Measurement Of DNA Content Research Articles

Poldip2 knockout results in perinatal lethality, reduced cellular growth and increased autophagy of mouse embryonic fibroblasts.

Polymerase-δ interacting protein 2 (Poldip2) is an understudied protein, originally described as a binding partner of polymerase delta and proliferating cell nuclear antigen (PCNA). Numerous roles for Poldip2 have been proposed, including mitochondrial elongation, DNA replication/repair and ROS production via Nox4. In this study, we have identified a novel role for Poldip2 in regulating the cell cycle. We used a Poldip2 gene-trap mouse and found that homozygous animals die around the time of birth. Poldip2−/− embryos are significantly smaller than wild type or heterozygous embryos. We found that Poldip2−/− mouse embryonic fibroblasts (MEFs) exhibit reduced growth as measured by population doubling and growth curves. This effect is not due to apoptosis or senescence; however, Poldip2−/− MEFs have higher levels of the autophagy marker LC3b. Measurement of DNA content by flow cytometry revealed an increase in the percentage of Poldip2−/− cells in the G1 and G2/M phases of the cell cycle, accompanied by a decrease in the percentage of S-phase cells. Increases in p53 S20 and Sirt1 were observed in passage 2 Poldip2−/− MEFs. In passage 4/5 MEFs, Cdk1 and CyclinA2 are downregulated in Poldip2−/− cells, and these changes are reversed by transfection with SV40 large T-antigen, suggesting that Poldip2 may target the E2F pathway. In contrast, p21CIP1 is increased in passage 4/5 Poldip2−/− MEFs and its expression is unaffected by SV40 transfection. Overall, these results reveal that Poldip2 is an essential protein in development, and underline its importance in cell viability and proliferation. Because it affects the cell cycle, Poldip2 is a potential novel target for treating proliferative conditions such as cancer, atherosclerosis and restenosis.

The cohesin acetyltransferase Eco1 coordinates rDNA replication and transcription

Eco1 is the acetyltransferase that establishes sister-chromatid cohesion during DNA replication. A budding yeast strain with an eco1 mutation that genocopies Roberts syndrome has reduced ribosomal DNA (rDNA) transcription and a transcriptional signature of starvation. We show that deleting FOB1—a gene that encodes a replication fork-blocking protein specific for the rDNA region—rescues rRNA production and partially rescues transcription genome-wide. Further studies show that deletion of FOB1 corrects the genome-wide replication defects, nucleolar structure, and rDNA segregation that occur in the eco1 mutant. Our study highlights that the presence of cohesin at the rDNA locus has a central role in controlling global DNA replication and gene expression.

DNA Content Variation and Its Significance in the Evolution of the Genus Micrasterias (Desmidiales, Streptophyta)

It is now clear that whole genome duplications have occurred in all eukaryotic evolutionary lineages, and that the vast majority of flowering plants have experienced polyploidisation in their evolutionary history. However, study of genome size variation in microalgae lags behind that of higher plants and seaweeds. In this study, we have addressed the question whether microalgal phylogeny is associated with DNA content variation in order to evaluate the evolutionary significance of polyploidy in the model genus Micrasterias. We applied flow-cytometric techniques of DNA quantification to microalgae and mapped the estimated DNA content along the phylogenetic tree. Correlations between DNA content and cell morphometric parameters were also tested using geometric morphometrics. In total, DNA content was successfully determined for 34 strains of the genus Micrasterias. The estimated absolute 2C nuclear DNA amount ranged from 2.1 to 64.7 pg; intraspecific variation being 17.4–30.7 pg in M. truncata and 32.0–64.7 pg in M. rotata. There were significant differences between DNA contents of related species. We found strong correlation between the absolute nuclear DNA content and chromosome numbers and significant positive correlation between the DNA content and both cell size and number of terminal lobes. Moreover, the results showed the importance of cell/life cycle studies for interpretation of DNA content measurements in microalgae.

“Mega” cytometry for a “mega” challenging cell type

“Mega” cytometry for a “mega” challenging cell type

Meiosis and Haploid Gametes in the Pathogen Trypanosoma brucei

SummaryIn eukaryote pathogens, sex is an important driving force in spreading genes for drug resistance, pathogenicity, and virulence [1]. For the parasitic trypanosomes that cause African sleeping sickness, mating occurs during transmission by the tsetse vector [2, 3] and involves meiosis [4], but haploid gametes have not yet been identified. Here, we show that meiosis is a normal part of development in the insect salivary glands for all subspecies of Trypanosoma brucei, including the human pathogens. By observing insect-derived trypanosomes during the window of peak expression of meiosis-specific genes, we identified promastigote-like (PL) cells that interacted with each other via their flagella and underwent fusion, as visualized by the mixing of cytoplasmic red and green fluorescent proteins. PL cells had a short, wide body, a very long anterior flagellum, and either one or two kinetoplasts, but only the anterior kinetoplast was associated with the flagellum. Measurement of nuclear DNA contents showed that PL cells were haploid relative to diploid metacyclics. Trypanosomes are among the earliest diverging eukaryotes, and our results support the hypothesis that meiosis and sexual reproduction are ubiquitous in eukaryotes and likely to have been early innovations [5].

Host Adaptation and Speciation through Hybridization and Polyploidy in Phytophthora

It is becoming increasingly evident that interspecific hybridization is a common event in phytophthora evolution. Yet, the fundamental processes underlying interspecific hybridization and the consequences for its ecological fitness and distribution are not well understood. We studied hybridization events in phytophthora clade 8b. This is a cold-tolerant group of plant pathogenic oomycetes in which six host-specific species have been described that mostly attack winter-grown vegetables. Hybrid characterization was done by sequencing and cloning of two nuclear (ITS and Ypt1) and two mitochondrial loci (Cox1 and Nadh1) combined with DNA content estimation using flow cytometry. Three different mtDNA haplotypes were recovered among the presumed hybrid isolates, dividing the hybrids into three types, with different parental species involved. In the nuclear genes, additivity, i.e. the presence of two alleles coming from different parents, was detected. Hybrid isolates showed large variations in DNA content, which was positively correlated with the additivity in nuclear loci, indicating allopolyploid hybridization followed by a process of diploidization. Moreover, indications of homeologous recombination were found in the hybrids by cloning ITS products. The hybrid isolates have been isolated from a range of hosts that have not been reported previously for clade 8b species, indicating that they have novel pathogenic potential. Next to this, DNA content measurements of the non-hybrid clade 8b species suggest that polyploidy is a common feature of this clade. We hypothesize that interspecific hybridization and polyploidy are two linked phenomena in phytophthora, and that these processes might play an important and ongoing role in the evolution of this genus.

Abstract PD4-7: Deep clonal profiling of primary breast cancer and distant lymph node metastases

Abstract Background: Breast and other solid tumors exhibit high and varying degrees of intra-tumor heterogeneity that make the management and treatment of these diseases rather difficult. In order to study clinically relevant tumor heterogeneity in detail we performed an in-depth analysis of the clonal architecture of primary chemoradiation-naive breast cancers and their lymph node metastases. We combined DNA content-based flow cytometry and ploidy analysis with high-definition array comparative genomic hybridization (aCGH) and next-generation sequencing technologies to interrogate the genomes and transcriptomes of multiple biopsies from the tumors and involved lymph nodes. Thus rather than inferring the presence of distinct tumor cell populations, our flow-sorting based approach of first identifying and defining the clonal populations and then interrogating their genomes provides a highly objective method of exploring the clinical significance of tumor heterogeneity in solid tumors. Material and methods: We used DAPI-based DNA content measurements and flow sorting to isolate nuclei from distinct clonal populations of diploid and aneuploid tumor cells in surgical tumor samples and distant lymph nodes from chemoradiation naive patients. In parallel, matching samples were processed for pathological, transcriptome, and IHC based assays. Each distinct sorted tumor cell population was then interrogated with aCGH and next generation sequencing. For each patient, the detailed profiles of ploidy, copy number aberrations and mutations were used to recreate and map the lineages present in the primary tumor and lymph. In one detailed case, we used 12 fresh frozen sections morphologically mapped from within a chemo-naïve primary invasive ductal carcinoma of histological grade 3 with lymph node involvement, 2-3 fresh frozen sections from 2 out of 5 lymph nodes with metastatic carcinoma, formalin-fixed paraffin-embedded (FFPE) tissue sections from 2 more of the involved lymph nodes and 5 morphologically mapped FFPE samples of normal tissue adjacent to the tumor. Results: We identified multiple coexisting aneuploid populations within biopsies from individual patients. Our clonal analysis determined the genomic aberrations arising in lineages that spread throughout the primary tumor and later bifurcated to distinct lymph nodes, revealing distinct therapeutic targets in the trunk and branches of the tumor. We identified driver events including high-level focal amplifications (e.g. SARC) and homozygous deletions (e.g. ROBO1 and ROBO2), that converge on distinct cellular signaling pathways. Conclusion: The approach highlighted here has broad implications in the study of tumor heterogeneity in breast and other solid tumors by providing a unique ultra-high resolution view of tumor heterogeneity that can advance more effective treatment and clinical management of patients with this disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-7.

The first report of an alloheptaploid from the genusFragaria(Rosaceae)

AbstractHybrid recognition in the genus Fragaria (strawberry, Rosaceae) is difficult when based on morphological characteristics since there is great similarity among Fragaria species, even between different ploidy levels. This study aimed to identify and characterise an alloheptaploid hybrid (2n = 7x = 49) Fragaria for the first time in the genus from Europe (Germany, Bavaria) using different techniques. The hybrid was documented based on the evidence obtained from ploidy levels using flow cytometric measurement of nuclear DNA content, identification of the parental origin using 14 RAPD primers, fertility assessment from artificial crosses and fruit set level. RAPD analysis showed that the hybrid shared five species-specific markers with diploid F. vesca, and three with hexaploid F. moschata, indicating these species to be the most likely parents. The mean fluorescent intensity reflected by nuclear DNA in the putative hybrid (703) fell within the range accepted for heptaploidy in Fragaria, while the corr...

A novel combination of nano-scaffolds with micro-scaffolds to mimic extracellularmatrices improve osteogenesis

To improve bone engineering for clinical applications, we coupled nanofiber-peptide hydrogel to nano-hydroxyapatite/collagen to form a bioactive scaffold (cnHAC) that mimics extracellular matrices. In comparison to nano-hydroxyapatite/collagen, we found that cnHAC promoted cell adhesion and spreading, and DNA content measurements, alkaline phosphatase activity assays, and reverse transcriptase-polymerase chain reaction analyses of osteogenic gene expression showed that cnHAC significantly improved cellular attachment, proliferation, and osteogenic differentiation in vitro (P < 0.05). In vivo models based on rat calvarial implants showed that cnHAC significantly enhanced bone regeneration (P < 0.05). In conclusion, we demonstrated that novel cnHAC scaffolds could potentially facilitate future bone regenerative medicine.

New fluorogenic dyes for analysis of cellular processes by flow cytometry and confocal microscopy

Fluorescent microscopy and fluorescent imaging by flow cytometry are two of the fastest growing areas in the medical and biological research. Innovations in fluorescent chemistry and synthesis of new dye probes are closely related to the development of service equipment such as light sources, and detection techniques. Among compounds known as fluorescent labels, the cyanine-based dyes have become widely used since they have high excitation coefficients, narrow emission bands and high fluorescence upon binding to nucleic acids.The key methods for evaluation of apoptosis and cell cycle allow measuring DNA content by several flow cytometric techniques.We have synthesized new monomethine cyanine dyes and have characterized their applicability for staining of live and/or apoptotic cells. Imaging experiments by flow cytometry and confocal laser scanning microscopy (CLSM) have been also performed. Two of the dyes have shown high-affinity binding to the nuclei at high dilutions, up to 10−9M. Flow cytometry and CLSM have confirmed that these dyes labeled selectively non-living, e.g. ethanol-fixed cells that makes them appropriate for estimations of cell viability and apoptosis. The novel structures proved to be appropriate also for analysis of the cell cycle.

Increased rate of atypical squamous cells of undetermined significance and declining high-risk human papillomavirus rates following implementation of ThinPrep Imaging System are associated with increased nuclear chromasia

The ThinPrep Imaging System ([Imager], Cytyc Corporation, Hologic, Inc.) for cervical cytology is commonly employed in laboratories. It uses a proprietary stain that allows measurement of cellular DNA content. We evaluated the initial effect of implementing the Imager stain on atypical squamous cells of undetermined significance (ASCUS) and squamous intraepithelial lesion (SIL) diagnoses, and high-risk human papillomavirus (hrHPV) rates. This study included 264 Imager-stained ASCUS cases from the first 5 months of Imager use. All cases were analyzed for hrHPV using Hybrid Capture II assay (Digene Corporation, Qiagen). ASCUS/SIL ratios and hrHPV-positive rates were calculated for the laboratory overall and for 3 cytopathologists. ASCUS/SIL ratios were also calculated for 5 cytotechnologists. These metrics were compared with those from the equivalent 5-month period 1 year prior. Slide review of all Imager-stained ASCUS cases was performed to assess for potential factors involved in ASCUS misinterpretation. The proportion of ASCUS diagnoses increased after Imager stain introduction, from 1.79% to 3.14% (P < 0.001). The proportion of SIL diagnoses also increased, from 0.69% to 1.1% (P < 0.001). However, the hrHPV-positive rate declined from 61.3% to 53.6% (P = 0.104). Review of Imager-stained ASCUS cases showed marked nuclear hyperchromasia, particularly in benign squamous metaplastic cells. Imager stain implementation, required for the ThinPrep Imaging System, led to significant increase in ASCUS rates while the hrHPV-positive rate declined. Cytopathologists will need to recalibrate thresholds for an ASCUS diagnosis when interpreting Imager-stained slides. In particular, nuclear hyperchromasia in squamous metaplastic cells should not be overdiagnosed as ASCUS.

Rapid regeneration and ploidy stability of 'cv IR36' indica rice (Oryza Sativa. L) confers efficient protocol for in vitro callus organogenesis and Agrobacterium tumefaciens mediated transformation.

BackgroundCereal crops are the major targets for transformation mediated crop improvement and IR36 is an early maturing, high yielding, insect and disease resistant rice variety however, it is abiotic stress sensitive. Hence, development of an efficient and reproducible micropropagation system via somatic embryogenesis and Agrobacterium tumefaciens mediated transformation is prerequisite to develop abiotic stress tolerant IR36. Further, Genetic stability of analysis of plantlets through RAPD and ISSR and Ploidy level through Flow cytometry (FCM) measurement of 2C DNA content is necessary for future application of transformed IR36.ResultsIn this study, Mature seeds inoculated on (Murashige and Skoog) MS medium with 11.31 μM 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.3 μM Kinetin (Kn) had highest callus induction frequency (98%). The highest regeneration frequency (80%) was observed in MS + 13.28 μM Benzyladenine (BA) with 8.06 μM α-naphthalene acetic acid (NAA). Randomly Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Flow Cytometry (FCM) analysis showed no significant variation in the 2C DNA (0.81 pg/2C) content and Ploidy level between wild type IR36 and in vitro maintained rice lines. Of the various OD bacterial culture, an optimum OD of 0.4 and inoculation duration of 10 min resulted in efficient Agrobacterium-mediated transformation. β-glucuronidase activity was maximum in callus (99.05%).ConclusionsThese results described here confirm the reliability of this protocol for micropropagation and delivery of desirable gene using A. tumefaciens into indica rice.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-47) contains supplementary material, which is available to authorized users.

Cytogenetic and molecular genetic analysis of gynogenesis in Megalobrama amblycephala using spermatozoa of Erythroculter ilishaeformis

In the present study we used both cytogenetics (measurement of DNA content, detection of chromosome number, observation of gonadal development)and molecular genetics(microsatellite analysis)to analyze the biological characteristics of gynogenetic M. amblycephala, which werecreatedthrough gynogenesis induced via UV-irradiated E. ilishaeformis spermatozoa to fertilize M. amblycephala eggs. The maternal genome was duplicated by cold shock in 0~4℃ cold water to form a population of M. amblycephala with 48 chromosomes whose DNA content was identical to the diploid maternal parent. Morphologically, this group of gynogenetic M. amblycephala was similar to the control group. All gynogenetic M. amblycephala were female, and no males were found in any of the examined gynogenetic M. amblycephala, providing cytogenetic evidence that our gynogenetic M. amblycephala are type XY. At the same time, microsatellite analysis showed that 63 alleles were amplified in the three test groups of gynogenetic M. amblycephala. Overall, the population of gynogenetic M. amblycephala observed heterozygosity average, and the expected average was significantly lower than the parental averages, demonstrating that after generation gynogenesis the gene homozygosity of M. amblycephala was significantly higher than the ordinary bream and E. ilishaeformis, making it a pure line. The genetic proximity of gynogenetic M. amblycephala to M. amblycephala demonstrates that gynogenesis passes on maternal DNA. Gynogenetic groups developed in this study may provide good genetic material for future breeding projects of M. amblycephala.

Body mass index affects proliferation and osteogenic differentiation of human subcutaneous adipose tissue-derived stem cells

BackgroundObesity is associated with a higher risk of developing cancer and co-morbidities that are part of the metabolic syndrome. Adipose tissue is recognized as an endocrine organ, as it affects a number of physiological functions, and contains adipose tissue-derived stem cells (ASCs). ASCs can differentiate into cells of multiple lineages, and as such are applicable to tissue engineering and regenerative medicine. Yet the question of whether ASC functionality is affected by the donor’s body mass index (BMI) still exists.ResultsASCs were isolated from patients having different BMIs (BMI-ASCs), within the ranges of 18.5-32.8. It was hypothesized that overweight BMI-ASCs would be more compromised in early adipogenic and osteogenic potential, and ability to form colonies in vitro. BMI was inversely correlated with ASC proliferation and colony forming potential as assessed by CyQUANT proliferation assay (fluorescence- based measurement of cellular DNA content), and colony forming assays. BMI was positively correlated with early time point (day 7) but not later time point (day 15) intracytoplasmic lipid accumulation as assessed by Oil-Red-O staining. Alizarin red staining and RT-PCR for alkaline phosphatase demonstrated that elevated BMI resulted in compromised ASC mineralization of extracellular matrix and decreased alkaline phosphatase mRNA expression.ConclusionsThese data demonstrate that elevated BMI resulted in reduced ASC proliferation, and potentially compromised osteogenic capacity in vitro; thus BMI is an important criterion to consider in selecting ASC donors for clinical applications.

Flow Cytometric Measurement of Aneuploid DNA Content Correlates with High S-Phase Fraction and Poor Prognosis in Patients with Non-Small-Cell Lung Cancer

Cellular DNA content (ploidy) and proliferation activity (e.g., S-phase fraction) measured by flow cytometry have been usually related to the biologic aggressiveness of various neoplasms. In this study, these parameters were analyzed in paraffin-embedded tumor specimens from 43 cases of resected non-small-cell lung cancer (NSCLC). Additionally, the correlation of them with both prognosis and a variety of clinic-pathological features were investigated. The stage and the appearance of both local recurrence and metastasis were related to overall survival of patients. Twenty-two tumors (51.2%) had a diploid DNA distribution, while 21 were aneuploid (48.8%). The mean of aneuploidy was 1.6% ± 0.3%. A correlation was found between ploidy and survival as well as with the appearance of local recurrence and/or metastasis. The mean values of S-phase fraction of diploid and aneuploid tumors were 16.7 ± 11.3% and 32.9 ± 12.1%, respectively, which were significantly different (P=0.0022). Similar results were obtained analyzing the proliferation index (sum of cells in S and G2/M phases of cell cycle) (P=0.0040). However, no correlation between these parameters and both overall survival of patients and clinicopathological features was observed. Our results could suggest the potential use of ploidy analysis as a useful complement of TNM stage in NSCLC.

NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, enhances the radiosensitivity of human glioma stem cells in vitro

NVP-BEZ235 is a novel dual PI3K/mTOR inhibitor and shows dramatic effects on gliomas. The aim of this study was to investigate the effects of NVP-BEZ235 on the radiosensitivity and autophagy of glioma stem cells (GSCs) in vitro. Human GSCs (SU-2) were tested. The cell viability and survival from ionizing radiation (IR) were evaluated using MTT and clonogenic survival assay, respectively. Immunofluorescence assays were used to identify the formation of autophagosomes. The apoptotic cells were quantified with annexin V-FITC/PI staining and flow cytometry, and observed using Hoechst 33258 staining and fluorescence microscope. Western blot analysis was used to analyze the expression levels of proteins. Cell cycle status was determined by measuring DNA content after staining with PI. DNA repair in the cells was assessed using a comet assay. Treatment of SU-2 cells with NVP-BEZ235 (10-320 nmol/L) alone suppressed the cell growth in a concentration-dependent manner. A low concentration of NVP-BEZ235 (10 nmol/L) significantly increased the radiation sensitivity of SU-2 cells, which could be blocked by co-treatment with 3-MA (50 μmol/L). In NVP-BEZ235-treated SU-2 cells, more punctate patterns of microtubule-associated protein LC3 immunoreactivity was observed, and the level of membrane-bound LC3-II was significantly increased. A combination of IR with NVP-BEZ235 significantly increased the apoptosis of SU-2 cells, as shown in the increased levels of BID, Bax, and active caspase-3, and decreased level of Bcl-2. Furthermore, the combination of IR with NVP-BEZ235 led to G1 cell cycle arrest. Moreover, NVP-BEZ235 significantly attenuated the repair of IR-induced DNA damage as reflected by the tail length of the comet. NVP-BEZ235 increases the radiosensitivity of GSCs in vitro by activating autophagy that is associated with synergistic increase of apoptosis and cell-cycle arrest and decrease of DNA repair capacity.

Abstract 3411: Mathematical model of the cell cycle to determine mechanism of action and optimize dosing schedule.

Abstract A mechanistic understanding of a drug's biological effect can have significant implications for selection of clinical dosing schedule, combination partners, as well as biomarker selection. In this context, a question often faced from target validation to the end of Phase I is how much of a drug's antitumor effect is through altering cell-cycle progression or through the induction of cell death. Here we demonstrate the utility of a mathematical model of the cell-cycle for determining the effect of a drug on cell-cycle kinetics, as an objective and quantiative interpretation of DNA flow cytometry and cellular growth rate data. The model uses periodic measurement of DNA content as analyzed by flow cytometry to determine the percentage of cells in G0/G1, S, and G2/M phases of the cell cycle, as well as continuous readouts of cell confluence over the course of treatment to assess growth rates. The model is then fitted to the data to extract effective cell cycle transition rates demonstrating the effect of the drug on cell cycle progression. To demonstrate of the approach, we applied the model to data acquired during treatment of A375 melanoma cells with the investigational drug TAK-733, an allosteric MEK 1/2 inhibitor hypothesized to cause a G1 arrest. Rather than an arrest at G1, the model predicted that the G1-to-S transition rate was reduced, and that much of the change in cell cycle data was due to an increase in the S-to-G2/M and G2/M-to-G0/G1 transition rates. These model predictions were then tested with video microscopy and both predictions (increased time spent in G0/G1 as well as reduced time spent in mitosis) were supported by experimental data. The model was then used to test whether the cell cycle activity of the compound has any implications for optimization of dosing schedule. By varying the schedule of drug treatment in the simulation, we find that for the biologically relevant concentration range the compound is schedule independent (i.e. effect is proportional to AUC). This finding suggests that there is no need to maintain constant pathway inhibition to see efficacy in tumor volume reduction. To test this prediction, we compare the results with the outcome of experiments in an A375 xenograft model with different dosing schedules and verify directly that AUC was indeed a better predictor of efficacy than Cmin. With this example, we demonstrate that mathematical modeling can be used to aid in interpretation of cell cycle data, often generated to describe the cell killing activity of oncology compounds. In addition, the model provides a method of interpreting the mechanism of action for early development decision making around schedule selection. This work demonstrates an approach that can be extended to other compounds to provide an integrated measure of the total contribution of cell death and/or cell cycle arrest, to help provide a mechanistic rationale for schedule selection with single agent and combination therapies. Citation Format: Jerome Mettetal, Derek Blair, Esha Gangoli, Patrick Vincent, Jeff Ecsedy, Wen Chyi Shyu, Arijit Chakravarty. Mathematical model of the cell cycle to determine mechanism of action and optimize dosing schedule. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3411. doi:10.1158/1538-7445.AM2013-3411

A novel in vitro sperm head decondensation protocol for rapid flow cytometric measurement of deoxyribonucleic acid content

To set up a novel protocol of sperm head invitro decondensation that obviates the problematic effect of the variable degree of sperm chromatin packaging on DNA staining needed for flow cytometric analysis. Development of a new cytofluorimetric assay. University laboratory. Semen specimens were obtained from normospermic healthy volunteers at the Department of Life and Environmental Sciences, Università Politecnica delle Marche. Setup of the novel invitro sperm head decondensation protocol; sperm were then stained and analyzed by flow cytometry to measure DNA content. Mean fluorescent channel, DNA content, percentage diploid sperm. Native nondecondensed fluorochrome-labeled sperm show significant under-staining, resulting in an underestimated C-value (approximately 1.4 pg). This protocol ensures stoichiometric staining of sperm DNA, which becomes fully reachable by fluorescent probes and makes the diploid (7.12 pg) over haploid (3.56 pg) sperm frequency quantification easier. This study establishes a simple method for invitro sperm head decondensation, which allows accurate detection of the real sperm DNA content.

Sicilian bottled natural waters: Major and trace inorganic components

Sixteen bottled waters of various Sicilian brands, 11 natural mineral waters and five normal drinking waters, were analyzed for major and trace inorganic components by ion chromatography (IC) and inductively coupled plasma-mass spectrometry (ICP-MS), respectively. The bottled waters represent a variety of water types with significantly different compositions in terms of salinity, major components and trace elements. Chemically, they range from Ca–HCO3 and Ca–SO4 to Na–HCO3 types. Total dissolved solids ranges from 54 to 433mg/L, total hardness from 25 to 238mg/L CaCO3, and measured Na content from 5.7 to 57mg/L. According to total dissolved ions, all the bottled waters were classified as oligomineral (50<TDS<500mg/L). All the bottled waters analyzed here had elemental concentrations which did not exceed the guideline and directive values, although a high concentration of Al was noted for one bottled water (O7, central Sicily) and high Rb and V in a bottled water (O1) from the Etna volcanic area. With regard to trace elements, the chemical quality of bottled waters was assessed by a metal index (MI). Chemical characteristics were compared with 10 tap water samples from private houses or public places, representative of the public water supply in Palermo, the largest and most densely populated city in Sicily. The municipal waters analyzed, belonging to HCO3-alkaline earth and Cl–SO4-alkaline earth waters, showed concentrations of chemical inorganic components well within drinking water limits. The data also indicated that the water supplied by the municipal authority is of fair quality, although fairly hard and high in Na concentration. Several considerations indicate that there is no sufficient reason to prefer bottled waters to tap waters.

A computational platform for robotized fluorescence microscopy (I): High‐content image‐based cell‐cycle analysis

Hardware automation and software development have allowed a dramatic increase of throughput in both acquisition and analysis of images by associating an optimized statistical significance with fluorescence microscopy. Despite the numerous common points between fluorescence microscopy and flow cytometry (FCM), the enormous amount of applications developed for the latter have found relatively low space among the modern high-resolution imaging techniques. With the aim to fulfill this gap, we developed a novel computational platform named A.M.I.CO. (Automated Microscopy for Image-Cytometry) for the quantitative analysis of images from widefield and confocal robotized microscopes. Thanks to the setting up of both staining protocols and analysis procedures, we were able to recapitulate many FCM assays. In particular, we focused on the measurement of DNA content and the reconstruction of cell-cycle profiles with optimal parameters. Standard automated microscopes were employed at the highest optical resolution (200 nm), and white-light sources made it possible to perform an efficient multiparameter analysis. DNA- and protein-content measurements were complemented with image-derived information on their intracellular spatial distribution. Notably, the developed tools create a direct link between image-analysis and acquisition. It is therefore possible to isolate target populations according to a definite quantitative profile, and to relocate physically them for diffraction-limited data acquisition. Thanks to its flexibility and analysis-driven acquisition, A.M.I.CO. can integrate flow, image-stream and laser-scanning cytometry analysis, providing high-resolution intracellular analysis with a previously unreached statistical relevance.