A gust of WNT: analysis of the canonical WNT pathway

The Wnt pathway is a signal-transduction cascade that mediates communication between cells; the Wnt pathway is involved in key steps during embryological development and in the maintenance of adult tissue homeostasis. Mutational dysregulation of Wnt cascade components has been observed in diverse human pathological conditions and in oncogenic transformations. For these reasons, the Wnt signalling pathway has acquired growing interest in scientific and medical research over recent years. This review outlines the biochemical and functional features of the Wnt cascade with particular emphasis on a detailed functional analysis of all key players. In this instance, the regulations of the pathway have also been covered, emphasizing novelty in this regard. Furthermore, past and present studies on Wnt have been included, as well as a prediction of scientific progress, which may be made in this rapidly evolving field, in the near future; the review also embraces considerations on how further understanding of the Wnt pathway will provide important insight into managing human diseases

DNA damage responses in the context of the cell division cycle

Pagination different from approved bound copy, but content the same.During my PhD, I have investigated aspects of the DNA damage response (DDR) in the context of three different cellular scenarios: DNA damage signalling in response to double-strand breaks during mitosis, coordination of DNA replication with DNA damage responses by regulation of the GINS complex, and checkpoint activation by the prototypical checkpoint protein Rad9. Here, I show that mitotic cells treated with DNA break-inducing agents activate a ‘primary’ DDR, including ATM and DNA-PK-dependent H2AX phosphorylation and recruitment of MDC1 and the MRN complex to damage sites. However, downstream DDR events and induction of a DNA damage checkpoint are inhibited in mitosis, with full DDR activation only ensuing when damaged mitotic cells enter G1. In addition, I provide evidence that induction of a primary DDR in mitosis is biologically important for cell viability. The GINS complex is an evolutionarily conserved component of the DNA replication machinery and may represent an ideal candidate for transferring the DNA damage signal to the replication apparatus. Here, I show the identification of a consensus ‘SQ’ PIKK phosphorylation motif at the carboxyl end of the GINS complex subunit, Psf1. In Saccharomyces cerevisiae, switching the conserved serine to a glutamic acid is lethal, indicating that the site is crucial for the protein’s function. Moreover, in human cells, I identified UV-DDB, a heterodimeric complex involved in NER repair, as a binding partner that specifically interacts with the Psf1 C-terminus in vitro. Finally, I discuss my findings in characterizing functional interactions between Rad9 and Chk1 in S. cerevisiae. I show that specific consensus CDK sites within Rad9 N-terminus are essential to enable Chk1 phosphorylation and activation, and that MCPH1, a human homologue of Rad9, may share a conserved function in binding and activating Chk1, underscoring the evolutionarily conservation of checkpoint activation mechanisms

SEA SURFACE TEMPERATURES RECONSTRUCTION OF THE LAST 16,000 YEARS IN THE EASTERN MEDITERRANEAN SEA

A detailed study has been performed on two eastern Mediterranean box cores (BC02 and BC06) and on a southern Adriatic piston core (AD91-17) on the alkenone unsaturation ratio, a molecular proxy for past sea surface temperatures. The aim was to identify climatic events of the last 16 Ky, with particular attention on the conditions during formation of sapropel S1. All three temperature curves lack evidence for cooling in the Younger Dryas stadial and warming in the Boelling/Alleroed interstadial events. Just prior to the sapropel S1 base, SST cooled and increased by about 5°C during the sapropel deposition interval. Within sapropel S1, SST show a marked warming followed by a clear cooling. In the topmost intervals of the cores SST are mostly constant, but a warming event is always observed. This warming phase may correspond to the Medieval climatic Optimum (in the AD91-17 core) and to the Roman Optimum (in the box cores)

DNA damage signaling in response to double-strand breaks during mitosis

Dividing cells can sense DNA damage and initiate a primary response, but repair isn’t completed until the cells enter G1

Epigenetics as an Evolutionary Tool for Centromere Flexibility

Centromeres are the complex structures responsible for the proper segregation of chromosomes during cell division. Structural or functional alterations of the centromere cause aneuploidies and other chromosomal aberrations that can induce cell death with consequences on health and survival of the organism as a whole. Because of their essential function in the cell, centromeres have evolved high flexibility and mechanisms of tolerance to preserve their function following stress, whether it is originating from within or outside the cell. Here, we review the main epigenetic mechanisms of centromeres’ adaptability to preserve their functional stability, with particular reference to neocentromeres and holocentromeres. The centromere position can shift in response to altered chromosome structures, but how and why neocentromeres appear in a given chromosome region are still open questions. Models of neocentromere formation developed during the last few years will be hereby discussed. Moreover, we will discuss the evolutionary significance of diffuse centromeres (holocentromeres) in organisms such as nematodes. Despite the differences in DNA sequences, protein composition and centromere size, all of these diverse centromere structures promote efficient chromosome segregation, balancing genome stability and adaptability, and ensuring faithful genome inheritance at each cellular generation

Visualization of the three-dimensional structure of the human centromere in mitotic chromosomes by super-resolution microscopy

The human centromere comprises large arrays of repetitive alpha-satellite DNA at the primary constriction of mitotic chromosomes. In addition, centromeres are epigenetically specified by the centromere-specific histone H3 variant CENP-A that supports kinetochore assembly to enable chromosome segregation. Since CENP-A is bound to only a fraction of the alpha-satellite elements within the megabase-sized centromere DNA, correlating the three-dimensional (3D) organization of alpha-satellite DNA and CENP-A remains elusive. To visualize centromere organization within a single chromatid, we used a combination of the Centromere Chromosome Orientation Fluorescent In Situ Hybridization (Cen-CO-FISH) technique together with Structured Illumination Microscopy (SIM). Cen-CO-FISH allows the differential labeling of the sister chromatids without the denaturation step used in conventional FISH that may affect DNA structure. Our data indicate that alpha-satellite DNA is arranged in a ring-like organization within prometaphase chromosomes, in presence or absence of spindle's microtubules. Using expansion microscopy (ExM), we found that CENP-A organization within mitotic chromosomes follows a rounded pattern similar to that of alpha-satellite DNA, often visible as a ring thicker at the outer surface oriented towards the kinetochore-microtubules interface. Collectively, our data provide a 3D reconstruction of alpha-satellite DNA along with CENP-A clusters that outline the overall architecture of the mitotic centromere. [Media: see text] [Media: see text] [Media: see text] [Media: see text

Xenon Upregulates Hypoxia Inducible Factor 1 Alpha in Neonatal Rat Brain under Normoxic Conditions

Xenon can induce cell and organ protection through different molecular mechanisms related to oxygen level. We explored the effect of xenon on oxygen-related signalling in the central nervous system via hypoxia inducible factor 1 alpha (HIF-1α) and mammalian target of rapamycin (mTOR). Methods. Postnatal day 7 (P7) Sprague Dawley rats were exposed to 25% oxygen/75% nitrogen (air group) or 25% oxygen/75% xenon (treatment group) for 120 min. Brains were collected immediately (transcript analysis—relative real-time polymerase chain reaction) or 24 hours (protein analysis—immunohistochemistry) after the 120-minute exposure period; peak anesthetic preconditioning has been previously identified at 24 hours post-exposure. Results. HIF-1α transcript and protein levels were found to be increased in xenon-exposed compared to air-exposed brains. Sustained nuclear translocation of the protein, accounting for an increased activity of HIF-1α, was also noted. mTOR transcript analysis revealed no significant difference between xenon-exposed and air-exposed brains immediately after the 120-minute exposure. Conclusion. Our data suggest that xenon induces the upregulation of HIF-1α transcription and translation, which may contribute to xenon's neuroprotective preconditioning effect. However, given that xenon exposure did not affect mTOR transcription, further investigation into other signalling cascades mediating xenon's effects on HIF-1α in developing brain is warranted

Xenon Upregulates Hypoxia Inducible Factor 1 Alpha in Neonatal Rat Brain under Normoxic Conditions

Xenon can induce cell and organ protection through different molecular mechanisms related to oxygen level. We explored the effect of xenon on oxygen-related signalling in the central nervous system via hypoxia inducible factor 1 alpha (HIF-1α) and mammalian target of rapamycin (mTOR). Methods. Postnatal day 7 (P7) Sprague Dawley rats were exposed to 25% oxygen/75% nitrogen (air group) or 25% oxygen/75% xenon (treatment group) for 120 min. Brains were collected immediately (transcript analysis—relative real-time polymerase chain reaction) or 24 hours (protein analysis—immunohistochemistry) after the 120-minute exposure period; peak anesthetic preconditioning has been previously identified at 24 hours post-exposure. Results. HIF-1α transcript and protein levels were found to be increased in xenon-exposed compared to air-exposed brains. Sustained nuclear translocation of the protein, accounting for an increased activity of HIF-1α, was also noted. mTOR transcript analysis revealed no significant difference between xenon-exposed and air-exposed brains immediately after the 120-minute exposure. Conclusion. Our data suggest that xenon induces the upregulation of HIF-1α transcription and translation, which may contribute to xenon's neuroprotective preconditioning effect. However, given that xenon exposure did not affect mTOR transcription, further investigation into other signalling cascades mediating xenon's effects on HIF-1α in developing brain is warranted

FANCD2 promotes mitotic rescue from transcription-mediated replication stress in SETX-deficient cancer cells

Replication stress (RS) is a leading cause of genome instability and cancer development. A substantial source of endogenous RS originates from the encounter between the transcription and replication machineries operating on the same DNA template. This occurs predominantly under specific contexts, such as oncogene activation, metabolic stress, or a deficiency in proteins that specifically act to prevent or resolve those transcription-replication conflicts (TRCs). One such protein is Senataxin (SETX), an RNA:DNA helicase involved in resolution of TRCs and R-loops. Here we identify a synthetic lethal interaction between SETX and proteins of the Fanconi anemia (FA) pathway. Depletion of SETX induces spontaneous under-replication and chromosome fragility due to active transcription and R-loops that persist in mitosis. These fragile loci are targeted by the Fanconi anemia protein, FANCD2, to facilitate the resolution of under-replicated DNA, thus preventing chromosome mis-segregation and allowing cells to proliferate. Mechanistically, we show that FANCD2 promotes mitotic DNA synthesis that is dependent on XPF and MUS81 endonucleases. Importantly, co-depleting FANCD2 together with SETX impairs cancer cell proliferation, without significantly affecting non-cancerous cells. Therefore, we uncovered a synthetic lethality between SETX and FA proteins for tolerance of transcription-mediated RS that may be exploited for cancer therapy

Modelling potential maize yield with climate and crop conditions around flowering

Abstract Understanding, and then modelling, the effects of sowing date and cultivar on maize yield is essential to develop appropriate climate change adaptation strategies. Here we test the WOFOST model and a hybrid model, based on physiological crop conditions around flowering, against observed data collected during 4 years of field experiments in a Mediterranean environment under fully irrigated conditions. We simulate sowing date and cultivar responses by using 45-year historical meteorological records from the experimental weather station and future climate conditions till 2060 as projected by a set of regional climate models. Both WOFOST and the hybrid approach reveal good performance in simulating average maize yield. However, the hybrid one outperforms WOFOST with respect to its responsiveness to changes in sowing date and cultivar. These findings, besides stressing the importance of crop conditions around flowering in determining maize yield, point to lower yields (14 %–17 %, average reduction) under future climate conditions. The estimated losses may only be partially offset by changes in phenology and sowing dates