Conformational States of Cytochrome P450 Oxidoreductase Evaluated by Förster Resonance Energy Transfer Using Ultrafast Transient Absorption Spectroscopy

NADPH-cytochrome P450 oxidoreductase (CYPOR) was shown to undergo large conformational rearrangements in its functional cycle. Using a new Förster resonance energy transfer (FRET) approach based on femtosecond transient absorption spectroscopy (TA), we determined the donor–acceptor distance distribution in the reduced and oxidized states of CYPOR. The unmatched time resolution of TA allowed the quantitative assessment of the donor–acceptor FRET, indicating that CYPOR assumes a closed conformation in both reduced and oxidized states in the absence of the redox partner. The described ultrafast TA measurements of FRET with readily available red–infrared fluorescent labels open new opportunities for structural studies in chromophore-rich proteins and their complexes

Monitoring of deforestation events in the tropics using multidimensional features of Sentinel 1 radar data

Many countries and regions are currently developing new forest strategies to better address the challenges facing forest ecosystems. Timely and accurate monitoring of deforestation events is necessary to guide tropical forest management activities. Synthetic aperture radar (SAR) is less susceptible to weather conditions and plays an important role in high-frequency monitoring in cloudy regions. Currently, most SAR image-based deforestation identification uses manually supervised methods, which rely on high quality and sufficient samples. In this study, we aim to explore radar features that are sensitive to deforestation, focusing on developing a method (named 3DC) to automatically extract deforestation events using radar multidimensional features. First, we analyzed the effectiveness of radar backscatter intensity (BI), vegetation index (VI), and polarization feature (PF) in distinguishing deforestation areas from the background environment. Second, we selected the best-performing radar features to construct a multidimensional feature space model and used an unsupervised K-mean clustering method to identify deforestation areas. Finally, qualitative and quantitative methods were used to validate the performance of the proposed method. The results in Paraguay, Brazil, and Mexico showed that (1) the overall accuracy (OA) and F1 score (F1) of 3DC were 88.1–98.3% and 90.2–98.5%, respectively. (2) 3DC achieved similar accuracy to supervised methods without the need for samples. (3) 3DC matched well with Global Forest Change (GFC) maps and provided more detailed spatial information. Furthermore, we applied the 3DC to deforestation mapping in Paraguay and found that deforestation events occurred mainly in the second half of the year. To conclude, 3DC is a simple and efficient method for monitoring tropical deforestation events, which is expected to serve the restoration of forests after deforestation. This study is also valuable for the development and implementation of forest management policies in the tropics

Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency

Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro, but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9. Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunction

A Novel Optical Path for Enhancing the Performance of High-Power Semiconductor Laser in Packaging

High-power semiconductor laser has more significant applications in long distance and high-reliability optical communication systems. It is noticed that the packaging plays an important role in the performance of high-power semiconductor laser, including high output power, high coupling efficiency, low relative intensity noise (RIN), and so on. Usually, in a symmetrical optical path, the light, which incident directly parallel to the optical axis of the lens, will be partially reflected back to the laser and cause noise. To solve this problem, a novel asymmetric optical path is designed and implemented to meet the requirement of using a high-power laser with low RIN in this work. By employing an isolator between the first and second lens, the laser with high beam quality and low reflection of the signal was achieved. Moreover, the optical focal length of the collimating lens and the angle of the inclined lens were optimized by simulation. The proposed laser exhibited high coupling efficiency with a RIN of −168.89 dB/Hz. According to theoretical and experimental analysis, the performance of the laser will be helpful in fabricating a high-power laser with low RIN for next-generation optical communication

Structural and Kinetic Studies of Asp632 Mutants and Fully Reduced NADPH-Cytochrome P450 Oxidoreductase Define the Role of Asp632 Loop Dynamics in the Control of NADPH Binding and Hydride Transfer

Conformational changes in NADPH-cytochrome P450 oxidoreductase (CYPOR) associated with electron transfer from NADPH to electron acceptors via FAD and FMN have been investigated via structural studies of the four-electron-reduced NADP⁺-bound enzyme and kinetic and structural studies of mutants that affect the conformation of the mobile Gly631–Asn635 loop (Asp632 loop). The structure of four-electron-reduced, NADP⁺-bound wild type CYPOR shows the plane of the nicotinamide ring positioned perpendicular to the FAD isoalloxazine with its carboxamide group forming H-bonds with N1 of the flavin ring and the Thr535 hydroxyl group. In the reduced enzyme, the C8–C8 atoms of the two flavin rings are ∼1 Å closer than in the fully oxidized and one-electron-reduced structures, which suggests that flavin reduction facilitates interflavin electron transfer. Structural and kinetic studies of mutants Asp632Ala, Asp632Phe, Asp632Asn, and Asp632Glu demonstrate that the carboxyl group of Asp632 is important for stabilizing the Asp632 loop in a retracted position that is required for the binding of the NADPH ribityl-nicotinamide in a hydride-transfer-competent conformation. Structures of the mutants and reduced wild type CYPOR permit us to identify a possible pathway for NADP­(H) binding to and release from CYPOR. Asp632 mutants unable to form stable H-bonds with the backbone amides of Arg634, Asn635, and Met636 exhibit decreased catalytic activity and severely impaired hydride transfer from NADPH to FAD, but leave interflavin electron transfer intact. Intriguingly, the Arg634Ala mutation slightly increases the cytochrome P450 2B4 activity. We propose that Asp632 loop movement, in addition to facilitating NADP­(H) binding and release, participates in domain movements modulating interflavin electron transfer

Noninvasive diagnosis of significant liver inflammation in patients with chronic hepatitis B in the indeterminate phase

ABSTRACTThe presence of significant liver inflammation is an important indication for antiviral treatment in patients with chronic hepatitis B (CHB) in the indeterminate phase. We aimed to establish a non-invasive nomogram to predict significant liver inflammation in these patients. A total of 195 CHB patients in the indeterminate phase were randomly split into training and validation sets. The least absolute shrinkage and selection operator and logistic regression were applied to identify risk factors and establish a predictive model. A calibration curve, decision curve analysis (DCA), and receiver operating characteristic (ROC) curve were applied to assess the performance of the nomogram. The median age was 42.0 y and 59.5% of the patients were male. Alkaline phosphatase, γ-glutamyl transpeptidase, and prothrombin time were independent predictors for significant liver inflammation and selected to establish the AGP-nomogram. The calibration plot demonstrated that the predicted results matched the actual values. The DCA showed a high net benefit when the threshold probability was 25-83% in the training set and 31-100% in the validation set. The areas under ROC curves of AGP-nomogram in predicting significant inflammation were significantly higher than ALT in the training set (0.744 vs. 0.642, P = 0.049) and validation set (0.766 vs. 0.660, P = 0.047). The ability of AGP-nomogram in predicting advanced inflammation was also superior to ALT. The AGP-nomogram can accurately identify significant inflammation in CHB patients in the indeterminate phase, and its application may reduce the need for liver biopsy and help identify candidates for antiviral treatment.Abbreviations: AASLD: American Association for the Study of Liver Diseases; ALB: albumin; ALP: alkaline phosphatase; ALT: alanine aminotransferase; APRI: aspartate aminotransferase-to-platelet ratio index; AST: aspartate aminotransferase; AUROC: area under the receiver operating characteristic curve; CHB: chronic hepatitis B; CI: confidence interval; DCA: decision curve analysis; FIB-4: fibrosis index based on the four factors; GLB: globulin; GGT: γ-glutamyl transpeptidase; HBcAb: hepatitis B core antibody; HBeAg: hepatitis B e antigen; HBsAg: hepatitis B surface antigen; HBV: hepatitis B virus; HCC: hepatocellular carcinoma; HIV: human immunodeficiency virus; INR: international-normalized ratio; IQR: interquartile range; LASSO: least absolute shrinkage and selection operator; LB: liver biopsy; LR: Likelihood ratio; NAFLD: non-alcoholic fatty liver disease; NPV: negative predictive value; PLT: platelets; PPV: positive predictive value; PT: prothrombin time; ROC: receiver operating characteristic; TB: total bilirubin; TE: transient elastography; ULN: upper limit of normal

Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency

Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro, but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9. Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunctions