Transcriptome changes of Apis mellifera female embryos with fem gene knockout by CRISPR/Cas9.

The sex of honey bees is decided by a regulatory cascade comprising of csd, fem and Amdsx. In order to further identify other genes involved in sex determination and differentiation of honey bees in the early stages of embryo development, the CRISPR/Cas9 method was used to knock out fem gene in the embryonic stage of diploid western honey bees, and RNA-seq was used to analyze gene expression changes in the embryo after fem knockout. Finally, we found that the bees had undergone gender changes due to fem knockout. A total of 155 differentially expressed genes (DEGs) were obtained, with 48 up-regulated and 107 down-regulated DEGs in the mutant group compared to the control group. Of them, many genes are related to sex development or differentiation. In addition, 1502 differentially expressed alternative splicing events (DEASEs) related to 1011 genes, including the main honey bee sex-determining genes csd, tra2, fem, and Amdsx, were identified between the mutant group and control group, indicating that fem regulates alternative splicing of a large number of downstream genes. Our results provide valuable clues for further investigating the molecular mechanism of sex determination and differentiation in honey bees.

Alterations in the jejunal microbiota and fecal metabolite profiles of rabbits infected with Eimeria intestinalis.

BACKGROUND: Rabbit coccidiosis is a major disease caused by various Eimeria species and causes enormous economic losses to the rabbit industry. Coccidia infection has a wide impact on the gut microbiota and intestinal biochemical equilibrium. In the present study, we established a model of Eimeria intestinalis infection in rabbits to evaluate the jejunal microbiota and fecal metabolite profiles. METHODS: Rabbits in the infected group were orally inoculated with 3 × 103 E. intestinalis oocysts. On the eighth day of infection, jejunal contents and feces were collected for 16S rRNA gene sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, respectively. Jejunum tissues were harvested for hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and immunohistochemistry (IHC) staining. RESULTS: Histopathological analysis showed that the whole jejunum was parasitized by E. intestinalis in a range of life cycle stages, and PAS staining showed that E. intestinalis infection caused extensive loss of goblet cells. IHC staining revealed that TNF-α expression was higher in the E. intestinalis infection group. Moreover, both the jejunal microbiota and metabolites significantly altered after E. intestinalis infection. At the genus level, the abundances of Escherichia and Enterococcus significantly increased in the infected group compared with the control group, while those of Oscillospira, Ruminococcus, Bacteroides, Akkermansia, Coprococcus, and Sarcina significantly decreased. In addition, 20 metabolites and two metabolic pathways were altered after E. intestinalis infection, and the major disrupted metabolic pathway was lipid metabolism. CONCLUSIONS: Eimeria intestinalis infection induced intestinal inflammation and destroyed the intestinal homeostasis at the parasitized sites, leading to significant changes in the gut microbiota and subsequent corresponding changes in metabolites.

Inflammatory Cytokines Changed in Patients With Depression Before and After Repetitive Transcranial Magnetic Stimulation Treatment.

Studies have found that repetitive transcranial magnetic stimulation rTMS can produce antidepressant effects by affecting inflammatory cytokines in patients with depression, which plays a key role in the therapeutic mechanism of antidepressants. This study aimed to explore the changes in inflammatory cytokine levels in patients with depression after 4 weeks of rTMS treatment to determine the possible antidepressant mechanism of rTMS. This prospective, double-blind, pseudo-stimulus-controlled study was conducted, and a total of 57 patients with depression and 30 healthy controls were recruited. Patients were randomly divided into the active rTMS (n = 29) and sham rTMS groups (n = 28). The Hamilton Depression Scale was used to evaluate depressive symptoms and their severity. The serum levels of seven inflammatory cytokines were measured using enzyme-linked immunosorbent assay. Inflammatory cytokines include high-sensitivity C-reactive protein (CRP-hc); tumor necrosis factor (TNF-α); interferon (IFN-γ); interleukin-2 (IL-2); interleukin-4 (IL-4); interleukin-6 (IL-6); and interleukin-8 (IL-8). At baseline, TNF-α (F = 36.699, p < 0.001), IFN-γ (F = 8.907, p < 0.001), IL-4 (F = 66.256, p < 0.001), and IL-2 (F = 9.162, p < 0.001) levels in the depression group were significantly different from those of healthy controls. In the self-control analysis of the active rTMS group, the levels of IL-2 and CRP-hc increased significantly after 2 and 12 weeks of treatment. In the sham-rTMS group, IFN-γ increased after 2 and 12 weeks of treatment. Our results revealed that the changes in inflammatory cytokines after rTMS treatment showed different patterns compared to the sham group, suggesting that the antidepressant effect of rTMS may be related to changes in inflammatory cytokines.

Fecal metabolomic analysis of rabbits infected with Eimeria intestinalis and Eimeria magna based on LC-MS/MS technique.

Rabbit coccidiosis is a common parasitic disease leading to economic losses in the rabbit industry. The intestinal flora plays a key role in pathogenesis of coccidiosis, and fecal metabolome mediates host-microbiome interactions as a functional readout of the gut microbiome. In this study, the E. intestinalis-infected and E. magna-infected rabbit models were established to investigate metabolic alterations and metabolic pathways based on LC-MS/MS technique for the first time. Multivariate OPLS-DA analysis was performed to explore differential metabolites. In total, 288 metabolites were detected from infected and uninfected rabbits. The level of 33 metabolites increased and 4 decreased in rabbits infected with E. intestinalis. Eight pathways were significantly perturbed during E. intestinalis infection including biosynthesis of unsaturated fatty acids, fatty acid biosynthesis, etc. After rabbits infected with E. magna, 13 metabolites were altered and 7 metabolic pathways were dysregulated. These metabolites and metabolic pathways were mainly involved in tuberculosis, parathyroid hormone synthesis, etc. Besides, 25 metabolites differed in abundance between E. intestinalis infection group and E. magna infection group, the major perturbed metabolic pathways were lipid metabolism and endocrine system, respectively. In general, it is confirmed that E. intestinalis and E. magna infection destroyed the intestinal flora, which caused corresponding changes in metabolites, and provide novel insights into the molecular mechanisms of rabbit-parasite interactions.

Effects of Continuous LPS Induction on Oxidative Stress and Liver Injury in Weaned Piglets.

Due to imperfections in their immune and digestive systems, weaned piglets are susceptible to invasions of the external environment and diseases, especially bacterial infections, which lead to slow growth, tissue damage, and even the death of piglets. Here, a model of weaned piglets induced by Escherichia coli lipopolysaccharide (LPS) was established to explore the effects of continuous low-dose LPS induction on the mechanism of liver injury. A total of forty-eight healthy 28-day-old weaned piglets (weight = 6.65 ± 1.19 kg) were randomly divided into two groups: the CON group and LPS group. During the experimental period of thirteen days, the LPS group was injected intraperitoneally with LPS (100 µg/kg) once per day, and the CON group was treated with the same volume of 0.9% NaCl solution. On the 1st, 5th, 9th, and 13th days, the serum and liver of the piglets were collected for the determination of serum biochemical indexes, an antioxidant capacity evaluation, and histopathological examinations. In addition, the mRNA expression levels of the TLR4 pathway and inflammatory cytokines were detected. The results showed that the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in the serum increased after LPS induction. The activities of total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) in the serum and liver homogenate of the LPS group were lower than those of the CON group, while the malondialdehyde (MDA) content in the serum and the activities of catalase (CAT) and superoxide dismutase (SOD) in the liver of the LPS group were higher than those in the CON group. At the same time, morphological impairment of the livers occurred, including hepatocyte caryolysis, hepatocyte vacuolization, karyopycnosis, and inflammatory cell infiltration, and the mRNA expression levels of TLR4, MyD88, NF-κB, TNF-α, IL-6, and IL-10 were upregulated in the livers after LPS induction. The above results were more obvious on the 1st and 5th days of LPS induction, while the trend during the later period was not significant. It was concluded that the oxidative stress and liver injury occurred at the early stage of LPS induction, while the liver damage weakened at the later stage. The weaned piglets probably gradually developed tolerance to the endotoxin after the continuous low-dose induction of LPS.

Rabbit microbiota across the whole body revealed by 16S rRNA gene amplicon sequencing.

BACKGROUND: Rabbit can produce meat, fur and leather, and serves as an important biomedical animal model. Understanding the microbial community of rabbits helps to raise rabbits healthily and better support their application as animal models. RESULTS: In this study, we selected 4 healthy Belgium gray rabbits to collect the microbial samples from 12 body sites, including skin, lung, uterus, mouth, stomach, duodenum, ileum, jejunum, colon, cecum, cecal appendix and rectum. The microbiota across rabbit whole body was investigated via 16S rRNA gene amplicon sequencing. After quality control, 46 samples were retained, and 3,148 qualified ASVs were obtained, representing 23 phyla and 264 genera. Based on the weighted UniFrac distances, these samples were divided into the large intestine (Lin), stomach and small intestine (SSin), uterus (Uter), and skin, mouth and lung (SML) groups. The diversity of Lin microbiota was the highest, followed by those of the SSin, Uter and SML groups. In the whole body, Firmicutes (62.37%), Proteobacteria (13.44%) and Bacteroidota (11.84%) were the most predominant phyla. The relative abundance of Firmicutes in the intestinal tract was significantly higher than that in the non-intestinal site, while Proteobacteria was significantly higher in the non-intestinal site. Among the 264 genera, 35 were the core microbiota distributed in all body sites. Sixty-one genera were specific in the SML group, while 13, 8 and 1 were specifically found in the Lin, SSin and Uter groups, respectively. The Lin group had the most difference with other groups, there were average 72 differential genera between the Lin and other groups. The functional prediction analysis showed that microbial function within each group was similar, but there was a big difference between the intestinal tracts and the non-intestinal group. Notably, the function of microorganism in uterus and mouth were the most different from those in the gastrointestinal sites; rabbit's coprophagy of consuming soft feces possibly resulted in little differences of microbial function between stomach and large intestinal sites. CONCLUSION: Our findings improve the knowledge about rabbit microbial communities throughout whole body and give insights into the relationship of microbial communities among different body sites in health rabbits.

The influence on oxidative stress markers, inflammatory factors and intestinal injury-related molecules in Wahui pigeon induced by lipopolysaccharide.

INTRODUCTION: The intestinal structure is the foundation for various activities and functions in poultry. An important question concerns the changes in the intestinal status under endotoxin stimulation. This study aimed to investigate the mechanism of intestinal injury induced by lipopolysaccharide (LPS) in Wahui pigeons. METHODS: Thirty-six 28-day-old healthy Wahui pigeons were randomly divided into two groups. The experimental group was injected with LPS (100 µg/kg) once per day for five days, and the control group was treated with the same amount of sterile saline. Blood and the ileum were collected from pigeons on the first, third, and fifth days of the experiment and used for oxidative stress assessment, inflammatory factor detection, histopathological examination, and positive cell localization. In addition, intestinal injury indices and mRNA expression levels (tight junction proteins, inflammatory cytokines, and factors related to autophagy and apoptosis) were evaluated. RESULTS: Villi in the ileum were shorter in the LPS group than in the control group, and D-lactic acid levels in the serum were significantly increased. Glutathione and catalase levels significantly decreased, but the malondialdehyde content in the serum increased. TNF-α and IL-10 were detected at higher levels in the serum, with stronger positive signals and higher mRNA expression levels, in the LPS group than in the control group. In addition, the levels of TLR4, MyD88, NF-κB, and HMGB1 in the inflammatory signaling pathway were also upregulated. Finally, the mRNA expression of Claudin3, Occludin, and ZO-1 was significantly decreased; however, that of Beclin1 and Atg5 was increased in the LPS group. CONCLUSION: Ileal pathological changes and oxidative stress were caused by LPS challenge; it is proposed that this triggering regulates the inflammatory response, causing excessive autophagy and apoptosis, promoting intestinal permeability, and leading to intestinal injury in Wahui pigeons.

Batch fluidized bed reactor based modified biosynthetic crystals: Optimization of adsorptive properties and application in fluoride removal from groundwater.

A batch fluidized bed reactor (BFBR) with modified biosynthetic crystals (MBC), derived from Pseudomonas sp. HXF1, was investigated for the treatment of the groundwater containing fluoride (F-). Impacts of different hydraulic retention time (HRT), pH, and initial F- concentration on F- removal were examined and the maximum defluorination efficiency was recorded as 95.20%. Moreover, recycling experiments were performed to evaluate the stability of repeated use. BFBR/MBC system showed a long-term effective treatment outcome with low fluctuation in the concentrations of residual Ca2+ and F-. The formed precipitates were characterized by SEM, XPS, XRD, and FTIR. The defluorination mechanisms of BFBR/MBC system were defined as the chemisorption and induced crystallization of Ca5(PO4)3F on the MBC surface. As a feasible, economical, and environment-friendly technique, the method has a long-term value, which suggests promising applications in F- removal and resourceful treatment.

Isovitexin Depresses Osteoarthritis Progression via the Nrf2/NF-κB Pathway: An in vitro Study.

PURPOSE: Osteoarthritis (OA) is a multifactorial joint disease and inflammatory processes contribute to joint destruction. Isovitexin (IVX) is a flavone component found in passion flower, Cannabis and, and the palm that is known for its anti-inflammatory properties. MATERIALS AND METHODS: This study investigated in vitro the role and underlying mechanism used by IVX in its regulation of OA development. Effects of IVX on the viability of chondrocytes were measured by CCK-8 assays. The phenotypes of extracellular matrix (ECM) degeneration and inflammation were measured by qPCR, Western blot, and ELISA; and NF-κB pathway was detected by immunofluorescence and Western blot. Molecular docking was applied to predict the interacting protein of IVX, while Nrf2 was knocked down by siRNAs to confirm its role. RESULTS: We demonstrated that IVX suppressed ECM degeneration and suppressed pro-inflammatory factors in IL-1ß-treated chondrocytes. Additionally, IVX impact on NF-κB signaling in IL-1ß-exposed chondrocytic cells; Mechanistically, it was also demonstrated in molecular docking and knock down studies that IVX might bind to Nrf2 to suppress NF-κB pathway. CONCLUSION: Our data suggest that IVX halts OA disease advancement through the Nrf2/NF-κB axis, suggesting a possibility of IVX as a target for OA therapy.

Apelin/APJ-Manipulated CaMKK/AMPK/GSK3ß Signaling Works as an Endogenous Counterinjury Mechanism in Promoting the Vitality of Random-Pattern Skin Flaps.

A random-pattern skin flap plays an important role in the field of wound repair; the mechanisms that influence the survival of random-pattern skin flaps have been extensively studied but little attention has been paid to endogenous counterinjury substances and mechanism. Previous reports reveal that the apelin-APJ axis is an endogenous counterinjury mechanism that has considerable function in protecting against infection, inflammation, oxidative stress, necrosis, and apoptosis in various organs. As an in vivo study, our study proved that the apelin/APJ axis protected the skin flap by alleviating vascular oxidative stress and the apelin/APJ axis works as an antioxidant stress factor dependent on CaMKK/AMPK/GSK3ß signaling. In addition, the apelin/APJ-manipulated CaMKK/AMPK/GSK3ß-dependent mechanism improves HUVECs' resistance to oxygen and glucose deprivation/reperfusion (OGD/R), reduces ROS production and accumulation, maintained the normal mitochondrial membrane potential, and suppresses oxidative stress in vitro. Besides, activation of the apelin/APJ axis promotes vascular migration and angiogenesis under relative hypoxia condition through CaMKK/AMPK/GSK3ß signaling. In a word, we provide new evidence that the apelin/APJ axis is an effective antioxidant and can significantly improve the vitality of random flaps, so it has potential be a promising clinical treatment.

Heterotrophic nitrification and biomineralization potential of Pseudomonas sp. HXF1 for the simultaneous removal of ammonia nitrogen and fluoride from groundwater.

Pseudomonas sp. HXF1, a strain capable of heterotrophic nitrification, aerobic denitrification (HNAD), and biomineralization was identified and employed for the simultaneous removal of ammonia nitrogen (NH4+-N) and fluoride (F-). It removed 99.2% of NH4+-N without accumulation of nitrous nitrogen (NO2--N) and nitrate nitrogen (NO3--N), while removed 87.3% of F-. Response surface methodology (RSM) was used to study the best removal conditions for NH4+-N and F-. The results of nitrogen balance experiments with NH4Cl, NaNO2, and NaNO3 as single nitrogen sources and amplification experiments of denitrification genes proved that the bacterial strains may remove NH4+-N through HNAD. The experimental results of Scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS), and X-ray diffractometer (XRD) indicated that the way of F- removal may be adsorption and co-precipitation. The results demonstrated that the strain HXF1 has great potential in the biological denitrification and F- removal of groundwater.

Study on the simultaneous removal of fluoride, heavy metals and nitrate by calcium precipitating strain Acinetobacter sp. H12.

The removal properties and mechanisms of fluoride (F-) and nickel (Ni2+) were studied by biomineralizing bacteria (Acinetobacter sp. H12). The results showed that the removal ratio of F-, Ca2+ and Ni2+ reached 75% (0.031 mg·L-1·h-1), 84.96% (2.123 mg·L-1·h-1), and 56.67% (0.024 mg·L-1·h-1) after 72 h, respectively. The removal ratio of nitrate (NO3-) reached 100% (0.686 mg·L-1·h-1) after 24 h. SEM and XRD images indicated that bioprecipitation of CaF2, Ca5(PO4)3F, Ca5(PO4)3(OH), NiCO3, CaCO3 and Ni were formed, and some of these precipitation used bacteria as nucleation sites to form biological crystal seeds. N2 was the primary product in gas chromatography analysis. Meanwhile, both the fluorescence spectroscopy and fourier transform near-infrared spectroscopy analysis proved that strain H12 had good ability to remove fluoride and nickel ions simultaneously.

Isolation of biosynthetic crystals by microbially induced calcium carbonate precipitation and their utilization for fluoride removal from groundwater.

Biosynthetic crystals (BC) were prepared through microbially induced calcium carbonate precipitation (MICP) for fluoride (F-) removal from the groundwater. Batch experiments were conducted to evaluate the fluoride adsorption capacity and the impacts of critical factors (organic matter, pH, initial fluoride concentration and BC dosage) on defluorination efficiency of BC. The maximum adsorption amount and defluorination efficiency were recorded as 5.10 mg g-1 and 98.24%, respectively. The adsorption kinetics and isotherms studies showed that pseudo-second-order kinetic model and Freundlich isotherm model were best fitting to the reaction. Adsorption thermodynamic parameters indicated a spontaneous, endothermic and thermodynamically favorable adsorption process. Moreover, the mechanism of F- removal by BC was further analyzed by SEM, XPS, XRD and FTIR. The method can cope with the problem of applying the external organic substances in MICP, and avoid the microbial safety risk in the effluent. As an economically and environmentally friendly adsorbent, BC can be used for F- removal from groundwater.

Inhibition of PI3K/Akt/NF-κB signaling by Aloin for ameliorating the progression of osteoarthritis: In vitro and in vivo studies.

Osteoarthritis (OA) is a progressive and degenerative joint disease. Aloin is a bitter and yellow-brown-coloured compound from the Aloe plant and is allowed for use in foods as a "natural flavour". In our study, we examined the protective effects of Aloin on the inhibition of OA development as well as its underlying mechanism in both in vitro and vivo experiments. In in-vitro experiments, the protective effect of aloin on the anabolism and catabolism of the extracellular matrix (ECM) induced by IL-1 ß in chondrocytes by inhibiting the expression of pro-inflammatory factors, including TNF-α (p = 0.016), IL-6 (p = 0.006), iNOS (p = 0.001) and COX-2 (p = 0.006). Mechanistically, Aloin suppressed the IL-1ß-induced activation of the PI3K/Akt/NF-κB signalling pathway cascades. Moreover, molecular docking studies demonstrated that Aloin bound strongly to PI3K. In vivo, Aloin ameliorated the OA process in the destabilization of the medial meniscus (DMM) model. In summary, our findings demonstrate that Aloin ameliorates the progression of OA via the PI3K/Akt/NF-κB signalling pathways, which supports Aloin as a promising therapeutic agent for the treatment of OA.

Effects of Repeated Lipopolysaccharide Treatment on Growth Performance, Immune Organ Index, and Blood Parameters of Sprague-Dawley Rats.

INTRODUCTION: The study was designed to investigate the effects of repeated lipopolysaccharide (LPS) treatment on growth performance, lymphoid organ indexes, and blood cells in Sprague-Dawley rats. MATERIAL AND METHODS: Forty healthy weaned Sprague-Dawley rats were randomly equally divided into LPS and control groups. Each rat in the LPS group was injected via the caudal vein with LPS (100 µg/kg b.w.) for 10 days, and the control group was treated with an equal volume of normal saline. On the 1st, 4th, 7th, and 10th days, growth performance, lymphoid organ indexes, and blood cells were evaluated in five necropsied rats. RESULTS: When rats were treated 3-10 times with LPS, their body weight and average daily gains increased more slowly than in the control group (P < 0.05). Repeated LPS treatment significantly increased spleen weight and the ratio of spleen to body weight (P < 0.05). White blood cells, neutrophils, and neutrophil percentage increased (P < 0.05) remarkably, but lymphocyte percentage, haemoglobin, and blood platelet counts decreased significantly (P < 0.05). CONCLUSION: LPS treatment obviously suppresses growth and promotes peripheral immune organ proliferation. It is indicated that host protective mechanism can be activated by multiple small doses of LPS and prevents organs from further damage during stress status.

Dynamic transcriptome landscape of Asian domestic honeybee (Apis cerana) embryonic development revealed by high-quality RNA sequencing.

BACKGROUND: Honeybee development consists of four stages: embryo, larva, pupa and adult. Embryogenesis, a key process of cell division and differentiation, takes 3 days in honeybees. However, the embryonic transcriptome and the dynamic regulation of embryonic transcription are still largely uncharacterized in honeybees, especially in the Asian honeybee (Apis cerana). Here, we employed high-quality RNA-seq to explore the transcriptome of Asian honeybee embryos at three ages, approximately 24, 48 and 72 h (referred to as Day1, Day2 and Day3, respectively). RESULTS: Nine embryo samples, three from each age, were collected for RNA-seq. According to the staging scheme of honeybee embryos and the morphological features we observed, our Day1, Day2 and Day3 embryos likely corresponded to the late stage four, stage eight and stage ten development stages, respectively. Hierarchical clustering and principal component analysis showed that same-age samples were grouped together, and the Day2 samples had a closer relationship with the Day3 samples than the Day1 samples. Finally, a total of 18,284 genes harboring 55,646 transcripts were detected in the A. cerana embryos, of which 44.5% consisted of the core transcriptome shared by all three ages of embryos. A total of 4088 upregulated and 3046 downregulated genes were identified among the three embryo ages, of which 2010, 3177 and 1528 genes were upregulated and 2088, 2294 and 303 genes were downregulated from Day1 to Day2, from Day1 to Day3 and from Day2 to Day3, respectively. The downregulated genes were mostly involved in cellular, biosynthetic and metabolic processes, gene expression and protein localization, and macromolecule modification; the upregulated genes mainly participated in cell development and differentiation, tissue, organ and system development, and morphogenesis. Interestingly, several biological processes related to the response to and detection of light stimuli were enriched in the first-day A. cerana embryogenesis but not in the Apis mellifera embryogenesis, which was valuable for further investigations. CONCLUSIONS: Our transcriptomic data substantially expand the number of known transcribed elements in the A. cerana genome and provide a high-quality view of the transcriptome dynamics of A. cerana embryonic development.

Covalent layer-by-layer assembly of hyperbranched polyether and polyethyleneimine: multilayer films providing possibilities for surface functionalization and local drug delivery.

A convenient and simple route to multifunctional surface coatings via the alternating covalent layer-by-layer (LBL) assembly of p-nitrophenyloxycarbonyl group-terminated hyperbranched polyether (HBPO-NO(2)) and polyethylenimine (PEI) is described. The in situ chemical reaction between HBPO-NO(2) and PEI onto aminolyzed substrates was rapid and mild. Results from ellipsometry measurements, contact angle measurements, and ATR-FTIR spectra confirmed the successful LBL assembly of the building blocks, and the surface reactivity of the multilayer films with HBPO-NO(2) as the outmost layer was demonstrated by the immobilization of an amine-functionalized fluorophore. Furthermore, a biomimetic surface was achieved by surface functionalization of the multilayer films with extracellular matrix protein collagen to promote the adhesion and growth of cells. The studies on the drug loading and in vitro release behaviors of the multilayer films demonstrated their application potentials in local delivery of hydrophilic and hydrophobic therapeutic agents.

Construction of multifunctional coatings via layer-by-layer assembly of sulfonated hyperbranched polyether and chitosan.

Layer-by-layer assembly has shown a great deal of promise in biomedical coatings, as well as local drug delivery systems. The poor loading capacity of hydrophobic drugs within the multilayers is a drawback in their potential applications. Herein, sulfonated hyperbranched polyether (HBPO-SO(3)) with a hydrophobic core was incorporated into LBL films to provide nanoreservoirs for hydrophobic guest molecules. HBPO-SO(3) was proven to form stable micelles in the sodium acetate and acetic acid buffer solution (HAc buffer) for LbL assembly. The QCM and ellipsometry experiments demonstrated that the LBL films can be fabricated via alternating deposition of HBPO-SO(3) micelles and chitosan. The fluorescence emission spectra verified that the hydrophobic pyrene can be incorporated both by pre-encapsulation in HBPO-SO(3) micelles and post-diffusion in preassembled multilayer films. Compared with the pre-encapsulation approach, the post-diffusion process was more efficient in incorporating hydrophobic guest molecules into the LbL films and carried out a much more controllable release of the guest molecules. A multifunctional coating with potential anticoagulation, antibacterial, and local release of hydrophobic drug Probucal, which has powerful antioxidant properties and can prevent restenosis after coronary angioplasty, was then developed via post-diffusion of the anti-restenosis agents into the multilayer films of HBPO-SO(3) and chitosan.

Generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza A H5N1 viruses.

BACKGROUND: The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs), AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model. CONCLUSIONS/SIGNIFICANCE: Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines.

[The differential expression of the human lung carcinoma cells infected with high pathogenic avian influenza virus A/Anhui/1/2005 (H5N1)].

OBJECTIVE: To identify genes in human cells infected with high pathogenic avian influenza viruses H5N1. METHODS: The lung carcinoma cells line A549 was infected with H5N1 and H1N1, respectively. We harvested the infected cells at the different time points after infection and screened the genes with differential expression via microarray technology. The candidate genes were selected and confirmed by quantitative real-time PCR. RESULTS: The spectrum of genes with the differential expression in the cells infected with H5N1 was obtained and 16 candidate genes were identified in the cellular apoptosis pathway, mTOR pathway, and the cellular immunity as well. CONCLUSIONS: Our results suggest that H5N1 exert a stronger impact on eliciting apoptosis of infected cells than the common influenza virus H1N1.