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Since the beginning of its existence mankind is using natural resources and applying them in construction techniques. The anthropogenic pressure induced by humans its clearly influencing the natural environment and so affecting its functional balance. Buildings are anthropogenic objects that enable mankind to inhabit its natural habitat according to their needs.This article discloses the use issues of buildings according to their anthropogenic pressure taken in consideration the example of a selected building.

This paper presents a performance evaluation of single and dual-band software-defined radio (SDR) front-ends using commercially available barium strontium titanate (BST) thin film to control SDR receivers dynamic range and mitigate interference. Firstly, an SDR front-end solution using a BST-based single-band bandpass filter (BPF) is developed to manage in-band attenuation according to the input power. Secondly, a dual-band SDR front-end to avoid high power interference with its spectral content on the below side of the second band is designed. This device is performed with a BST-based dual-band BPF providing independent control of the power limiting capabilities of the first and second bands. The BST component introduces a dependence on bias voltage, frequency, and input power. Consequently, by applying a 0 V bias, it is possible to target interferers between 700 MHz up to 1 GHz and attenuate them more than 7 dB at 40 dBm. The frequency dependence is defined by the second band, which retains its filtering performance while performing interference cancellation in the first band. This SDR solution implements a three functional dimension device in the upper MHz with a power-dependent insertion loss, high threshold power level, and frequency selectivity.

Nuclear pore complexes (NPCs) are large proteinaceous assemblies that mediate nuclear compartmentalization. NPCs undergo large-scale structural rearrangements during mitosis in metazoans and some fungi. However, our understanding of NPC remodeling beyond mitosis remains limited. Using time-lapse fluorescence microscopy, we discovered that NPCs undergo two mechanistically separable remodeling events during budding yeast meiosis in which parts or all of the nuclear basket transiently dissociate from the NPC core during meiosis I and II, respectively. Meiosis I detachment, observed for Nup60 and Nup2, is driven by Polo kinase-mediated phosphorylation of Nup60 at its interface with the Y-complex. Subsequent reattachment of Nup60-Nup2 to the NPC core is facilitated by a lipid-binding amphipathic helix in Nup60. Preventing Nup60-Nup2 reattachment causes misorganization of the entire nuclear basket in gametes. Strikingly, meiotic nuclear basket remodeling also occurs in the distantly related fission yeast, Schizosaccharomyces pombe. Our study reveals a conserved and developmentally programmed aspect of NPC plasticity, providing key mechanistic insights into the nuclear basket organization., Journal of Cell Biology, 222 (2), ISSN:0021-9525, ISSN:1540-8140

Embedded computing is a sector in strong growth, driven by the increasing offer in the internet of things. Since position information is typically one of the intrinsic features of a sensory platform, the present work considers and analyzes the embedded lossless compression of localization data. A structural data scheme is proposed based on the number of occurrences of the independent coordinate components, named as µJSON. It is shown that, when considering a low-level language implementation on an embedded processor acquiring GNSS data, the proposed schema implies lower compression rates than its counterparts. An embedded test-bench platform is assembled, and several real case scenarios are considered for effectiveness and validation purposes. GRANT_NUMBER: 32020 N/A

Bloom's syndrome is caused by inactivation of the BLM helicase, which functions with TOP3A and RMI1-2 (BTR complex) to dissolve recombination intermediates and avoid somatic crossing-over. We show here that crossover avoidance by BTR further requires the activity of cyclin-dependent kinase-1 (CDK1), Polo-like kinase-1 (PLK1), and the DDR mediator protein TOPBP1, which act in the same pathway. Mechanistically, CDK1 phosphorylates BLM and TOPBP1 and promotes the interaction of both proteins with PLK1. This is amplified by the ability of TOPBP1 to facilitate phosphorylation of BLM at sites that stimulate both BLM-PLK1 and BLM-TOPBP1 binding, creating a positive feedback loop that drives rapid BLM phosphorylation at the G2-M transition. In vitro, BLM phosphorylation by CDK/PLK1/TOPBP1 stimulates the dissolution of topologically linked DNA intermediates by BLM-TOP3A. Thus, we propose that the CDK1-TOPBP1-PLK1 axis enhances BTR-mediated dissolution of recombination intermediates late in the cell cycle to suppress crossover recombination and curtail genomic instability., Science Advances, 8 (5), ISSN:2375-2548

Meiotic defects derived from incorrect DNA repair during gametogenesis can lead to mutations, aneuploidies and infertility. The coordinated resolution of meiotic recombination intermediates is required for crossover formation, ultimately necessary for the accurate completion of both rounds of chromosome segregation. Numerous master kinases orchestrate the correct assembly and activity of the repair machinery. Although much less is known, the reversal of phosphorylation events in meiosis must also be key to coordinate the timing and functionality of repair enzymes. Cdc14 is a crucial phosphatase required for the dephosphorylation of multiple CDK1 targets in many eukaryotes. Mutations that inactivate this phosphatase lead to meiotic failure, but until now it was unknown if Cdc14 plays a direct role in meiotic recombination. Here, we show that the elimination of Cdc14 leads to severe defects in the processing and resolution of recombination intermediates, causing a drastic depletion in crossovers when other repair pathways are compromised. We also show that Cdc14 is required for the correct activity and localization of the Holliday Junction resolvase Yen1/GEN1. We reveal that Cdc14 regulates Yen1 activity from meiosis I onwards, and this function is essential for crossover resolution in the absence of other repair pathways. We also demonstrate that Cdc14 and Yen1 are required to safeguard sister chromatid segregation during the second meiotic division, a late action that is independent of the earlier role in crossover formation. Thus, this work uncovers previously undescribed functions of the evolutionary conserved Cdc14 phosphatase in the regulation of meiotic recombination., International Journal of Molecular Sciences, 22 (18), ISSN:1422-0067

To evaluate the effects of early anticoagulation on functional outcome, recurrent ischaemic events and haemorrhagic complications in Atrial Fibrillation (AF)-related acute ischaemic strokes (AIS). We retrospectively evaluated patients hospitalised in a Stroke Unit due to AF-related AIS. Patients were divided according to anticoagulation initiation timing (0–4 days, 5–14 days, no anticoagulation by the 14th day). We assessed the following outcomes at 3 months: favourable functional outcome [modified Rankin Scale (mRS) score 0–2 or equal to pre-stroke], recurrent ischaemic events and haemorrhagic complications after anticoagulation initiation. We included 395 patients. Anticoagulation was initiated at days 0–4 in 33.9% of patients, days 5–14 in 25.3% and not initiated by the day 14 in 40.8%. Factors associated with earlier anticoagulation included lower previous mRS, valvular AF and lower stroke severity. Favourable functional outcome occurred in 40.2% of patients, with lower odds in those anticoagulated at 5–14 versus 0–4 days (OR: 0.47, 95% CI: 0.23–0.94), independently of age, previous mRS and stroke severity. Recurrent ischaemic events occurred in 8.3% of patients, with higher odds in non-anticoagulated patients by the 14th day, compared to the remainder groups (OR: 3.26, 95% CI: 1.29–8.22 vs. 0–4 days and OR: 8.16, 95% CI: 1.76–37.9 vs. 5–14 days). In patients who started anticoagulation (n = 288), haemorrhagic complications occurred in 10.8%, being more frequent in those who started at 0–4 days vs. > 14 days. However, it did not abolish the 0–4-day initiation’s benefit on functional outcome. Early anticoagulation was associated with lower ischaemic recurrence and better functional outcome at 3 months. Additional studies are needed to better clarify its haemorrhagic risk.

Cell cycle tags allow to restrict target protein expression to specific cell cycle phases. Here, we present an advanced toolbox of cell cycle tag constructs in budding yeast with defined and compatible peak expression that allow comparison of protein functionality at different cell cycle phases. We apply this technology to the question of how and when Mus81-Mms4 and Yen1 nucleases act on DNA replication or recombination structures. Restriction of Mus81-Mms4 to M phase but not S phase allows a wildtype response to various forms of replication perturbation and DNA damage in S phase, suggesting it acts as a post-replicative resolvase. Moreover, we use cell cycle tags to reinstall cell cycle control to a deregulated version of Yen1, showing that its premature activation interferes with the response to perturbed replication. Curbing resolvase activity and establishing a hierarchy of resolution mechanisms are therefore the principal reasons underlying resolvase cell cycle regulation., eLife, 9, ISSN:2050-084X

CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1, using recently developed CRISPR-Cas-associated base editors2-4. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pahenu2 gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pahenu2 mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.

Multiparametric MRI represents the primary imaging modality to assess diffuse liver disease, both in a qualitative and in a quantitative manner. Diffusion-weighted imaging (DWI) is among the imaging techniques that can be used to assess fibrosis due to its unique capability to assess microstructural changes at the tissue level. DWI is based on water mobility patterns and has the potential to become a non-invasive and non-destructive virtual biopsy to assess diffuse liver disease, overcoming sampling bias errors due to its three-dimensional imaging capabilities. Parallel to DWI, another quantitative method called texture analysis may be used to assess early and advanced diffused liver disease through quantifying spatial relationships in a global and local level, applying to any type of digital imaging technique like MRI or CT. Initial results using texture analysis hold great promise. In the current paper, we will review the role of DWI and texture analysis using MR images in assessing diffuse liver disease.

SummaryDuring prophase of the first meiotic division, cells deliberately break their DNA. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables reciprocal exchange of DNA segments between homologous chromosomes, thus promoting genetic diversity in the progeny1. A successful completion of meiotic recombination requires nucleolytic processing of recombination intermediates. Genetic and cellular data implicated a pathway dependent on the putative MLH1-MLH3 (MutLγ) nuclease in generating crossovers, but mechanisms that lead to its activation were unclear2–4. Here, we have biochemically reconstituted key elements of this pro-crossover pathway. First, we show that human MSH4-MSH5 (MutSγ), which was known to support crossing over5–7, binds branched recombination intermediates and physically associates with MutLγ. This helps stabilize the ensemble at joint molecule structures and adjacent dsDNA. Second, we show that MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease, which demonstrates a novel and unexpected function for MutSγ in triggering crossing-over. Third, we find that MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Fourth, we also identify the replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) as additional components of the nuclease ensemble, and show thatS. cerevisiaestrains expressing PIP box-mutated MutLγ present striking defects in forming crossovers. Finally, we show that the MutLγ-MutSγ-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, the multilayered nuclease ensemble likely processes meiotic recombination intermediates by nicking dsDNA adjacent to junction points8. Since DNA nicking by MutLγ is dependent on its co-factors, the asymmetric distribution of MutSγ and RFC/PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This unique mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions within the meiotic chromosomal context3, 9.

Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible., eLife, 9, ISSN:2050-084X

Summary Homologous recombination (HR) is essential for high-fidelity DNA repair during mitotic proliferation and meiosis. Yet, context-specific modifications must tailor the recombination machinery to avoid (mitosis) or enforce (meiosis) the formation of reciprocal exchanges—crossovers—between recombining chromosomes. To obtain molecular insight into how crossover control is achieved, we affinity purified 7 DNA-processing enzymes that channel HR intermediates into crossovers or noncrossovers from vegetative cells or cells undergoing meiosis. Using mass spectrometry, we provide a global characterization of their composition and reveal mitosis- and meiosis-specific modules in the interaction networks. Functional analyses of meiosis-specific interactors of MutLγ-Exo1 identified Rtk1, Caf120, and Chd1 as regulators of crossing-over. Chd1, which transiently associates with Exo1 at the prophase-to-metaphase I transition, enables the formation of MutLγ-dependent crossovers through its conserved ability to bind and displace nucleosomes. Thus, rewiring of the HR network, coupled to chromatin remodeling, promotes context-specific control of the recombination outcome., Graphical Abstract, Highlights • Affinity proteomics reveals the composition and interaction landscape of HR enzymes • Interaction network rewiring of HR enzymes during mitotic proliferation and meiosis • Chd1, Rtk1, and Caf120 regulate meiotic crossing-over • Chd1 remodels chromatin to enable formation of MutLγ-Exo1-dependent crossovers, Wild et al. used affinity proteomics to characterize the composition and interaction landscape of 7 DNA repair enzymes during mitotic proliferation and meiosis. They report a concerted and context-specific rewiring of the interactomes and reveal meiosis-specific network components with roles in crossing-over.

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., The EMBO Journal, 36 (5), ISSN:0261-4189, ISSN:1460-2075

Summary The Bloom’s helicase ortholog, Sgs1, orchestrates the formation and disengagement of recombination intermediates to enable controlled crossing-over during meiotic and mitotic DNA repair. Whether its enzymatic activity is temporally regulated to implement formation of noncrossovers prior to the activation of crossover-nucleases is unknown. Here, we show that, akin to the Mus81-Mms4, Yen1, and MutLγ-Exo1 nucleases, Sgs1 helicase function is under cell-cycle control through the actions of CDK and Cdc5 kinases. Notably, however, whereas CDK and Cdc5 unleash nuclease function during M phase, they act in concert to stimulate Sgs1 activity during S phase/prophase I. Mechanistically, CDK-mediated phosphorylation enhances the velocity and processivity of Sgs1, which stimulates DNA unwinding in vitro and joint molecule processing in vivo. Subsequent hyper-phosphorylation by Cdc5 appears to reduce the activity of Sgs1, while activating Mus81-Mms4 and MutLγ-Exo1. These findings suggest a concerted mechanism driving orderly formation of noncrossover and crossover recombinants in meiotic and mitotic cells.

The Bloom’s helicase ortholog, Sgs1, orchestrates the formation and disengagement of DNA joint molecules to enable controlled crossing-over during meiotic and mitotic DNA repair. Whether its enzymatic activity is temporally regulated to implement formation of noncrossovers prior to the activation of crossover-nucleases is unknown. Here, we show that akin to the Mus81-Mms4, Yen1 and MutLγ-Exo1 nucleases, Sgs1 helicase function is under tight cell cycle control through the actions of CDK and Polo kinase Cdc5. Notably, however, whereas CDK and Cdc5 unleash nuclease function during M-phase, they collaboratively delimit Sgs1 activation to S-phase. Mechanistically, CDK-mediated phosphorylation enhances the velocity and processivity of Sgs1, which stimulates DNA unwinding in vitro and joint molecule processing in vivo. Subsequent hyper-phosphorylation by Cdc5 appears to reduce the activity of Sgs1, while activating Mus81-Mms4 and MutLγ-Exo1. These findings suggest a general mechanism that drives orderly formation of noncrossover and crossover recombinants in meiotic and mitotic cells.

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

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.

The formation of stable interactions between chromosomes of maternal and paternal origin-homologs-is required for their segregation during meiosis. To achieve this, cells take advantage of the recombination machinery, which promotes formation of reciprocal interhomolog exchanges, called crossovers, from the repair of self-inflicted DNA breaks. Important genetic studies led to the identification of key enzymes that control meiotic recombination. However, characterization of their biochemical properties when purified from meiotic cultures has been difficult to achieve. Here, we describe a simple approach to purify and characterize DNA repair enzymes from meiotic yeast cells. First, we provide a protocol to generate large-scale synchronous cultures. Second, we describe a general method to prepare meiotic extracts from which protein complexes can be immunoaffinity-purified. Finally, we detail how the purified material can be used for: (i) mass spectrometry-based analysis of interaction partners and posttranslational modifications, and (ii) monitoring enzymatic activities using synthetic DNA substrates.

The establishment of stable interactions between chromosomes underpins vital cellular processes such as recombinational DNA repair and bipolar chromosome segregation. On the other hand, timely disengagement of persistent connections is necessary to assure efficient partitioning of the replicated genome prior to cell division. Whereas great progress has been made in defining how cohesin-mediated chromosomal interactions are disengaged as cells prepare to undergo chromosome segregation, little is known about the metabolism of DNA joint molecules (JMs), generated during the repair of chromosomal lesions. Recent work on Mus81 and Yen1/GEN1, two conserved structure-selective endonucleases, revealed unforeseen links between JM-processing and cell cycle progression. Cell cycle kinases and phosphatases control Mus81 and Yen1/GEN1 to restrain deleterious JM-processing during S-phase, while safeguarding chromosome segregation during mitosis.

The formation of stable interactions between chromosomes of maternal and paternal origin-homologs-is required for their segregation during meiosis. To achieve this, cells take advantage of the recombination machinery, which promotes formation of reciprocal interhomolog exchanges, called crossovers, from the repair of self-inflicted DNA breaks. Important genetic studies led to the identification of key enzymes that control meiotic recombination. However, characterization of their biochemical properties when purified from meiotic cultures has been difficult to achieve. Here, we describe a simple approach to purify and characterize DNA repair enzymes from meiotic yeast cells. First, we provide a protocol to generate large-scale synchronous cultures. Second, we describe a general method to prepare meiotic extracts from which protein complexes can be immunoaffinity-purified. Finally, we detail how the purified material can be used for: (i) mass spectrometry-based analysis of interaction partners and posttranslational modifications, and (ii) monitoring enzymatic activities using synthetic DNA substrates.

The efficient separation of two equal DNA masses to the daughter cells is an essential step in mitosis. This process is dependent upon the removal of any remaining recombination or replication intermediates that link sister chromatids, and a failure to resolve these intermediates leads to genome instability. Similarly, a failure to resolve meiotic recombination intermediates that link homologous chromosomes can cause chromosome nondisjunction and aneuploidy. Cleavage of these potentially toxic replication/recombination intermediates requires the Mus81 endonuclease, which is active upon flaps, forks, and more complex secondary structures in DNA such as Holliday junctions. Recent studies of Mus81 revealed that it is regulated throughout the cell cycle: Mus81 activity is controlled in S-phase to limit the cleavage of replication fork structures, whereas it is activated at G2/M to ensure the cleavage of recombination and late replication intermediates. In this chapter, we describe a simple method that can monitor the activity of Mus81, which involves the immunoprecipitation of epitope-tagged Mus81 and use of an on-bead assay for nuclease activity.

Yeast is a powerful model for systems genetics. We present a versatile, time- and labor-efficient method to functionally explore the Saccharomyces cerevisiae genome using saturated transposon mutagenesis coupled to high-throughput sequencing. SAturated Transposon Analysis in Yeast (SATAY) allows one-step mapping of all genetic loci in which transposons can insert without disrupting essential functions. SATAY is particularly suited to discover loci important for growth under various conditions. SATAY (1) reveals positive and negative genetic interactions in single and multiple mutant strains, (2) can identify drug targets, (3) detects not only essential genes, but also essential protein domains, (4) generates both null and other informative alleles. In a SATAY screen for rapamycin-resistant mutants, we identify Pib2 (PhosphoInositide-Binding 2) as a master regulator of TORC1. We describe two antagonistic TORC1-activating and -inhibiting activities located on opposite ends of Pib2. Thus, SATAY allows to easily explore the yeast genome at unprecedented resolution and throughput., eLife, 6, ISSN:2050-084X