Salting-out Methods Research Articles

Skin Byproducts of Reinhardtius hippoglossoides (Greenland Halibut) as Ecosustainable Source of Marine Collagen

Collagen is a ubiquitous protein present in the extracellular matrix of all major metazoan animals, with approximately 28 different human collagen types described in the literature, each with unique physicochemical properties. Collagens found broad application in the cosmeceutical, pharmaceutical, and biomedical fields and can be isolated from environmentally sustainable sources such as marine byproducts, which are abundant in the fish processing industry and are highly appealing low-cost sources. In this study, marine collagen was isolated from the skins of Greenland halibut (Reinhardtius hippoglossoides), an unexplored byproduct from fish processing plants, using three different collagen extraction methods, due to the use of distinct salting-out methods using a solution of 2.6 M NaCl + 0.05 M Tris-HCl pH = 7.5, (method I); a combination of 0.7 M NaCl followed by a solution of 2.3 M NaCl + 0.05 M Tris-HCl pH = 7.5 (method II); and one method using only 0.9 M NaCl (method III), yielding COLRp_I, COLRp_II, and COLRp_III collagens. These extracted type I collagens were produced with a yield of around 2 and 4% and characterized regarding the physicochemical properties, considering possible biotechnological applications. This work evidenced that the typical triple helix structure conformation was preserved in all extraction methods, but influenced the thermal behavior, intrinsic morphology, and moisture capacity of the collagens, with interest for biotechnological application, as the incorporation as an ingredient in cosmetic formulation. Furthermore, the use of collagen isolated from skin byproducts represents a high economic value with decreasing collagen cost for industrial purposes and is also an environmentally sustainable source for industrial uses.

Evaluation of high molecular weight DNA extraction methods for long-read sequencing of Shiga toxin-producing Escherichia coli.

Next generation sequencing has become essential for pathogen characterization and typing. The most popular second generation sequencing technique produces data of high quality with very low error rates and high depths. One major drawback of this technique is the short reads. Indeed, short-read sequencing data of Shiga toxin-producing Escherichia coli (STEC) are difficult to assemble because of the presence of numerous mobile genetic elements (MGEs), which contain repeated elements. The resulting draft assemblies are often highly fragmented, which results in a loss of information, especially concerning MGEs or large structural variations. The use of long-read sequencing can circumvent these problems and produce complete or nearly complete genomes. The ONT MinION, for its small size and minimal investment requirements, is particularly popular. The ultra-long reads generated with the MinION can easily span prophages and repeat regions. In order to take full advantage of this technology it requires High Molecular Weight (HMW) DNA of high quality in high quantity. In this study, we have tested three different extraction methods: bead-based, solid-phase and salting-out, and evaluated their impact on STEC DNA yield, quality and integrity as well as performance in MinION long-read sequencing. Both the bead-based and salting-out methods allowed the recovery of large quantities of HMW STEC DNA suitable for MinION library preparation. The DNA extracted using the salting-out method consistently produced longer reads in the subsequent MinION runs, compared with the bead-based methods. While both methods performed similarly in subsequent STEC genome assembly, DNA extraction based on salting-out appeared to be the overall best method to produce high quantity of pure HMW STEC DNA for MinION sequencing.

Precipitant-Free Crystallization of Lysozyme and Glucose Isomerase by Drying

Protein crystallization is usually conducted by using precipitants, although the “salting-out” phenomenon is still unclear and complex. Moreover, the addition of precipitants sometimes results in irreversible disordered precipitation of protein molecules. Although precipitant-free lysozyme crystals obtained by centrifugal concentration showed significant changes in three-dimensional structure compared to the structure of salted-out crystals, it was rather difficult to mount crystals from a viscous dense liquid phase after centrifugal concentration, and the quality of the crystals often deteriorated during the mounting process. Here we present novel precipitant-free crystallization methods, which were effective for lysozyme and glucose isomerase. Tetragonal lysozyme crystals were successfully crystallized in a glass capillary simply by drying highly concentrated lysozyme solution in the presence of 0.01 M hydrochloric acid without using any precipitants. Glucose isomerase dissolved in ultra-pure water was also successfully crystallized in hanging drops by drying highly concentrated solution under low-humidity conditions. Oscillation images of the obtained crystals were safely collected without handling; they clearly indicated the crystals had a tetragonal form for lysozyme and an orthorhombic form for glucose isomerase, and their lattice parameters are similar to those of previously reported crystals obtained by salting-out methods.

Effects of the mTOR and AKT genes polymorphisms on systemic lupus erythematosus risk.

The systemic lupus erythematosus (SLE) is an autoimmune disease, leading to inflammatory response and systemic consequences. The mammalian target of rapamycin (mTOR) is a therapeutic target for autoimmune diseases like SLE. The aim of this study was to evaluate the effects of the mTOR rs2295080 and rs2536 polymorphisms and AKT1 rs2494732 gene polymorphism on SLE development. 2ml of peripheral blood was collected from 165 SLE patients and 170 controls in EDTA-containing tubes. The salting-out and PCR-RFLP methods were used for DNA extraction and genotype analysis, respectively. Based on the regression analysis, the frequency of TT genotype of mTOR rs2295080 polymorphism was significantly higher in the case group than that of the control group, with a 2.6-fold increased risk of SLE. There was also a significant difference between the two groups in terms of allelic distribution. No statistically significant association was found between TheAKT1 rs2494732 and mTORrs2536 polymorphisms and SLE development. Our results showed that the TT genotype and T allele ofmTOR rs2295080 polymorphism were risk factors for developing SLE. However, there was no significant association between mTORrs2536 and AKT1 rs2494732 polymorphisms and the SLE risk.

Differences in Quality of DNA Isolated from Fresh and Frozen Peripheral Blood with Salting-Out Methods

Differences in Quality of DNA Isolated from Fresh and Frozen Peripheral Blood with Salting-Out Methods

A method for improving the efficiency of DNA extraction from clotted blood samples.

BackgroundThe efficient and rapid extraction of high‐quality genomic DNA from clotted blood samples, which normally have a low yield and poor quality, is an important factor in genomic research. The objective of this study was to develop a simple and safe technique for dispersing the blood clots by the ball bearing metal shots. Normally, such clot samples may not have an acceptable yield by conventional DNA extraction methods. Also, in the present study, we have further investigated to improve salting‐out DNA extraction methods.MethodsInitially, 500 µL phosphate‐buffered saline (PBS) (1×) and two ball bearing metal shots were added to each tube of the clotted blood sample and then were gently rotated in an electric laboratory rotator for 1 hour at room temperature (18‐25°C). Genomic DNA was then extracted from samples using a modified salting‐out method and a modified QIAamp® DNA Blood Midi Kit and was compared with QIAamp® DNA Blood Midi Kit as a control. An assessment of the concentration and quality of the extracted DNA was performed using the UV‐visible spectrophotometer. The isolated DNA proved amenable to PCR amplification and gel electrophoresis.ResultsThe yield and purity of DNA obtained by these three methods were significantly different (P < 0.001), with a higher yield in the modified salting‐out method.ConclusionsOur proposed modified salting‐out method is simple and efficient for the isolation of DNA from old blood clot samples. It is both easy to use and is of low cost in routine laboratory tasks.

Collagen Extracted from Bigeye Tuna (Thunnus obesus) Skin by Isoelectric Precipitation: Physicochemical Properties, Proliferation, and Migration Activities.

Collagen was extracted from bigeye tuna (Thunnus obesus) skins by salting-out (PSC-SO) and isoelectric precipitation (PSC-IP) methods. The yield of the PSC-IP product was approximately 17.17% (dry weight), which was greater than the yield obtained from PSC-SO (14.14% dry weight). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that collagen from bigeye tuna skin belongs to collagen type I. Inductively coupled plasma mass spectrometry results indicate that the heavy metal abundance in PSC-IP was lower than the maximum acceptable amounts according to Chinese regulatory standards. In addition, results from a methylthiazolyldiphenyl-tetrazolium bromide assay and an in vitro scratch assay demonstrated that PSC-IP could promote the proliferation and migration of NIH-3T3 fibroblasts. Overall, results suggest PSC-IP could be used to rapidly extract collagen from marine by-products instead of traditional salting-out methods. Collagen from bigeye tuna skin may also have strong potential for cosmetic and biomedical applications.

Separation of immunoglobulins from colostrum using methods based on salting-out techniques

This study aimed to obtain a purified IgG fraction from bovine colostrum using salting-out precipitation methods as base techniques. As the first step after skimming of colostrum, isoelectric precipitation or rennet coagulation were used for casein removal. IgG concentrations in raw material and final products were determined using the ELISA method. SDS-PAGE electrophoresis was used for purity determination of the final fraction of IgG. According to the obtained results, the combination of precipitation, gel filtration, and cross-flow filtration techniques enables to separate the IgG fraction with purity up to 90% and yield up to 91%. Acid whey has a higher yield in comparison with sweet whey and both ammonium sulphate and sodium sulphate have similar effectivity for precipitation of IgG. The final concentrate of IgG can be used as a standalone product or as an additive to other food supplements.

Reward circuitry responsivity to food predicts future increases in body mass: Moderating effects of DRD2 and DRD4

ObjectiveTo determine whether responsivity of reward circuitry to food predicts future increases in body mass and whether polymorphisms in DRD2 and DRD4 moderate these relations. DesignThe functional magnetic resonance imaging (fMRI) paradigm investigated blood oxygen level dependent activation in response to imagined intake of palatable foods, unpalatable foods, and glasses of water shown in pictures. DNA was extracted from saliva samples using standard salting-out and solvent precipitation methods. ParticipantsForty-four adolescent female high school students ranging from lean to obese. Main OutcomeFuture increases in body mass index (BMI). ResultsWeaker activation of the frontal operculum, lateral orbitofrontal cortex, and striatum in response to imagined intake of palatable foods, versus imagined intake of unpalatable foods or water, predicted future increases in body mass for those with the DRD2 TaqIA A1 allele or the DRD4–7R allele. Data also suggest that for those lacking these alleles, greater responsivity of these food reward regions predicted future increases in body mass. DiscussionThis novel prospective fMRI study indicates that responsivity of reward circuitry to food increases risk for future weight gain, but that genes that impact dopamine signaling capacity moderate the predictive effects, suggesting two qualitatively distinct pathways to unhealthy weight gain based on genetic risk.

Versatility of three techniques for preparing ibuprofen-loaded methacrylic acid copolymer nanoparticles of controlled sizes

Comparative studies of the techniques used for preparing polymeric nanoparticles (NP) are rarely reported in the literature. To gain insight in this field, NP were prepared under different conditions using the salting-out, emulsification-diffusion and nanoprecipitation methods and their properties such as drug loading efficiency, residual poly(vinyl alcohol), thermal behavior and morphological characteristics were investigated. Methacrylic acid copolymer Type C (Eudragit L100-55) and ibuprofen (IBU) were used as polymer and model drug, respectively. The salting-out and emulsification-diffusion methods allowed the preparation of IBU-loaded NP with the mean sizes ranging from 140 to 650 nm. Drug loading efficiency was improved when the NP mean size increased and the aqueous phase pH decreased. Nanoprecipitation allowed production of NP with sub-140 nm mean size. IBU was more effectively incorporated in salting-out-NP than in emulsification-diffusion-NP. Basically, the inherent formulation parameters of the salting-out method – i.e. lower solubility of Eudragit L100-55 in acetone, miscibility of acetone with water and lower volume of water required to induce solvent diffusion – favored the polymer precipitation rate and solvent diffusion rate, promoting drug incorporation into the NP. Nanoprecipitation resulted in poor IBU incorporation into the NP. Differential scanning calorimetry studies revealed that IBU was present in an amorphous or molecularly dispersed state in the NP. Finally, it was observed that residual PVAL was directly proportional to the specific surface area of nanoparticles, as examined for the emulsion-based methods.

Extraction of thiamylal in serum using hydrophilic acetonitrile with subzero-temperature and salting-out methods.

For high-performance liquid chromatography (HPLC) of thiamylal, one of the barbiturates, the drug in serum samples was extracted by two alternative liquid-liquid extraction techniques using hydrophilic acetonitrile as a solvent and subzero-temperature and salting-out methods. Acetonitrile was mixed with the sample, separated by cooling at -20 degrees C or addition of sodium chloride, and injected directly into the HPLC apparatus. In both the methods, thiamylal was extracted effectively in the acetonitrile phase and pH adjustment of the sample was not required. The salting-out extraction method is rapid and would be suitable for quantitation of drugs in many samples. To avoid coextraction of added salt, the subzero-temperature extraction method was applied to identification of thiamylal by gas chromatography/mass spectrometry and liquid chromatography-tandem mass spectrometry.

II. Electrophoretic, nitrogen and lipide analyses of plasma and plasma fractions in disease

HAWES in the distribution and properties of the plasma proteins noted during disease are of considerable interest to clinicians and investigators. In a recent review of the literature dealing with plasma proteins in disease Gutman’ pointed out that distribution of these proteins is generally not specific for any one disease but is characterized by a decrease in albumin and an increase in various globulin components. Most of the available information is based on electrophoretic studies of plasma and plasma fractions obtained with neutral salt precipitation. The inability to correlate the changes in plasma proteins with a specific disease detracts from the diagnostic value of plasma protein fractionation by present salting-out methods. The carefully controlled fractionation procedures of Cohn and associates2 provide another approach to the study of this problem. Application of these technics to relatively small volumes of dog,” rat” and goat” plasmas reveal changes in the distribution of protein components after injury. In a previous paper6 the plasma proteins of normal young men were fractionated by the low temperature-ethanol procedures and standards were established for the rlectrophoretic. nitrogen and lipide distributions of the plasma fractions. In this investigation these same procedures were applied to the plasmas of patients with a variety of diseases. RESUI.TS

A Non-Specific Resistance Factor in the Albumin Residue Revealed By the Serial Cephalin Flocculation Curve

IN the last laboratory meeting of the Royal Society of Tropical Medicine, I demonstrated the various types of serial cephalin flocculation curves as obtained in health and disease with a special reference to their application in tropical derangements. In obtaining the curves, the serial dilution method was followed1, using ten tubes instead of Hanger's original one-tube method2. Serial cephalin flocculation curve I, shown in the accompanying figure, represents the normal range in fifty healthy volunteers. In a study of the mechanism of Hanger's test, which is comparable to the first tube in the serial dilution method, Kabat, Hanger et al.3 found the flocculating factor to be associated with the γ globulin. They failed, however, to find an inhibiting fraction in the albumins. Moore, Pierson et al.4 two years later, reported from the same laboratory the presence of an inhibiting fraction associated with the albumins. It is to be noted that these workers were using electrophoresis by means of which albumins appear homogeneous5. Dennis and Makari6, using salting-out methods, found the greatest concentrations of this inhibiting factor in the albumin residue. Since this inhibiting factor was found by the latter workers to be originally present in the serum in smaller amounts than the flocculating factor, the critical dilution-level of 0-04 mgm. is reached much earlier in the case of the inhibiting factor. The first zone representing the first five tubes in the figure presents, therefore, a delicate balance between flocculating factor (γ globulin) and inhibiting factor (albumin residue). In the second zone only the former is at work. This delicgite balance in this first zone represents as well a balance between the liver and reticulo-endothelial system.

Concentration of Pneumococcus Antibody

A method was given in a previous publication by which the protective substance of antipneumococcus serum could be concentrated with a resulting product practically free from chill-producing characteristics. It was also stated in the same publication that the steps outlined for the purification of the water-insoluble precipitate obtained by diluting potent serum with water were applicable to the water-insoluble precipitate obtained by the common salting-out methods, using ammonium sulfate or sodium sulfate. It is the purpose of this paper to outline a method of concentration with anhydrous sodium sulfate as the precipitating agent instead of the commonly used ammonium sulfate. Sodium sulfate, rather than ammonium sulfate, is given at present because, as judged from the troublesome reaction after intravenous administration, a better product has been obtained by its use. From the standpoint of concentration of the protective antibody alone, however, ammonium sulfate has not only been used in many experiments with the same result as with sodium sulfate but also it has the advantage of greater ease in handling. The objection to its use, from our experience, is the chill-producing reaction of the final product following intravenous administration. Attempts have been made to obviate this difficulty but so far they have met with only partial success. In the course of our investigation on the pneumococcus antibody, obtained from the serum of immunized horses, certain facts have been observed concerning the associated protein. Although details will be given in other publications a brief summary of our findings will be reported here. In the first place, in contradistinction to Avery's work,2 the antibody has been found by us to be associated only with a waterinsoluble protein; never with the water-soluble pseudoglobulin. This observation has been confirmed by Banzhaf.3 However, the possibility should not be overlooked that the antibody when isolated may be watersoluble and also that the manipulations in Avery's work may have been such that some of the protective antibody was found with the pseu-

Further observations on precipitin reaction of bence-jones protein

The studies of the immune reactions of Bence-Jones protein have not given concordant results. The earlier observers (Abderhalden and Rostoski,1 Boggs and Guthrie2) by injecting Bence-Jones proteins in rabbits obtained precipitins that reacted not only with this protein but also with human serum proteins. Massini3 noted a quantitative difference between Bence-Jones protein and blood serum proteins by means of complement fixation. He suggests that Bence-Jones is closely related yet different from the serum proteins, and that the fixation test is not suited for differentiation except with pure preparations. Micheli 4 studied a Bence-Jones protein in the urine, blood, and serous fluids of a patient with lymphosarcoma of the large intestine. Anaphylactic experiments and fixation tests with what he calls strictly pure BenceJones protein, as determined by careful chemical tests, and with human serum gave inter-reactions which Micheli explains as due to the presence in the Bence-Jones protein of two principal receptors, one with the same antigenic properties as the proteins in normal serum; the second, more abundant, being the specific Bence-Jones antigen. In the serum of rabbits injected with urine from cases of myeloma, Hektoen 5 demonstrated specific precipitin reacting with urine or serum containing Bence-Jones protein, but not with normal human serum. Bayne-Jones and Wilson6 found that while noncrystallin forms of Bence-Jones protein, isolated from urine by salting-out or other precipitation methods, contained traces of serum proteins, spontaneously crystallizing Bence-Jones protein, betrayed a specific protein, and was not acted on by antiserum to human serum. The claim by Hektoen 5 that Bence-Jones protein is a distinct antigen is based on the results of absorption experiments as illustrated in table 1.

THE ABSORPTION OF PROTEIN WITHOUT DIGESTION

The purpose of the experiments reported in this paper is to determine whether it is possible for a protein to be absorbed through the epithelium of the gastro-intestinal tract and appear in an unaltered form in the circulating blood. This question is not a new one in the physiology of digestion. It has been studied by many workers who have employed a variety of experimental methods. Most of the experiments now quoted in the literature were made before the biological specificity of protein was recognized. It is obviously impossible to distinguish sharply between closely allied animal proteins by purely chemical reactions, such as the measurement of the content of nitrogen, phosphorus, sulphur, or by means of the salting-out methods. The newer biological methods, such as the precipitin and anaphylactic reactions, have a high degree of biological specificity, and enable one to distinguish sharply between proteins so closely related as the