Palaeodistribution modelling and genetic evidence highlight differential post‐glacial range shifts of a rain forest conifer distributed across a latitudinal gradient

Aim We examine the range expansion/contraction dynamics during the last glacial cycle of the late‐successional tropical rain forest conifer Podocarpus elatus using a combination of modelling and molecular marker analyses. Specifically, we test whether distributional changes predicted by environmental niche modelling are in agreement with (1) the glacial maximum contractions inferred from the southern fossil record, and (2) population genetic‐based estimates of range disjunctions and demographic dynamics. In addition, we test whether northern and southern ranges are likely to have experienced similar expansion/contraction dynamics. Location Eastern Australian tropical and subtropical rain forests. Methods Environmental niche modelling was completed for three time periods during the last glacial cycle and was interpreted in light of the known palynology. We collected 109 samples from 32 populations across the entire range of P. elatus. Six microsatellite loci and Bayesian coalescence analysis were used to infer population expansion/contraction dynamics, and five sequenced loci (one plastid and four nuclear) were used to quantify genetic structure/diversity. Results Environmental niche modelling suggested that the northern and southern ranges of P. elatus experienced different expansion/contraction dynamics. In the northern range, the habitat suitable for P. elatus persisted in a small refugial area during the Last Glacial Maximum (LGM, 21 ka) and then expanded during the post‐glacial period. Conversely, in the south suitable habitat was widespread during the LGM but subsequently contracted. These differential dynamics were supported by Bayesian analyses of the population genetic data (northern dispersal) and are consistent with the greater genetic diversity in the south compared with the north. A contact zone between the two genetically divergent groups (corresponding to the Macleay Overlap Zone) was supported by environmental niche modelling and molecular analyses. Main conclusions The climatic fluctuations of the Quaternary have differentially impacted the northern and southern ranges of a broadly distributed rain forest tree in Australia. Recurrent contraction/expansion cycles contributed to the genetic distinction between northern and southern distributions of P. elatus. By combining molecular and environmental niche modelling evidence, this unique study undermines the general assumption that broadly distributed species respond in a uniform way to climate change.     

Frequency of the ΔF508 mutation in a sample of 175 Italian cystic fibrosis patients

Summary A sample of 175 Italian cystic fibrosis patients has been analysed for the presence of the ΔF508 mutation. The frequency of this mutation among 137 patients with pancreatic insufficiency is equal to 57%; in 23 patients with pancreatic sufficiency it is 26%. A high proportion of the unknown mutations is associated with the same rare haplotype found in association with ΔF508, suggesting that at least another mutation occurred on a chromosome characterized by the same haplotype.     

Reactivity of ferric Aplysia myoglobin towards anionic ligands in the acidic region: Proposal for a structural model

The equilibrium binding properties of ferric Aplysia myoglobin have been studied for a number of anionic ligands in the pH region from neutrality to ~4. For all the ligands studied, the intrinsic affinity of Aplysia metmyoglobin increases by more than one order of magnitude as the pH is lowered well below neutrality.The spectroscopic properties of the ligand-free and the ligand-bound molecules show a pH dependence with apparent pK values of 4.7 and 6.1, respectively.On the basis of temperature-jump experiments, a kinetic scheme has been proposed and rate constants have been measured for the binding of azide at pH 6 and pH 4.Kinetic and thermodynamic features match each other, suggesting that a single ionizing group is responsible for all the observed effects.By inspection of the three-dimensional structure, this group has been tentatively identified as the proximal imidazole. Protonation of the N_ε of proximal histidine would be associated to the rupture of the proximal bond, giving rise to the formation of a tetra-co-ordinate, ligand-free and penta-co-ordinate, ligand-bound molecule.     

Thermal aggregation of glycated bovine serum albumin

Aggregation and glycation processes in proteins have a particular interest in medicine fields and in food technology. Serum albumins are model proteins which are able to self-assembly in aggregates and also sensitive to a non-enzymatic glycation in cases of diabetes. In this work, we firstly reported a study on the glycation and oxidation effects on the structure of bovine serum albumin (BSA). The experimental approach is based on the study of conformational changes of BSA at secondary and tertiary structures by FTIR absorption and fluorescence spectroscopy, respectively. Secondly, we analysed the thermal aggregation process on BSA glycated with different glucose concentrations. Additional information on the aggregation kinetics are obtained by light scattering measurements. The results show that glycation process affects the native structure of BSA. Then, the partial unfolding of the tertiary structure which accompanies the aggregation process is similar both in native and glycated BSA. In particular, the formation of aggregates is progressively inhibited with growing concentration of glucose incubated with BSA. These results bring new insights on how aggregation process is affected by modification of BSA induced by glycation.     

A Permanent Automated Real-Time Passive Acoustic Monitoring System for Bottlenose Dolphin Conservation in the Mediterranean Sea

Within the framework of the EU Life+ project named LIFE09 NAT/IT/000190 ARION, a permanent automated real-time passive acoustic monitoring system for the improvement of the conservation status of the transient and resident population of bottlenose dolphin (Tursiops truncatus) has been implemented and installed in the Portofino Marine Protected Area (MPA), Ligurian Sea. The system is able to detect the simultaneous presence of dolphins and boats in the area and to give their position in real time. This information is used to prevent collisions by diffusing warning messages to all the categories involved (tourists, professional fishermen and so on). The system consists of two gps-synchronized acoustic units, based on a particular type of marine buoy (elastic beacon), deployed about 1 km off the Portofino headland. Each one is equipped with a four-hydrophone array and an onboard acquisition system which can record the typical social communication whistles emitted by the dolphins and the sound emitted by boat engines. Signals are pre-filtered, digitized and then broadcast to the ground station via wi-fi. The raw data are elaborated to get the direction of the acoustic target to each unit, and hence the position of dolphins and boats in real time by triangulation.     

Conservation genetics of an endangered rainforest tree (Fontainea oraria – Euphorbiaceae) and implications for closely related species

Four new eastern Australian Fontainea species have beenrecently described and all have a limited distribution. F.oraria is the rarest, being restricted to 10 adult individualswithin a single site in regrowth littoral rainforest. In order todevelop adequate management strategies, this study was aimed atsurveying the genetic variability remaining within the species by usingRAPD analysis. To assist with the correct interpretation of the results,a matching study was conducted on four populations of the closelyrelated F. australis. Similar amounts of within-populationgenetic diversity were recorded for both species. The RAPD-based studysuggested that adult plants are contributing unevenly to successivegenerations. RAPD analysis also recognised a close evolutionaryrelationship between F. oraria and F. australis.Sequencing of cpDNA (trnL-F) and nrDNA (ITS2) regions,confirmed recent divergence and possibly some historical reticulationbetween these two species and two other members of the genus. Ofparticular interest was the recognition that one of the F.australis populations (Limpinwood) represented a novel genotypiccombination in need of conservation attention. The implications of theRAPD and sequencing results are discussed in reference to theirinfluence upon the development of adequate conservation strategies forall important conservation units.     

Cardiac disease modeling using induced pluripotent stem cell-derived human cardiomyocytes

Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alterationhas been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes(CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells(i PSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since i PSC preserve the entire patients’ genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with i PSC.     

Use of Formalin-Fixed Paraffin-Embedded Samples for Gene Expression Studies in Breast Cancer Patients

To obtain gene expression profiles from samples collected in clinical trials, we conducted a pilot study to assess feasibility and estimate sample attrition rates when profiling formalin-fixed, paraffin-embedded specimens. Ten matched fresh-frozen and fixed breast cancer samples were profiled using the Illumina HT-12 and Ref-8 chips, respectively. The profiles obtained with Ref 8, were neither technically nor biologically reliable since they failed to yield the expected separation between estrogen receptor positive and negative samples. With the use of Affymetrix HG-U133 2.0 Plus chips on fixed samples and a quantitative polymerase chain reaction -based sample pre-assessment step, results were satisfactory in terms of biological reliability, despite the low number of present calls (M = 21%±5). Compared with the Illumina DASL WG platform, Affymetrix data showed a wider interquartile range (1.32 vs 0.57, P<2.2 E-16,) and larger fold changes. The Affymetrix chips were used to run a pilot study on 60 fixed breast cancers. By including in the workflow the sample pre-assessment steps, 96% of the samples predicted to give good results (44/46), were in fact rated as satisfactory from the point of view of technical and biological meaningfulness. Our gene expression profiles showed strong agreement with immunohistochemistry data, were able to reproduce breast cancer molecular subtypes, and allowed the validation of an estrogen receptor status classifier derived in frozen samples. The approach is therefore suitable to profile formalin-fixed paraffin-embedded samples collected in clinical trials, provided that quality controls are run both before (sample pre-assessment) and after hybridization on the array.     

Cerium Oxide Nanoparticles Reduce Microglial Activation and Neurodegenerative Events in Light Damaged Retina

The first target of any therapy for retinal neurodegeneration is to slow down the progression of the disease and to maintain visual function. Cerium oxide or ceria nanoparticles reduce oxidative stress, which is known to play a pivotal role in neurodegeneration. Our aim was to investigate whether cerium oxide nanoparticles were able to mitigate neurodegeneration including microglial activation and related inflammatory processes induced by exposure to high intensity light. Cerium oxide nanoparticles were injected intravitreally or intraveinously in albino Sprague-Dawley rats three weeks before exposing them to light damage of 1000 lux for 24 h. Electroretinographic recordings were performed a week after light damage. The progression of retinal degeneration was evaluated by measuring outer nuclear layer thickness and TUNEL staining to quantify photoreceptors death. Immunohistochemical analysis was used to evaluate retinal stress, neuroinflammatory cytokines and microglial activation. Only intravitreally injected ceria nanoparticles were detected at the level of photoreceptor outer segments 3 weeks after the light damage and electoretinographic recordings showed that ceria nanoparticles maintained visual response. Moreover, this treatment reduced neuronal death and “hot spot” extension preserving the outer nuclear layer morphology. It is noteworthy that in this work we demonstrated, for the first time, the ability of ceria nanoparticles to reduce microglial activation and their migration toward outer nuclear layer. All these evidences support ceria nanoparticles as a powerful therapeutic agent in retinal neurodegenerative processes.