105 research outputs found
Purification, profiling and bioengineering of extracellular vesicles
The nano-sized membrane enclosed extracellular vesicles (EVs) transfer macromolecular
information in the form of proteins, nucleic acids and lipids across cells. They are important
mediators of cell-to-cell communication, but their relatively small size makes EV isolation and
down-stream analysis challenging. In this thesis, we ventured through some of the current
challenges in the EV field touching upon purification, small RNA and protein content profiling
and ultimately characterization of engineered vesicles at the molecular level.
The isolation of EVs from the complex fluid they are surrounded by, represents the first
hindrance in studying these vesicles. Ideally, the purification method should preserve the
integrity and natural properties of the EVs and simultaneously deplete the vesicular portion
from unwanted components. In Paper I, we describe a novel liquid chromatography technique
for EV purification, that combines size separation with bind elution (BE-SEC) entrapping
molecules smaller than 700 kDa within the matrix core. The BE-SEC isolation method yields
high particles recovery in a reproducible and time-efficient way, without neither affecting the
EVs natural surface protein signature nor their physicochemical properties. By adding a prior
tangential flow filtration step, the BE-SEC could be scaled-up and the EV preparation further
depleted from unwanted non-vesicular proteins and RNAs.
Secondly, therapeutically engineered EVs are promising delivery vehicles and linking the
administered vesicular dose to the molecular cargo concentration is of extreme relevance to
achieve a desired response. Therefore, analytical methods focused on single vesicles
quantification rather than ‘bulk’ analysis and improved bioengineered vesicles are of utmost
importance for therapeutic applications. In Paper II, we extensively characterize a set of
fluorescently labelled EV-associated proteins, employing several qualitative and quantitative
methods. Using Fluorescence Correlation Spectroscopy, we quantify the number of fluorescent
molecules per single loaded vesicle. Different loading efficiencies were observed for the tested
proteins, with the tetraspanins (CD63, CD9 and CD81) showing the highest loading efficiency
with an average of 40-60 fluorescent molecules per vesicle. To summarize, we provide a
reference for selecting EV sorting domains that best fit the desired outcome, as well as an array
of quantitative and qualitative methodologies to support EV engineering.
In Paper III, we investigate the native RNA and protein content of EVs, with a focus on small
RNAs and RNA binding proteins respectively. Across different mouse and human cell-derived
EVs, a deficiency of miRNA sequences and relative depletion of ‘miRNA-related’ proteins
were observed. The majority of the RNA sequences detected in EVs was represented by rRNA-, coding- and tRNA fragments, reflecting the observations in the respective protein portion,
where ribosomal and translational proteins were predominantly identified.
In conclusion, this thesis explores and advances some of the challenges encountered in the EV
field by ameliorating the EV isolation workflow in terms of time and scalability, linking the
vesicular transcriptome and proteome of EVs derived from various cell lines, and
systematically comparing and quantifying the sorting efficiency of different proteins into EVs
An investigation on 4-thiazolidinone derivatives as dual inhibitors of aldose reductase and protein tyrosine phosphatase 1B, in the search for potential agents for the treatment of type 2 diabetes mellitus and its complications
Designed multiple ligands (DMLs), developed to modulate simultaneously a number of selected targets involved in etiopathogenetic mechanisms of a multifactorial disease, such as diabetes mellitus (DM), are considered a promising alternative to combinations of drugs, when monotherapy results to be unsatisfactory. In this work, compounds 1–17 were synthesized and in vitro evaluated as DMLs directed to aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two key enzymes involved in different events which are critical for the onset and progression of type 2 DM and related pathologies. Out of the tested 4-thiazolidinone derivatives, compounds 12 and 16, which exhibited potent AR inhibitory effects along with interesting inhibition of PTP1B, can be assumed as lead compounds to further optimize and balance the dual inhibitory profile. Moreover, several structural portions were identified as features that could be useful to achieve simultaneous inhibition of both human AR and PTP1B through binding to non-catalytic regions of both target enzymes
Total energy global optimizations using non orthogonal localized orbitals
An energy functional for orbital based calculations is proposed, which
depends on a number of non orthogonal, localized orbitals larger than the
number of occupied states in the system, and on a parameter, the electronic
chemical potential, determining the number of electrons. We show that the
minimization of the functional with respect to overlapping localized orbitals
can be performed so as to attain directly the ground state energy, without
being trapped at local minima. The present approach overcomes the multiple
minima problem present within the original formulation of orbital based
methods; it therefore makes it possible to perform calculations for an
arbitrary system, without including any information about the system bonding
properties in the construction of the input wavefunctions. Furthermore, while
retaining the same computational cost as the original approach, our formulation
allows one to improve the variational estimate of the ground state energy, and
the energy conservation during a molecular dynamics run. Several numerical
examples for surfaces, bulk systems and clusters are presented and discussed.Comment: 24 pages, RevTex file, 5 figures available upon reques
Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome
Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49-96%) corresponded to rRNA-, coding-and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics.Peer reviewe
Sense of smell in chronic rhinosinusitis: A multicentric study on 811 patients
Introduction: The impairment of the sense of smell is often related to chronic rhinosinusitis (CRS) with or without nasal polyps (CRSwNP, CRSsNP). CRSwNP is a frequent condition that drastically worsens the quality of life of those affected; it has a higher prevalence than CRSsNP. CRSwNP patients experience severe loss of smell with earlier presentation and are more likely to experience recurrence of their symptoms, often requiring revision surgery. Methods: The present study performed a multicentric data collection, enrolling 811 patients with CRS divided according to the inflammatory endotype (Type 2 and non-Type 2). All patients were referred for nasal endoscopy for the assessment of nasal polyposis using nasal polyp score (NPS); Sniffin' Sticks olfactory test were performed to measure olfactory function, and SNOT-22 (22-item sinonasal outcome test) questionnaire was used to assess patients' quality of life; allergic status was evaluated with skin prick test and nasal cytology completed the evaluation when available. Results: Data showed that Type 2 inflammation is more common than non-type 2 (656 patients versus 155) and patients suffer from worse quality of life and nasal polyp score. Moreover, 86.1% of patients with Type 2 CRSwNP were affected by a dysfunction of the sense of smell while it involved a lesser percentage of non-Type 2 patients. Indeed, these data give us new information about type-2 inflammation patients' characteristics. Discussion: The present study confirms that olfactory function weights on patients' QoL and it represents an important therapeutic goal that can also improve patients' compliance when achieved. In a future - and present - perspective of rhinological precision medicine, an impairment of the sense of smell could help the clinician to characterize patients better and to choose the best treatment available
Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles
Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.International Society for Advancement of Cytometry Marylou Ingram Scholar 2019-2023H2020 EXPERTSwedish foundation of Strategic Research (SSF-IRC; FormulaEx)ERC CoG (DELIVER)Swedish Medical Research CouncilAccepte
Diagnostic therapeutic assistance pathway (PDTA) of type 2 chronic rhinosinusitis
Chronic rhinosinusitis (CRS) is a complex and heterogeneous disorder whose etiopathogenetic picture is not yet completely known and is classically divided into CRS with (CRSwNP) and without nasal polyps (CRSsNP). But today the distinction is made with type 2 and nontype 2 variants. A rational and defined pathway for the diagnosis of chronic rhinosinusitis is an indispensable means to be able to arrive at a correct identification of the patient. This typing is essential to be able to arrive at the correct course of treatment, which turns out to be different for different types of patients. For this reason, the realization of a diagnostic therapeutic pathway represents a fundamental way for the otolaryngologist specialist but not only, since today diagnostics has a multidisciplinary framework. In the present work, precise indications have been developed to arrive at a correct diagnosis. The various diagnostic pathways and processes to arrive at a correct therapeutic framing have been highlighted. Therapy ranging from medical therapy to surgical therapy without neglecting the new biological therapies. It does not represent a guideline but a diagnostic method that can be adapted to all the various territorial realities
The association between insight and depressive symptoms in schizophrenia: Undirected and Bayesian network analyses
Background. Greater levels of insight may be linked with depressive symptoms among patients with schizophrenia, however, it would be useful to characterize this association at symptom-level, in order to inform research on interventions. Methods. Data on depressive symptoms (Calgary Depression Scale for Schizophrenia) and insight (G12 item from the Positive and Negative Syndrome Scale) were obtained from 921 community-dwelling, clinically-stable individuals with a DSM-IV diagnosis of schizophrenia, recruited in a nationwide multicenter study. Network analysis was used to explore the most relevant connections between insight and depressive symptoms, including potential confounders in the model (neurocognitive and social-cognitive functioning, positive, negative and disorganization symptoms, extrapyramidal symptoms, hostility, internalized stigma, and perceived discrimination). Bayesian network analysis was used to estimate a directed acyclic graph (DAG) while investigating the most likely direction of the putative causal association between insight and depression. Results. After adjusting for confounders, better levels of insight were associated with greater self-depreciation, pathological guilt, morning depression and suicidal ideation. No difference in global network structure was detected for socioeconomic status, service engagement or illness severity. The DAG confirmed the presence of an association between greater insight and self-depreciation, suggesting the more probable causal direction was from insight to depressive symptoms. Conclusions. In schizophrenia, better levels of insight may cause self-depreciation and, possibly, other depressive symptoms. Person-centered and narrative psychotherapeutic approaches may be particularly fit to improve patient insight without dampening self-esteem
The interplay among psychopathology, personal resources, context-related factors and real-life functioning in schizophrenia: stability in relationships after 4 years and differences in network structure between recovered and non-recovered patients
Improving real-life functioning is the main goal of the most advanced integrated treatment programs in people with schizophrenia. The Italian Network for Research on Psychoses previously explored, by using network analysis, the interplay among illness-related variables, personal resources, context-related factors and real-life functioning in a large sample of patients with schizophrenia. The same research network has now completed a 4-year follow-up of the original sample. In the present study, we used network analysis to test whether the pattern of relationships among all variables investigated at baseline was similar at follow-up. In addition, we compared the network structure of patients who were classified as recovered at follow-up versus those who did not recover. Six hundred eighteen subjects recruited at baseline could be assessed in the follow-up study. The network structure did not change significantly from baseline to follow-up, and the overall strength of the connections among variables increased slightly, but not significantly. Functional capacity and everyday life skills had a high betweenness and closeness in the network at follow-up, as they had at baseline, while psychopathological variables remained more peripheral. The network structure and connectivity of non-recovered patients were similar to those observed in the whole sample, but very different from those in recovered subjects, in which we found few connections only. These data strongly suggest that tightly coupled symptoms/dysfunctions tend to maintain each other's activation, contributing to poor outcome in schizophrenia. Early and integrated treatment plans, targeting variables with high centrality, might prevent the emergence of self-reinforcing networks of symptoms and dysfunctions in people with schizophrenia
The interplay among psychopathology, personal resources, context-related factors and real-life functioning in schizophrenia: stability in relationships after 4 years and differences in network structure between recovered and non-recovered patients
Improving real-life functioning is the main goal of the most advanced integrated treatment programs in people with schizophrenia. The Italian Network for Research on Psychoses previously explored, by using network analysis, the interplay among illness-related variables, personal resources, context-related factors and real-life functioning in a large sample of patients with schizophrenia. The same research network has now completed a 4-year follow-up of the original sample. In the present study, we used network analysis to test whether the pattern of relationships among all variables investigated at baseline was similar at follow-up. In addition, we compared the network structure of patients who were classified as recovered at follow-up versus those who did not recover. Six hundred eighteen subjects recruited at baseline could be assessed in the follow-up study. The network structure did not change significantly from baseline to follow-up, and the overall strength of the connections among variables increased slightly, but not significantly. Functional capacity and everyday life skills had a high betweenness and closeness in the network at follow-up, as they had at baseline, while psychopathological variables remained more peripheral. The network structure and connectivity of non-recovered patients were similar to those observed in the whole sample, but very different from those in recovered subjects, in which we found few connections only. These data strongly suggest that tightly coupled symptoms/dysfunctions tend to maintain each other's activation, contributing to poor outcome in schizophrenia. Early and integrated treatment plans, targeting variables with high centrality, might prevent the emergence of self-reinforcing networks of symptoms and dysfunctions in people with schizophrenia
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