Synthetic long oligonucleotides to generate artificial templates for use as positive controls in molecular assays: drug resistance mutations in influenza virus as an example

<p>Abstract</p> <p>Background</p> <p>Positive controls are an integral component of any sensitive molecular diagnostic tool, but this can be affected, if several mutations are being screened in a scenario of a pandemic or newly emerging disease where it can be difficult to acquire all the necessary positive controls from the host. This work describes the development of a synthetic oligo-cassette for positive controls for accurate and highly sensitive diagnosis of several mutations relevant to influenza virus drug resistance.</p> <p>Results</p> <p>Using influenza antiviral drug resistance mutations as an example by employing the utility of synthetic paired long oligonucleotides containing complementary sequences at their 3' ends and utilizing the formation of oligonucleotide dimers and DNA polymerization, we generated ~170bp dsDNA containing several known specific neuraminidase inhibitor (NAI) resistance mutations. These templates were further cloned and successfully applied as positive controls in downstream assays.</p> <p>Conclusion</p> <p>This approach significantly improved the development of diagnosis of resistance mutations in terms of time, accuracy, efficiency and sensitivity, which are paramount to monitoring the emergence and spread of antiviral drug resistant influenza strains. Thus, this may have a significantly broader application in molecular diagnostics along with its application in rapid molecular testing of all relevant mutations in an event of pandemic.</p

Increased variability in ApcMin/+ intestinal tissue can be measured with microultrasound

Altered tissue structure is a feature of many disease states and is usually measured by microscopic methods, limiting analysis to small areas. Means to rapidly and quantitatively measure the structure and organisation of large tissue areas would represent a major advance not just for research but also in the clinic. Here, changes in tissue organisation that result from heterozygosity in Apc, a precancerous situation, are comprehensively measured using microultrasound and three-dimensional high-resolution microscopy. Despite its normal appearance in conventionally examined cross-sections, both approaches revealed a significant increase in the variability of tissue organisation in Apc heterozygous tissue. These changes preceded the formation of aberrant crypt foci or adenoma. Measuring these premalignant changes using microultrasound provides a potential means to detect microscopically abnormal regions in large tissue samples, independent of visual examination or biopsies. Not only does this provide a powerful tool for studying tissue structure in experimental settings, the ability to detect and monitor tissue changes by microultrasound could be developed into a powerful adjunct to screening endoscopy in the clinic

A Systematic Molecular Pathology Study of a Laboratory Confirmed H5N1 Human Case

Autopsy studies have shown that human highly pathogenic avian influenza virus (H5N1) can infect multiple human organs other than just the lungs, and that possible causes of organ damage are either viral replication and/or dysregulation of cytokines and chemokines. Uncertainty still exists, partly because of the limited number of cases analysed. In this study, a full autopsy including 5 organ systems was conducted on a confirmed H5N1 human fatal case (male, 42 years old) within 18 hours of death. In addition to the respiratory system (lungs, bronchus and trachea), virus was isolated from cerebral cortex, cerebral medullary substance, cerebellum, brain stem, hippocampus ileum, colon, rectum, ureter, aortopulmonary vessel and lymph-node. Real time RT-PCR evidence showed that matrix and hemagglutinin genes were positive in liver and spleen in addition to positive tissues with virus isolation. Immunohistochemistry and in-situ hybridization stains showed accordant evidence of viral infection with real time RT-PCR except bronchus. Quantitative RT-PCR suggested that a high viral load was associated with increased host responses, though the viral load was significantly different in various organs. Cells of the immunologic system could also be a target for virus infection. Overall, the pathogenesis of HPAI H5N1 virus was associated both with virus replication and with immunopathologic lesions. In addition, immune cells cannot be excluded from playing a role in dissemination of the virus in vivo

Retrograde traffic in the biosynthetic-secretory route

In the biosynthetic-secretory route from the rough endoplasmic reticulum, across the pre-Golgi intermediate compartments, the Golgi apparatus stacks, trans Golgi network, and post-Golgi organelles, anterograde transport is accompanied and counterbalanced by retrograde traffic of both membranes and contents. In the physiologic dynamics of cells, retrograde flow is necessary for retrieval of molecules that escaped from their compartments of function, for keeping the compartments’ balances, and maintenance of the functional integrities of organelles and compartments along the secretory route, for repeated use of molecules, and molecule repair. Internalized molecules may be transported in retrograde direction along certain sections of the secretory route, and compartments and machineries of the secretory pathway may be misused by toxins. An important example is the toxin of Shigella dysenteriae, which has been shown to travel from the cell surface across endosomes, and the Golgi apparatus en route to the endoplasmic reticulum, and the cytosol, where it exerts its deleterious effects. Most importantly in medical research, knowledge about the retrograde cellular pathways is increasingly being utilized for the development of strategies for targeted delivery of drugs to the interior of cells. Multiple details about the molecular transport machineries involved in retrograde traffic are known; a high number of the molecular constituents have been characterized, and the complicated fine structural architectures of the compartments involved become more and more visible. However, multiple contradictions exist, and already established traffic models again are in question by contradictory results obtained with diverse cell systems, and/or different techniques. Additional problems arise by the fact that the conditions used in the experimental protocols frequently do not reflect the physiologic situations of the cells. Regular and pathologic situations often are intermingled, and experimental treatments by themselves change cell organizations. This review addresses physiologic and pathologic situations, tries to correlate results obtained by different cell biologic techniques, and asks questions, which may be the basis and starting point for further investigations

Preservation of microvascular barrier function requires CD31 receptor-induced metabolic reprogramming

Endothelial barrier (EB) breaching is a frequent event during inflammation, and it is followed by the rapid recovery of microvascular integrity. The molecular mechanisms of EB recovery are poorly understood. Triggering of MHC molecules by migrating T-cells is a minimal signal capable of inducing endothelial contraction and transient microvascular leakage. Using this model, we show that EB recovery requires a CD31 receptor-induced, robust glycolytic response sustaining junction re-annealing. Mechanistically, this response involves src-homology phosphatase activation leading to Akt-mediated nuclear exclusion of FoxO1 and concomitant \u3b2-catenin translocation to the nucleus, collectively leading to cMyc transcription. CD31 signals also sustain mitochondrial respiration, however this pathway does not contribute to junction remodeling. We further show that pathologic microvascular leakage in CD31-deficient mice can be corrected by enhancing the glycolytic flux via pharmacological Akt or AMPK activation, thus providing a molecular platform for the therapeutic control of EB response

Desenvolvimento e estado nutricional da beterraba em função da omisão de nutrientes Effect of macronutrient omission on growth and nutritional status on table beet

Com o objetivo de avaliar o efeito da omissão de macronutrientes no desenvolvimento e no estado nutricional da beterraba, assim como descrever sintomas visuais de deficiência nutricional, um experimento foi conduzido em casa de vegetação da UNESP, Campus de Jaboticabal, SP. O delineamento experimental foi inteiramente casualizado, com sete tratamentos, que corresponderam à solução completa (macro e micronutrientes) e à omissão individual de N, P, K, Ca, Mg e S, com três repetições. Avaliou-se a altura das plantas, o número de folhas, a área foliar, a matéria seca da parte aérea, da raiz e planta inteira, os teores dos macronutrientes da parte aérea e raiz e descritos as desordens nutricionais. As omissões individuais de N, P, K e Ca foram as mais limitantes para o crescimento vegetativo da beterraba, reduzindo consideravelmente a altura, o número de folhas e as matérias secas de parte aérea, raiz e planta inteira. Foram observados sintomas de deficiência nutricional de cada elemento. Os teores dos macronutrientes na parte aérea do tratamento completo e com omissão dos nutrientes foram respectivamente: N = 32,9 - 13,8; P = 9,0 - 0,8; K = 126,0 - 15,1; Ca = 12,0 - 1,0; Mg = 10,1 - 0,7; S = 3,6 - 1,2 g kg-1.<br>An experiment was conducted under greenhouse conditions, in Jaboticabal, São Paulo State, Brazil, to evaluate the effects of macronutrient omission on beet nutritional status and development, as well as to describe nutritional deficiency symptoms. The experiment was arranged in completely randomized design with three replications and seven treatments, corresponding to complete nutritive solution (macro and micronutrients) and individual N, P, K, Ca, Mg and S omission. Plant height, number of leaves, leaf area, shoot, root and whole plant dry mass and macronutrient levels were determined and nutritional deficiency symptoms were described. Individual omissions of N, P, K or Ca were the most limiting for beet growth, considerably reducing plant height, number of leaves, and shoot, root and whole plant dry mass. Nutritional deficiency symptoms were observed for each element. Shoot macronutrient levels in control and nutrient omission treatments were, respectively: N = 32.9 - 13.8; P = 9.0 - 0.8; K = 126.0 - 15.1; Ca = 12.0 - 1.0; Mg = 10.1 - 0.7; S = 3.6 - 1.2 g kg-1