Engineered cell entry links receptor biology with single-cell genomics.

Cells communicate with each other via receptor-ligand interactions. Here, we describe lentiviral-mediated cell entry by engineered receptor-ligand interaction (ENTER) to display ligand proteins, deliver payloads, and record receptor specificity. We optimize ENTER to decode interactions between T cell receptor (TCR)-MHC peptides, antibody-antigen, and other receptor-ligand pairs. A viral presentation strategy allows ENTER to capture interactions between B cell receptor and any antigen. We engineer ENTER to deliver genetic payloads to antigen-specific T or B cells to selectively modulate cellular behavior in mixed populations. Single-cell readout of ENTER by RNA sequencing (ENTER-seq) enables multiplexed enumeration of antigen specificities, TCR clonality, cell type, and states of individual T cells. ENTER-seq of CMV-seropositive patient blood samples reveals the viral epitopes that drive effector memory T cell differentiation and inter-clonal vs. intra-clonal phenotypic diversity targeting the same epitope. ENTER technology enables systematic discovery of receptor specificity, linkage to cell fates, and antigen-specific cargo delivery.

Specific CD4+ T cell phenotypes associate with bacterial control in people who 'resist' infection with Mycobacterium tuberculosis.

A subset of individuals exposed to Mycobacterium tuberculosis (Mtb) that we refer to as 'resisters' (RSTR) show evidence of IFN-γ- T cell responses to Mtb-specific antigens despite serially negative results on clinical testing. Here we found that Mtb-specific T cells in RSTR were clonally expanded, confirming the priming of adaptive immune responses following Mtb exposure. RSTR CD4+ T cells showed enrichment of TH17 and regulatory T cell-like functional programs compared to Mtb-specific T cells from individuals with latent Mtb infection. Using public datasets, we showed that these TH17 cell-like functional programs were associated with lack of progression to active tuberculosis among South African adolescents with latent Mtb infection and with bacterial control in nonhuman primates. Our findings suggested that RSTR may successfully control Mtb following exposure and immune priming and established a set of T cell biomarkers to facilitate further study of this clinical phenotype.

SlWRKY55 coordinately acts with SlVQ11 to enhance tomato thermotolerance by activating SlHsfA2.

High temperature (HT) severely restricts plant growth, development, and productivity. Plants have evolved a set of mechanisms to cope with HT, including the regulation of heat stress transcription factors (Hsfs) and heat shock proteins (Hsps). However, it is not clear how the transcriptional and translational levels of Hsfs and Hsps are controlled in tomato. Here, we reported that the HT-induced transcription factor SlWRKY55 recruited SlVQ11 to coordinately regulate defense against HT. SlWRKY55 directly bound to the promoter of SlHsfA2 and promoted its expression, which was increased by SlVQ11. Moreover, both SlWRKY55 and SlVQ11 physically interacted with SlHsfA2 to enhance the transcriptional activity of SlHsfA2. Thus, our results revealed a molecular mechanism that the SlWRKY55/SlVQ11-SlHsfA2 cascade enhanced thermotolerance and provided potential target genes for improving the adaptability of crops to HT.

Identification of NanoLuciferase Substrates Transported by Human ABCB1 and ABCG2 and their Zebrafish Homologs at the Blood-Brain Barrier.

ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, P-glycoprotein (P-gp, ABCB1) and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish where expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by P-gp, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disrpution. We transfected HEK-293 cells with both NanoLuc and human ABCB1 or ABCG2, or their zebrafish homologs Abcb4 and Abcg2a, which are functionally homologous to human P-gp and ABCG2, respectively, and expressed at the zebrafish BBB. We evaluated the brightness of ten NanoLuc substrates, then screened the eight brightest for their ability to be effluxed by the ABC transporters. We identified one ABCB1 substrate, two Abcb4 substrates, six ABCG2 substrates, and four Abcg2a substrates. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter. Significance Statement The ATP-Binding Cassette (ABC) transporters ABCB1 and ABCG2 at the blood-brain barrier (BBB) hinder pharmacological treatment of brain-related diseases. Consequently, there is a need for tools to identify BBB disruptors. We conducted a screen of ten NanoLuciferase substrates, identifying the brightest and those that were transported by human and zebrafish ABC transporters at the BBB. This work supports and complements our development of a transgenic zebrafish model, in which NanoLuciferase is expressed within glial cells, enabling detection of BBB disruption.

Jasmonic acid enhances osmotic stress responses by MYC2-mediated inhibition of protein phosphatase 2C1 and response regulators 26 transcription factor in tomato.

The jasmonic acid (JA) signaling pathway is involved in the plant response to drought stress. JA and other hormones synergistically regulate the drought response in plants. However, the molecular mechanism underlying this synergism remains poorly defined. In the present study, transcriptome analyses of guard cells and quantitative PCR experiments revealed that MYC2 negatively regulated the negative regulator of ABA signaling, SlPP2C1, and the type-B response regulator in the cytokinin pathway, SlRR26, and this negative regulation was direct. SlRR26 overexpression reduced drought tolerance in transgenic tomatoes, whereas slrr26cr lines were more tolerant to drought. SlRR26 negatively modulated reactive oxygen species levels in stomata and stomatal closure through RobhB. Moreover, SlRR26 overexpression counteracted JA-mediated stomatal closure, suggesting that SlRR26 played a negative role in the JA-mediated drought response. These findings suggest that MYC2 plays a key role in JA-regulated stomatal closure under drought stress by inhibiting SlPP2C1 and SlRR26.

Extracellular Vesicle-Derived Non-Coding RNAs: Key Mediators in Remodelling Heart Failure.

Heart failure (HF), a syndrome of persistent development of cardiac insufficiency due to various heart diseases, is a serious and lethal disease for which specific curative therapies are lacking and poses a severe burden on all aspects of global public health. Extracellular vesicles (EVs) are essential mediators of intercellular and interorgan communication, and are enclosed nanoscale vesicles carrying biomolecules such as RNA, DNA, and proteins. Recent studies have showed, among other things, that non-coding RNAs (ncRNAs), especially microRNAs (miRNAs), long ncRNAs (lncRNA), and circular RNAs (circRNAs) can be selectively sorted into EVs and modulate the pathophysiological processes of HF in recipient cells, acting on both healthy and diseased hearts, which makes them promising targets for the diagnosis and therapy of HF. This review aims to explore the mechanism of action of EV-ncRNAs in heart failure, with emphasis on the potential use of differentially expressed miRNAs and circRNAs as biomarkers of cardiovascular disease, and recent research advances in the diagnosis and treatment of heart failure. Finally, we focus on summarising the latest advances and challenges in engineering EVs for HF, providing novel concepts for the diagnosis and treatment of heart failure.

Sugar delivery at the tomato root and root galls after Meloidogyne incognita infestation.

Root-knot nematodes (RKNs) infect host plants and obtain nutrients such as sugars for their own development. Therefore, inhibiting the nutrient supply to RKNs may be an effective method for alleviating root-knot nematode disease. At present, the pathway by which sucrose is unloaded from the phloem cells to giant cells (GCs) in root galls and which genes related to sugar metabolism and transport play key roles in this process are unclear. In this study, we found that sugars could be unloaded into GCs only from neighboring phloem cells through the apoplastic pathway. With the development of galls, the contents of sucrose, fructose and glucose in the galls and adjacent tissue increased gradually. SUT1, SUT2, SWEET7a, STP10, SUS3 and SPS1 may provide sugar sources for GCs, while STP1, STP2 and STP12 may transport more sugar to phloem parenchyma cells. At the early stage of Meloidogyne incognita infestation, the sucrose content in tomato roots and leaves increased, while the glucose and fructose contents decreased. SWEET7a, SPS1, INV-INH1, INV-INH2, SUS1 and SUS3 likely play key roles in root sugar delivery. These results elucidated the pathway of sugar unloading in tomato galls and provided an important theoretical reference for eliminating the sugar source of RKNs and preventing root-knot nematode disease.

Extracellular vesicles in sepsis plasma mediate neuronal inflammation in the brain through miRNAs and innate immune signaling.

BACKGROUND: Neuroinflammation reportedly plays a critical role in the pathogenesis of sepsis-associated encephalopathy (SAE). We previously reported that circulating plasma extracellular vesicles (EVs) from septic mice are proinflammatory. In the current study, we tested the role of sepsis plasma EVs in neuroinflammation. METHODS: To track EVs in cells and tissues, HEK293T cell-derived EVs were labeled with the fluorescent dye PKH26. Cecal ligation and puncture (CLP) was conducted to model polymicrobial sepsis in mice. Plasma EVs were isolated by ultracentrifugation and their role in promoting neuronal inflammation was tested following intracerebroventricular (ICV) injection. miRNA inhibitors (anti-miR-146a, -122, -34a, and -145a) were applied to determine the effects of EV cargo miRNAs in the brain. A cytokine array was performed to profile microglia-released protein mediators. TLR7- or MyD88-knockout (KO) mice were utilized to determine the underlying mechanism of EVs-mediated neuroinflammation. RESULTS: We observed the uptake of fluorescent PKH26-EVs inside the cell bodies of both microglia and neurons. Sepsis plasma EVs led to a dose-dependent cytokine release in cultured microglia, which was partially attenuated by miRNA inhibitors against the target miRNAs and in TLR7-KO cells. When administered via the ICV, sepsis plasma EVs resulted in a marked increase in the accumulation of innate immune cells, including monocyte and neutrophil and cytokine gene expression, in the brain. Although sepsis plasma EVs had no direct effect on cytokine production or neuronal injury in vitro, the conditioned media (CM) of microglia treated with sepsis plasma EVs induced neuronal cell death as evidenced by increased caspase-3 cleavage and Annexin-V staining. Cytokine arrays and bioinformatics analysis of the microglial CM revealed multiple cytokines/chemokines and other factors functionally linked to leukocyte chemotaxis and migration, TLR signaling, and neuronal death. Moreover, sepsis plasma EV-induced brain inflammation in vivo was significantly dependent on MyD88. CONCLUSIONS: Circulating plasma EVs in septic mice cause a microglial proinflammatory response in vitro and a brain innate immune response in vivo, some of which are in part mediated by TLR7 in vitro and MyD88 signaling in vivo. These findings highlight the importance of circulating EVs in brain inflammation during sepsis.

SlVQ15 recruits SlWRKY30IIc to link with jasmonate pathway in regulating tomato defence against root-knot nematodes.

Tomato is one of the most economically important vegetable crops in the world and has been seriously affected by the devastating agricultural pest root-knot nematodes (RKNs). Current understanding of tomato resistance to RKNs is quite limited. VQ motif-containing family proteins are plant-specific regulators; however, whether and how tomato VQs regulate resistance to RKNs is unknown. Here, we found that SlVQ15 recruited SlWRKY30IIc to coordinately control tomato defence against the RKN Meloidogyne incognita without affecting plant growth and productivity. The jasmonate (JA)-ZIM domain (JAZ) repressors of the phytohormone JAs signalling associated and interfered with the interaction of SlVQ15 and SlWRKY30IIc. In turn, SlWRKY30IIc bound to SlJAZs promoters and cooperated with SlVQ15 to repress their expression, whereas this inhibitory effect was antagonized by SlJAZ5, forming a feedback regulatory mechanism. Moreover, SlWRKY30IIc expression was directly regulated by SlMYC2, a SlJAZ-interacting negative regulator of resistance to RKNs. In conclusion, our findings revealed that a regulatory circuit of SlVQ15-SlWRKY30IIc and the JA pathway fine-tunes tomato defence against the RKN M. incognita, and provided candidate genes and clues with great potential for crop improvement.

Discovery and Characterization of Putative Glycoprotein-Encoding Mycoviruses in the Bunyavirales.

Although segmented negative-sense RNA viruses (SNSRVs) have been frequently discovered in various fungi, most SNSRVs reported only the large segments. In this study, we investigated the diversity of the mycoviruses in the phytopathogenic fungus Fusarium asiaticum using the metatranscriptomic technique. We identified 17 fungal single-stranded RNA (ssRNA) viruses including nine viruses within Mitoviridae, one each in Narnaviridae, Botourmiaviridae, Hypoviridae, Fusariviridae, and Narliviridae, two in Mymonaviridae, and one trisegmented virus temporarily named Fusarium asiaticum mycobunyavirus 1 (FaMBV1). The FaMBV1 genome comprises three RNA segments, large (L), medium (M), and small (S) with 6,468, 2,639, and 1,420 nucleotides, respectively. These L, M, and S segments putatively encode the L protein, glycoprotein, and nucleocapsid, respectively. Phylogenetic analysis based on the L protein showed that FaMBV1 is phylogenetically clustered with Alternaria tenuissima negative-stranded RNA virus 2 (AtNSRV2) and Sclerotinia sclerotiorum negative-stranded RNA virus 5 (SsNSRV5) but distantly related to the members of the family Phenuiviridae. FaMBV1 could be vertically transmitted by asexual spores with lower efficiency (16.7%, 2/42). Comparison between FaMBV1-free and -infected fungal strains revealed that FaMBV1 has little effect on hyphal growth, pathogenicity, and conidium production, and its M segment is dispensable for viral replication and lost during subculture and asexual conidiation. The M and S segments of AtNSRV2 and SsNSRV5 were found using bioinformatics methods, indicating that the two fungal NSRVs harbor trisegmented genomes. Our results provide a new example of the existence and evolution of the segmented negative-sense RNA viruses in fungi. IMPORTANCE Fungal segmented negative-sense RNA viruses (SNSRVs) have been frequently found. Only the large segment encoding RNA-dependent RNA polymerase (RdRp) has been reported in most fungal SNSRVs, except for a few fungal SNSRVs reported to encode nucleocapsids, nonstructural proteins, or movement proteins. Virome analysis of the Fusarium spp. that cause Fusarium head blight discovered a novel virus, Fusarium asiaticum mycobunyavirus 1 (FaMBV1), representing a novel lineage of the family Phenuiviridae. FaMBV1 harbors a trisegmented genome that putatively encodes RdRp, glycoproteins, and nucleocapsids. The putative glycoprotein was first described in fungal SNSRVs and shared homology with glycoprotein of animal phenuivirus but was dispensable for its replication in F. asiaticum. Two other trisegmented fungal SNSRVs that also encode glycoproteins were discovered, implying that three-segment bunyavirus infections may be common in fungi. These findings provide new insights into the ecology and evolution of SNSRVs, particularly those infecting fungi.

Conical quasi-phase-matching second harmonic generation in a 2D photonic crystal with hybrid Ewald geometric method.

The direction variation of the fundamental wave in the same nonlinear photonic crystal would cause different pattern of harmonics generation. In a 2D/3D crystal with dense reciprocal lattice vectors, there will be large numbers of conical harmonic beams evolving with direction change of the fundamental wave. By rearranging the Ewald sphere and superposing it into the Ewald shell, we have a hybrid Ewald construction. It becomes a simple but useful geometric method to comprehensively depict the distribution of these quasi-phase-matching second harmonics and their conical form evolution. It presents conical second harmonic beams by their related reciprocal lattice vectors and simplifies the beams' distribution according to spatial arrangement of those reciprocal lattice vectors. It finds that the conical beams will create, annihilate, or get enhanced in specific order when fundamental waves change incident directions. We applied the method on a periodically poled 2D LiTaO3 crystal and all observed phenomena, meet the method's predictions. In our experiment, we observed that the conical beams distorted along the optic axis of the sample due to anisotropy, which was generally overlooked by earlier researches. The eccentricities of their ring projections suggest a potential auxiliary approach for crystal dispersion measurement.

Did Medicaid Reimbursements Shape the Effects of Medicaid Expansion on Access to Health Care Among the Low-Income Population?

BACKGROUND: Whether variation in Medicaid reimbursement fees influenced the impacts of the Medicaid expansions is not well understood. OBJECTIVE: We examine whether changes in health care access associated with Medicaid expansion are different in states with comparatively high Medicaid reimbursement rates compared against expanding in states with lower Medicaid reimbursement rates. DESIGN: Using a difference-in-difference-in-difference (DDD or triple-difference) regression approach, we compare relative differences in Medicaid expansion effects between lower and higher reimbursement states. PARTICIPANTS: 512,744 low-income adults aged 20-64 in the 2011-2019 Behavioral Risk Factor Surveillance System. MAIN MEASURES: Health insurance coverage status, unmet medical needs due to cost, regular source for health care, and a regular/scheduled checkup within the past year. KEY RESULTS: Medicaid expansion has significant and positive impacts on health coverage and access in both high- and low-fee states. In states with fee levels above the median Medicare-to-Medicaid ratios, expanding Medicaid eligibility reduced uninsurance rate by 15.2 percentage point (ppt, p < 0.01), shrank the cost-associated unmet medical need by 10.3 ppt (p < 0.01), improved access to usual source of care by 1.9 ppt (p < 0.1), and increased regular checkup by 14.4 ppt (p < 0.01), while such effects in low-fee states were 11.7 ppt (p < 0.01), 8.3 ppt (p < 0.01), 3.1 ppt (p < 0.1), and 12.3 ppt (p < 0.01), respectively. Our results suggest that Medicaid expansion effect on unmet medical need due to cost in higher-reimbursing states was 2.98 ppt (p < 0.05) larger than in lower-reimbursing states. Evidence suggests modest increases in health care access were more strongly associated with expansions in higher-fee states. CONCLUSIONS: Medicaid's fee structure should be considered as a factor influencing large-scale coverage expansions.

The value of pupillary diameter in evaluating pain perception after awakening in patients undergoing general anesthesia during orthopedic surgery.

BACKGROUND: The pupillary response to tetanic electrical stimulation reflects the balance between nociceptive stimulation and analgesia. Although pupillary pain index (PPI) was utilized to predict postoperative pain, it depended on tetanic stimulation and was complex. We aim to describe the potential relationship between PD in the presence of surgical stimulation and pain levels after awakening. METHODS: According to the Verbal Rating Scale (VRS) score after extubation, the patients were divided into painless group (VRS = 0) and pain group (VRS ≥ 1). Pupillary diameter (PD) and pupillary light reflex velocity (PLRV) were compared between two groups when patients entered the operating room (T1), before incision (T2), 10 s after incision (T3), 30 s after incision (T4), 1 h after incision (T5), at the end of surgery (T6), shortly after extubation (T7), and when patients expressed pain clearly (T8). The magnitude of PD change (ΔPD) compared to the baseline value after anesthesia induction (T2) was calculated. The correlations between pupillary parameters and pain after awakening were calculated. RESULTS: Patients with VRS ≥ 1 had greater PD than painless patients at T3-7 (P = 0.04, 0.04, 0.003, <0.001, <0.001), and it was positively correlated with VRS score after awakening at T4-7 (r = 0.188, 0.217, 0.684, 0.721). The ability of T6ΔPD to predict VRS ≥ 1 was strong [threshold: 20.53%, area under the curve (AUC): 0.93, 95% confidence interval (CI): 0.89-0.97 ]. CONCLUSION: Our study indicates that PD is a useful index to direct the individualized analgesics used during operation, to better avoid the occurrence of pain during the postoperative emergence period. TRIAL REGISTRATION: This study was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR2000040908, registration date: 15/12/2020).

Nanoparticle-enabled innate immune stimulation activates endogenous tumor-infiltrating T cells with broad antigen specificities.

Tumors are often infiltrated by T lymphocytes recognizing either self- or mutated antigens but are generally inactive, although they often show signs of prior clonal expansion. Hypothesizing that this may be due to peripheral tolerance, we formulated nanoparticles containing innate immune stimulants that we found were sufficient to activate self-specific CD8+ T cells and injected them into two different mouse tumor models, B16F10 and MC38. These nanoparticles robustly activated and/or expanded antigen-specific CD8+ tumor-infiltrating T cells, along with a decrease in regulatory CD4+ T cells and an increase in Interleukin-17 producers, resulting in significant tumor growth retardation or elimination and the establishment of immune memory in surviving mice. Furthermore, nanoparticles with modification of stimulating human T cells enabled the robust activation of endogenous T cells in patient-derived tumor organoids. These results indicate that breaking peripheral tolerance without regard to the antigen specificity creates a promising pathway for cancer immunotherapy.

First Report of Stem and Foliage Blight of Chrysanthemum morifolium cv. Fubaiju Caused by Stagonosporopsis chrysanthemi in China.

Chrysanthemum (Chrysanthemum morifolium cv. Fubaiju) is used as medicinal herb (Chen et al. 2020). In October 2021, a leaf spot disease was observed on leaves of C. morifolium in Huanggang, Hubei province. Disease incidence was approximately 40%. Leaf lesions manifested as necrotic spots, coalesced, and expanded to form brown-black spots, leading to wilting of the leaves. On stems, the lesions manifested as dark brown necrotic spots. To identify the pathogen, 29 pieces (5 × 5 mm) from lesion margins were surface sterilized in 1% NaOCl and rinsed three times with sterile water. The pieces were transferred onto potato dextrose agar (PDA) for incubation at 25℃ for 3 d in the dark. Fifteen fungal colonies were successfully isolated. The colony morphology with flat wavy edge, sparse aerial mycelia, and surface olivaceous black were observed at 7 days post incubation. Subglobular pycnidia were brown with a short beak, and pycnidia diameters were thick (212 to 265 × 189 to 363 µm, n = 20). Ovoid conidia were aseptate and hyaline, conidia diameters were thick (4.0 to 9.8 × 1.8 to 4.7 µm, n = 100). The morphological characters of these isolates were consistent with those of Stagonosporopsis chrysanthemi (Zhao et al. 2021). Pure culture of representative HGNU2021-18 isolated from the diseased leaves subjected to molecular identification. Sequences of the rDNA internal transcribed spacer (ITS) region, 28S large subunit ribosomal RNA (LSU), ß-tubulin (TUB2), actin (ACT), and partial RNA polymerase II largest subunit (RPB2) genes were amplified from genomic DNA of isolate HGNU2021-18 using the following primer pairs: ITS1/ITS4 (White et al. 1990), LR0R/LR5 (Rehner et al. 1994), Btub2Fd/Btub4Rd (Woudenberg et al. 2009), ACT512F/ACT783R (Carbone et al.1999), and RPB2-5F2 (Sung et al. 2007)/fRPB2-7cR (Liu et al. 1999), respectively. The PCR products were purified and then sequenced by Sangon Biotech (China). Nucleotide sequences of ITS (544 bp, OM346748), LSU (905 bp, OM758418), TUB2 (563 bp, OM945724), ACT (294 bp, OM793715), and RPB2 (957 bp, OM793716) amplified from the isolate HGNU2021-18 were subjected to BLASTn analysis. The results showed that ITS, LSU, TUB2, ACT, and RPB2 shared 100.00%, 99.45%, 99.20%, 100.00%, and 100.00% sequence identity to the five published sequences (MW810272.1, MH869953.1, MW815129.1, JN251973.1, and MT018012.1, respectively) of the S. chrysanthemi isolate CBS 500.63. Phylogenetic analysis of the multilocus sequences of ITS, LSU, RPB2, ACT, and TUB2 belonging to different Stagonosporopsis species was performed in MEGA 7.0 (Chen et al. 2015). Isolate HGNU2021-18 was placed in a clade with S. chrysanthemi with 99% bootstrap support. Thus, the results of morphological and molecular analyses indicated that the disease symptoms on chrysanthemum plants were caused by S. chrysanthemi. Under conditions of 25°C and 85% relative humidity, pathogenicity test was performed on 2-month-old healthy plants using isolate HGNU2021-18. The leaves were inoculated with 5 mm diameter mycelial plugs or with sterile agar plugs (control). Six plants were used in each treatment. Disease symptoms were observed on treated plants at 2 weeks post inoculation which were those previously observed in the field, while the control plants remained symptomless. The pathogen was re-isolated from the diseased plants, and S. chrysanthemi was confirmed as the causal pathogen. This is the first report of S. chrysanthemi causing stem and foliage blight of chrysanthemum in China.

The miR396a-SlGRF8 module regulates sugar accumulation in the roots via SlSTP10 during the interaction between root-knot nematodes and tomato plants.

Root-knot nematodes (RKNs; Meloidogyne spp.) are a serious threat to crop production. The competition between plants and pathogens for assimilates influences the outcome of their interactions. However, the mechanisms by which plants and nematodes compete with each other for assimilates have not been elucidated. In this study, we demonstrated that miR396a plays a negative role in defense against RKNs and a positive role in sugar accumulation in tomato roots. The overexpression of SlGRF8 (Solanum lycopersicum growth-regulating factor 8), the target of miR396a, decreased the sugar content of the roots and the susceptibility to RKNs, whereas the grf8-cr mutation had the opposite effects. Furthermore, we confirmed that SlGRF8 regulated the sugar content in roots by directly activating the transcription of SlSTP10 (Solanum lycopersicum sugar transporter protein 10) in response to RKN stress. Moreover, SlSTP10 was expressed primarily in the tissues surrounding giant cells, and the SlSTP10 knockout increased both the sugar content in the roots and the plant's susceptibility to RKNs. Overall, this study provides important insight into the molecular mechanism through which the miR396a-SlGRF8-SlSTP10 module regulates sugar allocation in roots under RKN stress.

[Non-targeted renal metabolomics reveals multi-target effects of Dahuang Zhechong Pills on renal aging in rats].

This study aims to observe the effects of the traditional Chinese medicine prescription Dahuang Zhechong Pills(DHZCP on renal aging and explore its potential multi-target effects. Rats were assigned into the normal, model, DHZCP, and vitamin E(VE)groups. Firstly, the rat model of D-galactose(D-gal)-induced renal aging was established. During the modeling period, the rats in the 4 groups were administrated with double distilled water, double distilled water, DHZCP suspension, and VE suspension, respectively,by gavage every day. On day 60 of intervention, the indicators of renal aging and injury in rats were measured, including the function,histopathological characteristics, senescence-associated ß-galactosidase( SA-ß-gal) staining, and expression levels of Klotho and proteins associated with cell cycle arrest and senescence-associated secretory phenotype(SASP) in the renal tissue. Moreover, nontargeted metabolomic analysis of the renal tissue was performed for the 4 groups of rats to screen out the potential biomarkers and metabolic pathways. Finally, the signaling pathways of key targets were preliminarily validated. The results showed that DHZCP and VE significantly improved the renal function, histopathological features of renal tubular/interstitial tissue, and degree of SA-ß-gal staining, up-regulated the expression level of Klotho, and down-regulated the expression levels of proteins associated with cell cycle arrest and SASP in the renal tissue of the aging rats. In addition, DHZCP and VE regulated the metabolites in the renal tissue of the aging rats. There were 21 common differential metabolites. Among them, 5 differential metabolites were significantly increased in the aging rats and recovered after DHZCP or VE treatment, and they were involved in the lipid metabolism and energy metabolism pathways. The areas under the curves of the groups in comparison varied within the range of 0. 88-1. DHZCP regulated multiple signaling pathways, such as the adenosine monophosphate-activated protein kinase(AMPK), cyclic guanosine monophosphate-protein kinase G( c GMP-PKG), cyclic adenylic acid( c AMP), phosphatidylinositol-3-kinase-protein kinase B( PI3K-Akt), mammalian target of rapamycin(mTOR), and autophagy signaling pathways. In addition, it affected the multiple metabolic pathways, such as renin secretion, longevity regulation pathway, diabetic cardiomyopathy, and niacin and nicotinamide metabolism. DHZCP and VE significantly up-regulated the expression level of the key proteins in the AMPK signaling pathway in the renal tissue of the aging rats. In all, DHZCP and VE could mitigate renal aging and injury. DHZCP exerted multi-target effects via multiple signaling pathways and metabolic pathways in the kidney, in which the AMPK signaling pathway may be one of the key targets for action.

Tomato defence against Meloidogyne incognita by jasmonic acid-mediated fine-tuning of kaempferol homeostasis.

Jasmonic acid (JA) is involved in the modulation of defence and growth activities in plants. The best-characterized growth-defence trade-offs stem from antagonistic crosstalk among hormones. In this study, we first confirmed that JA negatively regulates root-knot nematode (RKN) susceptibility via the root exudates (REs) of tomato plants. Omics and toxicological analyses implied that kaempferol, a type of flavonol, from REs has a negative effect on RKN infection. We demonstrated that SlMYB57 negatively regulated kaempferol contents in tomato roots, whereas SlMYB108/112 had the opposite effect. We revealed that JA fine-tuned the homeostasis of kaempferol via SlMYB-mediated transcriptional regulation and the interaction between SlJAZs and SlMYBs, thus ensuring a balance between lateral root (LR) development and RKN susceptibility. Overall, this work provides novel insights into JA-modulated LR development and RKN susceptibility mechanisms and elucidates a trade-off model mediated by JA in plants encountering stress.

SlVQ15 interacts with jasmonate-ZIM domain proteins and SlWRKY31 to regulate defense response in tomato.

Botrytis cinerea is one of the most widely distributed and harmful pathogens worldwide. Both the phytohormone jasmonate (JA) and the VQ motif-containing proteins play crucial roles in plant resistance to B. cinerea. However, their crosstalk in resistance to B. cinerea is unclear, especially in tomato (Solanum lycopersicum). In this study, we found that the tomato VQ15 was highly induced upon B. cinerea infection and localized in the nucleus. Silencing SlVQ15 using virus-induced gene silencing reduced resistance to B. cinerea. Overexpression of SlVQ15 enhanced resistance to B. cinerea, while disruption of SlVQ15 using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) technology increased susceptibility to B. cinerea. Furthermore, SlVQ15 formed homodimers. Additionally, SlVQ15 interacted with JA-ZIM domain proteins, repressors of the JA signaling pathway, and SlWRKY31. SlJAZ11 interfered with the interaction between SlVQ15 and SlWRKY31 and repressed the SlVQ15-increased transcriptional activation activity of SlWRKY31. SlVQ15 and SlWRKY31 synergistically regulated tomato resistance to B. cinerea, as silencing SlVQ15 enhanced the sensitivity of slwrky31 to B. cinerea. Taken together, our findings showed that the SlJAZ-interacting protein SlVQ15 physically interacts with SlWRKY31 to cooperatively control JA-mediated plant defense against B. cinerea.