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Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels.
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Imagem Molecular/métodos , Preservação de Tecido/métodos , Algoritmos , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Nervosas Mielinizadas/química , Proteômica , Substâncias Redutoras , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
An essential step toward understanding brain function is to establish a structural framework with cellular resolution on which multi-scale datasets spanning molecules, cells, circuits and systems can be integrated and interpreted1. Here, as part of the collaborative Brain Initiative Cell Census Network (BICCN), we derive a comprehensive cell type-based anatomical description of one exemplar brain structure, the mouse primary motor cortex, upper limb area (MOp-ul). Using genetic and viral labelling, barcoded anatomy resolved by sequencing, single-neuron reconstruction, whole-brain imaging and cloud-based neuroinformatics tools, we delineated the MOp-ul in 3D and refined its sublaminar organization. We defined around two dozen projection neuron types in the MOp-ul and derived an input-output wiring diagram, which will facilitate future analyses of motor control circuitry across molecular, cellular and system levels. This work provides a roadmap towards a comprehensive cellular-resolution description of mammalian brain architecture.
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Córtex Motor/anatomia & histologia , Córtex Motor/citologia , Neurônios/classificação , Animais , Atlas como Assunto , Feminino , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Glutamatos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroimagem , Neurônios/citologia , Neurônios/metabolismo , Especificidade de Órgãos , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
BACKGROUND: Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic, specifically proliferating Bifidobacilli and Lactobacilli and enhancing absorption of calcium and magnesium. The use of cellobiose 2-epimerase (CE) is considered an interesting alternative for industrial production of lactulose. CE reversibly converts D-glucose residues into D-mannose residues at the reducing end of unmodified ß-1,4-linked oligosaccharides, including ß-1,4-mannobiose, cellobiose, and lactose. Recently, a few CE 3D structure were reported, revealing mechanistic details. Using this information, we redesigned the substrate binding site of CE to extend its activity from epimerization to isomerization. RESULTS: Using superimposition with 3 known CE structure models, we identified 2 residues (Tyr114, Asn184) that appeared to play an important role in binding epilactose. We modified these residues, which interact with C2 of the mannose moiety, to prevent epimerization to epilactose. We found a Y114E mutation led to increased release of a by-product, lactulose, at 65 °C, while its activity was low at 37 °C. Notably, this phenomenon was observed only at high temperature and more reliably when the substrate was increased. Using Y114E, isomerization of lactose to lactulose was investigated under optimized conditions, resulting in 86.9 g/l of lactulose and 4.6 g/l of epilactose for 2 h when 200 g/l of lactose was used. CONCLUSION: These results showed that the Y114E mutation increased isomerization of lactose, while decreasing the epimerization of lactose. Thus, a subtle modification of the active site pocket could extend its native activity from epimerization to isomerization without significantly impairing substrate binding. While additional studies are required to scale this to an industrial process, we demonstrated the potential of engineering this enzyme based on structural analysis.
Assuntos
Carboidratos Epimerases/química , Carboidratos Epimerases/metabolismo , Celobiose/química , Celobiose/metabolismo , Bactérias Gram-Positivas/enzimologia , Engenharia de Proteínas/métodos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , Temperatura Alta , Microbiologia Industrial/métodos , Isomerismo , Lactose/genética , Lactose/metabolismo , Lactulose/biossíntese , Lactulose/química , Lactulose/metabolismo , Manose/metabolismo , Oligossacarídeos/metabolismo , Prebióticos , Domínios Proteicos , Especificidade por SubstratoRESUMO
We demonstrate that chikusetsusaponin IVa methyl ester (CME), a triterpenoid saponin from the root of Achyranthes japonica, has an anticancer activity. We investigate its molecular mechanism in depth in HCT116 cells. CME reduces the amount of ß-catenin in nucleus and inhibits the binding of ß-catenin to specific DNA sequences (TCF binding elements, TBE) in target gene promoters. Thus, CME appears to decrease the expression of cell cycle regulatory proteins such as Cyclin D1, as a representative target for ß-catenin, as well as CDK2 and CDK4. As a result of the decrease of the cell cycle regulatory proteins, CME inhibits cell proliferation by arresting the cell cycle at the G0/G1 phase. Therefore, we suggest that CME as a novel Wnt/ß-catenin inhibitor can be a putative agent for the treatment of colorectal cancers.
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Ciclo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ácido Oleanólico/análogos & derivados , Saponinas/farmacologia , beta Catenina/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA , Ésteres/química , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Transporte Proteico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saponinas/químicaRESUMO
Multiple stressors are linked to declines of insects and important pollinators, such as bees. Recently, interactive effects of multiple agrochemicals on bees have been highlighted, including fungicides, which increase toxicity of neonicotinoid insecticides. Here, we use a semi-field study across two seasons in controlled foraging tunnels to test the effects of a field application of a commercial fungicide product with two active ingredients (pyraclostrobin and metconazole) applied at label rates. We also examine its interactive effects with the neonicotinoid insecticide clothianidin, at a conservative field-realistic dose of 2.23 ppb, on 48 honey bee colonies. We found combined effects of pesticide exposure, including additive 2.93-fold increases in mortality, and an additional effect of increased infestation levels of the ectoparasitic mite, Varroa destructor. Pesticide treatments also reduced colony activity, reduced colony weight, and increased sugar consumption of whole colonies. These findings indicate that typical sublethal exposure levels to common, co-occurring agrochemicals in the field significantly affect the health of whole honey bee colonies, highlighting an unintended consequence of increasing pesticide applications.
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In response to the increasing demand for spheroid-based cancer research, the importance of developing integrated platforms that can simultaneously facilitate high-throughput spheroid production and multiplexed analysis is emphasized. In addition, the understanding of how the size and cellular composition of tumors directly influence their internal structures and functionalities underlines the critical need to produce spheroids of diverse sizes and compositions on a large scale. To address this rising demand, this work presents a configurable and linkable in vitro three-dimensional (3D) cell culture kit (CLiCK) for spheroids, termed CLiCK-Spheroid. This platform consists of three primary components: a hanging drop microarray (HDMA), a concave pillar microarray (CPMA), and gradient blocks. The HDMA alone produces a homogeneous spheroid array, while its combination with the gradient block enables one-step generation of a size-gradient spheroid array. Using the size-gradient spheroid arrays, the occurrence of necrotic cores based on spheroid size is demonstrated. Additionally, spheroids in a single batch can be conveniently compartmentalized and regrouped using a CPMA, enhancing the versatility of spheroid arrays and enabling multiplexed drug treatments. By combining the different assembly methods, this work has achieved high-throughput production of cell composition-gradient spheroid arrays, with noticeable variations in morphology and vascularization based on cell compositions.
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The rhythmic motor pathway activation by pacemaker neurons or circuits in the brain has been proposed as the mechanism for the timing of motor coordination, and the abnormal potentiation of this mechanism may lead to a pathological tremor. Here, we show that the potentiation of Ca(V)3.1 T-type Ca(2+) channels in the inferior olive contributes to the onset of the tremor in a pharmacological model of essential tremor. After administration of harmaline, 4- to 10-Hz synchronous neuronal activities arose from the IO and then propagated to cerebellar motor circuits in wild-type mice, but those rhythmic activities were absent in mice lacking Ca(V)3.1 gene. Intracellular recordings in brain-stem slices revealed that the Ca(V)3.1-deficient inferior olive neurons lacked the subthreshold oscillation of membrane potentials and failed to trigger 4- to 10-Hz rhythmic burst discharges in the presence of harmaline. In addition, the selective knockdown of Ca(V)3.1 gene in the inferior olive by shRNA efficiently suppressed the harmaline-induced tremor in wild-type mice. A mathematical model constructed based on data obtained from patch-clamping experiments indicated that harmaline could efficiently potentiate Ca(V)3.1 channels by changing voltage-dependent responsiveness in the hyperpolarizing direction. Thus, Ca(V)3.1 is a molecular pacemaker substrate for intrinsic neuronal oscillations of inferior olive neurons, and the potentiation of this mechanism can be considered as a pathological cause of essential tremor.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Núcleo Olivar/metabolismo , Tremor/metabolismo , Animais , Canais de Cálcio Tipo T/deficiência , Canais de Cálcio Tipo T/genética , Linhagem Celular , Modelos Animais de Doenças , Harmalina , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Núcleo Olivar/fisiopatologia , Interferência de RNA , Tremor/induzido quimicamente , Tremor/genética , Tremor/fisiopatologiaRESUMO
Profiling molecular expression in situ allows the integration of biomolecular and cellular features, enabling an in-depth understanding of biological systems. Multiplexed immunofluorescence methods can visualize tens to hundreds of proteins from individual tissue samples, but their application is usually limited to thin tissue sections. Multiplexed immunofluorescence of thick tissues or intact organs will enable high-throughput profiling of cellular protein expression within 3D tissue architectures (e.g., blood vessels, neural projections, tumors), opening a new dimension in diverse biological research and medical applications. We will review current multiplexed immunofluorescence methods and discuss possible approaches and challenges to achieve 3D multiplexed immunofluorescence.
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Proteínas , ImunofluorescênciaRESUMO
Nabis stenoferus is a zoophytophagous predator that lives in grasslands around agricultural fields. It is a candidate biological control agent for use via augmentation or conservation. To find a suitable food source for mass-rearing and to better understand this predator's biology, we compared the life history characteristics of N. stenoferus under the three different diets: aphids only (Myzus persicae), moth eggs only (Ephestia kuehniella), or a mixed diet of aphids and moth eggs. Interestingly, when only aphids were supplied, N. stenoferus developed to the adult stage but lacked normal levels of fecundity. There was a significant synergism of the mixed diet on N. stenoferus fitness in both the immature and adult stages, i.e., a 13% reduction in the nymphal developmental period and an 87.3-fold increase in fecundity, compared to aphid-only diet. Furthermore, the intrinsic rate of increase was significantly higher for the mixed diet (0.139) than either aphids only (0.022) or moth eggs only (0.097). These results show that M. persicae alone is not a complete diet for the mass-rearing of N. stenoferus, whereas this aphid can be a supplementary food when combined with E. kuehniella eggs. Implications and applications of these findings for biological control are discussed.
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Afídeos , Mariposas , Animais , Óvulo , Heterópteros , Dieta , FemininoRESUMO
Cell-generated mechanical forces drive many cellular and tissue-level movements and rearrangements required for the tissue or organ to develop its shape1, 2, 3, 4, 5. The prevalent view of tissue morphogenesis relies on epithelial folding resulting in compressed epithelial monolayers, overlooking the involvement of stroma in morphogenesis1, 4, 6, 7. Here, we report a giant web-like network formation of stromal cells in the epithelium-stroma interface, resulting from a multi-scale mechano-reciprocity between migrating cells and their extracellular environment. In multi-layered tissues, surface wrinkles form by a stromal cell-mediated tensional force exerted at the basement membrane. The topographical cue is transmitted to the stromal cell, directing its protrusion and migration along the wrinkles. This inductive movement of the cells conveys traction forces to its surrounding extracellular matrix, remodeling the local architectures of the stroma. In this manner, stromal cells and wrinkles communicate recursively to generate the cellular network. Our observation provides a rational mechanism for network formation in living tissues and a new understanding of the role of cellular-level tensional force in morphogenesis.
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In binocular animals that exhibit stereoscopic visual responses, the axons of retinal ganglion cells (RGCs) connect to brain areas bilaterally by forming a commissure called the optic chiasm (OC). Ventral anterior homeobox 1 (Vax1) contributes to the formation of the OC, acting endogenously in optic pathway cells and exogenously in growing RGC axons. Here, we generated Vax1AA/AA mice expressing the Vax1AA mutant, which is incapable of intercellular transfer. We found that RGC axons cannot take up Vax1AA protein from the Vax1AA/AA mouse optic stalk (OS) and grow slowly to arrive at the hypothalamus at a late stage. The RGC axons of Vax1AA/AA mice connect exclusively to ipsilateral brain areas after failing to access the midline, resulting in reduced visual acuity and abnormal oculomotor responses. Overall, our study provides physiological evidence for the necessity of intercellular transfer of Vax1 and the importance of the bilateral RGC axon projection in proper visuomotor responses.
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Neuropeptídeos , Quiasma Óptico , Camundongos , Animais , Quiasma Óptico/metabolismo , Células Ganglionares da Retina , Encéfalo/metabolismo , Camundongos Endogâmicos C57BL , Neuropeptídeos/metabolismo , Proteínas de Homeodomínio/metabolismoRESUMO
Hypoxic damage to the prefrontal cortex (PFC) has been implicated in the frontal lobe dysfunction found in various neuropsychiatric disorders. The underlying subcortical mechanisms, however, have not been well explored. In this study, we induced a PFC-specific hypoxia-like damage by cobalt-wire implantation to demonstrate that the role of the mediodorsal thalamus (MD) is critical for the development of frontal lobe dysfunction, including frontal lobe-specific seizures and abnormal hyperactivity. Before the onset of these abnormalities, the cross talk between the MD and PFC nuclei at theta frequencies was enhanced. During the theta frequency interactions, burst spikes, known to depend on T-type Ca(2+) channels, were increased in MD neurons. In vivo knockout or knockdown of the T-type Ca(2+) channel gene (Ca(V)3.1) in the MD substantially reduced the theta frequency MD-PFC cross talk, frontal lobe-specific seizures, and locomotor hyperactivity in this model. These results suggest a two-step model of prefrontal dysfunction in which the response to a hypoxic lesion in the PFC results in abnormal thalamocortical feedback driven by thalamic T-type Ca(2+) channels, which, in turn, leads to the onset of neurological and behavioral abnormalities. This study provides valuable insights into preventing the development of neuropsychiatric disorders arising from irreversible PFC damage.
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Canais de Cálcio Tipo T/metabolismo , Lobo Frontal/lesões , Lobo Frontal/fisiopatologia , Neurônios/metabolismo , Tálamo/metabolismo , Análise de Variância , Animais , Western Blotting , Condicionamento Clássico/fisiologia , Eletrofisiologia , Medo , Feminino , Corpos Estranhos , Lobo Frontal/metabolismo , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Masculino , Camundongos , Atividade Motora/fisiologiaRESUMO
A handheld confocal microscope using a rapid MEMS scanning mirror facilitates real-time optical biopsy for simple cancer diagnosis. Here we report a handheld confocal microscope catheter using high definition and high frame rate (HDHF) Lissajous scanning MEMS mirror. The broad resonant frequency region of the fast axis on the MEMS mirror with a low Q-factor facilitates the flexible selection of scanning frequencies. HDHF Lissajous scanning was achieved by selecting the scanning frequencies with high greatest common divisor (GCD) and high total lobe number. The MEMS mirror was fully packaged into a handheld configuration, which was coupled to a home-built confocal imaging system. The confocal microscope catheter allows fluorescence imaging of in vivo and ex vivo mouse tissues with 30â Hz frame rate and 95.4% fill factor at 256 × 256 pixels image, where the lateral resolution is 4.35 µm and the field-of-view (FOV) is 330 µm × 330 µm. This compact confocal microscope can provide diverse handheld microscopic applications for real-time, on-demand, and in vivo optical biopsy.
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Neuronal ensembles that hold specific memory (memory engrams) have been identified in the hippocampus, amygdala, or cortex. However, it has been hypothesized that engrams of a specific memory are distributed among multiple brain regions that are functionally connected, referred to as a unified engram complex. Here, we report a partial map of the engram complex for contextual fear conditioning memory by characterizing encoding activated neuronal ensembles in 247 regions using tissue phenotyping in mice. The mapping was aided by an engram index, which identified 117 cFos+ brain regions holding engrams with high probability, and brain-wide reactivation of these neuronal ensembles by recall. Optogenetic manipulation experiments revealed engram ensembles, many of which were functionally connected to hippocampal or amygdala engrams. Simultaneous chemogenetic reactivation of multiple engram ensembles conferred a greater level of memory recall than reactivation of a single engram ensemble, reflecting the natural memory recall process. Overall, our study supports the unified engram complex hypothesis for memory storage.
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Mapeamento Encefálico , Memória , Animais , Encéfalo , Medo/fisiologia , Hipocampo/fisiologia , Memória/fisiologia , CamundongosRESUMO
T-type Ca(2+) channels have been implicated in tremorogenesis and motor coordination. The α1 subunit of the Ca(V)3.1 T-type Ca(2+) channel is highly expressed in motor pathways in the brain, but knockout of the Ca(V)3.1 gene (α(1G)(-/-)) per se causes no motor defects in mice. Thus, the role of Ca(V)3.1 channels in motor control remains obscure in vivo. Here, we investigated the effect of the Ca(V)3.1 knockout in the null genetic background of α1 GABA(A) receptor (α1(-/-)) by generating the double mutants (α1(-/-)/α(1G)(-/-)). α1(-/-)/α(1G)(-/-) mice showed severer motor abnormalities than α1(-/-) mice as measured by potentiated tremor activities at 20Hz and impaired motor learning. Propranolol, an anti-ET drug that is known to reduce the pathologic tremor in α1(-/-) mice, was not effective for suppressing the potentiated tremor in α1(-/-)/α(1G)(-/-) mice. In addition, α1(-/-)/α(1G)(-/-) mice showed an age-dependent loss of cerebellar Purkinje neurons. These results suggest that α1(-/-)/α(1G)(-/-) mice are a novel mouse model for a distinct subtype of ET in human and that Ca(V)3.1 T-type Ca(2+) channels play a role in motor coordination under pathological conditions.
Assuntos
Canais de Cálcio Tipo T/genética , Ataxia Cerebelar/genética , Ataxia Cerebelar/patologia , Cerebelo/patologia , Tremor Essencial/genética , Tremor Essencial/patologia , Fatores Etários , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Modelos Genéticos , Atividade Motora/genética , Células de Purkinje/patologia , Receptores de GABA-A/genéticaRESUMO
Bemisia tabaci Mediterranean (Gennadius) (Hemiptera: Aleyrodidae) is an economically important insect pest worldwide. Previously, we have reported that most B. tabaci Mediterranean (MED) populations occurring in greenhouse tomatoes in Korea have been displaced from well-differentiated two genetic clusters (C1 and C2) to one (C2) during one-year period. To elucidate factors responsible for this phenomenon, we compared life history parameters of these two different genetic clusters through single and cross mating experiments on two different host plants, cucumber and tobacco, at 26°C. Intrinsic rate of increase (r), finite rate of increase (λ), and net reproductive rate (Ro) were significantly higher in the dominating cluster (C2) (0.247, 1.280, and 192.402, respectively on cucumber; 0.226, 1.253, and 133.792, respectively on tobacco) than in the other cluster (C1) (0.149, 1.161, and 50.539, respectively on cucumber; 0.145, 1.156, and 53.332, respectively on tobacco). Overall performances of cross mating groups, C2fC1m (C2 female × C1 male) and C1fC2m (C1 female × C2 male), were in-between those of C2 and C1, with C2fC1m performing better than C1fC2m. Thus, maternal inheritance appeared to be significantly associated with their life history parameters, with partial involvement of paternal inheritance. Our results demonstrated that the rapid displacement of genetic clusters of B. tabaci MED populations was clearly associated with differences in their life history parameters.
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Cruzamentos Genéticos , Hemípteros/crescimento & desenvolvimento , Hemípteros/genética , Estágios do Ciclo de Vida/genética , Família Multigênica , Comportamento Sexual Animal/fisiologia , Animais , Teorema de Bayes , Peso Corporal , Feminino , Fertilidade , Hemípteros/anatomia & histologia , Masculino , Oviposição , Razão de Masculinidade , Análise de SobrevidaRESUMO
The zoophytophagous mirid Nesidiocoris tenuis (Hemiptera: Miridae) is one of the biological control agents against the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), a major pest of greenhouse crops. The successful establishment of a biological control agent and its co-occurrence with the target pests increases the efficacy of biological control programs in greenhouses. In this study, we explored the effects of different wavelengths of LED light on establishment of N. tenuis in laboratory condition, with the goal of enhancing the biological control of B. tabaci in greenhouse crops. Nesidiocoris tenuis was most strongly attracted by LED light at a wavelength of 385 nm. This same wavelength was also highly attractive to B. tabaci in Y-tube experiments with lights of specific wavelengths provided is each arm of the apparatus. In trials in growth chambers, we verified the attraction of N. tenuis to 385 nm wavelength. When LED light at a wavelength of 385 nm was used in a growth chamber for 6 hours out of 24 hours, it significantly increased the remaining number of N. tenuis in growth chamber and level of predation compared to treatment with white LED light or without LED light. In conclusion, UV-LED light at a wavelength of 385 nm attracts both B. tabaci and N. tenuis. Thus, it would be used for enhancing early establishment of this mirid bug, better spatial congruence of both mirid bug and whitefly, and better control of the whitefly.
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Hemípteros , Controle Biológico de Vetores , Raios Ultravioleta , AnimaisRESUMO
Amblyseius eharai is a generalist predatory mite that consumes spider mites, rust mites, thrips, and pollen, with a high adaptability to various plants. To better understand ecological and behavioral aspects of this species, we investigated its functional response to different stages of two-spotted spider mite, Tetranychus urticae. Furthermore, we compared its environmental adaptability with that of other referenced phytoseiids using a temperature-dependent model of the intrinsic rate of increase. We were able to calculate the functional response parameters of both sexes of A. eharai when preying on eggs or larvae of T. urticae and, for females only, when preying on the deutonymph of T. urticae. Among the various combinations tested herein, A. eharai females preying on T. urticae larvae had the highest attack rate and shortest handling time. For eggs of T. urticae, A. eharai showed a lower attack rate; however, its handling time for eggs was significantly shorter compared to other phytoseiids. Using T. urticae larva as a prey, the attack rate of female A. eharai was higher and the handling time of both sexes of this species was shorter than those of other phytoseiid mites. Amblyseius eharai populations can show maximum performance quickly due to this species' lower optimal temperature for population growth (28.1°C) compared to other phytoseiid mites. Thus, we provided evidence that this predatory mite has the potential to be a new, effective biological control agent of greenhouse pests such as T. urticae due to its high predation capacity and low optimal temperature.
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Comportamento Alimentar , Ácaros/fisiologia , Controle Biológico de Vetores/métodos , Comportamento Predatório , Temperatura , Tetranychidae/fisiologia , Animais , Feminino , MasculinoRESUMO
The basal ganglia regulate a wide range of behaviors, including motor control and cognitive functions, and are profoundly affected in Parkinson's disease (PD). However, the functional organization of different basal ganglia nuclei has not been fully elucidated at the circuit level. In this study, we investigated the functional roles of distinct parvalbumin-expressing neuronal populations in the external globus pallidus (GPe-PV) and their contributions to different PD-related behaviors. We demonstrate that substantia nigra pars reticulata (SNr)-projecting GPe-PV neurons and parafascicular thalamus (PF)-projecting GPe-PV neurons are associated with locomotion and reversal learning, respectively. In a mouse model of PD, we found that selective manipulation of the SNr-projecting GPe-PV neurons alleviated locomotor deficit, whereas manipulation of the PF-projecting GPe-PV neurons rescued the impaired reversal learning. Our findings establish the behavioral importance of two distinct GPe-PV neuronal populations and, thereby, provide a new framework for understanding the circuit basis of different behavioral deficits in the Parkinsonian state.
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Globo Pálido/fisiopatologia , Vias Neurais/fisiopatologia , Neurônios/fisiologia , Transtornos Parkinsonianos/fisiopatologia , Animais , Feminino , Transtornos Neurológicos da Marcha/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Parvalbuminas , Reversão de Aprendizagem/fisiologiaRESUMO
CRM197, which retains the same inflammatory and immune-stimulant properties as diphtheria toxin but with reduced toxicity, has been used as a safe carrier in conjugated vaccines. Expression of recombinant CRM197 in E. coli is limited due to formation of inclusion bodies. Soluble expression attempts in Bacillus subtilis, P. fluorescens, Pichia pastoris, and E. coli were partially unsuccessful or did not generate yields sufficient for industrial scale production. Multiple approaches have been attempted to produce CRM197 in E. coli, which has attractive features such as high yield, simplicity, fast growth, etc., including expression of oxidative host, concurrent expression of chaperones, or periplasmic export. Recently, alternative methods for recovery of insoluble proteins expressed in E. coli were reported. Compared to traditional denaturation/refolding, these methods used the non-denaturing solubilization agent, N-lauroylsarkosine to obtain higher recovery yields of native proteins. Based on this work, here, we focused on solubilization of CRM197 from E. coli inclusion bodies. First, CRM197 was expressed as inclusion bodies by high-level expression of recombinant CRM197 in E. coli (126.8 mg/g dcw). Then bioactive CRM197 was isolated from these inclusion bodies with high yield (108.1 mg/g dcw) through solubilization with N-lauroylsarkosine including Triton X-100 and CHAPS, and purified by Ni-affinity chromatography and size-exclusion chromatography. In this study, we present a cost-effective alternative for the production of bioactive CRM197 and compare our recovery yield with yields in other production processes.