Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates

BACKGROUND: Anemia due to impaired erythropoietin (EPO) production is associated with kidney failure. Recombinant proteins are commonly administered to alleviate the symptoms of this dysfunction, whereas gene therapy approaches envisaging the delivery of EPO genes have been tried in animal models in order to achieve stable and long-lasting EPO protein production. Naked DNA intramuscular injection is a safe approach for gene delivery; however, transduction levels show high inter-individual variability in rodents and very poor efficiency in non-human primates. Transduction can be improved in several animal models by application of electric pulses after DNA injection. METHODS: We have designed a modified EPO gene version by changing the EPO leader sequence and optimizing the gene codon usage. This modified gene was electro-injected into mice, rabbits and cynomolgus monkeys to test for protein production and biological effect. CONCLUSIONS: The modified EPO gene yields higher levels of circulating transgene product and a more significant biological effect than the wild-type gene in all the species tested, thus showing great potential in clinically developable gene therapy approaches for EPO delivery.     

<Emphasis Type="Italic">Scenedesmus vacuolatus</Emphasis> cultures for possible combined laccase-like phenoloxidase activity and biodiesel production

A key aspect of the industrial development of microalgal production processes is the excessive cost of biomass production. A solution is a combination of biodiesel production and wastewater treatment. The microalga Scenedesmus has a high lipid content and a potential extracellular phenoloxidase activity, which could improve the phycoremediation of phenolic pollutants. In this work, the most suitable growth conditions to obtain this twofold aim were analyzed. First, different strains of Scenedesmus vacuolatus microalga were tested at different pH, salinity and CO₂ concentration in the gas phase. The two most promising strains were then cultivated in autotrophic and heterotrophic conditions, and were investigated in terms of efficient nitrogen removal, fatty acid profile and maximized extracellular phenoloxidase activity in the medium. The results showed two extreme conditions: (1) biomass productivity doubled when photobioreactors were sparged with 5% CO₂ supplemented air with respect to cultures sparged with air (the steady state values of strain 53 were 0.138 g L^?1 day^?1 in the presence of air, and 0.243 in the presence of CO₂ addition), and N-starvation under 5% CO₂enhanced the transesterified fraction of lipids (strain 53 FAME fraction in the presence of N-starvation was 33%, in the presence of nitrogen FAME fraction was 22%); (2) phenoloxidase activity was completely suppressed by presence of 5% CO₂ in the gas phase (strain 53 0.21 U mL^?1), indicating clear catabolite repression for the induction of this enzyme in the algal metabolism.     

Detrimental effects of melanocortin‐1 receptor (MC1R) variants on the clinical outcomes of BRAF V600 metastatic melanoma patients treated with BRAF inhibitors

Melanocortin‐1 receptor (MC1R) plays a key role in skin pigmentation, and its variants are linked with a higher melanoma risk. The influence of MC1R variants on the outcomes of patients with metastatic melanoma (MM) treated with BRAF inhibitors (BRAFi) is unknown. We studied the MC1R status in a cohort of 53 consecutive BRAF‐mutated patients with MM treated with BRAFi. We also evaluated the effect of vemurafenib in four ^V600BRAF melanoma cell lines with/without MC1R variants. We found a significant correlation between the presence of MC1R variants and worse outcomes in terms of both overall response rate (ORR; 59% versus 95%, P = 0.011 univariate, P = 0.028 multivariate analysis) and progression‐free survival (PFS) shorter than 6 months (72% versus 33%, P = 0.012 univariate, P = 0.027 multivariate analysis). No difference in overall survival (OS) was reported, probably due to subsequent treatments. Data in vitro showed a significant different phosphorylation of Erk1/2 and p38 MAPK during treatment, associated with a greater increase in vemurafenib IC50 in MC1R variant cell lines.     

Effect of NaCl on the conformational stability of the thermophilic γ-glutamyltranspeptidase from Geobacillus thermodenitrificans: Implication for globular protein halotolerance

The transpeptidation activity of γ-glutamyltranspeptidase from Geobacillus thermodenitrificans (GthGT) is negligible and the enzyme is highly thermostable. Here we have examined the effect of concentrated NaCl solutions on structure, stability, dynamics and enzymatic activity of GthGT. The protein exhibited hydrolytic activity over a broad range of NaCl concentrations. Even at 4.0 M NaCl, GthGT retained more than 90% of the initial activity and showed unaltered fluorescence emission, secondary structure and acrylamide quenching on tryptophan fluorescence. Furthermore, at 2.8 M and 4.0 M NaCl the temperature-induced unfolding profiles are dramatically changed with large (> 20 °C) positive shifts in the denaturation temperature. These features make GthGT an ideal system to be used in industrial processes that require high temperatures and high-salt environments. A general explanation of the NaCl effect by means of a statistical thermodynamic model is also provided, together with an analysis of residue distribution between protein surface and interior in 15 non-redundant families of halophilic and non-halophilic proteins. The results are in line with a comparative sequence and structural analysis between halophilic and non-halophilic γ-glutamyltranspeptidases which revealed that a major role in halotolerance should be played by solvent exposed negatively charged residues.     

Multimodality treatment of locally advanced soft-tissue sarcomas of the extremities

Background

Adjuvant radiotherapy (RTE) still has a fundamental role as a post-operative treatment of locally advanced soft tissues sarcomas of the extremities. Moreover the employment of combined modalities in locally advanced soft tissues sarcomas of the extremities allow to maximize the chance of local cure even in difficult presentation cases, and possibly improve survival, especially in high-risk disease patients. In patients with sarcomas of the extremities in which definitive surgery has not been radical (with positive or “close” margins) radiotherapy can improve the results in terms of Disease Free Survival (DFS) and, together with chemotherapy, of Overall Survival (OS). We recommend radiotherapy in case of deep tumor location, inadequate surgical margins and grade 3 tumour; for positive or “marginal (or close)” excision (that means inadequate surgery) or in selected patients with a bad prognosis, we believe that a multidisciplinary approach can be preferable.

Introduction

Adjuvant radiotherapy (RTE) still has a fundamental role as a post-operative treatment. In patients with sarcomas of the extremities in whom definitive surgery has been or not radical (positive or “close” margins), radiotherapy with chemotherapy can improve the results in terms of Disease Free Survival (DFS) and Overall Survival (OS), while RTE alone seems to improve local control.

Materials and methods

From 1/2000 to 12/2005 we treated 34 patients affected by locally advanced sarcomas of the upper or lower extremities with radiotherapy (doses ranging from 54 to 66 Gy) and chemotherapy in 18/34 with an adjuvant scheme that consisted in Epirubicine (120 mg/m²) plus Ifosfamide (7000–9000 mg/m²).

Results

Disease Free Survival (DFS) and the Overall Survival (OS) rates were 76% and 82%, respectively. Eighteen patients developed one or more long-term side effects. Most of these complications were mild: all patients experienced only erithema, edema, local sclerosis or moderate pain.

Conclusion

Radiotherapy has an important role as a post-operative treatment also when surgery was non-radical. It improves local control more in patients with high-grade sarcomas of the extremity with positive or close margins. It is still difficult to assess the role of adjuvant chemotherapy.     

Identification of the first archaeal oligopeptide-binding protein from the hyperthermophile Aeropyrum pernix

The archaeon Aeropyrum pernix grows optimally at 90°C and derives energy primarily from aerobic degradation of complex proteinaceous substrates. The ability of these nutrients to sustain growth is generally associated with the presence of oligopeptide transport systems, such as the well-known protein-dependent ATP-binding cassette (ABC) transporters. This study is concerned with the isolation and characterisation of the first archaeal oligopeptide-binding protein (OppA_Ap) from the extracellular medium of A. pernix. The protein shows a pI of 3.9 and a molecular mass of about 90 kDa under native conditions. By using a proteomic approach, the OppA_Ap-encoding gene was identified (APE1583) and about 55% of the protein amino-acid sequence was validated. The extracellular purified protein was able to efficiently bind oligopeptide substrates such as Xenopsin. The amount of a liganded peptide to OppA_Ap was about 70% at 90°C using a 1/100 (w/w) OppA_Ap/substrate ratio. Sequence comparisons showed a weak but significant similarity of OppA_Ap with bacterial oligopeptide binding proteins. Furthermore, APE1583 neighbouring genes encode for the cognate components of an ABC transport system, suggesting that these ORFs are organised in an operon-like structure, with OppA_Ap as the extracellular component for the uptake of oligopeptides.     

Biochemical and structural properties of gamma-glutamyl transpeptidase from Geobacillus thermodenitrificans: An enzyme specialized in hydrolase activity

Gamma-glutamyltranspeptidases (γ-GTs) catalyze the transfer of the gamma-glutamyl moiety of glutathione and related gamma-glutamyl amides to water (hydrolysis) or to amino acids and peptides (transpeptidation) and play a key role in glutathione metabolism. Recently, γ-GTs have been considered attractive pharmaceutical targets for cancer and useful tools to produce γ-glutamyl compounds. To find out γ-GTs with special properties we have chosen microorganisms belonging to Geobacillus species which are source of several thermostable enzymes of potential interest for biotechnology. γ-GT from Geobacillus thermodenitrificans (GthGT) was cloned, expressed in Escherichia coli, purified to homogeneity and characterized. The enzyme, synthesized as a precursor homotetrameric protein of 61-kDa per subunit, undergoes an internal post-translational cleavage of the 61 kDa monomer into 40- and 21-kDa shorter subunits, which are then assembled into an active heterotetramer composed of two 40- and two 21-kDa subunits. The kinetic characterization of the hydrolysis reaction using l-glutamic acid γ-(4-nitroanilide) as the substrate reveals that the active enzyme has K_m 7.6 μM and V_max 0.36 μmol min/mg. The optimum pH and temperature for the hydrolysis activity are 7.8 and 52 °C, respectively. GthGT hydrolyses the physiological antioxidant glutathione, suggesting an involvement of the enzyme in the cellular defense mechanism against oxidative stress. Unlike other γ-GTs, the mutation of the highly conserved catalytic nucleophile, Thr353, abolishes the post-translational cleavage of the pro-enzyme, but does not completely block the hydrolytic action. Furthermore, GthGT does not show any transpeptidase activity, suggesting that the enzyme is a specialized γ-glutamyl hydrolase. The GthGT homology-model structure reveals peculiar structural features, which should be responsible for the different functional properties of the enzyme and suggests the structural bases of protein thermostability.     

Role of histidine 148 in stability and dynamics of a highly fluorescent GFP variant

The armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pK_a values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein.