Polymersomes mediated intracellular delivery of antibodies: implication in anticancer therapy

Cancer is one of the leading causes of death in the developed world. Nevertheless, many pharmaceutical products available in the clinic lack of tissue specificity, and often have severe side effects. Nowadays, advances in molecular biology and biotechnology have allowed the development of biological therapeutic approaches aimed to give a step forward on cancer treatment. Biotherapy involves the use of biomolecules such as antibodies that have the potential to increase the specificity of anticancer treatments, thus limiting the side effects. Unfortunately, the effectiveness of such biomolecules is hindered by their pharmacokinetics, and their translation into patient care is heavily restricted by low solubility in water, instability / degradation in vivo and low efficiency. Besides that, such biomolecules require appropriate delivery strategies to penetrate cellular barriers, thus being able to interfere with pathways that can be involved in cancer development. This research project aims to bridge the lack of technology regarding the development of an effective and biocompatible delivery system. The pH sensitive PMPC-PDPA ((poly(2-(methacryloyloxy)ethyl phosphorylcholine) - poly(2-(diisopropylamino)ethyl methacrylate)) polymersome was employed as candidate for the intracellular delivery of functional therapeutic antibodies in live cells. The PMPC-PDPA diblock copolymer combines the ability to release the loaded cargo upon acidification within the endosomes with an overall biocompatibility. First, the antifouling proprieties of polymersomes were investigated, and then compared to micelles. Subsequently, electroporation was exploited as reliable technique to effectively encapsulate antibodies within polymersomes. The delivery of antibodies in live cells was assessed using anti γ-tubulin antibody as a model system. Finally, Ki-67 was explored as a possible target for anticancer therapy. Interestingly, relevant differences in the biological functions of this marker were revealed between cancerous and non-cancerous cells. Furthermore, antibodies against Ki-67 were delivered in live cells, and their activity was tested to explore the potential of Ki-67 as a target for anticancer therapy

The role of the two splice variants and extranuclear pathway on Ki-67 regulation in non-cancer and cancer cells

Ki-67 is a nuclear protein that has been used in cancer diagnostic because of its specific cell-cycle dependent expression profile. After quantifying and characterising the expression level of Ki-67, as a function of the cell cycle, we found out that the two main splice variants of the protein (i.e. α and β) are differently regulated in non-cancerous and cancerous cells both at mRNA and protein level. We were able to correlate the presence of the α variant of the protein with the progression through the interphase of cell cycle. We also observed that the different expression profiles correspond to different degradation pathways for non-cancerous and cancerous cells. Furthermore, Ki-67 is continuously regulated and degraded via proteasome system in both cell types, suggesting an active control of the protein. However we also observed a putative extranuclear elimination pathway of Ki-67 where it is transported to the Golgi apparatus. Our evidence in the different expression of the splice variants may represent a milestone for the development of new targets for cancer diagnostic and prognostic. Additionally, the unexpected extranuclear elimination of Ki-67 strongly suggests that this protein must be looked at also outside of the "nuclear box", as thought to date

Bifidobacteria and lactobacilli in the gut microbiome of children with non-alcoholic fatty liver disease: which strains act as health players?

Introduction: Non-alcoholic fatty liver disease (NAFLD), considered the leading cause of chronic liver disease in children, can often progress from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). It is clear that obesity is one of the main risk factors involved in NAFLD pathogenesis, even if specific mechanisms have yet to be elucidated. We investigated the distribution of intestinal bifidobacteria and lactobacilli in the stools of four groups of children: obese, obese with NAFL, obese with NASH, and healthy, age-matched controls (CTRLs). Material and methods: Sixty-one obese, NAFL and NASH children and 54 CTRLs were enrolled in the study. Anthropometric and metabolic parameters were measured for all subjects. All children with suspected NASH underwent liver biopsy. Bifidobacteria and lactobacilli were analysed in children’s faecal samples, during a broader, 16S rRNA-based pyrosequencing analysis of the gut microbiome. Results: Three Bifidobacterium spp. (Bifidobacterium longum, Bifidobacterium bifidum, and Bifidobacterium adolescentis) and five Lactobacillus spp. (L. zeae, L. vaginalis, L. brevis, L. ruminis, and L. mucosae) frequently recurred in metagenomic analyses. Lactobacillus spp. increased in NAFL, NASH, or obese children compared to CTRLs. Particularly, L. mucosae was significantly higher in obese (p = 0.02426), NAFLD (p = 0.01313) and NASH (p = 0.01079) than in CTRLs. In contrast, Bifidobacterium spp. were more abundant in CTRLs, suggesting a protective and beneficial role of these microorganisms against the aforementioned diseases. Conclusions: Bifidobacteria seem to have a protective role against the development of NAFLD and obesity, highlighting their possible use in developing novel, targeted and effective probiotics

Live cell imaging of membrane / cytoskeleton interactions and membrane topology

We elucidate the interaction between actin and specific membrane components, using real time live cell imaging, by delivering probes that enable access to components, that cannot be accessed genetically. We initially investigated the close interplay between Phosphatidylinositol 4,5-bisphosphate (PIP2) and the F-actin network. We show that, during the early stage of cell adhesion, PIP2 forms domains within the filopodia membrane. We studied these domains alongside cell spreading and observed that these very closely follow the actin tread-milling. We show that this mechanism is associated with an active transport of PIP2 rich organelles from the cell perinuclear area to the edge, along actin fibers. Finally, mapping other phospholipids and membrane components we observed that the PIP2 domains formation is correlated with sphingosine and cholesterol rafts

Proteomics boosts translational and clinical microbiology

The application of proteomics to translational and clinical microbiology is one of the most advanced frontiers in the management and control of infectious diseases and in the understanding of complex microbial systems within human fluids and districts. This new approach aims at providing, by dedicated bioinformatic pipelines, a thorough description of pathogen proteomes and their interactions within the context of human host ecosystems, revolutionizing the vision of infectious diseases in biomedicine and approaching new viewpoints in both diagnostic and clinical management of the patient.Indeed, in the last few years, many laboratories have matured a series of advanced proteomic applications, aiming at providing individual proteome charts of pathogens, with respect to their morph and/or cell life stages, antimicrobial or antimycotic resistance profiling, epidemiological dispersion. Herein, we aim at reviewing the current state-of-the-art on proteomic protocols designed and set-up for translational and diagnostic microbiological purposes, from axenic pathogens' characterization to microbiota ecosystems' full description. The final goal is to describe applications of the most common MALDI-TOF MS platforms to advanced diagnostic issues related to emerging infections, increasing of fastidious bacteria, and generation of patient-tailored phylotypes. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. © 2013 The Authors

Cases of cryptosporidiosis co-infections in AIDS patients: a correlation between clinical presentation and GP60 subgenotype lineages from aged formalin-fixed stool samples

Nine cases of cryptosporidiosis co-infections in AIDS patients were clinically categorised into severe (patients 1, 3, 8 and 9), moderate (patients 4 and 5) and mild (patients 2, 6 and 7). Formalin-fixed faecal specimens from these patients were treated to obtain high quality DNA competent for amplification and sequencing of the 60-kDa glycoprotein (GP60) gene. Sequence analysis revealed that one patient was infected with Cryptosporidium hominis whereas the remaining eight patients were infected with C. parvum. Interestingly, the patients showing severe cryptosporidiosis harboured two subtypes within the C. parvum allelic family IIc (IIcA5G3 and IIcA5G3R2), whereas patients with moderate or mild infections showed various subtypes of the C. parvum allelic family IIa (IIaA14G2R1, IIaA15G2R1, IIaA17G3R1 and IIaA18G3R1)

Network analysis of gut microbiome and metabolome to discover microbiota-linked biomarkers in patients affected by non-small cell lung cancer

Several studies in recent times have linked gut microbiome (GM) diversity to the pathogenesis of cancer and its role in disease progression through immune response, inflammation and metabolism modulation. This study focused on the use of network analysis and weighted gene co-expression network analysis (WGCNA) to identify the biological interaction between the gut ecosystem and its metabolites that could impact the immunotherapy response in non-small cell lung cancer (NSCLC) patients undergoing second-line treatment with anti-PD1. Metabolomic data were merged with operational taxonomic units (OTUs) from 16S RNA-targeted metagenomics and classified by chemometric models. The traits considered for the analyses were: (i) condition: disease or control (CTRLs), and (ii) treatment: responder (R) or non-responder (NR). Network analysis indicated that indole and its derivatives, aldehydes and alcohols could play a signaling role in GM functionality. WGCNA generated, instead, strong correlations between short-chain fatty acids (SCFAs) and a healthy GM. Furthermore, commensal bacteria such as Akkermansia muciniphila, Rikenellaceae, Bacteroides, Peptostreptococcaceae, Mogibacteriaceae and Clostridiaceae were found to be more abundant in CTRLs than in NSCLC patients. Our preliminary study demonstrates that the discovery of microbiota-linked biomarkers could provide an indication on the road towards personalized management of NSCLC patients

Network analysis of gut microbiome and metabolome to discover microbiota-linked biomarkers in patients affected by non-small cell lung cancer

Several studies in recent times have linked gut microbiome (GM) diversity to the pathogenesis of cancer and its role in disease progression through immune response, inflammation and metabolism modulation. This study focused on the use of network analysis and weighted gene co-expression network analysis (WGCNA) to identify the biological interaction between the gut ecosystem and its metabolites that could impact the immunotherapy response in non-small cell lung cancer (NSCLC) patients undergoing second-line treatment with anti-PD1. Metabolomic data were merged with operational taxonomic units (OTUs) from 16S RNA-targeted metagenomics and classified by chemometric models. The traits considered for the analyses were: (i) condition: disease or control (CTRLs), and (ii) treatment: responder (R) or non-responder (NR). Network analysis indicated that indole and its derivatives, aldehydes and alcohols could play a signaling role in GM functionality. WGCNA generated, instead, strong correlations between short-chain fatty acids (SCFAs) and a healthy GM. Furthermore, commensal bacteria such as Akkermansia muciniphila, Rikenellaceae, Bacteroides, Peptostreptococcaceae, Mogibacteriaceae and Clostridiaceae were found to be more abundant in CTRLs than in NSCLC patients. Our preliminary study demonstrates that the discovery of microbiota-linked biomarkers could provide an indication on the road towards personalized management of NSCLC patients

Longitudinal Multi-Omics Study of a Mother-Infant Dyad from Breastfeeding to Weaning: An Individualized Approach to Understand the Interactions Among Diet, Fecal Metabolome and Microbiota Composition

The development of the human gut microbiota is characterized by a dynamic sequence of events from birth to adulthood, which make the gut microbiota unique for everyone. Its composition and metabolism may play a critical role in the intestinal homeostasis and health. We propose a study on a single mother-infant dyad to follow the dynamics of an infant fecal microbiota and metabolome changes in relation to breast milk composition during the lactation period and evaluate the changes induced by introduction of complementary food during the weaning period. Nuclear Magnetic Resonance (NMR)-based metabolomics was performed on breast milk and, together with 16S RNA targeted-metagenomics analysis, also on infant stool samples of a mother-infant dyad collected over a period running from the exclusive breastfeeding diet to weaning. Breast milk samples and neonatal stool samples were collected from the 4th to the 10th month of life. Both specimens were collected from day 103 to day 175, while from day 219–268 only stool samples were examined. An exploratory and a predictive analysis were carried out by means of Common component and specific weight analysis and multi-block partial least squares discriminant analysis, respectively. Stools collected during breastfeeding and during a mixed fruit/breastfeeding diet were characterized by high levels of fucosyl-oligosaccharides and glycolysis intermediates, including succinate and formate. The transition to a semi-solid food diet was characterized by several changes in fecal parameters: increase in short-chain fatty acids (SCFAs) levels, including acetate, propionate and butyrate, dissapearance of HMOs and the shift in the community composition, mainly occurring within the Firmicutes phylum. The variations in the fecal metabolome reflected the infant’s diet transition, while the composition of the microbiota followed a more complex and still unstable behavior