The oral microbiome and breast cancer and nonmalignant breast disease, and its relationship with the fecal microbiome in the Ghana Breast Health Study.

The oral microbiome, like the fecal microbiome, may be related to breast cancer risk. Therefore, we investigated whether the oral microbiome was associated with breast cancer and nonmalignant breast disease, and its relationship with the fecal microbiome in a case-control study in Ghana. A total of 881 women were included (369 breast cancers, 93 nonmalignant cases and 419 population-based controls). The V4 region of the 16S rRNA gene was sequenced from oral and fecal samples. Alpha-diversity (observed amplicon sequence variants [ASVs], Shannon index and Faith's Phylogenetic Diversity) and beta-diversity (Bray-Curtis, Jaccard and weighted and unweighted UniFrac) metrics were computed. MiRKAT and logistic regression models were used to investigate the case-control associations. Oral sample alpha-diversity was inversely associated with breast cancer and nonmalignant breast disease with odds ratios (95% CIs) per every 10 observed ASVs of 0.86 (0.83-0.89) and 0.79 (0.73-0.85), respectively, compared to controls. Beta-diversity was also associated with breast cancer and nonmalignant breast disease compared to controls (P ≤ .001). The relative abundances of Porphyromonas and Fusobacterium were lower for breast cancer cases compared to controls. Alpha-diversity and presence/relative abundance of specific genera from the oral and fecal microbiome were strongly correlated among breast cancer cases, but weakly correlated among controls. Particularly, the relative abundance of oral Porphyromonas was strongly, inversely correlated with fecal Bacteroides among breast cancer cases (r = -.37, P ≤ .001). Many oral microbial metrics were strongly associated with breast cancer and nonmalignant breast disease, and strongly correlated with fecal microbiome among breast cancer cases, but not controls.

Associations of fecal microbial profiles with breast cancer and nonmalignant breast disease in the Ghana Breast Health Study.

The gut microbiota may play a role in breast cancer etiology by regulating hormonal, metabolic and immunologic pathways. We investigated associations of fecal bacteria with breast cancer and nonmalignant breast disease in a case-control study conducted in Ghana, a country with rising breast cancer incidence and mortality. To do this, we sequenced the V4 region of the 16S rRNA gene to characterize bacteria in fecal samples collected at the time of breast biopsy (N = 379 breast cancer cases, N = 102 nonmalignant breast disease cases, N = 414 population-based controls). We estimated associations of alpha diversity (observed amplicon sequence variants [ASVs], Shannon index, and Faith's phylogenetic diversity), beta diversity (Bray-Curtis and unweighted/weighted UniFrac distance), and the presence and relative abundance of select taxa with breast cancer and nonmalignant breast disease using multivariable unconditional polytomous logistic regression. All alpha diversity metrics were strongly, inversely associated with odds of breast cancer and for those in the highest relative to lowest tertile of observed ASVs, the odds ratio (95% confidence interval) was 0.21 (0.13-0.36; Ptrend < .001). Alpha diversity associations were similar for nonmalignant breast disease and breast cancer grade/molecular subtype. All beta diversity distance matrices and multiple taxa with possible estrogen-conjugating and immune-related functions were strongly associated with breast cancer (all Ps < .001). There were no statistically significant differences between breast cancer and nonmalignant breast disease cases in any microbiota metric. In conclusion, fecal bacterial characteristics were strongly and similarly associated with breast cancer and nonmalignant breast disease. Our findings provide novel insight into potential microbially-mediated mechanisms of breast disease.

Power of Microbiome Beta-Diversity Analyses Based on Standard Reference Samples.

A simple method to analyze microbiome beta-diversity computes mean beta-diversity distances from a test sample to standard reference samples. We used reference stool and nasal samples from the Human Microbiome Project and regressed an outcome on mean distances (2 degrees-of-freedom (df) test) or additionally on squares and cross-product of mean distances (5-df test). We compared the power of 2-df and 5-df tests with the microbiome regression-based kernel association test (MiRKAT). In simulations, MiRKAT had moderately greater power than the 2-df test for discriminating skin versus saliva and skin versus nasal samples, but differences were negligible for skin versus stool and stool versus nasal samples. The 2-df test had slightly greater power than MiRKAT for Dirichlet multinomial samples. In associating body mass index with beta-diversity in stool samples from the American Gut Project, the 5-df test yielded smaller P values than MiRKAT for most taxonomic levels and beta-diversity measures. Unlike procedures like MiRKAT that are based on the beta-diversity matrix, mean distances to reference samples can be analyzed with standard statistical tools and shared or meta-analyzed without sharing primary DNA data. Our data indicate that standard reference tests have power comparable to MiRKAT's (and to permutational multivariate analysis of variance), but more simulations and applications are needed to confirm this.

Variation in oral microbiome is associated with future risk of lung cancer among never-smokers.

OBJECTIVE: To prospectively investigate whether diversity in oral microbiota is associated with risk of lung cancer among never-smokers. DESIGN AND SETTING: A nested case-control study within two prospective cohort studies, the Shanghai Women's Health Study (n=74 941) and the Shanghai Men's Health Study (n=61 480). PARTICIPANTS: Lifetime never-smokers who had no cancer at baseline. Cases were subjects who were diagnosed with incident lung cancer (n=114) and were matched 1:1 with controls on sex, age (≤2 years), date (≤30 days) and time (morning/afternoon) of sample collection, antibiotic use during the week before sample collection (yes/no) and menopausal status (for women). MAIN OUTCOMES AND MEASURES: Metagenomic shotgun sequencing was used to measure the community structure and abundance of the oral microbiome in pre-diagnostic oral rinse samples of each case and control. Multivariable logistic regression models were used to estimate the association of lung cancer risk with alpha diversity metrics and relative abundance of taxa. The Microbiome Regression-Based Kernel Association Test (MiRKAT) evaluated the association between risk and the microbiome beta diversity. RESULTS: Subjects with lower microbiota alpha diversity had an increased risk of lung cancer compared with those with higher microbial alpha diversity (Shannon: ptrend=0.05; Simpson: ptrend=0.04; Observed Species: ptrend=0.64). No case-control differences were apparent for beta diversity (pMiRKAT=0.30). After accounting for multiple comparisons, a greater abundance of Spirochaetia (ORlow 1.00 (reference), ORmedium 0.61 (95% CI 0.32 to 1.18), ORhigh 0.42 (95% CI 0.21 to 0.85)) and Bacteroidetes (ORlow 1.00 (reference), ORmedium 0.66 (95% CI 0.35 to 1.25), ORhigh 0.31 (95% CI 0.15 to 0.64)) was associated with a decreased risk of lung cancer, while a greater abundance of the Bacilli class (ORlow 1.00 (reference), ORmedium 1.49 (95% CI 0.73 to 3.08), ORhigh 2.40 (95% CI 1.18 to 4.87)) and Lactobacillales order (ORlow 1.00 (reference), ORmedium 2.15 (95% CI 1.03 to 4.47), ORhigh 3.26 (95% CI 1.58 to 6.70)) was associated with an increased risk of lung cancer. CONCLUSIONS: Our prospective study of never-smokers suggests that lower alpha diversity was associated with a greater risk of lung cancer and the abundance of certain specific taxa was associated with altered risk, providing further insight into the aetiology of lung cancer in the absence of active tobacco smoking.

Comparison of fecal and oral collection methods for studies of the human microbiota in two Iranian cohorts.

BACKGROUND: To initiate fecal and oral collections in prospective cohort studies for microbial analyses, it is essential to understand how field conditions and geographic differences may impact microbial communities. This study aimed to investigate the impact of fecal and oral sample collection methods and room temperature storage on collection samples for studies of the human microbiota. RESULTS: We collected fecal and oral samples from participants in two Iranian cohorts located in rural Yazd (n = 46) and urban Gonbad (n = 38) and investigated room temperature stability over 4 days of fecal (RNAlater and fecal occult blood test [FOBT] cards) and comparability of fecal and oral (OMNIgene ORAL kits and Scope mouthwash) collection methods. We calculated interclass correlation coefficients (ICCs) based on 3 alpha and 4 beta diversity metrics and the relative abundance of 3 phyla. After 4 days at room temperature, fecal stability ICCs and ICCs for Scope mouthwash were generally high for all microbial metrics. Similarly, the fecal comparability ICCs for RNAlater and FOBT cards were high, ranging from 0.63 (95% CI: 0.46, 0.75) for the relative abundance of Firmicutes to 0.93 (95% CI: 0.89, 0.96) for unweighted Unifrac. Comparability ICCs for OMNIgene ORAL and Scope mouthwash were lower than fecal ICCs, ranging from 0.55 (95% CI: 0.36, 0.70) for the Shannon index to 0.79 (95% CI: 0.69, 0.86) for Bray-Curtis. Overall, RNAlater, FOBT cards and Scope mouthwash were stable up to 4 days at room temperature. Samples collected using FOBT cards were generally comparable to RNAlater while the OMNIgene ORAL were less similar to Scope mouthwash. CONCLUSIONS: As microbiome measures for feces samples collected using RNAlater, FOBT cards and oral samples collected using Scope mouthwash were stable over four days at room temperature, these would be most appropriate for microbial analyses in these populations. However, one collection method should be consistently since each method may induce some differences.

Effects of processed meat and drinking water nitrate on oral and fecal microbial populations in a controlled feeding study.

BACKGROUND: One mechanism that can explain the link between processed meat consumption and colorectal cancer (CRC) is the production of carcinogenic N-nitroso compounds (NOCs) in the gastrointestinal tract. Oral and gut microbes metabolize ingested proteins (a source of secondary and tertiary amines and amides) and can reduce nitrate to nitrite, generating potentially carcinogenic NOCs. OBJECTIVE: We evaluated whether nitrate/nitrite in processed meat or water influences the fecal or salivary microbiota. DESIGN: In this dietary intervention study, 63 volunteers consumed diets high in conventional processed meats for two weeks, switched to diets high in poultry for two weeks, and then consumed phytochemical-enriched conventional processed or low-nitrite processed meat diets for two weeks. During the intervention, they drank water with low nitrate concentrations and consumed a healthy diet with low antioxidants. Then the volunteers drank nitrate-enriched water for 1 week, in combination with one of the four different diets. We measured creatinine-adjusted urinary nitrate levels and characterized the oral and fecal microbiota using 16S rRNA amplicon sequencing. RESULTS: Using linear mixed models, we found that, compared to baseline, urinary nitrate levels were reduced during the phytochemical-enriched low-nitrite meat diet (p-value = 0.009) and modestly during the poultry diet (p-value = 0.048). In contrast, urinary nitrate increased after 1-week of drinking nitrate-enriched water (p-value<10-5). Nitrate-enriched water, but not processed meats with or without phytochemicals, altered the saliva microbial population (p-value ≤0.001), and significantly increased abundance of 8 bacterial taxa, especially genus Neisseria and other nitrate-reducing taxa. Meats, phytochemicals and nitrate-enriched water had no significant effects on saliva alpha diversity or any diversity parameter measured for the fecal microbiota. CONCLUSION: These findings support the hypothesis that drinking high nitrate water increases oral nitrate-reducing bacteria, which likely results in increased NOC. However, meat nitrate/nitrite at the levels tested had no effect on either the gut or oral bacteria. CLINICALTRIALS. GOV IDENTIFIER: NCT04138654.

Using standard microbiome reference groups to simplify beta-diversity analyses and facilitate independent validation.

Motivation: Comparisons of microbiome communities across populations are often based on pairwise distance measures (beta-diversity). Standard analyses (principal coordinate plots, permutation tests, kernel methods) require access to primary data if another investigator wants to add or compare independent data. We propose using standard reference measurements to simplify microbiome beta-diversity analyses, to make them more transparent, and to facilitate independent validation and comparisons across studies. Results: Using stool and nasal reference sets from the Human Microbiome Project (HMP), we computed mean distances (actually Bray-Curtis or Pearson correlation dissimilarities) to each reference set for each new sample. Thus, each new sample has two mean distances that can be plotted and analyzed with classical statistical methods. To test the approach, we studied independent (not reference) HMP subjects. Simple Hotelling tests demonstrated statistically significant differences in mean distances to reference sets between all pairs of body sites (stool, skin, nasal, saliva and vagina) at the phylum, class, order, family and genus levels. Using the distance to a single reference set was usually sufficient, but using both reference sets always worked well. The use of reference sets simplifies standard analyses of beta-diversity and facilitates the independent validation and combining of such data because others can compute distances to the same reference sets. Moreover, standard statistical methods for survival analysis, logistic regression and other procedures can be applied to vectors of mean distances to reference sets, thereby greatly expanding the potential uses of beta-diversity information. More work is needed to identify the best reference sets for particular applications. Availability and implementation: https://github.com/NCI-biostats/microbiome-fixed-reference. Supplementary information: Supplementary data are available at Bioinformatics online.

Postmenopausal breast cancer and oestrogen associations with the IgA-coated and IgA-noncoated faecal microbiota.

BACKGROUND: The diversity and composition of the gut microbiota may affect breast cancer risk by modulating systemic levels of oestrogens and inflammation. The current investigation tested this hypothesis in postmenopausal women by identifying breast cancer associations with an inflammation marker, oestrogen levels, and faecal microbes that were or were not coated with mucosal immunoglobulin A (IgA). METHODS: In this population-based study, we compared 48 postmenopausal breast cancer cases (75% stage 0-1, 88% oestrogen-receptor positive) to 48 contemporaneous, postmenopausal, normal-mammogram, age-matched controls. Microbiota metrics employed 16S rRNA gene amplicon sequencing from IgA-coated and -noncoated faecal microbes. High-performance liquid chromatography/mass spectrometry (HPLC/MS) and radioimmunoassay were used to quantify urine prostaglandin E metabolite (PGE-M), a possible marker of inflammation; urine oestrogens and oestrogen metabolites were quantified by HPLC/MS-MS. RESULTS: Women with pre-treatment breast cancer had non-significantly elevated oestrogen levels; controls' (but not cases') oestrogens were directly correlated with their IgA-negative microbiota alpha diversity (P=0.012). Prostaglandin E metabolite levels were not associated with case status, oestrogen levels, or alpha diversity. Adjusted for oestrogens and other variables, cases had significantly reduced alpha diversity and altered composition of both their IgA-positive and IgA-negative faecal microbiota. Cases' faecal microbial IgA-positive imputed Immune System Diseases metabolic pathway genes were increased; also, cases' IgA-positive and IgA-negative imputed Genetic Information Processing pathway genes were decreased (P⩽0.01). CONCLUSIONS: Compared to controls, breast cancer cases had significant oestrogen-independent associations with the IgA-positive and IgA-negative gut microbiota. These suggest that the gut microbiota may influence breast cancer risk by altered metabolism, oestrogen recycling, and immune pressure.

Phosphorylation of serine 51 regulates the interaction of human DNA ligase I with replication factor C and its participation in DNA replication and repair.

Human DNA ligase I (hLigI) joins Okazaki fragments during DNA replication and completes excision repair via interactions with proliferating cell nuclear antigen and replication factor C (RFC). Unlike proliferating cell nuclear antigen, the interaction with RFC is regulated by hLigI phosphorylation. To identity of the site(s) involved in this regulation, we analyzed phosphorylated hLigI purified from insect cells by mass spectrometry. These results suggested that serine 51 phosphorylation negatively regulates the interaction with RFC. Therefore, we constructed versions of hLigI in which serine 51 was replaced with either alanine (hLigI51A) to prevent phosphorylation or aspartic acid (hLigI51D) to mimic phosphorylation. hLigI51D but not hLigI51A was defective in binding to purified RFC and in associating with RFC in cell extracts. Although DNA synthesis and proliferation of hLigI-deficient cells expressing either hLig51A or hLig51 was reduced compared with cells expressing wild-type hLigI, cellular senescence was only observed in the cells expressing hLigI51D. Notably, these cells had increased levels of spontaneous DNA damage and phosphorylated CHK2. In addition, although expression of hLigI51A complemented the sensitivity of hLigI-deficient cells to a poly (ADP-ribose polymerase (PARP) inhibitor, expression of hLig151D did not, presumably because these cells are more dependent upon PARP-dependent repair pathways to repair the damage resulting from the abnormal DNA replication. Finally, neither expression of hLigI51D nor hLigI51A fully complemented the sensitivity of hLigI-deficient cells to DNA alkylation. Thus, phosphorylation of serine 51 on hLigI plays a critical role in regulating the interaction between hLigI and RFC, which is required for efficient DNA replication and repair.

Prospective and Cross-sectional Associations of the Rectal Tissue Microbiome with Colorectal Adenoma Recurrence.

BACKGROUND: The gut microbiome is plausibly associated with colorectal cancer risk; however, previous studies mostly investigated this association cross-sectionally. We investigated cross-sectional and prospective associations of the rectal tissue microbiome with adenoma recurrence in the Polyp Prevention Trial (PPT). METHODS: PPT is a 4-year randomized clinical trial of the effect of a dietary intervention on adenoma recurrence among community members. We extracted DNA from rectal biopsies at baseline, end of year 1, and end of year 4 among 455 individuals and sequenced the V4 region of the 16S rRNA gene. At each timepoint, we investigated associations of alpha diversity, beta diversity, and presence and relative abundance of select taxa with adenoma recurrence using multivariable logistic regression. RESULTS: Variation in beta diversity was primarily explained by subject and minimally by year of collection or time between biopsy and colonoscopy. Cross-sectionally, year 4 alpha diversity was strongly, inversely associated with adenoma prevalence [ORQ3 vs. Q1 Shannon index = 0.40 (95% confidence interval, CI: 0.21-0.76)]. Prospective alpha diversity associations (i.e., baseline/year 1 alpha diversity with adenoma recurrence 3-4 years later) were weak or null, as were cross-sectional and prospective beta diversity-adenoma associations. Bacteroides abundance was more strongly, positively associated with adenoma prevalence cross-sectionally than prospectively. CONCLUSIONS: Rectal tissue microbiome profiles may be associated with prevalent adenomas, with little evidence supporting prospective associations. IMPACT: Additional prospective studies, with serial fecal and tissue samples, to explore microbiome-colorectal cancer associations are needed. Eventually, it may be possible to use microbiome characteristics as intervenable risk factors or screening tools.

Stability of the Fecal and Oral Microbiome over 2 Years at -80°C for Multiple Collection Methods.

BACKGROUND: In prospective cohorts, biological samples are generally stored over long periods before an adequate number of cases have accrued. We investigated the impact of sample storage at -80°C for 2 years on the stability of the V4 region of the 16S rRNA gene across seven different collection methods (i.e., no additive, 95% ethanol, RNAlater stabilization solution, fecal occult blood test cards, and fecal immunochemical test tubes for feces; OMNIgene ORAL tubes and Scope mouthwash for saliva) among 51 healthy volunteers. METHODS: Intraclass correlation coefficients (ICC) were calculated for the relative abundance of the top three phyla, the 20 most abundant genera, three alpha-diversity metrics, and the first principal coordinates of three beta-diversity matrices. RESULTS: The subject variability was much higher than the variability introduced by the sample collection type, and storage time. For fecal samples, microbial stability over 2 years was high across collection methods (range, ICCs = 0.70-0.99), except for the samples collected with no additive (range, ICCs = 0.23-0.83). For oral samples, most microbiome diversity measures were stable over time with ICCs above 0.74; however, ICCs for the samples collected with Scope mouthwash were lower for two alpha-diversity measures, Faith's phylogenetic diversity (0.23) and the observed number of operational taxonomic units (0.23). CONCLUSIONS: Fecal and oral samples in most used collection methods are stable for microbiome analyses after 2 years at -80°C, except for fecal samples with no additive. IMPACT: This study provides evidence that samples stored for an extended period from prospective studies are useful for microbiome analyses.

Oral microbiome and risk of incident head and neck cancer: A nested case-control study.

OBJECTIVES: This nested case-control study in the NIH-AARP Diet and Health Study was carried out to prospectively investigate the relationship of oral microbiome with head and neck cancer (HNC). MATERIALS AND METHODS: 56 incident HNC cases were identified, and 112 controls were incidence-density matched to cases. DNA extracted from pre-diagnostic oral wash samples was whole-genome shotgun metagenomic sequenced to measure the overall oral microbiome. ITS2 gene qPCR was used to measure the presence of fungi. ITS2 gene sequencing was performed on ITS2 gene qPCR positive samples. We computed taxonomic and functional alpha-diversity and beta-diversity metrics. The presence and relative abundance of groups of red-complex (e.g., Porphyromonas gingivalis) and/or orange-complex (e.g., Fusobacterium nucleatum) periodontal pathogens were compared between cases and controls using conditional logistic regression models and MiRKAT. RESULTS: Participants with higher taxonomic microbial alpha-diversity had a non-statistically significant decreased risk of HNC. No case-control differences were found for beta diversity by MiRKAT model (all p > 0.05). A greater relative abundance of red-complex periodontal pathogens (OR = 0.51, 95 % CI = 0.26-1.00), orange-complex (OR = 0.38, 95 % CI = 0.18-0.83), and both complexes' pathogens (OR = 0.32, 95 % CI = 0.14-0.75), were associated with reduced risk of HNC. The presence of oral fungi was also strongly associated with reduced risk of HNC compared with controls (OR = 0.39, 95 % CI = 0.17-0.92). CONCLUSION: Greater taxonomic alpha-diversity, the presence of oral fungi, and the presence or relative abundance of multiple microbial species, including the red- and orange-complex periodontal pathogens, were associated with reduced risk of HNC. Future studies with larger sample sizes are needed to evaluate these associations.

Evaluation of alcohol-free mouthwash for studies of the oral microbiome.

Oral bacteria play important roles in human health and disease. Oral samples collected using ethanol-containing mouthwash are widely used for oral microbiome studies. However, ethanol is flammable and not ideal for transportation/storage in large quantities, and some individuals may avoid ethanol due to the burning sensation or due to various personal, medical, religious, and/or cultural factors. Here, we compared ethanol-free and ethanol-containing mouthwashes using multiple microbiome metrics and assessed the stability of the mouthwash samples stored up to 10 days before processing. Forty volunteers provided oral wash samples collected using ethanol-free and ethanol-containing mouthwashes. From each sample, one aliquot was immediately frozen, one was stored at 4°C for 5 days and frozen, while the third aliquot was stored for 5 days at 4°C and 5 days at ambient temperature to mimic shipping delays and then frozen. DNA was extracted, the 16S rRNA gene V4 region was amplified and sequenced, and bioinformatic processing was performed using QIIME 2. Microbiome metrics measured in the two mouthwash types were very similar, with intraclass correlation coefficients (ICCs) for alpha and beta diversity metrics greater than 0.85. Relative abundances of some taxa were significantly different, but ICCs of the top four most abundant phyla and genera were high (> 0.75) for the comparability of the mouthwashes. Stability during delayed processing was also high for both mouthwashes based on alpha and beta diversity measures and relative abundances of the top four phyla and genera (ICCs ≥ 0.90). These results demonstrate ethanol-free mouthwash performs similarly to ethanol-containing mouthwash for microbial analyses, and both mouthwashes are stable for at least 10 days without freezing prior to laboratory processing. Ethanol-free mouthwash is suitable for collecting and shipping oral wash samples, and these results have important implications for planning future epidemiologic studies of the oral microbiome.

Associations of Circulating Estrogens and Estrogen Metabolites with Fecal and Oral Microbiome in Postmenopausal Women in the Ghana Breast Health Study.

The human fecal and oral microbiome may play a role in the etiology of breast cancer through modulation of endogenous estrogen metabolism. This study aimed to investigate associations of circulating estrogens and estrogen metabolites with the fecal and oral microbiome in postmenopausal African women. A total of 117 women with fecal (N = 110) and oral (N = 114) microbiome data measured by 16S rRNA gene sequencing, and estrogens and estrogen metabolites data measured by liquid chromatography tandem mass spectrometry were included. The outcomes were measures of the microbiome and the independent variables were the estrogens and estrogen metabolites. Estrogens and estrogen metabolites were associated with the fecal microbial Shannon index (global P < 0.01). In particular, higher levels of estrone (ß = 0.36, P = 0.03), 2-hydroxyestradiol (ß = 0.30, P = 0.02), 4-methoxyestrone (ß = 0.51, P = 0.01), and estriol (ß = 0.36, P = 0.04) were associated with higher levels of the Shannon index, while 16alpha-hydroxyestrone (ß = -0.57, P < 0.01) was inversely associated with the Shannon index as indicated by linear regression. Conjugated 2-methoxyestrone was associated with oral microbial unweighted UniFrac as indicated by MiRKAT (P < 0.01) and PERMANOVA, where conjugated 2-methoxyestrone explained 2.67% of the oral microbial variability, but no other estrogens or estrogen metabolites were associated with any other beta diversity measures. The presence and abundance of multiple fecal and oral genera, such as fecal genera from families Lachnospiraceae and Ruminococcaceae, were associated with several estrogens and estrogen metabolites as indicated by zero-inflated negative binomial regression. Overall, we found several associations of specific estrogens and estrogen metabolites and the fecal and oral microbiome. IMPORTANCE Several epidemiologic studies have found associations of urinary estrogens and estrogen metabolites with the fecal microbiome. However, urinary estrogen concentrations are not strongly correlated with serum estrogens, a known risk factor for breast cancer. To better understand whether the human fecal and oral microbiome were associated with breast cancer risk via the regulation of estrogen metabolism, we conducted this study to investigate the associations of circulating estrogens and estrogen metabolites with the fecal and oral microbiome in postmenopausal African women. We found several associations of parent estrogens and several estrogen metabolites with the microbial communities, and multiple individual associations of estrogens and estrogen metabolites with the presence and abundance of multiple fecal and oral genera, such as fecal genera from families Lachnospiraceae and Ruminococcaceae, which have estrogen metabolizing properties. Future large, longitudinal studies to investigate the dynamic changes of the fecal and oral microbiome and estrogen relationship are needed.

IsoQuant: a software tool for stable isotope labeling by amino acids in cell culture-based mass spectrometry quantitation.

Accurate protein identification and quantitation are critical when interpreting the biological relevance of large-scale shotgun proteomics data sets. Although significant technical advances in peptide and protein identification have been made, accurate quantitation of high-throughput data sets remains a key challenge in mass spectrometry data analysis and is a labor intensive process for many proteomics laboratories. Here, we report a new SILAC-based proteomics quantitation software tool, named IsoQuant, which is used to process high mass accuracy mass spectrometry data. IsoQuant offers a convenient quantitation framework to calculate peptide/protein relative abundance ratios. At the same time, it also includes a visualization platform that permits users to validate the quality of SILAC peptide and protein ratios. The program is written in the C# programming language under the Microsoft .NET framework version 4.0 and has been tested to be compatible with both 32-bit and 64-bit Windows 7. It is freely available to noncommercial users at http://www.proteomeumb.org/MZw.html .

Dual modification of Alzheimer's disease PHF-tau protein by lysine methylation and ubiquitylation: a mass spectrometry approach.

In sporadic Alzheimer's disease (AD), neurofibrillary lesion formation is preceded by extensive post-translational modification of the microtubule associated protein tau. To identify the modification signature associated with tau lesion formation at single amino acid resolution, immunopurified paired helical filaments were isolated from AD brain and subjected to nanoflow liquid chromatography-tandem mass spectrometry analysis. The resulting spectra identified monomethylation of lysine residues as a new tau modification. The methyl-lysine was distributed among seven residues located in the projection and microtubule binding repeat regions of tau protein, with one site, K254, being a substrate for a competing lysine modification, ubiquitylation. To characterize methyl lysine content in intact tissue, hippocampal sections prepared from post mortem late-stage AD cases were subjected to double-label confocal fluorescence microscopy using anti-tau and anti-methyl lysine antibodies. Anti-methyl lysine immunoreactivity colocalized with 78 ± 13% of neurofibrillary tangles in these specimens. Together these data provide the first evidence that tau in neurofibrillary lesions is post-translationally modified by lysine methylation.

Reusing a prepaid health plan's fecal immunochemical tests for microbiome associations with colorectal adenoma.

An altered colonic microbiota probably increases colorectal adenoma (CRA) and cancer (CRC) risk, but large, unbiased fecal collections are needed to examine the relationship of gut microbiota diversity and composition to colorectal carcinogenesis. This study assessed whether fecal immunochemical tests (FITs) from CRA/CRC screening may fulfill this requirement. Using FIT, self-collected by members of Kaiser Permanente Hawaii (KPH), as well as interspersed quality control (QC) specimens, DNA was extracted and amplified to generate 16S rRNA microbiome profiles rarified at 10,000 reads. CRA/CRC were diagnosed by colonoscopy and histopathology. Covariates were from electronic KPH records. Of 921 participants' FIT devices, 538 (58%) yielded at least 10,000 rRNA reads and 1016 species-level variants mapped to 46 genera. Of the 538 evaluable participants, 63 (11.7%) were FIT-negative per protocol, and they were considered negative for CRA/CRC. Of the 475 FIT + participants, colonoscopy and pathologic review revealed that 8 (1.7%) had CRC, 71 (14.9%) had high-risk CRA, 107 (22.5%) had low-risk CRA, and 289 (60.8%) did not have CRA/CRC. Men were 2.27-fold [95% confidence interval (CI) 1.32-3.91] more likely than women to be FIT+ . Men also had 1.96-fold (CI 1.24-3.07) higher odds of low-risk CRA, with similar trends for high-risk CRA and CRC. CRA/CRC were not associated with overweight, obesity, diabetes, or antibiotic prescriptions in this study. QC analysis across 24 batches of FIT devices revealed QC outliers in four batches. With or without exclusion of the four QC-outlier batches, as well as lenient (1000-read) rarefaction, CRA/CRC had no consistent, statistically significant associations with fecal microbiome alpha diversity, beta diversity or genera relative abundance. CRA/CRC had expected associations with male sex but not with microbiome metrics. Fecal microbiome profiling using DNA extracted from at-home collected, re-used FIT devices is feasible, albeit with substantial challenges. Using FITs for prospective microbiome studies of CRA/CRC risk should consider the impact of the current findings on statistical power and requisite sample sizes.

The Oral Microbiome and Lung Cancer Risk: An Analysis of 3 Prospective Cohort Studies.

BACKGROUND: Previous studies suggested associations between the oral microbiome and lung cancer, but studies were predominantly cross-sectional and underpowered. METHODS: Using a case-cohort design, 1306 incident lung cancer cases were identified in the Agricultural Health Study; National Institutes of Health-AARP Diet and Health Study; and Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Referent subcohorts were randomly selected by strata of age, sex, and smoking history. DNA was extracted from oral wash specimens using the DSP DNA Virus Pathogen kit, the 16S rRNA gene V4 region was amplified and sequenced, and bioinformatics were conducted using QIIME 2. Hazard ratios and 95% confidence intervals were calculated using weighted Cox proportional hazards models. RESULTS: Higher alpha diversity was associated with lower lung cancer risk (Shannon index hazard ratio = 0.90, 95% confidence interval = 0.84 to 0.96). Specific principal component vectors of the microbial communities were also statistically significantly associated with lung cancer risk. After multiple testing adjustment, greater relative abundance of 3 genera and presence of 1 genus were associated with greater lung cancer risk, whereas presence of 3 genera were associated with lower risk. For example, every SD increase in Streptococcus abundance was associated with 1.14 times the risk of lung cancer (95% confidence interval = 1.06 to 1.22). Associations were strongest among squamous cell carcinoma cases and former smokers. CONCLUSIONS: Multiple oral microbial measures were prospectively associated with lung cancer risk in 3 US cohort studies, with associations varying by smoking history and histologic subtype. The oral microbiome may offer new opportunities for lung cancer prevention.

Stable isotope labeling with amino acids in cell culture based mass spectrometry approach to detect transient protein interactions using substrate trapping.

The analysis of protein interactors in protein complexes can yield important insight into protein function and signal transduction. Thus, a reliable approach to distinguish true interactors from nonspecific interacting proteins is of utmost importance for accurate data interpretation. Although stringent purification methods are critical, challenges still remain in the selection of criteria that will permit the objective differentiation of true members of the protein complex from nonspecific background proteins. To address these challenges, we have developed a quantitative proteomic strategy combining stable isotope labeling with amino acids in cell culture (SILAC), affinity substrate trapping, and gel electrophoresis followed by liquid chromatography-tandem mass spectrometry (geLC-MS/MS) protein quantitation. ATP hydrolysis-deficient vacuolar protein sorting-associated protein 4B (Vps4B) was used as the "bait" protein which served as a substrate trap since its lack of ATP hydrolysis enzymatic activity allows the stabilization of its transiently associated interacting proteins. A significant advantage of our approach is the use of our new in-house-developed software program for SILAC-based mass spectrometry quantitation, which further facilitates the differentiation between the bait protein, endogenous bait-interacting proteins, and nonspecific binding proteins based on their protein ratios. The strategy presented herein is applicable to the analysis of other protein complexes whose compositions are dependent upon the ATP hydrolysis activity of the bait protein used in affinity purification studies.

Cigarette Smoking and Opium Use in Relation to the Oral Microbiota in Iran.

Cigarettes and opium contain chemicals and particulate matter that may modify the oral microbiota. This study aimed to investigate the association between cigarette and opium use with the oral microbiota. A total of 558 participants were recruited from Iran between 2011 and 2015. Individuals were categorized as never cigarette nor opium users, ever cigarette-only smokers, ever opium-only users, and ever both cigarette and opium users. Participants provided saliva samples for 16S rRNA gene sequencing. Logistic regression, microbiome regression-based kernel association test (MiRKAT), and zero-inflated beta regression models were calculated. For every increase in 10 observed amplicon sequence variants (ASVs), the odds for being a cigarette-only smoker, opium-only user, and both user compared to never users decreased by 9% (odds ratio [OR] = 0.91; 95% confidence interval [95% CI] = 0.86 to 0.97), 13% (OR = 0.87; 95% CI = 0.75 to 1.01), and 12% (OR = 0.88; 95% CI = 0.80 to 0.96), respectively. The microbial communities differed by cigarette and opium use as indicated by MiRKAT models testing the three beta-diversity matrices (P < 0.05 for all). Three genera were less likely and one genus was more likely to be detected in cigarette-only smokers or opium-only users than in never users. The relative abundance of the phylum Actinobacteria (never, 14.78%; both, 21.20%) was higher and the phyla Bacteroidetes (never, 17.63%; both, 11.62%) and Proteobacteria (never, 9.06%; both, 3.70%) were lower in users of both cigarettes and opium, while the phylum Firmicutes (never, 54.29%; opium, 65.49%) was higher in opium-only users. Cigarette and opium use was associated with lower alpha-diversity, overall oral microbiota community composition, and both the presence and relative abundance of multiple taxa. IMPORTANCE Cigarette smoking and opium use are associated with periodontal disease caused by specific bacteria such as Porphyromonas gingivalis, which suggests a link between cigarette smoking and opium use and the oral microbiota. Alterations of the oral microbiota in cigarette smokers compared to nonsmokers have been reported, but this has not been studied across diverse populations. Additionally, the association of opium use with the oral microbiota has not been investigated to date. We conducted this study to investigate differences in the oral microbiota between ever users of cigarettes only, opium only, and both cigarettes and opium and never users of cigarettes and opium in Iran. Lower alpha-diversity, distinct overall oral microbial communities, and the presence and relative abundance of multiple taxa have been found for users of cigarettes and/or opium.