Tobacco use, self-reported professional dental cleaning habits, and lung adenocarcinoma diagnosis are associated with bronchial and lung microbiome alpha diversity.

RATIONALE: The lung microbiome is an inflammatory stimulus whose role in the development of lung malignancies is incompletely understood. We hypothesized that the lung microbiome associates with multiple clinical factors, including the presence of a lung malignancy. OBJECTIVES: To assess associations between the upper and lower airway microbiome and multiple clinical factors including lung malignancy. METHODS: We conducted a prospective cohort study of upper and lower airway microbiome samples from 44 subjects undergoing lung lobectomy for suspected or confirmed lung cancer. Subjects provided oral (2), induced sputum, nasopharyngeal, bronchial, and lung tissue (3) samples. Pathologic diagnosis, age, tobacco use, dental care history, lung function, and inhaled corticosteroid use were associated with upper and lower airway microbiome findings. MEASUREMENTS AND MAIN RESULTS: Older age was associated with greater Simpson diversity in the oral and nasopharyngeal sites (p = 0.022 and p = 0.019, respectively). Current tobacco use was associated with greater lung and bronchus Simpson diversity (p < 0.0001). Self-reported last profession dental cleaning more than 6 months prior (vs. 6 or fewer months prior) was associated with lower lung and bronchus Simpson diversity (p < 0.0001). Diagnosis of a lung adenocarcinoma (vs. other pathologic findings) was associated with lower bronchus and lung Simpson diversity (p = 0.024). Last professional dental cleaning, dichotomized as ≤ 6 months vs. >6 months prior, was associated with clustering among lung samples (p = 0.027, R2 = 0.016). Current tobacco use was associated with greater abundance of pulmonary pathogens Mycoplasmoides and Haemophilus in lower airway samples. Self-reported professional dental cleaning ≤ 6 months prior (vs. >6 months prior) was associated with greater bronchial Actinomyces and lung Streptococcus abundance. Lung adenocarcinoma (vs. no lung adenocarcinoma) was associated with lower Lawsonella abundance in lung samples. Inhaled corticosteroid use was associated with greater abundance of Haemophilus among oral samples and greater Staphylococcus among lung samples. CONCLUSIONS: Current tobacco use, recent dental cleaning, and a diagnosis of adenocarcinoma are associated with lung and bronchial microbiome α-diversity, composition (ß-diversity), and the abundance of several respiratory pathogens. These findings suggest that modifiable habits (tobacco use and dental care) may influence the lower airway microbiome. Larger controlled studies to investigate these potential associations are warranted.

Sputum microbiome α-diversity is a key feature of the COPD frequent exacerbator phenotype.

Background: The lung microbiome is an inflammatory stimulus whose role in COPD pathogenesis is incompletely understood. We hypothesised that the frequent exacerbator phenotype is associated with decreased α-diversity and increased lung inflammation. Our objective was to assess correlations between the frequent exacerbator phenotype, the microbiome and inflammation longitudinally during exacerbation-free periods. Methods: We conducted a case-control longitudinal observational study of the frequent exacerbator phenotype and characteristics of the airway microbiome. 81 subjects (41 frequent and 40 infrequent exacerbators) provided nasal, oral and sputum microbiome samples at two visits over 2-4 months. Exacerbation phenotype, relevant clinical factors and sputum cytokine values were associated with microbiome findings. Results: The frequent exacerbator phenotype was associated with lower sputum microbiome α-diversity (p=0.0031). This decrease in α-diversity among frequent exacerbators was enhanced when the sputum bacterial culture was positive (p<0.001). Older age was associated with decreased sputum microbiome α-diversity (p=0.0030). Between-visit ß-diversity was increased among frequent exacerbators and those who experienced a COPD exacerbation between visits (p=0.025 and p=0.014, respectively). Sputum cytokine values did not differ based on exacerbation phenotype or other clinical characteristics. Interleukin (IL)-17A was negatively associated with α-diversity, while IL-6 and IL-8 were positively associated with α-diversity (p=0.012, p=0.012 and p=0.0496, respectively). IL-22, IL-17A and IL-5 levels were positively associated with Moraxella abundance (p=0.027, p=0.0014 and p=0.0020, respectively). Conclusions: Even during exacerbation-free intervals, the COPD frequent exacerbator phenotype is associated with decreased sputum microbiome α-diversity and increased ß-diversity. Decreased sputum microbiome α-diversity and Moraxella abundance are associated with lung inflammation.

Investigation of Causal Effects of Protein Biomarkers on Cardiovascular Disease in Persons With HIV.

BACKGROUND: There is an incompletely understood increased risk for cardiovascular disease (CVD) among people with HIV (PWH). We investigated if a collection of biomarkers were associated with CVD among PWH. Mendelian randomization (MR) was used to identify potentially causal associations. METHODS: Data from follow-up in 4 large trials among PWH were used to identify 131 incident CVD cases and they were matched to 259 participants without incident CVD (controls). Tests of associations between 460 baseline protein levels and case status were conducted. RESULTS: Univariate analysis found CLEC6A, HGF, IL-6, IL-10RB, and IGFBP7 as being associated with case status and a multivariate model identified 3 of these: CLEC6A (odds ratio [OR] = 1.48, P = .037), HGF (OR = 1.83, P = .012), and IL-6 (OR = 1.45, P = .016). MR methods identified 5 significantly associated proteins: AXL, CHI3L1, GAS6, IL-6RA, and SCGB3A2. CONCLUSIONS: These results implicate inflammatory and fibrotic processes as contributing to CVD. While some of these biomarkers are well established in the general population and in PWH (IL-6 and its receptor), some are novel to PWH (HGF, AXL, and GAS6) and some are novel overall (CLEC6A). Further investigation into the uniqueness of these biomarkers in PWH and the role of these biomarkers as targets among PWH is warranted.

Chlorhexidine oral rinses for symptomatic COPD: a randomised, blind, placebo-controlled preliminary study.

OBJECTIVES: Determine the effect of twice-daily chlorhexidine oral rinses on oral and lung microbiota biomass and respiratory symptoms. SETTING: Single centre. PARTICIPANTS: Participants were aged 40-85 with chronic obstructive pulmonary disease (COPD) and chronic productive cough or COPD exacerbation within the last year. Exclusions included antibiotics in the previous 2 months and/or those with less than four teeth. Forty-four participants were recruited and 40 completed the study. INTERVENTION: Participants were randomised 1:1 to twice-daily 0.12% chlorhexidine oral rinses versus placebo for 2 months along with daily diaries. St. George's Respiratory Questionnaire (SGRQ), blood tests, oral rinse and induced sputum were collected at randomisation and the final visit. PRIMARY AND SECONDARY OUTCOMES: Primary outcome was a change in oral and sputum microbiota biomass. Secondary outcomes included: sputum and oral microbiota Shannon and Simpson diversity and taxonomy; inflammatory markers; Breathlessness, Cough and Sputum Scale and SGRQ scores. RESULTS: Neither the oral microbiota nor the sputum microbiota biomass decreased significantly in those using chlorhexidine compared with placebo (oral microbiota mean log10 difference (SE)=-0.103 (0.23), 95% CI -0.59 to 0.38, p=0.665; sputum microbiota 0.80 (0.46), 95% CI -0.15 to 1.75, p=0.096). Chlorhexidine decreased both oral and sputum microbiota alpha (Shannon) diversity (linear regression estimate (SE) oral: -0.349 (0.091), p=0.001; sputum -0.622 (0.169), p=0.001). Chlorhexidine use did not decrease systemic inflammatory markers compared with placebo (C reactive protein (chlorhexidine 1.8±7.5 vs placebo 0.4±6.8, p=0.467), fibrinogen (22.5±77.8 vs 10.0±77.0, p=0.406) or leucocytes (0.2±1.8 vs 0.5±1.8, p=0.560)). Chlorhexidine use decreased SGRQ scores compared with placebo (chlorhexidine -4.7±8.0 vs placebo 1.7±8.9, p=0.032). CONCLUSIONS: We did not detect a significant difference in microbiota biomass due to chlorhexidine use. Chlorhexidine decreased oral and sputum microbiota alpha diversity and improved respiratory health-related quality of life compared with placebo. TRIAL REGISTRATION: NCT02252588.

Calprotectin (S100A8/A9) Is an Innate Immune Effector in Experimental Periodontitis.

Upregulated in inflammation, calprotectin (complexed S100A8 and S100A9; S100A8/A9) functions as an innate immune effector molecule, promoting inflammation, and also as an antimicrobial protein. We hypothesized that antimicrobial S100A8/A9 would mitigate change to the local microbial community and promote resistance to experimental periodontitis in vivo. To test this hypothesis, S100A9-/- and wild-type (WT; S100A9+/+) C57BL/6 mice were compared using a model of ligature-induced periodontitis. On day 2, WT mice showed fewer infiltrating innate immune cells than S100A9-/- mice; by day 5, the immune cell numbers were similar. At 5 days post ligature placement, oral microbial communities sampled with swabs differed significantly in beta diversity between the mouse genotypes. Ligatures recovered from molar teeth of S100A9-/- and WT mice contained significantly dissimilar microbial genera from each other and the overall oral communities from swabs. Concomitantly, the S100A9-/- mice had significantly greater alveolar bone loss than WT mice around molar teeth in ligated sites. When the oral microflora was ablated by antibiotic pretreatment, differences disappeared between WT and S100A9-/- mice in their immune cell infiltrates and alveolar bone loss. Calprotectin, therefore, suppresses emergence of a dysbiotic, proinflammatory oral microbial community, which reduces innate immune effector activity, including early recruitment of innate immune cells, mitigating subsequent alveolar bone loss and protecting against experimental periodontitis.

Increased Likelihood of Detecting Ebola Virus RNA in Semen by Using Sample Pelleting.

Ebola virus RNA can reside for months or years in semen of survivors of Ebola virus disease and is probably associated with increased risk for cryptic sexual transmission of the virus. A modified protocol resulted in increased detection of Ebola virus RNA in semen and improved disease surveillance.

Adult and paediatric haematology and clinical chemistry laboratory reference limits for Liberia.

BACKGROUND: As more research is conducted in Liberia, there is a need for laboratory reference limits for common chemistry and haematology values based on a healthy population. Reference limits from the United States may not be applicable. OBJECTIVE: The aim of this study was to present laboratory reference ranges from a Liberian population and compare them to United States ranges. METHODS: Serum chemistry and haematology values from 2529 adults and 694 children and adolescents obtained from two studies conducted in Liberia between 2015 to 2017 were used to determine reference limits. After removing outliers, the reference limits defined by the 2.5th and 97.5th percentiles were determined by sex in three age groups (6-11, 12-17, and 18+ years). RESULTS: The median (interquartile range) of adults was 29 (23, 37) years; 44% were female. The median (interquartile range) for children and adolescents was 12 (9, 15) years; 53% were female. Several reference ranges determined using Liberian participants differed from those in the US. For chemistries, a high percentage of both adults and children/adolescents had high serum chloride levels based on United States ranges. For haematology, a high percentage of Liberian participants had haemoglobin and related assays below the lower limit of United States ranges. CONCLUSION: Chemistry and haematology reference intervals determined for a Liberian population of healthy individuals should be considered for establishing eligibility criteria and monitoring of laboratory adverse events for clinical trials as well as for use in clinical settings in Liberia and perhaps for other countries in Western Africa.

Chronic obstructive pulmonary disease upper airway microbiome is associated with select clinical characteristics.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disorder associated with lung microbiome dysbiosis. Although the upper airway microbiome is the source of the lung microbiome, the relationships between the oral, nasal, and sputum microbiota are incompletely understood. Our objective was to determine features that differentiate the oral, nasal, and sputum microbiome among subjects with stable COPD. METHODS: We recruited 15 current or former smokers to provide oral and sputum samples on day 1. On day 2, another oral sample and a nasal sample were obtained. Each sample and control underwent DNA extraction, 16S V4 rRNA amplification, 16S V4 sequencing, and qPCR of 16S rRNA. Data were analyzed using dada2 and R. RESULTS: Most (14 of 15) subjects were male with a mean age of 65.2. One subject had no pulmonary obstruction, while 5 had mild COPD, 7 had moderate COPD, and 2 had severe COPD. Three subjects (20%) were current tobacco users and 2 subjects (13%) used inhaled corticosteroids (ICS). Subjects had a mean of 49.1 pack-years of tobacco exposure. Bacterial biomass was associated with anatomic site, but no differences in biomass were observed with age, FEV1 percent predicted (FEV1pp), ICS use, smoking status, or edentulous state. Shannon index was associated with site (lower nasal diversity than oral and sputum diversity, p<0.001), but not age, ICS use, FEV1pp, tobacco use, or edentulous state. ß-diversity was illustrated by principal coordinate analysis using Bray-Curtis dissimilarity and PERMANOVA analyses, showing sample clustering by anatomic site (p = 0.001) with nasal samples forming a cluster separate from the combined oral wash samples and sputum samples. Clustering was also observed with ICS use (p = 0.029) and edentulous state (p = 0.019), while FEV1pp and current tobacco use were not significant. In an amplicon sequencing variant (ASV)-level analysis of oral samples using a linear regression model with Benjamini-Hochberg correction at an FDR<0.10, 10 ASVs were associated with age while no ASVs were associated with FEV1pp or smoking status. Sputum sample analysis demonstrated that 51 ASVs (25 unique genera) were associated with age, 61 ASVs (32 genera) were associated with FEV1pp, and no ASVs were associated with smoking status. In a combined dataset, the frequent exacerbator phenotype, rather than ICS use, was associated with decreased sputum Shannon diversity. CONCLUSIONS: Among the upper airway microbiota of COPD subjects, anatomic site was associated with bacterial biomass, Shannon diversity, and ß-diversity. ICS use and edentulous state were both associated with ß-diversity. Age was associated with taxa relative abundance in oral and sputum samples, while FEV1pp was associated with taxa relative abundance in sputum samples only.

Chronic obstructive pulmonary disease upper airway microbiota alpha diversity is associated with exacerbation phenotype: a case-control observational study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) frequent exacerbators (FE) suffer increased morbidity and mortality compared to infrequent exacerbators (IE). The association between the oral and sputum microbiota and exacerbation phenotype is not well defined. The objective of this study was to determine key features that differentiate the oral and sputum microbiota of FEs from the microbiota of IEs during periods of clinical stability. METHODS: We recruited 11 FE and 11 IE who had not used antibiotics or systemic corticosteroids in the last 1 month. Subjects provided oral wash and sputum samples, which underwent 16S V4 MiSeq sequencing and qPCR of 16S rRNA. Data were analyzed using Dada2 and R. RESULTS: FE and IE were similar in terms of age, FEV1 percent predicted (FEV1pp), pack-years of tobacco exposure, and St. George's Respiratory Questionnaire score. 16S copy numbers were significantly greater in sputum vs. oral wash (p = 0.01), but phenotype was not associated with copy number. Shannon diversity was significantly greater in oral samples compared to sputum (p = 0.001), and IE samples were more diverse than FE samples (p < 0.001). Sputum samples from FE had more Haemophilus and Moraxella compared to IE sputum samples, due to dominance of these COPD-associated taxa in three FE sputum samples. Amplicon sequencing variant (ASV)-level analysis of sputum samples revealed one ASV (Actinomyces) was significantly more abundant in IE vs. FE sputum (padj = 0.048, Wilcoxon rank-sum test), and this persisted after controlling for FEV1pp. Principal coordinate analysis using Bray-Curtis distance with PERMANOVA analyses demonstrated clustering by anatomic site, phenotype, inhaled corticosteroid use, current tobacco use, COPD severity, and last professional dental cleaning. CONCLUSIONS: FE have less diverse oral and sputum microbiota than IE. Actinomyces was significantly more abundant in IE sputum than FE sputum. The oral and sputum microbiota of COPD subjects cluster based on multiple clinical factors, including exacerbation phenotype. Even during periods of clinical stability, the frequent exacerbator phenotype is associated with decreased alpha diversity, beta-diversity clustering, and changes in taxonomic abundance.

HIV Care Trajectories as a Novel Longitudinal Assessment of Retention in Care.

Consistent engagement in care is associated with positive health outcomes among people living with HIV (PLWH). However, traditional retention measures ignore the evolving dynamics of engagement in care. To understand the longitudinal patterns of HIV care, we analyzed medical records from 2008 to 2015 of PLWH ≥ 18 years-old receiving care at a public, hospital-based HIV clinic (N = 2110). Using latent class analysis, we identified five distinct care trajectory classes: (1) consistent care (N = 1281); (2) less frequent care (N = 270); (3) return to care after initial attrition (N = 192); (4) moderate attrition (N = 163); and (5) rapid attrition (N = 204). The majority of PLWH in Class 1 (73.9%) had achieved sustained viral suppression (viral load ≤ 200 copies/mL at last test and > 12 months prior) by study end. Among the other care classes, there was substantial variation in sustained viral suppression (61.1% in Class 2 to 3.4% in Class 5). Care trajectories could be used to prioritize re-engagement efforts.

The lung tissue microbiota of mild and moderate chronic obstructive pulmonary disease.

BACKGROUND: Oral taxa are often found in the chronic obstructive pulmonary disease (COPD) lung microbiota, but it is not clear if this is due to a physiologic process such as aspiration or experimental contamination at the time of specimen collection. METHODS: Microbiota samples were obtained from nine subjects with mild or moderate COPD by swabbing lung tissue and upper airway sites during lung lobectomy. Lung specimens were not contaminated with upper airway taxa since they were obtained surgically. The microbiota were analyzed with 16S rRNA gene qPCR and 16S rRNA gene hypervariable region 3 (V3) sequencing. Data analyses were performed using QIIME, SourceTracker, and R. RESULTS: Streptococcus was the most common genus in the oral, bronchial, and lung tissue samples, and multiple other taxa were present in both the upper and lower airways. Each subject's own bronchial and lung tissue microbiota were more similar to each other than were the bronchial and lung tissue microbiota of two different subjects (permutation test, p = 0.0139), indicating more within-subject similarity than between-subject similarity at these two lung sites. Principal coordinate analysis of all subject samples revealed clustering by anatomic sampling site (PERMANOVA, p = 0.001), but not by subject. SourceTracker analysis found that the sources of the lung tissue microbiota were 21.1% (mean) oral microbiota, 8.7% nasal microbiota, and 70.1% unknown. An analysis using the neutral theory of community ecology revealed that the lung tissue microbiota closely reflects the bronchial, oral, and nasal microbiota (immigration parameter estimates 0.69, 0.62, and 0.74, respectively), with some evidence of ecologic drift occurring in the lung tissue. CONCLUSION: This is the first study to evaluate the mild-moderate COPD lung tissue microbiota without potential for upper airway contamination of the lung samples. In our small study of subjects with COPD, we found oral and nasal bacteria in the lung tissue microbiota, confirming that aspiration is a source of the COPD lung microbiota.

Biomarkers of inflammation in infants with cystic fibrosis.

BACKGROUND: There are urgent needs for clinically relevant biomarkers to identify children with cystic fibrosis (CF) at risk for more progressive lung disease and to serve as outcome measures for clinical trials. Our objective was to investigate three targeted biomarkers in a population of asymptomatic CF infants. METHODS: Urine, blood and lung function data were collected for 2 years from clinically stable infants diagnosed with CF by newborn screening. A subset of CF infants had bronchoscopy with lavage performed at 6 months and 1 year. Urine was collected quarterly from healthy control infants. Expectorated sputum and urine were collected quarterly for 2 years from clinically stable CF adults. Desmosine, club cell secretory protein (CCSP) and cathepsin B concentrations were measured and compared. Mixed effects models were used to identify associations between biomarker concentrations and clinical characteristics. Receiver operator characteristic curves were generated to investigate the sensitivity and specificity of the biomarkers. RESULTS: Urinary cathepsin B was significantly higher in CF infants compared to healthy infants (p = 0.005). CF infant airway and urinary cathepsin B concentrations were significantly lower compared to adult CF subjects (p = 0.002 & p = 0.022, respectively). CF infant airway CCSP was significantly higher than adult CF subjects (p < 0.001). There was a significant correlation between CF infant plasma CCSP and BALF CCSP (p = 0.046). BALF CCSP was negatively associated with IL-8 (p = 0.017). There was no correlation between biomarker concentration and FEV0.5. CONCLUSIONS: Cathepsin B and CCSP show promise as biomarkers of inflammation in CF infants. Further study is needed.

Potential Impact of Integrating HIV Surveillance and Clinic Data on Retention-in-Care Estimates and Re-Engagement Efforts.

Retention in care is essential to the health of people living with HIV and also for their communities. We sought to quantify the value of integrating HIV surveillance data with clinical records for improving the accuracy of retention-in-care estimates and the efficiency of efforts to re-engage out-of-care patients. Electronic medical records (EMRs) of HIV+ patients ≥18 years old from a public, hospital-based clinic in Minneapolis, MN between 2008 and 2014 were merged with state surveillance data on HIV-related laboratory tests, out-of-state relocation, and mortality. We calculated levels of retention and estimated the number of required case investigations to re-engage patients who appeared to be out of care over the study period with and without surveillance data integration. Retention was measured as the proportion of years in compliance with Health Resources and Services Administration (HRSA) guidelines (two clinical encounters >90 days apart annually) and the proportion of patients experiencing a gap in care >12 months. With data integration, retention estimates improved from an average HRSA compliance of 70.3% using EMR data alone to 77.5% with surveillance data, whereas the proportion of patients experiencing a >12-month gap in care decreased from 45.0% to 34.4%. If case investigations to re-engage patients were initiated after a 12-month gap in care, surveillance data would avoid 330 (29.3%) investigations over the study period. Surveillance data integration improves the accuracy of retention-in-care estimates and would avert a substantial number of unnecessary case investigations for patients who appear to be out of care but, in fact, receive care elsewhere or have died.

Synergistic reduction of HIV-1 infectivity by 5-azacytidine and inhibitors of ribonucleotide reductase.

Although many compounds have been approved for the treatment of human immunodeficiency type-1 (HIV-1) infection, additional anti-HIV-1 drugs (particularly those belonging to new drug classes) are still needed due to issues such as long-term drug-associated toxicities, transmission of drug-resistant variants, and development of multi-class resistance. Lethal mutagenesis represents an antiviral strategy that has not yet been clinically translated for HIV-1 and is based on the use of small molecules to induce excessive levels of deleterious mutations within the viral genome. Here, we show that 5-azacytidine (5-aza-C), a ribonucleoside analog that induces the lethal mutagenesis of HIV-1, and multiple inhibitors of the enzyme ribonucleotide reductase (RNR) interact in a synergistic fashion to more effectively reduce the infectivity of HIV-1. In these drug combinations, RNR inhibitors failed to significantly inhibit the conversion of 5-aza-C to 5-aza-2'-deoxycytidine, suggesting that 5-aza-C acts primarily as a deoxyribonucleoside even in the presence of RNR inhibitors. The mechanism of antiviral synergy was further investigated for the combination of 5-aza-C and one specific RNR inhibitor, resveratrol, as this combination improved the selectivity index of 5-aza-C to the greatest extent. Antiviral synergy was found to be primarily due to the reduced accumulation of reverse transcription products rather than the enhancement of viral mutagenesis. To our knowledge, these observations represent the first demonstration of antiretroviral synergy between a ribonucleoside analog and RNR inhibitors, and encourage the development of additional ribonucleoside analogs and RNR inhibitors with improved antiretroviral activity.

5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2'-Deoxycytidine.

5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2'-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography-tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTPin vitroat least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses.

Protein relative abundance patterns associated with sucrose-induced dysbiosis are conserved across taxonomically diverse oral microcosm biofilm models of dental caries.

BACKGROUND: The etiology of dental caries is multifactorial, but frequent consumption of free sugars, notably sucrose, appears to be a major factor driving the supragingival microbiota in the direction of dysbiosis. Recent 16S rRNA-based studies indicated that caries-associated communities were less diverse than healthy supragingival plaque but still displayed considerable taxonomic diversity between individuals. Metagenomic studies likewise have found that healthy oral sites from different people were broadly similar with respect to gene function, even though there was an extensive individual variation in their taxonomic profiles. That pattern may also extend to dysbiotic communities. In that case, shifts in community-wide protein relative abundance might provide better biomarkers of dysbiosis that can be achieved through taxonomy alone. RESULTS: In this study, we used a paired oral microcosm biofilm model of dental caries to investigate differences in community composition and protein relative abundance in the presence and absence of sucrose. This approach provided large quantities of protein, which facilitated deep metaproteomic analysis. Community composition was evaluated using 16S rRNA sequencing and metaproteomic approaches. Although taxonomic diversity was reduced by sucrose pulsing, considerable inter-subject variation in community composition remained. By contrast, functional analysis using the SEED ontology found that sucrose induced changes in protein relative abundance patterns for pathways involving glycolysis, lactate production, aciduricity, and ammonia/glutamate metabolism that were conserved across taxonomically diverse dysbiotic oral microcosm biofilm communities. CONCLUSIONS: Our findings support the concept of using function-based changes in protein relative abundance as indicators of dysbiosis. Our microcosm model cannot replicate all aspects of the oral environment, but the deep level of metaproteomic analysis it allows makes it suitable for discovering which proteins are most consistently abundant during dysbiosis. It then may be possible to define biomarkers that could be used to detect at-risk tooth surfaces before the development of overt carious lesions.

Lack of mutational hot spots during decitabine-mediated HIV-1 mutagenesis.

Decitabine has previously been shown to induce lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1). However, the factors that determine the susceptibilities of individual sequence positions in HIV-1 to decitabine have not yet been defined. To investigate this, we performed Illumina high-throughput sequencing of multiple amplicons prepared from proviral DNA that was recovered from decitabine-treated cells infected with HIV-1. We found that decitabine induced an ≈4.1-fold increase in the total mutation frequency of HIV-1, primarily due to a striking ≈155-fold increase in the G-to-C transversion frequency. Intriguingly, decitabine also led to an ≈29-fold increase in the C-to-G transversion frequency. G-to-C frequencies varied substantially (up to ≈80-fold) depending upon sequence position, but surprisingly, mutational hot spots (defined as upper outliers within the mutation frequency distribution) were not observed. We further found that every single guanine position examined was significantly susceptible to the mutagenic effects of decitabine. Taken together, these observations demonstrate for the first time that decitabine-mediated HIV-1 mutagenesis is promiscuous and occurs in the absence of a clear bias for mutational hot spots. These data imply that decitabine-mediated G-to-C mutagenesis is a highly effective antiviral mechanism for extinguishing HIV-1 infectivity.

HIV-1 and HIV-2 exhibit similar mutation frequencies and spectra in the absence of G-to-A hypermutation.

BACKGROUND: Human immunodeficiency virus type 2 (HIV-2) is often distinguished clinically by lower viral loads, reduced transmissibility, and longer asymptomatic periods than for human immunodeficiency virus type 1 (HIV-1). Differences in the mutation frequencies of HIV-1 and HIV-2 have been hypothesized to contribute to the attenuated progression of HIV-2 observed clinically. RESULTS: To address this hypothesis, we performed Illumina sequencing of multiple amplicons prepared from cells infected with HIV-1 or HIV-2, resulting in ~4.7 million read pairs and the identification of ~200,000 mutations after data processing. We observed that: (1) HIV-2 displayed significantly lower total mutation, substitution, and transition mutation frequencies than that of HIV-1, along with a mutation spectrum markedly less biased toward G-to-A transitions, (2) G-to-A hypermutation consistent with the activity of APOBEC3 proteins was observed for both HIV-1 and HIV-2 despite the presence of Vif, (3) G-to-A hypermutation was significantly higher for HIV-1 than for HIV-2, and (4) HIV-1 and HIV-2 total mutation frequencies were not significantly different in the absence of G-to-A hypermutants. CONCLUSIONS: Taken together, these data demonstrate that HIV-2 exhibits a distinct mutational spectrum and a lower mutation frequency relative to HIV-1. However, the observed differences were primarily due to reduced levels of G-to-A hypermutation for HIV-2. These findings suggest that HIV-2 may be less susceptible than HIV-1 to APOBEC3-mediated hypermutation, but that the fidelities of other mutational sources (such as reverse transcriptase) are relatively similar for HIV-1 and HIV-2. Overall, these data imply that differences in replication fidelity are likely not a major contributing factor to the unique clinical features of HIV-2 infection.

Glycerol Monolaurate Microbicide Protection against Repeat High-Dose SIV Vaginal Challenge.

Measures to prevent sexual mucosal transmission are critically needed, particularly to prevent transmission to young women at high risk in the microepidemics in South Africa that disproportionally contribute to the continued pandemic. To that end, microbicides containing anti-retroviral (ARV) agents have been shown to prevent transmission, but with efficacy limited both by adherence and pre-existing innate immune and inflammatory conditions in the female reproductive tract (FRT). Glycerol monolaurate (GML) has been proposed as a microbicide component to enhance efficacy by blocking these transmission-facilitating innate immune response to vaginal exposure. We show here in an especially rigorous test of protection in the SIV-rhesus macaque model of HIV-1 transmission to women, that GML used daily and before vaginal challenge protects against repeat high doses of SIV by criteria that include virological and immunological assays to detect occult infection. We also provide evidence for indirect mechanisms of action in GML-mediated protection. Developing a sustained formulation for GML delivery could contribute an independent, complementary protective component to an ARV-containing microbicide.

Metabolomics analysis identifies novel plasma biomarkers of cystic fibrosis pulmonary exacerbation.

BACKGROUND: Cystic fibrosis (CF) lung disease is characterized by infection, inflammation, lung function decline, and intermittent pulmonary exacerbations. However, the link between pulmonary exacerbation and lung disease progression remains unclear. Global metabolomic profiling can provide novel mechanistic insight into a disease process in addition to putative biomarkers for future study. Our objective was to investigate how the plasma metabolomic profile changes between CF pulmonary exacerbation and a clinically well state. METHODS: Plasma samples and lung function data were collected from 25 CF patients during hospitalization for a pulmonary exacerbation and during quarterly outpatient clinic visits. In collaboration with Metabolon, Inc., the metabolomic profiles of matched pair plasma samples, one during exacerbation and one at a clinic visit, were analyzed using gas and liquid chromatography coupled with mass spectrometry. Compounds were identified by comparison to a library of standards. Mixed effects models that controlled for nutritional status and lung function were used to test for differences and principal components analysis was performed. RESULTS: Our population had a median age of 27 years (14-39) and had a median FEV1 % predicted of 65% (23-105%). 398 total metabolites were identified and after adjustment for confounders, five metabolites signifying perturbations in nucleotide (hypoxanthine), nucleoside (N4-acetylcytidine), amino acid (N-acetylmethionine), carbohydrate (mannose), and steroid (cortisol) metabolism were identified. Principal components analysis provided good separation between the two clinical phenotypes. CONCLUSIONS: Our findings provide putative metabolite biomarkers for future study and allow for hypothesis generation about the pathophysiology of CF pulmonary exacerbation.