The Cochlostoma (Holcopoma) westerlundi group in Italy (Caenogastropoda: Cochlostomatidae)

Currently, the Cochlostoma (Holcopoma) westerlundi (Paulucci, 1879) group includes three subspecies inhabiting southern Italy up to southeastern Sicily. C. w. westerlundi (Paulucci, 1879) is limited to southern Calabria, C. w. yapigium (Westerlund, 1885) is widespread across the Salento (the southeastern extremity of the Apulia region) and C. w. dionysii (Paulucci, 1879) is endemic to the environs of Siracusa (SE Sicily). There is also a fourth taxon, C. paganum (Westerlund, 1885) described for Otranto (LE, Apulia), considered a synonym of C. w. yapigium. Up to now, the molecular genetics of C. westerlundi s.l. have been unknown and the morphological data of several populations are still lacking. Hence, the systematic classification of the group is tentative. Aiming at filling this gap, mtDNA (16S rDNA and COI) partial sequences were investigated and, in addition, the reproductive apparatus of C. w. westerlundi was described for the first time. Molecular sequences and anatomical data were used to test the taxonomic and phylogenetic status of the examined populations. Maximum Likelihood and Bayesian analysis revealed three clusters, strongly supported, corresponding to the three taxa. For the first time, synonymy between paganum and yapigium was confirmed by molecular evidence. Genetic distances between groups (D_xyJC) ranged from 2.6% to 5% (16S rDNA) and from 6.3% to 8.3% (COI). Molecular and morphological data led us to suggest elevating the three subspecies to the species rank.     

Molecular mechanisms generating and stabilizing terminal 22q13 deletions in 44 subjects with Phelan/McDermid syndrome

In this study, we used deletions at 22q13, which represent a substantial source of human pathology (Phelan/McDermid syndrome), as a model for investigating the molecular mechanisms of terminal deletions that are currently poorly understood. We characterized at the molecular level the genomic rearrangement in 44 unrelated patients with 22q13 monosomy resulting from simple terminal deletions (72%), ring chromosomes (14%), and unbalanced translocations (7%). We also discovered interstitial deletions between 17-74 kb in 9% of the patients. Haploinsufficiency of the SHANK3 gene, confirmed in all rearrangements, is very likely the cause of the major neurological features associated with PMS. SHANK3 mutations can also result in language and/or social interaction disabilities. We determined the breakpoint junctions in 29 cases, providing a realistic snapshot of the variety of mechanisms driving non-recurrent deletion and repair at chromosome ends. De novo telomere synthesis and telomere capture are used to repair terminal deletions; non-homologous end-joining or microhomology-mediated break-induced replication is probably involved in ring 22 formation and translocations; non-homologous end-joining and fork stalling and template switching prevail in cases with interstitial 22q13.3. For the first time, we also demonstrated that distinct stabilizing events of the same terminal deletion can occur in different early embryonic cells, proving that terminal deletions can be repaired by multistep healing events and supporting the recent hypothesis that rare pathogenic germline rearrangements may have mitotic origin. Finally, the progressive clinical deterioration observed throughout the longitudinal medical history of three subjects over forty years supports the hypothesis of a role for SHANK3 haploinsufficiency in neurological deterioration, in addition to its involvement in the neurobehavioral phenotype of PMS.     

A novel approach of homozygous haplotype sharing identifies candidate genes in autism spectrum disorder

Autism spectrum disorder (ASD) is a highly heritable disorder of complex and heterogeneous aetiology. It is primarily characterized by altered cognitive ability including impaired language and communication skills and fundamental deficits in social reciprocity. Despite some notable successes in neuropsychiatric genetics, overall, the high heritability of ASD (~90%) remains poorly explained by common genetic risk variants. However, recent studies suggest that rare genomic variation, in particular copy number variation, may account for a significant proportion of the genetic basis of ASD. We present a large scale analysis to identify candidate genes which may contain low-frequency recessive variation contributing to ASD while taking into account the potential contribution of population differences to the genetic heterogeneity of ASD. Our strategy, homozygous haplotype (HH) mapping, aims to detect homozygous segments of identical haplotype structure that are shared at a higher frequency amongst ASD patients compared to parental controls. The analysis was performed on 1,402 Autism Genome Project trios genotyped for 1 million single nucleotide polymorphisms (SNPs). We identified 25 known and 1,218 novel ASD candidate genes in the discovery analysis including CADM2, ABHD14A, CHRFAM7A, GRIK2, GRM3, EPHA3, FGF10, KCND2, PDZK1, IMMP2L and FOXP2. Furthermore, 10 of the previously reported ASD genes and 300 of the novel candidates identified in the discovery analysis were replicated in an independent sample of 1,182 trios. Our results demonstrate that regions of HH are significantly enriched for previously reported ASD candidate genes and the observed association is independent of gene size (odds ratio 2.10). Our findings highlight the applicability of HH mapping in complex disorders such as ASD and offer an alternative approach to the analysis of genome-wide association data.     

Pentosidine plasma levels and relation with metabolic control in diabetic patients.

Levels of plasma pentosidine, a well-known AGE, were measured in type 2 diabetic patients in varying states of metabolic control to verify possible relationships between this parameter and traditional metabolic control parameters such as HbA1c and plasma glucose levels. At baseline, mean values of fasting plasma glucose, HbA1c and pentosidine were significantly higher in diabetic patients than those of controls, confirming patients' poor glycemic control. After ten months, patients with good metabolic control achieved showed near-normal HbA1c levels and reduced but not normalized pentosidine levels. Significant differences were found in the mean percentage decrease in the parameters. Regarding linear correlation, HbA1c levels only showed a positive relationship with plasma glucose values at baseline. Patients affected by chronic complications showed higher levels of pentosidine than those without complications. Thus, pentosidine plasma levels may be used to evaluate very long-term metabolic control in diabetic patients. In addition, a period of ten months of acceptable metabolic control is not enough to normalize pentosidine levels in diabetics, thus emphasizing the need for a longer period of improved metabolic control to reduce both this parameter and the burden of chronic diabetic complications.     

Desaturase activities and metabolic control in type 2 diabetes

The aim of this study was to elucidate the effects of a poor glycemic control on fatty acid composition and desaturase activities in type 2 diabetic patients. Plasma phospholipid fatty acid composition and desaturase activities (estimated from fatty acid product to precursor ratios) were measured in 30 type 2 diabetic patients during poor metabolic control and after achieving a good metabolic control. Significant changes were recorded in the percentages of palmitic, stearic, dihomo-gamma-linolenic, docosatetraenoic and docosapentaenoic acid. The delta-5 desaturase activity was significantly higher with poor than with good metabolic control. The changes identified in plasma phospholipid fatty acid composition and the desaturase activity in type 2 diabetic patients go in the opposite direction to those described in similar conditions in type 1 diabetic patients and may be relevant to a better understanding of the role of metabolic control in the progression of chronic complications in type 2 diabetic patients.     

Niche-independent symmetrical self-renewal of a mammalian tissue stem cell

Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.     

Late Carnian (Tuvalian,Tropites dilleri zone) ostracods (Crustacea) from the Mufara Formation (Monte Scalpello,Central-Eastern Sicily,Italy)

Ostracod association from the Upper Triassic (Tropites dilleri zone of the Carnian stage) of the sedimentary succession (Mufara Formation) exposed along the east side of Monte Scalpello (Catenanuova, central eastern Sicily) has been studied for the first time. The specimens, silicified, are rare but well preserved and often consist of complete carapaces. They belong to eight families: Healdiidae, Cavellinidae, Bairdiidae, Acratiidae, Bythocyprididae, Pontocyprididae, Judahellidae, Glorianellidae. Twenty-three taxa have been listed; the family Bairdiidae is the most represented among all the others with fifteen species. Four species are new: Bairdia scaliae n. sp., Acratia maugerii n. sp., “Anchistrocheles” gemmellaroi n. sp. and Judahella? montanarii n. sp. Other species are left in open nomenclature because of the lack of specimens.     

In vitro metabolism of vitamin D3 by isolated liver cells

Summary Liver cells were prepared from rats fed a rachitogenic diet to investigate the hepatic metabolism of [ — 1,2 —³H₂] vitamin D₃. Rat hepatocytes suspended in Hanks medium rapidly took up labeled vitamin D₃ from the incubation medium and converted this sterol to various metabolites, including 25-hydroxy vitamin D₃ (25-OH-D₃). There was a steady increment in the cellular production of 25-OH-D₃ and of the more polar metabolites of vitamin D₃ over 3 hr of incubation as determined by thin layer chromatography. Neither the addition of cyclic nucleotides or dexamethasone to, nor the removal of calcium or phosphate from the medium resulted in changes in the rate of conversion of vitamin D₃ to its products. Rats pretreated with sodium diphenylhydantoin converted labeled vitamin D₃ to its metabolites at the same rate as control rats. These data indicate that isolated liver cells retain the capacity for vitamin D₃ hydroxylation, but suggest that the rate of this process does not undergo rapid changes in response to metabolic stimulation.Recipient of Research Career Development Award 1 K04 HL-00089.     

Innate immune dysfunction in HIV infection: effect of HIV envelope-NK cell interactions

We have previously described a number of NK cell dysfunctions in HIV-viremic individuals. In the present study, we performed DNA microarray analysis followed by phenotypic and functional characterization in an effort to investigate which HIV envelope glycoproteins (gp120) affect the physiologic functions of NK cells. Upon treatment of NK cells with HIV gp120, DNA microarray analyses indicated up-regulation of several categories of genes that are associated with apoptosis, suppression of both cellular proliferation and survival, as well as down-regulation of genes that play a vital role in cell proliferation, innate immune defense mechanism, and cell survival. Both subtypes of gp120 suppressed NK cell cytotoxicity, proliferation, and the ability to secrete IFN-gamma. NK cells exposed to X4-subtype HIV gp120 showed a significant decrease in the levels of CC chemokines, while exposure to R5-subtype HIV gp120 had minimal effect. Extended exposure to HIV gp120 resulted in apoptosis of NK cells, further validating the microarray data. Our data demonstrate that exposure of NK cells to HIV envelope proteins results in profound cellular abnormalities at the level of gene expression as well as generic cell functions. These findings are likely to be a consequence of a direct HIV gp120-mediated effect on NK cells. Identification of specific surface receptors on NK cells that interact with HIV envelope proteins might explain how HIV is capable of circumventing innate immune defense mechanisms and establishing infection in susceptible individuals.