Elizabeth A. Fogarty

A three-channel microfluidic device for generating static linear gradients and its application to the quantitative analysis of bacterial chemotaxis

We have developed a prototype three-channel microfluidic chip that is capable of generating a linear concentration gradient within a microfluidic channel and is useful in the study of bacterial chemotaxis. The linear chemical gradient is established by diffusing a chemical through a porous membrane located in the side wall of the channel and can be established without through-flow in the channel where cells reside. As a result, movement of the cells in the center channel is caused solely by the cells chemotactic response and not by variations in fluid flow. The advantages of this microfluidic chemical linear gradient generator are (i) its ability to produce a static chemical gradient, (ii) its rapid implementation, and (iii) its potential for highly parallel sample processing. Using this device, wildtype Escherichia coli strain RP437 was observed to move towards an attractant (e.g., l-asparate) and away from a repellent (e.g., glycerol) while derivatives of RP437 that were incapable of motility or chemotaxis showed no bias of the bacterias distribution. Additionally, the degree of chemotaxis could be easily quantified using this assay in conjunction with fluorescence imaging techniques, allowing for estimation of the chemotactic partition coefficient (CPC) and the chemotactic migration coefficient (CMC). Finally, using this approach we demonstrate that E. coli deficient in autoinducer-2-mediated quorum sensing respond to the chemoattractant l-aspartate in a manner that is indistinguishable from wildtype cells suggesting that chemotaxis is insulated from this mode of cell-cell communication.

Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli

ABSTRACT When eukaryotic proteins with multiple disulfide bonds are expressed at high levels in Escherichia coli, the efficiency of thiol oxidation and isomerization is typically not sufficient to yield soluble products with native structures. Even when such proteins are secreted into the oxidizing periplasm or expressed in the cytoplasm of cells carrying mutations in the major intracellular disulfide bond reduction systems (e.g., trxB gor mutants), correct folding can be problematic unless a folding modulator is simultaneously coexpressed. In the present study we explored whether the bacterial twin-arginine translocation (Tat) pathway could serve as an alternative expression system for obtaining appreciable levels of recombinant proteins which exhibit complex patterns of disulfide bond formation, such as full-length human tissue plasminogen activator (tPA) (17 disulfides) and a truncated but enzymatically active version of tPA containing nine disulfides (vtPA). Remarkably, targeting of both tPA and vtPA to the Tat pathway resulted in active protein in the periplasmic space. We show here that export by the Tat translocator is dependent upon oxidative protein folding in the cytoplasm of trxB gor cells prior to transport. Whereas previous efforts to produce high levels of active tPA or vtPA in E. coli required coexpression of the disulfide bond isomerase DsbC, we observed that Tat-targeted vtPA and tPA reach a native conformation without thiol-disulfide oxidoreductase coexpression. These results demonstrate that the Tat system may have inherent and unexpected benefits compared with existing expression strategies, making it a viable alternative for biotechnology applications that hinge on protein expression and secretion.

EFFECTS OF FREEZE–THAW EVENTS ON THE VIABILITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS IN SOIL

The effects of freeze–thaw events on the inactivation of Cryptosporidium parvum oocysts in soil were examined. Oocysts were inoculated into distilled water in microcentrifuge tubes or into chambers containing soil the water content of which was maintained at 3%, 43%, or 78% of the container capacity. The chambers and tubes were then embedded in 3 soil samples from different aspects of a hillside landscape (Experiments 1 and 2) and in 3 distinct soil types (Experiment 3) and frozen at −10 C. Containers were thawed every 3 days for a period of 24 hr in 1–9 freeze–thaw cycles over 27 days (Experiments 1 and 2) and 2–5 freeze–thaw cycles over 15 days (Experiment 3). Oocyst viability was measured using the fluorescent dyes 4′6-diaminidino-2-phenylindole and propidium iodide. Inactivation rates were greater in soils than in water and greater in dry soil than in moist and wet soils. Soil type showed no effect on inactivation. Oocysts subjected to freeze–thaw cycles had inactivation rates not significantly different from those of oocysts subjected to −10 C under static conditions. The results indicated that 99% of oocysts exposed to soils that are frozen at −10 C will become inactivated within 50 days whether or not freeze–thaw cycles occur.

Widespread alternative and aberrant splicing revealed by lariat sequencing

Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures.

Evolution of ligand specificity in vertebrate corticosteroid receptors

BackgroundCorticosteroid receptors include mineralocorticoid (MR) and glucocorticoid (GR) receptors. Teleost fishes have a single MR and duplicate GRs that show variable sensitivities to mineralocorticoids and glucocorticoids. How these receptors compare functionally to tetrapod MR and GR, and the evolutionary significance of maintaining two GRs, remains unclear.ResultsWe used up to seven steroids (including aldosterone, cortisol and 11-deoxycorticosterone [DOC]) to compare the ligand specificity of the ligand binding domains of corticosteroid receptors between a mammal (Mus musculus) and the midshipman fish (Porichthys notatus), a teleost model for steroid regulation of neural and behavioral plasticity. Variation in mineralocorticoid sensitivity was considered in a broader phylogenetic context by examining the aldosterone sensitivity of MR and GRs from the distantly related daffodil cichlid (Neolamprologus pulcher), another teleost model for neurobehavioral plasticity. Both teleost species had a single MR and duplicate GRs. All MRs were sensitive to DOC, consistent with the hypothesis that DOC was the initial ligand of the ancestral MR. Variation in GR steroid-specificity corresponds to nine identified amino acid residue substitutions rather than phylogenetic relationships based on receptor sequences.ConclusionThe mineralocorticoid sensitivity of duplicate GRs in teleosts is highly labile in the context of their evolutionary phylogeny, a property that likely led to neo-functionalization and maintenance of two GRs.

Effect of aerobic and anaerobic digestion on the viability of Cryptosporidium parvum oocysts and Ascaris suum eggs.

The viability of Cryptosporidium parvum oocysts and Ascaris suum eggs inoculated into aerobic and anaerobic digesters was measured. The digesters were maintained at 37°C, 47°C, and 55°C, with 10-day detention times. Eggs and oocysts were added to each digester in a single spike or in chambers placed in the digesters for varying periods. Oocysts were inactivated very rapidly in all systems as determined by a dye permeability assay, > 99% inactivated after 10 days at 37°C, 4 days at 47°C, and 2 days at 55°C. Eggs were more rapidly inactivated in anaerobic digesters than in aerobic digesters. At 55°C, eggs in both anaerobic and aerobic digesters were > 99% inactivated within 1 h. At 47°C, anaerobic digestion inactivated around 95% eggs in 2 days, but around 25% of the eggs were still viable after 10 days in aerobic digesters. At 37°C, anaerobic digestion inactivated more than 75% of the eggs after 10 days, but in the aerobic digester at 37°C, 10 days of treatment had no effect on viability. The oocysts and eggs added in chambers appeared to behave similarly to these pathogens added directly to the biosolids within the digesters.

Dgcr8 and Dicer are essential for sex chromosome integrity during meiosis in males

ABSTRACT Small RNAs play crucial roles in regulating gene expression during mammalian meiosis. To investigate the function of microRNAs (miRNAs) and small interfering RNAs (siRNAs) during meiosis in males, we generated germ-cell-specific conditional deletions of Dgcr8 and Dicer in mice. Analysis of spermatocytes from both conditional knockout lines revealed that there were frequent chromosomal fusions during meiosis, always involving one or both sex chromosomes. RNA sequencing indicates upregulation of Atm in spermatocytes from miRNA-deficient mice, and immunofluorescence imaging demonstrates an increased abundance of activated ATM kinase and mislocalization of phosphorylated MDC1, an ATM phosphorylation substrate. The Atm 3′UTR contains many potential microRNA target sites, and, notably, target sites for several miRNAs depleted in both conditional knockout mice were highly effective at promoting repression. RNF8, a telomere-associated protein whose localization is controlled by the MDC1–ATM kinase cascade, normally associates with the sex chromosomes during pachytene, but in both conditional knockouts redistributed to the autosomes. Taken together, these results suggest that Atm dysregulation in microRNA-deficient germ lines contributes to the redistribution of proteins involved in chromosomal stability from the sex chromosomes to the autosomes, resulting in sex chromosome fusions during meiotic prophase I. Highlighted Article: miRNA-deficient spermatocytes display frequent sex chromosome fusions and fail to progress through meiosis in a process that is probably mediated by dysregulation of Atm.

High-throughput discovery of post-transcriptional cis-regulatory elements

BackgroundPost-transcriptional gene regulation controls the amount of protein produced from an individual mRNA by altering rates of decay and translation. Many sequence elements that direct post-transcriptional regulation have been found; in mammals, most such elements are located within the 3′ untranslated regions (3′UTRs). Comparative genomic studies demonstrate that mammalian 3′UTRs contain extensive conserved sequence tracts, yet only a small fraction corresponds to recognized elements, implying that many additional novel elements exist. Despite a variety of computational, molecular, and biochemical approaches, identifying functional 3′UTRs elements remains difficult.ResultsWe created a high-throughput cell-based screen that enables identification of functional post-transcriptional 3′UTR regulatory elements. Our system exploits integrated single-copy reporters, which are expressed and processed as endogenous genes. We screened many thousands of short random sequences for their regulatory potential. Control sequences with known effects were captured effectively using our approach, establishing that our methodology was robust. We found hundreds of functional sequences, which we validated in traditional reporter assays, including verifying their regulatory impact in native sequence contexts. Although 3′UTRs are typically considered repressive, most of the functional elements were activating, including ones that were preferentially conserved. Additionally, we adapted our screening approach to examine the effect of elements on RNA abundance, revealing that most elements act by altering mRNA stability.ConclusionsWe developed and used a high-throughput approach to discover hundreds of post-transcriptional cis-regulatory elements. These results imply that most human 3′UTRs contain many previously unrecognized cis-regulatory elements, many of which are activating, and that the post-transcriptional fate of an mRNA is largely due to the actions of many individual cis-regulatory elements within its 3′UTR.

Estrogen Regulates JNK1 Genomic Localization to Control Gene Expression and Cell Growth in Breast Cancer Cells

Steroid hormone and MAPK signaling pathways functionally intersect, but the molecular mechanisms of this cross talk are unclear. Here, we demonstrate a functional convergence of the estrogen and c-Jun N-terminal kinase 1 (JNK1) signaling pathways at the genomic level in breast cancer cells. We find that JNK1 binds to many promoters across the genome. Although most of the JNK1-binding sites are constitutive, a subset is estrogen regulated (either induced on inhibited). At the estrogen-induced sites, estrogen receptor (ER)α is required for the binding of JNK1 by promoting its recruitment to estrogen response elements or other classes of DNA elements through a tethering mechanism, which in some cases involves activating protein-1. At estrogen-regulated promoters, JNK1 functions as a transcriptional coregulator of ERα in a manner that is dependent on its kinase activity. The convergence of ERα and JNK1 at target gene promoters regulates estrogen-dependent gene expression outcomes, as well as downstream estrogen-dependent cell growth responses. Analysis of existing gene expression profiles from breast cancer biopsies suggests a role for functional interplay between ERα and JNK1 in the progression and clinical outcome of breast cancers.

Do iodine water purification tablets provide an effective barrier against Cryptosporidium parvum

U.S. Army Iodine Water Purification Tablets were tested to determine their efficacy against Cryptosporidium parvum, a protozoan resistant to chemical disinfection. Purified oocysts in phosphate-buffered water were treated with varying concentrations of iodine or with iodine tablets as per U.S. Army protocol. Neonatal mouse pups were then each inoculated with 10,000 treated oocysts, and 1 week later scored as infected or uninfected. Using this methodology, iodine tablets were found to be inadequate against C. parvum because the Army doctrinal dose of 560 mg min/L, calculated as 16 mg of I2/L and 35 minutes of contact time, showed less than 1 log inactivation. A dose of 29 mg of I2/L at the same contact time was required to achieve a 2 log inactivation.