SNO1, identified previously in screens of the yeast knockout (YKO) library for mitotic fidelity genes. We found that the causative factor increasing mitotic error rate in the sno1Δ mutant is not loss of the Sno1 protein, but rather perturbation to the mRNA of the neighboring convergent gene, CTF13, encoding an essential component for forming the yeast kinetochore. This is caused by a combination of the Kanamycin resistance gene and the transcriptional terminator used in the YKO library affecting the CTF13 mRNA level and quality . We further provide a list of gene pairs potentially subjected to this artifact, which may be useful for accurate phenotypic interpretation of YKO mutants.To maintain genome stability, organisms depend on faithful chromosome segregation, a process affected by diverse genetic pathways, some of which are not directly linked to mitosis. In this study, we set out to explore one such pathway represented by an undercharacterized gene,  Cells must duplicate and equally partition their genomes with high fidelity. Diseases such as cancer are characterized by the disruption of processes involved in maintaining genome stability (Using budding yeast as a model eukaryotic cell to study CIN has advantages as this system is easily paired with whole genome screening. Considerable progress has been made toward identifying genes responsible for suppressing CIN using classic screening tools such as the chromosome transmission fidelity (CTF), A-like faker, and loss of heterozygosity assays (SNO1 (Saccharomyces Genome Database (SGD) with a predicted function in the pyridoxine biosynthesis pathway although its glutaminase activity has only been demonstrated in vitro  of mRNA from the neighboring convergent gene, CTF13, an essential protein involved in kinetochore formation. Of interest to the budding yeast research community, this YKO library artifact originates from the combination of the Kanamycin resistance gene with the associated TEF1 terminator sequence and has the potential to affect other convergent gene pairs in the genome-wide deletion library.One such gene that has consistently appeared in budding yeast screens related to altered CIN as well as genome instability is Yeast strain genotypes can be found in Supplementary Table S1. Polymerase chain reaction (PCR) and real-time quantitative polymerase chain reaction (qPCR) primers can be found in Supplementary Tables S2 and S3, respectively. Relevant plasmids are listed in Supplementary Table S4.ADH1 plasmid when noted) using 40–80 base pairs homology to the genomic region to be targeted. pFA6-KanMX4 and pFA6a-KanMX6 have identical KanMX sequences including the promoter, Kanamycin resistance gene, and the terminator . PCR products for homologous recombination transformations were generated in one of two ways depending on availability of preexisting mutants. If a mutant already existed such as a YKO collection mutant, genomic DNA (gDNA) was extracted from relevant YKO collection mutants and PCR was used to amplify the KO Kanamycin resistance (KanMX originating from pFA6-KanMX4) cassette with 400 base pairs up and downstream the open reading frame (ORF) using 5′/3′ UTR check primers designed using Primers-4-Yeast software  liquid media or solid plates prior to performing qCTF assays to maintain selection of the mini-chromosome (MC). During the 24-h nonselective period of the qCTF assay, cells were grown in SD-Complete liquid media . After transformant generation and selection for inserted selectable markers, all other (non-qCTF background) strains were grown in yeast-extract-peptone-dextrose (YPD) unless otherwise noted.The qCTF assay was performed as previously described (600 ∼0.4–0.6). Then the cultures were harvested to pellets in 2 mL screw-cap tubes and frozen in liquid nitrogen. Cell pellets were thawed and resuspended in 300 μL of lysis buffer  with 20 μL of 20% SDS and heated at 65°C for 1–3 min; 325 μL of 65°C preheated acid phenol  was added to samples and incubated while shaking for 15 min at 65°C. Samples were cooled for 5 min on ice and pelleted at 14,000 RPM for 10 min in a tabletop centrifuge at 4°C. The aqueous layer was added to 325 μL fresh room temperature acid phenol and vortexed for 30 s to 1 min followed by a second centrifugation under the same conditions. The aqueous layer was added to 325 μL chloroform  in a fresh 2 mL screw-cap tube. Samples were vortexed for 30 s at room temperature and centrifuged at maximum speed in a tabletop centrifuge for 2 min. The top aqueous layer was transferred into an RNAse-free tube and 3.0 M NaOAc pH 5.5 was added to bring the final concentration to 300 mM followed by addition of one volume of isopropanol to precipitate RNA at −80°C overnight. RNA was pelleted at 14,000 RPM in a tabletop centrifuge at 4°C for 30 min, washed in 70% ethanol, dried, and resuspended in 25–50 μL 10 mM Tris pH 8 on the same day the samples were to be used for cDNA generation.A hot acid–phenol chloroform extraction method was used to isolate total RNA. Overnight cultures of yeast were refreshed in 12–50 mL of media to mid-log phase  following dispensing into 384-well plates that were subsequently loaded into a BioRad CFX384 Touch Real-Time PCR Detection System. Minus reverse transcriptase (RT) controls were routinely performed and samples were only included in analysis if this control produced at least a five-cycle difference in average Cq value when compared to the samples with RT. qPCR primer sequences as well as validated efficiencies can be found in Supplementary Table S3. Data were analyzed using the comparative CT method as previously described  for 10–15 min at 37°C followed by the addition of 5 mM EDTA and heat inactivation at 65°C for 10 min. cDNA for qPCR was generated using random hexamer primers and the High-Capacity cDNA Reverse Transcription Kit . cDNA  and 130 nM each of forward and reverse primers were added to the 2X PerfeCTaescribed .T” primer (3′ rapid amplification of cDNA ends (3′ RACE) was performed as previously described with the following adjustments  supplemented with 40 mM DTT . Samples were boiled for 10 min and then vortexed at top speed with approximately 100 μL of 0.5 mm diameter glass beads  for 1 min and 30 s at 4°C. Lysates were boiled again for 10 min, spun down at maximum speed in a tabletop centrifuge for 1 min, and the supernatant was removed from the debris and glass beads into a fresh tube; 27 μL of this protein suspension was loaded for each sample into a 10-well 4–12% NuPAGE Bis-Tris gel  along with 10 μL Precision Plus Protein™ Dual Color Standard . SDS-PAGE gels were run according to manufacturer’s recommendation. The gel was next transferred to a PVDF membrane  using the iBlot™ 2 transfer system. Membranes were blocked in 5% bovine serum albumin  in 1x TBS with 0.1% Tween-20 (1x TBST) for 1 h at room temperature followed by overnight incubation in primary antibody in blocking solution at 4°C. Pgk1 mouse monoclonal antibody (1:3000 dilution) and HA rabbit monoclonal antibody (1:1000 dilution) were purchased from Invitrogen (Cat #: 459250) and Cell Signaling (Cat #: 3724S), respectively. Following eight washes in 1x TBST, the membrane was then incubated in a 1:5000 dilution in 1x TBST of the species appropriate HRP-linked secondary antibody  for 1 h at room temperature. After four 1x TBST washes, membranes were incubated in 2 mL of ECL reagent  for 1–5 min following membrane exposure on a LI-COR Odyssey® Fc Imaging system. Quantification was performed using Image Studio Lite software.Overnight cultures of cells were refreshed in YPD to log phase for 3.5–5 h at 30°C. Samples were then normalized to OD600 of 0.62 and 200 μL of each culture was pelleted, washed once with sterile H2O, resuspended in 200 μL sterile H2O, and transferred to a flat bottom 96-well plate; 20 μL of the most concentrated sample was then serially diluted into 180 μL of sterile H2O in a new well for a total of four 10-fold dilutions; 5 μL of each dilution was then dropped onto YPD or YPD plates supplemented with 200 mg/L Geneticin  and grown for 48 h at 30°C prior to image collection.Log phase cultures of cells were normalized to an ODP > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). If the result of the one-way ANOVA test was significant, then a Tukey’s post hoc test was performed to identify which groups significantly differed from the relevant control strain using the tukey_hsd function from the rstatix package (version 0.6.0) using the same significance cutoffs listed above. All significance cutoffs overlaid onto plots are generated from the Tukey’s test. Statistical analysis for qPCR and Western blot data was performed using the same statistical testing method. Unless otherwise noted, all qPCR experiments were performed using both biological and technical triplicates. Cq values for technical triplicates for each biological replicate were averaged and these Cq averages were used in the subsequent statistical analysis. Statistical analysis was only performed on qPCR data if there was a complete set of at least three biological replicates.Statistical analysis for the qCTF assay was performed via a one-way ANOVA test on the mean fold-change for samples using the stat_compare_means function from the ggpubR package (version 0.2.3) with default significance cutoffs . Supplementary Table S1 contains the genotypes of budding yeast strains used in this study and the sources of any strains not generated in the present study as well as the primer sets and templates DNA sequences used for homologous recombination transformations and aliases for relevant strains. Supplementary Table S2 contains a list of primer names and sequences used for PCR, 3′ RACE, and DNA sequencing. Supplementary Table S3 contains qPCR primer sequence sets, the ORFs they anneal to, and their experimentally calculated efficiencies. Supplementary Table S4 contains relevant plasmids developed and used during the course of this study. Supplementary Table S5 contains one of the tables generated from the source code for mining convergent gene pairs from the SGD. This table lists nonoverlapping convergent gene pairs, the number of base pairs between each stop codon, the chromosomal coordinates of each gene, and the chromosome they reside on. The source code also produces a table of overlapping convergent gene pairs. Supplemental Material is available at figshare: https://doi.org/10.25387/g3.14195846.All yeast strains and plasmids are available upon request. Source code for the convergent gene pair mining from the SGD is available online  strains through homologous recombination gene replacement that introduced a Kanamycin resistance (KanMX) gene in place of the target gene using the same PCR-based deletion strategy used to generate the YKO collection . Taken together, these data point to a lack of necessity for the Sno1p in maintaining proper chromosome segregation but rather suggest a potential artifact induced by the sno1Δ::KanMX mutant of the YKO collection.Three independent transformants of a ckground . To valihe Sno1p . We alsoTEF1 promoter and terminator from Ashbya gossypii was used to replace nonessential ORFs  or the Watson strand (sno1Δ-Watson”). Only the SNO1 deletion expressing KanMX from the Crick orientation causes a significant increase in the CIN rate compared to the control for this experiment . The remainder of elevated CIN in the sno1Δ6-Watson mutant points to an additional source of CIN independent of KanMX orientation within the sno1Δ6 background, consistent with the significantly different CIN rate between the sno1Δ and sno1Δ6 reported earlier . Together, these findings suggest a localized cis effect of the KanMX gene cassette on the CIN rate at the SNO1 locus with coding expression from the Crick strand, rather than a direct effect due to the absence of the Sno1p. This cis effect is present when KanMX is inserted at distances up to 1.3 kilobase pairs upstream of the SNO1 ORF as in the snz1Δ2 mutant, which was the farthest distance we tested  orientation with CTF13, such that the 3′ UTRs of both genes are overlapping with only 23 base pairs of intergenic sequence separating the two genes’ stop codons  to estimate the relative abundance CTF13 mRNA in the sno1Δ strain. When compared with the wild-type control, the sno1Δ strains showed significant reduction in CTF13 mRNA levels . Furthermore, we found this trend of reduced CTF13 mRNA to be consistent in the sno1Δ6 mutant, which also had increased CIN rates due to the KanMX insertion whereas the sno1Δ::URA3 mutant had wild-type abundance of CTF13 mRNA . These findings suggest that the KanMX sequence, inserted at varying positions in the SNO1 locus, perturbs the abundance CTF13 mRNA independent of an unaltered copy of the SNO1 ORF.Given that Sno1p is not responsible for preventing CIN, we next determined whether there may be neighboring elements that are disrupted in the  element . We notep codons . Given iA levels . This trsno1Δ mutant was due to reduced CTF13 mRNA, increased dosage of the CTF13 gene should prevent high CIN phenotype observed in this background. To test this, we examined the effect of adding an extra copy of the CTF13 gene in the sno1Δ background into an intergenic location on chromosome IV (native CTF13 is located on chromosome XIII). Adding the second copy of the CTF13 ORF to sno1Δ completely suppressed the high CIN back to the wild-type level (SNO1 ORF into the same location on chromosome IV in the sno1Δ background was not sufficient to rescue the high CIN phenotype (sno1Δ mutant is reduced CTF13 mRNA.If the CIN phenotype of the pe level . On the henotype . This suCTF13 mRNA is known to be affected by the nonsense-mediated decay (NMD) pathway such that inactivating NMD leads to increased CTF13 mRNA  led to a fivefold increase in the CTF13 mRNA when averaged across two independent CTF13 qPCR primer sets . We therefore reasoned that inactivating the NMD pathway may result in increased abundance of the CTF13 mRNA to a level sufficient to reduce the CIN phenotype in the sno1Δ YKO collection mutant. To this end, we constructed double mutants with sno1Δ combined with deletion of either UPF1 or UPF2, genes encoding two of the essential components of the NMD pathway. The resulting double mutants had markedly reduced CIN rates much closer to that of the control strain . NMD inactivation in either the sno1Δ2 or the sno1Δ6 mutant completely suppressed CIN back to the control levels, whereas the sno1Δ3 CIN rate was unaltered . NMD inactivation led to an increase in CTF13 mRNA compared to the single sno1Δ6 mutant , consistent with CIN suppression. This suggests that loss of the sequence uniquely deleted in the sno1Δ3 mutant has an additional effect on CTF13 gene expression unrelated to NMD (see Discussion).We also used several KanMX insertion mutants to determine whether inactivated NMD could also suppress the CIN in these backgrounds. In our high-throughput qCTF analysis, Δ mutant , but snoTEF1 terminator from the pFA6a-KanMX cassette used in the YKO library may be read bidirectionally such that the CTF13 transcript is prematurely terminated in the sno1Δ background. This hypothesis predicts two changes to the CTF13 mRNA in the sno1Δ background. First, the transcript would have a different length compared to the wild-type transcript. Second, the composition of the 3′ UTR would be altered. To address both of these predictions, we used 3′ RACE to map the sequence and length of the 3′ end of the CTF13 transcript in both the wild-type and sno1Δ background  and generated a novel sno1Δ mutant . Sequencing a PCR product from genomic DNA and 3′ RACE were used confirm the presence of the terminator sequence and that the CTF13 transcript was both truncated and polyadenylated . The presence of the ADH1CTF13-T sequence was sufficient to replenish the CTF13 mRNA back to wild-type levels in the sno1Δ6 background as well as reduce the CIN rate back to wild-type levels . This result suggests that the all of the CIN in the sno1Δ6 mutant can be explained by CTF13 mRNA defects.Given that the TEF1 sequence is necessary to cause elevated CIN when the KanMX gene is present in the Crick orientation at the SNO1 locus. To test whether TTEF1 sequence is sufficient to induce CIN, we generated SNO1 deletions using two other markers, pFA6a-NatMX and pFA6a-HphMX, which confer resistance to the drugs nourseothricin and hygromycin, respectively . Importantly, both of these KO cassettes use the same promoter as KanMX and TTEF1 immediately following the drug resistance gene as in the pFA6a-KanMX plasmid .The above results suggest that the TCTF13 mRNA in each of these strains was truncated . We also confirmed that the CTF13 mRNA was truncated in the sno1Δ::URA3 strain, indicating that PURA3 also functions bidirectionally . Surprisingly, neither the sno1Δ::Nat mutant nor the sno1Δ::Hph mutant had elevated CIN rates as was observed in the sno1Δ::Kan mutant , suggesting the TTEF1 sequence is insufficient to cause increased CIN. The sno1Δ::Nat mutant had wild-type levels of CTF13 mRNA , whereas the sno1Δ::Hyg mutant had reduced abundance of the CTF13 mRNA similar to what was observed in the sno1Δ::Kan mutant . We sequenced 3′ RACE cDNA ends to determine potential qualitative differences in CTF13 mRNA among these strains. All three biological replicates of the sno1Δ::Nat CTF13 mRNA had a consistent sequence at the end of the 3′ UTR  corresponding to TTEF1 sequence, whereas in the sno1Δ::Hyg mutant, two of three replicates matched the same CTF13 3′ UTR sequence of the sno1Δ::Nat mutant, while one replicate indicated a longer heterogenous CTF13 mRNA end . These data suggest that in the sno1Δ::Kan , the CTF13 transcript extends into the drug resistance marker ORF. This seems to correlate with transcript instability, but the causal relationship is unclear. This observation highlights the complexity of mRNA perturbation by TTEF1 and suggests that some sequence features of KanMX may also be involved.3′ RACE first confirmed that the sno1Δ YKO collection mutant and the ADH1sno1Δ::Kan-T mutant, we used negative selection to endogenously tag the CTF13 ORF with 3x hemagglutinin . Importantly, we inserted the 3xHA tag at the N-terminus of CTF13 to avoid adding any extra DNA sequence that may influence the natural CTF13 3′ UTR. Following sequencing-based confirmation of the in-frame 3xHA-CTF13 tag at the endogenous ORF , we further generated sno1Δ and ADH1sno1Δ::Kan-T deletions in the 3xHA-CTF13 background. Of note, although colonies formed following the initial generation of the 3xHA-CTF13/sno1Δ double mutant, we were unable to isolate single colonies on fresh selective agar plates and as such could not accurately genotype the isolates .To determine whether Ctf13p level may account for the different CIN rates between the 3xHA-CTF13 tag itself seems to cause a very mild growth defect compared to an untagged CTF13 allele. As such it is possible that the 3xHA tag reduces the efficiency of this essential kinetochore protein and or results in increased Ctf13p degradation, which may explain the pronounced defect in combination with a sno1Δ . In characterizing this double mutant, we noticed that releasing selective pressure allowed the 3xHA-CTF13/sno1Δ strain to grow with only a mild growth defect . It is possible that the sno1Δ is lethal in the 3xHA-CTF13 background and, if selection is removed, the cell population adapts, perhaps by shutting down expression of KanMX via mutation or epigenetic regulation. Furthermore, if any aneuploids exist in the original transformed population , these strains can have continued growth even in the presence of G418 selection. Unfortunately, we were unable to confirm the synthetic lethality of the 3xHA-CTF13/sno1Δ strain using tetrad dissection analysis because the close proximity of SNO1 and CTF13 would likely limit crossing over during meiosis.The 3xHA-CTF13/sno1Δ double mutant, we were able to generate multiple ADH13xHA-CTF13/sno1Δ::Kan-T double mutants, which grew qualitatively similarly to the 3xHA-CTF13 background strain and could grow well in the presence of selective pressure . Comparing the Ctf13p levels between wild-type and ADH1sno1Δ::Kan-T log phase cultures, Ctf13p levels were elevated in the ADH1sno1Δ::Kan-T in three repeated experiments  also disrupting the SNO1 ORF did not increase CIN. By altering independent portions of the KanMX construct, we were able to pinpoint the increased CIN to a previously unappreciated artifact present within the YKO collection. The transcriptional terminator TTEF1 has an ectopic terminator sequence that appears to be read from the opposite strand such that the neighboring gene arranged in a convergent orientation to the deleted gene may have perturbed mRNA levels. The mRNA depletion artifact only occurs when the Kanamycin resistance gene is present upstream of the TTEF1, as this phenotype was undetectable or not dominant in the NatMX or HphMX context, respectively.We began our study by characterizing a potential role for SNO1, the neighboring convergent gene CTF13 has truncated and reduced mRNA levels. This seems to underlie the increased CIN in a sno1Δ strain, as elevated CIN can be reduced by adding another copy of CTF13 in a separate genomic locus or by raising the CTF13 mRNA level via NMD inactivation. Furthermore, this effect appears to be specific to convergent gene pairs, as when we altered the KanMX orientation to be tandem with the CTF13 gene (as in the sno1Δ-Watson or sno1Δ6-Watson mutant), CIN was significantly reduced. This observation led us to focus on 3′ UTR composition and determine a difference in the quality of the 3′ ends of the CTF13 mRNA in the sno1Δ mutant, which is truncated and lacks a polyA tail, two factors likely affecting other aspects of the RNA regulation and translation. When CTF13 mRNA was polyadenylated as in the ADH1sno1Δ::Kan-T mutant, CIN was suppressed and CTF13 mRNA eventually reached wild-type levels, which correlated to elevated levels of the Ctf13p.In the case of CTF13 3′ UTR is naturally very long and likely encompasses the complete antisense SNO1 ORF sequence, it is tempting to speculate that the presence of the Sno1p may be irrelevant for chromosome segregation. Instead, the SNO1 ORF DNA sequence could act as a stabilizer or enhancer for the CTF13 mRNA. Recent studies have shed light on a novel mode of posttranscriptional control of convergent gene expression through the formation of RNA duplexes that sometimes contain the complete antisense ORF of one mRNA within the 3′ UTR of the convergent gene’s mRNA  have completely unaltered SNO1 ORFs but still experience significantly elevated CIN. Furthermore, when a copy of the CTF13 ORF was added into an endogenous locus in a sno1Δ background, CIN was suppressed independent of the presence of a complete SNO1 ORF. These findings together argue against the necessity for the SNO1 ORF sequence in maintaining proper chromosome segregation.Given the CTF13 mRNA level, as inactiving components of NMD increased the CTF13 mRNA level in wild-type, sno1Δ, ADH1sno1Δ::Kan-T, and sno1Δ6 backgrounds. Given that these mRNAs have different CTF13 3′ UTRs as evidenced by 3′ RACE, it is unlikely that each of them would be a direct substrate for NMD. In fact, the natural CTF13 mRNA was previously proposed to be an indirect substrate of NMD, as the mRNA’s half-life was unchanged in the presence and absence of NMD  but the ADH1/upf1Δsno1Δ::Kan-T double mutant has higher CTF13 mRNA than the wild-type strain. This difference may reflect that CTF13 transcription is increased equally in both strains, with extra transcripts retained efficiently in the ADH1sno1Δ::Kan-T strain due to the stable polyA tail, whereas extra transcripts are still subject to continuous instability in the sno1Δ strain.Our data confirm previous findings that NMD plays a role in the downregulation of sno1Δ3, which had an extra 97 base pair deletion compared to sno1Δ2 and sno1Δ6 combined. This 97 base pair region has predicted binding motifs for transcription factors encoded by ASH1, AZF1, CBF1, GCR1, PHO4, and PHO2 , a difference in the expression orientation of this marker, or a combination of both scenarios.Although the VPS36 and CDC73 on chrXII whose stop codons are separated by 203 base pairs of intergenic sequence. Deletion of all or portions of CDC73 with KanMX resulted in a subsequent decrease of VPS36 mRNA levels detected by qPCR (ADH1) is particularly useful for the community as it contains the universal primer sites, F1 and R1, present on commonly used pFA6a based plasmids that can be used for PCR-based gene replacement.The effect that we measured may be extrapolated to other gene pairs arranged in convergent orientations, as the two other example convergent gene pairs we tested had reduced mRNA levels of one gene when the other in the pair was deleted. There is at least one similar example in the literature with the convergent gene pair  by qPCR . This fiOf further interest to the yeast research community, our finding raises the possibility of the construction of new partially defective budding yeast mutants in which the KanMX construct is inserted directly after the stop codon in a convergent orientation with specific genes of interest. The concept for these mutants is similar to the Decreased Abundance by mRNA Perturbation (DAmP) mutants . The hypTEF1 terminator sequence, which can affect the mRNA of neighboring genes within convergent pairs and thus likely contributes to this neighboring gene effect. Without a full understanding of the underlying mechanisms, our findings offer a cautionary tale to the yeast research community.Finally, other researchers have suggested a concept known as the “neighboring gene effect” within the YKO collection and have created systematic ways to identify genes that may have incorrect annotations due to this problem (


By considering the bidirectional Poisson's effect and soft boundary conditions, a modified theoretical model depicting the relation between stress distributions and the basic characteristics was developed to reveal the mechanical mechanism of the regulation strategy. Furthermore, the resulting 2D ordered wrinkles can be used as a dynamic optical grating and a smart template to reversibly regulate the morphology of various functional materials. This study will pave the way for wrinkle regulation and guide fabrication technology for functional wrinkled surfaces.Regulating existing micro and nano wrinkle structures into desired configurations is urgently necessary yet remains challenging, especially modulating wrinkle direction and location on demand. In this work, we propose a novel light-controlled strategy for surface wrinkles, which can dynamically and precisely regulate all basic characteristics of wrinkles, including wavelength, amplitude, direction and location ( Wrinkles are widespread in nature and engineering, but hard to tune on demand. This article reports a light regulation strategy to realize ordered wrinkles with arbitrary configuration for various optical, electronic and sensor applications. A) can help arteries adapt to different blood pressures and become self-cleaning to prevent formation of the nidus of a blood clot [θ) and location (cL) of ridges on leaves and insect wings can transport necessary water and nutrients, offer mechanical support and even determine the shape of growth [λ), A, θ and cL.As the most ubiquitous surface pattern in both nature and engineering, wrinkles have attracted intense research interest for various applications, including flexible electronics , microfaood clot . The spef growth . HoweverConsiderable regulation techniques of buckling surface instability, such as via a prepattern template, prestretching-release , mechaniλ, A, θ and cL) of wrinkles on demand, where previous studies could modulate only λ and A [In this paper, we propose a novel light-controlled strategy to arbitrarily regulate wrinkle topographies by precisely controlling the modulus spatial distributions and boundary conditions of the top layer for bilayer systems. This work, for the first time, can modulate all basic characteristics . In the whole article, the ‘1D’ or ‘2D’ ordered wrinkles are used to describe the wrinkle patterns induced by a single or sequential exposure of UV light, respectively. And ‘1D’ or ‘2D’ ordered wrinkles are defined as wrinkles having one or multi-directions in a two-dimensional surface, respectively. The above sequential exposure strategy is lithography-compatible. As a result of sequential exposures when rotating the photomask, a repeatable unit of the top film/substrate system was divided into four rectangular domains with length L and width W, that is unexposed domain D1, first-exposure domain D2, second-exposure domain D3 and double-exposure domain D4  in design of material surface characteristics suggest that light treatment may be a suitable way to regulate the local internal stresses in a wrinkled bilayer system with arbitrary profiles. We first fabricated and regulated 2D ordered wrinkles on bilayer systems by controlling the modulus of the top photosensitive layer spatially and temporally via a sequential exposure of UV light Fig. . The bilhf resting on a compliant soft substrate with thickness sh, Young's modulus sE and Poisson's ratio sv. The mechanical properties of the films, such as the Young's modulus, Poisson's ratio and mechanical boundary conditions, can be spatially reconfigured and then used to regulate the stress distributions and subsequent morphological evolution of the surface film. It has been demonstrated that Poisson's effects can greatly affect the wrinkle patterns of strip-like samples [et al. [y direction can be induced by the thermal stress in the x direction and the additional strain x direction can also be induced by the thermal stress in the y direction . When considering bidirectional Poisson's effects, the corresponding thermal stresses of D4 along the x and y directions can be expressed asTo reveal the mechanical mechanism for the above intriguing phenomenon, a modified shear-lag model with bidirectional Poisson's effect is proposed to explain the evolution of the wrinkle patterns by the sequential exposure strategy Fig. b. As see samples ,38. The  samples . Wang etx and y directions because of the soft boundary conditions at x = ±L/2 or y = ±W/2 . The modified shear-lag model is proposed to depict the 2D stress relaxation behavior of the sequentially exposed film/substrate system with a soft constraint effect, as detailed in Section S2.2 in Supplementary data. According to Eq. (S17), the stress distributions of the film/substrate system in D4 can be rewritten ast and Then, the thermal-induced residual stresses can relax along both x direction at the soft boundary (x = ±L/2); therefore, y direction both experimentally and theoretically  , which is obtained from Eq. , the initial external thermal stress x and y axes when As theoretically predicted in Eq. , when thentclass1pt{minimaentclass1pt{minimaFigure entclass1pt{minimaentclass1pt{minimaλ = 532 nm), projected Bragg diffraction images with distinct 3rd or 4th order spots onto a black screen . This result is consistent with the 3D laser scanning confocal microscope (LSCM) images in Fig. Furthermore, the wrinkle morphology was also traced and checked by light diffraction to verify the validity of our theoretical model. The optically transparent wrinkle samples, which were transmitted by a green laser light λ = 53 nm, projeθ and exposure time. In the selective UV irradiation strategy, the samples were first placed under a strip photomask for 15 min and then irradiated under the same photomask arranged in different directions for the same time . When eθ = 45° and 60°, similar results are seen . These experimental results are almost consistent with the theoretical results of the geometric angle relationship: 24θ = 34θ = dθ. Therefore, when the exposure time is fixed, the first and second exposures are equivalent to regulate the wrinkles. In addition, the wrinkle morphology can be modulated again by heat treatment. When the samples were further heated at 110°C for 3 min, all the exposure domains generated evident wrinkles accompanied by a larger diffraction order , the directions of the wrinkles dominated by the value and direction of the principal stresses can be regulated by angle 2Δθ and 3Δθ) in D2 and D3 decrease when eθ increases  pθ of the single-exposure domain, which rotates the wrinkle orientation. Moreover, both 2Δθ and 3Δθ decrease with the increase of eθ. The reason is that the shear stress pθ decrease with increasing eθ. However, as seen in Fig. 4Δθ in D4 increases with eθ. The experimental 4Δθ in D4 is consistent with the theoretical principal angle pθ and can be given as: 4Δθ = pΔθ = 1/2eθ. The intrinsic mechanical explanation is that pθ in D4 increases with eθ. Based on the above analysis, we obtain the geometric relationship between 24θ, 2Δθ and 4Δθ as 24θ = 2Δθ+4Δθ.Here, we demonstrate that the values of orientation rotations for the wrinkles  of the wrinkle surface can be recorded by the characteristics  of the diffraction pattern. Furthermore, the wrinkle surface can be dynamically recorded by a green laser and a red laser simultaneously . This approach may offer a noncontact and efficient method to detect 3D microstructures of material surfaces without complicated experimental equipment and procedures.In addition, because the light diffraction properties are strictly correlated to the surface morphology of the wrinkles, we can easily characterize the surface topography by the diffraction patterns of the samples. When a green laser was moved back and forth over different domains with the ordered wrinkles, the diffraction pattern changed between 1D and 2D as the microstructure changed, accordingly . Therefore, the characteristics  as an elastomer substrate for the bilayer system . The theaterials ,43, but λ, A, θ and cL of the dynamic template, we can easily fabricate different periodic functionalized microstructures on demand. We believe that the three-layer or even multi-layer wrinkled platform enables various functional layers being ordered and being dynamic transformed on 2D surface. This powerful functionalized wrinkled platform may be used for flexible electronics, microfabrication, sensors and micro-/nanochannels.Furthermore, the bilayer system can be used as a dynamic template, which can deposit or transfer various functional molecules including conductor, semiconductor and insulator onto this bilayer to form a three-layer system: the upper layer, middle layer and substrate can act as a functional layer, a morphology control layer and a dynamic response layer, respectively . For example, we deposited 10 nm Au onto the bilayer, the wrinkle topography was still well regulated by UV light-responsive PAN-BA film and could be dynamically erased/restored by NIR light-responsive CNT-PDMS substrate  of 2D ordered wrinkles on a bilayer system comprising photocrosslinked PAN-BA as the top layer and CNT-PDMS as the substrate. The key point to the regulation of 2D ordered wrinkles is that the mechanical properties of the bilayer system can be precisely controlled by photodimerization. We further developed a modified shear-lag model with a bidirectional Poisson's effect and soft confinement effect to understand the 2D spatial distribution of the mechanical property for the top layer, which can determine the stress field and wrinkle topography. The theoretical stress analysis demonstrates that the wrinkle pattern evolution can be attributed to the competitive mechanism between the elastic modulus enhancement effect induced by sequential exposure and the thermal stress enhancement effect controlled by the external physical field. This study provides a general guideline to fabricate and regulate 2D ordered wrinkles on a surface and also offers a dynamic template to reversibly regulate the morphology of various functional materials. We envision that this light-induced dynamic and reconfigurable wrinkle pattern can be used as a smart surface platform for optical devices, sensors, flexible electronics, information storage and anticounterfeiting.In summary, we demonstrated a sequential exposure strategy to spatially and temporally regulate the morphology (3ONa), potassium iodide (KI), 9-anthracenemethanol (AN-OH) and 4-vinylbenzyl chloride (S-Cl) purchased from China National Pharmaceutical Group were used directly without further purification. n-butyl acrylate (BA) obtained from China National Pharmaceutical Group was washed with saturated sodium hydroxide solution to remove retarder and then dried with magnesium sulfate. Multiwalled carbon nanotubes (CNT) were purchased from J&K Scientific Ltd. and were directly used.Sodium methanolate . The silicone base and curing agents were mixed at a 10:1 mass ratio in a polystyrene Petri dish and stirred at room temperature. Then, the mixture was baked at 70°C for 4 h, and then, degassed for 0.5 h to yield a crosslinked PDMS elastomer substrate. CNT-PDMS sheets were prepared in the same way as PDMS sheets, except for premixing 0.05 wt% of multiwalled carbon nanotubes (CNT) into the silicone base.A 6% toluene solution of PAN-BA was first spin-coated on a PDMS (or CNT-PDMS) substrate and then irradiated with 365 nm UV light under a strip mask for the desired time, resulting in the relatively stiff domains on the top layer film. The coated PDMS substrate was heated to the desired temperature for 3 min and cooled down to room temperature to generate the ordered wrinkle patterns in the exposure domains. The ordered wrinkle patterns could be regulated by a second 365 nm UV light irradiation process for a certain time and orientation of the photomask.λ = 808 nm; LE-LS-808-1000TFCB, LEO Photonics). 1H NMR spectra were measured on a Varian Mercury Plus spectrometer (400 MHz) with deuterated chloroform (CDCl3) as the solvent and tetramethylsilane as an internal standard at room temperature.Static wrinkle patterns were recorded with a 3D laser scanning confocal microscope . The dynamic wrinkle pattern elimination/reappearance process was recorded with a profile measurement microscope . The Young's modulus of the top film was measured by atomic force microscopy nanomechanical mapping , where the oscillation frequency of the Z-piezo was 1.0 kHz and the peak force amplitude was set at 150 nm. The samples were scanned using Olympus microcantilevers with a spring constant of 3 N/m. The NIR light was produced by a laser diode controller (nwaa052_Supplemental_FilesClick here for additional data file.


CRISPR–Cas activator (CRISPRa) systems that selectively turn on transcription of a target gene are a potentially transformative technology for programming cellular function. While in eukaryotes versatile CRISPRa systems exist, in bacteria these systems suffer from a limited ability to activate different genes due to strict distance-dependent requirements of functional target binding sites, and require greater customization to optimize performance in different genetic and cellular contexts. To address this, we apply a rational protein engineering approach to create a new CRISPRa platform that is highly modular to allow for easy customization and has increased targeting flexibility through harnessing engineered Cas proteins. We first demonstrate that transcription activation domains can be recruited by CRISPR–Cas through noncovalent protein-protein interactions, which allows each component to be encoded on separate and easily interchangeable plasmid elements. We then exploit this modularity to rapidly screen a library of different activation domains, creating new systems with distinct regulatory properties. Furthermore, we demonstrate that by harnessing a library of circularly permuted Cas proteins, we can create CRISPRa systems that have different target binding site requirements, which together, allow for expanded target range. Owing to the ease of designing and synthesizing guide RNAs (gRNAs) that determine target specificity, CRISPR–Cas regulators have found broad utility for performing targeted gene perturbation studies to uncover gene function , which uses a nuclease-dead Cas9 (dCas9) and a single guide RNA (sgRNA) complex to sterically block transcription initiation and elongation ,28. ExteWhile in eukaryotes CRISPRa systems have provided robust tools for activating transcription that have resulted in the widespread use of these technologies, in bacteria they have not had the same impact because of several technical limitations. First, it is increasingly clear that currently available ADs offer distinct regulatory properties and trade-offs, and different ADs are likely to be optimal depending on the exact gene target or application. For example, some bacterial ADs, such as those derived from the transcription factor SoxS ,32 or RNBacterial CRISPRa systems represent a potentially transformative technology; however, work remains to be done to create a versatile tool. Towards this goal, we apply a rational protein engineering approach to create a new CRISPRa platform that is highly modular, allowing for facile exchange of new ADs, and presents expanded target range, through the exchange of different engineered dCas9 variants with different binding site requirements. First, recognizing that different ADs have distinct properties, we characterize a panel of ADs and identify the N-terminal domain of the alpha subunit of RNAP (αNTD) to robustly activate compared to previously reported ADs. Next, we explore modular approaches to recruit ADs to dCas9 through noncovalent protein-protein interaction sequences called SYNZIP domains ,40. Thistet, Pbad or a constitutive J23150 promoter in a p15a chloramphenicol resistant vector. sgRNAs were expressed with a J23119 promoter in a ColE1 ampicillin resistant vector. Constructs containing ADs fused to SYNZIP were expressed with J23106 promoter in a CloDF spectinomycin resistant vector. Reporter plasmids were cloned from pJF076Sa plasmid (addgene #113322) (All plasmids used in this study can be found in #113322)  or Golde#113322) .Escherichia coli strain MG1655, except for data shown in Figure E. coli K-12 BW25113 and E. coli K-12 BW25113 ΔrpoZ  was used (E. coli cells and plated on LB + Agar (Difco) plates containing combinations of 100 μg ml−1 carbenicillin, 34 μg ml−1 chloramphenicol, 25 μg ml−1 kanamycin and 50 μg ml−1 spectinomycin, depending on the plasmids used, and incubated ∼17 h overnight at 37 °C. In some experiments involving transformation of ≥3 plasmids, a subset of plasmids was first transformed, competent cells prepared from resulting colonies and the remaining plasmid transformed. Plates were taken out of the incubator and left at room temperature for ∼7 h. Single colonies were used to inoculate four cultures of 300 μl of LB containing antibiotics at the concentrations described above in a 2 ml 96-well block (Costar). 96-well blocks were incubated overnight in a Vortemp 56 (Labnet) incubator bench top shaker at 37 °C and 1000 RPM. 10 μl of the overnight culture were added to 290 μl of LB, with antibiotics and 1 ng ml−1 of anhydrotetracycline (aTc) inducer or 50 mM of arabinose when required. For N-(β-ketocaproyl)-l-homoserine lactone, Cayman chemical] solution was also added with a final concentration of 10 and 100 μM as indicated. After 8–10 h, 50 μl of culture were transferred to a 96-well plate containing 50 μl of phosphate buffered saline (PBS) solution. An Infinite m1000 Pro plate reader (Tecan) was used to measure optical density (OD) at 600 nm, and to measure RFP fluorescence (FL) (540 nm excitation and 600 nm emission). For time course experiments shown in Fluorescence characterization was performed using was used . Each exE. coli MG1655 cells transformed with a combination of the control plasmids pJEC101, pJEC102, pJEC103 and pJEC598 (blank cells), which only contained the antibiotic resistance cassette and thus not expressing the reporter gene  to display the bacterial species from which αNTD sequences were obtained for data shown in Figure An expanded multifurcated phylogenetic tree, based on the NCBI taxonomy database, was generated using phyloT  subunit of RNAP   , but no ne (AHL) , targetine (AHL) . Howevertet and Pbad promoters and the corresponding inducer was added. We observed that with Ptet, probably due to the leakiness of the promoter, more than 2-fold activation was obtained in the absence of the inducer and no further activation was achieved in the presence of the inducer. While Pbad promoter evidenced no activation in the absence of the inducer and >2-fold activation when the inducer was added. , Ruegeria pomeroyi and Rhodobacter capsulatus, and Gammaproteobacteria: Shewanella oneidensis, Pseudomonas fluorescens, Pseudomonas aeruginosa and Pseudomonas stutzeri . A plasmid encoding an N-terminal SYNZIP fusion was created and combined with a sgRNA plasmid library to target this CRISPRa system to different PAMs located upstream of the RFP reporter gene on the non-template strand  in which the original N- and C-termini were fused with a 20 amino acid linker and the order of the amino acid sequence changed to create new N- and C-termini . Our goae strand . Comparie strand . Followivariants  and charvariants . From thn Figure . From thx Figure  showed rx Figure . This suIn our work, we apply rational protein engineering to successfully generate and characterize a novel bacterial CRISPRa system presenting a high degree of modularity. Our system encodes the dCas9 and AD as plasmid-independent elements that can be easily exchanged, allowing each to be independently optimized and synergistically combined to create new CRISPRa systems with distinct properties. Through exchange of different ADs, we demonstrate that we can alter regulatory properties, such as fold activation, while exchanging dCas9 variants allows for distinct targeting range. As such, we believe the work described here enhances the current versatility of bacterial CRISPRa systems by creating a highly modular system with expanded target range.Our screening of different ADs identified the N-terminal domain of the α subunit of RNAP (αNTD) to be a robust AD, able to successfully activate transcription when connected to dCas9 through either covalent or noncovalent linker domains. The α subunit of RNAP is naturally composed of two independently folded domains: αNTD and αCTD. In the process of RNAP assembly, αNTD dimerizes with a separate αNTD to initiate assembly, while αCTD is not required for this process, but instead interacts with transcription factors to recruit the RNAP to specific promoter sequences. Interestingly, we observed that the αNTD appeared to be optimal when fused through its C-terminus, in effect, mimicking its natural configuration in which the αCTD interacts with transcription factors to recruit RNAP. In this case, αCTD was replaced by dCas9 which performs the role of recruitment to a target promoter sequence. This activation by recruitment is analogous to regulatory mechanisms found in natural bacterial transcription activators, which rely on the recruitment of RNAP by protein-protein interactions. Interestingly, upon screening a library of αNTDs derived from different bacterial species, we observed a surprising level of portability. This is likely due to the high degree of sequence conservation across different species ,59 and c70 promoter, we anticipate that this approach will allow activation of the large number of endogenous promoters containing PAM binding sites that are non-optimal for other CRISPRa systems .All source data for main and SI figures was deposited in Rice University's Rice Digital Scholarship Archive (gkab211_Supplemental_FileClick here for additional data file.


To assess the potential of the soluble transforming growth factor β receptor type III (sTGFβrIII), a key regulator in TGFβ signaling, as a biomarker for diagnosis and stratification of patients with acute pancreatitis (AP).In this small prospective pilot study, patients’ (N = 22) plasma samples were obtained at three time points: the first and fourth day of hospitalization and the day of hospital discharge. Healthy controls’ plasma (N = 25) was obtained at a single time point. Concentration of sTGFβrIII in plasma was determined by ELISA. Data were analyzed by fitting linear or linear mixed models.Plasma sTGFβrIII levels at presentation (day 1) were similar in AP patients and healthy participants, irrespectively of the disease severity. sTGFβrIII levels in patients were constant during hospital stay.These observations do not support further evaluation of plasma sTGFβrIII levels in this setting, but do not exclude a potential biological role of TGFβ and membrane-bound TGFβrIII in AP pathophysiology. Acute pancreatitis (AP) is an inflammatory condition of the pancreas most commonly caused by bile stones or excessive alcohol use . It has Transforming growth factor β (TGF β) is a pleiotropic cytokine involved in the regulation of vital cellular processes   as well This prospective observational study enrolled consecutive adults diagnosed with a first episode of AP graded as “mild” or “moderate,” admitted between December 10, 2019 and February 18, 2020 at a single tertiary center , and a sample of generally healthy volunteers. Patients provided blood samples for sTGFβrIII determination at presentation to the hospital (day 1), day 4 of hospital stay, and at discharge, while healthy participants provided a single blood sample for this purpose. The third time point (discharge day) was purposely chosen not to be a “fixed” day of hospital stay, but rather to represent significant clinical improvement – patients were discharged when the following was achieved: improvement of symptoms or no symptoms reported; adequate oral feeding; and no systemic complications and partial improvement or resolution of local complications. All participants provided a signed informed consent. The study was approved by the Ethics Committee of Dubrava University Hospital.All participants had to be free of any other acute or chronic inflammatory disease and have no medical history of malignancy. Acute pancreatitis was diagnosed and classified in line with the revised Atlanta criteria , and trePeripheral blood was drawn into citrated Vacutainer tubes (citrate to blood 1:9); plasma was immediately separated by centrifugation (15 minutes at 3000 g) and was kept at -80 °C until analysis. An indirect ELISA kit  was used to determine the plasma sTGFβrIII expression levels according to the manufacturer's instructions. All samples and standards were analyzed in duplicates, and the samples with an individual coefficient of variation (CV) greater than 25% were retested in duplicates.Data are summarized by health status, AP severity, and time point, and were analyzed by fitting linear or linear mixed models . Where required, the variables were transformed to achieve normality of residuals.A total of 22 AP patients  and 25 healthy participants were included . Plasma (Supplementary Table 1)The need for  readily available biochemical markers that would be useful aids to guide treatment and follow-up procedures in AP patients has been well recognized ,3, and aOverall, although limited by a small single-center sample, the present study does not support further evaluation of plasma sTGFβrIII levels as a potential diagnostic/prognostic aid in acute pancreatitis.


Escherichia coli rRNA operons in 15 lineages that were evolved for ∼13,750 generations with frequent single-cell bottlenecks that reduce the effects of selection. We identified 38 gene conversion events and estimated an overall rate of intragenomic recombination within the 16S and 23S genes between rRNA copies of 3.6 × 10−4 per genome per generation or 8.6 × 10−6 per rRNA operon per homologous donor operon per generation. This rate varied only slightly from random expectations at different sites within the rRNA genes and between rRNA operons located at different positions in the genome. Our accurate estimate of the rate of rRNA gene conversions fills a gap in our quantitative understanding of how ribosomal sequences and other multicopy elements diversify and homogenize during microbial genome evolution.Due to their universal presence and high sequence conservation, ribosomal RNA (rRNA) sequences are used widely in phylogenetics for inferring evolutionary relationships between microbes and in metagenomics for analyzing the composition of microbial communities. Most microbial genomes encode multiple copies of rRNA genes to supply cells with sufficient capacity for protein synthesis. These copies typically undergo concerted evolution that keeps their sequences identical, or nearly so, due to gene conversion, a type of intragenomic recombination that changes one copy of a homologous sequence to exactly match another. Widely varying rates of rRNA gene conversion have previously been estimated by comparative genomics methods and using genetic reporter assays. To more directly measure rates of rRNA intragenomic recombination, we sequenced the seven  Ribosomes perform some of the most highly conserved chemistry in cells—the translation of messenger RNAs into proteins—and ribosomal RNA (rRNA) genes are the most conserved genes across all domains of life  and is tThe need for new protein synthesis is often the dominant factor limiting cellular replication .rRNA genes and the intergenic regions between them tend to have extremely similar, if not identical, sequences within a single organism. This is thought to be due to a high rate of gene conversion, a type of non-reciprocal intragenomic homologous recombination between highly similar sequences that converts one into a precise copy of the other . In bactEscherichia coli rRNA operons range over many orders of magnitude  and the other including the rrl gene (23S) and the first downstream rrf gene copy (5S). PCR products for REL1206 and endpoint clones from 15 MA lineages were sequenced with the primers used for amplification and additional internal primers (Supplementary Table S1) to cover all heterologous rRNA sites. Sequencing was carried out at the UT Austin DNA Sequencing Facility on an ABI 3730xl DNA Analyzer (Thermo Fisher).The MA experiment has been described previously  using Geneious 6. We found that REL1206 differed from REL606 by a single gene conversion: the 23S–5S spacer in the The number of mutational events of a given type that occur in each MA experiment lineage is expected to follow a Poisson distribution with a time basis of 13,750 generations. However, unlike the case for calculating base substitution rates  and 23S + 5S (rrl+rrf) portions of each rRNA operon  that can reproduce the evolved rRNA sequences requires 38 gene conversions: 7 in the 16S subunit, 27 in the 23S subunit, and 4 in the 23S–5S spacer  and 3.0 × 10−4  per genome in the 16S and 23S genes, respectively. Though there is some evidence that these rates are different from one another , they were similar enough that we combined these observations to calculate an overall rate for these conversions, which all involve changing a small number of bases within mostly homologous sequences, of 3.6 × 10−4 per genome per generation . This corresponds to a rate of 8.6 × 10−6 per rRNA operon per homologous donor operon per generation. From the MA experiment observations, we can also estimate that conversions covering the 23S–5S intergenic spacer region, which can swap its sequence between the two divergent alleles found in E. coli, occur at a rate of 3.5 × 10−5 per genome per generation  or 8.3 × 10−7 per operon per donor per generation. This rate is similar to the one found for conversions within the 16S subunit, but somewhat lower than that found in 23S, which could be due to the smaller size of this region, the greater level of sequence divergence in the 23S–5S spacer than for any locus within these rRNA genes, or both.The most parsimonious model  is likely due to their use of counterselection against a sacB-neo cassette inserted into the rrsB gene as a means for recovering mutants. Insertion of this large (3825 bp) non-homologous region is expected to introduce a substantial barrier to conversion, as gene conversion rates fall sharply with reduced homology . The rrnC operon was the only exception: it was converted significantly more frequently than expected (adjusted P = 0.013). Genes located near the E. coli origin of replication are copied first. Therefore, they exist transiently in two daughter chromosome copies for longer during the cell cycle compared to genes located further from the origin , which is expected to increase the chances that it will undergo homologous recombination with those operons .We next examined whether certain rRNA operons were more likely to be converted than expected by chance . Of the e origin . As rrnClication , its eleP > 0.05 for all sites). However, we did notice that sites at one locus in the 23S subunit were converted 15 times in the MA experiment and 2.5-fold more frequently than nearby sites ∼200 bp away (rrnA) this site contains a sequence (5′-GCTCGTGG-3′) that differs by one base from a canonical E. coli Chi site (5′-GCTGGTGG-3′). Similar Chi-like sites retain up to 40% of the activity of a consensus Chi site in promoting homologous recombination . Sequence identity is only irreversibly ratcheted up by gene conversions when they completely eliminate an alternative allele from all copies of a gene, and the modest number of conversions that we observed was not enough to achieve this during the MA experiment. Therefore, this result underscores that homogenization via conversion is likely to only take place on long timescales, particularly for sequences with many copies like the E. coli rRNA operons.Gene conversions maintain homogeneity between rRNA operons on long evolutionary timescales , with noE. coli is ∼10−10 per base pair per generation  gene conversions that will occur in a lineage for every point mutation.The spontaneous rate of base substitution mutations in rrlH gene of the E. coli REL606 strain from which the ancestor of our MA lineages evolved  and the US Army Research Office (Grant No. W911NF-12-1-0390). Conflicts of interest: None declared.


Some of ultrafine-grained (UFG) metals including UFG twinning induced plasticity (TWIP) steels have been found to overcome the paradox of strength and ductility in metals benefiting from their unique deformation modes. Here, this study provides insights into the atomistic process of deformation twin nucleation at Σ3{111} twin boundaries, the dominant type of grain boundary in this UFG high manganese TWIP steel. In response to the applied tensile stresses, grain boundary sliding takes place which changes the structure of coherent Σ3{111} twin boundary from atomistically smooth to partly defective. High resolution transmission electron microscopy demonstrates that the formation of disconnection on Σ3{111} twin boundaries is associated with the motion of Shockley partial dislocations on the boundaries. The twin boundary disconnections act as preferential nucleation sites for deformation twin that is a characteristic difference from the coarse-grained counterpart, and is likely correlated with the lethargy of grain interior dislocation activities, frequently seen in UFG metals. The deformation twin nucleation behavior will be discussed based on in-situ TEM deformation experiments and nanoscale strain distribution analyses results. Similar to many other high strength metals and alloys, UFG metals and alloys commonly exhibit a high strength/hardness that is generally accompanied by poor ductility, known as the strength-ductility paradox, regardless of their crystal structure, for example, UFG aluminum (face-centered cubic)6, UFG iron and interstitial free steels (body-centered cubic)7, and nanograined copper alloys (face-centered cubic)2. The yield strength of materials increases monotonously with decreasing in the grain size, which is generally described by the Hall–Petch relationship9. The tensile ductility drops immediately when the average grain size becomes smaller than 1 µm, which is related to the characteristics of UFG microstructure, that is, fine grains leave very little space for dislocation dynamics leading to a less enhanced strain-hardening rate and resulting in the plastic instability during deformation11.Ultrafine-grained (UFG) and nanocrystalline metals and alloys have attracted intensive research interest since their birth because of their potential to achieve extraordinary properties over conventional coarse-grained (CG) counterparts. This has led to a series of studies regarding the synthesis, processing, characterization, and potential applications of UFG and nanocrystalline metals and alloys in the past few decades12, a UFG Fe–Ni–C metastable austenitic steel13, and UFG high manganese Twinning Induced Plasticity (TWIP) steels16. Experimental results of these materials indicate that nonconventional or reverse-order deformation modes were activated in addition to the normal dislocation slip, specifically, <c + a> dislocations activation in the UFG Mg alloy, martensitic transformation in the UFG metastable austenitic steel, and deformation twinning in the UFG TWIP steels. Tsuji et al.13 suggest that the sequential activation of different deformation modes would foster the regeneration of strain-hardening ability during plastic deformation and lead to a high strength and large ductility because of possible interactions between different deformation modes, in other words, overcoming the paradox of strength and ductility. However, the detailed mechanisms to activate such nonconventional deformation modes are not fully understood yet.Recently, a high strength and moderate ductility are simultaneously achieved in a UFG magnesium alloy14. Our results indicate that the activation takes place in the very early stage of plastic deformation like near the macroscopic yield point, and the grain size plays a major role in deformation mode alternation; deformation twin nucleation occurred at the grain boundaries in under-1 µm austenitic grains, while the normal in-grain slip was mostly observed in over-1 µm grains. This grain size dependence could be attributed to the lack of initial mobile dislocations and inactive in-grain dislocation sources; both are characteristics of the UFG microstructure. Because of its technical and scientific importance, the deformation twinning behavior in conjunction with mechanical properties has been extensively studied in conventional CG TWIP steels19. However, theses deformation twinning mechanisms based on the arrangement of highly coordinated Shockley partial dislocations on {111} slip planes24 appear to be insufficient to explain the grain boundary mediated deformation twinning in the UFG TWIP steels because the deformation twinning in UFG TWIP steels appears to be highly correlated with grain boundary structure.The authors have recently investigated the activation factors of different deformation modes in a UFG austenitic TWIP steel26. Our recent in-situ TEM deformation study has demonstrated that the deformation twin nucleation at a Σ3{111} twin boundary in the CG microstructure occurs only when a localized stress concentration field formed by dislocation pile-up at the grain boundary26. This deformation twin nucleation behavior is unlikely feasible in the UFG microstructure because the grain interior dislocation activities are no longer predominant thus limiting in-grain dislocation pile-up at a twin boundary 27. This leads an open question, that is, what mechanism would promote the deformation twin nucleation at a Σ3{111} twin boundary, which is the dominant boundary type in the UFG TWIP steels, when the localized stress concentration field cannot be generated by dislocation pile-up, or localized stress concentration field is no longer necessary for the deformation twin nucleation in the UFG TWIP steels.Regarding the correlation between grain boundary character and deformation twin mechanism, only handful studies have carried out even on the conventional CG TWIP steels28. The detailed deformed microstructure near Σ3{111} twin boundaries and their local strain level were investigated using transmission electron microscopy (TEM) and scanning transmission microscopy (STEM) techniques to shed a light on the atomistic process of deformation twin nucleation in the UFG microstructure.In this study, we aim to address the question by carefully observing the internal behavior of Σ3{111} twin boundaries subjected to external tensile stresses and revealing the deformation twin nucleation process on the boundaries in a UFG high manganese TWIP steel. A carbon-free UFG high-manganese steel was selected for this study to minimize possible additional effects such as the serrated flow that frequently takes place in carbon-containing TWIP steels−2)29, while the average grain size is about 20 times smaller than that of the CG one, 0.79 µm instead of 15.4 µm. Three grain boundary maps for the undeformed and two different tensile-strained samples are shown in Fig. The microstructure of both undeformed and tensile-strained UFG TWIP steel samples were firstly characterized by EBSD. The UFG TWIP steel has the identical chemical composition and stacking fault energy (SFE) with the CG TWIP steel we previously studied  maps for these three samples are shown in Fig. 31, and in fact, both boundaries were found to act as a nucleation site for deformation twin. The dark contrast indicated by the white arrows appears to be induced by a dislocation reaction within the grain boundary, i.e., a deformation twinning event associated with a grain-boundary dislocation dissociation. In Fig. In Fig. 32. The leading Shockley partial dislocations were continuously emitted from the Σ3{111} twin boundary  turned from dark to bright, i.e., two stacking faults (F1 and F2) were overlapping and showed the reversal fringe contrast. These spontaneously successive emission events occur rapidly at the Σ3{111} twin boundary and is found to be characteristically different from the one induced by in-grain dislocations− Σ3{111} twin boundary interaction in the CG counterpart in Supplementary Fig. S126. A Thompson tetrahedron inserted in Fig. 1 and b2 will glide on the twin plane of Figure fcc zone axis in the inset of Fig. matrix tensile axis causes the resolved shear stress applied on {111} 〈112 〉 leading Shockley partial dislocation to be larger than those applied on {111} 〈110 〉 perfect dislocations and {111} 〈112 〉 trailing Shockley partial dislocations in both Σ3{111} twin plane and deformation twin plane. For example, the Schmid factor for a/6 Σ21 tilt boundary45 or a [011] Σ9 tilt boundary , as shown in Fig. ies Fig. c–e, was 50 was applied as schematically illustrated in Fig. t1 to t8, i.e., the vectors from t1 to t4 and the vectors from t5 to t8 were defined with respect to the crystal frame of grain I (Twin) and grain II (Matrix), respectively. The Burgers vector of twin boundary dislocation can be determined by the summation of the lattice vectors when lattice vectors of t5 ~ t8 were coordinately transformed into the ones in crystal frame of twin. The Burgers vector of the twin boundary dislocation then can be expressed as the following:1 ~ t8 are listed in Fig. 1 and d3 are the DSC lattice vectors illustrated in the schematic of Fig. To determine the Burgers vector of the twin boundary dislocations associated with the twin boundary disconnection, the Burgers circuit approach52. In contrast, the disappearance of localized strain field in Fig. A quantitative analysis of nanoscale strain distribution indicates the correlation between local strain level and deformation twinning behavior. The strain level at/near the disconnection in Fig. 54. As a result, these disconnection propagations on Σ3{111} twin boundary results multiple disconnections, i.e., the atomistic step having few monolayers in height, on the originally atomistically smooth twin boundaries, that accommodates the misfit between two sides of grains across the twin boundary. The multiple disconnections emitted sequentially from triple junction and coexisted on the neighboring {111} twin plane are similar to the internal behavior observed by in situ atomistic resolution TEM experiment54 where a Σ11(113) coherent grain boundary showed a layer-by-layer migration behavior due to the sequential nucleation and lateral motion of grain boundary disconnections. This disconnection activity combination with shear coupled grain boundary migration was also observed in a nanocrystalline gold metal by in situ TEM deformation experiments and considered to be the dominant deformation mechanism55. The disconnections can act as preferential nucleation sites for stacking faults because the localized stress concentration field at/near disconnections could exceed the twinning stress during plastic deformation, thus allow in-grain dislocation slip transfer in the later stage of plastic deformation56. This grain boundary sliding process appears to be phenomenologically similar to the grain boundary migration mechanism originally proposed by Hirth57 and experimentally observed in the deformed microstructure of several nanograined coppers59.The mechanism to form a nucleation site for deformation twins is schematically illustrated in Fig. bR is the Burgers vector of residual twin boundary dislocation, bin is the Burgers vector of the dislocations within a disconnection, bout is the Shockley partial dislocation emitted from the disconnection. Based on the crystallographic orientation relationship between the Σ3{111} twin boundary and deformation twin, bin and bout can be designated to be 1/6 Σ9), and low-energy coherent Σ3{111} twin boundaries are all able to be a nucleation site for deformation twins.The formation of twin boundary disconnections appears to be governed by the motion of Shockley partial dislocations on the Σ3{111} twin boundary. These disconnections (defective steps) would accumulate strain and serve as the nucleation sites of deformation twins when the localized stress level exceeds the twinning stress.The mechanistic difference to generate localized strain concentrations on coherent Σ3{111} twin boundaries between the UFG and the CG alloys is concluded to be originated from a unique characteristic of the UFG microstructure. The grain size constraint suppresses the in-grain dislocation activities in the UFG steel and stimulates the twin boundary dislocation activities leading to the twin boundary disconnections which act as the deformation twin nucleation sites. On the other hand, the observed periodic contrast reversal of stacking fault during the deformation twin nucleation at Σ3{111} boundaries indicates that the deformation twin formation process is likely identical between the UFG and CG microstructures once nucleated, partly due to the identical chemistry and SFE.The deformation twin nucleation mechanism on Σ3{111} twin boundaries in a UFG Fe-31Mn-3Si-3Al austenitic TWIP steel was found to be characteristically different from that in the CG counterpart. The detailed microstructural investigation using in-situ TEM deformation, HRTEM and µP-STEM techniques has drawn the following conclusions:−2) was fabricated for this work29. The chemical composition of the steel was Fe-31Mn-3Al-3Si wt%. The as-received TWIP steel was cold rolled from 12 to 1 mm (92% reduction) by multi-pass cold rolling and then heat treated in a salt bath at 950 °C for 5 min followed by water quenching to obtain the aimed grain size.A carbon-free UFG (0.79 ± 0.39 μm) high-manganese austenitic steel TWIP steel with intermediate SFE  and transmission electron microscopy (TEM) techniques. The 150 µm thick sheets were tensile-deformed to 0.046 and 0.062 engineering strain using a testing machine (Kammrath and Weiss Module 200 N) at a strain rate of 4.6 × 10Specimens for post-deformation EBSD analyses were mechanically polished by abrasive paper up to 2000-grit then electropolished. The microstructural features including grain boundary maps and local strain distribution were examined using a TSL OIM EBSD system attached to a FEI Helios 600 dual-beam field emission gun (FEG) scanning electron microscope (SEM) / focused ion beam (FIB) system. EBSD maps were acquired at 30 kV acceleration voltage and 13 mm working distance. The scanning step size was 50 nm. No data clean-up was performed except for the removal of some points with low confidence value.The local strain distribution was evaluated from the kernel average misorientation (KAM). KAM is a local misorientation defined as the average misorientation of a point with its nearest neighbors in a grain. The average misorientation of a given point is calculated by taking that point as well as all of its nearest neighbors into account with a criterion that the misorientation exceeding threshold 3° will be discarded from the calculation, because these points are considered to belong to the adjacent grains.Specimens prepared for post-deformation TEM analyses were cut from the center of the deformed sheets. 2 × 2 mm square shape foils were taken then mechanically thinned to 70 μm thick. The final thinning to be partly electron transparent was achieved by using a twin-jet electropolisher (Fischione Model 110) with a 95% acetic acid—5% perchloric acid electrolyte maintained at 17 °C and the applied voltage of 38 V. The TEM characterization was performed using a JEOL JEM 2100 operated at 200 kV with Gatan Orius SC200D and Ultrascan 1000XP cameras.60 after thinned to be partly electron transparent. The strain rate was controlled at approximately 6.7 × 10–5 s−1 in this study. All videos were recorded in the bright field (BF) mode and a FEI Titan 80–300 S/TEM operated at 300 kV was used. Gatan Orius SC200D camera and Digital Micrograph with the high-resolution streaming video plug-in were used for video recording.Specimens for in-situ deformation tests were sliced from undeformed sheets; the shape of the foils, 2 × 2 mm square shape, is identical to the one used for TEM analysis. A square shape foil was fixed on a cartridge-type blade for SATO Holder Duo (Mel-Build Co.)The FEI Titan 80-300 V S/TEM also was used in this analysis. Strain mapping was performed using the parallel-nanoprobe scanning transmission electron microscopy (µP-STEM) technique. The µP-STEM employed a 10 µm second condenser aperture to reduce the beam semi-convergence angle to be 0.13 mrad and obtained less than 2 nm probe size.Supplementary Information 1.Supplementary Video 1.