Polyacrylamide Gel Research Articles

Rapid and reliable detection of Leishmania antibodies in canine serum with double-antigen sandwich homogeneous chemical luminescence

BackgroundLeishmaniasis, caused by Leishmania spp. parasites, is an important zoonotic disease globally, posing severe threats to humans and animals. In the absence of effective vaccines, reliable serological diagnostic methods are critical for disease control. However, the enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay have limitations due to complexity, time required and/or sensitivity. Therefore, our objective was to develop an accurate, rapid and user-friendly detection method of canine leishmania antibody based on double-antigen sandwich homogeneous chemical luminescence.MethodsHomogeneous chemiluminescent technology was employed, and expressed recombinant fusion proteins containing full-length K9, K39 and K26 repeat sequences were used as diagnostic antigens. To establish a dual-antigen sandwich serological assay capable of detecting various antibody types, a factorial design was used to optimize concentrations of diagnostic antigen-receptor microspheres and of biotinylated diagnostic antigens, as well as of reaction solution composition and reaction duration. To evaluate and validate this newly developed method, we collected 41 Leishmania-positive serum samples, 30 Leishmania-negative control serum samples and 78 clinical serum samples for which no diagnostic information was available. Comparative analyses were performed using parasitological testing and an indirect ELISA as reference methods, focusing on diagnostic sensitivity and specificity.ResultsSodium dodecyl sulfate–polyacrylamide gel electrophoresis confirmed the purification of the diagnostic antigens, which exhibited clear bands without impurities. Based on results from the 41 Leishmania-positive samples and 30 Leishmania-negative samples, there was sufficient sensitivity to detect samples diluted up to 256-fold, with analytical specificity of 100%. Overall diagnostic sensitivity was 100% and diagnostic specificity was 93.3%. Diagnostic performance was highly consistent between the newly developed method and the indirect ELISA (Kappa = 0.82, P < 0.01). Testing could be completed within 35 min with the new methodConclusionsWe have developed a novel double-antigen sandwich homogeneous chemical luminescence method to detect canine Leishmania antibodies, with high sensitively and specificity, a short incubation interval and a simple protocol. This streamlined approach not only offers a sensitive and efficient method for clinical diagnosis but also has great potential for use in automated testing.Graphical

Fruiting Characteristics and Molecular-Assisted Identification of Korla Fragrant Pear Bud Mutation Materials

Korla fragrant pear is a high-quality local pear variety native to Xinjiang, China. Currently, the low fruit-setting rate and low calyx shedding rate problems in Korla fragrant pears have been highlighted, which seriously affect the fruit yield and quality. It is of great significance to research the fruiting characteristics and molecular-assisted identification of Korla fragrant pear bud mutation materials for enriching the germplasm resources of Korla fragrant pear. In this research, a natural pollination group (YB) of Korla fragrant pear bud mutation materials and a natural pollination group (CK) of Korla fragrant pears were established. On the fruiting characteristics, the fruit-setting rate and calyx-removal rate of the two groups were investigated. In terms of fruit quality, the fruit shape index, fruit specific gravity, soluble solids content, sugar:acid ratio, soluble sugar content, and other indicators were measured. For the anatomical structure of the calyx tube, the detachment cells were observed. The formation time of the two groups of detached cells was compared. In the GBS simplified genome sequencing, a phylogenetic tree was constructed based on the obtained SNP sites. A principal component analysis, population genetic structure analysis, and genetic diversity index analysis were carried out. In the aspect of SSR molecular marker identification, the SSR types were counted. Polyacrylamide gel electrophoresis was performed. The results demonstrate the following: (1) the fruit-setting rate (30.87%) and calyx-removal rate (68.11%) in the YB group were significantly higher than those in the CK group (19.37%) and the calyx-removal rate (55.18%). (2) There was no significant difference in fruit quality indexes, such as average fruit weight (127.10–130.00 g) and soluble sugar content (9.47–9.56%) between the two groups. (3) Abscission-layer cells were observed at 2, 4, 6, 8, and 10 h after calyx tube discoloration in the YB group and at 48, 72, and 96 h after calyx tube discoloration in the CK group. (4) The genetic background of the YB group and the CK group was similar at the GBS level, but there were differences at the DNA level. This research finally shows that Korla fragrant pear bud mutation material is a good germplasm resource. This germplasm resource can promote the structural optimization of Korla fragrant pear varieties and the healthy development of the industry.

Aloe vera Cuticle: A Promising Organic Water-Retaining Agent for Agricultural Use

Water is an important resource for both human and environmental survival. However, due to current human practices, we are facing a serious crisis in accessing water. Thus, solutions must be explored to optimize the use of this resource. In the search for an organic water-retaining agent for agricultural use, the techno-functional properties of Aloe vera (Aloe barbadensis Miller) cuticle, an agro-industrial residue generated after gel extraction, were evaluated. The residue was dried and ground. The effects of particle size (180 µm and 250 µm), temperature (10 °C, 20 °C, 30 °C, and 40 °C), and pH (4.5, 6.0, and 7.0) on the solubility and water-holding capacity (WHC) of the obtained product (i.e., hydrogel) were then evaluated. The treatment with the highest WHC was selected and compared with the WHC of a commercial synthetic polyacrylamide gel widely used in agriculture. The effects of KNO3 and Ca(NO3)2 at different concentrations (10 g L−1, 20 g L−1, 30 g L−1, and 40 g L−1) on the WHC of the gels were assessed. Particle size, temperature, and pH interactions had statistically significant effects on solubility, while the WHC was affected by particle size × temperature and pH × temperature interactions. The highest product solubility (75%) was obtained at the smallest particle size (i.e., 180 µm), pH 4.5, and 20 °C. Meanwhile, the highest WHC (18 g g−1) was obtained at the largest particle size (i.e., 250 µm), pH 6.0, and 20 °C. This optimized gel kept its WHC across both salts and their concentrations. In contrast, the commercial gel significantly decreased its WHC with salt concentration. The product elaborated with A. vera cuticle could have bioeconomic potential as a water-retention agent for agricultural use, with the advantage that it is not affected by the addition of salts used for plant fertilization.

Pd nanoparticles supported on Fe3O4 particles grafted with poly(acrylamide) gel brush for simultaneous electrochemical‐sensing of dopamine and paracetamol

AbstractIn the present study, a glassy carbon electrode (GCE) modified with Pd nanoparticles deposited on poly(acrylamide) gel brush‐grafted magnetic particles (Pd/PAAmGB@Fe3O4) with core‐shell structure was fabricated for simultaneous electrochemical detection of dopamine (DA) and paracetamol (PA). Electrochemical performance of Pd/PAAmGB@Fe3O4/GCE was evaluated using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV). The fabricated sensor exhibited voltammetric sensing with a satisfactory linear determination range from 0.4–41 μM for DA and from 0.5–95 μM for PA. The limit of detection (LOD) was 0.1 μM for DA and 0.17 μM for PA based on signal to noise ratio of 3. The proposed sensor also had excellent reusability and selectivity, good reproducibility and stability, and satisfactory recovery in biomedical examples. It is believed that metal nanoparticles deposited on polymer gel brush composites may offer promising new horizons for sensor growth.

Effect of extracellular matrix stiffness on efficacy of Dapagliflozin for diabetic cardiomyopathy

BackgroundExtracellular matrix (ECM) stiffness is closely related to the progress of diabetic cardiomyopathy (DCM) and the response of treatment of DCM to anti-diabetic drugs. Dapagliflozin (Dapa) has been proven to have cardio-protective efficacy for diabetes and listed as the first-line drug to treat heart failure. But the regulatory relationship between ECM stiffness and treatment efficacy of Dapa remains elusive.Materials and methodsThis work investigated the effect of ECM stiffness on DCM progression and Dapa efficacy using both in vivo DCM rat model and in vitro myocardial cell model with high glucose injury. First, through DCM rat models with various levels of myocardial injury and administration with Dapa treatment for four weeks, the levels of myocardial injury, myocardial oxidative stress, expressions of AT1R (a mechanical signal protein) and the stiffness of myocardial tissues were obtained. Then for mimicking the stiffness of myocardial tissues at early and late stages of DCM, we constructed cell models through culturing H9c2 myocardial cells on the polyacrylamide gels with two stiffness and exposed to a high glucose level and without/with Dapa intervention. The cell viability, reactive oxygen species (ROS) levels and expressions of mechanical signal sensitive proteins were obtained.ResultsThe DCM progression is accompanied by the increased myocardial tissue stiffness, which can synergistically exacerbate myocardial cell injury with high glucose. Dapa can improve the ECM stiffness-induced DCM progression and its efficacy on DCM is more pronounced on the soft ECM, which is related to the regulation pathway of AT1R-FAK-NOX2. Besides, Dapa can inhibit the expression of the ECM-induced integrin β1, but without significant impact on piezo 1.ConclusionsOur study found the regulation and effect of biomechanics in the DCM progression and on the Dapa efficacy on DCM, providing the new insights for the DCM treatment. Additionally, our work showed the better clinical prognosis of DCM under early Dapa intervention.

Implications of monoclonal gammopathy and isoelectric focusing pattern 5 on the free light chain kappa diagnostics in cerebrospinal fluid.

Oligoclonal bands (OCB) analysis is the reference standard for detecting an intrathecal IgG synthesis. Alongside OCB, free light chains kappa (FLCκ) are considered an additional sensitive biomarker for determining patterns 2 or 3, indicating intrathecal Ig synthesis. However, kFLC IF is not suitable for detecting a monoclonal pattern 5. The primary aim of this study was to evaluate the impact of incorporating FLCκ analysis into routine cerebrospinal fluid (CSF) diagnostics instead of OCB testing on the rate of missed monoclonal IgG detection. A two-center retrospective biomarker study was conducted. OCB were identified using isoelectric focusing in polyacrylamide gels followed by silver staining or in agarose gels followed by immunofixation. FLCκ were quantified using nephelometry and FLCκ assay (Siemens). Out of a combined total of 17,755 OCB analyses conducted between 2011 and 2021, a subset of 269 cases (1.5 %) exhibited pattern 5. 98 samples (36 %), which included 18 samples with intrathecal inflammation as determined by additional OCB pattern 2 were included in the FLCκ analysis. Of those, 16 (89 %) had intrathecal FLCκ synthesis. While FLCκ offers a promising avenue for detecting an intrathecal inflammation, the pattern 5, though rare, remains a valuable additional finding of OCB analysis. A combined approach of FLCκ and OCB analysis is recommended for a comprehensive assessment of the humoral intrathecal immune response.

Extraction and characterization of collagen from the skin of Pterygoplichthys spp. (armored catfish) from Chiapas

Because of its biocompatibility and safety, collagen is a valuable biomaterial that is used in various industries. While traditionally sourced from terrestrial mammals, collagen extracted from fish waste is a promising alternative due to its chemical properties and low cost. In Mexico, Pterygoplichthys spp. is considered an invasive species, inedible, and of limited commercial importance due to the low muscle content and skin covered with rigid bony plates. This study aimed to extract and characterize collagen from the skin of Pterygoplichthys spp. obtained from two communities in Chiapas, Mexico. Our results show that the extracted collagen is type 1, with a yield of 43.0% (dry weight). Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed the presence of two alpha chains (α1, α2) and a beta and gamma component, consistent with type 1 collagen. Liquid chromatography coupled to mass spectrophotometry analysis identified peptide sequences homologous to those reported in other species. This study highlights the potential of Pterygoplichthys spp. skin collagen as a viable alternative to mammalian collagen. The efficiency of the extraction process and the identification of peptides resembling those of the other species underscore the feasibility of utilizing collagen from the skin of Pterygoplichthys spp. in industrial applications, offering a sustainable solution to environmental and economic challenges. Practical Application: Collagen extracted from the skin of Pterygoplichthys spp., an invasive fish species, can serve as a sustainable alternative to mammalian collagen, offering a cost-effective biomaterial with properties suitable for various industrial applications.

Blue Light Compromises Bacterial β-Lactamases Activity to Overcome β-Lactam Resistance.

In this study, we evaluated the effectiveness of antimicrobial blue light (aBL; 410 nm wavelength) against β-lactamase-carrying bacteria and the effect of aBL on the activity of β-lactamases. Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae strains carrying β-lactamases as well as a purified β-lactamase enzymes were studied. β-lactamase activity was assessed using a chromogenic cephalosporin hydrolysis assay. Additionally, we evaluated the role of porphyrins in the photoreaction, as well as protein degradation by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, we investigated the bactericidal effect of combined aBL-ceftazidime exposure against a metallo-β-lactamase expressing P. aeruginosa strain. Our study demonstrated that aBL effectively killed β-lactamase-producing bacteria and reduced β-lactamase activity. After an aBL exposure of 1.52 J/cm2, a 50% reduction in enzymatic activity was observed in P. aeruginosa. Additionally, we found a 40% decrease in the photoreaction activity of porphyrins following an aBL exposure of 64.8 J/cm2. We also revealed that aBL reduced β-lactamase activity via protein degradation (after 136.4 J/cm2). Additionally, aBL markedly improved the bactericidal effect of ceftazidime (by >4-log10) in the metallo-β-lactamase P. aeruginosa strain. Our results provide evidence that aBL compromises bacterial β-lactamase activity, offering a potential approach to overcome β-lactam resistance in bacteria.

Efficient adsorption of waste carbon nanotube by polyacrylamide gel to form wrinkled core-shell particles towards multifunctional composites for EMI shielding and sensing

Waste carbon nanotubes (CNTs) have always been a potential threat to the environment and biological health. However, the efficient recycling and utilization of CNTs has received little attention. Here, we used chemically cross-linked polyacrylamide (PAM) particles to adsorb waste CNTs in water to prepare PAM@CNT core-shell particles, which were blended with thermoplastic styrene-butadiene rubber (SBS) to prepare flexible composites. The concentration of residual CNTs in the supernatant was less than 3.5 μg mL−1, which meets the safety standards for discharge. The unique core-shell structure enables the composites with a thickness of 1 mm to exhibit high electromagnetic interference shielding effectiveness (53.3 dB) and low reflectivity (1.4 dB) at 30 wt% CNT content. In addition, at a CNT content of 10 wt%, the composites exhibit superior sensing properties concerning strain and solvents. Moreover, the composites prepared from different sources of waste CNTs have the same sensing properties. This work provides an effective strategy for the highly efficient recycling of waste CNTs for the preparation of green, high-performance multifunctional polymer composites for EMI shielding, human motion and solvent leakage monitoring, and solvent category differentiation.

Using ion exchange chromatography for the purification of glyoxylate reductase from corn leaves and studying its characteristics

The purpose of this study was to obtain a purified preparation of the studied enzyme from corn leaves and to study its characteristics. In this work, kinetic and regulatory parameters of glyoxylate reductase were calculated for leaves of 14-day-old corn (Zea mays) seedlings grown hydroponically at 25°C. The following methods were used during the study: sample homogenisation, four-step purification including ammonium sulphate for desalting, gel filtration on G-25 columns, and ion exchange chromatography using DEAE-sephacel, as well as electrophoresis on polyacrylamide gels and quantitative analysis of protein. To study the properties of the enzyme, we used electrophoretically homogenous preparations. The influence of pH, substrate concentration, and cofactor on the rate of the enzymatic reaction was determined by a series of measurements with different values of the enzymatic reaction rate. As a result of four-stage purification, we obtained a homogeneous preparation with a specific activity of167 E/mg of protein. Ion exchange chromatography was important for purification; we obtained 1 peak of enzyme activity upon desorption in 104 mM sodium chloride. Studying the properties of glyoxylate reductase showed a significant dependence of activity on pH. The optimal pH value was 6.5 units.

Influence of resveratrol on the sorption of phosphorylated tau protein and MAPT on the PVDF membrane in a mouse model of Alzheimer’s disease

Alzheimer’s disease is a complex neurodegenerative disease based on various processes associated with the accumulation and aggregation of defective proteins. Among them, the particularly important ones are the following: amyloid-β, which is formed by the breakdown of amyloid precursor protein, the accumulation of hyperphosphorylated tau proteins inside neurons that form neurofibrillary tangles, and aberrant aggregation and inclusion formation of microtubule-associated protein tau (MAPT). APP/PS1 transgenic mice act as a model of Alzheimer’s disease and express mutant human genes that cause the accumulation of amyloid-β peptides in the brain. The goal of this work was a quantitative assessment of the level of p-tau231 in the brain of transgenic mice with a model of AD using the sorption method. The objectives of the study also included testing the ability of the natural polyphenol resveratrol to reduce the concentration of p-tau231 in the brain of transgenic mice and improve their cognitive functions. Western blot is a widely used method for the immunodetection and in vitro quantitative determination of proteins. Western blot allows separating proteins based on their molecular weight with the further transfer to an adsorption membrane. In this case, the proteins are transferred from the gel to the PVDF membrane using electrophoretic elution. This method involves placing a protein-containing polyacrylamide gel in direct contact with a PVDF membrane represented by a linear polymer with repetitive links -(CF2-CH2)-. Proteins transferred to the membrane are well-retained on its surface during the whole immunodetection process due to a combination of dipole and hydrophobic interactions. Western blot showed that mice with impaired protein aggregation accumulated significantly more MAPT and phosphorylated tau protein in the brain as compared to wild mice. In addition, in the course of the Morris water maze test, these mice showed cognitive deficits, which manifested both in the difficulty of finding the platform and more anxious behaviour, which confirmed pronounced thigmotaxis. The natural polyphenol resveratrol partially reversed cognitive deficits, although this effect was not associated with decreased levels of phosphorylated tau and MAPT.

Impact of Co-60 γ-ray irradiation on the cross-linking and stability of fish collagen: Structural changes and digestibility

Irradiation can promote the cross-linking of collagen to enhance its biological properties, but the molecular changes in irradiated collagen have few been investigated. This study aimed to explore the impact of irradiation treatment on the molecular structure stabilization and cross-linking degree of skin collagen. The results showed that collagen in solution was more susceptible to Co-60 gamma irradiation than that in fish skin and in sponge forms. The solubility of collagen in solution decreased by more than 80% and the carbonyl content increased by more than 4 times compared with the control group after 12 kGy irradiation. Spectroscopic analysis and sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) showed that collagen in solution was cross-linked, especially at high irradiation doses. However, the triple helical structure of skin collagen and collagen sponge remained intact. Furthermore, irradiation also reduced the sensitivity of collagen to collagenase. Gastrointestinal digestion results showed that the antioxidant activity of collagen increased at 1–3 kGy doses and decreased at 9–12 kGy doses. Collagen irradiated at high doses (9–12 kGy) was more difficult to digest and had a larger average particle size. This study provides a potential new approach for the preparation of stable fishery-derived collagen with excellent antioxidant activity and resistance to digestion by irradiation treatment.

Determination of the molecular weight of native molecules of γ-hydroxybutyrate dehydrogenase isoforms obtained from maize (Zea mays L.) seedlings by gel chromatography

It is known that under oxygen deficiency the functioning of the tricarboxylic acid cycle is impaired, resulting in the activation of an alternative pathway, the GABA shunt. It maintains the functioning of the citric acid cycle by providing succinic acid. The key phase of this bypass is a reaction catalysed by succinate semialdehyde dehydrogenase (SSADH, EC 1.1.1.16). Under oxygen deficiency, SSADH ceases to function efficiently. This leads to the accumulation of succinic acid semialdehyde in the mitochondrial matrix and its high level adversely affects the plant cell metabolism. γ–hydroxybutyrate dehydrogenase (HBDH, EC 1.1.1.61) is an enzyme belonging to the group of oxidoreductases. It transforms γ–hydroxybutyrate into succinic acid semialdehyde, participating in the process of its detoxification, which is important in the maintenance of plant metabolism under oxygen deficiency. To date, unfortunately, there are no data on the biochemical and kinetic features of γ–hydroxybutyrate dehydrogenase. In this regard, our laboratory developed a method for purification of HBDH from green maize leaves, which makes it possible to study the physicochemical properties of this enzyme. During the study, we obtained γ-hydroxybutyrate dehydrogenase from 7-day-old seedlings of Zea mays L. by a five-stage purification. Homogenised plant material with extracted proteins was subjected to two-stage ammonium sulphate fractionation. The catalytic activity was determined spectrophotometrically at λ=340 nm by the amount of reduced NAD+. To remove ammonium salts, we used gel filtration through Sephadex G-25. Proteins were separated according to their charge by DEAE-Sephacel ion exchange chromatography. The enzyme was desorbed using a linear sodium chloride gradient (100-300 mM). The use of gel chromatography through Sephadex G-200 made it possible to determine the molecular mass of the purified enzyme isoforms. The homogeneity of enzyme preparations was confirmed by electrophoresis in polyacrylamide gel with universal AgNO3 staining. We used the tetrazolium method to confirm that the obtained protein preparations were γ-hydroxybutyrate dehydrogenase. As a result, homogeneous preparations of two isoforms of the enzyme (HBDH1 and HBDH2) were obtained. The first isoform of γ-hydroxybutyrate dehydrogenase was 185.7 times purified with a yield of 10% and had a specific activity of 343.6 U/mg of protein. The purification rate of the second isoform was 209 times with a yield of 7.74%. The specific activity of the obtained preparation was 386.7 U/mg of protein. Using gel chromatography through Sephadex G-200, we determined the molecular mass of native molecules of hydroxybutyrate dehydrogenase. We found that in 7-day-old maize seedlings the investigated enzyme was presented in low-molecular and high-molecular forms: for HBDH1 the Mr value was ⁓60.3 kDa, while for HBDH2 the molecular mass of the enzyme was 286 kDa.

Relative increase in production ratio of small dense low-density lipoprotein in acute coronary syndrome with high coronary plaque burden: an ex-vivo analysis.

The absolute value of small dense low-density lipoprotein (sd-LDL) including small LDL (s-LDL) and very small LDL (vs-LDL) has been shown to be associated with increased incidence of atherosclerosis. However, the impact of short-timeframe increases in sd-LDL on arteriosclerosis has not yet been elucidated. Therefore, we investigated the clinical roles of ex-vivo induced sd-LDL in acute coronary syndrome (ACS) using a novel method. This is a prospective, single-blind, and observational study that screened patients who underwent coronary angiography (CAG) for the treatment of ACS or investigation of heart-failure etiology between June 2020 and April 2022 (n = 247). After excluding patients with known diabetes mellitus and advanced renal disease, the patients were further divided into the ACS (n = 34) and control (non-obstructive coronary artery, n = 34) groups. The proportion of sd-LDL (s-LDL + vs-LDL) in total lipoproteins was observed before and after 2-h incubation at 37 ℃ (to approximate physiologic conditions) using 3% polyacrylamide gel electrophoresis. The coronary plaque burden was quantified upon CAG in the ACS group. There were no significant differences between the ACS and control groups in terms of clinical coronary risk factors. The baseline of large, medium, small, and very small LDL were comparable between the two groups. Following a 2-h incubation period, significant increases were observed in the ratios of s-LDL and vs-LDL in both the ACS and control groups (ACS, p = 0.01*; control, p = 0.01*). Notably, the magnitude of increase in sd-LDL was more pronounced in the ACS group compared to the control group, with s-LDL showing a significant difference (p = 0.03*) and vs-LDL showing a tread toward significance (p = 0.08). In addition, in both groups, there was a decrease in IDL and L-LDL, while M-LDL remained unchanged. The plaque burden index and rate of short-timeframe changes in both s-LDL (p = 0.01*) and vs-LDL (p = 0.04*) before and after incubation were significantly correlated in the ACS group. The enhanced production rate of sd-LDL induced under short-term physiologic culture in an ex-vivo model was greater in patients with ACS than in the control group. The increase in sd-LDL is positively correlated with coronary plaque burden. Short-timeframe changes in sd-LDL may serve as markers for the severity of coronary artery disease.

Combining SDS-PAGE to capillary zone electrophoresis-tandem mass spectrometry for high-resolution top-down proteomics analysis of intact histone proteoforms.

Mass spectrometry (MS)-based top-down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post-translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high-resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS-PAGE-based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI-MS) with capillary zone electrophoresis (CZE)-MS/MS for high-resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS-PAGE gel and follow-up cleanup as well as CZE-MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high-resolution separation and characterization of histone proteoforms. SDS-PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high-resolution separations of SDS-PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.

What are the main proteins in the hemolymph of Haemaphysalis flava ticks?

Haemaphysalis flava is a notorious parasite for humans and animals worldwide. The organs of H. flava are bathed in hemolymph, which is a freely circulating fluid. Nutrients, immune factors, and waste can be transported to any part of the body via hemolymph. The main soluble components in hemolymph are proteins. However, knowledge of the H. flava proteome is limited. The hemolymph was collected from fully engorged H. flava ticks by leg amputation. Hemolymph proteins were examined by both blue native polyacrylamide gel electrophoresis (BN-PAGE) and sodium dodecyl sulfate PAGE (SDS-PAGE). Proteins extracted from the gels were further identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two bands (380 and 520 kDa) were separated from tick hemolymph by BN-PAGE and were further separated into four bands (105, 120, 130, and 360 kDa) by SDS-PAGE. LC-MS/MS revealed that seven tick proteins and 13 host proteins were present in the four bands. These tick proteins mainly belonged to the vitellogenin (Vg) family and the α-macroglobulin family members. In silico structural analysis showed that these Vg family members all had common conserved domains, including the N-terminus lipid binding domain (LPD-N), the C-terminus von Willebrand type D domain (vWD), and the domain of unknown function (DUF). Additionally, two of the Vg family proteins were determined to belong to the carrier protein (CP) by analyzing the unique N-terminal amino acid sequences and the cleaving sites. These findings suggest that the Vg family proteins and α-macroglobulin are the primary constituents of the hemolymph in the form of protein complexes. Our results provide a valuable resource for further functional investigations of H. flava hemolymph effectors and may be useful in tick management.

GLUTENIN DIVERSITY OF FLOUR WHEAT (Triticum aestivum L.) FOR COOKIE QUALITY IN MEXICO

Glutenins are high- and low-molecular-weight proteins (HMW-G and LMW-G). They play a determining role in genetic improvement to define industrial quality in flour wheat, so understanding their variability is necessary for directing selection toward a specific quality. The objective of this research was to determine the diversity of HMW-G and LMW-G alleles of genotypes from the flour wheat breeding program of the National Institute of Forestry, Agricultural, and Livestock Research (INIFAP) useful for cookie quality. Twenty-five genotypes were used, including six Mexican cookie varieties, as well as 21 genotypes introduced from an international collection of accessions from the International Maize and Wheat Improvement Center (CIMMYT). HMW-G and LMW-G were identified based on their separation on polyacrylamide gels in the presence of sodium dodecyl sulfate. For HMW-G, the 2* variant in Glu-A1 was the most frequent variant for the trial and introduction nursery, with more than 80 %. At the Glu-B1 locus, the 7+9 and 7 variants were the most frequent, with 60 and 38.1 % in trial and nursery, respectively. For Glu-D1, the 2+12 variant was the most frequent in the trial with 64 %, and 5+10 with 85.7 % in nursery. The latter allele favors baking quality. For the Glu-A1 and Glu-D1 loci, it is necessary to introduce genotypes with null alleles or with the 2+121 variant in Glu-D1 associated with cookie quality. For the highest-frequency variants of LMW-G, c in Glu-A3, h in Glu-B3, and b in Glu-D3 are associated with baking quality. With the aforementioned, it is concluded that there is a low frequency and little variation in useful alleles of HMW-G and LMW-G for cookie quality in the introduced accessions analyzed, implying that new genetic sources that favor it should be explored.

Co-chaperonin GroES subunit exchange as dependent on time, pH, protein concentration, and urea

The discovery of a subunit exchange in some oligomeric proteins, implying short-term dissociation of their oligomeric structure, requires new insights into the role of the quaternary structure in oligomeric protein stability and function. Here we demonstrate the effect of pH, protein concentration, and urea on the efficiency of GroES heptamer (GroES7) subunit exchange. A mixture of equimolar amounts of wild-type (WT) GroES7 and its Ala97Cys mutant modified with iodoacetic acid (97-carboxymethyl cysteine or CMC-GroES7) was incubated in various conditions and subjected to isoelectric focusing (IEF) in polyacrylamide gel. For each sample, there are eight Coomassie-stained electrophoretic bands showing different charges that result from a different number of included mutant subunits, each carrying an additional negative charge. The intensities of these bands serve to analyze the protein subunit exchange. The protein stability is evaluated using the transverse urea gradient gel electrophoresis (TUGGE). At pH 8.0, the intensities of the initial bands corresponding to WT-GroES7 and CMC-GroES7 are decreased with a half-time of (23 ± 2) min. The exchange decreases with decreasing pH and seems to be strongly hindered at pH 5.2 due to the protonation of groups with pK ∼ 6.3, which stabilizes the protein quaternary structure. The destabilization of the protein quaternary structure caused by increased pH, decreased protein concentration, or urea accelerates the GroES subunit exchange. This study allows visualizing the subunit exchange in oligomeric proteins and confirms its direct connection with the stability of the protein quaternary structure.

Long-Term Biodegradation of Polyacrylamide Gel Residues in Mammary Glands: Physico-Chemical Analysis, Chromatographic Detection, and Implications for Chronic Inflammation.

In the past, polyacrylamide hydrogel was a popular choice for breast augmentation filler, and many women underwent mammoplasty with this gel. However, due to frequent complications, the use of polyacrylamide hydrogel in mammoplasty has been banned. Despite this ban, patients experiencing complications still seek medical treatment. The aim of this study was to investigate the fate of the polymer over a defined implantation period. Biopsies of breast implants were obtained from patients with 23 and 27 years of post-mammoplasty. These biopsies were meticulously purified from biological impurities and subjected to analysis using IR spectrometry, liquid chromatography-mass spectrometry, gas chromatography, and differential scanning calorimetry. The findings revealed the presence of polyacrylamide hydrogel residues, along with degradation products, within the infected material. Notably, the low-molecular-weight degradation products revealed via gas chromatography are aggressive and toxic substances capable of inducing chronic inflammation. This study sheds light on the long-term consequences of polyacrylamide hydrogel implantation, highlighting the persistence of harmful degradation products and their role in exacerbating patient complications.

Biochemical properties of polyphenol oxidase purified from Sarali plum (Prunus domestica)

Enzymatic browning, catalysed by the enzyme polyphenol oxidase in fruit and vegetables, limits the efficient use of natural resources and promote food waste. Plums are a popular fruit with consumers around the world and are considered an important raw material in the food industry. Plums are very susceptible to enzymatic browning due to their high phenolic compound content and climacteric nature. The aim of this study is to purify the polyphenol oxidase enzyme from Sarali plum (Prunus domestica) and to determine its biochemical properties, kinetic parameters, pH and thermal stability and inhibition. In this study, polyphenol oxidase enzyme was purified 22.54-fold by affinity chromatography using Sepharose-4B-l-Tyr-p-amino benzoic acid affinity gel. The purity and molecular mass of the enzyme were determined by SDS-PAGE and non-denaturing PAGE (native PAGE). The molecular mass of the enzyme was estimated to be 72.44 kDa by SDS-PAGE. The enzyme was confirmed as PPO by native PAGE as a single band. Kinetic characterization studies were conducted for both catechol and 4-methyl catechol substrates. The optimal pH and temperature for both substrates were found to be 7.0 and 20 °C, respectively. The thermal stability of PPO was investigated, and it retained about 90% of its activity for 90 min at 4 °C. The determination of KM and Vmax was carried out using the Lineweaver–Burk plot. The substrate specificity (Vmax/KM) values for catechol and 4-methyl catechol were determined as 790.91 ± 37.34 and 492.06 ± 13.75 respectively. The enzyme exhibited the best activity towards catechol substrate. IC50, Ki constant and inhibition types were determined for various anti-browning agents on PPO enzyme. Ascorbic acid, l-cysteine, citric acid, salicylic acid and tartaric acid effectively inhibited PPO activity.