Importin-β and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid

Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-α/β transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells. In digitonin-permeabilized mitotic cells, hKid was bound only to the spindle and not to the chromosomes themselves. Surprisingly, hKid bound to importin-α/β was efficiently targeted to mitotic chromosomes. The addition of Ran–guanosine diphosphate and an energy source, which generates Ran–guanosine triphosphate (GTP) locally at mitotic chromosomes, enhanced the importin-β–mediated chromosome loading of hKid. Our results indicate that the association of importin-β and -α with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP–mediated cargo release promotes the accumulation of hKid on chromosomes. Thus, this study demonstrates a novel nucleocytoplasmic transport factor–mediated mechanism for targeting proteins to mitotic chromosomes

Short-term changes in intracellular ROS localisation after the silver nanoparticles exposure depending on particle size

Silver nanoparticles (AgNPs) induce the production of reactive oxygen species (ROS) and apoptosis. These effects are enhanced by smaller particles. Using live-cell imaging, we show that AgNPs induced ROS production rapidly in a size-dependent manner after exposure of cells to 70-nm and 1-nm AgNPs (AgNPs-70, AgNPs-1), but not AgNO3. Exposure of cells to 5 μg/mL each of AgNPs-70, AgNPs-1 or AgNO3 for 1 h decreased the cell viability by approximately 40%, 100% and 20%, respectively. ROS were rapidly induced after 5 and 60 min by AgNPs-1 and AgNPs-70, respectively, whereas AgNO3 had no detectable effect. ROS production detected using the reporter dichlorodihydrofluorescein was observed in whole cells and mitochondria 5 and 60 min after exposure to AgNPs-1. The present study is the first, to our knowledge, to report the temporal expression and intracellular localisation of ROS induced by AgNPs

Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection.

The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method's sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2