Fabrication of nickel phytate modified bio-based polyol rigid polyurethane foam with enhanced compression-resistant and improved flame-retardant.

A bio-based flame retardant nickel phytate (PA-Ni) was synthesized and combined with soybean oil-based polyol (SO) to create a green rigid polyurethane foam (RPUF) with enhanced compressive strength, good thermal stability and flame retardant. The results showed that the RPUF-SO2/Ni3 with 3 wt% PA-Ni had the highest initial and termination temperature, maximum thermal decomposition rate temperature and carbon residue, and better thermal stability. Its limiting oxygen index was increased by 2.6% compared with RPUF-SO2 without PA-Ni added, and the peak heat release rate (PHRR) and total heat release rate (THR) were reduced by 14.92% and 19.92%, respectively. In addition, RPUF-SO2/Ni3 had the lowest Ds under the conditions of flame (18.90) and flameless (22.41), and had the best smoke suppression effect. And the compressive strength of RPUF-SO/Ni3 was significantly enhanced by the addition of PA-Ni. The results show that the improvement of flame retardancy of RPUF is mainly the result of the combined effect of gas-phase and condensed-phase flame retardancy, among which the flame retardancy of RPUF-SO/Ni3 was the best. The current findings offer a practical way to produce green and low-carbon RPUF as well as promising prospects for the material's safe application.

Functional and antigenic landscape of the Nipah virus receptor binding protein.

Nipah virus recurrently spills over to humans, causing fatal infections. The viral receptor-binding protein (RBP or G) attaches to host receptors and is a major target of neutralizing antibodies. Here we use deep mutational scanning to measure how all amino-acid mutations to the RBP affect cell entry, receptor binding, and escape from neutralizing antibodies. We identify functionally constrained regions of the RBP, including sites involved in oligomerization, along with mutations that differentially modulate RBP binding to its two ephrin receptors. We map escape mutations for six anti-RBP antibodies, and find that few antigenic mutations are present in natural Nipah strains. Our findings offer insights into the potential for functional and antigenic evolution of the RBP that can inform the development of antibody therapies and vaccines.

Structure and design of Langya virus glycoprotein antigens.

Langya virus (LayV) is a recently discovered henipavirus (HNV), isolated from febrile patients in China. HNV entry into host cells is mediated by the attachment (G) and fusion (F) glycoproteins which are the main targets of neutralizing antibodies. We show here that the LayV F and G glycoproteins promote membrane fusion with human, mouse, and hamster target cells using a different, yet unknown, receptor than Nipah virus (NiV) and Hendra virus (HeV) and that NiV- and HeV-elicited monoclonal and polyclonal antibodies do not cross-react with LayV F and G. We determined cryoelectron microscopy structures of LayV F, in the prefusion and postfusion states, and of LayV G, revealing their conformational landscape and distinct antigenicity relative to NiV and HeV. We computationally designed stabilized LayV G constructs and demonstrate the generalizability of an HNV F prefusion-stabilization strategy. Our data will support the development of vaccines and therapeutics against LayV and closely related HNVs.

Effects of low-tube voltage coronary CT angiography on plaque and pericoronary fat assessment: intraindividual comparison.

OBJECTIVES: To investigate the effects of low tube voltage on coronary plaques and pericoronary fat assessment, and to compare their extent among various levels of low voltage. MATERIALS AND METHODS: Patients were recommended for high-pitch low-tube voltage coronary computed tomography angiography (CCTA), and they were included if they had poor image quality and were referred to a conventional CCTA. The patients were classified into a low-voltage group (with 70-kV, 80-kV, and 90-kV subgroups) and a conventional group (100/120 kV). Their total plaque and subcomponent volumes and pericoronary fat attenuation index (FAI) were measured. RESULTS: A total of 1002 image slices (from 65 patients and 74 plaques) were included, including 21, 31, 13, 4, and 61 patients in the 70-kV, 80-kV, 90-kV, 100-kV, and 120-kV groups respectively. The CT values of noncalcified plaques in the conventional and low-voltage groups were 54.6 ± 21.3 HU and 31.5 ± 22.6 HU, respectively (p < 0.05). Compared with the conventional group, the necrotic core and calcification volume were increased, and the fibrolipid volume, periplaque, and right coronary artery FAI were decreased in the low-voltage group and its subgroups (p < 0.001). The magnitude of changes in fibrous and calcification volumes increased in the 70-kV subgroup compared with that in the 90-kV subgroup (p < 0.05). CONCLUSION: Low tube voltages, particularly of 70 kV, have a significant effect on coronary plaque and FAI. The effect of low voltage on plaque composition is characterized by a polarization pattern, i.e., a decrease in fibrolipid (medium density) and an increase in necrotic core (low density) and calcification (high density). CLINICAL RELEVANCE STATEMENT: Our results highlight the comparability and repeatability of plaque and pericoronary fat assessments facilitated by the same or a similar tube voltage. It is necessary to carry out studies on the specificity threshold of low tube voltage at each level. KEY POINTS: • Low tube voltage had a significant effect on coronary plaque and pericoronary fat, particularly 70 kV. • The effect of low tube voltage on plaque composition shows the shift from medium-density mixed components to low- and high-density components. • It is necessary to correct the specificity threshold or attenuation difference for low tube voltage at each level.

Segmental analysis of liver cirrhosis with different etiologies: a study based on iodine mixed imaging in port-venous phase.

BACKGROUND: Computed tomography (CT) in port-venous phase can display the intra-hepatic vessels, and may provide the possibility for segment function evaluation for cirrhosis. PURPOSE: To assess the value of iodine mixed imaging of dual-source dual-energy CT in port-venous phase in segmental evaluation of liver cirrhosis with different etiologies. MATERIAL AND METHODS: Patients diagnosed with liver cirrhosis were enrolled. Patients without cirrhosis were included as a control group. Each patient underwent iodine-contrast enhanced multi-phase dual-energy CT scanning. Parameters were analyzed by SPSS, version 22.0, and Medcalc. RESULTS: In total, 256 patients were investigated, including 114 Child-Pugh A, 51 Child-Pugh B, 41 Child-Pugh C and 50 control patients. Total iodine content (ICt)/body surface area (BSA) in the cirrhosis group was significantly lower than the control group (P < 0.05) and the standardized-iodine parameter (SI) of each segment decreased with cirrhosis progression. In Child-Pugh A and B, SI increased more significantly in the caudal and lateral segment in A (alcholism) than in the V (virus-related) and N (non-alcoholic steatohepatitis) groups (P < 0.001). ICt/BSA showed the best diagnosis power of cirrhosis with an area under the curve of 0.765, sensitivity of 76.0% and specificity of 71.8%. CONCLUSION: Blood flow compensated in the left lateral and caudal lobe in the early stage of liver cirrhosis. The compensation in alcoholism in the middle and early stages is significantly higher than that of V and N cirrhosis. Iodine mixed imaging in portal phase may provide the possibility of an incremental value in segmented blood flow perfusion and functional evaluation of liver cirrhosis on a morphological basis.

Structure and design of Langya virus glycoprotein antigens.

Langya virus (LayV) is a recently discovered henipavirus (HNV), isolated from febrile patients in China. HNV entry into host cells is mediated by the attachment (G) and fusion (F) glycoproteins which are the main targets of neutralizing antibodies. We show here that the LayV F and G glycoproteins promote membrane fusion with human, mouse and hamster target cells using a different, yet unknown, receptor than NiV and HeV and that NiV- and HeV-elicited monoclonal and polyclonal antibodies do not cross-react with LayV F and G. We determined cryo-electron microscopy structures of LayV F, in the prefusion and postfusion states, and of LayV G, revealing previously unknown conformational landscapes and their distinct antigenicity relative to NiV and HeV. We computationally designed stabilized LayV G constructs and demonstrate the generalizability of an HNV F prefusion-stabilization strategy. Our data will support the development of vaccines and therapeutics against LayV and closely related HNVs.

Potent monoclonal antibody-mediated neutralization of a divergent Hendra virus variant.

Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic Henipaviruses (HNVs) responsible for recurrent outbreaks in humans and domestic species of highly fatal (50 to 95%) disease. A HeV variant (HeV-g2) of unprecedented genetic divergence has been identified in two fatally diseased horses, and in two flying fox species in regions of Australia not previously considered at risk for HeV spillover. Given the HeV-g2 divergence from HeV while retaining equivalent pathogenicity and spillover potential, understanding receptor usage and antigenic properties is urgently required to guide One Health biosecurity. Here, we show that the HeV-g2 G glycoprotein shares a conserved receptor tropism with prototypic HeV and that a panel of monoclonal antibodies recognizing the G and F glycoproteins potently neutralizes HeV-g2­ and HeV G/F­mediated entry into cells. We determined a crystal structure of the Fab fragment of the hAH1.3 antibody bound to the HeV G head domain, revealing an antigenic site associated with potent cross-neutralization of both HeV-g2 and HeV. Structure-guided formulation of a tetravalent monoclonal antibody (mAb) mixture, targeting four distinct G head antigenic sites, results in potent neutralization of HeV and HeV-g2 and delineates a path forward for implementing multivalent mAb combinations for postexposure treatment of HNV infections.

Architecture and antigenicity of the Nipah virus attachment glycoprotein.

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness. The entry of HNVs into host cells requires the attachment (G) and fusion (F) glycoproteins, which are the main targets of antibody responses. To understand viral infection and host immunity, we determined a cryo-electron microscopy structure of the NiV G homotetrameric ectodomain in complex with the nAH1.3 broadly neutralizing antibody Fab fragment. We show that a cocktail of two nonoverlapping G-specific antibodies neutralizes NiV and HeV synergistically and limits the emergence of escape mutants. Analysis of polyclonal serum antibody responses elicited by vaccination of macaques with NiV G indicates that the receptor binding head domain is immunodominant. These results pave the way for implementing multipronged therapeutic strategies against these deadly pathogens.

Predictive value of spiral shape in coronary plaque progression: an intraindividual comparative study.

OBJECTIVE: We conducted a pilot study to explore the value of spiral-shaped sign of plaque from coronary computed tomographic angiography (CCTA) in predicting plaque progression by intraindividual comparison. METHODS: A total of 30 patients with a total of 60 plaques who received serial CCTA were retrospectively included and intraindividual compared. The spiral shape was defined as plaques coursing along the long axis of a coronary artery and encircling it at an angle of ≥ 180 degrees. The high-risk and other plaque signs were recorded. RESULTS: On baseline CCTA, the spiral shape (P < 0.01) and length (P < 0.05) of plaques were more frequently seen in the progression group than in the nonprogression group; however, there was no difference between two groups in terms of high-risk plaque signs. In the progression group, plaque length, volume, and napkin-ring sign on follow-up CCTA were significantly greater than at baseline (P < 0.05). In the nonprogression group, there were fewer low-attenuation and positive remodeling plaques on follow-up CCTA than at baseline (P < 0.05). The spiral shape (standardized ß = -4.55; P < 0.01) was an independent risk factor for plaque progression. There were 24 spiral plaques in the progression group, of which 16 (66.7%) had progression below the twist point of the spiral shape. CONCLUSIONS: The baseline spiral shape is more frequently found in those lesions that progress than in those that do not in patients with multiple coronary lesions, and the spiral shape is an independent predictor of which plaques will progress.

Broad sarbecovirus neutralization by a human monoclonal antibody.

The recent emergence of SARS-CoV-2 variants of concern1-10 and the recurrent spillovers of coronaviruses11,12 into the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here we describe a human monoclonal antibody designated S2X259, which recognizes a highly conserved cryptic epitope of the receptor-binding domain and cross-reacts with spikes from all clades of sarbecovirus. S2X259 broadly neutralizes spike-mediated cell entry of SARS-CoV-2, including variants of concern (B.1.1.7, B.1.351, P.1, and B.1.427/B.1.429), as well as a wide spectrum of human and potentially zoonotic sarbecoviruses through inhibition of angiotensin-converting enzyme 2 (ACE2) binding to the receptor-binding domain. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2X259 possesses an escape profile that is limited to a single substitution, G504D. We show that prophylactic and therapeutic administration of S2X259 protects Syrian hamsters (Mesocricetus auratus) against challenge with the prototypic SARS-CoV-2 and the B.1.351 variant of concern, which suggests that this monoclonal antibody is a promising candidate for the prevention and treatment of emergent variants and zoonotic infections. Our data reveal a key antigenic site that is targeted by broadly neutralizing antibodies and will guide the design of vaccines that are effective against all sarbecoviruses.

Combined coronary CT angiography with plain scan for diagnosis of ruptured plaque: comparison with optical coherence tomography.

To evaluate the diagnostic efficacy of CCTA + plain scan for ruptured plaques, with optical coherence tomography (OCT) as the reference, and to provide preliminary analysis of influential factors. Patients who underwent CCTA and OCT were retrospectively enrolled. The diagnostic standards for ruptured plaque on CCTA + plain scan were ulcer or intra-plaque dye penetration on CCTA, and a careful review of images from the plain scans to ensure areas of them were not calcification. The diagnosis of ruptured plaque was made by OCT. Total 65 patients with 71 plaques were included. There were 40 OCT-confirmed ruptured plaques in 38 patients and 31 OCT-confirmed non-ruptured plaques in 27 patients. CCTA + plain scan identified 27 ruptured plaques in 27 patients and 28 non-ruptured plaques in 24 patients. With OCT as the gold standard, the per-patient sensitivity, specificity, positive and negative predictive values, and accuracy of CCTA + plain scan for diagnosing ruptured plaque were 71%, 89%, 90%, 69%, and 78%, and there was good agreement (Kappa = 0.70) between CCTA + plain scan and OCT. Among 13 false negative ruptured plaques, 2 had calcifications close to the rupture, and the cavity depth in the remaining 11 was 0.46 ± 0.17 mm, versus 0.98 ± 0.26 mm in 27 true positive ruptured plaques (P < 0.01). CCTA + plain scan may identify morphological features of ruptured plaques. The cavity depth of the ruptured plaques and calcification at the rupture site seem major factors influencing the diagnostic accuracy for plaque rupture. Future perspective studied are needed to confirm these preliminary findings.

Structural basis for broad sarbecovirus neutralization by a human monoclonal antibody.

The recent emergence of SARS-CoV-2 variants of concern (VOC) and the recurrent spillovers of coronaviruses in the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here, we describe a human monoclonal antibody (mAb), designated S2X259, recognizing a highly conserved cryptic receptor-binding domain (RBD) epitope and cross-reacting with spikes from all sarbecovirus clades. S2X259 broadly neutralizes spike-mediated entry of SARS-CoV-2 including the B.1.1.7, B.1.351, P.1 and B.1.427/B.1.429 VOC, as well as a wide spectrum of human and zoonotic sarbecoviruses through inhibition of ACE2 binding to the RBD. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2X259 possesses a remarkably high barrier to the emergence of resistance mutants. We show that prophylactic administration of S2X259 protects Syrian hamsters against challenges with the prototypic SARS-CoV-2 and the B.1.351 variant, suggesting this mAb is a promising candidate for the prevention and treatment of emergent VOC and zoonotic infections. Our data unveil a key antigenic site targeted by broadly-neutralizing antibodies and will guide the design of pan-sarbecovirus vaccines.

Structures of MERS-CoV spike glycoprotein in complex with sialoside attachment receptors.

The Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and often lethal respiratory illness in humans, and no vaccines or specific treatments are available. Infections are initiated via binding of the MERS-CoV spike (S) glycoprotein to sialosides and dipeptidyl-peptidase 4 (the attachment and entry receptors, respectively). To understand MERS-CoV engagement of sialylated receptors, we determined the cryo-EM structures of S in complex with 5-N-acetyl neuraminic acid, 5-N-glycolyl neuraminic acid, sialyl-LewisX, α2,3-sialyl-N-acetyl-lactosamine and α2,6-sialyl-N-acetyl-lactosamine at 2.7-3.0 Å resolution. We show that recognition occurs via a conserved groove that is essential for MERS-CoV S-mediated attachment to sialosides and entry into human airway epithelial cells. Our data illuminate MERS-CoV S sialoside specificity and suggest that selectivity for α2,3-linked over α2,6-linked receptors results from enhanced interactions with the former class of oligosaccharides. This study provides a structural framework explaining MERS-CoV attachment to sialoside receptors and identifies a site of potential vulnerability to inhibitors of viral entry.

Image quality and diagnostic value of ultra low-voltage, ultra low-contrast coronary CT angiography.

OBJECTIVE: To explore the image quality (IQ) and diagnostic value of 70 kVp turbo high-pitch coronary CT angiography (THP-CCTA) using automated tube voltage selection (ATVS) and 30 mL of low-concentration contrast agent. METHODS: Patients who underwent 70 kVp THP-CCTA using ATVS with 30 mL of contrast agent (group A) were prospectively enrolled, and those who underwent conventional CCTA (100/120 kVp, prospective sequential mode with 65-75 mL of contrast agent) (group B) were retrospectively selected for study. IQ was assessed subjectively on a 5-point scale, and diagnostic value was assessed based on invasive coronary angiography as the gold standard. Heart rate (HR), HR fluctuation (HRF), body mass index (BMI), effective radiation dose (ED), and iodine uptake (IU) were recorded. RESULTS: A total of 796 patients (398/398 in groups A/B) were included. Between-group differences in age, gender, BMI, HR, HRF, and IQ values were not significant. The ED/IU values were 0.3 ± 0.1 mSv/9.0 ± 0.0 g and 5.8 ± 1.8 mSv/22.9 ± 1.0 g in groups A and B, respectively (p < 0.01). The sensitivity, specificity, positive and negative predictive values, and accuracy of THP-CCTA for the diagnosis of ≥ 50% stenosis were 94.8%, 97.5%, 92.0%, 98.4%, and 96.9% respectively. The mean HR and coronary calcium score were independent predictors of diagnostic image quality, and the best cutoff values were 71.5 bpm and 444.1 respectively. CONCLUSION: This third-generation dual-source CT imaging modality, a 70-kVp THP-CCTA system using ATVS with 30 mL of low-concentration contrast agent, produces high-quality images with high diagnostic accuracy for significant stenosis, with ultra low ED and IU. This technique was most promising in individuals with an HR < 71.5 bpm and coronary calcium score < 444.1. KEY POINTS: • Turbo high-pitch CCTA using 70 kVp via automated tube voltage selection and 30 mL of low-concentration contrast agent is feasible. • This protocol provides high diagnostic accuracy for significant coronary stenosis and reduces radiation doses and iodine uptake significantly. • This protocol was most promising in individuals with an HR < 71.5 bpm and coronary calcium score < 444.1.

Detection of Fungi from Low-Biomass Spacecraft Assembly Clean Room Aerosols.

Highly sensitive and rapid detection of airborne fungi in space stations is essential to ensure disease prevention and equipment safety. In this study, quantitative loop-mediated isothermal amplification (qLAMP) was used to detect fungi in the aerosol of the low-biomass environment of China's space station assembly clean room (CSSAC). A qLAMP primer set for detecting a wide range of aerosol fungi was developed by aligning 34 sequences of isolated fungal species and 17 space station aerosol-related fungal species. Optimization of sample pretreatment conditions of the LAMP reaction increased the quantitative results by 1.29-1.96 times. The results showed that our qLAMP system had high amplification specificity for fungi, with a quantifiable detection limit as low as 102. The detected fungal biomass in the aerosol of CSSAC was 9.59 × 102-2.20 × 105 28S rRNA gene copy numbers/m3. This qLAMP assay may therefore replace traditional colony-forming unit and quantitative PCR methods as an effective strategy for detecting fungi in space stations.

An aqueous fluorescent sensor for Pb2+ based on phenothiazine-polyamide.

A sensitive and selective fluorescent sensor for Pb2+ ion based on phenothiazine-polyamide was built (named sensor PP). Due to introducing of four diethanolamine groups to polyamide, this sensor was totally water soluble. PP could detect Pb2+ ion within 1 min in the presence of other metal ions in aqueous solution, the detect limit was 9.11 × 10-8 M.

Accuracy of cardiac CT in evaluating severity of left atrial appendage spontaneous echo contrast: comparison with transesophageal echocardiography.

To investigate the capacity of biphasic cardiac CT (CCT) for qualitative and quantitative evaluation of different grades of left atrial appendage spontaneous echo contrast (LAASEC). The study included 267 inpatients with confirmed atrial fibrillation who underwent both CCT and transesophageal echocardiography (TEE). CT numbers for LAA, ascending aorta (AA), and left atrium (LA) were identified, and ROC curves for LAA, LAA/AA, and LAA/LA were plotted. With TEE as the standard, the sensitivity, specificity, PPV, NPV, and accuracy of CCT for LAASEC grade ≥ 1 were 60.3, 92.9, 92.4, 64.8, and 75.7%; and for grade ≥ 2 were 100.0, 84.4, 71.4, 100.0, and 88.8% respectively. The values of LAA, LAA/AA, and LAA/LA were significantly larger in LAASEC grade 0 versus 1 and in grade 1 versus 2, but were similar in grades 2 and 3 or in grades 3 and 4. The values of LAA/AA were larger in grade 2 versus 4. When the cutoff value for LAA/AA = 0.897, sensitivity, specificity, PPV, NPV, and accuracy of CCT for LAASEC grade ≥ 1 was 89.6, 83.2, 87.9, 85.5, and 86.9% and with a cutoff for LAA/AA of 0.524, the sensitivity, specificity, PPV, NPV, and accuracy for LAASEC grade ≥ 2 was 98.7, 92.7, 84.1, 99.4, and 94.4%. Although CCT showed limited diagnostic accuracy for grade 1 LAASEC, grade ≥ 2 LAASEC could be excluded when there was no LAA filling defect on first-phase CCT, and TEE can be avoided. CCT has an excellent accuracy in diagnosing LAASEC, and quantitative analysis (in particular LAA/AA) is superior.

A Ratiometric Fluorescent Sensor for Cd2+ Based on Internal Charge Transfer.

This work reports on a novel fluorescent sensor 1 for Cd2+ ion based on the fluorophore of tetramethyl substituted bis(difluoroboron)-1,2-bis[(1H-pyrrol-2-yl)methylene]hydrazine (Me4BOPHY), which is modified with an electron donor moiety of N,N-bis(pyridin-2-ylmethyl)benzenamine. Sensor 1 has absorption and emission in visible region, at 550 nm and 675 nm, respectively. The long wavelength spectral response makes it easier to fabricate the fluorescence detector. The sensor mechanism is based on the tunable internal charge transfer (ICT) transition of molecule 1. Binding of Cd2+ ion quenches the ICT transition, but turns on the π - π transition of the fluorophore, thus enabling ratiometric fluorescence sensing. The limit of detection (LOD) was projected down to 0.77 ppb, which is far below the safety value (3 ppb) set for drinking water by World Health Organization. The sensor also demonstrates a high selectivity towards Cd2+ in comparison to other interferent metal ions.

Ascending aortic distensibility and target organ damage in primary hypertension without diabetes.

To investigate the relationship between ascending aortic distensibility (AAD) and hypertensive target organ damage (TOD) and its potential value in prediction. One hundred and sixty seven primary hypertension inpatients who underwent coronary CTA examination were enrolled into our study. Retrospective ECG-triggering scanning mode were applied and the images were reconstructed every 5% phase in the entire R-R interval. Maximum and minimum ascending aortic areas as well as the AAD value were calculated on the interested slice. AAD (P < 0.001) and brachial-ankle pulse wave velocity (baPWV, P < 0.05) were changed significantly as the deterioration of TOD. Multivariate logistic regression analysis between TOD and its possible influence factors indicated that AAD was the only independent risk factor for the presence and severity of TOD. One standard deviation decrease on AAD would increase the risk of TOD significantly: TOD1 (odds ratio 0.45, P < 0.05), TOD2 (odds ratio 0.23, P < 0.05), and TOD3 (odds ratio 0.01, P < 0.05). The odds ratio of TOD in the third tertile group was found 5.47 times higher than that in the second tertile group, and the second tertile group TOD odds ratio was 6.4 times higher than that in the first tertile group. Decline of AAD can be taken as the independent predict factor for TOD in primary hypertension patients, superior to baPWV method and other conventional predictors. Without additional contrast media consumption and radiation dose, AAD derived from coronary CTA may provide early detection for hypertensive TOD.