Search

Gingival mesenchymal stem cell-conditioned medium (GMSC-CM) is a conditioned medium obtained from cultured gingival mesenchymal stem cells . GMSCs are easily isolated from gingival tissue, whereas gingival tissue is easily obtained by minimally invasive techniques. On the other hand, C M contains proteins, cytokines, chemokine s and growth factors that play an important role in osteogenic differentiation. This study aim s to determine the total protein and calcium levels in GMSC-CM. GMSCs were grown in culture media with 10% of FBS. CM of GM SC was obtained from the media collection process with a 0.22 µm filter and concentrated with a centrifugal filter to obtain a concentrated GMSC-CM. The concentration of total protein was performed by bicinchoninic acid protein assay on GMSC-CM and concentrated GMSC-CM. Calcium level was performed by atomic absorption spectroscopy method. The results are that GMSC-CM had a total protein concentration of 2502±0.06 µg/ml, and the concentrated GMSC-CM was 1.912±0.08 μg/ml. The calcium level of GMSC-CM was 0.0 09 % and concentrated GMSC-CM was 0.0 09 %. It can be concluded that GMSC-CM had a high concentration of total protein and calcium. These parameters were used to develop bioprocesses to enhance the production of GMSC-CM, which will support the implementation of cell-free therapy for tissue regeneration.

Context. Proteins in the saliva are one of the defense mechanism factors that can protect the oral cavity from disease. However, smoking might affect the properties of saliva. Aim To determine the differences in salivary protein profiles and total concentrations in smokers and non-smokers and their correlation with dental caries severity as indicated by the Decayed, Missing, Filled-Teeth (DMF-T) scores. Methods and material This cross-sectional study included 25 smokers and 25 non-smokers. The DMF-T scores were recorded. The total salivary protein was measured by the Bradford method, and the profile proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results The average of salivary protein concentration in smokers was lower than that in non-smokers (551.486 µg/mL versus 765.361 µg/mL), but the difference was not statistically significant (P > 0.05). Further correlation analyses showed a negative correlation between the concentration of proteins based on the extent of smoking. A weak negative correlation was found between protein concentration and DMF-T scores (r = −0.239). Dominant salivary protein bands of 11.6 kDa and 54.5 kDa were found in smokers and 27 kDa, 60 kDa, and 94.5 kDa were found in non-smokers. Conclusion Different protein bands appeared in smokers and non-smokers. There was a weak correlation between protein concentration, DMF-T scores, and the extent of smoking.

The aim of our work was to develop a low-cost, portable device for the fast and easy determination of total protein content by using PDMS-based lab-in-a-syringe technology with removal of 3D-printed channels. We proposed two designs with a one-step PDMS curing and a two-step PDMS-curing fabrication procedure. The one-step PDMS microdevices were found to be the best in the view of preparation, repeatability, and stability of the reagent. This design was then applied for the determination of total protein content in biomedical products using the Bradford assay.

Objectives: This study aimed to obtain an optimum method for zincyeast production. Materials and Methods: Production of zinc-yeast was prepared using Yeast Extract Peptone Dextrose (YEPD) as a culture medium. Yeast that had been inoculated into culture media was incubated with a shaker at room temperature for 84 hrs, then zinc sulfate was added with various concentrations of 200, 300, and 400 mg/L, and then incubation continued for 24 hrs. The obtain yeast biomass was dried with a Freeze-dryer and analyzed for total accumulated zinc and protein content. Determination of zinc was using atomic absorption spectrophotometry (AAS) at a wavelength of 196 nm, whereas protein content was determined with Bradford method at a wavelength of 595 nm using UV-Vis spectrophotometer. Results: Zinc-yeast with the addition of 200 mg/L zinc sulfate obtained 0.3111 g that contains 2228.60 μg/g of zinc and 0.8515 mg/L of protein. While zinc-yeast with the addition of 300 mg/L zinc sulfate obtained 0.3704 g that contains 2458.30 μg/g of zinc and 0.8682 mg/L of protein. And the last, zinc-yeast with the addition of 400 mg/L zinc sulfate obtained 0.2370 g that contains 2648.43 μg/g of zinc and 0.8935 mg/L of protein. Conclusion: Production of zinc-yeast with the highest zinc and protein content can be obtained by adding 400 mg/L zinc sulfate solution to the stationary phase culture of S. cerevisiae. The modified zinc-yeast production method is simple and can be used for the production of functional food products.

Objectives: The objective of this study was to perform selenium yeast production and analyze the selenium and protein contained in selenium yeast obtained. Methods: Selenium yeast production was carried out by incubating the stationary phase culture of yeast with selenium solution for 24 hr, and yeast biomass was dried by Freeze-dryer to produce selenium yeast in powder form, then the selenium yeast was determined to the selenium content with atomic absorption spectrophotometer at a wavelength of 196 nm and protein content analyzing by the Bradford method at a wavelength of 595 nm using UV-Vis spectrophotometer. Results: Selenium yeast production reached 2.5 g of selenium yeast in powder form. The selenium and protein content of selenium yeast was 4258.0096 (±278.39) μg Se/g and 0.8505 (±0.0045) mg/mL, respectively. The method was easy, simple, and can prove that selenium yeast can still be produced and analyzed for the selenium and protein content. Conclusion: The present study demonstrated that production of selenium yeast in the form of dry powder could be carried out using the stationary phase culture of S. cerevisiae incubated with 30 μg/mL selenium solution, and the resulting selenium yeast can be analyzed for selenium content using atomic absorption spectrophotometer and protein content by Bradford method using UV-Vis spectrophotometer.

Developing accurate methods for the assessment of therapeutic protein release from polymer drug delivery systems (microcapsules, microspheres, nanoparticles, 3D-printed systems) is of paramount importance for new formulation development. The most straightforward method for protein release assessment is spectrophotometric analysis of the release medium surrounding the formulation. However, direct spectrophotometric analysis is inapplicable to formulations releasing interfering compounds (co-encapsulated drugs, additives) absorbing light in the same spectrum as proteins. Conventional protein release assays also require frequent release medium sampling and replacement, which reduces their accuracy. We propose a one-step method to assess protein release from core/shell microcapsules eliminating the need for sampling and allowing selective real-time protein quantitation in the release medium. To prevent spectral interferences, released protein is differentiated from interfering compounds by employing a colorimetric protein assay reagent, forming a colour complex selectively with the protein, as the release medium. To eliminate sampling, we employed a continuous flow closed loop set-up, where the release medium is constantly circulating between microcapsule-containing tank and spectrophotometer. A series of colorimetric protein assay reagents (bromocresol green, tetrabromophenol blue, eosin B, eosin Y, biuret) were evaluated in terms of their applicability as the release medium in described system. Only biuret reagent was found compatible with proposed method due to formation of color complex stable over extended period of time and low adsorption to microcapsules. Presented method allowed effective evaluation of albumin release from alginate-polyethersulfone microcapsules with accuracy equal to conventional ‘sample and separate’ technique. Albumin release followed first-order kinetics with plateau reached after 19 h.

In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification

The fabrication of sensitive protein microarrays such as PCR used in DNA microarray is challenging due to lack of signal amplification. The development of microarrays is utilized to improve the sensitivity and limitations of detection towards primal cancer detection. The sensitivity is enhanced by the use of ZnO-nanorods and is investigated as a substrate which enhance the florescent signal to diagnose the hepatocellular carcinoma (HCC) at early stages. The substrate for deposition of ZnO-nanorods is prepared by the conventional chemical bath deposition method. The resultant highly dense ZnO-nanorods enhance the fluorescent signal 7.2 times as compared to the substrate without ZnO-nanorods. The microarray showed sensitivity of 1504.7 ng ml-1 and limit of detection of 0.1 pg ml-1 in wide dynamic range of 0.05 pg-10 μg ml-1 for alpha fetoprotein (AFP) detection in 10% human serum. This immunoassay was successfully applied for human serum samples to detect tumor marker with good recoveries. The ZnO-nanorod substrate is a simple protein microarray which showed a great promise for developing a low-cost, sensitive, and high-throughput protein assay platform for several applications in both fundamental research and clinical diagnosis.

Objectives: To optimize an enzyme-linked immunosorbent assay (ELISA) for measuring the HspX protein (α-crystallin) levels and then evaluate its correlation with the accumulation of lipid bodies in Mycobacterium bovis (M. bovis) during hypoxia and exposure to nitric oxide. Methods: This study was conducted at Prince Sultan Military Medical City, Riyadh, Saudi Arabia between 2016 and 2017. We first optimized ELISA conditions for the detection of HspX. The optimization protocol focused on minimizing concentrations of the capture antibody, detection antibody, and conjugated secondary antibody, and determining the minimum detection limit of the antigen, HspX. Bacteria were grown either in shaking culture or in stationary flasks mimicking hypoxic environments. A standard Bradford assay was used to determine the total protein and HspX was detected using the optimized ELISA protocol. The effect of hypoxic environment and nitric oxide on the levels of HspX and lipid bodies, detected by staining with Nile red, was also evaluated. Results: An optimized ELISA protocol was established for the detection of HspX from M. bovis. Exposure to nitric oxide and hypoxic conditions led to an increase in the levels of HspX protein. The increase in HspX associated with nitric oxide treatment and hypoxic conditions correlated with higher levels of lipid bodies mainly found in pathogenic mycobacteria. Conclusions: The optimized ELISA protocol in this study can detect HspX protein levels in M. bovis growing in normal and hypoxic environments. Importantly, hypoxia led to enhanced expression of HspX protein, which correlated with the enhanced production of lipid bodies. Lipid body production is a survival strategy of pathogenic mycobacteria.

Poly(DL-lactic-co-glycolic acid) and poly(DL-lactic acid) are widely used for the preparation of nanoparticles due to favorable characteristics for medical use like biodegradability and controllable degradation behavior. The contact with different media like human plasma or serum leads to the formation of a protein corona that determines the NP's in vivo processing. In this study, the impact of surface end group identity, matrix polymer hydrophobicity, molecular weight, and incubation medium on the protein corona composition was evaluated. Corona proteins were quantified using Bradford assay, separated by SDS-PAGE, and identified via LC-MS/MS. The acquired data revealed that surface end group identity had the most profound effect on corona composition in both quantitative and qualitative terms. Regarding matrix polymer hydrophobicity, adsorption profiles on NP systems with similar physicochemical characteristics resembled each other. The molecular weight of the matrix polymers proved to impact quantity, but not quality of corona bound proteins. The corona of plasma incubated NP showed adsorption of incubation medium-specific proteins but resembled those of serum incubated NP in terms of protein function, average mass and isoelectric point. Overall, the NP physicochemical properties proved to be easily adjustable determining factors of protein corona formation in physiological environments.

Past investigations involving fingerprints have revolved heavily around the image of the fingerprint-including the minutiae, scarring, and other distinguishing features-to visually find a match to its originator. Recently, it has been proven that the biochemical composition can be used to determine originator attributes, such as sex, via chemical and enzymatic cascades. While this provides pertinent information about the originator's identity, it is not the only piece of information that can be provided. This research was designed with three goals in mind: (1) identify how long it would take before an aged female fingerprint could no longer be differentiated from a male fingerprint, (2) identify a correlation between the data collected and a specific time since deposition (TSD) time point, and (3) identify if a specific amino acid could be contributing to the decreasing response seen for the aging fingerprints. Using ultraviolet-visible (UV-vis) spectroscopy, aged fingerprints were evaluated over the course of 12 weeks via three chemical assays previously used for fingerprint analysis-the ninhydrin assay, the Bradford assay, and the Sakaguchi assay. As fingerprints age, the conditions they are exposed to cause the biochemical composition to decompose. As this occurs, there is less available to be detected by analytical means. This results in a less intense color production and, thus, a lower measured absorbance. The results displayed here afforded the ability to conclude that all three goals set forth for this research were accomplished-a female fingerprint can be differentiated from a male fingerprint for at least 12 weeks, UV-vis data collected from aged fingerprints can be correlated to a TSD range but not necessarily a specific time point, and the decomposition of at least a single amino acid can afford the ability to estimate the TSD of the fingerprint.

Introduction: Further development of magnetic-based detection techniques could be of significant use in increasing the sensitivity of detection and quantification of hepatitis B virus (HBV) infection. The present work addresses the fabrication and characterization of a new bio-nano composite based on the immobilization of goat anti-HBsAg antibody on modified core-shell magnetic nanoparticles (NPs) by (3-aminopropyl) triethoxysilane (APTES), named Fe3O4@SiO2/NH2, and magnetic NPs modified by chitosan (Fe3O4@CS). Methods: At the first step, Fe3O4 was modified with the silica and APTES (Fe3O4@SiO2/NH2) and chitosan (Fe3O4@CS) separately. The goat anti-HBsAg antibody was activated by two different protocols: Sodium periodate and EDC-NHS. Then the resulted composites were conjugated with activated goat anti-HBsAg IgG. An external magnet collected Bio-super magnetic NPs (BSMNPs) and the remained solution was analyzed by the Bradford method to check the amount of attached antibody to the surface of BSMNPs. Results: The findings indicated that activation of antibodies by sodium periodate method 15-17 µg antibody immobilized on 1 mg of super magnetic nanoparticles (SMNPs). However, in the EDC-NHS method, 8-10 µg of antibody was conjugated with 1 mg of SMNPs. The resulting bio-magnetic NPs were applied for interaction with the HBsAg target using enzyme-linked immunosorbent assay (ELISA). About 1 µg antigen attached to 1 mg SMNPs, which demonstrated that the fabricated materials are applicable in the detection scope of HBsAg. Conclusion: In the present study, we developed new antibody-conjugated magnetic NPs for the detection of HBsAg using an efficient conjugation strategy. The results demonstrated that the binding capacity of Fe3O4@SiO2/NH2 was comparable with commercially available products. Our designed method for conjugating anti-HBsAg antibody to a magnetic nanoparticle opens the way to produce a high capacity of magnetic NPs.

Lemna sp. is known to have several bioactive compounds and polysaccharide macromolecules that can function as immunomodulators to affect non-specific immune responses to increase the body's resistance to pathogens. This study aims to determine the potential of catfish eye extract as an immunostimulant by observing non-specific tilapia immune parameters. The extraction method used was 96% ethanol maceration for 2 days with a ratio of 1: 4. The experimental design used a Completely Randomized Design with 5 treatments (doses 0.2, 0.4, 0.6 mg/kg, control + and control -) and 3 replications. The non-specific parameters of immunity observed included total plasma protein (Bradford method), superoxide dismutase and lysozyme activity. The data obtained will be analyzed using ANOVA, if there is a significant difference, it will be further tested with Duncan Multiple. Range Test (DMRT). The results showed that the highest total plasma protein was found in treatment C (giving an extract of 0.3 mg/kg body weight) with an average total plasma protein after 12 days of maintenance of 4.99 g / dL. The extract dose of 0.3 mg/body weight showed a rapid decrease in SOD and increase Lysozyme activity.

Malaria is still an essential epidemiological disease worldwide, including in Indonesia. Several approaches are performed to control the disease, as well as vaccine development. The Cysteine-rich interdomain region α of Plasmodium falciparum erythrocyte membrane protein 1 (CIDRα-PfEMP1) is a pivotal domain in the malaria pathogenesis make it a malaria vaccine candidate. The development of the malaria vaccine is performed using recombinant technology. Recombinant protein production is an important step. The study aimed to determine the optimized condition for CIDRα-PfEMP1 recombinant protein expression in Escherichia coli BL21(DE3) expression system. Serial IPTG concentrations from 0.05, 0.1, 0.3, and 0.5 mM and two different incubation periods of 4 h and 8 h were optimized. The recombinant protein expression was visualized in SDS-PAGE, measured using the Bradford protein assay, and calculated using software Image J. SDS-PAGE visualization showed a 27 kDa band expressed CIDRα-PfEMP1 recombinant protein. The optimized condition for CIDRα-PfEMP1 recombinant protein expression was at 0.03 mM IPTG concentration and 8 h incubation period.

Yifan Wang,1– 3 Si Chen,1– 3 Xin Yang,1– 3 Shuang Zhang,1– 3 Chunying Cui1– 3 1Department of Pharmaceutics, School of Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China; 2Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Beijing, People’s Republic of China; 3Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Beijing, People’s Republic of ChinaCorrespondence: Chunying Cui; Shuang ZhangDepartment of Pharmaceutics, School of Pharmaceutical Science, Capital Medical University, Number 10 Youanmenwai Street, Fengtai, Beijing, 100069, People’s Republic of ChinaTel +86-10-8391-1668; +86-10-8391-1673Fax +86-10-8391-1668; +86-10-8391-1673Email ccy@ccmu.edu.cn; zshuang@ccmu.edu.cnBackground: siRNA brings hope for cancer therapy. However, there are many obstacles for application of siRNA in clinical. Because of the excellent biocompatibility, non-toxicity and non-immunogenicity of bovine serum albumin (BSA), BSA-based nanoparticles have been widely designed as a drug carrier system.Methods: The optimal formula for BSA NPs preparation was investigated by central composite design response surface methodology (CCD-RSM), BSA-based survivin-siRNA delivery system (BSA NPs/siRNA) was characterized by dynamic light scattering, atomic force microscope, transmission electron microscope and Bradford method. The in vitro anti-tumor effect and mechanism of BSA NPs were investigated by confocal microscopic imaging, MTT assay, RT-qPCR and ELISA analysis. Moreover, the anti-tumor effect, distribution and biosafety of BSA NPs were studied in vivo.Results: The optimal formula for BSA NPs was settled to be 20 mg/mL for BSA concentration, 9 for pH value, 136% for crosslinking degree and 1.6 mL/min for speed of ethanol addition. BSA NPs/siRNA could remain stable at 4°C for 4 weeks and protect siRNA from degradation by RNase A. Besides, BSA NPs/siRNA could maintain a sustained release of siRNA and promote the uptake of siRNA significantly. The survivin-mRNA level and the survivin-protein level were decreased by 55% ± 1.6% and 54% ± 1.6% separately. The in vivo tumor inhibition results suggested that the tumor inhibition rate of BSA NPs/siRNA-treated group was 54% ± 12% and was similar with that of DOX-treated group (57% ± 9.2%, P > 0.05). The biosafety results confirmed that BSA NPs/siRNA could not induce significant damages to the main organs and blood in vivo.Conclusion: These results demonstrated that CCD-RSM was an effective tool for preparation analysis, and the BSA NPs/siRNA was a promising system for siRNA-based gene therapy.Keywords: bovine serum albumin, BSA, CCD-RSM, survivin-siRNA, nanoparticles, RNAi

The presence of bio-macromolecules as major ingredients is a primary factor in marketing many biologically derived macromolecular supplements. Workflows for analyzing these supplements for quality assurance, adulteration, and other supply-chain difficulties must include a qualitative assessment of small-molecule and macromolecular components; however, no such integrated protocol has been reported for these bio-macromolecular supplements. Twenty whey protein supplements were analyzed using an integrated workflow to identify protein content, protein adulteration, inorganic elemental content, and macromolecular and small-molecule profiles. Orthogonal analytical methods were employed, including NMR profiling, LC-DAD-QToF analysis of small-molecule components, ICP-MS analysis of inorganic elements, determination of total protein content by a Bradford assay, SDS-PAGE protein profiling, and bottom-up shotgun proteomic analysis using LC-MS-MS. All 20 supplements showed a reduced protein content compared to the claimed content but no evidence of adulteration with protein from an unclaimed source. Many supplements included unlabeled small-molecule additives (but nontoxic) and significant deviations in metal content, highlighting the importance of both macromolecular and small-molecule analysis in the comprehensive profiling of macromolecular supplements. An orthogonal, integrated workflow allowed the detection of crucial product characteristics that would have remained unidentified using traditional workflows involving either analysis of small-molecule nutritional supplements or protein analysis.

Analysis and identification of seized doping-related products are important tasks for customs or forensic laboratories in order to prevent potentially dangerous and illegal compounds to go into circulation. At the Section of Forensic Chemistry in Copenhagen, we have a workflow consisting of four complimentary validated methods to identify common doping-related substances: liquid chromatography-ultraviolet (LC-UV), LC coupled with time of flight mass spectrometry (LC-TOF-MS), the colorimetric Bradford assay, and an immunoassay. The Bradford assay screens for peptide or proteins in the sample, and the immunoassay confirmed human chorionic gonadotropin (hCG). LC-UV was carried out with a C4 protein column for identification of peptides and proteins from a standard reference library, based on retention times and ratios between peak areas at 220, 254, and 280 nm. LC-TOF-MS was performed using a C18 column, and identification was based on comparison of the retention time and the accurate mass with those of reference standards. In 2019, we received 36 samples for peptide/protein analysis, all of which were tested using the LC-UV, LC-TOF-MS, and colorimetric method, and samples suspected of containing hCG were confirmed with an immunoassay. We found a total of 15 samples containing an illegal doping substance, 12 samples containing substances not prohibited by the Danish Doping List, and nine samples containing no peptides or proteins. In conclusion, the four complimentary methods constitute a suitable approach for identifying common peptide/protein doping substances in the day-to-day routine of a forensic laboratory, with limited sample preparation and interpretation of data.

Toad skin secretions are sources of complex mixtures of bioactive compounds, such as proteins and peptides. Rhinella jimi species is a common toad in the Brazilian northeast, considered by only a few known studies. The experimental design was applied to optimize the protein extraction method from R. jimi parotoid gland secretions. The optimum condition was using 100 mmol L−1 Tris-HCl buffer pH 7.2 under vortexing for 5 min. The FTIR analysis combined with PCA revealed high-protein purity of the extracts, confirming the success of the proposed extraction method. The total protein concentration by the Bradford method was 102.4 and 66.5 mg g−1 on toad poisons from Teresina and Picos, respectively. The comparative proteomic analysis using HPLC-SEC-DAD and 1D SDS-PAGE revealed significant differences in protein abundance. HMW biomolecules showed greater abundance in toads from Teresina, while LMW protein species were more abundant in toads from Picos. The significant difference in amphibian proteome can be attributed to the edaphoclimatic conditions of their habitat. The cytotoxicity of the protein extract from Teresina was higher on the tumor cell lines 4T1 and CT26.WT. These new findings are fundamental for future studies the on identity and biological activity of biomolecules from this noble sample.

The results of modelling the effect of cell sizes on the microorganism disintegration process are presented. Experiments were conducted in a horizontal bead mill for baker's yeast Saccharomyces cerevisiae. The kinetics of the cell disruption process was investigated by optical image analysis and the release of intracellular protein by Bradford method. A large variation in the size of yeast from one production cycle has been demonstrated. A significant increase in the constant rate of the process with an increase in the range of the cell size class was demonstrated. More beneficial values of the coefficient of determination were obtained for the spherical model of the cell. Worse results of the process description were obtained for the ellipsoidal model. They may result from large variations in cell volume in individual size classes. The greatest changes in the cell Elongation parameter within one size class ranged from 1.00 to 1.54.

The development of an effective and noninvasive early method for obtaining fetal cells is crucial to prenatal screening. Despite proving the presence of fetal cells in the reproductive tract, their use is limited due to their inability to properly isolate them from maternal cells. Magnetic-activated cell sorting (MACS) is a simple technique to separate cells. The present study aimed to develop a MACS-based platform for the isolation of the HLA-G expressing trophoblast cells. For this purpose, first, the triazine functionalized MNPs were synthesized and characterized. Then, MNPs were directly and indirectly conjugated by the MEM-G/9 antibodies targeting HLA-G+ cells. The antibody amount on the surface of the nanoparticles was determined with the Bradford assay. The cell capture efficiency was also investigated. Various characterization methods confirmed the successful nanoparticle synthesis and antibody conjugation. The optimal initial antibody amount for the immobilization was about 20 μg and the optimal time was 3 h. The antibody-nanoparticles by the indirect method had better targeting and capture efficiency than the direct method. The MNPs indirectly conjugated with antibodies are an efficient tool for cell isolation and present considerable potential to be applied in biomedical fields.

Background. Keratins are natural biopolymers with a wide range of applications in the field of biotechnology. Materials and Methods. Extraction of keratins was performed by a modified Nakamura method using 250 mM DTT. The protein concentration in the supernatant was determined by Bradford method. The protein composition was studied by their electro­phoretic separation in a polyacrylamide gel in the presence of sodium dodecyl sulfate. The films were made by casting. The surface characteristics of the films were determined using a scanning electron microscope REMMA-102. The elemental composition of the films was determined using an X-ray microanalyzer. Results . The protein concentration in the supernatant was 3.75 mg/mL. After using dithiothreitol in the extraction mixture, we obtained proteins of intermediate filaments with a molecular weight of 40–60 kDa and a low Sulfur content. In the low molecular weight region, we obtained keratin-associated proteins with a molecular weight of 10–30 kDa and a high content of Sulfur. These proteins belong to fibrillar proteins, which can be used as a matrix for the creation of new keratin-containing biocomposites with a wide range of applications in reparative medicine and tissue engineering. Based on the obtained keratin extract, polymer films with and without the addition of glycerol were made. Scanning electron microscopy revealed that glycerol provided the film structure with homogeneity and plasticity due to the accumulation of moisture after the fixation by water vapor. The X-ray microanalysis of films revealed such elements as Sodium, Silicon, Sulfur, Potassium. Among the detected elements, Sulfur has the largest share that is due to the large number of disulfide bonds in the keratin molecule. Conclusions. The polymer keratin films with the addition of glycerol demonstrated better mechanical properties and can be used in biomedicine. Keywords: keratins, hair, extraction, biopolymers Received 27 January, 2021 ● Revision 07 February, 2021 ● Accepted 17 March, 2021 ● Published 31 March, 2021

Extracellular vesicles (EVs) play essential roles in biological systems based on their ability to carry genetic and protein cargos, intercede in cellular communication and serve as vectors in intercellular transport. As such, EVs are species of increasing focus from the points of view of fundamental biochemistry, clinical diagnostics, and therapeutics delivery. Of particular interest are 30-200 nm EVs called exosomes, which have demonstrated high potential for use in diagnostic and targeted delivery applications. The ability to collect exosomes from patient biofluid samples would allow for comprehensive yet remote diagnoses to be performed. While several exosome isolation methods are in common use, they generally produce low recoveries, whose purities are compromised by concomitant inclusion of lipoproteins, host cell proteins, and protein aggregates. Those methods often work on lengthy timescales (multiple hours) and result in very low throughput. In this study, capillary-channeled polymer (C-CP) fiber micropipette tips were employed in a hydrophobic interaction chromatography (HIC) solid-phase extraction (SPE) workflow. Demonstrated is the isolation of exosomes from human urine, saliva, cervical mucus, serum, and goat milk matrices. This method allows for quick (

The human amniotic membrane (AM) is emerging as an interesting biomaterial for regenerative medicine due to its biological and mechanical proprieties. The beneficial effects of the AM are probably related to its bioactive factors produced by local cells and stored in the stromal matrix. However, the search for a preservation method capable of preserving AM properties remains a challenge. The aim of this study was to evaluate important features of 2 anatomical regions of the human AM (reflected and placental amnion) after different preservation methods. For this purpose, human placentas were harvested and processed for AM isolation and storage at 2 different conditions: room temperature for 18 h in DMEM (fresh AM) and −80°C in DMEM/glycerol solution for 30 days (cryopreserved AM). After the storage period, the structural integrity of the membrane was assessed by histological and Picrosirius polarization analysis, cellular viability analysis was performed using the MTT assay, and the soluble proteins were quantified with the Qubit Protein Assay Kit. Both preservation protocols reduced the cell viability, mainly in the placental amnion region of the AM, but preserved the morphology of epithelial and stromal layers, as well as the organization and distribution of collagen fibers. There was a reduction in soluble proteins only in fresh AM. Importantly, the cryopreserved AM group presented the same concentration as the control group. In conclusion, the cryopreservation using DMEM/glycerol was ideal for preserving the structural integrity and soluble protein content, indicating the feasibility of this method in preserving AM for its use in regenerative medicine.

Lectins arecarbohydrate-bindingproteinsthat are highly specific for sugarmoietiesof other molecules. They perform recognition on the cellular and molecular level and play numerous roles in biological recognition phenomena involving cells, carbohydrates, and proteins. Blood groups are inherited characters which give rise to antigen-antibody reaction. A total of 120 samples of local (Nigeria)Achatina achatinasnail specie were collected, authenticated at the Zoology Department of the University of Nigeria, Nsukka and 80mls of pooled crude lectin extract was obtained. Purifications were performed on 20mls of the crude extract in three steps viz, Ammonium sulphate precipitation and Dialysis (Partial purifications), Con A Sepharose 4B affinity chromatography column (Complete purification). The affinity purified lectin was used for all the actual tests conducted in this research. The crude, partially and complete/affinity purified lectin extracts were subjected to Haemagglutination tests, Protein Assay and Specific Sugar determinations. The molecular weight was assessed by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The results of the research showed as follows: On complete/affinity purification, 15mls of pure sample containing only the high molecular weight lectin was obtained. The respective haemagglutination tests on the crude, partially and affinity purified lectin showed on standardisation, preferential agglutination with Blood group A type. The Protein contents of the lectin was deduced to be as follows: The crude extract contains 13.5mg/dl, Dialysed precipitate – 5.7mg/dl, Dialysed supernatant – 5.0mg/dl and the Affinity purified Lectin – 0.422mg/dl. Galactose N-acetyl amine (Gal NAc) residue was determined to be its specific sugar. The SDS-PAGE analysis showed the molecular weight of the lectin to be 250 KDaltons. This research has therefore succeeded in the Purification, Characterisation and illustration of the lectinic properties of the local Nigeria snail -Achatina achatina.

Protein extraction is a preliminary step of protein purification which mainly focus on maximization of total protein yield. The heterogeneous properties cause diversification of protein; therefore, there is no absolute protocol in protein extraction. The ratio of buffer gives different protein concentrations in different types of mammalian tissues, and this condition leads to the study of optimization of buffer ratio to obtain a better total protein yield. The objectives of this study were to compare the total protein yield based on three different ratios of buffer used. The phosphate buffer saline (PBS), radioimmunoprecipitation assay (RIPA) buffer, and RIPA buffer with the addition of protease inhibitor (Pi) were used with the ratios of 1:1, 1:3, and 1:5. Fetal cotyledons removed from the placenta have undergone mechanical disruption, incubation, sonication, and centrifugation. The supernatant was retained and quantified with Bradford assay to determine the total protein yield based on the standard curve of bovine serum albumin (BSA). There was a statistically significant different between buffer ratio (p

Milk bioactive peptides are derivative of milk protein produced either through enzymatic activity, digestive processes, or fermentation, that give functional properties. The study aimed to obtain bioactive peptides fraction derived from goat’s milk casein through hydrolysis by papain, analyze the profiles of protein and peptides, and also test the antioxidative activity. The casein isolate was hydrolyzed by papain in a ratio of 100: 0.5 (v/v) at pH 7.0 and 50ºC. Analysis of protein concentration was carried out by the Bradford method and protein profile by SDS-PAGE. Antioxidant assay was carried out by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. A 1,000 ppm ascorbic acid solution was used as positive control. Peptide fractionation was done by membrane filtration with a cut off of 10 kD and 30 kDa. The protein concentration of casein hydrolysates decreased significantly after hydrolysis process with papain. The electrophoresis results showed six protein bands in casein with molecular weight of 7-33 kDa. After the hydrolysis process, all hydrolysates only contained two protein bands with molecular weights of 8 and 5 kDa. The hydrolysis process increased the antioxidant activity of the casein. P0 and P2 hydrolysates had the highest antioxidant activity, and fractions with the highest antioxidant activity were fraction

Artificial Insemination Center (AIC) produces frozen semen that was taken from superior bull. The selection of superior bull was based on breeding soundness evaluation. This study is aimed to evaluating the correlation between the molecular weight (MW) of seminal plasma protein and semen quality of Simmental bulls. Semen ejaculates from nine Simmental Bulls, that belong to AIC of Central Java, at Ungaran, were collected by using an artificial vagina. The sub population of sperm in ejaculates was evaluated through macroscopically and microscopically, in which the semen then centrifugated 6500 rpm for 30 minutes. The supernatant was collected and inserted into mini straw and stored in liquid nitrogen container. The concentration of seminal plasma protein was determined by the Bradford method. The Bradford protocol of analysis was suitable with User Guide Coomasize (Bradford) Protein Assay Kit. Thermo Skanlt RE for Multiskan Go Software, 3.2 version was applied for analyzing of the data, and protein characterization used was 1D-SDS-PAGE. The Coomassie Brilliant Blue was used for coloring the gels, and the proteins MW was determined by MW markers. The result demonstrated that there was no significant correlation between protein concentration and semen quality (P>0.05). Protein expressions based on MW were 62-48 kD, 100-71 kD found in this study. The analysis of correlation showed that the proteins with 100-71 kD MW correlated with sperm motility, normal sperm morphology, and sperm concentration. Semen quality in all parameters was significant (P˂0.05 and P>0.01) with 62-48 kD protein MW. The result concluded that seminal plasma protein had a correlation with semen quality and can be used as additional indicator for bull’s candidate selection.

Introduction Protein corona (PC) deposition on nanoparticles (NPs) in biological systems contributes to a great extent to NPs' fates; their targeting potential, the interaction with different biological systems and the subsequent functions. PC - when properly tuned - can serve as a potential avenue for optimization of NPs' use in cancer therapy. Methods Poly-lactic co-glycolic acid (PLGA)-based NPs exhibiting different physicochemical properties were fabricated and characterized. The PC makeup of these NPs were qualitatively and quantitatively analyzed by Western blot and Bradford assay, respectively. The effect of PC on the release of NPs' cargos and the intracellular uptake into B16F10 melanoma cells has been studied. Results The composition of NPs (polymeric PLGA NPs vs lipid-polymer hybrid NPs) and the conjugation of an active targeting ligand (cRGDyk peptide) represented the major determinants of the PC makeup of NPs. The in vitro release of the loaded cargos from the NPs depended on the PC and the presence of serum proteins in the release medium. Higher cumulative release has been recorded in the presence of proteins in the case of peptide conjugated NPs, cNPs, while the unconjugated formulations, uNPs, showed an opposite pattern. NPs intracellular uptake studies revealed important roles of distinct serum and cellular proteins on the extent of NPs' accumulation in melanoma cells. For example, the abundance of vitronectin (VN) protein from serum has been positively related to the intracellular accumulation of the NPs. Conclusion Careful engineering of nanocarriers can modulate the recruitment of some proteins suggesting a potential use for achieving endogenous targeting to overcome the current limitations of targeted delivery of chemotherapeutic agents.