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.

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.

Simultaneous detection of glycinin and ß-conglycinin in processed soybean products by high-performance liquid chromatography-tandem mass spectrometry with stable isotope-labeled standard peptides.

Glycinin and ß-conglycinin are the two main allergic proteins in soybean. Due to their complex structures and lack of protein standards, it is difficult to achieve quantitative determination of these proteins in soybeans. In this study, an HPLC-MS/MS method was developed for the simultaneous determination of five subunits of glycinin (G1, G2, G3, G4, and G5) and three subunits of ß-conglycinin (α, α', and ß) in processed soybean products based on 8 specific peptides and their stable isotope-labeled peptides. Here, each specific peptide was derived from one of the above 8 subunits. When soy protein was extracted and digested with trypsin, 8 specific peptides, and corresponding stable isotope-labeled peptides were analyzed by HPLC-MS/MS. The linear range for the specific peptides was between 3.2 and 1000 ng/mL (R2 > 0.9955). The recoveries of added peptides ranged from 83.4% to 117.8%, and the intra-day precisions (% CV) were below 17.4%. The limit of quantification of each subunit of glycinin and ß-conglycinin in processed soybean products (in terms of protein amount) was between 15.1 and 156.1 g/g. This method was successfully applied to the analysis of 8 subunits of glycinin and ß-conglycinin in 68 different processed soybean products, which provides technical support for processed product quality evaluation and monitoring soybean processing technology.

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.

Adverse outcomes in SARS-CoV-2-infected pregnant mice are gestational age-dependent and resolve with antiviral treatment.

SARS-CoV-2 infection during pregnancy is associated with severe COVID-19 and adverse fetal outcomes, but the underlying mechanisms remain poorly understood. Moreover, clinical studies assessing therapeutics against SARS-CoV-2 in pregnancy are limited. To address these gaps, we developed a mouse model of SARS-CoV-2 infection during pregnancy. Outbred CD1 mice were infected at E6, E10, or E16 with a mouse-adapted SARS-CoV-2 (maSCV2) virus. Outcomes were gestational age-dependent, with greater morbidity, reduced antiviral immunity, greater viral titers, and impaired fetal growth and neurodevelopment occurring with infection at E16 (third trimester equivalent) than with infection at either E6 (first trimester equivalent) or E10 (second trimester equivalent). To assess the efficacy of ritonavir-boosted nirmatrelvir, which is recommended for individuals who are pregnant with COVID-19, we treated E16-infected dams with mouse-equivalent doses of nirmatrelvir and ritonavir. Treatment reduced pulmonary viral titers, decreased maternal morbidity, and prevented offspring growth restriction and neurodevelopmental impairments. Our results highlight that severe COVID-19 during pregnancy and fetal growth restriction is associated with heightened virus replication in maternal lungs. Ritonavir-boosted nirmatrelvir mitigated maternal morbidity along with fetal growth and neurodevelopment restriction after SARS-CoV-2 infection. These findings prompt the need for further consideration of pregnancy in preclinical and clinical studies of therapeutics against viral infections.

A Stacked FKM/PU Triboelectric Nanogenerator for Discrete Mechanical Energy Harvesting.

To combine the advantages of elastic and nonelastic triboelectric materials, this work proposes a new type of triboelectric nanogenerator (TENG) based on stacking -the stacked FKM/PU TENG. By stacking the elastomer polyurethane (PU) and the nonelastomer fluororubber (FKM), the FKM/PU TENG combines the inherent triboelectric characteristics of both materials and the unique elasticity of PU to achieve an output performance that is much higher than that of the FKM-TENG or the PU-TENG. The maximum instantaneous open-circuit voltage and short-circuit current of the FKM/PU TENG reach 661 V and 71.2 µA, respectively. Under the limiting conditions of 3 Hz and maximum compression, this device can attain a maximum power density of 49.63 W/m3 and light more than 500 LEDs. Therefore, stacking materials with different properties gives the FKM/PU TENG high output performance and great application potential, which can contribute to future development of discrete mechanical energy harvesting.

Adverse outcomes in SARS-CoV-2 infected pregnant mice are gestational age-dependent and resolve with antiviral treatment.

SARS-CoV-2 infection during pregnancy is associated with severe COVID-19 and adverse fetal outcomes, but the underlying mechanisms remain poorly understood. Moreover, clinical studies assessing therapeutics against SARS-CoV-2 in pregnancy are limited. To address these gaps, we developed a mouse model of SARS-CoV-2 infection during pregnancy. Outbred CD1 mice were infected at embryonic day (E) 6, E10, or E16 with a mouse adapted SARS-CoV-2 (maSCV2) virus. Outcomes were gestational age-dependent, with greater morbidity, reduced anti-viral immunity, greater viral titers, and more adverse fetal outcomes occurring with infection at E16 (3rd trimester-equivalent) than with infection at either E6 (1st trimester-equivalent) or E10 (2nd trimester-equivalent). To assess the efficacy of ritonavir-boosted nirmatrelvir (recommended for pregnant individuals with COVID-19), we treated E16-infected dams with mouse equivalent doses of nirmatrelvir and ritonavir. Treatment reduced pulmonary viral titers, decreased maternal morbidity, and prevented adverse offspring outcomes. Our results highlight that severe COVID-19 during pregnancy and adverse fetal outcomes are associated with heightened virus replication in maternal lungs. Ritonavir-boosted nirmatrelvir mitigated adverse maternal and fetal outcomes of SARS-CoV-2 infection. These findings prompt the need for further consideration of pregnancy in preclinical and clinical studies of therapeutics against viral infections.

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.

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.

Maternal siRNA silencing of placental SAA2 mitigates preterm birth following intrauterine inflammation.

The placental inflammatory processes induced maternally result in preterm birth (PTB). Serum amyloid A (SAA) is a well-known biomarker of inflammation. The objective of this study was to investigate whether murine placental SAA isoforms (SAA1-4) participate in the mechanism of spontaneous PTB and whether maternal regulation of SAA production may serve as a therapeutic approach. During the gestation, all isoforms of SAA were detectable except SAA2. The mouse model of intrauterine inflammation was established using LPS infusion to the uterus. Following intrauterine inflammation, placental SAA2 increased significantly. Inhibition of Saa2, using siSaa2, markedly decreased PTB. The increased placental expression of pro-inflammatory cytokines Il1ß, Il6, and Tnfα were downregulated by siSaa2 treatment. Maternal inhibition of Saa2 did not change the expression of Saa1-4 in the fetal brain. Explant inflammatory culture of placentas with siSaa2 showed similar results to our in vivo experiments. This study demonstrates the highly expressed placental SAA2 as a novel therapeutic target, and maternal administration of siRNA as a promising approach to alleviate PTB.

Altered sialidase expression in human myeloid cells undergoing apoptosis and differentiation.

To gain insight into sialic acid biology and sialidase/neuraminidase (NEU) expression in mature human neutrophil (PMN)s, we studied NEU activity and expression in PMNs and the HL60 promyelocytic leukemic cell line, and changes that might occur in PMNs undergoing apoptosis and HL60 cells during their differentiation into PMN-like cells. Mature human PMNs contained NEU activity and expressed NEU2, but not NEU1, the NEU1 chaperone, protective protein/cathepsin A(PPCA), NEU3, and NEU4 proteins. In proapoptotic PMNs, NEU2 protein expression increased > 30.0-fold. Granulocyte colony-stimulating factor protected against NEU2 protein upregulation, PMN surface desialylation and apoptosis. In response to 3 distinct differentiating agents, dimethylformamide, dimethylsulfoxide, and retinoic acid, total NEU activity in differentiated HL60 (dHL60) cells was dramatically reduced compared to that of nondifferentiated cells. With differentiation, NEU1 protein levels decreased > 85%, PPCA and NEU2 proteins increased > 12.0-fold, and 3.0-fold, respectively, NEU3 remained unchanged, and NEU4 increased 1.7-fold by day 3, and then returned to baseline. In dHL60 cells, lectin blotting revealed decreased α2,3-linked and increased α2,6-linked sialylation. dHL60 cells displayed increased adhesion to and migration across human bone marrow-derived endothelium and increased bacterial phagocytosis. Therefore, myeloid apoptosis and differentiation provoke changes in NEU catalytic activity and protein expression, surface sialylation, and functional responsiveness.

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.

Downregulation of transcriptional activity, increased inflammation, and damage in the placenta following in utero Zika virus infection is associated with adverse pregnancy outcomes.

Zika virus (ZIKV) infection during pregnancy causes serious adverse outcomes to the developing fetus, including fetal loss and birth defects known as congenital Zika syndrome (CZS). The mechanism by which ZIKV infection causes these adverse outcomes and specifically, the interplay between the maternal immune response and ZIKV replication has yet to be fully elucidated. Using an immunocompetent mouse model of transplacental ZIKV transmission and adverse pregnancy outcomes, we have previously shown that Asian lineage ZIKV disrupts placental morphology and induces elevated secretion of IL-1ß. In the current manuscript, we characterized placental damage and inflammation during in utero African lineage ZIKV infection. Within 48 hours after ZIKV infection at embryonic day 10, viral RNA was detected in placentas and fetuses from ZIKA infected dams, which corresponded with placental damage and reduced fetal viability as compared with mock infected dams. Dams infected with ZIKV had reduced proportions of trophoblasts and endothelial cells and disrupted placental morphology compared to mock infected dams. While placental IL-1ß was increased in the placenta, but not the spleen, within 3 hours post infection, this was not caused by activation of the NLRP3 inflammasome. Using bulk mRNAseq from placentas of ZIKV and mock infected dams, ZIKV infection caused profound downregulation of the transcriptional activity of genes that may underly tissue morphology, neurological development, metabolism, cell signaling and inflammation, illustrating that in utero ZIKV infections causes disruption of pathways associated with CZS in our model.

Dendrimer-Based N-Acetyl Cysteine Maternal Therapy Ameliorates Placental Inflammation via Maintenance of M1/M2 Macrophage Recruitment.

Intrauterine inflammation (IUI) is the primary cause of spontaneous preterm birth and predisposes neonates to long-term sequelae, including adverse neurological outcomes. N-acetyl-L-cysteine (NAC) is the amino acid L-cysteine derivative and a precursor to the antioxidant glutathione (GSH). NAC is commonly used clinically as an antioxidant with anti-inflammatory properties. Poor bioavailability and high protein binding of NAC necessitates the use of high doses resulting in side effects including nausea, vomiting, and gastric disruptions. Therefore, dendrimer-based therapy can specifically target the drug to the cells involved in inflammation, reducing side effects with efficacy at much lower doses than the free drug. Towards development of the new therapies for the treatment of maternal inflammation, we successfully administered dendrimer-based N-Acetyl Cysteine (DNAC) in an animal model of IUI to reduce preterm birth and perinatal inflammatory response. This study explored the associated immune mechanisms of DNAC treatment on placental macrophages following IUI, especially on M1/M2 type macrophage polarization. Our results demonstrated that intraperitoneal maternal DNAC administration significantly reduced the pro-inflammatory cytokine mRNA of Il1ß and Nos2, and decreased CD45+ leukocyte infiltration in the placenta following IUI. Furthermore, we found that DNAC altered placental immune profile by stimulating macrophages to change to the M2 phenotype while decreasing the M1 phenotype, thus suppressing the inflammatory responses in the placenta. Our study provides evidence for DNAC therapy to alleviate IUI via the maintenance of macrophage M1/M2 imbalance in the placenta.

Dual quantum dot nanobeads-based fluorescence-linked immunosorbent assay for simultaneous detection of aflatoxin B1 and zearalenone in feedstuffs.

A novel dual quantum dot nanobeads-based fluorescence-linked immunosorbent assay (QBs-FLISA) was successfully developed for simultaneously detecting aflatoxin B1 (AFB1) and zearalenone (ZEN) in feedstuffs. Dual CdSe/ZnS quantum dot nanobeads with different diameters that emit red and green fluorescence were conjugated with anti-AFB1 and anti-ZEN monoclonal antibodies to prepare fluorescent probes, which greatly enhance analytical performance. Under the optimal conditions, the limits of detection for AFB1 and ZEN were 9.3 and 102.1 pg mL-1, respectively. The recoveries ranged from 82.50% to 116.21% with relative standard deviation less than 11.3%. Compared with traditional enzyme-linked immunosorbent assay, detection sensitivities of AFB1 and ZEN using QBs-FLISA were increased 20 and 5 folds, respectively. In addition, results of feedstuff samples analyzed by QBs-FLISA and liquid chromatography tandem mass spectrometry showed a good agreement (R2 = 0.99).

Quantification of lectin in soybeans and soy products by liquid chromatography-tandem mass spectrometry.

Lectin is one of the major anti-nutritional factors in soybeans and inhibits digestion of dietary protein. Here, an absolute quantification method was developed to detect lectin using synthetic peptide 183TTSWDLANNK192 as reference standard and corresponding isotope labeled peptide TTSWDLANNK (Alanine-13C3,15N) as internal standard to normalize results. After the ground soybeans and soy products were defatted with n-hexane and extracted with extraction buffer, the crude protein extract was digested on filter membrane by trypsin. Further, the enzymatic hydrolysis peptides were quantified using liquid chromatography-tandem mass spectrometry. The synthetic reference peptide showed a detection limit of 0.27 ng/mL and a linear relationship in the range of 3.2-1000 ng/mL (r2 > 0.997). Correspondingly, the detect limit of lectin in soybean samples was 35.5 µg/g. The results showed that the recoveries of the lectin in spiked samples ranged from 80.9% to 108.7% with intra-day precisions (% CV) less than 9%. The method was successfully used to evaluate lectin levels in hundreds of soybean seeds from different varieties and soy products from different soybean processing techniques. Furthermore, the method may provide a potential application as a general method for the ultrasensitive detection of various protein anti-nutritional factors in food.

Type 1 Cytotoxic T Cells Increase in Placenta after Intrauterine Inflammation.

CD8+ T cells recognize non-self antigen by MHC class I molecules and kill the target cells by the release of proinflammatory cytokines such as interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). Our group previously reported an increase of CD8+ T-cell trafficking in the placenta with exposure to Lipopolysaccharides (LPS). CD8+ cytotoxic T cells have been classified into distinct subsets based upon cytokine production: Tc1 cells produce IFN-γ, Tc2 cells produce interleukin 4 (IL-4). Accordingly, the purpose of this research is to analyze the subsets of placenta CD8+ T cells. We hypothesized that LPS injection would induce a change of properties of CD8+ T cell and Tc1/Tc2 ratio. We investigated the subsets of CD8+ T cell infiltration to placenta and their specific function in response to LPS-induced inflammation in a mouse model. At embryonic (E) day 17, pregnant CD-1 dams received an intrauterine injection of 25 µg LPS in100 µl PBS or 100 µl of PBS only. Flow cytometry was used to quantify CD8+ T cells, evaluate the phenotype and subtypes, and detect markers of Tc1 and Tc2 cells in placenta, at 6 hours and 24 hours post injection (hpi). Intracellular staining and flow cytometry were performed to characterize cytokines produced by CD8+ T cells. Standard statistical analysis were employed. After 6 and 24 hours of LPS injection, total CD8 T cells increased (P<0.05). Tc1 cells expanded (P<0.05) in LPS-treated dams compared with the PBS group. The Tc1/Tc2 ratio was significantly higher in the LPS group than the PBS group (P<0.05). The expression of TNF-α and IFN-γ were increased in LPS group both at 6hpi and 24 hpi (P<0.05). We identified functional placental CD8+ T cell subtypes and found a significant increase ratio of Tc1/Tc2. Following IUI, CD8+ T cells induced inflammatory response in the placenta primarily via the production of Type 1 cytokines such as IFN-γ and TNF-α. We have provided evidence of a Tc1-bias response and cytokines in the mouse model of IUI.

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.

[Progress of Researches on Brain Mechanism Underlying Regulatory Effect of Acupuncture Intervention on Visual Plasticity of Amblyopia].

Acupuncture therapy has a positive effect in the treatment of amblyopia. This article summarizes findings of the research on brain mechanisms underlying the regulatory effect of acupuncture on visual plasticity of amblyopia. In a multi-system and multi-level viewpoints, we elaborated brain mechanism underlying the regulatory effect of acupuncture on visual plasticity in amblyopia from the perspective of ultrastructure, plasticity, electrical activities, neural coding and visual microcirculation of the neurons of the visual cortex, and the targeting points from the visual center to the effector organ.