Physiological Ion Concentrations Research Articles

Different calcium ion concentrations affect epithelial mesenchymal transformation of human peritoneal mesothelial cells via endoplasmic reticulum stress

To study the effects of different calcium ion concentrations on epithelial mesenchymal transformation (EMT) of human peritoneal mesothelial cell (HPMC) via endoplasmic reticulum stress (ERS). HPMC cell line HMrSV5 was cultured in vitro and treated in groups. The cells in the control group, high calcium group 1, and high calcium group 2 were treated with medium containing calcium ion concentrations of 1.25, 1.75, and 2.25 mmol/L, respectively. The solvent control group was treated with medium containing 1.25 mmol/L physiological calcium ion concentration and 0.1% dimethyl sulfoxide (DMSO), the high calcium+solvent group was treated with medium containing 2.25 mmol/L calcium ion concentration and 0.1% DMSO, the high calcium+4-phenylbutyric acid (4-PBA) group was treated with medium containing 2.25 mmol/L calcium ion concentration and 1 mmol/L ERS inhibitor 4-PBA, and each group was treated for 48 hours. Morphological changes of cells in each group were observed under light microscope. The expressions of epithelial cell phenotype marker zonula occluden-1 (ZO-1) and mesenchymal cell phenotype marker α-smooth muscle actin (α-SMA) in the cells were observed by immunofluorescence staining. The expressions of EMT marker genes E-cadherin, ZO-1, α-SMA and Vimentin were detected by fluorescence quantitative polymerase chain reaction (PCR). The expressions of ERS marker proteins phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), transcription activating factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by Western blotting. Compared with the control group, the morphology of HMrSV5 cells became slender and fibrotic, the fluorescence intensity of ZO-1 increased, and the fluorescence intensity of α-SMA decreased in high calcium 1 and high calcium 2 groups, indicating that the cells transformed from epithelial cells to mesenchyme cells. The mRNA expressions of E-cadherin and ZO-1 were significantly decreased, while the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly increased, moreover, the expressions of the above marker genes or proteins in the high calcium 2 group was more obvious than those in the high calcium 1 group [E-cadherin mRNA (2-ΔΔCt): 0.53±0.05 vs. 0.75±0.09, ZO-1 mRNA (2-ΔΔCt): 0.42±0.06 vs. 0.69±0.06, α-SMA mRNA (2-ΔΔCt): 1.81±0.16 vs. 1.32±0.14, Vimentin mRNA (2-ΔΔCt): 2.05±0.22 vs. 1.48±0.16, p-PERK protein (p-PERK/β-actin): 0.81±0.09 vs. 0.59±0.06, p-eIF2α protein (p-eIF2α/β-actin): 0.87±0.10 vs. 0.50±0.06, ATF4 protein (ATF4/β-actin): 0.93±0.10 vs. 0.72±0.06, CHOP protein (CHOP/β-actin): 0.79±0.09 vs. 0.46±0.04, all P < 0.05]. Compared with the solvent control group, the morphological changes of cells, the expressions of EMT marker genes and ERS marker proteins after high calcium ion concentration of 2.25 mmol/L were consistent with those in the high calcium 2 group than control group. Compared with the high calcium+solvent group, the cell morphology recovered the characteristics of polygonal and pebble-like epithelial cells in the high calcium+4-PBA group, the fluorescence intensity of ZO-1 increased, the fluorescence intensity of α-SMA decreased, and the mRNA expressions of E-cadherin and ZO-1 in the cells were significantly increased [E-cadherin mRNA (2-ΔΔCt): 0.86±0.09 vs. 0.57±0.04, ZO-1 mRNA (2-ΔΔCt): 0.81±0.06 vs. 0.48±0.05, both P < 0.05], the mRNA expressions of α-SMA and Vimentin and the protein expressions of p-PERK, p-eIF2α, ATF4 and CHOP were significantly decreased [α-SMA mRNA (2-ΔΔCt): 1.21±0.13 vs. 1.77±0.15, Vimentin mRNA (2-ΔΔCt): 1.30±0.14 vs. 1.94±0.20, p-PERK protein (p-PERK/β-actin): 0.38±0.04 vs. 0.92±0.11, p-eIF2α protein (p-eIF2α/β-actin): 0.34±0.05 vs. 1.05±0.13, ATF4 protein (ATF4/β-actin): 0.57±0.06 vs. 0.97±0.11, CHOP protein (CHOP/β-actin): 0.51±0.04 vs. 0.90±0.12, all P < 0.05]. High calcium ion concentrations of 1.75 mmol/L and 2.25 mmol/L promote EMT of HPMC via activating ERS.

A SAM analogue-utilizing ribozyme for site-specific RNA alkylation in living cells

Post-transcriptional RNA modification methods are in high demand for site-specific RNA labelling and analysis of RNA functions. In vitro-selected ribozymes are attractive tools for RNA research and have the potential to overcome some of the limitations of chemoenzymatic approaches with repurposed methyltransferases. Here we report an alkyltransferase ribozyme that uses a synthetic, stabilized S-adenosylmethionine (SAM) analogue and catalyses the transfer of a propargyl group to a specific adenosine in the target RNA. Almost quantitative conversion was achieved within 1 h under a wide range of reaction conditions in vitro, including physiological magnesium ion concentrations. A genetically encoded version of the SAM analogue-utilizing ribozyme (SAMURI) was expressed in HEK293T cells, and intracellular propargylation of the target adenosine was confirmed by specific fluorescent labelling. SAMURI is a general tool for the site-specific installation of the smallest tag for azide-alkyne click chemistry, which can be further functionalized with fluorophores, affinity tags or other functional probes.

BopN is a Gatekeeper of the Bordetella Type III Secretion System.

The classical Bordetella species infect the respiratory tract of mammals. While B. bronchiseptica causes rather chronic respiratory infections in a variety of mammals, the human-adapted species B. pertussis and B. parapertussisHU cause an acute respiratory disease known as whooping cough or pertussis. The virulence factors include a type III secretion system (T3SS) that translocates effectors BteA and BopN into host cells. However, the regulatory mechanisms underlying the secretion and translocation activity of T3SS in bordetellae are largely unknown. We have solved the crystal structure of BopN of B. pertussis and show that it is similar to the structures of gatekeepers that control access to the T3SS channel from the bacterial cytoplasm. We further found that BopN accumulates at the cell periphery at physiological concentrations of calcium ions (2 mM) that inhibit the secretion of BteA and BopN. Deletion of the bopN gene in B. bronchiseptica increased secretion of the BteA effector into calcium-rich medium but had no effect on secretion of the T3SS translocon components BopD and BopB. Moreover, the ΔbopN mutant secreted approximately 10-fold higher amounts of BteA into the medium of infected cells than the wild-type bacteria, but it translocated lower amounts of BteA into the host cell cytoplasm. These data demonstrate that BopN is a Bordetella T3SS gatekeeper required for regulated and targeted translocation of the BteA effector through the T3SS injectisome into host cells. IMPORTANCE The T3SS is utilized by many Gram-negative bacteria to deliver effector proteins from bacterial cytosol directly into infected host cell cytoplasm in a regulated and targeted manner. Pathogenic bordetellae use the T3SS to inject the BteA and BopN proteins into infected cells and upregulate the production of the anti-inflammatory cytokine interleukin-10 (IL-10) to evade host immunity. Previous studies proposed that BopN acted as an effector in host cells. In this study, we report that BopN is a T3SS gatekeeper that regulates the secretion and translocation activity of Bordetella T3SS.

Designing double-site lipidated peptide amphiphiles as potent antimicrobial biomaterials to combat multidrug-resistant bacteria.

Rapidly evolving antimicrobial resistance and extremely slow development of new antibiotics have resulted in multidrug-resistant bacterial infections that present a serious threat to human health. Antimicrobial peptides (AMPs) provide promising substitutes, but more research is needed to address several of their present limitations, such as insufficient antimicrobial potency, high toxicity, and low stability. Here, we designed a series of novel double-site lipidated peptide amphiphiles based on a heptad repeat parent pentadecapeptide. The double-site lipidated peptide amphiphiles showed a broad spectrum of antimicrobial activities. Especially the double-site lipidated peptide amphiphile WL-C6 exhibited high potency to inhibit multidrug-resistant bacteria without significant toxicity toward mammalian cells. Furthermore, even at physiological salt ion concentrations, WL-C6 still exhibited outstanding antibacterial properties, and a sizeable fraction of it maintained its molecular integrity after being incubated with different proteases. Additionally, we captured the entire process of WL-C6 killing bacteria and showed that the rapid bacterial membrane disruption is the reason of bacterial death. Overall, WL-C6 shows great promise as a substitute for conventional antibiotics to combat the growing threat of multidrug-resistant bacterial infections.

Chromatin Liquid-Liquid Phase Separation (LLPS) Is Regulated by Ionic Conditions and Fiber Length.

The dynamic regulation of the physical states of chromatin in the cell nucleus is crucial for maintaining cellular homeostasis. Chromatin can exist in solid- or liquid-like forms depending on the surrounding ions, binding proteins, post-translational modifications and many other factors. Several recent studies suggested that chromatin undergoes liquid–liquid phase separation (LLPS) in vitro and also in vivo; yet, controversial conclusions about the nature of chromatin LLPS were also observed from the in vitro studies. These inconsistencies are partially due to deviations in the in vitro buffer conditions that induce the condensation/aggregation of chromatin as well as to differences in chromatin (nucleosome array) constructs used in the studies. In this work, we present a detailed characterization of the effects of K+, Mg2+ and nucleosome fiber length on the physical state and property of reconstituted nucleosome arrays. LLPS was generally observed for shorter nucleosome arrays (15-197-601, reconstituted from 15 repeats of the Widom 601 DNA with 197 bp nucleosome repeat length) at physiological ion concentrations. In contrast, gel- or solid-like condensates were detected for the considerably longer 62-202-601 and lambda DNA (~48.5 kbp) nucleosome arrays under the same conditions. In addition, we demonstrated that the presence of reduced BSA and acetate buffer is not essential for the chromatin LLPS process. Overall, this study provides a comprehensive understanding of several factors regarding chromatin physical states and sheds light on the mechanism and biological relevance of chromatin phase separation in vivo.

Calcium supplementation of bioinks reduces shear stress-induced cell damage during bioprinting

During bioprinting, cells are suspended in a viscous bioink and extruded under pressure through small diameter printing needles. The combination of high pressure and small needle diameter exposes cells to considerable shear stress, which can lead to cell damage and death. Approaches to monitor and control shear stress-induced cell damage are currently not well established. To visualize the effects of printing-induced shear stress on plasma membrane integrity, we add FM 1-43 to the bioink, a styryl dye that becomes fluorescent when bound to lipid membranes, such as the cellular plasma membrane. Upon plasma membrane disruption, the dye enters the cell and also stains intracellular membranes. Extrusion of alginate-suspended NIH/3T3 cells through a 200 µm printing needle led to an increased FM 1-43 incorporation at high pressure, demonstrating that typical shear stresses during bioprinting can transiently damage the plasma membrane. Cell imaging in a microfluidic channel confirmed that FM 1-43 incorporation is caused by cell strain. Notably, high printing pressure also impaired cell survival in bioprinting experiments. Using cell types of different stiffnesses, we find that shear stress-induced cell strain, FM 1-43 incorporation and cell death were reduced in stiffer compared to softer cell types and demonstrate that cell damage and death correlate with shear stress-induced cell deformation. Importantly, supplementation of the suspension medium with physiological concentrations of CaCl2 greatly reduced shear stress-induced cell damage and death but not cell deformation. As the sudden influx of calcium ions is known to induce rapid cellular vesicle exocytosis and subsequent actin polymerization in the cell cortex, we hypothesize that calcium supplementation facilitates the rapid resealing of plasma membrane damage sites. We recommend that bioinks should be routinely supplemented with physiological concentrations of calcium ions to reduce shear stress-induced cell damage and death during extrusion bioprinting.

Enzymatic Approach in Calcium Phosphate Biomineralization: A Contribution to Reconcile the Physicochemical with the Physiological View.

Biomineralization is the process by which organisms produce hard inorganic matter from soft tissues with outstanding control of mineral deposition in time and space. For this purpose, organisms deploy a sophisticated “toolkit” that has resulted in significant evolutionary innovations, for which calcium phosphate (CaP) is the biomineral selected for the skeleton of vertebrates. While CaP mineral formation in aqueous media can be investigated by studying thermodynamics and kinetics of phase transitions in supersaturated solutions, biogenic mineralization requires coping with the inherent complexity of biological systems. This mainly includes compartmentalization and homeostatic processes used by organisms to regulate key physiological factors, including temperature, pH and ion concentration. A detailed analysis of the literature shows the emergence of two main views describing the mechanism of CaP biomineralization. The first one, more dedicated to the study of in vivo systems and supported by researchers in physiology, often involves matrix vesicles (MVs). The second one, more investigated by the physicochemistry community, involves collagen intrafibrillar mineralization particularly through in vitro acellular models. Herein, we show that there is an obvious need in the biological systems to control both where and when the mineral forms through an in-depth survey of the mechanism of CaP mineralization. This necessity could gather both communities of physiologists and physicochemists under a common interest for an enzymatic approach to better describe CaP biomineralization. Both homogeneous and heterogeneous enzymatic catalyses are conceivable for these systems, and a few preliminary promising results on CaP mineralization for both types of enzymatic catalysis are reported in this work. Through them, we aim to describe the relevance of our point of view and the likely findings that could be obtained when adding an enzymatic approach to the already rich and creative research field dealing with CaP mineralization. This complementary approach could lead to a better understanding of the biomineralization mechanism and inspire the biomimetic design of new materials.

Preparation of Asymmetric Vesicles with Trapped CsCl Avoids Osmotic Imbalance, Non-Physiological External Solutions, and Minimizes Leakage.

The natural asymmetry of cellular membranes influences their properties. In recent years, methodologies for preparing asymmetric vesicles have been developed that rely on cyclodextrin-catalyzed exchange of lipids between donor lipid multilamellar vesicles and acceptor lipid unilamellar vesicles, and the subsequent separation of the, now asymmetric, acceptor vesicles from the donors. Isolation is often accomplished by preloading acceptor vesicles with a high concentration of sucrose, typically 25% (w/w), and separating from donor and cyclodextrin by sucrose gradient centrifugation. We found that when the asymmetric vesicles prepared using methyl-α-cyclodextrin exchange were dispersed under hypotonic conditions using physiological salt solutions, there was enhanced leakage of an entrapped probe, 6-carboxyfluorescein. Studies with symmetric vesicles showed this was due to osmotic pressure and was specific to hypotonic solutions. Inclusion of cholesterol partly reduced leakage but did not completely eliminate it. To avoid having to use hypotonic conditions or to suspend vesicles at nonphysiological solute concentrations to minimize leakage, a method for preparing asymmetric vesicles using acceptor vesicle-entrapped CsCl at a physiological ion concentration (100 mM) was developed. Asymmetric vesicles prepared with the entrapped CsCl protocol were highly resistant to 6-carboxyfluorescein leakage out of the vesicles.

Self-Assembling Peptide Dendron Nanoparticles with High Stability and a Multimodal Antimicrobial Mechanism of Action.

Self-assembling nanometer-scale structured peptide polymers and peptide dendrimers have shown promise in biomedical applications due to their versatile properties and easy availability. Herein, self-assembling peptide dendron nanoparticles (SPDNs) with potent antimicrobial activity against a range of bacteria were developed based on the nanoscale self-assembly of an arginine-proline repeat branched peptide dendron bearing a hexadecanoic acid chain. The SPDNs are biocompatible, and our most active peptide dendron nanoparticle, C16-3RP, was found to have negligible toxicity after both in vitro and in vivo studies. Furthermore, the C16-3RP nanoparticles showed excellent stability under physiological concentrations of salt ions and against serum and protease degradation, resulting in highly effective treatment in a mouse acute peritonitis model. Comprehensive analyses using a series of biofluorescence, microscopy, and transcriptome sequencing techniques revealed that C16-3RP nanoparticles kill Gram-negative bacteria by increasing bacterial membrane permeability, inducing cytoplasmic membrane depolarization and drastic membrane disruption, inhibiting ribosome biogenesis, and influencing energy generation and other processes. Collectively, C16-3RP nanoparticles show promising biocompatibility and in vivo therapeutic efficacy without apparent resistance development. These advancements may facilitate the development of peptide-based antibiotics in clinical settings.

Chelated Magnesium Logic Gate Regulates Riboswitch Pseudoknot Formation.

Magnesium plays a critical role in the structure, dynamics, and function of RNA. The precise microscopic effect of chelated magnesium on RNA structure is yet to be explored. Magnesium is known to act through its diffuse cloud around RNA, through the outer sphere (water-mediated), inner sphere, and often chelated ion-mediated interactions. A mechanism is proposed for the role of experimentally discovered site-specific chelated magnesium ions on the conformational dynamics of SAM-I riboswitch aptamers in bacteria. This mechanism is observed with atomistic simulations performed in a physiological mixed salt environment at a high temperature. The simulations were validated with phosphorothioate interference mapping experiments that help to identify crucial inner-sphere Mg2+ sites prescribing an appropriate initial distribution of inner- and outer-sphere magnesium ions to maintain a physiological ion concentration of monovalent and divalent salts. A concerted role of two chelated magnesium ions is newly discovered since the presence of both supports the formation of the pseudoknot. This constitutes a logical AND gate. The absence of any of these magnesium ions instigates the dissociation of long-range pseudoknot interaction exposing the inner core of the RNA. A base triple is the epicenter of the magnesium chelation effect. It allosterically controls RNA pseudoknot by bolstering the direct effect of magnesium chelation in protecting the functional fold of RNA to control ON and OFF transcription switching.

Ionic Environment Affects Biomolecular Interactions of Amyloid-β: SPR Biosensor Study.

In early stages of Alzheimer’s disease (AD), amyloid beta (Aβ) accumulates in the mitochondrial matrix and interacts with mitochondrial proteins, such as cyclophilin D (cypD) and 17β-hydroxysteroid dehydrogenase 10 (17β-HSD10). Multiple processes associated with AD such as increased production or oligomerization of Aβ affect these interactions and disbalance the equilibrium between the biomolecules, which contributes to mitochondrial dysfunction. Here, we investigate the effect of the ionic environment on the interactions of Aβ (Aβ1–40, Aβ1–42) with cypD and 17β-HSD10 using a surface plasmon resonance (SPR) biosensor. We show that changes in concentrations of K+ and Mg2+ significantly affect the interactions and may increase the binding efficiency between the biomolecules by up to 35% and 65% for the interactions with Aβ1–40 and Aβ1–42, respectively, in comparison with the physiological state. We also demonstrate that while the binding of Aβ1–40 to cypD and 17β-HSD10 takes place preferentially around the physiological concentrations of ions, decreased concentrations of K+ and increased concentrations of Mg2+ promote the interaction of both mitochondrial proteins with Aβ1–42. These results suggest that the ionic environment represents an important factor that should be considered in the investigation of biomolecular interactions taking place in the mitochondrial matrix under physiological as well as AD-associated conditions.

Novel degradation flow-through chamber for in vitro biomaterial characterization.

The characterization of degradation of biodegradable materials for tissue regeneration is classically carried out in three steps: in vitro degradation analysis, in vitro cell culture, and in vivo animal experiments. Each step involves an increasing complexity and should serve a more sophisticated material selection, which serves as an orientation to clinical studies and the final application in patients. Recently, the usefulness of degradation analyses is being discussed. In this context, the aim of this work is to increase the importance of in vitro degradation analysis by using flowing media to move closer to the in vivo situation. In the long term, this should lead to a more sensitive biomaterial characterization as well as to a replacement of time-consuming static or quasi-dynamic incubation experiments. The practicability of the novel chamber is demonstrated in context of a degradation study of silica/collagen/calcium phosphate composites in flowing media with physiological (2.4 mM) and lowered (0.5 mM) calcium ion concentrations. This is done by comparison with static and quasi-dynamic incubation experiments. In order to keep all media regimes comparable to each other, for the dynamic experiment, a flow rate was chosen equivalent to the medium exchange in quasi-dynamic incubation. Under flow-through conditions, there is a clearly decreased tendency to lower the calcium concentration, so that a concentration close to the physiological initial situation can be continuously maintained.

Differences in relative air humidity affect responses to soil salinity in freshwater and salt marsh populations of the dominant grass species Phragmites australis

Soil salinity diminishes the dominance of species and affects their distribution. Phragmites australis is a dominant ecosystem engineer with broad distribution, high intraspecific variation and great socio-economic importance. Coastal ecosystems inhabited by P. australis are threatened by salinization. Here, we investigated salinity tolerance of freshwater and salt marsh population of the species, grown under two soil salinities and in two common gardens. Salinity significantly affected the growth, biomass, leaf physiological parameters and ion concentration. Climate altered morphology and ion concentration of P. australis. In contrast to our hypothesis, the salt marsh population was not more salt tolerant than the freshwater population, and both showed a low degree of salt stress, maintaining their photosynthesis and chlorophyll concentration, and only showing small decreases in biomass and height when salt-treated. We therefore ruled out local adaptation to soil salinity. Instead, both populations acclimated by phenotypic plasticity of biomass, root: shoot ratio, stomatal conductance and ion content. The salinity tolerance strategy of both populations was ion concentration by tissue desiccation, which was most efficient in the drier climate common garden. Hence, plants utilizing tissue desiccation as salinity tolerance strategy will have an advantage in areas where climate change leads to drier air humidity.

Regulation of Protein Kinase R by Epstein-Barr Virus EBER1 RNA.

Protein kinase R (PKR) is a key antiviral component of the innate immune pathway and is activated by viral double-stranded RNAs (dsRNAs). Adenovirus-associated RNA 1 (VAI) is an abundant, noncoding viral RNA that functions as a decoy by binding PKR but not inducing activation, thereby inhibiting the antiviral response. In VAI, coaxial stacking produces an extended helix that mediates high-affinity PKR binding but is too short to result in activation. Like adenovirus, Epstein-Barr virus produces high concentrations of a noncoding RNA, EBER1. Here, we compare interactions of PKR with VAI and EBER1 and present a structural model of EBER1. Both RNAs function as inhibitors of dsRNA-mediated PKR activation. However, EBER1 weakly activates PKR whereas VAI does not. PKR binds EBER1 more weakly than VAI. Assays at physiological ion concentrations indicate that both RNAs can accommodate two PKR monomers and induce PKR dimerization. A structural model of EBER1 was obtained using constraints derived from chemical structure probing and small-angle X-ray scattering experiments. The central stem of EBER1 coaxially stacks with stem loop 4 and stem loop 1 to form an extended RNA duplex of ∼32 bp that binds PKR and promotes activation. Our observations that EBER1 binds PKR much more weakly than VAI and exhibits weak PKR activation suggest that EBER1 is less well suited to function as an RNA decoy.

Resveratrol Directly Controls the Activity of Neuronal Ryanodine Receptors at the Single-Channel Level

Calcium ion dyshomeostasis contributes to the progression of many neurodegenerative diseases and represents a target for the development of neuroprotective therapies, as reported by Duncan et al. (Molecules 15(3):1168-95, 2010), LaFerla (Nat Rev Neurosci 3(11):862-72, 2002), and Niittykoshi et al. (Invest Ophthalmol Vis Sci 51(12):6387-93, 2010). Dysfunctional ryanodine receptors contribute to calcium ion dyshomeostasis and potentially to the pathogenesis of neurodegenerative diseases by generating abnormal calcium ion release from the endoplasmic reticulum, according to Bruno et al. (Neurobiol Aging 33(5):1001 e1-6, 2012) and Stutzmann et al. (J Neurosci 24(2):508-13, 2004). Since ryanodine receptors share functional and structural similarities with potassium channels, as reported by Lanner et al. (Cold Spring Harb Perspect Biol 2(11):a003996, 2010), and small molecules with anti-oxidant properties, such as resveratrol (3,5,4'-trihydroxy-trans-stilbene), directly control the activity of potassium channels, according to Wang et al. (J Biomed Sci 23(1):47, 2016), McCalley et al. (Molecules 19(6):7327-40, 2014), Novakovic et al. (Mol Hum Reprod 21(6):545-51, 2015), Li et al. (Cardiovasc Res 45(4):1035-45, 2000), Gopalakrishnan et al. (Br J Pharmacol 129(7):1323-32, 2000), and Hambrock et al. (J Biol Chem 282(5):3347-56, 2007), we hypothesized that trans-resveratrol can modulate intracellular calcium signaling through direct binding and functional regulation of ryanodine receptors. The goal of our study was to identify and measure the control of ryanodine receptor activity by trans-resveratrol. Mechanisms of calcium signaling mediated by the direct interaction between trans-resveratrol and ryanodine receptors were identified and measured with single-channel electrophysiology. Addition of trans-resveratrol to the cytoplasmic face of the ryanodine receptor increased single-channel activity at physiological and elevated pathophysiological cytoplasmic calcium ion concentrations. The open probability of the channel increases after interacting with the small molecule in a dose-dependent manner, but remains also dependent on the concentration of its physiological ligand, cytoplasmic-free calcium ions. This study provides the first evidence of a direct functional interaction between trans-resveratrol and ryanodine receptors. Such functional control of ryanodine receptors by trans-resveratrol as a novel mechanism of action could provide additional rationales for the development of novel therapeutic strategies to treat and prevent neurodegenerative diseases.

Binding of tetracycline to its aptamer determined by 2D-correlated Mn2+ hyperfine spectroscopy.

The tetracycline-binding RNA aptamer (TC-aptamer) binds its cognate ligand the antibiotic tetracycline (TC) via a Mg2+ or Mn2+ ion with high affinity at high divalent metal ion concentrations (KD=800pM, ⩾10 mM). These concentrations lie above the physiological divalent metal ion concentration of ca. 1 mM and it is known from literature, that the binding affinity decreases upon decreasing the divalent metal ion concentration. This work uses a Mn2+ concentration of 1 mM and 1D-hyperfine experiments reveal two pronounced 31P couplings from the RNA besides the 13C signal of 13C-labeled TC. From these 1D-hyperfine data alone, however, no conclusions can be drawn on the binding of TC. Either TC may bind via Mn2+ to the aptamer or TC may form a free Mn-TC complex and some Mn2+ also binds to the aptamer. In this work, we show using 2D-correlated hyperfine spectroscopy at Q-band frequencies (34 GHz), that the 13C and 31P signals can be correlated; thus arising from a single species. We use THYCOS (triple hyperfine correlation spectroscopy) and 2D ELDOR-detected NMR (2D electron electron double resonance detected NMR) for this purpose showing that they are suitable techniques to correlate two different nuclear spin species (13C and 31P) on two different molecules (RNA and TC) to the same electron spin (Mn2+). Out of the two observed 31P-hyperfine couplings, only one shows a clear correlation to 13C. Although THYCOS and 2D EDNMR yield identical results, 2D EDNMR is far more sensitive. THYCOS spectra needed a time factor of ×20 in comparison to 2D EDNMR to achieve a comparable signal-to-noise.

A potent antibacterial activity of new short d-enantiomeric lipopeptide against multi drug resistant bacteria

The emergence of drug-resistant pathogenic bacteria threatens human health. Resistance to existing antibiotics is increasing, while the emergence of new antibiotics is slowing. Cationic antimicrobial peptides (CAMPs) are fascinating alternative antibiotics because they possess a broad spectrum of activity, being active against both Gram-positive and Gram-negative bacteria including those resistant to traditional antibiotics. However, low bioavailability resulting from enzymatic degradation and attenuation by divalent cations like Mg2+ and Ca2+ limits their use as antibiotic agents. Here, we report the design of new CAMPs showing both high antibacterial activity and serum stability under physiological ion concentrations. The peptides were designed by applying two approaches, the use of d-enantiomer and lipidation. Based on the sequence of the CopW (LLWIALRKK-NH2), a nonapeptide derived from coprisin, a series of novel d-form CopW lipopeptides with different acyl chain lengths (C6, C8, C10, C12, C14, and C16) were synthesized and evaluated with respect to their activity and salt sensitivity. Among the analogs, the d-form lipopeptide dCopW3 exhibited MIC values ranging from 1.25 to 5 μM against multidrug-resistant bacteria. Significantly, this compound did not induce bacterial resistance and was highly stable in human serum proteases. The results emphasize the potential of cationic d-form lipopeptide as therapeutically valuable antibiotics for treating drug-resistant bacterial infections.

Single Step Determination of Unlabeled Compound Kinetics Using a Competition Association Binding Method Employing Time-Resolved FRET.

The competition association binding method allows the characterization of the kinetics of unlabeled compounds and the calculation of receptor-drug affinity (K d). The K d value is defined as the ratio of the dissociation constant (or k off) of the receptor-bound ligand to its association rate constant (or k on) for a system at equilibrium. Traditionally, competition association binding experiments have been carried out using radiometric detection methods with limited assay throughput. Here we describe a novel method for the determination of unlabeled compound kinetics using the technique of time-resolved fluorescence resonance energy transfer (TR-FRET) performed at physiological temperature and sodium ion concentration. Based on a traditional screening format (10-point curves), up to 28 compounds can be tested on a single 384-well plate by this method.

Disordered N-Terminal Domain of Human Uracil DNA Glycosylase (hUNG2) Enhances DNA Translocation

Nuclear human uracil-DNA glycosylase (hUNG2) initiates base excision repair (BER) of genomic uracils generated through misincorporation of dUMP or through deamination of cytosines. Like many human DNA glycosylases, hUNG2 contains an unstructured N-terminal domain that encodes a nuclear localization signal, protein binding motifs, and sites for post-translational modifications. Although the N-terminal domain has minimal effects on DNA binding and uracil excision kinetics, we report that this domain enhances the ability of hUNG2 to translocate on DNA chains as compared to the catalytic domain alone. The enhancement is most pronounced when physiological ion concentrations and macromolecular crowding agents are used. These data suggest that crowded conditions in the human cell nucleus promote the interaction of the N-terminus with duplex DNA during translocation. The increased contact time with the DNA chain likely contributes to the ability of hUNG2 to locate densely spaced uracils that arise during somatic hypermutation and during fluoropyrimidine chemotherapy.

Dynamic distinctions in the Na+/Ca2+ exchanger adopting the inward- and outward-facing conformational states

Na+/Ca2+ exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na+ or Ca2+ specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.