DAPK1 interaction with NMDA receptor NR2B subunits mediates brain damage in stroke.

N-methyl-D-aspartate (NMDA) receptors constitute a major subtype of glutamate receptors at extrasynaptic sites that link multiple intracellular catabolic processes responsible for irreversible neuronal death. Here, we report that cerebral ischemia recruits death-associated protein kinase 1 (DAPK1) into the NMDA receptor NR2B protein complex in the cortex of adult mice. DAPK1 directly binds with the NMDA receptor NR2B C-terminal tail consisting of amino acid 1292-1304 (NR2B(CT)). A constitutively active DAPK1 phosphorylates NR2B subunit at Ser-1303 and in turn enhances the NR1/NR2B receptor channel conductance. Genetic deletion of DAPK1 or administration of NR2B(CT) that uncouples an activated DAPK1 from an NMDA receptor NR2B subunit in vivo in mice blocks injurious Ca(2+) influx through NMDA receptor channels at extrasynaptic sites and protects neurons against cerebral ischemic insults. Thus, DAPK1 physically and functionally interacts with the NMDA receptor NR2B subunit at extrasynaptic sites and this interaction acts as a central mediator for stroke damage.

Analysis of pollutant dispersion patterns in rivers under different rainfall based on an integrated water-land model.

In the context of rapid urban expansion, the interaction between humanity and nature has become more prominent. Urban land and rivers often exist as distinct entities with limited material exchange. However, during rainfall, these two systems interconnect, resulting in the transfer of land-derived pollutants into rivers. Such transfer significantly increases river pollutant levels, adversely affecting water quality. Therefore, developing a water quality simulation and prediction model is crucial. This model should effectively illustrate pollutant movement and dispersion during rain events. This study proposes a comprehensive model that merges the Storm Water Management Model (SWMM) with the Environmental Fluid Dynamics Code (EFDC). This integrated model assesses the spread and dispersion of pollutants, including Ammonia Nitrogen (NH3-N), Total Phosphorus (TP), Total Nitrogen (TN), and Chemical Oxygen Demand (COD), within urban water cycles for various rainfall conditions, thus offering critical theoretical support for managing the water environment. The application of this model under different rainfall intensities (light, moderate and heavy) provides vital insights. During light rainfall, the river's natural purification process can sustain surface water quality at Class IV. Moderate rainfall causes accumulation of pollutants, reducing water quality to Class V. Conversely, heavy rainfall rapidly increases pollutant concentrations due to higher inflow, pushing the river to a degraded Class V status, which is beyond its natural purification capacity, necessitating engineering solutions to reattain Class IV quality. Furthermore, pollutant accumulation in downstream river sections is more influenced by flow rate than by rainfall intensity. In summary, the SWMM-EFDC integrated model proves highly effective in predicting river water quality, thereby significantly aiding urban water pollution control.

Effects of growth years on ginsenoside biosynthesis of wild ginseng and cultivated ginseng.

BACKGROUND: Ginsenoside, as the main active substance in ginseng, has the function of treating various diseases. However, the ginsenosides content of cultivated ginseng is obviously affected by the growth years, but the molecular mechanism is not clear. In addition, there are significant differences in morphology and physiology between wild ginseng and cultivated ginseng, and the effect of growth years on ginsenoside synthesis not yet understood in wild ginseng. RESULTS: Transcriptome sequencing on the roots, stems and leaves of cultivated ginseng and wild ginseng with different growth years was performed in this study, exploring the effect of growth years on gene expression in ginseng. The number of differentially expressed genes (DEGs) from comparison groups in cultivated ginseng was higher than that in wild ginseng. The result of weighted gene co-expression network analysis (WGCNA) showed that growth years significantly affected the gene expression of Mitogen-activated protein kinases (MAPK) signaling pathway and terpenoid backbone biosynthesis pathway in cultivated ginseng, but had no effects in wild ginseng. Furthermore, the growth years had significant effects on the genes related to ginsenoside synthesis in cultivated ginseng, and the effects were different in the roots, stems and leaves. However, it had little influence on the expression of genes related to ginsenoside synthesis in wild ginseng. Growth years might affect the expression of genes for ginsenoside synthesis by influencing the expression of these transcription factors (TFs), like my elob lastosis (MYB), NAM, ATAF1 and 2, and CUC2 (NAC), APETALA2/ethylene-responsive factor (AP2/ERF), basic helix-loop-helix (bHLH) and WRKY, etc., thereby affecting the content of ginsenosides. CONCLUSIONS: This study complemented the gaps in the genetic information of wild ginseng in different growth periods and helped to clarify the potential mechanisms of the effect of growth years on the physiological state in wild ginseng and cultivated ginseng, which also provided a new insight into the mechanism of ginsenoside regulation.

Diversity and structure of the rhizosphere microbial communities of wild and cultivated ginseng.

BACKGROUND: The resources of wild ginseng have been reducing sharply, and it is mainly dependent on artificial cultivation in China, Korea and Japan. Based on cultivation modes, cultivated ginseng include understory wild ginseng (the seeds or seedlings of cultivated ginseng were planted under the theropencedrymion without human intervention) and farmland cultivated ginseng (grown in farmland with human intervention). Cultivated ginseng, can only be planted on the same plot of land consecutively for several years owing to soilborne diseases, which is mainly because of the variation in the soil microbial community. In contrast, wild ginseng can grow for hundreds of years. However, the knowledge of rhizosphere microbe communities of the wild ginseng is limited. RESULT: In the present study, the microbial communities in rhizosphere soils of the three types of ginseng were analyzed by high-throughput sequencing of 16 S rRNA for bacteria and internal transcribed spacer (ITS) region for fungi. In total, 4,381 bacterial operational taxonomic units (OTUs) and 2,679 fungal OTUs were identified in rhizosphere soils of the three types of ginseng. Among them, the shared bacterial OTUs was more than fungal OTUs by the three types of ginseng, revealing fungal communities were to be more affected than bacterial communities. In addition, the composition of rhizosphere microbial communities and bacterial diversity were similar between understory wild ginseng and wild ginseng. However, higher bacterial diversity and lower fungal diversity were found in rhizosphere soils of wild ginseng compared with farmland cultivated ginseng. Furthermore, the relative abundance of Chloroflexi, Fusarium and Alternaria were higher in farmland cultivated ginseng compared to wild ginseng and understory wild ginseng. CONCLUSIONS: Our results showed that composition and diversity of rhizosphere microbial communities were significantly different in three types of ginseng. This study extended the knowledge pedigree of the microbial diversity populating rhizospheres, and provided insights into resolving the limiting bottleneck on the sustainable development of P. ginseng crops, and even the other crops of Panax.

Fasting induces a form of autonomic synaptic plasticity that prevents hypoglycemia.

During fasting, activation of the counter-regulatory response (CRR) prevents hypoglycemia. A major effector arm is the autonomic nervous system that controls epinephrine release from adrenal chromaffin cells and, consequently, hepatic glucose production. However, whether modulation of autonomic function determines the relative strength of the CRR, and thus the ability to withstand food deprivation and maintain euglycemia, is not known. Here we show that fasting leads to altered transmission at the preganglionic → chromaffin cell synapse. The dominant effect is a presynaptic, long-lasting increase in synaptic strength. Using genetic and pharmacological approaches we show this plasticity requires neuropeptide Y, an adrenal cotransmitter and the activation of adrenal Y5 receptors. Loss of neuropeptide Y prevents a fasting-induced increase in epinephrine release and results in hypoglycemia in vivo. These findings connect plasticity within the sympathetic nervous system to a physiological output and indicate the strength of the final synapse in this descending pathway plays a decisive role in maintaining euglycemia.

Neuropeptide y gates a stress-induced, long-lasting plasticity in the sympathetic nervous system.

Acute stress evokes the fight-or-flight reflex, which via release of the catecholamine hormones affects the function of every major organ. Although the reflex is transient, it has lasting consequences that produce an exaggerated response when stress is reexperienced. How this change is encoded is not known. We investigated whether the reflex affects the adrenal component of the sympathetic nervous system, a major branch of the stress response. Mice were briefly exposed to the cold-water forced swim test (FST) which evoked an increase in circulating catecholamines. Although this hormonal response was transient, the FST led to a long-lasting increase in the catecholamine secretory capacity measured amperometrically from chromaffin cells and in the expression of tyrosine hydroxylase. A variety of approaches indicate that these changes are regulated postsynaptically by neuropeptide Y (NPY), an adrenal cotransmitter. Using immunohistochemistry, RT-PCR, and NPY(GFP) BAC mice, we find that NPY is synthesized by all chromaffin cells. Stress failed to increase secretory capacity in NPY knock-out mice. Genetic or pharmacological interference with NPY and Y1 (but not Y2 or Y5) receptor signaling attenuated the stress-induced change in tyrosine hydroxylase expression. These results indicate that, under basal conditions, adrenal signaling is tonically inhibited by NPY, but stress overrides this autocrine negative feedback loop. Because acute stress leads to a lasting increase in secretory capacity in vivo but does not alter sympathetic tone, these postsynaptic changes appear to be an adaptive response. We conclude that the sympathetic limb of the stress response exhibits an activity-dependent form of long-lasting plasticity.

NFATc1 marks articular cartilage progenitors and negatively determines articular chondrocyte differentiation.

The origin and differentiation mechanism of articular chondrocytes remain poorly understood. Broadly, the difference in developmental mechanisms of articular and growth-plate cartilage is still less elucidated. Here, we identified that the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) is a crucial regulator of articular, but not growth-plate, chondrocyte differentiation during development. At the early stage of mouse knee development (embryonic day 13.5), NFATc1-expressing cells were mainly located in the flanking region of the joint interzone. With development, NFATc1-expressing cells generated almost all articular chondrocytes but not chondrocytes in limb growth-plate primordium. NFATc1-expressing cells displayed prominent capacities for colony formation and multipotent differentiation. Transcriptome analyses revealed a set of characteristic genes in NFATc1-enriched articular cartilage progenitors. Strikingly, the expression of NFATc1 was diminished with articular chondrocyte differentiation, and suppressing NFATc1 expression in articular cartilage progenitors was sufficient to induce spontaneous chondrogenesis while overexpressing NFATc1 suppresses chondrogenesis. Mechanistically, NFATc1 negatively regulated the transcriptional activity of the Col2a1 gene. Thus, our results reveal that NFATc1 characterizes articular, but not growth-plate, cartilage progenitors during development and negatively determines articular chondrocyte differentiation at least partly through regulating COL2A1 gene transcription.

Within the body are about 300 joints connecting bones together. Many factors ­ including trauma, inflammation, aging, and genetic changes ­ can affect the cushion tissue covering the end of the bones in these joints known as articular cartilage. This can lead to diseases such as osteoarthritis which cause chronic pain, and in some cases disability. To treat such conditions, it is essential to know how cells in the articular cartilage are formed during development. In the embryo, most cells come from groups of progenitor cells that are programmed to produce specific types of tissue. But which progenitor cells are responsible for producing the main cells in articular cartilage, chondrocytes, and the mechanisms that govern this transformation are poorly understood. In 2016, a group of researchers found that the gene for the protein NFATc1, which is important for building bone, is also expressed in a group of progenitor cells at the site where ligaments insert into bone in mice. Inactivation of NFATc1 in these progenitor cells has also been shown to cause abnormal cartilage to form, a condition termed osteochondromas. Building on this work, Zhang, Wang et al. ­ including some of the researchers involved in the 2016 study ­ set out to find whether NFATc1 is also involved in the normal development of articular chondrocytes. To investigate, the team used genetically modified mice in which any cells with NFATc1 also had a green fluorescent protein, and tracked these cells and their progeny over the course of joint development. This led them to discover a group of NFATc1-containing progenitor cells that gave rise to almost all articular chondrocytes in the knee joint. Further experiments revealed that when NFATc1 was removed, this made the progenitors become articular chondrocytes very quickly. In contrast, when the cells had excess amounts of the protein, the formation of articular chondrocytes was significantly reduced. This suggests that the level of NFATc1 governs when progenitors develop into articular chondrocytes. These findings have provided a way to track the progenitors of articular chondrocytes throughout development and study how articular cartilage is formed. In the future, this work could help researchers develop treatment strategies for osteoarthritis and other cartilage-based diseases. However, before this can happen, further work is needed to confirm that the effects observed in this study also relate to humans.

The ß-Hydroxybutyrate-GPR109A Receptor Regulates Fasting-induced Plasticity in the Mouse Adrenal Medulla.

During fasting, increased sympathoadrenal activity leads to epinephrine release and multiple forms of plasticity within the adrenal medulla including an increase in the strength of the preganglionic → chromaffin cell synapse and elevated levels of agouti-related peptide (AgRP), a peptidergic cotransmitter in chromaffin cells. Although these changes contribute to the sympathetic response, how fasting evokes this plasticity is not known. Here we report these effects involve activation of GPR109A (HCAR2). The endogenous agonist of this G protein-coupled receptor is ß-hydroxybutyrate, a ketone body whose levels rise during fasting. In wild-type animals, 24-hour fasting increased AgRP-ir in adrenal chromaffin cells but this effect was absent in GPR109A knockout mice. GPR109A agonists increased AgRP-ir in isolated chromaffin cells through a GPR109A- and pertussis toxin-sensitive pathway. Incubation of adrenal slices in nicotinic acid, a GPR109A agonist, mimicked the fasting-induced increase in the strength of the preganglionic → chromaffin cell synapse. Finally, reverse transcription polymerase chain reaction experiments confirmed the mouse adrenal medulla contains GPR109A messenger RNA. These results are consistent with the activation of a GPR109A signaling pathway located within the adrenal gland. Because fasting evokes epinephrine release, which stimulates lipolysis and the production of ß-hydroxybutyrate, our results indicate that chromaffin cells are components of an autonomic-adipose-hepatic feedback circuit. Coupling a change in adrenal physiology to a metabolite whose levels rise during fasting is presumably an efficient way to coordinate the homeostatic response to food deprivation.

Overexpression of tomato thioredoxin h (SlTrxh) enhances excess nitrate stress tolerance in transgenic tobacco interacting with SlPrx protein.

The thioredoxin (Trx) system plays a vital function in cellular antioxidative defense. However, little is known about Trx in tomato under excess nitrate. In this study, we isolated the tomato gene encoding h-type Trx gene (SlTrxh). The mRNA transcript of SlTrxh in roots and leaves of tomato was induced incrementally under excess nitrate for 24 h. Subcellular localization showed that SlTrxh might localize in the cytoplasm, nucleus and plasma membrane. Enzymatic activity characterization revealed that SlTrxh protein possesses the disulfide reductase function and Cysteine (Cys) 54 is important for its activity. Overexpressing SlTrxh in tobacco resulted in increasing seed germination rate, root length and decreasing H2O2 and O2- accumulation, compared with the wild type (WT) tobacco under nitrate stress. While overexpressing SlTrxhC54S (Cysteine 54 mutated to Serine) in tobacco showed decreased germination rate and root length compared with the WT after nitrate treatment. After nitrate stress treatment, SlTrxh overexpressing transgenic tobacco plants have lower malonaldehyde (MDA), H2O2 contents and Reactive Oxygen Species (ROS) accumulation, and higher mRNA transcript level of NtP5CS, NtDREB2, higher ratio of ASA/DHA and GSH/GSSG, higher activities of ascorbate peroxidase and NADP thioredoxin reductase. Besides, SlTrxh overexpressing plants showed higher tolerance to Methyl Viologen (MV) in the seed germination and seedling stage. The yeast two-hybrid, pull-down, Co-immunoprecipitation and Bimolecular luciferase complementation assay confirmed that SlTrxh physically interacted with tomato peroxiredoxin (SlPrx). These results suggest that SlTrxh contributes to maintaining ROS homeostasis under excess nitrate stress interacting with SlPrx and Cys54 is important for its enzyme activity.

A Genomewide Scan for Genetic Structure and Demographic History of Two Closely Related Species, Rhododendron dauricum and R. mucronulatum (Rhododendron, Ericaceae).

Understanding the processes of divergence and speciation is an important task for evolutionary research, and climate oscillations play a pivotal role. We estimated the genetic structure and demographic history of two closely related species of Rhododendron, R. dauricum, and R. mucronulatum, distributed in northeastern China using 664,406 single nucleotide polymorphic loci of specific-locus amplified fragment sequencing (SLAF-seq) and 4 chloroplast DNA (cpDNA) fragments, sampling 376 individuals from 39 populations of these two species across their geographic distributions. The geographical distribution of cpDNA haplotypes revealed that R. dauricum and R. mucronulatum have different spatial genetic structures and haplotype diversity. Analysis of molecular variance (AMOVA) results showed that these two species have significant genetic differentiation and that the phylogeny demonstrates that these two species clustered a monophyletic group based on SLAF data, respectively, but not in cpDNA data. The evidence of significant gene flow was also detected from R. mucronulatum to R. dauricum. A deep divergence between the two species was observed and occurred during the early Oligocene. The niche models showed that the two species have different demographic histories. Thus, our results imply that geography and climate changes played important roles in the evolutionary process of R. dauricum and R. mucronulatum, and although there was an interspecific gene flow, the divergence was maintained by natural selection.

Recurrent hypoglycemia inhibits the counterregulatory response by suppressing adrenal activity.

Hypoglycemia activates the counterregulatory response (CRR), a neural-endocrine reflex that restores euglycemia. Although effective if occasionally activated, repeated induction of the CRR leads to a decline in responsiveness and prolonged exposure to hypoglycemia. The mechanism underlying this impairment is not known. We found that the reduction in epinephrine release that characterizes a suppressed CRR involves a long-lasting form of sympatho-adrenal synaptic plasticity. Using optogenetically evoked catecholamine release, we show that recurrent hypoglycemia reduced the secretory capacity of mouse adrenal chromaffin cells. Single activation of the CRR increased the adrenal levels of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine synthesis, but this was prevented by repeated activation. In contrast, the level of neuropeptide Y (NPY), an adrenal cotransmitter, remained elevated after recurrent hypoglycemia. Inhibition of NPY or Y1 signaling, either transgenically or pharmacologically, prevented the attenuation of both TH expression and epinephrine release. These results indicate that impairment of the CRR involves suppressed activity at the adrenal level. Interfering with the peripheral NPY-dependent negative feedback loop may provide a way to avoid the pathophysiological consequences of recurrent hypoglycemia which are common in the diabetic state.

AgRP-Expressing Adrenal Chromaffin Cells Are Involved in the Sympathetic Response to Fasting.

Fasting evokes a homeostatic response that maintains circulating levels of energy-rich metabolites and increases the drive to eat. Centrally, this reflex activates a small population of hypothalamic neurons that are characterized by the expression of AgRP, a neuropeptide with an extremely restricted distribution. Apart from the hypothalamus, the only other site with substantial expression is the adrenal gland, but there is disagreement about which cells synthesize AgRP. Using immunohistochemistry, flow cytometry, and reverse transcription-polymerase chain reaction, we show AgRP is present in the mouse adrenal medulla and is expressed by neuroendocrine chromaffin cells that also synthesize the catecholamines and neuropeptide Y. Short-term fasting led to an increase in adrenal AgRP expression. Because AgRP can act as an antagonist at MC3/4 receptors, we tested whether melanotan II, an MC3/4 receptor agonist, could regulate pre- and postsynaptic signaling within the adrenal medulla. Melanotan II decreased the paired-pulse ratio of evoked synaptic currents recorded in chromaffin cells; this effect was blocked by exogenous AgRP. In contrast, neither melanotan II nor AgRP altered the optogenetically evoked release of catecholamines from isolated chromaffin cells. These results are consistent with the idea that AgRP regulates the strength of the sympathetic input by modulation of presynaptic MC3/4 receptors located on preganglionic neurons. We conclude that a small population of neuroendocrine cells in the adrenal medulla, and the arcuate nucleus of the hypothalamus, express AgRP and neuropeptide Y and are functionally involved in the systemic response to fasting.