ROLE OF THE BACTERIAL CELL WALL GLYCO-CONJUGATES IN ELICITATION OR SUPPRESSION OF EUKARYOTIC INNATE IMMUNITY

The innate immune response plays an important role in eliciting host defence against microbial infections by the early recognition between pathogens’ structures arranged on the bacterial surface, called MAMPs; and their specific receptors in the host cells. Within this frame, my PhD project is focused on studying the role of bacterial glycoconjugates in the activation of immune response in plant cells. Glyco-conjugates represent a very heterogeneous group of biomolecules which includes lipopolysaccharides (LPS), peptidoglycan (PGN), glycolipids and glycoproteins. In Gram-negative bacteria, the majority of membrane glycoconjugates is constituted by lipopolysaccharides (LPS), amphiphilic macromolecules consisting of three different portions: a lipophilic domain called Lipid A, a hetero-oligosaccharide denominated Core, a hydrophilic hetero-polysaccharide called O-polysaccharide chain or O-chain. They are pivotal in mediating many processes of host-bacteria interaction like adhesion, recognition, pathogenesis, symbiosis. In order to understand the molecular basis of bacterium–host interaction, it is important to elucidate the structure of LPS and to identify how bacterium modifies the LPS in response to different environments and stimuli. It has been already demonstrated that a correct structure of LPS is required to establish a disease (pathogens) or to produce a beneficial outcome (symbiont) in host-microbe interaction. In particular, part of my PhD thesis is focused on bacteria involved in legume symbiotic nitrogen fixation by Rhizobiaceae, widespread in nature and able to fix nitrogen and symbiotic with plant roots. Rhizobiaceae, considered as biofertilizers, in agricultural system would decrease the need of chemical fertilizers, which are not only cost effective but also may create environmental problems

Autistic adults benefit from and enjoy learning via social interaction as much as neurotypical adults do

Background: Autistic people show poor processing of social signals (i.e. about the social world). But how do they learn via social interaction? // Methods: 68 neurotypical adults and 60 autistic adults learned about obscure items (e.g. exotic animals) over Zoom (i) in a live video-call with the teacher, (ii) from a recorded learner-teacher interaction video and (iii) from a recorded teacher-alone video. Data were analysed via analysis of variance and multi-level regression models. // Results: Live teaching provided the most optimal learning condition, with no difference between groups. Enjoyment was the strongest predictor of learning: both groups enjoyed the live interaction significantly more than other condition and reported similar anxiety levels across conditions. // Limitations: Some of the autistic participants were self-diagnosed—however, further analysis where these participants were excluded showed the same results. Recruiting participants over online platforms may have introduced bias in our sample. Future work should investigate learning in social contexts via diverse sources (e.g. schools). // Conclusions: These findings advocate for a distinction between learning about the social versus learning via the social: cognitive models of autism should be revisited to consider social interaction not just as a puzzle to decode but rather a medium through which people, including neuro-diverse groups, learn about the world around them. // Trial registration: Part of this work has been pre-registered before data collection https://doi.org/10.17605/OSF.IO/5PGA

Intensified high yield production of GMMA based vaccines for high burden neglected disease

Vaccines are among the most important public health interventions currently available. The global demand for vaccines is growing due to global population growth, ongoing global immunization campaigns and the increase of antibiotic-resistant bacteria. Vaccines are of high importance in Low- and Middle-Income Countries (LMIC) where millions of deaths occur annually due to vaccines-preventable diseases. Global coverage would need at least 200 million doses annually of a two-dose vaccine to cover the at-risk birth cohort, and considerably more for catch up vaccination during a roll out phase. Especially for vaccines against diseases in LMIC, there is a major added constraint: the vaccine production cost per unit and manufacturing facilities investments must be minimized to ensure the vaccines will be both viable and sustainable. For diseases caused by Gram-negative bacteria, the vaccine platform based on the GMMA technology is promising. GMMA are highly immunogenic outer membrane exosomes produced from genetically engineered bacteria modified for high yield hyper-blebbing that can be purified with minimal downstream processing. Simple robust scalable processes make GMMA ideal for cost-conscious vaccines. Please click Download on the upper right corner to see the full abstract

Serotype O:8 isolates in the Yersinia pseudotuberculosis complex have different O-antigen gene clusters and produce various forms of rough LPS

In Yersinia pseudotuberculosis complex, the O-antigen of LPS is used for the serological characterization of strains, and 21 serotypes have been identified to date. The O-antigen biosynthesis gene cluster and corresponding O-antigen structure have been described for 18, leaving O:8, O:13 and O:14 unresolved. In this study, two O:8 isolates were examined. The O-antigen gene cluster sequence of strain 151 was near identical to serotype O:4a, though a frame-shift mutation was found in ddhD, while No. 6 was different to 151 and carried the O:1b gene cluster. Structural analysis revealed that No. 6 produced a deeply truncated LPS, suggesting a mutation within the waaF gene. Both ddhD and waaF were cloned and expressed in 151 and No. 6 strains, respectively, and it appeared that expression of ddhD gene in strain 151 restored the O-antigen on LPS, while waaF in No. 6 resulted in an LPS truncated less severely but still without the O-antigen, suggesting that other mutations occurred in this strain. Thus, both O:8 isolates were found to be spontaneous O-antigen-negative mutants derived from other validated serotypes, and we propose to remove this serotype from the O-serotyping scheme, as the O:8 serological specificity is not based on the O-antigen.Peer reviewe

Analytical development to support manufacturing of a sustainable vaccine against Invasive Nontyphoidal Salmonellosis

GVGH is developing a candidate trivalent Salmonella vaccine to fight invasive nontyphoidal Salmonellosis (iNTS) and typhoid fever, especially aimed for sub-Saharan Africa to impact disease burden and to reduce anti-microbial resistance spread. This trivalent vaccine may be the only viable option for a sustainable iNTS vaccine in sub-Saharan Africa over the separate administration of Typhoid Conjugate Vaccines (TCV) and a vaccine against iNTS. GVGH generated the iNTS-TCV formulation by combining the GMMA technology for the iNTS components, S. Typhimurium (STm) and S. Enteritidis (SEn) GMMA adsorbed on Alhydrogel, and the Vi-CRM197 glycoconjugate, originally developed by GVGH and recently WHO prequalified as TCV TYPHIBEV by Biological E Ltd (Hyderabad, India). A set of analytical methods to support the vaccine lot release and characterization have been developed by GVGH. In particular, to quantify the key active ingredients of iNTS components a competitive ELISA-based method (FAcE, Formulated Alhydrogel competitive ELISA assay) has been setup and characterized in terms of specificity, accuracy and precision. Vi component is instead characterized by means of HPAEC-PAD method, able to specifically identify and quantify the total polysaccharide in the final drug product. With regard to safety assessment, a Monocyte Activation Test (MAT) has been developed as to monitor the intrinsic pyrogenicity of GMMA-based vaccines and applied as surveillance test for the Phase 1 clinical lot, with the plan to set release criteria based on clinical experience. In vivo potency assay has been set to characterize the immunogenicity of vaccine lots in comparison to freshly formulated material at the time of release and during real-time stability. A significant antibody response to each of the active ingredients of the trivalent vaccine is raised in mice and assessed by Parallel Line Assay. Overall, the applied analytical panel and the results support the development of an iNTS-TCV vaccine as a viable option for a sustainable iNTS vaccine in sub-Saharan Africa

The Role of Curcumin in Prostate Cancer Cells and Derived Spheroids

A major challenge in the clinical management of prostate cancer (PC) is to inhibit tumor growth and prevent metastatic spreading. In recent years, considerable efforts have been made to discover new compounds useful for PC therapy, and promising advances in this field were reached. Drugs currently used in PC therapy frequently induce resistance and PC progresses toward metastatic castration-resistant forms (mCRPC), making it virtually incurable. Curcumin, a commercially avail- able nutritional supplement, represents an attractive therapeutic agent for mCRPC patients. In the present study, we compared the effects of chemotherapeutic drugs such as docetaxel, paclitaxel, and cisplatin, to curcumin, on two PC cell lines displaying a different metastatic potential: DU145 (moder- ate metastatic potential) and PC-3 (high metastatic potential). Our results revealed a dose-dependent reduction of DU145 and PC-3 cell viability upon treatment with curcumin similar to chemotherapeutic agents (paclitaxel, cisplatin, and docetaxel). Furthermore, we explored the EGFR-mediated signaling effects on ERK activation in DU145 and PC-3 cells. Our results showed that DU145 and PC-3 cells overexpress EGFR, and the treatment with chemotherapeutic agents or curcumin reduced EGFR expression levels and ERK activation. Finally, chemotherapeutic agents and curcumin reduced the size of DU145 and PC-3 spheroids and have the potential to induce apoptosis and also in Matrigel. In conclusion, despite different studies being carried out to identify the potential synergistic curcumin combinations with chemopreventive/therapeutic efficacy for inhibiting PC growth, the results show the ability of curcumin used alone, or in combinatorial approaches, to impair the size and the viability of PC-derived spheroids

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment