Unveiling the mono-rhamnolipid and di-rhamnolipid mechanisms of action upon plasma membrane models

Rhamnolipids (RLs) are biosurfactants with significant tensioactive and emulsifying properties. They are mainly composed by mono-RL and di-RL components. Although there are numerous studies concerning their molecular properties, information is scarce regarding the mechanisms by which each of the two components interacts with cell membranes. Herein, we performed phase-contrast and fluorescence microscopy experiments on plasma membrane models represented by giant-unilamellar-vesicles (GUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-[[(E,2S,3R)-1,3-dihydroxy-2-(octadecanoylamino) octadec-4-enyl]peroxy-hydroxyphosphoryl]oxyethyl-trimethylazanium (sphingomyelin, SM) and (3β)-cholest-5-en-3-ol (cholesterol, CHOL) (1:1:1 M ratio), which present liquid-order (Lo) liquid-disorder (Ld) phase coexistence, in the presence of either mono-RL or di-RL in 0.06–0.25 mM concentration range. A new method has been developed to determine area and volume of GUVs with asymmetrical shape and a kinetic model describing GUV-RL interaction in terms of two mechanisms, RL-insertion and pore formation, has been worked out. Results show that the insertion of mono-RL in the membrane outer leaflet is the dominant process with no pore formation and a negligible effect in modifying membrane permeability, but induces lipid mixing. Conversely, the di-RL-GUV interaction begins with the insertion mechanism and, as the time passes by, the pore formation process occurs. The analyses of di-RL show that the whole process is only relevant in the Ld phase with a higher extent to 0.25 mM than to 0.06 mM

Guanosine Quadruplexes in Solution: A Small-Angle X-Ray Scattering Analysis of Temperature Effects on Self-Assembling of Deoxyguanosine Monophosphate

We investigated quadruplex formation in aqueous solutions of 2′-deoxyriboguanosine 5′-monophosphate, d(pG), which takes place in the absence of the covalent axial backbone. A series of in-solution small angle X-ray scattering experiments on d(pG) have been performed as a function of temperature in the absence of excess salt, at a concentration just above the critical one at which self-assembling occurs. A global fit approach has been used to derive composition and size distribution of the scattering particles as a function of temperature. The obtained results give thermodynamical justification for the observed phase-behavior, indicating that octamer formation is essential for quadruplex elongation. Our investigation shows that d(pG) quadruplexes are very suitable to assess the potential of G-quadruplex formation and to study the self-assembling thermodynamics

Unraveling the effect of proliferative stress in vivo in hematopoietic stem cell gene therapy mouse study

The hematopoietic system of patients enrolled in hematopoietic stem cells (HSC) gene therapy (GT) treatments is fully reconstituted upon autologous transplantation of engineered stem cells. HSCs highly proliferate up to full restoration of homeostasis and compete for niche homing and engraftment. The impact of the proliferation stress in HSC on genetic instability remains an open question that cured patients advocate for characterizing long-term safety and efficacy. The accumulation of somatic mutations has been widely used as a sensor of proliferative stress. Vector integration site (IS) can be used as a molecular tool for clonal identity, inherited by all HSC progeny, to uncover lineage dynamics in vivo at single-cell level. Here we characterized at single-clone granularity the proliferative stress of HSCs and their progeny over time by measuring the accumulation of mutations from the DNA of each IS. To test the feasibility of the approach, we set-up an experimental framework that combines tumor-prone Cdkn2a-/- and wild type (WT) mouse models of HSC-GT and molecular analyses on different hematopoietic cell lineages after transplantation of HSCs transduced with genotoxic LV (LV.SF.LTR) or GT-like non-genotoxic LV (SIN.LV.PGK). The Cdkn2a-/- mouse model provided the experimental conditions to detect the accumulation of somatic mutations, since the absence of p16INK4A and p19ARF enhances the proliferative potential of cells that have acquired oncogenic mutations. As expected, mice transplanted with Cdkn2a-/- Lin- cells marked with LV.SF.LTR (N=24) developed tumors significantly earlier compared to mock (N=20, p<0.0001), while mice treated with SIN. LV.PGK (N=23) did not. On the other side, mice that received WT Lin- cells treated with LV.SF.LTR (N=25) or SIN.LV.PGK (N=24) vector have not developed tumors. Given this scenario, we expect that Cdkn2a-/- Lin- cells transduced with LV.SF.LTR are associated with higher mutation rates compared to the SIN.LV.PGK group and wild type control mice. The composition of peripheral blood, lymphoid (B and T) and myeloid compartments was assessed by FACS on samples collected every 4 weeks and IS identification. More than 200,000 IS have been recovered. To identify the presence of somatic mutations, the genomic portions of sequencing reads flanking each different IS were analyzed with VarScan2. The accumulation rates of mutations have been evaluated by our new Mutation Index (MI) which normalizes the number of mutations by clones and coverage. Considering that a large portion of IS has been discarded since not covered by a minimum number of 5 unique reads (genomes), the remaining number of IS contained >90% of reads in each group. The MI increased over time in both LV.SF.LTR groups, with higher values for the Cdkn2a-/-. On the other hand, treatment with SIN.LV.PGK resulted in lower MI in both groups compared to LV.SF.LTR groups, reflecting the higher clonal composition of the cells treated with the SIN.LV.PGK and the phenomenon of insertional mutagenesis in the LV.SF.LTR. Moreover, the higher MI values of the SIN.LV.PGK Cdkn2a-/- group compared with the WT group proved the induction of DNA fragility. Our results showed that the analysis of the accumulation of somatic mutations at single clone unraveled HSC proliferation stress in vivo, combining for the first time the analysis of acquired mutations with IS. We are now applying our model to different clinical trials, and studying HSCs sub- clonal trees by symmetric divisions, previously indistinguishable by IS only. Our study will open the doors to in vivo long-term non-invasive studies of HSC stability in patients

Diagnostic testing for SARS-CoV-2 infection in HHT patients: nasopharyngeal versus oropharyngeal swab

On March 11, 2020, WHO has defined the novel coronavirus disease SARS-CoV-2 (COVID-19) outbreak as a pandemic that still today continues to affect much of the world. Among the reasons for the rapid spread of SARS-CoV-2 infection, there is the role of asymptomatic or minimally symptomatic carriers. Therefore diagnostic testing is central to contain the global pandemic. Up to now real-time reverse transcriptase polymerase chain reaction-based molecular assays for detecting SARS-CoV-2 in respiratory specimens is the current reference standard for COVID-19 diagnosis. Based on current knowledge regarding the sensitivity of the molecular test, the highest positive detection rate is from lower respiratory tract specimens; alternatively it is possible to perform a nasopharyngeal or oropharyngeal swab. Nasopharyngeal swab is the preferred choice for SARS-CoV-2 testing since it seems to have a greater sensitivity; however the procedure is not always free of complications and an epistaxis can occur. Among patients with greatest risk of massive nosebleed there are HHT patients. Hereditary hemorrhagic telangiectasia is an autosomal dominant disease that leads to multiregional mucocutanous telangiectases and visceral arteriovenous malformations. Clinically, the presence of telangiectases in nasal mucosa is the cause of recurrent epistaxis. In HHT patients the execution of the nasopharyngeal swab can determine from little or no consequences to a massive epistaxis leading to the necessity of nasal packing generally followed by hospital admission. In HHT patients undergoing a diagnostic test to evaluate the SARS-CoV-2 infection status, especially in those patients with frequent epistaxis with a history of anemia and repeated hospitalizations, it is therefore advisable to perform an oropharyngeal swab. This, compared to the nasopharyngeal swab, exposes to a lower risk of severe nosebleeds related treatments, such as blood transfusions or invasive procedures. According to the risk-benefit assessment and based on our experience, we consider that, despite a lower diagnostic sensitivity, oropharyngeal swab is preferable to nasopharyngeal swab for the diagnosis of SARS CoV-2 infection in patients with HHT

Structural–Functional Relationship of the Ribonucleolytic Activity of aIF5A from Sulfolobus solfataricus

The translation factor IF5A is a highly conserved protein playing a well-recognized and well-characterized role in protein synthesis; nevertheless, some of its features as well as its abundance in the cell suggest that it may perform additional functions related to RNA metabolism. Here, we have undertaken a structural and functional characterization of aIF5A from the crenarchaeal Sulfolobus solfataricus model organism. We confirm the association of aIF5A with several RNA molecules in vivo and demonstrate that the protein is endowed with a ribonuclease activity which is specific for long and structured RNA. By means of biochemical and structural approaches we show that aIF5A can exist in both monomeric and dimeric conformations and the monomer formation is favored by the association with RNA. Finally, modelling of the three-dimensional structure of S. solfataricus aIF5A shows an extended positively charged surface which may explain its strong tendency to associate to RNA in vivo

The dimer-monomer equilibrium of SARS-CoV-2 main protease is affected by small molecule inhibitors

The maturation of coronavirus SARS-CoV-2, which is the etiological agent at the origin of the COVID-19 pandemic, requires a main protease Mpro to cleave the virus-encoded polyproteins. Despite a wealth of experimental information already available, there is wide disagreement about the Mpro monomer-dimer equilibrium dissociation constant. Since the functional unit of Mpro is a homodimer, the detailed knowledge of the thermodynamics of this equilibrium is a key piece of information for possible therapeutic intervention, with small molecules interfering with dimerization being potential broad-spectrum antiviral drug leads. In the present study, we exploit Small Angle X-ray Scattering (SAXS) to investigate the structural features of SARS-CoV-2 Mpro in solution as a function of protein concentration and temperature. A detailed thermodynamic picture of the monomer-dimer equilibrium is derived, together with the temperature-dependent value of the dissociation constant. SAXS is also used to study how the Mpro dissociation process is affected by small inhibitors selected by virtual screening. We find that these inhibitors affect dimerization and enzymatic activity to a different extent and sometimes in an opposite way, likely due to the different molecular mechanisms underlying the two processes. The Mpro residues that emerge as key to optimize both dissociation and enzymatic activity inhibition are discussed

Characterization of epidemiological distribution and outcome of COVID-19 in patients with hereditary hemorrhagic telangiectasia: a nationwide retrospective multi-centre study during first wave in Italy

Background: Coronavirus Disease 2019 (COVID-19) continues to have a devastating impact across the world. A number of pre-existing common clinical conditions were reported to represent risk factors for more severe COVID-19 outcomes. Hereditary Hemorrhagic Telangiectasia (HHT) is a rare vascular heritable disorders, characterized by complications secondary to visceral Arterio-Venous Malformations. The impact of HHT, as well as for many Rare Diseases (RDs) on infection susceptibility profile and clinical adverse outcome risk is an unresolved issue. Objectives: The main objectives were: to assess the clinical features and outcomes of HHT patients infected with COVID-19; to compare the relative infection risk in these patients with the Italian general population throughout the first pandemic wave; to investigate the factors potentially associated with severe COVID-19 outcome in HHT patients, and the possible impact of COVID-19 infection on HHT-related symptoms/complications. Finally, we aimed to estimate how the lockdown-associated wearing of personal protective equipment/individual protection devices could affect HHT-related telangiectasia bleeding frequency. Methods: The study is a nation-wide questionnaire-based survey, with a multi-Center retrospective cross-sectional design, addressed to the whole Italian HHT population. COVID-19 cases, occurring throughout the first pandemic wave, were collected by a questionnaire-based semi-structured interview. Only the cases ascertained by laboratory confirmation (molecular/serological) were included for epidemiological estimates. Information concerning eventual SarS-Cov-2 infection, as well as regarding HHT-related manifestations and HHT-unrelated co-morbidities were collected by the questionnaire. Prevalence data were compared to Italian general population in the same period. Results: The survey disclosed 9/296 (3.04%) COVID-19 cases, 8/9 of them being resident in Lombardy, the main epidemic epicenter. Pneumonia was reported by 4/9 patients, which prompted hospital admission and intensive care management in 2 cases. No fatal outcome was recorded. After careful refinement of epidemiological analysis, the survey evidenced overlapping infection risk in HHT compared to general population. Conclusions: COVID-19 infection profile parallels geographical distribution of epidemic foci. COVID-19 in HHT patients can lead to highly variable clinical profile, likely overlapping with that of general population. The HHT disease does not seem to involve a different approach in terms of hospital admission and access to intensive care with respect to general population

Effect of Polydispersity and Anisotropy in Colloidal and Protein Solutions: an Integral Equation Approach

Application of integral equation theory to complex fluids is reviewed, with particular emphasis to the effects of polydispersity and anisotropy on their structural and thermodynamic properties. Both analytical and numerical solutions of integral equations are discussed within the context of a set of minimal potential models that have been widely used in the literature. While other popular theoretical tools, such as numerical simulations and density functional theory, are superior for quantitative and accurate predictions, we argue that integral equation theory still provides, as in simple fluids, an invaluable technique that is able to capture the main essential features of a complex system, at a much lower computational cost. In addition, it can provide a detailed description of the angular dependence in arbitrary frame, unlike numerical simulations where this information is frequently hampered by insufficient statistics. Applications to colloidal mixtures, globular proteins and patchy colloids are discussed, within a unified framework.Comment: 17 pages, 7 figures, to appear in Interdiscip. Sci. Comput. Life Sci. (2011), special issue dedicated to Prof. Lesser Blu

Knowledge and health care resource allocation: CME/CPD course guidelines-based efficacy.

BACKGROUND: Most health care systems consider continuing medical education a potential tool to improve quality of care and reduce disease management costs. Its efficacy in general practitioners needs to be further explored. OBJECTIVE: This study assesses the effectiveness of a one-year continuing medical education/continuing professional development course for general practitioners, regarding the improvement in knowledge of ARIA and GINA guidelines and compliance with them in asthma management. METHODS: Sixty general practitioners, covering 68,146 inhabitants, were randomly allocated to continuing medical education/continuing professional development (five residential events +four short distance-learning refresher courses over one year) or no training. Participants completed a questionnaire after each continuing medical education event; key questions were repeated at least twice. The Local Health Unit prescription database was used to verify prescription habits (diagnostic investigations and pharmacological therapy) and hospitalizations over one year before and after training. RESULTS: Fourteen general practitioners (46.7%) reached the cut-off of 50% attendance of the training courses. Knowledge improved significantly after training (p < 0.001, correct answers to key questions +13%). Training resulted in pharmaceutical cost containment (trained general practitioners +0.5% vs. controls +18.8%) and greater attention to diagnosis and monitoring (increase in spirometry +63.4%, p < 0.01). CONCLUSION: This study revealed an encouraging impact of educational events on improvement in general practitioner knowledge of guidelines and daily practice behavioral changes. Long-term studies of large populations are required to assess the effectiveness of education on the behavior of physicians in asthma management, and to establish the best format for educational events