RNA interference screening reveals host CaMK4 as a regulator of cryptococcal uptake and pathogenesis

ABSTRACT Cryptococcus neoformans , the causative agent of cryptococcosis, is an opportunistic fungal pathogen that kills over 200,000 individuals annually. This yeast may grow freely in body fluids, but it also flourishes within host cells. Despite extensive research on cryptococcal pathogenesis, host genes involved in the initial engulfment of fungi and subsequent stages of infection are woefully understudied. To address this issue, we combined short interfering RNA silencing and a high-throughput imaging assay to identify host regulators that specifically influence cryptococcal uptake. Of 868 phosphatase and kinase genes assayed, we discovered 79 whose silencing significantly affected cryptococcal engulfment. For 25 of these, the effects were fungus specific, as opposed to general alterations in phagocytosis. Four members of this group significantly and specifically altered cryptococcal uptake; one of them encoded CaMK4, a calcium/calmodulin-dependent protein kinase. Pharmacological inhibition of CaMK4 recapitulated the observed defects in phagocytosis. Furthermore, mice deficient in CaMK4 showed increased survival compared to wild-type mice upon infection with C. neoformans . This increase in survival correlated with decreased expression of pattern recognition receptors on host phagocytes known to recognize C. neoformans . Altogether, we have identified a kinase that is involved in C. neoformans internalization by host cells and in host resistance to this deadly infection. </jats:p

Space-Efficient Estimation of Statistics Over Sub-Sampled Streams

In many stream monitoring situations, the data arrival rate is so high that it is not even possible to observe each element of the stream. The most common solution is to subsample the data stream and use the sample to infer properties and estimate aggregates of the original stream. However, in many cases, the estimation of aggregates on the original stream cannot be accomplished through simply estimating them on the sampled stream, followed by a normalization. We present algorithms for estimating frequency moments, support size, entropy, and heavy hitters of the original stream, through a single pass over the sampled stream

Heads or tails: L1 insertion-associated 5' homopolymeric sequences

<p>Abstract</p> <p>Background</p> <p>L1s are one of the most successful autonomous mobile elements in primate genomes. These elements comprise as much as 17% of primate genomes with the majority of insertions occurring via target primed reverse transcription (TPRT). Twin priming, a variant of TPRT, can result in unusual DNA sequence architecture. These insertions appear to be inverted, truncated L1s flanked by target site duplications.</p> <p>Results</p> <p>We report on loci with sequence architecture consistent with variants of the twin priming mechanism and introduce dual priming, a mechanism that could generate similar sequence characteristics. These insertions take the form of truncated L1s with hallmarks of classical TPRT insertions but having a poly(T) simple repeat at the 5' end of the insertion. We identified loci using computational analyses of the human, chimpanzee, orangutan, rhesus macaque and marmoset genomes. Insertion site characteristics for all putative loci were experimentally verified.</p> <p>Conclusions</p> <p>The 39 loci that passed our computational and experimental screens probably represent inversion-deletion events which resulted in a 5' inverted poly(A) tail. Based on our observations of these loci and their local sequence properties, we conclude that they most probably represent twin priming events with unusually short non-inverted portions. We postulate that dual priming could, theoretically, produce the same patterns. The resulting homopolymeric stretches associated with these insertion events may promote genomic instability and create potential target sites for future retrotransposition events.</p

Internal priming: An opportunistic pathway for L1 and Alu retrotransposition in hominins

Retrotransposons, specifically Alu and L1 elements, have been especially successful in their expansion throughout primate genomes. While most of these elements integrate through an endonuclease-mediated process termed target primed reverse transcription, a minority integrate using alternative methods. Here we present evidence for one such mechanism, which we term internal priming and demonstrate that loci integrating through this mechanism are qualitatively different from classical insertions. Previous examples of this mechanism are limited to cell culture assays, which show that reverse transcription can initiate upstream of the 3′ poly-A tail during retrotransposon integration. To detect whether this mechanism occurs in vivo as well as in cell culture, we have analyzed the human genome for internal priming events using recently integrated L1 and Alu elements. Our examination of the human genome resulted in the recovery of twenty events involving internal priming insertions, which are structurally distinct from both classical TPRT-mediated insertions and non-classical insertions. We suggest two possible mechanisms by which these internal priming loci are created and provide evidence supporting a role in staggered DNA double-strand break repair. Also, we demonstrate that the internal priming process is associated with inter-chromosomal duplications and the insertion of filler DNA. © 2009 Elsevier B.V. All rights reserved

In search of polymorphic Alu insertions with restricted geographic distributions

Alu elements are transposable elements that have reached over one million copies in the human genome. Some Alu elements inserted in the genome so recently that they are still polymorphic for insertion presence or absence in human populations. Recently, there has been an increasing interest in using Alu variation for studies of human population genetic structure and inference of individual geographic origin. Currently, this requires a high number of Alu loci. Here, we used a linker-mediated polymerase chain reaction method to preferentially identify low-frequency Alu elements in various human DNA samples with different geographic origins. The candidate Alu loci were subsequently genotyped in 18 worldwide human populations (∼370 individuals), resulting in the identification of two new Alu insertions restricted to populations of African ancestry. Our results suggest that it may ultimately become possible to correctly infer the geographic affiliation of unknown samples with high levels of confidence without having to genotype as many as 100 Alu loci. This is desirable if Alu insertion polymorphisms are to be used for human evolution studies or forensic applications. © 2007 Elsevier Inc. All rights reserved

Insulin autoantibodies as determined by competitive radiobinding assay are positively correlated with impaired beta-cell function — The Ulm-Frankfurt population study

Out of a random population of 4208 non-diabetic pupils without a family history of Type I diabetes 44 (1.05%) individuals had islet cell antibody (ICA) levels greater or equal to 5 Juvenile Diabetes Foundation (JDF) units. 39 of these ICA-positives could be repeatedly tested for circulating insulin autoantibodies (CIAA) using a competitive radiobinding assay. The results were compared with the insulin responses in the intravenous glucose tolerance tests (IVGTT) and with HLA types. Six pupils were positive for CIAA. All of them had complement-fixing ICA, and 5 of them were HLA-DR4 positive. Three of the 6 showed a first-phase insulin response below the first percentile of normal controls. Our data indicate that in population-based studies CIAA can be considered as a high risk marker for impaired beta-cell function in non-diabetic ICA-positive individuals

The CER in Action: A Closer Look at Australia-NZ Trade Flows Since 1983

This paper employs statistical and analytical methods to examine the manner in which trade between Australia and New Zealand has developed since the Closer Economic Relations (CER) Agreement of 1983. The strength and weaknesses of their economic structures are highlighted through the examination of the evolution of mutual trade and trade with the world, as well as the changes thereto over time. Empirical analysis finds that the nature of economic reforms in both countries have affected their mutual and global trade relationships and policy directives have been drawn from the findings.Trade intensity, intra-industry trade, revealed comparative advantage, free trade area