The shift from aquatic to terrestrial phenotype in Lissotriton italicus: larval and adult remodelling of the skin

Morphology and ultrastructure of the skin of Lissotriton italicus (previously named Triturus italicus) have been described in different phases of its biological cycle: larval stage, metamorphic stage and adult stage with emphasis on modifications occurring between aquatic and terrestrial adults. In the present study, light microscopy and both scanning and transmission electron microscopy were employed to analyze the histological and cytological remodelling that occurs in the skin of L. italicus during metamorphosis. The ultrastructure of the larval epidermis is arranged into three principal layers comprising an external layer of pavement cells, a basal layer and 1-3 intermediate layers consisting of Leydig cells along with accessory cells and mitochondria-rich cells. By the onset of metamorphosis, morphological changes of the skin include stratification and flattening of epidermal layers and disappearance of typical larval cells. In both aquatic and terrestrial adult phases the thin, cornified epidermis shows the same general arrangement as found in other vertebrates with an external stratum corneum and a variable number of intermediate cell layers. During the terrestrial adult phase, the skin is characterized by the presence of numerous tubercles; moreover, the lower epithelium is thicker than in the aquatic phase. Ultrastructural analysis revealed no substantial differences in the cellular composition of the skin between aquatic and terrestrial phases.     

The second receptor binding site of the globular head of the Newcastle disease virus hemagglutinin-neuraminidase activates the stalk of multiple paramyxovirus receptor binding proteins to trigger fusion

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three distinct activities contributing to the ability of HN to promote viral fusion and entry: receptor binding, receptor cleavage (neuraminidase), and activation of the fusion protein. The relationship between receptor binding and fusion triggering functions of HN are not fully understood. For Newcastle disease virus (NDV), one bifunctional site (site I) on HN's globular head can mediate both receptor binding and neuraminidase activities, and a second site (site II) in the globular head is also capable of mediating receptor binding. The receptor analog, zanamivir, blocks receptor binding and cleavage activities of NDV HN's site I while activating receptor binding by site II. Comparison of chimeric proteins in which the globular head of NDV HN is connected to the stalk region of either human parainfluenza virus type 3 (HPIV3) or Nipah virus receptor binding proteins indicates that receptor binding to NDV HN site II not only can activate its own fusion (F) protein but can also activate the heterotypic fusion proteins. We suggest a general model for paramyxovirus fusion activation in which receptor engagement at site II plays an active role in F activation.     

Four amino acids of an insect densovirus capsid determine midgut tropism and virulence

Densoviruses are insect parvoviruses that are orally infectious for Lepidoptera. To assess the mechanisms underlying their specificity and their virulence, we investigated the role of eight candidate residues in the densovirus capsid. We showed that the substitutions of four amino acids were associated with decreased virulence due to a decreased ability to cross the host midgut epithelium, without an effect on viral replication in other tissues.     

Enzymatic decolorization of spent textile dyeing baths composed by mixtures of synthetic dyes and additives

The effects of different components of real dyeing bath formulations, such as the equalizing and fixing additives-acids, salts, and surfactants-on the decolorization catalyzed by Funalia trogii enzymatic extracts, were investigated to understand their influence on the recalcitrance to biodegradation of this type of wastewater. The decolorization of selected dyes and dye mixtures after tissue dyeing was performed in the presence/absence of auxiliary compounds. All spent dyeing baths were enzymatically decolorized to different extents, by the addition of extracts containing laccase only or laccase plus cellobiose dehydrogenase. Whereas surfactant auxiliaries, in some instances, inhibit the decolorization of spent dyeing baths, in several occurrences the acid/salt additives favor the enzymatic process. In general, the complete spent dyeing formulations are better degraded than those containing the dyes only. The comparison of extracellular extracts obtained from spent straws from the commercial growth of Pleurotus sp. mushrooms with those from F. trogii reveals similar decolorization extents thus allowing to further reduce the costs of bioremediation.     

Ascorbic acid metabolism during bilberry (Vaccinium myrtillus L.) fruit development

Bilberry (Vaccinium myrtillus L.) possesses a high antioxidant capacity in berries due to the presence of anthocyanins and ascorbic acid (AsA). Accumulation of AsA and the expression of the genes encoding the enzymes of the main AsA biosynthetic route and of the ascorbate-glutathione cycle, as well as the activities of the enzymes involved in AsA oxidation and recycling were investigated for the first time during the development and ripening of bilberry fruit. The results showed that the AsA level remained relatively stable during fruit maturation. The expression of the genes encoding the key enzymes in the AsA main biosynthetic route showed consistent trends with each other as well as with AsA levels, especially during the first stages of fruit ripening. The expression of genes and activities of the enzyme involved in the AsA oxidation and recycling route showed more prominent developmental stage-dependent changes during the ripening process. Different patterns of activity were found among the studied enzymes and the results were, for some enzymes, in accordance with AsA levels. In fully ripe berries, both AsA content and gene expression were significantly higher in skin than in pulp.     

A screening assay for neuraminidase inhibitors using neuraminidases N1 and N3 from a baculovirus expression system

Context: Development of inexpensive and safe enzymatic assays to screen for putative neuraminidase inhibitors. Objective: Validate the use of recombinant neuraminidase expressed in baculovirus located on the viral surface capsule to develop a neuraminidase inhibitor screening assay. Materials and methods: Recombinant baculovirus particles displaying neuraminidase N1 and N3 were used as enzyme sources. The assay set-up required the use of 2'-(4-methylumbelliferyl)-α-D-acetyl neuraminic acid as substrate and oseltamivir carboxylate as benchmark inhibitor. Results: The assay was set up in a standard 96-well plate. The within- and between-assay coefficients of variation were, on average, less than 10%. The 50% inhibitory concentration values of the inhibitor were in good agreement with those determined by independent kinetic experiments. Discussion and conclusions: The assay showed satisfactory within- and between-assay repeatability. The obtained results suggest that recombinant baculovirus expressing neuraminidase located on the virus membrane capsule can be used to set up affordable and reliable neuraminidase inhibitors screening assays.     

Determining resistance to Pseudomonas syringae in tomato, a comparison of different molecular markers

Pseudomonas syringae pv. tomato (Pst) is the causal agent of bacterial speck disease in tomato. Resistance to Pst is determined by Pto, a single resistance gene that belongs to a multi-gene family clustered on chromosome 5. Pst-resistant phenotypes in cultivated tomato are determined by a semi-dominant allele of Solanum pimpinellifolium, which was introgressed into Solanum lycopersicum in the past century. Seed companies, which are continuously interested in producing resistant varieties, can benefit from genetic markers closely linked to the Pto locus in breeding programs based on marker-assisted selection. In this research, three sequence characterised amplified region markers have been developed for identification of resistant and susceptible genotypes of Solanum lycopersicum. A cleaved amplified polymorphic sequence marker has been adapted to a real-time polymerase chain reaction platform using high-resolution melting curve analysis. Application to a tomato population for breeding programs is described. Advantages and disadvantages of the different markers are discussed.     

Botulinum neurotoxin a impairs neurotransmission following retrograde transynaptic transport

The widely used botulinum neurotoxin A (BoNT/A) blocks neurotransmission via cleavage of the synaptic protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Recent evidence demonstrating long-distance propagation of SNAP-25 proteolysis has challenged the idea that BoNT/A remains localized to the injection site. However, the extent to which distant neuronal networks are impacted by BoNT/A retrograde trafficking remains unknown. Importantly, no studies have addressed whether SNAP-25 cleavage translates into structural and functional changes in distant intoxicated synapses. Here we show that the BoNT/A injections into the adult rat optic tectum result in SNAP-25 cleavage in retinal neurons two synapses away from the injection site, such as rod bipolar cells and photoreceptors. Retinal endings displaying cleaved SNAP-25 were enlarged and contained an abnormally high number of synaptic vesicles, indicating impaired exocytosis. Tectal injection of BoNT/A in rat pups resulted in appearance of truncated-SNAP-25 in cholinergic amacrine cells. Functional imaging with calcium indicators showed a clear reduction in cholinergic-driven wave activity, demonstrating impairments in neurotransmission. These data provide the first evidence for functional effects of the retrograde trafficking of BoNT/A, and open the possibility of using BoNT/A fragments as drug delivery vehicles targeting the central nervous system.     

International cohort study of 73 anti-Ku-positive patients: association of p70/p80 anti-Ku antibodies with joint/bone features and differentiation of disease populations by using principal-components analysis

Introduction

An international cohort study of 73 anti-Ku-positive patients with different connective tissue diseases was conducted to differentiate the anti-Ku-positive populations of patients based on their autoantibody profile and clinical signs/symptoms and to establish possible correlations between antibodies against Ku p70 and Ku p80 with autoimmune diseases.

Methods

Sera of anti-Ku-positive patients were collected from six European centers and were all secondarily tested (in the reference center); 73 were confirmed as positive. Anti-Ku antibodies were detected with counter-immunoelectrophoresis (CIE), line immunoassay (LIA), and immunoblot analyses. All clinical and laboratory data were follow-up cumulative data, except for anti-Ku antibodies. Statistical analyses were performed by using R (V 2.12.1). The Fisher Exact test was used to evaluate the association between anti-Ku antibodies and diagnosis, gender, clinical signs, and other observed antibodies. The P values were adjusted for multiple testing. Separation of disease populations based on the presence of antibodies and clinical signs was investigated by principal-components analysis, which was performed by using thr// R's prcomp function with standard parameters.

Results

A 16% higher prevalence of anti-Ku p70 was found over anti-Ku p80 antibodies. In 41 (57%) patients, a combination of both was detected. Five (7%) patients, who were CIE and/or LIA anti-Ku positive, were negative for both subsets, as detected with the immunoblot; 31% of the patients had undifferentiated connective tissue disease (UCTD); 29% had systemic sclerosis (SSc); 18% had systemic lupus erythematosus (SLE); 11% had rheumatoid arthritis; 7% had polymyositis; and 3% had Sjögren syndrome.

Conclusions

A significant positive association was found between female patients with anti-Ku p70 and joint/bone features, and a significant negative association was found between female patients with anti-Ku p80 only and joint/bone features (P = 0.05, respectively). By using the first and the third components of the principal-component analysis (PCA) with 29 parameters evaluated, we observed that the anti-Ku-positive population of UCTD patients had overlapping parameters, especially with SLE, as opposed to SSc, which could be helpful in delineating UCTD patients.     

Role-Shifting PKCζ Fosters Its Own Proapoptotic Destruction by Complexing with Bcl10 at the Nuclear Envelope of Human Cervical Carcinoma Cells: A Proteomic and Biochemical Study

Many features of deadly human cervical cancers (HCCs) still require elucidation. Among HCC-derived cell lines, here we used the C4-I one since its quantitative gene expression pattern most closely mimics invasive HCCs, including protein kinase-Cζ (PKCζ) overexpression. Via proteomic, bioinformatic, and biochemical approaches we identified 31 and 33 proteins co-immunoprecipitating with PKCζ from nuclear membranes (NMs) of, respectively, untreated or VP-16-exposed C4-I cells. Such proteins belonged to eight functional groups, whose compositions and relative sizes changed with either context. Of the 56 proteins identified, only eight were shared between the two subproteomes, including Bcl10. Surprisingly, proteins known to associate with Bcl10, like Carma1/3 and Malt1, in so-called CBM signalosomes were absent. Notably, in VP-16-treated C4-I cells, PKCζ?Bcl10 complexes increasingly accrued at NMs, where PKCζ phosphorylated Bcl10, as PKCζ also did in vitro and in cell-free systems, both processes being thwarted by interfering RNA (iRNA) PKCζ depletion. Caspase-3 was associated with PKCζ?Bcl10 complexes and proteolyzed PKCζ leading to its inactivation/destruction; both events were prevented by Bcl10 iRNA suppression. Thus, PKCζ's molecular interactions and functional roles changed strikingly according to the untreated or apoptogen-treated cells context, and by complexing with Bcl10, PKCζ surprisingly favored its own demise, which suggests both proteins as HCCs therapeutic targets.