Testudinimyces gracilis gen. nov, sp. nov. and Astrotestudinimyces divisus gen. nov, sp. nov., two novel, deep-branching anaerobic gut fungal genera from tortoise faeces.

The anaerobic gut fungi (AGF, Neocallimastigomycota) represent a basal zoosporic phylum within the kingdom Fungi. Twenty genera are currently described, all of which were isolated from the digestive tracts of mammalian herbivores. Here, we report on the isolation and characterization of novel AGF taxa from faecal samples of tortoises. Twenty-nine fungal isolates were obtained from seven different tortoise species. Phylogenetic analysis using the D1/D2 region of the LSU rRNA gene, ribosomal internal transcribed spacer 1, and RNA polymerase II large subunit grouped all isolates into two distinct, deep-branching clades (clades T and B), with a high level of sequence divergence to their closest cultured relative (Khoyollomyces ramosus). Average amino acid identity values calculated using predicted peptides from the isolates' transcriptomes ranged between 60.80-66.21  % (clade T), and 61.24-64.83  % (clade B) when compared to all other AGF taxa; values that are significantly below recently recommended thresholds for genus (85%) and family (75%) delineation in the Neocallimastigomycota. Both clades displayed a broader temperature growth range (20-45 °C, optimal 30 °C for clade T, and 30-42 °C, optimal 39 °C for clade B) compared to all other AGF taxa. Microscopic analysis demonstrated that strains from both clades produced filamentous hyphae, polycentric rhizoidal growth patterns, and monoflagellated zoospores. Isolates in clade T were characterized by the production of unbranched, predominantly narrow hyphae, and small zoospores, while isolates in clade B were characterized by the production of multiple sporangiophores and sporangia originating from a single central swelling resulting in large multi-sporangiated structures. Based on the unique phylogenetic positions, AAI values, and phenotypic characteristics, we propose to accommodate these isolates into two novel genera (Testudinimyces and Astrotestudinimyces), and species (T. gracilis and A. divisus) within the order Neocallimastigales. The type species are strains T130AT (T. gracilis) and B1.1T (A. divisus).

Prevalence, intensity and pathology of the nasal parasite Nasicola hogansi in Atlantic bluefin tuna (Thunnus thynnus).

Ectoparasitic flatworms of Nasicola (Monogenoidea: Capsalidae), which infect nasal epithelium of true tunas (Thunnus spp.), are not well studied, nor have their impacts on the host's olfactory organ been evaluated. Infections of Nasicola hogansi on Atlantic bluefin tuna, Thunnus thynnus, were investigated with emphasis on the relationship between infection prevalence, abundance and mean intensity with bluefin tuna size, sex, body condition and capture month, as well as histopathological effects. Commercially caught Atlantic bluefin tuna (n = 161, 185-305 cm curved fork length) from the Gulf of Maine were sampled during June through August 2009 for infections by N. hogansi. A total of 247 specimens of N. hogansi were collected, with a prevalence of 45.3%, mean abundance of 1.57 (CI: 1.21-2.03) and mean intensity of 3.45 (CI: 2.91-4.22). Neither fish sex nor landing month had a significant effect on parasite parameters. Larger and better-conditioned Atlantic bluefin tuna had a higher mean intensity of infection. Pathology associated with infection by N. hogansi included extensive necrosis, sloughing of the nasal epithelium and associated inflammation of underlying connective tissues. Further epidemiological and pathological study of this host-parasite system is warranted since impaired olfaction, if present, could adversely affect spawning and migration of this top ocean predator.

Sailfish migrations connect productive coastal areas in the West Atlantic Ocean.

Isla Mujeres, Mexico is home to one of the most well-known aggregations of sailfish. Despite its fisheries prominence, little is known about this sailfish assemblage, or its relationship to other aggregation sites in the western Atlantic. In January 2012, April 2013 and 2014, we deployed 34 popup satellite archival tags on sailfish in order to study their behavior, population connectivity and biophysical interactions. Sailfish were monitored for up to one year, and displayed (1) predominantly shelf associated activity (2) occupancy of the Yucatán Current near Isla Mujeres for up to five months and (3) subsequent dispersals from the Yucatán to productive coastal areas in the Gulf of Mexico, the Caribbean Sea and along the South American coast. Tagged sailfish occupied a median temperature of 26.4°C (interquartile range, IQR = 2.5 °C; range = 12.3-33.3 °C) and median depth of 4.4 m (IQR = 19 m; range = 0-452 m). Diel activity was present and individuals made distinctive descents before sunrise and sunset. Tracking missions of sufficient duration (~1 year) revealed previously undetected connectivity between western Atlantic sailfish fisheries and pelagic longline catches, and highlighted how fishery independent tagging can improve understanding of sailfish migrations and behavior for assessment and management.

Declining Mercury Concentrations in Bluefin Tuna Reflect Reduced Emissions to the North Atlantic Ocean.

Tunas are apex predators in marine food webs that can accumulate mercury (Hg) to high concentrations and provide more Hg (∼40%) to the U.S population than any other source. We measured Hg concentrations in 1292 Atlantic bluefin tuna (ABFT, Thunnus thynnus) captured in the Northwest Atlantic from 2004 to 2012. ABFT Hg concentrations and variability increased nonlinearly with length, weight, and age, ranging from 0.25 to 3.15 mg kg-1, and declined significantly at a rate of 0.018 ± 0.003 mg kg-1 per year or 19% over an 8-year period from the 1990s to the early 2000s. Notably, this decrease parallels comparably reduced anthropogenic Hg emission rates in North America and North Atlantic atmospheric Hg0 concentrations during this period, suggesting that recent efforts to decrease atmospheric Hg loading have rapidly propagated up marine food webs to a commercially important species. This is the first evidence to suggest that emission reduction efforts have resulted in lower Hg concentrations in large, long-lived fish.

Evidence for a boat conformation at the transition state of GH76 α-1,6-mannanases--key enzymes in bacterial and fungal mannoprotein metabolism.

α-Mannosidases and α-mannanases have attracted attention for the insight they provide into nucleophilic substitution at the hindered anomeric center of α-mannosides, and the potential of mannosidase inhibitors as cellular probes and therapeutic agents. We report the conformational itinerary of the family GH76 α-mannanases studied through structural analysis of the Michaelis complex and synthesis and evaluation of novel aza/imino sugar inhibitors. A Michaelis complex in an (O) S2 conformation, coupled with distortion of an azasugar in an inhibitor complex to a high energy B2,5 conformation are rationalized through ab initio QM/MM metadynamics that show how the enzyme surface restricts the conformational landscape of the substrate, rendering the B2,5 conformation the most energetically stable on-enzyme. We conclude that GH76 enzymes perform catalysis using an itinerary that passes through (O) S2 and B2,5 (≠) conformations, information that should inspire the development of new antifungal agents.

Fibronectin conformation regulates the proangiogenic capability of tumor-associated adipogenic stromal cells.

BACKGROUND: Changes in fibronectin (Fn) matrix remodeling contribute to mammary tumor angiogenesis and are related to altered behavior of adipogenic stromal cells; yet, the underlying mechanisms remain unclear due in part to a lack of reductionist model systems that allow the inherent complexity of cell-derived extracellular matrices (ECMs) to be deciphered. In particular, breast cancer-associated adipogenic stromal cells not only enhance the composition, quantity, and rigidity of deposited Fn, but also partially unfold these matrices. However, the specific effect of Fn conformation on tumor angiogenesis is undefined. METHODS: Decellularized matrices and a conducting polymer device consisting of poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS) were used to examine the effect of Fn conformation on the behavior of 3T3-L1 preadipocytes. Changes in cell adhesion and proangiogenic capability were tested via cell counting and by quantification of vascular endothelial growth factor (VEGF) secretion, respectively. Integrin-blocking antibodies were utilized to examine varied integrin specificity as a potential mechanism. RESULTS: Our findings suggest that tumor-associated partial unfolding of Fn decreases adhesion while enhancing VEGF secretion by breast cancer-associated adipogenic precursor cells, and that altered integrin specificity may underlie these changes. CONCLUSIONS AND GENERAL SIGNIFICANCE: These results not only have important implications for our understanding of tumorigenesis, but also enhance knowledge of cell-ECM interactions that may be harnessed for other applications including advanced tissue engineering approaches. This article is part of a Special Issue entitled Organic Bioelectronics - Novel Applications in Biomedicine.

Religious and spiritual issues in DSM-5: matters of the mind and searching of the soul.

Religion, spirituality, and psychiatric illnesses share a complex relationship in the realm of diagnosis. Historically, however, these three constructs have existed in a very peripheral place in the diagnostic taxonomy for psychiatry in the United States. Given the important role that spirituality and religion play for many people in the experiences of coping with health and illness, it seems odd that such important elements are in the margins of the powerful and commanding nosology of the DSM. Explanations for understanding the glaring absence are complex and impacted by some very powerful political and sociological forces, including contributory elements from within the mental health disciplines. This article invites the reader to explore salient issues in the emergence of a broader recognition of religion, spirituality and psychiatric diagnosis in the DSM-5.

Implanted adipose progenitor cells as physicochemical regulators of breast cancer.

Multipotent adipose-derived stem cells (ASCs) are increasingly used for regenerative purposes such as soft tissue reconstruction following mastectomy; however, the ability of tumors to commandeer ASC functions to advance tumor progression is not well understood. Through the integration of physical sciences and oncology approaches we investigated the capability of tumor-derived chemical and mechanical cues to enhance ASC-mediated contributions to tumor stroma formation. Our results indicate that soluble factors from breast cancer cells inhibit adipogenic differentiation while increasing proliferation, proangiogenic factor secretion, and myofibroblastic differentiation of ASCs. This altered ASC phenotype led to varied extracellular matrix (ECM) deposition and contraction thereby enhancing tissue stiffness, a characteristic feature of breast tumors. Increased stiffness, in turn, facilitated changes in ASC behavior similar to those observed with tumor-derived chemical cues. Orthotopic mouse studies further confirmed the pathological relevance of ASCs in tumor progression and stiffness in vivo. In summary, altered ASC behavior can promote tumorigenesis and, thus, their implementation for regenerative therapy should be carefully considered in patients previously treated for cancer.

Stiffness of photocrosslinked RGD-alginate gels regulates adipose progenitor cell behavior.

Adipose progenitor cells (APCs) are widely investigated for soft tissue reconstruction following tumor resection; however, the long-term success of current approaches is still limited. In order to develop clinically relevant therapies, a better understanding of the role of cell-microenvironment interactions in adipose tissue regeneration is essential. In particular, the effect of extracellular matrix (ECM) mechanics on the regenerative capability of APCs remains to be clarified. We have used artificial ECMs based on photocrosslinkable RGD-alginate to investigate the adipogenic and pro-angiogenic potential of 3T3-L1 preadipocytes as a function of matrix stiffness. These hydrogels allowed us to decouple matrix stiffness from changes in adhesion peptide density or extracellular Ca(2+) concentration and provided a physiologically relevant 3D culture context. Our findings suggest that increased matrix rigidity promotes APC self-renewal and angiogenic capacity, whereas, it inhibits adipose differentiation. Collectively, this study advances our understanding of the role of ECM mechanics in adipose tissue formation and vascularization and will aid in the design of efficacious biomaterial scaffolds for adipose tissue engineering applications.

Tissue-engineered three-dimensional tumor models to study tumor angiogenesis.

Cell-microenvironment interactions play a critical role in the transformation of normal cells into cancer; however, the underlying mechanisms and effects are far from being well understood. Tissue Engineering provides innovative culture tools and strategies to study tumorigenesis under pathologically relevant culture conditions. Specifically, integration of biomaterials, scaffold fabrication, and micro/nano-fabrication techniques offers great promise to reveal the dynamic role of chemical, cell-cell, cell-extracellular matrix, and mechanical interactions in the pathogenesis of cancer. Due to the central importance of blood vessel formation in tumor growth, progression, and drug response, this review will discuss specific design parameters for the development of culture microenvironments to study tumor angiogenesis. Tumor engineering approaches have the potential to revolutionize our understanding of cancer, provide new platforms for testing of anti-cancer drugs, and may ultimately result in improved treatment strategies.

Parylene peel-off arrays to probe the role of cell-cell interactions in tumour angiogenesis.

Microenvironmental conditions impact tumour angiogenesis, but the role of cell-cell interactions in modulating the angiogenic capability of tumour cells is not well understood. We have microfabricated a peel-off cell-culture array (PeelArray) chip to spatiotemporally control interactions between tumour cells in a large array format and to analyse angiogenic factor secretion in response to these conditions. The PeelArray chip consists of a polyethylene glycol (PEG) treated glass coverslip coated with a parylene-C template that can be easily peeled off to selectively micropattern biomolecules and cells. We have designed the PeelArray chip to reproducibly deposit large uniform arrays of isolated single cells or isolated cell clusters on fibronectin features of defined surface areas. We have utilised this microfabricated culture system to study the secretion of angiogenic factors by tumour cells, in the presence or absence of cell-cell contact as controlled by micropatterning. Our results indicate that cell-cell interactions play a synergistic role in regulating the expression of angiogenic factors (i.e., vascular endothelial growth factor [VEGF] and interleukin-8 [IL-8]) in various cancer cell lines, independent of other more complex microenvironmental cues (e.g. hypoxia). Our PeelArray chip is a simple and adaptable micropatterning method that enables quantitative profiling of protein secretions and hence, a better understanding of the mechanisms by which cell-cell interactions regulate tumour cell behaviour and angiogenesis.

Membrane surface dynamics of DNA-threaded nanopores revealed by simultaneous single-molecule optical and ensemble electrical recording.

We describe a method for simultaneous single-molecule optical and electrical characterization of membrane-based sensors that contain ion-channel nanopores. The technique is used to study the specific and nonspecific interactions of streptavidin-capped DNA polymers with lipid bilayers composed of diphytanoyl phosphatidylcholine and diphytanoyl phosphatidylglycerol. Biotinylated DNA that is bound to fluorescently labeled streptavidin is electrophoretically driven into, or away from, the lumen of alpha hemolysin (alphaHL) ion channels by an external electric field. Confocal microscopy simultaneously captures single-molecule fluorescence dynamics from the membrane interface at different applied potentials. Fluorescence correlation analysis is used to determine the surface number density and diffusion constant of membrane-associated complexes. The dual optical and electrical approach can detect membrane-associated species at a surface coverage below 10(-5) monolayers of streptavidin, a sensitivity that surpasses most other in vitro surface analysis techniques. By comparing the change in transmembrane current to the number of fluorescent molecules leaving the bilayer when the electrical potential is reversed, we demonstrate the general utility of the approach within the context of nanopore-based sensing and discuss a mechanism by which DNA-streptavidin complexes can be nonspecifically retained at the membrane interface.