The S-layer protein DR_2577 binds deinoxanthin and under desiccation conditions protects against UV-radiation in Deinococcus radiodurans

Deinococcus radiodurans has the puzzling ability to withstand over a broad range of extreme conditions including high doses of ultraviolet radiation and deep desiccation. This bacterium is surrounded by a surface layer (S-layer) built of a regular repetition of several proteins, assembled to form a paracrystalline structure. Here we report that the deletion of a main constituent of this S-layer, the gene DR_2577, causes a decrease in the UVC resistance, especially in desiccated cells. Moreover, we show that the DR_2577 protein binds the carotenoid deinoxanthin, a strong protective antioxidant specific of this bacterium. A further spectroscopical characterization of the deinoxanthin-DR_2577 complex revealed features which could suggest a protective role of DR_2577. We propose that, especially under desiccation, the S-layer shields the bacterium from incident ultraviolet light and could behave as a first lane of defense against UV radiation

Solubilization method for isolation of photosynthetic mega- And super-complexes from conifer thylakoids

Photosynthesis is the main process by which sunlight is harvested and converted into chemical energy and has been a focal point of fundamental research in plant biology for decades. In higher plants, the process takes place in the thylakoid membranes where the two photosystems (PSI and PSII) are located. In the past few decades, the evolution of biophysical and biochemical techniques allowed detailed studies of the thylakoid organization and the interaction between protein complexes and cofactors. These studies have mainly focused on model plants, such as Arabidopsis, pea, spinach, and tobacco, which are grown in climate chambers even though significant differences between indoor and outdoor growth conditions are present. In this manuscript, we present a new mild-solubilization procedure for use with “fragile” samples such as thylakoids from conifers growing outdoors. Here, the solubilization protocol is optimized with two detergents in two species, namely Norway spruce (Picea abies) and Scots pine (Pinus sylvestris). We have optimized the isolation and characterization of PSI and PSII multimeric mega- and super-complexes in a close-to-native condition by Blue-Native gel electrophoresis. Eventually, our protocol will not only help in the characterization of photosynthetic complexes from conifers but also in understanding winter adaptation

Neutralizing antibody response during acute and chronic hepatitis C virus infection

Little is known about the role of Abs in determining the outcome of hepatitis C virus (HCV) infection. By using infectious retroviral pseudotypes bearing HCV glycoproteins, we measured neutralizing Ab (nAb) responses during acute and chronic HCV infection. In seven acutely infected health care workers, only two developed a nAb response that failed to associate with viral clearance. In contrast, the majority of chronically infected patients had nAbs. To determine the kinetics of strain-specific and crossreactive nAb emergence, we studied patient H, the source of the prototype genotype 1a H77 HCV strain. An early weak nAb response, specific for the autologous virus, was detected at seroconversion. However, neutralization of heterologous viruses was detected only between 33 and 111 weeks of infection. We also examined the development of nAbs in 10 chimpanzees infected with H77 clonal virus. No nAb responses were detected in three animals that cleared virus, whereas strain-specific nAbs were detected in six of the seven chronically infected animals after approximately 50 weeks of infection. The delayed appearance of high titer crossreactive nAbs in chronically infected patients suggests that selective mechanism(s) may operate to prevent the appearance of these Abs during acute infection. The long-term persistence of these nAbs in chronically infected patients may regulate viral replication

Structural analysis of the architecture and in situ localization of the main S-layer complex in Deinococcus radiodurans

Bacterial surface layers are paracrystalline assemblies of proteins that provide the first line of defense against environmental shocks. Here, we report the 3D structure, in situ localization, and orientation of the S-layer deinoxanthin-binding complex (SDBC), a hetero-oligomeric assembly of proteins that in Deinococcus radiodurans represents the main S-layer unit. The SDBC is resolved at 11-Å resolution by single-particle analysis, while its in situ localization is determined by cryo-electron crystallography on intact cell-wall fragments leading to a projection map at 4.5-Å resolution. The SDBC exhibits a triangular base with three comma-shaped pores, and a stalk departing orthogonally from the center of the base and oriented toward the intracellular space. Combining state-of-the-art techniques, results show the organization of this S-layer and its connection within the underlying membranes, demonstrating the potential for applications from nanotechnologies to medicine

An integrated care pathway for cancer patients with diabetes: A proposal from the Italian experience

Diabetes and cancer frequently coexist in the same subject, often with relevant clinical effects on the management and prognosis of the comorbid patient. The existing guidelines, however, do not appropriately address many clinical issues in this setting. Although collaboration between diabetologists and oncologists should play an important role in achieving appropriate levels of care, close coordination or agreement between these specialists is seldom offered. There is an urgent need for greater interdisciplinary integration between all specialists involved in this setting, for a shared approach ensuring that organisational silos are overcome. To this end, the Italian Associations of Medical Diabetologists (AMD) and the Italian Association of Medical Oncology (AIOM) recently established a dedicated Working Group on 'Diabetes and Cancer'. The working group outlined a diagnostic and therapeutic clinical pathway dedicated to hospitalised patients with diabetes and cancer. In this article, we describe the Italian proposal including some suggested measures to assess, monitor and improve blood glucose control in the hospital setting, to integrate different specialists from both areas, as well as to ensure discharge planning and continuity of care from the hospital to the territory

On farm agronomic and first environmental evaluation of oil crops for sustainable bioenergy chains.

Energy crops, and in particular oil crops, could be an important occasion for developing new non food production rows for a new multi-functional agriculture in Italy. In this view, the use of local biomass is a fundamental starting point for the development of a virtuous energy chain that should pursue not only agricultural profitability, but also chain sustainability and that is less dependent on the global market, characterized by instability in terms of biomass availability and price. From this perspective, particular attention must be paid to crop choice on the basis of its rusticity and of its adaptability to local growing conditions and to low input cropping systems. In this context, alike woody and herbaceous biomasses, oil crops such as sunflower and rapeseed should be able to support local agricultural bioenergy chain in Italy. In addition, in a local bioenergy chain, the role of the farmers should not be limited just to grain production; but also grain processing should be performed at farm or consortium level in oilseed extraction plants well proportioned to the cropped surface. In this way, by means of a simple power generator, farmer could thus produce its own thermal and electric energy from the oil, maximizing his profit. This objective could also be achieved through the exploitation of the total biomass, including crop residues and defatted seed meals, that may be considered as fundamental additional economic and/or environmental benefits of the chain. This paper reports some results of three-years on-farm experiments on oil crop chain carried out in the framework of "Bioenergie" project, that was focused to enhance farmers awareness of these criteria and to the feasibility at open field scale of low-input cultivation of rapeseed, sunflower and Brassica carinata in seven Italian regions. In several on-farm experiences, these crops produced more than 800 kg ha-1 of oil with good energy properties. Defatted seed meals could be interesting as organic fertilizers and, in the case of B. carinata, as a biofumigant amendment that could offer a total or partial alternative to some chemicals in agriculture. Furthermore, biomass soil incorporation could contribute to C sequestration, catching CO2 from atmosphere and sinking a part in soil as stable humus. Finally, four different open field experiences carried out again in the second year of the project, have been analysed in order to evaluate their energy and greenhouse gasses balance after cultivation phase

Genetic variability of hepatitis C virus before and after combined therapy of interferon plus ribavirin

We present an analysis of the selective forces acting on two hepatitis C virus genome regions previously postulated to be involved in the viral response to combined antiviral therapy. One includes the three hypervariable regions in the envelope E2 glycoprotein, and the other encompasses the PKR binding domain and the V3 domain in the NS5A region. We used a cohort of 22 non-responder patients to combined therapy (interferon alpha-2a plus ribavirin) for which samples were obtained before initiation of therapy and after 6 or/and 12 months of treatment. A range of 25-100 clones per patient, genome region and time sample were sequenced. These were used to detect general patterns of adaptation, to identify particular adaptation mechanisms and to analyze the patterns of evolutionary change in both genome regions. These analyses failed to detect a common adaptive mechanism for the lack of response to antiviral treatment in these patients. On the contrary, a wide range of situations were observed, from patients showing no positively selected sites to others with many, and with completely different topologies in the reconstructed phylogenetic trees. Altogether, these results suggest that viral strategies to evade selection pressure from the immune system and antiviral therapies do not result from a single mechanism and they are likely based on a range of different alternatives, in which several different changes, or their combination, along the HCV genome confer viruses the ability to overcome strong selective [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]

The Evolutionary Dynamics of a Rapidly Mutating Virus within and between Hosts: The Case of Hepatitis C Virus

Many pathogens associated with chronic infections evolve so rapidly that strains found late in an infection have little in common with the initial strain. This raises questions at different levels of analysis because rapid within-host evolution affects the course of an infection, but it can also affect the possibility for natural selection to act at the between-host level. We present a nested approach that incorporates within-host evolutionary dynamics of a rapidly mutating virus (hepatitis C virus) targeted by a cellular cross-reactive immune response, into an epidemiological perspective. The viral trait we follow is the replication rate of the strain initiating the infection. We find that, even for rapidly evolving viruses, the replication rate of the initial strain has a strong effect on the fitness of an infection. Moreover, infections caused by slowly replicating viruses have the highest infection fitness (i.e., lead to more secondary infections), but strains with higher replication rates tend to dominate within a host in the long-term. We also study the effect of cross-reactive immunity and viral mutation rate on infection life history traits. For instance, because of the stochastic nature of our approach, we can identify factors affecting the outcome of the infection (acute or chronic infections). Finally, we show that anti-viral treatments modify the value of the optimal initial replication rate and that the timing of the treatment administration can have public health consequences due to within-host evolution. Our results support the idea that natural selection can act on the replication rate of rapidly evolving viruses at the between-host level. It also provides a mechanistic description of within-host constraints, such as cross-reactive immunity, and shows how these constraints affect the infection fitness. This model raises questions that can be tested experimentally and underlines the necessity to consider the evolution of quantitative traits to understand the outcome and the fitness of an infection