Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping.

There is an urgent and unprecedented need for sensitive and high-throughput molecular diagnostic tests to combat the SARS-CoV-2 pandemic. Here we present a generalized version of the RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) assay, called "capture RASL-seq" (cRASL-seq), which enables highly sensitive (down to ~1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid purification or reverse transcription, steps that have introduced supply bottlenecks into standard assay workflows. Our simplified protocol additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

Predictors of Non-home Discharge after Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy.

BACKGROUND: Using a national database of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) recipients, we sought to determine risk factors for nonhome discharge (NHD) in a cohort of patients. METHODS: Patients undergoing CRS/HIPEC at any one of 12 participating sites between 2000 and 2017 were identified. Univariate analysis was used to compare the characteristics, operative variables, and postoperative complications of patients discharged home and patients with NHD. Multivariate logistic regression was used to identify independent risk factors of NHD. RESULTS: The cohort included 1593 patients, of which 70 (4.4%) had an NHD. The median [range] peritoneal cancer index in our cohort was 14 [0-39]. Significant predictors of NHD identified in our regression analysis were advanced age (odds ratio [OR], 1.09; 95% confidence interval [CI], 1.05-1.12; P < 0.001), an American Society of Anesthesiologists (ASA) score of 4 (OR, 2.87; 95% CI, 1.21-6.83; P = 0.017), appendiceal histology (OR, 3.14; 95% CI 1.57-6.28; P = 0.001), smoking history (OR, 3.22; 95% CI, 1.70-6.12; P < 0.001), postoperative total parenteral nutrition (OR, 3.14; 95% CI, 1.70-5.81; P < 0.001), respiratory complications (OR, 7.40; 95% CI, 3.36-16.31; P < 0.001), wound site infections (OR, 3.12; 95% CI, 1.58-6.17; P = 0.001), preoperative hemoglobin (OR, 0.81; 95% CI, 0.70-0.94; P = 0.006), and total number of complications (OR, 1.41; 95% CI, 1.16-1.73; P < 0.001). CONCLUSIONS: Early identification of patients at high risk for NHD after CRS/HIPEC is key for preoperative and postoperative counseling and resource allocation, as well as minimizing hospital-acquired conditions and associated health care costs.

Hierarchical assembly and modeling of DNA nanotube networks using Y-shaped DNA origami seeds.

DNA nanotechnology offers many means to synthesize custom nanostructured materials from the ground up in a hierarchical fashion. While the assembly of DNA nanostructures from small (nanometer-scale) monomeric components has been studied extensively, how the hierarchical assembly of rigid or semi-flexible units produces multi-micron scale structures is less understood. Here we demonstrate a mechanism for assembling micron-scale semi-flexible DNA nanotubes into extended structures. These nanotubes assemble from nanometer-scale tile monomers into materials via heterogeneous nucleation from rigid, Y-shaped DNA origami seeds to form Y-seeded nanotube architectures. These structures then assemble into networks via nanotube end-to-end joining. We measure the kinetics of network growth and find that the assembly of networks can be approximated by a model of hierarchical assembly that assumes a single joining rate between DNA nanotube ends. Because the number of nucleation sites on Y-seeds and their spatial arrangement can be systematically varied by design, this hierarchical assembly process could be used to form a wide variety of networks and to understand the assembly mechanisms that lead to different types of material architectures at length scales of tens to hundreds of microns.

Unbiased discovery of autoantibodies associated with severe COVID-19 via genome-scale self-assembled DNA-barcoded protein libraries.

Pathogenic autoreactive antibodies that may be associated with life-threatening coronavirus disease 2019 (COVID-19) remain to be identified. Here, we show that self-assembled genome-scale libraries of full-length proteins covalently coupled to unique DNA barcodes for analysis by sequencing can be used for the unbiased identification of autoreactive antibodies in plasma samples. By screening 11,076 DNA-barcoded proteins expressed from a sequence-verified human ORFeome library, the method, which we named MIPSA (for Molecular Indexing of Proteins by Self-Assembly), allowed us to detect circulating neutralizing type-I and type-III interferon (IFN) autoantibodies in five plasma samples from 55 patients with life-threatening COVID-19. In addition to identifying neutralizing type-I IFN-α and IFN-ω autoantibodies and other previously known autoreactive antibodies in patient plasma, MIPSA enabled the detection of as yet unidentified neutralizing type-III anti-IFN-λ3 autoantibodies that were not seen in healthy plasma samples or in convalescent plasma from ten non-hospitalized individuals with COVID-19. The low cost and simple workflow of MIPSA will facilitate unbiased high-throughput analyses of protein-antibody, protein-protein and protein-small-molecule interactions.

Neutralizing IFNL3 Autoantibodies in Severe COVID-19 Identified Using Molecular Indexing of Proteins by Self-Assembly.

Unbiased antibody profiling can identify the targets of an immune reaction. A number of likely pathogenic autoreactive antibodies have been associated with life-threatening SARS-CoV-2 infection; yet, many additional autoantibodies likely remain unknown. Here we present Molecular Indexing of Proteins by Self Assembly (MIPSA), a technique that produces ORFeome-scale libraries of proteins covalently coupled to uniquely identifying DNA barcodes for analysis by sequencing. We used MIPSA to profile circulating autoantibodies from 55 patients with severe COVID-19 against 11,076 DNA-barcoded proteins of the human ORFeome library. MIPSA identified previously known autoreactivities, and also detected undescribed neutralizing interferon lambda 3 (IFN-λ3) autoantibodies. At-risk individuals with anti- IFN-λ3 antibodies may benefit from interferon supplementation therapies, such as those currently undergoing clinical evaluation.

Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping.

The emergence of SARS-CoV-2 has caused the current COVID-19 pandemic with catastrophic societal impact. Because many individuals shed virus for days before symptom onset, and many show mild or no symptoms, an emergent and unprecedented need exists for development and deployment of sensitive and high throughput molecular diagnostic tests. RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) is a highly multiplexed technology for targeted analysis of polyadenylated mRNA, which incorporates sample barcoding for massively parallel analyses. Here we present a more generalized method, capture RASL-seq ("cRASL-seq"), which enables analysis of any targeted pathogen- (and/or host-) associated RNA molecules. cRASL-seq enables highly sensitive (down to ~1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid extraction or reverse transcription, steps that have caused testing bottlenecks associated with other assays. Our simplified workflow additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new surveillance technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

A simple and highly sensitive method for magnetic nanoparticle quantitation using 1H-NMR spectroscopy.

Iron oxide superparamagnetic nanoparticles (SPIONs) have drawn significant attention because of their potential impact on medical diagnosis and therapy. However, the difficulty of achieving reliable and standardized quantification of these nanoparticles has limited the uniform study of nanoparticle systems. Current measurement techniques have limited sensitivity, and are sophisticated and subject to individual instrumental settings. Here, a characterization method using proton nuclear magnetic resonance ((1)H-NMR) spectroscopy is presented that can quantify SPIONs regardless of surface modification. In addition to routine quantification of SPIONs during nanoparticle development, the method can also be used with in vitro nanoparticle assays and potentially with tissue samples for biodistribution studies. Specifically, measurement of water relaxivity shifts (R(1) or R(2)) of dissolved SPION samples is correlated with nanoparticle concentration. Unmodified and dextran- and poly(ethylene glycol)-coated SPIONs gave linear correlations between SPION concentration and R(1) and R(2) relaxivities over five orders of magnitude, to below 10 ppb iron. Quantification of SPION concentration was also demonstrated in the presence of RAW 264.7 macrophage cells. A linear correlation between the SPION concentration and relaxivities was observed to <10 ng Fe/mL. This method is a rapid and inexpensive approach for quantitation of SPIONs and exhibits a number of advantages over many of the current methods for quantitative SPION analysis.

PEI-PEG-Chitosan Copolymer Coated Iron Oxide Nanoparticles for Safe Gene Delivery: synthesis, complexation, and transfection.

Gene therapy offers the potential of mediating disease through modification of specific cellular functions of target cells. However, effective transport of nucleic acids to target cells with minimal side effects remains a challenge despite the use of unique viral and non-viral delivery approaches. Here we present a non-viral nanoparticle gene carrier that demonstrates effective gene delivery and transfection both in vitro and in vivo. The nanoparticle system (NP-CP-PEI) is made of a superparamagnetic iron oxide nanoparticle (NP), which enables magnetic resonance imaging, coated with a novel copolymer (CP-PEI) comprised of short chain polyethylenimine (PEI) and poly(ethylene glycol) (PEG) grafted to the natural polysaccharide, chitosan (CP), which allows efficient loading and protection of the nucleic acids. The function of each component material in this nanoparticle system is illustrated by comparative studies of three nanoparticle systems of different surface chemistries, through material property characterization, DNA loading and transfection analyses, and toxicity assessment. Significantly, NP-CP-PEI demonstrates an innocuous toxic profile and a high level of expression of the delivered plasmid DNA in a C6 xenograft mouse model, making it a potential candidate for safe in vivo delivery of DNA for gene therapy.

Inhibition of tumor-cell invasion with chlorotoxin-bound superparamagnetic nanoparticles.

Nanoparticles have been investigated as drug delivery vehicles, contrast agents, and multifunctional devices for patient care. Current nanoparticle-based therapeutic strategies for cancer treatment are mainly based on delivery of chemotherapeutic agents to induce apoptosis or DNA/siRNA to regulate oncogene expression. Here, a nanoparticle system that demonstrates an alternative approach to the treatment of cancers through the inhibition of cell invasion, while serving as a magnetic resonance and optical imaging contrast agent, is presented. The nanoparticle comprises an iron oxide nanoparticle core conjugated with an amine-functionalized poly(ethylene glycol) silane and a small peptide, chlorotoxin (CTX), which enables the tumor cell-specific binding of the nanoparticle. It is shown that the nanoparticle exhibits substantially enhanced cellular uptake and an invasion inhibition rate of approximately 98% compared to unbound CTX ( approximately 45%). Significantly, the investigation from flow cytometry analysis, transmission electron microscopy, and fluorescent imaging reveals that the CTX-enabled nanoparticles deactivated the membrane-bound matrix metalloproteinase 2 (MMP-2) and induced increased internalization of lipid rafts that contain surface-expressed MMP-2 and volume-regulating ion channels through receptor-mediated endocytosis, leading to enhanced prohibitory effects. Since upregulation and activity of MMP-2 have been observed in tumors of neuroectodermal origin, and in cancers of the breast, colon, skin, lung, prostate, ovaries, and a host of others, this nanoparticle system can be potentially used for non-invasive diagnosis and treatment of a variety of cancer types.

In vivo MRI detection of gliomas by chlorotoxin-conjugated superparamagnetic nanoprobes.

Converging advances in the development of nanoparticle-based imaging probes and improved understanding of the molecular biology of brain tumors offer the potential to provide physicians with new tools for the diagnosis and treatment of these deadly diseases. However, the effectiveness of promising nanoparticle technologies is currently limited by insufficient accumulation of these contrast agents within tumors. Here a biocompatible nanoprobe composed of a poly(ethylene glycol) (PEG) coated iron oxide nanoparticle that is capable of specifically targeting glioma tumors via the surface-bound targeting peptide, chlorotoxin (CTX), is presented. The preferential accumulation of the nanoprobe within gliomas and subsequent magnetic resonance imaging (MRI) contrast enhancement are demonstrated in vitro in 9L cells and in vivo in tumors of a xenograft mouse model. TEM imaging reveals that the nanoprobes are internalized into the cytoplasm of 9L cells and histological analysis of selected tissues indicates that there are no acute toxic effects of these nanoprobes. High targeting specificity and benign biological response establish this nanoprobe as a potential platform to aid in the diagnosis and treatment of gliomas and other tumors of neuroectodermal origin.

Blood vessel detection, localization and estimation using a smart laparoscopic grasper: a Monte Carlo study.

For centuries, surgeons have relied on their sense of touch to identify vital structures such as blood vessels in traditional open surgery. Over the past two decades, surgeons have shifted to minimally invasive surgical (MIS) approaches, including laparoscopic surgery, which include benefits such as less scarring, less risk for infection, and quicker recovery times. In fact, some surgeries such as cholecystectomies have seen more than an 80% adoption of this technique because of those benefits. However, due to the fundamental challenges associated with using laparoscopic surgery, there has been a lower adoption in more complex specialties, such as colorectal and thoracic surgery, where the field of surgery has bleeding, fat, scar tissue, and adhesions. These problems are exacerbated by complicating factors such as inflammation, cancer, chronic disease, obesity, and re-operations. Importantly, surgeons will often convert from laparoscopy to open surgery if they can no longer proceed using the minimally invasive approach because of issues described with these complicating factors, thereby negating the benefits that the patient would have seen. When the surgeon does attempt these procedures with those issues, the surgery takes on average 30 min - 1 hour longer. A new method by which surgeons can visualize structures like blood vessels could reduce the conversion rates and operating time, thereby driving a greater adoption of laparoscopic surgery in these complex procedures. Here, we show that by adding near infrared (NIR) LEDs and a linear image sensor onto the opposing jaws of the laparoscopic graspers, blood vessels that are embedded within tissues can be detected and localized efficiently, even those not visible using current imaging techniques. We show the results of Monte Carlo simulations to support our claim, including that blood vessels ranging from 2 to 6 mm and buried under up to 1 cm of tissue can be detected. We also report developing a smart grasper handheld prototype to run ex vivo experiments. The results of these experiments matched with those of the Monte Carlo simulations and the estimated blood vessel size showed a strong correlation with the actual size. This technology will be incorporated into already existing laparoscopic tools to assist surgeons during MIS procedures.

Intraoperative blood vessel detection and quantification: a Monte Carlo study.

With better surgical outcomes, quicker recovery times, decreased postoperative pain, and reduced scarring at the surgical site, the application of minimally invasive surgery (MIS) has gained a lot of prominence in the last 30 years. This change in surgical practice has taken away the ability of a surgeon to palpate for the presence of a blood vessel as would occur in an open procedure. They instead must rely on a laparoscopic video camera feed that unfortunately cannot detect the presence of a blood vessel hidden beneath tissue. In certain scenarios, a surgeon can accidentally cut a blood vessel, which can lead to severe, even fatal, complications. Here, we show that by adding a near-infrared LED and a photodiode onto the opposing jaws of laparoscopic graspers, blood vessels buried under tissue can be detected. We show the results of Monte Carlo simulations to support our theory that the blood vessels ranging from 3 to 6 mm buried under up to 1 cm of tissue can be detected and quantified. This technology could be added to already existing laparoscopic tools that have limited surface areas on the jaws to assist surgeons during MIS procedures.

Methotrexate-immobilized poly(ethylene glycol) magnetic nanoparticles for MR imaging and drug delivery.

We report the development of a biostable methotrexate-immobilized iron oxide nanoparticle drug carrier that may potentially be used for real-time monitoring of drug delivery through magnetic resonance imaging. Methotrexate (MTX) was immobilized on the nanoparticle surface via a poly(ethylene glycol) self-assembled monolayer (PEG SAM). The cytotoxicity of the nanoparticle-drug conjugate (NP-PEG-MTX) to target cells was studied with 9L glioma cells. Cellular uptake experiments showed that the uptake of NP-PEG-MTX conjugates by glioma cells was considerably higher than that of control nanoparticles. Magnetic resonance imaging in 9L cells cultured with NP-PEG-MTX of various concentrations showed significant contrast enhancement. NP-PEG-MTX demonstrated higher cytotoxicity in 9L cells to free MTX in vitro. Leucovorin, an MTX antidote, was used to rescue the cells that had been exposed to NP-PEG-MTX or free MTX, and the experiment verified the biocompatibility of NP-PEG-MTX conjugates and the MTX on NP-PEG-MTX conjugates to be the true source of the cytotoxicity to the target cells. TEM results showed that NP-PEG-MTX conjugates were internalized into the 9L cellular cytoplasm and retained its crystal structure therein for up to 144 h, as identified by electron diffraction. This prolonged particle retention may allow physicians to image tumor cells exposed to the NP-PEG-MTX conjugate over an extended therapeutic time course.

PEG-grafted chitosan as an injectable thermosensitive hydrogel for sustained protein release.

Thermosensitive polymer hydrogels that undergo a sol-to-gel transition in response to temperature changes are of great interest in therapeutic delivery and tissue engineering as injectable depot systems. A chitosan-based, injectable thermogel was prepared by grafting an appropriate amount of PEG onto the chitosan backbone and studied for drug release in vitro using bovine serum albumin (BSA) as a model protein. When more than approximately 40 wt.% of PEG was grafted to chitosan chains via covalent bonding, the aqueous solution of the resultant copolymer was an injectable liquid at low temperature and transformed to a semisolid hydrogel at body temperature. After an initial burst release in the first 5 h, a steady linear release of protein from the hydrogel was achieved for a period of approximately 70 h. Prolonged quasi-linear release of protein up to 40 days was achieved by crosslinking the hydrogel with genipin in situ, in a fashion suitable for protein encapsulation while maintaining the injectability of the hydrogel. The crosslinkage transformed the copolymer from a physical gel to an insoluble chemical gel and substantially reduced the initial burst release of protein. Both high performance liquid chromatography (HPLC) and gel electrophoresis indicated that the primary structure of BSA released from the hydrogels with or without genipin-crosslinking was generally conserved. The hydrogel can be prepared in solutions with a physiological pH, allowing the safe incorporation of bioactive molecules for a broad range of medical applications, particularly for sustained in vivo drug release and tissue engineering.

Adhesive and mechanical properties of hydrogels influence neurite extension.

Photopolymerizable polyethylene glycol (PEG) hydrogels conjugated with bioactive ligands were examined for their use as scaffolds in peripheral nerve regeneration applications. The bioactivity and mechanical properties of PEG hydrogels can be tailored through the integration of bioactive factors (adhesion ligands, proteolytic sites, growth factors) and the alteration of PEG concentrations, respectively. For peripheral nerve regeneration, it will be important to determine the type and concentration of the bioactive molecules required to improve neurite extension. In this study, cell adhesion ligands (RGDS, IKVAV, and YIGSR) were covalently attached to PEG hydrogels. Both the type and concentration of cell adhesion ligand used affected neurite extension. Extension from PC12 cells was greater on hydrogels with RGDS incorporated than IKVAV, and the optimal concentration for each ligand was different. Cells adhered to but did not extend neurites on hydrogels with YIGSR. Cells did not adhere to hydrogels containing RGES. Furthermore, different combinations of these ligands affected neurite extension to different degrees. The mechanical properties of the hydrogels also significantly affected neurite extension. PC12 cells grown on more flexible hydrogels exhibited the greatest degree of neurite extension. PEG hydrogels have thus been developed with varying biochemical and mechanical properties that may enhance nerve regeneration.

A pretargeted nanoparticle system for tumor cell labeling.

Nanoparticle-based cancer diagnostics and therapeutics can be significantly enhanced by selective tissue localization, but the strategy can be complicated by the requirement of a targeting ligand conjugated on nanoparticles, that is specific to only one or a limited few types of neoplastic cells, necessitating the development of multiple nanoparticle systems for different diseases. Here, we present a new nanoparticle system that capitalizes on a targeting pretreatment strategy, where a circulating fusion protein (FP) selectively prelabels the targeted cellular epitope, and a biotinylated iron oxide nanoparticle serves as a secondary label that binds to the FP on the target cell. This approach enables a single nanoparticle formulation to be used with any one of existing fusion proteins to bind a variety of target cells. We demonstrated this approach with two fusion proteins against two model cancer cell lines: lymphoma (Ramos) and leukemia (Jurkat), which showed 72.2% and 91.1% positive labeling, respectively. Notably, TEM analysis showed that a large nanoparticle population was endocytosed via attachment to the non-internalizing CD20 epitope.

Polyblend nanofibers for biomedical applications: perspectives and challenges.

Advances in disease treatment and tissue regeneration are buoyed by new, multifaceted materials that emulate and coercively interact with the local microenvironment. Polyblend nanofibers represent an emerging class of biomimetic nanostructures that can act as proxies of the native tissue, while providing topographical and biochemical cues that promote healing. These fibers are prepared with mixtures of synthetically and naturally derived polymers that can behave cooperatively to demonstrate unique combinations of mechanical, biochemical and structural properties. This flexibility has led to the application of polyblend nanofibers in a wide assortment of tissue engineering and drug delivery systems. In this review, we will examine design criteria and properties of polymer-blend nanofibers and their use in tissue engineering and local therapeutic delivery applications.

Chitosan-based hydrogels for controlled, localized drug delivery.

Hydrogels are high-water content materials prepared from cross-linked polymers that are able to provide sustained, local delivery of a variety of therapeutic agents. Use of the natural polymer, chitosan, as the scaffold material in hydrogels has been highly pursued thanks to the polymer's biocompatibility, low toxicity, and biodegradability. The advanced development of chitosan hydrogels has led to new drug delivery systems that release their payloads under varying environmental stimuli. In addition, thermosensitive hydrogel variants have been developed to form a chitosan hydrogel in situ, precluding the need for surgical implantation. The development of these intelligent drug delivery devices requires a foundation in the chemical and physical characteristics of chitosan-based hydrogels, as well as the therapeutics to be delivered. In this review, we investigate the newest developments in chitosan hydrogel preparation and define the design parameters in the development of physically and chemically cross-linked hydrogels.

Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging.

Magnetic nanoparticles (MNPs) represent a class of non-invasive imaging agents that have been developed for magnetic resonance (MR) imaging. These MNPs have traditionally been used for disease imaging via passive targeting, but recent advances have opened the door to cellular-specific targeting, drug delivery, and multi-modal imaging by these nanoparticles. As more elaborate MNPs are envisioned, adherence to proper design criteria (e.g. size, coating, molecular functionalization) becomes even more essential. This review summarizes the design parameters that affect MNP performance in vivo, including the physicochemical properties and nanoparticle surface modifications, such as MNP coating and targeting ligand functionalizations that can enhance MNP management of biological barriers. A careful review of the chemistries used to modify the surfaces of MNPs is also given, with attention paid to optimizing the activity of bound ligands while maintaining favorable physicochemical properties.