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The chaperone Hsp90 is a potential target for the development of drugs against fungal pathogens. Here the authors determine the structure of the Hsp90 nucleotide-binding domain from Candida albicans, which they use to design an inhibitor and demonstrate its selectivity for the fungal enzyme, both biochemically and in cells.

Disruption of protein quality control can be detrimental, having toxic effects on single cell organisms and contributing to neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s in humans. Here, we examined the effects of polyglutamine (polyQ) aggregation in a major fungal pathogen of humans, Candida albicans, with the goal of identifying new approaches to disable this fungus. However, we discovered that expression of polyQ stretches up to 230Q had no effect on C. albicans ability to grow and withstand proteotoxic stress. Bioinformatics analysis demonstrates that C. albicans has a similarly glutamine-rich proteome to the unicellular fungus Saccharomyces cerevisiae, which exhibits polyQ toxicity with as few as 72Q. Surprisingly, global transcriptional profiles indicated no significant change upon induction of up to 230Q. Proteomic analysis highlighted two key interactors of 230Q, Sis1 and Sgt2; however, loss of either protein had no additional effect on C. albicans toxicity. Our data suggest that C. albicans has evolved powerful mechanisms to overcome the toxicity associated with aggregation-prone proteins, providing a unique model for studying polyQ-associated diseases.

Species of the genus Eucalyptus (common name eucalyptus) are widely planted all across Ethiopia—including on large areas of land previously allocated to food production. In recent decades eucalyptus has also increasingly been planted on lands around and within “church forests,” sacred groves of old-aged Afromontane trees surrounding Ethiopian Orthodox Tewahido churches. These revered holy sites have long been recognized for their cultural values and also for their ecosystem services—including their potential to support species conservation and restoration, as church forests are some of the only remaining sanctuaries for many of Ethiopia’s indigenous and endemic plant and animal populations. Ethiopian Orthodox church communities have a long history of planting and nurturing indigenous tree seedlings to sustain church forest groves. However, due to the fast-growing nature of eucalyptuscombined with its widely recognized socio-economic benefits (as fuelwood, charcoal, construction wood, etc.), this introduced species has been widely plantedaround church forests—in some cases even replacing native tree species within church forests themselves. In many developing country contexts the introduction of exotic eucalyptus has been shown to have ecological impacts ranging from soil nutrient depletion, to lowering water tables, to allelopathic effects. In this study, we collected soil samples from indigenous forest fragments (church forests), adjacent eucalyptus plantations, and surrounding agricultural land to examine how eucalyptus plantations in Ethiopian Orthodox church communitiesmight impact soil quality relative to alternative land uses. Soil properties, including organic matter, pH, total nitrogen, and total phosphorus were measured in samples across 20 church forest sites in South Gondar, East Gojjam, West Gojjam, Awi, and Bahir Dar Liyu zones in the Amhara Region of the northern Ethiopian Highlands. Findings indicate that although soil in eucalyptus stands is more acidic and has lower organic matter and nutrient levels than nearby church forests, eucalyptusplantations also exhibit consistently higher organic matter and nutrient levels when compared to adjacent agricultural land. These findings suggest that eucalyptus planting could potentially benefit soil fertility on land that has been degraded by subsistence agriculture.

Invasive fungal infections caused by the pathogen Candida albicans have transitioned from a rare curiosity to a major cause of human mortality. This is in part due to the emergence of resistance to the limited number of antifungals available to treat fungal infections. Azoles function by targeting the biosynthesis of ergosterol, a key component of the fungal cell membrane. Loss-of-function mutations in the ergosterol biosynthetic gene ERG3 mitigate azole toxicity and enable resistance that depends upon fungal stress responses. Here, we performed a genome-wide synthetic genetic array screen in Saccharomyces cerevisiae to map ERG3 genetic interactors and uncover novel circuitry important for azole resistance. We identified nine genes that enabled erg3-mediated azole resistance in the model yeast and found that only two of these genes had a conserved impact on resistance in C. albicans. Further, we screened a C. albicans homozygous deletion mutant library and identified 13 genes for which deletion enhances azole susceptibility. Two of the genes, RGD1 and PEP8, were also important for azole resistance acquired by diverse mechanisms. We discovered that loss of function of retrograde transport protein Pep8 overwhelms the functional capacity of the stress response regulator calcineurin, thereby abrogating azole resistance. To identify the mechanism through which the GTPase activator protein Rgd1 enables azole resistance, we selected for mutations that restore resistance in strains lacking Rgd1. Whole genome sequencing uncovered parallel adaptive mechanisms involving amplification of both chromosome 7 and a large segment of chromosome 3. Overexpression of a transporter gene on the right portion of chromosome 3, NPR2, was sufficient to enable azole resistance in the absence of Rgd1. Thus, we establish a novel mechanism of adaptation to drug-induced stress, define genetic circuitry underpinning azole resistance, and illustrate divergence in resistance circuitry over evolutionary time.

The microbiome shapes diverse facets of human biology and disease, with the importance of fungi only beginning to be appreciated. Microbial communities infiltrate diverse anatomical sites as with the respiratory tract of healthy humans and those with diseases such as cystic fibrosis, where chronic colonization and infection lead to clinical decline. Although fungi are frequently recovered from cystic fibrosis patient sputum samples and have been associated with deterioration of lung function, understanding of species and population dynamics remains in its infancy. Here, we coupled high-throughput sequencing of the ribosomal RNA internal transcribed spacer 1 (ITS1) with phenotypic and genotypic analyses of fungi from 89 sputum samples from 28 cystic fibrosis patients. Fungal communities defined by sequencing were concordant with those defined by culture-based analyses of 1,603 isolates from the same samples. Different patients harbored distinct fungal communities. There were detectable trends, however, including colonization with Candida and Aspergillus species, which was not perturbed by clinical exacerbation or treatment. We identified considerable inter- and intra-species phenotypic variation in traits important for host adaptation, including antifungal drug resistance and morphogenesis. While variation in drug resistance was largely between species, striking variation in morphogenesis emerged within Candida species. Filamentation was uncoupled from inducing cues in 28 Candida isolates recovered from six patients. The filamentous isolates were resistant to the filamentation-repressive effects of Pseudomonas aeruginosa, implicating inter-kingdom interactions as the selective force. Genome sequencing revealed that all but one of the filamentous isolates harbored mutations in the transcriptional repressor NRG1; such mutations were necessary and sufficient for the filamentous phenotype. Six independent nrg1 mutations arose in Candida isolates from different patients, providing a poignant example of parallel evolution. Together, this combined clinical-genomic approach provides a high-resolution portrait of the fungal microbiome of cystic fibrosis patient lungs and identifies a genetic basis of pathogen adaptation.

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.

Identical genes in the same cellular environment are sometimes expressed differently. In some cases, including the immunoglobulin heavy chain (IgH) locus, this type of differential gene expression has been related to the absence of a transcriptional enhancer. To gain additional information on the role of the IgH enhancer, we examined expression driven by enhancers that were merely weakened, rather than fully deleted, using both mutations and insulators to impair enhancer activity. For this purpose we used a LoxP/Cre system to place a reporter gene at the same genomic site of a stable cell line. Whereas expression of the reporter gene was uniformly high in the presence of the normal, uninsulated enhancer and undetectable in its absence, weakened enhancers yielded variegated expression of the reporter gene; i.e., the average level of expression of the same gene differed in different clones, and expression varied significantly among cells within individual clones. These results indicate that the weakened enhancer allows the reporter gene to exist in at least two states. Subtle aspects of the variegation suggest that the IgH enhancer decreases the average duration (half-life) of the silent state. This analysis has also tested the conventional wisdom that enhancer activity is independent of distance and orientation. Thus, our analysis of mutant (truncated) forms of the IgH enhancer revealed that the 250 bp core enhancer was active in its normal position, approximately 1.4 kb 3' of the promoter, but inactive approximately 6 kb 3', indicating that the activity of the core enhancer was distance-dependent. A longer segment--the core enhancer plus approximately 1 kb of 3' flanking material, including the 3' matrix attachment region--was active, and the activity of this longer segment was orientation-dependent. Our data suggest that this 3' flank includes binding sites for at least two activators.

Web-based digital video (DV) applications such as Animoto, Magistro, and Videolicious allow users without technical experience to easily create quality video by matching up appropriate images with text and sound. DV's multimedia capabilities encourage students to become more proficient in new literacies as well as new technologies. This piece explores the potential classroom effectiveness of those digital film and animation creation services for facilitating reading and writing instruction, specifically analyzing poetry, building interdisciplinary connections, and teaching writing strategies.

Many recent publications describe writing as the neglected "r"; however, there is very little data on what writing instruction looks like in schools, especially in grades 4-6. The purpose of this article is to describe large urban school district literacy leaders' views on the state of writing instruction within their districts--and their projections for the future of writing instruction. Educators at all levels must re-think how they approach the teaching of writing, re-envisioning how to transform teaching practices to impact our students and their world for the next decade to come. (Contains 1 figure and 1 table.)

This study is a research collaborative conducted with multiple sites to examine the programmatic features within six literacy teacher education programs that have received the Certificate of Distinction of the International Reading Association (IRA). The objectives were to identify the features that were most highly ranked by internal and external experts and to delineate specific examples of how the features were actualized. A classical Delphi method was applied, and participants included leading literacy faculty members at each of the six institutions, the internal experts (n = 18), and members of program review teams identified by IRA, the external experts (n = 3). Analyses of results revealed that 14 programmatic features ranked higher in value than others at a statistically significant level. The study found that the internal and external literacy teacher education experts agreed on the most highly valued programmatic factors. These include the importance of relevant field experiences, the development of teacher candidates' abilities to teach and assess children through a wide variety of instructional strategies and assessment instruments, and ways to integrate literacy and language strategies throughout the curriculum. (Contains 5 tables.)

The purpose of this study was to analyze the effects of the most widely used literacy instructional approaches on the reading comprehension of Grade 2-6 students. Participants (N = 660) were enrolled in 4 districts in the United States; 53% were male (n = 348) and 47% were female (n = 312); 51% were Caucasian (n = 338), 23% were African American (n = 149), 21% were Hispanic (n = 138), and 5% represented other ethnic backgrounds (n = 35). Sixty-two percent came from low to low-middle socioeconomic status schools, and 38% came from middle to high socioeconomic status schools. The study was a quantified experimental versus controlled group comparison. Analyses of variance were used to determine the differences between literacy scores. Two-level hierarchical linear modeling analyses were used to examine the effects of school variables on academic achievement. The highest comprehension scores for all populations occurred through three approaches. When struggling readers received 20 min of instruction with 1 of these 3 approaches, their literacy growth was equal to or greater than that of their peers. Implications are that treatments using classroom books produced significantly higher comprehension scores than workbook practice or extending basal treatments. (Contains 7 tables.)

The purpose of this article is to document the legacy and continuing influence of Michael Pressley's work in the field of reading research and instruction. Descriptions are provided to demonstrate how he translated his data from the cognitive sciences into highly effective pedagogy. A second component of this article discusses how Michael Pressley, throughout his career, continuously collected converging evidence through multiple research methodologies to examine single, complex educational issues. The article concludes by presenting research agendas that Mike was pursuing near the end of his career and how these initiatives provide new directions for reading research, policy, and classroom practices today. His contemporary research focused on how to improve multiple comprehension strategy instruction so it could become an even greater, central component in K-12 curricula; how to expand the application of basic research in cognitive strategy instruction to classroom practices; and, how to assess reading comprehension more effectively. (Contains 2 tables.)

This article discusses a study to determine whether primary grade students can learn comprehension processes via hand motions to portray these mental processes. Comprehension Process Motions (CPMs) were designed to provide students with a way to make abstract comprehension processes more consciously accessible and also to give teachers a way to observe what students have (or have not) comprehended. Results were based on a 12-week study of 257 experimental and 256 control students in grades K-5. Comprehension scores on standardized, criterion-referenced, and norm-referenced tests were significantly higher for experimental students than for those who were not exposed to CPM instruction. These data provide evidence that even the youngest readers are capable of internalizing comprehension processes and knowing how to elicit them at specific points in a text when they are needed. (Contains 3 figures.)

The purpose of the study described in this article was to identify the qualities of teaching expertise that correlate with highly effective literacy instruction in grades 6 through 12. Findings draw on survey responses of secondary literacy supervisors at the local school district or state department of education level in every U.S. state. From these responses, 397 characteristics of teaching expertise were analyzed and collapsed into 38 categorical behaviors. After interrater reliabilities were computed, researchers returned to analyze and consolidate the data. This process yielded the following eight domains of secondary literacy teaching expertise: (1) Approaches to teaching (pedagogy); (2) Addressing diverse needs; (3) Personal characteristics; (4) Knowledge base; (5) Approaches to teaching (activities); (6) Professional development; (7) Appropriate relationships with students; and (8) Classroom management. Applications of these data for research, policy, and practice are discussed. (Contains 1 figure and 1 table.)

The purpose of this article is to provide teachers with examples of how to perform effective think-alouds. Twelve think-alouds are described. A rationale for their use in kindergarten through grade 5 is also included. In addition, this article was designed to provide teachers with lessons and activities to assist students in developing the ability to perform think-alouds independently, without teacher prompting. The last section of the article provides teachers with methods of assessing think-alouds.

Determines elementary teachers' knowledge of: current children's literature; children's books in six literary genres; and activities to promote students' recreational reading. Replicates a 1981 study to determine the level of knowledge possessed by today's teachers concerning children's literature and methods of increasing students' reading for pleasure. Attests to the lack of attention recreational reading receives in schools. (SG)