Highly Photoluminescent Nitrogen- and Zinc-Doped Carbon Dots for Efficient Delivery of CRISPR/Cas9 and mRNA.

Safe and efficient delivery of CRISPR/Cas9 systems is still a challenge. Here we report the development of fluorescent nitrogen- and zinc-doped carbon dots (N-Zn-doped CDs) using one-step microwave-aided pyrolysis based on citric acid, branched PEI25k, and different zinc salts. These versatile nanovectors with a quantum yield of around 60% could not only transfect large CRISPR plasmids (∼9 kb) with higher efficiency (80%) compared to PEI25k and lipofectamine 2000 (Lipo 2K), but they also delivered mRNA into HEK 293T cells with the efficiency 20 times greater than and equal to that of PEI25k and Lipo 2K, respectively. Unlike PEI25k, N-Zn-doped CDs exhibited good transfection efficiency even at low plasmid doses and in the presence of 10% fetal bovine serum (FBS). Moreover, these nanovectors demonstrated excellent efficiency in GFP gene disruption by transferring plasmid encoding Cas9 and sgRNA targeting GFP as well as Cas9/sgRNA ribonucleoproteins into HEK 293T-GFP cells. Hence, N-Zn-doped CDs with remarkable photoluminescence properties and high transfection efficiency in the delivery of both CRISPR complexes and mRNA provide a promising platform for developing safe, efficient, and traceable delivery systems for biological research.

Synthesis and characterization of vitamin D3-functionalized carbon dots for CRISPR/Cas9 delivery.

Aim: To develop a novel nanovector for the delivery of genetic fragments and CRISPR/Cas9 systems in particular. Materials & methods: Vitamin D3-functionalized carbon dots (D/CDs) fabricated using one-step microwave-aided methods were characterized by different microscopic and spectroscopic techniques. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry were employed to determine the cell viability and transfection efficiency. Results: D/CDs transfected CRISPR plasmid in various cell lines with high efficiency while maintaining their remarkable efficacy at high serum concentration and low plasmid doses. They also showed great potential for the green fluorescent protein disruption by delivering two different types of CRISPR/Cas9 systems. Conclusion: Given their high efficiency and safety, D/CDs provide a versatile gene-delivery vector for clinical applications.

Complex signal processing in synthetic gene circuits using cooperative regulatory assemblies.

Eukaryotic genes are regulated by multivalent transcription factor complexes. Through cooperative self-assembly, these complexes perform nonlinear regulatory operations involved in cellular decision-making and signal processing. In this study, we apply this design principle to synthetic networks, testing whether engineered cooperative assemblies can program nonlinear gene circuit behavior in yeast. Using a model-guided approach, we show that specifying the strength and number of assembly subunits enables predictive tuning between linear and nonlinear regulatory responses for single- and multi-input circuits. We demonstrate that assemblies can be adjusted to control circuit dynamics. We harness this capability to engineer circuits that perform dynamic filtering, enabling frequency-dependent decoding in cell populations. Programmable cooperative assembly provides a versatile way to tune the nonlinearity of network connections, markedly expanding the engineerable behaviors available to synthetic circuits.

Noble metal nanostructures in optical biosensors: Basics, and their introduction to anti-doping detection.

Nanotechnology has illustrated significant potentials in biomolecular-sensing applications; particularly its introduction to anti-doping detection is of great importance. Illicit recreational drugs, substances that can be potentially abused, and drugs with dosage limitations according to the prohibited lists announced by the World Antidoping Agency (WADA) are becoming of increasing interest to forensic chemists. In this review, the theoretical principles of optical biosensors based on noble metal nanoparticles, and the transduction mechanism of commonly-applied plasmonic biosensors are covered. We review different classes of recently-developed plasmonic biosensors for analytic determination and quantification of illicit drugs in anti-doping applications. The important classes of illicit drugs include anabolic steroids, opioids, stimulants, and peptide hormones. The main emphasis is on the advantages that noble metal nano-particles bring to optical biosensors for signal enhancement and the development of highly sensitive (label-free) biosensors. In the near future, such optical biosensors may be an invaluable substitute for conventional anti-doping detection methods such as chromatography-based approaches, and may even be commercialized for routine anti-doping tests.

Bacterial components as naturally inspired nano-carriers for drug/gene delivery and immunization: Set the bugs to work?

Drug delivery is a rapidly growing area of research motivated by the nanotechnology revolution, the ideal of personalized medicine, and the desire to reduce the side effects of toxic anti-cancer drugs. Amongst a bewildering array of different nanostructures and nanocarriers, those examples that are fundamentally bio-inspired and derived from natural sources are particularly preferred. Delivery of vaccines is also an active area of research in this field. Bacterial cells and their components that have been used for drug delivery, include the crystalline cell-surface layer known as "S-layer", bacterial ghosts, bacterial outer membrane vesicles, and bacterial products or derivatives (e.g. spores, polymers, and magnetic nanoparticles). Considering the origin of these components from potentially pathogenic microorganisms, it is not surprising that they have been applied for vaccines and immunization. The present review critically summarizes their applications focusing on their advantages for delivery of drugs, genes, and vaccines.

Hsf1 Phosphorylation Generates Cell-to-Cell Variation in Hsp90 Levels and Promotes Phenotypic Plasticity.

Clonal populations of cells exhibit cell-to-cell variation in the transcription of individual genes. In addition to this noise in gene expression, heterogeneity in the proteome and the proteostasis network expands the phenotypic diversity of a population. Heat shock factor 1 (Hsf1) regulates chaperone gene expression, thereby coupling transcriptional noise to proteostasis. Here we show that cell-to-cell variation in Hsf1 activity is an important determinant of phenotypic plasticity. Budding yeast cells with high Hsf1 activity were enriched for the ability to acquire resistance to an antifungal drug, and this enrichment depended on Hsp90, a known phenotypic capacitor and canonical Hsf1 target. We show that Hsf1 phosphorylation promotes cell-to-cell variation, and this variation, rather than absolute Hsf1 activity, promotes antifungal resistance. We propose that Hsf1 phosphorylation enables differential tuning of the proteostasis network in individual cells, allowing populations to access a range of phenotypic states.

Carbon nanotubes in microfluidic lab-on-a-chip technology: current trends and future perspectives.

Advanced nanomaterials such as carbon nano-tubes (CNTs) display unprecedented properties such as strength, electrical conductance, thermal stability, and intriguing optical properties. These properties of CNT allow construction of small microfluidic devices leading to miniaturization of analyses previously conducted on a laboratory bench. With dimensions of only millimeters to a few square centimeters, these devices are called lab-on-a-chip (LOC). A LOC device requires a multidisciplinary contribution from different fields and offers automation, portability, and high-throughput screening along with a significant reduction in reagent consumption. Today, CNT can play a vital role in many parts of a LOC such as membrane channels, sensors and channel walls. This review paper provides an overview of recent trends in the use of CNT in LOC devices and covers challenges and recent advances in the field. CNTs are also reviewed in terms of synthesis, integration techniques, functionalization and superhydrophobicity. In addition, the toxicity of these nanomaterials is reviewed as a major challenge and recent approaches addressing this issue are discussed.

Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation.

Heat shock factor (Hsf1) regulates the expression of molecular chaperones to maintain protein homeostasis. Despite its central role in stress resistance, disease and aging, the mechanisms that control Hsf1 activity remain unresolved. Here we show that in budding yeast, Hsf1 basally associates with the chaperone Hsp70 and this association is transiently disrupted by heat shock, providing the first evidence that a chaperone repressor directly regulates Hsf1 activity. We develop and experimentally validate a mathematical model of Hsf1 activation by heat shock in which unfolded proteins compete with Hsf1 for binding to Hsp70. Surprisingly, we find that Hsf1 phosphorylation, previously thought to be required for activation, in fact only positively tunes Hsf1 and does so without affecting Hsp70 binding. Our work reveals two uncoupled forms of regulation - an ON/OFF chaperone switch and a tunable phosphorylation gain - that allow Hsf1 to flexibly integrate signals from the proteostasis network and cell signaling pathways.

Microfluidic platform for controlling the differentiation of embryoid bodies.

Embryonic stem (ES) cells are pluripotent cells, which can differentiate into any cell type. This cell type has often been implicated as an eminent source of renewable cells for tissue regeneration and cellular replacement therapies. Studies on manipulation of the various differentiation pathways have been at the forefront of research. There are many ways in which ES cells can be differentiated. One of the most common techniques is to initiate the development of embryoid bodies (EBs) by in vitro aggregation of ES cells. Thereafter, EBs can be induced to undergo differentiation into various cell lineages. In this article, we present a microfluidic platform using biocompatible materials, which is suitable for culturing EBs. The platform is based on a Y-channel device with two inlets for two different culturing media. An EB is located across both streams. Using the laminar characteristics at low Reynolds number and high Peclet numbers, we have induced cell differentiation on half of the EB while maintaining the other half in un-induced stages. The results prove the potential of using microfluidic technology for manipulation of EBs and ES cells in tissue engineering.