Cell culture and in vivo analyses of cytopathic hepatitis C virus mutants

AbstractHCV-JFH1 yields subclones that develop cytopathic plaques (Sekine-Osajima Y, et al., Virology 2008; 371:71). Here, we investigated viral amino acid substitutions in cytopathic mutant HCV-JFH1 clones and their characteristics in vitro and in vivo. The mutant viruses with individual C2441S, P2938S or R2985P signature substitutions, and with all three substitutions, showed significantly higher intracellular replication efficiencies and greater cytopathic effects than the parental JFH1 in vitro. The mutant HCV-inoculated mice showed significantly higher serum HCV RNA and higher level of expression of ER stress-related proteins in early period of infection. At 8weeks post inoculation, these signature mutations had reverted to the wild type sequences. HCV-induced cytopathogenicity is associated with the level of intracellular viral replication and is determined by certain amino acid substitutions in HCV-NS5A and NS5B regions. The cytopathic HCV clones exhibit high replication competence in vivo but may be eliminated during the early stages of infection

Small Molecule Multi-Targeted Kinase Inhibitor RGB-286638 Triggers P53-Dependent and -Independent Anti-Multiple Myeloma Activity through Inhibition of Transcriptional CDKs

Small molecule multi-targeted CDK inhibitors (CDKIs) are of particular interest due to their potent antitumor activity independent of p53 gene alterations. P53 deletion is associated with a very poor prognosis in multiple myeloma (MM). In this regard, we tested the anti-MM activity of RGB-286638, an indenopyrazole-derived CDKI with Ki-nanomolar activity against transcriptional CDKs. We examined RGB-286638’s mode-of-action in MM cell lines with wild type (wt)-p53 and those expressing mutant p53. RGB-286638 treatment resulted in MM cytotoxicity in vitro associated with inhibition of MM tumor growth and prolonged survival in vivo. RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription. RGB-286638-triggered p53 accumulation via nucleolar stress and loss of Mdm2, accompanied by induction of p53 DNA binding activity. Additionally, RGB-286638 mediated p53-independent activity, which was confirmed by cytotoxicity in p53-knockdown and p53-mutant cells. We also demonstrated downregulation of oncogenic miR-19, miR-92a-1, and miR-21. Our data provide the rationale for the development of transcriptional CDK inhibitors as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells

Resectable hepatoblastoma with tumor thrombus extending into the right atrium after chemotherapy: A case report

AbstractHepatoblastoma with intraatrial tumor thrombus is relatively rare. We report a case of hepatoblastoma with tumor thrombus extending into the right atrium, which responded well to chemotherapy and was resected using extracorporeal circulation. A 4-year-old girl was referred to our hospital because of abdominal distention and tenderness. A computed tomography (CT) scan showed a large tumor occupying the left 3 segments of the liver with tumor thrombus extending into the right atrium. There was also a small intrahepatic metastasis in the right lobe of the liver. She was diagnosed with hepatoblastoma on the basis of the results of open biopsy. Neoadjuvant chemotherapy with an intense CDDP-based regimen was performed. The tumor responded well to chemotherapy, and intrahepatic metastasis became undetectable on CT scan, although the tumor thrombus remained in the right atrium. After 7 courses of chemotherapy, we performed resection using extracorporeal circulation. The postoperative course was uneventful, and adjuvant chemotherapy was started 10 days after the operation. Her serum alpha-fetoprotein (AFP) level decreased to the normal range, and she was free of disease for 1 year after the operation. Tumor resection using extracorporeal circulation can be performed safely and is justified in patients with intraatrial tumor thrombus

Cancer of Unknown Primary Site:A Review of 28 Cases and the Efficacy of Cisplatin/Docetaxel Therapy at a Single Institute in Japan

We evaluated the efficacy and toxicity of cisplatin/docetaxel (CDDP/TXT) chemotherapy and identified prognostic factors in Japanese patients with cancer of unknown primary site (CUP). Twenty-eight consecutive patients seen at a single institute were reviewed retrospectively. Sixteen patients were treated with TXT 80mg/m2, followed by CDDP 75mg/m2. The overall response rate to CDDP/TXT treatment was 62.5%, with a median survival time (MST) of 22.7 months. Common adverse reactions were myelosuppression and hyponatremia. The MST of all 28 patients with CUP was 8.3 months, and the 1-year overall survival rate was 45.6%. Univariate analysis identified 5 prognostic factors:performance status, liver involvement, bone involvement, pleural involvement, and lymph node involvement. In conclusion, CDDP/TXT chemotherapy is effective with tolerable toxicity in patients with CUP. Japanese patients with CUP might be chemosensitive and may survive longer

栽培方法，抽出条件の異なる緑茶抽出液によるin vitroでのビフィズス菌の増殖への影響

In this study green tea leaves harvested in Kyoto（June 2014）and grown with or without shading were extracted with boiled or 80℃ water, examining for their effects in vitro on the growth of Bifidobacterium bifidum. In addition, catechins and other components included in each sample were assayed by the HPLC method. The results demonstrated that all of the extracts increased the growth of the strain. Compared with water（control）, the growth-promoting effects on the strain were 517 times higher than those with the boiled-water extract from the leaves grown without shading, 322 times than those with the boiled-water extract from the leaves grown with shading, and 308 times than those with the 80℃ extract from the leaves grown with shading. The total amount of catechins in the extracts was 343.29 mg/100 ml, 240.57 mg/100 ml, and 128.87 mg/100 ml, respectively. Thus, the green tea extracts used in this experiment may have promoted an increase in strain growth in vitro, but the degree of the effects was not necessarily proportional to catechin content.研究ノー

Static and dynamic hypergravity responses of osteoblasts and osteoclasts in medaka scales

Fish scales are a form of calcified tissue similar to that found in human bone. In medaka scales, we detected both osteoblasts and osteoclasts and subsequently developed a new scale assay system. Using this system, we analyzed the osteoblastic and osteoclastic responses under 2-, 3-, and 4-gravity (G) loading by both centrifugation and vibration. After loading for 10 min, the scales from centrifugal and vibration loading were incubated for 6 and 24 hrs, respectively, after which the osteoblastic and osteoclastic activities were measured. Osteoblastic activity significantly increased under 2- to 4-G loading by both centrifugation and vibration. In contrast, we found that osteoclastic activity significantly decreased under 2- and 3-G loading in response to both centrifugation and vibration. Under 4-G loading, osteoclastic activity also decreased on centrifugation, but significantly increased under 4-G loading by vibration, concomitant with markedly increased osteoblastic activity. Expression of the receptor activator of the NF-αB ligand (RANKL), an activation factor of osteoclasts expressed in osteoblasts, increased significantly under 4-G loading by vibration but was unchanged by centrifugal loading. A protein sequence similar to osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, was found in medaka using our sequence analysis. The ratio of RANKL/OPG-like mRNAs in the vibration-loaded scales was significantly higher than that in the control scales, although there was no difference between centrifugal loaded scales and the control scales. Accordingly, medaka scales provide a useful model by which to analyze bone metabolism in response to physical strain. © 2013 Zoological Society of Japan

Daytime nap and nighttime breastfeeding are associated with toddlers\u27 nighttime sleep

The purpose of the present study is to examine the association between toddlers\u27 sleep arrangements and their nighttime sleep duration and other sleep variables. For this investigation, we performed a study in which child activity and sleep levels were recorded using actigraphy. The parents of 1.5-year-old toddlers (n = 106) were asked to attach an actigraphy unit to their child’s waist with an adjustable elastic belt and complete a sleep diary for 7 consecutive days. Questionnaires were used to assess the sleep arrangements of the toddlers. There was a significant negative correlation between nap duration and nighttime sleep duration, suggesting that longer nap sleep induces shorter nighttime sleep duration. Among the sleep arrangements, such as nighttime breastfeeding or co-sleeping, only nighttime breastfeeding predicted shorter nighttime sleep duration. Our findings indicate that shorter naps induce a longer nighttime sleep in 1.5-year-old toddlers while nighttime breastfeeding decreases their nighttime sleep duration

Phospholipase D Family Member 4, a Transmembrane Glycoprotein with No Phospholipase D Activity, Expression in Spleen and Early Postnatal Microglia

BACKGROUND: Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)(4)-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4. METHODOLOGY/PRINCIPAL FINDINGS: PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity. CONCLUSIONS/SIGNIFICANCE: Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells

Sleep maturation influences cognitive development of preterm toddlers

Our recent study on full-term toddlers demonstrated that daytime nap properties affect the distribution ratio between nap and nighttime sleep duration in total sleep time but does not affect the overall total amount of daily sleep time. However, there is still no clear scientific consensus as to whether the ratio between naps and nighttime sleep or just daily total sleep duration itself is more important for healthy child development. In the current study, to gain an answer to this question, we examined the relationship between the sleep properties and the cognitive development of toddlers born prematurely using actigraphy and the Kyoto scale of psychological development (KSPD) test. 101 premature toddlers of approximately 1.5 years of age were recruited for the study. Actigraphy units were attached to their waist with an adjustable elastic belt for 7 consecutive days and a child sleep diary was completed by their parents. In the study, we found no significant correlation between either nap or nighttime sleep duration and cognitive development of the preterm toddlers. In contrast, we found that stable daily wake time was significantly associated with better cognitive development, suggesting that sleep regulation may contribute to the brain maturation of preterm toddlers

Preterm toddlers have low nighttime sleep quality and high daytime activity.

A number of studies have been made on the sleep characteristics of children born preterm in an attempt to develop methods to address the sleep problems commonly observed among such children. However, the reported sleep characteristics from these studies vary depending on the observation methods used, i.e., actigraphy, polysomnography and questionnaire. In the current study, to obtain reliable data on the sleep characteristics of preterm-born children, we investigated the difference in sleep properties between 97 preterm and 97 term toddlers of approximately 1.5 years of age using actigraphy. Actigraphy units were attached to the toddlers’ waists with an adjustable elastic belt for 7 consecutive days, and a child sleep diary was completed by their parents. In the study, we found that preterm toddlers had more nocturnal awakenings and more daytime activity, suggesting that preterm-born children may have a different process of sleep development in their early development