Cytogenetic analysis of meiotic behaviour and stability in a trigeneric hybrid (triticale x trigopiro).

Trigeneric hybrids in Triticeae may help to establish evolutionary relationships among different genomes present in the same cellular genetic background and to transfer different alien characters into cultivated wheat. In the present study, a trigeneric hybrid involving species of Triticum, Secale, and Thinopyrum was synthesized by crossing hexaploid triticale with hexaploid trigopiro. The meiotic behaviour of chromosomes belonging to different genomes was analyzed, using routine and in situ hybridization techniques in F1, F2, and F3 generations of the trigeneric hybrid. The purpose of this study was to determine the chromosome number and genomic constitution and to discuss the mechanisms involved in the stabilization of the artificial tricepiro hybrids. The chromosome number of the trigeneric F1 hybrid was 2n = 42. Between 12 and 16 bivalents were observed in the central zone of the equatorial meiotic plate and between 9 and 18 univalents were found in the periphery of the MI equatorial plate. Seven of these univalents showed hybridization signals with rye DNA. Lagging rye and non-rye chromosomes and separation of sister chromatids were found in anaphase I. Tetrads with a maximum of six micronuclei, with and without hybridization signals of rye DNA, were observed. After three generations, meiotic cells revealed the presence of 42 chromosomes and 21 bivalents in diakinesis cells. The presence of 14 rye (Secale cereale) chromosomes and the complete pairing of chromosomes in F3 hybrids suggest that rye chromosomes would be preferentially transmitted to the progeny and that an elimination mechanism would act on chromosomes of Thinopyrum and wheat D genome.

ALS-associated VRK1 R321C mutation causes proteostatic imbalance and mitochondrial defects in iPSC-derived motor neurons.

Vaccinia-related kinase 1 (VRK1) is a gene which has been implicated in the pathological process of a broad range of neurodevelopmental disorders as well as neuropathies, such as Amyotrophic Lateral Sclerosis (ALS). Here we report a family presenting ALS in an autosomal recessive mode of inheritance, segregating with a homozygous missense mutation located in VRK1 gene (p.R321C; Arg321Cys). Proteomic analyses from iPSC-derived motor neurons identified 720 proteins eligible for subsequent investigation, and our exploration of protein profiles revealed significant enrichments in pathways such as mTOR signaling, E2F, MYC targets, DNA repair response, cell proliferation and energetic metabolism. Functional studies further validated such alterations, showing that affected motor neurons presented decreased levels of global protein output, ER stress and downregulation of mTOR signaling. Mitochondrial alterations also pointed to decreased reserve capacity and increased non-mitochondrial oxygen consumption. Taken together, our results present the main pathological alterations associated with VRK1 mutation in ALS.

Genome size in three species of Glandularia and their hybrids / Tamaño del genoma en tres especies de Glandularia y sus híbridos

In this work the relationship between genome size of Glandularia species and the meiotic configurations found in their hybrids are discussed. Glandularia incisa (Hook.) Tronc., growing in two localities of Corrientes and Córdoba provinces, Argentina, with different ecological conditions, showed inter-population variability of the 2C-value. The DNA content found in the Corrientes locality (2.41 pg) was higher than that obtained in the Córdoba locality (2.09 pg) which has more stressful environmental conditions than the former. These values are statistically different from those that were found in Glandularia pulchella (Sweet) Tronc. from Corrientes (1.43 pg) and in Glandularia perakii Cov. et Schn from Córdoba (1.47 pg). The DNA content of the diploid F1 hybrids, G. pulchella × G. incisa and G. perakii × G. incisa, differed statistically from the DNA content of the parental species, being intermediate between them. Differences in the frequency of pairing of homoeologous chromosomes were observed in the hybrids; these differences cannot be explained by differences in genome size since hybrids with similar DNA content differ significantly in their meiotic behavior. On the other hand, the differences in the DNA content between the parental species justify the presence of a high frequency of heteromorphic open and closed bivalents and univalents with different size in the hybrids.

En el presente trabajo se discute la relación entre el tamaño del genoma en especies de Glandularia y las configuraciones meióticas encontradas en sus híbridos. El valor 2C mostró variabilidad interpoblacional en muestras de Glandularia incisa (Hook.) Tronc. coleccionadas en dos localidades con diferentes condiciones ecológicas (provincias de Corrientes y Córdoba, Argentina). El contenido de ADN encontrado en Corrientes (2,41 pg) fue mayor que el obtenido en Córdoba (2,09 pg) donde se registran condiciones ambientales más estresantes. Estos valores son estadísticamente diferentes de los determinados en Glandularia pulchella (Sweet) Tronc. de Corrientes (1.43 pg) y en Glandularia perakii Cov. et Schn de Córdoba (1.47 pg). El contenido de ADN de los híbridos diploides F1, G. pulchella × G. incisa y G. perakii × G. incisa, difirió estadísticamente del contenido de ADN registrado en las especies parentales siendo intermedio entre ellas. Las diferencias observadas en la frecuencia de apareamiento de cromosomas homeólogos no pueden explicarse por diferencias en el tamaño del genoma, ya que híbridos con un contenido de ADN similar difieren significativamente en su comportamiento meiótico. Sin embargo, la diferencia en el contenido de ADN entre las especies parentales explica la presencia de una alta frecuencia de bivalentes heteromórficos tanto abiertos como cerrados y univalentes con diferentes tamaños.

Morphology, morphometry and chromatin distribution in llama sperm nuclei.

The objectives of this study were as follows: (i) to describe and evaluate the frequencies of different morphologies of llama sperm nuclei, (ii) to determine morphometric values of nuclear parameters, (iii) to describe and estimate the frequencies of different classes of chromatin distribution and (iv) to measure haploid DNA content and analyse its nuclear distribution. The study was performed using ejaculates collected from seven males, and sperm nuclei were stained with the Feulgen reaction. Normal morphology ranged from 78.36% to 93.92%, and abnormalities included short, small, large, pyriform, narrow, micro and round nuclei. Important differences in nuclei considered normal were found between some males. The following average values were obtained for each sperm nuclear morphometric parameter analysed: area 11.64 µm2 , perimeter 13.16 µm, length 5.12 µm, width 2.81 µm, ellipticity 1.85 and form 0.83. Differences between males were significant for all the parameters (p < .01). Light microscope observations and cytophotometric determinations allowed discriminating between three classes of chromatin distribution: homogeneous, diffuse and showing a clear band. Significant differences between males were found for the frequencies of the three classes (p < .01). Cluster analysis methods were used to estimate the resemblance between males according to the characteristics of their sperm nuclei. A great intermale variability was found for morphological, morphometric and chromatin distribution data. These parameters would have low dependence between them.

DNA content of Ovis musimon spermatozoa.

To our knowledge, the value of the haploid DNA content (C-value) of Ovis musimon (mouflon) has not been previously published. Therefore, the aim of the present work was to determine the C-value and the nuclear area of O. musimon sperm cells and compare both parameters with those of Ovis aries. Feulgen reaction, which is specific and stoichiometric for DNA, was carried out on semen smears. The C-value and sperm nuclear area were determined using microspectrophotometry and Gallus domesticus erythrocytes as standard species. The C-value of O. musimon was 3.02 ± 0.04 pg, and the sperm nuclear area was 23.92 ± 0.89 µm(2). The C-value and the sperm nuclear area of O. aries were 3.07 ± 0.03 pg and 22.98 ± 0.86 µm(2) respectively. The O. musimon C-value was not significantly different (P > 0.05) from that of O. aries, indicating that both species may have a very close phylogenetic relation.

Relationship between the nuclear morphology of the sperm of 10 bulls and their fertility.

The relationships between the fertility and nuclear morphology, chromatin maturity and chromatin condensation of the sperm of three bulls with a calving rate over a year of more than 65 per cent, four bulls with a calving rate between 65 per cent and 35 per cent, and three bulls with a calving rate of less than 35 per cent were studied. The sperm nuclei were stained with the Feulgen reaction, and chromatin condensation and maturation were evaluated in situ by staining with toluidine blue and acid aniline blue. Nuclear chromatin decondensation was induced with dithiothreitol; this showed that in the bulls with low fertility, more than 35 per cent of nuclei were decondensed, and that one of them had the lowest percentage of normal nuclei (64.9 per cent) and stronger positive reactions to the acid aniline blue and toluidine blue stains than the other bulls.

[Evaluation of the appropriateness of hospital use: the case of IRCCS Ospedale Maggiore di Milano, Italy]. / Valutazione di appropriatezza sull'utilizzo dell'Ospedale: il caso dell'IRCCS Ospedale Maggiore di Milano.

To measure the level of not appropriated hospital use for some DRG's a Lombardia Region AEP version was applied to two units in the Ospedale Maggiore in Milano. The aims of the study was to evaluate the level of appropriateness in Neurology and Dermatology wards for some DRG's which are at risk to be applied in not appropriated way, to study the reasons of not appropriated use, to educate medical doctors in neurology and dermatology to apply for the right level of care for they patients and to highlight some limits of the Lombardia Regional protocol.

[Evaluation of the appropriateness of hospital use: the case of IRCCS Ospedale Maggiore di Milano, Italy]. / Valutazione dell'appropriatezza dell'attività di ricovero dell'Ospedale Maggiore di Milano IRCCS mediante l'utilizzo del metodo PRUO.

In 1999 an Italian version of the AEP was applied to some units of the IRCCS Ospedale Maggiore di Milano and a modified AEP version was used to evaluate the day hospital activity. The main aims of the study were: to establish the appropriateness of the utilisation of beds and of day hospital activity and to calculate the costing of the misuse of hospital resources. 267 admission days were evaluated and 31.5% were not appropriated; whereas of 3,248 days of stay 37% were found not appropriated. The day hospital activity was evaluated through 300 record referred to 570 days of treatment and 13% of them were not appropriated. The cost of the 1,201 not appropriated hospital days was over 351.000. The not appropriated level of hospital utilisation in the IRCCS Ospedale Maggiore di Milano is similar to what is reported in national and international literature. Our study highlights some limits in the application of the AEP protocol at high specialised hospital units.

Chromatin cytophotometric analysis of abnormal bovine spermatozoa.

Sperm head morphology is basically conditioned by the nuclear structure. The aim of the present work was to study the relation between nuclear morphological features, DNA content and chromatin distribution in morphologically normal vs. abnormal bovine spermatozoa. To this end, individual Feulgen-reacted spermatozoa were cytophotometrically studied. Chromatin compactation was evaluated by means of nuclear area, as well as mean and maximal absorbance of each nucleus. Morphological abnormality analysed included large, small, pear, narrow and round shapes, together with presumably 'diploid' sperms. Both large and small spermatozoa have a DNA content that does not differ significantly from normal values, but their area and mean and maximal absorbance are significantly different. Size variation seems basically due to altered chromatin compactation. The pear shapes have a narrower neck and a significant increase in maximal absorbance alone, which is invariably recorded in the neck zone whose increase would indicate a change in distribution and/or compactation. The narrow and round shapes fail to present significant variations in studied parameters. The possible 'diploids' differ significantly from normal cells in all studied variables, with a little area increase.

Impaired circadian modulation of sympathovagal activity in diabetes. A possible explanation for altered temporal onset of cardiovascular disease.

BACKGROUND: Diabetic subjects have a high incidence of cardiovascular accidents, with an altered circadian distribution. Abnormalities in the circadian rhythm of autonomic tone may be responsible for this altered temporal onset of cardiovascular disease. METHODS AND RESULTS: To assess circadian changes of sympathovagal balance in diabetes, we performed 24-hour power spectral analysis of RR interval fluctuations in 54 diabetic subjects (age, 44 +/- 2 years) with either normal autonomic function or mild to severe autonomic neuropathy and in 54 age-matched control subjects. The power in the low-frequency (LF, 0.03-0.15 Hz) and high-frequency (HF, 0.18-0.40 Hz) bands was considered an index of relative sympathetic and vagal activity, respectively. Diabetic subjects with autonomic abnormalities showed a reduction in LF compared with control subjects (5.95 +/- 0.12 In-msec2 versus 6.73 +/- 0.11, p < 0.001) and an even greater reduction in LF, particularly during the night and the first hours after awakening (5.11 +/- 0.18 In-msec2 versus 6.52 +/- 0.14, p < 0.001). Day-night rhythm in sympathovagal balance was reduced or absent in diabetic subjects compared with control subjects. CONCLUSIONS: Diabetic subjects with or without signs of autonomic neuropathy have a decreased vagal activity (and hence a relatively higher sympathetic activity) during night hours and at the same time of the day, during which a higher frequency of cardiovascular accidents has been reported. These observations may provide insight into the increased cardiac risk of diabetic patients, particularly if autonomic neuropathy is present.

Relationship between fluctuations in heart rate and asymptomatic nocturnal ischaemia.

In order to quantify autonomic changes related to asymptomatic nocturnal myocardial ischaemia, we analyzed heart rate fluctuations recorded during Holter monitoring in 9 subjects with coronary heart disease (21 episodes) and in 11 age-matched controls. R-R interval spectral analysis was computed in sequences of 256 heart beats, taken during the ischaemic episode, 4, 8 and 60 minutes before, and 4 and 60 minutes after. Mean heart rate, R-R interval variability (assessed by R-R interval standard deviation), low and high (respiration-linked) frequency components of R-R interval spectrum were evaluated. Mean heart rate and R-R interval variability increased only during ischaemia (from 62.9 to 73.3 beats/minute, P less than 0.02, and from 39 to 88 msec, P less than 0.01, respectively). While high-frequency components of heart rate variability remained unchanged, low-frequency peak increased during ischaemia (from 9.4 to 43.3 sec2 X 10(-3)/Hz, P less than 0.01) and also 8 minutes (P less than 0.05) and 4 minutes before (P less than 0.05). Despite a moderate increase of heart rate occurring only during ischaemia, the early rearrangement of heart rate fluctuations suggests the occurrence of changes of autonomic tone before the electrocardiographic onset of ischaemia. Due to its limited amount, this phenomenon appears to be a consequence, most likely unspecific, of factors responsible for the genesis of myocardial ischaemia.