Although two related species may have extremely similar phenotypes, the genetic networks underpinning this conserved biology may have diverged substantially since they last shared a common ancestor. ...This is termed Developmental System Drift (DSD) and reflects the plasticity of genetic networks. One consequence of DSD is that some orthologous genes will have evolved different in vivo functions in two such phenotypically similar, related species and will therefore have different loss of function phenotypes. Here we report an RNAi screen in C. elegans and C. briggsae to identify such cases. We screened 1333 genes in both species and identified 91 orthologues that have different RNAi phenotypes. Intriguingly, we find that recently evolved genes of unknown function have the fastest evolving in vivo functions and, in several cases, we identify the molecular events driving these changes. We thus find that DSD has a major impact on the evolution of gene function and we anticipate that the C. briggsae RNAi library reported here will drive future studies on comparative functional genomics screens in these nematodes.
Most heritable traits, including disease susceptibility, are affected by interactions between multiple genes. However, we understand little about how genes interact because very few possible genetic ...interactions have been explored experimentally. We have used RNA interference in Caenorhabditis elegans to systematically test approximately 65,000 pairs of genes for their ability to interact genetically. We identify approximately 350 genetic interactions between genes functioning in signaling pathways that are mutated in human diseases, including components of the EGF/Ras, Notch and Wnt pathways. Most notably, we identify a class of highly connected 'hub' genes: inactivation of these genes can enhance the phenotypic consequences of mutation of many different genes. These hub genes all encode chromatin regulators, and their activity as genetic hubs seems to be conserved across animals. We propose that these genes function as general buffers of genetic variation and that these hub genes may act as modifier genes in multiple, mechanistically unrelated genetic diseases in humans.
T cell therapies require the removal and culture of T cells ex vivo to expand several thousand‐fold. However, these cells often lose the phenotype and cytotoxic functionality for mediating effective ...therapeutic responses. The extracellular matrix (ECM) has been used to preserve and augment cell phenotype; however, it has not been applied to cellular immunotherapies. Here, a hyaluronic acid (HA)‐based hydrogel is engineered to present the two stimulatory signals required for T‐cell activation—termed an artificial T‐cell stimulating matrix (aTM). It is found that biophysical properties of the aTM—stimulatory ligand density, stiffness, and ECM proteins—potentiate T cell signaling and skew phenotype of both murine and human T cells. Importantly, the combination of the ECM environment and mechanically sensitive TCR signaling from the aTM results in a rapid and robust expansion of rare, antigen‐specific CD8+ T cells. Adoptive transfer of these tumor‐specific cells significantly suppresses tumor growth and improves animal survival compared with T cells stimulated by traditional methods. Beyond immediate immunotherapeutic applications, demonstrating the environment influences the cellular therapeutic product delineates the importance of the ECM and provides a case study of how to engineer ECM‐mimetic materials for therapeutic immune stimulation in the future.
Significant improvements to cellular immunotherapy for cancer is achieved through engineering a novel T‐cell stimulating biomaterial hydrogel. Particularly interesting, the stiffness of the hydrogel drastically impacts mechanically sensitive T cell stimulation. Similarly, the hydrogel material, hyaluronic acid, contributes additional beneficial signaling to maintain a functional phenotype of stimulated T cells, demonstrating the importance of the environment on immune cell therapies.
Soil transmitted helminths (STHs) are major human pathogens that infect over a billion people. Resistance to current anthelmintics is rising and new drugs are needed. Here we combine multiple ...approaches to find druggable targets in the anaerobic metabolic pathways STHs need to survive in their mammalian host. These require rhodoquinone (RQ), an electron carrier used by STHs and not their hosts. We identified 25 genes predicted to act in RQ-dependent metabolism including sensing hypoxia and RQ synthesis and found 9 are required. Since all 9 have mammalian orthologues, we used comparative genomics and structural modeling to identify those with active sites that differ between host and parasite. Together, we found 4 genes that are required for RQ-dependent metabolism and have different active sites. Finding these high confidence targets can open up in silico screens to identify species selective inhibitors of these enzymes as new anthelmintics.
Due to the great biocompatibility of the aqueous two phase system (ATPS), biological cells have been widely encapsulated in ATPS microdroplets (diameter < 50 μm). However, the immobilization of ...relatively large multicellular organisms such as Caenorhabditis elegans in ATPS droplets remains challenging as the spontaneous generation of droplets greater than 200 μm is difficult without external perturbations. In this study, we utilize a microneedle-assisted coflow microfludic channel to passively form ATPS microdroplets larger than 200 μm and successfully entrap C. elegans in the microdroplets. We monitor the worm viability and its temporal stroke frequency up to 6 h. We study the effects of dextran (DEX)-to-polyethylene glycol (PEG) flow ratios and worm concentration on the droplet diameter, worm encapsulation efficiency, and the number of droplets containing individual worms. Larger ATPS microdroplets (>200 μm) form in the ranges of capillary number (Ca) between 0.020 to 0.20 and Weber number (We) between 10–5 and 10–3. An ATPS with the encapsulation ability and biocompatibility can offer an alternative immobilization tool for multicellular organisms to existing platforms such as water/oil droplets.
Normal development requires the right splice variants to be made in the right tissues at the right time. The core splicing machinery is engaged in all splicing events, but which precise splice ...variant is made requires the choice between alternative splice sites-for this to occur, a set of splicing factors (SFs) must recognize and bind to short RNA motifs in the pre-mRNA. In C. elegans, there is known to be extensive variation in splicing patterns across development, but little is known about the targets of each SF or how multiple SFs combine to regulate splicing. Here we combine RNA-seq with in vitro binding assays to study how 4 different C. elegans SFs, ASD-1, FOX-1, MEC-8, and EXC-7, regulate splicing. The 4 SFs chosen all have well-characterised biology and well-studied loss-of-function genetic alleles, and all contain RRM domains. Intriguingly, while the SFs we examined have varied roles in C. elegans development, they show an unexpectedly high overlap in their targets. We also find that binding sites for these SFs occur on the same pre-mRNAs more frequently than expected suggesting extensive combinatorial control of splicing. We confirm that regulation of splicing by multiple SFs is often combinatorial and show that this is functionally significant. We also find that SFs appear to combine to affect splicing in two modes-they either bind in close proximity within the same intron or they appear to bind to separate regions of the intron in a conserved order. Finally, we find that the genes whose splicing are regulated by multiple SFs are highly enriched for genes involved in the cytoskeleton and in ion channels that are key for neurotransmission. Together, this shows that specific classes of genes have complex combinatorial regulation of splicing and that this combinatorial regulation is critical for normal development to occur.
Thin adsorbed Nafion films, representing ionomer in the PEMFC catalyst layer, were prepared by spontaneous adsorption of Nafion on SiO
2 model substrate. The conductivity of the thin films was ...examined over a wide range of temperature and relative humidity by electrochemical impedance spectroscopy. The activation energy of proton conduction was found to be twice and three-and-half times that of Nafion 112 membrane at high RH (80%) and low RH (20%), respectively. These results suggest that the mechanism of proton transport in the PEMFC catalyst layer is different than that in the membrane.
►This work is the
first reported study on the conductivity of
adsorbed Nafion films. ►Intrinsic protonic conductivity of
adsorbed Nafion and membrane vary significantly. ►Activation energy for conduction in
adsorbed Nafion and membrane are different. ►Proton conduction mechanism in PEMFC catalyst layer and membrane are different.
The rate of RNA polymerase II (Pol II) elongation can influence splice site selection in nascent transcripts, yet the extent and physiological relevance of this kinetic coupling between transcription ...and alternative splicing (AS) is not well understood. We performed experiments to perturb Pol II elongation and then globally compared AS patterns with genome-wide Pol II occupancy. RNA binding and RNA processing functions were significantly enriched among the genes with Pol II elongation inhibition-dependent changes in AS. Under conditions that interfere with Pol II elongation, including cell stress, increased Pol II occupancy was detected in the intronic regions flanking the alternative exons in these genes, and these exons generally became more included. A disproportionately high fraction of these exons introduced premature termination codons that elicited nonsense-mediated mRNA decay (NMD), thereby further reducing transcript levels. Our results provide evidence that kinetic coupling between transcription, AS, and NMD affords a rapid mechanism by which cells can respond to changes in growth conditions, including cell stress, to coordinate the levels of RNA processing factors with mRNA levels.
The fundamental aim of genetics is to understand how an organism's phenotype is determined by its genotype, and implicit in this is predicting how changes in DNA sequence alter phenotypes. A single ...network covering all the genes of an organism might guide such predictions down to the level of individual cells and tissues. To validate this approach, we computationally generated a network covering most C. elegans genes and tested its predictive capacity. Connectivity within this network predicts essentiality, identifying this relationship as an evolutionarily conserved biological principle. Critically, the network makes tissue-specific predictions-we accurately identify genes for most systematically assayed loss-of-function phenotypes, which span diverse cellular and developmental processes. Using the network, we identify 16 genes whose inactivation suppresses defects in the retinoblastoma tumor suppressor pathway, and we successfully predict that the dystrophin complex modulates EGF signaling. We conclude that an analogous network for human genes might be similarly predictive and thus facilitate identification of disease genes and rational therapeutic targets.
Soil-transmitted helminths (STHs) are major pathogens infecting over a billion people. There are few classes of anthelmintics and there is an urgent need for new drugs. Many STHs use an unusual form ...of anaerobic metabolism to survive the hypoxic conditions of the host gut. This requires rhodoquinone (RQ), a quinone electron carrier. RQ is not made or used by vertebrate hosts making it an excellent therapeutic target. Here we screen 480 structural families of natural products to find compounds that kill Caenorhabditis elegans specifically when they require RQ-dependent metabolism. We identify several classes of compounds including a family of species-selective inhibitors of mitochondrial respiratory complex I. These identified complex I inhibitors have a benzimidazole core and we determine key structural requirements for activity by screening 1,280 related compounds. Finally, we show several of these compounds kill adult STHs. We suggest these species-selective complex I inhibitors are potential anthelmintics.
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