No abstract

The RNA polymerase (RNAP) binding protein A (RbpA) contributes to the formation of stable RNAP-promoter open complexes (RP) and is essential for viability in mycobacteria. Four domains have been identified in the RbpA protein, i.e., an N-terminal tail (NTT) that interacts with RNAP β' and σ subunits, a core domain (CD) that contacts the RNAP β' subunit, a basic linker (BL) that binds DNA, and a σ-interaction domain (SID) that binds group I and group II σ factors. Limited studies have been performed in mycobacteria, however, and how individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We investigated the roles of the RbpA structural domains in mycobacteria using a panel of mutants that target individual RbpA domains. The function of each RbpA domain was required for viability and optimal growth in We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP, indicating that the primary functions of the NTT and CD are not solely association with the RNAP. We show that the RbpA BL and SID are required for RP stabilization , while the NTT and CD antagonize this activity. Finally, RNA-sequencing analyses suggest that the NTT and CD broadly activate gene expression, whereas the BL and SID activate or repress gene expression in a gene-dependent manner for a subset of mycobacterial genes. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA. is the causative agent of tuberculosis and continues to be the most lethal infectious disease worldwide. Improved molecular understanding of the essential proteins involved in transcription, such as RbpA, could provide targets for much needed future therapeutic agents aimed at combatting this pathogen. In this study, we expand our understanding of RbpA by identifying the RbpA structural domains responsible for the interaction of RbpA with the RNAP and the effects of RbpA on transcription initiation and gene expression. These experiments expand our knowledge of RbpA while also broadening our understanding of bacterial transcription in general.

Two types of thiamine (vitamin B) salts, thiamine mononitrate (TMN) and thiamine chloride hydrochloride (TClHCl), are used to enrich and fortify food products. Both of these thiamine salt forms are sensitive to heat, alkali, oxygen, and radiation, but differences in stability between them have been noted. It was hypothesized that stability differences between the two thiamine salts could be explained by differences in solubility, solution pH, and activation energies for degradation. This study directly compared the stabilities of TMN and TClHCl in solution over time by documenting the impact of concentration and storage temperature on thiamine degradation and calculating reaction kinetics. Solutions were prepared containing five concentrations of each thiamine salt (1, 5, 10, 20, and 27 mg/mL), and three additional concentrations of TClHCl: 100, 300, and 500 mg/mL. Samples were stored at 25, 40, 60, 70, and 80 °C for up to 6 months. Degradation was quantified over time by high-performance liquid chromatography, and percent thiamine remaining was used to calculate reaction kinetics. First-order reaction kinetics were found for both TMN and TClHCl. TMN degraded significantly faster than TClHCl at all concentrations and temperatures. For example, in 27 mg/mL solutions after 5 days at 80 °C, only 32% of TMN remained compared to 94% of TClHCl. Activation energies and solution pHs were 21-25 kcal/mol and pH 5.36-6.96 for TMN and 21-32 kcal/mol and pH 1.12-3.59 for TClHCl. TClHCl degradation products had much greater sensory contributions than TMN degradation products, including intense color change and potent aromas, even with considerably less measured vitamin loss. Different peak patterns were present in HPLC chromatograms between TMN and TClHCl, indicating different degradation pathways and products. The stability of essential vitamins in foods is important, even more so when degradation contributes to sensory changes, and this study provides a direct comparison of the stability of the two thiamine salts used to fortify foods in environments relevant to the processing and shelf-life of many foods.

No abstract

The curriculum was associated with a short term increased knowledge regarding pediatric respiratory distress and failure for emergency helicopter transport providers and could be used as an alternative model to develop standardized ongoing medical education in pediatrics. Further work is needed to achieve knowledge retention in this learner population.

EV-D68 causes severe disease in the pediatric population, particularly in children with a history of asthma or recurrent wheezing. EV-D68-positive children are more likely to require therapy for refractory bronchospasm and may need intensive care unit- level care.

Structural biologists rely on X-ray crystallography as the main technique for determining the three-dimensional structures of macromolecules; however, in recent years, new methods that go beyond X-ray-based technologies are broadening the selection of tools to understand molecular structure and function. Simultaneously, national facilities are developing programming tools and maintaining personnel to aid novice structural biologists in de novo structure determination. The combination of X-ray free electron lasers (XFELs) and serial femtosecond crystallography (SFX) now enable time-resolved structure determination that allows for capture of dynamic processes, such as reaction mechanism and conformational flexibility. XFEL and SFX, along with microcrystal electron diffraction (MicroED), help side-step the need for large crystals for structural studies. Moreover, advances in cryogenic electron microscopy (cryo-EM) as a tool for structure determination is revolutionizing how difficult to crystallize macromolecules and/or complexes can be visualized at the atomic scale. This review aims to provide a broad overview of these new methods and to guide readers to more in-depth literature of these methods.

With the rising prevalence of multidrug-resistance, there is an urgent need to develop novel antibiotics. Many putative antibiotics demonstrate promising in vitro potency but fail in vivo due to poor drug-like qualities (e.g. serum half-life, oral absorption, solubility, toxicity). These drug-like properties can be modified through the addition of chemical protecting groups, creating “prodrugs” that are activated prior to target inhibition. Lipophilic prodrugging techniques, including the attachment of a pivaloyloxymethyl group, have garnered attention for their ability to increase cellular permeability by masking charged residues and the relative ease of the chemical prodrugging process. Unfortunately, pivaloyloxymethyl prodrugs are rapidly activated by human sera, rendering any membrane permeability qualities absent during clinical treatment. Identification of the bacterial prodrug activation pathway(s) will allow for the development of host-stable and microbe-targeted prodrug therapies. Here, we use two zoonotic staphylococcal species, S. schleiferi and S. pseudintermedius, to establish the mechanism of carboxy ester prodrug activation. Using a forward genetic screen, we identify a conserved locus in both species encoding the enzyme hydroxyacylglutathione hydrolase (GloB), whose loss-of-function confers resistance to carboxy ester prodrugs. We enzymatically characterize GloB and demonstrate that it is a functional glyoxalase II enzyme, which has the capacity to activate carboxy ester prodrugs. As GloB homologs are both widespread and diverse in sequence, our findings suggest that GloB may be a useful mechanism for developing species-or genus-level prodrug targeting strategies.