Mercoledì del PRIN n.4: Cinzia Giannini – Concetta Avitabile

Biomineralization in senescent human mesenchymal stem cells – an in-depth multi-modal and multi-scale supra-molecular and sub-molecular study (XMINE)

Dr. Cinzia Giannini

Biomineralization, the complex process leading to the formation of organized mineral crystals, persists throughout life and is fundamental in bone formation, repair, and remodeling. In human bone mesenchymal stem cells (bMSC), osteoblasts’ precursors, we have shown that biomineralization starts within the cell with the nucleation of Zn–hydroxyapatite, and then rapidly evolves toward hydroxyapatite crystals, with composition and structure similar to the one present in human bone. To this aim, Crystallography was jointly used with the Small and Wide Angle X-ray Scattering scanning microscope, available at the X-ray Micro-Imaging Laboratory, XMI-L@b (http://www.ic.cnr.it/ic4/x-ray-microimaging-laboratory-xmi-lb-ic-bari/), as in vitro diagnostic tools to study and classify the crystal and chemical nature of the Zn-hydroxyapatite calcifications.
bMSC, rare pluripotent cells that activate the genetic program leading to osteoblastogenesis in response to specific chemical or physical stimuli, can migrate into sites of injury, self-renew, and differentiate as well as release trophic and growth factors. For these reasons bMSC are prime candidates for use in regenerative medicine. Moreover, bMSC are critical for the osteointegration of orthopedic implants. With senescence, a numerical decline and a functional impairment of bMSC has been reported, resulting in bone diseases that are common in aged population and are among the main causes of disability and morbidity. The mechanisms of age-related changes in bMSC have not been fully characterized. It is known that senescent human bMSC are irreversibly growth arrested, genetically reprogrammed, morphologically altered, show increased activity of senescence-associated βgalactosidase, and lose osteogenic potential. Interestingly, in a murine model, senolytics improved osteogenic capacity of aged BMSC both in vitro and in vivo. Cellular senescence is a critical issue since it is a ubiquitous feature of cells derived from regenerative somatic tissue and a primary causal mechanism of ageing and age-related disease. Indeed, senescence is not a “tissue culture artefact”, and senescent cells are present in many different tissues in vivo.
The main goal of the Project is to study the in vitro behavior of young and senescent bMSC at the supra- and sub-molecular level to assign uniquely the correct identity of the inorganic calcium phosphate aggregates, deposited by the young, middle-aged and senescent cells. Indeed, all calcium-phosphate salts present similar composition, but have a different arrangement of the atoms in the crystal lattice. The specific role of some crucial metallic components in the hydroxyapatite formation – Mg, Zn and Cu – in young and senescent bMSC will be explored in order to interpret thoroughly the results obtained by microimaging.

Keywords:

1. Biomineralization
   2. Senescence
   3. Cell Imaging
   4. Metallomics
   5. X-ray techniques
   6. Bone

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Proline-rich antimicrobial peptides in the age of antibiotic-resistance: a new weapon to fight Klebsiella pneumoniae bloodstream infections – PrAMP4KleBI

Dr. Concetta Avitabile

Bloodstream infections require rapid and effective intervention because of their high mortality risk. However, the number of antibiotics used to treat these conditions has been reduced dramatically due to the diffusion of antibiotic-resistant pathogens. Bloodstream infections caused by antibiotic-resistant Klebsiella pneumoniae, with a worrisome mortality rate of between 40% and 50%, are representative of the current antibiotic crisis. The need for new antimicrobial drugs is evident. In this scenario, antimicrobial peptides (AMPs) gained interest as compounds to develop new anti-infectives. AMPs possess potent antimicrobial activity associated with low bacteria rate of resistance development. Unfortunately, most AMPs are also rather cytotoxic. The Proline-rich antimicrobial peptides (PrAMPs) are an exception. PrAMPs display specific and non-membranolytic antimicrobial mechanism of action. They kill bacteria by binding to the bacterial ribosomes and blocking protein synthesis. This makes PrAMPs selective for bacteria and generally well tolerated by eukaryotic cells. The aim of this project is to identify a lead compound from a group of selected PrAMPs suitable to fight antibiotic-resistant K. pneumoniae bloodstream infections. The project will identify the PrAMP that exhibits: i) the best in vitro antimicrobial activity against planktonic or sessile forms of multidrug-resistant clinical isolates of K. pneumoniae; ii) the best tolerability to human cells and possibly; iii) anti-inflammatory and LPS-inactivating properties. Experiments will be performed under conditions simulating the final use (e.g. in whole blood). Structure-activity relationship studies will help identify structural features of PrAMPs associated with desired biological activities. Finally, in vivo experiments in mouse models of a bloodstream K. pneumoniae infection and of LPS shock will determine the actual efficacy of the selected PrAMP as antimicrobial and/or immunomodulatory agent. The project, presented by three integrated and complementary units, will provide a robust preclinical evaluation of  PrAMPs, that will show whether these compounds can address the urgent need of new drugs to combat antibiotic-resistant K. pneumoniae bloodstream infections. In addition, the project could pave the way for the development of other further optimized antimicrobial drugs based on PrAMPs.

Keywords

1. Antimicrobial Peptides (AMP)
2. Bloodstream infections (BSIs)
3. Antibiotic resistance;
4. Klebsiella pneumoniae;
5. LPS-inflammation;
6. Proline rich Antimicrobial Peptides (PrAMP);