Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

<p>Abstract</p> <p>Background</p> <p>Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies.</p> <p>Results</p> <p>We developed a single-step cloning method, target-selective homologous recombination (TS-HR), in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector) with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into <it>E. coli</it>. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells.</p> <p>Conclusion</p> <p>The time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.</p

Suspension of proactive recommendations for HPV vaccination has led to a significant increase in HPV infection rates in young Japanese women : real-world data

Funding Information: We would like to thank Ms. Yuka Watanabe, Ms. Sachiko Ono, Ms. Anna Ishida, and administrator of Niigata city for their support in conducting the survey. This work was supported by the Japanese Agency for Medical Research and Development (JP15ck0106103).Peer reviewedPublisher PD

Procyanidins in Theobroma cacao Reduce Plasma Cholesterol Levels in High Cholesterol-Fed Rats

We evaluated the effect of cacao procyanidins (CP) on plasma lipid levels in high cholesterol-fed rats. Animals were divided into 4 groups, and each group was fed on either a normal diet, high cholesterol diet (HCD) containing 1% cholesterol (HCD without CP), HCD with 0.5% (HCD with 0.5% CP) or 1.0% CP (HCD with 1.0% CP) for 4 weeks. Plasma cholesterol level was significantly higher in the HCD without CP group than the normal diet group (p<0.01). Supplementation of CP significantly decreased plasma cholesterol (p<0.01) to levels similar to those of the normal diet group. The liver cholesterol and triglyceride levels in all HCD groups were significantly higher (p<0.01), but 1.0% CP feeding significantly reduced this increase. Fecal excretion of neutral sterol and triglyceride was significantly increased in all HCD groups (p<0.01), and the excreted amounts tended to be higher in the HCD with CP groups. The procyanidins dose-dependently reduced micellar solubility of cholesterol and this activity increased with increasing molecular weight. These results suggest that one of the mechanisms of CP to lower plasma cholesterol is inhibition of intestinal absorption of cholesterol

Internet Survey of Awareness and Behavior Related to HPV Vaccination in Japan

Funding Information: Funding: This work was supported by the Health and Labor Sciences Research Grant No. 26272001 and the Japanese Agency for Medical Research and Development (JP15ck0106103).Peer reviewedPublisher PD

Long-term effectiveness of HPV vaccination against HPV infection in young Japanese women : Real-world data.

ACKNOWLEDGEMENTS We would like to thank Ms. Yuka Watanabe, Ms. Sachiko Ono, Ms. Anna Ishida, and the administrator of Niigata city for their support in conducting the surveyPeer reviewedPublisher PD

Effectiveness of human papillomavirus vaccine against cervical precancer in Japan : Multivariate analyses adjusted for sexual activity.

ACKNOWLEDGMENTS We would like to thank Mr Kenshin Sekine and Mr Taishin Sekine for English editing, and Ms Yuka Watanabe, Ms Sachiko Ono, Ms Anna Ishida, and administrators of Niigata, Nagaoka, Joetsu, Shibata, Sanjo, Mitsuke city for their support in conducting the survey.Peer reviewedPublisher PD

Long-Term Effects of Human Papillomavirus Vaccination in Clinical Trials and Real-World Data : A Systematic Review

Peer reviewedPublisher PD

A new case of GABA transaminase deficiency facilitated by proton MR spectroscopy

BACKGROUND: Deficiency of 4-aminobutyrate aminotransferase (GABA-T) is a rare disorder of GABA catabolism, with only a single sibship reported. We report on a third case, a Japanese female infant with severe psychomotor retardation and recurrent episodic lethargy with intractable seizures, with the diagnosis facilitated by proton magnetic resonance (MR) spectroscopy ((1)H-MRS). METHODS: Neuroimaging was performed at the first episode of lethargy. For (1)H-MRS, locations were placed in the semioval center and the basal ganglia. Quantification of metabolite concentrations were derived using the LCModel. We confirmed the diagnosis subsequently by enzyme and molecular studies, which involved direct DNA sequence analysis and the development of a novel multiplex ligation-dependent probe amplification test. RESULTS: (1)H-MRS analysis revealed an elevated GABA concentration in the basal ganglia (2.9 mmol/l). Based on the results of quantitative (1)H-MRS and clinical findings, GABA-T deficiency was suspected and confirmed in cultured lymphoblasts. Molecular studies of the GABA-T gene revealed compound heterozygosity for a deletion of one exon and a missense mutation, 275G>A, which was not detected in 210 control chromosomes. CONCLUSIONS: Our results suggest that excessive prenatal GABA exposure in the central nervous system (CNS) was responsible for the clinical manifestations of GABA transaminase deficiency. Our findings suggest the dual nature of GABA as an excitatory molecule early in life, followed by a functional switch to an inhibitory species later in development. Furthermore, quantitative (1)H-MRS appears to be a useful, noninvasive tool for detecting inborn errors of GABA metabolism in the CNS