Enzymatic characterization of recombinant rat DDHD2, a soluble diacylglycerol lipase

学位記番号：医博甲1589

Resminostat in EGFR-mutated lung cancer

Drug-tolerant cells are mediators of acquired resistance. BIM-intron2 deletion polymorphism (BIM-del) is one of the mechanisms underlying the resistance to epidermal growth factor tyrosine kinase inhibitor (EGFR-TKI)-mediated apoptosis that induces drug tolerance. Here, we investigated whether resminostat, a histone deacetylase inhibitor, circumvents BIM-del-associated apoptosis resistance. The human EGFR-mutated non-small cell lung cancer (NSCLC) cell line PC-9 and its homozygous BIM-del-positive variant (PC-9 BIMi2-/-), established by editing with zinc finger nuclease, were used. In comparison with PC-9 cells, PC-9 BIMi2-/- cells were less sensitive to apoptosis mediated by EGFR-TKIs such as gefitinib and osimertinib. The combined use of resminostat and an EGFR-TKI preferentially induced the expression of the pro-apoptotic BIM transcript containing exon 4 rather than that containing exon 3, increased the level of pro-apoptotic BIM protein (BIMEL), and stimulated apoptosis in vitro. In a subcutaneous tumor model derived from PC-9 BIMi2-/- cells, gefitinib monotherapy decreased tumor size but retained residual lesions, indicative of the presence of tolerant cells in tumors. The combined use of resminostat and gefitinib increased BIMEL protein level and induced apoptosis, subsequently leading to the remarkable shrinkage of tumor. These findings suggest the potential of resminostat to circumvent tolerance to EGFR-TKIs associated with BIM deletion polymorphism

Dram1 regulates DNA damage-induced alternative autophagy

Autophagy is an evolutionarily conserved process that degrades subcellular constituents. Mammalian cells undergo two types of autophagy; Atg5-dependent conventional autophagy and Atg5-independent alternative autophagy, and the molecules required for the latter type of autophagy are largely unknown. In this study, we analyzed the molecular mechanisms of genotoxic stress-induced alternative autophagy, and identified the essential role of p53 and damage-regulated autophagy modulator (Dram1). Dram1 was sufficient to induce alternative autophagy. In the mechanism of alternative autophagy, Dram1 functions in the closure of isolation membranes downstream of p53. These findings indicate that Dram1 plays a pivotal role in genotoxic stress-induced alternative autophagy

Autophagy controls centrosome number by degrading Cep63

Centrosome number is associated with the chromosome segregation and genomic stability. The ubiquitin–proteasome system is considered to be the main regulator of centrosome number. However, here we show that autophagy also regulates the number of centrosomes. Autophagy-deficient cells carry extra centrosomes. The autophagic regulation of centrosome number is dependent on a centrosomal protein of 63 (Cep63) given that cells lacking autophagy contain multiple Cep63 dots that are engulfed and digested by autophagy in wild-type cells, and that the upregulation of Cep63 increases centrosome number. Cep63 is recruited to autophagosomes via interaction with p62, a molecule crucial for selective autophagy. In vivo, hematopoietic cells from autophagy-deficient and p62−/− mice also contained multiple centrosomes. These results indicate that autophagy controls centrosome number by degrading Cep63

Cell cycle control of telomere protection and NHEJ revealed by a ts mutation in the DNA-binding domain of TRF2

TRF2 is a component of shelterin, the telomere-specific protein complex that prevents DNA damage signaling and inappropriate repair at the natural ends of mammalian chromosomes. We describe a temperature-sensitive (ts) mutation in the Myb/SANT DNA-binding domain of TRF2 that allows controlled and reversible telomere deprotection. At 32°C, TRF2ts was functional and rescued the lethality of TRF2 deletion from conditional TRF2F/− mouse embryonic fibroblasts (MEFs). When shifted to the nonpermissive temperature (37°C), TRF2ts cells showed extensive telomere damage resulting in activation of the ATM kinase and nonhomologous end-joining (NHEJ) of chromosome ends. The inactivation of TRF2ts at 37°C was rapid and reversible, permitting induction of short periods (3–6 h) of telomere dysfunction in the G0, G1, and S/G2 phases of the cell cycle. The results indicate that both the induction of telomere dysfunction and the re-establishment of the protected state can take place throughout interphase. In contrast, the processing of dysfunctional telomeres by NHEJ occurred primarily in G1, being repressed in S/G2 in a cyclin-dependent kinase (CDK)-dependent manner

More subjects are required for ventrolateral than dorsolateral prefrontal TMS because of intolerability and potential drop-out.

Transcranial magnetic stimulation (TMS) of the human lateral prefrontal cortex, particularly the ventral region, often causes considerable discomfort to subjects. To date, in contrast to abundant literature on stimulations to the dorsolateral prefrontal cortex, the ventrolateral prefrontal cortex has been less frequently stimulated, partly because some subjects are intolerable of stimulation to the ventrolateral prefrontal cortex. To predict the additional number of subjects required for the stimulation of the dorsolateral and ventrolateral prefrontal cortices, 20 young healthy subjects reported two evaluation scores: the discomfort caused by TMS and the resulting intolerability to complete the TMS experiments. Single-pulse stimulation (SPS) or theta-burst stimulation (TBS) was administered to the lateral prefrontal cortex. The high-resolution extended 10-20 system was used to provide accurate estimation of the voxelwise scores. The discomfort ratings with the SPS and TBS were relatively higher in the ventrolateral prefrontal cortex than those in the dorsolateral prefrontal cortex. Both the SPS and TBS elicited maximal discomfort at the stimulation position F8. The SPS and TBS to F8 under the standard TMS protocols were intolerable for approximately one half (11 and 10, respectively) of the subjects. The intolerability was further calculated for all voxels in the lateral prefrontal cortex, which enabled us to estimate the additional number of subjects required for specific target areas. These results suggest that prior knowledge of subjects' discomfort during stimulation of the lateral prefrontal cortex can be of practical use in the experimental planning of the appropriate number of recruited subjects and provide the database for the probability of intolerability that can be used to predict the additional number of subjects

fmri_task_activation_parcelwise

The file "fmri_task_activation_parcelwise.zip" contains the data of parcel-based activation during the stop-signal task for each subject (contrast: Stop success minus Go success; Dataset 1-fMRI). The files in each subject folder can be viewed by using wb_view. The parcel-based task activation maps are displayed in each subject's original space