The ancient mechanisms that caused developmental gene regulatory networks to diversify among distantly related taxa are not well understood. Here we use ancestral protein reconstruction, biochemical experiments, and developmental assays of transgenic animals carrying reconstructed ancestral genes to investigate how the transcription factor Bicoid (Bcd) evolved its central role in anterior-posterior patterning in flies. We show that most of Bcd’s derived functions are attributable to evolutionary changes within its homeodomain (HD) during a phylogenetic interval >140 million years ago. A single substitution from this period (Q50K) accounts almost entirely for the evolution of Bcd’s derived DNA specificity in vitro. In transgenic embryos expressing the reconstructed ancestral HD, however, Q50K confers activation of only a few of Bcd’s transcriptional targets and yields a very partial rescue of anterior development. Adding a second historical substitution (M54R) confers regulation of additional Bcd targets and further rescues anterior development. These results indicate that two epistatically interacting mutations played a major role in the evolution of Bcd’s controlling regulatory role in early development. They also show how ancestral sequence reconstruction can be combined with in vivo characterization of transgenic animals to illuminate the historical mechanisms of developmental evolution. Animal body plans are established during development by interacting networks of transcription factors and other molecules, which organize temporal and spatial cascades of gene expression that control cell division, differentiation, and migration . How speConsiderable progress has been made toward this goal, particularly among closely related species and populations. Comparative analyses have identified differences in gene expression that correlate with phenotypic differences, and manipulative experiments have shown that altering expression of specific genes can disrupt or mimic specific forms of developmental diversity . TransgeFor deeper taxonomic divergences, it has been more difficult to establish the specific causes of evolutionary changes in gene regulation and development . DistantAncestral sequence reconstruction (ASR) can, in principle, help to address these difficulties. ASR uses statistical phylogenetic methods to infer the sequences of ancient genes or proteins, which can then be synthesized and experimentally characterized . This apASR is now widely used in evolutionary molecular biology and biochemistry  and has Drosophila melanogaster and other cyclorrhaphan flies – a large taxonomic group that appeared >140 Mya (– AP patterning depends on an anterior gradient of Bicoid (Bcd), a transcription factor translated in the embryo from maternally deposited, anteriorly localized mRNA  axis during embryonic development in flies . In Dros>140 Mya  – AP patzed mRNA . Embryoszed mRNA  by usingzed mRNA . Bcd alszed mRNA . In some factors .Cad translation , which is located on the recognition helix (RH) that directly contacts the DNA major groove, may have played a role. K50 is present in several HD proteins that bind canonical target sites similar to Bcd’s, but HDs that prefer Zen-like motifs contain a glutamine at this position (Q50). Biochemical studies have shown that swapping these two states between extant HDs affects relative affinity for these target sequences and suggest potential structural mechanisms for this effect (Numerous studies have addressed the sequence-structure function relations of Bcd and other HD proteins in s effect . Other rs effect . There hD. melanogaster Fushi tarazu (Ftz), a distantly related HD protein, and found that Q50K does not confer activation of any Bcd targets in fly embryos (D. melanogaster embryo and therefore masked the contribution of Q50K in this experiment. A recent study found that replacing the HD of D. melanogaster Bcd with that of orthodenticle (otd), which also contains K50 and many other sequence differences, abolishes activation of most of Bcd’s targets and does not rescue anterior development in transgenic embryos, suggesting that this residue is not sufficient to account for most of the regulatory functions of the Bcd HD  D. melanogaster and the bcd 3’UTR  rescued the E1bcd phenotype by all molecular and morphological criteria, and over 95% of embryos survived to adulthood (Drosophila Zen (DmZen) failed to rescue any aspect of the bcd-null phenotype  amino acid sequence of the HD at two key ancestral nodes: (1) AncZB, the common ancestor of all Zen and Bcd proteins in cyclorrhaphan flies, which represents the single ancestral sequence that existed just before the duplication and (2) AncBcd, one of AncZB’s daughter nodes, which represents the Bcd protein in the last common ancestor of all cyclorrhaphan flies . Both seD. melanogaster Bcd  consistTo characterize the evolutionary shift in preference for DNA motifs, we computed from the PBM data the additive effect of each possible nucleotide at each sequence position on the estimated binding energy of the protein-DNA complex . AncZB, To quantitatively characterize evolutionary changes in DNA preference that occurred between AncZB and AncBcd HDs, we used electrophoretic mobility shift assays (F-EMSA) to measure binding affinity to the PBM consensus Zen motif , the consensus Bcd motif  and five other noncanonical motifs to which Bcd has previously been shown to bind . We founWe further investigated DNA binding by the ancestral proteins using a surface plasmon resonance assay (SPR) . CorroboTaken together, these data indicate that during the phylogenetic interval immediately after the duplication of AncZB, a major change in DNA recognition and affinity occurred along the lineage leading to AncBcd. The ensemble of occupied DNA binding sites shifted dramatically, and the protein’s preference for canonical Zen vs. Bicoid motifs was inverted.bcd-null females. The reconstructed AncBcd HD yielded a phenotype indistinguishable from that of the DmBcd transgene, with essentially complete morphological rescue . We individually reverted each diagnostic residue in AncBcd HD to the state present in AncZB HD and measured the effect on DNA affinity using F-EMSA. Only one reversal – changing lysine 50 back to the ancestral glutamine (K50q) – shifted the protein’s preference for BM back toward ZM; this reversion caused a 38-fold change in relative affinity and restored the ancestral preference for ZM. Mutating arginine (R) to the ancestral methionine (m) at position 54 (R54m) – had no effect on relative preference, but it lowered affinity to both BM and ZM by a factor of ~15 . No otheTo test whether the substitutions at positions 50 and 54 were sufficient to cause the evolutionary change in DNA recognition in vitro, we introduced the derived states from AncBcd into the AncZB HD . SubstitWe next used PBMs to determine whether the evolution of Bcd’s global DNA specificity was also attributable primarily to substitution q50K. We compared the ensemble of DNA motifs bound by AncZB to those bound by AncZB-q50K. This substitution shifted the overall enrichment and de-enrichment across all oligomers to be extremely similar to those bound by AncBcd . SubstitThe other HD substitutions did not contribute to the evolutionary shift in global DNA specificity, as shown by a PBM comparison of AncZB to AncBcd-K50q, which is AncZB plus all 30 other derived states that evolved in the AncZB-AncBcd interval (except for q50K). The global DNA specificity of this protein remained strongly correlated to that of AncZB  completely abolished the rescue conferred by AncBcd HD: larval cuticles were similar to  embryos , express embryos , and Cad embryos . Substithb, kni, and eve stripe 2) were activated, but four  were not . During this interval, a dramatic shift occurred in the global set of DNA sequences to which AncBcd binds, and the relative preference for canonical BMs vs. ZMs changed by a factor of 250. This interval was also the key period for the evolution of Bcd’s in vivo functions: whereas AncZB-HD rescued no Bcd-specific functions, AncBcd-HD activated all seven Bcd target genes tested, inhibited Cad translation, and organized development of many anterior structures in the embryo, yielding functions and phenotypes indistinguishable from those conferred by the D. melanogaster Bcd HD.Our strategy was designed to directly characterize the causal contribution of historical substitutions to the acquisition of Bcd’s derived functions, both in vitro and in vivo, using the reconstructed context of the ancestral proteins in which they occurred. By functionally characterizing successive ancestral nodes on the phylogeny, we found that all the derived functions of the The sequence changes that occurred along this phylogenetic branch represent the candidate genetic causes for the evolution of Bcd HD’s derived functions. We focused on the contribution of 11 substitutions from this interval, because the derived states of these are conserved among all known Bcd proteins. When each of these was introduced singly into AncZB, only Q50K made a measurable contribution to the new specificity, and this substitution alone was sufficient to recapitulate the entire evolutionary shift in local specificity (affinity for BM and ZM) and global DNA affinity (as measured in a PBM), yielding in vitro functions indistinguishable from AncBcd. The other substitutions had no effect on motif preference, and only M54R had a significant effect on affinity, conferring a nonspecific increase in binding to both motifs; however, when M54R was introduced together with Q50K, it had no measurable effect on affinity for either motif, compared to Q50K alone. Thus, a single large-effect substitution appears to provide a nearly complete causal explanation for the evolution of the Bcd HD’s derived DNA specificity as measured in our in vitro assays.ems and btd), increasing expression levels and sharpening localization of the entire suite of targets, yielding anterior repression of Cad translation, and rescuing the full anterior developmental phenotype of AncBcd-HD. Further work will be required to determine the specific contribution of these substitutions to the evolution of Bcd function.By performing transgenic experiments with reconstructed ancestral HDs, we sought to directly assess the contribution of historical substitutions to the shift in embryonic gene expression and developmental phenotypes that occurred during Bcd evolution. We found that Q50K was a major evolutionary cause: introducing it into AncZB-HD conferred regulation of three of seven Bcd target genes and recovery of some aspects of anterior development. Unlike its minor role in vitro, M54R was an important causal contributor in vivo, conferring regulation of two additional targets and a more complete developmental rescue, but only when introduced together with Q50K. At least some of the 29 other substitutions that occurred during this interval made additional contributions, conferring activation of the last two transcriptional targets , it should be possible to use the data we present here to disentangle how Bcd’s regulatory effects on its various targets interact to specify AP polarity.Our results also have implications for understanding the evolution of Bcd’s role in cyclorrhaphan development. The difference we observed between the in vitro and in vivo contributions of the key historical substitutions indicates that the evolution of Bcd’s developmental functions cannot be explained entirely by a shift in binding to canonical ZM and BM. Q50K causes the entire shift in preference measured in vitro, but it accounts for a relatively small portion of the HD’s in vivo evolution – activation of three of seven major transcriptional target genes, and only a very limited rescue of anterior development; two of these, ains K50 . Adding ains K50 , but theains K50 , as are ains K50 . When thA major puzzle is how a new transcription factor could evolve a controlling role in a crucial process of early development, because intermediate evolutionary states that would disrupt AP development would presumably be strongly selected against. It has been suggested that Bcd may have usurped its role as a primary anterior organizing transcription factor from a more ancient protein with Bcd-like specificity, such as Otd, which plays a role in anterior organization in some wasps . In thisD. melanogaster Bcd with that of D. melanogaster Otd in transgenic flies yields activation of only a few Bcd targets and virtually no rescue of anterior development ;38F1 (w+). We cloned an injection plasmid (piattB40-Bcd) containing two inverted ΦC31-specific recombination sequences (AgeI/HindIII) for Recombination Mediated Cassette Exchange (RMCE), Gmr-GFP (HindIII/AscI), and a polylinker flanked by 1.9 kb bcd promoter and 0.8 kb 3’UTR (AgeI/AscI). The bcd and zen coding regions were amplified by PCR from pBS-SK + cDNA clones, digested with RsrII and AscI and ligated into piattB40-Bcd. We used standard cloning techniques to generate homeodomain swaps and residue changes. Gene Blocks coding for the ancestral HD and variant sequences together with the flanking Bcd coding sequence were obtained from Integrated DNA Technologies (IDT). They were digested with AscI and BspEI and ligated to the piattB40-Bcd vector digested with the same restriction enzymes. All transgenic lines were generated using the ΦC31 integration system, and constructs were integrated into the 38F1 landing site on the second chromosome , ±/romosome . Each trIn situ hybridizations were performed as previously described . BrieflyLonchoptera lutea, Platypeza consobrina and Rhajio tringarius (Protein sequences of Bicoid (Bcd), Zerknullt (Zen) and Hox3 orthologs were collected by BLAST against NCBI databases using Drosophila melanogaster Bcd and Zen HD sequences. Three zen cDNA sequences were reverse transcribed from insect species, ingarius . Proteiningarius . The aliingarius . Phylogeingarius  and LG mingarius  with gamingarius . Statistingarius .https://github.com/JoeThorntonLab/bcd_evolution; copy archived at https://github.com/elifesciences-publications/bcd_evolution). We identified all ambiguously reconstructed sites as those at which multiple states have posterior probability >0.20; we synthesized alternative ancestral reconstructions containing the state with the second highest likelihood at all such sites  and cloned into the pDEST15 vector through Gateway cloning (Life Technologies) to produce N-terminal GST fusion proteins. Ancestral HDs were expressed in BL21 (DE3) pLysS Rosetta cells.Ancestral HD cDNA sequences were synthesized (GeneScript). For SPR and F-EMSA experiments, HD cDNAs were cloned into the pETMALc-H10T vector  C-terminProtein expression was induced by 0.5 mM IPTG at A600 of 0.8–1.2. Cells were then grown overnight at 18°C, harvested and frozen at −20°C. Cells were lysed using BPER Protein Extraction Reagent Kit (ThermoScientific). For MBP-His fusion ancestral HDs, the supernatant of the cell lysate was loaded onto a pre-equilibrated 5 mL HisTrap HP column (GE) and eluted with a linear imidazole gradient (40 mM to 1 M) in 20 mM sodium phosphate and 200 mM NaCl buffer [pH 7.4] on AKTA FPLC system. Then, ancestral HDs were cleaved from MBP-His fusions using TEV protease in dialysis buffer consisting of 20 mM sodium phosphate and 250 mM NaCl [pH 7.0]. For GST fusion ancestral HDs, the supernatant of cell lysate was loaded onto a pre-equilibrated gravity Glutathione column (ThermoScientific), eluted with 10 mM glutathione, 125 mM Tris and 150 mM NaCl [pH 8.0], and dialysed in dialysis buffer [pH 6.0]. For the second purification step, samples were loaded onto a 5 mL HiPrep SP FF cation exchange column (GE) and eluted with a linear NaCl gradient (250 mM to 1 M) in 20 mM sodium phosphate buffer [pH 7.0]. Whenever needed, ancestral HDs were further purified on a HiPrep 16/60 Sephacryl S-100 HR size exclusion column (GE) with 20 mM Tris and 150 mM NaCl [pH 7.5]. Protein purity was assayed after purification by visualization on a 12% SDS-PAGE gel stained with Bio-Safe Coomassie G-250 stain (Bio-Rad). Purified protein aliquots were flash frozen in liquid nitrogen and stored at −80°C in 20 mM sodium phosphate, 150 mM NaCl, 1 mM DTT and 10% glycerol [pH7.5]. Protein folding and aggregation states were measured using circular dichroism (Jasco J-1500 CD) and dynamic light scattering (Wyatt DynaPro NanoStar) spectroscopy after freeze-thaw cycle. For biochemical assays, protein aliquots were thawed, centrifuged, and the supernatant was used for Quick StartTM Bradford Assay (BioRad) to quantify protein concentrations.D. melanogaster Bcd or Zen (Protein binding microarrays (PBM) were performed essentially as described previously  using a d or Zen .For heat-map and clustering analysis, E-scores for 8-mers scoring≥0.45 for at least one tested construct were clustered and plotted using the heatmap.2 function in the gplots R package, with the Manhattan distance metric. Clusters of 8-mers selected from the heatmaps were aligned as described , and seqPBM binding logos were derived from site-specific effects on binding energies estimated using a maximum likelihood method implemented in the BEEML-PBM algorithm . BEEML-PA). In Quantitative F-EMSAs were used to measure the binding affinities of ancestral HDs to 14 bp 5’ fluorescein-labeled dsDNA oligonucleotides (FAM-DNA). FAM-DNAs were produced by annealing equimolar complementary ssDNA strands . 5 µM annealed FAM-DNA aliquots were stored at −20°C. Typical reactions consisted of 2.5 nM FAM-DNA equilibrated with varying concentrations of protein in assay buffer for 30 min at room temperature . Immediately prior to loading, one-twentieth volume of 30% (v/v) glycerol, 0.01% (w/v) bromcresol green was added to each reaction as a dye marker. A 10 µl sample of each reaction was loaded onto a pre-run 8% native polyacrylamide gel in pre-chilled 0.5 × TBE buffer. The gels were run for 40 min at 100 volts then immediately scanned using a fluor-imager (Bio-Rad GelDoc) with a blue laser at 491 nm. The fluorescence intensity ratio of bound verse total FAM-DNA (F) was determined as a function of total protein concentration (P) and DNA concentration (D), and the data were fitted to a quadratic equation  to deteron and koff) were estimated by using BIAevaluation software (GE) to fit the observed data (R) to a function (0) at various time points (t) and correction terms for refractive index change (RRI), drifting baseline (cD) and local maximum response (Rmax).All SPR kinetic experiments were performed with a four-channel Biacore 3000 biosensor system (GE) at 25°C. 20 bp 3′-Biotin-labeled DNA oligonucleotides  were captured with streptavidin on CM5 sensor chips (GE) on which the carboxymethyl groups were activated and coupled with streptavidin to amine linkages. Flow cell #1 was left blank as a control while others contained a specific immobilized DNA sequence. Preliminary experiments were done to find appropriate buffer and surface regeneration conditions, DNA immobilization levels, protein concentrations, flow rate, association and dissociation contact time for each protein variant. Low levels of DNA immobilization, 100 ~ 150 RUs (response unit), and fast flow rate, 30 µl /min, were used to minimize mass transfer effects. Before each kinetic experiment, the system was stabilized for 3 min by flowing HBS-PE buffer at 30 µl /min . During the kinetic association phase, a protein dilution in modified HBS-PE buffer  was injected. Following a kinetic dissociation phase in which only running buffer was injected, DNA binding surface was regenerated by flowing 20 µl 0.05% SDS at 100 µl /min. The kinetic parameters . The other two reviewers remain anonymous.Thank you for submitting your work entitled "Ancient mechanism for the evolution of Bicoid function in fly development" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in Drosophila melanogaster, which does not allow for an analysis of the contributions of "trans" evolutionary changes in target sequences, and in interacting proteins. In the end, it was the lack of a significantly new advance in understanding BCD protein evolution/function that led to the decision.All three reviewers agree that the methodology is a significant strength of the paper. After discussion, however, the reviewers and editors felt that the results presented here are very much in line with what is already known about the functional changes brought about by evolutionary amino acid substitutions in the BCD homeodomain. Furthermore, the conclusions were based on testing variants in Reviewer #1:This study sought to characterize the amino acids changes that have evolved to confer functionality on Bicoid since the ancestral duplication that produced this gene in the lineage leading to cyclorrhaphan flies. The study focused on the homeodomain and used a transgenic rescue assay to test the effect of variants on target gene expression and overall ability to rescue in vivoin vivo. These experiments were complimented by in vitroin vitro analysis of binding site preferences of variants.The authors found that the Bcd homeodomain in an otherwise Zen protein could perform much like Bcd. They then used ancestral sequence reconstruction (ASR) to generate and test ancestral sequences. This allowed them to identify an ancestral-like sequence that could function like Bcd although it was unable to fully function like this transcription factor, in for example, repressing translation of caudal. The authors then sought to determine which Bcd specific amino acids in the homeodomain were responsible. They found that the K at position 50 conferred much of the characteristics of Bcd in vitroin vitro and in vivoin vivo and that the R at position 54 also contributed to Bcd function in vivoin vivo. None of the other amino acid differences tested made any detectable contribution, although since rescue was not complete they and other changes outside the homeodomain presumably also have minor roles.Overall the experiments are described clearly and the work confirms that the amino acids at positions 50 and 54 have made a major contribution to the evolution of Bcd functionality as inferred from several previous studies cited in the manuscript.A case is made in the Discussion sectionDiscussion section for how these amino acid changes may have facilitated the usurpation of the ancestral gene regulatory network for anterior patterning. However this study only assesses changes in the homeodomain of Bcd and therefore there appears to be an assumption that the binding sites in the target genes have not changed in sequence and nor has there been any other changes in trans. Therefore this speculation should be toned down or at least the caveats should be explained in more detail.It would also have been interesting to determine how Otd with a Bcd homeodomain and other variants with particular amino acid substitutions performed in these assays to further test what ancestral system Bcd replaced and how this was achieved.The Introduction sets out the case for the merits of the ASR approach to identify mutations underlying evolutionary differences. However, while ASR can be a powerful approach, I think the Introduction is too biased and there is an implication that horizontal comparisons do not generally allow the genetic changes underlying phenotypic differences to be identified  and continue to do so. Therefore a more balanced Introduction is needed.Reviewer #2:The article by Liu et al.et al. aims to understand the evolution of the very interesting fly protein Bicoid. They use a cutting edge tool for reconstructing the most likely ancestor of it and its paralog Zen, and testing the functions of variations on the modern and ancestral proteins in establishing and patterning the anterior posterior axis. I think the work is very well done, but in the end, does not advance our knowledge of the properties and evolution of Bicoid significantly. The importance of the transition from Q50 to K50 is well known and has been for around 30 years, and it is no surprise that it is far and away the most significant change in the protein. Position 54 is also well known to have important functions.., melanogaster have coevolved with the Drosophila Bcd homeodomain? I realize testing the latter hypothesis is outside the scope of the approach of this paper, but a more systematic probing of the potential of interactions among residues within the homeodomain with the two large effect changes could support or eliminate the intramolecular epistasis hypothesis, and could give more support to searching for other explanations.These results should be the starting point of the paper, not the main conclusions. The really interesting thing to me is why these are not sufficient to give complete Bicoid rescue? Is it still a question of intramolecular epistasis that prevents the combination of k50 and r54 from activating all bicoid targets? If so, which sites are involved? Or is something else going on? Perhaps the cis-regulatory elements in DAs it stands I feel that the field would not be advanced significantly by this work, since we are still at the point of knowing that q50 to k50 led to a big change in the sequences bound by Bicoid, but we have no further understanding of how this led to Bicoid becoming the anterior determinant of the cyclorrhaphans.Reviewer #3:I think this is an exemplary paper. As the authors explain in their very eloquent Introduction, incorporating explicit phylogenetic structure and ancestral character reconstruction into comparative studies produces much more rigorous results than simple pairwise comparisons between species. This is as true for development as it is for morphology and DNA sequences, but unfortunately the application of phylogenetic approaches is much rarer in evo-devo studies than in other sub-fields of evolutionary biology – – due at least in part to the difficulty of collecting appropriate data, but also perhaps to cultural factors. This paper raises the bar in both respects. The authors have performed a very nicely structured comparison of reconstructed ancestral proteins to investigate how their function evolved through time on a defined evolutionary lineage. At the same time, they have integrated a wealth of in vivoin vivo, in vitro, and biophysical data to characterize the functions of both extant and ancestral proteins. The result is a very thorough and rigorous paper. In some respects, the results are not entirely surprising – – the single mutation that was found to play by far the largest role in the evolution of protein function is the one that has been hypothesized to do just that for decades. However, in addition to confirming the role of this mutation, the authors were able to quantify its contribution to phenotypic change at different levels of biological organization , identify a second AA substitution that acts in concert with it, and to rule out the role of multiple other fixed AA changes . So the exceptionally laborious approach taken by the authors has paid off.[The decision letter after re-review follows.]As you know, the initial reviews and discussion identified this as interesting and unusually rigorous work demonstrating the phenotypic effects of lineage specific amino acid substitutions in flies. However, concerns were also expressed about the novelty of this work and impact on the field given that the amino acids tested had previously been suggested to be involved in the trait studied. After much debate, I decided initially to reject this work based on these grounds. You have done a great job highlighting the differences between this prior work and your new findings in the comprehensive response to reviewers and appeal letter. After consulting with all three of the original reviewers, we agree that the changes to the manuscript address our concerns. I am thus happy to now accept this work for publication. [Editors’ note: the author responses to the first round of peer review follow.]Thank you for the decision letter and reviews of our paper. As the letter states, all reviewers viewed the paper’s methodology as very strong, and there were no concerns about the quality of the experiments, the data, or their interpretation. The major comments pertained to the paper’s impact in relation to prior work and, to a lesser extent, to the way we framed a few issues in the introduction and discussion. We have revised the paper to address these concerns, as detailed below.1) Reviewers 1 and 2 commented that the impact of the paper was low, because the importance of sites 50 and 54 was already known. We believe this comment arises because we inadequately described previous work in our relation to our findings, because a close reading of the literature shows that the extent to which Q50K and M54R can account for the evolution of Bcd’s derived functions in vitro was not previously known, and in vivo, there was no evidence whatsoever to indicate that these substitutions can confer Bcd-like functions or played any role in Bcd evolution.none of Bcd’s derived functions, and the M54R substitution’s effects on Bcd functions was never previously assessed.As the reviewers pointed out, numerous previous studies showed that swapping lysine and glutamine at site 50 between several HD proteins strongly affects in vitro DNA preference for Bcd vs. Zen motifs, and other experiments studied the in vitro effects of various mutations at site 54. Thus, it was reasonable to hypothesize that these might have been involved Bcd evolution. However, these studies could not and did not quantify the extent to which the evolutionary acquisition of Bcd’s derived specificity was caused by the specific historical sequence changes that occurred at sites 50 and 54. That goal would require quantifying the historical shift in Bcd affinity for both kinds of target sequence and assessing the effects of the historical substitutions, in the ancestral background in which they occurred; none of this was previously done. in vivo, no studies have provided any evidence that these substitutions caused the evolution of Bcd’s role in gene regulation and development: the only relevant work found that introducing Q50K into a related HD confers Our findings are therefore unprecedented that (1) Q50K alone can confer significant  in vivo rescue of anterior gene regulation and development, (2) adding M54R yields a substantially more complete rescue of these phenotypes, (3) M54’s effect is epistatically dependent on co-introduction of Q50K, (4) Other substitutions from the same phylogenetic interval are required for complete acquisition of all of the Bcd HD’s regulatory activity and role in anterior development, and (5) the acquisition of Bcd’s derived DNA specificity in vitro is entirely attributable to Q50K.We have revised the Introduction and Discussion section to thoroughly discuss prior work and to explicitly place our strategy and the impact of our findings in the context of that work.D. melanogaster; this design, the comment says, did not allow us to test the evolutionary consequences of changes at Bcd’s regulatory targets or interacting proteins, which could also have been important in the evolution of its role in AP development. It is true that we focused on the evolutionary effect of changes in the HD, using D. melanogaster as a transgenic platform, but we believe this design is a strength of our study, so long as the interpretations are drawn appropriately. All transgenic experiments are tests of specific putative causal factors when introduced into a particular genetic background; they cannot address the contributions of other potential causes that are already “embedded” within the host organism, and they must be interpreted accordingly. In our study, changes at other loci could indeed have played a role in the evolution of Bcd’s developmental role, but our experiments quite decisively establish the developmental effects of the genetic changes within the Bcd HD that we tested. Further work will be necessary to study the impacts of changes at other loci.2) The decision letter and reviewer 2 expressed concern that our in vivo experiments were conducted by introducing variants of the ancestral Bcd into D. melanogaster transformed with its own DmBcd-HD and those transformed with the AncBcd-HD are essentially indistinguishable. Finally, our inferences involved tightly focused comparisons, in which we tested ancestral proteins from successive phylogenetic nodes and examined the effect of specific sequence changes on the phenotypes they produce; this design drastically limits the scope of the inferences that might plausibly be compromised by epistasis with the assay system, because the proteins we tested are so similar to each other. Only if epistasis differentially affects the specific substitutions Q50K, M54R, and the other differences between AncZB and AncBcd –which were assessed in otherwise identical proteins in a common genetic background –could our results be misleading.A second potential concern could relate to the possibility of an epistatic mismatch between the ancestral gene and the host organism into which it is introduced, potentially leading to artifactual results. This issue, which potentially affects all transgenic studies, is important to acknowledge. But a strength of our ancestral reconstruction strategy is that it can help to reduce this potential problem compared to horizontal transgenic experiments, because an ancestor and descendant differ only by the changes that occurred along only one branch, whereas the two extant species differ by all changes that occurred along both branches. Further, it is unlikely that our particular findings are attributable to epistasis between ancestral alleles and the transgenic host in which they were assayed, because the developmental and regulatory phenotypes of We have modified the DDiscussion section and Iintroduction to explicitly acknowledge the potential importance of evolutionary change at Bcd targets at interacting proteins and to carefully describe the issue that arise when ancestral genes are assessed by introducing them into an extant model organism.3) Reviewer 1 felt that our introduction was too dismissive of comparative strategies for identifying the genetic basis for the evolution of development. We agree that such approaches have been very important, particularly among closely related species among populations, and would not want imply otherwise. Among more distant taxa, they have also made significant contributions to our understanding, although typically at considerably lower resolution. We have modified the Introduction to acknowledge these contributions.4) Reviewer 1 suggested that we remove the phrase “higher taxa.” We have replaced it with “more distantly related taxa.”5) Reviewer 1 suggested that we refer to the Bcd motif using a longer sequence than the canonical 6-bp core sequence we described. We prefer the classic 6-bp core because it contains essentially all of the key information about Bcd’s mode of DNA recognition relative to Zen, which is the relevant change on the evolutionary timescale studied here. Further, it is consonant with our PBM and EMSA experiments, and it is far more accessible for the reader than a longer motif that includes a small amount of degenerate information at flanking sites. We have provided appropriate citations for the consensus sequence we use and have modified the text to acknowledge that there is some additional sequence preference at flanking sites.6) Reviewer 1 asked us to fix mismatches in the references and check all figure pointers; we did so.7) Reviewer 2 and reviewer 3 found aspects of our introductory description of the relationships among Hox3, Zen, and Bcd to be confusing. We have rewritten this section for accuracy and clarity.8) Reviewer 2 suggested we change description of the evolutionary relationship between Bcd and Ftz. We have done so, referring to Ftz as a distantly-related HD protein rather than a paralog.9) Reviewer 3 asked for more information about the constrained topology and why we used it. We have modified the methods section to more fully address this. Briefly, HD sequences are too short and have inadequate phylogenetic signal to resolve relationships among all the species with sequences in our alignment. These species relationships, however, are all well established by previous work using much larger datasets. We therefore imposed these relationships in a single tree topology, and then used maximum likelihood to optimize branch lengths and model parameters and infer ancestral sequences. We have explained this in the text.10) Reviewer 3 asked if including the Syrphid sequence affects the ancestral sequence. We checked, and the effect is trivial: reconstructions of AncBcd with or without EbaBcdHD have the same ML sequence, and the difference in their marginal average posterior probabilities is 0.006.11) Reviewer 3 asked about the correlation of DNA occupancy in PBM between Dn-Zen and AncZB. We cannot answer that question, because we did not perform a PBM for DmZen.


Indocyanine green fluorescent angiography (IcGA) has been used with success in guiding intraoperative management to prevent colorectal anastomotic complications. Prior studies in open and laparoscopic colorectal surgery, such as PILLAR II, have demonstrated a low anastomotic leak rate (1.4%). As the minimally invasive approach progresses from laparoscopic to robotic approach, the effect and safety of IcGA in assessing anastomotic perfusion in the latter deserve further investigation. The objective of the study was to determine the safety of IcGA in guiding intraoperative management of robotic assisted colorectal resection via perfusion assessment. The design was single-surgeon, retrospective case-control study. 74 patients underwent left-sided robotic assisted colorectal resection and anastomosis with IcGA guidance. 30 historical controls underwent left-sided robotic assisted colorectal resection and anastomosis without IcGA. Clinical, demographic, operative, and outcome variables were tabulated. In the control group, 1 patient suffered a postoperative anastomotic stricture requiring no surgery, and 1 patient suffered an anastomotic dehiscence requiring return to the operating room. There were no anastomotic complications in the IcGA group, including 4 patients who underwent a change in the chosen level of anastomosis based on intraoperative IcGA. IcGA is safe to use as demonstrated by the very low rate of complications in this case series. It is also safe to rely on to guide re-resection and recreation of an anastomosis intraoperatively by demonstration of blood flow. This may help offset the loss of tactile feedback and assessment of tension in the robotic platform. Indocyanine green (IcG) fluorescence angiography can assess perfusion of colorectal anastomoses in open and laparoscopic surgery , 2. Its As colorectal surgery evolves to incorporate the robotic platform, there is a need to study IcGA in this modality where haptic feedback and tactile sensation, critical to assessment of anastomotic integrity, are lacking. Therefore, outcomes for patients undergoing IcG fluorescence angiography (IcGA), including those with intraoperative anastomotic revision, were compared to historical controls without IcGA. This will allow us to assess safety.104 patients underwent left-sided robotic-assisted colorectal anastomosis by a single colorectal surgeon for various indications. Patient preoperative variables and outcomes were tabulated from a prospectively maintained database between January 2012 and April 2018. Informed consent was obtained from patients. Cases were nonemergent elective resections in patients with established health insurance coverage. A thorough preoperative history and physical was performed to ensure nutritional optimization and lack of frailty. Some LAR cases were done for nonrectal cancer disease due to the extent of resection as guided by IcGA for diverticulitis or prolapse.A standardized enhanced recovery pathway was faithfully adhered to for all patients, reducing historical bias within the case series. Demographic, clinical, operative, and outcome variables were tabulated Tables –4. All i104 patients were included in the final analysis. Median length of stay was 4 days. There were no mortalities. There were two postoperative anastomotic complications in the control group. A 68-year-old female who underwent LAR for diverticulitis suffered a postoperative stricture identified by postoperative colonoscopy at follow-up that did not require any therapeutic procedure. A 54-year-old female with a history of Crohn's colitis, multiple prior abdominal surgeries, and near-obstructing distal sigmoid stricture suffered an anastomotic dehiscence after LAR, lysis of adhesions, and diverting loop ileostomy. Despite the latter, the contamination of her leak resulted in emergent return to the operating due to a necrotizing soft tissue infection. She underwent washout, multiple debridements and end colostomy ultimately surviving to placement in rehab and eventual complete restoration of bowel continuity. Neither of these patients had prior pelvic irradiation.In the IcGA group, 4 patients underwent an intraoperative change in management based on the subjective assessment of the IcGA fluorescence. This change in management involved selecting a better fluorescing distal segment of bowel to use as part of the anastomosis. The number of IcGA needed to change management was 18. None of these patients suffered an anastomotic complication (stricture or dehiscence).The postoperative course was uneventful for 84% of patients; 16% had one or more postoperative complications . Two of There were two anastomotic complications in the control group and were none in the IcGA group, including four patients whose anastomosis was assessed and revised real time intraoperatively via IcGA. Therefore, it is safe to rely on IcGA in assessing and changing the anastomosis via perfusion assessment, fulfilling the objective of our study. Pelvic irradiation occurred more often in the control group; however, its use has not been conclusively shown to impact rectal anastomoses –9, and nAnastomotic complications are major contributors to morbidity and mortality in bowel surgery. Prevention relies on tissue integrity and vascularity . There iThe benefits of IcGA in laparoscopic LAR have been demonstrated by the PILLAR II study, reporting anastomotic complication rate of 1.4%  when it Additionally, our study corroborates the growing body of evidence supporting the safety of IcGA in revising an anastomosis real time, as demonstrated by Boni et al. in two papers , 19, oneIn contrast, a paper by Kin et al.  did not There are a few limitations that must be highlighted in our study. Our data was matched against historical controls. As with other papers utilizing historical controls, it compares the efforts of the surgeon in a less experienced time point. However the single-surgeon design limits the confounder of technical skill, which can vary significantly among surgeons within a group and account for differences in outcomes. The low rate of anastomotic complications reduces generalizability but occurred in the context of an elective, highly functional patient population. It is one of the largest studies evaluating the effects of IcGA on left-sided robotic assisted colorectal procedures to date and adds to the growing body regarding consequences of real time intraoperative guidance and revision.IcGA was safe in guiding operative management and has a potential role in reducing anastomotic complications in the robotic platform, where a surgeon may depend on it more exclusively than during laparoscopy or open procedures which have some guidance via tactile feedback and pulsatile assessment. Randomized controlled prospective may validate these findings in the near future.


Strikingly, we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 of recombinant nucleotide-bound Rab8A in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in transfected HEK293 cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and nuclear magnetic resonance structure of Ser111-phosphorylated Rab1B. The structures reveal that the phosphorylated SF3 motif does not induce any major changes, but may interfere with effector-Switch II interactions through intramolecular H-bond formation and/or charge effects with Arg79. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A indicating a potential cross-talk between PINK1-regulated mitochondrial homeostasis and LRRK2 signalling that requires further investigation in vivo.Loss of function mutations in the PTEN-induced kinase 1 (PINK1) kinase are causal for autosomal recessive Parkinson's disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies, we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor and GTPase activating protein. In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2  Autosomal recessive mutations in PTEN-induced kinase 1 (PINK1) represent the second most frequent cause of early onset Parkinson's disease (PD) ,3 as welPrevious work has shown that upon activation, PINK1 indirectly induces the phosphorylation of a subset of Rab GTPases including Rab 1B, 8A, 8B and 13 at a highly conserved Serine residue (Ser111 in Rab8A) that lies within the RabSF3 motif  ATP was from PerkinElmer, MLi-2 was obtained from Merck  ATP  in the absence or presence of MLi-2 (1 µM). Assays were incubated for 30 min at 30°C, with shaking at 1050 r.p.m. and quenched through addition of SDS sample loading buffer with 1% beta-mercaptoethanol and heating at 75°C for 10 min. Reaction mixtures were resolved by SDS–PAGE. Proteins were detected by Coomassie staining and gels imaged with an Epson scanner, prior to gel drying (Bio-Rad). Incorporation of [γ-32P] ATP into substrates was analyzed by autoradiography, using Amersham hyperfilm. Quantification of [γ-32P] ATP into substrates was performed by Cerenkov counting of SDS–PAGE gel bands.LRRK2 kinase assays were based on previous assay . Assays Crystallization trials of human pSer111-Rab8A:GDP were performed by the sitting-drop vapour diffusion method, using protein concentrations of 16 mg/ml and a protein:reservoir drop ratio of 1 : 1. Suitably sized crystals were grown in 0.2 M magnesium acetate, 0.1 M sodium cacodylate pH 6.5, 20% PEG 8000. Crystals were cryoprotected in reservoir solution supplemented with 20% glycerol prior to flash freezing in liquid nitrogen. Initial sparse-matrix screening of conditions yielded several hits for pSer111-Rab8A:GppNHp, which failed to produce well-diffracting crystals despite fine-screening efforts. Using numerous crystals grown in 0.1 M MES pH 6.5 and 25% PEG 6000/8000, and in 0.1 M Hepes pH 7.5 and 25% PEG 3000/4000/6000 as seeds, the method of random microseed matrix seeding  was usedλ = 1.0 Å) at the X06SA-beamline . X-ray intensities were assessed and data reduction was carried out with XDS , GFP-Rab8A WT or S111A were performed using the Polyethylenimine method -ATP-based screen of ∼140 recombinant kinases against wild-type (WT) or Ser111Ala (S111A) Rab8A in the GDP-bound conformation. We identified multiple kinases capable of phosphorylating wild-type Rab8A in vitro as judged by autoradiography including LRRK2 that has been reported to phosphorylate Rab8A at Thr72  antibody revealed that three kinases, TAK1, MST3 and MAP4K5 were capable of phosphorylating Rab8A at Thr72 equivalent to the LRRK2 site  and S2. in vitro  and S2. in vitro . In contin vitro . The scrin vitro  and in fUpon attachment to their target membranes, Rab GTPases act as molecular switches that are activated by a GEF that catalyzes the exchange of the bound guanosine-5′-diphosphate (GDP) for the more abundant guanosine-5′-triphosphate (GTP). Once active, the GTP-bound Rab can interact with specific effector proteins which mediate a variety of downstream functions including binding to cargoes, enabling vesicle formation and promoting vesicle fusion to target membranes . Rabs arWe, therefore, investigated the effect of the phosphorylation at Ser111 on Rab8A using preparative phosphorylated Rab8A. Employing genetic code expansion technologies for the incorporation of pSer65 into Ubiquitin , we wereWe next assessed the Switch II-dependent interaction of phosphorylated Rab8A with its GEF and GAP by measuring the nucleotide exchange and GTP hydrolysis rate, respectively. We first compared the Rabin8-mediated GDP–GTP exchange and the TBC1D20-mediated GTP hydrolysis rate in WT-Rab8A and pSer111-Rab8A by measuring the change in their intrinsic Trp fluorescence  upon nucRab1B shares many structural similarities with Rab8A  and can KD value of 923 nM for the binding affinity of WT-Rab8A and Mical-1, and a KD of 913 nM for pSer111 Rab8A with Mical-1, suggesting that the phosphorylation at Ser111 has no effect on Rab8-Mical-1 effector interaction  or pSer111 GDP or GTP-bound Rab8A  incubated with [γ-32P]-ATP and an activating LRRK2 mutant [G2019S]. Consistent with previous studies we observed that LRRK2 [G2019S] efficiently phosphorylated WT-Rab8A:GDP but was less able to phosphorylate WT-Rab8A:GppNHp as determined by measuring [γ-32P]-ATP incorporation via autoradiography and by immunoblotting of reactants with a pThr72 Rab8A phospho-specific antibody  as judged by autoradiography and immunoblotting of reactants with a p-Thr72 Rab1B phospho-specific antibody  and pSer111-Rab8A:GppNHp  (1H 15N Heteronuclear Single Quantum Coherence (HSQC) NMR data for wild-type and Ser111-phosphorylated Rab1B in the GDP and GppNHp states, most of the chemical shifts overlap well, indicating that Ser111 phosphorylation, probably does not lead to major structural alterations on Rab1B in solution. However, some of the resonances show shifts because of a change in the chemical environment close to the phosphate group  . The pSeD: 6STG) . InteresD: 6STG) . NeverthD: 6STG) . When cote group . Overallin vitro studies  under the accession codes 6STF and 6STG, respectively.


Bacillus spore consortium), and tap water as the control, on the resident microbiome of three common hospital surfaces . Pathogens, Escherichia coli and Staphylococcus aureus, were deposited and desiccated, and competitive exclusion was assessed for each microbiome. Cell survival was shown to be an incomplete tool for measuring microbial competitive exclusion. Biofilm competition offered a fuller understanding of competitive dynamics. A test for culturable cell survival showed that both plain soap and probiotic cleaner regimes established a surface microbiome that outcompeted the two pathogens. A different picture emerged when observing biofilms with a deposited and desiccated GFP-labeled pathogen, Pseudomonas aeruginosa. Competitive exclusion was again demonstrated. On surfaces cleaned with disinfectant the pathogen outcompeted the microbiomes. On surfaces cleaned with plain soap, the microbiomes outcompeted the pathogen. However, on surfaces cleaned with probiotic cleaner, despite the exponentially higher surface microbial loads, the microbiome did not completely outcompete the pathogen. Thus, the standard culturable cell test for survival on a surface confirmed the competitive advantage that is typically reported for probiotic cleaners. However, observation of competition in biofilms showed that the more diverse microbiome  established on a surface cleaned with plain soap had a better competitive advantage than the monoculture established by the probiotic cleaner. Therefore, microbial diversity appears to be as critical to the competitive exclusion principle as cell numbers. The study showed that both plain soap and probiotic cleaner fostered competitive exclusion far more effectively than disinfectant. Probiotic cleaners with microbial diversity could be worth considering for hospital cleaning.This study extends probiotic cleaning research to a built environment. Through an eight-month cleaning trial, we compared the effect of three cleaning products (disinfectant, plain soap, and a probiotic cleaner containing a patented  The link between ecological diversity and environmental balance is evident at all levels of life. In humans and animals, an imbalance of pathogens causes disease. On a global scale, such imbalances lead to plagues and ecological collapse. Understanding the effects of pathogen proliferation can inform approaches to human health and can be applied to environmental health policy. Since human and ecological health are interconnected, not independent, good hygiene policy connects inwards with microbiome health and outwards with environmental health.Hospitals are a particularly interesting biome, in terms of microbial diversity and homeostasis. Here, the built environment becomes a pathogen reservoir as a result of the heavy traffic of pathogen-laden patients and the liberal use of antibiotics ,2,3. ResApproximately 7% of patients in developed countries and 10% of patients in developing countries pick up nosocomial infections in general hospital wards , with soThis principle has leached into popular science as an awareness of the potential of probiotics, and has boosted the market for fermented foods such as kefir and kombucha . In thisIn this study we assessed competitive exclusion with the aim of fostering a better understanding of ecology in hospital cleaning regimes. The resident microbiomes established after an eight-month cleaning regime  were assessed for their competitive interaction with introduced desiccated pathogens. Competitive exclusion was evaluated by observing cell survival and biofilm dynamics. The cleaning regimes were applied to samples of three kinds of surface, i.e., ceramic, linoleum, and stainless steel, that were selected to represent surfaces common in hospitals, and were placed both indoors and outdoors to evaluate the effect of containment and to make the results representative of the wider hospital environment. In this paper, we use the term ”resident microbiome” to refer to the microbiome that had become established on the surfaces after the eight months’ cleaning process, consisting of any of the original microbes remaining, microbes that landed on the surface between the weekly cleanings, and microbes deposited via the cleaning process .We explored the competitive exclusion of microbiomes established on three different surfaces exposed to indoor and outdoor air microbiomes and subjected, for eight months, to distinct cleaning regimes, using three cleaning products and a control. First, we measured the culturable survival of pathogens desiccated on these surfaces, and then the competitive exclusion during the biofilm growth of the deposited pathogens, at physiological temperatures.Escherichia coli and Staphylococcus aureus strains  were inoculated in tryptic soy broth  within three days of isolation from a human host, and freeze cultures were prepared for storage after two passages . All strains were inoculated directly from freeze culture stocks and passaged twice prior to experimental inoculation onto surfaces, after eight months of surface cleaning . The clinical strains were not maintained as bench cultures at ambient temperatures, to prevent adaptation to laboratory conditions. Strains were characterized by plotting growth curves . MacConkey agar  was used for the identification of E. coli. Mannitol salt agar  was used for the identification of S. aureus. Clinical The resident microbiomes on three surface materials were subjected to four cleaning regimes , placed Bacillus spores. Probiotic cleaners generally contain only Bacillus spores, with some brands claiming, at most, “a variety of species or strains”. The blocks to be treated with probiotic cleaner were separated from the other blocks by at least 20 cm to prevent cross-contamination. Surfaces were cleaned twice a week for eight months, allowing a resident microbiome to become well established. The surfaces were thoroughly rinsed of disinfectant or soap after each cleaning, to prevent the residue having an impact on cell survival.Each treatment, surface, and placement combination was assessed in triplicate, with 72 control samples and 72 experimental samples. For all culturing and biofilm studies, small blocks of ceramic , linoleum (1.5 × 1.5 cm) and stainless steel (1.5 × 1.5 cm) were arranged randomly, indoors (in the Stellenbosch University Soil Sciences laboratory) and outdoors (on an exposed roof). For DNA extraction and molecular analysis of the resident surface microbiome diversity, large blocks of linoleum (45 × 30 cm) were arranged and cleaned according to the same four regimes . Four indoor cloths and four outdoor cloths were dedicated to each treatment , E. coli (MacConkey agar) and S. aureus  of the resident microbiome established at the end of the cleaning regimes. Baselines for each treatment, surface, and placement (indoors or outdoors) were assessed in triplicate. Each block was submerged face down in 5 mL sterile physiological saline solution , incubated at room temperature for ten minutes, and vortexed for one minute O is the final cell concentration per un-inoculated block (cfu·block−1), and [I] is the inoculum concentration per block (cfu·50 μL−1).A preliminary screening determined the most effective desiccation and disruption methods for consistent cell recovery . The method selected was as follows: Pseudomonas aeruginosa (PAO1-GFP) was grown under physiological conditions and deposited in saline (representing sputum) on the blocks and desiccated overnight. Competitive population dynamics were assessed with subsequent biofilm regrowth of the linoleum block microbiome and the desiccated pathogen, at a temperature simulating human physiology and favoring the adaptation of the pathogen (37 °C). The total microbiome was stained with a universal fluorescent dye, and the competitive survival of the deposited pathogen was visualized with GFP using fluorescence microscopy.Indoor linoleum blocks reserved for this experiment were subjected to each cleaning regime for eight months, and then removed to the laboratory. The pathogen P. aeruginosa strain of human origin , was maintained (passaged at least twice), grown (at 37 °C), harvested and washed as explained above , and desiccated under laminar flow in open Petri dishes, for three hours. Indoor linoleum blocks were selected for the biofilm study because they could be cut small enough to fit into flow cells, and the effect of the cleaning regime was the only variable assessed. Un-inoculated controls were also included, in triplicate for each treatment. The flow cells  were sterilized by soaking in sodium hypochlorite disinfectant solution , and washing thoroughly in a consecutive series of six sterile distilled H2O beakers (500 mL/beaker). Glass cover slips were briefly flame sterilized with ethanol, and small (3 cm) sections of tubing and connectors were autoclave sterilized. The inoculated and un-inoculated blocks were transferred into the respective channels, two blocks per channel, the coverslips glued onto the channels with marine silicone sealant (Bostik), and the tubing sections with connectors glued into each channel. The flow cells were left under laminar flow to cure for 24 h.The competitive exclusion of pathogenic biofilms by the resident microbiomes of the four indoor cleaning regimes was evaluated with confocal microscopy. A genome-integrated GFP-labeled ed above . Small , and washed with sterile distilled H2O (12 h). Growth medium (3 g·L−1 tryptic soy broth) was continuously passed through the system (3 h) directly prior to connecting the flow cells. The flow was then stopped and the cured flow cells containing linoleum pieces were connected after the bubble traps, ensuring aseptic transfer and connection. The channels were filled with tryptic soy broth and the flow stopped for 1 h to allow cell surface attachment in the liquid environment. The flow was, then, restarted and maintained for 48 h. The flow cells were disconnected and analyzed with confocal microscopy, comparing the proportion of GFP-labeled P. aeruginosa cells to the total biome, stained with Hoecsht 33342, a universal blue fluorescent bisbenzimide DNA-binding stain. The channels were stained for half an hour with 3 mL Hoecsht stain per channel, diluted in saline solution (5 μg/mL), and rinsed with saline solution. A Zeiss LSM 780 with ELYRA PS1 confocal microscope with an alpha Plan-Apochromat 100×/1.46 Oil DIC objective was used, according to de Waal et al. [Flow reactor systems were assembled and sterilized in a 37 °C incubator room prior to connecting the flow cells. Silicone tubing (inner diameter 1.575 mm) connected the medium reservoir to the waste receptacle, passing liquid through a 15 mL glass bubble trap via a Watson Marlow peristaltic pump. The system was sterilized with a continuous flow of a commercial hypochlorite disinfectant solution . Individual treatment effects relative to the controls were assessed in Excel with Student’s t tests for differences in independent means, with a confidence interval of 95% (p < 0.05). For competitive cell survival, a three-way analysis of variance (ANOVA) was done using the l m package in R  to test the effects of each treatment, surface, and placement on percentage survival, with a Fischer least significant difference (LSD) post hoc test. All means and standard deviations of triplicate values were determined in Excel and all graphs were generated in the Veusz scientific plotting application , on three surface materials , placed indoors and outdoors. After eight months of cleaning, the resident microbiome of each surface was assessed for cell concentration and diversity. Subsequently, the clinical pathogens were deposited on each surface and desiccated, and the competitive dynamics were quantified in terms of culturable cell survival and biofilm proliferation. Because surface-air interfaces are considered to be extreme environments  it is neTo understand the competitive survival of pathogens deposited and desiccated on a surface, we first needed to understand the resident surface microbiome of each eight-month cleaning regime. Therefore, we compared the cell concentrations (culturing) and the diversity (ARISA) of the resident microbiomes. The impact of a cleaning regime on competitive exclusion was the primary focus; however, we also compared the effects of the surface materials and of indoor and outdoor placement. p < 0.05, Student’s t-test of independent means). Probiotic cleaning established a surface cell population per block that was exponentially higher than for the other cleaning regimes, with the probiotic loads being much the same for indoor and outdoor placement. Probiotic cell numbers were lower on stainless steel (104 < 105 cfu/block) than on linoleum and ceramic (both 105 < 106 cfu/block) . The E. coli and S. aureus concentrations in the resident microbiome population on the surface materials were quantified as a baseline, for subtraction during inoculation and desiccation experiments. Baseline concentrations were sporadic, not higher than 101 < 102 cfu/block .Intuitively, total cell numbers followed an increasing trend, with the lowest cell numbers found for cleaning with disinfectant, followed by plain soap, the control, and the probiotic cleaner. Total cell numbers per block were consistently higher outdoors than indoors for all cleaning regimes except the probiotic cleaner  on the indoor linoleum blocks followed an unexpected trend in response to the cleaning regimes. The exponential Shannon diversity index and the inverse Simpson index showed that the microbiome diversity was lowest on surfaces cleaned with tap water, higher on those cleaned with plain soap, and highest on those that were disinfected . As expeBacillus spores was clearly visible on the linoleum surfaces, both indoor and outdoor, cleaned with probiotic cleaner (see The prokaryotic and eukaryotic beta diversity (differences in species diversity between ecosystems) also provided comparative information on the diversity of the surfaces, with tightly associated points on the graph representing lower diversity. Eukaryotic diversity was higher than prokaryotic diversity, as is evident in both the alpha  and betaaner see .E. coli and S. aureus strains in the four cleaning regime microbiomes. These pathogens were deposited and desiccated on the surfaces , indoors and outdoors, which had been cleaned for eight months with distinct regimes . This made it possible to differentiate between the viable survival responses of the clinical strains, adapted to physiological conditions, and those of the resident surface microbiome, adapted to ambient conditions. The results suggested that surface type had an impact on the pathogens’ tolerance of desiccation at the air-surface interface. E. coli survived best when desiccated on ceramic and S. aureus on stainless steel. The desiccation persistence of both the deposited pathogens was most pronounced on the control and disinfected surfaces (the latter thoroughly rinsed of disinfectant residue). The plain soap and the probiotic cleaner both limited the survival of the pathogens more than the disinfectant and tap water controls . In the probiotic case, the cell numbers were between one and five orders of magnitude higher than the surfaces treated with tap water, plain soap, or disinfectant, and had a relatively consistent microbial load . The surface microbial load was 10-fold lower on outdoor stainless-steel surfaces. This may have been due to metal ions on stainless steel surfaces influencing surface attachment, particularly with the added stress of ambient UV exposure and wind outdoors. It has been shown that spores such as Bacillus have a net negative charge similar to the net negative surface charge of stainless steel [In the case of the resident microbiomes, measured as surface microbial load, stainless steel had a more negative effect outdoors than indoors on the efficacy of the probiotic cleaner  was desiccated on the surfaces, and the total surface microbiomes were grown with the desiccated pathogen under biofilm conditions (37 °C). These microbiomes left on surfaces cleaned with the four cleaning regimes had distinct influences on the persistence of pathogens, measured by culturable survival (E. coli and S. aureus strains better than those on the surfaces cleaned with plain soap or probiotic cleaner (P. aeruginosa clinical pathogen was completely outcompeted in a biofilm by the resident control surface microbiome (The microbiomes on the surfaces cleaned with tap water promoted the quantifiable desiccation tolerance of clinical  cleaner , probabl cleaner . This prcrobiome A, which crobiome , supportE. coli and S. aureus better than those on the surfaces cleaned with plain soap and probiotic cleaner (P. aeruginosa (The microbiomes on the surfaces cleaned with disinfectant also promoted the quantifiable desiccation survival of  cleaner , but unl cleaner . This hyruginosa C. Scanniruginosa .P. aeruginosa outcompeted the probiotic spores (Cleaning surfaces with plain soap did not limit the resident microbial load as much as the disinfectant , and thec spores D. P. aeruginosa than the probiotic microbiome. The combined alpha and beta diversity indices of the probiotic microbiome showed a drastic limitation in diversity as compared with the other three treatments. Although the control and the probiotic sample had a similar alpha diversity, the beta diversity revealed that diversity was more limited in the probiotic samples. According to the competitive exclusion principle, the exponentially higher probiotic cell numbers should outcompete the pathogen more effectively than the microbiome of the surfaces treated with plain soap. Our results showed the opposite, suggesting that both diversity and cell numbers were important for competitive exclusion. The natural diversity and the cell concentrations established by a plain soap cleaning regime proved to be the most effective cleaning strategy within the limitations of this study. Although diversity has been proven to contribute to the competitive exclusion principle [Bacillus spore consortium or lactic acid bacteria [The probiotic and the plain soap microbiomes both reduced the number of culturable pathogens that survived; however, when grown under biofilm conditions, the plain soap microbiome was more effective against rinciple , this habacteria ,41,42,43bacteria , includiThis study showed that, by inhibiting the resident microbiome on surfaces, disinfectants strongly promote the survival of deposited pathogens. The use of probiotic cleaners to limit nosocomial infections is gaining interest and commercial traction, with numerous reports of their efficacy in forming stable surface biofilms, and thus excluding pathogens ,45. Bacillus spore consortium. Such a heavy predominance of one genus in a natural environment is rare. Studies have shown that Bacillus spores, even at high concentrations, are not an infection risk [Our cell survival results support these studies. We found that plain soap and probiotic cleaner were equally effective in excluding pathogens . Our proion risk . The priion risk ,42,47. NWe found that cleaning with plain soap promoted greater surface microbiome diversity than cleaning with the probiotic cleaner, which suggests that plain soap cleaning is better for promoting biofilm regrowth. To overcome the probiotic cleaner’s tendency to limit microbiome diversity, and to overcome antimicrobial resistance in hospitals, we suggest that diversity in probiotic cleaners should be investigated as a contributor to competitive exclusion. For general household cleaning, where antimicrobial resistance is not a factor, our study suggests it is best to use plain soaps, rather than disinfectants or probiotic cleaners. This study suggests a shift away from reliance on disinfectants, to plain or probiotic soaps in general hospital cleaning protocols. However, cleaning protocols are specific to national regulations, and hospital areas. Operating theatres must be disinfected. General waiting areas, bathrooms, walls, and floors all have distinct cleaning protocols, with unique disinfection frequencies. We selected a cleaning frequency to represent the general areas, walls, etc. This also reflects more general household and business cleaning regimes, widening the impact of the results. However, a more frequent cleaning regime might impact these conclusions. Thus, a controlled in situ study in general hospital areas, using in situ cleaning regimes, would add weight to these conclusions. We also conclude that a wider toolbox than culturable survival is necessary to assess the competitive exclusion of pathogens.Our measurements of the culturable survival of desiccated pathogens after our eight-month cleaning regimes supported the competitive exclusion principle, i.e., the resident microbiome had outcompeted the pathogens. Both the plain soap and the probiotic cleaner fostered competitive exclusion far more effectively than the disinfectant. However, when we looked at biofilm growth to measure the competitive exclusion of desiccated pathogens by the resident surface microbiomes, the picture that emerged showed that both microbial numbers and diversity were important for competitive exclusion. Although cleaning with the probiotic cleaner resulted in exponentially higher surface microbial loads than cleaning with the plain soap, the biofilm competitive exclusion was lower for this monoculture probiotic application. Therefore, we conclude that microbial diversity is as important as cell numbers for competitive exclusion, which is an important consideration when assessing methods for improving the microbial ecological health of the built environment, especially a hospital.


Osteosynthesis resorbable materials made of uncalcined and unsintered hydroxyapatite (u-HA) particles, poly-L-lactide (PLLA), are bioresorbable, and these materials have feasible bioactive/osteoconductive capacities. However, their strength and stability for fixation in mandibular condylar head fractures remain unclear. This in vitro study aimed to assess the biomechanical strength of u-HA/PLLA screws after the internal fixation of condylar head fractures. To evaluate their biomechanical behavior, 32 hemimandible replicas were divided into eight groups, each consisting of single-screw and double-screw fixations with titanium or u-HA/PLLA screws. A linear load was applied as vertical and horizontal load to each group to simulate the muscular forces in condylar head fractures. Samples were examined for 0.5, 1, 2, and 3-mm displacement loads. Two screws were needed for stable fixation of the mandibular condylar head fracture during biomechanical evaluation. After screw fixation for condylar head fractures, the titanium screws model was slightly more resistant to vertical and horizontal movement with a load for a small displacement than the u-HA/PLLA screws model. There was no statistically significant difference with load for large displacements. The u-HA/PLLA screw has a low mechanical resistance under small displacement loading compared with titanium within the limits of the mandibular head fracture model study. Mandibular condylar fractures are one of the most common types of maxillofacial fractures, and mandibular head fractures are a frequent disease that accounts for about half of all mandibular condylar fractures . Neff etAmong a large number of available types of fixators for the surgical treatment of condylar head fractures, screw fixation is the most widely used technique. It is minimally invasive, anatomically based, and has a number of advantages compared with the use of a plate system ,9. SurgiSince resorbable osteosynthesis material is degraded in vivo , seconda® . Forged composites of u-HA/PLLA were processed by machining or milling treatments to form screws, which contained 30% weight fractions of u-HA particles. u-HA is raw HA that contains neither calcined nor sintered material. Composite screws were filled with 30% weight fractions of u-HA particles. The screws used in this study were 2.0 mm in diameter. A 2.0-mm screw system  was used as the titanium screw. These titanium screws are alloys and titanium grade 4.In this study, we used the bioactive resorbable osteosynthesis systems, Super FIXSORB MXThe shear test jig in this study contained three parts. In the shear test, a hole of 2.5 mm diameter was used, and three parts were combined so that the holes cooperate with each other. The screw was first inserted to measure into the hole. Since the screws are sheared on both sides of the loading jig, the total test force at the two locations was measured. Therefore, the shear strength of the screw was half the test force. Using the method illustrated in We used 32 polyurethane replicas of human hemimandibles . Although a polyurethane mandible replicates the property of cancellous bone , this moOsteotomized mandibles were randomly divided into the following four groups and used for different fixation systems: (1) single titanium screw, (2) single u-HA/PLLA bioresorbable screw, (3) double titanium screws, and (4) double u-HA/PLLA bioresorbable screws.The screws used were titanium (2.0 mm diameter × 12 mm long) or u-HA/PLLA (2.0 mm diameter × 12 mm long). The fracture line and screw insertion direction were set according to the report of Guo et al. . The poiThe direction of passing through the midpoint of points 1 and 2 toward 3 was the direction of screw insertion. Double-screw fixation was moved parallel to the caudal side from this direction, and the other was inserted B.After screw fixation of the fracture segment, replicas were mounted on a testing machine  based on a biomechanical cantilever-bending model that simulates masticatory forces. Then, the mandibular ramus and angle areas of each replica were stabilized. The adaptation of the semi-fixed polyurethane to the machine was guaranteed by the metal support. Based on past biomechanical evaluation methods , the rept-tests for independent samples were performed to investigate whether the significance of differences between the mean values of the each groups. A p-value of <0.05 was considered statistically significant.Data were recorded and entered into an electronic database throughout the study using Microsoft Excel . Means and standard deviations (SD) were used where distribution was compatible with normality. The database was transferred to JMP version 14.2.0 software for Macintosh computers  for statistical analysis. Student’s p < 0.05). The u-HA/PLLA bioresorbable screw shear resistance was only 16.0% that of the titanium screw.The shear test force was 1156.0 N  for the titanium screw and 184.6 N (±18.7 N) for the u-HA/PLLA screw . In the Compared with u-HA/PLLA screws, both double-fixed screws showed statistically significantly higher strength at large displacements . In all A comparison of titanium and u-HA/PLLA screws with two screws in a horizontal load test is shown in The results of this experiment demonstrated that the mechanical resistances among osteosynthesis screws remained proportional to the amount of displacement. When comparing the single and double titanium screws and the u-HA/PLLA screw, the double-fixed screws showed significantly higher strength in titanium than the u-HA/PLLA screws . The meaThis study is the first report of an in vitro biomechanical evaluation using bioresorbable screws for the osteosynthesis of mandibular condylar head fractures. In this study, the bioactive u-HA/PLLA screw was lower in strength than conventional titanium screws. On the other hand, in a comparison after two-screw fixations for mandibular condylar head fractures, the bioresorbable screw model had slightly lower resistance to the vertical and horizontal loads for initial displacement than that for the titanium screw model in biomechanical evaluation. By contrast, there was no statistically significant difference in the load for large displacement. These results suggest that bioactive u-HA/PLLA screws have a slightly lower mechanical resistance compared with the titanium screws within the limitations of a mandibular condylar head model study.Screw osteosynthesis for mandibular joint fractures is a relatively minimally invasive operation and is the current standard surgical treatment for this type of fracture. The primary reason for this is that screw fixation provides better fixation stability, a smaller incision area, shorter surgery time, and less scarring of the temporomandibular joint than plate fixation . The secBioabsorbable materials are used in maxillofacial surgery, including orthognathic surgery , the ostIn our biomechanical test study, we examined the number of screws for fixation with one and two screws, respectively. Titanium double fixation showed a maximum load value that was about 1.3 times higher than the single fixation of their strength in vertical load and horizontal load tests. The u-HA/PLLA double fixation showed about 1.2–1.4 times higher resistance than single fixation in vertical load and horizontal load tests. This suggests that the double fixation increased the fixing force against loads in the both vertical and horizontal directions. As a noteworthy point, fixation with a single screw caused the bone fragments to turn under a high load in the horizontal load test. This is consistent with previous reports suggesting that using a single screw for fixation is not sufficient to provide rotational stability . The cliVertical load direction imitates the functional load at the time of mouth opening . The fixShear strength was presumed to be important for osteosynthesis devices in the mandibular condylar head fractures in this study. Therefore, the titanium screw showed higher strength than the bioresorbable screw even in the biomechanical test due to the excellent shear strength of the titanium screw itself. It is desirable to improve the shear strength to actively use the biomechanical screw in the future.In this study, biomechanical strength was assessed by measuring the load as a function of the amount of displacement. This study method was based on our previous experimental design in biomechanical studies of mandibular condylar fractures . Small dIt of great importance to determine the strength of the u-HA/PLLA materials used in this study. This is because the most common conventional resorbable material, pure PLLA, has disadvantages such as inability to bind directly to bone, unstable resorption and degradation processes, and long replacement times ,32. By cThis study has some limitations. As an in vitro biomechanical stress test, it does not fully reflect the dynamics of humans. However, the major functional load of mandibular movement has been reproduced experimentally. This is the first study of osteosynthesis for mandibular condylar head fracture using bioactive u-HA/PLLA screw, and it is important. Second, the mechanical model used in this study, the polyurethane model, is softer than the actual human mandible. The mandibular condylar head is a site with abundant bone marrow and close strength, but it is not enough to reproduce a faithful cortical bone in this model. Therefore, the results of this study suggest that the bioresorbable materials should not be used immediately for clinical treatment. Given the lower biomechanical strength of the u-HA/PLLA screw using this mandibular model, the actual clinical use of the resorbable screw should avoid early overload immediately after surgery. With the use of these u-HA/PLLA screws, patients with strong occlusal forces may need to be restricted by dietary guidance and elastic rubber to eat soft food after surgery.In this study, we show that a bioactive bioresorbable u-HA/PLLA screw has lower strength than the conventional titanium screw. Two screws were needed for stable fixation of the mandibular condylar head fracture. However, in a comparison after screw fixation for mandibular condylar head fractures, the titanium screw model was slightly more resistant to vertical and horizontal loads with a load for a small displacement than the u-HA/PLLA screws model in biomechanical evaluation. By contrast, there was no statistically significant difference in load for large displacements. These results suggest that the bioactive bioresorbable u-HA/PLLA screw has low mechanical resistance under small displacement loading compared with the titanium screw within the limits of the mandibular condylar head fracture model.