Multifocal fluorescence video-rate imaging of centimetre-wide arbitrarily shaped brain surfaces at micrometric resolution.

Fluorescence microscopy allows for the high-throughput imaging of cellular activity across brain areas in mammals. However, capturing rapid cellular dynamics across the curved cortical surface is challenging, owing to trade-offs in image resolution, speed, field of view and depth of field. Here we report a technique for wide-field fluorescence imaging that leverages selective illumination and the integration of focal areas at different depths via a spinning disc with varying thickness to enable video-rate imaging of previously reconstructed centimetre-scale arbitrarily shaped surfaces at micrometre-scale resolution and at a depth of field of millimetres. By implementing the technique in a microscope capable of acquiring images at 1.68 billion pixels per second and resolving 16.8 billion voxels per second, we recorded neural activities and the trajectories of neutrophils in real time on curved cortical surfaces in live mice. The technique can be integrated into many microscopes and macroscopes, in both reflective and fluorescence modes, for the study of multiscale cellular interactions on arbitrarily shaped surfaces.

Rapid detection of isocitrate dehydrogenase 1 mutation status in glioma based on Crispr-Cas12a.

The aim is to use Crispr-Cas12a for the rapid detection of the single nucleotide polymorphism (SNP) of isocitrate dehydrogenase 1 (IDH1)-R132H locus and explore the effectiveness and consistency of this method with direct sequencing method for detecting IDH1-R132H of glioma tissue samples. 58 previous frozen tissue and 46 recent fresh tissue samples of adult diffuse glioma were selected to detect IDH1-R132H using Crispr-Cas12a. The results of immunohistochemistry (IHC) and direct sequencing methods were analyzed. We calculated the efficiency index of Crispr-Cas12a and IHC, and analyzed the consistency among Crispr-Cas12a, IHC and direct sequencing method using paired Chi-sequare test and Kappa identity test. We accomplished the rapid detection of IDH1-R132H in 60 min using Crispr-Cas12a. Regarding direct sequencing method as the gold standard, the sensitivity, specificity and consistency rate of Crispr-Cas12a was 91.4%, 95.7% and 93.1% in the frozen sample group, while 96.1%, 89.7% and 92.0% in the fresh sample group, respectively. Kappa test showed good consistency between the two methods (k = 0.858). Crispr-Cas12a can quickly and accurately detect IDH1-R132H and has good stability. It is a promising method to detect IDH1 mutation status intraoperatively.

Cerebral cortex and hippocampus neural interaction during vagus nerve stimulation under in vivo large-scale imaging.

Objective: The purpose of this study was to study mechanisms of VNS modulation from a single neuron perspective utilizing a practical observation platform with single neuron resolution and widefield, real-time imaging coupled with an animal model simultaneously exposing the cerebral cortex and the hippocampus. Methods: We utilized the observation platform characterized of widefield of view, real-time imaging, and high spatiotemporal resolution to obtain the neuronal activities in the cerebral cortex and the hippocampus during VNS in awake states and under anesthesia. Results: Some neurons in the hippocampus were tightly related to VNS modulation, and varied types of neurons showed distinct responses to VNS modulation. Conclusion: We utilized such an observation platform coupled with a novel animal model to obtain more information on neuron activities in the cerebral cortex and the hippocampus, providing an effective method to further study the mechanisms of therapeutic effects modulated by VNS.

Interfacial-Shear-Mediated Snowball Assembly of Hotspot-Rich Silver Pompon Architectures for Tailored Surface-Enhanced Raman Scattering Responses.

To gain superior signal-enhanced performance, metal nanocrystals serving as building blocks can be collectively assembled into a hierarchically ordered structure for creating multiple hotspots. However, the collaborative assembly of anisotropic crystals to form a hotspot-rich structure remains a challenging task. In this study, controllable shear was introduced to a soft liquid-liquid interface to provide a unique environment for the snowball assembly of silver pompon architectures (Ag-PAs). Micrometer-scale 3D plasmonic Ag pompon architectures composed of densely packed nanoparticles (NPs) are fabricated using shear-mediating crystal growth dynamics. The crystal morphology and size are easily controlled by tuning the interfacial shear and diffusion pathways. The hotspot-rich Ag-PAs with high sensitivity (LOD = 1.1 × 10-13 mol/L) exhibit a superior Raman enhancement performance, which is comparable to some bimetals.

Transcriptome sequencing analysis of alfalfa reveals CBF genes potentially playing important roles in response to freezing stress

Abstract Alfalfa (Medicago sativa L.) is an important perennial forage, with high nutritional value, which is widely grown in the world. Because of low freezing tolerance, its distribution and production are threatened and limited by winter weather. To understand the complex regulation mechanisms of freezing tolerance in alfalfa, we performed transcriptome sequencing analysis under cold (4 °C) and freezing (-8 °C) stresses. More than 66 million reads were generated, and we identified 5767 transcripts differentially expressed in response to cold and/or freezing stresses. These results showed that these genes were mainly classified as response to stress, transcription regulation, hormone signaling pathway, antioxidant, nodule morphogenesis, etc., implying their important roles in response to cold and freezing stresses. Furthermore, nine CBF transcripts differentially expressed were homologous to CBF genes of Mt-FTQTL6 site, conferring freezing tolerance in M. truncatula, which indicated that a genetic mechanism controlling freezing tolerance was conservative between M. truncatula and M. sativa. In summary, this transcriptome dataset highlighted the gene regulation response to cold and/or freezing stresses in alfalfa, which provides a valuable resource for future identification and functional analysis of candidate genes in determining freezing tolerance.

Transcriptome sequencing analysis of alfalfa reveals CBF genes potentially playing important roles in response to freezing stress.

Alfalfa (Medicago sativa L.) is an important perennial forage, with high nutritional value, which is widely grown in the world. Because of low freezing tolerance, its distribution and production are threatened and limited by winter weather. To understand the complex regulation mechanisms of freezing tolerance in alfalfa, we performed transcriptome sequencing analysis under cold (4 °C) and freezing (-8 °C) stresses. More than 66 million reads were generated, and we identified 5767 transcripts differentially expressed in response to cold and/or freezing stresses. These results showed that these genes were mainly classified as response to stress, transcription regulation, hormone signaling pathway, antioxidant, nodule morphogenesis, etc., implying their important roles in response to cold and freezing stresses. Furthermore, nine CBF transcripts differentially expressed were homologous to CBF genes of Mt-FTQTL6 site, conferring freezing tolerance in M. truncatula, which indicated that a genetic mechanism controlling freezing tolerance was conservative between M. truncatula and M. sativa. In summary, this transcriptome dataset highlighted the gene regulation response to cold and/or freezing stresses in alfalfa, which provides a valuable resource for future identification and functional analysis of candidate genes in determining freezing tolerance.

Bioinformatics Analysis of MAPKKK Family Genes in Medicago truncatula.

Mitogen-activated protein kinase kinase kinase (MAPKKK) is a component of the MAPK cascade pathway that plays an important role in plant growth, development, and response to abiotic stress, the functions of which have been well characterized in several plant species, such as Arabidopsis, rice, and maize. In this study, we performed genome-wide and systemic bioinformatics analysis of MAPKKK family genes in Medicago truncatula. In total, there were 73 MAPKKK family members identified by search of homologs, and they were classified into three subfamilies, MEKK, ZIK, and RAF. Based on the genomic duplication function, 72 MtMAPKKK genes were located throughout all chromosomes, but they cluster in different chromosomes. Using microarray data and high-throughput sequencing-data, we assessed their expression profiles in growth and development processes; these results provided evidence for exploring their important functions in developmental regulation, especially in the nodulation process. Furthermore, we investigated their expression in abiotic stresses by RNA-seq, which confirmed their critical roles in signal transduction and regulation processes under stress. In summary, our genome-wide, systemic characterization and expressional analysis of MtMAPKKK genes will provide insights that will be useful for characterizing the molecular functions of these genes in M. truncatula.