Genomic and transcriptomic landscape of conjunctival melanoma.

Conjunctival melanoma (CJM) is a rare but potentially lethal and highly-recurrent cancer of the eye. Similar to cutaneous melanoma (CM), it originates from melanocytes. Unlike CM, however, CJM is relatively poorly characterized from a genomic point of view. To fill this knowledge gap and gain insight into the genomic nature of CJM, we performed whole-exome (WES) or whole-genome sequencing (WGS) of tumor-normal tissue pairs in 14 affected individuals, as well as RNA sequencing in a subset of 11 tumor tissues. Our results show that, similarly to CM, CJM is also characterized by a very high mutation load, composed of approximately 500 somatic mutations in exonic regions. This, as well as the presence of a UV light-induced mutational signature, are clear signs of the role of sunlight in CJM tumorigenesis. In addition, the genomic classification of CM proposed by TCGA seems to be well-applicable to CJM, with the presence of four typical subclasses defined on the basis of the most frequently mutated genes: BRAF, NF1, RAS, and triple wild-type. In line with these results, transcriptomic analyses revealed similarities with CM as well, namely the presence of a transcriptomic subtype enriched for immune genes and a subtype enriched for genes associated with keratins and epithelial functions. Finally, in seven tumors we detected somatic mutations in ACSS3, a possible new candidate oncogene. Transfected conjunctival melanoma cells overexpressing mutant ACSS3 showed higher proliferative activity, supporting the direct involvement of this gene in the tumorigenesis of CJM. Altogether, our results provide the first unbiased and complete genomic and transcriptomic classification of CJM.

Glucose-6-Phosphate Dehydrogenases: The Hidden Players of Plant Physiology.

Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes a metabolic hub between glycolysis and the pentose phosphate pathway (PPP), which is the oxidation of glucose-6-phosphate (G6P) to 6-phosphogluconolactone concomitantly with the production of nicotinamide adenine dinucleotide phosphate (NADPH), a reducing power. It is considered to be the rate-limiting step that governs carbon flow through the oxidative pentose phosphate pathway (OPPP). The OPPP is the main supplier of reductant (NADPH) for several "reducing" biosynthetic reactions. Although it is involved in multiple physiological processes, current knowledge on its exact role and regulation is still piecemeal. The present review provides a concise and comprehensive picture of the diversity of plant G6PDHs and their role in seed germination, nitrogen assimilation, plant branching, and plant response to abiotic stress. This work will help define future research directions to improve our knowledge of G6PDHs in plant physiology and to integrate this hidden player in plant performance.

The TIE1 transcriptional repressor controls shoot branching by directly repressing BRANCHED1 in Arabidopsis.

Shoot branching is a major determinant of plant architecture and is regulated by both endogenous and environmental factors. BRANCHED1 (BRC1) is a central local regulator that integrates signals controlling shoot branching. So far, the regulation of BRC1 activity at the protein level is still largely unknown. In this study, we demonstrated that TIE1 (TCP interactor containing EAR motif protein 1), a repressor previously identified as an important factor in the control of leaf development, also regulates shoot branching by repressing BRC1 activity. TIE1 is predominantly expressed in young axillary buds. The gain-of-function mutant tie1-D produced more branches and the overexpression of TIE1 recapitulated the increased branching of tie1-D, while disruption of TIE1 resulted in lower bud activity and fewer branches. We also demonstrated that the TIE1 protein interacts with BRC1 in vitro and in vivo. Expression of BRC1 fused with the C-terminus of the TIE1 protein in wild type caused excessive branching similar to that observed in tie1-D and brc1 loss-of-function mutants. Transcriptome analyses revealed that TIE1 regulated about 30% of the BRC1-dependent genes, including the BRC1 direct targets HB21, HB40 and HB53. These results indicate that TIE1 acts as a positive regulator of shoot branching by directly repressing BRC1 activity. Thus, our results reveal that TIE1 is an important shoot branching regulator, and provide new insights in the post-transcriptional regulation of the TCP transcription factor BRC1.

Correction: The TIE1 transcriptional repressor controls shoot branching by directly repressing BRANCHED1 in Arabidopsis.

[This corrects the article DOI: 10.1371/journal.pgen.1007296.].

Glial cells of the human fovea.

Purpose: The exact cellular types that form the human fovea remain a subject of debate, and few studies have been conducted on human macula to solve this question. The purpose of this study was to perform immunohistochemistry on fresh human samples to characterize the glial cells that form the human fovea. Methods: Immunohistochemistry was performed using antibodies against proteins expressed in astrocytes or in retinal Müller glial cells or both types of cells on six human macula obtained from eyes enucleated for peripheral intraocular tumors and on two postmortem eyes from healthy donors. The posterior poles of the enucleated eyes were cryosectioned and stained with antibodies against the glial proteins GFAP, vimentin, CRALBP, glutamine synthetase, and connexin 43. Results: A population of cells positive for GFAP and negative for glutamine synthetase and CRALBP that express connexin 43 were identified at the roof of the foveal pit. These cells are distinct from the Müller cone cells described by Yamada and Gass, suggesting that another type of foveal glial cells, most likely astrocytes, are present in the human fovea. Conclusions: This study showed that in humans, astrocytic glial cells cover the foveal pit. Their roles in macula homeostasis and mechanisms of macular diseases disease remain to be determined.

Comprehensive Genetic Landscape of Uveal Melanoma by Whole-Genome Sequencing.

Uveal melanoma (UM) is a rare intraocular tumor that, similar to cutaneous melanoma, originates from melanocytes. To gain insights into its genetics, we performed whole-genome sequencing at very deep coverage of tumor-control pairs in 33 samples (24 primary and 9 metastases). Genome-wide, the number of coding mutations was rather low (only 17 variants per tumor on average; range 7-28), thus radically different from cutaneous melanoma, where hundreds of exonic DNA insults are usually detected. Furthermore, no UV light-induced mutational signature was identified. Recurrent coding mutations were found in the known UM drivers GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. Other genes, i.e., TP53BP1, CSMD1, TTC28, DLK2, and KTN1, were also found to harbor somatic mutations in more than one individual, possibly indicating a previously undescribed association with UM pathogenesis. De novo assembly of unmatched reads from non-coding DNA revealed peculiar copy-number variations defining specific UM subtypes, which in turn could be associated with metastatic transformation. Mutational-driven comparison with other tumor types showed that UM is very similar to pediatric tumors, characterized by very few somatic insults and, possibly, important epigenetic changes. Through the analysis of whole-genome sequencing data, our findings shed new light on the molecular genetics of uveal melanoma, delineating it as an atypical tumor of the adult for which somatic events other than mutations in exonic DNA shape its genetic landscape and define its metastatic potential.

Excess mortality on the liver transplant waiting list: unintended policy consequences and Model for End-Stage Liver Disease (MELD) inflation.

UNLABELLED: The Model for End-Stage Liver Disease (MELD) allocation system for liver transplantation provides "exceptions" for diseases such as hepatocellular carcinoma (HCC). It was the aim of this study to assess equipoise between exception candidates and nonexception candidates on the waiting list and to assess if the exception system contributes to steadily increasing regional MELD at transplant. In all, 78,595 adult liver transplant candidates between January 2005 and December 2012 were analyzed. Yearly trends in waiting list characteristics and transplantation rates were analyzed for statistical association with MELD exceptions. Regional variations in these associations and the effect of exceptions on regional MELD scores at transplant were also analyzed. 27.29% of the waiting list was occupied by candidates with exceptions. Candidates with exceptions fared much better on the waiting list compared to those without exceptions in mean days waiting (HCC 237 versus non-HCC 426), transplantation rates (HCC 79.05% versus non-HCC 40.60%), and waiting list death rates (HCC 4.49% versus non-HCC 24.63%). Strong regional variation in exception use occurred but exceptions were highly correlated with waiting list death rates, transplantation rates, and MELD score at removal in all regions. In a multivariate model predicting MELD score at transplant within regions, the percentage of HCC MELD exceptions was the strongest independent predictor of regional MELD score at transplant. CONCLUSION: Liver transplant candidates with MELD exceptions have superior outcomes compared to nonexception candidates and the current MELD exception system is largely responsible for steadily increasing MELD scores at transplant independent of geography.

Angiogenic activity of breast cancer patients' monocytes reverted by combined use of systems modeling and experimental approaches.

Angiogenesis plays a key role in tumor growth and cancer progression. TIE-2-expressing monocytes (TEM) have been reported to critically account for tumor vascularization and growth in mouse tumor experimental models, but the molecular basis of their pro-angiogenic activity are largely unknown. Moreover, differences in the pro-angiogenic activity between blood circulating and tumor infiltrated TEM in human patients has not been established to date, hindering the identification of specific targets for therapeutic intervention. In this work, we investigated these differences and the phenotypic reversal of breast tumor pro-angiogenic TEM to a weak pro-angiogenic phenotype by combining Boolean modelling and experimental approaches. Firstly, we show that in breast cancer patients the pro-angiogenic activity of TEM increased drastically from blood to tumor, suggesting that the tumor microenvironment shapes the highly pro-angiogenic phenotype of TEM. Secondly, we predicted in silico all minimal perturbations transitioning the highly pro-angiogenic phenotype of tumor TEM to the weak pro-angiogenic phenotype of blood TEM and vice versa. In silico predicted perturbations were validated experimentally using patient TEM. In addition, gene expression profiling of TEM transitioned to a weak pro-angiogenic phenotype confirmed that TEM are plastic cells and can be reverted to immunological potent monocytes. Finally, the relapse-free survival analysis showed a statistically significant difference between patients with tumors with high and low expression values for genes encoding transitioning proteins detected in silico and validated on patient TEM. In conclusion, the inferred TEM regulatory network accurately captured experimental TEM behavior and highlighted crosstalk between specific angiogenic and inflammatory signaling pathways of outstanding importance to control their pro-angiogenic activity. Results showed the successful in vitro reversion of such an activity by perturbation of in silico predicted target genes in tumor derived TEM, and indicated that targeting tumor TEM plasticity may constitute a novel valid therapeutic strategy in breast cancer.

Molecular and social regulation of worker division of labour in fire ants.

Reproductive and worker division of labour (DOL) is a hallmark of social insect societies. Despite a long-standing interest in worker DOL, the molecular mechanisms regulating this process have only been investigated in detail in honey bees, and little is known about the regulatory mechanisms operating in other social insects. In the fire ant Solenopsis invicta, one of the most studied ant species, workers are permanently sterile and the tasks performed are modulated by the worker's internal state (age and size) and the outside environment (social environment), which potentially includes the effect of the queen presence through chemical communication via pheromones. However, the molecular mechanisms underpinning these processes are unknown. Using a whole-genome microarray platform, we characterized the molecular basis for worker DOL and we explored how a drastic change in the social environment (i.e. the sudden loss of the queen) affects global gene expression patterns of worker ants. We identified numerous genes differentially expressed between foraging and nonforaging workers in queenright colonies. With a few exceptions, these genes appear to be distinct from those involved in DOL in bees and wasps. Interestingly, after the queen was removed, foraging workers were no longer distinct from nonforaging workers at the transcriptomic level. Furthermore, few expression differences were detected between queenright and queenless workers when we did not consider the task performed. Thus, the social condition of the colony (queenless vs. queenright) appears to impact the molecular pathways underlying worker task performance, providing strong evidence for social regulation of DOL in S. invicta.

Oligopotent stem cells are distributed throughout the mammalian ocular surface.

The integrity of the cornea, the most anterior part of the eye, is indispensable for vision. Forty-five million individuals worldwide are bilaterally blind and another 135 million have severely impaired vision in both eyes because of loss of corneal transparency; treatments range from local medications to corneal transplants, and more recently to stem cell therapy. The corneal epithelium is a squamous epithelium that is constantly renewing, with a vertical turnover of 7 to 14 days in many mammals. Identification of slow cycling cells (label-retaining cells) in the limbus of the mouse has led to the notion that the limbus is the niche for the stem cells responsible for the long-term renewal of the cornea; hence, the corneal epithelium is supposedly renewed by cells generated at and migrating from the limbus, in marked opposition to other squamous epithelia in which each resident stem cell has in charge a limited area of epithelium. Here we show that the corneal epithelium of the mouse can be serially transplanted, is self-maintained and contains oligopotent stem cells with the capacity to generate goblet cells if provided with a conjunctival environment. Furthermore, the entire ocular surface of the pig, including the cornea, contains oligopotent stem cells (holoclones) with the capacity to generate individual colonies of corneal and conjunctival cells. Therefore, the limbus is not the only niche for corneal stem cells and corneal renewal is not different from other squamous epithelia. We propose a model that unifies our observations with the literature and explains why the limbal region is enriched in stem cells.

Notch1 functions as a tumor suppressor in mouse skin.

Notch proteins are important in binary cell-fate decisions and inhibiting differentiation in many developmental systems, and aberrant Notch signaling is associated with tumorigenesis. The role of Notch signaling in mammalian skin is less well characterized and is mainly based on in vitro studies, which suggest that Notch signaling induces differentiation in mammalian skin. Conventional gene targeting is not applicable to establishing the role of Notch receptors or ligands in the skin because Notch1-/- embryos die during gestation. Therefore, we used a tissue-specific inducible gene-targeting approach to study the physiological role of the Notch1 receptor in the mouse epidermis and the corneal epithelium of adult mice. Unexpectedly, ablation of Notch1 results in epidermal and corneal hyperplasia followed by the development of skin tumors and facilitated chemical-induced skin carcinogenesis. Notch1 deficiency in skin and in primary keratinocytes results in increased and sustained expression of Gli2, causing the development of basal-cell carcinoma-like tumors. Furthermore, Notch1 inactivation in the epidermis results in derepressed beta-catenin signaling in cells that should normally undergo differentiation. Enhanced beta-catenin signaling can be reversed by re-introduction of a dominant active form of the Notch1 receptor. This leads to a reduction in the signaling-competent pool of beta-catenin, indicating that Notch1 can inhibit beta-catenin-mediated signaling. Our results indicate that Notch1 functions as a tumor-suppressor gene in mammalian skin.

PlantACT! - how to tackle the climate crisis.

Greenhouse gas (GHG) emissions have created a global climate crisis which requires immediate interventions to mitigate the negative effects on all aspects of life on this planet. As current agriculture and land use contributes up to 25% of total GHG emissions, plant scientists take center stage in finding possible solutions for a transition to sustainable agriculture and land use. In this article, the PlantACT! (Plants for climate ACTion!) initiative of plant scientists lays out a road map of how and in which areas plant scientists can contribute to finding immediate, mid-term, and long-term solutions, and what changes are necessary to implement these solutions at the personal, institutional, and funding levels.

Identification of New Vulnerabilities in Conjunctival Melanoma Using Image-Based High Content Drug Screening.

Recent evidence suggests that numerous similarities exist between the genomic landscapes of both conjunctival and cutaneous melanoma. Since alterations of several components of the MAP kinases, PI3K/mTOR, and cell cycle pathways have been reported in conjunctival melanoma, we decided to assess the sensitivity of conjunctival melanoma to targeted inhibition mostly of kinase inhibitors. A high content drug screening assay based on automated fluorescence microscopy was performed in three conjunctival melanoma cell lines with different genomic backgrounds with 489 kinase inhibitors and 53 other inhibitors. IC50 and apoptosis induction were respectively assessed for 53 and 48 compounds. The genomic background influenced the response to MAK and PI3K/mTOR inhibition, more specifically cell lines with BRAF V600E mutations were more sensitive to BRAF/MEK inhibition, while CRMM2 bearing the NRASQ61L mutation was more sensitive to PI3k/mTOR inhibition. All cell lines demonstrated sensitivity to cell cycle inhibition, being more pronounced in CRMM2, especially with polo-like inhibitors. Our data also revealed new vulnerabilities to Hsp90 and Src inhibition. This study demonstrates that the genomic background partially influences the response to targeted therapy and uncovers a large panel of potential vulnerabilities in conjunctival melanoma that may expand available options for the management of this tumor.

Spatial control of potato tuberization by the TCP transcription factor BRANCHED1b.

The control of carbon allocation, storage and usage is critical for plant growth and development and is exploited for both crop food production and CO2 capture. Potato tubers are natural carbon reserves in the form of starch that have evolved to allow propagation and survival over winter. They form from stolons, below ground, where they are protected from adverse environmental conditions and animal foraging. We show that BRANCHED1b (BRC1b) acts as a tuberization repressor in aerial axillary buds, which prevents buds from competing in sink strength with stolons. BRC1b loss of function leads to ectopic production of aerial tubers and reduced underground tuberization. In aerial axillary buds, BRC1b promotes dormancy, abscisic acid responses and a reduced number of plasmodesmata. This limits sucrose accumulation and access of the tuberigen protein SP6A. BRC1b also directly interacts with SP6A and blocks its tuber-inducing activity in aerial nodes. Altogether, these actions help promote tuberization underground.

Evidence for degeneration of the Y chromosome in the dioecious plant Silene latifolia.

The human Y--probably because of its nonrecombining nature--has lost 97% of its genes since X and Y chromosomes started to diverge [1, 2]. There are clear signs of degeneration in the Drosophila miranda neoY chromosome (an autosome fused to the Y chromosome), with neoY genes showing faster protein evolution [3-6], accumulation of unpreferred codons [6], more insertions of transposable elements [5, 7], and lower levels of expression [8] than neoX genes. In the many other taxa with sex chromosomes, Y degeneration has hardly been studied. In plants, many genes are expressed in pollen [9], and strong pollen selection may oppose the degeneration of plant Y chromosomes [10]. Silene latifolia is a dioecious plant with young heteromorphic sex chromosomes [11, 12]. Here we test whether the S. latifolia Y chromosome is undergoing genetic degeneration by analyzing seven sex-linked genes. S. latifolia Y-linked genes tend to evolve faster at the protein level than their X-linked homologs, and they have lower expression levels. Several Y gene introns have increased in length, with evidence for transposable-element accumulation. We detect signs of degeneration in most of the Y-linked gene sequences analyzed, similar to those of animal Y-linked and neo-Y chromosome genes.

5-Hydroxymethylcytosine Loss in Conjunctival Melanoma.

AIMS: Conjunctival and cutaneous melanoma partially share similar clinical and molecular backgrounds. As 5-hydroxymethylcytosine (5-hmC) loss has been demonstrated in cutaneous melanoma, we decided to assess if similar changes were occurring in conjunctival melanoma. METHODS: 5-methylcytosine (5-mC), 5-hmC and TET2 were respectively identified by immunohistochemistry and RNA ISH in 40 conjunctival nevi and 37 conjunctival melanomas. Clinicopathological correlations were established. RESULTS: 5-mC, TET2 and 5-hmC were respectively identified in 67.5%, 95% and 100% of conjunctival nevi and in 81.1%, 35.1% and 54% of conjunctival melanomas. A significant 5-hmC and TET2 loss was identified in conjunctival melanoma comparing to nevus, as well as a significant correlation between TET2 and 5-hmC expression. In the melanomas, 5-hmC expression was only significantly associated with local lymphatic invasion, but not with other clinicopathological parameters. There was a correlation between TET2 expression and the localization of the tumors. 5-mC expression was not associated with any clinicopathological parameters. CONCLUSIONS: We identified a significant 5-hmC loss in conjunctival melanoma similar to cutaneous melanoma. This loss may possibly be attributed to TET2 loss or IDH1 mutations. 5-hmC loss in conjunctival melanoma may help in the differential diagnosis between atypical conjunctival nevus and conjunctival melanoma.

An in vitro Model of Human Retinal Detachment Reveals Successive Death Pathway Activations.

PURPOSE: was to create an in vitro model of human retinal detachment (RD) to study the mechanisms of photoreceptor death. METHODS: Human retinas were obtained through eye globe donations for research purposes and cultivated as explants. Cell death was investigated in retinas with (control) and without retinal pigment epithelium (RPE) cells to mimic RD. Tissues were studied at different time points and immunohistological analyses for TUNEL, Cleaved caspase3, AIF, CDK4 and the epigenetic mark H3K27me3 were performed. Human and monkey eye globes with retinal detachment served as controls. RESULTS: The number of TUNEL-positive cells, compared between 1 and 7 days, increased with time in both retinas with RPE (from 1.2 ± 0.46 to 8 ± 0.89, n = 4) and without RPE (from 2.6 ± 0.73 to 16.3 ± 1.27, p < 0.014). In the group without RPE, cell death peaked at day 3 (p = 0.014) and was high until day 7. Almost no Cleaved-Caspase3 signal was observed, whereas a transient augmentation at day 3 of AIF-positive cells was observed to be about 10-fold in comparison to the control group (n = 2). Few CDK4-positive cells were found in both groups, but significantly more in the RD group at day 7 (1.8 ± 0.24 vs. 4.7 ± 0.58, p = 0.014). The H3K27me3 mark increased by 7-fold after 5 days in the RD group (p = 0.014) and slightly decreased at day 7 and was also observed to be markedly increased in human and monkey detached retina samples. CONCLUSION: AIF expression coincides with the first peak of cell death, whereas the H3K27me3 mark increases during the cell death plateau, suggesting that photoreceptor death is induced by different successive pathways after RD. This in vitro model should permit the identification of neuroprotective drugs with clinical relevance.

Tp63-expressing adult epithelial stem cells cross lineages boundaries revealing latent hairy skin competence.

The formation of hair follicles, a landmark of mammals, requires complex mesenchymal-epithelial interactions and it is commonly believed that embryonic epidermal cells are the only cells that can respond to hair follicle morphogenetic signals in vivo. Here, we demonstrate that epithelial stem cells of non-skin origin (e.g. that of cornea, oesophagus, vagina, bladder, prostate) that express the transcription factor Tp63, a master gene for the development of epidermis and its appendages, can respond to skin morphogenetic signals. When exposed to a newborn skin microenvironment, these cells express hair-follicle lineage markers and contribute to hair follicles, sebaceous glands and/or epidermis renewal. Our results demonstrate that lineage restriction is not immutable and support the notion that all Tp63-expressing epithelial stem cells, independently of their embryonic origin, have latent skin competence explaining why aberrant hair follicles or sebaceous glands are sometimes observed in non-skin tissues (e.g. in cornea, vagina or thymus).

YODA Kinase Controls a Novel Immune Pathway of Tomato Conferring Enhanced Disease Resistance to the Bacterium Pseudomonas syringae.

Mitogen-activated protein kinases (MAPK) play pivotal roles in transducing developmental cues and environmental signals into cellular responses through pathways initiated by MAPK kinase kinases (MAP3K). AtYODA is a MAP3K of Arabidopsis thaliana that controls stomatal development and non-canonical immune responses. Arabidopsis plants overexpressing a constitutively active YODA protein (AtCA-YDA) show broad-spectrum disease resistance and constitutive expression of defensive genes. We tested YDA function in crops immunity by heterologously overexpressing AtCA-YDA in Solanum lycopersicum. We found that these tomato AtCA-YDA plants do not show developmental phenotypes and fitness alterations, except a reduction in stomatal index, as reported in Arabidopsis AtCA-YDA plants. Notably, AtCA-YDA tomato plants show enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and constitutive upregulation of defense-associated genes, corroborating the functionality of YDA in tomato immunity. This function was further supported by generating CRISPR/Cas9-edited tomato mutants impaired in the closest orthologs of AtYDA [Solyc08g081210 (SlYDA1) and Solyc03g025360 (SlYDA2)]. Slyda1 and Slyda2 mutants are highly susceptible to P. syringae pv. tomato DC3000 in comparison to wild-type plants but only Slyda2 shows altered stomatal index. These results indicate that tomato orthologs have specialized functions and support that YDA also regulates immune responses in tomato and may be a trait for breeding disease resistance.

Independent origin of sex chromosomes in two species of the genus Silene.

Here we introduce a new model species, Silene colpophylla, that could facilitate research of sex chromosome evolution and sex-determining systems. This species is related to the well-established dioecious plant model Silene latifolia. Our results show that S. colpophylla is, similarly to S. latifolia, a male heterogametic species, but its sex chromosomes have evolved from a different pair of autosomes than in S. latifolia. The results of our phylogenetic study and mapping of homologs of S. latifolia X-linked genes indicate that the sex determination system in S. colpophylla evolved independently from that in S. latifolia. We assert that this model species pair will make it possible to study two independent patterns of sex chromosome evolution in related species.