Identifying priority sites for whale shark ship collision management globally.

The expansion of the world's merchant fleet poses a great threat to the ocean's biodiversity. Collisions between ships and marine megafauna can have population-level consequences for vulnerable species. The Endangered whale shark (Rhincodon typus) shares a circumglobal distribution with this expanding fleet and tracking of movement pathways has shown that large vessel collisions pose a major threat to the species. However, it is not yet known whether they are also at risk within aggregation sites, where up to 400 individuals can gather to feed on seasonal bursts of planktonic productivity. These "constellation" sites are of significant ecological, socio-economic and cultural value. Here, through expert elicitation, we gathered information from most known constellation sites for this species across the world (>50 constellations and >13,000 individual whale sharks). We defined the spatial boundaries of these sites and their overlap with shipping traffic. Sites were then ranked based on relative levels of potential collision danger posed to whale sharks in the area. Our results showed that researchers and resource managers may underestimate the threat posed by large ship collisions due to a lack of direct evidence, such as injuries or witness accounts, which are available for other, sub-lethal threat categories. We found that constellations in the Arabian Sea and adjacent waters, the Gulf of Mexico, the Gulf of California, and Southeast and East Asia, had the greatest level of collision threat. We also identified 39 sites where peaks in shipping activity coincided with peak seasonal occurrences of whale sharks, sometimes across several months. Simulated collision mitigation options estimated potentially minimal impact to industry, as most whale shark core habitat areas were small. Given the threat posed by vessel collisions, a coordinated, multi-national approach to mitigation is needed within priority whale shark habitats to ensure collision protection for the species.

Species associated with whale sharks Rhincodontypus (Orectolobiformes, Rhincodontidae) in the Galapagos Archipelago.

Whale sharks Rhincodontypus frequently appear to interact or associate with other species, which vary depending on the community structure and the demographic of the whale sharks at each location globally. Here, we present the species sighted frequently around whale sharks in the Galapagos Archipelago and reported by dive guides and scientists and also in earlier publications. These associated species include cetacean species: bottlenose dolphins Tursiopstruncatus, other shark species: silky sharks Carcharhinusfalciformis, Galapagos sharks Carcharhinusgalapagensis, scalloped hammerhead sharks Sphyrnalewini, tiger sharks Galeocerdocuvier and teleost fish species: remoras Remora remora, yellowfin tuna Thunnusalbacares, almaco jacks Seriolarivoliana and black jacks Caranxlugubris. The recording of interspecies associations and interactions may lead to better understanding of the natural history of whale sharks and can show important symbiotic relationships or interdependence between different species.

Global collision-risk hotspots of marine traffic and the world's largest fish, the whale shark.

Marine traffic is increasing globally yet collisions with endangered megafauna such as whales, sea turtles, and planktivorous sharks go largely undetected or unreported. Collisions leading to mortality can have population-level consequences for endangered species. Hence, identifying simultaneous space use of megafauna and shipping throughout ranges may reveal as-yet-unknown spatial targets requiring conservation. However, global studies tracking megafauna and shipping occurrences are lacking. Here we combine satellite-tracked movements of the whale shark, Rhincodon typus, and vessel activity to show that 92% of sharks' horizontal space use and nearly 50% of vertical space use overlap with persistent large vessel (>300 gross tons) traffic. Collision-risk estimates correlated with reported whale shark mortality from ship strikes, indicating higher mortality in areas with greatest overlap. Hotspots of potential collision risk were evident in all major oceans, predominantly from overlap with cargo and tanker vessels, and were concentrated in gulf regions, where dense traffic co-occurred with seasonal shark movements. Nearly a third of whale shark hotspots overlapped with the highest collision-risk areas, with the last known locations of tracked sharks coinciding with busier shipping routes more often than expected. Depth-recording tags provided evidence for sinking, likely dead, whale sharks, suggesting substantial "cryptic" lethal ship strikes are possible, which could explain why whale shark population declines continue despite international protection and low fishing-induced mortality. Mitigation measures to reduce ship-strike risk should be considered to conserve this species and other ocean giants that are likely experiencing similar impacts from growing global vessel traffic.

Zoledronate.

Zoledronate is the most potent and most long-acting bisphosphonate in clinical use, and is administered as an intravenous infusion. Its major uses are in osteoporosis, Paget's disease, and in myeloma and cancers to reduce adverse skeletal related events (SREs). In benign disease, it is a first- or second-line treatment for osteoporosis, achieving anti-fracture efficacy comparable to that of the RANKL blocker, denosumab, over 3 years, and it reduces fracture risk in osteopenic older women. It is the preferred treatment for Paget's disease, achieving higher rates of remissions which are much more prolonged than with any other agent. Some trials have suggested that it reduces mortality, cardiovascular disease and cancer, but these findings are not consistent across all studies. It is nephrotoxic, so should not be given to those with significant renal impairment, and, like other potent anti-resorptive agents, can cause hypocalcemia in patients with severe vitamin D deficiency, which should be corrected before administration. Its most common adverse effect is the acute phase response, seen in 30-40% of patients after their first dose, and much less commonly subsequently. Clinical trials in osteoporosis have not demonstrated increases in osteonecrosis of the jaw or in atypical femoral fractures. Observational databases are currently inadequate to determine whether these problems are increased in zoledronate users. Now available as a generic, zoledronate is a cost-effective agent for fracture prevention and for management of Paget's disease, but wider provision of infusion facilities is important to increase patient access. There is a need to further explore its potential for reducing cancer, cardiovascular disease and mortality, since these effects could be substantially more important than its skeletal actions.

Correction: Association of whale sharks (Rhincodon typus) with thermo-biological frontal systems of the eastern tropical Pacific.

[This corrects the article DOI: 10.1371/journal.pone.0182599.].

Association of whale sharks (Rhincodon typus) with thermo-biological frontal systems of the eastern tropical Pacific.

Satellite tracking of 27 whale sharks in the eastern tropical Pacific, examined in relation to environmental data, indicates preferential occupancy of thermo-biological frontal systems. In these systems, thermal gradients are caused by wind-forced circulation and mixing, and biological gradients are caused by associated nutrient enrichment and enhanced primary productivity. Two of the frontal systems result from upwelling, driven by divergence in the current systems along the equator and the west coast of South America; the third results from wind jet dynamics off Central America. All whale sharks were tagged near Darwin Island, Galápagos, within the equatorial Pacific upwelling system. Occupancy of frontal habitat is pronounced in synoptic patterns of shark locations in relation to serpentine, temporally varying thermal fronts across a zonal expanse > 4000 km. 80% of shark positions in northern equatorial upwelling habitat and 100% of positions in eastern boundary upwelling habitat were located within the upwelling front. Analysis of equatorial shark locations relative to thermal gradients reveals occupancy of a transition point in environmental stability. Equatorial subsurface tag data show residence in shallow, warm (>22°C) water 94% of the time. Surface zonal current speeds for all equatorial tracking explain only 16% of the variance in shark zonal movement speeds, indicating that passive drifting is not a primary determinant of movement patterns. Movement from equatorial to eastern boundary frontal zones occurred during boreal winter, when equatorial upwelling weakens seasonally. Off Peru sharks tracked upwelling frontal positions within ~100-350 km from the coast. Off Central America, the largest tagged shark (12.8 m TL) occupied an oceanic front along the periphery of the Panama wind jet. Seasonal movement from waning equatorial upwelling to productive eastern boundary habitat is consistent with underlying trophic dynamics. Persistent shallow residence in thermo-biological frontal zones suggests the role of physical-biological interactions that concentrate food resources.

A General Strategy for Targeting Drugs to Bone.

Targeting drugs to their desired site of action can increase their safety and efficacy. Bisphosphonates are prototypical examples of drugs targeted to bone. However, bisphosphonate bone affinity is often considered too strong and cannot be significantly modulated without losing activity on the enzymatic target, farnesyl pyrophosphate synthase (FPPS). Furthermore, bisphosphonate bone affinity comes at the expense of very low and variable oral bioavailability. FPPS inhibitors were developed with a monophosphonate as a bone-affinity tag that confers moderate affinity to bone, which can furthermore be tuned to the desired level, and the relationship between structure and bone affinity was evaluated by using an NMR-based bone-binding assay. The concept of targeting drugs to bone with moderate affinity, while retaining oral bioavailability, has broad application to a variety of other bone-targeted drugs.

Whale shark (Rhincodon typus) seasonal presence, residence time and habitat use at darwin island, galapagos marine reserve.

The life history of the whale shark (Rhincodon typus), including its reproductive ecology, still remains largely unknown. Here, we present results from the first whale shark population study around Darwin Island, Galapagos Marine Reserve. Following a diversified approach we characterized seasonal occurrence, population structure and size, and described habitat use of whale sharks based on fine scale movements around the island. Whale shark presence at Darwin Island was negatively correlated with Sea Surface Temperature (SST), with highest abundance corresponding to a cool season between July and December over six years of monitoring. From 2011 to 2013 we photo-identified 82 whale sharks ranging from 4 to 13.1 m Total Length (TL). Size distribution was bimodal, with a great majority (91.5%) of adult female individuals averaging 11.35 m±0.12 m (TL±SE), all but one showing signs of a potential pregnancy. Population dynamics models for apparently pregnant sharks estimated the presence of 3.76±0.90 (mean ± SE) sharks in the study area per day with an individual residence time of 2.09±0.51 (mean ± SE) days. Movement patterns analysis of four apparently pregnant individuals tracked with acoustic tags at Darwin Island revealed an intense use of Darwin's Arch, where no feeding or specific behavior has been recorded, together with periodic excursions around the island's vicinity. Sharks showed a preference for intermediate depths (20-30 m) with occasional dives mostly to mid-water, remaining the majority of their time at water temperatures between 24-25°C. All of our results point to Darwin Island as an important stopover in a migration, possibly with reproductive purposes, rather than an aggregation site. Current studies carried out in this area to investigate regional scale movement patterns may provide essential information about possible pupping grounds for this enigmatic species.

The chemistry of bisphosphonates: from antiscaling agents to clinical therapeutics.

In the early 1960s, inorganic pyrophosphate (PPi) was found to be present in body fluids and to act as a natural inhibitor of calcification by its interaction with hydroxyapatite. In addition to inhibiting the formation of calcium phosphate, PPi also inhibited dissolution of hydroxyapatite crystals, which made it interesting for pharmacologic applications in the treatment of diseases associated with excessive bone resorption. However, PPi is metabolically unstable because of rapid hydrolysis of the P-O-P backbone by hydrolytic enzymes in the gastrointestinal tract. In the search for more stable analogues of PPi, attention turned to the chemical class of bisphosphonates (BPs). The first BPs were synthesized in the 19th century and widely used for industrial applications. Bisphosphonates are formally derived from PPi by replacement of the bridging oxygen atom by a carbon atom, resulting in a P-C-P moiety that is resistant to hydrolysis. In addition to its decisive role in stability, the central carbon atom also provides an attachment point for 2 additional substituents (R¹ and R²). While R¹ is preferentially a hydroxy group, allowing such derivatives to act as powerful tridentate ligands for calcium (bone hook), R² is mainly responsible for antiresorptive potency. The clinically available BPs can be divided into 2 subclasses based on their structure and molecular mechanism of action. The simple, non-nitrogen-containing derivatives can be incorporated into non-hydrolyzable cytotoxic ATP analogues. The more potent nitrogen-containing BPs inhibit FPPS, a key enzyme in the mevalonate pathway. Details of this crucial molecular interaction have recently been elucidated. Members of this class have a wide therapeutic window between therapeutic inhibition of bone resorption and undesired inhibition of bone formation, and several have found widespread use for the treatment of benign and malignant bone disease.

The backbone of progress--preclinical studies and innovations with zoledronic acid.

Bisphosphonates (BPs) are antiresorptive agents that block pathologic bone resorption by inhibiting osteoclast function and later inducing osteoclast apoptosis. These agents localize to bone and break the vicious cycle of bone resorption that results from cross-stimulation between cancer cells and the bone remodeling cells, thereby reducing cancer-induced osteolysis and the tumor burden in bone. Thus nitrogen-containing BPs (N-BPs) have well established clinical benefits in the treatment of bone metastases from solid tumors and bone lesions from multiple myeloma. Preclinical data indicate that N-BPs, especially zoledronic acid (ZOL), can exert antimyeloma activity both in vitro and in vivo. Studies show that N-BPs can inhibit multiple intracellular processes essential for cancer cell proliferation and invasion and induce apoptosis. Furthermore, clinically relevant doses of N-BPs inhibit tumor-associated angiogenesis and can modulate macrophage phenotype in vivo, which is likely to contribute to anticancer effects.

Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue.

The bisphosphonate zoledronic acid has antimyeloma activity in vivo by inhibition of protein prenylation.

Nitrogen-containing bisphosphonates (N-BPs) are effective antiosteolytic agents in patients with multiple myeloma. Preclinical studies have also demonstrated that these agents have direct antitumor effects in vitro and can reduce tumor burden in a variety of animal models, although it is not clear whether such effects are caused by direct actions on tumor cells or by inhibition of bone resorption. N-BPs prevent bone destruction in myeloma by inhibiting the enzyme farnesyl pyrophosphate synthase in osteoclasts, thereby preventing the prenylation of small GTPase signaling proteins. In this study, utilizing a plasmacytoma xenograft model without complicating skeletal lesions, treatment with zoledronic acid (ZOL) led to significant prolongation of survival in severe combined immunodeficiency mice inoculated with human INA-6 plasma cells. Following treatment with a clinically relevant dose of ZOL, histological analysis of INA-6 tumors from the peritoneal cavity revealed extensive areas of apoptosis associated with poly (ADP-ribose) polymerase cleavage. Furthermore, Western blot analysis of tumor homogenates demonstrated the accumulation of unprenylated Rap1A, indicative of the uptake of ZOL by nonskeletal tumors and inhibition of farnesyl pyrophosphate synthase. These studies provide, for the first time, clear evidence that N-BPs have direct antitumor effects in plasma cell tumors in vivo and this is executed by a molecular mechanism similar to that observed in osteoclasts.

Long-term protective effects of zoledronic acid on cancellous and cortical bone in the ovariectomized rat.

UNLABELLED: Current bisphosphonate therapies effectively prevent bone loss in postmenopausal women. We studied the effect of a single intravenous dose of ZOL in ovariectomized rats. Protection from bone loss was dose dependent, lasting for up to 32 weeks, supporting the rationale for an annual intravenous dosing regimen of ZOL for treatment of postmenopausal osteoporosis. INTRODUCTION: Once-yearly dosing with zoledronic acid (ZOL) 5 mg can increase BMD and reduce fracture rate in postmenopausal women with low BMD. The primary objective of this study was to determine the duration of bone protective effects of a single dose of ZOL in ovariectomized rats, an animal model of postmenopausal osteopenia. Secondary objectives were to determine the effects on bone turnover and mechanical properties. MATERIALS AND METHODS: Female Wistar rats (10 per group) received single intravenous doses of ZOL 0.8, 4, 20, 100, or 500 microg/kg, alendronate 200 microg/kg, or isotonic saline 4 days before bilateral ovariectomy. Sham-operated controls were pretreated with saline. Mass and density of cancellous and cortical bone (pQCT) were measured at 4-wk intervals for 32 wk. Bone architecture (microCT), bone formation dynamics (fluorochrome label-based histomorphometry), and biomechanical strength in compression testing were also assessed at 32 wk. RESULTS: Ovariectomy-associated BMD loss was significantly attenuated for 32 wk by ZOL >or=4 microg/kg for total BMD, ZOL >or=20 microg/kg for cortical BMD, and ZOL >or=4 microg/kg for cancellous BMD (p < 0.01 versus ovariectomized controls). Alendronate 200 microg/kg was of equivalent potency to ZOL 20 microg/kg. Ovariectomy-associated decreases in trabecular architectural parameters were dose-dependently attenuated by ZOL. Alendronate 200 microg/kg was equivalent to ZOL 20 microg/kg. The bone resorption marker TRACP5b indicated transient suppression of elevated osteoclast activity by ZOL relative to OVX-rats even at the lowest dose of 0.8 microg/kg, whereas at 100-500 microg/kg, the effect was significant relative to the OVX control for the entire duration of the study of 32 wk. Bone formation parameters were not significantly affected by ZOL 20 microg/kg but were significantly reduced by ZOL 100-500 microg/kg. Alendronate 200 microg/kg was equivalent to ZOL 100 microg/kg. ZOL produced dose-related improvements in bone strength parameters after ovariectomy. Alendronate 200 microg/kg was of similar potency to ZOL 20 microg/kg. CONCLUSIONS: The duration and magnitude of the bone-protecting effect of a single intravenous dose of ZOL in ovariectomized rats is dose dependent and lasts for up to 32 wk. Compared with alendronate, ZOL shows 10-fold higher potency in preventing bone loss. These data support the use of an annual intravenous ZOL dosing regimen for the treatment of osteoporosis.

Rapid adhesion of Stagonospora nodorum spores to a hydrophobic surface requires pre-formed cell surface glycoproteins.

Adhesion of fungal pathogens to leaf surfaces is an important first step in the infection process. Previous work on Stagonospora nodorum, a major necrotrophic pathogen of wheat and other cereals, has shown that conidia attach rapidly to a hydrophobic surface and this is followed by the active secretion of extracellular matrix material to consolidate adhesion. In this paper the role of pre-formed spore surface glycoproteins in the rapid adhesion of S. nodorum conidia to an artificial surface, polystyrene, has been investigated. Sodium dodecyl sulphate (SDS) and the enzymes chitinase and lyticase have been used to release cell wall glycoproteins from spores and these have been identified using SDS polyacrylamide gel electrophoresis (PAGE) and Western blotting. Labelling with fluorescently tagged lectins has also been used to study the spore surface. The results show that there are a small number of glycoproteins non-covalently and covalently attached to other components in the spore wall, which is not a uniform structure. The effects of proteases, lectins, and other treatments of spores in an adhesion assay have been used to show that pre-formed glycoproteins are involved in rapid adhesion to a hydrophobic surface. There is also evidence for a rapid release of glycoproteins by spores that is also involved in adhesion and this is not an active process.

Structural basis for the exceptional in vivo efficacy of bisphosphonate drugs.

To understand the structural basis for bisphosphonate therapy of bone diseases, we solved the crystal structures of human farnesyl pyrophosphate synthase (FPPS) in its unliganded state, in complex with the nitrogen-containing bisphosphonate (N-BP) drugs zoledronate, pamidronate, alendronate, and ibandronate, and in the ternary complex with zoledronate and the substrate isopentenyl pyrophosphate (IPP). By revealing three structural snapshots of the enzyme catalytic cycle, each associated with a distinct conformational state, and details about the interactions with N-BPs, these structures provide a novel understanding of the mechanism of FPPS catalysis and inhibition. In particular, the accumulating substrate, IPP, was found to bind to and stabilize the FPPS-N-BP complexes rather than to compete with and displace the N-BP inhibitor. Stabilization of the FPPS-N-BP complex through IPP binding is supported by differential scanning calorimetry analyses of a set of representative N-BPs. Among other factors such as high binding affinity for bone mineral, this particular mode of FPPS inhibition contributes to the exceptional in vivo efficacy of N-BP drugs. Moreover, our data form the basis for structure-guided design of optimized N-BPs with improved pharmacological properties.

A single intravenous administration of zoledronic acid prevents the bone loss and mechanical compromise induced by aromatase inhibition in rats.

Recent evidence has demonstrated that long-term estrogen deprivation using aromatase inhibitor therapy in postmenopausal women with breast cancer results in bone loss and increased fracture risk. Bisphosphonates are potent inhibitors of bone resorption and have demonstrated efficacy in preventing bone loss in postmenopausal women with low bone mineral density (BMD) and in patients with breast cancer receiving estrogen deprivation therapy. Therefore, this study investigated the effects of the bisphosphonate zoledronic acid on BMD and bone strength in rats treated with the aromatase inhibitor, letrozole. Peripheral quantitative computed tomography demonstrated that treatment of rats with daily oral letrozole (1 mg/kg) induced significant bone loss and cortical thinning compared with control animals (P < 0.01). A single prior intravenous dose of zoledronic acid dose dependently protected against letrozole-induced bone loss and cortical thinning, with the highest evaluated dose (20 microg/kg) resulting in BMD values that were not significantly different from controls over the 24 weeks of letrozole treatment. Furthermore, biomechanical testing of the distal femoral metaphysis demonstrated that zoledronic acid (20 microg/kg) significantly prevented the decrease in stiffness and elastic modulus induced by letrozole treatment. Taken together, these data support the use of zoledronic acid for the prevention of bone loss in women with breast cancer receiving aromatase inhibitor therapy.

Skeletal complications of prostate cancer: pathophysiology and therapeutic potential of bisphosphonates.

Patients with prostate cancer are at risk for skeletal complications resulting from treatment-induced bone loss and for bone metastases. The therapeutic potential of zoledronic acid for the treatment of prostate cancer has been demonstrated in both preclinical and clinical studies. In patients receiving androgen-deprivation therapy, zoledronic acid increases bone mineral density, and, in patients with bone metastases, it reduces the incidence of skeletal complications. Preclinical studies have also demonstrated the antitumor potential of bisphosphonates. Specifically, zoledronic acid inhibits proliferation and induces apoptosis of human prostate cancer cell lines in vitro and has enhanced antitumor activity when combined with taxanes. Animal models have further shown that bisphosphonates decrease tumor-induced osteolysis and reduce skeletal tumor burden. In a model of prostate cancer, zoledronic acid significantly inhibited growth of both osteolytic and osteoblastic tumors and reduced circulating levels of prostate-specific antigen. These studies suggest that zoledronic acid has the potential to inhibit bone metastasis and bone lesion progression in patients with prostate cancer.

Use of REMI and Agrobacterium-mediated transformation to identify pathogenicity mutants of the biocontrol fungus, Coniothyrium minitans.

Restriction enzyme mediated integration (REMI) and Agrobacterium-mediated transformation (ATMT) were used to transform protoplasts or germinated conidia of the mycoparasite Coniothyrium minitans to hygromycin resistance. Using REMI, up to 32 transformants mug DNA(-1) were obtained, while 37.8 transformants 5 x 10(5) germlings(-1) were obtained using ATMT. Single-copy integrations occurred in 8% and 40% of REMI and ATMT transformants, respectively. A novel microtitre plate-based test was developed to expedite screening of 4000 REMI and ATMT C. minitans transformants. Nine pathogenicity mutants that displayed reduced or no pathogenicity on sclerotia of Sclerotinia sclerotiorum were identified.