A hallmark of Huntington’s disease (HD) is a prolongedpolyglutaminesequence in the huntingtin protein and, correspondingly, an expandedcytosine, adenine, and guanine (CAG) triplet repeat region in themRNA. A majority of studies investigating disease pathology were concernedwith toxic huntingtin protein, but the mRNA moved into focus due toits recruitment to RNA foci and emerging novel therapeutic approachestargeting the mRNA. A hallmark of CAG-RNA is that it forms a stablehairpin in vitro which seems to be crucial for specific protein interactions.Using in-cell folding experiments, we show that the CAG-RNA is largelydestabilized in cells compared to dilute buffer solutions but remainsfolded in the cytoplasm and nucleus. Surprisingly, we found the samefolding stability in the nucleoplasm and in nuclear speckles underphysiological conditions suggesting that CAG-RNA does not undergoa conformational transition upon recruitment to the nuclear speckles.We found that the metabolite adenosine triphosphate (ATP) plays acrucial role in promoting unfolding, enabling its recruitment to nuclearspeckles and preserving its mobility. Using in vitro experiments andmolecular dynamics simulations, we found that the ATP effects canbe attributed to a direct interaction of ATP with the nucleobasesof the CAG-RNA rather than ATP acting as “a fuel” forhelicase activity. ATP-driven changes in CAG-RNA homeostasis couldbe disease-relevant since mitochondrial function is affected in HDdisease progression leading to a decline in cellular ATP levels. Yet, they all have a number of shared molecular propertiesand symptoms, including their mRNAs being capable of forming ribonuclearfoci that sequester proteins such as muscleblind protein 1 (MBNL1),leading to aberrant RNA splicing.8 In the context of HTT exon 1, base pairing with the CAGflanking sequences supports an ensemble of hairpin conformations notlimited to the CAG-repeat region.9 Remarkably,extended CAG-RNA is retained in the nucleus within RNA foci insidenuclear speckles, leading to the sequestration of different RNA bindingproteins.10 The bindingaffinity depends on the CAG-repeat length and, thus, the hairpin foldingstability11 and is associated with diseasepathology.11A key to such developments is acomprehensive understanding ofthe conformation and cellular distribution of HTT mRNA. In vitro experimentsand simulations showed that CAG-repeat RNAs form a stable hairpinwith increasing folding stability for higher repeat numbers due tothe repetitive stable pairing of G and C nucleotides.13 They are classified as membraneless phase-separated organelles thatexhibit liquid-like behavior and could play a functional role in thespatial organization of RNA splicing.14 Jain and Vale showed that CAG-repeat mRNA sequestered in nuclearspeckles is mobile.15 In vitro experimentsfurther revealed that CAG-repeat RNA itself forms clusters in condensatesat concentrations as low as 25 nM.15 However,in these assemblies, the RNA does not retain dynamic behavior, andthe condensates are thus classified as gels. Simulations suggest thatthis self-association behavior can be attributed to intermolecularbase pairing of  unfolded CAG hairpins.16 To explain the high mobility of CAG-RNA in nuclear specklescompared to its self-association in gels, Jain and Vale hypothesizedthat helicases in the nucleoplasm could remodel CAG-repeat RNA basepairing.15Nuclearspeckles contain high quantities of pre-mRNA splicing factors,transcription factors, and 3′-end RNA processing factors.The fact that nuclearspeckles have a liquid-like character andthe ability of CAG triplet repeat RNA to form gels suggest compellingresearch questions regarding CAG-RNA homeostasis: What is the moleculardriving force of CAG-RNA-condensate association? In which conformationis the CAG-RNA sequestered? Do aberrant CAG entanglements/aggregatesform preferentially in the nucleoplasm or in condensates? Is the sequestrationof potentially toxic RNA a cytoprotective mechanism, e.g., by preventingnon-native binding events in either of the environments? If this mechanismis exhausted, could nuclear speckles undergo pathogenic aberrant phasetransitions?In this study, we explore these questions by investigatingtheconformational equilibrium and mobility of CAG-RNA in different cellularenvironments and conditions.20 RNA into HeLa cells at a stock solution concentration of 100 μM (if not stated otherwise),yielding a cellular concentration of approximately 1.4 μM thatlargely exceeds the endogenous CAG-repeat RNA concentration.17 Colocalization with nuclear speckles was determined using the SC35nuclear speckle marker (20 RNA to a scrambled sequence[scrambled (CAG)20] and to randomized sequences of thesame length with a 30 and 50% GC content, respectively. We also includedHTT exon 1 sequences with 17, 49, and 72 CAG repeats. These were employedat lower stock solution concentrations (100–200 ng/μL)as yielded after in vitro transcription and stochastic fluorescentlabeling. While (CAG)20 RNA and all HTT exon 1 constructswere found to be predominantly localized in nuclear speckles, therandomized RNAs formed distinct small foci inside the nucleoplasm,and the scrambled RNA was mainly found in the nucleoli and the cytosol,with some small foci still being observed in the nucleoplasm [sampleimages: Figure S1].First,we investigated how the length, sequence, and flanking regions ofthe CAG repeats determine recruitment to nuclear speckles and howthese properties determine mobility. Therefore, we microinjected fluorophore-labeled(CAG)e marker 1A. We co21 .(CAG)20 RNA showed a significantly higher colocalizationshare with nuclear speckles (45 ± 13%) compared to the scrambledand randomized sequences   values. fmobile is determined by thefraction of fluorescence that recovers throughout the course of aFRAP experiment (20 RNA showed slightly higher mobility compared to the HTTexon 1 RNAs [(CAG)20: 46 ± 24%], and the randomizedand scrambled sequences of equal length showed significantly increasedmobility . Microinjectionsat different (CAG)20 stock concentrations  showed that the mobility measurements were independentof the cellular concentration .To further corroborate these results, we conductedfluorescencerecovery after photobleaching (FRAP) measurements in the differentfoci 1C. Wefoperiment 1C. This periment 1D. The (15 who measured a mobile fraction of 83 ±13% for a (CAG)49 sequence in nuclear speckles, the CAG-RNAsinvestigatedhere showed smaller mobile fractions . In their earlier study, Jain and Vale conjugated theCAG-RNA with 12 MS2 hairpin loops binding to yellow fluorescent protein-labeledMS2-binding protein. In comparison to the Alexa Fluor dyes used here,their bulky detection system could potentially affect the phase behavior,e.g., by diminishing intermolecular CAG interactions in the condensate.In comparison to earlier studies by Jain and Vale,In summary, the colocalization and FRAP studies both show thatan increased association of CAG-RNA sequences with nuclear specklesis accompanied by a decrease in mobility suggesting stronger associationwith the condensate. Association and interaction with nuclear specklesare specific to the CAG repetition motive and enhanced by the flankingsequences of HTT exon 1. For the here-studied HTT exon 1 constructs,these properties are independent of CAG-repeat length.20 RNA as a model system to analyze its conformational stabilityas a function of subcellular localization. We used fast relaxationimaging (FReI) that allows us to measure the folding kinetics andthermodynamics of biomolecules in cells with high spatio-temporalresolution by combining fluorescence resonance energy transfer (FRET)microscopy with fast temperature jumps induced by an infrared (IR)laser.24 The technique was previously used to study foldingand phase behavior of superoxide dismutase 1,26 conformational dynamics and aggregation of Huntingtin exon 1,28 or the folding stability of 4U-RNA in the nucleus and cytoplasmof cells.29Based on these results, we proceeded to study (CAG)30 we prepared (CAG)20 RNA for FReIexperiments by terminal FRET labeling using Alexa Fluor 488 at the5′-end and Alexa Fluor 594 at the 3′-end. Dyes werecovalently connected to the RNA backbone via C6 linkers and by alkyne–azideclick chemistry (5′-end) or amine coupling to a carbonic acidN-hydroxy succinimide ester (3′-end) (30 We found that the (CAG)20 RNA forms stable hairpins inDulbecco’s phosphate buffered saline  buffer solution ,30 whichwas in agreement with previous studies.31 However, thefolding stability was significantly decreased in the presence of cosolutesdue to various factors such as transient chemical interactions33 or a decrease in water activity,34 leading to the question whether(CAG)20 RNA remains folded in the densely crowded cellor upon sequestration in nuclear speckles.In preliminary work,(3′-end) 2A. We ch(3′-end) 2B, inset20 RNAinto HeLa cellsand measured its folding stability by FReI  , and the standard free energy of unfoldingat 37 °C: ΔGu°Cθ,37 = (310 K– Tm)·g(1).35 ΔGu°Cθ,37 was used to calculate the equilibrium constant K and folded and unfolded fractions of the RNA at 37 °C  with up to 1.5–2 μm of spatial resolution in live cells.We therefore microinjected (CAG) by FReI 2A. Analy by FReI 2B allowe20 RNA inDPBS under native and destabilizing conditions . Instead of the commonly used sigmoidal fittingprocedure which requires fully resolved folded and unfolded statebaselines, we used a previously established a kinetic method to analyzein-cell melting curves with a limited accessible temperature range.35 This method allows for a thermodynamic analysiswith only a few data points recorded beyond Tm (The model is based on the two-stateassumption which is supportedby single-molecule FRET measurements of (CAG)nditions S3. InsteTables S2 and S3, and ΔGu°Cθ,37 values arecompared in 20 RNA was largelydestabilized in the cellular environment compared to dilute DPBS buffersolution . Despite the major destabilization,it is important to note that the RNA was still mostly folded underphysiological conditions = 6.8 ± 2.1 kJ/mol, meaning that only 6.7% of the RNA was unfoldedat 37 °C). The decrease in ΔGu°Cθ,37 was also large compared to that under any in vitro conditionmeasured previously  = 18.0 ± 1.0 kJ/mol; ΔGu°Cθ,37(DPBS + 300 g/L ethylene glycol) = 12.0 ± 3.0 kJ/mol;ΔGu°Cθ,37(HeLa cell lysate)= 13.3 ± 0.5 kJ/mol).30 Thus, crowdingeffects such as the decrease in water activity or non-specific interactionsof the RNA and cosolutes could not solely account for the observeddecrease in folding stability.The results are summarized in Salmonella fourU RNA thermometer, was studiedfor comparison to CAG hairpins. The secondary structure in comparisonto that of a CAG hairpin is shown in Figure S4A,B. The RNA is located in the 5′-untranslated region (5′-UTR)of the aggregation–suppression protein and functions as a temperaturesensor in free-living microorganisms.37 lm4U* wasshown to fold in a two-state manner in HeLa cells,29 and since it is a prokaryotic RNA functioning as a temperature-sensitivecontrol element in free-living organisms, it is not engaged in specificcellular functions in HeLa cells. lm4U* is marginally stable in DPBSat 37 °C  = 0.3± 0.6 kJ/mol) and slightly destabilized in cells ΔGu°Cθ,37(cytosol) = −1.3 ± 1.0 kJ/mol, ΔGu°Cθ,37(nucleus) = −2.1 ± 0.8 kJ/mol) ]. We founda similar ΔGu°Cθ,37 in nuclear specklesand the bulk nucleoplasm = 6.0 ±1.4 kJ/mol; ΔGu°Cθ,37(nuclearspeckles) = 6.4 ± 1.6 kJ/mol (20 RNAassociates with nuclear speckles in the folded rather than unfoldedhairpin conformation (92.3% of the RNA is folded in nuclear speckles)and that condensate association is not associated with a conformationaltransition under physiological conditions.We then analyzed the folding stability in nuclearspeckles for(CAG)ondition S5]. We f20 RNA is enrichedin nuclear speckles upon stepwise heating . In particular, at temperatures beyond the meltingpoint where the RNA is mostly unfolded, strong migration to the fociwas observed . Wefound that the association of the RNA with nuclear speckles is partiallyirreversible as the partitioning coefficient (PC) between the nuclearspeckles and surrounding nucleoplasm does not recover its initialvalue at 37 °C . Sincethe RNA unfolds reversibly in a two-state manner in the cytoplasmand nucleoplasm and also under various crowded conditions in vitro,it is not expected that irreversibly misfolded CAG entanglements causethis effect. We analyzed single temperature jumps of FReI experimentsperformed in nuclear speckles at high temperatures .Despite this high temperature, (CAG)20 RNA unfolding showedno indication of self-association due to intermolecular FRET , which would be expected in thiscase due to the high ratio of labeled to endogenous CAG-RNA. In fact,FReI is very sensitive to such self-association events upon unfolding.41 Measurements of (CAG)20 RNA in concentrated adenosinetriphosphate (ATP) solutions (15 and 20 mM) led to microscopicallyvisible aggregates, showing that self-association could be indeeddetected for the (CAG)20 RNA construct . Thus, the data suggest that CAG-RNA entanglementsdo not form in speckles (or the nucleoplasm), even at high temperaturesand at concentrations much higher than those of endogenous CAG-RNAlevels.However, we observedthat the (CAG)15 we investigated the role of ATP in determining(CAG)20 RNA folding stability and mobility in specklesand the nucleoplasm. We first conducted in vitro experiments in theabsence of ATP-dependent helicases and other cofactors or bindingpartners. We found that (CAG)20 RNA is increasingly destabilizedwith rising ATP concentrations  = 20.7 ± 1.0 kJ/mol; ΔGu°Cθ,37(DPBS + 5 mM ATP) = 19.6 ± 0.5 kJ/mol; ΔGu°Cθ,37(DPBS + 7.5 mM ATP) = 17.1 ± 4.9 kJ/mol;and ΔGu°Cθ,37(DPBS + 10 mM ATP)= 12.5 ± 0.3 kJ/mol) (S4A). In comparison,lm4U* is slightly stabilized at 5 mM and also largely destabilizedat 10 mM  = 0.3 ±0.6 kJ/mol; ΔGu°Cθ,37(DPBS + 5 mM ATP)= 1.3 ± 1.0 kJ/mol; and ΔGu°Cθ,37(DPBS + 10 mM ATP) = −1.2 ± 0.9 kJ/mol) , kJ/mol) 3B. All eGu°Cθ,37 = 3.0 ± 0.3 kJ/mol; ΔGu°Cθ,37 = 2.4 ± 0.3 kJ/mol; see Figure S4), suggesting that the triphosphategroup is required to mediate the destabilization. In accordance withsimilar observations regarding proteins,42 the triphosphate could increase the solubility of ATP, thus facilitatingits interaction as a cosolute with the RNA. Furthermore, the extentof destabilization of (CAG)20 RNA by ATP is remarkablesince similar ΔGu°Cθ,37 values wereonly found for small cosolutes like ethylene glycol, PEG 200, or sucrose.30 However, this destabilization occurred at significantlylowered water activity and cosolute concentrations that are about50-fold higher. Neither crowding nor ATP destabilization alone couldmimic in-cell folding stability, but remarkably, we found that solutionscontaining crowding agents  in addition to 10 mM ATP ,44 and (CAG)20 RNA folding stability was assessed at theminimum ATP level. The normalized FRET ratio decreased from 1.0 to0.6, corresponding to a decrease in the cellular ATP concentrationby 2–3 mM.45 As ATP levels are estimatedto be 2–3 mM in human cells,46 itis assumed that ATP is completely depleted from the cells. Indeed,we found that ATP depletion caused a minor stabilization of (CAG)20 RNA by ΔΔGu°Cθ,37 ≈1.5 kJ/mol in both the cytosol and nucleus  = 8.5 ± 0.8 kJ/mol, ΔGu°Cθ,37 = 7.5 ± 1.5kJ/mol) of HeLa cells  ≈0.5 kJ/mol, ΔΔGu°Cθ,37(nucleus)≈ 1.1 kJ/mol) 20 and HTT exon 1 RNA recruitedto nuclear speckles  .15Moreover, ATP depletion led to a significant decreasein mobility(10–20%) of (CAG)8 ±15%] 3D. Remar15). Fromthis hypothesis, it can be deduced that unfolding of RNA hairpins,e.g., by RNA helicases should lead to decreased hairpin folding stability.However, since our in vitro results showed that ATP largely decreasesCAG hairpin folding stability as a cosolute in the absence of RNA-unwindinghelicases, we propose that the measured changes in both folding stabilityand mobility may be caused by direct interactions of ATP with theRNA.These results are in line withthe observations by Jain and Valewho found mobile fractions of 83 ± 13% under normal and 23 ±7% under ATP-depleted conditions. In their study, Jain and Vale suggestedthat ATP depletion led to a decrease in mobility due to a declinein activity of RNA chaperones with unwinding helicase activity 20 and lm4U* comprise 18 and 14 base pairs, respectively.In the case of (CAG)20, all of these are highly stableC–G pairs. Thus, it can be assumed that DEAD box chaperonesdo not modulate the folding stability and mobility of CAG repeat hairpins,especially not for the more elongated and stable hairpins linked tothe disease. Mobility and folding stability of these RNAs are rathergoverned by ATP as a cosolute.To further test this hypothesis, we performed in vitroexperimentsemploying three different DEAD box helicases .Indeed, we found that the helicases did not change RNA folding stabilitycompared to the respective buffer solution 4. This cTable S4) and different temperatures to reproduce the experimental conditions. Aseven small RNAs of 50–80 nucleic acids tend to fold on themillisecond regime,49 it is currently impossibleto simulate reversible (un)folding via conventional atomistic MD simulations,even though coarse-grained simulations are possible.50 Instead, we performed independent equilibrium MD simulationswith two different starting conformations—the native, foldedhairpin (named “native state” from here on) and a partiallyunfolded, unwound hairpin-like state . Final structuresfrom all simulations with ATP molecules surrounding the RNA are shownin S11. In the absence of ATP, native hairpin RNAwas stable at 300 K but destabilized at 340 and 380 K. Specifically,with increasing temperature, there was greater variation in the chitorsion angles which characterizes the relative nucleobase/riboseorientation . In the unwoundRNA, the presence of ATP led to fewer fluctuations in the root-mean-square-deviation(RMSD) with respect to the average nucleic backbone structure at 300K . The time required for theRMSD to plateau is reduced in the presence of ATP at 300 and 380 K.This indicates that the backbone of the unwound RNA stabilized fasterunder these conditions. At 340 K and the 5 mM ATP concentration, theunwound RNA backbone also stabilized faster. However, with a 10 mMATP concentration, the backbone RMSD stabilized after ∼450ns, similar to that observed in the absence of ATP  simulations. We used ATP and salt concentrations. To investigateand quantify this formation of hydrogen bonding interactions betweenATP and RNA moieties, we used a donor–acceptor distance cutoffof 0.4 nm and a minimum donor–hydrogen–acceptor angleof 120° as the criteria . In all cases, ATP formed,on average, more hydrogen bonds with the atoms involved in Watson–Crickbase pairing rather than with the RNA backbone’s phosphateor ribose moiety. Preferential formation of ATP–nucleobasehydrogen bonding interactions in the unwound state relative to thenative state suggests that ATP destabilizes the RNA hairpin. An exceptionis at 5 mM ATP and 380 K, where the average number of ATP–nucleobasehydrogen bonds for native and unwound states was roughly the same.However, the average number of ATP–ribose hydrogen bonds inthe unwound RNA was larger, and so the overall number of ATP-RNA interactionsincreased. The high local concentration of ATP close to the backbonealso affects the surrounding hydration shell and biomolecular stability,53 as the phosphate groups of the backbone offer direct hydration sidesand interactions sites for the counter ions and ATP.53 Quantifying these hydration shell changes would be exciting futurework by complementing, e.g., small-angle X-ray scattering of time-averagedcounterion distributions with MD simulations.54We also observed an opening of the hairpin andexposition of thenucleobases for intermolecular binding interactions with ATP see 5B. Previ20 RNA caused by preferential interactions of ATPwith the nucleobases in the unwound state, which is different froman entropically stabilized unwound state caused by elevated temperatures.This finding is in line with results reported in the literature andfurther corroborates our experimental observations.To summarize, the MD simulations show an enthalpicdestabilizationof (CAG)12 Here, we investigated CAG-RNA recruitment to nuclear speckles underdifferent cellular conditions as a function of RNA conformation andmobility. First, the comparison of (CAG)20 RNA with randomizedand scrambled CAG sequences corroborated the CAG-repeat sequence specificityfound in earlier studies.55 The comparison of (CAG)20 with HTT exon 1 RNAs showed that the flanking sequencesslightly increased the colocalization with nuclear speckles and decreasedthe mobility. This could be explained by previous studies that suggesteda profound role of the flanking sequences in HTT exon 1 folding, inparticular suppressing CAG hairpin formation at low CAG repeat lengths.9 The authors suggested that a GCUGC moiety locatedin the 5′ UTR and the (CCG)7-10 and GCUGCUGCmotifs in the flanking regions could provide additional binding valencyto form an intermolecular RNA network. A high binding valency is commonlyassociated with biomolecules that engage with biomolecular condensates.56 Given the high RNA concentration inside thenuclear speckles,57 this would explain the decreasedmobility and the increased association with nuclear speckles. Alternatively,the same binding valency could also be used to form extended hairpinstructures which could then be bound by hairpin-binding proteins suchas MBNL1 or MID1.11Sequestration and retention of RNA in nuclearspeckles are a hallmarkof several triplet repeat expansion diseases.n = 17) and above  the pathogenic threshold.This is, however, in agreement with fluorescence in situ hybridizationstudies reporting mostly small differences in the partitioning ofCAG-RNA between the nucleoplasm and nuclear speckles across the pathogenicthreshold.55 Significant differences were only observed for very high repeatexpansions (>120) that are only rarely found in patients.59 Our results thus suggest that CAG repeat RNA recruitment to nuclearspeckles itself may not be directly related to disease.55 However, pathogenic processes may still be inducedby elongated repeats in speckles. Furthermore, the repeat thresholdfor such processes may be increased to higher repeat numbers due tosomatic expansion and thus not detected in our experiments.60An unexpected result wasthat both the recruitment and mobilityof HTT exon 1 RNA were independent of CAG repeat length, with a similarbehavior of constructs with repeat numbers below  < 0) in comparison to thenucleoplasm (can be shown in a thermodynamic cycle). However, at physiologicaltemperature, we observed that folding stabilities were equal in bothcompartments with the RNAs being mostly folded (>90%), which suggeststhat no conformational transitions occur upon RNA migration. Heatingled to further recruitment of the (CAG)20 RNA to nuclearspeckles, increasing the PC, in particular near Tm withstrongly increasing fractions of unfolded RNA. Importantly, we couldnot observe any homotypic interactions of unfolded (CAG)20 RNA. Thus, in contrast to the hypothesisby Jain and Vale,15 we think that aggregationand gelation of the CAG-RNA  do not occur in cells and nuclear speckles.We suggest that CAG-RNA binding proteins like MBNL11 or MID111 could rather sequesterthe RNA in nuclear speckles. However, due to the high temperaturesapplied to the cells, this process could also be caused by non-physiologicalinteractions such as the binding of aberrant temperature-unfoldedproteins. Thus, experiments under different cell stresses are neededto further investigate this hypothesis.Next, we investigated the conformationsof CAG-repeat RNA recruitedto nuclear speckles. It was expected that RNA may unfold in nuclearspeckles due to its increased binding valency in the unfolded state.As such, previous studies found that single-stranded RNA20 and lm4U*] in the cell, witha CAG-specific function in enabling recruitment of the RNA to andits mobility in nuclear speckles. For the RNAs investigated here,MD simulations and in vitro experiments showed that the effect canbe attributed to a direct interaction of the nucleobases with ATPmolecules, rather than ATP acting as ‘a fuel’ for RNA-unwindinghelicase activity.15 However, on the cellularlevel, it is important to note that the results do not exclude synergisticunfolding by helicases or unfolding by ATP-dependent RNA-binding proteins.The metabolite ATP emergedas a crucial factor to promote unfoldingof RNAs [both for (CAG)46 exceeds the level requiredto sustain energy-consuming processes by a factor of up to 1000.61 In line with this hypothesis, previous studies showed that elevatedATP levels could increase protein solubility and maintain the liquid-likeproperties of nucleoli,64 stress granules,65 and, in general, the cytoplasm of eukaryotes and prokaryotes.67The function of ATP acting as a cosolute to maintain biomolecularprocesses in cells could explain why the cellular ATP concentration(5–10 mM)20 and lm4U* RNA. However,quantitatively, the effects on (CAG)20 were 5-fold larger,despite the two constructs having a similar number of base pairs.This could be explained by the profound mismatches in the (CAG)20 hairpin which provide more space for ATP to interact withthe RNA’s nucleobases. lm4U* does not contain such mismatchesas it consists of longer stretches of dsRNA. Especially at low concentrations,ATP mainly interacts with the cytosine and adenine nucleobases, furthercorroborating this assumption. A remarkable fact is that in the caseof lm4U*, the in-cell folding stability can be matched by cell-mimickingcrowding agents, whereas for (CAG)20, ATP is additionallyrequired. These findings suggest that ATP-induced destabilizationoccurs also in other hairpins. However, further studies are requiredto test this hypothesis and to investigate possible biological implications.Due to their comparable molecular composition,42 other nucleoside phosphates such as adenosine diphosphate,guanosine triphosphate, or cytidine 5′-triphosphate are expectedto show similar effects, but their impact is assumed to be smallerdue to their lower concentration in cells.In this study, similar trends forfolding stability in the presenceof ATP were observed for both (CAG)69 the changes in CAG-RNAhomeostasis could be linked to disease pathology. We hypothesize thatthe stabilized folded states of CAG-repeat RNA under ATP depletioncould preferentially bind to transcription and translation factorssuch as MBNL1 and MID1, resulting in an upregulation of the expressionof mutant HTT protein. In a self-amplifying mechanism, mutant HTTcould enhance mitochondrial dysfunction, leading to a further declinein ATP levels. In fact, this mechanism would be most detrimental tostriatal neurons (involved in HD disease pathology) with a high ATPcontent as these are most affected by decreasing ATP levels.69The recruitment of RNA to nuclear specklesis specific to CAG-repeatRNA. Although the CAG hairpin is largely destabilized in cells, itremains folded in the cytoplasm, the nucleoplasm, and in nuclear specklesunder physiological conditions. The metabolite ATP is crucial to destabilizethe hairpin and maintain its mobility and nuclear speckle associationby direct nucleobase interactions between ATP and RNA. Since mitochondrialfunction is affected in HD disease progression, leading to a declinein cellular ATP levels,


This binding is regulated by the presence of a second RNA binding protein, ADR-2, which when absent leads to reduced expression of both pqm-1 and downstream PQM-1 activated genes. Interestingly, we find that neural pqm-1 expression is sufficient to impact gene expression throughout the animal and affect survival from hypoxia; phenotypes that we also observe in adr mutant animals. Together, these studies reveal an important post-transcriptional gene regulatory mechanism that allows the nervous system to sense and respond to environmental conditions to promote organismal survival from hypoxia.The ability to alter gene expression programs in response to changes in environmental conditions is central to the ability of an organism to thrive. For most organisms, the nervous system serves as the master regulator in communicating information about the animal’s surroundings to other tissues. The information relay centers on signaling pathways that cue transcription factors in a given cell type to execute a specific gene expression program, but also provide a means to signal between tissues. The transcription factor PQM-1 is an important mediator of the insulin signaling pathway contributing to longevity and the stress response as well as impacting survival from hypoxia. Herein, we reveal a novel mechanism for regulating PQM-1 expression specifically in neural cells of larval animals. Our studies reveal that the RNA binding protein, ADR-1, binds to  Caenorhabditis elegans (C. elegans), the absence of oxygen can lead to a state of “suspended animation”   , daf-2(m-2(m596) , pqm-1(o1(ok485) . Neural Y75B8A.8 3’ UTR hairpin construct in prab3::GFP::unc-54 3’ UTR (pHH340); prab3::3XFLAG ADR-2 cDNA::unc-54 3’ UTR (pHH438)), HAH24 (BB20 + blmEx19(Y75B8A.8 3’ UTR hairpin construct in prab3::GFP::unc-54 3’ UTR Clone vector (pHH340)), HAH25 (BB19 + blmEx20(prab3::GFP::unc-54 3’ UTR (pHH21); prab3::3XFLAG ADR-1::unc-54 3’ UTR (pHH512))), HAH26 (BB21+ blmEx20(prab3::GFP::unc-54 3’ UTR(pHH21); prab3::3XFLAG ADR-1::unc-54 3’ UTR (pHH512))), HAH27 (adr-2(ok735), agIs6[dod-24::GFP]), HAH28 (BB20 + blmEx18(Y75B8A.8 3’ UTR hairpin construct in prab3::GFP::unc-54 3’ UTR (pHH340); prab3::3XFLAG ADR-2 cDNA::unc-54 3’ UTR (pHH438), agIs6[dod-24::GFP]), HAH29 (adr-2(ok735), daf-2(m596)), HAH30 (wildtype), HAH31 (daf-2(m596)), HAH32 (adr-2(ok735)), HAH33 (adr-2(ok735), daf-2(m596)), HAH42 (HAH38+ blmEx19(Y75B8A.8 3’ UTR hairpin construct in prab3::GFP::unc-54 3’ UTR (pHH340))), HAH43 (HAH38+blmEx21(Y75B8A.8 3’ UTR hairpin construct in prab3::GFP::unc-54 3’ UTR (pHH340); prab3::pqm-1::unc-54 3’ UTR (pHH549))), HAH47  CRISPR).Strains generated in this study include HAH23  used standard microinjection techniques and were passaged by selecting worms that contained the GFP co-injection marker. The injection mix contained 20 ng/μl of the co-injection marker and 1 ng/μl of the transgene of interest.rol-6 plasmid (HAH293) and 4μM of repair template ssODN (HH3089)  containing the mutation in ADR-1 dsRBD1(KKxxK-EAxxA). Genomic modifications were verified using Sanger sequencing and ADR-1 expression was verified using western blot.Animals created by CRISPR modification (HAH47) used standard microinjection techniques and were identified as rolling F1 progeny and non-rolling F2 progeny. Injection mix for the ADR-1 dsRBD1 mutant strain included 1.5 μM Cas9 , 4 μM tracrRNA (IDT), 4μM of crRNA (IDT)(HH3088) , 37 ng/μl dod-24::GFP] hermaphrodites to BB20 males, creation of HAH28 by crossing HAH27 hermaphrodites to HAH23 males, creation of HAH29 by crossing daf-2(m596) hermaphrodites to BB20 males, creation of HAH34 by crossing pqm-1(ok485) hermaphrodites to N2 males, creation of HAH35 (wildtype), HAH37 (adr-2(ok735)), HAH38 (pqm-1(ok485)), HAH39 (adr-2(ok735); pqm-1(ok485)) by crossing HAH34 hermaphrodites to BB20 males, creation of HAH40 (BB20 + blmEx19(Y75B8A.8 3’ UTR hairpin construct in prab3::GFP::unc-54 3’ UTR (pHH340))) and HAH41 (blmEx19(Y75B8A.8 3’ UTR hairpin construct in prab3::GFP::unc-54 3’ UTR (pHH340))) by crossing HAH24 hermaphrodites to N2 males, creation of HAH44 (BB20+ blmEx21(Y75B8A.8 3’ UTR hairpin construct in prab3::GFP::unc-54 3’ UTR (pHH340); prab3::pqm-1::unc-54 3’ UTR (pHH549))) by crossing HAH43 hermaphrodites to BB20 males, creation of HAH48 (wildtype), HAH49 (adr-2(ok735)) and HAH50 (ADR-1 dsRBD1 (KKxxK-EAxxA) CRISPR) by crossing HAH47 hermaphrodites to BB20 malesCrosses were performed by putting 9–10 males and 1 hermaphrodite on mating plates and genotyping was performed for the F1 progeny and F2 progeny using primers mentioned in Supplemental Table S2. The specific crosses performed included: creation of HAH27 by crossing agIs6-rab-3>cel_pqm-1, was constructed by VectorBuilder. The vector ID is VB221230–1025zug, which can be used to retrieve detailed vector information from vectorbuilder.com.The vector used to express adr-2(−) neural RNA-seq datasets.Gene set enrichment analysis was performed using the online WormCat software  for upre2PO4, 42.3 mM Na2HPO4, 85.6 mM NaCl, 1 mM MgSO4) solution thrice. The animals were incubated overnight in 1X M9 solution at 20°C. Next day, hatched L1 worms were spun down and washed again with 1X M9 solution thrice.Synchronized first larval stage (L1) animals were obtained by bleaching with 5M NaOH and Clorox solution. After bleach solution was added, animals were incubated on a shaker at 20°C for 7 minutes and then spun down to collect embryos. Collected embryos were washed with 1X M9 buffer  L1 neural datasets generated in . Indexing of the aligned bam files was performed using samtools (v1.3.1) and featureCounts (v2.0.1) was used to generate the raw read counts file. DESeq2 library (v1.26.0) on R studio [N2 and rated in  under acR studio  was used2) values were around 0.99 and at least 7 data points fell within the standard curve. Each cDNA measurement was performed in three technical replicates, and each experiment was performed in three biological replicates.RNA extraction was performed using TRIzol (Invitrogen) reagent and DNA contamination was removed by treatment with TURBO DNase (Ambion) followed by the RNeasy Extraction kit (Qiagen) and stored at – 80°C. Concentrations of the RNA samples and presence of any contamination with organic and protein components was determined using a Nanodrop (Fisher Scientific). For qPCR experiments using RNA from L1 animals, 2 ug of DNase-treated RNA was reverse transcribed into cDNA using Superscript III (Invitrogen) with random hexamers (Fisher Scientific) and oligo dT (Fisher Scientific) primers. For qPCR experiments using RNA from neural cells or COPAS sorted transgenic animals, the whole 12 ul was reverse transcribed into cDNA. Following reverse transcription of RNA from L1 animals, 20 μL of water was added to the cDNA. For RNA immunoprecipitation experiments, 200 ng RNA for inputs and the whole 12 ul of IP samples was reverse transcribed into cDNA and no water was added to the cDNA. Gene expression was determined using SybrFast Master Mix water and gene-specific primers  on a Thermofisher Quantstudio 3 instrument. The primers designed for qPCR  spanned an exon-exon junction to prevent detection of genomic DNA in the samples. Melting curves were generated for all primer pairs used to ensure high quality of qPCR products. For each gene analyzed, a standard curve of eight to ten samples of ten-fold serial dilutions of the amplified product were used to generate a standard curve of cycle threshold versus the relative concentration of amplified product. Standard curves were plotted on a logarithmic scale in relation to concentration and fit with a linear line. Fit  and the 10x objective. Each image was taken with the intestinal cells and head ganglia region in focus. For each trial, exposure time was calibrated to minimize the number of saturated pixels for that set of animals. ImageJ bundled with Java 8 software was used to quantify the total intensity of fluorescence per worm as measured by intensity of each pixel in the selected area of a frame (i.e. the worm).Synchronized L1 worms were washed with IP buffer  containing a mini EDTA-free cOmplete protease inhibitor tablet (Roche) and UV crosslinked (3 J/cm2) using the Spectrolinker (Spectronics). The worms were then frozen into pellets using liquid nitrogen and stored at −80°C. The frozen worm pellets were ground on dry ice with a cold mortar and pestle, and the cell lysate was centrifuged at maximum speed for 10 minutes to remove cellular debris. Protein concentration was measured using Bradford reagent (Sigma) and the entire L1 lysates were added to 25 ul magnetic Protein-G beads (Invitrogen) for preclearing. After incubation for 1 hour on a rotator at 4°C, 500 ug of the L1 lysate from the supernatant was added to 25 ul anti-FLAG magnetic beads (Sigma). After incubation for 1 hour on a rotator at 4°C, protein-bound beads were washed with wash buffer thrice  containing a mini EDTA-free cOmplete protease inhibitor tablet (Roche). A portion of the IP (2/5) was stored in 2X SDS loading buffer and used for immunoblotting. The remaining beads were incubated with 1 ul RNasin (Fisher) and 0.5 μl of 20 mg/ml Proteinase K (NEB) at 42°C and 1200 rpm for 15 minutes in a thermomixer. RNA was isolated and qPCR was performed as described above.Protein lysates were prepared as mentioned above. Protein lysates were boiled for five minutes and 100 ug of IP lysates and 10 ug of input lysates were subjected to SDS-PAGE. The same immunoblot was treated with antibodies against FLAG  and β-Actin  after cutting the blot. Protein bands were visualized using enhanced chemiluminescent detection SuperSignal West Femto Maximum Sensitivity Substrate (Fisher). The immunoblot images without saturation were acquired using Image Lab software (version 6.1.0 build 7) in the BIO-RAD ChemiDoc MP imaging system.2) powder (Sigma-Aldrich) was used to make a 0.1 M stock solution with distilled water and then working stocks of varying concentrations were prepared in 85 mM NaCl. Bleaching according to above mentioned conditions was performed to obtain L1-arrested animals. After washing the hatched animals twice with 85 mM NaCl, 5000 L1 animals per strain were exposed to CoCl2 of varying concentrations in a total volume of 500 ul for two hours. After exposure, the worms were washed twice with 85 mM NaCl to remove any residual CoCl2 and other debris. After washing, 40 worms per strain were transferred to 35 mm NGM plates seeded with OP50 and incubated at 20°C for twenty-four hours. After the incubation, alive and dead worms were counted in triplicates for each strain and three biological replicates for both the transgenic and non-transgenic sets of worms. Genotypes were blinded before the bleaching to reduce bias.These assays were performed as previously described  with sli


Drosophila has been a premier model organism for over a century and many discoveries in flies have furthered our understanding of human disease. Flies have been successfully applied to many aspects of health-based research spanning from behavioural addiction, to dysplasia, to RNA dysregulation and protein misfolding. Recently, Drosophila tissues have been used to study biomolecular condensates and their role in multicellular systems. Identified in a wide range of plant and animal species, biomolecular condensates are dynamic, non-membrane-bound sub-compartments that have been observed and characterised in the cytoplasm and nuclei of many cell types. Condensate biology has exciting research prospects because of their diverse roles within cells, links to disease, and potential for therapeutics. In this review, we will discuss processing bodies (P bodies), a conserved biomolecular condensate, with a particular interest in how Drosophila can be applied to advance our understanding of condensate biogenesis and their role in disease. Arabidopsis [Caenorhabditis elegans [Drosophila [Biomolecular condensates, commonly thought to form by phase separation, are self-organising regions of the cytoplasm or nucleoplasm . Having bidopsis ,9, Caeno elegans ,11,12, Dosophila ,14, miceosophila , and humosophila ,17. Lackosophila .Drosophila is particularly well suited to the study of P bodies in vivo  due to their conservation of key proteins and organ systems as well as their ability to model human disease.Work in yeast, mammalian cell lines (in cellulo), and in vitro systems have been foundational in our understanding of P bodies, detailing their composition ,18, prinDrosophila Schneider 2 (S2) cells [Drosophila cells and tissues to elucidate their function from the molecular to the organismal level.P bodies were first observed in cultured 2) cells ,26 and m2) cells . Whilst 2) cells . P bodieDrosophila intestinal stem cells, in vivo P bodies have been detected, but these were absent in the differentiated daughter enterocyte cells [In mammalian cultured stem cells, P bodies are generally thought to help balance between maintaining stemness and cell differentiation ,29. Simite cells . By reprte cells . P bodieDrosophila testes, the spermatogonia and spermatocytes, contain P bodies [nanos mRNA, which prevents the de-differentiation of the spermatogonia. However, the link between the function of Me31B and P bodies has not been explicitly investigated [In contrast, both the germinal stem cells as well as the differentiated cells in the P bodies ,31. The stigated . This fiDrosophila comes from studies in the egg chamber, containing the oocyte and nurse cells, and early embryo [To date, much of the developmental and organismal-based P body data in y embryo . More bry embryo . For exay embryo . Whilst y embryo , they arDrosophila. To elucidate P body biogenesis in Drosophila, it is critical to understand the entire protein complement of P bodies. While co-immunoprecipitation and interactome analysis of a key P body marker, Me31B, in the egg chamber [Drosophila , Squid (Sqd), Heterogeneous nuclear ribonucleoprotein at 27C (Hrb27C), Exuperantia (Exu), Ypsilon Schachtel (Yps), Lost, and Bruno 1 (Bru1) ,40,42.The CCR4/Not complex, which is localised to P bodies in yeast, mammalian cells, and S2 cells ,13,17, aThe miRNA machinery is localised to P bodies in S2 cells , but thiBelow, we focus on some notable data from Together, this highlights that the P body protein content is not necessarily consistent across cell and tissue types, and this presumably influences the recruitment and regulation of mRNAs.Drosophila P body proteins. Rules 1 and 2 are demonstrated for Drosophila P body proteins in Drosophila proteome [For in vivo phase separation to occur, the protein content of a granule must be governed by certain rules including: (1) a network of interactions between the proteins must exist that are dense and redundant; (2) a significant proportion of the proteins must be able to bind to RNA specifically or non-specifically; (3) a large proportion of proteins must have low-complexity sequences or high levels of intrinsic disorder to allow for weak, non-specific interactions between proteins and RNA . These tproteome .Importantly, there is significant tissue specificity in the protein complement of P bodies, and proteins could be recruited in a cell-dependent context to provide specific functions and likely provide additional contacts to increase the interaction density and RNA binding.Drosophila egg chamber provides a system to test the complexity of such in vivo P bodies. Examples of this diversity include the stoichiometry of resident P body proteins that appears to differ between the oocyte and nurse cells despite these cells being interconnected via cytoplasmic bridges [gurken (grk) mRNA translation only occurs at P bodies located at the dorso-anterior corner of the oocyte [Patient biopsies have shown that P bodies within the same tissue type in humans can also exhibit variation , and the bridges ,40. P bo bridges ,40,42. Se oocyte .Drosophila drive post-translational modifications and the re-organisation of the microtubule network in egg chambers, which leads to the aggregation of the P bodies [Developmental and environmental cues can also dramatically influence P body content, form, and function. In the early egg chamber, nurse cell P bodies have been shown to be rapidly and reversibly enlarged upon the addition of environmental stressors . SimilarP bodies ,49. ThisP bodies .bicoid (bcd) mRNA is localised to the oocyte anterior, where it is similarly stored and translationally repressed in stable P bodies. At this stage, the entirety of the bcd mRNA content is found in P bodies [In mature oocytes, P bodies ,42; thisP bodies . This suDrosophila development when the stable co-localisation between RNA and P bodies dramatically changes, which could help us to understand the properties of condensate disassembly. Egg activation, a universal event that ensures that the oocyte is competent to be fertilised and begin embryogenesis [bcd mRNA, the events of egg activation are hypothesised to release repressed mRNAs that are stored in the core of the P body, thus ensuring the correct spatiotemporal translation [In some diseases associated with RNA dysregulation, RNA can become disproportionately segregated into aggregates . There iogenesis , has beeogenesis ,42,53. Bnslation .oskar (osk) mRNA, and this correlates with the degradation of the mRNA [Later, in early embryogenesis, smaller and more dynamic P bodies reform , and thithe mRNA . Additiothe mRNA . OverallDrosophila neurones, differences in P body components are particularly evident [In mammalian and  evident ,58. A va evident ,60. The Nevertheless, these granules share some functional and material similarities to P bodies in oocytes and embryos and are similarly postulated to act as sites of translational repression until environmental cues lead to their disassembly ,62. NeurProteins are critical drivers of phase separation and P body formation. In vitro, protein properties, including valency ,65, regiDrosophila P bodies exist in multiple tissue types and often have specific protein contents. Disruption of P body proteins has revealed differences in the proteins required for P body formation between Drosophila tissues . Historically, Bruno 1 (Bru 1) has been shown to bind to sites in the 3′ UTR of osk mRNA and mediate oligomer formation [The ormation , which aormation . Recentlormation . Consistormation , Bru 1 iosk transport particles, also exemplify how the physical nature of the phase-separated granule influences its biological function. These particles have been shown to be ‘solid-like’ and this is required to maintain the RNA within the granule. When the condensate properties were experimentally altered to be more ‘liquid-like’, osk mRNA was prematurely released from the transport particle, resulting in mis-localised translation [Drosophila would be useful to identify whether the physical nature of the P body is necessary for their biological function in other contexts.The nslation . SimilarDrosophila S2 cells and nurse cells shows that these P bodies are also highly dependent on RNA for their integrity [Whilst often overlooked, RNA does appear to have a complex role in the biogenesis of biomolecular condensates . RNA hasntegrity ,14. Contntegrity ,44, suggntegrity .Drosophila [Unfortunately, there are only a few well-documented examples of RNAs that associate with P bodies in osophila ,54,95, abcd mRNA when stored in P bodies fits both criteria.Possible ways to identify additional RNAs that are associated with P bodies include particle sorting followed by transcriptomics, which has been successfully implemented in mammalian cells  and the Drosophila are optimal for the in vivo analysis of the sequence and structural properties of RNA that influence phase separation. This is exemplified by studies of polar granules, a specialised population of RNP granules that are required to specify the fate of the future germ cells in a variety of metazoans [Drosophila, these granules share significant protein overlap with P bodies [Once RNAs that localise to P bodies have been identified, etazoans . In DrosP bodies ,98 but, P bodies  as well P bodies . Over 20P bodies , and thoP bodies ,102.Drosophila will reveal whether these are conserved properties of RNA.However, similarly to oocyte and embryonic P bodies, recent research has shown that it is not the RNA but the proteins that are necessary to regulate the nucleation of the polar granules . DespiteWhile condensate assembly has received significant research focus, disassembly is now becoming an increasingly popular avenue of research due to the pathological implications and therapeutic potential. Again, the mature oocyte and early embryo provide an attractive in vivo system to explore the molecular mechanism of disassembly.In the mature oocyte, P bodies have been shown to exist in a stable state, which is thought to be essential for the long-term storage of RNA and sustained translational repression . The preIt seems likely that these mechanisms of disassembly are interconnected in vivo. For example, we know that the calcium rise triggers changes in the post-translational modifications of P body proteins , which cDrosophila enable the combination of genetic power with a wide array of visualisation techniques for proteins and RNAs. This provides an important model system to study the biological relevance of phase separation in vivo and this subsection will cover the available techniques for this investigation.Drosophila—for example, by altering protein binding sites [A wealth of publicly available resources enable the tissue- or cell-specific manipulation of most genes, including their ectopic expression, overexpression, knockdown, and knockout ,124,125.ng sites ,84; adding sites ; changinng sites ; disruptng sites ,102,104;Drosophila enable the high-resolution visualisation of the P body components and experimentation on the biological effect of altering the protein and RNA in P bodies : The premier method to visualise RNA in living cells is through the insertion of specific secondary structures (stem loops) into the RNA of interest . The second component, the coat protein, is conjugated to a fluorophore. When co-expressed in the same cell, the coat protein can bind to the stem loop with high affinity and thus decorate the mRNA in living cells . This teosophila  and has osophila ,129,130.Single-Molecule Fluorescent In Situ Hybridisation (smFISH): The gold standard for the visualisation of mRNA is smFISH. By creating multiple short nucleotide oligomer probes conjugated to a fluorophore , it is pMultiplexed smFISH: Two sets of smFISH probes can be created for the same RNA species: one allows for the visualisation of the 5′ UTR and the other the 3′ UTR. Full-length RNA and mRNA that have undergone 5′ to 3′ decay will have a different spectral signal, thus enabling decay intermediates to be spatially visualised ,132. HowDrosophila [Translating RNA Imaging by Coat Knock-Off (TRICK): This system simultaneously utilises the MS2 and PP7 RNA stem loops and their respective coat proteins. In a single RNA species, the PP7 RNA stem loops are engineered in the open reading frame and the MS2 stem loops are placed in the 3′ UTR. When co-expressed with the coat proteins, the untranslated RNA is labelled by both coat protein fluorophores. During the pioneer round of translation, the ribosome(s) knocks off the PP7 coat protein from the RNA but the MS2 coat protein remains bound. In this way, two spectrally different signals appear from untranslated RNA and one signal from translated RNA ,134. Thiosophila ,134.Drosophila and allows the visualisation of the nascent translation of proteins at the single molecule level. Multiple copies of the GCN4 epitope from yeast are placed at the start of the coding sequence for a protein of interest. Once translated the epitope is recognised by a constitutively expressed cytoplasmic binding partner conjugated to a fluorophore. Multiple copies of the epitope amplify the fluorescent signal, allowing for the visualisation of single molecules of the nascent transcript in real time [SunTag System : This teal time ,140,141.Drosophila has a strong history in modelling a myriad of human diseases granules ,157,158 granules ,161,162.Drosophila [Drosophila modified the α-synuclein-mediated toxicity and locomotor deficiency in adult flies [A major constituent of Lewy bodies, a pathological hallmark of Parkinson’s disease , α-synucosophila ,165,166,osophila . Moreovelt flies .Drosophila will be a useful model to study these and other neuronal diseases [In patients, a rare missense mutation in DDX6 (a core P body component) leads to intellectual disability, developmental delays, and similar dysmorphic features . Examinediseases ,168.Drosophila allows the straightforward expression of patient-relevant mutations and human disease genes. Mechanistically, changes in aggregation and phase separation can be identified and connected to observable neurodegeneration phenotypes at the individual neurone and whole organism level [Drosophila and may help to identify novel therapeutics [The genetic toolkit of sm level ,165,166.apeutics ,157,158.Drosophila are applicable to other complex human diseases, such as cancer, as they are well suited to experimentation on many of the hallmarks of tumorigenesis. At this time, the use of Drosophila in P body-specific cancer biology research is yet to be realised. What we do have evidence for shows that the link between P bodies and cancer is in its infancy, with the role of P bodies being highly dependent on the cell type and mutational history [Drosophila would be a valuable resource to test the role of P bodies in the EMT during normal development and pathogenesis.Similar approaches in  history . Early e history ,170. DroDrosophila [Due to the role of P bodies in RNA metabolism, they appear to be a target for a variety of viruses, which collectively have a devastating impact on human health worldwide ,181,182.osophila ,174,175.osophila ,180,181.osophila ,184 or tosophila .Drosophila could be used to probe for the insect and human anti-viral factors that act in the interplay between P bodies and viruses.A significant number of viruses that impact P bodies use arthropods as a vector—for example, the flaviviruses  , the bunDrosophila lacks the organ system complexity of their human counterparts and data interpretation should reflect this when modelling complex diseases. Despite this, human and Drosophila cells are observed to be strikingly similar in both normal and diseased conditions [Drosophila. Thus, they are particularly well suited to understanding the fundamental mechanisms underpinning intricate human diseases.However, the last common ancestor of humans and fruit flies lived over 600 million years ago  and thernditions . Over 75nditions ,193 and Drosophila is a good model to answer many outstanding questions about P bodies, RNP granules, biomolecular condensates, and phase separation in an in vivo context. This is due to the biologically relevant changes that P bodies undergo during development, the conservation of P body proteins, genetic malleability, and the high-resolution imaging techniques to examine RNA and proteins in living and fixed Drosophila tissue. Looking to the future, the following are major questions in the field of condensate biology that we feel Drosophila is particularly well suited to address:What regulates the assembly and disassembly of condensates in vivo?What effect do proteins, and their specific domains, have on condensate properties?What effect do post-translational modifications have on condensate integrity?How do RNAs, and certain motifs, contribute to the formation of RNP granules?Which RNA structures, sequences, and post-transcriptional modifications affect the ability of RNA to associate with RNP granules?Are the material properties of RNP granules intrinsic to their biological function?What are the biological functions of phase separation?Drosophila will be important in this endeavour.Time will tell whether biomolecular condensates fulfil their promise of having a major role in biology and human disease, and


Correction: BMC Plant Biol23, 527 (2023)https://doi.org/10.1186/s12870-023-04557-5Following publication of the original article , author Chang Liu1,2†, Mengya Cheng1,3†, Chao Ma4, Junfeng Chen1 and Hexin Tan1,2,5*1Department Chinese Medicine Authentication, College of Pharmacy, Naval Medical University , Shanghai, China2Department of Pharmacy, Shanghai Fourth People’s Hospital Affiliated to Tongji University School of Medicine, Shanghai, China3School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai, China4Department of Vascular Disease, Shanghai TCM-Integrated Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China5 Shanghai Key Laboratory for Pharmaceutical Metabolite Research, Shanghai, ChinaFurthermore, the font’s style and size of texts in the images of all figures are updated to be consistent. The correct Figs.


Adenosine deaminases acting on RNA (ADARs) have double-stranded RNA binding domains and a deaminase domain (DD). We used the MS2 system and specific guide RNAs to direct ADAR1-DD to target adenosines in the mRNA encoding-enhanced green fluorescence protein. Using this system in transfected HEK-293 cells, we evaluated the effects of changing the length and position of the guide RNA on the efficiency of conversion of amber (TAG) and ochre (TAA) stop codons to tryptophan (TGG) in the target. Guide RNAs of 19, 21 and 23 nt were positioned upstream and downstream of the MS2-RNA, providing a total of six guide RNAs. The upstream guide RNAs were more functionally effective than the downstream guide RNAs, with the following hierarchy of efficiency: 21 nt > 23 nt > 19 nt. The highest editing efficiency was 16.6%. Off-target editing was not detected in the guide RNA complementary region but was detected 50 nt downstream of the target. The editing efficiency was proportional to the amount of transfected deaminase but inversely proportional to the amount of the transfected guide RNA. Our results suggest that specific RNA editing requires precise optimization of the ratio of enzyme, guide RNA, and target RNA. Genome editing has gained considerable attention in recent years, especially since the development of the CRISPR-Cas9 system . AlthougThe conversion of adenosine (A) to inosine (I), which is read as guanosine (G) during translation, is induced by adenosine deaminase acting on RNA (ADAR) and is the most common form of RNA editing in mammals. ADARs harbor double-stranded RNA binding domains and a deaminase domain (DD). The majority of RNA editing events occur in the central nervous system , and altEGFP) to tryptophan (TGG), with no evidence of off-target editing [The MS2 system is used widely as an RNA binding system . Specifi editing . In subs editing  and upgr editing .EGFP mRNA. Guide RNAs of 19, 21, or 23 nt were positioned upstream and downstream of the MS2-RNA. The highest editing efficiency was 16.6%; however, one off-target editing site was detected in the EGFP mRNA and some editing of the target site occurred in the absence of a guide RNA. Our findings indicate that specific RNA editing requires precise optimization of the levels of deaminase enzyme, guide RNA, and target RNA. In addition, optimization of the promoters in the expression constructs and the tagged peptides adjacent to the MS2-ADAR1-DD might influence the functionality of this system for therapeutic applications.Balancing high-editing efficiency with a low rate of off-target effects is a major issue when considering therapeutic applications of RNA editing . SeveralThe MS2-ADAR1-DD construct used here was described in our previous studies ,16. The We designed 19, 21, and 23 nt guide RNAs that were complementary to the target RNA and positioned them upstream or downstream of the MS2-RNA . The MS25 cells/well. After incubation overnight at 37 °C and 5% CO2, the cells were transfected with varying amounts of the guide RNAs and deaminase using Lipofectamine 3000 , according to the manufacturer’s instructions. At 6 h post-transfection, the transfection medium was replaced with fresh DMEM  containing 10% fetal bovine serum . The amount of transfected EGFP reporter (10 ng) was constant in each experiment.HEK-293 cells were seeded into 24-well plates  at a density of 1.5 × 10At 72 h post-transfection, RNA was extracted from cells using TRIzol reagent (Invitrogen). Briefly, the culture medium was aspirated, the cells were washed gently with 1 × PBS, and then TRIzol (500 µL) was added to each well and the cells were homogenized by pipetting. Subsequently, chloroform (200 µL) was added to the homogenates, followed by mixing and incubation at room temperature for 5 min. The tubes were then centrifuged at 15,000 rpm for 20 min at 4 °C. The clear aqueous phase was transferred to a tube containing 200 µL of ice-cold isopropanol and the samples were mixed by inverting the tubes several times. Subsequently, the samples were centrifuged as described above and the supernatant was discarded. The RNA pellet was washed with 70% ethanol and air dried. Next, the extracted RNA was treated with DNase , according to the manufacturer’s instructions. Synthesis of cDNA was performed using EGFP-specific primers and Superscript III (Invitrogen), according to the manufacturer’s instructions. PCR amplification of the 324 bp product was performed using GoTaq polymerase (Promega), with annealing at 60 °C and extension at 72 °C for 30 s. A total of 40 PCR cycles were performed to generate the desired concentration of PCR product.®; Capitol Scientific, Inc., Austin, TX, USA; Catalog no. 749520-0090). Next, 0.1 × TE (10 µL) was added and the samples were vortexed for 10 min. After centrifugation for 10 min at high speed, the supernatant was collected for use in sequencing reactions. To assess the C/T peak height at the targeted adenosine, antisense sequencing was performed on a 3130xl genetic analyzer  using the reverse PCR primer. The efficiency of RNA editing (%) was calculated from the C/T peak height [PCR products were run on 6% polyacrylamide gels for 20 min in presence of 1 × TBE buffer. The required bands were excised from the gels under low-level, short UV transillumination. Subsequently, the gel pieces were frozen for 30 min at −80 °C and then crushed using a disposable pellet pestle/tissue grinder . Overall, the editing efficiency was higher for upstream guide RNAs than for their downstream equivalents .For the upstream guide RNAs, the editing efficiency was highest (16.6%) for the 21 nt guide RNA and lowest (12.7%) for the 19 nt upstream guide RNA. For the downstream guide RNAs, the editing efficiency was highest (13.2%) for the 23 nt guide RNA and lowest (12.4%) for the 19 nt guide RNA. In the absence of a guide RNA, the efficiency of target sequence editing was 7.5% .Next, we examined the effect of varying the amount of transfected guide RNA on target editing efficiency. HEK-293 cells were transfected with 500 ng of MS2-ADAR1-DD and 250, 500, or 1000 ng of the 21 nt upstream guide RNA. The editing efficiency was highest (12.9%) for 250 ng and lowest (4.4%) for 1000 ng of guide RNA.Off-target editing was also detected in cells transfected with 250 and 500 ng of guide RNA , although it was not detected in cells transfected with 1000 ng of guide RNA .We also examined the effect of varying the amount of deaminase on editing efficiency. HEK-293 cells were transfected with 500 ng of the 21 nt upstream guide RNA and 250, 500, or 1000 ng of MS2-ADAR1-DD. The editing efficiencies at 250, 500, and 1000 ng of deaminase were 3.8%, 8.4%, and 15.9%, respectively .At 500 and 1000 ng, off-target editing was detected 50 nt downstream of the target, with efficiencies of approximately 8.0% and 16.9%. By contrast, off-target editing was almost undetectable in cells transfected with 250 ng of deaminase .Next, we examined the editing efficiencies of different amounts of MS2-ADAR1-DD in the presence of 500 ng of a double-repeated 19 nt 2× upstream guide RNA. Cells were transfected with the EGFP construct, 500 ng of the double repeated guide RNA, and 250, 500, or 1000 ng of MS2-ADAR1-DD. In this case, the target editing efficiency was highest (16.2%) for 1000 ng of MS2-ADADR1-DD, whereas 250 ng of MS2-ADADR1-DD yielded an editing efficiency of 7.3% . Off-tarSubsequently, we kept the amount of deaminase constant at 500 ng and varied that of the double repeated 19 nt 2× upstream guide RNA .The editing efficiency was highest (7.8%) at 250 ng of the guide RNA and lowest (2.8%) at 1000 ng of the guide RNA . Off-tarAA) was edited with an efficiency of approximately 6.7%, whereas the 3′ adenosine (TAA) was edited with an efficiency of approximately 6.6%  of EGFP was examined. To this end, HEK-293 cells were transfected with 500 ng of MS2-ADAR1-DD and 250 ng of the 21 upstream guide RNA. We found that the 5′ adenosine  sites were located within the region surrounding the target site in our current analysis , Sanger In the presence of 500 ng of the 21 nt upstream guide RNA, 250 ng MS2-ADADR1-DD resulted in a target editing efficiency of 3.8% . When thWe found that when we kept the deaminase level constant at 500 ng, there was an inverse relationship between the guide RNA concentration and both the target and off-target editing efficiencies . This fiIn addition to examining the effects of guide RNA length on RNA editing efficiency, we also investigated the editing efficiency in cells transfected with a plasmid expressing a double-repeated 19 nt 2× upstream guide RNA. When 500 ng of the double-repeated 19 nt guide RNA was used alongside 250 ng of deaminase, the target editing efficiency was >7.8%  and it iAA) was edited more efficiently than the 3′ adenosine (TAA)  to an ochre stop codon (TAA), and confirmed that editing of TAA back to TGG switched on EGFP fluorescence in HEK-293 cells . In our ne (TAA) , suggestne (TAA) .The wild-type DD of ADAR2 is more specific than the mutated ADAR2-DD (E488Q), but has a lower editing efficiency ,23. In oRNA editing has promising prospects for gene therapy as it is efficient and has minimal side effects. Optimization of the concentrations of the effector molecules, including the guide RNA and enzymes, is needed for efficient RNA editing. In addition, fine-tuning of effector molecules is required to control off-target editing. Findings of this article shed light on the further optimization of in vivo approaches in mice model for the therapeutic purpose of human disease model as well. Gene delivery by the adenoviral vector can be utilized to deliver these editing factor at tissue level.


The role of immune checkpoints in gastrointestinal diseases.”Immunotherapy is one of the most cutting-edge fields in the current treatment of gastrointestinal (GI) diseases . Immune Qin et al. carried out a comprehensive meta-analysis to compare the efficacy and safety of the neoadjuvant use of ICIs combined with chemotherapy or chemoradiotherapy. Their results indicated that neoadjuvant immunotherapy could significantly improve the prognosis of patients with locally advanced EC, with acceptable toxicity. With regard to those with initially unresectable locally advanced EC, Huang et al. performed a real-world clinical trial and found that immunotherapy can offer patients a chance to receive a radical resection. Conversion surgery following immunochemotherapy was feasible and safe for these patients, with a better radiological and pathological response.Most of the studies in this Research Topic were related to cancer process. Esophageal cancer (EC) is one of the deadliest malignancies due to its late-stage diagnosis, and immunotherapies, represented by ICIs, has gained promising perspectives for the treatment of patients with EC . There iYang et al. reviewed recent findings about above achievements and proposed that adding immunotherapy into neoadjuvant therapy may change the treatment strategy of primary resectable or some metastatic CRC to reduce clinical stage but also to benefit patients to achieve a better local control. To test this hypothesis, the same group conducted a prospective, single-arm trial of long-course chemoradiotherapy combined with concurrent tislelizumab in locally advanced rectal cancer, to explore the safety and efficacy. Their results showed that long-course chemoradiotherapy combined with concurrent tislelizumab in patients with locally advanced low rectal cancer had favorable safety and efficacy, and did not increase the complication rate of surgery. Similar to these results, Chen et al. reviewed completed and ongoing clinical trials with ICIs in the area of gastroesophageal cancer (GEC). They found that ICIs combined with chemotherapy can be an effective first-line treatment and a monotherapy in second-line or more treatment and in maintenance therapy. To achieve a better response, Chen et al. also suggested that current biomarkers for predicting ICIs efficacy should be improved.How about results on the application of ICIs in other locally advanced GI cancer? As one of the most common malignant tumors over the world, treatment strategy involving ICIs has already started in CRC, which has shown favorable outcomes against deficient mismatch repair (dMMR)/high levels of microsatellite instability (MSI-H) CRC . Yang etPeng et al. explored this aspect and found that patients with HER-2-positive advanced gastroesophageal junction cancer received PD-1/CTLA-4 bispecific immunotherapy combined with chemotherapy could achieve a complete remission.In consistent with above notion, one research group explored the expression patterns of immune checkpoints on cancer tissue and peripheral blood T cells in patients with gastric cancer. They found that the expression levels of immunosuppressive markers were significantly increased in cancer tissues and peripheral blood T cells, suggesting that peripheral blood analysis may be an important tool for prognostic assessment of patients with gastric cancer. Based on the co-expression of immune checkpoint molecules on T cells, does combined use of immune checkpoint inhibitors represent a potential promising strategy to improve current efficacy of ICIs? In one case report study, Zhou et al. reviewed findings about the adverse events of ICIs, especially for ICI-related colitis. They proposed that the gut microbiota acted as an important regulator in the pathogenesis of ICI-related colitis, and microbiota modulations like probiotics and fecal microbiota transplantation might be potential therapeutic strategy to treat these adverse events of ICIs.Despite above aspects about the application of ICIs in treating GI cancers and strategies to predict and improve the efficacy of ICIs, toxicity is also a major problem to limit the use of ICIs . Zhou etIn summary, the 12 articles in this Research Topic explore or discuss the application of ICIs in treating GI diseases, and provide potential strategies to predict and/or improve the efficacy of ICIs. Based on the importance of gut microbiota in predicting the efficacy of ICIs and their regulation in ICI-related adverse events , more in


To investigate cost in working hours for initial integration of interictal EEG source localization (ESL) into clinical practice of a tertiary epilepsy center, and to examine concordance of results obtained with three different ESL pipelines.This prospective study covered the first year of using ESL in the Epilepsy‐Center Berlin‐Brandenburg. Patients aged ≥14 years with drug‐resistant focal epilepsy referred for noninvasive presurgical evaluation were included. Interictal ESL was based on low‐density EEG and individual head models. Source maxima were obtained from two freely available software packages and one commercial provider. One physician and computer scientist documented their working hours for setting up and processing ESL. Additionally, a survey was conducted among epilepsy centers in Germany to assess the current role of ESL in presurgical evaluation.P < 0.01. In addition, computer scientist and physician spent a total of 35.5 and 33.0 working hours on setting up the digital infrastructure, and on training and testing. Sublobar agreement between all three pipelines was 20%, mean measurement of agreement (kappa) 0.13. Finally, the survey revealed that 53% of epilepsy centers in Germany currently use ESL for presurgical evaluation.Of 40 patients included, 22 (55%) had enough interictal spikes for ESL. The physician's working times decreased from median 4.7 hours [interquartile range 3.9‐6.4] in the first third of cases to 2.0 hours [1.9‐2.4] in the remaining two thirds; This study provides information regarding expected effort and costs for integration of ESL into an epilepsy surgery program. Low result agreement across different ESL pipelines calls for further standardization. This study provides practical information for epilepsy centers considering clinical use of Electrical Source Localization (ESL).Although an initial substantial investment in personnel working hours is required, cost to benefit is predictable within 4 months.We report a relatively low concordance of results between different ESL programs, highlighting the need for more standardization.A survey in German centers found that half implemented ESL for presurgical evaluation, with costs listed as a main reason preventing use.1Electric source localization (ESL) is a computational analysis method integrating scalp electroencephalography (EEG) and magnetic resonance imaging (MRI) that assists in localizing epileptic EEG activity within a 3D model of the brain. ESL is most commonly applied to interictal epileptiform discharges (‘spikes’) but can also be used to localize ictal EEG patterns.Yet, despite these advantages, ESL is utilized in less than 50% of European epilepsy centers.Thus, this prospective study aimed at assessing the effort in both physician and IT working times required for first‐time integration of ESL into the presurgical workflow of a single tertiary epilepsy center. Additionally, we aimed at comparing the concordance of results between two freely available and one commercial software packages. Finally, we conducted an online survey regarding the current state of ESL implementation in all surgical epilepsy centers in Germany. The significance of this study is that it provides practical, real‐life information that may assist in the decision to integrate ESL in the presurgical routine.22.1We conducted a prospective analysis of patients that underwent noninvasive presurgical video EEG monitoring (VEM) between 1st July 2021 and 30th June 2022 at the Epilepsy‐Center Berlin‐Brandenburg  as part of the Epilepsy‐Center Berlin‐Brandenburg. The number of EEG surface electrodes was 33‐45, depending on the individual focus hypothesis before VEM. All electrodes recommended by the International Federation of Clinical Neurophysiology were included,Using average reference and bipolar longitudinal montages, interictal epileptic spikes were visually identified and marked. Homologous spikes, that is, those with the same configuration and the same topographical maximum, were determined visually and grouped to clusters. Per cluster, a minimum of 20‐30 single spikes was required.2.4http://surfer.nmr.mgh.harvard.edu/)All patients underwent 3‐Tesla structural MRI using a Siemens Magnetom Skyra scanner at Charité – Universitätsmedizin Berlin. 3D T1 images were exported in DICOM format and subjected to automated cortical reconstruction and volumetric segmentation using the freely available Freesurfer image analysis suite version 7.1.1.0.1 (2.5http://neuroimage.usc.edu/brainstorm).https://sites.google.com/site/cartoolcommunity). Epilog NV is a commercial provider of CE‐marked and FDA‐cleared EEG analyses seated in Ghent, Belgium (https://www.epilog.care/). All three approaches used individual head models and linear distributed inverse solutions.Interictal ESL was performed using three different approaches, based on two freely available academic software packages (Brainstorm and Cartool) and one commercial pipeline (Epilog). Brainstorm is a collaborative, open‐source application for EEG and MEG analyses which is documented and freely available for download online under the GNU general public license (https://de.mathworks.com/products/compiler/matlab‐runtime.html). Electrodes were co‐registered manually to the individual head surface. Individual boundary element model (BEM) surfaces were generated using the open‐source software OpenMEEG (http://openmeeg.github.io)Brainstorm version 3.210520 (20‐May‐2021) was running on the MatLab Runtime R2020a (9.8) that is freely available for download online (In Cartool version 3.91 (6638), electrodes were co‐registered manually to the head surface. A 3D grid of about 6000 solution points was distributed within the gray matter mask. The Locally Spherical Model with Anatomical Constraints (LSMAC)Continuous EEG files with markers from visual inspection (EDF) and MRI image files (DICOM) were uploaded to the internet portal of Epilog. Individual six‐layer finite‐differences head models (FDM)Interictal ESL with Brainstorm and Cartool was performed by BJV on a customary PC with 3.20 GHz processor, 8.00 GB installed RAM, Windows 10 Professional x64 bit system, and a single 24‐inch monitor. Since Brainstorm and Cartool are not licensed for clinical use, only Epilog results were regarded for clinical decision‐making.2.6Concordance of different interictal ESL pipelines were assessed by subjecting identical spikes to all three programs. To reduce bias, results were evaluated independently by different authors, with Brainstorm and Cartool results assessed by BJV and interictal Epilog results evaluated by GM. Ictal ESL was evaluated by KG. ESL results were visually assessed on sublobar (19 per hemisphere), lobar, and hemispheric levels.2.7Both authors BJV (neurologist) and TB (computer scientist) documented their working times for implementing ESL and analyzing all eligible cases during the 12‐month study period. Working times were recorded separately for the different software packages and for the different working steps. Tasks independent from patient cases included (1) installation of software, (2) training and testing, (3) troubleshooting, and (4) others. Working steps related to patient cases included (a) processing of the MRI, (b) forward and inverse modeling, (c) marking of spikes, (d) averaging and correction of spikes, (e) evaluating ESL results and preparing a slideshow with screenshots, and (f) others like loading, editing, and zipping files.Working times for each step were documented in minutes and could be rounded to 5‐minute blocks. To obtain results independent from computational power, only time effectively spent working on ESL was documented. Waiting times during computation that could be spent on other tasks were not counted as working times.https://neuroimage.usc.edu/brainstorm/Tutorials).Before June 2021, TB had not been working with ESL or with any of the software packages used. BJV had used Cartool2.8As an addendum to the main study, an online survey was conducted to evaluate the current state of ESL in presurgical epilepsy evaluation and common barriers in Germany in April 2022. Nineteen epilepsy centers with epilepsy surgery programs were identified through the website of the German Society of Epileptology (DGfE) and personal knowledge. An invitation to participate in the survey was sent via e‐mail to the head of each institution and to the head of the respective presurgical evaluation program. It was asked that one person per center completed the survey. Naming the institution was optional; otherwise, the survey was anonymous. If provided, centers' names were separated from all remaining answers before analysis. The survey was cleared by the data protection support team at Charité – Universitätsmedizin Berlin; survey structure and results are detailed in Table 2.9Pseudonymized patient data, ESL results, and working times were recorded and managed using Research Electronic Data Capture (REDCap) tools hosted at Charité – Universitätsmedizin Berlin.κ) for agreement between two software programs, and unweighted Fleiss' κ for determining agreement between three software programs. Values between 0‐0.2 reflect slight agreement, 0.21‐0.4 fair agreement, 0.41‐0.6 moderate agreement, 0.61‐0.8 substantial agreement, and 0.81‐1 almost perfect agreement.The measure of agreement between results of different ESL pipelines was calculated by using unweighted Cohen's kappa . To account for testing multiple variables, statistical significance was set at P < 0.01.Continuous variables are presented as median [interquartile range] and tested with Mann‐Whitney 2.10We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.33.1Forty consecutive patients were eligible for the study, and all agreed to participate. Nineteen were females; age at evaluation ranged from 15 to 61 years. For details, see Table 3.2P = 0.002 , an overall total of 147.1 working hours were spent on ESL by the physician (111.6 hours) and the computer scientist (35.5 hours). For successfully analyzed cases (n = 22), a total of 72.4 hours (median per case of 2.3 hours [2.0‐4.1]) was required by the physician. As the study progressed, processing times became significantly shorter, with the learning curve stabilizing after case #8. Accordingly, the first third of patients required a significantly longer processing time of 4.7 hours median [3.9‐6.4] compared to the remaining patients requiring 2.0 hours [1.9‐2.4]; 2 Figure . FurtherP = 0.002) and the computer scientist .In addition to time directly invested in patient analysis, 33 hours (median 0.9 hours [0.1‐3.3] per month) were spent by the physician in training, testing, and troubleshooting ESL software, and 35.5 hours (median 0.5 hours [0‐3.8] per month) were spent by the computer scientist in setting up system infrastructure and solving technical problems. Again, general ESL training and set‐up were largely done in the initial third of the study period for both the physician , whereas agreement on lobar and hemispheric levels were achieved in 18 (κ = 0.55) and 25 spikes (κ = 0.78), respectively. In examining agreement between different freeware and the commercial ESL pipeline, there were no statistically significant differences on the sublobar level , Cartool and Epilog 12 spikes (κ = 0.14), Cartool and Brainstorm 10 spikes (κ = 0.05)). For additional details, see Figure In the 22 patients eligible for interictal ESL, a total of 30 spike clusters were examined. The median number of single spikes per average was 28 [22‐33]. At the averaged spike's half‐rise, sublobar concordance of all three ESL software pipelines was achieved in six spikes (mean measurement of agreement κ = 0.50) and 16 (κ = 0.32) of 30 spikes on the lobar level, and in 22 (κ = 0.57) and 18 spikes (κ = 0.27) on the hemispheric level, respectively. Epilog results were concordant with visual analysis on the lobar level in 19 spikes (κ = 0.51) and on the hemispheric level in 21 spikes (κ = 0.49) , Epilog's automated spike detection resulted in identification of an additional spike type with a maximum >1 electrode away from that of a manually marked spike, possibly adding additional, previously undetected EEG information. Ictal ESL results, as well as concordance of results for interictal ESL performed at the peak of the averaged spike, are reported in the Appendix 3.4All 19 contacted surgical epilepsy centers in Germany completed the survey . Although all ESL approaches have been previously validated for clinical and research purposes,κ = 0.43 using distributed source models).Our study was neither aimed at nor powered for assessing the diagnostic accuracy of various ESL tools, and clinical outcome measures like postsurgical seizure outcome are not yet available for our cohort. Nevertheless, we compared the results obtained with the different pipelines to each other and found slight agreement only  used ESL in 2014.This study had several limitations. First, in‐house ESL was performed by a single physician (BJV) who had prior training in ESL for scientific purposes, while the commercial analyses were conducted externally in a blinded manner. A potential bias toward higher concordance between the two in‐house pipelines (Brainstorm and Cartool) compared to the external pipeline (Epilog) cannot be ruled out, although pairwise agreement rates were lowest between Brainstorm and Cartool. Second, because BJV had already been familiar with Cartool and Epilog, physician working hours are certainly underestimated. Nevertheless, a learning curve became evident, and working hours at the end of the study period can be considered reliable. Finally, only one head model and one distributed inverse solution were used per pipeline, whereas single dipole models were not regarded at all. This approach limits the generalizability of ESL results but reflects real‐life conditions, and systematic comparison of more variables would not have been realistic.In conclusion, this study shows that implementing ESL in presurgical epilepsy evaluation is feasible in formerly ESL‐naïve centers. One physician with basic understanding of ESL theory and practice, and one computer scientist seem sufficient. This study reports cost in personnel working hours required for initial clinical integration of ESL, providing helpful information for centers interested in use of this technology. Low concordance of results obtained with different software programs and heterogenous importance of the technology in surgical epilepsy centers in Germany highlight the need for additional evidence and more standardization.MH received speaker's honoraria and/or consultancy fees from Angelini, Bial, Desitin, Eisai, Jazz, Neuraxpharm, UCB, and Zogenix within the last 3 years. All other authors declare that they have no competing interests.Table S1Click here for additional data file.Table S2Click here for additional data file.Appendix S1Click here for additional data file.