The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides

Protoporphyrinogen oxidase (EC 1–3-3–4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the βαβ ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the K_m for protoporphyrinogen and the K_i for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains.     

Evaluación multimodal de la degeneración estructural de válvulas percutáneas en el seguimiento a largo plazo

Introduction and objectivesWe assessed the long-term hemodynamic performance of transcatheter heart valve (THV) by paired transthoracic echocardiography (TTE), and the incidence, characteristics and factors associated with THV structural valve degeneration (SVD).MethodsA total of 212 patients who underwent transcatheter aortic valve replacement and had a potential follow-up > 5 years with at least 1 TTE ≥ 1-year postprocedure were included. All patients had a TTE at 1 to 5 years and 36 had another one at 6 to 10 years. SVD was defined as subclinical (increase > 10 mmHg in mean transvalvular gradient +  decrease > 0.3 cm² in valve area and/or new-onset mild or moderate aortic regurgitation) and clinically relevant (increase > 20 mmHg in mean transvalvular gradient + decrease > 0.6 cm² in valve area and/or new-onset moderate-to-severe aortic regurgitation). Fifteen patients had a transesophageal echocardiography at the time of SVD diagnosis, and 85 an opportunistic computed tomography examination at 1 (0.5-2) years.ResultsTransvalvular mean gradient increased and valve area decreased over time (P < .01). At 8 years of follow-up, SVD occurred in 30.2% of patients (clinically relevant: 9.3%). Transesophageal echocardiography revealed thickened and reduced-mobility leaflets in 80% and 73% of SVD cases, respectively. No baseline or procedural factors were associated with SVD. THV underexpansion (3.5%) or eccentricity (8.2%) had no impact on valve hemodynamics/SVD at follow-up.ConclusionsA gradual THV hemodynamic deterioration occurred throughout a 10-year period, leading to SVD in ～30% of patients (clinically relevant in < 10%). Leaflet morphology/mobility were frequently impaired in SVD cases, but THV geometry did not influence valve hemodynamics or SVD.     

Genome-based design of a cell-free culture medium for Tropheryma whipplei

Empirical approaches have guided the development of bacterial cultures. The availability of sequenced genomes now provides opportunities to define culture media for growth of fastidious pathogens with computer modelling of metabolic networks. A key issue is the possibility of growing host-dependent bacteria in cell-free conditions. The sequenced Tropheryma whipplei genome was analysed to identify specific metabolic deficiencies. We used this information to design a comprehensive medium that allowed three established T whipplei strains from culture with human cells and one new strain from a clinical sample to grow axenically. Genomic information can, therefore, provide sufficient clues for designing axenic media for fastidious and uncultured pathogens.     

Benefit of treatment interruption in HIV-infected patients with multiple therapeutic failures: a randomized controlled trial (ANRS 097)

BACKGROUND: Both highly potent antiretroviral drug rescue therapy and treatment interruption have been suggested to be effective in patients with multiple treatment failure. OBJECTIVE: To assess both the benefits and risks of an 8-week treatment interruption associated with a six to nine-drug rescue regimen in patients with multiple treatment failures. DESIGN: A randomized comparative controlled trial in 19 university hospitals in France. PATIENTS: Sixty-eight HIV-infected patients with multiple previous treatment failures and CD4 cell counts less than 200 x 10(6) cells/l and plasma HIV-1-RNA levels of 50,000 copies/ml or greater. MEASUREMENTS: The primary efficacy outcome was the proportion of patients with at least a 1 log10 decrease (copies/ml) in the plasma HIV-1-RNA level after 12 weeks of therapy. RESULTS: Treatment interruption followed by multidrug salvage therapy led to a greater proportion of patients achieving virological success (i.e. 1 log10 decrease) at 12 weeks compared with patients receiving multidrug therapy alone (62 versus 26%, intent-to-treat analysis; P = 0.007). The median decrease in the HIV-1-RNA level was -1.91 and -0.37 log10 copies/ml (P = 0.008), respectively. Treatment interruption led to an increase in the number of sensitive drugs of the multidrug regimen (71 versus 35% of regimen with at least two sensitive drugs; P = 0.004). Factors associated with virological success were treatment interruption, the reversion of at least one mutation to wild type, adequate plasma drug concentration, and the use of lopinavir. CONCLUSION: Treatment interruption was beneficial for treatment-experienced HIV-infected patients with advanced HIV disease and multidrug-resistant virus.     

MEG and EEG Sensitivity in a Case of Medial Occipital Epilepsy

Interictal or ictal events in partial epilepsies may project on scalp EEG contralaterally to the side of the epileptogenic lesion. Such paradoxical lateralization can be observed in case of para-sagittal generators, and is likely due to the spatial orientation of the generator, presenting an oblique projection towards the midline. We present here a case of medial occipital epilepsy investigated using EEG, MEG and stereoelectroencephalography (SEEG). MRI displayed a focal cortical dysplasia in the superior margin of the right calcarine fissure. SEEG demonstrated bilateral medial occipital interictal spikes, with an inversion of polarity at the level of the lesion and a contralateral propagation occurring in 10 ms. Interictal iterative EEG cartographies showed a large posterior field, with a maximum contralateral to the initial generator (EEG paradoxical lateralization). With the same number of channels, interictal iterative MEG cartographies were more precise and more complex than EEG ones, indicating an onset accurately lateralized. A few milliseconds later, MEG cartographies were quadripolar, thus indicating two homotopic active generators. These MEG and EEG cartographies have been reproduced using BESA dipole simulator. Relative merits of MEG and EEG are still debated. With 151 channels, MEG source localizations indicated the right medial occipital area, as demonstrated by SEEG. An investigation with a corresponding number of EEG channels was not performed. After a down sampling to 64 sensors, this precision was lost. MEG and EEG source localization results, both with 64 channels, were quite comparable, indicating both medial occipital areas. However, a careful analysis of MEG/EEG iterative cartographies, performed with the same number of channels in both modalities, demonstrated that, in this configuration, MEG sensitivity was superior to the EEG one, allowing separating two medial occipital sources, characterized in SEEG by a time delay of 10 ms.     

Pre-Existing Cross-Reactive Antibodies to Avian Influenza H5N1 and 2009 Pandemic H1N1 in US Military Personnel

We studied cross-reactive antibodies against avian influenza H5N1 and 2009 pandemic (p) H1N1 in 200 serum samples from US military personnel collected before the H1N1 pandemic. Assays used to measure antibodies against viral proteins involved in protection included a hemagglutination inhibition (HI) assay and a neuraminidase inhibition (NI) assay. Viral neutralization by antibodies against avian influenza H5N1 and 2009 pH1N1 was assessed by influenza (H5) pseudotyped lentiviral particle-based and H1N1 microneutralization assays. Some US military personnel had cross-neutralizing antibodies against H5N1 (14%) and 2009 pH1N1 (16.5%). The odds of having cross-neutralizing antibodies against 2009 pH1N1 were 4.4 times higher in subjects receiving more than five inactivated whole influenza virus vaccinations than those subjects with no record of vaccination. Although unclear if the result of prior vaccination or disease exposure, these pre-existing antibodies may prevent or reduce disease severity.Outbreaks of 1997 avian influenza H5N1 and 2009 pandemic (p) H1N1 in humans have provided an opportunity to gain insight into cross-reactive immunity. The US military periodically collects and stores serum samples from service members linked to medical records.¹ We measured cross-reactive antibodies in stored serum to avian influenza H5N1 and 2009 pH1N1 from US military personnel and identified factors associated with presence of neutralizing antibodies.Two hundred archived serum samples were obtained from the US Department of Defense Serum Repository. They were representative of a wide cross-section of active military personnel at the times of collection, whereas specific geographic information was not available on the individual selected; the cohort represents the general US military population, which is deployed throughout the United States and globally. Fifty samples each were selected from four birth cohorts: (1) < 1949, (2) 1960–1965, (3) 1966–1971, and (4) 1972–1977. Within each cohort, 25 samples were collected in the year 2000 (before the introduction of intranasal live attenuated influenza vaccine [LAIV]), and 25 samples were collected in 2008 (where 51% of donors had received LAIV). It has been suggested that LAIV elicits cross-reactive immunity.^2,3 The samples were all collected before the outbreak of 2009 pH1N1, and there have not been any reported outbreaks of H5N1 in US military personnel.Assays used to measure antibodies included a hemagglutination inhibition (HI) assay and a neuraminidase inhibition (NI) assay.⁴ Viral neutralization by antibodies against H5N1 and 2009 pH1N1 was assessed by influenza (H5) pseudotyped lentiviral particle-based (H5pp)⁵ and microneutralization assays, respectively. Electronic medical and vaccination records from the Defense Medical Surveillance System (DMSS), which captured records before the serum sample date, were linked to samples and compared with the in vitro results.¹The odds ratios (ORs) and 95% confidence intervals (95% CIs) of univariate and multivariate binary logistic regression analyses were used to determine the association between donor characteristics and positive antibody responses. A multiple logistic regression model was constructed, and it included independent variables with a P value of < 0.05 in univariate logistic regression. A P value of < 0.05 was considered to indicate statistical significance. SPSS 12.0 for Windows (SPSS Inc., Chicago, IL) was used to perform all statistical analysis.Cross-reactivity is summarized in 5 and 22.5% for the NI assay. H5pp and NI antibody titers to H5N1 were evenly distributed among birth cohorts and did not differ substantially based on history of vaccination or prior respiratory infections. Of those individuals with neutralizing antibodies to H5N1 (N = 28), 32.1% also had neutralizing antibodies to pH1N1, whereas 19.3% of those individuals with any H5N1-specific antibody response also had neutralizing antibodies to pH1N1 (

Microparticle bearing tissue factor: A link between promyelocytic cells and hypercoagulable state

Patients with hematological malignancies have a 28-fold increased risk of venous thromboembolism (VTE). Among patients with acute myelogenous leukemia (AML), the 2-year cumulative incidence of VTE is 5.2%. Several studies suggest that microvesicles (MVs) harboring TF may play a role in VTE and disseminated intravascular coagulation (DIC) in acute promyelocytic leukemia (APL).