Computational framework for the prediction of transcription factor binding sites by multiple data integration

Background

Activation of extracellular signal-regulated protein kinase (ERK), a member of mitogen-activated protein kinase (MAPK) family, has been proposed to mediate neurite outgrowth-promoting effects of several neurotrophic factors in vitro. However, the precise activity of ERK during axonal regeneration in vivo remains unclear. Peripheral axotomy has been shown to activate ERK in the cell bodies of primary afferent neurons and associated satellite cells. Nevertheless, whether ERK is also activated in the axons and surrounded Schwann cells which also play a key role in the regeneration process has not been clarified.

Results

Phosphorylation of ERK in the sciatic nerve in several time-points after crush injury has been examined. Higher phosphorylation of ERK was observed in the proximal and distal nerve stumps compared to the contralateral intact nerve from one day to one month after crush. The activation of ERK was mainly localized in the axons of the proximal segments. In the distal segments, however, active ERK was predominantly found in Schwann cells forming Bungner's bands.

Conclusion

The findings indicate that ERK is activated in both the proximal and distal nerve stumps following nerve injury. The role of activated ERK in Wallerian degeneration and subsequent regeneration in vivo remains to be elucidated.     

Synthesis and Study the Effect of Donor-Acceptor Substituent on Fluorescence Behavior of Thieno[3, 2-c]pyridine Derivatives

4-Hetero-1-yl-2-bromothieno[3,2-c]pyridines 3(a–d) were synthesized by the reaction of 2-bromo-4-chlorothieno[3,2-c]pyridine (1) and cyclic amine 2(a–d), which on Suzuki coupling with substituted boronic acids 4(a–f) exclusively converted to corresponding 4-hetero -1-yl-2-arylthieno[3,2-c]pyridine 5(a–x) in good yields. The effect of donor-acceptor substituent on absorption emission properties and fluorescent quantum yield of new thienopyridine derivatives 5(a-x) were studied.     

Delta-opioid receptor endocytosis in spinal cord after dermenkephalin activation

Background

The delta(δ)-opioid receptors belong to the G protein-coupled receptors and in vitro studies have shown that δ-opioid receptors undergo an internalization process in response to agonist stimulation. The immediate consequence is the disappearance of receptors from the plasma membrane. This adaptation process reveals the cell's capacity to desensitize after a strong agonist stimulus. This process, if it occurs in vivo, could contribute to the tolerance phenomenon observed after opiate treatment. To study the mechanisms underlying regulation of the δ-opioid receptors in vivo, the effects of an application of the drug dermenkephalin, a potent and selective agonist of the δ-opioid receptor, were analysed in the rat spinal cord.

Results

Using immunocytochemistry and electron microscopy, we observed in control rats that membrane labelling was strictly localized at the interface between two neurites. Fifteen minutes after dermenkephalin stimulation, the plasma membrane labelling was associated with invaginated areas. Thirty minutes after stimulation, labelled vesicles were found in the cytoplasm confirming the internalization process.

Conclusions

The present findings support the view that δ-opioid receptors are internalized in response to prolonged exposure to dermenkephalin in vivo and confirm the presynaptic localization of δ-opioid receptors in the dorsal horn of the rat spinal cord.     

2-NBDG Fluorescence Imaging of Hypermetabolic Circulating Tumor Cells in Mouse Xenograft model of Breast Cancer

Objectives

To determine use of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) as a tracer for detection of hypermetabolic circulating tumor cells (CTC) by fluorescence imaging.

Procedures

Human breast cancer cells were implanted in the mammary gland fat pad of athymic mice to establish orthotopic human breast cancer xenografts as a mouse model of circulating breast cancer cells. Near-infrared fluorescence imaging of the tumor-bearing mice injected with 2-DeoxyGlucosone 750 (2-DG 750) was conducted to assess glucose metabolism of xenograft tumors. Following incubation with fluorescent 2-NBDG, circulating breast cancer cells in the blood samples collected from the tumor-bearing mice were collected by magnetic separation, followed by fluorescence imaging for 2-NBDG uptake by circulating breast cancer cells, and correlation of the number of hypermetabolic circulating breast cancer cells with tumor size at the time when the blood samples were collected.

Results

Human breast cancer xenograft tumors derived from MDA-MB-231, BT474, or SKBR-3 cells were visualized on near-infrared fluorescence imaging of the tumor-bearing mice injected with 2-DG 750. Hypermetabolic circulating breast cancer cells with increased uptake of fluorescent 2-NBDG were detected in the blood samples from tumor-bearing mice and visualized by fluorescence imaging, but not in the blood samples from normal control mice. The number of hypermetabolic circulating breast cancer cells increased along with growth of xenograft tumors, with the number of hypermetabolic circulating breast cancer cells detected in the mice bearing MDA-MB231 xenografts larger than those in the mice bearing BT474 or SKBR-3 xenograft tumors.

Conclusions

Circulating breast cancer cells with increased uptake of fluorescent 2-NBDG were detected in mice bearing human breast cancer xenograft tumors by fluorescence imaging, suggesting clinical use of 2-NBDG as a tracer for fluorescence imaging of hypermetabolic circulating breast cancer cells.     

Enhanced effect of microdystrophin gene transfection by HSV-VP22 mediated intercellular protein transport

Background

Duchenne musclar dystrophy (DMD) is an X-linked recessive disease caused by mutations of dystrophin gene, there is no effective treatment for this disorder at present. Plasmid-mediated gene therapy is a promising therapeutical approach for the treatment of DMD. One of the major issues with plasmid-mediated gene therapy for DMD is poor transfection efficiency and distribution. The herpes simplex virus protein VP22 has the capacity to spread from a primary transduced cell to surrounding cells and improve the outcome of gene transfer. To improve the efficiency of plasmid-mediated gene therapy and investigate the utility of the intercellular trafficking properties of VP22-linked protein for the treatment for DMD, expression vectors for C-terminal versions of VP22-microdystrophin fusion protein was constructed and the VP22-mediated shuttle effect was evaluated both in vitro and in vivo.

Results

Our results clearly demonstrate that the VP22-microdystrophin fusion protein could transport into C2C12 cells from 3T3 cells, moreover, the VP22-microdystrophin fusion protein enhanced greatly the amount of microdystrophin that accumulated following microdystrophin gene transfer in both transfected 3T3 cells and in the muscles of dystrophin-deficient (mdx) mice.

Conclusion

These results highlight the efficiency of the VP22-mediated intercellular protein delivery for potential therapy of DMD and suggested that protein transduction may be a potential and versatile tool to enhance the effects of gene delivery for somatic gene therapy of DMD.     

Use of ultraviolet-light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes

Background

As the eradication of tumor cells in vivo is most efficiently performed by cytolytic T lymphocytes (CTL), various methods for priming tumor-reactive lymphocytes have been developed. In this study, a method of priming CTLs with ultraviolet (UV)-irradiated tumor cells, which results in termination of tumor cell proliferation, apoptosis, as well as upregulation of heat shock proteins (HSP) expression is described.

Methods

Peripheral blood mononuclear cells (PBMC) were primed weekly with UV-irradiated or mitomycin-treated RPMI 8226 multiple myeloma cells. Following three rounds of stimulation over 21 days, the lymphocytes from the mixed culture conditions were analyzed for anti-MM cell reactivity.

Results

By day 10 of cultures, PBMCs primed using UV-irradiated tumor cells demonstrated a higher percentage of activated CD8+/CD4- T lymphocytes than non-primed PBMCs or PBMCs primed using mitomycin-treated MM cells. Cytotoxicity assays revealed that primed PBMCs were markedly more effective (p < 0.01) than non-primed PBMCs in killing RPMI 8226 MM cells. Surface expression of glucose regulated protein 94 (Grp94/Gp96) and Grp78 were both found to be induced in UV-treated MM cells.

Conclusion

Since, HSP-associated peptides are known to mediate tumor rejection; these data suggest that immune-mediated eradication of MM cells could be elicited via a UV-induced HSP process. The finding that the addition of 17-allylamide-17-demethoxygeldanamycin (17AAG, an inhibitor of HSP 90-peptide interactions) resulted in decreased CTL-induced cytotoxicity supported this hypothesis. Our study, therefore, provides the framework for the development of anti-tumor CTL cellular vaccines for treating MM using UV-irradiated tumor cells as immunogens.     

Generation and characterization of high affinity human monoclonal antibodies that neutralize staphylococcal enterotoxin B

Background

Staphylococcal enterotoxins are considered potential biowarfare agents that can be spread through ingestion or inhalation. Staphylococcal enterotoxin B (SEB) is a widely studied superantigen that can directly stimulate T-cells to release a massive amount of proinflammatory cytokines by bridging the MHC II molecules on an antigen presenting cell (APC) and the Vβ chains of the T-cell receptor (TCR). This potentially can lead to toxic, debilitating and lethal effects. Currently, there are no preventative measures for SEB exposure, only supportive therapies.

Methods

To develop a potential therapeutic candidate to combat SEB exposure, we have generated three human B-cell hybridomas that produce human monoclonal antibodies (HuMAbs) to SEB. These HuMAbs were screened for specificity, affinity and the ability to block SEB activity in vitro as well as its lethal effect in vivo.

Results

The high-affinity HuMAbs, as determined by BiaCore analysis, were specific to SEB with minimal crossreactivity to related toxins by ELISA. In an immunoblotting experiment, our HuMAbs bound SEB mixed in a cell lysate and did not bind any of the lysate proteins. In an in vitro cell-based assay, these HuMAbs could inhibit SEB-induced secretion of the proinflammatory cytokines (INF-γ and TNF-α) by primary human lymphocytes with high potency. In an in vivo LPS-potentiated mouse model, our lead antibody, HuMAb-154, was capable of neutralizing up to 100 μg of SEB challenge equivalent to 500 times over the reported LD₅₀ (0.2 μg), protecting mice from death. Extended survival was also observed when HuMAb-154 was administered after SEB challenge.

Conclusion

We have generated high-affinity SEB-specific antibodies capable of neutralizing SEB in vitro as well as in vivo in a mouse model. Taken together, these results suggest that our antibodies hold the potential as passive immunotherapies for both prophylactic and therapeutic countermeasures of SEB exposure.     

Measurement of regional cerebral glucose uptake by magnetic resonance spin-lock imaging

Purpose

The regional uptake of glucose in rat brain in vivo was measured at high resolution using spin-lock magnetic resonance imaging after infusion of the glucose analogue 2-deoxy-d-glucose (2DG). Previous studies of glucose metabolism have used ¹³C-labeled 2DG and NMR spectroscopy, ¹⁸F-labeled fluorodeoxyglucose (FDG) and PET, or chemical exchange saturation transfer (CEST) MRI, all of which have practical limitations. Our goal was to explore the ability of spin-lock sequences to detect specific chemically-exchanging species in vivo and to compare the effects of 2DG in brain tissue on CEST images.

Methods

Numerical simulations of R_1p and CEST contrasts for a variety of sample parameters were performed to evaluate the potential specificity of each method for detecting the exchange contributions of 2DG. Experimental measurements were made in tissue phantoms and in rat brain in vivo which demonstrated the ability of spin-lock sequences for detecting 2DG.

Results

R_1p contrast acquired with appropriate spin-lock sequences can isolate the contribution of exchanging protons in 2DG in vivo and appears to have better sensitivity and more specificity to 2DG–water exchange effects than CEST.

Conclusion

Spin-lock imaging provides a novel approach to the detection and measurement of glucose uptake in brain in vivo.     

Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations

Background

Minocycline, a second-generation tetracycline with anti-inflammatory and anti-apoptotic properties, has been shown to promote therapeutic benefits in experimental stroke. However, equally compelling evidence demonstrates that the drug exerts variable and even detrimental effects in many neurological disease models. Assessment of the mechanism underlying minocycline neuroprotection should clarify the drug's clinical value in acute stroke setting.

Results

Here, we demonstrate that minocycline attenuates both in vitro (oxygen glucose deprivation) and in vivo (middle cerebral artery occlusion) experimentally induced ischemic deficits by direct inhibition of apoptotic-like neuronal cell death involving the anti-apoptotic Bcl-2/cytochrome c pathway. Such anti-apoptotic effect of minocycline is seen in neurons, but not apparent in astrocytes. Our data further indicate that the neuroprotection is dose-dependent, in that only low dose minocycline inhibits neuronal cell death cascades at the acute stroke phase, whereas the high dose exacerbates the ischemic injury.

Conclusion

The present study advises our community to proceed with caution to use the minimally invasive intravenous delivery of low dose minocycline in order to afford neuroprotection that is safe for stroke.     

Fluorescent Styryl Dyes Based on Novel 4-Methoxy-9-Methyi-9H-Carbazole-3-Carbaldehyde—Synthesis,Photophysical Properties and DFT Computations

Novel carbazole based styryl derivatives (6a–6c) having styryl group at third position and a methoxy substitution were synthesized by condensing 4-methoxy-9-methyl-9H-carbazole-3-carbaldehyde 3 and different active methylene derivatives (5a–5c). Evaluated photophysical properties of these synthesized novel chromophores, studied the effect of solvent polarity on absorption, emission and quantum yield of these styryl derivatives. DFT and TD-DFT computations are carried out to study structural, molecular, electronic and photophysical parameters of dyes. The ratio of ground state to excited state dipole moment was calculated using Bakhshiev and Kawski-Chamma-Viallet correlations.     

In vivo assessment of the permeability of the blood--brain barrier and blood-retinal barrier to fluorescent indoline derivatives in zebrafish

ABSTRACT: BACKGROUND: Successful delivery of compounds to the brain and retina is a challenge in the development of therapeutic drugs and imaging agents. This challenge arises because internalization of compounds into the brain and retina is restricted by the blood--brain barrier (BBB) and blood-retinal barrier (BRB), respectively. Simple and reliable in vivo assays are necessary to identify compounds that can easily cross the BBB and BRB. METHODS: We developed six fluorescent indoline derivatives (IDs) and examined their ability to cross the BBB and BRB in zebrafish by in vivo fluorescence imaging. These fluorescent IDs were administered to live zebrafish by immersing the zebrafish larvae at 7--8 days post fertilization in medium containing the ID, or by intracardiac injection. We also examined the effect of multidrug resistance proteins (MRPs) on the permeability of the BBB and BRB to the ID using MK571, a selective inhibitor of MRPs. RESULTS: The permeability of these barriers to fluorescent IDs administered by simple immersion was comparable to when administered by intracardiac injection. Thus, this finding supports the validity of drug administration by simple immersion for the assessment of BBB and BRB permeability to fluorescent IDs. Using this zebrafish model, we demonstrated that the length of the methylene chain in these fluorescent IDs significantly affected their ability to cross the BBB and BRB via MRPs. CONCLUSIONS: We demonstrated that in vivo assessment of the permeability of the BBB and BRB to fluorescent IDs could be simply and reliably performed using zebrafish. The structure of fluorescent IDs can be flexibly modified and, thus, the permeability of the BBB and BRB to a large number of IDs can be assessed using this zebrafish-based assay. The large amount of data acquired might be useful for in silico analysis to elucidate the precise mechanisms underlying the interactions between chemical structure and the efflux transporters at the BBB and BRB. In turn, understanding these mechanisms may lead to the efficient design of compounds targeting the brain and retina.     

New spiro[benzotetraphene-fluorene] Derivatives: Synthesis and Application in Sky-Blue Fluorescent Host Materials

Blue light-emitting spiro[benzotetraphene-fluorene] (SBTF)-based host materials, 3-(1-naphthyl)-10-naphthylspiro[benzo[ij]tetraphene-7,9′-fluorene] (1), 3-(2-naphthyl)-10-naphthylspiro[benzo[ij]tetraphene-7,9′-fluorene] (2), and 3-[2-(6-phenyl)naphthyl]-10-naphthylspiro[benzo[ij]tetraphene-7,9′-fluorene] (3) were designed and prepared via multi-step Suzuki coupling reactions. Introducing various aromatic groups into SBTF core lead to a reduction in band gap and a determination of the color purity and luminescence efficiency. Typical sky-blue fluorescent organic light emitting diodes with the configuration of ITO/N,N′-di(1-naphthyl)-N,N′-bis[(4-diphenylamino)phenyl]-biphenyl-4,4′-diamie (60 nm)/N,N,N′,N′-tetra(1-biphenyl)-biphenyl-4,4′-diamine (30 nm)/host: dopant (30 nm, 5 %)/LG201 (electron transporting layer, 20 nm)/LiF/Al were developed using SBTF derivatives as a host material and p-bis(p-N,N-diphenyl-aminostyryl)benzene (DSA-Ph) as a sky-blue dopant material. A device obtained from three materials doped with DSA-Ph showed color purity of 0.148 and 0.239, a luminance efficiency of 7.91 cd/A, and an external quantum efficiency >4.75 % at 5 V.     

Enhanced expression of WD repeat-containing protein 35 (WDR35) stimulated by domoic acid in rat hippocampus: involvement of reactive oxygen species generation and p38 mitogen-activated protein kinase activation

Background

Domoic acid (DA) is an excitatory amino acid analogue of kainic acid (KA) that acts via activation of glutamate receptors to elicit a rapid and potent excitotoxic response, resulting in neuronal cell death. Recently, DA was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in vitro. We have reported that WDR35, a WD-repeat protein, may mediate apoptosis in several animal models. In the present study, we administered DA to rats intraperitoneally, then used liquid chromatography/ion trap tandem mass spectrometry (LC-MS/MS) to identify and quantify DA in the brains of the rats and performed histological examinations of the hippocampus. We further investigated the potential involvement of glutamate receptors, ROS, p38 MAPK, and WDR35 in DA-induced toxicity in vivo.

Results

Our results showed that intraperitoneally administered DA was present in the brain and induced neurodegenerative changes including apoptosis in the CA1 region of the hippocampus. DA also increased the expression of WDR35 mRNA and protein in a dose- and time-dependent manner in the hippocampus. In experiments using glutamate receptor antagonists, the AMPA/KA receptor antagonist NBQX significantly attenuated the DA-induced increase in WDR35 protein expression, but the NMDA receptor antagonist MK-801 did not. In addition, the radical scavenger edaravone significantly attenuated the DA-induced increase in WDR35 protein expression. Furthermore, NBQX and edaravone significantly attenuated the DA-induced increase in p38 MAPK phosphorylation.

Conclusion

In summary, our results indicated that DA activated AMPA/KA receptors and induced ROS production and p38 MAPK phosphorylation, resulting in an increase in the expression of WDR35 in vivo.     

A Novel Colorimetric and Off–On Fluorescent Chemosensor for Cr3+ in Aqueous Solution and its Application in Live Cell Imaging

A novel colorimetric and off–on fluorescent chemosensor 2 was designed and synthesized, which showed reversible and highly selective and sensitive recognition toward Cr³⁺ over other examined metal ions in aqueous solution. Upon addition of Cr³⁺, the solution of chemosensor 2 resulted in a color change from colorless to obvious pink color, these significant changes in color could be used for naked-eye detection. Chemosensor 2 exhibited a stable response for Cr³⁺ in the range 0–10 μM with a detection limit of 1 ppm. Furthermore, fluorescence imaging experiments of Cr³⁺ ions in living MGC803 cells demonstrated its value of practical applications in biological systems.     

Cingulate seizure-like activity reveals neuronal avalanche regulated by network excitability and thalamic inputs

Background

Cortical neurons display network-level dynamics with unique spatiotemporal patterns that construct the backbone of processing information signals and contribute to higher functions. Recent years have seen a wealth of research on the characteristics of neuronal networks that are sufficient conditions to activate or cease network functions. Local field potentials (LFPs) exhibit a scale-free and unique event size distribution (i.e., a neuronal avalanche) that has been proven in the cortex across species, including mice, rats, and humans, and may be used as an index of cortical excitability. In the present study, we induced seizure activity in the anterior cingulate cortex (ACC) with medial thalamic inputs and evaluated the impact of cortical excitability and thalamic inputs on network-level dynamics. We measured LFPs from multi-electrode recordings in mouse cortical slices and isoflurane-anesthetized rats.

Results

The ACC activity exhibited a neuronal avalanche with regard to avalanche size distribution, and the slope of the power-law distribution of the neuronal avalanche reflected network excitability in vitro and in vivo. We found that the slope of the neuronal avalanche in seizure-like activity significantly correlated with cortical excitability induced by γ-aminobutyric acid system manipulation. The thalamic inputs desynchronized cingulate seizures and affected the level of cortical excitability, the modulation of which could be determined by the slope of the avalanche size.

Conclusions

We propose that the neuronal avalanche may be a tool for analyzing cortical activity through LFPs to determine alterations in network dynamics.     

Synthesis and Spectroscopy of Novel Branched Fluorescent Dyes Containing Benzophenone Parts and the Possibility as Fluorescence Probes

This paper describes a new fluorescent family of branched dyes containing benzophenone unit including 4-N, N-diphenylamino-4??-phenacyl-stilbene (C1), 4,4??-di(4-benzoylphenylethylene)yl-triphenylamine (C2) and 4,4??,4??-tri(4-benzoylphenylethylene)yl-triphenylamine (C3). Benzophenone part is coupled with core through C?CC double bond. The chemical structures of the derivatives are characterized with ¹H and ¹³C nuclear magnetic resonance and elemental analysis. Strong ?ШC?? stacking interactions are discovered with the analysis of the X-ray crystallographic data of C1. The absorption maxima and emission maxima of the derivatives exhibit gradual bathochromic shift from C1 to C3. The optical density of C1, C2 and C3 are shown to be related to the number of branches. The changes of dipole moments between the excited and ground states for C1, C2 and C3 were estimated to be 4.356, 8.091 and 8.479 Derby, respectively by Lippert equation, confirming that the internal charge transfer (ICT) dominates the process of excited singlet state. The possibility as fluorescence probes of the derivatives on the estimation of what region of micelles interacting with samples was evaluated.     

Quantitative MR imaging of targeted SPIO particles on the cell surface and comparison to flow cytometry

Purpose

To detect anti-CEACAM5 targeted superparamagnetic iron oxide (SPIO) particles in vitro on the cell surface by quantitative magnetic resonance (MR) imaging and to compare with flow cytometry.

Materials and Methods

The monoclonal mouse antibody T84.1 and an appropriate IgG isotype antibody were conjugated to dextran-coated SPIO particles. HT29 cells expressing carcinoembryonic antigen (CEACAM5) were treated with antibody-conjugated SPIO particles. Purified cell samples were examined on a 3.0-T MR scanner using a multi-echo spin-echo sequence for MR relaxometry. Aliquots of the cell samples were further treated with a fluorescein isothiocyanate (FITC) anti-dextran antibody and an Alexa Fluor 488 anti-mouse antibody for the corresponding flow cytometry.

Results

MR relaxometry revealed a dose-dependent binding of T84.1-conjugated SPIO particles with a positive correlation between R₂ relaxation rate of cell samples and SPIO particle concentration during incubation (r=0.993, P<.01). Positive correlations were also observed between R₂ relaxation rate and flow cytometry (geometric mean) with both fluorescent antibodies (r=0.972 and r=0.953, both P<.01), respectively.

Conclusion

The study revealed the feasibility of quantitative MR imaging of targeted SPIO particles on the cell surface comparable to flow cytometry.     

Localization in the ground state of the ising model with a random transverse field

We study the zero-temperature behavior of the Ising model in the presence of a random transverse field. The Hamiltonian is given by $$H = - J\sum\limits_{\left\langle {x,y} \right\rangle } {\sigma _3 (x)\sigma _3 (y) - \sum\limits_x {h(x)\sigma _1 (x)} } $$ whereJ>0,x,y∈Z ^d, σ₁, σ₃ are the usual Pauli spin 1/2 matrices, andh={h(x),x∈Z ^d} are independent identically distributed random variables. We consider the ground state correlation function 〈σ₃(x)σ₃(y)〉 and prove:

1. Letd be arbitrary. For anym>0 andJ sufficiently small we have, for almost every choice of the random transverse fieldh and everyx∈Z ^d, that $$\left\langle {\sigma _3 (x)\sigma _3 (y)} \right\rangle \leqq C_{x,h} e^{ - m\left| {x - y} \right|} $$ for ally∈Z ^d withC _{x h} <∞.
2. Letd≧2. IfJ is sufficiently large, then, for almost every choice of the random transverse fieldh, the model exhibits long range order, i.e., $$\mathop {\overline {\lim } }\limits_{\left| y \right| \to \infty } \left\langle {\sigma _3 (x)\sigma _3 (y)} \right\rangle > 0$$ for anyx∈Z ^d.