Leucine-rich Repeat Research Articles

An LRR-RLK protein modulates drought- and salt-stress responses in maize

Maize (Zea mays), which is a vital source of food, feed, and energy feedstock globally, has significant potential for higher yields. However, environmental stress conditions, including drought and salt stress, severely restrict maize plant growth and development, leading to great yield losses. Leucine-rich repeat receptor-like kinases (LRR-RLKs) function in biotic and abiotic stress responses in the model plant Arabidopsis (Arabidopsis thaliana), but their roles in abiotic stress responses in maize are not entirely understood. In this study, we determine that the LRR-RLK ZmMIK2, a homolog of the Arabidopsis LRR-RK MALE DISCOVERER 1 (MDIS1)- INTERACTING RECEPTOR LIKE KINASE 2 (MIK2), functions in resistance to both drought and salt stress in maize. Zmmik2 plants exhibit enhanced resistance to both stresses, whereas overexpressing ZmMIK2 confers the opposite phenotypes. Furthermore, we identify C2-DOMAIN-CONTAINING PROTEIN 1 (ZmC2DP1), which interacts with the intracellular region of ZmMIK2. Notably, that region of ZmMIK2 mediates the phosphorylation of ZmC2DP1, likely by increasing its stability. Both ZmMIK2 and ZmC2DP1 are mainly expressed in roots. As with ZmMIK2, knockout of ZmC2DP1 enhanced resistance to both drought and salt stress. We conclude that ZmMIK2–ZmC2DP1 act as a negative regulatory module in maize drought- and salt-stress responses.

Recognition of a salivary effector by the TNL protein RCSP promotes effector-triggered immunity and systemic resistance in Nicotiana benthamiana.

Insects secret chemosensory proteins (CSPs) into plant cells as potential effector proteins during feeding. The molecular mechanisms underlying how CSPs activate plant immunity remain largely unknown. We show that CSPs from six distinct insect orders induce dwarfism when overexpressed in Nicotiana benthamiana. Agrobacterium-mediated transient expression of Nilaparvata lugens CSP11 (NlCSP11) triggered cell death and plant dwarfism, both of which were dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), neuregulin 1 (NRG1) and SENESCENCE-ASSOCIATED GENE 101 (SAG101), indicating the activation of effector-triggered immunity (ETI) in N. benthamiana. Overexpression of NlCSP11 led to stronger systemic resistance against Pseudomonas syringae DC3000 lacking effector HopQ1-1 and tobacco mosaic virus, and induced higher accumulation of salicylic acid (SA) in uninfiltrated leaves compared to another effector XopQ that is recognized by a Toll-interleukin-1 receptor (TIR) domain nucleotide-binding leucine-rich repeat receptor (TNL) called ROQ1 in N. benthamiana. Consistently, NlCSP11-induced dwarfism and systemic resistance, but not cell death, were abolished in N. benthamiana transgenic line expressing the SA-degrading enzyme NahG. Through large-scale virus-induced gene silencing screening, we identified a TNL protein that mediates the recognition of CSPs (RCSP), including aphid effector MP10 that triggers resistance against aphids in N. benthamiana. Co-immunoprecipitation, bimolecular fluorescence complementation and AlphaFold2 prediction unveiled an interaction between NlCSP11 and RCSP. Interestingly, RCSP does not contain the conserved catalytic glutamic acid in the TIR domain, which is required for TNL function. Our findings point to enhanced ETI and systemic resistance by a TNL protein via hyperactivation of the SA pathway. Moreover, RCSP is the first TNL identified to recognize an insect effector.

CK and LRRK2 Involvement in Neurodegenerative Diseases.

Neurodegenerative diseases (NDDs) are currently the most widespread neuronal pathologies in the world. Among these, the most widespread are Alzheimer's disease (AD), dementia, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD)-all characterized by a progressive loss of neurons in specific regions of the brain leading to varied clinical symptoms. At the basis of neurodegenerative diseases, an emerging role is played by genetic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene that cause increased LRRK2 activity with consequent alteration of neuronal autophagy pathways. LRRK2 kinase activity requires GTPase activity which functions independently of kinase activity and is required for neurotoxicity and to potentiate neuronal death. Important in the neurodegeneration process is the upregulation of casein kinase (CK), which causes the alteration of the AMPK pathway by enhancing the phosphorylation of α-synuclein and huntingtin proteins, known to be involved in PD and HD, and increasing the accumulation of the amyloid-β protein (Aβ) for AD. Recent research has identified CK of the kinases upstream of LRRK2 as a regulator of the stability of the LRRK2 protein. Based on this evidence, this review aims to understand the direct involvement of individual kinases in NDDs and how their crosstalk may impact the pathogenesis and early onset of neurodegenerative diseases.

VRAC Complex Modulates Mechano-Electrical Signal Responses in Human Airway Smooth Muscle Shortening.

Leucine-rich repeat containing 8A (LRRC8A) is an obligatory constituent of the volume-regulated anion channel (VRAC) that is fundamental to a wide range of biological processes, including regulating cell size, proliferation, and migration. Here we explored the physiological role for VRAC in excitation-contraction (E-C) coupling and shortening of human airway smooth muscle (HASM). In HASM cells, pharmacological inhibition of VRAC with DCPIB (0.1-10 μM) markedly attenuated swell-activated Cl- conductance and contractile agonist (histamine or carbachol)-induced cellular stiffening as measured by single-cell patch clamp and optical magnetic twisting cytometry, respectively. In addition, HASM cells treated with DCPIB or transfected with LRRC8A-targeting siRNA showed reduced agonist-induced phosphorylation of protein kinase B (AKT), paxillin, myosin phosphatase target subunit 1 (MYPT1), and myosin light chain (MLC). Consistent with the changes of these E-C coupling effectors, DCPIB appreciably decreased agonist-induced small airways narrowing in human precision-cut lung slices (hPCLS). Taken together, our findings shed a new light on the mechanistic link between HASM shortening and regulatory volume decrease via LRRC8A, revealing a previously unrecognized nodal point for modulation of the E-C coupling and acute airways constriction.

Novel autoantibody targets identified in patients with autoimmune hepatitis (AIH) by PhIP-Seq reveals pathogenic insights.

Autoimmune hepatitis (AIH) is a severe disease characterized by elevated immunoglobin levels. However, the role of autoantibodies in the pathophysiology of AIH remains uncertain. Phage Immunoprecipitation-Sequencing (PhIP-seq) was employed to identify autoantibodies in the serum of patients with AIH (n = 115), compared to patients with other liver diseases (metabolic associated steatotic liver disease (MASH) n = 178, primary biliary cholangitis (PBC), n = 26, or healthy controls, n = 94). Logistic regression using PhIP-seq enriched peptides as inputs yielded a classification AUC of 0.81, indicating the presence of a predictive humoral immune signature for AIH. Embedded within this signature were disease relevant targets, including SLA/LP, the target of a well-recognized autoantibody in AIH, disco interacting protein 2 homolog A (DIP2A), and the relaxin family peptide receptor 1 (RXFP1). The autoreactive fragment of DIP2A was a 9-amino acid stretch nearly identical to the U27 protein of human herpes virus 6 (HHV-6). Fine mapping of this epitope suggests the HHV-6 U27 sequence is preferentially enriched relative to the corresponding DIP2A sequence. Antibodies against RXFP1, a receptor involved in anti-fibrotic signaling, were also highly specific to AIH. The enriched peptides are within a motif adjacent to the receptor binding domain, required for signaling and serum from AIH patients positive for anti-RFXP1 antibody was able to significantly inhibit relaxin-2 singling. Depletion of IgG from anti-RXFP1 positive serum abrogated this effect. These data provide evidence for a novel serological profile in AIH, including a possible functional role for anti-RXFP1, and antibodies that cross react with HHV6 U27 protein.

Identification of the HbZAR1 Gene and Its Potential Role as a Minor Gene in Response to Powdery Mildew and Anthracnose of Hevea brasiliensis

Powdery mildew and anthracnose are the main diseases of rubber trees. In recent years, there have been large outbreaks in the rubber-planting areas of Asia, seriously affecting the yield and quality of rubber latex. ZAR1 is a conserved and distinctive coiled-coil nucleotide-binding leucine-rich (CNL) repeat in the plant kingdom, playing a crucial role in disease-resistance processes. To elucidate the function of the HbZAR1 gene in rubber trees (Hevea brasiliensis), three candidate HbZAR1 genes were identified using bioinformatics methods and comprehensively analyzed. The results indicate that the HbZAR1 protein is conserved in different plant species. Examination of cis-regulatory element sequences of HbZAR1genes reveals that the HbZAR1 gene promoter exhibits a remarkable enrichment of stress, light, and hormone elements. An expression analysis shows that the expression levels of the three HbZAR1 genes are highest in the bark and lowest in latex. Three HbZAR1 genes can respond to both rubber tree Erysiphe quercicola and Colletotrichum siamense infection; especially, HbZAR1.1 and HbZAR1.2 show significant upregulation in expression levels during the early stages of infection. These findings suggest that the three HbZAR1 genes may be involved in rubber tree susceptibility to E. quercicola and C. siamense through different immune mechanisms. Subcellular localization results indicate that the HbZAR1 genes are expressed in the nucleus and plasma membrane. This study also shows that the three HbZAR1 genes and activated mutant HbZAR1.1D481V do not induce stable ROS production and cell death, suggesting possible gene degradation, functional redundancy, or acting as minor genes in disease resistance. This research provides valuable insights for further studying the function of HbZAR1 genes in rubber trees and the mechanisms of immune molecules.

Evaluation of Osteogenic Phenotype in Postmenopausal Women Receiving Anabolic and Antiresorptive Osteoporosis Therapies.

Aging of the general population has led to a substantial increase in the prevalence of osteoporosis over the past decades. While there are effective pharmacological agents that increase bone formation, decrease bone resorption, and decrease fracture risk, they do not uniformly cure osteoporosis. This has prompted investigations to examine whether combination therapy (COMBO) with these agents can result in an additive benefit. Since concomitant therapy with denosumab and teriparatide has shown promise in this respect, investigations were undertaken to explore whether the changes in osteogenic phenotype could provide insight into the cellular and molecular mechanism of this effect. Investigations were performed in postmenopausal women receiving denosumab, teriparatide, or both for 3 months. Histomorphometric parameters were the primary outcome, while exploratory studies examined RNA expression in bone biopsies as well as in sorted and cultured bone marrow stromal cells (BMSCs). Osteogenic colony forming units of BMSCs were also evaluated. The studies demonstrated that COMBO results in an increase in osteoprogenitors, evidenced by an increase in osteoblastic colony-forming units. This was associated with an increased in BMSC expression of LGR6 (leucine-rich repeat containing G protein-coupled receptor 6), a stem cell marker and activator of the canonical Wnt signaling pathway. These data suggest that enhancement of canonical Wnt signaling contributes to the increase in osteoprogenitors and consequently an increase in bone density in postmenopausal women receiving COMBO for osteoporosis.

A novel LRR receptor-like kinase BRAK reciprocally phosphorylates PSKR1 to enhance growth and defense in tomato

Plants face constant threats from pathogens, leading to growth retardation and crop failure. Cell-surface leucine-rich repeat receptor-like kinases (LRR-RLKs) are crucial for plant growth and defense, but their specific functions, especially to necrotrophic fungal pathogens, are largely unknown. Here, we identified an LRR-RLK (Solyc06g069650) in tomato (Solanum lycopersicum) induced by the economically important necrotrophic pathogen Botrytis cinerea. Knocking out this LRR-RLK reduced plant growth and increased sensitivity to B. cinerea, while its overexpression led to enhanced growth, yield, and resistance. We named this LRR-RLK as BRAK (B. cinerea resistance-associated kinase). Yeast two-hybrid screen revealed BRAK interacted with phytosulfokine (PSK) receptor PSKR1. PSK-induced growth and defense responses were impaired in pskr1, brak single and double mutants, as well as in PSKR1-overexpressing plants with silenced BRAK. Moreover, BRAK and PSKR1 phosphorylated each other, promoting their interaction as detected by microscale thermophoresis. This reciprocal phosphorylation was crucial for growth and resistance. In summary, we identified BRAK as a novel regulator of seedling growth, fruit yield and defense, offering new possibilities for developing fungal disease-tolerant plants without compromising yield.

Soluble form of Lingo2, an autism spectrum disorder-associated molecule, functions as an excitatory synapse organizer in neurons

Autism Spectrum Disorder (ASD) is a developmental disorder characterized by impaired social communication and repetitive behaviors. In recent years, a pharmacological mouse model of ASD involving maternal administration of valproic acid (VPA) has become widely used. Newborn pups in this model show an abnormal balance between excitatory and inhibitory (E/I) signaling in neurons and exhibit ASD-like behavior. However, the molecular basis of this model and its implications for the pathogenesis of ASD in humans remain unknown. Using quantitative secretome analysis, we found that the level of leucine-rich repeat and immunoglobulin domain-containing protein 2 (Lingo2) was upregulated in the conditioned medium of VPA model neurons. This upregulation was associated with excitatory synaptic organizer activity. The secreted form of the extracellular domain of Lingo2 (sLingo2) is produced by the transmembrane metalloprotease ADAM10 through proteolytic processing. sLingo2 was found to induce the formation of excitatory synapses in both mouse and human neurons, and treatment with sLingo2 resulted in an increased frequency of miniature excitatory postsynaptic currents in human neurons. These findings suggest that sLingo2 is an excitatory synapse organizer involved in ASD, and further understanding of the mechanisms by which sLingo2 induces excitatory synaptogenesis is expected to advance our understanding of the pathogenesis of ASD.

Thiazolidine-4-one Analogues: Synthesis, In-Silico Molecular Modeling, and In-vivo Estimation for Anticonvulsant Potential.

Epilepsy is a critically deep-rooted CNS disorder affecting above 50 million people all over the world. Thus, a safe and effective treatment that proves its worth in this ailment is urgently needed. Thiazolidine-4-ones possess the molecules to be used as anticonvulsants. The thiazolidinedione is a cyclic analogue of thiosemicarbazides and thioureas as well as a (bio)isostere of hydantoin (imidazolidine-2,4-dione), which are recognized as novel anticonvulsant designs. This study aimed to develop and evaluate a novel thiazolidine-4-one derivative by three-component condensation in one pot reaction method. A novel thiazolidine-4-one derivative was formulated by three-component condensation. The selected OH (Alcohol) derivatives were found to be more potent; hence, a molecular docking study against a selected target LGI1 LRR domain was performed. Various analytical tests like FTIR and H1 NMR were accomplished. The FTIR was used to validate the existence of multiple functional moieties like C-S, O-H, C=O, C-N, N=O, C-NH, C-O in the wave region from 3075 cm-1 - 1236 cm-1 and H1 NMR was employed to ascertain if the synthesized analogues had the complete set of protons. Then, the anti-seizure activity of the selected compound was examined using PTZ models in mice at three successive doses, i.e., 25, 50, and 100mg/kg, and compared with standard ethosuximide. The docking simulations were initiated using PyMOL after the binding site was determined and the receptor and ligand were suitably prepared. It showed higher binding frequency in comparison to the standard marketed drug Ethosuximide. FTIR and H1 NMR spectroscopy were used to characterize the chemical components. Numerous functional groups, including O-H (alcohol), C=O (ketones), N=O, C-NH, C-N, C-S, and C-O bending stretching, were visible in the synthesized molecule accordingly. The synthesized compound was effective in inhibiting the convulsions at the concentration of 100 mg/kg. The novel thiazolidine-4-one derivative showed promising activity and could be considered for further investigation and dosage form preparation.

The leucine-rich repeat receptor kinase QSK1 regulates PRR-RBOHD complexes targeted by the bacterial effector HopF2Pto.

Plants detect pathogens using cell-surface pattern recognition receptors (PRRs) such as ELONGATION Factor-TU (EF-TU) RECEPTOR (EFR) and FLAGELLIN SENSING 2 (FLS2), which recognize bacterial EF-Tu and flagellin, respectively. These PRRs belong to the leucine-rich repeat receptor kinase (LRR-RK) family and activate the production of reactive oxygen species via the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD). The PRR-RBOHD complex is tightly regulated to prevent unwarranted or exaggerated immune responses. However, certain pathogen effectors can subvert these regulatory mechanisms, thereby suppressing plant immunity. To elucidate the intricate dynamics of the PRR-RBOHD complex, we conducted a comparative coimmunoprecipitation analysis using EFR, FLS2, and RBOHD in Arabidopsis thaliana. We identified QIAN SHOU KINASE 1 (QSK1), an LRR-RK, as a PRR-RBOHD complex-associated protein. QSK1 downregulated FLS2 and EFR abundance, functioning as a negative regulator of PRR-triggered immunity (PTI). QSK1 was targeted by the bacterial effector HopF2Pto, a mono-ADP ribosyltransferase, reducing FLS2 and EFR levels through both transcriptional and transcription-independent pathways, thereby inhibiting PTI. Furthermore, HopF2Pto transcriptionally downregulated PROSCOOP genes encoding important stress-regulated phytocytokines and their receptor MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2. Importantly, HopF2Pto requires QSK1 for its accumulation and virulence functions within plants. In summary, our results provide insights into the mechanism by which HopF2Pto employs QSK1 to desensitize plants to pathogen attack.

The CEP peptide-CRA2 receptor module promotes arbuscular mycorrhizal symbiosis

C-terminally encoded peptides (CEPs) are small secreted signaling peptides that promote nitrogen-fixing root nodulation symbiosis in legumes, depending on soil mineral nitrogen availability.1 In Medicago truncatula, their action is mediated by the leucine-rich repeat receptor-like protein kinase COMPACT ROOT ARCHITECTURE 2 (CRA2).2,3,4 Like most land plants, under inorganic phosphate limitation, M.truncatula establishes another root endosymbiotic interaction with arbuscular fungi, the arbuscular mycorrhizal symbiosis (AMS). Because this interaction is beneficial for the plant but has a high energetic cost, it is tightly controlled by host plants to limit fungal infections mainly depending on phosphate availability.5 We show in this study that the expression of a subset of CEP-encoding genes is enhanced in the low-phosphate conditions and that overexpression of the low-phosphate-induced MtCEP1 gene, previously shown to promote the nitrogen-fixing root nodulation symbiosis, enhances AMS from the initial entry point of the fungi. Conversely, a loss-of-function mutation of the CRA2 receptor required for mediating CEP peptide action2 decreases the endomycorrhizal interaction from the same initial fungal entry stage. Transcriptomic analyses revealed that the cra2 mutant is negatively affected in the regulation of key phosphate transport and response genes as well as in the biosynthesis of strigolactone hormones that are required for establishing AMS. Accordingly, strigolactone contents were drastically decreased in cra2 mutant roots. Overall, we showed that the CEP/CRA2 pathway promotes both root nodulation and AMS in legume plants, depending on soil mineral nutrient availability.

Fine mapping and identification of the downy mildew resistance gene BoDMR2 in Cabbage (Brassica oleracea L. var. capitata).

Cabbage (Brassica oleracea L. var. capitata) is an important crop within the Brassica oleracea species and is extensively cultivated worldwide. In recent years, outbreaks of downy mildew caused by Hyaloperonospora parasitica have resulted in substantial losses in cabbage production. Despite this, there have been limited studies on genes associated with resistance to downy mildew in cabbage. This study identified sister lines exhibiting significant differences in disease resistance and susceptibility. Using bulked segregant analysis followed by sequencing (BSA-seq) and linkage analysis, the cabbage resistance locus BoDMR2 was accurately mapped to an approximately 300kb interval on chromosome 7. Among the candidate genes identified, several single nucleotide polymorphisms (SNPs) and a 3-bp insertion were found within the conserved domain of the Bo7g117810 gene, encoding a leucine-rich repeat domain protein, in susceptible genotypes. Additionally, real-time quantitative polymerase chain reaction (RT‒qPCR) analysis revealed that the expression level of Bo7g117810 in resistant specimens was 2.5-fold higher than that in susceptible specimens. An insertion‒deletion (InDel) marker was designed based on the identified insertion in susceptible materials, facilitating the identification and selection of downy mildew-resistant cabbage cultivars. This study identifies Bo7g117810 as a potential candidate gene associated with adult-stage resistance to downy mildew in cabbage, supported by observed differences in gene sequence and expression levels. Furthermore, the development of an InDel marker I1-3, based on its mutation, provides valuable resources for breeding resistant cabbage cultivars.

High-intensity interval training and moderate-intensity continuous training alleviate vascular dysfunction in spontaneously hypertensive rats through the inhibition of pyroptosis

Evidence-based guidelines suggest that High-Intensity Interval Training (HIIT) is more beneficial than aerobic exercise for patients with cardiovascular disease, but the differences in underlying pathophysiological mechanisms require further confirmation. The comparison between HIIT and Moderate-Intensity Continuous Training (MICT) in regulating vascular dysfunction in spontaneously hypertensive rats (SHR), along with their underlying mechanisms, has not been previously reported. The purpose of this study is to provide an experimental basis for exercise prescription therapy in hypertensive patients. In this study, six-week-old male SHR were randomly assigned to a HIIT group, MICT group, or sedentary group. Wistar Kyoto rats (WKY) of the same age were used as the control group. The weight, heart rate, and blood pressure of the rats were monitored weekly throughout twelve weeks of treadmill training. At the end of the protocol, serum and aortic vascular tissues were collected for further vascular function tests and molecular and biochemical analyses. The results show that MICT is more favorable for weight control than HIIT, while both forms of exercise offer equal protection against hypertension. However, MICT demonstrates a greater benefit in preserving vascular morphology, whereas HIIT is more effective in reducing oxidative stress. Both HIIT and MICT ameliorate vascular dysfunction in SHR by suppressing nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 3 (NLRP3)-induced pyroptosis. The superior effect of HIIT on vascular dysfunction may be related to the inhibition of oxidative stress injury through AMPKα-SIRT1 activation. This study provides insight into the dose-effect relationship of exercise for cardiovascular health and offers foundational evidence for the development of exercise prescription therapies.

Activation of plant immunity through conversion of a helper NLR homodimer into a resistosome.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins can engage in complex interactions to detect pathogens and execute a robust immune response via downstream helper NLRs. However, the biochemical mechanisms of helper NLR activation by upstream sensor NLRs remain poorly understood. Here, we show that the coiled-coil helper NLR NRC2 from Nicotiana benthamiana accumulates in vivo as a homodimer that converts into a higher-order oligomer upon activation by its upstream virus disease resistance protein Rx. The cryo-EM structure of NbNRC2 in its resting state revealed intermolecular interactions that mediate homodimer formation and contribute to immune receptor autoinhibition. These dimerization interfaces have diverged between paralogous NRC proteins to insulate critical network nodes and enable redundant immune pathways, possibly to minimise undesired cross-activation and evade pathogen suppression of immunity. Our results expand the molecular mechanisms of NLR activation pointing to transition from homodimers to higher-order oligomeric resistosomes.

Effect of Heat Stress on the Expression of Circulating Cyto(chemo)kine and Inflammatory Markers in Broiler Chickens Selected for High- or Low-water Efficiency.

Water scarcity is a current, significant global concern that will only increase under the pressure of climate change. Improving water efficiency of poultry is a new and promising area to help temper agriculture's future impact on fresh water availability. Here, we explored the effects of acute heat stress (HS) on circulating stress and inflammatory markers in 2 lines of broilers divergently selected for water efficiency. Male chicks from low (LWE) and high water efficient (HWE) lines were raised in 12 environmental chambers (2 pens/chamber, 6 chambers/line, 20 birds/pen) under normal conditions until day 28. On day 29, birds were subjected to thermoneutral (TN, 25 °C) or HS (36 °C) conditions, resulting in four treatments (2 lines × 2 environmental conditions). After 3 h of HS, whole blood was collected (8 birds per line × environment) and analyzed for target gene expression and plasma cytokine levels. Data were analyzed by 2-way ANOVA, with line, environment, and their interaction as main factors, and means were compared using Tukey's multiple range test. Gene expression of heat shock protein (HSP) 27, HSP70, interleukin (IL)-6, IL-18, c-reactive protein (CRP), tumor necrosis factor-α (TNFα), C-C motif chemokine ligand 4 (CCL4), CCL20, nucleotide-binding domain, leucine-rich repeat (NLR) family pyrin domain containing 3 (NLRP3), NLR family CARD domain containing 5 (NLRC5), and NLR family member X1 (NLRX1) were increased by HS, with no differences between the lines. HSP70, IL-10, and NLRC3 were lower in the HWE as compared to the LWE lines. Additionally, there were interactive effects between line and environment for HSP90, IL-4, and CCL4, where HS induced HSP90 expression in the LWE only, and IL-4 and CCL4 in HWE only. Arginine vasopressin (AVP) gene expression was significantly lower in the whole blood of the HWE line; however, plasma protein levels were not different. Overall, most of the effects seen on cyto (chemokines) and inflammatory markers were due to acute HS, with only a few genes differentially regulated between the lines. This likely indicates that the divergent selection for water efficiency for four generations did not elicit changes in inflammation and stress molecular signatures.

LGR6 protects against myocardial ischemia-reperfusion injury via suppressing necroptosis

Regulated necrosis (necroptosis) and apoptosis are important biological features of ischemia-reperfusion (I/R) injury. However, the molecular mechanisms underlying myocardial necroptosis remain elusive. Leucine rich repeat containing G protein-coupled receptor 6 (LGR6) has been reported to play important roles in various cardiovascular disease. In this study, we aimed to determine whether LGR6 suppresses I/R-induced myocardial necroptosis and the underlying molecular mechanisms. We generated LGR6 knockout mice and used ligation of left anterior descending coronary artery to produce an in vivo I/R model. The effects of LGR6 and its downstream molecules were subsequently identified using RNA sequencing and CHIP assays. We observed significantly downregulated LGR6 expression in hearts post myocardial I/R and cardiomyocytes post hypoxia and reoxygenation (HR). LGR6 deficiency promoted and LGR6 overexpression inhibited necroptosis and acute myocardial injury after I/R. Mechanistically, in vivo and in vitro experiments suggest that LGR6 regulates the expression of STAT2 and ZBP1 by activating the Wnt signaling pathway, thereby inhibiting cardiomyocyte necroptosis after HR. Inhibiting STAT2 and ZBP1 effectively alleviated the aggravating effect of LGR6 deficiency on myocardial necroptosis after I/R. Furthermore, activating LGR6 with RSPO3 also effectively protected mice from acute myocardial I/R injury. Our findings reveal that RSPO3-LGR6 axis downregulates the expression of STAT2 and ZBP1 through the Wnt signaling pathway, thereby inhibiting I/R-induced myocardial injury and necroptosis. Targeting the RSPO3-LGR6 axis may be a potential therapeutic strategy to treat myocardial I/R injury.

Short Link N Modulates Inflammasome Activity in Intervertebral Discs Through Interaction with CD14.

Intervertebral disc degeneration and pain are associated with the nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) inflammasome activation and the processing of interleukin-1 beta (IL-1β). Activation of thehm inflammasome is triggered by Toll-like receptor stimulation and requires the cofactor receptor cluster of differentiation 14 (CD14). Short Link N (sLN), a peptide derived from link protein, has been shown to modulate inflammation and pain in discs in vitro and in vivo; however, the underlying mechanisms remain elusive. This study aims to assess whether sLN modulates IL-1β and inflammasome activity through interaction with CD14. Disc cells treated with lipopolysaccharides (LPS) with or without sLN were used to assess changes in Caspase-1, IL-1β, and phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). Peptide docking of sLN to CD14 and immunoprecipitation were performed to determine their interaction. The results indicated that sLN inhibited LPS-induced NFκB and Caspase-1 activation, reducing IL-1β maturation and secretion in disc cells. A significant decrease in inflammasome markers was observed with sLN treatment. Immunoprecipitation studies revealed a direct interaction between sLN and the LPS-binding pocket of CD14. Our results suggest that sLN could be a potential therapeutic agent for discogenic pain by mitigating IL-1β and inflammasome activity within discs.

Chlamydia trachomatis Inc Ct226 is vital for FLI1 and LRRF1 recruitment to the chlamydial inclusion.

The obligate intracellular pathogen, Chlamydia trachomatis, establishes an intracellular niche within a host membrane-derived vacuole called the chlamydial inclusion. From within this inclusion, C. trachomatis orchestrates numerous host-pathogen interactions, in part, by utilizing a family of type III secreted effectors, termed inclusion membrane proteins (Incs). Incs are embedded within the inclusion membrane, and some function to recruit host proteins to the inclusion. Two such recruited host proteins are leucine rich repeat Flightless-1 interacting protein 1 (LRRF1/LRRFIP1) and its binding partner Flightless 1 (FLI1/FLII). Previously, LRRF1 has been shown to interact with Inc protein Ct226/CTL0478. This is the first study to examine interactions of FLI1 with candidate Incs or with LRRF1 during infection. We hypothesized that FLI1 recruitment to the inclusion would be dependent on LRRF1 localization. We demonstrated that FLI1 co-immunoprecipitated with Ct226 but only in the presence of LRRF1. Furthermore, FLI1 localized to the inclusion when LRRF1 was depleted via small interfering RNA, suggesting that FLI1 may have an alternative recruitment mechanism. We further developed a series of CRISPRi knockdown and complementation strains in C. trachomatis serovar L2 targeting ct226 and co-transcribed candidate Incs, ct225 and ct224. Simultaneous knockdown of ct226, ct225, and ct224 prevented localization of both FLI1 and LRRF1 to the inclusion, and only complementation of ct226 restored their localization. Thus, we demonstrated Ct226 is critical for FLI1 and LRRF1 localization to the inclusion. Our results also indicate an LRRF1-independent localization mechanism for FLI1, which likely influence their mechanism(s) of action during chlamydial infection.IMPORTANCEChlamydia trachomatis is a leading cause of both bacterial sexually transmitted infections and preventable infectious blindness worldwide. As an obligate intracellular pathogen, C. trachomatis has evolved multiple ways of manipulating the host to establish a successful infection. As such, it is important to understand host-chlamydial protein-protein interactions as these reveal strategies that C. trachomatis uses to shape its intracellular environment. This study looks in detail at interactions of two host proteins, FLI1 and LRRF1, during chlamydial infection. Importantly, the series of CRISPR inference knockdown and complement strains developed in this study suggest these proteins have both independent and overlapping mechanisms for localization, which ultimately will dictate how these proteins function during chlamydial infection.

Parental assigned chromosomes for cultivated cacao provides insights into genetic architecture underlying resistance to vascular streak dieback.

Diseases of Theobroma cacao L. (Malvaceae) disrupt cocoa bean supply and economically impact growers. Vascular streak dieback (VSD), caused by Ceratobasidium theobromae, is a new encounter disease of cacao currently contained to southeast Asia and Melanesia. Resistance to VSD has been tested with large progeny trials in Sulawesi, Indonesia, and in Papua New Guinea with the identification of informative quantitative trait loci (QTLs). Using a VSD susceptible progeny tree (clone 26), derived from a resistant and susceptible parental cross, we assembled the genome to chromosome-level and discriminated alleles inherited from either resistant or susceptible parents. The parentally phased genomes were annotated for all predicted genes and then specifically for resistance genes of the nucleotide-binding site leucine-rich repeat class (NLR). On investigation, we determined the presence of NLR clusters and other potential disease response gene candidates in proximity to informative QTLs. We identified structural variants within NLRs inherited from parentals. We present the first diploid, fully scaffolded, and parentally phased genome resource for T. cacao L. and provide insights into the genetics underlying resistance and susceptibility to VSD.