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1.
Biomed Mater ; 2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32069444

RESUMO

Efficient attachment of magnetic nanoparticles to cell membranes plays an important role in the activation of cell membrane channels. Streptavidin (SA) was successfully modified to PEI-SPIONs to form SA/PEI-SPIONs which have high colloidal stability and low cytotoxicity. SA/PEI-SPIONs were incubated with PC-12 cells which had been firstly cultured in RPMI Medium 1640 containing 0.2 mg/L biotin for 12 h. The cells were observed by transmission electron microscopy (TEM), and the nanoparticles were clearly attached on the cell membrane, which can be attributed to the specific binding between SA and biotin sites on the cell surface. This work provides a simple way to attach SA modified nanoparticles on the membranes of cells by only culturing cells in biotin containing medium. This work makes possible the biomedical applications that need the nanoparticles to target cell membranes.

2.
J Mater Chem B ; 8(4): 758-766, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31897462

RESUMO

Effective attachment of magnetic nanoparticles to neuronal membranes has far-reaching significance in activating ion channels and treating neurodegenerative diseases. Superparamagnetic iron oxide nanoparticles (SPIONs) synthesized by the polyol pyrolysis method have the advantages of rich surface functional groups, excellent magnetic properties, controllable particle size and water dispersibility. We propose that perfusion of biotin into the targeted brain area should be initially performed because it tends to be adsorbed by cell membranes, followed by injection of streptavidin (SA)-modified SPIONs into the same area of the brain. By means of the strong binding force between SA and biotin, the SPIONs may subsequently adhere to the cell surfaces in the brain area. In this work, fluorescein isothiocyanate-streptavidin (FITC-SA) was modified on the surface of polyethylene imine (PEI)-SPIONs by the EDC-NHS method and stereotaxically injected into the biotin-supplemented substantia nigra of mice. The combination of fluorescence detection with transmission electron microscopy (TEM) confirmed that FITC-SA/PEI-SPIONs adhered to neuronal membranes in the substantia nigra of mice 24 h after injection. The results show that our strategy can promote the attachment of SPIONs to neuronal membranes.

3.
ACS Appl Mater Interfaces ; 12(5): 6788-6792, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31913014

RESUMO

Effective ultraviolet light-emitting diodes (LEDs) were fabricated by clamping the n-ZnO films on the top of p-hBN/p-GaN/sapphire substrates. An ultraviolet emission originating from ZnO was measured from the diode under a forward bias, the electroluminescence (EL) spectra of which show a peak wavelength of ∼376 nm with a narrow full-width at half maximum of ∼12 nm. Compared with the reference diode fabricated by directly growing n-ZnO on the p-hBN substrates using metal-organic chemical vapor deposition, the proposed diode showed a dramatic increment of the EL intensity; meanwhile, its emission onset lowered down considerably. The improved optical property of the proposed LED is mainly ascribed to suppressing the formation of the BNO-related layer at the n-ZnO/p-hBN interface. The present work provides a simple and feasible approach for developing advanced ZnO-based optoelectronic devices.

4.
IEEE Trans Cybern ; 50(3): 946-956, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30346302

RESUMO

This paper is concerned with passivity of a class of delayed neural networks. In order to derive less conservative passivity criteria, two Lyapunov-Krasovskii functionals (LKFs) with delay-dependent matrices are introduced by taking into consideration a second-order Bessel-Legendre inequality. In one LKF, the system state vector is coupled with those vectors inherited from the second-order Bessel-Legendre inequality through delay-dependent matrices, while no such coupling of them exists in the other LKF. These two LKFs are referred to as the coupled LKF and the noncoupled LKF, respectively. A number of delay-dependent passivity criteria are derived by employing a convex approach and a nonconvex approach to deal with the square of the time-varying delay appearing in the derivative of the LKF. Through numerical simulation, it is found that: 1) the coupled LKF is more beneficial than the noncoupled LKF for reducing the conservatism of the obtained passivity criteria and 2) the passivity criteria using the convex approach can deliver larger delay upper bounds than those using the nonconvex approach.

5.
MAbs ; 12(1): 1685814, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31774346

RESUMO

Therapeutic monoclonal antibodies (mAbs) are commonly administered to patients through intravenous (IV) infusion, which involves diluting the medication into an infusion solution (e.g., saline and 5% dextrose). Using the wrong diluent can cause product aggregation, which may compromise patient safety. We and others have shown that Herceptin® (trastuzumab) and Avastin® (bevacizumab) undergo rapid aggregation upon mixing with dextrose and human plasma in vitro. In this study, we evaluated the compatibility of a panel of 11 therapeutic mAbs with dextrose or saline and human serum. These mAbs were randomly selected for their distinct formulations and IgG isotypes (IgG1, IgG2, IgG4, and Fc-fusion protein). All the mAbs appeared to be compatible with saline and human serum. However, mAbs that were formulated at acidic pH (≤ 6.5) exclusively formed insoluble aggregates upon mixing with dextrose and serum. Such aggregation was not detected for the mAbs that are at neutral pH (7.2-7.5) or in buffers containing sodium chloride. Mass spectrometric analysis revealed that the insoluble aggregates were composed of mAb molecules and several serum proteins (e.g., complement proteins, apolipoprotein, fibronectin) that are characterized by an isoelectric point of pH 5.4-6.7. At proximate pH to the isoelectric point values, those abundant serum proteins appeared to undergo isoelectric precipitation with mAb molecules. Our observations highlight a potential risk of protein aggregation at the blood-IV interface if a diluent is incompatible with a specific mAb formulation. This information has implications in guiding the design of product formulations and the selection of the right diluent for intravenous infusion of therapeutic mAbs.Abbreviations: ADC: antibody-drug conjugate; D5W: 5% dextrose in water; IM: intramuscular; IV: intravenous; LC-MS/MS: liquid chromatography-tandem mass spectrometry; mAb: monoclonal antibody; SC: subcutaneous; pI: isoelectric point.

6.
J Nanosci Nanotechnol ; 20(4): 2018-2024, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31492207

RESUMO

Poly(ethylene glycol) (PEG)/poly(ethylene imine) (PEI) modified superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized by the thermal decomposition of iron (III) acetylacetonate (Fe(acac)3) in PEG containing PEI. Transferrin (Tf) was employed to functionalize SPIONs. The potential of Tf-SPIONs as brain magnetic resonance (MR) imaging contrast agents was explored by using Kunming mice as an animal model. The in vivo experiments revealed that Tf-SPIONs exhibited an enhanced contrast time as compared with the PEG-SPIONs and PEG/PEI-SPIONs. Tf-SPIONs exhibit promising potential for bioimaging applications because of their advantages of dispersibility in water, low cytotoxicity and long circulation time in blood.

7.
ACS Chem Neurosci ; 11(2): 197-204, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31867955

RESUMO

Localizing nanoparticles on or near cell membranes in vivo remains a big challenge. We present a cell membrane targeting complex based on chondroitin sulfate (CS)-conjugated superparamagnetic iron oxide nanoparticles (CS-SPIONs). After SPIONs were injected into the substantia nigra of rats, the subcellular distributions of SPIONs with and without CS modification have been evaluated by transmission electron microscopy (TEM) analysis. CS-SPIONs exhibited low toxicity and low endocytosis and were highly distributed in the extracellular spaces nearing neuronal cell bodies and synapses. This can be attributed to the nature of CS, one of the main components of perineuronal nets with the tendency to surround neuronal cell bodies, dendrites, and synapses. It is expected that CS-SPIONs have a great potential for therapies requiring targeting of or approach to cell membranes.

8.
Mol Phylogenet Evol ; 145: 106724, 2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31881327

RESUMO

Rhacophoridae are one of the most speciose and ecologically diverse families of amphibians. Resolution of their evolutionary relationships is key to understanding the accumulation of biodiversity, yet previous hypotheses based on Sanger sequencing exhibit much discordance amongst generic relationships. This conflict precludes the making of sound macroevolutionary conclusions. Herein, we conduct the first phylogenomic study using broad-scale sampling and sequences of 352 nuclear DNA loci obtained using anchored hybrid enrichment targeted sequencing. The robust time-calibrated phylogenetic hypothesis clarifies several long-disputed relationships and facilitates the testing of evolutionary hypotheses on spatiotemporal diversification and reproductive modes. The major extant lineages of Rhacophoridae appear to have radiated in mainland Asia, and the spatiotemporal process corresponds with several common accumulations of biodiversity in Asia. Analyses do not detect any case of "Out of Himalaya" in Rhacophoridae. All transitions of reproductive modes appear to have evolved in an ordered, gradual sequence associated with gaining independence of standing water for larval development. The different reproductive modes are phylogenetically conserved and the completion of their transitions appear to have occurred over a period of ~30 Ma, which does not fit a pattern of a rapid burst of diversification. Innovations in reproductive modes associate statistically with the uneven distribution of species-richness between clades, where higher diversification is linked to increased terrestrial modes of reproduction. These results strengthen the hypothesis that breeding innovations drive diversification by providing new opportunities for ecological release and dispersion.

9.
Mar Pollut Bull ; 144: 11-19, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31179976

RESUMO

In the present study, environmental parameters and macrozoobenthos in the Yangtze River estuary and its adjacent area, China, were investigated in summer and winter of 2015, and significant seasonal differences were found. Biodiversity was found to be lower in summer, which may result from the higher deposition rate. The relationship between the macrozoobenthic community and environmental parameters (especially dissolved oxygen) was revealed. Linear models describing the response of macrozoobenthic biodiversity indexes to mild oxygen deficiency were constructed. Mild seasonal oxygen deficiency was revealed significantly related to seasonal variations of the macrozoobenthic community, but this deficiency could not damage the community. In contrast, when mild oxygen deficiency occurred, natural predators of macrozoobenthos decreased, which may relieve the survival pressure of macrozoobenthos to some extent. Dissolved oxygen alone could not explain many variations of macrozoobenthos, and other environmental parameters, especially water depth, phosphate concentration and turbidity, also greatly contributed to those variations.


Assuntos
Organismos Aquáticos/efeitos dos fármacos , Monitoramento Ambiental/métodos , Estuários , Sedimentos Geológicos/química , Oxigênio/análise , Rios/química , Animais , Organismos Aquáticos/crescimento & desenvolvimento , Biodiversidade , China , Estações do Ano , Especificidade da Espécie
10.
Cell Death Discov ; 5: 102, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31231550

RESUMO

Doxorubicin is an important anticancer drug in the clinic. Unfortunately, it causes cumulative and dose-dependent cardiotoxic side effects. As the population of cancer survivors who have been exposed to treatment continues to grow, there is increased interest in assessing the long-term cardiac effects of doxorubicin and understanding the underlying mechanisms at play. In this study, we investigated doxorubicin-induced transcriptomic changes using RNA-sequencing (RNAseq) and a cellular model comprised of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Analyses of predicted upstream regulators identified the p53 protein as a key regulator of transcriptomic changes induced by doxorubicin. Clustering and pathway analyses showed that increased death receptor (DR) expression and enrichment of the extrinsic apoptotic pathway are significantly associated with doxorubicin-induced cardiotoxicity. Increased expression of p53 and DRs were confirmed via immunoblotting. Our data pinpoints increased DR expression as an early transcriptomic indicator of cardiotoxicity, suggesting that DR expression might function as a predictive biomarker for cardiac damage.

11.
Arterioscler Thromb Vasc Biol ; 39(6): 1212-1226, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31043075

RESUMO

Objective- IL (interleukin)-33 has been shown to play a role in endothelial dysfunction, but its role in atherosclerosis is controversial. Therefore, the purpose of this study is to examine its role in vascular wall remodeling following injury. Approach and Results- Thrombin induced IL-33 expression in a time-dependent manner in human aortic smooth muscle cells and inhibition of its activity by its neutralizing antibody suppressed thrombin induced human aortic smooth muscle cell migration but not DNA synthesis. In exploring the mechanisms, we found that Par1 (protease-activated receptor 1), Gαq/11 (Gα protein q/11), PLCß3 (phospholipase Cß3), NFATc1 (nuclear factor of activated T cells), E2F1 (E2F transcription factor 1), and LMCD1 (LIM and cysteine-rich domains protein 1) are involved in thrombin-induced IL-33 expression and migration. Furthermore, we identified an NFAT-binding site at -100 nt that mediates thrombin-induced IL-33 promoter activity. Interestingly, we observed that NFATc1, E2F1, and LMCD1 bind to NFAT site in response to thrombin and found that LMCD1, while alone has no significant effect, enhanced either NFATc1 or E2F1-dependent IL-33 promoter activity. In addition, we found that guidewire injury induces IL-33 expression in SMC and its neutralizing antibodies substantially reduce SMC migration and neointimal growth in vivo. Increased expression of IL-33 was also observed in human atherosclerotic lesions as compared to arteries without any lesions. Conclusions- The above findings reveal for the first time that thrombin-induced human aortic smooth muscle cell migration and injury-induced neointimal growth require IL-33 expression. In addition, thrombin-induced IL-33 expression requires LMCD1 enhanced combinatorial activation of NFATc1 and E2F1.

12.
Cancer Metastasis Rev ; 38(1-2): 297-305, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31053984

RESUMO

The presence of circulating tumor cells (CTCs) in the bloodstream signals the existence of a tumor and denotes risk of metastatic spread. CTCs can be isolated and analyzed to monitor cancer progression and therapeutic response. However, CTC isolation devices have shown considerable variation in detection rates, limiting their use as a routine diagnostic and monitoring tool. In this review, we discuss recent advances in CTC detection methodologies and associated clinical studies. We provide perspective on the future direction of CTC isolation and molecular characterization towards developing reliable biomarkers that monitor disease progression or therapeutic response.

13.
J Biomed Mater Res A ; 107(9): 1988-1998, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31067350

RESUMO

The impact of the surface modification on the subcellular distribution of nanoparticles in the brain remains elusive. The nanoparticles prepared by conjugating polyethylene glycol and maleic anhydride-coated superparamagnetic iron oxide nanoparticles (Mal-SPIONs) with bovine serum albumin (BSA/Mal-SPIONs) and with Arg-Gly-Asp peptide (RGD/Mal-SPIONs) were injected into the rat substantia nigra. Observation of transmission electron microscopy (TEM) samples obtained 24 h after perfusion showed that abundant RGD/Mal-SPIONs accumulated in the myelin sheath, dendrites, axon terminals and mitochondria, and on cell membranes in the brain tissue near the injection site. For rats injected with BSA/Mal-SPIONs, a few nanoparticles accumulated in the myelin sheath, axon terminals, endoplasmic reticulum, mitochondria, Golgi, and lysosomes of neurons and glial cells while least SPIONs in rats injected with Mal-SPIONs were found. TEM pictures showed some Mal-SPIONs were expelled out of the brain. RGD/Mal-SPIONs diffused extensively to the thalamus, frontal cortex, temporal lobe, olfactory bulb, and brain stem after injection. Only a few BSA/Mal-SPIONs diffused to the afore-mentioned brain areas. This work reveals different surface modifications on the iron oxide nanoparticles play crucial roles in their distribution and diffusion in the rat brains.

14.
ISA Trans ; 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31097192

RESUMO

This paper is concerned with guaranteed cost control for a hybrid-triggered networked system subject to stochastic cyber-attacks. First, a hybrid-triggered mechanism including time-triggered mechanism and event-triggered mechanism is proposed to mitigate the pressure of network transmission, in which the switching between two mechanisms satisfies Bernoulli distribution. Second, the closed-loop system subject to the hybrid communication scheme and stochastic cyber-attacks is modelled as a stochastic system with an interval time-varying delay. Then, based on the Lyapunov-Krasovskii functional approach, two theorems are presented for guaranteeing the mean-square stability of the studied system. Finally, the effectiveness of the proposed method is demonstrated through a numerical example.

15.
Cancers (Basel) ; 11(1)2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30650534

RESUMO

Circulating tumor cells (CTCs) in the peripheral blood are the precursors to distant metastasis but the underlying mechanisms are poorly understood. This study aims at understanding the molecular features within CTCs, in relation to their metastatic potential. Using in vitro CTC models, in which breast cancer cell lines were cultured in non-adherent conditions simulating the microenvironment in the blood stream, we found that the suspension culture resulted in resistance to TNF-related apoptosis inducing ligand (TRAIL)-mediated cell death. Such a resistance was directly correlated with a reduction in surface and total levels of DR5 protein. In the non-adherent state, the cells underwent a rapid autophagic flux, characterized by an accumulation of autophagosome organelles. Notably, DR5 was translocated to the autophagosomes and underwent a lysosomal degradation. Our data suggest that CTCs may evade the TNF cytokine-mediated immune surveillance through a downregulation of the death receptor (DR) expression. The data warrants further studies in cancer patients to find the status of DRs and other molecular features within primary CTCs, in relation to disease progression or chemoresistance.

16.
J Mater Sci Mater Med ; 30(1): 5, 2018 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-30569308

RESUMO

Glutathione-modified superparamagnetic iron oxide nanoparticles (GSH-SPIONs) were prepared by conjugating glutathione (GSH) on the surface of the PEG (Polyethylene glycol)/PEI (polyethyleneimine)-SPIONs which were synthesized by thermal decomposition method. Thermogravimetric analysis showed that the mass fraction of GSH on the surface of SPIONs was 30.64 wt%. GSH-SPIONs in PBS were injected into the substantia nigra of rat brains. The subcellular distributions of the nanoparticles in the brains was examined by the transmission electron microscope (TEM). A remarkable amount of GSH-SPIONs were found in vesicles inside cell bodies and axons, and in mitochondria. TEM pictures show that GSH-SPIONs enter the neuronal cells by endocytosis and travel through axoplasmic transport. GSH-SPIONs have great potential as drug delivery agents in the brain to treat diseases or study brain function via mitochondria-targeting way or axoplasmic transport way.


Assuntos
Glutationa/química , Glutationa/farmacocinética , Nanopartículas de Magnetita/química , Substância Negra/metabolismo , Animais , Sobrevivência Celular , Células PC12 , Ratos , Substância Negra/química , Distribuição Tecidual
17.
ACS Appl Mater Interfaces ; 10(38): 32289-32297, 2018 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-30183248

RESUMO

In this study, a dual-source vapor evaporation method was employed to fabricate the high-quality CsPbBr3 thin films with a good crystalline and high surface coverage. Temperature-dependent and excitation power-dependent photoluminescence measurements were performed to study the optical properties of the CsPbBr3 material. Further, based on the experimental data, the temperature sensitivity coefficient of band gap and exciton binding energy were estimated. More importantly, for the first time, we designed and prepared a hole-injection layer-free perovskite light-emitting diode (LED) based on the Au/MgO/CsPbBr3/n-MgZnO/n+-GaN structure, producing an intense green emission (∼538 nm) with a high purity. Besides, the device demonstrated a high luminance of 5025 cd/m2, an external quantum efficiency of 1.46%, a current efficiency of 1.92 cd/A, and a power efficiency of 1.76 lm/W. We studied in detail the current-voltage and electroluminescence properties of the prepared device and proposed the hole generation models and the carrier transport/recombination mechanisms to make these interesting characteristics certain. The results obtained would provide a new and effective strategy for the design and preparation of perovskite LEDs.

18.
Oncotarget ; 9(33): 23264-23273, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29796187

RESUMO

TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis by engaging its death receptors (DRs) 4 and/or 5 on targeted cells. Clinical attempts to stimulate this apoptotic pathway for cancer therapy, including the use of recombinant human TRAIL (rhTRAIL) or receptor agonistic antibodies, have been underway for over a decade. Unfortunately, these agents have only shown limited therapeutic effects due largely to tumor resistance arising from mechanisms yet to be defined. Here we show that intermediate filament proteins, keratin 8 and keratin 18 (K8/K18), negatively regulate TRAIL induced apoptosis. K8/K18 protein levels are consistently higher in TRAIL-resistant cells compared to TRAIL-sensitive cells in a panel of breast cancer cell lines. Blockade of K8 increased expression of DR5 on the surface of targeted cells and sensitized the cells to TRAIL-induced apoptosis. Conversely, ectopic expression of K8/K18 downregulated DR5 protein expression. K8/K18 appears to negatively regulate apoptosis signaling via DR5 in breast cancer cells. Our findings warrant additional studies to determine if K8/K18 could be a predictor of tumor resistance to DR5-targeted therapies.

19.
Curr Biol ; 28(10): R590-R592, 2018 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-29787716

RESUMO

Overexploitation, habitat destruction, human-driven climate change and disease spread are resulting in the extinction of innumerable species, with amphibians being hit harder than most other groups [1]. Few species of amphibians are widespread, and those that are often represent complexes of multiple cryptic species. This is especially true for range-restricted salamanders [2]. Here, we used the widespread and critically endangered Chinese giant salamander (Andrias davidianus) to show how genetically uninformed management efforts can negatively affect species conservation. We find that this salamander consists of at least five species-level lineages. However, the extensive recent translocation of individuals between farms, where the vast majority of extant salamanders now live, has resulted in genetic homogenization. Mitochondrial DNA (mtDNA) haplotypes from northern China now predominate in farms. Unfortunately, hybrid offspring are being released back into the wild under well-intentioned, but misguided, conservation management. Our findings emphasize the necessity of genetic assessments for seemingly well-known, widespread species in conservation initiatives. Species serve as the primary unit for protection and management in conservation actions [3], so determining the taxonomic status of threatened species is a major concern, especially for amphibians. The level of threat to amphibians may be underestimated, and existing conservation strategies may be inadvertently harmful if conducted without genetic assessment.


Assuntos
Conservação dos Recursos Naturais/métodos , Espécies em Perigo de Extinção , Variação Genética , Hibridização Genética , Urodelos , Animais , Ecossistema , Genética Populacional , Urodelos/classificação , Urodelos/genética
20.
Proc Natl Acad Sci U S A ; 115(22): E5056-E5065, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29760079

RESUMO

Tibetan frogs, Nanorana parkeri, are differentiated genetically but not morphologically along geographical and elevational gradients in a challenging environment, presenting a unique opportunity to investigate processes leading to speciation. Analyses of whole genomes of 63 frogs reveal population structuring and historical demography, characterized by highly restricted gene flow in a narrow geographic zone lying between matrilines West (W) and East (E). A population found only along a single tributary of the Yalu Zangbu River has the mitogenome only of E, whereas nuclear genes of W comprise 89-95% of the nuclear genome. Selection accounts for 579 broadly scattered, highly divergent regions (HDRs) of the genome, which involve 365 genes. These genes fall into 51 gene ontology (GO) functional classes, 14 of which are likely to be important in driving reproductive isolation. GO enrichment analyses of E reveal many overrepresented functional categories associated with adaptation to high elevations, including blood circulation, response to hypoxia, and UV radiation. Four genes, including DNAJC8 in the brain, TNNC1 and ADORA1 in the heart, and LAMB3 in the lung, differ in levels of expression between low- and high-elevation populations. High-altitude adaptation plays an important role in maintaining and driving continuing divergence and reproductive isolation. Use of total genomes enabled recognition of selection and adaptation in and between populations, as well as documentation of evolution along a stepped cline toward speciation.


Assuntos
Anuros/genética , Anuros/fisiologia , Fluxo Gênico/genética , Especiação Genética , Animais , Hibridização Genética , Metagenômica , Filogenia , Seleção Genética , Tibet
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