Biocatalytic characterization of Aspergillus oryzae β-galactosidase immobilized on functionalized multi-walled carbon nanotubes

The objectives of this work were to immobilize commercial Aspergillus oryzae β-galactosidase on functionalized multi-walled carbon nanotubes (MWCNTs) using different treatments and to characterize the products. Treatments were performed with glutaraldehyde, ethylenediamine and a mixture of concentrated H₂SO₄:HNO₃. The MWCNTs and their derivatives were characterized by thermogravimetric analysis. The immobilized enzymes were evaluated using inactivation kinetics, operating conditions, that is pH and temperature, kinetic parameters and lactose hydrolysis reusability. Immobilization yield and efficiency were significantly higher for β-galactosidase immobilized on MWCNTs functionalized by the acid mixture (Ac-Gal-MWCNTs). These values were 97% and 82%, respectively, after 3?h of immobilization. The activity of the Ac-Gal-MWCNTs was maintained at ～51% of their initial activity after being stored for 90 days at 4?°C. The Ac-Gal-MWCNTs retained more than 90% of their initial activity up to the fourth recycle. As the acid functionalization was the most efficient method tested for immobilizing A. oryzae β-galactosidase on MWCNTs, this method shows promise for industrial applications.     

A complex species complex: The controversial role of ecology and biogeography in the evolutionary history of Syllis gracilis Grube, 1840 (Annelida,Syllidae)

The cryptic diversity in the polychaete Syllis gracilis Grube, 1840, in the Mediterranean Sea was examined with an integrative morpho-molecular approach. Individuals of S. gracilis were collected at eleven Mediterranean localities to provide an insight into the role of brackish environments in inducing cryptic speciation. The examination of morphological features combined with a molecular genetic analysis based on a partial sequence of the 16S rRNA gene highlighted discrepancies between morphological and molecular diversity. Morphological data allowed to identify a morphotype with short appendages occurring in coralline algae communities and another one with long appendages observed in brackish-water environments and Sabellaria reefs. Multivariate analyses showed that sampling localities were the greatest source of morphological divergence, suggesting that phenotypic plasticity may play a role in local adaptations of S. gracilis populations. Molecular data showed the occurrence of four divergent lineages not corresponding to morphological clusters. Different species delimitation tests gave conflicting results, retrieving, however, at least four separated entities. Some lineages occurred in sympatry and were equally distributed in marine and brackish-water environments, excluding a biogeographic or ecological explanation of the observed pattern and suggesting instead ancient separation between lineages and secondary contact. The co-occurrence of different lineages hindered the identification of the lineage corresponding to S. gracilis sensu stricto. The discrepancy between morphological and molecular diversity suggests that different environmental and biogeographic features may interact in a complex and unpredictable way in shaping diversity patterns. An integrative approach is needed to provide a satisfactory insight on evolutionary processes in marine invertebrates.     

Oviduct histochemistry and site of synthesis of a 29.7 kDa jelly coat glycoprotein in the anuran Lepidobatrachus laevis

The oviduct of the anuran Lepidobatrachus laevis contains three morphological regions, each of which contains a histochemically distinct luminal mucosa. In the pars recta, the most anterior portion of the oviduct, there are periodic acid-Schiff base (PAS)-positive simple glands and epithelia. In the pars convoluta, there are alcian blue-positive, combined alcian blue- and PAS-positive and PAS-positive gland types. The most posterior region, the pars uterina, contains alcian blue-positive and alcian blue-negative epithelial cells. Previous work has shown that solubilized egg jelly contains a major 29.7 kDa glycoprotein subunit that was detected in oviduct tissue extracts from the pars convoluta in the present study. Rabbit antisera to the 29.7 kDa egg jelly glycoprotein of L. laevis reacted with the major pars convoluta glycoprotein and there were no immunoreactive components in the pars uterina. The slight immunoreactivity detected at 29.0–37.0 kDa in pars recta extracts is not believed to be the jelly molecule, based on low immunoreactivity and subunit molecular weight measurements. We conclude that the synthesis of the 29.7 kDa egg jelly glycoprotein is restricted to the pars convoluta region of the oviduct.     

miR146a up-regulation is involved in small HA oligosaccharides-induced pro-inflammatory response in human chondrocytes

BackgroundSmall HA fragments are produced during cartilage degradation and their role seems to be preponderant during pathologies in which cartilage injury contribute to trigger and perpetuate the inflammatory mechanism.Several reports have increasingly shown that MicroRNAs (miRs), a small non-coding mRNAs are involved in the regulation of multiple biological processes, including cell proliferation and inflammation response in different pathologies, among them miR146a seems to be involved in inflammatory processes.MethodsStarting by these evidences we investigated the levels of miR146a and its correlation with inflammatory mediators in an experimental model of 6-mer HA-induced inflammatory response in human cultured chondrocytes.ResultsTreatment of chondrocytes with 6-mer HA showed up-regulation in inflammation parameters such as TLR-4, and CD44 receptors activation, IL-6, IL-1β and MMP-13 mRNA expression and proteins production, as well as NF-kB activation. We also revealed an up-regulation of miR146a. Transfection with a miR146a mimic or miR146a inhibitor produced the following effects: chondrocytes receiving miR146a mimic and then 6-mer HA significantly reduced inflammatory cytokines and MMP-13, while exposition of chondrocytes with miR146a inhibitor and then the 6-mer HA incremented the activity of damaging cytokines and MMP13. Expression of CD44 receptor was not affected by miR-146a treatments, while TLR-4 expression and NF-kB activation were modified.ConclusionsWe concluded that up-regulation of miR146a occurred in 6-mer HA-induced inflammation response may reduce the inflammatory cascade by modulating TLR-4 and NF-kB activation.General significanceThese results could be useful in develop new therapeutic strategies with the aim to reduce OA and RA incidence.     

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

Small conductance Ca²⁺-sensitive potassium (SK2) channels are voltage-independent, Ca²⁺-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca²⁺ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca²⁺ and Ca²⁺-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca²⁺ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.     

No Evidence for a Difference in Neuropsychological Profile among Carriers and Noncarriers of the FMR1 Premutation in Adults under the Age of 50

The 5′ untranslated region of the fragile X mental retardation gene, FMR1, contains a polymorphic CGG repeat. Expansions of this repeat are associated with a spectrum of disorders. Full mutation alleles, repeats ≥ 200, are associated with fragile X syndrome. Premutation alleles, repeats of ~55–199, are associated with a tremor-ataxia syndrome most commonly in older males and primary ovarian insufficiency in females. However, the neuropsychological impact of carrying a premutation allele is presently unclear in younger adults. In this study, we analyzed neuropsychological scores for 138 males and 506 females ascertained from the general population and from families with a history of fragile X syndrome. Subjects were age 18–50 years and had varying repeat lengths. Neuropsychological scores were obtained from measures of general intelligence, memory, and executive functioning, including attention. Principal component analysis followed by varimax rotation was used to create independent factors for analysis. These factors were modeled for males and females separately via a general linear model that accounted for correlation among related subjects. All models were adjusted for potential confounders, including age at testing, ethnicity, and household income. Among males, no repeat length associations were detected for any factor. Among females, only a significant association with repeat length and self-report attention (p < 0.01) was detected, with premutation carriers self-reporting significantly more attention-related problems compared to noncarriers. No significant interactions between repeat length and age were detected. Overall, these results indicate the lack of a global neuropsychological impact of carrying a premutation allele among adults under the age of 50.     

Conflicting human interests over the re-introduction of endangered wild dogs in South Africa

In South Africa, a plan was launched to manage separate sub-populations of endangered African wild dogs (Lycaon pictus) in several small, geographically isolated, conservation areas as a single meta-population. This intensive management approach involves the re-introduction of wild dogs into suitable conservation areas and periodic translocations among them. To assess the attitudes towards re-introduced wild dogs, we conducted a questionnaire survey of multiple stakeholders—local community members, private landowners and tourists—in and around Hluhluwe-iMfolozi Park (HiP), one of the meta-population conservation areas. Here, we document conflicting human interests over the re-introduced wild dogs. Tourists in HiP, on the one hand, expressed overwhelmingly positive opinions about wild dogs across personal details of the respondents, but especially after having seen free-ranging wild dogs. On the other hand, we found misconceptions and perceptions that were more negative among the rural population around HiP, again largely independent of personal details of the participants, although educated respondents voiced more favourable views of wild dogs. These negative attitudes were in particular due to perceived and real threats of livestock losses. In a follow-up questionnaire survey, we also discovered apparent shortcomings of a previous short-lived conservation education programme among the local communities adjacent to HiP. Consequently, the mitigation of the conflict between wild dogs and rural people requires an understanding of the conditions under which livestock predation occurs, the encouragement of practices that prevent such predation, and increasing local tolerance of co-existence with wild dogs through both economic and non-monetary incentive schemes as well as continued conservation education.