmembrane (TM) Klotho contains two homologous domains, KL1 and KL2 with homology to glycosidases. After shedding by ADAM 10 and 17, a shed Klotho isoform is released into serum and urine by the kidney, and into the CSF by the choroid plexus. We previously reported that human Klotho contains two major cleavage sites. However, the exact cleavage site responsible for the cleavage between the KL1 and KL2 domains remains unknown for the human Klotho, and both sites are unknown for mouse Klotho. In this study, we aimed to identify the cleavage sites leading to the shed forms of human and mouse Klotho. Mutations in the region close to the TM domain of mouse Klotho result in the reduced shedding of the 130 kD (KL1+KL2) and 70 kD (KL1) fragments, suggesting that the cleavage site lies within the mutated region. We further identified the cleavage sites responsible for the cleavage between KL1 and KL2 of human and mouse Klotho. Moreover, mutated Klotho proteins have similar subcellular localization patterns as wild type Klotho. Finally, in an FGF23 functional assay, all Klotho mutants with a nine amino acid deletion can also function as an FGFR1 co-receptor for FGF23 signaling, however, the signaling activity was greatly reduced. The study provides new and important information on Klotho shedding, and paves the way for studies aimed to distinguish between the distinct roles of the various isoforms of Klotho.

Spatial analysis of RECK, MT1-MMP, and TIMP-2 proteins during early Xenopus laevis development

Proper cell-cell and cell-ECM interactions are vital for cell migration and patterning of the vertebrate embryo. MMPs and their inhibitors, RECK and TIMPs, are all differentially expressed during embryogenesis to regulate such ECM remodeling and cell interactions. MT1-MMP, RECK, and TIMP-2 are unique amongst other ECM-regulating proteins as they act in the pericellular space. Past studies have shown that RECK and TIMP-2 interact with MT1-MMP on the cell surface, thereby influencing cell behaviour as well as the microenvironment immediately surrounding the cells. We investigated the localization of RECK, TIMP-2, and MT1-MMP proteins throughout early X. laevis development using immunohistochemistry. We found that during neural tube formation, axis elongation, and organogenesis, RECK, TIMP-2, and MT1-MMP proteins show highly similar localization patterns, particularly in the ectoderm and in the dorsal-ventral differentiation of the neural tube. Our data suggests they function together during patterning events in early Xenopus development.

Topology highlights mesoscopic functional equivalence between imagery and perception: The case of hypnotizability

The functional equivalence (FE) between imagery and perception or motion has been proposed on the basis of neuroimaging evidence of large spatially overlapping activations between real and imagined sensori-motor conditions. However, similar local activation patterns do not imply the same mesoscopic integration of brain regions, which can be described by tools from Topological Data Analysis (TDA). On the basis of behavioral findings, stronger FE has been hypothesized in the individuals with high scores of hypnotizability scores (highs) with respect to low hypnotizable participants (lows) who differ between each other in the proneness to modify memory, perception and behavior according to specific imaginative suggestions. Here we present the first EEG evidence of stronger FE in highs. In fact, persistent homology shows that the highs EEG topological asset during real and imagined sensory conditions is significantly more similar than the lows. As a corollary finding, persistent homology shows lower restructuring of the EEG asset in highs than in lows during both sensory and imagery tasks with respect to basal conditions. Present findings support the view that greater embodiment of mental images may be responsible for the highs greater proneness to respond to sensori-motor suggestions and to report involuntariness in action. In addition, findings indicate hypnotizability-related sensory and cognitive information processing and suggest that the psycho-physiological trait of hypnotizability may modulate more than one aspect of the everyday life.

PID: a PDF-induced distance based on permutation cross-distribution entropy

In this paper, a PDF-induced distance (PID) based on permutation cross-distribution entropy is proposed to measure the dissimilarity between complex time series. It overcomes the effects of spatial distance by focusing on similar local fluctuation patterns. It also corrects the disadvantage of being insensitive to symmetric skewness distributions. We have applied PID to synthetic data and financial time series. The Euclidean distance is employed as a reference. In simulated experiments, eight signals generated from four models are detected, and results are presented by hierarchical clustering analysis via PID, which is correctly clustered and superior to other methods. Then, PID is applied to the real-world financial time series. Eight stocks in the global financial markets are employed. It reveals that they are clearly divided based on their financial backgrounds. The cophenetic correlation coefficient is used to measure the quality of solutions. It reveals that the PID is convincing and superior to the Euclidean distance. In addition, as a classic method for non-stationary time series, detrended cross-correlation analysis (DCCA) is adopted to compare the superiority of PID. Although experiments show that DCCA does perform well, it is inferior to PID. Also, we conduct additional experiments on the effects of non-Gaussian noises. Excitingly, PID can still cluster signals accurately after adding uniformly distributed noises, and the cophenetic correlation coefficient reaches 0.9788.

Chromokinesin Kif4 promotes proper anaphase in mouse oocyte meiosis.

Oocytes of many species lack centrioles and therefore form acentriolar spindles. Despite the necessity of oocyte meiosis for successful reproduction, how these spindles mediate accurate chromosome segregation is poorly understood. We have gained insight into this process through studies of the kinesin-4 family member Kif4 in mouse oocytes. We found that Kif4 localizes to chromosomes through metaphase and then largely redistributes to the spindle midzone during anaphase, transitioning from stretches along microtubules to distinct ring-like structures; these structures then appear to fuse together by telophase. Kif4’s binding partner PRC1 and MgcRacGAP, a component of the centralspindlin complex, have a similar localization pattern, demonstrating dynamic spindle midzone organization in oocytes. Kif4 knockdown results in defective midzone formation and longer spindles, revealing new anaphase roles for Kif4 in mouse oocytes. Moreover, inhibition of Aurora B/C kinases results in Kif4 mislocalization and causes anaphase defects. Taken together, our work reveals essential roles for Kif4 during the meiotic divisions, furthering our understanding of mechanisms promoting accurate chromosome segregation in acentriolar oocytes.

Characterization of Cks2 localization and interaction with Cdk1 in mitotic cells of Xenopus laevis embryos

Although mitotic regulation has been extensively studied, several of the mechanisms behind this process have yet to be fully understood. Our group is particularly interested in mechanisms regulating the timing of mitotic exit and how such mechanisms coordinate with other mitotic cues such as spindle orientation. Recent work in our lab has shown that spindle movements in epithelial cells of the developing frog embryo correlate with mitotic exit and perturbations in normal spindle movements lengthen metaphase. Further, these changes in mitotic progression are accompanied by increases in Cdk1 Tyr15 phosphorylation, specifically at cell junctions. We propose that the link between spindle movement and mitotic progression is an important part of the mechanisms influencing spindle orientation in the frog epithelium. Our current work examines the role of Cks2 in the aforementioned mechanism. Cks2 serves as a targeting subunit for the mitotic kinase Cdk1. Specifically, work in yeast suggests Cks2 may direct Cdk1 toward the anaphase‐promoting complex (APC) and interact with both complexes, facilitating the phosphorylation of Cdk1 and activation of the APC and thus help control mitotic exit. In this study, we characterize Cks2's mitotic function in Xenopus laevis. Our aim is to characterize Cks2 distribution throughout mitosis and determine whether perturbing Cks2 function alters mitotic progression in the gastrula‐stage epithelium. We hypothesize that if Cks2 influences Cdk1 function in mitosis, then the two proteins will exhibit similar localization patterns in mitotic cells. We visualized endogenous Cks2 and also expressed GFP‐tagged Cks2 in fixed epithelial tissues of Xenopus laevis embryos through confocal microscopy. Our early data show that Cks2 localization overlaps with that of a phosphorylated Cdk1(pY15) at cell junctions and overexpression of GFP‐tagged Cks2 results in increased levels of Cdk1‐pY15. In addition, the assay expressing a dominant negative version of Cks2 suggests that depletion of endogenous Cks2 activity may contribute to decreased Cdk1‐pY15 at cell junctions and increased cell area. Therefore, these data suggest a link between Cks2 and Cdk1 in mitotic regulation and are a first step toward a greater characterization of Cks2's role in regulating mitotic progression in vertebrate organisms.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mapping the sites of localization of epithelial sodium channel (ENaC) and CFTR in segments of the mammalian epididymis.

The sperm produced in the seminiferous tubules pass through the rete testis, efferent ducts, and epididymis. The epididymis has three distinct regions known as caput, corpus, and cauda. The transit through the epididymis is an essential process in sperm maturation. The lumen of each epididymal region has a unique fluid composition regulated by many ion channels and transporters in the epithelial cells. The objective of this study was to map the sites of localization of ion channels ENaC and CFTR along the length of the mouse and rat epididymis using confocal microscopic imaging. The integrity of the fine structure of the tissues was verified by fluorescent phalloidin staining of actin filaments visualized by high-resolution confocal microscopy. The 2D and 3D images showed preservation of the stereocilia. Based on these images we determined morphometric parameters of the epithelial cells and ducts. ENaC and CFTR immunofluorescence appeared almost continuously on the apical membrane of caput and in smooth muscle myoid cells. In cauda, CFTR expression was observed continuously in long stretches of epithelium interrupted by clusters of cells that showed no CFTR expression. Similar patterns of localization were observed in both mouse and rat samples. Mutations in the CFTR gene are known to result in male infertility. Based on the widespread presence of ENaC along the epididymis we suggest that mutations in ENaC subunits may also be associated with male infertility. The diverse phenotypes associated with CFTR mutations may be due to malfunction of CFTR at specific subcellular locations in the male reproductive system.

Topology highlights mesoscopic functional equivalence between imagery and perception

The functional equivalence between mental images and perception or motion has been proposed on the basis of neuroimaging evidence of large spatially overlapping activations between real and imagined sensori-motor conditions. However, similar local activation patterns do not imply the same mesoscopic integration of brain regions active during imagery and perception or action. Here we present the first EEG evidence of topological equivalence between functional network organization at intermediate and global scales during tasks. We show that the degree of functional equivalence varies in the population and is associated with different magnitudes in the restructuring of the functional connectivity between imagery and real tasks. In particular, changes observed during imagery with respect to basal conditions account for the cognitive effort experienced during imagery, and subjects characterized by stronger functional equivalence exhibit smaller topological deviations in the imagination tasks performed after real tasks, thus showing learning effects. Altogether, our findings point to different sensori-cognitive information processing in the subjects showing different functional equivalence. We anticipate our results to be a starting point for a novel dynamical description of functional equivalence, which will be relevant for socio-cognitive theories of embodiment and cognitive formulations of how different selves emerge from neurophysiological assets.

Mapping electrical properties heterogeneity of tumor using boundary informed electrical properties tomography (BIEPT) at 7T.

To develop and evaluate a boundary informed electrical properties tomography (BIEPT) technique for high-resolution imaging of tumor electrical properties (EPs) heterogeneity on a rodent tumor xenograft model. Tumor EP distributions were inferred from a reference area external to the tumor, as well as internal EP spatial variations derived from a plurality of relative transmit B1 measurements at 7T. Edge sparsity constraint was enforced to enhance numerical stability. Phantom experiments were performed to determine the imaging accuracy and sensitivity for structures of various EP values, as well as geometrical sizes down to 1.5 mm. Numerical simulation of a realistic rodent model was used to quantify the algorithm performance in the presence of noise. Eleven athymic rats with human breast cancer xenograft were imaged in vivo, and representative pathological samples were acquired for comparison. Reconstructed EPs of the phantoms correspond well to the ground truth acquired from dielectric probe measurements, with the smallest structure reliably detectable being 3 mm. EPs heterogeneity inside a tumor is successfully retrieved in both simulated and experimental cases. In vivo tumor imaging results demonstrate similar local features and spatial patterns to anatomical MRI and pathological slides. The imaged conductivity of necrotic tissue is higher than that of viable tissues, which agrees with our expectation. BIEPT enables robust detection of tumor EPs heterogeneity with high accuracy and sensitivity to small structures. The retrieved quantitative EPs reflect tumor pathological features (e.g., necrosis). These results provide strong rationale to further expand BIEPT studies toward pathological conditions where EPs may yield valuable, non-invasive biomarkers.

FBXW2 localizes with osteocalcin in bovine periosteum on culture dishes as visualized by double immunostaining

Osteocalcin (OC) is a well-known protein related to bone, however, the role of F-box and WD-40 domain-containing protein 2 (FBXW2) in bone remains unclear. In 2016, the presence of FBXW2 in bovine periosteum was reported. In this study, double immunostaining was used to investigate the relationship between OC and FBXW2. FBXW2 showed tubular structures, and OC showed a similar localization pattern as FBXW2. Double immunostaining findings suggested that FBXW2 tubes were coated with OC. To the author's knowledge, this is the first study to reveal the interaction between OC and FBXW2.

Strain localization in dry sheared granular materials: A compactivity-based approach.

Shear banding is widely observed in natural fault zones as well as in laboratory experiments on granular materials. Understanding the dynamics of strain localization under different loading conditions is essential for quantifying strength evolution of fault gouge and energy partitioning during earthquakes and characterizing rheological transitions and fault zone structure changes. To that end, we develop a physics-based continuum model for strain localization in sheared granular materials. The grain-scale dynamics is described by the shear transformation zone (STZ) theory, a nonequilibrium statistical thermodynamic framework for viscoplastic deformation in amorphous materials. Using a finite strain computational framework, we investigate the initiation and growth of complex shear bands under a variety of loading conditions and identify implications for strength evolution and the ductile to brittle transition. Our numerical results show similar localization patterns to field and laboratory observations and suggest that shear zones show more ductile response at higher confining pressures, lower dilatancy, and loose initial conditions. Lower pressures, higher dilatancy, and dense initial conditions favor a brittle response and larger strength drops. These findings shed light on a range of mechanisms for strength evolution in dry sheared granular materials and provide a critical input to physics-based multiscale models of fault zone instabilities.

Interactive Visual Exploration of Local Patterns in Large Scatterplot Spaces

AbstractAnalysts often use visualisation techniques like a scatterplot matrix (SPLOM) to explore multivariate datasets. The scatterplots of a SPLOM can help to identify and compare two‐dimensional global patterns. However, local patterns which might only exist within subsets of records are typically much harder to identify and may go unnoticed among larger sets of plots in a SPLOM. This paper explores the notion of local patterns and presents a novel approach to visually select, search for, and compare local patterns in a multivariate dataset. Model‐based and shape‐based pattern descriptors are used to automatically compare local regions in scatterplots to assist in the discovery of similar local patterns. Mechanisms are provided to assess the level of similarity between local patterns and to rank similar patterns effectively. Moreover, a relevance feedback module is used to suggest potentially relevant local patterns to the user. The approach has been implemented in an interactive tool and demonstrated with two real‐world datasets and use cases. It supports the discovery of potentially useful information such as clusters, functional dependencies between variables, and statistical relationships in subsets of data records and dimensions.

Epidermal growth factor receptor (EGFR) density may not be the only determinant for the efficacy of EGFR-targeted photoimmunotherapy in human head and neck cancer cell lines.

The aim of this study was to investigate the effects of targeted photoimmunotherapy (PIT) in vitro on cell lines with various expression levels of epidermal growth factor receptor (EGFR) using an anti-EGFR targeted conjugate composed of Cetuximab and IR700DX, phthalocyanine dye. Relative EGFR density and cell binding assay was conducted in three human head & neck cancer cell lines (scc-U2, scc-U8, and OSC19) and one reference cell line A431. After incubation with the conjugate for 1 or 24 hours, cellular uptake and localization were investigated by confocal laser scanning microscopy and quantified by image analysis. Cell survival was determined using the MTS assay and alamarBlue assay after PIT with a 690 nm laser to a dose of 7 J.cm-2 (at 5 mW.cm-2 ). The mode of cell death was examined with flow cytometry using apoptosis/necrosis staining by Annexin V/propidium iodide, together with immunoblots of anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL. A431 cells had the highest EGFR density followed by OSC19, and then scc-U2 and scc-U8. The conjugates were localized both on the surface and in the cytosol of the cells after 1- and 24-hour incubation. After 24-hour incubation the granular pattern was more pronounced and in a similar pattern of a lysosomal probe, suggesting that the uptake of conjugates by cells was via receptor-mediated endocytosis. The results obtained from the quantitative imaging analysis correlate with the level of EGFR expression. Targeted PIT killed scc-U8 and A431 cells efficiently; while scc-U2 and OSC19 were less sensitive to this treatment, despite having similar EGFR density, uptake and localization pattern. Scc-U2 cells showed less apoptotic cell dealth than in A431 after 24-hour targeted PIT. Immunoblots showed significantly higher expression of anti-apoptotic Bcl-2 and Bcl-xL proteins in scc-U2 cell lines compared to scc-U8. Our study suggests that the effectiveness of EGFR targeted PIT is not only dependent upon EGFR density. Intrinsic biological properties of tumor cell lines also play a role in determining the efficacy of targeted PIT. We have shown that in scc-U2 cells this difference may be caused by differences in the apoptopic pathway. Lasers Surg. Med. 50:513-522, 2018. © 2018 Wiley Periodicals, Inc.

Autophagy is activated and involved in cell death with participation of cathepsins during stress-induced microspore embryogenesis in barley.

Microspores are reprogrammed towards embryogenesis by stress. Many microspores die after this stress, limiting the efficiency of microspore embryogenesis. Autophagy is a degradation pathway that plays critical roles in stress response and cell death. In animals, cathepsins have an integral role in autophagy by degrading autophagic material; less is known in plants. Plant cathepsins are papain-like C1A cysteine proteases involved in many physiological processes, including programmed cell death. We have analysed the involvement of autophagy in cell death, in relation to cathepsin activation, during stress-induced microspore embryogenesis in Hordeum vulgare. After stress, reactive oxygen species (ROS) and cell death increased and autophagy was activated, including HvATG5 and HvATG6 up-regulation and increase of ATG5, ATG8, and autophagosomes. Concomitantly, cathepsin L/F-, B-, and H-like activities were induced, cathepsin-like genes HvPap-1 and HvPap-6 were up-regulated, and HvPap-1, HvPap-6, and HvPap-19 proteins increased and localized in the cytoplasm, resembling autophagy structures. Inhibitors of autophagy and cysteine proteases reduced cell death and promoted embryogenesis. The findings reveal a role for autophagy in stress-induced cell death during microspore embryogenesis, and the participation of cathepsins. Similar patterns of activation, expression, and localization suggest a possible connection between cathepsins and autophagy. The results open up new possibilities to enhance microspore embryogenesis efficiency with autophagy and/or cysteine protease modulators.

Systemic Israeli acute paralysis virus (IAPV) infection in bumblebees (Bombus terrestris) through feeding and injection.

Israeli acute paralysis virus (IAPV) can cause a systemic infection, resulting in mortality in both Apis and Bombus spp. bees. However, little is known about the virus infection dynamics within bee tissues. Here, we established systemic IAPV infections in reared bumblebee Bombus terrestris workers through feeding and injection and investigated the mortality, tissue tropism and viral localization. Injection of approximately 500 IAPV (IAPVinj stock) particles resulted in acute infection, viral loads within tissues that were relatively stable from bee to bee, and a distinctive tissue tropism, making this method suitable for studying systemic IAPV infection in bumblebees. Feeding with approximately 1 × 106 particles of the same virus stock did not result in systemic infection. A high-concentration stock of IAPV (IAPVfed stock) allowed us to feed bumblebees with approximately 1 × 109 viral particles, which induced both chronic and acute infection. We also observed a higher variability in viral titers within tissues and less clear tissue tropism during systemic infection, making feeding with IAPVfed stock less optimal for studying IAPV systemic infection. Strikingly, both infection methods and stocks with different viral loads gave a similar viral localization pattern in the brain and midgut of bumblebees with an acute infection. The implications of these findings in the study of the local immunity in bees and barriers to oral transmission are discussed. Our data provide useful information on the establishment of a systemic viral infection in bees.