Publications by authors named "Sean M Kelly"

28 Publications

  • Page 1 of 1

Single-dose combination nanovaccine induces both rapid and durable humoral immunity and toxin neutralizing antibody responses against Bacillus anthracis.

Vaccine 2021 06 2;39(29):3862-3870. Epub 2021 Jun 2.

Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, United States; Nanovaccine Institute, Ames, IA, United States. Electronic address:

Bacillus anthracis, the causative agent of anthrax, continues to be a prominent biological warfare and bioterrorism threat. Vaccination is likely to remain the most effective and user-friendly public health measure to counter this threat in the foreseeable future. The commercially available AVA BioThrax vaccine has a number of shortcomings where improvement would lead to a more practical and effective vaccine for use in the case of an exposure event. Identification of more effective adjuvants and novel delivery platforms is necessary to improve not only the effectiveness of the anthrax vaccine, but also enhance its shelf stability and ease-of-use. Polyanhydride particles have proven to be an effective platform at adjuvanting the vaccine-associated adaptive immune response as well as enhancing stability of encapsulated antigens. Another class of adjuvants, the STING pathway-targeting cyclic dinucleotides, have proven to be uniquely effective at inducing a beneficial inflammatory response that leads to the rapid induction of high titer antibodies post-vaccination capable of providing protection against bacterial pathogens. In this work, we evaluate the individual contributions of cyclic di-GMP (CDG), polyanhydride nanoparticles, and a combination thereof towards inducing neutralizing antibody (nAb) against the secreted protective antigen (PA) from B. anthracis. Our results show that the combination nanovaccine elicited rapid, high titer, and neutralizing IgG anti-PA antibody following single dose immunization that persisted for at least 108 DPI.
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http://dx.doi.org/10.1016/j.vaccine.2021.05.077DOI Listing
June 2021

Altered Mental Status in Patients Hospitalized with COVID-19: Perspectives from Neurologic and Psychiatric Consultants.

Harv Rev Psychiatry 2021 May 7. Epub 2021 May 7.

From the Department of Neurology, New York University School of Medicine (Drs. Talmasov, Kelly, Lewis, and Gurin); Harvard Medical School (Dr. Taylor); Department of Psychiatry, Brigham and Women's Hospital, Boston, MA (Dr. Taylor).

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http://dx.doi.org/10.1097/HRP.0000000000000298DOI Listing
May 2021

Synthesis and Characterization of Rapidly Degrading Polyanhydrides as Vaccine Adjuvants.

ACS Biomater Sci Eng 2020 01 3;6(1):265-276. Epub 2020 Jan 3.

Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011, United States.

There is a currently a need to develop adjuvants that are best suited to simultaneously enhance immune responses, induce immunologic memory, improve patient compliance (i.e., reduce doses and inflammation), and provide vaccine shelf stability for stockpiling and global deployment to challenging environments. Biodegradable polyanhydrides have been investigated extensively to overcome such challenges. It has been shown that controlling copolymer composition can result in chemistry-dependent immunomodulatory capabilities. These studies have revealed that copolymers rich in sebacic acid (SA) are highly internalized by antigen presenting cells and confer improved shelf stability of encapsulated proteins, while copolymers rich in 1,8-bis(-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) also exhibit enhanced internalization by and activation of antigen presenting cells (APCs), in addition to providing superior retention of protein stability following encapsulation and release. However, to date, CPTEG:SA copolymers have not been synthesized and described. In this work, we hypothesized that new copolymers composed of CPTEG and SA would combine the advantages of both monomers in terms of enhanced thermal properties, maintaining antigenicity of encapsulated proteins following nanoparticle synthesis, and superior cellular internalization and activation by APCs, demonstrated by the upregulation of costimulatory markers CD80, CD86, and CD40, as well as the secretion of proinflammatory cytokines IL-6, IL-1β, and TNF-α. Herein, we describe the synthesis and design of novel CPTEG:SA nanoparticles with improved thermal properties, payload stability, and internalization by antigen presenting cells for applications in vaccine delivery. The performance of these new CPTEG:SA formulations was compared to that of traditional polyanhydride copolymers.
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http://dx.doi.org/10.1021/acsbiomaterials.9b01427DOI Listing
January 2020

Polymeric Nanoparticle-Based Vaccine Adjuvants and Delivery Vehicles.

Curr Top Microbiol Immunol 2020 Nov 10. Epub 2020 Nov 10.

Departments of Chemical and Biological Engineering, Iowa State University, Ames, IA, 50011, USA.

As vaccine formulations have progressed from including live or attenuated strains of pathogenic components for enhanced safety, developing new adjuvants to more effectively generate adaptive immune responses has become necessary. In this context, polymeric nanoparticles have emerged as a promising platform with multiple advantages, including the dual capability of adjuvant and delivery vehicle, administration via multiple routes, induction of rapid and long-lived immunity, greater shelf-life at elevated temperatures, and enhanced patient compliance. This comprehensive review describes advances in nanoparticle-based vaccines (i.e., nanovaccines) with a particular focus on polymeric particles as adjuvants and delivery vehicles. Examples of the nanovaccine approach in respiratory infections, biodefense, and cancer are discussed.
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http://dx.doi.org/10.1007/82_2020_226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107186PMC
November 2020

Development of a subcutaneous ear implant to deliver an anaplasmosis vaccine to dairy steers.

J Anim Sci 2020 06;98(6)

Department of Anatomy and Physiology, Kansas State University, Manhattan, KS.

Bovine anaplasmosis is the most prevalent tick-transmitted disease of cattle worldwide and a major obstacle to profitable beef production. Use of chlortetracycline-medicated feed to control active anaplasmosis infections during the vector season has raised concerns about the potential emergence of antimicrobial resistance in bacteria that may pose a risk to human health. Furthermore, the absence of effectiveness data for a commercially available, conditionally licensed anaplasmosis vaccine is a major impediment to implementing anaplasmosis control programs. The primary objective of this study was to develop a single-dose vaccine delivery platform to produce long-lasting protective immunity against anaplasmosis infections. Twelve Holstein steers, aged 11 to 12 wk, were administered a novel 3-stage, single-dose vaccine against Anaplasma marginale, a major surface protein 1a. The vaccine consisted of a soluble vaccine administered subcutaneously (s.c.) for immune priming, a vaccine depot of a biodegradable polyanhydride rod with intermediate slow release of the vaccine for boosting immune response, and an immune-isolated vaccine platform for extended antigen release (VPEAR implant) deposited s.c. in the ear. Six calves were randomly assigned to 2 vaccine constructs (n = 3) that featured rods and implants containing a combination of 2 different adjuvants, diethylaminoethyl (DEAE)-Dextran and Quil-A (Group A). The remaining 6 calves were randomly assigned to 2 vaccine constructs (n = 3) that featured rods and implants containing the same adjuvant (either DEAE-Dextran or Quil A) (Group B). Twenty-one months post-implantation, calves were challenged intravenously with A. marginale stabilate and were monitored weekly for signs of fever, decreased packed cell volume (PCV) and bacteremia. Data were analyzed using a mixed-effects model and chi-squared tests (SAS v9.04.01, SAS Institute, Cary, NC). Calves in Group A had higher PCV than calves in Group B (P = 0.006) at day 35 post-infection. Calves in Group A were less likely to require antibiotic intervention compared with calves in Group B (P = 0.014). Results indicate that calves exhibited diminished clinical signs of anaplasmosis when antigen was delivered with a combination of adjuvants as opposed to a single adjuvant. This demonstrates the feasibility of providing long-lasting protection against clinical bovine anaplasmosis infections using a subcutaneous ear implant vaccine construct.
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http://dx.doi.org/10.1093/jas/skz392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271671PMC
June 2020

Stereocontrolled Synthesis of Arylomycin-Based Gram-Negative Antibiotic GDC-5338.

Org Lett 2019 11 31;21(22):9099-9103. Epub 2019 Oct 31.

Department of Small Molecule Process Chemistry , Genentech, Inc. , 1 DNA Way , South San Francisco , California 94080 , United States.

We report herein an efficient, stereocontrolled, and chromatography-free synthesis of the novel broad spectrum antibiotic . The route features the construction of a functionalized tripeptide backbone, a high-yielding macrocyclization via a Pd-catalyzed Suzuki-Miyaura reaction, and the late-stage elaboration of key amide bonds with minimal stereochemical erosion. Through extensive reaction development and analytical understanding, these key advancements allowed the preparation of in 17 steps, 15% overall yield, >99 A % HPLC, and >99:1 dr.
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http://dx.doi.org/10.1021/acs.orglett.9b03481DOI Listing
November 2019

Vitamin A deficiency impairs the immune response to intranasal vaccination and RSV infection in neonatal calves.

Sci Rep 2019 10 22;9(1):15157. Epub 2019 Oct 22.

Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, Agricultural Research Service, USDA, Ames, IA, USA.

Respiratory syncytial virus (RSV) infection is a leading cause of severe acute lower respiratory tract infection in infants and children worldwide. Vitamin A deficiency (VAD) is one of the most prevalent nutrition-related health problems in the world and is a significant risk factor in the development of severe respiratory infections in infants and young children. Bovine RSV (BRSV) is a primary cause of lower respiratory tract disease in young cattle. The calf model of BRSV infection is useful to understand the immune response to human RSV infection. We have previously developed an amphiphilic polyanhydride nanoparticle (NP)-based vaccine (i.e., nanovaccine) encapsulating the fusion and attachment proteins from BRSV (BRSV-NP). Calves receiving a single, intranasal dose of the BRSV-NP vaccine are partially protected from BRSV challenge. Here, we evaluated the impact of VAD on the immune response to the BRSV-NP vaccine and subsequent challenge with BRSV. Our results show that VAD calves are unable to respond to the mucosal BRSV-NP vaccine, are afforded no protection from BRSV challenge and have significant abnormalities in the inflammatory response in the infected lung. We further show that acute BRSV infection negatively impacts serum and liver retinol, rendering even well-nourished individuals susceptible to VAD. Our results support the use of the calf model for elucidating the impact of nutritional status on mucosal immunity and respiratory viral infection in infants and underline the importance of VA in regulating immunity in the respiratory mucosa.
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http://dx.doi.org/10.1038/s41598-019-51684-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805856PMC
October 2019

Single-dose combination nanovaccine induces both rapid and long-lived protection against pneumonic plague.

Acta Biomater 2019 12 11;100:326-337. Epub 2019 Oct 11.

Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, United States; Nanovaccine Institute, Iowa State University, Ames, IA, United States. Electronic address:

Yersinia pestis, the causative agent of pneumonic plague, induces a highly lethal infection if left untreated. Currently, there is no FDA-approved vaccine against this pathogen; however, USAMRIID has developed a recombinant fusion protein, F1-V, that has been shown to induce protection against pneumonic plague. Many F1-V-based vaccine formulations require prime-boost immunization to achieve protective immunity, and there are limited reports of rapid induction of protective immunity (≤ 14 days post-immunization (DPI)). The STimulator of INterferon Genes agonists cyclic dinucleotides (CDNs) have been shown to be promising vaccine adjuvants. Polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) have also shown to enhance immune responses due to their dual functionality as adjuvants and delivery vehicles. In this work, a combination nanovaccine was designed that comprised F1-V-loaded nanoparticles combined with the CDN, dithio-R,R-cyclic di-guanosine monophosphate, to induce rapid and long-lived protective immunity against pneumonic plague. All mice immunized with a single dose combination nanovaccine were protected from Y. pestis lethal challenge within 14 DPI and demonstrated enhanced protection over F1-V adjuvanted with CDNs alone at challenge doses ≥7000 CFU Y. pestis CO92. In addition, 75% of mice receiving the single dose of the combination nanovaccine were protected from challenge at 182 DPI, while maintaining high levels of antigen-specific serum IgG. ELISPOT analysis of vaccinated animals at 218 DPI revealed F1-V-specific long-lived plasma cells in bone marrow in mice vaccinated with CDN adjuvanted F1-V or the combination nanovaccine. Microarray analysis of serum from these vaccinated mice revealed the presence of serum antibody that bound to a broad range of F1 and V linear epitopes. These results demonstrate that combining the adjuvanticity of CDNs with a nanovaccine delivery system enables induction of both rapid and long-lived protective immunity against Y. pestis. STATEMENT OF SIGNIFICANCE: • Yersinia pestis, the causative agent of pneumonic plague, induces a highly lethal infection if left untreated. Currently, there is no FDA-approved vaccine against this biodefense pathogen. • We designed a combination nanovaccine comprising of F1-V antigen-loaded polyanhydride nanoparticles and a cyclic dinucleotide adjuvant to induce both rapid and long-lived protective immunity against pneumonic plague. • Animals immunized with the combination nanovaccine maintained high levels of antigen-specific serum IgG and long-lived plasma cells in bone marrow and the serum antibody showed a high affinity for a broad range of F1 and V linear epitopes. • The combination nanovaccine is a promising next-generation vaccine platform against weaponized Y. pestis based on its ability to induce both rapid and long-lived protective immunity.
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http://dx.doi.org/10.1016/j.actbio.2019.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012387PMC
December 2019

Sustained antigen release polyanhydride-based vaccine platform for immunization against bovine brucellosis.

Heliyon 2019 Aug 29;5(8):e02370. Epub 2019 Aug 29.

Infectious Bacterial Diseases Research Unit, National Animal Disease Centers, United States Department of Agriculture, 1920 Dayton Avenue, Ames, IA, 50010, USA.

Brucellosis is a bacterial zoonosis and a significant source of economic loss and a major public health concern, worldwide. Bovine brucellosis, as caused primarily by , is an important cause of reproductive loss in cattle. Vaccination has been the most effective way to reduce disease prevalence contributing to the success of control and eradication programs. Currently, there are no human vaccines available, and despite the success of commercial vaccines for livestock, such as strain RB51 (RB51), there is need for development of novel and safer vaccines against brucellosis. In the current study, we report the fabrication of and immune responses to an implantable single dose polyanhydride-based, methanol-killed RB51 antigen containing delivery platform (VPEAR) in cattle. In contrast to animals vaccinated with RB51, we did not observe measurable RB51-specific IFN-γ or IgG responses in the peripheral blood, following initial vaccination with VPEAR. However, following a subsequent booster vaccination with RB51, we observed an anamnestic response in both vaccination treatments (VPEAR and live RB51). The magnitude and kinetics of CD4+ IFN-γ-mediated responses and circulating memory T cell subpopulations were comparable between the two vaccination treatments. Additionally, IgG titers were significantly increased in animals vaccinated with VPEAR as compared to live RB51- vaccinated animals. These data demonstrate that killed antigen may be utilized to generate and sustain memory, IFN-γ-mediated, CD4+ T cell and humoral responses against Brucella in a natural host. To our knowledge, this novel approach to vaccination against intracellular bacteria, such as , has not been reported before.
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http://dx.doi.org/10.1016/j.heliyon.2019.e02370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728543PMC
August 2019

STING pathway stimulation results in a differentially activated innate immune phenotype associated with low nitric oxide and enhanced antibody titers in young and aged mice.

Vaccine 2019 05 12;37(20):2721-2730. Epub 2019 Apr 12.

Iowa State University, Department of Veterinary Microbiology and Preventative Medicine, United States; Nanovaccine Institute, Iowa State University, United States. Electronic address:

Background: One of the most concerning public health issues, related to vaccination and disease prevention, is the inability to induce durable immune responses following a single-dose immunization. In this regard, the nature of the inflammatory environment induced by vaccine adjuvants can negatively impact the resulting immune response. To address these concerns, new strategies to vaccine design are needed in order to improve the outcomes of immune responses, particularly in immunologically disadvantaged populations.

Methods: Comparisons of the scope of innate immune activation induced by TLR agonists versus cyclic dinucleotides (CDNs) was performed. Their effects on the activation characteristics (e.g., metabolism, cytokine secretion) of bone marrow derived dendritic cells (BMDCs) were studied. In addition, the differential effects on in vivo induction of antibody responses were measured.

Results: As compared to TLR ligands, the stimulation of BMDCs with CDNs induced distinctly different metabolic outcomes. Marked differences were observed in the production of nitric oxide (NO) and the cytokine BAFF. These distinct differences were correlated with improved (i.e., more rapid and persistent) vaccine antibody responses in both aged and young mice.

Conclusions: Our results illustrate that the innate immune pathway targeted by adjuvants can critically impact the outcome of the immune response post-vaccination. Specifically, CDN stimulation of APCs induced an activation phenotype that was characterized by decreased innate effector molecule production (e.g., NO) and increased BAFF. This was attributed to the induction of an innate inflammatory environment that enabled the host to make the most of the existing B lymphocyte potential. The use of adjuvants that differentially engage mechanisms of innate immune activation would be particularly advantageous for the generation of robust, single dose vaccines. The results of this study demonstrated that CDNs induced differential innate activation and enhanced vaccine induced antibody responses in both young and aged mice.
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http://dx.doi.org/10.1016/j.vaccine.2019.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499688PMC
May 2019

Radial Glial Lineage Progression and Differential Intermediate Progenitor Amplification Underlie Striatal Compartments and Circuit Organization.

Neuron 2018 07 12;99(2):345-361.e4. Epub 2018 Jul 12.

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. Electronic address:

The circuitry of the striatum is characterized by two organizational plans: the division into striosome and matrix compartments, thought to mediate evaluation and action, and the direct and indirect pathways, thought to promote or suppress behavior. The developmental origins of these organizations and their developmental relationships are unknown, leaving a conceptual gap in understanding the cortico-basal ganglia system. Through genetic fate mapping, we demonstrate that striosome-matrix compartmentalization arises from a lineage program embedded in lateral ganglionic eminence radial glial progenitors mediating neurogenesis through two distinct types of intermediate progenitors (IPs). The early phase of this program produces striosomal spiny projection neurons (SPNs) through fate-restricted apical IPs (aIPs) with limited capacity; the late phase produces matrix SPNs through fate-restricted basal IPs (bIPs) with expanded capacity. Notably, direct and indirect pathway SPNs arise within both aIP and bIP pools, suggesting that striosome-matrix architecture is the fundamental organizational plan of basal ganglia circuitry.
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http://dx.doi.org/10.1016/j.neuron.2018.06.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094944PMC
July 2018

Room Temperature Stable PspA-Based Nanovaccine Induces Protective Immunity.

Front Immunol 2018 2;9:325. Epub 2018 Mar 2.

Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, United States.

is a major causative agent of pneumonia, a debilitating disease particularly in young and elderly populations, and is the leading worldwide cause of death in children under the age of five. While there are existing vaccines against , none are protective across all serotypes. Pneumococcal surface protein A (PspA), a key virulence factor of , is an antigen that may be incorporated into future vaccines to address the immunological challenges presented by the diversity of capsular antigens. PspA has been shown to be immunogenic and capable of initiating a humoral immune response that is reactive across approximately 94% of pneumococcal strains. Biodegradable polyanhydrides have been studied as a nanoparticle-based vaccine (i.e., nanovaccine) platform to stabilize labile proteins, to provide adjuvanticity, and enhance patient compliance by providing protective immunity in a single dose. In this study, we designed a room temperature stable PspA-based polyanhydride nanovaccine that eliminated the need for a free protein component (i.e., 100% encapsulated within the nanoparticles). Mice were immunized once with the lead nanovaccine and upon challenge, presented significantly higher survival rates than animals immunized with soluble protein alone, even with a 25-fold reduction in protein dose. This lead nanovaccine formulation performed similarly to protein adjuvanted with Alum, however, with much less tissue reactogenicity at the site of immunization. By eliminating the free PspA from the nanovaccine formulation, the lead nanovaccine was efficacious after being stored dry for 60 days at room temperature, breaking the need for maintaining the cold chain. Altogether, this study demonstrated that a single dose PspA-based nanovaccine against induced protective immunity and provided thermal stability when stored at room temperature for at least 60 days.
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http://dx.doi.org/10.3389/fimmu.2018.00325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5863507PMC
March 2019

Efficacy of mucosal polyanhydride nanovaccine against respiratory syncytial virus infection in the neonatal calf.

Sci Rep 2018 02 14;8(1):3021. Epub 2018 Feb 14.

Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, Agricultural Research Service, USDA, Ames, IA, USA.

Human respiratory syncytial virus (HRSV) is a leading cause of severe acute lower respiratory tract infection in infants and children worldwide. Bovine RSV (BRSV) is closely related to HRSV and a significant cause of morbidity in young cattle. BRSV infection in calves displays many similarities to RSV infection in humans, including similar age dependency and disease pathogenesis. Polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) have shown promise as adjuvants and vaccine delivery vehicles due to their ability to promote enhanced immunogenicity through the route of administration, provide sustained antigen exposure, and induce both antibody- and cell-mediated immunity. Here, we developed a novel, mucosal nanovaccine that encapsulates the post-fusion F and G glycoproteins from BRSV into polyanhydride nanoparticles and determined the efficacy of the vaccine against RSV infection using a neonatal calf model. Calves receiving the BRSV-F/G nanovaccine exhibited reduced pathology in the lungs, reduced viral burden, and decreased virus shedding compared to unvaccinated control calves, which correlated with BRSV-specific immune responses in the respiratory tract and peripheral blood. Our results indicate that the BRSV-F/G nanovaccine is highly immunogenic and, with optimization, has the potential to significantly reduce the disease burden associated with RSV infection in both humans and animals.
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http://dx.doi.org/10.1038/s41598-018-21292-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5813012PMC
February 2018

A single dose polyanhydride-based vaccine platform promotes and maintains anti-GnRH antibody titers.

Vaccine 2018 02;36(7):1016-1023

Department of Veterinary Pathology, Iowa State University, Ames, IA 50011, USA; Nanovaccine Institute, Iowa State University, Ames, IA 50011, USA. Electronic address:

Traditionally, vaccination strategies require an initial priming vaccination followed by an antigen boost to generate adequate immunity. Here we describe vaccination against a self-peptide for reproductive sterilization utilizing a three-stage vaccine platform consisting of gonadotropin releasing hormone multiple antigenic peptide (GnRH-MAP) as a soluble injection coupled with subcutaneous administration of polyanhydride-immobilized GnRH-MAP and a cyto-exclusive implant containing GnRH-MAP dendrimer-loaded polyanhydride. This strategy generated and maintained cell-mediated and humoral immunity for up to 41 weeks after a single vaccination in mice with enhanced antibody avidity over time. All intact implants had a grossly visible tissue interface with neovascularization and lymphocytic aggregates. Despite detectable immunity, sterility was not achieved and the immune response did not lead to azoospermia in male mice nor prevent estrus and ovulation in female mice. However, the vaccine delivery device is tunable and the immunogen, adjuvants and release rates can all be modified to enhance immunity. This technology has broad implications for the development of long-term vaccination schemes.
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http://dx.doi.org/10.1016/j.vaccine.2017.12.050DOI Listing
February 2018

Chemoselective Copper-Catalyzed Ullmann-Type Coupling of Oxazolidinones with Bromoiodoarenes.

Org Lett 2017 06 22;19(11):3021-3024. Epub 2017 May 22.

Department of Small Molecule Process Chemistry, Genentech, Inc. , South San Francisco, California 94080, United States.

We describe the highly selective copper-catalyzed Ullmann-type coupling of bromoiodoarenes with oxazolidinones. 3,4,7,8-Tetramethyl-1,10-phenanthroline (MePhen) was identified as an optimal ligand promoting the desired C-N bond formation, while minimizing the competitive bromo-iodo exchange pathway that leads to formation of iodo-substituted and bis-coupled side products. The developed method is highly selective with a >98:2 ratio of the bromo- vs iodo-substituted compounds obtained in the isolated products.
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http://dx.doi.org/10.1021/acs.orglett.7b01304DOI Listing
June 2017

Nucleophilic Aromatic Substitution Reactions in Water Enabled by Micellar Catalysis.

Org Lett 2015 Oct;17(19):4734-7

Department of Chemistry & Biochemistry, University of California , Santa Barbara, California 93106, United States.

Given the huge dependence on dipolar, aprotic solvents such as DMF, DMSO, DMAc, and NMP in nucleophilic aromatic substitution reactions (SNAr), a simple and environmentally friendly alternative is reported. Use of a "benign-by-design" nonionic surfactant, TPGS-750-M, in water enables nitrogen, oxygen, and sulfur nucleophiles to participate in SNAr reactions. Aromatic and heteroaromatic substrates readily participate in this micellar catalysis, which takes place at or near ambient temperatures.
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http://dx.doi.org/10.1021/acs.orglett.5b02240DOI Listing
October 2015

Daclatasvir inhibits hepatitis C virus NS5A motility and hyper-accumulation of phosphoinositides.

Virology 2015 Feb 26;476:168-179. Epub 2014 Dec 26.

Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA. Electronic address:

Combinations of direct-acting antivirals (DAAs) against the hepatitis C virus (HCV) have the potential to revolutionize the HCV therapeutic regime. An integral component of DAA combination therapies is HCV NS5A inhibitors. It has previously been proposed that NS5A DAAs inhibit two functions of NS5A: RNA replication and virion assembly. In this study, we characterize the impact of a prototype NS5A DAA, daclatasvir (DCV), on HCV replication compartment formation. DCV impaired HCV replicase localization and NS5A motility. In order to characterize the mechanism behind altered HCV replicase localization, we examined the impact of DCV on the interaction of NS5A with its essential cellular cofactor, phosphatidylinositol-4-kinase III α (PI4KA). We observed that DCV does not inhibit PI4KA directly, nor does it impair early events of the NS5A-PI4KA interaction that can occur when NS5A is expressed alone. NS5A functions that are unaffected by DCV include PI4KA binding, as determined by co-immunoprecipitation, and a basal accumulation of the PI4KA product, PI4P. However, DCV impairs late steps in PI4KA activation that requires NS5A expressed in the context of the HCV polyprotein. These NS5A functions include hyper-stimulation of PI4P levels and appropriate replication compartment formation. The data are most consistent with a model wherein DCV inhibits conformational changes in the NS5A protein or protein complex formations that occur in the context of HCV polyprotein expression and stimulate PI4P hyper-accumulation and replication compartment formation.
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http://dx.doi.org/10.1016/j.virol.2014.12.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323755PMC
February 2015

Synthesis and characterization of proteoglycan-mimetic graft copolymers with tunable glycosaminoglycan density.

Biomacromolecules 2014 Oct 15;15(10):3772-80. Epub 2014 Sep 15.

School of Biomedical Engineering and ‡Department of Chemical and Biological Engineering, Colorado State University , 1370 Campus Delivery, Fort Collins, Colorado 80523, United States.

Proteoglycans (PGs) are important glycosylated proteins found on the cell surface and in the extracellular matrix. They are made up of a core protein with glycosaminoglycan (GAG) side chains. Variations in composition and number of GAG side chains lead to a vast array of PG sizes and functions. Here we present a method for the synthesis of proteoglycan-mimetic graft copolymers (or neoproteoglycans) with tunable GAG side-chain composition. This is done using three different GAGs: hyaluronan, chondroitin sulfate, and heparin. Hyaluronan is functionalized with a hydrazide-presenting linker. Either chondroitin sulfate or heparin is grafted by the reducing end on to the hyaluronan backbone through reductive amination. PG mimics with heparin or chondroitin sulfate side chains and four different ratios of GAG side chain result in graft copolymers with a wide range of sizes. The chemistry is confirmed through attentuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and (1)H NMR. Effective hydrodynamic diameter and zeta potential are determined using dynamic light scattering and electrophoretic mobility measurements. Graft copolymers were tested for their ability to bind and deliver basic fibroblast growth factor (FGF-2) to mesenchymal stem cells (MSCs). The chondroitin sulfate-containing graft copolymers successfully deliver FGF-2 to cells from surfaces. The lowest graft density of heparin-containing PG mimic also performs well with respect to growth factor delivery from a surface. This new method for preparation of GAG-based graft copolymers enables a wide range of graft density, and can be used to explore applications of PG mimics as new biomaterials with tunable biochemical and biomechanical functions.
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http://dx.doi.org/10.1021/bm501045kDOI Listing
October 2014

Chemoselective reductions of nitroaromatics in water at room temperature.

Org Lett 2014 Jan 16;16(1):98-101. Epub 2013 Dec 16.

Department of Chemistry & Biochemistry, University of California , Santa Barbara, California 93106, United States.

A robust and green protocol for the reduction of functionalized nitroarenes to the corresponding primary amines has been developed. It relies on inexpensive zinc dust in water containing nanomicelles derived from the commercially available designer surfactant TPGS-750-M. This mild process takes place at room temperature and tolerates a wide range of functionalities. Highly selective reductions can also be achieved in the presence of common protecting groups.
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http://dx.doi.org/10.1021/ol403079xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4013784PMC
January 2014

Diagnosis and management of hereditary haemochromatosis.

Br J Gen Pract 2013 Jun;63(611):331-2

York Teaching Hospital NHS Foundation Trust, York, UK.

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http://dx.doi.org/10.3399/bjgp13X668410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3662451PMC
June 2013

Hepatitis C virus stimulates the phosphatidylinositol 4-kinase III alpha-dependent phosphatidylinositol 4-phosphate production that is essential for its replication.

J Virol 2011 Sep 22;85(17):8870-83. Epub 2011 Jun 22.

Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA.

Phosphatidylinositol 4-kinase III alpha (PI4KA) is an essential cofactor of hepatitis C virus (HCV) replication. We initiated this study to determine whether HCV directly engages PI4KA to establish its replication. PI4KA kinase activity was found to be absolutely required for HCV replication using a small interfering RNA transcomplementation assay. Moreover, HCV infection or subgenomic HCV replicons produced a dramatic increase in phosphatidylinositol 4-phosphate (PI4P) accumulation throughout the cytoplasm, which partially colocalized with the endoplasmic reticulum. In contrast, the majority of PI4P accumulated at the Golgi bodies in uninfected cells. The increase in PI4P was not observed after infection with UV-inactivated HCV and did not reflect changes in PI4KA protein or RNA abundance. In an analysis of U2OS cell lines with inducible expression of the HCV polyprotein or individual viral proteins, viral polyprotein expression resulted in enhanced cytoplasmic PI4P production. Increased PI4P accumulation following HCV protein expression was precluded by silencing the expression of PI4KA, but not the related PI4KB. Silencing PI4KA also resulted in aberrant agglomeration of viral replicase proteins, including NS5A, NS5B, and NS3. NS5A alone, but not other viral proteins, stimulated PI4P production in vivo and enhanced PI4KA kinase activity in vitro. Lastly, PI4KA coimmunoprecipitated with NS5A from infected Huh-7.5 cells and from dually transfected 293T cells. In sum, these results suggest that HCV NS5A modulation of PI4KA-dependent PI4P production influences replication complex formation.
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http://dx.doi.org/10.1128/JVI.00059-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165839PMC
September 2011

Dinoxin B, a withanolide from Datura inoxia leaves with specific cytotoxic activities.

J Nat Prod 2011 Feb 31;74(2):267-71. Epub 2011 Jan 31.

United States Department of Agriculture, Agricultural Research Service, National Center of Agricultural Utilization Research, Functional Foods Research, 1815 N. University Street, Peoria, Illinois 61604, United States.

A new withanolide, dinoxin B (12,21-dihydroxy-1-oxowitha-2,5,24-trienolide-27-O-β-D-glucopyranoside, 1), was isolated from a methanol extract of Datura inoxia leaves, using bioassay-guided fractionation. The structure was determined by spectroscopic techniques, including (1)H, (13)C, and 2D NMR experiments as well as by HRMS. Extracts and the purified compound were tested for their antiproliferative activities toward a panel of human normal and cancer cell lines. Dinoxin B (1) and its aglycone (2) exhibited submicromolar IC(50) values against multiple human cancer cell lines. Among the most sensitive were several breast cancer cell lines. Dinoxin B (1) was found only in D. inoxia and was not detected in D. metel or D. stramonium. The accumulation of this compound was limited largely to leaf tissue, with little to none detected in extracts from the flowers, fruits, roots, or stems of D. inoxia.
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http://dx.doi.org/10.1021/np1004714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057138PMC
February 2011

SMAD signaling drives heart and muscle dysfunction in a Drosophila model of muscular dystrophy.

Hum Mol Genet 2011 Mar 6;20(5):894-904. Epub 2010 Dec 6.

Department of Pathology, The University of Chicago, Chicago, IL, USA.

Loss-of-function mutations in the genes encoding dystrophin and the associated membrane proteins, the sarcoglycans, produce muscular dystrophy and cardiomyopathy. The dystrophin complex provides stability to the plasma membrane of striated muscle during muscle contraction. Increased SMAD signaling due to activation of the transforming growth factor-β (TGFβ) pathway has been described in muscular dystrophy; however, it is not known whether this canonical TGFβ signaling is pathogenic in the muscle itself. Drosophila deleted for the γ/δ-sarcoglycan gene (Sgcd) develop progressive muscle and heart dysfunction and serve as a model for the human disorder. We used dad-lacZ flies to demonstrate the signature of TGFβ activation in response to exercise-induced injury in Sgcd null flies, finding that those muscle nuclei immediately adjacent to muscle injury demonstrate high-level TGFβ signaling. To determine the pathogenic nature of this signaling, we found that partial reduction of the co-SMAD Medea, homologous to SMAD4, or the r-SMAD, Smox, corrected both heart and muscle dysfunction in Sgcd mutants. Reduction in the r-SMAD, MAD, restored muscle function but interestingly not heart function in Sgcd mutants, consistent with a role for activin but not bone morphogenic protein signaling in cardiac dysfunction. Mammalian sarcoglycan null muscle was also found to exhibit exercise-induced SMAD signaling. These data demonstrate that hyperactivation of SMAD signaling occurs in response to repetitive injury in muscle and heart. Reduction of this pathway is sufficient to restore cardiac and muscle function and is therefore a target for therapeutic reduction.
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http://dx.doi.org/10.1093/hmg/ddq528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033181PMC
March 2011

Transcriptional coactivators PGC-1alpha and PGC-lbeta control overlapping programs required for perinatal maturation of the heart.

Genes Dev 2008 Jul;22(14):1948-61

Center for Cardiovascular Research, Washington University School of Medicine, St Louis, Missouri 63110, USA

Oxidative tissues such as heart undergo a dramatic perinatal mitochondrial biogenesis to meet the high-energy demands after birth. PPARgamma coactivator-1 (PGC-1) alpha and beta have been implicated in the transcriptional control of cellular energy metabolism. Mice with combined deficiency of PGC-1alpha and PGC-1beta (PGC-1alphabeta(-/-) mice) were generated to investigate the convergence of their functions in vivo. The phenotype of PGC-1beta(-/-) mice was minimal under nonstressed conditions, including normal heart function, similar to that of PGC-1alpha(-/-) mice generated previously. In striking contrast to the singly deficient PGC-1 lines, PGC-1alphabeta(-/-) mice died shortly after birth with small hearts, bradycardia, intermittent heart block, and a markedly reduced cardiac output. Cardiac-specific ablation of the PGC-1beta gene on a PGC-1alpha-deficient background phenocopied the generalized PGC-1alphabeta(-/-) mice. The hearts of the PGC-1alphabeta(-/-) mice exhibited signatures of a maturational defect including reduced growth, a late fetal arrest in mitochondrial biogenesis, and persistence of a fetal pattern of gene expression. Brown adipose tissue (BAT) of PGC-1alphabeta(-/-) mice also exhibited a severe abnormality in function and mitochondrial density. We conclude that PGC-1alpha and PGC-1beta share roles that collectively are necessary for the postnatal metabolic and functional maturation of heart and BAT.
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http://dx.doi.org/10.1101/gad.1661708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2492740PMC
July 2008

Regulation of ubiquitin-proteasome system mediated degradation by cytosolic stress.

Mol Biol Cell 2007 Nov 15;18(11):4279-91. Epub 2007 Aug 15.

Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239, USA.

ER-associated, ubiquitin-proteasome system (UPS)-mediated degradation of the wild-type (WT) gap junction protein connexin32 (Cx32) is inhibited by mild forms of cytosolic stress at a step before its dislocation into the cytosol. We show that the same conditions (a 30-min, 42 degrees C heat shock or oxidative stress induced by arsenite) also reduce the endoplasmic reticulum (ER)-associated turnover of disease-causing mutants of Cx32 and the cystic fibrosis transmembrane conductance regulator (CFTR), as well as that of WT CFTR and unassembled Ig light chain. Stress-stabilized WT Cx32 and CFTR, but not the mutant/unassembled proteins examined, could traverse the secretory pathway. Heat shock also slowed the otherwise rapid UPS-mediated turnover of the cytosolic proteins myoD and GFPu, but not the degradation of an ubiquitination-independent construct (GFP-ODC) closely related to the latter. Analysis of mutant Cx32 from cells exposed to proteasome inhibitors and/or cytosolic stress indicated that stress reduces degradation at the level of substrate polyubiquitination. These findings reveal a new link between the cytosolic stress-induced heat shock response, ER-associated degradation, and polyubiquitination. Stress-denatured proteins may titer a limiting component of the ubiquitination machinery away from pre-existing UPS substrates, thereby sparing the latter from degradation.
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http://dx.doi.org/10.1091/mbc.e07-05-0487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2043544PMC
November 2007

Inflammatory bowel disease in general practice.

Practitioner 2004 May;248(1658):344-6, 348, 350 passim

York Hospital, York.

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May 2004

N-linked glycosylation and sequence changes in a critical negative control region of the ASCT1 and ASCT2 neutral amino acid transporters determine their retroviral receptor functions.

J Virol 2003 Mar;77(5):2936-45

Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97201-3098, USA.

A widely dispersed interference group of retroviruses that includes the feline endogenous virus (RD114), baboon endogenous virus (BaEV), human endogenous virus type W (HERV-W), and type D primate retroviruses uses the human Na(+)-dependent neutral amino acid transporter type 2 (hASCT2; gene name, SLC1A5) as a common cell surface receptor. Although hamster cells are fully resistant to these viruses and murine cells are susceptible only to BaEV and HERV-W pseudotype viruses, these rodent cells both become highly susceptible to all of the viruses after treatment with tunicamycin, an inhibitor of protein N-linked glycosylation. A partial explanation for these results was recently provided by findings that the orthologous murine transporter mASCT2 is inactive as a viral receptor, that a related (ca. 55% identity) murine paralog (mASCT1; gene name, SLC1A4) mediates infections specifically of BaEV and HERV-W, and that N-deglycosylation of mASCT1 activates it as a receptor for all viruses of this interference group. Because the only two N-linked oligosaccharides in mASCT1 occur in the carboxyl-terminal region of extracellular loop 2 (ECL2), it was inferred that this region contributes in an inhibitory manner to infections by RD114 and type D primate viruses. To directly and more thoroughly investigate the receptor active sites, we constructed and analyzed a series of hASCT2/mASCT2 chimeras and site-directed mutants. Our results suggest that a hypervariable sequence of 21 amino acids in the carboxyl-terminal portion of ECL2 plays a critical role in determining the receptor properties of ASCT2 proteins for all viruses in this interference group. In addition, we analyzed the tunicamycin-dependent viral susceptibility of hamster cells. In contrast to mASCT1, which contains two N-linked oligosaccharides that partially restrict viral infections, hamster ASCT1 contains an additional N-linked oligosaccharide clustered close to the others in the carboxyl-terminal region of ECL2. Removal of this N-linked oligosaccharide by mutagenesis enabled hamster ASCT1 to function as a receptor for all viruses of this interference group. These results strongly suggest that combinations of amino acid sequence changes and N-linked oligosaccharides in a critical carboxyl-terminal region of ECL2 control retroviral utilization of both the ASCT1 and ASCT2 receptors.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC149750PMC
http://dx.doi.org/10.1128/jvi.77.5.2936-2945.2003DOI Listing
March 2003