Assessment of the Effect of Leonurine Hydrochloride in a Mouse Model of PCOS by Gene Expression Profiling.

Polycystic ovary syndrome (PCOS) is an endocrine disease commonly associated with metabolic disorders in females. Leonurine hydrochloride (Leo) plays an important role in regulating immunity, tumours, uterine smooth muscle, and ovarian function. However, the effect of Leo on PCOS has not been reported. Here, we used dehydroepiandrosterone to establish a mouse model of PCOS, and some mice were then treated with Leo by gavage. We found that Leo could improve the irregular oestros cycle of PCOS mice, reverse the significantly greater serum testosterone (T) and luteinising hormone (LH) levels, significantly reduce the follicle-stimulating hormone (FSH) level, and significantly increase the LH/FSH ratio of PCOS mice. Leo could also change the phenomenon of ovaries in PCOS mice presented with cystic follicular multiplication and a lacking corpus luteum. Transcriptome analysis identified 177 differentially expressed genes related to follicular development between the model and Leo groups. Notably, the cAMP signalling pathway, neuroactive ligand-receptor interactions, the calcium signalling pathway, the ovarian steroidogenesis pathway, and the Lhcgr, Star, Cyp11a, Hsd17b7, Camk2b, Calml4, and Phkg1 genes may be most related to improvements in hormone levels and the numbers of ovarian cystic follicles and corpora lutea in PCOS mice treated by Leo, which provides a reference for further study of the mechanism of Leo.

Systemic Pharmacology Reveals the Potential Targets and Signaling Mechanisms in the Adjuvant Treatment of Brucellosis with Traditional Chinese Medicine.

Human brucellosis is one of the world's most common zoonoses, caused by Brucella infection and characterized by induced inflammation, which in severe cases can lead to abortion and sterility in humans and animals. There is growing evidence that traditional Chinese medicine (TCM) is beneficial as an adjunct to the treatment of brucellosis. However, its specific targets of action and molecular mechanisms remain unclear. In this study, a systematic pharmacological approach was applied to demonstrate pharmacological targets, biological functions, and signaling pathways of TCM as an adjunct to the treatment of brucellosis (TCMTB). The results of network pharmacology were further verified by in vitro experiments. Network analysis revealed that 133 active ingredients and 247 targets were screened in TCMTB. Further data analysis identified 21 core targets and 5 core compounds in TCMTB, including beta-sitosterol, quercetin, kaempferol, luteolin, and paeoniflorin. Gene ontology and the Kyoto Encyclopedia of Gene and Genome analysis showed that TCMTB might actively treat brucellosis by regulating inflammatory response, enhancing immune function, and targeting signaling pathways such as tuberculosis and TNF. Molecular docking results showed that multiple compounds could bind to multiple targets. Further, in vitro experiments confirmed that quercetin, among the active compounds screened, induced the strongest immunomodulatory and pro-inflammatory cytokine production during Brucella abortus infection. Further, quercetin induced nitric oxide production, which attenuated the ability of B. abortus to internalize THP-1 cells as well as intracellular survival. This study reveals the mechanism by which TCMTB aids in the treatment of brucellosis through a synergistic multicomponent, multipathway, and multitarget action. The contribution of quercetin treatment to B. abortus infection was demonstrated for the first time, which may be related to the quercetin-induced production of nitric oxide and immunomodulatory and inflammatory cytokines. These predictions of the core compounds and targets may be used in the future for the clinical treatment of brucellosis.

The Flagellar Transcriptional Regulator FtcR Controls Brucella melitensis 16M Biofilm Formation via a betI-Mediated Pathway in Response to Hyperosmotic Stress.

The expression of flagellar proteins in Brucella species likely evolved through genetic transference from other microorganisms, and contributed to virulence, adaptability, and biofilm formation. Despite significant progress in defining the molecular mechanisms behind flagellar gene expression, the genetic program controlling biofilm formation remains unclear. The flagellar transcriptional factor (FtcR) is a master regulator of the flagellar system's expression, and is critical for B. melitensis 16M's flagellar biogenesis and virulence. Here, we demonstrate that FtcR mediates biofilm formation under hyperosmotic stress. Chromatin immunoprecipitation with next-generation sequencing for FtcR and RNA sequencing of ftcR-mutant and wild-type strains revealed a core set of FtcR target genes. We identified a novel FtcR-binding site in the promoter region of the osmotic-stress-response regulator gene betI, which is important for the survival of B. melitensis 16M under hyperosmotic stress. Strikingly, this site autoregulates its expression to benefit biofilm bacteria's survival under hyperosmotic stress. Moreover, biofilm reduction in ftcR mutants is independent of the flagellar target gene fliF. Collectively, our study provides new insights into the extent and functionality of flagellar-related transcriptional networks in biofilm formation, and presents phenotypic and evolutionary adaptations that alter the regulation of B. melitensis 16M to confer increased tolerance to hyperosmotic stress.

Arthrostoma leucurus sp. n. (Nematoda: Ancylostomatidae), A New Hookworm Species Isolated from Asian Badger in China.

PURPOSE: To date, ten validated Arthrostoma species were reported. Here, a new hookworm species was found from Asian badger (Meles leucurus). METHODS: Nineteen hookworms (9 males and 10 females) were collected from the small intestine of two Asian badgers in Xinjiang Uygur Autonomous Region, northwestern China. The hookworms were morphologically examined according to key taxonomic characters, such as anterior extremity direction, structures of oral opening (cutting plates or teeth), vulva location, buccal capsule anatomy (integrated or formed by articulating plates), the length of spicule and gubernaculum, number of plates of buccal capsule, and presence or absence of vulvar papillae. RESULTS: The hookworm species from Asian badger, here named as Arthrostoma leucurus sp. n., was different from the previously described ten Arthrostoma species. The phylogenetic tree based on the cox1 gene showed that Arthrostoma leucurus sp. n. formed a separate clade, as a sister group to Ancylostoma and Uncinaria species. CONCLUSION: Arthrostoma leucurus sp. n., the eleven validated Arthrostoma species, was identified from Asian badger in China.

Establishment and characterization of a sheep endometrial epithelial cell line.

Endometrial epithelial cells play a significant role in the "dialogue" between the embryo and the mother, but in vitro studies to clarify this are hampered by the limited lifespan of primary cells. As such, it is necessary to develop an in vitro model to study endometrial function. Morphological analysis showed that the pEECs were homogeneous, formed characteristic cobblestone monolayers, and expressed the epithelial cell-specific marker, cytokeratin 18. The isolated and purified cells were transfected with a plasmid encoding human telomerase reverse transcriptase (TERT) gene, pCI-neo-TERT, to establish an immortal endometrial epithelial cell line (iEECs). The transfected cells were cultured with G418 and monoclonal cells were selected for expanded culture. Expression of TERT mRNA was detected by RT-qPCR and protein was quantitated by Western blot. TERT expression was stable and continued to be active with no signs of aging. Assays for cell proliferation and apoptosis indicated higher proliferation and cellular activity in iEECs than pEECs. After stimulated by interferon tau (IFN-τ), both iEECs and pEECs showed similar upregulation levels in all the underlying genes. Taken together, these findings demonstrate that iEECs retained the basic morphology and function of pEECs, providing a robust in vitro model for study of the function of ovine endometrial epithelial cells.

Morphological and molecular analyses of Taenia and Mesocestoides species from red foxes (Vulpes vulpes) in northwestern China.

In this study, 263 tapeworms were collected from eight road-killed red foxes in Xinjiang Uygur Autonomous Region (XUAR, northwestern China). The tapeworms were analyzed based on morphological characters and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequences. Eighty-three Taenia and 180 Mesocestoides tapeworms were identified according to the presence or absence of rostellum, and the number, the length and the shape of the large rostellar hooks. The morphological and molecular analyses revealed that i) 180 Mesocestoides tapeworms, here named as Mesocestoides sp. (Vulpes vulpes), showed 99.21% (378/381 bp) identity to Mesocestoides sp. reported from red fox in Mongolia; and ii) 83 Taenia tapeworms belonged to three species. The first Taenia species [n = 16, named as Taenia sp. (Vulpes vulpes)], based on the length of large rostellar hooks (337-342 µm) and its cox1 sequence, was identified as a potentially novel species, which is phylogenetically close to Taenia laticollis. The second species [n = 54, named as Taenia sp. (Vulpes vulpes & Rhombomys opimus)], was morphologically similar to Taenia endothoracicus according to the number (n = 52), the length (319-332 µm) and the shape of the large rostellar hooks. This species, infecting three red foxes, shared 100% cox1 sequence identity with Taenia sp. (Rhombomys opimus) genotype C found previously in great gerbils (Rhombomys opimus) in the same region. The third species (n = 13, named as Taenia polyacantha-like), had shorter large rostellar hooks (178-180 µm) and showed 96.27% (361/375 bp) sequence identity to Taenia polyacantha reported from red fox in Italy. The "great gerbil-red fox" life cycle of Taenia sp. (Vulpes vulpes & Rhombomys opimus), belonging to the mitochondrial lineage of T. endothoracicus, is confirmed. The T. polyacantha-like species was firstly found in red fox in China. Taenia sp. (Vulpes vulpes) is a potentially novel species, which is close to T. laticollis based on its phylogenetic properties.

Taenia laticollis and a potentially novel Taenia species from the Eurasian lynx (Lynx) in Northwestern China.

The Eurasian lynx (Lynx) is a medium-sized wild cat species distributed throughout Eurasia. There has been no report on Taenia species (Cestoda: Cyclophyllidea) infecting this felid in China. In this study, 24 tapeworms were found in two Eurasian lynxes (#1 and #2) in Xinjiang Uygur Autonomous Region (XUAR), northwestern China. Based on the number, measurements and the shape of rostellar hooks, these tapeworms belong to two Taenia species. According to the number (n = 32) and length (185-194 µm) of small hooks, the first Taenia species (n = 1, found in #2 lynx) was identified as Taenia laticollis. Phylogenetically, this species was clustered with T. laticollis genotype C (JX860623) based on its cytochrome c oxidase subunit 1 (cox1) and 16S rDNA sequences. The second Taenia species (n = 23, provisionally named as "Taenia sp.") may represent a potentially novel tapeworm species, because of its obvious differences in the shape and lengths (174-182 µm, 98-113 µm) of large and small rostellar hooks in comparison with ten taxonomically related species. Molecular and phylogenetic analyses of the cox1 gene revealed that "Taenia sp." has the highest rate of sequence identity (92.93%, 368/396 bp) with Taenia hydatigena reported from sheep (Ovis aries) in Slovakia. To sum up, a potentially novel tapeworm species, "Taenia sp.", is found in Eurasian lynx. In addition, T. laticollis was found for the first time in China.

Rickettsiae in red fox (Vulpes vulpes), marbled polecat (Vormela peregusna) and their ticks in northwestern China.

BACKGROUND: Previously, twelve Rickettsia species were identified in ticks, fleas, sheep keds (Melophagus ovinus), bats (Pipistrellus pipistrellus) and a tick-bitten patient in the Xinjiang Uygur Autonomous Region (XUAR) in northwestern China. Here we aimed to molecularly detect rickettsial agents in red fox (Vulpes vulpes), marbled polecat (Vormela peregusna) and their ticks. METHODS: During 2018-2019, 12 red foxes, one marbled polecat and their ticks were sampled in two counties and a city of the XUAR. The heart, liver, spleen, lung and kidney of these 13 carnivores were dissected, followed by DNA extraction. Hard ticks were identified both morphologically and molecularly. All samples were examined for the presence of rickettsiae by amplifying four genetic markers (17-kDa, gltA, ompA, sca1). RESULTS: A total of 26 adult ticks and 28 nymphs (38 Ixodes canisuga, nine Ixodes kaiseri, six Haemaphysalis erinacei and one Dermacentor marginatus) were collected from red foxes, and four Ha. erinacei ticks were removed from the marbled polecat. Analysis of cytochrome c oxidase subunit I (COI) gene sequences indicated that 2-32 nucleotides differed between I. canisuga, I. kaiseri and Ha. erinacei from northwestern China and Europe. Rickettsia raoultii was detected in three red foxes, Candidatus Rickettsia barbariae in a red fox, Rickettsia sibirica in a red fox and a marbled polecat, and R. raoultii in two tick species (I. canisuga and D. marginatus). CONCLUSIONS: To the best of our knowledge, I. canisuga and I. kaiseri have not been previously reported from red foxes in China. The DNA of R. sibirica and R. raoultii was detected for the first time in the organs of red foxes, and R. sibirica in the organs of a marbled polecat. This is also the first molecular evidence for the presence of R. raoultii in I. canisuga. Our findings expand the range of tick-borne pathogens in wildlife species and associated ticks in China.

Effects of berberine on the growth performance, antioxidative capacity and immune response to lipopolysaccharide challenge in broilers.

This study evaluated the effects of berberine on growth performance, immunity, haematological parameters, antioxidant capacity, and the expression of immune response-related genes in lipopolysaccharide (LPS)-challenged broilers. We assigned 120 one-day-old male broilers (Ross 308) to two treatment groups; each group included two subgroups, each of which included six replicates of five birds per replicate. The experiment used a 2 × 2 factorial arrangement with berberine treatment (0 or 60 mg/kg dietary) and challenge status [injection of saline (9 g/L w/v) or LPS (1.5 mg/kg body weight)] as the main factors. On days 14, 16, 18 and 20, broilers were intraperitoneally injected with LPS or physiological saline. Blood and liver samples were collected on day 21. Dietary berberine supplementation significantly alleviated the compromised average daily gain and average daily feed intake (p < 0.05) caused by LPS. The LPS challenge led to increased lymphocyte and white blood cell (WBC) counts, malondialdehyde (serum and liver) content, and immunoglobulin G and M, tumour necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) expression (p < 0.05) and significantly reduced serum total superoxide dismutase (T-SOD) activity (p < 0.05). Dietary berberine significantly mitigated the LPS-induced decreases in the mRNA expression of nuclear factor-kappa B (NF-κB), TNF-α, IL-1ß, inducible nitrite synthase and cyclooxygenase-2 (p < 0.05) in the liver. In conclusion, berberine supplementation has a positive effect on LPS challenge, which may be related to the increase in antioxidant enzyme activity and inhibition of both NF-κB signalling and the expression of inflammatory mediators.

Dietary leonurine hydrochloride supplementation attenuates lipopolysaccharide challenge-induced intestinal inflammation and barrier dysfunction by inhibiting the NF-κB/MAPK signaling pathway in broilers.

This study was performed to evaluate the beneficial effects of dietary leonurine hydrochloride (LH) supplementation on intestinal morphology and barrier integrity and further illuminate its underlying antioxidant and immunomodulatory mechanisms in lipopolysaccharide (LPS)-treated broilers. A total of 120 1-d-old male broilers (Ross 308) were assigned to 4 treatment groups with 6 replicates of 5 birds per cage. The experiment was designed in a 2 × 2 factorial arrangement with LH (0 or 120 mg/kg) and LPS (injection of saline or 1.5 mg/kg body weight) as treatments. On days 14, 16, 18, and 20 of the trial, broilers were intraperitoneally injected with LPS or physiological saline. Compared with the control group, LPS-challenged broilers showed impaired growth performance (P < 0.05) from day 15 to day 21 of the trial, increased serum diamine oxidase (DAO) and D-lactic acid (D-LA) levels coupled with reduced glutathione (GSH) content and total superoxide dismutase (T-SOD) activity (duodenal and jejunal mucosa), reduced malondialdehyde (MDA) content (duodenal, jejunal, and ileal mucosa), and compromised morphological structure of the duodenum and jejunum. Additionally, LPS challenge increased (P < 0.05) the mRNA expression of proinflammatory cytokine genes and reduced tight junction (TJ) protein expression in the jejunum. However, dietary LH prevented LPS-induced reductions in average daily gain (ADG) and average daily feed intake (ADFI) in broilers. It also alleviated LPS challenge-induced increases in serum DAO levels, MDA content (duodenal and jejunal mucosa), and jejunal crypt depth (P < 0.05) but reduced villus height, GSH content (jejunal mucosa), and T-SOD activity (duodenal and jejunal mucosa) (P < 0.05). Additionally, LH supplementation significantly downregulated the mRNA expression of nuclear factor (NF)-κB, cyclooxygenase-2 (COX-2), and proinflammatory cytokines (TNF-α, IL-1ß, and IL-6) and upregulated the mRNA expression of zonula occludens-1 (ZO-1) and Occludin in the jejunal mucosa induced by LPS (P < 0.05). On the other hand, LH administration prevented LPS-induced activation of the p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) and attenuated IkB alpha (IκBα) phosphorylation and nuclear translocation of NF-κB (p65) in the jejunal mucosa. In conclusion, dietary LH supplementation attenuates intestinal mucosal disruption mainly by accelerating the expression of TJ proteins and inhibiting activation of the NF-κB/MAPK signaling pathway.

The anti-inflammatory and antioxidant effects of leonurine hydrochloride after lipopolysaccharide challenge in broiler chicks.

This study was carried out to investigate the protective effects of leonurine hydrochloride (LH, from Leonurus sibiricus) on lipopolysaccharide (LPS)-stimulated broiler chicks. A total of 120 one-day-old male Ross broilers were randomly divided into 4 treatment groups with 6 replicates of 5 birds per cage. The experiment was designed as a 2 × 2 factorial arrangement with LH (0 or 120 mg/kg) and LPS (injection of saline or 1.5 mg/kg body weight) levels as treatments. On days 14, 16, 18, and 20 of the trial, broilers were intraperitoneally injected with LPS or saline. Blood, spleen, and liver samples were collected on days 21 and 28 for analysis. The results showed that dietary LH had no effect on growth performance or immunoglobulin concentrations in the serum. However, dietary LH prevented LPS-induced reductions in average daily gain and average daily feed intake in the broilers on days 15-21 of the trial (P > 0.05). Dietary LH supplementation dramatically attenuated the LPS-induced increases in the spleen index, reduced glutathione (GSH) activity (serum and liver) and total superoxide dismutase (T-SOD) activity (serum and spleen), and significantly reduced malondialdehyde (MDA) levels (serum, spleen, and liver) on days 21 and 28 (P < 0.05). Additionally, LH supplementation significantly mitigated the LPS-induced increases in the tumor necrosis factor (TNF)-α (serum and spleen), interleukin (IL)-1ß (serum, spleen and liver), IL-2 (liver), IL-6 (serum, spleen and liver), toll-like receptor 4 (TLR4) (spleen and liver), and nuclear factor (NF)-κB (spleen and liver) levels on days 21 and 28 (P < 0.05). Therefore, this study revealed that LH could downregulate the expression of proinflammatory factors, mainly by inhibiting the expression of TLR4 and the activation of NF-κB. LH may be a potential feed additive with dual efficacy as an anti-inflammatory and antioxidant agent.

Selenylation modification: enhancement of the antioxidant activity of a Glycyrrhiza uralensis polysaccharide.

Selenium (Se) is an essential trace element for human beings and many other forms of life. Organic selenium from natural foods has greater bioavailability and is safer than inorganic selenium species. In this article, the structural properties and antioxidant activities of a Glycyrrhiza uralensis polysaccharide (GUP) after selenylation modification were investigated. The GUP was extracted by water decoction and ethanol precipitation and purified via protein elimination using the trichloroacetic acid method and column chromatography. The purified product was subsequently modified by the nitric acid-sodium selenite (HNO3-Na2SeO3) method. The selenized GUP (SeGUP) product was characterized by Fourier transform-infrared (FT-IR) spectroscopy, and its thermal stability, particle size, and antioxidant activities were investigated. FT-IR analysis indicated that the selenium in SeGUP existed mainly as O-Se-O. The thermal stability and particle size of SeGUP differed significantly from those of GUP. Moreover, compared to GUP, SeGUP exhibited greater antioxidant activities in vitro and in vivo. These results indicate that selenylation modification significantly enhances the antioxidant activity of SeGUP, increasing its potential for application as an antioxidant. Graphical abstract ᅟ.

Optimized Extraction, Preliminary Characterization, and In Vitro Antioxidant Activity of Polysaccharides from Glycyrrhiza Uralensis Fisch.

BACKGROUND This study performed optimized extraction, preliminary characterization, and in vitro antioxidant activities of polysaccharides from Glycyrrhiza uralensis Fisch. MATERIAL AND METHODS Three parameters (extraction temperature, ratio of water to raw material, and extraction time) were optimized for yields of G. uralensis polysaccharides (GUP) using response surface methodology with Box-Behnken design (BBD). The GUP was purified using DEAE cellulose 32-column chromatography. The main fraction obtained from G. uralensis Fisch was GUP-II, which was composed of rhamnose, arabinose, galactose, and glucose monosaccharide, was screened for antioxidant properties using DP Hand hydroxyl radical scavenging assays. In addition, immunological activity of GUP-II was determined by nitric oxide and lymphocyte proliferation assays. RESULTS Optimization revealed maximum GUP yields with an extraction temperature of 99°C, water: raw material ratio of 15: 1, and extraction duration of 2 h. GUP-II purified from G. uralensis Fisch had good in vitro DPPH and hydroxyl radical scavenging abilities. Immunologically, GUP-II significantly stimulated NO production in RAW 264.7 macrophages, and significantly enhanced LPS-induced lymphocyte proliferation. CONCLUSIONS Extraction of GUP from G. uralensis Fisch can be optimized with respect to temperature, extraction period, and ratio of water to material, using response surface methodology. The purified product (GUP-II) possesses excellent antioxidant and immunological activities.

Effect of Glycyrrhiza uralensis Fisch polysaccharide on growth performance and immunologic function in mice in Ural City, Xinjiang.

OBJECTIVE: To discuss the effect of Glycyrrhiza uralensis (G. uralensis) Fisch polysaccharide on growth performance and immunologic function in mice in Ural City, Xinjiang and to provide important data supporting the application of Glycyrrhiza polysaccharide. METHODS: A total of 100 Kunming mice aged 3 weeks old were randomly divided into 5 groups with 20 mice in each group (10 were females and 10 were males). About 0.5 mL normal saline was given to the mice of control group every day and 0.5 mL G. uralensis Fisch polysaccharide was given to the mice of other groups at the concentration of 1, 20, 50 and 100 mg/mL, respectively. The growth performance (average body weight, average daily feed intake and feed efficiency), immune organ indexes (spleen index and thymus index) and immunologic function (serum IL-2, CD4+/CD8+ and the activity of NK cells) of mice in each group were detected continuously. RESULTS: The average body weight, feed efficiency, serum IL-2, CD4+/CD8+ and the activity of NK cells of mice were increased with the increase of administrated time after administrating G. uralensis Fisch polysaccharide and were reached up the largest level on Day 28. At the same time, each index was proportional to the given dose and was significantly higher than those of control group and reached up the largest level at the administrated dose of 100 mg/mL. After administrating G. uralensis Fisch polysaccharide, the spleen index and thymus index of mice were increased with the increase of administrated dose and the spleen index and thymus index of mice administrated with the dose of 100 mg/mL were maximum which was more than 1.51 times and 1.43 times of that in control group, respectively and the comparative differences showed statistical significance (P < 0.05). The average daily feed intake of mice in each group was increased with the passage of time and at the same time, the comparison of average daily feed intake of mice in each group was not significantly different (P > 0.05). CONCLUSIONS: G. uralensis Fisch polysaccharide can significantly improve the growth performance and immunologic function of mice and laid a research basis for the clinical application of G. uralensis Fisch polysaccharide.

Expression and regulation of the ery operon of Brucella melitensis in human trophoblast cells.

Brucellosis is primarily a disease of domestic animals in which the bacteria localizes to fetal tissues such as embryonic trophoblast cells and fluids containing erythritol, which stimulates Brucella spp. growth. The utilization of erythritol is a characteristic of the genus Brucella. The ery operon contains four genes (eryA, eryB, eryC and eryD) for the utilization of erythritol, and plays a major role in the survival and multiplication of Brucella spp. The objective of the present study was to conduct a preliminary characterization of differential genes expression of the ery operon at several time points after Brucella infected embryonic trophoblast cells (HPT-8 cells). The result showed that the ery operon expression was higher in HPT-8 cells compared with the medium. The relative expression of eryA, eryB and eryC peaked at 2 h post-infection in HPT-8 cells, and eryD expression peaked at 3 h post-infection. The expression of eryA, eryB and eryC may be inhibited by increased eryD expression. However, the expression of the ery operon was stable in the presence of erythritol in cells. 2308Δery and 027Δery mutants of the ery operon were successfully constructed by homologous recombination, which were attenuated in RAW 264.7 murine macrophages. The characterization of the ery operon genes and their expression profiles in response to Brucella infection further contributes to our understanding of the molecular mechanisms of infection and the pathogenesis of brucellosis.

Loop-mediated isothermal amplification assay targeting the mpb70 gene for rapid differential detection of Mycobacterium bovis.

Loop-mediated isothermal amplification (LAMP) is a highly sensitive, rapid, cost-effective nucleic acid amplification method. Tuberculosis (TB) is widely popular in the world and it is difficult to cure. The fundamental treatment is to clear the types of TB pathogens such as Mycobacterium bovis (M. bovis), Mycobacterium tuberculosis (M. tuberculosis). In order to detect and diagnose TB early, we constructed the differential diagnostic method of TB. In this study, we used LAMP for detection of M. bovis, based on amplification of the mpb70 gene which is a unique gene in M. bovis strain. The LAMP assay was able to detect only seven copies of the gene per reaction, whereas for the conventional PCR, it was 70 copies. The LAMP was evaluated for its specificity using six strains of five Mycobacterium species and 18 related non-Mycobacterium microorganism strains as controls. The target three Mycobacterium strains were all amplified, and no cross-reaction was found with 18 non-Mycobacterium microorganism strains. TB was detected by two methods, LAMP and conventional PCR (based on mpb70 gene); the positive rates of the two methods were 9.55 and 7.01 %, respectively. Our results indicate that the LAMP method should be a potential tool with high convenience, rapidity, sensitivity and specificity for the diagnosis of TB caused by M. bovis. Most importance is that the use of LAMP as diagnostic method in association with diagnostic tests based on mpb70 gene would allow the differentiation between M. bovis and other Mycobacterium in humans or animals. The LAMP method is actually in order to detect human TB, and it can be used for differential diagnosis in this paper.

Effects of dietary protein and calcium on thymus apoptosis induced by fluoride in female rats (Wistar rats).

Our previous studies showed that excessive fluoride (F) ingestion seriously damaged the nonspecific immune function in rabbits. However, the underlying mechanisms of the F-induced damage to the immune system are unclear. The purpose of this study was to investigate whether F induces thymus apoptosis in female rats and its underlying mechanisms by monitoring ultrastructural changes and DNA fragmentation. The results showed that excessive F induced ultrastructural changes and significantly increased the tail length and tailing ratio in thymus lymphocytes. Protein (Pr) supplementation markedly decreased the tailing ratio in thymus lymphocytes in the case of malnutrition. Furthermore, molecular analysis showed that excessive F ingestion significantly up-regulated the expression levels of caspase-3 and caspase-9 mRNA, whereas Pr and calcium (Ca) supplementation down-regulated the expression levels induced by excessive F in the case of malnutrition. In conclusion, these results indicate that excessive F up-regulates the expression levels of caspase-3 and caspase-9 mRNA and induces thymus apoptosis in female rats. Pr and Ca play key roles in process of F-induced thymus apoptosis in malnourished female rats.