N-terminal serine/threonine motif has diverse and important effects on behavior of multiple AAV serotypes.

Adeno-associated virus (AAV) is a promising gene therapy vector, but questions remain regarding mechanisms of basic viral functions. We previously showed that a serine/threonine (S/T) triplet motif and its flanking residues, located in the overlapping N-terminus of VP1/VP2 and highly conserved across most AAV serotypes, are critical for viral transcript production in vitro. Here we generate a panel of S/T triplet mutants in AAV serotypes 2, 4, and 9 and characterize their behaviors in vitro and in vivo using next generation sequencing. We show that S/T triplet mutations can significantly hinder some stages of transduction in a serotype-dependent manner in vitro. Interestingly, these defects are largely overcome in C57BL/6 mice, with only one mutant displaying altered behavior in vivo. Taken together, our results identify a short N-terminal capsid motif with diverse roles across several AAV serotypes which better informs engineering efforts to improve AAV as a vector for gene therapy.

Alkali treatment facilitates functional nano-hydroxyapatite coating of 3D printed polylactic acid scaffolds.

Autografting is currently the gold standard for treatment of bone defects, but has shown disadvantages in the limited volume of and donor site morbidity associated with harvested bone. Customized bone scaffolds that mimic the mechanical and biological properties of native bone are needed to augment the currently limited bone regeneration strategies. To achieve this goal, a repeated cross-hatch structure with uniform cubic pores was designed and 3D printed using polylactic acid (PLA) via fused deposition modeling (FDM). PLA surfaces were modified by wet chemical (alkali) treatment for either 1 h (1hAT) or 6 h (6hAT), followed by coating with nano-hydroxyapatite (nHA). Our hypotheses were that: (i) 6-hour (but not 1-hour) alkali treatment would enhance nHA coating, (ii) the nHA coating on the 6-hour alkali-treated surface would increase hydrophilicity and cell attachment/proliferation, and (iii) stiffness, but not effective Young's modulus, would be reduced by 6-hour alkali treatment. The effects of AT and nHA coating on scaffold morphology was observed by scanning electron microscopy and quantified using a custom MATLAB script. Chemical composition and hydrophilicity were evaluated via energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy, and water contact angle analyses, respectively. Mechanical testing and in vitro cell culture were further employed to analyze compressive properties, and cell attachment and proliferation, respectively. As expected, 6hAT led to reduced strut width and stiffness, while improving the nHA coating and hydrophilicity. Interestingly, PLA/6hAT but not PLA/6hAT/nHA demonstrated a reduction in effective modulus compared to PLA and PLA/nHA scaffolds. From in vitro experiments, the combined PLA/6hAT/nHA modification resulted in the greatest extent of cell attachment but not proliferation. These results collectively demonstrate that the PLA/6hAT/nHA scaffold exhibits properties that may prove beneficial for cancellous bone regeneration.

Joint Image Encryption and Screen-Cam Robust Two Watermarking Scheme.

This paper proposes a joint encryption and screen-cam robust watermarking scheme. This method combines the advantages of smartphone, encryption and watermarking technologies, thereby achieving watermark extraction with a smartphone, partial decryption and tracking leakage from sneak shots. We design a dual watermarking algorithm to achieve watermark detection from both encrypted and decrypted images. First, a watermark is embedded in the discrete Fourier transform (DFT) domain to enable leakage tracking. Then, a second watermark is generated based on QR (Quick response) code encoding and inverse DFT to achieve high watermark capacity and error correction ability, where the secret key for decryption is included in the watermark message. By hiding this message carrying the watermark for the encrypted image in the changes caused by embedding the first watermark, we can improve imperceptibility and will not affect the effectiveness of the proposed scheme. Finally, to enhance the robustness of watermark after encryption, a chaotic mapping-based segment encryption algorithm is proposed. In the process of watermark detection, to cope with perspective correction, a frame locating based algorithm is employed to achieve watermark synchronization from a recaptured picture of the encrypted image. Considering the severe quality degradation, we use a noise component and local statistic feature-based method to extract the message bits. The experimental results show that the proposed scheme is secure, and highly robust, to screen-cam the process for both before and after decryption. Additionally, after decryption, the proposed scheme also has high robustness against common image processing attacks.

Constructing and evaluating caspase-activatable adeno-associated virus vector for gene delivery to the injured heart.

Adeno-associated virus (AAV) is a promising vector for gene therapy, but its broad tropism can be detrimental if the transgene being delivered is harmful when expressed ubiquitously in the body, i.e. in non-target tissues. Delivering the transgene of interest to target cells at levels high enough to be therapeutically effective while maintaining safety by minimizing delivery to off-target cells is a prevalent challenge in the field of gene therapy. We have developed a protease activatable vector (provector) platform based on AAV9 that can be injected systemically to deliver therapeutic transgenes site-specifically to diseased cells by responding to extracellular proteases present at the disease site. The provector platform consists of a peptide insertion into the virus capsid which disrupts the virus' ability to bind to cell surface receptors. This peptide contains a blocking motif (aspartic acid residues) flanked on either side by cleavage sequences that are recognized by certain proteases. Exposure to proteases cleaves the peptides off the capsid, activating or "switching ON" the provector. In response to the activation, the provectors regain their ability to bind and transduce cells. Here, we have designed a provector that is activated by cysteine aspartic proteases (caspases), which have roles in inflammation and apoptosis and thus are elevated at sites of diseases such as heart failure, neurodegenerative diseases, and ischemic stroke. This provector demonstrates a 200-fold reduction in transduction ability in the OFF state compared to AAV9, reducing the virus' ability to transduce off-target healthy tissue. Following exposure to and proteolysis by caspase-3, the provector shows a 95-fold increase in transduction compared to the OFF state. The switchable transduction behavior was found to be a direct result of the peptide insertion ablating the ability of the virus to bind to cells. In vivo studies were conducted to characterize the biodistribution, blood circulation time, neutralizing antibody formation, and targeted delivery ability of the caspase-activatable provector in a model of heart failure.

An essential N-terminal serine-rich motif in the AAV VP1 and VP2 subunits that may play a role in viral transcription.

Adeno-associated virus (AAV) is one of the most researched, clinically utilized gene therapy vectors. Though clinical success has been achieved, transgene delivery and expression may be hindered by cellular and tissue barriers. Understanding the role of receptor binding, entry, endosomal escape, cytoplasmic and nuclear trafficking, capsid uncoating, and viral transcription in therapeutic efficacy is paramount. Previous studies have shown that N-terminal regions of the AAV capsid proteins are responsible for endosomal escape and nuclear trafficking, however the mechanisms remain unknown. We identified a highly-conserved three-residue serine/threonine (S/T) motif in the capsid N-terminus, previously uncharacterized in its role in intracellular trafficking and transduction. Using alanine scanning mutagenesis, we found S155 and the flanking residues, D154 and G158, are essential for AAV2 transduction efficiency. Remarkably, specific capsid mutants show a 5 to 9-fold decrease in viral mRNA transcripts, highlighting a potential role of the S/T motif in transcription of the viral genome.

A low-damage plasma surface modification method of stacked graphene bilayers for configurable wettability and electrical properties.

In this work, we study surface functionalization effects of artificially stacked graphene bilayers (ASGBs) to control its wetting properties via low-damage plasma. The ASGBs were prepared on a SiO2/Si substrate by stacking two monolayer graphene, which was grown by chemical vapor deposition. As a result, the low-damage plasma functionalization of ASGBs could hold both the key characteristics of surface functionalization and electrical transport properties of graphene sheets. To characterize ASGBs, Raman and x-ray photoelectron spectroscopy (XPS) were used to determine the degree of defect formation and functionalization. Meanwhile, the degree of the wettability of the ASGBs surface was determined by optical contact angle (CA) measurements. Based on experimental results, the compositional ratio of C-OH + COOH was found to increase 67% based on the analysis of XPS spectra after low-damage plasma treatment. This treatment effect can also be found with 75.3% decrease in the CA of water droplet on graphene. In addition, we found that the ratio of 2D/(D + G') in Raman spectra shows strong correlation to the measured CA; it can be a reliable indicator of ASGBs surface wettability modification. This work showed that we obtained a higher degree functionalization of ASGBs without degrading the under-layer structure of ASGBs due to the moderate low-damage plasma treatment. The presented process technique of controllable wettability through low-damage plasma treatment can be employed for potential application in graphene-based sensors/devices.

Protease-Activatable Adeno-Associated Virus Vector for Gene Delivery to Damaged Heart Tissue.

Adeno-associated virus (AAV) has emerged as a promising gene delivery vector because of its non-pathogenicity, simple structure and genome, and low immunogenicity compared to other viruses. However, its adoption as a safe and effective delivery vector for certain diseases relies on altering its tropism to deliver transgenes to desired cell populations. To this end, we have developed a protease-activatable AAV vector, named provector, that responds to elevated extracellular protease activity commonly found in diseased tissue microenvironments. The AAV9-based provector is initially inactive, but then it can be switched on by matrix metalloproteinases (MMP)-2 and -9. Cryo-electron microscopy and image reconstruction reveal that the provector capsid is structurally similar to that of AAV9, with a flexible peptide insertion at the top of the 3-fold protrusions. In an in vivo model of myocardial infarction (MI), the provector is able to deliver transgenes site specifically to high-MMP-activity regions of the damaged heart, with concomitant decreased delivery to many off-target organs, including the liver. The AAV provector may be useful in the future for enhanced delivery of transgenes to sites of cardiac damage.

Physical, chemical, and synthetic virology: Reprogramming viruses as controllable nanodevices.

The fields of physical, chemical, and synthetic virology work in partnership to reprogram viruses as controllable nanodevices. Physical virology provides the fundamental biophysical understanding of how virus capsids assemble, disassemble, display metastability, and assume various configurations. Chemical virology considers the virus capsid as a chemically addressable structure, providing chemical pathways to modify the capsid exterior, interior, and subunit interfaces. Synthetic virology takes an engineering approach, modifying the virus capsid through rational, combinatorial, and bioinformatics-driven design strategies. Advances in these three subfields of virology aim to develop virus-based materials and tools that can be applied to solve critical problems in biomedicine and biotechnology, including applications in gene therapy and drug delivery, diagnostics, and immunotherapy. Examples discussed include mammalian viruses, such as adeno-associated virus (AAV), plant viruses, such as cowpea mosaic virus (CPMV), and bacterial viruses, such as Qß bacteriophage. Importantly, research efforts in physical, chemical, and synthetic virology have further unraveled the design principles foundational to the form and function of viruses. This article is categorized under: Diagnostic Tools > Diagnostic Nanodevices Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy.

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

Pluri-IQ: Quantification of Embryonic Stem Cell Pluripotency through an Image-Based Analysis Software.

Image-based assays, such as alkaline phosphatase staining or immunocytochemistry for pluripotent markers, are common methods used in the stem cell field to assess pluripotency. Although an increased number of image-analysis approaches have been described, there is still a lack of software availability to automatically quantify pluripotency in large images after pluripotency staining. To address this need, we developed a robust and rapid image processing software, Pluri-IQ, which allows the automatic evaluation of pluripotency in large low-magnification images. Using mouse embryonic stem cells (mESC) as a model, we combined an automated segmentation algorithm with a supervised machine-learning platform to classify colonies as pluripotent, mixed, or differentiated. In addition, Pluri-IQ allows the automatic comparison between different culture conditions. This efficient user-friendly open-source software can be easily implemented in images derived from pluripotent cells or cells that express pluripotent markers (e.g., OCT4-GFP) and can be routinely used, decreasing image assessment bias.

Local 3D matrix confinement determines division axis through cell shape.

How the division axis is determined in mammalian cells embedded in three-dimensional (3D) matrices remains elusive, despite that many types of cells divide in 3D environments. Cells on two-dimensional (2D) substrates typically round up completely to divide. Here, we show that in 3D collagen matrices, mammalian cells such as HT1080 human fibrosarcoma and MDA-MB-231 breast cancer cells exhibit division modes distinct from their Counterparts on 2D substrates, with a markedly higher fraction of cells remaining highly elongated through mitosis in 3D matrices. The long axis of elongated mitotic cells accurately predicts the division axis, independently of matrix density and cell-matrix interactions. This 3D-specific elongated division mode is determined by the local confinement produced by the matrix and the ability of cells to protrude and locally remodel the matrix via ß1 integrin. Elongated division is readily recapitulated using collagen-coated microfabricated channels. Cells depleted of ß1 integrin still divide in the elongated mode in microchannels, suggesting that 3D confinement is sufficient to induce the elongated cell-division phenotype.