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Foodborne pathogens such as Salmonella, Campylobacter, Escherichia coli, and Listeria are a major concern within the food industry due to their pathogenic potential to cause infection. Of these, Listeria monocytogenes, possesses a high mortality rate (approximately 20%) and is considered one of the most dangerous foodborne pathogens. Although the usual reservoirs for Listeria transmission have been extensively studied, little is known about the relationship between Listeria and live poultry production. Sporadic and isolated cases of listeriosis have been attributed to poultry production and Listeria spp. have been isolated from all stages of poultry production and processing. Farm studies suggest that live birds may be an important vector and contributor to contamination of the processing environment and transmission of Listeria to consumers. Therefore, the purpose of this review is to highlight the occurrence, incidence, and potential systemic interactions of Listeria spp. with poultry.

Optical coherence tomography provides high-resolution 3D imaging of the posterior segment of the eye. However, quantitative morphological analysis, particularly relevant in retinal degenerative diseases such as glaucoma, has been confined to simple sectorization and averaging with limited spatial sensitivity for detection of clinical markers. In this paper, we present point-wise analysis and visualization of the retinal nerve fiber layer and choroid from cross-sectional data using functional shapes (fshape) registration. The fshape framework matches two retinas, or generates a mean of multiple retinas, by jointly optimizing the surface geometry and functional signals mapped on the surface. We generated group-wise mean retinal nerve fiber layer and choroidal surfaces with the respective layer thickness mapping and showed the difference by age (normal, younger vs. older) and by disease (age-matched older, normal vs. glaucomatous) in the two layers, along with a more conventional sector-based analysis for comparison. The fshape results visualized the detailed spatial patterns of the differences between the age-matched normal and glaucomatous retinal nerve fiber layers, with the glaucomatous layers most significantly thinner in the inferior region close to Bruch's membrane opening. Between the young and older normal cases, choroid was shown to be significantly thinner in the older subjects across all regions, but particularly in the nasal and inferior regions. The results demonstrate a comprehensive and detailed analysis with visualization of morphometric patterns by multiple factors.

In some animals, the typical body temperature can be higher than humans, for example, 42°C in poultry and 40°C in rabbits which can be a potential thermal stress challenge for pathogens. Even in animals with lower body temperatures, when infection occurs, the immune system may increase body temperature to reduce the chance of survival for pathogens. However, some pathogens can still easily overcome higher body temperatures and/or rise in body temperatures through expression of stress response mechanisms. Salmonella is the causative agent of one of the most prevalent foodborne illnesses, salmonellosis, and can readily survive over a wide range of temperatures due to the efficient expression of the heat (thermal) stress response. Therefore, thermal resistance mechanisms can provide cross protection against other stresses including the non-specific host defenses found within the human body thus increasing pathogenic potential. Understanding the molecular mechanisms associated with thermal responses in Salmonella is crucial in designing and developing more effective or new treatments for reducing and eliminating infection caused by Salmonella that have survived heat stress. In this review, Salmonella thermal resistance is assessed followed by an overview of the thermal stress responses with a focus on gene regulation by sigma factors, heat shock proteins, along with the corresponding thermosensors and their association with virulence expression including a focus on a potential link between heat resistance and potential for infection.

Woodland caribou (Rangifer tarandus caribou) in Ontario are a threatened species that have experienced a substantial retraction of their historic range. Part of their decline has been attributed to increasing densities of anthropogenic linear features such as trails, roads, railways, and hydro lines. These features have been shown to increase the search efficiency and kill rate of wolves. However, it is unclear whether selection for anthropogenic linear features is additive or compensatory to selection for natural (water) linear features which may also be used for travel. We studied the selection of water and anthropogenic linear features by 52 resident wolves (Canis lupus x lycaon) over four years across three study areas in northern Ontario that varied in degrees of forestry activity and human disturbance. We used Euclidean distance-based resource selection functions (mixed-effects logistic regression) at the seasonal range scale with random coefficients for distance to water linear features, primary/secondary roads/railways, and hydro lines, and tertiary roads to estimate the strength of selection for each linear feature and for several habitat types, while accounting for availability of each feature. Next, we investigated the trade-off between selection for anthropogenic and water linear features. Wolves selected both anthropogenic and water linear features; selection for anthropogenic features was stronger than for water during the rendezvous season. Selection for anthropogenic linear features increased with increasing density of these features on the landscape, while selection for natural linear features declined, indicating compensatory selection of anthropogenic linear features. These results have implications for woodland caribou conservation. Prey encounter rates between wolves and caribou seem to be strongly influenced by increasing linear feature densities. This behavioral mechanism-a compensatory functional response to anthropogenic linear feature density resulting in decreased use of natural travel corridors-has negative consequences for the viability of woodland caribou.

Influenza virus imprinting is now understood to significantly influence the immune responses and clinical outcome of influenza virus infections that occur later in life. Due to the yearly cycling of influenza viruses, humans are imprinted with the circulating virus of their birth year and subsequently build a complex influenza virus immune history. Despite this knowledge, little is known about how the imprinting strain influences vaccine responses. To investigate the immune responses of the imprinted host to split-virion vaccination, we imprinted ferrets with a sublethal dose of the historical seasonal H1N1 strain A/USSR/90/1977. After a +60-day recovery period to build immune memory, ferrets were immunized and then challenged on Day 123. Antibody specificity and recall were investigated throughout the time course. At challenge, the imprinted vaccinated ferrets did not experience significant disease, while naïve-vaccinated ferrets had significant weight loss. Haemagglutination inhibition assays showed that imprinted ferrets had a more robust antibody response post vaccination and increased virus neutralization activity. Imprinted-vaccinated animals had increased virus-specific IgG antibodies compared to the other experimental groups, suggesting B-cell maturity and plasticity at vaccination. These results should be considered when designing the next generation of influenza vaccines.

The development of suitable separation technologies for the separation of carbon dioxide is a pressing technological requirement. The application of ion gel membranes for this purpose continues to stimulate a great deal of research, and in this study we focus on the chemical structure of the ionic liquid component in the ion gel, and its interactions with the sulfonated polyimide polymer. Whilst such membranes are known to give promising carbon dioxide separation properties together with mechanical strength and thin-film-processability, we further elaborate on how changing the cation of the ionic liquid from a typical imidazolium cation to a protic variant effects the physicochemical, thermal, and structural properties of the membranes, and how these changes further influence the carbon dioxide separation properties. We compare and contrast our findings with our earlier study on protic and aprotic ammonium-based ionic liquids, and highlight that for CO2 absorption behavior in the imidazolium systems, the importance of directionality of interactions (ion pairs exhibit a large energy stabilization only for a specific geometrical arrangement of cation and anion, e.g., hydrogen bonding rather than Coulombic interaction) between cation and anion applies not only to the protic system, but also to the nominally aprotic cation. Finally, we demonstrate that the phase separation behavior in the ion gels is an important factor in determining the carbon dioxide separation behavior.

Listeria monocytogenes is a Gram positive foodborne pathogen that can colonize the gastrointestinal tract of a number of hosts, including humans. These environments contain numerous stressors such as bile, low oxygen and acidic pH, which may impact the level of colonization and persistence of this organism within the GI tract. The ability of L. monocytogenes to establish infections and colonize the gastrointestinal tract is directly related to its ability to overcome these stressors, which is mediated by the efficient expression of several stress response mechanisms during its passage. This review will focus upon how and when this occurs and how this impacts the outcome of foodborne disease.

We present the complete mitochondrial genome of a newly described freshwater red alga Lympha mucosa. The genome was sequenced using the Illumina MiSeq platform. The circular mitochondrial genome is 25,191 bp, contains 46 genes (24 CDS, 20 tRNA, and 2 rRNA), and has an overall GC content of 27.5%. Phylogenetic analyses of the cox1 gene show the placement of Lympha mucosa within the strictly freshwater order Batrachospermales. The four mitochondrial genomes within the subclass Nemaliophycidae sequenced to date are highly conserved in terms of genome size, gene content, and gene synteny.

SUMMARY Fetal alcohol spectrum disorders (FASDs) are characterized by life-long changes in gene expression, neurodevelopment and behavior. What mechanisms initiate and maintain these changes are not known, but current research suggests a role for alcohol-induced epigenetic changes. In this study we assessed alterations to adult mouse brain tissue by assaying DNA cytosine methylation and small noncoding RNA (ncRNA) expression, specifically the microRNA (miRNA) and small nucleolar RNA (snoRNA) subtypes. We found long-lasting alterations in DNA methylation as a result of fetal alcohol exposure, specifically in the imprinted regions of the genome harboring ncRNAs and sequences interacting with regulatory proteins. A large number of major nodes from the identified networks, such as Pten signaling, contained transcriptional repressor CTCF-binding sites in their promoters, illustrating the functional consequences of alcohol-induced changes to DNA methylation. Next, we assessed ncRNA expression using two independent array platforms and quantitative PCR. The results identified 34 genes that are targeted by the deregulated miRNAs. Of these, four (Pten, Nmnat1, Slitrk2 and Otx2) were viewed as being crucial in the context of FASDs given their roles in the brain. Furthermore, ∼20% of the altered ncRNAs mapped to three imprinted regions (Snrpn-Ube3a, Dlk1-Dio3 and Sfmbt2) that showed differential methylation and have been previously implicated in neurodevelopmental disorders. The findings of this study help to expand on the mechanisms behind the long-lasting changes in the brain transcriptome of FASD individuals. The observed changes could contribute to the initiation and maintenance of the long-lasting effect of alcohol.

African Americans face institutional and social discrimination. The deleterious effects of discriminatory practices continue to be barriers to maintaining the family farm. Discriminatory lending is associated with farmland loss, such that the number of African American farmers in the United States has been falling at a much higher rate than that of White farmers. This community-based participatory research (CBPR) study sought to give voice to the experiences and perceptions of a small group of African American farmers in northeastern North Carolina. Researchers used Photovoice, a qualitative CBPR methodology, to identify strengths, concerns, and action steps in regards to farming and farmland loss in the community. This study revealed positive protective factors associated with farming, and long-lasting negative economic and psychological effects of discriminatory lending. Protective factors include increased self-reliance, strong work ethic, and hope for a new generation of African American farmers. Institutional discrimination remains a reported risk factor against maintaining generational family farming activities. Study participants reported a fear of further loss of the African American farming heritage as they perceive youth being deterred from the profession due to the combined effects of witnessing discrimination against their parents and the lure of fast, and often risky, money. This preliminary research revealed that African American farmers in the community of this study would benefit from innovative and engaging programs for youth, broadband internet access, and continued modification to current lending systems, including localized representation.

SUMMARY Myotubular myopathy (MTM) is a severe congenital muscle disease characterized by profound weakness, early respiratory failure and premature lethality. MTM is defined by muscle biopsy findings that include centralized nuclei and disorganization of perinuclear organelles. No treatments currently exist for MTM. We hypothesized that aberrant neuromuscular junction (NMJ) transmission is an important and potentially treatable aspect of the disease pathogenesis. We tested this hypothesis in two murine models of MTM. In both models we uncovered evidence of a disorder of NMJ transmission: fatigable weakness, improved strength with neostigmine, and electrodecrement with repetitive nerve stimulation. Histopathological analysis revealed abnormalities in the organization, appearance and size of individual NMJs, abnormalities that correlated with changes in acetylcholine receptor gene expression and subcellular localization. We additionally determined the ability of pyridostigmine, an acetylcholinesterase inhibitor, to ameliorate aspects of the behavioral phenotype related to NMJ dysfunction. Pyridostigmine treatment resulted in significant improvement in fatigable weakness and treadmill endurance. In all, these results describe a newly identified pathological abnormality in MTM, and uncover a potential disease-modifying therapy for this devastating disorder.

Paula Juliet Williams, Linda MorganHuman Genetics Research Group, University of Nottingham, Nottingham, UKAbstract: The pregnancy-specific condition pre-eclampsia not only affects the health of mother and baby during pregnancy but also has long-term consequences, increasing the chances of cardiovascular disease in later life. It is accepted that pre-eclampsia has a placental origin, but the pathogenic mechanisms leading to the systemic endothelial dysfunction characteristic of the disorder remain to be determined. In this review we discuss some key factors regarded as important in the development of pre-eclampsia, including immune maladaptation, inadequate placentation, oxidative stress, and thrombosis. Genetic factors influence all of these proposed pathophysiological mechanisms. The inherited nature of pre-eclampsia has been known for many years, and extensive genetic studies have been undertaken in this area. Genetic research offers an attractive strategy for studying the pathogenesis of pre-eclampsia as it avoids the ethical and practical difficulties of conducting basic science research during the preclinical phase of pre-eclampsia when the underlying pathological changes occur. Although pharmacogenomic studies have not yet been conducted in pre-eclampsia, a number of studies investigating treatment for essential hypertension are of relevance to therapies used in pre-eclampsia. The pharmacogenomics of antiplatelet agents, alpha and beta blockers, calcium channel blockers, and magnesium sulfate are discussed in relation to the treatment and prevention of pre-eclampsia. Pharmacogenomics offers the prospect of individualized patient treatment, ensuring swift introduction of optimal treatment whilst minimizing the use of inappropriate or ineffective drugs, thereby reducing the risk of harmful effects to both mother and baby.Keywords: pre-eclampsia, pharmacogenetics, placenta, trophoblast, genetics

This study explores the relationship between the Agulhas Current system and El Niño Southern Oscillation (ENSO) events. Specifically, it addresses monthly to yearly variations in Agulhas leakage where the Agulhas Current sheds waters into the Atlantic Ocean, in turn affecting meridional overturning circulation (MOC). Sea surface temperature (SST) data from the National Oceanic and Atmospheric Administration’s (NOAA) Advanced Very High Resolution Radiometer (AVHRR) combined with sea surface salinity (SSS) from Soil Moisture Ocean Salinity (SMOS) and Simple Ocean Data Assimilation (SODA) reanalysis are used to explore changes in Agulhas leakage dynamics. Agulhas leakage is anomalously warm in response to El Niño and anomalously cool in response to La Niña. The corresponding SSS signal shows both a primary and secondary signal response. At first, the SSS signal of Agulhas leakage is anomalously fresh in response to El Niño, but this primary signal is replaced by a secondary anomalously saline signal. In response to La Niña, the primary SSS signal of Agulhas leakage is anomalously saline, while the secondary SSS signal is anomalously fresh. The lag between the peak of ENSO and the response in SST and the corresponding primary SSS signal of Agulhas leakage is about 20 months, followed by the secondary SSS signal at a lag of about 26 months. In general, increasing ENSO strength increases the extremes of the resulting anomalous SST and SSS signal and impacts the Agulhas leakage region earlier during El Niño and slightly later during La Niña.

Morphological characters and molecular sequence data of the plastid-encoded RUBISCO large subunit gen (rbcL) confirmed the first record of Kumanoa mahlacensis (S. Kumano & W.A. Bowden-Kerby) Entwisle, M.L. Vis, W.B. Chiasson, Necchi & A.R. Sherwood in Europe from a Spanish Mediterranean coastal wetland. The genus Kumanoa has a worldwide distribution and inhabits in both lotic and lentic habitats, but information about the ecological requirements of species is scarce. In this study, the morphology and ecology of Spanish material is described in order to gain a broader insight of the distribution of species from Mediterranean areas.

In vitro disease modeling based on induced pluripotent stem cells (iPSCs) provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID), a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK) activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(D)J recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications.

Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80-90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum specimens should ensure that the techniques they are using are known to provide high-quality throughput DNA for later analysis.

Fire suppression and changing climate have resulted in increased large wildfire frequency and severity in the western United States, causing carbon cycle impacts. Forest thinning and prescribed burning reduce high-severity fire risk, but require removal of biomass and emissions of carbon from burning. During each fire a fraction of the burning vegetation and soil organic matter is converted into charcoal, a relatively stable carbon form. We sought to quantify the effects of pre-fire fuel load and type on charcoal carbon produced by biomass combusted in a prescribed burn under different thinning treatments and to identify more easily measured predictors of charcoal carbon mass in a historically frequent-fire mixed-conifer forest. We hypothesized that charcoal carbon produced from coarse woody debris (CWD) during prescribed burning would be greater than that produced from fine woody debris (FWD). We visually quantified post-treatment charcoal carbon content in the O-horizon and the A-horizon beneath CWD (> 30 cm diameter) and up to 60 cm from CWD that was present prior to treatment. We found no difference in the size of charcoal carbon pools from CWD (treatment means ranged from 0.3-2.0 g m-2 of A-horizon and 0.0-1.7 g m-2 of O-horizon charcoal) and FWD (treatment means ranged from 0.2-1.7 g m-2 of A-horizon and 0.0-1.5 g m-2 of O-horizon charcoal). We also compared treatments and found that the burn-only, understory-thin and burn, and overstory-thin and burn treatments had significantly more charcoal carbon than the control. Charcoal carbon represented 0.29% of total ecosystem carbon. We found that char mass on CWD was an important predictor of charcoal carbon mass, but only explained 18-35% of the variation. Our results help improve our understanding of the effects forest restoration treatments have on ecosystem carbon by providing additional information about charcoal carbon content.

Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy in need of new therapeutic options. Using unbiased analyses of super-enhancers (SEs) as sentinels of core genes involved in cell-specific function, here we uncover a druggable SE-mediated RNA-binding protein (RBP) cascade that supports PDAC growth through enhanced mRNA translation. This cascade is driven by a SE associated with the RBP heterogeneous nuclear ribonucleoprotein F, which stabilizes protein arginine methyltransferase 1 (PRMT1) to, in turn, control the translational mediator ubiquitin-associated protein 2-like. All three of these genes and the regulatory SE are essential for PDAC growth and coordinately regulated by the Myc oncogene. In line with this, modulation of the RBP network by PRMT1 inhibition reveals a unique vulnerability in Myc-high PDAC patient organoids and markedly reduces tumor growth in male mice. Our study highlights a functional link between epigenetic regulation and mRNA translation and identifies components that comprise unexpected therapeutic targets for PDAC.

Objective: Weight loss is a near societal obsession and many diet programs use significant calorie restriction (CR) including fasting/short term starvation to generate rapid effects. Fasting is also a well-recognized cause of immunosuppression especially within the innate immune system. In this study, we sought to determine if the IL-1 arm of the neuroimmune system was down-regulated by a 24 hr fast and how fasting might generate this effect. Design: Mice were allowed ad libitum access to food or had food withheld for 24 hrs. Expression of the endogenous IL-1 antagonists IL-1 receptor type 2 (IL-1R2) and IL-1 receptor antagonist (IL-1RA) were determined as were sickness behaviors before and after IL-1 administration.Results: Fasting markedly increased gene expression of IL-1R2 (83-fold in adipose tissue, 9.5-fold in liver) and IL-1RA (68-fold in liver). Fasted mice were protected from IL-1-induced weight loss, hypoglycemia, loss of locomotor and social anxiety. These protections were coupled to a large positive interaction of fasting and IL-1 on IL-1R2 gene expression in adipose tissue and liver (2.6-fold and 1.6-fold, respectively). Fasting not only increased IL-1RA and IL-1R2 protein 2.5-fold and 3.2-fold, respectively, in liver; but also increased IL-1R2 1.8-fold in adipose tissue. Fasting, in turn, triggered a 2.4-fold increase in plasma free-fatty acids (FFAs) and a 2.1-fold increase in plasma corticosterone. Inhibition, of glucocorticoid action with mifepristone did not impact fasting-dependent IL-1R2 or IL-1RA gene expression. Administration of the FFA, palmitate, to mice increased liver IL-1R2 and IL-1RA gene expression by 14-fold and 11-fold, respectively. Conclusion: These findings indicate that fasting augments expression of endogenous IL-1 antagonists inducing IL-1 resistance. Fasting-induced increases in plasma FFAs appears to be a signal that drives immunosuppression during fasting/short term starvation.

There is abundant evidence that prenatal alcohol exposure leads to a range of behavioural and cognitive impairments, categorized under the term fetal alcohol spectrum disorders (FASDs). These disorders are pervasive in Western cultures and represent the most common preventable source of neurodevelopmental disabilities. The genetic and epigenetic etiology of these phenotypes, including those factors that may maintain these phenotypes throughout the lifetime of an affected individual, has become a recent topic of investigation. This review integrates recent data that has progressed our understanding FASD as a continuum of molecular events, beginning with cellular stress response and ending with a long-term ‘footprint’ of epigenetic dysregulation across the genome. It reports on data from multiple ethanol-treatment paradigms in mouse models that identify changes in gene expression that occur with respect to neurodevelopmental timing of exposure and ethanol dose. These studies have identified patterns of genomic alteration that are dependent on the biological processes occurring at the time of ethanol exposure. This review also adds to evidence that epigenetic processes such as DNA methylation, histone modifications, and non-coding RNA regulation may underlie long-term changes to gene expression patterns. These may be initiated by ethanol-induced alterations to DNA and histone methylation, particularly in imprinted regions of the genome, affecting transcription which is further fine-tuned by altered microRNA expression. These processes are likely complex, genome-wide, and interrelated. The proposed model suggests a potential for intervention, given that epigenetic changes are malleable and may be altered by postnatal environment. This review accentuates the value of mouse models in deciphering the molecular etiology of FASD, including those processes that may provide a target for the ammelioration of this common yet entirely preventable disorder.

Rectal microbicides are being developed to prevent new HIV infections in both men and women. We focused our in vivo preclinical efficacy study on rectally-applied tenofovir. BLT humanized mice (n = 43) were rectally inoculated with either the primary isolate HIV-1JRCSF or the MSM-derived transmitted/founder (T/F) virus HIV-1THRO within 30 minutes following treatment with topical 1% tenofovir or vehicle. Under our experimental conditions, in the absence of drug treatment we observed 50% and 60% rectal transmission by HIV-1JRCSF and HIV-1THRO, respectively. Topical tenofovir reduced rectal transmission to 8% (1/12; log rank p = 0.03) for HIV-1JRCSF and 0% (0/6; log rank p = 0.02) for HIV-1THRO. This is the first demonstration that any human T/F HIV-1 rectally infects humanized mice and that transmission of the T/F virus can be efficiently blocked by rectally applied 1% tenofovir. These results obtained in BLT mice, along with recent ex vivo, Phase 1 trial and non-human primate reports, provide a critically important step forward in the development of tenofovir-based rectal microbicides.

White-nose syndrome (WNS) is an emerging infectious disease devastating hibernating North American bat populations that is caused by the psychrophilic fungus Geomyces destructans. Previous histopathological analysis demonstrated little evidence of inflammatory responses in infected bats, however few studies have compared other aspects of immune function between WNS-affected and unaffected bats. We collected bats from confirmed WNS-affected and unaffected sites during the winter of 2008-2009 and compared estimates of their circulating levels of total leukocytes, total immunoglobulins, cytokines and total antioxidants. Bats from affected and unaffected sites did not differ in their total circulating immunoglobulin levels, but significantly higher leukocyte counts were observed in bats from affected sites and particularly in affected bats with elevated body temperatures (above 20°C). Bats from WNS-affected sites exhibited significantly lower antioxidant activity and levels of interleukin-4 (IL-4), a cytokine that induces T cell differentiation. Within affected sites only, bats exhibiting visible fungal infections had significantly lower antioxidant activity and levels of IL-4 compared to bats without visible fungal infections. Overall, bats hibernating in WNS-affected sites showed immunological changes that may be evident of attempted defense against G. destructans. Observed changes, specifically elevated circulating leukocytes, may also be related to the documented changes in thermoregulatory behaviors of affected bats (i.e. increased frequencies in arousal from torpor). Alterations in immune function may reflect expensive energetic costs associated with these processes and intrinsic qualities of the immunocapability of hibernating bats to clear fungal infections. Additionally, lowered antioxidant activity indicates a possible imbalance in the pro- versus antioxidant system, may reflect oxidative tissue damage, and should be investigated as a contributor to WNS-associated morbidity and mortality.

We have previously reported a simple and customizable CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided Cas9 nuclease (RGN) system that can be used to efficiently and robustly introduce somatic indel mutations in endogenous zebrafish genes. Here we demonstrate that RGN-induced mutations are heritable, with efficiencies of germline transmission reaching as high as 100%. In addition, we extend the power of the RGN system by showing that these nucleases can be used with single-stranded oligodeoxynucleotides (ssODNs) to create precise intended sequence modifications, including single nucleotide substitutions. Finally, we describe and validate simple strategies that improve the targeting range of RGNs from 1 in every 128 basepairs (bps) of random DNA sequence to 1 in every 8 bps. Together, these advances expand the utility of the CRISPR-Cas system in the zebrafish beyond somatic indel formation to heritable and precise genome modifications.

The ability to direct human telomerase reverse transcriptase (hTERT) expression through either genetic control or tunable regulatory factors would advance not only our understanding of the transcriptional regulation of this gene, but also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and ZF nucleases (ZFNs) to target sequences within the hTERT promoter and exon-1. We were able to identify several active ZFTFs that demonstrate a broadly tunable response when screened by a cell-based transcriptional reporter assay. Using the same DNA-binding domains, we generated ZFNs that were screened in combinatorial pairs in cell-based extrachromosomal single-strand annealing (SSA) assays and in gene-targeting assays using stably integrated constructs. Selected ZFN pairs were tested for the ability to induce sequence changes in a Cel1 assay and we observed frequencies of genomic modification up to 18.7% at the endogenous hTERT locus. These screening strategies have pinpointed several ZFN pairs that may be useful in gene editing of the hTERT locus. Our work provides a foundation for using engineered ZF proteins (ZFPs) for modulation of the hTERT locus.