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Abstract The increasing availability of experimental and computational protein structures entices their use for function prediction. Here we develop an automated procedure to identify enzymes involved in metabolic reactions by assessing substrate conformations docked to a library of protein structures. By screening AlphaFold-modeled vitamin B6-dependent enzymes, we find that a metric based on catalytically favorable conformations at the enzyme active site performs best (AUROC Score=0.84) in identifying genes associated with known reactions. Applying this procedure, we identify the mammalian gene encoding hydroxytrimethyllysine aldolase (HTMLA), the second enzyme of carnitine biosynthesis. Upon experimental validation, we find that the top-ranked candidates, serine hydroxymethyl transferase (SHMT) 1 and 2, catalyze the HTMLA reaction. However, a mouse protein absent in humans (threonine aldolase; Tha1) catalyzes the reaction more efficiently. Tha1 did not rank highest based on the AlphaFold model, but its rank improved to second place using the experimental crystal structure we determined at 2.26 Å resolution. Our findings suggest that humans have lost a gene involved in carnitine biosynthesis, with HTMLA activity of SHMT partially compensating for its function.

Proper documentation is fundamental to providing structural health monitoring, damage identification and failure assessment for Cultural Heritage (CH). Three-dimensional models from photogrammetric and laser scanning surveys usually provide 3D point clouds that can be converted into meshes. The point clouds usually contain noise data due to different causes: non-cooperative material or surfaces, bad lighting, complex geometry and low accuracy of the instruments utilized. Point cloud denoising has become one of the hot topics of 3D geometric data processing, removing these noise data to recover the ground-truth point cloud and adding smoothing to the ideal surface. These cleaned point clouds can be converted in volumes with different algorithms, suitable for different uses, mainly for structural analysis. This paper aimed to analyse the geometric accuracy of algorithms available for the conversion of 3D point clouds into volumetric models that can be used for structural analyses through the FEA process. The process is evaluated, highlighting problems and difficulties that lie in poor reconstruction results of volumes from denoised point clouds due to the geometric complexity of the objects.

Abstract The aim of this study was to examine how running biomechanics (spatiotemporal and kinetic variables) adapt with exhaustion during treadmill runs at 90, 100, 110, and 120% of the peak aerobic speed (PS) of a maximal incremental aerobic test. Thirteen male runners performed a maximal incremental aerobic test on an instrumented treadmill to determine their PS. Biomechanical variables were evaluated at the start, mid, and end of each run until volitional exhaustion. The change of running biomechanics with fatigue was similar among the four tested speeds. Duty factor and contact and propulsion times increased with exhaustion (P ≤ 0.004; F ≥ 10.32) while flight time decreased (P = 0.02; F = 6.67) and stride frequency stayed unchanged (P = 0.97; F = 0.00). A decrease in vertical and propulsive peak forces were obtained with exhaustion (P ≤ 0.002; F ≥ 11.52). There was no change in the impact peak with exhaustion (P = 0.41; F = 1.05). For runners showing impact peaks, the number of impact peaks increased (P ≤ 0.04; $${\upchi }^{2}$$ χ 2 ≥ 6.40) together with the vertical loading rate (P = 0.005; F = 9.61). No changes in total, external, and internal positive mechanical work was reported with exhaustion (P ≥ 0.12; F ≤ 2.32). Results suggest a tendency towards a “smoother” vertical and horizontal running pattern with exhaustion. A smoother running pattern refers to the development of protective adjustments, leading to a reduction of the load applied to the musculoskeletal system at each running step. This transition seemed continuous between the start and end of the running trials and could be adopted by the runners to decrease the muscle force level during the propulsion phase. Despite these changes with exhaustion, there were no changes in either gesture speed (no alteration of stride frequency) or positive mechanical work, advocating that runners unconsciously organize themselves to maintain a constant whole-body mechanical work output.

Summary: Phosphonates—compounds containing a direct C–P bond—represent an important source of phosphorus in some environments. The most common natural phosphonate is 2-aminoethylphosphonate (AEP). Many bacteria can break AEP down through specialized “hydrolytic” pathways, which start with the conversion of AEP into phosphonoacetaldehyde (PAA), catalyzed by the transaminase PhnW. However, the substrate scope of these pathways is very narrow, as PhnW cannot process other common AEP-related phosphonates, notably N-methyl AEP (M1AEP). Here, we describe a heterogeneous group of FAD-dependent oxidoreductases that efficiently oxidize M1AEP to directly generate PAA, thus expanding the versatility and usefulness of the hydrolytic AEP degradation pathways. Furthermore, some of these enzymes can also efficiently oxidize plain AEP. By doing so, they surrogate the role of PhnW in organisms that do not possess the transaminase and create novel versions of the AEP degradation pathways in which PAA is generated solely by oxidative deamination.

Abstract The study aimed to compare the ankle muscles activation, biomechanics and energetics of running in male runners during submaximal level run using minimalist (MinRS) and traditional cushioned (TrdRS) running shoes. During 45-min running in MinRS and TrdRS, the ankle muscles pre- and co-activation, biomechanics, and energetics of running of 16 male endurance runners (25.5 ± 3.5 yr) were assessed using surface electromyography (tibialis anterior and gastrocnemius lateralis), instrumented treadmill and indirect calorimetry, respectively. The net energy cost of running (Cr) was similar for both conditions (P = 0.25) with a significant increase over time (P

Abstract Runners were classified using their duty factor (DF) and using their foot-strike pattern (FSP; rearfoot, midfoot, or forefoot strikers), determined from their foot-strike angle (FSA). High and low DF runners showed different FSPs but DF was assumed to not only reflect what happens at initial contact with the ground (more global than FSP/FSA). Hence, FSP and DF groups should not necessarily be constituted by the same runners. However, the relation between FSP and DF groups has never been investigated, leading to the aim of this study. One hundred runners ran at 9, 11, and 13 km/h. Force data (1000 Hz) and whole-body kinematics (200 Hz) were acquired by an instrumented treadmill and optoelectronic system and were used to classify runners according to their FSA and DF. Weak correlations were obtained between FSA and DF values and a sensitivity of 50% was reported between FSP and DF groups, i.e., only one in two runners was attributed to the DF group supposedly corresponding to the FSP group. Therefore, ‘local’ FSP/FSA and DF do not represent similar running pattern information when investigated at the individual level and DF should be preferred to FSP/FSA when evaluating the global running pattern of a runner.

Mammary carcinomas are the most common neoplasms observed in women and in female dogs. Canine mammary tumors show epidemiological, clinical, genetic, and prognostic characteristics comparable to human breast cancers. The recent introduction of next generation sequencing (NGS) technologies has greatly improved research and diagnostics for humans, while these new tools still need to be implemented in animal models. In this study we developed and validated an AmpliSeq Panel assay for the identification of BRCA variants in twenty-two different dogs. The amplicon mean coverage was 5499× and uniformity was higher than 98% in all samples. The results of germline single nucleotide variants (SNVs) and insertions/deletions (INDELs) were fully concordant regardless of the types of samples considered (blood, fresh and FFPE tissues). Moreover, despite the high DNA degradation observed in older FFPE blocks (>5 years), the assay allowed full coverage of all amplicons for downstream analyses. We consider the NGS panel developed in this study as a useful tool for expanding information on BRCA genes in the veterinary field and for human health from a comparative oncology perspective.

Polycomb-mediated gene regulation has been implicated in gliomas. Here the authors integrate transcriptomic and epigenomic analyses to define Polycomb-dependent networks that promote gliomagenesis, and find that the Polycomb-dependent silencing of the transcription factor Zfp423hinders survival.

Facilitation processes constitute basic elements of vegetation dynamics in harsh systems. Recent studies in tropical alpine environments demonstrated how pioneer plant species defined as "ecosystem engineers" are capable of enhancing landscape-level richness by adding new species to the community through the modification of microhabitats, and also provided hints about the alternation of different ecosystem engineers over time. Nevertheless, most of the existing works analysed different ecosystem engineers separately, without considering the interaction of different ecosystem engineers. Focusing on the altitudinal limit of Peruvian Dry Puna vegetation, we hypothesized that positive interactions structure plant communities by facilitation cascades involving different ecosystem engineers, determining the evolution of the microhabitat patches in terms of abiotic resources and beneficiary species hosted. To analyze successional mechanisms, we used a "space-for-time" substitution to account for changes over time, and analyzed data on soil texture, composition, and temperature, facilitated species and their interaction with nurse species, and surface area of engineered patches by means of chemical analyses, indicator species analysis, and rarefaction curves. A successional process, resulting from the dynamic interaction of different ecosystem engineers, which determined a progressive amelioration of soil conditions (e.g. nitrogen and organic matter content, and temperature), was the main driver of species assemblage at the community scale, enhancing species richness. Cushion plants act as pioneers, by starting the successional processes that continue with shrubs and tussocks. Tussock grasses have sometimes been found to be capable of creating microhabitat patches independently. The dynamics of species assemblage seem to follow the nested assemblage mechanism, in which the first foundation species to colonize a habitat provides a novel substrate for colonization by other foundation species through a facilitation cascade process.

This study aimed to compare two different maximal incremental tests with different time durations [a maximal incremental ramp test with a short time duration (8-12 min) (STest) and a maximal incremental test with a longer time duration (20-25 min) (LTest)] to investigate whether an LTest accurately assesses aerobic fitness in class II and III obese men. Twenty obese men (BMI≥35 kg.m-2) without secondary pathologies (mean±SE; 36.7±1.9 yr; 41.8±0.7 kg*m-2) completed an STest (warm-up: 40 W; increment: 20 W*min-1) and an LTest [warm-up: 20% of the peak power output (PPO) reached during the STest; increment: 10% PPO every 5 min until 70% PPO was reached or until the respiratory exchange ratio reached 1.0, followed by 15 W.min-1 until exhaustion] on a cycle-ergometer to assess the peak oxygen uptake [Formula: see text] and peak heart rate (HRpeak) of each test. There were no significant differences in [Formula: see text] (STest: 3.1±0.1 L*min-1; LTest: 3.0±0.1 L*min-1) and HRpeak (STest: 174±4 bpm; LTest: 173±4 bpm) between the two tests. Bland-Altman plot analyses showed good agreement and Pearson product-moment and intra-class correlation coefficients showed a strong correlation between [Formula: see text] (r=0.81 for both; p≤0.001) and HRpeak (r=0.95 for both; p≤0.001) during both tests. [Formula: see text] and HRpeak assessments were not compromised by test duration in class II and III obese men. Therefore, we suggest that the LTest is a feasible test that accurately assesses aerobic fitness and may allow for the exercise intensity prescription and individualization that will lead to improved therapeutic approaches in treating obesity and severe obesity.

This study aimed to compare fat oxidation, hormonal and plasma metabolite kinetics during exercise in lean (L) and obese (O) men. Sixteen L and 16 O men [Body Mass Index (BMI): 22.9 ± 0.3 and 39.0 ± 1.4 kg · m(-2)] performed a submaximal incremental test (Incr) on a cycle-ergometer. Fat oxidation rates (FORs) were determined using indirect calorimetry. A sinusoidal model, including 3 independent variables (dilatation, symmetry, translation), was used to describe fat oxidation kinetics and determine the intensity (Fat(max)) eliciting maximal fat oxidation. Blood samples were drawn for the hormonal and plasma metabolite determination at each step of Incr. FORs (mg · FFM(-1) · min(-1)) were significantly higher from 20 to 30% of peak oxygen uptake (VO2peak) in O than in L and from 65 to 85% VO2peak in L than in O (p ≤ 0.05). FORs were similar in O and in L from 35 to 60% VO2peak. Fat max was 17% significantly lower in O than in L (p

Aerobic exercise training performed at the intensity eliciting maximal fat oxidation (Fat(max)) has been shown to improve the metabolic profile of obese patients. However, limited information is available on the reproducibility of Fat(max) and related physiological measures. The aim of this study was to assess the intra-individual variability of: a) Fat(max) measurements determined using three different data analysis approaches and b) fat and carbohydrate oxidation rates at rest and at each stage of an individualized graded test. Fifteen healthy males [body mass index 23.1 ± 0.6 kg/m(2), maximal oxygen consumption (VO2max) 52.0 ± 2.0 ml/kg/min] completed a maximal test and two identical submaximal incremental tests on ergocycle (30-min rest followed by 5-min stages with increments of 7.5% of the maximal power output). Fat and carbohydrate oxidation rates were determined using indirect calorimetry. Fat(max) was determined with three approaches: the sine model (SIN), measured values (MV) and 3rd polynomial curve (P3). Intra-individual coefficients of variation (CVs) and limits of agreement were calculated. CV for Fat(max) determined with SIN was 16.4% and tended to be lower than with P3 and MV (18.6% and 20.8%, respectively). Limits of agreement for Fat(max) were -2 ± 27% of VO2max with SIN, -4 ± 32 with P3 and -4 ± 28 with MV. CVs of oxygen uptake, carbon dioxide production and respiratory exchange rate were 15%), regardless of the data analysis approach employed. Further research on the determinants of the variability of Fat(max) and fat oxidation rates is required.

Assessment of EGFR mutation in non-small cell lung cancer (NSCLC) patients is mandatory for optimization of pharmacologic treatment. In this respect, mutation analysis of circulating tumor cells (CTCs) may be desirable since they may provide real-time information on patient's disease status.Blood samples were collected from 37 patients enrolled in the TRIGGER study, a prospective phase II multi-center trial of erlotinib treatment in advanced NSCLC patients with activating EGFR mutations in tumor tissue. 10 CTC preparations from breast cancer patients without EGFR mutations in their primary tumors and 12 blood samples from healthy subjects were analyzed as negative controls. CTC preparations, obtained by the Veridex CellSearch System, were subjected to ultra-deep next generation sequencing (NGS) on the Roche 454 GS junior platform.CTCs fulfilling all Veridex criteria were present in 41% of the patients examined, ranging in number between 1 and 29. In addition to validated CTCs, potential neoplastic elements were seen in 33 cases. These included cells not fulfilling all Veridex criteria (also known as "suspicious objects") found in 5 (13%) of 37 cases, and isolated or clustered large naked nuclei with irregular shape observed in 33 (89%) cases. EGFR mutations were identified by NGS in CTC preparations of 31 (84%) patients, corresponding to those present in matching tumor tissue. Twenty-five (96%) of 26 deletions at exon 19 and 6 (55%) of 11 mutations at exon 21 were detectable (P = 0.005). In 4 (13%) cases, multiple EGFR mutations, suggesting CTC heterogeneity, were documented. No mutations were found in control samples.We report for the first time that the CellSearch System coupled with NGS is a very sensitive and specific diagnostic tool for EGFR mutation analysis in CTC preparations with potential clinical impact.

Microdeletions at exon 19 are the most frequent genetic alterations affecting the Epidermal Growth Factor Receptor (EGFR) gene in non-small cell lung cancer (NSCLC) and they are strongly associated with response to treatment with tyrosine kinase inhibitors. A series of 116 NSCLC DNA samples investigated by Sanger Sequencing (SS), including 106 samples carrying exon 19 EGFR deletions and 10 without deletions (control samples), were subjected to deep next generation sequencing (NGS). All samples with deletions at SS showed deletions with NGS. No deletions were seen in control cases. In 93 (88%) cases, deletions detected by NGS were exactly corresponding to those identified by SS. In 13 cases (12%) NGS resolved deletions not accurately characterized by SS. In 21 (20%) cases the NGS showed presence of complex (double/multiple) frameshift deletions producing a net in-frame change. In 5 of these cases the SS could not define the exact sequence of mutant alleles, in the other 16 cases the results obtained by SS were conventionally considered as deletions plus insertions. Different interpretative hypotheses for complex mutations are discussed. In 46 (43%) tumors deep NGS showed, for the first time to our knowledge, subpopulations of DNA molecules carrying EGFR deletions different from the main one. Each of these subpopulations accounted for 0.1% to 17% of the genomic DNA in the different tumors investigated. Our findings suggest that a region in exon 19 is highly unstable in a large proportion of patients carrying EGFR deletions. As a corollary to this study, NGS data were compared with those obtained by immunohistochemistry using the 6B6 anti-mutant EGFR antibody. The immunoreaction was E746-A750del specific. In conclusion, NGS analysis of EGFR exon 19 in NSCLCs allowed us to formulate a new interpretative hypothesis for complex mutations and revealed the presence of subpopulations of deletions with potential pathogenetic and clinical impact.

BackgroundFrequent somatic mutations have recently been identified in the ras-like domain of the heterotrimeric G protein alpha-subunit (GNAQ) in blue naevi 83%, malignant blue naevi (50%) and ocular melanoma of the uvea (46%). The mutations exclusively affect codon 209 and result in GNAQ constitutive activation which, in turn, acts as a dominant oncogene.MethodologyTo assess if the mutations are present in other tumor types we performed a systematic mutational profile of the GNAQ exon 5 in a panel of 922 neoplasms, including glioblastoma, gastrointestinal stromal tumors (GIST), acute myeloid leukemia (AML), blue naevi, skin melanoma, bladder, breast, colorectal, lung, ovarian, pancreas, and thyroid carcinomas.Principal findingsWe detected the previously reported mutations in 6/13 (46%) blue naevi. Changes affecting Q209 were not found in any of the other tumors. Our data indicate that the occurrence of GNAQ mutations display a unique pattern being present in a subset of melanocytic tumors but not in malignancies of glial, epithelial and stromal origin analyzed in this study.

Abstract Tissues and cells of hibernating mammals undergo striking seasonal modifications of their activity through a quiescence-reactivation cycle. During winter, the temperature drastically decreases, the cell timing greatly slows down, the mitotic index sharply falls, DNA, RNA and protein synthesis are drastically reduced; however, upon arousal, all metabolic and physiological activities are quickly restored at the euthermic levels. The physiological, biochemical and behavioural aspects of hibernation have been extensively studied, but data on the morpho-functional relationships of cell and tissue components during the euthermia-hibernation-arousal cycle are rare. In this review, an overview of cell and tissue structural modifications so far reported in hibernating dormice is given and the possible role in the adaptation to the hypometabolic state as well as in the rapid resumption of activities upon arousal is discussed. Riassunto Modificazioni strutturali di cellule e tessuti in Gliridi ibernanti I tessuti e le cellule dei mammiferi ibernanti subiscono profonde modificazioni stagionali della loro attività attraverso un ciclo di quiescenza-riattivazione. Durante l'inverno, la temperatura corporea si abbassa a valori vicini a quelli ambientali, il ciclo cellulare rallenta, l'indice mitotico si riduce notevolmente e la sintesi di DNA, RNA e proteine è drasticamente ridotta. Tuttavia, al risveglio, tutte le attività metaboliche e fisiologiche sono rapidamente ristabilite ai livelli eutermici. Mentre gli aspetti fisiologici, biochimici e comportamentali dell'ibernazione sono stati ampiamenti studiati, i dati sulle relazioni morfo-funzionali dei componenti cellulari e tessutali durante il ciclo eutermia-ibernazione-risveglio sono piuttosto rari. In questo articolo vengono riassunte le attuali conoscenze sulle modificazioni strutturali di cellule e tessuti nei Gliridi ibernanti e viene discusso il loro possibile ruolo nell'adattamento allo stato ipometabolico e nel rapido ripristino delle attività al risveglio.

Abstract In this paper the main Italian Pleistocene mammalofaunas are examined and a chronological sequence of the main deposits is proposed. Centers of spreading, times of first appearence in Italy and ranges through the peninsula of the more representative species are indicated, as far as possible. The insular faunas and the different degrees of endemism they show, are particularly discussed. Riassunto Vengono esaminate sinteticamente le principali faune a mammiferi del Pleistocene d'Italia e viene proposta una successione cronologica per i principali giacimenti. Per le specie più rappresentative vengono indicati, per quanto possibile, le aree di provenienza, il momento della comparsa e la loro diffusione nella penisola. Particolare attenzione viene posta alle forme insulari ed al loro carattere endemico.