A manager's guide to using eDNA metabarcoding in marine ecosystems.

Environmental DNA (eDNA) metabarcoding is a powerful tool that can enhance marine ecosystem/biodiversity monitoring programs. Here we outline five important steps managers and researchers should consider when developing eDNA monitoring program: (1) select genes and primers to target taxa; (2) assemble or develop comprehensive barcode reference databases; (3) apply rigorous site occupancy based decontamination pipelines; (4) conduct pilot studies to define spatial and temporal variance of eDNA; and (5) archive samples, extracts, and raw sequence data. We demonstrate the importance of each of these considerations using a case study of eDNA metabarcoding in the Ports of Los Angeles and Long Beach.

An alternative downstream translation start site in the non-TIR adaptor Scimp enables selective amplification of CpG DNA responses in mouse macrophages.

Toll-like receptor (TLR) signaling relies on Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor proteins that recruit downstream signaling molecules to generate tailored immune responses. In addition, the palmitoylated transmembrane adaptor protein family member Scimp acts as a non-TIR-containing adaptor protein in macrophages, scaffolding the Src family kinase Lyn to enable TLR phosphorylation and proinflammatory signaling responses. Here we report the existence of a smaller, naturally occurring translational variant of Scimp (Scimp TV1), which is generated through leaky scanning and translation at a downstream methionine.

LLAMA: a robust and scalable machine learning pipeline for analysis of large scale 4D microscopy data: analysis of cell ruffles and filopodia.

Background: With recent advances in microscopy, recordings of cell behaviour can result in terabyte-size datasets. The lattice light sheet microscope (LLSM) images cells at high speed and high 3D resolution, accumulating data at 100 frames/second over hours, presenting a major challenge for interrogating these datasets. The surfaces of vertebrate cells can rapidly deform to create projections that interact with the microenvironment.

Redescription of (Dana, 1853) (Crustacea, Isopoda, Sphaeromatidae), designation of neotype, and 16S-rDNA molecular phylogeny of the north-eastern Pacific species.

(Dana, 1852) is revised, a male neotype is designated, photographed, and illustrated; the species occurs from Vancouver British Columbia to the central California coast. 16S-rDNA sequences (~650 bp) for all available ethanol preserved species of were used to hypothesize their relationships. Our analyses revealed a sister taxon relationship between the fully marine and the brackish to freshwater species, .

Macropinocytosis: Insights from immunology and cancer.

Macropinocytosis is increasingly recognized for its versatile adaptations and functions as a highly conserved, ubiquitous pathway for the bulk uptake of fluid, particulate cargo, and membranes. Innate immune cells and transformed cancer cells share the capacity for both constitutive and induced macropinocytosis, which is used for immune surveillance, ingestion of pathogens, immune response shaping, and enhancement of scavenging for nutrients as fuel for cell survival and proliferation. Immunology and cancer biology are leading a resurgence of interest in defining the molecular and physiological regulation of macropinocytosis, partly in pursuit of ways to control macropinocytic uptake in disease settings.

Automated Analysis of Cell Surface Ruffling: Ruffle Quantification Macro.

Cell surface protrusions include F-actin rich, wave-like ruffles that are erected transiently in response to stimuli and during cell migration. Macrophages are innate immune cells that ruffle constitutively and more dramatically in cells activated by pathogens. Dorsal ruffles and their resulting macropinosomes are key sites for environmental sampling, pathogen detection and immune signaling.

SCIMP is a universal Toll-like receptor adaptor in macrophages.

In innate immune cells, pathogens and danger signals activate TLRs, unleashing potent and tailored inflammatory responses. Previously, we reported that an immune-specific transmembrane adaptor, SLP adaptor and CSK interacting membrane protein (SCIMP), interacts with TLR4 via direct binding to its cytoplasmic TIR domain. SCIMP scaffolds a Src family kinase, Lyn, for TLR4 phosphorylation and activation.

Rab8a localisation and activation by Toll-like receptors on macrophage macropinosomes.

Macropinocytosis is a prevalent and essential pathway in macrophages where it contributes to anti-microbial responses and innate immune cell functions. Cell surface ruffles give rise to phagosomes and to macropinosomes as multi-functional compartments that contribute to environmental sampling, pathogen entry, plasma membrane turnover and receptor signalling. Rapid, high resolution, lattice light sheet imaging demonstrates the dynamic nature of macrophage ruffling.

Guanine nucleotide exchange factors activate Rab8a for Toll-like receptor signalling.

Macrophages are important immune sentinels that detect and clear pathogens and initiate inflammatory responses through the activation of surface receptors, including Toll-like receptors (TLRs). Activated TLRs employ complex cellular trafficking and signalling pathways to initiate transcription for inflammatory cytokine programs. We have previously shown that Rab8a is activated by multiple TLRs and regulates downstream Akt/mTOR signalling by recruiting the effector PI3Kγ, but the guanine nucleotide exchange factors (GEF) canonically required for Rab8a activation in TLR pathways is not known.

Meeting report-Small GTPases in membrane processes: FASEB summer research conference.

In September 2018, conference organizers Nava Segev (University of Illinois, Chicago) and Marino Zerial (MPI, Dresden) hosted the 5th FASEB Meeting in Small GTPases in Membrane Processes: Trafficking, Autophagy and Disease at the National Conference Center in Leesburg, Virginia. With over 100 attendees from across the globe sharing their varied expertise and interests, we came together with the common goal of gaining a better understanding of how small GTPases and their regulators act in both canonical and non-canonical pathways to conduct a diversity of essential cellular functions. A broad range of disciplines was covered in this meeting, including the study of biophysical and structural properties of these proteins, functional studies to get at the roles of these proteins in various cellular contexts (eg, ciliary function, mitophagy, cell motility, cell cycle, and development), and translational approaches to understand the greater implications of small GTPases and their regulators in multicellular systems and disease pathology.

A new species of Alvinocaris (Crustacea: Decapoda: Caridea: Alvinocarididae) from Costa Rican methane seeps.

A new caridean shrimp, Alvinocaris costaricensis, is described from methane seeps in the eastern Pacific off Costa Rica. The new species is the 16th described species of the genus, and by molecular analysis appears closest to Alvinocaris komaii from the Lau Basin, southwestern Pacific, but shares certain morphological characters with A. lusca from the Galapagos Rift and A.

TLR Crosstalk Activates LRP1 to Recruit Rab8a and PI3Kγ for Suppression of Inflammatory Responses.

The multi-ligand endocytic receptor, low-density lipoprotein-receptor-related protein 1 (LRP1), has anti-inflammatory roles in disease. Here, we reveal that pathogen-activated Toll-like receptors (TLRs) activate LRP1 in human and mouse primary macrophages, resulting in phosphorylation of LRP1 at Y4507. In turn, this allows LRP1 to activate and recruit the guanosine triphosphatase (GTPase), Rab8a, with p110γ/p101 as its phosphatidylinositol 3-kinase (PI3K) effector complex.

Macropinosome formation by tent pole ruffling in macrophages.

Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure.

Worldwide Engagement for Digitizing Biocollections (WeDigBio): The Biocollections Community's Citizen-Science Space on the Calendar.

The digitization of biocollections is a critical task with direct implications for the global community who use the data for research and education. Recent innovations to involve citizen scientists in digitization increase awareness of the value of biodiversity specimens; advance science, technology, engineering, and math literacy; and build sustainability for digitization. In support of these activities, we launched the first global citizen-science event focused on the digitization of biodiversity specimens: Worldwide Engagement for Digitizing Biocollections (WeDigBio).

Development of SH2 probes and pull-down assays to detect pathogen-induced, site-specific tyrosine phosphorylation of the TLR adaptor SCIMP.

Protein tyrosine phosphorylation guides many molecular interactions for cellular functions. SCIMP is a transmembrane adaptor protein (TRAP) family member that mediates selective proinflammatory cytokine responses generated by pathogen-activated Toll-like receptor (TLR) pathways in macrophages. TLR activation triggers SCIMP phosphorylation and selective phosphorylation of distinct tyrosine residues on this adaptor offers the potential for regulating or biasing inflammatory responses.

Small GTPase Rab8a-recruited Phosphatidylinositol 3-Kinase γ Regulates Signaling and Cytokine Outputs from Endosomal Toll-like Receptors.

LPS-mediated activation of Toll-like receptor 4 (TLR4) in macrophages results in the coordinated release of proinflammatory cytokines, followed by regulatory mediators, to ensure that this potentially destructive pathway is tightly regulated. We showed previously that Rab8a recruits PI3Kγ for Akt-dependent signaling during TLR4 activation to limit the production of the proinflammatory cytokines IL-6 and IL-12p40 while enhancing the release of the regulatory/anti-inflammatory cytokine IL-10. Here we broaden the array of immune receptors controlled by Rab8a-PI3Kγ and further define the Rab-mediated membrane domains required for signaling.

SCIMP is a transmembrane non-TIR TLR adaptor that promotes proinflammatory cytokine production from macrophages.

Danger signals activate Toll-like receptors (TLRs), thereby initiating inflammatory responses. Canonical TLR signalling, via Toll/Interleukin-1 receptor domain (TIR)-containing adaptors and proinflammatory transcription factors such as NF-κB, occurs in many cell types; however, additional mechanisms are required for specificity of inflammatory responses in innate immune cells. Here we show that SCIMP, an immune-restricted, transmembrane adaptor protein (TRAP), promotes selective proinflammatory cytokine responses by direct modulation of TLR4.

Image-Based Analysis of Phagocytosis: Measuring Engulfment and Internalization.

The process of phagocytosis is crucial for fighting infection in innate immunity, for maintaining homeostasis through clearing cell debris, and for tissue remodeling in development. Here we describe two semi-automated image-based assays for the quantitative characterization of the early stages of phagocytosis and pathogen entry. A feature of these assays is the ability to detect and assess molecules or agents that subtly affect the stages both before and after cup closure or internalization.

Distinct Roles for APPL1 and APPL2 in Regulating Toll-like Receptor 4 Signaling in Macrophages.

Macrophages are activated by contact with pathogens to mount innate immune defenses against infection. Toll-like receptor 4 (TLR4) at the macrophage surface recognizes and binds bacterial lipopolysaccharide (LPS), setting off signaling and transcriptional events that lead to the secretion of pro- and anti-inflammatory cytokines; these in turn control inflammatory and antimicrobial responses. Although the complex regulatory pathways downstream of TLR4 have been extensively studied, further molecules critical for modulating the resulting cytokine outputs remain to be characterized.

Sequential recruitment of Rab GTPases during early stages of phagocytosis.

The phagocytosis and destruction of pathogens and dead cells by macrophages is important for innate immunity and tissue maintenance. Multiple Rab family GTPases engage effector molecules to coordinate the early stages of phagocytosis, which include rapid changes in actin polymerization, membrane phospholipids, trafficking and the activation of receptors. Defining the spatiotemporal, sequential recruitment of these Rabs is critical for insights into how phagocytosis is initiated and coordinated.

The murine neutrophil NLRP3 inflammasome is activated by soluble but not particulate or crystalline agonists.

Neutrophils express pattern recognition receptors (PRRs) and regulate immune responses via PRR-dependent cytokine production. An emerging theme is that neutrophil PRRs often exhibit cell type-specific adaptations in their signalling pathways. This prompted us to examine inflammasome signalling by the PRR NLRP3 in murine neutrophils, in comparison to well-established NLRP3 signalling pathways in macrophages.

Dynamic imaging of the recycling endosomal network in macrophages.

Recycling endosomes (REs) form an extensive and complex network of subcompartmentalized vesicular and tubular elements that connect with the cell surface and other endosomes in macrophages. As surveillance and defense cells of the innate immune system, macrophages are highly dependent on REs for their active and voluminous cell surface turnover and endocytic, exocytic, and recycling of membrane and cargo. Here we set out three approaches for imaging and analyzing REs in macrophages, based on the expression of fluorescently labeled RE-associated proteins and the uptake of fluorescent cargo.

Status of Exosphaeromaamplicauda (Stimpson, 1857), E.aphrodita (Boone, 1923) and description of three new species (Crustacea, Isopoda, Sphaeromatidae) from the north-eastern Pacific.

Exosphaeromaamplicauda (Stimpson, 1857) from the west coast of North America is reviewed and redescribed and revealed to be a group of closely related species. A neotype is designated and the species redescribed based on the neotype and topotypic specimens. Exosphaeromaamplicauda is known only from the coast of California, at Marin, Sonoma and San Mateo Counties.

Rab31 and APPL2 enhance FcγR-mediated phagocytosis through PI3K/Akt signaling in macrophages.

Membrane remodeling in the early stages of phagocytosis enables the engulfment of particles or pathogens and receptor signaling to activate innate immune responses. Members of the Rab GTPase family and their disparate effectors are recruited sequentially to regulate steps throughout phagocytosis. Rab31 (Rab22b) is known for regulating post-Golgi trafficking, and here we show in macrophages that Rab31-GTP is additionally and specifically recruited to early-stage phagosomes.