A Very Hungry Caterpillar: Polyethylene Metabolism and Lipid Homeostasis in Larvae of the Greater Wax Moth (Galleria mellonella).

Larvae of the greater wax moth (Galleria mellonella) possess the remarkable ability to consume and rapidly degrade low-density polyethylene. Previous studies have investigated the involvement of the animal's microbiome, but little is known about the host's actual role and if it benefits from biodegradation of this synthetic polymer. We used a combination of RNA sequencing and biochemical approaches to assess caterpillars fed honeycomb, fed polyethylene (PE), or starved for up to 72 h. Sequencing of gut transcripts revealed PE-fed larvae retain an expression profile consistent with normal intestinal function but also show distinct molecular signatures indicative of enhanced fatty acid metabolism (FAM). Further, quantification of total lipid content validated the impact of a PE diet on FAM; in contrast to lipid-depleted starved animals, PE-fed caterpillars maintain lipid reserves similar to honeycomb-fed larvae. Additionally, we found the activity of putative enzymes involved in lipid oxidation (e.g., alcohol dehydrogenase) are considerably higher in PE-fed larvae, indicating that on a functional level, these caterpillars are inducing pathways to effectively metabolize PE. Overall, we put forward a hypothesized model where the similarity in chemical structure between PE and its natural honeycomb diet has endowed larvae of G. mellonella with the extraordinary capability to derive energy from PE as an exclusive food source through pre-existing metabolic pathways.

Applications of Next-Generation Sequencing for Large-Scale Pathogen Diagnoses in Soybean.

Soybean (Glycine max) has become an important crop in Manitoba, Canada, with a 10-fold increase in dedicated acreage over the past decade. Given the rapid increase in production, scarce information about foliar diseases present in the province has been recorded. In order to describe the foliar pathogens affecting this legume, we harnessed next-generation sequencing (NGS) to carry out a comprehensive survey across Manitoba in 2016. Fields were sampled during the V2/3 (33 fields) and R6 (70 fields) growth stages, with at least three symptomatic leaves per field collected and subjected to RNA sequencing. We successfully detected several bacteria, fungi, and viruses known to infect soybean, including Pseudomonas savastanoi pv. glycinea, Septoria glycines, and Peronospora manshurica, as well as pathogens not previously identified in the province (e.g., Pseudomonas syringae pv. tabaci, Cercospora sojina, and Bean yellow mosaic virus). For some microorganisms, we were able to disentangle the different pathovars present and/or assemble their genome sequence. Since NGS generates data on the entire flora and fauna occupying a leaf sample, we also identified residual pathogens (i.e., pathogens of crops other than soybean) and multiple species of arthropod pests. Finally, the sequence information produced by NGS allowed for the development of polymerase chain reaction-based diagnostics for some of the most widespread and important pathogens. Although there are many benefits of using NGS for large-scale plant pathogen diagnoses, we also discuss some of the limitations of this technology.

Molecular evidence for the inhibition of cytochrome p450s and cholinesterases in ticks by the repellent DEET.

For more than 50 years DEET (N,N-Diethyl-m-toluamide) has been considered the gold standard of repellents. It is applied to the skin or clothing to deter mosquitoes and other blood-sucking invertebrate pests from approaching and/or settling, and ultimately it provides temporary protection from bites. Despite rampant global use, surprisingly little is understood about DEET's mode of action and the molecular targets of the active ingredient. Furthermore, the theories into its mechanism for repellency are largely based off fruit fly and mosquito research. Since ticks possess a unique sensory structure, the Haller's organ, the specific genes and pathways associated with DEET avoidance may differ from insects. In these studies, we collected American dog ticks (Dermacentor variabilis) from four natural populations within Manitoba, Canada. We first carried out behavior assays, which showed DEET effectively repelled the ticks. RNA sequencing revealed that DEET caused a rapid and substantial reduction in the abundance of transcripts encoding cytochrome P450 and acetylcholinesterase genes, which gradually recovered over the 24 h time course. Finally, enzymatic kinetics provided functional support for DEET's role as an effective inhibitor of P450 s. While many facets of its mode of action remain to be worked out, our study provides valuable insights into the molecular underpinnings of DEET's repellence in ticks.

Reconstitution of a 26-Subunit Human Kinetochore Reveals Cooperative Microtubule Binding by CENP-OPQUR and NDC80.

The approximately thirty core subunits of kinetochores assemble on centromeric chromatin containing the histone H3 variant CENP-A and connect chromosomes with spindle microtubules. The chromatin proximal 16-subunit CCAN (constitutive centromere associated network) creates a mechanically stable bridge between CENP-A and the kinetochore's microtubule-binding machinery, the 10-subunit KMN assembly. Here, we reconstituted a stoichiometric 11-subunit human CCAN core that forms when the CENP-OPQUR complex binds to a joint interface on the CENP-HIKM and CENP-LN complexes. The resulting CCAN particle is globular and connects KMN and CENP-A in a 26-subunit recombinant particle. The disordered, basic N-terminal tail of CENP-Q binds microtubules and promotes accurate chromosome alignment, cooperating with KMN in microtubule binding. The N-terminal basic tail of the NDC80 complex, the microtubule-binding subunit of KMN, can functionally replace the CENP-Q tail. Our work dissects the connectivity and architecture of CCAN and reveals unexpected functional similarities between CENP-OPQUR and the NDC80 complex.

The Mexican bean beetle (Epilachna varivestis) regurgitome and insights into beetle-borne virus specificity.

For nearly 400 million years, insects and plants have been embattled in an evolutionary arms race. Insects have developed diverse feeding strategies and behaviors in an effort to circumvent and overcome an extensive collection of plant defense tactics. Sap-sucking insects often inject saliva into hosts plants, which contains a suite of effector proteins and even microbial communities that can alter the plant's defenses. Lacking salivary glands, leaf-feeding beetles represent an interesting group of phytophagous insects. Feeding beetles regurgitate onto leaf surfaces and it is thought that these oral secretions influence insect-plant interactions and even play a role in virus-vector specificity. Since the molecular and biological makeup of the regurgitant is virtually unknown, we carried out RNA sequencing and 16S rDNA analysis on a major soybean pest, Epilachna varivestis, to generate the first ever beetle "regurgitome" and characterize its microbiome. Interestingly, the regurgitant is comprised of a rich molecular assortment of genes encoding putative extracellular proteins involved in digestion, molting, immune defense, and detoxification. By carrying out plant inoculation assays, we reinforced the fundamental role of the regurgitant in beetle-borne virus specificity. Ultimately, these studies begin to characterize the importance of regurgitant in virus transmission and beetle-plant interactions.

First Complete Genome Sequence of Tobacco necrosis virus D Isolated from Soybean and from North America.

We present the first complete genome sequence of the tombusvirus Tobacco necrosis virus D (TNV-D) from North America, obtained from an infected soybean plant. Compared with the three other TNV-D genomes isolated from different geographic regions and host plants, its nucleotide identities were between 83% and 93%.

Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells.

BACKGROUND: During metaphase clathrin stabilises the mitotic spindle kinetochore (K)-fibres. Many anti-mitotic compounds target microtubule dynamics. Pitstop 2™ is the first small molecule inhibitor of clathrin terminal domain and inhibits clathrin-mediated endocytosis. We investigated its effects on a second function for clathrin in mitosis. RESULTS: Pitstop 2 did not impair clathrin recruitment to the spindle but disrupted its function once stationed there. Pitstop 2 trapped HeLa cells in metaphase through loss of mitotic spindle integrity and activation of the spindle assembly checkpoint, phenocopying clathrin depletion and aurora A kinase inhibition. CONCLUSIONS: Pitstop 2 is therefore a new tool for investigating clathrin spindle dynamics. Pitstop 2 reduced viability in dividing HeLa cells, without affecting dividing non-cancerous NIH3T3 cells, suggesting that clathrin is a possible novel anti-mitotic drug target.

Drosophila Psf2 has a role in chromosome condensation.

The condensation state of chromosomes is a critical parameter in multiple processes within the cell. Failures in the maintenance of appropriate condensation states may lead to genomic instability, mis-expression of genes, and a number of disease states. During cell proliferation, replication of DNA represents an ongoing challenge for chromosome packaging as DNA must be unpackaged for replication and then faithfully repackaged. An integral member of the DNA replication machinery is the GINS complex which has been shown to stabilize the CMG complex which is required for processivity of the Mcm2-7 helicase complex during S phase. Through examination of the phenotypes associated with a null mutation in Psf2, a member of the evolutionarily conserved GINS complex, we find that Drosophila Psf2 likely has a role in establishing chromosome condensation and that the defects associated with this mis-condensation impact M phase progression, genomic stability, and transcriptional regulation.

Clathrin-mediated endocytic proteins are involved in regulating mitotic progression and completion.

A few proteins required for clathrin-mediated endocytosis (CME) are associated with successful completion of mitosis at distinct mitotic stages. Clathrin heavy chain (CHC) and epsin are required for chromosome segregation independent of their CME function and dynamin II (dynII) functions in the abscission stage of cytokinesis. In this study we screened for mitotic roles of eight CME proteins: CHC, α-adaptin, CALM, epsin, eps15, endophilin II (edpnII), syndapin II (sdpnII) and the GTPase dynII using a small interfering RNA targeting approach. All proteins, except for CALM, are associated with completion of the abscission stage of cytokinesis, suggesting that they function in this process in an endocytic-dependent manner. In support of this concept, overexpression of epsin(S357D), which blocks endocytosis, induced multinucleation. Moreover, six of them have a secondary role at earlier mitotic stages that is not dependent on their endocytic function: CHC, epsin and eps15 in chromosome segregation, and sdpnII, α-adaptin and CALM have a role in furrow ingression. Therefore, the role of endocytic proteins in mitosis is much broader than previously recognized.

Phosphorylation of dynamin II at serine-764 is associated with cytokinesis.

Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO(2) chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and Glutathione-S-Transferase-tagged-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.

The dynamin inhibitors MiTMAB and OcTMAB induce cytokinesis failure and inhibit cell proliferation in human cancer cells.

The endocytic protein dynamin II (dynII) participates in cell cycle progression and has roles in centrosome cohesion and cytokinesis. We have described a series of small-molecule inhibitors of dynamin [myristyl trimethyl ammonium bromides (MiTMAB)] that competitively interfere with the ability of dynamin to bind phospholipids and prevent receptor-mediated endocytosis. We now report that dynII functions specifically during the abscission phase of cytokinesis and that MiTMABs exclusively block this step in the cell cycle. Cells treated with MiTMABs (MiTMAB and octadecyltrimethyl ammonium bromide) and dyn-depleted cells remain connected via an intracellular bridge for a prolonged period with an intact midbody ring before membrane regression and binucleate formation. MiTMABs are the first compounds reported to exclusively block cytokinesis without affecting progression through any other stage of the cell cycle. Thus, MiTMABs represent a new class of antimitotic compounds. We show that MiTMABs are potent inhibitors of cancer cell growth and have minimal effect on nontumorigenic fibroblast cells. Thus, MiTMABs have toxicity and antiproliferative properties that preferentially target cancer cells. This suggests that dynII may be a novel target for pharmacologic intervention for the treatment of cancer.

The actin-binding and bundling protein, EPLIN, is required for cytokinesis.

Cytokinesis involves two phases: (1) membrane ingression followed by (2) membrane abscission. The ingression phase generates a cleavage furrow and this requires co-operative function of the actin-myosin II contractile ring and septin filaments. We demonstrate that the actin-binding protein, EPLIN, locates to the cleavage furrow during cytokinesis and this is possibly via association with the contractile ring components, myosin II and the septin, Sept2. Depletion of EPLIN results in formation of multinucleated cells and this is associated with inefficient accumulation of active myosin II (MRLC(S19)) and Sept2 and their regulatory small GTPases, RhoA and Cdc42, respectively, to the cleavage furrow during the final stages of cytokinesis. We suggest that EPLIN may function during cytokinesis to maintain local accumulation of key cytokinesis proteins at the furrow.