Leucine-rich Domain Research Articles

Plasma Zinc Levels in Patients with Diabetic Nephropathy: Is there a Relationship with NLRP3 Inflammasome Activation and Renal Prognosis?

Zinc is an essential trace element, and impaired zinc homeostasis may be associated with inflammation in patients with diabetic nephropathy (DN). We investigated the influence of zinc level on nod-like receptor nucleotide-binding domain and leucine-rich repeat pyrin-3 domain (NLRP3) inflammasome expression and renal prognosis in patients with DN. We recruited 90 patients definitively diagnosed with DN by renal biopsy and 40 healthy controls. Zinc, NLRP3, interleukin (IL)-1β, and IL-18 levels were detected in blood samples, and the correlations between these parameters were assessed. Receiver operating characteristic (ROC) curve and decision curve analysis (DCA) evaluated the predictive value of zinc and the NLRP3 inflammasome for DN. Furthermore, patients with DN were divided into low- and normal-zinc groups to observe differences in clinical indicators and identify expression of inflammatory-related factors in renal tissue. Kaplan-Meier survival curves predicted the impact of zinc levels on renal prognosis. We found thatthe plasma zinc concentration in patients with DN was lower, while NLRP3, IL-1β, and IL-18 levels were higher than were those in patients without DN (P < 0.05). Zinc level was negatively correlated with NLRP3, IL-1β, and IL-18 levels (P < 0.01). Zinc and the NLRP3 inflammasome were predictive of DN, but their combination improved the diagnostic value. The DCA curve demonstrated a good positive net benefit in the combined model. Compared to patients with low zinc levels, patients with normal zinc levels had lower expression of NLRP3 inflammasome and a better prognosis. Zinc has a protective effect on DN and may affect NLRP3 inflammasome activation.

TSH Receptor Oligomers Associated With the TSH Receptor Antibody Reactome.

The TSH receptor (TSHR) and its many forms are the primary antigens of Graves' disease as evidenced by the presence of TSHR antibodies of differing biological activity. The TSH holoreceptor undergoes complex posttranslational changes including cleavage of its ectodomain and oligomer formation. We have previously shown that the TSHR exists in both monomeric and dimeric structures in the thyroid cell membrane and have demonstrated, by modeling, that the transmembrane domains (TMD) can form stable dimeric structures. Based on these earlier simulations of the TSHR-TMD structure and our most recent model of the full-length TSHR, we have now built models of full-length TSHR multimers with and without TSH ligand in addition to multimers of the extracellular leucine-rich domain, the site of TSH and autoantibody binding. Starting from these models we ran molecular dynamics simulations of the receptor oligomers solvated with water and counterions; the full-length oligomers also were embedded in a dipalmitoylphosphatidylcholine bilayer. The full-length TSHR dimer and trimer models stayed in the same relative orientation and distance during 2000 ns (or longer) molecular dynamics simulation in keeping with our earlier report of TMD dimerization. Simulations were also performed to model oligomers of the leucine-rich domain alone; we found a trimeric complex to be even more stable than the dimers. These data provide further evidence that different forms of the TSHR add to the complexity of the immune response to this antigen that, in patients with autoimmune thyroid disease, generate an autoantibody reactome with multiple types of autoantibody to the TSHR.

Arabidopsis pollen prolyl-hydroxylases P4H4/6 are relevant for correct hydroxylation and secretion of LRX11 in pollen tubes.

Major constituents of the plant cell walls are structural proteins that belong to the hydroxyproline-rich glycoprotein (HRGP) family. Leucine-rich repeat extensin (LRX) proteins contain a leucine-rich domain and a C-terminal domain with repetitive Ser-Pro3-5 motifs that are potentially to be O-glycosylated. It has been demonstrated that pollen-specific LRX8-LRX11 from Arabidopsis thaliana are necessary to maintain the integrity of the pollen tube cell wall during polarized growth. In HRGPs, including classical extensins (EXTs), and probably in LRXs, proline residues are converted to hydroxyproline by prolyl-4-hydroxylases (P4Hs), thus defining novel O-glycosylation sites. In this context, we aimed to determine whether hydroxylation and subsequent O-glycosylation of Arabidopsis pollen LRXs are necessary for their proper function and cell wall localization in pollen tubes. We hypothesized that pollen-expressed P4H4 and P4H6 catalyze the hydroxylation of the proline units present in Ser-Pro3-5 motifs of LRX8-LRX11. Here, we show that the p4h4-1 p4h6-1 double mutant exhibits a reduction in pollen germination rates and a slight reduction in pollen tube length. Pollen germination is also inhibited by P4H inhibitors, suggesting that prolyl hydroxylation is required for pollen tube development. Plants expressing pLRX11::LRX11-GFP in the p4h4-1 p4h6-1 background show partial re-localization of LRX11-green fluorescent protein (GFP) from the pollen tube tip apoplast to the cytoplasm. Finally, immunoprecipitation-tandem mass spectrometry analysis revealed a decrease in oxidized prolines (hydroxyprolines) in LRX11-GFP in the p4h4-1 p4h6-1 background compared with lrx11 plants expressing pLRX11::LRX11-GFP. Taken together, these results suggest that P4H4 and P4H6 are required for pollen germination and for proper hydroxylation of LRX11 necessary for its localization in the cell wall of pollen tubes.

Abstract LB210: Identification of histone deacetylase 10 as a repressor of cancer cell migration

Abstract Histone deacetylases (HDACs) are a family of 18 enzymes with the potential to be epigenetic modifiers. Of the 4 classes of HDACs, HDAC6 and HDAC10 belong to the class IIb subgroup. Unlike HDAC6, HDAC10 contains two deacetylase-like domains, with one active deacetylase domain and one inactive leucine-rich domain, and a preference for a specific acetylated polyamine over other substrates. Aberrant expression of HDAC10 has been implicated in cancer and other diseases. However, the deacetylation targets and biological functions of HDAC10 have not been well characterized due to a lack of specific and potent HDAC10 inhibitors with cellular activity. We previously established CRISPR/Cas9-mediated HDAC10 knockout (KO) cell lines in HCT116 and HeLa S3 cells. In the current study, we used the HCT116 wildtype and KO pair for bulk RNA sequencing, differential gene expression, and gene set enrichment analyses (GSEA), which revealed an upregulation of gene markers and pathways potentially associated with metastasis and invasion in the HDAC10-KO cells, including S100A14, LAMA4, and TGF-beta. As the role of HDAC10 expression on cell migration and invasion has not been previously reported, we performed cell migration assays using our HDAC10 parental and knockout cell line pairs. In both HCT116 and HeLa systems, HDAC10 KO cells exhibited dramatically higher rates of cell migration in Boyden chamber and scratch assays. These same assays were performed in HDAC10-expressing cells in the presence of the HDAC10-specific inhibitor DKFZ-728, which similarly caused a dose-dependent increase in migration, strongly supporting a catalytic role for HDAC10 in repressing migration. In addition to these results, we present data investigating the role of HDAC10 in cell migration/invasion in representative prostate cancer cell lines (LNCAP and LAPC4), as the prostate tumor microenvironment is particularly rich in polyamines, including N 8-acetylated spermidine, the substrate of HDAC10. Finally, using our panel of HDAC10-KO cell lines and inhibitors, we analyze the contribution of commonly perturbed genes to the migratory phenotype with the goal of defining the precise mechanism of action. Overall, our studies provide functional evidence that loss of HDAC10 can increase cell migration, and thus, may lead to increased metastatic potential. These results emphasize the need for isoform-specific inhibitors and may have important clinical implications, as commonly utilized HDAC inhibitors, including vorinostat (SAHA), tubastatin A, and trichostatin A (TSA), also inhibit HDAC10. Our ongoing studies include incorporating HDAC10 proteolysis targeting chimeras (PROTACs) into our functional assays to examine differences between HDAC10 inhibition and PROTAC-mediated degradation, providing further rationale for modulating HDAC10 activity as an antineoplastic strategy. Citation Format: Ting-Ann Liu, Ashley Nwafor, Rajendra Kumar, Laura Sena, Raphael R. Steimbach, Aubry K. Miller, Robert A. Casero, Tracy Murray Stewart. Identification of histone deacetylase 10 as a repressor of cancer cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB210.

NLRP3/1-mediated pyroptosis: beneficial clues for the development of novel therapies for Alzheimer's disease.

The inflammasome is a multiprotein complex involved in innate immunity that mediates the inflammatory response leading to pyroptosis, which is a lytic, inflammatory form of cell death. There is accumulating evidence that nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome-mediated microglial pyroptosis and NLRP1 inflammasome-mediated neuronal pyroptosis in the brain are closely associated with the pathogenesis of Alzheimer's disease. In this review, we summarize the possible pathogenic mechanisms of Alzheimer's disease, focusing on neuroinflammation. We also describe the structures of NLRP3 and NLRP1 and the role their activation plays in Alzheimer's disease. Finally, we examine the neuroprotective activity of small-molecule inhibitors, endogenous inhibitor proteins, microRNAs, and natural bioactive molecules that target NLRP3 and NLRP1, based on the rationale that inhibiting NLRP3 and NLRP1 inflammasome-mediated pyroptosis can be an effective therapeutic strategy for Alzheimer's disease.

Identification and characterization of Toll-like receptor 14d in Northeast Chinese lamprey (Lethenteron morii).

Toll-like receptors (TLRs) play an important role in innate immunity of defense against bacterial or viral pathogens. To study the biological characteristics and functions of the TLR genes, TLR14d was identified from Northeast Chinese lamprey (Lethenteron morii) and named LmTLR14d. LmTLR14d coding sequence (cds) is 3285 bp in length and encodes 1094 amino acids (aa). The results showed that LmTLR14d has the typical structure of TLR molecule, which contains the extracellular domain of leucine-rich repeats (LRR), transmembrane domain, and intracellular domain of Toll/interleukin-1 receptor (TIR). The phylogenetic tree showed that LmTLR14d is a homologous gene of TLR14/18 in bony fish. Quantitative real-time PCR (qPCR) revealed that LmTLR14d was expressed in various healthy tissues, including immune and non-immune tissues. Pseudomonas aeruginosa infection up-regulated LmTLR14d in the supraneural body (SB), gill, and kidney tissues of infected Northeast Chinese lamprey. Immunofluorescence results showed that LmTLR14d was located in the cytoplasm of HEK 293T cells in clusters, and its subcellular localization was determined by the TIR domain. The immunoprecipitation results showed that LmTLR14d could recruit L.morii MyD88 (LmMyD88) but not L.morii TRIF (LmTRIF). Dual luciferase reporter results showed that LmTLR14d significantly enhanced the activity of L.morii NF-κβ (LmNF-κβ) promoter. Furthermore, co-transfection of LmTLR14d with MyD88 significantly enhanced the L.morii NF-κβ (LmNF-κβ) promoter activity. LmTLR14d can induce the expression of inflammatory cytokine genes il-6 and tnf-α downstream of NF-κB signal. This study suggested that LmTLR14d might play an important role in the innate immune signal transduction process of lamprey and revealed the origin and function of teleost-specific TLR14.

TRIM72 Alleviates Muscle Inflammation in mdx Mice via Promoting Mitophagy-Mediated NLRP3 Inflammasome Inactivation.

Chronic muscle inflammation exacerbates the pathogenesis of Duchenne muscular dystrophy (DMD), which is characterized by progressive muscle degeneration and weakness. NLRP3 (nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3) inflammasome plays a key role in the inflammatory process, and its abnormal activation leads to a variety of inflammatory or immune diseases. TRIM72 (MG53) is a protective myokine for tissue repair and regeneration. However, little is known about the potential impact of TRIM72 in the crosstalk between mitophagy and inflammatory process of DMD. Here, 10-week-old male mdx mice were injected intramuscularly with adeno-associated virus (AAV-TRIM72) to overexpress TRIM72 protein for 6 weeks. Then, skeletal muscle samples were collected, and relevant parameters were measured by histopathological analysis and molecular biology techniques. C2C12 cell line was transfected with lentivirus (LV-TRIM72) to overexpress or siRNA (si-TRIM72) to suppress the TRIM72 expression for the following experiment. Our data firstly showed that the TRIM72 expression was decreased in skeletal muscles of mdx mice. Then, we observed the increased NLRP3 inflammasome and impaired mitophagy in mdx mice compared with wild type mice. In mdx mice, administration of AAV-TRIM72 alleviated the accumulation of NLRP3 inflammasome and the consequent IL-18 and IL1β maturation by inducing autophagy, while this protective effect was reversed by chloroquine. Mitochondrial reactive oxygen species (mtROS), as a recognized activator for NLRP3 inflammasome, was attenuated by TRIM72 through the induction of mitophagy in C2C12 cells. Additionally, we proposed that the TRIM72 overexpression might promote mitophagy through both the early stage by PI3K-AKT pathway and the late stage by autolysosome fusion. In conclusion, the current study suggests that TRIM72 prevents DMD inflammation via decreasing NLRP3 inflammasomes and enhancing mitophagy. Collectively, our study provides insight into TRIM72 as a promising target for therapeutic intervention for DMD.

Structure of full-length TSH receptor in complex with antibody K1-70™.

Determination of the full-length thyroid-stimulating hormone receptor (TSHR) structure by cryo-electron microscopy (cryo-EM) is described. The TSHR complexed with human monoclonal TSHR autoantibody K1-70™ (a powerful inhibitor of TSH action) was detergent solubilised, purified to homogeneity and analysed by cryo-EM. The structure (global resolution 3.3 Å) is a monomer with all three domains visible: leucine-rich domain (LRD), hinge region (HR) and transmembrane domain (TMD). The TSHR extracellular domain (ECD, composed of the LRD and HR) is positioned on top of the TMD extracellular surface. Extensive interactions between the TMD and ECD are observed in the structure, and their analysis provides an explanation of the effects of various TSHR mutations on TSHR constitutive activity and on ligand-induced activation. K1-70™ is seen to be well clear of the lipid bilayer. However, superimposition of M22™ (a human monoclonal TSHR autoantibody which is a powerful stimulator of the TSHR) on the cryo-EM structure shows that it would clash with the bilayer unless the TSHR HR rotates upwards as part of the M22™ binding process. This rotation could have an important role in TSHR stimulation by M22™ and as such provides an explanation as to why K1-70™ blocks the binding of TSH and M22™ without activating the receptor itself.

Identification of a Leafy Head Formation Related Gene in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Leafy head formation is one of the most important characteristics of Chinese cabbage, and the process is regulated by a series of genes and environmental factors. In this study, a non-heading short leaf mutant slm was identified from an ethyl methane sulfonate mutagenesis (EMS) population of the heading Chinese cabbage line FT. The most significant phenotypic characteristics of slm was shortening leaves and increasing leaf numbers, which led to failure to form a leafy head. Genetic analysis showed that a single recessive gene Brslm was responsible for the mutant phenotype. Mutmap analysis suggested that Brslm was located on chromosome A07, and four candidate genes were predicted. KASP analysis demonstrated that BraA07g039390.3C was the target gene of the candidates. BraA07g039390.3C is a homologous to Arabidopsis CLV1 encoding receptor kinase with an extracellular leucine-rich domain. Sequencing analysis revealed that a single SNP from G to A occurred in 904th nucleotide of Brclv1, which resulted in the change of the 302nd amino acid from Asp to Asn. The SNP was co-segregated with the mutant phenotype in F2 individuals and located on the conserved domains. These results indicated that BrCLV1 was the mutant gene for slm which led to shortening leaves and increasing leaf numbers, disrupting the leafy heading formation in FT. These findings contribute to revealing the BrCLV1 function in leafy head formation in Chinese cabbage.

Olfactory marker protein contains a leucine-rich domain in the Ω-loop important for nuclear export

Olfactory marker protein (OMP) is a cytosolic protein expressed in mature olfactory receptor neurons (ORNs). OMP modulates cAMP signalling and regulates olfactory sensation and axonal targeting. OMP is a small soluble protein, and passive diffusion between nucleus and cytoplasm is expected. However, OMP is mostly situated in the cytosol and is only sparsely detected in the nuclei of a subset of ORNs, hypothalamic neurons and heterologously OMP-expressing cultured cells. OMP can enter the nucleus in association with transcription factors. However, how OMP is retained in the cytosol at rest is unclear. Because OMP is proposed to affect cell differentiation, it is important to understand how OMP is distributed between cytoplasm and nucleus. To elucidate the structural profile of OMP, we applied several bioinformatics methods to a multiple sequence alignment (MSA) of OMP protein sequences and ranked the evolutionarily conserved residues. In addition to the previously reported cAMP-binding domain, we identified a leucine-rich domain in the Ω-loop of OMP. We introduced mutations into the leucine-rich region and heterologously expressed the mutant OMP in HEK293T cells. Mutations into alanine increased the nuclear distribution of OMP quantified by immunocytochemistry and western blotting. Therefore, we concluded that OMP contains a leucine-rich domain important for nuclear transport.

Computational model of the full-length TSH receptor.

(GPCR)The receptor for TSH receptor (TSHR), a G protein coupled receptor (GPCR), is of particular interest as the primary antigen in autoimmune hyperthyroidism (Graves' disease) caused by stimulating TSHR antibodies. To date, only one domain of the extracellular region of the TSHR has been crystallized. We have run a 1000 ns molecular dynamic simulation on a model of the entire TSHR generated by merging the extracellular region of the receptor, obtained using artificial intelligence, with our recent homology model of the transmembrane domain, embedded it in a lipid membrane and solvated it with water and counterions. The simulations showed that the structure of the transmembrane and leucine-rich domains were remarkably constant while the linker region (LR), known more commonly as the 'hinge region,' showed significant flexibility, forming several transient secondary structural elements. Furthermore, the relative orientation of the leucine-rich domain with the rest of the receptor was also seen to be variable. These data suggest that this LR is an intrinsically disordered protein. Furthermore, preliminary data simulating the full TSHR model complexed with its ligand (TSH) showed that (a) there is a strong affinity between the LR and TSH ligand and (b) the association of the LR and the TSH ligand reduces the structural fluctuations in the LR. This full-length model illustrates the importance of the LR in responding to ligand binding and lays the foundation for studies of pathologic TSHR autoantibodies complexed with the TSHR to give further insight into their interaction with the flexible LR.

Review: The evolution of peptidergic signaling in Cnidaria and Placozoa, including a comparison with Bilateria.

Bilateria have bilateral symmetry and are subdivided into Deuterostomia (animals like vertebrates) and Protostomia (animals like insects and mollusks). Neuropeptides occur in both Proto- and Deuterostomia and they are frequently structurally related across these two lineages. For example, peptides belonging to the oxytocin/vasopressin family exist in both clades. The same is true for the G protein-coupled receptors (GPCRs) of these peptides. These observations suggest that these neuropeptides and their GPCRs were already present in the common ancestor of Proto- and Deuterostomia, which lived about 700 million years ago (MYA). Furthermore, neuropeptides and their GPCRs occur in two early-branching phyla that diverged before the emergence of Bilateria: Cnidaria (animals like corals and sea anemones), and Placozoa (small disk-like animals, feeding on algae). The sequences of these neuropeptides and their GPCRs, however, are not closely related to those from Bilateria. In addition, cnidarian neuropeptides and their receptors are not closely related to those from Placozoa. We propose that the divergence times between Cnidaria, Placozoa, and Bilateria might be too long for recognizing sequence identities. Leucine-rich repeats-containing GPCRs (LGRs) are a special class of GPCRs that are characterized by a long N-terminus containing 10-20 leucine-rich domains, which are used for ligand binding. Among the ligands for LGRs are dimeric glycoprotein hormones, and insulin-like peptides, such as relaxin. LGRs have been found not only in Proto- and Deuterostomia, but also in early emerging phyla, such as Cnidaria and Placozoa. Humans have eight LGRs. In our current review, we have revisited the annotations of LGRs from the sea anemone Nematostella vectensis and the placozoan Trichoplax adhaerens. We identified 13 sea anemone LGRs and no less than 46 LGRs from T. adhaerens. All eight human LGRs appear to have orthologues in sea anemones and placozoans. LGRs and their ligands, therefore, have a long evolutionary history, going back to the common ancestor of Cnidaria and Placozoa.

Abstract P3119: Selectively Expressing Sars-Cov-2 Spike Protein S1 Subunit In Cardiomyocytes Induces Cardiac Hypertrophy In Mice

Cardiac injury is common in hospitalized COVID-19 patients and portends poorer prognosis and higher mortality. To better understand how SARS-CoV-2 (CoV-2) damages the heart, it is critical to elucidate the biology of CoV-2 encoded proteins, each of which may play multiple pathological roles. HCoV-NL63 is another human coronavirus with a Spike protein (NL63-S) that also engages ACE2 for virus entry but is known to only cause moderate respiratory symptoms. By comparing the pathological effects of NL63-S and CoV-2 spike protein (CoV-2-S), we tested the hypothesis that CoV-2-S damages the heart by activating cardiomyocyte (CM) innate immune responses, which is independent of ACE2.We found that CoV-2-S and not NL63-S interacted with Toll-like receptor 4 (TLR4), a crucial pattern recognition receptor responsible for detecting pathogen and initiating innate immune responses. Our data show that the S1 subunit of CoV-2-S (CoV-2-S1) interacts with the extracellular leucine-rich repeats-containing domain of TLR4 and activates NF-kB. To investigate the possible pathological role of CoV-2-S1 in the heart, we generated constructs that express membrane-localized CoV-2-S1 (S1-TM) or HCoV-NL63-S1 (NLS1-TM). AAV9-mediated, selective expression of the S1-TM and not NLS1-TM in CMs caused heart dysfunction, induced hypertrophic remodeling, and elicited cardiac inflammation. In vitro, overexpressing S1-TM in cultured neonatal rat ventricular CMs induced hypertrophy, decreased Myh6 and increased IL-6 . In the ventricular biopsy of a deceased patient with COVID-19 associated myocarditis, Spike protein and TLR4 were detected in both CMs and non-CMs, and these two proteins were absent in a healthy pre-pandemic human heart. In the ventricular biopsy of a patient who died of modified RNA vaccine-associated myocarditis, the expression of Spike protein was only detected in a small fraction of CMs, and no obvious TLR4 signal was detected in CMs. Because CoV-2-S does not interact with murine ACE2, our study presents a novel ACE2-independent pathological role of CoV-2-S. Additionally, our data suggest that modified RNA vaccine unlikely causes myocarditis by directly transfecting the CMs.

Leucine-Rich Repeats and Transmembrane Domain 2 Controls Protein Sorting in the Striatal Projection System and Its Deficiency Causes Disturbances in Motor Responses and Monoamine Dynamics.

The striatum is involved in action selection, and its disturbance can cause movement disorders. Here, we show that leucine-rich repeats and transmembrane domain 2 (Lrtm2) controls protein sorting in striatal projection systems, and its deficiency causes disturbances in monoamine dynamics and behavior. The Lrtm2 protein was broadly detected in the brain, but it was enhanced in the olfactory bulb and dorsal striatum. Immunostaining revealed a strong signal in striatal projection output, including GABAergic presynaptic boutons of the SNr. In subcellular fractionation, Lrtm2 was abundantly recovered in the synaptic plasma membrane fraction, synaptic vesicle fraction, and microsome fraction. Lrtm2 KO mice exhibited altered motor responses in both voluntary explorations and forced exercise. Dopamine metabolite content was decreased in the dorsal striatum and hypothalamus, and serotonin turnover increased in the dorsal striatum. The prefrontal cortex showed age-dependent changes in dopamine metabolites. The distribution of glutamate decarboxylase 67 (GAD67) protein and gamma-aminobutyric acid receptor type B receptor 1 (GABABR1) protein was altered in the dorsal striatum. In cultured neurons, wild-type Lrtm2 protein enhanced axon trafficking of GAD67-GFP and GABABR1-GFP whereas such activity was defective in sorting signal-abolished Lrtm2 mutant proteins. The topical expression of hemagglutinin-epitope-tag (HA)-Lrtm2 and a protein sorting signal abolished HA-Lrtm2 mutant differentially affected GABABR1 protein distribution in the dorsal striatum. These results suggest that Lrtm2 is an essential component of striatal projection neurons, contributing to a better understanding of striatal pathophysiology.

No NLRP3 Inflammasome Expression in the Ouabain Animal Model of Bipolar Disorder

IntroductionInflammation is believed to play a role in both bipolar illness and unipolar depression. Markers of inflammation are elevated during acute mood episodes. Specifically, gene expressions of the nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3 (NLRP3)-related proteins in peripheral blood have been purported to be upregulated in patients with bipolar disorder. We examined the elaboration of NLRP3 in the ouabain animal model of bipolar disorder. MethodsThe frontal cortex, hippocampus, and basal ganglia tissue from young, male Sprague-Dawley rats who received intracerebroventricular (ICV) ouabain as a model of bipolar disorder or artificial cerebrospinal fluid (aCSF) were examined for NLRP3 utilizing protein immunoblot (Western) analysis. ResultsWe could not demonstrate any NLRP3 in rat brain, but NLRP3 was detected in control from mouse brain and lung.DiscussionThis study demonstrates that the manifestation of manic behavior in rats treated with ICV ouabain is not accompanied by elaboration of NLRP3 inflammasome. This raises the question of the primacy of inflammation in the pathophysiology of mania. If these findings are reproduced in this and other animal models of mania, they would raise important questions about whether inflammation is a primary or secondary phenomenon in the brains of subjects with bipolar disorder.

Association Mapping of Lathyrus sativus Disease Response to Uromyces pisi Reveals Novel Loci Underlying Partial Resistance.

Uromyces pisi ([Pers.] D.C.) Wint. is an important foliar biotrophic pathogen infecting grass pea (Lathyrus sativus L.), compromising their yield stability. To date, few efforts have been made to assess the natural variation in grass pea resistance and to identify the resistance loci operating against this pathogen, limiting its efficient breeding exploitation. To overcome this knowledge gap, the genetic architecture of grass pea resistance to U. pisi was investigated using a worldwide collection of 182 accessions through a genome-wide association approach. The response of the grass pea collection to rust infection under controlled conditions and at the seedling stage did not reveal any hypersensitive response but a continuous variation for disease severity, with the identification of promising sources of partial resistance. A panel of 5,651 high-quality single-nucleotide polymorphism (SNP) markers previously generated was used to test for SNP-trait associations, based on a mixed linear model accounting for population structure. We detected seven SNP markers significantly associated with U. pisi disease severity, suggesting that partial resistance is oligogenic. Six of the associated SNP markers were located in chromosomes 4 and 6, while the remaining SNP markers had no known chromosomal position. Through comparative mapping with the pea reference genome, a total of 19 candidate genes were proposed, encoding for leucine-rich repeat, NB-ARC domain, and TGA transcription factor family, among others. Results presented in this study provided information on the availability of partial resistance in grass pea germplasm and advanced our understanding of the molecular mechanisms of quantitative resistance to rust in grass pea. Moreover, the detected associated SNP markers constitute promising genomic targets for the development of molecular tools to assist disease resistance precision breeding.

Another One Fights the Dust: Targeting the NLRP3 Inflammasome for the Treatment of Silicosis.

Silicosis is a multifaceted lung disease, characterized by persistent inflammation and structural remodeling. Despite its poor prognosis, there are no treatments currently available for patients with silicosis. Recent preclinical findings in models of lung fibrosis have suggested a major role for the NLRP3 (nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3) inflammasome in silica-driven inflammation and fibrosis. This review outlines the beneficial effects of targeting the NLRP3 inflammasome in invitro cell experiments and in invivo animal models, whereby inflammation and fibrosis are abrogated after NLRP3 inflammasome inhibition. Although preclinical evidence is promising, studies that explore NLRP3 inflammasomes in the clinical setting are warranted. In particular, there is still a need to identify biomarkers that may be helpful for the early detection of silicosis and to fully elucidate mechanisms underlying these beneficial effects to further develop or repurpose existing anti-NLRP3 drugs as novel treatments that limit disease progression.

Prussian Blue Nanozyme as a Pyroptosis Inhibitor Alleviates Neurodegeneration.

Current pharmacological interventions for Parkinson's disease (PD) remain unsatisfactory in clinical settings. Inflammasome-mediated pyroptosis represents a potential therapeutic target for the alleviation of neurodegenerative diseases. The development of inflammasome-mediated pyroptosis agonists or antagonists may transform the treatment of neurodegenerative diseases. However, the identification of specific compounds that inhibit pyroptosis remains challenging. Herein, Prussian blue nanozyme (PBzyme) is revealed as a pyroptosis inhibitor to alleviate the neurodegeneration in mouse and cell models of PD. PBzyme protects the microglia and neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). PBzyme alleviates motor deficits, attenuates the damage of mitochondrial membrane potential, and rescues dopaminergic neurons. Furthermore, intra-cerebroventricular injection of PBzyme reduces dopaminergic degeneration and inhibits neuroinflammation in an MPTP-induced PD mouse model. Both in vitro and in vivo results demonstrate that PBzyme reduces the activation of microglial nucleotide-binding domain and leucine-rich repeat family pyrin domain containing 3 (NLRP3) inflammasomes and caspase-1 by scavenging reactive oxygen species, thereby downregulating gasdermin D (GSDMD) cleavage as well as inflammatory factor production, and eventually leading to the inhibition of microglia pyroptosis. Overall, this work highlights the neuroprotective effects of PBzyme as a pyroptosis inhibitor and provides valuable mechanistic insights and a potential therapeutic strategy for the treatment of PD.

Application of Synthetic Peptide CEP1 Increases Nutrient Uptake Rates Along Plant Roots.

The root system of a plant provides vital functions including resource uptake, storage, and anchorage in soil. The uptake of macro-nutrients like nitrogen (N), phosphorus (P), potassium (K), and sulphur (S) from the soil is critical for plant growth and development. Small signaling peptide (SSP) hormones are best known as potent regulators of plant growth and development with a few also known to have specialized roles in macronutrient utilization. Here we describe a high throughput phenotyping platform for testing SSP effects on root uptake of multiple nutrients. The SSP, CEP1 (C-TERMINALLY ENCODED PEPTIDE) enhanced nitrate uptake rate per unit root length in Medicago truncatula plants deprived of N in the high-affinity transport range. Single structural variants of M. truncatula and Arabidopsis thaliana specific CEP1 peptides, MtCEP1D1:hyp4,11 and AtCEP1:hyp4,11, enhanced uptake not only of nitrate, but also phosphate and sulfate in both model plant species. Transcriptome analysis of Medicago roots treated with different MtCEP1 encoded peptide domains revealed that hundreds of genes respond to these peptides, including several nitrate transporters and a sulfate transporter that may mediate the uptake of these macronutrients downstream of CEP1 signaling. Likewise, several putative signaling pathway genes including LEUCINE-RICH REPEAT RECPTOR-LIKE KINASES and Myb domain containing transcription factors, were induced in roots by CEP1 treatment. Thus, a scalable method has been developed for screening synthetic peptides of potential use in agriculture, with CEP1 shown to be one such peptide.

Characterization of P2X7 Receptors in Human Blood Cells.

P2X7 receptors are expressed in different circulating immune cells. Depending on the cell in which they are expressed, their function could vary from activation of the leucine-rich repeat receptor pyrin domain containing 3 (NLRP3) inflammasome or the shedding of different surface receptors. The P2X7 receptor has been involved in the evolution of several inflammatory diseases, and thus its characterization in blood samples is important to investigate its role in human diseases. In this chapter, we present different methods to detect the presence and the activation of P2X7 receptors in whole blood samples. These methods could be used to evaluate the degree of P2X7 receptor activation in blood samples from patients suffering different chronic inflammatory diseases.