Insects found at a crime scene can produce traces referred to as fly artifacts (FA) due to their movement over the corpse and the manner in which they feed upon it. These can be detrimental for carrying out criminal investigations. Confusing a FA with a genuine bloodspot can lead to misinterpretations, also taking into consideration that FA may contain a human DNA profile. The aim of the present study was to employ scanning electron microscopy (SEM) for the analysis of FA produced by Calliphora vomitoria on hard surfaces and fabrics that are commonly present at crime scenes. FA and control bloodstains were produced under experimental conditions on metal, glass, plaster, cotton, and polyester. After macroscopic analysis, FA were examined at standard low (20–40 ×), medium low (300–600 ×), and high ultrastructural (1200 ×) magnification through a SEM Stereoscan 360, Leica, Cambridge. SEM analysis enabled the identification of distinctive features of FA on hard surfaces, namely, amorphous crystals, micro-crystals with a morphology similar to those of uric or micro-crystals with a comparable morphology to cholesterol, absent in controls. Moreover, red blood cells (RBC) were absent in FA but were always present in controls. On cotton, for both FA and controls, the drop was almost completely absorbed and thus indistinguishable from the underlying fabric texture. On polyester, FA showed amorphous/crystal-like deposits and no RBC, as observed on hard surfaces, except for those showing a completely flat surface. SEM analysis appeared to be suitable for differential diagnosis between FA and genuine bloodstains on hard surfaces, although the results may be inconclusive on tested fabrics.

Among biological macromolecules, collagen enjoys quite a peculiar status. Making up as much as a third of the protein fraction of the body it is the main responsible for the functional properties of the extracellular matrix, which can be efficiently tuned and tailored by modifying the length, volume fraction, and spatial layout of its collagen content. The supramolecular aggregates of collagen are therefore subject to be investigation by several viewpoints and at different scales, from the finest interactions of individual collagen molecules to the spatial layout of fibril bundles. As a consequence, no treatise can pretend to be exhaustive about the several techniques that can be useful in different moments and/or for different purposes. So, in this chapter, we focus only on some applications of the transmission electron microscope (TEM), of the scanning electron microscope (SEM), and of the atomic force microscope (AFM).

Bloodstain pattern analysis has a key role in crime scene reconstruction; however, it can be hampered by diverse confounding factors, such as insect activity which may lead to the production of small artifactual bloodstains, commonly referred to as fly artifacts (FA). Although several techniques aimed at distinguishing human bloodstains and FA have been developed, actually, no standardized and reproducible methodology is available. The aim of our study was to test the use of scanning electron microscopy (SEM) to distinguish human bloodstains from FA produced by Sarcophaga carnaria. FA and bloodstains have been produced on five different deposition surfaces under experimental conditions. After visual analysis, bloodstains and FA were analyzed at standard low (× 40–× 300) and high (× 600–× 1200) magnification through a Philips SEM 515. Although differential diagnosis between bloodstains and FA resulted often inconclusive at visual analysis, SEM analysis allowed the identification of additional key distinctive morphological features. In particular, on the surface of FA, small crystal-like and/or amorphous material deposits were observed. Such deposits were absent on bloodstains which, on the other hand, displayed red blood cells stacked in “rouleaux.” Basing on these results and under our experimental conditions, SEM analysis resulted suitable to perform a differential diagnosis between bloodstains and FA produced from the insect activity of Sarcophaga carnaria.

During several gynecological retroperitoneal pelvic surgeries, portions of the pelvic auto- nomic nervous system can be accidentally damaged, in particular hypogastric nerves, leading to significant visceral dysfunctions, dramatically affecting woman’s quality of life. The aims of this study were to clarify the relationship of hypogastric nerve with definite anatomical land- marks and to assess any anatomical differences between the two sides of the pelvis. Detailed pelvic retroperitoneal dissection was performed in 5 nulliparous embalmed female cadavers and in 10 nulliparous women during in vivo laparoscopic surgery for rectosig- moid endometriosis without parametrial infiltration or radical hysterectomy (B1 according to Querleu-Morrow) for cervical cancer. On both hemipelvis, the closest distance between HNs and ureters, midsagittal plane, midcervical plane or uterosacral ligaments were documented. Comparison of anatomical data of the two hemipelvis were conducted. On cadavers and in vivo dissection, a right and left hypogastric nerves, covered by pre- hypogastric fascia, were identified in all specimens. Irrespective of the side, a wide anatomical variability was reported. Regarding differences between the two hemipelvis, we found that the right hypogastric nerve was further to the ureter and closer to the midsagittal plane than the left one. Mid-cervical plane was found 2.7 mm to the left of the midsagittal one. Right hypogas- tric nerve was found closer to mid-cervical plane and utero-sacral ligament than the left one. An accurate knowledge of the pelvic retroperitoneal anatomy and differences between the two sides of the pelvis are essential to preserve hypogastric nerve during surgical dissection. Because of the wide anatomical variability, the use of an interfascial approach between fascia propria recti and pre-hypogastric fascia could help to perform an efficient nerve-sparing sur- gery.

The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity.In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca++) and phosphorus (P5+) in culture medium were measured.We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca++.Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium.Statement of SignificanceThe novelty of the paper is the differentiation of human blood-derived osteoclast precursors, instead of mouse-derived macrophages as used in most studies, directly on biomimetic micro-nano structured HA-based surfaces, as triggered by osteoblast-produced factors (RANKL/OPG), and influenced by chemistry and topography of the substrate(s). Biomimetic HA-surfaces, like those obtained in calcium phosphate cements, are very different from the conventional calcium phosphate ceramics, both in terms of topography and ion exchange. The role of these factors in modulating precursors' differentiation and activity is analysed. The system is closely reproducing the physiological process of attachment of host cells and further maturation to osteoclasts toward resorption of the substrate, which occurs in vivo after filling bone defects with the calcium phosphate grafts. (C) 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The “Luigi Cattaneo” Wax Museum houses the skull and wax bust of Luigi Marchetti, a man with acromegaly who died in 1808 at the age of 47 years. His case aroused the interest of many famous anatomists and nineteenth century clinicians including Rudolf Virchow. Cesare Taruffi’s study of this case and the exemplary and symbolic comparison between the exact wax replica and the human skull show how traumatic and complex this anatomical-pathological paradigm must have been for nineteenth century medical science. It also marks the historical continuity between endocrinological research in the old Bologna Anatomy School from Mondino Dei Liuzzi to Marcello Malpighi and Giovan Battista Morgagni and present-day expertise in pituitary neurosurgery at Bologna University and the city’s “Carlo Alberto Pizzardi” Bellaria Hospital.

Inositide-dependent signalling pathways regulated by phosphoinositide-specific phospholi- pase C (PI-PLC) beta1 have been demonstrated to play important roles in MDS pathogenesis and in cell differentiation (1). Moreover, the MDS therapy aims at inducing myeloid and/or erythroid differentiation of MDS stem cells. Indeed, azacitidine is a demethylating agent that can induce myeloid differentiation. On the other hand, lenalidomide may restore a normal erythropoiesis. The exact molecular mechanisms underlying the effect of azacitidine and lenalidomide in MDS cells are still unclear, although it is clear that these therapies regulate stem cell proliferation, differentiation and apoptosis (2). The combination of azacitidine and lenalidomide in MDS therapy is now under considera- tion, given the capability of both drugs to balance proliferation and differentiation processes (3). In this study we analyzed the molecular effect of this combination therapy on PI-PLC isoenzymes, not only studying PI-PLCbeta1, but also PI-PLCgamma1, that can be associated with erythropoiesis. We analyzed 44 patients diagnosed with high-risk MDS who were given azacitidine and lenalidomide. Given the limited number of cells, we quantified the expression of these molecules by Real-Time PCR analyses and immunocytochemical experiments. Moreover, we carried out cell cycle analyses and studied both PI-PLCbeta1 methylation status and the expression of Globin genes. In our case series, 28/44 patients were evaluable, with an overall response rate of 78.6% (22/28 cases). At a molecular level, a significant increase of PI-PLCbeta1 and/or PI-PLCgamma1 expression was associated with a favourable clinical response to the combination therapy. Responder cases also showed an increase of Beta-globin expression, hinting at a specific contri- bution of lenalidomide on erythroid activation, whilst the frequent demethylation of PI-PLCbeta1 promoter could be specifically linked to azacitidine. Taken together, our results show that the combination of azacitidine and lenalidomide can be important for activating PI-PLC isoenzymes, therefore regulating myeloid and erythroid dif- ferentiation in MDS cells.

After a thirty-year gap, the Bologna University Anatomy School has resumed cadaver dissection as part of its anatomy teaching and research programme. The School is equipped with a modern dissecting room named after expert anatomist professor Giovanni Mazzotti, and boasts a rigorous body donor project. Anatomic dissection on human cadavers was the hallmark of Bologna’s Medical School in the early XIV century and paved the way to modern anatomy as it is known today. Human cadaver dissection was also the key feature in the second half of the XVIII century when the Bologna School made a major contribution to what was to become the conceptual framework of medical practice, the paradigm of anatomic pathology. This concept was to transform anatomy from a preparatory and subsidiary discipline into the cornerstone and foundation of the medical profession. Bologna’s “Luigi Cattaneo” Anatomical Wax Museum (part of the University Museum System), home to the “Cesare Taruffi” Pathological Anatomy Collection since 2002, plans to narrate the historical developments marking the 700 years linking the University’s worldwide renown to human cadaver anatomical dissection.

Akt is implicated in erythropoiesis, through PI-PLCgamma1, but is also connected with cell cycle and protein synthesis, via activation of the mTOR pathway (1). Lenalidomide is currently used in the treatment of low-risk MDS patients showing a del(5q) karyotype, as it induces erythropoiesis in non-del(5q) cells and selectively inhibits cell proliferation in del(5q) cells (2). However, the exact molecular mechanisms underlying the effect of Lenalidomide in del(5q) and non-del(5q) MDS cells are still unclear, although Lenalidomide demonstrated an effect on targeting signalling molecules involved in apoptosis, proliferation and differentiation, i.e. Akt (3). Here we analyzed the effect of Lenalidomide on two cell lines: Namalwa CSN.70, bearing a del(5q) karyotype, and U937, with a normal 5q chromosome. In particular, we analyzed several molecules implicated in inositide signalling, such as Akt, mTOR and PI-PLCgamma1, as well as Cyclins and Globin genes, in order to assess the effect of Lenalidomide on cell cycle and erythropoiesis. Moreover, we quantified the gene expression profile of 6 patients diagnosed with del(5q) Low-Risk MDS (IPSS: Low or Int-1) who were given Lenalidomide. In our case series, as well as in cell lines, erythropoiesis activation was associated with a response to Lenalidomide, with an induction of Akt/PLCgamma1 and an increase of Beta-Globin. Moreover, only in non-del(5q) cells, a normal proliferation was allowed, given that del(5q) cells showed a cell cycle arrest and a slight inhibition of the Akt/mTOR pathway, this latter being confirmed also by co-localization experiments performed on primary cells from MDS patients. Therefore, our data support the hypothesis of a specific activation of both inositide-dependent proliferation and erythroid differentiation pathways in response to Lenalidomide treatment in non-del(5q) cells, whereas in the del(5q) cell clone there is a cell cycle arrest and a slower erythroid differentiation. Taken together, these results hint to a specific activation of inositide-dependent signalling pathways during Lenalidomide administration and possibly pave the way to a larger investigation aiming to assess the role of these pathways during the therapy.

Thyroid carcinomas account for a minority of all malignant tumours but, after those of the gonads, they represent the most common forms of endocrine cancers. They include several types, among which the papillary thyroid cancer (PTC) and the anaplastic thyroid cancer (ATC) are the best known. The two hystotypes display significant biological and clinical differences: PTC is a well differentiated form of tumour with a high incidence and a good prognosis, while the ATC is less frequent but represents one of the most aggressive endocrine tumours with morphological features of an undifferentiated type. To date, as far as we know, no conclusive studies, useful to design arrays of molecular markers, have been published illustrating the phenotypic and proteomic differences between these two tumours. The aim of this work was to perform a comparative analysis of two thyroid cancer cell lines, derived respectively from papillary (BCPAP) and anaplastic (8505C) thyroid carcinomas. The comparative analysis included cell behaviour assays and proteomic analysis by 2D-PAGE and mass spectrometry. The results have highlighted a new proteomic signature for the anaplastic carcinoma-derived cells, consistent with their high proliferation rate, motility propensity and metabolic shift, in relation to the well-differentiated PTC cells.

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are extracellular matrix proteins produced by odontoblasts involved in the dentin mineralization. The aim this study was to compare the distribution of DMP1 and DSPP in human sound dentin vs human sclerotic dentin. Sixteen sound and sixteen carious human molars were selected, fixed in paraformaldehyde and processed for immunohistochemical detection of DMP1 and DSPP by means of light microscopy, transmission electron microscopy (TEM) and high-resolution field emission in-lens scanning electron microscopy (FEI-SEM). Specimens were submitted to a pre-embedding or a post-embedding immunolabeling technique using primary antibodies anti DMP1 and anti-DSPP and gold-conjugated secondary antibodies. Other samples were processed for the detection of DMP1 and DSPP levels. Dentin from these samples was mechanically fractured to powder, then a protein extraction and a protein level detection assay were performed. DMP1 and DSPP were more abundant in carious than in sound samples. Immunohistochemical analyses in sclerotic dentin disclosed a high expression of DMP1 and DSPP inside the tubules, suggesting an active biomineralization of dentin by odontoblasts. Furthermore, the detection of small amounts of these proteins inside the tubules far from the carious lesion, as shown in the present study, is consistent with the hypothesis of a preventive defense of all dentin after a noxious stimulus has undermined the tooth.

In the present pilot study, the authors morphologically investigated sandblasted, acid-etched surfaces (SLA) at very early experimental times. The tested devices were titanium plate-like implants with flattened wide lateral sides and jagged narrow sides. Because of these implant shape and placement site, the device gained a firm mechanical stability but the largest portion of the implant surface lacked direct contact with host bone and faced a wide peri-implant space rich in marrow tissue, intentionally created in order to study the interfacial interaction between metal surface and biological microenvironment. The insertion of titanium devices into the proximal tibia elicited a sequence of healing events. Newly formed bone proceeded through an early distance osteogenesis, common to both surfaces, and a delayed contact osteogenesis which seemed to follow different patterns at the two surfaces. In fact, SLA devices showed a more osteoconductive behavior retaining a less dense blood clot, which might be earlier and more easily replaced, and leading to a surface-conditioning layer which promotes osteogenic cell differentiation and appositional new bone deposition at the titanium surface. This model system is expected to provide a starting point for further investigations which clarify the early cellular and biomolecular events occurring at the metal surface.

In bone engineering, the adhesion, proliferation and differentiation of mesenchymal stromal cells rely on signaling from chemico-physical structure of the substrate, therefore prompting the design of mimetic “extracellular matrix”-like scaffolds. In this study, three-dimensional porous poly-L-lactic acid (PLLA)-based scaffolds have been mixed with different components, including single walled carbon nanotubes (CNT), micro-hydroxyapatite particles (HA), and BMP2, and treated with plasma (PT), to obtain four different nanocomposites: PLLA + CNT, PLLA + CNTHA, PLLA + CNT + HA + BMP2 and PLLA + CNT + HA + PT. Adult bone marrow mesenchymal stromal cells (MSCs) were derived from the femur of orthopaedic patients, seeded on the scaffolds and cultured under osteogenic induction up to differentiation and mineralization. The release of specific metabolites and temporal gene expression profiles of marrow-derived osteoprogenitors were analyzed at definite time points, relevant to in vitro culture as well as in vivo differentiation. As a result, the role of the different biomimetic components added to the PLLA matrix was deciphered, with BMP2-added scaffolds showing the highest biomimetic activity on cells differentiating to mature osteoblasts. The modification of a polymeric scaffold with reinforcing components which also work as biomimetic cues for cells can effectively direct osteoprogenitor cells differentiation, so as to shorten the time required for mineralization.

The collagen fibrils of cornea, blood vessel walls, skin, gut, interstitial tissues, the sheath of tendons and nerves, and other connective tissues are known to be made of helically wound subfibrils winding at a constant angle to the fibril axis. A critical aspect of this model is that it requires the axial microfibrils to warp, in an implausible way. This architecture lends itself quite naturally to an epitaxial layout where collagen microfibrils envelop a central core of a different nature. Here we demonstrate an axial domain in collagen fibrils from rabbit nerve sheath and tendon sheath by means of transmission electron microscopy after a histochemical reaction designed to evidence all polysaccharides and by tapping-mode atomic force microscopy. This axial domain was consistently found in fibrils with helical microfibrils but was not observed in tendon, whose microfibrils run longitudinal and parallel.

Force transmission and elastic recoil in tendon are related to fibre and fibrillar crimps. Tendon fibrils arrange in a network system in which forces are also laterally transferred between neighbouring fibrils through interfibrillar proteoglycans. The interfibrillar proteoglycan decorin with a dermatansulphate chain (DS) represents the 90% of tendon proteoglycans and seems to transfer forces between fibrils during tendon stretching. DS in decorin and biglycan may also play a role in packing fibrils forming crimps. Aim of this study was to investigate whether decorin and biglycan DS affects the microstructure/function of fibrillar crimps in tendon elastic recoil. Four relaxed Achilles tendons of 8 rats (group I) were immediately immersed for 6 hours in Chondroitinase-B solution, fixed, dehydrated and prepared for SEM. Other 4 tendons (group II) were fixed in a clamp, stretched 5 - 6 % in Chondroitinase-B solution, fixed in Karnovsky solution under stretching and processed as group I. Other 4 tendons (group III) were clamped, stretched in Chondroitinase-B solution for 6 hours, removed from the clamps to allow relaxation, fixed and processed as above. Other 4 tendons (group IV) were immersed in saline solution and mechanically disrupted to obtain isolated fibrils for TEM. Both enzymatic and mechanical removal of DS in relaxed tendons of group I and IV didn’t affect the morphology of fibre and fibrillar crimps. All collagen fibrils of group I showed crimped fibres showing particular knots or fibrillar crimps at the top of each fibre crimp: fibrils twisted leftwards first, changing their plane of running, and then sharply bent, changing their course on the new plane. Stretched tendons in Chondroitinase-B solution (group II) showed flattened crimps but regular fibrillar crimps were still present. Stretched tendons immersed in Chondroitinase-B solution, relaxed and fixed (group III) showed both regular fibre and fibrillar crimps: a fibril local leftward twisting and bending in the fibrillar crimp regions was observable like in group I. These data demonstrate that structure/function of fibrillar crimps in recoiling fibrils/fibres in tendon does not depend on DS, but seems to be related to the hierarchical alternating handedness of collagen structures.

Cross-Reacting Material 197 (CRM197) is a diphtheria toxin non-toxic mutant that has shown antitumor activity in mice and humans. It is still unclear whether this anti-tumorigenic effect depends on its strong inflammatory-immunological property, its ability to inhibit heparin-binding epidermal growth factor (HB-EGF), or even its possible weak toxicity. CRM197 is utilized as a specific inhibitor of HB-EGF that competes for the epidermal growth factor receptor (EGFR), overexpressed in colorectal cancer and implicated in its progression. In this study we evaluate the effects of CRM197 on HT-29 human colon cancer cell line behaviour and, for CRM197 recognized ability to inhibit HB-EGF, its possible influence on EGFR activation. In particular, while HT-29 does not show any reduction of viability after CRM197 treatment (MTT modified assay), or changes in cell cycle distribution (flow cytometry), in EGFR localization, phospho-EGFR detected signals (immunohistochemistry) or in morphology (scanning electron microscopy, SEM) they show a change in the gene expression profile by microarray analysis (cDNA microarray SS-H19k8). The overexpression of genes like protein phosphatase 2, catalytic subunit, alpha isozyme (PPP2CA), guanine nucleotide-binding protein G subunit alpha-1(GNAIl) and butyrophilin, subfamily 2, member A1 (BTN2A1) has been confirmed with real-time-qPCR. This is the first study where the CRM197 treatment on HT-29 shows a possible scarce implication of endogenous HB-EGF on EGFR expression and cancer cell development. At the same time, our results show the alteration of a specific and selected number of genes.