Zebrafish macroH2A variants have distinct embryo localization and function

Mouse and cell-based studies have shown that macroH2A histone variants predominantly associate with heterochromatin. Functional studies found that macroH2As are involved in gene repression, inhibiting the acquisition of pluripotency and preserving cell differentiation. However, only a few studies have analysed the role of macroH2A during early embryo development. We report the development of transgenic zebrafish lines expressing macroH2A isoforms (mH2A1 and mH2A2) fusion proteins (with GFP) under identified endogenous promoters. We found that mH2A1 and mH2A2 have different spatial and temporal expression patterns during embryonic development. mH2A1 is expressed mostly in the extraembryonic Yolk Syncytial Layer (YSL) starting before shield stage and decreasing once morphogenesis is completed. mH2A2 expression lags behind mH2A1, becoming evident at 24 hpf, within the whole body of the embryo proper. Our ChIP-seq analysis showed that mH2A1 and mH2A2 bind to different DNA regions, changing dramatically after gastrulation. We further analysed RNA-seq data and showed that there is not a general/unspecific repressing function of mH2A1 or mH2A2 associated with heterochromatin but a fine regulation depending on cell types and stage of development. mH2A1 downregulates DNA expression in specific cells and embryo stages and its effect is independent of heterochromatin formation but it is correlated with nucleus quiescence instead. Whereas mH2A2 DNA association correlates with upregulation of differentially expressed genes between 75% epiboly and 24 hpf stages. Our data provide information for underlying molecules that participate in crucial early developmental events, and open new venues to explore mH2A related mechanisms that involve cell proliferation, differentiation, migration and metabolism

Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta

Background - During natural fertilization, sperm fusion with the oocyte induces long lasting intracellular calcium oscillations which in turn are responsible for oocyte activation. PLCZ1 has been identified as the factor that the sperm delivers into the egg to induce such a response. We tested the hypothesis that PLCZ1 cRNA injection can be used to activate bovine oocytes. Results - Mouse and bovine PLCZ1 cRNAs were injected into matured bovine oocytes at different concentrations. Within the concentrations tested, mouse PLCZ1 injection activated bovine oocytes at a maximum rate when the pipette concentration of cRNA ranged from 0.25 to 1 μg/μL, while bovine PLCZ1 was optimal at 0.1 μg/μL. At their most effective concentrations, PLCZ1 induced parthenogenetic development at rates similar to those observed using other activation stimuli such as Ionomycin/CHX and Ionomycin/DMAP. Injection of mouse and bovine PLCZ1 cRNA induced dose-dependent sperm-like calcium oscillations whose frequency increased over time. Injection of bovine and mouse PLCZ1 cRNA also induced IP3R-1 degradation, although bovine PLCZ1 cRNA evoked greater receptor degradation than its mouse counterpart. Conclusion - Injection of PLCZ1 cRNA efficiently activated bovine oocytes by inducing a sperm-like calcium oscillatory pattern. Importantly, the high rate of aneuploidy encountered in parthenogenetic embryos activated by certain chemical means was not observed in PLCZ1 activated embryos

Telomere Dynamics in Human Cells Reprogrammed to Pluripotency

BACKGROUND:Human induced pluripotent stem cells (IPSCs) have enormous potential in the development of cellular models of human disease and represent a potential source of autologous cells and tissues for therapeutic use. A question remains as to the biological age of IPSCs, in particular when isolated from older subjects. Studies of cloned animals indicate that somatic cells reprogrammed to pluripotency variably display telomere elongation, a common indicator of cell "rejuvenation." METHODOLOGY/PRINCIPAL FINDINGS:We examined telomere lengths in human skin fibroblasts isolated from younger and older subjects, fibroblasts converted to IPSCs, and IPSCs redifferentiated through teratoma formation and explant culture. In IPSCs analyzed at passage five (P5), telomeres were significantly elongated in 6/7 lines by >40% and approximated telomere lengths in human embryonic stem cells (hESCs). In cell lines derived from three IPSC-teratoma explants cultured to P5, two displayed telomeres shortened to lengths similar to input fibroblasts while the third line retained elongated telomeres. CONCLUSIONS/SIGNIFICANCE:While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age

Reprogrammed Transcriptome in Rhesus-Bovine Interspecies Somatic Cell Nuclear Transfer Embryos

Global activation of the embryonic genome (EGA), one of the most critical steps in early mammalian embryo development, is recognized as the time when interspecies somatic cell nuclear transfer (iSCNT) embryos fail to thrive.In this study, we analyzed the EGA-related transcriptome of rhesus-bovine iSCNT 8- to 16-cell embryos and dissected the reprogramming process in terms of embryonic gene activation, somatic gene silencing, and maternal RNA degradation. Compared with fibroblast donor cells, two thousand and seven genes were activated in iSCNT embryos, one quarter of them reaching expression levels comparable to those found in in vitro fertilized (IVF) rhesus embryos. This suggested that EGA in iSCNT embryos had partially recapitulated rhesus embryonic development. Eight hundred and sixty somatic genes were not silenced properly and continued to be expressed in iSCNT embryos, which indicated incomplete nuclear reprogramming. We compared maternal RNA degradation in bovine oocytes between bovine-bovine SCNT and iSCNT embryos. While maternal RNA degradation occurred in both SCNT and iSCNT embryos, we saw more limited overall degradation of maternal RNA in iSCNT embryos than in SCNT embryos. Several important maternal RNAs, like GPF9, were not properly processed in SCNT embryos.Our data suggested that iSCNT embryos are capable of triggering EGA, while a portion of somatic cell-associated genes maintain their expression. Maternal RNA degradation seems to be impaired in iSCNT embryos. Further understanding of the biological roles of these genes, networks, and pathways revealed by iSCNT may expand our knowledge about cell reprogramming, pluripotency, and differentiation

Disciplina radnje, evokacija s fusnotama uloga vlaka, u školovanju

Tion. b) Pipette advanced into the oocyte; cytoplasm is aspirated to break the plasma membrane. c) Aspirated cytoplasm and Texas Red dextran are injected into the oocyte. d) Schematic representation of the microscope reticulum used as guide to control the injected volume. The oocyte is represented in yellow and the pipette in blue. The red lines indicate the volume introduced into the oocyte which, calculated measuring the pipette internal diameters at both ends, is 5.9 pL. e) An oil drop of the same size as the injected volume is shown next to an oocyte. f) Oocytes injected using Texas Red dextran. g) From left to right, oocyte injected 2X and 1X the normal volume of Texas Red dextran. h) Fluorescent intensity profile of the line shown in f. i) Fluorescent intensity profile of the line shown in g. j) Developmental rates of injected and uninjected bovine oocytes after activation using ionomycin/DMAP.<p><b>Copyright information:</b></p><p>Taken from "Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta"</p><p>http://www.biomedcentral.com/1471-213X/8/16</p><p>BMC Developmental Biology 2008;8():16-16.</p><p>Published online 19 Feb 2008</p><p>PMCID:PMC2266721.</p><p></p

Tissue engineering, stem cells, cloning, and parthenogenesis: new paradigms for therapy

Patients suffering from diseased and injured organs may be treated with transplanted organs. However, there is a severe shortage of donor organs which is worsening yearly due to the aging population. Scientists in the field of tissue engineering apply the principles of cell transplantation, materials science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Both therapeutic cloning (nucleus from a donor cell is transferred into an enucleated oocyte), and parthenogenesis (oocyte is activated and stimulated to divide), permit extraction of pluripotent embryonic stem cells, and offer a potentially limitless source of cells for tissue engineering applications. The stem cell field is also advancing rapidly, opening new options for therapy. The present article reviews recent progress in tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure

Parthenogenic Blastocysts Derived from Cumulus-Free In Vitro Matured Human Oocytes

Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis..Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer

Influence of the postoperative inflammatory response on cognitive decline in elderly patients undergoing on-pump cardiac surgery: a controlled, prospective observational study

BACKGROUND: The role of non-infective inflammatory response (IR) in the aetiology of postoperative cognitive dysfunction (POCD) is still controversial. The aim of this controlled, prospective observational study was to assess the possible relationship between the grade of IR, defined by procalcitonin (PCT) changes, and development of POCD related to cardiac surgery. METHODS: Forty-two patients, who were >/= 60 years of age and scheduled for elective cardiac surgery, were separated into the low inflammatory (LIR) and high inflammatory (HIR) response groups based on their PCT levels measured on the first postoperative day. A matched normative control group of 32 subjects was recruited from primary care practice. The PCT and C-reactive protein (CRP) levels were monitored daily during the first five postoperative days. The cognitive function and mood state were preoperatively tested with a set of five neurocognitive tests and two mood inventories and at the seventh postoperative day. The Reliable Change Index modified for practice (RCIp) using data from normative controls was applied to determine the significant decline in test performance. RESULTS: The LIR (n = 20) and HIR (n = 22) groups differed significantly in the PCT (p 0.05). Additionally, there was no difference in the mood states, anxiety levels and perioperative parameters known to influence the development of POCD. CONCLUSIONS: In this study, the magnitude of the non-infective inflammatory response generated by on-pump cardiac surgery did not influence the development of POCD in the early postoperative period in elderly patients

Cytokines as mediators of chemotherapy-associated cognitive changes: Current evidence, limitations and directions for future research

10.1371/journal.pone.0081234PLoS ONE812-POLN

Maltreated children use more grammatical negations

Many studies reveal a strong impact of childhood maltreatment on language development, mainly resulting in shorter utterances, less rich vocabulary, or a delay in grammatical complexity. However, different theories suggest the possibility for resilience – a positive adaptation to an otherwise adverse environment – in children who experienced childhood maltreatment. Here, we investigated different measures for language development in spontaneous speech, examining whether childhood maltreatment leads to a language deficit only or whether it can also result in differences in language use due to a possible adaptation to a toxic environment. We compared spontaneous speech during therapeutic peer-play sessions of 32 maltreated and 32 non-maltreated children from the same preschool and equivalent in gender, age (2 to 5 years), home neighborhood, ethnicity, and family income. Maltreatment status was reported by formal child protection reports, and corroborated by independent social service reports. We investigated general language sophistication (i.e., vocabulary, talkativeness, mean length of utterance), as well as grammatical development (i.e., use of plurals, tense, grammatical negations). We found that maltreated and non-maltreated children showed similar sophistication across all linguistic measures, except for the use of grammatical negations. Maltreated children used twice as many grammatical negations as non-maltreated children. The use of this highly complex grammatical structure shows an advanced linguistic skill, which shows that childhood maltreatment does not necessarily lead to a language deficit. The result might indicate the development of a negativity bias in the structure of spontaneous language due to an adaptation to their experiences