A transcriptional network required for bradyzoite development in Toxoplasma gondii is dispensable for recrudescent disease.

Identification of regulators of Toxoplasma gondii bradyzoite development and cyst formation is the most direct way to address the importance of parasite development in long-term persistence and reactivation of this parasite. Here we show that a T. gondii gene (named Regulator of Cystogenesis 1; ROCY1) is sufficient for T. gondii bradyzoite formation in vitro and in vivo. ROCY1 encodes an RNA binding protein that has a preference for 3' regulatory regions of hundreds of T. gondii transcripts, and its RNA-binding domains are required to mediate bradyzoite development. Female mice infected with ΔROCY1 parasites have reduced (>90%) cyst burden. While viable parasites can be cultivated from brain tissue for up to 6 months post-infection, chronic brain-resident ΔROCY1 parasites have reduced oral infectivity compared to wild type. Despite clear defects in bradyzoite formation and oral infectivity, ΔROCY1 parasites were able to reactivate with similar timing and magnitude as wild type parasites for up to 5 months post-infection. Therefore while ROCY1 is a critical regulator of the bradyzoite developmental pathway, it is not required for parasite reactivation, raising new questions about the persisting life stage responsible for causing recrudescent disease.

TMEM16A activation for the fast block to polyspermy in the African clawed frog does not require conventional activation of egg PLCs.

Fertilization of an egg by more than one sperm, a condition known as polyspermy, leads to gross chromosomal abnormalities and is embryonic lethal for most animals. Consequently, eggs have evolved multiple processes to stop supernumerary sperm from entering the nascent zygote. For external fertilizers, such as frogs and sea urchins, fertilization signals a depolarization of the egg membrane, which serves as the fast block to polyspermy. Sperm can bind to, but will not enter, depolarized eggs. In eggs from the African clawed frog, Xenopus laevis, the fast block depolarization is mediated by the Ca2+-activated Cl- channel TMEM16A. To do so, fertilization activates phospholipase C, which generates IP3 to signal a Ca2+ release from the ER. Currently, the signaling pathway by which fertilization activates PLC during the fast block remains unknown. Here, we sought to uncover this pathway by targeting the canonical activation of the PLC isoforms present in the X. laevis egg: PLCγ and PLCß. We observed no changes to the fast block in X. laevis eggs inseminated in inhibitors of tyrosine phosphorylation, used to stop activation of PLCγ, or inhibitors of Gαq/11 pathways, used to stop activation of PLCß. These data suggest that the PLC that signals the fast block depolarization in X. laevis is activated by a novel mechanism.

Large RNP granules in Caenorhabditis elegans oocytes have distinct phases of RNA-binding proteins.

The germ line provides an excellent in vivo system to study the regulation and function of RNP granules. Germ granules are conserved germ line-specific RNP granules that are positioned in the Caenorhabditis elegans adult gonad to function in RNA maintenance, regulation, and surveillance. In Caenorhabditis elegans, when oogenesis undergoes extended meiotic arrest, germ granule proteins and other RNA-binding proteins assemble into much larger RNP granules whose hypothesized function is to regulate RNA metabolism and maintain oocyte quality. To gain insight into the function of oocyte RNP granules, in this report, we characterize distinct phases for four protein components of RNP granules in arrested oocytes. We find that the RNA-binding protein PGL-1 is dynamic and has liquid-like properties, while the intrinsically disordered protein MEG-3 has gel-like properties, similar to the properties of the two proteins in small germ granules of embryos. We find that MEX-3 exhibits several gel-like properties but is more dynamic than MEG-3, while CGH-1 is dynamic but does not consistently exhibit liquid-like characteristics and may be an intermediate phase within RNP granules. These distinct phases of RNA-binding proteins correspond to, and may underlie, differential responses to stress. Interestingly, in oocyte RNP granules, MEG-3 is not required for the condensation of PGL-1 or other RNA-binding proteins, which differs from the role of MEG-3 in small, embryonic germ granules. Lastly, we show that the PUF-5 translational repressor appears to promote MEX-3 and MEG-3 condensation into large RNP granules; however, this role may be associated with regulation of oogenesis.

Imaging-associated stress causes divergent phase transitions of RNA-binding proteins in the Caenorhabditis elegans germ line.

One emerging paradigm of cellular organization of RNA and RNA-binding proteins is the formation of membraneless organelles. Examples of membraneless organelles include several types of ribonucleoprotein granules that form via phase separation. A variety of intracellular pH changes and posttranslational modifications, as well as extracellular stresses, can stimulate the condensation of proteins into granules. For example, the assembly of stress granules induced by oxidative stress, osmotic stress, and heat stress has been well characterized in a variety of somatic cell types. In the germ line, similar stress-induced condensation of proteins occurs; however, less is known about the role of phase separation during gamete production. Researchers who study phase transitions often make use of fluorescent reporters to study the dynamics of RNA-binding proteins during live cell imaging. In this report, we demonstrate that common conditions of live-imaging Caenorhabditis elegans can cause an inadvertent stress and trigger phase transitions of RNA-binding proteins. We show that this imaging-associated stress stimulates decondensation of multiple germ granule proteins and condensation of several P-body proteins. Proteins within larger ribonucleoprotein granules in meiotically arrested oocytes do not appear to be as sensitive to the stress as proteins in diakinesis oocytes of young hermaphrodites, with the exception of the germ granule protein PGL-1. Our results have important methodological implications for all researchers using live-cell imaging techniques. The data also suggest that the RNA-binding proteins within large ribonucleoprotein granules of arrested oocytes may have distinct phases, which we characterize in our companion article.

Evidence for a nuclear role for Drosophila Dlg as a regulator of the NURF complex.

Scribble (Scrib), Discs-large (Dlg), and Lethal giant larvae (Lgl) are basolateral regulators of epithelial polarity and tumor suppressors whose molecular mechanisms of action remain unclear. We used proximity biotinylation to identify proteins localized near Dlg in the Drosophila wing imaginal disc epithelium. In addition to expected membrane- and cytoskeleton-associated protein classes, nuclear proteins were prevalent in the resulting mass spectrometry dataset, including all four members of the nucleosome remodeling factor (NURF) chromatin remodeling complex. Subcellular fractionation demonstrated a nuclear pool of Dlg and proximity ligation confirmed its position near the NURF complex. Genetic analysis showed that NURF activity is also required for the overgrowth of dlg tumors, and this growth suppression correlated with a reduction in Hippo pathway gene expression. Together, these data suggest a nuclear role for Dlg in regulating chromatin and transcription through a more direct mechanism than previously thought.

Cancer Curriculum for Appalachian Kentucky Middle and High Schools.

BACKGROUND: Appalachian Kentucky faces the highest cancer incidence and mortality rates in the country due to poor health behaviors and lifestyle choices. These poor health behaviors are facilitated by a lack of cancer education. Youth represent a vulnerable population that could be greatly impacted by increased cancer education. Teachers have the power to facilitate this learning. PURPOSE: This study examined the need for cancer education curriculum in Appalachian Kentucky middle and high schools from the perspective of educators. METHODS: An online survey was conducted with science and health teachers (n=21) in Appalachian Kentucky, consisting of questions that investigated existing cancer education efforts, relevance of cancer education, and feasibility of such curriculum being delivered in the classroom. Content analysis was used to analyze teacher comments. A 3-part cancer education curriculum was developed that is culturally relevant and aligned with science and health education standards. RESULTS: All participating teachers agree that cancer education is important to students' lives. Teachers also agree that there is an inconsistent amount of cancer education within schools, and qualitative content analysis revealed that cancer education likely fits best in certain course subjects. Cancer education could feasibly be integrated into science and health classrooms, although the perception of needing to teach to the academic standards and having limited time to teach additional lessons outside of the standards are significant barriers. To combat this, a cancer curriculum that aligns with state and national science and health education standards was developed. IMPLICATIONS: Cancer education curriculum could play an important role in improving the cancer outlook in Appalachian Kentucky. Teachers have expressed a desire for increased cancer education in the classroom. By disseminating and implementing cancer curriculum in schools in the region and revising the curriculum -based on teacher and student feedback to better fit their needs, it has the potential to increase cancer literacy and improve related health behaviors and outcomes.

The Phenotypic Effects of Royal Jelly on Wild-Type D. melanogaster Are Strain-Specific.

The role for royal jelly (RJ) in promoting caste differentiation of honeybee larvae into queens rather than workers is well characterized. A recent study demonstrated that this poorly understood complex nutrition drives strikingly similar phenotypic effects in Drosophila melanogaster, such as increased body size and reduced developmental time, making possible the use of D. melanogaster as a model system for the genetic analysis of the cellular mechanisms underlying RJ and caste differentiation. We demonstrate here that RJ increases the body size of some wild-type strains of D. melanogaster but not others, and report significant delays in developmental time in all flies reared on RJ. These findings suggest that cryptic genetic variation may be a factor in the D. melanogaster response to RJ, and should be considered when attempting to elucidate response mechanisms to environmental changes in non-honeybee species.

Prickle isoforms control the direction of tissue polarity by microtubule independent and dependent mechanisms.

Planar cell polarity signaling directs the polarization of cells within the plane of many epithelia. While these tissues exhibit asymmetric localization of a set of core module proteins, in Drosophila, more than one mechanism links the direction of core module polarization to the tissue axes. One signaling system establishes a polarity bias in the parallel, apical microtubules upon which vesicles containing core proteins traffic. Swapping expression of the differentially expressed Prickle isoforms, Prickle and Spiny-legs, reverses the direction of core module polarization. Studies in the proximal wing and the anterior abdomen indicated that this results from their differential control of microtubule polarity. Prickle and Spiny-legs also control the direction of polarization in the distal wing (D-wing) and the posterior abdomen (P-abd). We report here that this occurs without affecting microtubule polarity in these tissues. The direction of polarity in the D-wing is therefore likely determined by a novel mechanism independent of microtubule polarity. In the P-abd, Prickle and Spiny-legs interpret at least two directional cues through a microtubule-polarity-independent mechanism.

Understanding psychosocial aspects of waterpipe smoking among college students.

OBJECTIVE: To gain an understanding of the psychosocial and environmental influences on waterpipe smoking among college students. Participants were 49 university students who were regular waterpipe smokers. METHODS: A series of in-depth, in-person, semi-structured qualitative interviews were conducted. RESULTS: It was a common belief that wa- ter in the waterpipe absorbed all the "impurities" in the tobacco and that it was safer and less addictive than smoking cigarettes. Main reasons for waterpipe smoking were social acceptance, peer influence, socializing with friends, perception of looking "cool" and physiological effects commonly referred to as "buzz." CONCLUSIONS: Interventions focusing on changing the perceptions of college students regarding health hazards associated with waterpipe smoking should be developed.

Prickle/spiny-legs isoforms control the polarity of the apical microtubule network in planar cell polarity.

Microtubules (MTs) are substrates upon which plus- and minus-end directed motors control the directional movement of cargos that are essential for generating cell polarity. Although centrosomal MTs are organized with plus-ends away from the MT organizing center, the regulation of non-centrosomal MT polarity is poorly understood. Increasing evidence supports the model that directional information for planar polarization is derived from the alignment of a parallel apical network of MTs and the directional MT-dependent trafficking of downstream signaling components. The Fat/Dachsous/Four-jointed (Ft/Ds/Fj) signaling system contributes to orienting those MTs. In addition to previously defined functions in promoting asymmetric subcellular localization of 'core' planar cell polarity (PCP) proteins, we find that alternative Prickle (Pk-Sple) protein isoforms control the polarity of this MT network. This function allows the isoforms of Pk-Sple to differentially determine the direction in which asymmetry is established and therefore, ultimately, the direction of tissue polarity. Oppositely oriented signals that are encoded by oppositely oriented Fj and Ds gradients produce the same polarity outcome in different tissues or compartments, and the tissue-specific activity of alternative Pk-Sple protein isoforms has been observed to rectify the interpretation of opposite upstream directional signals. The control of MT polarity, and thus the directionality of apical vesicle traffic, by Pk-Sple provides a mechanism for this rectification.

Cylindrospermopsis raciborskii dominates under very low and high nitrogen-to-phosphorus ratios.

In freshwater ecosystems, a variety of factors mediate phytoplankton community structure, including herbivore community structure, light availability, temperature, mixing, and absolute and relative nutrient concentrations (total nitrogen (TN), total phosphorus (TP)). Ecological stoichiometry examines how the nutrient content of organisms and their environment may mediate population-, community-, and ecosystem-level processes. The manipulation of N:P ratios is a widely regarded tool for managing phytoplankton species composition given that nitrogen-fixing cyanobacteria should dominate algal communities under relatively low N:P (<64:1, by atoms) given their ability to convert dissolved dinitrogen gas into organic nitrogen. However, due to the physiological expense of nitrogen fixation, diazotrophs should be outcompeted by non-nitrogen fixing phytoplankton under higher N:P when other environmental factors are similar. We tested this hypothesis in a field experiment using 2500-L limnocorrals installed in a eutrophic lake (ambient N:P ∼40:1 (by atoms); TN ∼1360 µgL(-1); TP ∼75 µgL(-1)). At the start of the experiment, we randomly assigned limnocorrals among the ambient (40:1) and low (7:1) or high (122:1) N:P treatments (n = 4 replicates/treatment), which were established by adding P or N at the start of the experiment, respectively. The phytoplankton community in the enclosures at the start of the experiment was diverse (i.e., 18 phytoplankton genera) and dominated by chlorophytes (including Coelastrum and Scenedesmus (30% and 13% of total biomass, respectively)) and cyanobacteria (including Anabaena and Cylindrospermopsis (23% and 17% of total biomass, respectively)). In contrast to predictions based on ecological stoichiometry, the phytoplankton community in all N:P treatments increased in abundance and was almost entirely composed of the nitrogen-fixing cyanobacterium, Cylindrospermopsis raciborskii, by the conclusion of the study. Moreover, concentrations of the cyanobacterial neurotoxin, saxitoxin, were enhanced under the two highest N:P conditions. The ability of C. raciborskii to dominate phytoplankton communities under such extreme N:P shows that short-term management of nutrient stoichiometry through fertilization is not likely to be effective for controlling blooms of this noxious cyanobacterium and may help to explain the rapid expansion of this invasive species to temperate latitudes.

A neuroprotective role for the DNA damage checkpoint in tauopathy.

ATM and p53, effectors of the DNA damage checkpoint, are generally considered pro-apoptotic in neurons. We show that DNA damage and checkpoint activation occurs in postmitotic neurons in animal models of tauopathy, neurodegenerative disorders that include Alzheimer's disease. Surprisingly, checkpoint attenuation potently increases neurodegeneration through aberrant cell cycle re-entry of postmitotic neurons. These data suggest an unexpected neuroprotective role for the DNA damage checkpoint in tauopathies.

Lysosomal dysfunction promotes cleavage and neurotoxicity of tau in vivo.

Expansion of the lysosomal system, including cathepsin D upregulation, is an early and prominent finding in Alzheimer's disease brain. Cell culture studies, however, have provided differing perspectives on the lysosomal connection to Alzheimer's disease, including both protective and detrimental influences. We sought to clarify and molecularly define the connection in vivo in a genetically tractable model organism. Cathepsin D is upregulated with age in a Drosophila model of Alzheimer's disease and related tauopathies. Genetic analysis reveals that cathepsin D plays a neuroprotective role because genetic ablation of cathepsin D markedly potentiates tau-induced neurotoxicity. Further, generation of a C-terminally truncated form of tau found in Alzheimer's disease patients is significantly increased in the absence of cathepsin D. We show that truncated tau has markedly increased neurotoxicity, while solubility of truncated tau is decreased. Importantly, the toxicity of truncated tau is not affected by removal of cathepsin D, providing genetic evidence that modulation of neurotoxicity by cathepsin D is mediated through C-terminal cleavage of tau. We demonstrate that removing cathepsin D in adult postmitotic neurons leads to aberrant lysosomal expansion and caspase activation in vivo, suggesting a mechanism for C-terminal truncation of tau. We also demonstrate that both cathepsin D knockout mice and cathepsin D-deficient sheep show abnormal C-terminal truncation of tau and accompanying caspase activation. Thus, caspase cleavage of tau may be a molecular mechanism through which lysosomal dysfunction and neurodegeneration are causally linked in Alzheimer's disease.

Two pathways of costimulation through CD28.

CD28 is recognized as the primary costimulatory molecule involved in the activation of naïve T cells. However, the biochemical signaling pathways that are activated by CD28 and how these pathways are integrated with TCR signaling are still not understood. We have recently shown that there are at least two independent activation pathways induced by CD28 costimulation. One is integrated with TCR signaling in the context of the immunological synapse and is mediated through transcriptional enhancement and the second is mediated through the induction of mRNA stability. Here, we review the immunological consequences and biochemical mechanisms associated with CD28 costimulation and discuss the major questions that need to be resolved to understand the molecular mechanisms that transduce CD28 costimulation.

Crystal structure of the heme-IsdC complex, the central conduit of the Isd iron/heme uptake system in Staphylococcus aureus.

Pathogens such as Staphylococcus aureus require iron to survive and have evolved specialized proteins to steal heme from their host. IsdC is the central conduit of the Isd (iron-regulated surface determinant) multicomponent heme uptake machinery; staphylococcal cell-surface proteins such as IsdA, IsdB, and IsdH are thought to funnel their molecular cargo to IsdC, which then mediates the transfer of the iron-containing nutrient to the membrane translocation system IsdDEF. The structure of the heme-IsdC complex reveals a novel heme site within an immunoglobulin-like domain and sheds light on its binding mechanism. The folding topology is reminiscent of the architecture of cytochrome f, cellobiose dehydrogenase, and ethylbenzene dehydrogenase; in these three proteins, the heme is bound in an equivalent position, but interestingly, IsdC features a distinct binding pocket with the ligand located next to the hydrophobic core of the beta-sandwich. The iron is coordinated with a tyrosine surrounded by several non-polar side chains that cluster into a tightly packed proximal side. On the other hand, the distal side is relatively exposed with a short helical peptide segment that acts as a lip clasping onto almost half of the porphyrin plane. This structural feature is argued to play a role in the mechanism of binding and release by switching to an open conformation and thus loosening the interactions holding the heme. The structure of the heme-IsdC complex provides a template for the understanding of other proteins, such as IsdA, IsdB, and IsdH, that contain the same heme-binding module as IsdC, known as the NEAT (near transporter) domain.

An induced fit conformational change underlies the binding mechanism of the heme transport proteobacteria-protein HemS.

Bacteria rely on their environment and/or host to acquire iron and have evolved specialized systems to sequester and transport heme. The heme uptake system HemRSTUV is common to proteobacteria, and a major challenge is to understand the molecular mechanism of heme binding and transfer between the protein molecules that underlie this heme transport relay process. In the Gram-negative pathogen Yersinia enterocolitica, the HemRSTUV system culminates with the cytoplasmic recipient HemS, which stores and delivers heme for cellular needs. HemS belongs to a family of proteins essential and unique to proteobacteria. Here we report on the binding mechanism of HemS based on structural data from its apo- and ligand-loaded forms. This heme carrier protein associates with its cargo through a novel, partly preformed binding pocket, formed between a large beta-sheet dome and a three-helix subdomain. In addition to a histidine interacting with the iron, the complex is stabilized by a distal non-coordinating arginine that packs along the porphyrin plane and extensive electrostatic contacts that firmly anchor the heme propionate groups within the protein. Comparison of apo- and ligand-bound HemS crystal structures reveals striking conformational changes that underlie a "heme-induced fit" binding mechanism. Local shifts in amino acid positions combine with global, rigid body-like domain movements, and together, these bring about a switch from an open, apo-form to a closed, bound state. This is the first report in which both liganded and unliganded forms of a heme transport protein are described, thus providing penetrating insights into its mechanism of heme binding and release.

Conformational mobility in the active site of a heme peroxidase.

Conformational mobility of the distal histidine residue has been implicated for several different heme peroxidase enzymes, but unambiguous structural evidence is not available. In this work, we present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A). In this variant, His-42 binds "on" to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing "off" the iron during catalysis. This conformational flexibility between the on and off forms is fully reversible and is used as a means to overcome the inherently unreactive nature of the on form toward peroxide, so that essentially complete catalytic activity is maintained. Contrary to the widely adopted view of heme enzyme catalysis, these data indicate that strong coordination of the distal histidine to the heme iron does not automatically undermine catalytic activity. The data add a new dimension to our wider appreciation of structure/activity correlations in other heme enzymes.

A close look at why one social norms campaign did not reduce student drinking.

The authors examined 3 possible explanations for the failure of a social norms campaign at a large public university. They administered an anonymous survey to 2 random samples of undergraduate classes: a baseline assessment of 616 students before the campaign's implementation and a follow-up survey of 723 students 4 academic years later. At follow-up, 66.5% of the students were aware of the campaign, yet the survey revealed no reduction in perceived drinking norms or alcohol use in this group. An analysis of the postcampaign sample revealed that (1) a majority of the students did not find the statistics used in the campaign messages credible, (2) higher levels of alcohol use predicted lower levels of perceived campaign credibility, and (3) only 38.5% of the students understood the campaign's intended purpose. If they are to influence personally relevant drinking norms, these campaigns must undergo further development to enhance message credibility and participants' understanding.

Analysis of a database of materials for HIV prevention program evaluation.

Program evaluation technology transfer is the transfer of information on program evaluation from research to practitioners. There have been anecdotal reports of a lack of technology transfer materials related to HIV prevention program evaluation, especially materials usable by persons without extensive training in evaluation. The Centers for Disease Control and Prevention reviewed available program evaluation materials relevant to HIV prevention and developed a database of those materials. Materials were classified according to appropriate audience, level of evaluation expertise required, steps in the evaluation process addressed, and other criteria. The database was queried to determine the number of materials available for various combinations of search criteria. These queries revealed that for certain audiences and steps in the evaluation process there are few materials, especially usable by individuals without evaluation experience. The conclusion is that for certain areas of program evaluation, and for certain audiences, more evaluation technology transfer materials are needed.