BACKGROUND

Inflammation is integral to the injury response. The inflammatory response is essential to the host defense against infection and also to tissue regeneration and repair. Toll-like receptors (TLRs) are critical activators of the innate immune response and present attractive therapeutic targets for inflammation-modulated tissue regeneration. The authors' previous study showed that depletion of TLR4 resulted in accelerated skull bone healing concurrent with increased expression of osteoclastogenic genes. As such, in the present study, the authors used various knockout mouse models for TLR4 and its associated signaling mediators as tools to further understand the role of Toll-like receptor-mediated inflammation in calvarial bone healing.

METHODS

Calvarial defects (1.8-mm diameter) were created in wild-type, TLR4 knockout (TLR4), TLR2, MyD88, TRIF, TLR4 knockout in myeloid cell (Lyz-TLR4), and TLR4 knockout in dendritic-lineage cell (CD11c-TLR4) mice. Bone healing was examined using micro-computed tomographic, histologic, and histomorphometric analyses.

RESULTS

Micro-computed tomographic and histomorphometric analyses revealed that TLR4-deficient mice (TLR4, Lyz-TLR4, and CD11c-TLR4) exhibited a faster intramembraneous healing response at postoperative day 7, whereas MyD88 and CD11c-TLR4 mice showed enhanced bone healing at day 28.

CONCLUSIONS

The authors' data suggest a detrimental role for TLR4 in CD11c cells, mediated by Myd88 signaling, during calvarial bone healing. The authors have demonstrated that Toll-like receptor signaling components affect calvarial bone healing, establishing a link between the skeletal and immune systems during craniofacial bone healing. Toll-like receptor signaling components might be used to initiate enhanced healing in bone defects to improve clinical outcomes.

Polyphenol can improve osteoporosis and is closely associated with gut microbiota, while the mechanism and the relationship among polyphenol, osteoporosis, and gut microbiota colonization remain unclear. Here, an osteoporosis rat model established by ovariectomy was employed to investigate the improving mechanism of arecanut (Areca catechu L.) seed polyphenol (ACP) on osteoporosis by regulating gut microbiota. We analyzed the bone microstructure, Paneth cells, regulating microbial protein (lysozyme (LYZ)), proinflammatory cytokines, macrophage infiltration levels, and gut microbial communities in a rat. ACP improved the trabecular microstructure compared to OVX, including the increased trabecular number (Tb.N) (P < 0.01) and trabecular thickness (Tb.Th) (P < 0.001) and decreased trabecular separation (Tb.Sp) (P < 0.01). At the phylum level, Bacteroidetes was increased after ovariectomy (P < 0.001) and Firmicutes and Proteobacteria were increased in ACP (P < 0.001). Antiosteoporosis groups with lower LYZ and Paneth cells (P < 0.001) showed that the microbiota Alistipes, which have a negative effect on bone metabolism were decreased in ACP (P < 0.001). Altogether, these studies showed that the estrogen deficiency could induce the shedding of Paneth cells, which leads to the decrease of LYZ, while ACP could increase the LYZ expression by maintaining the population of Paneth cells in an estrogen-deficient host, which were implicated in gut microbiota regulation and improved osteoporosis by controlling the inflammatory reaction.

Keywords: Areca catechu polyphenol; LYZ; Paneth cells; antiosteoporosis; gut microbiota; inflammatory reaction.

This study aimed to develop of a rapid and effective method to occlude dentinal tubules using carboxymethyl chitosan and lysozyme (CMC/LYZ) nanogels with encapsulated amorphous calcium phosphate (ACP) based on the transformation of ACP to HAP. In this work, CMC/LYZ was used to stabilize ACP and form CMC/LYZ-ACP nanogels, and then the nanogel-encapsulated ACP was applied to exposed dentinal tubule surfaces. The morphology of the nanogels was examined by transmission electron microscopy (TEM). Distribution and quantity of elements in CMC/LYZ-ACP nanogels were determined by element mapping and energy dispersive X-Ray spectroscopy (EDX). Scanning electron microscopy (SEM) images, XRD measurements and nanoindentation were applied to check the efficacy of tubular occlusion. TEM revealed that CMC/LYZ-ACP nanogels were spherical dense gel particles with size approximately 50-500 nm. Element mapping and EDX indicated that C, N, O, Ca, P, and S in the microspheres are thoroughly represented. SEM images shows that the thickness of the coating layer was approximately 1-2 μm and the depth to which the mineralized substance enters the dentinal tubule was approximately 4-8 μm. XRD measurements and nanoindentation indicated that the occluding mineralized substance observed were similar to nature dentin. CMC can form spherical dense nanogels loaded with ACP under the participation of lysozyme. The CMC/LYZ-ACP nanogels could increase the dentinal tubule occluding effectiveness. These results indicated that finding and developing novel nanomaterials of CMC/LYZ-ACP would be an effective strategy for the treatment of dentin hypersensitivity.

In this research, ionic liquid as functional monomer to prepare molecularly imprinted polymers for protein recognition was for the first time demonstrated, in which, 1-vinyl-3-butylimidazolium chloride was selected as functional monomer, acrylamide as co-functional monomer and lysozyme (Lyz) as template protein to synthesize imprinted polymers on the surface of multiwall carbon nanotubes in aqueous medium. The results indicated that ionic liquid was helpful to improve binding capacity of imprinted polymers, which had a maximum binding capacity of 763.36 mg/g in the optimum adsorption conditions. The prepared imprinted polymers had a fast adsorption rate and a much higher adsorption capacity than the corresponding non-imprinted polymers, with the difference in adsorption capacity up to 258.31 mg/g. The obtained polymer was evaluated by Lyz, bovine serum albumin (BSA), bovine hemoglobin (BHb), equine myoglobin (MB) and cytochrome c (Cyt c). The selectivity factor (β) for Lyz/BSA, Lyz/Mb, Lyz/BHb, and Lyz/Cyt c were 7.17, 2.12, 1.76 and 1.57, respectively, indicating the imprinted polymers had a good selectivity. Easy preparation of the imprinted polymers as well as high ability and selectivity to adsorb template proteins makes this polymer attractive and broadly applicable in biomacromolecular separation, biotechnology and sensors.

In this study, elucidation of protein adsorption mechanism is performed using dual polarization interferometry (DPI) and quartz crystal microbalance with dissipation (QCM-D) to study adsorption behaviors of bovine serum albumin (BSA) and lysozyme (LYZ) on poly (ethylene glycol) (PEG) layers. From the analysis of DPI, PEG2000 and PEG5000 show tight and loose mushroom conformations, respectively. Small amount of LYZ could displace the interfacial water surrounding the tight mushroomed PEG2000 chains by hydrogen bond attraction, leading to protein adsorption. The loose mushroomed PEG5000 chains exhibit a more flexible conformation and high elastic repulsion energy that could prevent protein adsorption of all BSA and most of LYZ. From the analysis of QCM, PEG2000 and PEG5000 show tight and extended brush conformations. The LYZ adsorbed mass has critical regions of PEG2000 (0.19 chain/nm(2)) and PEG5000 (0.16 chain/nm(2)) graft density. When graft density of PEG is higher than the critical region (brush conformations), the attraction of hydrogen bonds between PEG and LYZ is the dominant factor. When graft density of PEG is lower than the critical region (mushroom conformations), elastic repulsion between PEG and proteins is driven by the high conformation entropy of PEG chains, which is the dominant force of steric repulsion in PEG-protein systems. Therefore, the adsorption of BSA is suppressed by the high elastic repulsion energy of PEG chains, whereas the adsorption of LYZ is balanced by the interactions between the repulsion of entropy elasticity and the attraction of hydrogen bonds.

The immunostimulatory potential of the marine yeast Yarrowia lipolytica (D1 and N6 strains) administered orally was evaluated in the white shrimp Litopenaeus vannamei. Yeasts and commercial glucans were mixed with a commercial feed to formulate diets with a 1.1% concentration of immunostimulants. The shrimp were fed daily for a period of 21 days. Weekly determinations were performed for immunological parameters in hemolymph, such as total hemocyte count (THC), lysozyme activity (LYZ), prophenoloxidase activity, antioxidant enzymatic activities (superoxide dismutase [SOD], catalase [CAT], and peroxidases), and bactericidal activity against Vibrio parahaemolyticus. Expression profiles of penaeidin (PEN), lysozyme (LYZ), and prophenoloxidase (proPO) immune genes were evaluated in hemocytes. In general, an increase in the immune parameters was observed in shrimp fed yeast diet compared to glucan and the control diets. Yarrowia lipolytica, especially strain N6, provided maximum immunostimulatory effects evidenced by the increase of immune parameters (THC, LYZ, SOD, CAT) and gene expression profile. In conclusion, this study demonstrated that Y. lipolytica had immunostimulatory effects and increased bactericidal activity in hemocytes against V. parahaemolyticus in L. vannamei. These findings open the path for the potential application of Y. lipolytica-based immunostimulant for shrimp aquaculture.

Keywords: Aquaculture; Crustacea; Functional carbohydrates; Immunoprotection; Probiotic yeast.

Methionine (Met) is one of the 2 most limiting amino acids for milk production in dairy cow diets. The accepted "ideal" ratio of lysine (Lys) to Met (L:M) when formulating diets is 3:1. However, blood from cows fed corn silage-based diets without supplemental rumen-protected Met averages approximately 3.6:1 L:M. Recent in vivo research on cattle immunonutrition has revealed that the immune system could benefit from greater Met supply. To study more closely the effects of different L:M ratios, blood polymorphonuclear cells (PMN) were isolated from 5 Holstein cows in mid-lactation (238 ± 20 d postpartum, 33.8 ± 3.8 kg of milk/d; mean ± SD). The PMN were incubated at 3 different levels of L:M (3.6:1, 2.9:1, or 2.4:1) and stimulated with lipopolysaccharide (LPS) at either 0 or 50 μg/mL for 2 h at 37°C. Target genes were associated with cytokines, pathogen recognition, nuclear receptors, killing mechanisms, and Met and glutathione metabolism. Data were subjected to ANOVA using PROC MIXED in SAS, with L:M, LPS, and their interaction as fixed effects. Stimulation with LPS upregulated genes related to cytokines (IL1B, TNF, IL10 and IL6) and nuclear receptors, including nuclear factor kappa B (NFKB1) and glucocorticoid receptor (NR3C1), and downregulated the mRNA abundance of chemokine receptor 1 (CXCR1), lysozyme (LYZ) and glutathione reductase (GSR). A linear decrease was observed in the mRNA abundance of TNF when L:M was decreased. A similar response was observed for interleukin-1 receptor-associated kinase 1 (IRAK1) and NFKB1 abundance in cells stimulated with LPS (linear effect). A linear increase of LYZ mRNA expression as L:M decreased was detected in unstimulated cells. Furthermore, a decrease in L:M led to a linear decrease of superoxide dismutase 1 (SOD1) mRNA abundance in cells challenged with LPS. Overall, LPS challenge triggered the activation of isolated PMN from mid-lactation cows. However, data suggest the use of a shorter incubation time to capture the peak response and not the resolution of the inflammatory response as in the present study. Our results indicate a possible involvement of Met in modulating PMN inflammatory and oxidative stress status and in helping the resolution of inflammation after initial stimulation.

Due to its high proliferation capacity and rapid intracranial spread, glioblastoma (GBM) has become one of the least curable malignant cancers. Recently, the competing endogenous RNAs (ceRNAs) hypothesis has become a focus in the researches of molecular biological mechanisms of cancer occurrence and progression. However, there is a lack of correlation studies on GBM, as well as a lack of comprehensive analyses of GBM molecular mechanisms based on high-throughput sequencing and large-scale sample sizes. We obtained RNA-seq data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Further, differentially expressed mRNAs were identified from normal brain tissue and GBM tissue. The similarities between the mRNA modules with clinical traits were subjected to weighted correlation network analysis (WGCNA). With the mRNAs from clinical-related modules, a survival model was constructed by univariate and multivariate Cox proportional hazard regression analyses. Thereafter, we carried out Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Finally, we predicted interactions between lncRNAs, miRNAs and mRNAs by TargetScan, miRDB, miRTarBase and starBase. We identified 2 lncRNAs (NORAD, XIST), 5 miRNAs (hsa-miR-3613, hsa-miR-371, hsa-miR-373, hsa-miR-32, hsa-miR-92) and 2 mRNAs (LYZ, PIK3AP1) for the construction of a ceRNA network, which might act as a prognostic biomarker of GBM. Combined with previous studies and our enrichment analysis results, we hypothesized that this ceRNA network affects immune activities and tumour microenvironment variations. Our research provides novel aspects to study GBM development and treatment.

Keywords: co-expression network; competing endogenous RNA; glioblastoma; prediction; prognostic marker.

Myrtle (Myrtus communis L., Myrtaceae) is a significant plant which naturally distributed around the globe. Although numerous studies have demonstrated the benefits of myrtle in different species, studies using the oral route are rare in the literature. In the present study, we evaluated the effect of myrtle intake on the antioxidant, immune, appetite and growth related genes as well as mucosal immune responses in zebrafish (Danio rerio) model. Zebrafish were fed control or myrtle (5, 10 and 20 g kg-1 myrtle) supplemented diets for sixty days. The results showed that, oral administration of Myrtle significantly improved mucosal immune responses (the activity of lysozyme, total Ig and protease). Furthermore, fish fed 20 g kg-1 showed remarkably higher antioxidant (sod and cat) enzymes gene expression compared other treatment. There were significant difference between myrtle fed fish and control group regarding tnf-alpha and lyz expression. Also, evaluation of growth (gh and igf1) related genes revealed remarkable upregulation in 20 g kg-1 myrtle treatment compared other myrtle treatments and control group. Similar results was observed regarding the mRNA levels of appetite related genes (ghrl) in zebrafish fed 20 g kg-1 myrtle. The present results indicated that dietary administration of myrtle improved mucosal immune parameters and altered mRNA levels of selected genes. These results on zebrafish model also highlights the potential use of Myrtle supplements as additive in human diets.

Microcystins (MCs), produced by toxic cyanobacterial blooms that appeared world wildly in eutrophication waters, have often caused fish illness and even massive death cases. Among at least 90 structural variants, microcystin-LR (MC-LR) is the most common and toxic variant. In order to better understand innate immune responses in fish disrupted by environmental concentrations of MC-LR, male zebrafish (Danio rerio) were exposed to 0, 0.3, 1, 3, 10 and 30 μg/L MC-LR for 30 d, and the changes in splenic pathology and immunological gene expression as well as serum immune parameters were studied. In the low concentration groups (0.3, 1 and 3 μg/L), zebrafish displayed splenic inflammatory changes including the formation of melano-macrophage centers and the increase of macrophage pseudopodia, remarkable elevation of serum C3 levels, and significantly upregulated expression of innate immune-related genes (c3b, lyz, il1β, tnfα and ifnγ). In contrast, high concentrations of MC-LR (10 and 30 μg/L) resulted in the degeneration of splenic lymphocytes and macrophages, and down-regulation of immune-related genes as well as significant decreases in the level of serum C3. Furthermore, significant increases in the activity of serum ACP and ALP suggested that high concentrations of MC-LR increased permeability of macrophage plasma membrane or cellular necrosis, and subsequently decreased innate immune function. Our findings illustrated that sub-chronic exposure of MC-LR has dualistic influences on fish innate immune system with inflammatory activation at low exposure concentrations but turned to immune inhibition with the increases of exposure concentration.

The interface between nematic liquid crystal, 4-cyano-4'-pentylbiphenyl (5CB), and water in a transmission electron microscopy (TEM) grid cell coated with QP4VP-b-LCP (quaternized poly(4-vinylpyridine) (QP4VP) and poly(4-cyanobiphenyl-4'-oxyundecylacrylate) (LCP)) was examined for protein and DNA detection. QP4VP-b-LCP was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. Quaternization of P4VP with iodomethane (CH3I) made it a strong cationic polyelectrolyte and allowed QP4VP-b-LCP to form complexes with oppositely charged biological species. Several proteins, such as bovine serum albumin (BSA), hemoglobin (Hb), α chymotrypsinogen-A (ChTg), and lysozyme (LYZ), were tested for nonspecific protein detection. By injecting the protein solutions into the TEM grid cell, the initial homeotropic orientation of the TEM grid cell changed to a planar orientation above their isoelectric points (PIs) due to electrostatic interactions between QP4VP (+charge) and proteins (-charge), which did not occur below the PIs of the tested proteins. Their minimum concentrations at which the homeotropic to planar configurational change (H-P change) occurred were 0.01, 0.02, 0.03, and 0.04 wt.% for BSA, ChTg, Hb, and LYZ, respectively. One of the strong anionic polyelectrolytes, deoxyribonucleic acid (DNA) (due to the phosphate deoxyribose backbone) was also tested. A H-P change was observed with as little as 0.0013 wt.% salmon sperm DNA regardless of the pH of the cell. A H-P change occurred in 5CB and was observed by polarized optical microscopy. This simple and inexpensive setup for nonspecific biomaterial detection provides the basic idea for developing effective selective biosensors by introducing specific binding groups, such as the aptamer and antibody.

Dual responsive (temperature and pH) 4-cyano-4'-pentylbiphenyl (5CB) droplets were fabricated by coating with PNIPAM-b-LCP (PNIPAM: poly(N-isopropylacrylamide) and LCP: poly(4-cyanobiphenyl-4'-oxyundecylacrylate)) and sodium dodecyl sulfate (SDS) using a microfluidic method, and were tested for protein detection. The PNIPAM-b-LCP/SDS-functionalized 5CB droplets were effective in detecting proteins in water through a radial-to-bipolar (R-B) orientational change, with detection limits of 0.95, 1.1, 0.12, and 0.07 μM for bovine serum albumin (BSA), lysozyme (LYZ), hemoglobin (Hb), and chymotrypsinogen (ChTg), respectively. The R-B change of the 5CB droplet occurred above the lower critical solution temperature (LCST) of PNIPAM and at pH values below the pI values of the tested proteins, and was reversible by heating/cooling at the LCST of PNIPAM. These sensitive 5CB droplets are simple to prepare, cost-effective, easily detectable, and re-usable (by temperature control) for protein detection. Further, they can be applied to a biosensor after employing the selective units such as aptamers, antibodies, single-stranded DNA, proteins, peptides, and aptamer-binding RNA on the droplet.

Chorioamnionitis (CA) predisposes to preterm birth and affects the fetal mucosal surfaces (i.e., gut, lungs, and skin) via intra-amniotic (IA) inflammation, thereby accentuating the proinflammatory status in newborn preterm infants. It is not known if CA may affect more distant organs, such as the kidneys, before and after preterm birth. Using preterm pigs as a model for preterm infants, we investigated the impact of CA on fetal and neonatal renal status and underlying mechanisms. Fetal pigs received an IA dose of lipopolysaccharide (LPS), were delivered preterm by cesarean section 3 days later (90% gestation), and compared with controls (CON) at birth and at postnatal day 5. Plasma proteome and inflammatory targets in kidney tissues were evaluated. IA LPS-exposed pigs showed inflammation of fetal membranes, higher fetal plasma creatinine, and neonatal urinary microalbumin levels, indicating renal dysfunction. At birth, plasma proteomics revealed LPS effects on proteins associated with renal inflammation (up-regulated LRG1, down-regulated ICA, and ACE). Kidney tissues of LPS pigs at birth also showed increased levels of kidney injury markers (LRG1, KIM1, NGLA, HIF1A, and CASP3), elevated molecular traits related to innate immune activation (infiltrated MPO⁺ cells, complement molecules, oxidative stress, TLR2, TLR4, S100A9, LTF, and LYZ), and Th1 responses (CD3⁺ cells, ratios of IFNG/IL4, and TBET/GATA3). Unlike in plasma, innate and adaptive immune responses in kidney tissues of LPS pigs persisted to postnatal day 5. We conclude that prenatal endotoxin exposure induces fetal and postnatal renal inflammation in preterm pigs with both innate and adaptive immune activation, partly explaining the potential increased risks of kidney injury in preterm infants born with CA.

Keywords: acute kidney injury; chorioamnionitis; immune activation; intrauterine bacterial infection; plasma proteomics; renal inflammation.

A 8-weeks feeding trial was conducted to examine the effects of different levels (0, 0.5, 1 and 2%) of dietary Ferula (Ferula assafoetida) on expression of antioxidant enzymes (GSR, GPX and GSTA), immune (TNF-alpha, IL1B, IL- 8 and LYZ) and growth (GH, IGF1 and Ghrl) genes as well as cutaneous mucus and serum non-specific immune response in common carp. The results revealed Ferula significantly increased antioxidant gene expression (GSR and GSTA) in a dose dependent manner (P < 0.05). The expression of immune growth related genes were significantly higher in Ferula fed fish compared control group (P < 0.05). The effects of Ferula on expression of genes was more pronounced in higher doses. Feeding on Ferula supplemented diet remarkably increased skin mucus lysozyme activity (P < 0.05). However, evaluation of mucus total Ig and protease activity revealed no significant difference between control and treated groups (P>> 0.05). Regarding non-specific humoral response, serum total Ig, lysozyme and ACH50 showed no remarkable variation between Ferula fed carps and control group (P>> 0.05). These results indicated up-regulation of growth and health related genes in Ferula fed common carp. Further studies using pathogen or stress challenge is required to conclude that transcriptional modulation is beneficial in common carp.

Background: Renal cell carcinoma (RCC) is one of the most prevalent malignant tumors of the urinary system. Hypertension can cause hypertensive nephropathy (HN). Meanwhile, Hypertension is considered to be related to kidney cancer. We analyzed co-expressed genes to find out the relationship between hypertension and RCC and show possible biomarkers and novel therapeutic targets of hypertension-related RCC.

Methods: We identified the differentially expressed genes (DEGs) of HN and RCC through analyzing Gene Expression Omnibus (GEO) datasets GSE99339, GSE99325, GSE53757 and GSE15641 by means of bioinformatics analysis, respectively. Then we evaluated these genes with protein-protein interaction (PPI) networks, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and CTD database. Subsequently, we verified co-expressed DEGs with Gene Expression Profiling Interactive Analysis (GEPIA) database. Finally, corresponding predicted miRNAs of co-expressed DEGs were identified and verified via mirDIP database and Starbase, respectively.

Results: We identified 9 co-expressed DEGs, including BCAT1, CORO1A, CRIP1, ESRRG, FN1, LYZ, PYCARD, SAP30, and PTRF. CRIP1 and ESRRG and their corresponding predicted miRNAs, especially hsa-miR-221-5p, hsa-miR-205-5p, hsa-miR-152-3p and hsa-miR-137 may be notably related to hypertension-related RCC.

Conclusions: CRIP1 and ESRRG genes have great potential to become novel biomarkers and therapeutic targets concerning hypertension-related RCC.

Keywords: Biomarkers; computational biology; hypertension; renal cell carcinoma (RCC).

The present study was performed to investigate the effects of various levels of dietary ellagic acid (EA) on growth performance, haematological values, immune response, protection against Yersinia ruckeri infection, and oxidant/antioxidant status in rainbow trout, Oncorhynchus mykiss. Fish were fed with the control diet and three different experimental diets containing three graded levels of EA (50, 100 and 200 mg kg^-1 diet) for 8 weeks. At the end of the experiment, the growth performance [weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR)], haematological values [the red blood cell (RBC) count, haemoglobin (Hb) concentration, haematocrit (Ht) level and erythrocyte indices: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC)], immune response [white blood cell (WBC) count, oxidative radical production (nitroblue tetrazolium (NBT) assay), phagocytic activity (PA) and phagocytic index (PI), total protein (TP) and immunoglobulin M (IgM) levels, serum bactericidal activity (BA), lysozyme (LYZ) and myeloperoxidase (MPO) activities] and oxidant/antioxidant status [tissue malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities] were analysed. In addition, fish were challenged by Y. ruckeri and survival rate was recorded for 14 days. In the groups fed with EA the growth parameters such as WG, SGR, and FCR did not vary significantly. The RBC count, Hb concentration, and Ht level increased in the groups fed with EA when compared with the control group. However, there were no significant differences in the MCV, MCH and MCHC values among the groups. The results demonstrated enhancement in all the immunological parameters in the groups fed with EA compared to the control group. The results obtained from challenge with Y. ruckeri revealed reduction in the mortalities in the groups fed with EA. The dietary EA stimulated the SOD, CAT and GSH-Px activities in liver, head kidney and spleen as compared to the control group; however, a reverse trend was observed in the MDA levels of tissues. The present study suggest that EA can effectively enhance the haematological values, immune response, antioxidant capacity, and disease resistance in rainbow trout.

We aimed to introduce hydrophilic sulfobetaine-type zwitterionic groups to macromolecular chains of copolymers to construct novel copolymer hydrogels with anti-protein-fouling performance that could be used as soft contact lens (SCL) materials. Using hydroxyethyl methacrylate (HEMA), N-vinyl pyrrolidinone (NVP) and sulfobetaine methacrylate (SBMA) as comonomers, several copolymer hydrogels with different SBMA contents, poly(HEMA-NVP-SBMA), are synthesized via radical copolymerization in an aqueous phase. Surface chemistry, structural morphologies, water contact angle (WCA), equilibrium water content (EWC), visible light transmittance and tensile mechanical properties are investigated. The prepared hydrogels exhibit a closed-type porous structure. With increasing SBMA content in the comonomer mixture, the prepared copolymer hydrogel pore size gradually increases up to the micron level, WCA tends to decrease, EWC tends to increase, and visible light transmittance slightly increases, but their tensile mechanical properties decline. The amounts of protein Lyz and BSA adsorbed on the copolymer hydrogels and on commercially available EASY DAY® SCLs as a control are also determined by protein adsorption tests. The amount of protein adsorbed on the copolymer hydrogel decreases with increasing SBMA content. For the hydrogel prepared using the comonomer mixture with 5.0 wt % SBMA, the amount of adsorbed Lyz is 0.91 μg/cm2, which corresponds to only 56.8% of the amount adsorbed on EASY DAY® SCLs. Thus, novel SCL materials with high water content and excellent anti-protein-fouling performance were efficiently constructed by introducing sulfobetaine-type zwitterionic groups into a traditional polymer hydrogel system.

The binding of the model proteins HSA, LYZ and MYO to PEC nanoparticles is reported. PEC particles were prepared by mixing solutions of PDADMAC either with PSS or PMA-MS, followed by consecutive centrifugation. Monomodal anionic (PEC-1.50) and cationic (PEC-0.66) PEC particles were obtained using non-stoichiometric mixing ratios. PEC/protein conjugates were prepared by adding charged protein solutions to dispersions of respective like charged PEC particles, followed by one centrifugation step. Mixing proteins and PEC particles under attractive conditions led to flocculation of the dispersion. From CD, DLS and AFM the following trend for protein binding at PEC particles under repulsive conditions was obtained: HSA/PEC-1.50>> MYO/PEC-1.50>> LYZ/PEC-0.66. Protein uptakes up to 0.33 g x g(-1) (protein/PEC) (CD) and particle diameter enlargements up to 13 nm (AFM) were obtained at c(PROT) = 0.091 mg . mL(-1). Furthermore, novel spin coated films of PEC particles were interacted with proteins under both repulsive and attractive conditions. In-situ ATR FT-IR spectroscopy revealed that the adsorbed amount of HSA and LYZ under attractive conditions was significantly higher than under repulsive ones, which is analogous to protein adsorption at polyelectrolyte multilayers terminated either by polycation or polyanion. Similarly to the dispersed PEC/protein conjugates, under repulsive conditions the uptake of HSA was higher compared to LYZ. The shown protein uptake under repulsive conditions is related to concepts of mild enzyme or protein binding at nonbiogenic substrates.

Thiol group functionalized silica-coated magnetic nanoparticles (Si-MNPs@SH) were synthesized for rapid and selective magnetic field-based separation of mixed proteins. The highest adsorption efficiencies of binary proteins, bovine serum albumin (BSA; 66 kDa; pI = 4.65) and lysozyme (LYZ; 14.3 kDa; pI = 11) were shown at the pH values corresponding to their own pI in the single-component protein. In the mixed protein, however, the adsorption performance of BSA and LYZ by Si-MNPs@SH was governed not only by pH but also by the molecular weight of each protein in the mixed protein.

Understanding the interaction between protein-functionalized surfaces is an important subject in a variety of protein-related processes, ranging from coatings for biomedical implants to targeted drug carriers and biosensors. In this work, utilizing a total internal reflection microscope (TIRM), we have directly measured the interactions between micron-sized particles decorated with three types of common proteins concanavalin A (ConA), bovine serum albumin (BSA), lysozyme (LYZ), and glass surface coated with soy proteins (SP). Our results show that the protein adsorption greatly affects the charge property of the surfaces, and the interactions between those protein-functionalized surfaces depend on solution pH values. At pH 7.5-10.0, all these three protein-functionalized particles are highly negatively charged, and they move freely above the negatively charged SP-functionalized surface. The net interaction between protein-functionalized surfaces captured by TIRM was found as a long-range, nonspecific double-layer repulsion. When pH was decreased to 5.0, both protein-functionalized surfaces became neutral and double-layer repulsion was greatly reduced, resulting in adhesion of all three protein-functionalized particles to the SP-functionalized surface due to the hydrophobic attraction. The situation is very different at pH = 4.0: BSA-decorated particles, which are highly charged, can move freely above the SP-functionalized surfaces, while ConA- and LYZ-decorated particles can only move restrictively in a limited range. Our results quantify these nonspecific kT-scale interactions between protein-functionalized surfaces, which will enable the design of surfaces for use in biomedical applications and study of biomolecular interactions.

The influence of oxidative debris (OD) present in as-prepared graphene oxide (GO) suspensions on proteins and its toxicity to human embryonic kidney cells (HEK-293T) are reported here. The OD was removed by repeated washing with aqueous ammonia to produce the corresponding base-washed GO (bwGO). The loading (w/w) of bovine serum albumin (BSA) was increased by 85% after base washing, whereas the loading of hemoglobin (Hb) and lysozyme (Lyz), respectively, was decreased by 160% and 100%. The secondary structures of 13 different proteins bound to bwGO were compared with the corresponding proteins bound to GO using the UV circular dichroism spectroscopy. There was a consistent loss of protein secondary structure with bwGO when compared with proteins bound to GO, but no correlation between either the isoelectric point or hydrophobicity of the protein and the extent of structure loss was observed. All enzymes bound to bwGO and GO indicated significant activities, and a strong correlation between the enzymatic activity and the extent of structure retention was noted, regardless of the presence or absence of OD. At low loadings (<100 μg/mL) both GO and bwGO showed excellent cell viability but substantial cytotoxicity (~40% cell death) was observed at high loadings (>100 μg/mL). In control studies, OD by itself did not alter the growth rate even after a 48-h incubation. Thus, the presence of OD in GO played a very important role in controlling the chemical and biological nature of the protein-GO interface and the presence of OD in GO improved its biological compatibility when compared to bwGO.

Cross-linked layer-by-layer (LbL) assemblies with a precisely tuned surface ζ-potential were fabricated to control the adsorption of proteins, mammalian cells, and bacteria for different biomedical applications. Two weak polyions including a synthesized polyanion and polyethylenimine were assembled under controlled conditions and cross-linked to prepare three robust LbL films as model surfaces with similar roughness and water affinity but displaying negative, zero, and positive net charges at the physiological pH (7.4). These surfaces were tested for their abilities to adsorb proteins, including bovine serum albumin (BSA) and lysozyme (LYZ). In the adsorption tests, the LbL films bind more proteins with opposite charges but less of those with like charges, indicating that electrostatic interactions play a major role in protein adsorption. However, LYZ showed higher nonspecific adsorption than BSA, because of the specific behavior of LYZ molecules, such as stacked multilayer formation during adsorption. To exclude such stacking effects from experiments, protein molecules were covalently immobilized on AFM colloidal probes to measure the adhesion forces against the model surfaces utilizing direct protein molecule-surface contacts. The results confirmed the dominating role of electrostatic forces in protein adhesion. In fibroblast cell and bacteria adhesion tests, similar trends (high adhesion on positively charged surfaces, but much lower on neutral and negatively charged surfaces) were observed because the fibroblast cell and bacterial surfaces studied possess negative potentials. The cross-linked LbL films with improved stability and engineered surface charge described in this study provide an excellent platform to control the behavior of different charged objects and can be utilized in practical biomedical applications.

Informative microsatellites associated with two genes on HSA12 (lysozyme, LYZ; tumour necrosis factor receptor, TNFR) and one gene on HSA2 (glutamic acid decarboxylase 1, GAD1) were mapped in the US Meat Animal Research Center (MARC) swine reference population and the physical assignment of a-lactalbumin (LALBA) was determined. A comparative map for HSA2 and HSA12 with SSC15 and SSC5, respectively, was developed by combining the results from this study with published type I loci mapped in both species. One rearrangement between HSA2 and SSC15 was detected while the number of rearrangements between HSA12 and SSC5 were numerous. These results indicated that conservation of synteny does not imply a conservation of gene order and that additional type I markers need to be mapped in the pig to fully understand the chromosomal rearrangements that occurred during the evolution of mammals.