Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics.

Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism, pantothenate and CoA biosynthesis, glutathione metabolism, and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information, including Stemina Biomarker Discovery's predictive DevTox® model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity.

Teratogen-induced eye defects mediated by p53-dependent apoptosis.

BACKGROUND: Many birth defects are believed to involve gene-environment interactions, although the mechanisms involved are poorly understood. Apoptosis is a common effect of many kinds of environmental stresses on the developing embryo; therefore, mechanisms of teratogenesis may be approached within the context of the cell death program. The p53 tumor suppressor gene encodes a transcription factor which functions as a critical regulator of apoptosis in response to environmental stress. RESULTS: To investigate the relationship between p53-dependent apoptosis and teratogenesis, we subjected day 8 mouse embryos with different p53 gene backgrounds to a genotoxic stress, 2-chloro-2'-deoxyadenosine. Treatment rapidly stimulated nuclear p53 accumulation and triggered apoptosis in some (head-fold) but not other (primitive heart) developing structures. Induced cell death was p53 gene-dose dependent, as shown by the intermediate sensitivity of 4-5 somite stage embryos bearing only a single effective p53 allele and the lack of sensitivity of p53-null mutants. Abnormal development was manifested as eye defects by day 11, particularly lens agenesis. Overall the incidences of these defects at term were 73.3% for p53 wild-type fetuses, 52.5% for heterozygous mutants, and 2.2% for p53-null mutants. Statistical analysis indicated that the interaction between teratogen and genotype was highly significant (P < or = 0.001) for cell death on day 8 and eye defects on day 17. CONCLUSIONS: We conclude that teratogen induction of p53-dependent apoptosis in the developing embryo is positively coupled to the determination of congenital eye defects.

Insights into thymic purine metabolism and adenosine deaminase deficiency revealed by transgenic mice overexpressing ecto-5'-nucleotidase (CD73).

The adenosine producing enzyme ecto-5'-nucleotidase (5'-NT) is not normally expressed during thymocyte development until the medullary stage. To determine whether earlier expression would lead to adenosine accumulation and/or be deleterious for thymocyte maturation, thymic purine metabolism, and T cell differentiation were studied in lckNT transgenic mice overexpressing 5'-NT in cortical thymocytes under the control of the lck proximal promoter. In spite of a 100-fold elevation in thymic 5'-NT activity, transgenic adenosine levels were unchanged and T cell immunity was normal. Inosine, the product of adenosine deamination, was elevated more than twofold, however, indicating that adenosine deaminase (ADA) can prevent the accumulation of adenosine, even with a dramatic increase in 5'-NT activity, and demonstrating the availability of 5'-NT substrates in the thymus for the first time. Thymic adenosine concentrations of mice treated with the ADA inhibitor 2'-deoxycoformycin (dCF) were elevated over 30-fold, suggesting that high ADA activity, rather than an absence of 5'-NT, is mainly responsible for low thymic adenosine levels. The adenosine concentrations in dCF-treated mice are sufficient to cause adenosine receptor-mediated thymocyte apoptosis in vitro, suggesting that adenosine accumulation could play a role in ADA-deficient severe combined immunodeficiency.

Programming microphysiological systems for children's health protection.

Microphysiological systems (MPS) and computer simulation models that recapitulate the underlying biology and toxicology of critical developmental transitions are emerging tools for developmental effects assessment of drugs/chemicals. Opportunities and challenges exist for their application to alternative, more public health relevant and efficient chemical toxicity testing methods. This is especially pertinent to children's health research and the evaluation of complex embryological and reproductive impacts of drug/chemical exposure. Scaling these technologies to higher throughput is a key challenge and drives the need for in silico models for quantitative prediction of developmental toxicity to inform safety assessments. One example is cellular agent-based models, constructed from extant embryology, that produce data useful to simulate critical developmental transitions and thereby predict phenotypic consequences of disruption in silico. Biologically inspired MPS models built from human induced pluripotent stem (iPS)-derived cells and synthetic matrices that recapitulate organ-specific physiologies and native tissue architectures are providing exciting new research opportunities to advance the assessment of developmental toxicity and offer the possibility of deriving a full 'human on a chip' system, or a 'Homunculus.' Impact statement This 'commentary' summarizes research needs and opportunities for engineered MPS models for developmental and reproductive toxicity testing. Emerging concepts can be taken forward to a virtual tissue modeling framework for assessing chemical (and non-chemical) stressors on human development. These models will advance children's health research, both basic and translational and new ways to evaluate complex embryological and reproductive impacts of drug and chemical exposures to inform safety assessments.

Altered expression of mitochondrial 16S ribosomal RNA in p53-deficient mouse embryos revealed by differential display.

Inactivation of the tumor suppressor p53 is associated with neural tube defects and altered teratogenicity in early embryos. To gain insight into the function of p53 during early embryogenesis, RNA profiles of wild-type p53(+/+) and p53(-/-) null mutant mouse embryos were compared at the head-fold stage (day 8 post coitum) using HPLC-based mRNA differential display. The results of this screen revealed a deficiency of mitochondrial 16S ribosomal RNA in p53(-/-) embryos. RT-PCR showed abnormalities in 16S rRNA levels relative to some representative nuclear (COIV, beta-actin) and mitochondrial (COIII) transcripts in p53(-/-) embryos, and that 16S rRNA expression increased with development of p53(+/+) embryos during neurulation. Embryos that lack p53 also displayed weakened cytochrome c oxidase staining and reduced ATP content. During neurulation, the mouse embryo switches from an anaerobic (glycolytic) to an aerobic (oxidative) metabolism. The preliminary results of the present study suggest that p53 may be involved, directly or indirectly, in this transition.

Bcl-2 relieves deoxyadenylate stress and suppresses apoptosis in pre-B leukemia cells.

The influence of bcl-2 activity on 2'-deoxyadenosine-induced apoptosis was investigated in 697 human pre-B leukemia cells stably transfected with expression plasmid pHeBo-BCL-2alpha (697/BCL2 cells). Apoptosis was induced by the 2'-deoxyadenosine analogue, 2-chloro-2'-deoxyadenosine (Cl-dA), with the concentration for apoptosis in one-half of the cells at 24 hours (LD(50)) being 10 microM for 697 cells and 120 microM for 697/Bcl 2 cells. There was a strong positive correlation between Cl-dATP levels and apoptotic index (coefficient of determination, r(2)=0.95; P=0.027). When 697 cell and 697/Bcl 2 cell lines were treated with 5 microM Cl-dA, Cl-dATP did not significantly accumulate in the latter. The Cl-dATP/dATP ratio was 0.03 in Cl-dA treated 697/Bcl 2 cells but nearly 6 in treated 697 cells. Bcl 2 overproduction also suppressed the accumulation of dAMP, dADP and dATP in cells exposed to 2'-deoxyadenosine in the presence of pentostatin to abrogate the pronounced inversion of ATP/dATP pools associated with 2'-deoxyadenosine exposure. These results suggest that one consequence of bcl-2 activity is suppression of 2'-deoxyadenosine phosphorylation and elevation in the apoptotic target cells. Relief from deoxyadenylate stress imbalances implies a novel upstream site of bcl-2 activity.

Predictive value of soluble haemoglobin scavenger receptor CD163 serum levels for survival in verified tuberculosis patients.

Pre-treatment serum levels of sCD163 were measured in a cohort of 236 suspected tuberculosis (TB) cases from Guinea-Bissau, with a median follow-up period of 3.3 years (range 0-6.4 years). In 113 cases, the diagnosis of TB was verified by positive sputum microscopy and/or culture. Among the verified TB cases, a decreased survival rate was found in 27 patients with sCD163 levels above the upper reference limit (3.95 microg/mL). The difference in survival was significant during TB treatment (log rank, p<0.02) and after long-term follow-up (log rank, p<0.001). The decrease in survival rate during TB treatment remained significant in a multivariate Cox model controlling for human immunodeficiency virus (HIV) status, age and gender, with a mortality increase of 1.19 (95% CI, 1.04-1.36) per microg of sCD163, and a hazard ratio (HR) for sCD163 levels above the upper reference limit of 4.18 (95% CI, 1.06-16.4). The difference was not significant after excluding patients with concomitant HIV-1 and HIV-2 infection in Kaplan-Meier analyses (log rank, p 0.11). In contrast, the difference in survival remained significant in Kaplan-Meier analyses after long-term follow-up, even after excluding patients with concomitant HIV-1 and HIV-2 infection (log rank, p 0.002). In the Cox model, the mortality increase per microg of sCD163 was 1.27 (95% CI, 1.14-1.40), with an HR for elevated sCD163 levels of 2.85 (95% CI, 1.44-5.63). The HRs for concomitant HIV-1 and HIV-2 infection were 6.92 (95% CI, 3.28-14.58) and 2.48 (95% CI, 1.09-5.67), respectively. Thus, sCD163 levels appeared to be an independent predictor of survival in verified TB patients.

Murine ecto-5'-nucleotidase (CD73): cDNA cloning and tissue distribution.

The murine cDNA, encoding the purine catabolic enzyme, ecto-5'-nucleotidase (NT), was cloned and the tissue-specific distribution of both the mRNA and enzyme activity was examined. Starting with kidney RNA and primers based on the known rat sequence, reverse transcriptase-polymerase chain reaction (RT-PCR) was utilized to obtain the complete sequence for the translated portion of the murine cDNA. Murine NT is 94% identical to human NT at the amino acid (aa) level and 86% identical at the nucleotide (nt) level. NT enzyme assays revealed greater than tenfold more NT activity in mature vs. immature murine T- and B-lymphocytes. A similar increase in NT activity was also found when the pre-B-cell line, 70Z/3, was induced to produce surface kappa light chains with lipopolysaccharide (LPS) and gamma-interferon (gamma-IFN). Thus, culture systems in which murine lymphocytes mature may be useful for examining the mechanisms of NT gene regulation, as well as the function of NT in the immune system. In tissues, enzyme activity varied over 30-fold, from the lowest levels in skeletal muscle, thymus and spleen to highest in placenta, kidney and forestomach. Levels of mRNA, as determined by RNase protection assay, showed increased NT expression in the early gestation site, as compared to non-pregnant uterus, and in day-19.5 placenta, as compared to day-13 chorioallantoic placenta. Messenger RNA levels were in general proportional to enzyme activity, except in the lung and glandular stomach where mRNA levels were higher than expected, based on enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

A new multiplex PCR strategy for the simultaneous determination of four genetic polymorphisms affecting HIV-1 disease progression.

The CCR5 Delta32, CCR2 64I, SDF1 3'A, and CCR5 promoter 59029 polymorphisms have been suggested to influence HIV-1 disease progression. Furthermore, the CCR5 Delta32 and the CCR2 64I polymorphisms have been associated with various other diseases. The purpose of the present study was to develop a multiplex assay for the simultaneous determination of these four polymorphisms. Results obtained with the multiplex assay were compared to results obtained by conventional RFLP-PCR and no differences were observed. The multiplex assay offers a quick tool for the determination of the CCR5 Delta32, CCR2 64I, SDF1 3'A, and CCR5 promoter 59029 A/G polymorphisms.

Axonal guidance of adenosine deaminase immunoreactive primary afferent fibers in developing mouse spinal cord.

This study examined the precision of central fiber growth in a subpopulation of dorsal root ganglion neurons in developing mouse spinal cord. Immunohistochemical techniques using a monospecific, polyclonal antiserum to mouse adenosine deaminase (ADA) were utilized to label a population of primary sensory afferents that have been found to exclusively innervate laminae I and II of the dorsal horn in adult mice. Initial growth of ADA-immunoreactive (ADA-IR) primary afferents occurred very early in development, embryonic day 10 (E10), a time coincident with the earliest settling time of dorsal root ganglion neurons. Adenosine deaminase immunoreactive primary afferents were observed throughout the cross-sectional area of the primordial dorsal funiculus (DF) as early as E10. Immunostained fibers remained quiescent in the DF during its growth and separation into the tract of Lissauer and dorsal column pathway. By E15, the two pathways had formed and ADA-IR fibers were observed exclusively in the tract of Lissauer. This segregation of fibers remained throughout development and reflected the adult pattern. Growth was reinitiated at E16 when the fibers advanced into the dorsal horn and proceeded directly to laminae I and II mimicking their adult distribution. Exuberant fiber growth was not detected throughout their development. These results strongly suggest that ADA-IR fibers exhibit precise fiber guidance to a preferred pathway, the tract of Lissauer, and accurate laminar innervation of the dorsal horn.

Histochemical localization of glycosaminoglycans during morphogenesis of the secondary palate in mice.

The hydration of hyaluronic acid (HA) accumulated in the secondary palatal processes is expected to exert an intrinsic tissue pressure that could, in part, provide the impetus for shelf reorientation. Glycosaminoglycans were histochemically localized in the A/J mouse palate during development (days 12 to 15) by specific enzymatic degradation followed by preferential staining with alcian blue under differential pH or MgCl2 concentration. The presence of HA and chondroitin sulphates A and C (CS) was demonstrated in proportions that differed regionally. At the time of reorientation (days 14 to 15) HA was the predominant staining component, being distributed according to the relative prominence of extracellular spaces (ECS). HA was present in higher concentration in the anterior than the posterior part of the palate, particularly in an area of low cell density adjoining the CS-rich mesenchyme of the maxillary process. This arrangement suggests that the maxillary process might provide a resilient incompressible structural base for the palate as its HA-rich ECS expands. Sulphated GAG, with CS being the predominant component, was localized for the most part on the oral-side mesenchyme both in the anterior and posterior palate. The most intense staining of sulphated proteoglycans occurred in association with the basal lamina along the presumptive oral-side. Mesenchymal cells along this region appeared condensed and may have been stabilized by these sulphated GAG providing structural constraints which might function in palate morphogenesis.

Morphological analysis of abnormal digital chondrogenesis in the brachypod (bpH) mouse limb in organ culture.

Brachypod (bpH/bpH), an autosomal mutation in mice, is characterized by a shortening of the long bones and paws, and a delay or absence of ossification in some of the distal limb elements. The present study represents a detailed description of the brachypod phenotype in day 12 hindlimb buds maintained for 6 days in a submerged, serum-free organ culture system. Using this in vitro system, the proximal-to-distal effect on the severity of cartilage reduction was intensified in the brachypod explants with an intermediate expression in the heterozygotes. Immunofluorescent staining of the brachypod cartilage revealed a deficiency in and an abnormal distribution of the proteoglycans. Although there was no recognizable difference in the immunofluorescent staining for type II collagen between the mutant and wild-type, electron micrographs showed the presence of thick fibrils in the matrix. Other atypical structures in the brachypod cartilage included pleomorphic nuclei, reduced intracellular glycogen granules and profuse intercellular contacts. It is proposed that with the use of this in vitro system which supports the autonomous development of the individual limb elements, experiments concerning the pathogenesis of skeletal mutations such as brachypod should be more feasible.

[Spontaneous infrapatellar tendon rupture in a patient with systemic lupus erythematosus]. / Spontan infrapatellar seneruptur hos patient med systemisk lupus erythematosus.

A case is described of a 33-year old woman with systemic lupus erythematosus (SLE) in longterm treatment with corticosteroids who experienced spontaneous rupture of the left patellar tendon. A comparative study of 28 previously reported cases of SLE patients with spontaneous tendon rupture in weight bearing joints is performed. It is suggested that renal disease may be an etiological factor for spontaneous tendon rupture in patients with systemic lupus erythematosus.

Immediate and long-term consequences of vascular toxicity during zebrafish development.

Proper formation of the vascular system is necessary for embryogenesis, and chemical disruption of vascular development may be a key event driving developmental toxicity. In order to test the effect of environmental chemicals on this critical process, we evaluated a quantitative assay in transgenic zebrafish using angiogenesis inhibitors that target VEGFR2 (PTK787) or EGFR (AG1478). Both PTK787 and AG1478 exposure impaired intersegmental vessel (ISV) sprouting, while AG1478 also produced caudal and pectoral fin defects at concentrations below those necessary to blunt ISV morphogenesis. The functional consequences of vessel toxicity during early development included decreased body length and survival in juvenile cohorts developmentally exposed to inhibitor concentrations sufficient to completely block ISV sprouting angiogenesis. These data show that concentration-dependent disruption of the presumed targets for these inhibitors produce adverse outcomes at advanced life stages.

Release of targeted p53 from the mitochondrion as an early signal during mitochondrial dysfunction.

Increased accumulation of p53 tumor suppressor protein is an early response to low-level stressors. To investigate the fate of mitochondrial-sequestered p53, mouse embryonic fibroblast cells (MEFs) on a p53-deficient genetic background were transfected with p53-EGFP fusion protein led by a sense (m53-EGFP) or antisense (c53-EGFP) mitochondrial import signal. Rotenone exposure (100nM, 1h) triggered the translocation of m53-EGFP from the mitochondrion to the nucleus, thus shifting the transfected cells from a mitochondrial p53 to a nuclear p53 state. Antibodies for p53 serine phosphorylation or lysine acetylation indicated a different post-translational status of recombinant p53 in the nucleus and mitochondrion, respectively. These data suggest that cycling of p53 through the mitochondria may establish a direct pathway for p53 signaling from the mitochondria to the nucleus during mitochondrial dysfunction. PK11195, a pharmacological ligand of mitochondrial TSPO (formerly known as the peripheral-type benzodiazepine receptor), partially suppressed the release of mitochondria-sequestered p53. These findings support the notion that p53 function mediates a direct signaling pathway from the mitochondria to nucleus during mitochondrial dysfunction.

Differential programming of p53-deficient embryonic cells during rotenone block.

Mitochondrial dysfunction has been implicated in chemical toxicities. The present study used an in vitro model to investigate the differential expression of metabolic pathways during cellular stress in p53-efficient embryonic fibroblasts compared to p53-deficient cells. These cell lines differed with respect to NADH/NAD(+) balance. This ratio constitutes a driving force for NAD- and NADH-dependent reactions and is inversed upon exposure to Rotenone (complex I inhibitor). Rotenone perturbed the structure of the elongated fibrillar tubulin network and decreased mRNA expression of tubulin genes both suggesting reprogramming and reorganization of the cytoskeleton in both cell lines. These changes were reflected in the abundance of specific mRNA and microRNA (miRNA) species as determined from genome-based analysis. Changes in mRNA and miRNA expression profiles reflected differences in energy utilizing pathways, consistent with the notion that the p53 pathway influences the cellular response to mitochondrial dysfunction and that at least some control may be embedded within specific mRNA/miRNA networks in embryonic cells.

Gingival epithelial cells heterozygous for Toll-like receptor 4 polymorphisms Asp299Gly and Thr399ile are hypo-responsive to Porphyromonas gingivalis.

The Toll-like receptor (TLR)4 is the major sensor for bacterial lipopolysaccharide and its two common co-segregating polymorphisms, Asp299Gly and Thr399Ile, which occur at a frequency of between 6 and 10%, have been associated with infectious diseases, LPS hypo-responsiveness and cardiovascular disease. Porphyromonas gingivalis is a Gram-negative bacterium implicated in chronic periodontitis and is a known TLR4 and TLR2 agonist. We obtained two gingival epithelial cell primary cultures from subjects heterozygous for the TLR4 polymorphism Asp299Gly and compared response characteristics with similar cells from patients (four) with the wild-type TLR4 genes. Cytokine responses and transcriptome profiles of gingival epithelial cell primary culture cells to TNFalpha challenge were similar for all primary epithelial cell cultures. P. gingivalis challenge, however, gave markedly different responses for Asp299Gly heterozygous and wild-type epithelial cell cultures. The epithelial cells heterozygous for the TLR4 polymorphism Asp299Gly were functionally hypo-responsive, evidenced by differences in BD-2 mRNA expression, mRNA response profile by microarray analysis and by pro-inflammatory and chemokine cytokines at the protein and mRNA level. These findings emphasize variance in human epithelial cell TLRs, linked with Asp299Gly carriage, which results in a hypo-responsive epithelial cell phenotype less susceptible to Gram-negative diseases and associated systemic conditions.

Evidence for negative control of growth by adenosine in the mammalian embryo: induction of Hmx/+ mutant limb outgrowth by adenosine deaminase.

We investigated the growth-regulatory actions of adenosine and adenosine deaminase (ADA) during embryonic limb development in the mouse. Polydactylous outgrowth, an expression of the Hemimelia-extra toe (Hmx/+) mutant phenotype, was experimentally regulated in hindlimb buds explanted into a serum-free in vitro system at stage 18 of gestation. Its expression was promoted by exposure to 0.1 or 0.2 IU/ml exogenous ADA and suppressed by co-exposure to 10 nM (-)-N6-(R-phenylisopropyl)-adenosine (N6-PIA). Evidence that N6-PIA acted as a high-affinity agonist against the external adenosine receptor was provided by experiments in which 100 microM caffeine, a known antagonist, competitively blocked its effect. The endogenous adenosine content was analyzed by reversed-phase high-performance liquid chromatography with fluorometric detection following its conversion to the 1,N6-ethenoadenosine derivative. At stage 18, the adenosine levels were 0.5 pmol/micrograms DNA in whole embryos and 0.08 pmol/micrograms DNA in hindlimb buds. At the same stage, partially purified extracts of the embryonal plasma enriched fraction contained high levels of ADA activity (0.04-0.06 IU/embryo, or 0.7-1.0 IU/mg protein). In contrast, blood cells contained 0.0001 IU/embryo (or 0.01 IU/mg protein). This enzyme occurred as a single kinetic form with a molecular weight of 45000-47000 daltons and an apparent Km of 36-38 microM. Its presence in the embryonal plasma argues against an endocrine mechanism of adenosine secretion in favor of autocrine (self-regulatory) or paracrine (proximate-regulatory) mechanisms. Taken together, our results suggest that the in vitro outgrowth of the prospective polydactylous region is induced upon escape from the local growth-inhibitory influence of extracellular adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of morphogenetic cell death during abnormal limb-bud outgrowth in mice heterozygous for the dominant mutation Hemimelia-extra toe (Hmx).

A dominant mutation in the mouse, Hemimelia-extra toe (Hmx), induces congenital limb malformations in heterozygotes. Typical expression includes axial shortening of the radius, tibia and talus ('hemimelia'), with supernumerary metacarpals, metatarsals, and digits ('polydactyly'). Pathogenesis was investigated during developmental stages 16 through 22 (11th through 15th days of gestation). Full expression was apparent during stage 20 when the limb pattern was comprised of pre-cartilaginous anlagen. Formation of a pre-axial protrusion on the autopod during stage 17 or 18 was the earliest gross abnormality, and foreshadowed the development of supernumerary digits. Microscopically, there was an alteration in the pattern of physiologic cellular degeneration (PCD) programmed to occur within the zeugopod and autopod. The 'opaque patch' (mesodermal necrotic zone normally occurring between tibial and fibular anlagen) was overextended pre-axially causing resorption of the tibial precartilage. Additionally, PCD normally occurring within the basal cell layer of the apical ectodermal ridge (AER) and the 'foyer primaire préaxial' was not expressed in the mutant autopod. This occurred in association with outgrowth of the protrusion. The pre-axial portion of the AER remained in an abnormally thickened, viable, proliferative state, and did not undergo scheduled degression. This may have been the basis for prolonged induction of pre-axial outgrowth. Paucity of mesenchymal cell filopodial processes extended along the basal lamina, as well as a rarefaction of the filamentous material normally associated with the mesodermal face of the basal lamina, was detected at the pre-axial AER-mesenchymal interface on stage 18. A potential involvement of epithelial-mesenchymal interactions in the induction of epithelial PCD is discussed.