Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume

The functional landscape of mouse gene expression

BACKGROUND: Large-scale quantitative analysis of transcriptional co-expression has been used to dissect regulatory networks and to predict the functions of new genes discovered by genome sequencing in model organisms such as yeast. Although the idea that tissue-specific expression is indicative of gene function in mammals is widely accepted, it has not been objectively tested nor compared with the related but distinct strategy of correlating gene co-expression as a means to predict gene function. RESULTS: We generated microarray expression data for nearly 40,000 known and predicted mRNAs in 55 mouse tissues, using custom-built oligonucleotide arrays. We show that quantitative transcriptional co-expression is a powerful predictor of gene function. Hundreds of functional categories, as defined by Gene Ontology 'Biological Processes', are associated with characteristic expression patterns across all tissues, including categories that bear no overt relationship to the tissue of origin. In contrast, simple tissue-specific restriction of expression is a poor predictor of which genes are in which functional categories. As an example, the highly conserved mouse gene PWP1 is widely expressed across different tissues but is co-expressed with many RNA-processing genes; we show that the uncharacterized yeast homolog of PWP1 is required for rRNA biogenesis. CONCLUSIONS: We conclude that 'functional genomics' strategies based on quantitative transcriptional co-expression will be as fruitful in mammals as they have been in simpler organisms, and that transcriptional control of mammalian physiology is more modular than is generally appreciated. Our data and analyses provide a public resource for mammalian functional genomics

Short-course quinazoline drug treatments are effective in the Litomosoides sigmodontis and Brugia pahangi jird models

The quinazolines CBR417 and CBR490 were previously shown to be potent anti-wolbachials that deplete Wolbachia endosymbionts of filarial nematodes and present promising pre-clinical candidates for human filarial diseases such as onchocerciasis. In the present study we tested both candidates in two models of chronic filarial infection, namely the Litomosoides sigmodontis and Brugia pahangi jird model and assessed their long-term effect on Wolbachia depletion, microfilariae counts and filarial embryogenesis 16-18 weeks after treatment initiation (wpt). Once per day (QD) oral treatment with CBR417 (50 mg/kg) for 4 days or twice per day (BID) with CBR490 (25 mg/kg) for 7 days during patent L. sigmodontis infection reduced the Wolbachia load by >99% and completely cleared peripheral microfilaremia from 10-14 wpt. Similarly, 7 days of QD treatments (40 mg/kg) with CBR417 or CBR490 cleared >99% of Wolbachia from B. pahangi and reduced peritoneal microfilariae counts by 93% in the case of CBR417 treatment. Transmission electron microscopy analysis indicated intensive damage to the B. pahangi ovaries following CBR417 treatment and in accordance filarial embryogenesis was inhibited in both models after CBR417 or CBR490 treatment. Suboptimal treatment regimens of CBR417 or CBR490 did not lead to a maintained reduction of the microfilariae and Wolbachia load. In conclusion, CBR417 or CBR490 are pre-clinical candidates for filarial diseases, which achieve long-term clearance of Wolbachia endosymbionts of filarial nematodes, inhibit filarial embryogenesis and clear microfilaremia with treatments as short as 7 days

Role of Bacterial Effectors SopD and SopB in Pathogenicity of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that has evolved to take advantage of the eukaryotic host cells it inhabits during infection. It uses bacterial effectors translocated into the host cell cytosol to manipulate host cell machinery and establish a replicative niche. In this thesis I study the function of two of these effectors, SopD and SopB, which have been shown to act cooperatively to induce phenotypes associated with gastroenteritis (fluid secretion and neutrophil influx into the intestinal lumen). In addition to promoting gastroenteritis, SopD has also been implicated in systemic and persistent infection of mice. Although recently implicated in invasion, the precise function of SopD has remained elusive. Here I show that SopD affects membrane dynamics during S. Typhimurium invasion of epithelial cells. SopD promotes membrane sealing and macropinosome formation, events that may have important consequences for efficiency of bacterial cell entry in vivo. Furthermore, we demonstrate that SopD is recruited to the invasion site membranes through the phosphatase activity of SopB, suggesting a mechanism for their cooperative action during induction of gastroenteritis. Unlike SopD, SopB has been a focus of intense research efforts and its role in invasion as a phosphoinositide phosphatase is well documented. However, we have observed that SopB also inhibits fusion of lysosomes with Salmonella-containing vacuoles (SCVs) following invasion. This ability depends on SopB-mediated reduction of negative membrane charge of the SCV during invasion by hydrolysis of the phosphoinositide PI(4,5)P2. Membrane charge alterations driven by SopB result in removal of Rab GTPases from the SCV that depend on electrostatic interactions for their targeting. Two of these Rabs, Rab23 and Rab35 were previously shown to promote phagosome-lysosome fusion. Therefore their removal from the SCV may promote SCV trafficking away from the degradative endocytic pathway of host cells. This represents a new mechanism by which an invasion associated effector controls SCV maturation. Together, this work advances our knowledge of the interaction between S. Typhimurium and its host. This research also suggests a new mechanism by which pathogens other than S. Typhimurium could promote their intracellular survival.Ph

<i>F56A8</i>.<i>3</i> RNAi clone acts in the intestine, and the F56A8.3 protein localizes to lysosome-related organelles in the intestine.

<p>(A) Representative image of transgenic <i>C</i>. <i>elegans</i> expressing intestinal mCherry under control of the putative <i>F56A8</i>.<i>3</i> promoter. Scale bar = 100 μm. (B) Larval arrest of tissue-specific RNAi strains MGH167 (left, intestinal-specific) and SPC272 (right, muscle-specific) after <i>N</i>. <i>parisii</i> infection, measured as the percent animals reaching the adult stage at 3 dpi. Data are represented as mean values with SEM from two independent experiments (**p = 0.002; n.s. p = 0.26, unpaired two-tailed t-test). (C) Representative image of endogenous F56A8.3 localization in dissected intestines from wild-type N2 <i>C</i>. <i>elegans</i> (left) or from N2 treated with <i>F56A8</i>.<i>3</i> RNAi (right). F56A8.3 was detected with anti-F56A8.3 followed by goat anti-rabbit IgG conjugated to Cy3. Scale bar = 10 μm. (D) Representative image of endogenous F56A8.3 colocalization relative to CDF-2::GFP in the WU1236 transgenic strain. F56A8.3 was detected as in C; CDF-2 GFP was detected with anti-GFP followed by anti-mouse IgG conjugated to FITC. Scale bar = 10 μm.</p

Characterization of Microsporidia-Induced Developmental Arrest and a Transmembrane Leucine-Rich Repeat Protein in <i>Caenorhabditis elegans</i>

<div><p>Microsporidia comprise a highly diverged phylum of intracellular, eukaryotic pathogens, with some species able to cause life-threatening illnesses in immunocompromised patients. To better understand microsporidian infection in animals, we study infection of the genetic model organism <i>Caenorhabditis elegans</i> and a species of microsporidia, <i>Nematocida parisii</i>, which infects <i>Caenorhabditis</i> nematodes in the wild. We conducted a targeted RNAi screen for host <i>C</i>. <i>elegans</i> genes important for infection and growth of <i>N</i>. <i>parisii</i>, using nematode larval arrest as an assay for infection. Here, we present the results of this RNAi screen, and our analyses on one of the RNAi hits from the screen that was ultimately not corroborated by loss of function mutants. This hit was an RNAi clone against <i>F56A8</i>.<i>3</i>, a conserved gene that encodes a transmembrane protein containing leucine-rich repeats (LRRs), a domain found in numerous pathogen receptors from other systems. This RNAi clone caused <i>C</i>. <i>elegans</i> to be resistant to infection by <i>N</i>. <i>parisii</i>, leading to reduced larval arrest and lower pathogen load. Characterization of the endogenous F56A8.3 protein revealed that it is expressed in the intestine, localized to the membrane around lysosome-related organelles (LROs), and exists in two different protein isoforms in <i>C</i>. <i>elegans</i>. We used the CRISPR-Cas9 system to edit the <i>F56A8</i>.<i>3</i> locus and created both a frameshift mutant resulting in a truncated protein and a complete knockout mutant. Neither of these mutants was able to recapitulate the infection phenotypes of the RNAi clone, indicating that the RNAi-mediated phenotypes are due to an off-target effect of the RNAi clone. Nevertheless, this study describes microsporidia-induced developmental arrest in <i>C</i>. <i>elegans</i>, presents results from an RNAi screen for host genes important for microsporidian infection, and characterizes aspects of the conserved <i>F56A8</i>.<i>3</i> gene and its protein product.</p></div

Mutation of <i>F56A8</i>.<i>3a</i> does not recapitulate the larval arrest phenotype of <i>F56A8</i>.<i>3</i> RNAi.

<p>(A) <i>Top</i>: Schematic representation of the <i>F56A8</i>.<i>3a</i> and <i>F56A8</i>.<i>3b</i> pre-mRNA transcripts, with exons represented as black and gray blocks, indicated coding and non-coding sequences, respectively, and solid lines representing introns. The sequence covered by <i>F56A8</i>.<i>3</i> RNAi clone is indicated at the top as a dotted line. Image adapted from WormBase and based on EST data (WBGene00010139). <i>Bottom</i>: Schematic representation of F56A8.3a and F56A8.3b protein, with dotted lines showing the relative locations on the coding exons from which the main protein domains are derived (LRR is the leucine-rich repeat domain, CC is the coiled coil domain, TM is the transmembrane domain, and CTD is the C-terminal domain). (B) Representation of CRISPR-Cas9 genome editing of the 5'-most exon of the <i>F56A8</i>.<i>3</i> gene, with the <i>F56A8</i>.<i>3</i> start codon in bold, the sgRNA targeting sequence highlighted, and the protospacer adjacent motif (PAM) underlined (left). WT is the wild-type sequence found in N2, and -5 is a 5 bp deletion found in the mutant ERT327 (<i>jy4</i>), with representative 80 bp and 75 bp PCR products from the F1 screen shown (right). (C) Larval arrest of <i>eri-1</i> and <i>F56A8</i>.<i>3</i> frameshift mutant ERT360 <i>F56A8</i>.<i>3(jy4)</i> (in an <i>eri-1</i> background) on control or <i>F56A8</i>.<i>3</i> RNAi after <i>N</i>. <i>parisii</i> infection, measured as the percent animals reaching the L4 at 2 dpi. Data are represented as mean values with SEM from two independent experiments (*p = 0.013 (left), *p = 0.022 (right), unpaired two-tailed t-test). (D) F56A8.3 protein in N2 and <i>F56A8</i>.<i>3</i> frameshift mutation ERT327 <i>F56A8</i>.<i>3(jy4)</i> on either control (L4440) or <i>F56A8</i>.<i>3</i> RNAi. The top picture represents a single blot probed with anti-F56A8.3 antibodies, while the bottom represents a single blot probed with anti-actin. Indicated molecular weight markers are in kilodaltons (kD).</p

<i>F56A8</i>.<i>3</i> RNAi clone reduces the level of <i>N</i>. <i>parisii</i> infection at several stages of infection.

<p>(A) Pathogen load at 8 hpi on control or <i>F56A8</i>.<i>3</i> RNAi measured as the number of FISH-stained sporoplasms seen in intact <i>C</i>. <i>elegans</i> intestines. Data are represented as mean values with SEM from three independent, blinded experiments (**p = 0.002, paired two-tailed t-test). (B) Pathogen load at 30 hpi on control or <i>F56A8</i>.<i>3</i> RNAi measured as the fold change in <i>N</i>. <i>parisii</i> rDNA transcript by qRT-PCR relative to L4440 infected at the lowest dose. Data are represented as mean values with SEM from three independent experiments (*p = 0.033, two-way analysis of variation, testing RNAi treatment effecting pathogen load at all doses). (C) Pathogen load at 40 hpi with <i>C</i>. <i>elegans</i> infected at the L2 stage on control or <i>F56A8</i>.<i>3</i> RNAi measured as the average number of spores produced per animal. Data are represented as mean values with SEM from three independent experiments (***p = 0.0005, paired two-tailed t-test).</p