Comprehensive assessment of protein loop modeling programs on large-scale datasets: prediction accuracy and efficiency.

Protein loops play a critical role in the dynamics of proteins and are essential for numerous biological functions, and various computational approaches to loop modeling have been proposed over the past decades. However, a comprehensive understanding of the strengths and weaknesses of each method is lacking. In this work, we constructed two high-quality datasets (i.e. the General dataset and the CASP dataset) and systematically evaluated the accuracy and efficiency of 13 commonly used loop modeling approaches from the perspective of loop lengths, protein classes and residue types. The results indicate that the knowledge-based method FREAD generally outperforms the other tested programs in most cases, but encountered challenges when predicting loops longer than 15 and 30 residues on the CASP and General datasets, respectively. The ab initio method Rosetta NGK demonstrated exceptional modeling accuracy for short loops with four to eight residues and achieved the highest success rate on the CASP dataset. The well-known AlphaFold2 and RoseTTAFold require more resources for better performance, but they exhibit promise for predicting loops longer than 16 and 30 residues in the CASP and General datasets. These observations can provide valuable insights for selecting suitable methods for specific loop modeling tasks and contribute to future advancements in the field.

Targeted Affinity Purification and Mechanism of Action of Angiotensin-Converting Enzyme (ACE) Inhibitory Peptides from Sea Cucumber Gonads.

Protein hydrolysates from sea cucumber (Apostichopus japonicus) gonads are rich in active materials with remarkable angiotensin-converting enzyme (ACE) inhibitory activity. Alcalase was used to hydrolyze sea cucumber gonads, and the hydrolysate was separated by the ultrafiltration membrane to produce a low-molecular-weight peptide component (less than 3 kDa) with good ACE inhibitory activity. The peptide component (less than 3 kDa) was isolated and purified using a combination method of ACE gel affinity chromatography and reverse high-performance liquid chromatography. The purified fractions were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the resulting products were filtered using structure-based virtual screening (SBVS) to obtain 20 peptides. Of those, three noncompetitive inhibitory peptides (DDQIHIF with an IC50 value of 333.5 µmol·L-1, HDWWKER with an IC50 value of 583.6 µmol·L-1, and THDWWKER with an IC50 value of 1291.8 µmol·L-1) were further investigated based on their favorable pharmacochemical properties and ACE inhibitory activity. Molecular docking studies indicated that the three peptides were entirely enclosed within the ACE protein cavity, improving the overall stability of the complex through interaction forces with the ACE active site. The total free binding energies (ΔGtotal) for DDQIHIF, HDWWKER, and THDWWKER were -21.9 Kcal·mol-1, -71.6 Kcal·mol-1, and -69.1 Kcal·mol-1, respectively. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that HDWWKER could significantly decrease the systolic blood pressure (SBP) of SHRs after intravenous administration. The results showed that based on the better antihypertensive activity of the peptide in SHRs, the feasibility of targeted affinity purification and computer-aided drug discovery (CADD) for the efficient screening and preparation of ACE inhibitory peptide was verified, which provided a new idea of modern drug development method for clinical use.

Should sons breed independently or help? Local relatedness matters.

In cooperatively breeding birds, why do some individuals breed independently but others have to help at home? This question has been rarely addressed despite its fundamental importance for understanding the evolution of social cooperation. We address it using 15 years of data from Tibetan ground tits Pseudopodoces humilis where helpers consist of younger males. Since whether younger males successfully breed depends critically on their chances to occupy territories nearby home, our analytic strategy is to identify the determinants of individual differences in gaining territory ownership among these ready-to-breed males. Across widowed, last-year helper and yearling males, an age advantage was evident in inheriting resident territories, occupying adjacent vacancies and budding off part of adjacent territories, which left some last-year helpers and most yearling males to take the latter two routes. These males were more likely to acquire a territory if they were genetically related to the previous or current territory owners; otherwise they remained on natal territories as helpers. The relatedness effect can arise from the prior residence advantage established in the preceding winter when younger males followed their parents to perform kin-directed off-territory forays. Our research highlights the key role of local kinship in determining younger males' territory acquisition and thus their fate in terms of independent reproduction versus help. This finding provides insight into the formation of kin-based, facultative cooperative societies prevailing among vertebrates.

Affinity Purification and Molecular Characterization of Angiotensin-Converting Enzyme (ACE)-Inhibitory Peptides from Takifugu flavidus.

An affinity chromatography filler of CNBr-activated Sepharose 4B-immobilized ACE was used to purify ACE-inhibitory peptides from Takifugu flavidus protein hydrolysate (<1 kDa). Twenty-four peptides with an average local confidence score (ALC) ≥ 80% from bounded components (eluted by 1 M NaCl) were identified by LC-MS/MS. Among them, a novel peptide, TLRFALHGME, with ACE-inhibitory activity (IC50 = 93.5 µmol·L-1) was selected. Molecular docking revealed that TLRFALHGME may interact with the active site of ACE through H-bond, hydrophobic, and electrostatic interactions. The total binding energy (ΔGbinding) of TLRFALHGME was estimated to be -82.7382 kJ·mol-1 by MD simulations, indicating the favorable binding of peptides with ACE. Furthermore, the binding affinity of TLRFALHGME to ACE was determined by surface plasmon resonance (SPR) with a Kd of 80.9 µmol, indicating that there was a direct molecular interaction between them. TLRFALHGME has great potential for the treatment of hypertension.

Screening and Molecular Mechanisms of Novel ACE-Inhibitory Peptides from Gracilariopsis lemaneiformis.

Candidate peptides with novel angiotensin-I-converting enzyme (ACE) inhibitor activity were obtained from hydrolysates of Gracilariopsis lemaneiformis by virtual screening method. Our results showed that G. lemaneiformis peptides (GLP) could significantly lower blood pressure in spontaneously hypertensive rats (SHR). At least 101 peptide sequences of GLP were identified by LC-MS/MS analysis and subjected to virtual screening. A total of 20 peptides with the highest docking score were selected and chemically synthesized in order to verify their ACE-inhibitory activities. Among them, SFYYGK, RLVPVPY, and YIGNNPAKG showed good effects with IC50 values of 6.45 ± 0.22, 9.18 ± 0.42, and 11.23 ± 0.23 µmoL/L, respectively. Molecular docking studies revealed that three peptides interacted with the active center of ACE by hydrogen bonding, hydrophobic interactions, and electrostatic forces. These peptides could form stable complexes with ACE. Furthermore, SFYYGK, RLVPVPY, and YIGNNPAKG significantly reduced systolic blood pressure (SBP) in SHR. YIGNNPAKG exhibited the highest antihypertensive effect, with the largest decrease in SBP (approximately 23 mmHg). In conclusion, SFYYGK, RLVPVPY, and YIGNNPAKG can function as potent therapeutic candidates for hypertension treatment.

Production of Red Pigments by a Newly Isolated Talaromyces aurantiacus Strain with LED Stimulation for Screen Printing.

Microbial pigments have been widely applied to printing in food, textile, and paper industries as a sustainable alternative to synthetic dyes. Herein, we isolated a novel Talaromyces aurantiacus strain with a strong ability to produce red pigments. We further studied pigment production conditions, stability, screen printing application, and bioactivities. Our results showed that sucrose was a favourable carbon source and the addition of l-histidine significantly enhanced the production of red pigments. Pigment production was strictly photo-regulated with effective wavelengths around 450 nm (blue light). We mixed the red pigments with cellulosic materials and explored their application potentials for screen printing on paper, cotton fabrics, and polymeric carriers. The printing density was significantly improved from 0.3 to 0.7 by overlay printing. T. aurantiacus pigments could be stably stored at pH 5-11, temperature - 10 to 70 °C, and redox potential - 200 to 300 mV. Moreover, the stable ranges were extended to pH 1-11 and temperature over 100 °C after screen-printed on paper. The red pigments exhibited antioxidant activity towards 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (IC50 10.4 mg L-1 in solution). Our results further indicated the red pigments by T. aurantiacus was environmentally friendly based on acetylcholinesterase activity assay. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01008-x.

Global analysis of protein synthesis in Flavobacterium johnsoniae reveals the use of Kozak-like sequences in diverse bacteria.

In all cells, initiation of translation is tuned by intrinsic features of the mRNA. Here, we analyze translation in Flavobacterium johnsoniae, a representative of the Bacteroidetes. Members of this phylum naturally lack Shine-Dalgarno (SD) sequences in their mRNA, and yet their ribosomes retain the conserved anti-SD sequence. Translation initiation is tuned by mRNA secondary structure and by the identities of several key nucleotides upstream of the start codon. Positive determinants include adenine at position -3, reminiscent of the Kozak sequence of Eukarya. Comparative analysis of Escherichia coli reveals use of the same Kozak-like sequence to enhance initiation, suggesting an ancient and widespread mechanism. Elimination of contacts between A-3 and the conserved ß-hairpin of ribosomal protein uS7 fails to diminish the contribution of A-3 to initiation, suggesting an indirect mode of recognition. Also, we find that, in the Bacteroidetes, the trinucleotide AUG is underrepresented in the vicinity of the start codon, which presumably helps compensate for the absence of SD sequences in these organisms.

A Novel Angiotensin-I-Converting Enzyme (ACE) Inhibitory Peptide from Takifugu flavidus.

Alcalase, neutral protease, and pepsin were used to hydrolyze the skin of Takifugu flavidus. The T. flavidus hydrolysates (TFHs) with the maximum degree of hydrolysis (DH) and angiotensin-I-converting enzyme (ACE)-inhibitory activity were selected and then ultra-filtered to obtain fractions with components of different molecular weights (MWs) (<1, 1-3, 3-10, 10-50, and >50 kDa). The components with MWs < 1 kDa showed the strongest ACE-inhibitory activity with a half-maximal inhibitory concentration (IC50) of 0.58 mg/mL. Purification and identification using semi-preparative liquid chromatography, Sephadex G-15 gel chromatography, RP-HPLC, and LC-MS/MS yielded one new potential ACE-inhibitory peptide, PPLLFAAL (non-competitive suppression mode; IC50 of 28 µmmol·L-1). Molecular docking and molecular dynamics simulations indicated that the peptides should bind well to ACE and interact with amino acid residues and the zinc ion at the ACE active site. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that PPLLFAAL could significantly decrease the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHRs after intravenous administration. These results suggested that PPLLFAAL may have potential applications in functional foods or pharmaceuticals as an antihypertensive agent.

Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae. 1. Internal Structure of the Biofilm.

The Gram-negative bacterium Flavobacterium johnsoniae employs gliding motility to move rapidly over solid surfaces. Gliding involves the movement of the adhesin SprB along the cell surface. F. johnsoniae spreads on nutrient-poor 1% agar-PY2, forming a thin film-like colony. We used electron microscopy and time-lapse fluorescence microscopy to investigate the structure of colonies formed by wild-type (WT) F. johnsoniae and by the sprB mutant (ΔsprB). In both cases, the bacteria were buried in the extracellular polymeric matrix (EPM) covering the top of the colony. In the spreading WT colonies, the EPM included a thick fiber framework and vesicles, revealing the formation of a biofilm, which is probably required for the spreading movement. Specific paths that were followed by bacterial clusters were observed at the leading edge of colonies, and abundant vesicle secretion and subsequent matrix formation were suggested. EPM-free channels were formed in upward biofilm protrusions, probably for cell migration. In the nonspreading ΔsprB colonies, cells were tightly packed in layers and the intercellular space was occupied by less matrix, indicating immature biofilm. This result suggests that SprB is not necessary for biofilm formation. We conclude that F. johnsoniae cells use gliding motility to spread and maturate biofilms.

Molecular Mechanisms Underlying Inhibitory Binding of Alkylimidazolium Ionic Liquids to Laccase.

Water-miscible alkylimidazolium ionic liquids (ILs) are "green" co-solvents for laccase catalysis, but generally inhibit enzyme activity. Here, we present novel insights into inhibition mechanisms by a combination of enzyme kinetics analysis and molecular simulation. Alkylimidazolium cations competitively bound to the TI Cu active pocket in the laccase through hydrophobic interactions. Cations with shorter alkyl chains (C2~C6) entered the channel inside the pocket, exhibiting a high compatibility with laccase (competitive inhibition constant Kic = 3.36~3.83 mM). Under the same conditions, [Omim]Cl (Kic = 2.15 mM) and [Dmim]Cl (Kic = 0.18 mM) with longer alkyl chains bound with Leu296 or Leu297 near the pocket edge and Leu429 around TI Cu, which resulted in stronger inhibition. Complexation with alkylimidazolium cations shifted the pH optima of laccase to the right by 0.5 unit, and might, thereby, lead to invalidation of the Hofmeister series of anions. EtSO4- showed higher biocompatibility than did Ac- or Cl-, probably due to its binding near the TI Cu and its hindering the entry of alkylimidazolium cations. In addition, all tested ILs accelerated the scavenging of 2, 2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals, which, however, did not play a determining role in the inhibition of laccase.

Elizabethkingia anophelis: molecular manipulation and interactions with mosquito hosts.

Flavobacteria (members of the family Flavobacteriaceae) dominate the bacterial community in the Anopheles mosquito midgut. One such commensal, Elizabethkingia anophelis, is closely associated with Anopheles mosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements of E. anophelis have not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation of E. anophelis, including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function in E. anophelis. A flavobacterial expression system based on the promoter PompA was integrated into the E. anophelis chromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-tagged E. anophelis associated with mosquitoes at successive developmental stages and propagated in Anopheles gambiae and Anopheles stephensi but not in Aedes triseriatus mosquitoes. Feeding NanoLuc-tagged cells to A. gambiae and A. stephensi in the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected in Aedes triseriatus mosquitoes under the same conditions. Of the initial E. anophelis cells provided to larvae, 23%, 71%, and 85% were digested in A. stephensi, A. gambiae, and Aedes triseriatus, respectively, demonstrating that E. anophelis adapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells in A. stephensi significantly increased when arginine was added to a sugar diet, showing it to be an important amino acid for E. anophelis. Animal erythrocytes promoted E. anophelis growth in vivo and in vitro, indicating that this bacterium could obtain nutrients by participating in erythrocyte lysis in the mosquito midgut.

Radical Scavenging by Acetone: A New Perspective to Understand Laccase/ABTS Inactivation and to Recover Redox Mediator.

The biosynthetic utilization of laccase/mediator system is problematic because the use of organic cosolvent causes significant inhibition of laccase activity. This work explored how the organic cosolvent impacts on the laccase catalytic capacity towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in aqueous solution. Effects of acetone on the kinetic constants of laccase were determined and the results showed Km and Vmax varied exponentially with increasing acetone content. Acetone as well as some other cosolvents could transform ABTS radicals into its reductive form. The content of acetone in media significantly affected the radical scavenging rates. Up to 95% of the oxidized ABTS was successfully recovered in 80% (v/v) acetone in 60 min. This allows ABTS recycles at least six times with 70%-75% of active radicals recovered after each cycle. This solvent-based recovery strategy may help improve the economic feasibility of laccase/ABTS system in biosynthesis.

Ingestibility, digestibility, and engineered biological control potential of Flavobacterium hibernum, isolated from larval mosquito habitats.

Flavobacterium hibernum, isolated from larval habitats of the eastern tree hole mosquito, A. triseriatus, remained suspended in the larval feeding zone much longer (8 days) than other bacteria. Autofluorescent protein markers were developed for the labeling of F. hibernum with a strong flavobacterial expression system. Green fluorescent protein (GFP)-tagged F. hibernum cells were quickly consumed by larval mosquitoes at an ingestion rate of 9.5 × 10(4)/larva/h. The ingested F. hibernum cells were observed mostly in the foregut and midgut and rarely in the hindgut, suggesting that cells were digested and did not pass the gut viably. The NanoLuc luciferase reporter system was validated for quantitative larval ingestion rate and bacterial fate analyses. Larvae digested 1.87 × 10(5) cells/larva/h, and few F. hibernum cells were excreted intact. Expression of the GFP::Cry11A fusion protein with the P20 chaperone protein from Bacillus thuringiensis H-14 was successfully achieved in F. hibernum. Whole-cell bioassays of recombinant F. hibernum exhibited high larvicidal activity against A. triseriatus in microplates and in microcosms simulating tree holes. F. hibernum cells persisted in microcosms at 100, 59, 30, and 10% of the initial densities at days 1, 2, 3, and 6, respectively, when larvae were absent, while larvae consumed nearly all of the F. hibernum cells within 3 days of their addition to microcosms.

Extra-abdominal infections caused by Comamonas kerstersii: Case report.

RATIONALE: Comamonas kerstersii mainly causes intra-abdominal infections with favorable outcomes due to high antibiotic susceptibility. We report the first case of pneumonia caused by C Kerstersii, which promoted patient death, and a second urinary tract infection by C Kerstersii with extensive drug resistance. PATIENT CONCERNS: A 46-year-old male (Case 1) with craniocerebral injury underwent emergency decompressive craniectomy, but his condition deteriorated further and presented with discontinuous fever, small moist rales on both lungs, and respiratory failure. Retrospective average nucleotide identity (ANI) analysis of the genomic sequence of the sputum isolate identified it as C Kerstersii 12322-1, antimicrobial susceptibility testing (AST) revealed that it was sensitive to 18 of 21 tested antibiotics.An 82-year-old male (Case 2) with hypertrophic prostate experienced gradual obstruction during urination, and a urine test revealed WBC ++. Retrospective ANI analysis of the urine isolate identified it as C Kerstersii 121606, which was resistant to 18 of 21 tested antibiotics. DIAGNOSES: Case 1 was diagnosed empirically as pneumonia caused by C Kerstersii strain 12322-1 secondary to craniocerebral injury and confirmed by retrospective ANI analysis; case 2 was diagnosed empirically as urinary infection secondary to prostate hyperplasia caused by C Kerstersii strain 121606 confirmed by the retrospective ANI analysis. INTERVENTIONS: Case 1 was administered cefoxitin, cefodizime, imipenem-cilastatin sodium, and underwent comprehensive salvage management. Case 2 was administered doxycycline alone. OUTCOMES: Case 1 died partially because of untimely identification of the responsible bacteria-12322-1. Case 2 was cured even 121606 exhibited an extensive drug resistance feature. LESSONS: Except for intra-abdominal infections with good prognosis, we verified that C Kerstersii could also cause extra-abdominal infections, such as the first pneumonia case and urinary infection. It could promote patient death; actual infections were underestimated due to identification difficulties, posing a health threat due to the presence of extensive drug resistance.

Elizabethkingia anophelis MSU001 Isolated from Anopheles stephensi: Molecular Characterization and Comparative Genome Analysis.

Elizabethkingia anophelis MSU001, isolated from Anopheles stephensi in the laboratory, was characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToF/MS), biochemical testing, and genome sequencing. Average nucleotide identity analysis revealed 99% identity with the type species E. anophelis R26. Phylogenetic placement showed that it formed a clade with other mosquito-associated strains and departed from a clade of clinical isolates. Comparative genome analyses further showed that it shared at least 98.6% of genes with mosquito-associated isolates (except E. anophelis As1), while it shared at most 88.8% of common genes with clinical isolates. Metabolites from MSU001 significantly inhibited growth of E. coli but not the mosquito gut symbionts Serratia marcescens and Asaia sp. W12. Insect-associated E. anophelis carried unique glycoside hydrolase (GH) and auxiliary activities (AAs) encoding genes distinct from those of clinical isolates, indicating their potential role in reshaping chitin structure and other components involved in larval development or formation of the peritrophic matrix. Like other Elizabethkingia, MSU001 also carried abundant genes encoding two-component system proteins (51), transcription factor proteins (188), and DNA-binding proteins (13). E. anophelis MSU001 contains a repertoire of antibiotic resistance genes and several virulence factors. Its potential for opportunistic infections in humans should be further evaluated prior to implementation as a paratransgenesis agent (by transgenesis of a symbiont of the vector).

Case report: Sarcomatoid renal cell carcinoma masquerading as hydronephrosis.

Sarcomatoid renal cell carcinoma (SRCC), a manifestation of sarcomatoid dedifferentiation in renal cell carcinoma, is characterized by elevated invasiveness and a grim prognosis. Typically, SRCC patients present with advanced or metastatic conditions and survival rates rarely extend beyond one year. In this study, we describe a case of SRCC characterized by the patient exhibiting right flank pain without hematuria. Initially, imaging interpretations led to a diagnosis of severe hydronephrosis. Subsequently, an open right nephrectomy post-surgery confirmed the pathology of sarcomatoid renal cell carcinoma.

Discovery of Novel Inhibitors of BRD4 for Treating Prostate Cancer: A Comprehensive Case Study for Considering Water Networks in Virtual Screening and Drug Design.

Androgen receptor (AR) is the primary target for treating prostate cancer (PCa), which inevitably progresses due to drug-resistant mutations. Bromodomain-containing protein 4 (BRD4) has been a new potential drug target for PCa treatment. Herein, we report the rational design and discovery of novel BRD4 inhibitors through computer-aided drug design (CADD), and a hit compound SQ-1 (IC50 = 676 nM) was identified by structure-based virtual screening (SBVS) with the conserved water network. To optimize the structure of SQ-1, the free energy landscape was constructed, and the binding mechanism was explored by characterizing the water profile and the dissociation mechanism. Finally, the compound SQ-17 with improved inhibitory activity (IC50 < 100 nM) was discovered, which showed potent antiproliferative activity against LNCaP. These data highlighted a successful attempt to identify and optimize a small molecule by comprehensive CADD application and provided essential clues for developing novel therapeutics for PCa treatment.

Cell-type-dependent enzymatic hydrolysis of palm residues: chemical and surface characterization of fibers and parenchyma cells.

Chemical and surface characteristics of sulfite-pretreated royal palm sheath (RPS) fibers and parenchyma cells were investigated in order to study cell-type-dependent biomass hydrolysis by cellulase. Size, chemical composition, cellulose crystallinity and the exposure of cellulose microfibrils in pretreated RPS biomass affected the enzymatic accessibility and digestibility of different cell-type substrates.

Draft genome sequence of the emerging fish pathogen Flavobacterium tructae strain S12.

The draft genome of Flavobacterium tructae strain S12, isolated from hatchery-reared Chinook salmon (Oncorhynchus tshawytscha) fingerlings, consisted of 5,695,942 bp, a G + C content of 35.6%, 4,775 predicted open reading frames, a putative type IX secretion system, collagenase, and hemolysin. F. tructae strains can be used as models for emerging Flavobacterium pathogens.

Effect of bovine respiratory disease on the respiratory microbiome: a meta-analysis.

Background: Bovine respiratory disease (BRD) is the most devastating disease affecting beef and dairy cattle producers in North America. An emerging area of interest is the respiratory microbiome's relationship with BRD. However, results regarding the effect of BRD on respiratory microbiome diversity are conflicting. Results: To examine the effect of BRD on the alpha diversity of the respiratory microbiome, a meta-analysis analyzing the relationship between the standardized mean difference (SMD) of three alpha diversity metrics (Shannon's Diversity Index (Shannon), Chao1, and Observed features (OTUs, ASVs, species, and reads) and BRD was conducted. Our multi-level model found no difference in Chao1 and Observed features SMDs between calves with BRD and controls. The Shannon SMD was significantly greater in controls compared to that in calves with BRD. Furthermore, we re-analyzed 16S amplicon sequencing data from four previously published datasets to investigate BRD's effect on individual taxa abundances. Additionally, based on Bray Curtis and Jaccard distances, health status, sampling location, and dataset were all significant sources of variation. Using a consensus approach based on RandomForest, DESeq2, and ANCOM-BC2, we identified three differentially abundant amplicon sequence variants (ASVs) within the nasal cavity, ASV5_Mycoplasma, ASV19_Corynebacterium, and ASV37_Ruminococcaceae. However, no ASVs were differentially abundant in the other sampling locations. Moreover, based on SECOM analysis, ASV37_Ruminococcaceae had a negative relationship with ASV1_Mycoplasma_hyorhinis, ASV5_Mycoplasma, and ASV4_Mannheimia. ASV19_Corynebacterium had negative relationships with ASV1_Mycoplasma_hyorhinis, ASV4_Mannheimia, ASV54_Mycoplasma, ASV7_Mycoplasma, and ASV8_Pasteurella. Conclusions: Our results confirm a relationship between bovine respiratory disease and respiratory microbiome diversity and composition, which provide additional insight into microbial community dynamics during BRD development. Furthermore, as sampling location and sample processing (dataset) can also affect results, consideration should be taken when comparing results across studies.