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ACS Synthetic Biology May 2019Here, we introduce a one-pot method for the bottom-up assembly of complex single- and multicompartment synthetic cells. Cellular components are enclosed within giant...
Here, we introduce a one-pot method for the bottom-up assembly of complex single- and multicompartment synthetic cells. Cellular components are enclosed within giant unilamellar vesicles (GUVs), produced at the milliliter scale directly from small unilamellar vesicles (SUVs) or proteoliposomes with only basic laboratory equipment within minutes. Toward this end, we layer an aqueous solution, containing SUVs and all biocomponents, on top of an oil-surfactant mix. Manual shaking induces the spontaneous formation of surfactant-stabilized water-in-oil droplets with a spherical supported lipid bilayer at their periphery. Finally, to release GUV-based synthetic cells from the oil and the surfactant shell into the physiological environment, we add an aqueous buffer and a droplet-destabilizing agent. We prove that the obtained GUVs are unilamellar by reconstituting the pore-forming membrane protein α-hemolysin and assess the membrane quality with cryotransmission electron microscopy (cryoTEM), fluorescence recovery after photobleaching (FRAP), and zeta-potential measurements as well as confocal fluorescence imaging. We further demonstrate that our GUV formation method overcomes key challenges of standard techniques, offering high volumes, a flexible choice of lipid compositions and buffer conditions, straightforward coreconstitution of proteins, and a high encapsulation efficiency of biomolecules and even large cargo including cells. We thereby provide a simple, robust, and broadly applicable strategy to mass-produce complex multicomponent GUVs for high-throughput testing in synthetic biology and biomedicine, which can directly be implemented in laboratories around the world.
Topics: Cryoelectron Microscopy; Fluorescence Recovery After Photobleaching; Hemolysin Proteins; Magnesium Chloride; Oils; Surface-Active Agents; Synthetic Biology; Unilamellar Liposomes; Water
PubMed: 31042361
DOI: 10.1021/acssynbio.9b00034 -
Kidney International Jun 2001A new protein, named paracellin 1 (PCLN-1), expressed in human thick ascending limb (TAL) tight junctions, possibly plays a critical role in the control of magnesium and...
BACKGROUND
A new protein, named paracellin 1 (PCLN-1), expressed in human thick ascending limb (TAL) tight junctions, possibly plays a critical role in the control of magnesium and calcium reabsorption, since mutations of PCLN-1 are present in the hypomagnesemia hypercalciuria syndrome (HHS). However, no functional experiments have demonstrated that TAL magnesium and calcium reabsorption were actually impaired in patients with HHS.
METHODS
Genetic studies were performed in the kindred of two unrelated patients with HHS. Renal magnesium and calcium reabsorption in TAL were analyzed in one homozygous affected patient of each family, one patient with extrarenal hypomagnesemia (ERH), and two control subjects (CSs).
RESULTS
We found two yet undescribed mutations of PCLN-1 (Gly 162 Val, Ala 139 Val). In patients with HHS, renal magnesium and calcium reabsorptions were impaired as expected; NaCl renal conservation during NaCl deprivation and NaCl tubular reabsorption in diluting segment were intact. Furosemide infusion in CS markedly increased NaCl, Mg, and Ca urinary excretion rates. In HHS patients, furosemide similarly increased NaCl excretion, but failed to increase Mg and Ca excretion. Acute MgCl(2) infusion in CS and ERH patient provoked a dramatic increase in urinary calcium excretion without change in NaCl excretion. When combined with MgCl(2) infusion, furosemide infusion remained able to induce normal natriuretic response, but was unable to increase urinary magnesium and calcium excretion further. In HHS patients, calciuric response to MgCl(2) infusion was blunted.
CONCLUSION
This study is the first to our knowledge to demonstrate that homozygous mutations of PCLN-1 result in a selective defect in paracellular Mg and Ca reabsorption in the TAL, with intact NaCl reabsorption ability at this site. In addition, the study supports a selective physiological effect of basolateral Mg(2+) and Ca(2+) concentration on TAL divalent cation paracellular permeability, that is, PCLN-1 activity.
Topics: Adolescent; Adult; Calcium; Cations, Divalent; Child; Claudins; Diuretics; Family Health; Female; Furosemide; Genotype; Homozygote; Humans; Loop of Henle; Magnesium Chloride; Male; Membrane Proteins; Middle Aged; Natriuresis; Nephrocalcinosis; Pedigree; Phenotype; Point Mutation; Sodium Chloride
PubMed: 11380823
DOI: 10.1046/j.1523-1755.2001.00736.x -
Journal of Microbiological Methods Jan 2022Resuscitation and detection of stressed total coliforms in chlorinated water samples is needed to assess and prevent health effects from adverse exposure. In this study,...
Resuscitation and detection of stressed total coliforms in chlorinated water samples is needed to assess and prevent health effects from adverse exposure. In this study, we report that the addition of a growth enhancer mix consisting of trehalose, sodium pyruvate, magnesium chloride, and 1× trace mineral supplement improved growth of microorganisms from chlorinated secondary effluent in the base medium with Colilert-18. Improving growth of chlorine stressed microorganisms from secondary effluent is crucial to decreased detection time from 18 to 8 h.
Topics: Bacterial Load; Chlorine; Culture Media; Environmental Monitoring; Escherichia coli; Fluoridation; Magnesium Chloride; Pyruvates; Sewage; Trehalose; Water Microbiology
PubMed: 34774876
DOI: 10.1016/j.mimet.2021.106364 -
Clinical Cardiology Aug 1988To evaluate the effect of intravenous magnesium (Mg) treatment on the inotropic state of the heart and maximal work capacity, 9 healthy volunteers were entered in a... (Clinical Trial)
Clinical Trial Randomized Controlled Trial
To evaluate the effect of intravenous magnesium (Mg) treatment on the inotropic state of the heart and maximal work capacity, 9 healthy volunteers were entered in a double-blind, placebo-controlled, cross-over study. Separated by an interval of three weeks, the volunteers were tested twice, each time randomly allocated to receive either an intravenous injection of 10 mmol magnesium chloride dissolved in 100 ml isotonic sodium chloride or placebo of isotonic sodium chloride only. Before and after each infusion myocardial inotropism was evaluated by echocardiography. Mitral-septal distance (MSA) was used as a measure for ejection fraction. On each test day an ergometer bicycle exercise test was performed, and maximal work capacity was calculated. Magnesium treatment reduced the MSA (from 4.2 to 2.9 mm, p = 0.07), while no difference was found after placebo treatment. Likewise, a tendency toward increasing fractional shortening after magnesium treatment was detected, although this difference was not statistically significant (p = 0.1). No difference in maximal work capacity between the magnesium and placebo periods was found. Serum magnesium concentrations and placebo periods was found. Serum magnesium concentrations rose significantly after the infusions (from 0.82 to 1.38 mmol/l, p less than 0.001). It is concluded that intravenous magnesium does not exert a negative inotropic effect on the myocardium as previously stated. On the contrary, we found a tendency toward a positive inotropic effect. However, the observed differences are of borderline statistical significance and a more extended study, employing invasive measurements of cardiac inotropism appears to be necessary.
Topics: Adult; Double-Blind Method; Echocardiography; Exercise Test; Female; Humans; Magnesium; Magnesium Chloride; Male; Myocardial Contraction; Random Allocation; Stimulation, Chemical; Stroke Volume
PubMed: 3168339
DOI: 10.1002/clc.4960110807 -
Journal of Bacteriology Nov 2012The diarrheal pathogen Campylobacter jejuni and other gastrointestinal bacteria encounter changes in osmolarity in the environment, through exposure to food processing,...
The diarrheal pathogen Campylobacter jejuni and other gastrointestinal bacteria encounter changes in osmolarity in the environment, through exposure to food processing, and upon entering host organisms, where osmotic adaptation can be associated with virulence. In this study, growth profiles, transcriptomics, and phenotypic, mutant, and single-cell analyses were used to explore the effects of hyperosmotic stress exposure on C. jejuni. Increased growth inhibition correlated with increased osmotic concentration, with both ionic and nonionic stressors inhibiting growth at 0.620 total osmol liter(-1). C. jejuni adaptation to a range of osmotic stressors and concentrations was accompanied by severe filamentation in subpopulations, with microscopy indicating septum formation and phenotypic diversity between individual cells in a filament. Population heterogeneity was also exemplified by the bifurcation of colony morphology into small and large variants on salt stress plates. Flow cytometry of C. jejuni harboring green fluorescent protein (GFP) fused to the ATP synthase promoter likewise revealed bimodal subpopulations under hyperosmotic stress. We also identified frequent hyperosmotic stress-sensitive variants within the clonal wild-type population propagated on standard laboratory medium. Microarray analysis following hyperosmotic upshift revealed enhanced expression of heat shock genes and genes encoding enzymes for synthesis of potential osmoprotectants and cross-protective induction of oxidative stress genes. The capsule export gene kpsM was also upregulated, and an acapsular mutant was defective for growth under hyperosmotic stress. For C. jejuni, an organism lacking most conventional osmotic response factors, these data suggest an unusual hyperosmotic stress response, including likely "bet-hedging" survival strategies relying on the presence of stress-fit individuals in a heterogeneous population.
Topics: Animals; Bacterial Proteins; Bacteriological Techniques; Campylobacter jejuni; Chickens; Gene Expression Regulation, Bacterial; Glucose; Humans; Intestines; Magnesium Chloride; Osmotic Pressure; Potassium Chloride; Sodium Chloride; Stress, Physiological; Time Factors; Transcriptome
PubMed: 22961853
DOI: 10.1128/JB.01409-12 -
Scientific Reports Jul 2023Non-invasive delivery of hyaluronan into the stratum corneum (SC) is extremely difficult because of its high molecular weight and the strong barrier of the SC. We...
Non-invasive delivery of hyaluronan into the stratum corneum (SC) is extremely difficult because of its high molecular weight and the strong barrier of the SC. We developed a safe method of administering hyaluronan into the human SC and determined its penetration route. The amount of hyaluronan that penetrated into the SC was 1.5-3 times higher in the presence of magnesium chloride hexahydrate (MgCl) than other metal chlorides. The root-mean-square radius of hyaluronan in water decreased with the addition of MgCl. Moreover, MgCl solutions maintained their dissolved state on a plastic plate for a long time, suggesting that size compaction and inhibition of hyaluronan precipitation on the skin enhanced hyaluronan into the SC. Our results also strongly suggest that an intercellular route contributes to the penetration of hyaluronan from the upper to the middle layer of the SC. No disruption to the SC barrier was observed after continuous use once a day for 1 month, demonstrating the potential of our method for the safe, topical application of hyaluronan.
Topics: Humans; Magnesium Chloride; Hyaluronic Acid; Molecular Weight; Epidermis; Skin
PubMed: 37402762
DOI: 10.1038/s41598-023-37718-5 -
The Journal of Biological Chemistry Jan 2014Noncoding RNAs must function in the crowded environment of the cell. Previous small-angle x-ray scattering experiments showed that molecular crowders stabilize the...
Noncoding RNAs must function in the crowded environment of the cell. Previous small-angle x-ray scattering experiments showed that molecular crowders stabilize the structure of the Azoarcus group I ribozyme, allowing the ribozyme to fold at low physiological Mg(2+) concentrations. Here, we used an RNA cleavage assay to show that the PEG and Ficoll crowder molecules increased the biochemical activity of the ribozyme, whereas sucrose did not. Crowding lowered the Mg(2+) threshold at which activity was detected and increased total RNA cleavage at high Mg(2+) concentrations sufficient to fold the RNA in crowded or dilute solution. After correcting for solution viscosity, the observed reaction rate was proportional to the fraction of active ribozyme. We conclude that molecular crowders stabilize the native ribozyme and favor the active structure relative to compact inactive folding intermediates.
Topics: Azoarcus; Magnesium Chloride; Models, Chemical; Molecular Weight; Nucleic Acid Conformation; Protein Folding; RNA, Catalytic; Solutions; Viscosity
PubMed: 24337582
DOI: 10.1074/jbc.M113.527861 -
PloS One 2016The activated partial thromboplastin time (APTT) assay is a basic hemostatic assay based on the time it takes for clots to form in plasma samples after the addition of...
The activated partial thromboplastin time (APTT) assay is a basic hemostatic assay based on the time it takes for clots to form in plasma samples after the addition of calcium chloride. It is used to screen for various coagulation disorders. Several previous reports have suggested that magnesium (Mg) might contribute to coagulation reactions by binding to specific coagulation proteins. We investigated the effects of Mg on the APTT. In healthy controls, the APTT was significantly prolonged in proportion to the increase in the concentration of magnesium chloride in the range from 2.1 to 16.7 mmol/L. Among eight samples from patients with various disorders that exhibited prolonged APTT, two samples demonstrated shorter APTT when Mg was added, both of which were from patients that were positive for lupus anticoagulant. When we examined 206 clinical APTT samples, we found that Mg shortened the APTT of two samples. These two samples were also from lupus anticoagulant-positive patients (p-value: <0.003). Our findings regarding the unique effects of exogenous Mg on the APTT of lupus anticoagulant-positive patients might shed light on the role of Mg in APTT assays and lead to the development of a novel screening method for lupus anticoagulant.
Topics: Adult; Aged; Aged, 80 and over; Antiphospholipid Syndrome; Case-Control Studies; Female; Humans; Ions; Lupus Coagulation Inhibitor; Magnesium; Magnesium Chloride; Male; Middle Aged; Partial Thromboplastin Time
PubMed: 27355205
DOI: 10.1371/journal.pone.0157835 -
Journal of Clinical Microbiology Feb 1985Two temperate bacteriophages of differing morphology and host range were isolated by screening 94 isolates of Clostridium difficile. Phage 41 had a 300-nm flexible tail,...
Two temperate bacteriophages of differing morphology and host range were isolated by screening 94 isolates of Clostridium difficile. Phage 41 had a 300-nm flexible tail, whereas phage 56 had a shorter tail with a contractile sheath. Electron microscopy of phage 56 lysates exposed to elevated magnesium concentrations showed small virus-like particles which were 21 nm in diameter. The addition of MgCl2 to semisolid agar overlays enhanced both the titer and plaque size of phage 56. Phage 56 was more temperature labile than phage 41 and demonstrated unusual lability in buffer at pH 7.0. One-step growth and adsorption experiments revealed that both phages had latent periods of about 60 min, but phage 56 adsorbed to its indicator strain more efficiently. Phage 56, which was obtained from a toxigenic strain of C. difficile, was used to lysogenize its nontoxigenic indicator strain, but no conversion to toxigenicity was observed in this strain.
Topics: Bacteriophages; Cations, Divalent; Clostridium; Cytotoxins; Hydrogen-Ion Concentration; Magnesium; Magnesium Chloride
PubMed: 3972994
DOI: 10.1128/jcm.21.2.251-254.1985 -
Poultry Science May 1985Shells of hatching and nonhatching Large White turkey eggs were examined for magnesium, calcium, and phosphorus content. Eggshells containing embryos that hatched... (Comparative Study)
Comparative Study
Shells of hatching and nonhatching Large White turkey eggs were examined for magnesium, calcium, and phosphorus content. Eggshells containing embryos that hatched contained significantly (P less than .05) more magnesium than shells from nonhatching eggs. No differences were seen in their calcium content. Eggshells from pipped eggs contained significantly (P less than .05) more phosphorus than shells from eggs that were not pipped or those that hatched. Calcium and magnesium in turkey eggshells declined as time in lay increased. Injections of exogenous magnesium both at setting and transfer significantly (P less than .05) depressed hatchability, whereas exogenous calcium injected at transfer significantly (P less than .05) improved hatchability. Injections of exogenous calcium at the time of setting significantly (P less than .05) depressed hatchability. It was concluded that the mineral content of turkey eggshells may influence embryo physiology and hatchability.
Topics: Animals; Calcium; Calcium Chloride; Egg Shell; Embryo, Nonmammalian; Magnesium; Magnesium Chloride; Phosphorus; Turkeys
PubMed: 4001061
DOI: 10.3382/ps.0641020