Sensitive Detection of Single-Nucleotide Polymorphisms by Solid Nanopores Integrated With DNA Probed Nanoparticles.

Single-nucleotide polymorphisms (SNPs) are the abundant forms of genetic variations, which are closely associated with serious genetic and inherited diseases, even cancers. Here, a novel SNP detection assay has been developed for single-nucleotide discrimination by nanopore sensing platform with DNA probed Au nanoparticles as transport carriers. The SNP of p53 gene mutation in gastric cancer has been successfully detected in the femtomolar concentration by nanopore sensing. The robust biosensing strategy offers a way for solid nanopore sensors integrated with varied nanoparticles to achieve single-nucleotide distinction with high sensitivity and spatial resolution, which promises tremendous potential applications of nanopore sensing for early diagnosis and disease prevention in the near future.

Cis-2-dodecenoic Acid Mediates Its Synergistic Effect with Triazoles by Interfering with Efflux Pumps in Fluconazole-resistant Candida albicans.

OBJECTIVE: To evaluate the synergy of the Burkholderia signaling molecule cis-2-dodecenoic acid (BDSF) and fluconazole (FLU) or itraconazole (ITRA) against two azole-resistant C. albicans clinical isolates in vitro and in vivo. METHODS: Minimum inhibitory concentrations (MICs) of antibiotics against two azole-resistant C. albicans were measured by the checkerboard technique, E-test, and time-kill assay. In vivo antifungal synergy testing was performed on mice. Analysis of the relative gene expression levels of the strains was conducted by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS: BDSF showed highly synergistic effects in combination with FLU or ITRA with a fractional inhibitory concentration index of ⪕ 0.08. BDSF was not cytotoxic to normal human foreskin fibroblast cells at concentrations of up to 300 µg/mL. The qRT-PCR results showed that the combination of BDSF and FLU/ITRA significantly inhibits the expression of the efflux pump genes CDR1 and MDR1 via suppression of the transcription factors TAC1 and MRR1, respectively, when compared with FLU or ITRA alone. No dramatic difference in the mRNA expression levels of ERG1, ERG11, and UPC2 was found, which indicates that the drug combinations do not significantly interfere with UPC2-mediated ergosterol levels. In vivo experiments revealed that combination therapy can be an effective therapeutic approach to treat candidiasis. CONCLUSION: The synergistic effects of BDSF and azoles may be useful as an alternative approach to control azole-resistant Candida infections.

Protective Effects of cis-2-Dodecenoic Acid in an Experimental Mouse Model of Vaginal Candidiasis.

OBJECTIVE: To evaluate the efficacy of cis-2-dodecenoic acid (BDSF) in the treatment and prevention of vaginal candidiasis in vivo. METHODS: The activities of different concentrations of BDSF against the virulence factors of Candida albicans (C. albicans) were determined in vitro. An experimental mouse model of Candida vaginitis was treated with 250 µmol/L BDSF. Treatment efficiency was evaluated in accordance with vaginal fungal burden and inflammation symptoms. RESULTS: In vitro experiments indicated that BDSF attenuated the adhesion and damage of C. albicans to epithelial cells by decreasing phospholipase secretion and blocking filament formation. Treatment with 30 µmol/L BDSF reduced the adhesion and damage of C. albicans to epithelial cells by 36.9% and 42.3%, respectively. Treatment with 200 µmol/L BDSF completely inhibited phospholipase activity. In vivo mouse experiments demonstrated that BDSF could effectively eliminate vaginal infection and relieve inflammatory symptoms. Four days of treatment with 250 µmol/L BDSF reduced vaginal fungal loads by 6-fold and depressed inflammation. Moreover, BDSF treatment decreased the expression levels of the inflammatory chemokine-associated genes MCP-1 and IGFBP3 by 2.5- and 2-fold, respectively. CONCLUSION: BDSF is a novel alternative drug that can efficiently control vaginal candidiasis by inhibiting the virulence factors of C. albicans.

A new synthetic ligand that activates QscR and blocks antibiotic-tolerant biofilm formation in Pseudomonas aeruginosa.

Quorum sensing (QS) has been recognized to play an important role in many pathogenic bacteria and has become a novel target for the treatment of infectious disease. Pseudomonas aeruginosa is highly resistant to antibiotic treatment, largely due to its ability to form biofilms, and QS was found to be essential for the creation of mature, differentiated biofilms in this organism. A novel QS inhibitor, C2 (N-decanoyl-L-homoserine benzyl ester), can attenuate not only total protease and elastase activity, but also swarming motility and biofilm formation in the P. aeruginosa strain PAO1. We demonstrated that C2 showed a significant inhibitory effect on biofilm formation in a dose-dependent manner. Data from cDNA microarray showed that expression of 382 genes (∼6.4 %) was significantly different with C2 treatment, including downregulation of 215 genes (∼3.6 %) and upregulation of 167 genes (∼2.8 %). Real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that the gene qscR, which encodes the LuxR-type receptor QscR (quorum sensing control repressor), was significantly upregulated by 375.4 % during C2 treatment. The mechanism by which C2 inhibits biofilm formation may be through repression of Las and Rhl systems by QscR. C2 was shown to reduce biofilm formation; in combination with antibiotics, it abolishes biofilm formation completely. This result may pave the way for new treatments for biofilm-related infections and may be exploited for the general prevention of biofilm formation.

BDSF inhibits Candida albicans adherence to urinary catheters.

Cis-2-dodecenoic acid (BDSF) is a quorum-sensing signal molecule produced by the opportunistic pathogen Burkholderia cenocepacia and suppresses germ tube formation of Candida albicans. An in vitro model for biofilm formation evaluated the influence of BDSF on C. albicans. Biofilm morphology was observed using scanning electron microscopy, cell adherence was determined using polystyrene plates and siliconized urinary catheters, and the levels of expression of genes involved in adhesion were determined using Real-time Reverse-Transcriptase Polymerase Chain Reaction. BDSF inhibited initial biofilm formation by a clinical isolate of C. albicans and reduced its capability to adhere to the polystyrene surface. BDSF at concentrations up to 120 µM did not significantly affect the viability of C. albicans. BDSF (90 µM) inhibited cell adherence to plates and catheters by 4- and 25-fold. Compared with untreated yeasts, the level of expression of genes involved in adhesion, ALS1 and EAP1, were reduced by 4- and 0.25-fold, whereas that of YWP1 was increased at a 4-fold higher level. Here we show that BDSF effectively inhibited biofilm development as indicated by its ability to inhibit adherence. Thus, BDSF should be considered as a potential therapeutic agent to prevent disease caused by Candida species.

A new quorum-sensing inhibitor attenuates virulence and decreases antibiotic resistance in Pseudomonas aeruginosa.

Quorum sensing (QS) has been a novel target for the treatment of infectious diseases. Here structural analogs of Pseudomonas aeruginosa autoinducer N-acyl homoserine lactone (AHL) were investigated for QS inhibitor (QSI) activity and a novel QSI was discovered, N-decanoyl-L-homoserine benzyl ester (C2). Virulence assays showed that C2 down-regulated total protease and elastase activities, as well as the production of rhamnolipid, that are controlled by QS in P. aeruginosa wild-type strain PAO1 without affecting growth. C2 was also shown to inhibit swarming motility of PAO1. Using a microdilution checkerboard method, we identified synergistic interactions between C2 and several antibiotics, tobramycin, gentamycin, cefepime, and meropenem. Data from real-time RT-PCR suggested that C2 inhibited the expression of lasR (29.67%), lasI (21.57%), rhlR (28.20%), and rhlI (29.03%).

Transgenic sugarcane plants expressing high levels of modified cry1Ac provide effective control against stem borers in field trials.

To improve transgene expression level, we synthesized a truncated insecticidal gene m-cry1Ac by increasing its GC content from 37.4 to 54.8%, based on the codon usage pattern of sugarcane genes, and transferred it into two sugarcane cultivars (ROC16 and YT79-177) by microprojectile bombardment. The integration sites and expression pattern of the transgene were determined, respectively, by Southern, northern and western blot analyses. The transgenic sugarcane lines produced up to 50 ng Cry1Ac protein per mg soluble proteins, which was about fivefold higher than that produced by the partially modified s-cry1Ac (GC% = 47.5%). In greenhouse plant assay, about 62% of the transgenic lines exhibited excellent resistance to heavy infestation by stem borers. In field trials, the m-cry1Ac transgenic sugarcane lines expressing high levels of Cry1Ac were immune from insect attack. In contrast, expression of s-cry1Ac in transgenic sugarcane plants resulted in moderately decreased damages in internodes (0.4-1.7%) and stalks (13.3-26.7%) in comparison with the untransformed sugarcane controls, which showed about 4 and 26-40% damaged internodes and stalks, respectively. Significantly, these transgenic sugarcane lines with high levels of insect resistance showed similar agronomic and industrial traits as untransformed control plants. Taken together, the findings from this study indicate a promising potential of engineering an insect-resistant gene to tailor its protein expression levels in transgenic sugarcane to combat insect infestations.

H2O2-sensitive quantum dots for the label-free detection of glucose.

A novel label-free detection system based on CdTe/CdS quantum dots (QDs) was designed for the direct measurement of glucose. Herein we demonstrated that the photoluminescence (PL) of CdTe/CdS QDs was sensitive to hydrogen peroxide (H(2)O(2)). With d-glucose as a substrate, H(2)O(2) that intensively quenched the QDs PL can be produced via the catalysis of glucose oxidase (GOx). Experimental results showed that the decrease of the QDs PL was proportional to the concentration of glucose within the range of 1.8 microM to 1mM with the detection limit of 1.8 microM under the optimized experimental conditions. In addition, the QD-based label-free glucose sensing platform was adapted to 96-well plates for fluorescent assay, enhancing the capabilities and conveniences of this detection platform. An excellent response to the concentrations of glucose was found within the range of 2-30 mM. Glucose in blood and urine samples was effectively detected via this strategy. The comparison with commercialized glucose meter indicated that this proposed glucose assay system is not only simple, sensitive, but also reliable and suitable for practical application. The high sensitivity, versatility, portability, high-throughput and low cost of this glucose sensor implied its potential in point-of-care clinical diagnose of diabetes and other fields.

DNA-bridged bioconjugation of fluorescent quantum dots for highly sensitive microfluidic protein chips.

A DNA-bridged strategy is used to facilely conjugate streptavidin (STV) to luminescent semiconductor quantum dots (QDs), which leads to convenient and stable QD-DNA-biotin-STV conjugates that serve as fluorescent nanoprobes for ultrasensitive detection of cancer biomarkers with a microfluidic protein chip.

Regeneration of sugarcane elite breeding lines and engineering of stem borer resistance.

Five elite sugarcane breeding lines were tested for efficiency in embryogenesis and plant regeneration. All of them produced regenerative embryogenic calli but with varied efficiencies. To engineer strongly insect-resistant sugarcanes, the GC content of a truncated cry1Ac gene, which encodes the active region of Cry1Ac insecticidal delta-endotoxin, was increased from the original 37.4 to 47.5% following the sugarcane codon usage pattern. The synthetic cry1Ac gene (s-cry1Ac) was placed under the control of maize ubiquitin promoter and introduced by microprojectile bombardment into the embryogenic calli of sugarcane lines YT79-177 and ROC16. Southern blotting analysis showed that multicopies of s-cry1Ac were integrated into the genomes of transgenic sugarcane lines. Immunoblotting analysis identified 18 transgenic lines expressing detectable levels of s-Cry1Ac, which were estimated in the range of 1.8-10.0 ng mg(-1) total soluble proteins. Four transgenic and two parental lines were assayed for sugarcane stem borer resistance in leaf tissue feeding trials and greenhouse plant assays. The results showed that, while the untransformed control lines were severely damaged in both leaves and stems, the transgenic sugarcane lines expressing high levels of s-Cry1Ac proteins were highly resistant to sugarcane stem borer attack, resulting in complete mortality of the inoculated insects within 1 week after inoculation.

Molecular and conformational basis of a specific and high-affinity interaction between AlbA and albicidin phytotoxin.

The albA gene of Klebsiella oxytoca encodes a protein of 221 amino acids that binds the albicidin phytotoxin with a high affinity (dissociation constant = 6.4 x 10(-8) M). For this study, circular dichroism (CD) spectrometry and an alanine scanning mutagenesis approach were used in combination to investigate the molecular and conformational mechanisms of this high-affinity protein-ligand interaction. CD analysis revealed that AlbA contains a high-affinity binding site, and binding of the albicidin ligand to AlbA in a low-ionic-strength environment induced significant conformational changes. The ligand-dependent conformational changes of AlbA were specific and rapid and reached a stable plateau within seconds after the addition of the antibiotic. However, such conformational changes were not detected when AlbA and albicidin were mixed in the high-ionic-strength buffer that is required for maximal binding activity. Based on the conceptual model of protein-ligand interaction, we propose that a threshold ion strength allows AlbA to complete its conformational rearrangement and resume its original stable structure for accommodation of the bound albicidin. Mutagenesis analysis showed that the replacement of Lys106, Trp110, Tyr113, Leu114, Tyr126, Pro134, and Trp162 with alanine did not change the overall conformational structure of AlbA but decreased the albicidin binding activity about 30 to 60%. We conclude that these residues, together with the previously identified essential residue His125, constitute a high-affinity binding pocket for the ligand albicidin. The results also suggest that hydrophobic and electrostatic potentials of these key amino acid residues may play important roles in the AlbA-albicidin interaction.

Specificity and enzyme kinetics of the quorum-quenching N-Acyl homoserine lactone lactonase (AHL-lactonase).

N-Acyl homoserine lactone (AHL) quorum-sensing signals are the vital elements of bacterial quorum-sensing systems, which regulate diverse biological functions, including virulence. The AHL-lactonase, a quorumquenching enzyme encoded by aiiA from Bacillus sp., inactivates AHLs by hydrolyzing the lactone bond to produce corresponding N-acyl homoserines. To characterize the enzyme, the recombinant AHL-lactonase and its four variants were purified. Kinetic and substrate specificity analysis showed that AHL-lactonase had no or little residue activity to non-acyl lactones and noncyclic esters, but displayed strong enzyme activity toward all tested AHLs, varying in length and nature of the substitution at the C3 position of the acyl chain. The data also indicate that the amide group and the ketone at the C1 position of the acyl chain of AHLs could be important structural features in enzyme-substrate interaction. Surprisingly, although carrying a (104)HX- HXDH(109) short sequence identical to the zinc-binding motif of several groups of metallohydrolytic enzymes, AHL-lactonase does not contain or require zinc or other metal ions for enzyme activity. Except for the amino acid residue His-104, which was shown previously to not be required for catalysis, kinetic study and conformational analysis using circular dichroism spectrometry showed that substitution of the other key residues in the motif (His-106, Asp-108, and His-109), as well as His-169 with serine, respectively, caused conformational changes and significant loss of enzyme activity. We conclude that AHL-lactonase is a highly specific enzyme and that the (106)HXDH(109) approximately H(169) of AHL-lactonase represents a novel catalytic motif, which does not rely on zinc or other metal ions for activity.

A bacterial cell-cell communication signal with cross-kingdom structural analogues.

Extracellular signals are the key components of microbial cell-cell communication systems. This report identified a diffusible signal factor (DSF), which regulates virulence in Xanthomonas campestris pv. campestris, as cis-11-methyl-2-dodecenoic acid, an alpha,beta unsaturated fatty acid. Analysis of DSF derivatives established the double bond at the alpha,beta positions as the most important structural feature for DSF biological activity. A range of bacterial pathogens, including several Mycobacterium species, also displayed DSF-like activity. Furthermore, DSF is structurally and functionally related to farnesoic acid (FA), which regulates morphological transition and virulence by Candida albicans, a fungal pathogen. Similar to FA, which is also an alpha,beta unsaturated fatty acid, DSF inhibits the dimorphic transition of C. albicans at a physiologically relevant concentration. We conclude that alpha,beta unsaturated fatty acids represent a new class of extracellular signals for bacterial and fungal cell-cell communications. As prokaryote-eukaryote interactions are ubiquitous, such cross-kingdom conservation in cell-cell communication systems might have significant ecological and economic importance.

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics.

The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30%, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K. oxytoca ATCC 13182(T) to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His(78), His(125), His(141) and His(189)) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95 %. DEPC treatment increased absorbance at 240-244 nm by an amount indicating conversion to N-carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1 : 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that His(125) might be the residue required for albicidin binding. Mutation of His(125) to either alanine or leucine resulted in about 32 % loss of binding activity, and deletion of His(125) totally abolished binding activity. Mutation of His(125) to arginine and tyrosine had no effect. These results indicate that His(125) plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.