Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli

The expression of the recombinant diphtheria toxin mutant CRM197 in bacteria other than Corynebacterium diphtheriae has proven to be difficult. Here we propose a new and alternative procedure for the production of full-length CRM197 in Escherichia coli. The present study relates specifically to the expression of an artificial sequence and to a method for the isolation and purification of the corresponding protein. In particular, a synthetic gene coding for CRM197, bearing a short histidine tag and optimized for E. coli codon usage, was cloned in the pET9a vector. Accordingly, the over-expression of the protein was simply induced with arabinose in E. coli BL21AI. The recombinant protein was insoluble and always found inside protein aggregates, which were solubilised using urea. Surprisingly, the expression of CRM197, devoid of the short tag, always failed. Following a refolding step, the his-tagged CRM197 was purified by affinity and gel-filtration chromatography and the purity of the final preparation reached 95%. Interestingly, the recombinant protein features DNase activity, indicating that the presence of the tag is not affecting its biochemical properties. However, the removal of the synthetic tag could be easily obtained by incubating the target protein with a proper quantity of a commercial enterokinase.     

A serum fucose-binding lectin (DlFBL) from adult Dicentrarchus labrax is expressed in larva and juvenile tissues and contained in eggs

The purification, cloning, sequencing, molecular properties and expression of a fucose-binding lectin from the serum of Dicentrarchus labrax (DlFBL) have been previously reported. We now describe the distribution and expression of DlFBL during fish ontogeny. Immunohistochemistry and in situ hybridization assays were carried out at various developmental stages (from 10 days post-hatching larvae to juveniles). Another fucose-binding lectin, similar to DlFBL in biochemical, immunochemical and agglutinating properties, was extracted and purified from eggs and appeared to be localized in the embryo yolk sack residual. DlFBL was found in columnar and goblet cells of the intestinal epithelium of larvae (from 20 days post-hatching) and juveniles and in parenchymal tissue of juveniles. DlFBL mRNA and protein were detected in the intestinal epithelium and in hepatocytes. An amplification product from degenerate primers indicates that lectin isotypes with DlFBL epitopes are expressed in eggs and embryos. Whether the lectin fraction isolated from eggs and embryos includes DlFBL of maternal origin remains unclear.     

Models of climate associations and distributions of amphibians in Italy

Analyzing the relationships between the distribution of animal species and climatic variables is not only important for understanding which factors govern species distribution but also for improving our ability to predict future ecological responses to climate change. In the context of global climate change, amphibians are of particular interest because of their extreme sensitivity to the variation of temperature and precipitation regimes. We analyzed species–climate relationships for 17 amphibian species occurring in Italy using species distribution data at the 10 × 10 km resolution. A machine learning method, Random Forests, was used to model the distribution of amphibians in relation to a set of 18 climatic variables. The results showed that the variables which had the highest importance were those related to precipitation, indicating that precipitation is an important factor in determining amphibian distribution. Future projections showed a complex response of species distributions, emphasizing the potential severity of climate change on the distributions of amphibians in Italy. The species that will decrease the most are those occurring in mountainous and Mediterranean areas. Our results provide some preliminary information that could be useful for amphibian conservation, indicating if future conservation priorities for some species should be enhanced.     

Pinus sylvestris forest regeneration under different post-fire restoration practices in the northwestern Italian Alps

It is frequently believed that a post-fire environment requires immediate actions in order to be restored. Salvage logging followed by plantation is a common post-fire restoration practice in many forests of the northwestern Italian Alps.The objectives of this study were to assess the impact of active and passive management techniques on the restoration of a burned area of the Aosta Valley and to determine which approach is the most suitable for enhancing Pinus sylvestris regeneration after stand replacing wildfires.The influence of five management options (no intervention; salvage logging; broadleaves plantation; Larix decidua plantation; P. sylvestris or Pseudotsuga menziesii plantation) and environmental variables on natural regeneration structure and composition was evaluated through direct gradient analysis.Pinus sylvestris and Populus tremula were the dominant tree species (40 and 29%, respectively) in the regeneration layer. Density, size, and structural diversity of natural regeneration were higher in the no intervention area. The proximity to forest edge was found to be the most important environmental variable.This study provided evidence that taking advantage of natural restoration processes may be a suitable alternative strategy to the active restoration practices adopted according to the Aosta Valley policy of post-fire management.     

The effect of developmental stage on eggshell thickness variation in endangered falcons

We compared eggshell thickness of hatched eggs with that of non-developed eggs in endangered falcon taxa to explore the effect of embryo development on eggshell thinning. To our knowledge, this has never been examined before in falcons, despite the fact that eggshell thinning due to pollutants and environmental contamination is often considered the most common cause of egg failure in falcons. Because of the endangered nature of these birds, and the difficulty in gaining access to the nests and their eggs, there is a large gap in our knowledge regarding eggshell thickness variation and the factors affecting it. We used a linear mixed-effects (LME) model to explore the variation in eggshell thickness (n = 335 eggs) in relation to the developmental stage of the eggs, but also in relation to the falcon taxa, the laying sequence and the study zone. Female identity (n = 69) and clutch identity (n = 98) were also included in the LME model. Our results are consistent with the prediction that eggshell thickness decreases during incubation because of the important effect of calcium uptake by the embryo during development. Our results also show that eggs laid later in the sequence had significantly thinner eggshells. In this study, we provide the first quantitative data on eggshell thickness variation of hatched eggs in different falcon taxa that were not subjected to contamination or food limitation (i.e., bred under captive conditions). Because eggshell thickness strongly influences survival and because the species examined in this study are endangered, our data represent a valuable control for future studies on the effects of pollution on eggshells from wild populations and thus are an important contribution to the conservation of falcons.     

The identification of a series of novel, soluble non-peptidic neuropeptide Y Y2 receptor antagonists

The identification and subsequent optimisation of a selective non-peptidic NPY Y2 antagonist series is described. This led to the development of amine 2, a selective, soluble NPY Y2 receptor antagonist with enhanced CNS exposure.     

Monitoring liver alterations during hepatic tumorigenesis by NMR profiling and pattern recognition

Human hepatocellular carcinoma (HCC) is the most recurrent malignancy of the liver and represents one of the main causes of cancer death worldwide. Furthermore, the liver is the most frequent site of metastatic colonization, and hepatic metastases are far more common than primary cancers in Western countries. A possible way of investigating liver diseases is to study the tissue metabolic profiles. High-resolution nuclear magnetic resonance (NMR) spectroscopy of hepatic tissue extracts was combined with pattern-recognition and visualization techniques to uncover metabolic differences among analyzed tissue types. Extracts were from primary HCC, chronic hepatitis-C-virus related cirrhotic tissues, hepatic metastases from colorectal carcinomas, and non-cirrhotic normal liver tissues adjacent to metastases as controls. We identified all metabolites present in the NMR spectra, and after statistical evaluation of all spectral classes, we were able to define the metabolic changes underlying the different liver conditions and diseases. In particular, the lactate and the glucose tissue signals were found to primarily discriminate the different histological samples. We followed the biochemical changes of human hepatic lesions through primary (HCC) and secondary (metastases from colorectal carcinoma) liver tumors, cirrhotic tissues, and non-cirrhotic histologically-confirmed normal liver tissues adjacent to metastases, achieving a metabolic differentiation of the various pathological states based upon the variation of the intracellular lactate/glucose ratio. It is suggested that such a signal pattern may act as a potential marker for assessing pathological hepatic lesions.     

Mutations in the Small GTPase Gene RAB39B Are Responsible for X-linked Mental Retardation Associated with Autism, Epilepsy, and Macrocephaly

Human Mental Retardation (MR) is a common and highly heterogeneous pediatric disorder affecting around 3% of the general population; at least 215 X-linked MR (XLMR) conditions have been described, and mutations have been identified in 83 different genes, encoding proteins with a variety of function, such as chromatin remodeling, synaptic function, and intracellular trafficking. The small GTPases of the RAB family, which play an essential role in intracellular vesicular trafficking, have been shown to be involved in MR. We report here the identification of mutations in the small GTPase RAB39B gene in two male patients. One mutation in family X (D-23) introduced a stop codon seven amino acids after the start codon (c.21C > A; p.Y7X). A second mutation, in the MRX72 family, altered the 5′ splice site (c.215+1G > A) and normal splicing. Neither instance produced a protein. Mutations segregate with the disease in the families, and in some family members intellectual disabilities were associated with autism spectrum disorder, epileptic seizures, and macrocephaly. We show that RAB39B, a novel RAB GTPase of unknown function, is a neuronal-specific protein that is localized to the Golgi compartment. Its downregulation leads to an alteration in the number and morphology of neurite growth cones and a significant reduction in presynaptic buttons, suggesting that RAB39B is required for synapse formation and maintenance. Our results demonstrate developmental and functional neuronal alteration as a consequence of downregulation of RAB39B and emphasize the critical role of vesicular trafficking in the development of neurons and human intellectual abilities.     

Osmotic loading of spherical gels: a biomimetic study of hindered transport in the cell protoplasm

Osmotic loading of cells has been used to investigate their physicochemical properties as well as their biosynthetic activities. The classical Kedem-Katchalsky framework for analyzing cell response to osmotic loading, which models the cell as a fluid-filled membrane, does not generally account for the possibility of partial volume recovery in response to loading with a permeating osmolyte, as observed in some experiments. The cell may be more accurately represented as a hydrated gel surrounded by a semi-permeable membrane, with the gel and membrane potentially exhibiting different properties. To help assess whether this more elaborate model of the cell is justified, this study investigates the response of spherical gels to osmotic loading, both from experiments and theory. The spherical gel is described using the framework of mixture theory. In the experimental component of the study alginate is used as the model gel, and is osmotically loaded with dextran solutions of various concentrations and molecular weight, to verify the predictions from the theoretical analysis. Results show that the mixture framework can accurately predict the transient and equilibrium response of alginate gels to osmotic loading with dextran solutions. It is found that the partition coefficient of dextran in alginate regulates the equilibrium volume response and can explain partial volume recovery based on passive transport mechanisms. The validation of this theoretical framework facilitates future investigations of the role of the protoplasm in the response of cells to osmotic loading.     

The insulin-specific T cells of nonobese diabetic mice recognize a weak MHC-binding segment in more than one form

Several naturally occurring anti-insulin CD4 T cells were isolated from islet infiltrates of NOD mice. In accordance with the results of others, these T cells recognized the segment of the beta-chain from residues 9-23. Peptides encompassing the B:(9-23) sequence bound weakly to I-Ag7 in two main contiguous registers in which two residues at the carboxyl end, P20Gly and P21Glu, influenced binding and T cell reactivity. Naturally occurring insulin-reactive T cells exhibited differing reactivities with the carboxyl-terminal amino acids, although various single residue changes in either the flanks or the core segments affected T cell responses. The insulin peptides represent another example of a weak MHC-binding ligand that is highly immunogenic, giving rise to distinct populations of autoimmune T cells.