The Electrostatic Basis of Diacylglycerol Pyrophosphate—Protein Interaction

Diacylglycerol pyrophosphate (DGPP) is an anionic phospholipid formed in plants, yeast, and parasites under multiple stress stimuli. It is synthesized by the phosphorylation action of phosphatidic acid (PA) kinase on phosphatidic acid, a signaling lipid with multifunctional properties. PA functions in the membrane through the interaction of its negatively charged phosphomonoester headgroup with positively charged proteins and ions. DGPP, like PA, can interact electrostatically via the electrostatic-hydrogen bond switch mechanism but differs from PA in its overall charge and shape. The formation of DGPP from PA alters the physicochemical properties as well as the structural dynamics of the membrane. This potentially impacts the molecular and ionic binding of cationic proteins and ions with the DGPP enriched membrane. However, the results of these important interactions in the stress response and in DGPP’s overall intracellular function is unknown. Here, using 31P MAS NMR, we analyze the effect of the interaction of low DGPP concentrations in model membranes with the peptides KALP23 and WALP23, which are flanked by positively charged Lysine and neutral Tryptophan residues, respectively. Our results show a significant effect of KALP23 on the charge of DGPP as compared to WALP23. There was, however, no significant effect on the charge of the phosphomonoester of DGPP due to the interaction with positively charged lipids, dioleoyl trimethylammonium propane (DOTAP) and dioleoyl ethyl-phosphatidylcholine (EtPC). Divalent calcium and magnesium cations induce deprotonation of the DGPP headgroup but showed no noticeable differences on DGPP’s charge. Our results lead to a novel model for DGPP—protein interaction.

Claudin-10a Deficiency Shifts Proximal Tubular Cl- Permeability to Cation Selectivity via Claudin-2 Redistribution

Background The tight junction proteins claudin-2 and claudin-10a form paracellular cation and anion channels, respectively, and are expressed in the proximal tubule. However, the physiological role of claudin-10a in the kidney has been unclear. Methods To investigate the physiologic role of claudin-10a, we generated claudin-10a-deficient mice; confirmed successful knockout by Southern blot, Western blot, and immunofluorescence staining; and analyzed urine and serum of knockout and wild-type animals. We also used electrophysiologic studies to investigate the functionality of isolated proximal tubules, and studied compensatory regulation by pharmacologic intervention, RNA sequencing analysis, Western blot, immunofluorescence staining, and respirometry. Results Mice deficient in claudin-10a were fertile and without overt phenotypes. Upon knockout, claudin-10a was replaced by claudin-2 in all proximal tubule segments. Electrophysiology showed conversion from paracellular anion preference to cation preference and a loss of paracellular Cl- over HCO3- preference. As a consequence, there was tubular retention of calcium and magnesium, higher urine pH, and mild hypermagnesemia. A comparison of other urine and serum parameters under control conditions and sequential pharmacologic transport inhibition, as well as unchanged fractional lithium excretion, suggested compensative measures in proximal and distal tubular segments. Changes in proximal tubular oxygen handling and differential expression of genes regulating fatty acid metabolism indicated proximal tubular adaptation. Western blot and immunofluorescence revealed alterations in distal tubular transport. Conclusions Claudin-10a is the major paracellular anion channel in the proximal tubule and its deletion causes calcium and magnesium hyperreabsorption by claudin-2 redistribution. Transcellular transport in proximal and distal segments and proximal tubular metabolic adaptation compensate for loss of paracellular anion permeability.

The Influence of Prophylactic Calcium and Magnesium Supplementation on Postoperative Quality of Life and Hypocalcemia After Total Thyroidectomy: Study Protocol for a Randomized Controlled Trial

Background: We want to investigate if a routine preoperative dietary supplementation of calcium and magnesium prior to thyroidectomy for nodular goiter and graves' disease can influence patients' outcome with regards to hypocalcemia associated symptoms and quality of life in order to reduce the risk of post-thyroidectomy hypocalcemia and to improve patient's quality of life.Methods: The study will be conducted as a two-armed randomized controlled trial including patients scheduled for total thyroidectomy. Patients assigned to the intervention group will receive calcium carbonate and magnesium oxide starting 2 weeks preoperatively. Primary outcome is the postoperative quality of life measured by the ThyPRO-39 and EQ-5D questionnaires. Secondary outcome is the assessment of postoperative biochemical (calcium and PTH levels) and clinical hypocalcemia (symptoms as reported by the patient).Discussion: A prophylactic dietary supplementation with calcium and magnesium, which could easily be implemented in the preoperative setting, could potentially help to avoid or reduce hypocalcemia-associated symptoms and improve quality of life. In the event of a positive outcome, this preoperative procedure can be an inexpensive way to prepare patients scheduled for thyroidectomy and can possibly reduce disease-specific costs by reducing the postoperative complication rate.Clinical Trial Registration: DRKS00017195 in the German clinical trials register (Deutsches Register Klinischer Studien, DRKS) on the 22.05.2019.

The effect of storage period on calcium and magnesium level and antimicrobial activity of goat’s milk kefir

The purpose of this reasearch was to determine the effect of storage period goat’s milk kefir (GMK) on total LAB, total mineral levels included Ca and Mg and antimicrobial activity to pathogenic microbes. The materials used was GMK. This research was conducted using Completely Randomized Design with 4 treatments and 4 replications. The treatments were storage period that consist of D0 (0 day), D1 (7 days), D2 (14 days), and D3 (21 days). Total calculation of LAB used TPC (Total Plate Count). Samples were diluted from 10-1 to 10-8, then incoculated PCA medium by using pour plate method. Petri dish was incubated at 37ºC for 48 hours. Calcium and magnesium levels were calculed base AAS method. The result of this study was analyzed using analysis of variance (ANOVA), then followed by Duncan’s Multiple Range Test (DMRT). The storage time treatment has a significant effect on the total microbial count, mineral levels (Ca and Mg) and inhibition zone of Staphylococcus aureus, Escherichia coli and Salmonella enterica serovar typhi on goat's milk kefir. Average clear zone Staphylococcus aureus 2.60 – 3.18 mm. Average inhibition zone Escherichia coli 2.09 – 3.32 mm. Average clear zone Salmonella enterica serovar typhi 1.97 – 2.72 mm.

Reusing Flowback and Produced Water with Different Salinity to Prepare Guar Fracturing Fluid

Economical and environmental concerns have forced the oil and gas industry to consider reusing flowback and produced water for fracturing operations. The major challenge is that the high-salinity of flowback water usually prevents its compatibility with several fracturing fluid additives. In this paper, the authors explored an economic and effective method to prepare guar fracturing fluids with different salinity waters. The main research idea was to use chelating agents to mask metal ions, such as calcium and magnesium, that are harmful to crosslinking. Firstly, a complexometric titration test was conducted to measure the chelating ability of three chelating agents. Secondly, through viscosity, crosslinking, and hanging tests, it was verified that the complex masking method could cope with the problem of high-valence metal ions affecting crosslinking. Thirdly, the preferred chelating agent was mixed with several other additives, including thickeners, crosslinkers, and pH regulators, to prepare the novel guar fracturing fluid. The comprehensive performances of the novel fluid system were tested such as temperature and shear resistance, friction reduction, gel-breaking performance, and core damage rate. The results show that the organophosphate chelating agent (i.e., CA-5) had the greatest ability to chelate calcium and magnesium ions. There was a good linear relationship between the dosage of CA-5 and the total molar concentration of calcium and magnesium ions in brine water. The main mechanism was that the chelating agent formed a complex with calcium and magnesium ions at a chelation ratio of 1:5. The test results of the comprehensive performance evaluation indicate that the prepared guar fracturing fluid met the requirements for field application, and the lower the salinity of the flowback water, the more it is economical and effective.