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Alterations to the community structure and function of the microbiome are associated with changes to host physiology, including immune responses. However, the contribution of microbe‐derived RNAs carried by outer membrane vesicles (OMVs) to host immune responses remains unclear. This study investigated the role of OMVs and OMV‐associated small RNA (sRNA) species from pathogenic and commensal Bacteroides fragilis (ETBF and NTBF, respectively) in eliciting different immune responses from intestinal epithelial cells. To distinguish the differences in the sRNA profiles of the two strains and their OMVs, RNA‐seq, qRT‐PCR, and northern blotting were conducted to identify enrichment of discrete sRNA species in OMVs, which were also differentially expressed between the two strains. Specifically, coding and non‐coding RNAs were enriched in OMVs from NTBF and ETBF, with BF9343_RS22680 and BF9343_RS17870 being significantly enriched in ETBF OMVs compared to NTBF. To understand the effects of OMVs on pattern recognition receptors, reporter cells of Toll‐like receptor (TLR) activation were treated with OMVs, demonstrating activation of TLRs 2, 3, and 7. Treatment of Caco‐2 and HT29‐MTX cells with OMVs demonstrated increased expression of IL‐8. Surprisingly, we discovered that degradation of RNase‐accessible RNAs within ETBF OMVs, but not NTBF OMVs, resulted in vesicles with enhanced capacity to stimulate IL‐8 expression, indicating that these extravesicular RNAs exert an immunosuppressive effect. This suggests a dual role for OMV‐associated RNAs in modulating host immune responses, with implications for both bacterial pathogenesis and therapeutic applications. Outer membrane vesicles (OMVs) from Bacteroides fragilis carry distinct coding and non‐coding RNAs that influence host inflammatory signaling. Extravesicular RNAs from OMVs suppress IL‐8 expression in intestinal epithelial cells, while removal of these RNAs enhances pro‐inflammatory IL‐8 responses. These findings reveal dual immunomodulatory roles for OMV‐associated RNAs, highlighting a mechanism by which pathogenic B. fragilis may fine‐tune host immune responses.

The biological role of the bacterial chloramphenicol (Chl)–resistance enzyme, chloramphenicol acetyltransferase (CAT), has seen renewed interest due to the resurgent use of Chl against multi-drug-resistant microbes. This looming threat calls for more rationally designed antibiotic derivatives that have improved antimicrobial properties and reduced toxicity in humans. Herein, we utilize native ion mobility spectrometry–mass spectrometry (IMS-MS) to investigate the gas-phase structure and thermodynamic stability of the type I variant of CAT from Escherichia coli ( Ec CAT I ) and several Ec CAT I :ligand-bound complexes. Ec CAT I readily binds multiple Chl without incurring significant changes to its gas-phase structure or stability. A non-hydrolyzable acetyl-CoA derivative (S-ethyl-CoA, S-Et-CoA) was used to kinetically trap Ec CAT I and Chl in a ternary, ligand-bound state ( Ec CAT I :S-Et-CoA:Chl). Using collision-induced unfolding (CIU)-IMS-MS, we find that Chl dissociates from Ec CAT I :S-Et-CoA:Chl complexes at low collision energies, while S-Et-CoA remains bound to Ec CAT I even as protein unfolding occurs. Gas-phase binding constants further suggest that Ec CAT I binds S-Et-CoA more tightly than Chl. Both ligands exhibit negative cooperativity of subsequent ligand binding in their respective binary complexes. While we observe no significant change in structure or stability to Ec CAT I when bound to either or both ligands, we have elucidated novel gas-phase unfolding and dissociation behavior and provided a foundation for further characterization of alternative substrates and/or inhibitors of Ec CAT I . Graphical abstract

Characterizing glycans is analytically challenging since glycans are heterogeneous, branched polymers with different three-dimensional conformations. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been used to analyze native conformations and dynamics of biomolecules by measuring the mass increase of analytes as labile protons are replaced with deuterium following exposure to deuterated solvents. The rate of exchange is dependent on the chemical functional group, the presence of hydrogen bonds, pH, temperature, charge, and solvent accessibility. HDX-MS of carbohydrates is challenging due to the rapid exchange rate of hydroxyls. Here, we describe an observed HDX reaction associated with residual solvent vapors saturating electrospray sources. When undeuterated melezitose was infused after infusing D 2 O, samples with up to 73% deuterium exchange were detected. This residual solvent HDX was observed for both carbohydrates and peptides in multiple instruments and dependent on sample infusion rate, infusion time, and deuterium content of the solvent. This residual solvent HDX was observed over several minutes of sample analysis and persisted long enough to alter the measured deuterium labeling and possibly change the interpretation of the results. This work illustrates that residual solvent HDX competes with in-solution HDX for rapidly exchanging functional groups. Thus, we propose conditions to minimize this effect, specifically for top-down, in-electrospray ionization, and quench-flow HDX experiments. Graphical Abstract ᅟ

An understanding of how conformational dynamics modulates function and catalysis of human monoacylglycerol lipase (hMGL), an important pharmaceutical target, can facilitate the development of novel ligands with potential therapeutic value. Here, we report the discovery and characterization of an allosteric, regulatory hMGL site comprised of residues Trp-289 and Leu-232 that reside over 18 Å away from the catalytic triad. These residues were identified as critical mediators of long-range communication and as important contributors to the integrity of the hMGL structure. Nonconservative replacements of Trp-289 or Leu-232 triggered concerted motions of structurally distinct regions with a significant conformational shift toward inactive states and dramatic loss in catalytic efficiency of the enzyme. Using a multimethod approach, we show that the dynamically relevant Trp-289 and Leu-232 residues serve as communication hubs within an allosteric protein network that controls signal propagation to the active site, and thus, regulates active-inactive interconversion of hMGL. Our findings provide new insights into the mechanism of allosteric regulation of lipase activity, in general, and may provide alternative drug design possibilities.

Changes in these dynamical properties can affect binding with receptors. [...]the glycan distribution is a critical quality attribute that is carefully monitored during mAb manufacture [11-13]. HDX-MS studies have proved important for characterizing the dynamics of IgG1 glycoforms [13-20] and their interactions with receptors [15, 16]. [...]measurements of the differences in molecular dynamics of mAb glycoforms can provide information useful for evaluating similarities between an innovator biotherapeutic and a candidate biosimilar. SAF contains some small fractions of S1F glycan chains terminated with one Neu-5-Ac sialic acid. Since S1F and S2F were prepared using the a(2-6) linkage enzyme, human sialyltransferase, both sialylated structures have a(2-6) linkages. 3.3 Peptide Identifications from Mass Spectrometry Data Peptic peptides of soluble FcyRIa and aIL8hFc-control were generated by passing 20 pmol of protein through an Enzymate BEH pepsin digestion column (2.1 x 30 mm, 5 qm bead; Waters, Milford, MA, USA) and identified using MS/MS on the Thermo LTQ Orbitrap Elite mass spectrometer.

Transmembrane protein (TMP)-functionalized materials have resulted in powerful new methods in chemical analysis. Of particular interest is the development of high-throughput, TMP-functionalized stationary phases for affinity chromatography of complex mixtures of analytes. Several natural and synthetic phospholipids and lipid mimics have been used for TMP reconstitution, although the resulting membranes often lack the requisite chemical and temporal stability for long-term use, a problem that is exacerbated in flowing separation systems. Polymerizable lipids with markedly increased membrane stability and TMP functionality have been developed over the past two decades. More recently, these lipids have been incorporated into a range of analytical methods, including separation techniques, and are now poised to have a significant impact on TMP-based separations. Here, we describe current methods for preparing TMP-containing stationary phases and examine the potential utility of polymerizable lipids in TMP affinity chromatography.

The spreadsheet file reported herein provides centroid data, descriptive of deuterium uptake, for the FabFragment of NISTmAb (PDB: 5K8A) reference material, as measured by the bottom-up hydrogen-deuterium exchange mass spectrometry (HDX-MS) method. The protein sample was incubated in deuterium-rich solutions under uniform pH and salt concentrations between 3.6 oC and 25.4 oC for seven intervals ranging over (0 to 14,400) s plus a ∞pseudo s control. The deuterium content of peptic peptide fragments were measured by mass spectrometry. These data were reported by fifteen laboratories, which conducted the measurements using orbitrap and Q-TOF mass spectrometers. The cohort reported ≈ 78,900 centroids for 430 proteolytic peptide sequences of the heavy and light chains of NISTmAb, providing nearly 100 % coverage. In addition, some groups reported ≈ 10,900 centroid measurements for 77 peptide sequences of the Fc fragment. The instrumentation and physical and chemical conditions under which these data were acquired are documented.

Alterations to the community structure and function of the microbiome are associated with changes to host physiology, including immune responses. However, the contribution of microbe-derived RNAs carried by outer membrane vesicles (OMVs) to host immune responses remain unclear. This study investigated the role of OMVs and OMV-associated small RNA (sRNA) species from pathogenic and commensal (ETBF and NTBF respectively) in eliciting different immune responses from intestinal epithelial cells. To distinguish the differences in the sRNA profiles of the two strains and their OMVs, RNA-seq, qRT-PCR, and northern blotting were conducted to identify enrichment of discrete sRNA species in OMVs, which were also differentially expressed between the two strains. Specifically, both coding and non-coding RNAs were enriched in OMVs from NTBF and ETBF, with BF9343_RS22680 and BF9343_RS17870 being significantly enriched in ETBF OMVs compared to NTBF. To understand the effects of OMVs on pattern recognition receptors, reporter cells of Toll-like receptor (TLR) activation were treated with OMVs, demonstrating activation of TLRs 2, 3, and 7. Treatment of Caco-2 and HT29-MTX cells with OMVs demonstrated increased expression of IL-8. Surprisingly, we discovered that degradation of RNase-accessible RNAs within ETBF OMVs, but not NTBF OMVs, resulted in vesicles with enhanced capacity to stimulate IL-8 expression, indicating that these extravesicular RNAs exert an immunosuppressive effect. This suggests a dual role for OMV-associated RNAs in modulating host immune responses, with implications for both bacterial pathogenesis and therapeutic applications.

The goal of proteomics experiments is to identify proteins to observe changes in cellular processes and diseases. One challenge in proteomics is the removal of contaminants following protein extraction, which can limit protein identification. Single-pot, solid-phase-enhanced sample preparation (SP3) is a clean-up technique in which proteins are captured on carboxylate-modified particles through a proposed hydrophilic-interaction-liquid-chromatography (HILIC)-like mechanism. However, recent results have suggested that proteins are captured in SP3 due to a protein-aggregation mechanism. Thus, solvent precipitation, single-pot, solid-phase-enhanced sample preparation (SP4) is a newer clean-up technique that employs protein-aggregation to capture proteins without modified particles. SP4 has previously enriched low-solubility proteins, though differences in protein capture could affect which proteins are detected and identified. We hypothesize that the mechanisms of capture for SP3 and SP4 are distinct. Herein, we assess the proteins identified and enriched using SP3 versus SP4 for MCF7 subcellular fractions and correlate protein capture in each method to protein hydrophobicity. Our results indicate that SP3 captures more hydrophilic proteins through a combination of HILIC-like and protein-aggregation mechanisms, while SP4 captures more hydrophobic proteins through a protein-aggregation mechanism. From these results, we recommend clean-up techniques based on protein-sample hydrophobicity to yield high proteome coverage in biological samples.