Bicinchoninic Acid Assay Research Articles

Conjugation of Anti-EpCAM Antibody on Alginate–RIP MJ-30 Nanoparticle through Carbodiimide Reaction as a Model of Targeted Protein Therapy

Ribosome inactivating proteins from Mirabilis jalapa L. (RIP MJ) has shown higher cytotoxic activity when being formulated as a nanoparticle. However, the selectivity of the delivery system is also an important aspect when it comes to cytotoxic cell therapy. Epithelial cell adhesion molecule (EpCAM) is a monomeric glycoprotein which is overexpressed in epithelial cancer cells. This study aim was to develop a model of targeted protein delivery system by formulating the base fraction of RIP MJ (RIP MJ-30) into alginate nanoparticles and conjugating it with anti-EpCAM antibody. RIP MJ-30 was formulated in to nanoparticle using alginate and CaCl2 as cross-linker. Optimization of conjugation reaction condition was done in the pH variation of 4.5, 5.5, and 6.5. The success of conjugation was analyzed qualitatively using native polyacrylamide gel electrophoresis (native-PAGE) method and BCA assay. The optimum formula of RIP MJ-30 nanoparticles was produced using 0.3% alginate and 0.2% CaCl2. Results indicated that optimum conjugation reaction was carried out at pH level of 5.5. The optimum native-PAGE condition was by using 8% polyacrylamide gel in duration of 6h. Characterization of nanoparticle resulted in particle size of 205.0nm, zeta potential of -6.9mV, entrapment efficiency of 71.11±4.84%, and conjugation efficiency of 89.55±6.18%. It was concluded that RIP MJ-30 was successfully formulated into alginate nanoparticle and conjugated to anti-EpCAM antibody through carbodiimide reaction using 1-ethyl-(dimethylprophilamine) carbodiimide (EDAC).

Chemical characterization methods for the analysis of structural extracellular polymeric substances (EPS)

Biofilm structure and functionality depend on extracellular polymeric substances (EPS), but analytical methods for EPS often lack specificity which limits progress of biofilm research.EPS were extracted from aerobic granular sludge and analyzed with frequently applied colorimetric methods. The colorimetric methods were evaluated based on their applicability for EPS analysis. EPS fractions of interest were proteins, sugars, uronic acids and phenolic compounds. The applied methods (Lowry method, bicinchoninic acid assay, phenol sulfuric acid method, carbazole sulfuric acid method) were investigated in terms of their sensitivity towards the selected standard compound. Interference of compounds present in EPS with the colorimetric methods was further evaluated. All methods showed to be highly depending on the choice of standard compound and susceptible towards interference by compounds present in EPS.This study shows that currently used colorimetric methods are not capable of accurately characterizing EPS. More advanced methods are needed to be able to draw conclusions about biofilm composition, structure and functionality.

Lysozyme sorption by pure-silica zeolite MFI films

Nanostructured, porous materials, like zeolites, have been suggested as possible materials for biomedical applications in implantable device coatings, tissue engineering, and drug delivery systems due to their unique interactions with biomolecules and biological environments. Here, the fundamental sorption interactions between a pure-silica zeolite (MFI) with lysozyme, a positively charged enzyme, are discussed. Lysozyme sorption is considered a model for innate immunological processes in the body; high lysozyme sorption has been correlated with materials that are naturally antibiotic and act as cell guardians. The impact of three different parameters on the sorption of lysozyme was evaluated via enzyme-linked immunosorbent assays (ELISAs) and bicinchoninic acid assays (BCA assays), including the orientation of the film’s crystal structure (b-oriented or randomly-oriented), the lysozyme incubation volume (200 μL, 400 μL, 600 μL, 800 μL), and the lysozyme incubation time (1, 6, and 24 h). Additionally, the films were characterized via X-ray diffraction, scanning electron microscopy, and energy dispersive spectroscopy. In this work, we demonstrated that MFI films are capable of lysozyme sorption. Further, our observations suggested that, while crystal orientation did not play a significant role in the sorption process, incubation volume and time both impact sorption. The highest amounts of sorbed lysozyme were detected when films were incubated at intermediate volumes (400 and 600 μL) and shorter incubation times. The films’ ability to sorb differing amounts of lysozyme, depending on uptake parameters, make MFI films and coatings tailorable candidates for supports and/or coatings for implantable devices.

In vitro and in vivo evaluation of new PRP antibacterial moisturizing dressings for infectious wound repair

In this study, a solution chitosan fibroin emulsion with added Silver Nanoparticles (AgNPs) was freeze-dried to be the scaffold, and an asymmetric coating was formed on one side. PRP was loaded onto the composite scaffold using a secondary lyophilization technology to prepare the tissue engineering dressings. AgNPs were characterized using a transmission electron microscope. The morphologies of the composite dressing were examined under a scanning electron microscope. The silver content of the dressing was measured by inductively coupled plasma mass spectrometry. The asymmetric wettability of the composite dressing was demonstrated by water contact angle measurement. Relatively high porosity, favourable moisture retention capability and appropriate tensile strength were observed by measuring the physical and mechanical properties. Satisfactory antibacterial properties against various bacteria and microbial isolation performance were observed by the antibacterial effect analysis in vitro. The total protein slow-release property was measured using the BCA assay. Good biocompatibility and lower sensitization were examined both in vitro and in vivo. In addition, the healing effciency of the composite dressing on infected wound were examined in mice infected wound models. Analysis of wound healing rates, bacterial cultures of wound exudate, whole blood cell analysis and histological examination all showed satisfactory results. These results are demonstrated to provide a potential and possible pathway to promote wound tissue repair and regeneration.

Comparison of early posttreatment effects of two steroidal anti‐inflammatory ophthalmic drugs on the ocular inflammatory response induced by paracentesis in healthy canine eyes

We investigated the early posttreatment effects of two steroidal anti-inflammatory ophthalmic drugs on blood-aqueous barrier (BAB) breakdown by paracentesis in dogs. We studied 21 healthy beagles with normal eyes. Controlled anterior chamber paracentesis (0.5mL) was performed in one eye of each dog. Control group dogs (n=7) received no medication, whereas those in the treatment groups received a topical anti-inflammatory medication (difluprednate [DFBA] ophthalmic emulsion 0.05% [n=7] or betamethasone [BMZ] sodium phosphate ophthalmic solution 0.1% [n=7]) at 0, 15, 30, and 45minutes after initial paracentesis in the paracentesed eyes. Secondary aqueous humor (AH) was collected 60minutes after initial paracentesis. Protein and prostaglandin E2 (PGE2 ) concentrations in AH were determined using the bicinchoninic acid assay and commercially available immunoassay kit, respectively. All mean values in the three groups were compared using analysis of variance followed by Tukey's post hoc test. Aqueous protein and PGE2 concentrations were markedly increased at 60minutes following paracentesis. Both concentrations in the secondary AH of the DFBA group were significantly lower than those of the control group; however, treatment with BMZ had no significant effects. Early postparacentesis treatment with DFBA was more effective than that with BMZ for reducing aqueous protein and PGE2 contents in dogs with paracentesis-induced BAB breakdown. DFBA may be an appropriate treatment during the early stage of anterior uveitis caused by intraocular surgery in dogs.

Evaluation of exosomal miR-155, let-7g and let-7i levels as a potential noninvasive biomarker among refractory/relapsed patients, responsive patients and patients receiving R-CHOP

This study aimed to investigate and compare exosomal miR-155, let-7g and let-7i levels as a noninvasive biomarker among patients with refractory/relapsed or responsive DLBCL after R-CHOP treatment and patients receiving R-CHOP. Plasma was collected and exosomes were isolated from plasma. Exosomes confirmed by zeta-seizer, electron microscope, and western blot. Exosomes concentration was investigated by BCA assay. MiR-155, let-7g and let-7i levels were evaluated in plasma-derived exosomes by real-time PCR. Plasma IFN-γ and IL-4 level were measured by ELISA assay. We observed the significant increase in the exosomal miR-155 levels (p = .002) and exosomes concentration (p = .001) in refractory/relapsed patients compared to responsive patients and patients receiving R-CHOP. No association was not observed between exosomal miR-155 levels and IPI and disease stage. The significant decrease in IFN-γ levels was observed in patients receiving R-CHOP compared to refractory/relapsed or responsive patients (p=.001). Therefore, exosomal miR-155 might be useful as potential prognostic biomarkers to predict response to treatment in DLBCL patients.

MWCNT interactions with protein: surface-induced changes in protein adsorption and the impact of protein corona on cellular uptake and cytotoxicity.

PurposeProtein adsorption onto nanoparticles in the form of protein corona, affects properties of nanomaterials and their behavior in the biological milieu. This study aims at exploring the effects of multiwalled carbon nanotubes (MWCNTs) surface chemistry on bovine serum albumin (BSA) and immunoglobulin G (IgG), including their adsorption behavior and spatial configurations, as well as the impact on cellular uptake, cytotoxicity, and cellular responses.MethodsThree types of MWCNTs (pristine MWCNTs, MWCNTs-COOH, and MWCNTs-PEG) were synthesized by classical chemical reduction. The size, morphology, hydrodynamic size, and zeta potential were characterized using transmission electron microscopy and dynamic light scattering. MWCNTs were exposed to BSA and IgG solutions, then the amount of MWCNT absorption was performed by bicinchoninic acid assay, and the effects were assessed by utilizing fluorescence spectroscopy, circular dichroism (CD) spectroscopy. Quantitative measurement of MWCNTs uptake with or without protein corona was performed as turbidity method. CCK assay and a microdilution method were performed to evaluate the effects of protein corona on cytotoxicity and pro-inflammatory cytokines release.ResultsThe BSA and IgG adsorption capacities of MWCNTs followed the order pristine MWCNTs>MWCNTs-COOH and MWCNTs-PEG. MWCNT binding can cause fluorescence quenching and conformational changes in BSA and IgG, indicating that both the physicochemical properties of MWCNTs and protein properties play critical roles in determining their adsorption behavior. Further study showed time-dependent increases in MWCNT cellular uptake and internalization. Hydrophobicity is the major factor increasing cellular uptake of pristine MWCNTs, but a protein corona enriched with dysoposnins is the main factor reducing uptake of MWCNT-COOH by RAW264.7 cells. The cytotoxicity and pro-inflammatory response related to physicochemical properties of MWCNTs, and frustrated phagocytosis is a key initiating event in the pro-inflammatory response of MWCNT-exposed macrophages.ConclusionThese findings shed light on how functionalized MWCNTs interact with protein coronas and provide useful insight into the dramatic effect of protein coronas on different functionalized MWCNTs. These events affect cellular uptake and cytotoxicity, which could inform how to enhance MWCNT biocompatibility and develop approaches for managing MWCNT hazards.

Portable Smartphone-based Colorimetric Analyzer with Enhanced Gold Nanoparticles for On-site Tests of Seafood Safety.

Saxitoxin (STX) is one of the paralytic shellfish poisons (PSP) that endanger people's health. It is necessary to develop methods for the on-site rapid detection for STX in order to prevent safety accidents. An enzyme-linked immunosorbent assay (ELISA) is timesaving and effective, but it is not suitable for large-scale in-field tests due to the expensiveness of commercial ELISA kits and the bulkiness of a microtiter plate reader (MTPR). In this study, a portable smartphone-based colorimetric analyzer (SBCA) with a cost-effictive enhanced gold nanoparticle-based ELISA (EGNB-ELISA) was proposed for STX detection. In a bicinchoninic acid (BCA) protein assay (R2 = 0.9939) and a glucose assay (R2 = 0.9937), SBCA was shown to be in good agreement with MTPR. EGNB-ELISA had a 12.5-fold lower detection limit (0.4 ng/mL) and a lower detection range (1 - 50 ng/mL, Y = 0.4037X + 0.3564, R2 = 0.9797) than the classical ELISA. The recovery rate ranged over 89.1 - 112.2%. The whole detection system, combining both homemade SBCA and ENGB-ELISA, is expected to satisfy the needs of on-site STX sample tests to guarantee seafood safety.

UV Peroxidation Reduces Poloxamine T1107 Capability to Disaggregate Lysozyme

Poloxamers (poly-oxy-amers) and poloxamines (poly-oxy-amines) are amphiphilic multi-block polymer surfactants. Because their backbones are made of polyethylene oxide and polypropylene oxide blocks, they are susceptible to oxidative formation of hydroperoxide side groups that alter surfactant properties. Several poloxamers and poloxamines are able to prevent protein aggregation by binding to exposed hydrophobic domains. The purpose of this study was to determine the effect of oxidative degradation on T1107 catalysis of unfolded lysozyme. To peroxidate Poloxamine T1107 [Maroon Biotech], 2 mL of 15 mg/mL in phosphate-buffer saline (PBS) was exposed to a range of UV (254 nm, @∼2000 Gy/hr) radiation for 2, 4, 6, 8, 24, 32, 48, or 72 hours. DTT (5mM) reduced and aggregated hen egg white lysozyme (HEWL, 0.5mg/ml) [Sigma-Aldrich] was used to quantify T1107 (0.5mg/mL) catalyzed disaggregation at relevant temperatures (37°C). Aggregation was quantified by measuring absorption at 400 nm using a Beckman DU800 spectrophotometer (n=10). A reducing agent-compatible bicinchoninic acid (BCA-RAC) assay kit [Thermo Fisher] was used to measure the concentration of soluble native protein (n=3). A PeroxiDetect Kit [Sigma-Aldrich] was used to quantify the concentration of peroxides (n=6). P values <0.05 were considered statistically significant. Non-irradiated T1107 caused 6% disaggregation of DTT HEWL. T1107 irradiated for 72 hours was 99% as ineffective in disaggregating HEWL as no T1107. Increasing UV exposure was inversely related to T1107 ability to prevent aggregation. The non-irradiated T1107 indicated <5% loss in protein, while the most irradiated T1107 indicated 47% loss. CD showed highly irradiated T1107 and samples without T1107 did not preserve any secondary structures, whereas non-irradiated and less-irradiated T1107 did. 1H NMR (8-9 ppm), FTIR (1723cm−1), and PeroxiDetect showed the formation of peroxide groups. This study demonstrates that peroxidated Poloxamine 1107 is incapable of catalyzing proteins disaggregation.

Comparative Analysis of Protein Quantification Methods for the Rapid Determination of Protein Loading in Liposomal Formulations.

Advances in manufacturing processes provide the ability for the high throughput production of liposomes containing a range of moieties, from small molecules to large biologicals (including proteins and nucleic acids for prophylactic and therapeutic applications). Whilst rapid quantification methods for small molecules are generally well established, the ability to rapidly quantify liposomal entrapment of proteins is limited. Indeed, most standard protein quantification techniques (including the BCA assay and Reverse phase-high performance liquid chromatography (RP-HPLC)) measure protein encapsulation indirectly, by measuring the amount of non-incorporated drug, and subtracting from the initial amount of protein added. However, this can give inaccurate and misrepresentative results. To address this, we have developed a range of methods to directly quantify protein entrapment within liposomes. The encapsulation efficiency within neutral, anionic and cationic liposome formulations was determined by three techniques; BCA assay, RP-HPLC and HPLC coupled to an evaporative light scattering detector, (HPLC-ELSD). All three methods are reliable for the quantification of protein, with linear responses and correlation coefficients of 0.99, and LOQ for all three methods being less than 10 µg/mL. Here within, we provide three methods for the rapid and robust quantification of protein loading within liposomal (and other bilayer) vesicle systems.

Direct isolation and characterization of circulating exosomes from biological samples using magnetic nanowires

BackgroundTumor-derived exosomes are gaining attention as important factors that facilitate communication between neighboring cells and manipulate cellular processes associated with cancer development or progression. The conventional techniques for the isolation and detection of exosomes face several limitations, restricting their clinical applications. Hence, a highly efficient technique for the isolation and identification of exosomes from biological samples may provide critical information about exosomes as biomarkers and improve our understanding of their unique role in cancer research. Here, we describe the use of antibody cocktail-conjugated magnetic nanowires to isolate exosomes from plasma of breast and lung cancer patients.MethodsThe isolated exosomes were characterized based on size and concentration using nanoparticle tracking analysis. Levels of exosomal proteins were measured by bicinchoninic acid assay and enzyme-linked immunosorbent assay. Morphology was visualized by transmission electron microscopy. Immunoblotting (Western blotting) was used to detect the presence of exosomal markers.ResultsThe use of antibody cocktail-conjugated magnetic nanowires resulted in approximately threefold greater yield when compared to the conventional methods. The elongated feature of nanowires significantly improved the efficiency of exosome isolation, suggesting its potential to be translated in diverse clinical applications, including cancer diagnosis and treatment.ConclusionsThe nanowire-based method allows rapid isolation of homogeneous population of exosomes with relatively high yield and purity from even small amounts of sample. These results suggest that this method has the potential for clinical applications requiring highly purified exosomes for the analysis of protein, lipid, mRNA, and miRNA.

Bioactive characteristics of an implant surface coated with a pH buffering agent: an in vitro study.

PurposeThe purpose of this study was to evaluate the effectiveness of conventional sandblasted, large-grit, acid-etched (SLA) surface coated with a pH buffering solution based on surface wettability, blood protein adhesion, osteoblast affinity, and platelet adhesion and activation.MethodsTitanium discs and implants with conventional SLA surface (SA), SLA surface in an aqueous calcium chloride solution (CA), and SLA surface with a pH buffering agent (SOI) were prepared. The wetting velocity was measured by the number of threads wetted by blood over an interval of time. Serum albumin adsorption was tested using the bicinchoninic acid assay and by measuring fluorescence intensity. Osteoblast activity assays (osteoblast adhesion, proliferation, differentiation, mineralization, and migration) were also performed, and platelet adhesion and activation assays were conducted.ResultsIn both the wetting velocity test and the serum albumin adsorption assay, the SOI surface displayed a significantly higher wetting velocity than the SA surface (P=0.000 and P=0.000, respectively). In the osteoblast adhesion, proliferation, differentiation, and mineralization tests, the mean values for SOI were all higher than those for SA and CA. On the osteoblast migration, platelet adhesion, and activation tests, SOI also showed significantly higher values than SA (P=0.040, P=0.000, and P=0.000, respectively).ConclusionsSOI exhibited higher hydrophilicity and affinity for proteins, cells, and platelets than SA. Within the limits of this study, it may be concluded that coating an implant with a pH buffering agent can induce the attachment of platelets, proteins, and cells to the implant surface. Further studies should be conducted to directly compare SOI with other conventional surfaces with regard to its safety and effectiveness in clinical settings.

A Proposed Mechanism for Texture Property of Woody Breast in Broilers

ObjectivesWoody breast is a myopathy observed in chicken breast meat (Pectoralis major) characterized by its tough and rubbery texture. However, the exact causation of woody breast texture is still unknown. We hypothesize that sarcoplasmic reticulum (SR) dysfunctionality early postmortem results in rapid leakage of intracellular calcium may partially contribute to the abnormal meat texture observed in woody breast meat. The objective of this preliminary study was to investigate this hypothesis.Materials and MethodsFourteen Ross line broiler breast fillets (7 severe woody breast and 7 normal) were collected at 3 h postmortem from a commercial processing plant located in the southeast United States. The 7 woody breast samples also exhibited moderate to severe white striping. The 7 normal samples did not exhibit any signs of white striping or woody breast. Each sample was trimmed, weighed, vacuum packaged and frozen at –20°C at approximately 8 h postmortem. One 1.9 cm strip across the cranial end of each fillet was fabricated and pulverized in liquid nitrogen to measure sarcomere length (Laser Scan Confocal Microscope with a 100x/NA 1.4 objective), calpain activity (immunoblotting for µ-calpain autolysis), proteolysis (immunoblotting for troponin-T degradation) and collagen content (hydroxyproline content). Purge was also collected from each sample to evaluate protein (bicinchoninic acid assay) and free calcium concentration (atomic absorption).ResultsWoody breast fillets were heavier than normal chicken breast fillets (522.9 vs. 446.9g; P < 0.05). Woody breast samples tended to have shorter sarcomeres (1.70 vs. 2.02 µm; P = 0.0543) and less intact troponin-T compared to normal breast samples (relative intact troponin-T band density: 49.98 vs. 56.97%; P = 0.0515) at 8 h postmortem. It was interesting to note that no µ-calpain band was detected through immunoblotting for both the woody breast and normal samples at 8 h postmortem. Other studies have found similar results as poultry µ-calpain autolyzed at a much rapid rate than µ-calpain in mammalian species. In addition, the purge from woody breast samples also had higher levels of free calcium compared to normal samples (6.2 vs. 4.2 nmol calcium/mg protein; P < 0.05). Lastly, there was more collagen present in the woody breast samples compared to normal chicken breast samples (3.89 vs. 2.08 mg collagen/g muscle tissue; P < 0.05).ConclusionThe results indicated that the cause of texture abnormality of woody breast may be the combined effects of more calcium being released from the SR early postmortem resulting in shorter sarcomere length and more collagen being deposited in the chicken breast meat. Additional research with the focus on SR integrity and functionality as well as collagen crosslinks are needed to further elucidate the basic mechanism of woody breast texture formation.

Firefly Luciferase-Based Reporter Gene Assay for Investigating Nanoparticle-Mediated Nucleic Acid Delivery.

Investigation of nanoparticle-mediated nucleic acid delivery is a key step for the development of nucleic acid-based nanotherapeutics. Using luciferases as reporter genes, the efficiency of gene delivery can be quantified in a highly sensitive way based on bioluminescence measurements. Here we describe a robust assay to quantify the activity of exogenously produced firefly luciferase and its normalization by the total protein amount (bicinchoninic acid assay, BCA) in the cells. The method describes preparation of firefly luciferase assay buffer (F-LAB) along with BCA assay and employment of the optimized firefly luciferase assay for investigating in vitro gene delivery by polyplex and lipoplex nanoparticles. Reusability of F-LAB for ease of usage (by freezing and reusing it for luciferase assay) is also demonstrated.

Engineering T cell response to cancer antigens by choice of focal therapeutic conditions

Focal thermal therapy (Heat), cryosurgery (Cryo) and irreversible electroporation (IRE) are increasingly used to treat cancer. However, local recurrence and systemic spread are persistent negative outcomes. Nevertheless, emerging work with immunotherapies (i.e., checkpoint blockade or dendritic cell (DC) vaccination) in concert with focal therapies may improve outcomes. To understand the role of focal therapy in priming the immune system for immunotherapy, an in vitro model of T cell response after exposure to B16 melanoma cell lysates after lethal exposures was designed. Exposure included: Heat (50 °C, 30 min), Cryo (−80 °C, 30 min) and IRE (1250 V/cm, 99 pulses, 50 µs pulses with 1 Hz intervals). After viability assessment (CCK-8 assay), cell lysates were collected and assessed for protein release (BCA assay), protein denaturation (FTIR-spectroscopy), TRP-2 antigen release (western blot), and T cell activation (antigen-specific CD8 T cell proliferation). Results showed IRE released the most protein and antigen (TRP-2), followed by Cryo and Heat. In contrast, Cryo released the most native (not denatured) protein, compared to IRE and Heat. Finally, IRE dramatically outperformed both Cryo and Heat in T cell activation while Cryo modestly outperformed Heat. This study demonstrates that despite all focal therapies ability to destroy cells, the ‘quantity’ (i.e., amount) and ‘quality’ (i.e., molecular state) of tumor protein (including antigen) released can dramatically change the ensuing priming of the immune system. This suggests protein-based metrics whereby focal therapies can be designed to prime the immune system in concert with immunotherapies to eventually achieve improved and durable cancer treatment in vivo.

Optimization of Expression Condition, Two Dimensional And Melting Point-Based Characterization of Recombinant Human Interferon Alpha-2a Fusion and Non Fusion Forms

Recombinant Human Interferon Alpha-2a (rhIFNα-2a) is a therapeutic protein that used in hepatitis and cancer treatments. In our previous research, we developed higher molecular weight of the protein through human serum albumin fusion. The fusion and non fusion form of rhIFNα-2a were produced in Pichia pastoriswith 86 kDa and 19 kDa in size respectively. In previous research, protein yield was not reproducible due to unoptimized expression conditions. This reseach was aimed to optimize expression condition process and to characterize the fusion and non fusion forms of rhIFNα-2a. The parameters to observe in overproduction include nutrient (media and methanol concentration) and non nutrient (temperature andincubation period). Affinity and size exclusion cromatographicwere compared in protein purification. BCA assay was used to determine quantity of protein. Protein characterization was conducted using two-dimensional SDS PAGE and denaturation analyses. The optimal condition of expression was achieved using complex media with 1% of methanol for 3 day incubation period at 25°C. The protein yield was reproducible and higher comparing to previous research. Affinity chromatography resulted in higher purity of the proteins comparing to size exclusions. Characterization using two dimensional gel analysis revealed that isoelectric point of rhIFNα-2a is 6.5 for fusion form and 6.0 for non fusion form. The melting points of fusion protein were 56°C and 62°C whilst that of non fusion was 56°C.

Comparison of macronutrient content in human milk measured by mid-infrared human milk analyzer and reference methods

ObjectiveThe study aims at evaluating mid-infrared human milk analyzer (HMA) accuracy and precision, in human milk (HM).Study designRöse-Gottlieb, high-performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD), Kjeldahl and amino acid analysis (AA) were selected as references for total fat, lactose and total protein determination.ResultsNo significant difference was observed in lactose content between HMA and HPAEC-PAD. Significant differences were observed in fat and protein content between HMA and reference methods. However, the difference in fat content was lower than 12%, and therefore within the variability declared by supplier. For protein determination, the BCA protein assay was selected. No significant differences were observed in total protein content measured by BCA assay, Kjeldahl and AA methods.ConclusionsHMA was reliable for the quantification of total fat and lactose content, but not for total protein one. The latter was measured by BCA assay, which yielded comparable results to Kjeldahl and AA methods.

Intravenous anesthetic ketamine attenuates complete Freund's adjuvant-induced arthritis in rats via modulation of MAPKs/NF-κB.

The current study was intended to investigate the effect of ketamine (KET) on complete Freund's adjuvant (CFA)-induced arthritis in rats. The CFA was administered in the hind paw of the rats for the induction of adjuvant-induced arthritis. The paw swelling of experimental animals was measured as hind paw volume. Hematoxylin and eosin staining was estimated and pathological changes in the joint tissues were observed under a light microscope. Furthermore, the bicinchoninic acid assay was used for protein quantification. The antibody-reactive bands were visualized using enhanced chemiluminescence. The present study showed that KET significantly reduces the severity of arthritis in CFA mice. The therapeutic effects were linked with reduced joint swelling and destruction, as evidenced by analyzing rat paws. The KET also revealed to attenuate the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6). In western blot analysis, KET inhibit phosphorylation of MAPKs, IκBα and nuclear translocation NF-κB in the inflammatory joints of AIA rats. Moreover, KET showed to induce apoptosis via mitochondrial signalling pathways (Bcl2, Bax, cytochrome C, cleaved caspase-3 and cleaved caapse-9). Taken together, KET show significant anti-rheumatoid arthritis activity via multiple mechanisms and may thus have therapeutic benefits for RA.

Improvement of calcium phosphate scaffold osteogenesis in vitro via combination of glutamate-modified BMP-2 peptides

Alpha-tricalcium phosphate (α-TCP) based porous scaffolds have superior osteoconduction and osteoinduction in bone tissue engineering, furthermore, these 3D porous scaffolds can be used as efficient drug delivery carriers. In the concept of tissue engineering, the “drugs” could be defined as drug molecules or biomacromolecules, even cells. These “drugs” have endowed the scaffolds which were laden improved abilities compared with the blank scaffolds. In this study, we anchored osteogenic bone morphogenetic protein-2 (BMP-2) derived peptides to α-TCP 3D porous scaffolds by linking the E7 domain to the target peptides, constructed the modified active peptides (E7BMP-2 peptides) delivery system, which finally achieved the modified peptides sustaining release and enhanced rat bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation in vitro. The α-TCP 3D porous scaffolds had micropores and interconnected micropores which expanded surface area of the scaffolds. The release test testified the constructed the delivery system had realized long-term release in which the peptides dosage could be detected by the BCA protein assay kit after 10 days compared with BMP-2 proteins which absorbed on the same α-TCP 3D porous scaffolds. The constructed E7BMP-2 peptides delivery system supported rat BMSCs osteogenic differentiation in the form of improving the genes expression levels of Runx2, ALP and OCN. Based on electrostatic interactions, E7 domain fastened combination between the active BMP-2 derived peptides and the α-TCP 3D porous scaffolds, the sustaining E7BMP-2 peptides release promoted the BMSCs osteogenesis as BMP-2 proteins did, which endowed α-TCP 3D porous scaffolds enhanced osteoinductive abilities in vitro.

Measuring Protein Concentration with Absorbance, Lowry, Bradford Coomassie Blue, or the Smith Bicinchoninic Acid Assay Before Electrophoresis.

Measuring the concentration of proteins is an essential part of enzyme analysis or serves to monitor protein yields and losses during protein isolation procedures. Decisions on the usefulness of any protein isolation procedure depend on knowing the concentration of proteins before and after a procedure. Protein concentration in solution is generally measured with spectrophotometry in the UV range or in the presence of dyes or copper interacting with the protein. This review describes absorbance at 280nm, the Lowry, Bradford (Coomassie Blue), and Smith (bicinchoninic acid) assays for measuring protein and includes suggestions for optimizing each method.