Quantification of cytosolic plasmid DNA degradation using high‐throughput sequencing: implications for gene delivery

Background Although cytosolic DNA degradation plays an important role in decreasing transgene expression, the plasmid degradation pattern remains largely unexplored. Methods Illumina dye sequencing was employed to provide degradation site information for S1 and cytosolic nucleases. S1 nuclease provided a positive control for a comparison between the agarose gel method and sequencing approaches. Results The poly(A) region between the β‐lactamase gene and the cytomegalovirus (CMV) promoter was identified as the most likely cut site for polyplex‐treated cytosol. The second most likely site, at the 5' end of the β‐lactamase gene, was identified by gel electrophoresis and sequencing. Additional sites were detected in the OriC region, the SV40/poly(A) region, the luciferase gene and the CMV promoter. Sequence analysis of plasmid treated with cytosol from control cells showed the greatest cut activity in the OriC region, the β‐lactamase gene and the poly(A) region following the luciferase gene. Additional regions of cut activity include the SV40 promoter and the β‐lactamase poly(A) termination sequence. Both cytosolic nucleases and the S1 nuclease showed substantial activity at the bacterial origin of replication ( OriC ). Conclusions High‐throughput plasmid sequencing revealed regions of the luciferase plasmid DNA sequence that are sensitive to cytosolic nuclease degradation. This provides new targets for improving plasmid and/or polymer design to optimize the likelihood of protein expression. Copyright © 2014 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106947/1/jgm2761.pd

Animal-Based Measures for the On-Farm Welfare Assessment of Geese

Currently, no specific animal-based measures (ABMs) protocols are available for geese in commercial meat production systems. Following a critical review of the literature and consultation of experts, seven ABMs, potentially valid and feasible for the on-farm welfare assessment of geese, were identified and then tested in 12 farms in Poland to assess their inter-observer reliability. Two observers conducted the assessment, which was divided into two phases. First, a handling test assessed the human\u2013animal relationship (HAR), and a 100% inter-observer reliability was achieved by the observers when evaluating the attitudes of stockpeople and the reactions of geese to humans. Next, an animal inspection was conducted, and the observers simultaneously and independently visually evaluated 100 randomly selected geese per farm and assessed whether the selected ABMs could be identified. In terms of inter-observer reliability, high correlation coecients were found for plumage dirtiness (r = 0.745; p < 0.01), twisted wings (r = 0.890; p < 0.001), and broken/twisted wings (r = 0.858; p < 0.001). The results showed that plumage dirtiness, twisted wings, and broken/twisted wings are valid and reliable measures. Further research should address the reliability of ABMs of geese in other types of production systems

Universal behavior of quantum Green's functions

We consider a general one-particle Hamiltonian H = - \Delta_r + u(r) defined in a d-dimensional domain. The object of interest is the time-independent Green function G_z(r,r') = . Recently, in one dimension (1D), the Green's function problem was solved explicitly in inverse form, with diagonal elements of Green's function as prescribed variables. The first aim of this paper is to extract from the 1D inverse solution such information about Green's function which cannot be deduced directly from its definition. Among others, this information involves universal, i.e. u(r)-independent, behavior of Green's function close to the domain boundary. The second aim is to extend the inverse formalism to higher dimensions, especially to 3D, and to derive the universal form of Green's function for various shapes of the confining domain boundary.Comment: 46 pages, the shortened version submitted to J. Math. Phy

GLI1(+) progenitor cells in the adrenal capsule of the adult mouse give rise to heterotopic gonadal-like tissue

As certain strains of mice age, hyperplastic lesions resembling gonadal tissue accumulate beneath the adrenal capsule. Gonadectomy (GDX) accelerates this heterotopic differentiation, resulting in the formation of wedge-shaped adrenocortical neoplasms that produce sex steroids. Stem/progenitor cells that reside in the adrenal capsule and retain properties of the adrenogonadal primordium are thought to be the source of this heterotopic tissue. Here, we demonstrate that GLI1(+) progenitors in the adrenal capsule give rise to gonadal-like cells that accumulate in the subcapsular region. A tamoxifen-inducible Cre driver (Glil-creER(T2)) and two reporters (R26R-lacZ, R26R-confetti) were used to track the fate of GLI1(+) cells in the adrenal glands of B6D2F2 mice, a strain that develops both GDX-induced adrenocortical neoplasms and age-dependent subcapsular cell hyperplasia. In gonadectomized B6D2F2 mice GLI1(+) progenitors contributed to long-lived adrenal capsule cells and to adrenocortical neoplasms that expressed Gata4 and Foxl2, two prototypical gonadal markers. Pdgfra, a gene expressed in adrenocortical stromal cells, was upregulated in the GDX-induced neoplasms. In aged non-gonadectomized B6D2F2 mice GLI1(+) progenitors gave rise to patches of subcapsular cell hyperplasia. Treatment with GANT61, a small-molecule GLI antagonist, attenuated the upregulation of gonadal-like markers (Gata4, Foxl2) in response to GDX. These findings support the premise that GLI1(+) progenitor cells in the adrenal capsule of the adult mouse give rise to heterotopic tissue. (C) 2016 Elsevier Ireland Ltd. All rights reserved.Peer reviewe

Nasal Immunization with a Recombinant HIV gp120 and Nanoemulsion Adjuvant Produces Th1 Polarized Responses and Neutralizing Antibodies to Primary HIV Type 1 Isolates

ABSTRACT Epidemiological and experimental data suggest that both robust neutralizing antibodies and potent cellular responses play important roles in controlling primary HIV-1 infection. In this study we have investigated the induction of systemic and mucosal immune responses to HIV gp120 monomer immunogen administered intranasally in a novel, oil-in-water nanoemulsion (NE) adjuvant. Mice and guinea pigs intranasally immunized by the application of recombinant HIV gp120 antigen mixed in NE demonstrated robust serum anti-gp120 IgG, as well as bronchial, vaginal, and serum anti-gp120 IgA in mice. The serum of these animals demonstrated antibodies that cross-reacted with heterologous serotypes of gp120 and had significant neutralizing activity against two clade-B laboratory strains of HIV (HIVBaL and HIVSF162) and five primary HIV-1 isolates. The analysis of gp120-specific CTL proliferation, INF-γ induction, and prevalence of anti-gp120 IgG2 subclass antibodies indicated that nasal vaccination in NE also induced systemic, Th1-polarized cellular immune responses. This study suggests that NE should be evaluated as a mucosal adjuvant for multivalent HIV vaccines.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63251/1/aid.2007.0148.pd

The violation of the Hund's rule in semiconductor artificial atoms

The unrestricted Pople-Nesbet approach for real atoms is adapted to quantum dots, the man-made artificial atoms, under applied magnetic field. Gaussian basis sets are used instead of the exact single-particle orbitals in the construction of the appropriated Slater determinants. Both system chemical potential and charging energy are calculated, as also the expected values for total and z-component in spin states. We have verified the validity of the energy shell structure as well as the Hund's rule state population at zero magnetic field. Above given fields, we have observed a violation of the Hund's rule by the suppression of triplets and quartets states at the 1p energy shell, taken as an example. We also compare our present results with those obtained using the LS-coupling scheme for low electronic occupations. We have focused our attention to ground-state properties for GaAs quantum dots populated up to forty electrons.Comment: 5 pages, 2 figures, submitted to Semic. Sci. Techno

Pre-Clinical Evaluation of a Novel Nanoemulsion-Based Hepatitis B Mucosal Vaccine

Hepatitis B virus infection remains an important global health concern despite the availability of safe and effective prophylactic vaccines. Limitations to these vaccines include requirement for refrigeration and three immunizations thereby restricting use in the developing world. A new nasal hepatitis B vaccine composed of recombinant hepatitis B surface antigen (HBsAg) in a novel nanoemulsion (NE) adjuvant (HBsAg-NE) could be effective with fewer administrations.Physical characterization indicated that HBsAg-NE consists of uniform lipid droplets (349+/-17 nm) associated with HBsAg through electrostatic and hydrophobic interactions. Immunogenicity of HBsAg-NE vaccine was evaluated in mice, rats and guinea pigs. Animals immunized intranasally developed robust and sustained systemic IgG, mucosal IgA and strong antigen-specific cellular immune responses. Serum IgG reached > or = 10(6) titers and was comparable to intramuscular vaccination with alum-adjuvanted vaccine (HBsAg-Alu). Normalization showed that HBsAg-NE vaccination correlates with a protective immunity equivalent or greater than 1000 IU/ml. Th1 polarized immune response was indicated by IFN-gamma and TNF-alpha cytokine production and elevated levels of IgG(2) subclass of HBsAg-specific antibodies. The vaccine retains full immunogenicity for a year at 4 degrees C, 6 months at 25 degrees C and 6 weeks at 40 degrees C. Comprehensive pre-clinical toxicology evaluation demonstrated that HBsAg-NE vaccine is safe and well tolerated in multiple animal models.Our results suggest that needle-free nasal immunization with HBsAg-NE could be a safe and effective hepatitis B vaccine, or provide an alternative booster administration for the parenteral hepatitis B vaccines. This vaccine induces a Th1 associated cellular immunity and also may provide therapeutic benefit to patients with chronic hepatitis B infection who lack cellular immune responses to adequately control viral replication. Long-term stability of this vaccine formulation at elevated temperatures suggests a direct advantage in the field, since potential excursions from cold chain maintenance could be tolerated without a loss in therapeutic efficacy