Mg Of Bovine Serum Albumin Research Articles

#4868 MASS-SPECTROMETRY ANALYSIS OF CONGOPHILIC URINE AND ITS DETERGENT RESISTANT FRACTION IN PATIENTS WITH AMYLOID AND NON-AMYLOID NEPHROPATHIES

Abstract Background and Aims Being a well-known intrinsic property of amyloid, сongophilia was recently demonstrated in urine of patients with proteinuria of different etiology including renal amyloidosis (RA) and non-amyloid nephropathies (NANP) [1]. Urine proteins (UPs) responsible for congophilia in RA and NANP are supposed to have another property of amyloid such as resistance to ionic detergents and present in urine as detergent-resistant aggregates (DRA). In the pilot study we performed mass-spectrometry (MS) analysis of congophilic urine samples and its detergent-resistant fraction in patients with RA and NANP to investigate UPs and their specificity to the particular kidney disease. Method We collected first morning void urine samples from patients with RA (n = 4) and NANP (n = 4). Urine congophilia was assessed by Congo red Dot test as described previously [1]. To analyze bulk urine (BU) proteins, 30 μl of supernatant was precipitated with 80% acetone and boiled in 2% sodium dodecyl sulfate (SDS) for 30 minutes. Then 5 μg of UPs was separated in 10% polyacrylamide gel electrophoresis (PAGE) followed by Coomassie blue staining. The ultracentrifugation of 0.5-3 ml of urine at 300,000x g for 16h following by the treatment of precipitate with 3% sarcosyl in phosphate-buffered saline (PBS) for 10 min with subsequent washing of resistant aggregates in PBS were applied to obtain DRA. After boiling in SDS 8 μl of the sample was separated in 10% PAGE. UPs concentration in the DRA was estimated by densitometry regarding to 2 μg of bovine serum albumin. Then 6 μg of UPs was digested with trypsin, followed by purification on silicate (CDS Empore™ C18 Extraction Disks). Prepared samples of BU and DRA were analyzed by electrospray ionization tandem MS. We used MsFragger software to obtain lists of UPs for each sample. Most representative UPs in the sample were selected by the unique spectral count (USC). We considered the protein as having diagnosis-specific potential (DSP) if it was present in every sample of the particular disease group, i.e. AA, AL, NANP in BU or DRA, and was absent in any sample of another disease group. Results The patients had following characteristics: age 51±13 years, 3 male/ 5 female, eGFR = 45 (19; 95) ml/min/1.73 m2; 24 h proteinuria = 6.5 (4.5; 7.9) g. In PAGE analysis, BU proteins appeared to be similar in all 8 samples with 2 major bends in 70 kDa and 50 kDa regions, corresponding to albumin and immunoglobulin (Ig) heavy chains, respectively (Fig. 1A). The amount of protein in DRA was small and comprised 1.02 (0.71; 1.61) % of the total protein in the sample (4.7 (2.3; 5,1) g/l). Compared with BU PAGE analysis of DRA proteins revealed other bends with trace albumin bend, predominance of 45 kDA region bend and more apparent 30 kDa bend (Ig light chains) in the majority of patients (Fig. 1B). Results of MS analysis are shown on the Figure 2. There were more DSP revealed in DRA vs BU: 14 vs 5, 46 vs 2 and 4 vs 2 UPs in samples of AL, AA and NANP, respectively. When compare by a particular disease group, there were no DSP found either in BU or DRA in RA samples. One protein (aminopeptidase N) and 3 proteins (isoform 3 of unconventional myosin-LC, protein S100-A8, elongation factor 1-α 1) were detected as DSP in BU and DRA in AL, respectively. Alpha-1-acid glycoprotein 1 was only DSP for AA in DRA as well as serum paraoxonase 1 for NANP in BU. Venn-diagrams of shared and divergent UPs are present on the Figures 2D-G. Conclusion Although DRA represented a small portion of UPs, its composition significantly differed from BU and could contain more specific disease markers that makes urine detergent-resistant fraction promising for the future research. Understanding the role of DRA proteins in the pathogenesis of amyloid and non-amyloid renal disease and their diagnostic utility requires further studies.

Glutathione, Cysteine, and D-Penicillamine Role in Exchange of Silver Metal from the Albumin Metal Complex.

The purpose of this study is to investigate the exchange reaction taking place among the bovine serum albumin (BSA), 5,5′-dithiobis-(2-nitrobenzoic acid (ESSE), reduced glutathione, N-acetylcysteine, D-penicillamine (thiolates), and silver metal (AgI). For this purpose, stock solutions of BSA and Ellman's reagent were prepared by dissolving 264 mg of BSA in 5 ml of reaction buffer (0.1 M KH2PO4 at pH 7.8) and 23.8 mg of ESSE in 1.0 ml of reaction buffer which were mixed together. Mixture of BSA-AgI was prepared in a separate procedure by dissolving 0.17 mg of silver nitrate in 1 ml of reaction buffer and then dissolving BSA (200 mg) in the same solution of silver nitrate. Blocking of Cys-34 of BSA with AgI was confirmed by treating different dilutions of BSA-AgI (500 μM) solutions with the solutions of ESSE (85 μM) and ES− (85 μM) and recording the spectra (300-450) with a UV-visible spectrophotometer. The chromatographed AgI-modified BSA ((BSA-S)AgI)) samples (typically 500 μM) were subsequently mixed with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine). AgI and modified BSA (typically 500 μM each) were treated with these low molecular weight thiolates and allowed to react overnight followed by chromatographic separation (Sephadex G25). The redox reactions of AgI-modified BSA with various low molecular weight thiols revealed a mechanically important phenomenon. In the case of reduced glutathione and N-acetylcysteine, we observed the rapid release of a commensurate amount of Ellman's anion, indicating that an exchange has taken place and low molecular weight thiols (RSH) substituted AgI species at the Cys-34 of BSA eventually forming disulfide (BSA-SSR) at Cys-34. It can be anticipated from the phase of study involving bovine serum albumin that low molecular weight thiolates (reduced glutathione and N-acetylcysteine) take off AgI which are attached to proteins elsewhere in the physiological system, making these toxic metals free for toxic action.

Distribution of Coomassie Blue Stainable Particles in the Pearl River Estuary, China, Insight Into the Nitrogen Cycling in Estuarine System

Distributions of Coomassie Blue stainable particles (CSP), the sources and transports, as well as their implications for nitrogen biogeochemical cycles in the Pearl River estuary (PRE) were investigated during two cruises in August 2016 and January 2017. CSPcolor concentrations (CSP concentration determined spectrophotometrically) were 73.7–685.3 μg BSA eq L–1 [μg Bovine serum albumin (BSA) equivalent liter–1] in August 2016 and 100.6–396.4 μg BSA eq L–1 in January 2017, respectively. CSP concentrations were high in low-salinity waters (<5), and declined from the river to the middle estuary by 80% in the wet season and 55.6% in the dry season, respectively, then increased again in the lower estuary due to high primary production. CSP concentrations were mainly associated with chlorophyll a (Chl a) concentration except for the turbid mixing zone, suggesting that autochthonous phytoplankton production served as the primary source of CSP in the PRE. The concentrations of nitrogen (N) as CSP in the PRE were comparable to the nitrogen content of particulate hydrolysable amino acids (PHAA). Pictures of CSP taken by microscopy and the correlation between composition of PHAA and the ratio of Chl a/CSPcolor showed that CSP were relatively degraded due to delivery of old terrestrial protein to river section and extensive microbial degradation during mixing at the upper and middle parts of the estuary, whereas CSP in lower estuary appeared to be more labile due to higher fresh algal production. The contribution of CSP nitrogen (CSP-N) to the particulate nitrogen (PN) pool was 34.98% in summer and 30.8% in winter. The conservative estimate of CSP-N input flux in the Pearl River Delta was about 6 × 106 mol N d–1. These results suggested that CSP was a significant pool of organic nitrogen in the PRE. The study of CSP composition in terms of nitrogen provides new insight into the roles of CSP on nitrogen biogeochemical processes in the turbid and productive estuarine system.

Physicochemical nature of sodium dodecyl sulfate interactions with bovine serum albumin revealed by interdisciplinary approaches

To rigorously characterize the interactions of sodium dodecyl sulfate (SDS) with bovine serum albumin (BSA) a set of experimental methods, namely isothermal titration calorimetry, conductometric titration, steady-state fluorescence spectroscopy, differential scanning calorimetry and circular dichroism spectroscopy, supported by in silico analysis have been applied. The influence of pH and temperature on the binding mode has been revealed. At a low molar ratio of SDS to BSA up to ca. 16:1, there are at least two structurally distinct binding sites in BSA. The formation of SDS-BSA complexes is an enthalpy-driven process in which the van der Waals interactions play a crucial role. The first binding site, located close to the Trp-134 residue within the sub-domain IA, is pH-independent and binds two molecules of SDS per one molecule of BSA whereas the total number of SDS molecules bound to the second site of albumin is affected by temperature and pH. The saturation of the first binding site of BSA (ca. 0.009 mg of SDS per 1 mg of BSA) is sufficient to thermally stabilize the helical conformation of BSA. The presented results have important structural and thermodynamic implications to understand the influence of a widely used anionic surfactant on globular protein functionality in modern branches of chemistry.

A Full Set of In Vitro Assays in Chitosan/Tween 80 Microspheres Loaded with Magnetite Nanoparticles.

Microspheres have been proposed for different medical applications, such as the delivery of therapeutic proteins. The first step, before evaluating the functionality of a protein delivery system, is to evaluate their biological safety. In this work, we developed chitosan/Tween 80 microspheres loaded with magnetite nanoparticles and evaluated cell damage. The formation and physical–chemical properties of the microspheres were determined by FT-IR, Raman, thermogravimetric analysis (TGA), energy-dispersive X-ray spectroscopy (EDS), dynamic light scattering (DLS), and SEM. Cell damage was evaluated by a full set of in vitro assays using a non-cancerous cell line, human erythrocytes, and human lymphocytes. At the same time, to know if these microspheres can load proteins over their surface, bovine serum albumin (BSA) immobilization was measured. Results showed 7 nm magnetite nanoparticles loaded into chitosan/Tween 80 microspheres with average sizes of 1.431 µm. At concentrations from 1 to 100 µg/mL, there was no evidence of changes in mitochondrial metabolism, cell morphology, membrane rupture, cell cycle, nor sister chromatid exchange formation. For each microgram of microspheres 1.8 µg of BSA was immobilized. The result provides the fundamental understanding of the in vitro biological behavior, and safety, of developed microspheres. Additionally, this set of assays can be helpful for researchers to evaluate different nano and microparticles.

66 Effect of the co-culture system and the culture medium on invitro embryo development in alpacas (Vicugna pacos)

The aim was to evaluate 4 co-culture systems and 4 culture medium types to produce alpaca embryos by IVF. Gametes were obtained from ovaries and testes collected from a slaughterhouse. Oocytes were recovered by follicle aspiration using a 5-mL syringe. Oocytes with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured for 26h in an incubator (5% CO2 in air at 38.5°C), in TCM-199 supplemented with 10% fetal calf serum (FCS), 0.02IU mL−1 FSH, 1µg mL−1 oestradiol 17β, 0.2mM sodium pyruvate, and 50µg mL−1 gentamicin sulphate. After maturation, oocytes were placed in FERT-TALP (fertilization- Tyrode’s medium with albumin, lactate, and pyruvate) solution for 30min before IVF with epididymal sperm. Motile sperm were selected by swim-up method. Oocytes were co-cultured with 3×106 spermatozoa mL−1 for 18 to 20h under the same atmospheric conditions mentioned above. In Experiment 1, we evaluated 4 co-culture systems: ear fibroblasts (T1, n=224), fetal fibroblasts (T2, n=154), granulosa cells (T3, n=225), oviduct cells (T4, n=169) and synthetic oviductal fluid (SOF) invitro culture (IVC) (T5, n=116). As culture base, we used 0.5mL of SOF IVC medium supplemented with 10% FCS in all treatments. In Experiment 2, we evaluated 4 culture media: TCM-199+10% FCS (T1, n=137), CR1aa+3mg of bovine serum albumin (BSA) mL−1 (T2, n=85), KSOM medium+3mg of BSA mL−1 (T3, n=110), and SOF IVC+10% FCS (T4, n=66). The volume of culture medium used was 0.5mL in 4-well Nunc plates for each treatment. The time (8 days) and culture (38.5°C, 5% CO2 in air and high humidity) conditions, and change of medium (each 24h) were the same in both experiments. Statistical significance was determined using ANOVA. The mean and s.d. were calculated from the average of the percentages obtained in each repetition. In experiment 1, the cleavage rates were higher (P<0.05) in co-culture with fetal fibroblasts (46%±0.17), oviduct cells (50%±0.09), and ear fibroblasts (58%±0.17) than with granulosa cells (28%±0.12) and SOF IVC (30%±0.18). Also, the morula rates were higher (P<0.05) in co-culture with fetal fibroblasts (35%±0.16), oviduct cells (31%±0.01), and ear fibroblasts (35%±0.14) than with granulosa cells (11%±0.01) and SOF IVC (27%±0.17). In contrast, there were no differences in blastocyst rates between co-culture with granulosa cells (10%±0.04), SOF IVC (12%±0.09), and fetal fibroblasts (24%±0.14). However, there were differences between co-culture with oviduct cells (19%±0.06) and ear fibroblasts (32%±0.14). In Experiment 2, there were no differences in cleavage rates between TCM-199+FCS (28%±0.12), CR1aa+BSA (44%±0.24), KSOM+BSA (38%±0.19), and SOF-IVC+FCS (50%±0.20). However, there were differences in morula rates between CR1aa+BSA (7%±0.09) and SOF IVC+FCS (35%±0.21), TCM-199+FCS (23%±0.09), and KSOM+BSA (27%±0.15). We obtained a higher blastocyst rates in SOF IVC+BSA (24±0.12) and KSOM+BSA than with CR1aa+BSA (3±0.06) and TCM-199+FCS (9±0.03). In conclusion, KSOM and SOF-IVC were the best media for culture, and oviduct cells and ear and fetal fibroblasts were the best cells to produce alpaca embryos by IVF.

Overview of Portable Assays for the Detection of Mycotoxins, Allergens, and Sanitation Monitoring.

BackgroundMany food recalls are related to the presence of undeclared allergens and microorganisms in food products. To reduce these occurrences, portable diagnostic assay kits are available to quantitate mycotoxins, to detect allergens and gluten in foods and on environmental surfaces, and for sanitation monitoring.ObjectiveThis article reviews diagnostic kits that can detect sources of contamination in food and ingredients as well as on surfaces and clean-in-place rinses.MethodMycotoxins and gluten were detected using lateral flow diagnostic assays. Sanitation monitoring of surfaces was completed using a chemiluminescent assay to detect adenosine 5′-triphosphate disodium salt hydrate (ATP) and another assay to detect protein.ResultsGluten was detected at 10 ppm in spiked commodities and on wet and dry surfaces at 2.5 µg/100cm2. Deoxynivalenol was quantitated in dry distillers grains plus solubles and mean results were within two SDs of those determined by HPLC. The chemiluminescent assay had an LOD of 6 fmol of ATP and was able to detect a 1:10 000 dilution of orange juice from surfaces. The protein assay detected 5 µg of bovine serum albumin (BSA) directly applied to the sampler, 100 µg of BSA on surfaces, and detected 1:10 dilutions of Greek yogurt and raw beef from surfaces.ConclusionsPortable diagnostic kits evaluated in this work provided accurate, rapid, and sensitive results for detection of mycotoxins, gluten, proteins, and ATP. These methods can be used in facilities with minimal training and provide results that are important to ensure food safety.HighlightsPortable methods to detect gluten, mycotoxins, proteins, and ATP are presented.

Histopathological changes due to arsenic Kushta in testes of wistar rats.

Objectives: To see the detrimental effects of arsenic kushta (kushta-e-Sammulfur) induced toxicity in wistar rats. Study Design: Experimental Study. Setting: Department of Morbid Anatomy and Histopathology, University of Health Sciences, Lahore. Period: 02nd June 2016 to 30th September 2016. Material & Methods: This experimental study was conducted on a total of 48 wistar rats, each weighing approximately, 200-300grams. These rats were then randomly divided into four groups each of them comprising of 12 rats. Group I was taken as a control group which was given flour pellets. Group II was given a single dose of 180 mg/kg of arsenic kushta for 2 weeks, whereas Groups III was given 150 mg/kg of arsenic kushta only for 12 weeks and Group IV was also given dose of 150 mg/kg of arsenic kushta along with 75mg of Bovine Serum Albumin for 12 weeks. Histopathological changes were then seen in the germinal epithelium, stages of spermatogenesis and insterstitium of testes of rats. Results: Changes in germinal epithelium, stages of spermatogenesis and interstitium were seen in all the above groups except group 1 which was the control group. Maximum changes were seen in groups C and D which were given high doses of arsenic kushta along with injection of bovine serum albumin. Conclusion: Arsenic kushta preparation of kushta-e-Summulfur causes testicular toxicity in wistar rats and have similar toxic effects in human beings.

Anti-stress phytohormones impact on proteome profile of green gram (Vigna radiata) under salt toxicity

Anti-stress phytohormones impact on proteome profile of green gram (Vigna radiata) under salt toxicity

Generation of BSA-capsaicin Nanoparticles and Their Hormesis Effect on the Rhodotorula mucilaginosa Yeast.

Capsaicin is a chemical compound found in pungent chili peppers (Capsicum spp.). In biotechnology, capsaicin has been proposed as a pathogen control; however, its low solubility in water and high instability limits its uses. The aim of this work was to study the effect of high concentrations of capsaicin on the synthesis of nanoparticles and to evaluate their inhibitory effect on the growth of Rhodotorula mucilaginosa yeast. Bovine serum albumin (BSA)-capsaicin nanoparticles were formulated at 0, 16.2, 32.5, 48.7 and 65.0 µg of capsaicin per mg of BSA. Nanoparticle properties were evaluated and they were added to cultures of R. mucilaginosa to quantify their effect on cell viability. We found that increased capsaicin levels caused several changes to the physicochemical parameters, probably due to changes in the hydrophobicity sites of the albumin during the nanostructuration. The administration of nanoparticles to cultures of R. mucilaginosa produced a maximal viability with nanoparticles at 16.2 µg/mg; on the contrary, nanoparticles at 65.0 µg/mg caused maximal cell death. R. mucilaginosa cells displayed a hormesis effect in response to the nanoparticle dose concentration. The nanoparticles showed different responses during the uptake process, probably as a consequence of the nanostructural properties of capsaicin in the BSA molecules.

Adsorption of bovine serum albumin (BSA) by bare magnetite nanoparticles with surface oxidative impurities that prevent aggregation

Bare, uncoated magnetite nanoparticles, synthesized using an electrochemical surfactant-free synthesis, have highly oxidized surfaces that prevent aggregation. These particles have demonstrated highly intriguing biological activity showing extremely potent antibiotic activity against both gram-positive and gram-negative bacteria with little toxicity to rats. This difference in activity could be ascribed to the nature of the protein corona. The kinetics and thermodynamics of the binding of bovine serum albumin (BSA), used as a model serum protein, to these magnetite nanoparticles were analyzed. There is no significant change in particle diameter by dynamic light scattering following adsorption, indicating corona formation does not induce aggregation. The maximum adsorption capacity of the particles was determined to be 300 mg of BSA per gram of magnetite. The particles are able to adsorb 90% of the BSA at protein concentrations as high as 500 mg/L. The adsorption is best described using a pseudo second order model and a Langmuir Type III isotherm model. Thermodynamic analysis showed that the process is entropically driven and is spontaneous at all tested temperatures and conditions. However, it appears to be a weak to moderate physical adsorption. This moderate binding affinity could indicate the differential biological activity of these particles towards bacteria and mammalian cells and further support the contention that these are potentially useful new tools for targeting antibiotic-resistant bacteria.

Enhanced bovine serum albumin absorption on the N-hydroxysuccinimide activated graphene oxide and its corresponding cell affinity

By successively reacting with N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), the carboxyl on the graphene oxide (GO) surface was successfully activated into NHS active ester. In this study, bovine serum albumin (BSA) was selected as a model protein, used for studying the protein absorption capacity of the NHS activated GO (GO-EDC-NHS). Approximately 12.75mg of BSA could be covalent bonded onto the GO-EDC-NHS surface (BSA-CB-GO), whereas only 6.83mg of BSA physical absorbed onto the GO surface (BSA-NB-GO). With a 168h of phosphate buffer saline (PBS) soaking, the BSA accumulative desorption ratio, which was accordingly assigned to the BSA-NB-GO and the BSA-CB-GO, was separately 29.91wt% and 2.95wt%. Consequently, it proved GO-EDC-NHS exhibited more stable and stronger BSA absorption capacity. As compared to the mouse bone marrow mesenchymal stem cells (mBMSCs) cultivated on the BSA-NB-GO surface, the immunofluorescence staining images showed that more vinculins and integrin α5 were visible in the mBMSCs cultivated on the BSA-CB-GO surface, they also produced more distinct stress fibers and actin-containing microfilaments. In summary, BSA-CB-GO possesses an excellent cell affinity, which can be considered as a promising functional material used for promoting the bone remodeling.

Podocytes regulate the glomerular basement membrane protein nephronectin by means of miR-378a-3p in glomerular diseases

The pathophysiology of many proteinuric kidney diseases is poorly understood, and microRNAs (miRs) regulation of these diseases has been largely unexplored. Here, we tested whether miR-378a-3p is a novel regulator of glomerular diseases. MiR-378a-3p has two predicted targets relevant to glomerular function, the glomerular basement membrane matrix component, nephronectin (NPNT), and vascular endothelial growth factor VEGF-A. In zebrafish (Danio rerio), miR-378a-3p mimic injection or npnt knockdown by a morpholino oligomer caused an identical phenotype consisting of edema, proteinuria, podocyte effacement, and widening of the glomerular basement membrane in the lamina rara interna. Zebrafish vegf-A protein could not rescue this phenotype. However, mouse Npnt constructs containing a mutated 3'UTR region prevented the phenotype caused by miR-378a-3p mimic injection. Overexpression of miR-378a-3p in mice confirmed glomerular dysfunction in a mammalian model. Biopsies from patients with focal segmental glomerulosclerosis and membranous nephropathy had increased miR-378a-3p expression and reduced glomerular levels of NPNT. Thus, miR-378a-3p-mediated suppression of the glomerular matrix protein NPNT is a novel mechanism for proteinuria development in active glomerular diseases.

Size-resolved characterization of the polysaccharidic and proteinaceous components of sea spray aerosol

Dissolved organic polymers released by phytoplankton and bacteria abiologically self-assemble in surface ocean waters into nano-to micro-sized gels containing polysaccharides, proteins, lipids and other components. These gels concentrate in the sea surface microlayer (SML), where they can potentially contribute to sea spray aerosol (SSA). Sea spray is a major source of atmospheric aerosol mass over much of the earth's surface, and knowledge of its properties (including the amount and nature of the organic content), size distributions and fluxes are fundamental for determining its role in atmospheric chemistry and climate. Using a cascade impactor, we collected size-fractionated aerosol particles from ambient air and from freshly generated Sea Sweep SSA in the western North Atlantic Ocean together with biological and chemical characterization of subsurface and SML waters. Spectrophotometric methods were applied to quantify the polysaccharide-containing transparent exopolymer (TEP) and protein-containing Coomassie stainable material (CSM) in these particles and waters. This study demonstrates that both TEP and CSM in surface ocean waters are aerosolized with sea spray with the greatest total TEP associated with particles <180 nm in diameter and >5 000 nm. The higher concentrations of TEP and CSM in particles >5 000 nm most likely reflects collection of microorganism cells and/or fragments. The greater concentration of CSM in larger size particles may also reflect greater stability of proteinaceous gels compared to polysaccharide-rich gels in surface waters and the SML. Both TEP and CSM were measured in the ambient marine air sample with concentrations of 2.1 ± 0.16 μg xanthan gum equivalents (XG eq.) m−3 and 14 ± 1.0 μg bovine serum albumin equivalents (BSA eq.) m−3. TEP in Sea Sweep SSA averaged 4.7 ± 3.1 μg XG eq. m−3 and CSM 8.6 ± 7.3 μg BSA eq. m−3. This work shows the transport of marine biogenic material across the air-sea interface through primary particle emission and the first demonstration of particle size discriminated TEP and CSM characterization of SSA and ambient aerosol under field conditions.

FTIR characterization and release of bovine serum albumin from bioactive glasses.

Bioactive glass has attracted substantial interest in orthopedics, but it has been less explored as a drug carrier. This study investigated the bovine serum albumin (BSA) release from bioactive 13-93B0 and 13-93B3 glasses. Glass disks (13-93B0 and 13-93B3; n = 5) were loaded with 4 mg of BSA and coated under different chitosan-coating conditions. The amount of BSA released in phosphate-buffered saline (PBS) was evaluated, and a degradation study was performed to find out the weight loss and pH of PBS. Secondary structures of BSA on 13-93B0 were characterized by Fourier transform infrared (FTIR) spectroscopy. One hundred percent protein release occurred by 24 hours for all 13-93B3 groups. However, chitosan coating delayed 100% release up to 72 hours in 13-93B0 groups. The 13-93B3 glass showed higher degradation rates than 13-93B0 regardless of chitosan-coating status. Multilayer and sandwich chitosan coatings further delayed BSA release from 13-93B0. FTIR analysis revealed that α-helical structure was the highest among all groups and significantly higher in the 2% sandwich chitosan coating group (32.0% ± 2.1%), compared with uncoated and 4% chitosan groups. Chitosan coating can delay the burst release of BSA from 13-93B0 glass and be a potential coating on bioactive glass for drug delivery purposes.

Tandem Delivery Platform for Small Molecules and Biologic Pharmaceuticals Via Silicone Foam1

Tandem Delivery Platform for Small Molecules and Biologic Pharmaceuticals Via Silicone Foam1

A Novel PD-L1-targeting Antagonistic DNA Aptamer With Antitumor Effects.

The PD-1/PD-L1 axis is a major pathway involved in tumor immune evasion. Here, we report the novel PD-L1 antagonizing DNA aptamer (aptPD-L1) and demonstrate an integrated pipeline that expedites therapeutic aptamer development. Aptamer can exert antibody-mimic functions and is advantageous over antibody for its chemically synthetic nature, low immunogenicity, and efficient tissue penetration. Our results showed that aptPD-L1 blocked the binding between human PD-1 and PD-L1. Experiments using murine models showed that aptPD-L1 promoted in vitro lymphocyte proliferation and suppressed in vivo tumor growth without the induction of observable liver or renal toxicity. Analyses on the aptPD-L1-treated tumors further revealed elevated levels of infiltrating CD4+ and CD8+ T cells, intratumoral IL-2, TNF-α, interferon (IFN)-γ and the C-X-C motif chemokines, CXCL9 and CXCL10. The CD8+ T cells in the aptPD-L1-treated tumors had higher CXCR3 expression level compared to the random-sequence oligonucleotides-treated ones. Besides, the length and density of CD31+ intratumoral microvessels were significantly decreased in the aptPD-L1 treatment group. Collectively, our data suggested that aptPD-L1 helps T cell function restoration and modifies tumor microenvironment. These chemokines might orchestrate together to attract more T cells into the tumor tissues to form the positive amplification loop against tumor growth, indicating the translational potential of aptPD-L1 in cancer immunotherapy.

Blood Antioxidant Defenses and Sickle Cell Disease: Analyzing Cytochrome b Reductase 1 Protein Levels and Structure in Erythrocyte Membranes

Introduction: The pathology of sickle cell disease (SCD) includes ischemia / reperfusion events in the vasculature, compromised blood antioxidant defenses and elevated risk of cardiovascular complications. Vitamin C (ascorbate) is a major blood antioxidant; ascorbate inside red blood cells (RBC) furnishes an electron (e-) to recycle plasma ascorbate to its active, reduced state (Fig. 1). A key step, transmembrane e- transfer, involves cytochrome b reductase 1 (CYBRD1). Thus, a deficiency or defect in red cell CYBRD1 could plausibly increase oxidative stress and interconnected inflammatory processes, contributing to cardiovascular complications in SCD patients. Testing for CYBRD1 deficiency requires a quantitative assay for the protein in RBC, so we developed a suitable assay and used it to screen a small number of SCD patients and healthy individuals.Materials and methods: Blood samples were collected from steady state adult SCD patients, healthy African American adults, and steady state pediatric SCD patients before and 2-5 months on hydroxyurea (HU) therapy. Protocols were approved by institutional review.The CYBRD1 assay workflow is shown in Fig. 2. RBC membrane proteins were separated on two SDS-PAGE gels, along with a mixed standard (1.0 µg bovine serum albumin (BSA) + 1.9 ng of His6-tagged recombinant human CYBRD1 (rCYBRD1; PMID 21501687)). One gel was Coomassie-stained for quantitation of total membrane protein minus membrane-bound hemoglobin (Hb) (standard: BSA). Proteins in the other gel were transferred to a nitrocellulose filter, probed with CYBRD1 antibody (GeneTex #104793) and visualized with secondary antibody and chromogenic substrate for quantitation of CYBRD1 (standard: rCYBRD1), which was normalized to membrane protein. Gels, blots and calibrated density step tablets were scanned (transmittance mode) with an Epson V550; digitized images were analyzed using ImageJ. Two independent samples and paired t tests were used to compare between- and within-group mean differences in RBC CYBRD1 levels, respectively.Results: The Coomassie-stained gels exhibited the usual profile of RBC membrane proteins (Fig. 2). In the immunoblots, the rCYBRD1 standard showed the expected narrow monomer band at ~30 kDa and faint dimer band at ~60 kDa (Fig. 2). However, RBC membrane samples revealed several CYBRD1 monomer bands, indicating the presence of previously unsuspected isoforms of CYBRD1. Further, the CYBRD1 isoform pattern in RBC from healthy controls was quite different from those seen in SCD patients. The functional impact of these differences remains to be investigated.Assay of progressively diluted control RBC membranes produced a linear response for CYBRD1 from 0.7-7.2 ng (r2 = 0.975) and for total protein from 1 to 10 µg (r2 = 0.993). The sub-nanogram detection of CYBRD1 protein makes the assay usable with as little as 0.25 ml of blood. The coefficient of variation was ~15%, quite reasonable precision for quantitative immunoblotting.CYBRD1 levels in RBC from healthy African American adults averaged 0.68 ± 0.13 ng/µg membrane protein (N1 = 10; Fig. 3). CYBRD1 levels in RBC of adult SCD patients averaged 0.35 ± 0.14 ng/µg membrane protein (N2=7). This difference in means was highly statistically significant by two-tailed t-test (p < 0.0002), supporting our hypothesis that a deficiency in RBC CYBRD1 contributes to elevated oxidative stress in SCD patients.RBC CYBRD1 levels in pediatric SCD patients not on HU therapy averaged 0.27 ± 0.14 ng/µg protein (N3=12; Fig. 3). Pediatric patients on HU therapy averaged 0.41 ± 0.18 ng CYBRD1/µg membrane protein (N4=7). For the five pediatric patients examined both before and during HU therapy there was a marginally significant increased mean CYBRD1 level on HU (p < 0.099 by paired, two-tailed t test).Conclusions: Our newly developed assay allows quantitation of the RBC CYBRD1 protein level and analysis of the CYBRD1 isoform pattern using a small blood sample. The pilot screening results suggest that adult SCD patients have lower levels of CYBRD1 in their RBC, and a different pattern of CYBRD1 isoforms. Analysis of the CYBRD1 protein may be useful in assessing the state of individual SCD patients' blood antioxidant defenses and the antioxidant effects of HU. Thus, it will be worthwhile to examine the RBC CYBRD1 protein and activity in larger numbers of patients and controls, and to explore the origins of any differences between the groups. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dry powder pulmonary delivery of cationic PGA-co-PDL nanoparticles with surface adsorbed model protein

Pulmonary delivery of macromolecules has been the focus of attention as an alternate route of delivery with benefits such as; large surface area, thin alveolar epithelium, rapid absorption and extensive vasculature. In this study, a model protein, bovine serum albumin (BSA) was adsorbed onto cationic PGA-co-PDL polymeric nanoparticles (NPs) prepared by a single emulsion solvent evaporation method using a cationic surfactant didodecyldimethylammonium bromide (DMAB) at 2% w/w (particle size: 128.64±06.01nm and zeta-potential: +42.32±02.70mV). The optimum cationic NPs were then surface adsorbed with BSA, NP:BSA (100:4) ratio yielded 10.01±1.19μg of BSA per mg of NPs. The BSA adsorbed NPs (5mg/ml) were then spray-dried in an aqueous suspension of L-leucine (7.5mg/ml, corresponding to a ratio of 1:1.5/NP:l-leu) using a Büchi-290 mini-spray dryer to produce nanocomposite microparticles (NCMPs) containing cationic NPs. The aerosol properties showed a fine particle fraction (FPF, dae<4.46μm) of 70.67±4.07% and mass median aerodynamic diameter (MMAD) of 2.80±0.21μm suggesting a deposition in the respiratory bronchiolar region of the lungs.The cell viability was 75.76±03.55% (A549 cell line) at 156.25μg/ml concentration after 24h exposure. SDS-PAGE and circular dichroism (CD) confirmed that the primary and secondary structure of the released BSA was maintained. Moreover, the released BSA showed 78.76±1.54% relative esterolytic activity compared to standard BSA.

Preparation and characterization of genipin-cross-linked silk fibroin/chitosan sustained-release microspheres.

We report the effects of distinct concentrations of genipin and silk fibroin (SF):chitosan (CS) ratios on the formation of SF–CS composite microspheres. We selected microspheres featuring an SF:CS ratio of 1:1, encapsulated various concentrations of bovine serum albumin (BSA), and then compared their encapsulation efficiency and sustained-release rate with those of pure CS microspheres. We determined that the following five groups of microspheres were highly spherical and featured particle sizes ranging from 70 μm to 147 μm: mass ratio of CS:SF =1:0.5, 0.1 g or 0.5 g genipin; CS:SF =1:1, 0.05 g or 1 g genipin; and CS:SF =1:2, 0.5 g genipin. The microspheres prepared using 1:1 CS:SF ratio and 0.05 g genipin in the presence of 10 mg, 20 mg, and 50 mg of BSA exhibited encapsulation efficiencies of 50.16%±4.32%, 56.58%±3.58%, and 42.19%±7.47%, respectively. Fourier-transform infrared spectroscopy (FTIR) results showed that SF and CS were cross-linked and that the α-helices and random coils of SF were converted into β-sheets. BSA did not chemically react with CS or SF. Moreover, thermal gravimetric analysis (TGA) results showed that the melting point of BSA did not change, which confirmed the FTIR results, and X-ray diffraction results showed that BSA was entrapped in microspheres in a noncrystalline form, which further verified the TGA and FTIR data. The sustained-release microspheres prepared in the presence of 10 mg, 20 mg, and 50 mg of BSA burst release 30.79%±3.43%, 34.41%±4.46%, and 41.75%±0.96% of the entrapped BSA on the 1st day and cumulatively released 75.20%±2.52%, 79.16%±4.31%, and 89.04%±4.68% in 21 days, respectively. The pure CS microspheres prepared in the presence of 10 mg of BSA burst release 39.53%±1.76% of BSA on the 1st day and cumulatively released 83.57%±2.33% of the total encapsulated BSA in 21 days. The SF–CS composite microspheres exhibited higher sustained release than did the pure CS microspheres, and thus these composite microspheres might function as a superior drug carrier.