SDS-PAGE Research Articles

Characterisation and serodiagnostic evaluation of a recombinant 22.6-kDa tegument protein of Schistosoma spindale.

Schistosomosis in animals due to Schistosoma spindale significantly burdens India's livestock economy because of high prevalence and morbidity and is mostly underdiagnosed from the lack of sensitive tools for field-level detection. This study aimed to clone, express the 22.6-kDa tegument protein of S. spindale (rSs22.6kDa) and to utilise it in a dot enzyme-linked immunosorbent assay for serodiagnosis. RNA was extracted from adult worms recovered from the mesenteries of slaughtered cattle to amplify the gene encoding the 22.6-kDa protein. In silico analysis revealed the protein's secondary structure, consisting of 190 amino acids forming alpha helices (47.89%), extended strands (17.37%), beta turns (8.95%), and random coils (25.79%), with α helices and β sheets in the tertiary structure. Two conserved domains were noted: an EF-hand domain at the N-terminus and a dynein light-chain domain at the C-terminus. Phylogenetic studies positioned the S. spindale sequence as a sister clade to Schistosoma haematobium and Schistosoma bovis. The gene was cloned into a pJET vector and transformed into Escherichia coli Top 10 cells, with expression achieved using a pET28b vector, BL21 E. coli cells, and induction with 0.6 mM isopropyl-β-d-thiogalactopyranoside. The protein's soluble fraction was purified using nickel-chelating affinity chromatography, confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, identifying a distinct immunodominant 22.6-kDa protein. The diagnostic utility was validated using a dot enzyme-linked immunosorbent assay which demonstrated a of sensitivity of 89.47% and specificity of 100%. The study records for the first time the prokaryotic expression and evaluation of the 22.6-kDa tegumental protein of S. spindale, highlighting its potential as a diagnostic antigen for seroprevalence studies in bovine intestinal schistosomosis.

Phosphate and two-stage cooking: Impact on lipid oxidation and tenderness in sous-vide beef semitendinosus.

The study investigated phosphate's effects on tenderness and lipid oxidation in beef cooked sous-vide. Semitendinosus beef cuts were treated with 0.15% and 0.3% sodium tripolyphosphate (STPP), and plain water (0% STPP). These cuts were then sous-vide cooked at two different temperatures, 60 °C and 70 °C, for two different durations, 3 h and 6 h. Analytical techniques including Warner-Bratzler shear force (WBSF), pH, thiobarbituric acid reactive substances (TBARS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), scanning electron microscopy (SEM), and Fourier-transform infrared spectroscopy (FTIR) were employed. STPP concentration did not yield a statistically significant impact on the pH and FTIR spectrum of sous-vide beef (p > 0.05). However, STPP markedly enhanced tenderness by loosening the myofibril structure (observed in SEM) and weakening myosin and actin bands (observed in SDS-PAGE). This improvement led to lower WBSF values with 0.3% STPP, particularly noticeable at 60 °C for 6 h. Additionally, 0.3% STPP delayed lipid oxidation, evidenced by lower oxidation values after 4 weeks of refrigerated storage at 4 °C. These findings highlight STPP's potential to improve tenderness and lipid oxidation stability in sous-vide beef.

Synthesis of nanocellulose from Acalypha hispida leaves through enzymatic hydrolysis

The enzyme-hydrolysis of Acalypha hispida plant leaves for the synthesis of nanocellulose remains underexplored, yet it holds significant potential due to its biocompatibility, eco-friendliness, and the exceptional physical and biochemical properties. This innovative method employed the enzyme xylanase, produced by an indigenous strain of Bacillus pumilus, isolated from a compost pile sample and identified through 16S rRNA sequencing. The xylanolytic activity of the isolate was confirmed by the clear hydrolysis zones around bacterial colonies on xylan agar. The enzyme was purified using ammonium sulfate precipitation followed by dialysis, with a molecular weight of 45 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified xylanase was then utilized for enzyme-hydrolyzed nanocellulose (EHNC) synthesis. EHNC stands out for its green processing, biocompatibility, biodegradability, and low production cost. The resulting EHNC exhibited particle diameters ranging from 15 to 40 nm, with a zeta potential of −22.3 mV. The minimal enzyme concentrations and short incubation periods suggest significant potential for enzymatic applications in the production of nanofibrillated cellulose, offering an efficient and sustainable approach to manufacturing advanced nanocellulose-based materials.

Different polyphenols on cold plasma-induced covalent ovalbumin-polyphenols conjugates and affecting the antigenicity of OVA and its functionalities

The study investigated cold plasma (CP) treatment to modulate the antigenicity and functionality of ovalbumin (OVA) through CP-induced covalent conjugation with five structurally distinct dietary polyphenols, gallic acid (GA), syringic acid (SA), (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (EGC), and (-)-epicatechin (EC). Results indicated that a maximum polyphenol content of 176.63 ± 1.83 μmol/g in OVA-EGCG conjugates was obtained following CP treatment (60 s). The generation of covalent OVA-polyphenol conjugates was confirmed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The binding sites of the five polyphenols in OVA were identified using LC-MS/MS. Different regions and quantities of binding sites were observed for the various polyphenols. Seventeen binding sites were detected in OVA-EC conjugates, whereas only one site (His371) of EGC was found in OVA-EGC conjugates. Additionally, a maximum reduction in IgE binding capacity (63.13 ± 0.87%) of OVA was achieved after GA grafting, supported by western blotting (WB) and enzyme-linked immunosorbent assay (ELISA) analyses. The masking and steric-hindrance effect on linear epitopes by polyphenol grafting play key roles in reducing OVA’s antigenicity. Moreover, the emulsifying properties and antioxidant activity of OVA were substantially improved following polyphenol grafting. Correlation analysis revealed that the hydroxyl group (-OH) content in polyphenol molecules was the critical factor determining covalent binding efficiency between OVA and polyphenols, consequently resulting in antigenicity elimination and functionality improvement of OVA. This study demonstrates that CP-induced covalent grafting of polyphenols is an effective strategy for eliminating antigenicity and modulating the functionalities of proteins.

Menaquinone-7 and its therapeutic potential in type 2 diabetes mellitus based on a Zucker diabetic fatty rat model

BackgroundType 2 diabetes mellitus (T2DM) is marked by insulin resistance, low grade chronic inflammation, and endothelial dysfunction. Vitamin K2, especially menaquinone-7 (MK-7), might delay T2DM progression and alleviate its consequences. Hence, this study evaluated the effects of MK-7 on serum and urine markers of diabetes in an animal model of T2DM. MethodsHetero- (fa/+, control) and homozygous (fa/fa, diabetic) male Zucker diabetic fatty (ZDF) rats were supplemented or not with MK-7 for 12 weeks. After euthanasia, vitamin K1, menaquinone-4 and MK-7 serum concentrations were analyzed via reversed phase high pressure liquid chromatography. Glucose (serum), fructosamine (serum) and creatinine (serum and urine) levels were assessed photometrically, serum cystatin C and urinary total protein were turbidimetrically determined. Serum transforming growth factor beta1 (TGF-β1) and procollagen type III N-terminal peptide (PIIINP) were quantified with enzyme-linked immunosorbent assay. Urinary marker proteins were analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nephropathy was assessed histologically. ResultsSupplementation led to significantly elevated MK-7 serum levels and a significant reduction of PIIINP serum levels in both hetero- and homozygous ZDF rats. Additionally, not statistically significant reductions of TGF-β1 serum levels, proteinuria as well as the nephropathy score were observed. In vivo body mass, serum fructosamine, glucose, cystatin C and creatinine levels were unaffected. ConclusionMK-7 reduced serum markers of fibrosis, histological features of nephropathy and urinary protein excretion, but failed to affect serum markers of T2DM. The therapeutic potential of MK-7 in T2DM and its mode of action should be further investigated in more detail.

Effect of pre-acidification induction on the physicochemical features, myofibrillar protein microstructure, and headspace volatiles of ready-to-cook goose meat

This study examined the impact of pre-acidification induction on the quality attributes and flavor retention of ready-to-cook (RTC) goose meat products. The results demonstrated that pre-acidification could influence the eating qualities of RTC goose meat by effectively regulating the physicochemical properties of goose myofibrillar proteins (MP) including solubility and water-holding capacity. Elevated carbonyl content indicated an enhanced gel-forming capacity in RTC goose meat during storage, coupled with reduced total sulfhydryl content from enhanced protonation pretreatment and augmented lipid oxidation. Structural characterization of MP via sodium dodecyl sulfate–polyacrylamide gel electrophoresis, circular dichroism spectroscopy, and intrinsic fluorescence revealed the formation of a dense protein matrix under highly acidic conditions. Furthermore, the headspace concentration of aldehydes increased by 3.23 times upon enhancing the pre-acidification intensity, resulting in the production of esters and acidic flavor compounds with favorable aromas. Correlation analysis demonstrated the dependence of headspace concentrations of volatile constituents on the acidification-enhanced surface hydrophobicity of MP, attributed to the modified binding sites of proteins after pre-acidification. Current results have indicated both the positive and negative influence of pre-acidulation induction on the eating quality of goose meat products, suggesting the necessity of introducing extra processes to modulate the quality of prefabricated products.

How much do we know about nanobacteria?

Background/PurposeNanobacteria, known to date as self-replicating, nano-scale size organisms, smaller than bacteria. However, whether these are living organisms or agglomeration of biomolecules was one of the most controversial issues for many years. One of the reasons for debate is their lack of any genetic material. Although many studies investigate whether nanobacteria are the cause of diseases that affect human health, the discussions did not reach a conclusion. While most studies try to unravel the structures of nanobacteria, some studies investigated their effects on diseases. It has been proposed that pathological calcifications in kidney and dental pulp stones, as well as calcifications in heart valves and arteries, might be caused by nanobacteria. In this study, we investigated the effects and molecular structures of nanobacteria on the formation of atheroma plaques that cause atherosclerosis. MethodsNanobacteria grown from atheroma plaques in cell culture medium were analysed by scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDS). Macromolecules of nanobacteria were separated by SDS-polyacrylamide gel electrophoresis and their sensitivity to protease and nucleases was tested. After determining their resistance to nucleases and susceptibility to proteinase K, protein profiles were determined by quadrupole time-of-flight mass spectrometry (QTOF-MS). ResultsIn SEM, spherical structures with sizes ranging from 80 to 900 nm were visualized. EDS analyses showed that very low concentration of phosphorus (P: 0.3 %) indicated absence of nucleic acids. The resistance was observed to proteases. Also, protein profiles indicated human proteins, with a high percentage of albumin. ConclusionIt was determined that nanobacteria derived from atheroma plaque were structures composed of human proteins. The findings confirmed the theory that nanobacteria are non-living structures formed by a mechanism called biocrystallization. Although the accumulation of nanobacteria may be a contributing element to the creation of atheroma plaque, it is possible that the etiological components that initiate the formation of the plaque are not nanobacteria.

Self-induced anaerobiosis fermentation in coffees inoculated with yeast: Effect on key enzymes of the germination process and its relationship with the decrease in seed germination

Our objective was to monitor the main enzymes of coffee germinal metabolism and chemical composition during Self-Induced Anaerobiosis Fermentation (SIAF) with yeasts (Saccharomyces cerevisiae (CCMA0543), Candida parapsilosis (CCMA0544) and Torulospora delbrueckii (CCMA0684)) evaluating their relationship with seed germination. The starter cultures were assessed by qPCR. The organic acids were analyzed by liquid chromatography. Catalase (CAT), Esterase (EST), Alcohol dehydrogenase (ADH), and Isocitrate Lyase (ICL) enzyme activity was confirmed by the presence of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-Page) gel bands. The formation of a white halo confirmed the activity of the enzyme endo-β-mannanase, and its quantification was performed using the diameter of the halo of both the samples and the standard curve. At the end of the fermentation process, S. cerevisiae and T. delbrueckii presented the highest populations (>7 log10 cells/g). Succinic acids (average −1.11 g/kg) were consumed during SIAF. Lactic acid increased after 180 h in coffees fermented by the SIAF method (average 3.57 g/kg). CAT and EST showed high activity in the conventional process. ADH activity was detected in both processes after 180 h of the SIAF method. Yeast inoculation during the SIAF method increased the activity of ICL andshowed more intense activity in the first 96 h of fermentation, especially the pulped coffee. Endo-β-mannanase activity was intense during conventional coffee processing (9.89–10.99 pmol/min/g). Natural processing tends to preserve a higher percentage of viable seeds. Therefore, the processing and fermentation methods impact seed quality differently.

Trypsin from digestive tract of harpiosquillid mantis shrimp: Molecular characteristics and the inhibition by chitooligosaccharide and its catechin conjugate.

Trypsin from the digestive tract of harpiosquillid mantis shrimp (HMS) was purified using ammonium sulfate precipitation and a soybean trypsin inhibitor-CNBr-activated Sepharose 4B affinity column. The purified trypsin (PTRP-HMS) had a purity of 30.4-fold, and a yield of 14.5% was obtained. PTRP-HMS had the molecular weight of 23.0kDa as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and only one isoform was detected by native-PAGE. Its optimal temperature and pH were 55°C and 8.5, respectively. TLCK almost completely inhibited the activity of trypsin. The PTRP-HMS had a Michaelis-Menten constant (Km) and catalytic constant (Kcat) of 0.87mM and 13.04s-1, respectively, toward Nα-benzoyl-l-arginine 4-nitroanilide hydrochloride. When chitooligosaccharide (COS) and COS-catechin (COS-CAT) conjugates were examined for inhibition toward the PTRP, the latter exhibited higher efficacy in inhibiting the trypsin. Both COS and COS-CAT conjugate showed mixed-type inhibition kinetics. As a consequence, COS and COS-CAT conjugate could be used as natural additives for inhibiting trypsin in whole HMS, thus retarding the softening and lengthening the shelf-life of HMS during the iced storage. PRACTICAL APPLICATION: Harpiosquillid mantis shrimp (HMS) is of high demand due to its delicacy. However, its meat undergoes rapid softening within 2-3 days when stored in ice. Understanding causative proteolytic enzymes, especially trypsin from digestive tract, paves a way for preventing their negative impact on HMS eating quality. Employment of safe inhibitors, for example, chitooligosaccharide (COS) or COS conjugated with catechin, could inhibit HMS trypsin. Overall, softening of whole HMS containing trypsin in its digestive tract can be impeded, especially when treated with COS-CAT. This finding is beneficial for the HMS local vendor or exporter, in which HMS quality can be maintained.

Protein profile changes during priming explants to embryogenic response in Coffea canephora: identification of the RPN12 proteasome subunit involved in the protein degradation.

Plant somatic embryogenesis encompasses somatic cells switch into embryogenic cells that can later produce somatic embryos with the ability to produce plantlets. Previously, we defined in vitro culture settings for the somatic embryogenesis process of Coffea canephora that comprise adequate plantlets with auxin plus cytokinin followed by cut-leaf explant cultivation with cytokinin, producing embryos with the ability to regenerate plantlets. Here, we confirmed that cultivating cut-leaf explants with cytokinin is sufficient to promote somatic embryos proliferation and the high yield of somatic embryos in the protocol requires adequate plantlets with auxin plus cytokinin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels reveal auxin-plus cytokinin-dependent regulated proteins in plantlets with up and down abundance. Chitinase A class III, proteins involved in the metabolism and folding of proteins, photosynthesis, antioxidant activity, and chromatin organization were identified. The RPN12 protein, which is a subunit of the proteasome 26S, has an abundance that is not associated with transcript changes, suggesting post-translational regulation.

Whey proteins from camel's milk have higher in vitro wound-healing effect than whey proteins from cow's milk.

The effectiveness of whey obtained by either enzyme (sweet) or acid treatment on wound healing remains unclear. This study investigated the effectiveness of camel and bovine whey prepared enzymatically (CSW and BSW) or by pH reduction (CAW and BAW). After removing the cream from milk, HCl or rennet was used to remove casein, resulting in acid or sweet whey, respectively, followed by lactose removal using dialysis. Casein removal was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Wound-healing activity was measured in vitro on HT-29 cells by scratch assay. All four whey samples (0-1000 mg L-1) were applied on the cells, and the closure of the cell-free scratched areas was monitored for 48 h. All whey samples increased the cell migration significantly (P < 0.05) to help close the cell-free areas as an indication of wound healing compared to the negative control. However, the closure amounts between the highest dose (1000 mg L-1) and the control were not significantly different (P > 0.05). Acid whey samples significantly (P < 0.05) elevated the closure speed compared to the sweet whey samples. The highest closure percentage (64.69%) was achieved after treatment with 10 mg L-1 CAW for 48 h. Between the sweet whey samples, BSW was significantly (P < 0.05) more effective in closing the cell-free zone compared to CSW. This study investigated the wound-healing potential of camel and bovine whey in vitro by comparing their effects on HT-29 cell migration. CAW showed the greatest activity and may find uses as a treatment agent in wound healing. © 2024 Society of Chemical Industry.

A Systematic Workflow for Fragmentation Identification of Therapeutic Antibodies by Liquid Chromatography-Mass Spectrometry.

Fragmentation is a phenomenon ubiquitously observed during research and development of therapeutic antibodies. The clear identification of the cleavage site is vital for comprehending fragmentation mechanisms and optimizing processes. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is now widely used to detect and quantify fragments, while its peak identification is hindered by immature capillary electrophoresis-sodium dodecyl sulfate coupled with mass spectrometry techniques. In this study, we developed a systematic workflow for fragment characterization, which integrated direct identification, fragment enrichment, and fragmentation confirmation using multiple techniques, such as microscale peptide mapping (PM), PM of N-termini labeled sample, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in-gel extraction for molecular weight (MW) and PM measurements. By employing this innovative workflow, we successfully identified the cleavage sites of two therapeutic antibodies. In the first case, through direct liquid chromatography-mass spectrometry (LC-MS) characterization, the cleavages leading to the loss of biological function were identified on the linker of a bispecific antibody. In the second case involving a tetravalent antibody, direct LC-MS analysis failed. However, more sophisticated analysis nailed down the critical cleavage at the LC/HC: G105-R106 site in the CDR3 region of the antibody. Our systematic workflow provides a clear and accessible method for identifying cleavage sites with broad applicability across pharmaceutical laboratories.

Effect of transglutaminase cross-linking on the structure and emulsification performance of heated black bean protein isolate.

Transglutaminase (TGase) is a heat-resistant biocatalyst with strong catalytic activity, which functions effectively under moderate temperature and pH conditions, and is used widely in protein cross-linking and recombination. Transglutaminase cross-linking is a novel and specific modification method for black bean protein isolate (BBPI). This article investigates the effect of transglutaminase cross-linking on the structure and emulsification performance of heated BBPI. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that heated BBPI with TGase had a higher molecular weight than heated BBPI without TGase, and the protein bands widened with increasing enzyme activity, indicating that TGase cross-linking promoted protein molecule aggregation. A high molecular weight polymer can better stabilize the oil-water interface, preventing the emulsion from layering. Fourier transform infrared (FTIR) spectroscopy showed that the α-helix content decreased from 15.64% to 13.75%, and the β-sheet content increased from 48.13% to 54.08%. The decrease in α-helix content and increase in β-sheet content could make the structure more stable and improve the emulsifying properties of heated BBPI. When TGase was 20 U g-1, the protein emulsification activity index (EAI) reached its highest value of 1.87 m2 g-1, and the emulsification stability index (ESI) value was 0.27 min (P < 0.05); these figures were 0.19 m2 g-1, and 0.07 min higher, respectively, than in the sample without added TGase. In summary, transglutaminase cross-linking has a positive effect on the structure and emulsification performance of heated BBPI and can be used as an effective method for BBPI modification. © 2024 Society of Chemical Industry.

Insights into larval development and protein biochemical alterations of Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae) following Beauveria bassiana and Solanum lycopersicum treatments

BackgroundThe polyphagous notorious pest, black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), cause significant production losses due to its distinctive feeding and hiding behavior, making it particularly challenging to control it with conventional methods. Therefore, sustainable agriculture demands more effective and environmentally safe pest control solutions. This study aimed to investigate the toxicity of two insecticide alternatives, the entomopathogenic fungus (EPF) Beauveria bassiana and Solanum lycopersicum extract (Tomato plant crude extract, TPCE), using two bioassay methods: the poisoned bait method and the leaf dipping method. In addition, the impact of these biological tools on larval development and protein profiles was evaluated.ResultsThe bait application of both tested materials exhibited higher toxicity than the leaf dipping method, as indicated by the toxicity index. The LC50 values for B. bassiana were 1.6 × 10⁸ and 1.8 × 10⁶ conidia ml−1 using the leaf dipping method and poisoned baits method, respectively. For TPCE, the LC50 values were 4.35 and 1.51 mg ml−1 for the same methods, respectively. In addition, sublethal concentrations of both materials altered the larval and pupal durations. B. bassiana significantly reduced the concentration of larval hemolymph protein. A maximum of 12 protein bands in the control sample, with molecular weights (Mw) ranging between 35 and 120 kDa, were detected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). In B. bassiana-treated larvae, ten bands were detected with Mw ranging from 35 to 120 kDa. At least seven bands were detected in TPCE-treated larvae, with Mw ranging from 35 to 97 kDa.ConclusionsThe findings of this study can be integrated into management programs for A. ipsilon. In addition, the availability of B. bassiana and TPCE in Egypt and their cost-effectiveness as insecticide alternatives support their use in the management programs of this critical pest. These methods are particularly effective when applied in bait form.Graphical

Structure and functionality of Pleurotus geesteranus protein isolate as a function of pH.

Edible mushroom proteins hold great potential for food applications, but those extracted using the alkaline extraction-acid precipitation method typically exhibit poor solubility in neutral water, with the structural changes during acid precipitation remaining unclear. In this study, Pleurotus geesteranus protein isolate (PGPI) with high water solubility was prepared with alkaline extraction, followed by dialysis and freeze-drying, and the effects of pH on the structural and functional properties of PGPI were systematically investigated. PGPI was enriched in essential and aromatic amino acids, and the molecular weight of bands in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile was mainly distributed below 45kDa. The zeta potential of PGPI changed from +16.84 to 17.58mV when the pH increased from 2 to 9, with a pI of 4.3. At pH 7, PGPI showed a size of 232.7nm. Away from pH 7, the particle size of PGPI increased. When the pH decreased from 7 to 2, PGPI exhibited a lower α-helix structure content and a higher β-sheet content and a gradual decrease in fluorescence intensity. In addition, as the pH approached 4, H0 and the content of SS group increased to a peak. These results indicated that lowering the pH induced the development of more ordered protein structure, which could be the primary reason for the poor water solubility of P. geesteranus protein obtained through alkaline extraction and acid precipitation. Additionally, these structural changes result in alterations to its functional properties, including water-holding capacity, oil-holding capacity, foaming capacity, foaming stability, emulsion activity index, and emulsion stability index.

High-intensity ultrasound modification of techno-functional and structural properties of white finger millet protein fractions.

In this study, albumin, globulin, and glutelin were extracted from white finger millet, and their amino acid content, functional and structural properties were investigated. The protein concentration of albumin, globulin, and glutelin were 76.01%, 74.32%, and 69.55%, respectively. The results showed that all the fractions had a significant amount of essential amino acids. Aqueous protein dispersions (10%, w/v) were treated for 12min at different ultrasound power levels (100, 200, and 300W). The solubility, emulsifying, and foaming properties of albumin and glutelin were significantly (p<0.05) improved after ultrasound treatment (20kHz) which indicates that ultrasound could unfold protein aggregates. A decrease in particle size, increase in surface hydrophobicity, and zeta potential correlated with improved functional properties. Ultrasound treatment reduced the size of all proteins except for fractions at 300W and also sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a significant change in the molecular weight of albumin and glutelin at 300W. Scanning electron microscopy of treated protein fraction showed distinctive microstructure with irregular structure compared to untreated protein fraction. Although Fourier transform infrared spectroscopy spectra of proteins were similar after ultrasonication, a partial increase in the intensity of the Amide A band was observed. In conclusion, the ultrasound-treated protein fraction can be used as a high-value plant-based emulsifier.

The molecular consequences of FOXF1 missense mutations associated with alveolar capillary dysplasia with misalignment of pulmonary veins

BackgroundAlveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a fatal congenital lung disorder strongly associated with genomic alterations in the Forkhead box F1 (FOXF1) gene and its regulatory region. However, little is known about how FOXF1 genomic alterations cause ACD/MPV and what molecular mechanisms are affected by these mutations. Therefore, the effect of ACD/MPV patient-specific mutations in the FOXF1 gene on the molecular function of FOXF1 was studied.MethodsEpitope-tagged FOXF1 constructs containing one of the ACD/MPV-associated mutations were expressed in mammalian cell lines to study the effect of FOXF1 mutations on protein function. EMSA binding assays and luciferase assays were performed to study the effect on target gene binding and activation. Immunoprecipitation followed by SDS‒PAGE and western blotting were used to study protein‒protein interactions. Protein phosphorylation was studied using phos-tag western blotting.ResultsAn overview of the localization of ACD/MPV-associated FOXF1 mutations revealed that the G91-S101 region was frequently mutated. A three-dimensional model of the forkhead DNA-binding domain of FOXF1 showed that the G91-S101 region consists of an α-helix and is predicted to be important for DNA binding. We showed that FOXF1 missense mutations in this region differentially affect the DNA binding of the FOXF1 protein and influence the transcriptional regulation of target genes depending on the location of the mutation. Furthermore, we showed that some of these mutations can affect the FOXF1 protein at the posttranscriptional level, as shown by altered phosphorylation by MST1 and MST2 kinases.ConclusionMissense mutations in the coding region of the FOXF1 gene alter the molecular function of the FOXF1 protein at multiple levels, such as phosphorylation, DNA binding and target gene activation. These results indicate that FOXF1 molecular pathways may be differentially affected in ACD/MPV patients carrying missense mutations in the DNA-binding domain and may explain the phenotypic heterogeneity of ACD/MPV.

Enhanced characterization and utilization of cowpea protein isolate: rheological and textural properties of heat-induced gels for plant-based egg omelet formulation

This study aimed to evaluate the potential of cowpea isolate (CPI) as an effective plant-based egg substitute by comparing its composition and physicochemical properties with those of commercial isolates, including mung bean, soy, and pea proteins. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis identified a distinctive vicilin-like globulin in CPI that is critical for the unique functional properties of CPI. CPI has a smaller particle size, resulting in superior gelation capacity and water retention, which are essential for egg substitute applications. Thermal analysis revealed the high stability of CPI, which is advantageous for food processing. Notably, the unique heat-induced gelation behavior of CPI, characterized by a smooth transition from solid- to liquid-like states, provided a strength not observed in its commercial counterparts. Microstructural analysis showed that the CPI gel closely resembled the egg gel, underscoring the potential of CPI to mimic the texture of eggs, particularly in omelets.

Influence of protein in low paste viscosities of Bambara groundnut flours from heat-treated Bambara groundnut seeds

Heat treatment of Bambara groundnut seeds has been reported to cause low paste viscosities in resulting flours. Structural changes in Bambara groundnut protein, due to heat treatment, causes the protein to encapsulate starch, making it unavailable to paste causing low paste viscosities. In this study, trypsin was used to hydrolyze proteins in the flour and the microstructure analysis confirmed the disappearance of aggregates. Flour microstructure analysis confirmed hydrolysis of protein from previously aggregated status and showed liberated individual starch granules. Following the treatment of flours by trypsin, Confocal laser scanning microscopy did not show a protein signal. Hydrolysing Bambara groundnut proteins significantly increased the flour paste viscosities (P < 0.05). The final viscosities for flours from 20 % moisture-conditioned and infrared heat-treated seeds for 5 min were 733.9 mPa s before protein hydrolysis and 2081.71 mPa s after protein hydrolysis. The gelatinization temperature (81 °C) did not show a significant change following protein hydrolysis. Sodium dodecyl-sulphate polyacrylamide gel electrophoresis band intensity increased indicative of disulfide bonding and protein polymerisation when microwave and infrared heat treatment were combined. There were changes in Bambara groundnut protein secondary structures such as an increase of 57 % in β-sheet along with a 60 % reduction in the α-helix as shown by the Fourier-transform infrared spectroscopy. The changes in secondary structure of Bambara groundnut protein were caused by microwave and infrared heating. Heat treatment of Bambara groundnut seeds is partly responsible for the reduction in paste viscosities of their flours.

In-depth characterization of N-glycosylation and sialic acid content in fetal and adult fibrinogen

BackgroundFetal fibrinogen is a variant present in neonates. Blood products used in neonates are tailored for adults and do not seamlessly integrate into neonatal clots. Increased sialic acid content has been found in fetal fibrinogen compared with adult fibrinogen. However, the extent or location of sialic acids on fibrinogen remains unknown. ObjectivesTo investigate differences in glycosylation and sialic acid content between fetal and adult fibrinogen. MethodsGlycans were eluted from human cord blood-isolated fetal fibrinogen and commercially available adult fibrinogen using filter-aided N-linked glycan separation. A α, B β, and λ chains were isolated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in-gel enzymatic digestion was performed. Infrared matrix-assisted laser desorption electrospray ionization mass spectrometry was used for analysis. ResultsIn total, 39 and 22 glycans were detected in fetal and adult fibrinogen, respectively. Fetal fibrinogen glycans were most abundant in the lower molecular weight range <4 kDa. After isolating the Aα, Bβ, and λ chains, increased glycosylation and sialic acid content was found in fetal fibrinogen. Increased glycosylation was detected across all 3 chains, and increased sialic acid content was found in the Bβ chain. ConclusionSialylation in the Bβ chain of fetal fibrinogen supports previous findings showing more knob ‘B’ interactions occur in fetal fibrinogen than in adult fibrinogen during clot polymerization. This is also the first detection of glycosylation in the Aα chain of fibrinogen. By elucidating the fibrinogen N-linked glycome, this study found where sialic acid content differs the most between adult and fetal fibrinogen. This can ultimately be used to develop blood products that are neonatal-compatible.