Droplet digital PCR assay provides intrahepatic HBV cccDNA quantification tool for clinical application

The persistence of covalently closed circular DNA (cccDNA) poses a major obstacle to curing chronic hepatitis B (CHB). Here, we used droplet digital PCR (ddPCR) for cccDNA quantitation. The cccDNA-specific ddPCR showed high accuracy with the dynamic range of cccDNA detection from 101 to 105 copies/assay. The ddPCR had higher sensitivity, specificity and precisely than qPCR. The results of ddPCR correlated closely with serum HB core-related antigen and HB surface antigen (HBsAg) in 24 HBV-infected human-liver-chimeric mice (PXB-mice). We demonstrated that in 2 PXB-mice after entecavir treatment, the total cccDNA content did not change during liver repopulation, although the cccDNA content per hepatocyte was reduced after the treatment. In the 6 patients with HBV-related hepatocellular carcinoma, ddPCR detected cccDNA in both tumor and non-tumor tissues. In 13 HBeAg-negative CHB patients with pegylated interferon alpha-2a, cccDNA contents from paired biopsies were more significantly reduced in virological response (VR) than in non-VR at week 48 (p = 0.0051). Interestingly, cccDNA levels were the lowest in VR with HBsAg clearance but remained detectable after the treatment. Collectively, ddPCR revealed that cccDNA content is stable during hepatocyte proliferation and persists at quantifiable levels, even after serum HBsAg clearance

DDB Accumulates at DNA Damage Sites Immediately after UV Irradiation and Directly Stimulates Nucleotide Excision Repair

Damaged DNA-binding protein, DDB, is a heterodimer of p127 and p48 with a high specificity for binding to several types of DNA damage. Mutations in the p48 gene that cause the loss of DDB activity were found in a subset of xeroderma pigmentosum complementation group E (XP-E) patients and have linked to the deficiency in global genomic repair of cyclobutane pyrimidine dimers (CPDs) in these cells. Here we show that with a highly defined system of purified repair factors, DDB can greatly stimulate the excision reaction reconstituted with XPA, RPA, XPC.HR23B, TFIIH, XPF.ERCC1 and XPG, up to 17-fold for CPDs and approximately 2-fold for (6-4) photoproducts (6-4PPs), indicating that no additional factor is required for the stimulation by DDB. Transfection of the p48 cDNA into an SV40-transformed human cell line, WI38VA13, was found to enhance DDB activity and the in vivo removal of CPDs and 6-4PPs. Furthermore, the combined technique of recently developed micropore UV irradiation and immunostaining revealed that p48 (probably in the form of DDB heterodimer) accumulates at locally damaged DNA sites immediately after UV irradiation, and this accumulation is also observed in XP-A and XP-C cells expressing exogenous p48. These results suggest that DDB can rapidly translocate to the damaged DNA sites independent of functional XPA and XPC proteins and directly enhance the excision reaction by core repair factors

Performance Visualization for Hearing-Impaired Students

We have been teaching computer music to hearing impaired students of Tsukuba College of Technology for six years. Although students have hearing difficulties, almost all of them show an interest in music. Thus, this has been a challenging class to turn their weakness into enjoyment. We thought that performance visualization is a good method for them to keep their interest in music and try cooperative performances with others. In this paper, we describe our computer music class and the result of our preliminary experiment on the effectiveness of visual assistance. Though it was not a complete experiment with a sufficient number of subjects, the result showed that the show-ahead and selected-note-only types of performance visualization were necessary according to the purpose of the visual aid

Changes in Calcium, Magnesium and Phosphorus Contents of Eggshell during Stay in Oviduct Uterus in the Guineafowl and the Chicken

The amount of calcium, magnesium and phosphorus in the shell portion (shell with shell membrane) of eggs of guineafowl and chicken at various times during the stay in the uterus was measured by an atomic absorption spectrometry or colorimetric method. All of the minerals were found to increase during the stay in the uterus, but the aspect of the increase differs between the minerals : parabolic in calcium, linear in magnesium, and quadratic in phosphorus, and also differs between the guineafowl and the chicken

Calcitonin Directly Inhibits Luteinizing Hormone-Stimulated Progesterone Production in Granulosa Cells of the Largest Follicle of Hen

The present study was performed to demonstrate the presence of receptors for avian calcitonin (cCT) in ovarian granulosa cells of the largest follicle of laying hens and to determine the effect of cCT on LH-stimulated P4 production. The cCT binding in the granulosa cells of the largest follicle was determined using a [125I]CT binding assay. The binding properties in the granulosa cells satisfied the criteria of a receptor-ligand interaction in terms of specificity, saturation, high affinity and limited capacity. During incubation of ovarian granulosa cells in vitro, cCT alone did not affect the P4 production alone, but cCT decreased the amount of LH-stimulated P4 production and the sensitivity to LH. The present study suggests that cCT may act directly on the granulosa cell of the largest follicle via its receptor binding and inhibit P4 production in response to LH in hens