Emodin Alcohols: Design, Synthesis, Biological Evaluation and Multitargeting Studies with DNA, RNA, and HSA.

OBJECTIVE: A series of novel emodin alcohols were designed and prepared in an effort to overcome the increasing microorganism resistance. METHODS: Novel emodin alcohols were prepared from commercial emodin and different nitrogen-containing heterocycles via different synthetic strategies, such as O-alkylation and N-alkylation. The antimicrobial activity of synthesized emodin compounds was evaluated in vitro by a two-fold serial dilution technique. The interaction of emodin compound 3d with biomolecule was researched using UV-vis spectroscopic method and fluorescence spectroscopy. RESULTS: Emodin compound 3d containing 2-methyl-5-nitro imidazole ring showed relatively good antimicrobial activity. Notably, it exhibited equivalent activity against S. aureus in comparison to the reference drug norfloxacin (MIC = 4 g/mL). The combination of strong active compound 3d with reference drugs showed better antimicrobial activity with less dosage and a broader antimicrobial spectrum than their separate use. Further research displayed that emodin compound 3d could intercalate into S. aureus DNA to form the 3d-DNA complex, which might correlate with the inhibitory activity. The hydrogen bonds were found between S. aureus DNA gyrase and strong active compound 3d during the docking research, which were in accordance with the spectral experiment results. The interaction with yeast RNA of compound 3d could also form a complex via hydrogen bonds. The hydrogen bonds were found to play a major role in the transportation of emodin compound 3d by human serum albumin (HSA), as confirmed by molecular simulation. CONCLUSION: This work provides a promising starting point to optimize the structures of emodin derivatives as potent antimicrobial agents.

Encapsulation of bacteria in different stratified extracellular polymeric substances and its implications for performance enhancement and resource recovery.

Simultaneous recovery of biopolymers and enhanced bio-reactor performance are promising options for sustainable wastewater treatment, and the bioactivity of sludge after biopolymer extraction is thus critical for the performance of the system. To this end, stratified extracellular polymeric substances (EPS), including slime, loosely bound EPS (LB-EPS), and tightly bound EPS (TB-EPS), were extracted, and the bioactivities of the consequent extraction residues were assessed using aerobic respirogram, kinetic, and flow cytometry (FCM). After the initial weak extraction of slime, the particle size distribution of the sludge significantly decreased, and subsequent extractions of LB-EPS and TB-EPS produced an equivalent size distribution. In contrast, the fractal dimension decreased after each extraction, suggesting that LB-EPS and TB-EPS affected the compactness of flocs rather than the size. The aerobic bacteria distribution estimated using respirogram shows that slime mainly encapsulated heterotrophs while LB-EPS mainly encapsulated nitrifiers. In addition, the ammonia-nitrogen affinity coefficient decreased from 1.79 to 0.28 mg/L when slime was removed, thereby encouraging the activities of autotrophic nitrifiers. Further removal of LB-EPS induced high energy dispersion as the maintenance coefficient m and the metabolic dispersion index µ/m increased from 0.11 to 0.22 and 0.44 to 0.63, respectively. Meanwhile, the yield rate decreased from 0.77 to 0.66. Although pellets that resulted from TB-EPS extraction were not aerobically active as described by respirogram and growth curves, they were still metabolically active as measured by live/dead cell counting and redox sensor green signal. These pellets used more energy for maintenance as indicated by the high maintenance coefficient than those residual after either slime or LB-EPS extraction. In addition, the variation in bacteria community distribution across flocs was related to the variation in temperatures, suggesting that the inner part of a floc might be hotter than the outer side. Therefore, compared to bacteria in the raw sludge, the viable bacteria bounded in LB-EPS and TB-EPS convert more energy to heat rather than growth. These results indicate that energy was dispersed as metabolic heat for the LB-EPS extracted sludge, and removal of LB-EPS favored thermogenesis and sludge reduction. Based on the above findings, a simultaneously EPS-recovery and performance enhancement configuration is thus proposed, which holds great promise for the integration of next-generation wastewater treatment plants.

Investigation on polyphosphate accumulation in the sulfur transformation-centric EBPR (SEBPR) process for treatment of high-temperature saline wastewater.

This study investigated the polyphosphates accumulation rate in a novel sulfur transformation-centric enhanced biological phosphorus removal (SEBPR) process. The SEBPR system was continuously operated over 120 days in a sequencing batch reactor (SBR) that alternated between the anaerobic mode and the anoxic mode of operation (temperature: 30 °C and salinity: 6000 mg/L Cl-). In addition to the SBR, batch experiments were carried out to test the effect of two different sulfate concentrations on the system performance and sulfur-phosphorus transformations. The key intercellular polymers of polyphosphates and polysulfur (poly-S) were identified by employing advanced microscopes. Metagenomic analysis was performed to characterize the diversity of microbes and their functions enriched in the SEBPR system. Finally, several molecular techniques including flow cytometry cell sorting and 16S DNA high-throughput sequencing were applied to identify the phosphorus-accumulating organisms (PAOs). The amounts of P release and P uptake in the SEBPR increased gradually to nearly 18 ±â€¯6.4 mg P/L and 26.5 ±â€¯6.7 mg P/L respectively, yielding a net P removal efficiency of 84 ±â€¯25%. Batch tests indicated no polyhydroxyalkanate (PHA) synthesis, but P uptake was observed and it was correlated with the intracellular poly-S consumption, suggesting that the poly-S could act as an intracellular energy source for P uptake and polyphosphates formation. Moreover, CLSM and TEM micrographs clearly showed the presence of intercellular polyphosphates and poly-S respectively. Metagenomic analysis revealed that Proteobacteria (36.5%), Bacteroidetes (23.3%), Thermotogae (7.1%), Chloroflexi (4.5%) and Firmicutes (2.3%) were the dominant phyla in Bacteria. The conventional PAO of Candidatus Accumulibacter was found at a low abundance of 0.32% only; and an uncultured genus close to Rhodobacteraceae at the family level is speculated to be the putative sulfur PAO (SPAO). Finally, this research suggests that poly-S considerably impacts on polyphosphates accumulation in the SEBPR system when no PHAs are formed.

Multiple-cycle operation of sulphur-cycle-enhanced biological phosphorus removal to maintain stable performance at high temperatures.

This study investigated a new method of multiple-cycle operation of a sulphur-cycle-enhanced biological phosphorus (P) removal system to maintain good phosphorus removal performance at a high temperature (30 °C). The findings demonstrate that P removal was low and unstable under a normal cycle (77 ±â€¯18%), but multiple cycles resulted in a high and quite stable level of P removal (88 ±â€¯9%). Moreover, in the normal mode, the polyhydroxyalkanoate levels increased significantly from 2 to 15 mg C/g of VSS, the glycogen level doubled from 5 to 10 mg C/g of VSS and the polyhydroxyalkanoate and glycogen levels were maintained at considerably low levels after multiple cycles (only 5 C/g of VSS). The 16S rRNA high-throughput sequencing analysis revealed that the genera Thioalbus and Psychrobacter in the gamma-Proteobacteria class were the key functional communities. These findings suggest a high level of P removal with multiple cycles of sulphur-cycle enhanced biological phosphorus removal.

Translocation of enterohemorrhagic Escherichia coli effector Tir to the plasma membrane via host Golgi apparatus.

The translocated intimin receptor (Tir) is a canonical type III secretion system effector, secreted by the enterohemorrhagic Escherichia coli (E. coli). This receptor alters the regular cellular processing of host cells, to promote intracellular bacterial replication and evasion of the host immune system. Tir is translocated and integrated into the host cell plasma membrane, a process required for its pathogenic activity in these cells, however, the underlying mechanisms of how this occurs remain to be elucidated. The present study used immunofluorescence and immunoelectron microscopy to demonstrate that the Tir of enterohemorrhagic E. coli was localized to the plasma membrane and colocalized with the 58K Golgi protein of the host cells. Treatment with brefeldin A destroyed the Golgi structure, inhibited the formation of actin pedestal and blocked the localization of Tir on the host cell plasma membrane. The results of the present study suggested that Tir is translocated to the host plasma membrane in a Golgi­dependent manner. It may mimic the activities of eukaryotic secretory proteins in order to make use of the Golgi apparatus for transportation and integration into the plasma membrane. These findings reveal a novel trafficking pathway for the translocation of bacterial secretory effectors to their specific subcellular compartments.

[Identification and classification of disease severity of wheat stripe rust using near infrared spectroscopy technology].

Wheat stripe rust caused by Puccinia striiformis f. sp. tritici, is an economically important disease in the world. It is of great significance to assess disease severity of wheat stripe rust quickly and accurately for monitoring and controlling the disease. In this study, wheat leaves infected with stripe rust pathogen under different severity levels were acquired through artificial inoculation in artificial climate chamber. Thirty wheat leaves with disease severity equal to 1%, 5%, 10%, 20%, 40%, 60%, 80% or 100% were picked out, respectively, and 30 healthy leaves were chosen as controls. A total of 270 wheat leaves were obtained and then their near infrared spectra were measured using MPA spectrometer. According to disease severity levels, 270 near infrared spectra were divided into 9 categories and each category included 30 spectra. From each category, 7 or 8 spectra were randomly chosen to make up the testing set that included 67 spectra. The remaining spectra were treated as the training set. A qualitative model for identification and classification of disease severity of wheat stripe rust was built using near infrared reflectance spectroscopy (NIRS) technology combined with discriminant partial least squares (DPLS). The effects of different preprocessing methods of obtained spectra, ratios between training sets and testing sets, and spectral ranges on qualitative recognition results of the model were investigated. The optimal model based on DPLS was built using cross verification method in the spectral region of 4000-9000 cm(-1) when "centralization" was used as the preprocessing method of spectra and the spectra were divided into the training set and the testing set with the ratio equal to 3:1. Accuracy rate of the training set was 95.57% and accuracy rate of the testing set was 97.01%. The results showed that good recognition performance could be acquired using the model based on DPLS. The results indicated that the method using near infrared reflectance spectroscopy technology proposed in this study is feasible for identification and classification of disease severity of wheat stripe rust. A new method was provided for monitoring and assessment of wheat stripe rust.

Shiga toxins induce autophagic cell death in intestinal epithelial cells via the endoplasmic reticulum stress pathway.

Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A1, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.

[Detection of Puccinia strii formis f. sp. tritici latent infections in wheat leaves using near infrared spectroscopy technology].

To realize the early detection of P. strii formis f. sp. tritici latent infections in wheat leaves while no disease symptoms appear, a qualitative model for identification of the wheat leaves in the incubation period of stripe rust was built using near infrared reflectance spectroscopy (NIRS) technology combined with qualitative partial least squares (DPLS). In this study, 30 leaf samples infected with P. strii formis f. sp. tritici were collected each day during the eleven-day incubation period. And 30 healthy leaf samples and 30 leaf samples showing disease symptoms infected with P. strii formis f. sp. tritici, were also collected as controls. In total, there were 390 leaf samples that were divided into thirteen categories. Near infrared spectra of 390 leaf samples were obtained using MPA spectrometer and then a model to identify the categories of wheat leaves was built using cross verification method. The effects of different spectral ranges, samples for building the model, preprocessing methods of spectra and number of principal components on NIRS prediction results for qualitative identification were investigated. The optimal identification results were obtained for the model built in the combined spectral region of 5 400-6 600 and 7 600-8 900 cm(-1) when the spectra were divided into the training set and the testing set with the ratio equal to 4 : 1, "scatter correction" was used as the preprocessing method and the number of principal components was 14. Accuracy rate, misjudgment rate and confusion rate of the training set were 95.51%, 1.28% and 3.21%, respectively. And accuracy rate, misjudgment rate and confusion rate of the testing set were 100.00%, 0.00% and 0.00%, respectively. The result showed that using near infrared reflectance spectroscopy technology, P. strii formis f. sp. tritici latent infections in wheat leaves could be detected as early as one day after inoculation (or 11 days before symptoms appearing) and the number of days when the leaf has been infected could also be identified. The results indicated that the method using near infrared reflectance spectroscopy technology proposed in this study is feasible for the identification of wheat leaves latently infected by P. strii formis f. sp. tritici. A new method based on NIRS was provided for the early detection of wheat stripe rust in this study.

[Application of near infrared spectroscopy to qualitative identification and quantitative determination of Puccinia strii formis f. sp. tritici and P. recondita f. sp. tritici].

To realize qualitative identification and quantitative determination of Puccinia strii formis f. sp. tritici (Pst) and P. recondita f. sp. tritici (Prt), a qualitative identification model was built using near infrared reflectance spectroscopy (NIRS) combined with distinguished partial least squares (DPLS), and a quantitative determination model was built using NIRS combined with quantitative partial least squares (QPLS). In this study, 100 pure samples including 50 samples of Pst and 50 samples of Prt were obtained, and 120 mixed samples including three replicates of mixed urediospores of the two kinds of pathogen in different proportions (the content of Pst was within the range of 2. 5% 100% with 2. 5% as the gradient) were obtained. Then the spectra of the samples were collected using MPA spectrometer, respectively. Both pure samples and mixed samples were divided into training set and testing set with the ratio equal to 2:1. Qualitative identification model and quantitative determination model were built using internal cross-validation method in the spectral region 4,000--10,000 cm(-1) based on the training sets from pure samples and mixed samples, respectively. The results showed that the identification rates of the Pst-Prt qualitative identification model for training set and testing set were both up to 100. 00% when scatter correction was used as the preprocessing method of the spectra and the number of principal components was 3. When 'range normalization + scatter correction' was used as the preprocessing method of the spectra and the number of principal components was 6, determination coefficient (RZ), standard error of calibration (SEC) and average absolute relative deviation(AARD) of the Pst-Prt quantitative determination model for training set were 99.36%, 2.31% and 8.94%, respectively, and R2, standard error of prediction (SEP) and AARD for testing set were 99.37%, 2.29% and 5. 0%, respectively. The results indicated that qualitative identification and quantitative determination of Pst and Prt using near infrared spectroscopy technology are feasible and that the Pst-Prt qualitative identification model and the Pst-Prt quantitative determination model built in this study were reliable and stable. A new method based on NIRS was provided for qualitative identification and quantitative determination of plant pathogen in this study.

Simultaneous nitrogen and phosphorus removal in the sulfur cycle-associated Enhanced Biological Phosphorus Removal (EBPR) process.

Hong Kong has practiced seawater toilet flushing since 1958, saving 750,000 m(3) of freshwater every day. A high sulfate-to-COD ratio (>1.25 mg SO4(2-)/mg COD) in the saline sewage resulting from this practice has enabled us to develop the Sulfate reduction, Autotrophic denitrification and Nitrification Integrated (SANI(®)) process with minimal sludge production and oxygen demand. Recently, the SANI(®) process has been expanded to include Enhanced Biological Phosphorus Removal (EBPR) in an alternating anaerobic/limited-oxygen (LOS-EBPR) aerobic sequencing batch reactor (SBR). This paper presents further development - an anaerobic/anoxic denitrifying sulfur cycle-associated EBPR, named as DS-EBPR, bioprocess in an alternating anaerobic/anoxic SBR for simultaneous removal of organics, nitrogen and phosphorus. The 211 day SBR operation confirmed the sulfur cycle-associated biological phosphorus uptake utilizing nitrate as electron acceptor. This new bioprocess cannot only reduce operation time but also enhance volumetric loading of SBR compared with the LOS-EBPR. The DS-EBPR process performed well at high temperatures of 30 °C and a high salinity of 20% seawater. A synergistic relationship may exist between sulfur cycle and biological phosphorus removal as the optimal ratio of P-release to SO4(2-)-reduction is close to 1.0 mg P/mg S. There were no conventional PAOs in the sludge.

[Early diagnosis of wheat stripe rust and wheat leaf rust using near infrared spectroscopy].

In the present study, near-infrared reflectance spectroscopy (NIRS) technology was applied to implement early diagnosis of two kinds of wheat rusts, i. e. wheat stripe rust and wheat leaf rust, by detecting wheat leaves as disease symptom has not appeared. The wheat leaves were divided into five categories including healthy leaves, leaves in the incubation period infected with P. strii formis f. sp. tritici, leaves showing symptom infected with P. strii formis f. sp. tritici, leaves in the incubation period infected with P. recondita f. sp. tritici and leaves showing symptom infected with P. recondita f. sp. tritici. Near infrared spectra of 150 wheat leaves were obtained using MPA spectrometer and then a model to identify the categories of wheat leaves was built using distinguished partial least squares (DPLS). For building the model, second-order derivative method was regarded as the best preprocessing method of the spectra and the spectral region 4000 - 8000 cm(-1) was regarded as the optimal spectral region. Using the model with different training sets and testing sets, the average identification rate of the training sets was 96.56% and the average identification rate of the testing sets was 91.85%. The results proved the model's stability. The optimal identification rates were obtained while the ratio of training set to testing set was 2 : 1 and the number of principal components was 10. The identification rate of the training set was 97.00% and the identification rate of the testing set was 96.00%. The results indicated that the identification method based on the NIRS technology developed in this study is feasible for early diagnosis of wheat stripe rust and wheat leaf rust.

First genome sequence of a Burkholderia pseudomallei Isolate in China, strain BPC006, obtained from a melioidosis patient in Hainan.

Melioidosis, caused by Burkholderia pseudomallei, is considered to be endemic to Northern Australia and Southeast Asia, with high mortality and relapse rates, regardless of powerful antibiotic therapy. Here we report the first genome sequence of Burkholderia pseudomallei strain BPC006, obtained from a melioidosis patient in Hainan, China. The genome sizes of the 2 chromosomes were determined to be 4,001,777 bp and 3,153,284 bp.

Compromised autophagy by MIR30B benefits the intracellular survival of Helicobacter pylori.

Helicobacter pylori evade immune responses and achieve persistent colonization in the stomach. However, the mechanism by which H. pylori infections persist is not clear. In this study, we showed that MIR30B is upregulated during H. pylori infection of an AGS cell line and human gastric tissues. Upregulation of MIR30B benefited bacterial replication by compromising the process of autophagy during the H. pylori infection. As a potential mechanistic explanation for this observation, we demonstrate that MIR30B directly targets ATG12 and BECN1, which are important proteins involved in autophagy. These results suggest that compromise of autophagy by MIR30B allows intracellular H. pylori to evade autophagic clearance, thereby contributing to the persistence of H. pylori infections.

Identification of a novel linear epitope on EspA from enterohemorrhagic E. coli using a neutralizing and protective monoclonal antibody.

Enterohemorrhagic E. coli (EHEC) causes severe diseases in humans and animals via the production of Shiga toxins, and injection of effectors into epithelia using type III secretion system (TTSS). E. coli secreted protein A (EspA) forms the filamentous conduits of TTSS, which extends into the translocation pore embedded in host cell membranes and aids in the transportation of bacterial effectors. In addition, EspA is closely associated with initial bacterial adhesion and the formation of biofilms. EspA in its various forms elicits protective immune responses, although the epitope responsible has not to be identified. Here we report the presence of a linear, immunogenic, conserved and partially protective epitope E07 (100Lys-120Val) on EspA, which is recognized by the novel monoclonal antibody 1H10. This antibody blocks EHEC-induced actin polymerization and confers protection in mice. These findings provide a better understanding of EspA-induced immune responses and could lead to epitope-based vaccines and antibody-based therapies.

EspA is a novel fusion partner for expression of foreign proteins in Escherichia coli.

Escherichia coli secreted protein A (EspA) is a component of the type 3 secretion system (T3SS). The high level of expression when self-stimulated suggests that EspA may be used as a fusion partner. In the present study, EspA was used as a "fusion partner" to construct a fusion expression system, pEspA, in order to improve the expression and solubility of proteins from prokaryotes and eukaryotes. Target proteins were linked to the C-terminus of EspA by a linker containing a YAPQDP sequence, multiple cloning sites and an enterkinase cleavage site. Six proteins, IL-24, Stx2A1, Stx2B, S1, IntiminC300 and GFP, were expressed as EspA-fusion proteins using this vector. The expression level of each protein was enhanced by EspA and the majority of them (Stx2B, IntiminC300, GFP, Stx2A1, IL-24) were expressed in soluble form. EspA-fusion proteins can be purified by affinity chromatography (Sepharose chelated with EspA-specific monoclonal antibody) and by Ni(2+) affinity chromatography for they contain a 6× His tag at their C-terminus. In addition, IL-24 remains soluble and demonstrates certain anti-tumor activity after the removal of EspA by enterkinase. The EspA fusion expression system was efficient in enhancing expression levels and the solubility of target proteins.

[Disease index inversion of wheat stripe rust on different wheat varieties with hyperspectral remote sensing].

It is becoming more and more important to use mixed wheat varieties to control wheat stripe rust. Different wheat varieties were planted in field and stripe rust was caused by artificial inoculation. Disease index (DI) was assessed and the canopy reflection data of wheat canopy were obtained by ASD FieldSpec HandHeld FR(325-1 075 nm) made by ASD Company. The correlation analysis between DI and spectral data (reflectance and the first derivative) was conducted, and the estimation models between DI and reflection data (reflectance at 690 and 850 nm, SDr, NDVI and RVI) were built using linear regression method. The results showed that different combinations of wheat varieties had the similar variation at different disease index. DI has positive correlation with reflectance of wheat canopy in visible region, and has significant negative correlation in the near infrared region. DI has stable negative correlation with the first derivative in the region of 700-760 nm and with big fluctuation in other regions. The correlation was compared between DI and hyperspectral derivative index, and SDr has the best correlation with DI. DI estimation models were built based on the canopy reflectance at 690 and 850 nm, SDr, NDVI and RVI. The determinant coefficient of the models is between 0.588 and 0.855, 0.669 and 0.911, 0.534 and 0.773, and 0.587 and 0.751, respectively, and all the models were fit well. The results indicated that DI of wheat stripe rust could be inverted using hyperspectral remote sensing technique and that the inversion effect was hardly influenced by the different combinations of wheat varieties.

[Application of hyperspectral data to the classification and identification of severity of wheat stripe rust].

Wheat stripe rust, caused by Puccinia strii formis f. sp. tritici, is one of pandemic diseases causing severe losses in China. Monitoring and warning of this disease is principal for its precise prediction and for implementing effective measures to control it. The hyperspectral data used for analysis were attained from 88 leaves including healthy leaves and infected leaves over a range of disease severity levels. Support vector machine (SVM) was applied to classify and identify the severity of wheat leaves infected by the pathogen. The model was built based on 44 proof-read samples to estimate 44 proof-test samples. And the identification accuracy is totally 97%. So SVM can be used in the classification and identification of severity of wheat stripe rust based on attained hyperspectral data.