Life Science 2017, 1st Joint Annual Symposium of the Departments of Biology and Biotechnology, Molecular Medicine and CNR-­‐Institute of Molecular Genetics ABSTRACTS DBB Genetic variability in basic and clinical science The diffusion and evolution of multidrug-­‐resistant Klebsiella pneumoniae characterized by genomic epidemiology Sassera D Laboratory of Parasitology, Symbiosis, Genomic evolution, DBB UNIPV, Pavia The emergence of bacterial strains capable of resisisting to all the major classes of antibiotics is currently a sanitary concern of great importance worldwide. Among these feared 'superbugs' a prominent role is played by Klebsiella pneumoniae (Kp). Kp is an opportunistic nosocomial pathogen belonging to the Enterobacteriaceae family. Thanks to a highly plastic genome, and to the capacity to acquire novel plasmids, multiple strains of Kp have become resistant to first generation β-­‐lactams, extended spectrum cephalosporins, to the recently developed carbapenems and even to last resort drugs like colistin, causing thousands of deaths each year worldwide. The most successful strain of Kp is the clonal group 258 (CG258), which is responsible for most cases of panresistant Kp nosocomial infections in multiple countries, including an alarming percentage of the cases in Italian hospitals. The role of environmental strains in the evolution of panresistant isolates of Kp is currently being investigated with specific attention to animal isolates, subjected to strong selective pressures due to antibiotic abuse in livestock. Here I present three studies of genomics, focused at different spatial and temporal level, with the common goal to characterize Kp, understand its spread and evolution, and ultimately envision novel control strategies. The first study is the characterization of a local hospital outbreak of Kp that caused 15 infections and two deaths in an intensive care unit. In this work genomics allowed to characterize the isolates virulence profile and determine the relationship among the isolates, to assess that the spread of the outbreak followed a star-­‐
like radiation. The second study allowed to investigate the genomic variability of Kp in Italy through 89 full genome sequences, and to use these data to provide clues on the evolution of the CG258, highlighting the presence of a 1.3 megabases genomic recombination at the base of the CG258 clade, possibly instrumental in the evolution of this pandemic clonal complex. The third study, which will start in April 2017, envisions to provide an unprecedented level of characterization to the Kp population in a defined area, Pavia and its surroundings. By sequencing thousands of genomes and metagenomes from multiple hospitals, healthy carriers, livestock and from the environment, we will reconstruct gene flow through the different settings tracing the routes of transmission and evolution of this pathogen. A genome-­‐wide portrait of Italy Raveane A, Grugni V, Battaglia V, Capodiferro MR, Brandini S, Torroni A, Semino O, Achilli A, Olivieri A Laboratories of Genomics of Human and Animal Populations, DBB UNIPV, Pavia Surrounded by the sea and bounded by the Alps, Italy extends over more than 1,000 km along a North-­‐South axis and comprises the two largest islands of the Mediterranean, Sicily and Sardinia. The combination of this geographic complexity with a rich set of historical events and cultural dynamics has the potential to shape in a unique way the distribution of its genetic variation. An extensive genome-­‐wide analysis allowed us to dissect the structure and the admixture profile of Italian populations. Preliminary results confirmed the distinctiveness of Sardinia compared to the rest of Italy, while a more continuum distribution of genetic variation along a North-­‐South axis was observed for the remaining samples. A complex pattern of admixture episodes over the last few thousand years has been revealed. In particular, a strong component from the Caucasus along the entire Italian peninsula has been identified. This work is carried out in collaboration with the following research groups: Montinaro F, Capelli C -­‐ Dept. of Zoology, Univ. of Oxford, UK; Cardinali I, Lancioni H -­‐ Dept. of Chemistry, Biology and Biotechnology, Univ. of Perugia, Italy; Mulas A, Zoledziewska M -­‐ Istituto di Ricerca Genetica e Biomedica IRGB, CNR, Lanusei, Italy; Aneli S, Di Gaetano C, Guerrera S, Piazza A, Matullo G -­‐ Dept. of Medical Sciences, Univ of Turin, and HuGeF, Turin, Italy.; Barlera S -­‐ Dept.of Cardiovascular Research, IRCCS Mario Negri Institute for Pharmacological Research, Milan, Italy; Parolo S, Bione S -­‐ Computational Biology Unit, Institute of Molecular Genetics, IGM, CNR, Pavia, Italy, Boncoraglio G -­‐ Dept.of Cerebrovascular Diseases, IRCCS Istituto Neurologico Carlo, Besta, Milan, Italy, Brisighelli F, Pascali V-­‐ Catholic University of the Sacred Heart, Institute of Legal Medicine and Insurance, Rome, Italy; Di Blasio AM -­‐ Molecular Biology Laboratory, Istituto Auxologico Italiano, Milan, Italy, Pagani L-­‐ Div. of Biological Anthropology, Univ. of Cambridge, UK; Cucca F, Angius A -­‐ Istituto di Ricerca Genetica e Biomedica, IRGB, CNR, Monserrato, Italy; Metspalu M-­‐ Dept. of Evolutionary Biology, Estonian Biocentre and Univ. of Tartu, Estonia; Hellenthal G -­‐ UCL Genetics Institute, University College London, UK. Unravelling the genetic history of human populations: new insights on South America peopling Grugni V, Battaglia V, Bertoglio B, Brandini S, Capodiferro MR, Raveane A, Perego UA, Ferretti L, Olivieri A, Achilli A, Torroni A, Semino O Laboratories of Genomics of Human and Animal Populations, DBB UNIPV, Pavia With the aim of reconstructing the history and evolution of human populations, we study the geographic distribution of modern human genomic variation by using phylogeographic approaches associated with parametric and non-­‐parametric statistical analyses which allow us to: (i) identify ancestral components and founder haplotypes, (ii) evaluate modern and ancient admixture events, (iii) detect ancient migration routes and archaic introgressions, (iv) estimate migration times and (v) evaluate possible differences in migration rates between males and females. Results can be used in several scientific and cultural fields: from forensics to history, archaeology, linguistic, anthropology, education and public health. Presentations will focus on the following result: This presentation will focus on the Y-­‐chromosome variation of Native Americans. Deep Y-­‐chromosome sequencing and phylogeographic analyses within a common Native American haplogroup (Q) allowed us to start exploring the peopling of Meso-­‐ and South-­‐America from a male perspective. Results obtained, in agreement with recent archaeological findings in South America, are suggestive of a rapid initial growth after the initial human arrival (~10 thousand years ago, kya), followed by a relatively constant population size from 8 kya, when domestication slowly started in NW South America, and a more recent phase of exponential growth from 3 kya, when there was a shift to a predominantly sedentary and agricultural subsistence This work is carried out in collaboration with the following research group: Trombetta B, Cruciani F -­‐ Dip. di Biologia e Biotecnologie “C. Darwin”, Univ. La Sapienza, Rome, Italy. In vitro and in vivo models: dissecting disease mechanisms and developing novel therapeutic approaches Structural biology: dissecting cellular processes at molecular level Binda C, Magnani F, Forneris F, Mattevi A. Laboratories of Structural Biology, DBB UNIPV, Pavia The common theme in the projects ongoing in the Structural Biology groups of the Department of Biology and Biotechnology is the study of cellular processes at a molecular level by unravelling the structural properties of proteins and their interactions. The core of the research activity is represented by X-­‐ray crystallography and other biochemical and biophysical methodologies such as enzymatic assays, fluorescence and absorbance spectroscopy, surface plasmon resonance. An overview of few of the projects will be presented, which can be grouped in two main topics. One research line involves the study of proteins of medical relevance: the histone-­‐
modifying enzymes LSD1 and LSD2 and their interaction with chromatin partners and with inhibitors in the framework of the role of these enzymes in cancer; the mitochondrial-­‐membrane drug targets human monoamine oxidases MAO A and MAO B in the context of neurodegenerative and cardiovascular diseases; the human flavin monooxygenaseses FMO3 and FMO5 whose role in detoxification and bioactivation of specific pharmaceuticals and xenobiotics is emerging as comparable to that of the well-­‐known cytochrome P450. The other research line originates from a EU-­‐funded project based on a collaborative network involving both academic groups and industries whose aim is the study of enzymes used for biocatalytic applications. In particular, data will be presented on flavin-­‐dependent oxidases and monooxygenases that are being investigated to develop mutants with improved properties for industrial purposes. Innovative strategies for shaping a neuroprotective CNS microenvironment in amyotrophic lateral sclerosis (ALS) Peviani M1,2, Curti D1 1
Laboratory of Molecular & Cellular Neuropharmacology, DBB UNIPV, Pavia; 2Harvard Medical School, Dana Farber Cancer Institute -­‐ Boston's Children Hospital Cancer and blood disorders center, Boston, MA (USA) ALS is a progressive neurodegenerative disorder affecting motoneurons (MNs) in brain and spinal cord, resulting in paralysis and death. A functional crosstalk between neurons and non-­‐neuronal cells (including astrocytes, oligodendrocytes and microglia is crucial for the induction of MN death in ALS. Thus, therapeutic attempts should take into account not only strategies aimed at preserving MN function but also the modulation of glial reactivity to favor a neuroprotective, pro-­‐regenerative environment/milieu. In line with this observation, we recently highlighted that agonists of the Sigma-­‐1 receptor (S1R), a chaperone-­‐like protein localized at the Endoplasmic reticulum (ER)-­‐mitochondrion interface, can ameliorate the disease progression in ALS mouse models by exerting neuroprotective but also immunomodulatory effects involving the skewing of microglia reactivity towards a cytoprotective and neuro-­‐restorative phenotype. Aims of our research are to improve the protocols for preclinical and clinical investigations of drug candidates in ALS through: 1) the development of a MALDI-­‐MS-­‐based imaging system for simultaneous evaluation (ex vivo), in spinal cord slices, of drug distribution and efficacy readouts. S1R ligands are substrates for P-­‐glicoprotein (P-­‐gp) responsible for pharmacoresistance. The amount of drug able to reach the ALS vulnerable regions, at different disease stages and after treatment with or without a P-­‐gp inhibitor, shall be assessed in ALS mice to establish the correct dosage and/or to address the rationale design of molecules able to overcome this drawback. 2) the application of biodegradable, biocompatible nanoparticles (NPs) capable of targeting microglia cells in vivo. These NPs may be further improved to function as innovative multiple-­‐drug delivery platforms for the fine and selective tuning of microglia responses to counteract disease progression in ALS. To optimize the targeting for activated microglia, we are exploring the functionalization of NPs with Positron Emission Tomography (PET) tracers targeted to receptors upregulated in microglia cells during neuroinflammation. We designed our NPs platform to allow the use of paramagnetic compounds and obtain also traceability by Magnetic Resonance Imaging (MRI). Collaborations: Davoli E, Bendotti C, IRCCS Mario Negri, Milan; Collina S, Dept. Drug Sciences UNIPV, Pavia
Autophagy and celiac disease: a new way to”digest” gluten Comincini S1,Manai F1,Martinelli C1,Adrasto S1,Gabriele F1,Morandi M1,Biggiogera M1,Meazza C2, Bozzola M2 1
DBB UNIPV, Pavia; 2Dept. Internal Medicine and Therapeutics, Pediatrics and Adolescentology Unit, UNIPV, Fondazione IRCCS San Matteo, Pavia Celiac disease is a serious genetic autoimmune disease triggered by consuming gluten derived proteins, present in wheat, barley and rye. In celiac patients the immune system responds by damaging the finger-­‐like villi of the small intestine resulting in problems to absorb nutrients into the bloodstream. Additionally, celiac patients are at-­‐risk for serious health consequences, like other autoimmune diseases, osteoporosis, thyroid disease, and even certain cancers. It was recently proposed that the autophagy process (a physiological, regulated, destructive mechanism of the cell that disassembles unnecessary or dysfunctional components) might be impaired in particular cells within the gut of celiac patients. We have therefore initially explored the possibility to modulate the functionality of the autophagy process in presence of a typical digested product of the gluten, the gliadin peptide. To this purpose, we have evaluated the endocytic process of gliadin in different cell models, how these cells can catabolize the exogenous proteins and, if they can release gliadin into the extracellular space. As a general result, cells were not able to catabolize through autophagy the gliadin proteins: in fact, gliadin proteins were temporary store within autophagosomes but next released as large macroscopic aggregates outside cells. To activate the autophagy flux, we have assayed a starvation protocol to the cells, in presence of the gliadin proteins: we have therefore documented that, after autophagy activation, cells increased the rate of degradation and significantly reduced the release of undigested gliadin. Altogether, these preliminary results indicated that the modulation of the autophagy process might contribute to reduce the not proper immunomodulatory and toxic properties of the gliadin-­‐derived peptides, decreasing therefore the pathological inflammatory and autoimmune responses typical of the celiac disease. Identification of novel druggable sites in key players of oxidative stress Magnani F, Nenci S, Fananas EM, Ceccon M, Mattevi A Laboratories of Structural Biology, DBB UNIPV, Pavia During our talk, we will present the 3D-­‐structure of the enzymes involved in oxidative stress and in the insurgence of some diseases, such as cancer. Novel druggable sites for pharmacological intervention are identified and can now be exploited for the design of highly sought drugs. Development of “resistance resistant” antitubercular drugs by multitargeting strategy Chiarelli LR, Orena BS, Mori G, Riccardi G, Pasca MR Laboratory of Molecular Microbiology, DBB UNIPV, Pavia Tuberculosis (TB) still remains an urgent global health issue, with more than 10 million new cases and an estimated 1.4 million deaths in 2015, mostly due to the appearance of multidrug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (Mtb) strains. Indeed, the spreading of MDR Mtb, with about 480,000 cases in 2015, 10% of which have been reported as XDR, with high mortality rates, has led to an increased requirement of new drugs with novel mechanisms of action. Despite its great potential, target-­‐based approach proved to be mostly unsuccessful for anti-­‐TB drug discovery, mainly because of the difficulty in identifying compounds that cross the mycobacterial cell wall. By contrast, high-­‐throughput screening (HTS) has been the preferential method adopted for the identification of new hits from large compound libraries and led to the identification of several active molecules, as well as novel targets. Nonetheless, antibiotic resistance insurgence still remains a serious problem, leading to the necessity of drugs that might be more “resistance-­‐resistant”. A possible approach in this direction could be the multi-­‐targeting strategy, in which a single compound having more than one target, could be easier to develop than conventional combination therapies with two new drugs. In this context, through a whole cell screening we identified a series of prodrugs, activated by the EthA mono-­‐
oxigenase, which were demonstrated to efficiently inhibit the CTP synthetase (PyrG), thus validating this essential mycobacterial enzyme involved in pyrimidine biosynthesis as new antitubercular drug target. Moreover, by microbiological and biochemical approaches, the same compounds were demonstrated to target also the pantothenate kinase (PanK), thus suggesting an additional role of this enzyme in their mechanism of action. PanK is an essential enzyme involved in coenzyme A biosynthesis, and showing significant diversity with the eukaryotic pantothenate kinases, is considered a useful potential antitubercular drug target, and has already been exploited for target-­‐based screens. Being PyrG and PanK essential enzymes in Mtb, involved in different biosynthetic pathways (de novo pyrimidine synthesis and CoA synthesis, respectively), they have been recognized as potential targets for the development of antibacterials. Indeed, a further target-­‐based screening of a compounds library against PyrG and PanK activity, delivered new common inhibitors with antimycobacterial activity, confirming that the two enzymes are suitable tools to develop new multitargeting antitubercular compounds. Novel insights into the contribution of platelet-­‐derived microparticles to cancer metastasis Guidetti G, Zarà M, Boselli D, Villa C, Canobbio I, Visconte C, Canino J, Torti M. Laboratory of Platelet Biochemistry, DBB UNIPV, Pavia In addition to their fundamental contribution to thrombosis and haemostasis, blood platelets play important roles also in cancer. In particular, platelets are known to support metastasis through multiple, but only partially understood mechanisms. Platelet-­‐derived microparticles (PMPs) are small vesicles released by activated platelets and involved in several physiological processes, including blood coagulation, immunity, and cell-­‐to-­‐cell communication. Interestingly, increased levels of circulating PMPs are associated to several types of metastatic cancer, and it has recently been proposed that PMPs may represent novel players in the contribution of platelets to metastasis. This work aimed to investigate the involvement of PMPs in the interplay between human platelets and breast cancer, using as cellular models the cell lines MDA-­‐MB-­‐231 and MCF-­‐7, which are characterized by an intrinsic strong and poor invasiveness, respectively. In particular, we have analysed the ability of these cancer cells to induce the release of PMPs by platelets and explored whether cancer cell-­‐induced PMPs display specific pro-­‐
metastatic properties. Through flow cytometry, fluorescence microscopy and protein quantification experiments, we found that exposure of platelets to cancer cells caused a strong release of PMPs, with MDA-­‐MB-­‐231 cells being significantly more efficient than MCF-­‐7 cells in stimulating such response. The two types of cancer cell-­‐induced PMPs were then purified and incubated with MDA-­‐MB-­‐231 and MCF-­‐7 cells to determine their influence on invasiveness of these cell lines. Both types of PMPs were essentially unable to interact with MCF-­‐7 cells, whereas a strong association with MDA-­‐MB-­‐231 cells was observed. Confocal microscopy analysis revealed that cell-­‐vesicle association did not involve a simple interaction at membrane level, rather PMPs were efficiently internalized by MDA-­‐MB-­‐231 cells. The internalization of PMPs by MDA-­‐MB-­‐231 cells was accompanied by the phosphorylation of proteins involved in the regulation of cell motility, such as p38 MAPK and myosin light chain (MLC) and, importantly, this was associated to a significant stimulation of cell migration and invasiveness. Consistently with the results obtained in the interaction analysis, no significant effects of PMPs on MCF-­‐7 cells were observed. Altogether these results provide novel evidence about the mechanism that supports the increase of circulating PMPs in cancer patients and suggest that, depending on the nature of circulating cancer cells, PMPs can mediate pro-­‐metastatic signals that potentiate cancer invasiveness. Blood platelets as a link between vascular inflammation and Alzheimer’s disease Canobbio I, Visconte C, Canino J, Guidetti G, Zarà M, Pula G, Torti M Laboratory of Platelet Biochemistry, DBB UNIPV, Pavia Alzheimer’s disease (AD) is the most invalidating neurological disorder in the elderly. It is characterized by abnormal deposition of amyloid beta peptides in the brain parenchyma that disrupt synaptic signaling. Amyloid deposition is also found in cerebral vessels together with chronic inflammation. Emerging evidence points to a central role of cerebrovascular dysfunctions in the onset of AD. According to the “vascular hypothesis”, AD may be initiated by vascular dysfunctions that cause cerebral hypoperfusion and neuron suffering. Indeed, multiple bidirectional connections exist between AD and cerebrovascular dysfunctions. Vascular risks factors, such as diabetes, hypertension and hypercholesterolemia, predispose and accelerate AD and conversely AD patients show increased hemorragic or ischemic stroke risk. In this new scenario, blood platelets play a crucial role. Circulating platelets are responsible for haemostasis and thrombosis but are also implicated in neuroinflammation. Interestingly, blood platelets contain a high concentration of amyloid precursor protein (APP) and generate amyloid beta peptides. Following activation, platelets release amyloid beta peptides and inflammatory molecules in the circulation that in turn stimulate cell activation and exacerbate vascular inflammation. Altered morphology and metabolism and increased levels of activated platelets have been documented in AD patients and AD mouse models together with an augmented state of vascular inflammation. Therefore, targeting platelets in AD should aim at preventing their prothrombotic/proinflammatory action. In addition, platelets and platelet-­‐related proteins may represent a potential peripheral easy-­‐to-­‐access biomarkers for AD. All these aspects of the complex interplay between blood platelets, vascular inflammation and AD will be discussed. New approaches to fight Burkholderia cenocepacia, a very dangerous Cystic Fibrosis pathogen. Buroni S, Scoffone V, Chiarelli L, De Rossi E, Riccardi G. Laboratory of Molecular Microbiology, DBB UNIPV, Pavia Burkholderia cenocepacia is a Gram-­‐negative bacterium that is widespread in the environment and an opportunistic pathogen for Cystic Fibrosis (CF) patients. It causes an increased decline of lung function and can lead to a fatal pneumonia. B. cenocepacia shows resistance to numerous classes of antibiotics (aminoglycosides, cephalosporins, polymyxins, and β-­‐lactams). Different resistant mechanisms have been identified in these bacteria, including alteration of drug target site, enzymatic drug inactivation or modification, reduced membrane permeability, and/or activation of efflux systems. Our research is focused on the identification of new drugs and alternative targets to improve the clinical approach. Finding new molecules able to inhibit Burkholderia cenocepacia virulence in the host is of primary importance, together with the discovery of new antimicrobials. In this way, interfering with the activity of quorum sensing (QS) synthases may render the bacteria unable to produce virulence factors, making bacteria less able to colonize the host. Also, as therapies directed at inhibiting QS do not directly kill the bacteria the development of resistance is less likely. New antivirulence molecules able to inhibit the QS synthase CepI have been developed. These compounds have been shown to interfere with the protease and siderophore production of B. cenocepacia, and with its ability to form biofilm. Also, the survival of Caenorhabditis elegans infected with B. cenocepacia was greatly improved after treatment with the compounds, thus indicating a good in vivo activity against Burkholderia virulence. Finally, the two molecules are not toxic for human cells. These anti-­‐virulence drugs could potentially be used in combination with established or novel antimicrobials to improve the current therapy. In the struggle against B. cenocepacia it is essential to find also new antimicrobial compounds. A new promising bactericidal drug belonging to the 2,1,3-­‐benzothiadiazol-­‐5-­‐yl family, with an MIC of 8 µg/ml against B. cenocepacia. The compound is active against clinical isolates and other members of the Bcc, as well as against other Gram-­‐negative and -­‐positive bacteria. By a chemogenomic approach and super resolution microscopy, the mechanism of action of 10126109 was shown to rely on the inhibition of the cell division apparatus. Toxicity experiments carried out on CF epithelial bronchial cells and Caenorhabditis elegans revealed low toxicity of the compound. Evaluation of antibiotic effect in vivo is being performed in C. elegans infected with B. cenocepacia. Inhalable formulations will be developed in order to administer it to mice to evaluate its in vivo efficacy. Remodelling of the Ca2+ toolkit in tumors: focus on patients-­‐derived endothelial progenitor cells Zuccolo E1, Poletto V2, Scarpellino G1, Montagna D3, Laforenza U4, Rosti V2, Moccia F1 1
Laboratory of Calcium Signalling, DBB UNIPV, Pavia; 2Biotechnology Research Laboratory, Fondazione IRCCS Policlinico San Matteo, Pavia; 3Dip Science Clinico-­‐Chirurgiche, Diagnostiche e Pediatriche, UNIPV, Pavia; 4Dept Molecular Medicine, UNIPV, Pavia An increase in intracellular Ca2+ concentration ([Ca2+]i) is an established signalling mode whereby growth factors to stimulate angiogenesis and stimulate vascular remodelling in healthy tissues. Growing evidence has been provided to suggest that malignant remodelling of the Ca2+ toolkit in tumor endothelial cells contributes to the aberrant vascularisation of solid tumors and is involved in their resistance to anti-­‐cancer treatments. We focussed our recent work on the characterization of the Ca2+ toolkit in a patients-­‐derived endothelial progenitor cells (EPCs), which sustain the angiogenic switch during the early phases of tumor vascularisation. We focussed on endothelial progenitor cells (EPCs) isolated from individuals affected from renal cellular carcinoma (RCC) and breast cancer (BC). In both cases, we observed a dramatic decrease in endogenous Ca2+ levels, which resulted in a dramatic down-­‐regulation of the pro-­‐angiogenic effects of vascular endothelial growth factor (VEGF), which has long been regarded as the most suitable target for anti-­‐angiogenic therapies. Our results could help understand the failure of anti-­‐VEGF therapies in RCC and BC patients. Nevertheless, the pharmacological blockade of store-­‐operated Ca2+ entry (SOCE), the most important pathway for Ca2+ influx in non-­‐excitable cells, inhibited proliferation and in vitro tubulogenesis in both types of EPCs. These results will be discussed in terms of possible interactions with other members of the two Departments and the IMG-­‐CNR. 1
H-­‐NMR to evaluate the metabolome of BronchoAlveolar Lavage fluid (BALf) in Bronchiolitis Obliterans Syndrome (BOS): towards the development of a new approach for biomarker identification Fumagalli M 1, Viglio S 2, Bardoni A 2, Salvini R 2, DiVenere M 2, Cagnone M 2, Iadarola P 1 Laboratory of Structural and Functional Protein Biochemistry , 1 DBB and 2 Dept Molecular Medicine We have generated, for the first time, a profile of metabolites in human BronchoAlveolar Lavage fluid (BALf) that could be relevant for a better understanding of the biological activities involved in the transition from a state of stability to that of established Bronchiolitis Obliterans Syndrome (BOS) in patients after lung transplant. Metabolites in lung transplant recipients without BOS (stable, S), and with BOS at different degree of severity (BOS 0p; BOS I) have been profiled by NMR spectroscopy. Through the fine tuning of a number of parameters concerning both sample preparation/processing and variations of spectra acquisition modes, an efficient and reproducible protocol was designed for the screening of metabolites in a pulmonary fluid that should reflect the status of airway inflammation/injury. Exploiting the combination of mono and bi-­‐
dimensional NMR experiments, 38 polar metabolites, including amino acids, Krebs cycle intermediates, mono-­‐ and di-­‐saccharides, nucleotides and phospholipid precursors were unequivocally identified. To determine which signature could be correlated with the onset of BOS, metabolites’ content of the above recipients was analyzed by univariate and multivariate (PCA, PLS-­‐DA and OPLS-­‐DA) statistical methods. PCA analysis (almost) totally differentiated S from BOS I and this discrimination was significantly improved by the application of PLS-­‐
DA analysis. As a further confirmation, the model built from OPLS-­‐DA analysis was characterized by excellent fit and prediction values (R2=0.99 and Q2=0.88). The analysis of NMR profiles of S vs BOS 0p and of BOS 0p vs BOS I samples showed a clear discrimination of considered cohorts, although with a poorer efficiency compared to those measured for S vs BOS I patients. The differentiation between stable and BOS I patients is crucial for an early identification of the disorder onset. The fact that stable subjects can be separated much less efficiently from BOS 0p and BOS 0p from BOS I, rather than being a limitation, in our opinion confirms the reliability of the work. In fact, it shows how the method is “sensitive” to the change of clinical conditions that occur during the evolution from stability to the established disease. The data of this pilot work assess the suitability of NMR approach in monitoring different pathological lung conditions. Identification of these markers and mechanistic analysis of their role will open the way to gain insights on the molecular mechanisms that govern the evolution of the disease. Unravelling the key elements of the male ejaculate for fertility: the case of tsetse fly Scolari F1, Benoit JB2,3, Michalkova V3,4, Aksoy E3, Takac P4, Abd-­‐Alla AMM5, Aksoy S3, Attardo GM3, Malacrida AR1 1
Laboratory of Genomics and Biotechnlogies of Insects of Agronomy and Public Health Importance, , DBB UNIPV, Pavia; 2University of Cincinnati, McMicken School of Arts and Sciences, Dept of Biological Sciences, 45221, Cincinnati, OH, USA. 3Yale School of Public Health, Dept of Epidemiology of Microbial Diseases, 06520, New Haven, CT, USA. 4Section of Molecular and Applied Zoology, Institute of Zoology, Slovak Academy of Sciences, 845 06 SR, Bratislava, Slovakia. 5International Atomic Energy Agency, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, IPC Laboratory, A-­‐1400, Vienna, Austria Fertilization is one of the most fundamental biological processes in sexually reproducing animals and a measure of male reproductive success. Fertilization requires a complex cocktail of sperm and non-­‐sperm components transferred during copulation (termed male ejaculate), which plays key roles in the evolutionary ecology of mating. Ejaculate constituents contribute to sperm quality and thus male fertility, and impact the reproductive success of i) insects, which transmit diseases to humans, animals and plants, ii) humans, where male factors are a major cause of infertility and iii) livestock, where male factors affect sperm storage for breeding. Despite technological advancements in ejaculate research in many species, comprehensive knowledge of ejaculate composition, function and regulation, with direct effects on male fertility, is currently lacking. Identification of the key factors for male fertility requires an in-­‐depth interdisciplinary and integrated approach that links the flow of information from DNA to functional proteins and non-­‐coding RNAs. The viviparous physiology and associated anatomical adaptations of the tsetse fly, Glossina m. morsitans, provide an ideal experimental system to exploit. In these regards, we are exploring the make-­‐up of the male reproductive arsenal using a multidisciplinary approach combining state-­‐of-­‐the-­‐art ‘omics’ technologies with physiological experiments. In particular, we used a coupled transcriptomic-­‐proteomic approach to disentangle the composition of the spermatophore, a capsule formed in the female uterus during copulation, containing the ejaculate. We identified 287 proteins and their tissue of origin in the male. We found that male accessory glands produce a small number of abundant proteins with yet unknown functions, enzyme inhibitors and peptidase regulators. Testes contribute spermatozoa in addition to multiple less abundant proteins with binding, cytoskeletal and lipid/carbohydrate transporter functions. In addition, we approached a metabolomics analysis on male and female reproductive tissues to elucidate the metabolic pathways involved in the production/regulation of ejaculate components. A better understanding of identity and modes of action of male factors will boost applied research on innovative approaches to interfere in reproductive success of tsetse and, potentially, other insect disease vectors. A physiological approach to evaluate the antioxidant capacity of different matrices of grapefruit Molino S1, Verri M1, Dossena M1, Perez-­‐Burillo D2 , Buonocore D1 Rufiàn-­‐Henares JA2 1 Laboratory of Pharmacology & Toxicology, , DBB UNIPV, Pavia; 2 Dept of Nutrition and Bromatology, University of Granada, Campus Universitario de Cartuja, 18071 Granada, Spain Grapefruit is rich in polyphenols which exhibit antioxidant and antiflammatory properties. The study aimed at evaluating the influence of an enzymatic in vitro gastrointestinal digestion (GAR method1), followed by fermentation, on the antioxidant capacity of the different matrixes of grapefruit. This physiological-­‐resembling approach allows to extract antioxidants from the following grapefruit parts: zest, rind, pulp, and juice (obtained from red and white grapefruit), including two commercial juices. The analyses were conducted on the fresh soluble fractions obtained after in vitro digestion and fermentation (potentially absorbable fraction), and on the lyophilized solid fractions obtained after fermentation (non-­‐absorbable fraction). The antioxidant capacity was evaluated with ABTS, FRAP and Indigo Carmin Hydroxyls, AAPH, and Reduction methods. The results showed that rind possesses the highest antioxidant capacity, probably due to the elevate concentration of naringin. Of note, the soluble fractions derived from digestion possess higher scavenging activity rather than the soluble fractions from fermentation, which on the contrary have a considerable reducing ability. The insoluble fractions from fermentation showed no antioxidant activity in any of the assays. These preliminary data were corroborated through the analysis of polyphenols and their metabolites by means of mass spectrometry, in both fresh, digested and fermented soluble fractions. At the edge between fundamental research and biotechnological applications CodY-­‐mediated regulation of four B. subtilis extracellular protease genes Barbieri G1, Ferrari E1, Ursino E1, Sonenshein AL2, Belitsky BR2 , Albertini AM1. 1
Laboratory of Genetics, Genomics and Microbic Biotechnologies, DBB UNIPV, Pavia; 2Dept Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA CodY is a global transcriptional regulator of multiple metabolic pathways in most low-­‐G+C Gram-­‐positive bacteria, including the genera Bacillus, Listeria, Staphylococcus, Clostridium, Lactococcus, and Streptococcus. In pathogenic species, CodY is also a regulator of important virulence genes. In Bacillus subtilis it directly or indirectly controls the expression of more than two hundred genes involved in the adaptation to poor nutrients conditions. Its DNA binding ability is enhanced by the interaction with two classes of effectors, the branched-­‐chain amino acids and GTP, thus allowing modulation of gene expression in response to the nutritional status of the cell. We recently showed that the genes encoding four extracellular proteases -­‐ aprE, nprE, vpr and mpr -­‐ are negatively controlled by CodY. In all four cases, repression is exerted through binding of CodY at sites located in the vicinity of the transcription start points of the genes and, in for mpr, at an internal site within the coding sequence of the gene. Under conditions of maximal CodY activity, deletion of codY results in a 10-­‐fold increase in the expression of vpr and mpr, two minor exoproteases with a key role in swarming, biofilm formation and the maturation of some of the phosphatase regulators involved in quorum sensing. On the contrary, inactivation of CodY only mildly affects expression of the genes encoding the two major exoproteases of B. subtilis: only a less than 2-­‐fold increase in aprE expression and, surprisingly, a 2.2-­‐fold decrease of nprE transcript levels were observed in a codY mutant. The expression of these two genes is activated at the onset of stationary phase and is controlled by different transcriptional regulators, including ScoC. We reported that scoC is directly repressed by CodY. Consequently, a strong, direct role of CodY as a repressor of aprE and nprE can be observed only in the absence of ScoC or by impairing ScoC binding to the promoters of the exoprotease genes. Inactivation of CodY results in fact in an increase in scoC expression with a consequent stronger ScoC-­‐
mediated repression of aprE and nprE that compensates for the loss of CodY-­‐mediated repression. By repressing the protease genes while limiting the synthesis of a second repressor, CodY is both a direct negative regulator and an indirect positive regulator of aprE and nprE. Possible applications of the knowledge acquired on the regulation of B. subtilis promoters for the expression of genes encoding proteins of interest will be presented. The Natural Polymer γ-­‐PGA and its hydrolases Longanesi L1, Rama E, Cavaletti M, Astuto M, Seppi C, Calvio C1 1
Laboratory of Microbial Genetics, DBB UNIPV, Pavia -­‐ Cost-­‐competitive production of a natural biopolymer In collaboration with the Chemistry Dept. and the Electrical, Computer and Biomedical Engineering Dept. -­‐ UniPV Poly-­‐γ-­‐glutamic acid (γ-­‐PGA) is a homopolyamide composed of glutamic acid monomers connected by amide linkages between α-­‐amino and γ-­‐carboxyl groups. The polymer is mainly produced by Bacilli, although we recently expanded the list of producer species through a bioinformatics approach. In recent times the interest around γ-­‐PGA enormously raised for its suitability in a growing number of biotechnological applications: thickening gels, dietary food supplements, cream humectants, agriculture additives and metal flocculants based on γ-­‐PGA are already commercialized around the world, while intensive research is focused on the potential of γ-­‐PGA as drug carrier and biomaterial in several medical and technological fields. Regrettably the high production costs of the polymer limits its industrial development. In the past, genetic engineering allowed us to obtain a competitive γ-­‐PGA producer strain from the laboratory strain of Bacillus subtilis 168. Our current aim is cutting input costs by using low cost feedstocks in fermentation. We are working on the valorization of the organic fraction contained in two byproducts of different agro-­‐industrial processes through the improvement of the metabolic capacity of γ-­‐PGA producer strains. The RIVARIO project requires a strain with improved cellulolytic capabilities for exploitation of rice straw; the GlySyBi project requires tuning of the glycerol catabolic pathway to exploit crude glycerol derived from biodiesel plants and will be carried out applying synthetic biology concepts. Both research lines are sustained by the Cariplo Foundation. -­‐ Degradation of γ-­‐PGA In collaboration with the Molecular Medicine Dept. -­‐ UniPV Recently we found out that few unknown Bacillus genes indeed encode efficient and specific γ-­‐PGA degrading enzymes. Our aim is to better characterize such hydrolases and explore their potential use as therapeutics for the treatment of persistent infections caused by γ-­‐PGA-­‐producing pathogenic bacteria for which the polymer acts as fundamental virulence factor. -­‐GGT as biotechnological enzymes In collaboration with the Chemistry Dept. -­‐ UniMI The TailGluTran project sustained by the Cariplo Foundation is focused on the synthesis of γ-­‐glutamyl derivatives of naturally occurring and/or modified amino acids starting from bulk chemicals of biotechnological origin. The task of our unit is to obtain mutant GGT enzymes with enhanced activity and tailored for a selected acceptor compounds. Tyrosyl-­‐DNA phosphodiesterases and DNA damage response in plants Balestrazzi A, Macovei A, Araujo S, Pagano A, Carbonera D Laboratory of Plant Biotechnologies, DBB UNIPV, Pavia Genome integrity is a priority for plant survival under adverse environmental conditions and a strong challenge for preserving high crop productivity in the context of global climate change. Research at the Plant Biotechnology Laboratory focuses on the study of tyrosyl-­‐DNA phosphodiesterases (Tdps) which include Tdp1 and Tdp2 enzymes (EC number 3.1.4.-­‐) able to break the covalent 3'-­‐ and 5’-­‐phosphotyrosyl bonds between the DNA termini and the catalytic tyrosine residue of DNA topoisomerase I and II, respectively (Pommier et al. 2014, DNA Repair (Amst) 19: 114-­‐129). Tdps, responsible for the removal of topoisomerase-­‐mediated DNA damage, are emerging as multipurpose players in the DNA damage response (DDR). We have characterized the Tdp1 gene family from the model legume Medicago truncatula, including MtTdp1a and MtTdp1b genes, highlighting their role in the response to abiotic stress (Macovei et al. 2010, Planta 232: 393-­‐407). MtTdp1a-­‐
gene depletion in M. truncatula plants resulted in premature senescence, telomere shortening, and nucleolar disfunction (Donà et al. 2013, J. Exp. Bot. 64: 1941-­‐1951) as well as in reduced global cytosine methylation and altered TEs (transposable elements) expression patterns (Sabatini et al. 2016, J. Int. Plant Biol. 58: 618-­‐622). We are currently using NSC120686, an inhibitor of the human Tdp1 enzyme, as a tool for deciphering the peculiar roles of the plant Tdp1 genes. A parallel work has been carried out on Tdp2, which in human is also an essential component of proliferation/ apoptosis signaling pathways (Li et al. 2011, Cell Cycle 10: 1-­‐10). The lack of Tdp2 interferes with rRNA biogenesis in human cells and it has been hypothesized that the protein might be involved in the cross-­‐talk between cytoplasm and nucleolus under stress conditions (Vilotti et al. 2012, PLoS ONE 7: e35051). The overexpression of the MtTdp2α gene in Medicago truncatula plants enhances global DNA repair and tolerance to osmotic and heavy metal stress (Faè et al. 2014, Plant Cell Rep. 33: 1071-­‐1080). No information is currently available on the links between nucleolar function and Tdp2 in plants. Ultrastructural nucleolar morphology and ribogenesis are currently investigated in MtTdp2α-­‐overexpressing M. truncatula cell cultures. In addition, our research has highlighted the relevance of plant Tdp genes during early seed imbibition, when DDR is triggered to preserve genome integrity in embryo cells, promoting high seed vigor (Macovei et al. 2010, Planta 232: 393-­‐407). We are currently using Tdp genes as molecular indicators of seed quality in translational research projects involving Seed Companies. Extraction and quantification of nutraceutical and cosmeceutical compounds from fruit waste and vegetable products (I-­‐II-­‐III-­‐IV GAMMA). Buonocore D1, Doria E2, Boncompagni E 2, Dossena M1, Nielsen E 2 1
Laboratory of Pharmacobiochemistry and 2Laboratory of Plant Biochemistry, DBB UNIPV, Pavia Introduction. Aim of our reseach, supported by “Programma di Sviluppo Rurale 2007/2013 Regione Lombardia”, is to exploit the large amount of vegetable waste extracting Bioactive Compounds of nutraceutical and cosmeceutical value with consequent economic and ecological advantages. The vegetable waste results from the production of fresh plant fruits and vegetables (I gamma), modified plant products (II, III gamma) or fresh packaged plant products (IV gamma). Eight bioactive molecules have been selected till now for their interesting properties: Lycopene, from tomato mix wastes and red tomato peel; Phlorizin and Ursolic acid from stark apple peel; Kaempherol and Chlorogenic acids from leek leaves; Arbutin and Ursolic acid from pear peel and stumps; Lutein from catalonia herbs; Sulphoraphane from broccoli waste. Lycopene is a carotenoid abundant in tomato fruits with antioxidant and anticancer properties. Phloridzin, is considered a potential candidate for the treatment or prevention of diabetes and obesity. Ursolic acid appears to increase skeletal muscles and brown fat. Kaempferol is a flavonol with antioxidant and anti-­‐inflammatory activity able to inhibit the process of angiogenesis and cancer growth. Chlorogenic acid is an antioxidant phenolic compound possessing anticancer properties and inhibiting the growth of preadipocytes. Arbutin is used as a skin-­‐
lightening agent in cosmetics due to its ability to inhibit tyrosinases. Lutein is a xanthophyll pigment present in the membranes of macula lutea of the vision apparatus of primates; it is able to increase the macular pigment optical density. Sulforaphane is an isothiocyanate present in cruciferous vegetables, which is known to reduce cancer risk by different mechanisms. Methods. We applied different protocols using EU-­‐allowed solvents to extract selectively the above listed compounds of interest. The separation was carried out by reverse phase HPLC with spectrophotometric detection. Results and Conclusions. All the above-­‐cited bioactive compounds are extracted from the discarded fruits or wasted parts of different plants with satisfactory yields, not lower than those reported in literature in the case of extraction of the same molecules from eaten and fresh products of the same plants. So, extraction and purification of Bioactive Compounds from vegetable waste is to be considered as a promising innovative and sustainable process of special interest at industrial level where it might be possible to recover the chemical bond energy present in the residual biomass resulting from the extraction of Bioactive Compounds by means of anaerobic digestors producing biogas. Genotype and phenotype from the bench to bedside The epigenetic landscape of mammalian chromosomes: centromeres and telomeres. Giulotto E1, Raimondi E2, Nergadze SG1, Bensi M2, Cappelletti E1, Corbo M1, Gamba R1, Gozzo F1, Khoriauli L1, Mazzagatti A2, Piras FM1, Roberti A2, Santagostino M.1 1
Laboratory of Molecular & Cellular Biology; 2Laboratory of Molecular Cytogenetics, DBB UNIPV, Pavia Our research is mainly focused on the chromosomal elements responsible for the maintenance of genome integrity: centromeres and telomeres. Centromeres are required for chromosome segregation during cell division; their anomalous functioning contributes to cancer development and progression and to genome evolution. The centromeric function is related to epigenetic marks rather than to DNA sequences. The DNA component of centromeric chromatin is typically constituted by extended stretches of tandem repeats (satellite DNA); this organization hampered their molecular dissection. We discovered that equids are a powerful model system because a number of their centromeres is devoid of repetitive DNA thus allowing deep analysis of DNA/protein interactions. We are currently dissecting the architecture and functional organization of centromeric chromatin taking advantage of the equid model system. In addition, in the context of an international network, we are contributing to the FAANG (Functional Annotation of ANimal Genomes) project with the goal of analysing genome-­‐wide functional data on the horse species. Telomeres are nucleoprotein structures located at the ends of linear chromosomes which, in vertebrates, consist of tandem repetitions of the TTAGGG hexanucleotide. The relationship between telomere metabolism, aging and cancer is well documented. Following the demonstration that telomeres are transcriptionally active, we are studying the involvement of telomeric transcripts in human tumours and in the response to ionizing radiation. We are also studying the so called interstitial telomeres, which are telomeric-­‐like repeats localized at intrachromosomal sites. The role of interstitial telomeres in the regulation of gene expression is under investigation. Finally, thanks to the know how in cellular and molecular biology we set up efficient methods for high yield production of recombinant (normal and pathological) human proteins of medical interest in mammalian cells. These proteins maintain the post-­‐translational modifications, folding properties and functionality of the native counterparts representing valuable tools for structural and functional studies. The researchers involved possess complementary competences covering a wide spectrum of cellular, biomolecular, cytogenetic and bioinformatic techniques, including: basic recombinant DNA methodologies, next generation sequencing (ChIp-­‐seq, RNA-­‐seq, DNA methylation and histone modification analysis), establishment and immortalization of primary mammalian cells, construction of genetically modified mammalian cells, conventional molecular cytogenetics and single DNA molecule imaging (chromatin fiber analysis and DNA combing). Genetic predisposition to gastrointestinal tumors Marabelli M, Abou Khouzam R, Salvato I, Ranzani GN Laboratory of Human Genetics – Cancer Genetics, DBB UNIPV, Pavia The majority of colorectal cancers (CRCs) are classifiable as “sporadic”, i.e. patients do not show clear-­‐cut family history of the disease, while about 30% of cases are ascribable to some genetic predisposition. Known genetic susceptibility includes a series of common low-­‐penetrance risk variants, identified through GWAS, and a group of high-­‐penetrance pathogenic mutations causative of rare Mendelian syndromes (5% of all CRCs). The most common Mendelian predisposition to CRC is the Lynch syndrome, associated with germline defects of DNA mismatch repair genes. Other syndromes include: a dominant condition caused by germline defects of APC tumor suppressor gene (FAP); a recessive condition associated with MUTYH base excision repair gene mutations (MAP); a dominantly inherited polyposis (PPAP) due to mutations in POLE and POLD1 gene regions encoding the proofreading domain of DNA polymerases ε and δ, respectively. Our research work is aimed at: 1) investigating, by complementary molecular approaches on both DNA and RNA, genes that are known to be associated with the most common CRC syndromes; in particular, we are interested in characterizing “unconventional” mutations including intragenic deletions, splicing defects, allelic expression imbalance, and promoter alterations; 2) identifying, by an NGS approach, new susceptibility genes possibly underlying the inherited conditions that are genetically unexplained; 3) ascertaining, by different methods, the pathogenic significance of selected VUS (Variants of Uncertain Significance) emerged from NGS; 4) evaluating the contribution of low-­‐penetrance alleles to the risk of CRC in the Italian population. As is the case with CRC, the majority of gastric cancer (GC) cases occur sporadically. However, 10% show familiar clustering, and 1-­‐3% are strictly hereditary. In particular, the Hereditary Diffuse Gastric Cancer (HDGC) syndrome is a dominant condition associated with germline defects of CDH1 (E-­‐cadherin), including inactivating mutations, gene deletions and allelic expression imbalance. At any rate, in spite of experimental effort, no more than 10-­‐50% of HDGC cases are found to harbor CDH1 germline defects and a significant number of patients screened worldwide remains without a molecular explanation. The application of NGS has revealed the existence of mutations within new genes possibly predisposing to GC. However, each of the new genes proved to explain only very few CDH1 mutation-­‐negative families, indicating that the HDGC molecular basis still missing is likely distributed across many loci. Accordingly, we are currently investigating by NGS a series of selected Italian patients we demonstrated to be CDH1 mutation-­‐negative by a series of complementary molecular methods. Collaborations: IEO-­‐Milano; IST-­‐Genova; Istituto di Candiolo IRCCS; INT-­‐Milano; Dip. Med. Molecolare-­‐Univ. Pavia; IRST-­‐IRCCS Meldola; Helmholtz Zentrum München Germ and somatic pluripotent stem cell models of differentiation Garagna S., Zuccotti M, Merico V, Rebuzzini P. Laboratory of Developmental Biology, DBB UNIPV, Pavia Our laboratory adopts an ecological developmental biology approach to evaluate the interactions between organism and environment (exposome). We aim at understanding the cellular and molecular bases of differentiation of germ and somatic pluripotent stem cells. Two are the main pluripotent cell models employed: the mammalian gametes and embryonic stem cells (ESCs). While studying the mechanisms that regulate the differentiation of gametes we work to unravel the molecular signature of developmentally competent oocytes and sperm and when it is acquired during spermatogenesis or oogenesis. Also, we are studying how embryonic stem cells (ESCs) cardiac differentiation is dysregulated in the presence of environmental contaminants or therapeutic agents. The main aim of this project is the identification of the alterations induced by xenobiotics to the signaling mechanisms and to the phenotype during the differentiation from stem cells to cardiomyocytes. This ecological developmental biology approach gives new perspectives for the understanding of infertility and cardiac pathologies. In the Laboratory of Developmental Biology we have expertise on: histology; immunohistochemistry; immunoblotting; DNA and RNA extraction; PCR, RT-­‐PCR, real-­‐time PCR; Southern blotting; transfection; DNA and RNA in situ hybridisation; cytogenetics; cell culture including embryonic stem cells, iPS cells, oocytes and embryos, single cell manipulation and analysis; in vitro fertilisation; gamete and preimplantation embryos micromanipulation; nuclear transfer.