Quorum sensing: an emerging link between temperature and membrane biofouling in membrane bioreactors

Lab-scale membrane bioreactors (MBRs) were investigated at 12, 18, and 25?°C to identify the correlation between quorum sensing (QS) and biofouling at different temperatures. The lower the reactor temperature, the more severe the membrane biofouling measured in terms of the transmembrane pressure (TMP) during filtration. More extracellular polymeric substances (EPSs) that cause biofouling were produced at 18?°C than at 25?°C, particularly polysaccharides, closely associated with QS via the production of N-acyl homoserine lactone (AHL). However, at 12?°C, AHL production decreased, but the release of EPSs due to deflocculation increased the soluble EPS concentration. To confirm the temperature effect related to QS, bacteria producing AHL were isolated from MBR sludge and identified as Aeromonas sp., Leclercia sp., and Enterobacter sp. through a 16S rDNA sequencing analysis. Batch assays at 18 and 25?°C showed that there was a positive correlation between QS through AHL and biofilm formation in that temperature range.     

Colorimetric biosensor using dual-amplification of enzyme-free reaction through universal hybridization chain reaction system

On-site genetic detection needs to develop a sensitive and straightforward biosensor without special equipment, which can detect various genetic biomarkers. Hybridization chain reaction (HCR) amplifying signal isothermally could be considered as a good candidate for on-site detection. Here, we developed a novel genetic biosensor on the basis of enzyme-free dual-amplification of universal hybridization chain reaction (uHCR) and hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme. The uHCR is the strategy which enables simple design for multiple target detection by the introduction of target-specific trigger hairpin without changing the whole system according to a target change. Also, HRP-mimicking DNAzyme could produce a sensitive and quantitative colorimetric signal with increased stability with a limit of detection (LOD) of 5.67 nM. The universality of the uHCR biosensor was proven by the detection of four different targets (miR-21, miR-125b, KRAS-Q61K, and BRAF-V600E) for cancer diagnosis. The uHCR biosensor showed specificity that could discriminate single-nucleotide polymorphism. Moreover, the uHCR biosensor could detect targets in the diluted serum sample. Overall, the uHCR biosensor demonstrated the potential for field testing with a simple redesign without complicated steps or special equipment using a universal hairpin system and enzyme-free amplification. This strategy could enable stable and sensitive detection of a variety of targets. Therefore, it could be applied to urgent detection of various pathogens, remote diagnosis, and self-screening of diseases.     

Effects of Fluridone and Norflurazon on Conditioning and Germination of Striga asiatica Seeds

Inhibitors of carotenoid biosynthesis are known to prevent abscisic acid (ABA) biosynthesis and to affect germination and dormancy of seeds in many plants. In this study, the effects of three carotenoid biosynthesis inhibitors, fluridone, norflurazon and diflufenican, on the conditioning and germination of Striga asiatica seeds were examined. Fluridone and norflurazon shortened the conditioning period required before S. asiatica seeds would germinate after exposure to the germination stimulant strigol, and prevented the inhibitory effects of both light and supraoptimal temperature (40 °C) on seed germination. In addition, treatment with fluridone or norflurazon after conditioning in water induced seed germination in a manner similar to the effect of natural germination stimulants. Moreover, the seedlings developing after conditioned with fluridone formed haustorium-like structures without the involvement of haustorium inducing factors. In contrast, diflufenican had no effect on the conditioning and germination of S. asiatica seeds. These results indicate that fluridone and norflurazon have various effects on the germination of S. asiatica seeds and might be available for control of root parasites.     

Effective Treatment of Established GL261 Murine Gliomas through Picornavirus Vaccination-Enhanced Tumor Antigen-Specific CD8+ T Cell Responses

Glioblastoma (GBM) is among the most invasive and lethal of cancers, frequently infiltrating surrounding healthy tissue and giving rise to rapid recurrence. It is therefore critical to establish experimental model systems and develop therapeutic approaches that enhance anti-tumor immunity. In the current study, we have employed a newly developed murine glioma model to assess the efficacy of a novel picornavirus vaccination approach for the treatment of established tumors. The GL261-Quad system is a variation of the GL261 syngeneic glioma that has been engineered to expresses model T cell epitopes including OVA_257–264. MRI revealed that both GL261 and GL261-Quad tumors display characteristic features of human gliomas such as heterogeneous gadolinium leakage and larger T2 weighted volumes. Analysis of brain-infiltrating immune cells demonstrated that GL261-Quad gliomas generate detectable CD8+ T cell responses toward the tumor-specific K^b:OVA_257–264 antigen. Enhancing this response via a single intracranial or peripheral vaccination with picornavirus expressing the OVA_257–264 antigen increased anti-tumor CD8+ T cells infiltrating the brain, attenuated progression of established tumors, and extended survival of treated mice. Importantly, the efficacy of the picornavirus vaccination is dependent on functional cytotoxic activity of CD8+ T cells, as the beneficial response was completely abrogated in mice lacking perforin expression. Therefore, we have developed a novel system for evaluating mechanisms of anti-tumor immunity in vivo, incorporating the GL261-Quad model, 3D volumetric MRI, and picornavirus vaccination to enhance tumor-specific cytotoxic CD8+ T cell responses and track their effectiveness at eradicating established gliomas in vivo.     

Eugenol: A Phyto-Compound Effective against Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus Clinical Strain Biofilms

Background

Inhibition and eradication of Staphylococcus aureus biofilms with conventional antibiotic is difficult, and the treatment is further complicated by the rise of antibiotic resistance among staphylococci. Consequently, there is a need for novel antimicrobials that can treat biofilm-related infections and decrease antibiotics burden. Natural compounds such as eugenol with anti-microbial properties are attractive agents that could reduce the use of conventional antibiotics. In this study we evaluated the effect of eugenol on MRSA and MSSA biofilms in vitro and bacterial colonization in vivo.

Methods and Results

Effect of eugenol on in vitro biofilm and in vivo colonization were studied using microtiter plate assay and otitis media-rat model respectively. The architecture of in vitro biofilms and in vivo colonization of bacteria was viewed with SEM. Real-time RT-PCR was used to study gene expression. Check board method was used to study the synergistic effects of eugenol and carvacrol on established biofilms. Eugenol significantly inhibited biofilms growth of MRSA and MSSA in vitro in a concentration-dependent manner. Eugenol at MIC or 2×MIC effectively eradicated the pre-established biofilms of MRSA and MSSA clinical strains. In vivo, sub-MIC of eugenol significantly decreased 88% S. aureus colonization in rat middle ear. Eugenol was observed to damage the cell-membrane and cause a leakage of the cell contents. At sub-inhibitory concentration, it decreases the expression of biofilm-and enterotoxin-related genes. Eugenol showed a synergistic effect with carvacrol on the eradication of pre-established biofilms.

Conclusion/Major Finding

This study demonstrated that eugenol exhibits notable activity against MRSA and MSSA clinical strains biofilms. Eugenol inhibited biofilm formation, disrupted the cell-to-cell connections, detached the existing biofilms, and killed the bacteria in biofilms of both MRSA and MSSA with equal effectiveness. Therefore, eugenol may be used to control or eradicate S. aureus biofilm-related infections.     

Application of NMR Spectroscopy in the Assessment of Radiation Dose in Human Primary Cells

We employed the primary cell model system as a first step toward establishing a method to assess the influence of ionizing radiation by using a combination of common and abundant metabolites. We applied X‐ray irradiation amounts of 0, 1, and 5 Gy to the cells that were harvested 24, 48, or 72 h later, and profiled metabolites by 2D‐NMR spectroscopy to sort out candidate molecules that could be used to distinguish the samples under different irradiation conditions. We traced metabolites stemming from the input ¹³C‐glucose, identified twelve of them from the cell extracts, and applied statistical analysis to find out that all the metabolites, including glycine, alanine, and gluatamic acid, increased upon irradiation. The combinatorial use of the selected metabolites showed promising results where the product of signal intensities of alanine and lactate could differentiate samples according to the dose of X‐ray irradiation. We hope that this work can form a base for treating radiation‐poisoned patients in the future.     

Purification of anti‐colorectal cancer monoclonal antibody CO17‐1A from insect cell culture using a French press and sonication

Monoclonal antibody (mAb) CO17‐1A binds to GA733, which is a tumor‐associated glycoprotein antigen highly expressed on the colorectal cancer cell surface. Thus, mAb CO17‐1A is considered a useful biomolecule for diagnosis and treatment against colorectal cancer. Previously, we established a baculovirus–insect cell expression system for the production of mAb CO17‐1A. In order to use mAb CO17‐1A as a diagnostic and therapeutic tool, however, the antibody must be properly purified from the insect cells. In this study, our aim was to investigate effective purification processes of mAb CO17‐1A expressed in Spodoptera frugiperda (Sf9) insect cells, using a French press and sonication for cell disruption. SDS‐PAGE confirmed that both mAb CO17‐1A and mAb CO17‐1A fused to the KDEL endoplasmic reticulum (ER) retention signal (mAb CO17‐1AK) were expressed clearly in Sf9 insect cells. Western blot analysis showed that detection levels of mAb CO17‐1A and CO17‐1AK were higher when the insect cells were disrupted two times by the French press and then sonicated, compared to only one French press disruption plus sonication. Optical microscopy confirmed that insect cells treated with both the French press and sonication were properly disrupted. Analysis of gene sequence information on mAb CO17‐1A verified that a signal peptide is present but a transmembrane protein does not exist. These results suggest that cell disruption by the French press twice and sonication once is an effective method for improving purification efficiency.     

Mapping the genome of Miscanthus sinensis for QTL associated with biomass productivity

In light of rising energy costs, lignocellulosic ethanol has been identified as a renewable alternative to petroleum-based transportation fuels. In an attempt to reach government mandated ethanol production levels, potential plant biofeedstock candidates have been investigated, and cold-tolerant, perennial accessions within the C₄ grass genus Miscanthus have been identified as leading contenders in the Midwestern US. To facilitate the development of improved cultivars through marker-assisted breeding, a quantitative trait locus (QTL) study was conducted on a full-sib, F₁ mapping population segregating for flowering time, height, leaf width, and yield using a genetic map consisting of 846 segregating SNP and SSR markers. This was a 3 year study investigating the genetic architecture underlying traits important to biomass production in a population of 221 progeny from a cross between M. sinensis ‘Grosse Fountaine’ and M. sinensis ‘Undine’ established in the spring of 2010; 72 QTLs with LOD scores above the genome-wide, permuted threshold equivalent to a P-value of 0.05 were identified across 13 traits. Of the 36 QTLs identified in 2011, 22 were detected again the following year. Both the use of spring emergence and vigor rating as a covariate to account for variation related to differences in establishment increased the power to detect QTLs in the 2 year establishment period. Finally, a dry period in the middle of the 2012 growing season suggested that yield declines were due to a decrease in tiller diameter.