Comparison of different methods of RNA preparation from peripheral blood for nucleic acid amplification assay

Background: Nucleic acid amplification assays (NAAs), such as polymerase chain reaction or loop-mediated isothermal amplification (LAMP), are used for disease diagnosis. Current nucleic acid isolation kits require several hours for completion of protocol including the complicated handling steps. Objective: In this study, a simple and cost-effective nucleic acid preparation method was developed, and its performance was compared with those of commercial kits. Materials and Methods: RNA was prepared using our method and three commercial RNA isolation kits. The RNA quantity and quality were evaluated using the NanoDrop spectrophotometer and Agilent 2100 bioanalyser. Reverse transcription LAMP (RT-LAMP) reactions were performed to determine the usability of the RNA preparation methods. Results: The concentrations of RNA extracted from blood samples by four different methods were sufficient for use in NAAs. The RNA integrity number was >7.0 when RNA was isolated using other RNA isolation kits but lower when prepared using our method. The RT-LAMP reaction was successfully performed when RNA was prepared using any of the methods. Conclusions: These results demonstrate that despite the lower purity and integrity of RNA, our RNA preparation protocol is simple and rapid and shows reasonable performance in RT-LAMP.

Topical chemotherapy for giant ocular surface squamous neoplasia of the conjunctiva and cornea: Is surgery necessary?

Purpose: The purpose of this study is to report on the efficacy and safety of topical chemotherapy alone for giant ocular surface squamous neoplasia (OSSN). Methods: In this retrospective, interventional series, 10 eyes with giant OSSN underwent exfoliative biopsy to confirm the diagnosis followed by application of interferon alpha 2b (IFN ?2b) and/or 5 fluorouracil, 1% (5FU). Reported outcome measures were tumor response, visual acuity, recurrence, systemic metastasis, and treatment complications. Results: Ten patients (3 female, 7 male) had a mean age of 73 (median, 69; range 40–89) years. Mean tumor diameter was 13.1 (median, 12.3; range 8.2–19.4) mm. Five (50%) eyes were treated with IFN-?2b alone; 1 (10%) with 5-FU alone and 4 (40%) required both IFN-?2b and 5-FU. The mean duration of treatment was 3, 0.5, and 6.4 months for IFN-?2b alone, 5-FU alone, and both IFN-?2b and 5-FU respectively. Complete tumor response was observed in all 10 cases at mean follow-up of 12.8 (median, 11.5; range, 3–25) months. Complications noted were transient irritation and burning (n = 4), dry eyes (n = 2), and transient flu-like symptoms (n = 2). There was no evidence of chemotherapy-related symblepharon, stem cell deficiency, scleral thinning, or corneal opacity. There were no tumor recurrences, and no patient required surgical excision or cryotherapy. Conclusion: Topical chemotherapy was a safe and effective treatment, inducing complete regression in all cases of giant OSSN in this series. There were no sight-limiting complications.

Association of the Lipoprotein Lipase S447X and Neuropeptide Y Leu7Pro Genetic Polymorphisms with the Lipid Profiles of Individuals with and without Evidence of Coronary Artery Disease.

Background: Coronary artery disease (CAD) is a multifactorial disease whose etiogenesis involves a number of environmental, genetic and lifestyle-related factors. Genetic polymorphisms are noteworthy among these factors because they alter gene expression and, thus, the functions of the respective products. Methods: A case-control study was conducted in the Cardiology Institute, with 79 subjects classified as cases with CAD, and 96 subjects as controls without CAD or other disease. In this study, we evaluated the association between the single-nucleotide polymorphisms (SNPs) of S447X and Leu7Pro of the lipoprotein lipase (LPL) and neuropeptide Y (NPY) genes, respectively. Results: No differences were found in the frequencies of LPL SNP between the cases and controls. However, the LPL 447X allele carriers exhibited a near-significant difference in the triglycerides (p=0.086) and higher mean in the HDL-c (p=0.018). NPY polymorphisms proved to be infrequent in this study population, and no significant difference was observed between the groups. Conclusions: Our findings provide further support of the genetic polymorphisms effect on the lipid metabolism control. So, further studies are needed to assess the functional effect of this and other polymorphisms, on LPL and in the NPY activity, and their impact on CAD risk.

Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

Comparative fine structure of the epididymal spermatozoa from three Korean shrews with considerations on their phylogenetic relationships

This study examined the fine structures of epididymal spermatozoa on the lesser white-toothed shrew (Crocidura suaveolens), the Japanese white-toothed shrew (C. dsinezumi) and the big white-toothed shrew (C. lasiura) belonging to the subfamily Crocidurinae living in Korea. In the spermatozoa of C. suaveolens, the head has a large acrosome, a smooth inner acrosomal membrane and a wavy, finger-like, electron-dense apical body. The neck has a solid proximal centriole that is filled with electron-dense material. These results showed the spermatozoa of C. suaveolens possess the characteristics of both Crocidurinae and Soricinae. In C. dsinezumi and C. lasiura, the head has a large acrosome, a serrated inner acrosomal membrane and a common apical body. The neck has a fistulous proximal centriole with slightly dense elec tron granules. These results showed the typical characteristics of Crocidurinae. Although C. suaveolens belongs to the subfamily Crocidurinae, the spermatozoan morphology is different from C. dsinezumi and C. lasiurai because it has conserved characteristicsof the subfamily Soricinae.

Immunizing children to protect against the increasing risk of hepatitis A in adolescents and young adults in South Korea.

We evaluated the CR326F strain (VAQTA) derived hepatitis A vaccine in Korean children and adolescents >2 years of age to consider a future immunization program. In our study, the pediatric two-dose regimen of VAQTA was found to be generally well tolerated and resulted in 100% (95% CI 94.8, 100.0) seroconversion after 2 doses. Immunizing children with the HAV vaccine routinely should be considered in South Korea, particularly in areas where recent outbreaks have occurred.

Pectus excavatum in children: diagnostic significance for mitral valve prolapse.

Eighty-seven patients with pectus excavatum underwent cardiac examination and echocardiography (M-mode) to determine the diagnostic significance of pectus in children for mitral valve prolapse (MVP). Patients' ages ranged from 1 month to 18 years with a mean age of 5.4 years. Sixty-seven were males. Twenty of the 87 pectus patients (23%) had echocardiographic evidence of MVP, whereas 11 of these had auscultatory findings of a non-ejection click or late systolic murmur, and 4 had significant mitral insufficiency. Fourteen of the 77 patients (18%) with mild pectus, and 6 of the 10 patients (60%) with severe pectus had MVP. Two of the patients (3.4%) with mild pectus and 2 of the patients (20%) with severe pectus also had significant mitral insufficiency. Ten of the 23 patients (44%) older than 8 years of age and 10 of the 64 younger patients (16%) had MVP. Although MVP was present more frequently in females (30%) than in males (21%), the difference was not statistically significant. This study indicates the high incidence of MVP in children of 8 years of age and older, especially when pectus deformity is severe. This information is particularly helpful to heighten suspicion of MVP in children with pectus excavatum.