Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs.

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for future pandemics.

Protocol for a randomized controlled feasibility study of a coordinated parent/child weight loss intervention: Dyad Plus.

The prevalence of in youth with overweight and obesity is a global health concern, necessitating clinical interventions to treat obesity effectively through lifestyle modification. Interventions in adolescents have demonstrated improvements in healthy eating and physical activity with only modest weight loss outcomes. Consequently, there is growing interest in developing strategies to enhance the effectiveness of clinical interventions in adolescents. Targeting the family system can be an effective approach, but existing studies have failed to examine the impact of co-enrolling both the adolescent and adult in individually tailored weight loss programs and coordinating the adolescent/adult weight loss efforts. PURPOSE: This paper reports on the design and conceptual framework of the Dyad Plus study, which utilizes two weight loss clinics of the Wake Forest Baptist Medical Center: Brenner Families in training (Brenner FIT®; adolescents) and By Design (adults). Dyad Plus is a coordinated program designed to facilitate self-monitoring, positive communication, joint problem solving, and social support to increase physical activity, healthy eating, and weight loss relative to Brenner FIT alone. METHODS: A total of 45 parent/adolescent dyads are randomized to one of three conditions (n = 15 for each): Brenner FIT only, Dyad (adolescent and parent both enroll simultaneously in the age appropriate program), and Dyad Plus (both parent and adolescent enroll simultaneously, but with a coordinated component for adolescent and caregiver). This study aims to develop and pilot the coordinated intervention, establish feasibility of the intervention, and determine costs associated with implementation. RESULTS: The results of the study are expected in winter of 2021. CONCLUSION: If proven feasible and acceptable, Dyad Plus will be tested for effectiveness in a large-scale implementation-effectiveness clinical trial.

Styles of Adaptation: The Impact of Frequency and Valence of Adaptation on Preventing Substance Use.

Purpose: To be effective, evidence-based programs should be delivered as prescribed. This suggests that adaptations that deviate from intervention goals may limit a program's effectiveness. This study examines the impact that number and quality of adaptations have on substance use outcomes. Design: We examined 306 video recordings of teachers delivering 'All Stars', a middle school drug prevention program. Multiple observers coded each recording, noting the number and type of adaptation each teacher made. Each adaptation was given a valence rating. Adaptations that were deleterious to program goals received negative valence ratings; positive ratings were given for adaptations that were likely to facilitate achievement of program goals; neutral ratings were given to adaptations that were expected to have neither a positive nor negative impact on program goals. Findings: All teachers made adaptations. Teachers were consistent across time in the types of adaptations they made, suggesting each teacher has a personalized style of adapting. Those who made few adaptations, and whose average adaptation was rated as being positive had a higher percentage of students who remained non-drug users. In contrast, teachers who made many adaptations, whether their average valence rating was positive, neutral or negative, failed to have as many students remain non-drug users. Measures of fidelity, including quality of delivery and teacher understanding were related to valence of adaptations, with better performance related to making positive adaptations. Practical Implications: Through training and supervision, teachers should be guided and encouraged to follow programs directions, making few adaptations and ensuring that adaptations that are made advance the goals of intervention. Programs should define acceptable and unacceptable ways they may be adapted. Value: This study provides significant evidence about the challenges that face disseminated evidence-based programs.

Analysis of Cryptococcus neoformans sexual development reveals rewiring of the pheromone-response network by a change in transcription factor identity.

The fundamental mechanisms that control eukaryotic development include extensive regulation at the level of transcription. Gene regulatory networks, composed of transcription factors, their binding sites in DNA, and their target genes, are responsible for executing transcriptional programs. While divergence of these control networks drives species-specific gene expression that contributes to biological diversity, little is known about the mechanisms by which these networks evolve. To investigate how network evolution has occurred in fungi, we used a combination of microarray expression profiling, cis-element identification, and transcription-factor characterization during sexual development of the human fungal pathogen Cryptococcus neoformans. We first defined the major gene expression changes that occur over time throughout sexual development. Through subsequent bioinformatic and molecular genetic analyses, we identified and functionally characterized the C. neoformans pheromone-response element (PRE). We then discovered that transcriptional activation via the PRE requires direct binding of the high-mobility transcription factor Mat2, which we conclude functions as the elusive C. neoformans pheromone-response factor. This function of Mat2 distinguishes the mechanism of regulation through the PRE of C. neoformans from all other fungal systems studied to date and reveals species-specific adaptations of a fungal transcription factor that defies predictions on the basis of sequence alone. Overall, our findings reveal that pheromone-response network rewiring has occurred at the level of transcription factor identity, despite the strong conservation of upstream and downstream components, and serve as a model for how selection pressures act differently on signaling vs. gene regulatory components during eukaryotic evolution.

The role of teacher communicator style in the delivery of a middle school substance use prevention program.

We examine whether teachers' communicator style relates to student engagement, teacher-student relationships, student perceptions of teacher immediacy, as well as observer ratings of delivery skills during the implementation of All Stars, a middle school-based substance use prevention program. Data from 48 teachers who taught All Stars up to 3 consecutive years and their respective seventh-grade students (n = 2,240) indicate that having an authoritative communication style is negatively related to student engagement with the curriculum and the quality of the student-teacher relationship, while having an expressive communicator style improves teachers' immediacy to student needs. Adaptations made by a subsample of teachers (n = 27) reveal that those who were more expressive asked students more questions, used more motivational techniques, and introduced more new concepts than authoritarian teachers.

Draft genome sequence of Streptomyces sp. strain Wigar10, isolated from a surface-sterilized garlic bulb.

Streptomyces sp. strain Wigar10 was isolated from a surface-sterilized garlic bulb (Allium sativum var. Purple Stripe). Its genome encodes several novel secondary metabolite biosynthetic gene clusters and provides a genetic basis for further investigation of this strain's chemical biology and potential for interaction with its garlic host.

Mating pheromone in Cryptococcus neoformans is regulated by a transcriptional/degradative "futile" cycle.

Sexual reproduction in fungi requires induction of signaling pheromones within environments that are conducive to mating. The fungus Cryptococcus neoformans is currently the fourth greatest cause of infectious death in regions of Africa and undergoes mating in phytonutrient-rich environments to create spores with infectious potential. Here we show that under conditions where sexual development is inhibited, a ∼17-fold excess of MFα pheromone transcript is synthesized and then degraded by a DEAD box protein, Vad1, resulting in low steady state transcript levels. Transfer to mating medium or deletion of the VAD1 gene resulted in high level accumulation of MFα transcripts and enhanced mating, acting in concert with the mating-related HOG1 pathway. We then investigated whether the high metabolic cost of this apparently futile transcriptional cycle could be justified by a more rapid induction of mating. Maintenance of Vad1 activity on inductive mating medium by constitutive expression resulted in repressed levels of MFα that did not prevent but rather prolonged the time to successful mating from 5-6 h to 15 h (p < 0.0001). In sum, these data suggest that VAD1 negatively regulates the sexual cell cycle via degradation of constitutive high levels of MFα transcripts in a synthetic/degradative cycle, providing a mechanism of mRNA induction for time-critical cellular events, such as mating induction.

Allelic exchange of pheromones and their receptors reprograms sexual identity in Cryptococcus neoformans.

Cell type specification is a fundamental process that all cells must carry out to ensure appropriate behaviors in response to environmental stimuli. In fungi, cell identity is critical for defining "sexes" known as mating types and is controlled by components of mating type (MAT) loci. MAT-encoded genes function to define sexes via two distinct paradigms: 1) by controlling transcription of components common to both sexes, or 2) by expressing specially encoded factors (pheromones and their receptors) that differ between mating types. The human fungal pathogen Cryptococcus neoformans has two mating types (a and alpha) that are specified by an extremely unusual MAT locus. The complex architecture of this locus makes it impossible to predict which paradigm governs mating type. To identify the mechanism by which the C. neoformans sexes are determined, we created strains in which the pheromone and pheromone receptor from one mating type (a) replaced the pheromone and pheromone receptor of the other (alpha). We discovered that these "alpha(a)" cells effectively adopt a new mating type (that of a cells); they sense and respond to alpha factor, they elicit a mating response from alpha cells, and they fuse with alpha cells. In addition, alpha(a) cells lose the alpha cell type-specific response to pheromone and do not form germ tubes, instead remaining spherical like a cells. Finally, we discovered that exogenous expression of the diploid/dikaryon-specific transcription factor Sxi2a could then promote complete sexual development in crosses between alpha and alpha(a) strains. These data reveal that cell identity in C. neoformans is controlled fully by three kinds of MAT-encoded proteins: pheromones, pheromone receptors, and homeodomain proteins. Our findings establish the mechanisms for maintenance of distinct cell types and subsequent developmental behaviors in this unusual human fungal pathogen.

Three-year trajectory of teachers' fidelity to a drug prevention curriculum.

Little is known about the trajectories over time of classroom teachers' fidelity to drug prevention curricula. Using the "Concerns-Based Adoption Model" (C-BAM) as a theoretical framework, we hypothesized that teachers' fidelity would improve with repetition. Participants comprised 23 middle school teachers who videotaped their administration of three entire iterations of the All Stars curriculum. Investigators coded two key curriculum lessons, specifically assessing the proportion of activities of each lesson teachers attempted and whether they omitted, added, or changed prescribed content, or delivered it using new methods. Study findings provided only partial support for the C-BAM model. Considerable variability in teachers' performance over time was noted, suggesting that their progression over time may be nonlinear and dynamic, and quite possibly a function of their classroom and school contexts. There was also evidence that, by their third iteration of All Stars, teachers tended to regress toward the baseline mean. That is, the implementation quality of those that started out with high levels of fidelity tended to degrade, while those that started out with very low fidelity to the curriculum tended to improve. Study findings suggest the need for ongoing training and technical assistance, as well as "just in time" messages delivered electronically; but it is also possible that some prevention curricula may impose unrealistic expectations or burdens on teachers' abilities and classroom time.

Teachers' delivery skills and substance use prevention program outcomes: the moderating role of students' need for cognition and impulse decision making.

The purpose of this study was to extend the literature in both substance use implementation and persuasive health communication by examining the extent to which students' need for cognition and impulsive decision-making moderated the relationship between teachers' classroom communication behavior and program outcomes in an evidence-based middle school substance use prevention curriculum. Participants included 48 teachers and their respective 7th grade students who participated in a randomized trial testing the effectiveness of personal coaching as a means to improve the quality with which teachers implemented the All Stars curriculum. Need for cognition and impulse decision-making were both associated with positive changes in lifestyle incongruence and commitments to not use substances for students whose teachers displayed greater interactive teaching. Further, need for cognition was associated with lower alcohol use rates while impulse decision making related to lower rates of marijuana use in classes with interactive teaching.

Coaching to Enhance Quality of Implementation in Prevention.

PURPOSE: This study describes topics covered by coaches assisting teachers implementing a research-based drug prevention program and explores how coaching affected student outcomes. DESIGN: The All Stars drug prevention curriculum was implemented by 16 urban teachers who received four coaching sessions. Two coaches participated. Coaches were interviewed by investigators to assess topics covered. Students completed pretest-posttest measures of mediators and substance use behaviors. FINDINGS: The average teacher was coached on 11.7 different topics, out of a total of 23 topics. Coaching topics most heavily emphasized included: introduction and wrap up; time management; general classroom management; teacher's movement around the class; asking open-ended questions; using students' questions, comments and examples to make desired points; general preparation; engaging high-risk youth; reading from the curriculum; implementing activities correctly; focusing on objectives and goals; maintaining a focus on the task; and improving depth of understanding. Seven coaching topics were found to relate to changes in student mediators and behavior. RESEARCH LIMITATIONS/IMPLICATIONS: The current study was exploratory. Future research should explore how teachers develop the particular skills required by prevention programs and how coaches can assist them. PRACTICAL IMPLICATIONS: We postulate five levels of skill development which coaches may address: (1) fundamental teaching skills, (2) mechanics of program delivery, (3) development of an interactive teaching style, (4) effective response to student input, and (5) effective tailoring and adaptation. ORIGINALITY/VALUE: This represents one of a very few studies that explores how coaching impacts outcomes in substance abuse prevention.

Cognate Site Identifier analysis reveals novel binding properties of the Sex Inducer homeodomain proteins of Cryptococcus neoformans.

Homeodomain proteins function in fungi to specify cell types and control sexual development. In the meningoencephalitis-causing fungal pathogen Cryptococcus neoformans, sexual development leads to the production of spores (suspected infectious particles). Sexual development is controlled by the homeodomain transcription factors Sxi1alpha and Sxi2a, but the mechanism by which they act is unknown. To understand how the Sxi proteins regulate development, we characterized their binding properties in vitro, showing that Sxi2a does not require a partner to bind DNA with high affinity. We then utilized a novel approach, Cognate Site Identifier (CSI) arrays, to define a comprehensive DNA-binding profile for Sxi2a, revealing a consensus sequence distinct from those of other fungal homeodomain proteins. Finally, we show that the homeodomains of both Sxi proteins are required for sexual development, a departure from related fungi. Our findings support a model in which Sxi1alpha and Sxi2a control sexual development in a homeodomain-dependent manner by binding to DNA sequences that differ from those defined in previously established fungal paradigms.

Elucidating the pathogenesis of spores from the human fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans was first described as a human fungal pathogen more than a century ago. One aspect of the C. neoformans infectious life cycle that has been the subject of earnest debate is whether the spores are pathogenic. Despite much speculation, no direct evidence has been presented to resolve this outstanding question. We present evidence that C. neoformans spores are pathogenic in a mouse intranasal inhalation model of infection. In addition, we provide mechanistic insights into spore-host interactions. We found that C. neoformans spores were phagocytosed by alveolar macrophages via interactions between fungal beta-(1,3)-glucan and the host receptors Dectin-1 and CD11b. Moreover, we discovered an important link between spore survival and macrophage activation state: intracellular spores were susceptible to reactive oxygen-nitrogen species. We anticipate these results will serve as the basis for a model to further investigate the pathogenic implications of infections caused by fungal spores.

Calorie restriction on drinking days: an examination of drinking consequences among college students.

OBJECTIVE: This study examined the association between restricting calories on intended drinking days and drunkenness frequency and alcohol-related consequences among college students. PARTICIPANTS: Participants included a random sample of 4,271 undergraduate college students from 10 universities. METHODS: Students completed a Web-based survey regarding their high-risk drinking behaviors and calorie restriction on intended drinking days. RESULTS: Thirty-nine percent of past 30-day drinkers reported restricting calories on days they planned to drink alcohol, of which 67% restricted because of weight concerns. Restricting calories on drinking days was associated with greater odds of getting drunk in a typical week. Women who restricted were more likely to report memory loss, being injured, being taken advantage of sexually, and having unprotected sex while drinking. Men were more likely to get into a physical fight. CONCLUSIONS: These results highlight the importance of considering weight control behaviors in the examination of high-risk college drinking.

Isolation and characterization of Cryptococcus neoformans spores reveal a critical role for capsule biosynthesis genes in spore biogenesis.

Spores are essential particles for the survival of many organisms, both prokaryotic and eukaryotic. Among the eukaryotes, fungi have developed spores with superior resistance and dispersal properties. For the human fungal pathogens, however, relatively little is known about the role that spores play in dispersal and infection. Here we present the purification and characterization of spores from the environmental fungus Cryptococcus neoformans. For the first time, we purified spores to homogeneity and assessed their morphological, stress resistance, and surface properties. We found that spores are morphologically distinct from yeast cells and are covered with a thick spore coat. Spores are also more resistant to environmental stresses than yeast cells and display a spore-specific configuration of polysaccharides on their surfaces. Surprisingly, we found that the surface of the spore reacts with antibodies to the polysaccharide glucuronoxylomannan, the most abundant component of the polysaccharide capsule required for C. neoformans virulence. We explored the role of capsule polysaccharide in spore development by assessing spore formation in a series of acapsular strains and determined that capsule biosynthesis genes are required for proper sexual development and normal spore formation. Our findings suggest that C. neoformans spores may have an adapted cell surface that facilitates persistence in harsh environments and ultimately allows them to infect mammalian hosts.

The potential of coaching as a strategy to improve the effectiveness of school-based substance use prevention curricula.

Research-based substance use prevention curricula typically yield small effects when implemented by school teachers under real-world conditions. Using a randomized controlled trial, the authors examined whether expert coaching improves the effectiveness of the All Stars prevention curriculum. Although a positive effect on students' cigarette use was noted, this finding may be attributed to marked baseline differences on this variable across the intervention and control groups. No effects were found on students' alcohol or marijuana use or on any of several variables thought to mediate curriculum effects. The effects of coaching on teachers may not become evident until future years, when they have moved beyond an initial mechanical delivery of the curriculum.

Measuring quality of delivery in a substance use prevention program.

The purpose of this study was to develop and validate an observation measure designed to capture teachers' use of interactive teaching skills within the delivery of the All Stars substance use prevention program. Coders counted the number of times teachers praised and encouraged students, accepted and used students' ideas, asked questions, self-disclosed personal anecdotes, and corrected student misbehavior. These teacher behaviors loaded on three factors: classroom management, acknowledgment, and student-centered methods. Classroom management was negatively related to student engagement. Acknowledgment was negatively related to students' normative beliefs. Student-centered methods were positively related to student idealism and normative beliefs, and marginally predicted decreases in student marijuana use. Editors' Strategic Implications: The authors provide a promising approach to studying pedagogical prevention approaches, and they also link teaching processes to student outcomes. This study of program delivery should be of general interest (i.e., not limited to substance use prevention) to practitioners and researchers.

Formulation of a defined V8 medium for induction of sexual development of Cryptococcus neoformans.

For over 3 decades, sexual development in the human fungal pathogen Cryptococcus neoformans and other fungi has been initiated by growing compatible mating partners on V8 juice medium. Although this medium is an efficient inducer of sexual development, the mechanism by which it promotes the process is unknown. To understand how V8 juice medium induces sexual development, we attempted to purify inducing factors from V8 juice, and we carried out a complete compositional analysis of V8 juice. We discovered that no single factor is responsible for the effects of V8 juice medium. Rather, the unique composition of V8 juice medium provides the proper nutrient composition for inducing and sustaining complete sexual development. Utilizing these findings, we developed a defined V8 (DV8) medium that mimics V8 juice medium in sexual development assays. Then, using DV8 as a tool, we explored the roles that specific molecules play in enhancing sexual development. Surprisingly, we discovered that copper is a key factor, leading to an upregulation of the mating pheromone genes MFa and MFalpha, both required for the initial steps in sexual development. The utilization of DV8 to investigate the effects of copper on sexual development presented here is an example of how defining the conditions that induce sexual development will advance the study of C. neoformans.

Plasminogen alleles influence susceptibility to invasive aspergillosis.

Invasive aspergillosis (IA) is a common and life-threatening infection in immunocompromised individuals. A number of environmental and epidemiologic risk factors for developing IA have been identified. However, genetic factors that affect risk for developing IA have not been clearly identified. We report that host genetic differences influence outcome following establishment of pulmonary aspergillosis in an exogenously immune suppressed mouse model. Computational haplotype-based genetic analysis indicated that genetic variation within the biologically plausible positional candidate gene plasminogen (Plg; Gene ID 18855) correlated with murine outcome. There was a single nonsynonymous coding change (Gly110Ser) where the minor allele was found in all of the susceptible strains, but not in the resistant strains. A nonsynonymous single nucleotide polymorphism (Asp472Asn) was also identified in the human homolog (PLG; Gene ID 5340). An association study within a cohort of 236 allogeneic hematopoietic stem cell transplant (HSCT) recipients revealed that alleles at this SNP significantly affected the risk of developing IA after HSCT. Furthermore, we demonstrated that plasminogen directly binds to Aspergillus fumigatus. We propose that genetic variation within the plasminogen pathway influences the pathogenesis of this invasive fungal infection.