Prospective memory is important for our health and independence but declines with age. Hence, interventions to enhance prospective memory, for example by providing an incentive, may promote healthy ageing. The neuroanatomical correlates of prospective memory and the processing of incentive-related prospective memory changes in older adults are not fully understood. In an fMRI study, we will therefore test whether incentives improve prospective memory in older adults and how prospective memory is processed in the brain in general, and when incentives are provided. Since goals and interests change across adulthood, avoiding losses is becoming more important for older adults than achieving gains. We therefore posit that loss-related incentives will enhance prospective memory, which will be subserved by increased prefrontal and midbrain activity.n = 60 healthy older adults (60–75 years of age) in a randomized, single-blind, and parallel-group study. We will acquire 7T fMRI data in an incentive group and a control group . Before and after fMRI, all participants will complete questionnaires and cognitive tests to assess possible confounders .We will include The results of this study will clarify whether loss-related incentives can enhance prospective memory and how any enhancement is processed in the brain. In addition, we will determine how prospective memory is processed in the brain in general. The results of our study will be an important step towards a better understanding of how prospective memory changes when we get older and for developing interventions to counteract cognitive decline. Prospective memory is the ability to remember doing something at a specific point in time in the future  or when a specific event occurs  . WhetherRemembering prospective intentions is important for maintaining health and independence in older adults  , 10. FinThe aims of the current study are, therefore, threefold. First, we investigate the functional neuroanatomy of prospective memory with a particular emphasis on the question whether brain regions involved in time-based and event-based prospective memory overlap or differ. We expect them to differ when using a time-based prospective memory paradigm in which participants actively align their intentions with time. Second, we test whether event-based or time-based prospective memory can be modulated when participants have to avoid losing an incentive. We hypothesize that this will improve prospective memory. Third, we investigate the neuroanatomical correlates of incentive-related processing during prospective memory tasks. We expect increased prefrontal and midbrain activity when participants avoid losing an incentive and that the strength of activity increase will be associated with prospective memory task performance.n = 60 healthy older participants (60—75 years of age) will be included. Inclusion criteria will be fluency in German, no evidence of cognitive impairment on the Cognitive Telephone Screening , normal. During fMRI, time-based and event-based prospective memory will be tested, followed by a modified version of the monetary incentive delay task , which After MRI data acquisition, we will ask participants to describe the prospective memory task in their own words to assess whether they remember what they had to do during the prospective memory tasks. Then they will rate the perceived importance, motivation, and difficulty of the tasks. Finally, they will complete a German vocabulary test to test their verbal intelligence  as well To examine the neural substrates of event-based and time-based prospective memory, we will use a computerized task in the MR scanner , which wTo assess incentive-related processing during prospective memory tasks, participants in the incentive group will be informed that they are initially endowed with 5 Swiss Francs (per prospective memory task) that they may lose proportionally to the number of prospective memory cues they miss (0.50 Swiss Francs per missed cue). They will also be informed that half of their earnings will be donated to an organization of their choice .To examine the neural substrates of incentive-related processing in the absence of prospective memory, participants will complete a modified version of the monetary incentive delay task , 35 thatAt baseline, the participants will need to respond as fast and correctly as possible to triangles or squares and they will receive feedback . Each trial will start with a fixation cross presented in the centre of the screen for 0.6—1.0 s. Then, either of the two stimuli will appear for 0.8 s, followed by another fixation cross (for 1.0—1.4 s) and feedback (for 0.5 s). After 20 trials, the mean response time of all correct answers will be calculated. Next, the incentive block will follow, during which the participants will again respond to triangles or squares Fig. . In contSince we are primarily interested in whether incentives will enhance prospective memory, we will report accuracy and mean response times for the event-based task as well as accuracy and clock checking for the time-based task as primary outcomes.First, we will test whether incentives will improve task accuracy. We will use two-way mixed-design ANOVA on correctly remembered trials, with the within-subject factor ‘task type’ (event-based or time-based) and the between-subject factor ‘group’ (incentive or control). Next, we will test whether response times in the event-based task will become quicker with an incentive. We will use one-way ANOVA on response times for correctly remembered trials, with the between-subject factor ‘group’ (incentive or control). For time-based prospective memory, we will test whether an incentive influences how often participants check the clock. Similar to our previous study , we willp < 0.05 considered statistically significant. We will correct for multiple comparisons using Tukey’s method. Whenever the assumptions of normality or homogeneity are not met, data will be transformed and/or non-parametric alternatives will be used. In case of missing data, we will use linear-mixed effects models rather than ANOVAs.We will use R (Version 4.2.1) with Rstudio (Version 2022.02.3) for statistical analyses, with Task-based fMRI data will be pre-processed using Statistical Parametric Mapping , implemented in Matlab R2019b . Pre-processing will include realignment, slice-time correction, co-registration to the skull-stripped structural image, normalisation, and smoothing with a 6 mm FWHM Gaussian kernel. To remove low frequency noise from pre-processed data, a high-pass filter will be applied using SPM12’s default settings. For all first-level analyses, data will be analysed using a general linear model that models the time-series as a sequence of events convolved with the canonical hemodynamic response function provided by SPM12. We will analyse physiological data using either the CompCor  or RETROWe will first analyse which brain regions are involved in event-based vs. time-based prospective memory. For the first-level analysis, we will fit one model for each prospective memory task. Each model will include three regressors, the first for ongoing trials during the ongoing task only block, the second for ongoing trials during the prospective memory block, and the third for prospective memory trials. We will build regressors using the onset of every correct answer. Task instructions will be modelled as regressors of no interest. We will define different contrasts corresponding to different prospective memory phases: 1) to obtain brain activity that is associated with maintenance of an intention, we will create contrasts between ongoing trials during the prospective memory block vs. ongoing only trials, 2) to obtain brain activity corresponding to the detection of a target, retrieval of an intention and its’ execution, we will create contrasts between prospective memory trials vs. ongoing trials during the prospective memory block. For time-monitoring, we will fit a model with the number of clock checks during the different time intervals previously described in the behavioural analysis. The onset will be defined as the moment in which the participant presses the key to check the clock. For time-monitoring, we will define contrasts between the different time intervals such as: T-15 vs. T-30, T-15 vs. T + 15, T-15 vs. T + 30, T-30 vs. T + 15, T-30 vs. T + 30, T + 15 vs. T + 30. Again, we will include six nuisance regressors for the six movement parameters as well as regressors of physiological data.For second-level analyses, we will first use paired t-tests on first-level contrasts in the control group to test whether there would be differences in brain activity when comparing event-based to time-based prospective memory. Then, conjunction analysis will be performed to discern the neural substrates that are common in both task types. Then, we will calculate brain-behaviour correlations using beta estimates from significant regions correlated with task performance.Next, we will test which brain regions are associated with incentives in general . For first-level analysis, we will define a contrast between incentive and control trials during the anticipation phase of the monetary incentive delay task . We will include six nuisance regressors for the six movement parameters as well as regressors of physiological data. For second level analysis, we will perform a one-sample t-test on first-level contrast (incentive vs. control) to find out which brain regions are associated with the anticipation of an incentive. Regions that will be identified with this analysis will be used as regions-of-interest (ROI) in subsequent second-level analyses of prospective memory.Finally, we will test whether incentive-related processing during prospective memory tasks will be different to incentive-related processing during the monetary incentive delay task. We will first conduct two-sample t-tests on first-level contrast from the first analysis to test whether there are differences in brain activity with an incentive vs. without an incentive for event-based or time-based prospective memory. Next, we will use inclusive masks derived from the ROI analysis using the clusters identified in the monetary incentive delay task. Finally, we will use exclusive masking to test whether additional regions are active during incentive-related processing in prospective memory.p < 0.001 uncorrected, and the minimal cluster size threshold k to 15 voxels. For small regions , we will mask voxels inclusively using generated masks and will set the intensity threshold to p < 0.001, uncorrected, with a cluster-size threshold k of 3 voxels. This is to reduce Type II errors associated with weak fMRI signal changes in small brain areas.For all neuroimaging analyses, we will set the intensity threshold to f = 0.25) suggests that an inclusion of n = 54 participants would be needed to find such effects in a repeated measures design with two groups and five measurements with α = 0.01 and a power of 1-β = 0.99. To account for attrition, drop-out or exclusion due to movement artefacts, we plan to recruit N = 60 participants (n = 30 in each group).For the determination of sample size, we used G*power . We baseWe will pseudonymise all study data . The coding key will be stored separately and locked away. Each participant will be informed orally and in writing about the nature, usage, and storage of their data. Behavioural data will be stored in Dropbox folders encrypted with Boxcryptor. Neuroimaging data will be stored on GitLab hosted by servers of Bern University. Paper pencil data will be stored in folders that are locked away. Data processing will be done on personal computers/laptops and institutional servers. All computers will be password-protected and encrypted. The study team will be responsible for data management; data monitoring will be done by an independent researcher not involved in the study. At the end of the study, all personal data will be deleted. The procedures comply with Swiss data privacy laws.The results of our study will provide insight into three important aspects of prospective memory in older adults. First, our study will reveal whether time-based and event-based prospective memory differ neuroanatomically. Second, we will find out whether we can enhance prospective memory by using an incentive and third, we will determine how incentives are processed in the brain during such tasks.Older adults typically perform better in prospective memory tasks in which monitoring demands are low , 34, 41.Comparable to previous studies, we may find that incentives modulate event-based prospective memory. It has been shown, for example, that avoiding financial losses improved event-based prospective memory accuracy in older adults when it included a prosocial component  . We willFinally, our study will provide data on how incentives are processed in the brain during prospective memory tasks. So far, the monetary incentive delay task has been mainly used to study incentive-related processes in the brain. This task differentiates anticipation of an incentive from the actual outcome . This differentiation is not possible in a prospective memory task because any feedback would serve as a prospective memory cue and would therefore bias actual prospective memory abilities. It is, however, possible to assess the anticipation of an incentive since participants are informed in the beginning of the task that they may receive an incentive depending on their performance. During anticipation in the monetary incentive delay task, older adults showed less activity in the ventral and anterior insula as well as the dorsal striatum than younger adults did , 45–49. Our study may have some limitations. One limitation might be that the small amount of money that participants can earn might not be motivating enough. At least for donation, we highlighted the difference even a few Swiss Francs will make by showing pictures of aid organisations and examples of what they can do with small amounts of money. Another limitation might be that ventral and subcortical brain regions are in vicinity to bone and sinuses which makes them vulnerable to artefacts caused by magnetic field inhomogeneity, particularly with ultra-high field MRI . We willOur study will test the neuroanatomical correlates of event-based and time-based prospective memory in older adults. We will, in addition, examine whether incentives can enhance event-based and time-based prospective memory and how it is processed during such tasks. The results of our study will be an important step towards a better understanding of how memory changes when we get older and for developing interventions to counteract cognitive decline.Recruitment of participants started in January 2022. Data acquisition is ongoing, and we expect to finish data acquisition in autumn 2023.


C9orf72 gene represent the predominant genetic etiology underlying amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). The transcribed r(GGGGCC)n RNA repeats form RNA foci, which recruit RNA binding proteins and impede their normal cellular functions, ultimately resulting in fatal neurodegenerative disorders. Furthermore, the non-canonical translation of the r(GGGGCC)n sequence can generate dipeptide repeats, which have been postulated as pathological causes. Comprehensive structural analyses of r(GGGGCC)n have unveiled its polymorphic nature, exhibiting the propensity to adopt dimeric, hairpin, or G-quadruplex conformations, all of which possess the capacity to interact with RNA binding proteins. Small molecules capable of binding to r(GGGGCC)n have been discovered and proposed as potential lead compounds for the treatment of ALS and FTD. Some of these molecules function in preventing RNA–protein interactions or impeding the phase transition of r(GGGGCC)n. In this review, we present a comprehensive summary of the recent advancements in the structural characterization of r(GGGGCC)n, its propensity to form RNA foci, and its interactions with small molecules and proteins. Specifically, we emphasize the structural diversity of r(GGGGCC)n and its influence on partner binding. Given the crucial role of r(GGGGCC)n in the pathogenesis of ALS and FTD, the primary objective of this review is to facilitate the development of therapeutic interventions targeting r(GGGGCC)n RNA.The aberrant expansion of GGGGCC hexanucleotide repeats within the first intron of the  Amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD) are two neurodegenerative disorders characterized by progressive degeneration and dysfunction of neuronal architecture ,2,3,4,5.TARDBP mutations . Fi. Fi30]. toxicity ,34,35,36s system ,40,41,42ractions . The RNAractions , Zfp106 ractions , ADARB2 ractions , Purα . T. Tn. In tructure . Furtherce of K+ . Conversn have been successfully determined [3GGBrGG] (represented by PDB codes 2N2D)  sequence adopted an antiparallel G4 topology (High-resolution structures of d(GGGGCC)termined ,65. Janees 2N2D) . The inctopology b 67]..n have bn repeats. Their studies revealed distinct G4 folding patterns in the presence of K+ ions. Notably, d(GGGGCC)GGGG, d(GGGGCC)2, and d(GGGGCC)3 did not exhibit stable G4 structures. Instead, d(GGGGCC)2 and d(GGGGCC)3 displayed mixed forms of parallel and antiparallel G4 folding. On the other hand, d(GGGGCC)4 and d(GGGGCC)5 formed stable G4 structures. Specifically, d(GGGGCC)5 exhibited a combination of parallel and antiparallel G4 folds, while d(GGGGCC)4 adopted a homogeneous monomeric form characterized by a chair-type G4 structure . J. Jn repend 7ECG) d also coallel G4 f 59]..n repeatn can undergo phase separation both in vivo and in vitro . An. Ann in etention . These fn, serving as a histopathological marker in 97% of ALS cases and 45% of FTD cases.TAR DNA binding protein 43 (TDP-43), another member of the hnRNP family, possesses two RNA recognition motifs (RRMs), a nuclear localization signal (NLS), and a prion-like domain at the C-terminus . Numeroun, the pathogenic mechanism of TDP-43 in ALS/FTD is believed to involve its interaction with DPRs, which are non-ATG translation products of r(GGGGCC)n . W. Wn. NMR4 binding ligands . T. T19F mon and d(GGGGCC)n, the phase separation and transition of r(GGGGCC)n, the interactions of r(GGGGCC)n with RBPs, and the discovered ligands capable of inhibiting the non-ATG translations of r(GGGGCC)n and/or the interactions between r(GGGGCC)n and RBPs.In this review, we provide a comprehensive overview of the advancements in understanding the structure of r(GGGGCC)n RNA, and their interactions have garnered significant research attention. When the repeat number exceeds the threshold, r(GGGGCC)n RNA undergoes phase separation and transition, leading to the formation of nuclear RNA foci. These RNA foci recruit RBPs, disrupting the physiological functions of RNA splicing and maturation. Another pathogenic mechanism by which r(GGGGCC)n contributes to ALS or FTD is the cytotoxicity of repetitive dipeptide proteins generated through non-ATG translation. Aggregates of these repetitive dipeptide proteins, can recruit numerous 26S proteasome complexes and stabilize a transient substrate-processing conformation of the 26S proteasome, suggesting impaired degradation processes [The relationship between the fatal neurodegenerative diseases ALS/FTD, the structure of r(GGGGCC)rocesses .n RNA and elucidating the structure-function relationship are key areas of research in understanding the pathogenic causes. r(GGGGCC)n can adopt diverse structures, including hairpin and parallel G4 topologies, with equilibrium between them depending on solution conditions. However, the three-dimensional structures of r(GGGGCC)n RNA are still unknown. Achieving a dominant conformation for structural studies may require sequence and solution condition optimization. Another challenging aspect is determining the secondary structures of r(GGGGCC)n within RNA foci or gel-like states. Due to the non-crystalline solid state and heterogeneous nature of RNA foci, commonly used high-resolution structure determination methods such as X-ray crystallography or solution NMR are not applicable [Characterizing the repeat structure of r(GGGGCC)plicable ,117. To plicable ,119,120,n have been discovered to block RBP interactions, inhibit phase separation, and/or hinder non-ATG translation, as evidenced both in vivo and in vitro. Understanding the structural details of the interactions between r(GGGGCC)n RNA and ligands is crucial for facilitating the design of lead compounds to treat ALS/FTD. Similar to the challenges faced in studying r(GGGGCC)n RNA, the complex structure determination of r(GGGGCC)n RNA repeats and small molecules is lacking, necessitating further developments to gain insights into drug design.Several small molecules that bind to r(GGGGCC)C9orf72, have resulted in sustained reductions in RNA foci and dipeptide-repeat proteins, leading to the amelioration of behavioral deficits. These efforts have identified the gain of toxicity as a central disease mechanism caused by repeat-expanded C9orf72 and established the feasibility of ASO-mediated therapy [C9orf72 transcript or repeat expansion showed mitigation despite the presence of repeat-associated non-ATG translation products [n binding proteins into ALS/FTD cells has the potential to restore RBP functions by augmenting the intracellular pool of RBPs recruited by RNA foci. This approach represents an alternative strategy for treating ALS by targeting r(GGGGCC)n RNA. Lastly, gene editing system by CRISPR/Cas9 has successfully removed the GGGGCC repeat expansion in C9orf72, leading to reduction in RNA foci and DPR formations, proving a promising approach in ALS treatments.Another known treatment approach for ALS/FTD involves the use of antisense RNA. Single-dose injections of antisense oligonucleotides (ASOs) targeting repeat-containing RNAs, while preserving mRNA levels encoding  therapy . ALS braproducts . Moreove


The 2BC and 3AB1,2,3CD precursors undergo subsequent proteolysis to generate the proteins required for viral replication, including the enzymes 2C, 3Cpro, and 3Dpol. These precursors can be processed through both cis and trans  pathways, which are thought to be important for controlling virus replication. Our previous studies suggested that a single residue in the 3B3-3C junction has an important role in controlling 3AB1,2,3CD processing. Here, we use in vitro based assays to show that a single amino acid substitution at the 3B3-3C boundary increases the rate of proteolysis to generate a novel 2C-containing precursor. Complementation assays showed that while this amino acid substitution enhanced production of some nonenzymatic nonstructural proteins, those with enzymatic functions were inhibited. Interestingly, replication could only be supported by complementation with mutations in cis acting RNA elements, providing genetic evidence for a functional interaction between replication enzymes and RNA elements.Foot-and-mouth disease virus (FMDV) is a picornavirus, which infects cloven-hoofed animals to cause foot-and-mouth disease (FMD). The positive-sense RNA genome contains a single open reading frame, which is translated as a polyprotein that is cleaved by viral proteases to produce the viral structural and nonstructural proteins. Initial processing occurs at three main junctions to generate four primary precursors; LIMPORTANCE Foot-and-mouth disease virus (FMDV) is responsible for foot-and-mouth disease (FMD), an important disease of farmed animals, which is endemic in many parts of the world and can results in major economic losses. Replication of the virus occurs within membrane-associated compartments in infected cells and requires highly coordinated processing events to produce an array of nonstructural proteins. These are initially produced as a polyprotein that undergoes proteolysis likely through both cis and trans alternative pathways . The role of alternative processing pathways may help coordination of viral replication by providing temporal control of protein production and here we analyze the consequences of amino acid substitutions that change these pathways in FMDV. Our data suggest that correct processing is required to produce key enzymes for replication in an environment in which they can interact with essential viral RNA elements. These data further the understanding of RNA genome replication. Picornaviridae have limited genome size and therefore minimal coding capacity. These viruses have evolved several strategies to overcome this limitation, including the use of protein precursors that can perform different functions to the mature proteins. Furthermore, individual proteins and their precursors can also sometimes perform more than one function  and foot-and-mouth disease virus (FMDV). PV is responsible for the incapacitating  human disease poliomyelitis, while FMDV is the causative agent of the economically damaging foot-and-mouth disease, an acute vesicular disease of livestock, including cloven-hoofed ruminants and pigs. The FMDV genome contains one large open reading frame that encodes a ~250 kDa polyprotein  and 3AB1,2,3CD  , the protease 3Cpro, and the polymerase 3Dpol (The rmed P3) , 7. Lproyprotein , 8. The yprotein  before tecursors , 11. Prose 3Dpol , 13.pro is thought to occur through at least two separate pathways to generate mutually exclusive sets of precursors  can be processed to generate 3ABC and 3CD. Furthermore, it appears that this alternative processing may be temporally controlled and used to regulate virus replication. For example, previous studies with PV have demonstrated that later production of 3AB and 3CD can delay the initiation of viral RNA replication (pol (cis versus trans cleavage events). However, methods to differentiate between these cis versus trans cleavage events are challenging, and as a result, the mechanism(s) that controls this switch are not completely understood.Processing of the nonstructural polyprotein by 3Cecursors . For exalication . For FMDion  that flank the open reading frame (cis-acting replicative element (cre), a large stem-loop (termed the S-fragment) and a tandem series of pseudoknots  to generate the replicative primer, VPg-pUpU , 3BCD, and 3ABCD, and have been suggested to be important for controlling replication  expression over time, to investigate FMDV replication by mutation of the 3B proteins . We repo3, at the boundary with 3Cpro, dramatically changed processing of the FMDV 3AB1,2,3CD polyprotein and prevented release of active 3Dpol. However, through blind passage, a compensatory mutation was selected which restored replication and wild-type (WT) 3AB1,2,3CD processing. This was identified as a reversion of a lysine at the P2 residue of the 3B3-3C junction to the WT threonine  the reporter protein ptGFP replaced the structural proteins, allowing ptGFP expression to be used as an indicator of replicon replication  and ptGFP-3B3/2  (pol (ptGFP-3DGNN) were included as controls. The latter replicon serves as a negative control for ptGFP production from translation of the input transfected RNA, as we have previously described  . A WT ptescribed . RNAs frescribed .GNN control replicon, as previously reported  predicted to prevent its proteolytic activity , 3AB1,2,3 was also detected. Both 2B and 2C were present, in addition to a limited amount of the precursor 2BC at earlier time points. Compared to WT, the construct containing the 3B3T>K substitution resulted in greater amounts of the 3CD and 3AB1,2,3 precursors and less of the 3AB1,2,3CD precursor. There were also increased levels of the 2BC precursor in addition to a high molecular weight precursor, possibly 2BC3AB1,2,3, which was detected at early time points and gradually decreased over time. These data extended our previous observations demonstrating that the 3B3T>K substitution alone is sufficient to accelerate proteolysis at the 3B3-3C junction and so increases the relative amounts of 2BC, 3CD, and 3AB1,2,3.For the WT construct, the full-length 3ABin vitro translation experiments suggested that the 3B3T>K substitution increased the production of the 2BC precursor in addition to a larger molecular weight precursor not observed in the WT control. To confirm the identity of the 2C-containing precursors, the in vitro translation samples were immunoprecipitated using an anti-2C antibody. T7 constructs expressing the WT polyprotein or polyprotein containing the 3B3T>K substitution were used in in vitro coupled transcription/translation reactions with [35S] methionine/cysteine pulse/chase labeling. Samples were taken after 90 min and immunoprecipitation performed on half of the sample. Both pre- and post-immunoprecipitation protein samples were analyzed by SDS-PAGE  were immunoprecipitated with an anti-2C antibody following expression from the polyprotein containing the 3Btrans by 3Cpro and/or a 3Cpro-containing precursor. To explore the latter possibility, we adapted our in vitro assay to investigate trans-mediated cleavage. For simplicity, we adapted both the WT 3AB1,2,3CD precursor or precursor containing the 3B3T>K substitution to also contain the inactivating mutation in 3Cpro (3CC163A) to prevent self-proteolysis  were translated in vitro with [35S] methionine/cysteine before adding excess unlabeled methionine/cysteine and purified active 3Cpro to a duplicate set of reactions (plus 3Cpro). Samples were harvested at regular time points and processing of the [35S] labeled precursor was analyzed by SDS-PAGE ..3T>K pre3T>K precursor was also sensitive to cleavage by 3Cpro when this is present as part of a larger precursor molecule, we generated a 3AB1,2,3CD expression construct in which all the cleavage boundaries had been mutated. Thus, the protease activity of this precursor was retained but only in the context of a full-length 3AB1,2,3CD polyprotein. Our trans-cleavage assay was repeated using this new construct, termed 3AB1,2,3CproD, in place of the purified 3Cpro enzyme used above . Thus, driving rapid trans-mediated proteolysis at this junction results in overproduction of a specific set of viral precursor proteins.As before, the 3Ced above . This coin vitro polyprotein processing data above implies that the 3B3T>K substitution increases the rate of proteolysis at the 3B3-3C junction by stimulating trans-mediated proteolysis. In doing so, it drives the formation of 2BC, 3AB1,2,3, and 3CD precursors to the detriment of other products such as the enzymes 2C and 3Dpol. In our previous studies, we used trans-complementation assays to investigate protein function. These assays involve the co-transfection of two replication defective replicon constructs that express different fluorescent reporter genes, allowing replication to be differentially monitored. Co-transfection of the two replicons allows exchange of viral nonstructural proteins within replication complexes to permit replication of one (or both) of the input genomes  , which we have shown can be complemented in trans  . This coin trans . Co-tranin trans . Replicain trans . For brein trans  and C wi1,2,3Y3F replicon was significantly enhanced by the ptGFP-3B3T>K construct, as anticipated. The mCherry-2CGK>AA replicon was not recovered by any of the helper replicons, suggesting the functions of 2C cannot be provided in trans  from interacting with the template RNA, thus preventing its replication.A key observation from our 3T>K replicon RNA  was in vitro transcribed in the presence of BrUTP to generate BrU-labeled replicon RNA. Alongside, a WT ptGFP was transcribed with BrUTP and used to confirm that BrU labeling had no significant inhibitory effect on replicon replication (data not shown). The ptGFP-3B3T>K BrU replicon was co-transfected into BHK-21 cells seeded on coverslips together with a WT replicon in which 3Dpol had been labeled with a HA epitope (termed 3DHA). HA epitopes are derived from the human influenza virus hemagglutinin (HA) protein and we have previously demonstrated that tagging 3Dpol with HA in this manner does not affect replication  to study interactions between replicon RNA and components of the replication complex. First, ptGFP-3Blication . The covociation . The numociation .HA replicon generated PLA signals that were easily detectable, indicative of interactions between the ptGFP RNA and 3DHA protein as predicted from mixing of replication complex components. In contrast, little or no PLA signal was detected when a WT BrU labeled poliovirus replicon (BrU-PV) was co-transfected with the 3DHA helper replicon, with a ~40-fold decrease in the number of PLA foci/cell compared to the positive control. This suggests the PLA signal is generated through specific mixing of replication complexes. Co-transfection of the ptGFP-3B3T>K, ptGFP-3DGNN, or ptGFP-3B1,2,3Y3F BrU labeled replicon RNAs with the WT 3DHA replicon also generated PLA signals that were easily detectable, with ~40-, ~10-, and ~15-fold increase in PLA foci/cell compared to the BrU-PV control. This would suggest the 3B3T>K substitution does not prevent recruitment of its cognate RNA with a functional RNA polymerase.Co-transfection of the WT ptGFP BrU labeled replicon with the WT 3D3-3C cleavage junction prevents viral replication by disrupting polyprotein processing  prevented viral replication and in in vitro translation assays changed polyprotein processing, as predicted.All well-studied positive-sense RNA viruses produce polyproteins that help compensate for a relatively limited coding capacity. These polyproteins are processed by viral protease(s) to generate mature proteins via functional intermediates in a highly regulated manner that modulates viral replication. However, establishing how polyprotein processing regulates viral replication can be challenging due to the intricate nature of these interactions. In a previous study of the FMDV polyprotein, we showed that changing six amino acids at the 3Bocessing . We post3T>K construct changed polyprotein processing and understand the mechanism that underpinned this change, we employed a combination of in vitro translation, immunoprecipitation, and trans-cleavage assays in the context of the FMDV nonstructural polyprotein. We found that a 3B3-3C cleavage junction bearing this substitution was more efficiently cleaved by 3Cpro in trans compared to WT. Furthermore, the 3B3T>K substitution also rendered this cleavage junction sensitive to proteolysis by 3Cpro when the protease was present as part of a larger precursor . Thus, the consequence of introducing the 3B3T>K substitution was to generate a substrate that was more efficiently cleaved . This changed the subset of proteins produced; generating 2BC3AB1,2,3 . This would introduce another two cleavage sites that may subtly change the hierarchy of processing of the entire polyprotein.To build a more complete picture of how the 3Bnfection , 41), sieviously . Furtherpro which showed that charged residues in the P2 position of the cleavage junction are more efficiently recognized  and can give rise to alternative precursors, for example, 3AB1,2,3C and 3CD. It is clearly suggested from our data that trans cleavage of one 3AB1,2,3CD molecule by another polyprotein is possible. However, the WT precursor was not processed efficiently by 3AB1,2,3CD in trans, and the observation that 3Cpro in the context of 3CD is proteolytically active, but only able to cleave efficiently at 2B-2C and 2C-3A junctions, could suggest that other factors , are also involved. Picornavirus 3Cpro proteins are also implicated in RNA binding and lipid biogenesis , and therefore may act as co-factors to dictate processing pathways  was able to complement defective mutations in the 3B proteins but not 2C or 3Dpol, suggesting that this replicon can produce active primers for replication, 3B1, 3B2, and/or 3B3 but inactive replication enzymes, 2C and 3Dpol. Interestingly, the 3B3T>K replicon was only complemented by a replicon lacking cis-acting RNA replication elements  and when the protein components were provided as part of an entire polyprotein. However, the PLA data suggested that the 3B3T>K substitution does not prevent recruitment of its cognate RNA with 3Dpol in trans but instead potentially increases the association. The increased association between the 3B3T>K RNA and 3Dpol provided by a WT replicon in trans may suggest the formation of more stable RNA-protein complexes that are detrimental to replication.Using pol but not 3B). Hence, if processing of the precursor occurs too rapidly, it cannot associate correctly with these RNA elements required for replication, or generates a complex that is too stable . A similar mechanism has been suggested for PV where two molecules of 3ABCD are required for replication, one which produces 3CD that interacts with viral RNA structures while one produces enzymatically active 3Dpol  were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with glutamine (Sigma-Aldrich) supplemented with 10% FCS, 50 U/mL penicillin, and 50 μg/mL streptomycin.GNN, 3B3/2 and ΔS-fragment mutations (in vitro transcription and translation experiments pcDNA3.1(+) based expression plasmids were generated by PCR. Briefly, the relevant FMDV sequence was amplified to include flanking NotI restriction enzymes and upstream Kozak modified translational start site. The NotI digested PCR products were cloned into NotI digested pcDNA3.1(+) (Thermo Fisher Scientific). The sequence of all plasmids used in this study was confirmed by Sanger sequencing. The sequences of all primers and plasmids are available on request.Plasmids carrying wild-type FMDV replicons, pRep-mCherry and pRep-ptGFP, have already been described , 56, aloutations , 38, 39.in vitro transcription and translation assays were performed using the TNT Quick Coupled Transcription/Translation system (Promega) as described previously (35S] methionine/cysteine (PerkinElmer). Reactions were incubated at 30°C for 40 min chasing with 2 μL of 50 mg/mL unlabeled methionine/cysteine. Reactions were stopped at 20-min or hourly intervals by the addition of 2× Laemmli buffer. Samples were separated by SDS-PAGE before visualization of radiolabeled products by autoradiography.Coupled eviously . Reactiotrans-cleavage assays, the TNT reactions were supplemented with purified FMDV 3Cpro (a kind gift from Dr. Tobias Tuthill [pro-mediated proteolysis of radiolabeled precursor monitored by SDS-PAGE.For the  Tuthill ) to the AscI before being used to generate T7 in vitro transcribed RNA as previously described  as previously described (GNN transfection for input translation) were used to determine fluorescent thresholds and identify positive objects from background fluorescence. A positive object was determined as having an average fluorescent intensity of >8 green calibration units  and >2 RCU , which were kept constant throughout the experiments. The number of positive cells per well was determined from the average of up to nine nonoverlapping images per well. Unless stated otherwise, data are presented as mean fluorescent positive cells per well at 8 h posttransfection when replication was approximately maximal. For each experiment, the data were analyzed as both fluorescent cell counts per well and total fluorescent intensity per well. There was no difference observed when the data were analyzed in either way. Unless otherwise stated, statistical analysis was performed using a two-tailed unpaired t test.BHK-21 cells were seeded into 24-well tissue cultures vessels, allowed to adhere overnight for 16 h, before duplicate wells were transfected with 1 μg of each escribed . For co-escribed . Fluoresescribed . ControlBHK-21 cells seeded onto coverslips were co-transfected with replicon RNA before fixing in 4% paraformaldehyde and washing with PBS. Immunofluorescence was conducted as previously described . PrimaryImmunoprecipitation reactions were performed using Dynabeads Protein G (Invitrogen). To bind the antibody to magnetic beads, 5 μL of the FMDV 2C antibody (a kind gift from Francisco Sobrino) was mixed with 195 μL PBS and incubated at room temperature with 50 μL magnetic beads, shaken for 1 h, after which the supernatant was removed from the beads. Transcription and translation reaction samples were mixed with 200 μL PBS and incubated shaking at room temperature with 25 μL of Dynabeads as a preclear step. The tube was placed on the magnet and the supernatant removed. This was added to the 50 μL of Dynabeads with the 2C antibody bound and incubated at room temperature shaking for 1 h. The flow through was removed and added to 2× Laemmli buffer. The beads were washed three times with PBS pH 7.4 with 0.02% Tween 20 and each wash supernatant retained. Proteins were eluted from the beads by adding 50 μL of 2× Laemmli buffer and heating to 100°C.


Circular RNA (circRNA) is a newly discovered noncoding RNA that regulates gene transcription, binds to RNA-related proteins, and encodes protein microRNAs (miRNAs). The development of molecular biomarkers such as circRNAs holds great promise in the diagnosis and prognosis of clinical disorders. Importantly, circRNA-mediated maternal-fetus risk factors including environmental , maternal , placental, and fetal (preterm birth and low birth weight) factors are the early origins and likely to contribute to the occurrence and progression of developmental and pediatric cardiopulmonary disorders. Although studies of circRNAs in normal cardiopulmonary development and developmental diseases have just begun, some studies have revealed their expression patterns. Here, we provide an overview of circRNAs’ biogenesis and biological functions. Furthermore, this review aims to emphasize the importance of circRNAs in maternal-fetus risk factors. Likewise, the potential biomarker and therapeutic target of circRNAs in developmental and pediatric lung diseases are explored. Circular RNA (circRNA) was first described in RNA viruses/viroids as an error of endogenous RNA junctions . InitialThe lack of a 3′ and 5′ end in circRNA prevents it from being digested by ribonucleases such as RNase R. Hence, circRNA has a half-life ten times that of linear RNA. CircRNA sequences are highly conserved . CircRNACurrently over 20,000 circRNAs have been identified. In general, circRNAs are named based on their parental genes or specific functions. For instance, cerebellar degeneration-related protein antisense RNA (CDR1as) is also called ciRS-7 (circRNA sponge for miR-7) ,10. In mRemarkable advances have been achieved in circRNA biology over the past few decades, but the effect of regulatory networks on circRNA function and regulation remain largely unexplored in health and disease. Here, we briefly overview circRNA biogenesis and their mechanisms of action in general and important analytic approaches of circRNA profiling. Additionally, we will discuss the latest data regarding their role in mesendoderm differentiation, pulmonary bud formation, lung branching morphogenesis, and vascularization relevant to pulmonary development and related diseases. Finally, circRNA analysis in diagnostic and therapeutic contexts are discussed.mRNA precursers (pre-mRNA) typically include one to five exons and introns. Together, the combined sequence of both exons and introns can be as much as three times longer than linear processed mRNA alone . CircRNACircRNAs usually are composed of exons 2 and 3 of the gene locus. Most circular RNAs are generated at the expense of their linear counterparts since they are derived from constitutive exons. A large portion of circRNAs contain the canonical splice site motif, suggesting that the canonical spliceosome is involved. When transcription termination factors  or core spliceosome components  are inhibited, circRNAs can become the dominant RNA transcript. Transcripts that are read through can then be backspliced into downstream genes. CircRNAs can also be produced by exon skipping, through either the lariat-guided or intron-guided methods . CurrentAs research continues, more will be learned regarding the regulation of splicing and circularization to generate circRNAs. CircRNA biogenesis can contain the 5′ end of the pre-mRNA upstream exon coupled to the downstream exon at the 3′ end. CircRNAs have covalently closed loops with no 3′ cap and no 5′ end. Therefore, their resistance to fluid absorption and ribonuclease (RNase) degradation leads to the relative stability of circRNA in the body compared to linear RNA ,34. The Arthrobacter luteus) elements and inhibits the formation of circRNA [Several mechanisms have been discovered for regulating circRNA biogenesis. Inhibiting spliceosomes by depleting U2 small nuclear ribonucleoprotein components, can increase circular to linear RNA ratios . Alterna circRNA . Alu ele circRNA . In addi circRNA . ATP-dep circRNA .In the nucleus, circRNAs such as EIciRNAs can directly bind to elongated RNA Pol II binding sites or interact with the Pol II transcription complex after forming EIciRNA–U1 snRNP complexes through RNA–RNA interactions. Both interactions regulate the transcription of circRNA parent genes . In humamiRNA is noncoding RNA approximately 18 to 25 nucleotides long and an essential epigenetic regulator in eukaryotes. Mature miRNA can directly bind to target mRNAs, resulting in degradation of target mRNAs or suppression of target mRNA’s translation in lung development and diseases ,58,59. RRNA binding proteins (RBPs) can target RNA through RNA-binding domains to regulate gene expression in different cellular processes such as cell morphology and differentiation, cell proliferation, response to oxidative stress, aging, and apoptosis . CircRNASeveral aspects of circRNA sequence have raised the possibility of translation. Several in vitro and in vivo studies have shown that circular RNA have an intrinsic entry site (IRES) or a modification of N6-methyladenosine (m6A) that can facilitate translation into peptides  67,68,6,668,69. Pamudurti et al. in their study presented evidence supporting circRNA translation: 1. circRNA was associated explicitly with translation ribosome; 2. proteins were generated from circRNA minigenes; 3. ribosome footprint readings were supported by the stop codon in circMbl; 4. sequences were identified that promoted cap-independent translation on several circRNAs; 5. a novel isoform of a peptide was detected coinciding with circMbl. However, circRNAs associated with ribosomes were detected in lower abundance than free circRNAs and some minigenes did not lead to protein production, suggesting that a specific process promotes translation of a subset of circRNAs .Recent studies revealed that circRNA could compete with enzymes and influence the post-translational modifications of full-length proteins coded by parent genes. Circβ-catenin-coding β-catenin-370aa competitively interacted with GSK3β and inhibited its binding to β-catenin, leading to the antagonization of GSK3β-induced β-catenin degradation . AKT3-17Developmental and pediatric lung diseases are disorders of lung development affecting neonates, infants, children, and adolescents. The risk factors include environmental , maternal , placental, and fetal (preterm birth and low birth weight) factors. These risk factors are interrelated and interact during fetal development. They impact perinatal “plasticity”, lung development, as well as lung injury and repair processes. There is an increasing body of evidence that abnormal circRNA expression is associated with developmental diseases in humans and is associated with gene dysregulation ,79.In one animal model used to study the effects of high altitude (hypoxic) environmental stress and adaptation, circRNAs and mRNAs  were found to be downregulated in yak lungs exposed to high altitude . This stPreeclampsia is one of the leading causes of premature birth. As a result of premature birth, there are a number of complications that result in significant morbidity and mortality, including bronchopulmonary dysplasia (BPD), hyaline membrane disease, and pulmonary interstitial emphysema . A recenCurrent research into the role of circRNAs in developmental and pediatric lung disorders is restricted to BPD. The current definition of BPD describes a developmental and pediatric lung disease with less heterogeneity, simplified alveolar surface, and reduced and dysmorphic vascular bed . In pretAlthough non-coding RNAs (ncRNAs) were once considered a waste product, they are now recognized as molecules that regulate many lung disorders. Since circRNAs act as sponges in the human body for miRNAs and can alter the function of those miRNAs, presumably circRNA can influence the occurrence and progression of diseases through regulation of miRNAs  Figure .Recent discoveries revealing the profile of circRNA expression in preterm infants with BPD is beginning to elucidate the role of circRNA in BPD and its dysregulated biological processes. In the peripheral venous blood of neonates with BPD, 491 circRNAs were markedly altered . SignifiIn recent years, there has been considerable interest in exploring ncRNAs and miRNAs in BPD. Tao et al.  illustraIn a recent study, circRNA expression profiles were investigated in neonatal acute respiratory distress syndrome (ARDS) . In the There is growing evidence that exosome-noncoding RNAs likely contribute to lung disease. Research has demonstrated that exosomes isolated from tracheal aspirates of infants with severe BPD and bronchoalveolar lavage fluids from hyperoxia-treated newborn mice contain reduced miR-876 levels, a miRNA implicated in BPD development . A largeA substantial amount of research has been conducted in the field of cardiovascular diseases on stem cell exosome-based biomarkers, therapy strategies, and drug delivery . The useIn fascinating investigations, circRNAs have been engineered to act as sequestering sponges for miRNAs associated with human diseases ,119. ThiCircRNAs serve as scaffolds for proteins and miRNA sponges, and can affect translation, transcription, and degradation of specific mRNAs. There will likely be wide-ranging developments in the circulating RNA field in the coming years. CircRNA transport, localization, degradation, and biological function will be characterized in greater depth. Though newly emerging, circRNA has been identified as an essential player in lung development and developmental lung diseases. The role of circRNAs as miRNA sponges in the normal development of lung tissue and lung diseases is still being explored, and their precise mechanism of action is still unknown. It is, therefore, necessary to perform more analyses on samples collected from humans to identify circRNAs involved in cardiopulmonary development and related diseases. Further discovery of circRNA and circRNA-miRNA networks offer promising targets for therapies for developmental and pediatric cardiopulmonary disorders. As circRNAs are identified as potential biomarkers for developmental lung disease, more in-depth exploration is needed to demonstrate their relative accuracy and reliability in BPD as well as other developmental and pediatric lung diseases. Much is still unknown regarding the molecular function of circRNA in lung development and diseases. With our increased understanding of circRNA mechanisms, we expect increased opportunities for innovative treatments targeting the many circRNA-based roles in physiological and pathological processes, including those of the developing lung.


Alphaendornavirus are transmitted solely via seed and pollen and generally cause no apparent disease. It has been conjectured that certain plant endornaviruses may confer advantages on their hosts through improved performance  or resilience to abiotic or biotic insult. We recently characterised nine common bean (Phaseolus vulgaris L.) varieties that harboured either Phaseolus vulgaris endornavirus (PvEV1) alone, or PvEV1 in combination with PvEV2 or PvEV1 in combination with PvEV2 and PvEV3. Here, we investigated the interactions of these endornaviruses with each other, and with three infectious pathogenic viruses: cucumber mosaic virus (CMV), bean common mosaic virus (BCMV), and bean common mosaic necrosis virus (BCMNV).Plant viruses of the genus In lines harbouring PvEV1, PvEV1 and PvEV2, or PvEV1, PvEV2 plus PvEV3, the levels of PvEV1 and PvEV3 RNA were very similar between lines, although there were variations in PvEV2 RNA accumulation. In plants inoculated with infectious viruses, CMV, BCMV and BCMNV levels varied between lines, but this was most likely due to host genotype differences rather than to the presence or absence of endornaviruses. We tested the effects of endornaviruses on seed production and seedborne transmission of infectious pathogenic viruses but found no consistent relationship between the presence of endornaviruses and seed yield or protection from seedborne transmission of infectious pathogenic viruses.It was concluded that endornaviruses do not interfere with each other’s accumulation. There appears to be no direct synergy or competition between infectious pathogenic viruses and endornaviruses, however, the effects of host genotype may obscure interactions between endornaviruses and infectious viruses. There is no consistent effect of endornaviruses on seed yield or susceptibility to seedborne transmission of other viruses.The online version contains supplementary material available at 10.1186/s12985-023-02184-y. Alphaendornavirus [Endornaviruses accumulate in their hosts as non-enveloped double-stranded RNA molecules, although it is now thought that they evolved from positive-sense RNA viruses , 2. The rnavirus .Vicia faba, Phaseolus vulgaris), species of Capsicum, and cereals including rice and barley [P. vulgaris), the endornaviruses Phaseolus vulgaris endornavirus (PvEV) 1, PvEV2 and PvEV3 can occur singly or in combination in various lines, but they are not present in all lineages [Endornaviruses occur in certain lineages of many wild and cultivated plant species including, among others, members of the family Fabaceae , a double-stranded RNA (later confirmed to be an endornavirus) conferred a male sterility phenotype [It has been proposed that endornaviruses may provide benefits to their hosts , giving henotype , which ahenotype . HoweverCommon bean is an important crop in East and Central Africa, where it is an essential source of dietary protein and carbohydrates. Common bean is also rich in iron and zinc, which are of vital importance in this region, which has a high incidence of anaemia –19. Mixe−2 s−1 photosynthetically active radiation for 16 h per day. Plants used for measurement of the rate of viral seedborne transmission were grown in a glasshouse maintained at approximately 18 °C during the day and 15 °C at night, with supplementary lights activated between 04.00 and 20.00 when ambient light levels dropped below 150 W/m2.The common bean varieties used in this study were described in a previous paper and sourced through the bean research programmes of the Kenya Agricultural and Livestock Research Organisation (KALRO) in Kenya and the International Centre for Tropical Agriculture (CIAT) in Uganda . A list https://www.dsmz.de/collection/catalogue). Mechanical inoculation using infected plant sap (or mock inoculation with sterile water) onto the first two true leaves of bean plants has been described by Wamonje and colleagues [The infectious viruses bean common mosaic virus  isolate PV-0915, bean common mosaic necrosis virus  isolate PV-0413, and cucumber mosaic virus  isolate PV-0473 were obtained from the German Collection of Microorganisms and Cell Cultures  using a Mettler Toledo AX105 Analytical SemiMicro balance . Seeds were germinated and the first true leaves of the seedlings were sampled and used for DAS-ELISA to detect CMV and BCMV infections. Measurements of seed mass and numbers were analysed using R v.4.2.1in Rstudio  , using oPvActin 11 and PvUnknown 1 (NCBI GI 187435357), using primers designed specifically for RT-qPCR [RNA extractions were performed on samples of approximately 50 mg of trifoliate leaf tissue using the NORGEN Total RNA Purification Plus Kit . The concentrations and purity of RNA extracts were determined spectrophotometrically using a NanoDrop 2000 spectrophotometer . Total plant RNA was heated to 90 ºC and rapidly cooled on ice before being reverse transcribed using GoScript (Promega) with random primers. The cDNA samples were used for quantitative polymerase chain reaction (qPCR) assays using the BioLine SensiMix SYBR No-ROX kit in a CFX Connect Real-Time PCR System  using appropriate primers , including in lines that also harboured PvEV2  or PvEV2 plus PvEV3   , whilst CMV induced decreases in PvEV1 RNA levels in KK022 , SER16  and MCM2001 , and an increase in PvEV1 RNA levels in RWR2245  . However, PvEV2 RNA accumulation was diminished in CMV-infected KK022  and MCM2001  plants , whilst plants of MCM2001 exhibited a decline in PvEV3 RNA accumulation   CMV did not induce changes in PvEV1 RNA accumulation       and of line RWR2245, which contains PvEV1 and PvEV2. In RED40 plants, neither BCMV nor BCMNV affected PvEV1 RNA accumulation . However, when plant lines were grouped 1 to 3 in increasing order of CMV RNA accumulation , showed statistically significant differences between mean values for all three, with the mean value being an order of magnitude greater in Wairimu Dwarf, than in GLP24 Table . The levp = 0.0892).Seedborne transmission of acute viruses can occur at high frequencies in many common bean varieties  and prevPlants of line RED40 (which carries PvEV1) produced the highest numbers of seeds, compared to the other lines tested, and BCMV and CMV infection caused marked decreases in the number but not the mass of seeds Table . CMV infPvEV1 and PvEV3 levels were similar in all lines harbouring these endornaviruses, with PvEV2 being variable in its accumulation. The levels of RNA accumulation for PvEV1, PvEV2 and PvEV3 do not appear to be controlled by interactions with each other. Thus, one of our starting hypotheses, i.e., that synergy or interference occurs between endornaviruses appears not to be correct. This was surprising, since the RNA sequences of these viruses have extensive regions of similarity, which we thought might trigger RNA silencing , 33, andSynergism occurs in plants infected by two or more dissimilar viruses, leading to increased titres for at least one of the viruses, especially if one of the viruses possesses a strong RNA silencing suppressor . CMV encOryza sativa) showed that this virus accumulated to similar levels (c. 100 copies per cell) in various tissues. However, OsEV RNA levels were far higher (c. 1000 copies per cell) in actively dividing suspension cell cultures derived from these plants [Plant host developmental stage profoundly influences viral infection cycles . Our inve plants . It appee plants . In ricee plants , when enAll the viruses included in this investigation can be seed-transmitted. For the endornaviruses the rate is virtually 100% , while BI locus for BCMV resistance or lacking this locus [Our data on the effects of endornaviruses on seed yield contrast with the clear-cut results obtained by Khankhum and Valverde . These wis locus , 43. We is locus . FirstlyIf the second possibility is correct, it may be possible to investigate it further by generating a set of nearly isogenic lines containing various combinations of PvEV1, PvEV2 and PvEV3 for every common bean variety. However, conventional crossing and back-crossing for so many different lines would be time-consuming and impractical. In contrast, by using virus-induced gene silencing it should be feasible to obtain perfectly isogenic lines; for example, by expressing endornavirus-specific RNA silencing sequences using bean pod mottle virus (BPMV) vectors . BPMV woIn common bean the endornaviruses PvEV1, PvEV2 and PvEV3 appear not to inhibit or synergize each other, and only PvEV2 RNA showed distinct inter-line variation in its titre. It is likely that differences in accumulation of the acute viruses CMV, BCMV and BCMNV between lines were due to host genotype rather than to the presence of endornaviruses, and there seemed to be no direct synergy or competition between acute viruses and endornaviruses. Endornaviruses appeared to have no consistent effects on seed numbers or seed mass, or seedborne transmission of acute viruses.Additional file 1. Table S1 Lines used in this study, with information on their complement of endornaviruses. Table S2. Oligonucleotide primer pairs used for reverse transcription-could polymerase chain reactions for the quantification of viral RNA steady-state levels.Additional file 2. Spreadsheet S1 Raw Ct data.Additional file 3. Spreadsheet S2 Seed weight.


For about 30 years, SPEN has been the subject of research in many different fields due to its variety of functions and its conservation throughout a wide spectrum of species, like worms, arthropods, and vertebrates. To date, 216 orthologues have been documented. SPEN had been studied for its role in gene regulation in the context of cell signaling, including the NOTCH or nuclear hormone receptor signaling pathways. More recently, SPEN has been identified as a major regulator of initiation of chromosome-wide gene silencing during X chromosome inactivation (XCI) in mammals, where its function remains to be fully understood. Dependent on the biological context, SPEN functions via mechanisms which include different domains. While some domains of SPEN are highly conserved in sequence and secondary structure, species-to-species differences exist that might lead to mechanistic differences. Initiation of XCI appears to be different between humans and mice, which raises additional questions about the extent of generalization of SPEN’s function in XCI. In this review, we dissect the mechanism of SPEN in XCI. We discuss its subregions and domains, focusing on its role as a major regulator. We further highlight species-related research, specifically of mouse and human SPEN, with the aim to reveal and clarify potential species-to-species differences in SPEN’s function. Drosophila melanogaster as Split ends. Split ends was found to be involved in the development of axons in the peripheral neural system and received its name from a mutant phenotype that includes a distinctive pattern of veins in the wing margin, resembling the split ends of hair . A. A101]. LpSDSDD, SMRT: …YETLpSDSE) . As . As 6]. Considering that SPEN’s IDRs may undergo disorder-to-order transitions or changes in their conformational biases, the properties of this region could change under specific conditions. To gain a comprehensive understanding of this interaction region in the context of its surrounding IDRs, additional studies are required.Spen knockout in Dossin et al. (2020) [Spen deletion suggests a late embryonic lethality phenotype (E12.5), which is later than the embryonic lethality observed in mice with defective imprinted XCI (E8.5). The late lethality could be a consequence of residual truncated proteins, as this was not evaluated, and truncated transcripts were still detectable. Furthermore, the observed defects in the pancreas and heart of Spen mutant embryos and the timepoint of embryonic lethality in Kuroda et al. (2003) suggest an impairment of the NCoR/Smrt and/or Notch signaling pathway. Dossin et al. (2020) then demonstrated that zygotic Spen knockout impairs imprinted XCI. This observation highlighted Spen’s role in imprinted XCI in addition to its known necessity for random XCI in ESCs [Spen knockouts. The investigation of a null mutant embryo could resolve and potentially confirm the time point of embryonic lethality. Furthermore, it could also be compared to the phenotype obtained by the truncating Spen mutation of Kuroda et al. (2003) [Spen null mutants were to reveal a late lethality, it could suggest that in vivo, new and redundant factors might be compensating for the loss of Spen.Recent extensive research on SPEN has unveiled new functions and mechanisms, highlighting its significance in XCI. We summarize substantial in vitro and in vivo evidence, particularly in mice. This allows us to identify interesting areas for future investigation. When examining SPEN’s function in XCI, there remain gaps in our understanding of its mechanism, and its role in vivo is not yet fully clarified. Two targeted SPEN mutations have been engineered in mice, a truncated SPEN deletion in Kuroda et al. (2003) and a zygotic as well as a conditional oocyte-specific . (2020) ,71. The  in ESCs . HoweverWhen analyzing sub-regions of SPEN, it becomes clear that there are still some aspects that remain open for further investigation. The extension of RRM4 (xRRM4), for example, has not been investigated in regard of SPEN’s mechanism in XCI. The α-helix of many other xRRMs could be demonstrated to have a regulatory or direct involvement in the RNA binding ability and mechanism of the respective protein. Notably, mouse and human RRMs share 93.27–100% sequence identity . This hiSPEN’s binding to the H12-H13 hairpin substructure of SRA RNA reveals varying dependencies on its different RRMs. RRM1 and RRM2 are likely not involved, whereas RRM3 and RRM4 are both required for RNA interaction . HoweverRRM1 has been excluded from many functional and structural studies. Observations suggest that RRM1 is dispensable for the interactions studied. However, the high conservation of RRM1 implies that it is involved in the function of Spen and is possibly subject to positive evolutionary selection. Consequently, further investigations into the role of RRM1, especially in the context of XCI, would be interesting.Another open question is which mechanisms or dependencies underlie the specificity of SPEN for XIST RNA RepA. The sequence specificity of SPEN is still a matter of debate. Additionally, XIST RNA RepA exhibits structural features that are likely reoccurring throughout the RNA. Thus, the level of specificity for XIST RNA RepA, as exhibited by SPEN, may not necessarily be expected. Interestingly, RBM15 and RBM15B were demonstrated to not only bind RepA but also other regions of XIST RNA in HEK293 cells [Furthermore, additional factors might be involved in mediating this specific interaction, such as m6A writers WTAP/METTL3 or RBM15 and RBM15B, which also bind to XIST RNA RepA. Along with these factors, the role of m6A modifications of XIST RNA in regard to its binding abilities and structural consequences are not yet well understood. Additionally, it remains unclear if and how SPEN depends on WTAP-METTL3 recruitment or the presence of RBM15 and/or RBM15B for binding to XIST RNA RepA. A further interesting candidate in respect to the binding of XIST RNA RepA by SPEN is YTHDC1. Since YTHDC1 was shown to interact with XIST RNA, RBM15, RBM15B, and SPEN, there may be a direct or indirect role of YTHDC1 in the SPEN–RepA interaction. At present, a substantial knowledge gap remains regarding the exact mechanism underlying the specific binding of XIST RNA RepA by SPEN’s RRMs. Further research into the dependencies of these processes, as well as the interplay of the m6A machinery and SPEN’s role in XCI in both humans and mice, could contribute to filling this knowledge gap.The SPOC domain of SPEN evidently has a clear role in NCoR/SMRT recruitment, which, in turn, leads to the recruitment of HDAC3 for the subsequent deacetylation of the X chromosome. With an amino acid sequence identity of 99.4% between humans and mice , the SPOHdac3 knockout led to a delayed deacetylation of the Xi in mouse ESCs [Interestingly, Spen depletion or knockout was demonstrated to lead to a complete abolishment of XCI, while use ESCs ,30. HDACuse ESCs ,24. ThusCells with XIST RNA RepA deletion were demonstrated to still form a repressive compartment, and RNA PolII is still excluded from the area . Spen orIDRs have remained the least explored regions of SPEN. Notably, there is no study to this day investigating the IDRs of human SPEN. The disorder score of the IDRs is conserved between human and mouse SPEN by prediction. However, human and mouse amino acid sequences only share 63.26% identity . This neInterestingly, the phenotype of SPENΔIDR could be rescued to a large extent by fusing another protein’s IDR to the N-terminal end of SPENΔIDR in mouse cells . The repConcluding this research summary, present evidence suggests and supports a mechanism of SPEN which bridges the XIST RNA A-repeat to repressive machineries like NCoR/SMRT and HDAC3 . For tha


Membrane transporters are critical to the passage of molecules across biological membranes and compartments, including the extracellular and intracellular environments. For this reason, these proteins play a crucial role in a plethora of patho-physiological processes in all organisms and as such are of great interest from the biomedical and biotechnological viewpoints. They include primary and secondary active and passive transporters which exert their function through conformational changes induced by ligand binding or other mechanisms including, among others, allosteric gating, transmembrane potential variation, and ATP hydrolysis.This Research Topic collects scientific contributions concerning the study of membrane transporters, with a specific focus on the characterization of the relationships between their structure, dynamics, and mechanism of action through a combination of experimental and computational techniques. They also include studies on the interactions between transporters and membrane lipids and how these affect the transport mechanism.Hang and coworkers carried out a homology modeling and molecular dynamics study of Sxc−, a Na+-independent amino acid antiporter belonging to the Solute Carrier Family 7 of transporters (SLC7) that regulates the cellular uptake of cystine in exchange for glutamate across the plasma membrane. Sxc− is considered a possible target for the treatment of cancer and neurodegenerative diseases as it is overexpressed in several cancer types and plays a role in neurodegenerative diseases. The authors present an effort to construct high-quality structural models of this transporter in different conformations along the transport pathway, uncovering for the first time the structure of both N- and C- terminal domains, using multi-template homology modeling followed by refinement through molecular dynamics simulations. Validation of the approach has been successfully obtained through comparison with the Cryo-EM structures of Sxc− in the inward-open conformation, which, however, lacks structural information on the N- and C-termini exerting a regulatory role. Therefore, this is a good example highlighting the complementarity between experimental and computational techniques in providing a complete picture of the conformational transitions involved in the translocation of the ligands by membrane transporters.Mishra and colleagues provide additional insights into the mechanism underlying the different functions of two insertases, YidC1 and YidC2, expressed in Gram-positive bacteria. These proteins belong to the YidC/Oxa1/Alb3 family, which is conserved in all three domains of life. To date, the functional differences between the two paralogs have been attributed to their cytoplasmic domains. A combination of molecular modeling, coarse-grained molecular dynamics (CGMD) simulations, molecular genetics, and phenotypic characterization allowed the authors to investigate the coordinated cardiolipin-YidC2 activity in response to acid and envelope stress. In detail, cardiolipin (CL) molecules are proposed to establish different specific interactions with YidC1 and YidC2 dimers. Mutations of two specific orthologous residues affect both the paralog-specific function and the relative CL-interaction profiles. These new results enrich the current vision of the membrane protein insertion machinery of Gram-positive organisms, highlighting how the membrane composition could affect this process.Boakes and colleagues deals with equilibrium nucleoside transporters (ENTs), a major family of nucleoside transporters representing important pharmacological targets. The available crystal structure has several unsolved portions, but it revealed a peculiar outward-facing conformation similar to those of the major facilitator superfamily (MFS) members. However, unlike MFS transporters, ENTs have 11 rather than 12 transmembrane (TM) helices and lack the so-called A motif, which is essential for transport activity in many MFS transporters. Through stability and inhibitor assays, the authors investigate the human ENT isoform 1 (hENT1) variants, both unbound (apo-hENT) and bound to the NBMPR inhibitor. The introduction of a new protocol for bacmid DNA extraction is noteworthy. Variants on ICL6, one of the intracellular loops, and on the adjacent TM7 have been found to stabilize both apo- and NBMPR-bound-hENT1. These variants are proposed to contribute to the intracellular gating and to the interactions with the lipid bilayer. The evidence for the ICL6 importance, with the help of new structural data, could better explain the inhibitor and substrate sensitivity of hENT1.The article by Luo and coworkers reports the cryo-EM structure of Dispatched (Disp), a protein involved in the transport of hedgehog proteins, solved at 6.5 Å resolution. Disp belongs to the resistance-nodulation-division (RND) family, characterized by a pseudosymmetric arrangement of two-halves composed of 6 TMs and a large extracellular domain each. This article is an excellent example of the strategy generally employed for structural studies of membrane proteins, which are mostly difficult to crystallize. The authors first attempt to produce the mouse Disp; then, they turn to homologs from simpler organisms to obtain higher amounts of high-quality recombinant protein. In this case, the water bear Disp proved to be the best choice, and further optimization by deleting predicted flexible regions led to monodisperse particles that could be analyzed by cryo-EM. Structural features of water bear Disp appear to be slightly different from those reported for drosophila and human Disp at the TM regions, suggesting that the former might have been captured in a different conformation. This result is particularly intriguing, with the caveat of the low resolution, because obtaining snapshots of membrane transporters in different conformations is crucial for understanding their mechanism of action.The article by e.g., the lipid bilayer membrane) plays a critical role and has to be taken into account. This Research Topic also offers a view of the challenges involved in the study of these biological systems. Challenges that, these days, can be addressed by combining experimental and computational methods, with the latter being easily exploited to test and suggest new hypotheses.The contributions collected in this Research Topic are good examples of the research focused on the structure and function of membrane proteins, specifically membrane transporters, where the environment (


Narnaviridae. The present study filled a gap in the distribution of narnaviruses and implies their wide distribution in Stramenopiles.Current information on the diversity and evolution of eukaryotic RNA viruses is biased towards host lineages, such as animals, plants, and fungi. Although protists represent the majority of eukaryotic diversity, our understanding of the protist RNA virosphere is still limited. To reveal untapped RNA viral diversity, we screened RNA viruses from 30 marine protist isolates and identified a novel RNA virus named Haloplacidia narnavirus 1 (HpNV1). A phylogenetic analysis revealed that HpNV1 is a new member of the family  High-throughput sequencing (HTS) technology has expanded our knowledge of RNA virus diversity and evolution , Thecofilosea and Imbrecatea (Cercozoa), and Sagenista and Opalozoa  . To the g for 4 min. Taxonomic information on each protist is summarized in Detailed methods are described in the supplemental material. Briefly, 30 protists were cultured with Hemi medium  at 20°C,Since double‐stranded RNA (dsRNA) is a marker of RNA virus infection , dsRNA whttps://github.com/takakiy/FLDS), and cleaned reads were assembled de novo using CLC GENOMICS WORKBENCH version 11.0 (CLC Bio) (Raw sequence reads were processed as previously described (CLC Bio) . To obtaTo identify the host organisms of RNA viruses detected in pooled sequencing, we conducted a RT-PCR analysis targeting the virus sequences. Total nucleic acids were individually extracted from the cells of each isolate with SDS-phenol and used as the template. Two specific primer pairs were used in the RT-PCR analysis, and the products were applied to direct Sanger sequencing.The phylogenetic positions of the identified RNA viruses were elucidated based on a maximum likelihood-based phylogenetic tree using the deduced amino acid sequences of the RNA-dependent RNA polymerase (RdRp) gene. Details on this method and the accession numbers of the sequences used are shown in the supplemental material.Narnaviridae. To identify the host of this narnavirus from pool-2, RT-PCR was performed with two sets of specific primers named Narna-P1 and Narna-P2   B, and se–5) similarities to known protein sequences.To obtain the complete genome sequence of HpNV1, a FLDS analysis of strain YPF1522 was performed. The results obtained for HpNV1 revealed a bisegmented genome consisting of RNA1 and RNA2 A GenBanC. ConserNarnaviridae (Narnaviridae currently includes one genus (Narnavirus), and only two viruses (Saccharomyces 20S RNA narnavirus and Saccharomyces 23S RNA narnavirus) are defined as species in this genus by the International Committee on Taxonomy of Viruses . The phylogenetic tree revealed that HpNV1 was distantly related to the genus Narnavirus. However, HpNV1 clustered together with known viruses in the family Narnaviridae detected in isolates of Trypanosomatid (Euglenozoa), Peronosporomycetes (Stramenopiles), and Plasmodium (Alveolata) and in environmental samples of invertebrates, fungi, and Peronosporomycetes. This result suggests that HpNV1 is a new member of an undefined genus in the family Narnaviridae.To identify the phylogenetic position of HpNV1, we constructed a phylogenetic tree using the RdRp amino acid sequence of HpNV1 and viruses in the family aviridae . NarnaviRNA virus screening from various protist species was performed in the present study, and an RNA virus was detected from only one sample. Not all strains of the same species have RNA viruses, and the prevalence of RNA viruses differ among host species . This miHaloplacidia sp. YPF1522 is a heterotrophic marine protist belonging to Stramenopiles  The First Identification of a Narnavirus in Bigyra, a Marine Protist. https://doi.org/10.1264/jsme2.ME22077Supplementary Material


Studies have found that RNA encoding proteins only account for a small part of the total number, most RNA is non-coding RNA, and non-coding RNA may affect the occurrence and development of human cancers by affecting gene expression, therefore play an important role in human pathology. At present, ncRNAs studied include miRNA, circRNA, lncRNA, piRNA, and snoRNA, etc. After decades of research, the basic role of these ncRNAs in many cancers has been clear. As far as we know, the role of miRNAs in cancer is one of the hottest research directions, however, it is also found that the imbalance of ncRNAs will affect the occurrence of gastric cancer, breast cancer, lung cancer, meanwhile, it may also affect the prognosis of these cancers. Therefore, the study of ncRNAs in cancers may help to find new cancer diagnostic and treatment methods. Here, we reviewed the biosynthesis and characteristics of miRNA, cricRNA, and lncRNA etc., their roles in human cancers, as well as the mechanism through which these ncRNAs affect human cancers. Proteins in the human body are obtained by transcription and translation of RNA, but studies have shown that only 2–3% of the genes in the human body can encode proteins , 2. The MiRNA is one of the ncRNAs that are 22 nucleotides in length and can participate in many biological processes, such as cell cycle, differentiation, development, and metabolism via post-transcriptionally regulate gene expression, and its evolutionarily conserved single-stranded RNA , 4–6. AbCircRNAs are gene-regulatory RNA transcripts with covalently closed circular structures that are highly stable, especially in neural tissues circRNAs , 15. ThiLncRNAs are ncRNA greater than 200 nucleotides, which is relatively abundant in human body. Now thought to play a key role in many cellular processes , includiPiRNA is a single-stranded RNA that interacts with P-element-induced wimpy testis (PIWI) proteins and its about 26–31 nucleotides in length, the sequence is not conserved –45. FirsSnRNA is transcribed by RNA polymerase II (pol II), highly expressed in human and drosophila cell cycle and cell development, and usually exists in clusters in the genome . SnRNA cIn the nucleus, some snRNAs are identified as promoters of mRNA splicing and have nucleolar specificity and are named small nucleolar RNA (snoRNA).ApproximtRNA-derived small RNA(tsRNA)mostly produces stress conditions such as viral infection, ultraviolet radiation, oxidative stress, etc. tSRNA cDysregulation of ncRNAs can lead to a variety of human cancers. At present, some mechanisms have been clear, and most of the mechanisms still need to be further studied  and interferes with alternative splicing. The treatment for this disease is to target repetitive RNA and release MBNL1 from repeats using CUGrepeat-targeting [In addition, abnormal expression of miRNAs in some monogenic diseases, such as miR-145 and miR-146 deletions, can lead to 5q syndrome. Mutations in miR-96 can cause deafness and also coordinate glucose and fat utilization in skeletal muscle and serve as a diagnostic marker for gestational diabetes , 141. SnWith the deepening of ncRNA research, many diseases have ncRNA-based therapies. Compared with traditional therapy, this kind of therapy has many advantages, such as the drug can be delivered to the target cells, and the effect of the drug is more accurate. Traditional antitumor drugs have more adverse reactions such as nausea and bone marrow toxicity, while ncRNA-based drugs have fewer adverse reactions and are more easily accepted by patients. A variety of approaches are currently available, and a few are briefly described below: oligonucleotide-based therapeutics have attracted much attention as potential therapeutics. Compared with small molecule drugs, it is easier to find molecules that inhibit the function of proteins . AntisenThis article mainly introduces the influence of ncRNAs on the occurrence and development of several human cancers. NcRNA plays a very important role in cancers, and people are more and more interested in ncRNAs affected by human cancers. Therapists based on ncRNAs also encourage scientists to constantly explore the mechanism of ncRNAs playing their pathological role, but this is a long way to go, and the known mechanism is no more than the tip of the iceberg, which still needs continuous efforts. As the saying goes, “The road is long, and I will go up and down.“ One of the main challenges for further research is to identify functional ncRNAs in the human genome. The current second-generation sequencing methods, such as RNA sequencing, will provide detailed information on the ncRNA transcriptome . In addi