Redistribution of DNA topoisomerase II beta after in vitro stabilization of human erythroleukemic nuclei by heat or Cu++ revealed by confocal microscopy.

Using confocal laser scanning microscope and a monoclonal antibody we have examined by means of indirect immunofluorescence techniques the distribution of DNA topoisomerase II beta (the 180-kDa nucleolar isoform of topoisomerase II) following stabilization of isolated nuclei by exposure to moderate heat (37 degrees or 42 degrees C) or Cu++. In intact cells the antibody specifically decorated the nucleoli. The same pattern was maintained if nuclei were incubated at 0 degree C in a buffer containing spermine/spermidine/KCl or stabilized by means of 0.5 mM Cu++ for 10 minutes at 0 degree C in the same buffer. On the contrary, if stabilization was performed by incubating the nuclei either at 37 degrees or 42 degrees C, the immunoreactivity dispersed all over the nucleus, forming numerous speckles. This phenomenon was not detected if, in addition to spermine/spermidine/KCl, the incubation buffer also contained 5 mM Mg++ and the temperature was 37 degrees C. If the stabilization was performed at 42 degrees C, Mg++ failed to maintain the original distribution of DNA topoisomerase II beta, as seen in intact cells. The analysis on 2-D optical section showed the alteration of the nucleolar profile, particularly at 37 degrees C, even when the samples were treated with Mg++. The 3-D reconstruction figured out the irregularity of the surface at 37 degrees C and the variations of the volume occupied by the fluorescent figures. These were in close proximity to each other both in intact cells and in 0 degree C incubated nuclei; they showed a certain degree of shrinkage in 0 degree C plus Cu++ exposed samples (-20\% of the volume), and, on the contrary, the labeled structures were scattered in a volume increased two- or threefold when exposed to 37 degrees or 42 degrees C, respectively. The addition of Mg++ restored the original spatial relationship and volume at 37 degrees C, but not at 42 degrees C, where the volumetric analysis showed an increase of about 50\%. Our results demonstrate that heat stabilization of isolated nuclei in a buffer without Mg++ (i.e., a technique often employed to prepare the nuclear matrix or scaffold) cannot be considered an optimal procedure to maintain the original distribution of protein within the nucleus

Outlining the mission profile of agricultural tractors through CAN-BUS data analytics

Tractor manufacturers need to know how farmers use their agricultural tractors for an optimal machine design. Tractor usage is not easy to assess due to the large variability of field operations. However, modern tractors embed sensors integrated into the CAN-BUS network and their data is accessible through the ISO 11,783 protocol. Even though this technology has been available for a long time, the use of CAN-BUS data for outlining the tractor usage is still limited, because a proper post-processing method is lacking. This study aimed to present a novel classification scheme of CAN-BUS data which permits to outline the tractor usage. On a tractor, a CAN-BUS data logger and a GNSS receiver were installed, and real-world data were recorded for 579 h. Thus, data was obtained in the most realistic condition. Tractor positions were classified using GIS layers while operating conditions were classified depending on the usage of the tractor's subsystems. The method highlights that showed to be able to detect the 97% of the logged data and that the tractor operated on the field in working, on idle, and moving duties for 65%, 18% and 16% of the time, respectively. The method allows a far more precise outline of tractor usage opening opportunities to obtain large benefits from massively collected CAN-BUS data

Hyperglycemia, Type 2 Diabetes, and Depressive Symptoms: The British Whitehall II study

International audienceOBJECTIVE: To examine the recent suggestion that impaired fasting glucose may protect against depression, whereas a diagnosis of diabetes might then result in depression. RESEARCH DESIGN AND METHODS: Cross-sectional analysis of 4,228 adults (mean age 60.7 years, 73.0% men) who underwent oral glucose tolerance testing and completed the Center for Epidemiologic Studies Depression scale (CES-D). RESULTS: After adjustment for demographic factors, health behaviors, and clinical measurements (BMI, waist circumference, lipid profile, and blood pressure), there was a U-shaped association between fasting glucose and depression (P(curve) = 0.001), with elevated CES-D at low and very high glucose levels. This finding was replicable with 2-h postload glucose (P = 0.11) and A1C (P = 0.007). CONCLUSIONS: The U-shaped association between blood glucose and CES-D, with the lowest depression risk seen among those in the normoglycemic range of A1C, did not support the hypothesized protective effect of hyperglycemia

Rescue of heterochromatin organization in Hutchinson-Gilford progeria by drug treatment

Hutchinson-Gilford progeria (HGPS) is a premature aging syndrome associated with LMNA mutations. Progeria cells bearing the G608G LMNA mutation are characterized by accumulation of a mutated lamin A precursor (progerin), nuclear dysmorphism and chromatin disorganization. In cultured HGPS fibroblasts, we found worsening of the cellular phenotype with patient age, mainly consisting of increased nuclear-shape abnormalities, progerin accumulation and heterochromatin loss. Moreover, transcript distribution was altered in HGPS nuclei, as determined by different techniques. In the attempt to improve the cellular phenotype, we applied treatment with drugs either affecting protein farnesylation or chromatin arrangement. Our results show that the combined treatment with mevinolin and the histone deacetylase inhibitor trichostatin A dramatically lowers progerin levels, leading to rescue of heterochromatin organization and reorganization of transcripts in HGPS fibroblasts. These results suggest that morpho-functional defects of HGPS nuclei are directly related to progerin accumulation and can be rectified by drug treatment

Diabetes mellitus type II as a risk factor for depression: a lower than expected risk in a general practice setting

The aim of the present study was to determine whether a diagnosis of diabetes mellitus (DM) in a primary setting is associated with an increased risk of subsequent depression. A retrospective cohort design was used based on the Registration Network Family Practice (RNH) database. Patients diagnosed with diabetes mellitus at or after the age of 40 and who were diagnosed between 01-01-1980 and 01-01-2007 (N = 6,140), were compared with age-matched controls from a reference group (N = 18,416) without a history of diabetes. Both groups were followed for an emerging first diagnosis of depression (and/or depressive feelings) until January 1, 2008. 2.0% of the people diagnosed with diabetes mellitus developed a depressive disorder, compared to 1.6% of the reference group. After statistical correction for confounding factors diabetes mellitus was associated with an increased risk of developing subsequent depression (HR 1.26; 95% CI: 1.12–1.42) and/or depressive feelings (HR 1.33; 95% CI: 1.18–1.46). After statistical adjustment practice identification code, age and depression preceding diabetes, were significantly related to a diagnosis of depression. Patients with diabetes mellitus are more likely to develop subsequent depression than persons without a history of diabetes. Results from this large longitudinal study based on a general practice population indicate that this association is weaker than previously found in cross-sectional research using self-report surveys. Several explanations for this dissimilarity are discussed

Morphology on the cloud Virtual Campus, an integrated didactic platform for biomedical studies

The current Core Curricula of Degree courses in Biomedical areas has enormously compressed the hours dedicated to the student for self-learning in morphological subjects. The result is a reduced student attitude to integrate the information received by attending lectures and practical sessions, with the indispensable consultation of texts dealing with morphological and \u2018functional\u2019 subjects, a key experience to autonomously logically identify the rational of the morphology/function relationship in the human body, at the macroscopic and microscopic level. These changes are occurring at a time when new medical imaging technologies become more and more informative in both morphological and functional areas. As a consequence, we are modifying our way of organize lessons compared to the generations of colleagues who have preceded us. More and more frontal lessons are organized with a logical morpho-functional approach. For example, the reference to the anatomy of the living, displayed through invasive or not invasive imaging, is added to the necessary and traditional anatomy of the cadaver. The reference to the pathology helps to define how the alteration of morphological integrity is reflected on function, both at the macro and microscopic level, and so on. However, there are no organized easy-to-use guided tours for the student to allow, in the shortest possible time, to \u2018rationally see\u2019 what he has studied, in the various imaging contexts available at the macro- and microscopic level. At the same time, there are no \u2018data bank\u2019 of resources for the preparation of the lessons. That is why we have imagined \u2018virtual campus\u2019 an integrated digital learning platform for self-learning. The platform has been thought and realized thanks to a group of teachers of \u2018morphologic\u2019 and \u2018functional\u2019 biomedical subjects and computer engineers belonging to a publishing house. The presentation will explain the rationale behind the platform, its structure and the educational opportunities offered

Detection and characterisation of multi-drug resistance protein 1 (MRP-1) in human mitochondria

BACKGROUND: Overexpression of plasma membrane multi-drug resistance protein 1 (MRP-1) can lead to multidrug resistance. In this study, we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues. METHODS: MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased using retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-propidium iodide labelling of cells. RESULTS: MRP-1 was detected in the mitochondria of cancer and normal cells and tissues. The efflux activity of mitochondrial MRP-1 was more efficient (55-64%) than that of plasma membrane MRP-1 (11-22%; P<0.001). Induced MRP-1 expression resulted in a preferential increase in mitochondrial MRP-1, suggesting selective targeting to this organelle. Treatment with a non-lethal concentration of doxorubicin (0.85 nM, 8 h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. For the first time, we have identified MRP-1 with efflux activity in human mitochondria. CONCLUSION: Mitochondrial MRP-1 may be an exciting new therapeutic target where historically MRP-1 inhibitor strategies have limited clinical success

Autophagy is defective in collagen VI muscular dystrophies and its reactivation rescues myofiber degeneration

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Embryonic and adult isoforms of XLAP2 form microdomains associated with chromatin and the nuclear envelope

Laminin-associated polypeptide 2 (LAP2) proteins are alternatively spliced products of a single gene; they belong to the LEM domain family and, in mammals, locate to the nuclear envelope (NE) and nuclear lamina. Isoforms lacking the transmembrane domain also locate to the nucleoplasm. We used new specific antibodies against the N-terminal domain of Xenopus LAP2 to perform immunoprecipitation, identification and localization studies during Xenopus development. By immunoprecipitation and mass spectrometry (LC/MS/MS), we identified the embryonic isoform XLAP2γ, which was downregulated during development similarly to XLAP2ω. Embryonic isoforms XLAP2ω and XLAP2γ were located in close association with chromatin up to the blastula stage. Later in development, both embryonic isoforms and the adult isoform XLAP2β were localized in a similar way at the NE. All isoforms colocalized with lamin B2/B3 during development, whereas XLAP2β was colocalized with lamin B2 and apparently with the F/G repeat nucleoporins throughout the cell cycle in adult tissues and culture cells. XLAP2β was localized in clusters on chromatin, both at the NE and inside the nucleus. Embryonic isoforms were also localized in clusters at the NE of oocytes. Our results suggest that XLAP2 isoforms participate in the maintenance and anchoring of chromatin domains to the NE and in the formation of lamin B microdomains