Multiscale regulation of nutrient stress responses in Escherichia coli from chromatin structure to small regulatory RNAs.

Recent research has indicated the presence of heterochromatin-like regions of extended protein occupancy and transcriptional silencing of bacterial genomes. We utilized an integrative approach to track chromatin structure and transcription in E. coli K-12 across a wide range of nutrient conditions. In the process, we identified multiple loci which act similarly to facultative heterochromatin in eukaryotes, normally silenced but permitting expression of genes under specific conditions. We also found a strong enrichment of small regulatory RNAs (sRNAs) among the set of differentially expressed transcripts during nutrient stress. Using a newly developed bioinformatic pipeline, the transcription factors regulating sRNA expression were bioinformatically predicted, with experimental follow-up revealing novel relationships for 36 sRNA-transcription factors candidates. Direct regulation of sRNA expression was confirmed by mutational analysis for five sRNAs of metabolic interest: IsrB, CsrB and CsrC, GcvB, and GadY. Our integrative analysis thus reveals additional layers of complexity in the nutrient stress response in E. coli and provides a framework for revealing similar poorly understood regulatory logic in other organisms.

CsrA selectively modulates sRNA-mRNA regulator outcomes.

Post-transcriptional regulation, by small RNAs (sRNAs) as well as the global Carbon Storage Regulator A (CsrA) protein, play critical roles in bacterial metabolic control and stress responses. The CsrA protein affects selective sRNA-mRNA networks, in addition to regulating transcription factors and sigma factors, providing additional avenues of cross talk between other stress-response regulators. Here, we expand the known set of sRNA-CsrA interactions and study their regulatory effects. In vitro binding assays confirm novel CsrA interactions with ten sRNAs, many of which are previously recognized as key regulatory nodes. Of those 10 sRNA, we identify that McaS, FnrS, SgrS, MicL, and Spot42 interact directly with CsrA in vivo. We find that the presence of CsrA impacts the downstream regulation of mRNA targets of the respective sRNA. In vivo evidence supports enhanced CsrA McaS-csgD mRNA repression and showcases CsrA-dependent repression of the fucP mRNA via the Spot42 sRNA. We additionally identify SgrS and FnrS as potential new sRNA sponges of CsrA. Overall, our results further support the expanding impact of the Csr system on cellular physiology via CsrA impact on the regulatory roles of these sRNAs.

CsrA Shows Selective Regulation of sRNA-mRNA Networks.

Post-transcriptional regulation, by small RNAs (sRNAs) as well as the global Carbon Storage Regulator A (CsrA) protein, play critical roles in bacterial metabolic control and stress responses. The CsrA protein affects selective sRNA-mRNA networks, in addition to regulating transcription factors and sigma factors, providing additional avenues of cross talk between other stress-response regulators. Here, we expand the known set of sRNA-CsrA interactions and study their regulatory effects. In vitro binding assays confirm novel CsrA interactions with ten sRNAs, many of which are previously recognized as key regulatory nodes. Of those 10 sRNA, we identify that McaS, FnrS, SgrS, MicL, and Spot42 interact with CsrA in vivo. We find that the presence of CsrA impacts the downstream regulation of mRNA targets of the respective sRNA. In vivo evidence supports enhanced CsrA McaS-csgD mRNA repression and showcase CsrA-dependent repression of the fucP mRNA via the Spot42 sRNA. We additionally identify SgrS and FnrS as potential new sRNA sponges of CsrA. Overall, our results further support the expanding impact of the Csr system on cellular physiology via CsrA impact on the regulatory roles of these sRNAs.

Engineering Toehold-Mediated Switches for Native RNA Detection and Regulation in Bacteria.

RNA switches are versatile tools in synthetic biology for sensing and regulation applications. The discoveries of RNA-mediated translational and transcriptional control have facilitated the development of complex de novo designs of RNA switches. Specifically, RNA toehold-mediated switches, in which binding to the toehold sensing domain controls the transition between switch states via strand displacement, have been extensively adapted for coupling systems responses to specific trans-RNA inputs. This review highlights some of the challenges associated with applying these switches for native RNA detection in vivo, including transferability between organisms. The applicability and design considerations of toehold-mediated switches are discussed by highlighting twelve recently developed switch designs. This review finishes with future perspectives to address current gaps in the field, particularly regarding the power of structural prediction algorithms for improved in vivo functionality of RNA switches.

Pharmaceutical profiling and molecular dynamics simulations reveal crystallization effects in amorphous formulations.

Robust and reliable in vivo performance of medicines based on amorphous solid dispersions (ASDs) depend on maintenance of physical stability and efficient supersaturation. However, molecular drivers of these two kinetic processes are poorly understood. Here we used molecular dynamics (MD) simulations coupled with experimental assessments to explore supersaturation, nucleation, and crystal growth. The effect of drug loading on physical stability and supersaturation potential was highly drug specific. Storage under humid conditions influenced crystallization, but also resulted in morphological changes and particle fusion. This led to increased particle size, which significantly reduced dissolution rate. MD simulations identified the importance of nano-compartmentalization in the crystallization rate of the ASDs. Nucleation during storage did not inherently compromise the ASD. Rather, the poorer performance resulted from a combination of properties of the compound, nanostructures formed in the formulation, and crystallization.

Uncovering Transcriptional Regulators and Targets of sRNAs Using an Integrative Data-Mining Approach: H-NS-Regulated RseX as a Case Study.

Bacterial small RNAs (sRNAs) play a vital role in pathogenesis by enabling rapid, efficient networks of gene attenuation during infection. In recent decades, there has been a surge in the number of proposed and biochemically-confirmed sRNAs in both Gram-positive and Gram-negative pathogens. However, limited homology, network complexity, and condition specificity of sRNA has stunted complete characterization of the activity and regulation of these RNA regulators. To streamline the discovery of the expression of sRNAs, and their post-transcriptional activities, we propose an integrative in vivo data-mining approach that couples DNA protein occupancy, RNA-seq, and RNA accessibility data with motif identification and target prediction algorithms. We benchmark the approach against a subset of well-characterized E. coli sRNAs for which a degree of in vivo transcriptional regulation and post-transcriptional activity has been previously reported, finding support for known regulation in a large proportion of this sRNA set. We showcase the abilities of our method to expand understanding of sRNA RseX, a known envelope stress-linked sRNA for which a cellular role has been elusive due to a lack of native expression detection. Using the presented approach, we identify a small set of putative RseX regulators and targets for experimental investigation. These findings have allowed us to confirm native RseX expression under conditions that eliminate H-NS repression as well as uncover a post-transcriptional role of RseX in fimbrial regulation. Beyond RseX, we uncover 163 putative regulatory DNA-binding protein sites, corresponding to regulation of 62 sRNAs, that could lead to new understanding of sRNA transcription regulation. For 32 sRNAs, we also propose a subset of top targets filtered by engagement of regions that exhibit binding site accessibility behavior in vivo. We broadly anticipate that the proposed approach will be useful for sRNA-reliant network characterization in bacteria. Such investigations under pathogenesis-relevant environmental conditions will enable us to deduce complex rapid-regulation schemes that support infection.

Solvent-Assisted Secondary Drying of Spray-Dried Polymers.

PURPOSE: The purpose of this work is to introduce solvent-assisted secondary drying, a method used to accelerate the residual solvent removal from spray dried materials. Spray-drying is used to manufacture amorphous solid dispersions, which enhance the bioavailability of active pharmaceutical ingredients (APIs) with low aqueous solubility. In the spray-drying process, API and excipients are co-dissolved in a volatile organic solvent, atomized into droplets through a nozzle, and introduced to a drying chamber containing heated nitrogen gas. The product dries rapidly to form a powder, but small amounts of residual solvent (typically, 1 to 10 wt%) remain in the product and must be removed in a secondary-drying process. For some spray-dried materials, secondary drying by traditional techniques can take days and requires balancing stability risks with process time. METHODS: Spray-dried polymers were secondary dried, comparing the results for three state-of-the-art methods that employed a jacketed, agitated-vessel dryer: (1) vacuum-only drying, (2) water-assisted drying, or (3) methanol-assisted drying. Samples of material were pulled at various time points and analyzed by gas chromatography (GC) and Karl Fischer (KF) titration to track the drying process. RESULTS: Model systems were chosen for which secondary drying is slow. For all cases studied, methanol-assisted drying outperformed the vacuum-only and water-assisted drying methods. CONCLUSIONS: The observation that methanol-assisted drying is more effective than the other drying techniques is consistent with the free-volume theory of solvent diffusion in polymers.

A novel architecture for achieving high drug loading in amorphous spray dried dispersion tablets.

Although Amorphous Solid Dispersions (ASDs) effectively increase bioavailability, tablet mass can be high due to the large fraction of excipients needed to stabilize the amorphous drug in the solid state, extend drug supersaturation in solution and achieve robust manufacturability. The aim of this work was to reduce tablet mass of an ASD tablet comprising a low glass transition temperature (Tg), rapidly crystallizing drug without compromising these key attributes. In this approach, erlotinib (Tg = 42 °C, Tm/Tg = 1.4 K/K) was spray dried with the high Tg polymer poly(methyl methacrylate-co-methacrylic acid) (Eudragit® L100, Evonik) (Tg = 187 °C) to facilitate high drug loading while maintaining physical stability. Hydroxypropyl methylcellulose acetate succinate (HPMCAS) (AQOAT® HF, Shin-Etsu) was granulated with the ASD to extend supersaturation in solution. For comparison, a benchmark ASD was spray dried at a lower drug loading with HPMCAS-H (Tg = 119 °C). This High Loaded Dosage Form (HLDF) approach reduced tablet mass by 40%, demonstrated similar physical stability and in vitro performance as the benchmark and exhibited excellent downstream manufacturability. Strategically combining two different polymers in a tablet to maintain physical stability and sustain supersaturation in solution can decrease tablet mass of some low Tg, rapidly crystallizing amorphous drugs.

Effect of Spray-Dried Particle Morphology on Mechanical and Flow Properties of Felodipine in PVP VA Amorphous Solid Dispersions.

Amorphous solid dispersions (ASDs) are commonly used to enhance the oral absorption of drugs with solubility or dissolution rate limitations. Although the ASD formulation is typically constrained by physical stability and in vivo performance considerations, ASD particles can be engineered using the spray-drying process to influence mechanical and flow properties critical to tableting. Using the ASD formulation of 20% w/w felodipine dispersed in polyvinyl pyrrolidone vinyl acetate, spray-drying atomization and drying conditions were tuned to achieve 4 different powders with varying particle properties. The resulting particles ranged in volume moment mean diameter from 4 to 115 µm, bulk density from 0.05 to 0.38 g cm-3, and morphologies of intact, collapsed, and fractured hollow spheres. Powder flowability by shear cell ranged from poor to easy flowing, whereas mechanical property tests suggested all samples will produce strong tablets at reasonable solid fractions and compression pressures. In addition, Hiestand dynamic tableting indices showed excellent dynamic bonding for 3 powders, and low viscoelasticity with high brittleness for all powders. This work demonstrates the extent spray-dried ASD particle morphologies can be engineered to achieve desired powder flow and mechanical properties to mitigate downstream processing risks and increase process throughput.