'Phototaxis' in the absence of light? Locomotory patterns in unionid mussels.

Locomotion in benthic invertebrates can strongly affect habitat selection and ecosystem nutrient cycling. In the case of freshwater mussels, the drivers of locomotion are largely unresolved. Our aim was to assess the influence of light presence and intensity on the locomotory behaviour of freshwater mussels in controlled laboratory experiments. The species investigated in our study were Anodonta anatina and Unio pictorum, two widely distributed mussels in European lentic and lotic inland waters. At low algal concentrations, known to be associated with more frequent locomotory activities, we found that both species moved primarily in the absence of light (72.7% of all movements across experiments). However, the movements of both species were directed towards the light source, resembling a net-positive 'phototactic' response but in the absence of light. The distance to the light source, which was negatively correlated to light intensity, had a positive effect on the distance covered in locomotory activities by A. anatina but not by U. pictorum. Intraspecific variation in shell size had no impact on movement distance, indicating that the energetic costs of movement were not a limiting factor. We suggest that the observed movement towards brighter locations helps to enhance food quantity and quality, whilst movement in darkness mitigates predation risks.

The functional nasal anatomy of the pike, Esox lucius L.

Olfactory flow in fishes is a little-explored area of fundamental and applied importance. We investigated olfactory flow in the pike, Esox lucius, because it has an apparently simple and rigid nasal region. We characterised olfactory flow by dye visualisation and computational fluid dynamics, using models derived from X-ray micro-computed tomography scans of two preserved specimens. An external current induced a flow of water through the nasal chamber at physiologically relevant Reynolds numbers (200-300). We attribute this externally-induced flow to: the location of the incurrent nostril in a region of high static pressure; the nasal bridge deflecting external flow into the nasal chamber; an excurrent nostril normal to external flow; and viscous entrainment. A vortex in the incurrent nostril may be instrumental in viscous entrainment. Flow was dispersed over the olfactory sensory surface when it impacted on the floor of the nasal chamber. Dispersal may be assisted by: the radial array of nasal folds; a complementary interaction between a posterior nasal fold and the ventral surface of the nasal bridge; and the incurrent vortex. The boundary layer could delay considerably (up to ~ 3 s) odorant transport from the external environment to the nasal region. The drag incurred by olfactory flow was almost the same as the drag incurred by models in which the nasal region had been replaced by a smooth surface. The boundary layer does not detach from the nasal region. We conclude that the nasal bridge and the incurrent vortex are pivotal to olfaction in the pike.

Cochlear protein biomarkers as potential sites for targeted inner ear drug delivery.

The delivery of therapies to the cochlea is notoriously challenging. It is an organ protected by a number of barriers that need to be overcome in the drug delivery process. Additionally, there are multiple sites of possible damage within the cochlea. Despite the many potential sites of damage, acquired otologic insults preferentially damage a single location. While progress has been made in techniques for inner ear drug delivery, the current techniques remain non-specific and our ability to deliver therapies in a cell-specific manner are limited. Fortunately, there are proteins specific to various cell-types within the cochlea (e.g., hair cells, spiral ganglion cells, stria vascularis) that function as biomarkers of site-specific damage. These protein biomarkers have potential to serve as targets for cell-specific inner ear drug delivery. In this manuscript, we review the concept of biomarkers and targeted- inner ear drug delivery and the well-characterized protein biomarkers within each of the locations of interest within the cochlea. Our review will focus on targeted drug delivery in the setting of acquired otologic insults (e.g., ototoxicity, noise-induce hearing loss). The goal is not to discuss therapies to treat acquired otologic insults, rather, to establish potential concepts of how to deliver therapies in a targeted, cell-specific manner. Based on our review, it is clear that future of inner ear drug delivery is a discipline filled with potential that will require collaborative efforts among clinicians and scientists to optimize treatment of otologic insults. Graphical Abstract.

PD-L1 and MRN synergy in platinum-based chemoresistance of head and neck squamous cell carcinoma.

BACKGROUND: We have been investigating the molecular mechanisms of cisplatin-induced chemoresistance in head and neck squamous cell carcinoma (HNSCC). Based on our previous findings, the present study investigates how the Mre11, Rad50, and NBS1 (MRN) DNA repair complex interacts at the molecular level with the programmed cell death ligand 1 (PD-L1) in cisplatin-induced chemoresistance. METHODS: Human HNSCC cell lines were used to determine the role played by PD-L1 in cisplatin resistance. Initial experiments investigated PD-L1 expression levels in cells exposed to cisplatin and whether PD-L1 interacts directly with the MRN complex. Finally, in vitro studies and in vivo experiments on BALB/c nu/nu mice were performed to determine whether interference of PD-L1 or NBS1 synthesis modulated cisplatin resistance. RESULTS: Exposure to cisplatin resulted in PD-L1 being upregulated in the chemoresistant but not the chemosensitive cell line. Subsequent co-immunoprecipitation studies demonstrated that PD-L1 associates with NBS1. In addition, we found that the knockdown of either PD-L1 or NBS1 re-sensitised the chemoresistant cell line to cisplatin. Finally, but perhaps most importantly, synergy was observed when both PD-L1 and NBS1 were knocked down making the formerly chemoresistant strain highly cisplatin sensitive. CONCLUSIONS: PD-L1 plays a pivotal role in cisplatin resistance in chemoresistant human HNSCC cell lines.

Influence of accuracy, repeatability and detection probability in the reliability of species-specific eDNA based approaches.

Environmental DNA (eDNA) barcoding has a high potential to increase the cost-efficiency of species detection and monitoring in aquatic habitats. However, despite vast developments in the field, many published assays often lack detailed validation and there is little to no commonly (agreed upon) standardization of protocols. In this study, we evaluated the reliability of eDNA detection and quantification using published primers and assays targeting the Freshwater Pearl Mussel as a model organism. We first assessed limits of detection for two different target genes (COI and 16S) following the MIQE guidelines, and then tested the reliability of quantification in a double-blind mesocosm experiment. Our results reveal that different methodological indicators, namely accuracy, repeatability and detection probability affected the reliability of eDNA measurement at the different levels tested. The selection of the optimal analytical method was mainly determined by detection probability. Both the COI and 16S assays were highly specific for the targeted organism and showed similar accuracy and repeatability, whilst the limit of detection was clearly lower for the COI based approach. In contrast, the reliability of eDNA quantification hinged on repeatability, reflected by the scattering (r2 = 0.87) around the relationship between eDNA and mussel density in mesocosms. A bootstrapping approach, which allowed for the assignment of measures associated with repeatability of samples, revealed that variability between natural replicates (i.e. accuracy) strongly influenced the number of replicates required for a reliable species detection and quantification in the field.

Improved X-ray computed tomography reconstruction of the largest fragment of the Antikythera Mechanism, an ancient Greek astronomical calculator.

The Antikythera Mechanism is an extraordinarily complex ancient Greek astronomical calculating device whose mode of operation is now relatively well understood particularly since imaging studies in 2005 revealed gears and inscriptions which were previously illegible. Unfortunately, the highest resolution X-ray computed tomography image of the largest fragment had some errors which meant that the reconstructed images were not as clear as had been expected. Here, the original X-ray data have been reanalysed and reconstructed. The new X-ray computed tomography images have improved contrast and resolution, leading to better clarity and legibility. The improvement in image quality is characterised and some examples of writing on the Mechanism which can now be read with increased confidence are given.

New software protocols for enabling laboratory based temporal CT.

Temporal micro-computed tomography (CT) allows the non-destructive quantification of processes that are evolving over time in 3D. Despite the increasing popularity of temporal CT, the practical implementation and optimisation can be difficult. Here, we present new software protocols that enable temporal CT using commercial laboratory CT systems. The first protocol drastically reduces the need for periodic intervention when making time-lapse experiments, allowing a large number of tomograms to be collected automatically. The automated scanning at regular intervals needed for uninterrupted time-lapse CT is demonstrated by analysing the germination of a mung bean (vigna radiata), whilst the synchronisation with an in situ rig required for interrupted time-lapse CT is highlighted using a shear cell to observe granular segregation. The second protocol uses golden-ratio angular sampling with an iterative reconstruction scheme and allows the number of projections in a reconstruction to be changed as sample evolution occurs. This overcomes the limitation of the need to know a priori what the best time window for each scan is. The protocol is evaluated by studying barite precipitation within a porous column, allowing a comparison of spatial and temporal resolution of reconstructions with different numbers of projections. Both of the protocols presented here have great potential for wider application, including, but not limited to, in situ mechanical testing, following battery degradation and chemical reactions.

A novel nanoparticle delivery system for targeted therapy of noise-induced hearing loss.

Hearing loss is the most prevalent sensory disability worldwide and may be caused by age, drugs or exposure to excessive noise. We have previously developed a minimally-invasive nanohydrogel drug delivery system that successfully delivers nanoparticles into the inner ear. We have substantially extended this technique by functionalizing the nanoparticles and introducing a targeting peptide which recognizes prestin, a transmembrane electromotile protein uniquely expressed in outer hair cells (OHCs) of the inner ear. We demonstrate the successful delivery of molecules and plasmids specifically to OHCs. When compared to untargeted nanoparticles, the delivery of a c-Jun N-terminal kinase (JNK) inhibitor, D-JNKi-1, to OHCs by targeted nanoparticles improved protection from noise induced hearing loss (NIHL). This is the first demonstration of a protection from NIHL using a novel safe and controllable delivery system which is minimally-invasive to the inner ear and, as such, is an extremely appealing technique for use in many clinical applications.

Ontogeny of juvenile freshwater pearl mussels, Margaritifera margaritifera (Bivalvia: Margaritiferidae).

The gills of juvenile freshwater bivalves undergo a complex morphogenesis that may correlate with changes in feeding ecology, but ontogenic studies on juvenile mussels are rare. Scanning electron microscopy was used to examine the ultrastructure and ontogeny of 117 juvenile freshwater pearl mussels (Margaritifera margaritifera) ranging in age from 1-44 months and length from 0.49-8.90 mm. Three stages of gill development are described. In Stage 1 (5-9 inner demibranch filaments), only unreflected inner demibranch filaments were present. In Stage 2 (9-17 inner demibranch filaments), inner demibranch filaments began to reflect when shell length exceeded 1.13 mm, at 13-16 months old. Reflection began in medial filaments and then proceeded anterior and posterior. In Stage 3 (28-94 inner demibranch filaments), outer demibranch filaments began developing at shell length > 3.1 mm and about 34 months of age. The oral groove on the inner demibranch was first observed in 34 month old specimens > 2.66 mm but was never observed on the outer demibranch. Shell length (R2 = 0.99) was a better predictor of developmental stage compared to age (R2 = 0.84). The full suite of gill ciliation was present on filaments in all stages. Interfilamentary distance averaged 31.3 µm and did not change with age (4-44 months) or with size (0.75-8.9 mm). Distance between laterofrontal cirri couplets averaged 1.54 µm and did not change significantly with size or age. Labial palp primordia were present in even the youngest individuals but ciliature became more diverse in more developed individuals. Information presented here is valuable to captive rearing programmes as it provides insight in to when juveniles may be particularly vulnerable to stressors due to specific ontogenic changes. The data are compared with two other recent studies of Margaritifera development.

The Development of a Nano-based Approach to Alleviate Cisplatin-Induced Ototoxicity.

Cisplatin-induced hearing loss is experienced by a high percentage of patients with squamous cell carcinoma undergoing cisplatin chemotherapy. A novel nano-construct capable of sequestering extracellular cisplatin was developed to combat this problem. The nano-construct consisted of superparamagnetic iron oxide nanoparticles (SPIONs) entrapped within polymeric micelles, which were formed from a glutathione diethyl ester-conjugated amphiphilic diblock copolymer. The glutathione-micelles were analyzed at the cellular level and in an organotypic study for safety evaluation. All utilized methods indicated that the micelles do not cause cellular toxicity or organ damage. The micelles' ability to reduce cisplatin-induced cytotoxicity was then probed in an in vitro model. Cisplatin was pre-treated with the novel nano-construct before being added to growing cells. When compared to cells that were exposed to untreated cisplatin, cells in the pre-treated cisplatin group showed a significant increase in cell viability. This clearly demonstrates that the construct is able to protect the cells from cisplatin cytotoxicity and makes it highly likely that the novel nano-construct will be able to play a role in the protection of the inner ear from cisplatin-induced ototoxicity.

Challenges and opportunities in developing targeted molecular imaging to determine inner ear defects of sensorineural hearing loss.

The development of inner ear gene carriers and delivery systems has enabled genetic defects to be repaired and hearing to be restored in mouse models. Today, promising advances in translational therapies provide confidence that targeted molecular therapy for inner ear diseases will be developed. Unfortunately, the currently available non-invasive modalities, such as Computerized Tomography scan or Magnetic Resonance Imaging provide insufficient resolution to identify most pathologies of the human inner ear, even when the current generation of contrast agents is utilized. The development of targeted contrast agents may play a critical role in determining the cause of, and treatment for, sensorineural hearing loss. Such agents should be able to pass through the cochlea barriers, possess minimal cytotoxicity, and easily conjugate to a targeting agent, without distorting the anatomic details. This review focuses on a series of contrast agents which may fit these criteria for potential clinical application.

Motion-driven flow in an unusual piscine nasal region.

Fishes have several means of moving water to effect odorant transport to their olfactory epithelium ('olfactory flow'). Here we show that olfactory flow in the adult garpike Belone belone (Belonidae, Teleostei), a fish with an unusual nasal region, can be generated by its motion relative to water (swimming, or an external current, or both). We also show how the unusual features of the garpike's nasal region influence olfactory flow. These features comprise a triangular nasal cavity in which the olfactory epithelium is exposed to the external environment, a papilla situated within the nasal cavity, and an elongated ventral apex. To perform our investigation we first generated life-like plastic models of garpike heads from X-ray scans of preserved specimens. We then suspended these models in a flume and flowed water over them to simulate swimming. By directing filaments of dye at the static models, we were able to visualise flow in the nasal regions at physiologically relevant Reynolds numbers (700-2,000). We found that flow of water over the heads did cause circulation in the nasal cavity. Vortices may assist in this circulation. The pattern of olfactory flow was influenced by morphological variations and the asymmetry of the nasal region. The unusual features of the nasal region may improve odorant sampling in the garpike, by dispersing flow over the olfactory epithelium and by creating favourable conditions for odorant transport (e.g. steep velocity gradients). Unexpectedly, we found that the mouth and the base of the garpike's jaws may assist the sampling process. Thus, despite its apparent simplicity, the garpike's nasal region is likely to act as an effective trap for odorant molecules.

Application of infrared-based molecular imaging to a mouse model with head and neck cancer.

BACKGROUND: This study investigated whether near infrared (NIR) or visible fluorescent molecular imaging produced a better representation of a mouse model with head and neck squamous cell carcinoma (HNSCC). Additionally, the study explored whether epidermal growth factor receptor (EGFR)-targeted probes could play an important role in the diagnosis of HNSCC. METHODS: An orthotopic mouse model of HNSCC labeled with the NIR fluorophore, infrared fluorescent protein (iRFP), was developed and monitored noninvasively in real time. The tumors were further evaluated using tumor-specific EGFR-targeted probes conjugated with an NIR dye (IRDye800), or a visible fluorescent protein. RESULTS: The iRFP cell line produced better results than cells emitting visible light when studying local, distant, and deep tumors in the mouse model. The EGFR-targeted probe conjugated with IRDye800 accurately detected tumor perimeters. CONCLUSION: This model has great potential as a unique tool in the study of HNSCC tumor development. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1351-E1357, 2016.

Towards an Understanding of Molecule Capture by the Antennae of Male Beetles Belonging to the Genus Rhipicera (Coleoptera, Rhipiceridae).

Working on the hypothesis that an important function of the lamellate antennae of adult male beetles belonging to the genus Rhipicera is to detect scent associated with female conspecifics, and using field observations, anatomical models derived from X-ray microcomputed tomography, and scanning electron microscopy, we have investigated the behavioral, morphological, and morphometric factors that may influence molecule capture by these antennae. We found that male beetles fly upwind in a zigzag manner, or face upwind when perching, behavior consistent with an animal that is tracking scent. Furthermore, the ultrastructure of the male and female antennae, like their gross morphology, is sexually dimorphic, with male antennae possessing many more of a particular type of receptor-the sensillum placodeum-than their female counterparts (approximately 30,000 vs. 100 per antenna, respectively). Based on this disparity, we assume that the sensilla placodea on the male antennae are responsible for detecting scent associated with female Rhipicera beetles. Molecule capture by male antennae in their alert, fanned states is likely to be favoured by: (a) male beetles adopting prominent, upright positions on high points when searching for scent; (b) the partitioning of antennae into many small segments; (c) antennal morphometry (height, width, outline area, total surface area, leakiness, and narrow channels); (d) the location of the sensilla placodea where they are most likely to encounter odorant molecules; and (e) well dispersed sensilla placodea. The molecule-capturing ability of male Rhipicera antennae may be similar to that of the pectinate antennae of certain male moths.

Defining landscape resistance values in least-cost connectivity models for the invasive grey squirrel: a comparison of approaches using expert-opinion and habitat suitability modelling.

Least-cost models are widely used to study the functional connectivity of habitat within a varied landscape matrix. A critical step in the process is identifying resistance values for each land cover based upon the facilitating or impeding impact on species movement. Ideally resistance values would be parameterised with empirical data, but due to a shortage of such information, expert-opinion is often used. However, the use of expert-opinion is seen as subjective, human-centric and unreliable. This study derived resistance values from grey squirrel habitat suitability models (HSM) in order to compare the utility and validity of this approach with more traditional, expert-led methods. Models were built and tested with MaxEnt, using squirrel presence records and a categorical land cover map for Cumbria, UK. Predictions on the likelihood of squirrel occurrence within each land cover type were inverted, providing resistance values which were used to parameterise a least-cost model. The resulting habitat networks were measured and compared to those derived from a least-cost model built with previously collated information from experts. The expert-derived and HSM-inferred least-cost networks differ in precision. The HSM-informed networks were smaller and more fragmented because of the higher resistance values attributed to most habitats. These results are discussed in relation to the applicability of both approaches for conservation and management objectives, providing guidance to researchers and practitioners attempting to apply and interpret a least-cost approach to mapping ecological networks.

Using GPS telemetry to validate least-cost modeling of gray squirrel (Sciurus carolinensis) movement within a fragmented landscape.

In Britain, the population of native red squirrels Sciurus vulgaris has suffered population declines and local extinctions. Interspecific resource competition and disease spread by the invasive gray squirrel Sciurus carolinensis are the main factors behind the decline. Gray squirrels have adapted to the British landscape so efficiently that they are widely distributed. Knowledge on how gray squirrels are using the landscape matrix and being able to predict their movements will aid management. This study is the first to use global positioning system (GPS) collars on wild gray squirrels to accurately record movements and land cover use within the landscape matrix. This data were used to validate Geographical Information System (GIS) least-cost model predictions of movements and provided much needed information on gray squirrel movement pathways and network use. Buffered least-cost paths and least-cost corridors provide predictions of the most probable movements through the landscape and are seen to perform better than the more expansive least-cost networks which include all possible movements. Applying the knowledge and methodologies gained to current gray squirrel expansion areas, such as Scotland and in Italy, will aid in the prediction of potential movement areas and therefore management of the invasive gray squirrel. The methodologies presented in this study could potentially be used in any landscape and on numerous species.

Functional nasal morphology of chimaerid fishes.

Holocephalans (chimaeras) are a group of marine fishes comprising three families: the Callorhinchidae (callorhinchid fishes), the Rhinochimaeridae (rhinochimaerid fishes) and the Chimaeridae (chimaerid fishes). We have used X-ray microcomputed tomography and magnetic resonance imaging to characterise in detail the nasal anatomy of three species of chimaerid fishes: Chimaera monstrosa, C. phantasma and Hydrolagus colliei. We have shown that the nasal chamber of these three species is linked to the external environment by an incurrent channel and to the oral cavity by an excurrent channel via an oral groove. A protrusion of variable morphology is present on the medial wall of the incurrent channel in all three species, but is absent in members of the two other holocephalan families that we inspected. A third nasal channel, the lateral channel, functionally connects the incurrent nostril to the oral cavity, by-passing the nasal chamber. From anatomical reconstructions, we have proposed a model for the circulation of water, and therefore the transport of odorant, in the chimaerid nasal region. In this model, water could flow through the nasal region via the nasal chamber or the lateral channel. In either case, the direction of flow could be reversed. Circulation through the entire nasal region is likely to be driven primarily by the respiratory pump. We have identified several anatomical features that may segregate, distribute, facilitate and regulate flow in the nasal region and have considered the consequences of flow reversal. The non-sensory cilia lining the olfactory sensory channels appear to be mucus-propelling, suggesting that these cilia have a common protective role in cartilaginous fishes (sharks, rays and chimaeras). The nasal region of chimaerid fishes shows at least two adaptations to a benthic lifestyle, and suggests good olfactory sensitivity, with secondary folding enhancing the hypothetical flat sensory surface area by up to 70%.

A hypothetico-deductive approach to assessing the social function of chemical signalling in a non-territorial solitary carnivore.

The function of chemical signalling in non-territorial solitary carnivores is still relatively unclear. Studies on territorial solitary and social carnivores have highlighted odour capability and utility, however the social function of chemical signalling in wild carnivore populations operating dominance hierarchy social systems has received little attention. We monitored scent marking and investigatory behaviour of wild brown bears Ursus arctos, to test multiple hypotheses relating to the social function of chemical signalling. Camera traps were stationed facing bear 'marking trees' to document behaviour by different age sex classes in different seasons. We found evidence to support the hypothesis that adult males utilise chemical signalling to communicate dominance to other males throughout the non-denning period. Adult females did not appear to utilise marking trees to advertise oestrous state during the breeding season. The function of marking by subadult bears is somewhat unclear, but may be related to the behaviour of adult males. Subadults investigated trees more often than they scent marked during the breeding season, which could be a result of an increased risk from adult males. Females with young showed an increase in marking and investigation of trees outside of the breeding season. We propose the hypothesis that females engage their dependent young with marking trees from a young age, at a relatively 'safe' time of year. Memory, experience, and learning at a young age, may all contribute towards odour capabilities in adult bears.

Stimulating the substrate folding activity of a single ring GroEL variant by modulating the cochaperonin GroES.

In mediating protein folding, chaperonin GroEL and cochaperonin GroES form an enclosed chamber for substrate proteins in an ATP-dependent manner. The essential role of the double ring assembly of GroEL is demonstrated by the functional deficiency of the single ring GroEL(SR). The GroEL(SR)-GroES is highly stable with minimal ATPase activity. To restore the ATP cycle and the turnover of the folding chamber, we sought to weaken the GroEL(SR)-GroES interaction systematically by concatenating seven copies of groES to generate groES(7). GroES Ile-25, Val-26, and Leu-27, residues on the GroEL-GroES interface, were substituted with Asp on different groES modules of groES(7). GroES(7) variants activate ATP activity of GroEL(SR), but only some restore the substrate folding function of GroEL(SR), indicating a direct role of GroES in facilitating substrate folding through its dynamics with GroEL. Active GroEL(SR)-GroES(7) systems may resemble mammalian mitochondrial chaperonin systems.

Body mass index and outcomes from pancreatic resection: a review and meta-analysis.

INTRODUCTION: There are 1.6 billion adults worldwide who are overweight, with body mass indices (BMI) between 25 and 30, while more than 400 million are obese (BMI >30). Obesity predicts the incidence of and poor outcomes from pancreatic cancer. Obesity has also been linked to surgical complications in pancreatectomy, including increased length of hospital stay, surgical infections, blood loss, and decreased survival. However, BMI's impact on many complications following pancreatectomy remains controversial. METHODS: We performed a MEDLINE search of all combinations of "BMI" with "pancreatectomy," "pancreatoduodenectomy," or "pancreaticoduodenectomy." From included studies, we created pooled and weighted estimates for quantitative and qualitative outcomes. We used the PRISMA criteria to ensure this project's validity. RESULTS: Our primary cohort included 2,736 patients with BMI <30, 1,682 with BMI >25, and 546 with BMI between 25 and 30. Most outcomes showed no definitive differences across BMIs. Pancreatic fistula (PF) rates ranged from 4.7% to 31.0%, and four studies found multivariate association between BMI and PF (range odds ratio 1.6-4.2). Pooled analyses of PF by BMI showed significant association (p < 0.05). CONCLUSION: BMI increases the operative complexity of pancreatectomy. However, with aggressive peri- and post-operative care, increases in BMI-associated morbidity and mortality may be mitigated.