Comparative cytogenetics of three species of Dichotomius (Coleoptera, Scarabaeidae)

Meiotic and mitotic chromosomes of Dichotomius nisus, D. semisquamosus and D. sericeus were analyzed after conventional staining, C-banding and silver nitrate staining. In addition, Dichotomius nisus and D. semisquamosus chromosomes were also analyzed after fluorescent in situ hybridization (FISH) with an rDNA probe. The species analyzed had an asymmetrical karyotype with 2n = 18 and meta-submetacentric chromosomes. The sex determination mechanism was of the Xyp type in D. nisus and D. semisquamosus and of the Xy r type in D. sericeus. C-banding revealed the presence of pericentromeric blocks of constitutive heterochromatin (CH) in all the chromosomes of the three species. After silver staining, the nucleolar organizer regions (NORs) were located in autosomes of D. semisquamosus and D. sericeus and in the sexual bivalent of D. nisus. FISH with an rDNA probe confirmed NORs location in D. semisquamosus and in D. nisus. Our results suggest that chromosome inversions and fusions occurred during the evolution of the group

Molecular and clinical studies in five index cases with novel mutations in the GLA gene

Fabry disease is a metabolic and lysosomal storage disorder caused by the functional defect of the α-galactosidase A enzyme; this defect is due to mutations in the GLA gene, that is composed of seven exons and is located on the long arm of the X-chromosome (Xq21–22). The enzymatic deficit is responsible for the accumulation of glycosphingolipids in lysosomes of different cellular types, mainly in those ones of vascular endothelium. It consequently causes a cellular and microvascular dysfunction. In this paper, we described five novel mutations in the GLA gene, related to absent enzymatic activity and typical manifestations of Fabry disease. We identified three mutations (c.846_847delTC, p.E341X and p.C382X) that lead to the introduction of a stop codon in positions 297, 341 and 382. Moreover we found a missense mutation (p.R227P) in the exon 5 of the GLA gene and a single point mutation (c.639 + 5 G > T) occurring five base pairs beyond the end of the exon 4. These mutations have never been found in our group of healthy control subjects > 2300. The studied patients presented some clinical manifestations, such as cornea verticillata, hypo-anhidrosis, left ventricular hypertrophy, cerebrovascular disorders and renal failure, that, considering the null enzymatic activity, suggest that the new mutations reported here are related to the classic form of Fabry disease. The identification of novel mutations in patients with symptomatology referable to FD increases the molecular knowledge of the GLA gene and it gives clinicians an important support for the proper diagnosis of the disease

Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes

Cytogenetic analysis of Astylus antis using mitotic and meiotic cells was performed to characterize the haploid and diploid numbers, sex determination system, chromosome morphology, constitutive heterochromatin distribution pattern and chromosomes carrying nucleolus organizer regions (NORs). Analysis of spermatogonial metaphase cells revealed the diploid number 2n = 18, with mostly metacentric chromosomes. Metaphase I cells exhibited 2n = 8II+Xyp and a parachute configuration of the sex chromosomes. Spermatogonial metaphase cells submitted to C-banding showed the presence of small dots of constitutive heterochromatin in the centromeric regions of nearly all the autosomes and on the short arm of the X chromosome (Xp), as well as an additional band on one of the arms of pair 1. Mitotic cells submitted to double staining with base-specific fluorochromes (DAPI-CMA3 ) revealed no regions rich in A+T or G+C sequences. Analysis of spermatogonial mitotic cells after sequential Giemsa/AgNO 3 staining did not reveal any specific mark on the chromosomes. Meiotic metaphase I cells stained with silver nitrate revealed a strong impregnation associated to the sex chromosomes, and in situ hybridization with an 18S rDNA probe showed ribosomal cistrons in an autosomal bivalent

EpCAM^high and EpCAM^low circulating tumor cells in metastatic prostate and breast cancer patients.

The presence of high expressing epithelial cell adhesion molecule (EpCAMhigh) circulating tumor cells (CTC) enumerated by CellSearch® in blood of cancer patients is strongly associated with poor prognosis. This raises the question about the presence and relation with clinical outcome of low EpCAM expressing CTC (EpCAMlow CTC). In the EU-FP7 CTC-Trap program, we investigated the presence of EpCAMhigh and EpCAMlow CTC using CellSearch, followed by microfiltration of the EpCAMhigh CTC depleted blood. Blood samples of 108 castration-resistant prostate cancer patients and 22 metastatic breast cancer patients were processed at six participating sites, using protocols and tools developed in the CTC-Trap program. Of the prostate cancer patients, 53% had ≥5 EpCAMhigh CTC and 28% had ≥5 EpCAMlow CTC. For breast cancer patients, 32% had ≥5 EpCAMhigh CTC and 36% had ≥5 EpCAMlow CTC. 70% of prostate cancer patients and 64% of breast cancer patients had in total ≥5 EpCAMhigh and/or EpCAMlow CTC, increasing the number of patients in whom CTC are detected. Castration-resistant prostate cancer patients with ≥5 EpCAMhigh CTC had shorter overall survival versus those with high CTC (p = 0.000). However, presence of EpCAMlow CTC had no relation with overall survival. This emphasizes the importance to demonstrate the relation with clinical outcome when presence of CTC identified with different technologies are reported, as different CTC subpopulations can have different relations with clinical outcome

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks