Phaeogromids of the mesopelagic marine plankton: Temporal variability of concentrations and observations of feeding structures of four species from the mesopelagic in the Mediterranean Sea.

Challengerids, phaeogromids rhizarian protists, are emblematic protists of the deep sea but are also enigmatic as they occur in very low concentrations. In previous studies, we reported on temporal changes in abundance at a near-shore mesopelagic site, but only as part of sampling of the entire microplankton assemblage, not well-suited for examining phaeogromids. Consequently, we turned to using a closing plankton net to provide material from large volumes of seawater, thus allowing for more robust estimates of concentrations and material for observations of living cells, to our knowledge the first made. Here, we report our results on the four most commonly occurring species: Challengeranium diadon, Challengereron willemoesii, Challengeria xiphodon, and Euphysetta lucani. In contrast to our previous report, we found that changes in concentrations were not related to water column stratification, and the four species roughly co-varied with time. Observations of live cells revealed that all four species deploy tentacle-like pseudopods and also very large unstructured webs of fine pseudopods. The similarities in feeding webs suggest similar prey are exploited, and the similar temporal changes in abundances suggest a common factor or factors (unknown at this time) govern their concentrations. Films of live cells are provided in Supplementary Files.

Legumain Induces Oral Cancer Pain by Biased Agonism of Protease-Activated Receptor-2.

Oral squamous cell carcinoma (OSCC) is one of the most painful cancers, which interferes with orofacial function including talking and eating. We report that legumain (Lgmn) cleaves protease-activated receptor-2 (PAR2) in the acidic OSCC microenvironment to cause pain. Lgmn is a cysteine protease of late endosomes and lysosomes that can be secreted; it exhibits maximal activity in acidic environments. The role of Lgmn in PAR2-dependent cancer pain is unknown. We studied Lgmn activation in human oral cancers and oral cancer mouse models. Lgmn was activated in OSCC patient tumors, compared with matched normal oral tissue. After intraplantar, facial or lingual injection, Lgmn evoked nociception in wild-type (WT) female mice but not in female mice lacking PAR2 in NaV1.8-positive neurons (Par2Nav1.8), nor in female mice treated with a Lgmn inhibitor, LI-1. Inoculation of an OSCC cell line caused mechanical and thermal hyperalgesia that was reversed by LI-1. Par2Nav1.8 and Lgmn deletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR2-dependent hyperexcitability of trigeminal neurons from WT female mice. Par2 deletion, LI-1, and inhibitors of adenylyl cyclase or protein kinase A (PKA) prevented the effects of Lgmn. Under acidified conditions, Lgmn cleaved within the extracellular N terminus of PAR2 at Asn30↓Arg31, proximal to the canonical trypsin activation site. Lgmn activated PAR2 by biased mechanisms in HEK293 cells to induce Ca2+ mobilization, cAMP formation, and PKA/protein kinase D (PKD) activation, but not ß-arrestin recruitment or PAR2 endocytosis. Thus, in the acidified OSCC microenvironment, Lgmn activates PAR2 by biased mechanisms that evoke cancer pain.SIGNIFICANCE STATEMENT Oral squamous cell carcinoma (OSCC) is one of the most painful cancers. We report that legumain (Lgmn), which exhibits maximal activity in acidic environments, cleaves protease-activated receptor-2 (PAR2) on neurons to produce OSCC pain. Active Lgmn was elevated in OSCC patient tumors, compared with matched normal oral tissue. Lgmn evokes pain-like behavior through PAR2 Exposure of pain-sensing neurons to Lgmn decreased the current required to generate an action potential through PAR2 Inhibitors of adenylyl cyclase and protein kinase A (PKA) prevented the effects of Lgmn. Lgmn activated PAR2 to induce calcium mobilization, cAMP formation, and activation of protein kinase D (PKD) and PKA, but not ß-arrestin recruitment or PAR2 endocytosis. Thus, Lgmn is a biased agonist of PAR2 that evokes cancer pain.

A Pre-Existing Myogenic Temporomandibular Disorder Increases Trigeminal Calcitonin Gene-Related Peptide and Enhances Nitroglycerin-Induced Hypersensitivity in Mice.

Migraine is commonly reported among patients with temporomandibular disorders (TMDs), especially myogenic TMD. The pathophysiologic mechanisms related to the comorbidity of the two conditions remain elusive. In the present study, we combined masseter muscle tendon ligation (MMTL)-produced myogenic TMD with systemic injection of nitroglycerin (NTG)-induced migraine-like hypersensitivity in mice. Facial mechanical allodynia, functional allodynia, and light-aversive behavior were evaluated. Sumatriptan, an FDA-approved medication for migraine, was used to validate migraine-like hypersensitivity. Additionally, we examined the protein level of calcitonin gene-related peptide (CGRP) in the spinal trigeminal nucleus caudalis using immunohistochemistry. We observed that mice with MMTL pretreatment have a prolonged NTG-induced migraine-like hypersensitivity, and MMTL also enabled a non-sensitizing dose of NTG to trigger migraine-like hypersensitivity. Systemic injection of sumatriptan inhibited the MMTL-enhanced migraine-like hypersensitivity. MMTL pretreatment significantly upregulated the protein level of CGRP in the spinal trigeminal nucleus caudalis after NTG injection. Our results indicate that a pre-existing myogenic TMD can upregulate NTG-induced trigeminal CGRP and enhance migraine-like hypersensitivity.

Retention prediction using quantitative structure-retention relationships combined with the hydrophobic subtraction model in reversed-phase liquid chromatography.

The hydrophobic subtraction model (HSM) combined with quantitative structure-retention relationships (QSRR) methodology was utilized to predict retention times in reversed-phase liquid chromatography (RPLC). A selection of new analytes and new RPLC columns that had never been used in the QSRR modeling process were used to verify the proposed approach. This work is designed to facilitate early prediction of co-elution of analytes in pharmaceutical drug discovery applications where it is advantageous to predict whether impurities might be co-eluted with the active drug component. The QSRR models were constructed through partial least squares regression combined with a genetic algorithm (GA-PLS) which was employed as a feature selection method to choose the most informative molecular descriptors calculated using VolSurf+ software. The analyte hydrophobicity coefficient of the HSM was predicted for subsequent calculation of retention. Clustering approaches based on the local compound type and the local second dominant interaction were investigated to select the most appropriate training set of analytes from a larger database. Predicted retention times of five new compounds on five new RPLC C18 columns were compared with their measured retention times with percentage root-mean-square errors of 15.4 and 24.7 for the local compound type and local second dominant interaction clustering methods, respectively.

Past President's Address: Protists of the Mesopelagic and a Bit on the Long Path to the Deep Sea.

The deep sea has long been a mysterious and attractive habitat for protistologists. However, logistical difficulties severely limit sampling opportunities. Consequently, our knowledge of the protists in the deep sea, (arguably the largest habitat on earth), is relatively sparse. Here, we present a unique time-series concerning three different protist taxa that share only the characteristics of being relatively large, robust to sampling, and easily identifiable to species level using light microscopy: tintinnid ciliates, phaeogromid cercozoans (e.g. Challengerids) and amphisolenid dinoflagellates. We sampled a near-shore deep water site in the N.W. Mediterranean Sea at 250 m depth over a 2-yr period at approximately weekly intervals from January 2017 to December 2018. To our knowledge, no previous studies have employed sampling on a similar time scale. We found taxa that appear to be restricted to deep waters, distinct seasonal patterns of abundance in some taxa, and in others nonseasonal successional patterns. Based on data from sampling following a flash flood event, the Challengerid population appeared to respond positively to a pulse of terrigenous input. Some of the distinct mesopelagic tintinnid ciliates and amphisolinid dinoflagellates were also found in two samples from the North Atlantic mesopelagic gathered from near the Azores Islands in September 2018. We conclude that there are a variety of protist taxa endemic to the mesopelagic, that the populations are dynamic, and they may be widely distributed in the deep waters of the world ocean.

Retention Index Prediction Using Quantitative Structure-Retention Relationships for Improving Structure Identification in Nontargeted Metabolomics.

Structure identification in nontargeted metabolomics based on liquid-chromatography coupled to mass spectrometry (LC-MS) remains a significant challenge. Quantitative structure-retention relationship (QSRR) modeling is a technique capable of accelerating the structure identification of metabolites by predicting their retention, allowing false positives to be eliminated during the interpretation of metabolomics data. In this work, 191 compounds were grouped according to molecular weight and a QSRR study was carried out on the 34 resulting groups to eliminate false positives. Partial least squares (PLS) regression combined with a Genetic algorithm (GA) was applied to construct the linear QSRR models based on a variety of VolSurf+ molecular descriptors. A novel dual-filtering approach, which combines Tanimoto similarity (TS) searching as the primary filter and retention index (RI) similarity clustering as the secondary filter, was utilized to select compounds in training sets to derive the QSRR models yielding R2 of 0.8512 and an average root mean square error in prediction (RMSEP) of 8.45%. With a retention index filter expressed as ±2 standard deviations (SD) of the error, representative compounds were predicted with >91% accuracy, and for 53% of the groups (18/34), at least one false positive compound could be eliminated. The proposed strategy can thus narrow down the number of false positives to be assessed in nontargeted metabolomics.

Rapid Method Development in Hydrophilic Interaction Liquid Chromatography for Pharmaceutical Analysis Using a Combination of Quantitative Structure-Retention Relationships and Design of Experiments.

A design-of-experiment (DoE) model was developed, able to describe the retention times of a mixture of pharmaceutical compounds in hydrophilic interaction liquid chromatography (HILIC) under all possible combinations of acetonitrile content, salt concentration, and mobile-phase pH with R2 > 0.95. Further, a quantitative structure-retention relationship (QSRR) model was developed to predict retention times for new analytes, based only on their chemical structures, with a root-mean-square error of prediction (RMSEP) as low as 0.81%. A compound classification based on the concept of similarity was applied prior to QSRR modeling. Finally, we utilized a combined QSRR-DoE approach to propose an optimal design space in a quality-by-design (QbD) workflow to facilitate the HILIC method development. The mathematical QSRR-DoE model was shown to be highly predictive when applied to an independent test set of unseen compounds in unseen conditions with a RMSEP value of 5.83%. The QSRR-DoE computed retention time of pharmaceutical test analytes and subsequently calculated separation selectivity was used to optimize the chromatographic conditions for efficient separation of targets. A Monte Carlo simulation was performed to evaluate the risk of uncertainty in the model's prediction, and to define the design space where the desired quality criterion was met. Experimental realization of peak selectivity between targets under the selected optimal working conditions confirmed the theoretical predictions. These results demonstrate how discovery of optimal conditions for the separation of new analytes can be accelerated by the use of appropriate theoretical tools.

Benchmarking of Computational Methods for Creation of Retention Models in Quantitative Structure-Retention Relationships Studies.

Quantitative structure-retention relationship (QSRR) models are powerful techniques for the prediction of retention times of analytes, where chromatographic retention parameters are correlated with molecular descriptors encoding chemical structures of analytes. Many QSRR models contain geometrical descriptors derived from the three-dimensional (3D) spatial coordinates of computationally predicted structures for the analytes. Therefore, it is sensible to calculate these structures correctly, as any error is likely to carry over to the resulting QSRR models. This study compares molecular modeling, semiempirical, and density functional methods (both B3LYP and M06) for structure optimization. Each of the calculations was performed in a vacuum, then repeated with solvent corrections for both acetonitrile and water. We also compared Natural Bond Orbital analysis with the Mulliken charge calculation method. The comparison of the examined computational methods for structure calculation shows that, possibly due to the error inherent in descriptor creation methods, a quick and inexpensive molecular modeling method of structure determination gives similar results to experiments where structures are optimized using an expensive and time-consuming level of computational theory. Also, for structures with low flexibility, vacuum or gas phase calculations are found to be as effective as those calculations with solvent corrections added.

Community Structure of Tintinnid Ciliates of the Microzooplankton in the South West Pacific Ocean: Comparison of a High Primary Productivity with a Typical Oligotrophic Site.

Transient 'hot spots' of phytoplankton productivity occur in the generally oligotrophic Southern Pacific Ocean and we hypothesized that the population structure of tintinnid ciliates, planktonic grazers, would differ from that of a typical oligotrophic sites. Samples were collected over a 1-wk period at each of two sites between Fiji and Tahiti: one of elevated chlorophyll a concentrations and primary productivity with an abundance of N-fixing cyanobacteria Trichodesmium, and a distant oligotrophic site. Tintinnid abundance differed between the sites by a factor of 2. A single species (Favella sp.), absent from the oligotrophic site, highly dominated the 'hot spot' site. However, total species richness was identical (71 spp.) as well as short-term temporal variability (2-4 d). At both sites, species abundance distributions most closely fit a log-series or log-normal distribution and the abundance distributions of ecological types, forms of distinct lorica oral diameter, were the typical geometric. Morphological diversity was only slightly lower at the high productivity site. We found that communities of these plankton grazers in 'hot spots' of phytoplankton productivity in oligotrophic systems, although harboring different species, differ little from surrounding oligotrophic areas in community structure.

Molecular Phylogeny and Evolutionary Relationships between the Ciliate Genera Peniculistoma and Mytilophilus (Peniculistomatidae, Pleuronematida).

Peniculistoma mytili and Mytilophilus pacificae are placed in the pleuronematid scuticociliate family Peniculistomatidae based on morphology and ecological preference for the mantle cavity of mytiloid bivalves. We tested this placement with sequences of the small subunit rRNA (SSUrRNA) and cytochrome c oxidase subunit 1 (cox1) genes. These species are very closely related sister taxa with no distinct genetic difference in the SSUrRNA sequence but about 21% genetic difference for cox1, supporting their placement together but separation as distinct taxa. Using infection frequencies, M. pacificae, like its sister species P. mytili, does not interact with Ancistrum spp., co-inhabitants of the mantle cavity. On the basis of these ecological similarities, the fossil record of host mussels, and features of morphology and stomatogenesis of these two ciliates, we argue that M. pacificae derived from a Peniculistoma-like ancestor after divergence of the two host mussels. Our phylogenetic analyses of pleuronematid ciliates includes the SSUrRNA gene sequence of Sulcigera comosa, a Histiobalantium-like ciliate from Lake Baikal. We conclude: (i) that the pleuronematids are a monophyletic group; (ii) that the genus Pleuronema is paraphyletic; and (iii) that S. comosa is a Histiobalantium species. We transfer S. comosa to Histiobalantium and propose a new combination Histiobalantium comosa n. comb.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy.

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR(2), http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.

Novel animal models of acute and chronic cancer pain: a pivotal role for PAR2.

Targeted therapy to prevent the progression from acute to chronic pain in cancer patients remains elusive. We developed three novel cancer models in mice that together recapitulate the anatomical, temporal, and functional characteristics of acute and chronic head and neck cancer pain in humans. Using pharmacologic and genetic approaches in these novel cancer models, we identified the interaction between protease-activated receptor 2 (PAR2) and serine proteases to be of central importance. We show that serine proteases such as trypsin induce acute cancer pain in a PAR2-dependent manner. Chronic cancer pain is associated with elevated serine proteases in the cancer microenvironment and PAR2 upregulation in peripheral nerves. Serine protease inhibition greatly reduces the severity of persistent cancer pain in wild-type mice, but most strikingly, the development of chronic cancer pain is prevented in PAR2-deficient mice. Our results demonstrate a direct role for PAR2 in acute cancer pain and suggest that PAR2 upregulation may favor the development and maintenance of chronic cancer pain. Targeting the PAR2-serine protease interaction is a promising approach to the treatment of acute cancer pain and prevention of chronic cancer pain.

Morphological and ribosomal DNA-based characterization of six Antarctic ciliate morphospecies from the Amundsen Sea with phylogenetic analyses.

We characterized six tintinnid ciliates from Antarctic waters using molecular markers and morphological traits: Amphorellopsis quinquealata, Codonellopsis gaussi, Cymatocylis convallaria, Cy. calyciformis, Cy. drygalskii, and Laackmanniella prolongata. The 100% similarity in SSU-ITS1-5.8S rDNA-ITS2-partial LSU rDNA sequences among Cy. convallaria, Cy. calyciformis, and Cy. drygalskii is supportive of synonymy. Codonellopsis gaussi and L. prolongata also showed high levels of similarity in SSU rDNA (99.83%) and the D2 domain of LSU rDNA (95.77%), suggesting that they are closely related. Phylogenetic analysis placed Cymatocylis in the Rhabdonellidae, Amphorellopsis in the Tintinnidae and L. prolongata/Co. gaussi within the Dictyocystidae.

The saga of the false fossil foram Eozoon.

Eozoon canadense, 'the dawn animal of Canada', a large foraminifera, was announced in 1864 as the oldest fossil organism known. Camps soon formed into disbelievers of its fossil nature, agnostics, and "Eozoonists". Eozoon would number among its proponents major figures of the time. The saga of Eozoon, or more precisely the dispute as to its actual nature, spawned hundreds of publications. Here the story is told with a new focus, one on the stature and roles of the major personalities involved, and the evidence they presented. Eozoon is considered to have been 'de-bunked' in the late 19th century. However, it will be shown that it was never indisputably proven to be inorganic. Rather Eozoon simply faded away after its most ardent defenders died. As late as 1947, it was shown as the primordial organism in a biology textbook. The saga of Eozoon remains as a valuable cautionary tale. It is an example of an artifact accepted as fact because it filled a troubling void in knowledge, i.e., at that time, the first traces of life before Cambrian, and it endured because it was promoted by only a few, but powerful, figures in the scientific establishment of the era.

The odd couple of protistology: Edward Heron-Allen (1861-1943) and Arthur Earland (1866-1958).

Edward Heron-Allen and Arthur Earland were among the last great amateur foraminifera researchers. Their partnership began in 1907 and ended in 1932. While close in age to one another, they shared little more than a fascination for forams and a lack of any university training. In most other aspects, the two men were completely different. Heron-Allen was a famous upper class polymath, expert not only on forams, but also on the Persian language, violins, palm reading, history, asparagus, and barnacles. He was also an accomplished novelist and poet, who frequented literary circles. In contrast to the flamboyant Heron-Allen, Earland was a discrete civil servant who admitted to working on forams as a relief from the monotony of his job. Hence, the two were improbable partners. However, together they produced 39 substantial works on forams. Their studies concerned assemblages from Southern Ocean to the North Sea and they are today credited with the original description of 186 species. Here the distinct lives of the two men are presented, and their contributions to protistology, as partners as well as individuals, are reviewed. The case is made for considering Earland's work as neglected relative to that of Heron-Allen, except perhaps by foram taxonomists.

Sympathetic modulation of tumor necrosis factor alpha-induced nociception in the presence of oral squamous cell carcinoma.

ABSTRACT: Head and neck squamous cell carcinoma (HNSCC) causes more severe pain and psychological stress than other types of cancer. Despite clinical evidence linking pain, stress, and cancer progression, the underlying relationship between pain and sympathetic neurotransmission in oral cancer is unknown. We found that human HNSCC tumors and mouse tumor tissue are innervated by peripheral sympathetic and sensory nerves. Moreover, ß-adrenergic 1 and 2 receptors (ß-ARs) are overexpressed in human oral cancer cell lines, and norepinephrine treatment increased ß-AR2 protein expression as well as cancer cell proliferation in vitro. We have recently demonstrated that inhibition of tumor necrosis factor alpha (TNFα) signaling reduces oral cancer-induced nociceptive behavior. Norepinephrine-treated cancer cell lines secrete more TNFα which, when applied to tongue-innervating trigeminal neurons, evoked a larger Ca 2+ transient; TNF-TNFR inhibitor blocked the increase in the evoked Ca 2+ transient. Using an orthotopic xenograft oral cancer model, we found that mice demonstrated significantly less orofacial cancer-induced nociceptive behavior during systemic ß-adrenergic inhibitory treatment with propranolol. Furthermore, chemical sympathectomy using guanethidine led to a significant reduction in tumor size and nociceptive behavior. We infer from these results that sympathetic signaling modulates oral cancer pain through TNFα secretion and tumorigenesis. Further investigation of the role of neurocancer communication in cancer progression and pain is warranted.

Southern ocean biogeography of tintinnid ciliates of the marine plankton.

Ciliate microzooplankton are important grazers in most pelagic ecosystems and among them, tintinnids, with their largely species-specific loricas, allow relatively easy assessment of questions of diversity and distributions. Herein, we present the results of a survey of species records of tintinnids from the Southern Ocean (locations below 40°S) reported in 56 publications yielding 2,047 species records (synonyms included) from 402 locations. The 192 species reported can be parsed into two main groups: 32 endemic Southern Ocean species, known only from 40°S and further south, and a second group of 181 widespread species, forms with extensive geographic ranges extending into the Southern Ocean. Widespread species reported from the Southern Ocean can be further divided into a group of 81 species, each recorded multiple times in the Southern Ocean waters and 70 apparent "stray" species which have only been found but once. The endemic and widespread species of the Southern Ocean show both distinct distributional patterns and morphological differences. The assemblage of Southern Ocean endemics is found mostly within the Antarctic zone delimited by the average location of the Polar Front and contains a relatively large portion of wide-mouthed forms. We give suggestions for future study.

Re-visiting the ridiculed rival of Leeuwenhoek: Louis Joblot (1645-1723).

Louis Joblot published one of the first manuals of microscopy in 1718, just a few years before both he and Leeuwenhoek died. It contained Joblot's microscope designs and his extensive observations on microorganisms including experiments on spontaneous generation. Joblot's work and his observations have often been overlooked, misdated, and denigrated. This is due to attention given to a few apparently fanciful drawings of microorganisms, and the identification of his work as appearing in a posthumous 1754 edition. The second edition not only placed Joblot's work as decades after Leeuwenhoek's death, but was also expanded by the publisher to include unattributed material from famous sources. Here an attempt is made to shine a light on Joblot's work and bring it out of Leeuwenhoek's shadow.