Despite the importance of maintaining redox homeostasis for cellular viability, how cells control redox balance globally is poorly understood. Here we provide new mechanistic insight into how the ...balance between reduced and oxidized electron carriers is regulated at the level of gene expression by mapping the regulon of the response regulator ArcA from Escherichia coli, which responds to the quinone/quinol redox couple via its membrane-bound sensor kinase, ArcB. Our genome-wide analysis reveals that ArcA reprograms metabolism under anaerobic conditions such that carbon oxidation pathways that recycle redox carriers via respiration are transcriptionally repressed by ArcA. We propose that this strategy favors use of catabolic pathways that recycle redox carriers via fermentation akin to lactate production in mammalian cells. Unexpectedly, bioinformatic analysis of the sequences bound by ArcA in ChIP-seq revealed that most ArcA binding sites contain additional direct repeat elements beyond the two required for binding an ArcA dimer. DNase I footprinting assays suggest that non-canonical arrangements of cis-regulatory modules dictate both the length and concentration-sensitive occupancy of DNA sites. We propose that this plasticity in ArcA binding site architecture provides both an efficient means of encoding binding sites for ArcA, σ(70)-RNAP and perhaps other transcription factors within the same narrow sequence space and an effective mechanism for global control of carbon metabolism to maintain redox homeostasis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Nanoparticles exposed to biofluids become coated with proteins, thus making protein–nanoparticle interactions of particular interest. The consequence on protein conformation and activity depends upon ...the extent of protein adsorption on the nanoparticle surface. We report the interaction of bovine serum albumin (BSA) with gold nanostructures, particularly gold nanoparticles (GNP) and gold nanorods (GNR). The difference in the geometry and surface properties of nanoparticles is manifested during complexation in terms of different binding modes, structural changes, thermodynamic parameters, and the activity of proteins. BSA is found to retain native-like structure and properties upon enthalpy-driven BSA–GNP complexation. On the contrary, the entropically favored BSA–GNR complexation leads to substantial loss in protein secondary and tertiary structures with the release of a large amount of bound water, as indicated by isothermal calorimetry (ITC), circular dichroism (CD), and Fourier transform infrared (FTIR) and fluorescence spectroscopies. The esterase activity assay demonstrated a greater loss in BSA activity after complexation with GNR, whereas the original activity is retained in the presence of GNP. The formation of large assemblies (aggregates) and reduced average lifetime, as evidenced from dynamic light scattering and fluorescence decay measurements, respectively, suggest that GNR induces protein unfolding at its surface. The effect of temperature on the CD spectra of BSA–GNP was found to be similar to that of pristine BSA, whereas BSA–GNR shows distortion in CD spectra at lower wavelengths, strengthening the perception of protein unfolding. High binding constant and entropy change for BSA–GNR complexation determined by ITC are consistent with large surfacial interaction that may lead to protein unfolding. The present work highlights the differential response of a protein depending on the nature of the nanostructure and its surface chemistry, which need to be modulated for controlling the biological responses of nanostructures for their potential biomedical applications.
In biological fluids, nanoparticles are always surrounded by proteins. As the protein is adsorbed on the surface, the extent of adsorption and the effect on the protein conformation and stability are ...dependent on the chemical nature, shape, and size of the nanoparticle (NP). We have carried out a detailed investigation on the interaction of bovine serum albumin (BSA) with polyethyleneimine-functionalized ZnO nanoparticles (ZnO-PEI). ZnO-PEI was synthesized using a wet chemical method with a core size of ∼3–7 nm (from transmission electron microscopy). The interaction of BSA with ZnO-PEI was examined using a combination of calorimetric, spectroscopic, and computational techniques. The binding was studied by ITC (isothermal titration calorimetry), and the result revealed that the complexation is enthalpy-driven, indicating the possible involvement of electrostatic interaction. To investigate the nature of the interaction and the location of the binding site, a detailed domain-wise surface electrostatic potential calculation was performed using adaptive Poisson–Boltzmann software (APBS). The result shows that the protein surface can bind the nanoparticle. On binding ZnO-PEI, the protein gets destabilized to some extent, as displayed by CD (circular dichroism) and FTIR (Fourier transform infrared) spectroscopy. Chemical and thermal denaturation of BSA, when carried out in the presence of ZnO-PEI, also indicated a small perturbation in the protein structure. A comparison of the enthalpy and entropy components of binding with those derived for the interaction of BSA with ZnO nanoparticles explains the effect of hydrophilic cationic species attached on the NP surface. The effect of the NP surface modification on the structure and stability of BSA would find useful applications in nanobiotechnology.
Abstract
In modern era, wireless communications at ultrafast speed are need of the hour and search for its solution through cutting edge sciences is a new perspective. To address this issue, the data ...rates in order of terabits per second (TBPS) could be a key step for the realization of emerging sixth generation (6G) networks utilizing terahertz (THz) frequency regime. In this context, new class of transition metal dichalcogenides (TMDs) have been introduced as potential candidates for future generation wireless THz technology. Herein, a strategy has been adopted to synthesize high-quality monolayer of molybdenum di-sulfide (MoS
2
) using indigenously developed atmospheric pressure chemical vapor deposition (APCVD) set-up. Further, the time-domain transmission and sheet conductivity were studied as well as a plausible mechanism of terahertz response for monolayer MoS
2
has been proposed and compared with bulk MoS
2
. Hence, the obtained results set a stepping stone to employ the monolayer MoS
2
as potential quantum materials benefitting the next generation terahertz communication devices.
During transcription initiation, the TFIIH-kinase Kin28/Cdk7 marks RNA polymerase II (Pol II) by phosphorylating the C-terminal domain (CTD) of its largest subunit. Here we describe a ...structure-guided chemical approach to covalently and specifically inactivate Kin28 kinase activity in vivo. This method of irreversible inactivation recapitulates both the lethal phenotype and the key molecular signatures that result from genetically disrupting Kin28 function in vivo. Inactivating Kin28 impacts promoter release to differing degrees and reveals a “checkpoint” during the transition to productive elongation. While promoter-proximal pausing is not observed in budding yeast, inhibition of Kin28 attenuates elongation-licensing signals, resulting in Pol II accumulation at the +2 nucleosome and reduced transition to productive elongation. Furthermore, upon inhibition, global stabilization of mRNA masks different degrees of reduction in nascent transcription. This study resolves long-standing controversies on the role of Kin28 in transcription and provides a rational approach to irreversibly inhibit other kinases in vivo.
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•A general approach for covalent chemical inhibition of kinases in vivo•Targeted inhibition of Kin28 reveals an elusive elongation checkpoint in yeast•Varying impact on promoter escape and transition to productive elongation•Stabilization of existing mRNA buffers/masks reduction in nascent transcripts
Rodriguez-Molina et al. describe a general strategy for irreversible inhibition of kinases in vivo. Inhibition of Kin28/CDK7 reduces nascent transcription, increases stability of existing mRNA, and reveals an underappreciated role for Kin28 in priming Pol II for productive transcription elongation.
Pyrrole-imidazole (Py-Im) polyamides are synthetic molecules that can be rationally designed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro ...binding has been extensively studied, in vivo effects are often difficult to predict using current models of DNA binding. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their effective deployment in vivo. In this report we identified polyamide-DNA interaction sites across the entire genome, by covalently crosslinking and capturing these events in the nuclei of human LNCaP cells. This technique confirms the ability of two eight ring hairpin-polyamides, with similar architectures but differing at a single ring position (Py to Im), to retain in vitro specificities and display distinct genome-wide binding profiles.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Present study investigates the effect of pinned and fixed boundary conditions on deformation characteristics of silicone rubber pin‐reinforced polymer sandwich composite under rigid plate impact. ...These boundary conditions represent two different structural applications of the sandwich which consists of a soft polyurethane foam core, embedded with four silicone pins for reinforcement, placed in between two hard acrylic faceplates. Compression test were conducted on foam at different strain rates and the results obtained for quasi‐static case was later used in numerical analysis for procedure validation and impact simulation using finite element analysis software ABAQUS. Results for deformation, contact force generated and stresses induced are obtained. Quasi‐static compression results show good conformity between experimental and numerical results for foam. Impact results show that both boundary conditions have negligible effect on deformation and contact force but pinned condition produces much higher stresses than fixed one. The study also found that pin‐reinforcement significantly affects the impact behavior and the post‐impact recovery.
Effect of boundary support conditions on impact behavior of silicone pin‐reinforced polymer sandwich composite structure.
We have conjugated chloroquine, an anti-malarial, antiviral and anti-tumor drug, with thiol-functionalized gold nanoparticles and studied their binding interaction with bovine serum albumin (BSA) ...protein. Gold nanoparticles have been synthesized using sodium borohydride as reducing agent and 11-mercaptoundecanoic acid as thiol functionalizing ligand in aqueous medium. The formation of gold nanoparticles was confirmed from the characteristic surface plasmon absorption band at 522
nm and transmission electron microscopy revealed the average particle size to be ∼7
nm. Chloroquine was conjugated to thiolated gold nanoparticles by using EDC/NHS chemistry and the binding was analyzed using optical density measurement and Fourier transform infrared spectroscopy. The chloroquine–conjugated gold nanoparticles (GNP–Chl) were found to interact efficiently with BSA. Thermodynamic parameters suggest that the binding is driven by both enthalpy and entropy, accompanied with only a minor alteration in protein’s structure. Competitive drug binding assay revealed that the GNP–Chl bind at warfarin binding site I in subdomain IIA of BSA and was further supported by Trp212 fluorescence quenching measurements. Unraveling the nature of interactions of GNP–Chl with BSA would pave the way for the design of nanotherapeutic agents with improved functionality, enriching the field of nanomedicine.
We report the synthesis of chloroquine–conjugated gold nanoparticles and their binding to bovine serum albumin using biophysical and docking studies.
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► Synthesis and characterization of drug (chloroquine)–conjugated gold nanoparticles (GNP–Chl). ► Binding of GNP–Chl with bovine serum albumin (BSA) using biophysical techniques. ► Docking of drug with BSA.
Abstract
Spatial and temporal expression of genes is essential for maintaining phenotype integrity. Transcription factors (TFs) modulate expression patterns by binding to specific DNA sequences in ...the genome. Along with the core binding motif, the flanking sequence context can play a role in DNA-TF recognition. Here, we employ high-throughput in vitro and in silico analyses to understand the influence of sequences flanking the cognate sites in binding of three most prevalent eukaryotic TF families (zinc finger, homeodomain and bZIP). In vitro binding preferences of each TF toward the entire DNA sequence space were correlated with a wide range of DNA structural parameters, including DNA flexibility. Results demonstrate that conformational plasticity of flanking regions modulates binding affinity of certain TF families. DNA duplex stability and minor groove width also play an important role in DNA-TF recognition but differ in how exactly they influence the binding in each specific case. Our analyses further reveal that the structural features of preferred flanking sequences are not universal, as similar DNA-binding folds can employ distinct DNA recognition modes.
Nano-Tin oxide was synthesized using hydrothermal method at 150 °C for 6 h and then thin films were deposited by electrophoretic method at an optimized voltage of 100 V for 5 min on electropolished ...aluminum substrate. Spherical particles of about 30–50 nm diameters are observed with partial agglomeration when observed under electron microscope, which are tetragonal rutile structure. XPS results showed peaks related to Sn 4d, Sn 3d, O 1s & C 1s with spin–orbit splitting of 8.4 eV for Sn 3d. Feasibility studies of enzyme less urea sensing characteristics of nano-tin oxide thin films are exhibited herein. The deposited films have been used for enzyme less urea sensing from 1 to 20 mM concentration in buffer solution. The sensors were characterized electrochemically to obtain cyclic voltammogram as a function of urea concentration and scan rate. The sensitivity is estimated as 18.9 μA/mM below 5 mM and 2.31 μA/mM above 5 mM with a limit of detection of 0.6 mM.
•A simple method for synthesis of nano-tin oxide.•Enzyme less Urea sensing characteristics of synthesized material.•An attempt to design and develop enzyme less sensor and material.
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