Transcriptome Analysis of Entamoeba histolytica Trophozoites during in vivo Contact-independent Mediated Host-parasite Interaction: Putative Pathways Related to PCD

Aim: In the present study, we exploited DNA microarray-based transcriptome analysis and showed overall changes in gene expression in vivo of amoebic trophozoites that interact with animal soluble factors using an intraperitoneal dialysis bag model to elucidate putative molecular pathways and genes involved in this interaction. Study Design: We exploited DNA microarray-based transcriptome analysis. Results: An analysis from a network including the interactions of up-regulated genes and their neighbors revealed the presence of 11 functionally related modules. Six of the modules obtained were related to endoplasmic reticulum (ER) functions, such as degradation, stress, proteasome-ubiquitination, phosphorylation, lipid metabolism, and protein sorting. Furthermore, major transcriptional changes displayed by the parasite at the beginning of interaction were attributed to the response to the host defense. These data are consistent with the notion that the concerted expression of genes necessary for survival such as increment in protein synthesis, cytoskeleton rearrangement, vesicular traffic and genes involved in cell death including calcium imbalance and the ER signals associated with protein degradation (ERAD) is an overall landscape during the in vivo interaction between the amoebic trophozoites and animal soluble factors, and suggest that the ER stress is one of the main pathways leading to programmed cell death in E. histolytica. Conclusion: The present findings on the global transcriptional changes displayed by the parasite at the early stages of interaction with host environments in peritoneal implantation indicate that a substantial proportion of concerted changes in gene expression in amoebic trophozoites are attributable to the parasite’s response for cell death signals due to ER stress. A detailed knowledge of the underlying molecular mechanism might suggest the efficient elimination of this parasite by promoting their death pathways.

Association of FAT atypical cadherin 1 with clinicopathological parameters and prognosis in esophageal squamous cell carcinoma / 中华消化外科杂志

Objective:To investigate the association of FAT atypical cadherin 1 (FAT1) with clinicopathological parameters and prognosis in esophageal squamous cell carcinoma (ESCC).Methods:The retrospective cohort study was conducted. The clinicopathological data of 124 patients with ESCC who were admitted to Shanxi Cancer Hospital from January 2011 to December 2015 were collected. There were 85 males and 39 females, aged from 40 to 72 years, with a median age of 60 years. The ESCC tissues surgically removed and adjacent tissues specimens were collected to prepare tissue microarray for immunohistochemical staining. The 5 cases of ESCC tissues and adjacent tissues were analyzed by real-time quantitative polymerase chain reaction (qRT-PCR). Observation indicators: (1) the expression of FAT1 protein in ESCC and adjacent tissues; (2) the expression of FAT1 RNA in ESCC and adjacent tissues; (3) the expression of FAT1 protein in ESCC tissues and its association with clinicopathological parameters; (4) follow-up and survival. Follow-up using outpatient examination and telephone interview was conducted to detect survival of patients up to February 13, 2019. The survival time was from surgical date to tumor-related death or endpoint of follow-up. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was analyzed using the t test. Measurement data with skewed distribution were represented as M (range). Count data were described as absolute numbers or percentages, and comparison between groups was analyzed using the chi-square test. Comparison of ordinal data was analyzed using the non parameter rank sum test. The Kaplan-Meier method was used to calculate survival time, and Log-rank test was used for survival analysis. Results:(1) The expression of FAT1 protein in ESCC and adjacent tissues: of 124 specimens, the 107 cases of ESCC tissues and 93 cases of adjacent tissues were finally obtained because of exfoliative tissues. There were 76 cases of ESCC tissues and corresponding adjacent tissues matched. Results of immuno-histochemical staining showed that FAT1 protein was expressed in both ESCC and adjacent tissues and was brown after staining. FAT1 was located in cytomembrane, with high expression of FAT1 as ≥75 and low expression as <75. The relative expression levels of FAT1 protein in ESCC and adjacent tissues were 68±42 and 77±37, showing a significant difference between ESCC and adjacent tissues ( t=2.380, P<0.05). (2) The expression of FAT1 RNA in ESCC and adjacent tissues: results of qRT-PCR showed that the relative expression levels of FAT1 RNA in 5 cases of ESCC and adjacent tissues were 1.6±0.4 and 2.5±0.3, with a significant difference between them ( t=3.560, P<0.05). (3) The expression of FAT1 protein in ESCC tissues and its association with clinicopathological parameters: of the 107 ESCC patients, 58 cases had high expression of FAT1. There were 42 and 16 cases with high expression of FAT1 in 65 non-drinking patients and 42 drinking patients, respectively, showing a significant difference between them ( χ2=7.229, P<0.05). (4) Follow-up and survival: 96 of 107 ESCC patients were followed up for 38.0?94.9 months, with a median follow-up time of 45.9 months. Survival analysis showed that the survival time of patients with high FAT1 expression was 24 months, versus 22 months of patients with low FAT1 expression, indicating no significant difference between them ( χ2=1.773, P>0.05). Results of subgroup analysis showed that the survival time was 24 months and 21 months of female patients with high and low FAT1 expression, 23 months and 22 months of non-smoking patients with high FAT1 expression and low FAT1 expression, 23 months and 21 months of non-drinking patients with high FAT1 expression and low FAT1 expression, respectively, showing significant differences between them ( χ2=8.769, 12.827, 10.724, P<0.05). Conclusions:The expression of FAT1 in ESCC tissues is low. Female, non-smoking and non-drinking ESCC patients with high FAT1 expression have good survival.

Brain Gene Expression-DNA Methylation Correlation in Suicide Completers: Preliminary Results

Abstract Background: Gene expression alterations have been implicated in suicide pathology. However, the study of the regulatory effect of DNA methylation on gene expression in the suicidal brain has been restricted to candidate genes. Objective: The objective of the study was to identify genes whose expression levels are correlated with DNA methylation in the prefrontal cortex of suicides. Methods: Postmortem prefrontal cortex samples from 21 suicides and six non-suicides were collected. Transcriptomic and DNA methylation profiles were evaluated with microarrays; cis correlations between gene expression and CpG methylation were screened. We then analyzed the presence of transcription factor (TF) binding sites (TFBS) at CpG sites correlated with gene expression. Gene expression of TFs involved in neurodevelopmental binding to predicted TFBS was determined in the BrainSpan database. Results: We identified 22 CpG sites whose methylation levels correlated with gene expression in the prefrontal cortex of suicides. Genes annotated to identified CpG sites were involved in neurodevelopment (BBS4, NKX6-2, AXL, CTNND1, and MBP) and polyamine metabolism (polyamine oxidase [PAOX]). Such correlations were not detected in the non-suicide group. Nine TFs (USF1, TBP, SF1, NRF1, RFX1, SP3, PKNOX1, MAZ, and POU3F2) showed differential expression in pre- and post-natal developmental periods, according to BrainSpan database. Conclusions: The integration of different omic technologies provided novel candidates for the investigation of genes whose expression is altered in the suicidal brain and their potential regulatory mechanisms. (REV INVEST CLIN. 2020;72(5):283-92)

Gene expression analysis identifies hypothetical genes that may be critical during the infection process of Xanthomonas citri subsp. citri

Background: Gene expression analysis via microarray is widely used in phytobacteria to validate differential gene expression associated with virulence or to compare biological profiles of wild type and mutant strains. Here, we employed DNA microarrays to study the early stages of the infection process (24, 72 and 120 h post-inoculation) of Xanthomonas citri subsp. citri (Xac) infecting Citrus sinensis to interrogate the expression profiles of hypothetical genes. Results: Under infective conditions, 446 genes were up- and 306 downregulated. Outstanding among genes upregulated during infection were those involved in synthesizing the Type 3 Secretion System and effectors, xanthan gum and quorum-sensing induction, and flagellum synthesis and regulation. Additionally, 161 hypothetical genes were up- and 100 were downregulated, 49 of which are known to have a significant biological role. To understand hypothetical gene co-regulation or -expression, nine expression profiles including 158 genes were identified during the three infection phases. Of these, 47 hypothetical genes were identified as having expression profiles associated with at least one connected to a gene associated with adaptation and virulence. Conclusions: Expression patterns of six differentially expressed genes were validated by quantitative reverse transcription polymerase chain reaction, thus demonstrating the effectiveness of this tool in global gene expression analysis in Xac.

Insights into the signal transduction pathways of mouse lung type II cells revealed by transcription factor profiling in the transcriptome

Alveolar type II cells constitute a small fraction of the total lung cell mass. However, they play an important role in many cellular processes including trans-differentiation into type I cells as well as repair of lung injury in response to toxic chemicals and respiratory pathogens. Transcription factors are the regulatory proteins dynamically modulating DNA structure and gene expression. Transcription factor profiling in microarray datasets revealed that several members of AP1, ATF, NF-kB, and C/EBP families involved in diverse responses were expressed in mouse lung type II cells. A transcriptional factor signature consisting of Cebpa, Srebf1, Stat3, Klf5, and Elf3 was identified in lung type II cells, Sox9+ pluripotent lung stem cells as well as in mouse lung development. Identification of the transcription factor profile in mouse lung type II cells will serve as a useful resource and facilitate the integrated analysis of signal transduction pathways and specific gene targets in a variety of physiological conditions.

Complejidad de la expresión de genes asociados a obesidad en el tejido adiposo humano / Complexity of the expression of genes associated with obesity in human adipose tissue / Complexidade da expressão de genes associados à obesidade no tecido adiposo humano

Resumen Objetivo: Analizar la complejidad de la expresión génica en tejido adiposo de genes asociados con obesidad, mediante simulación computacional con diferentes herramientas bioinformáticas. Métodos: Después de una búsqueda bibliográfica en PubMed, se seleccionaron 37 genes asociados con obesidad con fold change mayor a 1,5. A partir del cálculo de valores de los z-score obtenidos de experimentos de micromatrices de ADN de muestras de tejido adiposo de personas obesas y de control, se construyó una red de interacción con el programa Cytoscape 3.2. La información detallada sobre las características genómicas de estos genes se extrajo de las bases de datos Genome Browser de la UCSC y del NCBI. Utilizando herramientas de análisis de multivariado, se hizo un análisis de componentes principales y uno de agrupación. Resultados: La red construida mostró que los genes con mayor número de interacciones fueron: 1) el factor nuclear respiratorio (NRF1), 2) el canal activado de potasio activado por calcio alfa 1 (KCNMA1) y 3) la sintasa de ácidos grasos (FASN). Los que tuvieron mayores valores de expresión fueron: 1) el factor de crecimiento endotelial vascular A (VEGFA), 2) la dioxigenasa dependiente de alfa-cetoglutarato (FTO) y 3) el regulador de crecimiento neuronal 1 (NEGR1). Las proteínas IL6, BDNF y HLC tuvieron los mayores valores de interacción con IL6R, NRF1 y ACACB, respetivamente. Las categorías ontológicas más importantes se relacionaron con procesos metabólicos de lipoproteínas, el ciclo de los ácidos tricarboxílicos, la activación de las MAP-quinasas y la cascada JNK. Conclusiones: En su conjunto los resultados obtenidos de sobreexpresión diferencial de genes asociados con el metabolismo de lípidos en el tejido adiposo de personas obesas podría ser un criterio para discriminar a nivel de diagnóstico esta patología.

Summary Objective: To analyze the complexity of gene expression in the adipose tissue of genes associated with obesity, by computer simulation with different bioinformatics tools. Methods: After conducting a PubMed literature search, 37 genes associated with obesity with a fold change greater than 1.5 were selected. An interaction network was cons tructed using the Cytoscape 3.2 program from the calculation of z-score values obtained from DNA microarray experiments of adipose tissue samples collected from obese and control people. Detailed information on the genomic characteristics of these genes was extracted from the UCSC and NCBI Genome Browser databases. Using multivariate analysis tools, a principal component analysis and a cluster analysis were carried out. Results: The constructed network showed that the genes with the greatest number of interactions were: 1) the nuclear respiratory factor (NRF1), 2) the activated channel of potassium activate by calcium alpha 1 (KCNMA1), and 3) the fatty acid synthase (FASN). Those with the higher expression values were: 1) vascular endothelial growth factor A (VEGFA), 2) alpha-ketoglutarate-dependent dioxygenase (FTO), and 3) neuronal growth regulator 1 (NEGR1). The IL6, BDNF and HLC proteins had the highest interaction values with IL6R, NRF1 and ACACB, respectively. The most important ontological categories were related to lipoprotein metabolic processes, the tricarboxylic acid cycle, the activation of the MAP kinases, and the JNK cascade. Conclusions: As a whole, the results obtained from differential overexpression of genes associated with lipid metabolism in the adipose tissue of obese people could be a criterion to discriminate this pathology at a diagnostic level.

Resumo Objetivo: Analisar a complexidade da expressão gênica no tecido adiposo de genes associados com obesidade, por meio de simulação por computador com diferentes ferramentas bioinformáticas. Métodos: Após uma busca bibliográfica em PubMed, foram selecionados 37 genes associados com obesidade com fold change superior a 1,5. A partir do cálculo de valores dos z-score obtidos de experimentos de micro matrizes de ADN de amostras de tecido adiposo de pessoas obesas e de controle, construiu-se uma rede de interação com o programa Cytoscape 3.2. A informação detalhada sobre as características genômicas destes genes foi obtida das bases de dados Genome Browser da UCSC e do NCBI. Utilizando ferramentas de análise de multivariado, fez-se uma análise de componentes principais e um de agrupação. Resultados: A rede construída mostrou que os genes com maior número de interações foram: 1) o fator nuclear respiratório (NRF1), 2) o canal ativado de potássio ativado por cálcio alfa 1 (KCNMA1) e 3) ácido graxo sintase (FASN). Os que tiveram maiores valores de expressão foram: 1) o fator de crescimento endo-telial vascular A (VEGFA), 2) a dioxigenase dependente de alfa-cetoglutarato (FTO) e 3) o regulador de crescimento neuronal 1 (NEGR1). As proteínas IL6, BDNF e HLC tiveram os maiores valores de interação com IL6R, NRF1 e ACACB, respectivamente. As categorias ontológicas mais importantes se relacionaram com processos metabólicos de lipoproteínas, o ciclo dos ácidos tri carboxílicos, a ativação das MAP-quinases e a cascata JNK. Conclusões: Em seu conjunto, os resultados obtidos de superexpressão diferencial de genes associados com o metabolismo de lipídios no tecido adiposo de pessoas obesas poderia ser um critério para discriminar a nível de diagnóstico esta patologia.

Diagnóstico clínico en saliva. Revisión

El odontólogo está llamado a convertirse en un elemento importante en el desarrollo de estudios de fenotipado de poblaciones para impulsar la aplicación de la medicina personalizada y la razón se debe a la saliva, que además de resultar un fluido imprescindible para la conservación de las funciones orales, se perfila con cualidades importantes para el diagnóstico clínico con base genética. Con el desarrollo de potentes técnicas de laboratorio, como el inmunoensayo, electroforesis, fluorescencia, cromatografía (HPLC y CG), espectrometría de masa (EM), la reacción en cadena de la polimerasa (PCR) y pruebas genéticas de fenotipado, se ha podido correlacionar la presencia de ciertos biomarcadores de daño tisular en la saliva con los niveles de esas especies en sangre. Estos biomarcadores constituyen señales del daño tisular o de respuestas del organismo a esas injurias, cuando aun no se pueden observar las evidencias clínicas del mismo. Esta aplicación de la saliva le confiere una importancia especial en lo referente al diagnóstico clínico. Al estudio de los biomarcadores orales se ha unido, de forma más reciente, el desarrollo de protocolos para la obtención de ADN genómico de la saliva que permite, por la viabilidad en la colección de muestras, su conservación y facilidades en su traslado, realizar estudios poblacionales para conocer la funcionalidad de ciertos genes que se relacionan con la biotransformación de los medicamentos, una causa importante de las variaciones interindividuales a los tratamientos farmacológicos. El conocimiento de polimorfismos en genes que expresan las enzimas que metabolizan fármacos específicos, permite realizar cambios en los principios activos o ajustes en las dosis de tratamientos individuales, que es el objetivo de la medicina personalizada o farmacogenética. Estos estudios también permiten conocer la predisposición genética de poblaciones al desarrollo de ciertas enfermedades, tanto orales como sistémicas, lo que propicia el establecimiento de nuevas políticas en la profilaxis y medicina preventiva. El uso de la saliva con estos fines abre nuevas perspectivas en la atención odontológica, y requiere de esfuerzos interdisciplinarios y cooperación en el equipo de salud.

The dentist is called to become an important element in the development of studies phenotyping of populations to advance the implementation of personalized medicine and the reason is because saliva, which besides being a prerequisite for the preservation of oral functions fluid , is emerging with important qualities for clinical diagnosis with genetic basis. With the development of powerful laboratory techniques, such as immunoassay, electrophoresis, fluorescence, chromatography (HPLC and GC), mass spectrometry (MS), polymerase chain reaction (PCR) and genetic testing phenotyping, it has been correlate the presence of certain biomarkers of tissue damage in saliva levels of these species in blood. These biomarkers are signs of tissue damage or agency responses to these injuries, when not even be seen clinical evidence of it. This application saliva attaches special importance with regard to clinical diagnosis. The study of oral biomarkers has joined, more recently, the development of protocols for obtaining genomic DNA from saliva that allows for viability in sample collection, preservation of the same and facilities during transport, conduct population studies to determine the function of certain genes that are related to biotransformation of drugs, a major cause of differences among individuals to drug treatments. Knowledge of polymorphisms in genes that express enzymes that metabolize specific drugs can make changes or adjustments active principles in doses of individual treatments, which is the goal of personalized medicine or pharmacogenetics. These studies also provide insight into the genetic predisposition of populations to the development of certain diseases, both oral and systemic, which favors the establishment of new policies in the prophylaxis and preventive medicine. The use of saliva for this purpose opens new perspectives in care dental and requires interdisciplinary efforts and cooperation in the health team.

Interactions of aminoglycosides with RNAs and proteins via carbohydrate microarray / 生物工程学报

Aminoglycosides are broad-spectrum antibacterials to treat bacterial infections, especially gram-negative bacteria infections. However, aminoglycosides are losing efficacy because of the increase in antibiotic resistance and their inherent toxicity, attracting more interests in developing new aminoglycosides. Several clinically used aminoglycosides are mainly exerted by inhibition of protein synthesis through binding to bacterial rRNA. The bacterial ribosome RNA is the most currently exploited RNA drug target. Identification of new compounds that target RNAs is indispensable to fight with the growing threat that bacteria pose to human safety. In this work, we used carbohydrate microarrays to probe interactions of low molecular weight ligands with RNAs and proteins. Carbohydrate microarrays, comprising hundreds to thousands of different glycan structures on surfaces in a spatially discrete pattern, are sensitive and versatile tools to study the interactions between biological macromolecules. Herein, aminoglycosides have been immobilized onto the modified glass microscope slides and their interactions with RNAs and proteins are then measured through the labeled fluorescence. The results displayed that microarray can be used to detect the binding of aminoglycosides with three types of target molecules, including the small RNA oligonucleotide mimics of aminoglycoside binding sites in the ribosome (rRNA A-site mimics), the large group I ribozyme RNA (approximately 400 nucleotide) and certain proteins (toxicity-causing enzymes, such as DNA polymerase and phospholipase C). For rRNA A-site mimics, the fluorescence intensities of 16S rRNA is stronger than that of 18S rRNA, illustrating that as a screen technique, the microarray method can not only determine the binding affinity to RNA but also detect the specific binding to bacterial rRNA mimic. The ability to screen group I ribozyme RNA can be helpful to the discovery of new RNA therapeutic targets. Binding of immobilized aminoglycosides to toxicity-causing proteins (DNA polymerase and phospholipase C) is a new method to study of aminoglycoside toxicity. These studies lay the foundation for rapid identification of new RNA-binding ligands with strong and specific binding affinity for their desired targets.

Chromosomal microarrays testing in children with developmental disabilities and congenital anomalies / Análise cromossômica por microarray em crianças com deficiências de desenvolvimento e anomalias congênitas

OBJECTIVES: Clinical use of microarray-based techniques for the analysis of many developmental disorders has emerged during the last decade. Thus, chromosomal microarray has been positioned as a first-tier test. This study reports the first experience in a Chilean cohort. METHODS: Chilean patients with developmental disabilities and congenital anomalies were studied with a high-density microarray (CytoScan(tm) HD Array, Affymetrix, Inc., Santa Clara, CA, USA). Patients had previous cytogenetic studies with either a normal result or a poorly characterized anomaly. RESULTS: This study tested 40 patients selected by two or more criteria, including: major congenital anomalies, facial dysmorphism, developmental delay, and intellectual disability. Copy number variants (CNVs) were found in 72.5% of patients, while a pathogenic CNV was found in 25% of patients and a CNV of uncertain clinical significance was found in 2.5% of patients. CONCLUSION: Chromosomal microarray analysis is a useful and powerful tool for diagnosis of developmental diseases, by allowing accurate diagnosis, improving the diagnosis rate, and discovering new etiologies. The higher cost is a limitation for widespread use in this setting. .

OBJETIVO: O uso clínico de técnicas baseadas em microarrays para a análise de transtornos de desenvolvimento tem surgido durante a última década. Assim, o microarray cromossômico tem sido posicionado como um teste de primeiro nível clínico. Relatamos a primeira experiência em uma coorte chilena. MÉTODOS: Pacientes chilenos com atraso de desenvolvimento e anomalias congênitas foram estudados com um microarray de alta densidade (CytoScan(tm) HD Array, Affymetrix, Inc., Santa Clara, CA, EUA). Pacientes tiveram estudos citogenéticos anteriores, ou um resultado normal ou de uma anomalia não bem caracterizada. RESULTADOS: Foram analisados 40 pacientes selecionados por dois ou mais critérios, incluindo: anomalias congênitas maiores, dismorfismo facial, atraso de desenvolvimento e deficiência intelectual. Uma variante do número de cópia (CNV) foi encontrada em 72,5% dos pacientes, enquanto que uma CNV patogênica foi encontrada em 25% dos pacientes e uma CNV de significado clínico incerto foi encontrada em 2,5% dos pacientes. CONCLUSÕES: A análise cromossômica microarray é uma ferramenta útil e poderosa em transtornos de desenvolvimento, permite um diagnóstico preciso, melhora a taxa de diagnóstico e descobre novas etiologias. O custo mais elevado é uma limitação para um uso difundido em nossa realidade. .

Microarray analysis of the in vitro granulomatous response to Mycobacterium tuberculosis H37Ra / Análisis con Micromatrices de la respuesta granulomatosa in vitro a Mycobacterium tuberculosis H37Ra

Background: The hallmark of tuberculosis is the granuloma, an organized cellular accumulation playing a key role in host defense against Mycobacterium tuberculosis. These structures sequester and contain mycobacterial cells preventing active disease, while long term maintenance of granulomas leads to latent disease. Clear understanding on mechanisms involved in granuloma formation and maintenance is lacking. Objective: To monitor granuloma formation and to determine gene expression profiles induced during the granulomatous response to M. tuberculosis (H37Ra). Methods: We used a previously characterized in vitro human model. Cellular aggregation was followed daily with microscopy and Wright staining for 5 days. Granulomas were collected at 24h, RNA extracted and hybridized to Affymetrix human microarrays. Results: Daily microscopic examination revealed gradual formation of granulomas in response to mycobacterial infection. Granulomatous structures persisted for 96 h, and then began to disappear. Conclusions: Microarray analysis identified genes in the innate immune response and antigen presentation pathways activated during the in vitro granulomatous response to live mycobacterial cells, revealing very early changes in gene expression of the human granulomatous response.

Antecedentes: La marca histológica de la tuberculosis es el granuloma, una acumulación celular organizada que cumple funciones claves en la defensa del hospedero contra Mycobacterium tuberculosis. Estas estructuras secuestran y confinan a las micobacterias previniendo el desarrollo de enfermedad activa; el mantenimiento a largo plazo de los granulomas conlleva al establecimiento de latencia. Un mejor entendimiento de los mecanismos involucrados en la formación y mantenimiento del granuloma es necesario. Objetivo: Monitorear la formación del granuloma y determinar los patrones de expresión génica inducidos durante la respuesta granulomatosa a M. tuberculosis (H37Ra). Métodos: En este estudio se empleó un modelo in vitro humano previamente caracterizado. La agregación celular fue examinada diariamente mediante microscopia óptica y tinción de Wright por 5 días. Para analizar la expresión génica, los granulomas fueron colectados a las 24 h, se extrajo el RNA sometiéndolo a hibridación a micromatrices de Affymetrix. Resultados: Se observó la formación gradual de granulomas en respuesta a la infección. Los granulomas persistieron por 96 h, y luego se desvanecieron. Conclusiones: Se identificaron genes de la respuesta inmune innata y vías de presentación antigénica activadas durante la respuesta granulomatosa in vitro a células micobacteriales vivas, lo cual reveló alteraciones tempranas de la expresión génica en el inicio de la respuesta granulomatosa humana.

Development of low-density oligonucleotide microarrays for detecting mutations causing wilson’s disease.

Background & objectives: Wilson’s disease (WD) is an autosomal recessive disorder caused by mutations in ATP7B, a copper transporter gene, leading to hepatic and neuropsychiatric manifestations due to copper accumulation. If diagnosed early, WD patients can be managed by medicines reducing morbidity and mortality. Diagnosis of this disease requires a combination of tests and at times is inconclusive due to overlap of the symptoms with other disorders. Genetic testing is the preferred alternative in such cases particularly for individuals with a family history. Use of DNA microarray for detecting mutations in ATP7B gene is gaining popularity because of the advantages it offers in terms of throughput and sensitivity. This study attempts to establish the quality analysis procedures for microarray based diagnosis of Wilson’s disease. Methods: A home-made microarrayer was used to print oligonucleotide based low-density microarrays for addressing 62 mutations causing Wilson’s disease reported from Indian population. Inter- and intra- array comparisons were used to study quality of the arrays. The arrays were validated by using mutant samples generated by site directed mutagenesis. Results: The hybridization reaction were found to be consistent across the surface of a given microarray. Our results have shown that 52 °C post-hybridization wash yields better reproducibility across experiments compared to 42 °C. Our arrays have shown > 80 per cent sensitivity in detecting these 62 mutations. Interpretation & conclusions: The present results demonstrate the design and evaluation of a low-density microarray for the detection of 62 mutations in ATP7B gene, and show that a microarray based approach can be cost-effective for detecting a large number of mutations simultaneously. This study also provides information on some of the important parameters required for microarray based diagnosis of genetic disorders.

Perspectivas actuales sobre el diagnóstico genómico en pediatría / Current perspectives on genome-based diagnostic tests in Pediatrics

El diagnóstico etiológico es un objetivo primordial para el manejo clínico de cada paciente. Algunos niños con cuadros clínicos complejos son objeto de una lista interminable de exámenes, lo que se ha denominado "odisea diagnóstica", y que, sin embargo, muchas veces no deja resultados concluyentes. En los últimos años, estamos siendo testigos de una verdadera revolución de la medicina genómica con la incorporación al ámbito clínico de tecnologías que prometen aumentar la capacidad de hacer diagnóstico y disminuir los tiempos. Con énfasis en pediatría, se actualizan conceptos sobre las principales ventajas y limitaciones del diagnóstico genómico, en contraposición con las metodologías usuales.

Etiological diagnosis is essential in the clinical management of individual patients. Some children with complex medical conditions are subjected to numerous testing, known as "diagnostic odyssey", which often gives no conclusive results. In recent years, a revolution in genomic medicine is underway with the use of technologies that promise to increase the ability to make a diagnosis and reduce the time involved. The main advantages and limitations of genomic diagnosis, as opposed to usual methodologies are reviewed with an emphasis on Pediatrics.

Multiple antibiotic susceptibility of polyphosphate kinase mutants (ppk1 and ppk2) from Pseudomonas aeruginosa PAO1 as revealed by global phenotypic analysis

BACKGROUND: Pseudomonas aeruginosa is known to be a multidrug resistant opportunistic pathogen. Particularly, P. aeruginosa PAO1 polyphosphate kinase mutant (ppk1) is deficient in motility, quorum sensing, biofilm formation and virulence. FINDINGS: By using Phenotypic Microarrays (PM) we analyzed near 2000 phenotypes of P. aeruginosa PAO1 polyP kinase mutants (ppk1 and ppk2). We found that both ppk mutants shared most of the phenotypic changes and interestingly many of them related to susceptibility toward numerous and different type of antibiotics such as Ciprofloxacin, Chloramphenicol and Rifampicin. CONCLUSIONS: Combining the fact that ppk1 mutants have reduced virulence and are more susceptible to antibiotics, polyP synthesis and particularly PPK1, is a good target for the design of molecules with anti-virulence and anti-persistence properties.

Gene Expression Profiling by Microarray during Tooth Development of Rats

Odontogenic cells express many genes spatiotemporally through complex and intricate processes during tooth formation. Therefore, investigating them during the tooth development has been an important subject for the better understanding of tooth morphogenesis. The present study was performed to identify the genetic profiles which are involved in the morphological changes during the different stages of rat tooth development using the Agilent Rat Oligonucleotide Microarrays. Morphologically, the maxillary 3rd molar germ at 10 days post-partum (dpp) was at the cap/bell stage. In contrast, the maxillary 2nd molar germ showed the root development stage. After microarray analysis, there were a considerable number of up- or down-regulated genes in the 3rd and the 2nd molar germ cells during tooth morphogenesis. Several differentially expressed genes for nerve supply were further studied. Among them, neuroligin 1 (Nlgn 1) was gradually downregulated during tooth development both at the transcription and the translation level. Also, Nlgn 1 was mostly localized in the dental sac, which is an important component yielding the nerve supply. This genetic profiling study proposed that many genes may be implicated in the biological processes for the dental hard tissue formation and, furthermore, may allow the identification of the key genes involved in the nerve supply to the dental sac.

From Faulty Biochemical Assumptions to Poor Diagnostic Performance: Reflections on the Failure of DNA Microarrays to Become a Reliable Clinical Diagnostic Tool.

Since their inception about two decades ago, DNA microarrays have been considered as a great hope in translational research and personalized medicine. Although DNA microarrays for gene expression profiling proved to be an indispensable tool in the laboratory settings, their applications as an instrument for clinical diagnostics have not yet produced tangible results. In this paper, we convey the idea that, apart from notoriously poor reproducibility and complexities of experimental validations, there exist other reasons hindering clinical application of DNA microarrays. These reasons are rooted in the very core of the DNA microarrays methodology, that is, in faulty biochemical assumptions underlying microarray measurements. A key premise the microarray measurements are based on is that mRNA abundances harvested from the eukaryotic cytoplasm are indicative of the activity levels of corresponding genes. There are at least two reasons why this premise is questionable. First, each transcription is supported by a number of transcription factors expressed by many genes. Due to this reason, relations between the transcription rates of genes and the mRNA abundances are the 'many-to-one', not the 'one-to-one'; therefore, abnormality in a certain mRNA abundance does not unequivocally indicate abnormality of the gene bearing its complimentary code. Second, mRNA copy numbers in cytoplasm are regulated by a number of epigenetic factors among which the post-transcriptional mRNA stability is of primary importance. Abnormal concentration of certain mRNA may result from deviant Opinion Article British Journal of Medicine & Medical Research, 4(27): 4511-4522, 2014 4512 mRNA stability, thus mimicking, but having nothing to do with, presumed abnormality in transcription rates of corresponding genes. An instrument built upon so poorly understood biochemical basis can hardly serve as a reliable tool in the delicate task of diagnosis of human disease in clinical settings.

Expressão gênica associada à produção de IFNG na resposta inicial à Leishmania braziliensis / Gene expression associated to IFNG production at initial response to infection with Leishmania braziliensis

Células de voluntários saudáveis, expostas in vitro a Leishmania, podem produzir altos níveis de IFNG na primeira exposição ao parasita, caracterizando os indivíduos como alto respondedores (AR), ou baixos níveis de IFNG, caracterizando os indivíduos baixo respondedores (BR). O objetivo deste estudo foi estudar o perfil de expressão gênica associado ao padrão de produção de IFNG de indivíduos AR e BR. Também avaliamos se as assinaturas gênicas identificadas nos indivíduos AR e BR se associam com o padrão de resposta imune observado em indivíduos de área endêmica identificada como subclínicos (SC) ou portadores de Leishmaniose Cutânea Localizada (LC). Inicialmente, Células Mononucleares do Sangue Periférico (CMSP) de voluntários saudáveis foram estimuladas in vitro com Leishmania braziliensis e identificamos indivíduos AR (com produção de IFNG acima de 330 pg/ml)...

Naïve volunteers exposed to Leishmania in vitro can be high IFNG producers, characterizing a high-responder (HR), or low IFNG producers, characterizing a low-responder (LR). The purpose of this work is to characterize the gene expression profile associated to IFNG production in HR and LR individuals. Formerly, we analyzed if the gene signature identified could be associated with the pattern of immune response in individuals from endemic areas identified as subclinical (SC), or patients with Localized Cutaneous Leishmaniasis (LC). Initially, we stimulated Peripheral Blood Mononuclear Cells (PBMCs) from healthy volunteers in vitro with Leishmania braziliensis where we identified HR (IFNG production above 330 pg/ml)...

Establishment of small molecular monoclonal antibody technology platform in Chinese materia medica / 中草药

Antibody is a tool of vital importance in modern bioscience research, small molecular antibody technology has a broad application prospect in the field of receptor binding analysis, enzyme assays, and quantitative and/or qualitative analytical techniques of Chinese materia medica (CMM) research. In this paper, combining with the research work carried out by our innovation team, we introduced the establishment background of the small molecular monoclonal antibody technology platform in CMM and technical difficulties in antibody preparation. In view of the technology products based on small molecular monoclonal antibody of CMM, such as ELISA test kit, immune affinity chromatography column, colloidal gold test paper, fluorescently labeled antibodies, and antibody microarrays, we explored and practised the various applications based on the small molecular monoclonal antibody technology looking forward to its scientific significances and application values in the field of CMM research.

Avaliação de marcadores de prognóstico no câncer de mama e análise funcional de CIP4 / Evaluation of prognostic markers in breast cancer and functional analysis of CIP4

O câncer de mama é uma doença extremamente heterogênea compreendendo diferentes subtipos moleculares que resultam em evoluções clínicas e condutas terapêuticas distintas. A maior gravidade desta patologia está associada a sua capacidade de formação de metástases Mudanças no padrão de expressão gênica têm sido associadas à manifestação do fenótipo metastático. Neste trabalho, utilizamos microarranjos de tecido (TMAs) para investigar a expressão de 8 biomarcadores candidatos (CIP4, PPIL1, ITGAV, AKAP14, MICA, FXYD1, ARPC3, ABG1) e avaliar seu potencial prognóstico em pacientes com carcinoma ductal invasivo da mama. Destes, ARPC3 PPIL1 e CIP4 mostraram associações estatisticamente significativas com a sobrevida câncer específica e/ou a probabilidade de desenvolvimento de metástases. Determinamos que a expressão aumentada de CIP4 nos tumores está associada a maior probabilidade de desenvolvimento de metástases. CIP4 é uma proteína adaptadora descrita na literatura como moduladora de migração e invasão celular e portanto selecionamos este candidato para caracterização funcional detalhada. Observamos que a expressão de CIP4 encontra-se aumentada em linhagens tumorais com características invasivas. A partir do silenciamento estável e regulado de CIP4 na linhagem metastática MDA-MB-231, determinamos que CIP4 modula positivamente a ativação de MAPK-p38 e a expressão de MMP2 , sugerindo que CIP4 participe em vias de sinalização importantes para a transição epitélio-mesenquima (EMT). O silenciamento de CIP4 resultou em uma redução de aproximadamente 50% da capacidade migratória e invasiva das células tumorais in vitro , e na diminuição da formação de metástases pulmonares in vivo. Coletivamente, nossos resultados indicam que CIP4 tem potencial como marcador de prognóstico assim como um possível alvo terapêutico no controle da disseminação de metástases nos tumores da mama

Breast cancer is an extremely heterogeneous disease comprising different molecular subtypes that result in different clinical outcomes and therapeutic procedures. The severity of this disease is mainly associated with its ability to produce metastasis. Changes in gene expression profile have been associated with the manifestation of the metastatic phenotype. In this study, we used tissue microarrays (TMAs) to investigate the expression of 8 candidate biomarkers (CIP4, PPIL1, ITGAV, AKAP14, MICA, FXYD1, ARPC3 e ABG1) and to evaluate their prognostic potential in patients with invasive ductal breast carcinoma. Among these, ARPC3, PPIL1 and CIP4 showed statistically significant associations with cancer specific survival and/or the patient's probability to develop metastasis. We found that increased expression of CIP4 in tumors is associated with a higher probability of developing metastasis. CIP4 is an adaptor protein described in the literature as a modulator of cell migration and invasion and therefore we selected this candidate for detailed functional characterization. We observed that CIP4 expression is increased in tumor cell lines with invasive characteristics. Following the stable and regulated knockdown of CIP4 in the metastatic line MDA-MB-231, we determined that it modulates positively the activation of MAPK-p38 and the expression of MMP2, suggesting that CIP4 participates in important signaling pathways required for the epithelial mesenchymal transition (EMT). CIP4 silencing resulted in an approximate 50% reduction of the migratory and invasive capacity of tumor cells in vitro and decreased the generation of lung metastases in vivo. Collectively, our results indicate that CIP4 has potential as a prognostic marker as well as a potential therapeutic target to control the metastatic dissemination of breast tumors

Investigating the Effects of Imputation Methods for Modelling Gene Networks Using a Dynamic Bayesian Network from Gene Expression Data

Background: Gene expression data often contain missing expression values. Therefore, several imputation methods have been applied to solve the missing values, which include k-nearest neighbour (kNN), local least squares (LLS), and Bayesian principal component analysis (BPCA). However, the effects of these imputation methods on the modelling of gene regulatory networks from gene expression data have rarely been investigated and analysed using a dynamic Bayesian network (DBN). Methods: In the present study, we separately imputed datasets of the Escherichia coli S.O.S. DNA repair pathway and the Saccharomyces cerevisiae cell cycle pathway with kNN, LLS, and BPCA, and subsequently used these to generate gene regulatory networks (GRNs) using a discrete DBN. We made comparisons on the basis of previous studies in order to select the gene network with the least error. Results: We found that BPCA and LLS performed better on larger networks (based on the S. cerevisiae dataset), whereas kNN performed better on smaller networks (based on the E. coli dataset). Conclusion: The results suggest that the performance of each imputation method is dependent on the size of the dataset, and this subsequently affects the modelling of the resultant GRNs using a DBN. In addition, on the basis of these results, a DBN has the capacity to discover potential edges, as well as display interactions, between genes.

Quick and inexpensive method to elaborate tissue punches useful in paraffin tissue microarrays / Método rápido y económico para la elaboración de dispositivos de punch útiles en microarreglos de tejidos en parafina

The tissue microarrays (TMAs) were first called multitumor block. In 1998 was described the current technique, that uses an innovated sampling method for more than 1,000 cylindrical paraffin tissue core biopsies in a single paraffin block. TMAs are now considered as a useful powerful research tool in Histology and Pathology laboratories, for the standardization of immunohistochemical techniques along with in situ hybridization. However, one disadvantage to its widespread use is the high cost of professional paraffin tissue punches, and the complexity in the development of homemade devices previously described in other studies. This study describes a step by step process to develop four different home-made devices made with materials that are common in hospitals and offices. These devices are useful in Histopathology laboratories to obtain paraffin blocks with until 360 samples of tissue, investing from two to fifteen dollars in the development of each device described.

Los microarreglos de tejido (TMAs) fueron llamados por primera vez como bloque multitumor. En 1998 se describió la técnica actual, que utiliza un novedoso método de muestreo para obtener más de 1,000 cilindros de biopsias de tejidos incluidos en un solo bloque de parafina. Actualmente, los TMAs se consideran una poderosa herramienta de investigación en laboratorios de Histología y Patología, para la estandarización de técnicas inmunohistoquímica e hibridación in situ entre otras. Sin embargo, uno de los inconvenientes para su uso generalizado es el alto costo de los dispositivos profesionales para tejidos en parafina, y la complejidad en la elaboración de los dispositivos caseros descritos previamente en otros estudios. Este estudio describe paso a paso el proceso de elaboración de cuatro dispositivos caseros útiles para la obtención de matrices de tejido elaborados con materiales que son comunes en hospitales y oficinas. Estos dispositivos son útiles en laboratorios de Histopatología con el fin de obtener bloques de parafina de hasta 360 muestras de tejido, con una inversión de 2 a 15 dólares en la elaboración de cada uno de los dispositivos descritos.