Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145–amino acid residues with extensive α-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells

Analysis of Clinical Outcome of Patients with Poorly Differentiated Thyroid Carcinoma

Background. We retrospectively analyzed whether poor differentiation is the independent prognostic factor for thyroid carcinoma or not. Methods. The subjects were 29 patients with PDTC who were treated between April 1996 and March 2006 to compare with those of well-differentiated papillary carcinoma patients (n = 227). Results. The relapse free (RFS), distant relapse-free survival and cause-specific survival, rates were significantly lower in patients with PDTC (P < .0001, P < .001, and P < .05). After classification into focal (<10%) and diffuse type (over 10%) of PDTC, there were no significant differences in RFS and cause-specific survival due to component type or proportion of poorly differentiated component. On multivariate analysis, poor differentiation (P < .0005, RR = 4.456, 95% CI; 1.953–10.167) and extrathyroidal infiltration (P < .05, RR = 2.898, 95% CI; 1.278–6.572) showed a significant impact on DFS, and poor differentiation (P < .05, RR = 9.343, 1.314–66.453) and age (P < .005, RR = 1.306, 1.103–1.547) significantly impacted cause-specific survival. Conclusion. Poor differentiation was an independent factor for survival. Distant relapse was significantly more common among PDTC patients, and systemic therapy might be warranted

Brazilian Propolis Suppresses Angiogenesis by Inducing Apoptosis in Tube-Forming Endothelial Cells through Inactivation of Survival Signal ERK1/2

We recently reported that propolis suppresses tumor-induced angiogenesis through tube formation inhibition and apoptosis induction in endothelial cells. However, molecular mechanisms underlying such angiogenesis suppression by propolis have not been fully elucidated. The aim of this study was to investigate the effects of ethanol extract of Brazilian propolis (EEBP) on two major survival signals, extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, and to elucidate whether changes in these signals were actually involved in antiangiogenic effects of the propolis. Detection by western blotting revealed that EEBP suppressed phosphorylation of ERK1/2, but not that of Akt. Pharmacological inhibition by U0126 demonstrated that ERK1/2 inactivation alone was enough to inhibit tube formation and induce apoptosis. It was also shown that EEBP and U0126 similarly induced activation of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP) and lamin A/C, all of which are molecular markers of apoptosis. These results indicate that inhibition of survival signal ERK1/2, and subsequent induction of apoptosis, is a critical mechanism of angiogenesis suppression by EEBP

Nymphaeol-A Isolated from Okinawan Propolis Suppresses Angiogenesis and Induces Caspase-Dependent Apoptosis via Inactivation of Survival Signals

Propolis, a resinous substance that honeybees collect to protect their beehive from enemies, is reported to have various biological activities. In our screening program to search for antiangiogenic compounds from propolis, the ethanol extracts of Okinawan propolis (EEOP) showed significant antiangiogenic activities in a tube formation assay with human umbilical vein endothelial cells (HUVECs) in vitro at 3.13 μg/mL and chorioallantoic membrane (CAM) assay in vivo at 25 μg/egg. To elucidate the active compounds of EEOP and their mode of action, we isolated some prenylated flavonoids from EEOP and found that nymphaeol-A had the strongest antiangiogenic activity among them. Nymphaeol-A significantly reduced in vivo neovessel formation in the CAM assay at 25 μg/egg. At the molecular level, nymphaeol-A markedly inactivated mitogen-activated protein kinase/ERK kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2), whose molecular activations signal new vessel formation in HUVECs. In addition, nymphaeol-A dose- and time-dependently induced caspase-dependent apoptosis in tube-forming HUVECs. Taken together, nymphaeol-A was shown to inhibit angiogenesis at least in part via inactivation of MEK1/2–ERK1/2 signaling and induction of caspase-dependent apoptosis. Okinawan propolis and its major component, nymphaeol-A, may be useful agents for preventing tumor-induced angiogenesis

Assessment of human stress and depression by DNA microarray analysis

Precise assessment of stress is an imminent issue to deal with stress-related social, medical and psychological problems. Psychological stress is known to stimulate the neuroendocrine, sympathetic nervous, and immune systems. By analyzing mRNA expression levels in leukocytes, which express receptors for hormones, neurotransmitters, growth factors, cytokines, and other stress related signals, levels of stress may be adequately measured. In a series of studies, our group has developed a cDNA microarray specifically designed to measure the mRNA levels of stress-related genes in peripheral blood leukocytes. This microarray enabled us to sensitively detect the response to psychological stress. In addition, our preliminary study suggests that the array could differentiate patients with depression from sex- and age-matched control subjects

Original Article Brazilian Propolis Suppresses Angiogenesis by Inducing Apoptosis in Tube-Forming Endothelial Cells through Inactivation of Survival Signal ERK1/2

We recently reported that propolis suppresses tumor-induced angiogenesis through tube formation inhibition and apoptosis induction in endothelial cells. However, molecular mechanisms underlying such angiogenesis suppression by propolis have not been fully elucidated. The aim of this study was to investigate the effects of ethanol extract of Brazilian propolis (EEBP) on two major survival signals, extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, and to elucidate whether changes in these signals were actually involved in antiangiogenic effects of the propolis. Detection by western blotting revealed that EEBP suppressed phosphorylation of ERK1/2, but not that of Akt. Pharmacological inhibition by U0126 demonstrated that ERK1/2 inactivation alone was enough to inhibit tube formation and induce apoptosis. It was also shown that EEBP and U0126 similarly induced activation of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP) and lamin A/C, all of which are molecular markers of apoptosis. These results indicate that inhibition of survival signal ERK1/2, and subsequent induction of apoptosis, is a critical mechanism of angiogenesis suppression by EEBP

An individual based computational model of intestinal crypt fission and its application to predicting unrestrictive growth of the intestinal epithelium.

Intestinal crypt fission is a homeostatic phenomenon, observable in healthy adult mucosa, but which also plays a pathological role as the main mode of growth of some intestinal polyps. Building on our previous individual based model for the small intestinal crypt and on in vitro cultured intestinal organoids, we here model crypt fission as a budding process based on fluid mechanics at the individual cell level and extrapolated predictions for growth of the intestinal epithelium. Budding was always observed in regions of organoids with abundant Paneth cells. Our data support a model in which buds are biomechanically initiated by single stem cells surrounded by Paneth cells which exhibit greater resistance to viscoelastic deformation, a hypothesis supported by atomic force measurements of single cells. Time intervals between consecutive budding events, as simulated by the model and observed in vitro, were 2.84 and 2.62 days, respectively. Predicted cell dynamics was unaffected within the original crypt which retained its full capability of providing cells to the epithelium throughout fission. Mitotic pressure in simulated primary crypts forced upward migration of buds, which simultaneously grew into new protruding crypts at a rate equal to 1.03 days-1 in simulations and 0.99 days-1 in cultured organoids. Simulated crypts reached their final size in 4.6 days, and required 40 6.2 days to migrate to the top of the primary crypt. The growth of the secondary crypt is independent of its migration along the original crypt. Assuming unrestricted crypt fission and multiple budding events, a maximal growth rate of the intestinal epithelium of 0.10 days-1 43 is predicted and thus approximately 22 days are required for a 10-fold increase of polyp size. These predictions are in agreement with the time reported to develop macroscopic adenomas in mice after loss of Apc in intestinal stem cells

Incidents of Violence and Verbal Abuse from Patients and Their Relatives against Nurses in Dokkyo Medical University Koshigaya Hospital

This study aimed to understand the nature and frequency of violence and verbal abuse toward nurses ina Japanese general hospital. Questionnaires concerning violence and verbal abuse against nurses were distributedto 440 nurses employed at a university hospital and a total of 438 nurses responded to the questionnaire.Over one third reported attempted incidents of violence, whereas further one quarter reported beingactually pinched, touched unnecessarily, or kicked. More than a half reported being yelled at, being complainedto unreasonably about a medical service, and experiencing overbearing voices. The most commonemotion experienced after facing violence or verbal abuse was embarrassment and discomfort, followed byanger. Nurses in the emergency and intensive care units and some surgical wards experienced higher levelsof violence and verbal abuse than those in other wards. These findings prove the urgent need for the developmentof preventive actions to address the problem of workplace adversity

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target