Thermal stability and aggregation of sulfolobus solfataricus b-glycosidase are dependent upon the N-e-methylation of specific lysyl residues: critical role of in vivo post-translational modifications.

Methylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of β-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native β-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of β-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of β-glycosidase from S. solfataricu

Modelling Hospital Medical Wards to Address Patient Complexity: A Case-Based Simulation-Optimization Approach

In this paper we focus on patient flows inside Internal Medicine Departments, with the aim of supporting new organizational models taking into account the patient relevant characteristics such as complexity and frailty. The main contribution of this paper is to develop a Discrete Event Simulation model to describe in detail the pathways of complex patients through medical hospital wards. The model has been applied to reproduce a case study of an Italian middle size hospital. The objective is quantifying the impact on resource use and outcome of introducing a new organizational model for medical departments. The re-organization is mainly focused on changing the available beds assignment among the wards to better address the complexity of care of patients with comorbidities. Following a patient-centered approach, patients are segmented considering the clinical characteristics (i.e. the pathology, proxy of Diagnoses Related Groups classification) and sub-grouped considering other characteristics, such as comorbidities and ward of admission. Then, an optimization component embedded into the model chooses the best pooling strategy to reorganize medical wards, determining the corresponding number of beds able to improve process indicators, such as length of stay. The simulation model is presented, and preliminary results are analyzed and discussed

A spectroscopic study on secondary structure and thermal unfolding of the plant toxin gelonin confirms some typical structural characteristics and unravels the sequence of thermal unfolding events

Gelonin from the Indian plant Gelonium multiflorum belongs to the type I ribosome-inactivating proteins (RIPs). Like other members of RIPs, this toxin glycoprotein inhibits protein synthesis of eukaryotic cells; hence, it is largely used in the construction of immunotoxins composed of cell-targeted antibodies. Lysosomal degradation is one of the main issues in targeted tumor therapies, especially for type I RIP-based toxins, as they lack the translocation domains. The result is an attenuated cytosolic delivery and a decrease of the antitumor efficacy of these plant-derived toxins; therefore, strategies to permit their release from endosomal vesicles or modifications of the toxins to make them resistant to degradation are necessary to improve their efficacy. Using infrared spectroscopy, we thoroughly analyzed both the secondary structure and the thermal unfolding of gelonin. Moreover, by the combination of two-dimensional correlation spectroscopy and phase diagram method, it was possible to deduce the sequence of events during the unfolding, confirming the typical characteristic of the RIP members to denature in two steps, as a sequential loss of tertiary and secondary structure was detected at 58 °C and at 65 °C, respectively. Additionally, some discrepancies in the unfolding process between gelonin and saporin-S6, another type I RIP protein, were detected