Validation of Normal Human Bronchial Epithelial Cells as a Model for Influenza A Infections in Human Distal Trachea

Primary normal human bronchial/tracheal epithelial (NHBE) cells, derived from the distal-most aspect of the trachea at the bifurcation, have been used for a number of studies in respiratory disease research. Differences between the source tissue and the differentiated primary cells may impact infection studies based on this model. Therefore, we examined how well-differentiated NHBE cells compared with their source tissue, the human distal trachea, as well as the ramifications of these differences on influenza A viral pathogenesis research using this model. We employed a histological analysis including morphological measurements, electron microscopy, multi-label immunofluorescence confocal microscopy, lectin histochemistry, and microarray expression analysis to compare differentiated NHBEs to human distal tracheal epithelium. Pseudostratified epithelial height, cell type variety and distribution varied significantly. Electron microscopy confirmed differences in cellular attachment and paracellular junctions. Influenza receptor lectin histochemistry revealed that α2,3 sialic acids were rarely present on the apical aspect of the differentiated NHBE cells, but were present in low numbers in the distal trachea. We bound fluorochrome bioconjugated virus to respiratory tissue and NHBE cells and infected NHBE cells with human influenza A viruses. Both indicated that the pattern of infection progression in these cells correlated with autopsy studies of fatal cases from the 2009 pandemic.     

LLY-507, a Cell-active,Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2

SMYD2 is a lysine methyltransferase that catalyzes the monomethylation of several protein substrates including p53. SMYD2 is overexpressed in a significant percentage of esophageal squamous primary carcinomas, and that overexpression correlates with poor patient survival. However, the mechanism(s) by which SMYD2 promotes oncogenesis is not understood. A small molecule probe for SMYD2 would allow for the pharmacological dissection of this biology. In this report, we disclose LLY-507, a cell-active, potent small molecule inhibitor of SMYD2. LLY-507 is >100-fold selective for SMYD2 over a broad range of methyltransferase and non-methyltransferase targets. A 1.63-Å resolution crystal structure of SMYD2 in complex with LLY-507 shows the inhibitor binding in the substrate peptide binding pocket. LLY-507 is active in cells as measured by reduction of SMYD2-induced monomethylation of p53 Lys³⁷⁰ at submicromolar concentrations. We used LLY-507 to further test other potential roles of SMYD2. Mass spectrometry-based proteomics showed that cellular global histone methylation levels were not significantly affected by SMYD2 inhibition with LLY-507, and subcellular fractionation studies indicate that SMYD2 is primarily cytoplasmic, suggesting that SMYD2 targets a very small subset of histones at specific chromatin loci and/or non-histone substrates. Breast and liver cancers were identified through in silico data mining as tumor types that display amplification and/or overexpression of SMYD2. LLY-507 inhibited the proliferation of several esophageal, liver, and breast cancer cell lines in a dose-dependent manner. These findings suggest that LLY-507 serves as a valuable chemical probe to aid in the dissection of SMYD2 function in cancer and other biological processes.     

Novel H7N9 Influenza Virus Shows Low Infectious Dose,High Growth Rate,and Efficient Contact Transmission in the Guinea Pig Model

The zoonotic outbreak of H7N9 subtype avian influenza virus that occurred in eastern China in the spring of 2013 resulted in 135 confirmed human cases, 44 of which were lethal. Sequencing of the viral genome revealed a number of molecular signatures associated with virulence or transmission in mammals. We report here that, in the guinea pig model, a human isolate of novel H7N9 influenza virus, A/Anhui/1/2013 (An/13), is highly dissimilar to an H7N1 avian isolate and instead behaves similarly to a human seasonal strain in several respects. An/13 was found to have a low 50% infectious dose, grow to high titers in the upper respiratory tract, and transmit efficiently among cocaged guinea pigs. The pH of fusion of the hemagglutinin (HA) and the binding of virus to fixed guinea pig tissues were also examined. The An/13 HA displayed a relatively elevated pH of fusion characteristic of many avian strains, and An/13 resembled avian viruses in terms of attachment to tissues. One important difference was seen between An/13 and both the H3N2 human and the H7N1 avian viruses: when inoculated intranasally at a high dose, only the An/13 virus led to productive infection of the lower respiratory tract of guinea pigs. In sum, An/13 was found to retain fusion and attachment properties of an avian influenza virus but displayed robust growth and contact transmission in the guinea pig model atypical of avian strains and indicative of mammalian adaptation.     

The Effects of an Animal-Assisted Intervention on Salivary Alpha-Amylase,Salivary Immunoglobulin A,and Heart Rate during Forensic Interviews in Child Sexual Abuse Cases

Animal-assisted invention (AAI) in gaining attention as a therapeutic modality; however, the effect of it has not been well studied in the child welfare system. The purpose of this study was to examine the effects of AAI on stress indicators (as measured by salivary alpha-amylase (sAA), immunoglobulin A (IgA), and heart rate) in children undergoing forensic interviews for alleged sexual abuse. A repeated measures design was used in this study. Upon receiving signed, informed consents and assents, children were enrolled in the study. Children (n = 42), whose ages ranged from 5 to 14 years (M = 8.91, SD = 2.33), were assigned to either the intervention condition (n = 19; AAI during forensic interview) or the control condition (n = 23; standard practice forensic interview). Each child's parent/guardian completed a demographic form, and saliva samples and heart rate measures were obtained from each child before and after the forensic interview. Mixed linear models were tested, with the level of significance set at p ≤ 0.05. There was an interactive effect regarding the duration of the interview and the presence of the dog on sAA after the forensic interview (p = 0.047). There also was a significant interaction between age and length of interview (p = 0.01). Salivary immunoglobulin A tended to be lower (p = 0.055) when the therapy dog was present during the forensic interview. Results further indicated that the drop in heart rate was greater in longer interviews and with older children (p = 0.02) when the dog was present. Individuals working in child welfare systems can use the results of this study to advocate for the use of therapy dogs as a therapeutic intervention. More research is needed to further examine the relationships among AAI, salivary biomarkers, and stress responses in children to improve child welfare.     

Elevated atmospheric CO2 stimulates soil fungal diversity through increased fine root production in a semiarid shrubland ecosystem

Soil fungal communities are likely to be central in mediating microbial feedbacks to climate change through their effects on soil carbon (C) storage, nutrient cycling, and plant health. Plants often produce increased fine root biomass in response to elevated atmospheric carbon dioxide (CO₂), but the responses of soil microbial communities are variable and uncertain, particularly in terms of species diversity. In this study, we describe the responses of the soil fungal community to free air CO₂ enrichment (FACE) in a semiarid chaparral shrubland in Southern California (dominated by Adenomstoma fasciculatum) using large subunit rRNA gene sequencing. Community composition varied greatly over the landscape and responses to FACE were subtle, involving a few specific groups. Increased frequency of Sordariomycetes and Leotiomycetes, the latter including the Helotiales, a group that includes many dark septate endophytes known to associate positively with roots, was observed in the FACE plots. Fungal diversity, both in terms of richness and evenness, increased consistently in the FACE treatment, and was relatively high compared to other studies that used similar methods. Increases in diversity were observed across multiple phylogenetic levels, from genus to class, and were distributed broadly across fungal lineages. Diversity was also higher in samples collected close to (5 cm) plants compared to samples in canopy gaps (30 cm away from plants). Fungal biomass correlated well with soil organic matter (SOM) content, but patterns of diversity were correlated with fine root production rather than SOM. We conclude that the fungal community in this ecosystem is tightly linked to plant fine root production, and that future changes in the fungal community in response to elevated CO₂ and other climatic changes will be primarily driven by changes in plant belowground allocation. Potential feedbacks mediated by soil fungi, such as soil C sequestration, nutrient cycling, and pathogenesis, are discussed.     

Microhabitat of critically endangered Lupinus aridorum (Fabaceae) at wild and introduced locations in Florida scrub

Elucidating microhabitat preferences of a rare species are critical for its conservation. Lupinus aridorum McFarlin ex Beckner (Fabaceae) is a critically endangered plant known only from a few locations in imperiled Florida scrub habitat and nothing is known about its preferred microhabitat. Our goals were threefold. First, determine whether L. aridorum has multiple cytotypes because this can influence its spatial distribution. Second, measure how microhabitat characteristics at locations supporting wild L. aridorum vary from random locations, which will provide information about microhabitat characteristics that influence the spatial distribution of individuals. Third, measure whether microhabitat characteristics differ between locations supporting wild or introduced plants, which will provide information about the realized and fundamental niche. Our research determined that L. aridorum is diploid and grew, on average, in areas closer to trees and shrubs, with lower soil moisture, and with a greater mixture of detritus than random locations. Some microhabitat characteristics at locations where L. aridorum were introduced were similar to microhabitat supporting wild L. aridorum, but multiple soil characteristics differed as did the plant community, which contained more nonnative plant species near introduced plants. Therefore, the realized niche is narrower than the fundamental niche. Overall, information about the microhabitat of L. aridorum can be used to design appropriate management programs to conserve and restore populations of this plant species and species that occupy a similar niche in imperiled Florida scrub.     

Phylum-wide comparative genomics unravel the diversity of secondary metabolism in Cyanobacteria

Background

Cyanobacteria are an ancient lineage of photosynthetic bacteria from which hundreds of natural products have been described, including many notorious toxins but also potent natural products of interest to the pharmaceutical and biotechnological industries. Many of these compounds are the products of non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways. However, current understanding of the diversification of these pathways is largely based on the chemical structure of the bioactive compounds, while the evolutionary forces driving their remarkable chemical diversity are poorly understood.

Results

We carried out a phylum-wide investigation of genetic diversification of the cyanobacterial NRPS and PKS pathways for the production of bioactive compounds. 452 NRPS and PKS gene clusters were identified from 89 cyanobacterial genomes, revealing a clear burst in late-branching lineages. Our genomic analysis further grouped the clusters into 286 highly diversified cluster families (CF) of pathways. Some CFs appeared vertically inherited, while others presented a more complex evolutionary history. Only a few horizontal gene transfers were evidenced amongst strongly conserved CFs in the phylum, while several others have undergone drastic gene shuffling events, which could result in the observed diversification of the pathways.

Conclusions

Therefore, in addition to toxin production, several NRPS and PKS gene clusters are devoted to important cellular processes of these bacteria such as nitrogen fixation and iron uptake. The majority of the biosynthetic clusters identified here have unknown end products, highlighting the power of genome mining for the discovery of new natural products.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-977) contains supplementary material, which is available to authorized users.     

Morning Surge of Ventricular Arrhythmias in a New Arrhythmogenic Canine Model of Chronic Heart Failure Is Associated with Attenuation of Time-Of-Day Dependence of Heart Rate and Autonomic Adaptation,and Reduced Cardiac Chaos

Patients with chronic heart failure (CHF) exhibit a morning surge in ventricular arrhythmias, but the underlying cause remains unknown. The aim of this study was to determine if heart rate dynamics, autonomic input (assessed by heart rate variability (HRV)) and nonlinear dynamics as well as their abnormal time-of-day-dependent oscillations in a newly developed arrhythmogenic canine heart failure model are associated with a morning surge in ventricular arrhythmias. CHF was induced in dogs by aortic insufficiency & aortic constriction, and assessed by echocardiography. Holter monitoring was performed to study time-of-day-dependent variation in ventricular arrhythmias (PVCs, VT), traditional HRV measures, and nonlinear dynamics (including detrended fluctuations analysis α1 and α2 (DFAα1 & DFAα2), correlation dimension (CD), and Shannon entropy (SE)) at baseline, as well as 240 days (240d) and 720 days (720d) following CHF induction. LV fractional shortening was decreased at both 240d and 720d. Both PVCs and VT increased with CHF duration and showed a morning rise (2.5-fold & 1.8-fold increase at 6 AM-noon vs midnight-6 AM) during CHF. The morning rise in HR at baseline was significantly attenuated by 52% with development of CHF (at both 240d & 720d). Morning rise in the ratio of low frequency to high frequency (LF/HF) HRV at baseline was markedly attenuated with CHF. DFAα1, DFAα2, CD and SE all decreased with CHF by 31, 17, 34 and 7%, respectively. Time-of-day-dependent variations in LF/HF, CD, DFA α1 and SE, observed at baseline, were lost during CHF. Thus in this new arrhythmogenic canine CHF model, attenuated morning HR rise, blunted autonomic oscillation, decreased cardiac chaos and complexity of heart rate, as well as aberrant time-of-day-dependent variations in many of these parameters were associated with a morning surge of ventricular arrhythmias.     

The Antipsychotic Olanzapine Interacts with the Gut Microbiome to Cause Weight Gain in Mouse

The second-generation antipsychotic olanzapine is effective in reducing psychotic symptoms but can cause extreme weight gain in human patients. We investigated the role of the gut microbiota in this adverse drug effect using a mouse model. First, we used germ-free C57BL/6J mice to demonstrate that gut bacteria are necessary and sufficient for weight gain caused by oral delivery of olanzapine. Second, we surveyed fecal microbiota before, during, and after treatment and found that olanzapine potentiated a shift towards an “obesogenic” bacterial profile. Finally, we demonstrated that olanzapine has antimicrobial activity in vitro against resident enteric bacterial strains. These results collectively provide strong evidence for a mechanism underlying olanzapine-induced weight gain in mouse and a hypothesis for clinical translation in human patients.