Assessment of telomere length during post-natal period in offspring produced by a bull and its fibroblast derived clone

Objective: To investigate the telomere length in bovine offspring produced by a cloned and control bull, and the telomerase activity in embryos produced with the same technology. Methods: Five daughters of a control and five daughters of a bull cloned using a fibroblast of the control were produced by IVF using sperm of the two bulls. Blood samples of the offspring were collected at 2, 6, and 12 months of age and the relative telomere length (RTL) was assessed by flow cytometry. At same time the body growth, hematological profile, and clinical biochemistry of the same progeny was extensively surveyed, and results have been reported in a previous work. Thereafter, the telomerase activity was assessed using a real time PCR quantitative assay in groups of embryos produced with the same technology. Results: The offspring of the clone exhibited a modest, but significant (P<0.05), shortening of the telomeres (21.36%, 20.56% and 20.56%) compared to that of the control (23.78%, 23.53% and 22.43%) as mean values determined at 2, 6 and 12 months, respectively. Shortening of telomeres in respect to the age was not significant. No statistical difference was reported between telomerase activity assessed in 144 cloned (3.4−03 ± 2.4−03 amoles/μL) and 80 control (2.1−03 ± 1.8−03 amoles/μL) embryos. Conclusions: The results have revealed a moderate shortening of telomeres in the offspring of the clone with respect to control. However, this study did not evidence differences in the two progenies that suggest welfare problems during the first year of life

OMEGA: a software tool for the management, analysis, and dissemination of intracellular trafficking data that incorporates motion type classification and quality control [preprint]

MOTIVATION: Particle tracking coupled with time-lapse microscopy is critical for understanding the dynamics of intracellular processes of clinical importance. Spurred on by advances in the spatiotemporal resolution of microscopy and automated computational methods, this field is increasingly amenable to multi-dimensional high-throughput data collection schemes (Snijder et al, 2012). Typically, complex particle tracking datasets generated by individual laboratories are produced with incompatible methodologies that preclude comparison to each other. There is therefore an unmet need for data management systems that facilitate data standardization, meta-analysis, and structured data dissemination. The integration of analysis, visualization, and quality control capabilities into such systems would eliminate the need for manual transfer of data to diverse downstream analysis tools. At the same time, it would lay the foundation for shared trajectory data, particle tracking, and motion analysis standards. RESULTS: Here, we present Open Microscopy Environment inteGrated Analysis (OMEGA), a cross-platform data management, analysis, and visualization system, for particle tracking data, with particular emphasis on results from viral and vesicular trafficking experiments. OMEGA provides easy to use graphical interfaces to implement integrated particle tracking and motion analysis workflows while keeping track of error propagation and data provenance. Specifically, OMEGA: 1) imports image data and metadata from data management tools such as Open Microscopy Environment Remote Objects (OMERO; Allan et al., 2012); 2) tracks intracellular particles moving across time series of image planes; 3) facilitates parameter optimization and trajectory results inspection and validation; 4) performs downstream trajectory analysis and motion type classification; 5) estimates the uncertainty associated with motion analysis; and, 6) facilitates storage and dissemination of analysis results, and analysis definition metadata, on the basis of our newly proposed Minimum Information About Particle Tracking Experiments (MIAPTE; Rigano & Strambio-De-Castillia, 2016; 2017) guidelines in combination with the OME-XML data model (Goldberg et al, 2005)

Operationalizing mild cognitive impairment criteria in small vessel disease: The VMCI-Tuscany Study

Introduction Mild cognitive impairment (MCI) prodromic of vascular dementia is expected to have a multidomain profile. Methods In a sample of cerebral small vessel disease (SVD) patients, we assessed MCI subtypes distributions according to different operationalization of Winblad criteria and compared the neuroimaging features of single versus multidomain MCI. We applied three MCI diagnostic scenarios in which the cutoffs for objective impairment and the number of considered neuropsychological tests varied. Results Passing from a liberal to more conservative diagnostic scenarios, of 153 patients, 5% were no longer classified as MCI, amnestic multidomain frequency decreased, and nonamnestic single domain increased. Considering neuroimaging features, severe medial temporal lobe atrophy was more frequent in multidomain compared with single domain. Discussion Operationalizing MCI criteria changes the relative frequency of MCI subtypes. Nonamnestic single domain MCI may be a previously nonrecognized type of MCI associated with SVD

An algorithm-centric Monte Carlo method to empirically quantify motion type estimation uncertainty in single-particle tracking [preprint]

Quantitative analysis of microscopy images is ideally suited for understanding the functional biological correlates of individual molecular species identified by one of the several available “omics” techniques. Due to advances in fluorescent labeling, microscopy engineering and image processing, it is now possible to routinely observe and quantitatively analyze at high temporal and spatial resolution the real-time behavior of thousands of individual cellular structures as they perform their functional task inside living systems. Despite the central role of microscopic imaging in modern biology, unbiased inference, valid interpretation, scientific reproducibility and results dissemination are hampered by the still prevalent need for subjective interpretation of image data and by the limited attention given to the quantitative assessment and reporting of the error associated with each measurement or calculation, and on its effect on downstream analysis steps (i.e., error propagation). One of the mainstays of bioimage analysis is represented by single-particle tracking (SPT)1–5, which coupled with the mathematical analysis of trajectories and with the interpretative modelling of motion modalities, is of key importance for the quantitative understanding of the heterogeneous intracellular dynamic behavior of fluorescently-labeled individual cellular structures, vesicles, virions and single-molecules. Despite substantial advances, the evaluation of analytical error propagation through SPT and motion analysis pipelines is absent from most available tools 6. This severely hinders the critical evaluation, comparison, reproducibility and integration of results emerging from different laboratories, at different times, under different experimental conditions and using different model systems. Here we describe a novel, algorithmic-centric, Monte Carlo method to assess the effect of experimental parameters such as signal to noise ratio (SNR), particle detection error, trajectory length, and the diffusivity characteristics of the moving particle on the uncertainty associated with motion type classification The method is easily extensible to a wide variety of SPT algorithms, is made widely available via its implementation in our Open Microscopy Environment inteGrated Analysis (OMEGA) software tool for the management and analysis of tracking data 7, and forms an integral part of our Minimum Information About Particle Tracking Experiments (MIAPTE) data model 8

Optical evaluation of the ionized EL2 fraction in proton (24 GeV) irradiated semi-insulating GaAs

Semi-insulating SI GaAs samples from a zone refined crystal were irradiated with high energy protons (24 GeV/c, fluences up to 1.64x10(14) p/cm(2)). Optical spectra in transmittance and reflectance were accurately measured in the energy range of 0.6-1.4 eV to determine, through the absorption coefficient, the concentrations of both neutral and ionized EL2 defects as a function of the proton fluence. Both these concentrations have been shown to increase linearly with the proton fluence; this behavior well explains the remarkable decrease of the charge collection efficiency observed in proton irradiated GaAs detectors at doses associated with high luminosity beams at a new particle collider accelerator (e.g., the LHC at the CERN laboratory)

Optical Evaluation of the Ionized EL2 Fraction in Proton (24 GeV) Irradiated Semi-Insulating GaAs

Semi-insulating SI GaAs samples from a zone refined crystal were irradiated with high energy protons (24 GeV/c, fluences up to 1.64×1014 p/cm2). Optical spectra in transmittance and reflectance were accurately measured in the energy range of 0.6–1.4 eV to determine, through the absorption coefficient, the concentrations of both neutral and ionized EL2 defects as a function of the proton fluence. Both these concentrations have been shown to increase linearly with the proton fluence; this behavior well explains the remarkable decrease of the charge collection efficiency observed in proton irradiated GaAs detectors at doses associated with high luminosity beams at a new particle collider accelerator (e.g., the LHC at the CERN laboratory)

In vitro fertilisation with frozen-thawed bovine sperm sexed by flow cytometry and validated for accuracy by real-time PCR

The methodologies used for cytometric sorting of fresh spermatozoa never allowed a clear resolution of sexual chromosomes of frozen-thawed semen. To devise a novel method for the production of bovine predefined sexed embryos using frozen-thawed semen, sorting efficiency of different protocols was studied using a new quantitative real-time PCR method to verify the purity of sexed semen. To this aim, after Percoll separation, frozen-thawed samples were stained at different temperatures and concentrations of Hoechst 33342 using a short-incubation time. The concentration of Hoechst 33342 of 500 mug/ml at a temperature of 37 degrees C provided good and stable fluorescence signals. Preventing the sperm clustering by adding 0.6% BSA in the 90% Percoll fraction led to X-bearing sperms purity of 91+/-2%. Thereafter, sorted sperms were used for in vitro fertilisation (IVF). Despite the lower cleavage rates reported in the sorted groups when compared with the control groups (40 vs 48%, P&lt;0.01), blastocyst formation in the sorted and control groups was not different (27 vs 24% of the cleaved respectively). The PCR analysis of 30 blastocysts confirmed 26 embryos to be correctly sexed (87%). Transfer of two embryos per recipient into six synchronised heifers resulted in four pregnancies. Two abortions occurred at day 60, while two pregnancies went to term delivering two female calves. In conclusion, high purity and repeatability of sorting was obtained with frozen-thawed bull semen that was subsequently used for IVF giving rise to viable embryos and offspring. In addition, real-time PCR revealed to be an optimal support for these studies, providing a rapid and reliable estimation of flow cytometric efficiency