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

Kir4.1 channels contribute to astrocyte CO2/H+-sensitivity and the drive to breathe

Astrocytes in the retrotrapezoid nucleus (RTN) stimulate breathing in response to CO2/H+, however, it is not clear how these cells detect changes in CO2/H+. Considering Kir4.1/5.1 channels are CO2/H+-sensitive and important for several astrocyte-dependent processes, we consider Kir4.1/5.1 a leading candidate CO2/H+ sensor in RTN astrocytes. To address this, we show that RTN astrocytes express Kir4.1 and Kir5.1 transcripts. We also characterized respiratory function in astrocyte-specific inducible Kir4.1 knockout mice (Kir4.1 cKO); these mice breathe normally under room air conditions but show a blunted ventilatory response to high levels of CO2, which could be partly rescued by viral mediated re-expression of Kir4.1 in RTN astrocytes. At the cellular level, astrocytes in slices from astrocyte-specific inducible Kir4.1 knockout mice are less responsive to CO2/H+ and show a diminished capacity for paracrine modulation of respiratory neurons. These results suggest Kir4.1/5.1 channels in RTN astrocytes contribute to respiratory behavior

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

Transcriptome analysis of the distal small intestine of Cftr null mice

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CFTR anion channel. Loss of CFTR function in pancreatic, biliary and intestinal epithelia, severely affects gastrointestinal function. Transcriptome analysis indicated the activation of an innate and adaptive immune response in the distal small intestine of Cftr null mice. Inflammation was associated with differential regulation of numerous genes involved in the transport and metabolism of nutrients and, particularly, lipids, that are targeted by ligand-dependent nuclear receptors and/or HNF4α. Among the most strongly down-regulated genes are the FXR targets Fgf15 and Nr0b2, the PPARα target Pdk4, and the PXR target Ces2a, whereas expression of the CF modifier gene Slc6a14 was strongly increased. Most changes in gene expression were reversed by bacterial containment. Our data suggest that the gut microbiota has a pervasive effect on gene expression in CF mice, affecting enterocyte maturation, lipid metabolism, and nutrient absorption in CF

The Influence of Moderate Hypercapnia on Neural Activity in the Anesthetized Nonhuman Primate

Hypercapnia is often used as vasodilatory challenge in clinical applications and basic research. In functional magnetic resonance imaging (fMRI), elevated CO2 is applied to derive stimulus-induced changes in the cerebral rate of oxygen consumption (CMRO2) by measuring cerebral blood flow and blood-oxygenation-level–dependent (BOLD) signal. Such methods, however, assume that hypercapnia has no direct effect on CMRO2. In this study, we used combined intracortical recordings and fMRI in the visual cortex of anesthetized macaque monkeys to show that spontaneous neuronal activity is in fact significantly reduced by moderate hypercapnia. As expected, measurement of cerebral blood volume using an exogenous contrast agent and of BOLD signal showed that both are increased during hypercapnia. In contrast to this, spontaneous fluctuations of local field potentials in the beta and gamma frequency range as well as multiunit activity are reduced by ∼15% during inhalation of 6% CO2 (pCO2 = 56 mmHg). A strong tendency toward a reduction of neuronal activity was also found at CO2 inhalation of 3% (pCO2 = 45 mmHg). This suggests that CMRO2 might be reduced during hypercapnia and caution must be exercised when hypercapnia is applied to calibrate the BOLD signal

Surface Hardness Impairment of Quorum Sensing and Swarming for Pseudomonas aeruginosa

The importance of rhamnolipid to swarming of the bacterium Pseudomonas aeruginosa is well established. It is frequently, but not exclusively, observed that P. aeruginosa swarms in tendril patterns—formation of these tendrils requires rhamnolipid. We were interested to explain the impact of surface changes on P. aeruginosa swarm tendril development. Here we report that P. aeruginosa quorum sensing and rhamnolipid production is impaired when growing on harder semi-solid surfaces. P. aeruginosa wild-type swarms showed huge variation in tendril formation with small deviations to the “standard” swarm agar concentration of 0.5%. These macroscopic differences correlated with microscopic investigation of cells close to the advancing swarm edge using fluorescent gene reporters. Tendril swarms showed significant rhlA-gfp reporter expression right up to the advancing edge of swarming cells while swarms without tendrils (grown on harder agar) showed no rhlA-gfp reporter expression near the advancing edge. This difference in rhamnolipid gene expression can be explained by the necessity of quorum sensing for rhamnolipid production. We provide evidence that harder surfaces seem to limit induction of quorum sensing genes near the advancing swarm edge and these localized effects were sufficient to explain the lack of tendril formation on hard agar. We were unable to artificially stimulate rhamnolipid tendril formation with added acyl-homoserine lactone signals or increasing the carbon nutrients. This suggests that quorum sensing on surfaces is controlled in a manner that is not solely population dependent

A Putative Homologue of CDC20/CDH1 in the Malaria Parasite Is Essential for Male Gamete Development

Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis

Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations

Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G > T, both reside in exon 13. Skipping exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2(armc1) mutants resulted in the production of usherin Delta exon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in induced pluripotent stem cell (iPSC)-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection and induced a detectable level of exon skipping until at least 6 months post-injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13