Extracellular vesicles incorporating retrovirus-like capsids for the enhanced packaging and systemic delivery of mRNA into neurons.

The blood-brain barrier (BBB) restricts the systemic delivery of messenger RNAs (mRNAs) into diseased neurons. Although leucocyte-derived extracellular vesicles (EVs) can cross the BBB at inflammatory sites, it is difficult to efficiently load long mRNAs into the EVs and to enhance their neuronal uptake. Here we show that the packaging of mRNA into leucocyte-derived EVs and the endocytosis of the EVs by neurons can be enhanced by engineering leucocytes to produce EVs that incorporate retrovirus-like mRNA-packaging capsids. We transfected immortalized and primary bone-marrow-derived leucocytes with DNA or RNA encoding the capsid-forming activity-regulated cytoskeleton-associated (Arc) protein as well as capsid-stabilizing Arc 5'-untranslated-region RNA elements. These engineered EVs inherit endothelial adhesion molecules from donor leukocytes, recruit endogenous enveloping proteins to their surface, cross the BBB, and enter the neurons in neuro-inflammatory sites. Produced from self-derived donor leukocytes, the EVs are immunologically inert, and enhanced the neuronal uptake of the packaged mRNA in a mouse model of low-grade chronic neuro-inflammation.

A Large Field-of-view, Single-cell-resolution Two- and Three-Photon Microscope for Deep Imaging.

In vivo imaging of large-scale neuron activity plays a pivotal role in unraveling the function of the brain's network. Multiphoton microscopy, a powerful tool for deep-tissue imaging, has received sustained interest in advancing its speed, field of view and imaging depth. However, to avoid thermal damage in scattering biological tissue, field of view decreases exponentially as imaging depth increases. We present a suite of innovations to overcome constraints on the field of view in three-photon microscopy and to perform deep imaging that is inaccessible to two-photon microscopy. These innovations enable us to image neuronal activities in a ~3.5-mm diameter field-of-view at 4 Hz with single-cell resolution and in the deepest cortical layer of mouse brains. We further demonstrate simultaneous large field-of-view two-photon and three-photon imaging, subcortical imaging in the mouse brain, and whole-brain imaging in adult zebrafish. The demonstrated techniques can be integrated into any multiphoton microscope for large-field-of-view imaging for system-level neural circuit research.

Serotonin neurons in mating female mice are activated by male ejaculation.

Sexual stimulation triggers changes in female physiology and behavior, including sexual satiety and preparing the uterus for pregnancy. Serotonin (5-HT) is an important regulator of reproductive physiology and sexual receptivity, but the relationship between sexual stimulation and 5-HT neural activity in females is poorly understood. Here, we investigated dorsal raphe 5-HT neural activity in female mice during sexual behavior. We found that 5-HT neural activity in mating females peaked specifically upon male ejaculation and remained elevated above baseline until disengagement. Artificial intravaginal mechanical stimulation was sufficient to elicit increased 5-HT neural activity but the delivery of ejaculatory fluids was not. Distal penis expansion ("penile cupping") at ejaculation and forceful expulsion of ejaculatory fluid each provided sufficient mechanical stimulation to elicit 5-HT neuron activation. Our study identifies a female ejaculation-specific signal in a major neuromodulatory system and shows that intravaginal mechanosensory stimulation is necessary and sufficient to drive this signal.

Serotonin neurons in mating female mice are activated by male ejaculation.

Sexual stimulation triggers changes in female physiology and behavior, including sexual satiety and preparing the uterus for pregnancy. Serotonin is an important regulator of reproductive physiology and sexual receptivity, but the relationship between sexual stimulation and serotonin neural activity in females is poorly understood. Here, we investigated dorsal raphe serotonin neural activity in females during sexual behavior. We found that serotonin neural activity in mating females peaked specifically upon male ejaculation, and remained elevated above baseline until disengagement. Artificial intravaginal mechanical stimulation was sufficient to elicit increased 5-HT neural activity but the delivery of ejaculatory fluids was not. Distal penis erectile enlargement ("penile cupping") at ejaculation and forceful expulsion of ejaculatory fluid each provided sufficient mechanical stimulation to elicit serotonin neuron activation. Our study identifies a female ejaculation-specific signal in a major neuromodulatory system and shows that intravaginal mechanosensory stimulation is necessary and sufficient to drive this signal.

Effect of Filtered Back-Projection Filters to Low-Contrast Object Imaging in Ultra-High-Resolution (UHR) Cone-Beam Computed Tomography (CBCT).

In this study, the effect of filter schemes on several low-contrast materials was compared using standard and ultra-high-resolution (UHR) cone-beam computed tomography (CBCT) imaging. The performance of the UHR-CBCT was quantified by measuring the modulation transfer function (MTF) and the noise power spectrum (NPS). The MTF was measured at the radial location around the cylindrical phantom, whereas the NPS was measured in the eight different homogeneous regions of interest. Six different filter schemes were designed and implemented in the CT sinogram from each imaging configuration. The experimental results indicated that the filter with smaller smoothing window preserved the MTF up to the highest spatial frequency, but larger NPS. In addition, the UHR imaging protocol provided 1.77 times better spatial resolution than the standard acquisition by comparing the specific spatial frequency (f50) under the same conditions. The f50s with the flat-top window in UHR mode was 1.86, 0.94, 2.52, 2.05, and 1.86 lp/mm for Polyethylene (Material 1, M1), Polystyrene (M2), Nylon (M3), Acrylic (M4), and Polycarbonate (M5), respectively. The smoothing window in the UHR protocol showed a clearer performance in the MTF according to the low-contrast objects, showing agreement with the relative contrast of materials in order of M3, M4, M1, M5, and M2. In conclusion, although the UHR-CBCT showed the disadvantages of acquisition time and radiation dose, it could provide greater spatial resolution with smaller noise property compared to standard imaging; moreover, the optimal window function should be considered in advance for the best UHR performance.

Intense threat switches dorsal raphe serotonin neurons to a paradoxical operational mode.

Survival depends on the selection of behaviors adaptive for the current environment. For example, a mouse should run from a rapidly looming hawk but should freeze if the hawk is coasting across the sky. Although serotonin has been implicated in adaptive behavior, environmental regulation of its functional role remains poorly understood. In mice, we found that stimulation of dorsal raphe serotonin neurons suppressed movement in low- and moderate-threat environments but induced escape behavior in high-threat environments, and that movement-related dorsal raphe serotonin neural dynamics inverted in high-threat environments. Stimulation of dorsal raphe Î³-aminobutyric acid (GABA) neurons promoted movement in negative but not positive environments, and movement-related GABA neural dynamics inverted between positive and negative environments. Thus, dorsal raphe circuits switch between distinct operational modes to promote environment-specific adaptive behaviors.

Exo-ethylene application mitigates waterlogging stress in soybean (Glycine max L.).

BACKGROUND: Waterlogging (WL) is a key factor hindering soybean crop productivity worldwide. Plants utilize various hormones to avoid various stress conditions, including WL stress; however, the physiological mechanisms are still not fully understood. RESULTS: To identify physiological mechanisms during WL stress, different phytohormones, such as ethephon (ETP; donor source of ethylene), abscisic acid, gibberellins, indole-3-acetic acid, kinetin, jasmonic acid, and salicylic acid were exogenously applied to soybean plants. Through this experiment, we confirmed the beneficial effects of ETP treatment. Thus, we selected ETP as a candidate hormone to mitigate WL. Further mechanistic investigation of the role of ETP in waterlogging tolerance was carried out. Results showed that ETP application mitigated WL stress, significantly improved the photosynthesis pigment, and increased the contents of endogenous GAs compared to those in untreated plants. The amino acid contents during WL stress were significantly activated by EPT treatments. The amino acid contents were significantly higher in the 100 µM ETP-treated soybean plants than in the control. ETP application induced adventitious root initiation, increased root surface area, and significantly increased the expressions of glutathione transferases and relative glutathione activity compared to those of non-ETP-treated plants. ETP-treated soybeans produced a higher up-regulation of protein content and glutathione S-transferase (GSTs) than did soybeans under the WL only treatment. CONCLUSIONS: In conclusion, the current results suggest that ETP application enabled various biochemical and transcriptional modulations. In particular, ETP application could stimulate the higher expression of GST3 and GST8. Thus, increased GST3 and GST8 induced 1) increased GSH activity, 2) decreased reactive oxygen species (ROS), 3) mitigation of cell damage in photosynthetic apparatus, and 4) improved phenotype consecutively.

Development of a chest digital tomosynthesis R/F system and implementation of low-dose GPU-accelerated compressed sensing (CS) image reconstruction.

PURPOSE: This work describes the hardware and software developments of a prototype chest digital tomosynthesis (CDT) R/F system. The purpose of this study was to validate the developed system for its possible clinical application on low-dose chest tomosynthesis imaging. METHODS: The prototype CDT R/F system was operated by carefully controlling the electromechanical subsystems through a synchronized interface. Once a command signal was delivered by the user, a tomosynthesis sweep started to acquire 81 projection views (PVs) in a limited angular range of ±20°. Among the full projection dataset of 81 images, several sets of 21 (quarter view) and 41 (half view) images with equally spaced angle steps were selected to represent a sparse view condition. GPU-accelerated and total-variation (TV) regularization strategy-based compressed sensing (CS) image reconstruction was implemented. The imaged objects were a flat-field using a copper filter to measure the noise power spectrum (NPS), a Catphan® CTP682 quality assurance (QA) phantom to measure a task-based modulation transfer function (MTFTask ) of three different cylinders' edge, and an anthropomorphic chest phantom with inserted lung nodules. The authors also verified the accelerated computing power over CPU programming by checking the elapsed time required for the CS method. The resultant absorbed and effective doses that were delivered to the chest phantom from two-view digital radiographic projections, helical computed tomography (CT), and the prototype CDT system were compared. RESULTS: The prototype CDT system was successfully operated, showing little geometric error with fast rise and fall times of R/F x-ray pulse less than 2 and 10 ms, respectively. The in-plane NPS presented essential symmetric patterns as predicted by the central slice theorem. The NPS images from 21 PVs were provided quite different pattern against 41 and 81 PVs due to aliased noise. The voxel variance values which summed all NPS intensities were inversely proportional to the number of PVs, and the CS method gave much lower voxel variance by the factors of 3.97-6.43 and 2.28-3.36 compared to filtered backprojection (FBP) and 20 iterations of simultaneous algebraic reconstruction technique (SART). The spatial frequencies of the f50 at which the MTFTask reduced to 50% were 1.50, 1.55, and 1.67 cycles/mm for FBP, SART, and CS methods, respectively, in the case of Bone 20% cylinder using 41 views. A variety of ranges of TV reconstruction parameters were implemented during the CS method and we could observe that the NPS and MTFTask preserved best when the regularization and TV smoothing parameters α and τ were in a range of 0.001-0.1. For the chest phantom data, the signal difference to noise ratios (SDNRs) were higher in the proposed CS scheme images than in the FBP and SART, showing the enhanced rate of 1.05-1.43 for half view imaging. The total averaged reconstruction time during 20 iterations of the CS scheme was 124.68 s, which could match-up a clinically feasible time (<3 min). This computing time represented an enhanced speed 386 times greater than CPU programming. The total amounts of estimated effective doses were 0.12, 0.53 (half view), and 2.56 mSv for two-view radiographs, the prototype CDT system, and helical CT, respectively, showing 4.49 times higher than conventional radiography and 4.83 times lower than a CT exam, respectively. CONCLUSIONS: The current work describes the development and performance assessment of both hardware and software for tomosynthesis applications. The authors observed reasonable outcomes by showing a potential for low-dose application in CDT imaging using GPU acceleration.

Indoleacetic acid production and plant growth promoting potential of bacterial endophytes isolated from rice (Oryza sativa L.) seeds.

Bacterial endophytes from the phyllosphere and rhizosphere have been used to produce bioactive metabolites and to promote plant growth. However, little is known about the endophytes residing in seeds. This study aimed to isolate and identify seed-borne bacterial endophytes from rice and elucidate their potential for phytohormone production and growth enhancement. The isolated endophytes included Micrococcus yunnanensis RWL-2, Micrococcus luteus RWL-3, Enterobacter soli RWL-4, Leclercia adecarboxylata RWL-5, Pantoea dispersa RWL-6, and Staphylococcus epidermidis RWL-7, which were identified using 16S rRNA sequencing and phylogenetic analysis. These strains were analyzed for indoleacetic acid (IAA) production by using GC-MS and IAA was found in the range of 11.50 ± 0.77 µg ml-1 to 38.80 ± 1.35 µg ml-1. We also assessed the strains for plant growth promoting potential because these isolates were able to produce IAA in pure culture. Most of the growth attributes of rice plants (shoot and root length, fresh and dry biomass, and chlorophyll content) were significantly increased by bacterial endophytes compared to the controls. These results show that IAA producing bacterial endophytes can improve hostplant growth traits and can be used as bio-fertilizers.

Bacillus aryabhattai SRB02 tolerates oxidative and nitrosative stress and promotes the growth of soybean by modulating the production of phytohormones.

Plant growth promoting rhizobacteria (PGPR) are diverse, naturally occurring bacteria that establish a close association with plant roots and promote the growth and immunity of plants. Established mechanisms involved in PGPR-mediated plant growth promotion include regulation of phytohormones, improved nutrient availability, and antagonistic effects on plant pathogens. In this study, we isolated a bacterium from the rhizospheric soil of a soybean field in Chungcheong buk-do, South Korea. Using 16S rRNA sequencing, the bacterium was identified as Bacillus aryabhattai strain SRB02. Here we show that this strain significantly promotes the growth of soybean. Gas chromatography-mass spectrometry analysis showed that SRB02 produced significant amounts of abscisic acid, indole acetic acid, cytokinin and different gibberellic acids in culture. SRB02-treated soybean plants showed significantly better heat stress tolerance than did untreated plants. These plants also produced consistent levels of ABA under heat stress and exhibited ABA-mediated stomatal closure. High levels of IAA, JA, GA12, GA4, and GA7, were recorded in SRB02-treated plants. These plants produced longer roots and shoots than those of control plants. B. aryabhattai SRB02 was found to be highly tolerant to oxidative stress induced by H2O2 and MV potentiated by high catalase (CAT) and superoxide dismutase (SOD) activities. SRB02 also tolerated high nitrosative stress induced by the nitric oxide donors GSNO and CysNO. Because of these attributes, B. aryabhattai SRB02 may prove to be a valuable resource for incorporation in biofertilizers and other soil amendments that seek to improve crop productivity.

Mitochondrial Genome Analysis of Wild Rice (Oryza minuta) and Its Comparison with Other Related Species.

Oryza minuta (Poaceae family) is a tetraploid wild relative of cultivated rice with a BBCC genome. O. minuta has the potential to resist against various pathogenic diseases such as bacterial blight (BB), white backed planthopper (WBPH) and brown plant hopper (BPH). Here, we sequenced and annotated the complete mitochondrial genome of O. minuta. The mtDNA genome is 515,022 bp, containing 60 protein coding genes, 31 tRNA genes and two rRNA genes. The mitochondrial genome organization and the gene content at the nucleotide level are highly similar (89%) to that of O. rufipogon. Comparison with other related species revealed that most of the genes with known function are conserved among the Poaceae members. Similarly, O. minuta mt genome shared 24 protein-coding genes, 15 tRNA genes and 1 ribosomal RNA gene with other rice species (indica and japonica). The evolutionary relationship and phylogenetic analysis revealed that O. minuta is more closely related to O. rufipogon than to any other related species. Such studies are essential to understand the evolutionary divergence among species and analyze common gene pools to combat risks in the current scenario of a changing environment.

BLA to vHPC inputs modulate anxiety-related behaviors.

The basolateral amygdala (BLA) and ventral hippocampus (vHPC) have both been implicated in mediating anxiety-related behaviors, but the functional contribution of BLA inputs to the vHPC has never been directly investigated. Here we show that activation of BLA-vHPC synapses acutely and robustly increased anxiety-related behaviors, while inhibition of BLA-vHPC synapses decreased anxiety-related behaviors. We combined optogenetic approaches with in vivo pharmacological manipulations and ex vivo whole-cell patch-clamp recordings to dissect the local circuit mechanisms, demonstrating that activation of BLA terminals in the vHPC provided monosynaptic, glutamatergic inputs to vHPC pyramidal neurons. Furthermore, BLA inputs exerted polysynaptic, inhibitory effects mediated by local interneurons in the vHPC that may serve to balance the circuit locally. These data establish a role for BLA-vHPC synapses in bidirectionally controlling anxiety-related behaviors in an immediate, yet reversible, manner and a model for the local circuit mechanism of BLA inputs in the vHPC.

Raf kinase inhibitory protein is required for cerebellar long-term synaptic depression by mediating PKC-dependent MAPK activation.

It was demonstrated previously that a positive feedback loop, including protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), is required for the gradual expression of cerebellar long-term depression (LTD). PKC and MAPK are mutually activated in this loop. MAPK-dependent PKC activation is likely to be mediated by phospholipase A2. On the other hand, it is not clear how PKC activates MAPK. Therefore, the entire picture of this loop was not fully understood. We here test the hypothesis that this loop is completed by the PKC substrate, Raf kinase inhibitory protein (RKIP). To test this hypothesis, we used a mutant form of RKIP that is not phosphorylated by PKC and thus constitutively inhibits Raf-1 and MEK, upstream kinases of MAPK. When this RKIP mutant was introduced into Purkinje cells of mouse cerebellar slices through patch-clamp electrodes, LTD was blocked, while wild-type (WT) RKIP had no effect on LTD. Physiological epistasis experiments demonstrated that RKIP works downstream of PKC and upstream of MAPK during LTD induction. Furthermore, biochemical analyses demonstrated that endogenous RKIP dissociates from Raf-1 and MEK during LTD induction in a PKC-dependent manner, suggesting that RKIP binding-dependent inhibition of Raf-1 and MEK is removed upon LTD induction. We therefore conclude that PKC-dependent regulation of RKIP leads to MAPK activation, with RKIP completing the positive feedback loop that is required for LTD.