SDS-PAGE Research Articles

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.

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

AbstractTrypsin 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 23.0 kDa 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 0.87 mM and 13.04 s−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 ApplicationHarpiosquillid 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.

Freshwater jellyfish in northeastern Argentina: a risk to human health.

Although cnidarians are mostly marine organisms, the occurrence of freshwater jellyfish frequently arouses the interest of ecologists, due to their sudden and unusual appearances in natural and artificial water bodies around the world. This study describes a series of cases compatible with cnidarian envenomation that occurred coincidentally in time and space with the presence of Craspedacusta sowerbii Lankester, 1880 jellyfish in the province of Misiones (El Saltito stream) in January 2022. A year later, its presence was confirmed in another watercourse of this province (Cazador stream). Based on these findings, its possible toxicological consequences in humans were evaluated by characterizing the jellyfish extract using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzymatic/toxic activities. We revealed the presence of proteolytic enzymes and cytolytic toxin(s), which-by means of activating the inflammatory cascade-could explain the stinging skin lesions observed in the suspected cases described. In addition to recording for the first time the occurrence of this cnidarian in northeastern Argentina, this work provides, for the first time, a toxinological explanation for the clinical observations after contact with the most widespread freshwater jellyfish species in the world, giving support to health professionals in the diagnosis and management of such accidents/envenomation.

Laccase purification and azo dye decolorization potential of Sparassis latifolia

Sparassis latifolia often referred to as Cauliflower mushroom possess both medicinal and edibility values. In this research work, first time laccase purification and dye colourisation efficacy of Sparassis latifolia’s purified laccase were assessed. Optimal laccase potential was noted after 12th day of incubation with 4 pH of medium at 45 °C. As supplementary nutritional source, sucrose and ammonium sulphate were unveiled as most effective synthetic carbon and nitrogen sources while wheat straw found as proficient lignocellulosic biomass for enhanced laccase production. Following optimisation of laccase production parameters, laccase was purified to 14.2 fold by ammonium sulphate precipitation, dialysis and anion exchange chromatography. Laccase molecular weight determined through SDS PAGE was found to be of 65 KDa. This purified laccase was tested for its ability to decolourise the Congo red at definite pH and temperature. It was found that this fungus has capability to decolourise up to 98.6% of Congo-red dye.

Contribution of haptoglobin phenotypic variation to the presence of hyperhomocysteinemia in type 2 diabetics with and without angiopathy.

The genetic polymorphism of haptoglobin (Hp) has been associated with several cardiovascular risk factors, but a possible relationship between Hp phenotypic variation and increased levels of homocysteine (Hcy) and cysteine (Cy) is still unknown. The objective of this study is to evaluate the relationship between the Hp polymorphism and hyperhomocysteinemia (HHcy) and hypercysteinemia (HCy) in type 2 diabetics (T2D) with and without angiopathy (AGP). A case-control study was carried out on 293 adults: Group I (GI) - 75 subjects with T2D and AGP; Group II (GII) - 75 subjects with T2D without AGP; Group III (GIII) - 143 controls. Plasma levels of Hcy, Cy and vitamin B6 were measured by high performance liquid chromatography (HPLC) and vitamins B9 and B12 determined by electrochemiluminescence (ECL). The Hp polymorphism was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and peroxidase staining. The results were analyzed in SPSS®, version 26.0 with a significance of 95%. Mean Hcy concentrations were significantly lower in carriers of the Hp2-2 phenotype (6.14 µM; p = 0.046) compared to the other genotypes. The presence of Hp2-1 is associated with an approximately 3.3 times greater probability of occurrence of HHcy (p = 0.015) and 3.7 times greater probability occurrence of HCy (p = 0.021) in T2D with AGP. The presence of the Hp2-1 phenotype is associated with the predisposition of HHcy and HCy in individuals with T2D and AGP, possibly through a positive heterosis mechanism. Carriers of the Hp2-2 phenotype appear to have a greater activation of the transsulfuration pathway in the Hcy cycle and consequent protection for its accumulation.

Rheological properties and in vitro digestibility of chicken breast myofibrillar protein containing various psyllium (Plantago ovata) husk concentrations

SummaryMost dietary fibres improved rheological properties but negatively impacted digestibility. However, the effect of psyllium husk powder (PHP) on digestibility has not been yet reported. Thus, this study aimed to evaluate the rheological properties and digestibility of chicken myofibrillar protein (MP) containing different concentrations of PHP (0%, 0.5%, 1.0%, 1.5%) at 0.45 m NaCl. To assess the rheological properties of MP, viscosity, hydrophobicity, disulphide bond, SDS–PAGE, cooking yield and gel strength were evaluated. The addition of PHP enhanced water retention capacity and protein–protein interactions in the MP system. To evaluate the effect of PHP on digestibility, protein digestibility, SDS–PAGE and microstructure observations were performed. It was confirmed that the addition of PHP improved the protein digestibility of MP by measuring the differences in protein content (%). Therefore, the addition of PHP into MP gel improved not only the rheological properties but also the protein digestibility during in vitro digestion.

Targeting the Plasmodium falciparum IspE Enzyme.

The enzyme IspE in Plasmodium falciparum is considered an attractive drug target, as it is essential for parasite survival and is absent in the human proteome. Yet it still has not been addressed by a small-molecule inhibitor. In this study, we conducted a high-throughput screening campaign against the PfIspE enzyme. Our approach toward a PfIspE inhibitor comprises in vitro screening, structure-activity relationship studies, examining the docking position using an AlphaFold model, and finally target verification through probe binding and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The newly synthesized probe containing a diazirine and an alkyne moiety (23) allowed us to demonstrate its binding to IspE in the presence of a lysate of human cells (HEK293 cells) and to get evidence that both probe 23 and the best inhibitor of the series (19) compete for the same IspE binding site.

Comparative Study on Protein Composition and Foam Characteristics of Barley and Wheat Beer.

Protein is an important component of beer, and its type, content and molecular weight directly affect the quality of beer, especially the foam quality of beer. Different brands of wheat beer and barley beer available in the market were used for this analysis. The differences in protein composition and foam performance between multi-sample barley and wheat beer were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-pressure size exclusion chromatography. Protein significantly influences beer quality, particularly its foam properties. Wheat beer (WB) has 9.52-84.10% more total protein content than barley beer (BB). The primary proteins in both beers are 6.9-20.1 kDa, with WB having 1.04 g/L more of this protein, 60.11% higher than that of BB. It is one of the main different proteins between WB and BB. WB also contains 66.67% more 20.1-32.4 kDa protein compared to BB. This is one of the main differences between WB and BB proteins. Both 6.9-20.1 kDa and 20.1-32.4 kDa proteins enhance beer viscosity and foam properties. Additionally, WB's > 32.4 kDa protein content is 246.67% higher than BB's, significantly improving beer hydrophobicity and foam performance. These protein differences are key factors in the superior foam quality of WB.

Evaluation of protective efficacy of recombinant Toxoplasma gondii DDX39 protein vaccine against acute and chronic T. gondii infection in mice

Toxoplasma gondii, a pervasive parasite responsible for toxoplasmosis, poses significant health risks to humans and animals. In this study, we investigated the immunogenicity and protective efficacy of the recombinant T. gondii DDX39 protein formulated with ISA201 adjuvant (rTgDDX39) as a candidate vaccine against toxoplasmosis. The full-length of TgDDX39 gene was successfully amplified, cloned into the pET-30a vector, and expressed in BL21 (DE3) competent cells, which was purified and identified as a 57.1 kDa protein via sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis confirmed that rTgDDX39 was specifically recognized by serum from T. gondii-infected mice. Furthermore, immunization of rats with rTgDDX39 generated antiserum that could specifically recognize the native TgDDX39 protein in T. gondii tachyzoite lysates. Immunofluorescence assay revealed that TgDDX39 was primarily located in the nucleus and perinuclear region of tachyzoites. Our vaccination strategy significantly increased T cell proliferation, with CD4+T cells rising by 21.9% and CD8+T cells by 57.8% by the sixth week compared to the adjuvant control group. Additionally, high titers of anti-rTgDDX39 IgG antibodies were detected in vaccinated mice, with a notable induction of IgG1 and IgG2a isotypes, and IgG1/IgG2a > 1 suggests a Th2-biased immune response.Furthermore, in vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize this parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a potential candidate vaccine against toxoplasmosis. The protective efficacy of the vaccine was evaluated in mice challenged with acute (RH) and chronic (PRU) strains of T. gondii, showing a survival time extended to 17 days in the acute model, compared to 13.5 and 14 days in the control groups, and a significant 34% reduction in cyst burden in the chronic model. Additionally, the survival rate in the PRU-infected mice increased from 15 to 20% in the control groups to 45% in the vaccinated group. In vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize the parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a candidate vaccine against toxoplasmosis.

A novel γ-chain mutation p.Asp318His in a Chinese family with dysfibrinogenemia.

Congenital dysfibrinogenemia is characterized by reduced fibrinogen activity, but normal immunoreactive fibrinogen levels. Here, we present a novel case with an elevated risk of thrombosis. Coagulation assays, gene analysis, in silico tools, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), fibrin polymerization, thrombin generation assay, and electron microscopy scanning were utilized to elucidate the pathogenic mechanism. The proband manifested with a normal immunologic fibrinogen (2.13 g/l) but reduced functional fibrinogen (0.39 g/l). Subsequent genetic analysis unveiled a novel heterozygous mutation, c.1030G>C (p.Asp318His), in the γ-chain D domain of fibrinogen, which was highly conserved in homologous species and led to enhanced thrombin generation capability. The ability of the proband's fibrinogen to polymerize was significantly impaired, with decreased final turbidity. Scanning electron microscopy indicated that the fibers of the proband were thinner than normal, with smaller pores. Thromboelastography (TEG) results demonstrated prolonged K time, decreased angle value, and a normal confidence interval value in the proband. We present a novel case displaying the γAsp318His mutation, which resulted in dysfibrinogenemia.