ResearchPad - signal-transduction https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[<i>Ehrlichia chaffeensis</i> TRP120-mediated ubiquitination and proteasomal degradation of tumor suppressor FBW7 increases oncoprotein stability and promotes infection]]> https://www.researchpad.co/article/elastic_article_13827 E. chaffeensis is an obligately intracellular bacterium that replicates in mononuclear phagocytes by secreting effectors that manipulate host cell processes and exploit evolutionarily conserved pathways. This investigation reveals the complex and expanding role of the E. chaffeensis TRP120 moonlighting effector as a ubiquitin (Ub) ligase targeting host nuclear proteins. Herein, we demonstrate that E. chaffeensis TRP120 HECT Ub ligase targets the nuclear tumor suppressor Skp1-cullin-1-FBOX E3 ubiquitin (Ub) ligase complex substrate recognition subunit, F-BOX and WD domain repeating-containing 7 (FBW7) for degradation. FBW7 is a central regulator of broadly acting host cell oncoproteins involved in cell proliferation and survival. The reduction in FBW7 through TRP120-mediated ubiquitination increases cellular oncoprotein levels and promotes E. chaffeensis infection. This study illuminates novel bacterial effector-host interactions, the importance and interplay of both host and bacterial Ub ligases and the Ub-proteasome system for infection, and mechanisms whereby evolutionarily conserved signaling pathways are hijacked by obligately intracellular pathogens.

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<![CDATA[Decyl caffeic acid inhibits the proliferation of colorectal cancer cells in an autophagy-dependent manner <i>in vitro</i> and <i>in vivo</i>]]> https://www.researchpad.co/article/elastic_article_13874 The treatment of human colorectal cancer (CRC) cells through suppressing the abnormal survival signaling pathways has recently become a significant area of focus. In this study, our results demonstrated that decyl caffeic acid (DC), one of the novel caffeic acid derivatives, remarkedly suppressed the growth of CRC cells both in vitro and in vivo. The inhibitory effects of DC on CRC cells were investigated in an in vitro cell model and in vivo using a xenograft mouse model. CRC cells were treated with DC at various dosages (0, 10, 20 and 40 μM), and cell survival, the apoptotic index and the autophagy level were measured using an MTT assay and flow cytometry analysis, respectively. The signaling cascades in CRC were examined by Western blot assay. The anti-cancer effects of DC on tumor growth were examined by using CRC HCT-116 cells implanted in an animal model. Our results indicated that DC differentially suppressed the growth of CRC HT-29 and HCT-116 cells through an enhancement of cell-cycle arrest at the S phase. DC inhibited the expression of cell-cycle regulators, which include cyclin E and cyclin A proteins. The molecular mechanisms of action were correlated to the blockade of the STAT3 and Akt signaling cascades. Strikingly, a high dosage of DC prompted a self-protection action through inducing cell-dependent autophagy in HCT-116 cells. Suppression of autophagy induced cell death in the treatment of DC in HCT-116 cells. DC seemed to inhibit cell proliferation of CRC differentially, and the therapeutic advantage appeared to be autophagy dependent. Moreover, consumption of DC blocked the tumor growth of colorectal adenocarcinoma in an experimental animal model. In conclusion, our results suggested that DC could act as a therapeutic agent through the significant suppression of tumor growth of human CRC cells.

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<![CDATA[Active Notch signaling is required for arm regeneration in a brittle star]]> https://www.researchpad.co/article/elastic_article_7845 Cell signaling pathways play key roles in coordinating cellular events in development. The Notch signaling pathway is highly conserved across all multicellular animals and is known to coordinate a multitude of diverse cellular events, including proliferation, differentiation, fate specification, and cell death. Specific functions of the pathway are, however, highly context-dependent and are not well characterized in post-traumatic regeneration. Here, we use a small-molecule inhibitor of the pathway (DAPT) to demonstrate that Notch signaling is required for proper arm regeneration in the brittle star Ophioderma brevispina, a highly regenerative member of the phylum Echinodermata. We also employ a transcriptome-wide gene expression analysis (RNA-seq) to characterize the downstream genes controlled by the Notch pathway in the brittle star regeneration. We demonstrate that arm regeneration involves an extensive cross-talk between the Notch pathway and other cell signaling pathways. In the regrowing arm, Notch regulates the composition of the extracellular matrix, cell migration, proliferation, and apoptosis, as well as components of the innate immune response. We also show for the first time that Notch signaling regulates the activity of several transposable elements. Our data also suggests that one of the possible mechanisms through which Notch sustains its activity in the regenerating tissues is via suppression of Neuralized1.

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<![CDATA[The qualitative assessment of optical coherence tomography and the central retinal sensitivity in patients with retinitis pigmentosa]]> https://www.researchpad.co/article/elastic_article_7697 To analyze the relationships between qualitative and quantitative parameters of spectral-domain optical coherence tomography (SD-OCT) and the central retinal sensitivity in patients with retinitis pigmentosa (RP).Materials and methodsNinety-three eyes of 93 patients were finally enrolled, with a median age (quartile) of 58 (24.5) years. We assessed the patients using SD-OCT and the 10–2 program of a Humphry Field Analyzer (HFA). As a qualitative parameter, two graders independently classified the patients’ SD-OCT images into five severity grades (grades 1–5) based on the severity of damage to the photoreceptor inner and outer segments (IS/OS) layer. As quantitative parameters, we measured the IS-ellipsoid zone (IS-EZ) width, IS/OS thickness, outer nuclear layer (ONL) thickness, central macular thickness (CMT, 1 and 3 mm) and macular cube (6 × 6 mm) volume and thickness. The central retinal sensitivity was defined by the best-corrected visual acuity (BCVA; logMAR), average sensitivities of the central 4 (foveal sensitivity [FS]) and 12 (macular sensitivity [MS]) points of the HFA 10–2 program and the mean deviation (MD) of the 10–2 program. Spearman’s correlation was used to assess the association between both qualitative and quantitative parameters and variables of the central retinal sensitivity. In addition, we performed a multiple regression analysis using these parameters to identify the parameters most strongly influencing the central retinal sensitivity.ResultsThe IS/OS severity grade was significantly correlated with the BCVA (ρ = 0.741, P < 0.001), FS (ρ = −0.844, P < 0.001), MS (ρ = −0.820, P < 0.001) and MD (ρ = −0.681, P < 0.001) and showed stronger correlations to them than any other quantitative parameters including the IS-EZ width, IS/OS thickness, ONL thickness, CMTs and macular cube volume/thickness. Furthermore, a step-wise multiple regression analysis indicated that the IS/OS severity grade was more strongly associated with the BCVA (β = 0.659, P < 0.001), FS (β = −0.820, P < 0.001), MS (β = −0.820, P < 0.001) and MD (β = −0.674, P < 0.001) than any other quantitative parameters. The intraclass correlation coefficient between two graders indicated substantial correlation (κ = 0.70).DiscussionThe qualitative grading of OCT based on the severity of the IS/OS layer was simple and strongly correlated with the central retinal sensitivity in patients with RP. It may be useful to assess the central visual function in patients with RP, although there is some variation in severity within the same severity grade. ]]> <![CDATA[A mathematical model coupling polarity signaling to cell adhesion explains diverse cell migration patterns]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be0154

Protrusion and retraction of lamellipodia are common features of eukaryotic cell motility. As a cell migrates through its extracellular matrix (ECM), lamellipod growth increases cell-ECM contact area and enhances engagement of integrin receptors, locally amplifying ECM input to internal signaling cascades. In contrast, contraction of lamellipodia results in reduced integrin engagement that dampens the level of ECM-induced signaling. These changes in cell shape are both influenced by, and feed back onto ECM signaling. Motivated by experimental observations on melanoma cells lines (1205Lu and SBcl2) migrating on fibronectin (FN) coated topographic substrates (anisotropic post-density arrays), we probe this interplay between intracellular and ECM signaling. Experimentally, cells exhibited one of three lamellipodial dynamics: persistently polarized, random, or oscillatory, with competing lamellipodia oscillating out of phase (Park et al., 2017). Pharmacological treatments, changes in FN density, and substrate topography all affected the fraction of cells exhibiting these behaviours. We use these observations as constraints to test a sequence of hypotheses for how intracellular (GTPase) and ECM signaling jointly regulate lamellipodial dynamics. The models encoding these hypotheses are predicated on mutually antagonistic Rac-Rho signaling, Rac-mediated protrusion (via activation of Arp2/3 actin nucleation) and Rho-mediated contraction (via ROCK phosphorylation of myosin light chain), which are coupled to ECM signaling that is modulated by protrusion/contraction. By testing each model against experimental observations, we identify how the signaling layers interact to generate the diverse range of cell behaviors, and how various molecular perturbations and changes in ECM signaling modulate the fraction of cells exhibiting each. We identify several factors that play distinct but critical roles in generating the observed dynamic: (1) competition between lamellipodia for shared pools of Rac and Rho, (2) activation of RhoA by ECM signaling, and (3) feedback from lamellipodial growth or contraction to cell-ECM contact area and therefore to the ECM signaling level.

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<![CDATA[Aspirin-triggered resolvin D1 attenuates PDGF-induced vascular smooth muscle cell migration via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdc0e7

Background and objectives

Resolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration.

Methods

VSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity.

Results

Treatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor.

Conclusions

Our results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.

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<![CDATA[Age-related transcriptional modules and TF-miRNA-mRNA interactions in neonatal and infant human thymus]]> https://www.researchpad.co/article/Ne5173bb6-5611-4e9c-b8d8-f6fe9062bcd6

The human thymus suffers a transient neonatal involution, recovers and then starts a process of decline between the 1st and 2nd years of life. Age-related morphological changes in thymus were extensively investigated, but the genomic mechanisms underlying this process remain largely unknown. Through Weighted Gene Co-expression Network Analysis (WGCNA) and TF-miRNA-mRNA integrative analysis we studied the transcriptome of neonate and infant thymic tissues grouped by age: 0–30 days (A); 31days-6 months (B); 7–12 months (C); 13–18 months (D); 19-31months (E). Age-related transcriptional modules, hubs and high gene significance (HGS) genes were identified, as well as TF-miRNA-hub/HGS co-expression correlations. Three transcriptional modules were correlated with A and/or E groups. Hubs were mostly related to cellular/metabolic processes; few were differentially expressed (DE) or related to T-cell development. Inversely, HGS genes in groups A and E were mostly DE. In A (neonate) one third of the hyper-expressed HGS genes were related to T-cell development, against one-twentieth in E, what may correlate with the early neonatal depletion and recovery of thymic T-cell populations. This genomic mechanism is tightly regulated by TF-miRNA-hub/HGS interactions that differentially govern cellular and molecular processes involved in the functioning of the neonate thymus and in the beginning of thymic decline.

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<![CDATA[Communication is key: Mother-offspring signaling can affect behavioral responses and offspring survival in feral horses (Equus caballus)]]> https://www.researchpad.co/article/Nfc9766a8-2564-4088-9a49-707302d05531

Acoustic signaling plays an important role in mother-offspring recognition and subsequent bond-formation. It remains unclear, however, if mothers and offspring use acoustic signaling in the same ways and for the same reasons throughout the juvenile stage, particularly after mutual recognition has been adequately established. Moreover, despite its critical role in mother-offspring bond formation, research explicitly linking mother-infant communication strategies to offspring survival are lacking. We examined the communicative patterns of mothers and offspring in the feral horse (Equus caballus) to better understand 1) the nature of mother-offspring communication throughout the first year of development; 2) the function(s) of mother- vs. offspring-initiated communication and; 3) the importance of mare and foal communication to offspring survival. We found that 1) mares and foals differ in when and how they initiate communication; 2) the outcomes of mare- vs. foal-initiated communication events consistently differ; and 3) the communicative patterns between mares and their foals can be important for offspring survival to one year of age. Moreover, given the importance of maternal activity to offspring behavior and subsequent survival, we submit that our data are uniquely positioned to address the long-debated question: do the behaviors exhibited during the juvenile stage (by both mothers and their young) confer delayed or immediate benefits to offspring? In summary, we aimed to better understand 1) the dynamics of mother-offspring communication, 2) whether mother-offspring communicative patterns were important to offspring survival, and 3) the implications of our research regarding the function of the mammalian juvenile stage. Our results demonstrate that we have achieved those aims.

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<![CDATA[Neuroprotective effects of exogenous erythropoietin in Wistar rats by downregulating apoptotic factors to attenuate N-methyl-D-aspartate-mediated retinal ganglion cells death]]> https://www.researchpad.co/article/N85685bba-c047-422b-abfc-358a98ed1fe7

The aim of this study was to investigate whether exogenous erythropoietin (EPO) administration attenuates N-methyl-D-aspartate (NMDA)-mediated excitotoxic retinal damage in Wistar rats. The survival rate of retinal ganglion cells (RGCs) were investigated by flat mount analysis and flow cytometry. A total of 125 male Wistar rats were randomly assigned to five groups: negative control, NMDA80 (i.e., 80 nmoles NMDA intravitreally injected), NMDA80 + 10ng EPO, NMDA80 + 50ng EPO, and NMDA80 + 250ng EPO. The NMDA80 + 50ng EPO treatment group was used to evaluate various administrated points (pre-/co-/post- administration of NMDA80). Meanwhile, the transferase dUTP Nick-End Labeling (TUNEL) assay of RGCs, the inner plexiform layer (IPL) thickness and the apoptotic signal transduction pathways of μ-calpain, Bax, and caspase 9 were assessed simultaneously using an immunohistochemical method (IHC). When EPO was co-administered with NMDA80, attenuated cell death occurred through the downregulation of the apoptotic indicators: μ-calpain was activated first (peak at ~18hrs), followed by Bax and caspase 9 (peak at ~40hrs). Furthermore, the images of retinal cross sections have clearly demonstrated that thickness of the inner plexiform layer (IPL) was significantly recovered at 40 hours after receiving intravitreal injection with NMDA80 and 50ng EPO. Exogenous EPO may protect RGCs and bipolar cell axon terminals in IPL by downregulating apoptotic factors to attenuate NMDA-mediated excitotoxic retinal damage.

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<![CDATA[Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer]]> https://www.researchpad.co/article/Naf611639-dea0-4cb3-8951-2157f0424339

Background

G protein-coupled estrogen receptor (GPER), or G protein-coupled receptor 30 (GPR30), is reported to mediate non-genomic estrogen signaling. GPR30 associates with breast cancer (BC) outcome and may contribute to tamoxifen resistance. We investigated the expression and prognostic significance of GPR30 in metachronous contralateral breast cancer (CBC) as a model of tamoxifen resistance.

Methods

Total GPR30 expression (GPR30TOT) and plasma membrane-localized GPR30 expression (GPR30PM) were analyzed by immunohistochemistry in primary (BC1; nBC1 = 559) and contralateral BC (BC2; nBC2 = 595), and in lymph node metastases (LGL; nLGL1 = 213; nLGL2 = 196). Death from BC (BCD), including BC death or death after documented distant metastasis, was used as primary end-point.

Results

GPR30PM in BC2 and LGL2 were associated with increased risk of BCD (HRBC2 = 1.7, p = 0.03; HRLGL2 = 2.0; p = 0.02). In BC1 and BC2, GPR30PM associated with estrogen receptor (ER)-negativity (pBC1<0.0001; pBC2<0.0001) and progesterone receptor (PR)-negativity (pBC1 = 0.0007; pBC2<0.0001). The highest GPR30TOT and GPR30PM were observed in triple-negative BC. GPR30PM associated with high Ki67 staining in BC1 (p<0.0001) and BC2 (p<0.0001). GPR30TOT in BC2 did not associate with tamoxifen treatment for BC1. However, BC2 that were diagnosed during tamoxifen treatment were more likely to express GPR30PM than BC2 diagnosed after treatment completion (p = 0.01). Furthermore, a trend was observed that patients with GPR30PM in an ER-positive BC2 had greater benefit from tamoxifen treatment.

Conclusion

PM-localized GPR30 staining is associated with increased risk of BC death when expressed in BC2 and LGL2. Additionally, PM-localized GPR30 correlates with prognostic markers of worse outcome, such as high Ki67 and a triple-negative subtype. Therefore, PM-localized GPR30 may be an interesting new target for therapeutic exploitation. We found no clear evidence that total GPR30 expression is affected by tamoxifen exposure during development of metachronous CBC, or that GPR30 contributes to tamoxifen resistance.

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<![CDATA[Role of MPK4 in pathogen-associated molecular pattern-triggered alternative splicing in Arabidopsis]]> https://www.researchpad.co/article/N4009e20f-330a-49f1-8a3f-309ba227a41c

Alternative splicing (AS) of pre-mRNAs in plants is an important mechanism of gene regulation in environmental stress tolerance but plant signals involved are essentially unknown. Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) is mediated by mitogen-activated protein kinases and the majority of PTI defense genes are regulated by MPK3, MPK4 and MPK6. These responses have been mainly analyzed at the transcriptional level, however many splicing factors are direct targets of MAPKs. Here, we studied alternative splicing induced by the PAMP flagellin in Arabidopsis. We identified 506 PAMP-induced differentially alternatively spliced (DAS) genes. Importantly, of the 506 PAMP-induced DAS genes, only 89 overlap with the set of 1950 PAMP-induced differentially expressed genes (DEG), indicating that transcriptome analysis does not identify most DAS events. Global DAS analysis of mpk3, mpk4, and mpk6 mutants in the absence of PAMP treatment showed no major splicing changes. However, in contrast to MPK3 and MPK6, MPK4 was found to be a key regulator of PAMP-induced DAS events as the AS of a number of splicing factors and immunity-related protein kinases is affected, such as the calcium-dependent protein kinase CPK28, the cysteine-rich receptor like kinases CRK13 and CRK29 or the FLS2 co-receptor SERK4/BKK1. Although MPK4 is guarded by SUMM2 and consequently, the mpk4 dwarf and DEG phenotypes are suppressed in mpk4 summ2 mutants, MPK4-dependent DAS is not suppressed by SUMM2, supporting the notion that PAMP-triggered MPK4 activation mediates regulation of alternative splicing.

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<![CDATA[Podocyte RNA sequencing reveals Wnt- and ECM-associated genes as central in FSGS]]> https://www.researchpad.co/article/Nff231b2e-f2d8-47eb-acf2-c510faf35a1a

Loss of podocyte differentiation can cause nephrotic-range proteinuria and Focal and Segmental Glomerulosclerosis (FSGS). As specific therapy is still lacking, FSGS frequently progresses to end-stage renal disease. The exact molecular mechanisms of FSGS and gene expression changes in podocytes are complex and widely unknown as marker changes have mostly been assessed on the glomerular level. To gain a better insight, we isolated podocytes of miR-193a overexpressing mice, which suffer from FSGS due to suppression of the podocyte master regulator Wt1. We characterised the podocytic gene expression changes by RNAseq and identified many novel candidate genes not linked to FSGS so far. This included strong upregulation of the receptor tyrosine kinase EphA6 and a massive dysregulation of circadian genes including the loss of the transcriptional activator Arntl. By comparison with podocyte-specific changes in other FSGS models we found a shared dysregulation of genes associated with the Wnt signaling cascade, while classical podocyte-specific genes appeared widely unaltered. An overlap with gene expression screens from human FSGS patients revealed a strong enrichment in genes associated with extra-cellular matrix (ECM) and metabolism. Our data suggest that FSGS progression might frequently depend on pathways that are often overlooked when considering podocyte homeostasis.

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<![CDATA[Distinct gradients of various neurotransmitter markers in caudate nucleus and putamen of the human brain]]> https://www.researchpad.co/article/N1dbc95ff-966f-4bb7-82cc-51b31f6a8b4c

Abstract

The caudate nucleus (CN) and the putamen (PUT) as parts of the human striatum are distinguished by a marked heterogeneity in functional, anatomical, and neurochemical patterns. Our study aimed to document in detail the regional diversity in the distribution of dopamine (DA), serotonin, γ‐aminobuturic acid, and choline acetyltransferase within the CN and PUT. For this purpose we dissected the CN as well as the PUT of 12 post‐mortem brains of human subjects with no evidence of neurological and psychiatric disorders (38–81 years old) into about 80 tissue parts. We then investigated rostro‐caudal, dorso‐ventral, and medio‐lateral gradients of these neurotransmitter markers. All parameters revealed higher levels, turnover rates, or activities in the PUT than in the CN. Within the PUT, DA levels increased continuously from rostral to caudal. In contrast, the lowest molar ratio of homovanillic acid to DA, a marker of DA turnover, coincided with highest DA levels in the caudal PUT, the part of the striatum with the highest loss of DA in Parkinson’s disease (N. Engl. J. Med., 318, 1988, 876). Highest DA concentrations were found in the most central areas both in the PUT and CN. We observed an age‐dependent loss of DA in the PUT and CN that did not correspond to the loss described for Parkinson’s disease indicating different mechanisms inducing the deficit of DA. Our data demonstrate a marked heterogeneity in the anatomical distribution of neurotransmitter markers in the human dorsal striatum indicating anatomical and functional diversity within this brain structure.

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<![CDATA[A graph-based algorithm for RNA-seq data normalization]]> https://www.researchpad.co/article/N0b813aa9-b155-4778-93ba-b0f37d26ae8a

The use of RNA-sequencing has garnered much attention in recent years for characterizing and understanding various biological systems. However, it remains a major challenge to gain insights from a large number of RNA-seq experiments collectively, due to the normalization problem. Normalization has been challenging due to an inherent circularity, requiring that RNA-seq data be normalized before any pattern of differential (or non-differential) expression can be ascertained; meanwhile, the prior knowledge of non-differential transcripts is crucial to the normalization process. Some methods have successfully overcome this problem by the assumption that most transcripts are not differentially expressed. However, when RNA-seq profiles become more abundant and heterogeneous, this assumption fails to hold, leading to erroneous normalization. We present a normalization procedure that does not rely on this assumption, nor prior knowledge about the reference transcripts. This algorithm is based on a graph constructed from intrinsic correlations among RNA-seq transcripts and seeks to identify a set of densely connected vertices as references. Application of this algorithm on our synthesized validation data showed that it could recover the reference transcripts with high precision, thus resulting in high-quality normalization. On a realistic data set from the ENCODE project, this algorithm gave good results and could finish in a reasonable time. These preliminary results imply that we may be able to break the long persisting circularity problem in RNA-seq normalization.

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<![CDATA[Restoration of Mal overcomes the defects of apoptosis in lung cancer cells]]> https://www.researchpad.co/article/N4678b2b4-03e3-4a62-aa79-1954dd96fe53

Background and aims

Cancer is one of the life-threatening diseases of human beings; the pathogenesis of cancer remains to be further investigated. Toll like receptor (TLR) activities are involved in the apoptosis regulation. This study aims to elucidate the role of Mal (MyD88-adapter-like) molecule in the apoptosis regulation of lung cancer (LC) cells.

Methods

The LC tissues were collected from LC patients. LC cells and normal control (NC) cells were isolated from the tissues and analyzed by pertinent biochemical and immunological approaches.

Results

We found that fewer apoptotic LC cells were induced by cisplatin in the culture as compared to NC cells. The expression of Fas ligand (FasL) was lower in LC cells than that in NC cells. FasL mRNA levels declined spontaneously in LC cells. A complex of FasL/TDP-43 was detected in LC cells. LC cells expressed less Mal than NC cells. Activation of Mal by lipopolysaccharide (LPS) increased TDP-43 expression in LC cells. TDP-43 formed a complex with FasL mRNA to prevent FasL mRNA from decay. Reconstitution of Mal or TDP-43 restored the sensitiveness of LC cells to apoptotic inducers.

Conclusions

LC cells express low Mal levels that contributes to FasL mRNA decay through impairing TDP-43 expression. Reconstitution of Mal restores sensitiveness of LC cells to apoptosis inducers that may be a novel therapeutic approach for LC treatment.

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<![CDATA[Prolyl isomerization of FAAP20 catalyzed by PIN1 regulates the Fanconi anemia pathway]]> https://www.researchpad.co/article/5c784fbdd5eed0c484007497

The Fanconi Anemia (FA) pathway is a multi-step DNA repair process at stalled replication forks in response to DNA interstrand cross-links (ICLs). Pathological mutation of key FA genes leads to the inherited disorder FA, characterized by progressive bone marrow failure and cancer predisposition. The study of FA is of great importance not only to children suffering from FA but also as a model to study cancer pathogenesis in light of genome instability among the general population. FANCD2 monoubiquitination by the FA core complex is an essential gateway that connects upstream DNA damage signaling to enzymatic steps of repair. FAAP20 is a key component of the FA core complex, and regulated proteolysis of FAAP20 mediated by the ubiquitin E3 ligase SCFFBW7 is critical for maintaining the integrity of the FA complex and FA pathway signaling. However, upstream regulatory mechanisms that govern this signaling remain unclear. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, regulates the integrity of the FA core complex, thus FA pathway activation. We demonstrate that PIN1 catalyzes cis-trans isomerization of the FAAP20 pSer48-Pro49 motif and promotes FAAP20 stability. Mechanistically, PIN1-induced conformational change of FAAP20 enhances its interaction with the PP2A phosphatase to counteract SCFFBW7-dependent proteolytic signaling at the phosphorylated degron motif. Accordingly, PIN1 deficiency impairs FANCD2 activation and the DNA ICL repair process. Together, our study establishes PIN1-dependent prolyl isomerization as a new regulator of the FA pathway and genomic integrity.

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<![CDATA[Furin, a transcriptional target of NKX2-5, has an essential role in heart development and function]]> https://www.researchpad.co/article/5c897793d5eed0c4847d307a

The homeodomain transcription factor NKX2-5 is known to be essential for both normal heart development and for heart function. But little is yet known about the identities of its downstream effectors or their function during differentiation of cardiac progenitor cells (CPCs). We have used transgenic analysis and CRISPR-mediated ablation to identify a cardiac enhancer of the Furin gene. The Furin gene, encoding a proprotein convertase, is directly repressed by NKX2-5. Deletion of Furin in CPCs is embryonic lethal, with mutant hearts showing a range of abnormalities in the outflow tract. Those defects are associated with a reduction in proliferation and premature differentiation of the CPCs. Deletion of Furin in differentiated cardiomyocytes results in viable adult mutant mice showing an elongation of the PR interval, a phenotype that is consistent with the phenotype of mice and human mutant for Nkx2-5. Our results show that Furin mediate some aspects of Nkx2-5 function in the heart.

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<![CDATA[Distinctive single-channel properties of α4β2-nicotinic acetylcholine receptor isoforms]]> https://www.researchpad.co/article/5c8acc7cd5eed0c48498f842

Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the α4β2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(α4β2)2β2- and LS-(α4β2)2α4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity α4(+)/(-)β2 subunit binding interfaces. However LS-(α4β2)2α4-nAChR also respond to higher concentrations of ACh, acting at a third α4(+)/(-)α4 subunit interface. To probe isoform functional differences further, HS- and LS-α4β2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(α4β2)2β2-nAChR exhibit a single conductance state, whereas LS-(α4β2)2α4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(α4β2)2α4-nAChR bursting and reduced closed times. Introduction of an α4(+)/(-)α4-interface loss-of-function α4W182A mutation abolished these changes, confirming this site’s role in mediating LS-(α4β2)2α4-nAChR responses. Small or large amplitude openings are highly-correlated within individual LS-(α4β2)2α4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by α4(+)/(-)α4 site ACh binding. These findings are consistent with α4(+)/(-)α4 subunit interface occupation resulting in allosteric potentiation of agonist actions at α4(+)/(-)β2 subunit interfaces, rather than independent induction of high conductance channel openings.

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<![CDATA[The role of TLR9 on Leishmania amazonensis infection and its influence on intranasal LaAg vaccine efficacy]]> https://www.researchpad.co/article/5c7d95ebd5eed0c484734fa1

Leishmania (L.) amazonensis is one of the etiological agents of cutaneous leishmaniasis (CL) in Brazil. Currently, there is no vaccine approved for human use against leishmaniasis, although several vaccine preparations are in experimental stages. One of them is Leishvacin, or LaAg, a first-generation vaccine composed of total L. amazonensis antigens that has consistently shown an increase of mouse resistance against CL when administered intranasally (i.n.). Since Toll-like receptor 9 (TLR9) is highly expressed in the nasal mucosa and LaAg is composed of TLR9-binding DNA CpG motifs, in this study we proposed to investigate the role of TLR9 in both L. amazonensis infection and in LaAg vaccine efficacy in C57BL/6 (WT) mice and TLR9-/- mice. First, we evaluated, the infection of macrophages by L. amazonensis in vitro, showing no significant difference between macrophages from WT and TLR9-/- mice in terms of both infection percentage and total number of intracellular amastigotes, as well as NO production. In addition, neutrophils from WT and TLR9-/- mice had similar capacity to produce neutrophil extracellular traps (NETs) in response to L. amazonensis. L. amazonensis did not activate dendritic cells from WT and TLR9-/- mice, analysed by MHCII and CD86 expression. However, in vivo, TLR9-/- mice were slightly more susceptible to L. amazonensis infection than WT mice, presenting a larger lesion and an increased parasite load at the peak of infection and in the chronic phase. The increased TLR9-/- mice susceptibility was accompanied by an increased IgG and IgG1 production; a decrease of IFN-γ in infected tissue, but not IL-4 and IL-10; and a decreased number of IFN-γ producing CD8+ T cells, but not CD4+ T cells in the lesion-draining lymph nodes. Also, TLR9-/- mice could not control parasite growth following i.n. LaAg vaccination unlike the WT mice. This protection failure was associated with a reduction of the hypersensitivity response induced by immunization. The TLR9-/- vaccinated mice failed to respond to antigen stimulation and to produce IFN-γ by lymph node cells. Together, these results suggest that TLR9 contributes to C57BL/6 mouse resistance against L. amazonensis, and that the TLR9-binding LaAg comprising CpG motifs may be important for intranasal vaccine efficacy against CL.

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<![CDATA[The seven transmembrane domain protein MoRgs7 functions in surface perception and undergoes coronin MoCrn1-dependent endocytosis in complex with Gα subunit MoMagA to promote cAMP signaling and appressorium formation in Magnaporthe oryzae]]> https://www.researchpad.co/article/5c7d95f6d5eed0c484735053

Regulator of G-protein signaling (RGS) proteins primarily function as GTPase-accelerating proteins (GAPs) to promote GTP hydrolysis of Gα subunits, thereby regulating G-protein mediated signal transduction. RGS proteins could also contain additional domains such as GoLoco to inhibit GDP dissociation. The rice blast fungus Magnaporthe oryzae encodes eight RGS and RGS-like proteins (MoRgs1 to MoRgs8) that have shared and distinct functions in growth, appressorium formation and pathogenicity. Interestingly, MoRgs7 and MoRgs8 contain a C-terminal seven-transmembrane domain (7-TM) motif typical of G-protein coupled receptor (GPCR) proteins, in addition to the conserved RGS domain. We found that MoRgs7, but not MoRgs8, couples with Gα MoMagA to undergo endocytic transport from the plasma membrane to the endosome upon sensing of surface hydrophobicity. We also found that MoRgs7 can interact with hydrophobic surfaces via a hydrophobic interaction, leading to the perception of environmental hydrophobiccues. Moreover, we found that MoRgs7-MoMagA endocytosis is regulated by actin patch-associated protein MoCrn1, linking it to cAMP signaling. Our studies provided evidence suggesting that MoRgs7 could also function in a GPCR-like manner to sense environmental signals and it, together with additional proteins of diverse functions, promotes cAMP signaling required for developmental processes underlying appressorium function and pathogenicity.

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