Exploration of YBX1 role in the prognostic value and immune characteristics by single-cell and bulk sequencing analysis for liver hepatocellular carcinoma.

BACKGROUND: Y-box binding protein 1 (YBX1) plays a variety of roles in progression of multiple tumors. However, the role of YBX1 in prognostic value and immune regulation for liver hepatocellular carcinoma (LIHC) remains unclear. The present study aimed to examine the effect of YBX1 on the regulation of tumor immunity and survival prediction in LIHC patients. METHODS: YBX1-related expression profiles and single-cell and bulk sequencing analysis were performed using online databases. YBX1 expression was validated by a quantitative real-time PCR (qRT-PCR), western blotting and immunohistochemistry. Univariate/multivariate Cox regression analysis was performed to determine independent predictors of overall survival (OS). The ESTIMATE (i.e., Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data) algorithm and Tumor Immune Dysfunction and Exclusion (TIDE) analysis were used to assess the relationships between YBX1 and LIHC immunity. RESULTS: YBX1 was over-expressed in LIHC tissues and cell lines. High YBX1 expression was significantly associated with poor OS. Univariate/multivariate Cox regression analysis revealed that YBX1 was an independent prognostic factor for LIHC. Gene set enrichment analysis revealed that YBX1 was associated with multiple signaling pathways correlated to LIHC. Additionally, YBX1 was expressed in multiple immune cells and was significantly correlated with immune cells, immune checkpoint markers and tumor immune microenvironment. The TIDE analysis demonstrated that LIHC patients with high YBX1 expression showed a higher T-cell dysfunction score and a higher exclusion score, as well as poorer immunotherapy response. CONCLUSIONS: YBX1 plays crucial oncogenic roles in LIHC and is closely associated with the immune defense system. YBX1 inhibition may serve as a potential treatment for LIHC.

TM-search: An Efficient and Effective Tool for Protein Structure Database Search.

The quickly increasing size of the Protein Data Bank is challenging biologists to develop a more scalable protein structure alignment tool for fast structure database search. Although many protein structure search algorithms and programs have been designed and implemented for this purpose, most require a large amount of computational time. We propose a novel protein structure search approach, TM-search, which is based on the pairwise structure alignment program TM-align and a new iterative clustering algorithm. Benchmark tests demonstrate that TM-search is 27 times faster than a TM-align full database search while still being able to identify ∼90% of all high TM-score hits, which is 2-10 times more than other existing programs such as Foldseek, Dali, and PSI-BLAST.

Inhibition of adult hippocampal neurogenesis induced by postoperative CD8 + T-cell infiltration is associated with cognitive decline later following surgery in adult mice.

BACKGROUND: Some patients show persistent cognitive decline for weeks, months or even years after surgery, which seriously affects their long-term prognosis and quality of life. However, most previous basic studies have focused mainly on the mechanisms of early postoperative cognitive decline, whereas cognitive decline in the longer term after surgery is less well-understood. The subgranular zone of the dentate gyrus exhibits life-long neurogenesis, supporting hippocampus-dependent learning and memory. MAIN TEXT: The aim of this study was to investigate whether adult hippocampal neurogenesis (AHN) involves in cognitive decline later following surgery and to further explore the roles of CD8 + T lymphocytes infiltrating the hippocampal parenchyma after surgery in this pathological process. Cognitive function was examined in adult mice that underwent laparotomy combined with partial hepatectomy, and the results showed that cognitive decline persisted in mice who underwent surgery during the first postoperative month, even though there was a trend toward continuous improvement over time. Significantly decreased numbers of DCX + cells, BrdU + cells, and BrdU + /DCX + cells were observed on day 8 after surgery, and a significantly decreased number of NeuN + /BrdU + cells was observed on day 28 after surgery, which indicated inhibition of AHN. After surgery, T lymphocytes, the majority of which were CD8 + T cells, infiltrated the hippocampus and secreted Interferon-γ (IFN-γ). Depletion of CD8 + T cells could inhibit the increase of IFN-γ synthesis, improve hippocampal neurogenesis, and improve postoperative cognitive function. Hippocampal microinjection of IFN-γ neutralizing antibody or adeno-associated virus to knock down IFN-γ receptor 1 (IFNGR1) could also partially attenuate the inhibition of AHN and improve postoperative cognitive function. CONCLUSIONS: These results demonstrate that postoperative infiltration of CD8 + T cells into the hippocampus and subsequent secretion of IFN-γ contribute to the inhibition of AHN and cognitive decline later following surgery.

Exosomes derived from hepatitis B virus-infected hepatocytes promote liver fibrosis via miR-222/TFRC axis.

Exosomal miRNAs activates hepatic stellate cell (HSC) and promote fibrosis. miR-222 was found to be increased in hepatitis B virus (HBV)-infected hepatocytes, and ferroptosis was reported to ameliorate liver fibrosis (LF). Although miR-222 and ferroptosis have been implicated in LF, the association between miR-222 and ferroptosis and how they coordinate to regulate LF are still not explicit. This study investigates the roles of miR-222 and transferrin receptor (TFRC) in LF. Lipid reactive oxygen species (ROS) level was analyzed by flow cytometry. FerroOrange staining was used to measure intracellular iron level. Luciferase reporter assay was adopted to confirm the binding of miR-222 and TFRC. Real-time quantitative PCR and immunoblots were applied to analyze gene and protein expression. The results showed that supplementation of exosomes derived from HBV-infected LO2 cells remarkably enhanced LX-2 cell activation, evidenced by elevated hydroxyprolin (Hyp) secretion and α-SMA and COL1A2 expression. miR-222 was significantly increased in HBV-Exo. Overexpressing miR-222 upregulated cell viability, secretion of Hpy, and expression of α-SMA and COL1A2, which were all blocked by overexpression of TFRC. Further study showed that TFRC was a target of miR-222, and miR-222 promoted LX-2 cell activation through suppressing TFRC-induced ferroptosis in LX-2 cells. Exosomal miR-222 derived from HBV-infected hepatocytes promoted LF through inhibiting TFRC and TFRC-induced ferroptosis. This study emphasizes the significance of miR-222/TFRC axis in LF and suggests new insights in clinical decision making while treating LF. Exosomes derived from HBV-infected LO2 cells promote LX-2 cell activation and liver fibrosis in mouse Exosomal miR-222 derived from HBV-infected LO2 cells promotes LX-2 cell activation TFRC is a target of miR-222 and inhibits LX-2 cell activation induced by miR-222 miR-222 promotes LX-2 cell activation through inhibiting TFRC-induced ferroptosis.

Clonal relationship of tet(X4)-positive Escherichia coli ST761 isolates between animals and humans.

OBJECTIVES: To characterize the relationship of tet(X4)-positive isolates from different hosts and environments. METHODS: PCR and MALDI-TOF MS were used to identify the tet(X4)-positive isolates. The MICs of 13 antimicrobial agents were determined by broth microdilution. Illumina technology was used to sequence all of the isolates. One isolate was randomly selected from Escherichia coli ST761 clones for long-read sequencing to obtain plasmid sequences. Bioinformatics analysis was used to determine the phylogeny of 46 tet(X4)-positive E. coli ST761 strains. RESULTS: A total of 12 tet(X4)-positive isolates, 8 E. coli and 4 Aeromonas simiae, were obtained from six lairages of a slaughterhouse. These isolates exhibited resistance to at least three classes of antimicrobials, including tigecycline. The majority of them, seven E. coli and three A. simiae, represent separate clonal groups. Notably, the seven E. coli isolates belonged to ST761, a common ST carrying the tet(X4) gene that has been identified in 39 isolates from animals, meat, wastewater and humans from seven Chinese provinces. All 46 tet(X4)-positive E. coli ST761 strains from various sources have a close phylogenetic relationship (0-72 SNPs), with a high nucleotide sequence similarity of resistance genes and the tet(X4)-carrying IncX1-IncFIA(HI1)-IncFIB(K) hybrid plasmid, indicating a clonal relationship of tet(X4)-positive E. coli ST761 among animals, food, the environment and humans. CONCLUSIONS: The clonal relationship of tet(X4)-positive E. coli ST761 between humans and animals poses a previously underestimated threat to public health. To the best of our knowledge, this is the first description of tet(X4)-positive A. simiae.

miR-30c inhibits angiogenesis by targeting delta-like ligand 4 in liver sinusoidal endothelial cell to attenuate liver fibrosis.

Liver fibrosis is a common feature of liver dysfunction during chronic liver diseases and is frequently associated with angiogenesis, a dynamic process that forms new blood vessels from preexisting vasculature. MicroRNAs (miRNAs), which act as posttranscriptional regulators of gene expression, have been shown to regulate liver fibrosis; however, how miRNAs regulate angiogenesis and its mechanism in fibrosis are not well understood. We aimed to elucidate the role and mechanism of miR-30c in attenuating liver fibrosis. Using miRNA profiling of fibrotic murine livers, we identified differentially regulated miRNAs and discovered that miR-30c is aberrantly expressed and targets endothelial delta-like ligand 4 (DLL4) in either carbon tetrachloride-treated or bile duct ligated fibrotic mice, as well as in patients with liver fibrosis. Using CCK-8, wound healing and Matrigel tube formation assays, we found that miR-30c inhibited liver sinusoidal endothelial cell (LSEC) proliferation, migration, and angiogenesis capacity by targeting DLL4 in vitro. Importantly, nanoparticle-based delivery of miR-30c to LSECs inhibited the DLL4/Notch pathway and angiogenesis, thereby ameliorating liver fibrosis in vivo. Collectively, our findings demonstrate a protective role of miR-30c in liver fibrosis by regulating DLL4/Notch signaling and angiogenesis. Thus, miR-30c may serve as a potential treatment for chronic liver diseases.

Study on the technology of efficient extraction of eleutheroside E from Acanthopanax senticosus by green solvent DES.

OBJECTIVES: Acanthopanax senticosus (Rupr. & Maxim.) Harms is a medicinal and edible plant which is clinically used for the recovery and treatment of cardiovascular and central diseases. As a characteristic active pharmaceutical ingredient of Acanthopanax senticosus, eleutheroside E is the core of the therapeutic effect. Organic solvent extraction has low selectivity, low extraction rate, difficulty in separation and purification and safety risks. The purpose of this study was to extract the effective component of Acanthopanax senticosus with a new green solvent. METHODS: In this article, two kinds of deep eutectic solvents (DESs) (DES-1 and DES-2) were synthesised by heating and stirring methods. Eleutheroside E was extracted by ultrasonic extraction with two kinds of DES as extractants and quantitatively analysed by Orbitrap-tandem mass spectrometry (MS/MS). RESULTS: The main results showed that the initial polarity of the DES was similar to that of 60 to 80% ethanol and hydrogen bond donors were the main factors affecting the polarity of DES. In the test, the viscosity of DES was higher than that of ethanol, and even the addition of a small amount of water (10%) caused intermolecular hydrogen bond disruption and redistribution of the solvent, resulting in a significant decrease in solvent viscosity. The solvents in the test group were stable after standing at 5°C in the dark for 100 days. The extraction rate of eleutheroside E by DES solvent was 5-6 times higher than that by ethanol. DES-1 and DES-2 can efficiently extract eleutheroside E. CONCLUSION: This study developed a new method for the application of the green extraction of eleutheroside E with certain practical significance.

The Diagnosis Value of a Novel Model with 5 Circulating miRNAs for Liver Fibrosis in Patients with Chronic Hepatitis B.

METHODS: Differential expression of five selected miRNAs (hsa-mir-1225-3p, hsa-mir-1238, hsa-miR-3162-3P, hsa-miR-4721, and hsa-miR-H7) was verified by qRT-PCR in the plasma of 83 patients and 20 healthy controls. The relative expression of these miRNAs was analyzed in different groups to screen target miRNA. A logistic regression analysis was performed to assess factors associated with fibrosis progression. The receiver operating characteristic (ROC) curve and discriminant analyses validated the ability of these predicted variables to discriminate the nonsignificant liver fibrosis group from the significant liver fibrosis group. Furthermore, the established models were compared with other prediction models to evaluate the diagnostic efficiency. RESULTS: These five tested miRNAs all had signature correlations with hepatic fibrotic level (p < 0.05), and the upregulation trends were consistent with miRNA microarray analysis previously. The multivariate logistic regression analysis identified that a model of five miRNAs (miR-5) had a high diagnostic accuracy in discrimination of different stages of liver fibrosis. The ROC showed that the miR-5 has excellent value in diagnosis of fibrosis, even better than the Forns score, FIB-4, S index, and APRI. GO functions of different miRNAs mainly involved in various biological processes were markedly involved in HBV and revealed signaling pathways dysregulated in liver fibrosis of CHB patients. CONCLUSIONS: It was validated that the combination of these five miRNAs was a new set of promising molecular diagnostic markers for liver fibrosis. The diagnosis model (miR-5) can distinguish significant and nonsignificant liver fibrosis with high sensitivity and specificity.

CXCL6-EGFR-induced Kupffer cells secrete TGF-ß1 promoting hepatic stellate cell activation via the SMAD2/BRD4/C-MYC/EZH2 pathway in liver fibrosis.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins in response to the inflammatory response that accompanies tissue injury, which at an advanced stage can lead to cirrhosis and even liver failure. This study investigated the role of the CXC chemokine CXCL6 (GCP-2) in liver fibrosis. The expression of CXCL6 was found to be elevated in the serum and liver tissue of high stage liver fibrosis patients. Furthermore, treatment with CXCL6 (100 ng/mL) stimulated the phosphorylation of EGFR and the expression of TGF-ß in cultured Kupffer cells (KCs). Although treatment with CXCL6 directly did not activate the hepatic stellate cell (HSC) line, HSC-T6, HSCs cultured with media taken from KCs treated with CXCL6 or TGF-ß showed increased expression of α-SMA, a marker of HSC activation. CXCL6 was shown to function via the SMAD2/BRD4/C-MYC/EZH2 pathway by enhancing the SMAD3-BRD4 interaction and promoting direct binding of BRD4 to the C-MYC promoter and CMY-C to the EZH2 promoter, thereby inducing profibrogenic gene expression in HSCs, leading to activation and transdifferentiation into fibrogenic myofibroblasts. These findings were confirmed in a mouse model of CCl4 -induced chronic liver injury and fibrosis in which the levels of CXCL6 and TGF-ß in serum and the expression of α-SMA, SMAD3, BRD4, C-MYC, and EZH2 in liver tissue were increased. Taken together, our results reveal that CXCL6 plays an important role in liver fibrosis through stimulating the release of TGF-ß by KCs and thereby activating HSCs.

Exosomes derived from miR-181-5p-modified adipose-derived mesenchymal stem cells prevent liver fibrosis via autophagy activation.

Proliferating hepatic stellate cells (HSCs) respond to liver damage by secreting collagens that form fibrous scar tissue, which can lead to cirrhosis if in appropriately regulated. Advancement of microRNA (miRNA) hepatic therapies has been hampered by difficulties in delivering miRNA to damaged tissue. However, exosomes secreted by adipose-derived mesenchymal stem cells (ADSCs) can be exploited to deliver miRNAs to HSCs. ADSCs were engineered to overexpress miRNA-181-5p (miR-181-5p-ADSCs) to selectively home exosomes to mouse hepatic stellate (HST-T6) cells or a CCl4-induced liver fibrosis murine model and compared with non-targeting control Caenorhabditis elegans miR-67 (cel-miR-67)-ADSCs. In vitro analysis confirmed that the transfer of miR-181-5p from miR-181-5p-ADSCs occurred via secreted exosomal uptake. Exosomes were visualized in HST-T6 cells using cyc3-labelled pre-miRNA-transfected ADSCs with/without the exosomal inhibitor, GW4869. The effects of miRNA-181-5p overexpression on the fibrosis associated STAT3/Bcl-2/Beclin 1 pathway and components of the extracellular matrix were assessed. Exosomes from miR181-5p-ADSCs down-regulated Stat3 and Bcl-2 and activated autophagy in the HST-T6 cells. Furthermore, the up-regulated expression of fibrotic genes in HST-T6 cells induced by TGF-ß1 was repressed following the addition of isolated miR181-5p-ADSC exosomes compared with miR-67-ADSCexosomes. Exosome therapy attenuated liver injury and significantly down-regulated collagen I, vimentin, α-SMA and fibronectin in liver, compared with controls. Taken together, the effective anti-fibrotic function of engineered ADSCs is able to selectively transfer miR-181-5p to damaged liver cells and will pave the way for the use of exosome-ADSCs for therapeutic delivery of miRNA targeting liver disease.

High Incidence of Escherichia coli Strains Coharboring mcr-1 and blaNDM from Chickens.

This study investigated the characteristics of Escherichia coli isolates carrying mcr-1-blaNDM from a chicken farm in China. Of the 78 E. coli isolates, 21 clonally unrelated isolates carried mcr-1-blaNDM Diverse IncI2 plasmids disseminated mcr-1, while the dissemination of blaNDM was mediated by diverse IncB/O plasmids. More striking was the colocalization of resistance genes mcr-1 and blaNDM-4 in an IncHI2/ST3 plasmid, which might pose a great challenge for public health.

Chromosome-Mediated mcr-3 Variants in Aeromonas veronii from Chicken Meat.

Two adjacent colistin resistance gene variants, termed mcr-3.3 and mcr-3-like, were identified in the chromosome of an Aeromonas veronii isolate obtained from retail chicken meat. The variants showed 95.20% and 84.19% nucleotide sequence identity, respectively, to mcr-3 from porcine Escherichia coli Functional cloning indicated that only mcr-3.3 conferred polymyxin resistance in both E. coli and Aeromonas salmonicida The mcr-3.3-mcr-3-like segment was also observed in other Aeromonas species, including A. media, A. caviae, and A. hydrophila.

CXCL6 promotes human hepatocyte proliferation through the CXCR1-NFκB pathway and inhibits collagen I secretion by hepatic stellate cells.

Hepatocyte proliferation and collagen I (COLI) secretion are important processes during liver regeneration. This study aimed to investigate the role of CXCL6 in hepatocyte proliferation and COLI secretion. Serum CXCL6 levels in patients with chronic hepatitis B (CHB) were examined and the effects of CXCL6 on the proliferation of L02 hepatocytes and the secretion of COLI from LX2 human hepatic stellate cells were evaluated. We found that serum CXCL6 levels increased gradually with disease progression of CHB, and there was positive correlation between serum CXCL6 level and alanine transaminase (ALT) and aspartate transaminase (AST). In vitro, CXCL6 promoted L02 proliferation but this was blocked upon CXCR1 knockdown. The level of phospho-IκBα was upregulated by CXCL6 but downregulated by CXCR1 siRNA in L02 cells. CXCL6 inhibited the secretion of COLI by LX2 cells, dependent on CXCR1 and CXCR2. Taken together, these data suggest that increased expression of CXCL6 during CHB could promote hepatocyte proliferation through the CXCR1-NFκB pathway and inhibit the secretion of COLI by hepatic stellate cells.

Isolation and Culture of Single Cell Types from Rat Liver.

There have been few reports on the simultaneous isolation of multiple liver cell populations thus far. As such, this study was aimed at establishing a protocol for the simultaneous separation of hepatocytes (HCs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) from the rat liver and assessing the in vitro culture of these cells. Single-cell suspensions from the liver were obtained by ethylene glycol tetraacetic acid/collagenase perfusion. After low-speed centrifugal separation of HCs, pronase was added to the nonparenchymal cell fraction to eliminate the remaining HCs. Subsequently, HSCs, LSECs and KCs were purified by two steps of density gradient centrifugation using Nycodenz and Percoll in addition to selective attachment. Pronase treatment increased the HSC yield (1.5 ± 0.2 vs. 0.7 ± 0.3 cells/g liver, p < 0.05) and improved LSEC purity (93.6 ± 3.6 vs. 82.5 ± 5.6%, p < 0.01). The isolated cells could also be cultured in vitro. LSEC apoptosis began on day 3 and reached a maximum on day 7. A few surviving LSECs began proliferating and split to form a cobblestone, sheet-like appearance on day 14. The LSECs on day 14 lost fenestrations but retained scavenger function. Thus, viable and purified liver cells were obtained with a high yield from the rat liver using the developed method, which may be useful for studying the physiology and pathology of the liver in the future.

[Predication analysis of microarray data to determine altered gene profiles in liver carcinoma related to HBV-related cirrhosis].

OBJECTIVE: To investigate whether gene expression profiles can be used to determine risk genes and predict HBV-related cirrhosis progression to liver carcinoma using Significance Analysis of Microarray (SAM) and Prediction Analysis of Microarray (PAM) methods. METHODS: The Affymetrix GeneChip was used to establish the gene expression profiles of liver tissues from 15 patients with chronic hepatitis B and cirrhosis or hepatocellular carcinoma (HCC). Differentially expressed genes (fold-change more than 2; P value less than 0.01) were selected by GeneSpring GX software. Risk genes related to cirrhosis and liver carcinoma were generated by SAM and PAM methods. Real-time PCR was used to verify the expression of risk genes in the liver tissues. RESULTS: Samples were clustered into the cirrhosis subgroup (n =15) or the HCC subgroup (n =15). A total of 497 differentially expressed genes were identified, SAM identified 162 significant genes, including 18 up-regulated genes and 144 down-regulated genes (fold-change:-1.46 to 1.28). PAM identified 22 genes with a "poor risk signature" (defined with a threshold of 5.5), which were associated with classifying cirrhosis and liver carcinoma; of these risk genes, 4 were down-regulated and 18 were up-regulated in the HCC group compared to the cirrhosis group (fold-change: 2.038 to 7.897, P value less than 0.01). The correction of classification was more than 80% . FOXP1, SPINK1 and KCNJ16 were verified by real-time PCR as differently expressed in the two subgroups (P value =0.011, 0.002 and 0.004, respectively). CONCLUSION: The altered gene profiles of carcinogenesis in HBV-related cirrhosis involves hundreds of genes. The combination of three "poor risk genes" may represent potential targets for diagnosis and prediction of liver carcinoma progression.

[Molecular allergen IgE tests show neosensitizaions occurring during house dust mite specific immunotherapy].

Objective:Neosensitizations may be occur during the allergen specific immunotherapy（AIT） due to the differences between allergen vaccine's content and a patient's molecular sensitization profile. This study investigates whether AIT with HDM extract changes the sensitization profile, whether de novo sensitization occurs, and the clinical importance of the neosensitization. Methods:Fifty-three patients with HDM allergic rhinitis ,with/without asthma, patients were received one year HDM subcutaneous AIT . Fourteen patients were recruited as control group and received only necessary medications. Serum samples were collected at baseline, 6thmoths and 12thof AIT, respectively. Serum samples were tested specific IgE against Der p, Der p 1/2/3 and Der f, Der f 1/2/3, as well as IgG4 against Der p, Der p 1/2 and Der f, Der f 1/2. VAS were collected at the time-points as well. Results:In AIT group, Der p, Der p 1/3, and Der f 1/3 specific IgE levels were significantly higher after one-year treatment, especially for Der p 3. There were 69.2%（18/26） patients whose Der p 3 specific IgE below 0.35 kU/L at baseline but became positive（>0.35 kU/L） after treatment, that is, neosensitization occurred. All tested allergen specific IgG4 level significantly increased after one year AIT treatment and the VAS declined dramatically. However, for patients with neosensitization and without neosensitization, there were no significantly changes concerning to IgG4 level and VAS. Conclusion:Patients undergoing AIT might have a risk of neosensitization to the allergen components in the vaccines. However, the clinical importance of the neosensitization remains unclear and warrants further studies.

4-Oxo-2-Nonenal- and Agitation-Induced Aggregates of α-Synuclein and Phosphorylated α-Synuclein with Distinct Biophysical Properties and Biomedical Applications.

α-Synuclein (α-syn) can form oligomers, protofibrils, and fibrils, which are associated with the pathogenesis of Parkinson's disease and other synucleinopathies. Both the lipid peroxidation product 4-oxo-2-nonenal (ONE) and agitation can induce aggregation of α-syn and phosphorylated α-syn. Thus, clarification of the characteristics of different α-syn species could help to select suitable aggregates for diagnosis and elucidate the pathogenesis of diseases. Here, we characterized ONE-induced wild-type (WT) α-syn aggregates (OW), ONE-induced phosphorylated α-syn (p-α-syn) aggregates (OP), agitation-induced α-syn preformed fibrils (PFF), and agitation-induced p-α-syn preformed fibrils (pPFF). Thioflavin T (ThT) dying demonstrated that OW and OP had fewer fibrils than the PFF and pPFF. Transmission electron microscopy revealed that the lengths of PFF and pPFF were similar, but the diameters differed. OW and OP had more compact structures than PFF and pPFF. Aggregation of p-α-syn was significantly faster than WT α-syn. Furthermore, OW and OP were more sodium dodecyl sulfate-stable and proteinase K-resistant, suggesting greater stability and compactness, while aggregates of PFF and pPFF were more sensitive to proteinase K treatment. Both ONE- and agitation-induced aggregates were cytotoxic when added exogenously to SH-SY5Y cells with increasing incubation times, but the agitation-induced aggregates caused cell toxicity in a shorter time and more p-α-syn inclusions. Similarly, p-proteins were more cytotoxic than non-p-proteins. Finally, all four aggregates were used as standard antigens to establish sandwich enzyme-linked immunosorbent assay (ELISA). The results showed that the recognition efficiency of OW and OP was more sensitive than that of PFF and pPFF. The OW- and OP-specific ELISA for detection of p-α-syn and α-syn in plasma samples of Thy1-α-syn transgenic mice showed that the content of aggregates could reflect the extent of disease. ONE and agitation induced the formation of α-syn aggregates with distinct biophysical properties and biomedical applications.

[The auxiliary diagnostic value of ECP and MPO expression in nasal secretions in different types of rhinitis].

Objective:To evaluate the expression of eosinophil cationic protein and myeloperoxidase in nasal secretions in different types of rhinitis, and to explore their values in the differential diagnosis of different types of rhinitis. Methods:Six hundred and eighty-four subjects were selected, including 62 subjects in the acute rhinitis group, 378 subjects in the allergic rhinitis group, 94 subjects in the vasomotor rhinitis group, 70 subjects in the eosinophilic non-allergic rhinitis group, and 80 subjects in the control group. Nasal secretion samples were collected from the five groups, and the percentages of inflammatory cells were counted by Rachel's staining, and the expression of ECP/MPO was detected by colloidal gold assay. The correlation between the clinical diagnosis, the inflammatory cells in the nasal secretions and the expression of ECP/MPO was analyzed. Results:Nasal cytological smears showed that compared with the control group, the percentage of eosinophils in the AR and NARES groups were significantly higher （P<0.05）, while the percentage of neutrophils was not different （P>0.05）; the percentage of neutrophils was significantly higher in the acute rhinitis group compared with the control group （P<0.05）, while the percentage of eosinophils was not statistically different （P>0.05）; in vasomotor rhinitis group, the eosinophils and neutrophils were not statistically different compared with the control group（P> 0.05）. The colloidal gold results showed that there were differences in the expression of ECP/MPO in different types of rhinitis, among which 49 cases （79.0%） in the acute rhinitis group expressed ECP+/MPO+; 267 cases （70.6%） in the AR group and 56 cases （75.7%） in the NARES group expressed ECP+/MPO-; 80 cases （85.1%） in the vasomotor rhinitis group and 69 cases （86.3%） in the control group expressed ECP-/MPO-. Conclusion:The differences in ECP and MPO expression between different types of rhinitis have certain reference value for the differential diagnosis of different types of rhinitis and the selection of treatment programs.

In vivo investigation of ceftiofur-loaded gelatin and PLGA microspheres in beagle dogs.

Drug delivery systems based on polymer microspheres have received considerable attention. Ceftiofur sodium and ceftiofur hydrochloride is widely used for the treatment of bacterial diseases in animals but the delivery in vivo has not been reported. In this paper, we report the synthesis of microspheres from gelatin and PLGA, two kinds of typical natural and artificial materials, for loading ceftiofur and the in vivo investigation of the pharmacokinetics in beagle dogs. By controlling the synthesis parameters, gelatin and PLGA microspheres with diameter between 5 and 35 microns were obtained. Assay procedures based on high performance liquid chromatography were evaluated and confirmed. The dogs were randomly divided into three groups, i.e., control group, gelatin group, and PLGA group and administrated via intravenous injection. Plasma concentrations of ceftiofur over time were measured and analyzed. Results indicate that the main kinetic parameters do not show significant difference for the gelatin group and control group, but the area under the curve, plasma half-life, apparent volume of distribution, and clearance ratio of PLGA group show significant difference from the gelatin group and the control group. The PLGA microspheres show a low area under the curve but long time release.