Involvement of p63 in the herpes simplex virus-1-induced demise of corneal cells

<p>Abstract</p> <p>Background</p> <p>The transcription factor p63 plays a pivotal role in the development and maintenance of epithelial tissues, including the ocular surface. In an effort to gain insight into the pathogenesis of keratitis caused by HSV-1, we determined the expression patterns of the p63 and Bax proteins in the Staatens Seruminstitute Rabbit Cornea cell line (SIRC).</p> <p>Methods</p> <p>SIRC cells were infected with HSV-1 at various multiplicities and maintained for different periods of time. Virus replication was measured by indirect immunofluorescence assay and Western blot analysis. Cell viability was determined by MTT assay. The apoptotic response of the infected cells was quantified by ELISA detecting the enrichment of nucleosomes in the cytoplasm. Western blot analysis was used to determine the levels of p63 and Bax proteins.</p> <p>Results</p> <p>Indirect immunofluorescence assays and Western blot analyses demonstrated the presence of HSV-1 glycoprotein D (gD) in the infected SIRC cell line, and the pattern of gD expression was consistent with efficient viral replication. The results of MTT and ELISA assays showed that HSV-1 elicited a strong cytopathic effect, and apoptosis played an important role in the demise of the infected cells. Mock-infected SIRC cells displayed the constitutive expression of ΔNp63α. The expressions of the Bax-β and TAp63γ isoforms were considerably increased, whereas the level of ΔNp63α was decreased in the HSV-1-infected SIRC cells. Experiments involving the use of acyclovir showed that viral DNA replication was necessary for the accumulation of TAp63γ.</p> <p>Conclusion</p> <p>These data suggest that a direct, virus-mediated cytopathic effect may play an important role in the pathogenic mechanism of herpetic keratitis. By disturbing the delicate balance between the pro-survival ΔN and the pro-apoptotic TA isoforms, HSV-1 may cause profound alterations in the viability of the ocular cells and in the tissue homeostasis of the ocular surface.</p

Propionibacterium acnes induces autophagy in keratinocytes: involvement of multiple mechanisms

Propionibacterium acnes is a dominant member of the cutaneous microbiota. Herein, we evaluate the effects of different P. acnes strains and propionic acid on autophagy in keratinocytes. Our results showed that P. acnes strain 889 altered the architecture of the mitochondrial network, elevated the levels of LC3B-II, Beclin-1 and phospho-AMPKalpha, stimulated autophagic flux, facilitated intracellular redistribution of LC3B, increased average number of autophagosomes per cell, and enhanced development of acidic vesicular organelles in the HPV-KER cell line. Propionic acid increased the level of phospho-AMPKalpha, enhanced lipidation of LC3B, stimulated autophagic flux, as well as facilitated translocation of LC3B into autophagosomes in HPV-KER cells. P. acnes strains 889, 6609 and heat-killed strain 889 also stimulated autophagosome formation in primary keratinocytes to varying degrees. These results indicate that cell wall components and secreted propionic acid metabolite of P. acnes evoke mitochondrial damage successively, thereby trigger AMPK-associated activation of autophagy, which in turn facilitates the removal of dysfunctional mitochondria and promotes survival of keratinocytes. Thus, we suggest that low-level colonization of hair follicles with non-invasive P. acnes strains, by triggering a local increase in autophagic activity, might exert a profound effect on several physiological processes responsible for the maintenance of skin tissue homeostasis

Herpes Simplex Virus Infection Alters the Immunological Properties of Adipose-Tissue-Derived Mesenchymal-Stem Cells

The proper functioning of mesenchymal stem cells (MSCs) is of paramount importance for the homeostasis of the body. Inflammation and infection can alter the function of MSCs, which can also affect the regenerative potential and immunological status of tissues. It is not known whether human herpes simplex viruses 1 and 2 (HSV1 and HSV2), well-known human pathogens that can cause lifelong infections, can induce changes in MSCs. In non-healing ulcers, HSV infection is known to affect deeper tissue layers. In addition, HSV infection can recur after initially successful cell therapies. Our aim was to study the response of adipose-derived MSCs (ADMSCs) to HSV infection in vitro. After confirming the phenotype and differentiation capacity of the isolated cells, we infected the cells in vitro with HSV1-KOS, HSV1-532 and HSV2 virus strains. Twenty-four hours after infection, we examined the gene expression of the cells via RNA-seq and RT-PCR; detected secreted cytokines via protein array; and determined autophagy via Western blot, transmission electron microscopy (TEM) and fluorescence microscopy. Infection with different HSV strains resulted in different gene-expression patterns. In addition to the activation of pathways characteristic of viral infections, distinct non-immunological pathways (autophagy, tissue regeneration and differentiation) were also activated according to analyses with QIAGEN Ingenuity Pathway Analysis, Kyoto Encyclopedia of Genes and Genome and Genome Ontology Enrichment. Viral infections increased autophagy, as confirmed via TEM image analysis, and also increased levels of the microtubule-associated protein light chain 3 (LC3B) II protein. We identified significantly altered accumulation for 16 cytokines involved in tissue regeneration and inflammation. Our studies demonstrated that HSV infection can alter the viability and immunological status of ADMSCs, which may have implications for ADMSC-based cell therapies. Alterations in autophagy can affect numerous processes in MSCs, including the inhibition of tissue regeneration as well as pathological differentiation

Vesicular stomatitis virus infection triggers apoptosis associated with decreased Delta Np63 alpha and increased Bax levels in the immortalized HaCaT keratinocyte cell line

In view of the powerful inherent oncolytic activity exhibited by the vesicular stomatitis virus (VSV) in several tumor types, we set Put to investigate the susceptibility of the immortalized HaCaT keratinocyte cell line to VSV, and analyzed the role of apoptosis in the VSV-mediated induction of cell death. Indirect immunofluorescence assays, Western blot analyses and plaque titrations demonstrated that the HaCaT cell line was permissive to VSV replication. The results of ELISA for detection of the enrichment of nucleosomes in the cytoplasm of apoptotic cells revealed that VSV infection elicits the apoptotic death of HaCaT cells. Mock-infected HaCaT cells displayed the endogenous expression of Delta Np63 alpha, p53 mutated on UV hot spots (p53(mt)), Bcl-2 and p21 Bax. The levels of Delta Np63 alpha and p53(mt) were decreased, Bcl-2 remained unaffected, while the expressions of p21 Bax and p18 Bax were increased in VSV-infected HaCaT cells. Together, these data demonstrate that VSV replicates efficiently and triggers apoptosis in the immortalized HaCaT keratinocyte cell line. The VSV-mediated alterations in the expressions of Delta Np63 alpha and Bax may be implicated in the apoptotic responses of infected cells and may also sensitize to other apoptotic stimuli. These findings may stimulate further studies with the goal of developing VSV-based virotherapy into an effective modality for the treatment of epithelial-derived malignant tumors of the skin. (c) 2007 Elsevier Masson SAS. All rights reserved

Interaction between Mo(VI) and siderophore models in aqueous solution

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Sensor-based IoT monitoring of Electronics Manufacturing in University Lab Environment

We present a sensor-based IoT monitoring of an electronics manufacturing laboratory in a university environment. The project focuses on the possibilities of retrofitting the current electronics manufacturing equipment (e.g., surface mount technology apparatus) with sensors to gather extensive in-situ information about the critical parameters of the machines and their ambient surroundings. The project is based on two different sensor nodes functioning as fixed and mobile measurement devices around the given points of the laboratory located at our university. The demonstration presents a cost-effective solution of retrofitting machines with IoT-based sensor nodes, enabling new data acquisition functionalities. Also, the demonstration shows an example of a system from where the students can learn about IoT and Industry 4.0 requirements in their educational environment and from remote access. The project also highlights the importance of operational safety and health (OSH) in such workplaces. The paper presents the block diagrams and the functionalities of the sensor nodes. Also, it presents the frontend and backend setup. The user interface layers are also presented, one, which is available through web access, and the other is a proprietary app for clear visualization of the recorded data. The two approaches share the same SQL database, which is stored on the server