TGF-β1 Induced Transdifferentiation of RPE Cells is Mediated by TAK1

Background and Aim

Proliferative vitreoretinopathy (PVR) is an active process that develops as a complication upon retinal detachment (RD), accompanied by formation of fibrotic tissue. The main cells involved in the development of fibrotic tissue during PVR are the retinal pigment epithelial (RPE) cells. The RPE cells undergo epithelial-mesenchymal transition (EMT) which leads to complex retinal detachment and loss of vision. Transforming growth factor-β1 (TGF-β1) is considered as the main player in the EMT of RPE cells, even though the mechanism is not fully understood. This study was performed to determine the possible involvement of transforming growth factor β activated kinase 1 (TAK1) in the EMT process of the RPE cells.

Methodology

ARPE-19 Cells were treated with 5Z-7 oxozeaenol (TAK1 inhibitor) or SB431542 (TGF-β1 receptor kinase inhibitor) followed by TGF-β1 stimulation. Immunofluorescence, scratch assay Real time PCR and collagen contraction assay assessed the EMT features. The phosphorylation of Smad2/3 and p38 was examined using western blots analysis.

Results

This study demonstrates that stimulation of RPE cells with TGF-β1 increases α-SMA expression, cell migration and cell contractility, all of which are EMT features. Remarkably, addition of TAK1 inhibitor abolishes all these processes. Furthermore, we show hereby that TAK1 regulates not only the activation of the non-canonical cascade of TGF-β1 (p38), but also the canonical cascade, the Smad2/3 activation. Thus, the outcome of the TGF-β response in RPE cells is TAK1 dependent.

Conclusions/Significance

This work demonstrated TAK1, a component of the non-canonical pathway of TGF-β1, is a key player in the EMT process, thus provides deep insight into the pathogenesis of PVR. The ability to halt the process of EMT in RPE cells may reduce the severity of the fibrotic response that occurs upon PVR, leading to a better prognosis and increase the probability of success in RD treatment.     

Optical characteristics and parameters of the plasma of a barrier discharge excited in a mixture of mercury dibromide vapor with nitrogen and helium

Results are presented from experimental and theoretical studies of the optical characteristics and parameters of the plasma of an atmospheric-pressure barrier discharge excited in a HgBr₂: N₂: He mixture, which was used as the working medium of a small-size (with a radiation area of 8 cm²) exciplex gas-discharge radiation source. The mean radiation power of 87 mW was achieved at the radiation wavelength λ_max = 502 nm. The electron energy distribution function, the transport characteristics, the specific energy lost in the processes involving electrons, the electron temperature and density, and the rate constants of elastic and inelastic electron scattering by the components of the working mixture were calculated as functions of the reduced field E/N. The plasma of a discharge excited in a HgBr₂: N₂: He mixture can be used as the working medium of a small-size blue-green radiation source. Such a source can find application in biotechnology, photonics, and medicine and can also be used to manufacture gas-discharge display panels.     

Novel Technique for Quantifying Adhesion of Metarhizium anisopliae Conidia to the Tick Cuticle

The present study describes an accurate quantitative method for quantifying the adherence of conidia to the arthropod cuticle and the dynamics of conidial germination on the host. The method was developed using conidia of Metarhizium anisopliae var. anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) and engorged Rhipicephalus annulatus (Say) (Arachnida: Ixodidae) females and was also verified for M. anisopliae var. acridum Driver et Milner (Hypocreales: Clavicipitaceae) and Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) larvae. This novel method is based on using an organic solvent (dichloromethane [DCM]) to remove the adhered conidia from the tick cuticle, suspending the conidia in a detergent solution, and then counting them using a hemocytometer. To confirm the efficacy of the method, scanning electron microscopy (SEM) was used to observe the conidial adherence to and removal from the tick cuticle. As the concentration of conidia in the suspension increased, there were correlating increases in both the number of conidia adhering to engorged female R. annulatus and tick mortality. However, no correlation was observed between a tick''s susceptibility to fungal infection and the amount of adhered conidia. These findings support the commonly accepted understanding of the nature of the adhesion process. The mechanism enabling the removal of the adhered conidia from the host cuticle is discussed.The entomopathogenic fungus Metarhizium anisopliae var. anisopliae (Metschn.) Sorokîn (1883) infects a broad range of arthropod hosts and can be used as a biopesticide against different insect and tick species (8, 22, 35, 36). The adhesion of the conidia of entomopathogenic fungi to the host cuticle is the initial stage of the pathogenic process and includes both passive and active events (5, 10). The hydrophobic epicuticular lipid layer plays an important role during both the attachment process and the germination of the conidia on the surface of the host (15, 19). According to Boucias et al. (7), the attachment of conidia to the host cuticle is based on nonspecific hydrophobic and electrostatic forces. The conidia of most entomopathogenic fungi, including M. anisopliae, have an outer cell layer made up of rodlets (6). The hydrophobins, specific proteins present in the rodlet layer, mediate the passive adhesion of conidia to hydrophobic surfaces, such as the cuticles of arthropods (16, 45, 46). However, as germination commences, the hydrophobins are replaced by an adhesion-like protein, Mad1, which promotes tighter and more-specific adhesion between the conidia and the host (44). Many factors may affect the adhesion and persistence of conidia on the host cuticle (i.e., characteristics of the pathogen, including its virulence [2, 18, 48], conditions under which the pathogen is cultured [17], type of spores [7, 16], topographical and chemical properties of the host cuticle [9, 38, 42], host surface hydrophobicity [15, 23], host behavior [31, 33], and environmental conditions [33]). Conidia of M. anisopliae have shown an affinity to cuticular regions containing setae or spines (7, 38) and to highly hydrophobic cuticle regions, such as mosquitoes'' siphon tubes (23) and intersegmental folds (43). Sites with higher numbers of adhered conidia varied among host species. However, in general, the membranous intersegmental regions were often particularly attractive sites for conidial attachment (26). Variation in the distribution of conidia across different anatomical regions has also been noted in studies of several tick species inoculated with entomopathogenic fungi (3, 21, 22). An evaluation of the attachment of Beauveria bassiana conidia to three tick species, Dermacentor variabilis, Rhipicephalus sanguineus, and Ixodes scapularis, demonstrated that the distribution patterns of the different conidia on the ticks'' bodies were not uniform (22). The density of the conidia and their germination varied dramatically across different anatomical regions of Amblyomma maculatum and A. americanum that had been inoculated with B. bassiana (21). Arruda et al. (3) demonstrated that mass adhesion of M. anisopliae conidia to engorged Boophilus microplus females occurs predominantly on ticks'' legs, suggesting its association with the presence of setae.There are a few approaches for assessing the adhesion of conidia to the host cuticle that are based on direct observation of the conidia on the arthropod cuticle. They involve examining a few areas on the surface of an arthropod by means of scanning electron microscopy (SEM) (11, 15, 30), transmission electron microscopy (TEM) (4), or fluorescence microscopy following vital staining of the conidia (2, 28, 29, 37). These methods are expensive, time-consuming, and relatively inaccurate due to the uneven distribution of conidia on the host surface.In this work, we describe a quantitative method for determining the total amount of conidia that have adhered to a whole host cuticle. This method is based on removing adhered conidia from the tick cuticle using an organic solvent, separating the conidia from the extract by centrifugation, resuspending the conidia in a detergent solution, and then counting the conidia in a hemocytometer. The efficacy of the method was evaluated by comparing the results of this procedure with those of a supplementary examination of conidial removal using SEM.The term “adhered” is often used to define conidia in different states: washed or unwashed after inoculation, present on the host cuticle immediately after inoculation, or kept for several hours (1, 2, 38). In this paper, the term “adhered conidia” refers to conidia that remained on the cuticle after washing by vortexing the inoculated and dried host in an aqueous solution of Triton X-100 and rinsing of the material under tap water.     

Chloroplasts in the epidermis of Sarracenia (the American pitcher plant) and their possible role in carnivory - An immunocytochemical approach

A photosynthetic apparatus is present in the epidermis of the bottom zone of the pitcher of Sarracenia purpurea L. ssp. purpurea. This has been demonstrated using conventional light and electron microscopy, as well as fluorescent and immunohistochemical techniques. Red intrinsic fluorescence by these chloroplasts indicates photochemical activity. Antibodies against the coupling factor of chloroplast ATPase and against the subunits of ribulose-bis-phosphate-carboxylase were bound to the epidermal chloroplasts. This has been visualized using a ferritin-isothiocyanate labeled second antibody. These results unequivocally prove the existence of the two main proteins which are associated with the photophosphorylation (membrane protein) and carbon dioxide fixation (stromal protein). The possible implication of this system to interrelationships between the carnivorous plant and aquatic insects inhabiting its leaves is discussed.     

HLA genotypes and HLA-linked genetic markers in Italian patients with classical 21-hydroxylase deficiency

Summary HLA genotype and HLA-linked marker data for 40 unrelated patients from central Italy and 2 unrelated patients from Sardinia with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OH-def) were analyzed. The results confirm that the HLA-linked 21-OH-def gene is associated with several different HLA determinants and complete HLA haplotypes, although the only determinant with significantly increased frequency was the complement C2 allele C2B. The HLA antigens B8 and DR3 were found in significantly decreased frequencies. The haplotype A3, Cw6, Bw47, BfF, DR7, which is exceptionally rare in the general population but which has been found in many other 21-OH-def patients from diverse geographical origins, was also found in one of the Italian patients. This and other HLA haplotype associations found among the Italian patients may represent mutations that have occurred on HLA haplotypes with genetic linkage disequilibrium or, alternatively, may represent mutations that have not yet had time to become randomly associated with different HLA complex determinants. The marked negative associations with B8 and DR3 could, however, result from an interaction between the gene products of the HLA complex and the 21-OH-def phenotype.     

The concordance of gene trees and species trees at two linked loci

The gene genealogies of two linked loci in three species are analyzed using a series of Markov chain models. We calculate the probability that the gene tree of one locus is concordant with the species tree, given that the gene tree of the other locus is concordant. We define a threshold value of the recombination rate, r*, to be the rate for which the difference between the conditional probability of concordance and its asymptotic value is reduced to 5% of the initial difference. We find that, although r* depends in a complicated way on the times of speciation and effective population sizes, it is always relatively small, <10/N4, where N4 is the effective size of the species represented by the internal branch of the species tree. Consequently, the concordance of gene trees of neutral loci with the species tree is expected to be on roughly the same length scale on the chromosome as the extent of significant linkage disequilibrium within species unless the effective size of contemporary populations is very different from the effective sizes of their ancestral populations. Both balancing selection and selective sweeps can result in much longer genomic regions having concordant gene trees.     

Efficient inhibition of bursts by bursts in the auditory system of crickets

In crickets, auditory information about ultrasound is carried bilaterally to the brain by the AN2 neurons. The ON1 neuron provides contralateral inhibitory input to AN2, thereby enhancing bilateral contrast between the left and right AN2s, an important cue for sound localization. We examine how the structures of the spike trains of these neurons affect this inhibitory interaction. As previously shown for AN2, ON1 responds to salient peaks in stimulus amplitude with bursts of spikes. Spike bursts, but not isolated spikes, reliably signal the occurrence of specific features of the stimulus. ON1 and AN2 burst at similar times relative to the amplitude envelope of the stimulus, and bursts are more tightly time-locked to stimulus feature than the isolated spikes. As a consequence, spikes that, in the absence of contralateral inhibition, would occur within AN2 bursts are more likely to be preceded by spikes in ON1 (mainly also in bursts) than are isolated AN2 spikes. This leads to a large decrease in the burst rate of the inhibited AN2. We conclude that the match in coding properties of ON1 and AN2 allows contralateral inhibition to be most efficient for those portions of the response that carry the behaviourally relevant information, i.e. for bursts. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Orthostatic panic as a key Vietnamese reaction to traumatic events: the case of September 11, 2001

This article discusses a culturally specific response to traumatic events: orthostatic panic attacks among Vietnamese refugees. We compared the rate and severity of orthostatic panic as well as the rates and severity of associated flashbacks a month before and a month after September 11, 2001. After that date, the rate and severity of orthostatic panic greatly increased, as did the rate and severity of associated flashbacks. The central role of orthostatic panic as a response to traumatic events is illustrated through a patient's vignette. An explanation of why September 11 so profoundly influenced this population is adduced, including an explanation of why it resulted in considerable worsening of orthostatic panic.     

The GacA global regulator of Vibrio fischeri is required for normal host tissue responses that limit subsequent bacterial colonization

Harmful and beneficial bacterium-host interactions induce similar host-tissue changes that lead to contrasting outcomes of association. A life-long association between Vibrio fischeri and the light organ of its host Euprymna scolopes begins when the squid collects bacteria from the surrounding seawater using mucus secreted from ciliated epithelial appendages. Following colonization, the bacterium causes changes in host tissue including cessation of mucus shedding, and apoptosis and regression of the appendages that may limit additional bacterial interactions. We evaluated whether delivery of morphogenic signals is influenced by GacA, a virulence regulator in pathogens, which also influences squid-colonization by V. fischeri. Low-level colonization by a GacA mutant led to regression of the ciliated appendages. However, the GacA mutant did not induce cessation of mucus shedding, nor did it trigger apoptosis in the appendages, a phenotype that normally correlates with their regression. Because apoptosis is triggered by lipopolysaccharide, we examined the GacA mutant and determined that it had an altered lipopolysaccharide profile as well as an increased sensitivity to detergents. GacA-mutant-colonized animals were highly susceptible to invasion by secondary colonizers, suggesting that the GacA mutant's inability to signal the full programme of light-organ responses permitted the prolonged recruitment of additional symbionts.