ASPM and Microcephalin, are related to the probability of speaking a tone language or not. The original study introducing this proposal used a cross-linguistic statistical approach, showing that these associations are “special” when compared with many other possible relationships between genetic variants and linguistic features. Recent experimental evidence supports strongly a negative effect of the “derived” allele of ASPM on tone perception and/or processing within individuals, but failed to find any effect for Microcephalin. Motivated by these experimental findings, I conduct here a cross-linguistic statistical test, using a larger and updated dataset of 175 samples from 129 unique (meta)populations, and a battery of methods including mixed-effects regression (Bayesian and maximum-likelihood), mediation and path analysis, decision trees and random forests, using permutations and restricted sampling to control for the confounding effects of genealogy (language families) and contact (macroareas). Overall, the results support a negative weak effect of ASPM-D against the presence of tone above and beyond the strong confounding influences of genealogy and contact, but they suggest that the original association between tone and MCPH1 might have been a false positive, explained by differences between populations and languages within and outside Africa. Thus, these cross-linguistic population-scale statistical results are fully consonant with the inter-individual-level experimental results, and suggest that the observed linguistic diversity may be, at least in some cases, partly driven by genetic diversity.While it is generally accepted that language and speech have genetic foundations, and that the widespread inter-individual variation observed in many of their aspects is partly driven by variation in genes, it is much less clear if differences between languages may also be partly rooted in our genes. One such proposal is that the population frequencies of the so-called “derived” alleles of two genes involved in brain growth and development,  The meta-information used for matching samples and (meta)populations is contained in the TAB-separated file ./data/genetics/input/populations.tsv, and is used in the pre-processing of the genetic data by the R script ./data/genetics/code/00_preprocesses_genetics.R. For ASPM-D, I added 111 new samples representing 84 unique (meta)populations, while for MCPH1-D, there are 107 new samples from 85 unique (meta)populations populations); the distribution of the original and new samples and (meta)populations is given in HTML analysis reports also plots their maps.The unit (the “group”) for which allele frequency information is given varies between, and even within, these data sources, and cannot be considered ASPM-D in the “Adygei” [PO000017I] sample SA001509P, there is information for A44871G from  and Russian  which correspond to sample SA004603N, or for Modern Hebrew , South Levantine Arabic  and Standard Arabic , which correspond to SA004371P. The full results are reported in the HTML analysis report, but they are very similar to those obtained using the whole set of genetic samples, suggesting that using all the available information is worth the cost of added noise.As this ambiguous mapping may introduce an extra level of uncertainty, for an extra analysis, I removed all the genetic samples that do not uniquely map to a single language. Please note that this procedure is very conservative, given that several “ambiguous” mappings are in fact concordant for tone, as is the case, for example, for WALS Online  is coded as “Moderately complex” in LAPSyD, but as “Simple” in WALS). Moreover, few languages are coded in all sources. Therefore, it would be preferable to arrive at an agreement coding of tone that (a) reconciles any existing conflicts and (b) can retain as many languages as possible. To this end, I implemented the following algorithm.While the agreement between these five sources is very good see , there aLAPSyD ≥ WALS ≥ DL2007 ≥ WPHON ≥ PHOIBLE. The actual rules are coded as patterns of information about a given language in the available sources and the actions to be taken ; in this way, the coding conflicts are implicitly solved by picking the “highest” available source.I implemented a set of rules based on a hierarchy of the sources and the pattern of agreements and disagreements between them; while still subjective, this is fully replicable, transparent and can be easily modified. I preferred to use manually-curated categorical classifications over the count-level sources, resulting in the following (rough) ordering in terms of precedence: LAPSyD ≥ WPHON ≥ PHOIBLE, with the added twist that for the last two (WPHON and PHOIBLE) I do not pick the “raw” value as given by the database itself, but instead a “corrected” value. More exactly, given that I consider the tone counts in LAPSyD as the “gold standard”, I performed first the quadratic regression of the LAPSyD counts on the WPHON counts and, separately, on the PHOIBLE counts, and used the (rounded) value predicted by these regression models from the “raw” value in the corresponding database. These regressions are:L is the predicted LAPSyD count from the “raw” WPHON (W) or PHOIBLE (P) count. This procedure attempts to “align” and “scale” the counts  so that they better map between sources. .I used a similar approach, in that the sources are ordered as With these, I obtained three agreement classifications , that agree very well with the original sources. These have the following distributions see also :binary (321): “No” (251), and “Yes” (70).3-way (314): “None” (248), “Simple” (39), and “Complex” (27).counts (314): “0” (249), “1” (26), “2” (23), “3” (6), “4” (5), “5” (3), and “6” (2).binary (321) versus 3-way and count (314) data because of the different patterns of missing data in the five original sources used, the type of coding they contain, and the possibility of deducing “coarser” codings from more “fine-grained” ones  but not the other way around ; 314 languages have data for all three codings.There are different numbers of languages with HTML analysis report for full details). This alternative can also be justified by the nature and coverage of the sources, and is, for binary and 3-way: WALS ≥ WPHON ≥ LAPSyD ≥ DL2007 ≥ PHOIBLE, and for counts: WPHON ≥ LaPSyD ≥ PHOIBLE. However, on the full dataset, these two agreement codings are very similar , and on the reduced datasets that also include the “derived” alleles they are virtually identical .While arguably justified, the hierarchy of the sources used to derive this agreement classification is only one of the many possible, and to check if it unduly biases the results I compared it to an alternative hierarchy of sources  vs “Yes” (70).tone2: this is a binary variable derived from the 3-way coding of tone by opposing “Complex” tone systems  vs “None” and “Simple” collapsed together .counts: the unchanged tone counts from above.tone tone1 is the direct equivalent of the binary presence/absence coding of tone used in the original paper )the distribution of the two “derived” alleles is also highly skewed geographically, being almost completely absent from Africa.Therefore,as “North America” and “South America” each have very few data points, I collapsed them into a single macroarea, “America”;fixed effects in the non-Bayesian approaches, as random effects (crossed with the families and (meta)populations) in a set of Bayesian mixed-effects regressions, and as a 2-dimensional Gaussian Process (with the families sand (meta)populations) in a different set of Bayesian mixed-effects regressions;in order to control for the influence of the macroareas, I modelled them as because the genetic data suggests that the major split is between “Africa” and the rest of the world, coupled with the fact that some methods cannot gracefully handle multi-valued factors, made me dichotomise, for some analyses, macroarea in into “Africa” vs “non-Africa”.Following , 10, andweak negative influence of the population frequency of ASPM-D on tone1 above and beyond the effects of shared ancestry (language family) and contact (macroarea);there is a no effect of MCPH1-D on tone1 independent of language family and macroarea, or it should be much smaller.there should be tone2 and tone counts), but we might expect a negative effect of ASPM-D:There are no clear predictions concerning the complexity of tone systems  and contact (macroarea)” are rather tricky in this particular case, and should be treated with care. These are indeed potential confounds in any cross-linguistic/cross-cultural/cross-population statistical studies and usually result in artificially inflated  associations , posterior probability p(β < 0) = 0.89, evidence ratio = 7.9, % HDI inside ROPE = 13.3%, pROPE = 0.12; model comparisons to the “null” model  suggest that they are roughly equivalent . For MCPH1-D: β = −0.63, 89%HDI = , posterior probability p(β < 0) = 0.83, evidence ratio = 5.0, % HDI inside ROPE = 15.2%, pROPE = 0.14; model comparisons to “null” suggest rough equivalence . Please note that the posterior probability for the directional hypothesis p(β < 0) should be as large as possible  and the associated evidence ratio as large as possible, the % HDI inside ROPE should be as small as possible , and that pROPE should also be as small as possible  and can be interpreted somewhat like a frequentist p-value by comparing it to a threshold of say, 0.05.Fitting a Bayesian mixed-effects logistic regression model of macroarea as a 2D Gaussian Processes in the Bayesian mixed-effects logistic regression model found that the interaction is not needed, but that each “derived” allele seems to have a negative effect on tone. For ASPM-D: β = −0.88, 89%HDI = , posterior probability p(β < 0) = 0.96, evidence ratio = 25, % HDI inside ROPE = 3.5%, pROPE = 0.065; model comparisons to the “null” model suggest that they are roughly equivalent . For MCPH1-D: β = −1.03, 89%HDI = , posterior probability p(β < 0) = 0.99, evidence ratio = 170, % HDI inside ROPE = 0%, pROPE = 0.011; model comparisons to “null” suggest moderate evidence for MCPH1-D .Modelling IV) = dichotomised macroarea (Africa vs the rest of the world), the outcome (DV) = tone1, and the mediator (MV) = either ASPM-D or MCPH1-D, respectively, as well as the “experimental” Bayesian simultaneous bi-mediated model  of macroarea on tone , p < 2 ⋅ 10−16; MCPH1-D: 0.50, 95%CI , p < 2 ⋅ 10−16), confirming that African languages tend to be more tonal in our database than languages outside Africa, as well as a significant positive average direct effect , p = 0.008; MCPH1-D: 0.55, 95%CI , p = 0.002). However, the average indirect effects (ACME), mediated by the “derived” allele frequencies, differ strongly between them: for ASPM-D, the mediated effect is significant and positive , p < 2 ⋅ 10−16), representing 44.9% of the total effect and resulting from a significant negative effect of being in Africa on ASPM-D , and a significant negative effect of ASPM-D on tone . For MCPH1-D, the mediated effect is not significant , p = 0.49), composed of a significant negative effect of being in Africa on MCPH1-D , and a non-significant effect of MCPH1-D on tone . Taken together, this shows that there’s a positive effect of being in Africa on tone, but that about half of it is mediated by the negative effect of ASPM-D, but not by MCPH1-D; moreover, the frequency of MCPH1-D is shaped much more by being in or outside Africa than that of ASPM-D.Using the full dataset and the “classic” approach, both main models find a significant positive ASPM-D mediating part of the influence of macroarea on tone, but not MCPH1-D; also, macroarea has a much stronger effect on the latter. This procedure does control for language family, but, on the other hand, drastically reduces the sample size to only N = 35 unique families, resulting in a low power of the individual mediation analyses, with relatively few effect sizes being large enough for statistical significance, but even so, there are many more significant indirect effects for ASPM-D  than for MCPH1-D .Restricted sampling  finds the same pattern see , with onz-scored) “derived” allele frequencies as well as the modelling of language family and (meta)population as random effects. It also found a positive TE , and a positive ADE . However, while there seems to be a positive ACME for ASPM-D  resulting from a negative effect of being in Africa on ASPM-D  and a negative effect of ASPM-D on tone , this is arguably absent for MCPH1-D . Simultaneously modelling the mediation of both “derived” alleles produces similar results  and ADE , with an arguably positive ACME for ASPM-D , resulting from a negative effect of being in Africa on ASPM-D  and a negative effect of ASPM-D on tone , but much less convincing evidence of an ACME for MCPH1-D  resulting from a negative effect of being in Africa on MCPH1-D  and no effect of MCPH1-D on tone .The Bayesian approach is more flexible and allows Beta regressions for the (non  results : positivIV) = dichotomised macroarea (Africa vs the rest of the world), the outcome (DV) = tone1, and the mediatorsMV1 = ASPM-D and MV2 = MCPH1-D, separately for the binary and the ordered codings of the IV and the DV .I ran path analyses see  with thep = 0.64; CFI = 1.00, TLI = 1.01, NNFI = 1.01 and RFI = 1.00), and, as shown in tone1, a significant negative effect on ASPM-D which has a negative significant effect on tone1, but while it has a stronger significant negative effect on MCPH1-D this has no effect on tone1. Please note that, for path analyses/SEM models, we want the χ2 goodness-of-fit test to be not significant, meaning that there is no reason to reject the hypothesis that the model fits the data; this is a binary decision. On the other hand, there are several fit indices , which are continuous, the closer to 1.00, the better the model fits to the data.Using the full dataset, the model fits the data very well , and generalises across 100 training/testing sets , and suggests that within Eurasia and Papunesia, the frequency of ASPM-D is a good predictor of tone1 (with p = 0.003). When macroarea is not included, then the fit for the whole dataset drops a bit  and generalises slightly less well ; now, ASPM-D is predictive overall (p < 0.001), and MCPH1-D is relevant (p = 0.004) only for low frequencies of ASPM-D , and the conditional random forests are even better . Of the 3 measures of variable importance, the mean decrease in accuracy for the random forests finds that macroarea > ASPM-D > MCPH1-D, the mean decrease in node impurity for the random forests finds that ASPM-D > MCPH1-D > macroarea, and the unconditional importance for the conditional random forests finds that ASPM-D > macroarea > MCPH1-D.If ASPM-D > MCPH1-D.Leaving macroarea out slightly reduces the fit , but now all three criteria agree that tone1, capturing the presence of any type of tone system, is negatively influenced by each of the two “derived” alleles by themselves , while controlling for these confounds still finds these negative influences but they are now much weaker. . The strengths of the effects of the two “derived” alleles are comparable and are affected in similar ways by controlling for the confounds. However, when properly modelling the difference between Africa and the rest of the world in allele frequencies and tone I found that only ASPM-D has a negative effect on tone above and beyond that due to macroarea and language family, suggesting that the frequency of ASPM-D partially mediates the influence of macroarea on tone. Thus, there seems to be a weak negative effect of ASPM-D on the presence/absence of tone above and beyond the confounding effects of contact and shared inheritance, but less convincingly for MCPH1-D.Thus, tone2 variable, where “Yes” represents complex tone systems.While the original hypothesis in  concernstone2, ASPM-D and MCPH1-D, there are 180 observations, distributed among 118 unique Glottolog codes (languages) in 35 families . There are 29 (16.1%) languages with complex tone (“Yes”) in the dataset (p < 2.2 ⋅ 10−16), and the distribution by macroarea is 37 (9 = 24.3% “Yes”) in Africa, 123 (18 = 14.6%) in Eurasia, 10 (1 = 10%) in America, and 10 (1 = 10%) in Papunesia (between macroareas p = 0.46); see When selecting all unique observations with non-missing data for tone2 and the population frequency of the two “derived” alleles is shown in ASPM-D has a low frequency, which seems to be the case also in Eurasia.The relationship between tone2 is almost completely clustered within families (ICC(tone2) = 95.6%). The interaction between ASPM-D and MCPH-D does not contribute (p = 0.76), macroarea does not predict tone2 , and neither do the two “derived” alleles individually .On the whole data and using the “classic” approach, ASPM-D has a negative effect on tone2 in all conditions, less clear when restricting the permutations within families, but MCPH1-D seems to have a negative effect only for unrestricted permutations when not controlling for macroarea.Permuting the observations , shows tASPM-D on tone2 even when controlling for macroarea and MCPH1-D population (nested within family) as random effects, found that the interaction is not needed, but that each “derived” allele seems to have a negative effect on tone. For ASPM-D: β = −1.27, 89%HDI = , posterior probability p(β < 0) = 0.93, evidence ratio = 14, % HDI inside ROPE = 6.1%, pROPE = 0.055; model comparisons to the “null” model suggest that they are roughly equivalent . For MCPH1-D: β = −0.91, 89%HDI = , posterior probability p(β < 0) = 0.85, evidence ratio = 5.5, % HDI inside ROPE = 10.9%, pROPE = 0.097; model comparisons to “null” suggest rough equivalence . Modelling macroarea as a 2D Gaussian Processes in the Bayesian mixed-effects logistic regression model found that the interaction is not needed, but that each “derived” allele seems to have a negative effect on tone. For ASPM-D: β = −1.15, 89%HDI = , posterior probability p(β < 0) = 0.97, evidence ratio = 33, % HDI inside ROPE = 0.6%, pROPE = 0.044; model comparisons to the “null” model suggest anecdotal evidence for ASPM-D . For MCPH1-D: β = −0.63, 89%HDI = , posterior probability p(β < 0) = 0.95, evidence ratio = 19, % HDI inside ROPE = 6.5%, pROPE = 0.099; model comparisons to “null” suggest that they are roughly equivalent .Fitting a Bayesian mixed-effects logistic regression model of ASPM-D there is a hint of a total effect (TE) of macroarea on tone , p = 0.078), but not for MCPH1-D , p = 0.12). This positive TE for ASPM-D is decomposed non-significant ADE of macroarea on tone2 , p = 0.43) and a positive ACME , p = 0.004), resulting from a significant negative effect of being in Africa on ASPM-D  and a significant negative effect of ASPM-D on tone .Using the full dataset and the “classic” approach, only for TE) and direct (ADE) effects, only ASPM-D shows a positive indirect effect (ACME), but pretty much none of these effects are individually significant.Restricted sampling , finds tTE for ASPM-D  and less clear for MCPH1-D , but arguably no ADE’s . However, while there seem to be a positive ACME for ASPM-D  resulting from a negative effect of being in Africa on ASPM-D  and a negative effect of ASPM-D on tone , this is arguably absent for MCPH1-D . Simultaneously modelling the mediation of both “derived” alleles produces similar results  and ADE , with an arguably positive ACME for ASPM-D , resulting from a negative effect of being in Africa on ASPM-D  and a negative effect of ASPM-D on tone , but much less convincing evidence of an ACME for MCPH1-D  resulting from a negative effect of being in Africa on MCPH1-D  and no effect of MCPH1-D on tone .The Bayesian approach found a possible positive  results : positivp = 0.55; CFI = 1.00, TLI = 1.01, NNFI = 1.01 and RFI = 0.99). As shown in ASPM-D which has a negative significant effect on complex tone; however, while it has a stronger significant negative effect on MCPH1-D, this has no effect on complex tone. The same pattern is also found by restricted sampling, except that here there is also a hint of a negative effect of MCPH1-D on complex tone .The decision trees including or excluding macroarea are the same and trivial in that they uniformly predict just the majority value “No” and have only one split depending on the frequency of macroarea is included as a predictor, the random forests fit the data well , and the conditional random forests are even better , and two measures of variable importance (mean decrease in accuracy and mean decrease in the Gini index) find that ASPM-D > MCPH1-D > macroarea, while the third  finds that ASPM-D > macroarea > MCPH1-D.If ASPM-D > MCPH1-D.Leaving macroarea out has very little effect on the fit , and all three criteria agree that tone2 is very skewed, and the results much less clear-cut than for tone1, but it seems that there is also a negative effect of ASPM-D on complex tone (while for MCPH1-D the evidence is much more ambiguous).Complex tone is much rarer than no tone and simple tone, so that the distribution of counts, ASPM-D and MCPH1-D, distributed among 121 unique Glottolog codes (languages) in 35 families ; see There are 184 unique observations with non-missing data for tone counts and the population frequency of the two “derived” alleles is shown in ASPM-D has a low frequency, but this does not seem to be a linear relationship.The relationship between the tone counts are strongly clustered within families (ICC(tone2) = 65.3%). The macroarea predicts the tone counts , and while ASPM-D has no significant effect by itself , MCPH1-D seemingly does , but this seems to fully overlap with that of macroarea.On the whole data and using the “classic” approach, the tone counts only when not controlling for macroarea and family.Permuting the observations , shows sASPM-D on tone counts even when controlling for macroarea and MCPH1-D population (nested within family) as random effects, found that the interaction is not needed, but that there might be a negative effect on tone for each “derived” allele. For ASPM-D: β = −0.25, 89%HDI = , posterior probability p(β < 0) = 0.93, evidence ratio = 5.2, % HDI inside ROPE = 21%, pROPE = 0.19; model comparisons to the “null” model suggest that they are roughly equivalent . For MCPH1-D: β = −0.24, 89%HDI = , posterior probability p(β < 0) = 0.80, evidence ratio = 4, % HDI inside ROPE = 20.4%, pROPE = 0.18; model comparisons to “null” suggest rough equivalence . Modelling macroarea as a 2D Gaussian Processes in the Bayesian mixed-effects logistic regression model found that the interaction is not needed, but that there might be a negative effect on tone for each “derived” allele, more convincing for MCPH1-D. For ASPM-D: β = −0.22, 89%HDI = , posterior probability p(β < 0) = 0.78, evidence ratio = 3.5, % HDI inside ROPE = 23.7%, pROPE = 0.21; model comparisons to the “null” model suggest rough equivalence . For MCPH1-D: β = −0.41, 89%HDI = , posterior probability p(β < 0) = 0.95, evidence ratio = 21, % HDI inside ROPE = 1.2%, pROPE = 0.058; model comparisons to “null” suggest that they are roughly equivalent .Fitting a Bayesian mixed-effects Poisson regression model of tone TE of macroarea on tone counts , p < 2 ⋅ 10−16; MCPH1-D: 0.69, 95%CI , p < 2 ⋅ 10−16), confirming that African languages tend to be have higher tone counts in our database than languages outside Africa, a non-significant ADE of macroarea on tone counts , p = 0.48; MCPH1-D: 0.44, 95%CI , p = 0.32). However, the ACMEs differ strongly between the two alleles: for ASPM-D, the mediated effect is significant and positive , p < 2 ⋅ 10−16), representing 100.0% of the total effect and resulting from a significant negative effect of being in Africa on ASPM-D , and a significant negative effect of ASPM-D on tone counts ; however, for MCPH1-D, the mediated effect is not significant , p = 0.53). Taken together, this shows that there’s a positive effect of being in Africa on tone counts, but that this is fully mediated by the negative effect of ASPM-D, but not by MCPH1-D.Using the full dataset and the “classic” approach, both models find a significant positive TE) and direct (ADE) effects, only ASPM-D shows a positive indirect effect (ACME), but very few of these effects are individually significant.Restricted sampling , finds tTE for ASPM-D  and less clear for MCPH1-D , clear positive ADE’s . However, while there seem to be a positive ACME for ASPM-D  resulting from a negative effect of being in Africa on ASPM-D  and a possible negative effect of ASPM-D on tone , this is arguably absent for MCPH1-D . Simultaneously modelling the mediation of both “derived” alleles produces similar results  and ADE , with an arguably positive ACME for ASPM-D , resulting from a negative effect of being in Africa on ASPM-D  and a possible negative effect of ASPM-D on tone , but much less convincing evidence of an ACME for MCPH1-D  resulting from a negative effect of being in Africa on MCPH1-D  and no effect of MCPH1-D on tone .The Bayesian approach found a possible positive  results : positivp = 0.59; CFI = 1.00, TLI = 1.01, NNFI = 1.01 and RFI = 1.00), and, as shown in ASPM-D which has a negative significant effect on tone counts, but while it has a stronger significant negative effect on MCPH1-D this has no effect on tone counts. The same pattern is also found by restricted sampling, except that here there is also a hint of a negative effect of MCPH1-D on complex tone .The actual number of tones/tone symbols has a skewed distribution, and the results are less clear, but it seems that there is a negative effect of tone1, tone2 and tone counts on the z-scored population frequency of each “derived” allele separately while controlling for family, (meta)population and macroarea as random effects  are sensitive to the particular choice of prior distributions used. This is a standard technique in Bayesian data analysis  prior for the intercept α and the slopes β, where μ is the location parameter (the median) and σ is the scale parameter, resulting in a relatively wide spread around 0 for z-scored predictors . To this, I compared a set of 13 other priors explicitly chosen for their properties and relevance , suggesting that indeed, the “derived” alleles probably have a negative effect on tone after controlling for family and macroarea. Second, the “default”, the “flat” and the “default normal” priors produce similar estimates for tone1 and tone counts  and slightly less so for tone2, suggesting that the “default” prior does not bias the results too much. Thus, there is enough information in the data to override even the extreme priors (and clearly the “default” prior used), and this information consistently points to a negative effect of the “derived” alleles on the three codings of tone.Given the importance of the Bayesian models in this analysis, I checked if the Bayesian regressions of s  that, fance see  by fittisimr package ASPM-D anS1 FigLAPSyD on the vertical axis and DL2007 on the horizontal axis); please note that the pairs are symmetric, so that the DL2007 vs LAPSyD panel is not shown; likewise, the identity panels  on the diagonal are also not shown. Each panel shows the number of languages with each possible combination of values from the two sources . The shade of blue varies between white (the lowest count) to light blue (highest count). A high agreement between two sources results in little discrepancy between corresponding values .Each panel shows a pair of sources Click here for additional data file.S2 FigThe same conventions as for (TIF)Click here for additional data file.S3 FigThe same conventions as for (TIF)Click here for additional data file.S4 FigThe same conventions as for (TIF)Click here for additional data file.S1 File(HTML)Click here for additional data file.


Acinetobacter baumannii (CRAB) is of great concern in clinical settings worldwide. It is urgent to develop new therapeutic agents against this pathogen. This study aimed to evaluate the therapeutic potentials of compound 62520, which has been previously identified as an inhibitor of the ompA promoter activity of A. baumannii, against CRAB isolates, both in vitro and in vivo. Compound 62520 was found to inhibit the ompA expression and biofilm formation in A. baumannii ATCC 17978 at sub-inhibitory concentrations in a dose-dependent manner. These inhibitory properties were also observed in clinical CRAB isolates belonging to sequence type (ST) 191. Additionally, compound 62520 exhibited a bacteriostatic activity against clinical clonal complex (CC) 208 CRAB isolates, including ST191, and ESKAPE pathogens. This bacteriostatic activity was not different between STs of CRAB isolates. Bacterial clearance was observed in mice infected with bioimaging A. baumannii strain 24 h after treatment with compound 62520. Compound 62520 was shown to significantly increase the survival rates of both immunocompetent and neutropenic mice infected with A. baumannii ATCC 17978. This compound also increased the survival rates of mice infected with clinical CRAB isolate. These results suggest that compound 62520 is a promising scaffold to develop a novel therapeutic agent against CRAB infections.The widespread of carbapenem-resistant  Acinetobacter baumannii is one of the problematic ‘ESKAPE’ pathogens, known to be the leading cause of nosocomial infections and to develop multidrug resistance (MDR) -n-butyric acid) was purchased from Enzo Life Science . Compound 62520 was synthesized at the Korea Research Institute of Chemical Technology (KRICT) [The chemical compounds used in this study were kindly provided by the Korea Chemical Bank ( (KRICT) .6 CFUs/well [A. baumannii ATCC 17978 was cultured with the compounds in MH broth without erythromycin to assess the growth inhibition of the WT strain. The same volume of dimethyl sulfoxide (DMSO)—used to dissolve the chemical compounds—was added into the wells as a control. The plates were incubated for 24 h and the growth of bacteria was measured using a microplate reader  with optical density set at 600 nm (OD600). To determine the growth inhibition of reporter strain OH101 by chemical compounds, the following equation was used: OD600 at 24 h − OD600 at 0 h in the presence of chemical compounds/OD600 at 24 h − OD600 at 0 h in the absence of chemical compounds [Ninety-five chemical compounds were selected based on their structural similarity with compound 62520 from 340,000 small molecules available from the Korea Chemical Bank. Reporter strain OH101 was cultured in LB for 20 h and then the cultured bacteria were inoculated into Mueller-Hinton (MH) broth supplemented with erythromycin (30 μg/mL) to obtain a final population to be transferred to 96-well plates at 6.0 × 10FUs/well . The cheompounds .A. baumannii ATCC 17978 was grown in LB containing compound 62520 with shaking for 24 h. Bacterial cells were harvested and then disrupted by sonication . The samples were centrifuged to remove bacterial debris and the supernatant was harvested by ultracentrifugation at 100,000× g for 1 h at 4 °C. The pellet was resuspended in 10 mM HEPES buffer with 2% sodium lauryl sarcosine  and was incubated for 30 min at room temperature. The samples were then centrifuged at 100,000× g for 1 h at 4 °C. The pellet containing outer membrane fractions was resuspended in a small volume of PBS. The outer membrane proteins (10 μg) were resuspended in the sample buffer used for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were boiled for 10 min to obtain denaturation. The proteins were separated on a 12% SDS-PAGE gel and stained with Coomassie brilliant blue R-250 ; they were then electroblotted onto nitrocellulose membranes to perform western blotting. The membranes were first incubated with a polyclonal anti-rabbit OmpA immune serum [ne serum , and theompA was determined through qPCR. Bacteria were cultured in MH broth containing compound 62520 for 24 h. Total RNA was obtained using the Qiagen RNeasy mini kit , according to the manufacturer’s instructions. Reverse transcription was performed to synthesize cDNA using 1.5 μg of total RNA, random hexamer primers, and M-MLV reverse transcriptase . The specific primers for the ompA and 16S rRNA genes were: 5′-TTG CAC TTG CTA CTA TGC TTG TTG-3′ and 5′-TGG CTG TCT TGG AAA GTG TAA CC-3′ for ompA, and 5′-ACT CCT ACG GGA GGC AGC AGT-3′ and 5′-TAT TAC CGC GGC TGC TGG C-3′ for 16S rRNA. The quantification of gene transcripts was performed using the TOPreal™ qPCR 2X PreMIX (SYBR Green with high ROX) (Enzynomics) on an ABI PRISM 7500 Real-Time System , according to the manufacturer’s instructions. Fold changes in gene expression were calculated using the comparative Ct method and samples were normalized to 16S rRNA expression. The assays were performed in three independent experiments.The expression of 600) was measured using the microplate reader. Planktonic bacteria were removed and the plate was washed twice with 200 μL of PBS. Biofilm cells were stained with 0.1% (w/v) crystal violet solution for 15 min. The biofilms were quantified at OD570 using a biofilm cell-associated dye and were normalized to bacterial growth (OD600). The biofilm assay was performed in triplicate in three independent experiments.The overnight cultures of bacteria, grown in LB without sodium chloride, were diluted 100-fold with the same medium, and then compound 62520 was added to the 200 μL of bacterial suspension in a 96-well plate. Two wells were used for each bacterial strain. Bacteria were incubated for 24 h at 30 °C under static conditions. Total bacterial growth  was acquired by a computer using Living Image Software (Perkin Elmer). All animal experiments were performed in accordance with regulations established by the Institutional Animal Care and Use Committee of Kyungpook National University.Eight-week-old female BALB/c mice were maintained under specific pathogen-free conditions. Mice were intraperitoneally infected with 1.0 × 10x strain . Subsequ9 CFUs and 5 × 108 CFUs of A. baumannii ATCC 17978, respectively, and, 2 h after bacterial injection, they were intraperitoneally injected with compound 62520 (100 μL of injection volume) in the opposite side of the abdomen. Neutropenia had been induced by intraperitoneal injections of cyclophosphamide (150 mg/kg) 4 and 1 day prior to the bacterial injection. The survival of mice was evaluated for 7 days after bacterial injection. To evaluate the effect of compound 62520 on clinical A. baumannii isolates, immunocompetent mice were intraperitoneally infected with 2.0 × 108 CFUs of A. baumannii 2140, and were then intraperitoneally injected with compound 62520 (5.6 mg/kg in 100 μL of PBS) 2 h after bacterial injection. All control mice were injected with the same volume of PBS.In order to evaluate the effect of compound 62520 on survival, immunocompetent and neutropenic eight-week-old female BALB/c mice were intraperitoneally infected with 1.0 × 10t-tests were performed to compare control and experimental groups. Kaplan-Meier curve analysis and log-rank test were conducted to assess the survival rate of mice treated with compound 62520. Differences at p < 0.05 were considered statistically significant.Statistical analyses were carried out using GraphPad Prism 5.0 software . One-way analysis of variance with Dunnett’s post-hoc analysis and Student’s


Enterobacterales (CRE) and Difficult-to-treat resistant (DTR)-P. aeruginosa (PA) present a therapeutic dilemma. Therapies are limited due to antibiotic resistance and inadequate CNS diffusion. Intraventricular polymyxins are utilized in this setting despite a lack in pharmacokinetic data after CNS injection. We describe the utilization of intravenous and intrathecal polymyxin E [colistimethate (CMS)] therapeutic drug monitoring (TDM) in 3 cases of post-neurosurgical meningitis. Central nervous system (CNS) infections caused by carbapenem-resistant Bacterial identification and susceptibility testing were performed using MicroScan. TDM was employed by dosing CMS at 125,000 IU  administered via external ventricular drain twice daily and 4.5 MIU (133.2 CBA or 360 mg) CMS administered over 30 minutes IV twice daily. Four pairs of CSF and blood samples were collected for each patient (Table 1). Samples were placed on ice to minimize in-vitro conversion of CMS to Colistin. Colistin binding in plasma and CSF was measured using ultracentrifugation. Concentrations of CMS and Colistin in CSF and human plasma were determined by liquid chromatography/mass spectrometry. Patients A, B and C received 20, 15, and 12 doses of CMS, respectively, prior to TDM.PA, blaKPCE. cloacae and blaOXA-48K. pneumoniae for patients A, B and C, respectively. Colistin minimum inhibitory concentrations (MIC) were 0.5 µg/ml, 0.125 µg/ml, and 0.125 µg/ml, respectively. The measured CSF and plasma concentrations of CMS, Colistin, and binding are shown in Table 1. Clinical resolution and microbiological cure were attained in all patients. Bacterial cultures revealed DTR Therapeutic Drug Monitoring of Unchanged CMS and Formed Colistin in CSF samples for patient A, B, and CTherapeutic Drug Monitoring of Unchanged CMS and Formed Colistin in Plasma Samples for patient A, B, and CFavorable concentrations of formed Colistin and CMS in CSF were achieved in 3 patients with complicated CNS infection. To the best of our knowledge, this is the first study to report the binding of Colistin in CSF in humans. A TDM method was effectively applied to demonstrate that Colistin achieves and maintains the PK/PD target (fAUC/MIC) [ratio of area under the plasma concentration curve of unbound drug to MIC] that best correlates with killing activity. Overall, our results support intraventricular polymyxins for treating DTR Gram-negative CNS infections.Robert A. Bonomo, MD, entasis (Research Grant or Support)Merck (Grant/Research Support)NIH (Grant/Research Support)VA Merit Award (Grant/Research Support)VenatoRx (Grant/Research Support)


Mus musculus and the first serological evidence of mammarenavirus and orthopoxvirus infections in Rattus norvegicus and M. musculus in the English-speaking Caribbean. Rodents may present a potential zoonotic and biosecurity risk for transmission of three human pathogens, namely orthohantaviruses, mammarenaviruses and orthopoxviruses in Barbados.Background: Rodents are reservoirs for several zoonotic pathogens that can cause human infectious diseases, including orthohantaviruses, mammarenaviruses and orthopoxviruses. Evidence exists for these viruses circulating among rodents and causing human infections in the Americas, but much less evidence exists for their presence in wild rodents in the Caribbean. Methods: Here, we conducted serological and molecular investigations of wild rodents in Barbados to determine the prevalence of orthohantavirus, mammarenavirus and orthopoxvirus infections, and the possible role of these rodent species as reservoirs of zoonotic pathogens. Using immunofluorescent assays (IFA), rodent sera were screened for the presence of antibodies to orthohantavirus, mammarenavirus (Lymphocytic choriomeningitis virus—LCMV) and orthopoxvirus (Cowpox virus—CPXV) infections. RT-PCR was then conducted on orthohantavirus and mammarenavirus-seropositive rodent sera and tissues, to detect the presence of viral RNA. Results: We identified antibodies against orthohantavirus, mammarenavirus, and orthopoxvirus among wild mice and rats  in Barbados. No orthohantavirus or mammarenavirus viral RNA was detected from seropositive rodent sera or tissues using RT–PCR. Conclusions: Key findings of this study are the first serological evidence of orthohantavirus infections in  Leptospira, orthohantaviruses, mammarenaviruses and orthopoxviruses ..30].M. musculus) were caught in comparison to wild rats (R. rattus and R. norvegicus) during the trapping survey , R. norvegicus  and R. rattus   were males compared to 45.3% (68/150) females, 56.8% (46/81) of the males had developed scrota indicative of reproductive maturity, whilst 43.2% (35/68) did not. Further, 17.6% (12/68) of the females were pregnant, 76.5% (52/68) were non-parous and with 5.9% (4/68) of the female M. musculus rodents the reproductive maturity was either indiscernible or not recorded  . Of the recorded .R. norvegicus rodents trapped, 12.5% (1/8) were males compared to 87.5% (7/8) females, 0% (0/1) of the males had developed scrota indicative of reproductive maturity whilst 100% (1/1) did not. Further, 0% (0/8) of the females were pregnant whilst 87.5% (7/8) were non-parous, and with 12.5% (1/8) the reproductive maturity was either indiscernible or not recorded  were orthohantavirus IFA-positive, while for both rat species, R. norvegicus and Rattus rattus, none of them (0% (0/8) and 0% (0/2) respectively) were orthohantavirus IFA-positive  rodents, where hearts were used to obtain the sera. Screening of sera from wild rodents trapped in seven different parishes in Barbados was conducted using IFA to identify seropositive rodents with orthohantavirus-specific IgG antibodies . Of the positive . The lack of orthohantavirus seropositivity among rats in this study is notable in comparison to a previous rodent survey in Barbados . The ortTissues and sera from seropositive wild rodents were then tested using orthohantavirus-specific RT-PCR. Molecular investigations failed to detect orthohantavirus vRNA among wild rodents in Barbados, as none of the seropositive rodent sera nor tissues yielded positive orthohantavirus-specific RT-PCR results .M. musculus rodents, 2.7% (4/150) were seropositive whilst 0% (0/8) of R. norvegicus and 0% (0/2) R. rattus rodents were seropositive for mammarenavirus infection  were orthopoxvirus IFA-positive, whilst for both rat species, R. norvegicus and R. rattus, 12.5% (1/8) and 50% (1/2) respectively were seropositive following orthopoxvirus IFA testing  were seropositive for orthopoxvirus infection . For mic testing . Among t testing .Orthohantavirus, mammarenavirus and orthopoxvirus studies on rodents in Caribbean countries have been sparse or non-existent, and we present the first serological evidence of multiple orthohantavirus, mammarenavirus and orthopoxvirus infections in wild rodent species in both Barbados and the English-speaking Caribbean. Seroprevalence rates of 3.8% for orthohantavirus, 2.5% for mammarenavirus and 7.5% for orthopoxvirus infections were observed among wild rodents in Barbados during this study. This has potential biosecurity implications for farms, hospitals, residential homes, businesses involved in recycling and persons working in and living around sugarcane fields.M. musculus rodents to test the hypothesis of M. musculus as possible rodent vectors of orthohantavirus infections in Barbados and represents the first serological evidence of orthohantavirus infection of M. musculus in the Caribbean and the first evidence of mammarenavirus infection and orthopoxvirus infection among wild rodents in the Caribbean.The last report of orthohantavirus infection among wild rodents in Barbados was in 2002, thus more updated data were required to understand the orthohantavirus infection status of wild rodents . This stRattus and Mus spp., SEOV infections would be expected given its worldwide distribution . Fo. Fo67]. ™ reagent , and RNA was extracted following the manufacturer’s instructions. RNA yield was determined using NanoDrop  based on absorbance measurements at 260 nm and 280 nm. Pan-orthohantavirus and mammarenavirus RT–PCRs were conducted as previously described [Lung and kidney samples from seropositive rodents were lysed and homogenized using Roche MagNA lyser  and Trizolescribed ,93.


Drosophila ovary. In ovarian follicle cells, loss of Nup54 and Nup58 results in compromised piRNA biogenesis exclusively from the flamenco locus, whereas knockdowns of other NPC subunits have widespread consequences. This provides evidence that some Nups can acquire specialised roles in tissue-specific contexts. Our findings consolidate the idea that the NPC has functions beyond simply constituting a barrier to nuclear/cytoplasmic exchange as genomic loci subjected to strong selective pressure can exploit NPC subunits to facilitate their expression.The nuclear pore complex (NPC) is the principal gateway between nucleus and cytoplasm that enables exchange of macromolecular cargo. Composed of multiple copies of ~30 different nucleoporins (Nups), the NPC acts as a selective portal, interacting with factors which individually license passage of specific cargo classes. Here we show that two Nups of the inner channel, Nup54 and Nup58, are essential for transposon silencing via the PIWI-interacting RNA (piRNA) pathway in the  Transposons are genetic sequences, which, when active, can move around the genome and insert themselves into new locations. This can potentially disrupt the information required for cells to work properly: in reproductive organs, for example, transposon activity can lead to infertility. Many organisms therefore have cellular systems that keep transposons in check.Animal cells comprise two main compartments: the nucleus, which contains the genetic information, and the cytosol, where most chemical reactions necessary for life take place. Molecules continually move between nucleus and cytosol, much as people go in and out of a busy train station. The connecting ‘doors’ between the two compartments are called Nuclear Pore Complexes (NPCs), and their job is to ensure that each molecule passing through reaches its correct destination.Recent research shows that the individual proteins making up NPCs  may play other roles within the cell. In particular, genetic studies in fruit flies suggested that some nucleoporins help to control transposon activity within the ovary – but how they did this was still unclear. Munafò et al. therefore set out to determine if the nucleoporins were indeed actively silencing the transposons, or if this was just a side effect of altered nuclear-cytosolic transport.Experiments using cells grown from fruit fly ovaries revealed that depleting two specific nucleoporins, Nup54 and Nup58, re-activated transposons with minimal effects on most genes or the overall health of the cells. This suggests that Nup54 and Nup58 play a direct role in transposon silencing.flamenco, was indeed affected. Normally, flamenco acts as a ‘master switch’ to turn off transposons. Without Nup54 and Nup58, the molecule encoded by flamenco could not reach its dedicated location in the cytosol, and thus could not carry out its task.Further, detailed analysis of gene expression in Nup54- and Nup58-lacking cells revealed that the product of one gene, These results show that, far from being mere ‘doorkeepers’ for the nucleus, nucleoporins play important roles adapted to individual tissues in the body. Further research will help determine if the same is true for other organisms, and if these mechanisms can help understand human diseases. The main gateway between the nucleus and the cytoplasm is the nuclear pore complex (NPC), a large multi-protein assembly spanning the nuclear envelope. The NPC is composed of multiple copies of ~30 proteins, termed nucleoporins (Nups), arranged into an eightfold symmetric ring . Small mDrosophila melanogaster, dual-strand clusters produce RNAs from both genomic strands in the nurse cells of the ovary  is the principal source of piRNAs in the somatic follicle cells that enclose egg chambers  silencing in animal gonads is accomplished primarily through the action of piRNAs . These 2he ovary  and relyhe ovary . Specifihe ovary . Uni-strhe ovary . Of the chambers  and was nsposons . Being axf1/Nxt1 . Upon exogenesis , though fs(1)Yb gene, which is exclusively expressed in somatic follicle cells , obtained through crosses between the two alleles. These mutants show strong de-repression of somatic, gypsy-family TEs (flam and Yb-bodies. Knockdowns of nxf1/nxt1 in ovarian somatic cells (OSCs), a cell line derived from the somatic compartment of the ovary that expresses a functional piRNA pathway , 42 in siNup54, and 42 in siNup58  (expanded (ex) gene on chromosome 2L , the scaffold protein Nup93-1 or the piRNA biogenesis factor Yb . Cells d by flam . Both th siNup58 . A substsiNup58) . One sucosome 2L . This stnup62 and nup93-1 also caused de-repression of some flam-regulated TEs, we found much more pronounced mis-expression of protein-coding genes, with 207 genes de-regulated in siNup62 and 417 in siNup93-1   , which c-element . Of notepression . Consideflam transcript upon nup54, nup58, nup62, nup93-1, and yb knockdowns in OSCs (flam as other somatic piRNA source loci (e.g. the protein-coding gene tj or the piRNA cluster 20A) were unaffected . Plotting fold-changes in these 1 kb bins following zuc knockdown, which prevents flam processing into piRNAs, showed an increased precursor abundance that was uniform across the entire locus  revealed one major transcription initiation peak, as expected  that reach the cytosol upon siNup54 and siNup58 might still be processed by Yb, although with lower efficiency than within properly formed Yb-bodies. This role of Nup54 and Nup58 could also, directly or indirectly, affect transcriptional elongation and termination of the piRNA cluster; however, further work will be required to test this hypothesis.Considered together, these results indicate that transposon silencing defects resulting from loss of Nup54 or Nup58 arise from their role in facilitating flam export. We therefore designed deletion mutants targeting Nup54 and Nup58 domains and assayed their ability to interact with other Nups and to rescue TE de-repression in OSCs  allele, produces a truncated protein lacking the carboxy-terminal region due to a Mi{ET1} transposon insertion (∆C) that is unable to sustain transposon repression in OSCs (nup54 knockdown in OSCs. Trans-heterozygous flies (MB/9B4nup54) carrying the MBnup54 allele over the 9B4nup54 deficiency spanning the entire nup54 locus , which rely on different, specialised transcription and export pathways, were unchanged, if not slightly higher, in the case of 80F (flam-regulated transposons (e.g. gypsy) was already evident in MB/w1118nup heterozygous flies  direct binding of Nup54 and/or Nup58 to flam RNA, or (3) an interaction between Nup54/Nup58 and flam RNP export complexes. Although the flam locus is at the nuclear periphery, we failed to detect any consistent correlation with the position of Yb-bodies on the opposite side of the nuclear envelope in both OSCs and ovaries  physical proximity of the  ovaries   thus thm export . Nxf1 wam export , therefoanscript , which wanscript . Since tflam export  physically interact with each other. We first searched for protein interactions between Yb and the NPC via BASU-mediated proximity labelling, followed by mass spectrometry (PL-MS) in OSCs  cells, which do not express a functional piRNA pathway and represent a convenient system to investigate protein-protein interactions that are not bridged by other piRNA pathway components. We used 3xFLAG-tagged Yb, Nup54, or Nup58 as baits and probed for the corresponding HA-tagged versions. As expected, Nup54 and Nup58 recovered a substantial amount of their respective partner Nup  and cytosolic (Yb) components of flam expression and thus suggest that these factors bridge nuclear export to cytosolic fate specification. Although several aspects of the molecular mechanism remain to be elucidated , we propose a tentative model whereby this Nxf1-NPC-Yb axis coordinates initiation, processivity, and directionality of flam export  and could represent a broader paradigm for co-option of FG-Nups for specific transcript trafficking routes. We envision that in principle any transcript subject to strong selective pressure could evolve a dedicated export machinery via adapting FG-Nup functions in a cell-type-specific manner. Such mechanisms may have gone unnoticed previously because of the general role of the NPC. Interestingly, several Nup genes in Drosophila show signs of rapid adaptive evolution that result in hybrid incompatibilities  and sonicated using a Bioruptor Pico . Sonicated lysates were diluted 2× in 50 mM Tris, pH 7.4, and cleared for 10 min at 16,500 g. Lysates were pre-cleared for 1 hr at 4°C with 100 μl of Protein A/G Dynabeads (Thermo Fisher Scientific 10015D) and the supernatant collected to a fresh tube. Biotinylated proteins were isolated by incubation with 200 μl of Dynabeads  overnight at 4°C. Beads were washed 2× in 2% SDS, 2× in Wash Buffer 1 −1-piperazineethanesulfonic acid, pH 7.5), 2× with Wash Buffer 2 , and 2× with 50 mM Tris. Beads were rinsed twice with 100 mM ammonium bicarbonate. BASU-Nup54, BASU-Nup58, and BASU-Yb pulldowns were subjected to TMT-labelling followed by quantitative mass spectrometry on a nano-ESI Fusion Lumos mass spectrometer (Thermo Fisher Scientific). On-bead Trypsin digestion, TMT chemical isobaric labelling and data analysis were performed by the CRUK-CI proteomics core as previously described  at 1% spectrum-level FDR  criteria using Percolator (University of Washington). MS1 mass tolerance was constrained to 20 ppm, and the fragment ion mass tolerance was set to 0.5 Da. TMT tags on lysine residues and peptide N termini (+229.163 Da) were set as static modifications. Oxidation of methionine residues (+15.995 Da), deamidation (+0.984) of asparagine and glutamine residues, and biotinylation of lysines and protein N-terminus (+226.078) were included as dynamic modifications. For TMT-based reporter ion quantitation, we extracted the signal-to-noise ratio for each TMT channel. Parsimony principle was applied for protein grouping, and the level of confidence for peptide identifications was estimated using the Percolator node with decoy database search. Strict FDR was set at q-value <0.01. Downstream data analysis was performed on R using the qPLEXanalyzer package  as descrS2 cells or OSCs were transfected with 3xFLAG- and HA-tagged constructs. After 48 hr, cells were lysed in 250 μl of Pierce IP lysis buffer supplemented with cOmplete protease inhibitors (Roche). Equal amounts of lysate for each sample were diluted to 1 ml with IP lysis buffer and incubated with 30 μl of anti-FLAG M2 magnetic beads (Sigma M8823) overnight at 4°C. For anti-HA pulldowns, lysates were incubated with 30 µl of anti-HA (Thermo Fisher Scientific 88836) beads overnight at 4°C. Beads were washed 3 × 15 min in 1× Tris-buffered Saline (TBS) with protease inhibitors, then resuspended in 2xNuPAGE LDS Sample Buffer (Thermo Fisher Scientific) and boiled for 3 min at 90°C to elute immunoprecipitated proteins.Western blots were carried out using standard protocols. Protein lysates were run on NuPAGE 4–12% pre-cast gels and transferred to nitrocellulose membranes using a dry blotting system (iBlot2.0). Membranes were blocked for 1 hr at RT with 1× LiCor blocking buffer diluted in Phosphate Buffered Saline (PBS) and primary antibodies incubated overnight at 4°C. Following 3× washes in PBS + 0.1%Tween, secondary antibodies conjugated to infrared dyes  were incubated for 45 min at room temperature (RT). Images were acquired on an Odyssey CLx scanner (LiCor). The following primary antibodies were used: anti-HA , anti-FLAG (Sigma #F1804), anti-Piwi , anti-YbCells were plated 1 day in advance on fibronectin-coated coverslips, fixed for 15 min in 4% Paraformaldehyde (PFA), permeabilised for 10 min in PBS, 0.2% Triton X-100, and blocked for 30 min in PBS, 0.1% Tween-20 (PBST), and 1% BSA. Primary antibodies were diluted 1:500 in PBST and 0.1%BSA) and incubated overnight at 4°C. After 3 × 5 min washes in PBST, secondary antibodies were incubated for 1 hr at RT. After 3 × 5 min washes in PBST, DAPI was incubated for 10 min at RT and washed twice in PBST. Coverslips were mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific #P36961) and imaged on a Leica SP8 confocal microscope (100× oil objective). For TurboID labelling, cell culture media was supplemented with 500 µM biotin for 1 hr. Detection was carried out using streptavidin conjugated to AlexaFluor-555 (Thermo Fisher Scientific). The following antibodies were used: anti-Piwi , anti-FLRNA-FISH was performed with hybridisation chain reaction (HCR), similar as reported . OSCs we20 primers. Real-time PCR (qPCR) experiments were performed with a QuantStudio Real-Time PCR Light Cycler (Thermo Fisher Scientific). Transposon levels were quantified using the ∆∆CT method , and reverse transcribed with the Superscript III First Strand Synthesis Kit (Thermo Fisher Scientific), using oligo-dTT method , normaliFly ovaries were dissected in ice-cold PBS, fixed for 15 min in 4% PFA at RT, and permeabilised with 3 × 10 min washes in PBS with 0.3% Triton X-100 (PBS-Tr). Samples were blocked in PBS-Tr with 1% BSA for 2 hr at RT and incubated overnight at 4°C with primary antibodies in PBS-Tr and 1% BSA. After 3 × 10 min washes at RT in PBS-Tr, secondary antibodies were incubated overnight at 4°C in PBS-Tr and 1% BSA. After 4 × 10 min washes in PBS-Tr at RT (DAPI was added during the third wash) and 2 × 5 min washes in PBS, samples were mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific #P36961) and imaged on a Leica SP8 confocal microscope. Images were deconvoluted using Huygens Professional. The following antibodies were used: anti-Piwi , anti-Ybflam RNA-FISH analysis, we identified nuclei from the lamin staining applying a difference of Gaussian filter. We then isolated the RNA-FISH spots and counted the number present inside the nuclear envelope versus the total amount. Cytoplasm was identified via oligo-dT staining. A similar number of images was processed for all samples.Acquired images were analysed on Fiji using custom scripts  and 2. BD. melanogaster rRNAs (siTOOLs Biotech), according to the manufacturer’s instructions. Fly riboPOOLs were hybridised to 1 µg of RNA by adding 1 µl of resuspended riboPOOLs (100 µM), 5 µl of hybridisation buffer , and 1 µl of RNAse Inhibitor Plus (Promega), incubating for 10 min at 68°C and cooling down slowly to 37°C. 80 µl of MyOne Streptavidin C1 beads (Thermo Fisher) for each sample were washed twice in 100 µl of Beads Resuspension Buffer  and twice in 100 µl of Beads Wash Buffer (0.1 M NaCl). The beads were resuspended in 160 µl of Depletion buffer  and divided into two 80 µl aliquots. Hybridised riboPOOLs were added to 80 µl of washed beads, mixed well, and incubated for 15 min at 37°C, followed by 5 min at 50°C. The supernatant was transferred to the second tube with 80 µl of washed beads and incubated for 15 min at 37°C, followed by 5 min at 50°C. rRNA-depleted samples were transferred to a fresh tube and purified using Agencourt RNAClean XP beads (Beckman Coulter A63987). RNA-seq libraries were prepared using the NEBNext Ultra Directional Library Prep Kit for Illumina (NEB #E7760), according to the manufacturer’s instructions for ribosome-depleted RNA. DNA libraries were quantified with KAPA Library Quantification Kit for Illumina (Kapa Biosystems) and sequenced on an Illumina HiSeq 4000 instrument.Ribosomal RNAs were depleted using riboPOOLs against Small RNA libraries from OSCs were generated as described previously with slight modifications . Briefly6 OSCs treated with siRNAs for 96 hr were used for nuclei isolation. Isolated nuclei were resuspended in storage buffer and stored at −80°C until further processing. Nuclear run-on reactions were performed with biotin-11-CTP and biotin-11-UTP and unlabelled ATP and GTP. Purified RNA samples were fragmented for 10 min on ice in 0.2 N NaOH and purified using Bio-Spin P30 columns (Bio-Rad). Biotin-labelled RNA was purified using MyOne Streptavidin C1 Dynabeads (Thermo Fisher Scientific), decapped using the RppH enzyme (NEB), and purified by phenol/chloroform extraction. 3′ linkers and then 5′ linkers  were ligated and biotinylated ligation products purified after each ligation using MyOne Streptavidin C1 Dynabeads. Reverse transcription was carried out using SuperScript III (Thermo Fisher Scientific), and libraries were amplified using HS Phusion Flex polymerase. The libraries were sequenced on an Illumina HiSeq 4000.PRO-seq was performed as described previously . 4 × 106DIP1 and flamenco genomic loci (chrX:21624796–21634619) using Oligominer  and crosslinked on ice with 400 mJ/cm2 at 254 nm. HALO-tag CLIP-seq was performed as previously described (1 × 10escribed . BrieflyAGATCGGAAGAGCACACGTCTGAACTCCAGTCA) keeping only reads with at least 23 bp length. The first and last four bases were trimmed using seqtk  . Only high-quality small RNA reads with a length between 23 and 29 bp were used for the piRNA genome browser shots. piRNA distribution was calculated and plotted in R.For small RNA-seq, adapters were clipped from raw fastq files with fastx_clipper (adapter sequence FlyBase)  using STFlyBase) . For trahttp://hannonlab.cshl.edu/fastx_toolkit/). After removal of reads mapping to Drosophila rRNA using STAR, high-quality reads were aligned to the D. melanogaster genome release 6  . For genes carrying ‘nearby transposon insertions’, we considered only gene promoters within −10 kb to +15 kb of a TE insertion on the same genomic strand. Coordinates used for the uni-strand piRNA clusters are chrX:21631891–22282863 (flam) and chrX:21520428–21556793 (20A). Coordinates used for the dual-strand piRNA clusters were from For RNA-seq, raw fastq files generated by Illumina sequencing were analysed by a pipeline developed in-house. In short, the first five bases of each 50 bp read were removed using fastx trimmer (FlyBase)  using STFlyBase) . For traepTools2  bamCoverepTools2  and plot2 fold-change between each knockdown and its control was calculated and plotted in R. The mappability was calculated as described in flam 100 kb bins analysis, the dm6 genome was divided into 100 kb sliding windows using 1 kb steps. Mappability for a window was defined as the fraction of all possible 50-mers derived from the window that aligned uniquely to it using STAR. Windows with mappability >0.05 and located fully within flam (n = 285) were kept for the analysis. For each sample, reads aligning uniquely to the sense strand and at least 50% within a window were counted and subsequently normalised to reads per 1 million uniquely aligned reads. The per-window log2 fold-change between each knockdown and its control was calculated using a pseudo-count of 1. Results from four individual replicates per knockdown were highly consistent with the results shown from the pooled analysis.For piRNA clusters 1 kb bin analysis, each cluster was divided into non-overlapping 1 kb bins and only those with a mappability score above 0.8 were retained. Uniquely mapped reads were counted using HTSeq , normalit-test using GraphPad. Box plots display median, first, and third quartiles (box) and highest/lowest value within 1.5 interquartile range (whiskers); dots represent potential outliers beyond 1.5 * interquartile range.Data visualisation and analyses were done using R and the following packages: ggplot2, DEseq2, qPLEXanalyzer. The UCSC genome browser was used to display high-throughput sequencing data and to prepare coverage plots shown in the article. n indicated in the figure legends refers to the number of independent biological replicates. Bar graphs display average of n biological replicates and standard deviation (SD), and p values were calculated with an unpaired  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:melanogaster subgroup of flies, the work is nonetheless important to the piRNA field generally, since the mechanisms that distinguish long non-coding RNAs from piRNA precursor transcripts are not understood in any animal.A central unsolved problem in the piRNA field is, what marks an RNA for piRNA production? Here, Czech and co-workers report that the nucleoporins Nup54 and Nup58 play a specialized role in specifying the flamenco transcript for processing into piRNAs, an essential step for silencing gypsy-family transposons in the somatic follicle cells of the fly ovary. Although it is not clear whether these findings will inform our understanding of piRNA biogenesis beyond the Decision letter after peer review:Drosophila" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Channel Nuclear Pore Complex subunits are required for transposon silencing in The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The three reviewers agree that this is overall a compelling study that provides novel, interesting and important observations. No major new experimentation is required but the data need to be more extensively described and the authors should also discuss alternative models. Detailed suggestions for changes are found in the constructive reviews but it is important that the authors improve the quality of some of their images, provide additional quantitative information and add better statistical analyses.Reviewer #1:Drosophila melanogaster OSC cells, a model for the somatic follicle cells that support oogenesis in the female fly. Detailed measurements of RNA production, piRNA production, and protein-protein interactions are a major strength of this work. In general, experiments using OSC cells are informative and conservatively interpreted. One weakness is the low quality confocal images (perhaps they were uploaded at too low a resolution?) make it difficult to evaluate the authors' descriptions of the location and abundance of specific subcellular components in cells. Only a small number (often just one) of representative images are shown, making it impossible to evaluate how representative any image is of the phenotype of RNAi depletion of a specific nucleoporin or other protein. Most importantly, the authors often ignore alternative explanations more consonant with the nucleopore literature. Interpretation of their genetic experiments is complicated by changes in the cell composition of mutant ovaries. The work will provide a starting point for the dissection of the flam RNA export pathway and the commitment of flam transcripts to piRNA biogenesis.The authors set out to identify nuclear pore components required for piRNA production in cultured 1. Please include a panel of representative images for mutant phenotypes in OSC and ovaries, not simply a single cell or field of view.2. Figure 2A: what is the source of the sense piRNAs whose abundance is unaltered by loss of Yb, Nup54, or Nup58? Do they map to cluster transcripts or elsewhere in the genome?3. The authors propose that Nup54/58 play a role in export of flam RNA. Given the bias towards loss of 3′ transcript coverage and piRNA abundance, together with both the involvement of the TREX complex in piRNA biogenesis and the role of nuclear pore proteins in transcriptional elongation, isn't it more likely that Nup54/58 promote transcriptional elongation across the entirety of flam or that they prevent premature termination in the 3′ region of the flam gene? The authors should at least discuss these alternative explanations, detailing the evidence supporting or refuting each.4. Page 18: since the mutant ovaries are small (please show ovary and ovariole images of what exactly this means!), then the cell composition of the controls and mutants will be quite different. Early stage ovaries will have a higher the ratio of follicle cells cytoplasm to nurse cells, since nurse cells will be smaller. Thus, it is not simple to normalize RNA or piRNA sequencing data to permit comparison between the two types of ovaries. This problem is exacerbated by the authors' finding that both TE and mRNA expression changes in nup54MB/9B4. A detailed description of the experimental evidence that explains why the authors' normalization strategy allows such a comparison is essential. Additionally, the authors need to show  that TE expression is in fact higher in vivo in identical cell types.Reviewer #2:The paper by Munafo et al. addresses the role of specific fly NPC proteins in the export and accumulation of flam transcripts in Yb bodies, condensates of the Yb RNA helicase, associated with the cytoplasmic side of NPCs in oocyte somatic cells (OSCs). Yb bodies are important for the biogenesis/maturation of flam piRNAs involved in the repression of specific transposable elements  in a still poorly understood process. Yb bodies depend on flam transcription and the Nxf1/Nxt1 nuclear export factors, but it is still unclear how the transcripts are directed to Yb-bodies and from there licensed for processing.In this paper, the authors show that in OSCs, the loss of two Nups, Nup54 and Nup58, which form a bigger complex with Nup62 and Nup93, specifically interferes with the export of flam RNAs but not other mRNAs. Nup54/58 physically interact with Nxf1/Nxt1 as well as Yb suggesting an export-coupled localization mechanism that specifies flam as a piRNA precursor.The study uses a wide variety of approaches, including transcriptomics, proteomics, biochemical analyses and imaging in different genetic backgrounds both in vitro using OSCs and in vivo using ovary tissue, to demonstrate the specific role of Nup54 and Nup58 in piRNA biogenesis. The paper presents a very large set of experiments with the following main observations:1. Knock down of Nup62 and Nup93 results in flam expression defects and upregulation of transposons as well as deregulation of multiple genes. In contrast, knock down of Yb, Nup54 and Nup58 leads to loss or dispersed Yb-bodies, decreased flam RNA levels as well as increased TE expression without substantially affecting overall gene expression or viability. The observations support that Nup54 and Nup58 have a dedicated role in flam RNA export and functional Yb-body formation.By fluorescence imaging approaches, they also show that Yb formation in OSCs depends on correct flam expression and functional Nxf1/Nxt1 export factors.2. RNA seq of cells depleted for Nup54 and Nup58 or Yb show reduced flam RNA levels. PROseq data show that these lower levels are due to loss of RNA stability rather than decreased transcription. Instability increases towards the 3'end of the flam locus, suggesting that Nup54/58 are required to ensure processivity of nuclear export of an otherwise unstable transcript. siNup54/58 specifically affect the stability of flam piRNAs while siYb affects the levels of different types of piRNAs indicating a specific role for Nup54/58 in flam piRNA biogenesis/maturation that is distinct from Yb.3. Loss of Nup54/58 affects flam RNA detection in the cytoplasm, but not in the nucleus where it is detected as a dot corresponding to the flam locus associated with the nuclear periphery. Loss of Nup54/58 also results in decrease and dispersion of Yb bodies. In contract siYb results in reduction of both nuclear and cytoplasmic flam foci supporting the different roles of Yb and Nup54/58 in flam piRNA biogenesis.Together, RNAseq/Proseq and imaging data support the view that Nup54/58 may ensure processivity of flam nuclear export, which otherwise gets degraded.4. Deletion mutants and complementation analyses in OSCs indicate that the Nup54/58 C-terminal domains, but not the FG-repeats, are important for efficient TE repression. The C-terminal domain is important for Nup54-Nup58 interaction and Yb body formation.Accordingly, a natural nup54MB C-terminal mutant shows decreased Yb body formation and increased TE expression in vivo.5. Finally the authors performed quite sophisticated mass spec analyses to identify factors interacting with either Yb or Nup54/58. Nup54/58 PL-MS identified Yb as well as Nxf1 as interactors. In contrast Yb PL-MS detected Nup214 but failed to detect Nxf1 or Nup54/58, which could possibly reflect the transient interaction of Yb with NPCs.The mass spec data were then validated by an important series of co-IP experiments between wild-type or mutant forms of the Nup54/58 and Yb proteins to define the interaction domains between these proteins key for flam piRNA biogenesis and TE repression.eLife and should be acceptable for publication provided the authors address few comments/questions.Overall, the experiments are well-controlled and the results, presented in multiple graphs with statistical analyses, are extremely convincing. The paper is clearly written and the data are on the whole supportive of the proposed model. This work is well suited for General remarks/questions:The paper is quite dense and sometimes difficult to follow when going back and forth between the multiple figures. It may be worse trying to simplify in order to strengthen the overall message.Figure 2 figure supplement 1G: this experiment suggests that flam transcription is affected in siYb, but not in sinup54/58. How could loss of Yb affect flam transcription efficiency?Figure 2 figure supplement 2G: this figure indicates that the Yb bodies and the flam DNA don't show an obvious proximity or correlation (on either side of the NPC). This is unexpected based on the model that Nup54/58 establish a physical connection between Nxf1/Nxt1-bound flam RNAs and their delivery to Yb bodies on the cytoplasmic side of the NPC. This observation is not really discussed in the text and should be clarified since it is somewhat questioning the model presented in Figure 4G.Figure 2 figure supplement 2H is shown too early since it is linked to Figure 3C.Figure 3A: the authors should indicate what the red domain in Nup54 corresponds to. Is it the same region lacking in the nup54MB mutant?Figure 3 figure supplement 1E: Yb staining shows quite clearly that the nup54MD presents reduced Yb body formation. However, it is not clear why DNA staining reveals larger foci in the nup54MD mutant?Figure 4 and supplements: the mass spec data indicate that Nup54/58 interact with Nxf1 and Yb, consistent with their proposed role in coupling flam RNA export and its addressing to Yb bodies. However, Yb does not efficiently interact with Nup54/58 nor Nxf1 by mass spec. It is unexpected that Yb does not interact with Nxf1, since Nxf1 is expected to transport flam RNAs to the cytoplasmic side of the NPC through interactions with Nup54/Nup58 in order to deliver the transcripts to Yb. co-IP experiments  then show that Yb weakly interacts with both Nup54/Nup58 and Nxt1. Why do the authors think that the weak interaction detected between Yb and Nup54/58 is more functional than the weak interaction between Yb and Nxt1?When does Yb bind Flam transcripts and wouldn't it be expected that Yb interacts with Nxf1/Nxt1 to take over the flam transcripts? This question should be more clearly addressed in the text or discussion.Reviewer #3:Drosophila ovaries. There is considerable interest in exploring how nucleoporins function outside of their canonical roles in nuclear transport. Thus, this study is relevant from a fundamental cell biological viewpoint but it also may illuminate how some nups appear to be uniquely required during embryonic development and why nup mutations sometimes result in tissue-specific disease. The paper is impressive in scope and in its use of systems-level, biochemical and cell biological approaches to explore changes to transcription, piRNA biogenesis and biochemical interactions. Further, in general, the data are of high quality although often missing statistical analyses to help evaluate highly variable data. The biggest challenge is that there is so much data crammed into a small number of figures that the paper is very frustrating to navigate. Thus, while it is thoroughly convincing that there is a unique requirement for Nup54 and Nup58 in piRNA biogenesis, specifically from the flam locus, a more deliberate and more detailed description of the data would vastly improve the work. Further, the proposed model may be premature and additional considerations regarding where nups might function in this pathway e.g. in the nucleus or cytoplasm should be more thoroughly considered.This work examines the role of two nucleoporins in the export of the flam RNA, which is a precursor for piRNA biogenesis necessary for transposon silencing in 1. There are so many experimental techniques and analyses used in this paper that, when coupled to the often obscure nomenclature of genes and mutant alleles, makes navigating this paper extremely frustrating, particularly for a non-expert. One (of many) example of this is the first figure call out, Figure 1, figure supplement 1, which refers to an 8 panel figure that is essentially not described. The strong suggestion is to expand the text to more fully describe all of the figures to do the work more justice and allow readers access.2. The strength of the work lies in the remarkable specificity exhibited by loss of function of Nup54/58 to piRNA biogenesis, particularly at the flam locus. However, the model proposed is not fully supported by the data. A key concern is that it is well established that nups can function outside of the NPC, both at genomic loci and also in the cytoplasm but this is not fully considered or ruled out. The suggestion is to be more circumspect with the proposed model and to consider alternative possibilities. The key may lie in the fact that it is the coiled-coil domains of the nups, and not their FG-motifs that appear to be important. Although one could argue that this supports their integration into the NPC, how these domains could connect to Nxf1 without FG-repeats remains difficult to rationalize.3. Invoking a model that suggests that Yb might act similarly to Dbp5 on the cytosolic filaments is certainly interesting, but the data do not yet support this. A clearer understanding of whether the interaction between Yb and Nup214 is direct would be necessary, further, it should be ruled out that DDX19 is not involved with the export of the flam RNA.Lastly, in general the data is well quantified but better descriptions of the plots and statistics are really needed for interpretation. Essential revisions:The three reviewers agree that this is overall a compelling study that provides novel, interesting and important observations. No major new experimentation is required but the data need to be more extensively described and the authors should also discuss alternative models. Detailed suggestions for changes are found in the constructive reviews but it is important that the authors improve the quality of some of their images, provide additional quantitative information and add better statistical analyses.We thank the reviewers for their positive evaluation of our manuscript. We have revised the text to expand the introduction, discuss alternative models, and provide more explanation of the data. Furthermore, we have added statistical analysis where relevant and clearly described the analysis in both the figure legends and in the Methods section . Please, find below our response to each of the points raised by the reviewers and detailed description of the changes made.Reviewer #1:[…] 1. Please include a panel of representative images for mutant phenotypes in OSC and ovaries, not simply a single cell or field of view.We have added additional representative images for OSC and ovary phenotypes and enlarged the panels where possible, see Figure 1—figure supplement 3, Figure 2—figure supplement 3A, Figure 3—figure supplement 2 and Figure 4—figure supplement 1C. Each knockdown/mutant is accompanied by the relative quantification of the size and number of Yb-bodies. We have provided high-resolution files for each figure, which should improve the image quality over the previous version seen by the reviewers . Individual images of each figure panel can be provided upon request.2. Figure 2A: what is the source of the sense piRNAs whose abundance is unaltered by loss of Yb, Nup54, or Nup58? Do they map to cluster transcripts or elsewhere in the genome?yb, nup54 and nup58 map to genomic TE insertions, predominantly of the gypsy retrotransposon. Since these sense piRNAs are relatively few in the siGFP control and their abundance correlates with the degree of TE up-regulation in the various knockdowns, we conclude that they are most likely derived from processing of re-activated transposon transcripts. The same analysis on antisense piRNAs is shown for comparison.We have added an additional panel to answer the reviewer’s question . Most sense piRNAs detected upon knockdown of 3. The authors propose that Nup54/58 play a role in export of flam RNA. Given the bias towards loss of 3′ transcript coverage and piRNA abundance, together with both the involvement of the TREX complex in piRNA biogenesis and the role of nuclear pore proteins in transcriptional elongation, isn't it more likely that Nup54/58 promote transcriptional elongation across the entirety of flam or that they prevent premature termination in the 3′ region of the flam gene? The authors should at least discuss these alternative explanations, detailing the evidence supporting or refuting each.yb leads to a mild reduction of PRO-seq signal over the entire flam cluster locus, possibly hinting towards a negative effect on transcriptional elongation in cases where this transcription-coupled export axis is disrupted. In conclusion, we believe that our data support the conservative hypothesis of Nup54 and Nup58 involvement in flam processive export, with destabilisation of the transcript upon their absence. Future work will shed light on the mechanisms underlying flam transcription, which is beyond the scope of the present study.We have added discussion of this potential explanation in the text. Overall, we agree with the reviewer that an effect on transcriptional elongation and/or termination is plausible, however our data are not sufficient to strongly support or refute either explanation. We noticed that knockdown of 4. Page 18: since the mutant ovaries are small (please show ovary and ovariole images of what exactly this means!), then the cell composition of the controls and mutants will be quite different. Early stage ovaries will have a higher the ratio of follicle cells cytoplasm to nurse cells, since nurse cells will be smaller. Thus, it is not simple to normalize RNA or piRNA sequencing data to permit comparison between the two types of ovaries. This problem is exacerbated by the authors' finding that both TE and mRNA expression changes in nup54MB/9B4. A detailed description of the experimental evidence that explains why the authors' normalization strategy allows such a comparison is essential. Additionally, the authors need to show  that TE expression is in fact higher in vivo in identical cell types.MBnup54 trans-heterozygote mutants. As one can appreciate, the mutant ovary morphology is grossly normal and only slightly smaller in size compared to controls, unlike other piRNA pathway mutants described in the literature. We did not observe any striking changes in the relative abundance of nurse or follicle cells, and therefore have no reason to question our normalisation strategy, which is routinely used in the field. We would like to emphasise that other published piRNA pathway mutants/knockdowns have far more rudimentary ovaries than our MB/9B4nup54, e.g. the piwi mutant and somatic knockdown of piwi in Olivieri et al., 2010. Another example is from our group , where we showed that germline knockdown of ctp leads to severe atrophy of the ovaries . The corresponding RNA-seq normalisation has been carried out similarly to the one we used in this study.We have now provided a brightfield image of ovaries from Reviewer #2:[…] General remarks/questions:The paper is quite dense and sometimes difficult to follow when going back and forth between the multiple figures. It may be worse trying to simplify in order to strengthen the overall message.We thank the reviewer for the feedback, we have now expanded the text to provide more detailed explanations and to clarify the overall message.Figure 2 figure supplement 1G: this experiment suggests that flam transcription is affected in siYb, but not in sinup54/58. How could loss of Yb affect flam transcription efficiency?flam RNA that is exported via Nup54/58 does not aggregate into discrete foci, due to absence of Yb, and is therefore hardly detectable by RNA-FISH. Second, our PRO-seq data shows a mild reduction in global flam nascent RNA levels upon loss of yb, which is more apparent towards the 3’ end of the locus. Although the molecular mechanism is not readily apparent, this might support the idea that flam transcriptional elongation is somehow dependent on its downstream processing into piRNAs. A minor fraction of flam-derived piRNAs is antisense to the cluster, so we cannot exclude that those might base pair with the nascent flam RNA and contribute to its transcriptional rate. Further investigation of the mechanisms underlying flam cluster definition and transcription will be required to clarify this.This result, which is in accordance with previous findings , might be explained in two possible ways. First, residual Figure 2 figure supplement 2G: this figure indicates that the Yb bodies and the flam DNA don't show an obvious proximity or correlation (on either side of the NPC). This is unexpected based on the model that Nup54/58 establish a physical connection between Nxf1/Nxt1-bound flam RNAs and their delivery to Yb bodies on the cytoplasmic side of the NPC. This observation is not really discussed in the text and should be clarified since it is somewhat questioning the model presented in Figure 4G.flam transcripts are exported, irrespective of their transcription site.This observation is in line with previous works . We suggest that the nucleation of Yb-bodies initiates wherever Figure 2 figure supplement 2H is shown too early since it is linked to Figure 3C.We thank the reviewer for this slip. Former panel 2H has now been moved to Figure 3—figure supplement 1A.Figure 3A: the authors should indicate what the red domain in Nup54 corresponds to. Is it the same region lacking in the nup54MB mutant?MBnup54 mutant. This information was already included in the legend of Figure 2A and we have now clarified the description of the MBnup54 allele also in the main text.The purple box indicates a Nup54-family domain, which is absent in the Figure 3 figure supplement 1E: Yb staining shows quite clearly that the nup54MD presents reduced Yb body formation. However, it is not clear why DNA staining reveals larger foci in the nup54MD mutant?MBnup54 allele is a hypomorph. Some ovarioles of the MBnup549B4/ trans-heterozygotes seem to have a less organised pattern of follicle cells, which often show slightly larger DNA foci. These ovarioles show the most pronounced decrease in Yb-body assembly, thus suggesting that they are the ones where the effects of the Nup54 mutation are most pronounced. However, this phenotype is not fully penetrant and we have now added a second representative image where the DNA foci are more similar to the control.The Figure 4 and supplements: the mass spec data indicate that Nup54/58 interact with Nxf1 and Yb, consistent with their proposed role in coupling flam RNA export and its addressing to Yb bodies. However, Yb does not efficiently interact with Nup54/58 nor Nxf1 by mass spec. It is unexpected that Yb does not interact with Nxf1, since Nxf1 is expected to transport flam RNAs to the cytoplasmic side of the NPC through interactions with Nup54/Nup58 in order to deliver the transcripts to Yb. co-IP experiments  then show that Yb weakly interacts with both Nup54/Nup58 and Nxt1. Why do the authors think that the weak interaction detected between Yb and Nup54/58 is more functional than the weak interaction between Yb and Nxt1?When does Yb bind Flam transcripts and wouldn't it be expected that Yb interacts with Nxf1/Nxt1 to take over the flam transcripts? This question should be more clearly addressed in the text or discussion.flam transcripts from Nxf1 to Yb is not entirely clear and may involve interactions between Yb and Nxf1/Nxt1 or other adaptor proteins that we have not yet identified. We believe that super-resolution imaging of individual NPCs might clarify the relative position of each component of this export route and shed light onto this point. We have now added a sentence on this to the discussion.Our co-immunoprecipitation experiments detected a weak interaction between Yb and Nxt1, but not between Yb and Nxf1. Since Nxf1 and Nxt1 are usually present as a heterodimer, we did not speculate further about this interaction being functional. We agree with the reviewer that the “handover” of Reviewer #3:Drosophila ovaries. There is considerable interest in exploring how nucleoporins function outside of their canonical roles in nuclear transport. Thus, this study is relevant from a fundamental cell biological viewpoint but it also may illuminate how some nups appear to be uniquely required during embryonic development and why nup mutations sometimes result in tissue-specific disease. The paper is impressive in scope and in its use of systems-level, biochemical and cell biological approaches to explore changes to transcription, piRNA biogenesis and biochemical interactions. Further, in general, the data are of high quality although often missing statistical analyses to help evaluate highly variable data. The biggest challenge is that there is so much data crammed into a small number of figures that the paper is very frustrating to navigate. Thus, while it is thoroughly convincing that there is a unique requirement for Nup54 and Nup58 in piRNA biogenesis, specifically from the flam locus, a more deliberate and more detailed description of the data would vastly improve the work. Further, the proposed model may be premature and additional considerations regarding where nups might function in this pathway e.g. in the nucleus or cytoplasm should be more thoroughly considered.This work examines the role of two nucleoporins in the export of the flam RNA, which is a precursor for piRNA biogenesis necessary for transposon silencing in 1. There are so many experimental techniques and analyses used in this paper that, when coupled to the often obscure nomenclature of genes and mutant alleles, makes navigating this paper extremely frustrating, particularly for a non-expert. One (of many) example of this is the first figure call out, Figure 1, figure supplement 1, which refers to an 8 panel figure that is essentially not described. The strong suggestion is to expand the text to more fully describe all of the figures to do the work more justice and allow readers access.We thank the reviewer for this feedback. We have expanded the text to provide more detailed explanations of the data.2. The strength of the work lies in the remarkable specificity exhibited by loss of function of Nup54/58 to piRNA biogenesis, particularly at the flam locus. However, the model proposed is not fully supported by the data. A key concern is that it is well established that nups can function outside of the NPC, both at genomic loci and also in the cytoplasm but this is not fully considered or ruled out. The suggestion is to be more circumspect with the proposed model and to consider alternative possibilities. The key may lie in the fact that it is the coiled-coil domains of the nups, and not their FG-motifs that appear to be important. Although one could argue that this supports their integration into the NPC, how these domains could connect to Nxf1 without FG-repeats remains difficult to rationalize.flam export. We agree that our data do not fully rule out a possible role of Nup54 and Nup58 outside of the NPC, perhaps within Yb-bodies. However, we believe that in such scenario they should have been detectable in our Yb PL-MS and more cytosolic signal would have emerged via immunofluorescence.We have adjusted the model description (now referred to as a “tentative model”) and clearly stated what knowledge gaps will need to be filled in order to draw a conclusive picture of 3. Invoking a model that suggests that Yb might act similarly to Dbp5 on the cytosolic filaments is certainly interesting, but the data do not yet support this. A clearer understanding of whether the interaction between Yb and Nup214 is direct would be necessary, further, it should be ruled out that DDX19 is not involved with the export of the flam RNA.We agree with the reviewer’s comment and we have removed the speculation about Yb acting similarly to Dbp5. Further experiments beyond this study are required to address this question.Lastly, in general the data is well quantified but better descriptions of the plots and statistics are really needed for interpretation.We have expanded the figure legends to provide better description of the plots and statistics and have added the exact individual p values to each measurement. All quantifications and statistical analyses are accurately described in the methods section.