Sensitive Detection and Cell Imaging of Ca2+ Based on a "Turn-On" Schiff Base Fluorescent Probe.

Ca2+ ion as a second messenger in signaling pathway plays many vital roles in many biological phenomena. Thus, it is of significance for developing effective probes to detect Ca2+ ion specifically. Herein, a new Schiff base fluorescent probe FPH, fluorescein monoaldehyde (2-aminomethylpyridine) hydrazone, was designed and synthesized to identify Ca2+ in DMSO aqueous solution. The probe FPH revealed significant responses to Ca2+ with a fluorescence enhancement at 540 nm, exhibiting an evident fluorescence change from ultraweak luminescence to bright green. Otherwise, the FPH displayed a good linear range of 0.67 × 10-6 to 3.33 × 10-6 mol/L with a lower detection limit at 7.02 × 10-8 mol/L. The probe FPH were further successfully utilized to detect Ca2+ in living cells by an increased bright green fluorescence.

Thermal stability of bivalent cation/phosphoinositide domains in model membranes.

As key mediators in a wide array of signaling events, phosphoinositides (PIPs) orchestrate the recruitment of proteins to specific cellular locations at precise moments. This intricate spatiotemporal regulation of protein activity often necessitates the localized enrichment of the corresponding PIP. We investigate the extent and thermal stabilities of phosphatidylinositol-4-phosphate (PI(4)P), phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2 and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) clusters with calcium and magnesium ions. We observe negligible or minimal clustering of all examined PIPs in the presence of Mg2+ ions. While PI(4)P shows in the presence of Ca2+ no clustering, PI(4,5)P2 forms with Ca2+ strong clusters that exhibit stablity up to at least 80°C. The extent of cluster formation for the interaction of PI(3,4,5)P3 with Ca2+ is less than what was observed for PI(4,5)P2, yet we still observe some clustering up to 80°C. Given that cholesterol has been demonstrated to enhance PIP clustering, we examined whether bivalent cations and cholesterol synergistically promote PIP clustering. We found that the interaction of Mg2+ or Ca2+ with PI(4)P remains extraordinarily weak, even in the presence of cholesterol. In contrast, we observe synergistic interaction of cholesterol and Ca2+ with PI(4,5)P2. Also, in the presence of cholesterol, the interaction of Mg2+ with PI(4,5)P2 remains weak. PI(3,4,5)P3 does not show strong clustering with cholesterol for the experimental conditions of our study and the interaction with Ca2+ and Mg2+ was not influenced by the presence of cholesterol.

Assembly and catalytic activity of short prion-inspired peptides.

Enzymes play a crucial role in biochemical reactions, but their inherent structural instability limits their performance in industrial processes. In contrast, amyloid structures, known for their exceptional stability, are emerging as promising candidates for synthetic catalysis. This article explores the development of metal-decorated nanozymes formed by short peptides, inspired by prion-like domains. We detail the rational design of synthetic short Tyrosine-rich peptide sequences, focusing on their self-assembly into stable amyloid structures and their metallization with biologically relevant divalent metal cations, such as Cu2+, Ni2+, Co2+ and Zn2+. The provided experimental framework offers a step-by-step guide for researchers interested in exploring the catalytic potential of metal-decorated peptides. By bridging the gap between amyloid structures and catalytic function, these hybrid molecules open new avenues for developing novel metalloenzymes with potential applications in diverse chemical reactions.

Abundant tannic acid modified gelatin/sodium alginate biocomposite hydrogels with high toughness, antifreezing, antioxidant and antibacterial properties.

The acidity of high tannic acid (TA) content solution can destroy the structure of protein, such as gelatin (G). This causes a big challenge to introduce abundant TA into the G-based hydrogels. Here, the G-based hydrogel system with abundant TA as hydrogen bonds provider was constructed by a "protective film" strategy. The protective film around the composite hydrogel was first formed by the chelation of sodium alginate (SA) and Ca2+. Subsequently, abundant TA and Ca2+ were successively introduced into the hydrogel system by immersing method. This strategy effectively protected the structure of the designed hydrogel. After treatment with 0.3 w/v TA and 0.06 w/v Ca2+ solutions, the tensile modulus, elongation at break and toughness of G/SA hydrogel increased about 4-, 2-, and 6-fold, respectively. Besides, G/SA-TA/Ca2+ hydrogels exhibited good water retention, anti-freezing, antioxidant, antibacterial properties and low hemolysis ratio. Cell experiments showed that G/SA-TA/Ca2+ hydrogels possessed good biocompatibility and could promote cell migration. Therefore, G/SA-TA/Ca2+ hydrogels are expected to be used in the field of biomedical engineering. The strategy proposed in this work also provides a new idea for improving the properties of other protein-based hydrogels.

Recent Advances in the Structural Biology of Mg2+ Channels and Transporters.

Magnesium ions (Mg2+) are the most abundant divalent cations in living organisms and are essential for various physiological processes, including ATP utilization and the catalytic activity of numerous enzymes. Therefore, the homeostatic mechanisms associated with cellular Mg2+ are crucial for both eukaryotic and prokaryotic organisms and are thus strictly controlled by Mg2+ channels and transporters. Technological advances in structural biology, such as the expression screening of membrane proteins, in meso phase crystallization, and recent cryo-EM techniques, have enabled the structure determination of numerous Mg2+ channels and transporters. In this review article, we provide an overview of the families of Mg2+ channels and transporters (MgtE/SLC41, TRPM6/7, CorA/Mrs2, CorC/CNNM), and discuss the structural biology prospects based on the known structures of MgtE, TRPM7, CorA and CorC.

Ion-dependent slow protein release from in vivo disintegrating micro-granules.

Through the controlled addition of divalent cations, polyhistidine-tagged proteins can be clustered in form of chemically pure and mechanically stable micron-scale particles. Under physiological conditions, these materials act as self-disintegrating protein depots for the progressive release of the forming polypeptide, with potential applications in protein drug delivery, diagnosis, or theragnosis. Here we have explored the in vivo disintegration pattern of a set of such depots, upon subcutaneous administration in mice. These microparticles were fabricated with cationic forms of either Zn, Ca, Mg, or Mn, which abound in the mammalian body. By using a CXCR4-targeted fluorescent protein as a reporter building block we categorized those cations regarding their ability to persist in the administration site and to sustain a slow release of functional protein. Ca2+ and specially Zn2+ have been observed as particularly good promoters of time-prolonged protein leakage. The released polypeptides result is available for selective molecular interactions, such as specific fluorescent labeling of tumor tissues, in which the protein reaches nearly steady levels.

Effect of different sample treatment methods on Low Endotoxin Recovery Phenomenon.

Endotoxin is a kind of lipopolysaccharide that exits on the cell wall of Gram-negative bacteria. It can cause fever, shock or even death when is delivered into human body. So, it is necessary to control the endotoxin contamination for biopharmaceutical products that are mainly administered by intravenous route. Limulus Amebocyte Lysate (LAL)-based tests are usually used to detect endotoxin content in biologics formulations. However, an undesirable phenomenon called "Low Endotoxin Recovery (LER)" often occurs in formulation buffers that usually contain chelating component, such as sodium citrate, and amphiphilic surfactant, such as Tween-20. The occurrence of this LER phenomenon may interfere with endotoxin detection and cause false negative results. In this study, we compared the effect of different sample treatment methods on endotoxin detection and found that the LER phenomenon was better controlled under the conditions of low pH (pH = 5.0), low temperature (2-8 °C) and in the presence of divalent cations in the solution. In addition, although the endotoxin activity was found to have decreased due to LER phenomenon, the particle size distribution of endotoxin determined by dynamic light scattering (DLS) in LER solution did not change obviously, which is different from previous hypothesis about LER phenomenon in literature that the particle size of endotoxin aggregates would decrease under LER conditions. These findings provide some insights into different sample treatment methods for endotoxin detection and give a better understanding and solution on minimizing the LER phenomenon.

DeepCys: Structure-based multiple cysteine function prediction method trained on deep neural network: Case study on domains of unknown functions belonging to COX2 domains.

Cysteine (Cys) is the most reactive amino acid participating in a wide range of biological functions. In-silico predictions complement the experiments to meet the need of functional characterization. Multiple Cys function prediction algorithm is scarce, in contrast to specific function prediction algorithms. Here we present a deep neural network-based multiple Cys function prediction, available on web-server (DeepCys) (https://deepcys.herokuapp.com/). DeepCys model was trained and tested on two independent datasets curated from protein crystal structures. This prediction method requires three inputs, namely, PDB identifier (ID), chain ID and residue ID for a given Cys and outputs the probabilities of four cysteine functions, namely, disulphide, metal-binding, thioether and sulphenylation and predicts the most probable Cys function. The algorithm exploits the local and global protein properties, like, sequence and secondary structure motifs, buried fractions, microenvironments and protein/enzyme class. DeepCys outperformed most of the multiple and specific Cys function algorithms. This method can predict maximum number of cysteine functions. Moreover, for the first time, explicitly predicts thioether function. This tool was used to elucidate the cysteine functions on domains of unknown functions belonging to cytochrome C oxidase subunit-II like transmembrane domains. Apart from the web-server, a standalone program is also available on GitHub (https://github.com/vam-sin/deepcys).

Biocompatible magnesium-doped biphasic calcium phosphate for bone regeneration.

Autologous bone grafting remains the gold standard for almost all bone void-filling orthopedic surgery. However, autologous bone grafting has several limitations, thus scientists are trying to identify an ideal synthetic material as an alternative bone graft substitute. Magnesium-doped biphasic calcium phosphate (Mg-BCP) has recently been in the spotlight and is considered to be a potential bone substitute. The Mg-BCP is a mixture of two bioceramics, that is, hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP), doped with Mg2+ , and can be synthesized through chemical wet-precipitation, sol-gel, single diffusion gel, and solid state reactions. Regardless of the synthesis routes, it is found that the Mg2+ preferentially accommodates in ß-TCP lattice instead of the HA lattice. The addition of Mg2+ to BCP leads to desirable physicochemical properties and is found to enhance the apatite-forming ability as compared to pristine BCP. In vitro results suggest that the Mg-BCP is bioactive and not toxic to cells. Implantation of Mg-BCP in in vivo models further affirmed its biocompatibility and efficacy as a bone substitute. However, like the other bioceramics, the optimum physicochemical properties of the Mg-BCP scaffold have yet to be determined. Further investigations are required regarding Mg-BCP applications in bone tissue engineering.

The role of surface oxide thickness and structure on the corrosion and nickel elution behavior of nitinol biomedical implants.

Biocompatibility is an important factor for metallic medical device implants, and corrosion resistance of implantable alloys is one aspect of biocompatibility. Corrosion behavior of nitinol is strongly dependent upon the nature of the surface oxide, which forms during processing. The surface oxide is comprised of a mixture of titanium and nickel oxides, and subsequent thermal exposure (e.g., during shape setting) and surface removal (e.g., electropolishing, mechanical polishing, etching, etc.) influence its structure. Corrosion behavior is often assessed through testing methods such as cyclic potentiodynamic polarization (e.g., ASTM F2129) and nickel ion release. Studies have suggested that a correlation exists between oxide thickness and nickel ion release, with thicker oxides eluting more nickel. It is hypothesized that the composition of the surface oxide, and not only its thickness, influences the corrosion performance of nitinol. To investigate this, nitinol wire samples were processed to produce surface oxides with different structures both in terms of thickness and composition. These samples were tested per ASTM F2129 and nickel ion release testing.

Anti-cancer and anti-inflammatory activities of a new family of coordination compounds based on divalent transition metal ions and indazole-3-carboxylic acid.

A new family of mononuclear coordination compounds has been synthetized and characterized: [M(3-ind)2(H2O)2] (M = Co (1), Ni (2), Zn (3), Fe (4), Mn (5); 3-ind = indazole-3-carboxylate). These materials are mononuclear coordination compounds that possess strong hydrogen bond interactions. The anti-inflammatory effects of these compounds were assayed in lipopolysaccharide activated RAW 264.7 macrophages by inhibition of NO production. Moreover, the cytotoxicity of the complexes and the ligand in RAW 264.7 cells were determined for the first time. The most significant results were obtained for the compounds 4 and 5 reaching values of NO inhibition close to 80% at 48 h, and above to 90% at 72 h of treatment. The highest inhibitory effects on NO production were showed at the range 7-23 µg/mL for compounds 4 and 5. As a consequence, compounds 4 and 5 could be potential drugs due to the interesting anti-inflammatory properties showed. The anti-cancer potential of these compounds has been also tested against different tumor cell lines. The cytotoxicity of the ligand and of compounds 2 and 3 were assayed in three cell lines: HT29, colon cancer cells, Hep-G2, hepatoma cells and B16-F10 melanoma cells. The best results have been achieved with compound 2 in HepG2 and B16-F10 cell lines, being between 1.5 and 2 times more effective that the ligand in HepG2 cells, and B16-F10 cells. All in all, indazole-3-carboxylic acid is a promising ligand for the formation of coordination compounds with biochemical properties.

A colorimetric aptamer-based method for detection of cadmium using the enhanced peroxidase-like activity of Au-MoS2 nanocomposites.

In this work, a colorimetric aptamer-based method for detection of cadmium using gold nanoparticles modified MoS2 nanocomposites as enzyme mimic is established. In short, biotinylated Cd2+ aptamers are immobilized by biotin-avidin binding on the bottoms of the microplate, the complementary strands of Cd2+ aptamers are connected to the Au-MoS2 nanocomposites which have the function of enhanced peroxidase-like activity. The csDNA-Au-MoS2 signal probe and target Cd2+ compete for binding Cd2+ aptamer, the color change can be observed by addition of chromogenic substrate, thereby realizing visual detection of Cd2+. The absorbance of the solution at 450 nm has a clear linear relationship with the Cd2+ concentration. The linear range is 1-500 ng/mL, and the limit of detection is 0.7 ng/mL. The assay was used to test white wine samples, the results are consistent with those of atomic absorption spectrometry; which prove that this method can be used for detection of Cd2+ in real samples.

Thermodynamic Basis for Conformational Coupling in an ATP-Binding Cassette Exporter.

ATP-binding cassette (ABC) transporters constitute one of the largest protein superfamilies, and they mediate the transport of diverse substrates across the membrane. The molecular mechanism for transducing the energy from ATP binding and hydrolysis into the conformational changes remains elusive. Here, we determined the thermodynamics underlying the ATP-induced global conformational switching for the ABC exporter TmrAB using temperature-resolved pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy. We show that a strong entropy-enthalpy compensation mechanism enables the closure of the nucleotide-binding domains (NBDs) over a wide temperature range. This is mechanically coupled with an outward opening of the transmembrane domains (TMDs) accompanied by an entropy gain. The conserved catalytic glutamate plays a key role in the overall energetics. Our results reveal the thermodynamic basis for the chemomechanical energy coupling in an ABC exporter and present a new strategy to explore the energetics of similar membrane protein complexes.

Half a century of amyloids: past, present and future.

Amyloid diseases are global epidemics with profound health, social and economic implications and yet remain without a cure. This dire situation calls for research into the origin and pathological manifestations of amyloidosis to stimulate continued development of new therapeutics. In basic science and engineering, the cross-ß architecture has been a constant thread underlying the structural characteristics of pathological and functional amyloids, and realizing that amyloid structures can be both pathological and functional in nature has fuelled innovations in artificial amyloids, whose use today ranges from water purification to 3D printing. At the conclusion of a half century since Eanes and Glenner's seminal study of amyloids in humans, this review commemorates the occasion by documenting the major milestones in amyloid research to date, from the perspectives of structural biology, biophysics, medicine, microbiology, engineering and nanotechnology. We also discuss new challenges and opportunities to drive this interdisciplinary field moving forward.

Cutting in-line with iron: ribosomal function and non-oxidative RNA cleavage.

Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg2+ as a primary mediator of ribosomal structure and function. Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosome's early evolution in a high Fe2+ environment, and the continued use of Fe2+ by obligate anaerobes inhabiting high Fe2+ niches. Here, we show that (i) Fe2+ cleaves RNA by in-line cleavage, a non-oxidative mechanism that has not previously been shown experimentally for this metal, (ii) the first-order in-line rate constant with respect to divalent cations is >200 times greater with Fe2+ than with Mg2+, (iii) functional ribosomes are associated with Fe2+ after purification from cells grown under low O2 and high Fe2+ and (iv) a small fraction of Fe2+ that is associated with the ribosome is not exchangeable with surrounding divalent cations, presumably because those ions are tightly coordinated by rRNA and deeply buried in the ribosome. In total, these results expand the ancient role of iron in biochemistry and highlight a possible new mechanism of iron toxicity.

Calcium enhances polyplex-mediated transfection efficiency of plasmid DNA in Jurkat cells.

Jurkat, an immortalized cell line derived from human leukemic T lymphocytes, has been employed as an excellent surrogate model of human primary T-cells for the advancement of T-cell biology and their applications in medicine. However, presumably due to its T-cell origin, Jurkat cells are very difficult to transfect. Thus, for the genetic modification of Jurkat cells, expensive and time-consuming viral vectors are normally required. Despite many previous efforts, non-viral vectors have not yet overcome the hurdles of low transfection efficiency and/or high toxicity in transfection of Jurkat cells. Here, we report that a simple addition of calcium ions (Ca2+) into culture media at optimal concentrations can enhance the efficiency of the polyplex-mediated transfection using poly(ethylene imine) (PEI) by up to 12-fold when compared to the polyplex-only control. We show that calcium enhances the association between polyplex and Jurkat, which is at least partially responsible for the increase in transmembrane delivery of polyplex and consequential enhancement in expression of transgene. Other cations, Mg2+ or Na+ did not show similar enhancement. Interestingly, addition of Ca2+ was rather detrimental for the transfection of lipoplex on Jurkat cells. Observation of significant enhancement in the transfection of non-viral vectors with a simple and physiologically relevant reagent like Ca2+ in the engineering of hard-to-transfect cells such as Jurkat warrants further investigation on similar strategies.

Self-recovering dual cross-linked hydrogels based on bioorthogonal click chemistry and ionic interactions.

The biocompatible, injectable and high water-swollen nature of hydrogels makes them a popular candidate to imitate the extracellular matrix (ECM) for tissue engineering both in vitro and in vivo. However, commonly used covalently cross-linked hydrogels, despite their stability and tunability, are elastic and deteriorate as bulk material degrades which would impair proper cell function. To improve these deficiencies, here, we present a self-recovering cross-linked hydrogel formed instantaneously with functionalized poly(ethylene glycol) as a basis. We combine covalent cross-links introduced via a strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction and non-covalent links between phosphonate groups and calcium ions. By adjusting the ratios of non-covalent and covalent cross-links, we synthesized these dual cross-linked (DC) hydrogels that displayed storage moduli below ∼2000 Pa and relaxation times from seconds to minutes. The gels recovered to 41-96% of their initial mechanical properties after two subsequent strain failures. Cryo-scanning electron microscopy revealed that DC hydrogels containing approximately equal amounts of covalent and non-covalent cross-links displayed phase separation. Finally, we functionalized the DC hydrogels by incorporating an integrin binding motif, RGDS, to provide a biocompatible environment for human mesenchymal stem cells (HMSCs) by facilitating adhesion inside the gel network. Inside these DC gels HSMCs displayed a viability up to 73% after five days of cell culture.

Methacrylated gellan gum and hyaluronic acid hydrogel blends for image-guided neurointerventions.

Cell-based therapies delivered via intrathecal injection are considered as one of the most promising solutions for the treatment of amyotrophic lateral sclerosis (ALS). Herein, injectable manganese-based biocompatible hydrogel blends were developed, that can allow image-guided cell delivery. The hydrogels can also provide physical support for cells during injection, and at the intrathecal space after transplantation, while assuring cell survival. In this regard, different formulations of methacrylated gellan gum/hyaluronic acid hydrogel blends (GG-MA/HA) were considered as a vehicle for cell delivery. The hydrogels blends were supplemented with paramagnetic Mn2+ to allow a real-time monitorization of hydrogel deposition via T1-weighted magnetic resonance imaging (MRI). The developed hydrogels were easily extruded and formed a stable fiber upon injection into the cerebrospinal fluid. Hydrogels prepared with a 75 : 25 GG-MA to HA ratio supplemented with MnCl2 at 0.1 mM showed controlled hydrogel degradation, suitable permeability, and a distinct MRI signal in vitro and in vivo. Additionally, human-derived adipose stem cells encapsulated in 75 : 25 GG-MA/HA hydrogels remained viable for up to 14 days of culture in vitro. Therefore, the engineered hydrogels can be an excellent tool for injectable image-guided cell delivery approaches.

Self-healing and easy-to-shape mineralized hydrogels for iontronics.

Hydrogel-based multifunctional materials have attracted much attention. In this work, novel mineralized hydrogels were fabricated through physically cross-linked polyvinylpyrrolidone (PVP) and CaCO3. The mineralized hydrogels were prepared by simply mixing CaCl2, Na2CO3, and PVP in aqueous solutions. The CO32- induced the aggregation of the PVP chains and the CaCO3 particles in situ generated in the aqueous solution worked as fillers to strengthen the hydrogels. Based on this method, other kinds of mineralized hydrogels were prepared by replacing the Ca2+ with different metal ions. The mineralized hydrogels displayed shapeable, self-healing and thixotropic properties. Moreover, the mineralized hydrogel-based sensor showed good and stable sensitivity to compressive pressure, and could be used to monitor human actions. This work presents a facile method for preparing mineralized hydrogels, which are promising for various applications due to their outstanding properties.

Molecular Mechanism of ISC Iron-Sulfur Cluster Biogenesis Revealed by High-Resolution Native Mass Spectrometry.

Iron-sulfur (Fe-S) clusters are ubiquitous protein cofactors that are required for many important biological processes including oxidative respiration, nitrogen fixation, and photosynthesis. Biosynthetic pathways assemble Fe-S clusters with different iron-to-sulfur stoichiometries and distribute these clusters to appropriate apoproteins. In the ISC pathway, the pyridoxal 5'-phosphate-dependent cysteine desulfurase enzyme IscS provides sulfur to the scaffold protein IscU, which templates the Fe-S cluster assembly. Despite their functional importance, mechanistic details for cluster synthesis have remained elusive. Recent advances in native mass spectrometry (MS) have allowed proteins to be preserved in native-like structures and support applications in the investigation of protein structure, dynamics, ligand interactions, and the identification of protein-associated intermediates. Here, we prepared samples under anaerobic conditions and then applied native MS to investigate the molecular mechanism for Fe-S cluster synthesis. This approach was validated by the high agreement between native MS and traditional visible circular dichroism spectroscopic assays. Time-dependent native MS experiments revealed potential iron- and sulfur-based intermediates that decay as the [2Fe-2S] cluster signal developed. Additional experiments establish that (i) Zn(II) binding stabilizes IscU and protects the cysteine residues from oxidation, weakens the interactions between IscU and IscS, and inhibits Fe-S cluster biosynthesis; and (ii) Fe(II) ions bind to the IscU active site cysteine residues and another lower affinity binding site and promote the intermolecular sulfur transfer reaction from IscS to IscU. Overall, these results support an iron-first model for Fe-S cluster synthesis and highlight the power of native MS in defining protein-associated intermediates and elucidating mechanistic details of enzymatic processes.