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Universal coatings with versatile surface adhesion, good mechanochemical robustness, and the capacity for secondary modification are of great scientific interest. However, incorporating these advantages into a system is still a great challenge. Here, we report a series of catechol-decorated polyallylamines (CPAs), denoted as pseudo-Mytilus edulis foot protein 5 (pseudo-Mefp-5), that mimic not only the catechol and amine groups but also the backbone of Mefp-5. CPAs can fabricate highly adhesive, robust, multifunctional polyCPA (PCPA) coatings based on synergetic catechol-polyamine chemistry as universal building blocks. Due to the interpenetrating entangled network architectures, these coatings exhibit high chemical robustness against harsh conditions (HCl, pH 1; NaOH, pH 14; H2O2, 30%), good mechanical robustness, and wear resistance. In addition, PCPA coatings provide abundant grafting sites, enabling the fabrication of various functional surfaces through secondary modification. Furthermore, the versatility, multifaceted robustness, and scalability of PCPA coatings indicate their great potential for surface engineering, especially for withstanding harsh conditions in multipurpose biomedical applications.
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The present study aimed to explore the effects of different exercise modes on neuromuscular junction (NMJ) and metabolism of skeletal muscle-related proteins in aging rats. Ten from 38 male Sprague-Dawley (SD) rats (3-month-old) were randomly selected into young (Y) group, while the rest were raised to 21 months old and randomly divided into elderly control (O), endurance exercise (EN) and resistance exercise (R) groups. After 8 weeks of corresponding exercises training, the gastrocnemius muscles of rats were collected, and the expression of S100B in Schwann cells was detected by immunofluorescence staining. Western blot was used to detect the protein expression levels of agglutinate protein (Agrin), low-density lipoprotein receptor-related protein 4 (Lrp4), muscle- specific kinase protein (MuSK), downstream tyrosine kinase 7 (Dok7), phosphorylated protein kinase B (p-Akt), phosphorylated mammalian target rapamycin (p-mTOR), and phosphorylated forkhead box O1 (p-FoxO1) in rat gastrocnemius muscles. The results showed that, endurance and resistance exercises increased the wet weight ratio of gastrocnemius muscle in the aging rats. The protein expression of S100B in the R group was significantly higher than those in the O and EN groups. Proteins related to NMJ function, including Agrin, Lrp4, MuSK, and Dok7 were significantly decreased in the O group compared with those in the Y group. Resistance exercise up-regulated these four proteins in the aging rats, whereas endurance exercise could not reverse the protein expression levels of Lrp4, MuSK and Dok7. Regarding skeletal muscle-related proteins, the O group showed down-regulated p-Akt, and p-mTOR protein expression levels and up-regulated p-FoxO1 protein expression level, compared to the Y group. Resistance and endurance exercises reversed the changes in p-mTOR and p-FoxO1 protein expression in the aging rats. These findings demonstrate that both exercise modes can enhance NMJ function, increase protein synthesis and reduce the catabolism of skeletal muscle-related proteins in aging rats, with resistance exercise showing a more pronounced effect.
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Envejecimiento , Músculo Esquelético , Unión Neuromuscular , Condicionamiento Físico Animal , Ratas Sprague-Dawley , Animales , Masculino , Envejecimiento/metabolismo , Envejecimiento/fisiología , Ratas , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal/fisiología , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiología , Proteínas Musculares/metabolismo , Entrenamiento de Fuerza/métodos , Proteína Forkhead Box O1RESUMEN
Thrombosis and infection are two major complications associated with central venous catheters (CVCs), which significantly contribute to morbidity and mortality. Antifouling coating strategies currently represent an efficient approach for addressing such complications. However, existing antifouling coatings have limitations in terms of both duration and effectiveness. Herein, we propose a durable zwitterionic polymer armor for catheters. This armor is realized by pre-coating with a robust phenol-polyamine film inspired by insect sclerotization, followed by grafting of poly-2-methacryloyloxyethyl phosphorylcholine (pMPC) via in-situ radical polymerization. The resulting pMPC coating armor exhibits super-hydrophilicity, thereby forming a highly hydrated shell that effectively prevents bacterial adhesion and inhibits the adsorption and activation of fibrinogen and platelets in vitro. In practical applications, the armored catheters significantly reduced inflammation and prevented biofilm formation in a rat subcutaneous infection model, as well as inhibited thrombus formation in a rabbit jugular vein model. Overall, our robust zwitterionic polymer coating presents a promising solution for reducing infections and thrombosis associated with vascular catheters.
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Worldwide, skin cancer prevalence necessitates accurate diagnosis to alleviate public health burdens. Although the application of artificial intelligence in image analysis and pattern recognition has improved the accuracy and efficiency of early skin cancer diagnosis, existing supervised learning methods are limited due to their reliance on a large amount of labeled data. To overcome the limitations of data labeling and enhance the performance of diagnostic models, this study proposes a semi-supervised skin cancer diagnostic model based on Self-feedback Threshold Focal Learning (STFL), capable of utilizing partial labeled and a large scale of unlabeled medical images for training models in unseen scenarios. The proposed model dynamically adjusts the selection threshold of unlabeled samples during training, effectively filtering reliable unlabeled samples and using focal learning to mitigate the impact of class imbalance in further training. The study is experimentally validated on the HAM10000 dataset, which includes images of various types of skin lesions, with experiments conducted across different scales of labeled samples. With just 500 annotated samples, the model demonstrates robust performance (0.77 accuracy, 0.6408 Kappa, 0.77 recall, 0.7426 precision, and 0.7462 F1-score), showcasing its efficiency with limited labeled data. Further, comprehensive testing validates the semi-supervised model's significant advancements in diagnostic accuracy and efficiency, underscoring the value of integrating unlabeled data. This model offers a new perspective on medical image processing and contributes robust scientific support for the early diagnosis and treatment of skin cancer.
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A reduced polyoxometalate-based organo-metallophosphate (MOPO) framework formulated as [Co4(PO4)(C7H8N4)6](PWVI10WV2O40) (Co-PO4-PW12) with an ultra-high CO production rate of 13 676 µmol g-1 h-1 has been presented through photocatalytic CO2 reduction investigations.
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Universal protein coatings have recently gained wide interest in medical applications due to their biocompatibility and ease of fabrication. However, the challenge persists in protein activity preservation, significantly complicating the functional design of these coatings. Herein, an active dual-protein surface engineering strategy assisted by a facile stepwise protein-protein interactions assembly (SPPIA) method for catheters to reduce clot formation and infection is proposed. This strategy is realized first by the partial oxidation of bovine serum albumin (BSA) and lysozyme (LZM) for creating stable nucleation platforms via hydrophobic interaction, followed by the assembly of nonoxidized BSA (pI, the isoelectric point, ≈4.7) and LZM (pI ≈11) through electrostatic interaction owing to their opposite charge under neutral conditions. The SPPIA method effectively preserves the conformation and functionality of both BSA and LZM, thus endowing the resultant coating with potent antithrombotic and bactericidal properties. Furthermore, the stable nucleation platform ensures the adhesion and durability of the coating, resisting thrombosis and bacterial proliferation even after 15 days of PBS immersion. Overall, the SPPIA approach not only provides a new strategy for the fabrication of active protein coatings but also shows promise for the surface engineering technology of catheters.
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Materiales Biocompatibles Revestidos , Muramidasa , Albúmina Sérica Bovina , Trombosis , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Trombosis/metabolismo , Trombosis/prevención & control , Animales , Materiales Biocompatibles Revestidos/química , Muramidasa/química , Propiedades de Superficie , Humanos , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Thrombus formation and tissue embedding significantly impair the clinical efficacy and retrievability of temporary interventional medical devices. Herein, we report an insect sclerotization-inspired antifouling armor for tailoring temporary interventional devices with durable resistance to protein adsorption and the following protein-mediated complications. By mimicking the phenol-polyamine chemistry assisted by phenol oxidases during sclerotization, we develop a facile one-step method to crosslink bovine serum albumin (BSA) with oxidized hydrocaffeic acid (HCA), resulting in a stable and universal BSA@HCA armor. Furthermore, the surface of the BSA@HCA armor, enriched with carboxyl groups, supports the secondary grafting of polyethylene glycol (PEG), further enhancing both its antifouling performance and durability. The synergy of robustly immobilized BSA and covalently grafted PEG provide potent resistance to the adhesion of proteins, platelets, and vascular cells in vitro. In ex vivo blood circulation experiment, the armored surface reduces thrombus formation by 95 %. Moreover, the antifouling armor retained over 60 % of its fouling resistance after 28 days of immersion in PBS. Overall, our armor engineering strategy presents a promising solution for enhancing the antifouling properties and clinical performance of temporary interventional medical devices.
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Protein and cell adhesion on temporary intravascular devices can lead to thrombosis and tissue embedment, significantly increasing complications and device retrieval difficulties. Here, we propose an endothelial glycocalyx-inspired dynamic antifouling surface strategy for indwelling catheters and retrievable vascular filters to prevent thrombosis and suppress intimal embedment. This strategy is realized on the surfaces of substrates by the intensely dense grafting of hydrolyzable endothelial polysaccharide hyaluronic acid (HA), assisted by an amine-rich phenol-polyamine universal platform. The resultant super-hydrophilic surface exhibits potent antifouling property against proteins and cells. Additionally, the HA hydrolysis induces continuous degradation of the coating, enabling removal of inevitable biofouling on the surface. Moreover, the dense grafting of HA also ensures the medium-term effectiveness of this dynamic antifouling surface. The coated catheters maintain a superior anti-thrombosis capacity in ex vivo blood circulation after 30 days immersion. In the abdominal veins of rats, the coated implants show inhibitory effects on intimal embedment up to 2 months. Overall, we envision that this glycocalyx-inspired dynamic antifouling surface strategy could be a promising surface engineering technology for temporary intravascular devices.
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Incrustaciones Biológicas , Trombosis , Ratas , Animales , Incrustaciones Biológicas/prevención & control , Proteínas , Ácido Hialurónico/química , Interacciones Hidrofóbicas e Hidrofílicas , Trombosis/prevención & control , Propiedades de SuperficieRESUMEN
A new green pathway of in situ electro-leaching coupled with electrochemically switched ion exchange (EL-ESIX) technology was developed for the separation and recovery of valuable metal ions from waste lithium batteries. By using the in situ electro-leaching, the leaching rates of Li+ and Co2+ from the prepared LiCoO2 film electrodes reached 100 % and 93.30 %, respectively, under the combined effect of the acidic microenvironment formed by the anodic electrolytic water and electrostatic repulsion. Subsequently, the Li+ in the electrolyte was further extracted by an electrochemically switched ion exchange (ESIX) process using LiMn2O4 as the film electrode, and Li+ was further enriched in the eluate by a cyclic adsorption and desorption process. The results indicate that the in situ electro-leaching has significant advantages over powder leaching, and for the recycling of waste lithium batteries, the final lithium recovery rate reached 94.51 % by using this in situ EL-ESIX technology.
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Litio , Metales , Intercambio Iónico , Reciclaje/métodos , Suministros de Energía Eléctrica , Iones , ElectrodosRESUMEN
Canopy interception loss affects the local water budget by removing a non-negligible proportion of rainfall from the terrestrial surface. Thus, quantifying interception loss is essential for thoroughly understanding the role of vegetation in the local hydrological cycle, especially in dryland ecosystems. However, sparse shrubs in dryland ecosystems have not been sufficiently studied, owing to time- and labor-intensive field experiments and challenging model parameterization. In this work, 4-year growing season field experiments on rainfall partitioning were conducted for three dominant shrub species (Haloxylon ammodendron, Nitraria sphaerocarpa, and Calligonum mongolicum) in an oasis-desert ecotone in northwestern China. The revised Gash analytical model was well parameterized, which reliably simulated the cumulative interception loss for sparse shrubs, and the validated model performed better for H. ammodendron, followed by C. mongolicum and N. sphaerocarpa, with relative errors of 8.4%, 15.4%, and 23.9%, respectively. The mean individual interception loss percentage for H. ammodendron (28.4%) was significantly higher than that for C. mongolicum (11.0%) and N. sphaerocarpa (10.9%) (p < 0.05), which could be ascribed to the higher canopy storage capacity and wet-canopy evaporation rate of H. ammodendron. For all shrub species, the majority proportion of interception loss occurred during canopy saturation and drying-out periods, accounting for approximately 79-85% of the cumulative interception loss. Overall, the mean local interception loss of three dominant shrub species in the ecotone removed nearly 17% of the corresponding cumulative rainfall during the growing season. These results not only provide methodological references for estimating the interception loss of sparse vegetation in dryland ecosystems, but also provide scientific insights for water resource management and ecosystem restoration in water-limited regions similar to the experimental site.
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Chenopodiaceae , Fabaceae , Ecosistema , Lluvia , Movimientos del Agua , AguaRESUMEN
Rainfall partitioning by the vegetation canopy represents a significant component of the local hydrological cycle by reshaping the amount and spatial distribution of rainfall. Measuring the components of rainfall partitioning, however, has been a challenging task due to laborious- and time-consuming field experiments. In this study, to probe the influences of long-term afforestation on dynamic patterns of rainfall partitioning, the dominant sand-stabilizing shrub Haloxylon ammodendron at three different ages was selected for field measurements during the 2020-2021 growing season. The throughfall percentage for young H. ammodendron (YH, 75.9 %) was significantly higher than that for middle-aged H. ammodendron (MAH, 63.4 %) and mature H. ammodendron (MH, 62.4 %) (p < 0.05 for all cases). However, the interception loss percentage of YH (22.3 %) was significantly lower than that for MAH (35.0 %) and MH (36.5 %) (p < 0.05 for all cases). No significant difference was found for stemflow percentage among YH (1.8 %), MAH (1.5 %) and MH (1.1 %). Smaller rainfall events contributed to a higher interception loss percentage and a lower net rainfall percentage for all ages. Both throughfall and stemflow percentage first showed increasing trends and then tended to be stable with increasing rainfall amount and duration, whereas interception loss percentage showed the opposite patterns. Rainfall partitioning was significantly correlated with the plant area index, stem basal area and canopy height (p < 0.05 for all cases), which may account for significant differences in rainfall partitioning patterns, as all shrubs experienced the same weather conditions. The average funneling ratio was 56.6, 26.7 and 17.9 for YH, MAH and MH, respectively. These results suggested that H. ammodendron afforestation can have a significant impact on rainfall partitioning by reducing net rainfall reaching the soil and may have some implications for local water budget and ecosystem management in oasis-desert ecotones.
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Ecosistema , Lluvia , Arena , Movimientos del Agua , ChinaRESUMEN
Hydrogen energy is a renewable and clean source, which makes a great difference in future sustainable energy systems. Visible-light-driven photocatalysis reaction involves harnessing the abundance of sunlight for hydrogen production among many catalytic technologies. However, the fabrication of photocatalysts that have distinctive performance in visible light is still the primary challenge. Herein, two new Cu-modified polyoxotungstate hybrids, {[Cu2(bim)4(H2O)2](HBW12O40)2·(H2bim)2·8H2O} (1) (bim = [1,1'-methylenebis(1H-imidazole)]) and {[Cu2(bim)4(H2O)2](H3PW10Ti2O40)2·(H2bim)2·8H2O} (2), have been successfully isolated by bridging two saturated Keggin polyoxotungstates and copper-azole complexes. Not surprisingly, 2 holds higher reduction activity due to the more negative charge and stronger basicity on the terminal oxygen of TiâO and bridge oxygen of Ti-O-W. The H2 yield was 17075 µmol g-1 h-1 for 2 in the tunable light-driven H2 production system, which is promising in the field of photocatalysis.
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BACKGROUND: Previously, a novel lamprey immune protein (LIP) was identified, which plays an important role in immunity and the regulation of growth and development in lampreys. However, the mechanism of how LIP regulates growth and development remains unclear. METHODS: In this study, a zebrafish model of LIP overexpression was established by delivering a transgenic plasmid to the fertilized egg. The biological function of LIP was explored in vivo through phenotypic characterization, comparative transcriptome sequencing, and physiological and biochemical analyses. RESULTS: LIP caused developmental toxicity in zebrafish, increased embryo mortality and exhibited strong teratogenic, lethal, and developmental inhibitory effects. Comparative transcriptome analysis showed that LIP-induced large-scale cell death by triggering ferroptosis. Furthermore, LIP-induced lipid peroxidation and caused pericardial edema. Direct inhibition of acsl4a and tfr1a, or silencing of acsl4a and tfr1a with specific siRNA suppressed ferroptosis and pericardial edema. CONCLUSIONS: Taken together, we confirmed that LIP can participate in growth and development via the regulation of lipid peroxidation and ferroptosis. This lays the foundation for future studies on the function of LIP in lampreys. Video Abstract.
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Ferroptosis , Lampreas , Animales , Edema , Desarrollo Embrionario , Pez CebraRESUMEN
Soil aggregates are the material basis of soil structure and important carriers of nutrients. Long-term application of organic and inorganic fertilizers can affect the composition of soil aggregates to varying degrees, which in turn affects the distribution and storage of soil nutrients. We report the results of a 15-year long-term field-based test of aeolian sandy soil and used the wet sieve method to analyze the stability of water-stable aggregates, as well as the distribution characteristics of nutrients in different particle size aggregates. Our results show that long-term application of organic fertilizer (M3) and combined organic−inorganic treatments (NPK1-M1, NPK1-M2, and NPK1-M3) help to increase the amount of organic carbon, inorganic carbon, and cation exchange in the macro-aggregates, and the improvement rates are 92−103%, 8−28%, and 74−85%, respectively. The organic content of the fertilizers also promotes the formation of macro-aggregates, and the stability of aggregates increase from 0.24 to 0.45. In contrast, the application of inorganic fertilizers (NPK1, NPK2, and NPK3) has no marked effect on the formation and stability of macro-aggregates; the application of inorganic fertilizers can merely maintain the organic carbon content of the soil. Correlation analysis shows that the application of organic fertilizers and chemical (inorganic) fertilizers containing phosphorus and potassium can markedly increase the content and reserves of available phosphorus and potassium across all aggregate sizes, and there is a significant positive correlation between these parameters and the amount of applied fertilizer (p < 0.05). Aggregates of various sizes in aeolian sandy soils in arid areas have the potential for greater nutrient storage. Therefore, organic fertilizers can be used in the agricultural production process to improve soil structure and fertility.
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Carbon dioxide (CO2) and the combustion of sulfide in gasoline are the main causes of air pollution. A great deal of attention has been paid to solving the problem and the catalytic reaction seems to be a decent choice. Due to the high-density of Lewis acidic active sites, polyoxometalates are undoubtedly an ideal choice for the sulfur oxidation reaction. With the reasons foregoing, two novel Zn-capped polyoxometalate-based organic-inorganic hybrids, {[α-PMoV2MoVI10O39(OH)Zn2][bbbm]3}·0.5C2H5OH (1) and TBA2{[ε-PMoV8MoVI4O37(OH)3Zn4][phim]3} (2) ((where bbbm = 1-(4-imidazol-1-ylbutyl) imidazole) and phim = 2-phenylimidazole) were successfully obtained by hydrothermal synthesis. In the two compounds, the N-donor ligands in a monodentate or bidentate coordination mode are directly connected to the Keggin anions by Zn-capped atoms, forming an extended one-dimensional chain. It is noteworthy that compound 2 ends up with an interesting spiral infinite chain possibly thanks to the TBA+ cations residing in gaps as structure-directing agents. Simultaneously, the catalytic properties indicate that compounds 1 and2 as efficient heterogeneous catalysts display a decent catalytic activity in the sulfur removal process. Especially, 2 enabled satisfying catalytic oxidation of dibenzothiophene (DBT) to produce more valuable dibenzothiophene sulfone (DBTO2) at 55 °C, and the conversion almost reached 99%. Besides, compound 2 also shows satisfactory catalytic effectiveness in the oxidation of various epoxides in the CO2 cycloaddition reaction, which suggests that compound 2 has the potential to function as a dual functional material with tremendous prospects in sulfur oxidation and carbon dioxide cycloaddition for the first time.
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As a critical evolutionary pivot between invertebrates and vertebrates, lampreys provide rich genetic information. Lamprey immune protein (LIP) is a key immune regulator. MicroRNAs, well-conserved in the response to immunological stress, remain understudied in lamprey immunity. We generated a lamprey microRNA expression atlas, using deep sequencing, upon Vibrio anguillarum infection. Using comparative methods, we found that miR-4561 potentially regulates innate immunity via interaction with lip. We found a sequence in the 3'-UTR region of LIP mRNA complementary to the miR-4561 seed region; miR-4561 expression was negatively correlated with LIP. During V. anguillarum infection, miR-4561 inhibited LIP expression and bacterial clearance. Notably, LIP expression in supraneural body cells was necessary for the Gram-negative immune response. Additionally, we observed that overexpression of miR-4561 induced apoptosis in embryonic cells, suggesting a role in embryonic development. Collectively, we show lamprey microRNAs may significantly affect gene regulation and provide new insights on LIP-mediated immune regulation.
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Enfermedades de los Peces/microbiología , Regulación de la Expresión Génica , Lampreas , MicroARNs/genética , Vibriosis/veterinaria , Vibrio/fisiología , Animales , Labio/metabolismo , Labio/microbiología , MicroARNs/metabolismo , Vibriosis/microbiologíaRESUMEN
Two captivating {P4Mo6}-based compounds, formulated as (H2bbi)2{[Co2(bbi)][Co2.33(H2O)4][H9.33CoP8Mo12O62]}·4H2O (1) and (H2bbi){[Zn(Hbbi)]2[Zn0.75(bbi)][K2Zn(H2O)4][H8.5ZnP8Mo12O62]} (2) [bbi = 1,1'-(1,4-butanediyl)bis(imidazole)], were successfully synthesized under hydrothermal conditions. Structural analysis demonstrates that compounds 1 and 2 are constructed from hourglass-shaped structures [M(P4Mo6O31)2]n- (M = Co, Zn), which are all made up of molybdophosphates and one transition metal ion as the central connecting node. Compounds 1 and 2 feature three-dimensional (3D) frameworks, which are all connected to form a 3D structure by metal ions and bbi ligands. More interestingly, compound 1 exhibits higher catalytic activity than 2 in CO2 photoreduction due to the suitable energy band structure of Co species in {P4Mo6} clusters. The CO yield was 3261 µmol g-1 with high selectivity in 8 h for compound 1 in photocatalytic CO2 reduction, which is highly promising in the photocatalytic field. Additionally, the photoluminescence properties of 2 were investigated.
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MOTIVATION: Malaria, a mosquito-borne infectious disease affecting humans and other animals, is widespread in tropical and subtropical regions. Microscopy is the most common method for diagnosing the malaria parasite from stained blood smear samples. However, this technique is time consuming and must be performed by a well-trained professional, yet it remains prone to errors. Distinguishing the multiple growth stages of parasites remains an especially challenging task. RESULTS: In this article, we develop a novel deep learning approach for the recognition of malaria parasites of various stages in blood smear images using a deep transfer graph convolutional network (DTGCN). To our knowledge, this is the first application of graph convolutional network (GCN) on multi-stage malaria parasite recognition in such images. The proposed DTGCN model is based on unsupervised learning by transferring knowledge learnt from source images that contain the discriminative morphology characteristics of multi-stage malaria parasites. This transferred information guarantees the effectiveness of the target parasite recognition. This approach first learns the identical representations from the source to establish topological correlations between source class groups and the unlabelled target samples. At this stage, the GCN is implemented to extract graph feature representations for multi-stage malaria parasite recognition. The proposed method showed higher accuracy and effectiveness in publicly available microscopic images of multi-stage malaria parasites compared to a wide range of state-of-the-art approaches. Furthermore, this method is also evaluated on a large-scale dataset of unseen malaria parasites and the Babesia dataset. AVAILABILITY: Code and dataset are available at https://github.com/senli2018/DTGCN_2021 under a MIT license.
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Aprendizaje Profundo , Malaria , Parásitos , Animales , Humanos , Malaria/diagnósticoRESUMEN
Polyoxovanadates (POVs), as one of the most prominent members of polyoxometalates (POMs), have been subject to extensive studies by virtue of their aesthetically intriguing structures and potential applications in catalysis, magnetism, and optics, among others. In recent years, organo-functionalized POVs have received considerable attention due to the combination of the advantages of POVs with the importance of organic species. In this review, the key developments of polyoxovanadates and, particularly, the achievements that are related to polyoxovanadates modified with organic ligands and transition metal-organic ligand are summarized. Herein, we systematically introduce the structural features of organo-functionalized POVs and their main applications involved in the magnetism and catalysis aspects. Finally, the current challenges and future prospects in the design, synthesis, and property investigation of polyoxovanadates are also discussed.