Visualizing Dynamics of Cell Signaling In Vivo with a Phase Separation-Based Kinase Reporter.

Visualizing dynamics of kinase activity in living animals is essential for mechanistic understanding of cell and developmental biology. We describe GFP-based kinase reporters that phase-separate upon kinase activation via multivalent protein-protein interactions, forming intensively fluorescent droplets. Called SPARK (separation of phases-based activity reporter of kinase), these reporters have large dynamic range (fluorescence change), high brightness, fast kinetics, and are reversible. The SPARK-based protein kinase A (PKA) reporter reveals oscillatory dynamics of PKA activities upon G protein-coupled receptor activation. The SPARK-based extracellular signal-regulated kinase (ERK) reporter unveils transient dynamics of ERK activity during tracheal metamorphosis in live Drosophila. Because of intensive brightness and simple signal pattern, SPARKs allow easy examination of kinase signaling in living animals in a qualitative way. The modular design of SPARK will facilitate development of reporters of other kinases.

Mechanisms Underlying the Overlooked Chiral Fungicide-Driven Enantioselective Proliferation of Antibiotic Resistance in Earthworm Intestinal Microbiome.

From a "One Health" perspective, the global threat of antibiotic resistance genes (ARGs) is associated with modern agriculture practices including agrochemicals application. Chiral fungicides account for a considerable proportion of wildly used agrochemicals; however, whether and how their enantiomers lead to differential proliferation of antibiotic resistance in agricultural environments remain overlooked. Focused on the soil-earthworm ecosystem, we for the first time deciphered the mechanisms underlying the enantioselective proliferation of antibiotic resistance driven by the enantiomers of a typical chiral fungicide mandipropamid (i.e., R-MDP and S-MDP) utilizing a multiomic approach. Time-series metagenomic analysis revealed that R-MDP led to a significant enhancement of ARGs with potential mobility (particularly the plasmid-borne ARGs) in the earthworm intestinal microbiome. We further demonstrated that R-MDP induced a concentration-dependent facilitation of plasmid-mediated ARG transfer among microbes. In addition, transcriptomic analysis with verification identified the key aspects involved, where R-MDP enhanced cell membrane permeability, transfer ability, biofilm formation and quorum sensing, rebalanced energy production, and decreased cell mobility versus S-MDP. Overall, the findings provide novel insights into the enantioselective disruption of microbiome and resistome in earthworm gut by chiral fungicides and offer significant contributions to the comprehensive risk assessment of chiral agrochemicals in agroecosystems.

Dysregulation of AMPK-mTOR signaling leads to comorbid anxiety in Dip2a KO mice.

Autism is often comorbid with other psychiatric disorders. We have previously shown that Dip2a knockout (KO) induces autism-like behaviors in mice. However, the role of Dip2a in other psychiatric disorders remains unclear. In this paper, we revealed that Dip2a KO mice had comorbid anxiety. Dip2a KO led to a reduction in the dendritic length of cortical and hippocampal excitatory neurons. Molecular mechanism studies suggested that AMPK was overactivated and suppressed the mTOR cascade, contributing to defects in dendritic morphology. Deletion of Dip2a in adult-born hippocampal neurons (Dip2a conditional knockout (cKO)) increased susceptibility to anxiety upon acute stress exposure. Application of (2R,6R)-hydroxynorketamine (HNK), an inhibitor of mTOR, rescued anxiety-like behaviors in Dip2a KO and Dip2a cKO mice. In addition, 6 weeks of high-fat diet intake alleviated AMPK-mTOR signaling and attenuated the severity of anxiety in both Dip2a KO mice and Dip2a cKO mice. Taken together, these results reveal an unrecognized function of DIP2A in anxiety pathophysiology via regulation of AMPK-mTOR signaling.

Distributed fiber mountain seismic monitoring and steady-state analysis under natural earthquakes.

Mountain dynamic response monitoring plays important roles in geological disaster evolution monitoring and warning. A distributed mountain seismic monitoring and steady-state analysis method is demonstrated with distributed acoustic sensing (DAS) and a natural earthquake stimulus. In the field test, the seismic detection capability is first verified by comparing the recorded seismic waveforms from DAS and existing seismic stations. The vibration signal difference between steady-state and unsteady-state mountain parts is apparent; the operational modal analysis method is utilized to extract the response difference and to monitor the disaster evolution process. The proposed method has many advantages, including being easy to deploy, all-weather online monitoring, etc. It is believed that the proposed method will broaden the DAS application scope and promote the development of geological disaster early warning such as landslides and collapses.

Autism candidate gene DIP2A regulates spine morphogenesis via acetylation of cortactin.

Dendritic spine development is crucial for the establishment of excitatory synaptic connectivity and functional neural circuits. Alterations in spine morphology and density have been associated with multiple neurological disorders. Autism candidate gene disconnected-interacting protein homolog 2 A (DIP2A) is known to be involved in acetylated coenzyme A (Ac-CoA) synthesis and is primarily expressed in the brain regions with abundant pyramidal neurons. However, the role of DIP2A in the brain remains largely unknown. In this study, we found that deletion of Dip2a in mice induced defects in spine morphogenesis along with thin postsynaptic density (PSD), and reduced synaptic transmission of pyramidal neurons. We further identified that DIP2A interacted with cortactin, an activity-dependent spine remodeling protein. The binding activity of DIP2A-PXXP motifs (P, proline; X, any residue) with the cortactin-Src homology 3 (SH3) domain was critical for maintaining the level of acetylated cortactin. Furthermore, Dip2a knockout (KO) mice exhibited autism-like behaviors, including excessive repetitive behaviors and defects in social novelty. Importantly, acetylation mimetic cortactin restored the impaired synaptic transmission and ameliorated repetitive behaviors in these mice. Altogether, our findings establish an initial link between DIP2A gene variations in autism spectrum disorder (ASD) and highlight the contribution of synaptic protein acetylation to synaptic processing.

Uptake, translocation, and metabolism of thiamethoxam in soil by leek plants.

Thiamethoxam (TMX) is commonly applied on leek plants by root irrigation. It might be taken up by leek plants and thus has lasting dietary risk. In this study, the uptake, translocation, and metabolism of TMX in leek plants were investigated. The results obtained from both the hydroponic and soil experiments indicated that TMX could be easily translocated upward and accumulated in leek shoots after being absorbed by roots. The total absorbed TMX amount (Mtotal) in leek plants from the tested soils varied greatly with its adsorption governed by soil characteristics. Interestingly, Mtotal was closely correlated with the concentration of TMX in in situ pore water, indicating that TMX in in situ pore water could be a useful approach to predict uptake of this chemical by leek plants from various soils. Profoundly, clothianidin (CLO) was detected with concentration of 0.07-1.54 mg/kg in roots and 0.27-4.12 mg/kg in shoots at 14 d, respectively, suggesting that TMX is easily converted into CLO in leek plants. The results showed that TMX used in soil is easily absorbed by leek and accumulated in edible parts accompanying with formation of CLO.

Continuous Quantitative Electroencephalogram (EEG) Monitoring for Early Detection of Brain Herniation in Large Hemispheric Infarction (LHI): A Case Report.

BACKGROUND: Computer-assisted electroencephalography (EEG) systems may improve the likelihood of detecting abnormal EEGs in adult patients with severe disease. CASE PRESENTATION: We implemented long-range EEG monitoring in a patient with large hemispheric infarction (LHI) and explored its real-time changes in reflecting the patient's brain function. The bands of Alpha, Beta, Delta, Theta, DAR (Delta/Alpha), DTABR (Delta+Theta/Alpha+Beta), and brain symmetry index (BSI) were calculated as a ratio of total power. The test results showed that this patient presents a progressive worsening trend and developed brain herniation. The sigh at the electrophysiological level of brain herniation could be seen 6 h in advance based on the quantitative EEG (QEEG) parameters test. We calculated QEEG at both C3 and C4, electrode locations simultaneously, and the results showed that the trend of QEEG at both electrodes was consistent with the global, affected, and unaffected side. CONCLUSIONS: QEEG parameters can reflect the trend of LHI patients in real-time and may predict the occurrence of LHI brain herniation. For LHI patients, monitoring with fewer EEG electrodes can be tried to predict the changes in conditions.

Uptake, Accumulation, and translocation of azoxystrobin by Vegetable plants in soils: influence of soil characteristics and plant species.

Although azoxystrobin has been widely applied on various crops, little is known about the bioavailability of azoxystrobin in the soil-vegetable system. In this study, the uptake, accumulation and translocation of azoxystrobin as affected by soil characteristics and plant species were respectively investigated. The accumulation amount of azoxystrobin in pakchoi increased as soil adsorption decreased and was positively associated with its concentration in pore water (Cpw), which was mainly affected by soil organic matter content. Therefore, Cpw could be a candidate for the estimation of azoxystrobin accumulation in pakchoi. In all the tested vegetables, azoxystrobin was mainly accumulated in roots, and its upward translocation was limited. Root lipid content was a major factor affecting the uptake and translocation of azoxystrobin in different vegetables.

Analysis of Dip2B Expression in Adult Mouse Tissues Using the LacZ Reporter Gene.

Disconnected (disco)-interacting protein 2 homolog B (Dip2B) is a member of the Dip2 superfamily and plays an essential role in axonal outgrowth during embryogenesis. In adults, Dip2B is highly expressed in different brain regions, as shown by in situ analysis, and may have a role in axon guidance. However, the expression and biological role of Dip2B in other somatic tissues remain unknown. To better visualize Dip2B expression and to provide insight into the roles of Dip2B during postnatal development, we used a Dip2btm1a(wtsi)komp knock-in mouse model, in which a LacZ-Neo fusion protein is expressed under Dip2b promoter and allowed Dip2B expression to be analyzed by X-gal staining. qPCR analyses showed that Dip2b mRNA was expressed in a variety of somatic tissues, including lung and kidney, in addition to brain. LacZ staining indicated that Dip2B is broadly expressed in neuronal, reproductive, and vascular tissues as well as in the kidneys, heart, liver, and lungs. Moreover, neurons and epithelial cells showed rich staining. The broad and intense patterns of Dip2B expression in adult mice provide evidence of the distribution of Dip2B in multiple locations and, thereby, its implication in numerous physiological roles.

Effect of low VEGF on lung development and function.

Vascular endothelial growth factor (VEGF) is important for lung development and function but ideal mouse models are limited to decipher the quantitative relationship between VEGF expression levels and its proper development and pathogenesis. Human SPC promoter has been used to faithfully express genes or cDNAs in the pulmonary epithelium in many transgenic mouse models. In the study, a mouse model of lung-specific and reversible VEGF repression (hspc-rtTRtg/+/VegftetO/tetO) was generated. Human SPC promoter was used to drive lung-specific rtTR expression, a cDNA coding for doxycycline-regulated transcription repression protein. By crossing with VegftetO/tetO mice, that has tetracycline operator sequences insertion in 5'-UTR region, it allows us to reversibly inhibit lung VEGF transcription from its endogenous level through doxycycline food, water or injection. The tissue-specific inhibition of VEGF is used to mimic abnormal expression levels of VEGF in lung. Reduced VEGF expression in lung is confirmed by quantitative real time PCR and immunoblotting. Lung development and structure was analyzed by histology analysis and found significantly affected under low VEGF. The pulmonary epithelium and alveolarization are found abnormal with swelling alveolar septum and enlargement of air space. Genome-wide gene expression analysis identified that immune activities are involved in the VEGF-regulated lung functions. The transgenic mouse model can be used to mimic human pulmonary diseases. The mouse model confirms the important regulatory roles of epithelial expressed VEGF in lung development and function. This mouse model is valuable for studying VEGF-regulated lung development, pathogenesis and drug screening under low VEGF expression.

Angiocrine FSTL1 (Follistatin-Like Protein 1) Insufficiency Leads to Atrial and Venous Wall Fibrosis via SMAD3 Activation.

OBJECTIVE: Angiocrine factors, mediating the endothelial-mural cell interaction in vascular wall construction as well as maintenance, are incompletely characterized. This study aims to investigate the role of endothelial cell-derived FSTL1 (follistatin-like protein 1) in vascular homeostasis. Approach and Results: Using conditional knockout mouse models, we show that loss of FSTL1 in endothelial cells (Fstl1ECKO) led to an increase of pulmonary vascular resistance, resulting in the heart regurgitation especially with tricuspid valves. However, this abnormality was not detected in mutant mice with Fstl1 knockout in smooth muscle cells or hematopoietic cells. We further showed that there was excessive αSMA (α-smooth muscle actin) associated with atrial endocardia, heart valves, veins, and microvessels after the endothelial FSTL1 deletion. There was also an increase in collagen deposition, as demonstrated in livers of Fstl1ECKO mutants. The SMAD3 (mothers against decapentaplegic homolog 3) phosphorylation (pSMAD3) was significantly enhanced, and pSMAD3 staining was colocalized with αSMA in vein walls, suggesting the activation of TGFß (transforming growth factor ß) signaling in vascular mural cells of Fstl1ECKO mice. Consistently, treatment with a TGFß pathway inhibitor reduced the abnormal association of αSMA with the atria and blood vessels in Fstl1ECKO mutant mice. CONCLUSIONS: The findings imply that endothelial FSTL1 is critical for the homeostasis of vascular walls, and its insufficiency may favor cardiovascular fibrosis leading to heart failure.

LncRNA PART1 facilitates the malignant progression of colorectal cancer via miR-150-5p/LRG1 axis.

Study has shown that long noncoding RNA (lncRNA) prostate androgen-regulated transcript 1 (PART1) was elevated in colorectal cancer tissues and cells, and the proliferation and metastasis of colorectal cancer cells were reduced after its downregulation. The tumor-suppressive role of microRNA-150-5p (miR-150-5p) has been shown in colorectal cancer. In this study, the association between PART1 and miR-150-5p in colorectal cancer was analyzed. Results revealed an increase of PART1, but a decrease of miR-150-5p in 56 colorectal cancer tissues. And there was a strong negative correlation between levels of PART1 and miR-150-5p in these cancer samples. Also, compared with 10 healthy controls, the level of PART1 was increased, whereas miR-150-5p expression was diminished in the serum of 10 colorectal cancer patients. Cell proliferation and migration, along with epithelial-mesenchymal transition, was promoted by PART1 overexpression. However, this lncRNA mitigated apoptosis of colorectal cancer cells. Whereas miR-150-5p mimic abrogated these effects caused by PART1 overexpression. The influences of PART1 knockdown on the above malignant characteristics of colorectal cancer cells were contrary to its overexpression. miR-150-5p inhibitor ablated the effects induced by PART1 knockdown. In xenograft mouse models, silencing of PART1 decreased tumor volume and weight. Our data supported that lncRNA PART1 may regulate leucine-rich α-2-glycoprotein-1 (LRG1) expression through a competing interaction mechanism that hindering miR-150-5p function. In conclusion, PART1 facilitates the malignant progression of colorectal cancer via miR-150-5p/LRG1 pathway. The study further clarified the molecular mechanism of PART1 in colorectal cancer. This study may provide a new approach to diagnose and treat colorectal cancer.

Preparation and Insights of Smart Foams with Phototunable Foamability Based on Azobenzene-Containing Surfactants.

Smart foams with tunable foamability exhibit superb applications in many fields such as colloidal and interface science. Herein, we have synthesized an azobenzene-containing surfactant with excellent photoresponsiveness by a simple thiol-maleimide click reaction between thioglycolic acid and 4-(N-maleimide) azobenzene (MAB). The structure and the photoresponsive behavior of the novel surfactant are characterized. Depending on the solution concentration, the synthesized surfactant demonstrated various speeds for the trans/cis photoisomerization varying from 9 to 24 s for the given concentration range and excellent reversible photoisomerization cycling stability (more than 20 cycles) upon light irradiation. Based on these conformational switches, a series of phototriggered obvious surface properties (e.g., critical micelle concentration (CMC), surface tension (γ), and surface excess concentration (Γ)) changes of the surfactant are achieved. More specifically, the smart foam system with tunable foamability is realized. As-formed smart foams with rapid photocontrolled reversible foaming/defoaming transition and excellent cycling stability make them very attractive candidates for applications in wastewater treatment, green textile, oil extraction, and emulsification.

Subcellular Imaging of Localization and Transformation of Silver Nanoparticles in the Oyster Larvae.

To accurately assess the behavior and toxicity of silver nanoparticles (AgNPs), it is essential to understand their subcellular distribution and biotransformation. We combined both nanoscale secondary ion mass spectrometry (NanoSIMS) and electron microscopy to investigate the subcellular localization of Ag and in situ chemical distribution in the oyster larvae Crassostrea angulata after exposure to isotopically enriched 109AgNPs. Oyster larvae directly ingested particulate Ag, and in vivo dissolution of AgNPs occurred. The results collectively showed that AgNPs were much less bioavailable than Ag+, and the intracellular Ag was mainly originated from the soluble Ag, especially those dissolved from the ingested AgNPs. AgNPs absorbed on the cell membranes continued to release Ag ions, forming inorganic Ag-S complexes extracellularly, while Ag-organosulfur complexes were predominantly formed intracellularly. The internalized Ag could bind to the sulfur-rich molecules (S-donors) in the cytosol and/or be sequestered in the lysosomes of velum, esophagus, and stomach cells, as well as in the digestive vacuoles of digestive cells, which could act as a detoxification pathway for the oyster larvae. Ag was also occasionally incorporated into the phosphate granules, rough endoplasmic reticulum, and mitochondria. Our work provided definite evidence for the partial sulfidation of AgNPs after interaction with oyster larvae and shed new light on the bioavailability and fate of nanoparticles in marine environment.

A-kinase interacting protein 1 as a potential biomarker of advanced tumor features and increased recurrence risk in papillary thyroid carcinoma patients.

BACKGROUND: This study aimed to detect the expression of A-kinase interacting protein 1 (AKIP1) and explore its correlation with clinicopathological features and clinical outcomes in papillary thyroid carcinoma (PTC) patients. METHODS: A total of 245 PTC patients treated by lobectomy or thyroidectomy were analyzed in this retrospective study. AKIP1 expression in tumor and adjacent tissue (from Specimen Room of our hospital) was detected by immunohistochemical (IHC) assay and then categorized as four grades: AKIP1 low (IHC score ≤3), high+ (IHC score 4-6), high++ (IHC score 7-9), and high+++ (IHC score 10-12). RESULTS: A-kinase interacting protein 1 low, high+, high++, and high+++ expression was 101 (41.2%), 101 (41.2%), 32 (13.1%), and 11 (4.5%) in tumor tissues, while was 173 (70.6%), 61 (24.9%), 9 (3.7%), and 2 (0.8%) in adjacent tissues. Further comparison analysis showed increased grade of AKIP1 expression in tumor tissues compared to adjacent tissue. Meanwhile, increased grade of tumor AKIP1 expression was correlated with larger tumor size, extrathyroidal invasion, increased pT stage, and higher pTNM stage. For prognosis, increased grade of tumor AKIP1 expression was correlated with shorter disease-free survival (DFS), while was not correlated with overall survival (OS). Forward stepwise multivariate Cox's regression revealed that higher tumor AKIP1 was an independent factor predicting worse DFS, but not OS. CONCLUSION: AKIP1 is upregulated in tumor tissue, and increased tumor AKIP1 expression correlates with advanced tumor features and increased recurrence risk in PTC patients, which suggest that AKIP1 severs as a potential marker for effective supervision of PTC progression.

Targeted Disruption of Mouse Dip2B Leads to Abnormal Lung Development and Prenatal Lethality.

Molecular and anatomical functions of mammalian Dip2 family members (Dip2A, Dip2B and Dip2C) during organogenesis are largely unknown. Here, we explored the indispensable role of Dip2B in mouse lung development. Using a LacZ reporter, we explored Dip2B expression during embryogenesis. This study shows that Dip2B expression is widely distributed in various neuronal, myocardial, endothelial, and epithelial cell types during embryogenesis. Target disruption of Dip2b leads to intrauterine growth restriction, defective lung formation and perinatal mortality. Dip2B is crucial for late lung maturation rather than early-branching morphogenesis. The morphological analysis shows that Dip2b loss leads to disrupted air sac formation, interstitium septation and increased cellularity. In BrdU incorporation assay, it is shown that Dip2b loss results in increased cell proliferation at the saccular stage of lung development. RNA-seq analysis reveals that 1431 genes are affected in Dip2b deficient lungs at E18.5 gestation age. Gene ontology analysis indicates cell cycle-related genes are upregulated and immune system related genes are downregulated. KEGG analysis identifies oxidative phosphorylation as the most overrepresented pathways along with the G2/M phase transition pathway. Loss of Dip2b de-represses the expression of alveolar type I and type II molecular markers. Altogether, the study demonstrates an important role of Dip2B in lung maturation and survival.

Repression of adipose vascular endothelial growth factor reduces obesity through adipose browning.

Obesity is the result of excessive energy accumulation and is associated with many diseases. We previously reported that universal repression of vascular endothelial growth factor (VEGF) leads to brown-like adipocyte development in white adipose tissues, and that these mice are resistant to obesity (Lu X et al. Endocrinology 153: 3123-3132, 2012). Using an adipose-specific VEGF repression mouse model (aP2-rtTR-krabtg/+/VEGFtetO/tetO), we show that adipose-specific VEGF repression can repeat the previous phenotypes, including adipose browning, increased energy consumption, and reduction in body weight. Expression of brown adipose-associated genes is increased, and white adipose-associated genes are downregulated under VEGF repression. Our study demonstrates that adipose-specific VEGF repression can lead to antiobesity activity through adipose browning and has potential clinical value.

Photocontrollable Wrinkle Morphology Evolution on Azo-Based Multilayers for Hierarchical Surface Micropatterns Fabrication.

Inspired by nature, comprehensive understanding and ingenious utilization of the self-organized wrinkling behaviors of the sandwiched multilayer bonded on substrates are important for engineering and/or functional laminated devices design. Herein, we report a facile and effective strategy to regulate the wrinkles morphology evolution and the resultant hierarchical surface micropatterns on azobenzene-based laminated multilayers by visible-light irradiation. Revealed by systematic experiments, the photocontrolled dynamic wrinkle evolutions are triggered by the reversible photoisomerization of azobenzene in the top azopolymer film and are strongly dependent on the intermediate photoinert layers (e.g., polystyrene and oxygen plasma-induced SiO x layer) with the wrinkle-reinforcing effect or the stress relaxation acceleration effect. Interestingly, large-area well-defined hierarchical surface wrinkle patterns could be fabricated on the multilayers upon selective exposure. In the unexposed region, the wrinkles evolved into highly oriented patterns, whereas in the exposed region, they were fully erased or evolved into smaller-wavelength wrinkles. This study not only sheds light on the morphological evolution of the wrinkling laminated composites in engineering and nature but also paves a new avenue to conveniently and controllably realize the hierarchical stimulus-responsive surface patterns.

Functional prediction and characterization of Dip2 gene in mice.

Disconnected interacting protein 2 (DIP2) is a highly conserved protein family among invertebrates and vertebrates, but its function remains unclear. In this paper, we summarized the conservation of gene sequences and protein domains of DIP2 family members and predicted that they may have a similar functional role in acetyl-coenzyme A (acetyl-CoA) synthesis. We then used the most characterized member, disconnected interacting protein 2 homolog A (DIP2A), for further study. DIP2A is a cytoplasmic protein that is preferentially localized to mitochondria, and its acetyl-CoA synthetase activity has been demonstrated in vitro. Furthermore, the level of acetyl-CoA in HEK293 cells overexpressing DIP2A was increased, which is consistent with its metabolically related function. Together, these data enrich the evolutionary and functional characterization of dip2 genes and provide significant insights into the identification and application of other homologs of DIP2.