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A finely tuned balance between excitation and inhibition (E/I) is essential for proper brain function. Disruptions in the GABAergic system, which alter this equilibrium, are a common feature in various types of neurological disorders, including autism spectrum disorders (ASDs). Mutations in Phosphatase and Tensin Homolog (PTEN) , the main negative regulator of the phosphatidylinositol 3-phosphate kinase/Akt pathway, are strongly associated with ASD. However, it is unclear whether PTEN deficiencies can differentially affect inhibitory and excitatory signaling. Using the Caenorhabditis elegans neuromuscular system, where both excitatory (cholinergic) and inhibitory (GABAergic) inputs regulate muscle activity, we found that daf-18 / PTEN mutations impact GABAergic (but not cholinergic) neurodevelopment and function. This selective impact results in a deficiency in inhibitory signaling. The defects observed in the GABAergic system in daf-18/PTEN mutants are due to reduced activity of DAF-16/FOXO during development. Ketogenic diets (KGDs) have proven effective for disorders associated with E/I imbalances. However, the mechanisms underlying their action remain largely elusive. We found that a diet enriched with the ketone body β-hydroxybutyrate during early development induces DAF-16/FOXO activity, therefore improving GABAergic neurodevelopment and function in daf-18/PTEN mutants. Our study provides valuable insights into the link between PTEN mutations and neurodevelopmental defects and delves into the mechanisms underlying the potential therapeutic effects of KGDs., Competing Interests: SG, MB, MD, DR No competing interests declared, (© 2024, Giunti et al.)

In budding yeast, the integrity of both the nuclear and mitochondrial genomes relies on dual-targeted isoforms of the conserved Pif1 helicase, generated by alternative translation initiation (ATI) of PIF1 mRNA from two consecutive AUG codons flanking a mitochondrial targeting signal. Here, we demonstrate that ribosomal leaky scanning is the specific ATI mechanism that produces not only these, but also novel, previously uncharacterized Pif1 isoforms. Both in-frame, downstream AUGs as well as near-cognate start codons contribute to the generation of these alternative isoforms. This has crucial implications for the rational design of genuine separation-of-function alleles and provides an explanation for the suboptimal behaviour of the widely employed mitochondrial- (pif1-m1) and nuclear-deficient (pif1-m2) alleles, with mutations in the first or second AUG codon, respectively. We have taken advantage of this refined model to develop improved versions of these alleles, which will serve as valuable tools to elucidate novel functions of this helicase and to disambiguate previously described genetic interactions of PIF1 in the context of nuclear and mitochondrial genome stability., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Homologous recombination involves the formation of branched DNA molecules that may interfere with chromosome segregation. To resolve these persistent joint molecules, cells rely on the activation of structure-selective endonucleases (SSEs) during the late stages of the cell cycle. However, the premature activation of SSEs compromises genome integrity, due to untimely processing of replication and/or recombination intermediates. Here, we used a biochemical approach to show that the budding yeast SSEs Mus81 and Yen1 possess the ability to cleave the central recombination intermediate known as the displacement loop or D-loop. Moreover, we demonstrate that, consistently with previous genetic data, the simultaneous action of Mus81 and Yen1, followed by ligation, is sufficient to recreate the formation of a half-crossover precursor in vitro. Our results provide not only mechanistic explanation for the formation of a half-crossover, but also highlight the critical importance for precise regulation of these SSEs to prevent chromosomal rearrangements., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Myelination is essential for neuronal function and health. In peripheral nerves, >100 causative mutations have been identified that cause Charcot-Marie-Tooth disease, a disorder that can affect myelin sheaths. Among these, a number of mutations are related to essential targets of the posttranslational modification neddylation, although how these lead to myelin defects is unclear. Here, we demonstrate that inhibiting neddylation leads to a notable absence of peripheral myelin and axonal loss both in developing and regenerating mouse nerves. Our data indicate that neddylation exerts a global influence on the complex transcriptional and posttranscriptional program by simultaneously regulating the expression and function of multiple essential myelination signals, including the master transcription factor EGR2 and the negative regulators c-Jun and Sox2, and inducing global secondary changes in downstream pathways, including the mTOR and YAP/TAZ signaling pathways. This places neddylation as a critical regulator of myelination and delineates the potential pathogenic mechanisms involved in CMT mutations related to neddylation.

Background: The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown., Methods: SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6-SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1-GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student's t-test, or one-way or two-way analysis of variance (ANOVA)., Results: We found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells., Conclusions: SUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells., (© 2024. The Author(s).)

There has been an intense focus to uncover the molecular mechanisms by which fasting triggers the adaptive cellular responses in the major organs of the body. Here, we show that in mice, hepatic S-adenosylmethionine (SAMe)-the principal methyl donor-acts as a metabolic sensor of nutrition to fine-tune the catabolic-fasting response by modulating phosphatidylethanolamine N-methyltransferase (PEMT) activity, endoplasmic reticulum-mitochondria contacts, β-oxidation, and ATP production in the liver, together with FGF21-mediated lipolysis and thermogenesis in adipose tissues. Notably, we show that glucagon induces the expression of the hepatic SAMe-synthesizing enzyme methionine adenosyltransferase α1 (MAT1A), which translocates to mitochondria-associated membranes. This leads to the production of this metabolite at these sites, which acts as a brake to prevent excessive β-oxidation and mitochondrial ATP synthesis and thereby endoplasmic reticulum stress and liver injury. This work provides important insights into the previously undescribed function of SAMe as a new arm of the metabolic adaptation to fasting., Competing Interests: Declaration of interests M.L.M.-C. advises for Mitotherapeutix., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Common bean ( Phaseolus vulgaris L.) is an important crop for food security and for national economics for several countries worldwide. One of the most important factors of risk in common bean production is the fungal disease anthracnose caused by Colletotrichum lindemuthianum , which, in some cases, causes complete yield losses; this kind of plant disease is usually managed through the application of chemical products such as fungicides that are commonly not accepted by society. This rejection is based on the relationship of pesticides with health damage and environmental contamination. In order to help in solving these drawbacks, the present work proposes the use of electrochemically activated salt solutions (EASSs) as a safer pathogen control agent in crops, due to it having shown an elicitor and biostimulant effect on plants. With this background, this manuscript presents in vitro results of the evaluation of the inhibitory effect for multiple bean pathogens and in vivo results of EASS in the common bean- Colletotrichum pathosystem by evaluation of the infection severity and defense activation, such as secondary metabolite production and antioxidant activity. EASS presence in growth media had a strong inhibitory effect at the beginning of experiments for some of the evaluated fungi. EASSs showed an effect against the development of the disease when applied in specific doses to prevent distress in plants.

Due to the increase in life expectancy worldwide, age-related disorders such as neurodegenerative diseases (NDs) have become more prevalent. Conventional treatments comprise drugs that only attenuate some of the symptoms, but fail to arrest or delay neuronal proteotoxicity that characterizes these diseases. Due to their diverse biological activities, imidazole rings are intensively explored as powerful scaffolds for the development of new bioactive molecules. By using C. elegans, our work aims to explore novel biological roles for these compounds. To this end, we have tested the in vivo anti-proteotoxic effects of imidazolium salts . Since NDs have been largely linked to impaired antioxidant defense mechanisms, we focused on 1-Mesityl-3-(3-sulfonatopropyl) imidazolium (MSI), one of the imidazolium salts that we identified as capable of improving iron-induced oxidative stress resistance in wild-type animals. By combining mutant and gene expression analysis we have determined that this protective effect depends on the activation of the Heat Shock Transcription Factor (HSF-1), whereas it is independent of other canonical cytoprotective molecules such as abnormal Dauer Formation-16 (DAF-16/FOXO) and Skinhead-1 (SKN-1/Nrf2). To delve deeper into the biological roles of MSI, we analyzed the impact of this compound on previously established C. elegans models of protein aggregation. We found that MSI ameliorates β-amyloid-induced paralysis in worms expressing the pathological protein involved in Alzheimer's Disease. Moreover, this compound also delays age-related locomotion decline in other proteotoxic C. elegans models, suggesting a broad protective effect. Taken together, our results point to MSI as a promising anti-proteotoxic compound and provide proof of concept of the potential of imidazole derivatives in the development of novel therapies to retard age-related proteotoxic diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Andersen, Veuthey, Blanco, Silbestri, Rayes and De Rosa.)

Yen1 and GEN1 are members of the Rad2/XPG family of nucleases that were identified as the first canonical nuclear Holliday junction (HJ) resolvases in budding yeast and humans due to their ability to introduce two symmetric, coordinated incisions on opposite strands of the HJ, yielding nicked DNA products that could be readily ligated. While GEN1 has been extensively characterized in vitro, much less is known about the biochemistry of Yen1. Here, we have performed the first in-depth characterization of purified Yen1. We confirmed that Yen1 resembles GEN1 in many aspects, including range of substrates targeted, position of most incisions they produce or the increase in the first incision rate by assembly of a dimer on a HJ, despite minor differences. However, we demonstrate that Yen1 is endowed with additional nuclease activities, like a nick-specific 5'-3' exonuclease or HJ arm-chopping that could apparently blur its classification as a canonical HJ resolvase. Despite this, we show that Yen1 fulfils the requirements of a canonical HJ resolvase and hypothesize that its wider array of nuclease activities might contribute to its function in the removal of persistent recombination or replication intermediates., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Most cancers are characterized by the somatic acquisition of genomic rearrangements during tumour evolution that eventually drive the oncogenesis. Here, using multiplatform sequencing technologies, we identify and characterize a remarkable mutational mechanism in human hepatocellular carcinoma caused by Hepatitis B virus, by which DNA molecules from the virus are inserted into the tumour genome causing dramatic changes in its configuration, including non-homologous chromosomal fusions, dicentric chromosomes and megabase-size telomeric deletions. This aberrant mutational mechanism, present in at least 8% of all HCC tumours, can provide the driver rearrangements that a cancer clone requires to survive and grow, including loss of relevant tumour suppressor genes. Most of these events are clonal and occur early during liver cancer evolution. Real-time timing estimation reveals some HBV-mediated rearrangements occur as early as two decades before cancer diagnosis. Overall, these data underscore the importance of characterising liver cancer genomes for patterns of HBV integration., (© 2021. The Author(s).)

Holliday junctions are four-way DNA structures that may arise during meiotic recombination, double-strand break repair, or postreplicative repair by the reciprocal exchange of single strands between two DNA molecules. Given their ability to effectively bridge two sister chromatids or homologous chromosomes, cells have implemented various pathways to ensure their timely removal. One of them is the nucleolytic processing of the Holliday junctions by specialized structure-selective endonucleases termed resolvases, which sever the connection between the linked molecules. These Holliday junction resolvases are essential tools of the DNA damage repair machinery to ensure accurate chromosomal segregation, whose activities can be modulated by posttranslational modifications like phosphorylation. Here, we describe a protocol to purify S. cerevisiae Yen1 resolvase in two different phosphorylation states (high and low) and to set up a biochemical assay to compare their ability to process a synthetic, oligonucleotide-based Holliday junction structures.

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editing induces aberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity during cell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The S phase checkpoint is crucial to maintain genome stability under conditions that threaten DNA replication. One of its critical functions is to prevent Exo1-dependent fork degradation, and Exo1 is phosphorylated in response to different genotoxic agents. Exo1 seemed to be regulated by several post-translational modifications in the presence of replicative stress, but the specific contribution of checkpoint-dependent phosphorylation to Exo1 control and fork stability is not clear. We show here that Exo1 phosphorylation is Dun1-independent and Rad53-dependent in response to DNA damage or dNTP depletion, and in both situations Exo1 is similarly phosphorylated at multiple sites. To investigate the correlation between Exo1 phosphorylation and fork stability, we have generated phospho-mimic exo1 alleles that rescue fork collapse in rad53 mutants as efficiently as exo1-nuclease dead mutants or the absence of Exo1, arguing that Rad53-dependent phosphorylation is the mayor requirement to preserve fork stability. We have also shown that this rescue is Bmh1-2 independent, arguing that the 14-3-3 proteins are dispensable for fork stabilization, at least when Exo1 is downregulated. Importantly, our results indicated that phosphorylation specifically inhibits the 5' to 3'exo-nuclease activity, suggesting that this activity of Exo1 and not the flap-endonuclease, is the enzymatic activity responsible of the collapse of stalled replication forks in checkpoint mutants., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

About half of all cancers have somatic integrations of retrotransposons. Here, to characterize their role in oncogenesis, we analyzed the patterns and mechanisms of somatic retrotransposition in 2,954 cancer genomes from 38 histological cancer subtypes within the framework of the Pan-Cancer Analysis of Whole Genomes (PCAWG) project. We identified 19,166 somatically acquired retrotransposition events, which affected 35% of samples and spanned a range of event types. Long interspersed nuclear element (LINE-1; L1 hereafter) insertions emerged as the first most frequent type of somatic structural variation in esophageal adenocarcinoma, and the second most frequent in head-and-neck and colorectal cancers. Aberrant L1 integrations can delete megabase-scale regions of a chromosome, which sometimes leads to the removal of tumor-suppressor genes, and can induce complex translocations and large-scale duplications. Somatic retrotranspositions can also initiate breakage-fusion-bridge cycles, leading to high-level amplification of oncogenes. These observations illuminate a relevant role of L1 retrotransposition in remodeling the cancer genome, with potential implications for the development of human tumors.

The role Beta-cyclodextrin (βCD) on improving biocompatibility on healthy cellular and animal models was studied upon a formulation obtained from the development of a simple coating procedure. The obtained nanosystems were thoroughly characterized by FTIR, TGA, atomic absorption spectroscopy, dynamic light scattering and zeta potential, TEM/HR-TEM and magnetic properties. βCD might interact with the magnetic core through hosting OA. It is feasible that the nanocomposite is formed by nanoparticles of MG@OA dispersed in a βCD matrix. The evaluation of βCD role on biocompatibility was performed on two healthy models. To this end, in vivo studies were carried out on Caenorhabditis elegans. Locomotion and progeny were evaluated after exposure animals to MG, MG@OA, and MG@OA-βCD (10 to 500 µg/mL). The influence of βCD on cytotoxicity was explored in vitro on healthy rat aortic endothelial cells, avoiding alteration in the results derived from the use of transformed cell lines. Biological studies demonstrated that βCD attaching improves MG biocompatibility.

An animal's stress response requires different adaptive strategies depending on the nature and duration of the stressor. Whereas acute stressors, such as predation, induce a rapid and energy-demanding fight-or-flight response, long-term environmental stressors induce the gradual and long-lasting activation of highly conserved cytoprotective processes 1-3 . In animals across the evolutionary spectrum, continued activation of the fight-or-flight response weakens the animal's resistance to environmental challenges 4,5 . However, the molecular and cellular mechanisms that regulate the trade-off between the flight response and long-term stressors are poorly understood. Here we show that repeated induction of the flight response in Caenorhabditis elegans shortens lifespan and inhibits conserved cytoprotective mechanisms. The flight response activates neurons that release tyramine, an invertebrate analogue of adrenaline and noradrenaline. Tyramine stimulates the insulin-IGF-1 signalling (IIS) pathway and precludes the induction of stress response genes by activating an adrenergic-like receptor in the intestine. By contrast, long-term environmental stressors, such as heat or oxidative stress, reduce tyramine release and thereby allow the induction of cytoprotective genes. These findings demonstrate that a neural stress hormone supplies a state-dependent neural switch between acute flight and long-term environmental stress responses and provides mechanistic insights into how the flight response impairs cellular defence systems and accelerates ageing.

Nematode parasites cause substantial morbidity to billions of people and considerable losses in livestock and food crops. The repertoire of effective anthelmintic compounds for treating these parasitoses is very limited, as drug development has been delayed for decades. Moreover, resistance has become a global concern in livestock parasites and is an emerging issue for human helminthiasis. Therefore, anthelmintics with novel mechanisms of action are urgently needed. Taking advantage of Caenorhabditis elegans as an established model system, we here screened the nematicidal potential of novel imidazolium and imidazole derivatives. One of these derivatives, diisopropylphenyl-imidazole (DII), is lethal to C. elegans at both mature and immature stages. This lethal effect appears to be specific because DII concentrations which prove to be toxic to C. elegans do not induce significant lethality on bacteria, Drosophila melanogaster, and HEK-293 cells. Our analysis of DII action on C. elegans mutant strains determined that, in the adult stage, null mutants of unc-29 are resistant to the drug. Muscle expression of this gene completely restores DII sensitivity. UNC-29 has been largely reported as an essential constituent of the levamisole-sensitive muscle nicotinic receptor (L-AChR). Nevertheless, null mutants in unc-63 and lev-8 (essential and non-essential subunits of L-AChRs, respectively) are as sensitive to DII as the wild-type strain. Therefore, our results suggest that DII effects on adult nematodes rely on a previously unidentified UNC-29-containing muscle AChR, different from the classical L-AChR. Interestingly, DII targets appear to be different between larvae and adults, as unc-29 null mutant larvae are sensitive to the drug. The existence of more than one target could delay resistance development. Its lethality on C. elegans, its harmlessness in non-nematode species and its novel and dual mechanism of action make DII a promising candidate compound for anthelmintic therapy., Competing Interests: The authors have declared that no competing interests exist.

During meiosis, crossover recombination promotes the establishment of physical connections between homologous chromosomes, enabling their bipolar segregation. To ensure that persistent recombination intermediates are disengaged prior to the completion of meiosis, the Yen1(GEN1) resolvase is strictly activated at the onset of anaphase II. Whether controlled activation of Yen1 is important for meiotic crossing-over is unknown. Here, we show that CDK-mediated phosphorylation of Yen1 averts its pervasive recruitment to recombination intermediates during prophase I. Yen1 mutants that are refractory to phosphorylation resolve DNA joint molecules prematurely and form crossovers independently of MutLγ, the central crossover resolvase during meiosis. Despite bypassing the requirement for MutLγ in joint molecule processing and promoting crossover-specific resolution, unrestrained Yen1 impairs the spatial distribution of crossover events, genome-wide. Thus, active suppression of Yen1 function, and by inference also of Mus81-Mms4(EME1) and Slx1-Slx4(BTBD12) resolvases, avoids precocious resolution of recombination intermediates to enable meiotic crossover patterning., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ten-eleven translocation (TET) proteins play key roles in the regulation of DNA-methylation status by oxidizing 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), which can both serve as a stable epigenetic mark and participate in active demethylation. Unlike the other members of the TET family, TET2 does not contain a DNA-binding domain, and it remains unclear how it is recruited to chromatin. Here we show that TET2 is recruited by the RNA-binding protein Paraspeckle component 1 (PSPC1) through transcriptionally active loci, including endogenous retroviruses (ERVs) whose long terminal repeats (LTRs) have been co-opted by mammalian genomes as stage- and tissue-specific transcriptional regulatory modules. We found that PSPC1 and TET2 contribute to ERVL and ERVL-associated gene regulation by both transcriptional repression via histone deacetylases and post-transcriptional destabilization of RNAs through 5hmC modification. Our findings provide evidence for a functional role of transcriptionally active ERVs as specific docking sites for RNA epigenetic modulation and gene regulation.

DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81-Mms4, this cell cycle stage-specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7-Dbf4 (DDK), targets Mus81-Mms4 in conjunction with Cdc5-both kinases bind to as well as phosphorylate Mus81-Mms4 in an interdependent manner. Moreover, DDK-mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which binds the Mus81-Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, CDK, Cdc5 and DDK Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution., (© 2017 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

While DNA replication and mitosis occur in a sequential manner, precisely how cells maintain their temporal separation and order remains elusive. Here, we unveil a double-negative feedback loop between replication intermediates and an M-phase-specific structure-selective endonuclease, MUS81-SLX4, which renders DNA replication and mitosis mutually exclusive. MUS81 nuclease is constitutively active throughout the cell cycle but requires association with SLX4 for efficient substrate targeting. To preclude toxic processing of replicating chromosomes, WEE1 kinase restrains CDK1 and PLK1-mediated MUS81-SLX4 assembly during S phase. Accordingly, WEE1 inhibition triggers widespread nucleolytic breakage of replication intermediates, halting DNA replication and leading to chromosome pulverization. Unexpectedly, premature entry into mitosis-licensed by unrestrained CDK1 activity during S phase-requires MUS81-SLX4, which inhibits DNA replication. This suggests that ongoing replication assists WEE1 in delaying entry into M phase and, indirectly, in preventing MUS81-SLX4 assembly. Conversely, MUS81-SLX4 activation during mitosis promotes targeted resolution of persistent replication intermediates, which safeguards chromosome segregation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Repair of DNA lesions through homologous recombination promotes the establishment of stable chromosomal interactions. Multiple helicases, topoisomerases and structure-selective endonucleases (SSEs) act upon recombining joint molecules (JMs) to disengage chromosomal connections and safeguard chromosome segregation. Recent studies on two conserved SSEs - MUS81 and Yen1/GEN1- uncovered multiple layers of regulation that operate to carefully tailor JM-processing according to specific cellular needs. Temporal restriction of SSE function imposes a hierarchy in pathway usage that ensures efficient JM-processing while minimizing reciprocal exchanges between the recombining DNAs. Whereas a conserved strategy of fine-tuning SSE functions exists in different model systems, the precise molecular mechanisms to implement it appear to be significantly different. Here, we summarize the current knowledge on the cellular switches that are in place to control MUS81 and Yen1/GEN1 functions.

DNA strand break repair by homologous recombination leads to the formation of intermediates in which sister chromatids are covalently linked. The efficient processing of these joint molecules, which often contain four-way structures known as Holliday junctions, is necessary for efficient chromosome segregation during mitotic division. Because persistent chromosome bridges pose a threat to genome stability, cells ensure the complete elimination of joint molecules through three independent pathways. These involve (1) BLM-Topoisomerase IIIα-RMI1-RMI2 (BTR complex), (2) SLX1-SLX4-MUS81-EME1 (SLX-MUS complex), and (3) GEN1. The BTR pathway promotes the dissolution of double Holliday junctions, which avoids the formation of crossover products, prevents sister chromatid exchanges, and limits the potential for loss of heterozygosity. In contrast to BTR, the other two pathways resolve Holliday junctions by nucleolytic cleavage to yield crossover and non-crossover products. To avoid competition with BTR, the resolution pathways are restrained until the late stages of the cell cycle. The temporal regulation of the dissolution/resolution pathways is therefore critical for crossover avoidance while also ensuring that all covalent links between chromosomes are resolved before chromosome segregation., (Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.)

The careful orchestration of cellular events such as DNA replication, repair, and segregation is essential for equal distribution of the duplicated genome into two daughter cells. To ensure that persistent recombination intermediates are resolved prior to cell division, the Yen1 Holliday junction resolvase is activated at anaphase. Here, we show that the master cell-cycle regulators, cyclin-dependent kinase (Cdk) and Cdc14 phosphatase, control the actions of Yen1. During S phase, Cdk-mediated phosphorylation of Yen1 promotes its nuclear exclusion and inhibits catalytic activity by reducing the efficiency of DNA binding. Later in the cell cycle, at anaphase, Cdc14 drives Yen1 dephosphorylation, leading to its nuclear relocalization and enzymatic activation. Using a constitutively activated form of Yen1, we show that uncontrolled Yen1 activity is detrimental to the cell: spatial and temporal restriction of Yen1 protects against genotoxic stress and, by avoiding competition with the noncrossover-promoting repair pathways, prevents loss of heterozygosity., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Homologous recombination leads to the formation of DNA joint molecules (JMs) that must be resolved to allow chromosome segregation, but how resolution is temporally coupled with chromosome segregation is unknown. Here, we have analyzed the role of the cell-cycle kinases Cdk and Cdc5 in coordinating these events through their involvement in the phosphoregulation of the Mus81-Mms4 nuclease. By identifying CDC5 and MMS4 mutants that uncouple Mus81-Mms4 activation from cell-cycle progression, we show that JM disengagement, prior to anaphase initiation, safeguards chromosome segregation. By simultaneously stimulating the cleavage of cohesin and activating Mus81-Mms4 at the G2/M transition, Cdk and Cdc5 coordinate the sequential elimination of all chromosomal interactions in preparation for chromosome segregation. Conversely, untimely Cdc5 expression increases crossover frequency due to premature activation of Mus81-Mms4. Therefore, temporal restriction of JM resolution, imposed by Cdk/Cdc5, minimizes the potential for loss of heterozygosity while preventing chromosome missegregation and aneuploidy., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Study Design: Prospective clinical study., Objectives: To assess fasting and postprandial (PP) perception of rectal distension and its correlation with symptoms in patients with spinal cord injury (SCI) and neurogenic bowel dysfunction compared to ten healthy subjects (HS)., Setting: Experimental Medicine and Motility Unit, Mexico General Hospital and National Institute of Rehabilitation., Methods: Twenty patients with complete SCI at cervical, thoracic and lumbar levels [American Spinal Injury Association (ASIA) A] were studied. Rectal sensitivity was evaluated with a barostat., Results: In SCI patients, while lower the rectal tone more time was used for defecate (R=0.50, P=0.048) and more PP episodes of fecal incontinence occur (R=0.54, P=0.030). The thresholds for non-noxious stimuli of first (23.6 mmHg, CI 19.5-27.7) vs 14.0 (CI 10.9-17.1), P=0.004; gas (27.9 mmHg, CI 19.9-35.8) vs 17.9 mmHg (CI 14.25-21.69), P=0.02 and urge-to-defecate sensation (33.2 mmHg, CI 27.5-38.8) vs 22.4 mmHg (CI 17.9-26.9), P=0.01 were reported by SCI patients at higher pressure than HS, respectively. SCI patients reported PP pain sensation at a lower pressure than controls (27.8 mmHg, CI 21.5-34.2 vs 36.5 mmHg, CI 31.8-41.2), P=0.04., Conclusion: SCI patients preserve rectal sensation, present rectal hyposensitivity for non-noxious stimuli and PP hypersensitivity. Lower rectal tone was related to the time used for defecate and with fecal incontinence. The results suggest that an intact neural transmission between the spinal cord and higher centres is indispensable for noxious stimulus, but not for non-noxious stimuli. Also, barostat sensitivity studies can complement ASIA criteria to verify a complete injury.

Objectives: To identify and classify disposable hospital products containing polyvinyl chloride (PVC), including the search and evaluation of cost-effective sustainable alternative products free of PVC., Methods: A descriptive observational analysis was performed, after classifying the latest research in major databases, and disposable products that could contain PVC. These were divided into 5 groups: cannulas, catheters, tubes, bags, and equipment, purchased in the period 2008-2009, differentiating between the technical and economic assessment of the materials., Results: In the analysis of the composition of 492 articles selected, 234 (47.5%) contained PVC, and 19.4% were considered PVC-free alternatives, with only 11.3% of these being economically viable., Conclusions: This study highlights the advantages of the classification of PVC products, by showing that safe and efficient alternatives exist for some product lines that are consistent with patient safety and quality in the work by doctors., (Copyright © 2011 SECA. Published by Elsevier Espana. All rights reserved.)

Most spontaneous DNA double-strand breaks (DSBs) arise during replication and are repaired by homologous recombination (HR) with the sister chromatid. Many proteins participate in HR, but it is often difficult to determine their in vivo functions due to the existence of alternative pathways. Here we take advantage of an in vivo assay to assess repair of a specific replication-born DSB by sister chromatid recombination (SCR). We analyzed the functional relevance of four structure-selective endonucleases (SSEs), Yen1, Mus81-Mms4, Slx1-Slx4, and Rad1, on SCR in Saccharomyces cerevisiae. Physical and genetic analyses showed that ablation of any of these SSEs leads to a specific SCR decrease that is not observed in general HR. Our work suggests that Yen1, Mus81-Mms4, Slx4, and Rad1, but not Slx1, function independently in the cleavage of intercrossed DNA structures to reconstitute broken replication forks via HR with the sister chromatid. These unique effects, which have not been detected in other studies unless double mutant combinations were used, indicate the formation of distinct alternatives for the repair of replication-born DSBs that require specific SSEs.

The efficient and timely resolution of DNA recombination intermediates is essential for bipolar chromosome segregation. Here, we show that the specialized chromosome segregation patterns of meiosis and mitosis, which require the coordination of recombination with cell-cycle progression, are achieved by regulating the timing of activation of two crossover-promoting endonucleases. In yeast meiosis, Mus81-Mms4 and Yen1 are controlled by phosphorylation events that lead to their sequential activation. Mus81-Mms4 is hyperactivated by Cdc5-mediated phosphorylation in meiosis I, generating the crossovers necessary for chromosome segregation. Yen1 is also tightly regulated and is activated in meiosis II to resolve persistent Holliday junctions. In yeast and human mitotic cells, a similar regulatory network restrains these nuclease activities until mitosis, biasing the outcome of recombination toward noncrossover products while also ensuring the elimination of any persistent joint molecules. Mitotic regulation thereby facilitates chromosome segregation while limiting the potential for loss of heterozygosity and sister-chromatid exchanges., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In this work, we examined the production of infective zoospores of Lagenidium giganteum in four culture media, and the larvicidal activity of the cultures was determined against Aedes aegypti larvae, as well as the effect of polymer encapsulation. Medium containing sunflower seed extract showed the greatest production of zoospores, 5.92×10(6) zoospores/ml after six days of fermentation at 25±2°C and 150rpm shaking. This culture tested against A. aegypti 1st stage larvae caused different mortality rates at 24, 48 and 72h posttreatment. The LC(50) obtained was 43.9, 41.1 and 42.9μl of total culture/ml, at 24, 48 and 72h posttreatment respectively, while the culture grown in medium with soybean meal showed 3-5 times higher LC(50) values. Finally, the total culture including mycelium, zoospores and presporangia formulated with 2.5% pectin showed significantly higher mortality rates, around 100% more than the unformulated culture, whose values were from 40 to 1% at 3, 6, 9, and 12d posttreatment in the bioassays carried out in the laboratory to determine residual activity., (2010 Elsevier B.V. All rights reserved.)

Objectives: This analysis of the Study for Monitoring Antimicrobial Resistance Trends (SMART) evaluated the susceptibility patterns of Enterobacteriaceae in Latin America in 2008, with emphasis on susceptibility trends of E. coli and K. pneumoniae., Methods: Clinical isolates were recovered from intra-abdominal infections (IAI) from 23 centers in 10 Latin American countries. Isolates were sent to a central laboratory for confirmation of identification, antimicrobial susceptibility and ESBL testing, following the Clinical Laboratory Standards Institute (CLSI) guidelines., Results: Of 1,003 Gram-negative bacilli collected from intra-abdominal infections, E. coli and K. pneumoniae were the most commonly isolated organisms, and 26.8% of E. coli and 37.7% of K. pneumoniae were ESBL positive. Ertapenem and imipenem were the most consistently active agents tested; 99% of ESBL-positive E. coli isolates were susceptible to ertapenem and 100% to imipenem as well, and 91% of ESBL-positive K. pneumoniae were susceptible to ertapenem and 98% to imipenem. Quinolones and cephalosporins were less active, achieving 1.5% to 76% inhibition against ESBL-producing E. coli and 3.5% to 61% inhibition against K. pneumoniae., Conclusions: Local and unit-specific surveillance data is particularly important for selection of empiric therapy and in community-acquired infections as they can help the clinician with antibiotic selection by providing guidance regarding the likely pathogens and their resistance profiles. Our data also confirm the increasing frequency with which ESBL-producing organisms are found in the community setting, with 31.4% of community-acquired and 24.9% of hospital-acquired infections found to produce ESBLs. Imipenem and ertapenem are the most active agents tested for ESBL-positive E. coli and K. pneumoniae.

Holliday junction (HJ) resolution is essential for chromosome segregation at meiosis and the repair of stalled/collapsed replication forks in mitotic cells. All organisms possess nucleases that promote HJ resolution by the introduction of symmetrically related nicks in two strands at, or close to, the junction point. GEN1, a member of the Rad2/XPG nuclease family, was isolated recently from human cells and shown to promote HJ resolution in vitro and in vivo. Here, we provide the first biochemical/structural characterization of GEN1, showing that, like the Escherichia coli HJ resolvase RuvC, it binds specifically to HJs and resolves them by a dual incision mechanism in which nicks are introduced in the pair of continuous (noncrossing) strands within the lifetime of the GEN1-HJ complex. In contrast to RuvC, but like other Rad2/XPG family members such as FEN1, GEN1 is a monomeric 5'-flap endonuclease. However, the unique feature of GEN1 that distinguishes it from other Rad2/XPG nucleases is its ability to dimerize on HJs. This functional adaptation provides the two symmetrically aligned active sites required for HJ resolution.

In eukaryotic cells, multiple DNA repair mechanisms respond to a wide variety of DNA lesions. Homologous recombination-dependent repair provides a pathway for dealing with DNA double-strand breaks and replication fork demise. A key step in this process is the resolution of recombination intermediates such as Holliday junctions (HJs). Recently, nucleases from yeast (Yen1) and human cells (GEN1) were identified that can resolve HJ intermediates, in a manner analogous to the E. coli HJ resolvase RuvC. Here, we have analyzed the role of Yen1 in DNA repair in S. cerevisiae, and show that while yen1Delta mutants are repair-proficient, yen1Delta mus81Delta double mutants are exquisitely sensitive to a variety of DNA-damaging agents that disturb replication fork progression. This phenotype is dependent upon RAD52, indicating that toxic recombination intermediates accumulate in the absence of Yen1 and Mus81. After MMS treatment, yen1Delta mus81Delta double mutants arrest with a G2 DNA content and unsegregated chromosomes. These findings indicate that Yen1 can act upon recombination/repair intermediates that arise in MUS81-defective cells following replication fork damage., (2009 Elsevier B.V. All rights reserved.)

Homologous recombination is a very important cellular process, as it provides a major pathway for the repair of DNA double-strand breaks. This complex process is affected by many factors within cells. Here, we have studied the effect of monovalent cations (K+, Na+, and NH4+) on the outcome of recombination events, as their presence affects the biochemical activities of the proteins involved in recombination as well as the structure of DNA. For this purpose, we used an in vitro recombination system that includes a protein nuclear extract, as a source of recombination machinery, and two plasmids as substrates for intramolecular homologous recombination, each with two copies of different alleles of the human minisatellite MsH43. We found that the presence of monovalent cations induced a decrease in the recombination frequency, accompanied by an increase in the fidelity of the recombination. Moreover, there is an emerging consensus that secondary structures of DNA have the potential to induce genomic instability. Therefore, we analyzed the effect of the sequences capable of forming G-quadruplex on the production of recombinant molecules, taking advantage of the capacity of some MsH43 alleles to generate these kinds of structure in the presence of K+. We observed that the MsH43 recombinants containing duplications, generated in the presence of K+, did not include the repeats located towards the 5'-side of the G-quadruplex motif, suggesting that this structure may be involved in the recombination events leading to duplications. Our results provide new insights into the molecular mechanisms underlying the recombination of repetitive sequences.

Four-way DNA intermediates, also known as Holliday junctions, are formed during homologous recombination and DNA repair, and their resolution is necessary for proper chromosome segregation. Here we identify nucleases from Saccharomyces cerevisiae and human cells that promote Holliday junction resolution, in a manner analogous to that shown by the Escherichia coli Holliday junction resolvase RuvC. The human Holliday junction resolvase, GEN1, and its yeast orthologue, Yen1, were independently identified using two distinct experimental approaches: GEN1 was identified by mass spectrometry following extensive fractionation of HeLa cell-free extracts, whereas Yen1 was detected by screening a yeast gene fusion library for nucleases capable of Holliday junction resolution. The eukaryotic Holliday junction resolvases represent a new subclass of the Rad2/XPG family of nucleases. Recombinant GEN1 and Yen1 resolve Holliday junctions by the introduction of symmetrically related cuts across the junction point, to produce nicked duplex products in which the nicks can be readily ligated.

Minisatellites are tandem repeats of short DNA units widely distributed in genomes. However, the information on their dynamics in a phylogenetic context is very limited. Here we have studied the organization of the MsH43 locus in several species of primates and from these data we have reconstructed the evolutionary history of this complex minisatellite. Overall, with the exception of gibbon, MsH43 has an organization that is asymmetric, since the distribution of repeats is distinct between the 5' and 3' halves, and heterogeneous since there are many different repeats, some of them characteristic of each species. Inspection of the MsH43 arrays showed the existence of many duplications and deletions, suggesting the implication of slippage processes in the generation of polymorphism. Concerning the evolutionary history of this minisatellite, we propose that the birth of MsH43 may be situated before the divergence of Old World Monkeys since we found the existence of some MsH43 repeat motifs in prosimians and New World Monkeys. The analysis of MsH43 in apes revealed the existence of an evolutionary breakpoint in the pathway that originated African great apes and humans. Remarkably, human MsH43 is more homologous to orang-utan than to the corresponding sequence in gorilla and chimpanzee. This finding does not comply with the evolutionary paradigm that continuous alterations occur during the course of genome evolution. To adjust our results to the standard phylogeny of primates, we propose the existence of a wandering allele that was maintained almost unaltered during the period that extends between orang-utan and humans.

In a previous work we used an in vitro system for the generation and analysis of double-strand breaks (DSBs) using nuclear extracts from rat testes as a source of DSB activity. Since the recombination process can be triggered by the formation of DSB, in the present study we developed a strategy to isolate and characterize recombinant molecules using the same in vitro system. Our results indicate that the mechanism for the formation of recombinants was non-homologous end-joining driven by microhomologies. The procedure described here represents an alternative to investigate the mechanisms of DNA end-joining and other forms of DNA repair.

Minisatellites are tandem repeat arrays of middle size (5-100 bp) repeat units widely distributed in eukaryotic genomes. They have been related to several important features of human genome biology, including gene regulation, chromosomal fragile sites, and imprinting. In this report, we have critically assessed and employed heteroduplex analysis (HA) for the identification of different human minisatellite MsH43 alleles. This minisatellite is organized as a repeat array of 5-6 bp units spanning 0.5 kbp. Our results demonstrate that this procedure is an easy, rapid, and reliable method to document allelic diversity for this locus. This work suggests that HA will also be a useful tool for studying the polymorphism of other minisatellites with small repeat units.

We have reported the existence in rat nuclear extracts of a specific cleavage activity on a DNA fragment containing the human minisatellite MsH42 region (minisatellite plus its flanking sequences). Here, we have developed a system to analyse the nature of the cleavage products from the MsH42 region generated by the nuclear extracts. Our results demonstrated the formation of DNA double-strand breaks (DSB) in the MsH42 region by two different enzymatic activities, and that their distribution along this fragment changes depending on the presence of Mg2+. In the assays with Mg2+, the DSB were located in the minisatellite and its 3'-flanking region, showing preference for G-rich stretches. Oligonucleotide mutagenesis analysis confirmed that this enzymatic activity has a strong preference for G-tracts and that the recognition site is polarized towards the 3' end. Moreover, this activity cuts GC palindromes efficiently. In contrast, in the experiments without Mg2+, most DSB were mapped within the minisatellite flanking sequences. The analysis with oligonucleotides showed that G-tracts are recognized by this endonuclease activity, but with differences in the cleavage behaviour with respect to the reactions observed with Mg2+. The existence of two separate activities (Mg2+-dependent and Mg2+-independent) for the production of DSB was confirmed by analysing the effect of EGTA, N-ethyl maleimide, ionic strength, and by preincubations of the nuclear extracts at different temperatures. The tissue distribution of both DSB-producing activities was also different. The in vitro system used in the present work may be a useful tool for studying the formation of DSB and for investigation of the mechanisms of DNA repair.

Predatory capacity and prey selectivity of nymphs of the dragonfly Pantala hymenaea (Odonata: Libellulidae) were evaluated on larvae of the mosquito Culex quinquefasciatus (Diptera: Culicidae) and larvae of the midge Chironomus plumosus (Diptera: Chironomidae) as prey. With functional response methodology, 7 larval densities were exposed to predator individuals in a glass jar under laboratory conditions. The study was performed in 2 experiments. The 1st was a test system with each prey species alone. The 2nd tested a mixture of both prey species in a 1:1 ratio. Prey selectivity and prey capacity were significantly greater on midge larvae than on mosquito larvae.

PCR preferential amplification consists of the inefficient amplification of one allele in a heterozygous sample. Here, we report the isolation of a GC-rich human minisatellite, MsH43, that undergoes allelic preferential amplification during PCR. This effect requires the existence of a (TGGGGC)(4) motif that is able to form a G-quadruplex in the presence of K(+). This structure interferes with the DNA synthesis of the alleles harbouring this motif during PCR The present results are the first demonstration that the formation of G-quadruplex can be one of the mechanisms involved in some kinds of preferential amplification.

Much work has been focused on the pathways that restore the integrity of the genome after different kinds of lesions, especially double-strand breaks. A classical method to investigate double-strand break repair is the incubation of a DNA substrate with cell-free extracts. In these end-joining assays, the DNA is efficiently ligated by the proteins present in the extract, generating circular molecules and/or multimers. In contrast, using a similar in vitro system, we detected DNA cleavage rather than end ligation. When comparing our results with previous works, a paradox emerges: lower amounts of DNA become multimerized instead of degraded and higher amounts of DNA are degraded rather than multimerized. Here, we have demonstrated that when the DNA/protein ratio is low enough, the DNA-binding proteins of the nuclear extract protect the DNA substrate, avoiding DNA degradation and vice versa. Therefore, the variation of the DNA/protein ratio is enough to switch the outcome of the experiment from a DNA cleavage assay to a typical end-joining assay.

One of the most exciting challenges in human biology is the understanding of how our genome was constructed during evolution. Here we explore the evolutionary history of the low polymorphic human minisatellite MsH42 and its flanking sequences. We show that the evolutionary birth of MsH42 took place within an intron, early in primate lineage evolution, more than 40 MYA. Then, single base-pair changes and duplications/deletions of repeat blocks by mispairing were probably the main forces governing the generation of this minisatellite and its polymorphism throughout primate evolution. Moreover, we detected several phylogenetic footprints at both sides of MsH42. We believe that our findings will contribute to the understanding of low-variability minisatellite evolution.

A strategy to increase residual activity of Bacillus thuringiensis serovar. israelensis (Bti) extract through slow-release formulations and protection from solar radiation was studied. The median lethal concentration (LC50) and 90% lethal concentration (LC90) levels of laboratory-reared early 4th-stage larval Aedes aegypti after exposure to Bti extract were determined. Formulations with 4 polymers and 1 solar protectant were prepared, and their shelf lives were evaluated for year-long storage at 20-35 degrees C and 50-80% relative humidity. Also, the effect of ultraviolet light on unformulated (extract) and formulated Bti larvicidal activity persistence was determined. Laboratory bioassays were conducted with larval Ae. aegypti introduced into treated and control containers at 1, 2, 7, 14, 21, 28, 35, and 42 days after treatment, and larval mortalities were checked 24 h after introduction. Probit analysis of Bti extract showed LC50 and LC90 values of 0.016 and 0.051 mg/liter, respectively. The polymer-based Bti formulations showed no significant loss of insecticidal activity after 8 months of storage. Ultraviolet irradiation reduced activity of unformulated Bti extract after different exposure times, up to 40-46%, whereas Bti formulated with gelatin or acacia gum showed lower variation in larvicidal activity than formulations with sodium alginate and paraffin for protecting the activity of Bti toxin. Residual activity against 4th-stage Ae. aegypti in the laboratory for the formulation containing acacia gum at 10% (w/w) was 80% mortality at 14 days after treatment, whereas the Bti formulation containing gelatin (10%, w/w) caused 65% mortality. In addition, Bti formulations made with paraffin at 5% (w/w) sustained up to 60% mortality for 21 days. Unformulated Bti showed only 2.6% mortality, and a commercial preparation maintained 37% mortality, both at 14 days after treatment.