Bradford Assay Research Articles

Cytotoxic and apoptosis-inducing properties of Staphylococcus aureus cytoplasmic extract on lung cancer cells: Insights from MTT assay and bax/bcl-2 gene expression analysis

BackgroundLung cancer ranks as the second most prevalent cancer in both men and women and stands as one of the most prevalent and lethal malignancies globally. In recent decades, the therapeutic potential of bacteria has gained recognition in pharmaceutical and therapeutic research. While bacteria have historically been associated with cancer causation, recent studies have unveiled their potential as efficacious agents for targeted cancer therapy. However, limited data exist on the characterization and impact of Staphylococcus aureus (S. aureus) extract on lung cancer cell lines. Consequently, this study aims to investigate the effect of S. aureus extract on inducing apoptosis in A549, a lung cancer cell line, and MRC-5, a lung normal cell line, by evaluating the expression levels of the bax and bcl-2 genes. MethodsInitially, cytoplasmic extract of S. aureus was prepared using the sonication technique. The protein concentration was determined via the Bradford assay, and the presence of proteins was confirmed using SDS-PAGE. A549, representing a lung cancer cell line, and MRC-5, representing a lung normal cell line, were exposed to varying concentrations of bacterial extract, and cell viability was assessed using the MTT assay. Subsequently, the expression levels of the bax and bcl-2 genes were quantified utilizing the Real-Time PCR method. ResultsCytoplasmic extract derived from S. aureus demonstrated the ability to modulate the expression levels of apoptotic genes. Relative to the control group, the bax gene exhibited a fivefold overexpression, while the expression of the bcl-2 gene decreased by more than half compared to the control. Furthermore, the results of the MTT assay indicated that the bacterial cytoplasmic extract exhibited concentration-dependent cytotoxicity on cancer cells, highlighting a significant increase in cell death with escalating concentrations. ConclusionsThis research highlights the potential of S. aureus extract for targeted lung cancer therapy by promoting cancer cell apoptosis while sparing normal cells. These findings open up exciting possibilities for innovative cancer treatments and improved patient outcomes, emphasizing the promise of S. aureus extract in the fight against lung cancer.

Purified Clinoptilolite-Tuff as an Efficient Sorbent for Food-Derived Peanut Allergens.

The avoidance of allergen intake is crucial for persons affected by peanut allergy; however, the cross-contamination of food is common and leads to unpredictable consequences after the consumption of supposedly "safe" food. The aim of the present study was to eliminate harmful traces of peanut allergens from food using purified clinoptilolite-tuff (PCT)-a specially processed zeolite material. Analyses were performed using a peanut ELISA and a Coomassie blue (Bradford) assay. Mimicking conditions of the human gastrointestinal tract demonstrated a higher efficacy of PCT in the intestine (pH 6.8) than in the stomach (pH 1.5). Adsorption rates were fast (<2 min) and indicated high capacities (23 µg and 40 µg per 1 mg of PCT at pH 1.5 and pH 6.8, respectively). Allergenically relevant peanut protein concentrations were sorbed in artificial fluids (32 µg/mL by 4 mg/mL of PCT at pH 1.5 and 80.8 µg/mL by 0.25 mg/mL of PCT at pH 6.8) when imitating a daily dose of 2 g of PCT in an average stomach volume of 500 mL. Experiments focusing on the bioavailability of peanut protein attached to PCT revealed sustained sorption at pH 1.5 and only minor desorption at pH 6.8. Accompanied by gluten, peanut proteins showed competing binding characteristics with PCT. This study therefore demonstrates the potential of PCT in binding relevant quantities of peanut allergens during the digestion of peanut-contaminated food.

The effect of programmed resistance training on the muscle myofibrillar proteome

Resistance training increases muscle size but little is known about the dynamic processes that underpin the gains in muscle mass. We investigated rat muscle during 30 days of unilateral programmed resistance training (PRT) using the stable isotope (deuterium oxide; D2O) in vivo and proteomics. Three-month-old, male Wistar rats (body weight 348 ± 20 g) were assigned to four groups (n = 4, in each), including a control group and experimental groups that received D2O for 10, 20 or 30 days. Under ethically approved procedures, deuterium was administered, and a stimulating device was surgically implanted to activate the left peroneal nerve and cause maximal contraction of the dorsiflexors (inc. tibialis anterior; TA) and partial contraction of the plantar flexors. PRT consisted of 1 bout per day of 5 sets of 10 repetitions (1 repetition consisted of 2 s stimulation at 100 Hz with 2 s rest between repetitions and 2.5 min between sets). One hour after the final training bout (i.e. day 10, 20, or 30) animals were killed and the left (stimulated) and right (contralateral control) TA were extracted and weighed. Muscles were fractionated into myofibrillar and sarcoplasmic components. Protein content was calculated via Bradford assay and proteomic analysis of myofibrillar proteins was conducted via liquid chromatography-tandem mass spectrometry of peptide digests. Statistical analysis was conducted using R (v4.3.2.) and functional annotation was performed using the STRING database. Two-way mixed ANOVA was used to investigate differences between condition (stimulated vs contralateral control) and time (0, 10, 20, or 30 days). The protein content of stimulated TA increased (P &lt; 0.05) from 0.579 ± 0.05 mg at day zero to 1.096 ± 0.15 mg after 30 days of PRT, whereas the protein content of control TA (0.618 ± 0.04 mg) was consistent across the 30-day experimental period. Proteomic analysis encompassed 244 proteins and the abundance of 45 proteins exhibited a significant (P &lt; 0.05) interaction between condition and time. Stimulated muscle was enriched in mitochondrial enzymes. For example, the beta-subunit of ATPase synthase (ATPB) increased from 14.6 ± 4.5 to 74 ± 15.6 μg in stimulated TA and was 17 ± 5 in control TA. PRT also increased the abundance of neonatal myosin heavy chain (MYH8), four-and-a-half LIM domains protein 1 (FHL1) and muscular LMNA interacting protein (MLIP). These findings suggest the energy requirements of daily PRT are met by mitochondrial metabolism and PRT may be associated with activation of the muscle developmental programme.

Using brix refractometry to measure the total protein level in chicken egg whites: Preliminary data

This study was conducted to investigate the feasibility of using Brix refractometry as a rapid method to measure the total protein level in chicken egg white samples. A total of 39 chicken eggs were purchased from local grocery stores and analyzed using three techniques: UV–Vis spectrophotometry, Brix refractometry, and the Bradford protein assay. Data shows that there is a strong positive correlation between the total protein levels as determined by UV–Vis absorbance values and the Brix values. No correlation (positive or negative) was found between the total protein levels as determined by the Bradford assay and either the Brix refractometry or the UV–Vis spectrophotometry. In comparison to UV–Vis spectrophotometry and the Bradford assay, Brix refractometry is both rapid and simple. It requires no sample processing steps, no special personnel training and can be conducted in any facility. We conclude that Brix refractometry is a suitable technique for rapid assessment of total protein level in chicken egg whites, although additional large-scale studies should be conducted to further validate the method.

Improving protein extraction and peptide production from Chlorella vulgaris using combined mechanical/physical and enzymatic pre-treatments

Chlorella vulgaris is a microalga rich in proteins with potential applications in food and feed industries. However, the presence of a cellulose-containing cell wall, which is a major barrier to protein extraction, together with fibroproteinaceous complexes, limits the bioaccessibility of nutritional and bioactive proteins and peptides from C. vulgaris biomass. Therefore, this study aimed to evaluate the effect of different mechanical/physical pre-treatments (bead milling, extrusion, freeze-drying, heating, microwave and sonication) combined or not with enzymatic treatments (commercial trypsin and pancreatin) on protein extraction and peptide formation from a C. vulgaris suspension. The amount of total protein and peptides released to the supernatant was quantified by Bradford and o-phthaldialdehyde assays, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was used to analyse the extracted protein fractions. The results showed that extrusion caused a 3-fold increase in total peptides (p < 0.001) compared to no-pretreatment, and trypsin increased peptides formed in bead-milled (p = 0.020) and freeze-dried (p = 0.021) microalga relative to those pre-treatments alone. Some pre-treatments, such as bead milling and microwave, were effective in releasing specific protein fractions, particularly those from 32 to 40 kDa (up to 1.2-fold), compared to control. Pancreatin combined with bead milling decreased 32 to 40 kDa- and 26 kDa-protein fractions (p < 0.010) compared with the sole use of mechanical treatment, whereas the same enzyme mixture associated with microwave produced a similar result for 26 kDa-protein fraction (p = 0.023). Pancreatin also effectively reduced the total protein fraction released after pre-treatment with sonication (p = 0.013). These findings suggest that combining different pre-treatments and enzymatic treatments could improve protein extraction from C. vulgaris biomass, providing a useful approach for the development of sustainable protein sources. The present results highlight the need for further studies to assess the efficacy of extrusion in improving the bioaccessibility of C. vulgaris proteins in monogastric animals' diets.

Comparison of morphology, protein concentration, and size distribution of bone marrow and Wharton's jelly-derived mesenchymal stem cells exosomes isolated by ultracentrifugation and polymer-based precipitation techniques

Exosomes which are tiny extracellular vesicles (30–150 nm), transport vital proteins and gene materials such as miRNA, mRNA, or DNA, whose role in cell communication and epithelia regulation is critical. Many techniques have been developed as a result of studying exosomes' biochemical and physicochemical properties, although there is still no standard method to isolate exosomes simply with high yield. Commercial kits have gained popularity for exosome extraction despite concerns about their effectiveness in scientific research. On the other hand, ultracentrifugation remains the gold standard isolation method. This study compares these two common exosome isolation methods to determine their impact on the quality and quantity of exosomes isolated from bone marrow (BM) and Wharton's jelly (WJ)-derived mesenchymal stem cells. Isolated exosomes from the two sources of the cell's conditioned medium by two methods (polymer kit and ultracentrifuge) were characterized using western blotting, scanning electron microscopy (SEM), dynamic light scattering (DLS), and the Bradford assay. Western blot analysis confirmed separation efficiency based on CD81 and CD63 markers, with the absence of calnexin serving as a negative control. The Morphology of exosomes studied by SEM image analysis revealed a similar round shape appearance and their sizes (30–150 nm) were the same in both isolation techniques. The DLS analysis of the sample results was consistent with the SEM ones, showing a similar size range and very low disparity. The exosome protein content concentration analysis revealed that exosomes isolated by the polymer-based kits contained higher protein concentration density and purity (p <0.001). In general, though the protein yield was higher when the polymer-based kits were used, there were no significant differences in morphology, or size between WJ-derived and BM-derived exosomes, regardless of the isolation method employed.

#1044 Quantitative analysis of dialytic protein loss with medium cut-off and high-flux membranes

Abstract Background and Aims Hemodialysis is an effective way for eliminating uremic retention products in patients with end-stage kidney disease. Medium cut-off membranes (MCO) were designed to enhance the dialytic removal of middle-sized molecules, such as beta-2 microglobulin (β2M). However, alongside these toxins, salutary proteins could also be lost. To better understand the spectrum of proteins cleared by MCO and high-flux (HF) dialyzers, we conducted qualitative and quantitative analysis of proteins in hemodialysis ultrafiltrate (UF). Method We established an ex vivo hemodialysis simulation system by using two Fresenius 2008T machines to concurrently dialyze a 2.5-liter human plasma reservoir for simultaneous testing of two dialyzers. We compared three dialyzers, including two HF dialyzers (Fresenius Medical Care Optiflux F180NR and FX CorAL 80), and an MCO membrane (Baxter Theranova 400; TH) (Fig. 1A). The blood flow rate was 400 mL/min, the dialysate flow rate was zero, and the ultrafiltration flow rate was 13 mL/min. UF samples were collected every 10 minutes from the UF line. Upon experiment completion at one-hour, residual filtrate was collected from the dialysate compartment. All samples were stored at −80°C until analysis. In total we conducted six dialysis simulations, each involving a concurrently tested dialyzer pair (Fig. 1B). UF samples underwent protein analysis (Bradford protein assay), albumin and creatinine measurement (Horiba® Pentra C400), liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomics (Agilent® Q-TOF 6546), and immune-based protein assay (Luminex® Magpix). We analyzed the average of four sets of data obtained from the same dialyzer at the same time point. Results The loss of total protein and albumin was significantly greater with TH compared to the two HF dialyzers (Fig. 1C). Creatinine removal was similar across all dialyzers (Fig. 1D). The overall protein loss through F180NR and CorAL was comparable, while the protein loss through TH was up to 14-fold higher (811.7 ± 149.5 mg; Fig. 1E). Similar trends were observed for albumin. Albumin comprised 62% of the protein content in UF from TH, whereas it accounts for approximately 30% for HF dialyzers. We next employed label-free LC-MS and LC-MS/MS to identify and quantify proteins in 12 UF samples collected at the 20-minute point. Using Spectrum Mill software, we identified 244 proteins in the UF, 113 were accurately semi-quantified. The protein intensity ratios of TH-to- F180NR, TH-to-CorAL, and CorAL-to-F180NR for these 113 proteins are presented in Fig. 2A-C. Each gray dot on the plot corresponds to a specific protein. In line with the total protein and albumin quantification, MS proteomics analysis consistently revealed a greater loss of every identified protein by TH compared to both HF dialyzers. The median protein intensity ratio of TH-to-F180NR and TH-to-CorAL was 9.14 and 8.25, respectively. Importantly, TH not only removed middle-molecules such as β2M, but also showed a higher loss of salutary proteins. For instance, our data indicate a 9- to 16-fold difference in the level of albumin, haptoglobin, and vitamin D-binding protein between TH and HF dialyzers. Protein loss did not differ between F180NR and CorAL. To validate the accuracy of the MS quantification, we quantified seven proteins using immune-based assays in all UF samples at the 20-minute point. The result revealed a strong consistency between MS data and other methods (Fig. 2D). Conclusion We established an ex vivo system to concurrently test two dialyzers and implemented a robust proteomics workflow for a direct comparison of protein loss in the UF. Our data comprehensively and consistently indicate a greater extent of protein loss with MCO compared to HF, including the loss of salutary proteins. Further research is warranted to explore the clinical consequences of the enhanced loss of salutary proteins.

Erythrocyte membrane-camouflaged mesoporous silica composite nanoparticles for loading and controlled release of the hepatoprotective agent silibinin: A-synthesis and physicochemical characterization.

Milk thistle has long been used in the treatment of liver and biliary disorders. In the present study, to make a long-acting delivery system for silibinin (SBN, a major active constituent of milk thistle seeds with antioxidant and hepatoprotective function), mesoporous silica composite nanoparticles (NC) were synthesized and coated with RBC membrane. A modified Stöber method was used for NC synthesis, which was then characterized using FE-SEM, DLS, TEM, FTIR, and EDAX techniques. A suitable lysis buffer was used to prepare RBC-ghost, and sonication was used to coat SBN-loaded NC (SBN-NC). The RBC-ghost coated SBN-NC (SBN-NC-RBCG) was evaluated by SDS-PAGE, Bradford, TEM, EDAX, and DLS methods. SBN release was then compared for the SBN-NC and SBN-NC-RBCG samples. the RBC membrane proteins were recovered from the coating of SBN-NC-RBCG, and SBN release was sustained over 24 h when compared with the SBN-NC. Overall, through prolonging circulation in the bloodstream and evading the immune system, the developed system can improve SBN bioavailability in liver inflammation and fibrosis conditions that need further research.

Mesenchymal stem cell-derived exosomes decrease Hyperplasia in Psoriasis by inducing transforming growth factor β2 (TGF-β2).

Psoriasis, a chronic inflammatory skin disease, is increasingly effectively managed with the targeted immunotherapy; however, long-term immunotherapy carries health risks, and loss of response. Therefore, we need to develop the alternative treatment strategies. Mesenchymal stem/stromal cell (M.S.C.) exosomes stand out for their remarkable immunomodulatory properties, gaining widespread recognition. This study investigated whether M.S.C. exosomes can reduce psoriasis-induced hyperplasia by inducing Transforming Growth Factor beta 2 (TGF-beta2) signaling. Exosomes were isolated from M.S.C.s by ultracentrifugation. Then, scanning electron microscopy was used for the morphology of exosomes. To ascertain the exosome concentration, the Bradford test was used. To ascertain the cellular toxicity of exosomes in Human Umbilical Vein Endothelial Cells ( H.U.V.E.C), an MTT experiment was then conducted. Real-time PCR was used to quantify TGF beta2 expression levels, whereas an ELISA immunosorbent assay was used to determine the protein concentration of TGF beta2. In this study, the exosomes of 15-30nm in size that were uniform, and cup-shaped were isolated. Moreover, the IC50 value for this Treatment was calculated to be 181.750µg/ml. The concentration of TGF-β2 gene in the target cells significantly increased following Treatment with the exosomes. Furthermore, the expression level of the studied gene significantly increased due to the Treatment. Upregulating the expression of TGF-β2 in psoriatic cells via TGF-β2 signaling is one way exosomes can help reduce hyperplasia.

Acorus calamus L. rhizome extract and its bioactive fraction exhibits antibacterial effect by modulating membrane permeability and fatty acid composition.

Ethnopharmacological relevanceIndia's ancient texts, the Charak Samhita and Sushruta Samhita, make reference to the traditional medicinal usage of Acorus calamus L. In India and China, it has long been used to cure stomach aches, cuts, diarrhea, and skin conditions. This ability of the rhizome is attributed to its antimicrobial properties. Research studies to date have shown its antimicrobial properties. However, scientific evidence on its mode of action is still lacking. Aim of the studyAcorus calamus L. rhizome extract and its bioactive fraction exhibits antibacterial effect by modulating membrane permeability and fatty acid composition. Material and methodThe secondary metabolites in the rhizome of A. calamus L. were extracted in hexane using Soxhlet apparatus. The ability of the extract to inhibit multidrug resistant bacterial isolates, namely Bacillus cereus, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa were evaluated using checkerboard assay. Further, the extract was purified using thin layer chromatography, gravity column chromatography, and combiflash chromatography. Structure elucidation of the active compound was done using GC-MS, FT-IR, and UV–Vis spectral scan. The mode of action of the bioactive fraction was determined. Bacterial membrane damage was analyzed using SEM, membrane permeability was determined using SYBR green I and PI dye, leakage of cytoplasmic contents were analyzed using Bradford assay and Fehling’s reagent. The ability to inhibit efflux pump of A. baumannii was determined using EtBr accumulation assay and β-lactamase inhibition was analyzed using nitrocefin as substrate. Also, the biofilm inhibition of B. cereus was determined using crystal violet dye. Moreover, the effect of the bioactive fraction on the fatty acid profile of the bacterial membrane was determined by GC-FAME analysis using 37 component FAME mix as standard. ResultsAcorus calamus L. rhizome hexane extract (AC-R-H) demonstrated broad-spectrum antibacterial activity against all the isolates tested. AC-R-H extract also significantly reduced the MIC of ampicillin against all tested bacteria, indicating its bacterial resistance modulating properties. The assay guided purification determined Asarone as the major compound present in the bioactive fraction (S-III-BAF). S-III-BAF was found to reduce the MIC of ampicillin against Escherichia coli (100–25 mg/mL), Pseudomonas aeruginosa (15–3.25 mg/mL), Acinetobacter baumannii (12.5–1.56 mg/ml), and Bacillus cereus (10–1.25 mg/mL). Further, it recorded synergistic activity with ampicillin against B. cereus (FICI = 0.365), P. aeruginosa (FICI = 0.456), and A. baumannii (FICI = 0.245). The mode of action of S-III-BAF can be attributed to its ability to disturb the membrane integrity, enhance membrane permeability, reduce biofilm formation, and possibly alter the fatty acid composition of the bacterial cell membranes. ConclusionThe bioactive fraction of AC-R-H extract containing Asarone as the active compound showed antibacterial activity and synergistic interactions with ampicillin against the tested bacterial isolates. Such activity can be attributed to the modulation of fatty acids present in bacterial membranes, which enhances membrane permeability and causes membrane damage.

A novel indirect ELISA for serodiagnosis of mucormycosis using antigens from Rhizopus arrhizus.

Due to a delay in diagnosis by conventional techniques and high mortality, the development of a standardised and rapid non-culture-based technique is an unmet need in pulmonary, gastrointestinal, and disseminated forms of mucormycosis. Though limited studies have been conducted for molecular diagnosis, there are no established serologic tests for this highly fatal infection. To develop and evaluate an indirect in-house enzyme-linked immunosorbent assay (ELISA) utilising antigens of Rhizopus arrhizus for detecting anti-Rhizopus antibodies (IgG and IgM) in sera of patients with mucormycosis. We extracted both secretory and mycelial Rhizopus antigens using standardised protocols. Bradford assay was used for protein quantification. We then standardised an indirect ELISA using R. arrhizus mycelial and secretory antigens (10.0 μg/mL in bicarbonate buffer pH 9.2) for detecting anti-Rhizopus IgG and IgM antibodies in patient sera. We included patients with mucormycosis, other fungal infections, and healthy controls. Antibody index value (E-value) was calculated for each patient sample. Asparagine broth culture filtrate utilising 85% ammonium sulphate salt fractionation and mycelial homogenate grown in yeast extract peptone dextrose (YPD) broth precipitated with trichloroacetic acid (TCA) yielded a large amount of good-quality protein for the assay. We included 55 patients with mucormycosis (rhino-orbito-cerebral mucormycosis [ROCM, n = 39], pulmonary [n = 15], gastrointestinal [n = 1]), 24 with other fungal infections (probable aspergillosis [n = 14], candidiasis [n = 10]), and healthy controls (n = 16). The sensitivity of the antibody test for diagnosing mucormycosis ranged from 83.6-92.7% for IgG and 72.7-87.3% for IgM, with a specificity of 91.7-92.5% for IgG and 80-82.5% for IgM. The sera from patients with other fungal infections and healthy individuals did not show significant cross-reactivity. The detection of anti-Rhizopus IgG antibody performed significantly better in comparison to IgM-based ELISA for diagnosing both ROCM (sensitivity of 84.6% vs. 69.2%) and pulmonary cases (86.6% vs. 80.0%). More extensive studies are required to confirm our findings.

Moderate intensity aerobic exercise prevents myopathic consequences of glucocorticoid administration in aged female mice

Elevated glucocorticoids induce a myopathy characterized by the loss of muscle mass and increased muscle fatiguability. The elderly is highly susceptible to the myopathic effects as they are more likely to experience elevated glucocorticoid levels. Aerobic exercise can prevent glucocorticoid-induced loss of muscle in younger rodents; however, it is unknown whether aerobic exercise can influence glucocorticoid myopathic features in aged muscle. Therefore, the purpose of this study was to define the extent to which moderate intensity aerobic exercise influenced glucocorticoid myopathy in aged muscle. 24-month-old female C57BL/6 mice were randomized into 4 groups of equal bodyweights. Two groups remained sedentary while the other 2 groups began treadmill aerobic exercise performed 5x/week at 10 m/min for 30 min at 5% grade. At the end of the first week of exercise, one group of sedentary mice and one group of exercising mice began receiving once daily subcutaneous injections of dexamethasone (1 mg/kg; DEX). The other group of sedentary mice and group of exercising mice began receiving once daily subcutaneous injections of saline only. All treatments (sedentary/exercise or DEX/saline) continued for an additional 2 weeks. At the end of week 3, the contractile properties of the triceps surae muscle complex were assessed in situ via electrical stimulation of the sciatic nerve. Specific measures included twitch force, sub tetanic force (60 Hz stimulation frequency), tetanic force (125 Hz stimulation frequency), and a fatigue protocol consisting of 60 high force contractions, with each contraction separated by 4 seconds. After testing, muscles of the triceps surae complex were extracted and weighed. Total protein content of the gastrocnemius was determined by the Bradford method. DEX lowered muscle mass, lowered gastrocnemius total protein content, and enhanced fatigue, with aerobic exercise preventing those changes. Interestingly, main effects were noted for DEX to increase twitch and 60 Hz absolute force, normalized force, and RFD in both sedentary and exercise trained mice. Neither DEX nor exercise altered half-relaxation time at any stimulation frequency, nor did they alter tetanic force at the 125 Hz stimulation. These data show that the detrimental effects of elevated glucocorticoids to skeletal muscle morphology and contractile function in aged muscle can be mitigated by moderate intensity aerobic exercise training. They also indicate that there may be some positive benefits to submaximal force generating capacity following a clinically relevant dose of glucocorticoids in aged females. This work was supported by a Doctoral grant from the American College of Sports Medicine (GRL) and NIH R03AG073445 (BSG). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effectiveness mutagenesis of Bacillus subtilis using EMS (Ethyl Methane Sulfonate) to increase xylanase enzyme activity

This research was conducted to determine the effect of mutagenizing Bacillus subtilis with Ethyl Methane Sulfonate (EMS) for xylanase production and evaluate the effect of different xylan concentrations from corn cobs. The xylanase enzyme is an enzyme that can reduce the xylan content which is an anti-nutrient in animal feed. The wild-type Bacillus subtilis was treated with 50 μg/ml and 100 μg/ml of EMS. The mutants generated were selected for xylanase production in a medium containing xylan from corn cobs as a carbon source. The parameters observed included: Total Plate Count (TPC) and reducing sugar (xylose). Protein concentration and xylanase enzymes were analyzed using the Bradford method and 3.5 Dinitro salicylic acid for reducing sugar according to Miller methods respectively. Five mutants developed from each of the EMS concentrations. Approximately 6 and 6.8 % of the mutants developed from 50 μg/ml and 100 μg/ml of EMS had higher xylanase activities than the wild type and protein and xylose concentrations were higher than the wild type. From the results of the research, it can be seen that the use of EMS compounds has the potential to increase enzyme activity so that it can be potential in reducing antinutrients in animal feedstuff.

Biochemical Separation of Cytoplasmic and Nuclear Fraction for Downstream Molecular Analysis.

Biochemical fractionation is a technique used to isolate and separate distinct cellular compartments, critical for dissecting cellular mechanisms and molecular pathways. Herein we outline a biochemical fraction methodology for isolation of ultra-pure nuclei and cytoplasm. This protocol utilizes hypotonic lysis buffer to suspend cells, coupled with a calibrated centrifugation strategy, for enhanced separation of cytoplasm from the nuclear fraction. Subsequent purification steps ensure the integrity of the isolated nuclear fraction. Overall, this method facilitates accurate protein localization, essential for functional studies, demonstrating its efficacy in separating cellular compartments. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Biochemical fractionation Support Protocol 1: Protein quantification using Bradford assay Support Protocol 2: SDS/PAGE and Western blotting.

Chemical composition and amino acids of Locusta migratoria (Orthoptera Acrididae) fed wheat seedlings (Triticum aestivum) or rape leaves (Brassica napus)

Abstract Locusts have high nutritional value and are considered alternative protein sources for humans and animals. Diet can strongly affect the nutritional composition of insects. In this study, the influences of diets – wheat (Triticum aestivum) seedlings and rape (Brassica napus) leaves – on the chemical composition and amino acids of the migratory locust (Locusta migratoria) were assessed. The crude protein, soluble carbohydrate and triglyceride were analyzed using the Bradford method, anthrone colorimetry, and glycerol kinase chromogenic method, respectively. Additionally, amino acids were determined using cation exchange chromatography. The study revealed that wheat seedlings had lower water content than rape leaves. Although the crude protein content was comparable, the soluble carbohydrates of wheat seedlings were approximately 2.5 times higher than those of rape leaves. The body weight (fresh weight and dry weight), crude protein and triglyceride of locusts significantly increased when fed wheat seedlings compared with when fed rape leaves, with values of 48.08%, 31.58%, 43.83% and 25.00%, respectively. The high water content of rape leaves resulted in the high water content of rape leaf-fed locusts. Plants also affect the amino acid composition of locusts. The eight amino acids (four essential amino acids and four non-essential amino acids) and the total amino acids of rape leaf-fed locusts were significantly enhanced compared with those of wheat seedlings. The essential amino acid index and biological value indicate that the protein quality of locusts fed rape leaves has been significantly improved. These results demonstrate that the nutritional composition of L. migratoria can be altered by manipulating the diet. Insects with controllable nutrients can be provided by insect farms and further studies will improve the development of insects as food or feed.

DTT protein equalization and Tryptophan protein quantification as a powerful tool in analytical proteomics.

Assessing total protein levels in biological samples is a common procedure in biochemistry and molecular biology. In this study, we compare tryptophan fluorescence (WF) with Bradford and BCA assays to determine total protein in serum samples. Our results indicate that tryptophan fluorescence spectrometry is an efficient, sensitive, and straightforward technique for quantifying proteins in serum. We observed minimal variation between the three methods: BCA de one with the lowers LOD and LOQ. The tryptophan method offers the possibility of reusing the intact sample that does not need colourimetric reagents for quantification. Consequently, free tryptophan serves as a reliable universal standard. This assay can be performed using a conventional fluorescence spectrometer with cuvettes or in a 96-well plate format with a plate reader. The method was successfully used as proof of concept, using serum from patients diagnosed with myeloma and serum from healthy donors.

Synthesis of multi-responsive poly(NIPA-co-DMAEMA)-PBA hydrogel nanoparticles in aqueous solution for application as glucose-sensitive insulin-releasing nanoparticles.

This study aimed to present an innovative method for synthesizing pH-thermo-glucose responsive poly(NIPA-co-DMAEMA)-PBA hydrogel nanoparticles via single-step aqueous free radical polymerization. The synthesis process involved free radical polymerization in an aqueous solution, and the resulting nanoparticles were characterized for their physical and chemical properties by 1H NMR, Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM). Insulin-loaded poly(NIPA-co-DMAEMA)-PBA hydrogel nanoparticles were prepared and evaluated for their insulin capture and release properties at different pH and temperature, in addition to different glucose concentrations, with the release profile of insulin quantitatively evaluated using the Bradford method. 1H NMR results confirmed successful PBA incorporation, and DLS outcomes consistently indicated a transition to a more hydrophobic state above the Lower Critical Solution Temperature (LCST) of NIPA and DMAEMA. While pH responsiveness exhibited variation, insulin release generally increased with rising pH from acidic to neutral conditions, aligning with the anticipated augmentation of anionic PBA moieties and increased hydrogel hydrophilicity. Increased insulin release in the presence of glucose, particularly for formulations with the lowest mol % PBA, along with a slight increase for the highest mol % PBA formulation when increasing glucose from 1 to 4mg/mL, supported the potential of this approach for nanoparticle synthesis tailored for glucose-responsive insulin release. This work successfully demonstrates a novel method for synthesizing responsive hydrogel nanoparticles and underscores their potential for controlled insulin release in response to glucose concentrations. The observed pH-dependent insulin release patterns and the influence of PBA content on responsiveness highlight the versatility and promise of this nanoparticle synthesis approach for applications in glucose-responsive drug delivery systems. Poly(NIPA) nanoparticles containing PBA moieties are normally synthesized in two or more steps in the presence of organic solvents. Here we propose a new method for the synthesis of multiresponsive hydrogel poly(NIPA-co-DMAEMA)-PBA nanoparticles in aqueous medium in a single reaction to provide a fast and effective strategy for the production of glucose-responsive multi-systems in aqueous media from free radical polymerization.

Composition analysis of Magnolia flower and their use for highly bright carbon dots

Colloidal carbon dots (C-dots) have excellent optical properties and they have been widely used for various types of applications. Compared to the C-dots prepared using chemicals, the Biomass-derived C-dots have attracted a great of attention because of their environmental friendliness and biocompatibility. However, currently obtained C-dots using biomass have low quantum yield. In this work, we demonstrated an efficient precursor of Magnolia flower for synthesizing high-quantum yield C-dots via hydrothermal approach. High performance liquid chromatography and Bradford assay were used to analyze the compositions of the Magnolia flower, which determine the quality of the C-dots. The typical emission bands of the as-prepared C-dots cover from 400 to 550 nm at different excitation wavelengths. The C-dots synthesized using violet Magnolia flower have the QY as high as 11.9%, which is higher than that of 10.4% (white Magnolia flower), because the violet color gives the higher content of the N. The as-obtained C-dots have excitation-dependent color and high quantum yield, showing a great potential for the use of the anti-counterfeiting system.

Variation in Nutritional Components and Antioxidant Capacity of Different Cultivars and Organs of Basella alba

Basella alba is a frequently consumed leafy vegetable. However, research on its nutritional components is limited. This study aimed to explore the variation in the nutritional components and antioxidant capacity of different cultivars and organs of Basella alba. Here, we primarily chose classical spectrophotometry and high-performance liquid chromatography (HPLC) to characterize the variation in nutritional components and antioxidant capacity among different organs (inflorescences, green fruits, black fruits, leaves, and stems) of eight typical cultivars of Basella alba. The determination indices (and methods) included the total soluble sugar (anthrone colorimetry), total soluble protein (the Bradford method), total chlorophyll (the ethanol-extracting method), total carotenoids (the ethanol-extracting method), total ascorbic acid (the HPLC method), total proanthocyanidins (the p-dimethylaminocinnamaldehyde method), total flavonoids (AlCl3 colorimetry), total phenolics (the Folin method), and antioxidant capacity (the FRAP and ABTS methods). The results indicated that M5 and M6 exhibited advantages in their nutrient contents and antioxidant capacities. Additionally, the inflorescences demonstrated the highest total ascorbic acid and total phenolic contents, while the green and black fruits exhibited relatively high levels of total proanthocyanidins and antioxidant capacity. In a comparison between the green and black fruits, the green fruits showed higher levels of total chlorophyll (0.77–1.85 mg g−1 DW), total proanthocyanidins (0.62–2.34 mg g−1 DW), total phenolics (15.28–27.35 mg g−1 DW), and ABTS (43.39–59.16%), while the black fruits exhibited higher levels of total soluble protein (65.45–89.48 mg g−1 DW) and total soluble sugar (56.40–207.62 mg g−1 DW) in most cultivars. Chlorophyll, carotenoids, and flavonoids were predominantly found in the leaves of most cultivars, whereas the total soluble sugar contents were highest in the stems of most cultivars. Overall, our findings underscore the significant influence of the cultivars on the nutritional composition of Basella alba. Moreover, we observed notable variations in the nutrient contents among the different organs of the eight cultivars, and proanthocyanidins may contribute significantly to the antioxidant activity of the fruits. On the whole, this study provides a theoretical basis for the genetic breeding of Basella alba and dietary nutrition and serves as a reference for the comprehensive utilization of this vegetable.

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.