Tumor derived Microvesicles enhance cross-processing ability of clinical grade Dendritic Cells

Tumor cells release extracellular microvesicles (MVs) in the microenvironment to deliver biological signals to neighbouring cells as well as to cells in distant tissues. Tumor-derived MVs appear to play contradictory role promoting both immunosuppression and tumor growth and both evoking tumor specific immune response. Recent evidences indicate that tumor-derived MVs can positively impact Dendritic Cells (DCs) immunogenicity by reprogramming DC antigen processing machinery and intracellular signaling pathways, thus promoting anti-tumor response. DCs are considered pivot cells of the immune system due to their exclusive ability to coordinate the innate and acquired immune responses, cross-present exogenous antigens and prime naïve T cells. DCs are required for the induction and maintenance of long-lasting anti-tumor immunity and their exploitation has been extensively investigated for the design of anti-tumor vaccines. However, the clinical grade culture conditions that are required to generate DCs for therapeutic use can strongly affect their functions. Here, we investigated the immunomodulatory impact of MVs carrying the MUC1 tumor glycoantigen (MVsMUC1) as immunogen formulation on clinical grade DCs grown in X-VIVO 15 (X-DCs). Results indicated that X-DCs displayed reduced performance of the antigen processing machinery in term of diminished phagocytosis and acidification of the phagosomal compartment suggesting an altered immunogenicity of clinical grade DCs. Pulsing DCs with MVsMUC1 restored phagosomal alkalinization, triggering ROS increase. This was not observed when a soluble MUC1 protein was employed (rMUC1). Concurrently, MVsMUC1 internalization by X-DCs allowed MUC1 cross-processing. Most importantly, MVsMUC1 pulsed DCs activated IFNγ response mediated by MUC1 specific CD8+ T cells. These results strongly support the employment of tumor-derived MVs as immunogen platforms for the implementation of DC-based vaccine

Tumor-derived microvesicles modulate antigen cross-processing via reactive oxygen species-mediated alkalinization of phagosomal compartment in dendritic cells

Dendritic cells (DCs) are the only antigen-presenting cells able to prime naïve T cells and cross-prime antigen-specific CD8+ T cells. Their functionality is a requirement for the induction and maintenance of long-lasting cancer immunity. Albeit intensively investigated, the in vivo mechanisms underlying efficient antigen cross-processing and presentation are not fully understood. Several pieces of evidence indicate that antigen transfer to DCs mediated by microvesicles (MVs) enhances antigen immunogenicity. This mechanism is also relevant for cross-presentation of those tumor-associated glycoproteins such as MUC1 that are blocked in HLA class II compartment when internalized by DCs as soluble molecules. Here, we present pieces of evidence that the internalization of tumor-derived MVs modulates antigen-processing machinery of DCs. Employing MVs derived from ovarian cancer ascites fluid and established tumor cell lines, we show that MV uptake modifies DC phagosomal microenvironment, triggering reactive oxygen species (ROS) accumulation and early alkalinization. Indeed, tumor MVs carry radical species and the MV uptake by DCs counteracts the chemically mediated acidification of the phagosomal compartment. Further pieces of evidence suggest that efficacious antigen cross-priming of the MUC1 antigen carried by the tumor MVs results from the early signaling induced by MV internalization and the function of the antigen-processing machinery of DCs. These results strongly support the hypothesis that tumor-derived MVs impact antigen immunogenicity by tuning the antigen-processing machinery of DCs, besides being carrier of tumor antigens. Furthermore, these findings have important implications for the exploitation of MVs as antigenic cell-free immunogen for DC-based therapeutic strategies

Desiccation Tolerance in Ramonda serbica Panc.: An Integrative Transcriptomic, Proteomic, Metabolite and Photosynthetic Study

The resurrection plant Ramonda serbica Panc. survives long desiccation periods and fully recovers metabolic functions within one day upon watering. This study aimed to identify key candidates and pathways involved in desiccation tolerance in R. serbica. We combined differential transcriptomics and proteomics, phenolic and sugar analysis, FTIR analysis of the cell wall polymers, and detailed analysis of the photosynthetic electron transport (PET) chain. The proteomic analysis allowed the relative quantification of 1192 different protein groups, of which 408 were differentially abundant between hydrated (HL) and desiccated leaves (DL). Almost all differentially abundant proteins related to photosynthetic processes were less abundant, while chlorophyll fluorescence measurements implied shifting from linear PET to cyclic electron transport (CET). The levels of H2O2 scavenging enzymes, ascorbate-glutathione cycle components, catalases, peroxiredoxins, Fe-, and Mn superoxide dismutase (SOD) were reduced in DL. However, six germin-like proteins (GLPs), four Cu/ZnSOD isoforms, three polyphenol oxidases, and 22 late embryogenesis abundant proteins (LEAPs; mainly LEA4 and dehydrins), were desiccation-inducible. Desiccation provoked cell wall remodeling related to GLP-derived H2O2/HO● activity and pectin demethylesterification. This comprehensive study contributes to understanding the role and regulation of the main metabolic pathways during desiccation aiming at crop drought tolerance improvemen

Integrative transcriptomic and TMT-based proteomic analysis reveals the desiccation tolerance in Ramonda serbica Panc.

Ramonda serbica Panc. is a resurrection plant that can survive long periods of desiccation and fully restores its metabolic functions just one day after watering. The aim of this study was to identify key candidates and metabolic pathways involved in R. serbica desiccation tolerance. We combined differential transcriptomics and proteomics with the analysis of phenolics, sugars, cell wall polymers and photosynthetic electron transport (PET) chain. TMT-based proteomic analysis allowed the relative quantification of 1192 different protein groups, 408 of which were differentially abundant between hydrated (HL) and desiccated leaves (DL). Almost all differentially abundant proteins and transcripts related to photosynthetic processes were downregulated in DL. Chlorophyll fluorescence measurements showed a shift from linear PET to cyclic electron transport (CET). The levels of H2O2-scavenging enzymes, ascorbate- glutathione cycle components, catalases, peroxiredoxins, Fe-, and Mn superoxide dismutase (SOD) were reduced in DL. However, six germin-like proteins (GLPs), four Cu/ZnSOD isoforms, three polyphenol oxidases, and 22 late embryogenesis abundant proteins (LEAPs; mainly LEA4 and dehydrins), were desiccation-inducible. Desiccation led to cell wall remodelling related to GLP-derived H2O2/HO● activity and pectin demethylesterification. This comprehensive study contributes to understanding the role and regulation of important metabolic pathways during desiccation with the final aim to help improving the drought tolerance in crops.Abstract: 5th Conference of the International Plant Proteomics Organization; May 22-25, 2022 | Porto Palace Hotel | Thessaloniki, Hella

Integration of differential transcriptomic and proteomic data in hydrated and desiccated leaves of Ramonda serbica Panc.

The resurrection plant Ramonda serbica Panc. survives long desiccation periods and fully recovers metabolic functions within one day upon watering. We aimed to identify key candidates and pathways involved in desiccation tolerance in R. serbica by employing a systems biology approach, combining transcriptomics and proteomics. A total of 68,694 differentially expressed genes (DEGs; p-value<0.005 and abs(log2FC)≥2) were obtained in R. serbica leaves upon desiccation. Among them, 23,935 and 26,169 genes were upregulated and downregulated in desiccated leaves (DL) and hydrated leaves (HL), respectively. By differential TMT-based proteomic analysis 1192 different protein groups were identified after filtering with at least two unique peptides per protein. In total, 229 protein groups were more abundant in HL and 179 in DL (p-value<0.05 and abs(FC)≥1.3). The majority of the DAPs and DEGs involved in photosynthesis, transport, secondary metabolism, and signaling, were less abundant in DL. On the other hand, proteins and transcripts associated with fermentation, N-metabolism, heme, protein synthesis, folding and assembly, C1- metabolism, and late embryogenesis abundant proteins, were more accumulated in DL. A poor correlation between proteomic and transcriptomic results was detected for mitochondrial electron transport and ATP production, gluconeogenesis, glycolysis, tricarboxylic acid cycle, and enzymatic H2O2 scavengers due to different mRNA half-life, protein turnover, dynamic posttranscriptional and posttranslational modifications. Finally, desiccation tolerance in R. serbica is a species-specific process orchestrated by several metabolic pathways that are temporally and compartmentally regulated at several levels.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Twenty different late embryogenesis abundant proteins (LEAPs) accumulate in desiccated Ramonda serbica leaves

Resurrection plant Ramonda serbica Panc. survives desiccation for a long period and fully recovers metabolic functions already within one day upon watering [1]. Besides osmotic stress, desiccation provokes the accelerated generation of reactive oxygen species. The aim of our study was obtaining more insight into the mechanisms of desiccation tolerance in R. serbica by TMT labelled comparative quantitative proteomics of hydrated (HL) and desiccated leaves (DL). After de novo transcriptome analysis, 189456 transcripts with 189003 unigenes were annotated with seven common databases. Proteomic analysis allowed for the relative quantification of 895 different protein groups, 321 with a statistically significant difference in abundance between FL and DL. Among them, 25% referred to chloroplast and almost the same percentage were associated with desiccation and oxidative stress. Almost all differentially abundant proteins related to photosynthetic processes were down-regulated in DL, while those required for protein translation were more abundant in HL. Within differentially abundant proteins involved in antioxidative defence, the levels of enzymes involved in ascorbate-glutathione cycle, peroxiredoxins, Fe and Mn superoxide dismutase (SOD) were all reduced in DL, while germin-like proteins, three Cu/Zn SOD isoforms and polyphenol oxidases were more abundant in DL compared with HL. The protein family with the highest number of members showing the greatest accumulation upon desiccation comprised twenty different late embryogenesis abundant proteins (LEAPs), similarly as found by differential transcriptomic analysis. Taken together, our results imply a key role of LEAPs and Cu/Zn SOD in protective mechanism against desiccation in R. serbica, that may have significant implications on drought-related studies of crops grown in arid areas. This work was supported by the Science Fund of the Republic of Serbia (PROMIS project LEAPSyn-SCI, grant number 6039663). M.V. wishes to acknowledge the support of COST Action BM1405 for approving STSM in Padua during 2017 and 2018

Twenty different late embryogenesis abundant proteins (LEAPs) accumulate in desiccated Ramonda serbica leaves

Resurrection plant Ramonda serbica Panc. survives desiccation for a long period and fully recovers metabolic functions already within one day upon watering [1]. Besides osmotic stress, desiccation provokes the accelerated generation of reactive oxygen species. The aim of our study was obtaining more insight into the mechanisms of desiccation tolerance in R. serbica by TMT labelled comparative quantitative proteomics of hydrated (HL) and desiccated leaves (DL). After de novo transcriptome analysis, 189456 transcripts with 189003 unigenes were annotated with seven common databases. Proteomic analysis allowed for the relative quantification of 895 different protein groups, 321 with a statistically significant difference in abundance between FL and DL. Among them, 25% referred to chloroplast and almost the same percentage were associated with desiccation and oxidative stress. Almost all differentially abundant proteins related to photosynthetic processes were down-regulated in DL, while those required for protein translation were more abundant in HL. Within differentially abundant proteins involved in antioxidative defence, the levels of enzymes involved in ascorbate-glutathione cycle, peroxiredoxins, Fe and Mn superoxide dismutase (SOD) were all reduced in DL, while germin-like proteins, three Cu/Zn SOD isoforms and polyphenol oxidases were more abundant in DL compared with HL. The protein family with the highest number of members showing the greatest accumulation upon desiccation comprised twenty different late embryogenesis abundant proteins (LEAPs), similarly as found by differential transcriptomic analysis. Taken together, our results imply a key role of LEAPs and Cu/Zn SOD in protective mechanism against desiccation in R. serbica, that may have significant implications on drought-related studies of crops grown in arid areas. This work was supported by the Science Fund of the Republic of Serbia (PROMIS project LEAPSyn-SCI, grant number 6039663). M.V. wishes to acknowledge the support of COST Action BM1405 for approving STSM in Padua during 2017 and 2018.Related to poster presentation: [https://cherry.chem.bg.ac.rs/handle/123456789/4424

Theranostic Microneedle Devices: Innovative Biosensing and Transdermal Drugs Administration

Biosensing systems based on microneedles can overcome the stratum corneum of the skin, i. e. the outer natural barrier of the human body, without any pain and detect the target analytes directly in the interstitial fluid. Moreover, microneedle-based devices (MNDs) can combine diagnostic sensing and therapeutic administration of drugs in one single tool. From this point of view, more than a painless door to the human body, a MND represents the a perfect example of theranostic instrument, since a single device could quantify the real value of a relevant biomolecule, such as glucose, and accurately deliver a drug, the insulin, if needed. MNDs could be integrated on printed circuit boards, flexible electronics and microfluidic channels, thus allowing a continuous monitoring of the physiological parameters with very low invasiveness, together with sustained and localized administration of drugs. MNDs can be designed for very specific applications, from the detection of skin cancer to the monitoring of metabolic pathways. Moreover, several fabrication approaches have been introduced, from laboratories to large-scale production. Finally MNDs can be properly functionalized to enhance analytical performances

HDV can constrain HBV genetic evolution in hbsag: Implications for the identification of innovative pharmacological targets

Chronic HBV + HDV infection is associated with greater risk of liver fibrosis, earlier hepatic decompensation, and liver cirrhosis hepatocellular carcinoma compared to HBV mono-infection. However, to-date no direct anti-HDV drugs are available in clinical practice. Here, we identified conserved and variable regions in HBsAg and HDAg domains in HBV + HDV infection, a critical finding for the design of innovative therapeutic agents. The extent of amino-acid variability was measured by Shannon-Entropy (Sn) in HBsAg genotype-D sequences from 31 HBV + HDV infected and 62 HBV mono-infected patients (comparable for demographics and virological-parameters), and in 47 HDAg genotype-1 sequences. Positions with Sn = 0 were defined as conserved. The percentage of conserved HBsAg-positions was significantly higher in HBV + HDV infection than HBV mono-infection (p = 0.001). Results were confirmed after stratification for HBeAg-status and patients’ age. A Sn = 0 at specific positions in the C-terminus HBsAg were correlated with higher HDV-RNA, suggesting that conservation of these positions can preserve HDV-fitness. Conversely, HDAg was characterized by a lower percentage of conserved-residues than HBsAg (p < 0.001), indicating higher functional plasticity. Furthermore, specific HDAg-mutations were significantly correlated with higher HDV-RNA, suggesting a role in conferring HDV replicative-advantage. Among HDAg-domains, only the virus-assembly signal exhibited a high genetic conservation (75% of conserved-residues). In conclusion, HDV can constrain HBsAg genetic evolution to preserve its fitness. The identification of conserved regions in HDAg poses the basis for designing innovative targets against HDV-infection