HLH Protein Research Articles

Assessment of ID family proteins expression in colorectal cancer of Iraqi patients.

Colorectal cancer (CRC) is the second most deathly worldwide and third most common cancer, CRC is a very heterogeneous disease where tumors can form by both environmental and genetic risk factors and includes epigenetic and genetic alternations. Inhibitors of DNA binding proteins (ID) are a class of helix-loop-helix transcription regulatory factors; these proteins are considered a family of four highly preserved transcriptional regulators (ID1-4), shown to play significant roles in many processes that are associated with tumor development. ID family plays as negatively dominant antagonists of other essential HLH proteins, concluding the creation of non-functional heterodimers and regulation of the transcription process. 120 Fresh tissue and blood samples Forty (40) samples of fresh tissue and blood were collected from patients diagnosed with CRC, twenty (20) samples were collected from a patient diagnosed as healthy. The (qRT-PCR) method is a sensitive technique for the quantifying of steady-state mRNA levels that used to evaluation the expression levels of ID (1-4) gene. The findings indicate downregulation in ID1 in tissue with a highly significant change between patients and control groups, where upregulation in the ID1 gene is shown in blood samples.ID2 gene also demonstrated high significant change where show upregulation in tissue and downregulation in blood sample. ID3 and ID4 genes show downregulation in tissue and blood samples with a significant change in ID3 blood samples between patient and blood groups. Because of the regulation function of the ID family in many processes, the up or down regulation of IDs genes in tumors Proves how important its tumor development, and therefore those proteins can be used as an indicator for CRC.

Juan Modolell (1937-2023).

Juan Modolell (1937-2023).

Insight Into Pituitary lncRNA and mRNA at Two Estrous Stages in Small Tail Han Sheep With Different FecB Genotypes.

The pituitary is a remarkably dynamic organ with roles in hormone (FSH and LH) synthesis and secretion. In animals with the FecB (fecundity Booroola) mutation, the pituitary experiences hormone fluctuations during the follicular–luteal transition, which is implicated in the expression and regulation of many genes and regulators. Long non-coding RNAs (lncRNAs) are a novel type of regulatory factors for the reproductive process. Nevertheless, the expression patterns of lncRNAs and their roles in FecB-mediated follicular development and ovulation remain obscure. Thus, we profiled the pituitary transcriptome during the follicular (F, 45 h after evacuation vaginal sponges) and luteal (L, 216 h after evacuation vaginal sponges) phases in FecB-mutant homozygous (BB) and wild-type (WW) Small Tail Han sheep. We identified 78 differentially expressed genes (DEGs) and 41 differentially expressed lncRNAs (DELs) between BB_F and BB_L, 32 DEGs and 26 DELs between BB_F and WW_F, 16 DEGs and 29 DELs between BB_L and WW_L, and 50 DEGs and 18 DELs between WW_F and WW_L. The results of real-time quantitative PCR (RT-qPCR) correlated well with the transcriptome data. In both the follicular and luteal phases, DEGs (GRID2, glutamate ionotropic receptor delta type subunit 2; ST14, ST14 transmembrane serine protease matriptase) were enriched in hormone synthesis, secretion, and action. MSTRG.47470 and MSTRG.101530 were the trans-regulated elements of ID1 (inhibitor of DNA binding 3, HLH protein) and the DEG ID3 (inhibitor of DNA binding 3, HLH protein), and EEF2 (eukaryotic translation elongation factor 2), respectively; these factors might be involved in melatonin and peptide hormone secretion. In the FecB-mediated follicular phase, MSTRG.125392 targeted seizure-related 6 homolog like (SEZ6L), and MSTRG.125394 and MSTRG.83276 targeted the DEG KCNQ3 (potassium voltage-gated channel subfamily Q member 3) in cis, while MSTRG.55861 targeted FKBP4 (FKBP prolyl isomerase 4) in trans. In the FecB-mediated luteal phase, LOC105613905, MSTRG.81536, and MSTRG.150434 modulated TGFB1, SMAD3, OXT, respectively, in trans. We postulated that the FecB mutation in pituitary tissue elevated the expression of certain genes associated with pituitary development and hormone secretion. Furthermore, this study provides new insights into how the pituitary regulates follicular development and ovulation, illustrated by the effect of the FecB mutation.

Regulation of lignin biosynthesis by an atypical bHLH protein CmHLB in Chrysanthemum.

Stem mechanical strength is one of the most important agronomic traits that affects the resistance of plants against insects and lodging, and plays an essential role in the quality and yield of plants. Several transcription factors regulate mechanical strength in crops. However, mechanisms of stem strength formation and regulation remain largely unexplored, especially in ornamental plants. In this study, we identified an atypical bHLH transcription factor CmHLB (HLH PROTEIN INVOLVED IN LIGNIN BIOSYNTHESIS) in chrysanthemum, belonging to a small bHLH sub-family - the PACLOBUTRAZOL RESISTANCE (PRE) family. Overexpression of CmHLB in chrysanthemum significantly increased mechanical strength of the stem, cell wall thickness, and lignin content, compared with the wild type. In contrast, CmHLB RNA interference lines exhibited the opposite phenotypes. RNA-seq analysis indicated that CmHLB promoted the expression of genes involved in lignin biosynthesis. Furthermore, we demonstrated that CmHLB interacted with Chrysanthemum KNOTTED ARABIDOPSIS THALIANA7 (CmKNAT7) through the KNOX2 domain, which has a conserved function, i.e. it negatively regulates secondary cell wall formation of fibres and lignin biosynthesis. Collectively, our results reveal a novel role for CmHLB in regulating lignin biosynthesis by interacting with CmKNAT7 and affecting stem mechanical strength in Chrysanthemum.

Epstein-Barr virus status of sporadic Burkitt lymphoma is associated with patient age and mutational features.

Sporadic Burkitt lymphoma (BL) is the most frequent tumour of children and adolescents but a rare subtype of lymphomas in adults. To date most molecular data have been obtained from lymphomas arising in the young. Recently, Epstein-Barr virus (EBV) positive and negative BL in young patients was shown to differ in molecular features. In the present study, we present a large age-overarching cohort of sporadic BL (n=162) analysed by immunohistochemistry, translocations of MYC proto-oncogene, basic helix-loop-helix transcription factor (MYC), B-cell leukaemia/lymphoma 2 (BCL2) and B-cell leukaemia/lymphoma 6 (BCL6) and by targeted sequencing. We illustrate an age-associated inter-tumoral molecular heterogeneity in this disease. Mutations affecting inhibitor of DNA binding 3, HLH protein (ID3), transcription factor 3 (TCF3) and cyclin D3 (CCND3), which are highly recurrent in paediatric BL, and expression of sex determining region Y-box transcription factor 11 (SOX11) declined with patient age at diagnosis (P=0·0204 and P=0·0197 respectively). In contrast, EBV was more frequently detected in adult patients (P=0·0262). Irrespective of age, EBV-positive sporadic BL showed significantly less frequent mutations in ID3/TCF3/CCND3 (P=0·0088) but more often mutations of G protein subunit alpha 13 (GNA13; P=0·0368) and forkhead box O1 (FOXO1; P=0·0044) compared to EBV-negative tumours. Our findings suggest that among sporadic BL an EBV-positive subgroup of lymphomas increases with patient age that shows distinct pathogenic features reminiscent of EBV-positive endemic BL.

Ectopic expression of the atypical HLH FaPRE1 gene determines changes in cell size and morphology

PACLOBUTRAZOL RESISTANCE (PRE) genes code atypical HLH transcriptional regulators characterized by the absence of a DNA-binding domain but present an HLH dimerization domain. In vegetative tissues, the function of these HLH proteins has been related with cell elongation processes. In strawberry, three FaPRE genes are expressed, two of them (FaPRE2 and FaPRE3) in vegetative tissues while FaPRE1 is fruit receptacle-specific. Ubiquitous FaPRE1 accumulation produced elongated flower receptacles and plants due to the elongation of the main aerial vegetative organs, with the exception of leaves. Histological analysis clearly demonstrated that the observed phenotype was due to significant changes in the parenchymal cell’s morphology. In addition, transcriptomic studies of the transgenic elongated flower receptacles allowed to identify a small group of differentially expressed genes that encode cell wall-modifying enzymes. Together, the data seem to indicate that, in the strawberry plant vegetative organs, FaPRE proteins could modulate the expression of genes related with the determination of the size and shape of the parenchymal cells.

The CIB1 transcription factor regulates light- and heat-inducible cell elongation via a two-step HLH/bHLH system.

Light and high temperature promote plant cell elongation. PHYTOCHROME INTERACTING FACTOR4 (PIF4, a typical basic helix-loop-helix [bHLH] transcriptional activator) and the non-DNA binding atypical HLH inhibitors PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and LONG HYPOCOTYL IN FAR-RED 1 (HFR1) competitively regulate cell elongation in response to light conditions and high temperature. However, the underlying mechanisms have not been fully clarified. Here, we show that in Arabidopsis thaliana, the bHLH transcription factor CRYPTOCHROME-INTERACTING BASIC HELIX-LOOP-HELIX 1 (CIB1) positively regulates cell elongation under the control of PIF4, PAR1, and HFR1. Furthermore, PIF4 directly regulates CIB1 expression by interacting with its promoter, and PAR1 and HFR1 interfere with PIF4 binding to the CIB1 promoter. CIB1 activates genes that function in cell elongation, and PAR1 interferes with the DNA binding activity of CIB1, thus suppressing cell elongation. Hence, two antagonistic HLH/bHLH systems, the PIF4-PAR1/HFR1 and CIB1-PAR1 systems, regulate cell elongation in response to light and high temperature. We thus demonstrate the important role of non-DNA binding small HLH proteins in the transcriptional regulation of cell elongation in plants.

A Gas-and-Brake Mechanism of bHLH Proteins Modulates Shade Avoidance.

Plants detect proximity of competitors through reduction in the ratio between red and far-red light that triggers the shade avoidance syndrome, inducing responses such as accelerated shoot elongation and early flowering. Shade avoidance is regulated by PHYTOCHROME INTERACTING FACTORs, a group of basic helix-loop-helix (bHLH) transcription factors. Another (b)HLH protein, KIDARI (KDR), which is non-DNA-binding, was identified in de-etiolation studies and proposed to interact with LONG HYPOCOTYL IN FAR-RED1 (HFR1), a (b)HLH protein that inhibits shade avoidance. Here, we established roles of KDR in regulating shade avoidance in Arabidopsis (Arabidopsis thaliana) and investigated how KDR regulates the shade avoidance network. We showed that KDR is a positive regulator of shade avoidance and interacts with several negative growth regulators. We identified KDR interactors using a combination of yeast two-hybrid screening and dedicated confirmations with bimolecular fluorescence complementation. We demonstrated that KDR is translocated primarily to the nucleus when coexpressed with these interactors. A genetic approach confirmed that several of these interactions play a functional role in shade avoidance; however, we propose that KDR does not interact with HFR1 to regulate shade avoidance. Based on these observations, we propose that shade avoidance is regulated by a three-layered gas-and-brake mechanism of bHLH protein interactions, adding a layer of complexity to what was previously known.

HLH-30/TFEB Is a Master Regulator of Reproductive Quiescence.

All animals have evolved the ability to survive nutrient deprivation, and nutrient signaling pathways are conserved modulators of health and disease. In C.elegans, late-larval starvation provokes the adult reproductive diapause (ARD), a long-lived quiescent state that enables survival for months without food, yet underlying molecular mechanisms remain unknown. Here, we show that ARD is distinct from other forms of diapause, showing little requirement for canonical longevity pathways, autophagy, and fat metabolism. Instead it requires the HLH-30/TFEB transcription factor to promote the morphological and physiological remodeling involved in ARD entry, survival, and recovery, suggesting that HLH-30 is amaster regulator of reproductive quiescence. HLH-30 transcriptome and genetic analyses reveal that Max-like HLH factors, AMP-kinase, mTOR, protein synthesis, and mitochondrial fusion are target processes that promote ARD longevity. ARD thus rewires metabolism to ensure long-term survival and may illuminate similar mechanisms acting in stem cell quiescence and long-term fasting.

Midkine-a Is Required for Cell Cycle Progression of Müller Glia during Neuronal Regeneration in the Vertebrate Retina

In the retina of zebrafish, Müller glia have the ability to reprogram into stem cells capable of regenerating all classes of retinal neurons and restoring visual function. Understanding the cellular and molecular mechanisms controlling the stem cell properties of Müller glia in zebrafish may provide cues to unlock the regenerative potential in the mammalian nervous system. Midkine is a cytokine/growth factor with multiple roles in neural development, tissue repair, and disease. In midkine-a loss-of-function mutants of both sexes, Müller glia initiate the appropriate reprogramming response to photoreceptor death by increasing expression of stem cell-associated genes, and entering the G1 phase of the cell cycle. However, transition from G1 to S phase is blocked in the absence of Midkine-a, resulting in significantly reduced proliferation and selective failure to regenerate cone photoreceptors. Failing to progress through the cell cycle, Müller glia undergo reactive gliosis, a pathological hallmark in the injured CNS of mammals. Finally, we determined that the Midkine-a receptor, anaplastic lymphoma kinase, is upstream of the HLH regulatory protein, Id2a, and of the retinoblastoma gene, p130, which regulates progression through the cell cycle. These results demonstrate that Midkine-a functions as a core component of the mechanisms that regulate proliferation of stem cells in the injured CNS.SIGNIFICANCE STATEMENT The death of retinal neurons and photoreceptors is a leading cause of vision loss. Regenerating retinal neurons is a therapeutic goal. Zebrafish can regenerate retinal neurons from intrinsic stem cells, Müller glia, and are a powerful model to understand how stem cells might be used therapeutically. Midkine-a, an injury-induced growth factor/cytokine that is expressed by Müller glia following neuronal death, is required for Müller glia to progress through the cell cycle. The absence of Midkine-a suspends proliferation and neuronal regeneration. With cell cycle progression stalled, Müller glia undergo reactive gliosis, a pathological hallmark of the mammalian retina. This work provides a unique insight into mechanisms that control the cell cycle during neuronal regeneration.

Inhibitor of DNA binding 2 (ID2) regulates key transcriptional programs in multiple myeloma

Dysregulation of transcriptional control is a common mechanism driving oncogenesis. Microenvironment is a critical factor promoting myelomagenesis, tumor growth and drug resistance by impacting myeloma transcriptome. Here, we used RNA-sequencing of multiple myeloma (MM) cells cultured with stromal cell line HS-5 to identify transcriptional changes enhancing MM cell proliferation. We identified a reproducible and significant downregulation of inhibitor of DNA binding 2 (ID2), a known regulator of transcription through heterodimerization with basic HLH (bHLH) proteins and inhibition of their binding to DNA, across various MM cell lines in presence of stroma.

Extramacrochaetae promotes branch and bouton number via the sequestration of daughterless in the cytoplasm of neurons.

The Class I basic helix-loop-helix (bHLH) proteins are highly conserved transcription factors that are ubiquitously expressed. A wealth of literature on Class I bHLH proteins has shown that these proteins must homodimerize or heterodimerize with tissue-specific HLH proteins in order to bind DNA at E-box consensus sequences to control tissue-specific transcription. Due to its ubiquitous expression, Class I bHLH proteins are also extensively regulated posttranslationally, mostly through dimerization. Previously, we reported that in addition to its role in promoting neurogenesis, the Class I bHLH protein daughterless also functions in mature neurons to restrict axon branching and synapse number. Here, we show that part of the molecular logic that specifies how daughterless functions in neurogenesis is also conserved in neurons. We show that the Type V HLH protein extramacrochaetae (Emc) binds to and represses daughterless function by sequestering daughterless to the cytoplasm. This work provides initial insights into the mechanisms underlying the function of daughterless and Emc in neurons while providing a novel understanding of how Emc functions to restrict daughterless activity within the cell.

OsHLH61-OsbHLH96 influences rice defense to brown planthopper through regulating the pathogen-related genes

BackgroundIn plants, basic helix-loop-helix (bHLH) proteins form the largest transcription factor (TF) family. Among them, HLH proteins are a small group of atypical members that lack the basic domain, and form dimers with bHLH proteins. Although bHLH proteins have been proved to play important roles in plant development and physiology, the function of HLH proteins is rarely studied, not to mention in plant biotic resistance. Brown planthopper (BPH) is a kind of rice-specific insect that causes devastating yield losses each year.ResultsIn this study, we identified OsHLH61 gene that encodes HLH protein. OsHLH61 gene could be highly induced by BPH infestation. Furthermore, Methyl Jasmonic acid (Me-JA) and cis-12-oxo- phytodienoic acid (OPDA) induced expression of OsHLH61, while SA repressed it. We knocked down expression of OsHLH61 by RNA interference (RNAi), the transgenic plants were susceptible to BPH infestation. RNA-seq analysis revealed that some pathogen-related (PR) genes in the Salicylic acid (SA) signaling pathway that mediate plant immunity were obviously down-regulated in the OsHLH61 RNAi plants. Meanwhile, yeast two-hybrid assay and bimolecular luciferase complementation (BiLC) analysis identified bHLH096 to be an interacting factor of OsHLH61. Also, some PR genes were down-regulated in the OsbHLH96 over expressing lines. Expression of OsbHLH96 was inhibited. Besides, OsbHLH96 might interact with Jasmonate Zim-Domain3 (OsJAZ3).ConclusionAltogether, we identified an OsHLH61–OsbHLH96 complex that might mediate defense to BPH through regulating PR genes. And OsHLH61–OsbHLH96 might be important in mediating SA and JA signaling crosstalk.

E proteins sharpen neurogenesis by modulating proneural bHLH transcription factors' activity in an E-box-dependent manner.

Class II HLH proteins heterodimerize with class I HLH/E proteins to regulate transcription. Here, we show that E proteins sharpen neurogenesis by adjusting the neurogenic strength of the distinct proneural proteins. We find that inhibiting BMP signaling or its target ID2 in the chick embryo spinal cord, impairs the neuronal production from progenitors expressing ATOH1/ASCL1, but less severely that from progenitors expressing NEUROG1/2/PTF1a. We show this context-dependent response to result from the differential modulation of proneural proteins' activity by E proteins. E proteins synergize with proneural proteins when acting on CAGSTG motifs, thereby facilitating the activity of ASCL1/ATOH1 which preferentially bind to such motifs. Conversely, E proteins restrict the neurogenic strength of NEUROG1/2 by directly inhibiting their preferential binding to CADATG motifs. Since we find this mechanism to be conserved in corticogenesis, we propose this differential co-operation of E proteins with proneural proteins as a novel though general feature of their mechanism of action.

Identification of open chromosomal regions and key genes in prostate cancer via integrated analysis of DNase‑seq and RNA‑seq data.

Prostate cancer is a type of adenocarcinoma arising from the peripheral zone of the prostate gland, and metastasized prostate cancer is incurable with the current available therapies. The present study aimed to identify open chromosomal regions and differentially expressed genes (DEGs) associated with prostate cancer development. The DNase sequencing data (GSE33216) and RNA sequencing data (GSE22260) were downloaded from the Gene Expression Omnibus database. DNaseI hypersensitive sites were detected and analyzed. Subsequently, DEGs were identified and their potential functions were enriched. Finally, upstream regulatory elements of DEGs were predicted. In LNCaP cells, following androgen receptor activation, 244 upregulated and 486 downregulated open chromosomal regions were identified. However, only 1% of the open chromosomal regions were dynamically altered. The 41genes with upregulated open chromosomal signals within their promoter regions were primarily enriched in biological processes. Additionally, 211upregulated and 150downregulated DEGs were identified in prostate cancer, including eight transcription factors (TFs). Finally, nine regulatory elements associated with prostate cancer were predicted. In particular, inhibitor of DNA binding1 HLH protein (ID1) was the only significantly upregulated TF which exhibited motif enrichment in the promoter regions of upregulated genes. CCCTC‑binding factor (CTCF) and ELK1 ETS transcription factor (ELK1), enriched in the open promoter regions of downregulated genes, were potential upstream regulatory elements. Furthermore, reverse transcription‑quantitative polymerase chain reaction analysis confirmed that ID1 expression was significantly upregulated in LNCaP cells and 5α‑dihydrotestosterone (DHT)‑treated LNCaP cells compared with that in BPH1 cells, while CTCF and ELK1 expression was significantly downregulated in LNCaP cells and DHT‑treated LNCaP cells. In conclusion, ID1, CTCF and ELK1 may be associated with prostate cancer, and may be potential therapeutic targets for the treatment of this disease.

Genetic Loci Controlling Carotenoid Biosynthesis in Diverse Tropical Maize Lines.

The discovery and use of genetic markers associated with carotenoid levels can help to exploit the genetic potential of maize for provitamin A accumulation more effectively. Provitamin A carotenoids are classes of carotenoids that are precursors of vitamin A, an essential micronutrient in humans. Vitamin A deficiency is a global public health problem affecting millions of people, especially in developing countries. Maize is one of the most important staple crops targeted for provitamin A biofortification to help alleviate vitamin A deficiency in developing countries. A genome-wide association study (GWAS) of maize endosperm carotenoids was conducted using a panel of 130 diverse yellow maize tropical inbred lines genotyped with Genotyping by Sequencing (GBS) SNP markers. Numerous significant association signals co-localizing with the known carotenoid biosynthesis genes crtRB1, lcyE and ZEP1 were identified. The GWAS confirmed previously reported large effects of the two major carotenoid biosynthesis genes lcyE and crtRB1. In addition, significant novel associations were detected for several transcription factors (e.g., RING zinc finger domain and HLH DNA-binding domain super family proteins) that may be involved in regulation of carotenoid biosynthesis in maize. When the GWAS was re-conducted by including the major effects of lcyE and crtRB1 genes as covariates, a SNP in a gene coding for an auxin response factor 20 transcription factor was identified which displayed an association with β-carotene and provitamin A levels. Our study provides a foundation for design and implementation of genomics-assisted selection strategies for provitamin A maize breeding in tropical regions, and advances efforts toward identification of additional genes (and allelic variants) involved in the regulation of carotenoid biosynthesis in plants.

Effect and mechanism of RNAi targeting WWTR1 on biological activity of gastric cancer cells SGC7901.

Gastric cancer (GC) is one of the most common malignancies in the world. It is essential to develop novel targets and therapeutic approaches for GC, which requires identification of novel functional molecules. WW-domain containing transcription regulator 1 (WWTR1) may activate many transcriptional factors and exhibit an important role in the development of various tissues in mammals. The results of the present study demonstrated that mRNA and protein levels of WWTR1 are increased in GC tissues and cell lines. The SGC7901 cell line was selected to perform RNA interference (RNAi) targeting WWTR1, and for subsequent study. Compared with control groups (cells without any treatment) and mock groups (cells treated with nonspecific siRNA), cell proliferation of siWWTR1 cells (cells treated with WWTR1 siRNA) was detected using a Cell Counting Kit-8 assay at 12, 24 and 48 h, and decreased in a time-dependent manner. Cell cycle and apoptosis status were determined by flow cytometry, and it was demonstrated that G1/S transition was blocked in the cell cycle and apoptosis promoted in siWWTR1 cells, compared with control and mock cells. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to detect the mRNA and protein levels of cell cycle and apoptosis-associated factors. The expression of Cyclin D1, cancer Myc and B cell lymphoma/leukemia-2 (Bcl-2) decreased and Bcl-2 associated X protein increased significantly in siWWRT1 cells, at the mRNA and protein level, compared with control and mock cells. With the exception of the Hippo pathway, siWWTR1 regulated downstream factors, including mothers against decapentaplegic homolog family member 3 (SMAD3) and inhibitor of DNA binding 1, HLH protein (ID1), HLH protein in the transforming growth factor (TGF)-β pathway. The expression of asparagine synthetase was decreased whereas ID1, SMAD3 (proteins that participate in intracellular TGF-β transduction) and betacellulin increased notably in siWWRT1 cells. In conclusion, WWTR1 promotes cell proliferation and inhibits apoptosis of GC cells by regulating cell cycle/apoptosis-associated factors, and effectors in the TGF-β pathway.

The Notch pathway regulates the Second Mitotic Wave cell cycle independently of bHLH proteins

Notch regulates both neurogenesis and cell cycle activity to coordinate precursor cell generation in the differentiating Drosophila eye. Mosaic analysis with mitotic clones mutant for Notch components was used to identify the pathway of Notch signaling that regulates the cell cycle in the Second Mitotic Wave. Although S phase entry depends on Notch signaling and on the transcription factor Su(H), the transcriptional co-activator Mam and the bHLH repressor genes of the E(spl)-Complex were not essential, although these are Su(H) coactivators and targets during the regulation of neurogenesis. The Second Mitotic Wave showed little dependence on ubiquitin ligases neuralized or mindbomb, and although the ligand Delta is required non-autonomously, partial cell cycle activity occurred in the absence of known Notch ligands. We found that myc was not essential for the Second Mitotic Wave. The Second Mitotic Wave did not require the HLH protein Extra macrochaetae, and the bHLH protein Daughterless was required only cell-nonautonomously. Similar cell cycle phenotypes for Daughterless and Atonal were consistent with requirement for neuronal differentiation to stimulate Delta expression, affecting Notch activity in the Second Mitotic Wave indirectly. Therefore Notch signaling acts to regulate the Second Mitotic Wave without activating bHLH gene targets.

Revisiting the phylogeny of phylum Ctenophora: a molecular perspective

The phylogenetic relationships of deep metazoans, specifically in the phylum Ctenophora (inside and outside the phylum), are not totally understood. Several loci (protein coding and ribosomal RNA) from organisms belonging to this phylum are currently available on public databases (e.g. GenBank). Previous studies take into account the ribosomal data and the protein data separately. In this study, we perform a meta-analysis of previously published data together. The published data of this phylum have been used in previous phylogenetic analyses inside the phylum and consist in nuclear ribosomal data, such as 18S, 5.8S, ITS1, ITS2, and protein-coding markers such as NFP (non-fluorescent protein). Previous studies concentrate their efforts toward the analyses of ribosomal data or the protein-coding marker separately. Now we take into account these markers together for an upgrade of the phylogenetic analysis of this phylum. We also test several markers such as 28S, IPNS, Tyrosine aminotransferase and HLH domaincontaining protein for the improvement of the study. This markers were analyzed by Bayesian Inference (MrBayes) and Maximum Likelihood (Garli and RAxML), individually and concatenated, showing improvement in the orders placement and presenting new interesting relationship between the paraphyletic order Cydippida and the other ctenophores. These analyses also include sequences from undescribed species that have been reported in GenBank which improved the alignment matrices and support values of some nodes. Adding the undescribed species suggests interesting and well supported clades, the posterior identification of this species would led to an improvement on the ctenophore’s taxonomy.

Spheroid growth in ovarian cancer alters transcriptome responses for stress pathways and epigenetic responses.

Ovarian cancer is the most lethal gynecological cancer, with over 200,000 women diagnosed each year and over half of those cases leading to death. These poor statistics are related to a lack of early symptoms and inadequate screening techniques. This results in the cancer going undetected until later stages when the tumor has metastasized through a process that requires the epithelial to mesenchymal transition (EMT). In lieu of traditional monolayer cell culture, EMT and cancer progression in general is best characterized through the use of 3D spheroid models. In this study, we examine gene expression changes through microarray analysis in spheroid versus monolayer ovarian cancer cells treated with TGFβ to induce EMT. Transcripts that included Coiled-Coil Domain Containing 80 (CCDC80), Solute Carrier Family 6 (Neutral Amino Acid Transporter), Member 15 (SLC6A15), Semaphorin 3E (SEMA3E) and PIF1 5'-To-3' DNA Helicase (PIF1) were downregulated more than 10-fold in the 3D cells while Inhibitor Of DNA Binding 2, HLH Protein (ID2), Regulator Of Cell Cycle (RGCC), Protease, Serine 35 (PRSS35), and Aldo-Keto Reductase Family 1, Member C1 (AKR1C1) were increased more than 50-fold. Interestingly, EMT factors, stress responses and epigenetic processes were significantly affected by 3D growth. The heat shock response and the oxidative stress response were also identified as transcriptome responses that showed significant changes upon 3D growth. Subnetwork enrichment analysis revealed that DNA integrity (e.g. DNA damage, genetic instability, nucleotide excision repair, and the DNA damage checkpoint pathway) were altered in the 3D spheroid model. In addition, two epigenetic processes, DNA methylation and histone acetylation, were increased with 3D growth. These findings support the hypothesis that three dimensional ovarian cell culturing is physiologically different from its monolayer counterpart.