Mycoplasma hyopneumoniae. Basic motifs like promoters and terminators have already been described, but no other cis-regulatory elements have been found. DNA repeat sequences have been shown to be an interesting potential source of cis-regulatory elements. In this work, a genome-wide search for tandem and palindromic repetitive elements was performed in the intergenic regions of all coding sequences from M. hyopneumoniae strain 7448. Computational analysis demonstrated the presence of 144 tandem repeats and 1,171 palindromic elements. The DNA repeat sequences were distributed within the 5’ upstream regions of 86% of transcriptional units of M. hyopneumoniae strain 7448. Comparative analysis between distinct repetitive sequences found in related mycoplasma genomes demonstrated different percentages of conservation among pathogenic and nonpathogenic strains. qPCR assays revealed differential expression among genes showing variable numbers of repetitive elements. In addition, repeats found in 206 genes already described to be differentially regulated under different culture conditions of M. hyopneumoniae strain 232 showed almost 80% conservation in relation to M. hyopneumoniae strain 7448 repeats. Altogether, these findings suggest a potential regulatory role of tandem and palindromic DNA repeats in the M. hyopneumoniae transcriptional profile.Transcriptional regulation, a multiple-step process, is still poorly understood in the important pig pathogen  Mycoplasma hyopneumoniae is a diminutive bacterium, characterized by a small genome (0.92 Mb) with a low GC content [M. hyopneumoniae have been sequenced [M. hyopneumoniae. The occurrence of transcription units [ content . It is cenome 0.9 Mb with  content . Until nequenced , 4–7 andequenced . Transcron units , promoteon units , 12 and on units  has alreDNA repeats, widespread and characterized in eukaryote genomes, can also play an important role in prokaryote genomic regulation . It is uNeisseria species, Haemophilus parainfluenzae, Moraxella catarrhalis [Mycoplasma hyorhinis [Mycoplasma spp., the presence of some repetitive elements has been described in Mycoplasma genitalium [Mycoplasma gallisepticum [Mycoplasma bovis [M. hyorhinis [Mycoplasma fermentans [Mycoplasma synoviae [Mycoplasma pneumoniae [Mycoplasma hominis [Mycoplasma penetrans [Mycoplasma pulmonis [Mycoplasma mobile [Mycoplasma mycoides [M. hyopneumoniae the presence of SSR elements [in silico prediction associated with experimental validation was performed, aiming to verify and associate the presence of tandem and palindromic repeats as transcriptional regulatory sites.DNA repeats can be found within coding-sequences (CDS), in intergenic regions, or in transposable elements, reflecting both regulatory and structural requirements for the bacterial chromosome , 18. Phaarrhalis  and Mycoyorhinis  involvinnitalium –24, Mycosepticum , 25, 26,ma bovis , M. hyoryorhinis , Mycoplarmentans , Mycoplasynoviae , 30, Myceumoniae , 24, 31, hominis , Mycoplaenetrans , Mycoplapulmonis , 24, 33,a mobile  and Mycomycoides , 34 genoelements  and someelements  have alrMycoplasma spp. [M. hyopneumoniae strain 7448 (NSDC AE017244.1) CDSs which were used as input for the software’s prediction. The annotation of the genome was manually cured, with confident translational start codons. For each type of repeat sequence, two independent software packages were used, as detailed below.Based on the most common repeats already described in prokaryote genomes, especially in sma spp. , two typIn the present work, SSR is defined as a sequence containing 2 to 10 repeated nucleotides as described by Huang et al. . The algSSRM is defined as a mononucleotide repeated at least 8 times in a head-to-tail orientation. The software packages used to predict SSRM were Tandem Repeat Finder (TRF)  and SSRIDR is defined as an element with 11 to 50 nucleotides repeated at least twice in a tandem way. TRF and ETANDEM from The European Molecular Biology Open Software Suite (EMBOSS)  were usePALG is an inverted repeat of 9 to 20 nucleotides containing additional gaps ranging from 5 to 15 bases. Palindrome from EMBOSS and Palindrome Search  were thePAL is an inverted repeat of 9 to 15 bases with no gaps. The same software was used to predict PAL and PALG, and only elements that had the maximum of 2 mismatches between the inverted repeat were considered.Just PALG and PAL elements that were located up to 300 bp distant from the ATG were analyzed, the remaining data was excluded. After the individual search of each element, the results were grouped and all overlaps were eliminated according to the following hierarchy: PAL, SSRM, DR, SSR and PALG.M. hyopneumoniae strain 7448 were classified based on the downstream CDS, ATG distance, association with promoter sequences and transcription unit distribution.All repetitive elements found in noncoding regions of A search for common motifs among repeat classes was performed through the web server MEME SUITE , using dM. hyopneumoniae strain 7448 transcription units [M. hyopneumoniae strain 7422 –pathogenic; M. hyopneumoniae strain J–nonpathogenic; and Mycoplasma flocculare ATCC 27716 –phylogenetically related to M. hyopneumoniae and nonpathogenic. Therefore, a BLAST [To validate the relevance of repetitive elements found, comparison analysis was performed using three different approaches. Initially, all tandem repeats  found upstream of the first gene of on units , 10 were a BLAST  search wM. hyopneumoniae strain 7448 were compared among the same mycoplasma genomes previously described. The repeat search was conducted directly upstream of the region of the orthologous gene or in the upstream region of the corresponding transcription unit. The comparison of PAL and PALG among mycoplasmas genomes were performed by Pattern Locator software [M. hyopneumoniae strain 7448 or 2) absent–the repetitive element PALG or PAL was not identified. The level of conservation (%) of palindromic repeats between mycoplasma genomes was calculated by dividing the number of conserved repeats found in the noncoding region of orthologue gene by the total number of repeats found in the respective M. hyopneumoniae strain 7448 gene .Culture conditions, RNA isolation and cDNA synthesis by Reverse Transcription (RT) were performed as described in Siqueira et al. . Target Quantitative PCR (qPCR) assay was performed using 1:50 cDNA as template and Platinum SYBR Green qPCR SuperMix-UDG  on 7500 Real-Time PCR Systems . The qPCR reactions were carried out at 90°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. A melting curve analysis for each primer pair was done to verify primer efficiency.M. hyopneumoniae strain 7448, M. hyopneumoniae strain J and M. flocculare ATCC 27716 RNAs. Relative expression of mRNA was calculated by the 2-ΔCt method [lon (MHP7448_0524). Reference gene was determined in a specific assay where expression profiles of several genes were evaluated in the three mycoplasma RNAs tested . The gene that showed no differential expression in all mycoplasma genomes tested was used in relative expression calculations. Three technical and two biological replicates were done for each target evaluated. Statistical analyses were performed using GraphPad Prism 6 software by One-way ANOVA followed by Tukey’s multiple comparison test (P < 0.05).Relative expression of each gene was evaluated in t method . The thrM. hyopneumoniae strain 7448. Among the tandem repeats, 272 SSRM, 55 SSR and 13 DR repeats were found. Palindromic elements were divided into 689 PAL and 1,190 PALG  and 1,171 palindromic repeats   . The pre98 PALG) .M. hyopneumoniae strain 7448 CDSs demonstrated an average of 3 PALG, 1 PAL, 1 SSRM, 1 SSR and 1 DR repeat per 5’ upstream region of a unique CDS  had putative promoter sequences   of M. hyr et al. ) or repeThe distribution of tandem and palindromic elements showed that individually, PALG were present in the majority of the genes 5’ upstream regions investigated (57%). The combination of PALG plus SSRM repeats was seen in 11% of genes 5’ upstream regions. PALG plus PAL elements were seen in 9% of genes 5’ upstream regions. None of the genes had exclusively DR repeats or a combination of all repeat types in a single 5’ upstream region .M. hyopneumoniae strain 7448, 149 (92%) of them contained a putative regulatory element (repeat or promoter) in the respective 5’ upstream region from the start gene of the transcription unit. Among the 149 transcription units, 56% contained palindromic elements associated with putative promoters; 21% showed the presence of the three elements , and 10% contained only palindromic sequences. The three situations mentioned above represented 87% of all possible cases of elements in the regulatory regions of the transcriptional units  and M. hyopneumoniae strain J (nonpathogenic), and also with the phylogenetically related nonpathogenic mycoplasma species, M. flocculare, was explored  than between different species (M. hyopneumoniae and M. flocculare)  compared with SSRM  . HoweverSSRM see . Also, ttion see .M. hyopneumoniae strain 7448 and M. hyopneumoniae strain 7422 orthologous genes and only those elements that fitted this feature were analysed in relation to M. hyopneumoniae strain J or M. flocculare orthologues. These comparisons were named “Pathogenic-non-Pathogenic species” (P-nP(sp)) and “Pathogenic-non-Pathogenic genera” (P-nP(gn)), respectively. Among M. hyopneumoniae strains, two DR repeats types, 12 SSR and 39 SSRM could be analysed. Higher conservation could be observed in P-nP(sp), and a reversed scenario was seen in P-nP(gn), where nonconserved and absence of tandem repeats were found  showed 40% of repeat conservation and 9% and 6% of nonconservation and absence, respectively. The P-nP(gn) analysis revealed that only 6% of repeats were conserved and 56% were nonconserved or absent among orthologous genes , palindromic elements were located directly in the CDS 5’ upstream region. However, this situation was not applied to MHP7448_0005, MHP7448_0006, MHP7448_0361 and MHP7448_0362 CDSs. Therefore, a search for repetitive elements was performed in the upstream region of Transcriptional Unit (TU)_01 (containing MHP7448_0005 and MHP7448_0006) and TU_63 (containing MHP7448_361 and MHP7448_362). Using this approach, PAL and PALG elements could be found. Comparative analysis revealed that all M. hyopneumoniae strain 7422 and M. hyopneumoniae strain J adhesin orthologous genes were present in the respective genome and a palindromic repeat conservation level of 87% and 69%, respectively, were established. Comparison between M. hyopneumoniae strain 7448 and M. flocculare demonstrated only 27% of conserved elements in 5’ upstream region of the orthologue genes . Some of these CDSs encode important proteins to bacterial survival, like transporters or signalling molecules, and others encode hypothetical proteins. Nine genes with palindromic sequences at the upstream region of putative adhesins proteins were also selected , were found in upstream region of the gene, nonsignificant differences in the basal transcription level were observed. In the same way, in genes with nonconserved SSR repeats in intergenic region (SSR_49_LI and SSR_30_LI in M. hyopneumoniae strain J) nonsignificant differences in gene expression were seen. However, when nonconserved SSR repeats were analysed in M. flocculare, differences in gene expression were observed. The absence of SSR element (SSR_05_LI) in M. hyopneumoniae strain J and M. flocculare upstream region of orthologous demonstrated different transcription profile  PAL and PALG present in MHP7448_0005 and MHP7448_0006 orthologues from observed . A detaiM. flocculare orthologous had gene expression variations under the tested conditions. In M. hyopneumoniae strain J only the classified absent repeats (SSR) demonstrate some potential bias in transcription. In relation to PAL and PALG the loss of some elements (lower conservation) could be related to the observed differences in gene expression. Orthologous of MHP7448_497, MHP7448_272 and MHP7448_272 in M. hyopneumoniae strain J and MHP7448_108, MHP7448_107, MHP7448_271 and MHP7448_272 could support this data  among the 5’ upstream regions of his data . All theM. hyopneumoniae strain 232 were compared against the M. hyopneumoniae strain 7448 genome  genes had exactly the same element found in M. hyopneumoniae strain 7448 and only 17 (10%) did not have any corresponding elements  compared with tandem repeats (144 elements). Similar results were also reported by Huang et al. [Mycoplasma agalactiae, M. bovis, M. fermentans and M. mycoides. Further analysis of the repetitive elements identified in our work demonstrated that common motifs between tandem or palindromic repetitive elements could not be established. The pattern frequently observed was the presence of AT-rich sequences in all elements investigated. In SSRM, for example, mononucleotides containing only Adenine (A) or Thymine (T) repeated 8 to 25 times were observed, in accordance with the AT-rich genome of M. hyopneumoniae strain 7448 [Prokaryote genomes are extremely diverse in terms of nucleotide composition and the presence of distinct patterns of repeat sequences that could affect the physical properties of DNA molecules . In thisg et al.  who inveM. hyopneumoniae strain 7448 transcriptional units demonstrating that different mechanisms of regulation can be considered , compared with nonpathogenic mycoplasmas (M. hyopneumoniae strain 7448 versus M. hyopneumoniae strain J and M. hyopneumoniae strain 7448 versus M. flocculare) as shown in M. hyopneumoniae strains and M. flocculare was performed with similar results as previous demonstrated for the tandem repeats. Adhesin-coding genes show greater differences in the number of palindromes in non-pathogenic strains (M. hyopneumoniae strain J and M. flocculare) than the two M. hyopneumoniae pathogenic strains (7448 and 7422). An example was seen in MHP7448_0107, MHP7448_0271 and MHP7448_497 genes, which showed 100% conservation in M. hyopneumoniae strain 7422 and a marked reduction in palindromic elements in M. hyopneumoniae strain J and M. flocculare. In this work tandem repeats (SSR and SSRM) were also reported in 3 of the 16 putative adhesin coding genes analysed . Previous authors have reported that tandem repeats, more precisely SSR, were able to influence adhesin gene expression [Combinations of palindromic elements, tandem repeats and promoter sequences could be detected in 92% of all ered see . Resultsered see . Previoupression , 25 but M. hyopneumoniae strains (7448 and J) and M. flocculare. Detailed analysis of comparative studies of the presence of tandem repeats  in different genes and palindromic elements found in putative adhesin-coding genes were the basis of the experimental investigation. In general, the results of qPCR assay revealed that conserved elements among distinct mycoplasmas seem to have no influence on gene expression, as observed in the MHP7448_0623 gene (M. hyopneumoniae strain J and M. flocculare). The same pattern was present in SSRM found in the 5’ upstream region of the glyA (glycine hydroxymethyltransferase) and sipS  genes, which are conserved in pathogenic mycoplasmas strains and nonconserved in nonpathogenic mycoplasmas  [M. gallisepticum (M9/pMGA adhesin proteins) [The adhesin-coding genes are important virulence factors and seem to be regulated depending on the presence or absence of palindromic repeats. Therefore, it is possible to suggest a relation between the differences in gene transcriptional level and the divergence of DNA repeats found in the 5’ upstream regions among mycoplasma orthologous genes, as observed in genes coding for adhesin proteins P102 (MHP7448_0271) and P97 (MHP7448_0272) and genes coding for proteins in the MgPa operon (MHP7448_0005 and MHP7448_0006). Similar results were previously demonstrated for genes coding adhesin in roteins)  and in Mroteins) , whereasM. genitalium genome represent hypervariable sites in the MgPa operon, which encodes two adhesin proteins that represent surface proteins required for the development of the terminal organelle structure and attachment of the organism to host epithelial cells. Such adhesin variation may allow this organism to evade the host immune response and to adapt to diverse host microenvironments, thus establishing persistent infection [M. gallisepticum, the GAA trinucleotide repeat region regulates M9/pMGA gene expression (which encodes adhesin(s) associated with haemagglutination). Depending on the copy number of GAA in the intergenic regions, gene expression can be inhibited [M. bovis, repetitive elements were involved in a family of phase- and size-variable membrane surface lipoprotein antigens [The repeats found in the nfection . In M. gnhibited . In M. bantigens .M. hyopneumoniae strain 7448 [Besides transcriptional regulation, DNA repeats could also be involved in translation regulation. Identification of transcription start site (TSS) was performed for 23 genes of ain 7448 . The anaM. hyopneumoniae 232 growing under several specific culture conditions [M. hyopneumoniae strain 7448 orthologues from differentially expressed genes of the M. hyopneumoniae strain 232 were selected and analysed for repeat conservation. This study revealed that almost all differentially expressed genes had palindromic elements in their 5’ upstream regions. Orthologous gene comparisons between the two M. hyopneumoniae strains (7448 and 232) showed that the PAL and PALG repeats were approximately 80% conserved in M. hyopneumoniae strain 232, reinforcing the putative regulatory role of these repetitive elements since temperature, pH and others factors can influence the formation of secondary structures, as well as could perturb the stability of the nucleotide bindings [Previous studies have demonstrated differential expression of genes from nditions –50, so abindings .M. hyopneumoniae strain 7448 CDSs was described. Relevance in transcriptional regulation of the DNA repeats found could be established through comparison analysis, demonstrating that repeats could be perpetuated among related mycoplasmas, and some of them could be involved in pathogenicity. Experimental assays revealed differential expression of genes differing in repetitive elements in the 5’ upstream region, reinforcing the putative regulatory role of palindromic and tandem repeats. Previously described differentially expressed genes show palindromic elements in the upstream region of the start codon and are conserved between two strains of M. hyopneumoniae. All of these findings suggest the importance of repetitive DNA elements in M. hyopneumoniae, and contribute to expand the source of regulatory sequences that can modulate gene expression in the important pig pathogen M. hyopneumoniae.In this work, the presence of SSR, SSRM, DR, PAL and PALG DNA repeats found in the 5’ upstream regions of S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(PDF)Click here for additional data file.


Pias3 gene, which resulted in complete loss of the Pias3 protein. −/−Pias3 mice did not show any overt phenotype, and retinal lamination appeared normal even at 18 months. We detected reduced photopic b-wave amplitude by electroretinography following green light stimulation of postnatal day (P)21 −/−Pias3 retina, suggesting a compromised visual response of medium wavelength (M) cones. No change was evident in response of short wavelength (S) cones or rod photoreceptors until 7 months. Increased S-opsin expression in the M-cone dominant dorsal retina suggested altered distribution of cone photoreceptors. Transcriptome profiling of P21 and 18-month-old −/−Pias3 retina revealed aberrant expression of a subset of photoreceptor genes. Our studies demonstrate functional redundancy in SUMOylation-associated transcriptional control mechanisms and identify a specific, though limited, role of Pias3 in modulating spatial patterning and optimal function of cone photoreceptor subtypes in the mouse retina.Protein inhibitor of activated Stat 3 (Pias3) is implicated in guiding specification of rod and cone photoreceptors through post-translational modification of key retinal transcription factors. To investigate its role during retinal development, we deleted exon 2-5 of the mouse  Summary: Loss of Pias3 in mice results in altered dorso-ventral patterning of retinal cone photoreceptors by modulating the expression of a subset of genes, but does not affect rod development. The vertebrate retina is designed to maximize the capture, integration, and transmission of visual information and consists of a stratified architecture with three cellular layers that include six neuronal cell types . The resThe differentiation of photoreceptors from multipotent retinal progenitor cells is orchestrated by the combinatorial and synergistic or antagonistic action of a small number of transcription factors . RetinalPost-translational modifications, such as phosphorylation and SUMOylation, can modulate the activity of transcription factors , includiPias3 gene (−/−Pias3) to elucidate Pias3 function during development. Despite a demonstrated role of Pias3 in multiple cellular pathways   staining of methacrylate sections revealed proper lamination and thickness of retinal cell layers, including photoreceptors, in both young [postnatal day (P)21] and old (18 month) mice (Pias3 was reported to control photoreceptor development (−/−Pias3 mice by electroretinography (ERG). Scotopic (rod-mediated) and UV (S-cone mediated) responses of −/−Pias3 retina did not differ significantly from the wild-type at P21 and begin to decline only by 7 months (−/−Pias3 mice to green stimuli (M-cone mediated) was impaired  and remained so at least until 12 months (Pias3.A targeting vector with LoxP sites spanning exon 2 to 5 of the th) mice A. Given elopment , 2010, w7 months . However2 months D and 3. −/−Pias3 retina showed altered dorsoventral gradient of S-opsin, with more S-opsin-positive cones identified in the dorsal region of both flat-mounted (−/−Pias3 retina (data not shown). A series of IHC stainings did not show any significant difference between wild-type (+/+Pias3) and −/−Pias3 retina for markers of rods , cones , Müller glia , activated Müller glia , bipolar cells , ganglion cells , or amacrine and horizontal cells  .Immunohistochemical (IHC) staining of P21 -mounted A and sec-mounted B retina., Calb1) A. Since , Calb1) B. No app−/−Pias3 retina (Table S1). We selected 28 uncharacterized or retina-related DEGs at P21 with FPKM >30 for qPCR validation and 24 of these were confirmed as being up- or down-regulated in both RNA-Seq and qPCR analyses (Opn1mw (M-opsin) and Opn1sw (S-opsin) were not identified as DEGs by our criteria though small differences were detected by qPCR . Using the same filtering criteria, whole transcriptome analysis of 18 month old −/−Pias3 retina identified only 69 DEGs (Table S2), including fewer rod- and slightly more cone-enriched genes compared to P21 analysis   of young (P21) and aged (18 month) wild-type and − retina  and 7. Uanalyses C. Opn1mw by qPCR C. Using Seq data , we obse−/− DEGs D. Furtheer genes E. Pathwaer genes F. Circul− retina G and som− retina H. To detctively) C. Furthectively) D. The nuPias proteins function as E3 SUMO ligases that facilitate, but are not required for, the covalent linkage of SUMO groups to lysine residues of target proteins . SUMOylain vivo electroporation was used to knockdown Pias3 in developing photoreceptors . In addition, analysis of published RNA-Seq data from flow-sorted photoreceptors of wild-type and cone-only retina showed that multiple E3 SUMO ligases are expressed in both rod and cone photoreceptors (Table S3)  . Such fuable S3) , and Trβable S3) . CompensPias1−/− , Pias2−/Pias2−/− , and PiaPias4−/−  mice, whPias4−/−  or SUMO-−/− mice . Breedin−/−Pias3 retina is intriguing. Pias3 is expressed in M-cones (Table S3). This explanation could also account for higher S-opsin expression in the dorsal −/−Pias3 retina, resulting from incomplete suppression of S-opsin in cells that are fated to be M-cones. Gene expression changes in M-cone population are likely masked because of their small numbers in the whole retina, and transcriptome profiling of isolated M- and S-cones from the −/−Pias3 retina would be necessary to test this hypothesis.The observation of an early M-cone defect in  M-cones  and migh−/−Pias3 retina suggest that Pias3 reinforces or augments gene expression changes rather than controls broader cell fate or developmental decisions. We propose that the addition of SUMO groups to target proteins, such as Nrl and Nr2e3, by Pias3 is a transient and dynamic process that serves to modulate (enhance or suppress) their activity in a context-dependent manner and that additional SUMO-conjugating enzymes are able to compensate its function in −/−Pias3 retina. Fine adjustments in gene expression patterns and redundancy in control mechanisms are necessary to establish and optimize spatial and functional organization of photoreceptors in the mammalian retina.The mild phenotype and seemingly unperturbed global SUMOylation status of Pias3 gene followed by neo cassette enclosed by FRT sites. Correctly targeted embryonic stem cell lines were identified by PCR at both ends designed to detect homologous recombination and used to generate chimeric mice. Original founders were mated twice with Actin-Flp recombinase to remove the neo cassette and were backcrossed to C57BL/6J mice. Specific deletion in the retina and the forebrain was achieved by crossing f/fPias3 mice with Rx-Cre-positive males . Wild-type and knockout mice of either sex were used for all experiments. All experiments were conducted according to protocols approved by a local Institutional Animal Care and Use Committee and adhered to the Association for Research in Vision and Ophthalmology Statement for animal use in ophthalmic and vision research.A gene-targeting vector was constructed to add LoxP sites flanking exons 2 to 5 of the ve males . HoweverTable S5). Alleles with Pias3 exon 2-5 deletion were detected in the same manner but using a reverse primer within exon 6 of the gene to amplify a 1 kb product generated by the knockout allele . Rx-Cre was detected by amplification of a 350 bp fragment .Genomic DNA was extracted from tail clippings and both wild-type and floxed alleles were detected via amplification using primers flanking either side of the 5′ LoxP site yielding products of 166 bp and 200 bp, respectively  and 20 mM N-ethylmaleimide (NEM) (Sigma-Aldrich). Supernatants were quantified by BCA protein assay according to manufacturer's protocol (Pierce) and solubilized in 4× Laemmli buffer+β-mercaptoethanol. After denaturation, 20 µg of the total protein extract was separated by SDS-PAGE and transferred to PVDF membrane (Bio-Rad). Standard immunoblot procedure was followed, as previously described .Table S4) diluted in blocking solution. The next day, they were washed 4× with 0.2% Triton X-100 in PBS then incubated at room temperature for 1 h with appropriate secondary antibodies (Table S4) diluted in blocking solution. After four subsequent washings, sections were stained for 5 min with DAPI in 1× PBS and washed again, mounted with Fluoromount-G  and imaged using a Zeiss LSM 700 confocal microscope.Eyeballs were enucleated and fixed in 4% PFA for 30 min prior to dissection. For cryosections, the eyecup was fixed for an additional 30 min and cryoprotected in 30% sucrose before being frozen in OCT embedding medium  then cut 12 µm thick. For whole mounts, the retina was fixed for an additional 30 min. Both cryosections and whole mounts were blocked for 1 h at room temperature in blocking solution , then incubated overnight at 4°C with primary antibodies .Total RNA was extracted by Trizol LS reagent (Thermo Fisher), according to the manufacturer's protocol, and its quality was assessed using an Agilent 2100 bioanalyzer and RNA 6000 Nano chip. Only RNA samples with RNA integrity number (RIN) of ≥7.9 were subjected to first-strand cDNA synthesis using SuperScript II Reverse Transcriptase (Life Technologies). cDNA samples then served as templates for quantitative PCR, per Fast SYBR Green Master Mix manufacturer's protocol (Applied Biosystems) performed in biological and technical triplicates. Primer sequences are provided in +/+Pias3 and −/−Pias3 mice at P21 and at 18 months of age. Total RNA was extracted by Trizol LS reagent (Thermo Fisher), according to the manufacturer's protocol, and its quality was assessed using an Agilent 2100 bioanalyzer and RNA 6000 Nano chip. Only RNA samples with RIN≥7.9 were used. Stranded RNA-seq libraries were constructed from 100 ng of total RNA using a modified TruSeq RNA Sample preparation kit protocol (http://pantherdb.org/).Whole transcriptome analysis was performed using three independent biological replicates from retina of protocol . Paired-protocol  and aligprotocol . Normaliprotocol . An FPKMt-test) was performed using GraphPad Prism 7 software.ERG responses to light stimulation were recorded simultaneously from both eyes on an Espion Electrophysiology System  as previously described for both photopic and scotopic conditions . A minim


We determined the proportion of perinatally HIV-exposed uninfected (HEU) newborns who were referred for additional hearing testing, and evaluated the association between in utero ARV exposures and newborn hearing screening results. Using a nested case-control design, we also examined congenital CMV infection in infants with and without screening referral. Congenital CMV infection was determined based on CMV DNA detection using a nested PCR assay in peripheral blood mononuclear cells obtained within 14 days of birth. Among the 1,435 infants , 45 (3.1%) did not pass the hearing screen and were referred for further hearing testing. Based on exact logistic regression models controlling for maternal use of tobacco and ototoxic medications, first trimester exposure to Tenofovir was associated with lower odds of a newborn hearing screening referral . Exposure to Atazanavir was linked to higher odds of newborn screening referral, although not attaining significance . Maternal ARV use may have varying effects on newborn hearing screenings. These results highlight the importance for audiologists to be knowledgeable of in utero ARV exposures in HEU children because of the possibility of higher referrals in these children.Perinatal HIV infection and congenital cytomegalovirus (CMV) infection may increase the risk for hearing loss. We examined 1,435 infants enrolled in the Surveillance Monitoring of ART Toxicities (SMARTT) study of the Pediatric HIV/AIDS Cohort Study (PHACS) network, a prospective study of the safety of  Late Studies of hearing screening results obtained from babies born to mothers infected with HIV are limited. Two studies have observed about two-fold higher risk of hearing loss for HIV-exposed uninfected (HEU) infants as compared to HIV-unexposed and uninfected (HUU) infants, although neither finding attained statistical significance due to the low prevalence of hearing loss ,10. In ain utero antiretroviral (ARV) exposures with newborn hearing screening results; 3) evaluate the association between newborn hearing screening results and other risk factors and 4) examine congenital CMV infection in a subset of infants with and without further hearing referrals as a result of the newborn screening.Early identification of newborn hearing loss has important implications for the child's speech, language and educational development. Since congenital CMV infection has been identified as a risk factor for permanent sensorineural hearing loss , screeniin utero antiretroviral (ARV) exposure on outcomes across several domains among HEU infants  were also evaluated, as were individual drug classes  and individual ARV agents. Demographic characteristics and maternal and birth characteristics were collected by questionnaire and medical record review and treated as possible risk factors. Substance use during pregnancy was self-reported by mothers as reported previously , and first trimester exposure to emtricitabine was also associated with lower odds of a referral , although not attaining statistical significance ; in addition, a similar magnitude of association was observed for infants with third trimester exposure to Atazanavir . There were no significant associations of newborn screening results with either demographic characteristics  or maternal and birth characteristics .Based on unadjusted exact logistic regression models, first trimester exposure to Tenofovir was associated with lower odds of a newborn hearing screening referral [odds ratio (OR) = 0.41, 95% CI 0.14-1.00, ificance . After c similar . ExposurAmong the 45 infants referred after the initial newborn hearing screening, 22 had acceptable PBMC samples for CMV testing and one (4.5%) was CMV-positive. Of the 1,390 infants who passed the hearing screening, 92 were tested and 5 (5.4%) were CMV-positive. Follow-up hearing testing was not available for the six CMV positive infants.Follow-up information on subsequent hearing tests for the 45 infants referred for further testing was evaluated through January 1, 2013. Median age at follow-up was three years with the oldest child under six years. Twelve of the 45 (27%) infants did not receive any follow-up hearing testing. Twenty-four (73%) of the remaining 33 infants with subsequent follow-up passed their hearing screenings. Another nine infants (27%) continued to have some degree of hearing problems on additional screenings or testing, including one (3%) with confirmed sensorineural hearing loss after an extensive hearing examination.Exposure to Atazanavir overall and specifically in the third trimester was linked with higher odds of a referral, but this association did not reach statistical significance. A recent report from within the same cohort found a significant association between exposure to Atazanavir and delayed language developmental . In thatConversely, exposure to Tenofovir in the first trimester was associated with significantly lower odds of a referral for further hearing testing. Further, first trimester exposure to emtricitabine, typically given in tablets co-formulated with Tenofovir was also associated with lower odds of a referral, but this did not attain statistical significance.Alternative approaches for evaluating associations between exposures and binary outcomes in the setting of rare events  have also been proposed, such as rare events logistic regression . UtiliziResults of newborn hearing screening in HEU infants in SMARTT showed that 3.1% (45/1435) were referred for further testing, which is lower than the approximate 10% first referral rate previously reported in one statewide program in Rhode Island of all newborns . This reBecause CMV-related hearing loss is not always present at birth, and can be progressive with time -14, the The Joint Committee on Infant Hearing  requiresAlthough HIV mother-to-child-transmission rates have declined dramatically in the U.S.,  the numbSupp Table 1Supp Table 2


Granulin epithelin precursor (GEP) is reported to function as a growth factor stimulating proliferation and migration, and conferring chemoresistance in many cancer types. However, the expression and functional roles of GEP in colorectal cancer (CRC) remain elusive. The aim of this study was thus to investigate the clinical significance of GEP in CRC and reveal the molecular mechanism of GEP in CRC initiation and progression.The mRNA expression of GEP in CRC cell lines were detected by qRT-PCR. The GEP protein expression was validated by immunohistochemistry in tissue microarray (TMA) including 190 CRC patient samples. The clinicopathological correlation analysis were achieved by GEP expression on TMA. Functional roles of GEP were determined by MTT proliferation, monolayer colony formation, cell invasion and migration and in vivo studies through siRNA/shRNA mediated knockdown assays. The cancer signaling pathway identification was acquired by flow cytometry, western blot and luciferase activity assays.The mRNA expression of GEP in CRC was significantly higher than it in normal colon tissues. GEP protein was predominantly localized in the cytoplasm and most of the CRC cases demonstrated abundant GEP protein compared with non-tumorous tissues. GEP overexpression was associated with non-rectal location, advanced AJCC stage, regional lymph node and distant metastasis. By Kaplan–Meier survival analysis, GEP abundance served as a prognostic marker for worse survival in CRC patients. GEP knockdown exhibited anti-cancer effect such as inhibiting cell proliferation, monolayer colony formation, cell invasion and migration in DLD-1 and HCT 116 cells and decelerating xenograft formation in nude mice. siGEP also induced G1 cell cycle arrest and apoptosis. Luciferase activity assays further demonstrated GEP activation was involved in MAPK/ERK signaling pathway.In summary, we compressively delineate the oncogenic role of GEP in colorectal tumorigenesis by activating MAPK/ERK signaling pathway. GEP might serve as a useful prognostic biomarker and therapeutic target for CRC.The online version of this article (10.1186/s12967-018-1530-7) contains supplementary material, which is available to authorized users. Colorectal cancer (CRC) is one of the major leading causes of cancer-related deaths in the world . AlthougGranulin epithelin precursor (GEP), also known as progranulin, acrogranin, proepithelin, and GP88/PC-cell derived growth factor, is a secreted glycoprotein composed of 7.5 repeats of cysteine-rich motif , 7. PhysAs the expression and functional role of GEP in CRC is unclear. In this study, the clinical significance and the function of GEP in CRC will be comprehensively revealed.Between 1999 and 2013, 190 patients undergoing resection of primary CRCs at Prince of Wales Hospital, the Chinese University of Hong Kong were recruited in the present study. None of the patients received any neo-adjuvant therapy. The age of the patients ranged from 34 to 92 years, with a median age of 67.4 years. There were 106 men and 84 women. Tumors were staged according to the pathological tumor-node-metastasis (pTNM) staging system, 7th version. Distribution of the pTNM stages and other clinicopathological features is listed in Table Human CRC cell lines Caco2, DLD-1, HCT 116, HT-29, LoVo, LS 180, SW480 and SW620 were all obtained American Type Culture Collection . All cell lines were cultured in media according to manufacturer’s instructions.For transient transfection, DLD-1 or HCT 116 cells were transfected using lipofectamine 2000  by siGEP or siRNA control  to interfere GEP expression. Procedures of transfection were performed according to the manufacturer’s instructions. Cells stably expressing downregulated GEP mediated by shRNA was established using retrovirus system. The shRNA information for GEP knockdown can be found in Additional file Total RNA from cells was extracted by Trizol (Thermo Fisher Scientific) according to the manufacturer’s instruction. cDNAs were synthesized using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) as protocol. Quantification was performed with the ABI 7500 Real Time PCR system . Primers and probes for GEP were GEP-forward (5′-CAA ATG GCC CAC AAC ACT GA-3′), GEP-reverse (5′-CCC TGA GAC GGT AAA GAT GCA-3′) and GEP-probe (5′-6FAM CCA CTG CTC TGC CGG CCA CTC MGBNFQ-3′). Primer and probe reagents for control 18s were ready-made reagents . All experiments were performed in a minimum of three replicates.Immunohistochemistry was performed on 5 µm sections cut from tissue microarray blocks using Dako Envision Plus System  following the manufacturer’s instruction with modifications. Briefly, antigen retrieval was followed by endogenous peroxidase blocking and the following antibody in accordance with the manufacturer’s recommendations: GEP  . Other primary antibodies were from Cell Signaling  commercially including Cyclin D1 , cleaved PARP (Asp214) , phospho-MAPK/ERK , MAPK/ERK , Caspase-8 , Cleaved Caspase-8 , Caspase-3 , Cleaved Caspase-3 . The secondary antibodies were anti-Mouse IgG-HRP  and anti-Rabbit IgG-HRP . The western blot bands were quantified by ImageJ.Cell proliferation assays were performed using 3--2,5-diphenylte-trazolium bromide  assay following the manufactures’ protocol. Briefly, CRC cells which have been transfected with siGEP or siControl were plated in 96-well plate. Cell viability was measured at 24, 48, 72, 96, and 120 h. Finally, the optical density was determined at 570 and 690 nm wavelength light absorption .3 to 5 × 103 for transfected cells. Cells were incubated at 37 °C for 14–21 days until colonies can obviously be observed. Colonies were fixed with methanol for 5 min and stained with 0.5% crystal violet. Colonies with cell numbers of more than 50 cells per colony were counted. The experiments were performed in duplicate wells in three independent experiments.Anchorage-dependent growth was assessed by monolayer colony formation. 6-well plates were used at a density of 1 × 10Cell invasion or migration activity were performed using 24 well biocoat matrigel invasion chambers or sterilized transwell insert chambers . Cells were harvested after transfected with siGEP or siControl for 24 h and re-plated with serum free medium in the upper chamber. The lower chamber was filled with culture medium containing 10% FBS as the chemoattractant. After 48 h, the cells that moved to the lower surface of polycarbonate membrane were stained with 0.5% crystal violet and counted at five random 200× fields.6 transfected CRC cells in 100 μl PBS were injected subcutaneously into the dorsal region of anaesthetized nude mice . When tumor was formed, tumor diameter was measured and documented every 3 days for 3 weeks. At the end of investigation, mice were sacrificed, and xenografts were collected for diameter check and weigh valuation. The animal handling and all experimental procedures were approved by the Department of Health of Hong Kong and the Animal Experimentation Ethics Committee, The Chinese University of Hong Kong.1 × 10Cells transfected with siGEP or siControl were harvested at 24, 48 or 72 h after transfection. Then live cells were incubated in the mixture of propidium iodide (PI) and annexin V-fluorescein isothiocyanate (Thermo Fisher Scientific) for apoptosis study. Harvested cells were fixed and incubated with PI and RNase A at 4 °C for cell cycle analysis. Finally, cells were determined by FACS Calibur Flow Cytometer  and CellQuest program.The firefly luciferase construct was co-transfected with Renilla luciferase vector (Qiagen) as a control into the siRNA treated cells. Dual Luciferase Reporter Assay System  was employed to check the luciferase activity after 48 h’ transfection. The results were expressed as the ratio of firefly luciferase activity to renilla luciferase activity. Experiments were repeated in triplicate.2 test was used to analyze the association of target expression with clinicopathological parameters. Survival curves were drawn using the Kaplan–Meier method and compared by means of the log-rank test. Univariable and multivariable Cox proportional hazard regression models were used to analyze independent prognostic factors. P < 0.05 was considered statistically significant. P < 0.01 was considered highly statistically significant.The statistical analysis was performed using IBM SPSS Statistics . The expression level of GEP in paired non-tumor and tumor tissues was compared with paired Student’s t-test. Independent Student’s t-test was used to compare the mean value of any two groups. The Pearson χhttp://www.oncomine.org/), an online collection of microarrays. Using the Ki colon dataset [P < 0.01, Fig. GEP mRNA expression was up-regulated in 6/8 CRC cell lines compared with the normal colon epithelium by qRT-PCR Fig. a. We ana dataset , we obseP = 0.032), AJCC stages (P < 0.01), lymph node involvement (P < 0.01) and distant metastasis status (P < 0.01). Specifically, a high GEP protein level more frequently occurred in tumors located on colon than that on rectum  and mean tumor weight (P < 0.01) in xenograft model , while S-phase cell percentage decreased after siGEP transfection  in these 2 cell lines .Additional file 6. Copy number change of GEP in ONCOMINE colon tissue datasets. .


Phoca vitulina) to aerial sounds was measured in the presence of typical environmental noise through auditory brainstem response measurements. A focus was put on the comparative hearing sensitivity at low frequencies. Low- and mid-frequency thresholds appeared to be elevated in both captive and free-ranging seals, but this is likely due to masking effects and limitations of the methodology used. The data also showed individual variability in hearing sensitivity with probable age-related hearing loss found in two old harbour seals. These results suggest that the acoustic sensitivity of free-ranging animals was not negatively affected by the soundscape they experienced in the wild.The hearing sensitivity of 18 free-ranging and 10 captive harbour seals ( Marine mammals have long been recognised as hearing specialists and perhaps the most vulnerable group of all aquatic animals to noise pollution at sea , and the overlap between their at-sea distribution and sound producing activities has led to concerns about the potential impacts of sound on this species  Act licence number 60/4009. Data on animal sex and weight were collected on site where ABR measurements were also taken. When possible, a tooth was extracted for aging purposes. Seals were aged by counting the growth layer groups in the cementum of an incisor tooth, using the method of Dietz et al. . In the ®, females 0.09–0.13 mg/kg, males 0.09–0.38 mg/kg), after ~10 min they were anaesthetised with an injection of ketamine  into the epidural sinus with a three and one half-inch spinal needle which was subsequently maintained in place. Additional intravenous doses of Ketaset® were administered to maintain the desired anaesthesia, and additional intravenous doses of midazolam were administered to control muscular tremors, a side effect of ketamine anaesthesia.Animals were given a premedication intramuscular injection of midazolam  to the animals in trials of 512–2048 stimulus repetitions with a 5-dB step size in descending order. Short tone pips consisting of five cycles of cosine-gated sine waves were used as stimuli, with a duration between 5 ms (at 1 kHz) and 1.8 ms (at 2.8 kHz). Additional frequencies were tested  if time allowed or no reproducible results could be achieved at the initial test frequencies. At a repetition rate of 33.3 stimuli per second, the polarity of successive stimuli was inverted to avoid stimulus artefacts.The signals emitted through the headphones were calibrated before the measurements using a frequency generator  for signal generation, an artificial ear  connected to a calibrated microphone  and a conditioning amplifier  as receiver. The signals were calibrated in terms of sound pressure level (SPL rms) over the duration of the tone pips. All signals were visually inspected for spectral quality (distortion) on a digital oscilloscope  over the tested frequency range. No signal distortion was documented for any of the frequencies and levels used in our study.The starting sound pressure level for the first animal tested was chosen to be approximately 30 dB above the hearing threshold of harbour seals based on previous publications. In subsequent measurements on the remaining animals, levels were adjusted to start 30 dB above levels determined in the first animal. In addition, background electrical noise was measured in the absence of stimuli. In all trials (with and without acoustic stimulation), the headphones were held in place over the animal’s ear openings by one of the researchers. The opening of the outer ear canals was regularly checked and acoustic stimuli were only played when the outer ear canal was visibly open.LZeq, unweighted) using a Casella CEL-6X0 handheld sound level meter   , Alachua, FL, USA]. The acoustic stimuli were generated using the TDT software SigGen at a digitisation rate of 50 kHz. The recorded electrode responses were amplified , passed through an anti-aliasing filter, and led to an A/D converter (TDT RA16). Subsequently, the response (digitisation rate 25 kHz) was digitally filtered , written to a memory buffer and tested for the presence of signal artefacts. We used the TDT software BioSig to average the resulting potentials to allow an assessment of artefacts that indicates successful reception of the signal.In all animals, the neuronal signals were measured with subdermal needle electrodes  which were placed along the dorsal midline of the head: the active electrode on the vertex, 2 cm in front of the line between both ear openings, the ground electrode in the nape of the neck  and the reference electrode another 10–15 cm further back. The input impedance between the electrodes was 1 kΩ or below during all measurements.Neuronal waveforms were measured over a period of 10 ms after acoustic stimulation and averaged over the total number of presentations. The peaks of the recorded neuronal waveforms are numbered (I–VII) according to their succession  were not included into the regression analysis.The hearing thresholds measured by Wolski et al.  represenHearing thresholds were measured for the target frequencies of 1.4, 2.0, and 2.8 kHz in all (18) free-ranging seals in The Wash in 2012 and in six of the ten animals tested in the zoos in 2013. Examples of the resulting neuronal waveforms measured after stimulation at 1.4 and 2.8 kHz are shown in Figs. In a few animals, additional frequencies covering the frequency range up to 22.4 kHz were measured. For details on sample sizes, see Table The maximum differences in hearing sensitivity at low frequencies between the 18 free-ranging animals ranged from 26 dB  to 41 dB , while the captive animals showed a maximum individual difference in hearing sensitivity between 13 dB  and 25 dB .The appropriate use of the ABR method to achieve auditory measures in harbour seals to low-frequency stimuli has not been previously demonstrated in seals. Wolski et al.  had succThe auditory measurements of harbour seals presented here revealed aerial hearing thresholds with only relatively small differences between the animals in both test settings (zoo and the wild). These differences may have resulted from the use of different equipment in each setting, differences in background noise levels, different stress levels evoked in the animals in the two settings or reflect population differences. The aerial hearing thresholds are in relatively good agreement with comparable ABR data (after conversion to SPL) published by Wolski et al. . HoweverMirounga angustirostris) for different immobilising drugs and over extended periods of time, but did not find any effect on the amplitude and latency of electrophysiological responses (wave V) measured in these species. Mulsow and Reichmuth  they tested. While these studies are not comprehensive enough to rule out any effect, it was considered being not substantial for the outcome of this study.There are other specific aspects which may explain some of the differences found. The volume of the headphone calibration system used is tailored to match the volume of the outer ear of humans, not of harbour seals and could potentially lead to a small offset in the received SPLs. However, as the ABR thresholds cannot be regarded as absolute thresholds, this offset was deemed negligible. Moreover, when comparing the audiometric results between individuals as in this study, this offset in threshold would be consistent for all animals tested. Anaesthesia and a possible change in body temperature during the test procedure have been shown to have an effect on the latency of neuronal responses in humans . This would effectively reduce the sound transmission to the middle and inner ear and lead to a decrease in sensitivity. However, such an effect should lead to a loss in hearing sensitivity over the entire hearing range and not decrease with increasing frequency.Despite the caveats listed above, this study is the first to compare auditory sensitivity at a selected range of low frequencies of a large number of captive and wild harbour seals. Analysing results achieved from several individuals under comparable environmental conditions, in the field as well as under laboratory conditions, indicates that there is both between-subject and within-subject variability in hearing sensitivity in this species (Lauter and Karzon Progressive hearing loss with increasing old age, presbyacusis, has been reported before in marine mammals (Schusterman et al. Theoretically, the poor hearing sensitivity in the free-ranging seals from The Wash could stem from exposure to intense underwater sound as found in seismic exploration, underwater explosions, shipping (Richardson et al.


P trend = 0.031). Further analysis by adenoma size and number revealed a pronounced association with a larger size (≥5 mm) and multiple numbers (≥2). These positive associations were reduced to non-significance following further adjustment for body mass index, and OR for the highest versus lowest quartile of plasma CRP became 1.12  in all subjects. In conclusion, this study suggests that obesity-related systemic low-grade inflammation may play an important role in the early stages of colorectal carcinogenesis.Epidemiologic studies have identified a positive association between obesity and colorectal neoplasia. Adiposity induces systemic low-grade inflammation, which is commonly assessed with a sensitive biomarker, C-reactive protein (CRP). To understand the molecular mechanisms of obesity in the etiology of colorectal neoplasia, the present study was conducted in 782 adenoma cases and 738 controls who underwent total colonoscopy, and their plasma CRP level was evaluated in relation to colorectal adenoma prevalence. A logistic regression model was used to compute odds ratios (OR) and 95% confidence intervals (CI) of adenoma according to quartile of plasma CRP. Plasma CRP level was positively associated with higher adenoma prevalence in all subjects (OR 1.30; 95% CI 0.94–1.79 for the highest versus lowest quartile;  Epidemiological evidence indicates that the risk of colorectal cancer is about 19% higher in obese people than in those of normal weight, and increases about 4–10% per 2-kg/m2 rise in body mass index (BMI)5. Similarly, for colorectal adenoma, a meta-analysis of 36 epidemiological studies which included a total of 29,860 cases found a 19% increase in adenoma risk associated with a 5-kg/m2 increase in BMI6. However, the biological link underlying this association has not been fully elucidated.Overweight and obesity are consistently associated with an increased risk of colorectal cancer and adenoma9. Excessive secretion of these cytokines from hypertrophic adipocytes following obesity is postulated to induce and maintain a systemic, low-grade inflammatory state, which may contribute to the initiation and progression of carcinogenesis9. In fact, experimental studies have consistently shown that oxidative stress due to systemic inflammation likely inhibits the function of tumor suppressor gene APC (adenomatous polyposis coli) and p53, which are closely related to the development of colorectal adenoma and progression to colorectal cancer, respectively10.Adipose tissue, although previously considered to be a passive storage organ, is now known to secrete a variety of inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)13 versus null results or even an inverse association in the others20. While all previous studies have used high-sensitivity CRP, the inconsistent results on this topic may be related to the influence of some studies with relatively smaller population sizes 20.C-reactive protein (CRP) is readily produced in response to inflammatory cytokines by hepatocytes, and accordingly serves as a useful biomarker of systemic inflammatory states. The importance of CRP in this role is further strengthened by its much higher circulating concentrations than individual inflammatory cytokines. Nonetheless, epidemiological studies evaluating the association between circulating CRP level and colorectal adenoma have been inconclusive, with 3 of the 10 studies showing a positive associationThus, this study was conducted in a relatively larger population of middle-aged and elderly Japanese. We quantified plasma concentrations of CRP using a highly sensitive method which enables the assessment of even low-grade inflammation, and evaluated the association between CRP level and the prevalence of colorectal adenoma.The Colorectal Adenoma Study in Tokyo was designed to investigate the role of dietary and lifestyle factors, including genetic susceptibility factors, in the early stage of colorectal carcinogenesis among healthy volunteer examinees of a colorectal cancer screening program.23. Briefly, eligible participants were defined as men aged 50–79 years and women aged 40–79 years who had undergone total colonoscopy from the anus to cecum, with no history of colorectal adenoma, any type of cancer, ulcerative colitis, Crohn’s disease, familial adenomatous polyposis, carcinoid tumor, or colectomy. Since a small number of men under the age of 50 years joined the cancer screening program, their age cut-off was set at 50 years. 2234 examinees satisfied these conditions, of whom 526 men and 256 women had at least one adenoma by the pit pattern classification of colorectal lesions and were included as cases. Of the remaining 1452 examinees without adenoma, 482 men and 721 women were identified as eligible controls, after exclusion of those with inflammatory polyps, diverticulitis, submucosal tumor, and bowel tuberculosis or hyperplastic polyps. Due to the lower number of male controls than male cases, all male controls were included in the study, while 256 potential female controls were randomly selected by frequency matching with respect to age category  and screening period  to cases. Thus, the study included a total of 782 cases and 738 controls. In this analysis, we further excluded 13 subjects with missing values for covariates and 88 nonsteroidal anti-inflammatory drug (NSAID) users. To limit the possibility of excessively high CRP levels due to acute inflammatory conditions such as infections or trauma, we also excluded 33 participants with the CRP value of 6 mg/L, meaning that they had CRP values above the upper detection limit. This left 729 cases (490 men and 239 women) and 657 controls (425 men and 232 women) for final analysis. All examinees provided written informed consent for the use of their screening data for medical research purposes. The study protocol, including all methods described in the present study, was approved by the institutional review board of the National Cancer Center , in accordance to relevant ethical guidelines for medical research in Japan.Between February 2004 and February 2005, a total of 3212 participants joined the cancer screening program of the Research Center for Cancer Prevention and Screening, National Cancer Center, Japan, and underwent colonoscopy examination. Inclusion criteria for the study have been detailed elsewhereFasting venous blood was collected before any screening examinations on the first day of screening. The collected blood samples were drawn into vacutainer tubes with ethylenediaminetetraacetic acid. The vacutainer tubes were centrifuged to obtain the plasma and buffy coat layers, and the specimens were stored at −80 °C until analysis.24.Plasma C-reactive protein concentrations were measured using a latex-enhanced high-sensitivity assay on a BN II nephelometer  at a commercial laboratory . All laboratory personnel were blinded with respect to case or control status. The mean intra-assay coefficient of variation from the quality control sample in our previous study was 2.7% (n = 10)2). Participants also completed a 138-item food frequency questionnaire (FFQ), with pre-specified standard portion sizes and nine intake frequency categories for most food items.Study subjects responded to a self-administered questionnaire before their screening examination which inquired about their demographics, lifestyle and socioeconomic characteristics, and personal and family medical history. Drinking status was initially categorized into three . Among current drinkers, per-week alcohol consumption was calculated from the frequency of alcohol drinking and number of standard units consumed per occasion for five different alcoholic beverages . Smoking status was first categorized the same way into three . Among ever smokers, lifetime exposure to cigarette smoking was measured by pack-years, in which one pack-year was defined as the smoking of 20 cigarettes every day for one year. The questionnaire also enquired about physical activities, including types of physical activity during working hours  and the frequency of physical activity during leisure time . The total sum of physical activity combining occupational and leisure-time physical activity was expressed in metabolic equivalent task hours per day (METs-hour/day). BMI was calculated for each subject as the weight in kilograms divided by the height in meters squared , fish (g/day), fiber (g/day), folate (μg/day), isoflavone (mg/day), fruits (g/day), and vegetables (g/day) were energy adjusted by the residual regression method25. In the association analysis, study subjects were divided into quartile categories based on the distribution of CRP values among the control group. Because men and women were selected by a different scheme, we used an unconditional logistic regression model to analyze both sexes together, and initially computed odds ratios (ORs) and 95% confidence intervals (CIs) with adjustment for sex, age , and screening period (model 1). Model 2 included further adjustment for family history of colorectal cancer, pack-years of smoking , drinking , physical activity , and history of diabetes mellitus. Because obesity-induced inflammation might be an intermediate marker between obesity and colorectal neoplasia, we adjusted for BMI  in a separate multivariate model. The linear trend test was performed by assigning ordinal values to the quartile categories of CRP and treating them as continuous variables in the regression model. Stratified analysis by sex and BMI  was conducted and characteristics of adenomas (size and number) were also taken into account. Effect modification by sex and BMI on the associations with plasma CRP was assessed by the likelihood ratio test which compared the model with and without product terms of these variables. Two-sided P values < 0.05 were set to show statistical significance. All statistical analyses were done using SAS version 9.3 .Case-control comparisons in mean, median, and proportions were tested with the Selected characteristics of cases and controls are summarized in Table Variations in some of the lifestyle and dietary factors among controls are shown in Table P trend = 0.031. When this analysis was stratified by gender, a similar pattern of non-linear positive association was also observed among men, whereas an attenuated pattern was seen in women; the corresponding OR was 1.41 (95% CI 0.93–2.13), with P trend = 0.018, in men and 0.99 (95% CI 0.58–1.70), with P trend = 0.62, in women. Effect modification by sex was not statistically significant (P interaction = 0.281). When stratified by BMI, the association between plasma CRP and colorectal adenoma did not differ according to BMI category (P interaction = 0.794).Plasma CRP concentrations was positively associated with the prevalence of colorectal adenoma, albeit in a nonlinear manner Table . The OR P trend = 0.25, in overall subjects and 1.22 (95% CI 0.80–1.88), with P trend = 0.11, in men after adjusting for BMI. Additional adjustment by energy-adjusted dietary factors, including total meat, fiber, folate, fruits, vegetables, isoflavone, and fish, did not substantially change the result from the BMI-adjusted model (results not shown).The observed positive association of plasma CRP level with colorectal adenoma overall as well as in male subjects, however, lost statistical significance when further adjusted for BMI. The OR of adenoma for the highest compared to the lowest quartile of plasma CRP was 1.12 (95% CI 0.80–1.56), with We further examined the association between plasma CRP level and colorectal adenoma by the size and number of adenomas Table . Of 729 In this study, we observed a positive association between plasma CRP concentration and the prevalence of colorectal adenoma. Given that CRP is a preferable biomarker of systemic inflammation and that a highly sensitive method can evaluate low-grade inflammation, our present finding of a positive association between higher CRP levels and an increased prevalence of adenoma supports the hypothesis that the development/growth of colorectal neoplasia likely involves a systemic, low-grade inflammatory state.28. After binding to its receptor, IL-6 triggers both the nuclear factor κB (NF-κB) cascade and STAT3 pathway, which are responsible for cell survival/tumor initiation and cell proliferation/tumor growth downstream, respectively29. In addition, TNF-α is involved in starting the NF-κB and activator protein-1 (AP-1) signaling pathways to promote cell proliferation and survival30. Thus, the low-grade inflammation established by obesity may play an important role in colorectal carcinogenesis, including its early stages.Of importance, the present study showed a clear influence of BMI on the association between plasma concentrations of CRP and colorectal adenoma. An increase in CRP levels was associated with an increased risk of colorectal adenoma only before BMI was taken into account, suggesting that the observed association can be ascribed to obesity-related inflammation. Indeed, inflammatory cytokines derived from adipose tissues, specifically IL-6 and TNF-α, have been well characterized to activate oncogenic transcription pathways of many types of cancer, including colorectal cancer32. More importantly, adenoma of an increased size and number are at a greatly elevated risk of malignant conversion. These findings underscore the importance of colorectal adenoma status in relation to colorectal cancer33. The Tennessee Colorectal Polyp Study (TCPS) in the US identified a positive association between circulating CRP and colorectal adenoma, but notably only for advanced and multiple adenomas13. Two other studies provide further evidence for the association between circulating CRP and advanced and synchronous adenoma12: Otake et al. found that the OR of circulating CRP associated with an adenoma size equal to or larger than 5 mm was more than 212, while Chiu et al. observed a positive association only in the presence of larger or multiple adenomas, highlighting the impact of size and number on the CRP-adenoma association. Interestingly, this positive association was retained in concurrent advanced and synchronous adenoma cases, which showed the further elevation of OR11.In our present observations, the CRP-adenoma association was more evident with larger and multiple adenomas than with smaller and single adenomas. Colorectal adenoma is widely considered to be a precursor to colorectal cancer, and the majority of colorectal cancers are known to arise from colorectal adenoma36, but less consistently in studies of colorectal adenoma37. Indeed, a meta-analysis on the association between BMI and colorectal cancer by Wang et al. found that colorectal cancer was the only one of a number of different cancer types to show a significant gender difference35. In the meantime, a Taiwanese cross-sectional study of circulating CRP and colorectal adenoma showed a non-linear positive increase in adenoma OR only in male subjects when stratified by gender11, which is consistent with our present findings. However, the gender difference in our study and in the Taiwanese study should be interpreted with caution as their sample sizes in women were nearly half those in men, and the resulting low statistical power may have obscured the true association in women. Evidence for this possible gender difference in colorectal adenoma in relation to circulating CRP remains limited, and further investigation is warranted.Although no significant interaction was observed, our findings showed a possible sex difference, in which the association between plasma CRP level and colorectal adenoma was relatively more apparent in men. A similar gender difference has been consistently observed in many prospective studies of obesity and colorectal cancer as well as their meta-analyses39. Colorectal adenoma cases in our study were not pathologically verified, and single measurement of plasma CRP may not have completely represented random variation within the subjects. Incorporating additional steps to reduce this possibility could have led to a further increase in the reliability of our results. Finally, as previously mentioned23, it is important to note that the present study was based on prevalent cases instead of incident cases, which suggests that the ORs in this study cannot necessarily be applied to infer the risk of adenoma “development”. Rather, they can only imply the risk of “having” an adenoma at a given time point, and should thus be interpreted carefully.A strength of our study is that all participants underwent total colonoscopy, reducing the possibility of misclassification of case and control status. Other strengths include our large sample size and adjustment for known and potential risk factors. Among limitations, one is the cross-sectional design of the study, which prevents any assignment of causality. Nevertheless, colorectal adenoma is unlikely to affect adiposity, as the underlying studies unanimously show the influence of BMI on low-grade systemic inflammation and adenoma, but not vice versaIn summary, this study in a middle-aged and elderly Japanese population showed a positive association between plasma CRP level and colorectal adenoma, notably in larger and multiple adenomas. However, the positive association was attenuated after adjustment for BMI, which likely suggests that plasma CRP level was largely correlated with BMI. Thus, our results may suggest the importance of an obesity-related systemic, low-grade inflammatory state in relation to colorectal carcinogenesis, at least in its early stages. Pathological evaluation of adenomas and measurement of circulating inflammatory cytokines can provide further insight into the molecular mechanisms of obesity in the etiology of colorectal neoplasia.


Cyclic nucleotide phosphodiesterases (PDEs) refer to a class of enzymes that degrade the phosphodiester bonds in cAMP and cGMP secondary messenger molecules  signaling pathway elucidated the existence and role that PDEs play in inner ear homeostasis. Most recently, PDE receptors were specified in the cochlea via reference to the guinea pig genome, which is up to 95% similar to the human genome. One study used the previously published full genome sequence of guinea pigs on the University of California, Santa Cruz (UCSC) genome bioinformatics browser to find PDEs present in humans, mice, and guinea pigs  demonstrates a heterogeneous genetic etiology. RP is a disorder characterized by gradual devolution of the retina and it affects between 1 in 3,000 and 8,000 people worldwide  in combination with PDE6 mutations. This “double hit” not only appears to contribute to a greater severity of retinal devolution, but it also demonstrates the coexistence of SNHL, ARRP, and a mutation in PDE. The aforementioned genetic syndromes, along with a growing number of disorders that are associated with PDE, all make the case that a successful PDE-treatment modality might not only be applied to multiple disorders, but is certain to have vast interdisciplinary effects.For autosomal recessive RP (ARRP), over 40 genes have been mapped, including  RetNet, . PDE6A aPDE-related molecular mechanisms underlying each disease. There is a need to explore the tissue specificity of different PDEs and how PDE mutations lead to phenotypic consequences. In addition, the careful analysis of genetic etiology is imperative in diagnosis and development of new treatment modalities. The advent of new genome editing technologies including clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 holds a great potential as a therapeutic strategy for genetic diseases including SNHL and RP (Hsu et al., NRL gene (Peng et al., While disorders such as SNHL, RP, and Usher syndrome encompass heterogeneous genetic etiologies, there is lack of information regarding All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hearing loss can affect the perception of emotional reaction to the music. The present study investigated whether the students with congenital hearing loss exposed to the deaf culture, percept the same emotion from the music as students with acquired hearing loss.Participants were divided into two groups; 30 students with bilaterally congenital moderate to severe hearing loss that were selected from deaf schools located in Tehran, Iran and 30 students with an acquired hearing loss with the same degree of hearing loss selected from Amiralam Hospital, Tehran, Iran and compared with the group of 30 age and gender-matched normal hearing subjects served our control in 2012. The musical stimuli consisted of three different sequences of music,  each with the duration of 60 sec. The students were asked to point to the lists of words that best matched with their emotions.P<0.001). There was no significant difference in the emotional perception of sadness, happiness, and fear among the group of acquired hearing loss and normal hearing group (P=0.75), (P=1) and (P=0.16) respectively. Emotional perception of sadness, happiness, and fear in congenital hearing loss children was significantly poorly than acquired hearing loss and normal hearing group (Neural plasticity induced by hearing assistant devises may be affected by the time when a hearing aid was first fitted and how the auditory system responds to the reintroduction of certain sounds via amplification. Therefore, children who experienced auditory input of different sound patterns in their early childhood will show more perceptual flexibility in different situations than the children with congenital hearing loss and Deaf culture  The music has always been considered to sound either happy or sad. Music has been though as a propellant of intense emotion. However, there are discrepancies in the literature about how the music can actually evoke emotions. The related emotions consist of a subjective feeling; however, others expand the definition to contain other aspects, such as cognition, physiologically arousal state motor and behavioral expressions, too. Two theories have been delineated about the possible links in between music and emotion, emotivist and cognitivist. Music can actually evoke or induce feeling in listeners. By contrast, some suggest that listeners simply feel positive or negative when they like or dislike the music. Listeners characterize the music as being happy or sad, originating from the fact that the music expresses happiness or sadness . . PerceEmotional prosody includes semantic information (differences between statement and question) and affective intent (differences between happiness and sadness) is crucial for achieving the meaning of speech and music . Deaf chThere is a relation between music and emotion. Some variables such as cultural circumstances may play a role in the judgment of emotional perception of music. On the other hands, some dimensions of music such as timbre and pitch may influence the listener’s perception of emotion of music. Neural structures that participate in the processing of auditory information are the other elements that incorporated in perceiving of the musically induced emotion. Any lesion in auditory system can disturb the perception of emotional reaction to the music. Deaf children were compared (those from a state school dedicated to the deaf where the deaf culture was supported strongly) to age-matched children who attended public schools. Deaf students had a different reaction to musically induced emotion. The authors presented the following facts for the justification of their finding: 1- In spite of using amplification, deaf students may not have accessibility to all auditory details that lead to the leakage of some auditory experiences. 2- Social environment has an important role in perceiving the specific emotion; there is little or no attention to give to sound in deaf school. 3- Familiarity with the type of presented music is an effective factor in recognizing emotion, so musical training can be a practical factor in perceiving the emotional content of music. Culture and experiences can affect the perception of music and suggested that musical therapy can increase the musical experiences of children within the same culture . Children, who experienced auditory input of different sound patterns in their early childhood, will show more perceptual flexibility in different situations. Thus, the purpose of the present study was to investigate whether the students with congenital hearing loss exposed to the deaf culture, percept the same emotion from the music as students with acquired hearing loss.The study participants were 60 students divided into two groups. The first group consisted of 30 students with bilaterally congenital moderate to severe hearing loss who were studying in deaf cultured high school from deaf schools and Amiralam Hospital Tehran, Iran in 2012. Mean (± standard deviation) of age was 16.77 (±1.25) yr and range, 15 – 19 yr, 16 boys, and 14 girls. Overall, 30 students with an acquired hearing loss of the same degree, who attending audiology clinic in a university hospital center participated as a second group. Mean (± SD) of age was 22.20 (± 1.32) yr and range, 20-25 yr 14 girls and 16 boys. A group of 30 age and gender-matched normal hearing subjects served our control group. Mean (± SD) of age was 19.83 (± 2.42) yr and range, 16-28 yr), 15 girls and 15 boys. Inclusion criteria were 1) the student with 15-30 yr old, 2) moderate to severe sensorineural hearing loss in each group of children with hearing loss, and 3) hearing threshold ≤ 25 dB in control group. Individuals with hearing loss  were all fitted by bilateral hearing aids.Musical stimuliThe musical stimuli used in this study consisted of three different sequences of music, each with the duration of 60 sec randomly ordered to depict one of the three basic emotions – happiness, sadness, and fear. We incorporated those musical pieces clearly intended their meaning by their composers. ProcedureParticipants were tested in a typical silent room. Individuals with hearing loss took their hearing aids off. They were seated in front of a table, there were three lists of words on the table, and each could express happiness, sadness or fear. Then they listened to the piece of music through the TDH39 headphone at their most comfortable level (MCL). After listening to each excerpt, they were asked to point to the lists of words that best matched with their emotions.Statistical analysisP-value≤0.05. Kolmogorov-Smirnov test was used to determine the normal distribution of variables. To compare different values between study and control groups, one-way ANOVA analysis variance test was used. SPSS software  was used for statistical evaluation. The criterion for statistical significance was defined as P>0.05). There was a significant difference between groups considering their performance on the variables of happiness, sadness, and fear; children with congenital hearing loss performed poorer than those with acquired hearing loss and control group. Emotional perception of sadness in congenital hearing loss children was significantly poorly than acquired hearing loss and normal hearing group . There was no significant difference in the emotional perception of sadness between the group of acquired hearing loss and normal hearing group (P=0.751). Additionally, perception of happiness and fear was significantly different between congenital and acquired hearing loss children  and  for happiness and fear, respectively. However, there were no significant differences between the group of acquired hearing loss and normal hearing group considering the perception of happiness and fear  as one of the rehabilitation approach, enable many deaf children to achieve more appropriate speech perception and perhaps enjoy the music. However, situation is very different for music perception, because with most CI signal processing strategies, pitch information may be transmitted insufficiently . The musP<0.001). In accordance with our findings, children using CI, could identify and repeat words correctly in both closed and open set tests, meaning that they had an access to the acoustic cues available in these words. They knew how to use this information to identify words presently acoustically, but these children had problems in perceiving an emotion presented by prosody in speech (In line with our research work, some studies have been encountered in the literature addressing emotional prosody perception in those with hearing loss. In a study , hearingn speech . In addin speech , 13. Chin speech . Some studies dealt with temporal resolution skills in those with cochlear hearing loss, which use cochlear implant. Their results showed abnormality in the frequency resolution skills, which lead to an abnormal music perception; because the accurate perception of music requires the precious discrimination of frequency modulations. The improvements in speech recognition by usage of a hearing devise overtime may not necessary result in improvement of music perception , 15. SenThe findings of the present study are in line with previous studies, where found no age and gender effects on musical perception. , 18, 19.In Conclusion, perception of musical emotion is depended on auditory experience and culture of someone. So the usage of amplification that introduce high resolution and fine temporal cues as soone as possible in children, can affect development and integration in central nervouse system


Cyclic nucleotide phosphodiesterases (PDEs) are the only enzymes that degrade the cyclic nucleotides cAMP and cGMP, and play a key role in modulating the amplitude and duration of the signal delivered by these two key intracellular second messengers. Defects in cyclic nucleotide signalling are known to be involved in several pathologies. As a consequence, PDEs have long been recognized as potential drug targets, and they have been the focus of intense research for the development of therapeutic agents. A number of PDE inhibitors are currently available for the treatment of disease, including obstructive pulmonary disease, erectile dysfunction, and heart failure. However, the performance of these drugs is not always satisfactory, due to a lack of PDE-isoform specificity and their consequent adverse side effects. Recent advances in our understanding of compartmentalised cyclic nucleotide signalling and the role of PDEs in local regulation of cAMP and cGMP signals offers the opportunity for the development of novel strategies for therapeutic intervention that may overcome the current limitation of conventional PDE inhibitors. Significant progress has been made since 1958, when Rall and Sutherland first described the involvement of adenosine 3′-5′-cyclic monophosphate (cAMP) in the cellular response to hormones . Today i2+ and is present in the cytosol and in several intracellular organelles and structures [cAMP is generated by the activation of adenylyl cyclases (ACs). This activation occurs by the binding of a first messenger (a hormone or neurotransmitter) to G protein-coupled receptors (GPCR), which triggers the release of the Gs α-subunit from the heterotrimeric G protein complex. The Gs α-subunit activates transmembrane ACs (tmACs) for the generation of cAMP by the conversion of ATP . cAMP caructures . The effl-arginine by nitric oxide synthases. Both particulate and soluble GCs generate cGMP by the conversion of GTP. The downstream effectors of cGMP are protein kinase G (PKG), ion channels, and PDEs.cGMP is generated by two different types of guanylyl cyclase (GC). Membrane-associated (or particulated) guanylyl cyclases are activated by the atrial natriuretic peptide, the B-type natriuretic peptide, and the C-type natriuretic peptide. Soluble GC is activated by nitric oxide, a gaseous signalling molecule endogenously synthesized from Non-canonical CN (such as cCMP and cUMP) can also be generated by the sGC and sAC, and share downstream effectors (PKA and PKG) with the classical CN, cAMP, and cGMP . cCMP an2+. This enzyme includes a Ca2+/calmodulin binding site at the N-terminus, and binding of Ca2+/CaM leads to 10-fold stimulation of its maximal velocity (Vmax) without changing its affinity for the substrate. However, the Michaelis constant Km for the substrate is reduced when the same site is phosphorylated by PKA [GMP-binding PDEs Anabaena adenylyl cyclase and Escherichia coliFh1A, which function as ligand-binding domains or facilitators of protein–protein interaction [PDEs are the only enzymes that degrade CN, and are grouped in a superfamily comprising 21 genes, which constitute 11 distinct PDE families, PDE1–11. Each PDE family may include several genes, and at least 100 different PDE isoforms can be generated by different splice variants or alternative transcription start sites . PDE1–3,d by PKA . PDE2, 6eraction ,18. Binderaction ,19,20. Teraction ,21. The eraction ,23.The model initially proposed for cAMP signalling was simple and linear: the first messenger activates a GPCR, and cAMP is generated, leading to the activation of PKA. The PKA-mediated phosphorylation of downstream protein targets then results in the required cellular effect . Howeverl-type calcium channels (LTCC) in response to β-adrenergic receptor stimulation [2+ re-uptake into the SR induced by adrenergic stimulation, confirming that the AKAP18δ/PKA/SERCA2/phospholamban signalosome provides a mechanism for the precise spatiotemporal control of phospholamban phosphorylation [Research over the past 30 years has clearly established that CN signalling is indeed compartmentalised . Compartmulation . LTCCs amulation . As anotrylation . Phosphorylation ,32,33.cGMP signalling presents mechanisms of spatial organisation similar to cAMP, yet less is known about the details of its compartmentalisation. A role for PDEs in local cGMP regulation has been demonstrated, in particular for PDE5 and PDE2. There is evidence that these enzymes are differently engaged in the degradation of cGMP, depending on whether cGMP is generated via particulate or soluble GCs, thus suggesting spatial segregation of these cGMP signalling components; PDE9A, unlike PDE5A, regulates cGMP produced via natriuretic peptide rather than nitric oxide stimulation ,34. PKG The key role of PDEs in the compartmentalisation of CN signalling was firmly established using fluorescence resonance energy transfer (FRET)-based genetically encoded fluorescent indicators that allow for cAMP ,38,39 orAs PDEs are the only route to CN degradation, the specific subcellular localisation of these enzymes represents a fundamental mechanism for the local regulation of the magnitude, duration, and specificity of CN-dependent signals. Without PDE-dependent control of local cAMP levels, stimulation of adenylyl cyclase activity by any hormone would fill up the cells with a uniform concentration of the second messenger. As a consequence, signal specificity would be lost, as all PKA subsets present in the cell would be activated . In thisPharmacological inhibition of PDEs is extensively used for experimental work, and it has enormously contributed to our understanding of the role of individual PDE families and the definition of the cAMP compartmentalisation model. For example, the PDE2 inhibitor Bay 60-7550 was used to show that PDE2 is responsible for the degradation of cGMP in hippocampal neurons and can improve memory functions by enhancing neuronal plasticity . The samA number of PDE inhibitors are used in the clinic. PDE3–5 family-selective inhibitors have been approved for clinical use. For example, roflumilast (a PDE4 selective inhibitor) is indicated for the treatment of chronic obstructive pulmonary disease (COPD). However, PDE4 inhibitors present adverse emetic effects due to the inhibition of PDE4 in the brain and the gut, limiting their use as therapeutics ,49,50. TAs discussed above, family-selective PDE inhibitors present limitations for clinical use, mainly due to lack of isoform selectivity, resulting in undesirable side effects. Studies are currently underway to circumvent this problem, and we briefly outline some of the existing isoform-selective inhibition strategies below.After the publication of the PDE4 crystal structure , new insSmall interfering RNA (siRNA) of individual PDEs is significantly more specific than most available pharmacological inhibitors , allowinIn a scenario where the compartmentalisation of CN signalling is crucial for cell function, techniques that allow the specific manipulation of local signalling provide an interesting opportunity to achieve a more targeted intervention. Displacement of specific isoforms of endogenous PDEs by the overexpression of their catalytically inactive mutant is one approach used in the laboratory. This strategy involves engineering specific PDE isoforms by introducing a single point mutation in their catalytic site. The overexpression of these constructs results in the displacement of the endogenous active PDE isoform from its original localisation within the cell. The technique was used to explore the role of specific anchored PDE isoforms in the compartmentalisation of CN signalling. Compared to the siRNA approach described above, the expression of catalytically inactive PDEs allows the role of PDE subcellular localisation in the regulation of a specific cellular function to be directly assessed. The first study to use this technique generated catalytically inactive PDE4A4, PDE4B1, PDE4C2, and PDE4D3 to identify the role of specific tethered PDE4 isoforms in regulating cAMP and PKA-dependent phosphorylation in resting COS1 cells. The study successfully showed that PDE4C2 and PDE4D3 isoforms are constitutively phosphorylated by PKA type-II, and as they are tethered in a complex including AKAP450 and PKA type-II, they play a central role in regulating the activation of AKAP450-bound PKA type-II at the perinuclear region . In anotAs previously discussed, different PDEs can localise to different subcellular sites—anchored to AKAPs or via interaction with other proteins. For example, PDE4D3, together with PKA, is localised by the mAKAP-organised signalosome in the perinuclear region , and viaCyclic nucleotide PDEs play an important role in cellular signalling by degrading cAMP and cGMP. Due to their diverse distribution in different tissues—at the cellular and subcellular levels—and their major role in regulating cellular function, PDEs have been the focus of intense research as potential therapeutic targets. The limitations with currently available pharmacological PDE inhibitors—mainly due to their lack of isoform selectivity—urge the development of new approaches to achieve effective and specific PDE inhibition for experimental work and clinical use. Emerging understanding of CN compartmentalised signalling, of the role of individual signalosomes, and the specific signalling and cellular responses regulated by individual PDE isoforms is providing the background knowledge and rationale for the development of new PDE targeting strategies, which in the future may deliver novel, more effective, and specific therapeutics.