UNC/NCSU Joint Department of Biomedical Engineering

CRISPR-Cas9 represents a promising platform for genome editing, yet means for its safe and efficient delivery remain to be fully realized. A novel vehicle that simultaneously delivers the Cas9 protein and single guide RNA (sgRNA) is based on DNA nanoclews, yarn-like DNA nanoparticles that are synthesized by rolling circle amplification. The biologically inspired vehicles were efficiently loaded with Cas9/sgRNA complexes and delivered the complexes to the nuclei of human cells, thus enabling targeted gene disruption while maintaining cell viability. Editing was most efficient when the DNA nanoclew sequence and the sgRNA guide sequence were partially complementary, offering a design rule for enhancing delivery. Overall, this strategy provides a versatile method that could be adapted for delivering other DNA-binding proteins or functional nucleic acids.

This review summarizes clinical studies in which glutathione was measured in tumor tissue from patients with brain, breast, gastrointestinal, gynecological, head and neck and lung cancer. Glutathione tends to be elevated in breast, ovarian, head and neck, and lung cancer and lower in brain and liver tumors compared to disease-free tissue. Cervical, colorectal, gastric, and esophageal cancers show both higher and lower levels of tumor glutathione. Some studies show an inverse relationship between patient survival and tumor glutathione. Based on this survey, we recommend approaches that may improve the clinical value of glutathione as a biomarker.

The modification of protein and nanoparticle therapeutics with polyethylene glycol ( PEG ), a flexible, uncharged, and highly hydrophilic polymer, is a widely adopted approach to reduce RES clearance, extend circulation time, and improve drug efficacy. Nevertheless, an emerging body of literature, generated by numerous research groups, demonstrates that the immune system can produce antibodies that specifically bind PEG , which can lead to the ‘accelerated blood clearance’ of PEGylated therapeutics. In animals, anti‐ PEG immunity is typically robust but short‐lived and consists of a predominantly anti‐ PEG IgM response. Rodent studies suggest that the induction of anti‐ PEG antibodies (α‐ PEG Abs) primarily occurs through a type 2 T‐cell independent mechanism. Although anti‐ PEG immunity is less well‐studied in humans, the presence of α‐ PEG Abs has been correlated with reduced efficacy of PEGylated therapeutics in clinical trials. The prevalence of anti‐ PEG IgG and reports of memory immune responses, as well as the existence of α‐ PEG Abs in healthy untreated individuals, suggests that the mechanism(s) and features of human anti‐ PEG immune responses may differ from those of animal models. Many questions, including the incidence rate of pre‐existing α‐ PEG Abs and immunological mechanism(s) of α‐ PEG Ab formation in humans, must be answered in order to fully address the potential complications of anti‐ PEG immunity. WIREs Nanomed Nanobiotechnol 2015, 7:655–677. doi: 10.1002/wnan.1339 This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials

Circulating antibodies (Ab) that specifically bind polyethylene glycol (PEG), a biocompatible polymer routinely used in protein and nanoparticle therapeutics, have been associated with reduced efficacy of and/or adverse reactions to therapeutics modified with or containing PEG. Unlike most antidrug antibodies that are induced following initial drug dosing, anti-PEG Ab can be found in treatment-naïve individuals (i.e., individuals who have never undergone treatment with PEGylated drugs but most likely have been exposed to PEG through other means). Unfortunately, the true prevalence, quantitative levels, and Ab isotype of pre-existing anti-PEG Ab remain poorly understood. Here, using rigorously validated competitive ELISAs with engineered chimeric anti-PEG monoclonal Ab standards, we quantified the levels of anti-PEG IgM and different subclasses of anti-PEG IgG (IgG1-4) in both contemporary and historical human samples. We unexpectedly found, with 90% confidence, detectable levels of anti-PEG Ab in ∼72% of the contemporary specimens (18% IgG, 25% IgM, 30% both IgG and IgM). The vast majority of these samples contained low levels of anti-PEG Ab, with only ∼7% and ∼1% of all specimens possessing anti-PEG IgG and IgM in excess of 500 ng/mL, respectively. IgG2 was the predominant anti-PEG IgG subclass. Anti-PEG Ab's were also observed in ∼56% of serum samples collected during 1970-1999 (20% IgG, 19% IgM, and 16% both IgG and IgM), suggesting that the presence of PEG-specific antibodies may be a longstanding phenomenon. Anti-PEG IgG levels demonstrated correlation with patient age, but not with gender or race. The widespread prevalence of pre-existing anti-PEG Ab, coupled with high Ab levels in a subset of the population, underscores the potential importance of screening patients for anti-PEG Ab levels prior to administration of therapeutics containing PEG.

Coating nanoparticles with polyethylene glycol (PEG), which reduces particle uptake and clearance by immune cells, is routinely used to extend the circulation times of nanoparticle therapeutics. Nevertheless, due to technical hurdles in quantifying the extent of PEG grafting, as well as in generating very dense PEG coatings, few studies have rigorously explored the precise PEG grafting density necessary to achieve desirable "stealth" properties. Here, using polymeric nanoparticles with precisely tunable PEG grafting, we found that, for a wide range of PEG lengths (0.6-20 kDa), PEG coatings at densities substantially exceeding those required for PEG to adopt a "brush" conformation are exceptionally resistant to uptake by cultured human macrophages, as well as primary peripheral blood leukocytes. Less than 20% of these nanoparticles were cleared from the blood after 2 h (t1/2 ∼ 14 h) in BALB/c mice, whereas slightly less densely PEGylated and uncoated control particles were both virtually eliminated within 2 h. Our results suggest that the stealth properties of PEG-coated nanoparticles are critically dependent on achieving PEG grafting at densities exceeding those required for brush conformation.

Two-photon polymerization (2PP) leverages the two-photon absorption (TPA) of near-infrared (NIR) radiation for additive manufacturing with sub-diffraction limit resolution within the bulk of a photosensitive material. This technology draws heavily on photosensitive polymers from the microelectronics industry, which were not optimized for TPA or for biocompatibility. 2PP with sub 100 nm resolution has been repeatedly demonstrated; however, this level of fabrication resolution comes at the expense of long fabrication times. Manufacturing of medical devices beyond surface texturing would be prohibitively slow using the current state of the art 2PP technology. Current research into TPA-sensitive photopolymers with good biocompatibility and holographic projections using spatial light modulators address current technological limitations by providing materials specifically formulated for biological applications and by making better use of available laser power for applications in which nanoscale resolution is not required.

The ability of bone-marrow-derived mesenchymal stem cells (MSCs) and adipose-derived stem cells (ASCs) to undergo chondrogenic differentiation has been studied extensively, and it has been suggested that the chondrogenic potential of these stem cells differ from each other. Here, we provide a comprehensive review and analysis of the various growth factor induction agents for MSC and ASC three-dimensional in vitro chondrogenic differentiation. In general, the most common growth factors for chondrogenic induction come from the transforming growth factor beta (TGFbeta) superfamily. To date, the most promising growth factors for chondrogenesis appear to be TGFbeta-3 and bone morphogenetic protein (BMP)-6. A thorough review of the literature indicates that human MSCs (hMSCs) appear to exhibit the highest chondrogenic potential in three-dimensional culture in the medium containing both dexamethasone and TGFbeta-3. Some reports indicate that the addition of BMP-6 to TFGbeta-3 and dexamethasone further increases hMSC chondrogenesis, but these results are still not consistently supported. Induction of human ASC (hASC) chondrogenesis appears most successful when dexamethasone, TGFbeta-3, and BMP-6 are used in combination. However, to date, current formulations do not always result in stable differentiation to the chondrocytic lineage by hMSCs and hASCs. Continued research must be performed to examine the expression cascades of the TFGbeta superfamily to further determine the effects of each growth factor alone and in combination on these stem cell lines.

UNLABELLED: Cervicovaginal mucus (CVM) can provide a barrier that precludes HIV and other sexually transmitted virions from reaching target cells in the vaginal epithelium, thereby preventing or reducing infections. However, the barrier properties of CVM differ from woman to woman, and the causes of these variations are not yet well understood. Using high-resolution particle tracking of fluorescent HIV-1 pseudoviruses, we found that neither pH nor Nugent scores nor total lactic acid levels correlated significantly with virus trapping in unmodified CVM from diverse donors. Surprisingly, HIV-1 was generally trapped in CVM with relatively high concentrations of d-lactic acid and a Lactobacillus crispatus-dominant microbiota. In contrast, a substantial fraction of HIV-1 virions diffused rapidly through CVM with low concentrations of d-lactic acid that had a Lactobacillus iners-dominant microbiota or significant amounts of Gardnerella vaginalis, a bacterium associated with bacterial vaginosis. Our results demonstrate that the vaginal microbiota, including specific species of Lactobacillus, can alter the diffusional barrier properties of CVM against HIV and likely other sexually transmitted viruses and that these microbiota-associated changes may account in part for the elevated risks of HIV acquisition linked to bacterial vaginosis or intermediate vaginal microbiota. IMPORTANCE: Variations in the vaginal microbiota, especially shifts away from Lactobacillus-dominant microbiota, are associated with differential risks of acquiring HIV or other sexually transmitted infections. However, emerging evidence suggests that Lactobacillus iners frequently colonizes women with recurring bacterial vaginosis, raising the possibility that L. iners may not be as protective as other Lactobacillus species. Our study was designed to improve understanding of how the cervicovaginal mucus barrier against HIV may vary between women along with the vaginal microbiota and led to the finding that the vaginal microbiota, including specific species of Lactobacillus, can directly alter the diffusional barrier properties of cervicovaginal mucus. This work advances our understanding of the complex barrier properties of mucus and highlights the differential protective ability of different species of Lactobacillus, with Lactobacillus crispatus and possibly other species playing a key role in protection against HIV and other sexually transmitted infections. These findings could lead to the development of novel strategies to protect women against HIV.

BACKGROUND: Design thinking is a problem-solving framework that has been used to enhance patient experiences, improve clinical outcomes, and refine medical curricula. This study reviewed the use of design thinking in health professions education. METHODS: A search yielded 169 articles, which were excluded if they were: (1) not related to education; (2) lacking an application of design thinking; or (3) not associated with healthcare. The final review yielded 15 articles, which were analyzed using qualitative methods. RESULTS: All articles were published in 2009 or later and were diverse in their context, participants, and approach. Six studies emphasized the early stages of design thinking, with inspiration and ideation stages fostered through a variety of activities, such as lectures, small group discussions, and workshops. Studies examined a range of outcomes, including self-efficacy, perceptions, and solutions to a specific problem. CONCLUSIONS: Our findings raise important considerations for health professions education, including the extent to which we should: 1) teach design thinking to students as a skill-based tool to prepare students for problem solving in complex healthcare environments; and 2) use design thinking to create, implement, and refine health professions curricula and educational programs. Despite the apparent benefits of design thinking, many questions for health professions education remain.

Robotic prostheses deliver greater function than passive prostheses, but we face the challenge of tuning a large number of control parameters in order to personalize the device for individual amputee users. This problem is not easily solved by traditional control designs or the latest robotic technology. Reinforcement learning (RL) is naturally appealing. The recent, unprecedented success of AlphaZero demonstrated RL as a feasible, large-scale problem solver. However, the prosthesis-tuning problem is associated with several unaddressed issues such as that it does not have a known and stable model, the continuous states and controls of the problem may result in a curse of dimensionality, and the human-prosthesis system is constantly subject to measurement noise, environmental change and human-body-caused variations. In this paper, we demonstrated the feasibility of direct heuristic dynamic programming, an approximate dynamic programming (ADP) approach, to automatically tune the 12 robotic knee prosthesis parameters to meet individual human users' needs. We tested the ADP-tuner on two subjects (one able-bodied subject and one amputee subject) walking at a fixed speed on a treadmill. The ADP-tuner learned to reach target gait kinematics in an average of 300 gait cycles or 10 min of walking. We observed improved ADP tuning performance when we transferred a previously learned ADP controller to a new learning session with the same subject. To the best of our knowledge, our approach to personalize robotic prostheses is the first implementation of online ADP learning control to a clinical problem involving human subjects.

The Focal Adhesion Analysis Server (FAAS) is a web-based implementation of a set of computer vision algorithms designed to quantify the behavior of focal adhesions in cells imaged in 2D cultures. The input consists of one or more images of a labeled focal adhesion protein. The outputs of the system include a range of static and dynamic measurements for the adhesions present in each image as well as how these properties change over time. The user is able to adjust several parameters important for proper focal adhesion identification. This system provides a straightforward tool for the global, unbiased assessment of focal adhesion behavior common in optical microscopy studies. The webserver is available at: http://faas.bme.unc.edu/.

The power of citizen science to contribute to both science and society is gaining increased recognition, particularly in physics and biology. Although there is a long history of public engagement in agriculture and food science, the term 'citizen science' has rarely been applied to these efforts. Similarly, in the emerging field of citizen science, most new citizen science projects do not focus on food or agriculture. Here, we convened thought leaders from a broad range of fields related to citizen science, agriculture, and food science to highlight key opportunities for bridging these overlapping yet disconnected communities/fields and identify ways to leverage their respective strengths. Specifically, we show that (i) citizen science projects are addressing many grand challenges facing our food systems, as outlined by the United States National Institute of Food and Agriculture, as well as broader Sustainable Development Goals set by the United Nations Development Programme, (ii) there exist emerging opportunities and unique challenges for citizen science in agriculture/food research, and (iii) the greatest opportunities for the development of citizen science projects in agriculture and food science will be gained by using the existing infrastructure and tools of Extension programmes and through the engagement of urban communities. Further, we argue there is no better time to foster greater collaboration between these fields given the trend of shrinking Extension programmes, the increasing need to apply innovative solutions to address rising demands on agricultural systems, and the exponential growth of the field of citizen science.

Cortical oscillations play a fundamental role in organizing large-scale functional brain networks. Noninvasive brain stimulation with temporally patterned waveforms such as repetitive transcranial magnetic stimulation (rTMS) and transcranial alternating current stimulation (tACS) have been proposed to modulate these oscillations. Thus, these stimulation modalities represent promising new approaches for the treatment of psychiatric illnesses in which these oscillations are impaired. However, the mechanism by which periodic brain stimulation alters endogenous oscillation dynamics is debated and appears to depend on brain state. Here, we demonstrate with a static model and a neural oscillator model that recurrent excitation in the thalamo-cortical circuit, together with recruitment of cortico-cortical connections, can explain the enhancement of oscillations by brain stimulation as a function of brain state. We then performed concurrent invasive recording and stimulation of the human cortical surface to elucidate the response of cortical oscillations to periodic stimulation and support the findings from the computational models. We found that (1) stimulation enhanced the targeted oscillation power, (2) this enhancement outlasted stimulation, and (3) the effect of stimulation depended on behavioral state. Together, our results show successful target engagement of oscillations by periodic brain stimulation and highlight the role of nonlinear interaction between endogenous network oscillations and stimulation. These mechanistic insights will contribute to the design of adaptive, more targeted stimulation paradigms.

The purpose of this paper is to provide the biomedical imaging community with details of a new high resolution contrast imaging approach referred to as "acoustic angiography." Through the use of dual-frequency ultrasound transducer technology, images acquired with this approach possess both high resolution and a high contrast-to-tissue ratio, which enables the visualization of microvascular architecture without significant contribution from background tissues. Additionally, volumetric vessel-tissue integration can be visualized by using b-mode overlays acquired with the same probe. We present a brief technical overview of how the images are acquired, followed by several examples of images of both healthy and diseased tissue volumes. 3D images from alternate modalities often used in preclinical imaging, contrast-enhanced micro-CT and photoacoustics, are also included to provide a perspective on how acoustic angiography has qualitatively similar capabilities to these other techniques. These preliminary images provide visually compelling evidence to suggest that acoustic angiography may serve as a powerful new tool in preclinical and future clinical imaging.

This paper aimed to develop and evaluate an environment-aware locomotion mode recognition system for volitional control of powered artificial legs. A portable terrain recognition (TR) module, consisting of an inertia measurement unit and a laser distance meter, was built to identify the type of terrain in front of the wearer while walking. A decision tree was used to classify the terrain types and provide either coarse or refined information about the walking environment. Then, the obtained environmental information was modeled as a priori probability and was integrated with a neuromuscular-mechanical-fusion-based locomotion mode (LM) recognition system. The designed TR module and environmental-aware LM recognition system was evaluated separately on able-bodied subjects and a transfemoral amputee online. The results showed that the TR module provided high quality environmental information: TR accuracy is above 98% and terrain transitions are detected over 500 ms before the time required to switch the prosthesis control mode. This enabled smooth locomotion mode transitions for the wearers. The obtained environmental information further improved the performance of LM recognition system, regardless of whether coarse or refined information was used. In addition, the environment-aware LM recognition system produced reliable online performance when the TR output was relatively noisy, which indicated the potential of this system to operate in unconstructed environment. This paper demonstrated that environmental information should be considered for operating wearable lower limb robotic devices, such as prosthetics and orthotics.

Here we report an ultra-long-acting tunable, biodegradable, and removable polymer-based delivery system that offers sustained drug delivery for up to one year for HIV treatment or prophylaxis. This robust formulation offers the ability to integrate multiple drugs in a single injection, which is particularly important to address the potential for drug resistance with monotherapy. Six antiretroviral drugs were selected based on their solubility in N-methyl-2-pyrrolidone and relevance as a combination therapy for HIV treatment or prevention. All drugs released with concentrations above their protein-adjusted inhibitory concentration and retained their physical and chemical properties within the formulation and upon release. The versatility of this formulation to integrate multiple drugs and provide sustained plasma concentrations from several weeks to up to one year, combined with its ability to be removed to terminate the treatment if necessary, makes it attractive as a drug delivery platform technology for a wide range of applications.

Osteoarthritis (OA) is a progressive degenerative disease of articular cartilage and surrounding tissues, and is associated with both advanced age and joint injury. Biomechanical factors play a critical role in the onset and progression of OA, yet the mechanisms through which physiologic or pathologic mechanical signals are transduced into a cellular response are not well understood. Defining the role of mechanosensory pathways in cartilage during OA pathogenesis may yield novel strategies or targets for the treatment of OA. The transient receptor potential vanilloid 4 (TRPV4) ion channel transduces mechanical loading of articular cartilage via the generation of intracellular calcium ion transients. Using tissue-specific, inducible Trpv4 gene-targeted mice, we demonstrate that loss of TRPV4-mediated cartilage mechanotransduction in adulthood reduces the severity of aging-associated OA. However, loss of chondrocyte TRPV4 did not prevent OA development following destabilization of the medial meniscus (DMM). These results highlight potentially distinct roles of TRPV4-mediated cartilage mechanotransduction in age-related and post-traumatic OA, and point to a novel disease-modifying strategy to therapeutically target the TRPV4-mediated mechanotransduction pathway for the treatment of aging-associated OA.

Rapid and accurate molecular blood analysis is essential for disease diagnosis and management. Field-effect transistor (FET) biosensors are a type of device that promise to advance blood point-of-care testing by offering desirable characteristics such as portability, high sensitivity, brief detection time, low manufacturing cost, multiplexing, and label-free detection. By controlling device parameters, desired FET biosensor performance is obtained. This review focuses on the effects of sensing environment, micro-/nanoscale device structure, operation mode, and surface functionalization on device performance and long-term stability.

Gelatin methacryloyl (GelMA) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator are commonly used in combination to produce a photosensitive polymer but there are concerns that must be addressed: the presence of unreacted monomer is well known to be cytotoxic, and lithium salts are known to cause acute kidney injury. In this study, acellular 10% GelMA hydrogels cross-linked with different LAP concentrations and cross-linking illumination times were evaluated for their cytotoxicity, photosensitizing potential, and elastic moduli. Alamar Blue and CyQuant Direct Cell viability assays were performed on human primary renal proximal tubule epithelial cells (hRPTECs) exposed to extracts of each formulation. UV exposure during cross-linking was not found to affect extract cytotoxicity in either assay. LAP concentration did not affect extract cytotoxicity as determined by the Alamar Blue assay but reduced hRPTEC viability in the CyQuant Direct cell assay. Photocatalytic activity of formulation extracts toward NADH oxidation was used as a screening method for photosensitizing potential; longer UV exposure durations yielded extracts with less photocatalytic activity. Finally, elastic moduli determined using nanoindentation was found to plateau to approximately 20–25 kPa after exposure to 342 mJ/cm2 at 2.87 mW of UV-A exposure regardless of LAP concentration. LAP at concentrations commonly used in bioprinting (<0.5% w/w) was not found to be cytotoxic although the differences in cytotoxicity evaluation determined from the two viability assays imply cell membrane damage and should be investigated further. Complete cross-linking of all formulations decreased photocatalytic activity while maintaining predictable final elastic moduli.

Symmetric ankle propulsion is the cornerstone of efficient human walking. The ankle plantar flexors provide the majority of the mechanical work for the step-to-step transition and much of this work is delivered via elastic recoil from the Achilles' tendon - making it highly efficient. Even though the plantar flexors play a central role in propulsion, body-weight support and swing initiation during walking, very few assistive devices have focused on aiding ankle plantarflexion. Our goal was to develop a portable ankle exoskeleton taking inspiration from the passive elastic mechanisms at play in the human triceps surae-Achilles' tendon complex during walking. The challenge was to use parallel springs to provide ankle joint mechanical assistance during stance phase but allow free ankle rotation during swing phase. To do this we developed a novel `smart-clutch' that can engage and disengage a parallel spring based only on ankle kinematic state. The system is purely passive - containing no motors, electronics or external power supply. This `energy-neutral' ankle exoskeleton could be used to restore symmetry and reduce metabolic energy expenditure of walking in populations with weak ankle plantar flexors (e.g. stroke, spinal cord injury, normal aging).