SDS-polyacrylamide Gel Electrophoresis Analysis Research Articles

Comparative study of a liposome and emulsion system with cinnamon essential oil on the quality and proteolysis of refrigerated minced pork.

Essential oils have been recognized for their strong antibacterial property, making them an innovative approach for preserving meat. However, their chemical instability and direct impact on meat proteins limit their application. To overcome these limitations, various loading systems have been explored. This study aimed to compare the effect of cinnamon essential oil (CEO) loaded in a liposome and emulsion system on the proteolysis of minced pork and to evaluate the advantages of each delivery system in preventing microorganism-induced quality deterioration of meat. Minced pork treated with CEO-liposomes exhibited lower pH, total volatile basic nitrogen (TVB-N), and total viable count (TVC) values than CEO-emulsions and provided better protection against microorganisms. SDS-polyacrylamide gel electrophoresis (PAGE) analysis confirmed that CEO-liposome was more effective in protecting proteins from degradation. Moreover, CEO-liposome produced lower amount of bitter amino acids and harmful biogenic amines. Antibacterial mechanisms indicated that CEO-liposome exhibited a stronger inhibitory effect against major spoilage bacteria in meat products by increasing cell membrane permeability. The membrane damage was further supported by an increase in conductivity and the leakage of nucleic acids. Compared to the CEO-emulsion system, CEO-liposome emerged as an effective preservative for minced pork. These results provided important theoretical support for using a bioactive compound delivery system to prevent microorganism-induced quality deterioration in meat.

Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosis.

Microscopic agglutination test (MAT) for the diagnosis of leptospirosis requires live cultures and is serovar-specific, while polymerase chain reaction (PCR) requires expensive equipment and sample preparation. The rLipL32 protein is conserved and can be used for the production of immunoglobulin G (IgG) anti-rLipL32 antibody, which can be used as a biomarker for leptospirosis diagnosis. This study aimed to produce and characterize an IgG anti-rLipL32 antibody as a biomarker for leptospirosis diagnosis. Escherichia coli rLipL32 was cultured and analyzed by PCR and sequencing. Cultures were used for rLipL32 protein expression and purification and the rLipL32 protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The rLipL32 protein was used to produce anti-rLipL32 serum and was analyzed by enzyme-linked immunosorbent assay (ELISA). Serum was purified to obtain IgG anti-rLipL32 antibody and characterized by SDS-PAGE and western blotting. PCR was able to amplify the LipL32 gene from E. coli rLipL32, and sequencing analysis showed 99.19% similarity with pathogenic Leptospira. SDS-PAGE analysis showed a 32-kDa band. ELISA results showed an increase in OD in anti-rLipL32 serum compared to preimmune serum. Western blotting results showed that the IgG anti-rLipL32 antibody was able to bind and cross-reacts with pathogenic Leptospira serovar but not with E. coli or Staphylococcus aureus. IgG anti-rLipL32 antibody has high specificity and sensitivity against Leptospira pathogens. These findings suggest that IgG anti-rLipL32 antibody is a promising biomarker for the diagnosis of leptospirosis.

Comparative study of the commonly used protein quantitation assays on different Hymenoptera venoms: A fundamental aspect of Hymenoptera venom proteome analysis

Determination of protein concentration in Hymenoptera venoms requires an accurate and reproducible assay as the results will be used to support subsequent proteomic techniques employed in their analyses. However, all protein assay techniques have inherent strengths and weaknesses, demanding their assessment before selecting the most suitable platform for sample analysis. In this study, protein profiles of ant, honeybee, and wasp venoms, and bovine serum albumin (BSA) and hyaluronidase standards were qualitatively assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Their amino acid and protein concentration were quantitatively determined via Amino Acid Analysis (AAA). Amino acid concentration was determined via hydrolysis, derivatization, and chromatographic quantification. Protein concentration was estimated using four different protein concentration assays. The ratios of protein concentration in venom samples to protein standards were calculated, and the accuracy of the protein concentration assays was analysed relative to the concentration determined from AAA. SDS-PAGE analysis showed that BSA contained several protein bands, while hyaluronidase contained a mixture of peptide and protein bands. Ant and honeybee venoms contained a higher proportion of peptide bands, while wasp venom contained more protein bands. As determined by AAA, the ratio of protein concentration in Hymenoptera venoms varied between 1.01 and 1.11 to BSA, and between 0.96 and 1.06 to hyaluronidase. Overall, the Bradford assay was found to be the least accurate and the BCA assay was the most accurate in estimating protein concentration in Hymenoptera venoms. There was no significant advantage in using hyaluronidase as a standard or increasing incubation temperature of BCA assay when analysing Hymenoptera venoms. Diluent solutions containing phenol and human serum albumin interfered with Lowry-based assays.

Proteolysis of dry-cured ham according to the type and temperature of starter mixed with Debaryomyces hansenii and Penicillium nalgiovense

This study analyzed the impact of yeast and mold on the proteolysis of dry-cured ham protein.Debaryomyces hansenii, isolated from Doenjang (DJ) and fermented sausage (FS), was used as a starter in the preparation. The experiment involved three repetitions, using 10 gluteus medius muscles each in the 2, 4, and 6-week aging periods. It assessed the following parameters: aging yield, moisture and crude protein content, pH, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profile, non-protein nitrogen (NPN) content, shear force, and in-vitro digestibility. SDS-PAGE analysis reve protein aled that proteolysis occurred as the aging period increased, and the treatment groups DJ25, FS25, and DF25 displayed thicker bands at 25 kDa than the treatment groups DJ20, FS20, and DF20. The NPN tended to increase as the aging period increased, and treatment groups consistently exhibited higher NPN levels than the control at the same aging temperature. The shear force DJ25, FS25, and DF25 tended to have lower shear force values than the other treatments in the final week. The in-vitro digestion profile showed an increased digestion rate of pepsin and trypsin as the aging period increased. DJ25, FS25, and DF25 exhibited significantly higher pepsin and trypsin digestion rates than DJ20, FS20, and DF20 (P < 0.05). In conclusion, the findings indicate that aging dry-cured ham at 25 °C yields higher proteolysis, lower shear force, and higher digestion rates, suggesting that such a process would result in enhanced consumer preference and superior product quality.

The RNA-binding protein Cpeb4 regulates splicing of the Id2 gene in osteoclast differentiation.

Cytoplasmic polyadenylation element-binding protein 4 (Cpeb4) is an RNA-binding protein that regulates posttranscriptional regulation, such as regulation of messengerRNA stability and translation. In the previous study, we reported that Cpeb4 localizes to nuclear bodies upon induction of osteoclast differentiation by RANKL. However, the mechanisms of the localization of Cpeb4 and osteoclastogenesis by Cpeb4 remain unknown. Here, we show that Cpeb4 localizes to the nuclear bodies by its RNA-binding ability and partially regulates normal splicing during osteoclast differentiation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisanalysis with Phos-tag® revealed that the phosphorylation levels of Cpeb4 were already high in the RAW264.7 cells and were not altered by RANKL treatment. Immunofluorescence showed that exogenous Cpeb4 in HEK293T cells without RANKL stimulation localized to the same foci as shown in RANKL-stimulated RAW264.7 cells. Furthermore, when nuclear export was inhibited by leptomycin B treatment, Cpeb4 accumulated throughout the nucleus. Importantly, RNA recognition motif (RRM) 7 of Cpeb4 was essential for the localization. In contrast, the intrinsically disordered region, RRM1, and zinc finger domain CEBP_ZZ were not necessary for the localization. The mechanistic study showed that Cpeb4 co-localized and interacted with the splicing factors serine/arginine-rich splicing factor5 (SRSF5) and SRSF6, suggesting that Cpeb4 may be involved in the splicing reaction. RNA-sequencing analysis revealed that the expression of genes related to cell proliferation processes, such as mitotic cell cycle and regulation of cell cycle processes, was elevated in osteoclasts depleted of Cpeb4. Interestingly, the splicing pattern of the inhibitor of DNA binding 2 (Id2) gene, which suppresses osteoclast differentiation, was altered by the depletion of Cpeb4. These results provide new insight into the role of Cpeb4 as a player of normal splicing of Id2 in osteoclast differentiation.

Determination of genetic diversity in Acacia modesta germplasm using SDS-PAGE.

Biochemical markers such as protein are very important to determine genetic diversity among plant species in a given population which in turn is very important for breeders and farmers as they can then easily select the most appropriate variety to grow in a given locality. In this connection, the present study is aimed to evaluate genetic diversity in Acacia modesta germplasm through Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) technique. About 40 genotypes were subjected to SDS-PAGE analysis where a total of 12 polypeptide bands were observed in electrophoretogram. Out of which 16.67% were monomorphic while the remaining 83.33% were polymorphic. Variation found in B-2, 4, 5, 6, 7, 8, 9, 10, 11 and 12, were 20, 22.50, 32.50, 10, 2.50, 22.50, 15, 5, 2.50 and 75% respectively. Locus contribution toward genetic disagreement was 83.33%. Cluster analysis sorted all the genotypes into 9 clusters. The genotypes in one cluster were identical regarding protein profiling and showed less intra-specific genetic variation whereas differences were find from other genotypes.

Variation in Antimicrobial Activity and Seed Storage Proteins in Three Species of the Medicinal Plant Alstonia

The family Apocynaceae comprises three species of the substantially important Alstonia plant, viz., A. scholaris, A. venenata, and A. macrophylla. The investigation of proteins contained within seeds, that has the potential to provide both precise details and a structural basis for characterizing diversity. The utilization of an electrophoretic technique for protein analysis has been observed in recent scholarly investigations. This implies that certain protein bands exhibit variability, with their presence or absence being detected across different seed arrangement levels in the gel. Furthermore, this implies that the protein bands have been segregated into distinct categories. A study was conducted to analyze the seed storage protein profiles of three distinct Alstonia species through the application of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The resolution of the seed storage protein of the Alstonia species using 15% SDS was found to alter the banding pattern of the polyacrylamide gel. The SDS-PAGE analysis revealed differential up regulation of proteins across distinct bands. Throughout the examination, it was determined that the three aforementioned species exhibited acommon band, in addition to a protein with a molecular weight of 34 kDa. The number of protein bands attached to A. venenata was the highest (ten bands), while the number of protein bands that adhered to A. scholaris was the lowest (five bands). Further, powdered leaves of A. scholaris, A. venenata, and A. macrophylla were investigated for antibacterial and the results, A. macrophylla leaf powder is most effective against Staphylococcus aureus, followed by A. venenata and A. scholaris at 1 μg/ mL. In addition, the findings support the inference that the A. scholaris leaf powder effectively inhibited the growth of Aspergillus niger. A. venenata and A. macrophylla, both at 1μg/mL, are, nonetheless, also effective against A. niger. A. macrophylla has the largest zone of inhibition (11.5 mm) against A. niger.

Two proteins prepared from defatted Antarctic krill (Euphausia superba) powder: Composition, structure and functional properties

Defatted krill powder, rich in protein, has emerged as a potential, novel food raw material. The present study aimed to prepare Antarctic krill protein from the supernatant (AKP-S) and precipitation (AKP-P) using the ultrasound-assisted alkali dissolution and acid precipitation method, and analyze their composition, structure, and functional properties were analyzed. The protein contents of AKP-P and AKP-S were more than 85% (dry weight), and their total amino acid content was 48.75 and 43.59 g/100 g of protein, respectively. The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and surface morphology analysis (SEM) results showed that the AKP-S was degraded during acid treatment and had a smaller particle size than AKP-P. The α-helix and random curled structures of AKP-P and AKP-S were few or even disappeared. The solubility of AKP-S was more than 70% in the pH range of 3–13, and the change in AKP-P solubility with pH was consistent with the trend of β-sheets. Except for pH 7.0–9.0, the solubility of AKP-P and AKP-S with pH was positively correlated with surface-hydrophobicity (H0). The emulsifying activity of AKP-P and AKP-S at different pH was approximately positively correlated with the solubility. The minimum gel-forming concentration of AKP-P was 8%, while AKP-S could not form gel at 14%. The gel water holding capacity reached more than 90% when the AKP-P concentration was 12%. The rheological results showed that the storage modulus (G′) predominated over the loss modulus (G″) of AKP-P in the range of 8%–14% concentration.

Protein A and CaptureSelect FcXP Affinity Resins Possess the Capability of Differentiating Hole-hole Homodimer Isoforms.

Knobs-into-holes (KiH) technology has been widely used in asymmetric bispecific antibody (bsAb) construction to promote heavy chain heterodimerization. However, despite the great improvement of heterodimer formation by this strategy, homodimers (especially the hole-hole homodimer) can still be generated at low levels. Consequently, hole-hole homodimer is a common byproduct associated with the production of KiH bsAbs. In addition, previous studies showed that hole-hole homodimer exists as two different isoforms. As the major difference between these two isoforms lies in the Fc region, we speculated that Protein A media, which bind IgG Fc region with high affinity, and CaptureSelect FcXP, a CH3 domain-specific affinity resin, may provide a certain resolution between these two conformational isoforms. The objective of this study was to study the capability of Protein A and CaptureSelect FcXP affinity resins in differentiating hole-hole homodimer isoforms. The hole-hole homodimer was produced in CHO cells by expressing the hole half-antibody. The homodimer, along with the half-antibody was initially captured by Protein A chromatography and was then further purified by size-exclusion chromatography (SEC), which separated the homodimer from the unpaired half-antibody. The purified hole-hole homodimer was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analytical hydrophobic interaction chromatography (HIC). The purified hole-hole homodimer was separately processed by columns packed with Protein A and CaptureSelect FcXP resins. The purified hole-hole homodimer was also analyzed by Protein A-high-performance liquid chromatography (HPLC). SDS-PAGE analysis and analytical HIC study confirmed that hole-hole homodimer exists as two conformational isoforms. When the hole-hole homodimer was processed by Protein A and CaptureSelect FcXP chromatography, the elution profiles contained two peaks, indicating that both affinity resins possess the capability of differentiating hole-hole homodimer isoforms. Our data suggest that Protein A and CaptureSelect FcXP affinity resins both possess the capability of differentiating hole-hole homodimer isoforms and, therefore, can be used for monitoring isoform conversion under various conditions.

How eluents define proteomic fingerprinting of protein corona on nanoparticles

Nanoparticles (NPs) have broad application prospects in the field of biomedicine due to their excellent physicochemical properties. When entering biological fluids, NPs inevitably encountered proteins and were subsequently surrounded by them, forming the termed protein corona (PC). As PC has been evidenced to have critical roles in deciding the biological fates of NPs, how to precisely characterize PC is vital to promote the clinical translation of nanomedicine by understanding and harnessing NPs’ behaviors. During the centrifugation-based separation techniques for the PC preparation, direct elution has been most widely used to strip proteins from NPs due to its simpleness and robustness, but the roles of multifarious eluents have never been systematically declared. Herein, seven eluents composed of three denaturants, sodium dodecyl sulfate (SDS), dithiothreitol (DTT), and urea (Urea), were applied to detach PC from gold nanoparticles (AuNPs) and silica nanoparticles (SiNPs), and eluted proteins in PC have been carefully characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and chromatography coupled tandem mass spectrometry (LC-MS/MS). Our results showed that SDS and DTT were the main contributors to the efficient desorption of PC on SiNPs and AuNPs, respectively. The molecular reactions between NPs and proteins were explored and verified by SDS-PAGE analysis of PC formed in the serums pretreated with protein denaturing or alkylating agents. The proteomic fingerprinting analysis indicated the difference of the eluted proteins brought by the seven eluents was the abundance rather than the species. The enrichment of some opsonins and dysopsonins in a special elution reminds us that the possibility of biased judgments on predicting NPs’ biological behaviors under different elution conditions. The synergistic effects or antagonistic effects among denaturants for eluting PC were manifested in a nanoparticle-type dependent way by integrating the properties of the eluted proteins. Collectively, this study not only underlines the urgent need of choosing the appropriate eluents for identifying PC robustly and unbiasedly, but also provides an insight into the understanding of molecular interactions during PC formation.

Assessment of protein and DNA polymorphisms in corn (Zea mays) under the effect of non-ionizing electromagnetic radiation

Many reports highlight biological responses of crop plants after non-ionizing electromagnetic radiation (EMR) exposure based on the phenotypic and physiological levels. So, this study aimed to estimate genetic alterations in proteins, isozymes, and DNA banding patterns as well as the extent of nuclear DNA damage of economic corn (Zea mays) under the stress of EMR using accurate and reliable bioassays like sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isozymes (Leucine- aminopeptidase, Esterases, Peroxidase, and Catalases), random amplified polymorphic DNA- polymerase chain reaction (RAPD-PCR), and Comet Assay, respectively. SDS-PAGE analysis showed distinct polymorphisms (96.66%) between EMR exposed and non-exposed corn seedlings depending on the number and type of bands, their intensities as well as molecular weight which ranged from (60.27 to 192.35 kDa), gain, and loss of bands. The four isozymes generated varies isozymatic polymorphisms based on relative front, zymogram number, and optical intensities. RAPD analysis generated 85 amplified DNA products with high polymorphism values ranged from 90.91 to 100% based on primers, band type, DNA sizes which ranged from 153 to 1008-bp, lose, gain, and intensity of DNA bands. Comet Assay scored highest extent of loosed DNA from nuclei (DNA damage) reached the value of (tailed ratio 20%) at EMR exposed corn nuclei for 5 days compared to non-exposed nuclei which reached the value of (tailed ratio 3%). This study concluded that each EMR exposure time had unique interaction with proteins, isozymes, and DNA of corn cells exhibiting wide range of genotoxic stress and subsequently, adversely effect on growth and yield of this sensitive crop plants.

Higher Protein Digestibility of Chicken Thigh than Breast Muscle in an In Vitro Elderly Digestion Model.

This study investigated the protein digestibility of chicken breast and thigh in an in vitro digestion model to determine the better protein sources for the elderly in terms of bioavailability. For this purpose, the biochemical traits of raw muscles and the structural properties of myofibrillar proteins were monitored. The thigh had higher pH, 10% trichloroacetic acid-soluble α-amino groups, and protein carbonyl content than the breast (p<0.05). In the proximate composition, the thigh had higher crude fat and lower crude protein content than the breast (p<0.05). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of myofibrillar proteins showed noticeable differences in the band intensities of tropomyosin α-chain and myosin light chain-3 between the thigh and breast. The intrinsic tryptophan fluorescence intensity of myosin was lower in the thigh than in the breast (p<0.05). Moreover, circular dichroism spectroscopy of myosin revealed that the thigh had higher α-helical and lower β-sheet structures than the breast (p<0.05). The cooked muscles were then chopped and digested in the elderly digestion model. The thigh had more α-amino groups than the breast after both gastric and gastrointestinal digestion (p<0.05). SDS-PAGE analysis of the gastric digesta showed that more bands remained in the digesta of the breast than that of the thigh. The content of proteins less than 3 kDa in the gastrointestinal digesta was also higher in the thigh than in the breast (p<0.05). These results reveal that chicken thigh with higher in vitro protein digestibility is a more appropriate protein source for the elderly than chicken breast.

Investigation of biological activities of two cultivars of Cicer arietinum proteins mass associated with Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, with some known classical factors. Cicer arietinum (Leguminosae) is a source of protein for humans and contains albumin, globulin, glutelin, and prolamin. The protein content of two cultivars of C. arietinum, Hashem and Mansour, was isolated to evaluate their inhibition activity against acetylcholinesterase (AChE), butyrylcholine esterase (BChE), and β-amyloid peptide (βA) aggregation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and molecular docking were also applied to evaluate the content and determine the potential of each chickpea protein to interact with AChE, respectively. Obtained data showed that proteins from both cultivars could inhibit AChE with IC50 of 17.73 (0.03) and 22.20 (0.06) μg/mL, respectively, with no activity on BChE. The 50 μg/mL protein concentration of each cultivar suppressed βA accumulation (Mansour: 25.66% and Hashem: 21.69%) and showed biometal chelating activity. SDS-PAGE analysis revealed relatively different protein patterns, though the Mansour cultivar contained some protein bands with molecular weights of 18, 24, and 70 kDa were estimated to belong to vicilin and legumin, which were absent in the Hashem protein mass. Molecular docking showed that legumin and especially vicilin have good potential to interact with AChE. The chickpea proteins showed inhibitory activity against AChE, which might be due to the vicilin and legumin fractions. The characterization of the inhibitory effect of each protein band could be promising in finding new therapeutic peptide candidates to treat Alzheimer's in the future, although more experimental work is needed in this issue.

Evaluation and Comparison of the Effects of Mature Silkworm (Bombyx mori) and Silkworm Pupae Extracts on Schwann Cell Proliferation and Axon Growth: An In Vitro Study.

Silkworm products were first used by physicians more than 8500 years ago, in the early Neolithic period. In Persian medicine, silkworm extract has several uses for treating and preventing neurological, cardiac, and liver diseases. Mature silkworms (Bombyx mori) and their pupae contain a variety of growth factors and proteins that can be used in many repair processes, including nerve regeneration. The study aimed to evaluate the effects of mature silkworm (Bombyx mori), and silkworm pupae extract on Schwann cell proliferation and axon growth. Silkworm (Bombyx mori) and silkworm pupae extracts were prepared. Then, the concentration and type of amino acids and proteins in the extracts were evaluated by Bradford assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and liquid chromatograph-mass spectrometer (LC-MS/MS). Also, the regenerative potential of extracts for improving Schwann cell proliferation and axon growth was examined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, electron microscopy, and NeuroFilament-200 (NF-200) immunostaining. According to the results of the Bradford test, the total protein content of pupae extract was almost twice that of mature worm extract. Also, SDS-PAGE analysis revealed numerous proteins and growth factors, such as bombyrin and laminin, in extracts that are involved in the repair of the nervous system. In accordance with Bradford's results, the evaluation of extracts using LC-MS/MS revealed that the number of amino acids in pupae extract was higher than in mature silkworm extract. It was found that the proliferation of Schwann cells at a concentration of 0.25 mg/mL in both extracts was higher than the concentrations of 0.01 and 0.05 mg/mL. When using both extracts on dorsal root ganglion (DRGs), an increase in length and number was observed in axons. The findings of this study demonstrated that extracts obtained from silkworms, especially pupae, can play an effective role in Schwann cell proliferation and axonal growth, which can be strong evidence for nerve regeneration, and, consequently, repairing peripheral nerve damage.

Seminal plasma protein profiles and testosterone levels as biomarker semen quality of candidate Madura bulls.

This study aims to determine the protein profile based on molecular weight (MW) and testosterone levels in seminal plasma (SP) that correlates to the semen quality of candidate Madura bulls. A total of 10 male candidate madura bulls underwent semen evaluation (motility, viability, membrane plasma integrity (MPI), and sperm concentration). The centrifuge was run at 1,200 rpm (4°C) for 20 min to collect SP. SP testosterone levels were measured using an Enzyme-linked immunosorbent assay. The characterization of SP proteins in Madura bulls was done using 1D sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. All parameters were analyzed using Pearson correlation analysis. The results of the SDS-PAGE analysis found eight protein bands with the highest MW of 110 kDa and the lowest of 12 kDa. The mean and SD of SP testosterone levels were 20.58 ± 8.56 ng/ml, motility 59.32% ± 20.14%, viability 67.45% ± 20.22%, MPI 32.77% ± 16.52%, and sperm concentration 1,002.64 ± 429.33 106/mm3. Proteins with MWs of 110 and 91 kDa significantly correlated with MPI, and 110 kDa negatively correlated with sperm concentration (p < 0.05). Proteins with MWs of 73 and 36 kDa significantly correlated with SP testosterone levels, while proteins with MWs of 29 kDa significantly correlated with sperm viability (p < 0.05). The expressed protein fraction based on MW is closely related to the quality of semen, so it has the potential to be a biomarker of semen quality. Further research is needed to determine the specific proteins in certain fractions.

Effects of in vitro digestion on protein degradation, phenolic compound release, and bioactivity of black bean tempeh.

The nutritional value and bioactivity of black beans are enhanced when fermented as tempeh, but their bioaccessibility and bioactivity after ingestion remain unclear. In this study, black bean tempeh and unfermented black beans were digested in vitro and changes in protein degradation, phenolic compound release, angiotensin I-converting enzyme (ACE)-inhibitory activity, and antioxidant activity between the two groups were compared. We observed that the soluble protein content of digested black bean tempeh was generally significantly higher than that of digested unfermented black beans at the same digestion stage (P < 0.05). The degree of protein hydrolysis and the content of <10 kDa peptides were also significantly higher in the digested black bean tempeh than in digested unfermented black beans (P < 0.05). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase high-performance liquid chromatography (RP-HPLC) analysis showed that most macromolecular proteins in tempeh had been degraded during fermentation and more of the small peptides were released from black bean tempeh during digestion, respectively. Compared to that of the unfermented black beans, the level of ACE inhibition of black bean tempeh was lower, but this significantly increased to 82.51% following digestion, closing the gap with unfermented black beans. In addition, the total respective levels of phenolics, flavonoids, and proanthocyanidins released from black bean tempeh were 1.21, 1.40, and 1.55 times those of unfermented black beans following in vitro digestion, respectively. Antioxidant activity was also significantly higher in digested black bean tempeh than in digested unfermented black beans and showed a positive correlation with phenolic compound contents (P < 0.05). The results of this study proved that, compared to unfermented black beans, black bean tempeh retained protein and phenolic compound bioaccessibility and antioxidant activity and showed an improved ACE-inhibitory activity even after consumption.

Enhancing SDS-PAGE Detection of Dilute Extracellular Polystyrene Degrading Enzymes Expressed by Bacillus megaterium Strain via Centrifugal Freeze Concentration Method

This study aims to enhance the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) detection and visualization of extracellular polystyrene degrading enzyme expressed by Bacillus megaterium as a result of utilizing polystyrene as a sole carbon source. Dilute polystyrene degrading enzyme expressed in the culture supernatant could not be directly visualized from SDS-PAGE stained with colloidal Coomassie blue stain as it falls below its sensitivity or detection limit level. To overcome this problem, the crude enzyme extract was concentrated via the centrifugal freeze concentration method (cryoconcentration). The extracellular enzyme profiles were determined by SDS-PAGE. The SDS-PAGE analysis of the concentrated enzyme extract revealed 6 visible protein bands ranging in size from 10 to 60 kDa. Three distinct protein bands at approximately 20, 45 and 60 kDa were observed to be highly expressed in the culture supernatant. Polystyrene degrading enzyme extracted from the culture supernatant was closely related to several polymer degrading enzymes which suggest that the extracted enzyme from Bacillus megaterium also belongs to the hydrolase’s enzyme family. GC-MS analysis of the extracted samples significantly contains benzene derivatives due to the breaking down of the long-chain aromatic hydrocarbon polymer. Hydroxylation of the aromatic ring formed phenolic substrate took place which suggests being facilitated by intracellular enzyme hydroxylases. Hence, the biodegradation of polystyrene by Bacillus megaterium was believed to incorporate both intracellular and extracellular enzymes.&#x0D; HIGHLIGHTS&#x0D; &#x0D; Bacillus megaterium strain was first reported to contribute to polystyrene biodegradation&#x0D; Polystyrene degrading enzyme expressed by Bacillus megaterium was detected through SDS-PAGE&#x0D; Cryoconcentration enhanced SDS-PAGE protein detection&#x0D; The protein bands were most distinct visualized at approximately 20, 45 and 60 kDa by SDS-PAGE&#x0D; GC-MS analysis detected benzene derivatives which confirmed enzymatic degradation activity&#x0D; &#x0D; GRAPHICAL ABSTRACT

Efficacy and Mechanisms of Copper Ion-Catalyzed Inactivation of Human Norovirus.

The antinoroviral effect of copper ions is well known, yet most of this work has previously been conducted in copper and copper alloy surfaces, not copper ions in solution. In this work, we characterized the effects that Cu ions have on human norovirus capsids’ and surrogates’ integrity to explain empirical data, indicating virus inactivation by copper alloy surfaces, and as means of developing novel metal ion-based virucides. Comparatively high concentrations of Cu(II) ions (>10 mM) had little effect on the infectivity of human norovirus surrogates, so we used sodium ascorbate as a reducing agent to generate unstable Cu(I) ions from solutions of copper bromide. We found that significantly lower concentrations of monovalent copper ions (∼0.1 mM) compared to divalent copper ions cause capsid protein damage that prevents human norovirus capsids from binding to cell receptors in vitro and induce a greater than 4-log reduction in infectivity of Tulane virus, a human norovirus surrogate. Further, these Cu(I) solutions caused reduction of GII.4 norovirus from stool in suspension, producing about a 2-log reduction of virus as measured by a reverse transcriptase-quantitative polymerase chain reaction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) data indicate substantial major capsid protein cleavage of both GI.7 and GII.4 norovirus capsids, and TEM images show the complete loss of capsid integrity of GI.7 norovirus. GII.4 virus-like particles (VLPs) were less susceptible to inactivation by copper ion treatments than GI.7 VLPs based upon receptor binding and SDS-PAGE analysis of viral capsids. The combined data demonstrate that stabilized Cu(I) ion solutions show promise as highly effective noroviral disinfectants in solution that can potentially be utilized at low concentrations for inactivation of human noroviruses.

Methods Validation of Pork Authentication in Processed Meat Products (Sausages) Through Densitometry Analysis

Analysis of the halalness of food products based on animal origin needs to be carried out, especially for critical food products such as processed meat products (sausages). Some processed meat products are found that they are contaminated by pork in the processing. This research was conducted to determine the validity of the analysis method of pork contamination in one of the processed products (beef, pork, and goat sausages) based on the characteristics of the protein profile. Beef, pork, and goat sausages that have been purchased from supermarkets in Jakarta and South Tangerang are used as the sample.The praparation of sample was preceded by extraction of the sausage protein in PBS buffer pH 7.2. Then, the dissolved protein content was tested, and the protein extract was separated by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). The protein profile result from SDS-PAGE separation was followed by densitometric analysis (ImageJ) to obtain biomarker protein candidates. The validation of the analytical method includes the precision (repeatability), test for accuracy, linearity, and robustness of the method by varying the extraction pH. The results show that the protein extract has the differences in protein content and composition for each sample. The SDS-PAGE analysis results show that biomarker protein candidates appeared below 50 kDa which were thought to be the protein fraction of actin. The precision and accuracy test results obtained for each sample have met the required standards, namely with a KV value &lt;5% and a percent recovery value&gt; 95%. The results of the linearity test and the toughness of the method also show that the test method is quite effective in testing the halalness of animal food products, especially in sausage products which are contaminated by pork.

Understanding the Anti-Diarrhoeal Properties of Incomptines A and B: Antibacterial Activity against Vibrio cholerae and Its Enterotoxin Inhibition.

Incomptines A (IA) and B (IB) are two sesquiterpene lactones with antiprotozoal, antibacterial, cytotoxic, antitumor, spermicidal, and phytotoxic properties. The antibacterial activity of IA and IB against bacteria causing diarrhoea have been reported; however, no information is available regarding their antibacterial activity on Vibrio cholerae. In this work, both compounds were evaluated for their anti-diarrhoeal potential using the bacterium V. cholerae, sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis on cholera toxin, and a cholera toxin-induced diarrhoea model in male Balb/c mice. In addition, a molecular docking study was carried out to understand the interaction of IA and IB with cholera toxin. In terms of antibacterial activity, IB was three times more active than IA on V. cholerae. In the case of SDS-PAGE analysis and the in silico study, IA was most effective, revealing its potential binding mode at a molecular level. In terms of anti-diarrhoeal activity, IA was 10 times more active than IB and racecadotril, an antisecretory drug used as positive control; the anti-diarrheal activity of IB was also closer than racecadotril. The results obtained from in vitro, in vivo, and computational studies on V. cholerae and cholera toxin support the potential of IA and IB as new anti-diarrhoeal compounds.