A Highly Stable Blood Meal Alternative for Rearing \u3cem\u3eAedes\u3c/em\u3e and \u3cem\u3eAnopheles\u3c/em\u3e Mosquitoes

We investigated alternatives to whole blood for blood feeding of mosquitoes with a focus on improved stability and compatibility with mass rearing programs. In contrast to whole blood, an artificial blood diet of ATP-supplemented plasma was effective in maintaining mosquito populations and was compatible with storage for extended periods refrigerated, frozen, and as a lyophilized powder. The plasma ATP diet supported rearing of both Anopheles and Aedes mosquitoes. It was also effective in rearing Wolbachia-infected Aedes mosquitoes, suggesting compatibility with vector control efforts

Estimating female malaria mosquito age by quantifying Y-linked genes in stored male spermatozoa

Vector control strategies are among the most effective measures to combat mosquito-borne diseases, such as malaria. These strategies work by altering the mosquito age structure through increased mortality of the older female mosquitoes that transmit pathogens. However, methods to monitor changes to mosquito age structure are currently inadequate for programmatic implementation. Female mosquitoes generally mate a single time soon after emergence and draw down spermatozoa reserves with each oviposition cycle. Here, we demonstrate that measuring spermatozoa quantity in female Anopheles mosquitoes is an effective approach to assess mosquito age. Using multiplexed qPCR targeted at male spermatozoa, we show that Y-linked genes in female mosquitoes are exclusively found in the spermatheca, the organ that houses spermatozoa, and the quantity of these gene sequences significantly declines with age. The method can accurately identify mosquitoes more than 10 days old and thus old enough to potentially transmit pathogens harbored in the salivary glands during blood feeding. Furthermore, mosquito populations that differ by 10% in daily survivorship have a high likelihood of being distinguished using modest sample sizes, making this approach scalable for assessing the efficacy of vector intervention control programs

Identification of Widespread Adenosine Nucleotide Binding in Mycobacterium tuberculosis

SummaryComputational prediction of protein function is frequently error-prone and incomplete. In Mycobacterium tuberculosis (Mtb), ∼25% of all genes have no predicted function and are annotated as hypothetical proteins, severely limiting our understanding of Mtb pathogenicity. Here, we utilize a high-throughput quantitative activity-based protein profiling (ABPP) platform to probe, annotate, and validate ATP-binding proteins in Mtb. We experimentally validate prior in silico predictions of >240 proteins and identify 72 hypothetical proteins as ATP binders. ATP interacts with proteins with diverse and unrelated sequences, providing an expanded view of adenosine nucleotide binding in Mtb. Several hypothetical ATP binders are essential or taxonomically limited, suggesting specialized functions in mycobacterial physiology and pathogenicity

Tracking a Medically Important Spider: Climate Change, Ecological Niche Modeling, and the Brown Recluse (Loxosceles reclusa)

Most spiders use venom to paralyze their prey and are commonly feared for their potential to cause injury to humans. In North America, one species in particular, Loxosceles reclusa (brown recluse spider, Sicariidae), causes the majority of necrotic wounds induced by the Araneae. However, its distributional limitations are poorly understood and, as a result, medical professionals routinely misdiagnose brown recluse bites outside endemic areas, confusing putative spider bites for other serious conditions. To address the issue of brown recluse distribution, we employ ecological niche modeling to investigate the present and future distributional potential of this species. We delineate range boundaries and demonstrate that under future climate change scenarios, the spider's distribution may expand northward, invading previously unaffected regions of the USA. At present, the spider's range is centered in the USA, from Kansas east to Kentucky and from southern Iowa south to Louisiana. Newly influenced areas may include parts of Nebraska, Minnesota, Wisconsin, Michigan, South Dakota, Ohio, and Pennsylvania. These results illustrate a potential negative consequence of climate change on humans and will aid medical professionals in proper bite identification/treatment, potentially reducing bite misdiagnoses

Experimental Characterization of <italic>Mycobacterium tuberculosis</italic> Adenosine Nucleotide Binding and Ser/Thr/Tyr Phosphosignaling

Thesis (Ph.D.)--University of Washington, 2014Tuberculosis (TB) is responsible for 1.3 million deaths each year and is a global health burden of overwhelming proportions. The success of Mycobacterium tuberculosis (Mtb), the causative agent of TB, lies in part with the pathogen's ability to persist under growth-limiting stress to cause disease months to years later. With drug resistant strains on the rise, the need for novel therapeutics is urgent. A fundamental problem for understanding Mtb metabolism and pathogenesis and for the development of novel therapeutics is the lack of reliable annotation for many Mtb proteins. In addition, our lack of understanding the molecular basis of persistence further hampers the development of effective therapies. Functional characterization of the proteome and the molecular networks that drive Mtb persistence is critical to inform novel therapeutic approaches. This dissertation will detail efforts to: 1. Annotate the Mtb ATP binding proteins (ATPome), 2. Characterize the role of serine/threonine protein kinases in growth-limiting adaptions that underlie persistence, and 3. Identify protein Tyr phosphorylation, a new Mtb phosphosignaling mechanism. The majority of functional annotation is based on in silico predictions; however, these inferences are often unreliable. In addition, ~25% of the Mtb protein-coding genes have no predicted function and are annotated as hypothetical proteins. To address the lack of functional annotation, we used a high-throughput quantitative activity-based protein profiling (ABPP) platform to probe, annotate, and validate the large family of ATP-binding proteins. We experimentally confirmed ~240 in silico predictions and identified 72 hypotheticals as ATP-binding proteins, a number of which are only found in mycobacteria and essential for in vitro growth or infection. These data help further define protein function in the Mtb proteome and show the diversity among ATP-binding proteins, which may be harnessed for the development of therapeutics. Mtb pathogenesis is characterized by adaptation to growth-limiting stress. The molecular mechanisms underlying adaptation are largely unknown; however, phosphosignaling has been suggested as a key mediator of stress response. To test this idea, we investigated a subset of ATP-binding proteins, the 11 eukaryotic-like serine/threonine protein kinases, to determine their role in mediating Mtb persistence under hypoxia, a growth-limiting stress induced by the host. Mtb adapts to hypoxia by entering reversible bacteriostasis until ample oxygen levels return and growth resumes. We found an essential serine/threonine protein kinase, PknB, to be a critical mediator of oxygen-dependent replication. Inhibition of PknB activity in aerated culture, and overexpression of PknB in hypoxia compromised Mtb viability. These data point to a model in which Mtb uses PknB to drive cell growth and division, but depletes PknB under hypoxic conditions to promote bacteriostasis and persistence. With serine/threonine protein kinases established as drug targets, these findings suggest PknB as a target in all stages of the Mtb life cycle, including in non-replicating bacilli thought to underlie persistence and latent infection. Bacteria drive cellular responses and physiological processes with a variety of ATP-dependent signaling molecules, including serine/threonine, histidine/aspartate, and tyrosine kinases. Mtb lacks canonical tyrosine kinases and no tyrosine phosphorylation was detected in previous phosphoproteomic studies, leading to the current notion that Mtb does not support protein Tyr phosphorylation. However, protein Tyr phosphorylation is widespread and found in most bacterial phyla, leading us to reevaluate the presence of Tyr phosphorylation in Mtb. We show that several serine/threonine protein kinases, including PknB, are in fact dual specificity kinases that are themselves regulated in part by tyrosine phosphorylation, and that Mtb supports extensive tyrosine phosphorylation in vivo. These data identify a previously unrecognized arm of Mtb phosphosignaling and may present new ways to target Mtb

Exercise and/or Genistein Do Not Revert 24-Week High-Fat, High-Sugar Diet-Induced Gut Microbiota Diversity Changes in Male C57BL/6J Adult Mice

The gut microbiota (GM) has been hypothesized to be a potential mediator in the health benefits of exercise and diet. The current literature is focused on the prevention effects of exercise and diet and could benefit from exploring whether these treatments alone or combined can treat obesity via the gut microbiome. This study aimed to explore the effects of genistein, exercise, and their synergistic effect to revert diet-induced obesity and gut microbiota changes. A total of 57 male adult C57BL/6 mice were randomized to 24 weeks of unpurified diet (chow) or a high-fat, high-sugar diet (HFD; 60% fat total energy). After the first 12 weeks, animals on the HFD were randomized into: HFD + chow, HFD, HFD + exercise (HFD + Exe), HFD + genistein (HFD + Gen), and HFD + Exe + Gen. We compared the body weight change between groups after 24 weeks. GM (&alpha;-diversity and &szlig;-diversity) was profiled after sequencing the 16S rRNA gene by Illumina MiSeq. HFD + Exe + Gen significantly (p &lt; 0.05) decreased weight gain relative to the HFD with only HFD + chow reverting the body weight change to that of chow. All diets including HFD reduced the GM richness (observed amplicon sequence variants) relative to chow with the HFD + Gen and HFD + Exe resulting in significantly lower phylogenetic diversity compared to the HFD. Data did not support an additive benefit to the GM for HFD + Gen + Exe. HFD + Exe + Gen showed a greater capacity to revert diet-induced obesity in adult male mice, but it was not as effective as switching from HFD to chow. Lifestyle treatment of HFD-induced obesity including exercise and genistein resulted in a reduction in weight gain and GM richness, but switching from HFD to chow had the greatest potential to revert these characteristics toward that of lean controls

Exercise and/or Genistein Treatment Impact Gut Microbiota and Inflammation after 12 Weeks on a High-Fat, High-Sugar Diet in C57BL/6 Mice

Genistein (Gen) and exercise (Exe) have been postulated as potential strategies to ameliorate obesity, inflammation, and gut microbiota (GM) with promising results. However, the impact of the combination of both Exe and Gen is yet to be investigated. We aimed to analyze the impacts of Exe, Gen, and their combined effects on GM and inflammation in mice after a 12-week high-fat, high-sugar diet (HFD). Eighty-three C57BL/6 mice were randomized to control, HFD, HFD + Exe, HFD + Gen, or HFD + Exe + Gen. The V4 region of the 16S rRNA gene was analyzed with Illumina MiSeq. Serum samples were used to analyze interleukin (Il)-6 and Tumor Necrosis Factor alpha (TNF-alpha). The HFD + Exe and HFD + Exe + Gen treatments resulted in significantly greater microbial richness compared to HFD. All the treatments had a significantly different impact on the GM community structure. Ruminococcus was significantly more abundant after the HFD + Exe + Gen treatment when compared to all the other HFD groups. Exe + Gen resulted in serum Il-6 concentrations similar to that of controls. TNF-alpha concentrations did not differ by treatment. Overall, Exe had a positive impact on microbial richness, and Ruminococcus might be the driving bacteria for the GM structure differences. Exe + Gen may be an effective treatment for preventing HFD-induced inflammation

A highly stable blood meal alternative for rearing Aedes and Anopheles mosquitoes

We investigated alternatives to whole blood for blood feeding of mosquitoes with a focus on improved stability and compatibility with mass rearing programs. In contrast to whole blood, an artificial blood diet of ATP-supplemented plasma was effective in maintaining mosquito populations and was compatible with storage for extended periods refrigerated, frozen, and as a lyophilized powder. The plasma ATP diet supported rearing of both Anopheles and Aedes mosquitoes. It was also effective in rearing Wolbachia-infected Aedes mosquitoes, suggesting compatibility with vector control efforts

<i>Mycobacterium tuberculosis</i> Ser/Thr Protein Kinase B Mediates an Oxygen-Dependent Replication Switch

<div><p>The majority of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) infections are clinically latent, characterized by drug tolerance and little or no bacterial replication. Low oxygen tension is a major host factor inducing bacteriostasis, but the molecular mechanisms driving oxygen-dependent replication are poorly understood. Here, we tested the role of serine/threonine phosphorylation in the <i>Mtb</i> response to altered oxygen status, using an <i>in vitro</i> model of latency (hypoxia) and reactivation (reaeration). Broad kinase inhibition compromised survival of <i>Mtb</i> in reaeration. Activity-based protein profiling and genetic mutation identified PknB as the kinase critical for surviving hypoxia. <i>Mtb</i> replication was highly sensitive to changes in PknB levels in aerated culture, and even more so in hypoxia. A mutant overexpressing PknB specifically in hypoxia showed a 10-fold loss in viability and gross morphological defects in low oxygen conditions. In contrast, chemically reducing PknB activity during hypoxia specifically compromised resumption of growth during reaeration. These data support a model in which PknB activity is reduced to achieve bacteriostasis, and elevated when replication resumes. Together, these data show that phosphosignaling controls replicative transitions associated with latency and reactivation, that PknB is a major regulator of these transitions, and that PknB could provide a highly vulnerable therapeutic target at every step of the <i>Mtb</i> life cycle—active disease, latency, and reactivation.</p></div

Effects of PknB dysregulation in hypoxia and reaeration.

<p>(A) <i>Mtb</i> growth (dashed line) is regulated by PknB (solid line) in response to oxygen tension. Under low oxygen conditions in hypoxia, PknB activity is decreased to facilitate growth arrest, but returns upon reaeration to support regrowth. PknB dysregulation by (B) overexpression or (C) inhibition interferes with survival at different stages. (D) <i>Mtb</i> growth is most vulnerable to PknB targeting in hypoxia and reaeration. Elevated PknB levels in hypoxia and reduced levels in reaeration are most detrimental.</p