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Abstract Nascent RNA profiling is growing in popularity; however, there is no standard analysis pipeline to uniformly process the data and assess quality. Here, we introduce PEPPRO, a comprehensive, scalable workflow for GRO-seq, PRO-seq, and ChRO-seq data. PEPPRO produces uniformly processed output files for downstream analysis and assesses adapter abundance, RNA integrity, library complexity, nascent RNA purity, and run-on efficiency. PEPPRO is restartable and fault-tolerant, records copious logs, and provides a web-based project report. PEPPRO can be run locally or using a cluster, providing a portable first step for genomic nascent RNA analysis.

Abstract A key challenge in epigenetics is to determine the biological significance of epigenetic variation among individuals. We present Coordinate Covariation Analysis (COCOA), a computational framework that uses covariation of epigenetic signals across individuals and a database of region sets to annotate epigenetic heterogeneity. COCOA is the first such tool for DNA methylation data and can also analyze any epigenetic signal with genomic coordinates. We demonstrate COCOA’s utility by analyzing DNA methylation, ATAC-seq, and multi-omic data in supervised and unsupervised analyses, showing that COCOA provides new understanding of inter-sample epigenetic variation. COCOA is available on Bioconductor ( http://bioconductor.org/packages/COCOA ).

Mammalian α-defensins are a family of abundant effector peptides of the mucosal innate immune system. Although primarily considered to be antimicrobial, α-defensins can increase rather than block infection by certain prominent bacterial and viral pathogens in cell culture and in vivo. We have shown previously that exposure of mouse and human adenoviruses (HAdVs) to α-defensins is able to overcome competitive inhibitors that block cell binding, leading us to hypothesize a defensin-mediated binding mechanism that is independent of known viral receptors. To test this hypothesis, we used genetic approaches to demonstrate that none of several primary receptors nor integrin co-receptors are needed for human α-defensin-mediated binding of HAdV to cells; however, infection remains integrin dependent. Thus, our studies have revealed a novel pathway for HAdV binding to cells that bypasses viral primary receptors. We speculate that this pathway functions in parallel with receptor-mediated entry and contributes to α-defensin-enhanced infection of susceptible cells. Remarkably, we also found that in the presence of α-defensins, HAdV tropism is expanded to non-susceptible cells, even when viruses are exposed to a mixture of both susceptible and non-susceptible cells. Therefore, we propose that in the presence of sufficient concentrations of α-defensins, such as in the lung or gut, integrin expression rather than primary receptor expression will dictate HAdV tropism in vivo. In summary, α-defensins may contribute to tissue tropism not only through the neutralization of susceptible viruses but also by allowing certain defensin-resistant viruses to bind to cells independently of previously described mechanisms.

Förster resonance energy transfer (FRET) microscopy is used in numerous biophysical and biomedical applications to monitor inter- and intramolecular interactions and conformational changes in the 2–10 nm range. FRET is currently being extended to in vivo optical imaging, its main application being in quantifying drug-target engagement or drug release in animal models of cancer using organic dye or nanoparticle-labeled probes. Herein, we compared FRET quantification using intensity-based FRET (sensitized emission FRET analysis with the three-cube approach using an IVIS imager) and macroscopic fluorescence lifetime (MFLI) FRET using a custom system using a time-gated-intensified charge-coupled device, for small animal optical in vivo imaging. The analytical expressions and experimental protocols required to quantify the product fDE of the FRET efficiency E and the fraction of donor molecules involved in FRET, fD, are described in detail for both methodologies. Dynamic in vivo FRET quantification of transferrin receptor-transferrin binding was acquired in live intact nude mice upon intravenous injection of a near-infrared-labeled transferrin FRET pair and benchmarked against in vitro FRET using hybridized oligonucleotides. Even though both in vivo imaging techniques provided similar dynamic trends for receptor-ligand engagement, we demonstrate that MFLI-FRET has significant advantages. Whereas the sensitized emission FRET approach using the IVIS imager required nine measurements (six of which are used for calibration) acquired from three mice, MFLI-FRET needed only one measurement collected from a single mouse, although a control mouse might be needed in a more general situation. Based on our study, MFLI therefore represents the method of choice for longitudinal preclinical FRET studies such as that of targeted drug delivery in intact, live mice.

Ecosystem alterations to Lake Michigan (LM), such as the invasion of dreissenid mussels, have decreased the abundance of phytoplankton and, subsequently, zooplankton, which has implications for Great Lake fisheries. The community composition of zooplankton has also been altered over the past 20 years in LM, shifting the summer dominance from cladoceran to calanoid taxa. However, most of our information on zooplankton community dynamics is from deeper pelagic zones. The nearshore beach (≤1 m) habitat, which serves as a critical nursery for some larvae and juvenile fish, is composed of different zooplankton taxa than deeper zones, but limited data are available for comparison. We conducted a standardized summer sampling campaign to characterize the zooplankton community across 32 beaches in LM. We found the lowest crustacean zooplankton density and biomass on northern LM beaches and a higher zooplankton density and biomass on Greater Green Bay and central LM beaches, which had warmer water temperatures. Smaller-bodied cladocera (mainly Bosmina) and cyclopoid taxa were the most abundant groups on beaches. Our results provide the first comprehensive characterization of zooplankton on beaches in the Great Lakes, with applications for other lake systems. These results may help identify potential “hot spots” of fish recruitment for management.

Species in the Laetiporus sulphureus species complex, also known as “chicken of the woods” mushrooms, are wood-decay fungi that cause brown rot within the heartwood of their tree hosts. The common name “chicken of the woods” is given to some species in this group because they are tasty edible mushrooms. Several Laetiporus species have been harvested to use as food colorants, to dye natural products such as wool, and for human consumption. This new 4-page publication of the UF/IFAS Plant Pathology Department, written by Brianna Benitez, Claudia A. Paez, Matthew E. Smith, and Jason A. Smith, describes these fungi as well as their ecology, management, and potential edibility. https://edis.ifas.ufl.edu/pp358

Biscogniauxia canker or dieback (formerly called Hypoxylon canker or dieback) is a common contributor to poor health and decay in a wide range of tree species (Balbalian & Henn 2014). This disease is caused by several species of fungi in the genus Biscogniauxia (formerly Hypoxylon). B. atropunctata or B. mediterranea are usually the species found on Quercus spp. and other hosts in Florida, affecting trees growing in many different habitats, such as forests, parks, green spaces and urban areas (McBride & Appel, 2009). Typically, species of Biscogniauxia are opportunistic pathogens that do not affect healthy and vigorous trees; some species are more virulent than others. However, once they infect trees under stress (water stress, root disease, soil compaction, construction damage etc.) they can quickly colonize the host. Once a tree is infected and fruiting structures of the fungus are evident, the tree is not likely to survive especially if the infection is in the tree's trunk (Anderson et al., 1995).

Ganoderma Karst. is a large and diverse genus of wood decay fungi that can rot the roots and/or lower trunk of many tree species. There are several laccate (varnished or polished) Ganoderma species that are found in the southeastern United States and this six-page fact sheet provides an overview of the different species. Written by Andrew L. Loyd, Jason A. Smith, Brantlee S. Richter, Robert A. Blanchette, and Matthew E. Smith and published by the Plant Pathology Department.­http://edis.ifas.ufl.edu/pp333

This 6-page fact sheet describes the emergence of these two pathogens of oak and grapevine in North America and Florida; the signs and symptoms of infection; mechanism of host disease and death; fungal morphology; origin, host range, and classification; and management options. Written by Sonja Mullerin and Jason A. Smith, and published by the UF Department of School of Forest Resources and Conservation, March 2015. (Photo: Jason Smith, UF/IFAS)

Armillaria root rot is a disease that decays the root system of many common trees and shrubs. It is caused by several species of Armillaria, fungi that can be recognized by the clusters of yellow to honey-colored mushrooms that emerge during moist conditions. The disease is often lethal, and infected trees may have wilting branches, branch dieback, and stunted growth. Infected trees and shrubs should be removed and replaced with resistant species. This 4-page fact sheet was written by Laura Sanagorski, Aaron Trulock, and Jason Smith, and published by the UF Department of Environmental Horticulture, July 2013. http://edis.ifas.ufl.edu/ep478

Thousand cankers disease of walnut is a tree disease of the edible nut-producing and ornamental tree, black walnut. The first diseased trees were observed in New Mexico in the 1990s. The disease has since spread to most of the western states. Until 2009, diseased black walnut trees were only found outside of the natural range of black walnut, which occurs from the mid-Atlantic states to just west of the Mississippi River. In 2009, it was found near Knoxville, Tennessee; it has since spread to Pennsylvania and Virginia. It is possible that within the next decade this disease could naturally spread to Florida. However, if people continue to move TCD infested walnut logs from place to place, this disease could arrive in Florida tomorrow. This 3-page fact sheet was written by Don Spence and Jason A. Smith, and published by the UF Department of School of Forest Resources and Conservation, February 2013. http://edis.ifas.ufl.edu/fr376

Forest health is an important topic for biology, agriculture, current issues, and environmental science classes. But conversations with educators suggested that they were not equipped to teach about it given their existing curricula. This 79-page educator guide includes six activities designed to help learners consider forest health from various viewpoints; understand interrelationships and feedback mechanisms in a forest system; visualize spatial and temporal mechanisms of forest system functions; appreciate the variety of threats to forest health; and consider their role as future forest stewards. Written by Geetha S. Iyer, Martha M. Monroe, and Jason A. Smith, and published by the UF Department of School of Forest Resources and Conservation, July 2011. FOR287/FR355: Beyond the Trees: A Systems Approach to Understanding Forest Health in the Southeastern United States (ufl.edu)

There is no question that forests provide important ecological services and economic resources to Floridians. Similarly, there is no doubt that the health of our forests is at the mercy of how we manage our landscape and make decisions. The activities in this supplement, with the original PLT Guide, help our students rise to these twin challenges. This 84-page handbook was written by Sarah L. Hicks, Martha C. Monroe, Geetha S. Iyer, and Jason A. Smith, and published by the UF Department of School of Forest Resources and Conservation, July 2011. FOR286/FR354: What Is a Healthy Forest? A Supplement to Florida Project Learning Tree (ufl.edu)

FOR276, a 5-page fact sheet by Jason A. Smith and Aaron Trulock, suggests a new understanding of the cause of decline and discusses current efforts to assess the population and develop a recovery plan. Includes references. Published by the UF Department of School of Forest Resources and Conservation, November 2010.

HS1179, a 4-page illustrated fact sheet by Jonathan H. Crane and Jason A. Smith, provides homeowners with an update on redbay ambrosia beetle-laurel wilt disease in Florida, how the beetle and pathogen are spread, symptoms of infestations, and recommendations for homeowners. Includes references. Published by the UF Department of Horticultural Sciences, May 2010.

HS-1137, a 6-page illustrated fact sheet by Albert E. Mayfield III, Jonathan H. Crane, and Jason A. Smith, describes for homeowners this fungal disease of trees in the laurel family that is carried by the non-native redbay ambrosia beetle. Includes descriptions of the beetles, plant hosts, impact, host symptoms, and management strategies. Includes references. Published by the UF Horticultural Sciences Department, March 2008.

Enteric alpha-defensins are potent effectors of innate immunity that are abundantly expressed in the small intestine. Certain enteric bacteria and viruses are resistant to defensins and even appropriate them to enhance infection despite neutralization of closely related microbes. We therefore hypothesized that defensins impose selective pressure during fecal-oral transmission. Upon passaging a defensin-sensitive serotype of adenovirus in the presence of a human defensin, mutations in the major capsid protein hexon accumulated. In contrast, prior studies identified the vertex proteins as important determinants of defensin antiviral activity. Infection and biochemical assays suggest that a balance between increased cell binding and a downstream block in intracellular trafficking mediated by defensin interactions with all of the major capsid proteins dictates the outcome of infection. These results extensively revise our understanding of the interplay between defensins and non-enveloped viruses. Furthermore, they provide a feasible rationale for defensins shaping viral evolution, resulting in differences in infection phenotypes of closely related viruses.

Development of long-term geographic information system (GIS) databases of species densities and distributions, combined with biological, ecological, and management-related metrics, can help guide research and management strategies. Here we summarize 3 decades of North American moose (Alces alces) population and harvest densities collected at the management unit scale for the years 1980, 1990, 2000, and 2010. A summary analysis of these data indicates that moose have both expanded and contracted along their southern range boundary in recent decades - including the Prairie Provinces and states, and a portion of the northeastern United States. A narrow band of relatively stable and high-density moose populations extends from central Alaska across the Prairie Provinces and east to the Maritime Provinces and upper northeastern states. Distributions in 2010 indicate that moose now occupy an area > 9,492,000 km2 in North America. We also identified that a core range of boreal habitat, only 30% of the occupied range across the continent, supports 89% of the estimated 1 million moose in North America. Time-series analyses can offer a simple and cost-effective approach to monitor the status of moose populations in North America, and might be particularly insightful given the current and predicted future influences of climate change on moose. Other analyses might address population dynamics, habitat, environmental constraints, and harvest management, among other issues. We encourage jurisdictions to cooperate strategically in implementing and coordinating GIS analyses to monitor, assess, and manage the North American moose population.

Understanding reproductive parameters of a population is vital to management, especially during periods of population fluctuation. Therefore, our objective was to provide the first estimates of reproductive parameters such as pregnancy rates, litter size, fecundity rates, conception dates, and fetal sex ratios for a moose (Alces alces) population in North Dakota, USA. Between 1978 and 1986, the North Dakota Game and Fish Department necropsied 54 hunter-killed cow moose which were all harvested after the rut (10 November to 12 December). Pregnancy rates for calves (n = 7), yearlings (1.5-years-old, n = 6), and adults (> 2.5-years-old, n = 41) were 0%, 100% and 95%, respectively. Mean conception date was 2 October. Overall, mean litter size was 1.76 fetuses, twinning rate was 73.3%, and mean fecundity was 1.66 fetuses and 0.85 female fetuses/cow. Fetal sex ratio did not differ from the expected 50:50 ratio, but the odds of producing at least one male calf increased with dam age, but not dam weight or litter size. This population displayed reproductive parameters consistent with an irruptive and expanding moose population.

The small intestinal epithelium produces numerous antimicrobial peptides and proteins, including abundant enteric α-defensins. Although they most commonly function as potent antivirals in cell culture, enteric α-defensins have also been shown to enhance some viral infections in vitro. Efforts to determine the physiologic relevance of enhanced infection have been limited by the absence of a suitable cell culture system. To address this issue, here we use primary stem cell-derived small intestinal enteroids to examine the impact of naturally secreted α-defensins on infection by the enteric mouse pathogen, mouse adenovirus 2 (MAdV-2). MAdV-2 infection was increased when enteroids were inoculated across an α-defensin gradient in a manner that mimics oral infection but not when α-defensin levels were absent or bypassed through other routes of inoculation. This increased infection was a result of receptor-independent binding of virus to the cell surface. The enteroid experiments accurately predicted increased MAdV-2 shedding in the feces of wild type mice compared to mice lacking functional α-defensins. Thus, our studies have shown that viral infection enhanced by enteric α-defensins may reflect the evolution of some viruses to utilize these host proteins to promote their own infection.

α-defensins are abundant antimicrobial peptides with broad, potent antibacterial, antifungal, and antiviral activities in vitro. Although their contribution to host defense against bacteria in vivo has been demonstrated, comparable studies of their antiviral activity in vivo are lacking. Using a mouse model deficient in activated α-defensins in the small intestine, we show that Paneth cell α-defensins protect mice from oral infection by a pathogenic virus, mouse adenovirus 1 (MAdV-1). Survival differences between mouse genotypes are lost upon parenteral MAdV-1 infection, strongly implicating a role for intestinal defenses in attenuating pathogenesis. Although differences in α-defensin expression impact the composition of the ileal commensal bacterial population, depletion studies using broad-spectrum antibiotics revealed no effect of the microbiota on α-defensin-dependent viral pathogenesis. Moreover, despite the sensitivity of MAdV-1 infection to α-defensin neutralization in cell culture, we observed no barrier effect due to Paneth cell α-defensin activation on the kinetics and magnitude of MAdV-1 dissemination to the brain. Rather, a protective neutralizing antibody response was delayed in the absence of α-defensins. This effect was specific to oral viral infection, because antibody responses to parenteral or mucosal ovalbumin exposure were not affected by α-defensin deficiency. Thus, α-defensins play an important role as adjuvants in antiviral immunity in vivo that is distinct from their direct antiviral activity observed in cell culture.

Human α-defensins are potent anti-microbial peptides with the ability to neutralize bacterial and viral targets. Single alanine mutagenesis has been used to identify determinants of anti-bacterial activity and binding to bacterial proteins such as anthrax lethal factor. Similar analyses of α-defensin interactions with non-enveloped viruses are limited. We used a comprehensive set of human α-defensin 5 (HD5) and human neutrophil peptide 1 (HNP1) alanine scan mutants in a combination of binding and neutralization assays with human adenovirus (AdV) and human papillomavirus (HPV). We have identified a core of critical hydrophobic residues that are common determinants for all of the virus-defensin interactions that were analyzed, while specificity in viral recognition is conferred by specific surface-exposed charged residues. The hydrophobic residues serve multiple roles in maintaining the tertiary and quaternary structure of the defensins as well as forming an interface for virus binding. Many of the important solvent-exposed residues of HD5 group together to form a critical surface. However, a single discrete binding face was not identified for HNP1. In lieu of whole AdV, we used a recombinant capsid subunit comprised of penton base and fiber in quantitative binding studies and determined that the anti-viral potency of HD5 was a function of stoichiometry rather than affinity. Our studies support a mechanism in which α-defensins depend on hydrophobic and charge-charge interactions to bind at high copy number to these non-enveloped viruses to neutralize infection and provide insight into properties that guide α-defensin anti-viral activity.

Defensins are effectors of the innate immune response with potent antibacterial activity. Their role in antiviral immunity, particularly for non-enveloped viruses, is poorly understood. We recently found that human alpha-defensins inhibit human adenovirus (HAdV) by preventing virus uncoating and release of the endosomalytic protein VI during cell entry. Consequently, AdV remains trapped in the endosomal/lysosomal pathway rather than trafficking to the nucleus. To gain insight into the mechanism of defensin-mediated neutralization, we analyzed the specificity of the AdV-defensin interaction. Sensitivity to alpha-defensin neutralization is a common feature of HAdV species A, B1, B2, C, and E, whereas species D and F are resistant. Thousands of defensin molecules bind with low micromolar affinity to a sensitive serotype, but only a low level of binding is observed to resistant serotypes. Neutralization is dependent upon a correctly folded defensin molecule, suggesting that specific molecular interactions occur with the virion. CryoEM structural studies and protein sequence analysis led to a hypothesis that neutralization determinants are located in a region spanning the fiber and penton base proteins. This model was supported by infectivity studies using virus chimeras comprised of capsid proteins from sensitive and resistant serotypes. These findings suggest a mechanism in which defensin binding to critical sites on the AdV capsid prevents vertex removal and thereby blocks subsequent steps in uncoating that are required for release of protein VI and endosomalysis during infection. In addition to informing the mechanism of defensin-mediated neutralization of a non-enveloped virus, these studies provide insight into the mechanism of AdV uncoating and suggest new strategies to disrupt this process and inhibit infection.

According to current models of retrovirus infection, receptor binding to the surface subunit (SU) of the envelope glycoprotein (Env) triggers a conformational change in the transmembrane subunit (TM) that mediates virus fusion to cell membranes. To understand how this occurs, we investigated the role of the receptor Tva in avian leukosis virus-A (ALV-A) infection. We find that Tva binding induced the formation of a reactive thiolate on Cys38 (Cys38-S- in SU. Both chemical and genetic inactivation of Cys38-S- completely abrogated ALV fusion and infection. Remarkably, Cys38-S- does not mediate isomerization of the SU-TM disulfide bond and is not required for Tva-induced activation of TM, including pre-hairpin association with membranes and low pH assembly of helical bundles. These findings indicate that, contrary to current models, receptor activation of TM is not sufficient for ALV fusion and infection and that formation of a reactive thiolate is an additional receptor-dependent step.