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.

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.

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.

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.

The impact of tumor immunogenicity on cancer pain phenotype using syngeneic oral cancer mouse models.

Head and neck squamous cell carcinoma (HNSCC) patients report severe function-induced pain at the site of the primary tumor. The current hypothesis is that oral cancer pain is initiated and maintained in the cancer microenvironment due to secretion of algogenic mediators from tumor cells and surrounding immune cells that sensitize the primary sensory neurons innervating the tumor. Immunogenicity, which is the ability to induce an adaptive immune response, has been widely studied using cancer cell transplantation experiments. However, oral cancer pain studies have primarily used xenograft transplant models in which human-derived tumor cells are inoculated in an athymic mouse lacking an adaptive immune response; the role of inflammation in oral cancer-induced nociception is still unknown. Using syngeneic oral cancer mouse models, we investigated the impact of tumor cell immunogenicity and growth on orofacial nociceptive behavior and oral cancer-induced sensory neuron plasticity. We found that an aggressive, weakly immunogenic mouse oral cancer cell line, MOC2, induced rapid orofacial nociceptive behavior in both male and female C57Bl/6 mice. Additionally, MOC2 tumor growth invoked a substantial injury response in the trigeminal ganglia as defined by a significant upregulation of injury response marker ATF3 in tongue-innervating trigeminal neurons. In contrast, using a highly immunogenic mouse oral cancer cell line, MOC1, we found a much slower onset of orofacial nociceptive behavior in female C57Bl/6 mice only as well as sex-specific differences in the tumor-associated immune landscape and gene regulation in tongue innervating sensory neurons. Together, these data suggest that cancer-induced nociceptive behavior and sensory neuron plasticity can greatly depend on the immunogenic phenotype of the cancer cell line and the associated immune response.

Jacob Whitman Bailey (1811-1857): Ehrenberg's ambassador to America.

Science in general, and microscopy in particular, lagged far behind Europe in early 19th century America. Jacob Bailey was one of the very few American microscopists. In eulogies he was called 'the Ehrenberg of America' and 'the founder of microscopical research'. He was a major figure in the scientific community of America in his time and instrumental in promoting microscopy and the study of microscopic organisms. His name maybe familiar to specialists who focus on diatoms, foraminifera, or radiolaria. However, Bailey's important contributions to protistology have received very little attention and he is rarely mentioned in histories of protistology. Here Jacob Bailey's life, his protistological works, as well as his roles in the development of microscopy in America and the 19th century debate over life in the deep sea, are reviewed.

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.

Oral cancer induced TRPV1 sensitization is mediated by PAR2 signaling in primary afferent neurons innervating the cancer microenvironment.

Oral cancer patients report sensitivity to spicy foods and liquids. The mechanism responsible for chemosensitivity induced by oral cancer is not known. We simulate oral cancer-induced chemosensitivity in a xenograft oral cancer mouse model using two-bottle choice drinking and conditioned place aversion assays. An anatomic basis of chemosensitivity is shown in increased expression of TRPV1 in anatomically relevant trigeminal ganglion (TG) neurons in both the xenograft and a carcinogen (4-nitroquinoline 1-oxide)-induced oral cancer mouse models. The percent of retrograde labeled TG neurons that respond to TRPV1 agonist, capsaicin, is increased along with the magnitude of response as measured by calcium influx, in neurons from the cancer models. To address the possible mechanism of TRPV1 sensitivity in tongue afferents, we study the role of PAR2, which can sensitize the TRPV1 channel. We show co-expression of TRPV1 and PAR2 on tongue afferents and using a conditioned place aversion assay, demonstrate that PAR2 mediates oral cancer-induced, TRPV1-evoked sensitivity in an oral cancer mouse model. The findings provide insight into oral cancer-mediated chemosensitivity.

On Charles Atwood Kofoid (1865-1947): The good, the bad, and the ugly.

Charles Atwood Kofoid was a scientist of considerable stature and a key figure in the development of protistology in the United States of America during first half of the 20th century. Today he is known mainly for his detailed taxonomic monographs on protists of the marine plankton, specifically dinoflagellates and tintinnid ciliates. Lesser known today is the wide range of Kofoid's work in protistology. Little known is his responsibility for one of the great mistakes of protistology: the existence of a protistan nervous system, the "neuromortorium". Largely unknown is Kofoid's enthusiastic involvement in the eugenics movement. Here, following a brief biography, I will provide a review of Kofoid's enduring contribution to protistology, "the good", then the story of the neuromotorium, "the bad", and finally an account of Kofoid's implication in the eugenics movement, "the ugly".

On Edouard Claparède and Johannes Lachmann (Clap & Lach) and their "Etudes sur les Infusoires et les Rhizopodes".

Accounts are given of the lives and careers of Edouard Claparède (1832-1871) and Johannes Lachmann (1832-1860), the authors of the landmark work of 19th century protistology "Etudes sur les Infusoires et les Rhizopodes", published in 3 parts in 1859, 1860 and 1861. Accounts are also given on the origin of the monograph, the relationship of Claparède and Lachmann with Ernst Haeckel, and Claparède's role as a promoter of Darwin's theories. Suggestions as to how to properly cite the monograph of Claparède and Lachmann are provided, as well as a supplementary file listing the protist species currently accepted as having been first described in their monograph.

Cathepsin S Evokes PAR2-Dependent Pain in Oral Squamous Cell Carcinoma Patients and Preclinical Mouse Models.

Oral squamous cell carcinoma (SCC) pain is more prevalent and severe than pain generated by any other form of cancer. We previously showed that protease-activated receptor-2 (PAR2) contributes to oral SCC pain. Cathepsin S is a lysosomal cysteine protease released during injury and disease that can activate PAR2. We report here a role for cathepsin S in PAR2-dependent cancer pain. We report that cathepsin S was more active in human oral SCC than matched normal tissue, and in an orthotopic xenograft tongue cancer model than normal tongue. The multiplex immunolocalization of cathepsin S in human oral cancers suggests that carcinoma and macrophages generate cathepsin S in the oral cancer microenvironment. After cheek or paw injection, cathepsin S evoked nociception in wild-type mice but not in mice lacking PAR2 in Nav1.8-positive neurons (Par2Nav1.8), nor in mice treated with LY3000328 or an endogenous cathepsin S inhibitor (cystatin C). The human oral SCC cell line (HSC-3) with homozygous deletion of the gene for cathepsin S (CTSS) with CRISPR/Cas9 provoked significantly less mechanical allodynia and thermal hyperalgesia, as did those treated with LY3000328, compared to the control cancer mice. Our results indicate that cathepsin S is activated in oral SCC, and that cathepsin S contributes to cancer pain through PAR2 on neurons.

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.