Polarized Cell Migration during Cell-to-Cell Transmission of Herpes Simplex Virus in Human Skin Keratinocytes

In addition to transmission involving extracellular free particles, a generally accepted model of virus propagation is one wherein virus replicates in one cell, producing infectious particles that transmit to the next cell via cell junctions or induced polarized contacts. This mechanism of spread is especially important in the presence of neutralizing antibody, and the concept underpins analysis of virus spread, plaque size, viral and host functions, and general mechanisms of virus propagation. Here, we demonstrate a novel process involved in cell-to-cell transmission of herpes simplex virus (HSV) in human skin cells that has not previously been appreciated. Using time-lapse microscopy of fluorescent viruses, we show that HSV infection induces the polarized migration of skin cells into the site of infection. In the presence of neutralizing antibody, uninfected skin cells migrate to the initial site of infection and spread over infected cells to become infected in a spatially confined cluster containing hundreds of cells. The cells in this cluster do not undergo cytocidal cell lysis but harbor abundant enveloped particles within cells and cell-free virus within interstitial regions below the cluster surface. Cells at the base and outside the cluster were generally negative for virus immediate-early expression. We further show, using spatially separated monolayer assays, that at least one component of this induced migration is the paracrine stimulation of a cytotactic response from infected cells to uninfected cells. The existence of this process changes our concept of virus transmission and the potential functions, virus, and host factors involved.     

CeO2 nanoparticles synthesized through green chemistry are biocompatible: In vitro and in vivo assessment

Widespread use of cerium oxide (CeO₂) nanoparticles (NPs) is found in almost all areas of research due to their distinctive properties. CeO₂ NPs synthesized via green chemistry have been characterized for antioxidant, phytochemical, and biological potential. Physical characterization through scanning electron microscopy, XRD, and TGA showed that the NPs are circular in shape, 20‐25 nm in size, and stable in a wide range of temperature. NPs display significant antioxidant (32.7% free radical scavenging activity) and antileishmanial (IC₅₀ 48 µg mL^?1) properties. In vitro toxicity tested against lymphocytes verified that NPs are biocompatible (99.38% viability of lymphocytes at 2.5 μg mL^?1). In vivo toxicity experiments showed no harmful effects on rat serum chemistry and histology of various organs and did not even change the concentration of antioxidative enzymes, total protein contents, lipid peroxidation, and nitrosative stress. These observations are in line with the statement that plant‐based synthesis of CeO₂ NPs lessens or nullifies in vitro and in vivo toxicity and hence CeO₂ NPs are regarded as a safe and biocompatible material to be used in drug delivery.     

The biological basis of degenerative disc disease: proteomic and biomechanical analysis of the canine intervertebral disc

IntroductionIn the present study, we sought to quantify and contrast the secretome and biomechanical properties of the non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP).MethodsWe used iTRAQ proteomic methods to quantify the secretome of both CD and NCD NP. Differential levels of proteins detected were further verified using immunohistochemistry, Western blotting, and proteoglycan extraction in order to evaluate the integrity of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan. Additionally, we used robotic biomechanical testing to evaluate the biomechanical properties of spinal motion segments from both CD and NCD canines.ResultsWe detected differential levels of decorin, biglycan, and fibronectin, as well as of other important extracellular matrix (ECM)-related proteins, such as fibromodulin and HAPLN1 in the IVD NP obtained from CD canines compared with NCD canines. The core proteins of the vital SLRPs decorin and biglycan were fragmented in CD NP but were intact in the NP of the NCD animals. CD and NCD vertebral motion segments demonstrated significant differences, with the CD segments having less stiffness and a more varied range of motion.ConclusionsThe CD NP recapitulates key elements of human degenerative disc disease. Our data suggest that at least some of the compromised biomechanical properties of the degenerative disc arise from fibrocartilaginous metaplasia of the NP secondary to fragmentation of SLRP core proteins and associated degenerative changes affecting the ECM. This study demonstrates that the degenerative changes that naturally occur within the CD NP make this animal a valuable animal model with which to study IVD degeneration and potential biological therapeutics.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0733-z) contains supplementary material, which is available to authorized users.     

Amniotic Fluid Derived Stem Cells with a Renal Progenitor Phenotype Inhibit Interstitial Fibrosis in Renal Ischemia and Reperfusion Injury in Rats

Objectives

Mesenchymal stem cells derived from human amniotic fluid (hAFSCs) are a promising source for cellular therapy, especially for renal disorders, as a subpopulation is derived from the fetal urinary tract. The purpose of this study was to evaluate if hAFSCs with a renal progenitor phenotype demonstrate a nephroprotective effect in acute ischemia reperfusion (I/R) model and prevent late stage fibrosis.

Methods

A total of 45 male 12-wk-old Wistar rats were divided into three equal groups;: rats subjected to I/R injury and treated with Chang Medium, rats subjected to I/R injury and treated with hAFSCs and sham-operated animals. In the first part of this study, hAFSCs that highly expressed CD24, CD117, SIX2 and PAX2 were isolated and characterized. In the second part, renal I/R injury was induced in male rats and cellular treatment was performed 6 hours later via arterial injection. Functional and histological analyses were performed 24 hours, 48 hours and 2 months after treatment using serum creatinine, urine protein to creatinine ratio, inflammatory and regeneration markers and histomorphometric analysis of the kidney. Statistical analysis was performed by analysis of variance followed by the Tukey’s test for multiple comparisons or by nonparametric Kruskal-Wallis followed by Dunn. Statistical significance level was defined as p <0.05.

Results

hAFSCs treatment resulted in significantly reduced serum creatinine level at 24 hours, less tubular necrosis, less hyaline cast formation, higher proliferation index, less inflammatory cell infiltration and less myofibroblasts at 48h. The treated group had less fibrosis and proteinuria at 2 months after injury.

Conclusion

hAFSCs contain a renal progenitor cell subpopulation that has a nephroprotective effect when delivered intra-arterially in rats with renal I/R injury, and reduces interstitial fibrosis on long term follow-up.     

Photoprotection Conferred by Changes in Photosynthetic Protein Levels and Organization during Dehydration of a Homoiochlorophyllous Resurrection Plant

During desiccation, homoiochlorophyllous resurrection plants retain most of their photosynthetic apparatus, allowing them to resume photosynthetic activity quickly upon water availability. These plants rely on various mechanisms to prevent the formation of reactive oxygen species and/or protect their tissues from the damage they inflict. In this work, we addressed the issue of how homoiochlorophyllous resurrection plants deal with the problem of excessive excitation/electron pressures during dehydration using Craterostigma pumilum as a model plant. To investigate the alterations in the supramolecular organization of photosynthetic protein complexes, we examined cryoimmobilized, freeze-fractured leaf tissues using (cryo)scanning electron microscopy. These examinations revealed rearrangements of photosystem II (PSII) complexes, including a lowered density during moderate dehydration, consistent with a lower level of PSII proteins, as shown by biochemical analyses. The latter also showed a considerable decrease in the level of cytochrome f early during dehydration, suggesting that initial regulation of the inhibition of electron transport is achieved via the cytochrome b₆f complex. Upon further dehydration, PSII complexes are observed to arrange into rows and semicrystalline arrays, which correlates with the significant accumulation of sucrose and the appearance of inverted hexagonal lipid phases within the membranes. As opposed to PSII and cytochrome f, the light-harvesting antenna complexes of PSII remain stable throughout the course of dehydration. Altogether, these results, along with photosynthetic activity measurements, suggest that the protection of retained photosynthetic components is achieved, at least in part, via the structural rearrangements of PSII and (likely) light-harvesting antenna complexes into a photochemically quenched state.Desiccation tolerance, the ability to survive absolute water contents down to approximately 0.1 g water g⁻¹ dry weight, is a trait found in some bacteria, algae, fungi, as well as animals and plants. In the plant kingdom, desiccation tolerance is common in ferns, mosses, and most seeds and pollen of flowering plants (angiosperms). Resurrection plants, a diverse group of approximately 300 angiosperm species, possess this trait also in their vegetative tissues. These plants are able to withstand prolonged periods of dehydration and to recover within hours to a few days once water is available. A major and interesting aspect in the study of desiccation tolerance in resurrection plants is how they protect themselves against oxidative damage during dehydration, which is often accompanied by conditions of high irradiance (for review, see Bartels and Hussain, 2011; Farrant and Moore, 2011; Morse et al., 2011).A decrease in water content quickly results in lowered leaf stomatal conductance and, consequently, decreased uptake of CO₂. This hinders and ultimately blocks the Calvin cycle. The light-driven reactions, however, typically continue well after the onset of water deficiency, with intact chlorophyll-protein complexes absorbing light energy. The imbalance between the light reactions and the downward biochemical pathways results in a lack of electron sinks and in the system becoming overenergized. This, in turn, leads to enhanced generation of reactive oxygen species (ROS), which inflict damage onto photosynthetic components as well as onto other chloroplast and cellular constituents. At times, the damage may be severe and lead to irreversible impairment and finally plant death (Dinakar et al., 2012).Resurrection plants minimize such potential ROS damage by shutting down photosynthesis during early stages of dehydration (Farrant, 2000; Farrant et al., 2007). There are two mechanisms whereby this is achieved. In poikilochlorophyllous resurrection plants, chlorophyll, along with photosynthetic protein complexes, are degraded, and thylakoids, the membranes that host the photosynthetic pigment-protein complexes, are dismantled. This straightforward mechanism prevents the formation of ROS, yet it comes at the cost of resynthesizing photosynthetic components de novo upon rehydration. On the other hand, homoiochlorophyllous species retain most of their photosynthetic complement and so must rely on other means to protect themselves from oxidative damage in the desiccated state. Some of these, such as leaf folding or curling, which minimize the exposure of inner leaves and/or of adaxial (upper) leaf surfaces to the light, and the accumulation of anthocyanins in leaf surfaces, which act as sunscreens, and the presence of reflective hairs and waxy cuticles, reduce the overall absorption of radiation and thus protect against photodamage (Sherwin and Farrant, 1998; Farrant, 2000; Bartels and Hussain, 2011; Morse et al., 2011). ROS that are generated are dealt with by antioxidants, ROS scavengers, and in some cases also by anthocyanins and other polyphenols (Moore et al., 2005; Kytridis and Manetas, 2006; Farrant et al., 2007). Nevertheless, all of these mechanisms are insufficient to completely prevent and/or detoxify all ROS that are formed, necessitating additional means to prevent or deal with possible damage that ROS may inflict during dehydration and while desiccated (Dinakar et al., 2012).The major photoprotective mechanism in plants and algae is nonphotochemical quenching (NPQ), in which excess light energy absorbed at the antennae of PSII is dissipated as heat. NPQ has been shown to be active in desiccation-tolerant bryophytes and pteridiophytes (Eickmeier et al., 1993; Oliver, 1996), in homoiochlorophyllous angiosperms (Alamillo and Bartels, 2001; Georgieva et al., 2009; Dinakar and Bartels, 2012; Huang et al., 2012), and during the initial stages of drying in poikilochlorophyllous angiosperms (Beckett et al., 2012). Photoinhibition, when damage to PSII (mainly to its D1 subunit) exceeds the repair capacity, typically under conditions of light stress, is also observed in homoiochlorophyllous resurrection plants (e.g. Georgieva and Maslenkova, 2006). Other ways to avoid ROS-induced damage include the rerouting of reducing equivalents to alternative electron sinks, such as the water-water cycle and/or photorespiration, as well as structural rearrangements of PSII and light-harvesting antenna (LHCII) complexes into energy-dissipating states (for review, see Dekker and Boekema, 2005; Yamamoto et al., 2014). These latter processes, in particular the ones pertaining to possible changes in PSII-LHCII macrostructure, have not yet been characterized in homoiochlorophyllous resurrection plants.To gain insight into the ways homoiochlorophyllous resurrection plants cope with dehydration while retaining most of their photosynthetic apparatus, we combined microscopic, spectroscopic, and biochemical approaches. Investigation of the supramolecular organization of photosynthetic complexes was carried out using cryoscanning electron microscopy (cryo-SEM) of high-pressure frozen, freeze-fractured leaf samples; to our knowledge, this combination of procedures has not been utilized previously to investigate thylakoid membranes within plant tissues.The studies reveal that during dehydration, the density of PSII in grana membranes gradually decreases. Notably, in the dehydrated state, in which photosynthetic activity is halted, PSII complexes are also observed to be arranged into rows and two-dimensional arrays. These arrangements are proposed to represent quenched PSII complexes that likely minimize the generation of ROS during desiccation. Furthermore, we observe inverted hexagonal (H_II) phases in this dry state, and these two structural rearrangements are correlated with the massive accumulation of Suc. Biochemical studies of thylakoid membrane fractions support the finding that the relative level of PSII proteins decreases during dehydration. These analyses also reveal that the level of the cytochrome f subunit of the cytochrome b₆f complex decreases quite dramatically and early during dehydration. This provides evidence for an additional level of regulation that inhibits/shuts down the photosynthetic light reactions during desiccation.     

Foliar Application of Phosphorus Enhances Photosynthesis and Biochemical Characteristics of Maize under Drought Stress

Water is essential for the growth period of crops; however, water unavailability badly affects the growth and physiological attributes of crops, which considerably reduced the yield and yield components in crops. Therefore, a pot experiment was conducted to investigate the effect of foliar phosphorus (P) on morphological, gas exchange, biochemical traits, and phosphorus use efficiency (PUE) of maize (Zea mays L.) hybrids grown under normal as well as water deficit situations at the Department of Agronomy, University of Agriculture Faisalabad, Pakistan in 2014. Two different treatments (control and P @ 8 kg ha⁻¹ ) and four hybrids (Hycorn, 31P41, 65625, and 32B33) of maize were tested by using a randomized complete block design (RCBD) with three replications. Results showed that the water stress caused a remarkable decline in total soluble protein (9.7%), photosynthetic rate (9.4%) and transpiration rate (13.4%), stomatal conductance (10.2%), and internal CO₂ rate (20.4%) comparative to well-watered control. An increase of 37.1%, 36.8%, and 24.5% were recorded for proline, total soluble sugar, and total free amino acid, respectively. However, foliar P application minimized the negative impact of drought by improving plant growth, physio-biochemical attributes, and PUE in maize plants under water stress conditions. Among the hybrids tested, the hybrid 6525 performed better both under stress and non-stress conditions. These outcomes confirmed that the exogenous application of P improved drought stress tolerance by modulating growth, physio-biochemical attributes, and PUE of maize hybrids.     

XRD studies of UV-irradiated chitin based polyurethane elastomers

Chitin based polyurethane (PU) elastomers constituted on 4,4´-diphenylmethane diisocyanate (MDI), poly(ε-caprolactone) (PCL) and extended with blends of chitin/1,4-butane diol were synthesized via two step polymerization technique. The synthesized samples were irradiated for 50, 100 and 200 h in an UV exposure chamber as such the spectral distribution of the light is good match for terrestrial solar radiation. The crystalline behavior of the irradiated PU samples were investigated by X-ray diffraction (XRD), differential scanning calorimetery (DSC) and dynamic mechanical thermal analysis (DMTA) techniques. The effect of irradiation time and chitin contents on crystallinity were studied and investigated. The maximum decrease in the crystalline behavior of samples after irradiation observed by XRD, DSC and tan δ peaks were found for the PU samples extended with lower contents of chitin (chitin/BDO; 0/100). In comparison with irradiation times the 200 h irradiation showed maximum change in the crystalline behavior.     

Characterization of Cylindrocarpon liriodendri Associated with Black Foot Disease of Grapevine in Iran

Eight Cylindrocarpon isolates recovered from the trunk bases of 10-year-old grapevines showing decline symptoms from two vineyards in Bavanat (Fars province, south-western Iran) were studied. Based on phenotypical characteristics, mating experiments and molecular data, they were identified as Cylindrocarpon liriodendri. Pathogenicity was confirmed with selected isolates inoculated into 8-month-old dormant rooted cuttings of grapevine rootstock cv. 110 Richter. This is the first report of C. liriodendri causing black foot disease of grapevines in Iran.     

Pro-oxidant Role of Heme Oxygenase in Mediating Glucose-induced Endothelial Cell Damage

Oxidative damage to the vascular endothelial cells may play a crucial role in mediating glucose-induced cellular dysfunction in chronic diabetic complications. The present study was aimed at elucidating the role of glucose-induced alteration of highly inducible heme oxygenase (HO) in mediating oxidative stress in the vascular endothelial cells. We have also investigated the interaction between HO and the nitric oxide (NO) system, and its possible role in alteration of other vasoactive factors.

Human umbilical vein endothelial cells (HUVECs) were exposed to low (5?mmol/l) and high (25?mmol/l) glucose levels. In order to determine the role of HO in endothelial dysfunction and to elucidate a possible interaction between the HO and NO systems, cells were exposed to HO inducer (hemin, 10?μmol/l), HO antagonist (SnPPIX, 10?μmol/l), and NO synthase blocker (l-NAME, 200?μmol/l) with or without NO donor (arginine, 1?mmol/l). mRNA expression of HO and NO isoforms was measured by real time RT-PCR. HO activity was measured by bilirubin production and cellular oxidative stress was assessed by 8-hydroxy-2′-deoxyguanosine (8-OHdG) and nitrotyrosine staining. We also determined the expression of vasoactive factors, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF).

In the endothelial cells, glucose caused upregulation of HO-1 expression and increased HO activity. A co-stimulatory relationship between HO and NO was observed. Increased HO activity also associated with oxidative DNA and protein damage in the endothelial cells. Furthermore, increased HO activity augmented mRNA expression of vasoactive factors, ET-1 and VEGF. These data suggest that HO by itself and via elaboration of other vasoactive factors may cause endothelial injury and functional alteration. These findings are of importance in the context of chronic diabetic complications.