Spectroscopic signatures of doorway-mediated intersystem crossing

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009.Vita.Includes bibliographical references (p. 229-239).Intersystem crossing in acetylene, C₂H₂, is described by a doorway-mediated model, where singlet-triplet mixing between the first excited singlet~ state, S₁, and the manifold of low-lying T₁,₂ states is controlled by special vibrational levels of the third excited triplet state, T₃. However, the influence of T₃ doorway states on the ~A¹Au <-- ~X¹[sigma]+g spectrum of acetylene is not well characterized for most S₁ vibrational levels. The doorway model is formulated to account for the LIF/SEELEM spectra of S₁ vibrational sublevels that are weakly perturbed by T₃ doorway states. The structure and properties of a doorway-mediated effective Hamiltonian are discussed, and a method for spectral deconvolution is presented for the doorway model. The dependence of fluorescence center-of-gravity on the time delay of a gated integration region is developed as an analysis tool for LIF spectroscopy. Triplet perturbations in the spectra of four S₁ vibrational sublevels are discussed in terms of the doorway model, using this new tool. IR-UV double resonance LIF/SEELEM spectroscopy is used to investigate the role of the torsional and "cis"-bending vibrational modes of S₁ in the promotion of vibrational overlap between levels of S₁ and T₃. A collisional method for the population of metastable molecules in a pulsed supersonic expansion is presented and discussed.by Kyle Lee Bittinger.Ph.D

An in vitro analysis of how lactose modifies the gut microbiota structure and function of adults in a donor-independent manner

IntroductionFollowing consumption of milk, lactose, a disaccharide of glucose and galactose, is hydrolyzed and absorbed in the upper gastrointestinal tract. However, hydrolysis and absorption are not always absolute, and some lactose will enter the colon where the gut microbiota is able to hydrolyze lactose and produce metabolic byproducts.MethodsHere, the impact of lactose on the gut microbiota of healthy adults was examined, using a short-term, in vitro strategy where fecal samples harvested from 18 donors were cultured anaerobically with and without lactose. The data were compiled to identify donor-independent responses to lactose treatment.Results and discussionMetagenomic sequencing found that the addition of lactose decreased richness and evenness, while enhancing prevalence of the β-galactosidase gene. Taxonomically, lactose treatment decreased relative abundance of Bacteroidaceae and increased lactic acid bacteria, Lactobacillaceae, Enterococcaceae, and Streptococcaceae, and the probiotic Bifidobacterium. This corresponded with an increased abundance of the lactate utilizers, Veillonellaceae. These structural changes coincided with increased total short-chain fatty acids (SCFAs), specifically acetate, and lactate. These results demonstrated that lactose could mediate the gut microbiota of healthy adults in a donor-independent manner, consistent with other described prebiotics, and provided insight into how dietary milk consumption may promote human health through modifications of the gut microbiome

Normalization and microbial differential abundance strategies depend upon data characteristics

BackgroundData from 16S ribosomal RNA (rRNA) amplicon sequencing present challenges to ecological and statistical interpretation. In particular, library sizes often vary over several ranges of magnitude, and the data contains many zeros. Although we are typically interested in comparing relative abundance of taxa in the ecosystem of two or more groups, we can only measure the taxon relative abundance in specimens obtained from the ecosystems. Because the comparison of taxon relative abundance in the specimen is not equivalent to the comparison of taxon relative abundance in the ecosystems, this presents a special challenge. Second, because the relative abundance of taxa in the specimen (as well as in the ecosystem) sum to 1, these are compositional data. Because the compositional data are constrained by the simplex (sum to 1) and are not unconstrained in the Euclidean space, many standard methods of analysis are not applicable. Here, we evaluate how these challenges impact the performance of existing normalization methods and differential abundance analyses.ResultsEffects on normalization: Most normalization methods enable successful clustering of samples according to biological origin when the groups differ substantially in their overall microbial composition. Rarefying more clearly clusters samples according to biological origin than other normalization techniques do for ordination metrics based on presence or absence. Alternate normalization measures are potentially vulnerable to artifacts due to library size. Effects on differential abundance testing: We build on a previous work to evaluate seven proposed statistical methods using rarefied as well as raw data. Our simulation studies suggest that the false discovery rates of many differential abundance-testing methods are not increased by rarefying itself, although of course rarefying results in a loss of sensitivity due to elimination of a portion of available data. For groups with large (~10×) differences in the average library size, rarefying lowers the false discovery rate. DESeq2, without addition of a constant, increased sensitivity on smaller datasets (&lt;20 samples per group) but tends towards a higher false discovery rate with more samples, very uneven (~10×) library sizes, and/or compositional effects. For drawing inferences regarding taxon abundance in the ecosystem, analysis of composition of microbiomes (ANCOM) is not only very sensitive (for &gt;20 samples per group) but also critically the only method tested that has a good control of false discovery rate.ConclusionsThese findings guide which normalization and differential abundance techniques to use based on the data characteristics of a given study

Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags

Intense interest centers on the role of the human gut microbiome in health and disease, but optimal methods for analysis are still under development. Here we present a study of methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene tags. We analyzed fecal samples from 10 individuals and compared methods for storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80°C, 2) storage on ice for 24 or 3) 48 hours. For DNA purification methods, we tested three commercial kits and bead beating in hot phenol. Variations due to the different methodologies were compared to variation among individuals using two approaches--one based on presence-absence information for bacterial taxa (unweighted UniFrac) and the other taking into account their relative abundance (weighted UniFrac). In the unweighted analysis relatively little variation was associated with the different analytical procedures, and variation between individuals predominated. In the weighted analysis considerable variation was associated with the purification methods. Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method. We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments. Based on our findings we present recommendations for protocols to collect, process and sequence bacterial 16S rDNA from fecal samples--some major points are 1) if feasible, bead-beating in hot phenol or use of the PSP kit improves recovery; 2) storage methods can be adjusted based on experimental convenience; 3) unweighted (presence-absence) comparisons are less affected by lysis method

Aktivitas Antibakteri Ekstrak Dan Fraksi Karang Lunak Lobophytum SP. Terhadap Bakteri Escherichia Coli Dan Staphylococcus Aureus

AKTIVITAS ANTIBAKTERI EKSTRAK DAN FRAKSI KARANG LUNAK Lobophytum sp. TERHADAP BAKTERI Escherichia coli DAN Staphylococcus aureus Queen Novika Hermina Loing1), Defny Silvia Wewengkang1), Jemmy Abidjulu 1) 1)Program Studi Farmasi FMIPA UNSRAT Manado ABSTRACT Indonesia is a tropical country which is have so many abundant biodiversity, and one of them is soft coral. This research aims to test the antibacterial activity of soft coral Lobophytum sp. fractions against the Escherichia coli and Staphylococcus aureus. Extraction was done by maceration and fractionation using n-hexane, chloroform, methanol and water as a solvent. The test of antibacterial activity was done by using agar diffusion method. The result shows that extract and fractions have an antibacterial activity. The results conclude that the extract and fractions of Lobophytum sp. effectively inhibit the Escherichia coli and Staphylococcus aureus, in medium and strong category. The Strong inhibitory capacity showed by chloroform fraction. Key words : Lobophytum sp, antibacterial activity, Escherichia coli, Staphylococcus aureus, fractionation, agar diffusion. ABSTRAK Indonesia merupakan negara tropis, yang mempunyai keanekaragaman hayati berlimpah salah satunya ialah karang lunak. Penelitian ini bertujuan untuk menguji aktivitas antibakteri fraksi karang lunak Lobophytum sp. terhadap bakteri Escherichia coli dan Stapylococcus aureus. Ekstraksi menggunakan metode maserasi dengan pelarut etanol dan fraksinasi menggunakan pelarut n-heksan, kloroform, metanol dan air. Pengujian antibakteri mengunakan metode difusi agar. Hasil penelitian menunjukkan ekstrak dan fraksi uji memiliki aktivitas sebagai antibakteri. Berdasarkan hasil penelitian, disimpulkan bahwa ekstrak dan fraksi karang lunak Lobophytum sp efektif dalam menghambat pertumbuhan bakteri Escherichia coli serta bakteri Staphylococus aureus dengan kategori sedang dan kuat. Daya hambat terbesar ditunjukkan oleh fraksi kloroform

Microbial lineages in sarcoidosis. A metagenomic analysis tailored for low–microbial content samples

RATIONALE: The etiology of sarcoidosis is unknown, but microbial agents are suspected as triggers. OBJECTIVES: We sought to identify bacterial, fungal, or viral lineages in specimens from patients with sarcoidosis enriched relative to control subjects using metagenomic DNA sequencing. Because DNA from environmental contamination contributes disproportionately to samples with low authentic microbial content, we developed improved methods for filtering environmental contamination. METHODS: We analyzed specimens from subjects with sarcoidosis (n = 93), control subjects without sarcoidosis (n = 72), and various environmental controls (n = 150). Sarcoidosis specimens consisted of two independent sets of formalin-fixed, paraffin-embedded lymph node biopsies, BAL, Kveim reagent, and fresh granulomatous spleen from a patient with sarcoidosis. All specimens were analyzed by bacterial 16S and fungal internal transcribed spacer ribosomal RNA gene sequencing. In addition, BAL was analyzed by shotgun sequencing of fractions enriched for viral particles, and Kveim and spleen were subjected to whole-genome shotgun sequencing. MEASUREMENTS AND MAIN RESULTS: In one tissue set, fungi in the Cladosporiaceae family were enriched in sarcoidosis compared with nonsarcoidosis tissues; in the other tissue set, we detected enrichment of several bacterial lineages in sarcoidosis but not Cladosporiaceae. BAL showed limited enrichment of Aspergillus fungi. Several microbial lineages were detected in Kveim and spleen, including Cladosporium. No microbial lineage was enriched in more than one sample type after correction for multiple comparisons. CONCLUSIONS: Metagenomic sequencing revealed enrichment of microbes in single types of sarcoidosis samples but limited concordance across sample types. Statistical analysis accounting for environmental contamination was essential to avoiding false positives

Antigen presenting ILC3 regulate T cell-dependent IgA responses to colonic mucosal-associated bacteria

Intestinal immune homeostasis is dependent upon tightly regulated and dynamic host interactions with the commensal microbiota. Immunoglobulin A (IgA) produced by mucosal B cells dictates the composition of commensal bacteria residing within the intestine. While emerging evidence suggests the majority of IgA is produced innately and may be polyreactive, mucosal-dwelling species can also elicit IgA via T cell-dependent mechanisms. However, the mechanisms that modulate the magnitude and quality of T cell-dependent IgA responses remain incompletely understood. Here we demonstrate that group 3 innate lymphoid cells (ILC3) regulate steady state interactions between T follicular helper cells (TfH) and B cells to limit mucosal IgA responses. ILC3 used conserved migratory cues to establish residence within the interfollicular regions of the intestinal draining lymph nodes, where they act to limit TfH responses and B cell class switching through antigen presentation. The absence of ILC3-intrinsic antigen presentation resulted in increased and selective IgA coating of bacteria residing within the colonic mucosa. Together these findings implicate lymph node resident, antigen-presenting ILC3 as a critical regulatory checkpoint in the generation of T cell-dependent colonic IgA and suggest ILC3 act to maintain tissue homeostasis and mutualism with the mucosal-dwelling commensal microbiota

Allometry and Ecology of the Bilaterian Gut Microbiome.

Classical ecology provides principles for construction and function of biological communities, but to what extent these apply to the animal-associated microbiota is just beginning to be assessed. Here, we investigated the influence of several well-known ecological principles on animal-associated microbiota by characterizing gut microbial specimens from bilaterally symmetrical animals (Bilateria) ranging from flies to whales. A rigorously vetted sample set containing 265 specimens from 64 species was assembled. Bacterial lineages were characterized by 16S rRNA gene sequencing. Previously published samples were also compared, allowing analysis of over 1,098 samples in total. A restricted number of bacterial phyla was found to account for the great majority of gut colonists. Gut microbial composition was associated with host phylogeny and diet. We identified numerous gut bacterial 16S rRNA gene sequences that diverged deeply from previously studied taxa, identifying opportunities to discover new bacterial types. The number of bacterial lineages per gut sample was positively associated with animal mass, paralleling known species-area relationships from island biogeography and implicating body size as a determinant of community stability and niche complexity. Samples from larger animals harbored greater numbers of anaerobic communities, specifying a mechanism for generating more-complex microbial environments. Predictions for species/abundance relationships from models of neutral colonization did not match the data set, pointing to alternative mechanisms such as selection of specific colonists by environmental niche. Taken together, the data suggest that niche complexity increases with gut size and that niche selection forces dominate gut community construction.IMPORTANCEThe intestinal microbiome of animals is essential for health, contributing to digestion of foods, proper immune development, inhibition of pathogen colonization, and catabolism of xenobiotic compounds. How these communities assemble and persist is just beginning to be investigated. Here we interrogated a set of gut samples from a wide range of animals to investigate the roles of selection and random processes in microbial community construction. We show that the numbers of bacterial species increased with the weight of host organisms, paralleling findings from studies of island biogeography. Communities in larger organisms tended to be more anaerobic, suggesting one mechanism for niche diversification. Nonselective processes enable specific predictions for community structure, but our samples did not match the predictions of the neutral model. Thus, these findings highlight the importance of niche selection in community construction and suggest mechanisms of niche diversification

Cervicovaginal Fungi and Bacteria Associated With Cervical Intraepithelial Neoplasia and High-Risk Human Papillomavirus Infections in a Hispanic Population

The human cervicovaginal microbiota resides at an interface between the host and the environment and may affect susceptibility to disease. Puerto Rican women have high human papillomavirus (HPV) infection and cervical cancer rates. We hypothesized that the population structure of the cervicovaginal bacterial and fungal biota changed with cervical squamous intraepithelial lesions and HPV infections. DNA was extracted from cervix, introitus, and anal sites of 62 patients attending high-risk San Juan clinics. The 16S rRNA V4 region and ITS-2 fungal regions were amplified and sequenced using Illumina technology. HPV genotyping was determined by reverse hybridization with the HPV SPF10-LiPA25 kit. HPV prevalence was 84% of which ∼44% subjects were infected with high-risk HPV, ∼35% were co-infected with as many as 9 HPV types and ∼5% were infected with exclusively low-risk HPV types. HPV diversity did not change with cervical dysplasia. Cervical bacteria were more diverse in patients with CIN3 pre-cancerous lesions. We found enrichment of Atopobium vaginae and Gardnerella vaginalis in patients with CIN3 lesions. We found no significant bacterial biomarkers associated with HPV infections. Fungal diversity was significantly higher in cervical samples with high-risk HPV and introitus samples of patients with Atypical Squamous Cells of Undetermined Significance (ASCUS). Fungal biomarker signatures for vagina and cervix include Sporidiobolaceae and Sacharomyces for ASCUS, and Malassezia for high-risk HPV infections. Our combined data suggests that specific cervicovaginal bacterial and fungal populations are related to the host epithelial microenvironment, and could play roles in cervical dysplasia

Disordered Microbial Communities in the Upper Respiratory Tract of Cigarette Smokers

Cigarette smokers have an increased risk of infectious diseases involving the respiratory tract. Some effects of smoking on specific respiratory tract bacteria have been described, but the consequences for global airway microbial community composition have not been determined. Here, we used culture-independent high-density sequencing to analyze the microbiota from the right and left nasopharynx and oropharynx of 29 smoking and 33 nonsmoking healthy asymptomatic adults to assess microbial composition and effects of cigarette smoking. Bacterial communities were profiled using 454 pyrosequencing of 16S sequence tags (803,391 total reads), aligned to 16S rRNA databases, and communities compared using the UniFrac distance metric. A Random Forest machine-learning algorithm was used to predict smoking status and identify taxa that best distinguished between smokers and nonsmokers. Community composition was primarily determined by airway site, with individuals exhibiting minimal side-of-body or temporal variation. Within airway habitats, microbiota from smokers were significantly more diverse than nonsmokers and clustered separately. The distributions of several genera were systematically altered by smoking in both the oro- and nasopharynx, and there was an enrichment of anaerobic lineages associated with periodontal disease in the oropharynx. These results indicate that distinct regions of the human upper respiratory tract contain characteristic microbial communities that exhibit disordered patterns in cigarette smokers, both in individual components and global structure, which may contribute to the prevalence of respiratory tract complications in this population