Individual Forks Research Articles

Reproductive Biology of Yellowfin Tuna (Thunnus albacares) in Tropical Western and Central Pacific Ocean

A total of 756 yellowfin tuna (Thunnus albacares) caught by a Chinese drifting longliner in the tropical western and central Pacific Ocean (WCPO) from May 2018 to March 2019 were investigated to describe the reproductive biology of the species. Generalized linear model and polytomous logistic regression for the ordinal response model were employed to assess the effects of biometric and spatiotemporal factors (such as individual fork length (FL), fishing depth, dissolved oxygen, and month) on the reproductive traits of yellowfin tuna. The results showed that FLs ranged from 87 to 163 cm, averaging 115.8 cm (SD = ±14.2) for females and 121.8 cm (SD = ±16.8) for males. The proportion of males in the sampled fish was 0.61 (SD = ±0.29), and larger males (>130 cm) were proportionally predominant. Analyses based on the monthly variation of the gonadosomatic index and monthly proportion of sexual maturity stages of the gonads showed that the main spawning period of yellowfin tuna lasts from September to December. In addition, the 50% first maturity FLs of males and females were 111.96 cm (SD = ±1.04) and 119.64 cm (SD = ±1.30), respectively. This study provides new information on the reproductive development of T. albacares in the tropical WCPO region. These reproductive parameters reduce uncertainty in current stock assessment models, which will ultimately assist the fishery in becoming sustainable for future generations.

Genome-wide mapping of individual replication fork velocities using nanopore sequencing

Little is known about replication fork velocity variations along eukaryotic genomes, since reference techniques to determine fork speed either provide no sequence information or suffer from low throughput. Here we present NanoForkSpeed, a nanopore sequencing-based method to map and extract the velocity of individual forks detected as tracks of the thymidine analogue bromodeoxyuridine incorporated during a brief pulse-labelling of asynchronously growing cells. NanoForkSpeed retrieves previous Saccharomyces cerevisiae mean fork speed estimates (≈2 kb/min) in the BT1 strain exhibiting highly efficient bromodeoxyuridine incorporation and wild-type growth, and precisely quantifies speed changes in cells with altered replisome progression or exposed to hydroxyurea. The positioning of >125,000 fork velocities provides a genome-wide map of fork progression based on individual fork rates, showing a uniform fork speed across yeast chromosomes except for a marked slowdown at known pausing sites.

Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

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 onMUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

Replication fork slowing and stalling are distinct, checkpoint-independent consequences of replicating damaged DNA.

In response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents—MMS, 4NQO and bleomycin—that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage.

Computational simulation of frontal impact of motorcycle telescopic fork

ABSTRACTThis paper presents modelling aspects of a high fidelity finite element model of a motorcycle telescopic fork. For validation purpose, a series of impact tests of which a frontal impact load imposed on the individual fork by a rigid striker was conducted using factorial experiment approach. The model was then used to simulate all the impacts accordingly. The outcomes were evaluated in terms of permanent deformations of the fork, energy dissipated in the impact, and the velocity and displacement time histories of the striker. The overall performance and sensitivity of the model was also assessed using the factorial analysis. The deviations of time histories data were quantified by mean error, mean absolute error and root mean square error. The key responses of the fork were found to be successfully simulated and compared well to the test results. The computed largest value among the respective metrics over the total errors was 6.2% of which corresponding to the root mean square error of the displacement time histories data, whereas others were less than 3%. It is thus concluded that the fork model has been successfully validated and the modelling methods has been established. Recommendations for improvements were also given in the paper.

Crosslinks and replication: the show must go on

Crosslinks and replication: the show must go on

Multivariate Regression Modeling of Parameters Affecting Punching Force during Random Velour Needling

Manufacturing of random velour needled fabrics comprises two separate needling operations. In the first stage a carded fibrous assembly is consolidated using a barbed needle. The consolidated assembly is opened-up due to action of fork needles of the second needling stage. In this special needling operation the mono-structure consolidated fibrous assembly is converted to a bi-structure fibrous assembly, comprised of base and pile layers. The extent of imparted structural changes influences physical and mechanical properties of the resultant random velour fabric. Fork needles which are the culprit of the structural alteration are subjected to relatively large forces. In this work the effect of random velour needling parameters and fiber characteristics on force exerted on the fork needle were investigated. Force measurement and data analysis were carried out using a purposely designed twin load cell force measuring unit and required software. Sixty four samples of polyester fabrics were prepared. Separate identification of the effect of principle independent parameters on the exerted force on individual fork needles was achieved using a linear multivariate regression analysis based on SAS 9.1 statistical software. Estimation of total average force (Frms) and peak average force (FPA) were used as response variables of the model. Results manifested that force exerted on the fork needle is profoundly affected by a large number of parameters. It was also found that, total fiber displacement “ TFD“, occurring during random-velour needling was the foremost factor directly influencing force on the fork needles.

Momentary measurement of force exerted on individual fork needles in random-velour needling

Random-velour needling can be regarded as a modified technique of conventional needling process. This modified technique results in the occurrence of fundamental changes in initial orientation of fibers within the preconsolidated fibrous assembly. As the consequence of these changes a dual fibrous structure comprising base and pile layer is created. It is evident that momentary analysis of forces exerted on fork needles is vitally important in the design of both needle and machine. In this research, momentary forces experienced by individual fork needles were precisely measured. A novel force measuring system enabling the momentary sensing of the forces was designed and developed. The results were analyzed by purposely written MATLAB-based software hereafter named as force analyzing software. Results showed that fork needle experiences various forces due to its vibration and inertia and also frictional resistances offered by preconsolidated fibrous assembly and brush conveyor during a complete needling cycle. Furthermore, fork needle is subjected to an additional force due to contact of fork needle with fibers of preconsolidated fibrous assembly during dwell of needle board at the bottom dead center. It is concluded that total force on the fork needle includes contact and noncontact components. Quantification of results showed that, noncontact forces are due to fork needle vibration and inertia. These comprise significant proportion of total force experienced by individual fork needles. Magnitudes of these forces are strongly dependent on stroke frequency of the needle board. It was found that contact component of the total force is dominated by preconsolidated fibrous assembly characteristics.

Continued DNA synthesis in replication checkpoint mutants leads to fork collapse.

Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1Δ and mrc1Δ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork "collapse point" in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1Δ and mrc1Δ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block.

Claspin and Chk1 regulate replication fork stability by different mechanisms

The checkpoint mediator protein Claspin facilitates the phosphorylation and activation of Chk1 by ATR and thus is required for efficient DNA replication. However, the physical association of Claspin homologues with replication factors and forks suggests that it might have additional functions in controlling DNA replication. DNA combing was used to examine the functions of Chk1 and Claspin at individual forks and to determine whether Claspin functions independently of Chk1. We find that Claspin, like Chk1, regulates fork stability and density in unperturbed cells. As expected, Chk1 regulates origin firing predominantly by controlling Cdk2-Cdc25 function. By contrast, Claspin functions independently of the Cdc25-Cdk2 pathway in mammalian cells. The findings support a model in which Claspin plays a role regulating replication fork stability that is independent of its function in mediating Chk1 phosphorylation.