Comparative analysis of different biofactories for the production of a major diabetes autoantigen

The 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major diabetes autoantigen that can be used for the diagnosis and (more recently) the treatment of autoimmune diabetes. We previously reported that a catalytically-inactive version (hGAD65mut) accumulated to tenfold higher levels than its active counterpart in transgenic tobacco plants, providing a safe and less expensive source of the protein compared to mammalian production platforms. Here we show that hGAD65mut is also produced at higher levels than hGAD65 by transient expression in Nicotiana benthamiana (using either the pK7WG2 or MagnICON vectors), in insect cells using baculovirus vectors, and in bacterial cells using an inducible-expression system, although the latter system is unsuitable because hGAD65mut accumulates within inclusion bodies. The most productive of these platforms was the MagnICON system, which achieved yields of 78.8 μg/g fresh leaf weight (FLW) but this was substantially less than the best-performing elite transgenic tobacco plants, which reached 114.3 μg/g FLW after six generations of self-crossing. The transgenic system was found to be the most productive and cost-effective although the breeding process took 3 years to complete. The MagnICON system was less productive overall, but generated large amounts of protein in a few days. Both plant-based systems were therefore advantageous over the baculovirus-based production platform in our hands.     

In-Vivo Real-Time Control of Protein Expression from Endogenous and Synthetic Gene Networks

We describe an innovative experimental and computational approach to control the expression of a protein in a population of yeast cells. We designed a simple control algorithm to automatically regulate the administration of inducer molecules to the cells by comparing the actual protein expression level in the cell population with the desired expression level. We then built an automated platform based on a microfluidic device, a time-lapse microscopy apparatus, and a set of motorized syringes, all controlled by a computer. We tested the platform to force yeast cells to express a desired fixed, or time-varying, amount of a reporter protein over thousands of minutes. The computer automatically switched the type of sugar administered to the cells, its concentration and its duration, according to the control algorithm. Our approach can be used to control expression of any protein, fused to a fluorescent reporter, provided that an external molecule known to (indirectly) affect its promoter activity is available.     

Clique of Functional Hubs Orchestrates Population Bursts in Developmentally Regulated Neural Networks

It has recently been discovered that single neuron stimulation can impact network dynamics in immature and adult neuronal circuits. Here we report a novel mechanism which can explain in neuronal circuits, at an early stage of development, the peculiar role played by a few specific neurons in promoting/arresting the population activity. For this purpose, we consider a standard neuronal network model, with short-term synaptic plasticity, whose population activity is characterized by bursting behavior. The addition of developmentally inspired constraints and correlations in the distribution of the neuronal connectivities and excitabilities leads to the emergence of functional hub neurons, whose stimulation/deletion is critical for the network activity. Functional hubs form a clique, where a precise sequential activation of the neurons is essential to ignite collective events without any need for a specific topological architecture. Unsupervised time-lagged firings of supra-threshold cells, in connection with coordinated entrainments of near-threshold neurons, are the key ingredients to orchestrate population activity.     

The PNPLA3 Ile148Met interacts with overweight and dietary intakes on fasting triglyceride levels

The Ile148Met (rs738409, G-allele) in the patatin-like phospholipase domain-containing protein 3 gene (PNPLA3) associates with liver fat content and may lead to loss-of-function (hydrolysis) or gain-of-function (CoA-dependent lysophosphatidic acid acyltransferase) defects. PNPLA3 is up-regulated by dietary carbohydrates, and interactions between rs738409 and carbohydrates, and sugar and ω6:ω3-polyunsaturated fatty acid (PUFA) ratio on hepatic fat accumulation have been reported. We examined interaction between rs738409 and overweight, and between rs738409 and dietary intakes (carbohydrates, sucrose and ω6:ω3-PUFA ratio), on fasting triglyceride levels. From the Malmo Diet and Cancer Study-Cardiovascular Cohort, 4,827 individuals without diabetes aged 58 ± 6 years, 2,346 with BMI ≤ 25 kg/m² and 2,478 with BMI > 25 kg/m², were included in cross-sectional analyses. Dietary data were collected by a modified diet history method. Overweight modified the association between rs738409 and fasting triglyceride levels (P_interaction = 0.003). G-allele associated with lower triglycerides only among overweight individuals (P = 0.01). Nominally, significant interaction on triglyceride levels was observed between rs738409 and sucrose among normal-weight individuals (P_interaction = 0.03). G-allele associated with lower triglycerides among overweight individuals in the lowest tertiles of carbohydrate and ω6:ω3-PUFA ratio (P = 0.04 and P = 0.001) and with higher triglycerides among normal-weight individuals in the highest tertile of sucrose (P = 0.001). We conclude that overweight and dietary sucrose may modify the association between rs738409 and fasting triglyceride levels.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0388-4) contains supplementary material, which is available to authorized users.     

Structure–function relationship and evolutionary history of the human selenoprotein M (SelM) found over-expressed in hepatocellular carcinoma

In humans we know 25 selenoproteins that play important roles in redox regulation, detoxification, immune-system protection and viral suppression. In particular, selenoprotein M (SelM) may function as thiol disulfide oxidoreductase that participates in the formation of disulfide bonds, and can be implicated in calcium responses. However, it presents a redox motif (CXXU), where U is a selenocysteine, and may also function as redox regulator because its decreased or increased expression regulated by dietary selenium alters redox homeostasis. No data are reported in literature about its involvement in cancer but only in neurodegenerative diseases. In this paper we evaluated the SelM expression in two hepatoma cell lines, HepG2 and Huh7, compared to normal hepatocytes. The results suggested its involvement in hepatocellular carcinoma (HCC) as well as its possible use to follow the progression of this cancer as putative marker. The aim of this study has been to analyze the structure–function relationships of SelM. Hence, firstly we studied the evolutionary history of this protein by phylogenetic analysis and GC content of genes from various species. So, we modeled the three-dimensional structure of the human SelM evaluating its energetic stability by molecular dynamics simulations. Moreover, we modeled some of its mutants to obtain structural information helpful for structure-based drug design.     

ZnFe2O4‐C/LiFePO4‐CNT: A Novel High‐Power Lithium‐Ion Battery with Excellent Cycling Performance

An innovative and environmentally friendly battery chemistry is proposed for high power applications. A carbon‐coated ZnFe₂O₄ nanoparticle‐based anode and a LiFePO₄‐multiwalled carbon nanotube‐based cathode, both aqueous processed with Na‐carboxymethyl cellulose, are combined, for the first time, in a Li‐ion full cell with exceptional electrochemical performance. Such novel battery shows remarkable rate capabilities, delivering 50% of its nominal capacity at currents corresponding to ≈20C (with respect to the limiting cathode). Furthermore, the pre‐lithiation of the negative electrode offers the possibility of tuning the cell potential and, therefore, achieving remarkable gravimetric energy and power density values of 202 Wh kg^?1 and 3.72 W kg^?1, respectively, in addition to grant a lithium reservoir. The high reversibility of the system enables sustaining more than 10 000 cycles at elevated C‐rates (≈10C with respect to the LiFePO₄ cathode), while retaining up to 85% of its initial capacity.     

iPathology cockpit diagnostic station: validation according to College of American Pathologists Pathology and Laboratory Quality Center recommendation at the Hospital Trust and University of Verona

Background

Validation of digital whole slide images is crucial to ensure that diagnostic performance is at least equivalent to that of glass slides and light microscopy. The College of American Pathologists Pathology and Laboratory Quality Center recently developed recommendations for internal digital pathology system validation. Following these guidelines we sought to validate the performance of a digital approach for routine diagnosis by using an iPad and digital control widescreen-assisted workstation through a pilot study.

Methods

From January 2014, 61 histopathological slides were scanned by ScanScope Digital Slides Scanner (Aperio, Vista, CA). Two independent pathologists performed diagnosis on virtual slides in front of a widescreen by using two computer devices (ImageScope viewing software) located to different Health Institutions (AOUI Verona) connected by local network and a remote image server using an iPad tablet (Aperio, Vista, CA), after uploading the Citrix receiver for iPad. Quality indicators related to image characters and work-flow of the e-health cockpit enterprise system were scored based on subjective (high vs poor) perception. The images were re-evaluated two weeks apart.

Results

The whole glass slides encountered 10 liver: hepatocarcinoma, 10 renal carcinoma, 10 gastric carcinoma and 10 prostate biopsies: adenocarcinoma, 5 excisional skin biopsies: melanoma, 5 lymph-nodes: lymphoma. 6 immuno- and 5 special stains were available for intra- and internet remote viewing. Scan times averaged two minutes and 54 seconds per slide (standard deviation 2 minutes 34 seconds). Megabytes ranged from 256 to 680 (mean 390) per slide storage. Reliance on glass slide, image quality (resolution and color fidelity), slide navigation time, simultaneous viewers in geographically remote locations were considered of high performance score. Side by side comparisons between diagnosis performed on tissue glass slides versus widescreen were excellent showing an almost perfect concordance (0.81, kappa index).

Conclusions

We validated our institutional digital pathology system for routine diagnostic facing with whole slide images in a cockpit enterprise digital system or iPad tablet. Computer widescreens are better for diagnosing scanned glass slide that iPad. For urgent requests, iPad may be used. Legal aspects have to be soon faced with to permit the clinical use of this technology in a manner that does not compromise patient care.

     

Chemo-orientation responses in hymenopteran parasitoids induced by substrate-borne semiochemicals

Hymenopteran parasitoids can utilize substrate-borne semiochemicals released by conspecifics or by their hosts, increasing the likelihood of successful mating and host location. According to the literature, two substrate-borne chemo-orientation patterns can occur: (1) biased random searching, a non-directional reaction toward the chemicals (kinesis), and (2) trail-following searching, a directional response toward the source emitting the chemical compounds (taxis). These two different strategies can be adopted by parasitoids to locate hosts and mates. In host location, random searching is induced by allelochemicals indirectly associated with the host, whereas trail-following behavior is induced by allelochemicals directly emitted by the target organism. In mate finding, sex pheromones emitted by conspecifics can induce either the random searching or the trail-following behavior, although the spatial distribution of virgin conspecifics could be an important factor driving the evolution of substrate-borne chemo-orientation patterns. The chemical nature of substrate-borne semiochemicals has not yet been fully elucidated. Most studies have shown that crude extracts are biologically active for eliciting parasitoid arrestment response, but few studies have clearly characterized their chemical nature. However, experimental evidence indicates that cuticular lipids located in the external layer of insects’ bodies play a role in parasitoid–parasitoid and host–parasitoid communication. The ecological role of parasitoid chemo-orientation in host and mate location is discussed from a biological control perspective.     

Looking for long-term changes in hydroid assemblages (Cnidaria,Hydrozoa) in Alboran Sea (South-Western Mediterranean): a proposal of a monitoring point for the global warming

In the last 20–30 years, the temperature of the Mediterranean Sea has increased and global warming is allowing the establishment of tropical-affinity species into more temperate zones. Sessile communities are particularly useful as a baseline for ecological monitoring; however, a lack of historical data series exists for sessile marine organisms without commercial interest. Hydroids are ubiquitous components of the benthic sessile fauna on rocky shores and have been used as bio-indicators of environmental conditions. In this study on the benthic hydroid assemblages of the Chafarinas Islands (Alboran Sea, South-Western Mediterranean), we characterized the hydroid assemblages, identified the bathymetric gradients, and compared them with a previous study carried out in 1991. Hydroid assemblages showed a significant difference both between year and among depths. Furthermore, eight species not present in 1991 were found, including two possible new species and the tropical and subtropical species Sertularia marginata. Due to its strategic position at the entrance of the Mediterranean and the existence of previous data on hydroid assemblages, the Chafarinas Islands are proposed as a possible monitoring point for entrance of Atlantic tropical species into the Mediterranean Sea.     

Correlation between interaction strengths drives stability in large ecological networks

Food webs have markedly non‐random network structure. Ecologists maintain that this non‐random structure is key for stability, since large random ecological networks would invariably be unstable and thus should not be observed empirically. Here we show that a simple yet overlooked feature of natural food webs, the correlation between the effects of consumers on resources and those of resources on consumers, substantially accounts for their stability. Remarkably, random food webs built by preserving just the distribution and correlation of interaction strengths have stability properties similar to those of the corresponding empirical systems. Surprisingly, we find that the effect of topological network structure on stability, which has been the focus of countless studies, is small compared to that of correlation. Hence, any study of the effects of network structure on stability must first take into account the distribution and correlation of interaction strengths.