GC-biased gene conversion drives accelerated evolution of ultraconserved elements in mammalian and avian genomes.

Ultraconserved elements (UCEs) are the most conserved regions among the genomes of evolutionarily distant species and are thought to play critical biological functions. However, some UCEs rapidly evolved in specific lineages, and whether they contributed to adaptive evolution is still controversial. Here, using an increased number of sequenced genomes with high taxonomic coverage, we identified 2191 mammalian UCEs and 5938 avian UCEs from 95 mammal and 94 bird genomes, respectively. Our results show that these UCEs are functionally constrained and that their adjacent genes are prone to widespread expression with low expression diversity across tissues. Functional enrichment of mammalian and avian UCEs shows different trends indicating that UCEs may contribute to adaptive evolution of taxa. Focusing on lineage-specific accelerated evolution, we discover that the proportion of fast-evolving UCEs in nine mammalian and 10 avian test lineages range from 0.19% to 13.2%. Notably, up to 62.1% of fast-evolving UCEs in test lineages are much more likely to result from GC-biased gene conversion (gBGC). A single cervid-specific gBGC region embracing the uc.359 allele significantly alters the expression of Nova1 and other neural-related genes in the rat brain. Combined with the altered regulatory activity of ancient gBGC-induced fast-evolving UCEs in eutherians, our results provide evidence that synergy between gBGC and selection shaped lineage-specific substitution patterns, even in the most constrained regulatory elements. In summary, our results show that gBGC played an important role in facilitating lineage-specific accelerated evolution of UCEs, and further support the idea that a combination of multiple evolutionary forces shapes adaptive evolution.

Preparation of Bispecific IgY-scFvs Inhibition Adherences of Enterotoxigenic Escherichia coli (K88 and F18) to Porcine IPEC-J2 Cell.

Enterotoxigenic Escherichia coli (ETEC) strains are significant contributors to postweaning diarrhea in piglets. Of the ETEC causing diarrhea, K88 and F18 accounted for 92.7%. Despite the prevalence of ETEC K88 and F18, there is currently no effective vaccine available due to the diversity of these strains. This study presents an innovative approach by isolating chicken-derived single-chain variable fragment antibodies (scFvs) specific to K88 and F18 fimbrial antigens from chickens immunized against these ETEC virulence factors. These scFvs effectively inhibited adhesion of K88 and F18 to porcine intestinal epithelial cells (IPEC-J2), with the inhibitory effect demonstrating a dose-dependent increase. Furthermore, a bispecific scFv was designed and expressed in Pichia pastoris. This engineered construct displayed remarkable potency; at a concentration of 25.08 µg, it significantly reduced the adhesion rate of ETEC strains to IPEC-J2 cells by 72.10% and 69.11% when challenged with either K88 or F18 alone. Even in the presence of both antigens, the adhesion rate was notably decreased by 57.92%. By targeting and impeding the initial adhesion step of ETEC pathogenesis, this antibody-based intervention holds promise as a potential alternative to antibiotics, thereby mitigating the risks associated with antibiotic resistance and residual drug contamination in livestock production. Overall, this study lays the groundwork for the development of innovative treatments against ETEC infections in piglets.

Convergent Genomic Signatures of Cashmere Traits: Evidence for Natural and Artificial Selection.

Convergent evolution provides powerful opportunities to investigate the genetic basis of complex traits. The Tibetan antelope (Pantholops hodgsonii) and Siberian ibex (Capra sibirica) belong to different subfamilies in Bovidae, but both have evolved similar superfine cashmere characteristics to meet the cold temperature in plateau environments. The cashmere traits of cashmere goats underwent strong artificial selection, and some traces of domestication also remained in the genome. Hence, we investigated the convergent genomic signatures of cashmere traits between natural and artificial selection. We compared the patterns of convergent molecular evolution between Tibetan antelope and Siberian ibex by testing positively selected genes, rapidly evolving genes and convergent amino acid substitutions. In addition, we analyzed the selected genomic features of cashmere goats under artificial selection using whole-genome resequencing data, and skin transcriptome data of cashmere goats were also used to focus on the genes involved in regulating cashmere traits. We found that molecular convergent events were very rare, but natural and artificial selection genes were convergent enriched in similar functional pathways (e.g., ECM-receptor interaction, focal adhesion, PI3K-Akt signaling pathway) in a variety of gene sets. Type IV collagen family genes (COL4A2, COL4A4, COL4A5, COL6A5, COL6A6) and integrin family genes (ITGA2, ITGA4, ITGA9, ITGB8) may be important candidate genes for cashmere formation and development. Our results provide a comprehensive approach and perspective for exploring cashmere traits and offer a valuable reference for subsequent in-depth research on the molecular mechanisms regulating cashmere development and fineness.

Quantum Dot Nanobead-Based Fluorescence-Linked Immunosorbent Assay for Detection of Glycinin in Soybeans and Soy Products.

Soybean glycinin, as a major soybean allergen, is difficult to accurately quantify due to its large molecular weight and complex structure. CdSe/ZnS quantum dot nanobead (QB) is a core/shell fluorescent nanomaterial with strong fluorescent signals and high sensitivity at 630 nm. An immunosorbent assay based on CdSe/ZnS quantum dot nanobeads (QBs-FLISA) was developed for the glycinin quantification in soybean and soybean products. Here, the purified glycinin was coated on the microporous plate to serve as the coating antigen, and CdSe/ZnS nanobead conjugated with anti-glycinin polyclonal antibodies was used as fluorescent detection probe. The target glycinin in the sample and the coated antigen on the plate competitively adsorbed the antibody labeled the CdSe/ZnS QBs probes. The limits of detection and quantitation for glycinin were 0.035 and 0.078 µg mL-1, respectively. The recoveries of the spiked samples ranged from 89.8% to 105.6%, with relative standard deviation less than 8.6%. However, compared with ELISA, the sensitivities of QBs-FLISA for the detection of glycinin were increased by 7 times, and the detection time was shortened by two-thirds. This QBs-FLISA method has been effectively applied to the detection of soybean seeds with different varieties and soy products with different processing techniques, which will provide a rapid screening method for soybean and soybean products with low allergens.

Neuraminidase 1-mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection.

Pseudomonas aeruginosa (Pa) expresses an adhesin, flagellin, that engages the mucin 1 (MUC1) ectodomain (ED) expressed on airway epithelia, increasing association of MUC1-ED with neuraminidase 1 (NEU1) and MUC1-ED desialylation. The MUC1-ED desialylation unmasks both cryptic binding sites for Pa and a protease recognition site, permitting its proteolytic release as a hyperadhesive decoy receptor for Pa. We found here that intranasal administration of Pa strain K (PAK) to BALB/c mice increases MUC1-ED shedding into the bronchoalveolar compartment. MUC1-ED levels increased as early as 12 h, peaked at 24-48 h with a 7.8-fold increase, and decreased by 72 h. The a-type flagellin-expressing PAK strain and the b-type flagellin-expressing PAO1 strain stimulated comparable levels of MUC1-ED shedding. A flagellin-deficient PAK mutant provoked dramatically reduced MUC1-ED shedding compared with the WT strain, and purified flagellin recapitulated the WT effect. In lung tissues, Pa increased association of NEU1 and protective protein/cathepsin A with MUC1-ED in reciprocal co-immunoprecipitation assays and stimulated MUC1-ED desialylation. NEU1-selective sialidase inhibition protected against Pa-induced MUC1-ED desialylation and shedding. In Pa-challenged mice, MUC1-ED-enriched bronchoalveolar lavage fluid (BALF) inhibited flagellin binding and Pa adhesion to human airway epithelia by up to 44% and flagellin-driven motility by >30%. Finally, Pa co-administration with recombinant human MUC1-ED dramatically diminished lung and BALF bacterial burden, proinflammatory cytokine levels, and pulmonary leukostasis and increased 5-day survival from 0% to 75%. We conclude that Pa flagellin provokes NEU1-mediated airway shedding of MUC1-ED, which functions as a decoy receptor protecting against lethal Pa lung infection.

NEU1 Sialidase Regulates Membrane-tethered Mucin (MUC1) Ectodomain Adhesiveness for Pseudomonas aeruginosa and Decoy Receptor Release.

Airway epithelia express sialylated receptors that recognize exogenous danger signals. Regulation of receptor responsiveness to these signals remains incompletely defined. Here, we explore the mechanisms through which the human sialidase, neuraminidase-1 (NEU1), promotes the interaction between the sialoprotein, mucin 1 (MUC1), and the opportunistic pathogen, Pseudomonas aeruginosa. P. aeruginosa flagellin engaged the MUC1 ectodomain (ED), increasing NEU1 association with MUC1. The flagellin stimulus increased the association of MUC1-ED with both NEU1 and its chaperone/transport protein, protective protein/cathepsin A. Scatchard analysis demonstrated NEU1-dependent increased binding affinity of flagellin to MUC1-expressing epithelia. NEU1-driven MUC1-ED desialylation rapidly increased P. aeruginosa adhesion to and invasion of the airway epithelium. MUC1-ED desialylation also increased its shedding, and the shed MUC1-ED competitively blocked P. aeruginosa adhesion to cell-associated MUC1-ED. Levels of desialylated MUC1-ED were elevated in the bronchoalveolar lavage fluid of mechanically ventilated patients with P. aeruginosa airway colonization. Preincubation of P. aeruginosa with these same ex vivo fluids competitively inhibited bacterial adhesion to airway epithelia, and MUC1-ED immunodepletion completely abrogated their inhibitory activity. These data indicate that a prokaryote, P. aeruginosa, in a ligand-specific manner, mobilizes eukaryotic NEU1 to enhance bacterial pathogenicity, but the host retaliates by releasing MUC1-ED into the airway lumen as a hyperadhesive decoy receptor.

The NEU1-selective sialidase inhibitor, C9-butyl-amide-DANA, blocks sialidase activity and NEU1-mediated bioactivities in human lung in vitro and murine lung in vivo.

Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung microvascular endothelia where it mediates multiple biological processes. We tested whether the NEU1-selective sialidase inhibitor, C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (C9-BA-DANA), inhibits one or more established NEU1-mediated bioactivities in human lung cells. We established the IC50 values of C9-BA-DANA for total sialidase activity in human airway epithelia, lung microvascular endothelia and lung fibroblasts to be 3.74 µM, 13.0 µM and 4.82 µM, respectively. In human airway epithelia, C9-BA-DANA dose-dependently inhibited flagellin-induced, NEU1-mediated mucin-1 ectodomain desialylation, adhesiveness for Pseudomonas aeruginosa and shedding. In lung microvascular endothelia, C9-BA-DANA reversed NEU1-driven restraint of cell migration into a wound and disruption of capillary-like tube formation. NEU1 and its chaperone/transport protein, protective protein/cathepsin A (PPCA), were differentially expressed in these same cells. Normalized NEU1 protein expression correlated with total sialidase activity whereas PPCA expression did not. In contrast to eukaryotic sialidases, C9-BA-DANA exerted far less inhibitory activity for three selected bacterial neuraminidases (IC50 > 800 µM). Structural modeling of the four human sialidases and three bacterial neuraminidases revealed a loop between the seventh and eighth strands of the ß-propeller fold, that in NEU1, was substantially shorter than that seen in the six other enzymes. Predicted steric hindrance between this loop and C9-BA-DANA could explain its selectivity for NEU1. Finally, pretreatment of mice with C9-BA-DANA completely protected against flagellin-induced increases in lung sialidase activity. Our combined data indicate that C9-BA-DANA inhibits endogenous and ectopically expressed sialidase activity and established NEU1-mediated bioactivities in human airway epithelia, lung microvascular endothelia, and fibroblasts in vitro and murine lungs in vivo.

Elevated expression of NEU1 sialidase in idiopathic pulmonary fibrosis provokes pulmonary collagen deposition, lymphocytosis, and fibrosis.

Idiopathic pulmonary fibrosis (IPF) poses challenges to understanding its underlying cellular and molecular mechanisms and the development of better therapies. Previous studies suggest a pathophysiological role for neuraminidase 1 (NEU1), an enzyme that removes terminal sialic acid from glycoproteins. We observed increased NEU1 expression in epithelial and endothelial cells, as well as fibroblasts, in the lungs of patients with IPF compared with healthy control lungs. Recombinant adenovirus-mediated gene delivery of NEU1 to cultured primary human cells elicited profound changes in cellular phenotypes. Small airway epithelial cell migration was impaired in wounding assays, whereas, in pulmonary microvascular endothelial cells, NEU1 overexpression strongly impacted global gene expression, increased T cell adhesion to endothelial monolayers, and disrupted endothelial capillary-like tube formation. NEU1 overexpression in fibroblasts provoked increased levels of collagen types I and III, substantial changes in global gene expression, and accelerated degradation of matrix metalloproteinase-14. Intratracheal instillation of NEU1 encoding, but not control adenovirus, induced lymphocyte accumulation in bronchoalveolar lavage samples and lung tissues and elevations of pulmonary transforming growth factor-ß and collagen. The lymphocytes were predominantly T cells, with CD8(+) cells exceeding CD4(+) cells by nearly twofold. These combined data indicate that elevated NEU1 expression alters functional activities of distinct lung cell types in vitro and recapitulates lymphocytic infiltration and collagen accumulation in vivo, consistent with mechanisms implicated in lung fibrosis.

[Clinical assessment of chronic prostatitis based on infrared imaging technology].

OBJECTIVE: To investigate the characteristic changes in the infrared thermogram of chronic prostatitis (CP) patients and find some evidence for the auxiliary diagnosis and therapeutic evaluation of the disease. METHODS: Fifty CP patients and 20 healthy male volunteers were included in this clinical trial. The infrared thermograms of the subjects were compared between the two groups for characteristic changes. The values obtained were used for the auxiliary diagnosis and therapeutic evaluation of the disease. RESULTS: Compared with the healthy males in the same age group, the CP patients showed extremely significant abnormal changes in the average temperature value in the hypogastrium (H), pubis (P), scrotum (S), and groin (G) (P < 0.01). The average H temperature value of the CP patients was correlated negatively with the CP symptom index (CPSI) (P < 0.01, Pearsons correlation coefficient = -0.519), while the S temperature positively with CPSI (P < 0.01, Pearsons correlation coefficient = 0.446). In addition to the H value, the P, S, and G values were all correlated in different degrees with CPSI (P < 0.01), which the S value exhibited the most significantly negative correlation (Pearson's correlation coefficient = -0.898). CONCLUSION: There are some characteristic changes in the hypogastrium temperature of CP patients in the infrared thermogram, which has a potential application value for the auxiliary diagnosis, symptom assessment, and therapeutic evaluation of CP.

NEU1 sialidase regulates the sialylation state of CD31 and disrupts CD31-driven capillary-like tube formation in human lung microvascular endothelia.

The highly sialylated vascular endothelial surface undergoes changes in sialylation upon adopting the migratory/angiogenic phenotype. We recently established endothelial cell (EC) expression of NEU1 sialidase (Cross, A. S., Hyun, S. W., Miranda-Ribera, A., Feng, C., Liu, A., Nguyen, C., Zhang, L., Luzina, I. G., Atamas, S. P., Twaddell, W. S., Guang, W., Lillehoj, E. P., Puché, A. C., Huang, W., Wang, L. X., Passaniti, A., and Goldblum, S. E. (2012) NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia. NEU1 restrains endothelial cell migration whereas NEU3 does not. J. Biol. Chem. 287, 15966-15980). We asked whether NEU1 might regulate EC capillary-like tube formation on a Matrigel substrate. In human pulmonary microvascular ECs (HPMECs), prior silencing of NEU1 did not alter tube formation. Infection of HPMECs with increasing multiplicities of infection of an adenovirus encoding for catalytically active WT NEU1 dose-dependently impaired tube formation, whereas overexpression of either a catalytically dead NEU1 mutant, NEU1-G68V, or another human sialidase, NEU3, did not. NEU1 overexpression also diminished EC adhesion to the Matrigel substrate and restrained EC migration in a wounding assay. In HPMECs, the adhesion molecule, CD31, also known as platelet endothelial cell adhesion molecule-1, was sialylated via α2,6-linkages, as shown by Sambucus nigra agglutinin lectin blotting. NEU1 overexpression increased CD31 binding to Arachis hypogaea or peanut agglutinin lectin, indicating CD31 desialylation. In the postconfluent state, when CD31 ectodomains are homophilically engaged, NEU1 was recruited to and desialylated CD31. In postconfluent ECs, CD31 was desialylated compared with subconfluent cells, and prior NEU1 silencing completely protected against CD31 desialylation. Prior CD31 silencing and the use of CD31-null ECs each abrogated the NEU1 inhibitory effect on EC tube formation. Sialyltransferase 6 GAL-I overexpression increased α2,6-linked CD31 sialylation and dose-dependently counteracted NEU1-mediated inhibition of EC tube formation. These combined data indicate that catalytically active NEU1 inhibits in vitro angiogenesis through desialylation of its substrate, CD31.

Human airway epithelia express catalytically active NEU3 sialidase.

Sialic acids on glycoconjugates play a pivotal role in many biological processes. In the airways, sialylated glycoproteins and glycolipids are strategically positioned on the plasma membranes of epithelia to regulate receptor-ligand, cell-cell, and host-pathogen interactions at the molecular level. We now demonstrate, for the first time, sialidase activity for ganglioside substrates in human airway epithelia. Of the four known mammalian sialidases, NEU3 has a substrate preference for gangliosides and is expressed at mRNA and protein levels at comparable abundance in epithelia derived from human trachea, bronchi, small airways, and alveoli. In small airway and alveolar epithelia, NEU3 protein was immunolocalized to the plasma membrane, cytosolic, and nuclear subcellular fractions. Small interfering RNA-induced silencing of NEU3 expression diminished sialidase activity for a ganglioside substrate by >70%. NEU3 immunostaining of intact human lung tissue could be localized to the superficial epithelia, including the ciliated brush border, as well as to nuclei. However, NEU3 was reduced in subepithelial tissues. These results indicate that human airway epithelia express catalytically active NEU3 sialidase.

Effect of Sini San freeze-dried powder on sleep-waking cycle in insomnia rats.

OBJECTIVE: To investigate the effects of the Sini San at different doses on each sleeping state [slow-wave sleep 1 (SWS 1), slow-wave sleep 2 (SWS2), rapid-eye-movement (REM), wakefulness (W)] in insomnia rats and to identify its mode of action for improving sleep. METHODS: The insomnia rats were randomly divided into a high-, medium- or low-dose group of Sini San (equal to crude drug 8.8, 4.4, or 2.2 g/kg, respectively) for seven consecutive days. RESULTS: Compared with pre-administration, SWS2 was significantly increased after administration of the low dose. Compared with pre-administration, W was significantly decreased and SWS1, SWS2, and the total sleeping time (TST) were markedly increased after administration of the medium dose. Compared with pre-administration, W was significantly decreased and SWS1, SWS2, rapid-eye-movement sleep, and TST were significantly longer after administration of the high dose. The effects of Sini San on sleep-wake cycle are dose-dependent. CONCLUSION: The results suggest that Sini San extends SWS1 and SWS2, which increases the total sleeping time.

Sedative and hypnotic effect of freeze-dried paeoniflorin and sini san freeze-dried powder in pentobarbital sodium-induced mice.

OBJECTIVE: To investigate the sedative and hypnotic activity of paeoniflorin and freeze-dried Sini San powder on mice and provide a reliable method for determining the pharmacodynamic material basis of Sini San. METHODS: Male adult mice weighing 20-22 g were used in this study. Three experiments were carried out. Synergism with pentobarbital was used as an index for hypnotic effect. Loss of the righting reflex was used to determine the start of sleep. Sleep latency and sleeping time were recorded in each experiment. RESULTS: The coefficient of variation of the suprathreshold dose (55 mg/kg) was significantly lower than that of the threshold dose. The sleep latency of mice was significantly decreased, and the sleeping time of mice was significantly prolonged. The effects of paeoniflorin and Sini San on prolonging the sleeping time of mice induced by pentobarbital sodium were significantly stronger than those in the control group. CONCLUSION: Paeoniflorin produces significant sedative and hypnotic effects, and there is an obvious dose-effect relationship.

Brain mechanism of acupuncture for children with anisometropic amblyopia: a resting functional magnetic resonance imaging study based on voxel-mirrored homotopic connectivity.

AIM: To explore the brain mechanism of acupuncture for children with anisometropic amblyopia using the voxel-mirror homotopic connectivity (VMHC) analysis method of resting functional magnetic resonance imaging (rs-fMRI) technology based on clinical effectiveness. METHODS: Eighty children with anisometropic monocular amblyopia were randomly divided into two groups: control (40 cases, 1 case of shedding) and acupuncture (40 cases, 1 case of shedding) groups. The control group was treated with glasses, red flash, grating, and visual stimulations, with each procedure conducted for 5min per time. Based on routine treatment, the acupuncture group underwent acupuncture of "regulating qi and unblocking meridians to bright eyes", Jingming (BL1), Cuanzhu (BL2), Guangming (GB37), Fengchi (GB20) acupoints were taken on both sides, with the needle kept for 30min each time. Both groups were treated once every other day, three times per week, for a total of 4wk. After the treatment, the overall curative effect of the two groups and the latency and amplitude changes of P100 wave of pattern visual-evoked potential were counted. At the same time, nine children with left eye amblyopia were randomly selected from the two groups and were scanned with rs-fMRI before and after treatment. The differences in the brain regions between the two groups were compared and analyzed with VMHC. RESULTS: Chi-square test showed a notable difference in the total efficiency rate between the acupuncture (94.87%) and control groups (79.49%). Regarding the P100 wave latency and amplitude, the acupuncture group had significantly shorter latency and higher amplitude of P100 wave than the control group. Moreover, the VMHC values of the bilateral temporal lobe, superior temporal gyrus, and middle temporal gyrus were notably increased in the acupuncture group after treatment. CONCLUSION: Acupuncture combined with conventional treatment can significantly improve the corrected visual acuity and optic nerve conduction in children with anisometropic amblyopia. Compared with the conventional treatment, the regulation of acupuncture on the functional activities of the relevant brain areas in the anterior cerebellum may be an effective acupuncture mechanism for anisometropic amblyopia.

A new triboelectric nanogenerator based on a multi-material stacking structure achieves efficient power conversion from discrete mechanical movement.

Due to its invaluable potential in discrete mechanical energy collection, TENG (triboelectric nanogenerator) is considered to satisfy the power requirements of intelligent electronic devices and drive the development of the Internet of Things (IoT). Nowadays, the promotion of TENGs has been hindered due to the limitation of their output performance and service life. Herein, a brand new triboelectric nanogenerator based on a multi-material stacking structure is proposed. By stacking various triboelectric materials in a specific order, a special charge balance state could be achieved inside the system such that the conductive layer generates more induced charges, and the output performance is significantly enhanced. Besides, due to the usage of the electropositive elastomer PU (polyurethane sponge), the design also effectively alleviates abrasion on the contact surface and adjusts its own output according to different compression environments. The experimental results show that the stacked PTFE/FKM/PU TENG (PFP-TENG) presents a more than 50% increase in transferred charge and almost 5 times the current output compared with the general contact-separation type TENG. When connected to the application circuit, the maximum output power reached 10.2 W m-2 and 145.2 W m-3, and more than 1400 LEDs could be easily lit. Finally, the PFP-TENG was also used to collect mechanical energy from simple motion and realize considerable power generation. This study not only provides new ideas for the design of TENGs by reasoning the theoretical model but also presents improved output performance, thus exemplifying the strong potential of this design in developing a power-generation device that can collect discrete mechanical energy.

TRAF6 protein couples Toll-like receptor 4 signaling to Src family kinase activation and opening of paracellular pathway in human lung microvascular endothelia.

Gram-negative bacteria release lipopolysaccharide (LPS) into the bloodstream. Here, it engages Toll-like receptor (TLR) 4 expressed in human lung microvascular endothelia (HMVEC-Ls) to open the paracellular pathway through Src family kinase (SFK) activation. The signaling molecules that couple TLR4 to the SFK-driven barrier disruption are unknown. In HMVEC-Ls, siRNA-induced silencing of TIRAP/Mal and overexpression of dominant-negative TIRAP/Mal each blocked LPS-induced SFK activation and increases in transendothelial [(14)C]albumin flux, implicating the MyD88-dependent pathway. LPS increased TRAF6 autoubiquitination and binding to IRAK1. Silencing of TRAF6, TRAF6-dominant-negative overexpression, or preincubation of HMVEC-Ls with a cell-permeable TRAF6 decoy peptide decreased both LPS-induced SFK activation and barrier disruption. LPS increased binding of both c-Src and Fyn to GST-TRAF6 but not to a GST-TRAF6 mutant in which the three prolines in the putative Src homology 3 domain-binding motif (amino acids 461-469) were substituted with alanines. A cell-permeable decoy peptide corresponding to the same proline-rich motif reduced SFK binding to WT GST-TRAF6 compared with the Pro → Ala-substituted peptide. Finally, LPS increased binding of activated Tyr(P)(416)-SFK to GST-TRAF6, and preincubation of HMVEC-Ls with SFK-selective tyrosine kinase inhibitors, PP2 and SU6656, diminished TRAF6 binding to c-Src and Fyn. During the TRAF6-SFK association, TRAF6 catalyzed Lys(63)-linked ubiquitination of c-Src and Fyn, whereas SFK activation increased tyrosine phosphorylation of TRAF6. The TRAF6 decoy peptide blocked both LPS-induced SFK ubiquitination and TRAF6 phosphorylation. Together, these data indicate that the proline-rich Src homology 3 domain-binding motif in TRAF6 interacts directly with activated SFKs to couple LPS engagement of TLR4 to SFK activation and loss of barrier integrity in HMVEC-Ls.

NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia: NEU1 restrains endothelial cell migration, whereas NEU3 does not.

The microvascular endothelial surface expresses multiple molecules whose sialylation state regulates multiple aspects of endothelial function. To better regulate these sialoproteins, we asked whether endothelial cells (ECs) might express one or more catalytically active sialidases. Human lung microvascular EC lysates contained heat-labile sialidase activity for a fluorogenic substrate, 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MU-NANA), that was dose-dependently inhibited by the competitive sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid but not its negative control. The EC lysates also contained sialidase activity for a ganglioside mixture. Using real time RT-PCR to detect mRNAs for the four known mammalian sialidases, NEU1, -2, -3, and -4, NEU1 mRNA was expressed at levels 2700-fold higher that those found for NEU2, -3, or -4. Western analyses indicated NEU1 and -3 protein expression. Using confocal microscopy and flow cytometry, NEU1 was immunolocalized to both the plasma membrane and the perinuclear region. NEU3 was detected both in the cytosol and nucleus. Prior siRNA-mediated knockdown of NEU1 and NEU3 each decreased EC sialidase activity for 4-MU-NANA by >65 and >17%, respectively, and for the ganglioside mixture by 0 and 40%, respectively. NEU1 overexpression in ECs reduced their migration into a wound by >40%, whereas NEU3 overexpression did not. Immunohistochemical studies of normal human tissues immunolocalized NEU1 and NEU3 proteins to both pulmonary and extrapulmonary vascular endothelia. These combined data indicate that human lung microvascular ECs as well as other endothelia express catalytically active NEU1 and NEU3. NEU1 restrains EC migration, whereas NEU3 does not.

NEU1 sialidase expressed in human airway epithelia regulates epidermal growth factor receptor (EGFR) and MUC1 protein signaling.

Epithelial cells (ECs) lining the airways provide a protective barrier between the external environment and the internal host milieu. These same airway epithelia express receptors that respond to danger signals and initiate repair programs. Because the sialylation state of a receptor can influence its function and is dictated in part by sialidase activity, we asked whether airway epithelia express catalytically active sialidase(s). Human primary small airway and A549 ECs expressed NEU1 sialidase at the mRNA and protein levels, and NEU1 accounted for >70% of EC sialidase activity. Blotting with Maackia amurensis and peanut agglutinin lectins established epidermal growth factor receptor (EGFR) and MUC1 as in vivo substrates for NEU1. NEU1 associated with EGFR and MUC1, and NEU1-EGFR association was regulated by EGF stimulation. NEU1 overexpression diminished EGF-stimulated EGFR Tyr-1068 autophosphorylation by up to 44% but enhanced MUC1-dependent Pseudomonas aeruginosa adhesion by 1.6-1.7-fold and flagellin-stimulated ERK1/2 activation by 1.7-1.9-fold. In contrast, NEU1 depletion increased EGFR activation (1.5-fold) and diminished MUC1-mediated bacterial adhesion (38-56%) and signaling (73%). These data indicate for the first time that human airway epithelia express catalytically active NEU1 sialidase that regulates EGFR- and MUC1-dependent signaling and bacterial adhesion. NEU1 catalytic activity may offer an additional level of regulation over the airway epithelial response to ligands, pathogens, and injurious stimuli.

[Decreasing effect of electro-acupuncture at Sanyin points on inflammatory cytokines in autoimmune prostatitis rats].

OBJECTIVE: To observe the changes of inflammatory cytokines in autoimmune prostatitis (AIP) rats treated by electro-acupuncture (EA) at Sanyin points. METHODS: We selected 40 Wistar male rats in this study, 10 as normal controls, and the other 30 made AIP models by intradermal injection of protein purification liquid from the prostate of allogeneic male rats with dual immune adjuvant. Then we randomly divided the AIP models into a model, a Cernilton control and an EA group of equal number, the latter two groups treated by Cernilton enema and EA, respectively. After 15 days of treatment, all the animals were sacrificed for detection of the levels of TNF-alpha, iNOS, MDA and T-AOC in the prostate tissue. RESULTS: Compared with the normal controls, the model rats showed significantly elevated TNF-alpha expression ([15.31 +/- 1.36] vs [32.20 +/- 1.65] pg/ml, P < 0.01), iNOS activity ([0.81 +/- 0.33] vs [1.25 +/- 0.23] U/ml, P < 0.01) and MDA content ([0.66 +/- 0.14] vs [0.91 +/- 0.21] nmol/ml, P < 0.05), but markedly reduced T-AOC activity ([1.56 +/- 0.16] vs [1.11 +/- 0.15] U/ml, P < 0.01). In comparison with the model group, the EA group exhibited significantly reduced levels of TNF-alpha ([17.32 +/- 2.69 ] pg/ml, P < 0.01), iNOS ([0.98 +/- 0.5 ] U/ml, P < 0.05) and MDA ([0.70 +/- 0.20] nmol/ml, P < 0.05), but remarkably increased level of T-AOC ([1.44 +/- 0.26] U/ml, P < 0.05). CONCLUSION: Electro-acupuncture at Sanyin points can protect the prostate tissue from morphological damage and reduce inflammatory reaction by decreasing pro-inflammatory cytokine activity, vascular permeability and inflammatory cell infiltration and increasing the activity of the antioxidant defense system.

Sex-specific differences in T-cell immune dysregulation and aberrant response to inflammatory stimuli in offspring exposed to maternal chronic inflammation.

PROBLEMS: Maternal chronic inflammation (MI) can adversely affect offspring's immune development resulting in dysregulation of splenic T cells. Interleukin 1 beta (IL-1ß) contributes to mediating inflammation in the placenta to induce fetal toxicity and cause long-term postnatal sequelae. In this study, we investigated how MI affects the T-cell immune development from the fetal to the neonatal period and how offspring responded to postnatal IL-1ß challenge when exposed to an adverse intrauterine environment. We also extend these studies to examine the sex-specific differences. METHODS OF STUDY: Time-pregnant CD1 dams were administrated with four consecutive injections of mouse recombinant Interleukin-1ß (rIL-1ß) or phosphate-buffered saline (PBS) from embryonic day (E)14 to E17. Pups were treated with rIL-1ß or PBS at postnatal day (PND)11 (pre-weaning) or PND24 (post-weaning). Pups' splenic immune cells were isolated and then characterized using flow cytometry. RESULTS: At PND12, no differences were observed either in Ctrl or MI offspring. At PND25, we observed elevated amount of CD8+ T cells, descending CD4+ /CD8+ and Treg/Teff ratio in MI offspring. Pre-weaning rIL-1ß administration did not affect T-cell subpopulation in Ctrl pups while post-weaning rIL-1ß administration increased T cells and CD8+ T cells and decreased CD4+ /CD8+ and Treg/Teff ratio in Ctrl offspring. Furthermore, pre-weaning rIL-1ß administration decreased the frequency of T cells and Treg/Teff ratio in MI pups while post-weaning rIL-1ß administration increased Tregs and Treg/Teff in MI pups. Regarding sex-specific changes, we observed that at PND12, MI females exhibited higher CD4+ /CD8+ and Treg/Teff ratio than Ctrl females. At PND25, we observed elevated amount of CD8+ T cells, descending CD4+ /CD8+ and Treg/Teff ratio in MI Females, while MI males did not show any changes in T-cell population. Pre-weaning rIL-1ß administration decreased T-cell frequency in both MI males and females and decreased Treg/Teff ratio only in MI females. Post-weaning rIL-1ß administration increased Tregs and Treg/Teff ratio, and decreased CD4+ /CD8+ ratio in MI females. CONCLUSIONS: Prenatal-inflammation-exposed offspring exhibited dysfunctional T-cell immunity and regulatory immune responses to postnatal challenges, showing both sex-specific and age-dependent differences. It could be speculated from our results that experiencing environmental challenges or adverse stimuli during the vulnerable intrauterine period, such as maternal chronic inflammation, stress, preterm birth, and chronic infections, might induce fetal immune reprogramming and potentially cause long-term adverse immune consequences, such as a predisposition to allergic diseases, autoimmune diseases, asthma and pediatric mortality of unknown etiology.