Whole genomes show contrasting trends of population size changes and genomic diversity for an Amazonian endemic passerine over the late quaternary.

The "Amazon tipping point" is a global change scenario resulting in replacement of upland terra-firme forests by large-scale "savannization" of mostly southern and eastern Amazon. Reduced rainfall accompanying the Last Glacial Maximum (LGM) has been proposed to have acted as such a tipping point in the past, with the prediction that terra-firme inhabiting species should have experienced reductions in population size as drier habitats expanded. Here, we use whole-genomes of an Amazonian endemic organism (Scale-backed antbirds - Willisornis spp.) sampled from nine populations across the region to test this historical demography scenario. Populations from southeastern Amazonia and close to the Amazon-Cerrado ecotone exhibited a wide range of demographic patterns, while most of those from northern and western Amazonia experienced uniform expansions between 400 kya and 80-60 kya, with gradual declines toward 20 kya. Southeastern populations of Willisornis were the last to diversify and showed smaller heterozygosity and higher runs of homozygosity values than western and northern populations. These patterns support historical population declines throughout the Amazon that affected more strongly lineages in the southern and eastern areas, where historical "tipping point" conditions existed due to the widespread replacement of humid forest by drier and open vegetation during the LGM.

Statistical Improvement of rGILCC 1 and rPOXA 1B Laccases Activity Assay Conditions Supported by Molecular Dynamics.

Laccases (E.C. 1.10.3.2) are glycoproteins widely distributed in nature. Their structural conformation includes three copper sites in their catalytic center, which are responsible for facilitating substrate oxidation, leading to the generation of H2O instead of H2O2. The measurement of laccase activity (UL-1) results may vary depending on the type of laccase, buffer, redox mediators, and substrates employed. The aim was to select the best conditions for rGILCC 1 and rPOXA 1B laccases activity assay. After sequential statistical assays, the molecular dynamics proved to support this process, and we aimed to accumulate valuable insights into the potential application of these enzymes for the degradation of novel substrates with negative environmental implications. Citrate buffer treatment T2 (CB T2) (pH 3.0 ± 0.2; λ420nm, 2 mM ABTS) had the most favorable results, with 7.315 ± 0.131 UL-1 for rGILCC 1 and 5291.665 ± 45.83 UL-1 for rPOXA 1B. The use of citrate buffer increased the enzyme affinity for ABTS since lower Km values occurred for both enzymes (1.49 × 10-2 mM for rGILCC 1 and 3.72 × 10-2 mM for rPOXA 1B) compared to those obtained in acetate buffer (5.36 × 10-2 mM for rGILCC 1 and 1.72 mM for rPOXA 1B). The molecular dynamics of GILCC 1-ABTS and POXA 1B-ABTS showed stable behavior, with root mean square deviation (RMSD) values not exceeding 2.0 Å. Enzyme activities (rGILCC 1 and rPOXA 1B) and 3D model-ABTS interactions (GILCC 1-ABTS and POXA 1B-ABTS) were under the strong influence of pH, wavelength, ions, and ABTS concentration, supported by computational studies identifying the stabilizing residues and interactions. Integration of the experimental and computational approaches yielded a comprehensive understanding of enzyme-substrate interactions, offering potential applications in environmental substrate treatments.

Structural Perspective into the Interaction of an Oncogenesis-Relevant pre-miRNA G-Quadruplex Ligand Carrier with the Protein Nucleolin.

The structural determinants of the interaction of the G-quadruplex (G4) motif found in precursor miRNA 149 (rG4) with the acridine orange derivative C8 , a G4 ligand stabilizer possessing anticancer activity, and the protein nucleolin (overexpressed in cancer cells) were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy. For the rG4/C8 complex, the results revealed a strong stabilizing interaction between the aromatic core and the iodinated ring of the C8 ligand with the rG4 structure. The NMR study revealed also different interaction patterns between nucleolin and rG4 and nucleolin and rG4/C8 complex. In the absence of the ligand, rG4 establishes interactions with polar residues of the protein while for the rG4/C8 complex, these contacts are mainly established with amino acids that have hydrophobic side chains. However, nucleolin chemical shift perturbation studies in the presence of rG4 or rG4/C8 reveal the same location between domains 1 and 2 of the protein, which suggests that the rG4 and rG4/C8 complex bind in this region. This puzzling structural study opens a new framework to study rG4/ligand/nucleolin complexes that might impact the biogenesis of miRNA 149.

A Shift in Pathotype Diversity and Complexity of Phytophthora sojae in Brazil.

Soybean root and stem rot caused by the oomycete Phytophthora sojae is a destructive disease worldwide that can affect plants at any growth stage. The use of resistant cultivars is the most effective method of controlling the disease. Therefore, monitoring changes in the population of P. sojae regarding the dynamics of avirulence genes capable of overcoming resistance genes (Rps) is important to reduce yield losses and to enhance the effectiveness of the Rps genes. Forty isolates of P. sojae sampled from a region of high incidence of soybean root and stem rot in Brazil were characterized using 14 soybean differentials, and 28 pathotypes were identified. Compared with a study conducted a decade ago, there was a major shift in pathotype diversity and complexity toward both higher numbers of different pathotypes and of avirulence genes in a given individual in the current population of P. sojae. Breeding programs aiming at developing soybean cultivars with resistance to root and stem rot should consider the high variability in the population of P. sojae and seek for strategic deployment of genes and germplasm.

Impact of Antibiotics as Waste, Physical, Chemical, and Enzymatical Degradation: Use of Laccases.

The first traces of Tetracycline (TE) were detected in human skeletons from Sudan and Egypt, finding that it may be related to the diet of the time, the use of some dyes, and the use of soils loaded with microorganisms, such as Streptomyces spp., among other microorganisms capable of producing antibiotics. However, most people only recognise authors dating between 1904 and 1940, such as Ehrlich, Domagk, and Fleming. Antibiotics are the therapeutic option for countless infections treatment; unfortunately, they are the second most common group of drugs in wastewaters worldwide due to failures in industrial waste treatments (pharmaceutics, hospitals, senior residences) and their irrational use in humans and animals. The main antibiotics problem lies in delivered and non-prescribed human use, use in livestock as growth promoters, and crop cultivation as biocides (regulated activities that have not complied in some places). This practice has led to the toxicity of the environment as antibiotics generate eutrophication, water pollution, nutrient imbalance, and press antibiotic resistance. In addition, the removal of antibiotics is not a required process in global wastewater treatment standards. This review aims to raise awareness of the negative impact of antibiotics as residues and physical, chemical, and biological treatments for their degradation. We discuss the high cost of physical and chemical treatments, the risk of using chemicals that worsen the situation, and the fact that each antibiotic class can be transformed differently with each of these treatments and generate new compounds that could be more toxic than the original ones; also, we discuss the use of enzymes for antibiotic degradation, with emphasis on laccases.

Lethal and sublethal toxicity of pesticides and vinasse used in sugarcane cultivation to Ceriodaphnia silvestrii (Crustacea: Cladocera).

With the growing use of agrochemicals in Brazil, there is also a growing need for more realistic toxicity assessments that aid in understanding the potential risks of environmental-realistic agrochemical (mixture) exposures in the natural ecosystems. The aim of the present study was therefore to evaluate the lethal and sublethal effects of environmental realistic (single and mixture) concentrations of the pesticides DMA® 806 BR (active ingredient - a.i. 2,4-D) and Regent® 800 WG (a.i. fipronil) and sugarcane vinasse to the Neotropical cladoceran Ceriodaphnia silvestrii. This evaluation was carried out through lethal (survival), sublethal (reproduction and intrinsic rates of population increase - r) and post-exposure (feeding rate and also reproduction) tests conducted in situ and with water from mesocosms contaminated with the recommended doses of these compounds. The results showed high acute toxicity for treatments containing fipronil and vinasse when acting in isolation, with survival rates only returning to control values on the last sampling day (75 days post application). Reproduction of surviving cladocerans was reduced in all treatments until the end of the experiment and were potentiated effect in the mixture of the three test compounds. The intrinsic rates of population increase were reduced in all treatments except the single 2,4-D treatment. Post-exposure feeding rate and reproduction, however, were not impaired under the conditions analyzed. The results show the high toxicity of recommended doses of fipronil and vinasse (and especially their mixture) and the importance of evaluating the risks of agrochemical mixtures at environmental-realistic concentrations.

Recognition of nucleolin through interaction with RNA G-quadruplex.

The development of novel biomarkers for early-stage diagnosis of prostate cancer (PCa) has attracted the attention of researchers in the last decade. Nucleolin (NCL) has emerged as a possible biomarker of PCa due to its high expression levels in the surface of PCa cells and affinity towards parallel G4s since it contains four RNA-binding domains (RBDs). Herein, we developed a novel strategy based on a microfluidic platform for the detection of NCL in biological samples, such as human plasma. The RNA G4 (rG4) sequence found in human precursor microRNA 92b (pre-miR-92b) was used as a molecular recognition probe since it forms a single dominant parallel rG4 conformation in the presence of 0.1 mM K+ as confirmed by NMR spectroscopy. The additional stability of the rG4 structure was provided by the acridine orange derivative ligand C8, which stabilizes the pre-miR-92b rG4 structure, as denoted by an increase in more than 30 °C of its melting temperature. FRET-melting assay revealed a remarkable synergistic effect of NCL RBD1,2 and C8 on the stabilization of the pre-miR-92b rG4. The binding of pre-miR-92b to NCL RBD1,2 was determined by in silico studies, which revealed a binding pocket formed by a 12-residue linker between RBD1 and RBD2. Both, pre-miR-92b rG4 and pre-miR-92b rG4/C8 complex demonstrated high affinity towards NCL RBD1,2, as proved by fluorimetric titrations (KD range between 10-12 and 10-9 M). The stability and nuclease resistance of pre-miR-92b rG4 and pre-miR-92b rG4/C8 complex were evaluated as molecular recognition probes to capture and detect NCL. Finally, the microfluidic platform detects NCL in complex biological samples, such as human plasma. Overall, this work demonstrates the usefulness of the microfluidic platform based on the pre-miR-92b to detect NCL and the possibility to be used as a valuable biomedical tool in PCa diagnosis.

Acute and chronic toxicity of 2,4-D and fipronil formulations (individually and in mixture) to the Neotropical cladoceran Ceriodaphnia silvestrii.

Brazil is the largest producer of sugarcane and the world's top pesticide market. Therefore, environmental consequences are of concern. The aim of the present study was to evaluate the acute and chronic toxicity of pesticide formulations largely used in sugarcane crops: the herbicide DMA® 806 BR (a.i. 2,4-D) and the insecticide Regent® 800 WG (a.i. fipronil), isolated and in mixture, to the Neotropical cladoceran Ceriodaphnia silvestrii. Toxicity tests with the individual formulated products indicated 48h-EC50 values of 169 ± 18 mg a.i./L for 2,4-D and 3.9 ± 0.50 µg a.i./L for fipronil. In the chronic tests, the 8d-EC50 values for reproduction were 55 mg a.i./L (NOEC/LOEC: 50/60 mg a.i./L) and 1.6 µg a.i./L (NOEC/LOEC: 0.40/0.80 µg a.i./L) for 2,4-D and fipronil, respectively. A significant decrease in reproduction of C. silvestrii in all concentrations tested of fipronil, except at the lowest, was observed. Regarding 2,4-D, the organisms had total inhibition of reproduction in the two highest concentrations. Probably your energy reallocation was focused (trade-off) only on its survival. The acute pesticide mixture toxicity (immobility) revealed a dose level dependent deviation with antagonism at low and synergism at high concentrations. For chronic mixture (reproduction) toxicity, antagonism occurred as a result of the interaction of the pesticides. Based on our results and concentrations measured in Brazilian water bodies, fipronil represents ecological risks for causing direct toxic effects on C. silvestrii. These results are worrisome given that agricultural production is likely to increase in the coming years.

Occurrence, antibiotic-resistance and virulence of E. coli strains isolated from mangrove oysters (Crassostrea gasar) farmed in estuaries of Amazonia.

Concentration of bacterial species indicative of fecal contamination in the gut of mangrove oysters (Crassostrea gasar) is a major concern for public health and food surveillance. Our work aimed to determine the occurrence, antibiotic-resistance, phylogenetic profile and virulence of Escherichia coli strains isolated from C. gasar farmed in four estuaries of Amazonia. Santo Antônio de Urindeua was the sampling point with the highest number of E. coli cells in oyster samples (104 per 100 g of sample). Twenty-four isolates (52.2%) showed resistance to cephalotin and 18 to amoxicillin (39.1%). Eighteen clonal populations were determined by rep-PCR and were mainly affiliated to the pathogenic and commensal phylo-groups B1 and D. The presence of elt genes suggests that 10 of these clones belong to the Enterotoxigenic Escherichia coli pathotype. Plasmids, mostly of the F incompatibility group, were detected in the majority of the strains. All isolates were susceptible to last-resort antibiotics.

Parrot Genomes and the Evolution of Heightened Longevity and Cognition.

Parrots are one of the most distinct and intriguing groups of birds, with highly expanded brains [1], highly developed cognitive [2] and vocal communication [3] skills, and a long lifespan compared to other similar-sized birds [4]. Yet the genetic basis of these traits remains largely unidentified. To address this question, we have generated a high-coverage, annotated assembly of the genome of the blue-fronted Amazon (Amazona aestiva) and carried out extensive comparative analyses with 30 other avian species, including 4 additional parrots. We identified several genomic features unique to parrots, including parrot-specific novel genes and parrot-specific modifications to coding and regulatory sequences of existing genes. We also discovered genomic features under strong selection in parrots and other long-lived birds, including genes previously associated with lifespan determination as well as several hundred new candidate genes. These genes support a range of cellular functions, including telomerase activity; DNA damage repair; control of cell proliferation, cancer, and immunity; and anti-oxidative mechanisms. We also identified brain-expressed, parrot-specific paralogs with known functions in neural development or vocal-learning brain circuits. Intriguingly, parrot-specific changes in conserved regulatory sequences were overwhelmingly associated with genes that are linked to cognitive abilities and have undergone similar selection in the human lineage, suggesting convergent evolution. These findings bring novel insights into the genetics and evolution of longevity and cognition, as well as provide novel targets for exploring the mechanistic basis of these traits.

Fluorescent light-up acridine orange derivatives bind and stabilize KRAS-22RT G-quadruplex.

KRAS is often found mutated in lethal cancers and should be an important target for anticancer drug development. However, no effective inhibitor has been reported so far, prompting the scientific community to describe the RAS proteins as nearly "undruggable". Recent approaches developed to modulate KRAS protein expression comprises the targeting of G-quadruplex (G4) structures formed within the nuclease hypersensitive element of KRAS promoter region, by designing small and specific ligands to stabilize the tertiary fold and reduce gene expression. In this work, we report in vitro and in silico studies of novel acridine orange (AO) derivatives (C3-C8), developed as G4 stabilizing agents. The results show that the ligands bind with high affinity and stabilize KRAS22-RT G4 with modest specificity over duplex DNA. The most promising ligand C8 stabilizes the structure by ≈ 40 °C. Molecular docking using NMR-derived distance restraints reveal atomic details about the ligand structural features in the interaction with KRAS22-RT G4. In vitro studies with HeLa cells show that the ligands are cytotoxic with IC50 values between 0.9 µM and 5.7 µM. Moreover, the ligands tend to localize in the nucleus as shown by confocal fluorescence microscopy. Overall, these results show that the reported AO ligands display favourable properties as G4 ligands and this study provides structural detail for the development of lead KRAS G4 ligands.

Reconstruction of the Fatty Acid Biosynthetic Pathway of Exiguobacterium antarcticum B7 Based on Genomic and Bibliomic Data.

Exiguobacterium antarcticum B7 is extremophile Gram-positive bacteria able to survive in cold environments. A key factor to understanding cold adaptation processes is related to the modification of fatty acids composing the cell membranes of psychrotrophic bacteria. In our study we show the in silico reconstruction of the fatty acid biosynthesis pathway of E. antarcticum B7. To build the stoichiometric model, a semiautomatic procedure was applied, which integrates genome information using KEGG and RAST/SEED. Constraint-based methods, namely, Flux Balance Analysis (FBA) and elementary modes (EM), were applied. FBA was implemented in the sense of hexadecenoic acid production maximization. To evaluate the influence of the gene expression in the fluxome analysis, FBA was also calculated using the log2⁡FC values obtained in the transcriptome analysis at 0°C and 37°C. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity of E. antarcticum B7 to de novo produce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contribute to better understanding of the bacterial adaptation at cold environments.

De novo synthesis of fatty acids is regulated by FapR protein in Exiguobacterium antarcticum B7, a psychrotrophic bacterium isolated from Antarctica.

BACKGROUND: FapR protein from the psychrotrophic species Exiguobacterium antarcticum B7 was expressed and purified, and subsequently evaluated for its capacity to bind to the promoter regions of the fabH1-fabF and fapR-plsX-fabD-fabG operons, using electrophoretic mobility shift assay. The genes that compose these operons encode for enzymes involved in the de novo synthesis of fatty acids molecules. In Bacillus subtilis, FapR regulates the expression of these operons, and consequently has influence in the synthesis of long or short-chain fatty acids. To analyze the bacterial cold adaptation, this is an important metabolic pathway because psychrotrophic microrganisms tend to synthesize short and branched-chain unsaturated fatty acids at cold to maintain cell membrane fluidity. RESULTS: In this work, it was observed that recombinant protein was able to bind to the promoter of the fully amplified fabH1-fabF and fapR-plsX-fabD-fabG operons. However, FapR was unable to bind to the promoter of fapR-plsX-fabD-fabG operon when synthesized only up to the protein-binding palindrome 5'-TTAGTACCAGATACTAA-3', thus showing the importance of the entire promoter sequence for the correct protein-DNA interaction. CONCLUSIONS: Through this observation, we demonstrate that the FapR protein possibly regulates the same operons as described for other species, which emphasizes its importance to cold adaptation process of E. antarcticum B7, a psychrotrophic bacterium isolated at Antarctica.

Corynebacterium pseudotuberculosis RNA-seq data from abiotic stresses.

Corynebacterium pseudotuberculosis causes significant loss to goat and sheep farmers because it is the causal agent of the infectious disease caseous lymphadenitis, which may lead to outcomes ranging from skin injury to animal death (Ruiz et al., 2011) [1]. This bacterium was grown under osmotic (2 M), acid (pH) and heat (50 °C) stress and under control (Normal-BHI brain heart infusion) conditions, which simulate the conditions faced by the bacteria during the infectious process. Subsequently, cDNA of each condition was sequenced by the SOLiD3 Plus platform using the RNA-Seq technique [2], [3], [4]. The data produced was processed to evaluate the differential gene expression, which is helpful to understand the adaptation mechanisms during the infection in the host. The sequencing data of all conditions are available in the European Bioinformatics Institute (EBI) repository under accession number E-MTAB-2017.

Exposure to an extremely low-frequency electromagnetic field only slightly modifies the proteome of Chromobacterium violaceumATCC 12472.

Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field.

The impact of quality filter for RNA-Seq.

BACKGROUND: With the emergence of large-scale sequencing platforms since 2005, there has been a great revolution regarding methods for decoding DNA sequences, which have also affected quantitative and qualitative gene expression analyses through the RNA-Sequencing technique. However, issues related to the amount of data required for the analyses have been considered because they affect the reliability of the experiments. Thus, RNA depletion during sample preparation may influence the results. Moreover, because data produced by these platforms show variations in quality, quality filters are often used to remove sequences likely to contain errors to increase the accuracy of the results. However, when reads of quality filters are removed, the expression profile in RNA-Seq experiments may be influenced. RESULT: The present study aimed to analyze the impact of different quality filter values for Corynebacterium pseudotuberculosis (sequenced by SOLiD platform), Microcystis aeruginosa and Kineococcus radiotolerans (sequenced by Illumina platform) RNA-Seq data. Although up to 47.9% of the reads produced by the SOLiD technology were removed after the QV20 quality filter is applied, and 15.85% were removed from K. radiotolerans data set using the QV30 filter, Illumina data showed the largest number of unique differentially expressed genes after applying the most stringent filter (QV30), with 69 genes. In contrast, for SOLiD, the acid stress condition with the QV20 filter yielded only 41 unique differentially expressed genes. Even for the highest quality M. aeruginosa data, the quality filter affected the expression profile. The most stringent quality filter generated a greater number of unique differentially expressed genes: 9 for high molecular weight dissolved organic matter condition and 12 for low P conditions. CONCLUSION: Even high-accuracy sequencing technologies are subject to the influence of quality filters when evaluating RNA-Seq data using the reference approach.

Genome Sequence of Corynebacterium pseudotuberculosis MB20 bv. equi Isolated from a Pectoral Abscess of an Oldenburg Horse in California.

The genome of Corynebacterium pseudotuberculosis MB20 bv. equi was sequenced using the Ion Personal Genome Machine (PGM) platform, and showed a size of 2,363,089 bp, with 2,365 coding sequences and a GC content of 52.1%. These results will serve as a basis for further studies on the pathogenicity of C. pseudotuberculosis bv. equi.

Omics profiles used to evaluate the gene expression of Exiguobacterium antarcticum B7 during cold adaptation.

BACKGROUND: Exiguobacterium antarcticum strain B7 is a Gram-positive psychrotrophic bacterial species isolated in Antarctica. Although this bacteria has been poorly studied, its genome has already been sequenced. Therefore, it is an appropriate model for the study of thermal adaptation. In the present study, we analyzed the transcriptomes and proteomes of E. antarcticum B7 grown at 0°C and 37°C by SOLiD RNA-Seq, Ion Torrent RNA-Seq and two-dimensional difference gel electrophoresis tandem mass spectrometry (2D-DIGE-MS/MS). RESULTS: We found expression of 2,058 transcripts in all replicates from both platforms and differential expression of 564 genes (absolute log2FC≥1, P-value<0.001) comparing the two temperatures by RNA-Seq. A total of 73 spots were differentially expressed between the two temperatures on 2D-DIGE, 25 of which were identified by MS/MS. Some proteins exhibited patterns of dispersion in the gel that are characteristic of post-translational modifications. CONCLUSIONS: Our findings suggest that the two sequencing platforms yielded similar results and that different omic approaches may be used to improve the understanding of gene expression. To adapt to low temperatures, E. antarcticum B7 expresses four of the six cold-shock proteins present in its genome. The cold-shock proteins were the most abundant in the bacterial proteome at 0°C. Some of the differentially expressed genes are required to preserve transcription and translation, while others encode proteins that contribute to the maintenance of the intracellular environment and appropriate protein folding. The results denote the complexity intrinsic to the adaptation of psychrotrophic organisms to cold environments and are based on two omic approaches. They also unveil the lifestyle of a bacterial species isolated in Antarctica.

Radiolabeled mannosylated dextran derivatives bearing an NIR-fluorophore for sentinel lymph node imaging.

Current methods for sentinel lymph node (SLN) mapping involve the use of radioactivity detection with technetium-99m sulfur colloid and/or visually guided identification using a blue dye. To overcome the kinetic variations of two individual imaging agents through the lymphatic system, we report herein on two multifunctional macromolecules, 5a and 6a, that contain a radionuclide ((99m)Tc or (68)Ga) and a near-infrared (NIR) reporter for pre- and/or intraoperative SLN mapping by nuclear and NIR optical imaging techniques. Both bimodal probes are dextran-based polymers (10 kDa) functionalized with pyrazole-diamine (Pz) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelating units for labeling with fac-[(99m)Tc(CO)3](+) or (68)Ga(III), respectively, mannose units for receptor targeting, and NIR fluorophore units for optical imaging. The probes allowed a clear visualization of the popliteal node by single-photon emission computed tomography (SPECT/CT) or positron emission tomography (PET/CT), as well as real-time optically guided excision. Biodistribution studies confirmed that both macromolecules present a significant accumulation in the popliteal node (5a: 3.87 ± 0.63% IA/organ; 6a: 1.04 ± 0.26% IA/organ), with minimal spread to other organs. The multifunctional nanoplatforms display a popliteal extraction efficiency >90%, highlighting their potential to be further explored as dual imaging agents.