RESUMO
Many insects utilise the polarisation pattern of the sky to adjust their travelling directions. The extraction of directional information from this sky-wide cue is mediated by specialised photoreceptors located in the dorsal rim area (DRA). While this part of the eye is known to be sensitive to the ultraviolet, blue or green component of skylight, the latter has only been observed in insects active in dim light. To address the functional significance of green polarisation sensitivity, we define the spectral and morphological adaptations of the DRA in a nocturnal ball-rolling dung beetle-the only family of insects demonstrated to orient to the dim polarisation pattern in the night sky. Intracellular recordings revealed polarisation-sensitive green photoreceptors in the DRA of Escarabaeus satyrus. Behavioural experiments verified the navigational relevance of this finding. To quantify the adaptive value of green sensitivity for celestial orientation at night, we also obtained the polarisation properties of the night sky in the natural habitat of the beetle. Calculations of relative photon catch revealed that under a moonlit sky the green-sensitive DRA photoreceptors can be expected to catch an order of magnitude more photons compared with the UV-sensitive photoreceptors in the main retina. The green-sensitive photoreceptors - which also show a range of morphological adaptations for enhanced sensitivity - provide E. satyrus with a highly sensitive system for the extraction of directional information from the night sky.
Assuntos
Besouros , Luz , Animais , Besouros/fisiologia , Visão Ocular , Células Fotorreceptoras , Retina/fisiologiaRESUMO
The relationship between pangolin-CoV and SARS-CoV-2 has been a subject of debate. Further evidence of a special relationship between the two viruses can be found by the fact that all known COVID-19 viruses have an abnormally hard outer shell (low M disorder, i.e., low content of intrinsically disordered residues in the membrane (M) protein) that so far has been found in CoVs associated with burrowing animals, such as rabbits and pangolins, in which transmission involves virus remaining in buried feces for a long time. While a hard outer shell is necessary for viral survival, a harder inner shell could also help. For this reason, the N disorder range of pangolin-CoVs, not bat-CoVs, more closely matches that of SARS-CoV-2, especially when Omicron is included. The low N disorder (i.e., low content of intrinsically disordered residues in the nucleocapsid (N) protein), first observed in pangolin-CoV-2017 and later in Omicron, is associated with attenuation according to the Shell-Disorder Model. Our experimental study revealed that pangolin-CoV-2017 and SARS-CoV-2 Omicron (XBB.1.16 subvariant) show similar attenuations with respect to viral growth and plaque formation. Subtle differences have been observed that are consistent with disorder-centric computational analysis.
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COVID-19 , Pangolins , SARS-CoV-2 , SARS-CoV-2/patogenicidade , Animais , COVID-19/virologia , COVID-19/transmissão , Pangolins/virologia , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Biologia Computacional/métodos , FosfoproteínasRESUMO
The behavioural state of animals has profound effects on neuronal information processing. Locomotion changes the response properties of visual interneurons in the insect brain, but it is still unknown if it also alters the response properties of photoreceptors. Photoreceptor responses become faster at higher temperatures. It has therefore been suggested that thermoregulation in insects could improve temporal resolution in vision, but direct evidence for this idea has so far been missing. Here, we compared electroretinograms from the compound eyes of tethered bumblebees that were either sitting or walking on an air-supported ball. We found that the visual processing speed strongly increased when the bumblebees were walking. By monitoring the eye temperature during recording, we saw that the increase in response speed was in synchrony with a rise in eye temperature. By artificially heating the head, we show that the walking-induced temperature increase of the visual system is sufficient to explain the rise in processing speed. We also show that walking accelerates the visual system to the equivalent of a 14-fold increase in light intensity. We conclude that the walking-induced rise in temperature accelerates the processing of visual information-an ideal strategy to process the increased information flow during locomotion.
Assuntos
Luz , Percepção Visual , Animais , Abelhas , Locomoção , Caminhada , Tempo de ReaçãoRESUMO
Spatial memory helps animals to navigate familiar environments. In insects, spatial memory has extensively been studied in central place foragers such as ants and bees. However, if butterflies memorize a spatial location remains unclear. Here, we conducted behavioural experiments to test whether monarch butterflies (Danaus plexippus) can remember and retrieve the spatial location of a food source. We placed several visually identical feeders in a flight cage, with only one feeder providing sucrose solution. Across multiple days, individual butterflies predominantly visited the rewarding feeder. Next, we displaced a salient landmark close to the feeders to test which visual cue the butterflies used to relocate the rewarding feeder. While occasional landmark displacements were ignored by the butterflies and did not affect their decisions, systematic displacement of both the landmark and the rewarding feeder demonstrated that the butterflies associated the salient landmark with the feeder's position. Altogether, we show that butterflies consolidate and retrieve spatial memory in the context of foraging.
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Borboletas , Abelhas , Animais , Memória Espacial , AlimentosRESUMO
OBJECTIVES: Breastfeeding is an energetically costly and intense form of human parental investment, providing sole-source nutrition in early infancy and bioactive components, including immune factors. Given the energetic cost of lactation, milk factors may be subject to tradeoffs, and variation in concentrations have been explored utilizing the Trivers-Willard hypothesis. As human milk immune factors are critical to developing immune system and protect infants against pathogens, we tested whether concentrations of milk immune factors (IgA, IgM, IgG, EGF, TGFß2, and IL-10) vary in response to infant sex and maternal condition (proxied by maternal diet diversity [DD] and body mass index [BMI]) as posited in the Trivers-Willard hypothesis and consider the application of the hypothesis to milk composition. METHODS: We analyzed concentrations of immune factors in 358 milk samples collected from women residing in 10 international sites using linear mixed-effects models to test for an interaction between maternal condition, including population as a random effect and infant age and maternal age as fixed effects. RESULTS: IgG concentrations were significantly lower in milk produced by women consuming diets with low diversity with male infants than those with female infants. No other significant associations were identified. CONCLUSIONS: IgG concentrations were related to infant sex and maternal diet diversity, providing minimal support for the hypothesis. Given the lack of associations across other select immune factors, results suggest that the Trivers-Willard hypothesis may not be broadly applied to human milk immune factors as a measure of maternal investment, which are likely buffered against perturbations in maternal condition.
Assuntos
Leite Humano , Estado Nutricional , Feminino , Lactente , Masculino , Humanos , Lactação/fisiologia , Aleitamento Materno , Fatores Imunológicos , Imunoglobulina GRESUMO
The shell disorder models have predicted that SARS-CoV-2 is of a specific but peculiar evolutionary nature. All coronaviruses (CoVs) closely related to SARS-CoV-2 have been found to have the hardest outer shells (M protein) among CoVs. This hard shell (low M percentage of intrinsic disorder (PID)) is associated with burrowing animals, for example, pangolins, and is believed to be responsible for the high contagiousness of SARS-CoV-2 because it will be more resistant to antimicrobial enzymes found in saliva/mucus. Incoming clinical and experimental data do support this along with a prediction based on another aspect of the shell (N, inner shell) disorder models that SARS-CoV-1 is more virulent than SARS-CoV-2 because SARS-CoV-2 produces fewer virus copies in vital organs even if large amounts of infections particles are shed orally and nasally. A phylogenetic study using M reveals a closer relationship of SARS-CoV to pangolin-CoVs than the bat-RaTG13 found in Yunnan, China. Previous studies may have been confused by recombinations that were poorly handled. The shell disorder models suggest that a pangolin-CoV strain may have entered the human population in 2017 or before as an attenuated virus, which could explain why SARS-CoV is found to be highly adapted to humans.
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COVID-19 , SARS-CoV-2 , Animais , China , Filogenia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Reward modulates the saliency of a specific drug exposure and is essential for the transition to addiction. Numerous human PET-fMRI studies establish a link between midbrain dopamine (DA) release, DA transporter (DAT) availability, and reward responses. However, how and whether DAT function and regulation directly participate in reward processes remains elusive. Here, we developed a novel experimental paradigm in Drosophila melanogaster to study the mechanisms underlying the psychomotor and rewarding properties of amphetamine (AMPH). AMPH principally mediates its pharmacological and behavioral effects by increasing DA availability through the reversal of DAT function (DA efflux). We have previously shown that the phospholipid, phosphatidylinositol (4, 5)-bisphosphate (PIP2), directly interacts with the DAT N-terminus to support DA efflux in response to AMPH. In this study, we demonstrate that the interaction of PIP2 with the DAT N-terminus is critical for AMPH-induced DAT phosphorylation, a process required for DA efflux. We showed that PIP2 also interacts with intracellular loop 4 at R443. Further, we identified that R443 electrostatically regulates DA efflux as part of a coordinated interaction with the phosphorylated N-terminus. In Drosophila, we determined that a neutralizing substitution at R443 inhibited the psychomotor actions of AMPH. We associated this inhibition with a decrease in AMPH-induced DA efflux in isolated fly brains. Notably, we showed that the electrostatic interactions of R443 specifically regulate the rewarding properties of AMPH without affecting AMPH aversion. We present the first evidence linking PIP2, DAT, DA efflux, and phosphorylation processes with AMPH reward.
Assuntos
Anfetamina , Proteínas da Membrana Plasmática de Transporte de Dopamina , Anfetamina/farmacologia , Animais , Sítios de Ligação , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Drosophila melanogaster , FosfatidilinositóisRESUMO
The sun is the most prominent source of directional information in the heading direction network of the diurnal, ball-rolling dung beetle Kheper lamarcki. If this celestial body is occluded from the beetle's field of view, the distribution of the relative weight between the directional cues that remain shifts in favour of the celestial pattern of polarised light. In this study, we continue to explore the interplay of the sun and polarisation pattern as directional cues in the heading direction network of K. lamarcki. By systematically altering the intensity and degree of the two cues, we effectively change the relative reliability as they appear to the dung beetle. The response of the beetle to these modifications allows us to closely examine how the weighting relationship of these two sources of directional information is influenced and altered in the heading direction network of the beetle. We conclude that the process by which K. lamarcki relies on directional information is very likely done based on Bayesian reasoning, where directional information conveying the highest certainty at a particular moment is afforded the greatest weight.
Assuntos
Besouros , Animais , Teorema de Bayes , Comportamento Animal/fisiologia , Besouros/fisiologia , Sinais (Psicologia) , Reprodutibilidade dos TestesRESUMO
South African ball-rolling dung beetles exhibit a unique orientation behavior to avoid competition for food: after forming a piece of dung into a ball, they efficiently escape with it from the dung pile along a straight-line path. To keep track of their heading, these animals use celestial cues, such as the sun, as an orientation reference. Here we show that wind can also be used as a guiding cue for the ball-rolling beetles. We demonstrate that this mechanosensory compass cue is only used when skylight cues are difficult to read, i.e., when the sun is close to the zenith. This raises the question of how the beetles combine multimodal orientation input to obtain a robust heading estimate. To study this, we performed behavioral experiments in a tightly controlled indoor arena. This revealed that the beetles register directional information provided by the sun and the wind and can use them in a weighted manner. Moreover, the directional information can be transferred between these 2 sensory modalities, suggesting that they are combined in the spatial memory network in the beetle's brain. This flexible use of compass cue preferences relative to the prevailing visual and mechanosensory scenery provides a simple, yet effective, mechanism for enabling precise compass orientation at any time of the day.
Assuntos
Comportamento Animal/fisiologia , Encéfalo/fisiologia , Besouros/fisiologia , Orientação Espacial/fisiologia , Animais , Atividade Motora/fisiologia , Sistema Solar , Memória Espacial/fisiologia , VentoRESUMO
Cilia are evolutionarily conserved hair-like structures with a wide spectrum of key biological roles, and their dysfunction has been linked to a growing class of genetic disorders, known collectively as ciliopathies. Many strides have been made towards deciphering the molecular causes for these diseases, which have in turn expanded the understanding of cilia and their functional roles. One recently-identified ciliary gene is ARL2BP, encoding the ADP-Ribosylation Factor Like 2 Binding Protein. In this study, we have identified multiple ciliopathy phenotypes associated with mutations in ARL2BP in human patients and in a mouse knockout model. Our research demonstrates that spermiogenesis is impaired, resulting in abnormally shaped heads, shortened and mis-assembled sperm tails, as well as in loss of axonemal doublets. Additional phenotypes in the mouse included enlarged ventricles of the brain and situs inversus. Mouse embryonic fibroblasts derived from knockout animals revealed delayed depolymerization of primary cilia. Our results suggest that ARL2BP is required for the structural maintenance of cilia as well as of the sperm flagellum, and that its deficiency leads to syndromic ciliopathy.
Assuntos
Proteínas de Transporte/genética , Ciliopatias/genética , Infertilidade Masculina/genética , Proteínas de Membrana Transportadoras/genética , Fotofobia/genética , Adulto , Animais , Cílios/patologia , Ciliopatias/patologia , Modelos Animais de Doenças , Feminino , Humanos , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Pessoa de Meia-Idade , Linhagem , Fotofobia/patologia , Motilidade dos Espermatozoides/genética , Cauda do Espermatozoide/patologia , Espermatogênese/genética , Síndrome , Fatores de TranscriçãoRESUMO
Human milk oligosaccharides (HMO), the third most abundant component of human milk, are thought to be important contributors to infant health. Studies have provided evidence that geography, stage of lactation, and Lewis and secretor blood groups are associated with HMO profile. However, little is known about how variation across the genome may influence HMO composition among women in various populations. In this study, we performed genome-wide association analyses of 395 women from 8 countries to identify genetic regions associated with 19 different HMO. Our data support FUT2 as the most significantly associated (P < 4.23-9 to P < 4.5-70) gene with seven HMO and provide evidence of balancing selection for FUT2. Although polymorphisms in FUT3 were also associated with variation in lacto-N-fucopentaose II and difucosyllacto-N-tetrose, we found little evidence of selection on FUT3. To our knowledge, this is the first report of the use of genome-wide association analyses on HMO.
Assuntos
Estudo de Associação Genômica Ampla , Leite Humano , Oligossacarídeos , Feminino , Humanos , Lactação , Leite Humano/química , Oligossacarídeos/químicaRESUMO
Insect declines have been attributed to several drivers such as habitat loss, climate change, invasive alien species and insecticides. However, in the global context, these effects remain patchy, whereas insect losses appear to be consistent worldwide. Increases in atmospheric CO2 concentrations are known to have indirect effects on herbivorous insects, but the effects on other insects are largely unexplored. We wondered if elevated atmospheric CO2 (eCO2 ) could influence the growth and survival of insects, not via rising temperature, nor through their changes in food quality, but by other means. Rearing tunnelling dung beetle Euoniticellus intermedius (Reiche, 1848) at pre-industrial (250 parts per million [ppm]), current (400 ppm) and eCO2 levels (600 and 800 ppm), we found that exposure to eCO2 resulted in longer developmental times and increased mortality. Elevated CO2 also caused reduction of adult size and mass which is detrimental to dung beetle fitness. Additional results showed associated increases in CO2 levels inside dung brood balls, dung pH and respiration rates of the soil surrounding the developing dung beetles (CO2 flux). We thus hypothesize that elevated CO2 increases competition for O2 and nutrients between soil microbiota and subterranean insects. Given that many insect orders spend at least part of their life underground, our findings indicate the possibility of a negative ubiquitous effect of eCO2 on a large portion of the earth's insect biota. These findings therefore suggest an important area for future research on the soil community in the context of atmospheric change.
Assuntos
Besouros , Microbiota , Animais , Dióxido de Carbono/análise , Mudança Climática , SoloRESUMO
This paper describes an investigation into how combined carbon nanotube doping and surface nanostructuring affects the surface properties of polystyrene. Multiwall carbon nanotubes (MWCNTs) have unique anisotropic electrical properties that can be utilized for light absorption, electromagnetic shielding and nanoscale electostatic forces. Polystyrene was doped with 5 wt% MWCNTs and the resulting composite was wetted onto a porous anodic alumina template to form a nanostructure surface of nanotubes. Scanning electron microscopy revealed a hierarchical surface structure with the composite nanotubes bundled together as the MWCNTs increased the attractive forces between the composite nanotubes. Water droplet testing revealed that this hierarchical surface structure was superhydrophobic. Though the presence of the MWCNTs caused a direct increase in absorption, the hierarchical surface structure increased reflection. The addition of 5 wt% of the anionic surfactant Sodium Dodecyl Benzene Sulfonate to ensure MWCNT dispersal did not significantly change hydrophobicity or light absorption despite the hierarchical surface structure becoming finer. The created composite has potential use as a surface layer on an organic surface cell as it provides reduced cleaning needs and electrical disturbance but further work is required to reduce the reflection.
RESUMO
Amyloidosis is a malignant pathology associated with the formation of proteinaceous amyloid fibrils that deposit in organs and tissues, leading to dysfunction and severe morbidity. More than 25 proteins have been identified as components of amyloid, but the most common form of systemic amyloidosis is associated with the deposition of amyloid composed of Ig light chains (AL). Clinical management of amyloidosis focuses on reducing synthesis of the amyloid precursor protein. However, recently, passive immunotherapy using amyloid fibril-reactive antibodies, such as 11-1F4, to remove amyloid from organs has been shown to be effective at restoring organ function in patients with AL amyloidosis. However, 11-1F4 does not bind amyloid in all AL patients, as evidenced by PET/CT imaging, nor does it efficiently bind the many other forms of amyloid. To enhance the reactivity and expand the utility of the 11-1F4 mAb as an amyloid immunotherapeutic, we have developed a pretargeting "peptope" comprising a multiamyloid-reactive peptide, p5+14, fused to a high-affinity peptide epitope recognized by 11-1F4. The peptope, known as p66, bound the 11-1F4 mAb in vitro with subnanomolar efficiency, exhibited multiamyloid reactivity in vitro and, using tissue biodistribution and SPECT imaging, colocalized with amyloid deposits in a mouse model of systemic serum amyloid A amyloidosis. Pretreatment with the peptope induced 11-1F4 mAb accumulation in serum amyloid A deposits in vivo and enhanced 11-1F4-mediated dissolution of a human AL amyloid extract implanted in mice.
Assuntos
Amiloidose/metabolismo , Amiloidose/terapia , Anticorpos Monoclonais/fisiologia , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Cadáver , Epitopos/metabolismo , Humanos , Cadeias Leves de Imunoglobulina/imunologia , Camundongos , Peptídeos/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Ligação Proteica , Proteína Amiloide A Sérica/metabolismo , Distribuição Tecidual , Resultado do TratamentoRESUMO
A model that predicts levels of coronavirus (CoV) respiratory and fecal-oral transmission potentials based on the shell disorder has been built using neural network (artificial intelligence, AI) analysis of the percentage of disorder (PID) in the nucleocapsid, N, and membrane, M, proteins of the inner and outer viral shells, respectively. Using primarily the PID of N, SARS-CoV-2 is grouped as having intermediate levels of both respiratory and fecal-oral transmission potentials. Related studies, using similar methodologies, have found strong positive correlations between virulence and inner shell disorder among numerous viruses, including Nipah, Ebola, and Dengue viruses. There is some evidence that this is also true for SARS-CoV-2 and SARS-CoV, which have N PIDs of 48% and 50%, and case-fatality rates of 0.5-5% and 10.9%, respectively. The underlying relationship between virulence and respiratory potentials has to do with the viral loads of vital organs and body fluids, respectively. Viruses can spread by respiratory means only if the viral loads in saliva and mucus exceed certain minima. Similarly, a patient is likelier to die when the viral load overwhelms vital organs. Greater disorder in inner shell proteins has been known to play important roles in the rapid replication of viruses by enhancing the efficiency pertaining to protein-protein/DNA/RNA/lipid bindings. This paper suggests a novel strategy in attenuating viruses involving comparison of disorder patterns of inner shells (N) of related viruses to identify residues and regions that could be ideal for mutation. The M protein of SARS-CoV-2 has one of the lowest M PID values (6%) in its family, and therefore, this virus has one of the hardest outer shells, which makes it resistant to antimicrobial enzymes in body fluid. While this is likely responsible for its greater contagiousness, the risks of creating an attenuated virus with a more disordered M are discussed.
Assuntos
Inteligência Artificial , Betacoronavirus , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Vacinas Virais , Betacoronavirus/química , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidade , COVID-19 , Vacinas contra COVID-19 , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Desenvolvimento de Medicamentos/métodos , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , SARS-CoV-2 , Carga Viral , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
A model to predict the relative levels of respiratory and fecal-oral transmission potentials of coronaviruses (CoVs) by measuring the percentage of protein intrinsic disorder (PID) of the M (Membrane) and N (Nucleoprotein) proteins in their outer and inner shells, respectively, was built before the MERS-CoV outbreak. With MPID = 8.6% and NPID = 50.2%, the 2003 SARS-CoV falls into group B, which consists of CoVs with intermediate levels of both fecal-oral and respiratory transmission potentials. Further validation of the model came with MERS-CoV (MPID = 9%, NPID = 44%) and SARS-CoV-2 (MPID = 5.5%, NPID = 48%) falling into the groups C and B, respectively. Group C contains CoVs with higher fecal-oral but lower respiratory transmission potentials. Unlike SARS-CoV, SARS-CoV-2 with MPID = 5.5% has one of the hardest outer shells among CoVs. Because the hard shell is able to resist the antimicrobial enzymes in body fluids, the infected person is able to shed large quantities of viral particles via saliva and mucus, which could account for the higher contagiousness of SARS-COV-2. Further searches have found that high rigidity of the outer shell is characteristic for the CoVs of burrowing animals, such as rabbits (MPID = 5.6%) and pangolins (MPID = 5-6%), which are in contact with the buried feces. A closer inspection of pangolin-CoVs from 2017 to 2019 reveals that pangolins provided a unique window of opportunity for the entry of an attenuated SARS-CoV-2 precursor into the human population in 2017 or earlier, with the subsequent slow and silent spread as a mild cold that followed by its mutations into the current more virulent form. Evidence of this lies in both the genetic proximity of the pangolin-CoVs to SARS-CoV-2 (â¼90%) and differences in N disorder. A 2017 pangolin-CoV strain shows evidence of higher levels of attenuation and higher fecal-oral transmission associated with lower human infectivity via having lower NPID (44.8%). Our shell disorder model predicts this to be a SARS-CoV-2 vaccine strain, as lower inner shell disorder is associated with the lesser virulence in a variety of viruses.
Assuntos
Betacoronavirus/química , Infecções por Coronavirus , Eutérios/virologia , Proteínas Intrinsicamente Desordenadas , Proteínas do Nucleocapsídeo , Pandemias , Pneumonia Viral , Animais , COVID-19 , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Proteínas do Nucleocapsídeo de Coronavírus , Humanos , Pandemias/veterinária , Fosfoproteínas , Pneumonia Viral/transmissão , Pneumonia Viral/veterinária , Pneumonia Viral/virologia , Coelhos/virologia , SARS-CoV-2 , Proteínas ViraisRESUMO
Several protein kinases, including protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and extracellular signal-regulated kinase, play key roles in the regulation of dopamine transporter (DAT) functions. These functions include surface expression, internalization, and forward and reverse transport, with phosphorylation sites for these kinases being linked to distinct regions of the DAT N terminus. Protein phosphatases (PPs) also regulate DAT activity, but the specific residues associated with their activities have not yet been elucidated. In this study, using co-immunoprecipitation followed by MS and immunoblotting analyses, we demonstrate the association of DAT with PP1 and PP2A in the mouse brain and heterologous cell systems. By applying MS in conjunction with a metabolic labeling method, we defined a PP1/2A-sensitive phosphorylation site at Thr-48 in human DAT, a residue that has not been previously reported to be involved in DAT phosphorylation. Site-directed mutagenesis of Thr-48 to Ala (T48A) to prevent phosphorylation enhanced dopamine transport kinetics, supporting a role for this residue in regulating DAT activity. Moreover, T48A-DAT displayed increased palmitoylation, suggesting that phosphorylation/dephosphorylation at this site has an additional regulatory role and reinforcing a previously reported reciprocal relationship between C-terminal palmitoylation and N-terminal phosphorylation.
Assuntos
Encéfalo/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Humanos , Lipoilação/genética , Camundongos , Camundongos Knockout , Fosforilação , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/genética , Treonina/genética , Treonina/metabolismoRESUMO
The rapid screening of plutonium from aqueous sources remains a critical challenge for nuclear nonproliferation efforts. The determination of trace-level Pu isotopes in water requires offsite sample preparation and analysis; therefore, new methods that combine plutonium purification, concentration, and isotopic screening in a fieldable detection system will provide an invaluable tool for nuclear safeguards. This contribution describes the development and characterization of thin polymer-ligand films for the isolation and concentration of waterborne Pu for direct spectroscopic analyses. Submicron thin films were prepared through spin coating onto Si wafers and consisted of combinations of polystyrene (PS) with dibenzoylmethane, thenoyltrifluoroacetone, and di(2-ethylhexyl)phosphoric acid (HDEHP). Pu uptake studies from solutions at pH from 2.3 to 6.3 indicated that only films containing HDEHP exhibited significant recovery of Pu. High alpha spectroscopy peak energy resolutions were achieved for PS-HDEHP films over a range of film thicknesses from 30 to 250 nm. A separate study was performed to evaluate uptake from a primarily Pu(V) solution where it was observed that doubling the HDEHP loading in the film increased uptake of Pu by an order of magnitude. X-ray photoelectron spectroscopy (XPS) analysis revealed that HDEHP was highly concentrated within the first few nanometers of the film at the higher loading. XPS analysis also revealed that, in the presence of water, HDEHP was stripped from the surface layer of the film at circumneutral pH. While significant losses of ligand were seen in all samples, higher loadings of HDEHP resulted in measurable amounts of ligand retained after a 12-h soak in water. Findings of this study are being used to guide the development of thin-film composite membrane-based detection methods for the rapid, fieldable analysis of Pu in water.
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Light chain-associated amyloidosis is characterized by the extracellular deposition of amyloid fibrils in abdominothoracic organs, skin, soft tissue, and peripheral nerves. Phagocytic cells of the innate immune system appear to be ineffective at clearing the material; however, human light chain amyloid extract, injected subcutaneously into mice, is rapidly cleared in a process that requires neutrophil activity. To better elucidate the phagocytosis of light chain fibrils, a potential method of cell-mediated dissolution, amyloid-like fibrils were labeled with the pH-sensitive dye pHrodo red and a near infrared fluorophore. After injecting this material subcutaneously in mice, optical imaging was used to quantitatively monitor phagocytosis and dissolution of fibrils concurrently. Histologic evaluation of the residual fibril masses revealed the presence of CD68+, F4/80+, ionized calcium binding adaptor molecule 1- macrophages containing Congo red-stained fibrils as well as neutrophil-associated proteins with no evidence of intact neutrophils. These data suggest an early infiltration of neutrophils, followed by extensive phagocytosis of the light chain fibrils by macrophages, leading to dissolution of the mass. Optical imaging of this novel murine model, coupled with histologic evaluation, can be used to study the cellular mechanisms underlying dissolution of synthetic amyloid-like fibrils and human amyloid extracts. In addition, it may serve as a test bed to evaluate investigational opsonizing agents that might serve as therapeutic agents for light chain-associated amyloidosis.