Comparative evaluation of multiple protein extraction procedures from three species of the genus Caulerpa

AbstractThe aim of this study was to define the simplest and least expensive protocol for total protein extraction for three different macroalgae of the genus Caulerpa (the invasive C. taxifolia and C. cylindracea and the autochthonous C. prolifera). Five multi-step protein extraction procedures, set up for other macroalgal species, were tested. For each of them, different pre-treatment and extraction conditions were simultaneously examined, according to a factorial design, considering the starting material, the solvent-to-biomass ratio, and the incubation temperature. Protein yield in the obtained extracts was estimated with the Bradford method. Further, polyacrylamide gel electrophoresis (SDS-PAGE) was used to resolve proteins, assessing their quality and integrity. Significant differences in protein yield were observed among the extraction protocols and the conditions tested, also in relation to the considered species. Profiles having an acceptable quality were obtained for C. prolifera and C. cylindracea, and from the obtained results, the best method to obtain high yield and quality protein extracts for the two above-mentioned species appears to require the use of a primary TCA/acetone extraction buffer followed by a lysis buffer with NaCl, KCl, urea, Triton, SDS and a protease inhibitor. The best results, in particular, were obtained starting from fresh pulped material with a buffer-to-biomass ratio of 10:1 and an incubation temperature of 4°C. For C. taxifolia, instead, none of the tested protocols produced satisfactory results and further studies will be required

The interactive effect of herbivory, nutrient enrichment and mucilage on shallow rocky macroalgal communities

This paper focuses on the interactive short and long-term effect of three different stressors on a macroalgal assemblage. Three stressors are considered: herbivory, nutrients and mucilage. The experiment was conducted in Tavolara Punta Coda Cavallo Marine Protected Area (Mediterranean Sea) during a bloom of the benthic mucilage-producing microalga Chrysophaeum taylorii (Pelagophyceae); this microalga is recently spreading in the Mediterranean Sea. On a rocky substratum, 36 plots 20 × 20 cm in size were prepared. Factorial combinations of three experimental treatments were applied in triplicate, including three grazing levels crossed with two nutrient enrichment and two mucilage removal treatments. Significant differences were observed among treatments 8 weeks later, at the end of summer. In particular, dark filamentous algae were more abundant in all enriched plots, especially where mucilage and macroalgae had been removed; a higher percent cover of crustose coralline algae was instead observed where nutrients had been increased and no grazing pressure acted. Furthermore, the abundance of Dictyota spp. and Laurencia spp. was significantly higher in enriched mucilage-free plots where the grazing pressure was null or low. However, the effects of the treatments on the overall assemblage of the macroalgal community were not long persistent (36 weeks later). These results illustrate the capacity of a shallow-water macroalgal community to quickly recover from the simultaneous impacts of herbivory, nutrient enrichment, and mucilage

The prognostic role of hemoglobin levels in patients undergoing concurrent chemo-radiation for anal cancer

Background: Concurrent chemo-radiation (CT-RT) is a standard therapy for squamous cell carcinoma of anal canal. Different clinical and biological factors may potentially affect outcome. We investigated the prognostic role of baseline hemoglobin (Hb) in a cohort of anal cancer patients submitted to CT-RT with 5-fluorouracil and mitomycin C. Methods: Up to 161 patients with clinical stage T1-T4/N0-N3/M0 were treated. Response was assessed at 6 weeks and thereafter at 3, 6 and 12 months. Two different approaches were used:a)simultaneous integrated boost following RTOG 05-29 indications;b)first sequence of 45Gy/25 fractions to the pelvis followed by 9-14.4 Gy/5-8 fractions to the macroscopic disease. Primary endpoints were progression-free survival (PFS) and overall survival (OS). Results: On multivariate analysis, pre-treatment Hb level had a significant correlation to OS (HR:0.53;95% CI:0.33-0.87; p = 0.001), but not to PFS (HR:0.78;95% CI:0.53-1.15; p = 0.12) Patients with pre-treatment Hb 65 12 g/dl had 5-year PFS and OS of 82.2%, compared to 29.3% and 32.8% for those below the threshold. The likelihood to achieve a complete remission increased by 5.6% for every single-unit (g/dl) increase in baseline Hb level over 11 g/dl. On multivariate analysis, response to treatment had a significant correlation to PFS (incomplete vs complete response - HR:5.43;95% CI:2.75-10.7; p < 0.0001) and OS (HR: 6.96;95% CI:2.96-16.5; p < 0.0001). Conclusions: We showed that baseline Hb level is a strong indicator for poor response to RT-CT in anal cancer patients. A close clinical monitoring for incomplete response to treatment should be advised in patients with low pre-treatment Hb. The hypothesis that the preservation of adequate Hb level during treatment may lead to a better outcome needs prospective evaluation

Global and Regional IUCN Red List Assessments: 1

In this contribution, the conservation status assessment of six plant species according to IUCN categories and criteria are presented. It includes the assessment at global level of Linaria tonzigii Lona, Allium garganicum Brullo, Pavone, Salmeri & Terrasi, Ferula arrigonii Bocchieri, Orchis patens Desf. subsp. patens and Armeria saviana Selvi and the assessment at regional level (Italy) of Viola jordanii Hanry

Parvoviruses in Blood Donors and Transplant Patients, Italy

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Decellularized Human Dermal Matrix as a Biological Scaffold for Cardiac Repair and Regeneration.

The complex and highly organized environment in which cells reside consists primarily of the extracellular matrix (ECM) that delivers biological signals and physical stimuli to resident cells. In the native myocardium, the ECM contributes to both heart compliance and cardiomyocyte maturation and function. Thus, myocardium regeneration cannot be accomplished if cardiac ECM is not restored. We hypothesize that decellularized human skin might make an easily accessible and viable alternate biological scaffold for cardiac tissue engineering (CTE). To test our hypothesis, we decellularized specimens of both human skin and human myocardium and analyzed and compared their composition by histological methods and quantitative assays. Decellularized dermal matrix was then cut into 600-mm-thick sections and either tested by uniaxial tensile stretching to characterize its mechanical behavior or used as three-dimensional scaffold to assess its capability to support regeneration by resident cardiac progenitor cells (hCPCs) in vitro. Histological and quantitative analyses of the dermal matrix provided evidence of both effective decellularization with preserved tissue architecture and retention of ECM proteins and growth factors typical of cardiac matrix. Further, the elastic modulus of the dermal matrix resulted comparable with that reported in literature for the human myocardium and, when tested in vitro, dermal matrix resulted a comfortable and protective substrate promoting and supporting hCPC engraftment, survival and cardiomyogenic potential. Our study provides compelling evidence that dermal matrix holds promise as a fully autologous and cost-effective biological scaffold for CTE

Prevalence of resistance-associated substitutions to NS3, NS5A and NS5B inhibitors at DAA-failure in hepatitis C virus in Italy from 2015 to 2019

: Despite the high efficacy of direct-acting antivirals (DAAs), the selection of resistance-associated substitutions (RASs) after virological failure of hepatitis C virus (HCV) DAAs can impair the cure of chronic HCV. The aim of the study was to characterize RASs after virological failure of DAAs in Italy over the years. Within the Italian network VIRONET-C, the change in prevalence of NS3/4A-NS5A-NS5B RASs was retrospectively evaluated in patients who failed a DAA regimen over the years 2015-2019. NS3, NS5A and NS5B Sanger sequencing was performed using homemade protocols and the geno2pheno system was used to define HCV-genotype/subtype and predict drug resistance. The changes in the prevalence of RASs over time were evaluated using the chi-square test for trend. Predictors of RASs at failure were analysed by logistic regression. Among 468 HCV-infected patients, HCV genotype 1 was the most prevalent (1b in 154, 33% and 1a in 109, 23%). DAA regimens were: ledipasvir (LDV)/sofosbuvir (SOF) in 131 patients (28%), daclatasvir (DCV)/SOF in 109 (23%), ombitasvir/paritaprevir/ritonavir+dasabuvir (3D) in 89 (19%), elbasvir (EBR)/grazoprevir (GRZ) in 52 (10.5%), velpatasvir (VEL)/SOF in 53 (11%), glecaprevir (GLE)/pibrentasvir (PIB) in 27 (6%) and ombitasvir/paritaprevir/ritonavir (2D) in 7 (1.5%); ribavirin was administered in 133 (28%). The NS5A fasta sequence was available for all patients, NS5B and NS3/4A both for 93%. The prevalence of NS5A and NS3/4A RASs significantly declined from 2015 to 2019; NS5B RAS remained stable. Independent predictors of any RASs included older age and genotype 1a (vs G2 and vs G4). Notably, at least partial susceptibility to all the agents included in the GLE/PIB and VEL/SOF/Voxilaprevir (VOX) combinations was predicted in >95% of cases. As RASs remain common at the failure of DAAs, their identification could play a crucial role in optimizing re-treatment strategies. In Italy RAS prevalence has been decreasing over the years and susceptibility to the latest developed drug combinations is maintained in most cases

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.publishedVersio

Immune inflammation indicators in anal cancer patients treated with concurrent chemoradiation: training and validation cohort with online calculator (ARC: Anal Cancer Response Classifier)

Background: In anal cancer, there are no markers nor other laboratory indexes that can predict prognosis and guide clinical practice for patients treated with concurrent chemoradiation. In this study, we retrospectively investigated the influence of immune inflammation indicators on treatment outcome of anal cancer patients undergoing concurrent chemoradiotherapy. Methods: All patients had a histologically proven diagnosis of squamous cell carcinoma of the anal canal/margin treated with chemoradiotherapy according to the Nigro's regimen. Impact on prognosis of pre-treatment systemic index of inflammation (SII) (platelet x neutrophil/lymphocyte), neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) were analyzed. Results: A total of 161 consecutive patients were available for the analysis. Response to treatment was the single most important factor for progression-free survival (PFS) and overall survival (OS). At univariate analysis, higher SII level was significantly correlated to lower PFS (p<0.01) and OS (p=0.046). NLR level was significantly correlated to PFS (p=0.05), but not to OS (p=0.06). PLR level significantly affected both PFS (p<0.01) and OS (p=0.02). On multivariate analysis pre-treatment, SII level was significantly correlated to PFS (p=0.0079), but not to OS (p=0.15). We developed and externally validated on a cohort of 147 patients a logistic nomogram using SII, nodal status and pre-treatment Hb levels. Results showed a good predictive ability with C-index of 0.74. An online available calculator has also been developed. Conclusion: The low cost and easy profile in terms of determination and reproducibility make SII a promising tool for prognostic assessment in this oncological setting