Knowledge-based identification of functional domains in proteins

The characterization of proteins and enzymes is traditionally organised according to the sequence-structure-function paradigm. The investigation of the inter-relationships between these three properties has motivated the development of several experimental and computational techniques, that have made available an unprecedented amount of sequence and structural data. The interest in developing comparative methods for rationalizing such copious information has, of course, grown in parallel. Regarding the structure-function relationship, for instance, the availability of experimentally resolved protein structures and of computer simulations have improved our understanding of the role of proteins' internal dynamics in assisting their functional rearrangements and activity. Several approaches are currently available for elucidating and comparing proteins' internal dynamics. These can capture the relevant collective degrees of freedom that recapitulate the main conformational changes. These collective coordinates have the potential to unveil remote evolutionary relationships between proteins, that are otherwise not easily accessible from purely sequence- or structure-based investigations. Starting from this premise, in the first chapter of this thesis I will present a novel and general computational method that can detect large-scale dynamical correlations in proteins by comparing different representative conformers. This is accomplished by applying dimensionality-reduction techniques to inter-amino acid distance fluctuation matrices. As a result, an optimal quasi-rigid domain decomposition of the protein or macromolecular assembly of interest is identified, and this facilitates the functionally-oriented interpretation of their internal dynamics. Building on this approach, in the second chapter I will discuss its systematic application to a class of membrane proteins of paramount biochemical interest, namely the class A G protein-coupled receptors. The comparative analysis of their internal dynamics, as encoded by the quasi-rigid domains, allowed us to identify recurrent patterns in the large-scale dynamics of these receptors. This, in turn, allowed us to single out a number of key functional sites. These were, for the most part, previously known -- a fact that at the same time validates the method, and gives confidence for the viability of the other, novel sites. Finally, for the last part of the thesis, I focussed on the sequence-structure relationship. In particular, I considered the problem of inferring structural properties of proteins from the analysis of large multiple sequence alignments of homologous sequences. For this purpose, I recasted the strategies developed for the dynamical features extraction in order to identify compact groups of coevolving residues, based only on the knowledge of amino acid variability in aligned primary sequences. Throughout the thesis, many methodological techniques have been taken into considerations, mainly based on concepts from graph theory and statistical data analysis (clustering). All these topics are explained in the methodological sections of each chapter

Ultrafast dynamics in unaligned MWCNTs decorated with metal nanoparticles

The relaxation dynamics of unaligned multi-walled carbon nanotubes decorated with metallic nanoparticles have been studied by using transient optical measurements. The fast dynamics due to the short-lived free-charge carriers excited by the pump are not affected by the presence of nanoparticles. Conversely, a second long dynamics, absent in bare carbon nanotubes, appears only in the decorated samples. A combination of experiment and theory allows us to ascribe this long dynamics to relaxation channels involving electronic states localized at the tube-nanoparticle interface

Management of PALB2-associated breast cancer: A literature review and case report

Key Clinical Message Germline pathogenic variants (PV) of the PALB2 tumor suppressor gene are associated with an increased risk of breast, pancreatic, and ovarian cancer. In previous research, PALB2-associated breast cancer showed aggressive clinicopathological phenotypes, particularly triple-negative subtype, and higher mortality regardless of tumor stage, type of chemotherapy nor hormone receptor status. The identification of this germline alteration may have an impact on clinical management of breast cancer (BC) from the surgical approach to the systemic treatment choice. We herein report the case of a patient with a germline PV of PALB2, diagnosed with locally advanced PD-L1 positive triple-negative BC, who progressed after an immune checkpoint inhibitor (ICI)-containing regimen and then experienced a pathologic complete response after platinum-based chemotherapy. This case report hints a major role of the germline PALB2 alteration compared to the PD-L1 expression as cancer driver and gives us the opportunity to extensively review and discuss the available literature on the optimal management of PALB2-associated BC. Overall, our case report and review of the literature provide additional evidence that the germline analysis of PALB2 gene should be included in routine genetic testing for predictive purposes and to refine treatment algorithms

IL-7 and IL-15 allow the generation of suicide gene–modified alloreactive self-renewing central memory human T lymphocytes

Abstract Long-term clinical remissions of leukemia, after allogeneic hematopoietic stem cell transplantation, depend on alloreactive memory T cells able to self-renew and differentiate into antileukemia effectors. This is counterbalanced by detrimental graft-versus-host disease (GVHD). Induction of a selective suicide in donor T cells is a current gene therapy approach to abrogate GVHD. Unfortunately, genetic modification reduces alloreactivity of lymphocytes. This associates with an effector memory (TEM) phenotype of gene-modified lymphocytes and may limit antileukemia effect. We hypothesized that alloreactivity of gene-modified lymphocytes segregates with the central memory (TCM) phenotype. To this, we generated suicide gene–modified TCM lymphocytes with a retroviral vector after CD28 costimulation and culture with IL-2, IL-7, or a combination of IL-7 and IL-15. In vitro, suicide gene–modified TCM cells self-renewed upon alloantigen stimulation and resisted activation-induced cell death. In a humanized mouse model, only suicide gene–modified T cells cultured with IL-7 and IL-15 persisted, differentiated in TEM cells, and were as potent as unmanipulated lymphocytes in causing GVHD. GVHD was halted through the activation of the suicide gene machinery. These results warrant the use of suicide gene–modified TCM cells cultured with IL-7 and IL-15 for the safe exploitation of the alloreactive response against cancer

Unifying view of mechanical and functional hotspots across class A GPCRs

G protein-coupled receptors (GPCRs) are the largest superfamily of signaling proteins. Their activation process is accompanied by conformational changes that have not yet been fully uncovered. Here, we carry out a novel comparative analysis of internal structural fluctuations across a variety of receptors from class A GPCRs, which currently has the richest structural coverage. We infer the local mechanical couplings underpinning the receptors' functional dynamics and finally identify those amino acids whose virtual deletion causes a significant softening of the mechanical network. The relevance of these amino acids is demonstrated by their overlap with those known to be crucial for GPCR function, based on static structural criteria. The differences with the latter set allow us to identify those sites whose functional role is more clearly detected by considering dynamical and mechanical properties. Of these sites with a genuine mechanical/dynamical character, the top ranking is amino acid 7x52, a previously unexplored, and experimentally verifiable key site for GPCR conformational response to ligand binding. \ua9 2017 Ponzoni et al

Immune Regulatory Neural Stem/Precursor Cells Protect from Central Nervous System Autoimmunity by Restraining Dendritic Cell Function

The systemic injection of neural stem/precursor cells (NPCs) provides remarkable amelioration of the clinico-pathological features of experimental autoimmune encephalomyelitis (EAE). This is dependent on the capacity of transplanted NPCs to engage concurrent mechanisms of action within specific microenvironments in vivo. Among a wide range of therapeutic actions alternative to cell replacement, neuroprotective and immune modulatory capacities of transplanted NPCs have been described. However, lacking is a detailed understanding of the mechanisms by which NPCs exert their therapeutic plasticity. This study was designed to identify the first candidate that exemplifies and sustains the immune modulatory capacity of transplanted NPCs.To achieve the exclusive targeting of the peripheral immune system, SJL mice with PLP-induced EAE were injected subcutaneously with NPCs and the treatment commenced prior to disease onset. NPC-injected EAE mice showed significant clinical improvement, as compared to controls. Exogenous NPCs lacking the expression of major neural antigens were reliably (and for long-term) found at the level of draining lymph nodes, while establishing sophisticated anatomical interactions with lymph node cells. Importantly, injected NPCs were never found in organs other than lymph nodes, including the brain and the spinal cord. Draining lymph nodes from transplanted mice showed focal up-regulation of major developmental stem cell regulators, such as BMP-4, Noggin and Sonic hedgehog. In lymph nodes, injected NPCs hampered the activation of myeloid dendritic cells (DCs) and steadily restrained the expansion of antigen-specific encephalitogenic T cells. Both ex vivo and in vitro experiments identified a novel highly NPC-specific–BMP-4-dependent–mechanism hindering the DC maturation.The study described herein, identifies the first member of the TGF β/BMP family of stem cell regulators as a novel tolerogenic factor released by NPCs. Full exploitation of this pathway as an efficient tool for vaccination therapy in autoimmune inflammatory conditions is underway

Identification of a 3-gene model as a powerful diagnostic tool for the recognition of ALK-negative anaplastic large-cell lymphoma

16siAnaplastic large-cell lymphomas (ALCLs) are a group of clinically and biologically heterogeneous diseases including the ALK+ and ALK+ systemic forms. Whereas ALK+ ALCLs are molecularly characterized and can be readily diagnosed, specific immunophenotypic or genetic features to define ALK- ALCL are missing, and their distinction from other T-cell non-Hodgkin lymphomas (T-NHLs) remains controversial. In the present study, we undertook a transcriptional profiling meta-analysis of 309 cases, including ALCL and other primary T-NHL samples. Pathway discovery and prediction analyses defined a minimum set of genes capable of recognizing ALK- ALCL. Application of quantitative RT-PCR in independent datasets from cryopreserved and formalin-fixed paraffin-embedded samples validated a 3-gene model (TNFRSF8, BATF3, and TMOD1) able to successfully separate ALK- ALCL from peripheral T-cell lymphoma not otherwise specified, with overall accuracy near 97%. In conclusion, our data justify the possibility of translating quantitative RT-PCR protocols to routine clinical settings as a new approach to objectively dissect T-NHL and to select more appropriate therapeutic protocols. © 2012 by The American Society of Hematology.openopenAgnelli L.; Mereu E.; Pellegrino E.; Limongi T.; Kwee I.; Bergaggio E.; Ponzoni M.; Zamo A.; Iqbal J.; Piccaluga P.P.; Neri A.; Chan W.C.; Pileri S.; Bertoni F.; Inghirami G.; Piva R.Agnelli, L.; Mereu, E.; Pellegrino, E.; Limongi, T.; Kwee, I.; Bergaggio, E.; Ponzoni, M.; Zamo, A.; Iqbal, J.; Piccaluga, P. P.; Neri, A.; Chan, W. C.; Pileri, S.; Bertoni, F.; Inghirami, G.; Piva, R

Isolated Bone Marrow Manifestation of HIV-Associated Hodgkin Lymphoma

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Predicting lymphoma in Sjögren's syndrome and the pathogenetic role of parotid microenvironment through precise parotid swelling recording

Objective: Parotid swelling (PSW) is a major predictor of non-Hodgkin lymphoma (NHL) in primary Sjögren's syndrome (pSS). However, since detailed information on the time of onset and duration of PSW is scarce, this was investigated to verify whether it may lead to further improved prediction. NHL localisation was concomitantly studied to evaluate the role of the parotid gland microenvironment in pSS-related lymphomagenesis. Methods: A multicentre study was conducted among patients with pSS who developed B cell NHL during follow-up and matched controls that did not develop NHL. The study focused on the history of salivary gland and lachrymal gland swelling, evaluated in detail at different times and for different durations, and on the localisation of NHL at onset. Results: PSW was significantly more frequent among the cases: at the time of first referred pSS symptoms before diagnosis, at diagnosis, and from pSS diagnosis to NHL. The duration of PSW was evaluated starting from pSS diagnosis, and the NHL risk increased from PSW of 2-12 months to > 12 months. NHL was prevalently localised in the parotid glands of the cases. Conclusion: A more precise clinical recording of PSW can improve lymphoma prediction in pSS. PSW as a very early symptom is a predictor, and a longer duration of PSW is associated with a higher risk of NHL. Since lymphoma usually localises in the parotid glands, and not in the other salivary or lachrymal glands, the parotid microenvironment appears to be involved in the whole history of pSS and related lymphomagenesis