The structural relationships that emerge can then be linked to microscopic properties of interest, which we exemplify for structural heterogeneity and tetrahedral density.Much of our understanding of complex structures is based on simplification: for example, metal–organic frameworks are often discussed in the context of “nodes” and “linkers”, allowing for a qualitative comparison with simpler inorganic structures. Here we show how such an understanding can be obtained in a systematic and quantitative framework, combining atom-density based similarity (kernel) functions and unsupervised machine learning with the long-standing idea of “coarse-graining” atomic structure. We demonstrate how the latter enables a comparison of vastly different chemical systems, and we use it to create a unified, two-dimensional structure map of experimentally known tetrahedral AB A coarse-graining approach enables structural comparisons across vastly different chemical spaces, from inorganic polymorphs to hybrid framework materials. The synthetic exploration of solid-state structural space has been documented in extensive databases,4 it is natural to ask whether they might help with the aforementioned challenges. ML algorithms can handle very large datasets, but are (deliberately) chemically agnostic, and it is not a priori clear whether they will discover the same relationships that a trained chemist identifies just by eye. In this context, “unsupervised” ML means that information is sought from a given set of data without labels5 – for example, from a mathematical representation of the atomic structure, for which reliable computational tools are now available.6With machine learning (ML) approaches currently burgeoning in materials chemistry,6 This approach builds a neighbour density for any given atom (using “smooth” Gaussian functions) and then evaluates the overlap between pairs of such neighbour densities, making use of an efficient mathematical approach;6 a short review is given in the Methods section. SOAP thereby quantifies how similar any two given atomic environments are, on an intuitive scale from zero to one. Initially used for fitting machine-learned force fields,7 it was suggested in 2016 that SOAP can be utilised also for visualising chemical space.8 Applications to date include known and hypothetical ice structures,9 the TiO2 polymorphs,10 molecular crystals,11 and hypothetical zeolites;12 an overview including several illustrative examples was given very recently.13 Once a SOAP-based structure map has been created, it can be used, e.g., to select the most representative structural motifs in a complex system for computational spectroscopy.14One such representation is given by the Smooth Overlap of Atomic Positions (SOAP) similarity function, or kernel.15 These materials are widely described in terms of their topology. Whilst extremely powerful, such approaches do not (by construction) include geometric arguments: two zeolites may differ in their bond lengths and angles yet share identical topologies, or conversely, they may have similar geometric features but different connectivity. SOAP combines all the characteristics of the neighbour environment up to a given cut-off: it thereby cannot reproduce the intuitive classification afforded by the well-known space-group or topology symbols, but in turn gives rise to a comprehensive geometric measure that incorporates bond angles, rings, and other subtleties.15Very recently, zeolites were studied with SOAP-based maps and assessed regarding synthesisability.2-type networks with tetrahedral-like [AB4] environments.Here, we generalise this approach such that it can make direct comparisons across vastly different families of chemical structures, and thereby we develop a framework in which geometric diversity can be quantified, visualised, and better understood. The key enabling step is the realisation that a density-based metric such as SOAP can be applied equally well to coarse-grained and uniformly scaled representations of chemical structures as to the structures themselves: this allows us to compare compounds with inherently different chemistries and bond lengths. With a long-term aim of discovering  structural relationships, we focus this proof-of-concept study on one notoriously diverse and important family of inorganic and hybrid frameworks: namely, the AB2 structures have been eminently useful,16 they have normally been limited to individual aspects either of the structure  or topology (thereby removing subtleties of the structure itself). For example, zeolitic imidazolate frameworks (ZIFs), such as ZIF-8  atoms that connect to the Zn2+ centres, as shown in We start by noting that whilst comparisons across ABas ZIF-8 ,17 have 2 solid is hydro-sodalite  are identified in inorganic solids and metal–organic frameworks.21 We refer to the resulting approach, including removal of guests, coarse-graining, and re-scaling, as “cg-SOAP” in the following.A classical inorganic example of a more complex ABsodalite .19 In thshion ESI. We also2-like materials, including zeolites, ices, and chain-like inorganic structures such as BeCl2. Among the data sources, we point out a review article on ZIFs by Yaghi and co-workers,18 a report on cadmium-based imidazolate frameworks (“CdIFs”) by Tian et al.,22 and a study of polymorphism in Zn(CN)2 by Chapman and co-workers.23 More structures were collected from the Cambridge Structural Database1 and the IZA Database of Zeolite Structures.1 Key information about this dataset is collected in e.g., because they contain non-tetrahedral environments) are given as ESI.†To test this idea on a much wider basis of experimentally validated structures, we assembled a dataset which includes diverse families of AB24 – an idea that originated in the field of chemical topology.25 This is a step of key importance, because otherwise the overlap of neighbour densities will be necessarily diminished as soon as there are different A–B distances k,6 on a scale from 0 to 1, obtained here using the openly available DScribe implementation.27 Details are given in the Methods section. In short, averaging over all combinations of A-site environments α in the i-th unit cell in our database and β in the j-th, we obtain a per-cell similarity, k̄. With this, one may then define a geometric distance (dissimilarity) between the i-th and j-th unit cell as8The SOAP kernel is a similarity measure between two atomic environments, equality .810,14 Our map is shown in cf.e.g., SiO2 polymorphs), molecular , and tetrahedral hybrid networks, viz. ZIFs and related cadmium-, boron-, or other cation based tetrahedral imidazolate frameworks (“TIFs”). We follow the naming conventions in the existing literature, accepting that the abbreviations will not always be entirely unambiguous – e.g., for cadmium-based species: Cd(Im)2-dia-c was labelled as a “ZIF” in 2-sod was initially reported as “CdIF-1” shortly thereafter.22We now progress to a much larger structure map that represents distances, obtained from 2 polymorphs and disordered ices (such as the common ice-I) are widely spread as well, whereas ordered ices are clustered closely together in the bottom left area. In addition to the absolute distribution across the map, we may quantify the relative distribution for each materials class, by which we mean the standard deviation of how far points are from their respective centre of mass – normalised such that the SiO2 polymorphs have a relative distribution of 1.0. ZIFs (zeolites) attain values of 1.20 (1.04), respectively. On the other hand, the ordered ices have a relative distribution of only 0.06, consistent with lower geometric flexibility in their strongly directional hydrogen-bonded networks.In the 2D space of  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.tbd" format="PBM" resolution="300" x-size="17" y-size="11" xml:space="preserve">0000000000000000000000000000000000000000000000000000111111111111111000000000000000000011111111111111100000000000000000001111111111111110000000000000000000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="23.636364pt" height="16.000000pt" viewBox="0 0 23.636364 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>N⋯Zn motif to a symmetric A–B–A link because of head-to-tail orientational disorder of the CN− linkers. Both phases are based on the same anticuprite structure, with no internal degrees of freedom; hence the two corresponding points coincide perfectly. LiCo(CO)4 adopts a lower-symmetry variant of the same structure type,30 with the CO ligand closer to Co than Li – its midpoint is shifted along  from x = 0.25 to 0.241. That structure is therefore almost, but not exactly, in the same location on the cg-SOAP map in We now walk through this map in clockwise direction, having labelled some more specific locations of interest with boldface numbers. In the lower left part, there is a point where two structures coincide exactly in the 2D map (1). One is disordered ice-VII, where we reduce the O–H⋯H–O bridge  to an A–B–A link. The other is the ambient polymorph of zinc cyanide, for which we also reduce the Zn⋯C2 networks. We move clockwise past more open framework structures, viz. the faujasite and Na–Y cages (3), and we find β-quartz near the top of the map (4). In the immediate vicinity, there is then a rather largely populated cluster of ZIFs and related structures . Of note is the cadmium-based framework, CdIF-1, which has sod topology 2 occupy exactly the same location; hexagonal ice-I is very close. We find a region of clathrate hydrates (6), related to the “empty” frameworks of the low-density ices III and XVI, reflected in close proximity in the cg-SOAP map. Separated clearly from the main area of the map, there is then an “island” of inorganic structures on the right-hand side (7): e.g., SiS2, which features chains of edge-sharing tetrahedra, very different from the compositionally homologous SiO2 polymorphs in which all tetrahedra are corner-sharing.β-Cristobalite is another high-symmetry structure with no internal degrees of freedom, located in the upper right part of the map (5). After coarse-graining and re-scaling, cubic ice-I and + or Cu+ cations in combination with B3+, and therefore are aliovalent equivalents to ZIFs (M2+).31 We re-iterate that even though we reduce the cation sites to a single type of “A” dummy atom, we do retain the relative differences in bond lengths around M+vs. B3+; therefore, the BIF-3 frameworks are not near other sod structures. Finally, near the bottom of the cg-SOAP map in 23 This particular one adopts the same topology as hexagonal ice-I (lon) – but in the Zn(CN)2 structure, the metal⋯cyanide distances are very dissimilar, about 1.6 and 2.0 Å respectively, and the data point is therefore away from ice-Ih in the 2D map of 32 this exemplifies a limit of our method in that it needs discrete positions for the “B” grains, and it cannot capture longer-range correlated disorder beyond the pairwise SOAP cut-off distance. Another limitation of the present approach is given by large and highly directional linkers such as [Au(CN)2]− which lead to incorrect A⋯A contacts, shorter than the shortest A–B ones, when dense interpenetrating networks are considered. An example, with six independent interpenetrating nets, is the structure of Zn[Au(CN)2]2;33 related issues will often occur for MOFs, where interpenetration is commonplace.In the lower right part of 3 The first quantity for which we test this question is again concerned with structural diversity. In e.g., in the BIFs, where different aliovalent cationic species occupy the A site, as mentioned above. This information can be visualised in a colour-coded version of our map, which is shown in An important aspect of a materials map is that it should be able to be correlated with relevant properties.15 In the context of the present work, a central such property is the tetrahedral (“T”) density: this is the simplest proxy for possible usefulness in catalysis, because low T densities indicate the presence of voids in the framework, which could be used for the absorption, diffusion, and transformation of guest molecules – noting that the T density of the re-scaled framework need not directly correspond to the accessible pore volume, nor indeed to the density of catalytically active sites. We show a colour-coded version of our map, illustrating the T density, in SOAP maps are beginning to be used to identify properties of application interest.2 polymorphs include locally heterogeneous environments  – but most of them do not, and neither do most other inorganic AB2 structures. In clathrates, on the other hand, we do not find any fully homogeneous structure . Disordered ices are overall more heterogeneous than ordered ones. Among the framework materials, CdIFs are the least locally heterogeneous, which is perhaps surprising given the large ionic radius and polarisability of Cd2+; BIFs show a large, and narrowly distributed, heterogeneity in The embedding of high-dimensional distances in 2D invariably leads to the loss of some information. It is therefore useful, in addition to the map, to look quantitatively at similarities and properties independent from where a given material is located in the 2D map. We quantify the distribution of A-site SOAP heterogeneities, separately for the different materials classes, in x = 0), but there are also two distinct regions of heterogeneity , dominated by zeolite structures (“+”). Generally, e.g., by exploiting solid-solution chemistry, both regarding isovalent or aliovalent cations, and combinations of different linkers.Finally, the information content of 34 hybrid perovskites,35 or to a more extensive range of MOFs,36 for example. Our approach is chemically agnostic on purpose  – although we note that the purely geometric SOAP kernel can be amended with terms that depend on the atomic numbers, or even with entirely different kernel definitions that capture, e.g., similarities in the electronic structure.37 Such combined models could then extend to application-related properties which are determined by geometry and chemistry . In regard to visualisation tools, we used one of the simplest , which already leads to appreciable results, but one might couple our approach to other, more involved dimensionality-reduction schemes such as the popular sketch-map scheme38 or t-stochastic neighbour embedding39 which are also beginning to be used with SOAP,8,15 and to openly available implementations which are beginning to emerge.13,40 To this end, our database of all coarse-grained representations will be made openly available online upon publication of this work, with the hope to enable future work in the community.We have shown how structural relationships across diverse material families can be understood by combining the idea of coarse-graining and scaling atomistic structure with a suitable atom-density based similarity metric . Our study has built on experimentally characterised structures and a carefully curated database of those, but similar approaches may now be extended to even larger sets of data: to hypothetical zeolites,6 here denoted α and β. To describe the environment of an atom α, an atomic density, ρα(r), is constructed by placing Gaussian functions, of broadness σ, on the atomic positions. The neighbour density is then expanded in a local basis set of suitable radial functions, Rn, and spherical harmonics, Ylm:nmax and lmax. This way, by collecting the combination coefficients, cnlmα) of pairs of atomic environments,https://github.com/SINGROUP/dscribe/),27 an expansion of the atomic neighbour density . We used a relatively large exponent for the “sharpness” of the kernel , e.g., using fewer descriptor vector entries in multispecies systems, but these differences are not expected to affect our conceptual findings or the interpretation of cg-SOAP maps. For the same reason, no numerical coordinates are given in the map in 8,10,24We computed SOAP vectors using the polynomial basis functions implemented in DScribe , asα (β) runs over all A sites in the i-th (j-th) cell, respectively, and the coarse-grained B sites are included in the respective neighbour densities of the A-sites α and β  have been proposed;8,10 the optimised choice of these definitions for cg-SOAP maps will be the subject of future, more technical work.We obtained the per-cell similarity, 42 The technique performs a least-squares minimisation of the stress, defined asdij is the SOAP distance between the i-th and j-th atomic environment in high-dimensional space  representation. The stress is zero if the original distances are fully respected. We obtained a stress value of 0.251, with convergence defined by a maximum change of 10−5. There is, hence, an appreciable loss of some part of the high-dimensional information, but this does not impair the validity of our 2D map  that form the basis for this work is given as ESI.There are no conflicts to declare.SC-011-D0SC03287E-s001


A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation. By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, we show that all the early hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoid’s partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate.For over 40 years, the Bicoid- Morphogen gradients are used by various organisms to establish polarity along embryonic axes or within organs. In these systems, positional information stems from the morphogen concentration detected by each cell in the target tissue and mediates the determination of cell identity through the expression of specific sets of target genes. While these processes ensure the reproducibility of developmental patterns and the emergence of properly proportioned individuals, the question of whether the morphogen itself directly contributes to this robustness or whether it requires the involvement of downstream cross-regulatory networks or cell-communication remains largely debated. This question becomes even more pressing with the recent discovery that when studied at the single-cell level, transcription is frequently observed to be an extremely noisy process, hardly suggestive of such precise control.bcd mRNA maternally provided and the number of functional bcd alleles in the females  morphogen system which initiates pattern formation along the antero-posterior (AP) axis in the young fruit fly embryo . The Bcd females .hunchback (hb) is expressed in a large domain spanning the whole anterior half of the embryo  bcd functional copies, the boundary shift of the Bcd-only synthetic reporter with 9 Bcd BS was small enough to set the Bcd system within the limits of an equilibrium model. Lastly, the shift observed for the hb-P2 reporter in 1 X vs 2 X bcd backgrounds was the same as for the synthetic reporters further supporting that the Bcd gradient is the main source of positional information for the early expression of hb.While quantitative models based on equilibrium binding of transcription factors to DNA shed lights on segmentation in osophila  or on thosophila , their iosophila  and the osophila . We showhb locus with all its Bcd-dependent enhancers did not affect the regulation of the wild-type gene   upstreamype gene , it is uhb intron  for all synthetic reporters  and hb-P2 (The expression of the B6 (6 Bcd BS), B9 (9 Bcd BS), and B12 (12 Bcd BS) reporters harbored similar features as expression of the hb-P2 reporter , which cb intron , Video 4b intron . For all AP axis , as obse AP axis . Thus, Bnd hb-P2 . This cond hb-P2  and whernd hb-P2  and the nd hb-P2  was onlyFor all reporters, the fraction of nuclei with MS2 signal during the cycle exhibited a sigmoid-like pattern along the AP axis reaching 100% in the anterior and 0% in the posterior . We fitthb pattern. Increasing this number up to nine is sufficient to recapitulate almost all spatial features of the hb-P2 pattern except for the steepness of the expression boundary. Of note, the Bcd-only reporters take much longer than the hb-P2 reporter to reach the final decision for boundary positioning suggesting that binding of additional transcription factors in the hb-P2 promoter likely contribute to speeding-up the process.Thus, even though 6 Bcd BS have been described in the hb-P2 promoter, having only 6 Bcd BS alone in a synthetic reporter is not sufficient to recapitulate the hb-P2) = 0.84 ± 0.008 was as high as for B9 or B12 . From the time trace of MS2 activity in each nucleus, the fluctuation of the transcription process   while thGiven that the expression patterns of hb-P2 and all Bcd-only reporters reached a plateau in the anterior where Bcd concentration is likely in excess, we compared the activation rates Despite the same number of Bcd BS in the B6 and hb-P2 reporters, their expression pattern and dynamics were very different . To deteWe first analyzed the impact of combining the Bcd BS with Hb BS. A H6 reporter containing only 6 Hb BS did not exhibit any MS2 signal from nc11 to nc13 (not shown). This indicated that the Hb protein alone, even with an abundance of Hb sites, could not activate transcription on its own. When combining 6 Hb BS with the 6 Bcd BS of B6  , with ve~0.95–1) . Meanwhi~0.95–1) .To model H6B6 activity, the same formalism, as applied to B9 and B12 reporters, was used starting from the parameter values imposed from the fitted model of B6 and then varying those parameters, either alone or in combination, to fit the H6B6 data. The simulations indicated that a moderate fit to the data was obtained when varying only the the data .Altogether, this suggests that Hb binding to the promoter accelerates the measurement of positional information by Bcd by improving both the unbinding kinetics of Bcd to its BS, which is consistent with the half reduction of the boundary steepness  and the As the hb-P2 promoter also contains Zld BS, we used our synthetic approach to investigate the role of Zld in the Bcd system. As a reporter with only 6 Zld BS (Z6) was strongly expressed along the whole AP axis , we had eporters , C-D buteporters . This sueporters . As obseeporters  when it eporters .The most striking feature of the Z2B6 reporter was the drastic posterior shift of its expression boundary by ~17.5 %EL when compared to B6 , C-D. ItPSpot  and the Spot  . As the amount of Bcd protein is produced from each bcd allele independently of any other allele in the genome . In such Bcd-2X and Bcd-1X embryos, we compared the fraction of expressing nuclei along the AP axis as modeled at the top of Since our synthetic Bcd-only reporters exclusively respond to Bcd, their expression boundary is exclusively dependent on specific thresholds of Bcd concentration, and this property was used to evaluate quantitatively the Bcd-activity gradient. For this, we reduced the amount of the Bcd protein by half in embryos from females, which were heterozygous for a CRISPR-induced deletion of the e genome  and as ce embryo , we assuvs Bcd-1X embryos, we observed that the shift was very consistent in the zone between B6 and Z2B6’s boundary regions (30% EL to 60% EL)  . Thus, i curves) . Lastly,boundary . As thisrs above , B-D, wehb in previous studies using the E1bcd allele   and reprl <0.05)  than theThus, lowering the decay length of the Bcd gradient to its effective value allows a more reliable fit of the model to the data and places back the Bcd system within the physical limits of an equilibrium model for concentration sensing.hb regulation by Bcd. The number and placement of TF BS in our MS2 reporters are not identical to those found on the endogenous hb promoter and the number of combinations tested was very limited when compared to the high throughput approaches mentioned above. Nevertheless, this synthetic approach combined with quantitative analyses and modeling sheds light on the mechanistic steps of transcription dynamics  involving each of the three TFs considered . Based on this knowledge from synthetic reporters and the known differences between them, we built an equilibrium model of transcription regulation which agrees with the data from the hb-P2 reporter expression.Recently, synthetic approaches have been used to understand how the details of gene regulation emerge from the plethora of binding sites for transcription factors buried in genomes. In developmental systems, these approaches are starting to help us unravel the evolution of gene regulatory modules  than the number of BS (1.5–2 times) suggesting a synergistic effect in transcription activation by Bcd.The burstiness of the Bcd-only reporters in regions with saturating amounts of Bcd, led us to build a model in two steps. The first step of this model accounts for the binding/unbinding of Bcd molecules to the BS arrays. It is directly related to the positioning and the steepness of the expression boundary and thus to the measurement of positional information. The second step of this model accounts for the dialog between the bound Bcd molecules and the transcription machinery. It is directly related to the fluctuation of the MS2 signals including the number of firing RNAP at a given time  and bursting . Interestingly, while the first step of the process is achieved with an extreme precision (10% EL) , the sechb expression pattern i.e. measurements of positional information. This is unexpected as the hb-P2 promoter is supposed to only carry 6 Bcd BS and leaves open the possibility that the number of Bcd BS in the hb promoter might be higher, as suggested previously (i) some of the Bcd binding sites in the hb-P2 promoter are also very close to each other (see ii) the binding of Bcd or other homeodomain containing proteins to two BS is generally increased by cooperativity when the sites are close to each other (as close as two base pairs for the paired homeodomain) compared to binding without cooperativity when they are separated by five base pairs or more  th or more .hb pattern establishment. Among these TFs, we focused on two known maternal partners of Bcd: Hb which acts in synergy with Bcd  information to the process. Interestingly, the B9 reporter is however much slower (2-fold) to reach the final boundary position than the hb-P2 reporter. This suggested that other maternally provided TFs binding to the hb-P2 promoter contribute to fast dynamics of the with Bcd  and Zld,with Bcd . Interesactivity .Zld functions as a pioneer factor by potentiating chromatin accessibility, transcription factor binding and gene expression of the targeted promoter . Zld hashb in the past, always giving a smaller shift than expected given the decay length of 20% EL for the Bcd protein gradient . Unless otherwise specified, all MS2 reporters were inserted at the vk33 docking site (Bloomington # 9750) via φC31-mediated integration system  from the previously used hb-MS2ΔZelda reporter  located just upstream start codon of Hunchback protein  , we firsAP axis)  for the the vk33 .To quantify the sharpness of the transcription patterns of MS2 reporters, we fit the patterns along the AP axis to a sigmoid function:In From the fitted pattern, we define the boundary width as the distance between two nuclei columns with We assume an exponential Bcd gradient with the decay length One should note that in In ficients .For a model of transcription regulation with We quantify the shift of the MS2 expression patterns along the AP axis from Bcd-2x to Bcd-1x flies in terms of the probability distribution of the shift Given this probability distribution as described in detail in the Appendix 4.We fit the models of Bcd binding/unbinding to binding sites and activation of transcription, each with a different value of the Bcd gradient decay length eporters  and hb-Peporters  as descr In this paper, the authors use synthetic transcriptional enhancers to probe the roles of three transcription factors, Bicoid, Hunchback and Zelda, in specifying the production of a sharp, accurately placed gene expression boundary in early fruit fly embryos. They find that Bicoid, which is expressed in an anterior-posterior gradient, is sufficient on its own to generate a boundary in the same location as a wild-type fly, but combinatorial regulation by Hunchback and Zelda is needed to ensure the boundary forms quickly enough. They further combine their experimental observations with modeling to conclude that Bicoid exists in active and inactive forms, and that an equilibrium model captures the relevant behaviors, implying energy expenditure during the binding of transcription factors to DNA or RNA polymerase is theoretically unnecessary. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:hunchback regulation by Bicoid" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Naama Barkai as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Timothy Saunders (Reviewer #1); Justin Crocker (Reviewer #3).Thank you for submitting your article "Synthetic deconstruction of The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. While all the reviewers found the study interesting, they also unanimously agree that more data and clarifications are required to substantiate the claims of the manuscript. Please do address all the major comments in the reviews below in your revision.Reviewer #1 (Recommendations for the authors):– My major concern regards the conclusions for the final section on the activity gradient. In the Introduction it is stated: "[the Bcd gradient has] an exponential AP gradient with a decay length of ~ 20% egg‐length (EL)". While this was the initial estimate , later measurements by the Gregor lab  found that "The mean length constant was reduced to 16.5 {plus minus} 0.7%EL after corrections for EGFP maturation". The original measurements by Houchmandzadeh et al. had issues with background control, that also led to the longer measured decay length. In later work, Durrieu et al., MSB 2018, found a similar scale for the decay length to Liu et al. Looking at Figure 5, a value of 16.5%EL for the decay length is fully consistent with the activity and protein gradients for Bcd being similar. So, either (1) the authors need to provide convincing evidence that, for some reason, in their specific lines the Bcd gradient is longer than expected; or (2) they need to alter the conclusions of the final sections. Indeed, the evidence to me is interesting as it shows that the activity gradient and protein gradient actually are similar (e.g. 15% EL activity vs 16.5% EL protein) which is not, as the authors highlight, a priori obvious. Now, this small shift may well be significant statistically and it is a worthy point in its own right. However, the strength of the conclusions clearly do not match the known gradient and should be substantially toned down.Related to the above, the abstract, intro and discussion all need parts to be rewritten to reflect more accurately the data interpretation. Given the current evidence presented in the manuscript, all conclusions on the activity gradient relative to the protein gradient are incorrect.– While the videos are nice, it would be very helpful to show some of the actual data in the figures. Clearly annotated time points would significantly enhance the readability of the first section. The heatmaps are insufficient. This is also true for Figure 2A – show some real data to highlight the burstiness point.– The axis scales are not helpful. I'm assuming that "0% EL" is meant to be the hb boundary. This is not defined in legend of figure 1, indeed there i simply says distance along embryo axis. Put 0% at one of the poles. This is convention and I see no reason to define 0% at a (somewhat arbitrary) position in the centre of the embryo. Please keep to standard nomenclature unless a very compelling reason. I think this also leads to confusion. E.g. line 242 states that the expression boundary is at 17.5% EL , yet the results in this figure only extend to 10%EL. Please clarify.– All of the experiments are performed in a background with the hb gene present. Does this impact on the readout, as the synthetic lines are essentially competing with the wild-type genes. What controls were done to account for this?– Further, the activity of the synthetic reporters depend on the location of insertion. Erceg et al. PLoS Genetics 2014 showed that the same synthetic enhancer can have different readout depending on its genomic location. I'm aware that the authors use a landing site that appears to replicate similar hb kinetics, but did they try random insertion or other landing site? In short, how robust are their results to the specific local genome site? This should have been tested, especially given the boldly written conclusions from the work.– Related to the above, it's also not obvious that readout is linear – i.e. as more binding sites are added, there could be cooperativity between binding domains. This may have been accounted for in the model but it is not clear to me how.eLife so that readers are given clearer context.– It would be good in the Introduction/Discussion to give a broader perspective on the advantages and disadvantages of the synthetic approach to study gene regulation. The intro only discusses Tran et al., Yet, there is a strong history of using this approach, which has also helped to reveal some of the approaches shortcoming. E.g. Gertz et al., Nature 2009 and Sharon et al., Nature Biotechnology 2012. Again, I may have missed, but from my reading I cannot see any critical analysis of the pros/cons of the synthetic approach in development. This is necessary for a broad journal such as Reviewer #3 (Recommendations for the authors):I think the authors should acknowledge more explicitly that Hb in the endogenous system acts in an autoregulatory fashion, which it does not do in the synthetic construct. This is relevant to two of the conclusions in particular:1) I agree that Hb's effects on bursting would increase patterning speed. However, in the B6+Hb BS construct an additional contribution to patterning dynamics could come from the fact that the endogenous Hb boundary is itself dynamic and forms faster than B6 alone. Unless we know how the reporter construct would act with autoregulation orthogonal to Hb, we can't say for sure whether adding Hb BS to B6 gives B6 access to higher concentrations of activator in the anterior than would be expected at the same time point in the endogenous autoregulatory loop.2) In lines 329-330 the authors state that "the effects of Zld and Hb are modeled implicitly by the kinetic rate constants". Is this valid? The Hb boundary is dynamic during the relevant time window, and because Hb in hb-p2 is autoregulatory even time-varying kinetic rates fixed to a measured dynamic trajectory for Hb concentration wouldn't be valid for modeling/drawing conclusions about the endogenous network.I find the logscale visualization in Figure 4 unnecessarily involved when the reader simply needs to know the approximate size of the boundary shift. I would move the plots to the supplement. Reviewer #1 (Recommendations for the authors):– My major concern regards the conclusions for the final section on the activity gradient. In the Introduction it is stated: "[the Bcd gradient has] an exponential AP gradient with a decay length of ~ 20% egg‐length (EL)". While this was the initial estimate , later measurements by the Gregor lab  found that "The mean length constant was reduced to 16.5 {plus minus} 0.7%EL after corrections for EGFP maturation". The original measurements by Houchmandzadeh et al. had issues with background control, that also led to the longer measured decay length. In later work, Durrieu et al., MSB 2018, found a similar scale for the decay length to Liu et al. Looking at Figure 5, a value of 16.5%EL for the decay length is fully consistent with the activity and protein gradients for Bcd being similar. So, either (1) the authors need to provide convincing evidence that, for some reason, in their specific lines the Bcd gradient is longer than expected; or (2) they need to alter the conclusions of the final sections. Indeed, the evidence to me is interesting as it shows that the activity gradient and protein gradient actually are similar (e.g. 15% EL activity vs 16.5% EL protein) which is not, as the authors highlight, a priori obvious. Now, this small shift may well be significant statistically and it is a worthy point in its own right. However, the strength of the conclusions clearly do not match the known gradient and should be substantially toned down.The reviewer is right: several studies aiming to quantitatively measure the Bicoid protein gradient ended-up with quite different decay lengths.A summary of the various decay lengths measured, and the method used for these measurements is given in vs fluorescent measurements on live sample) or to the type of protein detected (endogenous Bicoid vs fluorescently tagged).As indicated, these measurements are quite variable among the different studies and the differences can potentially be attributed to different methods of detection . Importantly, the new Hill coefficient allows us to set the Bcd system within the limits of an equilibrium model.We agree with the reviewer that given these discrepancies, the exact value of the Bcd protein gradient decay length is not known and that we only have measurements that put it in between 16 and 25 % EL see . Therefovs tagged protein might have different decay lengths due to potentially different diffusion coefficients or half-lives. Before drawing any conclusion on the exact value of the endogenous Bcd protein gradient decay length, it is essential to measure it again in conditions that correct for the background issues for immuno-staining as it was done in Liu et al., PNAS, 2013 for the Bcd-eGFP protein. In this study, the authors only measured the decay length of the Bcd fusion protein using immuno-staining for the Bcd protein. Unfortunately, in this study, the authors did not measure again the decay length of the endogenous Bcd protein gradient using immuno-staining and the same procedure for background control. Therefore, they do not firmly exclude the possibility that the endogenous vs tagged Bcd proteins might have different decay length.As mentioned by the reviewer, it is possible that the decay length of the protein gradient measured using antibody staining  was not correct due to background controls. Such measurements were also performed in Xu et al., (2015) which agree with the original measurements . As indicated in We thank the reviewer for his comment which helped us to clarify the message:– In the result section, we only focus on the decay length measurements of the Bcd activity gradient using the Bcd-only reporters, putting the emphasis on the fact that the decay length measured is short and places back the Bcd system within the physical limits of an equilibrium model.– In addition, as there is clearly an issue for the measurements of the Bcd protein gradient, we added an Appendix  and a Table (Supplementary File 3) describing the various decay length measured for the Bcd or the Bcd-fluorescently tagged protein gradients from previous studies.vs activity gradient , we also discuss the alternative possibility proposed by the reviewer which is that the protein vs activity gradients have the same decay lengths but that the decay length of the Bcd protein gradient was potentially not correctly evaluated.– Finally , in the discussion (lines 482 to lines 490), together with the possibility that there might be a protein Related to the above, the abstract, intro and discussion all need parts to be rewritten to reflect more accurately the data interpretation. Given the current evidence presented in the manuscript, all conclusions on the activity gradient relative to the protein gradient are incorrect.The sentences related to this aspect have been rephrased to be more accurate: in most cases (except in the discussion), we only focus on the decay length measurements of the Bcd activity gradient using the Bcd-only reporters, putting the emphasis on the fact that the decay length measured is short and places back the Bcd system within the physical limits of an equilibrium model.The modifications in the main text concerning this point are :– Introduction: line 99– Result section: everything concerning the decay length of the Bcd protein gradient has been removed from line 283 to the end of the section. The title of the section line 283 has been changed in “A Bicoid-Activity gradient with a short decay length”– Discussion: lines 482 to 490– While the videos are nice, it would be very helpful to show some of the actual data in the figures. Clearly annotated time points would significantly enhance the readability of the first section. The heatmaps are insufficient. This is also true for Figure 2A – show some real data to highlight the burstiness point.We completely agree with this comment which will help the reader to grasp even more what the MS2 videos bring.We added a new figure  to highlight these aspects. In this figure, we show :– A fluorescent trace (intensity of a locus as function of time) along a whole nc13 (Panel A) and series of snapshots at chosen times for this specific trace (Panel B) of the B6 reporter which is the most fluctuating one.– Kymographs of traces to highlight burstiness of all the reporters used: these are traces all obtained in nuclei located at the anterior (where Bcd is supposed to be in excess). 20 traces for each reporter used in this study are shown Panel C.– The axis scales are not helpful. I'm assuming that "0% EL" is meant to be the hb boundary. This is not defined in legend of figure 1, indeed there i simply says distance along embryo axis. Put 0% at one of the poles. This is convention and I see no reason to define 0% at a (somewhat arbitrary) position in the centre of the embryo. Please keep to standard nomenclature unless a very compelling reason. I think this also leads to confusion. E.g. line 242 states that the expression boundary is at 17.5% EL , yet the results in this figure only extend to 10%EL. Please clarify.17.5 %EL.As requested by the reviewer, the anterior pole has been set at 0% EL and the posterior at 100% EL in all figures, figure supplements and supplementary File 2. In line 270, we have clarified that the shift of the expression boundary (not the absolute position itself) between Z2B6 and B6’s boundary position is – All of the experiments are performed in a background with the hb gene present. Does this impact on the readout, as the synthetic lines are essentially competing with the wild-type genes. What controls were done to account for this?hb boundary where a nucleus has been shown to only contain ~ 700 Bicoid molecules . However, ~1000 Bicoid binding regions have been identified by ChIP seq experiments in nc14 embryos  and given that several Bcd binding sites are generally clustered together in a Bcd region, the number of Bcd binding sites in the fly genome is likely larger than 1000. It is much greater than the number of Bicoid binding sites in our synthetic reporters. Therefore, we think that it is unlikely that adding the synthetic reporters (which in the case of B12 only represents at most 1/100 of the Bcd binding sites in the genome) will severely alter the competition for Bcd binding between the other Bcd binding sites in the genome. Additionally, the insertion of a BAC spanning the endogenous hb locus with all its Bcd-dependent enhancers did not affect (as far as we can tell) the regulation of the wild-type gene .We agree with the reviewer that this concern might be particularly relevant at the We have added a sentence concerning this point in the main text (lines 110 to 113).– Further, the activity of the synthetic reporters depend on the location of insertion. Erceg et al., PLoS Genetics 2014 showed that the same synthetic enhancer can have different readout depending on its genomic location. I’m aware that the authors use a landing site that appears to replicate similar hb kinetics, but did they try random insertion or other landing site? In short, how robust are their results to the specific local genome site? This should have been tested, especially given the bo’dly written conclusions from the work.This concern of the reviewer has been tested and is addressed Figure 1—figure supplement 1 where we compare two random insertions of the hb-P2 transgene  and the insertion at the VK33 landing site that was used for the whole study. As shown Figure 1—figure supplement 1, the dynamics of transcription (kymographs) are very similar. In the main text, the reference Figure 1—figure supplement 1 is found in the result section (line 107) and in the Materials and methods section (line 529).– Related to the above, it’s also not obvious that readout is linear – i.e. as more binding sites are added, there could be cooperativity between binding domains. This may have been accounted for in the model but it is not clear to me how.The reviewer is totally correct. It is clear from our data that readout is not linear: comparing (increase of 1.5 X in the number of BS) B6 with B9 leads to a 4.5 X greater activation rate and this argues against independent activation of transcription by individual bound Bcd TF. There is almost no impact of adding 3 more sites when comparing B9 to B12 (even though it corresponds to an increase of 1.33 X in the number of BS).This issue has been rephrased in the main text (lines 205 to 208) and is further developed for the modeling aspects in the Appendix 3 and Figure 2—figure supplement 2. It is also discussed in the second paragraph of the discussion (lines 392 to 395).– It would be good in the Introduction/Discussion to give a broader perspective on the advantages and disadvantages of the synthetic approach to study gene regulation. The intro only discusses Tran et al. Yet, there is a strong history of using this approach, which has also helped to reveal some of the approaches shortcoming. E.g. Gertz et al., Nature 2009 and Sharon et al., Nature Biotechnology 2012. Again, I may have missed, but from my reading I cannot see any critical analysis of the pros/cons of the synthetic approach in development. This is necessary for a broad journal such as eLife so that readers are given clearer context.One sentence was added in the introduction concerning this point (lines 80 to 83).A short review concerning the synthetic approach in development has also been added at the beginning of the discussion (lines 359 to 371).Reviewer #3 (Recommendations for the authors):I think the authors should acknowledge more explicitly that Hb in the endogenous system acts in an autoregulatory fashion, which it does not do in the synthetic construct. This is relevant to two of the conclusions in particular:1) I agree that Hb's effects on bursting would increase patterning speed. However, in the B6+Hb BS construct an additional contribution to patterning dynamics could come from the fact that the endogenous Hb boundary is itself dynamic and forms faster than B6 alone. Unless we know how the reporter construct would act with autoregulation orthogonal to Hb, we can't say for sure whether adding Hb BS to B6 gives B6 access to higher concentrations of activator in the anterior than would be expected at the same time point in the endogenous autoregulatory loop.We totally agree that the endogenous Hb boundary formed thanks to the endogenous autoregulatory loop contributes to the dynamics of the H6B6 construct. In our synthetic approach, none of our reporters express the Hb protein, and thus we focused our work on understanding the role of each BS on transcription in the context of the endogenous autoregulation. Thus, comparing H6B6 and B6 let us understand the role of Hb BS on transcription in the same context of Hb protein dynamics. We did not focus our work on the combined effect of transcription and autoregulation, which would require an orthogonal system as noted by reviewer #3.2) In lines 329-330 the authors state that "the effects of Zld and Hb are modeled implicitly by the kinetic rate constants". Is this valid? The Hb boundary is dynamic during the relevant time window, and because Hb in hb-p2 is autoregulatory even time-varying kinetic rates fixed to a measured dynamic trajectory for Hb concentration wouldn't be valid for modeling/drawing conclusions about the endogenous network.hb transcription boundary is dynamic during the relevant time window. However, a time delay is expected concerning the corresponding increase in the nuclear concentration of the Hb protein (the activator) : the system requires nuclear export of the hb mRNA, its translation and the nuclear import of the translated Hb protein to affect the activity of the promoters. Given the extremely fast time scale of boundaries establishment (3 min), we don’t think that there would be a significant effect on patterning dynamics coming from endogenous hb transcribed at the same time over this time scale. This time delay has also to be considered in the context of the very dynamic nuclear division process. It is thus very likely that the nuclear Hb protein at work at nc13 was produced from mRNA expressed at earlier nuclear cycles. Therefore, we assumed that the Hb protein expression domain is well delimited by the position of the hb-P2 transcription boundary and that the amount of Hb protein expressed is constant both in its expression domain and along the period of the interphase nuclear cycle during which transcription occurs. As the boundaries of B6 and H6B6 are more anterior than the boundary of hb-P2, these boundaries are localized in a domain where the Hb protein is at its maximum and at a plateau.This assumption is clearly valid for Zelda which is homogeneously distributed all along the AP axis. For Hb, as noted by the reviewer, it is clear from our study that the I find the logscale visualization in Figure 4 unnecessarily involved when the reader simply needs to know the approximate size of the boundary shift. I would move the plots to the supplement.Δbcd strain). It became thus possible to merge the logscale figures for each of the 4 reporters. As these reporters have different boundary position along the AP axis and thus different threshold values of Bcd concentration for positioning their boundary, the determination of the effective decay length is obtained on a wider segment of the AP axis. The merged logscale has a wider horizontal region confirming that the effective activity gradient of Bcd is exponential at least in this part of the AP axis.We agree with the reviewer that as such this part of the Figure 4 was not very convincing nor did it add more than the measurements of the shifts and the fits with the position extracted from the model assuming the calculated decay length. We think that this was due to the fact that the logscale panels C and E of Figure 4 correspond to individual plots from the B9 (C) or the hb-P2 (E) reporters. Since the first submission of this manuscript, we were able to collect additional data for the B6 and Z2B6 reporters in the same experimental context  as well as the graphs obtained for the two additional reporters B6 (panel B) and Z2B6 (panel D). Of note, H6B6 cannot be used in this assay as the expression of the Hb protein varies in the 1X vs 2X


Staphylococcus species. Infective endocarditis is a rare but life-threatening complication of gonococcal bacteremia. We report the first case of a CIED pocket-site infection secondary to Neisseria gonorrhoeae (N. gonorrhoeae).  Cardiovascular implantable electronic devices (CIEDs) are being increasingly used in the primary and secondary prevention of malignant ventricular arrhythmias and conduction system disorders. Infectious complications associated with CIEDs include infective endocarditis, lead infections, and pocket-site infections, primarily involving N. gonorrhoeae. He underwent surgical debridement with complete ICD extraction and drainage of infected serosanguineous pocket fluid. Tissue cultures were negative, but isolation of N. gonorrhoeae in blood cultures confirmed it as the causative agent of the pocket-site infection in the absence of prior Gram-positive coverage. He was started on a prolonged course of ceftriaxone for 4 weeks with reimplantation of ICD at a different site after completion of treatment.  A 56-year-old male with a history of congestive heart failure status postimplantable cardioverter-defibrillator (ICD) insertion presented with a pocket-site swelling initially concerning for a hematoma which began to exhibit erythema and tenderness. The patient reported a history of high-risk sexual behavior. On presentation, he was afebrile and hemodynamically stable. Physical exam showed a 5 cm × 6 cm pocket-site swelling with overlying erythema. Labs revealed elevated ESR and CRP levels. Transthoracic and transesophageal echocardiography was concerning for infective endocarditis and lead vegetations. Blood cultures tested positive for  In patients with high-risk sexual behavior, gonococcal bacteremia can potentially lead to CIED infection. These individuals should be prudently evaluated for infective endocarditis or pocket-site infections as presenting complaints can be subtle. Staphylococcus species (Staphylococcus aureus and coagulase-negative Staphylococci) have been the usual culprit in infectious complications [N. gonorrhoeae bacteremia in an individual with a history of high-risk sexual behavior.The use of cardiovascular implantable electronic devices (CIEDs) has increased significantly for the prevention of life-threatening arrhythmias in coronary artery disease and congestive heart failure . CIEDs cications . We discStaphylococcus aureus (MRSA). Pocket-site ultrasound in clinic revealed a 4.5 cm × 2 cm × 2 cm fluid collection concerning for hematoma, prompting discontinuation of apixaban. The swelling progressively increased in size on a subsequent clinic visit. This, along with the development of pocket-site pain, erythema, and tenderness, in association with generalized malaise and fatigue prompted admission.A 56-year-old African-American male with past medical history of paroxysmal atrial fibrillation (on apixaban), chronic obstructive pulmonary disease (COPD), chronic kidney disease (CKD) stage III, and heart failure with reduced ejection fraction (EF: 25–30%) status post-ICD placement for primary prevention presented to the arrhythmia clinic with a pocket-site swelling. The patient reported that the swelling began recently a few days after lifting a heavy object at home. He had previously undergone complete ICD device and lead extraction and subsequent reimplantation 2 years prior due to a pocket-site infection which was secondary to methicillin-resistant 3) but elevated erythrocyte sedimentation rate: 34 mm/hr; C-reactive protein: 70.9 mg/dL; procalcitonin: 1.31 ng/ml; and INR: 1.3. Transthoracic echocardiography showed reduced EF (25–30%) with small mobile echodensities associated with the lead in the right ventricle which were concerning for vegetations. Transesophageal echocardiography showed no definitive ICD lead vegetations; however, the right atrium had a filamentous structure suspicious for an early infected vegetation. Meanwhile, blood cultures grew Gram-negative cocci in ¼ bottles. Since the patient had reported a prior penicillin allergy, aztreonam was initiated. The Gram-negative cocci speciated to N. gonorrhoeae on blood cultures, with the patient divulging a history of high-risk sexual behavior  in the recent past. Of note, he did not report any urogenital symptoms during the course of his illness. Further clarification of his penicillin allergy, which was limited to hives, allowed for transition to intravenous (IV) ceftriaxone on the second day of antibiotic therapy. Repeat blood cultures 24 hours later also grew gonococci but cleared thereafter. The patient underwent surgical debridement and complete device and lead extraction a week later which revealed infected and necrotic tissue at the pocket-site with drainage of serosanguineous fluid. The pocket site was washed with a triple-antibiotic solution and closed. Cultures from the tissue, fluid drained from the pocket, and the device failed to grow any organisms while Gram stain was negative for any pathogens as well. Infectious disease (ID) was consulted. Given the isolation of N. gonorrhoeae alone on blood cultures and clinical evidence of pocket-site infection, a diagnosis of device and pocket-site infection secondary to gonococcal bacteremia was made. In the absence of prior exposure to antibiotics with Gram-positive coverage, pocket-site infection with Gram-positive organisms, including the commonly implicated Staphylococcus or Streptococcus species, was effectively ruled out as the etiology of the infection. In accordance with ID recommendations, he continued to receive IV ceftriaxone for 4 weeks with gradual resolution of the pocket-site swelling. Since the ICD was implanted for primary prevention of ventricular arrhythmias, it was deemed prudent to wait until the patient had completed an appropriate course of antibiotics. He responded well to this course of therapy and was successfully discharged. He continued to follow with ID and cardiology as outpatient, eventually undergoing successful reimplantation of an ICD 1 month later at a different site without complications.On presentation, he was afebrile (98.3°F) and hemodynamically stable (pulse 93 bpm and blood pressure 115/82 mm Hg) with oxygen saturations of 96% on room air. Physical examination revealed a 5 cm × 6 cm precordial swelling exhibiting localized warmth and redness. A new-onset II/VI systolic murmur was auscultated in the tricuspid area. Relevant laboratory workup showed no leukocytosis  of cases, with Gram-negative bacilli and fungal pathogens causing the remainder [CIED infections encompass pocket-site and systemic (infective endocarditis or lead infection) subtypes. These subtypes are not mutually exclusive, as demonstrated in our patient who exhibited both a pocket-site infection and infective endocarditis (IE). Infections may also be classified as primary or secondary; our case exhibited CIED infection secondary to gonococcal bacteremia acquired as a sexually transmitted disease. emainder , 7. Cultemainder . In addiemainder , 9.Endocarditis may be reported in 1-2% of patients with disseminated gonococcal infection, making this a relatively rare entity with only a handful of cases reported in the medical literature , 11. SymN. gonorrhoeae endocarditis leads to significant valvular damage, necessitating surgical intervention in up to 70% of cases, compared to 25–50% when other organisms are involved [Corroborating prior reports, gonococcal growth on tissue and device cultures was negative in our case, likely due to prior antibiotic administration for a week, with the fastidious nature of the organism contributing to it as well. involved .CIED infections are managed with IV antibiotics, device explantation, debridement, and reimplantation at a different site . A proloN. gonorrhoeae should be part of the differential diagnoses in patients with high-risk sexual behavior presenting with constitutional symptoms and/or new-onset heart murmurs.Gonococcal bacteremia can lead to CIED pocket-site infection necessitating device removal, surgical debridement, and systemic antibiotics. Device infections and endocarditis with


Infectious bronchitis virus (IBV) infection causes significant economic losses to various sectors of the poultry industry worldwide. Over the past few years, the incidence of false layer syndrome in Eastern Canadian layer flocks has been associated with the increased prevalence of the IBV Delmarva (DMV)/1639 strain. In this study, 1-day-old specific-pathogen-free (SPF) hens were infected with the Canadian DMV/1639 strain and observed until 16 weeks of age in order to determine if the IBV DMV/1639 strain is causing false layer syndrome. Early after infection, the virus showed a wide tissue distribution with characteristic gross and histopathological lesions in the respiratory tract and kidney. Around 60–70% of the infected hens demonstrated continuous cloacal viral shedding until the end of the experiment (at 16 weeks) which was associated with high IBV genome loads detected in the cecal tonsils. The experiment confirmed the field observations that the Canadian DMV/1639 strain is highly pathogenic to the female reproductive tract causing marked cystic lesions in the oviduct. Moreover, significant histopathological damage was observed in the ovary. Our study provides a detailed description of the pathological consequences of the IBV DMV/1639 strain circulating in an important poultry production sector. Coronaviridae. IBV is primarily a respiratory pathogen where initial infection and replication occur within the epithelial layer of the upper respiratory tract of chickens causing signs such as gasping, coughing, sneezing, tracheal rales, and nasal discharge [Infectious bronchitis (IB) is currently endemic and poses an economic threat to most countries with large-scale poultry production ,2. The cischarge . Infecteischarge . IBV alsischarge  and monoischarge  and is bischarge . In addiischarge . The emeischarge ,10. Despischarge , the pooischarge .Nephropathogenicity is mostly described in broilers. Several QX-type IBV strains, identified throughout Asia, Europe, and Africa, are associated with renal pathology ,14,15. GInfection of the reproductive tract by certain IBV strains has been associated with adverse effects on egg production . Over thIn 2011, a new IBV variant designated as DMV/1639 was associated with multiple NIB outbreaks in the Delmarva peninsula, USA . Since 250) was calculated with the method of Reed and Muench [7 EID50/ mL and stored as aliquots of 0.1 mL allantoic fluid at −80 °C.The Canadian IBV DMV/1639 strain used in this study, designated as IBV/Ck/Can/17-036989, was described previously . This std Muench . The titWhite Leghorn SPF eggs, obtained from the Canadian Food Inspection Agency (CFIA), Ottawa, Ontario, were incubated and hatched in our laboratory, Foothills campus, University of Calgary. Hatching chicks were sexed, and day-old SPF females (n = 40) were housed in the Veterinary Science Research Station (VSRS) facility at the Spy Hill campus, University of Calgary. The feed and lightening system were adjusted according to the management guidelines recommended for White Leghorn growing pullets.6 EID50 by the oculo-nasal route [Day-old chicks were randomly divided into two groups (20 chicks in each) and housed in two separate negative pressure rooms. The chicks in the first group were infected with 100 μL of the challenge virus containing 1 × 10al route . The chiBirds in both groups were monitored twice daily for clinical signs, which were scored ranging from 0 to 3 as described previously . Briefly® Unitranz-RT® Transport System . Collected swabs were vortexed and aliquots of 250 µL of the transport medium were stored at −80 °C until processing.Oropharyngeal (OP) and cloacal (CL) swabs were collected weekly from all birds in both groups using the Puritan®  and Optimum Cutting Temperature (OCT) compound . Additionally, portions of trachea, lung, and kidney were collected in 10% neutral buffered formalin . At 14, 21, 28, 35, and 91 dpi, l ml of wing vein blood was collected from all birds in both groups.At 7 dpi, under isoflurane anesthesia, 1 mL of intracardiac blood was collected from four chicks of the infected group and five chicks of the control group followed by euthanasia through cervical dislocation. Portions of trachea, lung, kidney, ovary, oviduct, and cecal tonsils (CT) were collected in RNA SaveAt 112 dpi (16 weeks of age), all the remaining birds in both groups were euthanized by cervical dislocation under isoflurane anesthesia. Portions of kidney, ovary, oviduct, and cecal tonsils were collected in RNA Save. In addition, portions of previously mentioned tissues, except cecal tonsil, were fixed in 10% neutral buffered formalin.Tissues collected in RNA Save and OCT compound were preserved at −80 °C until processing. The gross anatomy of the birds that died or were euthanatized during the experiment was examined and recorded.To evaluate the developmental status of the reproductive systems in both groups, the weights of the ovaries and the lengths of oviducts were measured. The gross cystic lesions of the reproductive tract were estimated and scored using a four-grade-scoring: negative (no cyst), moderate , marked , and severe  .® reagent  following manufacturer’s guidelines. The concentration and quality of the extracted RNA was determined using a Nanodrop1000 spectrophotometer , with the absorbance at a 260/280 nm wavelength. One µg RNA of each swab sample and 2 µg RNA of each tissue sample were used to synthesize cDNA with the use of High-Capacity cDNA Reverse Transcription Kit  according to manufacturer’s guidelines.Total RNA was extracted from collected swabs and tissues using Trizol® Green Master Mix  in a reaction volume of 20 µL. Each reaction volume contained 10 µL of SYBR Green master mix, 100 ng of respective sample template, 0.5 µL of forward and reverse primers, and DNAse/RNAse-free water. Cycling conditions were 95 °C for 20 s; followed by 40 cycles of amplification/extension at 95 °C for 3 s, and 60 °C for 30 s; melting curve analysis was done at 95 °C for 10 s (Segment 1), 65 °C for 5 s (Segment 2) and 9 °C for 5 s (Segment 3). Fluorescent acquisition was done at 60 °C for 30 s.For IBV genome load determination in swab and tissue samples, a SYBR green-based qRT-PCR using primers targeting a conserved region within the IBV nucleocapsid gene (N) was used as previously described . All cDN™ Excel R.T.U. antibody kit Dylight 594  and incubated for 15 min followed by 30 min incubation with VectaFluor™ Dylight® Dye-labeled anti-goat IgG made in horse serum . Finally, coverslips were mounted on glass slides with Vectashield® antifade mounting medium with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) nuclear stain  and sealed with lacquer. The stained slides were examined using epifluorescent microscopy .Immunofluorescence staining was performed to detect IBV antigen. The tissues preserved in OCT compound were sectioned at 5 μm thickness using a freezing microtome  and adhered on to positively charged slides. The frozen sections were fixed in pre-cooled acetone for 5 min followed by air drying at room temperature for 10 min. Fixed cryosections were treated with 0.2% Triton X-100  for permeabilization. Blocking was done using 2.5% horse serum diluted in Trizma buffered saline (TBS) at room temperature for 1 h followed by incubation with a mouse monoclonal antibody against the nucleoprotein of IBV  for 30 min (1:400 dilution in blocking buffer). The tissue sections were then covered with amplifier antibody (goat anti-mouse antibody) from the VectaFluorCommercial ELISA kit  was used to measure the antibody-mediated immune response against IBV in serum (1:500 dilution). The assay was performed according to the manufacturer’s protocol. The antibody titers were calculated with the formula provided by IDEXX, and titers >396 (cut-off) were considered positive.Tissue samples collected at 7 and 112 dpi and preserved in 10% neutral buffered formalin were embedded in paraffin, sectioned at 5 μm, stained with hematoxylin and eosin (H&E) (Diagnostic Services Unit (DSU) at the University of Calgary), and examined with light microscopy  for lesions attributable to IBV infection.A Mixed-effects model followed by Sidak’s multiple comparisons test was used to identify the differences in clinical scores and anti-IBV antibody titers between infected and control groups and differences in IBV genome loads in the OP and CL swabs over different time points in the infected group. Friedman test followed by Dunn’s multiple comparisons test was used to identify differences in the IBV genome loads in the different tissues of the infected group in each time point. Differences in lengths of oviducts and weights of ovaries were analyzed using Mann–Whitney U test. The frequencies of lesions on oviduct were compared with the Fisher’s exact test. GraphPad Prism 9.2.0 Software  was used for the data analysis and to generate graphs.p < 0.05). The peak of the clinical signs was recorded at 6 dpi  than OP swabs at all time points except at 63 and 105 dpi .No anti-IBV antibody titer could be detected in the birds in the control group. In the infected group, 23–30% of the birds had positive serum antibody titers between 21 and 35 dpi. At 91 dpi, around 54% of birds showed positive titers. Positive mean titers in the infected group were 482 and 610 at 35 and 91 dpi, respectively. However, the mean titer in the infected group was significantly higher than the control group at 91 dpi only . Additionally, the number of birds with healthy oviducts in the infected group was significantly lower compared to the control group (p < 0.05) , 46% of the infected birds showed cystic lesions of varying sizes in the oviduct. The frequency of developing marked cystic lesions in the infected birds was significantly higher compared to the control birds ( < 0.05) . Cystic  < 0.05) . There wThe microscopic examination showed no significant pathological changes in the control group at 7 dpi a,c,f. ThNo significant pathological changes could be detected in the control group at 112 dpi a,c,g. InThe prevalence of the DMV/1639 IBV strain has increased significantly in Eastern Canada in recent years ,31, whicEarly after infection, clinical signs and pathological changes, indicative of a respiratory infection followed by a renal disease, induced by the Canadian DMV/1639 strain, were consistent with most IBV strains that have been shown to cause cystic oviduct lesions ,27,40,41A significantly higher cloacal compared to oropharyngeal viral shedding was recorded starting at 7 dpi, and this pattern persisted throughout the experiment. While the highest concentration of IBV can be detected in the trachea during the first 3–5 days post infection, the virus titer in the respiratory tract usually drops rapidly in the second week post infection . AdditioIn the present study, the cut-off level of the average ELISA titer was reached at 35 dpi and demonstrated a significant difference from the control group at 91 dpi. Similarly, infection of 1-day-old SPF chicks with seven different IBV strains of QX, Mass, and 793/B types induced positive ELISA titers between 21 and 42 dpi . The obsLong-term IBV persistence, particularly in the cecal tonsils and feces, has been previously reported ,49. In tA high incidence of oviduct cysts that contributes to the development of false layer syndrome was the most interesting finding of this study. Approximately 46% of the inoculated birds developed cystic formations of varying sizes in the oviduct. This result coincides with the field observations of flocks with false layers peaked more than 50% lower than the expected level of egg production . The obsOverall, our study confirms that the Canadian DMV/1639 strain is a highly pathogenic IBV strain that causes respiratory, renal, and reproductive pathologies.


The Tibetan Plateau is the highest plateau on Earth, with an average elevation of over 4,000 metres, and thus potentially provides very good opportunities for astronomy and particle astrophysics5. Here we report the results of three years of monitoring of testing an area at a local summit on Saishiteng Mountain near Lenghu Town in Qinghai Province. The altitudes of the potential locations are between 4,200 and 4,500 metres. An area of over 100,000 square kilometres surrounding Lenghu Town has a lower altitude of below 3,000 metres, with an extremely arid climate and unusually clear local sky (day and night)6. Of the nights at the site, 70 per cent have clear, photometric conditions, with a median seeing of 0.75 arcseconds. The median night temperature variation is only 2.4 degrees Celsius, indicating very stable local surface air. The precipitable water vapour is lower than 2 millimetres for 55 per cent of the night.On Earth’s surface, there are only a handful of high-quality astronomical sites that meet the requirements for very large next-generation facilities. In the context of scientific opportunities in time-domain astronomy, a good site on the Tibetan Plateau will bridge the longitudinal gap between the known best sites The results of site testing at a local summit near Lenghu Town in Qinghai Province on the Tibetan Plateau indicate that the site is suitable as an astronomical observing site. Light pollution is mainly the result of human activities. Qinghai Province on the Tibetan Plateau has a very low population; therefore, problematic artificial light sources are at present non-existent. However, this does not mean that industrial development will not occur in the future. If the local population were to grow with economic development, then control of light pollution could be lost. This potential conflict between scientific research and industry needs a resolution10. Owing to the enforceable and long-term night-sky protection policy issued by the local municipal government in 2017, a priori, such a potential threat to astronomical observations has been lifted. Night-sky protection in the whole area of Lenghu will be guaranteed by law.For any modern observatories for night optical/infrared astronomy and planetary sciences11, which has a wide passband from 400 nm to 600 nm centred at the Johnson V-band and accurately measures the integrated light of the entire visible sky, with the sensitivity optimized towards the zenith and a quick drop-off to less than 20% once the zenith angle is greater than 60°. The integrated full visible sky brightness is converted into zenith brightness in mag arcsec−2 (ref. 12). The night-sky brightness reaches 22.3 mag arcsec−2 during a fully clear new moon time, in the extreme case when the bright part of the Galactic Disk is far away from the local zenith. The average night-sky brightness is around 22.0 mag arcsec−2 when the Moon is below the horizon, comparable to the other three sites in Table First, we determined how dark the local night sky is. We monitored the night-sky brightness using a widely used commercial sky quality meter (SQM)13. All-sky images have been captured every 20 min during the day and every 5 min between dusk and dawn without interruption since March 2018, regardless of the weather conditions. Another measure to evaluate the observable time makes use of the same SQM14. The SQM reading changes smoothly with the rotation of the starry sky during a clear night, and any cloud passage through the visible sky modifies the sequence of SQM magnitudes, resulting in a chaotic light curve  with a 12-mm fish-eye lens customized for this site15. Measurements by DIMM are widely used and have become a standard assessment for integrated atmospheric optical turbulence16. A multi-aperture scintillation sensor can provide further information on how the total seeing is composed of different layers in the atmosphere above the site17. The same make of DIMM (the one provided by Alcor-System) applied in this study has also been used for many different site-selection campaigns19. Measurements are calculated for an average wavelength of 550 nm and corrected to the zenith (airmass unity).Seeing, the blurring of stars due to atmospheric turbulence along the light path, is one of the key parameters to assess the quality of observations at a site for seeing-limited scientific goals, and a good integrated seeing measurement using a DIMM is the starting point for advance observational modes applying adaptive optics−1 or lower, and the seeing measurement stops immediately when the wind speed exceeds 12 m s−1 for 3 min or 15 m s−1 for 1 min during observations. When the power or Internet connection was poor, and, of course, when weather turned bad, no DIMM data were taken. Seeing data were collected for 457 nights, evenly distributed during the whole period until 31 December 2020. We tested the temporal variation20 and the wind dependence of seeing and found that the seeing is stable for most of the observable time. The prevailing wind direction at the site is around 280° throughout the year, which is also the wind direction for the best median seeing. The median seeing is below 0.7 arcseconds between wind directions of 255° and 324° , and is clearly the best one on the Tibetan Plateau  and the median value of DIMM seeing (DMn) on each night . This method has been shown to be applicable for other sites on the Tibetan Plateau26. To anchor the modelled PWV to actual measurements, we applied Global Navigation Satellite Systems (GNSS) data27 from a week in January 2021. This cross-check shows that our modelling of PWV using onsite weather station data is consistent with the GNSS method. Figure 28, Lenghu has a different pattern, possibly due to different climate patterns. During 2019–2020, the Lenghu PWV was high in summer and very low in winter. At night, PWV at the Lenghu site is better than that at Mauna Kea, meaning that Lenghu has a much higher potential than other known sites for infrared wavelengths , Atacama (70.4042° W) and the Canary Islands (17.8577° W). This will form a perfect network of ground-based, high-quality observatories ready for great scientific discoveries, including searching signs of life on exoplanets, electromagnetic counterparts of gravitational wave outbursts, high-value transient events alerted by space-borne triggers that need to be done in very narrow time window35. Land transport from the site to the local supporting base, Lenghu Town, and then to the developed areas of China by road and railway networks is convenient. The nearest international airport, highway access and the cargo railway stations of Dunhuang are all within only 250 km of the town. The altitude of Lenghu Town is only 2,700 m and is 80 km away from the site, which provides comfortable conditions for a supporting base for the site. This infrastructure enables good logistics for future activity at the site ; when only the inner circle is clear, it is defined as ‘outer’ (spectroscopic).In Extended Data Table 13. Half of the AOT for Delingha  would be in blue (score 0.5) with the other half in brown (score 0.3), and the total score would be approximately 40%, which is typical for current existing classical observatories in China. For the Xinglong site (150 km from Beijing), where the Large Sky Area Multi-Object Fibre Spectroscopic Telescope (LAMOST) is hosted, the expected score would be even lower than the Delingha site for the same level of seeing, but with less observable time.Extended Data Table Using the publicly available seeing and AOT data from the European Southern Observatory (ESO) website, the sites at Cerro Paranal and La Silla are evaluated on the site quality matrix scale. Both the seeing and AOT data released on the ESO website are monthly average values. For the AOT of the ESO sites, a photometric night calls for six or more hours of consecutive photometric night. Therefore, only the third column of Table UT on 12 August 2020, and twice on 16 November 2020, at 11:18 UT and 23:44 UT. These balloon missions provided a vertical spatial resolution of 6.4 m. The mean potential temperature profile θ(h) is calculated byh is the altitude, T(h) is the temperature profile in K and P(h) is the pressure profile in hPa. The structure function of the temperature fluctuation 37. The refractive index structure constant To understand the local meteorological pattern at the mountain region where our site is located, three balloon experiments were conducted at the Lenghu weather station. Once at 23:15 −17.5 and 10−17 between 4 km and 11 km. On 16 November, the two turbulence profiles show a similar trend, but the turbulence strength at night (red profile) is lower than that in the morning (grey profile). At an altitude of 6–9 km, the turbulence profile shows a clear difference in August and November, which suggests possible seasonal changes.The turbulence profiles calculated using the parameters obtained during the balloon flights are shown in Extended Data Fig. ρ is the density of liquid water, g is the acceleration of gravity, pz is the pressure of the ground and q is the specific humidity. The value of q is calculated by the water vapour pressure e by the equation38. We used the temperature, pressure and humidity of the ground weather station to estimate the amount of PWV. Here we adopted a temperature drop rate of 6.5 K km−1, an exponential decay of air pressure with temperature, and the height of the tropopause is 11 km as measured by the balloon experiments  and Ngari  site26 (southwest part of the Tibetan Plateau) to re-estimate the PWV. Comparing the PWV estimates using the two sets of coefficients, our modelling of the PWV of the Lenghu site is consistent, but slightly overestimated by 0.15 mm and 0.01 mm, respectively.To explore the possible deviations of our PWV, we also adopted the empirical equation between PWV and specific pressure of water vapour, PWV = 10) density are 20.7 μg m−3 and 11.7 μg m−3, respectively. The ambient dust level of the Lenghu site is comparable to the sites in Atacama39. Twice in 2020, a high value of PM10 was recorded, of around 100 μg m−3, during sandstorms that originated from the Taklimakan and local Gobi deserts. Dust grain densities higher than 50 μg m−3 occurred 31 times in 2020, with a typical duration of several hours. Owing to the high altitude of the site, dust is less serious than at the La Palma site40, which suffers from proximity to the Sahara Desert, and precautions can be implemented at the Lenghu site to protect the equipment for the few days per year affected by dust.Dust and aerosol above an observing site can create problematic extinction for astronomical observations, and their presence in the ground layer can be troublesome for both optical surfaces and mechanical bearings. To measure local dust and aerosol, we implemented a dust meter (GRIMM EM180) in December 2019. We have so far collected a full year of uninterrupted data regarding the dust grains and aerosols of the site, with a temporal resolution of 5 min. The mean and median values of particulate matter with a diameter smaller than 10 μm (PMAny methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-021-03711-z.Peer Review File