The free-living flatworm, Macrostomum lignano has an impressive regenerative capacity. Following injury, it can regenerate almost an entirely new organism because of the presence of an abundant somatic stem cell population, the neoblasts. This set of unique properties makes many flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cellfate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of M. lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ∼75% of its sequence being comprised of simple repeats and transposon sequences. This has made high-quality assembly from Illumina reads alone impossible (N50 = 222 bp). We therefore generated 130× coverage by long sequencing reads from the Pacific Biosciences platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene-expression patterns during regeneration, examining pathways important to stem cell function.F latworms belong to the superphylum Lophotrochozoa, a vast assembly of protostome invertebrates (1, 2) (Fig. 1A). The evolutionary relationships within this clade are poorly resolved and the specific position of flatworms is currently debated (3, 4). Flatworms have attracted scientific attention for centuries because of their astonishing regenerative capabilities (5, 6), as well as their ability to "degrow" in a controlled way when starved (7). As far back as the early 1900s, Thomas Morgan recognized the potential of flatworms and conducted a number of fascinating regeneration experiments on planarian flatworms before his focus shifted to Drosophila genetics (8).Macrostomum lignano is (Fig. 1B), a free-living, regenerating flatworm isolated from the coast of the Mediterranean Sea. M. lignano is an obligatorily cross-fertilizing simultaneous hermaphrodite (9) that belongs to Macrostomorpha, whereas the other often-studied freeliving flatworms and human parasitic flatworms all belong to clades that are potentially more derived (less ancestral) in comparison with Macrostomorpha (2) (Fig. 1C).Many flatworms can regenerate nearly their entire body or amputated organs. This regenerative capacity is thought to be attributable to the presence of somatic stem cells, termed neoblasts (10, 11). In Schmidtea mediterranea (planarian flatworm), even a single transplanted neoblast has the ability to rescue, regenerate, and change the genotype of a fatally irradiated worm (12). M. lignano can regenerate every tissue, with the exception of the head region containing the brain (13,14).Neoblasts in M. lignano ( Fig. 1 D and E), in contrast to most vertebrate somatic stem cells, are plentiful, making up about ...
The ability to perceive and respond to harmful conditions is crucial for the survival of any organism. The transcription factor DAF-16/FOXO is central to these responses, relaying distress signals into the expression of stress resistance and longevity promoting genes. However, its sufficiency in fulfilling this complex task has remained unclear. Using C. elegans, we show that DAF-16 does not function alone but as part of a transcriptional regulatory module, together with the transcription factor HLH-30/TFEB. Under harmful conditions, both transcription factors translocate into the nucleus, where they often form a complex, co-occupy target promoters, and co-regulate many target genes. Interestingly though, their synergy is stimulus-dependent: They rely on each other, functioning in the same pathway, to promote longevity or resistance to oxidative stress, but they elicit heat stress responses independently, and they even oppose each other during dauer formation. We propose that this module of DAF-16 and HLH-30 acts by combinatorial gene regulation to relay distress signals into the expression of specific target gene sets, ensuring optimal survival under each given threat.
The strength of conclusions drawn from RNAi-based studies is heavily influenced by the quality of tools used to elicit knockdown. Prior studies have developed algorithms to design siRNAs. However, to date, no established method has emerged to identify effective shRNAs, which have lower intracellular abundance than transfected siRNAs and undergo additional processing steps. We recently developed a multiplexed assay for identifying potent shRNAs and have used this method to generate ~250,000 shRNA efficacy data-points. Using these data, we developed shERWOOD, an algorithm capable of predicting, for any shRNA, the likelihood that it will elicit potent target knockdown. Combined with additional shRNA design strategies, shERWOOD allows the ab initio identification of potent shRNAs that target, specifically, the majority of each gene’s multiple transcripts. We have validated the performance of our shRNA designs using several orthogonal strategies and have constructed genome-wide collections of shRNAs for humans and mice based upon our approach.
FNDC3B was recently identified in an oncogenomic screen for amplified oncogenes in hepatocellular carcinoma. It is located at 3q26 and is amplified in over 20% of cancers, usually as part of a broad amplified region encompassing the entire 3q arm. Consistent with an oncogenic role in multiple cancer types, we show here that overexpression of FNDC3B is capable of malignantly transforming mammary and kidney epithelial cells in addition to hepatocytes. To explore how FNDC3B transforms cells, we determined the cellular localization of its gene product and the cancer pathways that it activates. We found that the FNDC3B oncoprotein localizes to the Golgi network, and that its correct localization is essential for its transforming function. We found that overexpression of FNDC3B induces the epithelial-to-mesenchymal transition (EMT) and activates several cancer pathways, including PI3-kinase/Akt, Rb1 and TGFβ signaling. For TGFβ signaling, we analyzed the point in the pathway at which FNDC3B operates and obtained evidence that it induces expression of all three TGFβ ligands and also promotes TGFBR1 cell-surface localization. We found that RNAi-mediated knockdown of FNDC3B in cancer cells with 3q amplification suppressed their clonogenicity and tumorigenicity, but that the same RNAi knockdown had no effect on single-copy 3q cancer cells. These results indicate that FNDC3B is an important oncogenic driver gene of the 3q amplicon, adding to the growing list of oncogenic drivers within this commonly amplified region.
BACKGROUND: Hepatocellular carcinoma (HCC) is strongly associated with hepatitis B virus (HBV) infection. False-negative results are common in routine serological tests and quantitative real-time PCR because of HBV surface antigen (HBsAg) variation and low HBV copy number. Droplet digital PCR (ddPCR), a next generation digital PCR, is a novel, sensitive, and specific platform that can be used to improve HBV detection.
In C. elegans, the conserved transcription factor DAF-16/FOXO is a powerful aging regulator, relaying dire conditions into expression of stress resistance and longevity promoting genes. For some of these functions, including low insulin/IGF signaling (IIS), DAF-16 depends on the protein SMK-1/SMEK, but how SMK-1 exerts this role has remained unknown. We show that SMK-1 functions as part of a specific Protein Phosphatase 4 complex (PP4 SMK-1). Loss of PP4 SMK-1 hinders transcriptional initiation at several DAF-16-activated genes, predominantly by impairing RNA polymerase II recruitment to their promoters. Search for the relevant substrate of PP4 SMK-1 by phosphoproteomics identified the conserved transcriptional regulator SPT-5/SUPT5H, whose knockdown phenocopies the loss of PP4 SMK-1. Phosphoregulation of SPT-5 is known to control transcriptional events such as elongation and termination. Here we also show that transcription initiating events are influenced by the phosphorylation status of SPT-5, particularly at DAF-16 target genes where transcriptional initiation appears rate limiting, rendering PP4 SMK-1 crucial for many of DAF-16's physiological roles.
The Warburg effect is one of the important hallmarks of cancer. The activation of oncogene and inactivation of tumor suppressor gene contribute to the enhancement of glycolytic enzymes and the Warburg effect. The N-myc downstream regulated gene 2 (NDRG2) is a tumor suppressor gene and is frequently lost in various types of cancer. However, little is known about glycolytic function and therapeutic value of NDRG2 in hepatocellular carcinoma (HCC). In this study, we found that NDRG2 and lactate dehydrogenase A (LDHA) were aberrantly expressed in HCC and were closely related to the Warburg effect. The correlation between NDRG2 and LDHA expression predicted HCC prognosis and the clinical response to chemotherapy. NDRG2 expression was significantly decreased while LDHA expression was increased in HCC specimens. NDRG2 and LDHA expression was significantly correlated with differentiation status, vascular invasion, and TNM stage of HCC. NDRG2 inhibited LDHA expression, the Warburg effect and the growth of HCC cells. Furthermore, NDRG2 mediated gemcitabine-induced inhibition of LDHA expression and the Warburg effect in HCC cells. Taken together, our data suggest that NDRG2 plays an important role in inhibiting the Warburg effect and the malignant growth of HCC via LDHA. NDRG2 combined with LDHA might be powerful prognostic biomarkers and targets for chemotherapy treatment of HCC.
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