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The nucleolus, a membrane-less organelle, is responsible for ribosomal RNA transcription, ribosomal RNA processing, and ribosome assembly. Nucleolar size and number are indicative of a cell's protein synthesis rate and proliferative capacity, and abnormalities in the nucleolus have been linked to neurodegenerative diseases and cancer. In this study, we demonstrated that the nucleolar protein ZNF692 directly interacts with nucleophosmin 1 (NPM1). Knocking down ZNF692 resulted in the nucleolar redistribution of NPM1 in ring-like structures and reduced protein synthesis. Purified NPM1 forms spherical condensates in vitro but mixing it with ZNF692 produces irregular condensates more closely resembling living cell nucleoli. Our findings indicate that ZNF692, by interacting with NPM1, plays a critical role in regulating nucleolar architecture and function in living cells. [ABSTRACT FROM AUTHOR]

Transcription/processing of the ribosomal RNA (rRNA) precursor, as part of ribosome biosynthesis, is intensively studied and characterized in eukaryotic cells. Here, we constructed shRNA-based mouse cell lines partially silenced for the Upstream Binding Factor UBF, the master regulator of rRNA transcription and organizer of open rDNA chromatin. Full Ubf silencing in vivo is not viable, and these new tools allow further characterization of rRNA transcription and its coordination with cellular signaling. shUBF cells display cell cycle G1 delay and reduced 47S rRNA precursor and 28S rRNA at baseline and serum-challenged conditions. Growth-related mTOR signaling is downregulated with the fractions of active phospho-S6 Kinase and pEIF4E translation initiation factor reduced, similar to phosphorylated cell cycle regulator retinoblastoma, pRB, positive regulator of UBF availability/rRNA transcription. Additionally, we find transcription-competent pUBF (Ser484) severely restricted and its interacting initiation factor RRN3 reduced and responsive to extracellular cues. Furthermore, fractional UBF occupancy on the rDNA unit is decreased in shUBF, and expression of major factors involved in different aspects of rRNA transcription is severely downregulated by UBF depletion. Finally, we observe reduced RNA Pol1 occupancy over rDNA promoter sequences and identified unexpected regulation of RNA Pol1 expression, relative to serum availability and under UBF silencing, suggesting that regulation of rRNA transcription may not be restricted to modulation of Pol1 promoter binding/elongation rate. Overall, this work reveals that UBF depletion has a critical downstream and upstream impact on the whole network orchestrating rRNA transcription in mammalian cells. [ABSTRACT FROM AUTHOR]

Bacterial populations exhibit heterogeneity in gene expression, which facilitates their survival and adaptation to unstable and unpredictable environments through the bet-hedging strategy. However, unraveling the rare subpopulations and heterogeneity in gene expression using population-level gene expression analysis remains a challenging task. Single-cell RNA sequencing (scRNA-seq) has the potential to identify rare subpopulations and capture heterogeneity in bacterial populations, but standard methods for scRNA-seq in bacteria are still under development, mainly due to differences in mRNA abundance and structure between eukaryotic and prokaryotic organisms. In this study, we present a hybrid approach that combines random displacement amplification sequencing (RamDA-seq) with Cas9-based rRNA depletion for scRNA-seq in bacteria. This approach allows cDNA amplification and subsequent sequencing library preparation from low-abundance bacterial RNAs. We evaluated its sequenced read proportion, gene detection sensitivity, and gene expression patterns from the dilution series of total RNA or the sorted single Escherichia coli cells. Our results demonstrated the detection of more than 1000 genes, about 24% of the genes in the E. coli genome, from single cells with less sequencing effort compared to conventional methods. We observed gene expression clusters between different cellular proliferation states or heat shock treatment. The approach demonstrated high detection sensitivity in gene expression analysis compared to current bacterial scRNA-seq methods and proved to be an invaluable tool for understanding the ecology of bacterial populations and capturing the heterogeneity of bacterial gene expression. [ABSTRACT FROM AUTHOR]

• The transcription-reverse transcription concerted (TRC) method for Mycobacterium avium-intracellulare complex can consistently identify M. chimaera as M. intracellulare. • A clinical isolate identified as M. intracellulare by polymerase chain reaction did not react to TRC. • The 16S ribosomal RNA sequencing failed to precisely identify the isolate. • Whole-genome shotgun sequencing and average nucleotide identity analysis led to the identification as M. chimaera. • Mutations in 16S ribosomal RNA gene could have caused the false-negative reaction to TRC. To report an isolate of Mycobacterium intracellulare subsp. chimaera with multiple mutations in 16S ribosomal RNA (rRNA) gene, resulting in the false-negative reaction to the transcription-reverse transcription concerted (TRC) method for Mycobacterium avium-intracellulare complex. We used TRC, polymerase chain reaction (PCR), and Matrix-assisted laser desorption/ionization Time-of-Flight/Mass Spectrometry (MALDI-TOF/MS) methods to identify a clinical isolate in 2021. Due to the discordant results between TRC and PCR or MALDI-TOF MS methods, 16S rRNA sequencing, whole-genome shotgun (WGS) sequencing, and average nucleotide identity (ANI) analysis were employed to identify the isolate. A mycobacterial isolate from a sputum sample gave negative results for the detection of Mycobacterium tuberculosis complex or M. avium-intracellulare complex by the TRC method. However, the isolate was identified as M. intracellulare by both PCR method and MALDI-TOF MS method. WGS sequencing of 16S rRNA genome revealed eight substitution mutations and one insertion mutation within the region, which could hamper the correct reaction to TRC method. Subsequent ANI analysis between the isolate and various species of nontuberculosis mycobacteria revealed that the isolate could be identified as M. intracellulare subsp. chimaera. Rare mutations within the 16S rRNA genome resulted in the false-negative identification of Mycobacterium chimaera by the TRC method. WGS sequencing and ANI analysis was necessary to identify the isolate. [ABSTRACT FROM AUTHOR]

Bacterial pathogens like Mycobacterium tuberculosis (Mtb) employ transcription factors to adapt their physiology to the diverse environments within their host. CarD is a conserved bacterial transcription factor that is essential for viability in Mtb. Unlike classical transcription factors that recognize promoters by binding to specific DNA sequence motifs, CarD binds directly to the RNA polymerase to stabilize the open complex intermediate (RPo) during transcription initiation. We previously showed using RNA-sequencing that CarD is capable of both activating and repressing transcription in vivo. However, it is unknown how CarD achieves promoter-specific regulatory outcomes in Mtb despite binding indiscriminate of DNA sequence. We propose a model where CarD's regulatory outcome depends on the promoter's basal RPo stability and test this model using in vitro transcription from a panel of promoters with varying levels of RPo stability. We show that CarD directly activates full-length transcript production from the Mtb ribosomal RNA promoter rrnAP3 (AP3) and that the degree of transcription activation by CarD is negatively correlated with RPo stability. Using targeted mutations in the extended -10 and discriminator region of AP3, we show that CarD directly represses transcription from promoters that form relatively stable RPo. DNA supercoiling also influenced RPo stability and affected the direction of CarD regulation, indicating that the outcome of CarD activity can be regulated by factors beyond promoter sequence. Our results provide experimental evidence for how RNA polymerase-binding transcription factors like CarD can exert specific regulatory outcomes based on the kinetic properties of a promoter. [ABSTRACT FROM AUTHOR]

One of the first steps in ribosome biogenesis is transcription of the ribosomal DNA by RNA polymerase I (Pol I). Processing of the resultant rRNA begins cotranscriptionally, and perturbation of Pol I transcription elongation results in defective rRNA processing. Mechanistic insight regarding the link between transcription elongation and ribosome assembly is lacking because of limited in vivo methods to assay Pol I transcription. Here, we use native elongating transcript sequencing (NET-Seq) with a strain of Saccharomyces cerevisiae containing a point mutation in Pol I, rpa190-F1205H, which results in impaired rRNA processing and ribosome assembly. We previously demonstrated that this mutation caused a mild reduction in the transcription elongation rate of Pol I in vitro; however, transcription elongation by the mutant has not been characterized in vivo. Here, our findings demonstrate that the mutant Pol I has an increased pause propensity during processive transcription elongation both in vitro and in vivo. NET-Seq reveals that rpa190-F1205H Pol I displays alternative pause site preferences in vivo. Specifically, the mutant is sensitized to A/G residues in the RNA:DNA hybrid and at the last incorporated nucleotide position. Furthermore, both NET-Seq and EM analysis of Miller chromatin spreads reveal pileups of rpa190-F1205H Pol I throughout the ribosomal DNA, particularly at the 5' end of the 35S gene. This combination of in vitro and in vivo analyses of a Pol I mutant provides novel insights into Pol I elongation properties and indicates how these properties are crucial for efficient cotranscriptional rRNA processing and ribosome assembly. [ABSTRACT FROM AUTHOR]

Background Previously, a novel oat ready-to-use therapeutic food (o-RUTF) resulted in improved recovery from severe acute malnutrition (SAM) when compared to a standard RUTF (s-RUTF). The o-RUTF contained 18% oat, while the s-RUTF has no cereal ingredients. Objectives We determined the effects of o-RUTF on intestinal permeability, as measured by lactulose permeability, and the 16S ribosomal RNA (rRNA) fecal microbiome configuration of children with SAM. Methods This was a prospective, randomized, double-blinded, controlled clinical trial. Sierra Leonean children aged 6–59 mo with SAM, defined by a midupper arm circumference < 11.5 cm, were randomized to receive o-RUTF or s-RUTF. All children received 7 d of amoxicillin per guidelines. Lactulose permeability testing and fecal 16S rRNA sequencing were performed at baseline and after 4 wk of therapy. The change in lactulose permeability was the primary outcome, while the fecal 16S rRNA configuration at 4 wk was a secondary outcome. Results Of the 129 children enrolled, lactulose permeability testing was completed by 100 at baseline and 82 at week 4. After 4 wk of therapeutic feeding, there were no differences in lactulose permeability between the o-RUTF and s-RUTF groups (P  = 0.84), and over half of children had increased lactulose permeability (50% s-RUTF compared with 58% o-RUTF, mean difference = −7.5%; 95% CI: −29.2, 15.2; P  = 0.50). After 4 wk of feeding, there were no differences in the 16S rRNA configurations between the o-RUTF and s-RUTF groups (Permanova, 999 permutations; P  = 0.648; pseudo-F = 0.581), nor were there differences in α or β diversity. Conclusions Despite remarkably different compositions of o-RUTF and s-RUTF, no differences were identified in lactulose permeability or the fecal 16S rRNA configuration among children with SAM receiving these foods. These results suggest that the o-RUTF exerts its beneficial effects through mechanisms other than reducing intestinal permeability or altering the fecal 16S configuration. This trial was registered at clinicaltrials.gov as NCT04334538. [ABSTRACT FROM AUTHOR]

The SpoU-TrmD (SPOUT) methyltransferase superfamily was designated when structural similarity was identified between the transfer RNA-modifying enzymes TrmH (SpoU) and TrmD. SPOUT methyltransferases are found in all domains of life and predominantly modify transfer RNA or ribosomal RNA substrates, though one instance of an enzyme with a protein substrate has been reported. Modifications placed by SPOUT methyltransferases play diverse roles in regulating cellular processes such as ensuring translational fidelity, altering RNA stability, and conferring bacterial resistance to antibiotics. This large collection of S-adenosyl-L-methionine-dependent methyltransferases is defined by a unique α/β fold with a deep trefoil knot in their catalytic (SPOUT) domain. Herein, we describe current knowledge of SPOUT enzyme structure, domain architecture, and key elements of catalytic function, including S-adenosyl-L-methionine co-substrate binding, beginning with a new sequence alignment that divides the SPOUT methyl-transferase superfamily into four major clades. Finally, a major focus of this review will be on our growing understanding of how these diverse enzymes accomplish the molecular feat of specific substrate recognition and modification, as highlighted by recent advances in our knowledge of protein-RNA complex structures and the discovery of the dependence of one SPOUT methyltransferase on metal ion binding for catalysis. Considering the broad biological roles of RNA modifications, developing a deeper understanding of the process of substrate recognition by the SPOUT enzymes will be critical for defining many facets of fundamental RNA biology with implications for human disease. [ABSTRACT FROM AUTHOR]

Antibiotic resistance via epigenetic methylation of ribosomal RNA is one of the most prevalent strategies adopted by multidrug resistant pathogens. The erythromycin-resistance methyltransferase (Erm) methylates rRNA at the conserved A2058 position and imparts resistance to macrolides such as erythromycin. However, the precise mechanism adopted by Erm methyltransferases for locating the target base within a complicated rRNA scaffold remains unclear. Here, we show that a conserved RNA architecture, including specific bulge sites, present more than 15 Å from the reaction center, is key to methylation at the pathogenic site. Using a set of RNA sequences site-specifically labeled by fluorescent nucleotide surrogates, we show that base flipping is a prerequisite for effective methylation and that distal bases assist in the recognition and flipping at the reaction center. The Erm-RNA complex model revealed that intrinsically flipped-out bases in the RNA serve as a putative anchor point for the Erm. Molecular dynamic simulation studies demonstrated the RNA undergoes a substantial change in conformation to facilitate an effective protein-rRNA handshake. This study highlights the importance of unique architectural features exploited by RNA to impart fidelity to RNA methyltransferases via enabling allosteric crosstalk. Moreover, the distal trigger sites identified here serve as attractive hotspots for the development of combination drug therapy aimed at reversing resistance. [ABSTRACT FROM AUTHOR]

Recent advances have highlighted the significant roles of post-transcriptional modifications in rRNA in various cancers. Evidence suggests that dysregulation of rRNA modifications acts as a common denominator in cancer development, with alterations in these modifications conferring competitive advantages to cancer cells. Specifically, rRNA modifications modulate protein synthesis and favor the specialized translation of oncogenic programs, thereby contributing to the formation of a protumorigenic proteome in cancer cells. These findings reveal a novel regulatory layer mediated by changes in the deposition of rRNA chemical modifications. Moreover, inhibition of these modifications in vitro and in preclinical studies demonstrates potential therapeutic applications. The recurrence of altered rRNA modification patterns across different types of cancer underscores their importance in cancer progression, proposing them as potential biomarkers and novel therapeutic targets. This review will highlight the latest insights into how post-transcriptional rRNA modifications contribute to cancer progression and summarize the main developments and ongoing challenges in this research area. • Ribosomal RNA modifications are critical for ribosome function and their dysregulation is associated with pathogenesis. • 2'-O-me methylation patterns are highly altered in cancer and correlate with aggressiveness. • Impaired pseudouridine deposition in cancer leads to altered translation of key tumor supressor genes. • Aberrant deposition of base modifications in rRNA in various cancers modulate ribosome function to favor tumorigenesis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Identification of QSB based on comparing 16S rRNA gene sequences was developed. • QSB in activated sludge primarily belong to rare taxa and conditionally rare taxa. • QSB exhibited higher average clustering coefficient than non-QSB. • QSB keystones and QSB nodes had a positive impact on network complexity. • QSB communities directly contributed to maintaining network robustness. Quorum-sensing bacteria (QSB) are crucial factors for microbial communication, yet their ecological role in wastewater treatment plants (WWTPs) remains unclear. Here, we developed a method to identify QSB by comparing 16S rRNA gene sequences. QSB in 388 activated sludge samples collected from 130 WWTPs across China primarily were identified as rare taxa and conditionally rare taxa. A co-occurrence network shared by all sludge communities revealed that QSB exhibited higher average clustering coefficient (0.46) than non-QSB (0.15). Individual sludge networks demonstrated that quorum sensing microbiomes were positively correlated with network robustness and network complexity, including average clustering coefficient and link density. We confirmed that QSB keystones and QSB nodes have a positive impact on network complexity by influencing network modularity through a structural equation model. Meanwhile, QSB communities directly contributed to maintaining network robustness (r = 0.29, P < 0.05). Hence, QSB play an important role in promoting network complexity and stability. Furthermore, QSB communities were positively associated with the functional composition of activated sludge communities (r = 0.33, P < 0.01), especially the denitrification capacity (r = 0.45, P < 0.001). Overall, we elucidated the ecological significance of QSB and provided support for QS-based regulation of activated sludge microbial communities. [ABSTRACT FROM AUTHOR]

Elongating nuclear RNA polymerases (Pols) frequently pause, backtrack, and are then reactivated by endonucleolytic cleavage. Pol backtracking and RNA cleavage are also crucial for proofreading, which contributes to transcription fidelity. RNA polymerase I (Pol I) of the yeast Saccharomyces cerevisiae synthesizes exclusively 35S rRNA, the precursor transcript of mature ribosomal 5.8S, 18S, and 25S rRNA. Pol I contains the specific heterodimeric subunits Rpa34.5/49 and subunit Rpa12.2, which have been implicated in RNA cleavage and elongation activity, respectively. These subunits are associated with the Pol I lobe structure and encompass different structural domains, but the contribution of these domains to RNA elongation is unclear. Here, we used Pol I mutants or reconstituted Pol I enzymes to study the effects of these subunits and/or their distinct domains on RNA cleavage, backtracking, and transcription fidelity in defined in vitro systems. Our findings suggest that the presence of the intact C-terminal domain of Rpa12.2 is sufficient to support the cleavage reaction, but that the N-terminal domains of Rpa12.2 and the heterodimer facilitate backtracking and RNA cleavage. Since both N-terminal and C-terminal domains of Rpa12.2 were also required to faithfully incorporate NTPs in the growing RNA chain, efficient backtracking and RNA cleavage might be a prerequisite for transcription fidelity. We propose that RNA Pols containing efficient RNA cleavage activity are able to add and remove nucleotides until the matching nucleotide supports RNA chain elongation, whereas cleavage-deficient enzymes can escape this proofreading process by incorporating incorrect nucleotides. [ABSTRACT FROM AUTHOR]

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 20O-methylation, pseudouridylation, N6-methyladenosine (m6A), and N6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single m6A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m6A modification, the methyltransferase responsible for the 18S rRNA m6A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m6A modification at position 1832 of human 18S rRNA. Our work supports findings that TRMT112 is required for METTL5 stability and reveals that human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. We show that loss of METTL5 in human cancer cell lines and in mice regulates gene expression at the translational level; additionally, Mettl5 knockout mice display reduced body size and evidence of metabolic defects. While recent work has focused heavily on m6A modifications in mRNA and their roles in mRNA processing and translation, we demonstrate here that deorphanizing putative methyltransferase enzymes can reveal previously unappreciated regulatory roles for m6A in noncoding RNAs. [ABSTRACT FROM AUTHOR]

Background: Diet patterns are a significant and modifiable contributing factor to the composition of the human gut microbiota.Objectives: We set out to identify reproducible relationships between diet and gut microbial community composition in a diverse, healthy US adult cohort.Methods: We collected 2 to 3 automated self-administered 24-hour dietary recalls over 10-14 days, together with a single stool sample, from 343 healthy adults in a cross-sectional phenotyping study. This study examined a multi-ethnic cohort balanced for age (18-65 years), sex, and BMI (18.5-45 kg/m2). Dietary data were edited to a tree format according to published methods. The tree structure was annotated with the average total grams of dry weight, fat, protein, carbohydrate, or fiber from each food item reported. The alpha and beta diversity measurements, calculated using the tree structure, were analyzed relative to the microbial community diversity, determined by a Quantitative Insights Into Microbial Ecology (QIIME) 2 analysis of the bacterial 16S ribosomal RNA V4 region, sequenced from stool samples. K-means clustering was used to form groups of individuals consuming similar diets, and gut microbial communities were compared among groups using differential expression analysis for sequence count data.Results: The alpha diversity of diet dry weight was significantly correlated with the gut microbial community alpha diversity (r = 0.171). The correlation improved when diet was characterized using grams of carbohydrates (r = 0.186) or fiber (r = 0.213). Bifidobacterium was enriched with diets containing higher levels of total carbohydrate from cooked grains. Lachnospira, was enriched with diet patterns containing high consumption of fiber from fruits excluding berries.Conclusions: The tree structure, annotated with grams of carbohydrate, is a robust analysis method for comparing self-reported diet to the gut microbial community composition. This method identified consumption of fiber from fruit robustly associated with an abundance of pectinolytic bacterial genus, Lachnospira, in the guts of healthy adults. This trial was registered at clinicaltrials.gov as NCT02367287. [ABSTRACT FROM AUTHOR]

Scrub typhus (caused by Orientia tsutsugamushi) is a neglected and underdiagnosed disease due to its non-specific clinical presentation and challenging diagnostics. We document the first study of direct diagnosis of scrub typhus from the blood sample of a patient by full-length 16S ribosomal RNA gene amplicon analysis using Oxford Nanopore sequencing. • Diagnosis of scrub typhus (ST) is sometimes difficult. • We diagnosed ST using Nanopore 16S ribosomal RNA metagenomics from a blood sample. • Nanopore sequencing is a feasible option to diagnose ST in developing countries. [ABSTRACT FROM AUTHOR]

Antibiotic-resistant bacteria (ARB) are believed to have originated from pristine soil environments. However, the prevalence of ARB in the root-associated niches of plants grown in these environments, as well as the distribution patterns of ARB in the rhizosphere and rhizoplane—two critical root-associated niches—remains elusive. In this study, we investigated the prevalence and distribution of ARB in the rhizosphere and rhizoplane of two main plant species, Suaeda salsa and Sporobolus alterniflorus , grown in a pristine environment in Yancheng, China, to address these questions. In total,181 bacterial strains were isolated from the root-associated niches of the two plant species. Strong niche differentiation of the bacterial community between the rhizosphere and rhizoplane was identified by both bacterial isolation- and high-throughput 16S ribosomal RNA gene (rDNA) sequencing-based analyses. In total, 69.06 % of the isolated bacterial strains were classified as ARB, with strains resistant to β-lactam class antibiotics and polymyxin being predominant. The relative abundance of ARB in the rhizoplane was higher than that in the rhizosphere for both plant species. Furthermore, variations were noted in the prevalence and distribution of ARB in the root-associated niches between the two species. Several pathogenic ARBs, such as Stenotrophomonas maltophilia , were identified among the isolated strains. A representative S. maltophilia strain was sequenced, and several important antibiotic resistance- and pathogenicity-associated genes were identified in its genome. These findings provide novel insights into the prevalence and distribution patterns of ARB in the rhizosphere and rhizoplane of plants grown in pristine environments. [ABSTRACT FROM AUTHOR]

Body fluid traceability inferences can provide important clues to the investigation of forensic cases. Microbiome has been proven to be well applied in forensic body fluid traceability studies. Most of the specimens at crime scenes are often exposed to the external environment when collected, so it is extremely important to exploring the structure characteristics of microbial communities of body fluid samples under different exposure durations for tracing the origin of body fluids based on microorganisms. Full-length 16S rRNA sequencing technology and multiple data analysis methods were used to explore the microbial changes in three types of body fluid samples at five different exposure time points. With increasing exposure time, the Proteobacteria abundance gradually increased in the negative control and body fluid samples, and the Bacteroidetes and Firmicutes abundance decreased gradually, but the relative abundance of dominant genera in each body fluid remained dynamically stable. The microbial community structures of those samples from the same individual at different exposure durations were similar, and there were no significant differences in the microbial community structures among the different exposure time points. LEfSe and random forest analyses were applied to screen stable and differential microbial markers among body fluids, such as Streptococcus thermophilus, Streptococcus pneumoniae and Haemophilus parainfluenzae in saliva; Lactobacillus iners and Streptococcus agalactiae in vaginal fluid. There were no significant differences in microbial community structures of the three types of body fluid samples exposed to the environment for various time periods, although the relative abundance of some microbes in these samples would change. The exposed samples could still be traced back to their source of the body fluid samples using the microbial community structures. • The Proteobacteria gradually increased in the negative control and body fluid samples with increasing exposure time. • There was no significant difference in the microbial communities in the same type of body fluids at different exposure durations. • Exposed samples can be used for traceability studies of body fluids over time using the microbial community structure. [ABSTRACT FROM AUTHOR]

• Long-read sequencing is a suitable platform for tracking natural microbial dynamics. • Chlorophyll-a concentration shapes bacterioplankton communities. • Bloom's microbiome changes with the bloom stage and cyanobacteria species. Understanding spatial and temporal heterogeneity in ecosystems is essential to forecasting the effects of environmental changes. Freshwater microbes, including cyanobacteria, play a crucial role in food-web structures and biochemical processes, yet can exhibit substantial heterogeneity through space and time. They also act as powerful indicators of natural and human-induced stress due to their high metabolic and rapid response to environmental change. The formation of cyanobacteria blooms can be particularly important due to the potential production of toxins that are harmful to humans and wildlife. While high water temperatures and high nutrients are largely recognized as triggers of cyanobacterial bloom formation, there is growing evidence of the role of its associated microbiome in bloom formation. The inability to accurately forecast cyanobacteria blooms is challenged by uncertainty in the degree to which microbial diversity, and bloom forming taxa in particular, exhibit spatial heterogeneity and how spatial heterogeneity varies seasonally or between lakes spanning the trophic gradient. Here, we used long-read sequencing of the 16S rRNA gene to quantify variations in microbial spatiotemporal dynamics over the course of an ice-free season between two lakes that varied substantially in trophic status. Our results showed that the microbial community composition of eutrophic Chautauqua Lake was seasonally and spatially structured; however, during bloom events we observed lower diversity and a homogeneous community dominated by Microcystis and enriched with Gammaproteobacteria. In oligotrophic Lake George, seasonality rather than the basin of origin played a major role in structuring the microbial community; however, there was a significant difference between basins when controlling for the temporal effect and was linked to a South-to-North anthropogenic gradient. This study provides a solid foundation for exploiting long-read sequencing of prokaryotes and couples sequencing with traditional water quality monitoring to assess microbial dynamics (e.g., cyanobacteria bloom microbiome) and the effect of local and global stressors. [ABSTRACT FROM AUTHOR]

Cancer cells are dependent upon an abundance of ribosomes to maintain rapid cell growth and proliferation. The rate-limiting step of ribosome biogenesis is ribosomal RNA (rRNA) synthesis by RNA polymerase I (Pol I). Therefore, a goal of the cancer therapeutic field is to develop and characterize Pol I inhibitors. Here, we elucidate the mechanism of Pol I inhibition by a first-in-class small-molecule BMH-21. To characterize the effects of BMH-21 on Pol I transcription, we leveraged high-resolution in vitro transcription assays and in vivo native elongating transcript sequencing (NET-seq). We find that Pol I transcription initiation, promoter escape, and elongation are all inhibited by BMH-21 in vitro. In particular, the transcription elongation phase is highly sensitive to BMH-21 treatment, as it causes a decrease in transcription elongation rate and an increase in paused Pols on the ribosomal DNA (rDNA) template. In vivo NET-seq experiments complement these findings by revealing a reduction in Pol I occupancy on the template and an increase in sequence-specific pausing upstream of G-rich rDNA sequences after BMH-21 treatment. Collectively, these data reveal the mechanism of action of BMH-21, which is a critical step forward in the development of this compound and its derivatives for clinical use. [ABSTRACT FROM AUTHOR]

[Display omitted] • Thyroid cancer patients have reduced richness and diversity of gut microbiota. • A predictive model of 10 genera could distinguish thyroid cancer patients from healthy controls. • The loss of the short-chain fatty acid-producing bacteria may promote thyroid carcinoma. • The functional changes that occur in thyroid cancer patients affect the processing of genetic information. • A four-genus microbial signature may be able to distinguish thyroid carcinoma patients with metastatic lymphadenopathy from those without metastatic lymphadenopathy. Emerging evidence suggests that the essence of life is the ecological balance of the neural, endocrine, metabolic, microbial, and immune systems. Gut microbiota have been implicated as an important factor affecting thyroid homeostasis. This study aims to explore the relationship between gut microbiota and the development of thyroid carcinoma. Stool samples were collected from 90 thyroid carcinoma patients (TCs) and 90 healthy controls (HCs). Microbiota were analyzed using 16S ribosomal RNA gene sequencing. A cross-sectional study of an exploratory cohort of 60 TCs and 60 HCs was conducted. The gut microbiota signature of TCs was established by LEfSe, stepwise logistic regression, lasso regression, and random forest model analysis. An independent cohort of 30 TCs and 30 HCs was used to validate the findings. Functional prediction was achieved using Tax4Fun and PICRUSt2. TC patients were subsequently divided into subgroups to analyze the relationship between microbiota and metastatic lymphadenopathy. In the exploratory cohorts, TCs had reduced richness and diversity of gut microbiota compared to HCs. No significant difference was found between TCs and HCs on the phylum level, though 70% of TCs had increased levels of Proteobacteria- types based on dominant microbiota typing. A prediction model of 10 genera generated with LEfSe analysis and lasso regression distinguished TCs from HCs with areas under the curves of 0.809 and 0.746 in the exploration and validation cohorts respectively. Functional prediction suggested that the microbial changes observed in TCs resulted in a decline in aminoacyl-tRNA biosynthesis, homologous recombination, mismatch repair, DNA replication, and nucleotide excision repair. A four-genus microbial signature was able to distinguish TC patients with metastatic lymphadenopathy from those without metastatic lymphadenopathy. Our study shows that thyroid carcinoma patients demonstrate significant changes in gut microbiota, which will help delineate the relationship between gut microbiota and TC pathogenesis. [ABSTRACT FROM AUTHOR]

Bromodomain adjacent to zinc finger domain protein 2A (BAZ2A) (also called transcription termination factor-1 interacting protein 5), a key component of the nucleolar remodeling complex, recruits the nucleolar remodeling complex to ribo-somal RNA genes, leading to their transcriptional repression. In addition to its tandem plant homeodomain-bromodomain that is involved in binding to acetylated histone H4, BAZ2A also contains a methyl-CpG-binding domain (MBD)-like Tip5/ ARBP/MBD (TAM) domain that shares sequence homology with the MBD. In contrast with the methyl-CpG-binding ability of the canonical MBD, the BAZ2A TAM domain has been shown to bind to promoter-associated RNAs of ribosomal RNA genes and promoter DNAs of other genes independent of DNA methylation. Nevertheless, how the TAM domain binds to RNA/DNA mechanistically remains elusive. Here, we characterized the DNA-/RNA-binding basis of the BAZ2A TAM domain by EMSAs, isothermal titration calorimetry binding assays, mutagenesis analysis, and X-ray crystallography. Our results showed that the TAM domain of BAZ2A selectively binds to dsDNA and dsRNA and that it binds to the backbone of dsDNA in a sequence nonspecific manner, which is distinct from the base-specific binding of the canonical MBD. Thus, our results explain why the TAM domain of BAZ2A does not specifically bind to mCG or TG dsDNA like the canonical MBD and also provide insights for further biological study of BAZ2A acting as a transcription factor in the future. [ABSTRACT FROM AUTHOR]

Background: At a population level, the relation between dairy consumption and gut microbiome composition is poorly understood.Objectives: We sought to study the cross-sectional associations between individual dairy foods (i.e., milk, yogurt, and cheese), as well as total dairy intake, and the gut microbiome composition in a large, representative sample of men living in south-eastern Australia.Methods: Data on 474 men (mean ± SD: 64.5 ± 13.5 y old) from the Geelong Osteoporosis Study were used to assess the cross-sectional association between dairy consumption and gut microbiome. Information on dairy intake was self-reported. Men were categorized as consumers and nonconsumers of milk, yogurt, cheese, and high- and low-fat milk. Milk, yogurt, and cheese intakes were summed to calculate the total dairy consumed per day and categorized into either low (<2.5 servings/d) or high (≥2.5 servings/d) total dairy groups. Fecal samples were analyzed using bacterial 16S ribosomal RNA (rRNA) gene sequencing. After assessment of α and β diversity, differential abundance analysis was performed to identify bacterial taxa associated with each of milk, yogurt, and cheese consumption compared with nonconsumption, low compared with high total dairy, and low- compared with high-fat milk consumption. All analyses were adjusted for potential confounders.Results: α Diversity was not associated with consumption of any of the dairy groups. Differences in β diversity were observed between milk and yogurt consumption compared with nonconsumption. Taxa belonging to the genera Ruminococcaceae UCG-010 and Bifidobacterium showed negative and weak positive associations with milk consumption, respectively. A taxon from the genus Streptococcus was positively associated with yogurt consumption, whereas a taxon from the genus Eisenbergiella was negatively associated with cheese consumption. No specific taxa were associated with low- compared with high-fat milk nor low compared with high total dairy consumption.Conclusions: In men, community-level microbiome differences were observed between consumers and nonconsumers of milk and yogurt. Bacterial taxon-level associations were detected with milk, yogurt, and cheese consumption. Total dairy consumption was not associated with any microbiome measures, suggesting that individual dairy foods may have differential roles in shaping the gut microbiome in men. [ABSTRACT FROM AUTHOR]

• Q fever is the most commonly reported zoonotic disease in Australia. • Endocarditis is one of the most common manifestations of chronic infection. • Q fever–related valvular damage may make patients susceptible to other pathogens. • We recommend screening for Q fever in endemic areas among patients with endocarditis with valvulopathies. Coxiella burnetti is the causative organism of the zoonotic infection Q fever, of which endocarditis is one of the most common manifestations of the chronic form. Polymicrobial endocarditis with Q fever is extremely rare and is yet to be described among an Australasian cohort. We present the case of a 32-year-old gardener with culture-negative chronic Q fever prosthetic valve endocarditis concomitant with another bacterial pathogen, leading to aortic root abscess formation, requiring a Bentall procedure, extracorporeal membrane oxygenation, and prolonged antimicrobial therapy, with a fatal outcome. Unique to our case, Q fever was identified early, and the second pathogen was only detected on 16S ribosomal RNA (rRNA) polymerase chain reaction of explanted valvular tissue. Given the high risk for morbidity, we recommend that screening for Q fever in endemic areas among patients with infective endocarditis from other etiologies be considered. In addition, this case highlights the role for Q fever vaccination of the at-risk population with underlying valvulopathy. Furthermore, clinicians should be aware of polymicrobial infective endocarditis and suspicious in case of patients with atypical clinical features. [ABSTRACT FROM AUTHOR]

Background: Diet affects the human gastrointestinal microbiota. Blood and urine samples have been used to determine nutritional biomarkers. However, there is a dearth of knowledge on the utility of fecal biomarkers, including microbes, as biomarkers of food intake.Objectives: This study aimed to identify a compact set of fecal microbial biomarkers of food intake with high predictive accuracy.Methods: Data were aggregated from 5 controlled feeding studies in metabolically healthy adults (n = 285; 21-75 y; BMI 19-59 kg/m2; 340 data observations) that studied the impact of specific foods (almonds, avocados, broccoli, walnuts, and whole-grain barley and whole-grain oats) on the human gastrointestinal microbiota. Fecal DNA was sequenced using 16S ribosomal RNA gene sequencing. Marginal screening was performed on all species-level taxa to examine the differences between the 6 foods and their respective controls. The top 20 species were selected and pooled together to predict study food consumption using a random forest model and out-of-bag estimation. The number of taxa was further decreased based on variable importance scores to determine the most compact, yet accurate feature set.Results: Using the change in relative abundance of the 22 taxa remaining after feature selection, the overall model classification accuracy of all 6 foods was 70%. Collapsing barley and oats into 1 grains category increased the model accuracy to 77% with 23 unique taxa. Overall model accuracy was 85% using 15 unique taxa when classifying almonds (76% accurate), avocados (88% accurate), walnuts (72% accurate), and whole grains (96% accurate). Additional statistical validation was conducted to confirm that the model was predictive of specific food intake and not the studies themselves.Conclusions: Food consumption by healthy adults can be predicted using fecal bacteria as biomarkers. The fecal microbiota may provide useful fidelity measures to ascertain nutrition study compliance. [ABSTRACT FROM AUTHOR]

The RNA-dependent RNA polymerase (NSP12) of COVID-19 plays a significant role in the viral infection process, which promotes viral RNA replication by cooperating with NSP7 and NSP8, but little is known about its regulation on the function of host cells. We firstly found that overexpression of NSP12 had little effect on host mRNAs transcription. Using iCLIP technology, we found that NSP12 can bind a series of host RNAs with the conserved binding motif G(C/A/G)(U/G/A)UAG, especially ribosomal RNA. We found that NSP12 could directly bind to eEF1A factor via the NIRAN domain of NSP12 and N-terminal domain of eEF1A. NSP12 colocalized with eEF1A to inhibit type I interferon expression upon virus infection. In order to prove that NSP12 regulates the translation level of host cells, we found that NSP12 significantly affected the translation efficiency of many host mRNAs (such as ISG15, NF-κB2, ILK and SERPINI2) via ribosome profiling experiment, and the genes with significant upregulation in translation efficiency were mainly enriched in positive regulation of ubiquitin-dependent proteasomal process and NIK/NF-κB signaling pathway (such as NF-κB2, ILK), and negative regulation of type I interferon production, protein level of these genes were further confirmed in HEK293T and Calu3 cells upon NSP12 overexpression. These results indicate that NSP12 of SARS-CoV-2 can hijack the eEF1A factor to regulate translation efficiency of host mRNAs, which provides a new idea for us to evaluate the impact of SARS-CoV2 virus on the host and study the potential drug targets. [Display omitted] • NSP12 have little effect on mRNA transcription of host cells. • NSP12 can bind a series of host RNAs with the conserved binding motif. • NSP12 hijack eEF1A factor to inhibit type I IFN expression and play pro-viral role. • NSP12 hijack eEF1A factor to regulate the translation level of host mRNAs. [ABSTRACT FROM AUTHOR]

• Human cells carry ∼ 400 rRNA gene copies, which are epigenetically regulated via DNA methylation and histone modifications. • Dysregulation of rRNA synthesis and ribosome biogenesis are characteristic features of cancers. • Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers globally. • We show that rRNA genes are downregulated and their promoters are hypermethylated in multiple HNSCC cell lines. • Our results show correlation between rRNA gene downregulation and rDNA promoter hypermethylation in the HNSCC cell lines. Eukaryotes carry hundreds of ribosomal RNA (rRNA) genes as tandem arrays, which generate rRNA for protein synthesis. Humans carry ∼ 400 rRNA gene copies and their expression is epigenetically regulated. Dysregulation of rRNA synthesis and ribosome biogenesis are characteristic features of cancers. Targeting aberrant rRNA expression for cancer therapy is being explored. Head and neck squamous cell carcinoma (HNSCC) is among the most prevalent cancers globally. Using quantitative PCR and bisulfite sequencing, we show that rRNA genes are downregulated and their promoters are hypermethylated in HNSCC cell lines. These findings may have relevance for prognosis and diagnosis of HNSCC. [ABSTRACT FROM AUTHOR]

• Zooplankton community was studied in the western Pacific Warm Pool. • COI metabarcoding detected 2.3 times more zooplankton taxa than microscopy. • Sequence counts were significantly correlated to ZooScan data. • COI metabarcoding detected greater seasonality under stable environment. The environment of the western Pacific Warm Pool is considered to be pronouncedly uniform and stable compared with other tropical ocean environments. Knowledge of the seasonal variations in the zooplankton community structure in this region with a high plankton species diversity remains limited. In this study, three methods, including identification using microscopy, ZooScan digital imaging, and DNA metabarcoding, were used to analyze zooplankton samples collected in three seasons, and to detect the seasonal variations in the community. The results show that the metabarcoding method based on the mitochondrial cytochrome c oxidase I (COI) marker detected up to 2.3 times more zooplankton taxa than identification using microscopy. In contrast, the small subunit ribosomal RNA (18S) marker revealed fewer taxa than microscopy. Significantly positive correlations were observed between the sequence counts and three types of quantitative data, including the biovolume, dry weight biomass, and carbon biomass in all three seasons. The COI sequence counts exhibited a stronger correlation with the dry weight biomass than the biovolume and carbon biomass. Although both the microscopic and molecular methods detected seasonal variability of the zooplankton community, the COI metabarcoding method was more sensitive in detecting seasonal variations in the community, which was positively correlated with the taxonomic resolution of the different methods. In summary, the results of this study suggest that COI metabarcoding is an ideal tool for monitoring the seasonal variations in the diversity and community structure of zooplankton in the tropical open ocean regions with a stable environment all year round. [ABSTRACT FROM AUTHOR]

[Display omitted] • nZVI exposure increased the antibiotic resistome in anaerobic digestion. • Efflux pump and rRNA methyltransferase shaped the antibiotic resistome variation. • Improved adaptability of efflux pump benefited the anaerobes withstanding nZVI. • Increased acquisition of rRNA methyltransferase preserved the structure of protein. • Polyspecificity of multidrug resistance drove the ARGs evolution in anaerobes. Nano zero-valent iron (nZVI) assisted anaerobic digestion (AD) is considered an effective approach for mediating antibiotic resistance genes (ARGs) dissemination in activated sludge. However, little is known about the mechanisms that drive antibiotic resistomes evolution in relation with their physiological roles distinct from protection of bacteria against antimicrobials. Metagenomic sequencing showed that efflux pumps and rRNA methyltransferase genes were the most prevalent ARGs in anaerobes, being responsible for the variations of antibiotic resistome upon nZVI addition. Particularly, to withstand the oxidative stimuli derived from initially 9 days nZVI exposure, genes involved in the two component systems (PhoRB , BaeSR , EvgSA) and global regulator networks (MarA , SoxS) were increased, activating the chromosomally encoded intrinsic efflux pumps expression. Furthermore, to benefit for survival, the polyspecific efflux pumps were involved in the microbial physiological adaptations of enhanced siderophores, lipid moieties, and quorum sensing molecules exportation, which induced the adaptative efflux pumps expression. On the other hand, to maintain the protein synthesis under oxidative condition, genes encoding Rsm, Erm, and Rlu family methyltransferases were notably up-regulated via horizontal transfer, inducing the acquired rRNA methylation resistance. Importantly, after prolonged exposure to nZVI for 18 and 27 days, the antibiotic resistomes were measurably decreased, due to the recovered oxidative state and increased membrane rigidity. Overall, these findings provide a deep understanding on the necessity of antibiotic resistome evolution for anaerobes to adapt to nZVI exposure for survival, which can be introduced to improve the ARGs risks analysis. [ABSTRACT FROM AUTHOR]

Ticks are vectors of a variety of pathogens that can infect humans and animals. Ticks also harbor non-pathogenic microbiota. This study characterized the microbiota of the ticks infesting beef cattle in Thailand. Two species of ticks; Rhipicephalus microplus (n = 15) and Haemaphysalis bispinosa (n = 5), were collected in seven provinces in northeastern Thailand. Microbial community profile of ticks was examined based on sequences of the V3-V4 region of 16S rRNA gene. Proteobacteria (Pseudomonadota) was the most abundant phylum, followed by Firmicutes (Bacillota), and Actinobacteriota. Coxiella -like endosymbiont was the most abundant bacterial taxon overall (49% of sequence reads), followed by Anaplasma (8.5%), Corynebacterium (5.5%), Ehrlichia (3.9%), and Castellaniella (3.4%). Co-infections of the pathogenic bacteria Ehrlichia and Anaplasma were detected in 19/20 (95%) female ticks. The tick with the lowest number of bacteria had the lowest abundance of the Coxiella -like endosymbiont, and the pathogenic bacteria Anaplasma and Ehrlichia were absent. This study provides baseline information of the microbiota of cattle ticks in northeastern Thailand, suggesting that ticks carry a few dominant bacterial taxa that are primarily non-pathogenic but can co-occur with pathogenic microorganisms. The information obtained is useful for monitoring disease outbreaks in the future and informing prevention and control strategies against cattle tick-borne diseases. • Cattle-tick microbiota analyzed in northeastern Thailand. • First reported microbiota of R. microplus and H. bispinosa in the region. • Coxiella -like endosymbiont is the most abundant bacterial taxon of hard ticks. • Co-infections with the pathogens Ehrlichia and Anaplasma were detected. [ABSTRACT FROM AUTHOR]

Recent reports provide evidence that the platinum chemotherapeutic oxaliplatin causes cell death via ribosome biogenesis stress, while cisplatin causes cell death via the DNA damage response (DDR). Underlying differences in mechanisms that might initiate disparate routes to cell death by these two broadly used platinum compounds have not yet been carefully explored. Additionally, prior studies had demonstrated that cisplatin can also inhibit ribosome biogenesis. Therefore, we sought to directly compare the initial influences of oxaliplatin and cisplatin on nucleolar processes and on the DDR. Using pulse-chase experiments, we found that at equivalent doses, oxaliplatin but not cisplatin significantly inhibited ribosomal RNA (rRNA) synthesis by Pol I, but neither compound affected rRNA processing. Inhibition of rRNA synthesis occurred as early as 90 min after oxaliplatin treatment in A549 cells, concurrent with the initial redistribution of the nucleolar protein nucleophosmin (NPM1). We observed that the nucleolar protein fibrillarin began to redistribute by 6 h after oxaliplatin treatment and formed canonical nucleolar caps by 24 h. In cisplatin-treated cells, DNA damage, as measured by ΔH2AX immunofluorescence, was more extensive, whereas nucleolar organization was unaffected. Taken together, our results demonstrate that oxaliplatin causes early nucleolar disruption via inhibition of rRNA synthesis accompanied by NPM1 relocalization and subsequently causes extensive nucleolar reorganization, while cisplatin causes early DNA damage without significant nucleolar disruption. These data support a model in which, at clinically relevant doses, cisplatin kills cells via the canonical DDR, and oxaliplatin kills cells via ribosome biogenesis stress, specifically via rapid inhibition of rRNA synthesis. [ABSTRACT FROM AUTHOR]

Elongin A (EloA) is an essential transcription factor that stimulates the rate of RNA polymerase II (Pol II) transcription elongation in vitro. However, its role as a transcription factor in vivo has remained underexplored. Here we show that in mouse embryonic stem cells, EloA localizes to both thousands of Pol II transcribed genes with preference for transcription start site and promoter regions and a large number of active enhancers across the genome. EloA deletion results in accumulation of transcripts from a subset of enhancers and their adjacent genes. Notably, EloA does not substantially enhance the elongation rate of Pol II in vivo. We also show that EloA localizes to the nucleoli and associates with RNA polymerase I transcribed ribosomal RNA gene, Rn45s. EloA is a highly disordered protein, which we demonstrate forms phaseseparated condensates in vitro, and truncation mutations in the intrinsically disordered regions (IDR) of EloA interfere with its targeting and localization to the nucleoli. We conclude that EloA broadly associates with transcribed regions, tunes RNA Pol II transcription levels via impacts on enhancer RNA synthesis, and interacts with the rRNA producing/processing machinery in the nucleolus. Our work opens new avenues for further investigation of the role of this functionally multifaceted transcription factor in enhancer and ribosomal RNA biology. [ABSTRACT FROM AUTHOR]

Protein biosynthesis is fundamental to cellular life and requires the efficient functioning of the translational machinery. At the center of this machinery is the ribosome, a ribonucleoprotein complex that depends heavily on Mg21 for structure. Recent work has indicated that other metal cations can substitute for Mg21, raising questions about the role different metals may play in the maintenance of the ribosome under oxidative stress conditions. Here, we assess ribosomal integrity following oxidative stress both in vitro and in cells to elucidate details of the interactions between Fe21+ and the ribosome and identifyMn21+ as a factor capable of attenuating oxidant-induced Fe21-mediated degradation of rRNA. We report that Fe21 promotes degradation of all rRNA species of the yeast ribosome and that it is bound directly to RNA molecules. Furthermore, we demonstrate that Mn21+ competes with Fe21 for rRNA-binding sites and that protection of ribosomes from Fe21+-mediated rRNA hydrolysis correlates with the restoration of cell viability. Our data, therefore, suggest a relationship between these two transitionmetals in controlling ribosome stability under oxidative stress. [ABSTRACT FROM AUTHOR]

Microbiome research based on high-throughput sequencing has grown exponentially in recent years, but methodological variations can easily undermine the reproducibility across studies. To systematically evaluate the comparability of sequencing results of 16S rRNA gene sequencing (16Ss)- and shotgun metagenomic sequencing (SMs)-based microbial community profiling in laboratories under routine conditions. We designed a multicenter study across 35 participating laboratories in China using designed mock communities and homogenized fecal samples. A wide range of practices and approaches was reported by the participating laboratories. The observed microbial compositions of the mock communities in 46.2% (12/26) of the 16Ss and 82.6% (19/23) of the SMs laboratories had significant correlations with the expected result (Spearman r>0.59, P <0.05). The results from laboratories with near-identical protocols showed slight interlaboratory deviations. However, a high degree of interlaboratory deviation was found in the observed abundances of specific taxa, such as Bacteroides spp. (range: 0.3%-53.5%), Enterococci spp. (range: 0.8%-43.9%) and Fusobacterium spp. (range: 0.1%-39.8%). SMs performed better than 16Ss in detecting low-abundance bacteria (B. bifidum). The differences in DNA extraction methods, amplified regions and bioinformatics analysis tools (taxonomic classifiers and database) were important factors causing interlaboratory deviations. Addressing laboratory contamination is an urgent task because various sources of unexpected microbes were found in negative control samples. Well-defined control samples, such as the mock communities in this study, should be routinely used in microbiome research for monitoring potential biases. The findings in this study will provide guidance in the choice of more reasonable operating procedures to minimize potential methodological biases in revealing human microbiota composition. [ABSTRACT FROM AUTHOR]

Background: Recent meta-analyses suggest that the consumption of fermented dairy products reduces type 2 diabetes and cardiovascular disease (CVD) risk, although the underlying mechanisms remain unclear.Objective: We evaluated whether dairy protein products modulated gut microbiota and cardiometabolic features in mouse models of diet-induced obesity and CVD.Methods: Eight-week-old C57BL/6J wild-type (WT) and LDLr-/-ApoB100/100 (LRKO) male mice were fed for 12 and 24 wk, respectively, with a high-fat/high-sucrose diet [66% kcal lipids, 22% kcal carbohydrates (100% sucrose), 12% kcal proteins]. The protein sources of the 4 diets were 100% nondairy protein (NDP), or 50% of the NDP energy replaced by milk (MP), milk fermented by Lactobacillus helveticus (FMP), or Greek-style yogurt (YP) protein. Fecal 16S rRNA gene-based amplicon sequencing, intestinal gene expression, and glucose tolerance test were conducted. Hepatic inflammation and circulating adhesion molecules were measured by multiplex assays.Results: Feeding WT mice for 12 wk led to a 74% increase in body weight, whereas after 24 wk the LRKO mice had a 101.5% increase compared with initial body weight. Compared with NDP and MP, the consumption of FMP and YP modulated the gut microbiota composition in a similar clustering pattern, upregulating the Streptococcus genus in both genotypes. In WT mice, feeding YP compared with NDP increased the expression of genes involved in jejunal (Reg3b, 7.3-fold, P = 0.049) and ileal (Ocln, 1.7-fold, P = 0.047; Il1-β,1.7-fold, P = 0.038; Nos2, 3.8-fold, P = 0.018) immunity and integrity. In LRKO mice, feeding YP compared with MP improved insulin sensitivity by 65% (P = 0.039). In LRKO mice, feeding with FMP versus NDP attenuated hepatic inflammation (monocyte chemoattractant protein 1, 2.1-fold, P ˂ 0.0001; IL1-β, 5.7-fold, P = 0.0003; INF-γ, 1.7-fold, P = 0.002) whereas both FMP [vascular adhesion molecule 1 (VCAM1), 1.3-fold, P = 0.0003] and YP (VCAM1, 1.04-fold, P = 0.013; intracellular adhesion molecule 1, 1.4-fold, P = 0.028) decreased circulating adhesion molecules.Conclusion: Both fermented dairy protein products reduce cardiometabolic risk factors in diet-induced obese mice, possibly by modulating the gut microbiota. [ABSTRACT FROM AUTHOR]

Background: The root of Platycodon grandiflorus (PG) has a long-standing tradition in the Asian diet and herbal medicine, because of its anti-inflammatory and antiobesity effects. Changes in the gut microbiota can have dietary effects on host health, which suggests a relation between the 2.Objectives: The aim of our study was to investigate the relation between PG-mediated suppression of obesity and the composition and functioning of the gut microbiota.Methods: Six-week-old male C57BL/6J mice were fed either a control diet (CON, 10% kcal from fat), a high-fat diet (HFD, 60% kcal from fat), or a PG-supplemented HFD for 18 wk. PG was administered by oral gavage at 2 g · kg body weight-1 · d-1. Body weight and food intake were monitored. Lipid metabolism, inflammation, and intestinal barrier function were determined. Amplicon sequencing of the bacterial 16S ribosomal RNA gene was used to explore gut microbiota structure, and nontargeted metabolomics analysis was performed to investigate metabolite concentrations in fecal samples.Results: We found that PG significantly ameliorated HFD-induced inflammation, recovered intestinal barrier integrity (reduced permeability by 39% , P = 0.008), reduced fat accumulation by 26% (P = 0.009), and changed the expression of key genes involved in the development of white adipose tissue (P < 0.05) in HFD-fed mice to similar levels in CON mice. Moreover, PG attenuated HFD-induced changes in the gut microbiota; it especially increased Allobaculum (7.3-fold, P = 0.002) relative to HFD, whereas CON was 15.2-fold of HFD (P = 0.002). These changes by PG were associated with an increase in the production of SCFAs (butyrate and propionate, P < 0.001) and other carbohydrate-related metabolites known to have a major role in disease suppression.Conclusions: Our study demonstrated that PG beneficially changed the gut microbiota and the gut metabolome in HFD-fed mice, and suggests that the antiobesity effects of PG may be mediated via changes in gut microbiota composition and metabolic activity. [ABSTRACT FROM AUTHOR]

rRNA-modifying enzymes participate in ribosome assembly. However, whether the catalytic activities of these enzymes are important for theribosomeassemblyandother cellular processes is not fully understood. Here, we report the crystal structure of WT human dimethyladenosine transferase 1 (DIMT1), an 18S rRNA N6,6-dimethyladenosine (m26,6A) methyltransferase, and results obtained with a catalytically inactive DIMT1 variant. We found that DIMT11/2 heterozygous HEK 293T cells have a significantly decreased 40S fraction and reduced protein synthesis but no major changes in m2 6,6A levels in 18S rRNA. Expression of a catalytically inactive variant, DIMT1-E85A, in WTand DIMT11/2 cells significantly decreasedm2 6,6A levels in 18SrRNA,indicatingadominant-negativeeffectofthisvarianton m2 6,6A levels. However, expression of the DIMT1-E85A variant restored the defects in 40S levels. Of note, unlike WT DIMT1, DIMT1-E85A could not revert the defects in protein translation. We found that the differences between this variant and the WT enzyme extended to translation fidelity and gene expression patterns in DNA damage response pathways. These results suggest that the catalytic activity ofDIMT1 is involved in protein translation and that the overall protein scaffold of DIMT1, regardless of the catalytic activity on m2 6,6A in 18S rRNA, is essential for 40S assembly. [ABSTRACT FROM AUTHOR]

The RNA exosome is a multisubunit protein complex involved in RNA surveillance of all classes of RNA, and is essential for pre-rRNA processing. The exosome is conserved throughout evolution, present in archaea and eukaryotes from yeast to humans, where it localizes to the nucleus and cytoplasm. The catalytically active subunit Rrp44/Dis3 of the exosome in budding yeast (Saccharomyces cerevisiae) is considered a protein present in these two subcellular compartments, and here we report that it not only localizes mainly to the nucleus, but is concentrated in the nucleolus, where the early pre-rRNA processing reactions take place. Moreover, we show by confocal microscopy analysis that the core exo-some subunits Rrp41 and Rrp43 also localize largely to the nucleus and strongly accumulate in the nucleolus. These results shown here shed additional light on the localization of the yeast exosome and have implications regarding the main function of this RNase complex, which seems to be primarily in early pre-rRNA processing and surveillance. [ABSTRACT FROM AUTHOR]

Ribosome profiling (RIBO-Seq) has improved our understanding of bacterial translation, including finding many unannotated genes. However, protocols for RIBO-Seq and corresponding data analysis are not yet standardized. Here, we analyzed 48 RIBO-Seq samples from nine studies of Escherichia coli K12 grown in lysogeny broth medium and particularly focused on the size-selection step. We show that for conventional expression analysis, a size range between 22 and 30 nucleotides is sufficient to obtain protein-coding fragments, which has the advantage of removing many unwanted rRNA and tRNA reads. More specific analyses may require longer reads and a corresponding improvement in rRNA/tRNA depletion. There is no consensus about the appropriate sequencing depth for RIBOSeq experiments in prokaryotes, and studies vary significantly in total read number. Our analysis suggests that 20 million reads that are not mapping to rRNA/tRNA are required for global detection of translated annotated genes. We also highlight the influence of drug-induced ribosome stalling, which causes bias at translation start sites. The resulting accumulation of reads at the start site may be especially useful for detecting weakly expressed genes. As different methods suit different questions, it may not be possible to produce a "one-size-fits-all" ribosome profiling data set. Therefore, experiments should be carefully designed in light of the scientific questions of interest. We propose some basic characteristics that should be reported with any new RIBO-Seq data sets. Careful attention to the factors discussed should improve prokaryotic gene detection and the comparability of ribosome profiling data sets. [ABSTRACT FROM AUTHOR]

RNA sequencing (RNA-seq)-based whole transcriptome analysis (WTA) using ever-evolving next-generation sequencing technologies has become a primary tool for coding and/or noncoding transcriptome profiling. As WTA requires RNA-seq data for both coding and noncoding RNAs, one key step for obtaining high-quality RNA-seq data is to remove ribosomal RNAs, which can be accomplished by using various commercial kits. Nonetheless, an ideal rRNA removal method should be efficient, user-friendly and cost-effective so it can be adapted for homemade RNA-seq library construction. Here, we developed a novel reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) method. We demonstrated that RTR2D was simple and efficient, and depleted human or mouse rRNAs with high specificity without affecting coding and noncoding transcripts. RNA-seq data analysis indicated that RTR2D yielded highly correlative transcriptome landscape with that of NEBNext rRNA Depletion Kit at both mRNA and lncRNA levels. In a proof-of-principle study, we found that RNA-seq dataset from RTR2D-depleted rRNA samples identified more differentially expressed mRNAs and lncRNAs regulated by Nutlin3A in human osteosarcoma cells than that from NEBNext rRNA Depletion samples, suggesting that RTR2D may have lower off-target depletion of non-rRNA transcripts. Collectively, our results have demonstrated that the RTR2D methodology should be a valuable tool for rRNA depletion. [ABSTRACT FROM AUTHOR]

Jiaoke is a traditional Mongolian fermented dairy product that is nutritious and has a unique taste. It is made from the fat separated from fermented milk. In this study, we collected 24 jiaoke samples from the Xilingol region of Inner Mongolia. The microbiota composition of the collected samples was analyzed using 16S rRNA small-molecule real-time sequencing, and the lactic acid bacteria (LAB) population was enumerated and isolated by laboratory culture techniques. We used an electronic tongue device to assess the taste quality of the products. One hundred fifty LAB isolates (5 genera and 14 species) were recovered and identified by 16S rRNA sequencing across all samples. Lactococcus lactis and Lactobacillus plantarum accounted for 51.33% and 10.67% of the total isolates, respectively. The small-molecule real-time sequencing of full-length 16S rRNAs revealed an overall bacterial microbiota composition of 10 phyla, 121 genera, and 186 species, largely represented by sequences of Lactococcus (68.46%) and Lactococcus lactis (52.92%) at the genus and species levels, respectively. The electronic tongue analysis revealed that the sweetness, bitterness, sourness, and saltiness of jiaoke varied greatly between samples. The presence of Lactococcus lactis correlated positively with bitter aftertaste; the presence of Lactococcus piscium correlated positively with umami and negatively with astringent and bitter aftertastes; and the presence of Lactobacillus helveticus correlated positively with sourness and negatively with other taste qualities. These results suggest that the microbiota composition and product taste are closely related. The novel LAB strains collected in this work represent valuable natural microbial resources. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA gene expression is coordinately regulated both pre- and post-transcriptionally, and its perturbation can lead to human pathologies. Mitochondrial rRNAs (mtrRNAs) undergo a series of nucleotide modifications after release from polycistronic mitochondrial RNA precursors, which is essential for mitochondrial ribosomal biogenesis. Cytosine N4-methylation (m4C) at position 839 (m4C839) of the 12S small subunit mt-rRNA was identified decades ago; however, its biogenesis and function have not been elucidated in detail. Here, using several approaches, including immunofluorescence, RNA immunoprecipitation and methylation assays, and bisulfite mapping, we demonstrate that human methyltransferaselike 15 (METTL15), encoded by a nuclear gene, is responsible for 12S mt-rRNA methylation at m4C839 both in vivo and in vitro. We tracked the evolutionary history of RNA m4C methyltransferases and identified a difference in substrate preference between METTL15 and its bacterial ortholog rsmH. Additionally, unlike the very modest impact of a loss of m4C methylation in bacterial small subunit rRNA on the ribosome, we found that METTL15 depletion results in impaired translation of mitochondrial protein-coding mRNAs and decreases mitochondrial respiration capacity. Our findings reveal that human METTL15 is required for mitochondrial function, delineate the evolution of methyltransferase substrate specificities and modification patterns in rRNA, and highlight a differential impact of m4C methylation on prokaryotic ribosomes and eukaryotic mitochondrial ribosomes. [ABSTRACT FROM AUTHOR]

• Dysbiosis persists for at least 1 year after hematopoietic stem cell transplantation (HSCT). • An increased abundance of Enterococcus indicates intestinal devastation in HSCT. • 16S rRNA analysis of intestinal flora at 1 month post-HSCT predicts long-term outcome. Intestinal flora plays an essential role in regulating immune responses. Changes in the gut flora are associated with poor prognosis after allogeneic hematopoietic stem cell transplantation (HSCT). We aimed to investigate the impact of diverse intestinal flora on survival after allogeneic HSCT. Using next-generation sequencing of the bacterial 16S ribosomal RNA (rRNA) gene, we found that the intestinal microbiota of patients undergoing allogeneic HSCT differed significantly from that of healthy controls. Furthermore, dysbiosis persisted for at least 1 year after transplantation. Interestingly, increased abundance of the genus Enterococcus detected by 16S rRNA sequencing as early as 1 month after transplantation was correlated with poor survival (overall survival at 2 years post-HSCT, 83.9% for patients with <1% relative abundance of Enterococcus and 47.6% for those with ≥1% relative abundance of Enterococcus), which was undetectable by conventional standard stool culture. These findings suggest that detection of Enterococcus by 16S rRNA analysis reflects compromised intestinal flora and may be a promising prognostic indicator. [ABSTRACT FROM AUTHOR]

RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I-associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I-specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity. [ABSTRACT FROM AUTHOR]

A new nitrogen-containing compound, trichothioneic acid, was discovered from the metabolites of fungal strain FKI-7573 using a mass spectrometry screening method guided by odd number of molecular weights, which indicates compounds that contain an odd number of nitrogen atoms. Strain FKI-7573 was isolated from soil collected in Obihiro, Hokkaido, Japan, and identified as Trichoderma virens by a sequence analysis of the internal transcribed spacer region, including 5.8S ribosomal RNA. The structure of trichothioneic acid was determined by mass spectrometry, nuclear magnetic resonance spectroscopy, electronic circular dichroism spectra, and chemical degradation analyses. These analyses revealed that trichothioneic acid consists of heptelidic acid and l -ergothioneine, and contains three nitrogen atoms. Trichothioneic acid exhibited hydroxyl radical-scavenging and singlet oxygen-quenching activities. [ABSTRACT FROM AUTHOR]

Xylose, the main component of xylan, is the second most abundant sugar in nature after glucose. Consequently, xylose represents an attractive feedstock for the production of value-added compounds such as biosurfactants (BSs), which are produced by various bacteria and yeasts. In this study, we screened and isolated yeast strains that synthesize BSs using xylose as the sole carbon source. We applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to screen for BS-producing yeasts and isolated eight strains as the liamocin producers. Two of the eight strains, AS37 and SK25, were identified as Aureobasidium melanogenum , which is known as black yeasts, by based on 26S ribosomal RNA gene sequences. Both strains produced a wide variety of liamocin structures from not only xylose but also glucose and sucrose. According to the MALDI-TOF MS analysis, signals corresponding to sodium ion adducts of di-, tri-, tetra-, penta- and hexa-acylated C6-liamocins and di-, tri- and tetra-acylated C5-liamocins were detected. In addition, their mono-acetylated form was also detected. The dominant sugar component of liamocins produced by strains AS37 and SK25 is mannitol as estimated by HPLC analysis. This is the first report to describe the screening of liamocins-producing yeasts using xylose as the sole carbon source. [ABSTRACT FROM AUTHOR]

The host-associated gut microbiota is considered critical for the occurrence and progression of colorectal cancer (CRC); however, systematic evaluations of the changes in the biodiversity and richness of mucosa-associated gut microbiota with the development of CRC have been limited. Twenty-three paired samples from colorectal tumor sites and the surrounding non-tumor tissues were collected from stage I to IV CRC patients. The microbial compositions of the samples were analyzed by Illumina MiSeq sequencing of the V4 region of the 16S rRNA gene. Gut bacterial alterations at the tumor sites and surrounding healthy tissue sites collected from the different stages of CRC patients were analyzed. No significant differences were observed in the overall microbial richness and biodiversity between the CRC tissue and surrounding non-CRC tissue samples, however, composition and community segregation of the gut microbiota with the progression of CRC were observed. A general increasing trend of Bacteroidetes, Firmicutes, and Fusobacteria and decreasing trend of Proteobacteria were observed at the phylum level with the development of CRC. Further analysis revealed that thirty-four taxa differed significantly with the progression of CRC. Conclusively, our findings provide a comprehensive view of the human mucosa-associated gut microbiota, in association with the different stages of CRC. [ABSTRACT FROM AUTHOR]

Background: It is unclear whether the favorable effects of walnuts on the gut microbiota are attributable to the fatty acids, including α-linolenic acid (ALA), and/or the bioactive compounds and fiber.Objective: This study examined between-diet gut bacterial differences in individuals at increased cardiovascular risk following diets that replace SFAs with walnuts or vegetable oils.Methods: Forty-two adults at cardiovascular risk were included in a randomized, crossover, controlled-feeding trial that provided a 2-wk standard Western diet (SWD) run-in and three 6-wk isocaloric study diets: a diet containing whole walnuts (WD; 57-99 g/d walnuts; 2.7% ALA), a fatty acid-matched diet devoid of walnuts (walnut fatty acid-matched diet; WFMD; 2.6% ALA), and a diet replacing ALA with oleic acid without walnuts (oleic acid replaces ALA diet; ORAD; 0.4% ALA). Fecal samples were collected following the run-in and study diets to assess gut microbiota with 16S rRNA sequencing and Qiime2 for amplicon sequence variant picking.Results: Subjects had elevated BMI (30 ± 1 kg/m2), blood pressure (121 ± 2/77 ± 1 mmHg), and LDL cholesterol (120 ± 5 mg/dL). Following the WD, Roseburia [relative abundance (RA) = 4.2%, linear discriminant analysis (LDA) = 4], Eubacterium eligensgroup (RA = 1.4%, LDA = 4), LachnospiraceaeUCG001 (RA = 1.2%, LDA = 3.2), Lachnospiraceae UCG004 (RA = 1.0%, LDA = 3), and Leuconostocaceae (RA = 0.03%, LDA = 2.8) were most abundant relative to taxa in the SWD (P ≤ 0.05 for all). The WD was also enriched in Gordonibacter relative to the WFMD. Roseburia (3.6%, LDA = 4) and Eubacterium eligensgroup (RA = 1.5%, LDA = 3.4) were abundant following the WFMD, and Clostridialesvadin BB60group (RA = 0.3%, LDA = 2) and gutmetagenome (RA = 0.2%, LDA = 2) were most abundant following the ORAD relative to the SWD (P ≤ 0.05 for all). Lachnospiraceae were inversely correlated with blood pressure and lipid/lipoprotein measurements following the WD.Conclusions: The results indicate similar enrichment of Roseburia following the WD and WFMD, which could be explained by the fatty acid composition. Gordonibacter enrichment and the inverse association between Lachnospiraceae and cardiovascular risk factors following the WD suggest that the gut microbiota may contribute to the health benefits of walnut consumption in adults at cardiovascular risk. This trial was registered at clinicaltrials.gov as NCT02210767. [ABSTRACT FROM AUTHOR]

Background: Diet is a determinant of gut microbiota. Both diet and gut microbiota have been linked to metabolic diseases.Objective: We aimed to examine data-driven food patterns in relation to the prevalence of prediabetes and gut microbiota composition and food pattern-associated bacteria in relation to prediabetes.Methods: Food patterns were extracted using principal component analysis in 1726 individuals (aged 18-71 y, 55% women, mean BMI = 25.5 kg/m2) without diabetes from the population-based Malmö Offspring Study. The gut (fecal) microbiota was analyzed by sequencing the 16S ribosomal RNA gene (V1-V3 region). Prediabetes classification was based on fasting glucose ≥6.0 mmol/L and/or glycated hemoglobin ≥42 mmol/L at baseline and/or type 2 diabetes diagnosis during follow-up (0-3.8 y). Logistic regression was used to investigate cross-sectional associations with prediabetes, and the general linear model to examine associations between food patterns and bacterial genera.Results: Two food patterns, the Health-conscious and the Sugar and High-Fat Dairy patterns, were identified. Adherence to the Health-conscious pattern was associated with a lower prevalence of prediabetes (OR comparing highest quintile with lowest: 0.54; 95% CI: 0.32, 0.92; P-trend = 0.03) and with the abundance of several gut bacterial genera, of which the most robust findings were with a higher abundance of Roseburia and Lachnospira and with a lower abundance of Eubacterium. Roseburia was also associated with a lower prevalence of prediabetes (OR comparing highest quintile with lowest: 0.56; 95% CI: 0.35, 0.92; P-trend = 0.01) and the association between the Health-conscious pattern and prediabetes was attenuated after adjustment for abundance of Roseburia and BMI. Adherence to the Sugar and High-Fat Dairy pattern was associated with a higher prevalence of prediabetes in women (P-trend across food pattern quintiles = 0.03).Conclusions: In this Swedish population-based study, a Health-conscious food pattern showed an inverse association with the prevalence of prediabetes. Potential underlying explanations may involve links between healthy diet and BMI, as well as gut microbiota, especially a higher abundance of Roseburia. [ABSTRACT FROM AUTHOR]

The identification of milk microbial communities in ruminants is relevant for understanding the association between milk microbiota and health status. The most common approach for studying the microbiota is amplifying and sequencing specific hypervariable regions of the 16S rRNA gene using massive sequencing techniques. However, the taxonomic resolution is limited to family and, in some cases, genus level. We aimed to improve taxonomic classification of the water buffalo milk microbiota by amplifying and sequencing the full-length 16S rRNA gene (1,500 bp) using Nanopore sequencing (single-molecule sequencing). When comparing with short-read results, we improved the taxonomic classification, reaching species level. We identified the main microbial agents of subclinical mastitis at the species level that were in accordance with the microbiological culture results. These results confirm the potential of single-molecule sequencing for in-depth analysis of microbial populations in dairy animals. [ABSTRACT FROM AUTHOR]

Diet-induced milk fat depression (MFD) is a condition marked by a reduction in milk fat yield experimentally achieved by increasing dietary unsaturated fatty acids and fermentable carbohydrates. 2-Hydroxy-4-(methylthio) butanoate (HMTBa) is a methionine analog observed to reduce diet-induced MFD in dairy cows. We hypothesize that the reduction in diet-induced MFD by HMTBa is due to changes in the rumen microbiota. To test this, 22 high-producing cannulated Holstein dairy cows were placed into 2 groups using a randomized block design and assigned to either control or HMTBa supplementation (0.1% of diet dry matter). All cows were then exposed to 3 different diets with a low risk (32% neutral detergent fiber, no added oil; fed d 1 to 7), a moderate risk (29% neutral detergent fiber and 0.75% soybean oil; fed d 8 to 24), or a high risk (29% neutral detergent fiber and 1.5% soybean oil; fed d 25 to 28) for diet-induced MFD. Rumen samples were collected on d 0, 14, 24, and 28, extracted for DNA, PCR-amplified for the V1–V2 region of the 16S rRNA gene, sequenced on an Illumina MiSeq (Illumina, San Diego, CA), and subjected to bacterial diversity analysis using the QIIME pipeline. The α diversity estimates (species richness and Shannon diversity) were decreased in the control group compared with the HMTBa group. Bacterial community composition also differed between control and HMTBa groups based on both weighted UniFrac (relative abundance of commonly detected bacteria) and unweighted UniFrac (presence/absence) distances. Within the HMTBa group, no differences were observed in bacterial community composition between d 0 and d 14, 24, and 28; however, in the control group, d 0 samples were different from d 14, 24, and 28. Certain bacterial genera including Dialister , Megasphaera , Lachnospira , and Sharpea were increased in the control group compared with the HMTBa group. Interestingly, these genera were positively correlated with milk fat trans- 10, cis- 12 conjugated linoleic acid and trans -10 C18:1, fatty acid isomers associated with biohydrogenation-induced MFD. It can be concluded that diet-induced MFD is accompanied by significant alterations in the rumen bacterial community and that HMTBa supplementation reduces these microbial perturbations. [ABSTRACT FROM AUTHOR]