Thermal Stability of RNA Phage Virus-Like Particles Displaying Foreign Peptides

<p>Abstract</p> <p>Background</p> <p>To be useful for genetic display of foreign peptides a viral coat protein must tolerate peptide insertions without major disruption of subunit folding and capsid assembly. The folding of the coat protein of RNA phage MS2 does not normally tolerate insertions in its AB-loop, but an engineered single-chain dimer readily accepts them as long as they are restricted to one of its two halves.</p> <p>Results</p> <p>Here we characterize the effects of peptide insertions on the thermal stabilities of MS2 virus-like particles (VLPs) displaying a variety of different peptides in one AB-loop of the coat protein single-chain dimer. These particles typically denature at temperatures around 5-10°C lower than unmodified VLPs. Even so, they are generally stable up to about 50°C. VLPs of the related RNA phage PP7 are cross-linked with intersubunit disulfide bonds and are therefore significantly more stable. An AB-loop insertion also reduces the stability of PP7 VLPs, but they only begin to denature above about 70°C.</p> <p>Conclusions</p> <p>VLPs assembled from MS2 single-chain dimer coat proteins with peptide insertions in one of their AB-loops are somewhat less stable than the wild-type particle, but still resist heating up to about 50°C. Because they possess disulfide cross-links, PP7-derived VLPs provide an alternate platform with even higher stability.</p

Asymmetric interactions in the adenosine-binding pockets of the MS2 coat protein dimer

BACKGROUND: The X-ray structure of the MS2 coat protein-operator RNA complex reveals the existence of quasi-synmetric interactions of adenosines -4 and -10 in pockets formed on different subunits of the coat protein dimer. Both pockets utilize the same five amino acid residues, namely Val29, Thr45, Ser47, Thr59, and Lys61. We call these sites the adenosine-binding pockets. RESULTS: We present here a heterodimer complementation analysis of the contributions of individual A-pocket amino acids to the binding of A-4 and A-10 in different halves of the dimer. Various substitutions of A-pocket residues were introduced into one half of single-chain coat protein heterodimers where they were tested for their abilities to complement Y85H or T91I substitutions (defects in the A-4 and A-10 half-sites, respectively) present in the other dimer half. CONCLUSIONS: These experiments provide functional tests of interactions predicted from structural analyses, demonstrating the importance of certain amino acid-nucleotide contacts observed in the crystal structure, and showing that others make little or no contribution to the stability of the complex. In summary, Val29 and Lys61 form important stabilizing interactions with both A-4 and A-10. Meanwhile, Ser47 and Thr59 interact primarily with A-10. The important interactions with Thr45 are restricted to A-4

Stability and assembly in vitro of bacteriophage PP7 virus-like particles

<p>Abstract</p> <p>Background</p> <p>The stability of a virus-like particle (VLP) is an important consideration for its use in nanobiotechnology. The icosahedral capsid of the RNA bacteriophage PP7 is cross-linked by disulfide bonds between coat protein dimers at its 5-fold and quasi-6-fold symmetry axes. This work determined the effects of these disulfides on the VLP's thermal stability.</p> <p>Results</p> <p>Measurements of the thermal denaturation behavior of PP7 VLPs in the presence and absence of a reducing agent show that disulfide cross-links substantially stabilize them against thermal denaturation. Although dimers in the capsid are linked to one another by disulfides, the two subunits of dimers themselves are held together only by non-covalent interactions. In an effort to confer even greater stability a new cross-link was introduced by genetically fusing two coat protein monomers, thus producing a "single-chain dimer" that assembles normally into a completely cross-linked VLP. However, subunit fusion failed to increase the thermal stability of the particles, even though it stabilized the isolated dimer. As a step toward gaining control of the internal composition of the capsid, conditions that promote the assembly of PP7 coat protein dimers into virus-like particles <it>in vitro </it>were established.</p> <p>Conclusion</p> <p>The presence of inter-dimer disulfide bonds greatly stabilizes the PP7 virus-like particle against thermal denaturation. Covalently cross-linking the subunits of the dimers themselves by genetically fusing them through a dipeptide linker sequence, offers no further stabilization of the VLP, although it does stabilize the dimer. PP7 capsids readily assemble <it>in vitro </it>in a reaction that requires RNA.</p

A malaria vaccine candidate based on an epitope of the Plasmodium falciparum RH5 protein

BACKGROUND: The Plasmodium falciparum protein RH5 is an adhesin molecule essential for parasite invasion of erythrocytes. Recent studies show that anti-PfRH5 sera have potent invasion-inhibiting activities, supporting the idea that the PfRH5 antigen could form the basis of a vaccine. Therefore, epitopes recognized by neutralizing anti-PfRH5 antibodies could themselves be effective vaccine immunogens if presented in a sufficiently immunogenic fashion. However, the exact regions within PfRH5 that are targets of this invasion-inhibitory activity have yet to be identified. METHODS: A battery of anti-RH5 monoclonal antibodies (mAbs) were produced and screened for their potency by inhibition of invasion assays in vitro. Using an anti-RH5 mAb that completely inhibited invasion as the selecting mAb, affinity-selection using random sequence peptide libraries displayed on virus-like particles of bacteriophage MS2 (MS2 VLPs) was performed. VLPs were sequenced to identify the specific peptide epitopes they encoded and used to raise specific antisera that was in turn tested for inhibition of invasion. RESULTS: Three anti-RH5 monoclonals (0.1 mg/mL) were able to inhibit invasion in vitro by >95%. Affinity-selection with one of these mAbs yielded a VLP which yielded a peptide whose sequence is identical to a portion of PfRH5 itself. The VLP displaying the peptide binds strongly to the antibody, and in immunized animals elicits an anti-PfRH5 antibody response. The resulting antisera against the specific VLP inhibit parasite invasion of erythrocytes more than 90% in vitro. CONCLUSIONS: Here, data is presented from an anti-PfRH5 mAb that completely inhibits erythrocyte invasion by parasites in vitro, one of the few anti-malarial monoclonal antibodies reported to date that completely inhibits invasion with such potency, adding to other studies that highlight the potential of PfRH5 as a vaccine antigen. The specific neutralization sensitive epitope within RH5 has been identified, and antibodies against this epitope also elicit high anti-invasion activity, suggesting this epitope could form the basis of an effective vaccine against malaria

Design Evolution of the Wide Field Infrared Survey Telescope Using Astrophysics Focused Telescope Assets (WFIRST-AFTA) and Lessons Learned

The design of the Wide Field Infrared Survey Telescope using Astrophysics Focused Telescope Assets (WFIRST-AFTA) continues to evolve as each design cycle is analyzed. In 2012, two Hubble sized (2.4 m diameter) telescopes were donated to NASA from elsewhere in the Federal Government. NASA began investigating potential uses for these telescopes and identified WFIRST as a mission to benefit from these assets. With an updated, deeper, and sharper field of view than previous design iterations with a smaller telescope, the optical designs of the WFIRST instruments were updated and the mechanical and thermal designs evolved around the new optical layout. Beginning with Design Cycle 3, significant analysis efforts yielded a design and model that could be evaluated for Structural-Thermal-Optical-Performance (STOP) purposes for the Wide Field Imager (WFI) and provided the basis for evaluating the high level observatory requirements. Development of the Cycle 3 thermal model provided some valuable analysis lessons learned and established best practices for future design cycles. However, the Cycle 3 design did include some major liens and evolving requirements which were addressed in the Cycle 4 Design. Some of the design changes are driven by requirements changes, while others are optimizations or solutions to liens from previous cycles. Again in Cycle 4, STOP analysis was performed and further insights into the overall design were gained leading to the Cycle 5 design effort currently underway. This paper seeks to capture the thermal design evolution, with focus on major design drivers, key decisions and their rationale, and lessons learned as the design evolved

The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers.

Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma exhibit a 10,000-fold greater affinity for human hepatocellular carcinoma than for hepatocytes, endothelial cells or immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, small interfering RNA and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a 10(6)-fold improvement over comparable liposomes

BMQ

BMQ: Boston Medical Quarterly was published from 1950-1966 by the Boston University School of Medicine and the Massachusetts Memorial Hospitals

NIMBUS: The Near-Infrared Multi-Band Ultraprecise Spectroimager for SOFIA

We present a new and innovative near-infrared multi-band ultraprecise spectroimager (NIMBUS) for SOFIA. This design is capable of characterizing a large sample of extrasolar planet atmospheres by measuring elemental and molecular abundances during primary transit and occultation. This wide-field spectroimager would also provide new insights into Trans-Neptunian Objects (TNO), Solar System occultations, brown dwarf atmospheres, carbon chemistry in globular clusters, chemical gradients in nearby galaxies, and galaxy photometric redshifts. NIMBUS would be the premier ultraprecise spectroimager by taking advantage of the SOFIA observatory and state of the art infrared technologies. This optical design splits the beam into eight separate spectral bandpasses, centered around key molecular bands from 1 to 4 microns. Each spectral channel has a wide field of view for simultaneous observations of a reference star that can decorrelate time-variable atmospheric and optical assembly effects, allowing the instrument to achieve ultraprecise calibration for imaging and photometry for a wide variety of astrophysical sources. NIMBUS produces the same data products as a low-resolution integral field spectrograph over a large spectral bandpass, but this design obviates many of the problems that preclude high-precision measurements with traditional slit and integral field spectrographs. This instrument concept is currently not funded for development.Comment: 14 pages, 9 figures, SPIE Astronomical Telescopes and Instrumentation 201

Use of the Behavior Assessment Tool in 18 Pilot Residency Programs

Background: The purpose of this study was to determine the feasibility and evaluate the effectiveness of the American Board of Orthopaedic Surgery Behavior Tool (ABOSBT) for measuring professionalism. Methods: Through collaboration between the American Board of Orthopaedic Surgery and American Orthopaedic Association\u27s Council of Residency Directors, 18 residency programs piloted the use of the ABOSBT. Residents requested assessments from faculty at the end of their clinical rotations, and a 360° request was performed near the end of the academic year. Program Directors (PDs) rated individual resident professionalism (based on historical observation) at the outset of the study, for comparison to the ABOSBT results. Results: Nine thousand eight hundred ninety-two evaluations were completed using the ABOSBT for 449 different residents by 1,012 evaluators. 97.6% of all evaluations were scored level 4 or 5 (high levels of professional behavior) across all of the 5 domains. In total, 2.4% of all evaluations scored level 3 or below reflecting poorer performance. Of 431 residents, the ABOSBT identified 26 of 32 residents who were low performers (2 or more \u3c level 3 scores in a domain) and who also scored below expectations by the PD at the start of the pilot project (81% sensitivity and 57% specificity), including 13 of these residents scoring poorly in all 5 domains. Evaluators found the ABOSBT was easy to use (96%) and that it was an effective tool to assess resident professional behavior (81%). Conclusions: The ABOSBT was able to identify 2.4% low score evaluations ( Level of Evidence: Level II

The validity of using ICD-9 codes and pharmacy records to identify patients with chronic obstructive pulmonary disease

Background: Administrative data is often used to identify patients with chronic obstructive pulmonary disease (COPD), yet the validity of this approach is unclear. We sought to develop a predictive model utilizing administrative data to accurately identify patients with COPD. Methods: Sequential logistic regression models were constructed using 9573 patients with postbronchodilator spirometry at two Veterans Affairs medical centers (2003-2007). COPD was defined as: 1) FEV1/FVC <0.70, and 2) FEV1/FVC < lower limits of normal. Model inputs included age, outpatient or inpatient COPD-related ICD-9 codes, and the number of metered does inhalers (MDI) prescribed over the one year prior to and one year post spirometry. Model performance was assessed using standard criteria. Results: 4564 of 9573 patients (47.7%) had an FEV1/FVC < 0.70. The presence of ≥1 outpatient COPD visit had a sensitivity of 76% and specificity of 67%; the AUC was 0.75 (95% CI 0.74-0.76). Adding the use of albuterol MDI increased the AUC of this model to 0.76 (95% CI 0.75-0.77) while the addition of ipratropium bromide MDI increased the AUC to 0.77 (95% CI 0.76-0.78). The best performing model included: ≥6 albuterol MDI, ≥3 ipratropium MDI, ≥1 outpatient ICD-9 code, ≥1 inpatient ICD-9 code, and age, achieving an AUC of 0.79 (95% CI 0.78-0.80). Conclusion: Commonly used definitions of COPD in observational studies misclassify the majority of patients as having COPD. Using multiple diagnostic codes in combination with pharmacy data improves the ability to accurately identify patients with COPD.Department of Veterans Affairs, Health Services Research and Development (DHA), American Lung Association (CI- 51755-N) awarded to DHA, the American Thoracic Society Fellow Career Development AwardPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/84155/1/Cooke - ICD9 validity in COPD.pd