Complement-Opsonized Nano-Carriers Are Bound by Dendritic Cells (DC) via Complement Receptor (CR)3, and by B Cell Subpopulations via CR-1/2, and Affect the Activation of DC and B-1 Cells

The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses.     

Tyrp1 Mutant Variants Associated with OCA3: Computational Characterization of Protein Stability and Ligand Binding

Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder caused by mutations in the TYRP1 gene. Tyrosinase-related protein 1 (Tyrp1) is involved in eumelanin synthesis, catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA) to 5,6-indolequinone-2-carboxylic acid (IQCA). Here, for the first time, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally to understand Tyrp1 protein stability and catalytic activity. Using the Tyrp1 crystal structure (PDB:5M8L), global mutagenesis was conducted to evaluate mutant protein stability. Consistent with the foldability parameter, C30R and H215Y should exhibit greater instability, and two other mutants, D308N and R326H, are expected to keep a native conformation. SDS-PAGE and Western blot analysis of the purified recombinant proteins confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability. Further, the mutant variant structures were built and simulated for 100 ns to generate free energy landscapes and perform docking experiments. Free energy landscapes formed by Y362, N378, and T391 indicate that the binding clefts of C30R and H215Y mutants are larger than the wild-type Tyrp1. In docking simulations, the hydrogen bond and salt bridge interactions that stabilize DHICA in the active site remain similar among Tyrp1, D308N, and R326H. However, the strengths of these interactions and stability of the docked ligand may decrease proportionally to mutation severity due to the larger and less well-defined natures of the binding clefts in mutants. Mutational perturbations in mutants that are not unfolded may result in allosteric alterations to the active site, reducing the stability of protein-ligand interactions.     

Novel Biomolecules in the Pathogenesis of Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM) is one of the most common metabolic diseases in pregnant women. Its early diagnosis seems to have a significant impact on the developing fetus, the course of delivery, and the neonatal period. It may also affect the later stages of child development and subsequent complications in the mother. Therefore, the crux of the matter is to find a biopredictor capable of singling out women at risk of developing GDM as early as the very start of pregnancy. Apart from the well-known molecules with a proven and clear-cut role in the pathogenesis of GDM, e.g., adiponectin and leptin, a potential role of newer biomolecules is also emphasized. Less popular and less known factors with different mechanisms of action include: galectins, growth differentiation factor-15, chemerin, omentin-1, osteocalcin, resistin, visfatin, vaspin, irisin, apelin, fatty acid-binding protein 4 (FABP4), fibroblast growth factor 21, and lipocalin-2. The aim of this review is to present the potential and significance of these 13 less known biomolecules in the pathogenesis of GDM. It seems that high levels of FABP4, low levels of irisin, and high levels of under-carboxylated osteocalcin in the serum of pregnant women can be used as predictive markers in the diagnosis of GDM. Hopefully, future clinical trials will be able to determine which biomolecules have the most potential to predict GDM.     

Qualitative and Quantitative Molecular Testing Methodologies and Traceability Systems for Commercialised Bt Cotton Events and Other Bt Crops Under Field Trials in India

Qualitative and quantitative PCR assays were developed for detection of commercialised Bt cotton events, i.e. MON531, MON15985 and other Bt crops, which are under different stages of field trials in India, i.e. Bt brinjal, Bt rice, Bt cauliflower, Bt potato and Bt okra. Multiplex PCR assays simultaneously detecting specific cry1Ac, cry1Ab, cry2Ab genes, Cauliflower Mosaic Virus 35S promoter, nptII marker gene along with species- or taxon-specific endogenous gene in these Bt crops have been developed. The quantitative real-time PCR assays were also reported for cry1Ac gene using designed primers and TaqMan probe. The sensitivity of developed assays for detection of specific transgene was established up to 0.01%. The analytical methods developed in the present report will be of immense use for qualitative screening and detection of Bt crops along with the quantitative analysis of inserted cry1Ac gene to meet the threshold level for regulatory compliance.     

Strength of pure alumina ceramics above 1 GPa

Up to now, commercially available alumina ceramics were claimed to have strength between 400 and 550 MPa. However, our study shows strength ~ 2 times higher for commercially available alumina than commonly believed. The average and characteristic strength, measured on 31 pure alumina ceramic discs by ball on three balls (B3B) test, were 1205 ± 93 MPa and 1257 MPa, respectively, with a Weibull modulus of m = 11.8. Tested specimens were in form of discs with a diameter of 5 mm and thickness 0.5 mm. The grain size distribution of the alumina is bimodal with an average grain size of ~ 850 nm measured at the surface. The fracture reveals a mixed transgranular / intergranular failure mode. To avoid incorporation of additional flaws, the discs were tested as sintered. The characteristic flexural strength measured in B3B was recalculated according to Weibull theory for standard 4-point bending bars of size 3 × 4 × 45 mm as bend 856 MPa. The measured strength of nearly 900 MPa shows the potential of strength for high purity alumina ceramics.     

Interleukins 6 and 15 Levels Are Higher in Subcutaneous Adipose Tissue,but Obesity Is Associated with Their Increased Content in Visceral Fat Depots

Excess adiposity is associated with chronic inflammation, which takes part in the development of obesity-related complications. The aim of this study was to establish whether subcutaneous (SAT) or visceral (VAT) adipose tissue plays a major role in synthesis of pro-inflammatory cytokines. Concentrations of interleukins (IL): 1β, 6, 8 and 15 were measured at the protein level by an ELISA-based method and on the mRNA level by real-time PCR in VAT and SAT samples obtained from 49 obese (BMI > 40 kg/m²) and 16 normal-weight (BMI 20–24.9 kg/m²) controls. IL-6 and IL-15 protein concentrations were higher in SAT than in VAT for both obese (p = 0.003 and p < 0.0001, respectively) and control individuals (p = 0.004 and p = 0.001, respectively), while for IL-1β this was observed only in obese subjects (p = 0.047). What characterized obese individuals was the higher expression of IL-6 and IL-15 at the protein level in VAT compared to normal-weight controls (p = 0.047 and p = 0.016, respectively). Additionally, obese individuals with metabolic syndrome had higher IL-1β levels in VAT than did obese individuals without this syndrome (p = 0.003). In conclusion, concentrations of some pro-inflammatory cytokines were higher in SAT than in VAT, but it was the increased pro-inflammatory activity of VAT that was associated with obesity and metabolic syndrome.     

Kinetic studies of dyes sorption from aqueous solutions onto the strongly basic anion-exchanger Lewatit MonoPlus M-600

The sorption of Tartrazine, Allura Red, Sunset Yellow and Indigo Carmine from aqueous solutions onto the strongly basic anion-exchanger (Lewatit MonoPlus M-600) of dimethylethanolamine functional groups and styrene–divinylbenzene matrix was investigated. The experimental data obtained at 50, 100, 200, 300 and 500 mg/dm³ initial concentrations at 20 °C were applied to the pseudo-first order, pseudo-second order and Weber–Morris kinetic models. The calculated sorption capacities (q_1,cal) and the rate constant of the first-order adsorption (k₁) were determined. The pseudo-second order kinetic constants (k₂) and capacities (q_2,cal) were calculated from the plots of t/q_t vs. t, 1/q_t vs. 1/t, 1/t vs. 1/q_t, q_t/t vs. q_t and 1/q₂ − q_t vs. t for type 1, type 2, type 3, type 4 and type 5 of the pseudo-second order expression, respectively.     

The Use of a Novel Ferulic Acid Esterase from Lactobacillus acidophilus K1 for the Release of Phenolic Acids from Brewer's Spent Grain

In this work, an extracellular ferulic acid esterase was produced in bioreactor cultivations of Lactobacillus acidophilus K1 strain. The enzyme was partially purified using ultrafiltration (10 kDa), dialysis (4–6 kDa) and Fast Protein Liquid Chromatography (Sepharose CM, Sephacryl S‐300). A considerable increment of enzyme activity (31‐fold) in the final preparation was achieved. Two distinct bands (approx. 21.5 kDa and 39 kDa) were obtained after SDS‐PAGE. A high similarity of the purified enzyme (LC‐MS/MS analysis) to tannase and ferulic acid esterase from Burkholderia ambifaria MEX‐5 was obtained. The optimal pH and temperature for the enzyme activity were 6.3 and 37°C, respectively. The enzyme preparation effectively released phenolic acids (mainly ferulic and p‐coumaric acid) from brewer's spent grain. This novel enzyme preparation can be used for the utilisation of a valuable and inexpensive by‐product of the brewing industry.