CNS4 causes subtype-specific changes in agonist efficacy and reversal potential of permeant cations in NMDA receptors.

The NMDA subtype of glutamate receptor serves as an attractive drug target for the treatment of disorders evolving from hyper- or hypoglutamatergic conditions. Compounds that optimize the function of NMDA receptors are of great clinical significance. Here, we present the pharmacological characterization of a biased allosteric modulator, CNS4. Results indicate that CNS4 sensitizes ambient levels of agonists and reduces higher-concentration glycine & glutamate efficacy in 1/2AB receptors, but minimally alters these parameters in diheteromeric 1/2A or 1/2B receptors. Glycine efficacy is increased in both 1/2C and 1/2D, while glutamate efficacy is decreased in 1/2C and unaltered in 1/2D. CNS4 does not affect the activity of competitive antagonist binding at glycine (DCKA) and glutamate (DL-AP5) sites; however, it decreases memantine potency in 1/2A receptors but not in 1/2D receptors. Current-voltage (I-V) relationship studies indicate that CNS4 potentiates 1/2A inward currents, a phenomenon that was reversed in the absence of permeable Na+ ions. In 1/2D receptors, CNS4 blocks inward currents based on extracellular Ca2+ concentration. Further, CNS4 positively modulates glutamate potency on E781A_1/2A mutant receptors, indicating its role at the distal end of the 1/2A agonist binding domain interface. Together, these findings reveal that CNS4 sensitizes ambient agonists and allosterically modulates agonist efficacy by altering Na+ permeability based on the GluN2 subunit composition. Overall, the pharmacology of CNS4 aligns with the need for drug candidates to treat hypoglutamatergic neuropsychiatric conditions such as loss function GRIN disorders and anti-NMDA receptor encephalitis.

The relationship between bacterial outer membrane vesicles and halophilic adaptation.

Many cells are known to actively release nano-sized outer membrane vesicles (OMVs) that contain bioactive proteins, lipids, and nucleic acids into the extracellular environment. These vesicles have been associated with adaptation to environmental stress in other species, but their role in halophilic salt stress adaptation is not known. This study aimed to isolate and characterize the OMVs of Halomonas caseinilytica KB2 at various salt concentrations [6% (KB2-6), 12% (KB2-12), and 18% (KB2-18)] and to identify the patterns of adaptations to increasing salinity in its structure, protein composition, and expression. Also, a comparison with the composition of OMVs of E. coli, a mesophilic bacterium, was performed. Bioinformatics and statistical analysis were carried out to elucidate the underlying proteome differences that may exist as a result of increasing salinity. The results show that OMV production in H. caseinilytica KB2 is promoted by a decrease in salinity. OMVs also revealed possible structural and metabolic changes happening in the cells which led to the deduction that cells become more stable with increasing salt concentrations. Cell wall integrity, protein expression and folding are important. Although H. caseinilytica KB2 OMVs show cellular changes with changing salt concentration, they may not play a direct role in adaptation to changing salinity.

Respiratory management of spinal muscular atrophy type 1 patients treated with Nusinersen.

INTRODUCTION: The recent development of disease-modifying treatments in spinal muscular atrophy (SMA) type 1 shifted these patients' management from palliative to proactive. The aim of this study was to assess patients' nocturnal gas exchanges before noninvasive ventilation (NIV) initiation and their clinical evolution to determine if capnia is a good criterion to decide when to introduce respiratory support. PATIENTS AND METHODS: This multicentric retrospective study reports the respiratory management and evolution of 17 SMA type 1 children (10 females) for whom treatment with Nusinersen was initiated between 2016 and 2018. RESULTS: Median [interquartile range-IQR] age at diagnosis and at first Nusinersen injection was of 4 [3;8] and 4 [3;9] months, respectively. Patients were followed during 38 [24;44] months. Thirteen (76%) patients were started on NIV at a median [IQR] age of 12 [9;18] months. Repeated hospitalizations and intensive care unit admissions were needed for 11 of them. Blood gas and nocturnal gas exchange recordings performed before NIV initiation were always normal. 9/13 X-ray performed before NIV showed atelectasis and/or acute lower respiratory tract infections. There was a significant decrease in the total number of hospital admissions between the first and second year of treatment (p = 0.04). CONCLUSION: This study shows that patients do not present with nocturnal hypoventilation before respiratory decompensations and NIV initiation, and suggests that a delay in NIV initiation might result in respiratory complications. There is a need for disease-centered guidelines for the respiratory management of these patients, including NIV indications.

Cranial manipulation affects cholinergic pathway gene expression in aged rats.

CONTEXT: Age-dependent dementia is a devastating disorder afflicting a growing older population. Although pharmacological agents improve symptoms of dementia, age-related comorbidities combined with adverse effects often outweigh their clinical benefits. Therefore, nonpharmacological therapies are being investigated as an alternative. In a previous pilot study, aged rats demonstrated improved spatial memory after osteopathic cranial manipulative medicine (OCMM) treatment. OBJECTIVES: In this continuation of the pilot study, we examine the effect of OCMM on gene expression to elicit possible explanations for the improvement in spatial memory. METHODS: OCMM was performed on six of 12 elderly rats every day for 7 days. Rats were then euthanized to obtain the brain tissue, from which RNA samples were extracted. RNA from three treated and three controls were of sufficient quality for sequencing. These samples were sequenced utilizing next-generation sequencing from Illumina NextSeq. The Cufflinks software suite was utilized to assemble transcriptomes and quantify the RNA expression level for each sample. RESULTS: Transcriptome analysis revealed that OCMM significantly affected the expression of 36 genes in the neuronal pathway (false discovery rate [FDR] <0.004). The top five neuronal genes with the largest-fold change were part of the cholinergic neurotransmission mechanism, which is known to affect cognitive function. In addition, 39.9% of 426 significant differentially expressed (SDE) genes (FDR<0.004) have been previously implicated in neurological disorders. Overall, changes in SDE genes combined with their role in central nervous system signaling pathways suggest a connection to previously reported OCMM-induced behavioral and biochemical changes in aged rats. CONCLUSIONS: Results from this pilot study provide sufficient evidence to support a more extensive study with a larger sample size. Further investigation in this direction will provide a better understanding of the molecular mechanisms of OCMM and its potential in clinical applications. With clinical validation, OCMM could represent a much-needed low-risk adjunct treatment for age-related dementia including Alzheimer's disease.

Comparison of Prognostic Scoring Systems to Predict Durable Pain Relief After Microvascular Decompression for Trigeminal Neuralgia.

BACKGROUND: Microvascular decompression (MVD) is an effective treatment for trigeminal neuralgia, but pain recurs in a substantial minority of patients. Two recently published scoring systems by Hardaway et al. and Panczykowski et al. use simple preoperative clinical and imaging features to predict durable pain relief following MVD, but their predictive performance has not been independently validated. This study aimed to compare predictive performance of the Hardaway et al. score (HS) and Panczykowski et al. score (PS) for 1-year, 3-year, and long-term pain-free outcomes after MVD for trigeminal neuralgia. METHODS: HS and PS were computed for a retrospective, single-institution cohort of 68 patients with trigeminal neuralgia who underwent MVD. Primary outcome was pain recurrence after MVD. Predictive performance of HSs and PSs was evaluated with area under the curve sensitivity analysis and regression models for survival analyses at 1 year, 3 years, and last follow-up. RESULTS: Area under the curve for predicting pain-free outcome was higher for PS versus HS at 1 year (0.873 vs. 0.775) and 3 years (0.793 vs. 0.704). Cox proportional hazard models showed that PS better predicted long-term pain-free outcomes compared with HS (P < 0.05). One-year pain-free outcome was best predicted by pain type; longer-term outcomes were better predicted by presence and degree of neurovascular compression on preoperative imaging. CONCLUSIONS: PS is superior to HS in predicting pain-free outcomes after MVD, which may aid in patient selection and counseling. Overall, more significant neurovascular compression of the trigeminal nerve root, and to a lesser extent classical paroxysmal pain, are good predictors of durable pain relief after MVD.

A glutamate concentration-biased allosteric modulator potentiates NMDA-induced ion influx in neurons.

Precisely controlled synaptic glutamate concentration is essential for the normal function of the N-methyl D-aspartate (NMDA) receptors. Atypical fluctuations in synaptic glutamate homeostasis lead to aberrant NMDA receptor activity that results in the pathogenesis of neurological and psychiatric disorders. Therefore, glutamate concentration-dependent NMDA receptor modulators would be clinically useful agents with fewer on-target adverse effects. In the present study, we have characterized a novel compound (CNS4) that potentiates NMDA receptor currents based on glutamate concentration. This compound alters glutamate potency and exhibits no voltage-dependent effect. Patch-clamp electrophysiology recordings confirmed agonist concentration-dependent changes in maximum inducible currents. Dynamic Ca2+ and Na+ imaging assays using rat brain cortical, striatal and cerebellar neurons revealed CNS4 potentiated ion influx through native NMDA receptor activity. Overall, CNS4 is novel in chemical structure, mechanism of action and agonist concentration-biased allosteric modulatory effect. This compound or its future analogs will serve as useful candidates to develop drug-like compounds for the treatment of treatment-resistant schizophrenia and major depression disorders associated with hypoglutamatergic neurotransmission.

Mitochondrial activity of human umbilical cord mesenchymal stem cells.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) serve as a potential cell-based therapy for degenerative disease. They provide immunomodulatory and anti-inflammatory properties, multipotent differentiation potential and are harvested with no ethical concern. It is unknown whether MSCs collected from different areas of the human umbilical cord elicit more favorable effects than others. Three MSC populations were harvested from various regions of the human umbilical cord: cord lining (CL-MSCs), perivascular region (PV-MSCs), and Wharton's jelly (WJ-MSCs). Mesenchymal markers (CD90 and CD73) were expressed by all three cell populations. Stemness marker (OCT4), endothelial cell adhesion molecular marker (CD146), and monocyte-macrophage marker (CD14) were expressed by WJ-MSCs, PV-MSCs, and CL-MSCs, respectively. Stroke presents with oxygen and glucose deprivation and leads to dysfunctional mitochondria and consequently cell death. Targeting the restoration of mitochondrial function in the stroke brain through mitochondrial transfer may be effective in treating stroke. In vitro exposure to ambient and OGD conditions resulted in CL-MSCs number decreasing the least post-OGD/R exposure, and PV-MSCs exhibiting the greatest mitochondrial activity. All three hUC-MSC populations presented similar metabolic activity and survival in normal and pathologic environments. These characteristics indicate hUC-MSCs potential as a potent therapeutic in regenerative medicine.

NMDA receptor modulation and severe acute respiratory syndrome treatment.

N-Methyl-D-aspartate (NMDA) subtype of glutamate receptors is expressed in the human lungs and central nervous system.  NMDA receptor potentiation could increase calcium ion influx and promote downstream signaling mechanisms associated with cellular contractions that are disrupted in severe acute respiratory syndrome. Pharmacological effects generated by triggering glutamate receptor function in the brain, coupled with concurrent stimulation of the respiratory tract, may produce a synergetic effect, improving the airway smooth muscle function. A novel multipronged intervention to simultaneously potentiate NMDA receptors expressed both in the central nervous system and airway muscles would be helpful for the treatment of severe acute respiratory syndrome that deteriorates peripheral and central nervous system function before causing death in humans.

Catalytic Metal-free Allylic C-H Amination of Terpenoids.

The selective replacement of C-H bonds in complex molecules, especially natural products like terpenoids, is a highly efficient way to introduce new functionality and/or couple fragments. Here, we report the development of a new metal-free allylic amination of alkenes that allows the introduction of a wide range of nitrogen functionality at the allylic position of alkenes with unique regioselectivity and no allylic transposition. This reaction employs catalytic amounts of selenium in the form of phosphine selenides or selenoureas. Simple sulfonamides and sulfamates can be used directly in the reaction without the need to prepare isolated nitrenoid precursors. We demonstrate the utility of this transformation by aminating a large set of terpenoids in high yield and regioselectivity.

Cytotoxic diterpenoids from the leaves and stem bark of Croton haumanianus (Euphorbiaceae).

The leaf extract of Croton haumanianus J. Léonard (Euphorbiaceae) yielded twenty-six compounds, including eight previously reported ent-kauranes and an ent-labdane and eight undescribed ent-kauranes, ent-16R-kauran-17-al, ent-3ß-hydroxy-16R-kauran-17-al, ent-16S,17-epoxykauran-19-ol, ent-16S,17-epoxykauran-3ß-ol, ent-17-palmityloxykaurane-3ß,16ß-diol, ent-17-palmityloxykauran-16ß-ol, ent-3α,18-cyclokaurane-16ß,17-diol and 19-nor-16α,17-dihydroxy-ent-kaur-4(18)-ene and three undescribed ent-clerodanes, dimethyl ent-15,16-epoxy-6ß-hydroxy-1,3,13(16),14-clerodatetraen-20,12S-olide-18,19-dioate (saniolide A), dimethyl ent-15,16-epoxy-6ß-hydroxy-1,3,13(16),14-clerodatetraen-20,12R-olide-18,19-dioate (12-epi-saniolide A), methyl ent-15,16-epoxy-1,3,13(16),14-clerodatetraen-18,6R:20,12S-diolide-19-oate (saniolide B). The stem bark extract yielded the ent-clerodane crotocorylifuran, and five undescribed ent-isopimaranes, ent-isopimara-8(14),15-dien-18-al, ent-18-hydroxyisopimara-8(14),15-dien-7-one, ent-isopimara-7,15-dien-18-oic acid, ent-isopimara-7,15-dien-18-ol and ent-isopimara-8,15-dien-7-oxo-18-oic acid. Three compounds, ent-kaurane-3ß,16ß,17-triol, ent-17-palmityloxykaurane-3ß,16ß-diol and ent-17-palmityloxykauran-16ß-ol, showed selective activity against three of the NCI 60 cancer cell lines, the colon (HCT-116), the melanoma (M14) and the renal (786-0) cancer cell lines at a concentration of 10-5 M.

[Pregabalin and risk of addiction: A new care issue?] / Prégabaline et risque d'addiction : une nouvelle demande de soin ?

PURPOSE: Pregabalin (PRG) is a gamma-aminobutyric acid (GABA) analogue used for treatment of epilepsy, neuropathic pain, generalised anxiety disorder and currently being studied for other indications. Supported by the results of case studies and a limited number of studies, there is an ongoing debate about the addictive potential of PRG. However, evidence is scarce and no definitive assessment on the potential for abuse and dependence to PRG is available. The objective of our study was to identify the number of cases of abuse or dependence to PRG published and to study potential risk factors of addiction to PRG. METHODS: We have identified on PubMed and ScienceDirect published case studies of PRG abuse or dependence and analysed these cases on the basis of several clinical parameters. RESULTS: A total of 118 cases of PRG abuse or dependence were identified, including 21 isolated cases (mean age 33 years, 67 % men). The mean daily dose of PRG was 2,9 g. Current or past polydrug abuse was present in the majority of cases. Psychiatric diagnoses, other than substance-related disorders, were reported in as many patients, and almost all patients experienced withdrawal symptoms when PRG was discontinued. CONCLUSION: Current literature suggests an important and growing concern for the abuse of PRG. Male sex, psychiatric and/or addiction history, including opioid addiction, may be potential risk factors for the development of addictive behaviours associated with PRG.

Mechanoceutics Alters Alzheimer's Disease Phenotypes in Transgenic Rats: A Pilot Study.

Current advancements in neurovascular biology relates a mechanoceutics treatment, known as cranial osteopathic manipulation (COM), Alzheimer's disease (AD). COM could be used as an evidence-based treatment strategy to improve the symptoms of AD if molecular mechanisms, which currently remain unclear, are elucidated. In the present pilot study, using transgenic rats, we have identified COM mediated changes in behavioral and biochemical parameters associated with AD phenotypes. We expect these changes may have functional implications that might account for improved clinical outcomes of COM treatment. Further investigations on COM will be helpful to establish an adjunct treatment for AD.

Ent-abietane and ent-pimarane diterpenoids from Croton mubango (Euphorbiaceae).

Twelve ent-abietane and two ent-pimarane diterpenoids were isolated from the leaves of Croton mubango Müll. Arg. (Euphorbiaceae) collected in the Democratic Republic of the Congo. 2ß-Hydroxy-ent-abieta-7,13-dien-3-one, 15-hydroxy-ent-abieta-7,13-dien-3-one, 13α,15-dihydroxy-ent-abieta-8(14)-en-3-one, 2ß,9,13-trihydroxy-ent-abieta-7-en-3-one, 2ß,7ß-dihydroxy-ent-abieta-8,11,13-trien-3-one, 15-hydroxy-ent-abieta-8,11,13-trien-3-one and ent-pimara-8(14),15-dien-3-one and the ent-forms of the previously reported normal series diterpenoids, ent-abieta-8,11,13-trien-3-one, 7ß-hydroxy-ent-abieta-8,11,13-trien-3-one, 3α-hydroxy-ent-abieta-8,11,13-triene, 15-hydroxy-ent-abieta-8,11,13-triene and 6ß-hydroxy-ent-abieta-8,11,13-triene are reported here for the first time. Structures were established using HRESIMS, FTIR, NMR, DP4+ probability calculations and by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. Ent-pimara-8(14), 15-dien-3-one, showed antiproliferative activity against melanoma (MALME-3M), renal (UO-31) and ovarian cancer cell lines (IGROV1) at a concentration of 10-5 M in the NCI 60 screen.

Effect of Osteopathic Cranial Manipulative Medicine on an Aged Rat Model of Alzheimer Disease.

CONTEXT: In the aging brain, reduction in the pulsation of cerebral vasculature and fluid circulation causes impairment in the fluid exchange between different compartments and lays a foundation for the neuroinflammation that results in Alzheimer disease (AD). The knowledge that lymphatic vessels in the central nervous system play a role in the clearance of brain-derived metabolic waste products opens an unprecedented capability to increase the clearance of macromolecules such as amyloid ß proteins. However, currently there is no pharmacologic mechanism available to increase fluid circulation in the aging brain. OBJECTIVE: To demonstrate the influence of an osteopathic cranial manipulative medicine (OCMM) technique, specifically, compression of the fourth ventricle, on spatial memory and changes in substrates associated with mechanisms of metabolic waste clearance in the central nervous system using the naturally aged rat model of AD. RESULTS: Significant improvement was found in spatial memory in 6 rats after 7 days of OCMM sessions. Live animal positron emission tomographic imaging and immunoassays revealed that OCMM reduced amyloid ß levels, activated astrocytes, and improved neurotransmission in the aged rat brains. CONCLUSION: These findings demonstrate the molecular mechanism of OCMM in aged rats. This study and further investigations will help physicians promote OCMM as an evidence-based adjunctive treatment for patients with AD.

Investigation of the structural requirements for N-methyl-D-aspartate receptor positive and negative allosteric modulators based on 2-naphthoic acid.

The N-methyl-D-aspartate receptor (NMDAR), a ligand-gated ion channel activated by L-glutamate and glycine, plays a major role in the synaptic plasticity underlying learning and memory. NMDARs are involved in neurodegenerative disorders such as Alzheimer's and Parkinson's disease and NMDAR hypofunction is implicated in schizophrenia. Herein we describe structure-activity relationship (SAR) studies on 2-naphthoic acid derivatives to investigate structural requirements for positive and negative allosteric modulation of NMDARs. These studies identified compounds such as UBP684 (14b), which act as pan potentiators by enhancing NMDAR currents in diheteromeric NMDAR tetramers containing GluN1 and GluN2A-D subunits. 14b and derivatives thereof are useful tools to study synaptic function and have potential as leads for the development of drugs to treat schizophrenia and disorders that lead to a loss of cognitive function. In addition, SAR studies have identified a series of styryl substituted compounds with partial NAM activity and a preference for inhibition of GluN2D versus the other GluN2 subunits. In particular, the 3-and 2-nitrostyryl derivatives UBP783 (79i) and UBP792 (79h) had IC50s of 1.4 µM and 2.9 µM, respectively, for inhibition of GluN2D but showed only 70-80% maximal inhibition. GluN2D has been shown to play a role in excessive pain transmission due to nerve injury and potentially in neurodegenerative disorders. Partial GluN2D inhibitors may be leads for the development of drugs to treat these disorders without the adverse effects observed with full NMDAR antagonists.

Positive Modulatory Interactions of NMDA Receptor GluN1/2B Ligand Binding Domains Attenuate Antagonists Activity.

N-methyl D-aspartate receptors (NMDAR) play crucial role in normal brain function and pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDAR contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that include six different gene products; four GluN2 (A-D) and two GluN3 (A-B) subunits. The heterogeneity of subunit combination facilities the distinct function of NMDARs. All GluN subunits contain an extracellular N-terminal Domain (NTD) and ligand binding domain (LBD), transmembrane domain (TMD) and an intracellular C-terminal domain (CTD). Interaction between the GluN1 and co-assembling GluN2/3 subunits through the LBD has been proven crucial for defining receptor deactivation mechanisms that are unique for each combination of NMDAR. Modulating the LBD interactions has great therapeutic potential. In the present work, by amino acid point mutations and electrophysiology techniques, we have studied the role of LBD interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, and allosteric modulators. The results reveal that agonists (glycine and glutamate) potency was altered based on mutant amino acid sidechain chemistry and/or mutation site. Most antagonists inhibited mutant receptors with higher potency; interestingly, clinically used NMDAR channel blocker memantine was about three-fold more potent on mutated receptors (N521A, N521D, and K531A) than wild type receptors. These results provide novel insights on the clinical pharmacology of memantine, which is used for the treatment of mild to moderate Alzheimer's disease. In addition, these findings demonstrate the central role of LBD interactions that can be exploited to develop novel NMDAR based therapeutics.

Synthesis of a Series of Novel 3,9-Disubstituted Phenanthrenes as Analogues of Known NMDA Receptor Allosteric Modulators.

9-Substituted phenanthrene-3-carboxylic acids have been reported to have allosteric modulatory activity at the NMDA receptor. This receptor is activated by the excitatory neurotransmitter L-glutamate and has been implicated in a range of neurological disorders such as schizophrenia, epilepsy and chronic pain and neurodegenerative disorders such as Alzheimer's disease. Herein, the convenient synthesis of a wide range of novel 3,9-disubstituted phenanthrene derivatives starting from a few common intermediates is described. These new phenanthrene derivatives will help to clarify the structural requirements for allosteric modulation of the NMDA receptor.

Identification of novel allosteric modulator binding sites in NMDA receptors: A molecular modeling study.

The dysfunction of N-methyl-d-Aspartate receptors (NMDARs), a subtype of glutamate receptors, is correlated with schizophrenia, stroke, and many other neuropathological disorders. However, not all NMDAR subtypes equally contribute towards these disorders. Since NMDARs composed of different GluN2 subunits (GluN2A-D) confer varied physiological properties and have different distributions in the brain, pharmacological agents that target NMDARs with specific GluN2 subunits have significant potential for therapeutic applications. In our previous research, we have identified a family of novel allosteric modulators that differentially potentiate and/or inhibit NMDARs of differing GluN2 subunit composition. To further elucidate their molecular mechanisms, in the present study, we have identified four potential binding sites for novel allosteric modulators by performing molecular modeling, docking, and in silico mutations. The molecular determinants of the modulator binding sites (MBS), analysis of particular MBS electrostatics, and the specific loss or gain of binding after mutations have revealed modulators that have strong potential affinities for specific MBS on given subunits and the role of key amino acids in either promoting or obstructing modulator binding. These findings will help design higher affinity GluN2 subunit-selective pharmaceuticals, which are currently unavailable to treat psychiatric and neurological disorders.

Receptor-mediated exopolysaccharide perception controls bacterial infection.

Surface polysaccharides are important for bacterial interactions with multicellular organisms, and some are virulence factors in pathogens. In the legume-rhizobium symbiosis, bacterial exopolysaccharides (EPS) are essential for the development of infected root nodules. We have identified a gene in Lotus japonicus, Epr3, encoding a receptor-like kinase that controls this infection. We show that epr3 mutants are defective in perception of purified EPS, and that EPR3 binds EPS directly and distinguishes compatible and incompatible EPS in bacterial competition studies. Expression of Epr3 in epidermal cells within the susceptible root zone shows that the protein is involved in bacterial entry, while rhizobial and plant mutant studies suggest that Epr3 regulates bacterial passage through the plant's epidermal cell layer. Finally, we show that Epr3 expression is inducible and dependent on host perception of bacterial nodulation (Nod) factors. Plant-bacterial compatibility and bacterial access to legume roots is thus regulated by a two-stage mechanism involving sequential receptor-mediated recognition of Nod factor and EPS signals.