Role of Brain Derived Neurotrophic Factor and Related Therapeutic Strategies in Central Post-Stroke Pain.

Brain-derived neurotrophic factor (BDNF) is vital for synaptic plasticity, cell persistence, and neuronal development in peripheral and central nervous systems (CNS). Numerous intracellular signalling pathways involving BDNF are well recognized to affect neurogenesis, synaptic function, cell viability, and cognitive function, which in turn affects pathological and physiological aspects of neurons. Stroke has a significant psycho-socioeconomic impact globally. Central post-stroke pain (CPSP), also known as a type of chronic neuropathic pain, is caused by injury to the CNS following a stroke, specifically damage to the somatosensory system. BDNF regulates a broad range of functions directly or via its biologically active isoforms, regulating multiple signalling pathways through interactions with different types of receptors. BDNF has been shown to play a major role in facilitating neuroplasticity during post-stroke recovery and a pro-nociceptive role in pain development in the nervous system. BDNF-tyrosine kinase receptors B (TrkB) pathway promotes neurite outgrowth, neurogenesis, and the prevention of apoptosis, which helps in stroke recovery. Meanwhile, BDNF overexpression plays a role in CPSP via the activation of purinergic receptors P2X4R and P2X7R. The neuronal hyperexcitability that causes CPSP is linked with BDNF-TrkB interactions, changes in ion channels and inflammatory reactions. This review provides an overview of BDNF synthesis, interactions with certain receptors, and potential functions in regulating signalling pathways associated with stroke and CPSP. The pathophysiological mechanisms underlying CPSP, the role of BDNF in CPSP, and the challenges and current treatment strategies targeting BDNF are also discussed.

Enhanced isradipine sensitivity in vascular smooth muscle cells due to hypoxia-induced Cav1.2 splicing and RbFox1/Fox2 downregulation.

Calcium influx via the L-type voltage-gated Cav1.2 calcium channel in smooth muscle cells regulates vascular contraction. Calcium channel blockers (CCBs) are widely used to treat hypertension by inhibiting Cav1.2 channels. Using the vascular smooth muscle cell line, A7r5 and primary culture of cerebral vascular smooth muscle cells, we found that the expression and function of Cav1.2 channels are downregulated during hypoxia. Furthermore, hypoxia induces structural changes in Cav1.2 channels via alternative splicing. The expression of exon 9* is upregulated, whereas exon 33 is downregulated. Such structural alterations of Cav1.2 channels are caused by the decreased expression of RNA-binding proteins RNA-binding protein fox-1 homolog 1 and 2 (RbFox1 and RbFox2). Overexpression of RbFox1 and RbFox2 prevents hypoxia-induced exon 9* inclusion and exon 33 exclusion. Importantly, such structural alterations of the Cav1.2 channel partly contribute to the enhanced sensitivity of Cav1.2 to isradipine (a CCB) under hypoxia. Overexpression of RbFox1 and RbFox2 successfully reduces isradipine sensitivity in hypoxic smooth muscle cells. Our results suggest a new strategy to manage ischemic diseases such as stroke and myocardial infarction.

Synthesis, structural characterization, analytical profiling, and application to authentic samples of synthesized Phase I metabolites of the synthetic cannabinoid 5F-MDMB-PICA.

5F-MDMB-PICA, an indole-type synthetic cannabinoid (SC), was classified illicit globally in 2020. Although the extensive metabolism of 5F-MDMB-PICA in the human body warrants the development of robust analytical methods for metabolite detection and quantification, a current lack of reference standards for characteristic metabolites hinders such method creation. This work described the synthesis of 18 reference standards for 5F-MDMB-PICA and its possible Phase I metabolites, including three hydroxylated positional isomers R14 to R16. All the compounds were systematic characterized via nuclear magnetic resonance, Fourier transform infrared spectroscopy, and high-resolution mass spectrometry. Furthermore, two methods were developed for the simultaneous detection of all standards using liquid chromatography-tandem mass spectrometry and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. By comparison with authentic samples, R17 was identified as a suitable urine biomarker for 5F-MDMB-PICA uptake.

Effects of clitorienolactones from Clitoria ternatea root on calcium channel mediating hippocampal long-term potentiation in rats induced chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion (CCH) is a common mechanism of acute brain injury due to impairment of blood flow to the brain. Moreover, a prolonged lack of oxygen supply may result in cerebral infarction or global ischemia, which subsequently causes long-term memory impairment. Research on using Clitoria ternatea root extract for treating long-term memory has been studied extensively. However, the bioactive compound contributing to its neuroprotective effects remains uncertain. In the present study, we investigate the effects of clitorienolactone A (CLA) and B (CLB) from the roots of Clitoria ternatea extract on hippocampal neuroplasticity in rats induced by CCH. CLA and CLB were obtained using column chromatography. The rat model of CCH was induced using two-vessel occlusion surgery (2VO). The 2VO rats were given 10 mg/kg of CLA and CLB orally, followed by hippocampal neuroplasticity recording using in vivo electrophysiological. Rats received CLA and CLB (10 mg/kg) significantly reversed the impairment of long-term potentiation following 2VO surgery. Furthermore, we investigate the effect of CLA and CLB on the calcium channel using the calcium imaging technique. During hypoxia, CLA and CLB sustain the increase in intracellular calcium levels. We next predict the binding interactions of CLA and CLB against NMDA receptors containing GluN2A and GluN2B subunits using in silico molecular docking. Our result found that both CLA and CLB exhibited lower binding affinity against GluN2A and GluN2B subunits. Our findings demonstrated that bioactive compounds from Clitoria ternatea improved long-term memory deficits in the chronic cerebral hypoperfusion rat model via calcium uptake. Hence, CLA and CLB could be potential therapeutic tools for treating cognitive dysfunction.

In vivo evaluation of monoclonal antibody M4M using a humanised rat model of stroke demonstrates attenuation of reperfusion injury via blocking human TRPM4 channel.

BACKGROUND: Blocking Transient Receptor Potential Melastatin 4 (TRPM4) in rodents by our antibody M4P has shown to attenuate cerebral ischaemia-reperfusion injury. Since M4P does not interact with human TRPM4, the therapeutic potential of blocking human TRPM4 remains unclear. We developed a monoclonal antibody M4M that inhibited human TRPM4 in cultured cells. However, M4M has no effect on stroke outcome in wild-type rats. Therefore, M4M needs to be evaluated on animal models expressing human TRPM4. METHODS: We generated a humanised rat model using the CRISPR/Cas technique to knock-in (KI) the human TRPM4 antigen sequence. RESULTS: In primary neurons from human TRPM4 KI rats, M4M binds to hypoxic neurons, but not normoxic nor wild-type neurons. Electrophysiological studies showed that M4M blocked ATP depletion-induced activation of TRPM4 and inhibited hypoxia-associated cell volume increase. In a stroke model, administration of M4M reduced infarct volume in KI rats. Rotarod test and Neurological deficit score revealed improvement following M4M treatment. CONCLUSION: M4M selectively binds and inhibits hypoxia-induced human TRPM4 channel activation in neurons from the humanised rat model, with no effect on healthy neurons. Use of M4M in stroke rats showed functional improvements, suggesting the potential for anti-human TRPM4 antibodies in treating acute ischaemic stroke patients.

Effect of agricultural management practices on rice yield and greenhouse gas emissions in the rice-wheat rotation system in China.

Agricultural management practices (AMPs) have the potential to significantly enhance crop yield, albeit with the possible side effect of escalating greenhouse gas emissions. Few studies have undertaken a comprehensive quantification of the impact of AMPs on crop production and soil GHG, particularly in identifying the optimal AMPs for rice cultivation within rice-wheat rotation system. Here, we combined data analysis and keyword search methods on 1433 individual experimental observations from 172 studies on diverse soil types in the subtropical monsoon climate zone of China to assess the impact of AMPs on rice yield, CH4 and N2O emissions, total greenhouse gas emissions (TGHGE). We focused on four key AMPs: mineral N fertilizer management (including ordinary N fertilizer and slow-/controlled-release fertilizer (SCRF)), organic material management (incorporating organic fertilizer, biochar amendment, and straw return), water-saving irrigation, and no-tillage. Our result showed the rice yield ranged from 2525 to 31,196 kg ha-1, and mineral N fertilizer and organic material management boosted rice yield by 2.84-16.19 % and 2.47-8.52 %, respectively. In terms of N2O emissions, biochar amendment resulted in a decrease of 13.05 %, while ordinary N fertilizer, organic fertilizer, and water-saving irrigation led to increases of 63.16 %, 136.66 %, and 37.41 %, respectively. The implementation of SCRF, water-saving irrigation, and no-tillage significantly curtailed CH4 (6.83 %-35.91 %) and TGHGE (6.22 %-20.59 %). Conversely, organic fertilizer and straw return significantly escalated CH4 emissions by 102.20 % and 33.64 % and TGHGE by 85.03 % and 32.40 %. Rice yield and GHG emissions are mainly influenced by variables such as soil bulk density, pH, soil organic carbon, soil texture, mean annual temperature, and total nitrogen. Our study demonstrates that the application of SCRF, water-saving irrigation, and no-tillage can effectively reduce GHG without compromising yield. These practices are particularly effective under climatic and soil conditions of rice-wheat rotation systems in China, thereby contributing to the sustainable rice farming.

TRPM4 blocking antibody reduces neuronal excitotoxicity by specifically inhibiting glutamate-induced calcium influx under chronic hypoxia.

Excitotoxicity arises from unusually excessive activation of excitatory amino acid receptors such as glutamate receptors. Following an energy crisis, excitotoxicity is a major cause for neuronal death in neurological disorders. Many glutamate antagonists have been examined for their efficacy in mitigating excitotoxicity, but failed to generate beneficial outcome due to their side effects on healthy neurons where glutamate receptors are also blocked. In this study, we found that during chronic hypoxia there is upregulation and activation of a nonselective cation channel TRPM4 that contributes to the depolarized neuronal membrane potential and enhanced glutamate-induced calcium entry. TRPM4 is involved in modulating neuronal membrane excitability and calcium signaling, with a complex and multifaceted role in the brain. Here, we inhibited TRPM4 using a newly developed blocking antibody M4P, which could repolarize the resting membrane potential and ameliorate calcium influx upon glutamate stimulation. Importantly, M4P did not affect the functions of healthy neurons as the activity of TRPM4 channel is not upregulated under normoxia. Using a rat model of chronic hypoxia with both common carotid arteries occluded, we found that M4P treatment could reduce apoptosis in the neurons within the hippocampus, attenuate long-term potentiation impairment and improve the functions of learning and memory in this rat model. With specificity to hypoxic neurons, TRPM4 blocking antibody can be a novel way of controlling excitotoxicity with minimal side effects that are common among direct blockers of glutamate receptors.

FPGA-based CCD signal acquisition and transmission system design.

In order to facilitate the analysis and processing of optical signals, an FPGA-based CCD signal acquisition and data transmission system is designed in this work. The system uses an FPGA as the main control device, the TCD1304DG/AP chip as the optical signal detector, and the CYUSB3KIT-003 development board product by Cypress for data transmission. Verilog and Python languages are employed for modular design and on-board verification. Through the coordination of each module, the system successfully achieves CCD signal data acquisition and transmission.

Anaerobic digestion of liquid dairy manure pretreated by the microwave-enhanced advanced oxidation process.

The microwave-enhanced advanced oxidation process (MW-AOP) was utilized for the treatment of liquid dairy manure, prior to anaerobic digestion (AD). A significant amount of soluble chemical oxygen demand (SCOD) was formed in the treated solution, but there was no or little increase in volatile fatty acid (VFA). In this study, up-flow anaerobic sludge blanket (UASB) reactors were used in both the conventional AD system and as the methane phase reactor in a two-phase anaerobic digestion (TPAD) system. Both AD systems were capable of operating at very short hydraulic retention times (HRTs) to as low as about 3 days, and very high methane production rates were achieved. However, much higher methane production was obtained in the TPAD system. The phase separation of the acidogenesis and the methanogenesis in the TPAD system not only increased methane production but also maintained reactor stability throughout the experimental period. Thus, the combination of MW-AOP treatment and TPAD appears to be an effective means of energy recovery from dairy manure.

Visualization and Characterization of the Brain Regional Heterogeneity of Astrocyte-Astrocyte Structural Interactions by Using Improved Iontophoresis with Dual-Fluorescent Dyes.

Astrocytes are morphologically intricate cells and actively modulate the function of the brain. Through numerous fine processes, astrocytes come into contact with neurons, blood vessels, and other glia cells. Emerging evidence has shown that astrocytes exhibit brain regional diversity in their morphology, transcriptome, calcium signaling, and functions. However, little is known about the brain regional heterogeneity of astrocyte-astrocyte structural interaction. So far, the visualization and characterization of the morphological features of adjacent astrocytes have been difficult, and as a result, it is still well-accepted that astrocytes in the adult brain share non-overlapped territory. In contrast, employing an approach that combines viral labeling with dual-fluorescent dyes iontophoresis under brightfield and imaging using confocal microscopy allows for the efficient and specific labeling of adjacent astrocytes, enabling a comprehensive visualization of their fine processes and the degree of their territorial overlap. Our study in the hypothalamic regions of the brain revealed a marked spatial overlap among adjacent astrocytes, which differs from the conventional understanding based on more extensively studied regions, like the hippocampus. Additionally, we revealed the heterogeneity of the astrocyte-neuron ratio across brain regions and conducted an assessment of the photostability and labeling efficiency of fluorescent dyes used for labeling adjacent astrocytes. Our study provides new insights for studying the morphological heterogeneity of astrocytes across the central nervous system.

Mitigating ammonia volatilization in rice cultivation: The impact of partial organic fertilizer substitution.

Optimizing water and nitrogen management to minimize NH3 volatilization from paddy fields has been extensively studied. However, there is limited research on the combined effect of different rates of organic fertilizer substitution (OFS) and irrigation methods in rice cultivation, exploring an effective water and nitrogen combination is beneficial to mitigate NH3 volatilization. To address this gap, we conducted a two-year field experiment to investigate NH3 volatilization under different OFS rates (0%, 25%, and 50%) combined with continuous flooding irrigation (CF) and alternate wet and dry irrigation (AWD). Our findings revealed that NH3 fluxes exhibited similar emission patterns after each fertilization event and significantly decreased with increasing rates of OFS during the basal stage. Compared to no substitution (ON0), the low (ON25) and high (ON50) rates of OFS reduced cumulative NH3 emissions by 18.9% and 16.6%, and lowed NH3 emission factors (EFs) by 26.7% and 23.3%, respectively. Although OFS resulted in a slight reduction in rice yield, yield-scaled NH3 emissions were significantly reduced by 11.9% and 6.5% under the low and high substitution rates, respectively. This reduction was mainly attributed to the slight yield reduction observed at the low substitution rate. Furthermore, when combined with ON0, AWD irrigation had the potential to increase NH3 volatilization. However, this increase was not observed when combined with ON25 and ON50. During each fertilization stage, floodwater + concentration emerged as the prominent environmental factor influencing NH3 volatilization, showing a stronger and more positive correlation compared to other factors such as floodwater pH, soil pH, and NH4+ concentration. Based on our findings, we recommend implementing effective water and nitrogen management strategies to minimize NH3 volatilization in rice cultivation. This involves applying a lower rate of organic fertilizer substitution during the basal stage, maintaining high water levels during fertilization, and implementing mild AWD irrigation during non-fertilization periods.

Effects and mechanisms of tanshinone IIA on PTSD-like symptoms.

BACKGROUND: In recent years, Salvia miltiorrhiza and its active substances have remarkably progressed in treating central neurological disorders. Tanshinone IIA (TSA) is an active ingredient derived from the rhizome of Salvia miltiorrhiza that has been found to alleviate the symptoms of several psychiatric illnesses. Post-traumatic stress disorder (PTSD) is a mental disorder that results after experiencing a serious physical or psychological injury. The currently used drugs are not satisfactory for the treatment of PTSD. However, it has been reported that TSA can improve PTSD-like symptoms like learning and memory, cognitive disorder, and depression through multi-target regulation. PURPOSE: This paper discusses the ameliorative effects of TSA on PTSD-like symptoms and the possible mechanisms of action in terms of inhibition of neuronal apoptosis, anti-neuroinflammation, and anti-oxidative stress. Based on the pathological changes and clinical observations of PTSD, we hope to provide some reference for the clinical transformation of Chinese medicine in treating PTSD. METHODS: A large number of literatures on tanshinone in the treatment of neurological diseases and PTSD were retrieved from online electronic PubMed and Web of Science databases. CONCLUSION: TSA is a widely studied natural active ingredient against mental illness. This review will contribute to the future development of TSA as a new clinical candidate drug for improving PTSD-like symptoms.

SLC26A11 Inhibition Reduces Oncotic Neuronal Death and Attenuates Stroke Reperfusion Injury.

Neuronal swelling is a pathological feature of stroke which contributes to the formation of cytotoxic edema. Under hypoxic condition, aberrant accumulation of sodium and chloride ions inside neurons increases osmotic pressure, leading to cell volume increase. Sodium entry pathway in neurons has been studied extensively. Here, we determine whether SLC26A11 is the major chloride entry pathway under hypoxia and could be the target for protection against ischemic stroke. In this study, electrophysiological properties of chloride current in primary cultured neurons were characterized using low chloride solution, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, and SLC26A11-specific siRNA under physiological conditions or ATP-depleted conditions. In vivo effect of SLC26A11 was evaluated on a rat stroke reperfusion model. We found that SLC26A11 mRNA in primary cultured neurons was upregulated as early as 6 h after oxygen glucose deprivation, and later, the protein level was elevated accordingly. Blockade of SLC26A11 activity could reduce chloride entry and attenuate hypoxia-induced neuronal swelling. In the animal stroke model, SLC26A11 upregulation was mainly located in surviving neurons close to the infarct core. SLC26A11 inhibition ameliorates infarct formation and improves functional recovery. These findings demonstrate that SLC26A11 is a major pathway for chloride entry in stroke, contributing to neuronal swelling. Inhibition of SLC26A11 could be a novel therapeutic strategy for stroke.

A combination of organic fertilizers partially substitution with alternate wet and dry irrigation could further reduce greenhouse gases emission in rice field.

Alternate wet and dry (AWD) irrigation and organic fertilizers substitution (OFS) have contrasting effects on CH4 and N2O emissions in rice cultivation. Combining these two practices may be feasible for simultaneous reduction of CH4 and N2O emission from paddy. Hence, we conducted a two-year field experiment to explore the reduction of greenhouse gases under the combination of AWD and OFS. The field experiment which was designed with two irrigation methods (continuous flooding (CF) irrigation and AWD irrigation), and five nitrogen regimes (N0, N135, and N180 represent 0, 135, and 180 kg N ha-1, respectively, ON25 and ON50 represent 25% and 50% OFS for inorganic fertilizer, respectively). The results showed a single-peak emission for CH4 fluxes during the whole rice growing season in all water and nitrogen treatments while the N2O fluxes peak only observed during tillering period with AWD irrigation. AWD irrigation and OFS showed a limited reduction in global warming potential (GWP). These were owing to that AWD irrigation reduced 38.3% CH4 emissions while increase 145.9% N2O emissions when compared to CF irrigation, and the low rate (25%) OFS only reduced CH4 emission by 29.4% while high rate (50%) only reduce N2O emission by 38.8% when compared to conventional inorganic nitrogen fertilizer (N180). Combined AWD and ON25 could maximize the reduction in GWP and yield-scaled GWP, which were reduce 58.0% and 52.5%, respectively, compare to the conventional water and nitrogen management (CF and N180). Furthermore, the structural equation modelling (SEM) indicated that the soil dissolved organic carbon (DOC) and rice aboveground biomass showed a significant positive effect on CH4 fluxes while soil NH4+ with a negative effect, and the soil NH4+, nitrification potential, denitrification potential significant affected N2O fluxes with a positive effect while DOC with a negative effect. These results investigated that 25% OFS rate for inorganic fertilizer could further reduce warming potential in AWD irrigation rice field.

The role of RNA splicing factor PTBP1 in neuronal development.

Alternative pre-mRNA splicing, which produces various mRNA isoforms with distinct structures and functions from a single gene, is regulated by specific RNA-binding proteins and is an essential method for regulating gene expression in mammals. Recent studies have shown that abnormal change during neuronal development triggered by splicing mis-regulation is an important feature of various neurological diseases. Polypyrimidine tract binding protein 1 (PTBP1) is a kind of RNA-binding proteins with extensive biological functions. As a well-known splicing regulator, it affects the neuronal development process through its involvement in axon formation, synaptogenesis, and neuronal apoptosis, according to the most recent studies. Here, we summarized the mechanism of alternative splicing, structure and function of PTBP1, and the latest research progress on the role of alternative splicing events regulated by PTBP1 in axon formation, synaptogenesis and neuronal apoptosis, to reveal the mechanism of PTBP1-regulated changes in neuronal development process.

Percent body fat, but not body mass index, is associated with cardiometabolic risk factors in children and adolescents.

Background: The epidemic of overweight and obesity has become a worldwide public health problem. Cardiometabolic diseases may originate in childhood. We investigated the association between percent body fat (PBF) measured by the bioelectrical impedance assay and cardiometabolic risk (CMR) in pediatrics. Methods: This cross-sectional study involved 3819 subjects (6-17 years old) in Shanghai. We assessed the association between PBF and body mass index (BMI) with multiple CMR factors. We examined the risk for cardiometabolic abnormalities attributable to overweight and obesity based on age- and sex-specific PBF Z-scores and BMI Z-scores, respectively. Results: PBF, but not BMI, was positively associated with multiple CMR factors in males and females except for total cholesterol in females (all p < 0.05). Compared with the non-overweight group based on PBF, overweight and obese subjects had increasingly higher odds ratio of dyslipidemia (2.90 (1.99-4.23), 4.59 (2.88-7.32) for males and 1.82 (1.20-2.75), 2.46 (1.47-4.11) for females) and elevated blood pressure (BP) (3.26 (2.35-4.51), 4.55 (2.92-7.09) for males and 1.59 (1.07-2.34), 3.98 (2.27-6.17) for females). Obesity females showed a higher likelihood for hyperglycemia (2.19 (1.24-3.84)) than non-overweight females. In both sexes, the predictive effect of PBF on dyslipidemia and elevated BP in adolescents was better than that in children. For hyperglycemia, the predictive effect of PBF was better in male adolescents and female children. There was no risk difference for cardiometabolic abnormalities attributable to BMI-based obesity categories. Conclusions: PBF but not BMI was associated with CMR. Overweight and obesity categories based on PBF had an increased risk for cardiometabolic abnormalities in children and adolescents.

Syntaphilin mediates axonal growth and synaptic changes through regulation of mitochondrial transport: a potential pharmacological target for neurodegenerative diseases.

Mitochondria are a crucial energy source for maintaining neuronal growth and synaptic function. Neurons possess unique morphological characteristics, which make the proper regulation of mitochondrial transport essential for meeting their energy demands. Syntaphilin (SNPH) is capable of specifically targeting the outer membrane of axonal mitochondria, anchoring them to microtubules, and thereby preventing their transport. SNPH also interacts with other mitochondrial proteins to regulate mitochondrial transport. The regulation of mitochondrial transport and anchoring mediated by SNPH is indispensable for axonal growth during neuronal development, maintenance of ATP levels during neuronal synaptic activity, and regeneration of mature neurons following damage. Precise blocking of SNPH may be an effective therapeutic strategy for neurodegenerative diseases and related mental disorders.

TRPM4 Blocking Antibody Protects Cerebral Vasculature in Delayed Stroke Reperfusion.

Reperfusion therapy for acute ischemic stroke aims to restore the blood flow of occluded blood vessels. However, successful recanalization is often associated with disruption of the blood-brain barrier, leading to reperfusion injury. Delayed recanalization increases the risk of severe reperfusion injury, including severe cerebral edema and hemorrhagic transformation. The TRPM4-blocking antibody M4P has been shown to alleviate reperfusion injury and improve functional outcomes in animal models of early stroke reperfusion. In this study, we examined the role of M4P in a clinically relevant rat model of delayed stroke reperfusion in which the left middle cerebral artery was occluded for 7 h. To mimic the clinical scenario, M4P or control IgG was administered 1 h before recanalization. Immunostaining showed that M4P treatment improved vascular morphology after stroke. Evans blue extravasation demonstrated attenuated vascular leakage following M4P treatment. With better vascular integrity, cerebral perfusion was improved, leading to a reduction of infarct volume and animal mortality rate. Functional outcome was evaluated by the Rotarod test. As more animals with severe injuries died during the test in the control IgG group, we observed no difference in functional outcomes in the surviving animals. In conclusion, we identified the potential of TRPM4 blocking antibody M4P to ameliorate vascular injury during delayed stroke reperfusion. If combined with reperfusion therapy, M4P has the potential to improve current stroke management.

Rubiginosin B selectively inhibits Treg cell differentiation and enhances anti-tumor immune responses by targeting calcineurin-NFAT signaling pathway.

BACKGROUND: The accumulation of CD4+Foxp3+ regulatory T cells (Tregs) in the tumor microenvironment (TME) dampens anti-tumor immune responses and promotes tumor progression. Therefore, the elimination of Tregs has become a strategy to enhance the efficacy of tumor immunotherapy, although it is still a daunting challenge. Rhododendron brachypodum (R. brachypodum) is a perennial shrub mainly distributed in Southwestern China, whereas the chemical constituents in this plant remain elusive. PURPOSE: To identify small-molecule inhibitors of Tregs from R. brachypodum. METHODS: Meroterpenoids in R. brachypodum were isolated by column chromatography under the guidance of LCMS analyses. The structures of isolates were identified by spectroscopic data and quantum calculations. The activities of compounds were first evaluated on CD4+ T cell differentiation by flow cytometry in Th1, Th2, Th17, and Treg polarizing conditions, and then on CT26 and MC38 murine colorectal carcinoma cells-allografted mice models. The mechanism of action was first investigated by determining Foxp3 degradation in Jurkat T cells transfected with pLVX-TetOne-Puro-Foxp3-tGFP, and then through analyses of Foxp3 expression on several pre-transcriptional signaling molecules. RESULTS: Two new prenylated phenolic acids (1 and 2) and a chromane meroterpenoid, rubiginosin B (RGB, 3) were obtained from R. brachypodum. The structure of S-anthopogochromene C (1) was rectified according to the electronic circular dichroism (ECD) experiment, and rhodobrachypodic acid (2) was proposed as the precursor of RGB by photochemical transformation. In this investigation, we first found that RGB (3) selectively suppressed the de novo differentiation of TGFß-induced CD4+Foxp3+ regulatory T cells (iTregs), overcome the immunosuppressive TME, and consequently inhibited the growth of tumor in mouse models. The mechanistic study revealed that RGB could target calcineurin, inhibited the nuclear factor of activated T cells (NFAT) dephosphorylation, and down-regulated Foxp3 expression. The hypothetical binding modes of RGB with calcineurin were predicted by molecular docking, and the interactions were mainly hydrophobic effects and hydrogen bonds. CONCLUSION: These results suggest that RGB enhances anti-tumor immune responses by inhibiting Treg cell differentiation through calcineurin-NFAT signaling pathway, and therefore RGB or its analogs may be used as adjuvant agents meriting further investigation.

The interaction between the lemon ribosomal protein ClRPS9-2 and citrus yellow vein clearing virus coat protein affects viral infection and gene silencing suppressor activity.

Citrus yellow vein clearing virus (CYVCV) is an emerging virus that causes serious economic damage to the lemon industry worldwide. The coat protein (CP) of CYVCV is a strong RNA silencing suppressor and is associated with the severity of symptoms in citrus, yet the interaction between CP and host factors remains unknown. In this study, the 40S ribosomal subunit protein S9-2 (ClRPS9-2) was identified as a CP-binding partner using the yeast two-hybrid system from a lemon (cv. Eureka) cDNA library, and the interaction between CP and ClRPS9-2 was demonstrated by in vivo methods. The results suggest that the N-terminal 8-108 amino acid sequence of ClRPS9-2 is crucial for its interaction with CP and may be associated with the nuclear localization of ClRPS9-2. The accumulation and silencing suppressor activity of CP were reduced by transient expression of ClRPS9-2 in Nicotiana benthamiana. Reverse transcription-quantitative PCR analysis showed that the content of CYVCV in ClRPS9-2 transgenic Eureka lemon plants was approximately 50% of that in CYVCV-infected wild-type plants 1 month after inoculation, and mild yellowing and vein clearing symptoms were observed in the transgenic plants. These findings demonstrate that ClRPS9-2 plays a role in host defensive reactions, and the enhanced resistance of transgenic plants to CYVCV may be associated with the up-regulation of salicylic acid-related and R genes.