Search Results (7)

Linear unconstrained DNA cannot harbor supercoils since these supercoils can diffuse and be eliminated by free rotation of the DNA strands at the end of the molecule. Mammalian telomeres, despite constituting the ends of linear chromosomes, can hold supercoils and be subjected to topological stress. While negative supercoiling was previously observed, thus proving the existence of telomeric topological constraints, positive supercoils were never probed due to the lack of an appropriate tool. Indeed, the few tools available currently could only investigate unwound (Trioxsalen) or overwound (GapR) DNA topology (variations in twist) but not the variations in writhe (supercoils and plectonemes). To address this question, we have designed innovative tools aimed at analyzing both positive and negative DNA writhe in cells. Using them, we could observe the build-up of positive supercoils following replication stress and inhibition of Topoisomerase 2 on telomeres. TRF2 depletion caused both telomere relaxation and an increase in positive supercoils while the inhibition of Histone Deacetylase I and II by TSA only caused telomere relaxation. Moving outside telomeres, we also observed a build-up of positive supercoils on the FRA3B fragile site following replication stress, suggesting a topological model of DNA fragility for this site. Full article

Aims: Pathogenic variants in the CYP21A2 gene are related to the classic and non-classic forms of congenital adrenal hyperplasia (CAH). However, the role of CAH carrier status in the clinical presentation of polycystic ovarian syndrome (PCOS) is still unclear. Moreover, the possible associations of different CYP21A2 gene polymorphisms with metabolic and reproductive abnormalities in PCOS have not been investigated. Therefore, the present study aims to examine the prevalence of the most common CYP21A2 pathogenic variant IVS2-13A/C>G (c.293-13A/C>G) in Eastern European women with PCOS and to evaluate the associations between common intron 2 genetic polymorphisms and the clinical symptoms of the patients. Methods: Sixty consecutively recruited women with PCOS were genotyped for the CYP21A2 intron 2 IVS2-13A/C>G genetic variant. Additionally, CYP21A2 intron 2 polymorphic variants rs6453 (c.293-44G>T), rs6451 (c.293-67C>A/G), rs369651496 (c.293-104del), and rs6474 (c.308G>A/p.R103L) were tested and described. The clinical and hormonal characteristics were compared in women with PCOS and with polymorphic and wild-type genotypes. Results: The heterozygous CYP21A2 pathogenic variant IVS2-13A/C>G was found in one of the investigated PCOS patients (1.67%) with a non-hyperandrogenic type of PCOS. The presence of the rs6453 (c.293-44G>T) T-allele was associated with increased levels of DHEAS (15.18 vs. 9.14 µmol/L, p = 0.003) compared to the wild-type genotype in the investigated group. The rs6451 (c.293-67C>A/G) minor alleles were associated with an earlier age of menarche in the patients (12.0 vs. 13.0 years, p = 0.007). The polymorphic rs369651496 minor 6G allele was related to a better lipid profile in the women with PCOS, while the rs6474 variant modulated the blood pressure of the patients. Conclusions: The presence of CYP21A2 genetic minor alleles of rs6467 (IVS2-13A/C, c.293-13A/C), rs6453 (c.293-44G>T), rs6451 (c.293-67C>A/G), rs369651496 (c.293-104del), and rs6474 (c.308G>A/p.R103L) might modulate the adrenal androgens, age of menarche, and metabolic features in women with PCOS. Further studies on 21-hydroxylase genetic variants (pathogenic and polymorphisms) in different ethnic groups might help reveal the influence of adrenal steroidogenesis on PCOS development, clinical manifestations, and lifelong cardiovascular risks. Full article

The effector proteins secreted by a pathogen not only promote the virulence and infection of the pathogen but also trigger plant defense response. Lasiodiplodia theobromae secretes many effectors that modulate and hijack grape processes to colonize host cells, but the underlying mechanisms remain unclear. Herein, we report LtGAPR1, which has been proven to be a secreted protein. In our study, LtGAPR1 played a negative role in virulence. By co-immunoprecipitation, 23 kDa oxygen-evolving enhancer 2 (NbPsbQ2) was identified as a host target of LtGAPR1. The overexpression of NbPsbQ2 in Nicotiana benthamiana reduced susceptibility to L. theobromae, and the silencing of NbPsbQ2 enhanced L. theobromae infection. LtGAPR1 and NbPsbQ2 were confirmed to interact with each other. Transiently, expressed LtGAPR1 activated reactive oxygen species (ROS) production in N. benthamiana leaves. However, in NbPsbQ2-silenced leaves, ROS production was impaired. Overall, our report revealed that LtGAPR1 promotes ROS accumulation by interacting with NbPsbQ2, thereby triggering plant defenses that negatively regulate infection. Full article

Multi-skilled resources have brought more flexibility to resource scheduling and have been a key factor in the research of resource-constrained project scheduling problems. However, existing studies are mainly limited to deterministic problems and neglect some uncertainties such as resource breakdowns, while resource availability may change over time due to unexpected risks such as the COVID-19 pandemic. Therefore, this paper focuses on the multi-skilled project scheduling problem with uncertainty in resource availability. Different from previous assumptions, multi-skilled resources are allowed a switch in their skills, which we call dynamic skill assignment. For this complex problem, a nested dynamic scheduling algorithm called GA-PR is proposed, which includes three new priority rules to improve the solving efficiency. Moreover, the algorithm’s effectiveness is verified by an example, and the modified Project Scheduling Problem Library (PSPLIB) is used for numerical experimental analysis. Numerical experiments show that when the uncertainty in resource availability is considered, the more skills the resource has and the more resources are supplied, the better the dynamic scheduling method performs; on the other hand, the higher the probability of resource unavailability and the more skills are required, the worse the dynamic scheduling method performs.The results are helpful for improved decision making. Full article

The nucleoid-associated protein GapR found in Caulobacter crescentus is crucial for DNA replication, transcription, and cell division. Associated with overtwisted DNA in front of replication forks and the 3′ end of highly-expressed genes, GapR can stimulate gyrase and topo IV to relax (+) supercoils, thus facilitating the movement of the replication and transcription machines. GapR forms a dimer-of-dimers structure in solution that can exist in either an open or a closed conformation. It initially binds DNA through the open conformation and then undergoes structural rearrangement to form a closed tetramer, with DNA wrapped in the central channel. Here, we show that the DNA binding domain of GapR (residues 1–72, GapR^ΔC17) exists as a dimer in solution and adopts the same fold as the two dimer units in the full-length tetrameric protein. It binds DNA at the minor groove and reads the spatial distribution of DNA phosphate groups through a lysine/arginine network, with a preference towards AT-rich overtwisted DNA. These findings indicate that the dimer unit of GapR has an intrinsic DNA binding preference. Thus, at the initial binding step, the open tetramer of GapR with two relatively independent dimer units can be more efficiently recruited to overtwisted regions. Full article

The idea that amyloid fibrils and other types of protein aggregates are toxic for cells has been challenged by the discovery of a variety of functional aggregates. However, an identification of crucial differences between pathological and functional aggregation remains to be explored. Functional protein aggregation is often reversible by nature in order to respond properly to changing physiological conditions of the cell. In addition, increasing evidence indicates that fast fibril growth is a feature of functional amyloids, providing protection against the long-term existence of potentially toxic oligomeric intermediates. It is becoming clear that functional protein aggregation is a complexly organized process that can be mediated by a multitude of biomolecular factors. In this overview, we discuss the roles of diverse biomolecules, such as lipids/membranes, glycosaminoglycans, nucleic acids and metal ions, in regulating functional protein aggregation. Our studies on the protein GAPR-1 revealed that several of these factors influence the amyloidogenic properties of this protein. These observations suggest that GAPR-1, as well as the cysteine-rich secretory proteins, antigen 5 and pathogenesis-related proteins group 1 (CAP) superfamily of proteins that it belongs to, require the assembly into an amyloid state to exert several of their functions. A better understanding of functional aggregate formation may also help in the prevention and treatment of amyloid-related diseases. Full article

Key DNA transactions, such as genome replication and transcription, rely on the speedy translocation of specialized protein complexes along a double-stranded, right-handed helical template. Physical tethering of these molecular machines during translocation, in conjunction with their internal architectural features, generates DNA topological strain in the form of template supercoiling. It is known that the build-up of transient excessive supercoiling poses severe threats to genome function and stability and that highly specialized enzymes—the topoisomerases (TOP)—have evolved to mitigate these threats. Furthermore, due to their intracellular abundance and fast supercoil relaxation rates, it is generally assumed that these enzymes are sufficient in coping with genome-wide bursts of excessive supercoiling. However, the recent discoveries of chromatin architectural factors that play important accessory functions have cast reasonable doubts on this concept. Here, we reviewed the background of these new findings and described emerging models of how these accessory factors contribute to supercoil homeostasis. We focused on DNA replication and the generation of positive (+) supercoiling in front of replisomes, where two accessory factors—GapR and HMGA2—from pro- and eukaryotic cells, respectively, appear to play important roles as sinks for excessive (+) supercoiling by employing a combination of supercoil constrainment and activation of topoisomerases. Looking forward, we expect that additional factors will be identified in the future as part of an expanding cellular repertoire to cope with bursts of topological strain. Furthermore, identifying antagonists that target these accessory factors and work synergistically with clinically relevant topoisomerase inhibitors could become an interesting novel strategy, leading to improved treatment outcomes. Full article