Organoclay-assisted interfacial polymerization for microfluidic production of monodisperse PEG-microdroplets and in situ encapsulation of E. coli

We developed a novel one-pot synthetic strategy for preparing monodisperse polyethylene glycol diacrylate (PEGDA) microdroplets via organoclay-assisted interfacial polymerization approach for Escherichia coli encapsulation. Based on the mechanism of spontaneous and rapid polymerization of PEGDA precursor solution with Mg-organoclay, the prepared PEGDA microdroplets have uniform size and fine round shape, with size range of 74-118 μm. The size of microdroplets can be controlled through the changing continuous phase flow rate. Organoclay-assisted polymerization method provides a unique environment to produce non-toxic ways of fabricating microorganism encapsulated microdroplets and to prohibit microdroplets merge during the processes. Furthermore, we successfully carried out to entrap E. coli inside of the PEGDA microdroplets. E. coli expressing a green fluorescent protein shows a good viability inside the PEGDA microdroplets. The in situ microfluidic synthetic method provides a novel approach for the preparation of monodisperse PEGDA microdroplets via a one-pot route.     

Development of multilayered cell‐hydrogel composites using an acoustic focusing technique

Multilayered composites, composed of mammalian cells arranged in a hydrogel, have been prepared using an acoustic focusing technique. Acoustic focusing is a simple, nonchemical technique that allows for the fast arrangement of cells in matrices where the control of cell geometry is beneficial. Breast cancer cells (MDA‐MB231) were dispersed in a 30 wt % solution of poly(ethylene glycol) diacrylate (PEGDA) of molecular weight 400 at a density of 5 × 10⁶ cells/mL of PEGDA solution. An ultrasonic field was used to organize the cells before polymerization of PEGDA. Disk‐shaped hydrogel composites, typically 1 cm in diameter and 2‐mm thick were prepared based on a PEGDA solution volume of 130 μL. At an acoustic frequency of 2.32 MHz, composites having cells positioned within concentric cylindrical shells interspersed with zones of cell‐free hydrogel were produced. The cells were located in annuli approximately 80‐μm thick and about 300 μm apart. The structure and viability of the cells within these constructs were studied using a fluorescent LIVE/DEAD assay. The viability of the cells was on the order of 50%. For the conditions used in this study, cell death was primarily attributed to exposure of cells to the PEGDA solution prior to polymerization, rather than adverse effects of polymerization or the sound field itself. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Enhancement of cell internalization and photostability of red and green emitter quantum dots upon entrapment in novel cationic nanoliposomes

Two quantum dots (QDs), a green emitter, CdSe and a red emitter, CdSe with ZnS shell are encapsulated into novel liposomes in two different formulations including cationic liposomes. Quantum dots have proven themselves as powerful inorganic fluorescent probes, especially for long‐term, multiplexed imaging and detection. Upon delivery into a cell, in endocytic vesicles such as endosomes, their fluorescence is quenched. We have investigated the potential toxic effects, photophysical properties and cell internalization of QDs in new formulation of liposomes as an in vitro vesicle model. Entrapment of QDs into liposomes is brought about with a decrease in their intrinsic fluorescence and toxicities and an increase in their photostability and lifetime. The biomimetic lipid bilayer of liposomes provides high biocompatibility, thereby enhancing the effectiveness of fluorescent nanoparticles for biological recognition in vitro and in vivo. The prepared lipodots could effectively prevent QDs from photo‐oxidation during storage and when exposed to ultraviolet (UV) light. Moreover, the flow cytometry of HEK 293 T cells showed that the cell internalization of encapsulated QDs in (DSPC/CHO/DOPE/DOAB) liposome is enhanced 10 times compared with non‐encapsulated QD (bare QDs).     

Fluorescence optical detection in situ for real‐time monitoring of cytochrome P450 enzymatic activity of liver cells in multiple microfluidic devices

We describe an in situ fluorescence optical detection system to demonstrate real‐time and non‐invasive detection of reaction products in a microfluidic device while under perfusion within a standard incubator. The detection system is designed to be compact and robust for operation inside a mammalian cell culture incubator for quantitative detection of fluorescent signal from microfluidic devices. When compared to a standard plate reader, both systems showed similar biphasic response curves with two linear regions. Such a detection system allows real‐time measurements in microfluidic devices with cells without perturbing the culture environment. In a proof‐of‐concept experiment, the cytochrome P450 1A1/1A2 activity of a hepatoma cell line (HepG2/C3A) was monitored by measuring the enzymatic conversion of ethoxyresorufin to resorufin. The hepatoma cell line was embedded in Matrigel^TM construct and cultured in a microfluidic device with medium perfusion. The response of the cells, in terms of P450 1A1/1A2 activity, was significantly different in a plate well system and the microfluidic device. Uninduced cells showed almost no activity in the plate assay, while uninduced cells in Matrigel^TM with perfusion in a microfluidic device showed high activity. Cells in the plate assay showed a significant response to induction with 3‐Methylcholanthrene while cells in the microfluidic device did not respond to the inducer. These results demonstrate that the system is a potentially useful method to measure cell response in a microfluidic system. Biotechnol. Bioeng. 2009; 104: 516–525 © 2009 Wiley Periodicals, Inc.     

Non‐genetic diversity modulates population performance

Biological functions are typically performed by groups of cells that express predominantly the same genes, yet display a continuum of phenotypes. While it is known how one genotype can generate such non‐genetic diversity, it remains unclear how different phenotypes contribute to the performance of biological function at the population level. We developed a microfluidic device to simultaneously measure the phenotype and chemotactic performance of tens of thousands of individual, freely swimming Escherichia coli as they climbed a gradient of attractant. We discovered that spatial structure spontaneously emerged from initially well‐mixed wild‐type populations due to non‐genetic diversity. By manipulating the expression of key chemotaxis proteins, we established a causal relationship between protein expression, non‐genetic diversity, and performance that was theoretically predicted. This approach generated a complete phenotype‐to‐performance map, in which we found a nonlinear regime. We used this map to demonstrate how changing the shape of a phenotypic distribution can have as large of an effect on collective performance as changing the mean phenotype, suggesting that selection could act on both during the process of adaptation.     

Rapid one‐step purification of single‐cells encapsulated in alginate microcapsules from oil to aqueous phase using a hydrophobic filter paper: Implications for single‐cell experiments

By virtue of the biocompatibility and physical properties of hydrogel, picoliter‐sized hydrogel microcapsules have been considered to be a biometric signature containing several features similar to that of encapsulated single cells, including phenotype, viability, and intracellular content. To maximize the experimental potential of encapsulating cells in hydrogel microcapsules, a method that enables efficient hydrogel microcapsule purification from oil is necessary. Current methods based on centrifugation for the conventional stepwise rinsing of oil, are slow and laborious and decrease the monodispersity and yield of the recovered hydrogel microcapsules. To remedy these shortcomings we have developed a simple one‐step method to purify alginate microcapsules, containing a single live cell, from oil to aqueous phase. This method employs oil impregnation using a commercially available hydrophobic filter paper without multistep centrifugal purification and complicated microchannel networks. The oil‐suspended alginate microcapsules encapsulating single cells from mammalian cancer cell lines (MCF–7, HepG2, and U937) and microorganisms (Chlorella vulgaris) were successfully exchanged to cell culture media by quick (～10 min) depletion of the surrounding oil phase without coalescence of neighboring microcapsules. Cell proliferation and high integrity of the microcapsules were also demonstrated by long‐term incubation of microcapsules containing a single live cell. We expect that this method for the simple and rapid purification of encapsulated single‐cell microcapsules will attain widespread adoption, assisting cell biologists and clinicians in the development of single‐cell experiments.     

Luminescent gelatin nanospheres by encapsulating CdSe quantum dots

Quantum dots (QDs) have been encapsulated within gelatin nanoparticles (GNPs), which gives GNPs fluorescent properties and improves the biocompatibility of QDs. Hydrophilic CdSe QDs were produced through thermodecomposition following the ligand‐exchange method, and were then encapsulated in GNPs. The results of high‐resolution transmission electron microscopy and transmission electron microscopy show that CdSe QDs and QDs‐encapsulated GNPs (QDs‐GNPs) have average diameters of 5 ± 1 and 150 ± 10 nm, respectively. Results of both high‐resolution transmission electron microscopy and confocal laser scanning microscopy indicate that CdSe QDs are successfully encapsulated within GNPs. The QDs‐GNPs have distinctive fluorescent properties with maximum emission at 654 nm, with a 24 nm red‐shift comapred with hydrophilic mercaptoundecanoic acid (MUA)‐modified QDs. In addition, an in vitro cytotoxicity test shows that QDs‐GNPs do not have any toxic effect on cells. It is expected that QDs‐GNPs might be an excellent candidate as a contrast agent in bio‐imaging. Copyright © 2013 John Wiley & Sons, Ltd.     

Microfluidic device‐assisted etching of p‐HEMA for cell or protein patterning

The construction of biomaterials with which to limit the growth of cells or to limit the adsorption of proteins is essential for understanding biological phenomena. Here, we describe a novel method to simply and easily create thin layers of poly (2‐hydroxyethyl methacrylate) (p‐HEMA) for protein and cellular patterning via etching with ethanol and microfluidic devices. First, a cell culture surface or glass coverslip is coated with p‐HEMA. Next, a polydimethylsiloxane (PDMS) microfluidic is placed onto the p‐HEMA surface, and ethanol is aspirated through the device. The PDMS device is removed, and the p‐HEMA surface is ready for protein adsorption or cell plating. This method allows for the fabrication of 0.3 µm thin layers of p‐HEMA, which can be etched to 10 µm wide channels. Furthermore, it creates regions of differential protein adhesion, as shown by Coomassie staining and fluorescent labeling, and cell adhesion, as demonstrated by C2C12 myoblast growth. This method is simple, versatile, and allows biologists and bioengineers to manipulate regions for cell culture adhesion and growth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:243–248, 2018     

Polymer composite fluorescent hydrogel film based on nitrogen‐doped carbon dots and their application in the detection of Hg2+ ions

A simple microwave‐assisted solvothermal method was used to prepare fluorescent nitrogen‐doped carbon dots (N‐CDs) with high fluorescence quantum yield (79.63%) using citric acid and N‐(2‐hydroxyethyl)ethylenediamine as starting materials. The PVAm‐g‐N‐CDs grafted products were synthesized by amide bond formation between the carboxylic groups of N‐CDs and amine groups of polyvinylamine (PVAm). Fluorescent hydrogel films (PVAm‐g‐N‐CDs/PAM) were synthesized by interpenetration polymer network polymerization of PVAm‐g‐N‐CDs and acrylamide (AM). When used for ion detection, we found that the fluorescence of the hydrogel films was clearly quenched by addition of Hg²⁺. Repeatability tests on using the hydrogel films for Hg²⁺ detection showed that they could be applied at least three times. The PVAm‐g‐N‐CDs/PAM could serve as an effective fluorescent sensing platform for sensitive detection of Hg²⁺ ions with a detection limit of 0.089 μmol/L. This work may offer a new approach for developing recoverable and sensitive N‐CDs‐based sensors for biological and environmental applications.     

Development of a full‐length human protein production pipeline

There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high‐throughput (HT) methods, we transferred the genes of 31 full‐length proteins into three expression vectors, and expressed the collection as N‐terminal HaloTag fusion proteins in Escherichia coli and two commercial cell‐free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip^® GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from E. coli, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full‐length human proteins in these three expression systems.     

Studying the non‐thermal effects of terahertz radiation on E. coli/pKatG‐GFP biosensor cells

Studies of the impact of terahertz radiation on living objects present a significant interest since its use for security systems is currently considered promising. We studied the non‐thermal impact of terahertz radiation on E. coli/pKatG‐gfp biosensor cells. The Novosibirsk free electron laser (NovoFEL), which currently has the world's highest average and peak power, was used as the source of terahertz radiation. We demonstrated that exposure to terahertz radiation at the wavelengths of 130, 150, and 200 µm and a power of 1.4 W/cm² induces changes in green fluorescent protein (GFP) fluorescence values and thus induces the expression of GFP in E. coli/pKatG‐gfp biosensor cells. Possible mechanisms of the E. coli response to non‐thermal exposure to terahertz radiation are discussed. Bioelectromagnetics 34:15–21, 2013. © 2012 Wiley Periodicals, Inc.     

Production of N‐Acetyl‐Phosphinothricin: A Substance used for Inducing Male Sterility in Transgenic Plants

The non‐toxic compound N‐acetyl‐L‐phosphinothricin (N‐Ac‐L‐PPT) is used in a so‐called deacetylation system to induce male sterility in transgenic plants by tapetum specific deacetylation to the herbicide L‐phosphinothricin (L‐PPT). A procedure was developed to produce pure racemic and L‐isomeric N‐Ac‐PPT containing less than 30 ppm residual PPT. Experiments applied to wild type tobacco and PPT‐resistant tobacco showed that the maximal tolerated N‐Ac‐PPT concentration would be less than 45 mM of the L‐isomer. Otherwise unspecific deacetylation by several acylases, as well as by environmental conditions like higher temperatures or pHs beyond neutrality, increased the residual L‐PPT content to toxic concentrations. In contrast, N‐acetyl‐L‐phosphinothricyl‐alanyl‐alanine (N‐Ac‐L‐PPTT), a substance also occurring during the biosynthesis of phosphinothricyl‐alanyl‐alanine (PPTT) by some Streptomyces species, was tolerated up to 274 mM by wild type tobacco plants. However, the ArgE deacatylase from Escherichia coli originally used in the deacetylation system, as well as some other acylases, showed no activity towards N‐Ac‐L‐PPTT.     

Rationally engineered biotransformation of p‐nitrophenol

An operon encoding enzymes responsible for degradation of the EPA priority contaminant para‐nitrophenol (PNP) from Pseudomonas sp. ENV2030 contains more genes than would appear to be necessary to mineralize PNP. To determine some necessary genes for PNP degradation, the genes encoding the proposed enzymes in the degradation pathway (pnpADEC) were assembled into a broad‐host‐range, BioBricks‐compatible vector under the control of a constitutive promoter. These were introduced into Escherichia coli DH10b and two Pseudomonas putida strains, one with a knockout of the aromatic transport TtgB and the parent with the native transporter. The engineered strains were assayed for PNP removal. E. coli DH10b harboring several versions of the refactored pathway was able to remove PNP from the medium up to a concentration of 0.2 mM; above which PNP was toxic to E. coli. A strain of P. putida harboring the PNP pathway genes was capable of removing PNP from the medium up to 0.5 mM. When P. putida harboring the native PNP degradation cluster was exposed to PNP, pnpADEC were induced, and the resulting production of β‐ketoadipate from PNP induced expression of its chromosomal degradation pathway (pcaIJF). In contrast, pnpADEC were expressed constitutively from the refactored constructs because none of the regulatory genes found in the native PNP degradation cluster were included. Although P. putida harboring the refactored construct was incapable of growing exclusively on PNP as a carbon source, evidence that the engineered pathway was functional was demonstrated by the induced expression of chromosomal pcaIJF. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Maternal consumption of non‐toxic Microcystis by Daphnia magna induces tolerance to toxic Microcystis in offspring

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Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

The soil bacterium Pseudomonas putida KT2440 has gained increasing biotechnological interest due to its ability to tolerate different types of stress. Here, the tolerance of P. putida KT2440 toward eleven toxic chemical compounds was investigated. P. putida was found to be significantly more tolerant toward three of the eleven compounds when compared to Escherichia coli. Increased tolerance was for example found toward p‐coumaric acid, an interesting precursor for polymerization with a significant industrial relevance. The tolerance mechanism was therefore investigated using the genome‐wide approach, Tn‐seq. Libraries containing a large number of miniTn5‐Km transposon insertion mutants were grown in the presence and absence of p‐coumaric acid, and the enrichment or depletion of mutants was quantified by high‐throughput sequencing. Several genes, including the ABC transporter Ttg2ABC and the cytochrome c maturation system (ccm), were identified to play an important role in the tolerance toward p‐coumaric acid of this bacterium. Most of the identified genes were involved in membrane stability, suggesting that tolerance toward p‐coumaric acid is related to transport and membrane integrity.

     

Chemiluminescence of non‐differentiated THP‐1 promonocytes: developing an assay for screening anti‐inflammatory milk proteins and peptides

Human leukemic THP‐1 promonocytes are widely used as a model for peripheral blood monocytes. However, superoxide production during respiratory burst (RB) of non‐differentiated THP‐1 (nd‐THP‐1) cells is very low. Here we present a rapid and low‐cost method for measuring the chemiluminescence (CL) of opsonized zymosan (OZ) induced RB which allows detection of Escherichia coli lipopolysaccharide (LPS) induced priming of nd‐THP‐1 cells on the basis of CL reaction kinetics. Maximum CL intensity obtained was 2.20 ± 0.25 and 1.30 ± 0.11 relative light units, while CL peak time was achieved at 18.1 ± 2.6 and 28.7 ± 1.3 min in primed and non‐primed cells, respectively. The priming of nd‐THP‐1 cells with LPS evoked typical TNF‐α and IL‐6 production. We tested the effects of bovine lactoferrin and protein fractions from Lactobacillus helveticus BGRA43 fermented milk for potential anti‐inflammatory effects on LPS primed nd‐THP‐1 cells. Four fractions were found to inhibit the OZ‐induced CL in a dose‐dependent manner (IC₅₀ 3–30 µg/mL), while lactoferrin inhibited CL to a lesser extent (IC₅₀ 270 µg/mL). These results suggest that measuring CL response of nd‐THP‐1 cells can serve as a method for screening anti‐inflammatory compounds which could be used in reducing the risk of phagocyte‐mediated inflammatory diseases. Copyright © 2010 John Wiley & Sons, Ltd.     

The pattern‐recognition molecule mindin binds integrin Mac‐1 to promote macrophage phagocytosis via Syk activation and NF‐κB p65 translocation

Mindin has a broad spectrum of roles in the innate immune system, including in macrophage migration, antigen phagocytosis and cytokine production. Mindin functions as a pattern‐recognition molecule for microbial pathogens. However, the underlying mechanisms of mindin‐mediated phagocytosis and its exact membrane receptors are not well established. Herein, we generated mindin‐deficient mice using the CRISPR‐Cas9 system and show that peritoneal macrophages from mindin‐deficient mice were severely defective in their ability to phagocytize E  coli. Phagocytosis was enhanced when E  coli or fluorescent particles were pre‐incubated with mindin, indicating that mindin binds directly to bacteria or non‐pathogen particles and promotes phagocytosis. We defined that ¹³¹I‐labelled mindin binds with integrin Mac‐1 (CD11b/CD18), the F‐spondin (FS)‐fragment of mindin binds with the α_M‐I domain of Mac‐1 and that mindin serves as a novel ligand of Mac‐1. Blockade of the α_M‐I domain of Mac‐1 using either a neutralizing antibody or si‐Mac‐1 efficiently blocked mindin‐induced phagocytosis. Furthermore, mindin activated the Syk and MAPK signalling pathways and promoted NF‐κB entry into the nucleus. Our data indicate that mindin binds with the integrin Mac‐1 to promote macrophage phagocytosis through Syk activation and NF‐κB p65 translocation, suggesting that the mindin/Mac‐1 axis plays a critical role during innate immune responses.     

2‐Picoline‐borane: A non‐toxic reducing agent for oligosaccharide labeling by reductive amination

Analysis of N‐glycans is often performed by LC coupled to fluorescence detection. The N‐glycans are usually labeled by reductive amination with a fluorophore containing a primary amine to allow fluorescence detection. Moreover, many of the commonly applied labels also allow improved mass spectrometric detection of oligosaccharides. For reductive amination, the amine group of the label reacts with the reducing‐end aldehyde group of the oligosaccharide to form a Schiff base, which is reduced to a secondary amine. Here, we propose the use of 2‐picoline‐borane as the reducing agent, as a non‐toxic alternative to the extensively used, but toxic sodium cyanoborohydride. Using dextran oligosaccharides and plasma N‐glycans, we demonstrate similar labeling efficacies for 2‐picoline‐borane and sodium cyanoborohydride. Therefore, 2‐picoline‐borane is a non‐toxic alternative to sodium cyanoborohydride for the labeling of oligosaccharides.     

A unique Ni2+ ‐dependent and methylglyoxal‐inducible rice glyoxalase I possesses a single active site and functions in abiotic stress response

The glyoxalase system constitutes the major pathway for the detoxification of metabolically produced cytotoxin methylglyoxal (MG) into a non‐toxic metabolite d ‐lactate. Glyoxalase I (GLY I) is an evolutionarily conserved metalloenzyme requiring divalent metal ions for its activity: Zn²⁺ in the case of eukaryotes or Ni²⁺ for enzymes of prokaryotic origin. Plant GLY I proteins are part of a multimember family; however, not much is known about their physiological function, structure and metal dependency. In this study, we report a unique GLY I (OsGLYI‐11.2) from Oryza sativa (rice) that requires Ni²⁺ for its activity. Its biochemical, structural and functional characterization revealed it to be a monomeric enzyme, possessing a single Ni²⁺ coordination site despite containing two GLY I domains. The requirement of Ni²⁺ as a cofactor by an enzyme involved in cellular detoxification suggests an essential role for this otherwise toxic heavy metal in the stress response. Intriguingly, the expression of OsGLYI‐11.2 was found to be highly substrate inducible, suggesting an important mode of regulation for its cellular levels. Heterologous expression of OsGLYI‐11.2 in Escherichia coli and model plant Nicotiana tabacum (tobacco) resulted in improved adaptation to various abiotic stresses caused by increased scavenging of MG, lower Na⁺/K⁺ ratio and maintenance of reduced glutathione levels. Together, our results suggest interesting links between MG cellular levels, its detoxification by GLY I, and Ni²⁺ – the heavy metal cofactor of OsGLYI‐11.2, in relation to stress response and adaptation in plants.