Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis

The crustacean stomatogastric ganglion (STG) is modulated by both locally released neuroactive compounds and circulating hormones. This study presents mass spectrometric characterization of the complement of peptide hormones present in one of the major neurosecretory structures, the pericardial organs (POs), and the detection of neurohormones released from the POs. Direct peptide profiling of Cancer borealis PO tissues using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) revealed many previously identified peptides, including proctolin, red pigment concentrating hormone (RPCH), crustacean cardioactive peptide (CCAP), several orcokinins, and SDRNFLRFamide. This technique also detected corazonin, a well-known insect hormone, in the POs for the first time. However, most mass spectral peaks did not correspond to previously known peptides. To characterize and identify these novel peptides, we performed MALDI postsource decay (PSD) and electrospray ionization (ESI) MS/MS de novo sequencing of peptides fractionated from PO extracts. We characterized a truncated form of previously identified TNRNFLRFamide, NRNFLRFamide. In addition, we sequenced five other novel peptides sharing a common C-terminus of RYamide from the PO tissue extracts. High K+ depolarization of isolated POs released many peptides present in this tissue, including several of the novel peptides sequenced in the current study.     

Hormone effects on male sex behavior in adult South African clawed frogs, Xenopus laevis

Intact, adult male Xenopus laevis were injected with human chorionic gonadotropin (HCG) and tested with intact HCG-primed females. Under these conditions, males displayed high levels of sex behavior (clasping of females). By 2 weeks after castration, these males had ceased clasping. Testosterone and dihydrotestosterone reinstated clasping in male castrates. Following removal of testosterone or dihydrotestosterone pellets, castrated males ceased to clasp. No male was ever observed to clasp following estradiol implanted in pellets or in silastic capsules. In experiments on castrated, adult, female Xenopus laevis, both testosterone and testosterone propionate pellets reliably produced male sex behavior in the form of clasping. The clasping of testosterone-implanted female and male castrates was very similar in form and duration. The behavioral effectiveness of testosterone in both sexes and the ineffectiveness of estradiol in eliciting clasping is paralleled by autoradiographic localization of sex steroids in brain where the distribution of testosterone-concentrating, cells is the same for males and females, but different from the distribution of estradiol-concentrating cells.     

P2X7 receptor agonists pre- and postcondition the heart against ischemia-reperfusion injury by opening pannexin-1/P2X₇ channels

Protection of the heart from ischemia-reperfusion injury can be achieved by ischemic preconditioning and ischemic postconditioning. Previous studies revealed that a complex of pannexin-1 with the P2X(7) receptor forms a channel during ischemic preconditioning and ischemic postconditioning that results in the release of endogenous cardioprotectants. ATP binds to P2X(7) receptors, inducing the formation of a channel in association with pannexin-1. We hypothesized that this channel would provide a pathway for the release of these same cardioprotectants. Preconditioning-isolated perfused rat hearts with 0.4 μM ATP preceding 40 min of ischemia minimized infarct size upon subsequent reperfusion (5% of risk area) and resulted in >80% recovery of left ventricular developed pressure. Postconditioning with ATP after ischemia during reperfusion was also protective (6% infarct and 72% recovery of left ventricular developed pressure). Antagonists of both pannexin-1 (carbenoxolone and mefloquine) and P2X(7) receptors (brilliant blue G and A438079) blocked ATP pre- and postconditioning, indicating that ATP protection was elicited via the opening of a pannexin-1/P2X(7) channel. An antagonist of binding of the endogenous cardioprotectant sphingosine 1-phosphate to its G protein-coupled receptor diminished protection by ATP, which is also consistent with an ATP-dependent release of cardioprotectants. Suramin, an antagonist of binding of ATP (and ADP) to P2Y receptors, was without effect on ATP protection. Benzoyl benzoyl-ATP, a more specific P2X(7) agonist, was also a potent pre- and postconditioning agent and sensitive to blockade by pannexin-1/P2X(7) channel antagonists. The data point out for the first time the potential of P2X(7) agonists as cardioprotectants.     

Comparison of a Salmonella typhimurium proteome defined by shotgun proteomics directly on an LTQ-FT and by proteome pre-fractionation on an LCQ-DUO.

Shotgun proteomics is rapidly becoming one of the most efficient and popular tools to examine protein expression in cells. Numerous laboratories now have a wide array of low- and high-performance mass spectrometry instrumentation necessary to complete proteome-wide projects. Often these laboratories have time and financial constraints that prohibit all projects from being conducted on high-performance state-of-the-art mass spectrometers. Here, we compare shotgun proteomic results using a direct 'lyse, digest and analyse' approach on a high-performance mass spectrometer (i.e. the LTQ-FT) with the results from a much lower-performance instrument (i.e. the LCQ-DUO) where, for the latter, various traditional protein pre-fractionation steps and gas-phase fractionation were used to increase the proteome coverage. Our results demonstrate that shotgun proteomic analyses conducted on the lower-performance LCQ-DUO mass spectrometer could adequately characterize a PhoP constitutive strain of Salmonella typhimurium if proteome pre-fractionation steps and gas-phase fractionation were included.     

High-throughput screening of chromatographic separations: III. Monoclonal antibodies on ceramic hydroxyapatite

High-throughput screening (HTS) of chromatography resins for identifying optimal protein purification conditions is becoming an integral part of industrial process development. In this work, ceramic hydroxyapatite (cHA) chromatography of 15 humanized monoclonal antibodies (mAbs) was examined by HTS. MAb binding, as quantified by partition coefficient (K(p)), was measured under 92 combinations of sodium chloride, phosphate, and pH. Binding varied inversely with these variables for all mAbs tested. However, the magnitudes of binding among mAbs under identical conditions varied significantly, showing a >1.5 log range in K(p). Analysis of variance (ANOVA) techniques were used to describe the binding of each mAb as a function of the three screen variables. Linear models relating log K(p) to the pH, log[sodium chloride], and log[phosphate] fit the data for each antibody with 93-96% accuracy. From these models, characteristic charge values for the cation exchange and metal coordination components of the multi-modal mAb/cHA interaction varied twofold across the mAbs, reflecting inherent variability in the number of contacts between a particular mAb and the cHA surface. Furthermore, we reduced the number of test conditions required from 92 to 8 while maintaining an accurate representation of the full binding response surface. This eight-point modeling method accurately predicted the binding behavior of mAbs as well as mAb aggregates, a common impurity in crude mAb preparations. Using this eight-point modeling method, binding and selectivity information for mAb and aggregate can be obtained from less than two milligrams of protein, making the method attractive for early manufacturability assessments.     

Phylogenetic classification of short environmental DNA fragments

Metagenomics is providing striking insights into the ecology of microbial communities. The recently developed massively parallel 454 pyrosequencing technique gives the opportunity to rapidly obtain metagenomic sequences at a low cost and without cloning bias. However, the phylogenetic analysis of the short reads produced represents a significant computational challenge. The phylogenetic algorithm CARMA for predicting the source organisms of environmental 454 reads is described. The algorithm searches for conserved Pfam domain and protein families in the unassembled reads of a sample. These gene fragments (environmental gene tags, EGTs), are classified into a higher-order taxonomy based on the reconstruction of a phylogenetic tree of each matching Pfam family. The method exhibits high accuracy for a wide range of taxonomic groups, and EGTs as short as 27 amino acids can be phylogenetically classified up to the rank of genus. The algorithm was applied in a comparative study of three aquatic microbial samples obtained by 454 pyrosequencing. Profound differences in the taxonomic composition of these samples could be clearly revealed.     

Exploration of the Canyon-Incised Continental Margin of the Northeastern United States Reveals Dynamic Habitats and Diverse Communities

The continental margin off the northeastern United States (NEUS) contains numerous, topographically complex features that increase habitat heterogeneity across the region. However, the majority of these rugged features have never been surveyed, particularly using direct observations. During summer 2013, 31 Remotely-Operated Vehicle (ROV) dives were conducted from 494 to 3271 m depth across a variety of seafloor features to document communities and to infer geological processes that produced such features. The ROV surveyed six broad-scale habitat features, consisting of shelf-breaching canyons, slope-sourced canyons, inter-canyon areas, open-slope/landslide-scar areas, hydrocarbon seeps, and Mytilus Seamount. Four previously unknown chemosynthetic communities dominated by Bathymodiolus mussels were documented. Seafloor methane hydrate was observed at two seep sites. Multivariate analyses indicated that depth and broad-scale habitat significantly influenced megafaunal coral (58 taxa), demersal fish (69 taxa), and decapod crustacean (34 taxa) assemblages. Species richness of fishes and crustaceans significantly declined with depth, while there was no relationship between coral richness and depth. Turnover in assemblage structure occurred on the middle to lower slope at the approximate boundaries of water masses found previously in the region. Coral species richness was also an important variable explaining variation in fish and crustacean assemblages. Coral diversity may serve as an indicator of habitat suitability and variation in available niche diversity for these taxonomic groups. Our surveys added 24 putative coral species and three fishes to the known regional fauna, including the black coral Telopathes magna, the octocoral Metallogorgia melanotrichos and the fishes Gaidropsarus argentatus, Guttigadus latifrons, and Lepidion guentheri. Marine litter was observed on 81% of the dives, with at least 12 coral colonies entangled in debris. While initial exploration revealed the NEUS region to be both geologically dynamic and biologically diverse, further research into the abiotic conditions and the biotic interactions that influence species abundance and distribution is needed.     

Ultrastructure of neurons and synapses in the tentacle gastrodermis of the sea anemone Calliactis parasitica

Little is known about gastrodermal neurons and synapses in the tentacles of sea anemones. Using transmission electron microscopy of serial thin sections of Calliactis parasitica, we have identified both a sensory cell and a ganglion cell with granular vesicles originating from the Golgi complex and have identified four types of synapses in the tentacular gastrodermal nerve plexus. The sensory cell has a recessed apical cilium with a basal body and a perpendicularly oriented centriole, below which are several strands of striated rootlets surrounded by mitochondria. The ganglion cell lacks a cilium and resembles a bipolar neuron, with oppositely directed processes lying parallel to the basally located circular smooth muscle. Both one-way and two-way interneuronal synapses are present with 60- to 90-nm granular vesicles of various densities aligned at the paired electron-dense membranes and fine cross filaments in the intervening 13-nm cleft. Two types of neuroeffector synapses have been located. Dense granular vesicles are present at neuromuscular synapses, whereas less dense vesicles are present at neuroglandular synapses. Most of the synaptic vesicles range from 60 to 120 nm in diameter. Two types of nerve cells and a variety of synaptic loci provide morphological substrates for the spontaneous SS2 conduction pulses in the tentacular gastrodermis of C. parasitica. J Morphol 231:217–223, 1997. © 1997 Wiley-Liss, Inc.     

A Call for More Nuanced Dialogues About Trophy Hunting

Reported effects of trophy harvest often are controversial. The subject is nuanced and many studies lack details necessary to place their results in context. Consequently, many studies are misunderstood or their conclusions misapplied. We propose that all dialogues about trophy hunting include a definition of how they use the term trophy, details of variables measured and why they were selected, and explanations of temporal and spatial scales employed. Only with these details can potential effects of trophy hunting be understood in context and used for management and policy decisions. © 2021 The Wildlife Society.