Heterologous booster vaccination enhances antibody responses to SARS-CoV-2 by improving Tfh function and increasing B-cell clonotype SHM frequency.

Heterologous prime-boost has broken the protective immune response bottleneck of the COVID-19 vaccines. however, the underlying mechanisms have not been fully elucidated. Here, we investigated antibody responses and explored the response of germinal center (GC) to priming with inactivated vaccines and boosting with heterologous adenoviral-vectored vaccines or homologous inactivated vaccines in mice. Antibody responses were dramatically enhanced by both boosting regimens. Heterologous immunization induced more robust GC activation, characterized by increased Tfh cell populations and enhanced helper function. Additionally, increased B-cell activation and antibody production were observed in a heterologous regimen. Libra-seq was used to compare the differences of S1-, S2- and NTD-specific B cells between homologous and heterologous vaccination, respectively. S2-specific CD19+ B cells presented increased somatic hypermutations (SHMs), which were mainly enriched in plasma cells. Moreover, a heterologous booster dose promoted the clonal expansion of B cells specific to S2 and NTD regions. In conclusion, the functional role of Tfh and B cells following SARS-CoV-2 heterologous vaccination may be important for modulating antibody responses. These findings provide new insights for the development of SARS-CoV-2 vaccines that induce more robust antibody response.

Isotope Evidence for Rice Accumulation of Newly Deposited and Soil Legacy Cadmium: A Three-Year Field Study.

Biogeochemical processes of atmospherically deposited cadmium (Cd) in soils and accumulation in rice were investigated through a three-year fully factorial atmospheric exposure experiment using Cd stable isotopes and diffusive gradients in thin films (DGT). Our results showed that approximately 37-79% of Cd in rice grains was contributed by atmospheric deposition through root and foliar uptake during the rice growing season, while the deposited Cd accounted for a small proportion of the soil pools. The highly bioavailable metals in atmospheric deposition significantly increased the soil DGT-measured bioavailable fraction; yet, this fraction rapidly aged following a first-order exponential decay model, leading to similar percentages of the bioavailable fraction in soils exposed for 1-3 years. The enrichment of light Cd isotopes in the atmospheric deposition resulted in a significant shift toward lighter Cd isotopes in rice plants. Using a modified isotopic mass balance model, foliar and root uptake of deposited Cd accounted for 47-51% and 28-36% in leaves, 41-45% and 22-30% in stems, and 45-49% and 26-30% in grains, respectively. The implications of this study are that new atmospheric deposition disproportionately contributes to the uptake of Cd in rice, and managing emissions thus becomes very important versus remediation of impacted soils.

Interleukin-22 receptor 1-mediated stimulation of T-type Ca2+ channels enhances sensory neuronal excitability through the tyrosine-protein kinase Lyn-dependent PKA pathway.

BACKGROUND: Interleukin 24 (IL-24) has been implicated in the nociceptive signaling. However, direct evidence and the precise molecular mechanism underlying IL-24's role in peripheral nociception remain unclear. METHODS: Using patch clamp recording, molecular biological analysis, immunofluorescence labeling, siRNA-mediated knockdown approach and behavior tests, we elucidated the effects of IL-24 on sensory neuronal excitability and peripheral pain sensitivity mediated by T-type Ca2+ channels (T-type channels). RESULTS: IL-24 enhances T-type channel currents (T-currents) in trigeminal ganglion (TG) neurons in a reversible and dose-dependent manner, primarily by activating the interleukin-22 receptor 1 (IL-22R1). Furthermore, we found that the IL-24-induced T-type channel response is mediated through tyrosine-protein kinase Lyn, but not its common downstream target JAK1. IL-24 application significantly activated protein kinase A; this effect was independent of cAMP and prevented by Lyn antagonism. Inhibition of PKA prevented the IL-24-induced T-current response, whereas inhibition of protein kinase C or MAPK kinases had no effect. Functionally, IL-24 increased TG neuronal excitability and enhanced pain sensitivity to mechanical stimuli in mice, both of which were suppressed by blocking T-type channels. In a trigeminal neuropathic pain model induced by chronic constriction injury of the infraorbital nerve, inhibiting IL-22R1 signaling alleviated mechanical allodynia, which was reversed by blocking T-type channels or knocking down Cav3.2. CONCLUSION: Our findings reveal that IL-24 enhances T-currents by stimulating IL-22R1 coupled to Lyn-dependent PKA signaling, leading to TG neuronal hyperexcitability and pain hypersensitivity. Understanding the mechanism of IL-24/IL-22R1 signaling in sensory neurons may pave the way for innovative therapeutic strategies in pain management.

The Role of Alpha-7 Nicotinic Acetylcholine Receptors in Pain: Potential Therapeutic Implications.

Chronic pain represents a prevalent and costly medical challenge globally. Nicotinic acetylcholine receptors (nAChRs), one type of ligand-gated ion channels found extensively in both the central and peripheral nervous systems, have emerged as promising therapeutic targets for chronic pain. Although there are currently no FDA-approved analgesics specifically targeting nAChRs, accumulating preclinical and clinical evidence suggest that selective ligands for alpha 7 (α7) nAChRs show potential for treating chronic pain, boasting a reduced incidence of side effects compared with other nicotinic receptor types. The recent structural resolution of human α7 nAChRs has confirmed their negative association with heightened pain, providing a valuable foundation for the development of targeted medications. This review presents a comprehensive overview, encompassing insights into the roles of α7 nAChRs derived from structural and functional studies, recent advancements in pharmacology, and investigations into their involvement in the pathophysiology of chronic pain. Moreover, the review addresses the variability in analgesic effects based on the type of receptor agonist and highlights the current research limitations. As such, this review offers potential therapeutic approaches for the development of innovative strategies for chronic pain management.

FOXD3-mediated transactivation of ALKBH5 promotes neuropathic pain via m6A-dependent stabilization of 5-HT3A mRNA in sensory neurons.

The N6-methyladenosine (m6A) modification of RNA is an emerging epigenetic regulatory mechanism that has been shown to participate in various pathophysiological processes. However, its involvement in modulating neuropathic pain is still poorly understood. In this study, we elucidate a functional role of the m6A demethylase alkylation repair homolog 5 (ALKBH5) in modulating trigeminal-mediated neuropathic pain. Peripheral nerve injury selectively upregulated the expression level of ALKBH5 in the injured trigeminal ganglion (TG) of rats. Blocking this upregulation in injured TGs alleviated trigeminal neuropathic pain, while mimicking the upregulation of ALKBH5 in intact TG neurons sufficiently induced pain-related behaviors. Mechanistically, histone deacetylase 11 downregulation induced by nerve injury increases histone H3 lysine 27 acetylation (H3K27ac), facilitating the binding of the transcription factor forkhead box protein D3 (FOXD3) to the Alkbh5 promoter and promoting Alkbh5 transcription. The increased ALKBH5 erases m6A sites in Htr3a messenger RNA (mRNA), resulting in an inability of YT521-B homology domain 2 (YTHDF2) to bind to Htr3a mRNA, thus causing an increase in 5-HT3A protein expression and 5-HT3 channel currents. Conversely, blocking the increased expression of ALKBH5 in the injured TG destabilizes nerve injury-induced 5-HT3A upregulation and reverses mechanical allodynia, and the effect can be blocked by 5-HT3A knockdown. Together, FOXD3-mediated transactivation of ALKBH5 promotes neuropathic pain through m6A-dependent stabilization of Htr3a mRNA in TG neurons. This mechanistic understanding may advance the discovery of new therapeutic targets for neuropathic pain management.

Epigenetic regulation of beta-endorphin synthesis in hypothalamic arcuate nucleus neurons modulates neuropathic pain in a rodent pain model.

Although beta-endorphinergic neurons in the hypothalamic arcuate nucleus (ARC) synthesize beta-endorphin (ß-EP) to alleviate nociceptive behaviors, the underlying regulatory mechanisms remain unknown. Here, we elucidated an epigenetic pathway driven by microRNA regulation of ß-EP synthesis in ARC neurons to control neuropathic pain. In pain-injured rats miR-203a-3p was the most highly upregulated miRNA in the ARC. A similar increase was identified in the cerebrospinal fluid of trigeminal neuralgia patients. Mechanistically, we found histone deacetylase 9 was downregulated following nerve injury, which decreased deacetylation of histone H3 lysine-18, facilitating the binding of NR4A2 transcription factor to the miR-203a-3p gene promoter, thereby upregulating miR-203a-3p expression. Further, increased miR-203a-3p was found to maintain neuropathic pain by targeting proprotein convertase 1, an endopeptidase necessary for the cleavage of proopiomelanocortin, the precursor of ß-EP. The identified mechanism may provide an avenue for the development of new therapeutic targets for neuropathic pain treatment.

Independent Protection and Influence of the Spike-Specific Antibody Response of SARS-CoV-2 Nucleocapsid Protein (N) in Whole-Virion Vaccines.

Of all of the components in SARS-CoV-2 inactivated vaccines, nucleocapsid protein (N) is the most abundant and highly conserved protein. However, the function of N in these vaccines, especially its influence on the targeted spike protein's response, remains unknown. In this study, the immunization of mice with the N protein alone was shown to reduce the viral load, alleviating pulmonary pathological lesions after challenge with the SARS-CoV-2 virus. In addition, co-immunization and pre-immunization with N were found to induce higher S-specific antibody titers rather than compromise them. Remarkably, the same trend was also observed when N was administered as the booster dose after whole inactivated virus vaccination. N-specific IFN-γ-secreting T cell response was detected in all groups and exhibited a certain relationship with S-specific IgG antibody improvements. Together, these data indicate that N has an independent role in vaccine-induced protection and improves the S-specific antibody response to inactivated vaccines, revealing that an interplay mechanism may exist in the immune responses to complex virus components.

Stable Isotope Ratios Trace the Rice Uptake of Cadmium from Atmospheric Deposition via Leaves and Roots.

Cadmium (Cd) stable isotopes provide a novel technique to investigate the fate of Cd in the environment, but challenges exist for tracing the sources in the plants. We performed individual rice leaf and root exposures to dry and wet deposition using customized open-top chambers (OTCs) in the greenhouse and in the field next to a smelter, respectively. The field experiment also included a control without Cd deposition and a "full" treatment. The exposure experiments and isotope signatures showed that leaves can directly take up atmospheric Cd and then translocate within rice plants to other tissues, contributing 52-70% of Cd in grains, which exceeded the contribution (30-48%) by root exposure. The Cd isotopes in leaves, nodes, internodes, and grains demonstrate that roots preferentially take up Cd from wet deposition, but leaves favor uptake of Cd from dry deposition. The Cd uptake by leaves is redistributed via nodes, allowing for upward transport to the grains but preventing downward transport to the roots. Leaves favor uptake of heavy isotopes from atmospheric deposition (ΔCd114/110Leaf-Dust: 0.10 ± 0.02‰) but retain light isotopes and transport heavy isotopes to the nodes and further to grains. These findings highlight the contribution of atmospheric deposition to rice and Cd isotopes as a useful tracer for quantifying sources in plants when different isotopic compositions are in sources.

Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice.

BACKGROUND: Adipokines, including adiponectin, are implicated in nociceptive pain; however, the underlying cellular and molecular mechanisms remain unknown. METHODS: Using electrophysiological recording, immunostaining, molecular biological approaches and animal behaviour tests, we elucidated a pivotal role of adiponectin in regulating membrane excitability and pain sensitivity by manipulating Cav3.2 channels in trigeminal ganglion (TG) neurons. RESULTS: Adiponectin enhanced T-type Ca2+ channel currents (IT) in TG neurons through the activation of adiponectin receptor 1 (adipoR1) but independently of heterotrimeric G protein-mediated signaling. Coimmunoprecipitation revealed a physical association between AdipoR1 and casein kinase II alpha-subunits (CK2α) in the TG, and inhibiting CK2 activity by chemical inhibitor or siRNA targeting CK2α prevented the adiponectin-induced IT response. Adiponectin significantly activated protein kinase C (PKC), and this effect was abrogated by CK2α knockdown. Adiponectin increased the membrane abundance of PKC beta1 (PKCß1). Blocking PKCß1 pharmacologically or genetically abrogated the adiponectin-induced IT increase. In heterologous expression systems, activation of adipoR1 induced a selective enhancement of Cav3.2 channel currents, dependent on PKCß1 signaling. Functionally, adiponectin increased TG neuronal excitability and induced mechanical pain hypersensitivity, both attenuated by T-type channel blockade. In a trigeminal neuralgia model induced by chronic constriction injury of infraorbital nerve, blockade of adipoR1 signaling suppressed mechanical allodynia, which was prevented by silencing Cav3.2. CONCLUSION: Our study elucidates a novel signaling cascade wherein adiponectin stimulates TG Cav3.2 channels via adipoR1 coupled to a novel CK2α-dependent PKCß1. This process induces neuronal hyperexcitability and pain hypersensitivity. Insight into adipoR-Cav3.2 signaling in sensory neurons provides attractive targets for pain treatment.

Trace amine-associated receptor 1 regulation of Kv1.4 channels in trigeminal ganglion neurons contributes to nociceptive behaviors.

BACKGROUND: Trace amines, such as tyramine, are endogenous amino acid metabolites that have been hypothesized to promote headache. However, the underlying cellular and molecular mechanisms remain unknown. METHODS: Using patch-clamp recording, immunostaining, molecular biological approaches and behaviour tests, we elucidated a critically functional role of tyramine in regulating membrane excitability and pain sensitivity by manipulating Kv1.4 channels in trigeminal ganglion (TG) neurons. RESULTS: Application of tyramine to TG neurons decreased the A-type K+ current (IA) in a manner dependent on trace amine-associated receptor 1 (TAAR1). Either siRNA knockdown of Gαo or chemical inhibition of ßγ subunit (Gßγ) signaling abrogated the response to tyramine. Antagonism of protein kinase C (PKC) prevented the tyramine-induced IA response, while inhibition of conventional PKC isoforms or protein kinase A elicited no such effect. Tyramine increased the membrane abundance of PKCθ in TG neurons, and either pharmacological or genetic inhibition of PKCθ blocked the TAAR1-mediated IA decrease. Furthermore, PKCθ-dependent IA suppression was mediated by Kv1.4 channels. Knockdown of Kv1.4 abrogated the TAAR1-induced IA decrease, neuronal hyperexcitability, and pain hypersensitivity. In a mouse model of migraine induced by electrical stimulation of the dura mater surrounding the superior sagittal sinus, blockade of TAAR1 signaling attenuated mechanical allodynia; this effect was occluded by lentiviral overexpression of Kv1.4 in TG neurons. CONCLUSION: These results suggest that tyramine induces Kv1.4-mediated IA suppression through stimulation of TAAR1 coupled to the Gßγ-dependent PKCθ signaling cascade, thereby enhancing TG neuronal excitability and mechanical pain sensitivity. Insight into TAAR1 signaling in sensory neurons provides attractive targets for the treatment of headache disorders such as migraine.

Zinc Supply Affects Cadmium Uptake and Translocation in the Hyperaccumulator Sedum Plumbizincicola as Evidenced by Isotope Fractionation.

This study employs stable isotope analysis to investigate the mechanisms of cadmium (Cd) and zinc (Zn) interaction in the metal hyperaccumulating plant species Sedum plumbizincicola. To this end, the Cd and Zn isotope compositions of root, stem, leaf, and xylem sap samples were determined during metal uptake and translocation at different Cd and Zn concentrations. The enrichment of light isotopes of both elements in plants during uptake was less pronounced at low metal supply levels, likely reflecting the switch from a low-affinity to a high-affinity transport system at lower levels of external metal supply. The lower δ114/110Cd values of xylem sap when treated with a metabolic inhibitor decreasing the active Cd uptake further supports the preference of heavier Cd isotopes during high-affinity transport. The Δ66Znplant-initial solution or Δ66Znplant-final solution values were similar at different Cd concentrations, indicating negligible interaction of Cd in the Zn uptake process. However, decreasing Zn supply levels significantly increased the enrichment of light Cd isotopes in plants (Δ114/110Cd = -0.08‰) in low-Cd treatments but reduced the enrichment of light Cd isotopes in plants (Δ114/110Cd = 0.08‰) under high Cd conditions. A systematic enrichment of heavy Cd and light Zn isotopes was found in root-to-shoot translocation of the metals. The Cd concentrations of the growth solutions thereby had no significant impact on Zn isotope fractionation during root-to-shoot translocation. However, the Δ114/110Cdtranslocation values hint at possible competition between Cd and Zn for transporters during root-to-shoot transfer and this may impact the transport pathway of Cd. The stable isotope data demonstrate that the interactions between the two metals influenced the uptake and transport mechanisms of Cd in S. plumbizincicola but had little effect on those of Zn.

Adaptation to low nitrogen and salt stresses in the desert poplar by effective regulation of nitrogen assimilation and ion balance.

As a main desert plant from arid regions of Central Asia, Populus euphratica always encounters with nitrogen shortage in its long life, apart from salt or drought stress. However, it remains unknown how this species responds to low nitrogen and combined stresses of low nitrogen and salinity. Thus, saplings of P. euphratica with uniform size were exposed to normal or low nitrogen condition (150 and 15 ppm ammonium nitrate separately) individually or in combination with salinity. Under low nitrogen conditions we found a positive effect on P. euphratica root growth, which could be associated to high level of nitrogen allocation to support root growth and effective regulation of nitrogen assimilation in comparison with the other poplar species reported before. Under salt stress the root growth of P. euphratica was significantly inhibited, with the side effects of oxidative stress, as saplings stored higher Na+ and Cl- contents in roots. Under the combined stressors of both salinity and low nitrogen, P. euphratica undergo a risky strategy, as stimulated root growth is accompanied by further oxidative stress.The concentrations of root K+ and whole plant NO3- were increased to support the tolerance of the combined stressors in P. euphratica, showing same characteristics with halophytes. Overall, our results provide evidence that the desert poplar can adapt to the salt stress/low nitrogen bundle, by effective regulation of nitrogen assimilation and ion homoeostasis.

Long-term prognosis of patients with stroke associated with middle cerebral artery occlusion. Single-centre registration study.

Introduction: Large artery intracranial occlusive disease including middle cerebral artery (MCA) is a major contributor to the incidence of stroke in China. The data on the prognosis of symptomatic atherosclerotic MCA occlusions (MCAO) are limited. We aimed to investigate the related factors of unfavorable outcomes in patients with stroke associated with MCAO. Material and methods: A total of 119 patients with MCAO symptom were enrolled in this retrospective longitudinal cohort study. All patients met inclusion criteria of cerebral angiography by CT angiography or magnetic resonance angiography. Stroke severity was assessed on admission using the National Institutes of Health Stroke Scale (NIHSS) and modified Rankin Scale (mRS). Results: We showed an average follow-up time of 46.8 months, within which 20 (19.6%) cases died and 14 (13.7%) cases had stroke recurrence. Using mRS as an evaluation index, the patients were divided into an unfavourable outcome group (mRS > 2, 48 cases) and a favourable outcome group (mRS ≤ 2, 54 cases). Logistic regression analysis suggests that age and NIHSS score were independent risk factors for a poor outcome value. Coexisting other cerebral vascular occlusion was an independent risk factor for stroke recurrence. Age was an independent risk factor for death. Conclusions: The prognosis of patients with MCAO was generally optimistic, with higher survival rate and longer survival time. As compared, elder age and higher NIHSS score both tend to be associated with worse prognosis of survival. MCAO patients with other extracranial or intracranial vascular occlusion have higher risk of recurrent stroke. Death rate increases with age among the MCAO patients.

Will People Accept a Third Booster Dose of the COVID-19 Vaccine? A Cross-Sectional Study in China.

Objective: The coronavirus disease 2019 (COVID-19) vaccines are considered to be an effective way to prevent the spread of the infection. Our previous study has shown that about 75% of healthcare workers (HCWs) in China were willing to receive the vaccine when it became available. Here, we examined the acceptance of a third booster dose among Chinese people and identified the influencing factors. Methods: A cross-sectional online survey was conducted and the snowball sampling method was utilized. An online questionnaire was provided to all the participants in the form of a quick response (QR) code. The questionnaire included general demographic information, views on vaccines, the General Health Questionnaire-12 (GHQ-12), and the Depression, Anxiety, and Stress Scale-21 (DASS-21). The univariate analysis was done between all the variables and our dependent variable. Then, we used the multivariate logistic regression model to examine the influencing factors of the third booster dose acceptance. Results: We collected 1,062 complete answers. Of these, 90.39% (n = 960) declared that they would accept the booster dose. Knowing more about the vaccine and recognizing the efficacy of vaccines were significantly associated with greater acceptance of the booster dose. People willing to take the booster dose had better psychological health. A belief that the booster dose could prevent severe infection caused by COVID-19 and enhance the effectiveness of the first two doses were the main contributing factors to vaccine acceptance. Vaccine hesitancy was mainly due to a low perceived risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and rapid mutation of SARS-CoV-2. Conclusion: This study revealed that Chinese people were very receptive to the third booster dose, which is an inspiring result. More positive attitudes regarding COVID-19 vaccination were supported by its efficacy and few side effects.

Potential of Polyethyleneimine as an Adjuvant To Prepare Long-Term and Potent Antifungal Nanovaccine.

Background: Candida albicans infections are particularly prevalent in immunocompromised patients. Even with appropriate treatment with current antifungal drugs, the mortality rate of invasive candidiasis remains high. Many positive results have been achieved in the current vaccine development. There are also issues such as the vaccine's protective effect is not persistent. Considering the functionality and cost of the vaccine, it is important to develop safe and efficient new vaccines with long-term effects. In this paper, an antifungal nanovaccine with Polyethyleneimine (PEI) as adjuvant was constructed, which could elicit more effective and long-term immunity via stimulating B cells to differentiate into long-lived plasma cells. Materials and Methods: Hsp90-CTD is an important target for protective antibodies during disseminated candidiasis. Hsp90-CTD was used as the antigen, then introduced SDS to "charge" the protein and added PEI to form the nanovaccine. Dynamic light scattering and transmission electron microscope were conducted to identify the size distribution, zeta potential, and morphology of nanovaccine. The antibody titers in mice immunized with the nanovaccine were measured by ELISA. The activation and maturation of long-lived plasma cells in bone marrow by nanovaccine were also investigated via flow cytometry. Finally, the kidney of mice infected with Candida albicans was stained with H&E and PAS to evaluate the protective effect of antibody in serum produced by immunized mice. Results: Nanoparticles (NP) formed by Hsp90-CTD and PEI are small, uniform, and stable. NP had an average size of 116.2 nm with a PDI of 0.13. After immunizing mice with the nanovaccine, it was found that the nano-group produced antibodies faster and for a longer time. After 12 months of immunization, mice still had high and low levels of antibodies in their bodies. Results showed that the nanovaccine could promote the differentiation of B cells into long-lived plasma cells and maintain the long-term existence of antibodies in vivo. After immunization, the antibodies in mice could protect the mice infected by C. albicans. Conclusion: As an adjuvant, PEI can promote the differentiation of B cells into long-lived plasma cells to maintain long-term antibodies in vivo. This strategy can be adapted for the future design of vaccines.

Histone methylation-mediated microRNA-32-5p down-regulation in sensory neurons regulates pain behaviors via targeting Cav3.2 channels.

microRNA (miRNA)­mediated gene regulation has been studied as a therapeutic approach, but its functional regulatory mechanism in neuropathic pain is not well understood. Here, we identify that miRNA-32-5p (miR-32-5p) is a functional RNA in regulating trigeminal-mediated neuropathic pain. High-throughput sequencing and qPCR analysis showed that miR-32-5p was the most down-regulated miRNA in the injured trigeminal ganglion (TG) of rats. Intra-TG injection of miR-32-5p agomir or overexpression of miR-32-5p by lentiviral delivery in neurons of the injured TG attenuated established trigeminal neuropathic pain. miR-32-5p overexpression did not affect acute physiological pain, while miR-32-5p down-regulation in intact rats was sufficient to cause pain-related behaviors. Nerve injury increased the methylated histone occupancy of binding sites for the transcription factor glucocorticoid receptor in the miR-32-5p promoter region. Inhibition of the enzymes that catalyze H3K9me2 and H3K27me3 restored the expression of miR-32-5p and markedly attenuated pain behaviors. Further, miR-32-5p­targeted Cav3.2 T-type Ca2+ channels and decreased miR-32-5p associated with neuropathic pain caused an increase in Cav3.2 protein expression and T-type channel currents. Conversely, miR-32-5p overexpression in injured TG suppressed the increased expression of Cav3.2 and reversed mechanical allodynia. Together, we conclude that histone methylation-mediated miR-32-5p down-regulation in TG neurons regulates trigeminal neuropathic pain by targeting Cav3.2 channels.

Interleukin 33-mediated inhibition of A-type K+ channels induces sensory neuronal hyperexcitability and nociceptive behaviors in mice.

Background: Interleukin-33 (IL-33) has been implicated in nociceptive pain behaviors. However, the underlying molecular and cellular mechanisms remain unclear. Methods: Using electrophysiological recording, immunoblot analysis, immunofluorescence labeling, reverse transcription-PCR, siRNA-mediated knockdown approach and behavior tests, we determined the role of IL-33 in regulating sensory neuronal excitability and pain sensitivity mediated by A-type K+ channels. Results: IL-33 decreased A-type transient outward K+ currents (IA) in small-sized DRG neurons in a concentration-dependent manner, whereas the delayed rectifier currents (IDR) remained unaffected. This IL-33-induced IA decrease was dependent on suppression of the tumorigenicity 2 (ST2) receptor and was associated with a hyperpolarizing shift in the steady-state inactivation. Antagonism of Syk abrogated the IL-33-induced IA response, while inhibition of JAK2 and PKA elicited no such effect. Exposure of DRG cells to IL-33 increased the activity of Akt, but surprisingly, neither Akt nor PI3K influenced the IL-33-induced IA response. IL-33 increased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK). Chemical inhibition of p38 and genetic siRNA knockdown of p38 beta (p38ß), but not p38α, abrogated the IA response induced by IL-33. Moreover, IL-33 increased neuronal excitability of DRG neurons and facilitated peripheral pain sensitivity in mice; both of these effects were occluded by IA blockade. Conclusions: Our present study reveals a novel mechanism by which IL-33/ST2 suppresses IA via a Syk-dependent p38ß signaling pathway. This mechanism thereby increases DRG neuronal excitability and pain sensitivity in mice. Targeting IL-33/ST2-mediated p38ß signaling may represent a therapeutic approach to ameliorate pain behaviors.

Evaluation of the in vitro Activity and in vivo Efficacy of Anidulafungin-Loaded Human Serum Albumin Nanoparticles Against Candida albicans.

Recent decades have seen a significant increase in invasive fungal infections, resulting in unacceptably high mortality rates. Anidulafungin (AN) is the newest echinocandin and appears to have several advantages over existing antifungals. However, its poor water solubility and burdensome route of administration (i.e., repeated, long-term intravenous infusions) have limited its practical use. The objective of this study was to develop anidulafungin-loaded Human Serum Albumin (HSA) nanoparticles (NP) so as to increase both its solubility and antifungal efficacy. HSA was reduced using SDS and DTT, allowing liberation of free thiols to form the intermolecular disulfide network and nanoassembly. Reduced HSA was then added to MES buffer (0.1 M, pH 4.8) and magnetically stirred at 350 rpm and 25°C with AN (m/m 50:1) for 2 h to form nanoparticles (AN NP). We next performed routine antifungal susceptibility testing of Candida strains (n = 31) using Clinical and Laboratory Standards Institute (CLSI) methodologies. Finally, the in vivo efficacy of both AN and AN NP was investigated in a murine model of invasive infection by one of the most common fungal species-C. albicans. The results indicated that our carrier formulations successfully improved the water solubility of AN and encapsulated AN, with the latter having a particle size of 29 ± 1.5 nm with Polymer dispersity index (PDI) equaling 0.173 ± 0.039. In vitro AN NP testing revealed a stronger effect against Candida species (n = 31), with Minimum Inhibitory Concentration (MIC) values 4- to 32-fold lower than AN alone. In mice infected with Candida and having invasive candidiasis, we found that AN NP prolonged survival time (P < 0.005) and reduced fungal burden in kidneys compared to equivalent concentrations of free drug (P < 0.0001). In conclusion, the anidulafungin nanoparticles developed here have the potential to improve drug administration and therapeutic outcomes for individuals suffering from fungal diseases.

Neuromedin B receptor stimulation of Cav3.2 T-type Ca2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity.

Background: Neuromedin B (Nmb) is implicated in the regulation of nociception of sensory neurons. However, the underlying cellular and molecular mechanisms remain unknown. Methods: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of Nmb on the sensory neuronal excitability and peripheral pain sensitivity mediated by Cav3.2 T-type channels. Results: Nmb reversibly and concentration-dependently increased T-type channel currents (IT) in small-sized trigeminal ganglion (TG) neurons through the activation of neuromedin B receptor (NmbR). This NmbR-mediated IT response was Gq protein-coupled, but independent of protein kinase C activity. Either intracellular application of the QEHA peptide or shRNA-mediated knockdown of Gß abolished the NmbR-induced IT response. Inhibition of protein kinase A (PKA) or AMP-activated protein kinase (AMPK) completely abolished the Nmb-induced IT response. Analysis of phospho-AMPK (p-AMPK) revealed that Nmb significantly activated AMPK, while AMPK inhibition prevented the Nmb-induced increase in PKA activity. In a heterologous expression system, activation of NmbR significantly enhanced the Cav3.2 channel currents, while the Cav3.1 and Cav3.3 channel currents remained unaffected. Nmb induced TG neuronal hyperexcitability and concomitantly induced mechanical and thermal hypersensitivity, both of which were attenuated by T-type channel blockade. Moreover, blockade of NmbR signalling prevented mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain, and this effect was attenuated by siRNA knockdown of Cav3.2. Conclusions: Our study reveals a novel mechanism by which NmbR stimulates Cav3.2 channels through a Gßγ-dependent AMPK/PKA pathway. In mouse models, this mechanism appears to drive the hyperexcitability of TG neurons and induce pain hypersensitivity.

Pretreating poplar cuttings with low nitrogen ameliorates salt stress responses by increasing stored carbohydrates and priming stress signaling pathways.

Soil salinity is a widespread stress in semi-arid forests worldwide, but how to manage nitrogen (N) nutrition to improve plant saline tolerance remains unclear. Here, the cuttings of a widely distributed poplar from central Asia, Populus russikki Jabl., were exposed to either normal or low nitrogen (LN) concentrations for two weeks in semi-controlled greenhouse, and then they were added with moderate salt solution or not for another two weeks to evaluate their physiological, biochemical, metabolites and transcriptomic profile changes. LN-pretreating alleviated the toxicity caused by the subsequent salt stress in the poplar plants, demonstrated by a significant reduction in the influx of Na+ and Cl- and improvement of the K+/Na+ ratio. The other salt-stressed traits were also ameliarated, indicated by the variations of chlorophyll content, PSII photochemical activity and lipid peroxidation. Stress alleviation resulted from two different processes. First, LN pretreatment caused a significant increase of non-structural carbohydrates (NSC), allowed for an increased production of osmolytes and a higher potential fueling ion transport under subsequent salt condition, along with increased transcript levels of the cation/H+ ATPase. Second, LN pretreatment enhanced the transcript levels of stress signaling components and phytohormones pathway as well as antioxidant enzyme activities. The results indicate that early restrictions of N supply could enhance posterior survival under saline stress in poplar plants, which is important for plantation programs and restoration activities in semi-arid areas.