Lysis Reaction Research Articles

Deep insights into the enhanced waste active sludge lysis by MoS2 and Fe2+ co-activated persulfate

The lysis treatment of waste active sludge (WAS) is significant to sludge dewatering/reduction and resource utilization. However, the low lysis efficiency of WAS is still a main limiting factor for the engineering application. Herein, MoS2 reinforced Fe2+/peroxydisulfate (PDS) oxidation system was unprecedentedly proposed to expedite the WAS cytolysis. The MoS2/PDS system was more effective than Fe2+/PDS system in reducing the water content of WAS, are 70.79 % and 76.38 %, respectively, at the PDS and activator dosages of 1.2 and 1.5 mmol/g volatile suspended solids. The concentrations of total organic carbon and DNA were highest in the MoS2/PDS system, were 115.12 ± 0.57 mg/L and 110.55 ± 1.65 ng/μL, respectively. The best lysis was obtained in the MoS2 + Fe2+/PDS system, which enabled a strong oxidation to excessively destroy the released organic matters. The outward discharge of organics from the intracellular and tightly bound-extracellular polymers (EPS) to loosely bound-EPS and supernatant slime-EPS was achieved with no Mo residue in the liquid phase. Of the identified active species ·OH, 1O2 and SO4•−, ·OH was found to play the most critical role in the fragmentation process for the MoS2 + Fe2+/PDS system. The MoS2-mediated Fe2+/Fe3+ cycle is favorable for the PDS activation and the formation of highly reactive radicals to accelerate the lysis reaction, thus contributing to the WAS reduction.

CFD and experimental microfluidic study of the interaction between a natural compound nanoemulsion and E. coli bacterium considering reaction-diffusion-convection model

Essential oils have recently emerged as potent natural antibacterial agents that are effective against pathogenic bacteria. The antibacterial efficacy of a stabilized nanoemulsion of carvacrol (with a mean droplet size of 123.51 nm ± 4.2 nm) on a Gram-negative Escherichia coli (E. coli ATCC 25922) was experimentally and numerically studied utilizing a continuous-flow microfluidic system. The morphology of the destroyed bacteria was examined visually and, bacteria lysis was confirmed by measuring the release of cellular contents. After establishing the antibacterial capability kinetics of the carvacrol nanoemulsions, the acquired data from the time killing assay was used to characterize the E. coli lysis. Moreover, a computational fluid dynamics (CFD) model was implemented to evaluate the influence of altering key parameters such as the Reynolds number, flow rate ratio and inlet carvacrol concentration on the unlysed bacteria percentage at the outlet (Y1) and the mass flow rate (μg/s) of the lysed bacteria at the outlet (Y2). The maximum lysis reaction rate observed in the device was 0.96s-1 corresponding to an inlet carvacrol nanoemulsion concentration of 160μg/mL. The reaction rate (r) was established based on the carvacrol concentration (Aμg/mL) as r=-0.149A0.373. The results obtained from numerical simulations indicated that the Reynolds number causes the most changes in the average value of Y1, while carvacrol concentration impacted most on the average value of Y2 response. The findings presented in this article provide insight into the probable kinetics of carvacrol nanoemulsion antibacterial activity in the microfluidics system.

Preclinical evaluation of a gel composition based on a flavonoid complex for the treatment of periodontal diseases in orthodontic patients

The aim of the work was a preclinical assessment of acute toxicity, skin resorptive, irritant effects, cumulative and catalase activity, as well as sensitizing properties of the local gel composition “Benzidaflaziverdine” (GCB) used for the treatment of periodontal diseases in orthodontic patients. Materials and methods. 119 animals were involved in the experiment, assigned to seven main and two control groups. GCB was administered intragastrically in doses of 300–600 mg/kg and intradermally of 200 μg into the outer surface of the ear. The native solution of GCB was applied to the skin and mucous membranes, administered orally by the method of “subchronic toxicity” and to the surface of the chorioallantoic membrane (CAM) of chicken embryos. The intensity of lipid peroxidation (LPO) was assessed by the level of diene conjugates (DCs) and malondialdehyde (MDA), and the antioxidant system by catalase activity. The specific leukocyte agglomeration reaction (SLAR), the specific leukocyte lysis reaction, and neutrophil damage indicators were used. Results. The median lethal dose LD50 for rats and mice of both sexes exceeded 5000 mg/kg. The irritant effect of GCB on the mucous membranes was manifested by hyperemia on the second day. Symptoms of irritation disappeared after 3–4 days without medical intervention. An analysis of the CAM blood vessels after exposure to GCB in two observations at the 120th second showed the beginning of hemorrhages. In one observation, GCB caused minor hemorrhages at the 300th second of the experiment. It was found that the coefficient of GCB irritant action was 5 (the mean score of Me (Q1; Q3) was 5 (4; 5)). The coefficient of cumulation (Kcum) exceeded 8.2. An insignificant increase in the median or mean values of catalase enzyme activity, DCs, and the amount of LPO end product such as MDA was observed compared to the control group animals. The SLAR test indicated the development of a delayed-type allergic reaction under the influence of GCB in a 1:10 dilution. One-hundred-fold dilution did not cause significant changes in the indicator in the main group compared to the control one. Conclusions. GCB belongs to the 4th class of toxicity – practically non-toxic substances, does not have sex- and species sensitivity, has weak cumulative activity, minimal effect on the system of LPO. GCB can be recommended for the use in clinical periodontology for medical support of orthodontic patients.

Dual-RNAseq Analysis Unravels Virus-Host Interactions of MetSV and Methanosarcina mazei.

Methanosarcina spherical virus (MetSV), infecting Methanosarcina species, encodes 22 genes, but their role in the infection process in combination with host genes has remained unknown. To study the infection process in detail, infected and uninfected M. mazei cultures were compared using dual-RNAseq, qRT-PCRs, and transmission electron microscopy (TEM). The transcriptome analysis strongly indicates a combined role of virus and host genes in replication, virus assembly, and lysis. Thereby, 285 host and virus genes were significantly regulated. Within these 285 regulated genes, a network of the viral polymerase, MetSVORF6, MetSVORF5, MetSVORF2, and the host genes encoding NrdD, NrdG, a CDC48 family protein, and a SSB protein with a role in viral replication was postulated. Ultrastructural analysis at 180 min p.i. revealed many infected cells with virus particles randomly scattered throughout the cytoplasm or attached at the cell surface, and membrane fragments indicating cell lysis. Dual-RNAseq and qRT-PCR analyses suggested a multifactorial lysis reaction in potential connection to the regulation of a cysteine proteinase, a pirin-like protein and a HicB-solo protein. Our study's results led to the first preliminary infection model of MetSV infecting M. mazei, summarizing the key infection steps as follows: replication, assembly, and host cell lysis.

Combustion characteristic of 2,3,3,3-tetrafluroropropene (R1234yf)

To describe the combustion process of hydrofluoroolefins (HFOs) 2,3,3,3-tetrafluroropropene (R1234yf), a kinetics mechanism was developed. On the M06–2X/6–311++G(d,p) level, dynamic calculations have been performed using the density function theory (DFT) method. Proposed R1234yf kinetic reactions included unimolecular decomposition and intermolecular reactions, such as collisions with O2, OH, and H radicals. The results indicated that R1234yf combustion is a step-by-step chain reaction involving chain initiation (unimolecular lysis and collision reaction with oxygen), chain transmission (collision reactions with free radicals), and chain termination. In conjunction with the priority rule of kinetics reactions, a schematic diagram of the primary microscopic combustion pathways for R1234yf was created at 298.15 K and 1 atm. All the simulation results could provide a data basis to establish the kinetic model of hydrofluoroolefin pyrolysis and combustion. In addition, to verify the product direction of the reaction path, experiments were conducted to determine the properties of the R1234yf combustion products.

Low-allergenic hydrolysates of whey proteins with natural bioactive peptides

Hydrolysate of whey protein concentrate (WPC) has been obtained under conditions that ensure retention of natural bioactive peptides. Prior to this, the WPC was characterised by electrophoresis, which revealed the presence of major whey proteins that can cause allergies and be precursors of bioactive peptides. The electrophoretic studies have allowed establishing that by the 120th minute, the proteolysis of the main protein allergens was almost complete. That is why this sample of WPC hydrolysate was used for further studies. Sephadex G-50 gel filtration has shown that 23.4% to 27.5% of proteolytic products soluble in trichloroacetic acid are low-molecular-weight peptides with a molecular weight up to 1500 Da, while the control WPC sample contains less than 3 % of them. The hydrolysate obtained under physiological conditions was tested for allergenicity. The study was conducted in 18 rats divided into three groups. Animals of the first group (control) were given water, the second group whey protein concentrate, the third group pancreatin hydrolysate of whey proteins. According to the results of the experiment, the concentration of IgE in the 2nd group is significantly higher compared with the control (49%), and in the 3rd group, does not differ from the control values. To detect possible sensitisation in the experimental animals, we used the specific leucocyte agglomeration reaction, the leucocyte specific lysis reaction, the values of the change in the concentration of circulating immune complexes, and the neutrophil damage index. The studies have shown that in the animals receiving WPC hydrolysate, no signs of an allergic reaction were detected, while the animals sensitised with WPC developed type I hypersensitivity (by the value of the IgE content).

Оценка сенсибилизирующего действия дезинфицирующего средства на основе глутарового альдегида при ингаляции

When investigating new disinfectants, it is necessary to assess the degree of their danger by modeling the conditions of use. As is known, the main danger of disinfectants, especially volatiles, is posed by the inhalation route of entry into the body, exerting an irritating effect on the respiratory organs, as well as a general toxic and sensitizing effect. Despite reasonable restrictions on the use of disinfectants from the aldehyde class for decontamination of the surfaces of premises and equipment, state registration receives disinfectants based on glutaraldehyde (GA), intended for wide use in various areas, including everyday life, which is unacceptable. Experiments were carried out to confirm the danger of using GA-based disinfectants. Sensitizing effect of GA-based disinfectant under conditions of surface treatment by rubbing method is studied. Significant release of GA into the air after surface treatment (27.7±4.1 mg/m3) was detected. The concentration of GA in the air decreased by 4.5 times after 6 hours of ventilation. The sensitizing effect of the GA-based agent was determined on guinea pigs by a provocative ear swelling test and a leukocyte specific lysis reaction. The results of the study indicate a pronounced sensitizing effect of the agent. Keywords: glutaraldehyde, toxicity, disinfectants, disinfection, sensitization, inhalation, guinea pigs.

Nanophotonic Cell Lysis and Polymerase Chain Reaction with Gravity-Driven Cell Enrichment for Rapid Detection of Pathogens.

Rapid and precise detection of pathogens is a critical step in the prevention and identification of emergencies related to health and biosafety as well as the clinical management of community-acquired urinary tract infections or sexually transmitted diseases. However, a conventional culture-based pathogen diagnostic method is time-consuming, permitting physicians to use antibiotics without ample clinical data. Here, we present a nanophotonic Light-driven Integrated cell lysis and polymerase chain reaction (PCR) on a chip with Gravity-driven cell enrichment Health Technology (LIGHT) for rapid precision detection of pathogens (<20 min). We created the LIGHT, which has the three functions of (1) selective enrichment of pathogens, (2) photothermal cell lysis, and (3) photonic PCR on a chip. We designed the gravity-driven cell enrichment via a nanoporous membrane on a chip that allows an effective bacterial enrichment of 40 000-fold from a 1 mL sample in 2 min. We established a light-driven photothermal lysis of preconcentrated bacteria within 1 min by designing the network of nanoplasmonic optical antenna on a chip for ultrafast light-to-heat conversion, created the nanoplasmonic optical antenna network-based ultrafast photonic PCR on a chip, and identified Escherichia coli. Finally, we demonstrated the end-point detection of up to 103 CFU/mL of E. coli in 10 min. We believe that our nanophotonic LIGHT will provide rapid and precise identification of pathogens in both developing and developed countries.

TOXICOLOGICAL EVALUATION OF BENZOPHENONE AS A BASIS OF ITS HYGIENIC NORMALIZATION IN THE AIR OF THE WORKING ZONE

For the purpose of hygienic rationing of benzophenone in the air of the working zone its toxic properties have been studied. DL50 for male rats, male and female mice amounted to 3000, 2000 and 1300 mg/kg, respectively. Benzophenone refers to moderately hazardous substances (hazard class III). The clinical picture of the acute poisoning with this substance was characterized by the low mobility, myorelaxation, shaky gait, breathing loss, weakened response to external stimuli and death on days 1 to 3 after the poisoning. There were no significant differences in the species and gender susceptibility of animals to the substance: coefficients of species differences and gender susceptibility ≈1.5. Benzophenone has an average cumulative capacity: a cumulation factor accounts for 3.2. It does not have a local irritant effect on the skin and skin-resorptive effect. It has a mild irritant effect on the mucous membranes of the eyes. There was no sensitizing effect in benzophenone: the specific leukocyte lysis reaction, the specific agglomeration of leukocytes and the eosinophil content in the blood of the experimental guinea pigs did not differ significantly from the control. After single 4-hour inhalation primers of benzophenone at concentrations of 35.2, 94.6 and 200 mg/m3, there were no violations of the synthetic and enzymatic functions of the liver. The threshold of acute inhalation action is 94.6 mg/ m3 (for decreasing body temperature, increasing the summation threshold and leukocytosis). Approximate safe level of exposure to benzophenone in the air in the working area is 2 mg/m3 (aerosol).

The ePrep-System: A new electrophoretic approach for DNA isolation from biological samples.

We present a new free-flow electrophoretic separation system that extends the established concepts of nucleic acid migration in an electric field to a broadly applicable preparative scale. The system comprises a disposable flow tube in which the target nucleic acids are separated from impurities by a balanced combination of electrophoretic migration and counter-streaming electroosmotic flow under the influence of an applied external electric field. Despite the complex theoretical background the introduced electrophoretic technology offers simple hardware setup and handling protocols. A variable number of small and disposable flow tubes can be processed in parallel, which largely eliminates the cumulative increase in extraction times inherent to batch processing methods and allows faster throughput of intermediate sample numbers. We demonstrate easy isolation of nucleic acids without user interaction during the run by using existing and well established lysis chemistries. Sample loading is realized by concentrated transfer of DNA-loaded magnetic beads from a lysis reaction into the extraction flow tube. The present study centers on the development of a functional model for the device and the flow tube as well as a preliminary standard extraction protocol. The system is compatible with a broad range of sample types and we present proof of principle data demonstrating its suitability for biomarker detection in translational research applications.

Abstract 367: Microfluidic devices for the interrogation of single circulating tumor cells

Abstract Introduction: Genetic and phenotypic characterization of Circulating Tumor Cells (CTC) offer the opportunity for a “real time liquid biopsy”. However, heterogeneity and rarity of CTC command the need for individual cell characterization. Following an enrichment procedure of CTC from blood, the identification, isolation and manipulation of single CTC for further analysis without cell loss remains challenging. Here, we present microfluidic devices for parallel single cell whole genome amplification (psc WGA) and parallel probing of drug response of single cancer cells (psc probing). Method: Microfluidic devices were designed using AUTOCAD software, and fabricated using PDMS multilayer soft-lithography. Cells from the SKBR-3 and MCF-7 breast cancer cell lines were used in the devices and identified using fluorescence microscopy after immunofluorescence staining. For pscWGA, the GE Single Cell GenomiPhi DNA Amplification kit was used under isothermal conditions. Results: In the 1st scWGA device, single cancer cells were addressed in 16 individual reaction chambers, subsequently lyzed, and their DNA amplified on a chip. We successfully amplified DNA of single cancer cells in a ca. 23 nanoliter reaction volume. 1,000-fold amplified DNAs were validated using qPCR targeting a set of genes on different chromosomes. For WGA of CTC present in a large number of other cells, we developed a 2nd scWGA platform by combining a self-sorting microwell cell sorter and a microfluidic device. After filtration of a cell suspension using a microwell plate, cancer cells were identified using fluorescence microscopy at the bottom of the plate. Cells of interest were subsequently punched into the open-well structures of the microfluidic device for further analysis. The lysis and WGA reaction buffers were loaded using peristaltic pumping of integrated micro-valves. After cell lysis, DNA was amplified in the open-well reaction chamber. For validation ∼ 100 ng of DNA was pipetted out of the well. We also developed microfluidic devices to study drug-dose response of single cancer cells. This device is capable of capturing single cells, dosing various concentrations of drugs and exposing the cells to the drugs. We optimized the capturing efficiency using different sizes of beads (3 μm, 6 μm, and 15 μm) as well as MCF-7 cells. We demonstrated that single cancer cells could be exposed to different drug candidates in the reagent chambers and their response measured. Conclusion: We successfully developed various microfluidic devices for individual cell characterization to be applied for CTC analysis. For genetic make-up, whole genome amplification of single cells either in suspension or in a self-sorting microwell plate was demonstrated. On-chip cell lysis and DNA amplification were performed and validated by qPCR targeting specific genes. In addition microfluidic devices were designed and tested to investigate single cell response to cancer drugs. Citation Format: Yoonsun Yang, Hoon Suk Rho, Joost F. Swennenhuis, Michiel Stevens, Arjan GJ Tibbe, Séverine Le Gac, Han Gardeniers, Leon WMM Terstappen. Microfluidic devices for the interrogation of single circulating tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 367. doi:10.1158/1538-7445.AM2015-367

Disposable on‐chip microfluidic system for buccal cell lysis, DNA purification, and polymerase chain reaction

This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off-chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip.

Experimental investigation and modeling of innovative five-tank anaerobic-anoxic/oxic process

A five-tank anaerobic-anoxic/oxic (A2/O) activated sludge process was investigated as innovative configuration in the activated sludge process technologies which has advantage of save energy power, cost and enhance nitrogen and phosphorus removal whereas it does not need equipment for sludge and mixed liquor recycle and also it required small land for construction. The five-tank process achieved 89.1%±1.37%, 87.78%±1.15%, 73.62%±2.13%, and 83.78%±0.92% of chemical oxygen demand, NH4+–N, TN, and total phosphorus (TP) removal efficiencies, respectively, during a 16-month operation with the effluent meeting Chinese sewage discharge standard GB18918-Grade A. A computer program was built based on activated sludge model No. 2d for simulating the performance of each compartment in five-tank process. The difficulty of simulation is coming from the system operation condition where it is operated under unsteadily state condition in all its compartments .The results showed that the growth rate constant of autotrophic organisms was 2.4day−1 and yield coefficient was 0.14. According to simulation results, heterotrophic organism; phosphate accumulating organism, and autotrophic organism are decreased in the anaerobic compartments because of the lysis reaction. Then these organisms are increased in the aerobic compartments due to aerobic growth. The heterotrophic organism; phosphate accumulating organism; and autotrophic organism are increased in quantities by about 56.6%, 36.12% and 74.31% in compartment one due to change the operation condition from anaerobic to aerobic and decreased in quantities by about 20.21%, 44.18%, and 0.142% in the compartment three due to change from aerobic to anoxic. The total nitrifying species to total active biomass was fluctuated between 1–11.89% in the process reactor.

Modeling and particular application of ASM2d model for describing organic matter and nutrient removal in a novel anaerobic-anoxic/oxic eight-phased system

An eight-phased AA/O process has advantages of saving energy power, cost, and enhancing nitrogen and phosphorus removal; it does not need equipment for sludge and mixed liquor recycle and also it required small land for construction. A computer program was built based on activated sludge model No. 2d (ASM2d) for simulating the performance of multi-tank AA/O activated sludge process in Wuxi campus, Southeast university. The difficulty of simulation is the system operation with unsteadily state condition. The results indicated that the growth rate constant of ammonia oxidizing bacteria was 1.4 day−1 and yield coefficient was 0.14. According to simulation, heterotrophic organism XH, phosphate accumulating organism XPAO, and ammonia oxidizing bacteria XAOB decreased in the anaerobic tanks because of the lysis reaction. Then the XH, XPAO, and XA increased in the aerobic tanks due to aerobic growth. The heterotrophic microorganism; phosphorus accumulating organism; and autotrophic bacteria concentrations increased in quantities by about 56, 36, and 74% in tank one due to changes in the environmental state condition from anaerobic to aerobic and decreased in quantities by about 20, 44, and 0.14% in the tank three due to changes in the environmental state condition from aerobic to anoxic. The ratio of total nitrifying species to total active biomass varied between 1 and 12% in multi-tank AA/O process. The multi-tank AA/O system achieved 89 ± 1.3%, 87.7 ± 1.1%, 73.6 ± 2.1%, and 83.7 ± 0.9% of chemical oxygen demand, NH4+ -N, TN, and total phosphorus (TP) removal efficiencies, respectively, during a six-month operation with the effluent meeting Chinese sewage discharge standard GB18918-Grade A.

Preparation of Genomic DNA from Mammalian Sperm

Semen consists of spermatozoa and the seminal fluid, also called seminal plasma. This fluid is important for the survival of the spermatozoa, but may decrease the purity and thus quality of the DNA due to its fructose and protein content. In this protocol, spermatozoa are washed with ethanol to remove the fluid. The spermatozoa themselves are protected by a membrane rich in disulfide bonds, which impede cell lysis and thus hamper DNA isolation. To break disulfide bonds a strong antioxidant, such as dithiothreitol (DDT), is necessary. Similar to other protocols for DNA isolation from other mammalian tissues, proteinase K and SDS are included in the lysis reaction. After lysis of the sperm cells, the DNA is precipitated using ethanol and then redissolved in TE-buffer or ddH(2)O.

Microfluidic Cytometer for the Characterization of Cell Lysis

Blood cytometry and intercellular analysis typically requires lysis as a preparatory step, which can alter the results of downstream analyses. We fabricated a microfluidic cytometer to characterize erythrocyte lysis kinetics. Forward light scatter from erythrocytes was used for enumeration at specific locations on a microfluidic chip. Diffusive transport coupled with laminar flow was used to control the concentration and exposure time of the lysis reagent Zap-OGLOBIN II to erythrocytes. Standard clinical practice is to expose erythrocytes to lysis reagent for 10 min. Under optimum conditions, we achieved complete erythrocyte lysis of a blood sample in 0.7 s. A maximum lysis reaction rate of 1.55 s(-1) was extrapolated from the data. Lysis began after 0.2 s and could be initiated with a lysis reagent concentration of 1.0% (68.5 mM). An equation that related lysis reagent concentration, [A], to erythrocyte lysis, [B], was determined to be [B] = -0.77[A](0.29)t.

Prevalence of imp type metallo-β-lactamase in carbapenem resistant pseudomonas aeruginosa and correlation of genotype to phenotype

Background Pseudomonas aeruginosa is a ubiquitous organism that is easily recovered from moist environments. It is encountered as a nosocomial pathogen and has become one of the most serious causes of nosocomial bacterial infections particularly in the immunocompromised and burned hosts as well as in patients with cystic fibrosis. Increasingly, Pseudomonas aeruginosa that are multidrug or pan-resistant are being isolated from clinical specimens. Metallo-b-lactamases (MBLs) which belong to Ambler class B b-lactamase have emerged as a therapeutic threat, as they often confer high-level resistance to all b-lactams except aztreo-nam. Aim The aim of this study is to identify the number of isolates that are carbapenem resistant due to the presence of an IMP type MBL from a storage collection in Queensland and to review the characteristics and epidemiology of these isolates. The secondary aim is to analyse whether the MBL genotypes correlate well with phenotypic detection. Method Previously stored carbapenem resistant Pseudomonas aeruginosa clinical isolates were retrieved. The minimum inhibitory concentrations (MICs) to two carbapenems (meropenem and imipenem) were defined using E-test (bioMerieux, France). Template DNA was prepared by heat lysis and polymerase chain reaction (PCR) was performed using IMP-1A and IMP-1B primers. Phenotypic testing was done using the same subculture by the modified Hodge test (MHT) and double disc synergy testing (DDST) methods. Results A total of 69 isolates was included in this study of which 14.5% (10 isolates) were positive for IMP type MBL. Of the 10 isolates, five were collected from intensive care units, three from medical wards and one each from the surgical ward and outpatients department. Phenotypic testing of the 10 isolates with the IMP type MBL showed negative results on MHT but was positive for the DDST. Conclusion This is the first study from Australia looking at the prevalence of IMP type MBL in carbapenem resistant Pseudomonas aeruginosa . The prevalence of IMP type MBL is 14.5% in carbapenem resistant Pseudomonas aeruginosa isolates in Queensland from as early back as 2002. Laboratory screening is important in identifying and preventing the further spread of MBL-producing organisms. The DDST is a relatively simple and economic test for this purpose. A positive screening test will need molecular confirmation to identify the MBL involved.

Development of PCR Microchip for Early Cancer Risk Prediction

An integrated continuous-flow microfluidic chip was fabricated on glass substrate with polydimethylsiloxane (PDMS)-based microchannels, cell lysis and Polymerase chain reaction (PCR) modules on the same chip. While gold-microelectrode was used for electrochemical cell lysis, indium-tin-oxide (ITO) microheater was used for thermal cycling during PCR reaction. The fabricated device was used for PCR amplification of pancreatic cancer DNA marker (SMAD4) from non-tumorigenic MCF10a human cell lines. The PCR product (193 bp) was verified for MCF10a cells by agarose gel electrophoresis after 20 cycles of reaction on the microchip, whereas no product was detected in case of tumorigenic MCF7 cells. The total time required for the entire reaction was less than 45 min. Therefore, the proposed microchip can be helpful in predicting the risk of metastatic cancer by analysis of genetic tumor markers from human samples and can also be used for other genetic analysis involving PCR reaction.

Analysis of gene expression at the single-cell level using microdroplet-based microfluidic technology

In the present work, we have measured the messenger RNA expression of specific genes both from total RNA and cells encapsulated in droplets. The microfluidic chip introduced includes the following functionalities: RNA∕cell encapsulation, lysis, reverse transcription and real-time polymerase chain reaction. We have shown that simplex and duplex gene expression measurements can be carried out over a population of 100 purified RNA samples encapsulated simultaneously in 2 nl droplets in less than 2 h. An analysis of 100 samples containing one to three cells has shown excellent consistency with standard techniques regarding average values. The cell-to-cell distributions of the E-cadherin expression suggest fluctuations on the order of 80% in the number of transcripts, which is highly consistent with the general findings from the literature. A mathematical model has also been introduced to strengthen the interpretation of our results. The present work paves the way for the systematic acquisition of such information in biological and biomedical studies.

상피세포 시료 전처리용 마이크로바이오칩에 관한 연구

This paper describes a separable microfluidic device fabricated with PDMS (polydimethylsiloxane) and glass. The device is used for sample preparation involving cell lysis and the DNA purification process. The cell lysis was performed for 2 min at 80°C in a serpentine-type microreactor (20 l) using a Au microheater that was integrated with μ a thermal microsensor on a glass substrate. The DNA that was mixed with other residual products during the cell lysis process was then filtered through a new filtration system composed of microbeads (diameter: 50 m) and PDMS pillars. μ Since the entire process (sample loading, cell lysis reaction, DNA purification, and sample extraction) was performed within 5 min in a microchip, we could reduce the sample preparation time in comparison with that for the conventional methods used in biochemistry laboratories. Finally, we verified the performance of the sample preparation chip by conducting PCR (polymerase chain reaction) analysis of the chip product.