Microstructure Evolution, Hot Deformation Behavior and Processing Maps of an FeCrAl Alloy.

The deteriorated plasticity arising from the insoluble precipitates may lead to cracks during the rolling of FeCrAl alloys. The microstructure evolution and hot deformation behavior of an FeCrAl alloy were investigated in the temperature range of 750-1200 °C and strain rate range of 0.01-10 s-1. The flow stress of the FeCrAl alloy decreased with an increasing deformation temperature and decreased strain rate during hot working. The thermal deformation activation energy was determined to be 329.49 kJ/mol based on the compression test. Then, the optimal hot working range was given based on the established hot processing maps. The hot processing map revealed four small instability zones. The optimal working range for the material was identified as follows: at a true strain of 0.69, the deformation temperature should be 1050-1200 °C, and the strain rate should be 0.01-0.4 s-1. The observation of key samples of thermally simulated compression showed that discontinuous dynamic recrystallization started to occur with the temperate above 1000 °C, leading to bended grain boundaries. When the temperature was increased to 1150 °C, the dynamic recrystallization resulted in a microstructure composed of fine and equiaxed grains.

Ame-miR-980-3p participates in autophagy-mediated midgut remodelling in Apis mellifera via targeting Atg2B.

Autophagy is a process that serves to degrade damaged proteins and organelles, thereby promoting cell homeostasis, differentiation, development and survival. Many miRNAs have been found to have regulatory roles in autophagy. In insects, it has been shown that autophagy is involved in hormone-regulated programmed cell death during metamorphic midgut remodelling. However, whether this is also true during the remodelling of the honey bee midgut is unclear. In the present study, we explored the relationship between autophagy and midgut remodelling and sought to identify miRNAs involved in this physiological process. We found that autophagy occurred during midgut remodelling and that the inhibition of autophagy resulted in midgut dysplasia in prepupae. Differentially expressed miRNAs enriched in the autophagy signalling pathway during midgut remodelling were identified by small RNA-seq. Ame-miR-980-3p, which targets the autophagy-related gene Atg2B, was screened out. Furthermore, abnormal expression of ame-miR-980-3p in the pupal stage led to the thinning of the midgut wall of newly emerged bees (NE). When ame-miR-980-3p expression was inhibited, the intestinal villi of NE bees became significantly shorter and sparse, and the lipid signal in the peritrophic matrix of Pb almost disappeared, indicating that the adult midgut was underdeveloped and the lipid absorption ability was weakened. Taken together, ame-miR-980-3p targeted Atg2B to participate in the regulation of midgut autophagy in the pupae, and the abnormal expression of ame-miR-980-3p would interfere with cell proliferation and death in the process of midgut remodelling, hinder the formation of adult midgut and eventually lead to adult midgut dysplasia and affect the lipid absorption function of the midgut in Apis mellifera.

Molecular characterization and immunological properties of Echinococcus granulosus sensu stricto (G1) ADK1 and ADK8.

Adenylate kinases (ADKs) are one of the important enzymes regulating adenosine triphosphate (ATP) metabolism in Echinococcus granulosus sensu lato. The objective of the present study was to explore the molecular characteristics and immunological properties of E. granulosus sensu stricto (G1) adenylate kinase 1 (EgADK1) and adenylate kinase 8 (EgADK8). EgADK1 and EgADK8 were cloned and expressed, and the molecular characteristics of EgADK1 and EgADK8 were analyzed through different bioinformatics tools. Western blotting was used to examine the reactogenicity of recombinant adenylate kinase 1 (rEgADK1) and recombinant adenylate kinase 8 (rEgADK8) and to evaluate their diagnostic value. The expression profiles of EgADK1 and EgADK8 in 18-day-old strobilated worms and protoscoleces were analyzed by quantitative real-time PCR, and their distribution in 18-day-old strobilated worms, the germinal layer, and protoscoleces was determined by immunofluorescence localization. EgADK1 and EgADK8 were successfully cloned and expressed. Bioinformatics analysis predicted that EgADK1 and EgADK8 have multiple phosphorylation sites and B-cell epitopes. Compared with EgADK8, EgADK1 and other parasite ADKs have higher sequence similarity. In addition, both cystic echinococcosis (CE)-positive sheep sera and Cysticercus tenuicollis-infected goat sera could recognize rEgADK1 and rEgADK8. EgADK1 and EgADK8 were localized in protoscoleces, the germinal layer, and 18-day-old strobilated worms. EgADK1 and EgADK8 showed no significant difference in their transcription level in 18-day-old strobilated worms and protoscoleces, suggesting that EgADK1 and EgADK8 may play an important role in the growth and development of E. granulosus sensu lato. Since EgADK1 and EgADK8 can be recognized by other parasite-positive sera, they are not suitable as candidate antigens for the diagnosis of CE.

Seroprevalence of Neospora caninum infection and associated risk factors in cattle in Shanxi Province, north China.

Neospora caninum is an obligate intracellular parasitic protozoan that can cause abortions in cattle and pose considerable economic losses to the cattle industry. As a major livestock province, little is known of N. caninum infection in cattle in Shanxi Province, north China. In order to investigate the seroprevalence of N. caninum in cattle in Shanxi Province, 978 cattle serum samples were collected from 11 cities in three representative geographical locations in Shanxi Province, and the N. caninum-specific IgG antibodies were examined using an indirect enzyme linked immunosorbent assay (ELISA) kit commercially available. The results showed that 133 of the 978 examined cattle serum samples (13.60%, 95% CI = 11.45-15.75) were positive for N. caninum antibodies, and the seroprevalence in different cities ranged from 0 to 78.89%. The geographical location and management mode were the risk factors associated with N. caninum infection in cattle herds in Shanxi Province. Cattle in Northern and Central Shanxi Province as well as cattle whose management mode is that of large-scale cattle farming companies are more susceptible to N. caninum infection. This was the first large-scale survey of N. caninum seroprevalence and assessment of associated risk factors in cattle in Shanxi Province, which provided baseline information for the prevention and control of N. caninum infection in cattle in Shanxi Province, north China.

Retinoic Acid Receptor Gamma (RARγ) Promotes Cartilage Destruction through Positive Feedback Activation of NF-κB Pathway in Human Osteoarthritis.

Osteoarthritis (OA) is a severe inflammation-related disease which leads to cartilage destruction. The retinoic acid receptor gamma (RARγ) has been indicated to be involved in many inflammation processes. However, the role and mechanism of RARγ in cartilage destruction caused by inflammation in OA are still unknown. Here, we demonstrated that the RARγ was highly expressed in chondrocytes of OA patients compared with healthy people and was positively correlated with the damage degree of cartilage in OA. Cytokine TNF-α promoted the transcription and expression of RARγ through activating the NF-κB pathway in OA cartilage. In addition, the overexpression of RARγ resulted in the upregulation of matrix degradation and inflammation associated genes and downregulation of differentiation and collagen production genes in human normal chondrocyte C28/I2 cells. Mechanistically, overexpression of RARγ could increase the level of p-IκBα and p-P65 to regulate the expression of downstream genes. RARγ and IκBα also could interact with each other and had the same localization in C28/I2 cells. Moreover, the SD rats OA model induced by monosodium iodoacetate indicated that CD437 (RARγ agonist) and TNF-α accelerated the OA progression, including more severe cartilage layer destruction, larger knee joint diameter, and higher serum ALP levels, while LY2955303 (RARγ inhibitor) showed the opposite result. RARγ was also highly expressed in OA group and even higher in TNF-α group. In conclusion, RARγ/NF-κB positive feedback loop was activated by TNF-α in chondrocyte to promote cartilage destruction. Our data not only propose a novel and precise molecular mechanism for OA disease but also provide a prospective strategy for the treatment.

[Cloning and Prokaryotic Expression of Fusion Gene of Group II Allergen Der p2 T Cell Epitope from Dermatophagoides pteronyssinus].

OBJECTIVE: To express and purify the T cell epitope fusion peptide of the major allergen Der p2 from Dermatophagoides pteronyssinus. METHODS: Nucleotide sequences reported to encode four T-cell epitopes (T1-T4) of Der p2 of D. pteronyssinus were linked in the rank of T1-T2-T3-T4. In this way, the chimeric gene was synthesized, named as Der p2 T. The gene of Der p2 T was amplified by PCR, purified, and cloned into the pET-28a (+) vector, forming the prokaryotic recombinant expression vector pET-28a (+)-Der p2 T. This formation was verified by double digestion. The pET-28a (+)-Der p2 T vector was transfected into E. coli strain BL-21, and its expression was induced by addition of IPTG. The recombinant protein was purified and collected by Ni-NTA affinity chromatography, and prepared for SDS-PAGE and Western blotting analysis. ELISA was used to evaluate the binding ability of Der p2 T cell epitope fusion peptide to serum IgE from patients with house dust mite allergy. RESULTS: Double digestion results confirmed the construction of the pET-28a (+)-Der p2 T vector. SDS-PAGE revealed the expression of recombinant Der p2 T cell epitope fusion peptide with M, of 10,000. Western blotting confirmed the purification of Der p2 T cell epitope fusion peptide. The binding ability of Der p2 T cell epitope fusion peptide to serum IgE from patients with house dust mite allergy [(37.70±9.89) µg/ml] decreased significantly in comparison to that of Der p2 [(85.89±9.63) µg/ml] (P<0.01). CONCLUSION: The Der p2 T cell epitope fusion peptide is prepared, and its binding ability to serum IgE from patients with house dust mite allergy significantly decreases than that of Der p2.

[Immunotherapy effect of ploypeptide vaccine of allergen group 1 from Dermatophagoides farinae on asthma mice].

OBJECTIVE: To investigate the enzymatic hydrolysates of ProDer f land their hydrolytic products for specific immunotherapy. METHODS: The asthma models of mice made by ProDer f 1 allergen were treated by using two kinds of hydrolysates as vaccine for analyzing their effects of immunotherapy. Fifty female BALB/c mice were randomly divided into 5 groups (n = 10 for each), i.e., a PBS group, an asthma group, an immunotherapy group by ProDer f 1 protein, an immunotherapy groups by papain hydrolysates and trypsin hydrolysates. On day 0, 7 and 14, the mice were intraperitoneally injected with 10 µg of ProDer f 1 allergen, which was dissolved in 100 µl PBS containing 2% (W/V) Al (OH)3 suspension. At day 21, the animals were caged in the airway challenge apparatus, and challenged by nebulized inhalation of allergen suspension (0.5 µg/ml) for 30 min for 7 successive days. The mice were undergone allergen specific immunotherapy (ASIT) by intraperitoneal injection and of the immunotherapy group by ProDer f 1 protein, immunotherapy groups by papain hydrolysates and trypsin hydrolysates (100 µg/ml) in a dose of 200 µl of reactive allergens, 30 min prior to the inhalation treatment at day 25, 26 and 27, respectively. The PBS group was managed with both intraperitoneal injection and aerosol of PBS. Twenty-four hours after the last challenge, all the mice were sacrificed. The bronchoalveolar lavage fluid (BALF) and sera were collected, and the splenocytes were cultured. The levels of IL-4, IL-10, IL-17 and IFN-γ in BALF and supernatant of splenocytes cultured (SSCC) were detected by ELISA, and the serum levels of specific IgE and IgG2a antibodies were also detected by ELISA. RESULTS: Compared with the asthma group, the histo- logic examination of the lungs taken from the mice in the immunotherapy group by ProDer f 1 protein, immunotherapy groups by papain hydrolysates and trypsin hydrolysates showed alleviated peribronchial and perivascular inflammatory infiltration, absently of eosinophils. The normal lung architectures were also exhibited, particularly, the epithelium was of normal size and morphology, similar to that of the PBS-challenged group. The levels of IL-4 in BLAF of the ASIT groups and asthma group were (231.61 ± 11.73), (206.20 ± 14.33), (200.44 ± 9.34), (299.68 ± 12.46) pg/ml; the levels of IL-10 in BLAF were (361.87 ± 13.62), (376.27 ± 20.57), (413.57 ± 12.98), (171.28 ± 19.79) pg/ml; the levels of IL-17 in BLAF were (142.12 ± 5.01), (128.27 ± 5.34), (130.79 ± 6.30), (273.59 ± 11.56) pg/ml; the levels of IFN-γ in BLAF were (229.60 ± 11.32), (269.13 ± 11.98), (282.25 ± 19.65), (147.76 ± 11.36) pg/ml. The levels of IL-4 in SCCS of the ASIT groups and asthma group were (218.54 ± 12.62), (220.21 ± 10.73), (201.59 ± 18.54), (256.86 ± 15.53) pg/ml; the levels of IL-10 were (360.45 ± 13.10), (383.41 ± 19.81), (413.51±13.14), (173.50 ± 20.25) pg/ml; the levels of IL-17 were (154.23 ± 5.18), (137.72 ± 6.66), (141.01 ± 7.35), (297.55 ± 8.97) pg/ml, the levels of IFN-γ were (243.22 ± 25.01), (275.20 ± 14.65), (284.67 ± 25.87), (154.54 ± 17.45) pg/ml. The levels of antigen-specific IgE antibody of the ASIT groups and asthma group were (309.66 ± 13.56), (256.61 ± 40.64), (248.83 ± 10.51), (359.60 ± 29.48) µg/ml, and the antigen-specific IgG2a antibody levels were (8.87 ± 0.82), (9.15 ± 0.83), (10.56 ± 1.68), (7.04 ± 0.42) µg/ml. The resulting serum antigen-specific IgE antibody levels suggested that the IgE levels in the immunotherapy group by ProDer f 1 protein, immunotherapy groups by papain hydrolysates and trypsin hydrolysates were significantly lower than that in the asthma group. Conversely, the level of antigen-specific IgG2a in sera was significantly higher in the immunotherapy group by ProDer f 1 protein, immunotherapy groups by papain hydrolysates and trypsin hydrolysates than that in the asthmatic group. The levels of IL-4, IL- 17 in BALF and SCCS in the immunotherapy group by ProDer f 1 protein, immunotherapy groups by papain hydrolysates and trypsin hydrolysates significantly decreased, compared with that in the asthma group. However, the levels of IL-10 and IFN-γ in BALF and SCCS in the immunotherapy group by ProDer f 1 protein, immunotherapy groups by papain hydrolysates and trypsin hydrolysates were increased dramatically in the immunotherapy group by ProDer f 1 protein, immunotherapy groups by papain hydrolysates and trypsin hydrolysates than that in the asthma group. CONCLUSION: The hydrolytic products above-mentioned can alleviate asthmatic symptoms effectively after the antigen-specific immunotherapy in murine asthma models.

[Investigation of natural foci of Metorchis taiwanensis in Wuhu area].

OBJECTIVE: To explore the existence of natural foci of Metorchis taiwanensis in Wuhu area. METHODS: The ecological environment and daily living habits of residents were investigated; the eggs of M. taiwanensis in the dung of ducks in local region were collected by the sedimentation method; the Parafossarulus striatulus were disposed by the tabletting method and examined with a microscope to isolate the rediae and cercariae; the metacercariae in Pseudorasbora parve were isolated by using the homogenation. The duckling were fed directively by metacercaria of M. taiwanensis or by P. parve infected with metacercaria of M. taiwanensis for artificial infection, and M. taiwanensis were separated from ducks by dissecting the gallbladder and bile ducts. RESULTS: The survey revealed that there were staggered lake river, rich vegetation, overgrown shrubs, aquatic plants, rich variety of freshwater snails and fish resources in Wuhu area, and it was home to a variety of waterfowl and duck. The average detection rate of eggs of M. tanwanensis in stool of ducks was 33.33% (10/30); that of rediae and cercarriae in P. striatulus was 1.17%(7/600); and that of encysted metacercaria in P. parve was 13.33% (8/60); the number of M. taiwanensis detected in the infected ducklings in 3 groups were 31, 8 and 0, respectively, the natural M. tanwanensis infection rate of ducks was 23.33% (7/30). CONCLUSION: We have confirmed the existence of natural foci of M. tanwanensis in Wuhu area.

[Investigation of Acaroid mites breeding in university canteen condiments].

OBJECTIVE: To investigate the condition of Acaroid mite breeding in university canteen common condiments. METHODS: Thirteen kinds of condiments of the public cafeteria and 29 kinds of condiments of the canteen warehouse storage were collected. The mites were separated by the method of water film microscopy and an electric powder set, and then the film production; mite identification and counting were performed under a microscope. RESULTS: There were 13 spices with acaroid mites in canteen warehouse storage condiments tested and the detection rate was 62.07%, and there were 13 species, 10 genera, 5 families of mites. There were 9 spices with Acaroid mites in canteen condiments tested and the detection rate was 72.73%, and there were 11 species, 9 genera, 4 families of mites. CONCLUSION: The college canteen common condiments are seriously polluted with acaroid mites, which should be paid attention to.

[Morphology observation of Histiostoma feroniarum hypopus].

OBJECTIVE: To observe the external morphology of Histiostoma feroniarum hypopus under light microscope. METHODS: The samples were collected in a mushroom cultivation base, and the Histiostoma feroniarum hypopus was isolated and purified. The slide samples were prepared and observed under an optical microscope. RESULTS: The back body of the Histiostoma feroniarum hypopus was flat with tiny bristles, the epidermis was of significant ossification, the ventral had four pairs of slender feet stretched, the sucker plate was prosperous in the end of the body, and the sucker plate had eight suckers. The gnathosoma was thin, long and highly specialized. CONCLUSION: The light microscopy shows the morphological characteristics of Histiostomaferoniarum hypopus, providing the basis for identifying the hypopus.

[Detection of DNA-protein crosslinks with modified comet assay].

Modifications of the comet assay were introduced to measure crosslinks and the effect of formaldehyde in liver cells of the tested animals was investigated by the new method to see whether the method is feasible. Since exposure of slides to proteinase K can increase DNA migration of the treated cells, the presence of DNA-protein crosslinks can be indicated by compare of the tail moment before and after the proteinase K added. The results showed that the modified protocol of the alkaline comet assay is a fast, inexpensive and sensitive tool for the detection of potent crosslinkers-induced DNA-protein crosslinks at single cell level. Due to its specific advantages, the modified comet assay seems to be a useful tool as a DNA crosslink potency indicator.

Cloning and Sequencing of Rhizobium huakuii exoA Gene Encoding a Glucosyl-transferase.

From exoR'-11 which could complement two Exo(-) mutants of R. huakuii 107: NA03 and NA10, a 2.0kb BglI fragment was subcloned in pRK415. The resulted plasmid pJB-H701 could restore the Exo(-) phenotype of NA03 and NA10. The complete nucleotide sequence of the fragment was determined, which contains the structural genes of a glucosyl-transferase, as well as the 5'- and 3'- flanking regions. An open reading frame of 984 base pairs was identified as R. huakuii exoA gene. The MW of ExoA, as deduced from the nucleotide sequence, was estimated as 35 kD. Comparison of the nucleotide sequences revealed a high similarity between exoA genes of R. huakuii and R. meliloti. The deduced amino acid sequence of R. huakuii ExoA also showed a high similarity with that of R. meliloti ExoA. Furthermore, the exoA-lacZ transcription fusion gene was constructed and the expression of exoA-lacZ was analyzed.

Analysis of the Exo(-) Mutant of Rhizobium huakuii and Their Exopolysaccharide Component.

We have subcloned two plasmids, pWS-6BP1 and pWS-6BP2, from the 8.5 kb DNA fragment of plasmid pJB22-B6. The plasmid of pWS-6BP1, with a 5.8 kb DNA fragment, can complement the exopolysaccharide-deficient mutant NA06 and NA12 of Rhizobium huakuii strain 107, but the plasmid pWS-6BP2, with a 2.6 kb DNA fragment can only complement NA12. The effective nodules formed by the wild type strain 107 (Rh107) contain lots of bacteriods; the ineffective nodules formed by the mutant NA06 and NA12 contain few bacteroids. Although the nodule formed by Exo(+) transconjugants are ineffective, the bacteria can be successfully released into the cells and form lots of bacteroids. The Rh107 produces acidic exopolysaccharides (EPS) of high molecular weight (HMW) in large amount with only small amount of low molecular weight (LMW) forms. EPS secreted by mutants NA06 and NA12 are just the other way round with LMW in higher amount than HMW ones. They also produce an EPS with molecular weight in between. The EPS secreted by the Exo(+) transconjugants is more like the EPS of Rh107 than that of mutants, although their component and structure are still different from that of Rh107.

Sequence Analysis of the exo1 Gene Involved in the Exopolysaccharide Synthesis of Rhizobium huakuii.

The 2.6 kb fragment which can complement the Exo(-)Ndv(-)Fix(-) mutants of R. huakuii to Exo(+)Nod(+)Fix(+) was sequenced. The result revealed the presence of an open reading frame exo1, which encodes 340 amino acids. Analysis of the hydrophobicity plot of the putative protein indicated that it was a cytoplasmic protein. Exo1 displayed strong homology to the ExoU protein of R. meliloti, which was a glucosyltransferase. A transcriptional fusion to lacZ using the promoterless vector pMP221 showed that there were two regions with promoter activity in exo1. Pexo1a was identified upstream of exo1, and Pexo1b in the middle of exo1. Pexo1a probably contains the promoter of exo1 gene.

The Nucleotide Sequence of gltD Gene Encoding the Small Subunit of Rhodobacter sphaeroides Glutamate Synthase.

We have determined the complete nucleotide sequence of a 2 387 bp chromosomal SalI-EcoRI fragment, which contains the structural gene (gltD) for the small subunit of Rhodobacter sphaeroides glutamate synthase, as well as the 5'- and 3'-flanking regions. An open reading frame of 1 242 base pairs was identified as the R. sphaeroides gltD gene. The MW of the small subunit, as deduced from the nucleotide sequence, was estimated to be 44 kD. A comparison of the nucleotide sequence revealed a high similarity among gltD genes of R. sphaeroides, Azospirillum brasilense and Escherichia coli. The deduced amino acid sequence of R. sphaeroides GltD showed a high similarity with that of A. brasilense GltD. The analysis of the binding domains of R. sphaeroides GltD was also carried out. The gltD-lacZ fusion was observed in the presence of 15 mM leucine.

The Characteristics of DCPIPH(2) right curved arrow MV Electron Transport in Rb. sphaeroides 601.

The absorption spectrum and fluorescence emission spectrum of RS601 were found to keep the typical characteristics of those of the purple nonsulfide bacteria Rb. sphaeroides. Under illumination, methyl viologen was reduced by RS601 chromatophores in the presence of DCPIPH(2) as the electron donor, setting up a standard noncyclic electron transport. o-phenanthroline with I(50) of 1.0 mM inhibited the DCPIPH(2) right curved arrow MV electron transport. Antimycin A did not inhibit the DCPIPH(2) right curved arrow MV electron transport and had no I(50). The results suggested that the exact site where methyl viologen accepted electron should locate between the secondary electron acceptor, Q(B), and cyt b, but not at the Q(A) binding site as indicated before. The difference of electron transport between reduced sides of reaction centers of Rb. sphaeroides and Rs. rubrum was discussed.

Comparison of Different Effects of Chloroacetates on Electron Transports in PS II and in the Reaction Center of Rb. sphaeroides 601.

Chloroacetates displayed different effects on electron transports in the photosynthetic reaction center of purple bacteria (Rb.sphaeroides 601) and in the photosystem II (PS II) of higher plants. Decays of chlorophyII a fluorescence measured after actinic flashes show that chloroacetates inhibit the electron transport from Q(A)(-) to Q(B) (Q(B)(-)) and the equilibrium between Q(A)(-)Q(B) (Q(B)(-)) and Q(A)Q(B)(-) (Q(B)(2-)), acting on electron transport as well as proton transduction. The study on PSII electron transport indicates another inhibition site of chloroacetate at the oxidation side of PSII. Chloroacetates up to 500 mM have no inhibition on the DCPIPH(2) right curved arrow MV electron transport in the RS601's chromatophores. Dissociation constants of OP and trichloroacetates from RS601 reaction center were assessed to be 1.1x10(-3) M and from Dixon curve respectively. The differences on aspects of structures and functions between RS601 reaction center and PS II were discussed.