Assessment of genotyping markers in the molecular characterization of a population of clinical isolates of Fusarium in Colombia / Evaluación de marcadores de genotipificación en la caracterización molecular de una población de aislamientos clínicos de Fusarium en Colombia

Introduction: Fusarium is a very heterogeneous group of fungi, difficult to classify, with a wide range of living styles, acting as saprophytes, parasites of plants, or pathogens for humans and animals. Prevalence of clinical fusariosis and lack of effective treatments have increased the interest in the precise diagnosis, which implies a molecular characterization of Fusarium populations. Objective: We compared different genotyping markers in their assessment of the genetic variability and molecular identification of clinical isolates of Fusarium. Materials and methods: We evaluated the performance of the fingerprinting produced by two random primers: M13, which amplifies a minisatellite sequence, and (GACA)4, which corresponds to a simple repetitive DNA sequence. Using the Hunter Gaston Discriminatory Index (HGDI), an analysis of molecular variance (AMOVA), and a Mantel test, the resolution of these markers was compared to the reference sequencing-based and PCR genotyping methods. Results: The highest HGDI value was associated with the M13 marker followed by (GACA)4. AMOVA and the Mantel tests supported a strong correlation between the M13 classification and the reference method given by the partial sequencing of the transcription elongation factor 1-alpha (TEF1-α) and rDNA 28S. Conclusion: The strong correlation between the M13 classification and the sequencing-based reference together with its higher resolution demonstrates its adequacy for the characterization of Fusarium populations.

Introducción. Fusarium es un grupo heterogéneo de hongos, difícil de clasificar y con una amplia gama de estilos de vida, que actúa como saprófito, parásito de plantas o patógeno de humanos y animales. La prevalencia de la fusariosis clínica y la falta de tratamientos han incrementado el interés en su diagnóstico preciso, lo que conlleva la caracterización molecular de las poblaciones. Objetivo. Comparar marcadores de genotipificación en la evaluación de la variabilidad genética e identificación de aislamientos clínicos de Fusarium. Materiales y métodos. Se evaluó la huella genética producida por dos cebadores aleatorios: M13, que amplifica una secuencia minisatélite, y (GACA)4, que corresponde a una secuencia repetitiva de ADN. Utilizando el índice discriminatorio de Hunter Gaston (HGDI), el análisis de varianza molecular (AMOVA) y una prueba de Mantel, se comparó la resolución de estos marcadores con métodos de genotipificación basados en secuenciación y PCR. Resultados. El mayor HGDI se asoció con el marcador M13, seguido de (GACA)4. Las pruebas AMOVA y Mantel mostraron correlación entre las clasificaciones obtenidas con M13 y la referencia basada en la secuenciación parcial del factor de elongación de transcripción 1-alfa (TEF1-α) y el ADNr 28S. Conclusión. La fuerte correlación entre la clasificación obtenida con M13 y el método de referencia, así como su alta resolución, demuestran su idoneidad para la caracterización de poblaciones de Fusarium.

Triple tandem mimotope peptide of epidermal growth factor receptor displaying on the surface of M13 phage induces anti-tumor response in mice tumor model

Epidermal growth factor receptor [EGFR] has been shown to play a critical role in tumor cell growth and its over expression has been observed in many epithelial tumors. In the field of cancer vaccine research, displaying the peptide mimotope on the surface of phage particles has shown promising results. In this study using m13-PVIII phage display system, two constructions were prepared: triple tandem repeat of EGFR mimotpe displaying particles [3M] and single EGFR mimotope displaying phage particle [1M]. To investigate the anti-tumor properties of phage vaccine, C57BL/6 mice Lewis lung carcinoma xenograft model was established and treated with 3M phage vaccine, 1M phage vaccine and control agents. Immunization of mice with these phage-based vaccines showed strong immune response against phage-mimotope. 3M phage vaccine showed more potency against tumor in comparison with control groups. Also the survival time was extended in phage vaccine treated tumor-bearing mice compared with untreated mice. Our findings suggest that mimotope-displaying phage vaccine can induce specific antibodies with antitumoral activity, which its potential as a candidate vaccine for EGFR-specific cancer immunotherapy needs to be more investigated in future studies

Molecular Typing of Cryptococcus neoformans Isolated from Korean Patients

Cyptococcosis is generally caused by Cryptococcus neoformans, the opportunistic agent which has two species such as C. neoformans and C. gattii. Both C. neoformans and C. gattii species contain a number of genetically diverse subgroups that can be differentiated by various molecular typing methods. We conducted a molecular epidemiological analysis of 30 clinical isolates of the C. neoformans from cryptococcosis patients who had been hospitalized between 2008 and 2010 in medical centers located in Seoul and Busan in Korea. To determine the genetic diversity, 30 strains of C. neoformans were typed using PCR fingerprinting with the microsatellite specific primer of the phage M13 and the restriction fragment length polymorphism (RFLP) of orotidine monophosphosphate pyrophosphorylase (URA5) gene. All isolates were identified as serotype A, mating type MATa and molecular type VNI. The random amplified polymorphic DNA (RAPD) profiles obtained by using two primers revealed a single pattern. Our study shows that 30 strains of clinical C. neoformans are genetically homogeneous, with all of the isolates were molecular type VN1, serotype A, mating type MATa.

Molecular Characterization of Clinical and Environmental Strains of Cryptococcus neoformans Isolated from Busan, Korea

Cryptocococcus neoformans is an encapsulated yeast that can cause life-threatening infections in immunocompromised patients. In this study, the genetic variability and epidemiological relationships of clinical and environmental isolates of C. neoformans from Busan, Korea, 2000~2005 were investigated. A total of 12 strains of C. neoformans, 7 clinical and 5 environmental isolates were analyzed by random amplified polymorphic DNA (RAPD) using three different primers and PCR-fingerprinting with a minisatellite-specific core sequence of phage M13. All strains belonged to C. neoformans serotype A and mating type MATa. Two different RAPD profiles (I and II) and a single pattern by M13 PCR-fingerprinting were identified. The major RAPD profile was pattern I (8 of 12 strains) and pattern II was identified from 2 clinical and 2 environmental strains, which clearly distinguished among isolates. Clinical strains with pattern II were isolated from the patients with HIV positive. Taken together, molecular patterns provide a good characterization of strains of C. neoformans as a heterogeneous group and epidemiological relationships in clinical and environmental strains.

Immune responses of selected phagotopes from monoclonal antibodies of Burkholderia pseudomallei.

Random peptide libraries displayed by bacteriophage T7 and M13 were employed to identify mimotopes from 4 monoclonal antibodies (MAbs) specific to Burkholderia pseudomallei. Insert DNA sequences of bound phages selected from four rounds of panning with each MAb revealed peptide sequences corresponding to B. pseudomallei K96243 hypothetical protein BPSL2046, hypothetical protein BpseP_02000035, B. pseudomallei K96243 hypothetical protein BPSS0784, B. pseudomallei 1710b hypothetical protein BURPS1710b_1104, and B. cenocepacia H12424 TonB-dependent siderophore receptor, all located at the outer membrane. The immune responses from all selected phagotopes were significantly higher than that of lipopolysaccharide. The study demonstrates the feasibility of identifying mimotopes through screening of phage-displayed random peptide libraries with B. pseudomallei MAbs.

Application of optical proteinchip in detecting phage M13KO7 / 生物工程学报

Avidin layer was bound on the substrate surface of Silicon wafer modified with aldehyde. The interaction between avidin and biotin was adopted for the immobilization of mouse monoclonal biotin-anti-M13 (antibody GP3)-labeled biotin. The surface was incubated in a solution containing phage M13KO7, which was trapped by the antibody GP3 with the interaction between phage M13KO7 and antibody GP3, resulting in a variation of layer thickness that was detected by imaging ellipsometry. The results showed a saturated layer of antibody GP3 with a thickness about 6.9 nm on the surface of the silicon wafer. The specific interaction between phage M13KO7 and antibody GP3 resulted in a variation of the layer thickness. The layer of phage M13KO7 bound with antibody GP3 was 17.5 nm in the concentration of 1.1 x 10(10) pfu/mL. Each variation of layer thickness corresponded to a concentration of phage M13KO7 in the range of 0.1 x 10(10) approximately 2.5 x 10(10) pfu/mL, with the sensitivity of 10(9) pfu/mL. Compared with other methods, the optical protein-chip requires only short measurement time, is label free, is a quantitative test, and can be visualized. This study could be significant on the investigation of interactions between the antibody and virus, and shows potential in the early diagnosis of virosis.

Puzzling peptides from a phage display library

The commercial availability of random peptide libraries displayed on the M 13 phage increased their use for studies on epitope identification, enzyme inhibitors and receptor ligands. We planed two experiments for selection of peptides. First, with sheep antibodies, the positive selector was IgG, prepared on Protein G columns from a pool of 11 sheep protected with a vaccine prepared from larvae of Lucilia cuprina, against blowfly strike and the negative selectors were the IgG from the same sheep before vaccination and IgG from vaccinated sheep which were not protected. Four rounds of positive and negative selection were done respectively. In the second experiment, human IgG again prepared on new Protein G columns. Four rounds of positive selection with either IgG from MS or schizophrenia patients were done. They were alternated with two negative selections of schizophrenia IgG on the MS associated peptides and vice versa and two negative selections on each with the IgG from people who had died from non-nervous system diseases as control. After the fourth negative selection of each experiment, the phage were amplified and random closes were picked for nucleotide sequencing. A total of 44 peptides were sorted with the PILEUP program. From those, 15 peptides had a large number with either 4 or 5 methionines

Identification of Genes Involved in Liver Cancer Cell Growth Using an Antisense Library of Phage Genomic DNA / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Genes involved in liver cancer cell growth have been identified using an antisense library of large circular (LC-) genomic DNA of a recombinant M13 phage. MATERIALS AND METHODS: A subtracted cDNA library was constructed by combining procedures of suppression subtractive hybridization (SSH) and unidirectional cloning of the subtracted cDNA into an M13 phagemid vector. Utilizing the life cycle of M13 bacteriophages, LC-antisense molecules derived from 1, 200 random cDNA clones selected by size were prepared from the culture supernatant of bacterial transformants. The antisense molecules were arrayed for transfection on 96-well plates preseeded with HepG2. RESULTS: When examined for growth inhibition after antisense transfection, 153 out of 1, 200 LC-antisense molecules showed varying degrees of growth inhibitory effect to HepG2 cells. Sequence comparison of the 153 clones identified 58 unique genes. The observations were further extended by other cell-based assays. CONCLUSION: These results suggest that the LC-antisense library offers potential for unique high-throughput screening to find genes involved in a specific biological function, and may prove to be an effective target validation system for gene-based drug discovery.

Construction and characterization of M13 bacteriophages displaying gp120 binding domains of human CD4.

The envelope glycoprotein, gp120, on the surface of HIV interacts with the human CD4 molecule and thus helps the virus in gaining entry into the T-helper cells. To display the gp120 binding domains of human CD4 on the surface of the bacteriophage M13, two types of vectors have been constructed. In these, the first 176 amino acids of the human CD4 have been fused with the minor coat protein, gIIIp, of M13 bacteriophage for surface display. The Western blot analysis revealed that using the phage based vector, M13CD41923, all the copies of gIIIP (3-5 per virion) were present as fusion protein indicating multivalent display. In the phagemid based vector, phage particles were produced only upon infection of the cells carrying pVCCD43426, with the helper phage, M13KO7. Thus these phage particles carried both, the fusion protein as well as the unfused gIIIp, as shown by Western blot analysis. The presence of large amount of unfused gIIIp ensured that the phage particles did not display more than one fusion protein per phage particle, thus leading to monovalent display. Phage particles produced by both vectors could be captured on immobilized gp120, thereby showing that the displayed CD4 domains were functional.