Using Network Analysis and Predictive Functional Analysis to Explore the Fluorotelomer Biotransformation Potential of Soil Microbial Communities.

Microbial transformation of per- and polyfluoroalkyl substances (PFAS), including fluorotelomer-derived PFAS, by native microbial communities in the environment has been widely documented. However, few studies have identified the key microorganisms and their roles during the PFAS biotransformation processes. This study was undertaken to gain more insight into the structure and function of soil microbial communities that are relevant to PFAS biotransformation. We collected 16S rRNA gene sequencing data from 8:2 fluorotelomer alcohol and 6:2 fluorotelomer sulfonate biotransformation studies conducted in soil microcosms under various redox conditions. Through co-occurrence network analysis, several genera, including Variovorax, Rhodococcus, and Cupriavidus, were found to likely play important roles in the biotransformation of fluorotelomers. Additionally, a metagenomic prediction approach (PICRUSt2) identified functional genes, including 6-oxocyclohex-1-ene-carbonyl-CoA hydrolase, cyclohexa-1,5-dienecarbonyl-CoA hydratase, and a fluoride-proton antiporter gene, that may be involved in defluorination. This study pioneers the application of these bioinformatics tools in the analysis of PFAS biotransformation-related sequencing data. Our findings serve as a foundational reference for investigating enzymatic mechanisms of microbial defluorination that may facilitate the development of efficient microbial consortia and/or pure microbial strains for PFAS biotransformation.

Assessment of a SARS-CoV-2 wastewater monitoring program in El Paso, Texas, from November 2020 to June 2022.

The border city of El Paso, Texas, and its water utility, El Paso Water, initiated a SARS-CoV-2 wastewater monitoring program to assess virus trends and the appropriateness of a wastewater monitoring program for the community. Nearly weekly sample collection at four wastewater treatment facilities (WWTFs), serving distinct regions of the city, was analyzed for SARS-CoV-2 genes using the CDC 2019-Novel coronavirus Real-Time RT-PCR diagnostic panel. Virus concentrations ranged from 86.7 to 268,000 gc/L, varying across time and at each WWTF. The lag time between virus concentrations in wastewater and reported COVID-19 case rates (per 100,00 population) ranged from 4-24 days for the four WWTFs, with the strongest trend occurring from November 2021 - June 2022. This study is an assessment of the utility of a geographically refined SARS-CoV-2 wastewater monitoring program to supplement public health efforts that will manage the virus as it becomes endemic in El Paso.

Aerobic biotransformation of 6:2 fluorotelomer sulfonate in soils from two aqueous film-forming foam (AFFF)-impacted sites.

Although 6:2 fluorotelomer sulfonate (6:2 FTS) is a common ingredient in aqueous film-forming foam (AFFF) formulations, its environmental fate at AFFF-impacted sites remains poorly understood. This study investigated the biotransformation of 6:2 FTS in microcosms prepared with soils collected from two AFFF-impacted sites; the former Loring Air Force Base (AFB) and Robins AFB. The half-life of 6:2 FTS in Loring soil was 43.3 days; while >60 mol% of initially spiked 6:2 FTS remained in Robins soil microcosms after a 224-day incubation. Differences in initial sulfate concentrations and the depletion of sulfate over the incubation likely contributed to the different 6:2 FTS biotransformation rates between the two soils. At day 224, stable transformation products, i.e., C4C7 perfluoroalkyl carboxylates, were formed with combined molar yields of 13.8 mol% and 1.2 mol% in Loring and Robins soils, respectively. Based on all detected transformation products, the biotransformation pathways of 6:2 FTS in the two soils were proposed. Microbial community analysis suggests that Desulfobacterota microorganisms may promote 6:2 FTS biotransformation via more efficient desulfonation. In addition, species from the genus Sphingomonas, which exhibited higher tolerance to elevated concentrations of 6:2 FTS and its biotransformation products, are likely to have contributed to 6:2 FTS biotransformation. This study demonstrates the potential role of biotransformation processes on the fate of 6:2 FTS at AFFF-impacted sites and highlights the need to characterize site biogeochemical properties for improved assessment of 6:2 FTS biotransformation behavior.

COVID-19 and brain-heart-lung microbial fingerprints in Italian cadavers.

Introduction: The fact that SARS-CoV-2, the coronavirus that caused COVID-19, can translocate within days of infection to the brain and heart and that the virus can survive for months is well established. However, studies have not investigated the crosstalk between the brain, heart, and lungs regarding microbiota that simultaneously co-inhabit these organs during COVID-19 illness and subsequent death. Given the significant overlap of cause of death from or with SARS-CoV-2, we investigated the possibility of a microbial fingerprint regarding COVID-19 death. Methods: In the current study, the 16S rRNA V4 region was amplified and sequenced from 20 COVID-19-positive and 20 non-COVID-19 cases. Nonparametric statistics were used to determine the resulting microbiota profile and its association with cadaver characteristics. When comparing non-COVID-19 infected tissues versus those infected by COVID-19, there is statistical differences (p < 0.05) between organs from the infected group only. Results: When comparing the three organs, microbial richness was significantly higher in non-COVID-19-infected tissues than infected. Unifrac distance metrics showed more variance between control and COVID-19 groups in weighted analysis than unweighted; both were statistically different. Unweighted Bray-Curtis principal coordinate analyses revealed a near distinct two-community structure: one for the control and the other for the infected group. Both unweighted and weighted Bray-Curtis showed statistical differences. Deblur analyses demonstrated Firmicutes in all organs from both groups. Discussion: Data obtained from these studies facilitated the defining of microbiome signatures in COVID-19 decedents that could be identified as taxonomic biomarkers effective for predicting the occurrence, the co-infections involved in its dysbiosis, and the evolution of the virus.

HPV-related anal cancer is associated with changes in the anorectal microbiome during cancer development.

Background: Squamous cell carcinoma of the anus (SCCA) is a rare gastrointestinal cancer. Factors associated with progression of HPV infection to anal dysplasia and cancer are unclear and screening guidelines and approaches for anal dysplasia are less clear than for cervical dysplasia. One potential contributing factor is the anorectal microbiome. In this study, we aimed to identify differences in anal microbiome composition in the settings of HPV infection, anal dysplasia, and anal cancer in this rare disease. Methods: Patients were enrolled in two prospective studies. Patients with anal dysplasia were part of a cross-sectional cohort that enrolled women with high-grade lower genital tract dysplasia. Anorectal tumor swabs were prospectively collected from patients with biopsy-confirmed locally advanced SCCA prior to receiving standard-of-care chemoradiotherapy (CRT). Patients with high-grade lower genital tract dysplasia without anal dysplasia were considered high-risk (HR Normal). 16S V4 rRNA Microbiome sequencing was performed for anal swabs. Alpha and Beta Diversity and composition were compared for HR Normal, anal dysplasia, and anal cancer. Results: 60 patients with high-grade lower genital tract dysplasia were initially enrolled. Seven patients had concurrent anal dysplasia and 44 patients were considered HR Normal. Anorectal swabs from 21 patients with localized SCCA were included, sequenced, and analyzed in the study. Analysis of weighted and unweighted UniFrac distances demonstrated significant differences in microbial community composition between anal cancer and HR normal (p=0.018). LEfSe identified that all three groups exhibited differential enrichment of specific taxa. Peptoniphilus (p=0.028), Fusobacteria (p=0.0295), Porphyromonas (p=0.034), and Prevotella (p=0.029) were enriched in anal cancer specimens when compared to HR normal. Conclusion: Although alpha diversity was similar between HR Normal, dysplasia and cancer patients, composition differed significantly between the three groups. Increased anorectal Peptoniphilus, Fusobacteria, Porphyromonas, and Prevotella abundance were associated with anal cancer. These organisms have been reported in various gastrointestinal cancers with roles in facilitating the proinflammatory microenvironment and neoplasia progression. Future work should investigate a potential role of microbiome analysis in screening for anal dysplasia and investigation into potential mechanisms of how these microbial imbalances influence the immune system and anal carcinogenesis.

Cervicovaginal Microbiota Profiles in Precancerous Lesions and Cervical Cancer among Ethiopian Women.

Although high-risk human papillomavirus infection is a well-established risk factor for cervical cancer, other co-factors within the local microenvironment may play an important role in the development of cervical cancer. The current study aimed to characterize the cervicovaginal microbiota in women with premalignant dysplasia or invasive cervical cancer compared with that of healthy women. The study comprised 120 Ethiopian women (60 cervical cancer patients who had not received any treatment, 25 patients with premalignant dysplasia, and 35 healthy women). Cervicovaginal specimens were collected using either an Isohelix DNA buccal swab or an Evalyn brush, and ribosomal RNA sequencing was used to characterize the cervicovaginal microbiota. Shannon and Simpson diversity indices were used to evaluate alpha diversity. Beta diversity was examined using principal coordinate analysis of weighted UniFrac distances. Alpha diversity was significantly higher in patients with cervical cancer than in patients with dysplasia and in healthy women (p < 0.01). Beta diversity was also significantly different in cervical cancer patients compared with the other groups (weighted UniFrac Bray-Curtis, p < 0.01). Microbiota composition differed between the dysplasia and cervical cancer groups. Lactobacillus iners was particularly enriched in patients with cancer, and a high relative abundance of Lactobacillus species was identified in the dysplasia and healthy groups, whereas Porphyromonas, Prevotella, Bacteroides, and Anaerococcus species predominated in the cervical cancer group. In summary, we identified differences in cervicovaginal microbiota diversity, composition, and relative abundance between women with cervical cancer, women with dysplasia, and healthy women. Additional studies need to be carried out in Ethiopia and other regions to control for variation in sample collection.

Assessing aerobic biotransformation of 8:2 fluorotelomer alcohol in aqueous film-forming foam (AFFF)-impacted soils: Pathways and microbial community dynamics.

Production of 8:2 fluorotelomer alcohol (8:2 FTOH) for industrial and consumer products, including aqueous film-forming foams (AFFFs) used for firefighting, has resulted in its widespread occurrence in the environment. However, the fate of 8:2 FTOH at AFFF-impacted sites remains largely unknown. Using AFFF-impacted soils from two United States Air Force Bases, microcosm experiments evaluated the aerobic biotransformation of 8:2 FTOH (extent and byproduct formation) and the dose-response on microbial communities due to 8:2 FTOH exposure. Despite different microbial communities, rapid transformation of 8:2 FTOH was observed during a 90-day incubation in the two soils, and 7:2 secondary fluorotelomer alcohol (7:2 sFTOH) and perfluorooctanoic acid (PFOA) were detected as major transformation products. Novel transformation products, including perfluoroalkane-like compounds (1H-perfluoroheptane, 1H-perfluorohexane, and perfluoroheptanal) were identified by liquid chromatography-high resolution mass spectrometry (LC-HRMS) and used to develop aerobic 8:2 FTOH biotransformation pathways. Microbial community analysis suggests that species from genus Sphingomonas are potential 8:2 FTOH degraders based on increased abundance in both soils after exposure, and the genus Afipia may be more tolerant to and/or involved in the transformation of 8:2 FTOH at elevated concentrations. These findings demonstrate the potential role of biological processes on PFAS fate at AFFF-impacted sites through fluorotelomer biotransformation.

Metagenomes of rectal swabs in larger, advanced stage cervical cancers have enhanced mucus degrading functionalities and distinct taxonomic structure.

BACKGROUND: Gut microbiome community composition differs between cervical cancer (CC) patients and healthy controls, and increased gut diversity is associated with improved outcomes after treatment. We proposed that functions of specific microbial species adjoining the mucus layer may directly impact the biology of CC. METHOD: Metagenomes of rectal swabs in 41 CC patients were examined by whole-genome shotgun sequencing to link taxonomic structures, molecular functions, and metabolic pathway to patient's clinical characteristics. RESULTS: Significant association of molecular functions encoded by the metagenomes was found with initial tumor size and stage. Profiling of the molecular function abundances and their distributions identified 2 microbial communities co-existing in each metagenome but having distinct metabolism and taxonomic structures. Community A (Clostridia and Proteobacteria predominant) was characterized by high activity of pathways involved in stress response, mucus glycan degradation and utilization of degradation byproducts. This community was prevalent in patients with larger, advanced stage tumors. Conversely, community B (Bacteroidia predominant) was characterized by fast growth, active oxidative phosphorylation, and production of vitamins. This community was prevalent in patients with smaller, early-stage tumors. CONCLUSIONS: In this study, enrichment of mucus degrading microbial communities in rectal metagenomes of CC patients was associated with larger, more advanced stage tumors.

Biotransformation of 8:2 Fluorotelomer Alcohol in Soil from Aqueous Film-Forming Foams (AFFFs)-Impacted Sites under Nitrate-, Sulfate-, and Iron-Reducing Conditions.

The environmental fate of per- and polyfluoroalkyl substances (PFAS) in aqueous film-forming foams (AFFFs) remains largely unknown, especially under the conditions representative of natural subsurface systems. In this study, the biotransformation of 8:2 fluorotelomer alcohol (8:2 FTOH), a component of new-generation AFFF formulations and a byproduct in fluorotelomer-based AFFFs, was investigated under nitrate-, iron-, and sulfate-reducing conditions in microcosms prepared with AFFF-impacted soils. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (HRMS) were employed to identify biotransformation products. The biotransformation was much slower under sulfate- and iron-reducing conditions with >60 mol % of initial 8:2 FTOH remaining after ∼400 days compared to a half-life ranging from 12.5 to 36.5 days under nitrate-reducing conditions. Transformation products 8:2 fluorotelomer saturated and unsaturated carboxylic acids (8:2 FTCA and 8:2 FTUA) were detected under all redox conditions, while 7:2 secondary fluorotelomer alcohol (7:2 sFTOH) and perfluorooctanoic acid (PFOA) were only observed as transformation products under nitrate-reducing conditions. In addition, 1H-perfluoroheptane (F(CF2)6CF2H) and 3-F-7:3 acid (F(CF2)7CFHCH2COOH) were identified for the first time during 8:2 FTOH biotransformation. Comprehensive biotransformation pathways for 8:2 FTOH are presented, which highlight the importance of accounting for redox condition and the related microbial community in the assessment of PFAS transformations in natural environments.

Microbiome Dynamics During Chemoradiation Therapy for Anal Cancer.

PURPOSE: Patients with localized squamous cell carcinoma of the anus (SCCA) who experience treatment toxicity or recurrences have few therapeutic options. Investigation into the microbiome's influence on treatment toxicity and its potential use as a predictive biomarker could improve these patients' outcomes. Our study presents the first longitudinal characterization of the SCCA tumor microbiome and its associations with treatment-related toxicities. METHODS AND MATERIALS: This prospective cohort study included patients with nonmetastatic SCCA receiving standard-of-care chemoradiation therapy. Anorectal swabs of the tumor site were collected before, during, and after treatment. Patient-reported quality-of-life metrics were collected at similar time points. 16S rRNA gene sequencing was used to perform diversity and taxonomic characterization of the SCCA microbiome. Wilcoxon signed-rank tests were used to compare microbial diversity and abundance over time. Wilcoxon rank-sum tests were used to compare microbial profiles of high and low toxicity groups. RESULTS: Twenty-two patients with SCCA were included in this study with a median age of 58.5 years (range, 39-77 years), and 18 (82%) were women. Alpha diversity remained relatively stable throughout chemoradiation therapy except for decreases in the Observed Features (P = .03) index at week 5 relative to baseline. Tumor microbial compositions changed significantly by the end of treatment (P = .03). Differential enrichment of bacteria at specific time points occurred during treatment, including the enrichment of Clostridia at follow-up (vs week 5, q = 0.019) and Corynebacterium at week 5 (vs baseline, q = 0.015; vs follow-up, q = 0.022). Patients experiencing high toxicity at week 5 had higher relative counts of Clostridia, Actinobacteria, and Clostridiales at baseline (P = .03 for all). CONCLUSIONS: The tumor microbiome changes during and after chemoradiation therapy, and patient-reported toxicity levels are associated with patients' microbial profiles. Further studies into these microbial characterizations and toxicity associations will elucidate the tumor microbiome's role in predicting treatment-related outcomes for patients with SCCA.

Gut Microbiome Profiles and Associated Metabolic Pathways in HIV-Infected Treatment-Naïve Patients.

The normal composition of the intestinal microbiota is a key factor for maintaining healthy homeostasis, and accordingly, dysbiosis is well known to be present in HIV-1 patients. This article investigates the gut microbiota profile of antiretroviral therapy-naive HIV-1 patients and healthy donors living in Latin America in a cohort of 13 HIV positive patients (six elite controllers, EC, and seven non-controllers, NC) and nine healthy donors (HD). Microbiota compositions in stool samples were determined by sequencing the V3-V4 region of the bacterial 16S rRNA, and functional prediction was inferred using PICRUSt. Several taxa were enriched in EC compared to NC or HD groups, including Acidaminococcus, Clostridium methylpentosum, Barnesiella, Eubacterium coprostanoligenes, and Lachnospiraceae UCG-004. In addition, our data indicate that the route of infection is an important factor associated with changes in gut microbiome composition, and we extend these results by identifying several metabolic pathways associated with each route of infection. Importantly, we observed several bacterial taxa that might be associated with different viral subtypes, such as Succinivibrio, which were more abundant in patients infected by HIV subtype B, and Streptococcus enrichment in patients infected by subtype C. In conclusion, our data brings a significant contribution to the understanding of dysbiosis-associated changes in HIV infection and describes, for the first time, differences in microbiota composition according to HIV subtypes. These results warrant further confirmation in a larger cohort of patients.

Diversity and composition of gut microbiome of cervical cancer patients: Do results of 16S rRNA sequencing and whole genome sequencing approaches align?

BACKGROUND: Next generation sequencing has progressed rapidly, characterizing microbial communities beyond culture-based or biochemical techniques. 16S ribosomal RNA gene sequencing (16S) produces reliable taxonomic classifications and relative abundances, while shotgun metagenome sequencing (WMS) allows higher taxonomic and functional resolution at greater cost. The purpose of this study was to determine if 16S and WMS provide congruent information for our patient population from paired fecal microbiome samples. RESULTS: Comparative indices were highly congruent between 16S and WMS. The most abundant genera for 16S and WMS data did not overlap. Overlap was observed at the Phylum level, as expected. However, relative abundances correlated poorly between the two methodologies (all P-value>0.05). Hierarchical clustering of both sequencing analyses identified overlapping enterotypes. Both approaches were in agreement with regard to demographic variables. CONCLUSION: Diversity, evenness and richness are comparable when using 16S and WMS techniques, however relative abundances of individual genera are not. Clinical associations with diversity and evenness metrics were similarly identified with WMS or 16S.

A prospective study of the adaptive changes in the gut microbiome during standard-of-care chemoradiotherapy for gynecologic cancers.

BACKGROUND: A diverse and abundant gut microbiome can improve cancer patients' treatment response; however, the effect of pelvic chemoradiotherapy (CRT) on gut diversity and composition is unclear. The purpose of this prospective study was to identify changes in the diversity and composition of the gut microbiome during and after pelvic CRT. MATERIALS AND METHODS: Rectal swabs from 58 women with cervical, vaginal, or vulvar cancer from two institutions were prospectively analyzed before CRT (baseline), during CRT (weeks 1, 3, and 5), and at first follow-up (week 12) using 16Sv4 rRNA gene sequencing of the V4 hypervariable region of the bacterial 16S rRNA marker gene. 42 of these patients received antibiotics during the study period. Observed operational taxonomic units (OTUs; representative of richness) and Shannon, Simpson, Inverse Simpson, and Fisher diversity indices were used to characterize alpha (within-sample) diversity. Changes over time were assessed using a paired t-test, repeated measures ANOVA, and linear mixed modeling. Compositional changes in specific bacteria over time were evaluated using linear discriminant analysis effect size. RESULTS: Gut microbiome richness and diversity levels continually decreased throughout CRT (mean Shannon diversity index, 2.52 vs. 2.91; all P <0.01), but were at or near baseline levels in 60% of patients by week 12. Patients with higher gut diversity at baseline had the steepest decline in gut microbiome diversity. Gut microbiome composition was significantly altered during CRT, with increases in Proteobacteria and decreases in Clostridiales, but adapted after CRT, with increases in Bacteroides species. CONCLUSION: After CRT, the diversity of the gut microbiomes in this population tended to return to baseline levels by the 12 week follow-up period, but structure and composition remained significantly altered. These changes should be considered when designing studies to analyze the gut microbiome in patients who receive pelvic CRT for gynecologic cancers.

Gut microbiome diversity is an independent predictor of survival in cervical cancer patients receiving chemoradiation.

Diversity of the gut microbiome is associated with higher response rates for cancer patients receiving immunotherapy but has not been investigated in patients receiving radiation therapy. Additionally, current studies investigating the gut microbiome and outcomes in cancer patients may not have adjusted for established risk factors. Here, we sought to determine if diversity and composition of the gut microbiome was independently associated with survival in cervical cancer patients receiving chemoradiation. Our study demonstrates that the diversity of gut microbiota is associated with a favorable response to chemoradiation. Additionally, compositional variation among patients correlated with short term and long-term survival. Short term survivor fecal samples were significantly enriched in Porphyromonas, Porphyromonadaceae, and Dialister, whereas long term survivor samples were significantly enriched in Escherichia Shigella, Enterobacteriaceae, and Enterobacteriales. Moreover, analysis of immune cells from cervical tumor brush samples by flow cytometry revealed that patients with a high microbiome diversity had increased tumor infiltration of CD4+ lymphocytes as well as activated subsets of CD4 cells expressing ki67+ and CD69+ over the course of radiation therapy. Modulation of the gut microbiota before chemoradiation might provide an alternative way to enhance treatment efficacy and improve treatment outcomes in cervical cancer patients.

Effect of Antibiotics on Gut and Vaginal Microbiomes Associated with Cervical Cancer Development in Mice.

Antibiotics affect microbial diversity in the gut, leading to dysbiosis and impaired immunity. However, the impact of antibiotics on microbial communities at other sites, such as vagina is less understood. It is also not clear whether changes induced by antibiotics in both microbiomes affect the development of cervical cancer. In this study, we utilized the murine model to evaluate these questions. We show that oral application of broad-spectrum antibiotics in mice changed not only diversity, but composition and sharing of gut and vaginal microbiomes in mice and influenced cervical cancer development in an orthotopic tumor model. Antibiotics decreased richness and diversity indexes in the gut but increased them in the vagina. Some beneficial taxa, such as Bacteroides, Ruminococcaceae, and Lachnospiraceae increased their abundance in the vagina while other pathogenic species, such as Proteobacteria, were decreased. As a result of the changes, mice with greater richness and diversity of the vaginal microbiome after antibiotics exposure were less likely developed tumors. No association between richness and diversity of the gut microbiome and tumor development was identified.

Tumor microbial diversity and compositional differences among women in Botswana with high-grade cervical dysplasia and cervical cancer.

INTRODUCTION: We characterized the cervical 16S rDNA microbiome of patients in Botswana with high-grade cervical dysplasia and locally advanced cervical cancer. METHODS: This prospective study included 31 patients: 21 with dysplasia and 10 with cancer. The Shannon diversity index was used to evaluate alpha (intra-sample) diversity, while the UniFrac (weighted and unweighted) and Bray-Curtis distances were employed to evaluate beta (inter-sample) diversity. The relative abundance of microbial taxa was compared among samples using linear discriminant analysis effect size. RESULTS: Alpha diversity was significantly higher in patients with cervical cancer than in patients with cervical dysplasia (P<0.05). Beta diversity also differed significantly (weighted UniFrac Bray-Curtis, P<0.01). Neither alpha diversity (P=0.8) nor beta diversity (P=0.19) varied by HIV status. The results of linear discriminant analysis effect size demonstrated that multiple taxa differed significantly between patients with cervical dysplasia vs cancer. Lachnospira bacteria (in the Clostridia class) were particularly enriched among cervical dysplasia patients, while Proteobacteria (members of the Firmicutes phyla and the Comamonadaceae family) were enriched in patients with cervical cancer. DISCUSSION: The results of our study suggest that differences exist in the diversity and composition of the cervical microbiota between patients with cervical dysplasia and patients with cervical cancer in Botswana. Additional studies are warranted to validate these findings and elucidate their clinical significance among women living in sub-Saharan Africa, as well as other regions of the world.

Impact of DNA Extraction Method on Variation in Human and Built Environment Microbial Community and Functional Profiles Assessed by Shotgun Metagenomics Sequencing.

Both the host microbiome and the microbiome of the built environment can have profound impacts on human health. While prior studies have suggested that the variability introduced by DNA extraction method is less than typical biologic variation, most studies have focused on 16S rRNA amplicon sequencing or on high biomass fecal samples. Shotgun metagenomic sequencing provides advantages over amplicon sequencing for surveying the microbiome, but is a challenge to perform in lower microbial biomass samples with high human DNA content such as sputum or vacuumed dust. Here we systematically evaluate the impact of four different extraction methods (phenol:choloroform, and three high-throughput kit-based approaches, the Promega Maxwell gDNA, Qiagen MagAttract PowerSoil DNA, and ZymoBIOMICS 96 MagBead). We report the variation in microbial community structure and predicted microbial function assessed by shotgun metagenomics sequencing in human stool, sputum, and vacuumed dust obtained from ongoing cohort studies or clinical trials. The same beadbeating protocol was used for all samples to focus our evaluation on the impact of kit chemistries on sequencing results. DNA yield was overall highest in the phenol:choloroform and Promega approaches. Only the phenol:choloroform approach showed evidence of contamination in negative controls. Bias was evaluated using mock community controls, and was noted across all extraction methods, although Promega exhibited the least amount of bias. The extraction method did not impact the proportion of human reads, although stool had the lowest proportion of human reads (0.1%) as compared to dust (44.1%) and sputum (80%). We calculated Bray-Curtis dissimilarity and Aitchison distances to evaluate the impact of extraction method on microbial community structure by sample type. Extraction method had the lowest impact in stool (extraction method responsible for 3.0-3.9% of the variability), the most impact in vacuumed dust (12-16% of the variability) and intermediate values for sputum (9.2-12% variability). Similar differences were noted when evaluating microbial community function. Our results will inform investigators planning microbiome studies using diverse sample types in large clinical studies. A consistent DNA extraction approach across all sample types is recommended, particularly with lower microbial biomass samples that are more heavily influenced by extraction method.

Microbial Diversity and Composition Is Associated with Patient-Reported Toxicity during Chemoradiation Therapy for Cervical Cancer.

PURPOSE: Patients receiving pelvic radiation for cervical cancer experience high rates of acute gastrointestinal (GI) toxicity. The association of changes in the gut microbiome with bowel toxicity from radiation is not well characterized. METHODS AND MATERIALS: Thirty-five patients undergoing definitive chemoradiation therapy (CRT) underwent longitudinal sampling (baseline and weeks 1, 3, and 5) of the gut microbiome and prospective assessment of patient-reported GI toxicity. DNA was isolated from stool obtained at rectal examination and analyzed with 16S rRNA sequencing. GI toxicity was assessed with the Expanded Prostate Cancer Index Composite instrument to evaluate frequency, urgency, and discomfort associated with bowel function. Shannon diversity index was used to characterize alpha (within sample) diversity. Weighted UniFrac principle coordinates analysis was used to compare beta (between sample) diversity between samples using permutational multivariate analysis of variance. Linear discriminant analysis effect size highlighted microbial features that best distinguish categorized patient samples. RESULTS: Gut microbiome diversity continuously decreased over the course of CRT, with the largest decrease at week 5. Expanded Prostate Cancer Index Composite bowel function scores also declined over the course of treatment, reflecting increased symptom burden. At all individual time points, higher diversity of the gut microbiome was linearly correlated with better patient-reported GI function, but baseline diversity was not predictive of eventual outcome. Patients with high toxicity demonstrated different compositional changes during CRT in addition to compositional differences in Clostridia species. CONCLUSIONS: Over time, increased radiation toxicity is associated with decreased gut microbiome diversity. Baseline diversity is not predictive of end-of-treatment bowel toxicity, but composition may identify patients at risk for developing high toxicity.

Intermittent fasting from dawn to sunset for 30 consecutive days is associated with anticancer proteomic signature and upregulates key regulatory proteins of glucose and lipid metabolism, circadian clock, DNA repair, cytoskeleton remodeling, immune system and cognitive function in healthy subjects.

Murine studies showed that disruption of circadian clock rhythmicity could lead to cancer and metabolic syndrome. Time-restricted feeding can reset the disrupted clock rhythm, protect against cancer and metabolic syndrome. Based on these observations, we hypothesized that intermittent fasting for several consecutive days without calorie restriction in humans would induce an anticarcinogenic proteome and the key regulatory proteins of glucose and lipid metabolism. Fourteen healthy subjects fasted from dawn to sunset for over 14 h daily. Fasting duration was 30 consecutive days. Serum samples were collected before 30-day intermittent fasting, at the end of 4th week during 30-day intermittent fasting, and one week after 30-day intermittent fasting. An untargeted serum proteomic profiling was performed using ultra high-performance liquid chromatography/tandem mass spectrometry. Our results showed that 30-day intermittent fasting was associated with an anticancer serum proteomic signature, upregulated key regulatory proteins of glucose and lipid metabolism, circadian clock, DNA repair, cytoskeleton remodeling, immune system, and cognitive function, and resulted in a serum proteome protective against cancer, metabolic syndrome, inflammation, Alzheimer's disease, and several neuropsychiatric disorders. These findings suggest that fasting from dawn to sunset for 30 consecutive days can be preventive and adjunct therapy in cancer, metabolic syndrome, and several cognitive and neuropsychiatric diseases. SIGNIFICANCE: Our study has important clinical implications. Our results showed that intermittent fasting from dawn to sunset for over 14 h daily for 30 consecutive days was associated with an anticancer serum proteomic signature and upregulated key regulatory proteins of glucose and lipid metabolism, insulin signaling, circadian clock, DNA repair, cytoskeleton remodeling, immune system, and cognitive function, and resulted in a serum proteome protective against cancer, obesity, diabetes, metabolic syndrome, inflammation, Alzheimer's disease, and several neuropsychiatric disorders. Importantly, these findings occurred in the absence of any calorie restriction and significant weight loss. These findings suggest that intermittent fasting from dawn to sunset can be a preventive and adjunct therapy in cancer, metabolic syndrome and Alzheimer's disease and several neuropsychiatric diseases.

Residual sugar from microalgae biomass harvested from phycoremediation of swine wastewater digestate.

The present study assessed the carbohydrate and sugar production from Chlorella spp. biomass harvested from a field scale reactor simulating phycoremediation of swine wastewater. The microalgae biomass was mainly composed by (%): carbohydrates (41 ± 0.4), proteins (50 ± 0.4), and lipids (1.3 ± 0.5). The residual sugar present in the biomass was extracted via acid hydrolysis. Among different concentrations of sulfuric acid tested (i.e., 47, 94, 188, 281 and 563 mM), significantly higher sugar content was obtained with 188 mM (0.496 g-sugar g-1 microalgae-DW). The concentration of sugar present in the microalgae did not differ significantly between the biomasses harvested by either centrifugation or coagulation-flocculation. Two commercially available strains of yeast (i.e., Saccharomyces cerevisiae and S. cerevisiae chardonnay) were tested for their capability to ferment sugar from lyophilized microalgae biomass. S. cerevisiae chardonnay showed a significantly faster consumption of sugar during the exponential growth phase. Both strains of yeast were capable of consuming most of the sugar added ≅ 8 g L-1 within 24 h. Overall, the results suggest that carbohydrate-rich microalgae biomass obtained from the phycoremediation of swine wastewaters can play an important role in green design for industries seeking alternative sources of feedstock rich in sugar.