RosettaDDGPrediction for high-throughput mutational scans: From stability to binding

Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein–protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction .     

The primary structure of D-amino acid oxidase from Rhodotorula gracilis

Summary The amino acid sequence of D-amino acid oxidase from Rhodotorula gracilis was determined by automated Edman degradation of peptides generated by enzymatic and chemical cleavage. The enzyme monomer contains 368 amino acid residues and its sequence is homologous to that of other known D-amino acid oxidases. Six highly conserved regions appear to have a specific role in binding of coenzyme FAD, in active site topology and in peroxisomal targeting. Moreover, Rhodotorula gracilis D-amino acid oxidase contains a region with a cluster of basic amino acids, probably exposed to solvent, which is absent in other D-amino acid oxidases.     

Seed germination ecology and salt stress response in eight Mediterranean populations of Sarcopoterium spinosum (L.) Spach

This study aims to deepen the analysis of seed germination ecology and salinity tolerance of Sarcopoterium spinosum (Rosaceae). Germination tests were conducted to evaluate the effect of the fruit’s spongy tissue and the intraspecific variability in seed germination among eight populations of the species on responses to light and total darkness, constant and alternating temperatures, salt stress and germination recovery. The effect of the presence of the spongy tissue varied among populations, with significant results for seed germination. For all populations, optimum germination temperatures were observed in the range of 10–20°C, indicating that S. spinosum and its germination in the field might occur preferably in the period between autumn and early spring. The high water availability due to rainfall during this period could be a considerable advantage for the seed germination of this species. Seeds of S. spinosum showed the ability to germinate in up to 250 mM NaCl in the substrate, and their ability to recover after salt exposure may be interpreted as adaptation to the coastal habitats in which they generally grow. These results give this species a halo-tolerant character. Great inter-population variability is detected in this study in several aspects, which indicated that the Mediterranean populations of S. spinosum differ considerably and are adapted to their local conditions. This study provides new information about S. spinosum seed ecology, which could help to preserve and apply effective conservation measures for this species, which in several areas of its distribution range is endangered.     

Synthesis and biological evaluation of [6]-gingerol analogues as transient receptor potential channel TRPV1 and TRPA1 modulators

In order to explore the structural determinants for the TRPV1 and TRPA1 agonist properties of gingerols, a series of nineteen analogues (1b-5) of racemic [6]-gingerol (1a) was synthesized and tested on TRPV1 and TRPA1 channels. The exploration of the structure-activity relationships, by modulating the three pharmacophoric regions of [6]-gingerol, led to the identification of some selective TRPV1 agonists/desensitizers of TRPV1 channels (3a, 3f, and 4) and of some full TRPA1 antagonists (2c, 2d, 3b, and 3d).     

Gallstone disease is associated with more severe liver damage in patients with non-alcoholic Fatty liver disease

Background

Nonalcoholic fatty liver disease (NAFLD) and gallstone disease (GD) are both highly prevalent in the general population and associated with obesity and insulin resistance. We aimed to evaluate the prevalence of GD in a cross sectional study of NAFLD patients and to define whether the presence of GD is associated with diabetes and predicts more severe liver disease.

Methodology/Principal Findings

We merged databases of four Liver Units, comprising 524 consecutive biopsy-proven NAFLD (373 males) observed between January 2003 and June 2010. GD was diagnosed in 108 (20%), and 313 cases (60%) were classified by liver biopsy as nonalcoholic steatohepatitis (NASH). The GD subgroup was characterized by a significantly higher prevalence of females, prediabetes/diabetes, abdominal obesity and metabolic syndrome, older age, higher BMI, fasting glucose, HOMA-IR and lower ALT. The prevalence of GD progressively increased with advancing fibrosis and with the severity of necroinflammatory activity (p for trend  = 0.0001 and  = 0.01, respectively), without differences in the severity of steatosis. At multivariate analysis GD was associated with female gender (OR 1.37, 95% CI 1.04–1.8), age (OR 1.027, 95% CI1.003–1.05), fasting glucose (OR 1.21, 95% CI 1.10–1.33) and NASH (OR 1.40,95% CI 1.06–1.89), whereas ALT levels were associated with a lower GD risk (OR 0.98, 95% CI 0.97–0.99). When subjects with cirrhosis were excluded from analysis, the association between GD and fasting glucose, female gender, and NASH was maintained.

Conclusion

Patients with NAFLD have a high prevalence of GD, which characterizes subjects with altered glucose regulation and more advanced liver disease.     

Signal-to-noise ratio of diffusion weighted magnetic resonance imaging: Estimation methods and in vivo application to spinal cord

Diffusion tensor imaging (DTI) and tractographic reconstruction may be applied for in vivo clinical spinal cord studies. However, this structure represents a challenge to current acquisition and reconstruction strategies, due to its small size, motion artifacts, partial volume effects and low signal-to-noise-ratio (SNR). Aims of this work were to select the best approach for the estimate of SNR and to use it for spinal cord diffusion weighted (DW) sequence optimization.Seven methods for the estimate of SNR were compared on uniform phantom DW images, and the best performing approach (single ROI for signal and noise, difference of images—SNR_diff) was applied for the following in vivo sequence evaluations.Fifteen sequences with different parameters (voxel size, repetition (TR) and echo (TE) times) were compared according to SNR, resolution, fractional anisotropy (FA) and tractography performances on three healthy volunteers. In vivo optimization of DW sequences resulted in: axial sequence, with voxel size = 1.5 mm × 1.5 mm × 3.5 mm, TR = 3200 ms and TE = 89 ms, sagittal sequence with voxel size = 2.2 mm × 2.2 mm × 2 mm, TR = 3000 ms and TE = 84 ms.An objective method tested on phantom and a practical index for in vivo spinal cord DTI SNR estimation allowed to obtain axial and sagittal optimized sequences, providing excellent tractographic results, with acceptable acquisition times for in vivo clinical applications.     

Levels of uroporphyrinogen decarboxylase (URO-D) in erythrocytes of Italian porphyria cutanea tarda patients.

Porphyria cutanea tarda (PCT) is a human metabolic disorder due to the acquired or genetic impairment of uroporphyrinogen decarboxylase (URO-D) activity, the fifth enzyme of the heme biosynthetic pathway. A classification of inherited and non-inherited forms is based on the enzyme activity levels in red blood cells (RBC). Clinical manifestations of PCT are often precipitated by triggering factors such as alcohol, drug abuse, estrogens, virus infections, hepatotoxic chemicals and hepatic siderosis. We measured URO-D activity in RBC from a large sample of Italian PCT patients in order to define the enzyme activity distribution and to attempt a correlation among activity, risk factors and clinical outcome. Three classes of patients with low, normal and over-normal URO-D activity were defined according to control values. Low URO-D levels were present in 25.8% of patients, suggesting the familial form of PCT (type II). In this group, the outcome of PCT seems to be less influenced by risk factors. Patients with over-normal URO-D activity in RBC deserve further investigation.     

Guardians' Perceptions of Dogs' Welfare and Behaviors Related to Visiting the Veterinary Clinic

A large survey of Italian dog guardians (n = 906) was conducted to assess dog behavior and welfare at the veterinary clinic and to investigate how guardians and veterinarians affect them. This study confirmed that the veterinary clinic is a source of stress for most dogs, who showed impaired welfare in all phases: in the waiting room, entering the examination room, on the examination table, and when approached by the vet. This study also characterizes some factors related to the guardians' and veterinarians' behavior that affect the dogs' behavior and welfare during the veterinary examination. If dogs had not been examined by a vet since puppyhood, if they did not accept treatments by their guardians, and if they were scolded when refusing a treatment, the risk for having problems with dog welfare and behavior at the veterinary clinic increased. The attention paid by the vet to the dog was found to be positively related with a good response of the dog to the vet. Prevention seems to be the key for the protection of dog welfare related to veterinary care.