Dynamic variable selection in SNP genotype autocalling from APEX microarray data

Background  

Single nucleotide polymorphisms (SNPs) are DNA sequence variations, occurring when a single nucleotide – adenine (A), thymine (T), cytosine (C) or guanine (G) – is altered. Arguably, SNPs account for more than 90% of human genetic variation. Our laboratory has developed a highly redundant SNP genotyping assay consisting of multiple probes with signals from multiple channels for a single SNP, based on arrayed primer extension (APEX). This mini-sequencing method is a powerful combination of a highly parallel microarray with distinctive Sanger-based dideoxy terminator sequencing chemistry. Using this microarray platform, our current genotype calling system (known as SNP Chart) is capable of calling single SNP genotypes by manual inspection of the APEX data, which is time-consuming and exposed to user subjectivity bias.     

Generation and Initial Characterization of FDD Knock In Mice

Background

Mutations in the integral membrane protein 2B [1], also known as BRI₂ [2], a type II trans-membrane domain protein cause two autosomal dominant neurodegenerative diseases, Familial British and Danish Dementia [3]. In these conditions, accumulation of a C-terminal peptide (ABri and ADan) cleaved off from the mutated precursor protein by the pro-protein convertase furin [4], leads to amyloid deposition in the walls of blood vessels and parenchyma of the brain. Recent advances in the understanding of the generation of amyloid in Alzheimer''s disease has lead to the finding that BRI₂ interacts with the Amyloid Precursor Protein (APP), decreasing the efficiency of APP processing to generate Aβ [5], [6], [7]. The interaction between the two precursors, APP and BRI₂, and possibly between Aβ and ABri or ADan, could be important in influencing the rate of amyloid production or the tendency of these peptides to aggregate.

Methodology/Principal Findings

We have generated the first BRI₂ Danish Knock-In (FDD_KI) murine model of FDD, expressing the pathogenic decamer duplication in exon 6 of the BRI₂ gene. FDD_KI mice do not show any evident abnormal phenotype, with normal brain histology and no detectable amyloid deposition in blood vessel walls or parenchyma.

Conclusions/Significance

This new murine mouse model will be important to further understand the interaction between APP and BRI₂, and to provide insights into the molecular basis of FDD.     

Lack of the receptor for advanced glycation end-products attenuates E. coli pneumonia in mice

Background

The receptor for advanced glycation end-products (RAGE) has been suggested to modulate lung injury in models of acute pulmonary inflammation. To study this further, model systems utilizing wild type and RAGE knockout (KO) mice were used to determine the role of RAGE signaling in lipopolysaccharide (LPS) and E. coli induced acute pulmonary inflammation. The effect of intraperitoneal (i.p.) and intratracheal (i.t.) administration of mouse soluble RAGE on E. coli injury was also investigated.

Methodology/Principal Findings

C57BL/6 wild type and RAGE KO mice received an i.t. instillation of LPS, E. coli, or vehicle control. Some groups also received i.p. or i.t. administration of mouse soluble RAGE. After 24 hours, the role of RAGE expression on inflammation was assessed by comparing responses in wild type and RAGE KO. RAGE protein levels decreased in wild type lung homogenates after treatment with either LPS or bacteria. In addition, soluble RAGE and HMGB1 increased in the BALF after E. coli instillation. RAGE KO mice challenged with LPS had the same degree of inflammation as wild type mice. However, when challenged with E. coli, RAGE KO mice had significantly less inflammation when compared to wild type mice. Most cytokine levels were lower in the BALF of RAGE KO mice compared to wild type mice after E. coli injury, while only monocyte chemotactic protein-1, MCP-1, was lower after LPS challenge. Neither i.p. nor i.t. administration of mouse soluble RAGE attenuated the severity of E. coli injury in wild type mice.

Conclusions/Significance

Lack of RAGE in the lung does not protect against LPS induced acute pulmonary inflammation, but attenuates injury following live E. coli challenge. These findings suggest that RAGE mediates responses to E. coli-associated pathogen-associated molecular pattern molecules other than LPS or other bacterial specific signaling responses. Soluble RAGE treatment had no effect on inflammation.     

Microfluidics-based diagnostics of infectious diseases in the developing world

One of the great challenges in science and engineering today is to develop technologies to improve the health of people in the poorest regions of the world. Here we integrated new procedures for manufacturing, fluid handling and signal detection in microfluidics into a single, easy-to-use point-of-care (POC) assay that faithfully replicates all steps of ELISA, at a lower total material cost. We performed this 'mChip' assay in Rwanda on hundreds of locally collected human samples. The chip had excellent performance in the diagnosis of HIV using only 1 μl of unprocessed whole blood and an ability to simultaneously diagnose HIV and syphilis with sensitivities and specificities that rival those of reference benchtop assays. Unlike most current rapid tests, the mChip test does not require user interpretation of the signal. Overall, we demonstrate an integrated strategy for miniaturizing complex laboratory assays using microfluidics and nanoparticles to enable POC diagnostics and early detection of infectious diseases in remote settings.     

Bacterial infection-mediated mucosal signalling induces local renal ischaemia as a defence against sepsis

Ascending urinary tract infections can cause extensive damage to kidney structure and function. We have used a number of advanced techniques including multiphoton microscopy to investigate the crucial early phases of uropathogenic Escherichia coli induced pyelonephritis within a living animal. Our results reveal a previously undescribed innate vascular response to mucosal infection, allowing isolation and eradication of the pathogen. The extremely rapid host response to mucosal infection was highlighted by the triggering of a cascade of events within 3-4 h. Epithelial signalling produced an increase in cellular O(2) consumption and affected microvascular flow by clotting, causing localized ischaemia. Subsequent ischaemic damage affected pathophysiology with actin re-arrangement and epithelial sloughing leading to paracellular bacterial movement. A denuded tubular basement membrane is shown to hinder immediate dissemination of bacteria, giving the host time to isolate the infection by clotting. Suppression of clotting by heparin treatment caused fatal urosepsis. Clinically these findings may be relevant in antibiotics delivery in pyelonephritis patients and to the use of anticoagulants in sepsis.     

CD8 T cells are sufficient to mediate allorecognition and allograft rejection

Different T cell subsets may play different roles in allorecognition and allograft rejection. It has been suggested that CD8 T cells can only initiate rejection with help from CD4 T cells. Since CD8 T cells may have different requirements for allorecognition and for costimulation, it is important to clarify the role of CD8 cells in rejection. We examined the role of CD8 cells in allorecognition using a TCR transgenic mouse transplantation model. In our study, CD8 cells were able to recognize alloantigens and reject allografts in the absence of help from CD4 T cells. Furthermore our study provides a model to study the mechanisms of CD8-mediated allograft rejection. It may be important in the future, to consider that CD8 T cells may need to be targeted independently of CD4 T cells in strategies used to prevent rejection and induce tolerance.     

Effect of mutation in the aromatic amino acid pathway on sporulation of Saccharomyces cerevisiae.

Mutations in ARO1 and ARO2 genes coding for enzymes involved in the common part of the aromatic amino acid pathway completely block the sporulation of Saccharomyces cerevisiae when in a homozygous state, whereas mutations in all the other genes of the same pathway do not. This effect is not due to the lack of any intermediate metabolite but rather to the accumulation of a metabolite preceding chorismic acid. Shikimic acid or one of its precursors was identified as the possible inhibitor. The presence of the three aromatic amino acids in the sporulation medium restores the ability to undergo meiosis. This seems not to be due to a feedback inhibition of the first enzymes of the pathway but rather to a competition between aromatic amino acids and the inhibitor on a site specific for the meiotic process. The inhibition of sporulation seems to occur at a very early step in meiosis, as indicated by the lack of premeiotic DNA synthesis in aro1 and aro2 mutants.