Fabio Quondamatteo

AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation

Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene.

The mesocolon: a histological and electron microscopic characterization of the mesenteric attachment of the colon prior to and after surgical mobilization.

Background:Colonic mobilization requires separation of mesocolon from underlying fascia. Despite the surgical importance of planes formed by these structures, no study has formally characterized their microscopic features. The aim of this study was to determine the histological and electron microscopic appearance of mesocolon, fascia, and retroperitoneum, prior to and after colonic mobilization. Methods:In 24 cadavers, samples were taken from right, transverse, descending, and sigmoid mesocolon. In 12 cadavers, specimens were stained with hematoxylin and eosin (3 sections) or Masson trichrome (3 sections). In the second 12 cadavers, lymphatic channels were identified by staining immunohistochemically for podoplanin. The ascending mesocolon was assessed with scanning electron microscopy. The above process was first conducted with the mesocolon in situ. The mesocolon was then surgically mobilized, and the process was repeated on remaining structures. Results:The microscopic structure of mesocolon and associated fascia was consistent from ileocecal to mesorectal level. A surface mesothelium and underlying connective tissue were evident throughout. Fibrous septae separated adipocyte lobules. Where apposed to retroperitoneum, 2 mesothelial layers separated mesocolon and underlying retroperitoneum. A connective tissue layer occurred between these (ie, Toldts fascia). Lymphatic channels were evident both in mesocolic connective tissue and Toldts fascia. After surgical separation of mesocolon and fascia both remained contiguous, the fascia remained in situ and the retroperitoneum undisturbed. Conclusions:The findings demonstrate that the contiguous mesocolon and retroperitoneum are separated by mesothelial and connective tissue layers. These properties generate the surgical planes (ie, meso- and retrofascial planes) exploited in colonic and mesocolic mobilization.

NADPH oxidase complex-derived reactive oxygen species, the actin cytoskeleton, and Rho GTPases in cell migration

SIGNIFICANCEnRho GTPases are historically known to be central regulators of actin cytoskeleton reorganization. This affects many processes including cell migration. In addition, members of the Rac subfamily are known to be involved in reactive oxygen species (ROS) production through the regulation of NADPH oxidase (Nox) activity. This review focuses on relationships between Nox-regulated ROS, Rho GTPases, and cytoskeletal reorganization, in the context of cell migration.nnnRECENT ADVANCESnIt has become clear that ROS participate in the regulation of certain Rho GTPase family members, thus mediating cytoskeletal reorganization.nnnCRITICAL ISSUESnThe role of the actin cytoskeleton in providing a scaffold for components of the Nox complex needs to be examined in the light of these new advances. During cell migration, Rho GTPases, ROS, and cytoskeletal organization appear to function as a complex regulatory network. However, more work is needed to fully elucidate the interactions between these factors and their potential in vivo importance.nnnFUTURE DIRECTIONSnUltrastructural analysis, that is, electron microscopy, particularly immunogold labeling, will enable direct visualization of subcellular compartments. This in conjunction with the analysis of tissues lacking specific Rho GTPases, and Nox components will facilitate a detailed examination of the interactions of these structures with the actin cytoskeleton. In combination with the analysis of ROS production, including its subcellular location, these data will contribute significantly to our understanding of this intricate network under physiological conditions. Based on this, in vivo and in vitro studies can then be combined to elucidate the signaling pathways involved and their targets.

A detailed appraisal of mesocolic lymphangiology – an immunohistochemical and stereological analysis

Inadequate resection of the adjoining mesentery is associated with adverse outcome for colon cancer. Disruption of the integrity of the mesenteric lymphatic package has been implicated in this, though not proven. Recent studies have determined mesenteric anatomy and histology and now provide an opportunity to determine accurately the distribution of lymphatic vessels. The aim of this study was to characterise the distribution of the lymphatic vessels (LV) within the small intestinal and colonic mesentery, and in Toldts fascia, which lies between the mesocolon and underlying retroperitoneum. Mesenteric samples were harvested from 12 human cadavers. Samples were taken from the small bowel mesentery, ascending, transverse, descending mesocolon and from both apposed and non‐apposed portions of the mesosigmoid. Serial sections were stained immunohistochemically with monoclonal antibody D2‐40 (podoplanin), and Massons Trichrome. Lymphatic vessel (LV) density and radius of diffusion were determined using a stereological approach. A lymphatic network was embedded within the mesenteric connective tissue lattice throughout each mesenteric region. LV were identifiable within the submesothelial connective tissue where they measured 10.2 ± 4.1 μm in diameter and had an average radius of diffusion of 174.72 ± 97.68 μm. Unexpectedly, LV were identified in Toldts fascia, where they measured 4.3 ± 3.1 μm in diameter and had a radius of diffusion of 165.12 ± 66.26 μm. This is the first study systematically to determine and quantify the distribution of lymphatic vessels within the mesenteric organ and to demonstrate the presence of such vessels within Toldts fascia. A rich lymphatic network occupies all levels of the mesenteric connective tissue lattice. Within the latter, they are found within 0.1 mm of peritonealised mesenteric surfaces and are separated by an average distance of 0.17 mm and may be particularly vulnerable during surgery.

Mesenteric-Based Surgery Exploits Gastrointestinal, Peritoneal, Mesenteric and Fascial Continuity from Duodenojejunal Flexure to the Anorectal Junction--A Review.

Introduction: It is now well established that mesenteric-based colorectal surgery is associated with superior outcomes. Recent anatomic observations have demonstrated that the mesenteric organ is contiguous from the duodenojejunal to the anorectal junction. This led to similar observations in relation to associated peritoneum and fascia. The aim of this review was to demonstrate the relevance of the contiguity principle to resectional colorectal surgery. Methods: All literature in relation to mesenteric anatomy was reviewed from 1873 to the present, without language restriction. Results: Mesenteric-based surgery (i.e. complete mesocolic excision, total mesocolic and mesorectal excision) requires division of the peritoneal reflection (i.e. peritonotomy), and mesenteric mobilisation in the mesofascial plane. These are the fundamental technical elements of mesenterectomy. Mesenteric, peritoneal and fascial contiguity mean that in resectional surgery, these technical elements can be reproducibly applied at all levels from the origin at the superior mesenteric root, to the anorectal junction. Conclusions: The goals of complete mesocolic, total mesocolic and mesorectal excision can be universally achieved at any level from duodenojejunal flexure to anorectal junction, by adopting technical elements based on mesenteric, peritoneal and fascial contiguity.

Skin and diabetes mellitus: what do we know?

Diabetes mellitus (DM) is becoming increasingly prevalent worldwide. Although major complications of this condition involve kidney, retina and peripheral nerves, the skin of diabetic patients is also frequently injured. Hence, interest is mounting in the definition of the structural and molecular profile of non-complicated diabetic skin, i.e., before injuries occur. Most of the available knowledge in this area has been obtained relatively recently and, in part, derives from various diabetic animal models. These include both insulin-dependent and insulin-resistant models. Structural work in human diabetic skin has also been carried out by means of tissue samples or of non-invasive methods. Indications have indeed been found for molecular/structural changes in diabetic skin. However, the overall picture that emerges is heterogeneous, incomplete and often contradictory and many questions remain unanswered. This review aims to detail, as much as possible, the various pieces of current knowledge in a systematic and synoptic manner. This should aid the identification of areas in which key questions are still open and more research is needed. A comprehensive understanding of this field could help in determining molecular targets for the prevention and treatment of skin injuries in DM and markers for the monitoring of cutaneous and systemic aspects of the disease. Additionally, with the increasing development of non-invasive optics-based deep-tissue-imaging diagnostic technologies, precise knowledge of cutaneous texture and molecular structure becomes an important pre-requisite for the use of such methods in diabetic patients.

Preclinical trial of a novel surface architecture for improved primary fixation of cementless orthopaedic implants

BACKGROUNDnA new surface architecture for cementless orthopaedic implants (OsteoAnchor), which incorporates a multitude of tiny anchor features for enhancing primary fixation, was tested in an ovine hemi-arthroplasty pilot study.nnnMETHODSnTest animals were implanted with a hip stem component incorporating the OsteoAnchor surface architecture produced using additive layer manufacturing and control animals were implanted with stems containing a standard plasma sprayed titanium coating.nnnFINDINGSnIntra-operative surgeon feedback indicated that superior primary fixation was achieved for the OsteoAnchor stems and rapid return to normal gait and load bearing was observed post-operation. Following a 16-week recovery time, histological evaluation of the excised femurs revealed in-growth of healthy bone into the porous structure of the OsteoAnchor stems. Bone in-growth was not achieved for the plasma sprayed stems.nnnINTERPRETATIONnThese results indicate the potential for the OsteoAnchor surface architecture to enhance both the initial stability and long term lifetime of cementless orthopaedic implants.

Protrusion-guided extracellular vesicles mediate CD30 trans-signalling in the microenvironment of Hodgkin's lymphoma.

Classical Hodgkins lymphoma (cHL)‐affected lymphoid tissue contains only a few malignant Hodgkin and Reed–Sternberg (HRS) cells, which are disseminated within a massive infiltrate of reactive cells. In particular, the innate immune infiltrate is deemed to support tumour growth by direct cell–cell interaction. Since they are rarely found in close proximity to the malignant cells in situ, we investigated whether cHL‐derived extracellular vesicles might substitute for a direct cell–cell contact. We studied the crosstalk of the transmembrane proteins CD30 and CD30 ligand (CD30L) because they are selectively expressed on HRS and innate immune cells, respectively. Here, we showed that HRS cells released both the ectodomain as a soluble molecule (sCD30) and the entire receptor on the surface of extracellular vesicles. The vesicle diameter was 40–800 nm, as determined by cryo‐ and immune electron microscopy. In addition to CD30, typical extracellular vesicle markers were detected by mass spectrometry and flow cytometry, including tetraspanins, flotillins, heat shock proteins and adhesion molecules. In contrast to sCD30, vesicles caused a CD30‐dependent release of interleukin‐8 in CD30L+ eosinophil‐like EoL‐1 cells and primary granulocytes from healthy donors, underscoring the functionality of CD30 on vesicles. In extracellular matrix (ECM)‐embedded culture of HRS cells, a network of actin and tubulin‐based protrusions guided CD30+ vesicles into the micro‐environment. This network targeted CD30+ vesicles towards distant immune cells and caused a robust polarization of CD30L. Confocal laser scanning microscopy of 30 µm sections showed a CD30 vesicle‐containing network also in cHL‐affected lymphoid tissue of both mixed‐cellularity and nodular sclerosing subtypes. This network might facilitate the communication between distant cell types in cHL tissue and allow a functional CD30–CD30L interaction in trans. The tubulin backbone of the network may provide a target for the therapy of cHL with antitubulin‐based CD30 antibody constructs. Copyright

Changes in the electrical properties of the electrode–skin–underlying tissue composite during a week-long programme of neuromuscular electrical stimulation

Particular neuromuscular electrical stimulation (NMES) applications require the use of the same electrodes over a long duration (>1xa0day) without having access to them. Under such circumstance the quality of the electrode-skin contact cannot be assessed. We used the NMES signal itself to assess the quality of the electrode-skin contact and the electrical properties of the underlying tissues over a week. A 14% decrease in the skins stratum corneum resistance (from 20 to 17 kΩ) and a 15% decrease in the resistance of the electrodes and underlying tissues (from 550 to 460 Ω) were observed in the 14 healthy subjects investigated. A follow-on investigation of the effect of exercise-induced sweating on the electrical properties of the electrode-skin-underlying tissue composite during NMES indicated a correlation between the decrease in the resistance values observed over the course of the week and the accumulation of sweat at the electrode-skin interface. The value of the capacitance representing the dielectric properties of the skins stratum corneum increased after exercise-induced sweating but did not change significantly over the course of the week. We conclude that valuable information about the electrode-skin-underlying tissue composite can be gathered using the NMES signal itself, and suggest that this is a practical, safe and relatively simple method for monitoring these electrical properties during long-term stimulation.

An appraisal of the computed axial tomographic appearance of the human mesentery based on mesenteric contiguity from the duodenojejunal flexure to the mesorectal level

AbstractObjectiveThe human mesentery is now regarded as contiguous from the duodenojejunal (DJ) to anorectal level. This interpretation prompts re-appraisal of computed tomography (CT) images of the mesentery.MethodsA digital model and reference atlas of the mesentery were generated using the full-colour data set of the Visible Human Project (VHP). Seventy one normal abdominal CT images were examined to identify mesenteric regions. CT appearances were correlated with cadaveric and histological appearances at corresponding levels.ResultsAscending, descending and sigmoid mesocolons were identifiable in 75xa0%, 86xa0% and 88xa0% of the CTs, respectively. Flexural contiguity was evident in 66xa0%, 68xa0%, 71xa0% and 80xa0% for the ileocaecal, hepatic, splenic and rectosigmoid flexures, respectively. A posterior mesocolic boundary corresponding to the anterior renal fascia was evident in 40xa0% and 54xa0% of cases on the right and left, respectively. The anterior pararenal space (in front of the boundary) corresponded to the mesocolon.ConclusionsUsing the VHP, a mesenteric digital model and reference atlas were developed. This enabled re-appraisal of CT images of the mesentery, in which contiguous flexural and non-flexural mesenteric regions were repeatedly identifiable. The anterior pararenal space corresponded to the mesocolon.Key Points• The Visible Human Project (VHP) allows direct identification of mesenteric structures.n • Correlating CT and VHP allows identification of flexural and non-flexural mesenteric components.n • Radiologic appearance of intraperitoneal structures is assessed, starting from a mesenteric platform.