The Coordination of Centromere Replication, Spindle Formation, and Kinetochore–Microtubule Interaction in Budding Yeast

The kinetochore is a protein complex that assembles on centromeric DNA to mediate chromosome–microtubule interaction. Most eukaryotic cells form the spindle and establish kinetochore–microtubule interaction during mitosis, but budding yeast cells finish these processes in S-phase. It has long been noticed that the S-phase spindle in budding yeast is shorter than that in metaphase, but the biological significance of this short S-phase spindle structure remains unclear. We addressed this issue by using ask1-3, a temperature-sensitive kinetochore mutant that exhibits partially elongated spindles at permissive temperature in the presence of hydroxyurea (HU), a DNA synthesis inhibitor. After exposure to and removal of HU, ask1-3 cells show a delayed anaphase entry. This delay depends on the spindle checkpoint, which monitors kinetochore–microtubule interaction defects. Overproduction of microtubule-associated protein Ase1 or Cin8 also induces spindle elongation in HU-arrested cells. The spindle checkpoint-dependent anaphase entry delay is also observed after ASE1 or CIN8 overexpression in HU-arrested cells. Therefore, the shorter spindle in S-phase cells is likely to facilitate proper chromosome–microtubule interaction

FL118, a novel camptothecin derivative, is insensitive to ABCG2 expression and shows improved efficacy in comparison with irinotecan in colon and lung cancer models with ABCG2-induced resistance

International audienceAbstractBackgroundIrinotecan is a camptothecin analogue currently used in clinical practice to treat advanced colorectal cancer. However, acquired resistance mediated by the drug efflux pump ABCG2 is a recognized problem. We reported on a novel camptothecin analogue, FL118, which shows anticancer activity superior to irinotecan. In this study, we sought to investigate the potency of FL118 versus irinotecan or its active metabolite, SN-38, in both in vitro and in vivo models of human cancer with high ABCG2 activity. We also sought to assess the potency and ABCG2 affinity of several FL118 analogues with B-ring substitutions.MethodsColon and lung cancer cells with and without ABCG2 overexpression were treated with FL118 in the presence and absence of Ko143, an ABCG2-selective inhibitor, or alternatively by genetically modulating ABCG2 expression. Using two distinct in vivo human tumor animal models, we further assessed whether FL118 could extend time to progression in comparison with irinotecan. Lastly, we investigated a series of FL118 analogues with B-ring substitutions for ABCG2 sensitivity.ResultsBoth pharmacological inhibition and genetic modulation of ABCG2 demonstrated that, in contrast to SN-38, FL118 was able to bypass ABCG2-mediated drug resistance. FL118 also extended time to progression in both in vivo models by more than 50% compared with irinotecan. Lastly, we observed that FL118 analogues with polar substitutions had higher affinity for ABCG2, suggesting that the nonpolar nature of FL118 plays a role in bypassing ABCG2-mediated resistance.ConclusionsOur results suggest that in contrast to SN-38 and topotecan, FL118 is a poor substrate for ABCG2 and can effectively overcome ABCG2-mediated drug resistance. Our findings expand the uniqueness of FL118 and support continued development of FL118 as an attractive therapeutic option for patients with drug-refractory cancers resulting from high expression of ABCG2

Pre-Treatment with Melatonin Enhances Therapeutic Efficacy of Cardiac Progenitor Cells for Myocardial Infarction

Background/Aims: Melatonin possesses many biological activities such as antioxidant and anti-aging. Cardiac progenitor cells (CPCs) have emerged as a promising therapeutic strategy for myocardial infarction (MI). However, the low survival of transplanted CPCs in infarcted myocardium limits the successful use in treating MI. In the present study, we aimed to investigate if melatonin protects against oxidative stress-induced CPCs damage and enhances its therapeutic efficacy for MI. Methods: TUNEL assay and EdU assay were used to detect the effects of melatonin and miR-98 on H2O2-induced apoptosis and proliferation. MI model was used to evaluate the potential cardioprotective effects of melatonin and miR-98. Results: Melatonin attenuated H2O2-induced the proliferation reduction and apoptosis of c-kit+ CPCs in vitro, and CPCs which pretreated with melatonin significantly improved the functions of post-infarct hearts compared with CPCs alone in vivo. Melatonin was capable to inhibit the increase of miR-98 level by H2O2 in CPCs. The proliferation reduction and apoptosis of CPCs induced by H2O2 was aggravated by miR-98. In vivo, transplantation of CPCs with miR-98 silencing caused the more significant improvement of cardiac functions in MI than CPCs. MiR-98 targets at the signal transducer and activator of the transcription 3 (STAT3), and thus aggravated H2O2-induced the reduction of Bcl-2 protein. Conclusions: Pre-treatment with melatonin protects c-kit+ CPCs against oxidative stress-induced damage via downregulation of miR-98 and thereby increasing STAT3, representing a potentially new strategy to improve CPC-based therapy for MI

Loss of Function of the Cik1/Kar3 Motor Complex Results in Chromosomes with Syntelic Attachment That Are Sensed by the Tension Checkpoint

The attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The spindle checkpoint can sense the lack of tension on chromosomes as well as detached kinetochores to prevent anaphase onset. In budding yeast Saccharomyces cerevisiae, tension checkpoint proteins Aurora/Ipl1 kinase and centromere-localized Sgo1 are required to sense the absence of tension but are dispensable for the checkpoint response to detached kinetochores. We have found that the loss of function of a motor protein complex Cik1/Kar3 in budding yeast leads to syntelic attachments. Inactivation of either the spindle or tension checkpoint enables premature anaphase entry in cells with dysfunctional Cik1/Kar3, resulting in co-segregation of sister chromatids. Moreover, the abolished Kar3-kinetochore interaction in cik1 mutants suggests that the Cik1/Kar3 complex mediates chromosome movement along microtubules, which could facilitate bipolar attachment. Therefore, we can induce syntelic attachments in budding yeast by inactivating the Cik1/Kar3 complex, and this approach will be very useful to study the checkpoint response to syntelic attachments

PaTAVTT: A Hardware-in-the-Loop Scaled Platform for Testing Autonomous Vehicle Trajectory Tracking

With the advent of autonomous vehicles, in particular its adaptability to harsh conditions, the research and development of autonomous vehicles attract significant attention by not only academia but also practitioners. Due to the high risk, high cost, and difficulty to test autonomous vehicles under harsh conditions, the hardware-in-the-loop (HIL) scaled platform has been proposed as it is a safe, inexpensive, and effective test method. This platform system consists of scaled autonomous vehicle, scaled roadway, monitoring center, transmission device, positioning device, and computers. This paper uses a case of the development process of tracking control for high-speed U-turn to build the tracking control function. Further, a simplified vehicle dynamics model and a trajectory tracking algorithm have been considered to build the simulation test. The experiment results demonstrate the effectiveness of the HIL scaled platform

Intercropping of wheat changed cucumber rhizosphere bacterial community composition and inhibited cucumber Fusarium wilt disease

Enhancing the plant rhizosphere microbial community function by increasing plant diversity in the field is a promising strategy for enhancing agricultural sustainability. This study evaluated the effectiveness of the intercropping of wheat in controlling cucumber Fusarium wilt disease. Bacterial community diversity and abundance in cucumber rhizosphere were estimated by high-throughput amplicon sequencing and quantitative polymerase chain reaction (PCR). Results showed that the intercropping of wheat inhibited the severity of cucumber seedling Fusarium wilt disease, increased the alpha diversity and altered the composition of the bacterial community in cucumber rhizosphere. Compared with monocropped cucumber, intercropped cucumber had higher relative abundances of Anaerolineae, Deltaproteobacteria, Phycisphaerae and Planctomycetacia, and lower Alphaproteobacteria and Cyanobacteria. Moreover, the intercropping of wheat promoted bacterial genera with plant-beneficial potential (e.g. Pseudomonas, Haliangium and Archangium spp.) in cucumber rhizosphere. Quantitative PCR confirmed that Pseudomonas spp. abundance was higher in intercropped cucumber rhizosphere than in monocropped cucumber rhizosphere. Overall, the intercropping of wheat decreased the severity of Fusarium wilt of cucumber and promoted potential plant-beneficial microbes in cucumber rhizosphere

Fin1-PP1 Helps Clear Spindle Assembly Checkpoint Protein Bub1 from Kinetochores in Anaphase

Summary: The spindle assembly checkpoint (SAC) monitors chromosome attachment defects, and the assembly of SAC proteins at kinetochores is essential for its activation, but the SAC disassembly process remains unknown. We found that deletion of a 14-3-3 protein, Bmh1, or hyperactivation of Cdc14 early anaphase release (FEAR) allows premature SAC silencing in budding yeast, which depends on a kinetochore protein Fin1 that forms a complex with protein phosphatase PP1. Previous works suggest that FEAR-dependent Fin1 dephosphorylation promotes Bmh1-Fin1 dissociation, which enables kinetochore recruitment of Fin1-PP1. We found persistent kinetochore association of SAC protein Bub1 in fin1Δ mutants after anaphase entry. Therefore, we revealed a mechanism that clears SAC proteins from kinetochores. After anaphase entry, FEAR activation promotes kinetochore enrichment of Fin1-PP1, resulting in SAC disassembly at kinetochores. This mechanism is required for efficient SAC silencing after SAC is challenged, and untimely Fin1-kinetochore association causes premature SAC silencing and chromosome missegregation. : Bokros et al. examine the process of spindle assembly checkpoint (SAC) disassembly and find that the kinetochore protein Fin1, in complex with the protein phosphatase PP1, localizes to the kinetochores during anaphase. Fin1-PP1 promotes the dissociation of the SAC protein Bub1 from kinetochores, leading to SAC disassembly. Keywords: spindle assembly checkpoint, Bmh1, Fin1, PP1, FEAR pathwa