METHODS: Caprine islets were isolated and purified. Islets were handpicked and the diameter of the islets was recorded using light microscopy. Viablility of the islets was analyzed by confocal microscopy. Insulin secretion assay was carried out and analyzed by ELISA.
RESULTS: When tested at 48 h after isolation, these small islets were 29.3% more viable compared to the large-sized islets. Large islets showed a high ratio (P
METHODS: For identification of the minimal selective domain for apoptosis, the wild-type Apoptin gene had been reconstructed by PCR to generate segmental deletions at the N' terminal and linked with nuclear localization sites (NLS1 and NLS2). All the constructs were fused with maltose-binding protein gene and individually expressed by in vitro Rapid Translation System. Standardized dose of proteins were delivered into human breast adenocarcinoma MCF-7 cells and control human liver Chang cells by cytoplasmic microinjection, and subsequently observed for selective apoptosis effect.
RESULTS: Three of the truncated Apoptin proteins with N-terminal deletions spanning amino acid 32-83 retained the cancer selective nature of wild-type Apoptin. The proteins were successfully translocated to the nucleus of MCF-7 cells initiating apoptosis, whereas non-toxic cytoplasmic retention was observed in normal Chang cells. Whilst these truncated proteins retained the tumour-specific death effector ability, the specificity for MCF-7 cells was lost in two other truncated proteins that harbor deletions at amino acid 1-31. The detection of apoptosing normal Chang cells and MCF-7 cells upon cytoplasmic microinjection of these proteins implicated a loss in Apoptin's signature targeting activity.
CONCLUSIONS: Therefore, the critical stretch spanning amino acid 1-31 at the upstream of a known hydrophobic leucine-rich stretch (LRS) was strongly suggested as one of the prerequisite region in Apoptin for cancer targeting. Identification of this selective domain provides a platform for developing small targets to facilitating carrier-mediated-transport across cellular membrane, simultaneously promoting protein delivery for selective and effective breast cancer therapy.
MATERIALS AND METHODS: A prospective study was conducted in a tertiary referral cen re using the Scoliosis Research Society-22 (SRS-22) questionnaire during the patients' annual follow up, betwee February to April 2014. Thirtyseven patients who met the inclusion criteria were enrolled.
RESULTS: The mean pre-operative Cobb's angles were 57.8o ± 12.7o and mean post-operative angle of 20.0o ± 10.4o, resulting in average correction of 65.9 ± 14.4%. Mean preoperative rib hump was 61.1 ± 15.4 mm with mean postoperative rib hump of 15.8 ± 17.8 mm, resulting in average reduction of 77.7 ± 23.7%. Mean of post-operative total SRS score was 4.1 ± 0.5. Using Spearman rank correlation, the percentage of Cobb's angle correction versus the SRS-22 score showed correlation of 0.17 (P=0.33) while the percentage of rib hump reduction versus SRS-22 score showed a correlation of 0.11 (P=0.53).
CONCLUSION: In this study, the average total SRS-22 score was 4.1 ± 0.5 (range, 3.1-4.9) post-operatively indicating very high satisfaction rate overall. Despite attempts at greater curve correction and rib hump reduction, there is no direct correlation between patient satisfaction and radiographic parameters.