Systematic vitamin D supplementation is associated with improved outcomes and reduced thyroid adverse events in patients with cancer treated with immune checkpoint inhibitors: results from the prospective PROVIDENCE study.

BACKGROUND: Hypovitaminosis D can have a negative prognostic impact in patients with cancer. Vitamin D has a demonstrated role in T-cell-mediated immune activation. We hypothesized that systematic vitamin D repletion could impact clinical outcomes in patients with cancer receiving immune-checkpoint inhibitors (ICIs). METHODS: We planned a prospective observational study (PROVIDENCE) to assess serum vitamin D levels in patients with advanced cancer receiving ICIs (cohort 1 at treatment initiation, cohort 2 during treatment) and the impact of systematic repletion on survival and toxicity outcomes. In an exploratory analysis, we compared the clinical outcomes of cohort 1 with a control cohort of patients followed at the participating centers who did not receive systematic vitamin D repletion. RESULTS: Overall, 164 patients were prospectively recruited in the PROVIDENCE study. In cohort 1, consisting of 101 patients with 94.1% hypovitaminosis (≤ 30 ng/ml) at baseline, adequate repletion with cholecalciferol was obtained in 70.1% at the three months re-assessment. Cohort 2 consisted of 63 patients assessed for vitamin D at a median time of 3.7 months since immunotherapy initiation, with no patients having adequate levels (> 30 ng/ml). Even in cohort 2, systematic supplementation led to adequate levels in 77.8% of patients at the three months re-assessment. Compared to a retrospective control group of 238 patients without systematic vitamin D repletion, PROVIDENCE cohort 1 showed longer overall survival (OS, p = 0.013), time to treatment failure (TTF, p = 0.017), and higher disease control rate (DCR, p = 0.016). The Inverse Probability of Treatment Weighing (IPTW) fitted multivariable Cox regression confirmed the significantly decreased risk of death (HR 0.55, 95%CI: 0.34-0.90) and treatment discontinuation (HR 0.61, 95%CI: 0.40-0.91) for patients from PROVIDENCE cohort 1 in comparison to the control cohort. In the context of longer treatment exposure, the cumulative incidence of any grade immune-related adverse events (irAEs) was higher in the PROVIDENCE cohort 1 compared to the control cohort. Nevertheless, patients from cohort 1 experienced a significantly decreased risk of all grade thyroid irAEs than the control cohort (OR 0.16, 95%CI: 0.03-0.85). CONCLUSION: The PROVIDENCE study suggests the potential positive impact of early systematic vitamin D supplementation on outcomes of patients with advanced cancer receiving ICIs and support adequate repletion as a possible prophylaxis for thyroid irAEs.

Ontogenetic Plasticity in Shoaling Behavior in a Forage Fish under Warming.

Shoaling behavior is known to increase survival rates during attacks from predators, minimize foraging time, favor mating, and potentially increase locomotor efficiency. The onset of shoaling typically occurs during the larval phase, but it is unclear how it may improve across ontogenetic stages in forage fishes. Warming is known to increase metabolic rates during locomotion in solitary fish, and shoaling species may adjust their collective behavior to offset the elevated costs of swimming at higher temperatures. In this study, we quantified the effects of warming on shoaling performance across the ontogeny of a small forage fish, zebrafish (Danio rerio) at different speeds. Shoals of larval, juvenile, and adult zebrafish were acclimated at two temperatures (28°C and 32°C), and metabolic rates were quantified prior to and following nonexhaustive exercise at high speed. Shoals of five individuals were filmed in a flow tank to analyze the kinematics of collective movement. We found that zebrafish improve shoaling swimming performance from larvae to juveniles to adults. In particular, shoals become more cohesive, and both tail beat frequency (TBF) and head-to-tail amplitude decrease with ontogeny. Early life stages have higher thermal sensitivity in metabolic rates and TBF especially at high speeds, when compared to adults. Our study shows that shoaling behavior and thermal sensitivity improve as zebrafish shift from larval to juvenile to adult stages.

Immunotherapy for Metastatic Non-Small Cell Lung Cancer: Therapeutic Advances and Biomarkers.

Immunotherapy has revolutionized the treatment paradigm of non-small cell lung cancer and improved patients' prognosis. Immune checkpoint inhibitors have quickly become standard frontline treatment for metastatic non-oncogene addicted disease, either as a single agent or in combination strategies. However, only a few patients have long-term benefits, and most of them do not respond or develop progressive disease during treatment. Thus, the identification of reliable predictive and prognostic biomarkers remains crucial for patient selection and guiding therapeutic choices. In this review, we provide an overview of the current strategies, highlighting the main clinical challenges and novel potential biomarkers.

EcoPhysioMechanics: Integrating energetics and biomechanics to understand fish locomotion under climate change.

Ecological physiologists and biomechanists have been broadly investigating swimming performance in a diversity of fishes, however the connection between form, function and energetics of locomotion has been rarely evaluated in the same system and under climate change scenarios. In this perspective I argue that working within the framework of 'EcoPhysioMechanics', i.e., integrating energetics and biomechanics tools, to measure locomotor performance and behavior under different abiotic factors, improves our understanding of the mechanisms, limits and costs of movement. To demonstrate how ecophysiomechanics can be applied to locomotor studies, I outline how linking biomechanics and physiology allows us to understand how fishes may modulate their movement to achieve high speeds or reduce the costs of locomotion. I also discuss how the framework is necessary to quantify swimming capacity under climate change scenarios. Finally, I discuss current dearth of integrative studies and gaps in empirical datasets that are necessary to understand fish swimming under changing environments.

Multi-animal pose estimation, identification and tracking with DeepLabCut.

Estimating the pose of multiple animals is a challenging computer vision problem: frequent interactions cause occlusions and complicate the association of detected keypoints to the correct individuals, as well as having highly similar looking animals that interact more closely than in typical multi-human scenarios. To take up this challenge, we build on DeepLabCut, an open-source pose estimation toolbox, and provide high-performance animal assembly and tracking-features required for multi-animal scenarios. Furthermore, we integrate the ability to predict an animal's identity to assist tracking (in case of occlusions). We illustrate the power of this framework with four datasets varying in complexity, which we release to serve as a benchmark for future algorithm development.

Fish-inspired segment models for undulatory steady swimming.

Many aquatic animals swim by undulatory body movements and understanding the diversity of these movements could unlock the potential for designing better underwater robots. Here, we analyzed the steady swimming kinematics of a diverse group of fish species to investigate whether their undulatory movements can be represented using a series of interconnected multi-segment models, and if so, to identify the key factors driving the segment configuration of the models. Our results show that the steady swimming kinematics of fishes can be described successfully using parsimonious models, 83% of which had fewer than five segments. In these models, the anterior segments were significantly longer than the posterior segments, and there was a direct link between segment configuration and swimming kinematics, body shape, and Reynolds number. The models representing eel-like fishes with elongated bodies and fishes swimming at high Reynolds numbers had more segments and less segment length variability along the body than the models representing other fishes. These fishes recruited their anterior bodies to a greater extent, initiating the undulatory wave more anteriorly. Two shape parameters, related to axial and overall body thickness, predicted segment configuration with moderate to high success rate. We found that head morphology was a good predictor of its segment length. While there was a large variation in head segments, the length of tail segments was similar across all models. Given that fishes exhibited variable caudal fin shapes, the consistency of tail segments could be a result of an evolutionary constraint tuned for high propulsive efficiency. The bio-inspired multi-segment models presented in this study highlight the key bending points along the body and can be used to decide on the placement of actuators in fish-inspired robots, to model hydrodynamic forces in theoretical and computational studies, or for predicting muscle activation patterns during swimming.

Convergence of undulatory swimming kinematics across a diversity of fishes.

Fishes exhibit an astounding diversity of locomotor behaviors from classic swimming with their body and fins to jumping, flying, walking, and burrowing. Fishes that use their body and caudal fin (BCF) during undulatory swimming have been traditionally divided into modes based on the length of the propulsive body wave and the ratio of head:tail oscillation amplitude: anguilliform, subcarangiform, carangiform, and thunniform. This classification was first proposed based on key morphological traits, such as body stiffness and elongation, to group fishes based on their expected swimming mechanics. Here, we present a comparative study of 44 diverse species quantifying the kinematics and morphology of BCF-swimming fishes. Our results reveal that most species we studied share similar oscillation amplitude during steady locomotion that can be modeled using a second-degree order polynomial. The length of the propulsive body wave was shorter for species classified as anguilliform and longer for those classified as thunniform, although substantial variability existed both within and among species. Moreover, there was no decrease in head:tail amplitude from the anguilliform to thunniform mode of locomotion as we expected from the traditional classification. While the expected swimming modes correlated with morphological traits, they did not accurately represent the kinematics of BCF locomotion. These results indicate that even fish species differing as substantially in morphology as tuna and eel exhibit statistically similar two-dimensional midline kinematics and point toward unifying locomotor hydrodynamic mechanisms that can serve as the basis for understanding aquatic locomotion and controlling biomimetic aquatic robots.

Sharks surf the slope: Current updrafts reduce energy expenditure for aggregating marine predators.

An animal's energy landscape considers the power requirements associated with residing in or moving through habitats. Within marine environments, these landscapes can be dynamic as water currents will influence animal power requirements and can change rapidly over diel and tidal cycles. In channels and along slopes with strong currents, updraft zones may reduce energy expenditure of negatively buoyant fishes that are also obligate swimmers. Despite marine predators often residing within high-current area, no study has investigated the potential role of the energetic landscape in driving such habitat selectivity. Over 500 grey reef sharks Carcharhinus amblyrhynchos reside in the southern channel of Fakarava Atoll, French Polynesia. We used diver observations, acoustic telemetry and biologging to show that sharks use regions of predicted updrafts and switch their core area of space use based on tidal state (incoming versus outgoing). During incoming tides, sharks form tight groups and display shuttling behaviour (moving to the front of the group and letting the current move them to the back) to maintain themselves in these potential updraft zones. During outgoing tides, group dispersion increases, swimming depths decrease and shuttling behaviours cease. These changes are likely due to shifts in the nature and location of the updraft zones, as well as turbulence during outgoing tides. Using a biomechanical model, we estimate that routine metabolic rates for sharks may be reduced by 10%-15% when in updraft zones. Grey reef sharks save energy using predicted updraft zones in channels and 'surfing the slope'. Analogous to birds using wind-driven updraft zones, negatively buoyant marine animals may use current-induced updraft zones to reduce energy expenditure. Updrafts should be incorporated into dynamic energy landscapes and may partially explain the distribution, behaviour and potentially abundance of marine predators.

Family History of Cancer as Potential Prognostic Factor in Stage III Colorectal Cancer: a Retrospective Monoinstitutional Study.

BACKGROUND AND AIMS: The prognostic role of family history of cancer (FHC) in resected colorectal cancer (CRC) is controversial. The aim of the current study was to evaluate its impact in a monoinstitutional series of stage III CRC patients. METHODS: This single institution retrospective analysis is aimed at evaluating whether FHC affects overall survival (OS) and disease-free survival (DFS) in stage III CRC patients. Moreover, the role of both colorectal FHC (FHCRC, in patients with at least one relative with CRC) and FHC "burden" have been investigated; patients were classified according to FHC in FHC negative, FHC-low (one "familial cluster" among parents/children/grandparents, brothers/sisters, uncles/cousins), and FHC-high (at least two clusters of those above mentioned). RESULTS: From October 2000 to March 2019, 112 consecutive stage III CRC patients have been evaluated. Median age was 67 years (range 24-89); male/female ratio was 64/48. Fifty-three (47.3%) patients were FHC-negative while 59 (52.7%) patients were FHC-positive, 18 (16.1%) of whom were FHCRC-positive. Thirty-three (29.5%) patients were FHC-low, and 10 (8.9%) were FHC-high. At a median follow-up of 41.9 months, no statistically significant differences in DFS were found. FHC-positive patients had a significantly longer OS than FHC-negative (HR = 0.32 [95% CI 0.12-0.84], p = 0.0210), and a significant trend towards improved OS according to the FHC burden was found (p = 0.0255). No statistically significant differences were found in DFS and OS according to FHCRC. CONCLUSION: In this retrospective analysis, FHC-positive stage III CRC patients had a significantly longer OS compared to FHC-negative. Moreover, this survival benefit seems to increase according to the FHC burden. Further prospective studies, with longer follow-up and larger sample size, are necessary to confirm FHC as prognostic factor in this setting.

Ocean acidification and warming affect skeletal mineralization in a marine fish.

Ocean acidification and warming are known to alter, and in many cases decrease, calcification rates of shell and reef building marine invertebrates. However, to date, there are no datasets on the combined effect of ocean pH and temperature on skeletal mineralization of marine vertebrates, such as fishes. Here, the embryos of an oviparous marine fish, the little skate ( Leucoraja erinacea), were developmentally acclimatized to current and increased temperature and CO2 conditions as expected by the year 2100 (15 and 20°C, approx. 400 and 1100 µatm, respectively), in a fully crossed experimental design. Using micro-computed tomography, hydroxyapatite density was estimated in the mineralized portion of the cartilage in jaws, crura, vertebrae, denticles and pectoral fins of juvenile skates. Mineralization increased as a consequence of high CO2 in the cartilage of crura and jaws, while temperature decreased mineralization in the pectoral fins. Mineralization affects stiffness and strength of skeletal elements linearly, with implications for feeding and locomotion performance and efficiency. This study is, to my knowledge, the first to quantify a significant change in mineralization in the skeleton of a fish and shows that changes in temperature and pH of the oceans have complex effects on fish skeletal morphology.

Thermal tolerance of the invasive red-bellied pacu and the risk of establishment in the United States.

Indigenous red-bellied pacu, Piaractus brachypomus, populations are in decline due to overfishing. Once ignored by aquaculturists because of their perceived low economic value, renewed aquaculture efforts in Central and South America aim to relieve fishing pressures on natural pacu populations. In the southern United States pacu aquaculture for the aquarium trade has raised concerns that accidental release could lead to establishment of overwintering populations outside captivity-a threat accentuated by the average 6 °C increase in shallow-water temperatures predicted by the end of the century. In the present study, Critical and Chronic Thermal Methodology was used to quantify red-bellied pacu thermal tolerance niche requirements. The data suggest that red-belllied pacu are a thermophilic species capable of tolerating low and high chronic temperatures of 16.5 °C and 35 °C, respectively. Critical thermal minimum and maximum temperatures of fish acclimated near their chronic limits are 10.3 and 44.4 °C. Red-bellied pacu aquaculture in the United States is concentrated in subtropical Florida regions that encourage rapid growth and reproduction, but carry an increased risk of establishing reproducing populations in local freshwater systems. The thermal niche data show that the risk of bioinvasion can be reduced or eliminated by adopting an approach whereby aquaculture potential is integrated with environmental temperature constraints.

Understanding Fish Linear Acceleration Using an Undulatory Biorobotic Model with Soft Fluidic Elastomer Actuated Morphing Median Fins.

Although linear accelerations are an important common component of the diversity of fish locomotor behaviors, acceleration is one of the least-understood aspects of propulsion. Analysis of acceleration behavior in fishes with both spiny and soft-rayed median fins demonstrates that fin area is actively modulated when fish accelerate. We implemented an undulatory biomimetic robotic fish model with median fins manufactured using multimaterial three-dimensional printing-a spiny-rayed dorsal fin, soft-rayed dorsal/anal fins, and a caudal fin-whose stiffnesses span three orders of magnitude. We used an array of fluidic elastomeric soft actuators to mimic the dorsal/anal inclinator and erector/depressor muscles of fish, which allowed the soft fins to be erected or folded within 0.3 s. We experimentally show that the biomimetic soft dorsal/anal fin can withstand external loading. We found that erecting the soft dorsal/anal fins significantly enhanced the linear acceleration rate, up to 32.5% over the folded fin state. Surprisingly, even though the projected area of the body (in the lateral plane) increased 16.9% when the median fins were erected, the magnitude of the side force oscillation decreased by 24.8%, which may have led to significantly less side-to-side sway in the robotic swimmer. Visualization of fluid flow in the wake of median fins reveals that during linear acceleration, the soft dorsal fin generates a wake flow opposite in direction to that of the caudal fin, which creates propulsive jets with time-variant circulations and jet angles. Erectable/foldable fins provide a new design space for bioinspired underwater robots with structures that morph to adapt to different locomotor behaviors. This biorobotic fish model is also a potentially promising system for studying the dynamics of complex multifin fish swimming behaviors, including linear acceleration, steady swimming, and burst and coast, which are difficult to analyze in freely swimming fishes.

High postural costs and anaerobic metabolism during swimming support the hypothesis of a U-shaped metabolism-speed curve in fishes.

Swimming performance is considered a key trait determining the ability of fish to survive. Hydrodynamic theory predicts that the energetic costs required for fishes to swim should vary with speed according to a U-shaped curve, with an expected energetic minimum at intermediate cruising speeds and increasing expenditure at low and high speeds. However, to date no complete datasets have shown an energetic minimum for swimming fish at intermediate speeds rather than low speeds. To address this knowledge gap, we used a negatively buoyant fish, the clearnose skate Raja eglanteria, and took two approaches: a classic critical swimming speed protocol and a single-speed exercise and recovery procedure. We found an anaerobic component at each velocity tested. The two approaches showed U-shaped, though significantly different, speed-metabolic relationships. These results suggest that (i) postural costs, especially at low speeds, may result in J- or U-shaped metabolism-speed curves; (ii) anaerobic metabolism is involved at all swimming speeds in the clearnose skate; and (iii) critical swimming protocols might misrepresent the true costs of locomotion across speeds, at least in negatively buoyant fish.

Batoid locomotion: effects of speed on pectoral fin deformation in the little skate, Leucoraja erinacea.

Most batoids have a unique swimming mode in which thrust is generated by either oscillating or undulating expanded pectoral fins that form a disc. Only one previous study of the freshwater stingray has quantified three-dimensional motions of the wing, and no comparable data are available for marine batoid species that may differ considerably in their mode of locomotion. Here, we investigate three-dimensional kinematics of the pectoral wing of the little skate, Leucoraja erinacea, swimming steadily at two speeds [1 and 2 body lengths (BL) s-1]. We measured the motion of nine points in three dimensions during wing oscillation and determined that there are significant differences in movement amplitude among wing locations, as well as significant differences as speed increases in body angle, wing beat frequency and speed of the traveling wave on the wing. In addition, we analyzed differences in wing curvature with swimming speed. At 1 BL s-1, the pectoral wing is convex in shape during the downstroke along the medio-lateral fin midline, but at 2 BL s-1 the pectoral fin at this location cups into the flow, indicating active curvature control and fin stiffening. Wing kinematics of the little skate differed considerably from previous work on the freshwater stingray, which does not show active cupping of the whole fin on the downstroke.

Phototactic guidance of a tissue-engineered soft-robotic ray.

Inspired by the relatively simple morphological blueprint provided by batoid fish such as stingrays and skates, we created a biohybrid system that enables an artificial animal--a tissue-engineered ray--to swim and phototactically follow a light cue. By patterning dissociated rat cardiomyocytes on an elastomeric body enclosing a microfabricated gold skeleton, we replicated fish morphology at 1/10 scale and captured basic fin deflection patterns of batoid fish. Optogenetics allows for phototactic guidance, steering, and turning maneuvers. Optical stimulation induced sequential muscle activation via serpentine-patterned muscle circuits, leading to coordinated undulatory swimming. The speed and direction of the ray was controlled by modulating light frequency and by independently eliciting right and left fins, allowing the biohybrid machine to maneuver through an obstacle course.

Progressive hypoxia decouples activity and aerobic performance of skate embryos.

Although fish population size is strongly affected by survival during embryonic stages, our understanding of physiological responses to environmental stressors is based primarily on studies of post-hatch fishes. Embryonic responses to acute exposure to changes in abiotic conditions, including increase in hypoxia, could be particularly important in species exhibiting long developmental time, as embryos are unable to select a different environment behaviourally. Given that oxygen is key to metabolic processes in fishes and aquatic hypoxia is becoming more severe and frequent worldwide, organisms are expected to reduce their aerobic performance. Here, we examined the metabolic and behavioural responses of embryos of a benthic elasmobranch fish, the little skate (Leucoraja erinacea), to acute progressive hypoxia, by measuring oxygen consumption and movement (tail-beat) rates inside the egg case. Oxygen consumption rates were not significantly affected by ambient oxygen levels until reaching 45% air saturation (critical oxygen saturation, S crit). Below S crit, oxygen consumption rates declined rapidly, revealing an oxygen conformity response. Surprisingly, we observed a decoupling of aerobic performance and activity, as tail-beat rates increased, rather than matching the declining metabolic rates, at air saturation levels of 55% and below. These results suggest a significantly divergent response at the physiological and behavioural levels. While skate embryos depressed their metabolic rates in response to progressive hypoxia, they increased water circulation inside the egg case, presumably to restore normoxic conditions, until activity ceased abruptly around 9.8% air saturation.

Skating by: low energetic costs of swimming in a batoid fish.

We quantify the oxygen consumption rates and cost of transport (COT) of a benthic batoid fish, the little skate, Leucoraja erinacea, at three swimming speeds. We report that this species has the lowest mass-adjusted swimming metabolic rate measured for any elasmobranch; however, this species incurs a much higher COT at approximately five times the lowest values recorded for some teleosts. In addition, because skates lack a propulsive caudal fin and could not sustain steady swimming beyond a relatively low optimum speed of 1.25 body lengths s(-1), we propose that the locomotor efficiency of benthic rajiform fishes is limited to the descending portion of a single COT-speed relationship. This renders these species poorly suited for long-distance translocation and, therefore, especially vulnerable to regional-scale environmental disturbances.

Intraspecific variation in physiological performance of a benthic elasmobranch challenged by ocean acidification and warming.

Elucidating the combined effects of increasing temperature and ocean acidification on performance of fishes is central to our understanding of how species will respond to global climate change. Measuring the metabolic costs associated with intense and short activities, such as those required to escape predators, is key to quantifying changes in performance and estimating the potential effects of environmental stressors on survival. In this study, juvenile little skate Leucoraja erinacea from two neighboring locations (Gulf of Maine, or northern location, and Georges Bank, or southern location) were developmentally acclimatized and reared at current and projected temperatures (15, 18 or 20°C) and acidification conditions (pH 8.1 or 7.7), and their escape performance was tested by employing a chasing protocol. The results from this study suggest countergradient variation in growth between skates from the two locations, while the optimum for escape performance was at a lower temperature in individuals from the northern latitudes, which could be related to adaptation to the local thermal environment. Aerobic performance and scope declined in skates from the northern latitudes under simulated ocean warming and acidification conditions. Overall, the southern skates showed lower sensitivity to these climatic stressors. This study demonstrates that even mobile organisms from neighboring locations can exhibit substantial differences in energetic costs of exercise and that skates from the northern part of the geographic range may be more sensitive to the directional increase in temperature and acidification expected by the end of the century.

Complex Effects of Ecosystem Engineer Loss on Benthic Ecosystem Response to Detrital Macroalgae.

Ecosystem engineers change abiotic conditions, community assembly and ecosystem functioning. Consequently, their loss may modify thresholds of ecosystem response to disturbance and undermine ecosystem stability. This study investigates how loss of the bioturbating lugworm Arenicola marina modifies the response to macroalgal detrital enrichment of sediment biogeochemical properties, microphytobenthos and macrofauna assemblages. A field manipulative experiment was done on an intertidal sandflat (Oosterschelde estuary, The Netherlands). Lugworms were deliberately excluded from 1× m sediment plots and different amounts of detrital Ulva (0, 200 or 600 g Wet Weight) were added twice. Sediment biogeochemistry changes were evaluated through benthic respiration, sediment organic carbon content and porewater inorganic carbon as well as detrital macroalgae remaining in the sediment one month after enrichment. Microalgal biomass and macrofauna composition were measured at the same time. Macroalgal carbon mineralization and transfer to the benthic consumers were also investigated during decomposition at low enrichment level (200 g WW). The interaction between lugworm exclusion and detrital enrichment did not modify sediment organic carbon or benthic respiration. Weak but significant changes were instead found for porewater inorganic carbon and microalgal biomass. Lugworm exclusion caused an increase of porewater carbon and a decrease of microalgal biomass, while detrital enrichment drove these values back to values typical of lugworm-dominated sediments. Lugworm exclusion also decreased the amount of macroalgae remaining into the sediment and accelerated detrital carbon mineralization and CO2 release to the water column. Eventually, the interaction between lugworm exclusion and detrital enrichment affected macrofauna abundance and diversity, which collapsed at high level of enrichment only when the lugworms were present. This study reveals that in nature the role of this ecosystem engineer may be variable and sometimes have no or even negative effects on stability, conversely to what it should be expected based on current research knowledge.