CHROP APPROACH DISSECTS THE DYNAMIC PROFILING OF CHROMATOME AT ENHANCERS OF INFLAMMATORY GENES

Inflammatory stimuli drive a fine rearrangement of cell-specific chromatin determinants at cis-regulatory regions of inflammatory genes. Although a few determinants are known (i.e. H3K4me1 and Pu.1), a global picture of the enhancers\u2019 molecular signature and how specific factors dynamically synergize during the inflammatory response remain incomplete. The aim of my thesis is the global characterization of the enhancers\u2019 determinants and their dynamic profiling during inflammatory response. I addressed this issue by employing the chromatin proteomics approach (ChroP), which combines chromatin immunoprecipitation and mass spectrometry-based proteomics, to dissect histone post-translational modifications (modificome) and chromatin-binding proteins (interactome) associated with a specific chromatin region. We used H3K4me1 and Pu.1 antibodies to specifically enrich enhancers from macrophage-derived RAW264.7 cells. Native chromatin and formaldehyde-fixed chromatin from SILAC-labeled cells were used for the hPTMs profiling and the identification of chromatin binding proteins, respectively. Furthermore, a triple-SILAC setup was used in time-course experiments to profile the gene transcriptional activation triggered by lipopolysaccharide. Our findings suggest that enhancers have in basal condition an overall higher-ordered structure that is maintained during the inflammation; while a subset of proteins displays a dynamic behavior, i.e. the PBAF complex and Dnmt1, that seem to synergize with specific hPTMs to set an environment permissive to transcription. Interestingly, a number of factors are newly recruited at enhancers, suggesting a role in fine-tuning the appropriate gene expression: among them, Junb and Stat1 are well characterized to be involved in inflammatory response, while others (i.e. DDIT3 and Ifi204) are novel, very promising and under further investigation

Expanding the Circuitry of Pluripotency by Selective Isolation of Chromatin-Associated Proteins

Maintenance of pluripotency is regulated by a network of transcription factors coordinated by Oct4, Sox2, and Nanog (OSN), yet a systematic investigation of the composition and dynamics of the OSN protein network specifically on chromatin is still missing. Here we have developed a method combining ChIP with selective isolation of chromatin-associated proteins (SICAP) followed by mass spectrometry to identify chromatin-bound partners of a protein of interest. ChIP-SICAP in mouse embryonic stem cells (ESCs) identified over 400 proteins associating with OSN, including several whose interaction depends on the pluripotent state. Trim24, a previously unrecognized protein in the network, converges with OSN on multiple enhancers and suppresses the expression of developmental genes while activating cell cycle genes. Consistently, Trim24 significantly improved efficiency of cellular reprogramming, demonstrating its direct functionality in establishing pluripotency. Collectively, ChIP-SICAP provides a powerful tool to decode chromatin protein composition, further enhanced by its integrative capacity to perform ChIP-seq

Chromatin proteomics reveals novel combinatorial histone modification signatures that mark distinct subpopulations of macrophage enhancers

The integrated activity of cis-regulatory elements fine-tunes transcriptional programs of mammalian cells by recruiting cell type-specific as well as ubiquitous transcription factors (TFs). Despite their key role in modulating transcription, enhancers are still poorly characterized at the molecular level, and their limited DNA sequence conservation in evolution and variable distance from target genes make their unbiased identification challenging. The coexistence of high mono-methylation and low tri-methylation levels of lysine 4 of histone H3 is considered a signature of enhancers, but a comprehensive view of histone modifications associated to enhancers is still lacking. By combining chromatin immunoprecipitation (ChIP) with mass spectrometry, we investigated cis-regulatory regions in macrophages to comprehensively identify histone marks specifically associated with enhancers, and to profile their dynamics after transcriptional activation elicited by an inflammatory stimulation. The intersection of the proteomics data with ChIP-seq and RNA-seq analyses revealed the existence of novel subpopulations of enhancers, marked by specific histone modification signatures: specifically, H3K4me1/K36me2 marks transcribed enhancers, while H3K4me1/K36me3 and H3K4me1/K79me2 combinations mark distinct classes of intronic enhancers. Thus, our MS analysis of functionally distinct genomic regions revealed the combinatorial code of histone modifications, highlighting the potential of proteomics in addressing fundamental questions in epigenetics

BCAT1 redox function maintains mitotic fidelity

The metabolic enzyme branched-chain amino acid transaminase 1 (BCAT1) drives cell proliferation in aggressive cancers such as glioblastoma. Here, we show that BCAT1 localizes to mitotic structures and has a non-metabolic function as a mitotic regulator. Furthermore, BCAT1 is required for chromosome segregation in cancer and induced pluripotent stem cells and tumor growth in human cerebral organoid and mouse syngraft models. Applying gene knockout and rescue strategies, we show that the BCAT1 CXXC redox motif is crucial for controlling cysteine sulfenylation specifically in mitotic cells, promoting Aurora kinase B localization to centromeres, and securing accurate chromosome segregation. These findings offer an explanation for the well-established role of BCAT1 in promoting cancer cell proliferation. In summary, our data establish BCAT1 as a component of the mitotic apparatus that safeguards mitotic fidelity through a moonlighting redox functionality

Worsening of Cardiomyopathy Using Deflazacort in an Animal Model Rescued by Gene Therapy

We have previously demonstrated that gene therapy can rescue the phenotype and extend lifespan in the delta-sarcoglycan deficient cardiomyopathic hamster. In patients with similar genetic defects, steroids have been largely used to slow down disease progression. Aim of our study was to evaluate the combined effects of steroid treatment and gene therapy on cardiac function. We injected the human delta-sarcoglycan cDNA by adeno-associated virus (AAV) 2/8 by a single intraperitoneal injection into BIO14.6 Syrian hamsters at ten days of age to rescue the phenotype. We then treated the hamsters with deflazacort. Treatment was administered to half of the hamsters that had received the AAV and the other hamsters without AAV, as well as to normal hamsters. Both horizontal and vertical activities were greatly enhanced by deflazacort in all groups. As in previous experiments, the AAV treatment alone was able to preserve the ejection fraction (70±7% EF). However, the EF value declined (52±14%) with a combination of AAV and deflazacort. This was similar with all the other groups of affected animals. We confirm that gene therapy improves cardiac function in the BIO14.6 hamsters. Our results suggest that deflazacort is ineffective and may also have a negative impact on the cardiomyopathy rescue, possibly by boosting motor activity. This is unexpected and may have significance in terms of the lifestyle recommendations for patients

Surgical treatment of sacral chordoma: survival and prognostic factors

Sacral chordoma is a rare low-to-intermediate grade malignant tumour. The mainstay of treatment is still surgery with en bloc and wide resection margins, which can grant the best chances of a long-term control or cure of this disease. The first aim of this paper is to collect data about survival, time to local recurrence and metastasis among patients affected by sacral chordoma and primarily treated with surgery. The second aim is to analyze the influence of level resection, tumor volume and surgical margins on local recurrence

The performance of human periodontal ligament mesenchymal stem cells on xenogenic biomaterials

Mesenchymal stem cells from periodontal ligament (PDL-MSCs) hold great promise for bone regeneration. Most studies regarding the osteogenic differentiation of stem cells from periodontal tissue suggest that PDL cells may have many osteoblast-like properties, including the ability to form calcified nodules in vitro. This study investigated the morphological and histochemistry aspects of human PDL-MSCs, induced for osteogenic differentiation and seeded on a xenogenic porcine bone substitute in vitro, at different times of incubation. This biomaterial seems physically identical to human bone, and it has been reported to be osteoconductive. Our results indicated that the cells had a high affinity for the three-dimensional biomaterials; in fact, cellular proliferation and colonization was evident, and after 21 days the adherent cells started to detach themselves from the substrate, and at 30 days of incubation in differentiation medium, the cells completely lost the adhesion to the Petri's disk, englobing all bioparticles. In conclusion, the in vitro behaviour of PDL-MSCs and their relationship with three-dimensional scaffold biomaterials encourage in vivo investigations for their use in dental tissue regeneration

Spatiotemporal proteomics uncovers cathepsin-dependent macrophage cell death during Salmonella infection

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined Click chemistry with pulsed stable isotope labelling of amino acids in cell culture to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen Salmonella enterica Typhimurium. We monitored newly synthesized proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified that active cathepsins re-traffic to the nucleus and that these are linked to cell death. Pharmacological cathepsin inhibition and nuclear targeting of a cellular cathepsin inhibitor (stefin B) suppressed S. enterica Typhimurium-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolution of host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.Bio-organic Synthesi

Segmental transverse colectomy. Minimally invasive versus open approach: results from a multicenter collaborative study

The role of minimally invasive surgery in the treatment of transverse colon cancer is still controversial. The aim of this study is to investigate the advantages of a totally laparoscopic technique comparing open versus laparoscopic/robotic approach. Three hundred and eighty-eight patients with transverse colon cancer, treated with a segmental colon resection, were retrospectively analyzed. Demographic data, tumor stage, operative time, intraoperative complications, number of harvested lymph nodes and recovery outcomes were recorded. Recurrences and death were also evaluated during the follow-up. No differences were found between conventional and minimally invasive surgery, both for oncological long-term outcomes (recurrence rate p = 0.28; mortality p = 0.62) and postoperative complications (overall rate p = 0.43; anemia p = 0.78; nausea p = 0.68; infections p = 0.91; bleeding p = 0.62; anastomotic leak p = 0.55; ileus p = 0.75). Nevertheless, recovery outcomes showed statistically significant differences in favor of minimally invasive surgery in terms of time to first flatus (p = 0.001), tolerance to solid diet (p = 0.017), time to first mobilization (p = 0.001) and hospital stay (p = 0.004). Compared with laparoscopic approach, robotic surgery showed significantly better results for time to first flatus (p = 0.001), to first mobilization (p = 0.005) and tolerance to solid diet (p = 0.001). Finally, anastomosis evaluation confirmed the superiority of intracorporeal approach which showed significantly better results for time to first flatus (p = 0.001), to first mobilization (p = 0.003) and tolerance to solid diet (p = 0.001); moreover, we recorded a statistical difference in favor of intracorporeal approach for infection rate (p = 0.04), bleeding (p = 0.001) and anastomotic leak (p = 0.03). Minimally invasive approach is safe and effective as the conventional open surgery, with comparable oncological results but not negligible advantages in terms of recovery outcomes. Moreover, we demonstrated that robotic approach may be considered a valid option and an intracorporeal anastomosis should always be preferred