An Excel fitting routine for correcting protein absorption spectra for scatter.

Protein concentrations are often determined in a non-destructive manner by measuring the absorbance at 280 nm. However, light scattering in protein samples can complicate such assessment. We here describe a simple Excel Solver-based fitting routine to correct full protein UV absorption spectra for both Rayleigh and Mie scattering. Using samples displaying various degrees of natural and artificially induced scattering, we show that our multi-wavelength fitting method is not only capable of aiding in the determination of protein concentrations but can also be employed in the spectral analysis of protein structural changes that are accompanied by alterations in scatter intensity.

Bovine Serum Albumin Interaction with Polyanionic and Polycationic Brushes: The Case Theoretical Study.

We apply a coarse-grained self-consistent field Poisson-Boltzmann framework to study interaction between Bovine Serum Albumin (BSA) and a planar polyelectropyte brush. Both cases of negatively (polyanionic) and positively (polycationic) charged brushes are considered. Our theoretical model accounts for (1) re-ionization free energy of the amino acid residues upon protein insertion into the brush; (2) osmotic force repelling the protein globule from the brush; (3) hydrophobic interactions between non-polar areas on the globule surface and the brush-forming chains. We demonstrate that calculated position-dependent insertion free energy exhibits different patterns, corresponding to either thermodynamically favourable BSA absorption in the brush or thermodynamically or kinetically hindered absorption (expulsion) depending on the pH and ionic strength of the solution. The theory predicts that due to the re-ionization of BSA within the brush, a polyanionic brush can efficiently absorb BSA over a wider pH range on the "wrong side" of the isoelectric point (IEP) compared to a polycationic brush. The results of our theoretical analysis correlate with available experimental data and thus validate the developed model for prediction of the interaction patterns for various globular proteins with polyelectrolyte brushes.

Aluminum Foil vs. Gold Film: Cost-Effective Substrate in Sandwich SERS Immunoassays of Biomarkers Reveals Potential for Selectivity Improvement.

The first application of aluminum foil (Al F) as a low-cost/high-availability substrate for sandwich immunoassay using surface-enhanced Raman spectroscopy (SERS) is reported. Untreated and unmodified Al F and gold film are used as substrates for sandwich SERS immunoassay to detect tuberculosis biomarker MPT64 and human immunoglobulin (hIgG) in less than 24 h. The limits of detection (LODs) for tuberculosis (TB) biomarker MPT64 on Al foil, obtained with commercial antibodies, are about 1.8-1.9 ng/mL, which is comparable to the best LOD (2.1 ng/mL) reported in the literature for sandwich ELISA, made with fresh in-house antibodies. Not only is Al foil competitive with traditional SERS substrate gold for the sandwich SERS immunoassay in terms of LOD, which is in the range 18-30 pM or less than 1 pmol of human IgG, but it also has a large cost/availability advantage over gold film. Moreover, human IgG assays on Al foil and Si showed better selectivity (by about 30-70% on Al foil and at least eightfold on Si) and a nonspecific response to rat or rabbit IgG, in comparison to the selectivity in assays using gold film.

The effect of Bacillus coagulans Unique IS-2 supplementation on plasma amino acid levels and muscle strength in resistance trained males consuming whey protein: a double-blind, placebo-controlled study.

PURPOSE: The aim of the present study was to assess the effect of Bacillus coagulans Unique IS-2 supplementation on absorption and utilization of protein in resistance-trained males. METHODS: In this double blind, placebo-control trial, resistance-trained males (21.08 ± 2.84 years) were randomized to consume, either 20 g of whey protein powder {80% whey protein concentrate (WPC80), amounting to 15.4 g protein} with 2 billion CFU Bacillus coagulans Unique IS-2 (supplemental group) or 20 g of whey protein powder and lactose instead of Bacillus coagulans (placebo group) once daily for 60 days with a controlled resistance exercise protocol. The whey protein concentrate (WPC-80) given to both groups had a lactose content of 6.8%. Plasma-free amino acids (PFAAs) were determined at baseline, at 30 and 60 days of supplementation. Muscle strength, hypertrophy, VO2 max, and body composition, and other biochemical parameters were assessed at baseline and end line. RESULTS: A positive effect of probiotic Bacillus coagulans Unique IS-2 supplementation was observed on protein absorption as evidenced by an increase in total PFAA by + 16.1% (p = 0.004). Branched chain amino acids (BCAA) comprising isoleucine (p = 0.016), leucine (p = 0.001), and valine (p = 0.002) were increased by + 33.1% in ITT analysis as compared to placebo after 60 days. At 30 days an increase in isoleucine by + 35% (p = 0.113), leucine by + 43% (p = 0.032), and valine by + 32% (p = 0.017) was observed in ITT analysis. Probiotic effect was shown on exercise performance as evidenced by an increase in one RM of leg press and vertical jump power by + 16.61% (p = 0.024) and + 7.86% (p = 0.007), respectively. CONCLUSION: Significantly increased absorption of BCAA with supplementation of B. coagulans Unique IS-2 along with whey protein and improvement in leg press and vertical jump power was noted indicating the positive effect of the probiotic on muscle power in the lower body. TRIAL REGISTRATION NUMBER: CTRI/2017/03/008117; Date:16.03.2017.

Postprandial amino acid availability after intake of intact or hydrolyzed meat protein in a mixed meal in healthy elderly subjects: a randomized, single blind crossover trial.

The absorption of dietary proteins affects the anabolic response, among others protein synthesis. For elderly, optimal amino acid absorption is warranted to preserve the amino acid pool of the body, especially skeletal muscle proteins. Therefore, the aim of this study was to characterize if hydrolyzing meat protein (HMP) would improve the amino acid absorption after ingestion of meat compared to equal amounts of the same meat proteins but present in a different structure; steak or minced meat. With a crossover study design on 12 healthy older adults (> 65 years of age, BMI 18.5-30), the amino acid absorption kinetics were explored by ingesting 0.55 g protein/kg LBM as a mixed meal together with intrinsically [2H5]phenylalanine labeled meat proteins prepared as a STEAK, MINCED meat, or HMP. Plasma [2H5]phenylalanine enrichment as well as AA concentrations were measured by mass spectrometry from blood samples drawn during a 5-h postprandial period. After HMP ingestion, [2H5]phenylalanine was faster absorbed in the initial 2 h compared to STEAK and MINCED. The peak time in AA concentrations was faster in HMP compared to STEAK and MINCED. However, the peak AA concentrations were not different between STEAK, MINCED, and HMP. Although HMP showed to have the fastest initial amino acid appearance in older adults, the peak EAA concentrations were similar after ingesting meal with either STEAK, MINCED, or HMP in the 5-h postprandial period.

Multiphysics Modeling and Simulation of Subcutaneous Injection and Absorption of Biotherapeutics: Model Development.

PURPOSE: Many monoclonal antibodies (mAbs) are administered via subcutaneous (SC) injection. Local transport and absorption kinetics and mechanisms, however, remain poorly understood. A multiphysics computational model was developed to simulate the injection and absorption processes of a protein solution in the SC tissue. METHODS: Quantitative relationships among tissue properties and transport behaviors of an injected solution were described by respective physical laws. SC tissue was treated as a 3-dimensional homogenous, poroelastic medium, in which vasculatures and lymphatic vessels were implicitly treated. Tissue deformation was considered, and interstitial fluid flow was modeled by Darcy's law. Transport of the drug mass was described based on diffusion and advection, which was integrated with tissue mechanics and interstitial fluid dynamics. RESULTS: Injection and absorption of albumin and IgG solutions were simulated. Upon injection, a sharp rise in tissue pressure, porosity, and fluid velocity could be observed at the injection tip. Largest tissue deformation appeared at the model surface. Transport of drug mass out of the injection zone was minimal. Absorption by local lymphatics was found to last several weeks. CONCLUSIONS: A bottom-up method was developed to simulate drug transport and absorption of protein solutions in skin tissue base on physical principles. The results appear to match experimental observations.

Multiphysics Modeling and Simulation of Subcutaneous Injection and Absorption of Biotherapeutics: Sensitivity Analysis.

PURPOSE: A multiphysics simulation model was recently developed to capture major physical and mechanical processes of local drug transport and absorption kinetics of subcutaneously injected monoclonal antibody (mAb) solutions. To further explore the impact of individual drug attributes and tissue characteristics on the tissue biomechanical response and drug mass transport upon injection, sensitivity analysis was conducted and reported. METHOD: Various configurations of injection conditions, drug-associated attributes, and tissue properties were simulated with the developed multiphysics model. Simulation results were examined with regard to tissue deformation, porosity change, and spatiotemporal distributions of pressure, interstitial fluid flow, and drug concentration in the tissue. RESULTS: Injection conditions and tissue properties were found influential on the mechanical response of tissue and interstitial fluid velocity to various extents, leading to distinct drug concentration profiles. Intrinsic tissue porosity, lymphatic vessel density, and drug permeability through the lymphatic membrane were particularly essential in determining the local absorption rate of an mAb injection. CONCLUSION: The sensitivity analysis study may shed light on the product development of an mAb formulation, as well as on the future development of the simulation method.

Overcoming Nanoparticle-Mediated Complement Activation by Surface PEG Pairing.

Many PEGylated nanoparticles activate the complement system, which is an integral component of innate immunity. This is of concern as uncontrolled complement activation is potentially detrimental and contributes to disease pathogenesis. Here, it is demonstrated that, in contrast to carboxyPEG2000-stabilized poly(lactic-co-glycolic acid) nanoparticles, surface camouflaging with appropriate combinations and proportions of carboxyPEG2000 and methoxyPEG550 can largely suppress nanoparticle-mediated complement activation through the lectin pathway. This is attributed to the ability of the short, rigid methoxyPEG550 chains to laterally compress carboxyPEG2000 molecules to become more stretched and assume an extended, random coil configuration. As supported by coarse-grained molecular dynamics simulations, these conformational attributes minimize statistical protein binding/intercalation, thereby affecting sequential dynamic processes in complement convertase assembly. Furthermore, PEG pairing has no additional effect on nanoparticle longevity in the blood and macrophage uptake. PEG pairing significantly overcomes nanoparticle-mediated complement activation without the need for surface functionalization with complement inhibitors.

Short- and long-term low-salinity acclimation effects on the branchial and intestinal gene expression in the European seabass (Dicentrarchus labrax).

The European seabass (Dicentrarchus labrax) is a teleost remarkably adapted to a wide range of water salinity, through osmoregulatory mechanisms, mainly operating in the gills and the intestine. As an important aquaculture species, its rearing in low-salinity conditions offers benefits for its inland culture. However, this demands a full comprehension of the European seabass osmoregulatory mechanisms and its response to acclimation protocols. The purpose of this study was to evaluate different acclimation protocols in terms of osmoregularity and stress response, following transferring of European seabass juveniles from seawater to freshwater. In addition, nutrient absorption was also examined since drinking rates are sensitive to salinity. The acclimation challenge was applied through three protocols: direct transfer (0 h) to freshwater, gradual transfer during 3 h and during 72 h. The short- (1 h after complete change to freshwater) and long-term effects (after 2 months) of each acclimation protocol were evaluated by assessing the expression of 1. The osmoregulatory genes: Na+/K+-ATPase α1, Na+/K+/2Cl- 1 co-transporter, aquaporins 1 and 3, and the cystic fibrosis transmembrane conductance regulator; 2. The heat shock protein 70 gene; 3. The peptide transporter genes corresponding to PepT1a, PepT1b and PepT2. The short-term acclimation response was pronounced in both gills and the intestine affecting stress-, osmoregulatory- and nutrient-related gene expression. Long-term effects were only evident in the intestine. Direct transfer in freshwater mainly induced a long-term stress response, while the short-term effect was more pronounced in the 3 h-transfer, potentially due to handling. Our results suggest that although the European seabass can withstand direct transfer to low-salinity conditions, a gradual transfer is recommended to prevent long-term stress effects.

Molecular Dynamic Simulations for Biopolymers with Biomedical Applications.

Computational modeling (CM) is a versatile scientific methodology used to examine the properties and behavior of complex systems, such as polymeric materials for biomedical bioengineering. CM has emerged as a primary tool for predicting, setting up, and interpreting experimental results. Integrating in silico and in vitro experiments accelerates scientific advancements, yielding quicker results at a reduced cost. While CM is a mature discipline, its use in biomedical engineering for biopolymer materials has only recently gained prominence. In biopolymer biomedical engineering, CM focuses on three key research areas: (A) Computer-aided design (CAD/CAM) utilizes specialized software to design and model biopolymers for various biomedical applications. This technology allows researchers to create precise three-dimensional models of biopolymers, taking into account their chemical, structural, and functional properties. These models can be used to enhance the structure of biopolymers and improve their effectiveness in specific medical applications. (B) Finite element analysis, a computational technique used to analyze and solve problems in engineering and physics. This approach divides the physical domain into small finite elements with simple geometric shapes. This computational technique enables the study and understanding of the mechanical and structural behavior of biopolymers in biomedical environments. (C) Molecular dynamics (MD) simulations involve using advanced computational techniques to study the behavior of biopolymers at the molecular and atomic levels. These simulations are fundamental for better understanding biological processes at the molecular level. Studying the wide-ranging uses of MD simulations in biopolymers involves examining the structural, functional, and evolutionary aspects of biomolecular systems over time. MD simulations solve Newton's equations of motion for all-atom systems, producing spatial trajectories for each atom. This provides valuable insights into properties such as water absorption on biopolymer surfaces and interactions with solid surfaces, which are crucial for assessing biomaterials. This review provides a comprehensive overview of the various applications of MD simulations in biopolymers. Additionally, it highlights the flexibility, robustness, and synergistic relationship between in silico and experimental techniques.