Some Southern Waters

Some Southern Waters is a microbudget feature-length film written, directed, and produced by Julian Baner as part of the requirements for earning a Master of Fine Arts in Entrepreneurial Digital Cinema from the University of Central Florida. The film is a black and white, semi-abstract narrative mainly in the mystery genre, with elements of thriller, horror, and black comedy throughout. This thesis is a description of the creative impetus behind the project, as well as the technical process from pre-production through production, and post-production

A dual-tag microarray platform for high-performance nucleic acid and protein analyses

DNA microarrays serve to monitor a wide range of molecular events, but emerging applications like measurements of weakly expressed genes or of proteins and their interaction patterns will require enhanced performance to improve specificity of detection and dynamic range. To further extend the utility of DNA microarray-based approaches we present a high-performance tag microarray procedure that enables probe-based analysis of as little as 100 target cDNA molecules, and with a linear dynamic range close to 105. Furthermore, the protocol radically decreases the risk of cross-hybridization on microarrays compared to current approaches, and it also allows for quantification by single-molecule analysis and real-time on-chip monitoring of rolling-circle amplification. We provide proof of concept for microarray-based measurement of both mRNA molecules and of proteins, converted to tag DNA sequences by padlock and proximity probe ligation, respectively

"Mi vereda en plástico" un estudio desde la ecología política de la transformación de los usos del suelo rural y resistencia del campesinado frente a estos procesos en la vereda Guapante de Guarne Antioquia

RESUMEN: En este escrito se mostrarán algunos de los principales usos del suelo tradicional y actual en la vereda Guapante, perteneciente al municipio de Guarne, Antioquia, Colombia, haciendo referencia principalmente al monocultivo como un uso del suelo moderno que ha implicado importantes cambios en aspectos políticos, económicos, sociales, culturales y ambientales en dicho territorio. También se establecerá un paralelo entre la agricultura tradicional de la década de los 50 y la agricultura moderna, haciendo énfasis en el impacto que tuvo la revolución verde en la transición, que se traduce en pérdida de la diversidad biológica agrícola y la autonomía del campesinado. Adicionalmente se evidencia la incompatibilidad de los modelos tecnificados y la sostenibilidad ambiental, los cuales han ocasionado formas visibles y cotidianas de resistencia campesina en el territorio.ABSTRACT: This writing shows some of the main uses of the traditional and current land in the countryside of Guapante which belongs to Guarne, Antioquia, Colombia. This is done by making reference principally to monocultures as a use of the modern land that has produced important changes in political, economic, social, cultural and environmental aspects in this territory. A parallel between the traditional agriculture in the 50s and the modern agriculture will be drawn, by emphasizing the impact of the green revolution in the transition which refers to the loss of biological and agricultural diversity as well as the autonomy of the peasantry. Additionally, the incompatibility of the technical models and the environmental sustainability which have caused visible and everyday ways of peasant resistance in the territory are evidenced

Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations – particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.25% of total population) and which takes a single working day from purified plasmid to library transformation. We demonstrate its efficacy with whole gene codon reassignment of chloramphenicol acetyl transferase, mutating 19 codons in a single reaction in KOD DNA polymerase and generating high quality, multiple-site libraries in T7 RNA polymerase and Tgo DNA polymerase. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation

Proyecto de análisis de datos de servicios de telecomunicaciones evaluado con rúbricas analíticas

La vinculación profesional entre la universidad y la industria es objetivo de nuestra labor académica. Es interés del trabajo registrar un proyecto de articulación basado en el análisis de datos de servicios de telecomunicaciones. El desempeño de los estudiantes de ingeniería que lo implementan se evalúa a partir de rúbricas analíticas elaboradas para este caso. Los procesos de transformación digital originan profundos cambios, entre otros en la generación de bienes y servicios. En este contexto la obtención de información relevante a partir de datos disponibles resulta un aporte de valor para las organizaciones. El proyecto se basa en un estudio exploratorio de datos reales, que utiliza como herramienta la plataforma Python y sus diferentes librerías, para construir un perfil de cliente con alto potencial de abandono en los servicios de telecomunicaciones. Se pretende contribuir al fomento de actividades que integren la academia con la industria reflejando el valor que la aplicación de nuevas herramientas de evaluación pueden aportar a estudiantes y docentes.Red de Universidades con Carreras en Informátic

High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

BACKGROUND: Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods. RESULTS: A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point. CONCLUSIONS: Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample

Multiplex cDNA quantification method that facilitates the standardization of gene expression data

Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h

High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. RESULTS: SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. CONCLUSIONS: Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring

Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets

The current arsenal of molecular tools for site-directed cleavage of single-stranded DNA (ssDNA) is limited. Here, we describe a method for targeted DNA cleavage that requires only the presence of an A nucleotide at the target position. The procedure involves hybridization of a complementary oligonucleotide probe to the target sequence. The probe is designed to create a deliberate G:A mismatch at the desired position of cleavage. The DNA repair enzyme MutY glycosylase recognizes the mismatch structure and selectively removes the mispaired A from the duplex to create an abasic site in the target strand. Addition of an AP-endonuclease, such as Endonuclease IV, subsequently cleaves the backbone dividing the DNA strand into two fragments. With an appropriate choice of an AP-cleaving enzyme, the 3′- and 5′-ends of the cleaved DNA are suitable to take part in subsequent enzymatic reactions such as priming for polymerization or joining by DNA ligation. We define suitable standard reaction conditions for glycosylase/AP-cleaving enzyme (G/AP) cleavage, and demonstrate the use of the method in an improved scheme for in situ detection using target-primed rolling-circle amplification of padlock probes