Single-stranded DNA ligation and XLF-stimulated incompatible DNA end ligation by the XRCC4-DNA ligase IV complex: influence of terminal DNA sequence

The double-strand DNA break repair pathway, non-homologous DNA end joining (NHEJ), is distinctive for the flexibility of its nuclease, polymerase and ligase activities. Here we find that the joining of ends by XRCC4-ligase IV is markedly influenced by the terminal sequence, and a steric hindrance model can account for this. XLF (Cernunnos) stimulates the joining of both incompatible DNA ends and compatible DNA ends at physiologic concentrations of Mg2+, but only of incompatible DNA ends at higher concentrations of Mg2+, suggesting charge neutralization between the two DNA ends within the ligase complex. XRCC4-DNA ligase IV has the distinctive ability to ligate poly-dT single-stranded DNA and long dT overhangs in a Ku- and XLF-independent manner, but not other homopolymeric DNA. The dT preference of the ligase is interesting given the sequence bias of the NHEJ polymerase. These distinctive properties of the XRCC4-DNA ligase IV complex explain important aspects of its in vivo roles

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Discussion on the Stiffness of the Drive Chain in the Legs of Biped Robots

Biped robots’ locomotion is realized by driving the joint motion via a drive chain. Therefore, the stiffness of the drive chain is an important factor that affects the drive performance and can influence the locomotion behavior of the biped robot. This work focused on the influence of the stiffness of the leg’s drive chain using a mass-spring model based on the biped robot AIRO built in Zhejiang Lab. Methods for determination of the parameters in the proposed model were presented, including the use of ANSYS Workbench to determine the stiffness parameters and the determination of the inertia parameters by dynamic modelling of the biped robot. Simulation results show that special attention should be paid to the stiffness of the drive train of the leg when designing a biped robot to ensure the walking capability of the robot. Using the model proposed in this work, relations between the executed accuracy of the joint trajectories and the stiffness can be analyzed; after that, the stiffness parameters can be optimized. In addition, simulation results also showed that attention should be paid to manufacturing tolerances to ensure the symmetry of the legs of the bipedal robot in order to reduce the vibration of the robot body. Experiments were conducted on AIRO for validating the proposed model and the simulation analysis

Single-stranded DNA ligation and XLF-stimulated

incompatible DNA end ligation by the XRCC4-DNA ligase IV complex: influence of terminal DNA sequenc

Stable and Fast Planar Jumping Control Design for a Compliant One-Legged Robot

Compliant bipedal robots demonstrate a potential for impact resistance and high energy efficiency through the introduction of compliant elements. However, it also adds to the difficulty of stable control of the robot. To motivate the control strategies of compliant bipedal robots, this work presents an improved control strategy for the stable and fast planar jumping of a compliant one-legged robot designed by the authors, which utilizes the concept of the virtual pendulum. The robot was modeled as an extended spring-loaded inverted pendulum (SLIP) model with non-negligible torso inertia, leg inertia, and leg damping. To enable the robot to jump forward stably, a foot placement method was adopted, where due to the asymmetric feature of the extended SLIP model, a variable time coefficient and an integral term with respect to the forward speed tracking error were introduced to the method to accurately track a given forward speed. An energy-based leg rest length regulation method was used to compensate for the energy dissipation due to leg damping, where an integral term, regarding jumping height tracking error, was introduced to accurately track a given jumping height. Numerical simulations were conducted to validate the effectiveness of the proposed control strategy. Results show that stable and fast jumping of compliant one-legged robots could be achieved, and the desired forward speed and jumping height could also be accurately tracked. In addition to that, using the proposed control strategy, the robust jumping performance of the robot could be observed in the presence of disturbances from state variables or uneven terrain

XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps

XRCC4 and DNA ligase IV form a complex that is essential for the repair of all double-strand DNA breaks by the nonhomologous DNA end joining pathway in eukaryotes. We find here that human XRCC4:DNA ligase IV can ligate two double-strand DNA ends that have fully incompatible short 3′ overhang configurations with no potential for base pairing. Moreover, at DNA ends that share 1–4 annealed base pairs, XRCC4:DNA ligase IV can ligate across gaps of 1 nt. Ku can stimulate the joining, but is not essential when there is some terminal annealing. Polymerase mu can add nucleotides in a template-independent manner under physiological conditions; and the subset of ends that thereby gain some terminal microhomology can then be ligated. Hence, annealing at sites of microhomology is very important, but the flexibility of the ligase complex is paramount in nonhomologous DNA end joining. These observations provide an explanation for several in vivo observations that were difficult to understand previously

Optimizing the method for generation of integration-free induced pluripotent stem cells from human peripheral blood

Abstract Background Generation of induced pluripotent stem cells (iPSCs) from human peripheral blood provides a convenient and low-invasive way to obtain patient-specific iPSCs. The episomal vector is one of the best approaches for reprogramming somatic cells to pluripotent status because of its simplicity and affordability. However, the efficiency of episomal vector reprogramming of adult peripheral blood cells is relatively low compared with cord blood and bone marrow cells. Methods In the present study, integration-free human iPSCs derived from peripheral blood were established via episomal technology. We optimized mononuclear cell isolation and cultivation, episomal vector promoters, and a combination of transcriptional factors to improve reprogramming efficiency. Results Here, we improved the generation efficiency of integration-free iPSCs from human peripheral blood mononuclear cells by optimizing the method of isolating mononuclear cells from peripheral blood, by modifying the integration of culture medium, and by adjusting the duration of culture time and the combination of different episomal vectors. Conclusions With this optimized protocol, a valuable asset for banking patient-specific iPSCs has been established

MicroRNA-383 Regulates the Apoptosis of Tumor Cells through Targeting Gadd45g

<div><p><i>Background</i></p><p>MicroRNAs (miRNAs) are a class of small non-coding single-stranded RNA molecules that inhibit gene expression at post-transcriptional level. Gadd45g (growth arrest and DNA-damage-inducible 45 gamma) is a stress-response protein, which has been implicated in several biological processes, including DNA repair, the cell cycle and cell differentiation.</p><p><i>Results</i></p><p>In this work, we found that miR-383 is a negative regulator of Gadd45g. Forced expression of miR-383 decreased the expression of Gadd45g through binding to the 3′ untranslated region (3′-UTR), whereas inhibition of miR-383 increased Gadd45g expression. The presence of miR-383 increased the cellular sensitivity to DNA damage in breast cancer cells, which was rescued by ectopic expression of Gadd45g without the 3′-UTR. miR-383 also regulates the expression of Gadd45g in embryonic stem (ES) cells, but not their apoptosis under genotoxic stress. miR-383 was further showed to negatively regulate ES cell differentiation via targeting Gadd45g, which subsequently modulates the pluripotency-associated genes. Taken together, our study demonstrates that miR-383 is a negative regulator of Gadd45g in both tumor cells and ES cells, however, has distinct function in regulating cell apoptosis. miR-383 may be used as antineoplastic agents in cancer chemotherapy.</p><p><i>Conclusion</i></p><p>We demonstrate for the first time that miR-383 can specifically regulates the expression of Gadd45g by directly targeting to the 3-UTR region of Gadd45g mRNA, a regulatory process conserved in human tumor cells and mouse embryonic stem cells. These two compotents can be potentially used as antineoplastic agents in cancer chemotherapy.</p></div

The plant hormone abscisic acid stimulates megakaryocyte differentiation from human iPSCs in vitro

In the clinic, the supply of platelets is frequently insufficient to meet transfusion needs. To address this issue, many scientists have established the derivation of functional platelets from CD34+ cells or human pluripotent stem cells (PSCs). However, the yield of platelets is still far below what is required. Here we found that the plant hormone abscisic acid (ABA) could increase the generation of megakaryocytes (MKs) and platelets from human induced PSCs (hiPSCs). During platelet derivation, ABA treatment promoted the generation of CD34+/CD45+ HPCs and CD41+ MKs on day 14 and then increased CD41+/CD42b+ MKs and platelets on day 19. Moreover, we found ABA-mediated activation of Akt and ERK1/2 signal pathway through receptors LANCL2 and GRP78 in a PKA-dependent manner on CD34+/CD45+ cells. In conclusion, our data suggest that ABA treatment can promote CD34+/CD45+ HPC proliferation and CD41+ MK differentiation