Agrobacterium-Mediated Co-transformation of Multiple Genes in Metarhizium robertsii

Fungi of the Metarhizium genus are a very versatile model for understanding pathogenicity in insects and their symbiotic relationship with plants. To establish a co-transformation system for the transformation of multiple M. robertsii genes using Agrobacterium tumefaciens, we evaluated whether the antibiotic nourseothricin has the same marker selection efficiency as phosphinothricin using separate vectors. Subsequently, in the two vectors containing the nourseothricin and phosphinothricin resistance cassettes were inserted eGFP and mCherry expression cassettes, respectively. These new vectors were then introduced independently into A. tumefaciens and used to transform M. robertsii either in independent events or in one single co-transformation event using an equimolar mixture of A. tumefaciens cultures. The number of transformants obtained by co-transformation was similar to that obtained by the individual transformation events. This method provides an additional strategy for the simultaneous insertion of multiple genes into M. robertsii.

Incidence and Risk Factors for Leptomeningeal Carcinomatosis in Breast Cancer Patients with Parenchymal Brain Metastases

OBJECTIVE: The objective of study is to evaluate the incidence of leptomeningeal carcinomatosis (LMC) in breast cancer patients with parenchymal brain metastases (PBM) and clinical risk factors for the development of LMC. METHODS: We retrospectively analyzed 27 patients who had undergone surgical resection (SR) and 156 patients with whole brain radiation therapy (WBRT) as an initial treatment for their PBM from breast cancer in our institution and compared the difference of incidence of LMC according to clinical factors. The diagnosis of LMC was made by cerebrospinal fluid cytology and/or magnetic resonance imaging. RESULTS: A total of 27 patients (14%) in the study population developed LMC at a median of 6.0 months (range, 1.0-50). Ten of 27 patients (37%) developed LMC after SR, whereas 17 of 156 (11%) patients who received WBRT were diagnosed with LMC after the index procedure. The incidence of LMC was significantly higher in the SR group compared with the WBRT group and the hazard ratio was 2.95 (95% confidence interval; 1.33-6.54, p<0.01). Three additional factors were identified in the multivariable analysis : the younger age group (<40 years old), the progressing systemic disease showed significantly increased incidence of LMC, whereas the adjuvant chemotherapy reduce the incidence. CONCLUSION: There is an increased risk of LMC after SR for PBM from breast cancer compared with WBRT. The young age (<40) and systemic burden of cancer in terms of progressing systemic disease without adjuvant chemotherapy could be additional risk factors for the development of LMC.

Comparison of Two Enzyme-Linked Immunosorbent Assays for Detecting Parasitic Diseases / 대한임상미생물학회지

BACKGROUND: Serologic tests for specific antibody nowadays are widely employed for the diagnosis of parasitic diseases. Recently, an increasing numbers of kits have adopted enzyme-linked immunosorbent assay (ELISA) for the detection of parasitic antibodies. In this study, we evaluated two ELISA reagents for the diagnosis of parasitic diseases. METHODS: A total of 553 serum and 156 CSF samples were assayed using an in-house micro-ELISA and Genedia(R) Ab ELISA (Green cross PBM, Korea) for Cysticercus, Paragonimus westermani, Clonorchis sinensis, and Sparganum. We reviewed the medical records of all patients. The results from Genedia(R) Ab ELISA kit were compared with those from the in- house micro-ELISA method. RESULTS: The overall concordance rate between the two ELISA tests was 95.5%. When compared with the clinical information, the sensitivity, specificity, positive predictive value, and negative predictive value of the in-house micro-ELISA were 100%, 99.0~99.6%, 82.4~96.4%, and 100%, and the respective figures for Genedia(R) Ab ELISA kit were 92.9~100%, 88.0~97.3%, 41.7~50%, and 99.9~100% with kappa agreement of 0.53-0.63. Comparison of two ELISA methods showed a significant difference (P<0.05). Retesting of 85 discordant samples showed that the concordance rate of the in-house ELISA was 97.7% and that of Genedia(R) Ab ELISA was 28.2%. CONCLUSION: Genedia(R) Ab ELISA kit showed an intermediate level of kappa agreement compared with the in-house ELISA. Further studies are necessary to improve the concordance rate of the two methods, and a careful interpretation of these results is required for a precise diagnosis.

Introduction of three independent selection markers in Leishmania

The pLE2SCX vector was developed for the stable expression of exogenous genes in the protozoan parasite Leishmania. The pLE2SCX construct contains three independent selection markers: herpes simplex virus thymidine kinase [HSV-TK], cytosine deaminase [CD] and streptothericin acetyltransferase gene [sat] in multiple cloning site, flanking by 5' and 3' untranslated regions of the previously cloned Leishmania major hexa-binding protein gene. Selection was based on resistance to the nourseothericin [Ns] which corresponds to sat gene. The two negative selection; HSV-TK and CD genes, make the transformed cell sensitive to ganciclovir [GCV] and 5-fluorocytosine [5-FC]. The vector was introduced into Leishmania promastigotes by electroporation and maintained as circular form. The selected transfectants were not grown on media with GCV or 5-FC. Using two drug sensitive selectable markers together on a vector is a novel strategy in gene cloning in Leishmania. This stable transfection vector has allowed the permanently expression of several different exogenous genes at the same time in Leishmania