dSTORM microscopy evidences in HeLa cells clustered and scattered γH2AX nanofoci sensitive to ATM, DNA-PK, and ATR kinase inhibitors.

In response to DNA double-strand breaks (DSB), histone H2AX is phosphorylated around the lesion by a feed forward signal amplification loop, originating γH2AX foci detectable by immunofluorescence and confocal microscopy as elliptical areas of uniform intensity. We exploited the significant increase in resolution (~ × 10) provided by single-molecule localization microscopy (SMLM) to investigate at nanometer scale the distribution of γH2AX signals either endogenous (controls) or induced by the radiomimetic bleomycin (BLEO) in HeLa cells. In both conditions, clustered substructures (nanofoci) confined to γH2AX foci and scattered nanofoci throughout the remnant nuclear area were detected.

Association of microtubules and axonal RNA transferred from myelinating Schwann cells in rat sciatic nerve.

Transference of RNAs and ribosomes from Schwann cell-to-axon was demonstrated in normal and regenerating peripheral nerves. Previously, we have shown that RNAs transfer is dependent on F-actin cytoskeleton and Myosin Va. Here, we explored the contribution of microtubules to newly synthesized RNAs transport from Schwann cell nuclei up to nodal microvilli in sciatic nerves.

Familial primary ovarian insufficiency associated with an SYCE1 point mutation: defective meiosis elucidated in humanized mice.

More than 50% of cases of primary ovarian insufficiency (POI) and nonobstructive azoospermia in humans are classified as idiopathic infertility. Meiotic defects may relate to at least some of these cases. Mutations in genes coding for synaptonemal complex (SC) components have been identified in humans, and hypothesized to be causative for the observed infertile phenotype.

Revealing stage-specific expression patterns of long noncoding RNAs along mouse spermatogenesis.

The discovery of a large number of long noncoding RNAs (lncRNAs), and the finding that they may play key roles in different biological processes, have started to provide a new perspective in the understanding of gene regulation. It has been shown that the testes express the highest amount of lncRNAs among different vertebrate tissues. However, although some studies have addressed the characterization of lncRNAs along spermatogenesis, an exhaustive analysis of the differential expression of lncRNAs at its different stages is still lacking.

PARP-1/2 Inhibitor Olaparib Prevents or Partially Reverts EMT Induced by TGF-β in NMuMG Cells.

Poly- adenosine diphosphate (ADP)-ribose (PAR) is a polymer synthesized as a posttranslational modification by some poly (ADP-ribose) polymerases (PARPs), namely PARP-1, PARP-2, tankyrase-1, and tankyrase-2 (TNKS-1/2). PARP-1 is nuclear and has also been detected in extracellular vesicles. PARP-2 and TNKS-1/2 are distributed in nuclei and cytoplasm.

γH2AX prefers late replicating metaphase chromosome regions.

DNA damage response (DDR) constitutes a protein pathway to handle eukaryotic DNA lesions in the context of chromatin. DDR engages the recruitment of signaling, transducer, effector, chromatin modifiers and remodeling proteins, allowing cell cycle delay, DNA repair or induction of senescence or apoptosis. An early DDR-event includes the epigenetic phosphorylation of the histone variant H2AX on serine 139 of the C-termini, so-called gammaH2AX.

Intact DNA purified from flow-sorted nuclei unlocks the potential of next-generation genome mapping and assembly in species.

Next-generation genome mapping through nanochannels (Bionano optical mapping) of plant genomes brings genome assemblies to the 'nearly-finished' level for reliable and detailed gene annotations and assessment of structural variations. Despite the recent progress in its development, researchers face the technical challenges of obtaining sufficient high molecular weight (HMW) nuclear DNA due to cell walls which are difficult to disrupt and to the presence of cytoplasmic polyphenols and polysaccharides that co-precipitate or are covalently bound to DNA and might cause oxidation and/or affect the access of nicking enzymes to DNA, preventing downstream applications. Here we describe important improvements for obtaining HMW DNA that we tested on crops and wild relatives.

Transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage.

Background: Spermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis.

The methylating agent streptozotocin induces persistent telomere dysfunction in mammalian cells.

We analyzed chromosomal aberrations involving telomeres in the progeny of mammalian cells exposed to the methylating agent and antineoplastic/diabetogenic drug streptozotocin (STZ), to test whether it induces long-term telomere instability (by chromosome end loss and/or telomere dysfunction). Rat cells (ADIPO-P2 cell line, derived from Sprague-Dawley rat adipose cells) were treated with a single concentration of STZ (2mM). Chromosomal aberrations were analyzed 18h, 10 days, and 15 days after treatment, using PNA-FISH with a pan-telomeric probe [Cy3-(CCCTAA)3] to detect (TTAGGG)n repeats.

Bleomycin-induced γH2AX foci map preferentially to replicating domains in CHO9 interphase nuclei.

Exposure to DNA damaging agents triggers phosphorylation of histone variant H2AX (generating γH2AX) in large chromatin regions flanking DNA lesions, allowing their immunodetection as nuclear foci. Even though a predominance of γH2AX foci in euchromatin has been postulated, foci positioning when DNA insult occurs in replicating eu- or heterochromatin regions has not been extensively explored. Labeling of interphase nuclei with 5-ethynyl-2'-deoxyuridine (EdU) pulses has revealed that DNA replication is temporarily and spatially regulated: euchromatin replicates in early S (ES) and heterochromatin along mid and late S (MS/LS) phases.

Burst speciation processes and genomic expansion in the neotropical annual killifish genus Austrolebias (Cyprinodontiformes, Rivulidae).

The extent to which genome sizes and other nucleotypic factors influence the phyletic diversification of lineages has long been discussed but remains largely unresolved. In the present work, we present evidence that the genomes of at least 16 species of the neotropical rivulid killifish genus Austrolebias are unusually large, with an average DNA content of about 5.95 ± 0.

Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye.

Availability of purified or highly enriched fractions representing the various spermatogenic stages is a usual requirement to study mammalian spermatogenesis at the molecular level. Fast preparation of high quality testicular cell suspensions is crucial when flow cytometry (FCM) is chosen to accomplish the stage/s purification. Formerly, we reported a method to rapidly obtain good quality rodent testicular cell suspensions for FCM analysis and sorting.

Preferential localization of γH2AX foci in euchromatin of retina rod cells after DNA damage induction.

DNA damage may lead to cell transformation, senescence, or death. Histone H2AX phosphorylation, immunodetected as γH2AX foci, is an early response to DNA damage persisting even after DNA repair. In cycling mammalian cells with canonical nuclear architecture, i.

Simple and efficient technique for the preparation of testicular cell suspensions.

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses. Various strategies such as Staput, centrifugal elutriation, and flow cytometry (FC) have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies.

High-purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells.

Mammalian spermatogenesis is still nowadays poorly understood at the molecular level. Testis cellular heterogeneity is a major drawback for spermatogenic gene expression studies, especially when research is focused on stages that are usually very short and poorly represented at the cellular level such as initial meiotic prophase I (i.e.

Close encounters: RIDGEs, hyperacetylated chromatin, radiation breakpoints and genes differentially expressed in tumors cluster at specific human chromosome regions.

Through analysis of published data we positioned along human chromosome idiograms (850 bands) hyperacetylated H4 chromatin (H4(+a)), regions of increased gene expression (RIDGEs), antiRIDGEs, ionizing radiation breakpoints, integration sites of highly expressed GFP reporter constructs and candidate genes differentially expressed in tumor tissues. Highly expressed regions of the human genome (especially RIDGEs) seem to be more sensitive to radiation damage. Comparatively, antiRIDGEs appear as radiation resistant.

Karyological Features of Achyrocline (Asteraceae, Gnaphalieae): stable karyotypes, low DNA content variation and linkage of rRNA genes.

Many Achyrocline (Asteraceae, tribe Gnaphalieae) species are widely used in Argentina, Brazil and Uruguay as popular medicinal and aromatic plants. Achyrocline flaccida, A. satureioides, A.

Impact of EMS outreach: successful developments in Latin America.

This collection of articles was inspired by the long-standing relationship between the Environmental Mutagen Society and Latin American scientists, and by the program for the 39th Environmental Mutagen Society meeting in Puerto Rico in 2008, which included a symposium featuring "South of the border" scientists. This collection, compiled by Graciela Spivak and Ofelia Olivero, both originally from Argentina, highlights scientists who work in or were trained in Latin American countries and in Puerto Rico in a variety of scientific specialties related to DNA repair and cancer susceptibility, genomic organization and stability, genetic diversity, and environmental contaminants.

Ultra-fast and optimized method for the preparation of rodent testicular cells for flow cytometric analysis.

Unlabelled: Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling.

Nuclear architecture, chromosome domains and genetic damage.

The interphase nucleus structure is radially organised into three-dimensional discrete chromosome territories or domains (CTs) surrounded by a channel network named the interchromatin compartment (IC) which harbours factors involved in DNA replication or repair as well as RNA transcription and processing. Gene-rich chromosomes are centrally located whereas gene-poor ones are bound to the nuclear outskirts. Chromatin dynamics also reflect nuclear compartment organisation.