Co-fibrillogenesis of Wild-type and D76N β2-Microglobulin: THE CRUCIAL ROLE OF FIBRILLAR SEEDS*

The amyloidogenic variant of β₂-microglobulin, D76N, can readily convert into genuine fibrils under physiological conditions and primes in vitro the fibrillogenesis of the wild-type β₂-microglobulin. By Fourier transformed infrared spectroscopy, we have demonstrated that the amyloid transformation of wild-type β₂-microglobulin can be induced by the variant only after its complete fibrillar conversion. Our current findings are consistent with preliminary data in which we have shown a seeding effect of fibrils formed from D76N or the natural truncated form of β₂-microglobulin lacking the first six N-terminal residues. Interestingly, the hybrid wild-type/variant fibrillar material acquired a thermodynamic stability similar to that of homogenous D76N β₂-microglobulin fibrils and significantly higher than the wild-type homogeneous fibrils prepared at neutral pH in the presence of 20% trifluoroethanol. These results suggest that the surface of D76N β₂-microglobulin fibrils can favor the transition of the wild-type protein into an amyloid conformation leading to a rapid integration into fibrils. The chaperone crystallin, which is a mild modulator of the lag phase of the variant fibrillogenesis, potently inhibits fibril elongation of the wild-type even once it is absorbed on D76N β₂-microglobulin fibrils.     

Cytokine expression and ultrastructural alterations in fresh-frozen,freeze-dried and γ-irradiated human amniotic membranes

The aim of this work was to compare the effects on human amniotic membrane of freeze-drying and γ-irradiation at doses of 10, 20 and 30 kGy, with freezing. For this purpose, nine cytokines (interleukin 10, platelet-derived growth factor-AA, platelet-derived growth factor-BB, basic fibroblast growth factor, epidermal growth factor, transforming growth factor beta 1, and tissue inhibitors of metalloproteinase-1, -2, and -4) were titrated in 5 different preparations for each of 3 amniotic membranes included in the study. In addition, the extracellular matrix structure of each sample was assessed by transmission electron microscopy. While freeze-drying did not seem to affect the biological structure or cytokine content of the different amniotic membrane samples, γ-irradiation led to a significant decrease in the tissue inhibitors of metalloproteinase-4, basic fibroblast growth factor and epidermal growth factor, and induced structural damage to the epithelium, basement membrane and lamina densa. The higher the irradiation dose the more severe the damage to the amniotic membrane structure. In conclusion, the Authors recommend processing amniotic membrane under sterile conditions to guarantee safety at every step rather than final sterilization with γ-irradiation, thereby avoiding alteration to the biological characteristics of the amniotic membrane.     

Clinical, cytogenetic and molecular evaluation in a dog with bilateral cryptorchidism and hypospadias

The aim of this study was to estimate prognostic factors in a Dalmatian dog with bilateral cryptorchidism and hypospadias. Cytogenetic and molecular analyses revealed a normal karyotype (2n = 78,XY) and the presence of SRY, INSL3 and RXFP2 genes with a normal DNA sequence for SRY and RXFP2, while the INSL3 sequence differed slightly from the normal one due to a heterozygous nucleotide change involving amino acid 22 of the INSL3 dog precursor protein. Levels of plasmatic testosterone were only 0.01 ng/ml, while FSH and LH serum levels were not detectable. After the human chorionic gonadotropin (hCG) test, the serum testosterone level was 0.01 ng/ml. Therefore, the phenotypic aetiology of this subject can not be well-defined because cryptorchidism and hypospadias were frequent clinical features with high genetic heterogeneity.     

The anatomy and ultrastructure of the suctorial organ of Solifugae (Arachnida)

Solifugae possess an evertable, adhesive pedipalpal organ (suctorial organ) at the tip of the distal tarsus of each pedipalp that is unique among arachnids. When inverted inside the pedipalp, the suctorial organ is covered with two cuticular lips, a dorsal upper lip and a ventral lower lip, but it can be protruded rapidly in order to facilitate grasping prey or climbing on bushes or even climbing on smooth surfaces due to its remarkable adhesive properties. In this study, the suctorial organs of different species from old world families Galeodidae and Karschiidae and new world families Ammotrechidae and Eremobatidae were investigated by means of light microscopy, scanning and transmission electron microscopy. In all representatives, the suctorial organ is formed by an evertable, cuticular pad with a complex internal stabilizing structure. The procuticle of this pad consists of a lattice-like basal plate and numerous stalked structures connected to this basal plate. The shafts of the stalked structures are regularly organized and ramify apically. The surface of the suctorial organ is constituted of a very thin epicuticle overlaying the ramifying apices forming ridges and furrows on the ventral side of the suctorial organ.     

Chelicerae as male grasping organs in scorpions: sexual dimorphism and associated behaviour

Specialised structures that enable males to grasp females during sexual interactions are highly susceptible to selection and thus diverge relatively rapidly over evolutionary time. These structures are often used to test hypotheses regarding sexual selection such as sexually antagonistic co-evolution and sexual selection by female choice. In the present study, we determine whether there is a relationship between a novel record of scorpion sexual dimorphism, the sexual dimorphism of chelicerae (CSD), and the presence of the mating behaviour termed “cheliceral grip” (CG). The presence of both traits in the order Scorpiones is also reviewed from a phylogenetic perspective. The results confirm a strong relationship between CSD and the presence of CG. The morphological and behavioural patterns associated with “CSD–CG” are opposed to the predictions postulated by the hypothesis of sexually antagonistic co-evolution. However, if the female shows resistance after the deposition of the spermatophore, the possibility that the male exerts pressure as a “cryptic form” of coercion to prevent the interruption of mating cannot be ruled out completely. Female choice by “mechanical fit” could be another explanation for some aspects of the CG's contact zone. The possibility that the “CG–CSD” complex has evolved under natural selection in order to ensure sperm transfer is also considered.     

Development and psychometric testing of a theory-based tool to measure self-care in diabetes patients: the Self-Care of Diabetes Inventory

Background

Self-care is essential for patients with diabetes mellitus. Both clinicians and researchers must be able to assess the quality of that self-care. Available tools have various limitations and none are theoretically based. The aims of this study were to develop and to test the psychometric properties of a new instrument based on the middle range-theory of self-care of chronic illness: the Self-Care of Diabetes Inventory (SCODI).

Methods

Forty SCODI items (5 point Likert type scale) were developed based on clinical recommendations and grouped into 4 dimensions: self-care maintenance, self-care monitoring, self-care management and self-care confidence based on the theory. Content validity was assessed by a multidisciplinary panel of experts. A multi-centre cross-sectional study was conducted in a consecutive sample of 200 type 1 and type 2 diabetes patients. Dimensionality was evaluated by exploratory factor analyses. Multidimensional model based reliability was estimated for each scale. Multiple regression models estimating associations between SCODI scores and glycated haemoglobin (HbA1c), body mass index, and diabetes complications, were used for construct validity.

Results

Content validity ratio was 100%. A multidimensional structure emerged for the 4 scales. Multidimensional model-based reliabilities were between 0.81 (maintenance) and 0.89 (confidence). Significant associations were found between self-care maintenance and HbA1c (p?=?0.02) and between self-care monitoring and diabetes complications (p?=?0.04). Self-care management was associated with BMI (p?=?0.004) and diabetes complications (p?=?0.03). Self-care confidence was a significant predictor of self-care maintenance, monitoring and management (all p?<?0.0001).

Conclusion

The SCODI is a valid and reliable theoretically-grounded tool to measure self-care in type 1 and type 2 DM patients.

     

Incidence of X-Y aneuploidy in sperm of two indigenous cattle breeds by using dual color fluorescent in situ hybridization (FISH)

The present study reports on the incidence of X-Y aneuploidy in the sperm population of two indigenous cattle breeds reared in Italy for beef purposes, the Podolian and Maremmana. Totally, more than 50 000 sperm nuclei from 10 subjects (5 from each breed) have been fluorescent in situ hybridization (FISH) analyzed by using Xcen- and Y-chromosome-specific painting probes. In both breeds, the fraction of Y-bearing sperm was significantly higher (P < 0.01) compared with the X-counterpart. The rates of X-Y aneuploidy were 0.180% and 0.200%, respectively, in the Podolian and Maremmana. No significant interindividual differences were found. Average frequencies of disomic and diploid sperm were 0.149% and 0.031% in the former and 0.098% and 0.102% in the latter. Significant differences (P < 0.05) were found among the XX-XY and YY-disomy classes in both breeds, while diploidy classes were uniformly represented. In the Podolian breed, disomies were more frequent than diploidies (P < 0.05), whereas in the Maremmana they showed similar frequencies. In both breeds disomies arising from errors in meiosis I (X-Y disomies) were more represented than those arising in meiosis II (XX and YY), while this difference was not detected for diploidies. The present study provides specific information on the incidence of X-Y sperm aneuploidy in two indigenous breeds of cattle, in order to establish a breed-specific ‘aneuploidy data-base' that could be used as reference for genetic improvement and future monitoring of the reproductive health of the breed.     

Biophysical Characterization of Met-G-CSF: Effects of Different Site-Specific Mono-Pegylations on Protein Stability and Aggregation

The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation. In particular, we found that the two mono-pegylations of Met-G-CSF at the N-terminal methionine and at glutamine 135 increase the protein thermal stability, reduce the aggregation propensity, preventing also protein precipitation, as revealed by circular dichroism (CD), Fourier transform infrared (FTIR), intrinsic fluorescence spectroscopies and dynamic light scattering (DLS). Interestingly, the two pegylation strategies were found to drastically reduce the polydispersity of Met-G-CSF, when incubated under conditions favouring protein aggregation, as indicated by DLS measurements. Our in vitro results are in agreement with preclinical studies, underlining that preliminary biophysical analyses, performed in the early stages of the development of new biopharmaceutical variants, might offer a useful tool for the identification of protein variants with improved therapeutic values.