miR-181a regulates multiple pathways in hypopharyngeal squamous cell carcinoma

MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that play essential roles in a variety of biological processes including apoptosis, proliferation and differentiation. Altered expression of miRNAs in head and neck cancer has been reported, but the consequences of aberrant expression of specific miRNAs on cancer pathways remain vague. Therefore, it is important to identify and elucidate pathways that may be regulated by specific miRNAs in this disease. miR-181a was up-regulated in head and neck versus normal tissues, thus pathway analysis was performed to profile the changes in the activities of ten signaling pathways relevant to cancer influenced by down-regulation of miR-181a. Expression of four pathway reporters were significantly increased (p53/DNA damage, TGFβ, MAPK/ERK and MAPK/JNK), while expression of two pathway reporters were decreased (Wnt and NFkB) upon miR-181a down-regulation. Notch, Myc/Max, hypoxia and cell cycle/pRB-E2F pathways were not significantly affected by miR-181a down-regulation. This study provides insight into the understanding of miRNA regulation on major cancer pathways.Key words: MicroRNA, head and neck cancer, miR-181a, pathway analysis, luciferase assay, FaDu cell line, transfection, qRT-PCR

Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion

Alveolar proteins stabilize cortical microtubules in Toxoplasma gondii

Single-celled protists use elaborate cytoskeletal structures, including arrays of microtubules at the cell periphery, to maintain polarity and rigidity. The obligate intracellular parasite Toxoplasma gondii has unusually stable cortical microtubules beneath the alveoli, a network of flattened membrane vesicles that subtends the plasmalemma. However, anchoring of microtubules along alveolar membranes is not understood. Here, we show that GAPM1a, an integral membrane protein of the alveoli, plays a role in maintaining microtubule stability. Degradation of GAPM1a causes cortical microtubule disorganisation and subsequent depo-lymerisation. These changes in the cytoskeleton lead to parasites becoming shorter and rounder, which is accompanied by a decrease in cellular volume. Extended GAPM1a depletion leads to severe defects in division, reminiscent of the effect of disrupting other alveolar proteins. We suggest that GAPM proteins link the cortical microtubules to the alveoli and are required to maintain the shape and rigidity of apicomplexan zoites

Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages

Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

The present study was funded by were funded by the Biotechnology and Biological Sciences Research Council (BB/R008612/1, BB/S004343/1 to RH and RG; grant BB/R008973/1 to SM and CD) and the Institute Strategic Programme Grants (BBS/E/D/20002172, BBS/E/D/30002275 and BBS/E/D/10002070, to RH and RG). The funders had no roles in the study design, data collection and analysis, decision to publish or preparation of the manuscript.Peer reviewedPublisher PD