Characterizing the Human Mycobiota: A Comparison of Small Subunit rRNA, ITS1, ITS2, and Large Subunit rRNA Genomic Targets

Interest in the human microbiome has increased dramatically in the last decade. However, much of this research has focused on bacteria, while the composition and roles of their fungal counterparts remain less understood. Furthermore, a variety of methodological approaches have been applied, and the comparability between studies is unclear. This study compared four primer pairs targeting the small subunit (SSU) rRNA (18S), ITS1, ITS2, and large subunit (LSU) rRNA (26S) genomic regions for their ability to accurately characterize fungal communities typical of the human mycobiota. All four target regions of 21 individual fungal mock community taxa were capable of being amplified adequately and sequenced. Mixed mock community analyses revealed marked variability in the ability of each primer pair to accurately characterize a complex community. ITS target regions outperformed LSU and SSU. Of the ITS regions, ITS1 failed to generate sequences for Yarrowia lipolytica and all three Malassezia species when in a mixed community. These findings were further supported in studies of human sinonasal and mouse fecal samples. Based on these analyses, previous studies using ITS1, SSU, or LSU markers may omit key taxa that are identified by the ITS2 marker. Of methods commonly used in human mycobiota studies to date, we recommend selection of the ITS2 marker. Further investigation of more recently developed fungal primer options will be essential to ultimately determine the optimal methodological approach by which future human mycobiota studies ought to be standardized

Inflammatory Endotypes and Microbial Associations in Chronic Rhinosinusitis

A complex mix of inflammatory and microbial associations underscores the chronic inflammatory condition chronic rhinosinusitis (CRS), and the etiology remains poorly understood. Recent work has begun to delineate between variants (endotypes) of CRS on the basis of inflammatory biomarkers. This study aimed to assess inflammatory patterns in CRS phenotypes, identify putative endotypes of CRS, and to assess inflammatory associations with the sinonasal microbiota. Ten cytokines and six inflammatory cell types were assessed in mucosal biopsies from 93 CRS subjects and 17 controls via cytometric bead array and immunohistochemical techniques. Putative endotypes were identified via cluster analysis of subjects on the basis of inflammatory markers and comorbidities including polyposis, asthma, and aspirin sensitivity. Finally, previously published bacterial data for this cohort were reanalyzed to evaluate associations with inflammatory markers and CRS subtypes. Inflammatory patterns were highly variable within standard CRS phenotypes. Cluster analysis identified eight subject clusters, with strong delineation on the basis of polyposis and asthma, but also subtle distinctions in inflammatory markers. An association was also identified between depletion of several “health-associated” bacterial taxa, reduced bacterial diversity and increased overall bacterial load, with markers of inflammation and clinical severity. This study contributes to ongoing efforts to define distinct endotypes of CRS on the basis of underlying inflammatory processes, and also offers compelling evidence of a link between bacterial community dysbiosis and inflammation in CRS. Further resolving the heterogeneity of CRS is vital to inform clinical management and personalized treatment approaches

Sequencing, Mapping, and Analysis of 27,455 Maize Full-Length cDNAs

Full-length cDNA (FLcDNA) sequencing establishes the precise primary structure of individual gene transcripts. From two libraries representing 27 B73 tissues and abiotic stress treatments, 27,455 high-quality FLcDNAs were sequenced. The average transcript length was 1.44 kb including 218 bases and 321 bases of 5′ and 3′ UTR, respectively, with 8.6% of the FLcDNAs encoding predicted proteins of fewer than 100 amino acids. Approximately 94% of the FLcDNAs were stringently mapped to the maize genome. Although nearly two-thirds of this genome is composed of transposable elements (TEs), only 5.6% of the FLcDNAs contained TE sequences in coding or UTR regions. Approximately 7.2% of the FLcDNAs are putative transcription factors, suggesting that rare transcripts are well-enriched in our FLcDNA set. Protein similarity searching identified 1,737 maize transcripts not present in rice, sorghum, Arabidopsis, or poplar annotated genes. A strict FLcDNA assembly generated 24,467 non-redundant sequences, of which 88% have non-maize protein matches. The FLcDNAs were also assembled with 41,759 FLcDNAs in GenBank from other projects, where semi-strict parameters were used to identify 13,368 potentially unique non-redundant sequences from this project. The libraries, ESTs, and FLcDNA sequences produced from this project are publicly available. The annotated EST and FLcDNA assemblies are available through the maize FLcDNA web resource (www.maizecdna.org)

Regulatory role of orexin on postnatal maturation of the central vestibular system in rats

Orexin is a hypothalamic neuropeptide which is associated with motor function in the vestibular nucleus (VN) complex. Deficiency of orexin in adult rats results in a sudden loss of muscle strength as in cataplexy and deterioration in the performance of vestibular-mediated behaviors. To evaluate whether early postnatal perturbation of orexinergic circuits in the VN exerts any effects on the vestibular-related behaviors, we perturbed the activities of orexin receptors in the VN of postnatal day (P) 1 rat by implanting polymer (Elvax) slices loaded with orexin, orexin receptor antagonist (SB334867) or agonist ([Ala11, D-Leu15]-orexin-B) onto the dorsal surface of VN for slow release of the drug to the underlying VN. Specific behavioral tests including negative geotaxis, surface righting, air righting, balanced beam, rotarod, tail suspension, and dead reckoning were performed on these rats at different postnatal ages. Improvement of vestibular-related behavior was observed in rats pretreated with orexin receptor antagonist, while derangement of vestibular-related behaviors were observed in rats pretreated with orexin or its agonist. Interestingly, perturbation delivered to the VN at P14 did not impose any effect on animals’ behavior, indicating the presence of a critical period of orexinergic perturbation. To ascertain the source of orexinergic projection, we conducted behavioral experiments in rats which had received stereotaxic injection of a designed shOrexin adeno-associated virus (AAV) into the lateral hypothalamus (LH) of rat pups at P4. The virus expresses designed shRNA cocktails which reduces orexin mRNA and protein expression by more than 2 folds as indicated in results of western blot and quantitative polymerase chain reaction. Similar alternation in vestibular behavior were also observed in the experiments of air righting, tail suspension and dead reckoning, thereby providing further evidence with regard to the role of orexin on functional maturation of the VN complex during postnatal development. Given that glutamatergic receptors at the VN constitutes to the coordination of motor function, we hypothesized that behavioral alternation upon early orexinergic perturbation may be associated with glutamatergic circuits in the VN. Significant decrease in protein levels of AMPA receptor subunit GluA1 and GluR2 as well as NMDA receptor GluR1 were observed in the medial vestibular nucleus (MVN) of P6 rats pretreated at P1 with orexin and its agonist. In addition, significant increase in spine density and maturation were observed in the MVN of P14 rats pretreated at P1 with orexin and its agonist. Such change in morphology of dendritic spine among MVN neurons may account for the delay in behavioral emergence of vestibular-related responses. Altogether, our findings indicate that orexin modulates the glutamatergic circuits within the VN, and thereby regulate the expression of vestibular behavior and dendritic spine morphology of the MVN neuron. These provide us insights for development of novel therapeutic approach in children patients suffered from cataplexy and vestibular disorder.published_or_final_versionBiological SciencesMasterMaster of Philosoph

Alpha diversity measurements (mean ± SEM) for triplicate data from each of the four DNA extraction methods, amplified with 16S rRNA gene primers (A) and ITS1 primers (B).

<p>Alpha diversity measurements (mean ± SEM) for triplicate data from each of the four DNA extraction methods, amplified with 16S rRNA gene primers (A) and ITS1 primers (B).</p

Summary of DNA extraction methods examined in this study.

<p>Summary of DNA extraction methods examined in this study.</p

A role for triglyceride lipase \u3ci\u3ebrummer\u3c/i\u3e in the regulation of sex differences in \u3ci\u3eDrosophila\u3c/i\u3e fat storage and breakdown

Triglycerides are the major form of stored fat in all animals. One important determinant of whole-body fat storage is whether an animal is male or female. Here, we use Drosophila, an established model for studies on triglyceride metabolism, to gain insight into the genes and physiological mechanisms that contribute to sex differences in fat storage. Our analysis of triglyceride storage and breakdown in both sexes identified a role for triglyceride lipase brummer (bmm) in the regulation of sex differences in triglyceride homeostasis. Normally, male flies have higher levels of bmm mRNA both under normal culture conditions and in response to starvation, a lipolytic stimulus. We find that loss of bmm largely eliminates the sex difference in triglyceride storage and abolishes the sex difference in triglyceride breakdown via strongly male-biased effects. Although we show that bmm function in the fat body affects whole-body triglyceride levels in both sexes, in males, we identify an additional role for bmm function in the somatic cells of the gonad and in neurons in the regulation of whole-body triglyceride homeostasis. Furthermore, we demonstrate that lipid droplets are normally present in both the somatic cells of the male gonad and in neurons, revealing a previously unrecognized role for bmm function, and possibly lipid droplets, in these cell types in the regulation of whole-body triglyceride homeostasis. Taken together, our data reveal a role for bmm function in the somatic cells of the gonad and in neurons in the regulation of male–female differences in fat storage and breakdown and identify bmm as a link between the regulation of triglyceride homeostasis and biological sex

Differentially Regulated Host Proteins Associated with Chronic Rhinosinusitis Are Correlated with the Sinonasal Microbiome

The chronic inflammatory nature of chronic rhinosinusitis (CRS) makes it a morbid condition for individuals with the disease and one whose pathogenesis is poorly understood. To date, proteomic approaches have been applied successfully in a handful of CRS studies. In this study we use a multifaceted approach, including proteomics (iTRAQ labeling) and microbiome (bacterial 16S rRNA gene sequencing) analyses of middle meatus swabs, as well as immune cell analysis of the underlying tissue, to investigate the host-microbe interaction in individuals with CRS (n = 10) and healthy controls (n = 9). Of the total 606 proteins identified in this study, seven were significantly (p &lt; 0.05) more abundant and 104 were significantly lower in the CRS cohort compared with healthy controls. The majority of detected proteins (82% of proteins identified) were not significantly correlated with disease status. Elevated levels of blood and immune cell proteins in the CRS cohort, together with significantly higher numbers of B-cells and macrophages in the underlying tissue, confirmed the inflammatory status of CRS individuals. Protein PRRC2C and Ras-related protein (RAB14) (two of the seven elevated proteins) showed the biggest fold difference between the healthy and CRS groups. Validation of the elevated levels of these two proteins in CRS samples was provided by immunohistochemistry. Members of the bacterial community in the two study cohorts were not associated with PRRC2C, however members of the genus Moraxella did correlate with RAB14 (p &lt; 0.0001, rho = −0.95), which is a protein involved in the development of basement membrane. In addition, significant correlations between certain members of the CRS bacterial community and 33 lower abundant proteins in the CRS cohort were identified. Members of the genera Streptococcus, Haemophilus and Veillonella were strongly correlated with CRS and were significantly associated with a number of proteins with varying functions. The results from this study reveal a strong association between the host and microbes in the sinonasal cavity. Proteins identified as associated with CRS could be new targets for drug therapies and biomarkers for assessment of treatment efficacy