ResearchPad - confocal-microscopy https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[<i>Ehrlichia chaffeensis</i> TRP120-mediated ubiquitination and proteasomal degradation of tumor suppressor FBW7 increases oncoprotein stability and promotes infection]]> https://www.researchpad.co/article/elastic_article_13827 E. chaffeensis is an obligately intracellular bacterium that replicates in mononuclear phagocytes by secreting effectors that manipulate host cell processes and exploit evolutionarily conserved pathways. This investigation reveals the complex and expanding role of the E. chaffeensis TRP120 moonlighting effector as a ubiquitin (Ub) ligase targeting host nuclear proteins. Herein, we demonstrate that E. chaffeensis TRP120 HECT Ub ligase targets the nuclear tumor suppressor Skp1-cullin-1-FBOX E3 ubiquitin (Ub) ligase complex substrate recognition subunit, F-BOX and WD domain repeating-containing 7 (FBW7) for degradation. FBW7 is a central regulator of broadly acting host cell oncoproteins involved in cell proliferation and survival. The reduction in FBW7 through TRP120-mediated ubiquitination increases cellular oncoprotein levels and promotes E. chaffeensis infection. This study illuminates novel bacterial effector-host interactions, the importance and interplay of both host and bacterial Ub ligases and the Ub-proteasome system for infection, and mechanisms whereby evolutionarily conserved signaling pathways are hijacked by obligately intracellular pathogens.

]]>
<![CDATA[Confocal Raman microscopy to identify bacteria in oral subgingival biofilm models]]> https://www.researchpad.co/article/elastic_article_7675 The study of oral disease progression, in relation to the accumulation of subgingival biofilm in gingivitis and periodontitis is limited, due to either the ability to monitor plaque in vitro. When compared, optical spectroscopic techniques offer advantages over traditional destructive or biofilm staining approaches, making it a suitable alternative for the analysis and continued development of three-dimensional structures. In this work, we have developed a confocal Raman spectroscopy analysis approach towards in vitro subgingival plaque models. The main objective of this study was to develop a method for differentiating multiple oral subgingival bacterial species in planktonic and biofilm conditions, using confocal Raman microscopy. Five common subgingival bacteria (Fusobacterium nucleatum, Streptococcus mutans, Veillonella dispar, Actinomyces naeslundii and Prevotella nigrescens) were used and differentiated using a 2-way orthogonal Partial Least Square with Discriminant Analysis (O2PLS-DA) for the collected spectral data. In addition to planktonic growth, mono-species biofilms cultured using the ‘Zürich Model’ were also analyzed. The developed method was successfully used to predict planktonic and mono-species biofilm species in a cross validation setup. The results show differences in the presence and absence of chemical bands within the Raman spectra. The O2PLS-DA model was able to successfully predict 100% of all tested planktonic samples and 90% of all mono-species biofilm samples. Using this approach we have shown that Confocal Raman microscopy can analyse and predict the identity of planktonic and mono-species biofilm species, thus enabling its potential as a technique to map oral multi-species biofilm models.

]]>
<![CDATA[A case of recurrent lentigo maligna diagnosed with precise reflectance confocal microscopy–guided biopsy technique]]> https://www.researchpad.co/article/Nc9400d41-942d-4efc-8c8f-fd3f0a9c50e0 <![CDATA[Basal cell carcinoma and balloon cell nevus collision mimicking a melanoma on reflectance confocal microscopy]]> https://www.researchpad.co/article/N44292095-0bc3-4a6b-a978-14c58a9db49a ]]> <![CDATA[A conserved regulatory mechanism mediates the convergent evolution of plant shoot lateral organs]]> https://www.researchpad.co/article/N38f7a2a5-9838-4ae0-b206-f959ee03524f

Land plant shoot structures evolved a diversity of lateral organs as morphological adaptations to the terrestrial environment, with lateral organs arising independently in different lineages. Vascular plants and bryophytes (basally diverging land plants) develop lateral organs from meristems of sporophytes and gametophytes, respectively. Understanding the mechanisms of lateral organ development among divergent plant lineages is crucial for understanding the evolutionary process of morphological diversification of land plants. However, our current knowledge of lateral organ differentiation mechanisms comes almost entirely from studies of seed plants, and thus, it remains unclear how these lateral structures evolved and whether common regulatory mechanisms control the development of analogous lateral organs. Here, we performed a mutant screen in the liverwort Marchantia polymorpha, a bryophyte, which produces gametophyte axes with nonphotosynthetic scalelike lateral organs. We found that an Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 and Oryza G1 (ALOG) family protein, named M. polymorpha LATERAL ORGAN SUPRESSOR 1 (MpLOS1), regulates meristem maintenance and lateral organ development in Marchantia. A mutation in MpLOS1, preferentially expressed in lateral organs, induces lateral organs with misspecified identity and increased cell number and, furthermore, causes defects in apical meristem maintenance. Remarkably, MpLOS1 expression rescued the elongated spikelet phenotype of a MpLOS1 homolog in rice. This suggests that ALOG genes regulate the development of lateral organs in both gametophyte and sporophyte shoots by repressing cell divisions. We propose that the recruitment of ALOG-mediated growth repression was in part responsible for the convergent evolution of independently evolved lateral organs among highly divergent plant lineages, contributing to the morphological diversification of land plants.

]]>
<![CDATA[Efficacy of liposomal amphotericin B and anidulafungin using an antifungal lock technique (ALT) for catheter-related Candida albicans and Candida glabrata infections in an experimental model]]> https://www.researchpad.co/article/5c75ac7dd5eed0c484d088b2

Objective

The aims of this study were as follows. First, we sought to compare the in vitro susceptibility of liposomal amphotericin B (LAmB) and anidulafungin on Candida albicans and Candida glabrata biofilms growing on silicone discs. Second, we sought to compare the activity of LAmB versus anidulafungin for the treatment of experimental catheter-related C. albicans and C. glabrata infections with the antifungal lock technique in a rabbit model.

Methods

Two C. albicans and two C. glabrata clinical strains were used. The minimum biofilm eradication concentration for 90% eradication (MBEC90) values were determined after 48h of treatment with LAmB and anidulafungin. Confocal microscopy was used to visualize the morphology and viability of yeasts growing in biofilms. Central venous catheters were inserted into New Zealand rabbits, which were inoculated of each strain of C. albicans and C. glabrata. Then, catheters were treated for 48h with saline or with antifungal lock technique using either LAmB (5mg/mL) or anidulafungin (3.33mg/mL).

Results

In vitro: anidulafungin showed greater activity than LAmB against C. albicans and C. glabrata strains. For C. albicans: MBEC90 of anidulafungin versus LAmB: CA176, 0.03 vs. 128 mg/L; CA180, 0.5 vs. 64 mg/L. For C. glabrata: MBEC90 of anidulafungin versus LAmB: CG171, 0.5 vs. 64 mg/L; CG334, 2 vs. 32 mg/L. In vivo: for C. albicans species, LAmB and anidulafungin achieved significant reductions relative to growth control of log10 cfu recovered from the catheter tips (CA176: 3.6±0.3 log10 CFU, p≤0.0001; CA180: 3.8±0.1 log10 CFU, p≤0.01). For C. glabrata, anidulafungin lock therapy achieved significant reductions relative to the other treatments (CG171: 4.8 log10 CFU, p≤0.0001; CG334: 5.1 log10 CFU, p≤0.0001)

Conclusions

For the C. albicans strains, both LAmB and anidulafungin may be promising antifungal lock technique for long-term catheter-related infections; however, anidulafungin showed significantly higher activity than LAmB against the C. glabrata strains.

]]>
<![CDATA[MUC1 is a receptor for the Salmonella SiiE adhesin that enables apical invasion into enterocytes]]> https://www.researchpad.co/article/5c61e93fd5eed0c48496fa96

The cellular invasion machinery of the enteric pathogen Salmonella consists of a type III secretion system (T3SS) with injectable virulence factors that induce uptake by macropinocytosis. Salmonella invasion at the apical surface of intestinal epithelial cells is inefficient, presumably because of a glycosylated barrier formed by transmembrane mucins that prevents T3SS contact with host cells. We observed that Salmonella is capable of apical invasion of intestinal epithelial cells that express the transmembrane mucin MUC1. Knockout of MUC1 in HT29-MTX cells or removal of MUC1 sialic acids by neuraminidase treatment reduced Salmonella apical invasion but did not affect lateral invasion that is not hampered by a defensive barrier. A Salmonella deletion strain lacking the SiiE giant adhesin was unable to invade intestinal epithelial cells through MUC1. SiiE-positive Salmonella closely associated with the MUC1 layer at the apical surface, but invaded Salmonella were negative for the adhesin. Our findings uncover that the transmembrane mucin MUC1 is required for Salmonella SiiE-mediated entry of enterocytes via the apical route.

]]>
<![CDATA[Metagenomic quorum quenching enzymes affect biofilm formation of Candida albicans and Staphylococcus epidermidis]]> https://www.researchpad.co/article/5c58d622d5eed0c4840316f5

Biofilm formation in the clinical environment is of increasing concern since a significant part of human infections is associated, and caused by biofilm establishment of (opportunistic) pathogens, for instance Candida albicans and Staphylococcus epidermidis. The rapidly increasing number of antibiotic-resistant biofilms urgently requires the development of novel and effective strategies to prevent biofilm formation ideally targeting a wide range of infectious microorganisms. Both, synthesis of extracellular polymeric substances and quorum sensing are crucial for biofilm formation, and thus potential attractive targets to combat undesirable biofilms.We evaluated the ability of numerous recently identified metagenome-derived bacterial quorum quenching (QQ) proteins to inhibit biofilm formation of C. albicans and S. epidermidis. Here, proteins QQ-5 and QQ-7 interfered with the morphogenesis of C. albicans by inhibiting the yeast-to-hyphae transition, ultimately leading to impaired biofilm formation. Moreover, QQ5 and QQ-7 inhibited biofilm formation of S. epidermidis; in case of QQ7 most likely due to induced expression of the icaR gene encoding the repressor for polysaccharide intercellular adhesin (PIA) synthesis, the main determinant for staphylococcal biofilm formation. Our results indicate that QQ-5 and QQ-7 are attractive potential anti-biofilm agents in the prevention and treatment of C. albicans and S. epidermidis mono-species biofilms, and potentially promising anti-biofilm drugs in also combating multi-species infections.

]]>
<![CDATA[Recruitment of Vps34 PI3K and enrichment of PI3P phosphoinositide in the viral replication compartment is crucial for replication of a positive-strand RNA virus]]> https://www.researchpad.co/article/5c3fa5f5d5eed0c484caa846

Tombusviruses depend on subversions of multiple host factors and retarget cellular pathways to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus (CIRV) recruit the cellular Vps34 phosphatidylinositol 3-kinase (PI3K) into the large viral replication compartment. The kinase function of Vps34 is critical for TBSV replication, suggesting that PI(3)P phosphoinositide is utilized by TBSV for building of the replication compartment. We also observed increased expression of Vps34 and the higher abundance of PI(3)P in the presence of the tombusviral replication proteins, which likely leads to more efficient tombusvirus replication. Accordingly, overexpression of PI(3)P phosphatase in yeast or plants inhibited TBSV replication on the peroxisomal membranes and CIRV replication on the mitochondrial membranes. Moreover, the purified PI(3)P phosphatase reduced TBSV replicase assembly in a cell-free system. Detection of PI(3)P with antibody or a bioprobe revealed the enrichment of PI(3)P in the replication compartment. Vps34 is directly recruited into the replication compartment through interaction with p33 replication protein. Gene deletion analysis in surrogate yeast host unraveled that TBSV replication requires the vesicle transport function of Vps34. In the absence of Vps34, TBSV cannot efficiently recruit the Rab5-positive early endosomes, which provide PE-rich membranes for membrane biogenesis of the TBSV replication compartment. We found that Vps34 and PI(3)P needed for the stability of the p33 replication protein, which is degraded by the 26S proteasome when PI(3)P abundance was decreased by an inhibitor of Vps34. In summary, Vps34 and PI(3)P are needed for providing the optimal microenvironment for the replication of the peroxisomal TBSV and the mitochondrial CIRV.

]]>
<![CDATA[Third harmonic generation imaging and analysis of the effect of low gravity on the lacuno-canalicular network of mouse bone]]> https://www.researchpad.co/article/5c3667cbd5eed0c4841a6455

The lacuno-canalicular network (LCN) hosting the osteocytes in bone tissue represents a biological signature of the mechanotransduction activity in response to external biomechanical loading. Using third-harmonic generation (THG) microscopy with sub-micrometer resolution, we investigate the impact of microgravity on the 3D LCN structure in mice following space flight. A specific analytical procedure to extract the LCN characteristics from THG images is described for ex vivo studies of bone sections. The analysis conducted in different anatomical quadrants of femoral cortical bone didn’t reveal any statistical differences between the control, habitat control and flight groups, suggesting that the LCN connectivity is not affected by one month space flight. However, significant variations are systematically observed within each sample. We show that our current lack of understanding of the extent of the LCN heterogeneity at the organ level hinders the interpretation of such investigations based on a limited number of samples and we discuss the implications for future biomedical studies.

]]>
<![CDATA[Generating Vegfr3 reporter transgenic mouse expressing membrane-tagged Venus for visualization of VEGFR3 expression in vascular and lymphatic endothelial cells]]> https://www.researchpad.co/article/5c366797d5eed0c4841a5c74

Vascular endothelial growth factor receptor 3 (Vegfr3) has been widely used as a marker for lymphatic and vascular endothelial cells during mouse embryonic development and in adult mouse, making it valuable for studying angiogenesis and lymphangiogenesis under normal and pathological conditions. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a membrane-localized fluorescent reporter protein, Gap43-Venus, under the control of the Vegfr3 regulatory sequence. Vegfr3-Gap43-Venus BAC Tg recapitulated endogenous Vegfr3 expression in vascular and lymphatic endothelial cells during embryonic development and tumor development. Thus, this Tg mouse line contributes a valuable model to study angiogenesis and lymphangiogenesis in physiological and pathological contexts.

]]>
<![CDATA[Danger signals activate a putative innate immune system during regeneration in a filamentous fungus]]> https://www.researchpad.co/article/5c0ae430d5eed0c4845891aa

The ability to respond to injury is a biological process shared by organisms of different kingdoms that can even result in complete regeneration of a part or structure that was lost. Due to their immobility, multicellular fungi are prey to various predators and are therefore constantly exposed to mechanical damage. Nevertheless, our current knowledge of how fungi respond to injury is scarce. Here we show that activation of injury responses and hyphal regeneration in the filamentous fungus Trichoderma atroviride relies on the detection of two danger or alarm signals. As an early response to injury, we detected a transient increase in cytosolic free calcium ([Ca2+]c) that was promoted by extracellular ATP, and which is likely regulated by a mechanism of calcium-induced calcium-release. In addition, we demonstrate that the mitogen activated protein kinase Tmk1 plays a key role in hyphal regeneration. Calcium- and Tmk1-mediated signaling cascades activated major transcriptional changes early following injury, including induction of a set of regeneration associated genes related to cell signaling, stress responses, transcription regulation, ribosome biogenesis/translation, replication and DNA repair. Interestingly, we uncovered the activation of a putative fungal innate immune response, including the involvement of HET domain genes, known to participate in programmed cell death. Our work shows that fungi and animals share danger-signals, signaling cascades, and the activation of the expression of genes related to immunity after injury, which are likely the result of convergent evolution.

]]>
<![CDATA[Function and regulation of the Caenorhabditis elegans Rab32 family member GLO-1 in lysosome-related organelle biogenesis]]> https://www.researchpad.co/article/5bf3295ed5eed0c4843beab4

Cell type-specific modifications of conventional endosomal trafficking pathways lead to the formation of lysosome-related organelles (LROs). C. elegans gut granules are intestinally restricted LROs that coexist with conventional degradative lysosomes. The formation of gut granules requires the Rab32 family member GLO-1. We show that the loss of glo-1 leads to the mistrafficking of gut granule proteins but does not significantly alter conventional endolysosome biogenesis. GLO-3 directly binds to CCZ-1 and they both function to promote the gut granule association of GLO-1, strongly suggesting that together, GLO-3 and CCZ-1 activate GLO-1. We found that a point mutation in GLO-1 predicted to spontaneously activate, and function independently of it guanine nucleotide exchange factor (GEF), localizes to gut granules and partially restores gut granule protein localization in ccz-1(-) and glo-3(-) mutants. CCZ-1 forms a heterodimeric complex with SAND-1(MON1), which does not function in gut granule formation, to activate RAB-7 in trafficking pathways to conventional lysosomes. Therefore, our data suggest a model whereby the function of a Rab GEF can be altered by subunit exchange. glo-3(-) mutants, which retain low levels of GLO-3 activity, generate gut granules that lack GLO-1 and improperly accumulate RAB-7 in a SAND-1 dependent process. We show that GLO-1 and GLO-3 restrict the distribution of RAB-7 to conventional endolysosomes, providing insights into the segregation of pathways leading to conventional lysosomes and LROs.

]]>
<![CDATA[Role of critical elements in botulinum neurotoxin complex in toxin routing across intestinal and bronchial barriers]]> https://www.researchpad.co/article/5b4a1929463d7e428027f88f

The highly potent botulinum neurotoxin serotype A (BoNT/A) inhibits neurotransmitter release at neuromuscular junctions resulting in flaccid muscle paralysis, respiratory arrest and death. In order to reach their neuronal cell targets, BoNT/A must cross epithelial cell barriers lining the intestines and airways. The toxin is produced as a large protein complex comprised of the neurotoxin and non-toxic neurotoxin-associated proteins (NAPs). Although NAPs are known to protect the toxin from harsh environments, their role in the movement of BoNT/A across epithelial barriers has not been fully characterized. In the current study, movement of the toxin across epithelial cells was examined macroscopically using a sensitive near infrared fluorescence transcytosis assay and microscopically using fluorescently labeled toxin and confocal microscopy. The studies show that the BoNT/A complex internalizes more rapidly than the pure toxin. The studies also show that one NAP protein, hemaglutinin 33 (Hn33), enhanced both the binding and movement of a deactivated recombinant botulinum neurotoxin A (DrBoNT) across epithelial cell monolayers and that the toxin associates with Hn33 on the cell surface. Collectively, the data demonstrate that, in addition to their protective role, NAPs and Hn33 play an important role in BoNT/A intoxication.

]]>
<![CDATA[Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques]]> https://www.researchpad.co/article/5989d9e7ab0ee8fa60b6bb88

This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera’s collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response.

]]>
<![CDATA[Theranostic Properties of a Survivin-Directed Molecular Beacon in Human Melanoma Cells]]> https://www.researchpad.co/article/5989db00ab0ee8fa60bc654e

Survivin is an inhibitor of apoptosis overexpressed in different types of tumors and undetectable in most terminally differentiated normal tissues. In the current study, we sought to evaluate the in vitro theranostic properties of a molecular beacon-oligodeoxynucleotide (MB) that targets survivin mRNA. We used laser scanning confocal microscopy to study MB delivery in living cells and real-time PCR and western blot to assess selective survivin-targeting in human malignant melanoma cells. We further assess the pro-apoptotic effect of MB by measuring internucleosomal DNA fragmentation, dissipation of mitochondrial membrane potential (MMP) and changes in nuclear morphology. Transfection of MB into A375 and 501 Mel cells generated high signal intensity from the cytoplasm, while no signal was detected in the extracellular environment and in survivin-negative cells (i.e., human melanocytes and monocytes). MB time dependently decreased survivin mRNA and protein expression in melanoma cells with the maximum effect reached at 72 h. Treatment of melanoma cells with MB induced apoptosis by significant changes in MMP, accumulation of histone-complexed DNA fragments in the cytoplasm and nuclear condensation. MB also enhanced the pro-apoptotic effect of standard chemotherapeutic drugs tested at clinically relevant concentrations. The MB tested in the current study conjugates the ability of imaging with the pharmacological silencing activity against survivin mRNA in human melanoma cells and may represent an innovative approach for cancer diagnosis and treatment.

]]>
<![CDATA[ICln: A New Regulator of Non-Erythroid 4.1R Localisation and Function]]> https://www.researchpad.co/article/5989da3aab0ee8fa60b87bc3

To optimise the efficiency of cell machinery, cells can use the same protein (often called a hub protein) to participate in different cell functions by simply changing its target molecules. There are large data sets describing protein-protein interactions (“interactome”) but they frequently fail to consider the functional significance of the interactions themselves. We studied the interaction between two potential hub proteins, ICln and 4.1R (in the form of its two splicing variants 4.1R80 and 4.1R135), which are involved in such crucial cell functions as proliferation, RNA processing, cytoskeleton organisation and volume regulation. The sub-cellular localisation and role of native and chimeric 4.1R over-expressed proteins in human embryonic kidney (HEK) 293 cells were examined. ICln interacts with both 4.1R80 and 4.1R135 and its over-expression displaces 4.1R from the membrane regions, thus affecting 4.1R interaction with ß-actin. It was found that 4.1R80 and 4.1R135 are differently involved in regulating the swelling activated anion current (ICl,swell) upon hypotonic shock, a condition under which both isoforms are dislocated from the membrane region and thus contribute to ICl,swell current regulation. Both 4.1R isoforms are also differently involved in regulating cell morphology, and ICln counteracts their effects. The findings of this study confirm that 4.1R plays a role in cell volume regulation and cell morphology and indicate that ICln is a new negative regulator of 4.1R functions.

]]>
<![CDATA[Syndecan-1 in the Mouse Parietal Peritoneum Microcirculation in Inflammation]]> https://www.researchpad.co/article/5989dab0ab0ee8fa60bab0e3

Background

The heparan sulfate proteoglycan syndecan-1 (CD138) was shown to regulate inflammatory responses by binding chemokines and cytokines and interacting with adhesion molecules, thereby modulating leukocyte trafficking to tissues. The objectives of this study were to examine the expression of syndecan-1 and its role in leukocyte recruitment and chemokine presentation in the microcirculation underlying the parietal peritoneum.

Methods

Wild-type BALB/c and syndecan-1 null mice were stimulated with an intraperitoneal injection of Staphylococcus aureus LTA, Escherichia coli LPS or TNFα and the microcirculation of the parietal peritoneum was examined by intravital microscopy after 4 hours. Fluorescence confocal microscopy was used to examine syndecan-1 expression in the peritoneal microcirculation using fluorescent antibodies. Blocking antibodies to adhesion molecules were used to examine the role of these molecules in leukocyte-endothelial cell interactions in response to LTA. To determine whether syndecan-1 co-localizes with chemokines in vivo, fluorescent antibodies to syndecan-1 were co-injected intravenously with anti-MIP-2 (CXCL2), anti-KC (CXCL1) or anti-MCP-1 (CCL2).

Results and Conclusion

Syndecan-1 was localized to the subendothelial region of peritoneal venules and the mesothelial layer. Leukocyte rolling was significantly decreased with LPS treatment while LTA and TNFα significantly increased leukocyte adhesion compared with saline control. Leukocyte-endothelial cell interactions were not different in syndecan-1 null mice. Antibody blockade of β2 integrin (CD18), ICAM-1 (CD54) and VCAM-1 (CD106) did not decrease leukocyte adhesion in response to LTA challenge while blockade of P-selectin (CD62P) abrogated leukocyte rolling. Lastly, MIP-2 expression in the peritoneal venules was not dependent on syndecan-1 in vivo. Our data suggest that syndecan-1 is expressed in the parietal peritoneum microvasculature but does not regulate leukocyte recruitment and is not necessary for the presentation of the chemokine MIP-2 in this tissue.

]]>
<![CDATA[Early wound healing and refractive response of different pocket configurations following presbyopic inlay implantation]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdbd8a

Background

Presbyopic inlays have mostly been implanted under a corneal flap. Implantation in a pocket has advantages including less postoperative dry eye and neurotrophic effect, and better biomechanical corneal stability. This study investigated the effect of different pocket and flocket dimensions on corneal stability and refractive power after Raindrop™ implantation, and the associated wound healing response.

Methodology

Ten New Zealand White rabbits had bilateral pocket Raindrop™ implantation. Eyes were allocated to 4 groups: pockets with 4mm, 6mm, and 8mm diameters, and 8mm flocket. They were examined pre-operatively, at day 1, weeks 1, 2, 3 and 4 post-surgery with anterior segment optical coherence tomography, corneal topography and in-vivo confocal microscopy. After euthanasia (week 4), CD11b, heat shock protein (HSP) 47 and fibronectin corneal immunohistochemistry was performed.

Results

Corneal thickness (mean±SD) increased from 360.0±16.2μm pre-operatively to 383.9±32.5, 409.4±79.3, 393.6±35.2, 396.4±50.7 and 405±20.3μm on day 1, weeks 1,2,3 and 4 respectively (p<0.008, all time-points). Corneal refractive power increased by 11.1±5.5, 7.5±2.5, 7.5±3.1, 7.0±3.6 and 6.3±2.9D (p<0.001). Corneal astigmatism increased from 1.1±0.3D to 2.3±1.6, 1.7±0.7, 1.8±1.0, 1.6±0.9 and 1.6±0.9D respectively (p = 0.033). CT, refractive power change and astigmatism were not different between groups. The 8mm pocket and 8mm flocket groups had the least stromal keratocyte reflectivity. CD11b, fibronectin or HSP47 weren’t detected.

Conclusions

Anatomical and refractive stability was achieved by 1 week; the outcomes were not affected by pocket or flocket configuration. No scarring or inflammation was identified. The 8mm pocket and flocket showed the least keratocyte activation, suggesting they might be the preferred configuration.

]]>
<![CDATA[Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus]]> https://www.researchpad.co/article/5989dad2ab0ee8fa60bb67bf

Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs). The VRCs built by Tomato bushy stunt virus (TBSV) are enriched with phosphatidylethanolamine (PE) through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP)-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5–positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.

]]>