Identifying pathological biomarkers: histochemistry still ranks high in the omics era

In recent years, omic analyses have been proposed as possible approaches to diagnosis, in particular for tumours, as they should be able to provide quantitative tools to detect and measure abnormalities in gene and protein expression, through the evaluation of transcription and translation products in the abnormal vs normal tissues. Unfortunately, this approach proved to be much less powerful than expected, due to both intrinsic technical limits and the nature itself of the pathological tissues to be investigated, the heterogeneity deriving from polyclonality and tissue phenotype variability between patients being a major limiting factor in the search for unique omic biomarkers. Especially in the last few years, the application of refined techniques for investigating gene expression in situ has greatly increased the diagnostic/prognostic potential of histochemistry, while the progress in light microscopy technology and in the methods for imaging molecules in vivo have provided valuable tools for elucidating the molecular events and the basic mechanisms leading to a pathological condition. Histochemical techniques thus remain irreplaceable in pathologist's armamentarium, and it may be expected that even in the future histochemistry will keep a leading position among the methodological approaches for clinical pathology

Interfacial properties of most monofluorinated bile acids deviate markedly from the natural congeners: studies with the Langmuir-Pockels surface balance

We characterized the air-water interfacial properties of four monofluorinated bile acids alone and in binary mixtures with a common lecithin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), using an automated Langmuir-Pockels surface balance. We compared 7alpha-fluoromurocholic acid (FMCA), 7alpha-fluorohyodeoxycholic acid (FHDCA), 6alpha-fluoroursodeoxycholic acid (FUDCA), and 6alpha-fluorochenodeoxycholic acid (FCDCA) with their natural dihydroxy homologs, murocholic acid (MCA), hyodeoxycholic acid (HDCA), ursodeoxycholic acid (UDCA), and chenodeoxycholic acid (CDCA). For further comparison, two trihydroxy bile acids, 3alpha,6beta,7alpha-trihydroxycholanoic acid [alpha-muricholic acid (alpha-MCA)] and 3alpha,6alpha,7beta-trihydroxycholanoic acid [omega-muricholic acid (omega-MCA)], with isologous OH polar functions to FMCA and FUDCA were also studied. Pressure-area isotherms of MCA, HDCA, UDCA, CDCA, and FMCA displayed sharp collapse points. In contrast, FHDCA, FUDCA, and FCDCA formed monolayers that were less stable than the trihydroxy bile acids, displaying second-order phase transitions in their isotherms. All natural and fluorinated bile acids condensed mixed monolayers with POPC, with maximal effects at molar bile acid concentrations between 30 and 50 mol%. Examination of molecular models revealed that the 7alpha-F atom of the interfacially stable FMCA projects away from the 6beta-OH function, resulting in minimal steric interactions, whereas in FHDCA, FUDCA, and FCDCA, close vicinal interactions between OH and F polar functions result in progressive bulk solubility upon monolayer compression. These results provide a framework for designing F-modified bile acids to mimic or diverge from the natural compounds in vivo

Rasch analysis of the Fatigue Severity Scale in Italian subjects with multiple sclerosis.

To perform a psychometric analysis of the Fatigue Severity Scale (FSS) using Rasch analysis in a sample of Italian subjects with multiple sclerosis

Routinely frozen biopsies of human skeletal muscle are suitable for morphological and immunocytochemical analyses at transmission electron microscopy

The aim of the present investigation was to evaluate whether routinely frozen biopsies of human skeletal muscle may be suitable for morphological and immunocytochemical analyses at transmission electron microscopy. The fixation/embedding protocols we successfully used for decades to process fresh mammalian tissues have been applied to frozen muscle biopsies stored for one to four years in liquid nitrogen. After 2.5% glutaraldehyde -2% paraformaldehyde - 1% OsO4 fixation and embedding in epoxy resin, the ultrastructural morphology of myofibres and satellite cells as well as of their organelles and inclusions proved to be well preserved. As expected, after 4% paraformaldehyde - 0.5% glutaraldehyde fixation and embedding in LR White resin, the morphology of membrane-bounded organelles was relatively poor, although myofibrillar and sarcomeric organization was still recognizable. On the contrary, the myonuclei were excellently preserved and, after conventional staining with uranyl acetate, showed an EDTA-like effect, i.e. the bleaching of condensed chromatin, which allows the visualization of RNP-containing structures. These samples proved to be suitable for immunocytochemical analyses of both cytoskeletal and nuclear components, whereas the poor mitochondrial preservation makes unreliable any in situ investigation on these organelles

Cultured myoblasts from patients affected by myotonic dystrophy type 2 exhibit senescence-related features: ultrastructural evidence

Myotonic dystrophy type 2 (DM2) is an autosomal dominant disorder caused by the expansion of the tetranucleotidic repeat (CCTG)n in the first intron of the Zinc Finger Protein-9 gene. In DM2 tissues, the expanded mutant transcripts accumulate in nuclear focal aggregates where splicing factors are sequestered, thus affecting mRNA processing. Interestingly, the ultrastructural alterations in the splicing machinery observed in the myonuclei of DM2 skeletal muscles are reminiscent of the nuclear changes occurring in age-related muscle atrophy. Here, we investigated in vitro structural and functional features of satellite cell-derived myoblasts from biceps brachii, in the attempt to investigate cell senescence indices in DM2 patients by ultrastructural cytochemistry. We observed that in satellite cell-derived DM2 myoblasts, cell-senescence alterations such as cytoplasmic vacuolization, reduction of the proteosynthetic apparatus, accumulation of heterochromatin and impairment of the pre-mRNA maturation pathways occur earlier than in myoblasts from healthy patients. These results, together with preliminary in vitro observations on the early onset of defective structural features in DM2 myoblast derived-myotubes, suggest that the regeneration capability of DM2 satellite cells may be impaired, thus contributing to the muscular dystrophy in DM2 patients

Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP)-containing components (PANA, hnRNP-core proteins, fibrillarin) or RNP-associated nuclear proteins (SC-35 splicing factor). Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures

Investigation of marmoset hybrids (Cebuella pygmaea x Callithrix jacchus) and related Callitrichinae (Platyrrhini) by cross-species chromosome painting and comparative genomic hybridization

We report on the cytogenetics of twin offspring from an interspecies cross in marmosets (Callitrichinae, Platyrrhini), resulting from a pairing between a female Common marmoset (Callithrix jacchus, 2n = 46) and a male Pygmy marmoset (Cebuella pygmaea, 2n = 44). We analyzed their karyotypes by multi-directional chromosome painting employing human, Saguinus oedipus and Lagothrix lagothricha chromosome-specific probes. Both hybrid individuals had a karyotype with a diploid chromosome number of 2n = 45. As a complementary tool, interspecies comparative genomic hybridization (iCGH) was performed in order to screen for genomic imbalances between the hybrids and their parental species, and between Callithrix argentata and S. oedipus, respectively. Copyright (C) 2005 S. Karger AG, Basel

The Golgi apparatus is a primary site of intracellular damage after photosensitization with Rose Bengal acetate

The aim of the present investigation was to elucidate whether the Golgi apparatus undergoes photodamage following administration of the fluorogenic substrates Rose Bengal acetate (RBAc) and irradiation at the appropriate wavelength. Human HeLa cells were treated in culture and the changes in the organization of the Golgi apparatus were studied using fluorescence confocal microscopy and electron microscopy, after immunocytochemical labeling. To see whether the cytoskeletal components primarily involved in vescicle traffic (i.e., microtubules) might also be affected, experiments of tubulin immunolabeling were performed. After treatment with RBAc and irradiation, cells were allowed to grow in drug-free medium for different times. 24hr after irradiation, the cisternae of the Golgi apparatus became packed, and after 48-72 hr they appeared more fragmented and scattered throughout the cytoplasm; these changes in the organization of the Golgi cisternae were confirmed at electron microscopy. Interestingly enough, apoptosis was found to occur especially 48-72h after irradiation, and apoptotic cells exhibited a dramatic fragmentation of the Golgi membranes. The immunolabeling with anti-tubulin antibody showed that microtubules were also affected by irradiation in RBAc-treated cells