A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R² = 0.81, p < .001, biofouling R² = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.     

Regulation of Ergothioneine Biosynthesis and Its Effect on Mycobacterium tuberculosis Growth and Infectivity

Ergothioneine (EGT) is synthesized in mycobacteria, but limited knowledge exists regarding its synthesis, physiological role, and regulation. We have identified Rv3701c from Mycobacterium tuberculosis to encode for EgtD, a required histidine methyltransferase that catalyzes first biosynthesis step in EGT biosynthesis. EgtD was found to be phosphorylated by the serine/threonine protein kinase PknD. PknD phosphorylates EgtD both in vitro and in a cell-based system on Thr²¹³. The phosphomimetic (T213E) but not the phosphoablative (T213A) mutant of EgtD failed to restore EGT synthesis in a ΔegtD mutant. The findings together with observed elevated levels of EGT in a pknD transposon mutant during in vitro growth suggests that EgtD phosphorylation by PknD negatively regulates EGT biosynthesis. We further showed that EGT is required in a nutrient-starved model of persistence and is needed for long term infection of murine macrophages.     

The cytoplasmic tail of glycoprotein M (gpUL100) expresses trafficking signals required for human cytomegalovirus assembly and replication

The virion envelope of human cytomegalovirus (HCMV) is complex and consists of an incompletely defined number of glycoproteins. The gM/gN protein complex is the most abundant protein component of the envelope. Studies have indicated that deletion of the viral gene encoding either gM or gN is a lethal mutation. Analysis of the amino acid sequence of gM disclosed a C-terminal acidic cluster of amino acids and a tyrosine-containing trafficking motif, both of which are well-described trafficking/sorting signals in the cellular secretory pathway. To investigate the roles of these signals in the trafficking of the gM/gN complex during virus assembly, we made a series of gM (UL100 open reading frame) mutants in the AD169 strain of HCMV. Mutant viruses that lacked the entire C-terminal cytoplasmic tail of gM were not viable, suggesting that the cytoplasmic tail of gM is essential for virus replication. In addition, the gM mutant protein lacking the cytoplasmic domain exhibited decreased protein stability. Mutant viruses with a deletion of the acidic cluster or alanine substitutions in tyrosine-based motifs were viable but exhibited a replication-impaired phenotype suggestive of a defect in virion assembly. Analysis of these mutant gMs using static immunofluorescence and fluorescence recovery after photobleaching demonstrated delayed kinetics of intracellular localization of the gM/gN protein to the virus assembly compartment compared to the wild-type protein. These data suggest an important role of the glycoprotein gM during virus assembly, particularly in the dynamics of gM trafficking during viral-particle assembly.     

Assessing the effects of repeated handling on the physiology and condition of semi‐precocial nestlings

Repeated exposure to elevated levels of glucocorticoids during development can have long‐term detrimental effects on survival and fitness, potentially associated with increased telomere attrition. Nestling birds are regularly handled for ecological research, yet few authors have considered the potential for handling‐induced stress to influence hormonally mediated phenotypic development or bias interpretations of subsequent focal measurements. We experimentally manipulated the handling experience of the semi‐precocial nestlings of European Storm Petrel Hydrobates pelagicus to simulate handling in a typical field study and examined cumulative effects on physiology and condition in late postnatal development. Neither baseline corticosterone (the primary glucocorticoid in birds), telomere length nor body condition varied with the number of handling episodes. The absence of a response could be explained if Storm Petrels did not perceive handling to be stressful or if there is dissociation of the hypothalamic–pituitary–adrenal axis from stressful stimuli in early life. Eliciting a response to a stressor may be maladaptive for cavity‐dwelling young that are unable to escape or defend themselves. Furthermore, avoiding elevated overall glucocorticoid exposure may be particularly important in a long‐lived species, in which accelerated early‐life telomere erosion could impact negatively upon longevity. We propose that the level of colony‐wide disturbance induced by investigator handling of young could be important in underlining species‐specific responses. Storm Petrel nestlings appear unresponsive to investigator handling within the limits of handling in a typical field study and handling at this level should not bias physiological and morphological measurements.     

Detection of viral hemorrhagic septicemia in round gobies in New York State (USA) waters of Lake Ontario and the St. Lawrence River

In May 2006 a large mortality of several thousand round gobies Neogobius melanostomus (Pallas, 1814) occurred in New York waters of the St. Lawrence River and Lake Ontario. Necropsies of sampled fish from these areas showed pallor of the liver and gills, and hemorrhagic areas in many organs. Histopathologic examination of affected tissues revealed areas of necrosis and hemorrhage. Inoculations of fathead minnow Pimephales promelas (Rafinesque, 1820) cell cultures with dilutions of tissue samples from the necropsied gobies produced a cytopathic effect within 5 d post-inoculation. Samples of cell culture supernatant were tested using RT-PCR and confirmed the presence of viral hemorrhagic septicemia virus (VHSV). Sequence analysis of the VHSV isolate resulted in its assignment to the type-IVb subgroup. The detection of VHSV in a relatively recent invasive fish species in the Great Lakes and the potential impact of VHSV on the ecology and economy of the area will require further investigation and careful management considerations.     

A 1-D model to explore the effects of tissue loading and tissue concentration gradients in the revised Starling principle

The recent experiments in Hu et al. (Am J Physiol Heart Circ Physiol 279: H1724-H1736, 2000) and Adamson et al. (J Physiol 557: 889-907, 2004) in frog and rat mesentery microvessels have provided strong evidence supporting the Michel-Weinbaum hypothesis for a revised asymmetric Starling principle in which the Starling force balance is applied locally across the endothelial glycocalyx layer rather than between lumen and tissue. These experiments were interpreted by a three-dimensional (3-D) mathematical model (Hu et al.; Microvasc Res 58: 281-304, 1999) to describe the coupled water and albumin fluxes in the glycocalyx layer, the cleft with its tight junction strand, and the surrounding tissue. This numerical 3-D model converges if the tissue is at uniform concentration or has significant tissue gradients due to tissue loading. However, for most physiological conditions, tissue gradients are two to three orders of magnitude smaller than the albumin gradients in the cleft, and the numerical model does not converge. A simpler multilayer one-dimensional (1-D) analytical model has been developed to describe these conditions. This model is used to extend Michel and Phillips's original 1-D analysis of the matrix layer (J Physiol 388: 421-435, 1987) to include a cleft with a tight junction strand, to explain the observation of Levick (Exp Physiol 76: 825-857, 1991) that most tissues have an equilibrium tissue concentration that is close to 0.4 lumen concentration, and to explore the role of vesicular transport in achieving this equilibrium. The model predicts the surprising finding that one can have steady-state reabsorption at low pressures, in contrast to the experiments in Michel and Phillips, if a backward-standing gradient is established in the cleft that prevents the concentration from rising behind the glycocalyx.     

Role of vinculin in regulating focal adhesion turnover

Although vinculin (-/-) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in vinculin null compared to wild-type cells. Expression of vinculin rescued the spreading defect and resulted in larger and more stable FAs. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a role in vinculin activation by relieving an intramolecular association between the vinculin head (Vh) and tail (Vt) that masks the ligand binding sites in Vh and Vt. To investigate the role of the vinculin/PIP2 interaction in FA dynamics, we used a vinculin mutant lacking the C-terminal arm (residues 1053-1066) and referred to as the deltaC mutation. This mutation reduced PIP2 binding to a Vt deltaC polypeptide by >90% compared to wild type without affecting binding to Vh or F-actin. Interestingly, cells expressing the vinculin deltaC mutant assembled remarkably stable FAs. The results suggest that vinculin inhibits cell migration by stabilising FAs, and that binding of inositol phospholipids to Vt plays an important role in FA turnover.     

Recent pulsed EPR studies of the photosystem II oxygen-evolving complex: implications as to water oxidation mechanisms

The pulsed electron paramagnetic resonance (EPR) methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the structure of the Photosystem II oxygen-evolving complex (OEC), including the paramagnetic manganese cluster and its immediate surroundings. Recent unpublished results from the pulsed EPR laboratory at UC-Davis are discussed, along with aspects of recent publications, with a focus on substrate and cofactor interactions. New data on the proximity of exchangeable deuterons around the Mn cluster poised in the S(0)-state are presented and interpreted. These pulsed EPR results are used in an evaluation of several recently proposed mechanisms for PSII water oxidation. We strongly favor mechanistic models where the substrate waters bind within the OEC early in the S-state cycle. Models in which the O-O bond is formed by a nucleophilic attack by a Ca(2+)-bound water on a strong S(4)-state electrophile provide a good match to the pulsed EPR data.