Search

This paper proposes a deep learning method for phase retrieval from two interferograms. The proposed method converts phase retrieval into the Zernike coefficient extraction problem, which can achieve Zernike coefficient extraction from two interferograms with random phase shifts. After knowing Zernike coefficients, the phase distribution can be retrieved using Zernike polynomials. The pre-filtering and phase unwrapping process are not required using the proposed method. The simulated data are analyzed, and the root mean square (RMS) of phase error reaches 0.01 λ. The effectiveness of the method is verified by the measured data.

More than half of the world's food is provided by cereals, as humans obtain60% of daily calories from grains. Producing more carbohydrates is always the final target of crop cultivation. The carbohydrate partitioning pathway directly affects grain yield, but the molecular mechanisms and biological functions are poorly understood, including rice (Oryza sativa L.), one of the most important food sources. Here, we reported a prolonged grain filling duration mutant 1 (gfd1), exhibiting a long grain filling duration, less grain number per panicle, and bigger grain size without changing grain weight. Map-based cloning and molecular biological analyses revealed that GFD1 encoded a MATE transporter and expressed high in vascular tissues of the stem, spikelet hulls, and rachilla, but low in the leaf, controlling carbohydrate partitioning in the stem and grain but not in the leaf. GFD1 protein was partially localized on the plasma membrane and in the Golgi apparatus, and was finally verified to interact with two sugar transporters, OsSWEET4 and OsSUT2. Genetics analyses showed that GFD1 might control grain filling duration through OsSWEET4, adjust grain size with OsSUT2, and synergistically modulate grain number per panicle with both OsSUT2 and OsSWEET4. Together, our work proved that, the three transporters, which are all initially classified in the MFS family, could control starch storage in both the primary sink (grain) and temporary sink (stem), and affect carbohydrate partitioning in the whole plant through physical interaction, giving a new vision of sugar transporter interactome and providing a tool for rice yield improvement.

As an important agronomic trait in rice (Oryza sativa), moderate leaf rolling helps to maintain the erectness of leaves and minimize shadowing between leaves, leading to improved photosynthetic efficiency and grain yield. However, the molecular mechanisms underlying rice leaf rolling still need to be elucidated. Here, we isolated a rice mutant, rl89, showing adaxially rolled leaf phenotype due to decreased number and size of bulliform cells. We confirmed that the rl89 phenotypes were caused by a single nucleotide substitution in OsDRB2 (LOC_Os10g33970) gene encoding DOUBLE-STRANDED RNA-BINDING2. This gene was constitutively expressed, and its encoded protein was localized to both nucleus and cytoplasm. Yeast two-hybrid assay showed that OsDRB2 could interact with DICER-LIKE1 (DCL1) and OsDRB1-2 respectively. qRT-PCR analysis of 29 related genes suggested that defects of the OsDRB2-miR166-OsHBs pathway could play an important role in formation of the rolled leaf phenotype of rl89, in which OsDRB2 mutation reduced miR166 accumulation, resulting in elevated expressions of the class III homeodomain-leucine zipper genes (such as OsHB1, 3 and 5) involved in leaf polarity and/or morphology development. Moreover, OsDRB2 mutation also reduced accumulation of miR160, miR319, miR390, and miR396, which could cause the abnormal leaf development in rl89 by regulating expressions of their target genes related to leaf development.

Summary Circadian clock, an endogenous time‐setting mechanism, allows plants to adapt to unstable photoperiod conditions and induces flowering with proper timing. In Arabidopsis, the central clock oscillator was formed by a series of interlocked transcriptional feedback loops, but little is known in rice so far. By MutMap technique, we identified the candidate gene OsLHY from a later flowering mutant lem1 and further confirmed it through genetic complementation, RNA interference knockdown, and CRISPR/Cas9‐knockout. Global transcriptome profiling and expression analyses revealed that OsLHY might be a vital circadian rhythm component. Interestingly, oslhy flowered later under ≥12 h day length but headed earlier under ≤11 h day length. qRT‐PCR results exhibited that OsLHY might function through OsGI‐Hd1 pathway. Subsequent one‐hybrid assays in yeast, DNA affinity purification qPCR, and electrophoretic mobility shift assays confirmed OsLHY could directly bind to the CBS element in OsGI promoter. Moreover, the critical day length (CDL) for function reversal of OsLHY in oslhy (11–12 h) was prolonged in the double mutant oslhy osgi (about 13.5 h), indicating that the CDL set by OsLHY was OsGI dependent. Additionally, the dual function of OsLHY entirely relied on Hd1, as the double mutant oslhy hd1 showed the same heading date with hd1 under about 11.5, 13.5, and 14 h day lengths. Together, OsLHY could fine‐tune the CDL by directly regulating OsGI, and Hd1 acts as the final effector of CDL downstream of OsLHY. Our study illustrates a new regulatory mechanism between the circadian clock and photoperiodic flowering.

The circadian clock and histone modifications could form a feedback loop in Arabidopsis; whether a similar regulatory mechanism exists in rice is still unknown. Previously, we reported that SDG724 and OsLHY are two rice heading date regulators in rice. SDG724 encodes a histone H3K36 methyltransferase, and OsLHY is a vital circadian rhythm transcription factor. Both could be involved in transcription regulatory mechanisms and could affect gene expression in various pathways. To explore the crosstalk between the circadian clock and histone methylation in rice, we studied the relationship between OsLHY and SDG724 via the transcriptome analysis of their single and double mutants, oslhy, sdg724, and oslhysdg724. Screening of overlapped DEGs and KEGG pathways between OsLHY and SDG724 revealed that they could control many overlapped pathways indirectly. Furthermore, we identified three candidate targets (OsGI, OsCCT38, and OsPRR95) of OsLHY and one candidate target (OsCRY1a) of SDG724 in the clock pathway. Our results showed a regulatory relationship between OsLHY and SDG724, which paved the way for revealing the interaction between the circadian clock and histone H3K36 methylation.

Metal micro-droplet 3D printing is an effective additive manufacturing technique to form micro pillar structures. However, the control mechanism of the pillar contour is still unclear. To form a pillar with uniform radius, it is essential to investigate the dependence of processing parameters on pillar contours. In this study, a 3D numerical model was employed to simulate and analyze the successive pileup processes of multiple droplets. The solidification angle of solidified new-coming droplets is defined to characterize the pillar contour. An analytic equation of solidification angle was established. In this case, the solidified morphology of the droplets could be feasibly predicted through thermophysical parameters and initial conditions. This work provides good physical understanding of the complicated mechanisms for fabricating a pillar structure by means of the successive pileup of molten metal droplets, and paves the way for pillar contour control.

In vivo fundus imaging offers non-invasive access to neuron structures and biochemical processes in the retina. However, optical aberrations of the eye degrade the imaging resolution and prevent visualization of subcellular retinal structures. We developed an adaptive optics two-photon excitation fluorescence microscopy (AO-TPEFM) system to correct ocular aberrations based on a nonlinear fluorescent guide star and achieved subcellular resolution for in vivo fluorescence imaging of the mouse retina. With accurate wavefront sensing and rapid aberration correction, AO-TPEFM permits structural and functional imaging of the mouse retina with submicron resolution. Specifically, simultaneous functional calcium imaging of neuronal somas and dendrites was demonstrated. Moreover, the time-lapse morphological alteration and dynamics of microglia were characterized in a mouse model of retinal disorder. In addition, precise laser axotomy was achieved, and degeneration of retinal nerve fibres was studied. This high-resolution AO-TPEFM is a promising tool for non-invasive retinal imaging and can facilitate the understanding of a variety of eye diseases as well as neurodegenerative disorders in the central nervous system., Adaptive-Optics Fluorescence Microscopy: Seeing clearly into eyes An advance in Two-Photon Excitation Fluorescence Microscopy (TPEFM) allows greatly improved subcellular imaging of the retina, promising better understanding of the eye and the central nervous system in health and disease. Researchers led by Jianan Y. Qu at Hong Kong University of Science and Technology used adaptive optics techniques to correct the optical aberrations that have limited application of TPEFM in living eyes. They demonstrated their innovation using fluorescent detector molecules to image the activity of calcium ions in mouse retinal neurons. They also monitored subcellular structures in microglial cells, and guided laser light to cut the axons that transmit nerve signals. This technology could help investigate the development of neurodegenerative diseases, since the eye offers a window into nerves of the central nervous system that link the eye with the brain.

The iron-sulfur subunit (SDH2) of succinate dehydrogenase plays a key role in electron transport in plant mitochondria. However, it is yet unknown whether SDH2 genes are involved in leaf senescence and yield formation. In this study, we isolated a late premature senescence mutant, lps1, in rice (Oryza sativa). The mutant leaves exhibited brown spots at late tillering stage and wilted at the late grain-filling stage and mature stage. In its premature senescence leaves, photosynthetic pigment contents and net photosynthetic rate were reduced, chloroplasts and mitochondria were degraded. Meanwhile, lps1 displayed small panicles, low seed-setting rate and dramatically reduced grain yield. Gene cloning and complementation analysis suggested that the causal gene for the mutant phenotype was OsSDH2-1 (LOC_Os08g02640), in which single nucleotide mutation resulted in an amino acid substitution in the encoded protein. OsSDH2-1 gene was expressed in all organs tested, with higher expression in leaves, root tips, ovary and anthers. OsSDH2-1 protein was targeted to mitochondria. Furthermore, reactive oxygen species (ROS), mainly H2O2, was excessively accumulated in leaves and young panicles of lps1, which could cause premature leaf senescence and affect panicle development and pollen function. Taken together, OsSDH2-1 plays a crucial role in leaf senescence and yield formation in rice.

Spinal cord, as part of the central nervous system, accounts for the main communication pathway between the brain and the peripheral nervous system. Spinal cord injury is a devastating and largely irreversible neurological trauma, and can result in lifelong disability and paralysis with no available cure. In vivo spinal cord imaging in mouse models without introducing immunological artifacts is critical to understand spinal cord pathology and discover effective treatments. We developed a minimal-invasive intervertebral window by retaining ligamentum flavum to protect the underlying spinal cord. By introducing an optical clearing method, we achieved repeated two-photon fluorescence and stimulated Raman scattering imaging at subcellular resolution with up to 16 imaging sessions over 167 days and observed no inflammatory response. Using this optically cleared intervertebral window, we studied the neuron-glia dynamics following laser axotomy and observed strengthened contact of microglia with the nodes of Ranvier during axonal degeneration. By enabling long-term, repetitive, stable, high-resolution and inflammation-free imaging of mouse spinal cord, our method provides a reliable platform in the research aiming at understanding and treatment of spinal cord pathology.

High-resolution optical imaging of deep tissue in-situ such as the living brain is fundamentally challenging because of the aberration and scattering of light. In this work, we develop an innovative adaptive optics three-photon microscope based on direct focus sensing and shaping that can accurately measure and effectively compensate for both low- and high-order specimen-induced aberrations and recover near-diffraction-limited performance at depth. A conjugate adaptive optics configuration with remote focusing enables in vivo imaging of fine neuronal structures in the mouse cortex through the intact skull up to a depth of 750 µm below pia, making high-resolution microscopy in cortex near non-invasive. Functional calcium imaging with high sensitivity and accuracy, and high-precision laser-mediated microsurgery through the intact skull were demonstrated. Moreover, we also achieved in vivo high-resolution imaging of the deep cortex and subcortical hippocampus up to 1.1 mm below pia within the intact brain.

Mg-protoporphyrin IX monomethyl ester cyclase (MPEC) plays an essential role in chlorophyll biosynthesis. Further study on the key enzyme will provide us more comprehensive understanding about its function. In the present study, we identified a yellow and spotted leaf 1 (ysl1) mutant in rice exhibiting a yellow-green leaf phenotype throughout the whole developmental stage and a spotted leaf phenotype at seedling and tillering stages. Genetic analysis indicated that a single recessive gene was responsible for the phenotype of ysl1 mutant. Map-based cloning showed that the mutation site was located at the region of 105.4-kb between RM572 and L3 on the short arm of chromosome 1. Sequence analysis revealed that a point mutation (C-to-T) happened in the coding region of candidate gene YSL1 (LOC_Os01g17170) which caused an amino acid change in MPEC. YSL1 was expressed mainly in photosynthetic tissues and organs, and its encoded protein was transported into the chloroplast with a signal peptide. The phenotype of ysl1 returned to normal phenotype of wild type after introducing the wild-type YSL1 gene. Based on the evidence from different levels, we confirmed that the phenotype of ysl1 was caused by a single nucleotide substitution of YSL1 which is allelic to YGL8, OsCRD1, and YL-1, and demonstrated the spotted leaf at seedling and tillering stages as a novel phenotype of the MPEC mutants in rice.

High-resolution optical imaging deep in tissues is challenging because of optical aberrations and scattering of light caused by the complex structure of living matter. Here we present an adaptive optics three-photon microscope based on analog lock-in phase detection for focus sensing and shaping (ALPHA-FSS). ALPHA-FSS accurately measures and effectively compensates for both aberrations and scattering induced by specimens and recovers subcellular resolution at depth. A conjugate adaptive optics configuration with remote focusing enables in vivo imaging of fine neuronal structures in the mouse cortex through the intact skull up to a depth of 750 µm below the pia, enabling near-non-invasive high-resolution microscopy in cortex. Functional calcium imaging with high sensitivity and high-precision laser-mediated microsurgery through the intact skull were also demonstrated. Moreover, we achieved in vivo high-resolution imaging of the deep cortex and subcortical hippocampus up to 1.1 mm below the pia within the intact brain.

Background Tetrapyrroles play indispensable roles in various biological processes. In higher plants, glutamate 1-semialdehyde 2,1-aminomutase (GSAM) converts glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA), which is the rate-limiting step of tetrapyrrole biosynthesis. Up to now, GSAM genes have been successively identified from many species. Besides, it was found that GSAM could form a dimeric protein with itself by x-ray crystallography. However, no mutant of GSAM has been identified in monocotyledonous plants, and no experiment on interaction of GSAM protein with itself has been reported so far. Result We isolated a yellow leaf mutant, ys53, in rice (Oryza sativa). The mutant showed decreased photosynthetic pigment contents, suppressed chloroplast development, and reduced photosynthetic capacity. In consequence, its major agronomic traits were significantly affected. Map-based cloning revealed that the candidate gene was LOC_Os08g41990 encoding GSAM protein. In ys53 mutant, a single nucleotide substitution in this gene caused an amino acid change in the encoded protein, so its ALA-synthesis ability was significantly reduced and GSA was massively accumulated. Complementation assays suggested the mutant phenotype of ys53 could be rescued by introducing wild-type OsGSAM gene, confirming that the point mutation in OsGSAM is the cause of the mutant phenotype. OsGSAM is mainly expressed in green tissues, and its encoded protein is localized to chloroplast. qRT-PCR analysis indicated that the mutation of OsGSAM not only affected the expressions of tetrapyrrole biosynthetic genes, but also influenced those of photosynthetic genes in rice. In addition, the yeast two-hybrid experiment showed that OsGSAM protein could interact with itself, which could largely depend on the two specific regions containing the 81th–160th and the 321th–400th amino acid residues at its N- and C-terminals, respectively. Conclusions We successfully characterized rice GSAM gene by a yellow leaf mutant and map-based cloning approach. Meanwhile, we verified that OsGSAM protein could interact with itself mainly by means of the two specific regions of amino acid residues at its N- and C-terminals, respectively.

In this paper, chemically bonded phosphate ceramic coatings (CBPCs) with the addition of carbon nanotubes (CNTs) were prepared on the mild steel (Q235) through sol gel method. The wear and corrosion performance of CBPCs with the addition of CNTs were evaluated by tribological and electrochemical tests combining the analysis of microstructure, phase characterization and curing behavior. Results indicate CNTs nanofiller significantly enhances the wear and corrosion behavior of CBPCs. The enhancement of wear performance for CBPCs with CNTs nanofiller is because CNTs can effectively hinder the dislocation movement through the coating and substrate system and the occurrence of plastic deformation owing to their remarkable mechanical and lubricating properties. The enhancement of corrosion performance for CBPCs with CNTs nanofiller is because that CNTs can help CBPCs to get denser and more compact microstructure, which is contributed to prolong the electrolyte diffusion path. Besides, CNTs exhibit excellent hydrophobicity and can absorb oxygen molecules, which prevents the corrosion process on the anode and cathode sections of coating and substrate system.

Significance Synaptic plasticity in the hippocampus is important for learning and memory formation. In particular, homeostatic synaptic plasticity enables neurons to restore their activity levels in response to chronic neuronal activity changes. While astrocytes modulate synaptic functions via the secretion of factors, the underlying molecular mechanisms remain unclear. Here, we show that suppression of hippocampal neuronal activity increases cytokine IL-33 release from astrocytes in the CA1 region. Activation of IL-33 and its neuronal ST2 receptor complex promotes functional excitatory synapse formation. Moreover, IL-33/ST2 signaling is important for the neuronal activity blockade-induced increase of CA1 excitatory synapses in vivo and spatial memory formation. This study suggests that astrocyte-secreted IL-33 acts as a negative feedback control signal to regulate hippocampal homeostatic synaptic plasticity., Hippocampal synaptic plasticity is important for learning and memory formation. Homeostatic synaptic plasticity is a specific form of synaptic plasticity that is induced upon prolonged changes in neuronal activity to maintain network homeostasis. While astrocytes are important regulators of synaptic transmission and plasticity, it is largely unclear how they interact with neurons to regulate synaptic plasticity at the circuit level. Here, we show that neuronal activity blockade selectively increases the expression and secretion of IL-33 (interleukin-33) by astrocytes in the hippocampal cornu ammonis 1 (CA1) subregion. This IL-33 stimulates an increase in excitatory synapses and neurotransmission through the activation of neuronal IL-33 receptor complex and synaptic recruitment of the scaffold protein PSD-95. We found that acute administration of tetrodotoxin in hippocampal slices or inhibition of hippocampal CA1 excitatory neurons by optogenetic manipulation increases IL-33 expression in CA1 astrocytes. Furthermore, IL-33 administration in vivo promotes the formation of functional excitatory synapses in hippocampal CA1 neurons, whereas conditional knockout of IL-33 in CA1 astrocytes decreases the number of excitatory synapses therein. Importantly, blockade of IL-33 and its receptor signaling in vivo by intracerebroventricular administration of its decoy receptor inhibits homeostatic synaptic plasticity in CA1 pyramidal neurons and impairs spatial memory formation in mice. These results collectively reveal an important role of astrocytic IL-33 in mediating the negative-feedback signaling mechanism in homeostatic synaptic plasticity, providing insights into how astrocytes maintain hippocampal network homeostasis.

Imaging of the brain in its native state at high spatial resolution poses major challenges to visualization techniques. Two-photon microscopy integrated with the thinned-skull or optical clearing skull technique provides a minimally invasive tool for in vivo imaging of the cortex of mice without activating immune response and inducing brain injury. However, the imaging contrast and spatial resolution are severely compromised by the optical heterogeneity of the skull, limiting the imaging depth to the superficial layer. In this work, an optimized configuration of an adaptive optics two-photon microscope system and an improved wavefront sensing algorithm are proposed for accurate correction for the aberrations induced by the skull window and brain tissue. Using this system, we achieved subcellular resolution transcranial imaging of layer 5 pyramidal neurons up to 700 μm below pia in living mice. In addition, we investigated microglia–plaque interaction in living brain of Alzheimer’s disease and demonstrated high-precision laser dendrotomy and single-spine ablation.

Optical deep brain imaging in vivo at high resolution has remained a great challenge over the decades. Two-photon endomicroscopy provides a minimally invasive approach to image buried brain structures, once it is integrated with a gradient refractive index (GRIN) lens embedded in the brain. However, its imaging resolution and field of view are compromised by the intrinsic aberrations of the GRIN lens. Here, we advance two-photon endomicroscopy by adding adaptive optics(AO) based on the direct wavefront sensing of the descanned fluorescent guide star created by two-photon excitation, which enables effective correction of the aberrations and recovery of diffraction-limited resolution over a much extended imaging volume (300 μm on each side). Benefiting from a precompensation strategy based on a lookup table, we achieved in vivo structural imaging of pyramidal neurons at synaptic resolution across all layers of the mouse hippocampus CA1. Moreover, by combining the AO endomicroscope system with a rapid multiplane imaging technique, we demonstrated simultaneous calcium imaging of hippocampal neuronal somata and dendrites in awake behaving mice. Our study holds great promise to facilitate brain research by enabling large-volume deep brain imaging in vivo at high resolution.

An innovative imaging tool enables in vivo high-resolution visualization of deep brain structures and functions over large volume., Optical deep-brain imaging in vivo at high resolution has remained a great challenge over the decades. Two-photon endomicroscopy provides a minimally invasive approach to image buried brain structures, once it is integrated with a gradient refractive index (GRIN) lens embedded in the brain. However, its imaging resolution and field of view are compromised by the intrinsic aberrations of the GRIN lens. Here, we develop a two-photon endomicroscopy by adding adaptive optics based on direct wavefront sensing, which enables recovery of diffraction-limited resolution in deep-brain imaging. A new precompensation strategy plays a critical role to correct aberrations over large volumes and achieve rapid random-access multiplane imaging. We investigate the neuronal plasticity in the hippocampus, a critical deep brain structure, and reveal the relationship between the somatic and dendritic activity of pyramidal neurons.

Two-photon microscopy has become the method of choice for in vivo brain imaging in neuroscience research during the past decades owing to its inherent sectioning capability and large penetration depth in scattering tissues. By integrating with a gradient refractive index (GRIN) lens that implanted into the brain, two-photon microendoscopy further extends the imaging depth down to subcortical regions. However, the imaging resolution and field of view (FOV) are compromised due to the intrinsic aberrations of the inserted GRIN lens. Here, we developed an adaptive optics (AO) two-photon microendoscopy based on direct wavefront sensing that can measure and correct the aberrations of GRIN lens during in vivo brain imaging. Using our system, the diffraction-limited resolution was restored and the fine structures such as dendritic spines of hippocampal CA1 neurons can be clearly resolved over a much extended FOV.

Chronic in vivo optical imaging of the spinal cord is an effective way to study the biological processes during and after spinal cord injury (SCI) in mouse models. It normally relies on an implanted spinal chamber to provide continuous optical access to the spinal cord. However, the chronic window consists of multiple layers of transparent materials with various optical properties and irregular thickness, which induce large optical aberration. Therefore, the image quality of multiphoton microscopy as well as the precision of femtosecond laser axotomy were dramatically degraded. In this work, we developed an adaptive optics (AO) microscope system integrating stimulated Raman scattering (SRS) and twophoton excited fluorescence (TPEF). Using our system, the aberrations induced by the spinal cord window were measured and compensated accordingly, enabling both high-resolution imaging and precise laser axotomy of the mouse spinal cord.

Lipid droplets are highly associated with obesity, diabetes, inflammatory disorders, and cancer. A reliable two-photon dye for specific lipid droplets imaging in live cells and live tissues at ultralow concentration has rarely been reported. In this work, four new aggregation-induced emission luminogens (AIEgens) based on the naphthalene core were designed and synthesized for specific two-photon lipid droplet staining. The new molecules, namely, NAP AIEgens, exhibit large Stokes shift (>110 nm), high solid-state fluorescence quantum yield (up to 30%), good two-photon absorption cross section (45–100 GM at 860 nm), high biocompatibility, and good photostability. They could specifically stain lipid droplets at ultralow concentration (50 nM) in a short time of 15 min. Such ultralow concentration is the lowest value for lipid droplets staining in live cells reported so far. In vitro and ex vivo two-photon imaging of lipid droplets in live cells and live mice liver tissues were successfully demonstrated. In ad...

Summary Neuronal activation is often accompanied by the regulation of cerebral hemodynamics via a process known as neurovascular coupling (NVC) which is essential for proper brain function and has been observed to be disrupted in a variety of neuropathologies. A comprehensive understanding of NVC requires imaging capabilities with high spatiotemporal resolution and a field-of-view that spans different orders of magnitude. Here, we present an approach for concurrent multi-contrast mesoscopic and two-photon microscopic imaging of neurovascular dynamics in the cortices of live mice. We investigated the spatiotemporal correlation between sensory-evoked neuronal and vascular responses in the auditory cortices of living mice using four imaging modalities. Our findings unravel drastic differences in the NVC at the regional and microvascular levels and the distinctive effects of different brain states on NVC. We further investigated the brain-state-dependent changes of NVC in large cortical networks and revealed that anesthesia and sedation caused spatiotemporal disruption of NVC., Graphical abstract, Highlights • Concurrent mesoscopic and microscopic imaging of neurovascular dynamics • Spatiotemporal characteristics of neurovascular responses across multiple scales • Distinct effects of anesthesia and sedation on neurovascular coupling • Cortex-wide correlation of neuronal activity and cerebral hemodynamics, Small animal imaging; Optical imaging; Techniques in neuroscience

Background Calvin cycle plays a crucial role in carbon fixation which provides the precursors of organic macromolecules for plant growth and development. Currently, no gene involved in Calvin cycle has been identified in monocotyledonous plants through mutant or/and map-based cloning approach. Results Here, we isolated a low-tillering mutant, c6635, in rice (Oryza sativa). The mutant displayed light green leaves and intensely declined pigment contents and photosynthetic capacity at early growth stage. Moreover, its individual plant showed a much smaller size, and most individuals produced only two tillers. At mature stage, its productive panicles, grain number and seed setting rate were significantly decreased, which lead to a sharp reduction of the grain yield. We confirmed that a single nucleotide mutation in LOC_Os04g16680 gene encoding sedoheptulose 1,7-bisphosphatase (SBPase) involved in Calvin cycle was responsible for the mutant phenotype of c6635 through map-based cloning, MutMap analysis and complementation experiments. Sequence analysis suggested that the point mutation caused an amino acid change from Gly-364 to Asp at the C-terminal of SBPase. In addition, OsSBPase gene was mainly expressed in leaf, and the encoded protein was located in chloroplast. The mutation of OsSBPase could significantly affect expression levels of some key genes involved in Calvin cycle. Conclusions We successfully identified a SBPase gene in monocotyledonous plants. Meanwhile, we demonstrated that a single nucleotide substitution at the 3′-end of this gene severely affects plant growth and grain yield, implying that the Gly-364 at the C-terminal of SBPase could play an important role in SBPase function in rice.

Additional file 3: Figure S2. Expression pattern of the OsSBPase gene by semiquantitative analysis (two replications in a gel). R: Root, S: Stem, L: Leaf blade, LS: leaf sheath, P: young panicle.

Additional file 2: Figure S1. PCR test of PCR-positive transgenic lines. M: DL-2000 marker; 1 and 18: PCR-positive control (pC2300-Actin-OsSBPase plasmid); 2: PCR-negative control (c6635 mutant); 3–6, 8–17, 19, 21, 22: PCR-positive transgenic lines; 7 and 20: PCR-negative transgenic lines.

Additional file 1: Table S1. The information of fourteen single nucleotide polymorphisms (SNPs) identified by MutMap in the target region.

BackgroundPhytyl residues are the common side chains of chlorophyll (Chl) and tocopherols. Geranylgeranyl reductase (GGR), which is encoded byCHLPgene, is responsible for phytyl biosynthesis. The light-harvesting like protein LIL3 was suggested to be required for stability of GGR and protochlorophyllide oxidoreductase in Arabidopsis.ResultsIn this study, we isolated a yellow-green leaf mutant,637ys, in rice (Oryza sativa). The mutant accumulated majority of Chls with unsaturated geranylgeraniol side chains and displayed a yellow-green leaf phenotype through the whole growth period. The development of chloroplasts was suppressed, and the major agronomic traits, especially No. of productive panicles per plant and of spikelets per panicle, dramatically decreased in637ys. Besides, the mutant exhibited to be sensitive to light intensity and deficiency of tocopherols without obvious alteration in tocotrienols in leaves and grains. Map-based cloning and complementation experiment demonstrated that a point mutation on theOsLIL3gene accounted for the mutant phenotype of637ys.OsLIL3is mainly expressed in green tissues, and its encoded protein is targeted to the chloroplast. Furthermore, the637ys 502ys(lil3 chlp) double mutant exclusively accumulated geranylgeranyl Chl and exhibited lethality at the three-leaf stage.ConclusionsWe identified theOsLIL3gene through a map-based cloning approach. Meanwhile, we demonstrated that OsLIL3 is of extreme importance to the function of OsGGR, and that the complete replacement of phytyl side chain of chlorophyll by geranylgeranyl chain could be fatal to plant survival in rice.

Summary: Impairment of microglial clearance activity contributes to beta-amyloid (Aβ) pathology in Alzheimer’s disease (AD). While the transcriptome profile of microglia directs microglial functions, how the microglial transcriptome can be regulated to alleviate AD pathology is largely unknown. Here, we show that injection of interleukin (IL)-33 in an AD transgenic mouse model ameliorates Aβ pathology by reprogramming microglial epigenetic and transcriptomic profiles to induce a microglial subpopulation with enhanced phagocytic activity. These IL-33-responsive microglia (IL-33RMs) express a distinct transcriptome signature that is highlighted by increased major histocompatibility complex class II genes and restored homeostatic signature genes. IL-33-induced remodeling of chromatin accessibility and PU.1 transcription factor binding at the signature genes of IL-33RM control their transcriptome reprogramming. Specifically, disrupting PU.1-DNA interaction abolishes the microglial state transition and Aβ clearance that is induced by IL-33. Thus, we define a PU.1-dependent transcriptional pathway that drives the IL-33-induced functional state transition of microglia, resulting in enhanced Aβ clearance. : Lau et al. show that interleukin-33 (IL-33) enhances microglial amyloid-beta clearance by inducing a subpopulation of MHC-II+ phagocytic microglia, which is, in turn, controlled by PU.1-dependent transcriptome reprogramming. Thus, the authors reveal an IL-33-PU.1 axis involved in transcriptional regulation that promotes beneficial microglial functions in Alzheimer’s disease. Keywords: chemotaxis, phagocytosis, interleukins, beta-amyloid, disease-associated microglia, MHC-II, epigenetics, PU.1, single-cell RNA-sequencing, chromatin accessibility

In this work, the metabolic characteristics of adipose tissues in live mouse model were investigated using a multiphoton redox ratio and fluorescence lifetime imaging technology. By analyzing the intrinsic fluorescence of metabolic coenzymes, we measured the optical redox ratios of adipocytes in vivo and studied their responses to thermogenesis. The fluorescence lifetime imaging further revealed changes in protein bindings of metabolic coenzymes in the adipocytes during thermogenesis. Our study uncovered significant heterogeneity in the cellular structures and metabolic characteristics of thermogenic adipocytes in brown and beige fat. Subgroups of brown and beige adipocytes were identified based on the distinct lipid size distributions, redox ratios, fluorescence lifetimes and thermogenic capacities. The results of our study show that this label-free imaging technique can shed new light on in vivo study of metabolic dynamics and heterogeneity of adipose tissues in live organisms.

Background: Phytyl residues are the common side chains of chlorophyll (Chl) and tocopherols. Geranylgeranyl reductase (GGR), which is encoded by CHL P gene, is responsible for phytyl biosynthesis. The light-harvesting like protein LIL3 was suggested to be required for stability of GGR and protochlorophyllide oxidoreductase in Arabidopsis. Results: In this study, we isolated a yellow-green leaf mutant, 637ys, in rice (Oryza sativa). The mutant accumulated majority of Chls with unsaturated geranylgeraniol side chains and displayed a yellow-green leaf phenotype through the whole growth period. The development of chloroplasts was suppressed, and the major agronomic traits, especially No. of productive panicles per plant and of spikelets per panicle, dramatically decreased in 637ys. Besides, the mutant exhibited to be sensitive to light intensity and deficiency of tocopherols without obvious alteration in tocotrienols in leaves and grains. Map-based cloning and complementation experiment demonstrated that a point mutation on OsLIL3 gene accounted for the mutant phenotype of 637ys. OsLIL3 is mainly expressed in green tissues and its encoded protein is targeted to the chloroplast. Furthermore, the 637ys 502ys (lil3 chl p) double mutant exclusively accumulated geranylgeranyl Chl and exhibited lethality at the three-leaf stage. Conclusions: Taken together, we succeeded to identify OsLIL3 gene through a map-based cloning approach. Meanwhile, we demonstrated that OsLIL3 is of extreme importance to the function of OsGGR, and that the complete replacement of phytyl side chain of chlorophyll by geranylgeranyl chain could be fatal to plant survival in rice.

Two-photon microscopy is a powerful tool for in vivo brain imaging that has greatly facilitated the neuroscience research in the past few decades. However, it still remains a challenge to image deep inside the brain with near diffraction-limited resolution due to the optical aberrations induced by the biological tissue and the cranial window. Here, we used an adaptive optics approach based on direct wavefront sensing to correct the aberration induced by the thinned skull window and achieved minimally invasive imaging of cerebral cortex with near-diffraction-limit resolution. Besides, by compensating the intrinsic aberration of a miniature gradient-index lens that implanted into the brain, two-photon imaging of hippocampal dendritic spines was realized over an extended field of view. The improvement in fluorescence intensity and imaging resolution enabled us to resolve the fine structures in live mouse brain such as dendritic spines that were invisible without the help of adaptive optics.

Additional file 9: Figure S5. Predicted transmembrane domain of the OsLIL3 (LOC_Os02g03330) protein. (PDF 201 kb)

Quantitative methods to precisely measure cellular states in vivo have become increasingly important and desirable in modern biology. Recently, stimulated Raman scattering (SRS) microscopy has emerged as a powerful tool to visualize small biological molecules tagged with alkyne (C≡C) or carbon-deuterium (C-D) bonds in the cell-silent region. In this study, we developed a technique based on SRS microscopy of vibrational tags for quantitative imaging of lipid synthesis and lipolysis in live animals. The technique aims to overcome the major limitations of conventional fluorescent staining and lipid extraction methods that do not provide the capability of in vivo quantitative analysis. Specifically, we used three bioorthogonal lipid molecules (the alkyne-tagged fatty acid 17-ODYA, deuterium-labeled saturated fatty acid PA-D31, and unsaturated fatty acid OA-D34) to investigate the metabolic dynamics of lipid droplets (LDs) in live Caenorhabditis elegans ( C. elegans). Using a hyperspectral SRS (hsSRS) microscope and subtraction method, the interfering non-Raman background was eliminated to improve the accuracy of lipid quantification. A linear relationship between SRS signals and fatty acid molar concentrations was accurately established. With this quantitative analysis tool, we imaged and determined the changes in concentration of the three fatty acids in LDs of fed or starved adult C. elegans. Using the hsSRS imaging mode, we also observed the desaturation of fatty acids in adult C. elegans via spectral analysis on the SRS signals from LDs. The results demonstrated the unique capability of hsSRS microscopy in quantitative analysis of lipid metabolism in vivo.

Abnormal deposition of brain amyloid is a major hallmark of Alzheimer's disease (AD). The toxic extracellular amyloid plaques originating from the aberrant aggregation of beta-amyloid (Aβ) protein are considered to be the major cause of clinical deficits such as memory loss and cognitive impairment. Two-photon excited fluorescence (TPEF) microscopy provides high spatial resolution, minimal invasiveness, and long-term monitoring capability. TPEF imaging of amyloid plaques in AD transgenic mice models has greatly facilitated studies of the AD pathological mechanism. However, the imaging of deep cortical layers is still hampered by the conventional amyloid probes with short excitation/emission wavelength. In this work, we report that a near-infrared (NIR) probe, named CRANAD-3, is far superior for deep in vivo TPEF imaging of brain amyloid in comparison with the commonly used short-wavelength probe. Our findings show that the major interference for TPEF signal of the NIR probe is from the autofluorescence of lipofuscin, the "aging-pigment" in the brain. To eliminate the interference, we characterized the lipofuscin fluorescence in the aged brains of AD mice and found that it has unique broad emission and short lifetime. The lipofuscin signal can be clearly separated from the fluorescence of CRANAD-3 and fluorescent protein via a ratio-based unmixing method. Our results demonstrate the great advantages of NIR probes for in vivo deep-tissue imaging of amyloid plaques in AD.