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 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.

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.

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.

A naturally occurring soluble isoform of murine Fas generated by alternative splicing.

We report a soluble isoform of mouse Fas, which is generated by alternative splicing of Fas mRNA to a newly identified exon located between exons 2 and 3 of the previously published Fas sequence. This splicing event creates a novel Fas transcript, Fas beta, with the potential to encode a truncated form of the extracellular domain, termed Fas B. In vitro, P815 mastocytoma cells transfected with Fas B become resistant to Fas ligand-induced apoptosis, and the resistance is mediated by a secreted product of the transfected cells. In vivo, Fas beta mRNA expression is correlated inversely with apoptosis among subsets of intrahepatic T lymphocytes, a cell population in which activation-induced T cell apoptosis occurs. We propose that Fas B is a new cytokine that acts physiologically to limit apoptosis induced by Fas ligand.

T cells with gamma/delta T cell receptors (TCR) of intestinal type are preferentially expanded in TCR-alpha-deficient lpr mice.

Fas-mediated apoptosis is essential for activation-induced cell death of alpha/beta T cells, but it is not clear what role, if any, it plays in regulating other components of the immune system. To study the role of Fas in gamma/delta T cell development, Fas-deficient lpr mice were bred with T cell receptor alpha gene-ablated (TCR-alpha-/-) mice to generate mice deficient in one or both genes. The TCR-alpha-/-, lpr/lpr mice had a nearly 10-fold increase in total lymph node cell (LNC) number compared with Fas-intact TCR-alpha-/- mice, because of expansion of TCR-gamma/delta+ and TCR-beta+ cells. In Fas-intact TCR-alpha-/- mice, approximately one third of the LNCs expressed TCR-gamma/delta. These were evenly divided between the CD4-, CD8-alpha+ and the CD4-, CD8- subsets, and rarely expressed the B220 epitope of CD45. In contrast, in TCR-alpha-/-, lpr/lpr mice, TCR-gamma/delta+ cells comprised half of the LNCs and were primarily CD4-, CD8-, and B220+. Moreover, Fas deficiency in TCR-alpha-/- mice caused a preferential expansion of gamma/delta T cells expressing variable region genes characteristic of intestinal intraepithelial lymphocytes. These results demonstrate a role for Fas in regulating the gamma/delta T cell contribution to peripheral lymph nodes. This mechanism may be most important in limiting the access of activated intestinal intraepithelial lymphocytes to the peripheral lymphoid system.

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.

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.

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.

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.

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."

An investigation into the distribution of different collagen types within adult and juvenile porcine pancreata.

One of the difficulties of porcine islet isolation is their fragility during collagenase digestion. The object of this study was to determine the distribution of 4 different collagen types within adult and juvenile porcine pancreata. Five different areas from each pancreas were analysed by light microscopy. The distribution of collagen types I, IV, V and VI were measured within the interlobar, intralobular (acinar), peri-insular and intra-islet regions. Their was an abundance of collagen type VI compared to I, IV and V in both the interlobar and intralobular septa in both juveniles (P<0.001) and adults (P<0.001). The periinsular collagen content also showed diversity. This was mainly attributable to the distribution of collagen type I (weak) and type VI (intense) in both adults and juveniles. In general, the peri-insular capsule was fragmentary and contained less than 50% of the total islet circumference. The latter finding taken together with the distributions of different collagen types can partly explain some of the variability of porcine islet isolation.

Different treatment regimens to reduce microbial contamination of purified porcine islets of Langerhans.

BACKGROUND: Different treatment regimens for minimizing microbial contamination of purified porcine islets were analyzed. METHODS: Purified islets, prepared from abattoir-excised pancreata (n=26), were cultured in Hams F-12 with or without antibiotics after islet isolation. Aliquots of pancreas transport media were all contaminated with microbes (100%). Of those islets cultured with no antibiotics, 70% were contaminated, compared with 42% of the islets cultured in penicillin and streptomycin (P/S) (P>0.05). A further 20 consecutive pancreata were then randomized to saline washing (N=10) before islet isolation. Islets were then cultured in either Hams F-12 with P/S (groups C and E) or Hams F-12 with gentamicin, penicillin, and amphotericin B (G/P/A) (groups D and F). RESULTS: None of those purified islets prepared from washed pancreata and then undergoing culture in Hams F-12 with G/P/A (group F) were contaminated. This compared with 30% contamination when islets were cultured in P/S (group E). Neither antibiotic regimen compromised stimulated insulin release (P=0.13). CONCLUSIONS: In conclusion pancreas washing in saline and culture in G/P/A was shown to eradicate detectable microbial contamination of purified porcine islets isolated from abattoir-excised pancreata.

A preliminary study of the activation of endogenous pancreatic exocrine enzymes during automated porcine islet isolation.

The activation of endogenous pancreatic enzymes during automated pancreas digestion may be detrimental to islet isolation. In this report we assessed the activation of trypsin, chymotrypsin, elastase, carboxypeptidases A and B, phospholipase A2, and lipase using a porcine model. Four islet isolations were examined. Duplicate aliquots were taken from the automated circuit at 5-min time intervals up to the completion of pancreas digestion (approx 60 min). One aliquot was activated in vitro with exogenous trypsin in order to convert the enzymes into their active non-"proform," with the exception of trypsinogen, which was activated with exogenous enterokinase. This was done to assess the percentage activation of each individual enzyme (total potentially activatable enzyme release). The extent of activation between isolations was extremely variable. During the closed (recirculating) circuit phase of pancreas digestion there were both gradual and rapid increases in the levels of enzymes released. Peak activity of enzyme activation varied from 13 to 30 min; similarly, total potentially activatable peaks occurred between 13 and 38 min. Lipase and carboxypeptidase B showed greater than 70% activation, chymotrypsin, carboxypeptidase A, and phospholipase A2 between 50% and 70% activation, and trypsin and elastase less than 20%. There were up to 30-fold differences between the four islet preparations. In summary, it is unlikely that poor islet yields are soley explained by variations between collagenases; the variable activation of endogenous pancreatic exocrine enzymes is also likely to be influential to porcine islet yields.

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.

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.

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.