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FASEB Journal : Official Publication of... Jul 2023Leucine-rich repeat containing 8A (LRRC8A) volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis...
Leucine-rich repeat containing 8A (LRRC8A) volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A associates with NADPH oxidase 1 (Nox1) and supports extracellular superoxide production. We tested the hypothesis that VRACs modulate TNFα signaling and vasomotor function in mice lacking LRRC8A exclusively in vascular smooth muscle cells (VSMCs, Sm22α-Cre, Knockout). Knockout (KO) mesenteric vessels contracted normally but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). Forty-eight hours of ex vivo exposure to TNFα (10 ng/mL) enhanced contraction to norepinephrine (NE) and markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 μM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 33 proteins that interacted with LRRC8A. Among them, the myosin phosphatase rho-interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots. siLRRC8A or CBX treatment decreased RhoA activity in VSMCs, and MYPT1 phosphorylation was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure. Interaction of LRRC8A with MPRIP may allow redox regulation of the cytoskeleton by linking Nox1 activation to impaired vasodilation. This identifies VRACs as potential targets for treatment or prevention of vascular disease.
Topics: Animals; Mice; Acetylcholine; Anions; Membrane Proteins; Mice, Knockout; Myosin-Light-Chain Phosphatase; Signal Transduction; Tumor Necrosis Factor-alpha; Muscle, Smooth, Vascular
PubMed: 37310356
DOI: 10.1096/fj.202300561R -
Chemical Record (New York, N.Y.) Aug 2023Bio-compatible ionic liquids (Bio-ILs) represent a class of solvents with peculiar properties and exhibit huge potential for their applications in different fields of... (Review)
Review
Bio-compatible ionic liquids (Bio-ILs) represent a class of solvents with peculiar properties and exhibit huge potential for their applications in different fields of chemistry. Ever since they were discovered, researchers have used bio-ILs in diverse fields such as biomass dissolution, CO sequestration, and biodegradation of pesticides. This review highlights the ongoing research studies focused on elucidating the microscopic structure of bio-ILs based on cholinium cation ([Ch] ) and amino acid ([AA] ) anions using the state-of-the-art and classical molecular dynamics (MD) simulations. The microscopic structure associated with these green ILs guides their suitability for specific applications. ILs of this class differ in the side chain of the amino acid anions, and varying the side chain significantly affects the structure of these ILs and thus helps in tuning the efficiency of biomass dissolution. This review demonstrates the central role of the side chain on the morphology of choline amino acid ([Ch][AA]) bio-ILs. The seemingly matured field of bio-ILs and their employment in various applications still holds significant potential, and the insights on their microscopic structure would steer the field of target specific application of these green ILs.
Topics: Amino Acids; Ionic Liquids; Choline; Molecular Dynamics Simulation; Anions
PubMed: 36960931
DOI: 10.1002/tcr.202200295 -
Journal of the American Society For... Aug 2023Peptide identification by positive electrospray ionization (ES+) tandem mass spectrometry (MS/MS) is a well-established strategy in proteomics. Several research groups...
Peptide identification by positive electrospray ionization (ES+) tandem mass spectrometry (MS/MS) is a well-established strategy in proteomics. Several research groups reported the usefulness of negative electrospray ionization (ES-) for gaining complementary structural information on peptides and their post-translational modifications (PTM) compared to ES+. Fragmentation of citrullinated peptides has not been previously explored in ES-. In this study, 9 peptides containing citrulline residues were investigated in ES- by stepwise collision energy-dependent measurements on a QTOF instrument and a Q-Orbitrap instrument. Our results of high resolution and mass accuracy show the favored citrulline-selective loss of HNCO from these peptide precursors and their fragments─similarly to that in ES+─along with y-NH/z, c, c-NH/b sequence ions. Loss of HNCO from citrullinated peptides in ES- and a proposed mechanism for the reaction have been described here for the first time. HNCO loss intensities from precursors were generally even higher than that in ES+. Interestingly, the most intense fragments corresponded to neutral losses from sequence ions while intact sequence ions were usually minor components of the spectra. High-intensity ions related to cleavages -terminal to Asp and Glu residues that have been previously reported were also observed. On the other hand, a relatively high number of peaks were observed, possibly due to internal fragmentation and/or scrambling events. While (ES-) MS/MS spectra always require manual inspection and the annotation may be ambiguous, the favorable loss of HNCO and the preferable cleavage -terminal to Asp residues can be used to differentiate between citrullinated/deamidated sequences.
Topics: Tandem Mass Spectrometry; Citrulline; Spectrometry, Mass, Electrospray Ionization; Peptides; Anions; Ions
PubMed: 37414397
DOI: 10.1021/jasms.3c00044 -
International Journal of Molecular... Oct 2023Antimicrobial resistance is a silent pandemic considered a public health concern worldwide. Strategic therapies are needed to replace antibacterials that are now...
Antimicrobial resistance is a silent pandemic considered a public health concern worldwide. Strategic therapies are needed to replace antibacterials that are now ineffective. One approach entails the use of well-known antibacterials along with adjuvants that possess non-antibiotic properties but can extend the lifespan and enhance the effectiveness of the treatment, while also improving the suppression of resistance. In this regard, a group of uniform materials based on organic salts (GUMBOS) presents an alternative to this problem allowing the combination of antibacterials with adjuvants. Fluoroquinolones are a family of antibacterials used to treat respiratory and urinary tract infections with broad-spectrum activity. Ciprofloxacin and moxifloxacin-based GUMBOS were synthesized via anion exchange reactions with lithium and sodium salts. Structural characterization, thermal stability and octanol/water partition ratios were evaluated. The antibacterial profiles of most GUMBOS were comparable to their cationic counterparts when tested against Gram-positive and Gram-negative , except for deoxycholate anion, which demonstrated the least effective antibacterial activity. Additionally, some GUMBOS were less cytotoxic to L929 fibroblast cells and non-hemolytic to red blood cells. Therefore, these agents exhibit promise as an alternative approach to combining drugs for treating infections caused by resistant bacteria.
Topics: Fluoroquinolones; Salts; Escherichia coli; Staphylococcus aureus; Anti-Bacterial Agents; Anions; Microbial Sensitivity Tests
PubMed: 37958698
DOI: 10.3390/ijms242115714 -
International Journal of Molecular... Sep 2023Studies in human colonic cell lines and murine intestine suggest the presence of a Ca-activated anion channel, presumably TMEM16a. Is there a potential for fluid...
The Anion Channel TMEM16a/Ano1 Modulates CFTR Activity, but Does Not Function as an Apical Anion Channel in Colonic Epithelium from Cystic Fibrosis Patients and Healthy Individuals.
Studies in human colonic cell lines and murine intestine suggest the presence of a Ca-activated anion channel, presumably TMEM16a. Is there a potential for fluid secretion in patients with severe cystic fibrosis transmembrane conductance regulator () mutations by activating this alternative pathway? Two-dimensional nondifferentiated colonoid-myofibroblast cocultures resembling transit amplifying/progenitor (TA/PE) cells, as well as differentiated monolayer (DM) cultures resembling near-surface cells, were established from both healthy controls (HLs) and patients with severe functional defects in the gene (PwCF). F508del mutant and CFTR knockout (null) mice ileal and colonic mucosa was also studied. HL TA/PE monolayers displayed a robust short-circuit current response (ΔI) to UTP (100 µM), forskolin (Fsk, 10 µM) and carbachol (CCH, 100 µM), while ΔI was much smaller in differentiated monolayers. The selective TMEM16a inhibitor Ani9 (up to 30 µM) did not alter the response to luminal UTP, significantly decreased Fsk-induced ΔI, and significantly increased CCH-induced ΔI in HL TA/PE colonoid monolayers. The PwCF TA/PE and the PwCF differentiated monolayers displayed negligible agonist-induced ΔI, without a significant effect of Ani9. When TMEM16a was localized in intracellular structures, a staining in the apical membrane was not detected. TMEM16a is highly expressed in human colonoid monolayers resembling transit amplifying cells of the colonic cryptal neck zone, from both HL and PwCF. While it may play a role in modulating agonist-induced CFTR-mediated anion currents, it is not localized in the apical membrane, and it has no function as an apical anion channel in cystic fibrosis (CF) and healthy human colonic epithelium.
Topics: Animals; Humans; Mice; Anions; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelium; Uridine Triphosphate
PubMed: 37762516
DOI: 10.3390/ijms241814214 -
Nature Communications Nov 2023The anion channel SLAC1 functions as a crucial effector in the ABA signaling, leading to stomata closure. SLAC1 is activated by phosphorylation in its intracellular...
The anion channel SLAC1 functions as a crucial effector in the ABA signaling, leading to stomata closure. SLAC1 is activated by phosphorylation in its intracellular domains. Both a binding-activation model and an inhibition-release model for activation have been proposed based on only the closed structures of SLAC1, rendering the structure-based activation mechanism controversial. Here we report cryo-EM structures of Arabidopsis SLAC1 WT and its phosphomimetic mutants in open and closed states. Comparison of the open structure with the closed ones reveals the structural basis for opening of the conductance pore. Multiple phosphorylation of an intracellular domain (ICD) causes dissociation of ICD from the transmembrane domain. A conserved, positively-charged sequence motif in the intracellular loop 2 (ICL2) seems to be capable of sensing of the negatively charged phosphorylated ICD. Interactions between ICL2 and ICD drive drastic conformational changes, thereby widening the pore. From our results we propose that SLAC1 operates by a mechanism combining the binding-activation and inhibition-release models.
Topics: Arabidopsis; Arabidopsis Proteins; Cryoelectron Microscopy; Abscisic Acid; Plant Stomata; Membrane Proteins; Anions
PubMed: 37963863
DOI: 10.1038/s41467-023-43193-3 -
Environmental Research Dec 2023Layered double hydroxides (LDHs) are well-known and important class of hydrotalcite-type anionic clays (HTs) materials that are cost-effective with additional advantages... (Review)
Review
Layered double hydroxides (LDHs) are well-known and important class of hydrotalcite-type anionic clays (HTs) materials that are cost-effective with additional advantages of facile synthesis, composition, tenability, and reusability. These convincing characteristics are liable for their applications in various fields related to energy, environment, catalysis, biomedical, and biotechnology. HTs/LDHs are generally synthesized from low cost abundantly available chemical precursors through the aqueous synthetic pathways under mild reaction conditions. These materials can be termed green materials based on their non-toxic nature, availability of precursors, facile and low-cost production using aqueous medium conditions with less hazardous effluents. Diverse and fascinating characteristics have been attributed to HTs/LDHs like anion exchange ability, surface basicity, biocompatibility, controlled release of the anion specific area, porosity, easy surface modification, and pH dependent biodegradability. Hence, HTs/LDHs and their modified and/or functionalized nanohybrids/nanocomposites are reported as the potential drug delivery carriers with a capability to stabilize the susceptible bioactive molecules, may enhance the solubility of poorly soluble drugs along with controlled drug/bioactive molecule release and delivery. These clay and bioactive hybrid materials have good biocompatibility, less cytotoxicity, and better site-targeting with improved cellular uptake than that of free parent biomolecules. These lamellar solids of micro/nanostructure are compatible, host-guest materials and able to fabricate with drugs/cosmeceutical/bio- or synthetic polymers without any change in their molecular structure and reactivity along with improvement in their stabilities. Other important features are facile synthesis, basicity, high stability with easy storage, and efficient administration with low bio-toxicity. This study enlightens the applications of HTs/LDHs along with their hybrids/composites in the field of drug/cosmeceutical/gene delivery systems of natural/synthetic biomolecules.
Topics: Synthetic Drugs; Cosmeceuticals; Hydroxides; Water; Nanocomposites
PubMed: 37734578
DOI: 10.1016/j.envres.2023.117171 -
Science Advances Aug 2023DNA computing harnesses the immense potential of DNA molecules to enable sophisticated and transformative computational processes but is hindered by low computing speed....
DNA computing harnesses the immense potential of DNA molecules to enable sophisticated and transformative computational processes but is hindered by low computing speed. Here, we propose freeze-thaw cycling as a simple yet powerful method for high-speed DNA computing without complex procedures. Through iterative cycles, we achieve a substantial 20-fold speed enhancement in basic strand displacement reactions. This acceleration arises from the utilization of eutectic ice phase as a medium, temporarily increasing the effective local concentration of molecules during each cycle. In addition, the acceleration effect follows the Hofmeister series, where kosmotropic anions such as sulfate (SO) reduce eutectic phase volume, leading to a more notable enhancement in strand displacement reaction rates. Leveraging this phenomenon, freeze-thaw cycling demonstrates its generalizability for high-speed DNA computing across various circuit sizes, achieving up to a remarkable 120-fold enhancement in reaction rates. We envision its potential to revolutionize molecular computing and expand computational applications in diverse fields.
Topics: Acceleration; DNA; Recombination, Genetic; Sulfates
PubMed: 37624882
DOI: 10.1126/sciadv.aax7983 -
International Journal of Molecular... Mar 2024The linear undecapeptide KKLFKKILKYL-NH (BP100) highlights for its antibacterial activity against Gram-negative bacteria and its low toxicity. These excellent biological... (Review)
Review
The linear undecapeptide KKLFKKILKYL-NH (BP100) highlights for its antibacterial activity against Gram-negative bacteria and its low toxicity. These excellent biological properties prompted the investigation of its mechanism of action, which were undertaken using spectroscopic techniques, biophysical analysis, microscopy, and molecular dynamic simulations. Studies were conducted in different membrane environments, such as anionic, zwitterionic, and mixed membranes, as well as in vesicles (LUVs and GUVs) and bacteria. The findings suggest that BP100 exhibits a preference for anionic membranes, and its mechanism of action involves charge neutralization and membrane permeabilization. In these membranes, BP100 transitions from an unstructured state in water to an α-helix with the axis parallel to the surface. MD simulations suggest that after electrostatic interaction with the membrane, BP100 flips, facilitating the insertion of its hydrophobic face into the membrane bilayer. Thus, BP100 adopts an almost vertical transmembrane orientation with lysine side chains snorkelling on both sides of the membrane. As a result of the rotation, BP100 induces membrane thinning and slow lipid diffusion and promotes water penetration, particularly in anionic lipid membranes. These investigations pointed towards a carpet-like mechanism and are aligned with the biological activity profile described for BP100. This review covers all the studies carried out on the mechanism of action of BP100 published between 2009 and 2023.
Topics: Lipid Bilayers; Antimicrobial Peptides; Oligopeptides; Anti-Bacterial Agents; Water
PubMed: 38542427
DOI: 10.3390/ijms25063456 -
Organic & Biomolecular Chemistry Jul 2023The synthesis, acid-base behaviour and anion recognition of neurotransmitters (dopamine, tyramine and serotonin) in aqueous solution of different aza-scorpiand ligands...
The synthesis, acid-base behaviour and anion recognition of neurotransmitters (dopamine, tyramine and serotonin) in aqueous solution of different aza-scorpiand ligands functionalized with hydroxyphenyl and phenyl moieties (L1-L3 and L4, respectively) have been studied by potentiometry, NMR, UV-Vis and fluorescence spectroscopy and isothermal titration calorimetry (ITC). The analysis of the potentiometric results shows the selective recognition of serotonin at physiological pH ( = 8.64 × 10) by L1. This selectivity has an entropic origin probably coming from a fine pre-organization of the interacting partners. Thus, the complementarity of the receptor and the substrate allows the reciprocal formation of hydrogen bonds, π-π and cation-π interactions, stabilizing the receptors and slowing the rate of oxidative degradation, and satisfactory results are obtained at acidic and neutral pH values. NMR and molecular dynamics studies reveal the rotation blockage in the neurotransmitter side chain once complexed with L1.
Topics: Ligands; Serotonin; Water; Anions; Oxidation-Reduction
PubMed: 37335019
DOI: 10.1039/d3ob00562c