Метод лабораторного определения параметров устройства гидроимпульсного воздействия

Дана стаття описує лабораторний метод, що визначає: мету, умови, обсяг і порядок проведення досліджень параметрів пристрою гідроімпульсної дії.This article describes the laboratory method that defines: the purpose, conditions, effort and procedure of the researching the device settings of hydroimpulsive impact

Comprehensive Profiling of Mutations to Influenza Virus PB2 That Confer Resistance to the Cap-Binding Inhibitor Pimodivir

Antivirals are used not only in the current treatment of influenza but are also stockpiled as a first line of defense against novel influenza strains for which vaccines have yet to be developed. Identifying drug resistance mutations can guide the clinical deployment of the antiviral and can additionally define the mechanisms of drug action and drug resistance. Pimodivir is a first-in-class inhibitor of the polymerase basic protein 2 (PB2) subunit of the influenza A virus polymerase complex. A number of resistance mutations have previously been identified in treated patients or cell culture. Here, we generate a complete map of the effect of all single-amino-acid mutations to an avian PB2 on resistance to pimodivir. We identified both known and novel resistance mutations not only in the previously implicated cap-binding and mid-link domains, but also in the N-terminal domain. Our complete map of pimodivir resistance thus enables the evaluation of whether new viral strains contain mutations that will confer pimodivir resistance

Licensing of primordial germ cells for gametogenesis depends on genital ridge signaling

In mouse embryos at mid-gestation, primordial germ cells (PGCs) undergo licensing to become gametogenesis-competent cells (GCCs), gaining the capacity for meiotic initiation and sexual differentiation. GCCs then initiate either oogenesis or spermatogenesis in response to gonadal cues. Germ cell licensing has been considered to be a cell-autonomous and gonad-independent event, based on observations that some PGCs, having migrated not to the gonad but to the adrenal gland, nonetheless enter meiosis in a time frame parallel to ovarian germ cells -- and do so regardless of the sex of the embryo. Here we test the hypothesis that germ cell licensing is cell-autonomous by examining the fate of PGCs in Gata4 conditional mutant (Gata4 cKO) mouse embryos. Gata4, which is expressed only in somatic cells, is known to be required for genital ridge initiation. PGCs in Gata4 cKO mutants migrated to the area where the genital ridge, the precursor of the gonad, would ordinarily be formed. However, these germ cells did not undergo licensing and instead retained characteristics of PGCs. Our results indicate that licensing is not purely cell-autonomous but is induced by the somatic genital ridge

Retinoic acid activates two pathways required for meiosis in mice

In all sexually reproducing organisms, cells of the germ line must transition from mitosis to meiosis. In mice, retinoic acid (RA), the extrinsic signal for meiotic initiation, activates transcription of Stra8, which is required for meiotic DNA replication and the subsequent processes of meiotic prophase. Here we report that RA also activates transcription of Rec8, which encodes a component of the cohesin complex that accumulates during meiotic S phase, and which is essential for chromosome synapsis and segregation. This RA induction of Rec8 occurs in parallel with the induction of Stra8, and independently of Stra8 function, and it is conserved between the sexes. Further, RA induction of Rec8, like that of Stra8, requires the germ-cell-intrinsic competence factor Dazl. Our findings strengthen the importance of RA and Dazl in the meiotic transition, provide important details about the Stra8 pathway, and open avenues to investigate early meiosis through analysis of Rec8 induction and function

MEIOC is expressed throughout most of meiotic prophase I in the male and female germline.

<p>(A) Immunohistochemistry for MEIOC (brown) in adult testis, with hematoxylin counterstaining to enable identification of germ cell types by nuclear morphology [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006704#pgen.1006704.ref057" target="_blank">57</a>]. Low magnification image shows MEIOC staining in the majority of meiotic cell populations. Background staining was also observed in mature sperm in center of the tubule. High magnification images show that MEIOC was detected in meiotic germ cells at preleptotene (pL), leptotene (L), zygotene (Z), and pachytene (P) stages; it was not detected in meiotic germ cells at diplotene (D) stage, in cells undergoing meiotic metaphase (MI), or in postmeiotic round spermatids (rs). Low magnification scale bar = 100 μm, high magnification scale bar = 10 μm. (B) Immunohistochemistry for STRA8 (brown) in adult testis, counterstained with hematoxylin. In contrast to MEIOC, STRA8 expression is limited to germ cells initiating meiosis (preleptotene and leptotene stages) as well as differentiating spermatogonia. Scale bar = 100 μm. (C) Immunofluorescence staining for MEIOC in ovary at E13.5, E14.5, E16.5, E18.5, P0, P2, P15, and adult (>8 weeks). Mouse Vasa Homolog (MVH) costaining identifies germ cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006704#pgen.1006704.ref058" target="_blank">58</a>]. Nuclei stained by DAPI. MEIOC is detected in germ cells at all stages. From E13.5 to E18.5, corresponding to premeiotic to late pachytene stages, MEIOC is detected predominantly in cytoplasm. Towards the end of this period, MEIOC is detected in nucleus (arrowheads), and continues to be expressed in nuclei of germ cells at postnatal timepoints. Scale bar = 10 μm.</p

<i>Meioc</i>-deficient testicular and ovarian germ cells express molecular markers of meiotic prophase, but do not progress past early zygotene.

<p>(A) Immunofluorescence staining for STRA8, MVH, and EdU incorporation, in wild-type and <i>Meioc</i> -/- P10 testis and E14.5 ovary sections. Insets: Higher magnification, STRA8 and EdU staining. In wild-type P10 testis, STRA8 expression is seen in a subset of MVH+ germ cells (arrowhead and inset), indicative of these cells initiating meiosis. STRA8+ germ cells are also observed in the <i>Meioc</i> -/- P10 testis. In wild-type and <i>Meioc</i> -/- E14.5 ovaries, STRA8 expression is visible in most MVH+ germ cells (arrowhead and inset), indicative of germ cells synchronously initiating meiosis. In wild-type and <i>Meioc</i> -/- ovaries of both sexes, some STRA8+ cells are also EdU+ (arrowhead and inset), reflecting premeiotic DNA synthesis. Scale bar = 10 μm. (B) Immunofluorescence staining for DMC1, SYCP3, and MVH, in wild-type and <i>Meioc</i> -/- P15 testis and E16.5 ovary sections. Insets: Higher magnification, SYCP3 staining. In wild-type P15 testis, we expected to observe germ cells in leptotene, zygotene, and pachytene (empty arrowhead and inset) stages of meiotic prophase. DMC1 expression and SYCP3 localization along the chromosomes is consistent with these stages. In <i>Meioc</i> -/- P15 testis, expression of both DMC1 and SYCP3 is seen, but the pattern of SYCP3 localization does not progress beyond what is typical of early zygotene, and is often accompanied by SYCP3 aggregates (empty arrowhead and inset). Additionally, some germ cells contain only SYCP3 aggregates (filled arrowhead and inset). In wild-type E16.5 ovary, we expected most germ cells to be in pachytene of meiotic prophase. DMC1 expression and SYCP3 localization along the chromosomes are consistent with pachytene stage (empty arrowhead and inset). In <i>Meioc</i> -/- E16.5 ovary, DMC1 expression and SYCP3 expression are also observed, but the pattern of SYCP3 localization does not progress beyond what is typical of early zygotene, and is often accompanied by SYCP3 aggregates (empty arrowhead and inset). Some germ cells contain only SYCP3 aggregates (filled arrowhead and inset). Scale bar = 10 μm. (C) Immunofluorescence staining for DMC1, γH2AX, and SYCP3 in chromosome spreads of wild-type and <i>Meioc</i> -/- germ cells from P15 testis. DNA stained by DAPI. In wild-type germ cells, we observed DMC1, γH2AX, and SYCP3 localization consistent with leptotene, zygotene, and pachytene stages of meiotic prophase. In some <i>Meioc</i> -/- germ cells, we observed DMC1, γH2AX, and SYCP3 staining indicative of leptotene and early zygotene stages. In metaphase-like cells, we observed SYCP3 localization at the ends of chromosomes, likely at centromeres. (D) Immunofluorescence staining for SYCP3 and SYCP1 in chromosome spreads of wild-type and <i>Meioc</i> -/- zygotene stage germ cells from P15 testis. In both wild-type and <i>Meioc</i> -/- germ cells, SYCP3 localizes along the entire length of chromosomes, and SYCP1 localizes to regions of synapsis. (E) Immunofluorescence staining for SYCP3 and REC8 in chromosome spreads of wild-type and <i>Meioc</i> -/- germ cells from P15 testis. DNA stained by DAPI. In both wild-type and <i>Meioc</i> -/- zygotene stage germ cells, SYCP3 and REC8 localize along the entire length of chromosomes. In metaphase-like cells, SYCP3 and REC8 localize, respectively, to the ends of chromosomes and to the condensed chromosomes. This localization of SYCP3 and REC8 is similar to that observed in wild-type metaphase I germ cells adult testes.</p

<i>Sycp3</i> induction precedes negative regulation of <i>Rec8</i> along the anterior-posterior axis of the E14.5 ovary.

<p>(A) Representative scatterplot of transcript densities of <i>Sycp3</i> against <i>Rec8</i> in E14.5 wild-type ovarian germ cells. (B) Scatterplots of transcript densities in germ cells from (A), divided by location in the posteriormost, mid-posterior, mid-anterior, and anteriormost quarters of the ovary. (C) The trajectory of gene expression for an individual germ cell as inferred from gene expression at posterior, middle, and anterior positions.</p

<i>Meioc</i>-deficient testicular and ovarian germ cells express molecular markers of metaphase.

<p>Immunofluorescence staining for CENPA and α-TUB in wild-type adult testicular germ cells in metaphase I, as well as wild-type and <i>Meioc</i> -/- P15 testis and E16.5 ovary sections. Nuclei stained by DAPI; MVH immunostains germ cells. Inset: CENPA and α-TUB staining together, and separately. In wild-type adult testicular germ cells in metaphase I, CENPA localizes to the metaphase plate and a bipolar spindle is formed. In wild-type P15 testis and E16.5 ovary, CENPA localizes to periphery of nuclei in meiotic cells, and no spindle is observed. In <i>Meioc</i> -/- P15 testis and E16.5 ovary, CENPA does not localize to periphery of nuclei, and instead localizes to ends of a disorganized, radiating spindle. Scale bar = 10 μm.</p

RNA-seq analysis of <i>Dazl</i> and <i>Stra8</i>-deficient fetal gonads reveals <i>Stra8</i>-independent regulation of meiotic prophase genes.

<p>Relative expression of 104 meiotic prophase-specific genes in E14.5 wild-type, <i>Stra8</i>-deficient, and <i>Dazl</i>-deficient ovary. Gene expression was measured by RNA-seq and represented as log transformed and mean centered FPKM. Genes (rows) are arranged from least to most down-regulated in the <i>Stra8</i>-deficient ovary relative to the <i>Dazl</i>-deficient ovary, with the exception of the bottom four genes which were not significantly down-regulated in the <i>Dazl</i>-deficient ovary (q > 0.05), and were expressed at < 5 FPKM in wild-type, <i>Dazl</i>-deficient, and <i>Stra8</i>-deficient ovaries. Thirteen genes, listed to the right of the gene expression heat map, were selected for subsequent smFISH analysis. Source data for the 104 meiotic prophase genes, as well as all Refseq annotated genes, is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005531#pgen.1005531.s008" target="_blank">S2 Table</a>.</p

Identification of MEIOC-interacting proteins by quantitative mass spectrometry.

<p>Identification of MEIOC-interacting proteins by quantitative mass spectrometry.</p