Digital data storage on DNA tape using CRISPR base editors.

While the archival digital memory industry approaches its physical limits, the demand is significantly increasing, therefore alternatives emerge. Recent efforts have demonstrated DNA's enormous potential as a digital storage medium with superior information durability, capacity, and energy consumption. However, the majority of the proposed systems require on-demand de-novo DNA synthesis techniques that produce a large amount of toxic waste and therefore are not industrially scalable and environmentally friendly. Inspired by the architecture of semiconductor memory devices and recent developments in gene editing, we created a molecular digital data storage system called "DNA Mutational Overwriting Storage" (DMOS) that stores information by leveraging combinatorial, addressable, orthogonal, and independent in vitro CRISPR base-editing reactions to write data on a blank pool of greenly synthesized DNA tapes. As a proof of concept, this work illustrates writing and accurately reading of both a bitmap representation of our school's logo and the title of this study on the DNA tapes.

Digital data storage on DNA tape using CRISPR base editors.

While the archival digital memory industry approaches its physical limits, the demand is significantly increasing, therefore alternatives emerge. Recent efforts have demonstrated DNA's enormous potential as a digital storage medium with superior information durability, capacity, and energy consumption. However, the majority of the proposed systems require on-demand de-novo DNA synthesis techniques that produce a large amount of toxic waste and therefore are not industrially scalable and environmentally friendly. Inspired by the architecture of semiconductor memory devices and recent developments in gene editing, we created a molecular digital data storage system called "DNA Mutational Overwriting Storage" (DMOS) that stores information by leveraging combinatorial, addressable, orthogonal, and independent in vitro CRISPR base-editing reactions to write data on a blank pool of greenly synthesized DNA tapes. As a proof of concept, we wrote both a bitmap representation of our school's logo and the title of this study on the DNA tapes, and accurately recovered the stored data.

Dark-Field Microscopic Study of Cellular Uptake of Carbon Nanodots: Nuclear Penetrability.

Carbon nanodots are fascinating candidates for the field of biomedicine, in applications such as bioimaging and drug delivery. However, the nuclear penetrability and process are rarely studied and lack understanding, which limits their applications for drug carriers, single-molecule detection and live cell imaging. In this study, we attempt to examine the uptake of CNDs in cells with a focus on the potential nuclear penetrability using enhanced dark-field microscopy (EDFM) associated with hyperspectral imaging (HSI) to quantitatively determine the light scattering signals of CNDs in the cells. The effects of both CND incubation time and concentration are investigated, and plausible nuclear penetration involving the nuclear pore complex (NPC) is discussed. The experimental results and an analytical model demonstrate that the CNDs' uptake proceeds by a concentration-dependent three-stage behavior and saturates at a CND incubation concentration larger than 750 µg/mL, with a half-saturated concentration of 479 µg/mL. These findings would potentially help the development of CNDs' utilization in drug carriers, live cell imaging and other biomedical applications.

Tuning Microbial Activity via Programmatic Alteration of Cell/Substrate Interfaces.

A wide portfolio of advanced programmable materials and structures has been developed for biological applications in the last two decades. Particularly, due to their unique properties, semiconducting materials have been utilized in areas of biocomputing, implantable electronics, and healthcare. As a new concept of such programmable material design, biointerfaces based on inorganic semiconducting materials as substrates introduce unconventional paths for bioinformatics and biosensing. In particular, understanding how the properties of a substrate can alter microbial biofilm behavior enables researchers to better characterize and thus create programmable biointerfaces with necessary characteristics on demand. Herein, the current status of advanced microorganism-inorganic biointerfaces is summarized along with types of responses that can be observed in such hybrid systems. This work identifies promising inorganic material types along with target microorganisms that will be critical for future research on programmable biointerfacial structures.

Cell Rupture and Morphogenesis Control of the Dimorphic Yeast Candida albicans by Nanostructured Surfaces.

Nanostructured surfaces control microbial biofilm formation by killing mechanically via surface architecture. However, the interactions between nanostructured surfaces (NSS) and cellular fungi have not been thoroughly investigated and the application of NSS as a means of controlling fungal biofilms is uncertain. Cellular yeast such as Candida albicans are structurally and biologically distinct from prokaryotic microbes and therefore are predicted to react differently to nanostructured surfaces. The dimorphic opportunistic fungal pathogen, C. albicans, is responsible for most cases of invasive candidiasis and is a serious health concern due to the rapid increase of drug resistance strains. In this paper, we show that the nanostructured surfaces from a cicada wing alter C. albicans' viability, biofilm formation, adhesion, and morphogenesis through physical contact. However, the fungal cell response to the NSS suggests that nanoscale mechanical interactions impact C. albicans differently than prokaryotic microbes. This study informs on the use of nanoscale architecture for the control of eukaryotic biofilm formation and illustrates some potential caveats with the application of NSS as an antimicrobial means.

Characterization of Hydrothermal Deposition of Copper Oxide Nanoleaves on Never-Dried Bacterial Cellulose.

Bacterial cellulose (BC) has attracted a great deal of interest due to its green synthesis and biocompatibility. The nanoscale dimension of BC nanofibers generates an enormous surface area that enhances interactions with water and soluble components within aqueous solution. Recent work has demonstrated that BC is a versatile platform for the formation of metal/metal oxide nanocomposites. Copper oxide (CuO) is a useful material to compare nanomaterial deposition on BC with other cellulosic materials because of copper's colorimetric reaction as it forms copper hydroxide (Cu(OH)2) and transitions to CuO. In this research, we found that never-dried BC readily deposits CuO into its matrix in a way that does not occur on cotton, dried BC, or regenerated cellulose fibers. We conclude that hydroxyl group availability does not adequately explain our results and that intrafibrillar pores in never-dried BC nanofibers play a critical role in CuO deposition.

Behavior of E. coli with Variable Surface Morphology Changes on Charged Semiconductor Interfaces.

Bacterial behavior is often controlled by structural and composition elements of their cell wall. Using genetic mutant strains that change specific aspects of their surface structure, we modified bacterial behavior in response to semiconductor surfaces. We monitored the adhesion, membrane potential, and catalase activity of the Gram-negative bacterium Escherichia coli (E. coli) that were mutant for genes encoding components of their surface architecture, specifically flagella, fimbriae, curli, and components of the lipopolysaccharide membrane, while on gallium nitride (GaN) surfaces with different surface potentials. The bacteria and the semiconductor surface properties were recorded prior to the biofilm studies. The data from the materials and bioassays characterization supports the notion that alteration of the surface structure of the E. coli bacterium resulted in changes to bacterium behavior on the GaN medium. Loss of specific surface structure on the E. coli bacterium reduced its sensitivity to the semiconductor interfaces, while other mutations increase bacterial adhesion when compared to the wild-type control E. coli bacteria. These results demonstrate that bacterial behavior and responses to GaN semiconductor materials can be controlled genetically and can be utilized to tune the fate of living bacteria on GaN surfaces.

Nanosphere Lithography of Chitin and Chitosan with Colloidal and Self-Masking Patterning.

Complex surface topographies control, define, and determine the properties of insect cuticles. In some cases, these nanostructured materials are a direct extension of chitin-based cuticles. The cellular mechanisms that generate these elaborate chitin-based structures are unknown, and involve complicated cellular and biochemical "bottom-up" processes. We demonstrated that a synthetic "top-down" fabrication technique-nanosphere lithography-generates surfaces of chitin or chitosan that mimic the arrangement of nanostructures found on the surface of certain insect wings and eyes. Chitin and chitosan are flexible and biocompatible abundant natural polymers, and are a sustainable resource. The fabrication of nanostructured chitin and chitosan materials enables the development of new biopolymer materials. Finally, we demonstrated that another property of chitin and chitosan-the ability to self-assemble nanosilver particles-enables a novel and powerful new tool for the nanosphere lithographic method: the ability to generate a self-masking thin film. The scalability of the nanosphere lithographic technique is a major limitation; however, the silver nanoparticle self-masking enables a one-step thin-film cast or masking process, which can be used to generate nanostructured surfaces over a wide range of surfaces and areas.

Characterization of Pseudomonas aeruginosa Films on Different Inorganic Surfaces before and after UV Light Exposure.

The changes of the surface properties of Au, GaN, and SiO x after UV light irradiation were used to actively influence the process of formation of Pseudomonas aeruginosa films. The interfacial properties of the substrates were characterized by X-ray photoelectron spectroscopy and atomic force microscopy. The changes in the P. aeruginosa film properties were accessed by analyzing adhesion force maps and quantifying the intracellular Ca2+ concentration. The collected analysis indicates that the alteration of the inorganic materials' surface chemistry can lead to differences in biofilm formation and variable response from P. aeruginosa cells.

Noninvasive Stimulation of Neurotypic Cells Using Persistent Photoconductivity of Gallium Nitride.

The persistent photoconductivity (PPC) of the n-type Ga-polar GaN was used to stimulate PC12 cells noninvasively. Analysis of the III-V semiconductor material by atomic force microscopy, Kelvin probe force microscopy, photoconductivity, and X-ray photoelectron spectroscopy quantified bulk and surface charge, as well as chemical composition before and after exposure to UV light and cell culture media. The semiconductor surface was made photoconductive by illumination with UV light and experienced PPC, which was utilized to stimulate PC12 cells in vitro. Stimulation was confirmed by measuring the changes in intracellular calcium concentration. Control experiments with gallium salt verified the stimulation of neurotypic cells. Inductively coupled plasma mass spectrometry data confirmed the lack of gallium leaching and toxic effects during the stimulation.

Bioelectronics communication: encoding yeast regulatory responses using nanostructured gallium nitride thin films.

Baker's yeast, S. cerevisiae, is a model organism that is used in synthetic biology. The work demonstrates how GaN nanostructured thin films can encode physiological responses in S. cerevisiae yeast. The Ga-polar, n-type, GaN thin films are characterized via Photocurrent Measurements, Atomic Force Microscopy and Kelvin Probe Force Microscopy. UV light is used to induce persistent photoconductivity that results in charge accumulation on the surface. The morphological, chemical and electronic properties of the nanostructured films are utilized to activate the cell wall integrity pathway and alter the amount of chitin produced by the yeast. The encoded cell responses are induced by the semiconductor interfacial properties associated with nanoscale topography and the accumulation of charge on the surface that promotes the build-up of oxygen species and in turn cause a hyperoxia related change in the yeast. The thin films can also alter the membrane voltage of yeast. The observed modulation of the membrane voltage of the yeast exposed to different GaN samples supports the notion that the semiconductor material can cause cell polarization. The results thus define a strategy for bioelectronics communication where the roughness, surface chemistry and charge of the wide band gap semiconductor's thin film surface initiate the encoding of the yeast response.

Variably doped nanostructured gallium nitride surfaces can serve as biointerfaces for neurotypic PC12 cells and alter their behavior.

Neurotypic PC12 cells behavior was studied on nanostructured GaN and rationalized with respect to surface charge, doping level, and chemical functionalization. The semiconductor analysis included atomic force microscopy, Kelvin probe force microscopy, and X-ray photoelectron spectroscopy. The semiconductor surfaces were then evaluated as biointerfaces, and the in vitro cell behavior was quantified based on cell viability, reactive oxygen species production, as well as time dependent intracellular Ca concentration, [Ca2+]i, a known cell-signaling molecule. In this work, we show that persistent photoconductivity (PPC) can be used to alter the surface properties prior to chemical functionalization, the concentration of dopants can have some effect on cellular behavior, and that chemical functionalization changes the surface potential before and after exposure to UV light. Finally, we describe some competing mechanisms of PPC-induced [Ca2+]i changes, and how researchers looking to control cell behavior non-invasively can consider PPC as a useful control knob.

Alternative SiO2 Surface Direct MDCK Epithelial Behavior.

The mechanical interactions of cells are mediated through adhesive interactions. In this study, we examined the growth, cellular behavior, and adhesion of MDCK epithelial cells on three different SiO2 substrates: amorphous glass coverslips and the silicon oxide layers that grow on ⟨111⟩ and ⟨100⟩ wafers. While compositionally all three substrates are almost similar, differences in surface energy result in dramatic differences in epithelial cell morphology, cell-cell adhesion, cell-substrate adhesion, actin organization, and extracellular matrix (ECM) protein expression. We also observe striking differences in ECM protein binding to the various substrates due to the hydrogen bond interactions. Our results demonstrate that MDCK cells have a robust response to differences in substrates that is not obviated by nanotopography or surface composition and that a cell's response may manifest through subtle differences in surface energies of the materials. This work strongly suggests that other properties of a material other than composition and topology should be considered when interpreting and controlling interactions of cells with a substrate, whether it is synthetic or natural.

Peristalsis in the junction region of the Drosophila larval midgut is modulated by DH31 expressing enteroendocrine cells.

BACKGROUND: The underlying cellular and molecular mechanisms that coordinate the physiological processes in digestion are complex, cryptic, and involve the integration of multiple cellular and organ systems. In all intestines, peristaltic action of the gut moves food through the various stages of digestion from the anterior end towards the posterior, with the rate of flow dependent on signals, both intrinsic and extrinsic to the gut itself. RESULTS: We have identified an enteroendocrine cell type that regulates gut motility in the Drosophila melanogaster larval midgut. These cells are located at the junction of the anterior and the acidic portions of the midgut and are a group of enteroendocrine cells that express the peptide hormone Diuretic Hormone 31 in this region of the gut. Using cell ablation and ectopic activation via expression of the Chlamydomonas reinhardtii blue light-activated channelopsin, we demonstrate that these enteroendocrine cells are both necessary and sufficient for the peristalsis in the junction region of the midgut and require the Diuretic Hormone 31 to affect normal peristalsis in this region. Within the same junction region of the midgut, we have also identified morphological features suggesting that this region acts as a valve that regulates the transit of food from the anterior midgut into the acidic portion of the gut. CONCLUSIONS: We have characterized and described a set of enteroendocrine cells called the Midgut Junction DH31 expressing cells that are required for peristaltic movement in the junction region between the anterior portion and acidic region of the larval midgut of Drosophila melanogaster. We have shown that the Midgut Junction DH31 expressing cells are necessary and sufficient for motility and that the peptide hormone DH31 is required for peristalsis in the junction region of the midgut. The Drosophila model system will allow for a further dissection of the digestion process and provide a better understanding of the mechanisms that regulate digestion in all organisms.

Modeling early Epstein-Barr virus infection in Drosophila melanogaster: the BZLF1 protein.

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with several forms of cancer, including lymphomas and nasopharyngeal carcinoma. The EBV immediate-early protein BZLF1 functions as a transcriptional activator of EBV early gene expression and is essential for the viral transition between latent and lytic replication. In addition to its role in the EBV life cycle, BZLF1 (Z) also has profound effects upon the host cellular environment, including disruption of cell cycle regulation, signal transduction pathways, and transcription. In an effort to understand the nature of Z interactions with the host cellular environment, we have developed a Drosophila model of early EBV infection, where we have expressed Z in the Drosophila eye. Using this system, we have identified a highly conserved interaction between the Epstein-Barr virus Z protein and shaven, a Drosophila homolog of the human Pax2/5/8 family of genes. Pax5 is a well-characterized human gene involved with B-cell development. The B-cell-specific Pax5 also promotes the transcription of EBV latent genes from the EBV Wp promoter. Our work clearly demonstrates that the Drosophila system is an appropriate and powerful tool for identifying the underlying genetic networks involved in human infectious disease.

Epstein-Barr virus immediate-early proteins BZLF1 and BRLF1 alter mitochondrial morphology during lytic replication.

Epstein-Barr virus (EBV) is a human DNA virus that is responsible for the syndrome infectious mononucleosis, and is associated with several forms of cancer. During both lytic and latent viral infection, viral proteins manipulate the host's cellular components to aid in viral replication and maintenance. Here, it is demonstrated that induction of EBV lytic replication results in a dramatic reorganization of mitochondria accompanied by a significant alteration of mitochondrial membrane potential and a rapid and transient increase in the microtubular cytoskeleton. Moreover, we show that expression of the EBV immediate-early genes BZLF1 and BRLF1 contributes to the mitochondrial alteration but not the increase in the microtubule cytoskeleton, suggesting that the mechanism for the observed cytoplasmic restructuring involves a number of coordinated viral and host proteins.