Characterization of the internal IRES element of the zebrafish connexin55.5 reveals functional implication of the polypyrimidine tract binding protein

<p>Abstract</p> <p>Background</p> <p>Connexin55.5 (Cx55.5) is a gap junction protein with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the receptor-horizontal cell complex in form of hemichannels where light-dependent plasticity occurs. This connexin is the first example of a gap junction protein processed to form two protein isoforms from a monocistronic message by an IRES mediated process. The nuclear occurrence of a carboxy-terminal fragment of this protein provides evidence that this gap junction protein may participate in a putative cytoplasmic to nuclear signal transfer.</p> <p>Results</p> <p>We characterized the IRES element of Cx55.5 in terms of sequence elements necessary for its activity and protein factor(s), which may play a role for its function. Two stretches of polypyrimidine tracts designated PPT1 and PPT2 which influence the IRES activity of this neuronal gap junction protein were identified. Selective deletion of PPT1 results in an appreciable decrease of the IRES activity, while the deletion of PPT2 results in a complete loss. RNA-EMSA and UV-cross linking experiments showed that protein complexes bind to this IRES element, of which the polypyrimidine tract binding protein (PTB) was identified as one of the interacting partners with influence on IRES activity. These results indicate that PTB conveys a role in the regulation of the IRES activity of Cx55.5.</p> <p>Conclusion</p> <p>Our findings indicate that the activity of the IRES element of the neuronal gap junction protein Cx55.5 is subject of regulation through flanking polypyrimidine tracts, and that the non-canonical trans-activation factor PTB plays an essential role in this process. This observation is of considerable importance and may provide initial insight into molecular-functional relationships of electrical coupling in horizontal cells.</p

Implication of Connexin 43 as a Tumor Suppressor in Pathogenesis of Breast Cancer

Breast cancer (BC) is a global public health burden, constituting the highest cancer incidence in women worldwide. Connexins 43 proteins propagate intercellular communication, gap junction intercellular communication (GJIC), remarkably expressed in several tumor types including liver, prostate, and breast. This domain of Cx43 possesses functionally critical sites identical to those involved in gating of channel and phosphorylation sites for various kinases. However, the mechanism by which Cx43 down regulation occurs in breast cancer is far from clear. Several mechanisms like Cx43 promoter hyper-methylation or a cancer-specific reduction of Cx43 expression/trafficking by the modulation of various components of the Cx43 life cycle give the idea to be involved in the down regulation of Connexins in mammary glands, but irreversible mutational alterations have not yet been proved to be among them. Summarily, the efficacy or specificity of these drugs can be increased by a combinatory approach considering an effect on both the Connexins and their regulatory molecules. This chapter will summarize the knowledge about the connexins and gap junction activities in breast cancer highlighting the differential expression and functional dynamics of connexins in the pathogenesis of the disease

IRES-mediated translation of the carboxy-terminal domain of the horizontal cell specific connexin Cx55.5 in vivo and in vitro

<p>Abstract</p> <p>Background</p> <p>Changes of the interneuronal coupling mediated by electrical synapse proteins in response to light adaptation and receptive field shaping are a paramount feature in the photoreceptor/horizontal cell/bipolar cell (PRC/HC/BPC) complex of the outer retina. The regulation of these processes is not fully understood at the molecular level but they may require information transfer to the nucleus by locally generated messengers. Electrical synapse proteins may comprise a feasible molecular determinant in such an information-laden signalling pathway.</p> <p>Results</p> <p>Connexin55.5 (Cx55.5) is a connexin with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the PRC/HC/BPC complex in form of hemichannels where light-dependent plasticity occurs. Here we provide evidence for the generation of a carboxy-terminal domain of Cx55.5. The protein product is translated from the Cx55.5 mRNA by internal translation initiation from an in-frame ATG codon involving a putative internal ribosome entry site (IRES) element localized in the coding region of Cx55.5. This protein product resembling an 11 kDa domain of Cx55.5 is partially located in the nucleus <it>in vivo </it>and <it>in vitro</it>.</p> <p>Conclusion</p> <p>Our results demonstrate the generation of a second protein from the coding region of Cx55.5 by an IRES mediated process. The nuclear occurrence of a fraction of this protein provides first evidence that this electrical synapse protein may participate in a putative cytoplasmic to nuclear signal transfer. This suggests that Cx55.5 could be involved in gene regulation making structural plasticity at the PRC/HC/BPC complex feasible.</p

EUV Emission and Scattered Light Diagnostics of Equatorial Coronal Holes as Seen by Hinode/EIS

Spectroscopic diagnostics of solar coronal plasmas critically depends on the uncertainty in the measured line intensities. One of the main sources of uncertainty is instrumental scattered light, which is potentially most important in low-brightness areas. In the solar corona, such areas include polar and equatorial coronal holes, which are the source regions of the solar wind; instrument-scattered light must thus pose a significant obstacle to studies of the source regions of the solar wind. In this paper we investigate the importance of instrument-scattered light on observations of equatorial coronal holes made by the Hinode/EIS spectrometer in two different phases of the solar cycle. We find that the instrument-scattered light is significant at all temperatures, and in both regions it amounts to approximately 10% of the average intensity of the neighboring quiet Sun regions. Such contribution dominates the measured intensity for spectral lines formed at temperatures larger than Log T = 6.15 K, and has deep implications for spectroscopic diagnostics of equatorial coronal hole plasmas and studies of the source regions of a large portion of the solar wind which reaches Earth. Our results suggest that the high temperature tail of in the coronal hole plasma distribution with temperature, however small, is an artifact due to the presence of scattered light.Comment: 11 pages, 6 figure

Genetic Alterations of RET: Possible Implications and Clinical Correlations in Thyroid Carcinogenesis

Thyroid cancers are malignant tumors in the thyroid gland. DNA polymorphisms are playing a decisive role in unscrambling the genomic basis of tumor formation and development in cancer. Thyroid cancer is influenced in a polygenic and low-penetrance manner by RET gene polymorphisms and this part of the world (North India) has not recorded any study regarding RET alterations in this very cancer. We assessed RET G691S (rs1799939), L769L (rs1800861) and S904S (rs1800863) polymorphisms by restriction fragment length polymorphism (RFLP) in order to explain their potential role in the diagnosis and prognosis of Papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). In RET G691S polymorphism, the total dissemination of variant alleles (GA + AA) was 62.9% in cases as related to 44.5% in controls (P < 0.05). RET L769L variant alleles (TG + GG) was 70% in cases versus 88% in controls (P < 0.05). In RET S904S, occurrence of variant alleles (CG + GG) was 56% in cases versus 44% in controls (P < 0.05). G691S and L769L polymorphism advocate a “Dominant mode of inheritance”. The S904S polymorphism approves an “Additive mode of inheritance”. In conclusion, there was an over-representation of RET G691S/S904S polymorphisms and under-representation of L769L polymorphism in PTC and FTC patients. Additionally, our data suggest that some haplotypes (A T G, G T G and A T C) of RET may act as low penetrance alleles for predisposition of thyroid cancer

Rapamycin inhibition of baculovirus recombinant (BVr) ribosomal protein S6 kinase (S6K1) is mediated by an event other than phosphorylation

<p>Abstract</p> <p>Background</p> <p>Ribosomal protein S6 kinase 1(S6K1) is an evolutionary conserved kinase that is activated in response to growth factors and viral stimuli to influence cellular growth and proliferation. This downstream effector of target of rapamycin (TOR) signaling cascade is known to be directly activated by TOR- kinase mediated hydrophobic motif (HM) phosphorylation at Threonine 412 (T412). Selective loss of this phosphorylation by inactivation of TOR kinase or activation/recruitment of a phosphatase has accordingly been implicated in mediating inhibition by rapamycin.</p> <p>Findings</p> <p>We present evidence that baculovirus driven expression of S6K1 in insect cells (Sf9) fails to activate the enzyme and instead renders it modestly active representing 4-6 folds less activity than its fully active mammalian counterpart. Contrary to the contention that viral infection activates TOR signaling pathway, we report that BVr enzyme fails to exhibit putative TOR dependent phosphorylation at the HM and the resultant phosphorylation at the activation loop (AL) of the enzyme, correlating with the level of activity observed. Surprisingly, the BVr enzyme continued to exhibit sensitivity to rapamycin that remained unaffected by mutations compromised for TOR phosphorylation (T412A) or deletions compromised for TOR binding (ΔNH _2-46/ΔCT₁₀₄).</p> <p>Conclusions</p> <p>These data together with the ability of the BVr enzyme to resist inactivation by phosphatases indicate that inhibition by rapamycin is not mediated by any phosphorylation event in general and TOR dependent phosphorylation in particular.</p

Molecular Alterations and Expression Dynamics in the Etiopathogenesis of Thyroid Cancer

Thyroid carcinoma is the most prevalent endocrine malignancy and accounts for 2% of all human cancers. In the past decade, knowledge of genetic alterations of thyroid cancer (TC) has rapidly expanded, which has provided new insights into thyroid cancer etiology and has offered novel diagnostic tools and prognostic markers that enable improved and personalized management of thyroid cancer patients. Alterations in key signaling effectors seem to be the hallmark of distinct forms of thyroid neoplasia. Mutations or rearrangements in genes that encode Mitogen activated protein kinase (MAPK) pathway effectors seem to be required for transformation. Mutations in BRAF were the most recently identified MAPK effector in thyroid cancer. BRAF V600E is the most common alteration in sporadic papillary carcinoma. Three RAS proto-oncogenes (NRAS, HRAS & KRAS) are implicated in human thyroid tumorigenesis. High incidence of thyroid cancer worldwide indicates the importance of studying genetic alterations that lead to its carcinogenesis. BRAF and RAS alterations represent a novel indicator of the progression and aggressiveness of thyroid carcinogenesis. The GSα-adenylyl cyclase-cyclic AMP (cAMP) cascade is effected in thyroid cancer. Promoter hypermethylation of multiple genes especially TSHR has been identified to play a role in thyroid cancers, in particular showing a close association with BRAF mutational status. So, the main aim of the study was to elucidate the involvement of BRAF and RAS gene mutations along with BRAF expression and thyroid-stimulating hormone receptor (TSHR) hypermethylation in North Indian patients and investigate their association with clinicopathological characteristics

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries