Search

An academic, anatomist, and Lombrosian psychiatrist active at the University of Parma in Italy at the end of the 19th century, Lorenzo Tenchini produced ceroplastic‐like masks that are unique in the anatomical Western context. These were prepared from 1885 to 1893 with the aim of ‘cataloguing’ the behaviour of prison inmates and psychiatric patients based on their facial surface anatomy. Due to the lack of any reference to the procedure used to prepare the masks, studies were undertaken by our group using X‐ray scans, infrared spectroscopy, bioptic sampling, and microscopy analysis of the mask constituents. Results showed that the masks were stratified structures including plaster, cotton gauze/human epidermis, and wax, leading to a fabrication procedure reminiscent of ‘additive layer manufacturing’. Differences in the depths of these layers were observed in relation to the facial contours, suggesting an attempt to reproduce, at least partially, the three‐dimensional features of the facial soft tissues. We conclude the Tenchini masks are the first historical antecedent of the experimental method for face reconstruction used in the early 2000s to test the feasibility of transferring a complete strip of face and scalp from a deceased donor to a living recipient, in preparation for a complete face transplant. In addition, the layering procedure adopted conceptually mimics that developed only in the late 20th century for computer‐aided rapid prototyping, and recently applied to bioengineering with biomaterials for a number of human structures including parts of the skull and face. Finally, the masks are a relevant example of mixed ceroplastic‐cutaneous preparations in the history of anatomical research for clinical purposes.

We investigated the scavenging behavior of some vertebrate species inhabiting a mountainous area of Eastern Tuscany (Italy). Fieldwork was conducted by using camera traps from July 2010 to June 2013. The red fox (Vulpes vulpes) resulted to be the main scavenger species, followed by the wolf (Canis lupus), while the wild boar (Sus scrofa) resulted to be one of the less efficient scavengers, exploiting, in groups, only two carcasses. The wolf seemed to use carcasses mostly during the summer season, while no differences in red fox seasonal use of and visit to carcasses were detected. All the wild scavenger species showed a peak of activity between dusk and dawn, during both summer and winter, likely to minimize human disturbance. The red fox used carcasses whenever available, and in most cases it also was the first species arriving on a new carcass, with a very limited searching time. This paper represents the first Italian study on carrion use by Mediterranean vertebrate communities.

Hybridization between wolves ( Canis lupus ) and domestic dogs ( Canis familiaris ) can represent a threat to wild populations via genetic introgression and ecological competition. Therefore understanding the ecological role of hybrids may be crucial for developing appropriate conservation strategies. The Italian wolf population has a peculiar genetic composition due to a long-lasting geographic isolation. Nowadays, however, its genetic integrity is threatened by the spread of canine genes as a result of the hybridization with stray dogs in the wild. The aim of the present study was to gain insights into the ecological role of free-ranging wolf–dog hybrids by investigating their winter food habits in comparison with wolves in a mountain area of Central Italy. Levels of genetic introgression from the dogs were assessed in two adjacent areas occupied by up to five different packs by analyzing non-invasive samples and carcasses collected therein with a set of uniparental and bi-parental molecular markers. The obtained results enabled us to classify the two areas as ‘hybrid’ and ‘wolf’ areas based on their level of genetic introgression. Trophic niche and similarity/dissimilarity analyses did not detect significant difference in the diet between the two areas: in both of them, wild boar was the main prey, followed by roe deer. Furthermore, the same age/body mass classes of the two ungulates were selected by wolves and hybrids. Our findings confirmed wolf–dog hybrids as potential competitors for wolves. Further studies on other aspects of their biology and ecology are recommended in order to better estimate the impact of hybridization on natural wolf populations.

The arterial, venous, and lymphatic vessels as well as sensory and motor nerves of the human scalp depict a very consistent and stable topographical anatomy. They are organized as “stripes” of neurovascular structures with well-defined medial-lateral and cranial-caudal orientation, whose distribution on the surface of the head and contiguous facial and neck areas can be predicted in relation to the major anatomical bone landmarks of the face and cranial vault in anterior, lateral, posterior, and superior projections. As such, anatomo-surgical dissection of each neurovascular pedicle can be promptly achieved. In addition, two main neurovascular fields can be identified by a frontal plane passing just ahead of the auricles, whose anterior one is provided exclusively by branches of the internal carotid artery (with related veins and lymphatics) and trigeminal nerve whereas the posterior one by branches of the external carotid artery (with related veins and lymphatics) but both the trigeminal nerve and cervical plexus. Finally, the same motor innervation involves both anterior and posterior neurovascular fields, is provided by branches of the facial nerve, and concerns the epicranius muscle.

Anthropogenic hybridization is widely perceived as a threat to the conservation of biodiversity. Nevertheless, to date, relevant policy and management interventions are unresolved and highly convoluted. While this is due to the inherent complexity of the issue, we hereby hypothesize that a lack of agreement concerning management goals and approaches, within the scientific community, may explain the lack of social awareness on this phenomenon, and the absence of effective pressure on decision-makers. By focusing on wolf x dog hybridization in Europe, we hereby (a) assess the state of the art of issues on wolf x dog hybridization within the scientific community, (b) assess the conceptual bases for different viewpoints, and (c) provide a conceptual framework aiming at reducing the disagreements. We adopted the Delphi technique, involving a three-round iterative survey addressed to a selected sample of experts who published at Web of Science listed journals, in the last 10 years on wolf x dog hybridization and related topics. Consensus was reached that admixed individuals should always be defined according to their genetic profile, and that a reference threshold for admixture (i.e., q-value in assignment tests) should be formally adopted for their identification. To mitigate hybridization, experts agreed on adopting preventive, proactive and, when concerning small and recovering wolf populations, reactive interventions. Overall, experts' consensus waned as the issues addressed became increasingly practical, including the adoption of lethal removal. We suggest three non-mutually exclusive explanations for this trend: (i) value-laden viewpoints increasingly emerge when addressing practical issues, and are particularly diverging between experts with different disciplinary backgrounds (e.g., ecologists, geneticists); (ii) some experts prefer avoiding the risk of potentially giving carte blanche to wolf opponents to (illegally) remove wolves, based on the wolf x dog hybridization issue; (iii) room for subjective interpretation and opinions result from the paucity of data on the effectiveness of different management interventions. These results have management implications and reveal gaps in the knowledge on a wide spectrum of issues related not only to the management of anthropogenic hybridization, but also to the role of ethical values and real-world management concerns in the scientific debate.

Few data are available on the effect of biomaterials on surface antigens of mammalian bone marrow-derived, adult mesenchymal stromal cells (MSCs). Since poly(L-lactic acid) or PLLA is largely used in tissue engineering of human bones, and we are developing a reverse engineering program to prototype with biomaterials the vascular architecture of bones for their bioartificial reconstruction, both in humans and animal models, we have studied the effect of porous, flat and smooth PLLA scaffolds on the immunophenotype of in vitro grown, rat MSCs in the absence of any coating, co-polymeric enrichment, and differentiation stimuli. Similar to controls on plastic, we show that our PLLA scaffold does not modify the distribution of some surface markers in rat MSCs. In particular, the maintained expression of CD73 and CD90 on two different sub-populations (small and large cells) is consistent with their adhesion to the PLLA scaffold through specialized appendages, and to their prominent content in actin. In addition, our PLLA scaffold favours retention of the intermediate filament desmin, believed a putative marker of undifferentiated state. Finally, it preserves all rat MSCs morphotypes, and allows for their survival, adhesion to the substrate, and replication. Remarkably, a subpopulation of rat MSCs grown on our PLLA scaffold exhibited formation of membrane protrusions of uncertain significance, although in a size range and morphology compatible with either motility blebs or shedding vesicles. In summary, our PLLA scaffold has no detrimental effect on a number of features of rat MSCs, primarily the expression of CD73 and CD90.

Bioartificial endocrine organs are a recent promise of regenerative medicine, offering a translational perspective for the cure by transplantation of a number of endocrine and metabolic disorders. In 2007 we have designed with biocompatible material a 3D scaffold - bioreactor unit mimiking the 3D macro-microscopic geometry (here coined as “organomorphism”) of the human thyroid gland, choosen as a suitable model of endocrine soft tissue organ, with the intent of bioengineering directly “on the laboratory bench” (i.e. ex situ) a complete and functional bioartificial thyroid. This unit reproduces the architecture of the inner, human thyroid stromal / vascular scaffold (also called matrix), and replicates the original thyrocyte / vascular interface. It is now under intense investigation as an experimental tool to test cellular 3D auto-assembly of both heterologous and autologous thyroid tissue, and related vascular system, up to the ex situ generation of a 3D macroscopic thyroid gland. We believe that these studies will lay the groundwork for an innovative concept in regenerative medicine of endocrine organs, i.e. that the organomorphism of a biocompatible scaffold - bioreactor complex is essential to guide the 3D organization of seeded stem / precursor cells. Specifically, organomorphism may favour phenotypic differentiation of seeded cells towards glandular and vascular elements, eventually leading to a physiologically-competent and immunotolerant bioconstruct, macroscopically suitable for transplantation and clinical applications. We also envisage potential applications of this principle to the ex situ reconstruction of other endocrine glands, like the anterior pituitary and adrenals, and to complex soft tissue viscera including the thymus, and parts of the central nervous system.

Wolf (Canis lupus) diet is determined by several ecological factors which can differ with latitude and human impact on the environment. Here we aim to compare Northern and Southern Europe with respect to wolf feeding habits. Scats were collected and analysed for nine years in South-central Scandinavia and four years in Tuscany, Italy, where prey density, predator-prey size relation and habitat heterogeneity, were compared in different ecological perspectives. Consumption followed prey density in Scandinavia but not in Tuscany and the main prey species, moose and wild boar respectively, were more seasonally age diversified in Scandinavia than in Tuscany. Most likely, the risk of injury was an important factor in prey age selection, especially in Tuscany. Diet composition in Scandinavia showed a negligible variance while in Tuscany, temporal and spatial variation were clearly recognised. The underlying mechanism is most likely related to the limited ecological diversity of landscape in Scandinavia contrasted with the higher variability of South European landscapes resulting in higher variation in prey abundance and consequently prey choice.

Novel biomaterials are of paramount importance for bone regrowth. In this study, we investigated human adipose stem cells (hASCs) for osteogenic, osteoconductivity, and osteoinductivity effects of an innovative collagen/hydroxylapatite hybrid scaffold. In hASCs that were grown on this scaffold, osteogenic genes were analyzed for their expression profiles, together with adhesion and extracellular matrix genes. In hASC integrins, basement membrane constituents and collagens were up-regulated, together with cell proliferation. In addition, expression of osteopontin and activated focal adhesion kinase was studied at the protein level. Our in vitro data indicate that hASCs, together with hybrid biomaterial, is an important model of study to investigate in vitro bone induction.-Mazzoni, E., D'Agostino, A., Manfrini, M., Maniero, S., Puozzo, A., Bassi, E., Marsico, S., Fortini, C., Trevisiol, L., Patergnani, S., Tognon, M. Human adipose stem cells induced to osteogenic differentiation by an innovative collagen/hydroxylapatite hybrid scaffold.

Red fox ( Vulpes vulpes ) and wolf ( Canis lupus ) are two widespread opportunistic predators living in simpatry in many areas. Nonetheless, scarce information are available on their trophic interactions. We investigated food habits of these two carnivores in a mountain area in Italy and assessed the extent of their trophic niche overlap, focusing on the consumption of wild ungulates. Thereby we analyzed the content of 669 red fox scats and 253 wolf scats collected between May 2008 and April 2009. Red foxes resulted to have a more than three times higher niche breadth than wolves. Vegetables, small mammals, wild ungulates, and invertebrates were major items (altogether 92% of volume) of the red fox annual diet. On the contrary wolf annual diet relied on wild ungulates (94% of volume) with wild boar ( Sus scrofa ) being the main food item. The degree of trophic niche overlap between the two species was found to be low (Pianka's O = 0.356). Diet variation between the warm and the cold seasons was limited in both species, and higher in red fox than in wolf. The two canids appeared to use wild ungulates unevenly being the former more selective for younger preys, smaller in size (newborn piglets and roe deer Capreolus capreolus fawns), whereas the latter exhibited a preference for medium-sized and large ungulates (10–35 kg wild boar and adult roe deer). Even if wild ungulates represent the main shared food category, the different use of age/weight classes by the two predators, together with their possible consumption as carrions by red fox, suggests a very limited trophic competition between wolf and red fox.This study represents a contribution to the knowledge of trophic interaction in predator–prey systems where sympatric carnivores are present.

Antisense-mediated exon skipping has proven to be efficacious for subsets of Duchenne muscular dystrophy mutations. This approach is based on targeting specific splicing motifs that interfere with the spliceosome assembly by steric hindrance. Proper exon recognition by the splicing machinery is thought to depend on exonic splicing enhancer sequences, often characterized by purine-rich stretches, representing potential targets for antisense-mediated exon skipping. We identified and functionally characterized two purine-rich regions located within dystrophin intron 11 and involved in splicing regulation of a pseudo-exon. A functional role for these sequences was suggested by a pure intronic DMD deletion causing X-linked dilated cardiomyopathy through the prevalent cardiac incorporation of the aberrant pseudo-exon, marked as Alu-exon, into the dystrophin transcript. The first splicing sequence is contained within the pseudo-exon, whereas the second is localized within its 3' intron. We demonstrated that the two sequences actually behave as splicing enhancers in cell-free splicing assays because their deletion strongly interferes with the pseudo-exon inclusion. Cell-free results were then confirmed in myogenic cells derived from the patient with X-linked dilated cardiomyopathy, by targeting the identified motifs with antisense molecules and obtaining a reduction in dystrophin pseudo-exon recognition. The splicing motifs identified could represent target sequences for a personalized molecular therapy in this particular DMD mutation. Our results demonstrated for the first time the role of intronic splicing sequences in antisense modulation with implications in exon skipping-mediated therapeutic approaches.

Exon skipping using antisense oligonucleotides (AONs) has successfully been used to reframe the mRNA in various Duchenne muscular dystrophy patients carrying deletions in the DMD gene. In this study we tested the feasibility of the exon skipping approach for patients with small mutations in in-frame exons. We first identified 54 disease-causing point mutations. We selected five patients with nonsense or frameshifting mutations in exons 10, 16, 26, 33, and 34. Wild-type and mutation specific 2′OMePS AONs were tested in cell-free splicing assays and in cultured cells derived from the selected patients. The obtained results confirm cell-free splicing assay as an alternative system to test exon skipping propensity when patients' cells are unavailable. In myogenic cells, similar levels of exon skipping were observed for wild-type and mutation specific AONs for exons 16, 26, and 33, whereas for exon 10 and exon 34 the efficacy of the AONs was significantly different. Interestingly, in some cases skipping efficiencies for mutated exons were quite dissimilar when compared with previous reports on the respective wild-type exons. This behavior may be related to the effect of the mutations on exon skipping propensity, and highlights the complexity of identifying optimal AONs for skipping exons with small mutations. Hum Mutat 30:1–8, 2009. © 2009 Wiley-Liss, Inc.

It is currently known that a number of human vascular systems have a fractal geometry. Since we have recently developed a technique to prototype single arterial branches of human soft tissue organs by additive layer manufacturing (AM), we have explored the possibility that auto-similarity in vessel branching represents a key variable for accurate computational modeling of the organ three-dimensional (3D), macro / microscopic anatomy, and its reproduction by inverse engineering. To this purpose, ramification features of the intra-lobar arteries of the human thyroid were studied using injection-corrosion casts of a cadaveric gland. Vessel diameters, ramification angles and branch lengths were measured by light microscopic, computer-aided optical metrology. Distribution of morphological variables was considered on a cumulative basis, and special focus was given to the branching laws. To reduce the bias of vascular distortion due to the pressure of intra-vascular resin injection, measures were made dimensionless through the use of a scaling parameter set on the vascular caliber of major afferent arteries. In addition, using high resolution micro-tomography (mCT Skyscan 1172, Bruker micro-CT) equipped with CTAn software and the Otsu algorithm for segmentation, spaces occupied by vascular branches (referred to as Volume of Interests, VOI) were selected, and their planar fractal dimension calculated (Mandelbrot 1982). Finally, a computational simulation of the vascular tree was achieved using a mixed, stochastic / deterministic algorithm, based on diffusion limited aggregation (DLA; Witten and Sander, 1981), constrained by mean values of vascular variables. Ratios among decreasing cast calibers, ramification angles and branch lengths, respectively, were found strictly interrelated, mCT-VOI depicted fractal dimensions, and DLA simulation led to a fractal-like organization consistent with real data morphometrics. In summary, thyroid arterial geometry reliably exhibited a degree of auto-similarity, suggesting that fractality is a key feature for computational modeling and eventual AM of 3D vascular networks of the human thyroid.

Introduction Monitoring large carnivores is a central issue in conservation biology. The wolf (Canis lupus) is the most studied large carnivore in the world. After a massive decline and several local extinctions, mostly due to direct persecutions, wolves are now recolonizing many areas of their historical natural range. One of the main monitoring techniques is the howling survey, which is based on the wolves’ tendency to use vocalisations to mark territory ownership in response to howls of unknown individuals. In most cases wolf howling sessions are useful for the localisation of the pack, but they provide only an aural estimation of the chorus size. We tested and present a new bioacoustic approach to estimate chorus size by recording wolves’ replies and visualising choruses through spectrograms and spectral envelopes. To test the methodology, we compared: a) the values detected by visual inspections with the true chorus size to test for accuracy; b) the bioacoustic estimations of a sample of free-ranging wolves’ replies developed by different operators to test for precision of the method; c) the aural field estimation of chorus size of a sample of free-ranging wolves’ replies with the sonogram analysis of the same recordings to test for difference between methods. Results Visual inspection of the chorus by spectrogram and spectrum proved to be useful in determining the number of concurrent voices in a wolf chorus. Estimations of chorus size were highly correlated with the number of wolves counted in a pack, and 92 % of 29 known chorus sizes were recognized by means of bioacoustic analysis. On the basis of spectrographic evidence, it was also possible to identify up to seven concurrent vocalisations in a chorus of nine wolves. Spectral analysis of 37 free ranging wolves’ replies showed a high correlation between the chorus size estimations of the different operators (92.8 %), but a low correlation with the aural estimation (59.2 %). Conclusions Wolf howling monitoring technique could be improved by recording wolves’ replies and by using bioacoustic tools such as spectrograms and spectral envelopes to determine the size of the wolf chorus. Compared with other monitoring techniques (i.e., genetic analysis), bioacoustic analysis requires widely available informatic tools (i.e., sound recording set of devices and sound analysis software) and a low budget. Information obtained by means of chorus analysis can also be combined with that provided by other techniques. Moreover, howls can be recorded and stored in audio file format with a good resolution (i.e. in “Wave” format), thus representing a useful tool for future listening and investigations, which can be countlessly employed without risks of time deterioration. Electronic supplementary material The online version of this article (doi:10.1186/s12983-015-0114-0) contains supplementary material, which is available to authorized users.

Duchenne muscular dystrophy (DMD) is a severe hereditary neuromuscular disorder caused by mutations in the dystrophin gene. Antisense-mediated targeted exon skipping has been shown to restore dystrophin expression both in DMD patients and in the mdx mouse, the murine model of DMD, but the ineffective delivery of these molecules limits their therapeutic use. We demonstrated that PMMA/N-isopropil-acrylamide (ZM2) nanoparticles (NPs), administered both intraperitoneally and orally, were able to deliver 2'OMePS antisense inducing various extents of dystrophin restoration in the mdx mice. Defining NP biodistribution is crucial to improve effects on target and dose regimens; thus, we performed in vivo studies of novel ZM4 NPs. ZM4 are conjugated with NIR fluorophores as optical probes suitable for studies on the Odyssey Imaging System. Our results indicate that NPs are widely distributed in all body muscles, including skeletal muscles and heart, suggesting that these vehicles are appropriate to deliver antisense oligonucleotides for targeting striated muscles in the DMD animal model, thus opening new horizons for Duchenne therapy.

We have previously demonstrated that intraperitoneal injections of 2′-O-methyl-phosphorothioate (2′OMePS) antisense oligoribonucleotides adsorbed onto a cationic core-shell nanoparticles (NPs), termed ZM2, provoke dystrophin restoration in the muscles ofmdxmice. The aim of the present work was to evaluate the oral route as an alternative way of administration for ZM2-antisense oligoribonucleotides complexes. The biodistribution and elimination of nanoparticles were evaluated after single and multiple oral doses of IR-dye conjugated nanoparticles. Labeled nanoparticles were trackedin vivoas well as in tissue cryosections, urines and feces by Odyssey infrared imaging system, and revealed a permanence in the intestine and abdominal lymph nodes for 72 hours to 7 days before being eliminated. We subsequently tested alginate-free and alginate-encapsulated ZM2-antisense oligoribonucleotides (AON) complexes orally administered 2 and 3 times per week, respectively, inmdxmice for a total of 12 weeks. Treatment with alginate ZM2-AON induced a slight dystrophin rescue in diaphragm and intestine smooth muscles, while no dystrophin was detected in alginate-free ZM2-AON treated mice. These data encourage further experiments on oral administration testing of NP and AON complexes, possibly translatable in oligoribonucleotides-mediated molecular therapies.

The impact of predation on prey populations has long been a focus of ecologists, but a firm understanding of the factors influencing prey selection, a key predictor of that impact, remains elusive. High levels of variability observed in prey selection may reflect true differences in the ecology of different communities but might also reflect a failure to deal adequately with uncertainties in the underlying data. Indeed, our review showed that less than 10% of studies of European wolf predation accounted for sampling uncertainty. Here, we relate annual variability in wolf diet to prey availability and examine temporal patterns in prey selection; in particular, we identify how considering uncertainty alters conclusions regarding prey selection.Over nine years, we collected 1,974 wolf scats and conducted drive censuses of ungulates in Alpe di Catenaia, Italy. We bootstrapped scat and census data within years to construct confidence intervals around estimates of prey use, availability and selection. Wolf diet was dominated by boar (61.5 ± 3.90 [SE] % of biomass eaten) and roe deer (33.7 ± 3.61%). Temporal patterns of prey densities revealed that the proportion of roe deer in wolf diet peaked when boar densities were low, not when roe deer densities were highest. Considering only the two dominant prey types, Manly's standardized selection index using all data across years indicated selection for boar (mean = 0.73 ± 0.023). However, sampling error resulted in wide confidence intervals around estimates of prey selection. Thus, despite considerable variation in yearly estimates, confidence intervals for all years overlapped. Failing to consider such uncertainty could lead erroneously to the assumption of differences in prey selection among years. This study highlights the importance of considering temporal variation in relative prey availability and accounting for sampling uncertainty when interpreting the results of dietary studies.

We have tested different types of polymeric cationic core–shell nanoparticles (NPs) for delivering 2- O -methyl-phosphorothioate antisense oligoribonucleotides (AONs), in mdx mice. Both T1 and ZM2 NP bind and convey AONs: intraperitoneal (IP) injections of low doses (52.5 mg/kg) of NP–AON complex restored dystrophin protein synthesis in skeletal and cardiac muscles, allowing protein localization in up to 40% of muscle fibers with skipping level up to 20%. We have tested in vivo (mdx) the tissue biodistribution and elimination timing of NPs by Odyssey, using an infrared dye conjugated ZM2 NP. Administration mode was both IP and oral. Elimination by feces was up to 80% after 22 days of a single injection treatment in mdx treated by intraperitoneal doses, and close to 100% in those treated orally after 72 h of a single dose treatment. Evaluation of NP biodistribution, measured by Odyssey, in organs/tissues cryosections of sacrificed mdx revealed an intense positivity of labeled NP in muscles, heart, intestine, and all organs. We also demonstrated that NP–AON formulations passes the gastric barrier and induce dystrophin rescue in the intestinal smooth muscles as well as in the diaphragm. However the intense positivity at biodistribution of NPs in the heart (and skeletal muscles) conflicts with the relatively low efficacy of the compound in terms of skipping and dystrophin rescue. We are therefore performing ELISA assay to dose the antisense in the treated mice muscles in order to define the release kinetic of the AON from NPs. This is crucial in order to further proceed with other NP studies.

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of2′-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules.

Potentially viable therapeutic approaches for Duchenne muscular dystrophy (DMD) are now within reach. Indeed, clinical trials are currently under way. Two crucial aspects still need to be addressed: maximizing therapeutic efficacy and identifying appropriate and sensible outcome measures. Nevertheless, the end point of these trials remains painful muscle biopsy to show and quantify protein restoration in treated boys. In this study we show that PMMA/N-isopropil-acrylamide+ (NIPAM) nanoparticles (ZM2) bind and convey antisense oligoribonucleotides (AONs) very efficiently. Systemic injection of the ZM2-AON complex restored dystrophin protein synthesis in both skeletal and cardiac muscles of mdx mice, allowing protein localization in up to 40% of muscle fibers. The mdx exon 23 skipping level was up to 20%, as measured by the RealTime assay, and dystrophin restoration was confirmed by both reverse transcription-PCR and western blotting. Furthermore, we verified that dystrophin restoration also occurs in the smooth muscle cells of the dorsal skin arrector pili, an easily accessible histological structure, in ZM2-AON-treated mdx mice, with respect to untreated animals. This finding reveals arrector pili smooth muscle to be an appealing biomarker candidate and a novel low-invasive treatment end point. Furthermore, this marker would also be suitable for subsequent monitoring of the therapeutic effects in DMD patients. In addition, we demonstrate herein the expression of other sarcolemma proteins such as alpha-, beta-, gamma- and delta-sarcoglycans in the human skin arrector pili smooth muscle, thereby showing the potential of this muscle as a biomarker for other muscular dystrophies currently or soon to be the object of clinical trials.

For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2′-O-methyl-phosphorothioate (2′OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine.

Background The commonest pathogenic DMD changes are intragenic deletions/duplications which make up to 78% of all cases and point mutations (roughly 20%) detectable through direct sequencing. The remaining mutations (about 2%) are thought to be pure intronic rearrangements/mutations or 5'-3' UTR changes. In order to screen the huge DMD gene for all types of copy number variation mutations we designed a novel custom high density comparative genomic hybridisation array which contains the full genomic region of the DMD gene and spans from 100 kb upstream to 100 kb downstream of the 2.2 Mb DMD gene. Results We studied 12 DMD/BMD patients who either had no detectable mutations or carried previously identified quantitative pathogenic changes in the DMD gene. We validated the array on patients with previously known mutations as well as unaffected controls, we identified three novel pure intronic rearrangements and we defined all the mutation breakpoints both in the introns and in the 3' UTR region. We also detected a novel polymorphic intron 2 deletion/duplication variation. Despite the high resolution of this approach, RNA studies were required to confirm the functional significance of the intronic mutations identified by CGH. In addition, RNA analysis identified three intronic pathogenic variations affecting splicing which had not been detected by the CGH analysis. Conclusion This novel technology represents an effective high throughput tool to identify both common and rarer DMD rearrangements. RNA studies are required in order to validate the significance of the CGH array findings. The combination of these tools will fully cover the identification of causative DMD rearrangements in both coding and non-coding regions, particularly in patients in whom standard although extensive techniques are unable to detect a mutation.

Fractal properties have been demonstrated in literature for several human vascular systems. In the frame of the investigation of additive manufacturing (AM) as a viable solution to prototype single arterial branches of human soft tissue organs, the paper provides a fractal analysis of the arterial tree of the human thyroid gland. The possibility that the thyroid arterial structure may be described as auto-similar is investigated, by studying injection-corrosion casts of the cadaveric gland. Vessel branching is analyzed by measuring branch diameters, ramification angles, and vessel lengths with the use of an optical microscope. Metrological results are made dimensionless by applying, as a scaling parameter, the caliber of major arteries. Data are then studied on a cumulative basis and processed to infer general rules for vessel branching. High resolution microtomography (mCT) is used to determine the spaces occupied by vascular branches and calculate their planar fractal dimension. Finally, the vascular tree has been simulated by a mixed, stochastic / deterministic algorithm based on diffusion limited aggregation (DLA), in which mean values of vascular variables are set as constraints. The purpose of this research is to understand if fractality can be reliably assumed for computational modeling of the organ anatomy, in order to be able to produce, by AM, more representative physical prototypes and scaffolds. The finding allow to affirm that the human thyroid arterial structure exhibits a degree of auto similarity.