Phase-field modeling of constrained interactive fungal networks.

Fungi develop structures that interact with their surroundings and evolve adaptively in the presence of geometrical constraints, finding optimal solutions for complex combinatorial problems. The pathogenic fungus Ophiocordyceps constitutes a perfect model for the study of constrained interactive networks. Modeling these networks is challenging due to the highly coupled physics involved and their interaction with moving boundaries. In this work, we develop a computational phase-field model to elucidate the mechanics of the emerging properties observed in fungal networks. We use a variational approach to derive the equations governing the evolution in time of the mycelium biomass and the nutrients in the medium. We present an extensive testing of our model, reproduce growing and decaying phenomena, and capture spatial and temporal scales. We explore the variables interplay mechanism that leads to different colony morphologies, and explain abrupt changes of patterns observed in the laboratory. We apply our model to simulate analogous processes to the evolution of Ophiocordyceps as it grows through confined geometry and depletes available resources, demonstrating the suitability of the formulation to study this class of biological networks.

Zombie-ant fungi cross continents: II. Myrmecophilous hymenostilboid species and a novel zombie lineage.

Ophiocordyceps species infecting ants are globally distributed, with diversity concentrated in the tropics and decreasing with increasing latitude. Among these myrmecophilous species, the ones exhibiting the ability to manipulate host behavior, the so-called "zombie-ant fungi" of the O. unilateralis clade, have been studied progressively over the last decade. However, we know very little about other myrmecophilous groups, such as species within the Ophiocordyceps subgenus Neocordyceps. Species within this group exhibit Hymenostilbe asexual morphs with the ascospores readily breaking into part-spores and regularly kill their hosts on the forest floor, with few records of behavioral manipulation. Here, we describe five new species of Ophiocordyceps belonging to the subgenus Neocordyceps infecting ants in the rainforests of the Brazilian Amazon and Ghana and analyze their ability to manipulate host behavior. We also propose a new status for a species previously described as a variety, providing its phylogenetic placement for the first time. The species proposed herein can readily be separated using classic taxonomic criteria, and this is further supported by ecological and molecular multiloci data.

Zombie-ant fungi across continents: 15 new species and new combinations within Ophiocordyceps. I. Myrmecophilous hirsutelloid species.

Ophiocordyceps species infecting ants - the so-called zombie-ant fungi - comprise one of the most intriguing and fascinating relationships between microbes and animals. They are widespread within tropical forests worldwide, with relatively few reports from temperate ecosystems. These pathogens possess the ability to manipulate host behaviour in order to increase their own fitness. Depending on the fungal species involved the infected ants are manipulated either to leave the nest to ascend understorey shrubs, to die biting onto vegetation, or descend from the canopy to die at the base of trees. Experimental evidence has demonstrated that the behavioural change aids spore dispersal and thus increases the chances of infection, because of the existing behavioural immunity expressed inside ant colonies that limits fungal development and transmission. Despite their undoubted importance for ecosystem functioning, these fungal pathogens are still poorly documented, especially regarding their diversity, ecology and evolutionary relationships. Here, we describe 15 new species of Ophiocordyceps with hirsutella-like asexual morphs that exclusively infect ants. These form a monophyletic group that we identified in this study as myrmecophilous hirsutelloid species. We also propose new combinations for species previously described as varieties and provide for the first time important morphological and ecological information. The species proposed herein were collected in Brazil, Colombia, USA, Australia and Japan. All species could readily be separated using classic taxonomic criteria, in particular ascospore and asexual morphology.

Epitypification and re-description of the zombie-ant fungus, Ophiocordyceps unilateralis (Ophiocordycipitaceae).

The type of Ophiocordyceps unilateralis (Ophiocordycipitaceae, Hypocreales, Ascomycota) is based on an immature specimen collected on an ant in Brazil. The host was identified initially as a leaf-cutting ant (Atta cephalotes, Attini, Myrmicinae). However, a critical examination of the original illustration reveals that the host is the golden carpenter ant, Camponotus sericeiventris (Camponotini, Formicinae). Because the holotype is no longer extant and the original diagnosis lacks critical taxonomic information - specifically, on ascus and ascospore morphology - a new type from Minas Gerais State of south-east Brazil is designated herein. A re-description of the fungus is provided and a new phylogenetic tree of the O. unilateralis clade is presented. It is predicted that many more species of zombie-ant fungi remain to be delimited within the O. unilateralis complex worldwide, on ants of the tribe Camponotini.

Diversity of Entomopathogenic Fungi: Which Groups Conquered the Insect Body?

The entomopathogenic fungi are organisms that evolved to exploit insects. They comprise a wide range of morphologically, phylogenetically, and ecologically diverse fungal species. Entomopathogenic fungi can be found distributed among five of the eight fungal phyla. Entomopathogens are also present among the ecologically similar but phylogenetically distinct Oomycota or water molds, which belong to a different kingdom, the Stramenopila. As a group of parasites, the entomopathogenic fungi and water molds infect a wide range of insect hosts, from aquatic larvae to adult insects from high canopies in tropical forests or even deserts. Their hosts are spread among 20 of the 31 orders of insects, in all developmental stages: eggs, larvae, pupae, nymphs, and adults. Such assortment of niches has resulted in these parasites evolving a considerable morphological diversity, resulting in enormous biodiversity, the majority of which remains unknown. Here we undertake a comprehensive survey of records of these entomopathogens in order to compare and contrast both their morphologies and their ecological traits. Our findings highlight a wide range of adaptations that evolved following the evolutionary transition by the fungi and water molds to infect the most diverse and widespread animals on Earth, the insects.

Disease Dynamics in Ants: A Critical Review of the Ecological Relevance of Using Generalist Fungi to Study Infections in Insect Societies.

It is assumed that social life can lead to the rapid spread of infectious diseases and outbreaks. In ants, disease outbreaks are rare and the expression of collective behaviors is invoked to explain the absence of epidemics in natural populations. Here, we address the ecological approach employed by many studies that have notably focused (89% of the studies) on two genera of generalist fungal parasites (Beauveria and Metarhizium). We ask whether these are the most representative models to study the evolutionary ecology of ant-fungal parasite interactions. To assess this, we critically examine the literature on ants and their interactions with fungal parasites from the past 114years (1900-2014). We discuss how current evolutionary ecology approaches emerged from studies focused on the biological control of pest ants. We also analyzed the ecological relevance of the laboratory protocols used in evolutionary ecology studies employing generalist parasites, as well as the rare natural occurrence of these parasites on ants. After a detailed consideration of all the publications, we suggest that using generalist pathogens such as Beauveria and Metarhizium is not an optimal approach if the goal is to study the evolutionary ecology of disease in ants. We conclude by advocating for approaches that incorporate greater realism.

From So Simple a Beginning: The Evolution of Behavioral Manipulation by Fungi.

Parasites can manipulate the behavior of their hosts in ways that increase either their direct fitness or transmission to new hosts. The Kingdom Fungi have evolved a diverse array of strategies to manipulate arthropod behavior resulting in some of the most complex and impressive examples of behavioral manipulation by parasites. Here we provide an overview of these different interactions and discuss them from an evolutionary perspective. We discuss parasite manipulation within the context of Niko Tinbergen's four questions (function, phylogeny, causation, and ontogeny) before detailing the proximate mechanisms by which fungi control arthropod behavior and the evolutionary pathways to such adaptations. We focus on some systems for which we have recently acquired new knowledge (such as the zombie ant fungus, Ophiocordyceps unilateralis s.l.), but a major goal is also to highlight how many interesting examples remain to be discovered and investigated. With this in mind, we also discuss likely examples of manipulated spiders that are largely unexplored ("zombie spiders"). Armed with advanced tools in evolutionary biology (from serial block face SEM to RNAseq) we can discover how the fungi, a group of microbes capable of coordinated activity, have evolved the ability to direct animal behavior. In short, we have the ability to understand how the organism without the brain controls the one with the brain. We hope such a goal, coupled with the knowledge that many diverse examples of control exist, will inspire other organismal biologists to study the complex adaptations that have arisen from "so simple a beginning."

Familial Hypercholesterolaemia in the Era of Genetic Testing.

Familial hypercholesterolaemia (FH) is a relatively common autosomal dominant genetic condition leading to premature ischaemic vascular disease and mortality if left untreated. Currently, a universal consensus on the diagnostic criteria of FH does not exist but the diagnosis of FH largely relies on the evaluation of low density lipoprotein-cholesterol (LDL-C) levels, a careful documentation of family history, and the identification of clinical features. Diagnosis based purely on lipid levels remains common but there are several limitations to this method of diagnosis both practically and in the proportion of false-negatives and false-positives detected, resulting in substantial under-diagnosis of FH. In some countries, diagnostic algorithms are supplemented with genetic testing of the index case as well as genetic and lipid testing of relatives of the index case. Such "cascade" screening of families following identification of index cases appears to not only improve the rate of diagnosis but is also cost-effective. Currently, we observe a great variation in the excess mortality among patients with FH, which likely reflects a combination of additional genetic and environmental effects on risk overlaid on the risk associated with FH. Current accepted drug therapies for FH include statins and PSCK9 inhibitors. Further work is required to evaluate the cardiovascular disease risk in patients with genetically diagnosed FH and to determine whether a risk-based approach to the treatment of FH is appropriate.

Mifamurtide in metastatic and recurrent osteosarcoma: a patient access study with pharmacokinetic, pharmacodynamic, and safety assessments.

PURPOSE: This non-randomized, patient-access protocol, assessed both safety and efficacy outcomes following liposomal muramyl-tripeptide-phosphatidylethanolamine (L-MTP-PE; mifamurtide) in patients with high-risk, recurrent and/or metastatic osteosarcoma. METHODS: Patients received mifamurtide 2 mg/m(2) intravenously twice-weekly ×12 weeks, then weekly ×24 weeks with and without chemotherapy. Serum concentration-time profiles were collected. Adverse events within 24 hours of drug administration were classified as infusion-related adverse events (IRAE); other AEs and overall survival (OS) were assessed. RESULTS: The study began therapy in January 2008; the last patient completed therapy in October 2012. Two hundred five patients were enrolled; median age was 16.0 years and 146/205 (71%) had active disease. Mifamurtide serum concentrations declined rapidly in the first 30 minutes post-infusion, then in a log-linear manner 2-6 hours post-dose; t1/2 was 2 hours. There were no readily apparent relationships between age and BSA-normalized clearance, half-life, or pharmacodynamic effects, supporting the dose of 2 mg/m(2) mifamurtide across the age range. Patients reported 3,679 IRAE after 7,482 mifamurtide infusions. These were very rarely grade 3 or 4 and most commonly included chills + fever or headache + fatigue symptom clusters. One- and 2-year OS was 71.7% and 45.9%. Patients with initial metastatic disease or progression approximated by within 9 months of diagnosis (N = 40) had similar 2-year OS (39.9%) as the entire cohort (45.9%) CONCLUSIONS: Mifamurtide had a manageable safety profile; PK/PD of mifamurtide in this patient access study was consistent with prior studies. Two-year OS was 45.9%. A randomized clinical trial would be required to definitively determine impact on patient outcomes.

Regulation of NOTCH signaling by reciprocal inhibition of HES1 and Deltex 1 and its role in osteosarcoma invasiveness.

The highly conserved NOTCH signaling pathway has many essential functions in the development of diverse cells, tissues and organs from Drosophila to humans, and dysregulated NOTCH signaling contributes to several disorders, including vascular and bone defects, as well as several cancers. Here we describe a novel mechanism of NOTCH regulation by reciprocal inhibition of two NOTCH downstream effectors: Deltex1 and HES1. This mechanism appears to regulate invasion of osteosarcoma cells, as Deltex1 blocks osteosarcoma invasiveness by downregulating NOTCH/HES1 signaling. The inhibitory effect of endogenous Deltex1 on NOTCH signaling is mediated through binding with the intracellular domain of NOTCH and ubiquitination and degradation of NOTCH receptors. Conversely, we show that the NOTCH target gene HES1 causes transcriptional inhibition of Deltex1 by directly binding to the promoter of Deltex1. An HES1 binding site is identified 400 bp upstream of the transcription start site of Deltex1. HES1-mediated repression of Deltex1 requires the C-terminal H3/H4 and WRPW domains of HES1, which associate with the TLE/Groucho corepressors. Taken together, we define a molecular mechanism regulating NOTCH signaling by reciprocal inhibition of the NOTCH target genes HES1 and Deltex1 in mammalian cells. This mechanism may have important clinical implications for targeting NOTCH signaling in osteosarcoma and other cancers.

Extended phenotype: nematodes turn ants into bird-dispersed fruits.

A recent study has discovered a novel extended phenotype of a nematode which alters its ant host to resemble ripe fruit. The infected ants are in turn eaten by frugivorous birds that disperse the nematode's eggs.

Genetically engineered T cells expressing a HER2-specific chimeric receptor mediate antigen-specific tumor regression.

In this report, we developed a chimeric receptor (N29gamma chR) involving the single chain Fv (scFv) derived from N29 monoclonal antibody (mAb) specific for p185HER2 and characterized the therapeutic efficacy of primary T cells engineered to express N29gamma chR in two histologically distinct murine tumor models. Murine breast (MT901) and fibrosarcoma (MCA207) cancer cell lines were engineered to express human HER2 as targets. Administration of N29gamma chR-expressing T cells eliminated 3-day pulmonary micrometastases of MT901/HER2 and MCA207/HER2 but not parental tumor cells. A 5 to 8-fold increased dose of N29gamma T cells was required to mediate regression of advanced 8-day macrometastases. Exogenous administration of interleukin-2 (IL-2) after N29gamma T-cell transfer was dispensable for treatment of 3-day micrometastases, but was required for mediating regression of well-established 8-day macrometastases. Moreover, fractionated CD8 T cells expressing N29gamma chR suppressed HER2-positive tumor cell growth after adoptive transfer independent of CD4(+) cells. These data indicate that genetically modified T cells expressing a HER2-targeting chimeric receptor can mediate antigen-specific regression of preestablished metastatic cancers in a cell dose-dependent fashion. Systemic administration of IL-2 augments the therapeutic efficacy of these genetically engineered T cells in advanced diseases. These results are relevant to the implication of genetically redirected T cells in clinical cancer immunotherapy.

Hairworm anti-predator strategy: a study of causes and consequences.

One of the most fascinating anti-predator responses displayed by parasites is that of hairworms (Nematomorpha). Following the ingestion of the insect host by fish or frogs, the parasitic worm is able to actively exit both its host and the gut of the predator. Using as a model the hairworm, Paragordius tricuspidatus, (parasitizing the cricket Nemobius sylvestris) and the fish predator Micropterus salmoïdes, we explored, with proteomics tools, the physiological basis of this anti-predator response. By examining the proteome of the parasitic worm, we detected a differential expression of 27 protein spots in those worms able to escape the predator. Peptide Mass Fingerprints of candidate protein spots suggest the existence of an intense muscular activity in escaping worms, which functions in parallel with their distinctive biology. In a second step, we attempted to determine whether the energy expended by worms to escape the predator is traded off against its reproductive potential. Remarkably, the number of offspring produced by worms having escaped a predator was not reduced compared with controls.

Fate and function of anti-CD3/CD28-activated T cells following adoptive transfer: IL-2 promotes development of anti-tumor memory T cells in vivo.

BACKGROUND: Adoptive immunotherapy with T cells activated through CD3 alone requires exogenous IL-2 for T-cell function and survival after transfer, but the in vivo cytokine requirement of T cells activated through CD3 and CD28 is unknown. We hypothesized that CD3/CD28-activated T cells, unlike those activated through CD3 alone, might develop into long-lived memory T cells, either with or without systemic IL-2. METHODS: We used MHC class I-restricted TCR transgenic T cells from the OT-1 mouse, specific for the surrogate tumor Ag ovalbumin (OVA), to assess the trafficking kinetics, antigenic responsiveness and anti-tumor efficacy of dual-activated T cells in vivo as a function of IL-2 administration. At days 7, 14, and 28 after transfer, lymph node cells and splenocytes were examined for donor cell persistence and antigenic responsiveness by FACS and ELISA, respectively. RESULTS: In IL-2-treated mice, donor CD8+ T cells persisted and developed a memory phenotype, based on CD44 and Ly6c expression at day 28, while mice given no IL-2 had fewer donor cells at all time points. OVA-specific release of IFN-gamma was higher from lymphocytes of IL-2-treated mice compared with no-IL-2 mice (P<0.02 at all time points). In mice challenged with an OVA-bearing subline of the AML leukemia model C1498, IL-2 did not confer added protection from tumor challenge at 1 or 2 weeks after adoptive transfer, but gave improved survival at 4 weeks post-transfer. DISCUSSION: We conclude that exogenous IL-2 is not required for anti-tumor activity of CD3/CD28-activated CD8+ cells early after adoptive transfer, but promotes T-cell persistence that confers disease protection at more remote times.

Tiny genomes and endoreduplication in Strepsiptera.

Using flow cytometry, the genome sizes of two species of Strepsiptera were studied: that of male Caenocholax fenyesi texensis Kathirithamby & Johnston (Myrmecolacidae) at 108 Mb, which is the smallest insect genome documented to date; and those of male and female Xenos vesparum Rossi (Stylopidae), which are 1C = 130 and 133 Mb, respectively. The genome sizes of the following were analysed for comparative purposes: (a) the Hessian fly, Mayetiola destructor (Say), which was previously reported to be the smallest among insects: the male measured at 1C = 121 Mb and the female at 1C = 158 Mb; and (b) the female parasitic, haplodiploid, microhymenopteran wasp, Trichogramma brassicae Bezdenko, which measured at 1C = 246 Mb. The hosts of the strepsipterans were also measured: male Solenopsis invicta Buren, the red imported fire ant (host of male C. f. texensis), which is 1C = 753.3 Mb, and female Polistes dominulus Christ, the paper wasp (host of X. vesparum), is 1C = 301.4 Mb. Endoreduplication (4C) of the genome of the thorax of the male strepsipteran, and higher levels of endoduplication (4, 8, 16C) in the body of the larger female was observed. In contrast, little or no endoreduplication was observed, either in the Hessian fly, or in the parasitic wasp.

Correction of DiGeorge anomaly with EBV-induced lymphoma by transplantation of organ-cultured thymus and Epstein-Barr-specific cytotoxic T lymphocytes.

A young woman with DiGeorge anomaly showed normal immune tests as a child and did not experience the symptoms of profound T cell immunodeficiency. However, she had chronic pulmonary infections which led to bronchiectasis. At age 14, she developed an Epstein-Barr virus-induced lymphoma and her T cell function tests were markedly abnormal. After intensive chemotherapy, she received an organ-cultured thymus transplant but because of an abnormally high EBV DNA titer was also given autologous EBV-specific cytotoxic T cells, prepared prior to transplant. Titers fell from 80,000 genome copies/mg DNA to 2000 within 6 weeks. She was clinically well and her T cell tests improved. Sixteen months after the transplant, however, her tumor returned; EBV DNA levels had risen to 40,000 copies/mg DNA. She again received autologous EBV-specific cytotoxic T lymphocytes and valcyclovir and Cytogam as well. Her tumor resolved on this therapy and she has remained well to this date, 29 months after the recurrence. T cell tests, which had deteriorated with the recurrence of the tumor, now show normal responses. This experience records a number of unique features of thymus transplantation. This is the first recorded successful thymus transplant in a patient with partial T cell immunity and thus expands the potential of this treatment modality to patients other than infants with complete DiGeorge anomaly. The patient demonstrates cytotoxic activity against mouse cells, demonstrating the ability to respond to a new antigen which requires host antigen presenting cells. Measurement of alpha 1 TRECs (T cell receptor excision circles) shows evidence of increasing and sustained thymopoiesis since the transplant at a level consistent with the age of the transplant donor rather than that of the recipient.

Evidence for heterotypic interaction between the receptor tyrosine kinases TIE-1 and TIE-2.

The orphan receptor tyrosine kinase Tie-1 is expressed in endothelial cells and is essential for vascular development. Nothing is known about the signaling pathways utilized by this receptor. In this study we have used chimeric receptors composed of the TrkA ectodomain fused to the transmembrane and intracellular domains of Tie-1, or the related receptor Tie-2, to examine Tie-1 signaling capacity. In contrast to TrkA/Tie-2, the Tie-1 chimera was unable to phosphorylate cellular proteins or undergo autophosphorylation. Consistent with this Tie-1 exhibited negligible kinase activity. Co-immunoprecipitation analysis revealed Tie-1 was present in endothelial cells bound to Tie-2. Full-length Tie-1 and truncated receptor, formed by regulated endoproteolytic cleavage, were found to complex with Tie-2. Association was mediated by the intracellular domains of the receptors and did not require Tie-1 to be membrane-localized. Tie-1 bound to Tie-2 was not tyrosine-phosphorylated under basal conditions or following Tie-2 stimulation. This study provides the first evidence for the existence of a pre-formed complex of Tie-1 and Tie-2 in endothelial cells. The data suggest Tie-1 does not signal via ligand-induced kinase activation involving homo-oligomerization. The physical association between Tie-1 and Tie-2 is consistent with Tie-1 having a role in modulating Tie-2 signaling.

Tie-1 receptor tyrosine kinase endodomain interaction with SHP2: potential signalling mechanisms and roles in angiogenesis.

The endothelial receptor tyrosine kinase plays an essential role in vascular development where it is thought to be required for vessel maturation and stabilization. The ligands responsible for activating Tie-1, its signalling pathways and specific cellular functions are however not known. As with some other receptor tyrosine kinases, Tie-1 is subject to extracellular proteolytic cleavage generating a membrane bound receptor fragment comprising the intracellular and transmembrane domains. Here we examine the signalling potential of this Tie-1 endodomain. We show that the Tie-1 endodomain has poor ability to induce tyrosine phosphorylation. However, on formation the endodomain physically associates with a number of tyrosine phosphorylated signalling intermediates including the tyrosine phosphatase and adaptor protein SHP2. The assembly of this multimolecular complex is consistent with the endodomain having a ligand-independent signalling role in the endothelial cell. The potential roles of ectodomain cleavage and cleavage activated signalling in regulating microvessel stability in angiogenesis, vessel remodelling and regression are considered.

The risks of total pancreatectomy and splenic islet autotransplantation.

The intraportal site is the most common site for islet transplantation. Many other sites have been tried experimentally, including the spleen, which has successfully lead to insulin independence in a number of animal models. Nevertheless, there are no detailed reports of total pancreatectomy and splenic islet autotransplantation in humans. Five patients underwent total pancreatectomy and splenic islet autotransplantation for chronic pancreatitis. Four patients had a pylorus-preserving total pancreatectomy and one patient a duodenal-preserving pancreatectomy. In three cases islets were embolized into both the portal vein and spleen. Two patients received splenic islet transplants alone. Islets were transplanted by retrograde venous infusion via the short gastric veins (n = 3), splenic vein stump (n = 1), and the left gastroepiploic vein (n = 1). The total volumes of transplanted pancreatic digest in those receiving combined intraportal and splenic autografts (n = 3) were 15.8, 13.0, and 13.5 ml. The volumes in those receiving a splenic-alone autograft (n = 2) were 12.0 and 5 ml. The mean rise in portal pressure was 18 cm of water. Complications related to the splenic autograft included a wedge splenic infarct, an emergency splenectomy, and a portal vein thrombosis in one patient having a combined intraportal and splenic autograft. Two patients developed insulin independence. two patients were still insulin independent at 1-year follow-up, and all had normal HbA1c levels (mean 5.6, range 5.2-6.3). Splenic islet autotransplantation, after total pancreatectomy, does lead to insulin independence. However, in our experience the combined procedure has a high morbidity because of splenic infarction and venous thrombosis.