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Background: Peptides have shown anti-sickling effect and are able to reduce oxidative stress, thus contributing to the management of sickle cell disease. Objectives: The present study was undertaken to characterize and to assess the antisickling and antioxidant properties of albumins and globulins fractions from black bean seed (Phaseolus vulgaris L.) used to manage sickle cell disease in West Region of Cameroon. Methods: NaCl 5% (1:10 w/v) and dialysis were used for extracting albumins and globulins respectively. The concentration of proteins was determined by the Bradford method. Molecular size was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In vitro antisickling properties, investigate was carried up using microscopic enumeration. Membrane stability effect and antioxidant potentials were determined using colorimetric method. Results: In non-reducing buffer showed both albumin and globulin in native form, had the main proteins with dense band of about 130 and 37 kDa. Antisickling tests revealed the best inhibition (79.15 ± 3%) and reversibility (73.49 ± 6%) rates with globulin fraction at 3.82 mg/ml. Hemolysis decreased significantly with albumin and globulin fractions at different concentrations showing the stability effect on the membranes of erythrocytes. However the concentration of globulin at 3.82 mg/ml has shown the best activity (from 100 to 4.62%). The FRAP test has shown that the proteins extracted from black bean seeds (Phaseolus vulgaris L.) have a global antioxidant power of 48.38 ± 5 mg FeII/100g (albumin fraction) and 7.26 ± 4 mg Fell/100g (globulin fraction). The value obtained from the albumin fraction was significantly lower (p

Lipase from Candida rugosa was immobilized on MgO·SiO2 hybrid grafted with amine, thiol, cyano, phenyl, epoxy and carbonyl groups. The products were analyzed using Fourier transform infrared spectroscopy, nuclear magnetic resonance, low-temperature N2 sorption and elemental analysis. Additionally, the degree of coverage of the oxide material surface with different functional groups and the number of surface functional groups were estimated. The Bradford method was used to determine the quantity of immobilized enzyme. The largest quantity of enzyme (25-28 mg/g) was immobilized on the hybrid functionalized with amine and carbonyl groups. On the basis of hydrolysis reaction of p-nitrophenyl palmitate to p-nitrophenol, it was determined how the catalytic activity of the obtained biocatalysts is affected by pH, temperature, storage time, and repeated reaction cycles. The best results for catalytic activity were obtained for the lipase immobilized on MgO∙SiO2 hybrids with amine and carbonyl groups. The biocatalytic system demonstrated activity above 40% in the pH range 4–10 and in the temperature range 30–70 °C. Lipase immobilized on the MgO∙SiO2 systems with amine and epoxy groups retains, respectively, around 80% and 60% of its initial activity after 30 days of storage, and approximately 60–70% after 10 reaction cycles.

A quantitative protein assay and label-free immunodetection based on dye-doped liquid crystal (DDLC) was developed in this study. The black dye incorporated as the dopant in the DDLC mixture features a high dichroic ratio and wide visible-light absorption band so that the level of disturbance in DDLC orientation in the presence of biomolecules can be readily observed with the naked eye. Due to the absorption and anisotropic features of DDLC, transmission spectrometry was exploited to establish a quantitative method for DDLC-based biosensing. Our results indicate that the transmittance of DDLC decreased with increasing concentration of bovine serum albumin (BSA), a common protein standard, and the detection sensitivity can be improved by using linearly polarized light during spectral measurement. A linear correlation between the spectral parameters of DDLC and BSA concentration was thus derived to facilitate protein quantitation. As a demonstration of clinical relevance, a label-free DDLC-based immunoassay for the cancer biomarker CA125 was manifested by reacting CA125 with immobilized anti-CA125 antibody. The transmittance of DDLC decreased with increasing concentration of CA125, but when a nonspecific protein such as BSA was reacted with the anti-CA125 antibody, the transmission spectrum was similar to that in the absence of CA125, suggesting that immunodetection via the DDLC-based biosensing platform was specific. It is evident from this study that with its unique dichroic and optical properties, DDLC is a potential biosensing material with capabilities of both qualitative detection as required in rapid screening as well as quantitative bioassay as in cancer screening.

In this work, two kinds of monolith-based immobilized enzyme microreactors (IMER) were compared in terms of monolith properties and digestion performance. Two monoliths were prepared involving poly (tetraethoxysiLane-co-3-aminopropyltriethoxysilane) (poly (TEOS-co-APTES)) monolith and poly (N-acryloxysuccinimide-co-acrylamide-co-ethylene dimethacrylate) (poly (NAS-co-AA-co-EDMA)) monolith for covalent trypsin immobilization. A higher chromatographic permeability constant 66.7 was obtained from the poly (TEOS-co-APTES) monolith in comparison with only 4.37 for poly (NAS-co-AA-co-EDMA) monolith, and the porous property of monolith measured by N2 adsorption/desorption and mercury intrusion porosimetry also indicated that poly (TEOS-co-APTES) monolith has more pores than poly (NAS-co-AA-co-EDMA) monolith. On the other hand, the digestion results demonstrated that the more efficient IMER was obtained by immobilizing trypsin on the poly (TEOS-co-APTES) monolith even if a less amount of immobilized trypsin determined by the Bradford method than poly (NAS-co-AA-co-EDMA) monolith. Therefore, the capacity of trypsin immobilization of monolith was not the exclusive factor to determine the digestion performance of IMER. Physical properties-penetrability and pore distribution of monolith showed a crucial effect on the digestion performance of IMER.

Sensitive and specific detection of protein is of great significance for early diagnosis and prognosis of many diseases. However, great challenges remain unsolved including relative low sensitivity, high cost, long testing time, complicated instrument and laborious operation. To improve the performance of protein detection methods, development of fine reaction interface for recognition and signal amplification is of great importance. In this work, we construct a novel mode of DNA assembly at electrode interface based on a tripodal surface anchor and an electrochemical aptasensor for protein assay is developed. The orientation of the immobilized DNA is optimized, which promises the efficiency of protein recognition. In addition, hybridization chain reaction is employed for further signal amplification. Therefore, this detection method shows high sensitivity with excellent specificity. The strategy can be universally applicable by simply modifying the sequences of used DNA probes.

Here, we report a simple yet effective surface-modification approach to imparting hydrophobic surfaces with superhydrophilicity using ultralow fouling/functionalizable carboxybetaine (CB) copolymers via a dip-coating technique. A new series of CB random copolymers with varying amphiphilicities were synthesized and coated on hydrophobic polypropylene (PP) and polystyrene (PS) surfaces. The nonfouling capability of each coating was screened by an enzyme-linked immunosorbent assay (ELISA) and further comprehensively assessed against 100% human serum by a Micro BCA protein assay kit. The random copolymer containing ∼30 mol % CB units showed superhydrophilicity with the highest air contact angle of more than 165° in DI water and the best nonfouling capability against 100% human blood serum. Surfaces of a 96-well plate coated with the optimal CB random copolymer had a significantly better nonfouling capability than those of a commercial 96-well plate with an ultralow attachment surface. The adhesion of mouse embryonic fibroblast cells (NIH3T3) was completely inhibited on surfaces coated with CB random copolymers. Furthermore, the optimal nonfouling CB copolymer surface was functionalized with an antigen via covalent bonding where its specific interactions with its antibody were verified. Thus, this CB random copolymer is capable of imparting both ultralow fouling and functionalizable capabilities to hydrophobic surfaces for blood-contacting devices.

The Cu/Co-α-NiMoO4 nanorods were fabricated by microwave hydrothermal process and characterized by various techniques. According to XRD/HRTEM/SAED/EDS analysis, the dopant ions were existed in the lattice of α-NiMoO4.The Cu/Co-α-NiMoO4 samples have capacity to inactivate the multidrug resistant Pseudomonas aeruginosa in visible light within 180 min. Also, they revealed the excellent stability with reusability. The scavengers test indicates that H2O2/OH /O2 −/h+ play a crucial work for pathogen inactivation. Also, NBT assay suggested the enhancement of intracellular O2 − production in Cu (33 folds) and Co doped (29 folds) α-NiMoO4. The FESEM analysis revealed dead bacteria cells by attack of active species on cell wall. Also, DNA fragmentation (gel electrophoresis) and protein degradation (Bradford assay) confirmed the inactivation of bacteria by active species. The evidence of superior biofilm penetration ability (69%) by doped photocatalyst was exhibited by well plate based crystal violet assay and TEM/EDS mapping. The band gap reduction, surface plasmon resonance effect, oxygen vacancy, electron-hole trapping by Cu2+/Co2+/Co3+, formation of intermediate energy band, and efficient separation of electron-hole pair were contributed for enhancement of the pathogen inactivation in visible light by doping of Cu and Co in α-NiMoO4. Also, Cu doped photocatalyst has slightly more pathogen inactivation efficacy than Co doped photocatalyst. The toxicity of photocatalyst was evaluated by using RAW 264.7 cells via MTT assay and the result revealed the toxicity. In conclusion, the Cu/Co-α-NiMoO4 photocatalyst can be applied for efficient inactivation of multidrug resistant pathogen in biological waste water treatment.

Human growth hormone (hGH) is synthesized by the anterior pituitary gland and promotes cell proliferation and growth. This protein has been authorized to use for the treatment of various human growth disorders and until recently, substantial efforts have been made to upgrade the previous introduced strategies. Due to the small size of hGH and absence of posttranslational modifications, Escherichia coli is the ideal host for hGH production. In the present work, we employed a previously established cleavable self-aggregating tag (cSAT) for the expression and purification of hGH in BL21 (DE3) strain of E. coli to evaluate its effectiveness. The tag is composed of a self-cleavable intein and a self-assembling peptide ELK16 (Mxe GyrA intein-ELK16). At the first step, an active insoluble aggregate of the recombinant hGH-Mxe GyrA intein-ELK16 fusion protein was expressed through an efficient T7 based expression system and then purified with a simple centrifugation. The thiol reagent dithiothreitol (DTT) was then added to induce the intein-mediated cleavage and as a result the peptide released into the soluble fraction. Afterward, the hGH production was determined by SDS-PAGE and then the final concentration of released hGH was measured by the Bradford assay (4.96 mg/ml). Furthermore, the bioactivity of purified hGHs was confirmed by calculating its growth-stimulating effect using Nb2 cell line proliferation assay. All in all, the current study offers a straightforward and fast procedure for the production of pure and bioactive hGH in E. coli.

The target-triggered DNA assembling probe is presented for highly selective protein detection. Target-triggered DNA assembling is used in an amplification strategy based on affinity binding for identification and determination of proteins in general. Specifically, it was applied to the platelet derived growth factor-BB (PDGF-BB). A hairpin DNA (H-DNA) probe was designed containing (a) an aptamer domain for protein recognition and (b) a blocked DNAzyme domain for DNAzyme cleavage. An assistant DNA (A-DNA) probe containing aptamer and complementary domains was also employed to recognize protein and to induce DNA assembly. Once H-DNA and A-DNA recognize the same protein, H-DNA and A-DNA are in close proximity to each other. This induces DNA assembling for protein-triggered complex (Protein-Complex) with free DNAzyme domains. The free DNAzymes trigger the circular cleavage of molecular beacons for amplified signals. The assay is performed by fluorometry at an excitation wavelength of 980 nm and by collecting fluorescence at 545 nm. The platelet derived growth factor-BB (PDGF-BB) was accurately identified and selectively determined by this assay with a 22 pM detection limit (using the 3σ criterion). The responses for PDGF-BB is nearly 6-fold higher than for PDGF-AB, and 16-fold higher than PDGF-AA. This upconversion assay avoids any interference by the autofluorescence of biological fluids. Graphical abstractSchematic representation of the principle of the target-triggered DNA assembling probes mediated amplification strategy based on affinity binding for PDGF-BB. The UCNP probe is used for the quantitation of PDGF-BB with high selectivity.

The development of J93-463-1-16-10 cell culture by sequential adaptation method in serum-free media of Hybridoma-SFM medium and CD Hybridoma medium to produce the monoclonal antibody specific to Japanese encephalitis virus was shown that the viabilities of J93-463-1-16-10 were more than 70%. Then the supernatant from the cell culture was purified after precipitation with ammonium sulfate until the final concentration was 50% in order to separate the IgG. After that, the protein concentrations were measured by using the Bradford assay. Next, the proteins were separated, which made each protein purer with SDS-PAGE method in order to find the molecular weights of the separated proteins. The putative IgG and its heavy chains and light chains were divided in two sizes which were approximate 23-25 kDa and 50-53 kDa respectively. Later, the type of immunoglobulin will be identified by the Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) method and its antibody characteristic to Japanese encephalitis virus which will be done further.In the future, a monoclonal antibody in this research could be used to study the expression of the envelope protein of Japanese encephalitis virus in green cos and red cos, and to develop the vaccine for Japanese encephalitis virus in swine.Keywords: Japanese encephalitis virus, J93-463-1-16-10, serum-free media, MALDI-TOF

The study about the activity of the enzyme amylase from amylolytic bacterial isolates from wet sagoo starch and its traditional provision environment had been done in Jayapura. The purposes of this study were to determine the activity of amylase enzyme and to identify the bacteria isolated from wet sagoo starch and its processing environment in Jayapura district. The method used was an experimental laboratorium in which isolation of amylolytic bacteria was performed by using nutrient agar medium with 1% soluble starch on spreed pour plate method. The enzyme activity was detected with 0.2% iodine in 2% potassium iodide which were able to form a clear zone. The protein content of the crude enzyme extract was determined by the Bradford method using bovine serum albumin (BSA). Amylase enzyme activity was determined by the formula: DUN/ml = [(R0-R1)/R0] [dilution factor] DUN/ml (dextrinizing units per ml). The results showed that there were 15 isolates amylolytic bacteria. Four (4) bacterial isolates have amylolytic power of more than 30 mm. The amilase activity of amylolytic bacterial of all isolates were quite high: which were 35 577, 18 903, 32 106 and 46 600 U/mg for SU4, SU13, SU23 and SU40 respectively. The identification of isolates indicated that the three isolates are members of the Bacillus cereus ATCC 14 579 types with a similarity value of 71.70% to 81.10%, and one isolate is Bacillus subtilis ATCC 6501 members with a similarity value of 94.30%. Keywords: Amylolytic bacteria, amylase activity, characterization, sago flour.

Small molecules are not only intermediates of metabolism, but also play important roles in signaling and in controlling cellular metabolism, growth, and development. Although a few systematic studies have been conducted, the true extent of protein-small molecule interactions in biological systems remains unknown. PROtein-metabolite interactions using size separation (PROMIS) is a method for studying protein-small molecule interactions in a non-targeted, proteome- and metabolome-wide manner. This approach uses size-exclusion chromatography followed by proteomics and metabolomics liquid chromatography-mass spectrometry analysis of the collected fractions. Assuming that small molecules bound to proteins would co-fractionate together, we found numerous small molecules co-eluting with proteins, strongly suggesting the formation of stable complexes. Using PROMIS, we identified known small molecule-protein complexes, such as between enzymes and cofactors, and also found novel interactions. © 2019 The Authors. Basic Protocol 1: Preparation of native cell lysate from plant material Support Protocol: Bradford assay to determine protein concentration Basic Protocol 2: Separation of molecular complexes using size-exclusion chromatography Basic Protocol 3: Simultaneous extraction of proteins and metabolites using single-step extraction protocol Basic Protocol 4: Metabolomics analysis Basic Protocol 5: Proteomics analysis.

International audience; Determination of protein concentrations prior to (sodium dodecyl sulfate) SDS electrophoresis is made difficult by the simultaneous presence of SDS and reducers in the buffers used for protein extraction. Reducers interfere with the copper-based assays, while SDS interferes with the dye-binding assays. The combined use of cyclodextrins with a commercial Bradford reagent concentrate, described in this chapter, allows to determine protein concentrations in a Laemmli-type buffer, containing both SDS and reducers, in a single step (without any precipitation) with a simple spectrophotometric assay. The use of various cyclodextrins brings compatibility not only with SDS but also with other nonionic and ionic detergents such as sodium deoxycholate or detergents of the Triton type.

Gold nanoparticles (AuNPs) functionalized with antibodies have the potential to improve biosensing technology because of the unique optical properties of AuNPs and the specificity of antibody-antigen interactions. Critical to the development and optimization of these AuNP-enabled sensing technologies is the immobilization of the antibody onto the AuNP. The development of novel immobilization strategies that optimize antibody loading and orientation in an effort to enhance antibody activity, and therefore assay performance, has been the focus of many recent studies. However, few analytical methods exist to accurately quantify the activity of conjugated antibodies and reliably compare different immobilization strategies. Herein, we describe an enzyme-mediated assay to quantify the fraction of the immobilized antibodies that is accessible for antigen binding. Anti-horseradish peroxidase (anti-HRP) antibody is mixed with AuNPs to allow for conjugation, and the unbound, excess antibody is quantified with a modified Bradford assay to determine antibody loading onto AuNPs. The conjugates are then mixed with excess HRP to saturate all accessible binding sites, and bound HRP is quantified based on enzymatic reaction rate. This analytical scheme was used to compare two common immobilization strategies, nonspecific adsorption and protein A-mediated immobilization. We found that the antibody surface coverage is greater for direct adsorption than protein A-mediated binding; however, 23 ± 6% of the directly adsorbed antibodies were active, whereas 91 ± 19% of the antibodies bound through protein A were active. In addition to establishing this method as quantitatively precise and accurate, our results emphasize the need to quantify both antibody loading and antibody activity upon conjugation to gain greater insight into differences in immobilization chemistries and identify optimum protein conjugation strategies to maximize immunoassay performance.

DOI: 10.26650/EurJBiol.2018.0004Objective: Chitosan is a natural polysaccharide derived from chitin by deacetylation. It is a non-toxic, biocompatible and biodegradable polymer, and has attracted considerable interest in a wide range of pharmaceutical applications including drug and vaccine delivery. The immune-stimulating activity of chitosan microparticles such as the activation of macrophages and polymorphonuclear leukocytes has been reported. In this work, we have investigated the releasing properties of influenza virus antigens from the chitosan microparticles and beads.Materials and Methods: Chitosan microparticles and beads were prepared by coacervation and ionotropic gelation method, respectively. The microparticles and beads were loaded with the viral antigens by passive adsorption and/or entrapment into the microparticles. The titration of the viruses was defined by haemagglutination assay or by quantitation of viral proteins using the Bradford method.Results: The results showed that the loading efficiency and the loading capacity of chitosan microparticles/beads with the viral antigens and the releasing profiles of the antigens from the particles changed depending on the type of chitosan, the pH of the loading buffer and the methods used to prepare the particles. The influenza viral antigens, passively adsorbed onto microparticles/beads, were released within 2 hours to 5 days. In contrast, the viral antigens entrapped into the chitosan microparticles were released more slowly and continued for up to 30 days.Conclusion: It was concluded from the viral antigen releasing profiles of chitosan particles that the viral antigens entrapped into the microparticles are more suitable for in vivo applications as a potential mucosal vaccine., DOI:10.26650/EurJBiol.2018.0004Objective: Chitosan is a naturalpolysaccharide derived from chitin by deacetylation. It is a non-toxic,biocompatible and biodegradable polymer, and has attracted considerableinterest in a wide range of pharmaceutical applications including drug andvaccine delivery. The immune-stimulating activity of chitosan microparticlessuch as the activation of macrophages and polymorphonuclear leukocytes has beenreported. In this work, we have investigated the releasing properties ofinfluenza virus antigens from the chitosan microparticles and beads.Materials and Methods: Chitosanmicroparticles and beads were prepared by coacervation and ionotropic gelationmethod, respectively. The microparticles and beads were loaded with the viralantigens by passive adsorption and/or entrapment into the microparticles. Thetitration of the viruses was defined by haemagglutination assay or byquantitation of viral proteins using the Bradford method. Results: The results showed that theloading efficiency and the loading capacity of chitosan microparticles/beadswith the viral antigens and the releasing profiles of the antigens from theparticles changed depending on the type of chitosan, the pH of the loadingbuffer and the methods used to prepare the particles. The influenza viralantigens, passively adsorbed onto microparticles/beads, were released within 2hours to 5 days. In contrast, the viral antigens entrapped into the chitosanmicroparticles were released more slowly and continued for up to 30 days.Conclusion: It was concluded from the viralantigen releasing profiles of chitosan particles that the viral antigensentrapped into the microparticles are more suitable for in vivo applications asa potential mucosal vaccine.

Background: Proteomics is considered a new era in neurophysiological/ neuropathological research including brain tumors. Gliomas which are derived from glial cells are the most common type of brain tumor in humans. Methods: In the present study the total protein content of healthy cells of the brain and brain tumor cells was extracted, purified and quantified by Bradford assay. Two-dimensional electrophoresis were used for protein separation followed by statistical analysis. Primary protein detection was performed based on the differences in isoelectric pH, molecular weight of proteins and protein data banks, which was further confirmed by MALDI-TOF/TOF mass spectrometry (MS). Results: Our results showed elevated levels of beta-actin protein expression in glioma brain tumor cells. It is important to know when a cell is transformed and when it becomes malignant. Here we evaluated the beta-actin expression in malignant cells. Conclusion: Since structural changes are highly involved in tumor cell malignancy, beta-actin elevations can contribute in glioma tumor cell invasiveness.

Herein, a general protein conversion and analysis strategy was developed for homogeneous, label-free, and sensitive protein detection, on the basis of the affinity binding-induced Hg2+ release for protein conversion, and the succeeding Hg2+ doping-induced ZnSe quantum dot (QD) photoluminescence for signal readout. Two DNA motifs were designed, each of which was conjugated with a protein-specific recognition ligand. The mercury ions were initially introduced into one DNA motif by T-Hg2+-T interaction. The Hg2+ releasing was then accomplished after protein recognition-initiated strand exchange reaction between two DNA motifs. Then, the simultaneous incorporation of the released Hg2+ into ZnSe QD resulted in a doping-dependent fluorescence emission at 560 nm correlated with protein analysis. The protein assay is outperformed only by a simple one-step mixing operation but no separation or washing steps. Also, the use of doped QD as a fluorogenic reporter can avoid the fluorophore and/or quencher labeling, and...

Three-dimensional (3D) printing is a novel process used to manufacture bone tissue engineered scaffolds. This process allows for easy control of the architecture at the micro structure. However, the scaffold properties are typically limited in terms of cellular activity at the scaffold surface due to the printed materials properties. In this study, we developed a polycaprolactone (PCL) blended with polyethylene glycol (PEG) 3D printed scaffold using a rapid prototyping system. The manufactured scaffolds were then washed out to form small pores on the surface in order to improve the scaffolds hydrophilicity. We analyzed the resultant material by using Scanning Electron Microscopy (SEM), water absorption, water contact angle, in vitro WST-1, and the Bradford assay. Additionally, cells incubated on the fabricated scaffolds were visualized by Confocal Laser Scanning Microscopy (CLSM). The developed scaffolds exhibited small pores on the strand surface which served to increase hydrophilicity as well as improve cellular proliferation and increase total protein content. Our findings suggest that the presence of small pores on the scaffolds can be used as an effective tool for improving implant cellular interaction. This research indicates that these modified scaffolds can be considered useful for bone tissue engineering applications to improve human health.

OBJECTIVE To use proteomic analysis to determine the protein constituents of synovial fluid samples from the stifle joints of dogs with and without osteoarthritis secondary to cranial cruciate ligament rupture (CCLR). ANIMALS 12 dogs with clinically normal stifle joints (controls) and 16 dogs with osteoarthritis secondary to CCLR. PROCEDURES A synovial fluid sample was obtained from all dogs. Synovial fluid total protein concentration was determined by the Bradford assay. Proteins were separated by use of a 1-D SDS-PAGE to detect protein bands that differed between dogs with and without osteoarthritis. Those protein bands then underwent trypsin digestion and were analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry, the results of which were compared with a curated protein sequence database for protein identification. One of the most frequently identified proteins, apoprotein (apo) A-I, was then quantified in all synovial fluid samples by use of a competitive-inhibition ELISA. Results were compared between dogs with and without osteoarthritis. RESULTS Median synovial fluid total protein and apo A-I concentrations for dogs with osteoarthritis were significantly greater than those for control dogs. The most abundant proteins identified in the synovial fluid were albumin and apo A-I. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that quantification of synovial fluid total protein and apo A-I concentrations might facilitate diagnosis of osteoarthritis secondary to CCLR in dogs. Further research and validation of synovial fluid apo A-I concentration as a biomarker for osteoarthritis in dogs are necessary before it can be recommended for clinical use.

The aim of the current investigation was to determine the antibacterial and antibiofilm potential of MgO nanoparticles (NPs) against antibiotic-resistant clinical strains of bacteria. MgO NPs were synthesized by a wet chemical method and further characterized by scanning electron microscopy and energy dispersive X-ray. Antibacterial activity was determined by broth microdilution and agar diffusion methods. The Bradford method was used to assess cellular protein leakage as a result of loss of membrane integrity. Microtiter plate assay following crystal violet staining was employed to determine the effect of MgO NPs on biofilm formation and removal of established biofilms. MIC values ranged between 125 and 500 μg/mL. Moreover, treatment with MgO NPs accelerated rate of membrane disruption, measured as a function of leakage of cellular proteins. Leakage of cellular protein content was greater among gram-negative bacteria. Cell adherence assay indicated 25.3-49.8% inhibition of bacterial attachment to plastic surfaces. According to a static biofilm method, MgO NPs reduced biofilm formation potential from 31% to 82.9% in a time-dependent manner. Moreover, NPs also significantly reduced the biomass of 48, 72, 96 and 120 hr old biofilms (P < 0.05). Cytotoxicity experiments using a neutral red assay revealed that MgO NPs are non-toxic to HeLa cells at concentrations of 15-120 μg/mL. These data provide in vitro scientific evidence that MgO NPs are effective and safe antibiofilm agents that inhibit adhesion, biofilm formation and removal of established biofilms of multidrug-resistant bacteria.

There is a need to adapt cell bioassays to 384-well and 1536-well formats instead of the traditional 96-well format as high-throughput screening (HTS) demands increase. However, the sensitivity and performance of the bioassay must be re-verified in these higher micro-well plates, and verification of cell health must also be HT (high-throughput). We have adapted two commonly used human breast luciferase transactivation cell bioassays, the recently re-named estrogen agonist/antagonist screening VM7Luc4E2 cell bioassay (previously designated BG1Luc4E2) and the androgen/glucocorticoid screening MDA-kb2 cell bioassay, to 384-well formats for HTS of endocrine-active substances (EASs). This cost-saving adaptation includes a fast, accurate, and easy measurement of protein amount in each well via the fluorescamine assay with which to normalize luciferase activity of cell lysates without requiring any transfer of the cell lysates. Here we demonstrate that by accounting for protein amount in the cell lysates, antagonistic agents can easily be distinguished from cytotoxic agents in the MDA-kb2 and VM7Luc4E2 cell bioassays. Additionally, we demonstrate via the fluorescamine assay improved interpretation of luciferase activity in wells along the edge of the plate (the so-called "edge effect"), thereby increasing usable wells to the entire plate, not just interior wells.

Summary Being attractive for insects, non-genetically modified rapeseed is valuable for maintaining environmental biodiversity. Primarily, the rapeseed is an important industrial crop which is used for production of vegetable oil. Oil extraction from rapeseeds results in the generation of substantial amounts of rapeseed meal which is used either as a protein rich feed additive or as a source for preparation of protein containing ingredients for food industry. Both applications require frequent evaluation of protein content. Although Kjeldahl method is considered standard, it is not appropriate for routine evaluation of protein content in protein extracts. The aim of the study was to evaluate suitability of biuret and Bradford methods for protein quantification in rapeseed meal extracts. After consecutive triple extraction of proteins with water, 5% NaCl, 70% ethyl alcohol and 0.1 N NaOH, protein evaluation of each albumin, globulin, prolamin and glutelin extraction aliquot demonstrated overall lower protein content by Bradford method compared to biuret method. The most pronounced differences in protein content were observed with prolamin fraction where three fold higher protein concentrations in each extraction aliquot was observed when biuret method was applied for the evaluation. Comparative quantification of the total protein of each of the four fractions followed a similar trend of lower protein content evaluation by Bradford method. Overall results indicated biuret method as more suitable for protein quantification in rapeseed meal extracts which was confirmed by comparison with data obtained by Kjeldahl method.

To quantify and compare the amounts of surfactant proteins SP-A, SP-B, SP-C and SP-D in the tear fluid collected from patients with dry eye syndrome and from individuals with a healthy ocular surface.Schirmer strips were used to collect tear fluid from both eyes of 241 volunteers (99 men, 142 women; age range: 18-87 years). Dry eye syndrome was diagnosed by ophthalmologists in 125 patients, whereas the healthy control group comprised 116 individuals. The total protein concentration was determined via Bradford assay. The relative concentration of surfactant proteins SP-A through -D was measured by enzyme-linked immuno-sorbent assay (ELISA).The mean relative concentrations of SP-A, SP-C and SP-D were significantly higher in the dry eye group as compared to the healthy controls (p0.05, one-way ANOVA). SP-B was also detected at a higher concentration in the dry eye group, but the difference to the control group was not statistically significant.The upregulation of SP-A and SP-D in the dry eye group is probably related to these proteins' known antimicrobial and immunomodulatory effects at the ocular surface. It may represent a pathophysiological response to the inflammatory condition of the ocular surface in dry eye. The upregulation of SP-B and SP-C may represent an effort of the lacrimal system to reduce surface tension and thus to counteract the increased tendency of the tear film to tear in dry eye.

Dye liquid crystals (DLCs) have been extensively investigated in the field of liquid crystal display, but their potential in biosensing has not been explored. To provide insights into the potential of DLCs as a means of biosensing material, a DLC-based biosensor was developed for protein detection and quantitation. Unlike dye-doped LCs, the DLC used in this study contains an azobenzene LC molecule chemically modified with two azo groups as chromophores, enabling the DLC to exhibit unique optical anisotropy and dichroic absorption at 470 nm, which were exploited in the quantitation of bovine serum albumin (BSA). The change in orientation of DLC molecules from the homeotropic to a disrupted state by BSA immobilized on the aligning-layer surface was evaluated in terms of the relative loss in transmittance, expressed by the standard parameter S%, due to the absorption as well as scattering of light. The DLC-based protein assay was capable of determining the BSA concentration, with which S% varied linearly between 1 μg/ml and 7.5 μg/ml. When the DLC molecules were reoriented from a BSA-disrupted state to the homeotropic alignment by an externally applied electric field, a linear correlation between S% and BSA concentration could be also derived from the voltage–transmittance plots. The unique electro-optical properties of the intrinsically dichroic DLC are promising toward the development of a simple and reliable sensing platform for the quantitative analysis of biomolecules.

A new modular nanoswitch was described for versatile, rapid (within 1 h), homogeneous, and sensitive protein detection. The system employs two hairpins (HP1 and HP2) that can be reciprocally recognized through the apical loop-loop interaction. HP2 possesses a conformation-switching stem-loop structure, with appended single-stranded tails on each end, which can hybridize with the recognition-element-conjugated DNA strands to construct a protein-responsive HP2 scaffold. It works according to a simple mix-and-detect assay format, with the first formation of a kissing complex between HP1 and HP2 scaffolds for fluorescence quenching, and then cascade propagation from steric strain through protein binding to the dissociation of the kissing complex for fluorescence recovery. The detection universality of such a modular nanoswitch was demonstrated by using three multivalent proteins, including anti-digoxigenin (Anti-Dig) antibody, streptavidin, and thrombin, with detection limits of 0.33, 0.17, and 0.5 nm, respectively.

Background and Objective: Mannanase is a mannan-degrading enzyme that can be produced by microorganisms such as bacteria. Mannanase is widely used in the animal feed industry with the aim of improving the quality of feed ingredients containing high levels of mannan, such as palm kernel meal. This study aimed to isolate, characterize and produce mannanase from bacteria found in the hindgut of termites. Materials and Methods: Bacteria were isolated by culturing on solid media containing 0.5% locust bean gum for 24 h at 30°C. The mannanolytic index was then measured by the addition of 2% congo red, which was then washed with 1 M NaCl. Mannanase activity was measured by the dinitrosalicylic acid method, while the protein content was measured by the Bradford method. Results: A total of 8 isolates were obtained. The 3 isolates with the highest mannanolytic indices were selected (isolates V9, AZ and AD). Isolate V9 exhibited the highest mannanase activity of 29.50 U mL–1 after 88 h of incubation with a specific activity of 110.32 U mg–1. The activity of the mannanase was optimal at pH 8 and at an incubation temperature of 50°C. Based on 16S-DNA analysis, isolate V9 was identified as Bacillus cereus. Conclusion: Bacillus cereus, which is isolated from the hindgut of termites, has fairly high mannanase activity and has the potential to hydrolyse mannan in feed ingredients such as palm kernel meal.

Detection of molecular biomarkers with high specificity and sensitivity from biological samples requires both sophisticated sample preparation and subsequent analysis. These tasks are often carried out on separate platforms which increases required sample volumes and the risk of errors, sample loss, and contamination. Here, we present an optofluidic platform which combines an optical detection section with single nucleic acid strand sensitivity, and a sample processing unit capable of on-chip, specific extraction and labeling of nucleic acid and protein targets in complex biological matrices. First, on-chip labeling and detection of individual lambda DNA molecules down to concentrations of 8 fM is demonstrated. Subsequently, we demonstrate the simultaneous capture, fluorescence tagging and detection of both Zika specific nucleic acid and NS-1 protein targets in both buffer and human serum. We show that the dual DNA and protein assay allows for successful differentiation and diagnosis of Zika against cross-reacting species like dengue.

Exosome-like small-extracellular vesicles (sEVs) are extracellular vesicles that act in intercellular communication and are involved in several biologic and pathologic processes. While sEVs increase the stability of their cargo molecules, there is still a need for standardization of sampling and isolation of these microvesicles. We aimed to determine the best sampling method for isolation of sEVs from peripheral blood from reproductive-aged women. Material and Methods: We included samples of plasma from our biobank collected in 2014 by venipuncture in heparin tubes and stored at -80°C. We also included blood samples collected in heparin tubes and Ethylenediamine tetraacetic acid (EDTA) tubes and stored at -80°C for one to two weeks prior processing. All blood samples were collected from the same nine reproductive-aged female volunteers. sEVs were isolated from plasma by ultracentrifugation and filtration and indirectly quantified using Pierce BCA Protein Assay kit. Transmission electron microscopy (TEM) and Nano Tracking Analysis (NTA) were performed to confirm the isolation of sEVs. Results and Discussion: TEM and NTA confirmed the isolation of sEVs. Protein concentration of short-time stored heparin samples was not statistically different from long-time stored heparin samples (1847.2 ± 651.4 vs. 2363.2 ± 1025.1, p = 0.14). There was no difference between heparin and EDTA plasma samples recently collected (2363.2 ± 1025.1 vs. 2044.8 ± 653.2, p = 0.44). In conclusion, blood samples may be collected using heparin or EDTA for isolation of sEVs. Long-time stored plasma samples maintain sEVs integrity and may be used, especially in comparative studies.

In this work, we observed the effect of grape juice (% concentrated juice/% concentrated camel milk: GJ20/80, GJ50/50) and pomegranate juice (PJ20/80, PJ40/60) fortification on camel milk (CM) protein solubility and digestibility. Proteins were dissolved in sodium phosphate buffer to 50 mg/ml and defatted prior Bradford assay of protein concentration, then analyzed by Size Exclusion-Ultra High-Performance Liquid chromatography (SE-UHPLC). The CM protein aggregation and their stability were further monitored at different pH 2.0, 4.0, and 7.5 via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Freeze dried CM (FDCM) was the reference sample and our results showed that GJ50/50 and PJ40/60 with the highest fruit juice ratio had the lowest protein content in the supernatant, hence the decreased solubility. SE-UHPLC of supernatants showed a slight decrease in retention times of 11 kDa and 62 kDa proteins for GJ50/50 and PJ40/60 suggesting a possibility of adduct formation due to fortification leading to higher molecular weight. The simulated static in vitro gastrointestinal digestion of samples revealed that most soluble proteins were readily digested by pepsin, trypsin and chymotrypsin enzymes leading to small peptides. However, the SDS PAGE of pellets showed the partial resistance of casein and α-lactalbumin against peptic digestion. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4747]

Glomalin-related soil protein (GRSP), a ubiquitous microbial product, plays a crucial role in particle aggregation and metal adsorption, but the underlying mechanisms remain unknown. Here, GRSP fraction was extracted from estuarine ecosystems and systematically characterized to elucidate the aggregation mechanisms and its impact on coastal environment improvement. We found that GRSP fraction (gravimetric mass of extracted GRSP, 5.1–24.3 mg g−1) was a globally relevant novel bioflocculant and that protein (linked to Bradford protein assay, 1.64–4.37 mg g−1) was the active flocculant constituent. The zeta potential, FTIR, XPS, and 13C NMR analyses identified its key constituents and structure, and revealed that the charge neutralization and bridging were GRSP fraction aggregation mechanisms. Thermogravimetric-infrared spectrometry analysis showed that GRSP fraction was highly thermostable, and the main volatile pyrolysis products included H2O, CO2, CO, and CH4. The SEM-EDX and XPS Fe valence spectroscopy suggested that GRSP fraction contained rich Fe (11.91 ± 0.48%) and could form Fe-rich flocs with particles. We also found that GRSP fraction has a high adsorption capacity (76–95%) for Cu, Zn, Pb and Cd, and its flocculation properties provide new insights into metal adsorption. The analysis of particle aggregation mechanism and its metal adsorption capacity is of great significance to elucidate the role of GRSP fraction in coastal environment improvement.

Deep-ultraviolet laser ablation with a pulsed 193 nm ArF excimer laser was used to remove localized regions from tissue sections from which proteins were extracted for spatially resolved proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). The ability to capture intact proteins by ablation at 193 nm wavelength was verified by matrix-assisted laser desorption ionization (MALDI) of the protein standard bovine serum albumin (BSA), which showed that BSA was ablated and captured without fragmentation. A Bradford assay of the ablated and captured proteins indicated 90% efficiency for transfer of the intact protein at a laser fluence of 3 kJ/m2. Rat brain tissue sections mounted on quartz microscope slides and ablated in transmission mode yielded 2 μg protein per mm2 as quantified by the Bradford assay. Tissue areas ranging from 0.06 mm2 to 1 mm2 were ablated and the ejected material was collected for proteomic analysis. Extracted proteins were digested and the resulting peptides were analyzed by LC-MS/MS. The proteins extracted from the ablated areas were identified and the average number of identified proteins ranged from 85 in the 0.06 mm2 area to 2400 in the 1 mm2 area of a 50 μm thick tissue . In comparison to infrared laser ablation of equivalent sampled areas, both the protein mass and number of proteins identified using DUV laser ablation sampling were approximately four times larger.

Anacystis nidulans is a small, rod-shaped, unicellular, colonial, obligatory phototrophic microalga isolated from Sambhar Lake, Jaipur (Rajasthan). To find out the best inorganic composition cultures were grown in five different defined inorganic medium such as Modified BG-11 medium (pH 7.31), BG-11 medium (7.1), CHU-10 (pH 7.65), Zarrouk’s medium (pH 10.2) and Kratz & Myer medium (pH 9.5) and kept at the temperature of 25 ± 2°C, illuminated with white fluorescent lamps at a light intensity of 2.5 Klux with 12:12 hours light/dark photoperiod in departmental laboratory. Protein content is determined by Bradford assay and qualitatively by SDS-PAGE. Protein expression levels were determined through densitometry. Highest protein and amino acid content were obtained in Modified BG-11 medium as compared to other medium. Two polypeptides of 54.3 and 56.2 kDa were uniquely observed, but the genotype of 35.8 kDa polypeptide was completely degraded under Modified BG-11 inorganic formulation. 35.8, 54.3, 56.2 and 61.8 kDa polypeptides were completely degraded in Zarrouk’s as well as Kratz and Myer medium. The expression of some polypeptides of 14.0, 34.1, 42.3, 45.9, 49.5 and 75.0 kDa were greatly reduced and expressed only 1mm level in Zarrouk’s and Kratz and Myer medium. Quantity of free amino acids maximum was in Modified BG-11 medium and minimum was in the Kratz and Myer Medium. Total 17amino acids were observed in the HPLC chromatogram. No detectable amount of asparagine, glutamine and tryptophan were found throughout the course of the algal life cycle

A series of polyacrylates with different n-alkyl side chain lengths (1 to 6, and 12 carbons) and a ω-methoxy terminal group (poly(ω-methoxyalkyl acrylate): PMCxA) were prepared to study their nonthrombogenicity using human platelet adhesion, micro bicinchoninic acid (micro BCA) protein assay, and enzyme-linked immunosorbent assay (ELISA) tests. In all cases, human platelet adhesion was suppressed on the PMCxA-coated substrates, and the number of human platelets adhered to the PMC3A (poly(3-methoxypropyl acrylate))-coated surface was comparable to that of commercialized nonthrombogenic coating agent poly(2-methoxyethyl acrylate) (PMEA, equal to PMC2A). The amount of protein adsorbed onto the PMCxA was investigated by micro BCA using bovine serum albumin (BSA) and human fibrinogen (hFbn), revealing that PMC3A exhibited significantly high resistance to nonspecific BSA adsorption. Additionally, the amount of hFbn adsorbed onto the PMC3A was suppressed to the same extent as PMEA. The exposure degree of platelet adhesion sites in adsorbed hFbn was evaluated using an ELISA test, and the degree on the PMCxA with three to six methylene carbons was comparable to the PMEA. The hydration water structure in the hydrated PMCxA was also characterized using differential scanning calorimetry (DSC). The amount of intermediate water, which is the hydration water molecules that moderately interact with the polymer matrix, was maximum in the PMEA with two methylene run lengths, whereas the amount decreased by increasing the number of methyelnes in the side chain. The amount of adsorbed protein increased with a decrease in the amount of intermediate water, suggesting that the protein adsorption amount is tunable by simply changing the number of methylene carbons in the side chain. The present study revealed that poly(ω-methoxyalkyl acrylate)s are useful for blood-contacting medical devices, and PMC3A is the best mode of PMCxA to apply as an antiprotein adsorption coating agent.

This study aims to find a way for the creation of polyethyleneimine-modified biofunctionalized zinc oxide (ZnO) nanoparticles, stable in phosphate buffered saline (PBS). Biofunctionalized ZnO nanoparticles are promising for bioanalitycal applications due to a combination of diverse physico-chemical ZnO properties and selectivity of biomolecules. ZnO nanoparticles were synthesized in diethylene glycol media at 150°C. Different strategies were utilized for ZnO nanoparticle modification in order to disclose the role of polyethylenimine (PEI) in stability of colloidal system. Synthesized and modified ZnO nanoparticles were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and infrared spectroscopy measurements, and stability of colloidal system was investigated using DLS data. Finally, ability of ZnO nanoparticles to attach proteins for potential analytical applications was proved using Bradford protein assay. Among different strategies of modification, ZnO nanoparticles modified by trisodium citrate, PEI and glutaraldehyde (GA) have showed the best stability in PBS while preserving low aggregation level and high positive surface charge.

In the present study, the disruptive effects of epigallocatechin-3-gallate (EGCG) and A-type dimeric epigallocatechin-3-gallate (A-type EGCG dimer) on the preformed bovine insulin amyloid fibrils were studied by several biophysical methods including thioflavin-T (ThT) fluorescence assay, 1-anilinonaphthalene-8-sulfonic (ANS) fluorescence assay, Congo red (CR) binding assay, dynamic light scattering (DLS), transmission electron microscopy (TEM), Gel electrophoresis (SDS-PAGE) and Bradford assay. Our results demonstrated that A-type EGCG dimer showed significantly more potential disaggregative effects on the bovine insulin amyloid fibrils than EGCG. A-type EGCG dimer could not only dramatically promote the disaggregation of the preformed bovine insulin amyloid fibrils, but also restructure the amyloid fibrils into amorphous aggregates. While, EGCG could only shorten and thin the fibrils, but induce no small amorphous aggregates. Our present results provided additional evidence for the more potent disaggregation effects of dimeric polyphenols than monomeric polyphenols and suggested that A-type EGCG dimer seems to have potential application as an excellent anti-amyloidogenic agent.

This study aimed to provide a novel and highly sensitive protein assay based on the biuret reaction and using chromeazurol B, a metal chelate compound. The method consists of two reagents and an automated analyzer. First, a complex of copper and protein (biuret reaction) is formed. Second, a chelating reagent containing chromeazurol B forms a three-dimensional complex of protein, copper, and chromeazurol B at neutral pH, resulting in highly sensitive coloration. The intra-assay (n = 20) variation for the three levels was 3.54 % or lower at each concentration. Each response with α, β-, and γ-globulin was 103.8 % and 104.3 %, respectively, against albumin. The molar absorption coefficient (e) of the present method was 2.5 × 105 m2/mol against human albumin, higher than that of the commercially available Lowry method (e = 8.7 × 104 m2/mol), which is based on the same principle. The correlation test for the pyrogallol method with 30 urine samples showed good performance (r = 0.961). The method described here (the Biuret-based CAB method) is a more sensitive and rapid assay than the Lowry method, and it may also be applied to biological samples because of its similar reactivity towards various proteins.

A highly selective, sensitive, and long-term stable glucose biosensor was developed, based on β-cyclodextrins (β-CDs) grafted onto magnetite/polydopamine (Fe3O4/PDA) with adsorbed glucose oxidase (GOx) from Aspergillus niger. The properties of the nanoplatform were determined by transmission electron microscopy (TEM), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), Bradford assay, electrokinetic analysis (zeta potential), non-invasive light scattering (NIBS) and measurement of the polydispersity index (PdI). The biosensor’s operating conditions were optimized, and the optimal conditions were used in tests on model and real glucose solution. Results for the biosensor showed a broad linear range (1–26 mM glucose), with sensitivity as high as 115.74 μA mM−1 cm−2 and a limit of detection (LOD) of 1.55 μM. The results show the biosensor to be an interesting potential tool for use in many areas where glucose measurement is essential. Moreover, the proposed system demonstrated long-term stability, up to 9 months. Furthermore, excellent selectivity against ascorbic acid, uric acid, L-cysteine and sugars such as saccharose, maltose and fructose was confirmed.

High nutrient loading in aquatic environment has become the main causative of harmful algae blooms (HABs) in water resources particularly pond, lake and river. HABs are mostly dominated by microalgae derived from the group of blue-green algae which are capable of releasing harmful toxins. Therefore, this study aims to investigate the inhibitory effects of thiourea derivatives on the growth of such blue-green algae. Thiourea derivatives have been proven to exhibit antifungal and antibacterial effects. However, there is still limited study had been conducted on the effect of thiourea derivatives toward blue-green algae species in recent years. In this research, a species of blue-green algae from Kenyir Lake, Terengganu, Malaysia was successfully isolated using morphological characters and molecularly identified as Synechoccus elongatus. Four new thiourea derivative compounds were also successfully synthesised. The compounds were designed with variation on different R-substitution group and characterised using Nuclear Magnetic Resonance (NMR) to confirm their molecular structure. Those compounds were characterised as 1-Benzyl-3-(3,5-dimethoxy-benzoyl)-thiourea (C1), 1-(3-Chloro-benzyl)-3-(3,5-dimethoxy-benzoyl)-thiourea (C2), 1-(3,5-Dimethoxy-benzoyl)-3-(3-methyl-benzyl)-thiourea (C3) and 1-(3,5-Dimethoxy-benzoyl)-3-(3-trifluoromethyl-benzyl)-thiourea (C4). For the inhibition assessment, S. elongatus were treated with C1–C4 for 5 day at concentration of 2, 5, 10 and 20 μg/ml, respectively. C3 compound showed the highest inhibition percentage with 98% of inhibition after 5 days treatment. By using Bradford method, protein extraction of S. elongatus was conducted at the highest inhibition percentage. Protein concentration of treated species was observed with 3.28 μg/ml as compared to protein concentration of control with 6.48 μg/ml. This result indicated the reduction of protein content after the treatment. Protein band pattern was identified intensed after the treatment SDS PAGE was carried out. The thiourea derivatives compound proved to have successfully inhibited the growth of blue-green algae. Hence, further study should be carried out to ensure the compound can be practically utilized in the pond and in natural environment.

Objective To prepare specific IgY against A. fumigatus and verify its specificity and antifungal effect on A. fumigatus keratitis. Method Lay hens were immunized with the suspension of inactivated A. fumigatus hyphae which mixed with Freund's complete adjuvant or incomplete Freund's adjuvant. The IgY protein specific for A. fumigatus was extracted by ammonium sulfate salting-out method at the fifth to eighth week after immunization. Bradford method and indirect ELISA were used to determine the concentration and titer of IgY. To verify the inhibitory effect of specific IgY on fungal growth, 1 × 105 CFU/mL A. fumigatus hyphae suspension and specific IgY of different concentrations were mixed and cultured for 24 h, 48 h and 72 h to measure the absorbance. Using specific IgY to treat A. fumigatus keratitis in mice, we observed the cornea under a slit lamp at 24 h, 72 h, and 120 h after treatment. Clinical score was used to assess the disease severity of fungal keratitis in mice cornea. The indirect ELISA method was used to determine the titer of specific IgY stored at room temperature and 4 °C for 1, 2, 3, 4, 5, 6, 7, 8, and 9 months. Results The protein concentrations of specific IgY at the fifth, sixth, seventh and eighth weeks after immunization were 5.46 mg/mL, 5.79 mg/mL, 26.98 mg/mL, 28.71 mg/mL. The titer of the specific IgY of A. fumigatus can reach 1:10000, and the antifungal effect of the specific IgY is dose dependent within a certain range. Specific IgY treatment alleviated the severity of fungal keratitis of mice and reduced the clinical score. Moreover, there were no significant change in the titer of specific IgY after storage at room temperature for 2 months and storage at 4 °C for 6 months. Conclusion The specific IgY can be successfully prepared by ammonium sulfate salting-out method. And it has excellent stability and significant antifungal effect on A. fumigatus keratitis.

Background: Worldwide, chicken meat is widely consumed due to its low cost, high nutritional value and non-interference with religious or cultural beliefs. However, during animal husbandry chickens are exposed to many chemical substances, including tetracyclines and β-lactams, which are used to prevent and cure several infections. Some residues of these compounds may bioaccumulate and be present in chicken meat after slaughtering, promoting oxidative reactions. Methods: In order to evaluate in vitro carbonylation induced by tetracyclines and β-lactams residues, a proteomic approach was used. For this, chicken muscle was individually contaminated with tetracyclines (tetracycline, chlortetracycline, oxytetracycline, and doxycycline) and β-lactams (ampicillin, benzathine penicillin, dicloxacillin and oxacillin) at 0.5, 1.0 and 1.5 times their maximum residue level (MRL). Then, sarcoplasmic, myofibrillar and insoluble proteins were extracted and their content were measured using the Bradford method. Protein carbonylation was measured using the 2,4-Dinitrophenylhydrazine alkaline method. Results: Residues of tetracyclines and β-lactams induced in vitro carbonylation on sarcoplasmic, myofibrillar and insoluble proteins even at 0.5MRL concentrations (p). When comparing the carbonylation induced by both antibiotics no differences were found (p>0.05). Variables such as the partition coefficient (log P) and the concentration of these antibiotics showed a high correlation with the oxidative capacity of tetracyclines and β-lactams on chicken breast proteins. Conclusions: This study shows that the presence of tetracyclines and β-lactams residues at MRLs concentrations promotes in vitro carbonylation on chicken breast proteins. Our results provide important insights about the impact of antibiotics on the integrity of meat proteins intended for human consumption.

Cholesteric liquid crystal (CLC) is characterized by the selective reflection in the Bragg condition for constructive interference. Instead of assembling a LC cell with a pair of glass substrates, only a single glass substrate modified with vertical alignment reagent and layered with a CLC film of 3.4 ± 0.2 μm in thickness was utilized in this study for the detection of bovine serum albumin (BSA). By fine-tuning the ratio of the thickness d to the helical pitch P of the CLC film, both qualitative and quantitative biosensing capabilities were demonstrated with an E7/R811 CLC (d/P close to but smaller than unity) and an E7/R5011 CLC (d/P > > 1) film, respectively. The E7/R811 film was in the unwound homeotropic state in the absence of BSA, but transitioned to the fingerprint state at 10−9 to 10−6-g/ml BSA and to the planar state between 10−5 and 10−2-g/ml BSA. Quantitative protein assay based on E7/R5011 CLCs was established through transmission spectrometric analysis, and detection sensitivity was optimized by adjusting the Bragg reflection wavelength. A limit of detection of 5.9 × 10−9 g/ml was achieved for the CLC film consisting of 2.0-wt% R5011 in the nematic host E7 and exhibiting a Bragg reflection wavelength of 700 nm. As a majority of clinical assays are performed on a single solid substrate, the CLC-based, single-substrate biosensing platform allows for better integration with current technologies. Besides, the protein array employed in this work can be further miniaturized and converted to a protein microarray format to facilitate high-throughput analysis.

Rice protein has attracted considerable attention recently due to its physiological effects. This study extracted the proteins from paddy rice (PR) and germinated paddy rice (GPR) using three methods i.e., alkaline, sodium dodecyl sulfate (SDS) reagent and enzymatic extractions. The extracted proteins or protein fractions were assessed for their properties using various techniques. Data were analyzed by 2′3 factorial design experiment. It was found that germination and extraction methods significantly affected the concentration of protein fractions when analyzed by Bradford assay. Average protein fraction concentration of the GPR was lower than that of PR. SDS-PAGE patterns of protein fractions obtained from PR and GPR using any extraction method displayed similar protein profiles. Three major protein bands at about 13 kDa (prolamin), 22–23 kDa (basic glutelin) and 37–39 kDa (acidic glutelin) with small amount of 57 kDa proglutelin were observed. For amino acid profile, germination increased the content of most amino acids, resulting in the higher content of amino acids in GPR, excepted for some amino acids. When processed with in vitro digestion, protein fractions from GPR exhibited a higher level of digestibility than those from PR as evidenced by the less intensity of the protein bands obtained from SDS-PAGE. Alkaline and SDS reagent extractions provided more digestible protein fractions than enzymatic extraction. Extraction methods also influenced phase transition of protein fractions as investigated by a DSC. Alkaline extraction resulted in protein fractions with higher phase transition temperature than the other methods. For antioxidant capacity, extraction methods as well as germination significantly affected antioxidant capacity of the protein fractions. Enzymatic extraction provided protein fractions with the best antioxidant capacity.

Measurement of total protein in urine is key to monitoring kidney health in diabetes. However, most total protein assays are performed using large, expensive laboratory chemistry analyzers that are not amenable to point-of-care analysis or home monitoring and cannot provide real-time readouts. We developed a miniaturized optoelectronic biosensor using a vertical cavity surface-emitting laser (VCSEL), coupled with a fast protein assay based on protein-induced fluorescence enhancement (PIFE), that can dynamically measure protein concentrations in protein-spiked buffer, serum, and urine in seconds with excellent sensitivity (urine LOD = 0.023 g/L, LOQ = 0.075 g/L) and over a broad range of physiologically relevant concentrations. Comparison with gold standard clinical assays and standard fluorimetry tools showed that the sensor can accurately and reliably quantitate total protein in clinical urine samples from patients with diabetes. Our VCSEL biosensor is amenable to integration with miniaturized electronics, which could afford a portable, low-cost, easy-to-use device for sensitive, accurate, and real-time total protein measurements from small biofluid volumes.

The article presents a comparative analysis of four methods for quantifying the protein-peptide complexes content in extracts obtained from animal raw materials, as well as the low- and highmolecular weight extract fractions: the direct spectrophotometric determination at wavelengths of 260 and 280 nm with subsequent calculation by the Kalckar formula; the biuret reaction by the Kingsley-Weichselbaum method; the method with Bradford reagent and the standard Lowry method. Experimental data analysis demonstrates that in case of the extract that contains protein-peptidic complexes in different molecular weights range, the Kingsley-Weichselbaum method shows the highest quality of protein concentration determination; while studying highmolecular weight fraction (more than 30 kDa), it is possible to obtain more information by combining the spectrophotometric method and the Kingsley-Weichselbaum method. Low-molecular weight fractions (less than 30 kD) should be investigated by complex methods including the spectrophotometric method, Lowry and Bradford methods. These methods make it possible to presumably estimate protein molecules size ranges (by amount of peptide bonds), and also to determine hydrophobic and aromatic amino acids presence.

Glycomacropeptide (GMP) is a nutraceutical additive, which is released during cheese preparation. In this study, the protein content in a laboratory GMP preparation and two commercial GMP samples, which was estimated by the Bradford, biuret, Lowry and bicinchoninic acid (BCA) assays, were compared using the Kjeldahl method. There were apparent differences in the values of estimated proteins using the different methods. An absence of dose response in the Bradford method and a low dose response using the Lowry method indicated the unsuitability of both these methods. Absorbance in the biuret and BCA methods linearly increased with increasing GMP content. The BCA method was the most sensitive of these methods.

This research displayed the differences of formation process, morphology, degradation, and release behaviors between PCL and PCL-mPEG microspheres. Lysozyme loaded microspheres were prepared by double emulsion evaporation method, and the double droplets transformation process was observed and compared under microscope. Microspheres morphology, in vitro degradation and release behaviors were evaluated by scanning electron microscope, gel permeation chromatography, and bicinchoninic acid protein assay kit. PCL-mPEG microspheres got a rough surface and the outer water phase penetrated into PCL-mPEG double droplet easier than PCL during the transformation, accompanied with a high broken percentage and size swelling. PCL-mPEG microspheres get a faster degradation/erosion rate than PCL microspheres, and the rate was in direct proportion to mPEG chain length. In vitro release analysis showed that PCL-mPEG microspheres get a faster release rate than PCL microspheres, and the rate was also in direct proportion to mPEG chain length.

Culture broth of Ganoderma lucidum was determined for antioxidant, antibacterial and α-amylase inhibitory activities. The culture broth contained protein as determined by Bradford method equaled to 0.2 mg/ml and total phenol content as 0.078 mg GAE/mg protein (0.557 mg GAE/g extract). It exhibited radicals scavenging activities against ABTS+· and DPPH· radicals with a half maximal inhibitory concentration (IC50) less than 1.70 ± 0.02 and 2.28 ± 0.02 µg protein/ml, respectively and reducing power equaled to 4.38 ± 0.02 µmol Trolox/µg protein as investigated by ferric ion reducing antioxidant power method. The culture broth experimented into two approached; (1) treated with pronase and (2) filtered through a membrane with 10 kDa molecular weight cut-off (MWCO). The pronase-treated culture broth exhibited insignificant lower antioxidant activities, but the retained culture broth 10 kDa MWCO resulted in significant decrease in antioxidant activities suggesting that the small proteins might play the key role in antioxidant activity. The culture broth could protect DNA damage from hydroxyl radicals (·OH) generated by Fenton’s reaction. This culture broth showed antibacterial activity towards pathogenic strains Staphylococcus epidermidis and Pseudomonas aeruginosa and also had an interesting α-amylase inhibitory activity. This study suggested that apart from the fruiting bodies and the mycelial of G. lucidum, its culture broth also had potential applications as a value-added ingredient in the product such as in cosmetics and in nutraceuticals.

Background: Cytokines and growth factors were reported to play an important role in stimulating fibroblast proliferation. In vitro culture, fibroblast is mostly culture in medium containing fetal bovine serum (FBS). Human umbilical cord blood (hUCB) has been reported to have low immunogenic property and potential in wound healing, so therefore hUCB serum (hUCBS) could be potential and were investigated in current study. Materials and Methods : Five hUCBs were collected from healthy volunteers with normal delivering procedure. hUCB was ex utero immediately collected from umbilical vein in vacutainers and processed. NIH3T3 cells were cultured in DMEM with 10% FBS or 5-20% hUCBS for 48 hours. Cells were then quantified using MTT assay. Protein concentration of FBS and hUCBS were quantified using Bradford assay. Results: NIH3T3 cells density grown in DMEM with 10% FBS was the lowest. NIH3T3 cells densities were increased along with the increment of hUCBS concentrations. MTT results showed that average number of NIH3T3 cells grown in DMEM with 10% FBS was 6,185±1,243. Meanwhile average numbers of NIH3T3 cells grown in DMEM with 5%, 10% and 20% hUCBS were 8,126±628, 9,685±313 and 12,200±304, respectively. Average numbers of NIH3T3 cells grown in DMEM with 5% hUCBS were significantly higher than the ones with 10% FBS ( p =0.000). Bradford results showed that concentration of hUCBS was significantly higher than the one of FBS ( p =0.000). Conclusion: hUCBS could induce higher proliferation rate of NIH3T3 cells than FBS. Hence hUCBS could be suggested as an alternate of FBS in inducing fibroblast. Keywords: NIH3T3, fibroblast, UCB, serum, FBS, proliferation

A Maillard type reaction in the dry state was utilized to create conjugates between whey protein isolate (WPI) and sugar beet pectin (SBP) to achieve improved functional properties including solubility, colloidal stability and oil-in-water emulsion stability. To optimize the reaction conditions, mixtures of WPI and SBP at varying weight ratios, 3:1, 2:1 and 1:1, were heated at 60 °C, 79% RH for 72 h. Changes in the physical properties, i.e. solubility, and chemical compositions, i.e. free sulfhydryl and amine contents were assessed using chemical assays. Bradford assay results demonstrated that the protein solubility of WPI increased significantly, ∼20% by reacting with lower levels of SBP (3:1 and 2:1) whereas the total powder solubility was not affected until an equal ratio of SBP was used, and the reduction was only ∼15%. The free sulfhydryl (SH) and primary (NH2) and secondary amine (NH) contents of WPI were also mitigated by conjugating with SBP. The formation of the covalent conjugates between WPI and SBP was confirmed by SDS-PAGE gel electrophoresis through staining for both proteins and glycoproteins. The UV–VIS spectroscopy showed significant changes in the molecular electronic transition states of both WPI and the feruloyl moieties of SBP upon interaction and conjugation. Measurements of ζ-potential, particles size distribution and average particle size demonstrated that the WPI-SBP conjugates prepared at the weight ratio of 3:1 were most effective at stabilizing oil-in-water emulsions than other ratios used in this work.