The Role of Prep1 in the Regulation of Mesenchymal Stromal Cells.

Molecular mechanisms governing cell fate decision events in bone marrow mesenchymal stromal cells (MSC) are still poorly understood. Herein, we investigated the homeobox gene Prep1 as a candidate regulatory molecule, by adopting Prep1 hypomorphic mice as a model to investigate the effects of Prep1 downregulation, using in vitro and in vivo assays, including the innovative single cell RNA sequencing technology. Taken together, our findings indicate that low levels of Prep1 are associated to enhanced adipogenesis and a concomitant reduced osteogenesis in the bone marrow, suggesting Prep1 as a potential regulator of the adipo-osteogenic differentiation of mesenchymal stromal cells. Furthermore, our data suggest that in vivo decreased Prep1 gene dosage favors a pro-adipogenic phenotype and induces a "browning" effect in all fat tissues.

3D fiber deposited polymeric scaffolds for external auditory canal wall.

The external auditory canal (EAC) is an osseocartilaginous structure extending from the auricle to the eardrum, which can be affected by congenital, inflammatory, and neoplastic diseases, thus reconstructive materials are needed. Current biomaterial-based approaches for the surgical reconstruction of EAC posterior wall still suffer from resorption (biological) and extrusion (synthetic). In this study, 3D fiber deposited scaffolds based on poly(ethylene oxide terephthalate)/poly(butylene terephthalate) were designed and fabricated to replace the EAC wall. Fiber diameter and scaffold porosity were optimized, leading to 200 ± 33 µm and 55% ± 5%, respectively. The mechanical properties were evaluated, resulting in a Young's modulus of 25.1 ± 7.0 MPa. Finally, the EAC scaffolds were tested in vitro with osteo-differentiated human mesenchymal stromal cells (hMSCs) with different seeding methods to produce homogeneously colonized replacements of interest for otologic surgery. This study demonstrated the fabrication feasibility of EAC wall scaffolds aimed to match several important requirements for biomaterial application to the ear under the Tissue Engineering paradigm, including shape, porosity, surface area, mechanical properties and favorable in vitro interaction with osteoinduced hMSCs. This study demonstrated the fabrication feasibility of outer ear canal wall scaffolds via additive manufacturing. Aimed to match several important requirements for biomaterial application to ear replacements under the Tissue Engineering paradigm, including shape, porosity and pore size, surface area, mechanical properties and favorable in vitro interaction with osteo-differentiated mesenchymal stromal cells.

3D enamel thickness in Neandertal and modern human permanent canines.

Enamel thickness figures prominently in studies of human evolution, particularly for taxonomy, phylogeny, and paleodietary reconstruction. Attention has focused on molar teeth, through the use of advanced imaging technologies and novel protocols. Despite the important results achieved thus far, further work is needed to investigate all tooth classes. We apply a recent approach developed for anterior teeth to investigate the 3D enamel thickness of Neandertal and modern human (MH) canines. In terms of crown size, the values obtained for both upper and lower unworn/slightly worn canines are significantly greater in Neandertals than in Upper Paleolithic and recent MH. The 3D relative enamel thickness (RET) is significantly lower in Neandertals than in MH. Moreover, differences in 3D RET values between the two groups appear to decrease in worn canines beginning from wear stage 3, suggesting that both the pattern and the stage of wear may have important effects on the 3D RET value. Nevertheless, the 3D average enamel thickness (AET) does not differ between the two groups. In both groups, 3D AET and 3D RET indices are greater in upper canines than in lower canines, and overall the enamel is thicker on the occlusal half of the labial aspect of the crown, particularly in MH. By contrast, the few early modern humans investigated show the highest volumes of enamel while for all other components of 3D enamel, thickness this group holds an intermediate position between Neandertals and recent MH. Overall, our study supports the general findings that Neandertals have relatively thinner enamel than MH (as also observed in molars), indicating that unworn/slightly worn canines can be successfully used to discriminate between the two groups. Further studies, however, are needed to understand whether these differences are functionally related or are the result of pleiotropic or genetic drift effects.

A Neanderthal deciduous human molar with incipient carious infection from the Middle Palaeolithic De Nadale cave, Italy.

OBJECTIVES: The aim of the study is the assessment of Nadale 1, a Neanderthal deciduous tooth recently discovered in Northeastern Italy in the De Nadale cave (Middle Palaeolithic). Together with the clear archaeological context of the site, this study brings new insight on Neanderthal behavior and dental morphological variability. MATERIALS AND METHODS: We used microCT data to provide a morphological description and morphometric analysis (diameter measurements and dental tissue volumes) of the Nadale 1 human tooth. Microwear analysis, taphonomical investigation and caries identification were performed using a stereomicroscope and Scanning Electron Microscope. RESULTS: In terms of morphology (i.e., incipient tuberculum molare, marked mesial marginal ridge and well-developed mid-trigonid crest connecting the protoconid and the metaconid, deep anterior fovea) and size, Nadale 1 presents features frequently observed in Neanderthal lower first deciduous molars. Microscope investigations reveal the presence of a small pit which could be correlated to an incipient caries. CONCLUSION: Nadale 1 expands the Italian Middle Palaeolithic fossil record and provides further information on Neanderthal dm1s in terms of dimensional and morphological variability. Furthermore, the presence of an incipient caries brings further data on Neanderthal diet.

The dawn of dentistry in the late upper Paleolithic: An early case of pathological intervention at Riparo Fredian.

OBJECTIVES: Early evidence for the treatment of dental pathology is found primarily among food-producing societies associated with high levels of oral pathology. However, some Late Pleistocene hunter-gatherers show extensive oral pathology, suggesting that experimentation with therapeutic dental interventions may have greater antiquity. Here, we report the second earliest probable evidence for dentistry in a Late Upper Paleolithic hunter-gatherer recovered from Riparo Fredian (Tuscany, Italy). MATERIALS AND METHODS: The Fredian 5 human consists of an associated maxillary anterior dentition with antemortem exposure of both upper first incisor (I1 ) pulp chambers. The pulp chambers present probable antemortem modifications that warrant in-depth analyses and direct dating. Scanning electron microscopy, microCT and residue analyses were used to investigate the purported modifications of external and internal surfaces of each I1 . RESULTS: The direct date places Fredian 5 between 13,000 and 12,740 calendar years ago. Both pulp chambers were circumferentially enlarged prior to the death of this individual. Occlusal dentine flaking on the margin of the cavities and striations on their internal aspects suggest anthropic manipulation. Residue analyses revealed a conglomerate of bitumen, vegetal fibers, and probable hairs adherent to the internal walls of the cavities. DISCUSSION: The results are consistent with tool-assisted manipulation to remove necrotic or infected pulp in vivo and the subsequent use of a composite, organic filling. Fredian 5 confirms the practice of dentistry-specifically, a pathology-induced intervention-among Late Pleistocene hunter-gatherers. As such, it appears that fundamental perceptions of biomedical knowledge and practice were in place long before the socioeconomic changes associated with the transition to food production in the Neolithic.

Maternal high-fat feeding leads to alterations of brain glucose metabolism in the offspring: positron emission tomography study in a porcine model.

AIMS/HYPOTHESIS: Maternal obesity negatively affects fetal development. Abnormalities in brain glucose metabolism are predictive of metabolic-cognitive disorders. METHODS: We studied the offspring (aged 0, 1, 6, 12 months) of minipigs fed a normal vs high-fat diet (HFD), by positron emission tomography (PET) to measure brain glucose metabolism, and ex vivo assessments of brain insulin receptors (IRß) and GLUT4. RESULTS: At birth, brain glucose metabolism and IRß were twice as high in the offspring of HFD-fed than control mothers. During infancy and youth, brain glucose uptake, GLUT4 and IRß increased in the offspring of control mothers and decreased in those of HFD-fed mothers, leading to a 40-85% difference (p < 0.05), and severe glycogen depletion, lasting until adulthood. CONCLUSIONS/INTERPRETATION: Maternal high-fat feeding leads to brain glucose overexposure during fetal development, followed by long-lasting depression in brain glucose metabolism in minipigs. These features may predispose the offspring to develop metabolic-neurodegenerative diseases.

Guidelines to PET measurements of the target occupancy in the brain for drug development.

This guideline summarizes the current view of the European Association of Nuclear Medicine Drug Development Committee. The purpose of this guideline is to guarantee a high standard of PET studies that are aimed at measuring target occupancy in the brain within the framework of development programs of drugs that act within the central nervous system (CNS drugs). This guideline is intended to present information specifically adapted to European practice. The information provided should be applied within the context of local conditions and regulations.

Middle paleolithic human deciduous incisor from Grotta del Cavallo, Italy.

OBJECTIVES: In this contribution, we present a morphological description and comparative morphometric analysis of Cavallo D, a human tooth unearthed from the Mousterian FIII sublayer of Grotta del Cavallo (Apulia, Italy). MATERIALS AND METHODS: We used microCT data to provide a detailed morphological description and morphometric analysis of the Cavallo D human tooth based on traditional diameter measurements and 3D enamel thickness. Moreover, new AMS radiocarbon dating of charcoals from layers FII was carried out. RESULTS: Morphological features observed in Cavallo D align the tooth to Neandertals. Similarly, the large size of the tooth (e.g., BL diameter) and the relatively thinner enamel thickness are typical Neandertal traits. 14 C datings of layer FII attribute the tooth to a time range of 45,600-42,900 cal BP (at 68% level of probability). DISCUSSION: Up to now, the Rdi1 Cavallo D represents the most recent Neandertal human remain in southern Italy related to a radiocarbon dated stratigraphy. Moreover, since deciduous teeth have been less investigated than the permanent ones, this contribution brings new data to increase our knowledge on the variability of the Neandertal deciduous dentition.

[Integrated patient-centered care in Community Health Centers in Italy: performance evaluation]. / La casa della salute: integrazione professionale per la centralità del cittadino. Valutazione performances.

In Italy, a "Casa della Salute" (or "House of Health"), translated for the purposes of this article as Community Health Center (CHC), is a public center (of the national public healthcare system), where one can find allocated all primary community healthcare services centered on prevention, treatment and rehabilitation of the citizen in the same physical space, including general practitioner offices, outpatient specialist clinics, and social services. CHCs assure patient-centered primary care services and are the ideal place for applying the Chronic Care Model. We compared inpatient admission rates for all diseases and for chronic diseases, pharmaceutical and specialist expenditure, spending for implants and emergency room admissions, among persons assigned to General Practitioners (GPs) who work in CHCs compared with those in the same geographical area followed by GPs not working in a CHC. Subjects cared for in CHCs showed lower levels of hospital admission and emergency room access. Slight increases in the consumption of drugs and specialist resources, including consultation and diagnostic tests, were observed, due to increased taking charge of chronic cases in CHCs.

Design, synthesis and preliminary evaluation of (18)F-labelled 1,8-naphthyridin- and quinolin-2-one-3-carboxamide derivatives for PET imaging of CB2 cannabinoid receptor.

In the present work, we report the synthesis of new aryliodonium salts used as precursors of single-stage nucleophilic (18)F radiofluorination. The corresponding unlabelled fluorinated derivatives showed to be CB2 cannabinoid receptor specific ligands, with Ki values in the low nanomolar range and high CB2/CB1 selectivity. The radiolabelled compound [(18)F]CB91, was successfully formulated for in vivo administration, and its preliminary biodistribution was assessed with microPET/CT. This tracer presented a reasonable in vivo stability and a preferential extraction in the tissues that constitutionally express CB2 cannabinoid receptor. The results obtained indicate [(18)F]CB91 as a possible candidate marker of CB2 cannabinoid receptor distribution. This study would open the way to further validation of this tracer for assessing pathologies for which the expression of this receptor is modified.

Piscivory in a Miocene Cetotheriidae of Peru: first record of fossilized stomach content for an extinct baleen-bearing whale.

Instead of teeth, modern mysticetes bear hair-fringed keratinous baleen plates that permit various bulk-filtering predation techniques (from subsurface skimming to lateral benthic suction and engulfment) devoted to various target prey (from small invertebrates to schooling fish). Current knowledge about the feeding ecology of extant cetaceans is revealed by stomach content analyses and observations of behavior. Unfortunately, no fossil stomach contents of ancient mysticetes have been described so far; the investigation of the diet of fossil baleen whales, including the Neogene family Cetotheriidae, remains thus largely speculative. We report on an aggregate of fossil fish remains found within a mysticete skeleton belonging to an undescribed late Miocene (Tortonian) cetotheriid from the Pisco Formation (Peru). Micro-computed tomography allowed us to interpret it as the fossilized content of the forestomach of the host whale and to identify the prey as belonging to the extant clupeiform genus Sardinops. Our discovery represents the first direct evidence of piscivory in an ancient edentulous mysticete. Since among modern mysticetes only Balaenopteridae are known to ordinarily consume fish, this fossil record may indicate that part of the cetotheriids experimented some degree of balaenopterid-like engulfment feeding. Moreover, this report corresponds to one of the geologically oldest records of Sardinops worldwide, occurring near the Tortonian peak of oceanic primary productivity and cooling phase. Therefore, our discovery evokes a link between the rise of Cetotheriidae; the setup of modern coastal upwelling systems; and the radiation of epipelagic, small-sized, schooling clupeiform fish in such highly productive environments.

Three-dimensional parametric mapping in quantitative micro-CT imaging of post-surgery femoral head-neck samples: preliminary results.

Osteoporosis and pathological increased occurrence of fractures are an important public health problem. They may affect patients' quality of life and even increase mortality of osteoporotic patients, and consequently represent a heavy economic burden for national healthcare systems. The adoption of simple and inexpensive methods for mass screening of population at risk may be the key for an effective prevention. The current clinical standards of diagnosing osteoporosis and assessing the risk of an osteoporotic bone fracture include dual-energy X-ray absorptiometry (DXA) and quantitative computed tomography (QCT) for the measurement of bone mineral density (BMD). Micro-computed tomography (micro-CT) is a tomographic imaging technique with very high resolution allowing direct quantification of cancellous bone microarchitecture. The Authors performed micro-CT analysis of the femoral heads harvested from 8 patients who have undergone surgery for hip replacement for primary and secondary degenerative disease to identify possible new morphometric parameters based on the analysis of the distribution of intra-subject microarchitectural parameters through the creation of parametric images. Our results show that the micro-architectural metrics commonly used may not be sufficient for the realistic assessment of bone microarchitecture of the femoral head in patients with hip osteoarthritis. The innovative micro-CT approach considers the entire femoral head in its physiological shape with all its components like cartilage, cortical layer and trabecular region. The future use of these methods for a more detailed study of the reaction of trabecular bone for the internal fixation or prostheses would be desirable.

Independent effects of circulating glucose, insulin and NEFA on cardiac triacylglycerol accumulation and myocardial insulin resistance in a swine model.

AIMS/HYPOTHESIS: Cardiac steatosis and myocardial insulin resistance elevate the risk of cardiac complications in obesity and diabetes. We aimed to disentangle the effects of circulating glucose, insulin and NEFA on myocardial triacylglycerol (TG) content and myocardial glucose uptake. METHODS: Twenty-two pigs were stratified according to four protocols: low NEFA + low insulin (nicotinic acid), high NEFA + low insulin (fasting) and high insulin + low NEFA ± high glucose (hyperinsulinaemia-hyperglycaemia or hyperinsulinaemia-euglycaemia). Positron emission tomography, [U-(13)C]palmitate enrichment techniques and tissue biopsies were used to assess myocardial metabolism. Heart rate and rate-pressure product (RPP) were monitored. RESULTS: Myocardial glucose extraction was increased by NEFA suppression and was similar in the hyperinsulinaemia-hypergylcaemia, hyperinsulinaemia-euglycaemia and nicotinic acid groups. Hyperglycaemia enhanced myocardial glucose uptake due to a mass action. Myocardial TG content was greatest in the fasting group, whereas hyperinsulinaemia had a mild effect. Heart rate and RPP increased in hyperinsulinaemia-euglycaemia, in which cardiac glycogen content was reduced. Heart rate correlated with myocardial TG and glycogen content. CONCLUSIONS/INTERPRETATION: Elevated NEFA levels represent a powerful, self-sufficient promoter of cardiac TG accumulation and are a downregulator of myocardial glucose uptake, indicating that the focus of treatment should be to 'normalise' adipose tissue function to lower the risk of cardiac TG accumulation and myocardial insulin resistance. The observation that hyperinsulinaemia and nicotinic acid led to myocardial fuel deprivation provides a potential explanation for the cardiovascular outcomes reported in recent intensive glucose-lowering and NEFA-lowering clinical trials.

A historical case of amelogenesis imperfecta: Giovanna of Austria, Grand Duchess of Tuscany (1547-1578).

The skeletal remains of Giovanna of Austria (1547-1578), daughter of the Emperor Ferdinand I of Habsburg (1503-1564) and first wife of the Grand Duke of Tuscany, Francesco I (1541-1587), exhumed from the Basilica of San Lorenzo in Florence, were submitted to paleopathological study. Examination of the dentition, which was in a good state of preservation, showed maxillary retrognathism, together with a caries lesion, moderate periodontal disease, malposition of the upper second premolars and tooth wear. Furthermore, several horizontal grooves were observed in both the buccal and the lingual crown surfaces of almost all teeth, especially the anterior ones. The orthopantomogram showed hypomineralized enamel and alveolar bone loss. Two third-molar teeth were investigated using micro-computed tomography (micro-CT) analysis, revealing highly irregular enamel caps with reduced average thickness. The observed features suggest a diagnosis of hypoplastic amelogenesis imperfecta, a developmental condition affecting enamel formation.

Proof of concept of a new plasma complement Factor H from waste plasma fraction.

Introduction: Complement factor H (FH) is a major regulator of the complement alternative pathway, its mutations predispose to an uncontrolled activation in the kidney and on blood cells and to secondary C3 deficiency. Plasma exchange has been used to correct for FH deficiency and although the therapeutic potential of purified FH has been suggested by in vivo experiments in animal models, a clinical approved FH concentrate is not yet available. We aimed to develop a purification process of FH from a waste fraction rather than whole plasma allowing a more efficient and ethical use of blood and plasma donations. Methods: Waste fractions from industrial plasma fractionation (pooled human plasma) were analyzed for FH content by ELISA. FH was purified from unused fraction III and its decay acceleration, cofactor, and C3 binding capacity were characterized in vitro. Biodistribution was assessed by high-resolution dynamic PET imaging. Finally, the efficacy of the purified FH preparation was tested in the mouse model of C3 glomerulopathy (Cfh-/- mice). Results: Our purification method resulted in a high yield of highly purified (92,07%), pathogen-safe FH. FH concentrate is intact and fully functional as demonstrated by in vitro functional assays. The biodistribution revealed lower renal and liver clearance of human FH in Cfh-/- mice than in wt mice. Treatment of Cfh-/- mice documented its efficacy in limiting C3 activation and promoting the clearance of C3 glomerular deposits. Conclusion: We developed an efficient and economical system for purifying intact and functional FH, starting from waste material of industrial plasma fractionation. The FH concentrate could therefore constitute possible treatments options of patients with C3 glomerulopathy, particularly for those with FH deficiency, but also for patients with other diseases associated with alternative pathway activation.

MicroPET/CT imaging of αvß3 integrin via a novel 68Ga-NOTA-RGD peptidomimetic conjugate in rat myocardial infarction.

PURPOSE: The αvß3 integrin is expressed in angiogenic vessels and is a potential target for molecular imaging of evolving pathological processes. Its expression is upregulated in cancer lesions and metastases as well as in acute myocardial infarction (MI) as part of the infarct healing process. The purpose of our study was to determine the feasibility of a new imaging approach with a novel (68)Ga-2,2',2″-(1,4,7-triazonane-1,4,7-triyl)triacetic acid (NOTA)-arginine-glycine-aspartic acid (RGD) construct to assess integrin expression in the evolving MI. METHODS: A straightforward labelling chemistry to attach the radionuclide (68)Ga to a NOTA-based chelating agent conjugated with a cyclic RGD peptidomimetic is described. Affinity for αvß3 integrin was assessed by in vitro receptor binding assay. The proof-of-concept in vivo studies combined the (68)Ga-NOTA-RGD with the flow tracer (13)N-NH3 imaging in order to obtain positron emission tomography (PET)/CT imaging of both integrin expression and perfusion defect at 4 weeks after infarction. Hearts were then processed for immunostaining of integrin ß3. RESULTS: NOTA-RGD conjugate displayed a binding affinity for αvß3 integrin of 27.9 ± 6.8 nM. (68)Ga-NOTA-RGD showed stability without detectable degradation or formation of by-products in urine up to 2 h following injection in the rat. MI hearts exhibited (68)Ga-NOTA-RGD uptake in correspondence to infarcted and border zone regions. The tracer signal drew a parallel with vascular remodelling due to ischaemia-induced angiogenesis as assessed by immunohistochemistry. CONCLUSION: As compared to similar imaging approaches using the (18)F-galacto-derivative, we documented for the first time with microPET/CT imaging the (68)Ga-NOTA-RGD derivative that appears eligible for PET imaging in animal models of vascular remodelling during evolving MI. The simple chemistry employed to synthesize the (68)Ga-based radiotracer may greatly facilitate its translation to a clinical setting.

[Translational perspectives in molecular imaging: methodological evolution and nanostructured materials]. / Prospettive traslazionali dell'imaging molecolare: evoluzione delle metodiche e materiali nanostrutturati.

Molecular imaging techniques play an increasingly important role in the deep understanding of pathologies. They represent a direct spotlight on the molecular correlates of diseases and can be used for assessing earlier the state of health and decide the treatment of each patient in a personalized way. This article will show the basis of several imaging techniques, and give examples on the application and development of molecular imaging tracers. Particular attention will be pointed on the use of nanostructured materials, that has a promising role in the finding of new tracers, on the use of novel methodological approaches (multimodality, theranostics, pretargeting) and on the possibility of translational applications.

[Hybrid imaging: present and future of non-invasive diagnosis]. / Imaging ibrido: presente e futuro della diagnostica non invasiva.

The integration of complementary information from different medical imaging techniques opened, since the 90s, new scenarios and possibilities for non-invasive diagnosis. The anatomo-functional information obtained with the image fusion (i.e., by composing PET and CT or SPECT and CT), is greater than the sum of information given by each modality, and allows quantitative evaluation of functional parameters through the application of specific model-based image processing. Before the introduction of integrated multimodal scanners from most manufacturers (hardware fusion), many software techniques have been used to allow image fusion from different modalities; such methods of software fusion is still important for all modalities that cannot by merged in a single scanner, or whenever the patient movement can introduce inter-modality misalignments within the same exam in an integrated scanner or if each modality is utilized at different times and on separate scanners. This article summarizes the software and hardware evolution of clinical and preclinical hybrid imaging, and discusses the outcome of this technology in the clinical environment with particular emphasis on the cardiovascular application.

Computer tomography and magnetic resonance for multimodal imaging of fossils and mummies.

The study of fossils and mummies has largely benefited from the use of modern noninvasive and nondestructive imaging technologies and represents a fast developing area. In this review, we describe the emerging role of imaging based on Magnetic Resonance (MR) and Computer Tomography (CT) employed for the study of ancient remains and mummies. For each methodology, the state of the art in paleoradiology applications is described, by emphasizing new technologies developed in the field of both CT, such as micro- and nano-CT, dual-energy and multi-energy CT, and MR, with the description of novel dedicated sequences, radiofrequency coils and gradients. The complementarity of CT and MR in paleoradiology is also discussed, by pointing out what MR provides in addition to CT, with an overview on the state of the art of emerging strategies in the use of CT/MR combination for the study of a sample following a multimodal integrated approach.