Elevation of Hyaluronan Synthase by Magnesium Supplementation Mediated through the Activation of GSK3 and CREB in Human Keratinocyte-Derived HaCaT Cells

Skin barrier damage is present in the patients with hereditary disorders of the magnesium channel, but the molecular mechanism has not been fully understood. We found that the expressions of hyaluronan synthase (HAS), HAS2 and HAS3 are influenced by MgCl2 concentration in human keratinocyte-derived HaCaT cells. The exposure of cells to a high concentration (5.8 mM) of MgCl2 induced the elevation of HAS2/3 expression, which was inhibited by mRNA knockdown of nonimprinted in Prader-Willi/Angelman syndrome-like domain containing 4 (NIPAL4). Similarly, the content of hyaluronic acid (HA) was changed according to MgCl2 concentration and the expression of NIPAL4. The MgCl2 supplementation increased the reporter activities of HAS2/3, which were inhibited by NIPAL4 knockdown, indicating that the expressions of HAS2/3 are up-regulated at the transcriptional level. The reporter activities and mRNA levels of HAS2/3, and the production of HA were inhibited by CHIR-99021, a glycogen synthase kinase-3 (GSK3) inhibitor, and naphthol AS-E, a cyclic AMP-response element binding protein (CREB) inhibitor. Furthermore, the mutation in putative CREB-binding sites of promoter region in HAS2/3 genes inhibited the MgCl2 supplementation-induced elevation of promoter activity. Our results indicate that the expressions of HAS2/3 are up-regulated by MgCl2 supplementation in HaCaT cells mediated through the activation of GSK3 and CREB. Magnesium may play a pivotal role in maintaining the skin barrier function and magnesium supplementation may be useful to enhance moisturization and wound repair in the skin.

Assay optimization for measuring the alternate complement pathway activity in Asian seabass (Lates calcarifer)

Hastuti SD, Barton MD, Pyecroft SB, Costabile M. 2020. Assay optimization for measuring the alternate complement pathway activity in Asian seabass (Lates calcarifer). Biodiversitas 21: 3034-3040. Complement proteins are one component of innate immunity present in fish. The measurement of complement activity in fish can be used to monitor the health status of fish. This is particularly important in Asian seabass (Lates calcarifer) aquaculture, where disease can impact on productivity. We have found an optimal condition assay for measuring the alternate complement pathway (ACP) activity of Asian seabass which includes Magnesium chloride (MgCl2) concentration, buffer pH, incubation temperature and incubation time. The assay was optimized using pooled serum of Asian seabass, diluted in Magnesium ethylenediamine tetraacetic acid gelatine veronal buffer (Mg-EDTA-GVB) and added with Rabbit red blood cells (RRBC) suspension. Subsequently, the suspension was incubated and centrifuged. The supernatant was removed and transferred to a well plate and the optical density (OD) was measured at 540 nm. The optimal condition obtained included a 7.5 mM MgCl2, pH optimum of 7.5, 25°C incubation temperature, and a 30 minutes incubation period. The presently developed assay was robust, rapid, and reliable to be used in monitoring the health status of Asian seabass in aquaculture farms. It can be used as guidance in further immunological studies on this fish.

MAGNESIA CORROSION OF GROUTING MATERIALS

The issue of magnesia corrosion of grouting materials in oil and gas wells is very relevant in the construction of oil and gas wells since magnesia salts can lead to the destruction of Portland cement-based cement stone within few months. When fastening powerful intervals of salt deposits represented by magnesium salts, the use of magnesia cements is effective. However, individual layers and interlayers containing dissolved magnesium salts are not individually cemented, but overlap over the entire interval of the open hole with cement Portland cement, which can be destroyed due to magnesia corrosion. The main aim of the paper is to analyze the corrosion of Portland cement stone in aggressive environments of magnesia. As the quantitative indicators characterizing the degree of stone damage, the thickness of the damaged layer and the stone resistance coefficient are taken, characterized by the ratio of the compressive strength or bending strength of stone samples after being in an aggressive environment to the strength of control samples at the same hardening time. The corrosion resistance of cement stone was assessed after 8 weeks in an aggressive magnesia environment. Also, the role of MgCl2 concentration on the mechanism of corrosion damage to cement stone was studied. The use of reducing the water-cement ratio and adding palygorskite clay to reduce the porosity of cement stone and reduce the rate of corrosion damage is proposed. The kinetics and the main factors affecting the corrosion process were considered, an X-ray diffraction analysis of corrosion products and unaffected cement stone was carried out.

Study of corrosion stability of a cement stone in magnesia aggressive environment

One of the main tasks in the construction of oil and gas wells is to ensure the high quality of well casing. It is especially difficult to get it in wells, in sections of which there are salt-bearing strata. From the salts, the most dangerous are magnesia salts, which can lead to the destruction of the stone based on portland cement within a few months. The report presents the results of experimental studies on the corrosion of cement stone in aggressive magnesia media.The quantitative indicators reflecting the degree of damage to the stone are taken as the thickness of the damaged layer and the coefficient of stone resistance, characterized by the ratio of the ultimate strength of stone samples for compression or bending stored in an aggressive environment to the strength of control samples at the same time of hardening. In the course of the research, the corrosion resistance of the cement stone to the magnesian corrosive environment was assessed, after 8 weeks in a medium with a constant concentration of MgCl2. In addition, the effect of MgCl2 concentration on the cement stone corrosion mechanism was investigated. The use of the palygorskit additive and the reduction of water cement ratio to reduce the porosity of the cement stone and reduce the rate of corrosion damage are proposed. The kinetics and the main factors affecting the corrosion process are considered, and the x-ray structural analysis of corrosion products and unaffected cement stone is carried out.

Building a molecular typing protocol for rs1801133 based on real-time PCR HRM technique

rs1801133 is a single nucleotide polymorphism (SNP) located in the sequence of MTHFR on human chromosome 1. The alleles of this SNP affect the activity of the MTHFR enzyme. People bearing C/T genotype have 66% activity of MTHFR while people with T/T genotype have only 25% activity. These reduced activities of MTHFR cause homocysteinemia. There are several publications on the relationship between homocysteinemia and human diseases such as cardiovascular disease, neurological diseases, abnormal fetus, infertility and cancer. In this study, we built a molecular protocol for genotyping rs1801133 using real-time PCR HRM technique. This protocol could be used for diagnosis of molecular mechanism of homocysteinemia causing the mentoned above diseases as well as for the study of the relationship between rs1801133 and other human diseases. We successfully designed the primer pairs for genotyping and nucleotide sequencing rs1801133 by real-time PCR HRM and Sanger sequencing method. We also examined the optimal MgCl2 concentration for clear differentiation of three rs1801133 genotypes. Performance characteristics of the real-time PCR HRM protocol included of specificity, repeatability, reproducibility was evaluated and it showed good results. Comparison of genotyping results of rs1801133 between the realtime PCR HRM method and the Sanger nucleotide sequencing method showed good concordances. Finally, this real-time PCR HRM protocol for rs1801133 genotyping was applied on 100 human DNA samples to evaluate the clinical utility of the protocol.

Optimization of Cellulase Production by Aspergillus niger ITBCC L74 with Bagasse as Substrate using Response Surface Methodology

Cellulase is a very important enzyme for lignocelluloses based ethanol production. Bagasse contains mainly cellulose (57.76%), hemicellulose (12.44%), lignin (21.34%), and others (7.96%). Lignocellulosic material has been considered as the good option for cellulase production because it is cheap and already available in a huge amount. The objective of this research was to produce cellulase enzyme and to optimize it by using response surface methodology. The bagasse with water content of 80% was incubated with 2 ml inoculum of Aspergillus niger ITBCC L74 in a 250 ml Erlenmeyer flask. After reaching the specified time the enzyme was extracted and then determined for its activity. Effect of process parameters such as pH, urea and MgCl2 addition were studied. The optimal cellulase activity was achieved at urea concentration of 4.5% (w/w), MgCl2 concentration of 1 mM and pH of 3.5, with maximum enzyme activity was 0.630 U/gr.

An optimized HRM method for diagnosis of G6PD deficiency in kinh Vietnamese via viangchan mutation

With Glucose-6-phosphate dehydrogenase (G6PD) deficiency being the most common enzyme disorder in human, there have been 184 discovered point mutations and several methods that have been applied for diagnosing this disease. However, these techniques often pose several major problems such as being time-consuming, low sensitivity and high cost. Recently, the High Resolution Melting (HRM) has been studied and proven to be effective for DNA genotyping, mutation scanning and sequence matching. Therefore, HRM has been chosen for diagnosing G6PD deficiency via Viangchan mutation in this study. In this study, a total of 56 dried blood spot samples (including six control samples which were known the exact genotype by sequencing and fifty unknown samples) were collected and extracted DNA by using QIAamp DNA Blood Mini Kit. Primers for HRM analysis were designed through by the Umelt software. Then HRM optimization was carried out for annealing temperature of primers (Ta) and MgCl2 concentration on six control samples. The optimized HRM protocol with 2.5 μM of MgCl2 and Ta at 62oC was applied for fifty G6PD samples and then comparing with ARMS-PCR genotyping results for the validation process. In the final step, genotyping results were confirmed by sequencing. In a results, both sensitivity and specificity of this technique reached 100%. Based on these favorable outcomes, this study has successfully optimized the HRM conditions for diagnosing fifty G6PD samples. It was such an essential precondition that showed HRM could be applied for other types of G6PD through other types of mutations such as Canton mutation or continues to be developed for HRM-Multiplex reactions.

Purification and optimization of conditions for DNA polymerase isolated from thermophile bacteria Bacillus caldolyticus

Тhermophilic bacteria Bacillus caldolyticus isolated from the hot spring in Bansko, Republic of Macedonia, were used for the isolation of DNA polymerase. Bacterial cells were disrupted by sonication and the first step in the purification of DNA polymerase was 40% ammonium sulfate precipitation. This was followed by chromatographic procedures on Sephadex G-50, DE-52 and CM-52 cellulose. DNA polymerase activity was analyzed at each step of purification using the incorporation of 3H dATP in activated calf thymus DNA. The purity of the DNA polymerase was analyzed on SDS PAGE. Optimal conditions of activity were determined for temperature, pH, dNTP and Mg++ concentration. In addition, the effect of ethanol and EDTA as possible inhibitors of polymerase activity was also analyzed. The optimal temperature of DNA polymerase was 66 ºC; the optimal pH was 7.2, optimal MgCl2 concentration was 2.5 mM, and the optimal substrate concentration was 2.5 × 10–6 M dNTP. The inhibitory effect of EDTA and ethanol on DNA polymerase was above 10 mM and 10%, respectively.

Optimizing the PCR HRM for initial study of the association between rs10941679 and breast cancer in Vietnamese population

As the second most popular cancer in the world, breast cancer affects millions of people and causes a large number of deaths every year. Identifying the genetic marker is one of approaches of early diagnosis for following cancer and give a correct treatment. Many recent studies have shown several SNPs including rs10941679 (located at the uptream of MRPS30 gene) are strongly associated with breast cancer in European and other populations. The association between rs10941679 and breast cancer in Vietnamese population has been investigated in this study using High Resolution Melt (HRM) method as a tool for genotyping. HRM was designed on Umelt sofware and was optimized based on annealing temperature and MgCl2 concentration gradient in order to have 3 distinct melting curves from 3 genotypes of rs10941679. The optimal HRM conditions were selected with Tm=58 oC and MgCl2=3.0 mM and 100 Cases/control samples were genotyped by the optimimal HRM condition. The results showed that the frequency of the risk allele G in case and control group were 48.5 % and 54.0 %, respectively. Regression analysis on the presense of the risk allele and risk allele-containing genotypes has shown no association between rs10941679 and breast cancer in Vietnamese population (OR = 0.80; 95% CI = [0.54 – 1.19]; PG = 0.27; PGG/GA = 0.56). The power of this result was estimated to be 12.02 %. To obtain the power up to 90 %, the sample size up to 1691 cases/controls is needed. Due to its low posibility in the assocation with breast cancer in Vietnamese population, rs1094169 is not recommended for further study in finding the genetic for markers cancer in Vietnamese population.

Optimization of PCR Conditions for DNA Amplification of Common Buckwheat Using EST Primers

Under optimal conditions the PCR reaction is very efficient; microgram quantities may be synthesized from a single molecule of substrate DNA. DNA of four lines of common buckwheat (Kyusu, Canada, Miyazaki and Botansoba) was used to optimize PCR reaction and cycling program of 26 primers for DNA amplification of common buckwheat. Annealing temperature (Ta), PCR cycle number and MgCl2 concentration were considered optimum if the single clear band was observed. Of the 26 primers Ta of only 10 primers could be optimized. Three primer pairs performed best at Ta of 54°C. The optimum concentration of MgCl2 was found to be 1.5mM for all primer pairs. Similarly the number of PCR cycles was found to be 40 for all 10 primer pairs except for primer pair 57. Optimized PCR conditions were used for subsequent studies such as transferability of EST primers to other Fagopyrum species and construction of linkage map.Nepal Agric. Res. J. Vol. 8, 2007, pp. 1-6DOI: http://dx.doi.org/10.3126/narj.v8i0.11563