Polyacrylamide Gel Research Articles

DELINEATING GENETIC DIVERSITY IN SOYBEAN (GLYCINE MAX L.) GENOTYPES: INSIGHTS FROM A-PAGE ANALYSIS OF GLOBULIN RESERVE PROTEINS

This study explores the genetic diversity of soybean (Glycine max) genotypes using polyacrylamide gel electrophoresis (A-PAGE) to analyze globulin reserve proteins. The research utilized 51 soybean genotypes from various origins, including Canada, Turkey, Uzbekistan and others. The A-PAGE method was employed to extract and analyze the globulin storage proteins in the grains of soybean samples. The proteins were categorized into ω, γ, β and α zones, revealing distinct electrophoretic patterns among the genotypes. A total of 22 spectra and 107 patterns were identified, demonstrating polymorphism based on the frequency of occurrence. The genetic diversity index, calculated using Nei’s formula, indicated varying levels of genetic diversity across the different zones.

Robust peroxidase from Bacillus mojavensis TH309: Immobilization on walnut shell hydrochar and evaluation of its potential in dye decolorization

Peroxidases have received considerable attention as a cost-effective and environmentally friendly catalyst for bioremediation. Their rapid activity loss under harsh environmental conditions and inability to be used repetitively limit their exploitation in real-world wastewater treatment. First, a peroxidase was produced extracellularly by Bacillus mojavensis TH309 and purified 8.12-fold with a final yield of 47.10 % using Sephadex G-100 superfine resin. The pure peroxidase (BmPer) possessed a relatively low molecular weight of ∼21 kDa and was active against L-DOPA on acrylamide gel after electrophoresis. BmPer was immobilized by adsorption functionalized walnut shell hydrochar (WsH) with 61.99 ± 1.34 % efficiency and 37.07 ± 4.16 % activity loss. BmPer and its immobilized form (WsH-BmPer) exhibited maximum activity at 50 °C and pH 9. WsH-BmPer exhibited 3.23-, 2.37-, 1.65-, and 2.25-fold longer half-life than BmPer at 50, 60, 70, and 80 °C, respectively. Immobilization significantly enhanced the stability of the enzyme under acidic conditions. BmPer and WsH-BmPer showed maximal activity in the presence of 1 % salt and retained more than 85 % of their activity even after pre-incubation with 2.5 M salt for 60 min at 50 °C. Their catalytic efficiency was significantly stimulated by pre-incubation with Triton X-100 (1 mM), Tween20 (1 mM), and Mg2+ (1 and 10 mM). Immobilization strongly reduced the loss of activity caused by inhibitors including Ba2+, Hg2+, and Cu2+. Moreover, both forms of the enzyme were compatible with solvents. The Michaelis constant (Km) values of BmPer and WsH-BmPer were 0.88 and 2.66 mM for 2,4 DCP, respectively. WsH-BmPer peroxidase maintained about 82 % and 85 % of its activity when stored at 4 °C for 30 days and reused for up to 10 cycles, respectively. Furthermore, it decolorized Cibacron red (CR), Poly R-478 (PR), Remazol Brilliant Blue R (RBBR), and Methyl red (MR) dyes by 60.13 %, 91.34 %, 86.41 %, and 50.51 % within 60 min, respectively.

Oligomerization of protein arginine methyltransferase 1 and its effect on methyltransferase activity and substrate specificity.

Proper protein arginine methylation by protein arginine methyltransferase 1 (PRMT1) is critical for maintaining cellular health, while dysregulation is often associated with disease. How the activity of PRMT1 is regulated is therefore paramount, but is not clearly understood. Several studies have observed higher order oligomeric species of PRMT1, but it is unclear if these exist at physiological concentrations and there is confusion in the literature about how oligomerization affects activity. We therefore sought to determine which oligomeric species of PRMT1 are physiologically relevant, and quantitatively correlate activity with specific oligomer forms. Through quantitative western blotting, we determined that concentrations of PRMT1 available in a variety of human cell lines are in the sub-micromolar to low micromolar range. Isothermal spectral shift binding data were modeled to a monomer/dimer/tetramer equilibrium with an EC50 for tetramer dissociation of ~20 nM. A combination of sedimentation velocity and Native polyacrylamide gel electrophoresis experiments directly confirmed that the major oligomeric species of PRMT1 at physiological concentrations would be dimers and tetramers. Surprisingly, the methyltransferase activity of a dimeric PRMT1 variant is similar to wild type, tetrameric PRMT1 with some purified substrates, but dimer and tetramer forms of PRMT1 show differences in catalytic efficiencies and substrate specificity for other substrates. Our results define an oligomerization paradigm for PRMT1, show that the biophysical characteristics of PRMT1 are poised to support a monomer/dimer/tetramer equilibrium in vivo, and suggest that the oligomeric state of PRMT1 could be used to regulate substrate specificity.

Regulation of mitochondrial metabolism by hibernating bear serum: Insights into seasonal metabolic adaptations

Hibernating animals undergo a unique and reversible decrease in their whole-body metabolism, which is often accompanied by a suppression of mitochondrial respiration. However, the precise mechanisms underlying these seasonal shifts in mitochondrial metabolism remain unclear. In this study, the effect of the serum from active and hibernating Japanese black bears on mitochondrial respiration was assessed. Stromal–vascular cells were obtained from bear white adipose tissue and cultured with or without an adipocyte differentiation cocktail. When the oxygen consumption was measured in the presence of bear serum, the hibernating bear serum reduced maximal respiration by 15.5 % (p < 0.05) and spare respiratory capacity by 46.0 % (p < 0.01) in the differentiated adipocytes in comparison to the active bear serum. Similar reductions of 23.4 % (p = 0.06) and 40.6 % (p < 0.05) respectively were observed in undifferentiated cells, indicating the effect is cell type-independent. Blue native PAGE analysis revealed that hibernating bear serum suppressed cellular metabolism independently of the assembly of mitochondrial respiratory chain complexes. RNA-seq analysis identified 1094 differentially expressed genes (fold change>1.5, FDR<0.05) related to insulin signaling and glucose metabolism pathways. These findings suggest that the rapid alterations in mitochondrial metabolism during hibernation are likely induced by a combination of reduced insulin signaling and suppressed mitochondrial function, rather than changes in respiratory complex assembly.

Recyclable NaCl template assisted preparation of N/O co-doped porous carbon for zinc-ion hybrid capacitor

The traditional strong alkaline activation method for preparing porous carbon faces with complex preparation processes, equipment corrosion, excessive disordered microporous structures, and environmental pollution. Hence, a green, facile and sustainable method is proposed for synthesizing N/O co-doped porous carbon derived from polyacrylamide gel with the assistance of recyclable sodium chloride (NaCl) template. NaCl template is dissolved in gel with water and can give full play to the template effect after recrystallization. Due to a suitable specific surface area (499.8 m2 g−1) and massive N (11.38 at. %) and O (8.71 at. %) functional groups, the optimized NPC-5 electrode possesses a high specific capacitance of 283.3 F g−1 at 0.5 A g−1, superior rate characteristics and excellent cycle stability in 6 M KOH aqueous electrolyte. In addition, the constructed NPC-5//NPC-5 symmetric supercapacitor shows a high energy density of 34.0 Wh kg−1 in 1 M ZnSO4 aqueous electrolyte. More importantly, the assembled Zn//ZnSO4//NPC-5 hybrid capacitor shows a high capacity of 115.44 mAh g−1 and an energy density of 85.3 Wh kg−1.

Phleum pratense-pollen adaptive variations and pollen microbiome investigation under different climatic regions and prospects of allergenicity.

Phleum pratense is an allergenic grass that pollinates in spring in Pakistan. Databases Allergenonline.org and Allergen.org record ten P. pratense allergens and their isoforms. Phl P 1, Phlp 5, and Phl p 11 are major P. pratense-pollen allergens with demonstrated basophil activity and skin test reactivity. Little is known about P. pratense pollen adaptive variations in different climatic regions and pollen-associated microbial diversity. In this study, we collected P. pratense-pollen and soils in the spring season 2022. Samples were collected from three climatic regions in Pakistan (R1, R2 and R3) with differences in mean monthly air temperature, mean monthly precipitation and elevation. The morphology of pollen was observed by light microscopy, scanning electron microscopy (SEM), biochemical fingerprint analysis, and composition of pollen were investigated by fourier-transform infrared spectroscopy (FTIR). The pollen-associated bacterial populations were identified through a Biolog GEN III microplate system. The pollen water-soluble proteins were isolated and stabilized in phosphate buffer saline (PBS) and tested for allergenicity responses through dot blots and western blots analysis. The morphology study found difference in pollen biochemical composition. Biolog identified Brevibacterium epidermidis and Pantoea agglomerans from P. pratense pollen. Protein extract quantification and sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) gel found decreased protein expression in R1 region pollen compared to R2 and R3 region pollen. Allergenicity studies found differential expression of beta-expansin and profilin allergens in pollen obtained from the three regions. Beta-expansin and profilin were suppressed in R1 pollen and expressed in compared to R2 and R3 pollen. This is the first study to identify B. epidermidis and P. agglomerans growth on P. pratense pollen. Variable allergen expression in P. pratense pollen has also been observed in different regions. Soil pH, an increase in mean monthly temperature and a decrease in mean monthly precipitation correlated with pollen biochemical composition, and reduced beta-expansin and profilin expression involved in pollen growth and development. The findings of this research are unique, which enhances basic knowledge and understanding of P. pratense-pollen associated microbiota and climate change impacts on the pollen allergen expression.

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.

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.

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.

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)

AbstractEnzymatic 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.