Single-cell RNA-Seq (scRNA-Seq) profiles gene expression of individual cells. Recent scRNA-Seq datasets have incorporated unique molecular identifiers (UMIs). Using negative controls, we show UMI ...counts follow multinomial sampling with no zero inflation. Current normalization procedures such as log of counts per million and feature selection by highly variable genes produce false variability in dimension reduction. We propose simple multinomial methods, including generalized principal component analysis (GLM-PCA) for non-normal distributions, and feature selection using deviance. These methods outperform the current practice in a downstream clustering assessment using ground truth datasets.
The cocaine vaccine dAd5GNE is comprised of a disrupted serotype 5 adenovirus gene therapy vector covalently conjugated to the cocaine analog GNE. The vaccine evokes a high titer of circulating ...anti-cocaine antibodies that prevent cocaine from reaching its cognate receptors in the central nervous system. Prior studies have demonstrated the efficacy of dAd5GNE in models of occasional, moderate cocaine use. However, previous studies have not sufficiently evaluated the efficacy of dAd5GNE in models of the repetitive and high-dose "binge" use patterns common in human addicts. In the present study, we evaluated the capacity of dAd5GNE vaccination to protect against "binge" cocaine use and circumstances where vaccinated addicts attempt to override the vaccine. We modeled repetitive daily cocaine use in vaccinated Balb/c mice and African green monkeys, and evaluated high-dose "binge" scenarios in Balb/c mice. In each model of daily use the dAd5GNE vaccine prevented cocaine from reaching the central nervous system. In the high-dose "binge" model, vaccination decreased cocaine-induced hyperactivity and reduced the number of cocaine-induced seizures. Based on this data and our prior data in rodents and nonhuman primates, we have initiated a clinical trial evaluating the dAd5GNE anti-cocaine vaccine as a potential therapy for cocaine addicts who wish to stop cocaine use. If dAd5GNE vaccination is safe and produces high anti-cocaine antibody titers in the clinic, we hypothesize that the vaccine will restrict the access of cocaine to the central nervous system and inhibit cocaine-induced "highs" even in the context of moderate daily and high-dose "binge" use that might otherwise cause a drug-induced overdose.
Additive manufacturing through material extrusion, often termed three-dimensional (3D) printing, is a burgeoning method for manufacturing thermoplastic components. However, a key obstacle facing ...3D-printed plastic parts in engineering applications is the weak weld between successive filament traces, which often leads to delamination and mechanical failure. This is the chief obstacle to the use of thermoplastic additive manufacturing. We report a novel concept for welding 3D-printed thermoplastic interfaces using intense localized heating of carbon nanotubes (CNTs) by microwave irradiation. The microwave heating of the CNT-polymer composites is a function of CNT percolation, as shown through in situ infrared imaging and simulation. We apply CNT-loaded coatings to a 3D printer filament; after printing, microwave irradiation is shown to improve the weld fracture strength by 275%. These remarkable results open up entirely new design spaces for additive manufacturing and also yield new insight into the coupling between dielectric properties and radio frequency field response for nanomaterial networks.
This project is to develop therapies to bypass challenges to effective and continuous drug delivery to the brain, for the treatment of glioblastoma multiforme (GBM). Currently, individuals diagnosed ...with GBM have a short life expectancy of 12‐14 months. Our approach has the potential to deliver one single dose of gene therapy directly to the GBM tumor environment and block the production of cancer‐driving genes. Epidermal growth factor receptor (EGFR) is dysregulated in 57% of all GBM. Our approach uses an adeno‐associated virus gene transfer vector encoding RNA therapeutics targeting critical elements of the EGFR pre‐mRNA transcript. We have examined the ‘pre‐mRNA structurome’ of EGFR to evaluate the accessibility of targetable regions. To advance our therapeutic strategy, we have analyzed the secondary structure of the EGFR transcript using selective 2’ hydroxyl acylation and primer extension followed by mutational profiling (SHAPE‐MaP). SHAPE‐MaP reactivity profiles were generated revealing the structure of splicing and cryptic polyadenylation signal (PAS) elements within the targeted region. We identified enhancer binding motifs surrounding the 5’ splice site and hidden elements of a cryptic polyadenylation signal. Based on these structural profiles, we generated RNA therapies that interact with structural elements to unravel the hidden polyadenylation signal with the potential to activate expression of the short therapeutic isoform. In this project, we cloned these therapies into our therapeutic delivery platform and tested their efficacy to alter EGFR gene expression in tissue culture cells. Currently, we are evaluating in vitro, the therapeutic RNA interaction with the target sequence of the EGFR pre‐mRNA transcript.
Undergraduate biology students often graduate without exposure to authentic research experiences. Laboratory courses follow a one or two week fail‐proof experiment resembling a cookbook recipe, ...lacking the uncertainty of genuine research. Techniques in molecular biology cover an array of skills essential to succeed in a biotechnological laboratory today. I have been developing an evolving molecular biology lab course based on the teaching of concepts while imparting the skills and applications of modern techniques, providing students with theoretical concepts and laboratory skills. We prepare students to carry‐out scientific protocols that can be applied to a future workforce setting. Students are immersed in a 10‐week series of labs with the objective to use molecular cloning to make a novel gene therapy vector; therapies are designed to inhibit the expression of genes of the virus that causes Covid‐19, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Students are introduced to PubMed and GenBank to research the background of SARS‐CoV‐2 and its target genes; the Spike gene (S), the membrane gene (M) the nucleocapsid gene (N) and the envelope gene (E). Students use the DNA software, Serial Cloner, as a tool to evaluate DNA sequences and mFOLD to analyze RNA structure. Students generate and visualize the design of an antisense gene therapy directed against one of the target viral genes. Using our gene therapy platform, students generate a vector with a unique target. Subsequently student‐generated vectors were transfected into mammalian tissue culture cells that express one of the target genes (S, M, N or E) and RNA and protein was collected to measure the efficacy of the gene therapy vector to reduce the expression of the target viral gene. In our initial experiments, we observed greater than 2.5‐fold reduction in the Spike Covid mRNA. Currently, students are testing additional therapies to maximize efficacy.
Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) control many pathophysiological processes and are ...the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NK
R) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NK
R in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane-targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and β-arrestin blocked SP-induced NK
R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal-regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NK
R antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NK
R antagonists. These results reveal a critical role for endosomal signaling of the NK
R in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.
Emerging viral diseases have increased in recent decades. In December 2019, an epidemic with low respiratory infections emerged in Wuhan, China. The disease, Covid‐19 was found to be caused by a ...novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). As of October 14, 2021, WHO has confirmed greater than 239,000,000 global cases, nearly 5 million deaths worldwide, including greater than 720,000 in the USA. Fortunately, a vaccine has been approved and distributed, yet there are no approved therapeutics for infected individuals, and the threat of emerging vaccine‐resistant strains remain. From advances in biotechnology, the genome and structure of SARS‐CoV‐2 is known. Three proteins are anchored in the viral envelope, Spike (S), Envelope (E), and Membrane (M), which is linked to the Nucleocapsid (N) protein connecting to the viral RNA genome. Our lab is developing an innovative therapy that delivers multiple therapeutic microRNAs to block the expression of distinct Covid viral proteins. The design of the anti‐Covid microRNAs 1) mimics human microRNA cluster 17‐92a structural stability, 2) forms guide‐RNA substrates for the RNA induced silencing complex, and 3) are complementary to specific regions of the SARS‐CoV‐2 RNA genome without off‐targets effects in the human genome. Twenty‐one microRNA sequences were designed to target the S gene, six for N, two for M, and one for E. These were cloned into our microRNA‐17‐92 therapy vector which expresses six distinct anti‐Covid RNA therapeutics simultaneously. We have transfected the S gene into our tissue culture model to measure the efficacy of the anti‐Covid microRNA therapy to down‐regulate the S gene expression. In our preliminary experiments we show a significant 2.5‐fold reduction in the relative abundance of the Spike mRNA in the treated cells (p < 0.05). We are currently testing additional therapies and verifying changes in spike protein levels. Next steps are to examine the secondary structure of our RNA therapy using SHAPE‐MAP to optimize RNA therapeutic stability in comparison to the stable structure based on the human gene, microRNA Cluster 17‐92a.
Individuals diagnosed with glioblastoma multiforme (GBM) have a short life expectancy of 12‐15 months. This project is to develop therapies for effective and continuous drug delivery to the brain, ...targeting cancer‐driving genes. Tumor cell proliferation in GBM is often stimulated by epidermal growth factor receptor (EGFR) and is important for tumor cell survival. In our lab, we are developing RNA therapies to alter the splicing mechanism of EGFR to block its activation, thus stop tumor cell growth. Our approach uses an adeno‐associated virus gene transfer vector encoding RNA therapeutics targeting critical elements of the EGFR pre‐mRNA transcript. In this project, we cloned therapies into our therapeutic delivery platform and tested their efficacy to alter EGFR gene expression in tissue culture cells. We have found that our therapies have led to a shift in Intron 10A retention increasing alternative intronic polyadenylation generating a short soluble therapeutic decoy. Our antisense therapies targeted the EGFR pre‐mRNA transcript, specifically, regions of exon 10 and intron 10. Therapies were either designed to enhance recognition of the alternative intronic polyadenylation signal or block recognition of canonical exon 10/exon 11 splicing. We targeted the Exonic Splicing Enhancer (ESE) wild‐type 5’ Splice Site (wt‐5’SS) of exon 10, and the regions surrounding the alternative intronic polyadenylation signal. Each target showed a significant increase in intron retention. Targeting the ESE showed a 5‐fold increase in intron retention (p < 0.04), the wt‐5’SS was 15‐fold (p < 0.001), and the region surrounding the alternative intronic polyadenylation signal showed the strongest effect, with greater than 60‐fold increase in intron retention (p < 0.002). Currently, we are examining additional antisense therapies, as well as generation of the therapeutic soluble decoy protein using ELISA.
Glioblastoma multiforme (GBM) is the most common and aggressive primary intracranial brain tumor in adults with a mean survival of 14 to 15 months. Aberrant activation of the epidermal growth factor ...receptor (EGFR) plays a significant role in GBM progression, with amplification or overexpression of EGFR in 60% of GBM tumors. To target EGFR expressed by GBM, we have developed a strategy to deliver the coding sequence for cetuximab, an anti-EGFR antibody, directly to the CNS using an adeno-associated virus serotype rh.10 gene transfer vector. The data demonstrates that single, local delivery of an anti-EGFR antibody by an AAVrh.10 vector coding for cetuximab (AAVrh.10Cetmab) reduces GBM tumor growth and increases survival in xenograft mouse models of a human GBM EGFR-expressing cell line and patient-derived GBM. AAVrh10.CetMab-treated mice displayed a reduction in cachexia, a significant decrease in tumor volume and a prolonged survival following therapy. Adeno-associated-directed delivery of a gene encoding a therapeutic anti-EGFR monoclonal antibody may be an effective strategy to treat GBM.
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