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Engineering of genetic networks with artificial signaling pathways (ASPs) can reprogram cellular responses and phenotypes under different circumstances for a variety of diagnostic and therapeutic purposes. However, construction of ASPs between originally independent endogenous genes in mammalian cells is highly challenging. Here we report an amplifiable RNA circuit that can theoretically build regulatory connections between any endogenous genes in mammalian cells. We harness the system of catalytic hairpin assembly with combination of controllable CRISPR‐Cas9 function to transduce the signals from distinct messenger RNA expression of trigger genes into manipulation of target genes. Through introduction of these RNA‐based genetic circuits, mammalian cells are endowed with autonomous capabilities to sense the changes of RNA expression either induced by ligand stimuli or from various cell types and control the cellular responses and fates via apoptosis‐related ASPs. Our design provides a generalized platform for construction of ASPs inside the genetic networks of mammalian cells based on differentiated RNA expression. [ABSTRACT FROM AUTHOR]

• Tumor-derived EVs serve as delivery for chemotherapeutic drugs and dual genes manipulation tools. • EVs-based gene editing tool delivery strategies are efficient both for glioblastoma. • Dual genes manipulation enhanced chemotherapy potentiates antitumor immunity. Glioblastoma is one of the most malignant tumors in the central nervous system, which the conventional therapeutic modalities fail to cure the disease but improve the treatment outcome slightly. In the past decades, immune checkpoint blockade (ICB) and cytokine-regulated immunotherapy have ushered in a new era for glioblastoma treatment. However, the immunosuppressive tumor microenvironment hinders and opposites the therapeutic outcomes. Therefore, the increased importance and necessary for better modality in urgent. Moreover, many novel methods and strategies are searching for more effectively therapeutic outcomes. In this study, the glioblastoma cells derived extracellular vesicles (EVs) were adopted as a delivery platform for the chemotherapy agent (doxorubicin, Dox), gene editing platform of CD47 downregulation and IL-9 overexpression (EVs@Dox/sgCD47/IL-9), due to its excellent blood brain barrier (BBB) crossing rate and specifically targeting capability. The as-prepared EVs@Dox/sgCD47/IL-9 was capable to deliver the Dox and gene editing platform into glioblastoma tissue and was assimilated by cancer cells efficiently, where the Dox induce the immunogenic cell death (ICD) of glioblastoma cells, the CRISPR-Cas9 platform down-regulated the expression of CD47 and the over-expression system up-regulated the expression of IL-9, both enhanced the immunity of biological system. Collectively, EVs@Dox/sgCD47/IL-9 demonstrated remarkable anti-tumor effects and reshaped the immunosuppressive tumor microenvironment that polarized the tumor-associated macrophages and activated CD8+ T cells with good biosafety. Thus, it establishes a strong foundation for the development of more effective strategies against glioblastoma. [ABSTRACT FROM AUTHOR]

Comparative ecophysiologists strive to understand physiological problems in non-model organisms, but molecular tools such as RNA interference (RNAi) are under-used in our field. Here, we provide a framework for invertebrate ecophysiologists to use RNAi to answer questions focused on physiological processes, rather than as a tool to investigate gene function. We specifically focus on non-model invertebrates, in which the use of other genetic tools (e.g. , genetic knockout lines) is less likely. We argue that because RNAi elicits a temporary manipulation of gene expression, and resources to carry out RNAi are technically and financially accessible, it is an effective tool for invertebrate ecophysiologists. We cover the terminology and basic mechanisms of RNA interference as an accessible introduction for "non-molecular" physiologists, include a suggested workflow for identifying RNAi gene targets and validating biologically relevant gene knockdowns, and present a hypothesis-testing framework for using RNAi to answer common questions in the realm of invertebrate ecophysiology. This review encourages invertebrate ecophysiologists to use these tools and workflows to explore physiological processes and bridge genotypes to phenotypes in their animal(s) of interest. [Display omitted] • RNAi is an accessible genetic tool that is underused in invertebrate physiology. • The RNAi target should be chosen to disrupt a physiological process of interest. • To appropriately interpret RNAi experiments, validation of the RNAi is required. • Careful timing of RNAi can provide strong insight into physiological processes. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized (Cno) Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (Cno CAS9 or "CnoCAS9") reliably enabled genome editing in the widely used KN99a C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans. [ABSTRACT FROM AUTHOR]

Na+/H+ exchangers are directly involved in a variety of an animal's essential physiological processes such as ionoregulation, acid-base regulation, nitrogenous waste excretion, and nutrient absorption. While nine NHX isoforms have been identified in Caenorhabditis elegans , the physiological importance of each isoform is not understood. The current study aimed to further our knowledge of NHX-3 which has previously been suggested to be involved in the movement of ammonia and acid-base equivalents across the nematode's hypodermis. Although NHX-3 knockout mutant nematodes exported H+ and imported Na+ at slower rates than wild-type nematodes, attempts to inhibit the NHX activity of mutant nematodes using amiloride and EIPA caused an unexpected increase in hypodermal H+ export and did not impact Na+ fluxes suggesting that the different H+ and Na+ transport profiles of the nematodes are likely due to compensatory changes in the mutants in response to the NHX-3 knockout, rather than the loss of NHX-3's physiological function. Significant changes in the mRNA expression of 7 other NHX isoforms, 2 Na+/H+ antiporter isoforms, and the V-type H+-ATPase were detected between wild-type and mutant nematodes. Furthermore, mutant nematodes possessed significantly reduced rates of cytochrome C oxidase activity and ammonia excretion rates, indicating the knockout of NHX-3 induced fundamental changes in metabolism that could impact the nematode's need to eliminate metabolic end-products like H+ and ammonia that relate to NHX transport. While C. elegans is a popular genetic model with cheap and accessible commercial mutants, our findings suggest caution in interpretation of results in studies using mutants to study physiological traits and the biological significance of specific transporters. [Display omitted] • ΔNHX-3 mutants show a lower rate of H+ secretion compared to N2 wild-type nematodes. • ΔNHX-3 mutants display substantial compensatory responses of other cation/H+ exchangers. • ΔNHX-3 mutant have a lower metabolic capacity compared to N2 wild-type nematodes. • ΔNHX-3 and N2 wild-type nematodes show similar Na+ uptake rates. [ABSTRACT FROM AUTHOR]

Congenital long QT syndrome (LQT) is a group of cardiac disorders associated with the dysfunction of cardiac ion channels. It is characterized by prolongation of the QT-interval, episodes of syncope and even sudden death. Individuals may remain asymptomatic for most of their lives while others present with severe symptoms. This heterogeneity in phenotype makes diagnosis difficult with a greater emphasis on more targeted therapy. As a means of understanding the molecular mechanisms underlying LQT syndrome, evaluating the effect of modifier genes on disease severity as well as to test new therapies, the development of model systems remains an important research tool. Mice have predominantly been the animal model of choice for cardiac arrhythmia research, but there have been varying degrees of success in recapitulating the human symptoms; the mouse cardiac action potential (AP) and surface electrocardiograms exhibit major differences from those of the human heart. Against this background, the zebrafish is an emerging vertebrate disease modelling species that offers advantages in analysing LQT syndrome, not least because its cardiac AP much more closely resembles that of the human. This article highlights the use and potential of this species in LQT syndrome modelling, and as a platform for the in vivo assessment of putative disease-causing mutations in LQT genes, and of therapeutic interventions. [ABSTRACT FROM AUTHOR]

Proteome analysis, and more recently DNA chip technology, has led to the identification of a large number of genes that are implicated in the anaerobic response of plants. As a result, an increasingly complex picture of the response in terms of biochemical and regulatory processes is emerging. A challenge is to find out more about the function of these newly identified gene products, and how they contribute to flooding tolerance. Our approach has been to manipulate levels of candidate gene products (using sense and antisense constructs) in the model system Arabidopsis thaliana, combined with biochemical and phenotypic analysis of the resulting transgenic plants. [ABSTRACT FROM PUBLISHER]

Genetic improvement of bacterial ability to accumulate phosphate (Pi) was investigated using Escherichia coli as a test organism. High levels of Pi accumulation were achieved by (i) modifying the genetic regulation and increasing the dosage of the E. coli genes encoding polyphosphate kinase (ppk), acetate kinase (ackA), and the phosphate inducible transport system (pstS, pstC, pstA, and pstB) and (ii) genetically inactivating ppx encoding exopolyphosphatase. Acetate kinase was employed as an ATP regeneration system for polyphosphate synthesis. The best recombinant strain, which contained both pBC29 (ppk) and pEP02.2 (pst genes) accumulated approximately 10-fold more Pi than did the control strain. The phosphorus content of this recombinant reached a maximum of 16 % on the dry weight basis (49 % as phosphate). About 65 % of the cellular phosphorus was stored as polyphosphate. [ABSTRACT FROM AUTHOR]