Sixiu Liu

Isolation and algae-lysing characteristics of the algicidal bacterium B5

Water blooms have become a worldwide environmental problem. Recently, algicidal bacteria have attracted wide attention as possible agents for inhibiting algal water blooms. In this study, one strain of algicidal bacterium B5 was isolated from activated sludge. On the basis of analysis of its physiological characteristics and 16S rDNA gene sequence, it was identified as Bacillus fusiformis. Its algaelysing characteristics on Microcystis aeruginosa, Chlorella and Scenedesmus were tested. The results showed that: (1) the algicidal bacterium B5 is a Gram-negative bacterium. The 16S rDNA nucleotide sequence homology of strain B5 with 2 strains of B. fusiformis reached 99.86%, so B5 was identified as B. fusiformis; (2) the algal-lysing effects of the algicidal bacterium B5 on M. aeruginosa, Chlorella and Scenedesmus were pronounced. The initial bacterial and algal cell densities strongly influence the removal rates of chlorophyll-a. The greater the initial bacterial cell density, the faster the degradation of chlorophyll-a. The greater the initial algal cell density, the slower the degradation of chlorophyll-a. When the bacterial cell density was 3.6 x 10(7) cells/ml, nearly 90% of chlorophyll-a was removed. When the chlorophyll-a concentration was less than 550 microg/L, about 70% was removed; (3) the strain B5 lysed algae by secreting metabolites and these metabolites could bear heat treatment.

Microfluidic Device for Efficient Airborne Bacteria Capture and Enrichment

Highly efficient capture and enrichment is always the key for rapid analysis of airborne pathogens. Herein we report a simple microfluidic device which is capable of fast and efficient airborne bacteria capture and enrichment. The device was validated with Escherichia coli (E. coli) and Mycobacterium smegmatis. The results showed that the efficiency can reach close to 100% in 9 min. Compared with the traditional sediment method, there is also great improvement with capture limit. In addition, various flow rate and channel lengths have been investigated to obtain the optimized condition. The high capture and enrichment might be due to the chaotic vortex flow created in the microfluidic channel by the staggered herringbone mixer (SHM) structure, which is also confirmed with flow dynamic mimicking. The device is fabricated from polydimethylsiloxane (PDMS), simple, cheap, and disposable, perfect for field application, especially in developing countries with very limited modern instruments.

Microfluidic chip integrating high throughput continuous-flow PCR and DNA hybridization for bacteria analysis

Rapid identification of clinical pathogens is the initial and essential step for antimicrobial therapy. Herein, we successfully developed a microfluidic device which combines high-throughput continuous-flow PCR and DNA hybridization for the detection of various bacterial pathogens. Universal primers were designed based on the conserved regions of bacterial 16S ribosomal DNA (16S rDNA), and specific probes were designed from a variable region of 16S rDNA within the amplicon sequences. In the chip operation, after the continuous flow PCR was achieved in the first microfluidic chip, the product was directly introduced into a hybridization chip integrated with microarray containing the immobilized DNA probes. The target-probe hybridization was completed within 1h at 55 °C, and fluorescence signals were obtained as the readout. The presented device is simple, versatile and with less sample consumption compared with traditional instruments. It can perform high-throughput bacteria detections continuously in a single assay, which makes it a promising platform for clinical bacteria identifications.

Microfluidic platform for direct capture and analysis of airborne Mycobacterium tuberculosis.

Airborne Mycobacterium tuberculosis is the main source of tuberculosis infection, which is known as one of the worldwide infectious diseases. Direct capture and analysis of airborne Mycobacterium tuberculosis is essential for disease prevention and control. At present, low concentration of pathogens directly collected from the air is the major drawback for rapid analysis. Herein an integrated microfluidic system capable of airborne Mycobacterium tuberculosis capture, enrichment, and rapid bacteriological immunoassay was developed. The whole detection time was decreased to less than 50 min including 20 min of enrichment and 30 min of immunoreaction analysis. It had the advantages of low detection limit, fast detection speed, and low reagent consumption compared with conventional techniques, showing the potential to become a new airborne pathogen analysis platform.

An integrated microfluidic device for rapid serodiagnosis of amebiasis

A microfluidic device was successfully fabricated for the rapid serodiagnosis of amebiasis. A micro bead-based immunoassay was fabricated within integrated microfluidic chip to detect the antibody to Entamoeba histolytica in serum samples. In this assay, a recombinant fragment of C terminus of intermediate subunit of galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica (C-Igl, aa 603-1088) has been utilized instead of the crude antigen. This device was validated with serum samples from patients with amebiasis and showed great sensitivity. The serodiagnosis can be completed within 20 min with 2 μl sample consumption. The device can be applied for the rapid and cheap diagnosis of other infectious disease, especially for the developing countries with very limited medical facilities.

Molecular cloning and characterization of a novel ice gene from Capsella bursa-pastoris

A new ice gene (designated as Cbice53, an inducer of CBF expression) was cloned from Capsella bursa-pastoris by rapid amplification of cDNA ends (RACE). The full-length cDNA of Cbice53 was 1811 bp long, with a 1476-bp open reading frame (ORF) encoding a Myc-like protein of 492 amino acids. The predicted CbICE53 protein contained a potential basic helix-loop-helix domain, a nuclear localization signal (NLS), an RNA-binding region (RGG box), and N-glycosylation and kinase phosphorylation sites. Bioinformatic analysis showed that CbICE53 was highly homologous to ICE1 from Arabidopsis thaliana. Transcription of Cbice53 gene was induced transiently during salt and cold treatments, suggesting that it was somehow involved in cold acclimation. The results of our study indicate that the Cbice53 gene is a new member of the ice gene family and may have a role in cold and salt responsiveness in C. bursa-pastoris.

Immuno-laser capture microdissection of frozen prolactioma sections to prepare proteomic samples

Laser capture microdissection (LCM) technology combined with immunohistochemistry (immuno-LCM) is a valuable tool to obtain specific target cell populations and therefore this technique enables more accurate proteomic profile. In this study, we optimized the regular immuno-LCM technique to isolate and stain pure prolactin cells from either normal human pituitary (n=6) or prolactioma (n=11). Compared with the routine procedure, more intense and specific staining could be obtained when sections were pretreated with 0.2% Triton X-100 for 4 min. Interestingly, longer pretreatment (0.2% Triton X-100 for 10 min) or higher concentration (2% Triton X-100 for 4 and 10 min) greatly impaired labeling intensity and cell shape. Further scanning electron microscope study revealed that the component extracted from the cell surface by Triton X-100 was lipid. Using the optimized immuno-LCM technique, more pure prolactin cells could be isolated and prepared for further proteomic analysis. Taken together, we reported an optimized immuno-LCM technique that could effectively dissect pure target cells in different type pituitary adenomas for further proteomics analysis.

The Algicidal Characteristics of One Algae-Lysing FDT5 Bacterium on Microcystis aeruginosa

One strain of algicidal bacterium which can inhibit Harmful algal blooms (HABs), FDT5, was isolated from activated sludge and found to have good algicidal effects on Microcystis aeruginosa. It was revealed that: The FDT5 was a Gram-negative bacterium and identified as Ochrobactrum sp.; The greater the initial bacterial cell density, the faster the degradation of chlorophyll a.; The algicidal efficiency was evaluated at the most favorable conditions which were a temperature of 30–35°C, a pH of 7.6 and complete darkness; The FDT5 strain lysed Microcystis aeruginosa not directly but by secreting metabolites which could withstand high temperatures and pressure.

Microfluidic chip for isolation of viable circulating tumor cells of hepatocellular carcinoma for their culture and drug sensitivity assay

ABSTRACT Circulating tumor cells (CTCs) have been proposed to be an active source of metastasis or recurrence of hepatocellular carcinoma (HCC). The enumeration and characterization of CTCs has important clinical significance in recurrence prediction and treatment monitoring in HCC patients. We previously developed a unique method to separate HCC CTCs based on the interaction of the asialoglycoprotein receptor (ASGPR) expressed on their membranes with its ligand. The current study applied the ligand-receptor binding assay to a CTC-chip in a microfluidic device. Efficient capture of HCC CTCs originates from the small dimensions of microfluidic channels and enhanced local topographic interactions between the microfluidic channel and extracellular extensions. With the optimized conditions, a capture yield reached > 85% for artificial CTC blood samples. Clinical utility of the system was further validated. CTCs were detected in all the examined 36 patients with HCC, with an average of 14 ± 10/2 mL. On the contrary, no CTCs were detected in healthy, benign liver disease or non-HCC cancer subjects. The current study also successfully demonstrated that the captured CTCs on our CTC-chip were readily released with ethylene diamine tetraacetic acid (EDTA); released CTCs remained alive and could be expanded to form a spheroid-like structure in a 3-dimensional cell culture assay; furthermore, sensitivity of released CTCs to chemotherapeutic agents (sorafenib or oxaliplatin) could be effectively tested utilizing this culture assay. In conclusion, the methodologies presented here offer great promise for accurate enumeration and easy release of captured CTCs, and released CTCs could be cultured for further functional studies.

A SIMPLE, FAST, SOLVENT-FREE METHOD FOR THE DETERMINATION OF VOLATILE COMPOUNDS IN MAGNOLIA GRANDIFLORA LINN

Magnolia grandiflora Linn belonging to the Magnoliaceae family has been used to treat hypertension for many years in China. Based on microwave-assisted extraction (MAE) and headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-mass spectrometry (GC-MS), the volatile compounds of Magnolia grandiflora L. were determined. Results indicated that the optimum conditions of the determination of the volatile compounds in Magnolia grandiflora L. were achieved with fiber coating of carboxen/polidimethylsiloxane, microwave power of 700 W and irradiation time of 4 min. Under the optimal conditions, for the first time, 48 volatile compounds were separated and identified from the fresh leaves of Magnolia grandiflora L. The highest content component of 48 compounds was γ-elemene (15.67%). Relative standard deviation (RSD) values less than 11% shows that the present method has good precision. The experimental results demonstrate that MAE-HS-SPME followed by GC-MS is a simple, time-saving solvent-free method, and it is a potential analytic tool for the determination of the volatile compounds in plant materials.