The Defective in Autoregulation (DAR) gene of Medicago truncatula encodes a protein involved in regulating nodulation and arbuscular mycorrhiza.

BACKGROUND: Legumes utilize a long-distance signaling feedback pathway, termed Autoregulation of Nodulation (AON), to regulate the establishment and maintenance of their symbiosis with rhizobia. Several proteins key to this pathway have been discovered, but the AON pathway is not completely understood. RESULTS: We report a new hypernodulating mutant, defective in autoregulation, with disruption of a gene, DAR (Medtr2g450550/MtrunA17_Chr2g0304631), previously unknown to play a role in AON. The dar-1 mutant produces ten-fold more nodules than wild type, similar to AON mutants with disrupted SUNN gene function. As in sunn mutants, suppression of nodulation by CLE peptides MtCLE12 and MtCLE13 is abolished in dar. Furthermore, dar-1 also shows increased root length colonization by an arbuscular mycorrhizal fungus, suggesting a role for DAR in autoregulation of mycorrhizal symbiosis (AOM). However, unlike SUNN which functions in the shoot to control nodulation, DAR functions in the root. CONCLUSIONS: DAR encodes a membrane protein that is a member of a small protein family in M. truncatula. Our results suggest that DAR could be involved in the subcellular transport of signals involved in symbiosis regulation, but it is not upregulated during symbiosis. DAR gene family members are also present in Arabidopsis, lycophytes, mosses, and microalgae, suggesting the AON and AOM may use pathway components common to other plants, even those that do not undergo either symbiosis.

Joint resource optimization and flexible QoS provision using hybrid optical core node architecture.

All-optical networks (AON) provide large bandwidth, high-speed switching, and quality of service (QoS). However, the emergence of recent applications generating flexible traffic demands affects AON resource utilization and QoS management. These concerns are handled by AON using core nodes with large wavelength converters (WC) and fiber delay line (FDL) spaces, resulting in a high cost of implementation and operation. This paper proposes a hybrid core node architecture, upgrading existing nodes with limited effective modification to minimize physical topology costs. This hybrid architecture increases WC and FDL availability and reduces wavelength utilization according to traffic behavior. These new node functions are enabled using an advanced algorithm integrating a novel cost formulation merging WC and FDL with lightpath deflection (LD). This algorithm activates resource availabilities using a flexible QoS scheme considering the required and real-time traffic loss and blocking delay. The experimental results indicate that the proposed approach optimizes AON resources while guaranteeing required traffic demands compared to the existing techniques.

CRISPR/Cas9-Mediated Knock-Out of the MtCLE35 Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions.

Legume plants have the ability to establish a symbiotic relationship with soil bacteria known as rhizobia. The legume-rhizobium symbiosis results in the formation of symbiotic root nodules, where rhizobia fix atmospheric nitrogen. A host plant controls the number of symbiotic nodules to meet its nitrogen demands. CLE (CLAVATA3/EMBRYO SURROUNDING REGION) peptides produced in the root in response to rhizobial inoculation and/or nitrate have been shown to control the number of symbiotic nodules. Previously, the MtCLE35 gene was found to be upregulated by rhizobia and nitrate treatment in Medicago truncatula, which systemically inhibited nodulation when overexpressed. In this study, we obtained several knock-out lines in which the MtCLE35 gene was mutated using the CRISPR/Cas9-mediated system. M. truncatula lines with the MtCLE35 gene knocked out produced increased numbers of nodules in the presence of nitrate in comparison to wild-type plants. Moreover, in the presence of nitrate, the expression levels of two other nodulation-related MtCLE genes, MtCLE12 and MtCLE13, were reduced in rhizobia-inoculated roots, whereas no significant difference in MtCLE35 gene expression was observed between nitrate-treated and rhizobia-inoculated control roots. Together, these findings suggest the key role of MtCLE35 in the number of nodule numbers under high-nitrate conditions, under which the expression levels of other nodulation-related MtCLE genes are reduced.

DMD antisense oligonucleotide mediated exon skipping efficiency correlates with flanking intron retention time and target position within the exon.

Mutations in the DMD gene are causative for Duchenne muscular dystrophy (DMD). Antisense oligonucleotide (AON) mediated exon skipping to restore disrupted dystrophin reading frame is a therapeutic approach that allows production of a shorter but functional protein. As DMD causing mutations can affect most of the 79 exons encoding dystrophin, a wide variety of AONs are needed to treat the patient population. Design of AONs is largely guided by trial-and-error, and it is yet unclear what defines the skippability of an exon. Here, we use a library of phosphorodiamidate morpholino oligomer (PMOs) AONs of similar physical properties to test the skippability of a large number of DMD exons. The DMD transcript is non-sequentially spliced, meaning that certain introns are retained longer in the transcript than downstream introns. We tested whether the relative intron retention time has a significant effect on AON efficiency, and found that targeting an out-of-frame exon flanked at its 5'-end by an intron that is retained in the transcript longer ('slow' intron) leads to overall higher exon skipping efficiency than when the 5'-end flanking intron is 'fast'. Regardless of splicing speed of flanking introns, we find that positioning an AON closer to the 5'-end of the target exon leads to higher exon skipping efficiency opposed to targeting an exons 3'-end. The data enclosed herein can be of use to guide future target selection and preferential AON binding sites for both DMD and other disease amenable by exon skipping therapies.

BlockmiR AONs as Site-Specific Therapeutic MBNL Modulation in Myotonic Dystrophy 2D and 3D Muscle Cells and HSALR Mice.

The symptoms of Myotonic Dystrophy Type 1 (DM1) are multi-systemic and life-threatening. The neuromuscular disorder is rooted in a non-coding CTG microsatellite expansion in the DM1 protein kinase (DMPK) gene that, upon transcription, physically sequesters the Muscleblind-like (MBNL) family of splicing regulator proteins. The high-affinity binding occurring between the proteins and the repetitions disallow MBNL proteins from performing their post-transcriptional splicing regulation leading to downstream molecular effects directly related to disease symptoms such as myotonia and muscle weakness. In this study, we build on previously demonstrated evidence showing that the silencing of miRNA-23b and miRNA-218 can increase MBNL1 protein in DM1 cells and mice. Here, we use blockmiR antisense technology in DM1 muscle cells, 3D mouse-derived muscle tissue, and in vivo mice to block the binding sites of these microRNAs in order to increase MBNL translation into protein without binding to microRNAs. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and highly specific transcriptomic expression. The blockmiRs are well tolerated in 3D mouse skeletal tissue inducing no immune response. In vivo, a candidate blockmiR also increases Mbnl1/2 protein and rescues grip strength, splicing, and histological phenotypes.

Long-range functional loops in the mouse olfactory system and their roles in computing odor identity.

Elucidating the neural circuits supporting odor identification remains an open challenge. Here, we analyze the contribution of the two output cell types of the mouse olfactory bulb (mitral and tufted cells) to decode odor identity and concentration and its dependence on top-down feedback from their respective major cortical targets: piriform cortex versus anterior olfactory nucleus. We find that tufted cells substantially outperform mitral cells in decoding both odor identity and intensity. Cortical feedback selectively regulates the activity of its dominant bulb projection cell type and implements different computations. Piriform feedback specifically restructures mitral responses, whereas feedback from the anterior olfactory nucleus preferentially controls the gain of tufted representations without altering their odor tuning. Our results identify distinct functional loops involving the mitral and tufted cells and their cortical targets. We suggest that in addition to the canonical mitral-to-piriform pathway, tufted cells and their target regions are ideally positioned to compute odor identity.

Novel Exon-Skipping Therapeutic Approach for the DMD Gene Based on Asymptomatic Deletions of Exon 49.

Exon skipping is a promising therapeutic approach. One important condition for this approach is that the exon-skipped form of the gene can at least partially perform the required function and lead to improvement of the phenotype. It is therefore critical to identify the exons that can be skipped without a significant deleterious effect on the protein function. Pathogenic variants in the DMD gene are responsible for Duchenne muscular dystrophy (DMD). We report for the first time a deletion of the in-frame exon 49 associated with a strikingly normal muscular phenotype. Based on this observation, and on previously known therapeutic approaches using exon skipping in DMD for other single exons, we aimed to extend the clinical use of exon skipping for patients carrying truncating mutations in exon 49. We first determined the precise genomic position of the exon 49 deletion in our patients. We then demonstrated the feasibility of skipping exon 49 using an in vitro AON (antisense oligonucleotide) approach in human myotubes carrying a truncating pathogenic variant as well as in healthy ones. This work is a proof of concept aiming to expand exon-skipping approaches for DMD exon 49.

Progress in the Self-Regulation System in Legume Nodule Development-AON (Autoregulation of Nodulation).

The formation and development of legumes nodules requires a lot of energy. Legumes must strictly control the number and activity of nodules to ensure efficient energy distribution. The AON system can limit the number of rhizobia infections and nodule numbers through the systemic signal pathway network that the aboveground and belowground parts participate in together. It can also promote the formation of nodules when plants are deficient in nitrogen. The currently known AON pathway includes four parts: soil NO3- signal and Rhizobium signal recognition and transmission, CLE-SUNN is the negative regulation pathway, CEP-CRA2 is the positive regulation pathway and the miR2111/TML module regulates nodule formation and development. In order to ensure the biological function of this important approach, plants use a variety of plant hormones, polypeptides, receptor kinases, transcription factors and miRNAs for signal transmission and transcriptional regulation. This review summarizes and discusses the research progress of the AON pathway in Legume nodule development.

Restoring SMN Expression: An Overview of the Therapeutic Developments for the Treatment of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder and one of the most common genetic causes of infant death. It is characterized by progressive weakness of the muscles, loss of ambulation, and death from respiratory complications. SMA is caused by the homozygous deletion or mutations in the survival of the motor neuron 1 (SMN1) gene. Humans, however, have a nearly identical copy of SMN1 known as the SMN2 gene. The severity of the disease correlates inversely with the number of SMN2 copies present. SMN2 cannot completely compensate for the loss of SMN1 in SMA patients because it can produce only a fraction of functional SMN protein. SMN protein is ubiquitously expressed in the body and has a variety of roles ranging from assembling the spliceosomal machinery, autophagy, RNA metabolism, signal transduction, cellular homeostasis, DNA repair, and recombination. Motor neurons in the anterior horn of the spinal cord are extremely susceptible to the loss of SMN protein, with the reason still being unclear. Due to the ability of the SMN2 gene to produce small amounts of functional SMN, two FDA-approved treatment strategies, including an antisense oligonucleotide (AON) nusinersen and small-molecule risdiplam, target SMN2 to produce more functional SMN. On the other hand, Onasemnogene abeparvovec (brand name Zolgensma) is an FDA-approved adeno-associated vector 9-mediated gene replacement therapy that can deliver a copy of the human SMN1. In this review, we summarize the SMA etiology, the role of SMN, and discuss the challenges of the therapies that are approved for SMA treatment.

In Vitro Delivery of PMOs in Myoblasts by Electroporation.

Antisense oligonucleotides (AONs) are small synthetic molecules of therapeutic interest for a variety of human disease. Their ability to bind mRNA and affect its splicing gives AONs potential use for exon skipping therapies aimed at restoring the dystrophin transcript reading frame for Duchenne muscular dystrophy (DMD) patients. The neutrally charged phosphorodiamidate morpholino oligomers (PMOs) are a stable and relatively nontoxic AON modification. To assess exon skipping efficiency in vitro, it is important to deliver them to target cells. Here, we describe a method for the delivery of PMOs to myoblasts by electroporation. The described protocol for the Amaxa 4D X unit nucleofector system allows efficient processing of 16 samples in one nucleocuvette strip, aiding in high-throughput PMO efficacy screens.

Evolutionary and expression dynamics of LRR-RLKs and functional establishment of KLAVIER homolog in shoot mediated regulation of AON in chickpea symbiosis.

Chickpea shoot exogenously treated with cytokinin showed stunted phenotype of root, shoot and significantly reduced nodule numbers. Genome-wide identification of LRR-RLKs in chickpea and Medicago resulted in 200 and 371 genes respectively. Gene duplication analysis revealed that LRR-RLKs family expanded through segmental duplications in chickpea and tandem duplications in Medicago. Expression profiling of LRR-RLKs revealed their involvement in cytokinin signaling and plant organ development. Overexpression of KLAVIER ortholog of chickpea, Ca_LRR-RLK147, in roots revealed its localization in the membrane but showed no effect on root nodulation despite increased cle peptide levels. Two findings (i) drastic effect on nodule number by exogenous cytokinin treatment to only shoot and restoration to normal nodulation by treatment to both root and shoot tissue and (ii) no effect on nodule number by overexpression of Ca_LRR-RLK147 establishes the fact that despite presence of cle peptides in root, the function of Ca_LRR-RLK147 was shoot mediated during AON.

Sinorhizobium medicae WSM419 Genes That Improve Symbiosis between Sinorhizobium meliloti Rm1021 and Medicago truncatula Jemalong A17 and in Other Symbiosis Systems.

Some soil bacteria, called rhizobia, can interact symbiotically with legumes, in which they form nodules on the plant roots, where they can reduce atmospheric dinitrogen to ammonia, a form of nitrogen that can be used by growing plants. Rhizobium-plant combinations can differ in how successful this symbiosis is: for example, Sinorhizobium meliloti Rm1021 forms a relatively ineffective symbiosis with Medicago truncatula Jemalong A17, but Sinorhizobium medicae WSM419 is able to support more vigorous plant growth. Using proteomic data from free-living and symbiotic S. medicae WSM419, we previously identified a subset of proteins that were not closely related to any S. meliloti Rm1021 proteins and speculated that adding one or more of these proteins to S. meliloti Rm1021 would increase its effectiveness on M. truncatula A17. Three genes, Smed_3503, Smed_5985, and Smed_6456, were cloned into S. meliloti Rm1021 downstream of the E. coli lacZ promoter. Strains with these genes increased nodulation and improved plant growth, individually and in combination with one another. Smed_3503, renamed iseA (increased symbiotic effectiveness), had the largest impact, increasing M. truncatula biomass by 61%. iseA homologs were present in all currently sequenced S. medicae strains but were infrequent in other Sinorhizobium isolates. Rhizobium leguminosarum bv. viciae 3841 containing iseA led to more nodules on pea and lentil. Split-root experiments with M. truncatula A17 indicated that S. meliloti Rm1021 carrying the S. medicae iseA is less sensitive to plant-induced resistance to rhizobial infection, suggesting an interaction with the plant's regulation of nodule formation. IMPORTANCE Legume symbiosis with rhizobia is highly specific. Rhizobia that can nodulate and fix nitrogen on one legume species are often unable to associate with a different species. The interaction can be more subtle. Symbiotically enhanced growth of the host plant can differ substantially when nodules are formed by different rhizobial isolates of a species, much like disease severity can differ when conspecific isolates of pathogenic bacteria infect different cultivars. Much is known about bacterial genes essential for a productive symbiosis, but less is understood about genes that marginally improve performance. We used a proteomic strategy to identify Sinorhizobium genes that contribute to plant growth differences that are seen when two different strains nodulate M. truncatula A17. These genes could also alter the symbiosis between R. leguminosarum bv. viciae 3841 and pea or lentil, suggesting that this approach identifies new genes that may more generally contribute to symbiotic productivity.

Shoot-derived miR2111 controls legume root and nodule development.

Legumes control their nodule numbers through the autoregulation of nodulation (AON). Rhizobia infection stimulates the production of root-derived CLE peptide hormones that are translocated to the shoot where they regulate a new signal. We used soybean to demonstrate that this shoot-derived signal is miR2111, which is transported via phloem to the root where it targets transcripts of Too Much Love (TML), a negative regulator of nodulation. Shoot perception of rhizobia-induced CLE peptides suppresses miR2111 expression, resulting in TML accumulation in roots and subsequent inhibition of nodule organogenesis. Feeding synthetic mature miR2111 via the petiole increased nodule numbers per plant. Likewise, elevating miR2111 availability by over-expression promoted nodulation, while target mimicry of TML induced the opposite effect on nodule development in wild-type plants and alleviated the supernodulating and stunted root growth phenotypes of AON-defective mutants. Additionally, in non-nodulating wild-type plants, ectopic expression of miR2111 significantly enhanced lateral root emergence with a decrease in lateral root length and average root diameter. In contrast, hairy roots constitutively expressing the target mimic construct exhibited reduced lateral root density. Overall, these findings demonstrate that miR2111 is both the critical shoot-to-root factor that positively regulates root nodule development and also acts to shape root system architecture.

Capturing a Comprehensive Picture of Biological Events From Adverse Outcome Pathways in the Drug Exposome.

Background: The chemical part of the exposome, including drugs, may explain the increase of health effects with outcomes such as infertility, allergies, metabolic disorders, which cannot be only explained by the genetic changes. To better understand how drug exposure can impact human health, the concepts of adverse outcome pathways (AOPs) and AOP networks (AONs), which are representations of causally linked events at different biological levels leading to adverse health, could be used for drug safety assessment. Methods: To explore the action of drugs across multiple scales of the biological organization, we investigated the use of a network-based approach in the known AOP space. Considering the drugs and their associations to biological events, such as molecular initiating event and key event, a bipartite network was developed. This bipartite network was projected into a monopartite network capturing the event-event linkages. Nevertheless, such transformation of a bipartite network to a monopartite network had a huge risk of information loss. A way to solve this problem is to quantify the network reduction. We calculated two scoring systems, one measuring the uncertainty and a second one describing the loss of coverage on the developed event-event network to better investigate events from AOPs linked to drugs. Results: This AON analysis allowed us to identify biological events that are highly connected to drugs, such as events involving nuclear receptors (ER, AR, and PXR/SXR). Furthermore, we observed that the number of events involved in a linkage pattern with drugs is a key factor that influences information loss during monopartite network projection. Such scores have the potential to quantify the uncertainty of an event involved in an AON, and could be valuable for the weight of evidence assessment of AOPs. A case study related to infertility, more specifically to "decrease, male agenital distance" is presented. Conclusion: This study highlights that computational approaches based on network science may help to understand the complexity of drug health effects, with the aim to support drug safety assessment.

An fNIRS Study of Brain Lateralization During Observation and Execution of a Fine Motor Task.

Brain activity in the action observation network (AON) is lateralized during action execution, with greater activation in the contralateral hemisphere to the side of the body used to perform the task. However, it is unknown whether the AON is also lateralized when watching another person perform an action. In this study, we use fNIRS to measure brain activity over the left and right cortex while participants completed actions with their left and right hands and watched an actor complete action with their left and right hands. We show that while activation is lateralized when the participants themselves are moving, brain lateralization is not affected by the side of the body when the participant is observing another person's action. In addition, we demonstrate that individual differences in hand preference and dexterity between the right and left hands are related to brain lateralization patterns.

Chitosan-Shelled Nanobubbles Irreversibly Encapsulate Morpholino Conjugate Antisense Oligonucleotides and Are Ineffective for Phosphorodiamidate Morpholino-Mediated Gene Silencing of DUX4.

Orphan drugs, including antisense oligonucleotides (AONs), siRNAs/miRNAs, Cas9 nuclease, and recombinant genes, have recently been made available for rare diseases. However, the main bottleneck for these new therapies is delivery. Drugs/synthetic genes need to reach the affected tissues with minimal off-target effects and immune reactions. AON molecules are currently delivered as backboned naked compounds or via viral vectors. Nanocarriers are considered promising vehicles, able to improve drug distribution by organ targeting and limiting safety issues. We tested perfluoropentane-based nanobubbles (NBs) as vehicles for loading phosphorodiamidate morpholino (PMO) AON to suppress DUX4 expression in a facioscapulohumeral muscular dystrophy cell model. In vitro cell-free analysis demonstrated a good loading capacity of PMO into NBs, while experiments in cell cultures showed lack of therapeutic effect since expression of DUX4 and its targets remained unmodified. We conclude that these types of chitosan-shelled NBs do not release PMO-AON and are therefore not ideal for PMO AON-related therapies.

Superior Facilitation of an Action Observation Network by Congruent Character Movements in Brain-Computer Interface Action-Observation Games.

Action observation (AO) is a promising strategy for promoting motor function in neural rehabilitation. Recently, brain-computer interface (BCI)-AO game rehabilitation, which combines AO therapy with BCI technology, has been introduced to improve the effectiveness of rehabilitation. This approach can improve motor learning by providing feedback, which can be interactive in an observation task, and the game contents of the BCI-AO game paradigm can affect rehabilitation. In this study, the effects of congruent rather than incongruent feedback in a BCI-AO game on mirror neurons were investigated. Specifically, the mu suppression with congruent and incongruent BCI-AO games was measured in 17 healthy adults. The mu suppression in the central motor cortex was significantly higher with the congruent BCI-AO game than with the incongruent one. In addition, the satisfaction evaluation results were excellent for the congruent case. These results support the fact that providing feedback congruent with the motion of an action video facilitates mirror neuron activity and can offer useful guidelines for the design of BCI-AO games for rehabilitation.

Shoot Extracts from Two Low Nodulation Mutants Significantly Reduce Nodule Number in Pea.

E107 and E132 are pea mutants that nodulate poorly. Because they have a shoot-controlled nodulation phenotype, we asked if their mutated genes were implicated in the autoregulation of nodulation (AON), a mechanism which consists of two systemic circuits, the positive CEP/CRA2 and the negative CLE/SUNN, coordinated via NIN and miR2111. We further characterized the mutants' phenotype by studying nodule distribution and nodulation efficiency. E107 was similar to wild-type (WT) in its nodule distribution, but E132 had an extended nodulation zone with nodules forming distally on its lateral roots. Moreover, we tested whether their shoots produced a compound inhibitory to nodulation. We made ethyl-acetate extracts of roots and shoots of both mutants and WT, which we applied to rhizobia-inoculated WT seedlings and to pure rhizobial cultures. Whereas free-living bacteria were unaffected by any of the extracts, WT treated with shoot extracts from either inoculated mutant had fewer nodules than that of control. E107 and E132 shoot extracts led to a 50% and a 35% reduction in nodule number, respectively. We propose that E107 and E132 belong to a new sub-class of AON mutants, i.e., hypo-nodulators, and that their respective gene products are acting in the AON descending branch, upstream of TML signaling.

Antisense Oligonucleotide- and CRISPR-Cas9-Mediated Rescue of mRNA Splicing for a Deep Intronic CLRN1 Mutation.

Mutations in CLRN1 cause Usher syndrome (USH) type III (USH3A), a disease characterized by progressive hearing impairment, retinitis pigmentosa, and vestibular dysfunction. Due to the lack of appropriate disease models, no efficient therapy for retinitis pigmentosa in USH patients exists so far. In addition, given the yet undefined functional role and expression of the different CLRN1 splice isoforms in the retina, non-causative therapies such as gene supplementation are unsuitable at this stage. In this study, we focused on the recently identified deep intronic c.254-649T>G CLRN1 splicing mutation and aimed to establish two causative treatment approaches: CRISPR-Cas9-mediated excision of the mutated intronic region and antisense oligonucleotide (AON)-mediated correction of mRNA splicing. The therapeutic potential of these approaches was validated in different cell types transiently or stably expressing CLRN1 minigenes. Both approaches led to substantial correction of the splice defect. Surprisingly, however, no synergistic effect was detected when combining both methods. Finally, the injection of naked AONs into mice expressing the mutant CLRN1 minigene in the retina also led to a significant splice rescue. We propose that both AONs and CRISPR-Cas9 are suitable strategies to initiate advanced preclinical studies for treatment of USH3A patients.

Characterization of odor-evoked neural activity in the olfactory peduncle.

The tenia tecta is extensively interconnected with the main olfactory bulb and olfactory cortical areas and is well positioned to contribute to olfactory processing. However, little is known about odor representation within its dorsal (DTT) and ventral (VTT) components. To address this need, spontaneous and odor-evoked activity of DTT and VTT neurons was recorded from urethane anesthetized mice and compared to activity recorded from adjacent areas within adjacent caudomedial aspects of the anterior olfactory nucleus (AON). Neurons recorded from DTT, VTT, and AON exhibited odor-selective alterations in firing rate in response to a diverse set of monomolecular odorants. While DTT and AON neurons exhibited similar tuning breadth, selectivity, and response topography, the proportion of odor-selective neurons was substantially higher in the DTT. These findings provide evidence that the tenia tecta may contribute to the encoding of specific stimulus attributes. Further work is needed to fully characterize functional organization of the tenia tecta and its contribution to sensory representation and utilization.