Human phosphodiesterase 4D7 (PDE4D7) expression is increased in TMPRSS2-ERG-positive primary prostate cancer and independently adds to a reduced risk of post-surgical disease progression.

BACKGROUND: There is an acute need to uncover biomarkers that reflect the molecular pathologies, underpinning prostate cancer progression and poor patient outcome. We have previously demonstrated that in prostate cancer cell lines PDE4D7 is downregulated in advanced cases of the disease. To investigate further the prognostic power of PDE4D7 expression during prostate cancer progression and assess how downregulation of this PDE isoform may affect disease outcome, we have examined PDE4D7 expression in physiologically relevant primary human samples. METHODS: About 1405 patient samples across 8 publically available qPCR, Affymetrix Exon 1.0 ST arrays and RNA sequencing data sets were screened for PDE4D7 expression. The TMPRSS2-ERG gene rearrangement status of patient samples was determined by transformation of the exon array and RNA seq expression data to robust z-scores followed by the application of a threshold>3 to define a positive TMPRSS2-ERG gene fusion event in a tumour sample. RESULTS: We demonstrate that PDE4D7 expression positively correlates with primary tumour development. We also show a positive association with the highly prostate cancer-specific gene rearrangement between TMPRSS2 and the ETS transcription factor family member ERG. In addition, we find that in primary TMPRSS2-ERG-positive tumours PDE4D7 expression is significantly positively correlated with low-grade disease and a reduced likelihood of progression after primary treatment. Conversely, PDE4D7 transcript levels become significantly decreased in castration resistant prostate cancer (CRPC). CONCLUSIONS: We further characterise and add physiological relevance to PDE4D7 as a novel marker that is associated with the development and progression of prostate tumours. We propose that the assessment of PDE4D7 levels may provide a novel, independent predictor of post-surgical disease progression.

The cAMP phosphodiesterase-4D7 (PDE4D7) is downregulated in androgen-independent prostate cancer cells and mediates proliferation by compartmentalising cAMP at the plasma membrane of VCaP prostate cancer cells.

BACKGROUND: Isoforms of the PDE4 family of cAMP-specific phosphodiesterases (PDEs) are expressed in a cell type-dependent manner and contribute to underpinning the paradigm of intracellular cAMP signal compartmentalisation. Here we identify the differential regulation of the PDE4D7 isoform during prostate cancer progression and uncover a role in controlling prostate cancer cell proliferation. METHODS: PDE4 transcripts from 19 prostate cancer cell lines and xenografts were quantified by qPCR. PDE4D7 expression was further investigated because of its significant downregulation between androgen-sensitive (AS) and androgen-insensitive (AI) samples. Western blot analysis, PDE activity assay, immunofluorescent staining and cAMP responsive FRET assays were used to investigate the sub-plasma membrane localisation of a population of PDE4D7 in VCaP (AS) and PC3 (AI) cell lines. Disruption of this localisation pattern using dominant-negative protein expression and siRNA knockdown showed that PDE4D7 acts in opposition to proliferative signalling as assessed by electrical impedance-based proliferation assays. RESULTS: Here we identify the differential regulation of the PDE4D7 isoform during prostate cancer progression. PDE4D7 is highly expressed in AS cells and starkly downregulated in AI samples. The significance of this downregulation is underscored by our finding that PDE4D7 contributes a major fraction of cAMP degrading PDE activity tethered at the plasma membrane and that displacement of PDE4D7 from this compartment leads to an increase in the proliferation of prostate cancer cells. PDE4D7 mRNA expression is not, however, directly regulated by the androgen receptor signalling axis despite an overlapping genomic structure with the androgen responsive gene PART1. PDE4D7, which locates to the plasma membrane, acts to supress aberrant non-steroidal growth signals within the prostate or AS metastasis. CONCLUSIONS: PDE4D7 expression is significantly downregulated between AS and AI cell phenotypes. This change in expression potentially provides a novel androgen-independent biomarker and manipulation of its activity or its expression may provide therapeutic possibilities and insights into contributory aspects of the complex molecular pathology of prostate cancer.

Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator.

The protein EP300 and its paralog CREBBP (CREB-binding protein) are ubiquitously expressed transcriptional co-activators and histone acetyl transferases. The gene EP300 is essential for normal cardiac and neural development, whereas CREBBP is essential for neurulation, hematopoietic differentiation, angiogenesis and skeletal and cardiac development. Mutations in CREBBP cause Rubinstein-Taybi syndrome, which is characterized by mental retardation, skeletal abnormalities and congenital cardiac defects. The CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) binds EP300 and CREBBP with high affinity and regulates gene transcription. Here we show that Cited2-/- embryos die with cardiac malformations, adrenal agenesis, abnormal cranial ganglia and exencephaly. The cardiac defects include atrial and ventricular septal defects, overriding aorta, double-outlet right ventricle, persistent truncus arteriosus and right-sided aortic arches. We find increased apoptosis in the midbrain region and a marked reduction in ErbB3-expressing neural crest cells in mid-embryogenesis. We show that CITED2 interacts with and co-activates all isoforms of transcription factor AP-2 (TFAP2). Transactivation by TFAP2 isoforms is defective in Cited2-/- embryonic fibroblasts and is rescued by ectopically expressed CITED2. As certain Tfap2 isoforms are essential in neural crest, neural tube and cardiac development, we propose that abnormal embryogenesis in mice lacking Cited2 results, at least in part, from its role as a Tfap2 co-activator.

Non-canonical Wnt signalling regulates scarring in biliary disease via the planar cell polarity receptors.

The number of patients diagnosed with chronic bile duct disease is increasing and in most cases these diseases result in chronic ductular scarring, necessitating liver transplantation. The formation of ductular scaring affects liver function; however, scar-generating portal fibroblasts also provide important instructive signals to promote the proliferation and differentiation of biliary epithelial cells. Therefore, understanding whether we can reduce scar formation while maintaining a pro-regenerative microenvironment will be essential in developing treatments for biliary disease. Here, we describe how regenerating biliary epithelial cells express Wnt-Planar Cell Polarity signalling components following bile duct injury and promote the formation of ductular scars by upregulating pro-fibrogenic cytokines and positively regulating collagen-deposition. Inhibiting the production of Wnt-ligands reduces the amount of scar formed around the bile duct, without reducing the development of the pro-regenerative microenvironment required for ductular regeneration, demonstrating that scarring and regeneration can be uncoupled in adult biliary disease and regeneration.

Re-evaluation of hypoplastic left heart syndrome from a developmental and morphological perspective.

BACKGROUND: Hypoplastic left heart syndrome (HLHS) covers a spectrum of rare congenital anomalies characterised by a non-apex forming left ventricle and stenosis/atresia of the mitral and aortic valves. Despite many studies, the causes of HLHS remain unclear and there are conflicting views regarding the role of flow, valvar or myocardial abnormalities in its pathogenesis, all of which were proposed prior to the description of the second heart field. Our aim was to re-evaluate the patterns of malformation in HLHS in relation to recognised cardiac progenitor populations, with a view to providing aetiologically useful sub-groupings for genomic studies. RESULTS: We examined 78 hearts previously classified as HLHS, with subtypes based on valve patency, and re-categorised them based on their objective ventricular phenotype. Three distinct subgroups could be identified: slit-like left ventricle (24%); miniaturised left ventricle (6%); and thickened left ventricle with endocardial fibroelastosis (EFE; 70%). Slit-like ventricles were always found in combination with aortic atresia and mitral atresia. Miniaturised left ventricles all had normally formed, though smaller aortic and mitral valves. The remaining group were found to have a range of aortic valve malformations associated with thickened left ventricular walls despite being described as either atresia or stenosis. The degree of myocardial thickening was not correlated to the degree of valvar stenosis. Lineage tracing in mice to investigate the progenitor populations that form the parts of the heart disrupted by HLHS showed that whereas Nkx2-5-Cre labelled myocardial and endothelial cells within the left and right ventricles, Mef2c-AHF-Cre, which labels second heart field-derived cells only, was largely restricted to the endocardium and myocardium of the right ventricle. However, like Nkx2-5-Cre, Mef2c-AHF-Cre lineage cells made a significant contribution to the aortic and mitral valves. In contrast, Wnt1-Cre made a major contribution only to the aortic valve. This suggests that discrete cardiac progenitors might be responsible for the patterns of defects observed in the distinct ventricular sub-groups. CONCLUSIONS: Only the slit-like ventricle grouping was found to map to the current nomenclature: the combination of mitral atresia with aortic atresia. It appears that slit-like and miniature ventricles also form discrete sub-groups. Thus, reclassification of HLHS into subgroups based on ventricular phenotype, might be useful in genetic and developmental studies in investigating the aetiology of this severe malformation syndrome.

Cardiovascular defects associated with abnormalities in midline development in the Loop-tail mouse mutant.

Loop-tail (Lp) is a naturally occurring mouse mutant that develops severe neural tube defects. In this study, we describe complex cardiovascular defects in Lp homozygotes, which include double-outlet right ventricle, with obligatory perimembranous ventricular septal defects, and double-sided aortic arch, with associated abnormalities in the aortic arch arteries. Outflow tract and aortic arch defects are often related to abnormalities in the cardiac neural crest, but using molecular and anatomic markers, we show that neural crest migration is normal in Lp/Lp embryos. On the other hand, the heart fails to loop normally in Lp/Lp embryos, in association with incomplete axial rotation and reduced cervical flexion. As a consequence, the ventricular loop is shifted posteromedially relative to its position in wild-type embryos. This suggests that the observed cardiac alignment defects in the Lp mutant may be secondary to failure of neural tube closure and incomplete axial rotation. Double-sided aortic arch is a rare finding among mouse models. In humans, it is usually an isolated malformation, only rarely occurring in combination with other cardiac defects. We suggest that the double-sided arch arises as a primary defect in the Lp mutant, unrelated to the alignment defects, perhaps reflecting a role for the (as-yet-unknown) Lp gene in maintenance/regression of the aortic arch system.

Over-expression of the chondroitin sulphate proteoglycan versican is associated with defective neural crest migration in the Pax3 mutant mouse (splotch).

Splotch mice, which harbour mutations in the Pax3 gene, exhibit neural crest-related abnormalities including pigmentation defects, reduced or absent dorsal root ganglia and failure of cardiac outflow tract septation in homozygotes. Although splotch neural crest cells fail to colonise target tissues, they initiate migration in vivo and appear to migrate as well as wild type neural crest cells in vitro, suggesting that the neural crest abnormality in splotch may reside not in the neural crest cells themselves, but rather in the extracellular environment through which they migrate. We have examined the expression of genes encoding extracellular matrix molecules in Sp2H homozygous embryos and find a marked over-expression of transcripts for the chondroitin sulphate proteoglycan versican in the pathways of neural crest cell migration. Use of cadherin-6 expression as a marker for neural crest demonstrates a striking correlation between up-regulation of versican expression and absence of migrating neural crest cells, both in the mesenchyme lateral to the neural tube and in the lower branchial arches of Sp2H homozygotes. Pax3 and versican have mutually exclusive expression patterns in normal embryos whereas, in Sp2H homozygotes, versican is generally over-expressed with 'infilling' in regions that would normally express functional Pax3. Versican, like other chondroitin sulphate proteoglycans, is non-permissive for migration of neural crest cells in vitro, and we suggest that over-expression of this molecule leads to the arrest of neural crest cell migration in splotch embryos. Pax3 may serve to negatively regulate versican expression during normal development, thereby guiding neural crest cells into their pathways of migration.

RhoB is expressed in migrating neural crest and endocardial cushions of the developing mouse embryo.

RhoB mRNA expression was examined in the developing mouse embryo between E8.5 and E11.5. Specific expression was found in migrating neural crest (NC) cells, from the first stages of their migration at E9.5, throughout the migration period. Expression is maintained in NC derivatives for at least one embryonic day after they reach their final destinations, but is then down-regulated. RhoB is also expressed in non NC-derived neural tissues, including motor neurones and the floor plate of the neural tube. RhoB mRNA expression is also found in the developing endocardial cushions of the atrioventricular and outflow regions of the developing heart.

Development of a lethal congenital heart defect in the splotch (Pax3) mutant mouse.

OBJECTIVE: The splotch (Sp2h) mutation disrupts the Pax3 gene and is lethal in homozygotes. The aim of the present study was to investigate the cause of lethality. METHODS AND RESULTS: Using the splotch (Sp2H) mouse mutant, we demonstrated that approximately 60% of Sp2H homozygotes die in utero at 13.5-14.5 days of gestation. All these embryos have cardiac malformations involving partial or complete failure of septation of the outflow tract. Although the cause of death in utero is unknown, the dying embryos are edematous, their superior caval veins are over-expanded, and the fetal liver is enlarged and engorged with blood, all signs of cardiac failure. The remaining Sp2H homozygotes die around the time of birth, and these embryos have grossly normal hearts. All Sp2H homozygotes have neural tube defects, either spina bifida, exencephaly, or both. Although these defects clearly do not cause death in utero, they are very likely responsible for the perinatal death of homozygotes that survive to late gestation. There is no correlation between the presence or absence of a cardiac defect and the type of neural tube defect. On the other hand, there is a striking correlation between presence of a cardiac defect and reduction or absence of dorsal root ganglia, which are derivatives of the neural crest. CONCLUSIONS: In this paper, we show that the lethality has a biphasic pattern, and the data strongly suggests that mid-gestation lethality is due to cardiac defects and not the associated neural tube defects. This finding supports the idea that 'conotruncal' cardiac defects involving the ventricular outflow tracts develop as a result of failure of the 'cardiac' neural crest to colonise the developing heart in the mid-gestation embryo, and that the resulting heart defects are solely responsible for the observed mortality.

The effect of age on antipyrine metabolism by liver microsomes isolated from phenobarbital-treated rats.

A study was performed to test the hypothesis that ageing influences the activities of diverse forms or populations of cytochrome P-450 isoenzymes in different ways. The formation of antipyrine metabolites in induced rats is mediated by such different forms or populations of cytochrome P-450 isoenzymes. To test the hypothesis, the formation rates of antipyrine metabolites by liver microsomes isolated from phenobarbital-treated rats of different ages was determined. After phenobarbital induction in vitro, the maximal velocity for norantipyrine formation decreased from 12 to 24 months and then showed a tendency to increase with age. Hydroxymethylantipyrine formation did not change with age. 4-Hydroxyantipyrine increased between 3 and 12 months and remained constant afterwards. This is in agreement with data obtained in vivo in uninduced male BN/BiRij rats. It can be concluded that age does indeed influence the activities of different forms or populations of the cytochrome P-450 isoenzymes in different ways. Consequently, determining the overall clearance of antipyrine, which is metabolized by several isoenzymes, especially in the induced situation, is not to be recommended for measuring the activity of cytochrome P-450 isoenzymes as a function of age.

Physical and psychological follow-up on offspring of renal allograft recipients.

In light of concern regarding the effect of pregnancy on renal allograft recipients and the lack of information about their offspring, we studied ten infants born to eight allograft recipients (five female and three male). In all five female recipients, allograft function remained normal throughout pregnancy and the puerperium. Complications of pregnancy were minimal. Four patients were delivered by cesarean section, three for obstetrical reasons and one electively. Three of ten infants were premature births. There were no significant neonatal complications. At time of follow-up study, the offspring were 4 months to 6 8/12 years of age; physical examination was normal on all offspring. Developmental test results ranged from 82 to 131. Parents tended to be overly cautious and sought medical attention at the first sign of illness. Our data indicated no adverse effect on renal allograft function of the female recipients and no physical or developmental abnormalities in the offspring.

Coma from hyponatremia following transurethral resection of prostate.

Even though hyponatremia may occur following transurethral resection of the prostate (TURP), only 14 patients of a large TURP population deteriorated to a comatose state as a result of hyponatremia. These patients were generally older, with larger prostates, and longer resection times than the average for transurethral resection of the prostate. They also consistently had serum sodium levels postoperatively of near 120 mEq./L. or below. It was noted that obtundation can occur immediately or be delayed several hours. Even though no deaths occurred, awareness of the possibility of post-TURP hyponatremia and prompt treatment with hypertonic saline were shown to reduce morbidity significantly.

Management of hydrocephalus secondary to intraventricular hemorrhage in the preterm infant with a subcutaneous ventricular catheter reservoir.

To control hydrocephalus resulting from massive intraventricular hemorrhage in premature neonates with respiratory distress syndrome, we inserted a specially designed low profile subcutaneous ventricular catheter reservoir (reservoir) by the 12th day of life (average; range, 3 to 30 days) in 20 neonates whose mean birth weight was 1110 +/- 270 g (28.7 +/- 1.6 weeks of gestation). The reservoir was repeatedly aspirated over 10 to 48 days. No cerebrospinal fluid infection, reservoir obstruction, or breakdown of the skin overlying the reservoir occurred. Serial computed tomographic scans documented control of the hydrocephalus and an increase in the thickness of the cortical mantle of the survivors. No mortality was associated with placement of the reservoir or its subsequent conversion, if necessary, to a ventriculoperitoneal shunt. However, only 7 of the 20 infants survived. On follow-up 3 to 5 years later, 2 of the 7 have normal intellectual and motor development. Two infants are normal intellectually, but have a motor deficit. The remaining 3 patients have both significant intellectual and motor developmental delay. The use of the reservoir is offered as a safe and effective alternative to repeated ventricular punctures, external ventricular drainage, or initial shunting. Aggressive management of hydrocephalus secondary to intraventricular hemorrhage may improve neurological function in some surviving neonates.

Drug abuse and incarcerated women. A research review.

Drug abuse is the primary reason women enter prison and is the primary health problem of women in prison. There has been little research conducted specifically with this population; information must be drawn from studies with nonincarcerated addicted women and incarcerated addicted men. The purpose of this paper is to review what is known about the treatment and aftercare needs of this group (including relapse and recidivism prevention) and to propose an agenda for future research.

Temporal changes in soil properties at an upland Scottish site between 1956 and 1997.

The aim of this study was to examine the frequency with which soil samples require to be taken in order to determine significant temporal changes in soil properties. The examination was carried out using data from Glensaugh Research Station in north-east Scotland where podzolic soils were sampled in 1956, 1977 and 1997, and by re-analysis of archived material. Significant differences in chemistry due to storage were detected, particularly decreases in pH of air-dried organic soils. In these cases original data were used for statistical analysis to establish changes between 1956 and 1997. Temporal changes were found for exchangeable Ca and Mg which generally decreased with time throughout the soil profile, whereas exchangeable H increased. Derived data, such as percent base saturation, declined dramatically due to decreases in exchangeable base cations. Similar podzolic soils were sampled at an adjacent Environmental Change Network (ECN) site in 1993. Application of statistical techniques to the ECN soil chemistry data allowed an estimation of the detectable change between any two years. These data along with the rates of temporal change from 1956 to 1997 allowed the calculation of the number of years required for measurable changes to be achieved. These changes and sampling intervals vary among different horizons and chemistries. Although they are site-specific, they do confirm that the current ECN protocols of a 5-year and 20-year sampling would be appropriate in order to detect changes in soil properties over time at this site.

Consciousness raising in participatory research: method and methodology for emancipatory nursing inquiry.

Nursing practice and research that are grounded in emancipatory inquiry challenge the inequalities and injustices that affect health in this society. Increasing numbers of nurse scholars have explored the use of emancipatory inquiries (particularly feminist theory and critical theory) as philosophic bases for nursing practice and as theoretical or methodologic perspectives for nursing research. This article presents participatory research as a methodology and consciousness raising as a method for nursing research that addresses the emancipatory goals of feminist and critical theories. An example of consciousness raising in a participatory nursing research project is offered. It is argued that participatory research can produce a revolutionary emancipatory knowledge for the future of nursing and health care.

3-Pyridyl ethers as SPECT radioligands for imaging nicotinic acetylcholine receptors.

To develop a suitable single photon emission computed tomography (SPECT) radioligand for neuronal nicotinic acetylcholine receptors (nAChRs) that displays faster in vivo kinetics than 5-[123I]iodo-A-85380, we synthesised the radioiodinated analogue of A-84543. 5-[123I]Iodo-A-84543 was prepared by electrophilic iododestannylation in a modest yield of 23%. In the baboon brain, 5-[123I]iodo-A-85380 displayed a profile consistent with the known distribution of nAChRs, however, 5-[123I]iodo-A-84543 displayed a homogenous uptake with no preferential localisation in regions known to contain nAChRs. To examine the effect of halogen substitution on the 3-pyridyl ether, A-84543, the 5-chloro, 5-bromo and 5-iodo analogues were synthesised and evaluated with respect to nAChR binding. In vitro binding data revealed that halogen substitution at the 5-position of A-84543 was not well tolerated with an increase in halogen size resulting in lower binding towards nAChRs. The 5-chloro analogue 4 displayed highest affinity, Ki =1.3 nM, compared to the 5-bromo and 5-iodo compounds, 5 Ki =3.3 nM and 3 Ki =40.8 nM, respectively. Taken together, these results clearly indicate that 5-[123I]iodo-A-84543 is not suitable for the study of nAChRs in vivo using SPECT.

HIV infection: risks to health care workers and infection control.

The risk for occupational transmission of HIV-1 in the health setting is extremely small. Current data from a number of prospective studies of health care workers sustaining adverse exposure to blood or other body fluids from patients infected with HIV-1 demonstrate that the rate for transmission of infection following a needlestick injury is approximately 0.5%. Similar data regarding the risk for occupational transmission of hepatitis B virus infection, however, indicates that the rate of transmission of infection with this virus following a needlestick injury may be as high as 30% and the risk for transmission of other blood-borne infections is poorly defined. Infection control precautions designed to minimize the risk for HIV-1 infection were recommended by CDC shortly following the first reported case of AIDS in the United States. These measures were implemented for patients diagnosed with or suspected to be infected with HIV-1, determined by history-taking or serologic evaluation. The inability of these mechanisms to accurately identify all infected individuals, coupled with the occurrence of undiagnosed or unrecognized blood-borne infections, emphasizes the need for health care workers to consider all patients as potentially infected with HIV-1 or other blood-borne pathogens. Implementation and enforcement of universal precautions should minimize exposure of health care personnel to blood and body fluids and thus substantially reduce the risk for occupational transmission of HIV-1 and other blood-borne infections in the health care environment.