Development of methods for the microbiome analysis of formalin fixed paraffin embedded tissue specimens

For Synthetic Biology to reach its potential, it necessitates foundational knowledge of the organisms that can be engineered. The remarkable influence our microbiome has on our health status has made it a focus of attention for engineering possibilities aiming at its modulation. As the field of the human microbiome expands, it necessitates access to high-quality nucleic acid samples which are truly representative of the community of bacteria under study. Formalin-fixed, paraffin-embedded (FFPE) samples represent the most comprehensive collections of patient materials in hospital pathology archives. However, for this sample to become reliably accessible for microbiome studies, the effects of FFPE processing on bacteria must be considered. Any sample processing method should be based upon specific study aims, target organisms and sample types. It is only through a holistic understanding of FFPE-induced changes to the bacterial cellular structure and its DNA content, that a reliable method can be developed It is hypothesised here that with a sample-prep workflow considering the effects of FFPE on bacterial cells, their DNA content and the overall contamination introduced, a reliable and reproducible analysis of the microbiome of FFPE samples could be achieved. As such, the overall aim of this thesis was to characterise FFPE induced changes to the bacterial cell walls/membranes and their DNA content, and with this information, to propose strategies for purifying and repairing DNA suitable for microbiome analysis, while also characterising the common contaminants found in samples processed in this manner. To achieve this, an appropriate FFPE bacterial study model was first developed. With this in place, a thorough characterisation of the state of bacterial FFPE DNA was performed and strategies to reduce this damage assessed. Finally, to develop an appropriate method for bacterial DNA extraction from FFPE samples (unavailable at the time of writing), the state of the bacterial cell wall/membrane was assessed and strategies for a uniform bacterial lysis and host depletion evaluated. Chapter 2 Describes methods for creating a mock bacterial FFPE block (Protoblock) that serves as a standard for FFPE samples. The Protoblock is a cell matrix which can be populated with cell types and numbers as desired, so as to resemble those of the FFPE tissue specimens. Its accuracy for representing bacterial load and cell architecture was validated by microscopy. With this model, the performance of the human gold-standard FFPE kit for microbiome analysis of FFPE samples was evaluated and found unsuitable for microbiome research. Additionally, the Protoblock permitted the characterisation of bacterial FFPE DNA, where it was found to be highly fragmented, a poor PCR template and featured significant sequence alterations. Finally, this model also permitted the characterisation of contaminants originating from the FFPE process, the most common being Xanthomonadaceae, Pseudomonadaceae and Clostridiaceae. Chapter 3 Makes a thorough investigation of the state of bacterial FFPE DNA in terms of PCR readability, formalin crosslinking, and the presence of sequence artefacts. Here, bacterial FFPE DNA was found to be highly fragmented, with a significant inverse correlation between fragment size and PCR recovery and a log-fold reduction between the recovery of 200 bp and 500 bp fragments. It was also evident that 95-97% of DNA present in these samples was crosslinked and that the most evident sequence artefacts were those derived from oxidative damage. Two strategies to reduce this damage were investigated. (1) An optimised decrosslinking procedure (10 oC lower than current methods) significantly reduced sequence artefacts. (2) The in vitro reconstitution of the Base Excision Repair pathway targeting oxidative DNA damage, using FPG and Endo VIII DNA glycosylases. Samples treated with both strategies showed a 3X increase in fragment length and a significant reduction in sequence chimeras and SNPs, leading to a significant improvement in sequencing readability. Chapter 4 Investigates the state of the bacterial cell wall/envelope and mammalian membrane to assess the state of their permeabilisation in FFPE samples. In this chapter, mammalian and Gram-negative bacterial cells were found to be impermeable to molecules with dimensions of 3-5 nm. A host depletion strategy was devised using a combination of Saponin and DNAse (Benzonase). It was also found that FFPE bacterial cells require a lysis strategy, and the use of a mix of bacterial-lytic enzymes was found to provide a uniform cross-taxa bacterial lysis. The collection of methods developed were tested by 16S rRNA gene sequence analysis of protoblocks, murine FFPE faeces and human breast tumour samples. The collection of methods provided an overall increase in recovery of 16S PCR amplicons, a higher uniformity in bacterial lysis, and a higher bacterial to host DNA ratio in high biomass models. However, these improvements were obscured for low biomass samples, where contaminants dominated the sequencing reads. It is concluded from this work that to unlock the potential of FFPE specimens for the microbiome field, a full dedicated workflow, comprising not only sample-prep, but also QC, 16S PCR and 16S sequencing, needs to be in place. This workflow should be directed by a robust QC system. In addition, a database for known FFPE derived common contaminants is essential to inform future strategies for the biological removal of contaminants from these samples

OPTIMIZACIÓN DE RUTA CORTA USANDO ALGORITMO GENÉTICO GENERACIONAL

El presente artículo de investigación tiene como objetivo utilizar algoritmo genético generacional, propio de la inteligencia artificial, donde se aprovecha el proceso evolutivo para optimizar el recorrido de los n puntos o nodos, a un coste de computador menor. Una de las ventajas de este método evolutivo es que no compiten todos contra todos, sinoque se crea y procesa una porción de la población total, con el objetivo de encontrar la posible mejor ruta, o también llamada búsqueda local. Se ha considerado para el método de la ruleta, cruce por intercambio de 2 puntos, mutación por intercambio y método de parada de acuerdo a la cantidad de generaciones. En un computador de regular característica selogró implementar el algoritmo genético generacional en lenguaje Matlab 8.3, tomando como ejemplo las distancias de cada ciudad. Se obtuvieron como resultados para n puntos o nodos, en cada evaluación, las posibles mejores rutas basándose en el modelo evolutivo. Las pruebas realizadas desde 100 hasta 1000 ciudades resultaron en tiempos de 3,2006 s y 27,924  s, respectivamente. De acuerdo al conjunto de pruebas, demuestra un incremento de manera polinómica de nivel 2; por lo que esta investigación se centra en el incremento de los dos métodos de parada a una secuencia lineal

Microbiome analysis as a platform R&D tool for parasitic nematode disease management

The relationship between bacterial communities and their host is being extensively investigated for the potential to improve the host’s health. Little is known about the interplay between the microbiota of parasites and the health of the infected host. Using nematode co-infection of lambs as a proof-of-concept model, the aim of this study was to characterise the microbiomes of nematodes and that of their host, enabling identification of candidate nematode-specific microbiota member(s) that could be exploited as drug development tools or for targeted therapy. Deep sequencing techniques were used to elucidate the microbiomes of different life stages of two parasitic nematodes of ruminants, Haemonchus contortus and Teladorsagia circumcincta, as well as that of the co-infected ovine hosts, pre- and post infection. Bioinformatic analyses demonstrated significant differences between the composition of the nematode and ovine microbiomes. The two nematode species also differed significantly. The data indicated a shift in the constitution of the larval nematode microbiome after exposure to the ovine microbiome, and in the ovine intestinal microbial community over time as a result of helminth co-infection. Several bacterial species were identified in nematodes that were absent from their surrounding abomasal environment, the most significant of which included Escherichia coli/Shigella. The ability to purposefully infect nematode species with engineered E. coli was demonstrated in vitro, validating the concept of using this bacterium as a nematode-specific drug development tool and/or drug delivery vehicle. To our knowledge, this is the first description of the concept of exploiting a parasite’s microbiome for drug development and treatment purposes

Estrategia de optimización con algoritmo genético para ruta corta sin corte en el espacio finito

Al contar con diferentes puntos en un espacio específico, surge la necesidad de recorrerlas tomando como referencia el problema del agente viajero, así también surge otro problema en el recorrido en el espacio, que es el riesgo en que los caminos se intersecten en el espacio, por tanto la presenta investigación para solucionarlo aplica una estrategia al algoritmo genético para evitar estos cortes, donde se aprovechó la ventaja de no competir todos contra todos, si no que a partir de una pequeña población se recorre el universo de casos, encontrando la posible mejor ruta en el espacio evitando estos cortes. Tomando en cuenta las funciones propias del algoritmo genético se resolvieron estos problemas usando la estrategia de traer de la generación anterior un par de mejores individuos a la generación actual. Considerando si se tiene 50 nodos en el espacio se logró resolver en 15.1 seg, generando una secuencia de duración y en función de los x nodos en la ecuación lineal de y = 0.3134x + 0.733, con R2 = 0.978, así también para el método de la varianza refleja la ecuación y = 0,0009x3 - 0,1256x2 + 6,1963x - 36,563, con R2 = 0,9349. Logrando encontrar la mejor ruta optima en el espacio finito solucionando los problemas encontrados

"Estrategia para la detección de tipos de enfermedades oculares usando red neuronal SOM"

"This research aims to cover a need to be able to classify according to the funds of eyes in diabetic retinopathy disease, how to convert to gray tone, perform an equalization, apply the canny edge highlighting algorithm and apply morphological operations so that a SOM (self-organization ma p ) neural network can be entered and classified. To achieve this, it is classified as 0 to diabetic retinopathy, 1 to glaucoma and 3 to healthy eyes. To corroborate this strategy, a public database of Fundus-images has been taken, being 45 images of eyes for training and for tests 15 images that were not part of the training were used and for the tests 3 images that were not part of the training were used and each grayscale image is scaled to a dimension of 256x256 pixels, managing to demonstrate with this strategy an affectivity of 93.7% certainty in the identification of class of eye disease

Casos de estudio en México y Latinoamérica

Ante la presencia de conflictos sociales y territoriales, las comunidades organizadas buscan estrategias de solución y confrontación. Es el estudio de dichos movimientos, lo que motiva a la publicación de éste libro: Respuestas comunitarias ante conflictos ambientales. Casos de estudio en México y Latinoamérica, reúne catorce textos que analizan las respuestas sociales y documentan la acción colectiva de comunidades que se han organizado para autogestionar soluciones ante conflictos territoriales, económicos y ambientales, en su propio entorno. Las aportaciones de investigadores y activistas, desde enfoques teóricos y metodológicos particulares, exponen casos de estudio sobre organizaciones formales e informales que se han conformado para afrontar los retos que representan proyectos productivos como fábricas cementeras, mineras, puertos, productores de energía, entre otros. Los enfoques analíticos tocan también temas nodales en el estudio de la acción colectiva como la ética, el despojo, los derechos humanos y las estrategias de comunicación y visibilización. Este material, que reúne estudios hechos en distintos lugares de México y Latinoamérica, es un compendio de métodos de investigación y un acercamiento al estudio de los movimientos sociales.UAEMEX, CONACyT, SE

CoARA y la reforma de la evaluación científica

[ES]Comunicación presentada a la «Jornada sobre Ciencia Abierta y nuevos procesos de evaluación de la investigación» celebrada en la Universidad de Salamanca el 24 de octubre de 2023 con motivo de la Semana Internacional del Acceso Abiert

Dinámica de conservación en el lugar de las variedades criollas de maíz en la aldea El Trapiche, Choluteca, Honduras

Diversity and conservation of traditional crops have been scarcely studied. The objective of this study is to describe the in situ conservation dynamics of corn crops in a rural village of Honduras, through the analysis of management practices and the morphological characterization of native corn varieties that are being used at this location, which permits the evaluation of key characteristics for its identification.This work was carried out in four communities in the village of El Trapiche, Choluteca in southern Honduras. Consultations were conducted through a survey of 26 corn growers in the area, about the characteristics that allow them to identify the cultivated varieties of corn, agricultural practices and planting material exchanges. We also carried out a preliminary assessment of the varieties they grow through the measurements in situ of morphological characteristics of the plant and corncob.The results demonstrate how traditional management creates an overtime dynamic process in situ for grown corn germplasm. Farmers reported seven identified varieties, five native and one foreign, and the commercial variety donated by the Honduran government. The identified varieties were: young corn, yellow, ground, cuarenteño, black and maizón (considered a foreign and improved variety).Most of the varieties are white grain, which is intended for human consumption, the yellow variety is used mainly for animal feed. The planting strategy reports an absence of animal labor, 50% of respondents report using Gramoxone for land clearing and 38.5 % of respondents indicate the use urea as soil fertilizer. Regarding the exchange of sowing seeds, 53% of respondents report sharing process material through purchase and gifts from family and friends. Clearly the persistence of a traditional corn can be observed in their practices and the use of local seeds that has been adapted to rocky soils which hinders agricultural modernization and the use of improved seeds in the area.Revista Ciencia y Tecnología No. 17, December 2015 pp.79-93La diversidad y la conservación de los cultivos tradicionales han sido poco estudiadas, por eso el objetivo de este estudio es describir la dinámica de conservación en el lugar del cultivo de maíz en una aldea rural de Honduras, a través del análisis de las prácticas de manejo y la caracterización morfológica de las variedades criollas de maíz que son utilizadas en esta localidad, lo cual permite evaluar algunas de las características claves en su identificación.Este trabajo se llevó a cabo en cuatro caseríos pertenecientes a la aldea El Trapiche, municipio de Choluteca, en la región sur de Honduras. Se consultó a través de una encuesta a 26 cultivadores de maíz de esta zona sobre las características que les permiten identificar las variedades criollas de maíz que cultivan, prácticas agrícolas y el intercambio de material de siembra. Además, se realizó una caracterización morfológica de las variedades criollas que cultivan mediante la medición de caracteres morfológicos de planta y mazorca.Los resultados demuestran cómo las actividades de manejo tradicional originan un proceso dinámico de conservación en el sitio a través del tiempo para el germoplasma de maíz cultivado. Los agricultores reportaron siete variedades: cinco criollas, una variedad foránea y la variedad comercial donada por el Gobierno de Honduras. Las variedades identificadas fueron: maicito, amarillo, planta baja, cuarenteño, negro y maizón; esta última considerada una variedad foránea y mejorada. La mayoría de ellas son de grano blanco y destinada para la alimentación humana, la variedad amarillo es utilizada principalmente para la alimentación animal. La estrategia de siembra es a bordón, no se reporta uso de trabajo animal, el 50 % de los encuestados manifiestan el uso de gramoxone para la limpieza del terreno; mientras que el 38.5 % indican que usan urea para fertilizar el suelo. Con respecto al intercambio de semilla para siembra, el 53 % de los encuestados notifica el intercambio de material a través de procesos de compra y regalo de familiares y amigos. Es evidente la persistencia de un sistema tradicional en el cultivo del maíz, lo que se observó en sus prácticas y el uso de semillas locales adaptadas a suelos pedregosos que dificulta la modernización agrícola y el uso de semillas mejoradas en esta zona.Revista Ciencia y Tecnología No. 17, diciembre 2015 pp.79-9

Function2Form Bridge - Towards synthetic protein holistic performance-prediction

Protein engineering and synthetic biology stand to benefit immensely from recent advances in in silico tools for structural and functional analyses of proteins. In the context of designing novel proteins, current in silico tools inform the user on individual parameters of a query protein, with output scores/metrics unique to each parameter. In reality, proteins feature multiple â partsâ /functions, and modification of a protein aimed at altering a given part, typically has collateral impact on other protein parts. A system for prediction of the combined effect of design parameters on the overall performance of the final protein does not exist. Function2Form Bridge (F2F-Bridge), attempts to address this by combining the scores of different design parameters pertaining to the protein being analysed into a single easily interpreted output describing overall performance. The strategy comprises 1. A mathematical strategy combining data from a myriad of in silico tools into an OP-score (a singular score informing on a user-defined overall performance); 2. The F2F-Plot, a graphical means of informing the wetlab biologist holistically on designed construct suitability in the context of multiple parameters, highlighting scope for improvement. F2F predictive output was compared with wetlab data from a range of synthetic proteins designed, built and tested for this study. Statistical/machine learning approaches for predicting overall performance, for use alongside the F2F plot, were also examined. Comparisons between wetlab performance and F2F predictions demonstrated close and reliable correlations. This user-friendly strategy represents a pivotal enabler in increasing accessibility of synthetic protein building and de novo protein design

Assessment of HPV infection among female university students in Honduras via Roche linear array

Contains fulltext : 96652.pdf (publisher's version ) (Closed access)OBJECTIVE: To determine the prevalence of specific types of human papillomavirus (HPV), and the association with possible risk factors, among female university students at university in Honduras. METHODS: In a cross-sectional study, cervical samples from 400 women aged 18-35years were tested using a Roche HPV linear array to differentiate 37 genotypes of HPV. Associations with risk factors were assessed. RESULTS: Of the 400 participants, 393 completed the study. HPV DNA was detected in 45% of these women, of whom 73% were infected with high-risk types of HPV and 46% had multiple infections. Overall, 36 HPV genotypes were identified, of which HPV types 16, 51, 84, 66, and 39 were the most common. There was a marked decrease in the prevalence of multiple and high-risk infections with age. The factors that were independently associated with risk of being infected were related to sexual behavior and smoking habits. CONCLUSION: The study showed that genital HPV infection is common among sexually active women at university in Honduras. In addition, the Roche linear array was shown to be a valuable tool for HPV genotyping, which will be useful for monitoring the future effectiveness of an HPV vaccine in the population