3D Printing of Covalent Functionalized Graphene Oxide Nanocomposite via Stereolithography.

A major challenge for many industries wanting to adopt 3D printing technologies for rapid prototyping, customized parts, and low-volume manufacturing depends on the availability and functionality of the input materials to suit specific requirements. A well-studied nanofiller because of its distinct properties and wide range of applications, graphene oxide (GO) proves to be a good choice in the development of new materials. However, as a filler in a polymer matrix, GO has its own unique set of problems enough to make certain constraints in achieving an optimum reinforcement in the targeted polymer matrix. The need for a matrix-filler interaction is critical because reinforcement occurs only when the external load applied to the material can be successfully transmitted from the matrix to the filler, which will only happen if the interfacial adhesion between the matrix and the filler is strong. This study demonstrates the synthesis of the covalently linked GO-methacrylate (MA) nanocomposite materials through 3D printing via stereolithography (SL). Spectral analysis using Fourier-transform infrared confirms the successful functionalization of GO and ascertains the presence of the functionalized GO (fGO) in the 3D-printed nanocomposite specimens. Likewise, further validation using thermogravimetric analysis and differential scanning calorimetry also affirms the formation of fGO for use as a functional filler, activating a stronger interfacial bonding with the MA polymer. Excellent attributes of GO will become futile because of premature fracturing of the material simply because of an oversight to consider robustness during the early stages of design. Hence, different mechanical and thermal properties of the new 3D-printed MA-fGO nanocomposite material are characterized and presented in the discussion. This work demonstrates the first successful 3D printing of the functionalized GO nanocomposite via SL, forming a complex structure with consistently high fidelity and enhanced material properties with potential for various industrial applications.

Plant proton pumping pyrophosphatase: the potential for its pyrophosphate synthesis activity to modulate plant growth.

Cellular pyrophosphate (PPi) homeostasis is vital for normal plant growth and development. Plant proton-pumping pyrophosphatases (H+ -PPases) are enzymes with different tissue-specific functions related to the regulation of PPi homeostasis. Enhanced expression of plant H+ -PPases increases biomass and yield in different crop species. Here, we emphasise emerging studies utilising heterologous expression in yeast and plant vacuole electrophysiology approaches, as well as phylogenetic relationships and structural analysis, to showcase that the H+ -PPases possess a PPi synthesis function. We postulate this synthase activity contributes to modulating and promoting plant growth both in H+ -PPase-engineered crops and in wild-type plants. We propose a model where the PPi synthase activity of H+ -PPases maintains the PPi pool when cells adopt PPi-dependent glycolysis during high energy demands and/or low oxygen environments. We conclude by proposing experiments to further investigate the H+ -PPase-mediated PPi synthase role in plant growth.

18F-FDG PET/contrast enhanced CT in the standard follow-up of patients with lymphoma. / 18F-FDG PET/TC con contraste intravenoso en el seguimiento estandarizado de pacientes con linfoma.

AIM: To assess the diagnostic accuracy of 18F-FDG PET/contrast enhanced computed tomography (ceCT) in the detection of asymptomatic recurrences in patients with lymphoma. MATERIAL AND METHODS: Patients with lymphoma and clinical complete remission underwent 18F-FDG PET/ceCT for standard follow-up.18F-FDG PET and ceCT were evaluated blindly by two independent observers, and classified as positive or negative for recurrence. Additionally a combined evaluation of both techniques was performed. The final diagnosis was established by histopathological analysis or a clinical follow-up longer than 6 months. Statistical diagnostic parameters and concordance levels between both diagnostic techniques were calculated. RESULTS: A total of 114 explorations on 90 patients were analyzed. Only 4 patients were diagnosed as asymptomatic recurrence during the follow-up. 18F-FDG PET/ceCT, 18F-FDG PET and ceCT showed an association with the final diagnosis (p=0.002 and χ2=11.96; p<0.001 and χ2=15.60; p=0.001 and χ2=11.96, respectively). The concordance between 18F-FDG PET and ceCT was moderate/high and significant (kappa=0.672; p<0.001). A sensitivity and specificity of 50% and 88% was obtained for the 18F-FDG PET/ceCT civ, 50% and 93% for the 18F-FDG PET, and 50% and 91% for the ceCT. CONCLUSION: The combined use of 18F-FDG PET/ceCT did not offer any advantage compared to any isolated diagnostic technique in the detection of asymptomatic lymphoma recurrence.

Projected Impact of Climate Change on Hydrological Regimes in the Philippines.

The Philippines is one of the most vulnerable countries in the world to the potential impacts of climate change. To fully understand these potential impacts, especially on future hydrological regimes and water resources (2010-2050), 24 river basins located in the major agricultural provinces throughout the Philippines were assessed. Calibrated using existing historical interpolated climate data, the STREAM model was used to assess future river flows derived from three global climate models (BCM2, CNCM3 and MPEH5) under two plausible scenarios (A1B and A2) and then compared with baseline scenarios (20th century). Results predict a general increase in water availability for most parts of the country. For the A1B scenario, CNCM3 and MPEH5 models predict an overall increase in river flows and river flow variability for most basins, with higher flow magnitudes and flow variability, while an increase in peak flow return periods is predicted for the middle and southern parts of the country during the wet season. However, in the north, the prognosis is for an increase in peak flow return periods for both wet and dry seasons. These findings suggest a general increase in water availability for agriculture, however, there is also the increased threat of flooding and enhanced soil erosion throughout the country.

Basal (18)F-FDG PET/CT in follicular lymphoma: A comparison of metabolic and clinical variables in the prognostic assessment.

AIM: To analyze the relationship of clinical variables related to prognosis and tumor burden, with metabolic variables obtained in the staging (18)F-FDG PET/CT, and their value in the prognosis in follicular lymphoma (FL). METHODS: 82 patients with FL, a (18)F-FDG PET/CT at diagnosis and a follow-up for a minimum of 12 months, were retrospectively enrolled in the present study. Clinical variables (Tumor grade, Follicular Lymphoma International Prognostic Index (FLIPI) and Tumor burden) were evaluated. Metabolic variables such as SUVmax in the highest hypermetabolic lesion, extralymphatic locations, number of involved lymph node locations, bone marrow (BM) involvement, PET stage and diameter of the biggest hypermetabolic lesion, were analyzed in order to establish a PET score and classify the studies in low, intermediate and high metabolic risk. Clinical and metabolic variables (included metabolic risk) were compared. The relation among all variables and disease-free survival (DFS) was studied. RESULTS: The 28% of patients had a high-grade tumor. The 30.5% had FLIPI risk low, 29.3% intermediate y 40.2% high. The 42.7% presented a high tumor burden. The PET/CT was positive in 94% of patients. The tumor grade did not show significant relation with metabolic variable. FLIPI risk and tumor burden showed statistical relations with the SUV max and the PET score (p<0.008 and p=0.003 respectively). With respect to DFS, significant differences were detected for the PET stage and FLIPI risk (p=0.015 and p=0.047 respectively). FLIPI risk was the only significant predictor in Cox regression analysis, with a Hazard Ratio of 5.13 between high risk and low risk. CONCLUSION: The present research highlights the significant relation between metabolic variables obtained with FDG PET/CT and clinical variables although their goal as an independent factor of prognosis was not demonstrated in the present work.

[Quantitative and qualitative evaluation of the interim PET/CT in lymphoma treatment in the prediction of complete metabolic response]. / Evaluación cuantitativa y cualitativa de la PET/TC a mitad del tratamiento en linfomas en la predicción de respuesta metabólica completa.

OBJECTIVE: To compare two different methods for the interpretation of interim PET/CT (PET/CT-i) in lymphomas, and to establish which one best predicts a complete metabolic response (CMR) in the PET/CT study at the end of treatment (PET/CT-et). MATERIAL AND METHODS: Retrospective longitudinal analysis of the PET/CT studies for staging (PET/CT-s), PET/CT-i and PET/CT-et of 65 patients, 35 Hodgkin's lymphoma (HL) and 30 Non-HL. The PET/CT-i was performed between the second and fourth chemotherapy cycle. It was interpreted using two different criteria: qualitative criteria (5 point visual scale), semiquantitative criteria (percentage difference between the lesion with more SUVmax in the PET/CT-s and PET/CT-i). We analyzed the likelihood of obtaining a CMR in the PET/CT-et according to the results obtained on the PET/CT-i with these two criteria. RESULTS: We obtained sensitivity (S), specificity (Sp), positive predictive values (PPV), negative predictive values (NPV) and likelihood ratio (LR) for the qualitative/semiquantitative method of 91%/80%, 76.2%/67%, 88.9%/83.3%, 80%/60.9% and 32%/7.8%, respectively, to predict a CMR in the PET/CT-et. There were no statistically significant differences between the LR of both methods (p=0.1942). CONCLUSION: We found clear differences in S, Sp, PPV and NPV between both interpretation criteria for the PET/CT-i to predict a CMR in the PET/CT-et. Nevertheless, we cannot confirm the superiority of the qualitative method over the semiqualitative method for this purpose as no statistically significance differences were found in their LR in our study.

18F-FDG-PET/CT in the surveillance of patients with lymphoma: detection of asymptomatic recurrences.

AIM: To assess the diagnostic accuracy of (18)F-FDG-PET/CT in detecting asymptomatic recurrences in patients with lymphoma. To define uptake patterns of recurrence indicative of recurrence. MATERIAL AND METHODS: Those patients with lymphoma who fulfilled the following inclusion criteria of clinical complete remission and negative PET/CT study were included retrospectively and longitudinally. Conventional surveillance of these patients was performed only by (18)F-FDG PET/CT following a standardized procedure. Pathologic locations (supra- and infradiaphragmatic) and their character (single or multiple) were analyzed in order to determine reliable metabolic patterns of recurrence. The final diagnosis was established by histopathological analysis or clinical follow-up greater than 8 months. RESULTS: A total of 199 explorations belonging to 106 patients with lymphoma were included. Of these patients, 59 had Hodgkin's lymphoma and 47 non-Hodgkin's lymphoma. There was suspicion of relapse from the metabolic point of view in 27 of the PET/CT scans. Of these, 14 (10 patients) were false positive (FP), and 13 (8 patients) true positive. The remaining studies were true negative, no false negatives being detected. The pattern most frequently related to recurrence was infradiaphragmatic lymph node involvement while most of the FP had isolated supradiaphragmatic involvement. Sensitivity, specificity, PPV, NPV and diagnostic accuracy of PET/CT parameters for the study were 100%, 92%, 48%, 100% and 93%, respectively. CONCLUSION: (18)F-FDG-PET/CT is a sensitive technique in the detection of asymptomatic recurrences in patients with lymphoma during their follow-up. Multiple character and infradiaphragmatic locations were the patterns that best correlated to the diagnosis of recurrence.

Oligomerization paths of the nucleoprotein of influenza A virus.

The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP) and is associated with the polymerase complex into ribonucleoprotein (RNP) particles. Despite its importance in the virus life cycle, the interactions between the NP and the genome are not well understood. Here, we studied the assembly process of NP-RNA oligomers and analyzed how the oligomeric/monomeric status of RNA-free NP affects RNA binding and oligomerization. Recombinant wild-type NP purified in low salt concentrations and a derived mutant engineered for oligomerization deficiency (R416A) were mainly monomeric in RNA-free solutions as shown by biochemical and electron microscopy techniques. NP monomer formed with RNA a fast 1/1 complex characterized by surface plasmon resonance. In a subsequent and slow process that depended on the RNA length, oligomerization of NP was mediated by RNA binding. In contrast, preparations of wild-type NP purified in high salt concentrations as well as mutant Y148A engineered for deficiency in nucleic acid binding were partly or totally oligomeric in RNA-free solutions. These trimer/tetramer NP oligomers bind directly as oligomers to RNA with a higher affinity than that of the monomers. Both oligomerization routes we characterized could be exploited by cellular or viral factors to modulate or control viral RNA encapsidation by NP.

[PET-CT with intravenous contrast in the evaluation of patients with lymphoma. Contribution to diagnostic indications]. / PET-TAC con contraste intravenoso en la valoración de pacientes con linfoma. Aproximación hacia las indicaciones diagnósticas.

AIM: To define the utility of intravenous contrast administration in the PET-CT (PET-CTc) in patients with lymphoma in order to determine its possible indications. MATERIAL AND METHODS: 78 patients with lymphoma were prospectively evaluated. All underwent simultaneous PET-CTc scans in a hybrid system for staging (8), evaluation of response to treatment (29), suspicion of recurrence (9) and complete remission control (48). The PET scan was acquired by a conventional method and the diagnostic CT scan was performed according to radiological protocol. Both examinations were evaluated blinded and independently, analyzing 28 anatomical locations in order to determine the degree of agreement. Final diagnosis was established by the clinician based on the histological study, results of other diagnostic techniques or clinical follow-up. RESULTS: The final result of both techniques were concordant in 87/94 studies (92.5%). A total of 158 (36 FP) pathological locations were detected with PET-CT and 189 (71 FP) with CTc, with 72 locations being discordant between both techniques. Global sensitivity, specificity, PPV and NPV were 93%, 98%, 77% and 99%; and 94%, 97%, 62% and 99%, respectively. CONCLUSIONS: Administration of intravenous contrast does not seem to provide any advantage in the determination of nodal and extranodal disease in lymphoma patients. The low prevalence of disease probably accounts for the limited PPV of both techniques. An increase of our sample size, with a greater homogeneity of the groups, should offer more reliable results.

Error growth patterns in systems with spatial chaos: from coupled map lattices to global weather models.

Error growth in spatiotemporal chaotic systems is investigated by analyzing the interplay between temporal and spatial dynamics. The spatial correlation and localization of relative fluctuations grow and decay indicating two different regimes, before and after saturation by nonlinear effects. This general behavior is shown to hold both in simple coupled map lattices and in global weather models. This explains the increasing or decreasing trends previously observed in the exponential growth rate of these spatiotemporal systems.

Establishing the impact of acid mine drainage through metal bioaccumulation and taxa richness of benthic insects in a tropical Asian stream (The Philippines).

The Boac-Makulapnit River (The Philippines) is contaminated with heavy metals primarily due to acid mine drainage (AMD). Heavy-metal levels in river water, sediment, and algae suggest that Cu is the primary contaminant in this system. Copper concentrations decrease longitudinally, indicating a contamination gradient away from the mine. This is also reflected in the whole-body metal concentration of the caddisfly Hydropsyche, wherein higher Cu levels are observed in upstream stations (677-980 microg/g in 2001). In 2002, AMD formation was observed in a midstream station resulting from the oxidation of mine tailings in floodplains deposited during a 1996 tailings spill event. This consequently increased heavy-metal concentrations locally in environmental media and is likewise perceived to have caused the absence of aquatic insects in the midstream reach. Copper concentrations also increased in Hydropsyche found downstream of the new AMD site during this year. Furthermore, even the lower Cu concentrations in Hydropsyche observed in downstream stations are still at least six times higher than the established regional baseline value (29.9 microg/g), which is calculated from samples from clean tributary streams. These tributary streams also showed higher taxa richness for aquatic insect community in riffle zones. Both biomonitoring tools suggest that the whole length of the river is severely impacted. Rehabilitation projects will be underway next year; therefore, such monitoring programs will truly be beneficial when gauging river habitat recovery.

Modulation of RNA function by oligonucleotides recognizing RNA structure.

Numerous RNA structures are responsible for regulatory processes either because they constitute a signal, like the hairpins or pseudoknots involved in ribosomal frameshifting, or because they are binding sites for proteins such as the trans-activating responsive RNA element of the human immunodeficiency virus whose binding to the viral protein Tat and cellular proteins allows full-length transcription of the retroviral genome. Selective ligands able to bind with high affinity to such RNA motifs may serve as tools for dissecting the molecular mechanisms in which they are involved. Such ligands might also constitute prototypes of therapeutic agents when RNA structures play a role in the expression of dysfunctional genes or in the multiplication of pathogens. Different classes of ligands (aminoglycosides, interacalating agents, peptides) are of interest to this aim. However, oligonucleotides deserve particular consideration. They have been extensively used in the frame of the antisense strategy. The apparent simplicity of this rational approach is, at first sight, very attractive. Indeed, numerous successful studies have been published describing the efficient inhibition of translation, splicing, or reverse transcription in cell-free systems, in cultured cells, or in vivo by oligomers complementary to an RNA region. However, RNA structures restrict the access of the target site to the antisense sequence: The competition between the intramolecular association of RNA regions weakens or even abolishes the antisense effect. Various possibilities have been developed to circumvent this limitation. This includes both rational and combinatorial strategies. High-affinity oligomers were designed to invade the RNA structure. Alternatively, triplex-forming oligonucleotides (TFO) and aptamers may recognize the folded RNA motif. Whereas the use of TFOs is rather limited owing to the strong sequence constraints for triple-helix formation, in vitro selection offers a way to explore vast oligoribo or oligodeoxyribo libraries to identify strong, selective oligonucleotide binders. The candidates (aptamers) selected against the TAR RNA element of HIV-1, which form stable loop-loop (kissing) complexes with the target, provide interesting examples of oligonucleotides recognizing a functional RNA structure through an important contribution of tertiary interactions.

RNA and N3'-->P5' kissing aptamers targeted to the trans-activation responsive (TAR) RNA of the human immunodeficiency virus-1.

We used in vitro selection to identify RNA aptamers able to selectively bind to the TAR RNA motif of HIV-1, an unperfect RNA hairpin involved in the transcription of the retroviral genome. We selected aptameric RNA hairpins giving rise to kissing complexes with TAR. The N3'-->P5' phosphoramidate variant of the aptamer bind to TAR with a Kd in the low nanomolar range. However, only the RNA-RNA loop-loop complex is recognized by the Rop protein of E. coli which is specific for kissing complexes.

Is a closing "GA pair" a rule for stable loop-loop RNA complexes?

RNA hairpin aptamers specific for the trans-activation-responsive (TAR) RNA element of human immunodeficiency virus type 1 were identified by in vitro selection (Ducongé, F., and Toulmé, J. J. (1999) RNA 5, 1605-1614). The high affinity sequences selected at physiological magnesium concentration (3 mm) were shown to form a loop-loop complex with the targeted TAR RNA. The stability of this complex depends on the aptamer loop closing "GA pair" as characterized by preliminary electrophoretic mobility shift assays. Thermal denaturation monitored by UV-absorption spectroscopy and binding kinetics determined by surface plasmon resonance show that the GA pair is crucial for the formation of the TAR-RNA aptamer complex. Both thermal denaturation and surface plasmon resonance experiments show that any other "pairs" leads to complexes whose stability decreases in the order AG > GG > GU > AA > GC > UA >> CA, CU. The binding kinetics indicate that stability is controlled by the off-rate rather than by the on-rate. Comparison with the complex formed with the TAR* hairpin, a rationally designed TAR RNA ligand (Chang, K. Y., and Tinoco, I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8705-8709), demonstrates that the GA pair is a key determinant which accounts for the 50-fold increased stability of the TAR-aptamer complex (K(d) = 2.0 nm) over the TAR-TAR* one (K(d) = 92. 5 nm) at physiological concentration of magnesium. Replacement of the wild-type GC pair next to the loop of RNA I' by a GA pair stabilizes the RNA I'-RNA II' loop-loop complex derived from the one involved in the control of the ColE1 plasmid replication. Thus, the GA pair might be the preferred one for stable loop-loop interactions.

Maintaining abstinence from cigarette smoking: effectiveness of group counselling and factors predicting outcome.

The aim of the study was to assess the effectiveness of worksite group counselling interventions designed to prevent smoking relapse after abstinence has been achieved following 3 months therapy using group support and/or transdermal nicotine replacement therapy. After 3 months, abstinent subjects were randomly allocated either to a counselling group led by professional psychologists (PG), to a counselling group led by former smokers (SG) or to no intervention group (NG). The 3 and 12 months abstinence were defined, respectively, as a sustained smoking cessation during the last month, and the last 9 months. Complete abstinence was confirmed by expired carbon monoxide and by urine cotinine concentrations. The abstinence rate at 3 months was 35.1%. After 12 months abstinence rates were not statistically different in the PG, the SG and the NG (respectively 57.8, 53.4 and 49.6% of those randomised). In multivariate analyzes, baseline variables associated with 12 months abstinence were non-smoking family, gender (male), lower daily intake of nicotine and better psychological adjustment. Mean weight gain at 3 months in abstinent versus relapsed subjects, was respectively, 4.1 and 2.4 kg. Baseline variables associated with weight gain at 3 months were higher Fagerström score, gender (male) and professional status (blue collar worker). Group support after abstinence has been achieved did not significantly improve the abstinence. This study shows the difficulty of preventing smoking relapse with monthly group counselling. The results indicate the need to investigate further specific programmes focusing on factors such as gender, family, nicotine dependence, psychological and weight concerns/issues which may precipitate relapse.

[The decision to breastfeed: grandmother's view] / A decisão de amamentar da nutriz: percepção de sua mãe.

OBJECTIVE: This study is the result of an inquiry into the grandmother's role in the decision as well as the duration of her daughter's choice to breast-feed. METHOD: This project uses a phenomenological theoretical background which leads one to understand breast-feeding based on the view of the grandmother. Thus, this project analyzes the discourse of 10 women by using the following guiding questions: Howdid you feed your children when they were born? What did you think about your daughter's choice to breast-feed? Why do you think your daughter decided to breast-feed? RESULTS: The analysis of the discourses as a whole reveals that for these women breast-feeding is something intrinsical to a mother's role as well as an experience transferred from mothers to daughters. This project also reveals that breast-feeding is a family tradition which can be influenced by advice and support. CONCLUSION: From this perspective, a new focus is revealed by giving advice, supporting and encouraging breast-feeding.

Photoacoustic calorimetry of proteins.

We have described two examples of time-resolved photoacoustic calorimetry for the study of heme protein transient intermediates. Before photoacoustic calorimetry, determining thermodynamic information on short-lived intermediates was difficult. Along with being sensitive to enthalpic and volume changes, photoacoustic calorimetry can detect conformational changes in a time-resolved manner. In complex protein systems, the interpretation of the structural origins of a conformational change is sometimes difficult. Site-directed mutagenesis has been used successfully to identify the residues that play important roles in the ligand binding to both Mb and cytochrome P450cam. In both systems the hydration state of salt bridges gave rise to volume changes that were identified through mutagenesis of the residues involved. With its increasing popularity and the power of site-directed mutagenesis, time-resolved photoacoustic calorimetry is fast becoming a technique to probe conformational dynamics in proteins.

Improving cancer prevention at the worksite: how are Belgian companies dealing with smoking regulations?

Little is known about the impact of smoking policy on companies' policies related to smoking in Belgium. The study was designed in order to compare the companies' policies related to smoking (CPRS) in 1990 and in 1993. During this 3 year period, numerous information and regulation inputs (media, brochures) were aimed at companies to regulate smoking behavior in the workplace, so as to reduce the risks of passive smoking. A short questionnaire was mailed to 3543 Belgian companies in October 1990 and in June 1993 to assess: designation of smoke free areas (SFA); willingness to offer a worksite information program (WIP); willingness to offer a worksite smoking cessation program (WSCP); willingness to subsidize a WSCP; willingness to offer a WSCP during working hours; willingness to offer a meeting room for a WSCP and actual organization of a WSCP. In 1990, 773 companies (22%) and in 1993, 890 companies (25%) responded to the questionnaire. The results showed that small companies and companies with a high blue/white collar ratio were less able to implement health policy recommendations. A total of 325 (9%) companies responded to the 1990 and 1993 questionnaires. Comparison of 1990 and 1993 dataset showed that during this 3 year period, no major changes occurred for a majority of companies. Amongst the small positive changes, only the designation of smoke free areas and the organization of a small number of WSCP was observed. The conclusion is that despite inputs, little impact on smoking policies is noticeable. Strategies designed to intensify inputs and to promote implementation of regulation of WSCP should therefore further be studied.

Dynamics of carbon monoxide binding with cytochromes P-450.

The dynamics of CO rebinding with cytochromes P-450cam, P-450scc, and P-450LM2 after laser flash photolysis have been investigated from 293 to 77 K, and the distribution functions of the rate parameters P(k) and of the activation enthalpy P(H) were determined using the maximum entropy method. In a fluid solvent, geminate rebinding is nonexponential, presumably because of a spectral shift induced by protein relaxation on the same time scale. Substrate binding increases the yield of the bimolecular process and decreases the bimolecular rate by 1 or 2 orders of magnitude. The amplitude of these effects seems to correlate with substrate specificity. In a rigid environment at low temperature, cytochromes P-450 exhibit a bimodal distribution of activation enthalpy; P(H) consists of two distinct bands which are in a thermal equilibrium even at 77 K. The results lead to a scheme in which a common structural perturbation splits the conformational substates of cytochromes P-450 into pairs of "doublet" substates with different dynamic properties. The hierarchy of conformational substates of cytochromes P-450 thus contrasts with that of oxygen-binding hemoproteins such as myoglobin.