bFourteen Holstein cows of similar ages were monitored through their first two lactation cycles, during which ruminal solids and liquids, milk samples, production data, and feed consumption data were collected for each cow during early (76 to 82 days in milk [DIM]), middle (151 to 157 DIM), and late (251 to 257 DIM) lactation periods. The bacterial community of each ruminal sample was determined by sequencing the region from V6 to V8 of the 16S rRNA gene using 454 pyrosequencing. Gross feed efficiency (GFE) for each cow was calculated by dividing her energy-corrected milk by dry matter intake (ECM/DMI) for each period of both lactation cycles. Four pairs of cows were identified that differed in milk production efficiency, as defined by residual feed intake (RFI), at the same level of ECM production. The most abundant phyla detected for all cows were Bacteroidetes (49.42%), Firmicutes (39.32%), Proteobacteria (5.67%), and Tenericutes (2.17%), and the most abundant genera included Prevotella (40.15%), Butyrivibrio (2.38%), Ruminococcus (2.35%), Coprococcus (2.29%), and Succiniclasticum (2.28%). The bacterial microbiota between the first and second lactation cycles were highly similar, but with a significant correlation between total community composition by ruminal phase and specific bacteria whose relative sequence abundances displayed significant positive or negative correlation with GFE or RFI. These data suggest that the ruminal bacterial community is dynamic in terms of membership and diversity and that specific members are associated with high and low milk production efficiency over two lactation cycles.R uminants, such as cattle, rely upon a rich and diverse community of symbiotic ruminal microbes to digest their feed. These symbionts are capable of fermenting host-indigestible feed into nutrient sources usable by the host, such as volatile fatty acids (1, 2). Ruminants are born without a functional rumen and are thought to acquire their digestive microbes from the environment as the rumen develops (3-6). Although the means by which ruminants acquire this ruminal microbial community remains unclear, the membership and stability of the ruminal community can have a direct and measurable impact on host function and health (7). Importantly, the host requires ruminal fermentation products for body maintenance and growth (8) and milk production (9).Of particular interest, both scientifically and agriculturally, is the impact of the ruminal microbial community on host milk production efficiency in dairy cattle. Two major methods are widely used to calculate milk production efficiency: gross feed efficiency (GFE) and residual feed intake (RFI). GFE is a more traditional measure that is based on the yield of milk produced (corrected to a constant-energy basis) per unit of intake of dietary dry matter (DM) (10). RFI was first applied to weight gain in steers but has more recently been adapted for use in dairy production (11); it is defined as the difference in feed consumption relative to that of other animals on the...

Here, we develop and apply a semi-quantitative method for the high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identify many known sites of pseudouridylation and uncover previously unreported uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites are validated using 1000-mer synthetic RNA controls bearing a single pseudouridine in the center position, demonstrating systematic under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our workflow allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data and multiple modifications on the same strand.

Summary The genus Fibrobacter contains cellulolytic bacteria originally isolated from the rumen. Culture-independent investigations have since identified Fibrobacter populations in gastrointestinal tracts of numerous hindgut-fermenting herbivores, but their physiology is poorly characterized due to few representative axenic cultures. To test the hypothesis that novel Fibrobacter diversity exists in hindgut fermenters, we performed culture and 16S rRNA gene amplicon sequencing on samples collected from phylogenetically diverse herbivorous hosts. Using a unique approach for recovering axenic Fibrobacter cultures, we isolated 45 novel strains from 11 different hosts. Full-length 16S rRNA gene sequencing of these isolates identified nine discrete phylotypes (cutoff = 0.03%) among them, including several that were only isolated from hindgut-fermenting hosts, and four previously unrepresented by axenic cultures. Our phylogenetic analysis indicated that six of the phylotypes are more closely related to previously described subspecies of Fibrobacter succinogenes, while the remaining three were more closely related to F. intestinalis. Culture-independent bacterial community profiling confirmed that most isolates were representative of numerically dominant phylotypes in their respective samples and strengthened the association of certain phylotypes with either ruminants or hindgut-fermenters. Despite considerable phylogenetic diversity observed among the Fibrobacter strains isolated here, phenotypic characterization suggests a conserved specialization for growth on cellulose.

Objectives The gastric H+/K+ ATPase proton pump has previously been shown to be expressed in the human larynx, however its contribution to laryngopharyngeal reflux (LPR) signs, symptoms and associated diseases such as laryngeal cancer is unknown. Proton pump expression in the larynx of patients with LPR and laryngeal cancer was investigated herein. A human hypopharyngeal cell line expressing the proton pump was generated to investigate its effects. Study Design In‐vitro translational. Methods Laryngeal biopsies were obtained from three LPR and eight LSCC patients. ATP4A, ATP4B and HRPT1 were assayed via qPCR. Human hypopharyngeal FaDu cell lines stably expressing proton pump were created using lentiviral transduction and examined via transmission electron microscopy and qPCR for genes associated with inflammation or laryngeal cancer. Results Expression of ATP4A and ATP4B was detected in 3/3 LPR, 4/8 LSCC‐tumor and 3/8 LSCC‐adjacent specimens. Expression of ATP4A and ATP4B in FaDu elicited mitochondrial damage and expression of IL1B, PTGS2, and TNFA (P < .0001); expression of ATP4B alone did not. Conclusions Gastric proton pump subunits are expressed in the larynx of LPR and LSCC patients. Mitochondrial damage and changes in gene expression observed in cells expressing the full proton pump, absent in those expressing a single subunit, suggest that acid secretion by functional proton pumps expressed in upper airway mucosa may elicit local cell and molecular changes associated with inflammation and cancer. Level of Evidence NA Laryngoscope, 131:130–135, 2021

Objectives/Hypothesis Cell culture models are valuable tools for investigation of the molecular pathogenesis of diseases including otitis media (OM). Previous study indicates that age‐, sex‐, and race‐associated differences in molecular signaling may impact disease pathophysiology. Currently, a singular immortalized middle ear epithelial (MEE) cell line exists, HMEEC‐1, derived from an adult without known middle ear disease. In this study, HMEEC‐1 and primary MEE cultures from pediatric patients with and without OM were stimulated with inflammatory cytokines or OM‐pathogenic bacterial lysates to examine differences in the response of molecules associated with OM pathogenesis. Study Design Case–control series. Methods MEE cultures were established from patients aged <6 years: two with recurrent OM (ROM), two with OM with effusion (OME), and one patient without OM who was undergoing cochlear implant surgery control undergoing cochlear implantation (Peds CI). Primary MEE cultures and HMEEC‐1 cells were stimulated with tumor necrosis factor‐α, interleukin (IL)‐1β, or nontypeable Haemophilus influenzae lysate. TNFA, IL1B, IL6, IL8, IL10, and MUC5B were assayed via quantitative polymerase chain reaction. IL‐8 was assayed by enzyme‐linked immunosorbent assay. Results Gene/protein target expressions were frequently higher in pediatric OM lines than in HMEEC‐1 and Peds CI. HMEEC‐1 cells were frequently less responsive to stimuli than all pediatric lines. OME lines were often more responsive than ROM lines. Conclusions OM may be associated with specific molecular phenotypes that are retained in primary cell culture. Adult‐derived HMEEC‐1 cells differ significantly in baseline expression and response of OM‐associated molecules relative to pediatric MEE cells. Work is underway to immortalize pediatric OM MEE cultures as improved tools for the OM research community. Level of Evidence 4 Laryngoscope, 131:410–416, 2021

The current therapeutic approach to asthma focuses exclusively on targeting inflammation and reducing airway smooth muscle force to prevent the recurrence of symptoms. However, even when inflammation is brought under control, airways in an asthmatic can still hyper-constrict when exposed to a low dose of agonist. This suggests that there are mechanisms at play that are likely triggered by inflammation and eventually become self-sustaining so that even when airway inflammation is brought back under control, these alternative mechanisms continue to drive airway hyperreactivity in asthmatics. In this study, we hypothesized that stiffening of the airway extracellular matrix is a core pathological change sufficient to support excessive bronchoconstriction even in the absence of inflammation. To test this hypothesis, we increased the stiffness of airway extracellular matrix by photo-crosslinking collagen fibers within the airway wall of freshly dissected bovine rings using riboflavin (vitamin B2) and Ultraviolet-A radiation. In our experiments, collagen crosslinking led to a two-fold increase in the stiffness of the airway extracellular matrix. This change was sufficient to cause airways to constrict to a greater degree, and at a faster rate when they were exposed to 10-5M acetylcholine for 5 minutes. Our results show that stiffening of the extracellular matrix is sufficient to drive excessive airway constriction even in the absence of inflammatory signals.

Nanopore direct RNA sequencing (DRS) enables measurements of native RNA modifications. Modification-free transcripts are an important control for DRS. Additionally, it is advantageous to have canonical transcripts from multiple cell lines to better account for human transcriptome variation. Here we generated and analyzed Nanopore DRS datasets for five human cell lines using in vitro transcribed (IVT) RNA. We compared performance statistics amongst biological replicates. We also documented nucleotide and ionic current level variation across cell lines. These data will serve as a resource to the community for RNA modification analysis.

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