Association of maternal hypertension and diabetes with variants of the NKX2-5, LEFTY1 and LEFTY2 genes in children with congenital heart defects: a case-control study from Pakistani Population.

BACKGROUND: Globally, congenital heart defect (CHD) is the most common congenital malformation, responsible for higher morbidity and mortality in the pediatric population. It is a complex multifactorial disease influenced by gene-environment and gene-gene interactions. The current study was the first attempt to study these polymorphisms in common clinical phenotypes of CHD in Pakistan and the association between maternal hypertension and diabetes with single nucleotide polymorphisms (SNPs) in children. METHODS: A total of 376 subjects were recruited in this current case-control study. Six variants from three genes were analyzed by cost-effective multiplex PCR and genotyped by minisequencing. Statistical analysis was done by GraphPad prism and Haploview. The association of SNPs and CHD was determined using logistic regression. RESULTS: The risk allele frequency was higher in cases as compared to healthy subjects, but the results were not significant for rs703752. However, stratification analysis suggested that rs703752 was significantly associated with the tetralogy of Fallot. The rs2295418 was significantly associated with maternal hypertension (OR = 16.41, p = 0.003), while a weak association was present between maternal diabetes and rs360057 (p = 0.08). CONCLUSION: In conclusion, variants in transcriptional and signaling genes were associated with Pakistani pediatric CHD patients that showed varied susceptibility between different clinical phenotypes of CHD. In addition, this study was the first report regarding the significant association between maternal hypertension and the LEFTY2 gene variant.

Single-molecule tracking of Nodal and Lefty in live zebrafish embryos supports hindered diffusion model.

The hindered diffusion model postulates that the movement of a signaling molecule through an embryo is affected by tissue geometry and binding-mediated hindrance, but these effects have not been directly demonstrated in vivo. Here, we visualize extracellular movement and binding of individual molecules of the activator-inhibitor signaling pair Nodal and Lefty in live developing zebrafish embryos using reflected light-sheet microscopy. We observe that diffusion coefficients of molecules are high in extracellular cavities, whereas mobility is reduced and bound fractions are high within cell-cell interfaces. Counterintuitively, molecules nevertheless accumulate in cavities, which we attribute to the geometry of the extracellular space by agent-based simulations. We further find that Nodal has a larger bound fraction than Lefty and shows a binding time of tens of seconds. Together, our measurements and simulations provide direct support for the hindered diffusion model and yield insights into the nanometer-to-micrometer-scale mechanisms that lead to macroscopic signal dispersal.

Nodal is a short-range morphogen with activity that spreads through a relay mechanism in human gastruloids.

Morphogens are signaling molecules that convey positional information and dictate cell fates during development. Although ectopic expression in model organisms suggests that morphogen gradients form through diffusion, little is known about how morphogen gradients are created and interpreted during mammalian embryogenesis due to the combined difficulties of measuring endogenous morphogen levels and observing development in utero. Here we take advantage of a human gastruloid model to visualize endogenous Nodal protein in living cells, during specification of germ layers. We show that Nodal is extremely short range so that Nodal protein is limited to the immediate neighborhood of source cells. Nodal activity spreads through a relay mechanism in which Nodal production induces neighboring cells to transcribe Nodal. We further show that the Nodal inhibitor Lefty, while biochemically capable of long-range diffusion, also acts locally to control the timing of Nodal spread and therefore of mesoderm differentiation during patterning. Our study establishes a paradigm for tissue patterning by an activator-inhibitor pair.

Role of EBAF/Nodal/p27 signaling pathway in development of placenta in normal and diabetic rats.

Placentas control the maternal-fetal transport of nutrients and gases. Placental reactions to adverse intrauterine conditions affect fetal development. Such adverse conditions occur in pregnancies complicated by diabetes, leading to alterations in placental anatomy and physiology. In this study, streptozocin (STZ) injection produced sustained hyperglycemia during pregnancy in rats. Hyperglycemic pregnant rats had gained significantly less weight than normal pregnant rats on embryonic day 15.5. We investigated the influence of diabetes on placental anatomy and physiology. Compared with controls, the diabetic group had a markedly thicker junctional zone at embryonic day 15.5. To explore a mechanism for this abnormality, we examined Nodal expression in the junctional zone of control and diabetic groups. We found lower expression of Nodal in the diabetic group. We then investigated the expression of its target gene p27Kip1 (p27), which is related to cell proliferation. In vitro, Nodal overexpression up-regulated p27 protein levels while interfered EBAF up-regulated p27. In vivo, the expression of p27 was lower in diabetic compared with normal rats, and localization was similar between the two groups. In contrast, a higher expression of PCNA was found in diabetic versus normal placenta. Endometrial bleeding associated factor (EBAF), an up-stream molecular regulator of Nodal, was expressed at higher levels in placenta from diabetic versus normal rats. Based on these results, we speculate that the EBAF/Nodal/p27 signaling pathway plays a role in morphological change of diabetic placenta.

Anti-inflammatory potential of Lactobacillus reuteri LM1071 via eicosanoid regulation in LPS-stimulated RAW264.7 cells.

AIMS: To investigate anti-inflammatory effects of Lactobacillus reuteri LM1071 in lipopolysaccharides (LPS)-induced inflammation RAW264.7 cells. METHODS AND RESULTS: To evaluate anti-inflammatory activities of L. reuteri LM1071, LPS-stimulated RAW264.7 cells were used. Gene expression levels of eight immune-associated genes including IL-1ß, IL-6 and TNF-α and protein production levels of COX-1 and COX-2 were analysed. Moreover, the production of eicosanoids as important biomarkers for anti-inflammation was determined. CONCLUSIONS: The current study demonstrates that L. reuteri LM1071 has anti-inflammatory potential by inhibiting the production of inflammation mediators such as NO, eicosanoids such as PGE1 & PGE2, pro-inflammatory cytokines and COX proteins. It can also enhance the production of inflammatory associated genes such as IL-11, BMP4, LEFTY2 and EET metabolite. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus reuteri is one of the crucial bacteria for food fermentation. It can be found in the gastrointestinal system of human and animals. Several studies have shown that L. reuteri has valuable effects on host health. The current study firstly demonstrated that L. reuteri has a beneficial effect on the inflammation containing the variation of eicosanoids (PGE1 and PGE2) which are one of the most important biomarkers and moreover eicosanoid-associated genes as well as proteins (COX-2).

Compositional Analysis of Extracellular Aggregates in the Eyes of Patients With Exfoliation Syndrome and Exfoliation Glaucoma.

Purpose: Exfoliation syndrome (XFS) is a condition characterized by the production of insoluble fibrillar aggregates (exfoliation material; XFM) in the eye and elsewhere. Many patients with XFS progress to exfoliation glaucoma (XFG), a significant cause of global blindness. We used quantitative mass spectrometry to analyze the composition of XFM in lens capsule specimens and in aqueous humor (AH) samples from patients with XFS, patients with XFG and unaffected individuals. Methods: Pieces of lens capsule and samples of AH were obtained with consent from patients undergoing cataract surgery. Tryptic digests of capsule or AH were analyzed by high-performance liquid chromatography-mass spectrometry and relative differences between samples were quantified using the tandem mass tag technique. The distribution of XFM on the capsular surface was visualized by SEM and super-resolution light microscopy. Results: A small set of proteins was consistently upregulated in capsule samples from patients with XFS and patients with XFG, including microfibril components fibrillin-1, latent transforming growth factor-ß-binding protein-2 and latent transforming growth factor-ß-binding protein-3. Lysyl oxidase-like 1, a cross-linking enzyme associated with XFS in genetic studies, was an abundant XFM constituent. Ligands of the transforming growth factor-ß superfamily were prominent, including LEFTY2, a protein best known for its role in establishing the embryonic body axis. Elevated levels of LEFTY2 were also detected in AH from patients with XFG, a finding confirmed subsequently by ELISA. Conclusions: This analysis verified the presence of suspected XFM proteins and identified novel components. Quantitative comparisons between patient samples revealed a consistent XFM proteome characterized by strong expression of fibrillin-1, lysyl oxidase-like-1, and LEFTY2. Elevated levels of LEFTY2 in the AH of patients with XFG may serve as a biomarker for the disease.

Podocalyxin promotes an impermeable epithelium and inhibits pro-implantation factors to negatively regulate endometrial receptivity.

Embryo implantation is a key step in establishing pregnancy and a major limiting factor in IVF. Implantation requires a receptive endometrium but the mechanisms governing receptivity are not well understood. We have recently discovered that podocalyxin (PCX or PODXL) is a key negative regulator of human endometrial receptivity. PCX is expressed in all endometrial epithelial cells in the non-receptive endometrium but selectively down-regulated in the luminal epithelium at receptivity. We have further demonstrated that this down-regulation is essential for implantation because PCX inhibits embryo attachment and penetration. However, how PCX confers this role is unknown. In this study, through RNAseq analysis of Ishikawa cell line stably overexpressing PCX, we discovered that PCX suppresses expression of genes controlling cell adhesion and communication, but increases those governing epithelial barrier functions, especially the adherens and tight junctions. Moreover, PCX suppresses multiple factors such as LIF and signaling pathways including Wnt and calcium signaling that support receptivity but stimulates anti-implantation genes such as LEFTY2. Functional studies confirmed that PCX promotes epithelial barrier functions by increasing key epithelial junction proteins such as E-cadherin and claudin 4. PCX thus promotes an anti-adhesive and impermeable epithelium while impedes pro-implantation factors to negatively control endometrial receptivity for implantation.

Expression of Cytokine-Coding Genes BMP8B, LEFTY1 and INSL5 Could Distinguish between Ulcerative Colitis and Crohn's Disease.

Ulcerative colitis (UC) and Crohn's disease (CD) are characterized by an imbalance between pro-inflammatory and anti-inflammatory cytokines, interfering with the resolution of inflammation. Due to the crucial role of cytokines, new insights into their profiles in UC and CD would help to improve our understanding of pathogenesis and enable the development of new treatment modalities. We provide an expression profile of cytokines in UC and CD, using bioinformatics approach, and experimental validation of expression of the selected genes. We retrieved data and analyzed the cytokine gene expression profiles of UC and CD. From ten genes with inverse expression, common to CD and UC, BMP8B, LEFTY1 and INSL5 were selected for gene expression experimental validation. Experimentally, BMP8B and INSL5 were down-regulated in both CD and UC but followed the bioinformatics trend. The expression of genes LEFTY1 and BMP8B was statistically significant when comparing UC and CD in colon and the expression of gene LEFTY1 showed statistical significance when CD in ileum and colon were compared. Using the bioinformatics approach and experimental validation, we found differences in expression profiles between UC and CD for INSL5, LEFTY1 and BMP8B. These three promising candidate genes need to be further explored at different levels, such as DNA methylation and protein expression, to provide more evidence on their potential diagnostic role in CD and UC.

LEFTY-PITX2 signaling pathway is critical for generation of mature and ventricular cardiac organoids in human pluripotent stem cell-derived cardiac mesoderm cells.

The generation of mature ventricular cardiomyocytes (CMs) resembling adult CMs from human pluripotent stem cells (hPSCs) is necessary for disease modeling and drug discovery. To investigate the effect of self-organizing capacity on the generation of mature cardiac organoids (COs), we generated cardiac mesoderm cell-derived COs (CMC-COs) and CM-derived COs (CM-COs) and evaluated COs. CMC-COs exhibited more organized sarcomere structures and mitochondria, well-arranged t-tubule structures, and evenly distributed intercalated discs. Increased expressions of ventricular CM, cardiac metabolic, t-tubule formation, K+ ion channel, and junctional markers were confirmed in CMC-COs. Mature ventricular-like function such as faster motion vector speed, decreased beats per min, increased peak-to-peak duration, and prolonged APD50 and APD90 were observed in CMC-COs. Transcriptional profiling revealed that extracellular matrix-integrin, focal adhesion, and LEFTY-PITX2 signaling pathways are upregulated in CMC-COs. LEFTY knockdown affected ECM-integrin-FA signaling pathways in CMC-COs. Here, we found that high self-organizing capacity of CMCs is critical for the generation of mature and ventricular COs. We also demonstrated that LEFTY-PITX2 signaling plays key roles for CM maturation and specification into ventricular-like CM subtype in CMC-COs. CMC-COs are an attractive resource for disease modeling and drug discovery.

Lefty1 Ameliorates Post-infarction Fibrosis by Suppressing p-Smad2 and p-ERK1/2 Signaling Pathways.

Transforming growth factor-ß1 signaling pathways are known to involve in the development of post-infarction fibrosis, a process characterized by the aberrant activation, proliferation, and differentiation of fibroblasts, as well as the unbalanced turnover of extracellular matrix proteins. Recent studies have shown that Lefty1, a novel member of TGF-ß superfamily, acts as a brake on the TGF-ß signaling pathway in non-cardiac tissues. However, its role in myocardial infarction (MI)-induced fibrosis and left ventricular remodeling has not been fully elucidated. Here, for the first time, we reported that Lefty1 alleviated post-MI fibroblast proliferation, differentiation, and secretion through suppressing p-Smad2 and p-ERK1/2 signaling pathways in vivo and in vitro. In MI mice or TGF-ß1-treated neonatal rat cardiac fibroblasts (CFBs), the expression of Lefty1 was upregulated. Adenovirus-mediated overexpression of Lefty1 significantly attenuated TGF-ß1-induced CFBs' proliferation, differentiation, and collagen production. Using the adeno-associated virus approach, we confirmed that Lefty1 attenuates MI-induced cardiac injury, as evidenced by the decreased infarct size and preserved cardiac function. These results highlight the importance of Lefty1 in the prevention of post-MI fibrosis and may help identify potential targets for therapeutic intervention of cardiac fibrosis. Graphical abstract.

Right, left and cilia: How asymmetry is established.

The initial breaking of left-right (L-R) symmetry in the embryo is controlled by a motile-cilia-driven leftward fluid flow in the left-right organiser (LRO), resulting in L-R asymmetric gene expression flanking the LRO. Ultimately this results in left- but not right-sided activation of the Nodal-Pitx2 pathway in more lateral tissues. While aspects of the initial breaking event clearly vary between vertebrates, events in the Lateral Plate Mesoderm (LPM) are conserved through the vertebrate lineage. Evidence from model systems and humans highlights the role of cilia both in the initial symmetry breaking and in the ability of more lateral tissues to exhibit asymmetric gene expression. In this review we concentrate on the process of L-R determination in mouse and humans.

Zebrafish Cdx1b modulates epithalamic asymmetry by regulating ndr2 and lft1 expression.

Nodal signaling is essential for mesoderm and endoderm formation, as well as neural plate induction and establishment of left-right asymmetry. However, the mechanisms controlling expression of Nodal pathway genes in these contexts are not fully known. Previously, we showed that Cdx1b induces expression of downstream Nodal signaling factors during early endoderm formation. In this study, we show that Cdx1b also regulates epithalamic asymmetry in zebrafish embryos by modulating expression of ndr2 and lft1. We first knocked down cdx1b with translation-blocking and splicing-blocking morpholinos (MOs). Most embryos injected with translation-blocking MOs showed absent ndr2, lft1 and pitx2c expression in the left dorsal diencephalon during segmentation and pharyngula stages accompanied by aberrant parapineal migration and habenular laterality at 72 â€‹h post fertilization (hpf). These defects were less frequent in embryos injected with splicing-blocking MO. To confirm the morphant phenotype, we next generated both zygotic (Z)cdx1b-/- and maternal zygotic (MZ)cdx1b-/- mutants by CRISPR-Cas9 mutagenesis. Expression of ndr2, lft1 and pitx2c was absent in the left dorsal diencephalon of a high proportion of MZcdx1b-/- mutants; however, aberrant dorsal diencephalic pitx2c expression patterns were observed at low frequency in Zcdx1b-/- mutant embryos. Correspondingly, dysregulated parapineal migration and habenular laterality were also observed in MZcdx1b-/- mutant embryos at 72 hpf. On the other hand, Kupffer's vesicle cilia length and number, expression pattern of spaw in the lateral plate mesoderm and pitx2c in the gut as well as left-right patterning of various visceral organs were not altered in MZcdx1b-/- mutants compared to wild-type embryos. Chromatin immunoprecipitation revealed that Cdx1b directly regulates ndr2 and lft1 expression. Furthermore, injection of cdx1b-vivo MO1 but not cdx1b-vivo 4 â€‹mm MO1 in the forebrain ventricle at 18 hpf significantly downregulated lft1 expression in the left dorsal diencephalon at 23-24 â€‹s stages. Together, our results suggest that Cdx1b regulates transcription of ndr2 and lft1 to maintain proper Nodal activity in the dorsal diencephalon and epithalamic asymmetry in zebrafish embryos.

Requirements of LEFTY and Nodal overexpression for tumor cell survival under hypoxia in glioblastoma.

Glioblastomas (GBM) contain numerous hypoxic foci associated with a rare fraction of glioma stem cells (GSCs). Left-right determination factor (LEFTY) and Nodal, members of the transforming growth factor ß (TGF-ß) superfamily, have glycogen synthase kinase 3ß (GSK-3ß) phosphorylation motifs and are linked with stemness in human malignancies. Herein, we investigated the roles of LEFTY and Nodal in GBM hypoxic foci. In clinical samples, significantly higher expression of LEFTY, Nodal, phospho (p) GSK-3ß, pSmad2, and Nestin, as well as higher apoptotic and lower proliferation rates, were observed in nonpseudopalisading (non-Ps) perinecrotic lesions as compared to Ps and non-necrotic tumor lesions, with a positive correlation between LEFTY, Nodal, pGSK-3ß, or pSmad2 scores. In KS-1, a GBM cell line that lacks endogenous Nodal expression, treatment with the hypoxic mimetic CoCl2 increased LEFTY, pGSK-3ß, and pSmad2 levels, but decreased pAkt levels. Moreover, the promoter for LEFTY, but not Nodal, was activated by Smad2 or TGF-ß1, suggesting that overexpression of LEFTY and Nodal may be due to Akt-independent GSK-3ß inactivation, with or without cooperation of the TGF-ß1/Smad2 axis. LEFTY and Nodal overexpression increased proliferation rates and reduced susceptibility to CoCl2 -induced apoptosis, and increased the expression of epithelial-mesenchymal transition (EMT)/GSC-related markers. An increased ALDH1high population and more efficient spheroid formation was also observed in LEFTY-overexpressing cells. These findings suggest that LEFTY and Nodal may contribute to cell survival in non-Ps GBM perinecrotic lesions, leading to alterations in apoptosis, proliferation, or EMT/GCS features.

LEFTY2 alleviates hepatic stellate cell activation and liver fibrosis by regulating the TGF-ß1/Smad3 pathway.

Activated hepatic stellate cells (HSCs) are the major cell type involved in the deposition of extracellular matrix (ECM) during the development of hepatic fibrosis. In this study, we revealed that left-right determination factor 2 (LEFTY2), one of the proteins belonging to the transforming growth factor-ß (TGF-ß) protein superfamily, was remarkedly decreased in human hepatic fibrosis tissues and in a carbon tetrachloride (CCl4)-induced liver fibrosis mouse model. In addition, TGF-ß1 treatment markedly reduced the level of LEFTY2 in HSCs. Importantly, overexpression of LEFTY2 suppressed the activation and proliferation of HSCs. LEFTY2 inhibited the expression of TGF-ß1-induced fibrosis-associated genes (α-SMA and COL1a1) in human (LX-2) and rat (HSC-T6) HSC cell lines in vitro. Mechanistically, we demonstrated, for the first time, the role of LEFTY2 in inhibiting TGF-ß1/Smad3 signaling, suggesting that there is a mutual antagonism between LEFTY2 and TGF-ß1/Smad3 signaling during liver fibrosis. Similarly, we observed that LEFTY2 has a negative effect on its downstream genes, including c-MYC, CDK4, and cyclin D1, in liver fibrosis. Collectively, our data strongly indicated that LEFTY2 plays an important role in controlling the proliferation and activation of HSCs in the progression of liver fibrosis and this could be a potential therapeutic target for its treatment.

LEFTY2/endometrial bleeding-associated factor up-regulates Na+ Coupled Glucose Transporter SGLT1 expression and Glycogen Accumulation in Endometrial Cancer Cells.

LEFTY2 (endometrial bleeding associated factor; EBAF or LEFTYA), a cytokine released shortly before menstrual bleeding, is a negative regulator of cell proliferation and tumour growth. LEFTY2 down-regulates Na+/H+ exchanger activity with subsequent inhibition of glycolytic flux and lactate production in endometrial cancer cells. Glucose can be utilized not only for glycolysis but also for glycogen formation. Both glycolysis and glycogen formation require cellular glucose uptake which could be accomplished by the Na+ coupled glucose transporter-1 (SGLT1; SLC5A1). The present study therefore explored whether LEFTY2 modifies endometrial SGLT1 expression and activity as well as glycogen formation. Ishikawa and HEC1a cells were exposed to LEFTY2, SGLT1 and glycogen synthase (GYS1) transcript levels determined by qRT-PCR. SGLT1, GYS1 and phospho-GYS1 protein abundance was quantified by western blotting, cellular glucose uptake from 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) uptake, and cellular glycogen content utilizing an enzymatic assay and subsequent colorimetry. As a result, a 48-hour treatment with LEFTY2 significantly increased SGLT1 and GYS1 transcript levels as well as SGLT1 and GYS1 protein abundance in both Ishikawa and HEC1a cells. 2-NBDG uptake and cellular glycogen content were upregulated significantly in Ishikawa (type 1) but not in type 2 endometrial HEC1a cells, although there was a tendency of increased 2-NBDG uptake. Further, none of the effects were seen in human benign endometrial cells (HESCs). Interestingly, in both Ishikawa and HEC1a cells, a co-treatment with TGF-ß reduced SGLT1, GYS and phospho-GYS protein levels, and thus reduced glycogen levels and again HEC1a cells had no significant change. In conclusion, LEFTY2 up-regulates expression and activity of the Na+ coupled glucose transporter SGLT1 and glycogen synthase GYS1 in a cell line specific manner. We further show the treatment with LEFTY2 fosters cellular glucose uptake and glycogen formation and TGF-ß can negate this effect in endometrial cancer cells.

Lefty-1 inhibits renal epithelial-mesenchymal transition by antagonizing the TGF-ß/Smad signaling pathway.

Epithelial-mesenchymal transition (EMT) is a biological process in which tubular epithelial cells lose their phenotypes, and new mesenchymal feature are obtained. In particular, type II EMT possibly contributes to renal tissue fibrogenesis. Recent studies indicate that Lefty-1, a novel member of the TGF-ß superfamily with pleiotropical and biological regulation characteristics on TGF-ß and other signaling pathways, is considered to have potential fibrotic effects. However, its role in EMT, which is often a long-term consequence of renal tubulointerstitial fibrosis, remains unknown. In this study, we found that Lefty-1 alleviates EMT induction through antagonizing TGF-ß/Smad pathway in vivo and in vitro. In unilateral ureteral obstruction (UUO) model mice, administration of adenovirus-mediated overexpression of Lefty-1 (Ad-Lefty-1) significantly reduced TGF-ß1/Smad expression and alleviated the phenotypic transition of epithelial cells to mesenchymal cells and extracellular matrix (ECM) accumulation. In high glucose-induced rat renal tubular duct epithelial cell line (NRK-52E), EMT and ECM synthesis were alleviated with Lefty-1 treatment, which significantly inhibited TGF-ß1/Smad pathway activation in UUO mice and high glucose-treated NRK-52E cells. Thus, Lefty-1 can alleviate EMT and renal interstitial fibrosis in vivo and in vitro through antagonizing the TGF-ß/Smad pathway, and Lefty-1 might have a potential novel therapeutic effect on fibrotic kidney diseases.

Expression of Lefty predicts Merkel cell carcinoma-specific death.

BACKGROUND: Lefty and Nodal are transforming growth factor ß-related proteins, which, beside their role in determination of laterality during embryogenesis, have also been linked with cancer progression. OBJECTIVES: Prompted by the observed significant left-sided laterality of Merkel cell carcinoma (MCC), we addressed whether Lefty and Nodal are expressed in MCC and correlated expression patterns with clinical parameters such as MCC laterality and patient outcome. METHODS: Expression of Lefty and Nodal in primary MCC was assessed in 29 patients by immunohistochemistry. The histology (H-)score was calculated and correlated with clinical parameters. RESULTS: The median (range) H-score of Lefty and Nodal was 17.6 (0-291) and 74.9 (0.7-272), respectively. There was a significant correlation between Lefty expression and Nodal expression (correlation coefficient of 0.60, P = 0.0006). There was no significant correlation between Lefty expression and Nodal expression with either tumour laterality, gender, age, Merkel cell polyomavirus status, disease stage, anatomical localization of primary tumours or disease relapse. On univariate analysis, low Lefty expression and Nodal expression were significantly associated with MCC-specific death (P = 0.010 and P = 0.019, respectively). On univariate analysis, low Lefty expression was the only significant independent predictor for MCC-specific death (P = 0.025) as indicated by an odds ratio of 14 (95% CI: 1.43-137.33). CONCLUSIONS: Lefty and Nodal are frequently expressed in MCC, but not correlated with tumour laterality. Importantly, our data suggest that a low level of Lefty expression in primary MCC is a strong predictor of MCC-specific death.

Chemokine signaling links cell-cycle progression and cilia formation for left-right symmetry breaking.

Zebrafish dorsal forerunner cells (DFCs) undergo vigorous proliferation during epiboly and then exit the cell cycle to generate Kupffer's vesicle (KV), a ciliated organ necessary for establishing left-right (L-R) asymmetry. DFC proliferation defects are often accompanied by impaired cilia elongation in KV, but the functional and molecular interaction between cell-cycle progression and cilia formation remains unknown. Here, we show that chemokine receptor Cxcr4a is required for L-R laterality by controlling DFC proliferation and KV ciliogenesis. Functional analysis revealed that Cxcr4a accelerates G1/S transition in DFCs and stabilizes forkhead box j1a (Foxj1a), a master regulator of motile cilia, by stimulating Cyclin D1 expression through extracellular regulated MAP kinase (ERK) 1/2 signaling. Mechanistically, Cyclin D1-cyclin-dependent kinase (CDK) 4/6 drives G1/S transition during DFC proliferation and phosphorylates Foxj1a, thereby disrupting its association with proteasome 26S subunit, non-ATPase 4b (Psmd4b), a 19S regulatory subunit. This prevents the ubiquitin (Ub)-independent proteasomal degradation of Foxj1a. Our study uncovers a role for Cxcr4 signaling in L-R patterning and provides fundamental insights into the molecular linkage between cell-cycle progression and ciliogenesis.

Embryo presence regulates NODAL/LEFTY2 system in the rat oviduct in vivo.

To gain further insight in the mechanisms of the embryo-maternal dialog in the oviduct, expression of members of the transforming growth factor-ß superfamily, NODAL, its inhibitor, LEFTY2, and their coreceptor, CFC1, were studied in the oviduct of 3-day post copula (3 dpc) females with and without embryos (E and NE), pseudopregnant rats (SP3), and in 3-day embryos. Nodal transcripts in SP3 oviducts showed a steady-state relative abundance when compared with proestrus stage and the 3 dpc. In contrast, Lefty2 and Cfc1 relative abundance levels in proestrus and 3 dpc were higher. When comparing E with NE oviducts, Nodal and Lefty2 expression levels decreased, while Cfc1 expression increased in the presence of embryos. Nodal messenger RNA (mRNA) was observed in the embryo, but Lefty2 and Cfc1 transcripts were not found. In addition, an increase in Lefty2 expression coincided with increased levels of matrix metalloproteinases 9 mRNA and protein in the oviduct and in the oviductal fluid, respectively. These observations have shed new light on the relevance of the NODAL/LEFTY2 pathway in the oviduct during early embryo development and the role of the embryo in modulating this pathway.