Host cell death in Legionella pneumophila pathogenesis and immunity

Legionella pneumophila is an intracellular pathogen that causes a severe, atypical pneumonia termed Legionnaires\u27 disease. Upon entering the host cell, L. pneumophila resides in a membrane-bound vacuole, in which the bacterium evades lysosomal fusion and replicates. The establishment of the vacuole requires the Dot/Icm (Defect in organelle trafficking/ intracellular multiplication) transport system, which translocates a large number of substrates into host cells to re-orchestrate various cellular processes, such as intracellular trafficking, protein synthesis and host cell death pathways. Therefore, a key step in understanding the biology of Legionella is to dissect the mechanisms of action of the Dot/Icm substrates. By fusing the gene of interest with β-lactamase, we determined the transfer of the fusions into mammalian cells using the β-lactamase reporter substrate CCF4-AM. 164 Dot/Icm substrates were identified in this screening, which led to the expansion of the list of known Dot/Icm substrates by 70 effectors. These efforts have facilitated future studies on the biology of L. pneumophila and its interaction with hosts.^ Taking advantage of the expanded list of L. pneumophila Dot/Icm effectors, we initiated a screening to survey all known effectors for the ability to activate caspase-3. Our screening led to the discovery that at least five Dot/Icm substrates, Lpg0716, Lpg0898, Lpg1831, Lpg1625 and Lpg2716, activate caspase-3 when ectopically expressed in mammalian cells. We further demonstrated that one of these effectors, VipD (lpg2831), is a phospholipase A2 that hydrolyzes the phosphatidylethanolamine (PE) and phosphatidylcholine (PC) on the mitochondrial membrane in a manner that appears to require host cofactor(s). The lipase activity is essential to its ability to activate caspase-3 via the production of free fatty acids and 2-lysophospholipids, which destabilize the mitochondrial membranes. Mitochondria membrane destabilization may contribute to cytochrome c release and subsequent activation of caspase-3. ^ To investigate how primary macrophages respond to Legionella environmental isolates, which are responsible for most of the Legionnaires\u27 disease outbreaks so far, we analyzed the intracellular growth of a few such isolates in A/J mouse macrophages, which is permissive to most, if not all, laboratory strains. None of the Legionella environmental isolates were able to replicate in these macrophages. Further investigation into one of the isolates, LPE509, revealed that such growth restriction is not due to the lack of important pathogenic determinants, as this strain is competent of replication in two protozoan hosts and the human macrophage U937 cell. Moreover, the inability of LPE509 to replicate intracellularly is accompanied by host cell death of unique features, as it is independent of most cell death signaling components known to control Legionella infection. These results together with the conservation of rpsL gene across diverse bacterial species prompted us to test whether the RpsL protein per se is responsible for the induction of host cell death. Indeed, delivery of RpsL into mouse macrophages elicits a noncanonical form of cell death involved in the permeabilization of lysosomal membranes. Upon the loss of lysosome membrane integrity, various hydrolases are released into the cytosol, where they process diverse substrates and cause cell death. Amongst these hydrolases, cathepsin B plays an important role in RpsL induced cell death, as genetic ablation of this gene dampens host cell death induced by recombinant RpsLWT or Legionella expressing wild type RpsL. Taken together, our results suggested that RpsL is a microbial associated molecular pattern (MAMP) that triggers a unique cell death pathway in mouse macrophages, which limits the spread of invading bacterial pathogens. (Abstract shortened by UMI.

Data Driven Search in the Displaced bbˉb\bar{b} Pair Channel for a Higgs Boson Decaying to Long-Lived Neutral Particles

This article presents a proposal for a new search channel for the Higgs boson decaying to two long-lived neutral particles, each of which decays to $b\bar b$ at a displaced vertex. The decay length considered is such that the decay takes place within the LHC beampipe. We present a new data-driven analysis using jet substructure and properties of the tracks from the highly-displaced vertices. We consider a model with a 125 GeV Higgs boson with a significant branching fraction to decay via this mode, with the long-lived neutral particle having a mass in the range of 15--40 GeV and a decay length commensurate with the beam pipe radius. Such a signal can be readily observed with an integrated luminosity of 19.5 fb$^{-1}$ at 8TeV at the LHC.Comment: 16 pages, 8 figures, Submitted to JHE

Evaluation of the Theoretical Uncertainties in the W to Lepton and Neutrino Cross Sections at the LHC

We study the sources of systematic errors in the measurement of the W to lepton and neutrino cross-sections at the LHC. We consider the systematic errors in both the total cross-section and acceptance for anticipated experimental cuts. We include the best available analysis of QCD effects at NNLO in assessing the effect of higher order corrections and PDF and scale uncertainties on the theoretical acceptance. In addition, we evaluate the error due to missing NLO electroweak corrections and propose which MC generators and computational schemes should be implemented to best simulate the events.Comment: 33 pages, 109 eps figures, uses JHEP3.cls, rotating.sty Version 2 corrects an error in Table 1, adds some references, and updates an author addres

Using Crowd-Based Software Repositories to Better Understand Developer-User Interactions

Software development is a complex process. To serve the final software product to the end user, developers need to rely on a variety of software artifacts throughout the development process. The term software repository used to denote only containers of source code such as version control systems; more recent usage has generalized the concept to include a plethora of software development artifact kinds and their related meta-data. Broadly speaking, software repositories include version control systems, technical documentation, issue trackers, question and answer sites, distribution information, etc. The software repositories can be based on a specific project (e.g., bug tracker for Firefox), or be crowd-sourced (e.g., questions and answers on technical Q&A websites). Crowd-based software artifacts are created as by-products of developer-user interactions which are sometimes referred to as communication channels. In this thesis, we investigate three distinct crowd-based software repositories that follow different models of developer-user interactions. We believe through a better understanding of the crowd-based software repositories, we can identify challenges in software development and provide insights to improve the software development process. In our first study, we investigate Stack Overflow. It is the largest collection of programming related questions and answers. On Stack Overflow, developers interact with other developers to create crowd-sourced knowledge in the form of questions and answers. The results of the interactions (i.e., the question threads) become valuable information to the entire developer community. Prior research on Stack Overflow tacitly assume that questions receives answers directly on the platform and no need of interaction is required during the process. Meanwhile, the platform allows attaching comments to questions which forms discussions of the question. Our study found that question discussions occur for 59.2% of questions on Stack Overflow. For discussed and solved questions on Stack Overflow, 80.6% of the questions have the discussion begin before the accepted answer is submitted. The results of our study show the importance and nuances of interactions in technical Q&A. We then study dotfiles, a set of publicly shared user-specific configuration files for software tools. There is a culture of sharing dotfiles within the developer community, where the idea is to learn from other developers’ dotfiles and share your variants. The interaction of dotfiles sharing can be viewed as developers sources information from other developers, adapt the information to their own needs, and share their adaptations back to the community. Our study on dotfiles suggests that is a common practice among developers to share dotfiles where 25.8% of the most stared users on GitHub have a dotfiles repository. We provide a taxonomy of the commonly tracked dotfiles and a qualitative study on the commits in dotfiles repositories. We also leveraged the state-of-the-art time-series clustering technique (K-shape) to identify code churn pattern for dotfile edits. This study is the first step towards understanding the practices of maintaining and sharing dotfiles. Finally, we study app stores, the platforms that distribute software products and contain many non-technical attributes (e.g., ratings and reviews) of software products. Three major stakeholders interacts with each other in app stores: the app store owner who governs the operation of the app store; developers who publish applications on the app store; and users who browse and download applications in the app store. App stores often provide means of interaction between all three actors (e.g., app reviews, store policy) and sometimes interactions with in the same actor (e.g., developer forum). We surveyed existing app stores to extract key features from app store operation. We then labeled a representative set of app store collected by web queries. K-means is applied to the labeled app stores to detect natural groupings of app stores. We observed a diverse set of app stores through the process. Instead of a single model that describes all app stores, fundamentally, our observations show that app stores operates differently. This study provide insights in understanding how app stores can affect software development. In summary, we investigated software repositories containing software artifacts created from different developer-user interactions. These software repositories are essential for software development in providing referencing information (i.e., Stack Overflow), improving development productivity (i.e., dotfiles), and help distributing the software products to end users (i.e., app stores)

Bacillus anthracis genome organization in light of whole transcriptome sequencing

Emerging knowledge of whole prokaryotic transcriptomes could validate a number of theoretical concepts introduced in the early days of genomics. What are the rules connecting gene expression levels with sequence determinants such as quantitative scores of promoters and terminators? Are translation efficiency measures, e.g. codon adaptation index and RBS score related to gene expression? We used the whole transcriptome shotgun sequencing of a bacterial pathogen Bacillus anthracis to assess correlation of gene expression level with promoter, terminator and RBS scores, codon adaptation index, as well as with a new measure of gene translational efficiency, average translation speed. We compared computational predictions of operon topologies with the transcript borders inferred from RNA-Seq reads. Transcriptome mapping may also improve existing gene annotation. Upon assessment of accuracy of current annotation of protein-coding genes in the B. anthracis genome we have shown that the transcriptome data indicate existence of more than a hundred genes missing in the annotation though predicted by an ab initio gene finder. Interestingly, we observed that many pseudogenes possess not only a sequence with detectable coding potential but also promoters that maintain transcriptional activity

Ab initio Gene Identification in Metagenomic Sequences

© The Author(s) 2010. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited.DOI: 10.1093/nar/gkq275We describe an algorithm for gene identification in DNA sequences derived from shotgun sequencing of microbial communities. Accurate ab initio gene prediction in a short nucleotide sequence of anonymous origin is hampered by uncertainty in model parameters. While several machine learning approaches could be proposed to bypass this difficulty, one effective method is to estimate parameters from dependencies, formed in evolution, between frequencies of oligonucleotides in protein-coding regions and genome nucleotide composition. Original version of the method was proposed in 1999 and has been used since for (i) reconstructing codon frequency vector needed for gene finding in viral genomes and (ii) initializing parameters of self-training gene finding algorithms. With advent of new prokaryotic genomes en masse it became possible to enhance the original approach by using direct polynomial and logistic approximations of oligonucleotide frequencies, as well as by separating models for bacteria and archaea. These advances have increased the accuracy of model reconstruction and, subsequently, gene prediction. We describe the refined method and assess its accuracy on known prokaryotic genomes split into short sequences. Also, we show that as a result of application of the new method, several thousands of new genes could be added to existing annotations of several human and mouse gut metagenome

Head3D: Complete 3D Head Generation via Tri-plane Feature Distillation

Head generation with diverse identities is an important task in computer vision and computer graphics, widely used in multimedia applications. However, current full head generation methods require a large number of 3D scans or multi-view images to train the model, resulting in expensive data acquisition cost. To address this issue, we propose Head3D, a method to generate full 3D heads with limited multi-view images. Specifically, our approach first extracts facial priors represented by tri-planes learned in EG3D, a 3D-aware generative model, and then proposes feature distillation to deliver the 3D frontal faces into complete heads without compromising head integrity. To mitigate the domain gap between the face and head models, we present dual-discriminators to guide the frontal and back head generation, respectively. Our model achieves cost-efficient and diverse complete head generation with photo-realistic renderings and high-quality geometry representations. Extensive experiments demonstrate the effectiveness of our proposed Head3D, both qualitatively and quantitatively