Single-cell sequencing provides insights into the landscape of ovary in PCOS and alterations induced by CUMS.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder related to psychological distress. However, the mechanism underlying increased prevalence of depression in PCOS remained unclear. This study aimed to explore the unique transcriptional landscape of ovary and offered a platform to explore the mechanism of PCOS, as well as the influences caused by depression. The PCOS rat model was established by letrozole whereas PCOS rat model with depression was established by letrozole combined with chronic unpredicted mild stress (CUMS). Then single-cell RNA sequencing (scRNA-Seq) was applied to analyze the transcriptional features of rat ovaries. Granulosa cells (GCs) and fibroblasts (Fibros) accounted for the top two clusters of total 12 cell types. There were nine clusters in GCs, related to inflammatory response, endoplasmic reticulum (ER) stress, and steroidogenesis. The expression of differentially expressed genes (DEG) Hes1 was higher in PCOS and PCOS + CUMS groups, exhibiting enhanced expression by pseudotime and positively related to inflammation. Pseudotemporal analysis revealed that inflammation contributed to the different GCs distributions. Moreover, analysis of DEGs and gene ontology (GO) function enrichment revealed CUMS aggravated inflammation in PCOS GCs possibly via interferon signaling pathway. In theca cells (TCs), nine clusters were observed and some of them were relevant to inflammation, ER stress, and lipid metabolism. DEGs Ass1, Insl3, and Ifi27 were positively related to Cyp17a1, and Ces1d might contribute to the different trajectory of TCs. Subsequent scRNA-seq revealed a signature profile of endothelial cells (ECs) and Fibros, which suggest that inflammation-induced damage of ECs and Fibro, further exacerbated by CUMS. Finally, analysis of T cells and mononuclear phagocytes (MPs) revealed the existence of immune dysfunction, among which interferon signaling played a critical role. These findings provided more knowledge for a better understanding PCOS from the view of inflammation and identified new biomarkers and targets for the treatment of PCOS with psychological diseases.NEW & NOTEWORTHY In this study, we mapped the landscape of polycystic ovary syndrome (PCOS) ovary with rat model induced by letrozole and provided a novel insight into the molecular mechanism of PCOS accompanied by chronic unpredicted mild stress (CUMS) at single-cell transcriptomic level. These observations highlight the importance of inflammation in the pathogenesis of PCOS, which might also be the bridge between PCOS and psychological diseases.

Thermal fogging with disinfectants and antifreezes enables effective industrial disinfection in subzero cold-chain environment.

AIM: During several local COVID-19 outbreaks in China in 2020, SARS-CoV-2 or its RNA was isolated or detected from frozen food or packages, revealing the lack of effective disinfection measures in the frozen food chain and risk of transmission. We explored the possibility that disinfectant plus antifreeze could be delivered as thermal fog to realize effective disinfection at subzero temperatures. METHODS AND RESULTS: We selected two disinfectant-antifreeze combinations, didecyl dimethyl ammonium bromide (DDAB) - propylene glycol (PPG) and peracetic acid (PAA) - triethylene glycol (TEG), and each combination is used with a custom-optimized thermal fogging machine. The two fogs were tested in -20°C freezer warehouses for their disinfection efficacy against a coronavirus porcine epidemic diarrhoea virus (PEDV) field strain, a swine influenza virus (SIV) field strain, and three indicator bacteria, Escherichia coli, Staphylococcus aureus and Bacillus subtilis endospores. At -20°C, the DDAB-PPG or PAA-TEG thermal fogs settle within 3.5 to 4.5 h and effectively inactivated PEDV with median tissue culture infective dose of 10-3.5 0.1 ml-1 and SIV-H1N1 with hemagglutination titre of 26  ml-1 within 15-60 min. DDAB-PPG could inactivate S. aureus and E. coli vegetative cells (106  cfu ml-1 ) within 15-60 min but not effective on B. subtilis spores, while PAA-TEG could disinfect B. subtilis spores more effectively than for S. aureus and E. coli. CONCLUSIONS: We showed that a practical subzero temperature disinfection technology was effective in killing enveloped viruses and vegetative bacteria or bacterial spores. DDAB-PPG or PAA-TEG thermal fogging may be a practical technology for cold-chain disinfection. SIGNIFICANCE AND IMPACT OF THE STUDY: This subzero temperature disinfection technology could help to meet the urgent public health need of environmental disinfection in frozen food logistics against pandemic and other potential pathogens and to enhance national and international biosecurity.

Molecular mechanism of the increased tissue uptake of trivalent inorganic arsenic in mice with type 1 diabetes mellitus.

Arsenic is associated with several adverse health outcomes, and people with diabetes may be more susceptible to arsenic. In this study, we found that arsenic levels in some tissues such as liver, kidney, and heart but not lung of type 1 diabetes mellitus (T1DM) mice were higher than in those of normal mice after a single oral dose of arsenic trioxide for 2 h. However, little is known about the molecular mechanism of the increased tissue uptake of trivalent inorganic arsenic in mice with T1DM. This study aimed to investigate the expression of the mammalian arsenic transporters aquaglyceroporins (AQPs) and glucose transporter 1 (GLUT1) in T1DM mice and compare them with those in normal mice. Results showed that the levels of AQP9 and GLUT1 mRNA and protein were higher in T1DM mouse liver than in the normal one. The levels of AQP7 mRNA and protein were higher in T1DM mouse kidney. In the heart, we observed that the levels of AQP7 and GLUT1 mRNA and protein were higher in T1DM mice, but the levels of AQP9 mRNA and protein in the lung had no significant difference between both mice. These results suggested that T1DM may increase the expression of transporters of trivalent inorganic arsenic and thus increase the arsenic uptake in specific tissues.

Arsenic trioxide mediates HAPI microglia inflammatory response and subsequent neuron apoptosis through p38/JNK MAPK/STAT3 pathway.

Arsenic is a widely distributed toxic metalloid all over the world. Inorganic arsenic species are supposed to affect astrocytic functions and to cause neuron apoptosis in CNS. Microglias are the key cell type involved in innate immune responses in CNS, and microglia activation has been linked to inflammation and neurotoxicity. In this study, using ELISA, we showed that Arsenic trioxide up-regulated the expression and secretion of IL-1ß in a dose-dependent manner and a time-dependent manner in cultured HAPI microglia cells. The secretion of IL-1ß caused the apoptosis of SH-SY5Y. These pro-inflammatory responses were inhibited by the STAT3 blocker, AG490 and P38/JNK MAPK blockers SB202190, SP600125. Further, Arsenic trioxide exposure could induce phosphorylation and activation of STAT3, and the translocation of STAT3 from the cytosol to the nucleus in this HAPI microglia cell line. Thus, the STAT3 signaling pathway can be activated after Arsenic trioxide treatment. However, P38/JNK MAPK blockers SB202190, SP600125 also obviously attenuated STAT3 activation and transnuclear transport induced by Arsenic trioxide. In concert with these results, we highlighted that the secretion of IL-1ß and STAT3 activation induced by Arsenic trioxide can be mediated by elevation of P38/JNK MAPK in HAPI microglia cells and then induced the toxicity of neurons.

ß-Actin protein expression differs in the submandibular glands of male and female mice.

ß-actin, a cytoskeletal protein, is the most widely used housekeeping gene. Although housekeeping genes are expressed in all tissues, the ß-actin gene is expressed in certain cell types because of differential binding of transcriptional factors to the regulatory elements of the gene. The expression and localization of ß-actin protein in the submandibular glands (SMG) of mice were investigated in this study, using Western blot analysis and immunohistochemistry. In ICR and C57BL/6J mice, the levels of ß-actin protein in the SMG of females are significantly higher than those in the SMG of males. ß-actin protein is majorly distributed in acinar cells of SMG. There is no significant difference in the expression level of ß-actin protein between females and castrated males. After castrated male ICR mice are treated with 10 mg/kg/day testosterone propionate (TP) for 3 weeks, the levels of ß-actin protein in SMG decrease. The numbers of duct per unit area increase, whereas the numbers of acinus per unit area decrease after TP administration. These data suggest that ß-actin protein is mainly distributed in acinar cells of SMG and results in a marked sexual dimorphism in mice.

Arsenic trioxide mediates HAPI microglia inflammatory response and the secretion of inflammatory cytokine IL-6 via Akt/NF-κB signaling pathway.

Arsenic is a widely distributed toxic metalloid in around the world. Inorganic arsenic species are deemed to affect astrocytes functions and to cause neuron apoptosis. Microglia are the key cell type involved in innate immune responses in CNS, and microglia activation has been linked to inflammation and neurotoxicity. In this study, using ELISA and reverse transcriptase PCR (RT-PCR), we showed that Arsenic trioxide up-regulated the expression and secretion of IL-6 in a dose-dependent manner and a time-dependent manner in cultured HAPI microglia cells. These pro-inflammatory responses were inhibited by the Akt blocker, LY294002. Further, Arsenic trioxide exposure could induce phospho rylationand degradation of IкBα, and the translocation of NF-κB p65 from the cytosol to the nucleus in this HAPI microglia cell line. Thus, the NF-кB signaling pathway can be activated after Arsenic trioxide treatment. Besides, Akt blocker LY294002 also obviously attenuated NF-кB activation and transnuclear induced by Arsenic trioxide. In concert with these results, we highlighted that the secretion of pro-inflammatory cytokine and NF-кB activation induced by Arsenic trioxide can be mediated by elevation of p-Akt in HAPI microglia cells.

Targeting the cGAS-STING Pathway Inhibits Peripheral T-cell Lymphoma Progression and Enhances the Chemotherapeutic Efficacy.

Peripheral T-cell lymphoma (PTCL) is a highly heterogeneous group of mature T-cell malignancies. The efficacy of current first-line treatment is dismal, and novel agents are urgently needed to improve patient outcomes. A close association between the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway and tumor promotion exists, revealing prospective therapeutic targets. This study, investigates the role of the cGAS-STING pathway and its underlying mechanisms in PTCL progression. Single-cell RNA sequencing showes that the cGAS-STING pathway is highly expressed and closely associated with PTCL proliferation. cGAS inhibition suppresses tumor growth and impaires DNA damage repair. Moreover, Cdc2-like kinase 1 (CLK1) is critical for residual tumor cell survival after treatment with cGAS inhibitors, and CLK1 suppression enhances sensitivity to cGAS inhibitors. Single-cell dynamic transcriptomic analysis indicates reduced proliferation-associated nascent RNAs as the underlying mechanism. In first-line therapy, chemotherapy-triggered DNA damage activates the cGAS-STING pathway, and cGAS inhibitors can synergize with chemotherapeutic agents to kill tumors. The cGAS-STING pathway is oncogenic in PTCL, whereas targeting cGAS suppresses tumor growth, and CLK1 may be a sensitivity indicator for cGAS inhibitors. These findings provide a theoretical foundation for optimizing therapeutic strategies for PTCL, especially in patients with relapsed/refractory disease.

Epidemiology and Genetic Characteristics of Porcine Reproductive and Respiratory Syndrome Virus in the Hunan and Hebei Provinces of China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant threat to the pig industry in China. However, the epidemiological characteristics of PRRSV after the outbreak of African swine fever in China were not thoroughly investigated. In the present study, the serological and epidemiological investigations of PRRSV in pigs from the Hunan and Hebei provinces of China were assessed. The results showed that 73.12% (95% CI 71.74-74.49) of pigs were positive for PRRSV-special antibody by enzyme-linked immunosorbent assay. Out of 5799 samples, 482 (8.31%, 95% CI 7.60-9.02) samples were positive for PRRSV nucleic acids. The positive rates of PRRSV in healthy pigs from farms and slaughterhouses were 2.27% (47/2072) and 7.70% (217/2818), which were lower than that in diseased pigs (23.98%, 218/909). Furthermore, the full-length OFR5 gene sequences of 43 PRRSV strains were sequenced and analysed. Phylogenetic analysis revealed that 43 isolates were classified into three lineages, namely lineage 1 (n = 24), lineage 8 (n = 15), and lineage 3 (n = 4). Lineage 1 could be further divided into sublineage 1.5 (n = 2) and sublineage 1.8 (n = 22), and lineage 8 was classified into sublineage 8.1 (n = 3) and sublineage 8.7 (n = 12). Collectively, our findings revealed the severe prevalence of PRRSV in the Hunan and Hebei provinces, where sublineage 1.8 and sublineage 8.7 predominated. The present study provides the update information of the epidemiological and genetic characteristics of PRRSV in the investigated regions, which will be beneficial for PRRS control.

Identification of a cell wall-associated subtilisin-like serine protease involved in the pathogenesis of Streptococcus suis serotype 2.

Streptococcus suis is an important swine and human pathogen, and also an emerging zoonotic agent. A surface-associated subtilisin-like serine protease (SspA) of S. suis was identified by screening a genomic expression library as fragments of this protein reacted most strongly with convalescent-phase pig sera. The sspA gene is present in 29 of 33 S. suis serotypes reference strains and is expressed on the surface of S. suis. Relative real-time quantitative PCR assay demonstrated that sspA mRNA expression in vivo was several thousand fold of that in vitro. A sspA(-) mutant was generated from a S. suis serotype 2 strain SC19 by allelic exchange. The mutant was not different from the wild type strain in subcellular structures and in hemolytic phenotype. However, the virulence of the sspA(-) mutant was markedly lower than the wild type in pigs as demonstrated in experimental infections. These data indicated that the surface-associated protein SspA is a conserved virulence factor of S. suis and is involved in the pathogenesis of S. suis.

Chronic exposure to low dose of bisphenol A causes follicular atresia by inhibiting kisspeptin neurons in anteroventral periventricular nucleus in female mice.

Bisphenol-A (BPA) is an estrogenic chemical extensively used in industrial and household applications. The present study was conducted to investigate the effect of chronic exposure to BPA on the adult female neuroendocrine system. Herein, we found that expose of adult female mice to BPA (50 µg/kg) by oral gavage for 60 days (BPA mice) prolonged diestrus and decreased serum 17ß-estradiol (E2) concentration by reducing the number of antral follicles and corpora luteum. In comparison with controls, the levels of serum luteinizing hormone (LH), follicle stimulating hormone (FSH), hypothalamic gonadotrophin releasing hormone (GnRH) and the expression of kisspeptin in anteroventral periventricular nucleus (AVPV) decreased in BPA mice, which could be reversed by injecting kisspeptin-10 (i.c.v.). Treatment with BPA or estrogen receptor α (ERα) antagonist MPP, but not ERß antagonist PHTPP inhibited E2-induced AVPV-kisspeptin expression in ovariectomized mice. Use of ERα agonist PPT rather than ERß agonist DPN enhanced AVPV-kisspepetin expression, which decreased after treatment with BPA. The amplitude of the proestrus LH surge decreased in mice exposed to BPA, but was recovered by administering kisspeptin-10. The present study provides in vivo evidence that chronic exposure to a low dose of BPA suppressed ERα-induced activation of AVPV-kisspeptin neurons, leading to prolonged diestrus and reduced ovulation in adult female mice.

The Not so Good, the Bad and the Ugly: Differential Bacterial Adhesion and Invasion Mediated by Salmonella PagN Allelic Variants.

While advances in genomic sequencing have highlighted significant strain variability between and within Salmonella serovars, only a few protein variants have been directly related to evolutionary adaptation for survival, such as host specificity or differential virulence. The current study investigated whether allelic variation of the Salmonella adhesin/invasin PagN influences bacterial interaction with their receptors. The Salmonella enterica, subspecies enterica serovar Typhi (S. Typhi) allelic variant of PagN was found to bind significantly better to different enterocytes as well as to the extracellular matrix protein laminin than did the major Salmonella enterica, subspecies enterica serovar Typhimurium (S. Typhimurium) allele. The two alleles differed at amino acid residues 49 and 109 in two of the four predicted PagN surface loops, and residue substitution analysis revealed that a glutamic acid at residue 49 increased the adhesive and invasive properties of S. Typhi PagN. PagN sequence comparisons from 542 Salmonella strains for six representative S. enterica serovars and S. diarizonae further supported the role of glutamic acid at residues 49 and 109 in optimizing adhesion to cells and laminin, as well as for cell invasion. In summary, this study characterized unique residues in allelic variants of a virulence factor that participates in the colonization and invasive properties of different Salmonella stains, subspecies and serovars.

Tanshinone IIA attenuates estradiol-induced polycystic ovarian syndrome in mice by ameliorating FSHR expression in the ovary.

Tanshinone IIA (TSIIA) is a major component of Salvia miltiorrhiza, a Chinese herb that exhibits a therapeutic effect on polycystic ovary syndrome (PCOS). The present study replicated PCOS via the neonatal treatment of estradiol in mice. Estrous cycles, body and ovarian weight, serum levels of testosterone and estradiol were determined. Histological examination of ovaries was performed. The mRNA and protein levels of aromatase luteinizing hormone receptor and follicle-stimulating hormone (FSHR) in ovaries and granule cells were assayed by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. TSIIA was revealed to reverse all disorders induced by estradiol treatment, including prolonged estrous cycles, increased body and ovarian weight, increased atretic cyst-like follicles and decreased corpus luteum, large antral follicles and preovulatory follicles. These improvements in PCOS as a result of TSIIA treatment are likely due to the revised testosterone/estradiol balance, as TSIIA reversed the decrease in aromatase mRNA, the enzyme that converts androgen to estrogen. As the expression of aromatase is regulated by the FSH pathway, TSIIA-mediated elevation in FSHR expression may lead to the upregulation of aromatase. Therefore, TSIIA revises the balance of androgen and estrogen by rescuing the reduced expression of FSHR and aromatase, thus attenuating murine PCOS. The current study aimed to further the application of natural drugs in the treatment of PCOS to confront the side effects of hormone drugs and expand the use of TSIIA.

Crocetin attenuates DHT-induced polycystic ovary syndrome in mice via revising kisspeptin neurons.

Polycystic ovarian syndrome (PCOS), the most common endocrine disorder of women in reproductive age, is typical with hyperandrogenism and disturbance of the hypothalamus-pituitary-ovary (HPO) axis, i.e. abnormal expression of hypothalamic gonadotropin-releasing hormone (GnRH) followed by the elevated ratio of serum luteinizing hormone (LH) level to follicle-stimulating hormone (FSH) level. This derangement might have a close relationship with hypothalamic kisspeptin expression that is thought to be a key regulator of GnRH. Crocetin, one of the main components of Saffron clinically used as traditional medicine in gynecology diseases, was evaluated for its therapeutic effects on PCOS induced by prenatally exposure to dihydrotestosterone (DHT) in mice. Herein, we found that DHT-treated mice showed a similar phenotype to human PCOS such as heavier ovary, prolonged diestrus, multiple enlarged follicles with fewer corpus luteum, and higher LH and testosterone levels. Kisspeptin expression was lower in anteroventral periventricular nucleus (AVPV) but higher in arcuate nucleus (ARC). Treatment of crocetin prevented the prolongation of diestrus and reduction in corpora luteum, recover the levels of GnRH, FSH, LH, progesterone (P4), estradiol (E2) and testosterone (T), and increase the kisspeptin level in AVPV but reduce that in ARC. The present study provides in vivo evidence that crocetin improved the PCOS in mice via increasing AVPV-kisspeptin and reducing ARC-kisspeptin expression.

Wogonin Attenuates Isoprenaline-Induced Myocardial Hypertrophy in Mice by Suppressing the PI3K/Akt Pathway.

Many studies have focused on identifying therapeutic targets of myocardial hypertrophy for the treatment of correlative cardiac events. Wogonin is a natural flavonoid compound that displays a potent anti-hypertrophic effect. Knowledge of its pharmacological mechanisms might reveal an effective way to search for therapeutic targets. Myocardial hypertrophy was replicated by the subcutaneous implantation of an isoprenaline mini-pump in mice or isoprenaline treatment of H9C2 cells. Pathologic changes in cardiac structure were assessed by echocardiographic and histological examinations. The signaling transduction in hypertrophy-promoting pathways and the genes involved were detected by western blot and RT-qPCR. Wogonin significantly attenuated isoprenaline-induced myocardial hypertrophy in vivo and in vitro by suppressing phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) hypertrophy-promoting pathway. Wogonin promoted the ubiquitination and degradation of PI3K catalytic subunit alpha (Pik3ca), the catalytic subunit of PI3K, which was upregulated by isoprenaline treatment. Wogonin also increased the expression of neural precursor cells expressing developmentally down-regulated gene 4-like (Nedd4l), the ubiquitin E3 ligase of Pik3ca. Therefore, wogonin targets Nedd4l to induce the degradation of Pik3ca, which reverses the over-activation of the PI3K/Akt pathway and consequently relieves the isoprenaline-induced myocardial hypertrophy.

Characterization of smoke and dust episode over West Africa: comparison of MERRA-2 modeling with multiwavelength Mie-Raman lidar observations.

Observations of multiwavelength Mie-Raman lidar taken during the SHADOW field campaign are used to analyze a smoke-dust episode over West Africa on 24-27 December 2015. For the case considered, the dust layer extended from the ground up to approximately 2000 m while the elevated smoke layer occurred in the 2500-4000 m range. The profiles of lidar measured backscattering, extinction coefficients, and depolarization ratios are compared with the vertical distribution of aerosol parameters provided by the Modern-Era Retrospective Analysis for Research and Applications, version 2 (MERRA-2). The MERRA-2 model simulated the correct location of the near-surface dust and elevated smoke layers. The values of modeled and observed aerosol extinction coefficients at both 355 and 532 nm are also rather close. In particular, for the episode reported, the mean value of difference between the measured and modeled extinction coefficients at 355 nm is 0.01 km-1 with SD of 0.042 km-1. The model predicts significant concentration of dust particles inside the elevated smoke layer, which is supported by an increased depolarization ratio of 15 % observed in the center of this layer. The modeled at 355 nm the lidar ratio of 65 sr in the near-surface dust layer is close to the observed value (70 ± 10) sr. At 532 nm, however, the simulated lidar ratio (about 40 sr) is lower than measurements (55 ± 8 sr). The results presented demonstrate that the lidar and model data are complimentary and the synergy of observations and models is a key to improve the aerosols characterization.

Development and evaluation of a DAS-ELISA for rapid detection of avian influenza viruses.

Rapid detection of avian influenza virus (AIV) infection is critical for control of avian influenza (AI) and for reducing the risk of pandemic human influenza. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for this purpose. The method employed a monoclonal antibody (MAb) as the capture antibody and rabbit polyclonal IgG labeled with horseradish peroxidase as the detector antibody, and both antibodies were against type-specific influenza A nucleoprotein (NP). The DAS-ELISA could detect minimally 2.5 ng of influenza viral protein in virus preparations treated with Triton X-100, which is equvilent to 2.5 x 10(2) EID50 virus particles. This DAS-ELISA could detect all 15n AIV subtypes (H1-H15) and did not cross react with other avian pathogens tested. The DAS-ELISA were directly compared with virus isolation (VI) in embryonated chicken eggs, the current standard of influenza virus detection, for 805 chicken samples. The DAS-ELISA results correlated with VI results for 98.6% of these samples, indicating a sensitivity of 97.4% and specificity of 100%. The method was further tested with H5N1 and H9N2 AIV experimentally infected chickens, ducks, and pigeons, as well as field samples obtained from central China in 2005. The DAS-ELISA method has demonstrated application potential as an AIV screening tool and as a supplement for virus isolation in Asia.

Development of a specific latex agglutination test based on a recombinant hemagglutinin protein to detect antibodies to H5 avian influenza viruses.

A latex agglutination test (LAT) was developed for rapid detection of antibodies to H5 avian influenza viruses (AIVs). The hemagglutinin protein of H5 AIV was covalently linked to carboxylated latex by ethyl-dimethyl-amino-propyl carbodiimide to prepare the sensitized latex beads. The LAT was evaluated with the hemagglutination inhibition (HI) assay as the reference test. The H5-LAT showed a sensitivity of 87.0% and specificity of 88.9% in detecting 126 serum samples from experimentally infected chickens and a sensitivity of 82.5% and specificity of 86% in detecting 587 field chicken serum samples from mostly vaccinated chickens. The agreement ratio between H5-LAT and HI was found to be 87.3% and 83.1% for the two groups of samples, respectively. Difficulty with background agglutination in stored chicken sera was overcome by serum pretreatment with either dried chicken liver powder or dilution buffer containing detergent Tween-20. The H5-LAT has advantages over a previously reported whole-virus LAT in terms of biosafety in preparation, chemical stability, and higher specificity. It is a rapid and simple test suitable for field monitoring of antibodies to H5-type AIV.

[Blood leptin, orexins and NPY levels and their relations in obese children].

OBJECTIVE: To investigate the concentration levels of leptin, orexins and neuropeptide Y (NPY) in the blood of obese children, and to analysed the relationship between these substances. METHODS: RIA methods were used to measure the concentrations of leptin, orexinA, orexinB, and NPY in the blood of 98 obese children [BMI: male (29.24 +/- 1.87) kg/mZ, female (28.12 +/- 2.30) kg/m2] and in 104 normal children [BMI: male (20.49 +/- 1.95) kg/m2, female (19.59 +/- 1.51) kg/m2] as the control group. RESULTS: The leptin concentrations in obese children [male (26.00 +/- 14.66) ng/mL; female (33.59 +/- 14.63) ng/mL] were higher than those in the control group [male (6.65 +/- 44.49) ng/mL; female [10.48 +/- 5.52) ng/mL P < 0.013]. The concentrations of plasma orexinA in obes children [male (3.23 +/- 1.86) pg/mL; female (3.38 +/- 1.80) pg/mL] were lower than those in the control group [male (4.52 +/- 1.52) pg/mL; female (4.71 +/- 1.53) pg/mL P < 0.05]; Negative correlations between leptin and NPY were noted in the obesity group (r = -0.302) and the control group (r = -0.310, P < 0.01), while the slopes in the two groups were different (control group -2.969; obese group -0.809). A positive correlation between NPY and orexinA was noted (r = 0.207, P < 0.05). The fluctuation range of orexinA in obese children was markedly narrowed when compared with that in the control group. CONCLUSION: The concentration level of peripheral orexinA and leptin in the obese and non-obese children change inversely. The obesity in children correlates with the concentrations of orexinA, leptin, NPY as well as with their interactions.

Identification and characterization of novel immunogenic outer membrane proteins of Haemophilus parasuis serovar 5.

Haemophilus parasuis is the aetiological agent of Glässer's disease, which is responsible for cases of fibrinous polyserositis, polyarthritis and meningitis in young pigs. To develop more effective vaccines, an immunoproteome-based approach was used to analyze the outer membrane proteins of H. parasuis serovar 5. A total of 15 proteins with high immunogenicity were identified and all were showed to be immunogens for the first time in H. parasuis. Further analyses of 8 selected proteins revealed that (1) significantly higher level of serum antibodies against 6 proteins was detected with convalescent sera and immunized sera; (2) antisera against 5 of the selected proteins could effectively inhibit H. parasuis growth in mouse blood; and (3) 4 proteins could induce protective response of the vaccinated mice against H. parasuis. The results suggest these 4 proteins (PalA, Omp2, D15 and HPS_06257) have strong potential to be vaccine candidates.

[Preparation antibodies against recombinant bovine IFN-gamma and development of sandwich ELISA for bovine IFN-gamma detection].

This study was aimed to establish ELISA for recombinant bovine IFN-gamma (BovIFN-gamma) detection and provide a new method for diagnosis of pathogenic infection. The total RNA was isolated from peripheral blood leucocytes cultured with PHA mitogen stimulation. Then bovine IFN-gamma (BovIFN-gamma) gene cDNA was amplified by RT-PCR and cloned into pET28a to obtain the expression plasmid designated as pETBovIFN-gamma. The pETBovlFN-gamma was further transformed into competent E. coli BL21 cells and a 18kD His-tagged protein as expected was expressed after IPTG induction. By using purified recombinant BovIFN-gamma as antigen and lymphocyte-hybridoma technique, four hybridoma cell lines which stably secreted monoclonal antibodies against rBovIFN-gamma were generated, designated as A7, A10, G6, and G10. The immunoglobin subset was identified as IgG1 . Western-blotting analysis and ELISA demonstrated that the monoclonal antibodies secreted by all the four hybridoma cell lines could react specifically to the recombinant BovIFN-gamma, but not irrelative proteins such as Ag85B, ESAT-6-CFP-10 and GM-CSF, suggesting that the four hybridoma cell lines were rBovIFN-gamma specific monoclonal antibodies. A sandwich ELISA was established by using A10 secreted monoclonal antibody and rabbit polyclonal antibodies against BovIFN-gamma, HRP labeled goat anti-rabbit IgG. The results indicated that the sensitivity was 2ng/mL. This sandwich ELISA to detect BovIFN-gamma paved the way to develop a sensitive method for specific infection detection such as bovine tuberculosis diagnosis.