Inhibiting DNA methylation as a strategy to enhance adipose-derived stem cells differentiation. Focus on the role of Akt/mTOR and Wnt/β-catenin pathways on adipogenesis

Adipose-derived mesenchymal stem cells (ASCs) represent a valid therapeutic option for clinical application in several diseases, due to their ability to repair damaged tissues and to mitigate the inflammatory/immune response. A better understanding of the underlying mechanisms regulating ASC biology might represent the chance to modulate their in vitro characteristics and differentiation potential for regenerative medicine purposes. Herein, we investigated the effects of the demethylating agent 5-azacytidine (5-aza) on proliferation, clonogenicity, migration, adipogenic differentiation and senescence of ASCs, to identify the molecular pathways involved. Through functional assays, we observed a detrimental effect of 5-aza on ASC self-renewal capacity and migration, accompanied by actin cytoskeleton reorganization, with decreased stress fibers. Conversely, 5-aza treatment enhanced ASC adipogenic differentiation, as assessed by lipid accumulation and expression of lineage-specific markers. We analyzed the involvement of the Akt/mTOR, MAPK and Wnt/beta-catenin pathways in these processes. Our results indicated impairment of Akt and ERK phosphorylation, potentially explaining the reduced cell proliferation and migration. We observed a 5-aza-mediated inhibition of the Wnt signaling pathway, this potentially explaining the pro-adipogenic effect of the drug. Finally, 5-aza treatment significantly induced ASC senescence, through upregulation of the p53/p21 axis. Our data may have important translational implications, by helping in clarifying the potential risks and advantages of using epigenetic treatment to improve ASC characteristics for cell-based clinical approaches

A Multicentre Study: The Use of Micrografts in the Reconstruction of Full-Thickness Posttraumatic Skin Defects of the Limbs - A Whole Innovative Concept in Regenerative Surgery

The skin graft is a surgical technique commonly used in the reconstructive surgery of the limbs, in order to repair skin loss, as well as to repair the donor area of the flaps and cover the dermal substitutes after engraftment. The unavoidable side effect of this technique consists of unaesthetic scars. In order to achieve the healing of posttraumatic ulcers by means of tissue regeneration and to avoid excessive scarring, a new innovative technology based on the application of autologous micrografts, obtained by Rigenera technology, was reported. This technology was able to induce tissue repair by highly viable skin micrografts of 80 micron size achieved by a mechanical disaggregation method. The specific cell population of these micrografts includes progenitor cells, which in association with the fragment of the Extracellular Matrix (ECM) and growth factors derived by patients' own tissue initiate biological processes of regeneration enhancing the wound healing process. We have used this technique in 70 cases of traumatic wounds of the lower and upper limbs, characterized by extensive loss of skin substance and soft tissue. In all cases, we have applied the Rigenera protocol using skin micrografts, achieving in 69 cases the complete healing of wounds in a period between 35 and 84 days. For each patient, the reconstructive outcome was evaluated weekly to assess the efficacy of this technique and any arising complication. A visual analogue scale (VAS) was administered to assess the amount of pain felt after the micrografts' application, whereas we evaluated the scars according to the Vancouver scale and the wound prognosis according to Wound Bed Score. We have thus been able to demonstrate that Rigenera procedure is very effective in stimulating skin regeneration, while reducing the outcome scar

MULTICUBE: Multi-objective design space exploration of multi-core architectures

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One year of surgical mask testing at the University of Bologna labs:Lessons learned from data analysis

The outbreak of SARS-CoV-2 pandemic highlighted the worldwide lack of surgical masks and personal protective equipment, which represent the main defense available against respiratory diseases as COVID-19. At the time, masks shortage was dramatic in Italy, the first European country seriously hit by the pandemic: aiming to address the emergency and to support the Italian industrial reconversion to the production of surgical masks, a multidisciplinary team of the University of Bologna organized a laboratory to test surgical masks according to European regulations. The group, driven by the expertise of chemical engineers, microbiologists, and occupational physicians, set-up the test lines to perform all the functional tests required. The laboratory started its activity on late March 2020, and as of the end of December of the same year 435 surgical mask prototypes were tested, with only 42 masks compliant to the European standard. From the analysis of the materials used, as well as of the production methods, it was found that a compliant surgical mask is most likely composed of three layers, a central meltblown filtration layer and two external spunbond comfort layers. An increase in the material thickness (grammage), or in the number of layers, does not improve the filtration efficiency, but leads to poor breathability, indicating that filtration depends not only on pure size exclusion, but other mechanisms are taking place (driven by electrostatic charge). The study critically reviewed the European standard procedures, identifying the weak aspects; among the others, the control of aerosol droplet size during the bacterial filtration test results to be crucial, since it can change the classification of a mask when its performance lies near to the limiting values of 95 or 98%

Clinical outcome of instrumented fusion for the treatment of failed back surgery syndrome: a case series of 100 patients

Scientific Assessment and Innovation in Neurosurgical Treatment Strategie

The largest multicentre data collection on prepectoral breast reconstruction: The iBAG study

Background and Objectives: In the last years, prepectoral breast reconstruction has increased its popularity, becoming a standard reconstructive technique by preserving pectoralis major anatomy and functionality. Nevertheless, the lack of solid and extensive data negatively impacts on surgeons\u2019 correct information about postoperative complication rates and proper patient selection. This study aims to collect the largest evidence on this procedure. Methods: A multicentre retrospective audit, promoted by the Barcelona Hospital, collected the experience of 30 centers on prepectoral breast reconstruction with Braxon ADM. The study had the scientific support of INPECS and IIB societies which provided the online database Clinapsis. Results: A total of 1450 procedures were retrospectively collected in a 6-year period. Mean age 52.4 years, BMI 23.9, follow-up 22.7 months. Reconstruction was carried out after a tumor in 77.1% of the cases, 20.1% had prophylactic surgery, 2.8% had revisions. Diabetes, smoke, and immunosuppression had an influence on complications occurrence, as well as implant weight. Capsular contracture was associated with postoperative radiotherapy, but the overall rate was low (2.1%). Complications led to implant loss in 6.5% of the cases. Conclusions: The international Braxon Audit Group multicentre data collection represents a milestone in the field of breast reconstruction, extensively improving the knowledge of this procedure

Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action

Genome-wide identification of FoxO-dependent gene networks in skeletal muscle during C26 cancer cachexia

BACKGROUND: Evidence from cachectic cancer patients and animal models of cancer cachexia supports the involvement of Forkhead box O (FoxO) transcription factors in driving cancer-induced skeletal muscle wasting. However, the genome-wide gene networks and associated biological processes regulated by FoxO during cancer cachexia are unknown. We hypothesize that FoxO is a central upstream regulator of diverse gene networks in skeletal muscle during cancer that may act coordinately to promote the wasting phenotype. METHODS: To inhibit endogenous FoxO DNA-binding, we transduced limb and diaphragm muscles of mice with AAV9 containing the cDNA for a dominant negative (d.n.) FoxO protein (or GFP control). The d.n.FoxO construct consists of only the FoxO3a DNA-binding domain that is highly homologous to that of FoxO1 and FoxO4, and which outcompetes and blocks endogenous FoxO DNA binding. Mice were subsequently inoculated with Colon-26 (C26) cells and muscles harvested 26 days later. RESULTS: Blocking FoxO prevented C26-induced muscle fiber atrophy of both locomotor muscles and the diaphragm and significantly spared force deficits. This sparing of muscle size and function was associated with the differential regulation of 543 transcripts (out of 2,093) which changed in response to C26. Bioinformatics analysis of upregulated gene transcripts that required FoxO revealed enrichment of the proteasome, AP-1 and IL-6 pathways, and included several atrophy-related transcription factors, including Stat3, Fos, and Cebpb. FoxO was also necessary for the cancer-induced downregulation of several gene transcripts that were enriched for extracellular matrix and sarcomere protein-encoding genes. We validated these findings in limb muscles and the diaphragm through qRT-PCR, and further demonstrate that FoxO1 and/or FoxO3a are sufficient to increase Stat3, Fos, Cebpb, and the C/EBPβ target gene, Ubr2. Analysis of the Cebpb proximal promoter revealed two bona fide FoxO binding elements, which we further establish are necessary for Cebpb promoter activation in response to IL-6, a predominant cytokine in the C26 cancer model. CONCLUSIONS: These findings provide new evidence that FoxO-dependent transcription is a central node controlling diverse gene networks in skeletal muscle during cancer cachexia, and identifies novel candidate genes and networks for further investigation as causative factors in cancer-induced wasting.R01 AR060217 - NIAMS NIH HHS; R01 AR060209 - NIAMS NIH HHS; T32 HD043730 - NICHD NIH HHS; R00 HL098453 - NHLBI NIH HHS; R00HL098453 - NHLBI NIH HHS; R01AR060209 - NIAMS NIH HHS; R01AR060217 - NIAMS NIH HH