Vacuum/Compression Valving (VCV) Using Parrafin-Wax on a Centrifugal Microfluidic CD Platform

This paper introduces novel vacuum/compression valves (VCVs) utilizing paraffin wax. A VCV is implemented by sealing the venting channel/hole with wax plugs (for normally-closed valve), or to be sealed by wax (for normally-open valve), and is activated by localized heating on the CD surface. We demonstrate that the VCV provides the advantages of avoiding unnecessary heating of the sample/reagents in the diagnostic process, allowing for vacuum sealing of the CD, and clear separation of the paraffin wax from the sample/reagents in the microfluidic process. As a proof of concept, the microfluidic processes of liquid flow switching and liquid metering is demonstrated with the VCV. Results show that the VCV lowers the required spinning frequency to perform the microfluidic processes with high accuracy and ease of control.     

Culture and Motion Analysis of Diatom Bacillaria paradoxa on a Microfluidic Platform

We proved the feasibility of using a microfluidic chip to culture diatom Bacillaria paradoxa, and analyzed the gliding characteristics of its self-organized colony in detail. The optimal cultivation parameters of B. paradoxa for the designed chip made with polydimethylsiloxane are as follows: the preferable cells injecting rate for keeping the cells alive is 0.2 mL/h, the initial cell density for fast reproduction is 5.5 × 10⁴ cells/mL, and the optimal replacement period of culture medium is 4 days. B. paradoxa tends to form a colony during their growth, and the colony can glide with a steady period of 29 ± 3 s along its axial direction in a constant stream, the amplitude of the colony will not decay (e.g., 24 μm of two-cell colony at 1.1 mm/s flow rate), and the colony rapidly adjusts its direction of gliding to the direction of water flow. The successful culture of diatoms on a microfluidic platform may be used for biosensing chips and the creation of gasoline-producing diatom solar panels.     

植物微流芯片——一种实时定量监测生长发育的 高通量整合分析平台

生长发育是一个复杂的动态过程, 了解其发生细节是生命科学研究的重要内容。最新发展起来的微流芯片技术为实现这个目标提供了新的途径。动物及微生物中的应用表明, 该技术兼有实时定量监测和高通量整合处理的优势。在植物研究领域, 用针对根生长特点和要求设计的根微流芯片结合荧光共振能量转移探针已经成功地检测出拟南芥(Arabidopsis thaliana)根细胞内葡萄糖和游离的Ca²⁺、Zn²⁺的浓度。随着各种底物特异的荧光共振能量转移探针的开发和应用, 根微流芯片还可以用来检测植物细胞内激素或其它代谢中间产物的浓度及其动态变化过程。不仅如此, 以微流芯片为基础发展起来的Plant Chip和Tip Chip则为研究植物与微生物的相互作用以及植物花粉管极性生长和细胞分裂分化提供了理想的平台。作为了解遗传因素或环境刺激导致细胞生命活动变化细节的有力工具, 微流芯片技术有望为植物研究领域带来更多新的进展和突破。     

Peptide Bond Synthesis by a Mechanism Involving an Enzymatic Reaction and a Subsequent Chemical Reaction

We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and l-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which includes many kinds of enzymes, including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(d-alanyl)-l-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-l-cysteine as an intermediate via its “enzymatic activity” and (ii) subsequent “chemical” S → N acyl transfer in the intermediate, resulting in peptide formation. Step ii is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step i is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in native chemical ligation. Together with these findings, the same mechanism-dependent formation of N-acyl compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate; thus, not only the corresponding peptides but also new types of amide compounds can be formed.     

Effects of Prechilling and Sequential Washing on Enumeration of Microorganisms from Refuse

Techniques were evaluated for formation of a liquid inoculum from shredded municipal refuse, including chilling the refuse at 4°C prior to blending and multiple washing and blending cycles. The average count of cellulolytic bacteria from six different detachment treatments was 5.1 × 10⁴ cells per g (dry weight) of refuse with a range of 0.7 × 10⁴ to 12.7 × 10⁴ cells per g (dry weight). The liquid obtained from blending the refuse in phosphate buffer followed by hand squeezing was the selected detachment procedure. The inoculum formation procedure was validated by the addition of ruminal cellulolytic bacteria to refuse and recovery of the cellulolytic bacteria by most-probable-number enumerations. The ratio of measured to expected cell counts among tests in which different volumes of ruminal fluid were added to refuse ranged from 2.7 to 14.4. There was no evidence of anaerobic cellulolytic fungi in a refuse sample.     

Detection of Antigen-antibody Reaction Using a Fluorescent Probe and Its Application to Homogeneous Competitive-type Immunoassay for Insulin

The interaction of bovine insulin with anti-human insulin antibody (mAb) was examined using a fluorescent probe. The fluorescence intensity of fluoresceinthiocarbamyl (FTC)-insulin was increased by adding mAb, and the increase was saturated at 53% at a molar ratio of FTC-insulin to mAb of 2.0. Based on the change in fluorescence intensity, a standard curve of the homogeneous competitive-type immunoassay was constructed, and the detection range of insulin was found to be 50–400 nM.     

Inspection of an Acyl-Enzyme Intermediate in a Lipase Reaction by Gas Chromatography-Mass Spectrometry and Modelling of the Reaction Mechanism

An acyl-enzyme intermediate proposed in the reaction mechanism of lipase was inspected by the exchange of oxygen between substrate (oleic acid) and solvent (¹⁸O-labelled water). Gas chromatography-mass spectrometry analysis supported the formation of an acyl-enzyme intermediate in the reaction mechanism through the observed incorporation of ¹⁸O into oleic acid. The incorporation did not occur in the absence of the lipase. When Ser residues were modified with diisopropylfluorophosphate, the activity of lipase OF 360 was markedly decreased. Photooxidation of His residues also resulted in a decrease in the activity of the lipase. Chemical modification studies suggested the existence of a charge relay system (Ser-His-Asp/Glu) in the active site. Based on these results, a model of the active site and reaction mechanism of the lipase are presented.     

Structural Evidence for a Sequential Release Mechanism for Activation of Heterotrimeric G Proteins

Heptahelical G-protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors couple to heterotrimeric G proteins to relay extracellular signals to intracellular signaling networks, but the molecular mechanism underlying guanosine 5′-diphosphate (GDP) release by the G protein α-subunit is not well understood. Amino acid substitutions in the conserved α5 helix of G_i, which extends from the C-terminal region to the nucleotide-binding pocket, cause dramatic increases in basal (receptor-independent) GDP release rates. For example, mutant Gα_i1-T329A shows an 18-fold increase in basal GDP release rate and, when expressed in culture, it causes a significant decrease in forskolin-stimulated cAMP accumulation. The crystal structure of Gα_i1-T329A·GDP shows substantial conformational rearrangement of the switch I region and additional striking alterations of side chains lining the catalytic pocket that disrupt the Mg⁺² coordination sphere and dislodge bound Mg⁺². We propose a “sequential release” mechanism whereby a transient conformational change in the α5 helix alters switch I to induce GDP release. Interestingly, this mechanistic model for heterotrimeric G protein activation is similar to that suggested for the activation of the plant small G protein Rop4 by RopGEF8.     

Cloud-Enabled Microscopy and Droplet Microfluidic Platform for Specific Detection of Escherichia coli in Water

We report an all-in-one platform – ScanDrop – for the rapid and specific capture, detection, and identification of bacteria in drinking water. The ScanDrop platform integrates droplet microfluidics, a portable imaging system, and cloud-based control software and data storage. The cloud-based control software and data storage enables robotic image acquisition, remote image processing, and rapid data sharing. These features form a “cloud” network for water quality monitoring. We have demonstrated the capability of ScanDrop to perform water quality monitoring via the detection of an indicator coliform bacterium, Escherichia coli, in drinking water contaminated with feces. Magnetic beads conjugated with antibodies to E. coli antigen were used to selectively capture and isolate specific bacteria from water samples. The bead-captured bacteria were co-encapsulated in pico-liter droplets with fluorescently-labeled anti-E. coli antibodies, and imaged with an automated custom designed fluorescence microscope. The entire water quality diagnostic process required 8 hours from sample collection to online-accessible results compared with 2–4 days for other currently available standard detection methods.     

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15–1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.     

Multi-Channel Microfluidic Biosensor Platform Applied for Online Monitoring and Screening of Biofilm Formation and Activity

Bacterial colonization of surfaces and interfaces has a major impact on various areas including biotechnology, medicine, food industries, and water technologies. In most of these areas biofilm development has a strong impact on hygiene situations, product quality, and process efficacies. In consequence, biofilm manipulation and prevention is a fundamental issue to avoid adverse impacts. For such scenario online, non-destructive biofilm monitoring systems become important in many technical and industrial applications. This study reports such a system in form of a microfluidic sensor platform based on the combination of electrical impedance spectroscopy and amperometric current measurement, which allows sensitive online measurement of biofilm formation and activity. A total number of 12 parallel fluidic channels enable real-time online screening of various biofilms formed by different Pseudomonas aeruginosa and Stenotrophomonas maltophilia strains and complex mixed population biofilms. Experiments using disinfectant and antibiofilm reagents demonstrate that the biofilm sensor is able to discriminate between inactivation/killing of bacteria and destabilization of biofilm structures. The impedance and amperometric sensor data demonstrated the high dynamics of biofilms as a consequence of distinct responses to chemical treatment strategies. Gene expression of flagellar and fimbrial genes of biofilms grown inside the microfluidic system supported the detected biofilm growth kinetics. Thus, the presented biosensor platform is a qualified tool for assessing biofilm formation in specific environments and for evaluating the effectiveness of antibiofilm treatment strategies.     

Metastability of Papain and the Molecular Mechanism for its Sequential Acid-Denaturation

Acid unfolding of non-inhibited papain at pH 2 was studied by means of spectroscopic and electrophoresis techniques as well as activity assays. We found a molten globule like species (A state) similar to that previously reported for bromelain and S-carboxy-methyl-papain. We demonstrated that this A state is not thermodynamically stable but a metastable conformer which decays into an unfolded conformation in a few hours. The mechanism of acid unfolding to the A state proved to be completely irreversible, with a biphasic time evolution of spectroscopic signals characteristic of the existence of a kinetic intermediate. This latter species showed properties in-between native and A state such as secondary structure, exposition of hydrophobic area and tryptophan environment, but a native like hydrodynamic radius. Native papain seems to unfold at acid pH through at least two kinetic barriers, being its proregion mandatory to conduct and stabilize its active structure. Computer simulations of acid unfolding, followed by ANS docking, identified three regions of cavity formation induced by acid media which might be used as regions to be fortified by protein engineering in the quest for extreme-resistant proteases or as hot-spots for protease inactivation.     

A Novel Strategy for Detection and Enumeration of Circulating Rare Cell Populations in Metastatic Cancer Patients Using Automated Microfluidic Filtration and Multiplex Immunoassay

Size selection via filtration offers an antigen-independent approach for the enrichment of rare cell populations in blood of cancer patients. We evaluated the performance of a novel approach for multiplex rare cell detection in blood samples from metastatic breast (n = 19) and lung cancer patients (n = 21), and healthy controls (n = 30) using an automated microfluidic filtration and multiplex immunoassay strategy. Captured cells were enumerated after sequential staining for specific markers to identify circulating tumor cells (CTCs), circulating mesenchymal cells (CMCs), putative circulating stem cells (CSCs), and circulating endothelial cells (CECs). Preclinical validation experiments using cancer cells spiked into healthy blood demonstrated high recovery rate (mean = 85%) and reproducibility of the assay. In clinical studies, CTCs and CMCs were detected in 35% and 58% of cancer patients, respectively, and were largely absent from healthy controls (3%, p = 0.001). Mean levels of CTCs were significantly higher in breast than in lung cancer patients (p = 0.03). Fifty-three percent (53%) of cancer patients harbored putative CSCs, while none were detectable in healthy controls (p<0.0001). In contrast, CECs were observed in both cancer and control groups. Direct comparison of CellSearch^® vs. our microfluidic filter method revealed moderate correlation (R² = 0.46, kappa = 0.47). Serial blood analysis in breast cancer patients demonstrated the feasibility of monitoring circulating rare cell populations over time. Simultaneous assessment of CTCs, CMCs, CSCs and CECs may provide new tools to study mechanisms of disease progression and treatment response/resistance.     

Assessing Adhesion Forces of Type I and Type IV Pili of Xylella fastidiosa Bacteria by Use of a Microfluidic Flow Chamber

Xylella fastidiosa, a bacterium responsible for Pierce's disease in grapevines, possesses both type I and type IV pili at the same cell pole. Type IV pili facilitate twitching motility, and type I pili are involved in biofilm development. The adhesiveness of the bacteria and the roles of the two pili types in attachment to a glass substratum were evaluated using a microfluidic flow chamber in conjunction with pilus-defective mutants. The average adhesion force necessary to detach wild-type X. fastidiosa cells was 147 ± 11 pN. Mutant cells possessing only type I pili required a force of 204 ± 22 pN for removal, whereas cells possessing only type IV pili required 119 ± 8 pN to dislodge these cells. The experimental results demonstrate that microfluidic flow chambers are useful and convenient tools for assessing the drag forces necessary for detaching bacterial cells and that with specific pilus mutants, the role of the pilus type can be further assessed.     

Sequential effects of prostaglandins and interferon-gamma on differentiation of CD8+ suppressor cells

We have previously demonstrated that differentiation of CD8+ Tp44- suppressor cells in pokeweed mitogen (PWM)-stimulated cultures requires soluble factors elaborated by CD4+ cells and monocytes, and that the monocyte signal for such differentiation can be replaced by prostaglandin E2 (PGE2). In this study, we explored the ability of interleukin 2 (IL 2) and interferon-gamma (IFN-gamma) to replace the CD4+ signal. When IL 2 or IFN-gamma was used at concentrations equivalent to those present in supernatants of PWM-pulsed cultures of CD4+ cells, no effect on differentiation of CD8+ cells was observed. However, a potent suppressor inducing activity was detected when IFN-gamma, but not IL 2, was mixed with supernatants derived from cultures of PWM-pulsed purified monocytes (M phi sup) or with 10(-8) M PGE2. Differentiated CD8+ suppressor cells (Ts) inhibited both PWM-stimulated proliferative response of CD4+ cells and immunoglobulin production by B cells. The signals mediated by the M phi sup or PGE2 and IFN-gamma were shown to act sequentially. That is, M phi sup or PGE2 was required initially, followed by an IFN-gamma-dependent differentiative step. These studies thus suggest a cascade of cellular interactions involving monocytes, CD4+ cells, and CD8+ Ts precursors that are required for the differentiation of CD8+ suppressor effector cells.     

紫外线辐照聚苯乙烯微孔板用于酶免疫测定的研究与表征

以重组人钙调素（rhCaM）、亲环素（rhCyP）、心磷脂和双链DNA（dsDNA）为包被抗原,建立了检测针对上述4种抗原自身抗体的间接ELISA方法,并对聚苯乙烯微孔板（PS）紫外线（UV）辐照前后进行了对比研究.结果发现：PS板经UV-辐照后,可显著改善酶免疫分析的测定效果,自身抗体的测定敏感度和重复性均有显著提高.原子力学显微镜（AFM）表征结果则提供了改善酶免疫分析的直接证据,抗原分子均匀平铺于UV-辐照的PS基底表面,而未经辐照的PS板则抗原分子的吸附率低,且分布不均并有成团聚集现象.X射线光电子能谱（XPS）分析表明,PS板经UV-辐照后,基底表面发生了氧化并引入了含氧的活性基团,O/C元素比较辐照前提高了6.9倍,改善了对抗原生物分子的亲水和化学反应性能,此亦即UV-辐照PS板改善对抗原分子固定效果的主要原因.     

Thermal and Spectroscopic Characterization of a Proton Pumping Rhodopsin from an Extreme Thermophile

So far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. In this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus, which lives in a hot spring at around 75 °C. The gene for the retinylidene protein, named thermophilic rhodopsin (TR), was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of Escherichia coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans-retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 °C, and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (200 ×), Haloquadratum walsbyi bacteriorhodopsin (by 10-times), and Gloeobacter rhodopsin (100 ×). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have high stability. TR should allow the investigation of the molecular mechanisms of ion transport and protein folding.     

Development and Validation of an Immunoassay for Quantification of Topoisomerase I in Solid Tumor Tissues

Background

Topoisomerase I (Top1) is a proven target for cancer therapeutics. Recent data from the Fluorouracil, Oxaliplatin, CPT-11: Use and Sequencing (FOCUS) trial demonstrated that nuclear staining of Top1 correlates with chemotherapeutic efficacy. Such a correlation may help identify patients likely to respond to Top1 inhibitors and illuminate their mechanism of action. Cellular response to Top1 inhibitors is complex, but Top1 target engagement is a necessary first step in this process. This paper reports the development and validation of a quantitative immunoassay for Top1 in tumors.

Methodology/Principal Findings

We have developed and validated a two-site enzyme chemiluminescent immunoassay for quantifying Top1 levels in tumor biopsies. Analytical validation of the assay established the inter-day coefficient of variation at 9.3%±3.4% and a 96.5%±7.3% assay accuracy. Preclinical fit-for-purpose modeling of topotecan time- and dose-effects was performed using topotecan-responsive and -nonresponsive xenografts in athymic nude mice. Higher baseline levels of Top1 were observed in topotecan-responsive than -nonresponsive tumors. Top1 levels reached a maximal decrease 4 to 7 hours following treatment of engrafted mice with topotecan and the indenoisoquinoline NSC 724998.

Conclusions/Significance

Our analysis of Top1 levels in control and treated tumors supports the previously proposed mechanism of action for Top1 inhibitor efficacy, wherein higher baseline Top1 levels lead to formation of more covalent-complex-dependent double-strand break damage and, ultimately, cell death. In contrast, xenografts with lower baseline Top1 levels accumulate fewer double-stand breaks, and may be more resistant to Top1 inhibitors. Our results support further investigation into the use of Top1 levels in tumors as a potential predictive biomarker. The Top1 immunoassay described in this paper has been incorporated into a Phase I clinical trial at the National Cancer Institute to assess pharmacodynamic response in tumor biopsies and determine whether baseline Top1 levels are predictive of response to indenoisoquinoline Top1 inhibitors.