Magnetic resonance spectroscopy outperforms perfusion in distinguishing between pseudoprogression and disease progression in patients with glioblastoma.

Background: There is a need to establish biomarkers that distinguish between pseudoprogression (PsP) and true tumor progression in patients with glioblastoma (GBM) treated with chemoradiation. Methods: We analyzed magnetic resonance spectroscopic imaging (MRSI) and dynamic susceptibility contrast (DSC) MR perfusion data in patients with GBM with PsP or disease progression after chemoradiation. MRSI metabolites of interest included intratumoral choline (Cho), myo-inositol (mI), glutamate + glutamine (Glx), lactate (Lac), and creatine on the contralateral hemisphere (c-Cr). Student T-tests and area under the ROC curve analyses were used to detect group differences in metabolic ratios and their ability to predict clinical status, respectively. Results: 28 subjects (63 ± 9 years, 19 men) were evaluated. Subjects with true progression (n = 20) had decreased enhancing region mI/c-Cr (P = .011), a marker for more aggressive tumors, compared to those with PsP, which predicted tumor progression (AUC: 0.84 [0.76, 0.92]). Those with true progression had elevated Lac/Glx (P = .0009), a proxy of the Warburg effect, compared to those with PsP which predicted tumor progression (AUC: 0.84 [0.75, 0.92]). Cho/c-Cr did not distinguish between PsP and true tumor progression. Despite rCBV (AUC: 0.70 [0.60, 0.80]) and rCBF (AUC: 0.75 [0.65, 0.84]) being individually predictive of tumor response, they added no additional predictive value when combined with MRSI metabolic markers. Conclusions: Incorporating enhancing lesion MRSI measures of mI/c-Cr and Lac/Glx into brain tumor imaging protocols can distinguish between PsP and true progression and inform patient management decisions.

How does the close surgical margin impact recurrence and survival when treating oral squamous cell carcinoma?

PURPOSE: The surgical margin is the main prognostic factor over which the surgeon has control during resection of oral squamous cell carcinoma (OSCC). This study examined the association between surgical excision margins of patients with OSCC and outcomes of disease-free and overall survival. MATERIALS AND METHODS: The authors implemented a retrospective cohort study. The sample was composed of patients with OSCC having resection as their initial treatment. The predictor variable was the pathologic surgical margin, defined as clear (>5 mm), close (1 to 5 mm), or involved (<1 mm). The outcome variables were disease-free (absence of locoregional recurrence) and overall survival. Data were analyzed using Kaplan-Meier survival curves and Cox regression hazard model. RESULTS: The sample was composed of 54 patients with a mean age of 60.5 years (range, 19 to 85 yr) and 26% were women. The 2- and 5-year overall survival rates were 59 and 50%, respectively. The clear surgical margin group showed higher disease-free survival rates than patients with close and involved margins (5-yr probability, 0.78 vs 0.43 and 0.29; P = .014) and a trend toward increased overall survival at 2 and 5 years (P = .093). CONCLUSION: The results suggest that the presence of a close surgical margin (1 to 5 mm) is an adverse risk feature comparable to an involved margin and therefore is associated with decreased disease-free and overall survival. Future studies are needed to replicate these findings before they can be used as a basis for clinical recommendations.

Mutations in HIV-1 reverse transcriptase cause misfolding and miscleavage by the viral protease.

Previous work on mutations in the thumb of HIV-1 reverse transcriptase (RT) showed that the majority of the mutant RTs were degraded (by the viral protease) to various extents in virions. This degradation was, in most cases, temperature sensitive, and presumably was due to a partial unfolding of the protein at 37°C. We used recombinant proteins to investigate the effects of the mutations on the thermal stability and proteolytic degradation of RT. Both subunits contribute to the stability of RT. In general, the differences in stability between the mutants and WT were greater if the mutation was in p51 rather than p66. Expressing the Pol polyprotein containing the RT mutants in Escherichia coli produced results similar to what was seen in virions; the mutant RTs were misfolded and/or degraded at 37°C, but were better folded and processed at 30°C.

Solution properties of murine leukemia virus gag protein: differences from HIV-1 gag.

Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV), a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of ∼7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed.

Foamy retrovirus integrase contains a Pol dimerization domain required for protease activation.

Unlike orthoretroviruses, foamy retroviruses (FV) synthesize Pol independently of Gag. The FV Pol precursor is cleaved only once between reverse transcriptase (RT) and integrase (IN) by the protease (PR), resulting in a PR-RT and an IN protein. Only the Pol precursor, not the cleaved subunits, is packaged into virions. Like orthoretroviral PRs, FV PR needs to dimerize to be active. Previously, we showed that a Pol mutant lacking IN has defects in PR activity and Pol packaging into virions. We now show that introduction of a leucine zipper (zip) dimerization motif in an IN truncation mutant can restore PR activity, leading to Pol processing in cells. However, these zip mutants neither cleave Gag nor incorporate Pol into virions. We propose that IN is required for Pol dimerization, which is necessary for the creation of a functional PR active site.

Corrective lens use and refractive error among United States Air Force aircrew.

Corrective lens use by military aviators is an important consideration in the design of head-mounted equipment. The United States Air Force (USAF) has periodically monitored lens use by aviators; however, it has been over a decade since the last study. We provide an update on the prevalence of corrective lenses and refractive error among USAF aircrew based on eyeglass orders processed through the Spectacle Request Transmission System (SRTS). Currently, 41% of active duty USAF pilots and 54% of other aircrew require corrective lenses to perform flight duties. Refractive errors are characterized by low to moderate levels of myopia with a mean spherical equivalent power of -1.01 diopters (D) for pilots and -1.68 D for others. Contact lenses, and more recently refractive surgery, reduce the number of aircrew that must rely on spectacles when flying; however, spectacle compatibility remains an important consideration in the cockpit.

Purification and protective efficacy of monomeric and modified Yersinia pestis capsular F1-V antigen fusion proteins for vaccination against plague.

The F1-V vaccine antigen, protective against Yersinia pestis, exhibits a strong tendency to multimerize that affects larger-scale manufacture and characterization. In this work, the sole F1-V cysteine was replaced with serine by site-directed mutagenesis for characterization of F1-V non-covalent multimer interactions and protective potency without participation by disulfide-linkages. F1-V and F1-V(C424S) proteins were overexpressed in Escherichia coli, recovered using mechanical lysis/pH-modulation and purified from urea-solubilized soft inclusion bodies, using successive ion-exchange, ceramic hydroxyapatite, and size-exclusion chromatography. This purification method resulted in up to 2mg/g of cell paste of 95% pure, mono-disperse protein having < or =0.5 endotoxin units per mg by a kinetic chromogenic limulus amoebocyte lysate reactivity assay. Both F1-V and F1-V(C424S) were monomeric at pH 10.0 and progressively self-associated as pH conditions decreased to pH 6.0. Solution additives were screened for their ability to inhibit F1-V self-association at pH 6.5. An L-arginine buffer provided the greatest stabilizing effect. Conversion to >500-kDa multimers occurred between pH 6.0 and 5.0. Conditions for efficient F1-V adsorption to the cGMP-compatible alhydrogel adjuvant were optimized. Side-by-side evaluation for protective potency against subcutaneous plague infection in mice was conducted for F1-V(C424S) monomer; cysteine-capped F1-V monomer; cysteine-capped F1-V multimer; and a F1-V standard reported previously. After a two-dose vaccination with 2 x 20 microg of F1-V, respectively, 100%, 80%, 80%, and 70% of injected mice survived a subcutaneous lethal plague challenge with 10(8) LD(50)Y. pestis CO92. Thus, vaccination with F1-V monomer and multimeric forms resulted in significant, and essentially equivalent, protection.

Interactions between HIV-1 Gag molecules in solution: an inositol phosphate-mediated switch.

Retrovirus particle assembly is mediated by the Gag protein. Gag is a multi-domain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) is mixed with nucleic acid, it assembles into virus-like particles (VLPs) in a fully defined system in vitro. However, this assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon the interactions between Gag protein molecules in solution. Our data indicate that basic regions at both ends of the protein contribute to IP6 binding. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, when IP6 is added, Gag is in monomer-trimer rather than monomer-dimer equilibrium. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the "dimer interface" is essential for the trimeric interaction in IP6. We discuss possible explanations for these results, including a change in conformation within the capsid domain induced by the binding of IP6 to other domains within the protein. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in three-dimensional space; direct support for this conclusion is provided in a companion manuscript. As Gag is an extended rod in immature virions, this apparent proximity of the ends in solution implies that it undergoes a major conformational change during particle assembly.

Conformation of the HIV-1 Gag protein in solution.

A single multi-domain viral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. We have purified the human immunodeficiency virus type 1 (HIV-1) Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) from bacteria. This protein is capable of assembly into virus-like particles in a defined in vitro system. We have reported that it is in monomer-dimer equilibrium in solution, and have described a mutant Gag protein that remains monomeric at high concentrations in solution. We report that the mutant protein retains several properties of wild-type Gag. This mutant enabled us to analyze solutions of monomeric protein. Hydrodynamic studies on the mutant protein showed that it is highly asymmetric, with a frictional ratio of 1.66. Small-angle neutron scattering (SANS) experiments confirmed its asymmetry and yielded an R(g) value of 34 A. Atomic-level structures of individual domains within Gag have previously been determined, but these domains are connected in Gag by flexible linkers. We constructed a series of models of the mutant Gag protein based on these domain structures, and tested each model computationally for its agreement with the experimental hydrodynamic and SANS data. The only models consistent with the data were those in which Gag was folded over, with its N-terminal matrix domain near its C-terminal nucleocapsid domain in three-dimensional space. Since Gag is a rod-shaped molecule in the assembled immature virion, these findings imply that Gag undergoes a major conformational change upon virus assembly.

HIV-1 reverse transcriptase structure with RNase H inhibitor dihydroxy benzoyl naphthyl hydrazone bound at a novel site.

The rapid emergence of drug-resistant variants of human immunodeficiency virus, type 1 (HIV-1), has limited the efficacy of anti-acquired immune deficiency syndrome (AIDS) treatments, and new lead compounds that target novel binding sites are needed. We have determined the 3.15 A resolution crystal structure of HIV-1 reverse transcriptase (RT) complexed with dihydroxy benzoyl naphthyl hydrazone (DHBNH), an HIV-1 RT RNase H (RNH) inhibitor (RNHI). DHBNH is effective against a variety of drug-resistant HIV-1 RT mutants. While DHBNH has little effect on most aspects of RT-catalyzed DNA synthesis, at relatively high concentrations it does inhibit the initiation of RNA-primed DNA synthesis. Although primarily an RNHI, DHBNH binds >50 A away from the RNH active site, at a novel site near both the polymerase active site and the non-nucleoside RT inhibitor (NNRTI) binding pocket. When DHBNH binds, both Tyr181 and Tyr188 remain in the conformations seen in unliganded HIV-1 RT. DHBNH interacts with conserved residues (Asp186, Trp229) and has substantial interactions with the backbones of several less well-conserved residues. On the basis of this structure, we designed substituted DHBNH derivatives that interact with the NNRTI-binding pocket. These compounds inhibit both the polymerase and RNH activities of RT.

Identification of glycosylation sites in the SU component of the Avian Sarcoma/Leukosis virus Envelope Glycoprotein (Subgroup A) by mass spectrometry.

We used enzymatic digestion and mass spectrometry to identify the sites of glycosylation on the SU component of the Avian Sarcoma/Leukosis virus (ASLV) Envelope Glycoprotein (Subgroup A). The analysis was done with an SU(A)-rIgG fusion protein that binds the cognate receptor (Tva) specifically. PNGase F removed all the carbohydrate from the SU(A)-rIgG fusion. PNGase F is specific for N-linked carbohydrates; this shows that all the carbohydrate on SU(A) is N-linked. There are 10 modified aspargines in SU(A) (N17, N59, N80, N97, N117, N196, N230, N246, N254, and N330). All conform to the consensus site for N-linked glycosylation NXS/T. There is one potential glycosylation site (N236) that is not modified. Removing most of the carbohydrate from the mature SU(A)-rIgG by PNGase F treatment greatly reduces the ability of the protein to bind Tva, suggesting that carbohydrate may play a direct role in receptor binding.

Remediation of MTBE from drinking water: air stripping followed by off-gas adsorption.

The widespread use of methyl tertiary butyl ether (MTBE) as an oxygenate in gasoline has resulted in the contamination of a large number of ground and surface water sources. Even though air stripping has been proven to be an effective treatment technology for MTBE removal, off-gas treatment often is required in conjunction with it. This study evaluated the combined treatment technologies of air stripping followed by off-gas adsorption on a pilot scale for the treatment of MTBE-contaminated water. The effect of air/water ratios on the treatment efficiency was studied, and the mass transfer coefficient was determined. Air/water ratios of 105:1, 151:1, 177:1, 190:1, 202:1, and 206:1 were used, and a treatment efficiency of >99% was achieved for all the runs conducted. The depth of packing required to achieve maximum treatment efficiency decreased with increasing air/water ratio. Relative humidity (RH) impacts on the MTBE adsorption capacity of a granular activated carbon (GAC) and carbonaceous resin were determined from pilot plant studies. Breakthrough profiles obtained from the pilot plant studies conducted at 20, 30, and 50% RH indicated that GAC has a higher adsorptive capacity than resin. The adsorptive capacity of GAC decreased with increasing RH, whereas RH did not impact the resin adsorptive capacity.

Characterization of the polymerase and RNase H activities of human foamy virus reverse transcriptase.

Foamy virus (FV) replication, while related to that of orthoretroviruses, differs at a number of steps. Several of these differences involve the reverse transcriptase (RT). There appear to be fewer RTs present in FV than in orthoretroviruses; we previously proposed that the polymerase of FV RT was more active than orthoretroviral RTs to compensate for the numerical difference. Here we present further characterization of the RT of FV. The polymerase activity of FV RT was greater than that of human immunodeficiency virus type 1 RT in a variety of assays. We also examined the RNase H activity of FV RT, and we propose that FV RT has a basic loop in the RNase H domain. Although the sequence of the basic loop of FV RT is different from the basic loop of either Moloney leukemia virus RNase H or Escherichia coli RNase H, the FV RT basic loop appears to have a similar function.