CuCo2O4 Nanoflowers with Multiple Enzyme Activities for Treating Bacterium-Infected Wounds via Cuproptosis-like Death.

Nanozyme-driven catalytic therapy provides a promising treatment strategy for bacterial biofilm-infected wounds. However, the single functionality and limited catalytic efficiency of nanozyme-based materials often restrict the effectiveness of wound infection treatment. In this study, CuCo2O4 nanoflowers with multiple enzymatic activities were prepared for antibacterial/antibiofilm treatment by cuproptosis-like death. CuCo2O4 exhibited peroxidase-like (POD-like) and oxidase-like (OXD-like) dual enzyme activities that generated large amounts of •OH and O2•-. Moreover, the glutathione peroxidase-like (GSH-Px-like) activity of CuCo2O4 was able to reduce the overexpression of GSH in the wound microenvironment, enhancing the therapeutic effects of reactive oxygen species (ROS). The morphology of CuCo2O4 was modified using a hydrothermal method with PEG4000 as the solvent, resulting in the exposure of more active center sites and a significant improvement in enzyme catalytic activity. The in vitro results demonstrated the pronounced disruption effect of CuCo2O4 on biofilms formed by bacteria. In vivo, CuCo2O4 significantly promoted angiogenesis, collagen deposition, and cell proliferation. Transcriptome sequencing revealed that elevated ROS levels in bacteria led to cell membrane damage and metabolic disruption. In addition, Cu2+ overload in bacteria induces lipid peroxidation accumulation and disrupts the respiratory chain and tricarboxylic acid (TCA) cycle, ultimately leading to bacterial cuproptosis-like death. This therapeutic strategy, which combines the synergistic effects of multiple enzyme-like activities with cuproptosis-like death, provides an approach for treating biofilm infections.

The plant rhabdovirus viroporin P9 facilitates insect-mediated virus transmission in barley.

Potassium (K+) plays crucial roles in both plant development and immunity. However, the function of K+ in plant-virus interactions remains largely unknown. Here, we utilized Barley yellow striate mosaic virus (BYSMV), an insect-transmitted plant cytorhabdovirus, to investigate the interplay between viral infection and plant K+ homeostasis. The BYSMV accessory P9 protein exhibits viroporin activity by enhancing membrane permeability in Escherichia coli. Additionally, P9 increases K+ uptake in yeast (Saccharomyces cerevisiae) cells, which is disrupted by a point mutation of Glycine 14 to Threonine (P9G14T). Furthermore, BYSMV P9 forms oligomers and targets to both the viral envelope and the plant membrane. Based on the recombinant BYSMV-green fluorescent protein (BYGFP) virus, a P9-deleted mutant (BYGFPΔP9) was rescued and demonstrated infectivity within individual plant cells of Nicotiana benthamiana and insect vectors. However, BYGFPΔP9 failed to infect barley plants after transmission by insect vectors. Furthermore, infection of barley plants was severely impaired for BYGFP-P9G14T lacking P9 K+ channel activity. In vitro assays demonstrate that K+ facilitates virion disassembly and the release of genome RNA for viral mRNA transcription. Altogether, our results show that the K+ channel activity of viroporins is conserved in plant cytorhabdoviruses and plays crucial roles in insect-mediated virus transmission.

New monoterpene phenol dimers from the fruits of Psoralea corylifolia.

Three new monoterpene phenol dimers, bisbakuchiols V-X (1-3), and two bakuchiol ethers (4 and 5), along with four known compounds (6-9) were isolated from the fruits of Psoralea corylifolia. Their structures were elucidated based on extensive spectral analysis. The absolute configurations of 1, 2, 4, and 5 were specified by quantum chemical calculations of ECD spectra.

Chiral separation and bioactivities of six pairs of enantiomeric dilignans from Magnolia officinalis var. biloba.

Six pairs of enantiomeric dilignans, (+)/(-)-magdiligols A-F, have been isolated from an ethanolic extract of the barks of Magnolia officinalis var. biloba. Their chemical structures were elucidated by extensive spectroscopic analyses, NMR calculation with DP4+ analysis, and the electronic circular dichroism spectra calculation. (+)/(-)-1-3 possessed a dihydrobenzopyran ring, while a propyl chain of 1 was linked via ether bond. (+)/(-)-Magdiligols D and E ((+)/(-)-4 and 5) were dilignans possessing a furan ring. (+)-Magdiligol B ((+)/(-)-2), (+)/(-)-magdiligol C ((+)/(-)-3), and racemes 2, 3, and 5 showed potential hepatoprotective effects against APAP-induced HepG2 cell damage, increased the cell viability from 65.4% to 72.7, 78.7.76.6, 73.9, 77.9 and 73.2%, via decreasing the level of the live enzymes ALH and LDH consistently. (+)/(-)-Magdiligols B-D ((+)/(-)-2-4) and (+)/(-)-magdiligol F ((+)/(-)-6) exhibited significant antioxidative activity. (+)/(-)-Magdiligols B-C ((+)/(-)-2 and 3), (-)-magdiligol D ((-)-4), and (+)-magdiligol E ((+)-5) displayed significant PTP1B inhibitory activity with IC50 values 1.41-3.42 µM. (+)/(-)-Magdiligol B ((+)/(-)-2), and its raceme (2) demonstrated α-glucosidase inhibitory activity with the IC50 values 1.47, 2.88 and 1.85 µM, respectively.

Activation of the TNF-α-Necroptosis Pathway in Parvalbumin-Expressing Interneurons of the Anterior Cingulate Cortex Contributes to Neuropathic Pain.

The hyperexcitability of the anterior cingulate cortex (ACC) has been implicated in the development of chronic pain. As one of the key causes of ACC hyperexcitation, disinhibition of the ACC may be closely related to the dysfunction of inhibitory parvalbumin (PV)-expressing interneurons (PV-INs). However, the molecular mechanism underlying the ACC PV-INs injury remains unclear. The present study demonstrates that spared sciatic nerve injury (SNI) induces an imbalance in the excitation and inhibition (E/I) of the ACC. To test whether tumor necrosis factor-α (TNF-α) upregulation in the ACC after SNI activates necroptosis and participates in PV-INs damage, we performed a differential analysis of transcriptome sequencing using data from neuropathic pain models and found that the expression of genes key to the TNF-α-necroptosis pathway were upregulated. TNF-α immunoreactivity (IR) signals in the ACCs of SNI rats were co-located with p-RIP3- and PV-IR, or p-MLKL- and PV-IR signals. We then systematically detected the expression and cell localization of necroptosis-related proteins, including kinase RIP1, RIP3, MLKL, and their phosphorylated states, in the ACC of SNI rats. Except for RIP1 and MLKL, the levels of these proteins were significantly elevated in the contralateral ACC and mainly expressed in PV-INs. Blocking the ACC TNF-α-necroptosis pathway by microinjecting TNF-α neutralizing antibody or using an siRNA knockdown to block expression of MLKL in the ACC alleviated SNI-induced pain hypersensitivity and inhibited the upregulation of TNF-α and p-MLKL. Targeting TNF-α-triggered necroptosis within ACC PV-INs may help to correct PV-INs injury and E/I imbalance in the ACC in neuropathic pain.

A plant cytorhabdovirus modulates locomotor activity of insect vectors to enhance virus transmission.

Transmission of many plant viruses relies on phloem-feeding insect vectors. However, how plant viruses directly modulate insect behavior is largely unknown. Barley yellow striate mosaic virus (BYSMV) is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus). Here, we show that BYSMV infects the central nervous system (CNS) of SBPHs, induces insect hyperactivity, and prolongs phloem feeding duration. The BYSMV accessory protein P6 interacts with the COP9 signalosome subunit 5 (LsCSN5) of SBPHs and suppresses LsCSN5-regulated de-neddylation from the Cullin 1 (CUL1), hereby inhibiting CUL1-based E3 ligases-mediated degradation of the circadian clock protein Timeless (TIM). Thus, virus infection or knockdown of LsCSN5 compromises TIM oscillation and induces high insect locomotor activity for transmission. Additionally, expression of BYSMV P6 in the CNS of transgenic Drosophila melanogaster disturbs circadian rhythm and induces high locomotor activity. Together, our results suggest the molecular mechanisms whereby BYSMV modulates locomotor activity of insect vectors for transmission.

Hypoglycemic oligostilbenes from the stems of Caragana sinica.

Six new oligostilbenes, carastilphenols A-E (1-5) and (-)-hopeachinol B (6), with three reported oligostilbenes were obtained from the stems of Caragana sinica. The structures of compounds 1-6 were determined by comprehensive spectroscopy analysis, and their absolute configurations were determined by electronic circular dichroism calculations. Thus, natural tetrastilbenes were determined as absolute configuration for the first time. Also, we did several pharmacological essays. In the antiviral tests, compounds 2, 4 and 6 showed moderate anti-coxsackie virus B3 type (CVB3) effect on Vero cells activities in vitro with IC50 values of 19.2 âˆ¼ 69.3 µM; and compounds 3 and 4 showed different levels of anti-respiratory syncytial virus (RSV) effect on Hep2 cells activities in vitro with IC50 values of 23.1 and 33.3 µM, respectively. As for hypoglycemic activity, compounds 6-9 (10 µM) showed the inhibition of α-glucosidase in vitro with IC50 values of 0.1 âˆ¼ 0.4 µM; and compound 7 showed significant inhibition (88.8%, 10 µM) of protein tyrosine phosphatase 1B (PTP1B) with IC50 value of 1.1 µM in vitro.

A pyrolysis-free Ni/Fe bimetallic electrocatalyst for overall water splitting.

Catalysts capable of electrochemical overall water splitting in acidic, neutral, and alkaline solution are important materials. This work develops bifunctional catalysts with single atom active sites through a pyrolysis-free route. Starting with a conjugated framework containing Fe sites, the addition of Ni atoms is used to weaken the adsorption of electrochemically generated intermediates, thus leading to more optimized energy level sand enhanced catalytic performance. The pyrolysis-free synthesis also ensured the formation of well-defined active sites within the framework structure, providing ideal platforms to understand the catalytic processes. The as-prepared catalyst exhibits efficient catalytic capability for electrochemical water splitting in both acidic and alkaline electrolytes. At a current density of 10 mA cm-2, the overpotential for hydrogen evolution and oxygen evolution is 23/201 mV and 42/194 mV in 0.5 M H2SO4 and 1 M KOH, respectively. Our work not only develops a route towards efficient catalysts applicable across a wide range of pH values, it also provides a successful showcase of a model catalyst for in-depth mechanistic insight into electrochemical water splitting.

NLRP3-Mediated Piezo1 Upregulation in ACC Inhibitory Parvalbumin-Expressing Interneurons Is Involved in Pain Processing after Peripheral Nerve Injury.

The anterior cingulate cortex (ACC) is particularly critical for pain information processing. Peripheral nerve injury triggers neuronal hyper-excitability in the ACC and mediates descending facilitation to the spinal dorsal horn. The mechanically gated ion channel Piezo1 is involved in the transmission of pain information in the peripheral nervous system. However, the pain-processing role of Piezo1 in the brain is unknown. In this work, we found that spared (sciatic) nerve injury (SNI) increased Piezo1 protein levels in inhibitory parvalbumin (PV)-expressing interneurons (PV-INs) but not in glutaminergic CaMKⅡ+ neurons, in the bilateral ACC. A reduction in the number of PV-INs but not in the number of CaMKⅡ+ neurons and a significant reduction in inhibitory synaptic terminals was observed in the SNI chronic pain model. Further, observation of morphological changes in the microglia in the ACC showed their activated amoeba-like transformation, with a reduction in process length and an increase in cell body area. Combined with the encapsulation of Piezo1-positive neurons by Iba1+ microglia, the loss of PV-INs after SNI might result from phagocytosis by the microglia. In cellular experiments, administration of recombinant rat TNF-α (rrTNF) to the BV2 cell culture or ACC neuron primary culture elevated the protein levels of Piezo1 and NOD-like receptor (NLR) family pyrin domain containing 3 (NLRP3). The administration of the NLRP3 inhibitor MCC950 in these cells blocked the rrTNF-induced expression of caspase-1 and interleukin-1ß (key downstream factors of the activated NLRP3 inflammasome) in vitro and reversed the SNI-induced Piezo1 overexpression in the ACC and alleviated SNI-induced allodynia in vivo. These results suggest that NLRP3 may be the key factor in causing Piezo1 upregulation in SNI, promoting an imbalance between ACC excitation and inhibition by inducing the microglial phagocytosis of PV-INs and, thereby, facilitating spinal pain transmission.

Neuroimmune Mechanisms Underlying Neuropathic Pain: The Potential Role of TNF-α-Necroptosis Pathway.

The neuroimmune mechanism underlying neuropathic pain has been extensively studied. Tumor necrosis factor-alpha (TNF-α), a key pro-inflammatory cytokine that drives cytokine storm and stimulates a cascade of other cytokines in pain-related pathways, induces and modulates neuropathic pain by facilitating peripheral (primary afferents) and central (spinal cord) sensitization. Functionally, TNF-α controls the balance between cell survival and death by inducing an inflammatory response and two programmed cell death mechanisms (apoptosis and necroptosis). Necroptosis, a novel form of programmed cell death, is receiving increasing attraction and may trigger neuroinflammation to promote neuropathic pain. Chronic pain is often accompanied by adverse pain-associated emotional reactions and cognitive disorders. Overproduction of TNF-α in supraspinal structures such as the anterior cingulate cortex (ACC) and hippocampus plays an important role in pain-associated emotional disorders and memory deficits and also participates in the modulation of pain transduction. At present, studies reporting on the role of the TNF-α-necroptosis pathway in pain-related disorders are lacking. This review indicates the important research prospects of this pathway in pain modulation based on its role in anxiety, depression and memory deficits associated with other neurodegenerative diseases. In addition, we have summarized studies related to the underlying mechanisms of neuropathic pain mediated by TNF-α and discussed the role of the TNF-α-necroptosis pathway in detail, which may represent an avenue for future therapeutic intervention.

Large-scale multiplexed mosaic CRISPR perturbation in the whole organism.

Here, we report inducible mosaic animal for perturbation (iMAP), a transgenic platform enabling in situ CRISPR targeting of at least 100 genes in parallel throughout the mouse body. iMAP combines Cre-loxP and CRISPR-Cas9 technologies and utilizes a germline-transmitted transgene carrying a large array of individually floxed, tandemly linked gRNA-coding units. Cre-mediated recombination triggers expression of all the gRNAs in the array but only one of them per cell, converting the mice to mosaic organisms suitable for phenotypic characterization and also for high-throughput derivation of conventional single-gene perturbation lines via breeding. Using gRNA representation as a readout, we mapped a miniature Perturb-Atlas cataloging the perturbations of 90 genes across 39 tissues, which yields rich insights into context-dependent gene functions and provides a glimpse of the potential of iMAP in genome decoding.

A rhabdovirus accessory protein inhibits jasmonic acid signaling in plants to attract insect vectors.

Plant rhabdoviruses heavily rely on insect vectors for transmission between sessile plants. However, little is known about the underlying mechanisms of insect attraction and transmission of plant rhabdoviruses. In this study, we used an arthropod-borne cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), to demonstrate the molecular mechanisms of a rhabdovirus accessory protein in improving plant attractiveness to insect vectors. Here, we found that BYSMV-infected barley (Hordeum vulgare L.) plants attracted more insect vectors than mock-treated plants. Interestingly, overexpression of BYSMV P6, an accessory protein, in transgenic wheat (Triticum aestivum L.) plants substantially increased host attractiveness to insect vectors through inhibiting the jasmonic acid (JA) signaling pathway. The BYSMV P6 protein interacted with the constitutive photomorphogenesis 9 signalosome subunit 5 (CSN5) of barley plants in vivo and in vitro, and negatively affected CSN5-mediated deRUBylation of cullin1 (CUL1). Consequently, the defective CUL1-based Skp1/Cullin1/F-box ubiquitin E3 ligases could not mediate degradation of jasmonate ZIM-domain proteins, resulting in compromised JA signaling and increased insect attraction. Overexpression of BYSMV P6 also inhibited JA signaling in transgenic Arabidopsis (Arabidopsis thaliana) plants to attract insects. Our results provide insight into how a plant cytorhabdovirus subverts plant JA signaling to attract insect vectors.

Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury.

BACKGROUND: Peripheral nerve inflammation or lesion can affect contralateral healthy structures, and thus result in mirror-image pain. Supraspinal structures play important roles in the occurrence of mirror pain. The anterior cingulate cortex (ACC) is a first-order cortical region that responds to painful stimuli. In the present study, we systematically investigate and compare the neuroimmune changes in the bilateral ACC region using unilateral- (spared nerve injury, SNI) and mirror-(L5 ventral root transection, L5-VRT) pain models, aiming to explore the potential supraspinal neuroimmune mechanism underlying the mirror-image pain. METHODS: The up-and-down method with von Frey hairs was used to measure the mechanical allodynia. Viral injections for the designer receptors exclusively activated by designer drugs (DREADD) were used to modulate ACC glutamatergic neurons. Immunohistochemistry, immunofluorescence, western blotting, protein microarray were used to detect the regulation of inflammatory signaling. RESULTS: Increased expressions of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and chemokine CX3CL1 in ACC induced by unilateral nerve injury were observed on the contralateral side in the SNI group but on the bilateral side in the L5-VRT group, representing a stronger immune response to L5-VRT surgery. In remote ACC, both SNI and L5-VRT induced robust bilateral increase in the protein level of Nav1.6 (SCN8A), a major voltage-gated sodium channel (VGSC) that regulates neuronal activity in the mammalian nervous system. However, the L5-VRT-induced Nav1.6 response occurred at PO 3d, earlier than the SNI-induced one, 7 days after surgery. Modulating ACC glutamatergic neurons via DREADD-Gq or DREADD-Gi greatly changed the ACC CX3CL1 levels and the mechanical paw withdrawal threshold. Neutralization of endogenous ACC CX3CL1 by contralateral anti-CX3CL1 antibody attenuated the induction and the maintenance of mechanical allodynia and eliminated the upregulation of CX3CL1, TNF-α and Nav1.6 protein levels in ACC induced by SNI. Furthermore, contralateral ACC anti-CX3CL1 also inhibited the expression of ipsilateral spinal c-Fos, Iba1, CD11b, TNF-α and IL-6. CONCLUSIONS: The descending facilitation function mediated by CX3CL1 and its downstream cascade may play a pivotal role, leading to enhanced pain sensitization and even mirror-image pain. Strategies that target chemokine-mediated ACC hyperexcitability may lead to novel therapies for the treatment of neuropathic pain.

Meroterpenoids with unknown skeletons from the leaves of Psidium guajava including one anti-inflammatory and anticoagulant compound: psidial F.

Four unknown meroterpenoids named as psidials D-G (1-4) together with 5 known compounds (5-9) had been obtained from the leaves of Psidium guajava. Their absolute structures were elucidated by spectral and calculated methods. Psidials DF (1-3) represented unknown carbon skeleton of the 3,5-diformylbenzyl phloroglucinol-coupled sesquiterpenoid. The possible biosynthetic pathway for 1-3 was postulated. In the bioactivity assay, psidial F (3) was found to possess anti-inflammatory and anticoagulant activities.

Developing reverse genetics systems of northern cereal mosaic virus to reveal superinfection exclusion of two cytorhabdoviruses in barley plants.

Recently, reverse genetics systems of plant negative-stranded RNA (NSR) viruses have been developed to study virus-host interactions. Nonetheless, genetic rescue of plant NSR viruses in both insect vectors and monocot plants is very limited. Northern cereal mosaic virus (NCMV), a plant cytorhabdovirus, causes severe diseases in cereal plants through transmission by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. In this study, we first developed a minireplicon system of NCMV in Nicotiana benthamiana plants, and then recovered a recombinant NCMV virus (rNCMV-RFP), with a red fluorescent protein (RFP) insertion, in SBPHs and barley plants. We further used rNCMV-RFP and green fluorescent protein (GFP)-tagged barley yellow striate mosaic virus (rBYSMV-GFP), a closely related cytorhabdovirus, to study superinfection exclusion, a widely observed phenomenon in dicot plants rarely studied in monocot plants. Interestingly, cellular superinfection exclusion of rBYSMV-GFP and rNCMV-RFP was observed in barley leaves. Our results demonstrate that two insect-transmitted cytorhabdoviruses are enemies rather than friends at the cellular level during coinfections in plants.

Host casein kinase 1-mediated phosphorylation modulates phase separation of a rhabdovirus phosphoprotein and virus infection.

Liquid-liquid phase separation (LLPS) plays important roles in forming cellular membraneless organelles. However, how host factors regulate LLPS of viral proteins during negative-sense RNA (NSR) virus infection is largely unknown. Here, we used barley yellow striate mosaic virus (BYSMV) as a model to demonstrate regulation of host casein kinase 1 (CK1) in phase separation and infection of NSR viruses. We first found that the BYSMV phosphoprotein (P) formed spherical granules with liquid properties and recruited viral nucleotide (N) and polymerase (L) proteins in vivo. Moreover, the P-formed granules were tethered to the ER/actin network for trafficking and fusion. BYSMV P alone formed droplets and incorporated the N protein and the 5' trailer of genomic RNA in vitro. Interestingly, phase separation of BYSMV P was inhibited by host CK1-dependent phosphorylation of an intrinsically disordered P protein region. Genetic assays demonstrated that the unphosphorylated mutant of BYSMV P exhibited condensed phase, which promoted viroplasm formation and virus replication. Whereas, the phosphorylation-mimic mutant existed in diffuse phase state for virus transcription. Collectively, our results demonstrate that host CK1 modulates phase separation of the viral P protein and virus infection.

A Versatile Expression Platform in Insects and Cereals Based on a Cytorhabdovirus.

In recent years, plant virus-based vectors have been widely applied to express heterologous proteins for genomic studies and commercial production. Among these versatile RNA viral vectors, the barley yellow striate mosaic virus (BYSMV)-based expression vector system has outstanding capability to express large and multiple heterologous proteins. Here we describe a detailed protocol for expression of heterologous proteins using BYSMV expression systems in monocot plants and insects.

A cytorhabdovirus-based expression vector in Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera.

The brown planthopper (BPH, Nilaparvata lugens), the small brown planthopper (SBPH, Laodelphax striatellus), and the white-backed planthopper (WBPH, Sogatella furcifera) are problematic insect pests and cause severe yield losses through phloem sap-sucking and virus transmission. Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, has been developed as versatile expression platforms in SBPHs and cereal plants. However, bio-safe overexpression vectors based on recombinant BYSMV (rBYSMV) remain to be developed and applied to the three kinds of planthoppers. Here, we found that rBYSMV was able to infect SBPHs, BPHs and WBPHs through microinjection with crude extracts from rBYSMV-infected barley leaves. To ensure bio-safety of the rBYSMV vectors, we generated an rBYSMV mutant by deleting the accessory protein P3, a putative viral movement protein. As expected, the resulting mutant abolished viral systemic infection in barley plants but had no effects on BYSMV infectivity in insect vectors. Subsequently, we used the modified rBYSMV vector to overexpress iron transport peptide (ITP) in the three kinds of planthoppers and revealed the potential functions of ITP. Overall, our results provide bio-safe overexpression platforms to facilitate functional genomics studies of planthoppers.

Pyrolysis-Free Synthesized Catalyst towards Acidic Oxygen Reduction by Deprotonation.

Acidic oxygen reduction is vital for renewable energy devices such as fuel cells. However, many aspects of the catalytic process are still uncertain-especially the large difference in activity in acidic and alkaline media. Thus, the design and synthesis of model catalysts to determine the active centers and the inactivation mechanism are urgently needed. We report a pyrolysis-free synthesis route to fabricate a catalyst (CPF-Fe@NG) for oxygen reduction in acidic conditions. By introducing a deprotonation process, we extended the oxygen reduction reaction (ORR) activity from alkaline to acidic conditions. CPF-Fe@NG demonstrated outstanding performance with a half-wave potential of 853 mV (vs. RHE) and good stability after 10000 cycles in 1 M HClO4 . The pyrolysis-free route could also be used to assemble fuel cells, with a maximum power density of 126 mW cm-2 . Our findings offer new insights into the ORR process to optimize catalysts for both mechanistic studies and practical applications.