Mechanical Cell Lysis Research Articles

Turbolysis: A low-cost, small footprint alternative to commercial bead beaters for cell lysis

turboLysis is a novel mechanical cell lysis device that utilizes small beads to efficiently lyse tough cells like Mycobacterium, Saccharomyces, and Arabidopsis. We compared turboLysis to bead beating using the BeadBug 6 for several concentrations of Mycobacterium tuberculosis roughly correlated to the bacterial load commonly seen in patient samples. turboLysis performed similarly to the BeadBug at low bacterial concentrations and outperformed it at high concentrations above 2x105 CFU/ml (p < 0.005). Thus, turboLysis offers good cell lytic performance in a small form factor at a low cost.

An optimised protocol for the expression and purification of adenovirus core protein VII

Adenovirus protein VII (pVII) is a highly basic core protein, bearing resemblance to mammalian histones. Despite its diverse functions, a comprehensive understanding of its structural intricacies and the mechanisms underlying its functions remain elusive, primarily due to the complexity of producing a good amount of soluble pVII. This study aimed to optimise the expression and purification of recombinant pVII from four different adenoviruses with a simple vector construct. This study successfully determined the optimal conditions for efficiently purifying pVII across four adenovirus species, revealing the differential preference for bacterial expression systems. The One Shot BL21 Star (DE3) proved favourable over Rosetta 2 (DE3) pLysS with consistent levels of expression between IPTG-induced and auto-induction. We demonstrated that combining chemical and mechanical cell lysis is possible and highly effective. Other noteworthy benefits were observed in using RNase during sample processing. The addition of RNase has significantly improved the quality and quantity of the purified protein as confirmed by chromatographic and western blot analyses. These findings established a solid groundwork for pVII purification methodologies and carry the significant potential to assist in unveiling the core structure of pVII, its arrangement within the core, DNA condensation intricacies, and potential pathways for nuclear transport.

Combination of direct boiling and glass beads increases the purity and accuracy of bacterial DNA extraction.

Extraction of DNA is a key step in molecular biology experiments and important for counting tiny microbial individuals. Direct boiling and mechanical cell lysis like glass beads are two independent physical extraction methods, thus crossing the barriers of thresholds of magnitude in popular reagent kits or traditional spread plate method when non-equilibrium phenomenon is ongoing. The two approaches above were combined to generate a new one. In three typical microbial species, direct boiling with glass beads significantly increased the purity of DNA solution compared with some other methods (p<0.05). The qPCR results of them were closer to direct microscopy counting than some other methods. Therefore, it provides a new choice in extracting bacterial DNA for specific circumstances.

Acoustofluidic lysis of cancer cells and Raman spectrum profiling.

The lysis of cancer cells inside a sessile droplet was performed using traveling surface acoustic waves (SAWs) without any chemical reagents. Raman spectrum profiling was then carried out to explore detailed cell-derived data. The Rayleigh waves formed by an interdigital transducer were made to propagate along the surface of an LiNbO3 substrate. Polystyrene microparticles (PSMPs) were used to establish mechanical cell lysis effectively, and gold nanoparticles (AuNPs) were added to enhance the Raman signals from the lysed cells by SAWs. The lysis efficiency was evaluated according to the size and concentration of the PSMPs in experiments where the frequency was varied. Lysis occurred mainly by mechanical collision using PSMPs in a high-frequency domain, and the lysis efficiency was improved by increasing the application time and the energy density of the SAWs. Raman signals from the lysed cells were greatly enhanced by nanogaps formed by the AuNPs, which were evenly distributed irrespective of the SAWs through the frequency-independent behavior of the AuNPs. Finally, detailed Raman spectra of MDA-MB-231, malignant breast cancer cells, were acquired, and various organic matter-derived peaks were observed. The 95% confidence region for cells subjected to lysis was more widely distributed than that of cells not subjected to lysis. The proposed SAW platform is expected to facilitate the detection of small quantities and to be applied in biomedical applications.

Advanced unified monophasic lipid extraction protocol with wide coverage on the polarity scale optimized for large-scale untargeted clinical lipidomics analysis of platelets

Lipid extraction is a critical step in sample preparation of lipidomics studies. Biphasic liquid-liquid extraction protocol with methyl tert-butyl ether (MTBE)/methanol (MeOH) as organic solvents are widely adopted by researchers nowadays as an eco-friendly replacement of classic Folch, and Bligh&Dyer protocols. Yet, it has some limitations such as suboptimal performance for the most polar lipids (e.g. acylcarnitines), complicated handling as it requires phase separation, and is therefore non-ideal for large-scale clinical studies. To advance the extraction protocol for large-scale clinical lipidomics, in this study we explored i) 6 different extraction solvent systems, ii) distinct processing procedures (sonication, mechanical cell lysis and bead homogenizer), and iii) also 7 different reconstitution solvents. The extraction systems investigated included biphasic systems MTBE/MeOH/H2O and Hexane/2-propanol (IPA)/1 M acetic acid, and monophasic systems MTBE/MeOH/CHCl3, IPA/H2O (90% IPA), MeOH/MTBE/IPA, and IPA/H2O/MTBE as solvent system for lipid extraction of human platelets. Extraction recovery was investigated by repeated extraction cycles. Subcellular extraction efficiency was assessed by the mitochondria-specific cardiolipins. It turned out that monophasic extraction with MeOH/MTBE/IPA (1.3:1:1, v/v/v), bead homogenizer for cell disruption and MeOH/MTBE (1:1, v/v) as reconstitution solvent provide optimal cellular and subcellular extraction efficiencies for both polar (e.g. acylcarnitines) and apolar lipids (e.g. triglycerides). It is simplified (no phase separation), eco-friendly (reduced solvent consumption and no halogenated ones), fast (5 min for 24 samples in parallel), and can be easily adapted for cells, plasma, and tissue. Therefore, it has great potential for large-scale clinical lipidomics studies.

Comparison Analysis of Different DNA Extraction Methods on Suitability for Long-Read Metagenomic Nanopore Sequencing.

Metagenomic next-generation sequencing (mNGS) is a novel useful strategy that is increasingly used for pathogens detection in clinic. Some emerging mNGS technologies with long-read ability are useful to decrease sequencing time and increase diagnosed accuracy, which is of great significance in rapid pathogen diagnosis. Reliable DNA extraction is considered critical for the success of sequencing; hence, there is thus an urgent need of gentle DNA extraction method to get unbiased and more integrate DNA from all kinds of pathogens. In this study, we systematically compared three DNA extraction methods (enzymatic cell lysis based on MetaPolyzyme, mechanical cell lysis based on bead beating, and the control method without pre–cell lysis, respectively) by assessing DNA yield, integrity, and the microbial diversity based on long-read nanopore sequencing of urine samples with microbial infections. Compared with the control method, the enzymatic-based method increased the average length of microbial reads by a median of 2.1-fold [Inter Quartile Range (IQR), 1.7–2.5; maximum, 4.8) in 18 of the 20 samples and the mapped reads proportion of specific species by a median of 11.8-fold (Inter Quartile Range (IQR), 6.9–32.2; maximum, 79.27]. Moreover, it provided fully (20 of 20) consistent diagnosed results to the clinical culture and more representative microbial profiles (P < 0.05), which all strongly proves the excellent performance of enzymatic-based method in long-read mNGS–based pathogen identification and potential diseases diagnosis of microbiome related.

Optimizing Nanopore Sequencing for Rapid Detection of Microbial Species and Antimicrobial Resistance in Patients at Risk of Surgical Site Infections.

ABSTRACTSurgical site infections (SSI) are a significant burden to patients and health care systems. We evaluated the use of Nanopore sequencing (NS) to rapidly detect microbial species and antimicrobial resistance (AMR) genes present in intraoperative bile aspirates. Bile aspirates from 42 patients undergoing pancreatic head resection were included. Three methods of DNA extraction using mechanical cell lysis or protease cell lysis were compared to determine the optimum method of DNA extraction. The impact of host DNA depletion, sequence run duration, and use of different AMR gene databases was also assessed. To determine clinical value, NS results were compared to standard culture (SC) results. NS identified microbial species in all culture positive samples. Mechanical lysis improved NS detection of cultured species from 60% to 76%, enabled detection of fungal species, and increased AMR predictions. Host DNA depletion improved detection of streptococcal species and AMR correlation with SC. Selection of AMR database influenced the number of AMR hits and resistance profile of 13 antibiotics. AMR prediction using CARD and ResFinder 4.1 correctly predicted 79% and 81% of the bile antibiogram, respectively. Sequence run duration positively correlated with detection of AMR genes. A minimum of 6 h was required to characterize the biliary microbes, resulting in a turnaround time of 14 h. Rapid identification of microbial species and AMR genes can be achieved by NS. NS results correlated with SC, suggesting that NS may be useful in guiding early antimicrobial therapy postsurgery.IMPORTANCE Surgical site infections (SSI) are a significant burden to patients and health care systems. They increase mortality rates, length of hospital stays, and associated health care costs. To reduce the risk of SSI, surgical patients are administered broad-spectrum antibiotics that are later adapted to target microbial species detected at the site of surgical incision. Use of broad-spectrum antibiotics can be harmful to the patient. We wanted to develop a rapid method of detecting microbial species and their antimicrobial resistance phenotypes. We developed a method of detecting microbial species and predicting resistance phenotypes using Nanopore sequencing. Results generated using Nanopore sequencing were similar to current methods of detection but were obtained in a significantly shorter amount of time. This suggests that Nanopore sequencing could be used to tailor antibiotics in surgical patients and reduce use of broad-spectrum antibiotics.

A Flexible Microdevice for Mechanical Cell Stimulation and Compression in Microfluidic Settings

Evidence continues to emerge that cancer is a disease not only of genetic mutations, but also of altered mechanobiological profiles of the cells and microenvironment. This mutation-independent element might be a key factor in promoting development and spread of cancer. Biomechanical forces regulate tumor microenvironment by solid stress, matrix mechanics, interstitial pressure, and flow. Compressive stress by tumor growth and stromal tissue alters cell deformation and recapitulates the biophysical properties of cells to grow, differentiate, spread, or invade. Such solid stress can be introduced externally to change the cell response and to mechanically induce cell lysis by dynamic compression. In this work, we report a microfluidic cell culture platform with an integrated, actively modulated actuator for the application of compressive forces on cancer cells. Our platform is composed of a control microchannel in a top layer for introducing external force and a polydimethylsiloxane (PDMS) membrane with monolithically integrated actuators. The integrated actuator, herein called micro-piston, was used to apply compression on SKOV-3 ovarian cancer cells in a dynamic and controlled manner by modulating applied gas pressure, localization, shape, and size of the micro-piston. We report fabrication of the platform, characterization of the mechanical actuator experimentally and computationally, and cell loading and culture in the device. We further show the use of the actuator to perform both repeated dynamic cell compression at physiological pressure levels and end point mechanical cell lysis, demonstrating suitability for mechanical stimulation to study the role of compressive forces in cancer microenvironments. Finally, we extend cell compression applications in our device to investigating mechanobiologically related protein and nuclear profiles in cyclically compressed cells.

Optimized protocol proposal to extract eDNA from oligotrophic and degraded water samples

Aquifer represents an oligotrophic environment that sustains a relatively small amount of microbial cells, mostly non-culturable. Due to this dominance of unculturable microorganisms in natural ecosystems, studying microbial communities and their functionality should include culture-independent approaches based on molecular techniques using DNA analysis (Purswani et al. 2011). For practical reasons, aquifer routine analyses focus on groundwater samples, while solid aquifer samples are typically not included (Ritalahti et al. 2010). The amount of groundwater collected, together with the types and concentrations of inhibitory compounds if present, determine the abundance of the target biomarker(s) available for subsequent analyses. Hence, filtering large volumes of groundwater seems beneficial, but for practical purposes, 0.5–2 L of water are typically collected, depending on groundwater characteristics (Ritalahti et al. 2010). In this work, environmental DNA (eDNA) was extracted from groundwater samples, filtering three different initial volumes (1000 ml, 500 ml, and 250 ml) of water samples, using 0.22 µm membranes. Also, two DNA extraction commercial kits were tested, DNeasy PowerWater Kit and DNeasy PowerSoil kit (Qiagen, Germantown, MD) specific for water samples and solid matrix, respectively. For DNeasy PowerWater Kit the standard protocol was carried out, whereas a modified protocol equipped with the Inhibitor Removal Technology® (IRT) was selected for testing DNeasy PowerSoil kit. In order to minimize operator bias, both the protocols were made semi-automated by using a QIAcube provided by Qiagen for genomic DNA extraction. Additional steps to the PowerSoil IRT protocol were performed for optimizing chemical and mechanical cell lysis processes and facilitating the sample dispersion into the buffer solutions. eDNA was verified via electrophoresis and quantified fluorometrically. eDNA extracted from 250 ml of groundwater sample using the DNeasy PowerSoil kit with modified IRT protocol was also tested in downstream applications, including Polymerase Chain Reactions (PCRs) with specific primer pairs for the identification of microbial targets. Results suggest that the PowerSoil modified IRT protocol was the best performing one, allowing a higher eDNA yield from all the water sample volumes tested. In addition, plotting on a graph eDNA concentration values against sample volumes filtered, the yield of PowerSoil modified IRT protocol appeared more similar to an ideal direct proportionality than the yield of PowerWater standard protocol. eDNA quality was suitable for PCR analyses and the identification of bacterial targets, including bacterial subgroups (α and β-proteobacteria) and single species of interest, such as Shewanella oneidensis capable of hexavalent chromium reduction (Tumolo et al. 2020). Under these good performances, the PowerSoil optimized IRT protocol was also applied in a further experiment about bioremediation to extract eDNA from 50 ml of water spiked with 1000 µg/l of hexavalent chromium. The resulting genomic material was successfully used in quantitative PCR (qPCR) assays for monitoring the relative abundance of Shewanella oneidensis during the bioremediation process, allowing to highlight the hexavalent chromium inhibitor effect on the selected microbial target (Ancona et al. 2020). Downstream applications of eDNA obtained using DNeasy PowerSoil kit resulted in positive outcomes for both the experiments previously described. In light of this, it is possible to conclude that this kit combined with the protocol adjustments proposed in this work, can be a performing tool for eDNA extraction, also from small amounts of water sample collected from oligotrophic or degraded environments. Further investigations will be oriented to optimize eDNA extraction from the aquifer solid portion characterized by few nutrients and microbial cells to better understand how microbial populations can distribute themselves between solid and aqueous phases.

In vitro Analysis of O-Antigen-Specific Bacteriophage P22 Inactivation by Salmonella Outer Membrane Vesicles.

Bacteriophages use a large number of different bacterial cell envelope structures as receptors for surface attachment. As a consequence, bacterial surfaces represent a major control point for the defense against phage attack. One strategy for phage population control is the production of outer membrane vesicles (OMVs). In Gram-negative host bacteria, O-antigen-specific bacteriophages address lipopolysaccharide (LPS) to initiate infection, thus relying on an essential outer membrane glycan building block as receptor that is constantly present also in OMVs. In this work, we have analyzed interactions of Salmonella (S.) bacteriophage P22 with OMVs. For this, we isolated OMVs that were formed in large amounts during mechanical cell lysis of the P22 S. Typhimurium host. In vitro, these OMVs could efficiently reduce the number of infective phage particles. Fluorescence spectroscopy showed that upon interaction with OMVs, bacteriophage P22 released its DNA into the vesicle lumen. However, only about one third of the phage P22 particles actively ejected their genome. For the larger part, no genome release was observed, albeit the majority of phages in the system had lost infectivity towards their host. With OMVs, P22 ejected its DNA more rapidly and could release more DNA against elevated osmotic pressures compared to DNA release triggered with protein-free LPS aggregates. This emphasizes that OMV composition is a key feature for the regulation of infective bacteriophage particles in the system.

Candida albicans Culture, Cell Harvesting, and Total RNA Extraction.

Transcriptional analysis has become a cornerstone of biological research, and with the advent of cheaper and more efficient sequencing technology over the last decade, there exists a need for high-yield and efficient RNA extraction techniques. Fungi such as the human pathogen Candida albicans present a unique obstacle to RNA purification in the form of the tough cell wall made up of many different components such as chitin that are resistant to many common mammalian or bacterial cell lysis methods. Typical in vitro C. albicans cell harvesting methods can be time consuming and expensive if many samples are being processed with multiple opportunities for product loss or sample variation. Harvesting cells via vacuum filtration rather than centrifugation cuts down on time before the cells are frozen and therefore the available time for the RNA expression profile to change. Vacuum filtration is preferred for C. albicans for two main reasons: cell lysis is faster on non-pelleted cells due to increased exposed surface area, and filamentous cells are difficult to pellet in the first place unlike yeast or bacterial cells. Using mechanical cell lysis, by way of zirconia/silica beads, cuts down on time for processing as well as overall cost compared to enzymatic treatments. Overall, this method is a fast, efficient, and high-yield way to extract total RNA from in vitro cultures of C. albicans.

Antimicrobial potential of a hydrolyzed protein extract of the microalgae Nannochloropsis sp

Microalgae are photosynthetic microorganisms widely used at the industrial level because of their high content of compounds with bioactive properties such as antimicrobial peptides. These compounds have a wide range of action mechanisms, do not produce the resistance of conventional antibiotics and could be effective against bacteria, fungi, viruses, parasites, and even tumor cells. In this work, by mechanical cell lysis treatment, a protein extract of dry biomass of Nannochloropsis sp was obtained, subsequently, it was hydrolyzed with an enzyme complex of proteases, papain, and pancreatin, for 6 hours, at 44 ° C and pH 7. Finally, the antimicrobial activity of the fraction of hydrolyzed extract with a size less than 3 kDa was evaluated against Staphylococcus aureus, Escherichia coli, and Candida albicans, obtaining a percentage of growth inhibition of 93.1%, 96.6%, and 14.4% respectively, which represents a peptic extract with bacteriostatic and fungistatic properties.

Passive cell disruption lipid extraction methods of microalgae for biofuel production – A review

The conventional method of lipid extraction mandatorily includes cell lysis which is considered an essential process for the enhancement of extraction efficiency. However, cell lysis method especially those incorporating mechanical and physical methods such as homogenization, sonication, microwave techniques, etc., are energy intensive and drastically escalates the biofuel production cost. Therefore, the development of an alternate route of extraction skipping the conventional mechanical cell lysis step remains a challenge to be accomplished. In this context, we discuss the reports involving passive techniques that are able to extract lipid from microalgae without significant cell wall shearing. This includes methods such as in-situ transesterification, direct saponification, supercritical fluid extraction, organic solvent extraction, etc. Moreover, the review discusses the employment of passive lipid extraction methods in a biorefinery set-up, outlining the various bioproducts that could be generated along with lipid. The review concludes with the analysis of the economics of microalgal lipid extraction using passive disruption methods and compares with the processes incorporating cell lysis steps.

Analysis of centrifugal homogenization and its applications for emulsification & mechanical cell lysis

We detail the analysis of centrifugal homogenization process by a hydrodynamic model and the model-guided design of a low-cost centrifugal homogenizer. During operation, centrifugal force pushes a multiphase solution to be homogenized through a thin nozzle, consequently homogenizing its contents. We demonstrate and assess the homogenization of coarse emulsions into relatively monodisperse emulsions, as well as the application of centrifugal homogenization in the mechanical lysis of mpkCCD mouse kidney cells. To gain insight into the homogenization mechanism, we investigate the dependence of emulsion droplet size on geometrical parameters, centrifugal acceleration, and dispersed phase viscosity. Our experimental results are in qualitative agreement with models predicting the droplet size. Furthermore, they indicate that high shear rates kept constant throughout operation produce more monodisperse droplets. We show this ideal homogenization condition can be realized through hydrodynamic model-guided design minimizing transient effects inherent to centrifugal homogenization. Moreover, we achieved power densities comparable to commercial homogenizers by model guided optimization of homogenizer design and experimental conditions. Centrifugal homogenization using the proposed homogenizer design thus offers a low-cost alternative to existing technologies as it is constructed from off-the-shelf parts (Falcon tubes, syringe, needles) and used with a centrifuge, readily available in standard laboratory environment.

Piezoelectric Microchip for Cell Lysis through Cell-Microparticle Collision within a Microdroplet Driven by Surface Acoustic Wave Oscillation.

Cell lysis is an important and crucial step for the detection of intracellular secrets. Usually, cell lysis is based on strong ultrasonic waves or toxic chemical regents, which require a large amount of cell suspension. To obtain high efficiency cell lysis for a small amount of sample, a mechanical cell lysis method based on a surface acoustic wave (SAW) microchip is proposed. The microchip simply consists of a piece of LiNbO3 crystal substrate, interdigitated transducers (IDTs) with 80 pairs of parallel electrodes and 3M Magic Tapes. The modulated input electrical signal is coupled into the substrate through IDTs, which produces an acoustic stream in the droplet on the surface of a substrate. When a biofluid droplet containing cells and microparticles is dropped on the surface of the microchip, the cells and microparticles are accelerated and collide with each other. The fluorescence staining results illustrate that the cell membrane is efficiently destroyed and that proteins as well as nucleic acids inside the cell are released. The experimental results show that this method has a high efficiency and low sample consumption. The potential application is the pretreatment of a small amount of tested sample in a hospital or biolab.

Distinct molecular signatures in dissolved organic matter produced by viral lysis of marine cyanobacteria.

Dissolved organic matter (DOM) plays a central role in the microbial ecology and biogeochemistry of aquatic environments, yet little is known about how the mechanism of DOM release from its ultimate source, primary producer biomass, affects the molecular composition of the inputs to the dissolved pool. Here we used a model marine phytoplankton, the picocyanobacterium Synechococcus WH7803, to compare the composition of DOM released by three mechanisms: exudation, mechanical cell lysis and infection by the lytic phage S-SM1. A broad, untargeted analytical approach reveals the complexity of this freshly sourced DOM, and comparative analysis between DOM produced by the different mechanisms suggests that exudation and viral lysis are sources of unsaturated, oxygen-rich and possibly novel biomolecules. Furthermore, viral lysis of WH7803 by S-SM1 releases abundant peptides derived from specific proteolysis of the major light-harvesting protein phycoerythrin, raising the possibility that phage infection of these abundant cyanobacteria could be a significant source of high molecular weight dissolved organic nitrogen compounds.

Use of the Precellys® Evolution Homogenizer For Unbiased DNA Extraction From The Zymobiomics™ microbial Community Standard

Microbiome profiling via Next Generation Sequencing (NGS) techniques is rapidly changing the landscape of microbiology. However, to ensure accurate microbiome measurements, it is essential to ensure the microbiome workflow is unbiased and robust [1]. For instance, it has been reported that bias in microbiomics analyses can be introduced at every step [2-4] including the DNA extraction step if microbial cells are not uniformly and completely lysed. Mechanical cell lysis using beads of various composition (bead beating) is a generally accepted method used to lyse various microbes [5] and, although a variety of mechanical lysis devices and instruments are available, their performance greatly varies [6-8]. Until recently, there has not been a convenient way to assess the efficiency of lysis afforded by any particular instrument, however, with the release of the ZymoBIOMICS™ Microbial Community Standard (Zymo Research Corp, Irvine, CA, USA) this is now possible. The ZymoBIOMICS Microbial Community is a mock microbial community standard comprised of ten well-characterized organisms (i.e., 5 grampositive, 3 gram-negative, and 2 yeast) that are mixed at defined proportions. The standard contains microbes of different cell wall recalcitrance and of different cell size making it ideal for evaluating cell lysis efficiency. In this study, the efficiency of microbial lysis with the ZymoBIOMICS Microbial Community Standard was assessed using the Precellys® Evolution tissue homogenizer combined with Cryolys® Evolution cooling unit (Bertin Instruments, Rockville, MD, USA).

Isolation of high molecular weight and humic acid-free metagenomic DNA from lignocellulose-rich samples compatible for direct fosmid cloning.

Activity-based screening of metagenomic DNA libraries is a promising approach to fish out genes encoding novel bioactive compounds/enzymes of industrial importance. The starting point of such functional screening in fosmid vectors is isolation of high molecular weight (HMW) DNA of sufficient purity from diverse environments. Metagenomic DNA isolation protocols mostly employ mechanical cell lysis that yields fragmented DNA. Those established for HMW DNA using enzymatic lysis have not considered samples with high lignocellulose or humic acid content. Enzymes from such environments are in great demand for bioenergy, paper, and related industries. Thus, an improved method was standardized that has three key features, i.e., use of harvested microbial biomass instead of raw samples, removal of humic substances prior to cell lysis by aluminum sulfate flocculation, and enzymatic/chemical lysis of cells with a lysozyme, mutanolysin, proteinase K, and SDS cocktail followed by phenol-chloroform extraction and precipitation of DNA by polyethylene glycol and NaCl. HMW DNA (~ 40kb) was efficiently isolated from garden and forest soils, rice straw compost, and degrading wood from a hypersaline lake. The humic acid removal efficiency across samples was 96-98%. The isolated DNA was of high quality/purity and could be successfully used in downstream applications like PCR, ligation, and fosmid cloning. In fact, the DNA was directly used without any size selection, for fosmid library preparation with 70-90% efficiency as compared to the control insert. Thus, the method could suitably be used for HMW DNA isolation for the functional screening of enzymes from diverse humic acid-/lignocellulose-rich environments.

Isolation and characterization of human CapG expressed and post-translationally modified in Pichia pastoris

CapG is an actin-binding protein, which is overexpressed in a variety of tumors, i.e. breast, ovarian, pancreatic and lung carcinoma. We successfully expressed human CapG in the wild type strain X-33 of the methylotrophic yeast Pichia pastoris (P. pastoris), which does not express endogenous CapG, in order to characterize this protein in more detail. After mechanical cell lysis, debris was centrifuged and the soluble protein was precipitated with ammonium sulfate. This protein pellet was dialyzed and used for CapG purification. Ca2+-dependent exposure of hydrophobic sites allowed single step and selective elution from a Phenyl Sepharose™ matrix. 3.5 mg CapG/10 g wet biomass were isolated and showed a Ca2+-sensitive and dose-dependent capping activity of actin in a fluorometric assay. In P. pastoris, CapG is located at actin patches, actin cables and arranges along the budding neck. The proliferation rate and morphology of the yeast cells are not influenced by the interaction of CapG with actin. The modification pattern of human CapG from P. pastoris and human carcinoma cells is highly similar. We validated most of the known post-translational modifications and found three new phosphorylation and nine new acetylation sites by mass spectrometry. The N-terminus is acetylated or truncated. Truncated CapG is not phosphorylated at the residues S10, T212 and S337. First mutagenesis experiments indicate an N-terminal acetylation dependent C-terminal phosphorylation.

On-chip Extraction of Intracellular Molecules in White Blood Cells from Whole Blood.

The extraction of virological markers in white blood cells (WBCs) from whole blood—without reagents, electricity, or instruments—is the most important first step for diagnostic testing of infectious diseases in resource-limited settings. Here we develop an integrated microfluidic chip that continuously separates WBCs from whole blood and mechanically ruptures them to extract intracellular proteins and nucleic acids for diagnostic purposes. The integrated chip is assembled with a device that separates WBCs by using differences in blood cell size and a mechanical cell lysis chip with ultra-sharp nanoblade arrays. We demonstrate the performance of the integrated device by quantitatively analyzing the levels of extracted intracellular proteins and genomic DNAs. Our results show that compared with a conventional method, the device yields 120% higher level of total protein amount and similar levels of gDNA (90.3%). To demonstrate its clinical application to human immunodeficiency virus (HIV) diagnostics, the developed chip was used to process blood samples containing HIV-infected cells. Based on PCR results, we demonstrate that the chip can extract HIV proviral DNAs from infected cells with a population as low as 102/μl. These findings suggest that the developed device has potential application in point-of-care testing for infectious diseases in developing countries.