The UV-light-induced shift in DNA-binding preferences of transcription factors, impacting both consensus and non-consensus DNA sites, holds crucial implications for their regulatory and mutagenic functions within the cellular framework.
Regular fluid flow is a ubiquitous feature of cells in natural settings. Despite this, the vast majority of experimental platforms rely on batch cell cultures, failing to account for the influence of flow-driven processes on cellular behavior. Our microfluidic and single-cell imaging study uncovered a transcriptional response in the human pathogen Pseudomonas aeruginosa, where the interplay of chemical stress and physical shear rate (a measure of fluid flow) played a critical role. Hydrogen peroxide (H2O2), a ubiquitous chemical stressor, is rapidly removed from the media by cells in batch cell cultures, thereby safeguarding themselves. Spatial gradients of hydrogen peroxide are a consequence of cell scavenging, as observed in microfluidic settings. High shear rates lead to the replenishment of H2O2, the removal of any gradients, and the creation of a stress response. Combining computational simulations with biophysical experiments, we find that the action of flow causes a phenomenon analogous to wind chill, making cells significantly more susceptible to H2O2 concentrations 100 to 1000 times lower than those conventionally studied in batch cultures. To one's astonishment, the shear rate and hydrogen peroxide concentration required to initiate a transcriptional response are strikingly similar to their respective levels within the human bloodstream. Hence, the outcomes of our study offer an explanation for the longstanding divergence in H2O2 levels between experimental setups and those existing in the host. In conclusion, we provide evidence that the shear forces and hydrogen peroxide levels characteristic of the human circulatory system induce genetic responses in the blood-borne pathogen Staphylococcus aureus, hinting that blood flow renders bacteria more sensitive to chemical stressors in vivo.
Sustained and passive drug release, facilitated by degradable polymer matrices and porous scaffolds, addresses a broad range of diseases and conditions relevant to treatments. Active pharmacokinetic control, customized for patient-specific needs, is seeing heightened interest. This is enabled by programmable engineering platforms, which integrate power sources, delivery systems, communication hardware, and related electronics, normally requiring surgical removal following a defined usage period. see more This self-powered, light-controlled technology, addressing the critical weaknesses of earlier systems, adopts a bioresorbable design structure. The system's programmability is realized by illuminating an implanted, wavelength-sensitive phototransistor with an external light source. This causes a short circuit in the electrochemical cell structure's anode, which is a metal gate valve. Subsequent electrochemical corrosion, removing the gate, causes a dose of drugs to diffuse passively into surrounding tissues, thereby accessing an underlying reservoir. A strategy of wavelength-division multiplexing facilitates programming the release from any single reservoir or any arbitrary grouping of reservoirs situated within an integrated device. Studies on bioresorbable electrode materials serve to identify essential factors and direct the development of optimized designs. see more Live demonstrations of lidocaine's programmed release adjacent to sciatic nerves in rat models exemplify its utility in pain management, a vital element of patient care enhanced by the presented data.
Investigations into transcriptional initiation mechanisms in diverse bacterial taxa showcase a multiplicity of molecular controls over this initial gene expression step. Within Actinobacteria, the WhiA and WhiB factors are required to express cell division genes, and are crucial in notable pathogens such as Mycobacterium tuberculosis. Within Streptomyces venezuelae (Sven), the WhiA/B regulons' binding sites have been determined, exhibiting a cooperative effect on sporulation septation activation. Still, the complex molecular interactions among these factors are not understood. Cryo-electron microscopy structures of Sven transcriptional regulatory complexes are presented here, displaying the intricate interplay between RNA polymerase (RNAP) A-holoenzyme and the regulatory proteins WhiA and WhiB, complexed with their target promoter, sepX. Examination of these structures reveals that WhiB binds to A4, a portion of the A-holoenzyme, creating a link between its interaction with WhiA and its non-specific interaction with the DNA stretch preceding the -35 core promoter element. The WhiA C-terminal domain (WhiA-CTD) establishes base-specific interactions with the conserved WhiA GACAC motif, distinct from the interaction between the N-terminal homing endonuclease-like domain of WhiA and WhiB. The structure of the WhiA-CTD and its interactions with the WhiA motif demonstrate remarkable parallels with the interactions between A4 housekeeping factors and the -35 promoter element; this indicates an evolutionary connection. The structure-guided mutagenesis strategy employed to disrupt protein-DNA interactions effectively curtails or abolishes developmental cell division in Sven, establishing their importance. Lastly, we juxtapose the architecture of the WhiA/B A-holoenzyme promoter complex against the unrelated yet illustrative CAP Class I and Class II complexes, demonstrating that WhiA/WhiB represents a novel mechanism within bacterial transcriptional activation.
For metalloprotein activity, the precise redox state of transition metals is crucial and can be manipulated via coordination chemistry or by separating them from the bulk solvent environment. Human methylmalonyl-CoA mutase (MCM) employs 5'-deoxyadenosylcobalamin (AdoCbl) as a metallocofactor to catalyze the isomerization of methylmalonyl-CoA into succinyl-CoA. Catalysis occasionally results in the escape of the 5'-deoxyadenosine (dAdo) moiety, leaving the cob(II)alamin intermediate susceptible to hyperoxidation into the difficult-to-repair hydroxocobalamin. Through bivalent molecular mimicry, ADP in this study is shown to utilize 5'-deoxyadenosine and diphosphate as cofactor and substrate components, respectively, to thwart cob(II)alamin overoxidation on the MCM platform. EPR and crystallographic studies unveil that ADP's effect on metal oxidation state is predicated on a conformational shift that isolates the metal from solvent, in contrast to a change in coordination of five-coordinate cob(II)alamin to the more air-stable four-coordinate state. The off-loading of cob(II)alamin from methylmalonyl-CoA mutase (MCM) to adenosyltransferase for repair is promoted by the subsequent attachment of methylmalonyl-CoA (or CoA). This research uncovers an atypical approach to managing metal redox states. A plentiful metabolite, by obstructing access to the active site, is crucial for maintaining and regenerating a rare, yet essential, metal cofactor.
The ocean's role in releasing nitrous oxide (N2O), a greenhouse gas and ozone-depleting substance, into the atmosphere is substantial. The process of ammonia oxidation, frequently conducted by ammonia-oxidizing archaea (AOA), yields nitrous oxide (N2O) as a trace side product; these archaea are numerically dominant in most marine ammonia-oxidizing communities. The mechanisms behind N2O production and their associated kinetics, however, are not fully understood. In this study, 15N and 18O isotopes are used to track the kinetics of N2O production and the origin of the nitrogen (N) and oxygen (O) atoms in the N2O product from a model marine ammonia-oxidizing archaea, Nitrosopumilus maritimus. In ammonia oxidation, the apparent half-saturation constants for nitrite and nitrous oxide generation are similar, suggesting both reactions are tightly linked through enzymatic mechanisms at low ammonia concentrations. Via multiple reaction sequences, the constituent atoms of N2O are produced from the chemical compounds ammonia, nitrite, oxygen, and water molecules. Although ammonia is the main source of nitrogen atoms in N2O, the magnitude of its involvement varies according to the ratio of ammonia to nitrite. The presence of different substrates alters the ratio of 45N2O to 46N2O (single or double nitrogen labeling), generating a wide spectrum of isotopic signatures in the resulting N2O pool. The diatomic oxygen molecule, O2, is the principal provider of oxygen atoms, O. Along with the previously demonstrated hybrid formation pathway, our findings highlight a considerable contribution from hydroxylamine oxidation, rendering nitrite reduction a minor contributor to N2O formation. The innovative use of dual 15N-18O isotope labeling in our study provides crucial insights into the complex N2O production pathways in microbes, offering significant implications for elucidating marine N2O sources and regulatory mechanisms.
CENP-A histone H3 variant enrichment acts as the epigenetic signature of the centromere, triggering kinetochore assembly at that location. Mitosis depends on the kinetochore, a multi-component complex, for the precise binding of microtubules to the centromere and the subsequent accurate separation of sister chromatids. In order for CENP-I, a kinetochore constituent, to reside at the centromere, the presence of CENP-A is mandatory. Nevertheless, the precise mechanisms by which CENP-I influences CENP-A localization and centromeric characterization remain uncertain. Direct interaction between CENP-I and centromeric DNA was observed in this study. This interaction is markedly selective for AT-rich DNA sequences, driven by a contiguous DNA-binding surface comprised of conserved charged residues at the terminus of the N-terminal HEAT repeats. see more CENP-I mutants, incapable of DNA binding, still showed interaction with CENP-H/K and CENP-M; however, a notable decrease in CENP-I's centromeric localization and mitosis chromosome alignment was observed. Beyond that, the DNA binding of CENP-I is critical for the centromeric incorporation of the newly generated CENP-A.