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Despite the plethora of studies investigating listening effort and the amount of research concerning music perception by cochlear implant (CI) users, the investigation of the influence of background noise on music processing has never been performed. Given the typical speech in noise recognition task for the listening effort assessment, the aim of the present study was to investigate the listening effort during an emotional categorization task on musical pieces with different levels of background noise. The listening effort was investigated, in addition to participants' ratings and performances, using EEG features known to be involved in such phenomenon, that is alpha activity in parietal areas and in the left inferior frontal gyrus (IFG), that includes the Broca's area. Results showed that CI users performed worse than normal hearing (NH) controls in the recognition of the emotional content of the stimuli. Furthermore, when considering the alpha activity corresponding to the listening to signal to noise ratio (SNR) 5 and SNR10 conditions subtracted of the activity while listening to the Quiet condition-ideally removing the emotional content of the music and isolating the difficulty level due to the SNRs- CI users reported higher levels of activity in the parietal alpha and in the homologous of the left IFG in the right hemisphere (F8 EEG channel), in comparison to NH. Finally, a novel suggestion of a particular sensitivity of F8 for SNR-related listening effort in music was provided.

BackgroundAnterior cruciate ligaments (ACL) injuries represent a major risk factor for early osteoarthritis (OA).PurposeTo evaluate the prevalence and 4-year progression of knee OA measured with 3T MR-imaging in individuals with ruptured, reconstructed or normal ACL and to assess the impact of thigh muscle characteristics.MethodsA total of 54 knees (23/54 male, 31/54 female) were recruited from the Osteoarthritis Initiative (OAI). At baseline, 15/54 subjects had prevalent ACL ruptures and 15/54 subjects had prevalent ACL reconstruction (24/54 normal ACL). Western Ontario and McMasters Universities Arthritis Index (WOMAC) scores, Physical Activity Scores of the Elderly (PASE) and thigh muscle characteristics including strength, fat infiltration (Goutallier score) and thigh muscle cross-sectional area (CSA) MR measurements were obtained at baseline. Whole-organ MR-imaging Scores (WORMS) were obtained at baseline and at a 4-year follow-up time-point. Multivariate regression models, adjusting for covariates (age, gender, body mass index), were used for statistical analysis.ResultsAt baseline, subjects with prevalent ACL ruptures had worse WORMS total scores (mean±SEM, 44.1±3.5) than subjects with ACL reconstruction (30.8±4.0; P = 0.015) and worse than subjects with normal ACL (21.3±3.0; P

We have reviewed over 60 years of studies on healthcare education outreach programs that are aimed to support first-generation, low-income, as well as underrepresented racial and ethnic minority groups (historically marginalized students) to pursue pre-health professions. As a systematic literature review, we present the challenges studies on healthcare education outreach programs had as three main categories: 1) Design, 2) Evaluation, and 3) Analysis. 1) Designs of studies on healthcare education outreach programs often lacked theoretical foundations whereby a) the interventions did not present theories underlying a causal mechanism of inequity in health professions; and/or 1b) the defined outcome measures were not clearly aligned with the problem the intervention tried to address. 2) Evaluations of studies on healthcare education outreach programs were not always conducted effectively whereby: 2a) controlled groups were commonly absent for comparison with the intervention group; and/or 2b) post measures were solely used without pre-measures. 3) Analyses of studies on healthcare education outreach programs were not adequate whereby: 3a) the response rates and effect size were commonly low; and/or 3b) qualitative results commonly did not supplement quantitative results. Overall, our findings reveal studies on healthcare education outreach programs have common challenges that hinder the reliability of their effects supporting historically marginalized students in pursuing pre-health professions. To address such challenges with studies on healthcare education outreach programs aimed at supporting historically marginalized students, we created a decision flow chart for researchers to ask themselves: 1) how is the design guided by theoretical goals; 2) how are measurements used to evaluate success; and 3) how does the analysis lead to reliable results?

ObjectivePolycystic ovary syndrome (PCOS) is a heterogeneous endocrinopathy, which etiology encompasses complex genetic traits associated with epigenetic factors, including differences in microRNA (miRNA) expression in a variety of tissues. The circulating form of these molecules is raising attention in the syndrome not only as potential biomarkers of PCOS but also as possible therapeutic targets. The aim of this study was to explore the circulating miRNA profiles present in a cohort of Brazilian women with and without PCOS and to evaluate the potential role of miRNAs in the pathophysiology of the syndrome.MethodsCross-sectional study of 36 well-characterized PCOS women and 16 healthy controls. Clinical, hormone and metabolic data were recorded and evaluated. The expression profile of the 201 circulating miRNA selected were analyzed by taqman quantitative real time polymerase chain reactions (RT-PCR) using a customized Open Array platform. Statistical and bioinformatic analyzed were performed.ResultsCirculating miR-21-5p, miR-23a-3p and miR-26a-5p were upregulated, and miR-103a-3p, miR-376a-3p, miR-19b-3p and miR-222-3p were downregulated in women with PCOS compared to healthy normo-ovulatory controls. miR-21-5p, miR-103a-3p and miR-376a-3p levels correlated positively with androgen levels. These miRNAs, in combination, were related to pathways involved in insulin signaling, steroids biosynthesis and endothelial regulation as well as in folliculogenesis.ConclusionIn this study, we identified a specific circulating miRNA signature in Brazilian women with PCOS. According to our data, circulating miR-21-5p, miR-23a-3p, miR-26a-5p, miR-103a-3p, miR-376a-3p, miR-19b-3p and miR-222-3p may represent potential candidates for differential diagnosis of PCOS in the future.

The Tomb of the Diver has been subject for many decades of fierce debate among archaeologists and classicists. Since its discovery in 1968, some scholars have considered it a unique example of the lost tradition of Greek painting, others have emphasized Etruscan or Italic parallels. More recently, a possible local production has been suggested. With the aim of trying to solve the archaeological question, an archaeometric comparison among this well-known artwork and several frescoed tombs coming from Hellenistic and Lucan necropolis was carried out. The multi-analytical study was focused on the identification of peculiar features of executive techniques and raw materials since the first period of the archaeological site. The analytical investigation has been preliminary based on a non-destructive approach, performed in-situ by portable equipment including imaging diagnostics and compositional spectroscopic techniques for identifying pigments and the conservation state of original painted surface; subsequently, a further deepening by using destructive techniques was performed in-lab for the mortar-based supports characterization. Archaeometric study suggested that technological choices slightly changed in a time span of about two centuries, highlighting important markers that allow clustering the contemporary artistic productions. Moreover, a comparison with mortars from temples decorations was provided to better understand the whole artistic context. The archaeometric data showed that the Tomb of the Diver could be traced back to a local artisanal tradition and therefore is neither Etruscan nor Greek, but the first and foremost an expression of the local elite culture of Paestum.

AIM:The study aimed to establish how granulocytes, monocytes and macrophages contribute to the development of bronchopulmonary dysplasia (BPD). MATERIALS AND METHODS:Study A: samples of blood and tracheal aspirates (TAs) collected from preterm newborn infants during the first 3 days of life were investigated by flow cytometry, and testing for white blood cells (WBCs), neutrophils and neutrophil extracellular traps (NETs). Maternal blood samples were also collected. Study B: data from previously-tested samples of TAs collected from preterm newborn infants were re-analyzed in the light of the findings in the new cohort. RESULTS:Study A: 39 preterm newborn infants were studied. A moderate correlation emerged between maternal WBCs and neutrophils and those of their newborn in the first 3 days of life. WBCs and neutrophils correlated in the newborn during the first 8 days of life. Decision rules based on birth weight (BW) and gestational age (GA) can be used to predict bronchopulmonary dysplasia (BPD). Neutrophil levels were lower in the TAs from the newborn with the lowest GAs and BWs. Study B: after removing the effect of GA on BPD development, previously-tested newborn were matched by GA. Monocyte phenotype 1 (Mon1) levels were lower in the blood of newborn with BPD, associated with a higher ratio of Monocyte phenotype 3 (Mon3) to Mon1. Newborn infants from mothers with histological chorioamnionitis (HCA) had lower levels of classically-activated macrophages (M1) and higher levels of alternatively-activated macrophages (M2) in their TAs than newborn infants from healthy mothers. CONCLUSION:Immune cell behavior in preterm newborn infants was examined in detail. Surprisingly, neutrophil levels were lower in TAs from the newborn with the lowest GA and BW, and no correlation emerged between the neutrophil and NET levels in TAs and the other variables measured. Interestingly, monocyte phenotype seemed to influence the onset of BPD. The rise in the ratio of Mon 3 to Mon 1 could contribute to endothelial dysfunction in BPD.

The vast majority of rabies deaths occur in developing countries and rural areas. Due to the absence of surveillance and the lack of reliable information, many endemic countries are not able to assess their rabies burden and implement appropriate solutions. This study reports the incidence of animal bites considered at risk of rabies transmission, along with rates and determinants of the adherence to post-exposure prophylaxis (PEP) between 2008 and 2014 in Dodoma Region, Tanzania. A retrospective analysis of rabid animal bites considered at risk of rabies transmission at Dodoma Regional Referral Hospital (DRRH) during 2008-2014 was conducted. Data were collected from the registers of patients presenting to the hospital because of a potential rabies exposure. The patients were assessed by a trained health worker and each bite was considered as "at risk of rabies" based on the victim's description of the event. Overall, 10,771 patients coming from Dodoma Region attended DRRH because of a bite from a suspected rabid animal, giving a mean incidence of 74 bites at risk of rabies transmission per 100,000 persons per year. Overall, only 46.0% of people exposed received a complete course of PEP and 61.6% attended the clinic within 48 hours after the bite. Multivariate analysis shows that people age >15 years, residence in rural areas and occurrence during the rainy season were independently associated to delayed access to care. Male gender, age below 15 years. and bites occurring during the dry season were associated with completion of PEP. In this area with a high rate of at-risk bites, several factors-mainly related to health care access and to the affordability and delivery of rabies vaccines-still need to be addressed in order to reduce gender and social inequalities in rabies prevention and control. Further efforts are required to establish an efficient rabies surveillance system in Dodoma Region.

Transplantation of ovarian tissue for the preservation of fertility in oncological patients is becoming an accepted clinical practice. However, the risk of re-introducing tumour cells at transplantation has stirred an increased interest for complete in vitro folliculogenesis. This has not yet been achieved in humans possibly for the lack of knowledge on the environmental milieu that orchestrates folliculogenesis in vivo. The main aim of this study was to investigate the effect of oxygen availability on follicle health and growth during in vitro culture of ovarian tissue strips. To this end, a model was developed to predict the dissolved oxygen concentration in tissue under varying culture conditions. Ovarian cortical strips of bovine, adopted as an animal model, and human tissue were cultured in conventional (CD) and gas permeable (PD) dishes under different media column heights and gaseous oxygen tensions for 3, 6 and 9 days. Follicle quality, activation of primordial follicles to the primary stage, and progression to the secondary stage were analysed through histology. Follicle viability was assessed through a live-dead assay at the confocal scanning laser microscope. Findings showed a higher follicle quality and viability after culture of bovine ovarian strips in PD in adequate medium height and oxygen tensions. The best culture conditions found in the bovine were adopted for human ovarian strip culture and promoted a higher follicle quality, viability and progression. Overall, data demonstrated that modulation of oxygen availability in tissue plays a key role in maintaining follicles' health and their ability to survive and progress to the secondary stage during ovarian tissue in vitro culture. Such culture conditions could increase the yield of healthy secondary follicles for subsequent dissection and individual culture to obtain competent oocytes.

Health-enhancing potential bioactive peptide (BP) has driven an interest in food proteins as well as in the development of predictive methods. Research in this area has been especially active to use them as components in functional foods. Apparently, BPs do not have a given biological function in the containing proteins and they do not evolve under independent evolutionary constraints. In this work we performed a large-scale mapping of BPs in sequence and structural space. Using well curated BP deposited in BIOPEP database, we searched for exact matches in non-redundant sequences databases. Proteins containing BPs, were used in fold-recognition methods to predict the corresponding folds and BPs occurrences were mapped. We found that fold distribution of BP occurrences possibly reflects sequence relative abundance in databases. However, we also found that proteins with 5 or more than 5 BP in their sequences correspond to well populated protein folds, called superfolds. Also, we found that in well populated superfamilies, BPs tend to adopt similar locations in the protein fold, suggesting the existence of hotspots. We think that our results could contribute to the development of new bioinformatics pipeline to improve BP detection.

Bis-dehydroxycurcumin tert-butyl ester (K2T23) is a derivative of the natural spice curcumin. Curcumin is widely studied for its multiple therapeutic properties, including photosensitized cytotoxicity. However, the full exploitation of curcumin phototoxic potential is hindered by the extreme instability of its excited state, caused by very efficient non radiative decay by means of transfer of the enolic proton to the nearby keto oxygen. K2T23 is designed to exhibit a tautomeric equilibrium shifted toward the diketo conformers with respect to natural curcumin. This property should endow K2T23 with superior excited-state stability when excited in the UVB band, i.e., in correspondence of the diketo conformers absorption peaks, making this compound an interesting candidate for topical photodynamic therapy of, e.g., skin tumors or oral infections. In this work, the tautomeric equilibrium of K2T23 between the keto-enolic and diketo conformers is assessed in the ground state in several organic solvents by UV-visible absorption and by nuclear magnetic resonance. The same tautomeric equilibrium is also probed in the excited-state in the same environments by means of steady-state fluorescence and time-correlated single-photon counting measurements. These techniques are also exploited to elucidate the excited state dynamics and excited-state deactivation pathways of K2T23, which are compared to those determined for several other curcuminoids characterized in previous works of ours. The ability of K2T23 in photosensitizing the production of singlet oxygen is compared with that of curcumin.

For the assessment of glucose tolerance from IVGTT data in Zucker rat, minimal model methodology is reliable but time- and money-consuming. This study aimed to validate for the first time in Zucker rat, simple surrogate indexes of insulin sensitivity and secretion against the glucose-minimal-model insulin sensitivity index (SI) and against first- (Φ1) and second-phase (Φ2) β-cell responsiveness indexes provided by C-peptide minimal model. Validation of the surrogate insulin sensitivity index (ISI) and of two sets of coupled insulin-based indexes for insulin secretion, differing from the cut-off point between phases (FPIR3-SPIR3, t = 3 min and FPIR5-SPIR5, t = 5 min), was carried out in a population of ten Zucker fatty rats (ZFR) and ten Zucker lean rats (ZLR). Considering the whole rat population (ZLR+ZFR), ISI showed a significant strong correlation with SI (Spearman's correlation coefficient, r = 0.88; P

A C-peptide-based assessment of β-cell function was performed here in the Zucker fatty rat, a suitable animal model of human metabolic syndrome. To this aim, a 90-min intravenous glucose tolerance test (IVGTT) was performed in seven Zucker fatty rats (ZFR), 7-to-9 week-old, and seven age-matched Zucker lean rats (ZLR). The minimal model of C-peptide (CPMM), originally introduced for humans, was adapted to Zucker rats and then applied to interpret IVGTT data. For a comprehensive evaluation of glucose tolerance in ZFR, CPMM was applied in combination with the minimal model of glucose kinetics (GKMM). Our results showed that the present CPMM-based interpretation of data is able to: 1) provide a suitable fit of C-Peptide data; 2) achieve a satisfactory estimation of parameters of interest 3) quantify both insulin secretion by estimating the time course of pre-hepatic secretion rate, SR(t), and total insulin secretion, TIS, and pancreatic sensitivity by means of three specific indexes of β-cell responsiveness to glucose stimulus (first-phase, Ф(1), second-phase, Ф(2), and steady-state, Ф(ss), never assessed in Zucker rats before; 4) detect the significant enhancement of insulin secretion in the ZFR, in face of a severe insulin-resistant state, previously observed only using a purely experimental approach. Thus, the methodology presented here represents a reliable tool to assess β-cell function in the Zucker rat, and opens new possibilities for the quantification of further processes involved in glucose homeostasis such as the hepatic insulin degradation.

Although African horse sickness (AHS) can cause up to 95% mortality in horses, naïve animals can be protected by vaccination against the homologous AHSV serotype. Genome segment 2 (Seg-2) encodes outer capsid protein VP2, the most variable of the AHSV proteins. VP2 is also a primary target for AHSV specific neutralising antibodies, and consequently determines the identity of the nine AHSV serotypes. In contrast VP1 (the viral polymerase) and VP3 (the sub-core shell protein), encoded by Seg-1 and Seg-3 respectively, are highly conserved, representing virus species/orbivirus-serogroup-specific antigens. We report development and evaluation of real-time RT-PCR assays targeting AHSV Seg-1 or Seg-3, that can detect any AHSV type (virus species/serogroup-specific assays), as well as type-specific assays targeting Seg-2 of the nine AHSV serotypes. These assays were evaluated using isolates of different AHSV serotypes and other closely related orbiviruses, from the 'Orbivirus Reference Collection' (ORC) at The Pirbright Institute. The assays were shown to be AHSV virus-species-specific, or type-specific (as designed) and can be used for rapid, sensitive and reliable detection and identification (typing) of AHSV RNA in infected blood, tissue samples, homogenised Culicoides, or tissue culture supernatant. None of the assays amplified cDNAs from closely related heterologous orbiviruses, or from uninfected host animals or cell cultures.

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories.

The ability of detecting the subtle variations occurring, among different individuals, within specific DNA sequences encompassed in highly polymorphic genes discloses new applications in genomics and diagnostics. DQB1 is a gene of the HLA-II DQ locus of the Human Leukocyte Antigens (HLA) system. The polymorphisms of the trait of the DQB1 gene including codons 52-57 modulate the susceptibility to a number of severe pathologies. Moreover, the donor-receiver tissue compatibility in bone marrow transplantations is routinely assessed through crossed genotyping of DQB and DQA. For the above reasons, the development of rapid, reliable and cost-effective typing technologies of DQB1 in general, and more specifically of the codons 52-57, is a relevant although challenging task. Quantitative assessment of the fluorescence resonance energy transfer (FRET) efficiency between chromophores labelling the opposite ends of gene-specific oligonucleotide probes has proven to be a powerful tool to type DNA polymorphisms with single-nucleotide resolution. The FRET efficiency can be most conveniently quantified by applying a time-resolved fluorescence analysis methodology, i.e. time-correlated single-photon counting, which allows working on very diluted template specimens and in the presence of fluorescent contaminants. Here we present a full in-vitro characterization of the fluorescence responses of two probes when hybridized to oligonucleotide mixtures mimicking all the possible genotypes of the codons 52-57 trait of DQB1 (8 homozygous and 28 heterozygous). We show that each genotype can be effectively tagged by the combination of the fluorescence decay constants extrapolated from the data obtained with such probes.

BackgroundImmunological non-responders (INRs) lacked CD4 increase despite HIV-viremia suppression on HAART and had an increased risk of disease progression. We assessed immune reconstitution profile upon intensification with maraviroc in INRs.MethodsWe designed a multi-centric, randomized, parallel, open label, phase 4 superiority trial. We enrolled 97 patients on HAART with CD4+ResultsBy W48 both groups displayed a CD4 increase without a significant inter-group difference. A statistically significant change in CD8 favored patients in arm HAART+maraviroc versus HAART at W12 (p=.009) and W48 (p=.025). The CD4>200/µL and CD4>200/µL + CD4 gain ≥ 25% end-points were not satisfied at W12 (p=.24 and p=.619) nor at W48 (p=.076 and p=.236). Patients continuing HAART displayed no major changes in parameters of T-cell homeostasis and activation. Maraviroc-receiving patients experienced a significant rise in circulating IL-7 by W48 (p=.01), and a trend in temporary reduction in activated HLA-DR+CD38+CD4+ by W12 (p=.06) that was not maintained at W48.ConclusionsMaraviroc intensification in INRs did not have a significant advantage in reconstituting CD4 T-cell pool, but did substantially expand CD8. It resulted in a low rate of treatment discontinuations.Trial registrationClinicalTrials.gov NCT00884858 http://clinicaltrials.gov/show/NCT00884858.

Amyotrophic lateral sclerosis (ALS) is a fatal progressive motor neuron disease, for which there are still no diagnostic/prognostic test and therapy. Specific molecular biomarkers are urgently needed to facilitate clinical studies and speed up the development of effective treatments.We used a two-dimensional difference in gel electrophoresis approach to identify in easily accessible clinical samples, peripheral blood mononuclear cells (PBMC), a panel of protein biomarkers that are closely associated with ALS. Validations and a longitudinal study were performed by immunoassays on a selected number of proteins. The same proteins were also measured in PBMC and spinal cord of a G93A SOD1 transgenic rat model. We identified combinations of protein biomarkers that can distinguish, with high discriminatory power, ALS patients from healthy controls (98%), and from patients with neurological disorders that may resemble ALS (91%), between two levels of disease severity (90%), and a number of translational biomarkers, that link responses between human and animal model. We demonstrated that TDP-43, cyclophilin A and ERp57 associate with disease progression in a longitudinal study. Moreover, the protein profile changes detected in peripheral blood mononuclear cells of ALS patients are suggestive of possible intracellular pathogenic mechanisms such as endoplasmic reticulum stress, nitrative stress, disturbances in redox regulation and RNA processing.Our results indicate that PBMC multiprotein biomarkers could contribute to determine amyotrophic lateral sclerosis diagnosis, differential diagnosis, disease severity and progression, and may help to elucidate pathogenic mechanisms.

BACKGROUND:Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease, and protein aggregation has been proposed as a possible pathogenetic mechanism. However, the aggregate protein constituents are poorly characterized so knowledge on the role of aggregation in pathogenesis is limited. METHODOLOGY/PRINCIPAL FINDINGS:We carried out a proteomic analysis of the protein composition of the insoluble fraction, as a model of protein aggregates, from familial ALS (fALS) mouse model at different disease stages. We identified several proteins enriched in the detergent-insoluble fraction already at a preclinical stage, including intermediate filaments, chaperones and mitochondrial proteins. Aconitase, HSC70 and cyclophilin A were also significantly enriched in the insoluble fraction of spinal cords of ALS patients. Moreover, we found that the majority of proteins in mice and HSP90 in patients were tyrosine-nitrated. We therefore investigated the role of nitrative stress in aggregate formation in fALS-like murine motor neuron-neuroblastoma (NSC-34) cell lines. By inhibiting nitric oxide synthesis the amount of insoluble proteins, particularly aconitase, HSC70, cyclophilin A and SOD1 can be substantially reduced. CONCLUSION/SIGNIFICANCE:Analysis of the insoluble fractions from cellular/mouse models and human tissues revealed novel aggregation-prone proteins and suggests that nitrative stress contribute to protein aggregate formation in ALS.

Muscle wasting occurs during various chronic diseases and precedes death in humans as in mice. The evaluation of the degree of muscle atrophy in diseased mouse models is often overlooked since it requires the sacrifice of the animals for muscle examination or expensive instrumentation and highly qualified personnel, such as Magnetic Resonance Imaging (MRI). Very often behavioral tests for muscle strength evaluation are used as an outcome measurement in preclinical therapeutic trials. However, these tests are easy to perform serially, but not enough sensitive to detect early muscle changes during disease progression. Monitoring muscle loss in living animals could allow to perform more informative preclinical trials with a better evaluation of therapeutic benefit with respect to muscle wasting. We developed a non-invasive procedure based on micro-computed tomography (micro-CT) without contrast agents to monitor hind limb muscle wasting in mouse models of amyotrophic lateral sclerosis (ALS) and cancer cachexia: the transgenic SOD1G93A mouse and the colon adenocarcinoma C26-bearing mouse, respectively. We established the scanning procedure and the parameters to consider in the reconstructed images to calculate the Index of Muscle Mass (IMM). The coefficient of variance for the whole procedure was 2.2%. We performed longitudinally micro-CT scan of hind limbs in SOD1G93A mice at presymptomatic and symptomatic stages of the disease and calculated the IMM. We found that IMM in SOD1G93A mice was lower than age-matched controls even before symptom onset. We also detected a further decrease in IMM as disease progresses, most markedly just before disease onset. We performed the same analyses in the C26-based mouse model losing quickly body and muscle mass because of cancer cachexia. Overall, we found that the reduced muscle content detected by micro-CT mirrored the reduced muscle weight in both disease models. We developed a fast, precise and easy-to-conduct imaging procedure to monitor hind limb muscle mass, useful in therapeutic preclinical trials but also in proof-of-principle studies to identify the onset of muscle wasting. This method could be widely applied to other disease models characterized by muscle wasting, to assist drug development and search for early biomarkers of muscle atrophy. Moreover, reducing the number of mice needed for the experiments and being less distressing are in line with the 3R principle embodied in national and international directives for animal research.

Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site. As H/D exchange kinetics followed by FTIR spectroscopy can provide information on the conformational changes in proteins where solvent accessibility is affected, here the amide I region was used to measure the exchange rates of the different elements of the secondary structure for aromatase in the ligand-free form and in the presence of the substrate androstenedione and the inhibitor anastrozole. Biphasic exponential functions were found to fit the H/D exchange data collected as a function of time. Two exchange rates were assigned to two populations of protons present in different flexible regions of the protein. The addition of the substrate androstenedione and the inhibitor anastrozole lowers the H/D exchange rates of the α-helices of the enzyme when compared to the ligand-free form. Furthermore, the presence of the inhibitor anastrozole lowers exchange rate constant (k1) for β-sheets from 0.22±0.06 min(-1) for the inhibitor-bound enzyme to 0.12±0.02 min(-1) for the free protein. Dynamics effects localised in helix F were studied by time resolved fluorescence. The data demonstrate that the fluorescence lifetime component associated to Trp224 emission undergoes a shift toward longer lifetimes (from ≈5.0 to ≈5.5 ns) when the substrate or the inhibitor are present, suggesting slower dynamics in the presence of ligands. Together the results are consistent with different degrees of flexibility of the access channel and therefore different conformations adopted by the enzyme in the free, substrate- and inhibitor-bound forms.