Prostaglandin actions in established insect cell lines

Prostaglandins (PGs) are oxygenated metabolites of arachidonic acid (AA) and two other C20 polyunsaturated fatty acids that serve as biochemical signals mediating physiological functions. We reported that PGs influence protein expression in insect cell lines, which prompted the question: do PGs influence cell proliferation or viability in insect cell lines? Here, we report on the outcomes of experiments designed to address the question in cell lines from three insect orders: Hemiptera (squash bug, Anasa tristis, BCIRL-AtE-CLG15A), Coleoptera (red flour beetle, Tribolium castaneum, BCIRL-TcA-CLG1), and Lepidoptera (tobacco budworm, Heliothis virescens, BCIRL-HvAM1). Treating the insect cell lines with PGA₁, PGA₂, or PGD₂ led to dose-dependent reductions in cell numbers. All three cell lines were sensitive to PGA₁ and PGA₂ (IC₅₀s = 9.9 to 26.9 μM) and were less sensitive to PGD₂ (IC₅₀s = 31.6 to 104.7 μM). PG treatments also led to cell death at higher concentrations, as seen in mammalian cell lines. PGE₁, PGE₂, and PGF_2α treatments did not influence AtE-CLG15A or HvAM1 cell numbers at lower concentrations, but led to dose-related reductions in TcA-CLG1 cells at higher concentrations. Similar treatments with pharmaceutical inhibitors of PG biosynthesis also led to reduced cell numbers: MAFP (inhibits phospholipase A₂), indomethacin (inhibits PG biosynthesis), and esculetin (inhibits lipoxygenase). Because these pharmaceuticals are used to relieve inflammation and other medical issues in human medicine, they are not toxic to animal cells. We infer PGs are necessary in optimal quantities for ongoing homeostatic functions in established cell lines; in quantities outside the optimal concentrations, PGs are deleterious.     

Diatrack particle tracking software: Review of applications and performance evaluation

Object tracking is an instrumental tool supporting studies of cellular trafficking. There are three challenges in object tracking: the identification of targets; the precise determination of their position and boundaries; and the assembly of correct trajectories. This last challenge is particularly relevant when dealing with densely populated images with low signal‐to‐noise ratios—conditions that are often encountered in applications such as organelle tracking, virus particle tracking or single‐molecule imaging. We have developed a set of methods that can handle a wide variety of signal complexities. They are compiled into a free software package called Diatrack. Here we review its main features and utility in a range of applications, providing a survey of the dynamic imaging field together with recommendations for effective use. The performance of our framework is shown to compare favorably to a wide selection of custom‐developed algorithms, whether in terms of localization precision, processing speed or correctness of tracks.      

Differences in sperm morphology in foam‐nesting leptodactyline frogs (Anura,Leptodactylidae)

Sperm morphology is diverse among vertebrates and is influenced by the reproductive strategies adopted by species. In anurans, sperm morphology is associated with reproductive modes and mating systems. Here, we describe the sperm morphology of 11 frog species in the genus Leptodactylus and that of Lithodytes lineatus and discuss the relationship between sperm morphology and species' mating systems. We observed two distinct sperm morphotypes among the leptodactyline species, which differed mostly in head morphology. Type I sperm had triangular head, discrete acrosome vesicle with posterior margin not clearly visible; type II sperm had elongated head, clear acrosomal vesicle with posterior margin clearly visible. These sperm types do not seem to be associated with phylogeny; instead, type II sperm was observed in all polyandrous species analysed and in species with evidences of polyandry. Moreover, sperm of all species presented tail with undulating membrane connected to the axial fibre. We suggest that differences in sperm morphology might be associated with sperm competition to what polyandrous species are subjected. However, natural history observations on polyandrous mating in some species presenting type II sperm and phylogenetic comparative studies are need to elucidate the role of mating systems in the evolution of sperm morphology in leptodactylines.     

Integrated gut/liver microphysiological systems elucidates inflammatory inter‐tissue crosstalk

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut‐liver tissue interactions under normal and inflammatory contexts, via an integrative multi‐organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long‐term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut‐liver crosstalk. Moreover, significant non‐linear modulation of cytokine responses was observed under inflammatory gut‐liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA‐seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut‐liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut‐liver interaction also negatively affected tissue‐specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi‐tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. Biotechnol. Bioeng. 2017;114: 2648–2659. © 2017 Wiley Periodicals, Inc.     

SAMHD1 protects cancer cells from various nucleoside-based antimetabolites

Recently, we demonstrated that sterile α motif and HD domain containing protein 1 (SAMHD1) is a major barrier in acute myelogenous leukemia (AML) cells to the cytotoxicity of cytarabine (ara-C), the most important drug in AML treatment. Ara-C is intracellularly converted by the canonical dNTP synthesis pathway to ara-CTP, which serves as a substrate but not an allosteric activator of SAMHD1. Using an AML mouse model, we show here that wild type but not catalytically inactive SAMHD1 reduces ara-C treatment efficacy in vivo. Expanding the clinically relevant substrates of SAMHD1, we demonstrate that THP-1 CRISPR/Cas9 cells lacking a functional SAMHD1 gene showed increased sensitivity to the antimetabolites nelarabine, fludarabine, decitabine, vidarabine, clofarabine, and trifluridine. Within this Extra View, we discuss and build upon both these and our previously reported findings, and propose SAMHD1 is likely active against a variety of nucleoside analog antimetabolites present in anti-cancer chemotherapies. Thus, SAMHD1 may constitute a promising target to improve a wide range of therapies for both hematological and non-haematological malignancies.     

Plant functional types broadly describe water use strategies in the Caatinga,a seasonally dry tropical forest in northeast Brazil

1. In seasonally dry tropical forests, plant functional type can be classified as deciduous low wood density, deciduous high wood density, or evergreen high wood density species. While deciduousness is often associated with drought‐avoidance and low wood density is often associated with tissue water storage, the degree to which these functional types may correspond to diverging and unique water use strategies has not been extensively tested.
2. We examined (a) tolerance to water stress, measured by predawn and mid‐day leaf water potential; (b) water use efficiency, measured via foliar δ¹³C; and (c) access to soil water, measured via stem water δ¹⁸O.
3. We found that deciduous low wood density species maintain high leaf water potential and low water use efficiency. Deciduous high wood density species have lower leaf water potential and variable water use efficiency. Both groups rely on shallow soil water. Evergreen high wood density species have low leaf water potential, higher water use efficiency, and access alternative water sources. These findings indicate that deciduous low wood density species are drought avoiders, with a specialized strategy for storing root and stem water. Deciduous high wood density species are moderately drought tolerant, and evergreen high wood density species are the most drought tolerant group.
4. Synthesis. Our results broadly support the plant functional type framework as a way to understand water use strategies, but also highlight species‐level differences.

     

Characterisation of novel functionality within the Blastocystis tryptophanase gene

In recent years, the human gut microbiome has been recognised to play a pivotal role in the health of the host. Intestinal homeostasis relies on this intricate and complex relationship between the gut microbiota and the human host. While much effort and attention has been placed on the characterization of the organisms that inhabit the gut microbiome, the complex molecular cross-talk between the microbiota could also exert an effect on gastrointestinal conditions. Blastocystis is a single-cell eukaryotic parasite of emerging interest, as its beneficial or pathogenic role in the microbiota has been a subject of contention even to-date. In this study, we assessed the function of the Blastocystis tryptophanase gene (BhTnaA), which was acquired by horizontal gene transfer and likely to be of bacterial origin within Blastocystis. Bioinformatic analysis and phylogenetic reconstruction revealed distinct divergence of BhTnaA versus known bacterial homologs. Despite sharing high homology with the E. coli tryptophanase gene, we show that Blastocystis does not readily convert tryptophan into indole. Instead, BhTnaA preferentially catalyzes the conversion of indole to tryptophan. We also show a direct link between E. coli and Blastocystis tryptophan metabolism: In the presence of E. coli, Blastocystis ST7 is less able to metabolise indole to tryptophan. This study examines the potential for functional variation in horizontally-acquired genes relative to their canonical counterparts, and identifies Blastocystis as a possible producer of tryptophan within the gut.     

Extinction of anciently associated gut bacterial symbionts in a clade of stingless bees

Animal-microbe symbioses are often stable for millions of years. An example is the clade consisting of social corbiculate bees—honeybees, bumblebees, and stingless bees—in which a shared ancestor acquired specialized gut bacteria that subsequently diversified with hosts. This model may be incomplete, however, as few microbiomes have been characterized for stingless bees, which are diverse and ecologically dominant pollinators in the tropics. We surveyed gut microbiomes of Brazilian stingless bees, focusing on the genus Melipona, for which we sampled multiple species and biomes. Strikingly, Melipona lacks Snodgrassella and Gilliamella, bacterial symbionts ubiquitous in other social corbiculate bees. Instead, Melipona species harbor more environmental bacteria and bee-specific Starmerella yeasts. Loss of Snodgrassella and Gilliamella may stem from ecological shifts in Melipona or the acquisition of new symbionts as functional replacements. Our findings demonstrate the value of broadly sampling microbiome biodiversity and show that even ancient symbioses can be lost.Subject terms: Symbiosis, Microbiome, Microbial ecology, Metagenomics, Microbial ecology