Farmland nutrient pollution and its evolutionary relationship with plantation economic development in China.

Contradiction between growing plantation economic demand and agro-ecological degradation has always restricted sustainable development of agricultural countries. This study applied the unit inventory analysis to evaluate the productions and discharges of farmland non-point source (FNPS) nitrogen (TN) and phosphorus (TP) among China's nine national-level agricultural districts over 1999-2019. On this basis, we quantified the evolutionary relationship between plantation economic output and FNPS pollution based on optimal regression fitting. The results showed that over 1999-2019, farmland cumulative TN and TP discharges for the whole China were approximately 15807 × 104 t and 1312 × 104 t, with prominent district heterogeneity. According to FNPS discharge magnitudes, China's agricultural districts can be classified into three categories: high, moderate and slight discharge zones. Huang-Huai-Hai Plain and Middle-lower Yangtze Plain were identified as the main severely-polluted districts. Mineral fertilizer is the primary contributor to FNPS pollution. Annual FNPS load showed a trend of increasing followed by decreasing, and the peak interval was recorded in 2014-2016. Spatiotemporal dynamics in FNPS discharge intensities were disparate from that in discharge magnitudes. SC has the highest TN discharge intensity, with an annual average intensity of 0.068 t/ha, followed by MLYP (0.044 t/ha) and HHHP (0.041 t/ha). HHHP has the highest TP discharge intensity, with an annual average intensity of 0.0051 t/ha, followed by SC (0.0038 t/ha) and MLYP (0.0031 t/ha). District-based agro-ecological restoration strategies were accordingly proposed considering FNPS discharge magnitude and intensity concurrently. In most agricultural districts, with the growing economic output in plantation, the FNPS load showed an increase followed by a decrease or to leveling off. Furthermore, with the increasing TN/TP economic partial productivity, the FNPS TN/TP discharge intensities reached the climax, then declined or tended to be flattening out.

Antiviral activity and mechanism of the antifungal drug, anidulafungin, suggesting its potential to promote treatment of viral diseases.

BACKGROUND: The severe fever with thrombocytopenia syndrome disease (SFTS), caused by the novel tick-borne SFTS virus (SFTSV), was listed among the top 10 priority infectious disease by World Health Organization due to the high fatality rate of 5-30% and the lack of effective antiviral drugs and vaccines and therefore raised the urgent need to develop effective anti-SFTSV drugs to improve disease treatment. METHODS: The antiviral drugs to inhibit SFTSV infection were identified by screening the library containing 1340 FDA-approved drugs using the SFTSV infection assays in vitro. The inhibitory effect on virus entry and the process of clathrin-mediated endocytosis under different drug doses was evaluated based on infection assays by qRT-PCR to determine intracellular viral copies, by Western blot to characterize viral protein expression in cells, and by immunofluorescence assays (IFAs) to determine virus infection efficiencies. The therapeutic effect was investigated in type I interferon receptor defective A129 mice in vivo with SFTSV infection, from which lesions and infection in tissues caused by SFTSV infection were assessed by H&E staining and immunohistochemical analysis. RESULTS: Six drugs were identified as exerting inhibitory effects against SFTSV infection, of which anidulafungin, an antifungal drug of the echinocandin family, has a strong inhibitory effect on SFTSV entry. It suppresses SFTSV internalization by impairing the late endosome maturation and decreasing virus fusion with the membrane. SFTSV-infected A129 mice had relieving symptoms, reduced tissue lesions, and improved disease outcomes following anidulafungin treatment. Moreover, anidulafungin exerts an antiviral effect in inhibiting the entry of other viruses including SARS-CoV-2, SFTSV-related Guertu virus and Heartland virus, Crimean-Congo hemorrhagic fever virus, Zika virus, and Herpes simplex virus 1. CONCLUSIONS: The results demonstrated that the antifungal drug, anidulafungin, could effectively inhibit virus infection by interfering with virus entry, suggesting it may be utilized for the clinical treatment of infectious viral diseases, in addition to its FDA-approved use as an antifungal. The findings also suggested to further evaluate the anti-viral effects of echinocandins and their clinical importance for patients with infection of viruses, which may promote therapeutic strategies as well as treatments and improve outcomes pertaining to various viral and fungal diseases.

Urban expansion dynamic and its potential effects on dry-wet circumstances in China's national-level agricultural districts.

Although urbanization has been widely examined in individual city and urban agglomeration scales, urban expansion patterns and dynamics in large-scale agricultural districts remain absent. In this study, multifaceted characteristics in urban expansion were quantified in China's nine national-level agricultural districts, and responses of dry-wet circumstances to urban sprawl were evaluated. From 1980 to 2018, China has undergone an extensive urban sprawl. Huang-Huai-Hai Plain (HHHP) has the maximum urban coverage extent, followed by Middle-lower Yangtze Plain (MLYP) and Southern China (SC). The largest annual increase was recorded in MLYP, reaching 816.12 km2; followed by HHHP, with an annual increase of 725.22 km2. There are prominent heterogeneities in expansion rate and direction among various districts. The dominating growth patterns were edge- and leapfrogging-expansion, accompanying by a less percentage of infilling-expansion. Accompanying by urbanization, connectedness in urban landscapes gradually improved, while separation degree decreased. Upon many occasions, holistic average dry-wet circumstances in non-urbanized areas are superior to those in urban areas, although this is not absolute for all the districts or periods. In urbanization progress, the development of leapfrogging-expansion has a potential to ameliorate dry-wet circumstances in both urban and non-urban zones, while infilling- and edge-expansion would constitute an inverse effect. In comparison to urban zones, leapfrogging-expansion would cause a more prominent effect on dry-wet environment in non-urbanized zones. Increased connectivity in urbanized landscapes would improve dry-wet environments, especially for urbanized zones. Inversely, increased spatial separated extent among urban landscapes would perform an opposite effect. This study provides a potential for understanding the dynamic features of urban expansion in large-scale agricultural districts. Moreover, the results can also provide a potential opportunity for optimizing dry-wet environments by regulating urbanization pattern and landscape configuration.

Progress on Poxvirus E3 Ubiquitin Ligases and Adaptor Proteins.

Poxviruses have evolved a variety of innate immunity evasion mechanisms, some of which involve poxvirus-encoded E3 ubiquitin ligases and adaptor proteins. Based on their functional domains and ubiquitin transfer mechanisms, these poxvirus-encoded E3 ubiquitin ligases and adaptor proteins can be divided into five categories: PRANC, ANK/BC, BBK, P28/RING, and MARCH proteins. Although the substrates of many poxvirus E3 ubiquitin ligases remain to be discovered, most of the identified substrates are components of the innate immune system. In this review, we discuss the current research progress on poxvirus-encoded E3 ubiquitin ligases and adaptor proteins to provide mechanistic insights into the interplay between these viruses and their hosts.

Tomato SlPUB24 enhances resistance to Xanthomonas euvesicatoria pv. perforans race T3.

Solanum lycopersicum var. cerasiforme accession PI 114490 has broad-spectrum resistance to bacterial spot caused by several species of Xanthomonas. Resistance is quantitatively inherited, and a common quantitative trait locus QTL-11B on chromosome 11 has been identified previously. In this study, the SlPub24 gene was characterized in QTL-11B. SlPub24 in PI 114490 was upregulated by infection with X. euvesicatoria pv. perforans race T3, but its transcription was low in the susceptible line OH 88119 whether or not it was infected by the pathogen. The differential expression of SlPub24 between PI 114490 and OH 88119 was due to great sequence variation in the promoter region. The promoter of SlPub24 in OH 88119 had very low activity and did not respond to pathogen infection. Transgenic lines of OH 88119 overexpressing SlPub24 isolated from PI 114490 showed significantly enhanced resistance, while mutants of Slpub24 generated by CRISPR/Cas9 editing showed more susceptibility to race T3 and to other races. The mutants also showed spontaneous cell death in leaves. The expression of the salicylic acid (SA) pathway gene phenylalanine ammonia-lyase (PAL) and signaling-related genes pathogenesis-related (PR1) and nonexpresser of PR1 (NPR1) were influenced by SlPub24. The content of SA in tomato plants was consistent with the level of SlPub24 expression. Furthermore, SlPUB24 interacted with the cell wall protein SlCWP and could regulate the degradation of SlCWP. The expression levels of SlCWP and SlCWINV1, a cell wall invertase gene, showed opposite patterns during pathogen infection. The activity of SlCWINV1 was lower in mutants than in PI 114490. The results are discussed in terms of the roles of the abovementioned genes, and a potential model for SlPUB24-mediated resistance to bacterial spot is proposed.

Tomato protein Rx4 mediates the hypersensitive response to Xanthomonas euvesicatoria pv. perforans race T3.

Bacterial spot, which is caused by several Xanthomonas species, is an economically important disease in tomato (Solanum lycopersicum). Great efforts have been made for the identification of resistant sources and the genetic analysis of resistance. However, the development of resistant commercial varieties is slow due to the existence of multiple species of the pathogen and a poor understanding of the resistance mechanism in tomato. The current study revealed that the Rx4 gene encodes a nucleotide-binding leucine-rich repeat protein in the wild tomato species Solanum pimpinellifolium and specifically recognizes and confers a hypersensitive response (HR) to Xanthomonas euvesicatoria pv. perforans race T3 expressing the AvrXv3 avirulence protein. Complementation of the Rx4 gene in the susceptible tomato line Ohio 88119 using a transgenic approach resulted in HR, whereas knockout of the gene through CRISPR/Cas9 editing in resistant lines Hawaii 7981 and PI 128216 led to non-HR to race T3. Transcription of Rx4 was not induced by the presence of race T3. Furthermore, the Rx4 protein did not show physical interaction with AvrXv3 but interacted with SGT1-1 and RAR1. Virus-induced gene silencing of SGT1-1 and RAR1 in the resistant line PI128216 suppressed the HR to race T3. Taken together, our study confirms Rx4 is the gene conferring the HR to bacterial spot race T3 and reveals the potential roles of SGT1-1 and RAR1 as signals in the Rx4-mediated HR. This discovery represents a step forward in our understanding of the mechanism of resistance to bacterial spot in tomato and may have important implications for understanding plant-bacterial interactions.

Oxidative damage mechanism in Saccharomyces cerevisiae cells exposed to tetrachlorobisphenol A.

Tetrachlorobisphenol A (TCBPA) can promote intracellular reactive oxygen species (ROS) accumulation. However, limited attention has been given to mechanisms underlying TCBPA exposure-associated ROS accumulation. Here, such mechanisms were explored in the simple eukaryotic model organism Saccharomyces cerevisiae exposed to multiple concentrations of TCBPA. Addition of diphenyleneiodonium, a specific inhibitor of NADPH oxidase, blocked TCBPA treatment-associated intracellular ROS accumulation. NADPH oxidase can be activated by calcineurin, mitogen-activated protein kinase (MAPK), and tyrosine kinase. Therefore, corresponding specific inhibition respectively on these three kinases was performed and results suggested that the Ca2+ signaling pathway, MAPK pathway, and tyrosine kinase pathway all contributed to the TCBPA exposure-associated intracellular ROS accumulation. In addition, TCBPA exposure-associated up-regulation of genes involved in ROS production and down-regulation of catalase promoted ROS accumulation in S. cerevisiae. To sum up, our current results provide insights into the understanding of TCBPA exposure-associated ROS accumulation.

Regulation of trehalose, a typical stress protectant, on central metabolisms, cell growth and division of Saccharomyces cerevisiae CEN.PK113-7D.

Trehalose could protect the typical food microorganism Saccharomyces cerevisiae cell against environmental stresses; however, the other regulation effects of trehalose on yeast cells during the fermentation are still poorly understood. In this manuscript, different concentrations (i.e., 0, 2 and 5% g/v) of trehalose were respectively added into the medium to evaluate the effect of trehalose on growth, central metabolisms and division of S. cerevisiae CEN.PK113-7D strain that could uptake exogenous trehalose. Results indicated that addition of trehalose could inhibit yeast cell growth in the presence or absence of 8% v/v ethanol stress. Exogenous trehalose inhibited the glucose transporting efficiency and reduced intracellular glucose content. Simultaneously, increased intracellular trehalose content destroyed the steady state of trehalose cycle and caused the imbalance between the upper glycolysis part and the lower part, thereby leading to the dysfunction of glycolysis and further inhibiting the normal yeast cell growth. Moreover, energy metabolisms were impaired and the ATP production was reduced by addition of trehalose. Finally, exogenous trehalose-associated inhibition on yeast cell growth and metabolisms delayed cell cycle. These results also highlighted our knowledge about relationship between trehalose and growth, metabolisms and division of S. cerevisiae cells during fermentation.

Application of a cellulose filter aid in municipal sewage sludge dewatering and drying: Jar, pilot, and factory scale.

Cellulose filter aid (CFA), a skeleton builder, has been studied for sludge conditioning at laboratory scale because of its function in reducing compressibility of sludge cake. However, there are few practical applications of CFA at the pilot and factory scales. In this study, we combined CFA with an advanced sludge dewatering technology, in practical applications. For jar test, the optimum CFA dose was 3 wt.% dry solid (DS); furthermore, the combination of CFA with polyaluminum chloride (PAC) and polyacrylamide (PAM) resulted in the lowest moisture content (73.92%) and the highest calorific value (13,628 kJ/kg) of the sludge cake. In the pilot test, the 5 wt.% DS of the CFA resulted in the optimal sludge feeding amount. In the factory test, the optimal CFA dose was further increased to 10%, and the amount of sludge feeding in a single batch was 20.5% higher than that without CFA. The optimal dose of CFA in the jar-pilot-factory test increased, which may occur because the CFA more easily penetrated the cake and performed better in building the skeleton when the sludge cake was thin. This study could provide a reference for the application of CFA and other skeleton builders in sludge dewatering and drying. PRACTITIONER POINTS: An advanced sludge treatment process-low-temperature vacuum dewatering and drying was tested. Cellulose filter aid showed good effect in jar test, pilot test, and factory test. When the scale of the experiment was from jar to factory, the optimal dosage of the cellulose filter aid was magnified. The amplification phenomenon of optimal dosage was related to the distribution pattern of cellulose filter aid in different thickness mud cakes.

Maghemite (γ-Fe2O3) nanoparticles enhance dissimilatory ferrihydrite reduction by Geobacter sulfurreducens: Impacts on iron mineralogical change and bacterial interactions.

Microbially mediated bioreduction of iron oxyhydroxide plays an important role in the biogeochemical cycle of iron. Geobacter sulfurreducens is a representative dissimilatory iron-reducing bacterium that assembles electrically conductive pili and cytochromes. The impact of supplementation with γ-Fe2O3 nanoparticles (NPs) (0.2 and 0.6 g) on the G. sulfurreducens-mediated reduction of ferrihydrite was investigated. In the overall performance of microbial ferrihydrite reduction mediated by γ-Fe2O3 NPs, stronger reduction was observed in the presence of direct contact with γ-Fe2O3 NPs than with indirect contact. Compared to the production of Fe(II) derived from biotic modification with ferrihydrite alone, increases greater than 1.6- and 1.4-fold in the production of Fe(II) were detected in the biotic modifications in which direct contact with 0.2 g and 0.6 g γ-Fe2O3 NPs, respectively, occurred. X-ray diffraction analysis indicated that magnetite was a unique representative iron mineral in ferrihydrite when active G. sulfurreducens cells were in direct contact with γ-Fe2O3 NPs. Because of the sorption of biogenic Fe(II) onto γ-Fe2O3 NPs instead of ferrihydrite, the addition of γ-Fe2O3 NPs could also contribute to increased duration of ferrihydrite reduction by preventing ferrihydrite surface passivation. Additionally, electron microscopy analysis confirmed that the direct addition of γ-Fe2O3 NPs stimulated the electrically conductive pili and cytochromes to stretch, facilitating long-range electron transfer between the cells and ferrihydrite. The obtained findings provide a more comprehensive understanding of the effects of iron oxide NPs on soil biogeochemistry.

Improved bauxite residue dealkalization by combination of aerated washing and electrodialysis.

Bauxite residue, a major by-product of the alumina-producing Bayer process, is a serious environmental pollutant due to its high alkalinity. Here, we reported an operation system designed in our laboratory that included washing and electrodialysis dealkalization systems with aeration pipes. Washing with aeration releases a substantial amount of free alkali and attached alkali into water and increases the dealkalization efficiency. The washing liquid was treated with five steps of batch-mode electrodialysis. The average removal of total dissolved solids (TDS) after the aeration and non-aeration electrodialysis processes were 61.30% and 39.61%, respectively. The average removal of OH- under aeration conditions was 76.62%, a value that was greater than the value produced under non-aeration conditions (68.48%). This efficiency was also higher than that of some other reports (64.90-68.50%). Aeration decreased the energy consumption to a greater extent than the non-aeration condition. NaOH was recovered in terms of the concentration chamber, and the NaAl(OH)4 present in the dilution chamber was separated for the electrodialysis treatment. Membrane scaling was generated to a lesser amount under aeration conditions than that of non-aeration conditions, which would improve the dealkalization efficiency. The high repeatability of the experiments was indicated by the intraclass correlation coefficient (P < 0.05).

Using drug-loaded pH-responsive poly(4-vinylpyridine) microspheres as a new strategy for intelligent controlling of Lactobacillus plantarum contamination in bioethanol fermentation.

Bioethanol fermentation is usually contaminated by bacteria, especially lactic acid bacteria (LAB), thereby leading to decrease of bioethanol yield and serious economic losses. Nisin is safer for controlling of bacterial contamination than antibiotics that are widely applied in industry. Moreover, in LAB contaminative bioethanol fermentation system, consistently decreased pH value provides opportunity to realize pH value responsive material-based release of anti-bacteria substances for intelligent and persistent controlling of bacterial contamination. In this study, nisin was embedded into pH-sensitive poly(4-vinylpyridine) (P4VP) microspheres synthesized by suspension polymerization to realize intelligent controlling of Lactobacillus plantarum contamination in bioethanol fermentation. Chloramphenicol with the highest antimicrobial activity and excellent stability was chosen as the model drug to be embedded into P4VP microspheres to test the drug release behavior. The drug release curve of chloramphenicol-loaded P4VP microspheres showed sustained and pH-responsive release properties. The diameters of the microspheres ranged from 40 to 100 µm. The encapsulation efficiency of nisin into P4VP microspheres was 47.67% and the drug-loading capacity of nisin was 2.5%. Nisin-loaded P4VP microspheres were added into the simulated contaminative fermentation system, and successfully reversed the decline of bioethanol yield secondary to L. plantarum contamination. The results in this study indicated that L. plantarum contamination in bioethanol fermentation could be effectively controlled by nisin-loaded P4VP microspheres.

Oxidative stress and cytotoxicity induced by tetrachlorobisphenol A in Saccharomyces cerevisiae cells.

Tetrachlorobisphenol A (TCBPA), which is widely used as flame retardant, can be released into various environments, thereby being absorbed by wildlife or human beings through food chain's bio-magnification and causing some adverse influences on wildlife or human beings. However, limited data are currently available on TCBPA-associated cytotoxicity and related mechanisms. Here, the cytotoxicity induced by different concentrations of TCBPA (i.e., 5, 10 and 20 µM) was studied using Saccharomyces cerevisiae, a simple eukaryotic model organism. TCBPA treatment inhibited the growth and survival rate of yeast cell in a dose-dependent manner. Moreover, TCBPA promoted the increasing of intracellular oxidative stress by enhancing accumulation of intracellular reactive oxygen species (ROS). Meanwhile, lipid peroxidation degree (represented by malondialdehyde (MDA) content) and DNA damage degree (represented by 8-hydroxy deoxyguanosine (8-oxodG) content) in yeast cell also increased after TCBPA treatment. However, yeast cell mitochondrial membrane potential (Δψm) decreased after TCBPA treatment. It was noteworthy that there was no significant inhibitory effect on yeast cell growth or survival rate in 5 µM TCBPA-treated cells, but the intracellular MDA content and Δψm level changed significantly, suggesting the potential cell damage secondary to the relative low dose of TCBPA exposure. Results presented here would highlight our knowledge about TCBPA-associated cytotoxicity in organisms.

Analysis of EF-Hand Proteins in Soybean Genome Suggests Their Potential Roles in Environmental and Nutritional Stress Signaling.

Calcium ion (Ca2+) is a universal second messenger that plays a critical role in plant responses to diverse physiological and environmental stimuli. The stimulus-specific signals are perceived and decoded by a series of Ca2+ binding proteins serving as Ca2+ sensors. The majority of Ca2+ sensors possess the EF-hand motif, a helix-loop-helix structure which forms a turn-loop structure. Although EF-hand proteins in model plant such as Arabidopsis have been well described, the identification, classification, and the physiological functions of EF-hand-containing proteins from soybean are not systemically reported. In this study, a total of at least 262 genes possibly encoding proteins containing one to six EF-hand motifs were identified in soybean genome. These genes include 6 calmodulins (CaMs), 144 calmodulin-like proteins (CMLs), 15 calcineurin B-like proteins, 50 calcium-dependent protein kinases (CDPKs), 13 CDPK-related protein kinases, 2 Ca2+- and CaM-dependent protein kinases, 17 respiratory burst oxidase homologs, and 15 unclassified EF-hand proteins. Most of these genes (87.8%) contain at least one kind of hormonal signaling- and/or stress response-related cis-elements in their -1500 bp promoter regions. Expression analyses by exploring the published microarray and Illumina transcriptome sequencing data revealed that the expression of these EF-hand genes were widely detected in different organs of soybean, and nearly half of the total EF-hand genes were responsive to various environmental or nutritional stresses. Quantitative RT-PCR was used to confirm their responsiveness to several stress treatments. To confirm the Ca2+-binding ability of these EF-hand proteins, four CMLs (CML1, CML13, CML39, and CML95) were randomly selected for SDS-PAGE mobility-shift assay in the presence and absence of Ca2+. Results showed that all of them have the ability to bind Ca2+. This study provided the first comprehensive analyses of genes encoding for EF-hand proteins in soybean. Information on the classification, phylogenetic relationships and expression profiles of soybean EF-hand genes in different tissues and under various environmental and nutritional stresses will be helpful for identifying candidates with potential roles in Ca2+ signal-mediated physiological processes including growth and development, plant-microbe interactions and responses to biotic and abiotic stresses.

Nano-sized Fe2O3/Fe3O4 facilitate anaerobic transformation of hexavalent chromium in soil-water systems.

The purpose of this study is to investigate the effects of nano-sized or submicro Fe2O3/Fe3O4 on the bioreduction of hexavalent chromium (Cr(VI)) and to evaluate the effects of nano-sized Fe2O3/Fe3O4 on the microbial communities from the anaerobic flooding soil. The results indicated that the net decreases upon Cr(VI) concentration from biotic soil samples amended with nano-sized Fe2O3 (317.1±2.1mg/L) and Fe3O4 (324.0±22.2mg/L) within 21days, which were approximately 2-fold of Cr(VI) concentration released from blank control assays (117.1±5.6mg/L). Furthermore, the results of denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing indicated a greater variety of microbes within the microbial community in amendments with nano-sized Fe2O3/Fe3O4 than the control assays. Especially, Proteobacteria occupied a predominant status on the phylum level within the indigenous microbial communities from chromium-contaminated soils. Besides, some partial decrease of soluble Cr(VI) in abiotic nano-sized Fe2O3/Fe3O4 amendments was responsible for the adsorption of nano-sized Fe2O3/Fe3O4 to soluble Cr(VI). Hence, the presence of nano-sized Fe2O3/Fe3O4 could largely facilitate the mobilization and biotransformation of Cr(VI) from flooding soils by adsorption and bio-mediated processes.

Repetitive extragenic palindromic DNA sequences from Brucella melitensis stimulate Toll-like receptor 9 signaling in macrophages.

Brucella DNA activates the host innate immune system via the intracellular Toll-like receptor 9 (TLR9). However, the Brucella DNA sequences which are responsible for these immunostimulatory effects remain to be elucidated. The present study demonstrated that repetitive extragenic palindromic (REPs) sequences present in Brucella DNA were able to stimulate macrophages through TLR9. The induction of interferon-α (IFN-α) production by Brucella REPs was detected in cultured RAW264.7 mouse macrophages as well as in Wistar rats. Knockdown of TLR9 expression by siRNA in macrophages led to a reduction in IFN-α production following REPs stimulation. In addition, it was confirmed that the activating capacity of Brucella REPs is CpG dependent. Induction of IFN-α by Brucella REPs was completely abrogated when REP sequences were transformed into non-CpG sequences or by C-methylated modifications. Furthermore, it was observed that REPs-initiated TLR9/NF-κB and TLR9/MAPK signaling pathways contributed to the production of IFN-α. The identification of Brucella REPs as natural TLR9 agonists may be useful for the development of novel therapeutic applications.

[The effects of repetitive extragenic palindromic sequences from Brucella melitensis DNA on the toll-like receptor 9-mediated interferon-α production].

OBJECTIVE: To screen the repetitive extragenic palindromic sequences with activation of toll-like receptor 9 (TLR9) activity from Brucella melitensis DNA, providing new ideas and new targets for prevention and treatment of brucellosis. METHODS: Bioinformatics methods were used to detect repetitive extragenic palindromic (REP) sequences from Brucella melitensis DNA. The studied REPs were selected and synthesized. RAW264.7 was cultured and transfected with REPs mediated by lipofectamine 3000. Additionally, TLR9-siRNA was used to downregulate TLR9 expression. The content of interferon-α (IFN-α) in the supernatant was then measured by ELISA. RESULTS: A total of 2 200 REP sequences in Brucella melitensis DNA were identified. Twelve REP sequences were synthesized for further detecting of the TLR9 agonistic activity. IFN-α expression in RAW264.7 treated with M2, M3, M4, M5, M6, M7, M9, M12 were (26.944 ± 1.868), (46.461 ± 2.562), (34.980 ± 2.055), (43.016 ± 2.162), (62.533 ± 4.031), (67.125 ± 5.069), (18.908 ± 1.633), (39.572 ± 2.465) pg/ml respectively, which significantly increased when compared with the negative control group [(12.594 ± 1.338) pg/ml, t=10.817, 20.295, 15.812, 20.724, 20.365, 18.016, 5.180, 16.660, all P<0.05]. Additionally, TLR9-siRNA can significantly decrease the levels of IFN-α in RAW264.7 treated with M6. CONCLUSION: REP sequences presented in Brucella melitensis DNA are able to induce IFN-α expression through TLR9, which can be helpful for the understanding of pathogenesis and immunity of Brucella melitensis.

Polymerase chain reaction-based assays for the diagnosis of human brucellosis.

Polymerase chain reaction (PCR) is an in vitro technique for the nucleic acid amplification, which is commonly used to diagnose infectious diseases. The use of PCR for pathogens detection, genotyping and quantification has some advantages, such as high sensitivity, high specificity, reproducibility and technical ease. Brucellosis is a common zoonosis caused by Brucella spp., which still remains as a major health problem in many developing countries around the world. The direct culture and immunohistochemistry can be used for detecting infection with Brucella spp. However, PCR has the potential to address limitations of these methods. PCR are now one of the most useful assays for the diagnosis in human brucellosis. The aim of this review was to summarize the main PCR techniques and their applications for diagnosis and follow-up of patients with brucellosis. Moreover, advantages or limitation of the different PCR methods as well as the evaluation of PCR results for treatment and follow-up of human brucellosis were also discussed.