Two spatially distinct kinesin-14 proteins, Pkl1 and Klp2, generate collaborative inward forces against kinesin-5 Cut7 in S. pombe

Kinesin motors play central roles in bipolar spindle assembly. In many eukaryotes, spindle pole separation is driven by kinesin-5, which generates outward force. This outward force is balanced by antagonistic inward force elicited by kinesin-14 and/or dynein. In fission yeast, two kinesin-14 proteins, Pkl1 and Klp2, play an opposing role against the kinesin-5 motor protein Cut7. However, how the two kinesin-14 proteins coordinate individual activities remains elusive. Here, we show that although deletion of either pkl1 or klp2 rescues temperature-sensitive cut7 mutants, deletion of only pkl1 can bypass the lethality caused by cut7 deletion. Pkl1 is tethered to the spindle pole body, whereas Klp2 is localized along the spindle microtubule. Forced targeting of Klp2 to the spindle pole body, however, compensates for Pkl1 functions, indicating that cellular localizations, rather than individual motor specificities, differentiate between the two kinesin-14 proteins. Interestingly, human kinesin-14 (KIFC1 or HSET) can replace either Pkl1 or Klp2. Moreover, overproduction of HSET induces monopolar spindles, reminiscent of the phenotype of Cut7 inactivation. Taken together, this study has uncovered the biological mechanism whereby two different Kinesin- 14 motor proteins exert their antagonistic roles against kinesin-5 in a spatially distinct manner.This work was supported by the Japan Society for the Promotion of Science (JSPS) [KAKENHI Scientific Research (A) 16H02503 to T.T., a Challenging Exploratory Research grant 16K14672 to T.T., Scientific Research (C) 16K07694 to M.Y.], the Naito Foundation (T.T.) and the Uehara Memorial Foundation (T.T)

Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors

Many human cancer cells contain more than two centrosomes, yet these cancer cells can form pseudo-bipolar spindles through the mechanism, called centrosome clustering, and survive, instead of committing lethal multipolar mitoses. Kinesin-14/HSET, a minus end-directed motor, plays a crucial role in centrosome clustering. Accordingly, HSET is deemed to be a promising chemotherapeutic target to selectively kill cancer cells. Recently, three HSET inhibitors (AZ82, CW069 and SR31527) have been reported, but their specificity and efficacy have not been evaluated rigorously. This downside partly stems from the lack of robust systems for the assessment of these drugs. Yeasts and filamentous fungi provide not only powerful models for basic and applied biology but also versatile tools for drug discovery and evaluation. Here we show that these three inhibitors on their own are cytotoxic to fission yeast, suggesting that they have off-targets in vivo except for kinesin-14. Nonetheless, intriguingly, AZ82 can neutralize otherwise toxic overproduced HSET; this includes a substantial reduction in the percentage of HSET-driven abnormal mitotic cells and partial suppression of its lethality. SR31527 also displays modest neutralizing activity, while we do not detect such activity in CW069. As an experimental proof-of-principle study, we have treated HSET-overproducing fission yeast cells with extracts prepared from various plant species and found activities that rescue HSET-driven lethality in those from Chamaecyparis pisifera and Toxicodendron trichocarpum. This methodology of protein overproduction in fission yeast, therefore, provides a convenient, functional assay system by which to screen for not only selective human kinesin-14 inhibitors but also those against other molecules of interest.This work was supported by the Japan Society for the Promotion of Science (JSPS) (KAKENHI Scientific Research (A) 16H02503 to T.T., a Challenging Exploratory Research grant 16K14672 to T.T., Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers (S2902) to T.T. and S.A. and Scientific Research (C) 16K07694 to M.Y.), the Naito Foundation (T.T.) and the Uehara Memorial Foundation (T.T)

VLBI Detections of Parsec-Scale Nonthermal Jets in Radio-Loud Broad Absorption Line Quasars

We conducted radio detection observations at 8.4 GHz for 22 radio-loud broad absorption line (BAL) quasars, selected from the Sloan Digital Sky Survey (SDSS) Third Data Release, by a very-long-baseline interferometry (VLBI) technique. The VLBI instrument we used was developed by the Optically ConnecTed Array for VLBI Exploration project (OCTAVE), which is operated as a subarray of the Japanese VLBI Network (JVN). We aimed at selecting BAL quasars with nonthermal jets suitable for measuring their orientation angles and ages by subsequent detailed VLBI imaging studies to evaluate two controversial issues of whether BAL quasars are viewed nearly edge-on, and of whether BAL quasars are in a short-lived evolutionary phase of quasar population. We detected 20 out of 22 sources using the OCTAVE baselines, implying brightness temperatures greater than 10^5 K, which presumably come from nonthermal jets. Hence, BAL outflows and nonthermal jets can be generated simultaneously in these central engines. We also found four inverted-spectrum sources, which are interpreted as Doppler-beamed, pole-on-viewed relativistic jet sources or young radio sources: single edge-on geometry cannot describe all BAL quasars. We discuss the implications of the OCTAVE observations for investigations for the orientation and evolutionary stage of BAL quasars.Comment: 10 pages, no figure, 3 tables, accepted for publication in PAS

Fundamental Parameters of the Milky Way Galaxy Based on VLBI astrometry

We present analyses to determine the fundamental parameters of the Galaxy based on VLBI astrometry of 52 Galactic maser sources obtained with VERA, VLBA and EVN. We model the Galaxy's structure with a set of parameters including the Galaxy center distance R_0, the angular rotation velocity at the LSR Omega_0, mean peculiar motion of the sources with respect to Galactic rotation (U_src, V_src, W_src), rotation-curve shape index, and the V component of the Solar peculiar motions V_sun. Based on a Markov chain Monte Carlo method, we find that the Galaxy center distance is constrained at a 5% level to be R_0 = 8.05 +/- 0.45 kpc, where the error bar includes both statistical and systematic errors. We also find that the two components of the source peculiar motion U_src and W_src are fairly small compared to the Galactic rotation velocity, being U_src = 1.0 +/- 1.5 km/s and W_src = -1.4 +/- 1.2 km/s. Also, the rotation curve shape is found to be basically flat between Galacto-centric radii of 4 and 13 kpc. On the other hand, we find a linear relation between V_src and V_sun as V_src = V_sun -19 (+/- 2) km/s, suggesting that the value of V_src is fully dependent on the adopted value of V_sun. Regarding the rotation speed in the vicinity of the Sun, we also find a strong correlation between Omega_0 and V_sun. We find that the angular velocity of the Sun, Omega_sun, which is defined as Omega_sun = Omega_0 + V_sun/R_0, can be well constrained with the best estimate of Omega_sun = 31.09 +/- 0.78 km/s/kpc. This corresponds to Theta_0 = 238 +/- 14 km/s if one adopts the above value of R_0 and recent determination of V_sun ~ 12 km/s.Comment: 14 pages, 6 figures, PASJ in pres

Up-Regulation of Pain Behavior and Glial Activity in the Spinal Cord after Compression and Application of Nucleus Pulposus onto the Sciatic Nerve in Rats

Study DesignExperimental animal study.PurposeTo evaluate pain-related behavior and changes in glial activity in the spinal dorsal horn after combined sciatic nerve compression and nucleus pulposus (NP) application in rats.Overview of LiteratureMechanical compression and inflammation caused by prostaglandins and cytokines at disc herniation sites induce pain. Structural changes and pain-associated cytokines in the dorsal root ganglia and spinal dorsal horn contribute to prolonged pain. Glial cells in the spinal dorsal horn may also function in pain transmission.MethodsThe sciatic nerve was compressed with NP for 2 seconds using forceps in the NP+nerve compression group; the sham-operated group received neither compression nor NP; and the control group received no operation. Mechanical hyperalgesia was measured for 3 weeks using von Frey filaments. Glial activity in the spinal dorsal horn was examined 7 days and 14 days postsurgery using anti-glial fibrillary acidic protein and anti-Ionized calcium binding adaptor molecule-1 antibodies to detect astrocytes and microglia, respectively.Results Mechanical hyperalgesia was detected throughout the 14-day observation in the NP+nerve compression group, but not in control or sham-operated groups (p<0.05). Both astrocytes and microglia were significantly increased in the spinal dorsal horn of the NP+nerve compression group compared to control and sham groups on days 7 and 14 (p<0.05).ConclusionsNerve compression with NP application produces pain-related behavior, and up-regulates astrocytes and microglia in the spinal dorsal horn, suggesting that these glia may be related to pain transmission

Evaluation of Behavior and Expression of Receptor Activator of Nuclear Factor-Kappa B Ligand in Dorsal Root Ganglia after Sciatic Nerve Compression and Application of Nucleus Pulposus in Rats

Study DesignExperimental animal study.PurposeTo evaluate pain-related behavior and changes in nuclear factor-kappa B (NF-kB), receptor activator of NF-kB (RANK), and ligand (RANKL) in dorsal root ganglia (DRG) after combined sciatic nerve compression and nucleus pulposus (NP) application in rats.Overview of LiteratureThe pathological mechanisms underlying pain from lumbar-disc herniation have not been fully elucidated. RANKL are transcriptional regulators of inflammatory cytokines. Our aim was to evaluate pain-related behavior and RANKL expression in DRG after sciatic-nerve compression and application of NP in rats.MethodsMechanical hyperalgesia and RANKL expression were assessed in three groups of rats: NP+sciatic nerve compression (2 seconds), sham-operated, and controls (n=20 each). Mechanical hyperalgesia was measured every other day for 3 weeks using von Frey filaments. RANKL expression in L5 DRGs was examined at five and ten days after surgery using immunohistochemistry.ResultsMechanical hyperalgesia was observed over the 12-day observation period in the NP+nerve compression group, but not in the control and sham-operated animal groups (p<0.05). RANKL immunoreactivity was seen in the nuclei of L5 DRG neurons, and its expression was significantly upregulated in NP+nerve compression rats compared with control and sham-operated rats (p<0.01).ConclusionsThe exposure of sciatic nerves to mechanical compression and NP produces pain-related behavior and up-regulation of RANKL in DRG neurons. RANKL may play an important role in mediating pain after sciatic nerve injury with exposure to NP

Interspinous Ligament Lidocaine and Steroid Injections for the Management of Baastrup's Disease: A Case Series

Study DesignProspective study.PurposeTo examine the long-term effects of interspinous ligament injections of local anesthetics and steroids for the treatment of Baastrup's diseases.Overview of LiteratureBaastrup's disease is associated with axial low back pains. Baastrup's disease has been more recently described as the "kissing spinous processes" disease. Several authors have reported methods for the diagnosis and treatment of the disease. However, there has been only one report of patients receiving interspinous ligament injections of agents for the treatment of Baastrup's disease.MethodsSeventeen patients showed severe low back pains between spinous processes at L3-L4 or L4-L5. X-ray imaging, computed tomography, and magnetic resonance imaging revealed kissing spinous processes, consolidation of spinous process, or inflammation of an interspinous ligament. Pain reliefs after lidocaine and dexamethasone administration into interspinous ligament as therapy for low back pains were being examined and followed up.ResultsLow back pain scores significantly improved immediately after injection of the agents into interspinous ligaments. At final follow-up (1.4 year), low back pain scores significantly improved as compared with before the treatment.ConclusionsFindings from the current study indicate that lidocaine and dexamethasone administration into interspinous ligament in patients diagnosed with Baastrup's disease is effective for managing the pain associated with this disease

Influence of Skeletal Muscle Mass and Spinal Alignment on Surgical Outcomes for Lumbar Spinal Stenosis

Study Design Retrospective observational study. Purpose We considered the relationship between spinal alignment and skeletal muscle mass on clinical outcomes following a surgery for lumbar spinal stenosis (LSS). Overview of Literature There are no reports of preoperative factors predicting residual low back pain following surgery for LSS. Methods Our target population included 34 women (mean age, 74.4 years) who underwent surgery for LSS. Prior to and 6 months after the surgery, systemic bone mineral density and lean soft tissue mass were measured using dual-energy X-ray absorptiometry. Skeletal muscle mass index (SMI) was calculated as the sum of the arm and leg lean mass in kilograms divided by height in meters squared. The spinal alignment was also measured. Clinical outcomes were evaluated using the Japanese Orthopedic Association scoring system, leg and low back pain Visual Analog Scale, and Roland–Morris Disability Questionnaire (RDQ). Additionally, we examined the bone mineral density, skeletal muscle mass, and spinal alignment before and after the surgery. We used the Spearman correlation coefficient to examine the associations among clinical outcomes, preoperative muscle mass, and spinal alignment. Results Sarcopenia (SMI 6.12), RDQ was significantly higher in subjects with sarcopenia (p =0.04). RDQ was significantly negatively correlated with SMI (r =−0.42, p <0.05). There was a significant positive correlation between postoperative RDQ and pelvic tilt (PT; r =0.41, p <0.05). SMI and PT were significantly negatively correlated (r =−0.39, r <0.05). Conclusions Good postoperative outcomes were negatively correlated with low preoperative appendicular muscle mass, suggesting that postoperative outcomes were inferior in cases of decreased appendicular muscle mass (sarcopenia). Posterior PT due to decreased limb muscle mass may contribute to postoperative back pain, showing that preoperatively reduced limb muscle mass and posterior PT are predictive factors in the persistence of postoperative low back pain