Scientific Reports
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All preprints, ranked by how well they match Scientific Reports's content profile, based on 3612 papers previously published here. The average preprint has a 2.95% match score for this journal, so anything above that is already an above-average fit. Older preprints may already have been published elsewhere.
Toloza, J.; Opazo, J. C.
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In catarrhine primates, trichromatic color vision is associated with the presence of three opsin genes that absorb light at three different wavelengths. The OPN1LW and OPN1MW genes are found on the X chromosome. Their proximity and similarity suggest that they originated from a duplication event in the catarrhine ancestor. In this study we use the primate genomes available in public databases to study the duplicative history of the OPN1LW and OPN1MW genes and characterize their spectral sensitivity. Our results reveal a phylogenetic tree that shows a clade containing all X-linked opsin paralogs found in Old World monkeys to be related to a clade containing all X-linked opsin paralogs identified in apes, suggesting that routine trichromacy originated independently in apes and Old World monkeys. Also, we found spectral variability in the X-linked opsin gene of primates. Our study presents a new perspective for the origin of trichromatic color vision in apes and Old World monkeys, not reported so far.
Aparicio-Rodriguez, G.; Ruiz-Navalon, D.; Manubens, P.; Sanchez-Jimenez, A.; Calvo-Tapia, C.; Villacorta-Atienza, J. A.
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In nature, survival requires coping with complex time-changing situations in real time. In this process, memory plays a major role since the retrieval of critical information is key to rapid and reliable decision making. This work explores modulation of human memory under the hypothesis that in dynamic scenarios such critical information is encoded as a static map of future interactions. Specifically, the reported results show that dynamic visual stimuli that contain future interactions are better recalled than equivalent stimuli that do not. This is in line with the proposed hypothesis since the former type of stimulus would be encoded in a more simplified way than the latter. Moreover, dynamic stimuli with future interactions are better recalled than simpler dynamic stimuli, which reinforces that the former are processed by a static representation - their map of interactions. This cognitive strategy seems to be modulated by the complexity of the stimulus, since in simple situations differences in recall appear only in men, whereas when complexity increases, such differences do not show gender bias. Therefore, this work proposes an answer to how memory can help us reliably cope with dynamic situations, demonstrating that those critical for survival (such as fighting, chasing, fleeing, etc., which involve interactions) are better remembered, allowing more efficient learning and decision making, essential to deal with our complex and changing world.
Barrionuevo, P. A.; Diaz Barrancas, F.
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Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) play a critical role in regulating physiological and behavioral responses to light. However, little is known about how melanopsin and ipRGC signals are shaped by the statistical properties of real-world environments. Here, we analyzed statistics of melanopsin, ipRGC codification of extrinsic and intrinsic photoresponses, and luminance using hyperspectral images of natural and human-made scenes under daylight illumination. The statistics were obtained simulating receptive fields from current knowledge about ipRGCs anatomy and physiology. Our findings reveal that human-made environments exhibit significantly higher melanopsin, luminance, and ipRGC excitations compared to natural environments. In natural scenes, luminance contrasts were higher than melanopsin and ipRGC contrasts across most of the range. Melanopsin contrast was largely independent of excitation, and was significantly reduced for larger receptive fields. Differences between ipRGC codification models suggest an interaction between input weighting and environmental structure. These results indicate that modifications of natural regularities by human-made environments could affect ipRGC-driven physiology in everyday life.
Fortunato, A.; Taylor, J.; Scirone, J.; Aktipis, A.; Maley, C.
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There are no reports of cancer in sponges, despite them having somatic cell turnover, long lifespans and no specialized adaptive immune cells. In order to investigate whether sponges are cancer resistant, we exposed a species of sponge, Tethya wilhelma, to X-rays. We found that T. wilhelma can withstand 600 Gy of X-ray radiation. That is approximately 100 times the lethal dose for humans. A single high dose of X-rays did not induce cancer in sponges, providing the first experimental evidence of cancer resistance in the phylum, Porifera. Following X-ray exposure, we found an overexpression of genes involved in DNA repair, signaling transduction pathways and epithelial to mesenchymal transition. Sponges have the highest level of radiation resistance that has yet been observed in animals that have sustained somatic cell turnover. This may make them an excellent model system for studying cancer resistance and developing new approaches for cancer prevention and treatment.
Arun, I.; Lazar, L.
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The influence of language on perceptual processes, referred to as the Whorfian hypothesis, has been a contentious issue. Cross-linguistic research and lab-based experiments have shown that verbal labels can facilitate perceptual and discriminatory processes, mostly in visual and auditory modalities. Here, we investigated whether verbal labels improve performance in a tactile texture discrimination task using natural textures. We also explored whether the grammatical category of these verbal labels plays a role in discrimination ability. In our experiments, we asked the participants to discriminate between pairs of textures presented to the fingertip after a five-day training phase. During the training phase, the tactile textures and English pseudowords were co-presented consistently in the congruent (experimental) condition and inconsistently in the incongruent (control) condition, allowing them to form implicit associations only in the former condition. The pseudoword verbal labels belonged to two grammatical categories, verb-like and noun-like. We found an improvement in the texture discrimination ability only for the congruent condition, irrespective of the grammatical category.
Reimann, M.; Aloui, J.; Obländer, N.; Andresen, N.; Hohlbaum, K.; Hellwich, O.; Reiske, P.
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Animal welfare is a central aspect in animal-based research where mice are most commonly used. Their facial expression can be analyzed to assess their well-being status using the Mouse Grimace Scale. However, its manual application becomes increasingly impractical when used on a large number of animals. This lead to the ongoing integration of computer vision methods to automate the analysis. While such methods have proven effective qualitatively, a systematic assessment to verify their reliability largely remains an open research gap. In this work, we attempted to close this gap as we evaluated three dominant paradigms (i.e., classification from supervised learning features, self-supervised learning features, or landmark locations) for the binary (i.e., well-being un-/impaired) classification of facial mouse images. Our quantitative results showed that such methods can be employed successfully with as low as 16% type II error rates. For qualitative assessment, we visualized the decision-making process and demonstrated that mainly pixels associated with the mouse rather than its environment are used. We further discovered that visual characteristics of the mice beyond those described by the Mouse Grimace Scale contributed to the classification. Our work showed that the automated well-being status assessment in mice is trustworthy and urges towards widespread adoption.
Rashmi, R.; Nandi, C.; Majumdar, S.
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THAP9 is a transposable element-derived gene that encodes the THAP9 protein, which is homologous to the Drosophila P-element transposase (DmTNP) and can cut and paste DNA. However, the exact functional role of THAP9 is unknown. Here, we perform evolutionary analysis and extensive in silico characterization of THAP9, including predicting domains and putative post-translational modification sites. We predict previously unreported mammalian-specific post-translational modification sites that may play a role in the subcellular localization of THAP9. We also observe that although THAP9 has evolved under a strong pervasive purifying selection, yielding high conservation of THAP9, there are distinct class-specific conservation patterns of key functional residues in certain domains. Furthermore, investigation of THAP9 expression profiles in various cancer and matched normal datasets demonstrated underexpression and overexpression in testicular cancers and thymic epithelial tumors, respectively, thus suggesting a possible role of THAP9 in cancer.
Guglielmi, J.; D'Andrea, G.; Graslin, F.; Chatti, K.; Schiazza, A.; Lindenthal, S.; Darcourt, J.; Cambien, B.; Pourcher, T.
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BackgroundThe main basic aspects of the regulation of thyroid metabolism by iodine are known, but given the complexity of the mechanisms involved, further analyzes in living animals are still required. Here, we provided new insights into iodine physiology but also into the optimization of radiotherapy with iodine, as well as effective countermeasures in the case of an exposure to radioactive iodine. MethodsWe performed Single Photon Emission Computed Tomography (SPECT) coupled to an X-ray scanner to record radiotracers in living mice and rats. Our imaging system was similar to that routinely used in nuclear medicine but was specifically designed for studies with small animals. Different modalities of administration of radioactive iodine or its radioactive analogues combined with a low or high iodine diet have been studied in pregnant, lactating and control animals. To optimize countermeasures against acute or chronic iodine exposure, the protective effects of potassium iodide (KI) administration protocols were analyzed. Perchlorate was administered to study the iodine metabolism in the kidney and stomach. ResultsOur results showed how the various organs capable of iodine uptake adapt to an iodine-deficient diet. Indeed, the uptake capacity of the thyroid gland, but also that of the salivary glands was significantly increased on a low iodine diet. In contrast, the iodine uptake capacity of the thyroid and lactating mammary glands was reduced on an iodide-rich diet. Our results also showed the physiological role of the kidneys in controlling excess circulating iodide. In addition, they revealed an active iodine cycle in the stomach. We also investigated the protective effects of daily KI administration during radioactive iodine exposure and found that the overall protection was better in rats (85%) than in mice (65%). We also included pregnant females and newborns, and we revealed the existence of specific mechanisms for the inhibition of the fetal thyroid by circulating iodine. Indeed, an iodine-rich diet or repeated daily administration of KI led to a strong inhibition of the iodide uptake capacity of the fetal thyroid. ConclusionsOur study contributes to a better understanding of iodine metabolism and its regulation in the thyroid and in non-thyroidal organs in adult, fetal and newborn animals. Extrapolated to humans, our results not only provide better understanding of iodide withdrawal as a clinical preparatory measure for patients with differentiated thyroid cancer, but also help to optimize countermeasures in the case of an exposure to radioactive iodine.
Fujibayashi, M.; Abe, K.
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Understanding animal behavior is crucial in behavioral neuroscience, which aims to unravel the mechanism driving these behaviors. A milestone in this field is the analysis of behavioral reactions among animals engaging in social interactions. Although many studies have revealed the fundamental roles of social interaction in social learning, the behavioral aspects of these interactions remain poorly understood, largely due to the lack of tools for analyzing complex behaviors and the attention of subjects in naturalistic, free-moving conditions. Here, we introduce a high-precision system for behavior analysis in songbirds using a marker-based motion capture technique. This system accurately tracks the body location and head direction of freely moving finches and is applicable to multiple subjects during social interaction. With this system, we have quantitatively analyzed behaviors of zebra finches (Taeniopygia guttata) related to visual attention. Our analysis revealed variations in the use of right and left eyes, as well as the duration of sight, among the individuals presented. Further analysis and comparison of their behaviors during both virtual and live presentation identified the similarities and differences in their behavioral reactions. Additionally, we observed changes in their behavioral reactions during a conditioned learning paradigm. This system provides an efficient and easy-to-use tool for advanced behavioral analysis in songbirds, providing an objective method to infer their focus of attention.
Lorenzi, E.; Perrino, M.; Zanon, M.; Messina, A.; Vallortigara, G.
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Whether non-symbolic encoding of quantity is predisposed at birth with dedicated hard-wired neural circuits is debated. Here we presented newly-hatched visually naive chicks with stimuli (flashing dots) of either identical or different numerousness (with a ratio 1:3) with their continuous physical appearance (size, contour length, density, convex hull) randomly changing. Chicks spontaneously tell apart the stimuli on the basis of the number of elements. Upon presentation of either fixed or changing numerousness chicks also showed different expression of early gene c-fos in the visual Wulst, the hippocampal formation, the intermediate medial mesopallium, and the caudal part of the nidopallium caudolaterale. The results support the hypothesis that the ability to discriminate quantities does not require any specific instructive experience. Evidence for innateness of non-symbolic numerical cognition have implications for both neurobiology and philosophy of mathematics.
Barumerli, R.; Geronazzo, M.; Cesari, P.
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Our brain maps the space immediately surrounding the body, the peripersonal space (PPS), to sharpen sensory-motor coordination whenever an object enters it. Within PPS, past research demonstrated how several factors influence motor readiness: from exogenous factors, such as body-object distance and stimulus semantics, to endogenous traits like personality traits. Nevertheless, most paradigms rely on vision or touch, relegating hearing to a supporting role and leaving auditory-only contributions unclear. Here, we tested whether affective content and individual traits modulate motor planning for looming sounds that stop within PPS. Thirty-three adults completed three auditory-only tasks in which positive, negative, or neutral sounds halted at five simulated distances from the participants ears (0.3-0.7 m). We recorded anticipatory postural adjustments, distance estimates, affective ratings, and sensory suggestibility via a questionnaire. Motor responses were largely anticipated as sounds stopped nearer the body, while delayed and less precise for semantic (positive or negative) than neutral sounds. Higher suggestibility predicted longer and more variable premotor latencies, particularly for non-semantic sounds. These findings show that auditory cues alone engage flexible sensorimotor mechanisms within PPS, where exogenous (distance, semantics) and endogenous (suggestibility) factors jointly shape motor readiness and spatial perception.
Macedo, G.; McKenna, B.; Peters, S.; Nowicki, S.; Lipshutz, S.
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Birdsong mediates territory acquisition and mate choice. In agonistic interactions, local songs generally elicit stronger responses than songs from more distant populations. However, the molecular mechanisms associated with differential responses to local vs. foreign songs are poorly understood. We addressed this knowledge gap by combining behavioral assays in the field with blood transcriptomic analysis, using a within-subjects design to ask whether male song sparrows (Melospiza melodia) show differential gene expression when exposed to playback of local and foreign songs. Transcriptomic profiles reflected the difference in behavioral response to local vs. foreign songs, with individuals exposed to local songs showing greater expression of genes associated with song perception and production, anti-inflammatory responses and energy metabolism. Our study suggests that changes in expression of key molecular pathways correlate with behavioral responses to geographic song variation, providing insight into the potential mechanisms regulating signal recognition and response to social challenges. HighlightsO_LIGene expression in sparrow blood was measured after simulated territorial intrusion. C_LIO_LIStronger response to local songs was associated with differential gene expression. C_LIO_LISong-associated genes (FOXP2, NRXN1) had higher expression when birds heard local songs. C_LIO_LIGene expression in the blood contains potential biomarkers of song recognition. C_LI
Stepnicka, Z.; Piorkowska, N.; Brozyna, M.; Matys, T.; Junka, A.
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Invertebrate and larval model organisms such as Drosophila melanogaster, Caenorhabditis elegans, Danio rerio larvae, and Galleria mellonella are increasingly employed in biomedical, toxicological, and ecological research. Their behavioral responses serve as sensitive indicators of functional changes, yet traditional methods of observation remain low-throughput, subjective, and poorly scalable. Artificial intelligence (AI), including machine learning (ML) and deep learning (DL), has emerged as a powerful alternative, enabling automated and unbiased analysis of highly dimensional behavioral data. Here, we present the first systematic review comprehensively mapping the use of AI in behavioral analysis of invertebrate and larval organisms. Following PRISMA 2020 guidelines, we screened literature published between 2015 and May 2025. A total of 97 eligible studies were analyzed for model organisms investigated, AI methods applied, input data characteristics, preprocessing pipelines, model architectures, and evaluation metrics. We observed a steep increase in publications, from only 2 in 2015 to 97 by mid-2025, with the majority originating from the USA, China, and Germany. The most frequently studied organisms included D. melanogaster, C. elegans, and zebrafish larvae, alongside aquaculture and pest species. Since 2021, DL models, particularly convolutional neural networks (CNNs), including YOLO models, and pose estimation frameworks such as DeepLabCut have dominated the field, while supervised ML remains common for classification tasks, and unsupervised learning is primarily applied in exploratory clustering. Input data were typically video or image recordings, but reporting practices were highly inconsistent regarding resolution, frame rate, preprocessing steps, and model training details. Evaluation metrics also varied widely, limiting reproducibility and cross-study comparisons. To address these gaps, we propose a standardized reporting framework encompassing input data specifications, preprocessing pipelines, model architecture, and evaluation metrics. Such standardization will enhance transparency, reproducibility, and comparability across laboratories. AI-driven behavioral analysis has the potential to accelerate drug discovery, toxicology, and environmental monitoring while reducing reliance on vertebrate models in preclinical research.
Shimojo, K.; Shimojo, E.; Katsuragi, R.; Akashi, T.; Shimojo, S.
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Planarians are aquatic worms with powerful regenerative and memory retention abilities. This paper examines whether a dissected tail half of a Planarian (Dugesia Dorotocephala) can retain and exhibit a previously-conditioned response, possibly before the regeneration of the head and the ganglia. We conditioned intact Planarians in a Pavlovian procedure with an electric shock (ES) as the unconditioned stimulus and weak ultraviolet (UV) light as the conditioned stimulus. Then, we dissected their bodies into halves, keeping the dissected tail halves. Starting from the 2nd day after dissection, we presented the same UV light 3 times daily while video-recording the responses. The recorded responses were then classified by a DNN: a VGG16 model was pre-trained by ImageNet for extracting features from images and additionally trained with 211 responses to ES and 118 to UV light before conditioning/dissection to categorize planarians reactions into "UV-induced" or "ES-induced" reactions. The cross-validated accuracy in categorization was 83.6%. We then let the DNN analyze 99 recorded responses to UV from 20 individual conditioned tail halves. 96.8 % of their reactions were classified as "ES-induced" (against 22.0% wrongly classified as "ES-induced" for unconditioned samples under UV), indicating they have shown the "Conditioned Response" (p<3.06E-30). This provides evidence that planarians can conserve and reveal a learned response even without the head/ganglia, as it takes approximately 7 days for the head/ganglia to regenerate versus the given 2-3 days. Although similar findings have been reported repeatedly in the literature, this is the first positive evidence with automated procedures and DNN classification. The result implies the presence of a decentralized nervous structure outside of its head/ganglia that allows a tail half to retain memory and execute motion accordingly, despite their cephalization.
Zamani, J.; Sadr, A.; Javadi, A.-H.
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AbstractsIdentifying individuals with early mild cognitive impairment (EMCI) can be an effective strategy for early diagnosis and delay the progression of Alzheimers disease (AD). Many approaches have been devised to discriminate those with EMCI from healthy control (HC) individuals. Selection of the most effective parameters has been one of the challenging aspects of these approaches. In this study we suggest an optimization method based on five evolutionary algorithms that can be used in optimization of neuroimaging data with a large number of parameters. Resting-state functional magnetic resonance imaging (rs-fMRI) measures, which measure functional connectivity, have been shown to be useful in prediction of cognitive decline. Analysis of functional connectivity data using graph measures is a common practice that results in a great number of parameters. Using graph measures we calculated 1155 parameters from the functional connectivity data of HC (n=36) and EMCI (n=34) extracted from the publicly available database of the Alzheimers disease neuroimaging initiative database (ADNI). These parameters were fed into the evolutionary algorithms to select a subset of parameters for classification of the data into two categories of EMCI and HC using a two-layer artificial neural network. All algorithms achieved classification accuracy of 94.55%, which is extremely high considering single-modality input and low number of data participants. These results highlight potential application of rs-fMRI and efficiency of such optimization methods in classification of images into HC and EMCI. This is of particular importance considering that MRI images of EMCI individuals cannot be easily identified by experts.
Degen, J.; Storms, M.; Lee, C. B.; Jechow, A.; Stoeckl, A. L.; Hoelker, F.; Jakhar, A.; Walter, T.; Walter, S.; Mitesser, O.; Hovestadt, T.; Degen, T.
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One of the most dramatic changes occurring on our planet in recent decades is the ever-increasing extensive use of artificial light at night, which drastically altered the environment nocturnal animals are adapted to 1,2. One nocturnal species group experiencing marked declines are moths, which are not only of great importance for species conservation, but also for their key role in food webs and in ecosystem services such as nocturnal plant pollination 3,4. Light pollution has been identified as a driver in the dramatic insect decline of the past years 5-7, yet little is known about its impact on natural insect orientation behaviour. Using harmonic radar tracking, we show that the orientation of several species of moths is significantly affected by streetlights, although only 4 % of individuals showed flight-to-light behaviour. We reveal a species-specific barrier effect of streetlights on lappet moths whenever the moon was not available as a natural celestial cue. Furthermore, streetlights increased the tortuosity of flight trajectories for both hawk moths and lappet moths. Our results provide the first spatially resolved experimental evidence for the fragmentation of landscapes by streetlights and demonstrate that light pollution affects movement patterns of moths beyond previously assumed extend, potentially affecting their reproductive success and hampering a vital ecosystem service.
Wokke, M. E.; Ridderinkhof, K. R.; Padding, L.
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Creativity is considered to be the driving force behind innovation and progress, yet the mechanisms supporting creative thought remain elusive. In the current study, we investigated whether fluctuations in top-down control are related to creative thinking. Here, participants performed a caption this task in which they had to provide an original and apt caption to accompany a presented picture, while EEG signals were recorded. To assess changing levels of top-down control, we made use of the strong relationship between mid frontal oscillatory activity in the theta range (4-7 HZ) and top-down control. Results demonstrate that specifically during the process of optimization and implementation of creative solutions, lower levels of mid frontal theta resulted in higher levels of creativity. In addition, increased creativity related to enhanced functional connectivity between occipital and mid frontal cortex. Together, our findings indicate that creativity benefits from a top-down induced shift towards an internally-oriented state during idea optimization and evaluation.
Nakul, E.; Deroualle, D.; Montava, M.; Lavieille, J.-P.; Lopez, C.
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Vestibular inputs from the inner ear are at the basis of the vestibulo-spinal and vestibulocollic reflexes involved in balance control. Studies have focused on how attentional load and emotions influence balance, but low-level social cues, such as observing human bodies in motion, have been neglected. Yet, individuals observing another person in a challenging posture or in motion can experience imbalance, indicating that sensorimotor resonance between self and others is involved. The present study examines how the observation of videos depicting human bodies in motion modulates well-established neurophysiological signatures of vestibular information processing. The excitability of vestibulocollic reflexes was assessed by analyzing the waveform of vestibular-evoked myogenic potentials (VEMPs) over the sternocleidomastoid and trapezius muscles of 25 healthy participants (13 females, 12 males). Here we show that observing human bodies undergoing passive whole-body rotations reduced the VEMPs amplitude when compared to observing an object. Importantly, the modulation depended on the person depicted in the video as VEMPs were reduced when observing oneself, compared to someone else being moved. Direction-specific effects and electromyography recordings ruled out non-specific emotional and attentional effects. These results show that the vestibular system is sensitive to observing human bodies in motion, establishing new connections between social neuroscience and vestibular neurophysiology. Significance StatementVestibulocollic reflexes are thought to be consistent and of short latency. Yet, previous results show that observing conspecifics influences balance. We combined approaches from social neuroscience and vestibular electrophysiology to describe how the observation of self and other bodies in motion influences vestibular information processing. The results show that observing human bodies in motion reduces the amplitude of vestibulocollic reflexes involved in the stabilization of the head and balance. These results establish new relations between the sense of balance and social cognition and challenge classical views in vestibular neuroscience.
Kim, N.; Truty, T.; Han, S. D.; Heo, M.; Buchman, A. S.; Bennett, D. A.; Tasaki, S.
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The Mini-Mental State Examination (MMSE) is a widely employed assessment tool for measuring the severity of cognitive impairment. Among the MMSE items, the pentagon copying test (PCT) requires participants to accurately replicate a sample of two interlocking pentagons. While the PCT are traditionally scored on a binary scale, there has been limited developments of granular scoring scale to assess task performance. In this paper, we present a novel three-stage algorithm, called Quantification of Interlocking Pentagons (QIP), which quantifies PCT performance by computing the areas of individual pentagons and their intersection areas, and a balance ratio between the areas of the two individual pentagons. The three stages of the QIP algorithm include: (1) detection of line segments, (2) unraveling of the interlocking pentagons, and (3) quantification of areas. The QIP algorithm was applied to 497 cases from 84 participants. Analysis of the quantified data revealed a significant inverse relationship between age and balance ratio between two pentagon areas (beta = -0.49, p = 0.0033), indicating that older age was associated with a smaller balance ratio. The QIP algorithm enhanced the scoring of performance in the PCT. It can serve as a useful tool for granular level scoring of PCT.
Mabilleau, G.; Boorman, D.; Diniz, J.
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Raman imaging combined with scanning electron microscopy (SEM) is a powerful technique that allows for topographical, chemical and structural correlative multi-scale imaging. It provides the perfect tool to determine which of the Raman mineral-to-matrix ratios represent the best parameter to accurately measure the degree of mineralization of the bone matrix using quantitative backscattered electron imaging (qBEI) as the reference methodology. Indeed, previous studies evidenced that the v2PO4 and v4PO4 vibrational modes were less sensitive to laser polarization than the v1PO4. However, using the v2PO4 or v4PO4 requires a longer acquisition time or lower spectral resolution. In the present study, we evaluated the correlation between mineral-to-matrix ratios computed from v1PO4 and v2PO4 in a human bone sample retrieved from orthopaedic surgery during hip replacement and wt% mineral / wt% organic matrix obtained from qBEI using the inLux SEM Raman interface. We reported here that all mineral-to-matrix ratios were significantly linearly correlated with wt% mineral / wt% organic matrix and that v1PO4/CH2 exhibited the strongest correlation coefficient (r=0.880). This study suggests that the v1PO4 is still a valid Raman peak to estimate the mineral-to-matrix ratio in bone samples and can be used to diagnose bone fragility disorders.