Three-Dimensional Hydrodynamic Focusing Method for Polyplex Synthesis

Successful intracellular delivery of nucleic acid therapeutics relies on multiaspect optimization, one of which is formulation. While there has been ample innovation on chemical design of polymeric gene carriers, the same cannot be said for physical processing of polymer–DNA nanocomplexes (polyplexes). Conventional synthesis of polyplexes by bulk mixing depends on the operators’ experience. The poorly controlled bulk mixing process may also lead to batch-to-batch variation and consequent irreproducibility. Here, we synthesize polyplexes by using a three-dimensional hydrodynamic focusing (3D-HF) technique in a single-layered, planar microfluidic device. Without any additional chemical treatment or postprocessing, the polyplexes prepared by the 3D-HF method show smaller size, slower aggregation rate, and higher transfection efficiency, while exhibiting reduced cytotoxicity compared to the ones synthesized by conventional bulk mixing. In addition, by introducing external acoustic perturbation, mixing can be further enhanced, leading to even smaller nanocomplexes. The 3D-HF method provides a simple and reproducible process for synthesizing high-quality polyplexes, addressing a critical barrier in the eventual translation of nucleic acid therapeutics

Shape-Controlled Synthesis of Hybrid Nanomaterials <i>via</i> Three-Dimensional Hydrodynamic Focusing

Shape-controlled synthesis of nanomaterials through a simple, continuous, and low-cost method is essential to nanomaterials research toward practical applications. Hydrodynamic focusing, with its advantages of simplicity, low-cost, and precise control over reaction conditions, has been used for nanomaterial synthesis. While most studies have focused on improving the uniformity and size control, few have addressed the potential of tuning the shape of the synthesized nanomaterials. Here we demonstrate a facile method to synthesize hybrid materials by three-dimensional hydrodynamic focusing (3D-HF). While keeping the flow rates of the reagents constant and changing only the flow rate of the buffer solution, the molar ratio of two reactants (<i>i.e.</i>, tetrathiafulvalene (TTF) and HAuCl₄) within the reaction zone varies. The synthesized TTF–Au hybrid materials possess very different and predictable morphologies. The reaction conditions at different buffer flow rates are studied through computational simulation, and the formation mechanisms of different structures are discussed. This simple one-step method to achieve continuous shape-tunable synthesis highlights the potential of 3D-HF in nanomaterials research

Detection of Single Enzymatic Events in Rare or Single Cells Using Microfluidics

In the present study we demonstrate highly sensitive detection of rare, aberrant cells in a population of wild-type human cells by combining a rolling-circle-enhanced enzyme activity single-molecule detection assay with a custom-designed microfluidic device. Besides reliable detection of low concentrations of aberrant cells, the integrated system allowed multiplexed detection of individual enzymatic events at the single cell level. The single cell sensitivity of the presented setup relies on the combination of single-molecule rolling-circle-enhanced enzyme activity detection with the fast reaction kinetics provided by a picoliter droplet reaction volume and subsequent concentration of signals in a customized drop-trap device. This setup allows the fast reliable analyses of enzyme activities in a vast number of single cells, thereby offering a valuable tool for basic research as well as theranostics

<i>Arabidopsis</i> HFR1 Is a Potential Nuclear Substrate Regulated by the <i>Xanthomonas</i> Type III Effector XopD_{<i>Xcc</i>8004}

<div><p>XopD_{<i>Xcc</i>8004}, a type III effector of <i>Xanthomonas campestris</i> pv. <i>campestris</i> (<i>Xcc</i>) 8004, is considered a shorter version of the XopD, which lacks the N-terminal domain. To understand the functions of XopD_{<i>Xcc</i>8004}, <i>in planta</i>, a transgenic approach combined with inducible promoter to analyze the effects of XopD_{<i>Xcc</i>8004} in <i>Arabidopsis</i> was done. Here, the expression of XopD_{<i>Xcc</i>8004}, in <i>Arabidopsis</i> elicited the accumulation of host defense-response genes. These molecular changes were dependent on salicylic acid and correlated with lesion-mimic phenotypes observed in <i>XVE::XopD_Xcc8004</i> transgenic plants. Moreover, XopD_{<i>Xcc</i>8004} was able to desumoylate HFR1, a basic helix-loop-helix transcription factor involved in photomorphogenesis, through SUMO protease activity. Interestingly, the <i>hfr1-201</i> mutant increased the expression of host defense-response genes and displayed a resistance phenotype to <i>Xcc</i>8004. These data suggest that HFR1 is involved in plant innate immunity and is potentially regulated by XopD_{<i>Xcc</i>8004}.</p></div

XopD_{<i>Xcc</i>8004} interacts with HFR1.

<p>(a) Investigation of the interaction between HFR1 and XopD_{<i>Xcc</i>8004} by yeast two-hybrid analysis. Yeast strains transformed with two plasmids (prey and bait) were plated onto synthetic-defined (SD) minimal medium lacking tryptophan/leucine or tryptophan/leucine/histidine. (b) <i>Nicotiana benthamiana</i> leaves were co-infiltrated with agrobacterium carrying <i>35S::XopD</i>_{<i>Xcc8004</i>}<i>-YFP</i> and <i>35S::HFR1-CFP</i>. Fluorescence and differential interference contrast (DIC) images were obtained by confocal laser scanning microscopy. <i>35S::YFP</i> and <i>35S::CFP</i> were used for control. Scale bars: 15 μm. (c) Investigation of the interaction between HFR1 and XopD_{<i>Xcc</i>8004} by using pull-down assays. Briefly, 2 μg of GST or GST fusion proteins were used to pull down 2 μg of MBP or MBP fusion proteins, and an anti-MBP antibody was used to detect the associated proteins by western blotting (the left panel). The right panels present the input proteins examined by anti-MBP or anti-GST antibodies. The asterisk indicates the protein signal of MBP-HFR1.</p

Salicylic acid-dependent defense responses were elicited by the expression of XopD_{<i>Xcc</i>8004} in <i>Arabidopsis</i>.

<p>(a) Schematic representation of XopD proteins. (b) Morphological examination and trypan blue staining of two-week-old leaves of <i>Arabidopsis XVE::XopD</i>_{<i>Xcc8004</i>} transgenic plants. Lesion-mimic phenotypes were indicated by arrows. Scale bar: 1 mm. (c) Translated products of <i>XopD</i>_{<i>Xcc8004</i>} and <i>XopD</i>_{<i>Xcc8004</i>}<i>(C355A)</i> were examined by western blotting using a specific antibody against XopD_{<i>Xcc</i>8004}. Anti-tubulin was used for loading control. (d) The expression levels of genes involved in the SA-mediated defense signaling network were examined by qRT-PCR and normalized to <i>EF1α</i>. The relative expression levels of each gene in the DMSO control were set at 1.</p

K72 in HFR1 is desumoylated by XopD_{<i>Xcc</i>8004}<i>in vitro</i>.

<p>(a) The deduced amino acid sequence of HFR1 from 65 to 80 a.a. The arrow indicates a putative sumoylation site at K72 of the consensus motif ΨKxE/D of HFR1. (b) An <i>in vitro</i> sumoylation system was established by using purified recombinant proteins, including <i>Arabidopsis</i> SAE1, SAE2, SCE1, and AtSUMO1. MBP-HFR1 or MBP-HFR1(K72A) were used as potential substrates for sumoylation and detected with an anti-MBP antibody. (c) Together with SAE1, SAE2, SCE1, AtSUMO1, and MBP-HFR1, XopD_{<i>Xcc</i>8004} was added to the reaction mixture to investigate the sumoylation of HFR1. XopD_{<i>Xcc</i>8004}(C355A), a SUMO protease mutant, was used as control. Asterisks indicate sumoylated MBP-HFR1 proteins. Arrowheads indicate unmodified MBP-HFR1 proteins. Signals below the unmodified MBP-HFR1 proteins were degraded products of purified MBP-HFR1 proteins.</p

Expression of XopD_{<i>Xcc</i>8004} induces a long hypocotyl phenotype in <i>Arabidopsis</i>.

<p>(a) Phenotypes of 12-day-old <i>Arabidopsis</i> transgenic plants carrying a <i>XopD</i>_{<i>Xcc8004</i>} gene driven by the inducible <i>XVE</i> promoter. Scale bars: 8 mm. (b) Average hypocotyl lengths of seedlings grown on medium containing DMSO or 20 μM β-estradiol. Statistically significant differences were determined using one-way ANOVA (** indicates <i>p</i> < 0.005).</p

Synthesis of Fluorosurfactants for Emulsion-Based Biological Applications

Microemulsion represents an attractive platform for fundamental and applied biomedical research because the emulsified droplets can serve as millions of compartmentalized micrometer-sized reactors amenable to high-throughput screening or online monitoring. However, establishing stable emulsions with surfactants that are compatible with biological applications remains a significant challenge. Motivated by the lack of commercially available surfactants suitable for microemulsion-based biological assays, this study describes the facile synthesis of a biocompatible fluorosurfactant with nonionic tris(hydroxymethyl)methyl (Tris) polar head groups. We have further demonstrated compatibility of the developed surfactant with diverse emulsion-based applications, including DNA polymeric nanoparticle synthesis, enzymatic activity assay, and bacterial or mammalian cell culture, in the setup of both double- and multiphases of emulsions

Additional file 1 of Stabilization and improved functionality of three-dimensional perfusable microvascular networks in microfluidic devices under macromolecular crowding

Supplementary Material