Low Calcium Ion Concentrations Research Articles

Supramolecular Composite Hydrogel Loaded with CaF2 Nanoparticles Promotes the Recovery of Periodontitis Bone Resorption.

Treatments to reduce periodontal inflammation and rescue periodontitis bone resorption have been of interest to researchers. Bone tissue engineering materials have been gradually used in the treatment of bone defects, but periodontal bone tissue regeneration still faces challenges. Considering the biocompatibility factor, constructing bionic scaffolds with natural extracellular matrix properties is an ideal therapeutic pathway. Based on the pathological mechanism of periodontitis, in this study, short peptide and nanometer inorganic particles were comingled to construct NapKFF-nano CaF2 supramolecular composite hydrogels with different ratios. Material characterization experiments confirmed that the composite hydrogel had suitable mechanical properties and a three-dimensional structure that can function in the resorption region of the alveolar bone and provide spaces for cell proliferation and adhesion. The release of low concentrations of fluoride and calcium ions has been shown to have positive biological effects in both in vivo and in vitro experiments. Vitro experiments confirmed that the composite hydrogel had good biocompatibility and promoted osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Microbiological experiments confirmed that the composite hydrogel inhibited the activity of periodontal pathogenic bacteria. In animal studies, composite hydrogel applied to periodontitis rats in vivo can effectively repair alveolar bone resorption. This composite hydrogel has a simple preparation method and is inexpensive to produce, yet it has antibacterial and osteogenesis-promoting incremental effects, which makes it well suited for the treatment of periodontitis bone resorption, providing a new strategy for periodontal bone tissue engineering.

Fabrication, characterization, and antibacterial properties of sodium alginate/chito-oligosaccharide gel beads

Developing sodium alginate (SA)/chito-oligosaccharide (COS) beads with antibacterial properties is highly appealing for food industrial applications. Herein, in this paper, the different viscosity-average molecular weight of SA/COS polyelectrolyte (SCP) gel beads were prepared by adding a low concentration of calcium ions (Ca2+) as crosslinker and their size, mechanical property, microscopic morphology, elemental composition, and thermal stability of the gel beads were characterized. Furthermore, the morphological changes of gel beads in in vitro simulated digestion were investigated. A time-dependent co-culture assay was used to explore the antibacterial effect of gel beads on Staphylococcus aureus and Escherichia coli. The results showed that as the viscosity-average molecular weight (Mv) of SA decreased, the bursting strength faded, the thermal stability decreased, and the swelling index increased. Similarly, increasing the Ca2+ content could also regulate the characteristics of the gel beads, such as diminishing size, increasing the bursting strength, making the surfaces rough, and producing the network structure in the cross-section. In addition, Ca2+ competes with COS in cross-linking with the carboxyl groups of SA, which leads to a decrease in the content of N element on the surfaces of the gel beads and to a weakening of the antibacterial effect. After the addition of COS, the gel beads obtained antimicrobial effects and a denser cross-sectional structure. The present study could provide a theoretical basis for the design and development of novel gel foods in the food industry.

Synthesis of high-value CaCO3 via indirect CO2 fixation utilized blast furnace slag

Ultrasonic and bubbling carbonation method achieved precipitation of CaCO3 was described as a particularly advantageous method for synthesizing low-dispersity vaterite CaCO3. Herein, high-purity vaterite CaCO3 microspheres with regular morphology was synthesized from blast furnace slag (BFS) through mineral phase leaching, precipitation impurity removal, and CO2 solidification in ultrasonic reaction field. The results demonstrated that the method employed for distributing bubbling CO2 under an ultrasonic field aided both CO2 fixation and indeed the formation of homogenized vaterite. And vaterite synthesis is further aided by factors such as low initial calcium ion concentration, low reaction temperature, high CO2 concentration, and short aging time. Furthermore, a sphere-dumbbell-sphere mechanism has been proposed as a possible growth mechanism for the spheroidal structures. Finally, large-scale production of high value-added vaterite CaCO3 would eventually be attained at carbonation temperature of 20 ℃, Ca2+ concentration of 0.1 mol·L−1, CO2 proportion in the gas mixture of 70 %.

Calcium carbonate crystallization process from the mineralization of calcium chloride waste

The distillation waste stream from the ammonia-alkali process contains 90–120 g/L of calcium chloride. Using calcium chloride as raw material to mineralize carbon dioxide can achieve efficient utilization of calcium chloride waste and CO2 emission reduction. This article is based on a two-step synthesis method, using ammonium carbonate and calcium chloride as raw materials to synthesize calcium carbonate. The changes in pH of the mineralized solution with time were investigated under factors such as temperature, ammonium carbonate concentration, feeding method, and retention time. The crystallization mechanism of carbonation process shows that the pH of solution will affect the degree of supersaturation of calcium carbonate, and high supersaturation is conducive to the precipitation of metastable aragonite and vaterite phases. The experimental results showed that when ammonium carbonate was passed into calcium chloride at a rate of 10 mL/min at different temperatures and different ammonium carbonate concentrations, the pH change of the mineralized solution could be divided into three stages: a rapid pH decrease stage (9.5–7), a pH stabilization stage (∼7) and a pH rebound stage (7–8.5). Increasing the temperature is conducive to the formation of submicron calcite type calcium carbonate, changing the concentration of ammonium carbonate, and the crystal phase and size of the product are almost unaffected. If calcium chloride is added to ammonium carbonate, under low calcium ion concentration conditions, calcium carbonate tends to form aragonite phase.

Utilization of local food on the prevention of dental caries among the elementary school children in the fishing community

The potential benefits of local food derived from rebon shrimp (Acetes sp.) as a preventive measure for dental caries are described in this study, which focused on Bagan Serdang village. Situated in the coastal area of Pantai Labu subdistrict, Deli Serdang Regency, this village relies mainly on fisheries for income resources, with rebon shrimp being one of the leading marine products. The high abundance of calcium in rebon shrimp is well-known, with 2,306 mg of shrimp paste equivalent to 16 times the calcium content of 100 g of dietary milk. This preliminary study aimed to investigate the potential of rebon shrimp as a preventive measure for dental caries in school children aged 8-12 years from public elementary school 106448 in Bagan Serdang village. The study utilized Cariogram software to assess caries risk factors, including caries experience, pH and saliva secretion, plaque index, diet contents and frequency, fluoride exposure program, and clinical judgment. Atomic Absorbed Spectrophotometer (AAS) method was used to measure salivary calcium ions. In addition, the calcium content in rebon shrimp was measured in the laboratory of the industrial research center in Medan. The concentration of salivary calcium ions was found to be very low, with a mean of 0.75±0.32 mmol/L. The Cariogram assessment revealed a high caries risk of 54.12% among elementary school children. The laboratory results showed that dried rebon shrimp had a calcium content of 1.50% (w/w), while fresh rebon shrimp had a calcium content of 1.54% (w/w). Given the high prevalence and caries risk profile of elementary school children in Bagan Serdang village, the study suggests that the low concentration of salivary calcium ions and the high availability of calcium-enriched rebon shrimp can be used as a preventive strategy against dental caries in this population.

Discovery of Highly Active Derivatives of Daptomycin by Assessing the Effect of Amino Acid Substitutions at Positions 8 and 11 on a Daptomycin Analogue.

Daptomycin is an important antibiotic used for treating serious infections caused by Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci. Establishing structure-activity relationships of daptomycin is important for developing new daptomycin-based antibiotics with expanded clinical applications and for tackling the ever-increasing problem of antimicrobial resistance. Toward this end, Dap-K6-E12-W13, an active analogue of daptomycin in which the uncommon amino acids in daptomycin are replaced with their common counterparts, was used as a model system for studying the effect of amino acid variation at positions 8 and 11 on in vitro biological activity against a model organism, Bacillus subtilis, and calcium-dependent insertion into model membranes. None of the new peptides were more active than Dap-K6-E12-W13; however, substitution at positions 8 and/or 11 with cationic residues resulted in little or no loss of activity, and some of these analogues were able to insert into model membranes at lower calcium ion concentrations than the parent peptide. Incorporation of these cationic residues into positions 8 and/or 11 of daptomycin itself yielded some derivatives that exhibited lower minimum inhibitory concentrations than daptomycin against B. subtilis 1046 as well as comparable and sometimes superior activity against clinical isolates of MRSA.

Bone Tissue Response to Different Grown Crystal Batches of Octacalcium Phosphate in Rat Long Bone Intramedullary Canal Area.

The microstructure of biomaterials influences the cellular and biological responses in the bone. Octacalcium phosphate (OCP) exhibits higher biodegradability and osteoconductivity than hydroxyapatite (HA) during the conversion process from OCP to HA. However, the effect of the microstructure of OCP crystals on long tubular bones has not been clarified. In this study, two types of OCPs with different microstructures, fine-OCP (F-OCP) and coarse-OCP (C-OCP), were implanted in rat tibia for 4 weeks. F-OCP promoted cortical bone regeneration compared with C-OCP. The osteoclasts appearance was significantly higher in the C-OCP group than in the control group (defect only) at 1-week post-implantation. To investigate whether the solubility equilibrium depends on the different particle sizes of OCPs, Nano-OCP, which consisted of nanometer-sized OCPs, was prepared. The degree of supersaturation (DS) tended to decrease modestly in the order of C-OCP, F-OCP, and Nano-OCP with respect to HA and OCP in Tris-HCl buffer. F-OCP showed a higher phosphate ion concentration and lower calcium ion concentration after immersion in the buffer than C-OCP. The crystal structures of both OCPs tended to be converted to HA by rat abdominal implantation. These results suggest that differences in the microstructure of OCPs may affect osteoclastogenesis and result in osteoconductivity of this material in long tubular bone by altering dissolution behavior.

Abnormal Coagulation Function of Patients With COVID-19 Is Significantly Related to Hypocalcemia and Severe Inflammation.

This study aimed to detect, analyze, and correlate the clinical characteristics, blood coagulation functions, blood calcium levels, and inflammatory factors in patients with mild and severe COVID-19 infections. The enrolled COVID-19 infected patients were from Wuhan Jin Yin-tan Hospital (17 cases, Wuhan, China), Suzhou Infectious Disease Hospital (87 cases, Suzhou, China), and Xuzhou Infectious Disease Hospital (14 cases, Xuzhou, China). After admission, basic information was collected; X-ray and chest CT images were obtained; and data from routine blood tests, liver and kidney function, myocardial enzymes, electrolytes, blood coagulation function, (erythrocyte sedimentation rate) ESR, C-reactive protein (CRP), IL-6, procalcitonin (PCT), calcitonin, and other laboratory tests were obtained. The patients were grouped according to the clinical classification method based on the pneumonia diagnosis and treatment plan for new coronavirus infection (trial version 7) in China. The measurements from mild (56 cases) and severe cases (51 cases) were compared and analyzed. Most COVID-19 patients presented with fever. Chest X-ray and CT images showed multiple patchy and ground glass opacities in the lungs of COVID 19 infected patients, especially in patients with severe cases. Compared with patients with mild infection, patients with severe infection were older (p = 0.023) and had a significant increase in AST and BUN. The levels of CK, LDH, CK-MB, proBNP, and Myo in patients with severe COVID-19 infection were also increased significantly compared to those in patients with mild cases. Patients with severe COVID-19 infections presented coagulation dysfunction and increased D-dimer and fibrin degradation product (FDP) levels. Severe COVID-19 patients had low serum calcium ion (Ca2+) concentrations and high calcitonin and PCT levels and exhibited serious systemic inflammation. Ca2+ in COVID-19 patients was significantly negatively correlated with PCT, calcitonin, D-dimer, PFDP, ESR, CRP and IL-6. D-dimer in COVID-19 patients was a significantly positively correlated with CRP and IL-6. In conclusion, patients with severe COVID-19 infection presented significant metabolic dysfunction and abnormal blood coagulation, a sharp increase in inflammatory factors and calcitonin and procalcitonin levels, and a significant decrease in Ca2+. Decreased Ca2+ and coagulation dysfunction in COVID-19 patients were significantly correlated with each other and with inflammatory factors.

Decoupling salinity and carbonate chemistry: low calcium ion concentration rather than salinity limits calcification in Baltic Sea mussels

Abstract. The Baltic Sea has a salinity gradient decreasing from fully marine (> 25) in the west to below 7 in the central Baltic Proper. Habitat-forming and ecologically dominant mytilid mussels exhibit decreasing growth when salinity < 11; however, the mechanisms underlying reduced calcification rates in dilute seawater are not fully understood. Both [HCO3-] and [Ca2+] also decrease with salinity, challenging calcifying organisms through CaCO3 undersaturation (Ω≤1) and unfavourable ratios of calcification substrates ([Ca2+] and [HCO3-]) to the inhibitor (H+), expressed as the extended substrate–inhibitor ratio (ESIR). This study combined in situ monitoring of three southwest Baltic mussel reefs with two laboratory experiments to assess how various environmental conditions and isolated abiotic factors (salinity, [Ca2+], [HCO3-] and pH) impact calcification in mytilid mussels along the Baltic salinity gradient. Laboratory experiments rearing juvenile Baltic Mytilus at a range of salinities (6, 11 and 16), HCO3- concentrations (300–2100 µmol kg−1) and Ca2+ concentrations (0.5–4 mmol kg−1) reveal that as individual factors, low [HCO3-], pH and salinity cannot explain low calcification rates in the Baltic Sea. Calcification rates are impeded when Ωaragonite ≤ 1 or ESIR ≤ 0.7 primarily due to [Ca2+] limitation which becomes relevant at a salinity of ca. 11 in the Baltic Sea. Field monitoring of carbonate chemistry and calcification rates suggest increased food availability may be able to mask the negative impacts of periodic sub-optimal carbonate chemistry, but not when seawater conditions are permanently adverse, as observed in two Baltic reefs at salinities < 11. Regional climate models predict a rapid desalination of the southwest and central Baltic over the next century and potentially a reduction in [Ca2+] which may shift the distribution of marine calcifiers westward. It is therefore vital to understand the mechanisms by which the ionic composition of seawater impacts bivalve calcification for better predicting the future of benthic Baltic ecosystems.

Polypyrrole-BaFe2O4 sensing layer for detection of strontium ion in aqueous solution using surface plasmon resonance curves fitting

The low concentration of strontium, calcium, and barium were measured with a surface plasmon resonance curve fitting technique. Polypyrrole-BaFe2O4 nanocomposite was prepared using electro-polymerization of pyrrole in the presence of magnetic nanoparticles. The thickness of the layer in the range of 6.3 and 62.2 nm the analytical methods such as Fourier transform infrared spectroscopy, energy dispersive spectroscopy, and field emission scanning electron microscopy authenticated the polypyrrole-BaFe2O4 nanocomposite formed on the surface of the gold layer. The prepared layers were used to detect and measure the low concentration of strontium, calcium, and barium ions in aqueous solution using surface plasmon resonance technique and the optical parameter, angle of resonance, resonance angle shift and affinity constant were achieved using curve fitting based on minimizing the sum of squared errors. Consequently, the degree of adsorption for the detection of strontium is about 91.3%, and the sensitivity of the polypyrrole-BaFe2O4 nanocomposite layer for the detection of strontium is about 0.006° that is more than other ions.

Reducing anionic surfactant adsorption using polyacrylate as sacrificial agent investigated by QCM-D

Surfactant losses by adsorption to rock surfaces make surfactant-based enhanced oil recovery economically less feasible. We investigated polyacrylate (PA) as a sacrificial agent in the reduction of anionic surfactant adsorption with focus on calcite surfaces by using quartz crystal microbalance with dissipation monitoring. It was found that the adsorption of the anionic surfactant alcohol alkoxy sulfate (AAS) followed a Langmuir adsorption isotherm, and the adsorbed amount reached saturation above its critical micellar concentration. Adsorption of PA was a much slower process compared to AAS adsorption. Increasing the calcium ion concentration also increased the amount of AAS adsorbed as well as the mass increase rate of PA adsorption. Experimental results combined with density functional theory calculations indicated that calcium cation bridging was important for anionic surfactant AAS and PA adsorption to calcite surfaces. To effectively reduce the amount of surfactant adsorption, it was needed to preflush with PA, rather than by a simultaneous injection. Preflushing with 30 ppm of PA gave a reduction of AAS adsorption of 30% under high salinity (HS, 31,800 ppm) conditions, compared to 8% reduction under low salinity (LS, 3180 ppm) conditions. In the absence of PA, the amount of adsorbed AAS was reduced by already 50% upon changing from HS to LS conditions. Lower calcium ion concentrations, as under LS conditions, contributed to this observation. On different mineral surfaces, PA reduced the AAS adsorption in the order of alumina > calcite > silica. These results offer important insights into mitigating surfactant adsorption using PA polyelectrolyte as sacrificial agent and contribute to improved flooding strategies with reduced surfactant loss.

A Ca2+-Binding Protein that Regulates the Lifetime of the Transducin Active State

Abstract—It has been previously shown in the study of the kinetic behavior of cGMP-specific phosphodiesterase in preparations of the outer segments of bovine retinal rods, which was temporarily activated by 2 μM GTP at high and low concentrations of free ions of Ca2+ (100 μM and <10 nm, respectively), that the lifetime of cGMP-specific phosphodiesterase in the active state significantly decreased (by ≈2 times) when low concentrations of free calcium ions were used. This suggested that there is a Ca2+-binding protein in the outer segments of bovine retinal rods that can interact with the so-called free transducin and regulate the rate of hydrolysis of GTP, which is bound in its active site, depending on the concentration of free Ca2+ ions. This paper is devoted to a discussion of the known Ca2+-binding proteins that occur in the outer segments of bovine retinal rods that could play the role of a potential regulator.

Single-molecule nanomechanical spectroscopy shows calcium ions contribute to chain association and structural flexibility of blood clotting factor VIII

Blood coagulation factor VIII (FVIII) can bind calcium ions and ion-protein interactions appear central importance for both their structure and function in coagulation cascade. However, the mechanism and details of how calcium dependent structure change of proteins to fulfill their function remain to be fully defined. In this work, PeakForce Quantitative Nanomechanics (PF-QNM) mode atomic force microscopy (AFM) was used to map the topography and mechanical properties of FVIII with single protein resolution under different calcium concentrations. The obtained nanomechanical spectroscopy showed that calcium ions play dual roles in the chain association and structural flexibility of FVIII. Low concentration of calcium ions prefer to bind isolated chains and increase their mechanical properties, whereas they link the heavy and light chains to keep the protein re-association under higher ions concentration. Our results provide a novel insight into the mechanistic details of the metal ions on the stability and function of blood clotting proteins.

The Effect of the Arg91Gly and Glu139del Mutations in β-Tropomyosin Associated with Congenital Myopathy of Human Skeletal Muscles on Actin–Myosin Interaction

The structural changes in proteins of the contractile apparatus of muscle fiber and the violation of their function due to point mutations in these proteins can be a cause of many hereditary diseases of human muscular tissue. Some such diseases are cap-myopathy and distal arthrogryposis, which may be connected with tropomyosin mutations. The deletion of glutamic-acid residue at position 139 of β-tropomyosin leads to the development of cap-myopathy, and the replacement of arginine at position 91 with glycine in this protein is linked to distal arthrogryposis. To understand how the Arg91Gly and Glu139del mutations disrupt the coordinated work of the contractile system of muscle fibers, recombinant wild-type and mutant β-tropomyosins were overexpressed and incorporated into thin filaments of ghost-muscle fiber. Fluorescent probes of 1,5-IAEDANS or FITC-phalloidin were specifically linked to the Cys707 of the myosin subfragment-1 and the three neighboring actin monomers, respectively. The polarized-microfluorimetry technique was used to study the spatial arrangements of actin and myosin in mimicking different stages of the ATPase cycle (in the presence of ADP or ATP and in the absence of a nucleotide) at low and high concentration of calcium ions. Both mutations were shown to change the conformational rearrangements of the myosin head and actin in the ATP hydrolysis cycle, which may be caused by abnormal behavior of the mutant tropomyosins during regulation. The altered work of the contractile system may be a cause of muscle weakness in congenital myopathies associated with these mutations.

Aircraft Measurements of Total Mercury and Monomethyl Mercury in Summertime Marine Stratus Cloudwater from Coastal California, USA.

Water samples from marine stratus clouds were collected during 16 aircraft flights above the Pacific Ocean near the Central California coast during the summer of 2016. These samples were analyzed for total mercury (THg), monomethyl mercury (MMHg), and 32 other chemical species in addition to aerosol physical parameters. The mean concentrations of THg and MMHg in the cloudwater samples were 9.2 ± 6.0 ng L-1 (2.3-33.8 ng L-1) ( N = 97) and 0.87 ± 0.66 ng L-1 (0.17-2.9 ng L-1) ( N = 22), respectively. This corresponds to 9.5% (3-21%) MMHg as a fraction of THg. Low and high nonsea salt calcium ion (nss-Ca2+) concentrations in cloudwater were used to classify flights as "marine" and "continental", respectively. Mean [MMHg]marine was significantly higher ( p < 0.05) than [MMHg]continental consistent with an ocean source of dimethyl Hg (DMHg) to the atmosphere. Mean THg in cloudwater was not significantly different between the two categories, indicating multiple emissions sources. Mean [THg]continental was correlated with pH, CO, NO3-, NH4+, and other trace metals, whereas [THg]marine was correlated with MMHg and Na+. THg concentrations were negatively correlated with altitude, consistent with ocean and land emissions, coupled with removal at the cloud-top due to drizzle formation.

EFFECT OF DIFFERENT CALCIUM CONCENTRATIONS IN SOIL ON SURVIVAL PERCENT AND UPTAKE OF Na+ AND Cl- IONS BY RICE PLANT

Salinity is a stress factor affecting the production of crop in many regions. Calcium can reduce Na+ transport to shoots in rice. Two greenhouse experiments were conducted in Faculty of Agriculture, Cairo University, Egypt, during 2015 growing season of rice to evaluate the effect of different calcium concentrations on survival percent along with uptake of Na+ and Cl- ions by two varieties of rice (Oryza sativa L.) differing in salt-tolerance. The first experiment was undertaken to study the effect of different calcium concentrations on survival percent of IR28 (salt-sensitive) and Nona Bokra (Salt-tolerant) seedlings which were transferred to salinized nutrient solution containing 0.5% NaCl and a variable calcium concentrations at 4, 40, 100 and 200 ppm; plants were grown up to 40 days. The second experiment investigated the effect of different calcium concentrations on growth, uptake and transport of Na+ and Cl- ions in the two rice varieties differing in salt-tolerance. The seedlings were transferred to salinized nutrient solution containing 0.5% NaCl and calcium ion concentrations at two levels, 4 and 40 ppm. Plants were harvested at 0, 1, 3, 5 and 7 days from salinization. The results indicated that the salt-tolerant variety (Nona Bokra) survived for more than 40 days under exposure to 0.5% NaCl when calcium concentration of the culture solution ranged from 40 to 200 ppm Ca++. The low calcium ion concentration (4 ppm) depressed the growth of plants at 5 and 7 days after salinization. In Nona Bokra, the shoot had less sodium and Cl than the root. This implies that the salt tolerance of Nona Bokra may be attributed to the restricted translocation of Na+ and Cl- from the root to the shoot. Sodium as well as cloride content in the shoot of IR28 was more than twice that of Nona Bokra. An adequate amount of Ca+2 tended to lower the salt injury caused by high levels of salinity in rice plants. The effect of calcium ion on salt tolerance varied greatly between Nona Bokra and IR28 varieties.

Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi.

Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties. We applied iterative rounds of molecular evolution to develop FGCaMP, a novel green calcium indicator. It includes the circularly permuted version of the enhanced green fluorescent protein (EGFP) sandwiched between the fungal CaM and a fragment of CaM-dependent kinase. FGCaMP is an excitation-ratiometric indicator that has a positive and an inverted fluorescence response to calcium ions when excited at 488 and 405 nm, respectively. Compared with the GCaMP6s indicator in vitro, FGCaMP has a similar brightness at 488 nm excitation, 7-fold higher brightness at 405 nm excitation, and 1.3-fold faster calcium ion dissociation kinetics. Using site-directed mutagenesis, we generated variants of FGCaMP with improved binding affinity to calcium ions and increased the magnitude of FGCaMP fluorescence response to low calcium ion concentrations. Using FGCaMP, we have successfully visualized calcium transients in cultured mammalian cells. In contrast to the limited mobility of GCaMP6s and G-GECO1.2 indicators, FGCaMP exhibits practically 100% molecular mobility at physiological concentrations of calcium ion in mammalian cells, as determined by photobleaching experiments with fluorescence recovery. We have successfully monitored the calcium dynamics during spontaneous activity of neuronal cultures using FGCaMP and utilized whole-cell patch clamp recordings to further characterize its behavior in neurons. Finally, we used FGCaMP in vivo to perform structural and functional imaging of zebrafish using wide-field, confocal, and light-sheet microscopy.

Denervated aortic smooth muscle tension and cellular membrane current in variations of calcium ion concentration in iso-osmotic solution

Objective To study the influence of high calcium ion environment on the membrane current of denervated aortic smooth muscles in iso-osmotic condition, as well as study the myogenic spontaneous vasomotion of the aortic smooth muscles under different pre-load state and intervention of calcium-channel blockers. Methods Smooth muscle samples were taken from the aortic smooth muscle layer of Kunming mice. The samples were fixed at two-ends in relaxed state and infiltrated in Ringer's solution. After stabilization, a glass microelectrode was adsorbed on the smooth muscle membrane to form gigaohm-seal. The current state of the membrane was observed under the physiological osmotic pressure. Then, the calcium ions concentration in the Ringer's solution was increased from 0.9 mol/L to 1.2 mol/L, and the immediate change of membrane current was observed after changing of osmotic pressure. Finally, the membrane potential change in iso-osmotic condition was observed when the pre-load was increased to 1 g, and the spontaneous vasomotion curves of the samples were recorded. Results In the relaxation state, the membrane current of the smooth muscle was increased significantly, i.e. (10.25±1.34) pA vs. (24.91±3.27) pA (P<0.05), when the calcium ion concentration was increased from 0.9 mol/L to 1.2 mol/L in iso-osmotic condition. When the pre-load was increased, the variation amplitude of the membrane current was increased in iso-osmotic condition, i.e. (10.25±1.34) pA to (15.33±4.33) pA (P<0.05) for the lower calcium ion concentration, and (24.91±3.27) pA to (33.31±7.25) pA (P<0.05) for the higher calcium ion concentration. If only increasing the pre-load, the myogenic spontaneous vasomotion could be increased by 175% for the condition of lower calcium ion concentration (0.9 mol/L). When the calcium ion concentration was increased to 1.2 mol/L, the spontaneous vasomotion frequency of the specimen was increased, and the range of the vasomotion could be further increased by 40%. After pretreatment with the calcium-channel blocker (0.5 g/L nitrendipine), the membrane current and myogenic spontaneous vasomotion were significantly decreased in the higher calcium ion environment, which indicated that the membrane potential and spontaneous vasomotion was dominated by calcium ions. Conclusions The increase of extracellular calcium ion concentration not only can improve the excitability of the aorta smooth muscle cells, but also can cause significant spontaneous vasomotion, and can improve the compliance of the smooth muscle tissue in pre-load variation. The calcium-channel blockers can inhibit calcium-dominated transmembrane currents, reduce myogenic spontaneous vasomotion, and enhance the stiffness of smooth muscle, which may have negative effects on windkessel vessel function. Key words: Denervation; Quick stretching; Overloading; Spontaneous vasomotion; Membrane current

Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer

Surface layers (S-layers) are paracrystalline, proteinaceous structures found in most archaea and many bacteria. Often the outermost cell envelope component, S-layers serve diverse functions including aiding pathogenicity and protecting against predators. We report that the S-layer of Caulobacter crescentus exhibits calcium-mediated structural plasticity, switching irreversibly between an amorphous aggregate state and the crystalline state. This finding invalidates the common assumption that S-layers serve only as static wall-like structures. In vitro, the Caulobacter S-layer protein, RsaA, enters the aggregate state at physiological temperatures and low divalent calcium ion concentrations. At higher concentrations, calcium ions stabilize monomeric RsaA, which can then transition to the two-dimensional crystalline state. Caulobacter requires micromolar concentrations of calcium for normal growth and development. Without an S-layer, Caulobacter is even more sensitive to changes in environmental calcium concentration. Therefore, this structurally dynamic S-layer responds to environmental conditions as an ion sensor and protects Caulobacter from calcium deficiency stress, a unique mechanism of bacterial adaptation. These findings provide a biochemical and physiological basis for RsaA’s calcium-binding behavior, which extends far beyond calcium’s commonly accepted role in aiding S-layer biogenesis or oligomerization and demonstrates a connection to cellular fitness.

Expression, purification and identification of a thermolysin-like protease, neutral protease I, from Aspergillus oryzae with the Pichia pastoris expression system

Neutral proteases are widely used in the textile, food and medical industries. This study was designed to obtain high expression levels of neutral protease I from Aspergillus oryzae 3.042 by using Pichia pastoris GS115 as the host strain for industrial purposes. The coding sequence of the target gene was modified, synthesized, and then cloned into the expression vector pHBM905BDM, which harbored the d1+2 × 201 AOX1 promoter and the MF4I leader sequence. The recombinant plasmid was transformed into Pichia pastoris GS115. The recombinant strain was used for high-density fermentation in a 4-L fermenter. The yield of the target protein reached 12.87 mg/mL, and the enzyme activity was approximately 49370 U/mL, which indicated that this enzyme was expressed in Pichia pastoris at a high level. The target protein was purified and characterized. Its optimum temperature and pH were 55 °C and 8.0, respectively. This enzyme was extremely sensitive to EDTA, which is consistent with the previous reports that it is a zinc-dependent metalloprotease. Our results indicated that low concentrations of zinc, calcium and magnesium ions stimulated the enzyme activity, whereas high concentrations inhibited its activity. In addition, calcium and magnesium ions increased the thermostability of the enzyme. All of the evidence indicated that this protease is a thermolysin-like peptidase.