SDS-PAGE Gel Electrophoresis Research Articles

Black soldier fly (Hermetia illucens L.) whole and fractionated larvae: In vitro protein digestibility and effect of lipid and chitin removal

Protein quality, which can be defined by amino acid profile and protein digestibility, is of paramount importance when assessing a novel protein source. As the presence of chitin might impair insect protein digestion, and as there is little to no clarity as to how different insect fractions influence the overall protein digestibility, this study aimed at assessing the influence of lipids and chitin removal on the protein digestibility of black soldier fly larvae. The samples underwent an in vitro simulated gastro-intestinal digestion following the INFOGEST method, commonly used for humans, and both undigested matrices and digesta were characterized by means of amino acid composition, SDS-PAGE gel electrophoresis, and proteomic/peptidomic approaches. Protein solubilization, degree of hydrolysis (DH%) after digestion, and digestible indispensable amino acid (DIAA) contents were also determined. The results highlighted that the presence of chitin hindered protein digestion, as expected: in fact, the protein isolate showed the highest solubilized protein (84.0%), DH% (61.1%), and number of peptides and proteins detected by high resolution mass spectrometry (64 and 16, respectively), while the chitin-rich fraction the lowest (38.4% solubilized protein, 41.2% DH%, 37 peptides and 6 proteins detected, respectively). Additionally, the chitin-rich fraction had the lowest DIAAS. Interestingly, the preferred C-terminal cleavage sites for all samples were in line with the specificity of the enzymes used, meaning that insect proteins, compared to other matrices, do not change the enzymatic behavior in terms of their specificity.

LC-MS characterization of N/O-glycans of α- and β-subunits of chicken ovomucin separated by SDS-PAGE

As the main active glycoprotein of egg white, the biological functions of chicken ovomucin α- and β-subunit are closely related to the structure of glycans. However, the exact composition and structure of the subunit glycans are still unknown. We obtained highly pure chicken ovomucin α-subunit and β-subunit protein bands by the strategy combined with two-step isoelectric precipitation and SDS-PAGE gel electrophoresis. The ammonia-catalyzed one-pot procedure was then used to release and capture α-and β-subunit protein glycans with 1-phenyl- 3-Methyl-5-pyrazolone (PMP). The N/O-glycans of bis-PMP derivatives were purified and analyzed by LC-MS. More importantly, an effective dual modification was performed to accurately quantify neutral and sialylated O-glycans through methylamidation of sialic acid residues and simultaneously through carbonyl condensation reactions of reducing ends with PMP. We first showed that the α-subunit protein has only N-glycosylation modification, and the β-subunit only O-glycosylation, a total of 22 N-glycans and 20 O-glycans were identified in the α- and β-subunit, respectively. In addition, the complex N-glycan (47 %) and the sialylated O-glycan (77 %) are each major types of the above subunits. Such findings in this study provide a basis for studying the functional and biological activities of chicken ovomucin glycans.

Gas Cluster Ion Beam-Assisted Deposition for Fabricating Dry Multilayers Containing Enzyme and Substrate with On-Demand Release.

Gas cluster ion beam (GCIB)-assisted deposition is used to build multilayered protein-based structures. In this process, Ar3000-5000+ clusters bombard and sputter molecules from a reservoir (target) to a collector, an operation that can be sequentially repeated with multiple targets. The process occurs under a vacuum, making it adequate for further sample conservation in the dry state, since many proteins do not have long-term storage stability in the aqueous state. First of all, the stability in time and versatility in terms of molecule selection are demonstrated with the fabrication of peptide multilayers featuring a clear separation. Then, lysozyme and trypsin are used as protein models to show that the activity remaining on the collector after deposition is linearly proportional to the argon ion dose. The energy per atom (E/n) of the Ar clusters is a parameter that was also changed for lysozyme deposition, and its increase negatively affects activity. The intact detection of larger protein molecules by SDS-PAGE gel electrophoresis and a bioassay (trypsin at ≈25 kDa and glucose oxidase (GOx) at ≈80 kDa) is demonstrated. Finally, GOx and horseradish peroxidase, two proteins involved in the same enzymatic cascade, are successively deposited on β-d-glucose to build an on-demand release material in which the enzymes and the substrate (β-d-glucose) are combined in a dry trilayer, and the reaction occurs only upon reintroduction in aqueous medium.

Quality evaluation of highly purified human menopausal gonadotropin preparations by means of gel electrophoresis and mass spectrometry.

Human gonadotropins are glycoprotein hormones with a highly complex structure, which demands the application of sophisticated analytical methodologies to assess their quality. The principal objective of this study was a comparative evaluation of gel electrophoretic techniques and mass spectrometry-based methods for the quality study of the two urinary-derived, highly purified, human menopausal gonadotropin preparations, Menopur 75/75 I. U. and Meriofert 75 I. U. Molecular mass (Mr), isoelectric point (pI), and isoform pattern of studied compounds were estimated via SDS-PAGE and 2D gel electrophoresis, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for the downstream characterization of peptides obtained after in-gel tryptic digestion of selected protein spots. Additionally, for the estimation of the glycosylation pattern of these biologics, the enzymatic release of oligosaccharides was performed, and the isoform pattern was studied. Gel electrophoresis showed a typical electrophoretic behaviour for protein biotherapeutics medicines consisting of extremely complex spot patterns migrating at different masses and pIs. MS analysis proved to be a powerful tool for the identification and detailed characterization of the gonadotropins and the relevant peptides were identified with high sequence coverages. The results of this study are not only useful for the quality assessment of this class of complex biopharmaceuticals but may also serve as a supporting platform for further development of biopharmaceuticals based on modulation of the glycosylation pattern to enhance efficacy or reduce side effects.

Oral sericin ameliorates type 2 diabetes through passive intestinal and bypass transport into the systemic circulation

Ethnopharmacological relevanceBoiled silkworm cocoons have been used to treat 'Xiaoke disease' (diabetes mellitus) recorded in Chinese medicine for over 800 years. In recent years, it has been found that the active substance silk sericin (SS) has therapeutic benefits in treating type 2 diabetes mellitus (T2DM). SS promotes pancreatic islet signalling, the proliferation of pancreatic islet cells, and insulin secretion. It is inferred that SS enters the bloodstream after oral administration and plays a role in the body's circulation. As a natural protein, SS needs to resist digestion by proteases in the gastrointestinal tract and cross the gastrointestinal barrier after oral administration. It is currently unclear how SS crosses the gastrointestinal barrier and whether it exerts therapeutic effects on T2DM by entering the circulation. Aim of the studyTo study how SS crosses the gastrointestinal barrier and whether it enters the body circulation to exert a therapeutic effect on T2DM. Materials and methodsSS was extracted from silkworm cocoons using an alkaline method with sodium carbonate. The antidigestive capacity of SS was detected using SDS-PAGE gel electrophoresis experiments. The mode of uptake and translocation of orally consumed SS in vivo was analysed using the AP-side to BL-side and BL-side-AP-side translocations, apparent Permeability coefficient (Papp), and Exocytosis rates (ER). The study compared the differences between the adSS group and the adSS + EDTA group by using Ethylenediaminetetraacetic acid (EDTA) to separate the tight junctions between Caco-2 cells. The aim was to analyze whether the transport mode of oral filaggrin proteins in vivo could be absorbed by bypass transport. By administering SS through oral and intraperitoneal injection to type 2 diabetic mice, we measured its concentration in the blood, as well as blood glucose and insulin levels, to determine its effectiveness in treating diabetes and its ability to enter the body's circulation for treatment. ResultsThe molecular weight of SS decreased from 10k∼25 kDa to 10k∼15 kDa after in vitro simulated gastrointestinal fluid digestion, indicating its good antidigestive properties. The apparent Papp was greater than 1 × 10−6 cm·s-1, and the ER was between 0.5 and 1.5, indicating that adSS was well-absorbed and mainly passively transported. The Caco-2 cell model showed that the addition of EDTA promoted the transport of adSS, resulting in significantly larger Papp and ER values, indicating that adSS was absorbed by bypass transport. After oral administration of SS, the concentration of SS in the blood was lower than after intraperitoneal injection, which is 60% of intraperitoneal administration. Mice with a T2DM model who were administered SS for 5 weeks showed significant improvement in insulin resistance and glucose tolerance. Additionally, the pancreatic tissue appeared more regular. In the treatment of T2DM, injections of SS have been shown to be more effective than oral administration. Both oral and intraperitoneal injections have been partially involved in the circulation. ConclusionsSS is enzymatically cleaved by proteolytic enzymes in the gastrointestinal tract. The smaller molecules are partially absorbed into the body's circulation through passive and paracrine transport, exerting a therapeutic effect on T2DM.

Tumor Targeting with Apatinib-loaded Nanoparticles and Sonodynamic Combined Therapy.

This study implies the enhancement of apatinib killing effect in 4T1 tumor cells through constructing drug-loaded nanoparticles apatinib/Ce6@ZIF- 8@Membranes (aCZM) to enhance tumor therapeutic targeting and reduce toxic side following sonodynamic therapy (SDT). apatinib/Ce6@ZIF-8 (aCZ) were synthesized by in situ encapsulation, and aCZM were constructed by encapsulating the nanoparticles with extracted breast cancer 4T1 cell membranes. aCZM were characterized and tested for the stability by electron microscopy, and the membrane proteins on the nanoparticles' surface were assessed using SDS-PAGE gel electrophoresis. The cell viability of 4T1 cells following treatment with aCZM was tested using cell counting kit-8 (CCK-8). The uptake of nanoparticles was detected by laser confocal microscopy and flow cytometry, and the SDT-mediated production of reactive oxygen species (ROS) was verified by singlet oxygen sensor green (SOSG), electron spin resonance (ESR), and DCFH-DA fluorescent probes. The CCK-8 assay and flow cytometry using Calcein/PI were used to assess the antitumoral effect of aCZM nanoparticles under SDT. The biosafety of aCZM was further verified in vitro and in vivo using the hemolysis assay, routine blood test and H&E staining of vital organs in Balb/c mice. aCZM with an average particle size of about 210.26 nm were successfully synthesized. The results of the SDS-PAGE gel electrophoresis experiment showed that aCZM have a band similar to that of pure cell membrane proteins. The CCK-8 assay demonstrated the absence of effects on cell viability at a low concentration range, and the relative cell survival rate reached more than 95%. Laser confocal microscopy and flow cytometry analysis showed that aCZM treated group has the strongest fluorescence and the highest cellular uptake of nanoparticles. SOSG, ESR, and DCFH-DA fluorescent probes all indicated that the aCZM + SDT treated group has the highest ROS production. The CCK-8 assay also showed that when the ultrasound intensity was fixed at 0.5 W/cm2, the relative cell survival rates in the medium concentration group (10 μg/ml) (5.54 ± 1.26%) and the high concentration group (20 μg/ml) (2.14 ± 1.63%) were significantly lower than those in the low concentration group (5 μg/ml) (53.40 ± 4.25%). Moreover, there was a concentration and intensity dependence associated with the cellkilling effect. The mortality rate of the aCZM in the ultrasound group (44.95 ± 3.03%) was significantly higher than that of the non-ultrasound (17.00 ± 2.26%) group and aCZ + SDT group (24.85 ± 3.08%) (P<0.0001). The live and dead cells' staining (Calcein/PI) also supported this result. Finally, in vitro hemolysis test at 4 and 24 hours showed that the hemolysis rate of the highest concentration group was less than 1%. The blood routine, biochemistry, and H&E staining results of major organs in Balb/c mice undergoing nano-treatments showed no obvious functional abnormalities and tissue damage in 30 days. In this study, a multifunctional bionic drug delivery nanoparticles (aCZM) system with good biosafety and compatibility in response to acoustic dynamics was successfully constructed and characterized. This system enhanced apatinib killing effect on tumor cells and reduced toxic side effects under SDT.

Identification of antimicrobial peptides from Enterococcus faecium IP5-2a isolated from tilapia gut using mass spectrometry

Antimicrobial peptides are small molecules that exhibit strong antagonistic activity against pathogenic microorganisms and are synthesized by a wide variety or organisms, including probiotic bacteria. The aim of the study was to identify peptides with antimicrobial activity in the Enterococcus faecium IP5-2a strain previously isolated from the intestinal tract of Nile tilapia (Oreochromis niloticus). Cellular and extracelullar proteins extraction was performed, followed by 1D SDS-PAGE gel electrophoresis and MALDI-TOF/TOF mass spectrometry. The results revealed peptides with antimicrobial activity, including P54 (SagA) protein, a bacteriocin-like protein and a lantibiotic. These proteins play an important role in the antagonistic capacity of the E. faecium IP52a strain, allowing it to be considered as a potential probiotic in the aquaculture industry.

Changes in the saliva proteome analysed by gel-proteomics in horses diagnosed with equine gastric ulcer syndrome (EGUS) at diagnosis and after successful treatment

Equine gastric ulcer syndrome (EGUS) is currently one of the more frequent diseases in horses. We aimed to identify changes in the salivary proteome in horses with EGUS at diagnosis and after successful treatment by using gel proteomics. Saliva samples were collected from nine horses with EGUS before and after treatment and nine matched healthy controls. SDS-PAGE (1DE) and two-dimensional gel electrophoresis (2DE) were performed, and significantly different protein bands and spots were identified by mass spectrometry. Horses with EGUS had increases in proteins such as adenosine deaminase (ADA), triosephosphate isomerase, keratins and immuno-globulin heavy constant mu and decreases in carbonic anhydrase (CA), albumin and prolactin-induced protein. These changes would indicate various physiopathological mechanisms involved in this disease, such as the activation of the immune system, decreased stomach defence mechanisms and inflammation. The treated horses presented lower expression levels of thioredoxin (TRX) after a successful treatment, in proteomics analysis and also measured with a commercially available ELISA kit. Overall, horses with EGUS have protein changes in their saliva when measured with gel proteomics compared with healthy horses, and they also showed changes after successful treatment. These proteins could be potential biomarkers for detection and monitoring treatment response in EGUS.

A promising microbial α-amylase production, and purification from Bacillus cereus and its assessment as antibiofilm agent against Pseudomonas aeruginosa pathogen

Background and aimThe purpose of the current study is to isolate a heavily amylase-producing bacteria of the genus Bacillus from soil samples, optimize the production of the enzyme, purify it, and evaluate its activity against biofilm-producing bacteria. A total of 12 soil samples were collected and screened for promising Bacillus species with good amylolytic activity. Isolation was done by serial dilution and plating technique and amylolytic activity was determined by starch agar plate method. Among the 12 Bacillus isolates recovered from soil samples, 7 showed positive α-amylase production. The best isolate that recorded the greatest amylolytic activity was selected for further studies. This isolate was identified by 16S rRNA sequencing as Bacillus cereus and registered under gene bank accession number OP811897. Furthermore, the α-amylase enzyme was produced by a submerged fermentation technique using best production media and partially purified by ammonium sulfate and chilled ethanol and molecular weight had been determined by SDS-PAGE gel electrophoresis. The production of α-amylase was optimized experimentally by one-factor at a time protocol and statistically by Plackett–Burman design as well as RSM CCD design. Data obtained from OFAT and CCD revealed that α-amylase activities were 1.5- and twofold respectively higher as compared to un-optimized conditions. The most significant factors had been identified and optimized by CCD design.ResultsAmong the eleven independent variables tested by PBD, glucose, peptone, (NH4)2SO4, and Mg SO4 were the most significant parameters for α-amylase production with an actual yield of 250U/ml. The best physical parameters affecting the enzyme production were incubation time at 35 °C, and pH 5.5 for 48 h. The partially purified enzyme with 60% ammonium sulphate saturation with 1.38- fold purification showed good stability characteristics at a storage temperature of 4 °C and pH up to 8.5 for 21 days. Antibiofilm activity of purified α-amylase was determined against Pseudomonas aeruginosa (ATCC 35659) by spectrophotometric analysis and CLSM microscopic analysis. Results demonstrated biofilm inhibition by 84% of the formed Pseudomonas biofilm using a microtiter plate assay and thickness inhibition activity by 83% with live/Dead cells percentage of 17%/83% using CLSM protocol.ConclusionsA highly stable purified α-amylase from B. cereus showed promising antibiofilm activity against one of the clinically important biofilm-forming MDR organisms that could be used as a cost-effective tool in pharmaceutical industries.

Optimization of Affinity Chromatography Based on Sepharose 4B- chitin for Rapid Purification of Urtica dioica Agglutinin.

Today, numerous antimicrobial and anticancer properties have been reported for plant lectins due to their ability to bind to carbohydrates. The Urtica dioica agglutinin (UDA lectin) is a monomeric, small, and low molecular weight glycoprotein. It has attracted the attention of many researchers for identification, treatment, and other clinical purposes. The aim of this study is the optimization of the chitin affinity chromatography based on Sepharose 4B (CNBr-activated Sepharose 4B) for the rapid purification of UDA lectin from Urtica dioica rhizome. The chitin ligands were dissolved in 40% Trichloroacetic acid and attached to Sepharose 4B according to the Amersham-Biosciences instructions. The attachment of the ligand to the Sepharose 4B beads was investigated by Fourier transform infrared (FTIR) spectroscopy. An acidic crude extract of nettle rhizome passes from chromatographic columns in two sizes with dimensions: 24 x 0.51 cm and 8.44 x 0.86 cm. Quantity and quality of purified lectin were calculated by the Bradford microplate method: SDS-PAGE gel electrophoresis and human erythrocyte cell (RBC) hemagglutination, respectively. The analysis of FTIR spectrograms showed that major changes were observed in the fingerprint regions. Besides, due to the dissolution of Sepharose 4B and chitin in the aqueous phase, this difference was not significant in the Imine and Nitrile regions. On the other hand, the comparative results of purification chromatograms showed that increasing the column length causes a smaller half-width and increases the length of the purified peak. Also, it leads to high-quality purified UDA lectin, with a molecular weight of almost 12.5 kDa in gel electrophoresis. Hemagglutination activity on trypsinized red blood cells was displayed, and agglutination of purified UDA lectin started at least at 300 μg.mL-1 concentration. According to our findings, we suggested that dissolving chitin in the polar solvent of Trichloroacetic acid, using Sepharose 4B as the beads of a matrix, and increasing the column length might lead to a decrease in the half-width of the peak. These can increase the purity and concentration of purified UDA lectin, and speed up the purification process. These findings could be used by researchers to accelerate the purification of UDA lectin in other studies, dealing with drug delivery systems, ELISA techniques, and cell growth.

Proteomic profile of stallion seminal plasma

Proteins mediate all physiological changes. Seminal plasma (SP), the product of testes, epididymis, and accessory sex glands, is a fluid released during ejaculation and represents up to 98% of the voluminous stallion ejaculate. The SP plays an essential role in reproduction as a transit medium and a source of energy, antioxidants, enzymes, and minerals. This study aimed to investigate proteomic profile of the seminal plasma of stallions. Twenty-four Criollo stallions with a known reproductive history and per-cycle concept rate data calculated from at least 30 inseminations were used. One ejaculate was collected from each stallion during the breeding season. Pregnancy rates ranged at day 16 after artificial insemination from 20.2% to 95.6%. The animals were divided into two groups: High Pregnancy (HP), with a pregnancy rate per cycle ≥51%, and Low Pregnancy (LP), with a pregnancy rate of <50%. Sperm concentration, kinetics, morphology, and plasma membrane integrity and functionality were evaluated. The sample was centrifuged at 400xg for 10min. SP was then transferred to a 2 mL microcentrifuge tube and centrifuged again (10.000xg, 60min, 4°C). Proteins were separated using 2D SDS PAGE gel electrophoresis in duplicate. Spots in at least 80% of the gels in one of the groups, with significant abundance (P<0.05) and at least a 1.5-fold magnitude difference between groups, were selected. Thespectra were acquired in a nano LC-MS/MS spectrometer, and proteins were identified by the MASCOT application and validated by SCAFFOLD. From a total of 716 analyzed spots, four were selected. For non-parametric data the Kruskal Wallis and for parametric data Anova and t-test were used. The cysteine-rich secretory protein 3 (CRISP3) and SP protein HSP-1-like (HSP1) showed higher abundance in the SP in group LP than HP (P< 0.05). However, kallikrein-1E2 (KLK) and phospholipase A2 (PLA2) showed higher abundance in HP than LP (P<0.05). In the horse, CRISP-3 is synthesized in great amounts in the accessory sexual glands, ampulla and seminal vesicle. HSP1 has an essential role as a molecular chaperone for sperm motility and viability, in the formation of the oviductal sperm reservoir, in the regulation of cell volume, and possibly in the interaction between gametes. KLK is one of the major proteins found in seminal equine plasma. The kallikreins are serine proteases, and the glandular kallikrein is regulated by androgens and secreted by the prostate. The PLA2 plays an essential role in the late maturational events of spermatozoa, the acrosomal reaction, and sperm egg fusion. In conclusion, the proteins are essential to the biological processes related to stallion fertility, providing insight into SP proteins that could serve as biomarkers for fertility in stallions.

Preparation of Nanoparticle-Loaded Extracellular Vesicles Using Direct Flow Filtration.

Extracellular vesicles (EVs) have shown great potential as cell-free therapeutics and biomimetic nanocarriers for drug delivery. However, the potential of EVs is limited by scalable, reproducible production and in vivo tracking after delivery. Here, we report the preparation of quercetin-iron complex nanoparticle-loaded EVs derived from a breast cancer cell line, MDA-MB-231br, using direct flow filtration. The morphology and size of the nanoparticle-loaded EVs were characterized using transmission electron microscopy and dynamic light scattering. The SDS-PAGE gel electrophoresis of those EVs showed several protein bands in the range of 20-100 kDa. The analysis of EV protein markers by a semi-quantitative antibody array confirmed the presence of several typical EV markers, such as ALIX, TSG101, CD63, and CD81. Our EV yield quantification suggested a significant yield increase in direct flow filtration compared with ultracentrifugation. Subsequently, we compared the cellular uptake behaviors of nanoparticle-loaded EVs with free nanoparticles using MDA-MB-231br cell line. Iron staining studies indicated that free nanoparticles were taken up by cells via endocytosis and localized at a certain area within the cells while uniform iron staining across cells was observed for cells treated with nanoparticle-loaded EVs. Our studies demonstrate the feasibility of using direct flow filtration for the production of nanoparticle-loaded EVs from cancer cells. The cellular uptake studies suggested the possibility of deeper penetration of the nanocarriers because the cancer cells readily took up the quercetin-iron complex nanoparticles, and then released nanoparticle-loaded EVs, which can be further delivered to regional cells.

Production and Characterization of ACE Inhibitory and Anti-Diabetic Peptides from Buffalo and Camel Milk Fermented with Lactobacillus and Yeast: A Comparative Analysis with In Vitro, In Silico, and Molecular Interaction Study.

The investigation aimed at assessing a comparative study on the production and characterization of ACE inhibitory, anti-diabetic, and anti-inflammatory activities, along with the production of ACE inhibitory and anti-diabetic peptides through the fermentation of buffalo and camel milk by Limosilactobacillus fermentum (KGL4) and Saccharomyces cerevisiae (WBS2A). The angiotensin-converting enzyme (ACE) inhibitory and anti-diabetic properties were evaluated at particular time intervals (12, 24, 36, and 48 h) at 37 °C, and we discovered maximum activity at 37 °C after 48 h of incubation. The maximum ACE inhibitory, lipase inhibitory activities, alpha-glucosidase inhibitory, and alpha-amylase inhibitory activities were found in the fermented camel milk (77.96 ± 2.61, 73.85 ± 1.19, 85.37 ± 2.15, and 70.86 ± 1.02), as compared to the fermented buffalo milk (FBM) (75.25 ± 1.72, 61.79 ± 2.14, 80.09 ± 0.51, and 67.29 ± 1.75). Proteolytic activity was measured with different inoculation rates (1.5%, 2.0%, and 2.5%) and incubation times (12, 24, 36, and 48 h) to optimize the growth conditions. Maximum proteolysis was found at a 2.5% inoculation rate and at a 48 h incubation period in both fermented buffalo (9.14 ± 0.06) and camel milk (9.10 ± 0.17). SDS-PAGE and 2D gel electrophoresis were conducted for protein purification. The camel and buffalo milk that had not been fermented revealed protein bands ranging from 10 to 100 kDa and 10 to 75 kDa, respectively, whereas all the fermented samples showed bands ranging from 10 to 75 kDa. There were no visible protein bands in the permeates on SDS-PAGE. When fermented buffalo and camel milk were electrophoresed in 2D gel, 15 and 20 protein spots were detected, respectively. The protein spots in the 2D gel electrophoresis ranged in size from 20 to 75 kDa. To distinguish between different peptide fractions, water-soluble extract (WSE) fractions of ultrafiltration (3 and 10 kDa retentate and permeate) of fermented camel and buffalo milk were employed in RP-HPLC (reversed-phase high-performance liquid chromatography). The impact of fermented buffalo and camel milk on inflammation induced by LPS (lipopolysaccharide) was also investigated in the RAW 264.7 cell line. Novel peptide sequences with ACE inhibitory and anti-diabetic properties were also analyzed on the anti-hypertensive database (AHTDB) and bioactive peptide (BIOPEP) database. We found the sequences SCQAQPTTMTR, EMPFPK, TTMPLW, HPHPHLSFMAIPPK, FFNDKIAK, ALPMHIR, IPAVFK, LDQWLCEK, and AVPYPQR from the fermented buffalo milk and the sequences TDVMPQWW, EKTFLLYSCPHR, SSHPYLEQLY, IDSGLYLGSNYITAIR, and FDEFLSQSCAPGSDPR from the fermented camel milk.

Changes in the Saliva Proteome of Pigs with Diarrhoea Caused by Escherichia coli

Escherichia coli represents the main cause of diarrhoea in pigs. Saliva can provide information about the pathophysiology of diseases and be a source of biomarkers. We aimed to identify changes in the salivary proteome of pigs with diarrhoea caused by E. coli. Saliva samples were collected from 10 pigs with this disease and 10 matched healthy controls. SDS-PAGE (1DE) and two-dimensional gel electrophoresis (2DE) were performed, and significantly different protein bands and spots were identified by mass spectrometry. For validation, adenosine deaminase (ADA) was measured in 28 healthy and 28 diseased pigs. In 1DE, increases in lipocalin and IgA bands were observed for diseased pigs, whereas bands containing proteins such as odorant-binding protein and/or prolactin-inducible protein presented decreased concentrations. Two-dimensional gel electrophoresis (2DE) results showed that saliva from E. coli animals presented higher expression levels of lipocalin, ADA, IgA and albumin peptides, being ADA activity increased in the diseased pigs in the validation study. Spots containing alpha-amylase, carbonic anhydrase VI, and whole albumin were decreased in diseased animals. Overall, pigs with diarrhoea caused by E. coli have changes in proteins in their saliva related to various pathophysiological mechanisms such as inflammation and immune function and could potentially be biomarkers of this disease.

In Vitro Anti-Venom Potentials of Aqueous Extract and Oils of Toona ciliata M. Roem against Cobra Venom and Chemical Constituents of Oils.

There are high mortality and morbidity rates from poisonous snakebites globally. Many medicinal plants are locally used for snakebite treatment in Uganda. This study aimed to determine the in vitro anti-venom activities of aqueous extract and oils of Toona ciliata against Naja melanoleuca venom. A mixture of venom and extract was administered intramuscularly in rats. Anticoagulant, antiphospholipase A2 (PLA2) inhibition assay, and gel electrophoresis for anti-venom activities of oils were done. The chemical constituents of the oils of ciliata were identified using Gas chromatography-tandem mass spectroscopy (GC-MS/MS). The LD50 of the venom was 0.168 ± 0.21 µg/g. The venom and aqueous extract mixture (1.25 µg/g and 3.5 mg/g) did not cause any rat mortality, while the control with venom only (1.25 µg/g) caused death in 1 h. The aqueous extract of T. ciliata inhibited the anticoagulation activity of N. melanoleuca venom from 18.58 min. to 4.83 min and reduced the hemolytic halo diameter from 24 to 22 mm. SDS-PAGE gel electrophoresis showed that oils completely cleared venom proteins. GC-MS/MS analysis showed that the oils had sesquiterpene hydrocarbons (60%) in the volatile oil (VO) and oxygenated sesquiterpenes (48.89%) in the non-volatile oils (NVO). Some major compounds reported for the first time in T. ciliata NVOs were: Rutamarin (52.55%), β-Himachalol (9.53%), Girinimbine (6.68%) and Oprea1 (6.24%). Most compounds in the VO were reported for the first time in T. ciliata, including the major ones Santalene (8.55%) and Himachal-7-ol (6.69%). The result showed that aqueous extract and oils of T. ciliata have anti-venom/procoagulant activities and completely neutralized the venom. We recommend a study on isolation and testing the pure compounds against the same venom.

Process optimisation of defatted wheat germ protein extraction in a novel alkaline-based deep eutectic solvent (DES) via Box–Behnken experimental design (BBD)

This is the first study to provide a preliminary investigation into the recovery of protein from wheat germ and the prediction of their extraction conditions in microwave cavity using a novel DES solvent. The response surface methodology was used to predict the microwaved protein extraction conditions of the DWG. The effects of DES buffer-concentration (X1: 0.01–1.00 g/mL), microwave power (X2: 50–250 W), irradiation time (X3: 1–5 min) and sample-to-buffer ratio (X4: 1:10–1:50) were tested using a single factor and Box–Behnken experimental design. Under the optimized conditions (X1 = 0.52 g/mL, X2 = 186 W, X3 = 3.28 min, and X4 = 1:39) protein yield and absorbed microwave were obtained at the optimal value of 33.00 % and 677 J/min, respectively with no denaturation of the protein as validated from the SDS-PAGE gel electrophoresis profile. Consequently, this investigation provides a practical approach for the extraction of bioactive protein from DWG using a novel deep eutectic solvent.

Lactoferrin and the development of salivary stones: a pilot study

Salivary stones (sialoliths) are calcified structures located in the ductal system of the major salivary glands. Their exact cause is not clear but in general they are characterized by concentric inorganic (hydroxyapatite) layers. The formation is a slow intermittent process which may result in enlargement of the sialolith causing obstruction of saliva secretion resulting in mealtime related pain and swelling of the affected salivary gland. Various studies reported the presence of organic material such as proteins and lipids in the core of sialoliths. In the present study the protein composition of twenty submandibular sialoliths was analyzed. It was found that proteins contributed on average 5% to the dry weight of submandibular stones whereby small salivary stones contained more extractable proteins than large salivary stones. Using a combination of SDS-PAGE gel electrophoresis and Western blotting, we identified α-amylase (in all stones; 100%), lysozyme (95%), lactoferrin (85%), secretory-IgA (75%), MUC7 (60%), complement C4 (60%) and C-reactive protein (35%). The presence, and the combinations, of lactoferrin, lysozyme, s-IgA and α-amylase in sialoliths was confirmed by ELISA. The gradually increasing size of a sialolith might provoke a local inflammatory response in the duct of the submandibular gland whereby the relatively low concentrations of lactoferrin and lysozyme may originate from neutrophils. The interaction of lactoferrin with s-IgA could contribute to the accumulation of lactoferrin in sialoliths. In summary, these results suggest a new pathophysiological role for lactoferrin, in the formation of sialoliths.

Biochemical characterization of alkaline phosphatase in midgut of Cry2Ab dosed, pink bollworm, Pectinophora gossypiella (Saunders)

Aim: The holistic management of pink bollworm is possible only after uncovering the underlying mechanism/s of pink bollworm resistance. The present study was carried out for investigating the effect of Cry2Ab toxin on alkaline phosphatase activity and expression of its isoforms in midgut of Pectinophora gossypiella. Methodology: Resistant and susceptible pink bollworm larval populations were subjected to Cry2Ab bioassays using F1 larvae in a 21 days diet-incorporation method. The total midgut protein was estimated by Bradford method. Alkaline phosphatase activity was estimated in midgut homogenates, Non-denaturing SDS-PAGE and Native polyacrylamide gel electrophoresis was performed by the following standard procedures. Results: The study showed increased alkaline phosphatase activity in pink bollworm midgut is correlated with insect resistance in response to Cry2Ab. The non-denaturing SDS-PAGE analysis of pink bollworm midgut alkaline phosphatase isozymes had differential banding patterns in resistant and susceptible populations. Yavatmal (R1), Hingoli (R4) and Parbhani (R5) showed similar banding patterns ranging from 65-95 kDa; Guntur (R2) and Hingoli (R3) showed near similar banding patterns ranging from 70-95 kDa; whereas susceptible population showed banding pattern in the range of 50-70 kDa. Interpretation: The expression profiles of alkaline phosphatase isomers in resistant and susceptible populations can be utilized as a biomarker to aid for the screening of PBW infected Bt -cotton fields and development of pink bollworm management strategies. Key words: Alkaline Phosphatase, Cry2Ab, Insect resistance, Pectinophora gossypiella, SDS-PAGE

Postponement of the opacification of lentoid bodies derived from human induced pluripotent stem cells after lanosterol treatment-the first use of the lens aging model in vitro in cataract drug screening.

Purpose: Our previous study observed that human induced pluripotent stem cell (HiPSC)-derived lentoid bodies (LBs) became cloudy with extended culture time, partially mimicking the progress of human age-related cataracts (ARCs) in a dish. In the present study, lanosterol, a potential anticataract drug, was used to further verify the value of this model in drug screening for cataract treatment. Methods: Mature LBs on day 25, which were differentiated from HiPSCs using the “fried egg” method, were continually cultured and treated with either dimethyl sulfoxide (control) or lanosterol. The LBs’ shape and opacity alterations were examined using light microscopy and mean gray value evaluation. The soluble and insoluble proteins were examined through SDS-PAGE gel electrophoresis combined with Coomassie blue staining. The protein aggregations were examined with immunofluorescence. Results: The mature LBs became cloudy with an extended culture time, and the opacification of the LBs was partially prevented by lanosterol treatment. There was less increase in insoluble proteins in the lanosterol-treated LBs than in the control group. There were also fewer cells containing aggregated protein (αA‐crystallin and αB‐crystallin) puncta in the lanosterol-treated LBs than in the control LBs. Conclusion: It was found that the opacification of LBs could be delayed by lanosterol treatment, which could be achieved by reducing protein aggregation, suggesting a promising HiPSC-derived drug-screening model for Age-related cataract.

Purification and Characterization of Novel Antihypertensive and Antioxidative Peptides From Whey Protein Fermentate: In Vitro, In Silico, and Molecular Interactions Studies

Objective The goal of this research was to purify and characterize the novel angiotensin-converting enzyme (ACE)–inhibitory and antioxidant peptides from fermented whey protein concentrate produced by Lactobacillus paracasei and Saccharomyces cerevisiae in a co-fermentation system. Method Whey protein fermented with lactic acid bacteria and yeast culture was analyzed for antioxidative, ACE inhibition, as well as anti-inflammatory activity followed by SDS-PAGE, isoelectric focusing, and 2-dimensional (2D) analysis. Anti-inflammatory activity of whey protein fermentate was also studied on the RAW 264.7 cell line. The bioactive peptides were separated from the whey protein fermentate using reverse-phase high-performance liquid chromatography (RP-HPLC) and reverse-phase liquid chromatography mass spectrometry (RPLC/MS), and thus identification and characterization of purified bioactive peptide was performed. Results Whey protein fermentate samples’ bioactivity was analyzed at specific time intervals at 12, 24, 36, and 48 hours at 37 °C for M11 and at 25 °C for WBS2A. The development settings (incubation time [12, 24, 36, and 48 hours) and inoculation rates [1.5%, 2.0%, and 2.5%]) were optimized for peptide synthesis via the o-phthaldialdehyde (OPA) method (proteolytic activity). Maximum proteolytic activity was observed at 37 °C for M11 (6.50 mg/mL) and at 25 °C for WBS2A (8.59 mg/mL) for 48 hours of incubation. Protein profiling was carried out using SDS-PAGE and 2D gel electrophoresis, in which Sodium dodecyl-sulfate (SDS) exhibited protein bands in the 10- to 55-kDa range, while 2D showed protein bands varying from 10 to 70 kDa. Every spot from 2D was digested by trypsin and identified by RPLC/MS. Protein fractionations (3- and 10-kDa permeates) were carried out employing RP-HPLC. Whey protein fermentate has anti-inflammatory action in RAW 264.7 macrophages that have been exposed to lipopolysaccharide. A molecular docking system was also used to investigate the interactions of peptides (AFLDSRTR, ILGAFIQIITFR) with human myeloperoxidase enzyme. Conclusions The antihypertensive and antioxidative peptides discovered from whey protein fermentate may be helpful in the design of pharmacologically active healthy ingredients in the upcoming years.