In this work we construct a novel atomicallythin, all van der Waals SQUID, in which current flows between NbSe2 contacts through two parallel graphene weak links. The 2Dplanar SQUID remains uniquely stable at high in-plane field, whichenables tracing critical current interference patterns as a functionof the field up to 4.5 T. From these we extract the evolution of thecurrent distribution up to high fields, demonstrating sub-nanometersensitivity to deviation of current flow from a perfect atomic planeand observing a field-driven transition in which supercurrent redistributesto a narrow channel. We further suggest a new application of the asymmetricSQUID geometry to directly probe the current density in the absenceof phase information.Thin NbSe NbSe2 has been coupled laterally to grapheneto realize NS junctions.6 Devices consisting ofNbSe2 flakes coupled on both sides of a narrow graphenechannel , constructed exclusivelyfrom van der Waals (vdW) materials by transferring a cracked NbSe2 flake on top of graphene, are unique in retaining a Josephsoneffect at high parallel magnetic fields.It is useful to incorporate thin TMD superconductors in devicesthat utilize their unique properties at high channel 1aare we2 contacts through parallelmonolayer graphene (MLG) and few-layer graphene (FLG) weak links (see IC(B⊥) interference patterns up to B∥ = 4.5 T. We reconstruct the distributionof current flow from IC(B⊥), presenting a new method utilizing the asymmetricSQUID geometry without the need for phase retrieval. At high B∥, we observe a qualitatively apparenttransition, indicating a narrowing of the current channel in the MLG.Furthermore, we find that our SQUID is highly sensitive to the nanometer-scaleheight difference between the MLG and FLG current planes.In this work, we extend the all-vdW 2DJJ conceptto a SQUID geometry,with current flowing between NbSeinks see 1b. In thinks see 1c, ensur2 flake, ofapproximately 13 nm thickness, as seen in the cross-sectional TEMmeasurement (see the Supporting Information); the length of both junctions, imposed by the NbSe2 crack,is d = 140 nm in the direction of the current flow.We begin by showcasing the basic propertiesof 2D SQUID in hreshold 2a. The tB⊥), the superconducting order parameter Δeiφ acquires a position-dependent phaseand undergoes interference. This leads to a Fourier relation betweenthe critical current IC(B⊥) and the maximal local critical current density J0(x)9x1, x2 and extendingacross the junction length d into the superconductorsup to the London penetration depth λ encloses a magnetic fluxof k(x2– x1)/2π in units of Φ0  magneticfield perpendicular to the junction (f Φ0 see 1d.IC(B⊥) at zero gate voltage and B∥ =0 T is shown in IC, witha magnetic field period ΔB = Φ0ASQ ≈ 380μT, reflect the area of the SQUID ASQ = 2δ(2λ + d) = 5.4 μm2. This area implies an effective penetration length λ = 930nm, longer than λL ≈ 200 nm yet shorter thanthe Pearl length Λ = 2λL2/t ≈ 6 μm. TheSQUID oscillations are modulated by an envelope which derives fromthe areas of the MLG and FLG channels, as illustrated by a schematicof two channel interference in B∥ = 0 T pattern is not perfectly symmetrical with respect to B⊥. This could be a signature of varioussymmetry-breaking effects16 but is most likelydue to vortices in the vicinity of the junction or a small trappedparallel flux.The interferencepattern of B∥ dramaticallychanges this pattern. At B∥ = 2.8T  in the SQUID, we maketwo simplifying assumptions: (i) the phase dynamics are local and(ii) the current–phase relation is sinusoidal. Both assumptionsare typical for graphene-based JJs. However, ballistic graphene JJsmay exhibit measurable skewness in the current–phase relation,12 while in ultrathin superconducting contacts,the Pearl length Λ = 2λL2/t replaces λL as the relevant field decay scale and the dynamics become potentiallynonlocal.13 Our smaller than bulk SQUIDperiodicity ΔB, discussed above, might hintat nonlocal dynamics; however, the typical nonlocal geometric relationΔB = 1.8Φ0/WS2,13 with WS being thewidth of the superconductor, is too large for our measured periodicity.This raises the need for future geometry-controlled experiments.To gain initial insight into the expected 14  generating Fraunhofer-like envelopes . VG trace extracted from the fit. For 2λ + d =2 μm, we find that fit parameters WM, WF, δ agree with the dimensionsdetermined from SEM measurements shown in Supporting Information). The extracted currentdensities in We fit these curves using the analytical modeldescribed above,where the free parameters are the MLG and FLG widths s 2δ see 1e, and tB∥. In IC, at VG = 0 V. This interference plotis extremely stable, up to B∥ =4 T, barring minor flux jumps around B∥ = 2.8 T, likely due to vortices entering the vicinity of the junction(see the Supporting Information). B∥ is kept strictly aligned to the SQUIDplane by careful compensation of the out-of-plane coil. This precisealignment procedure utilizes the phase of the fast SQUID oscillationsand allows us to avoid flux jumps up to higher fields than in previousworks.27B⊥ isdefined as geometrically perpendicular to B∥. Note that we control the field along the axes of the lab magnets,which are not exactly aligned with the SQUID plane; we describe thecompensation and alignment procedure in detail in the Supporting Information.We now turn to a detailedinvestigation of the field-driven transitionshown in B⊥, evident in a shift of the maximal IC and of the first Fraunhofer nodes. This diagonalis due to deviation from a perfect planar geometry: the step height h between MLG and FLG planes creates an angle θ betweenthe SQUID plane and the MLG. With B∥ aligned exactly to the SQUID plane, it contributes a component B∥(sinθ) of flux perpendicular tothe MLG and FLG flakes. The condition of zero flux through the MLG,for which the Fraunhofer envelope function is maximal, thus driftstoward negative values of B⊥, followingthe linear relation B⊥(max(IC)) = −B∥(sinθ) to compensate . The angle θ ≈0.025° extracted from the fit indicates a step height of around1 nm, while the TEM measured FLG thickness is 2.4 nm; this probablyindicates a distribution of current throughout the FLG, with the meanSQUID plane being determined by the center of the FLG flake. Therecould also be an additional step or curvature in the hBN outside therange of the TEM. The 2DJJ SQUID is thus an extremely sensitive toolfor tracking deviations from the atomic planar geometry.The data exhibit a diagonal driftof the MLG envelope toward negative B∥ = 0, 2, and 2.5T, together with the best fit of our analytical model. The currentdensities corresponding to the fit appear in B∥ increases.Theeffect of field-driven current redistribution is apparent inthe transition to a SQUID-like interference pattern. 17The analytical curves fit the data reasonably well for the centrallobes of the interference patterns see 3d and 4dN discrete spatial points and subject to known physicalconstraints to calculate the critical current via a forward Fouriertransform. The current density profile is then adjusted to obtainthe best fit of the calculated interference pattern to the data, asin ref .We use an approach that we term “the maximum entropymethod”suitable for reproducing current distributions with no symmetry requirements.The method postulates a current density profile sampled at sin ref , with anB∥ = 0 T anddifferent gate voltages  in To demonstrate the maximum entropy method, we returnto the interferencepatterns measured at voltages 3, and exC(l) are Fouriertransform pairs. In our case, |IC(B⊥)|2 is the energy spectraldensity of J0(x) andthusThe fit obtained by the maximum entropy method is remarkablysuccessful.Nevertheless, it is a complex method with many algorithmic as wellas physical parameters. Since we are interested in circumventing thephase-retrieval problem altogether, we introduce a new method thatharnesses our asymmetric SQUID geometry. We employ the narrower FLGjunction as a direct probe of the current density in the wider MLGjunction. This method leans on the Wiener–Khinchin theorem,which states that the energy spectral density of a function and itsautocorrelation J0(x) also provides direct information about J0(x) itself. Consider an ideal two-channel device,where the current density in the one channel is extremely narrow,approximated by the Dirac delta function, whereas the current in theother channel is widely distributed. The separation between the centersof the two channels is larger than their combined widths. The autocorrelationin this case contains a term equal to the current density in the widerchannel .Note that here, to calculate the autocorrelation,we performaforward Fourier transform of the energy spectral density, which doesnot require any knowledge of the phase. This calculation is alwayspossible; however, only for a specific asymmetric SQUID geometry,the autocorrelation of l =−2δ equal to the distance between the centers of thetwo channels. l = –2.7 μm is qualitatively similar in its form to the extractedcurrent density in In our device, the FLG is a few times narrower than theMLG andcarries a similar total current. In this instance, the autocorrelationconvolves the FLG and MLG densities, resulting in a feature whichqualitatively resembles the MLG current density “smeared”at the scale of the FLG width and centered at IC data set (see the Supporting Information). This map allows us to visualize how the currentdensity in the MLG redirects into a narrow channel. The interferencepatterns produced by the maximum entropy procedure fit most of themeasured patterns closely, as shown for selected values of B∥ = 0, 2, and 2.5 T in B∥ increases.We now apply these methods to extract a visual pictureof the evolutionof the current density with a parallel magnetic field. l = 2.7 μm , is qualitatively similarto the maximum entropy MLG current density shown in B∥ = 2 T.This phenomenology is apparent alsoin the current density extractedby autocorrelation. The sideband marked by pink lines in 2 junctions also undergo a transition toSQUID-like interference patterns where all lobes are of similar heightat high B∥.8 In that work, the patterns were too disordered to fit to 2 substrate.8 In the present work, the device is flat due tothe use of an hBN substrate. In addition, the signal is sufficientlystable to allow a quantitative fitting. All models, assuming an experimentalgeometry corroborated by SEM and TEM, yield a clear transition betweena distributed current density in the MLG at low fields, to a narrowsupercurrent channel at high B∥.The transition toward narrow supercurrentchannels has alreadybeen hinted at in our previous work—indeed, multiple diffusiveMLG–NbSeB∥, seen by Suominen etal., was attributed to suppression of supercurrent in the bulk ofthe JJ due to a magnetic dipole formed by tilted flux lines.21 In our geometry, however, SQUID-like interferenceindicates one channel in the MLG, not necessarily on the edge. Theflux focusing effect is also weaker in thin NbSe2 electrodes,where the tilt of the field lines is minimal due to a long Londonpenetration length.We note that a similar effect of SQUID-like interferencepatternsat high B∥. The suppression of a 2DJJ supercurrent vs B∥ is determined by the interplay of the Thouless andZeeman energy scales,22 the Thouless energy being a transportenergy scale determined by the inverse of the traversal time of thejunction.23 Hence, the superior resilienceof a single channel could be the consequence of a higher Thoulessenergy if a particular channel allows faster traversal of the junction.This could be, for example, a guided edge mode or a shorter channelin a nonuniform junction geometry. However, the presence of a similareffect in a number of devices in ref  pickup technique35 to pick up first the NbSe2 and then the graphenestrips oriented perpendicular to the crack and finally deposited thestack on the hBN substrate. We applied standard e-beam lithographyand e-beam evaporation to create Ti/Au contacts to the NbSe2, removing surface oxide using in situ argon ion milling prior toevaporation. Four-probe measurements were conducted in a BluFors dilutioncryostat with a 3 T/9 T vector magnet and a base temperature of 20mK.We exfoliated hbN on markered SiO


Staphylococcus aureus. This interaction is necessary and sufficient for prophage activation after SOS-mediated CI auto-cleavage, defining the final stage in the prophage induction cascade. Our findings unveil unexpected roles of bacterial protease ClpX in phage biology.Bacteriophages (phages) are the most abundant biological entities on Earth, exerting a significant influence on the dissemination of bacterial virulence, pathogenicity, and antimicrobial resistance. Temperate phages integrate into the bacterial chromosome in a dormant state through intricate regulatory mechanisms. These mechanisms repress lytic genes while facilitating the expression of integrase and the CI master repressor. Upon bacterial SOS response activation, the CI repressor undergoes auto-cleavage, producing two fragments with the N-terminal domain (NTD) retaining significant DNA-binding ability. The process of relieving CI NTD repression, essential for prophage induction, remains unknown. Here we show a specific interaction between the ClpX protease and CI NTD repressor fragment of phages Ф11 and 80α in  Staphylococcus aureus is controlled by the ClpX protease, unveiling and unexpected role for ClpX in phage biology.Prophage induction is a fundamental process in the phage life cycle. Here the authors show that the final stage of prophage induction in  Temperate phages, such as the model phage λ establish a complex relationship with their bacterial host, where they can either replicate and kill their host cell (lytic cycle) or integrate into their host’s genome and be passed on vertically (lysogenic cycle) until they become reactivated upon defined environmental cues. Lysogeny is controlled by a phage repressor called CI in λ, which represses the transcription of genes required for phage excision, replication, packaging, and host cell lysis as well as its own transcription4. Upon induction, the prophage produces the lytic regulator protein Cro, which stabilises the lytic cycle by blocking transcription from the PRM promoter driving cI transcription3. The expression levels of CI and Cro are finely balanced to ensure tight repression of the phage lytic cycle while enabling rapid activation upon sensing the appropriate stimulus.Bacteriophages, or phages, are the most abundant biological entities on Earth6. In the absence of DNA damage, the bacterial LexA repressor binds to operator sequences within the promoter of SOS response genes (SOS boxes/Cheo boxes in Bacillus subtilis)8. LexA binds to these boxes as a dimer with each monomer comprising a C-terminal dimerisation domain (CTD) and an N-terminal DNA-binding domain (NTD)9. DNA damage results in the formation of single-stranded DNA fragments to which RecA binds resulting in an activated RecA-nucleoprotein filaments (RecA*)10. RecA* in turn promotes the autocatalytic cleavage of unbound LexA11, reducing the cellular LexA pool and allowing the progressive activation of SOS-genes according to their LexA binding affinities. Cleavage of the LexA repressor separates its CTD and NTD, revealing latent ClpX-recognition motifs leading to the degradation of both fragments by the ClpXP protease complex12. Even though both fragments of the E. coli LexA protein are recognised by ClpX, ClpX is mainly involved in the degradation of the LexA NTD in vivo12, while the Lon protease is responsible for degrading the LexA CTD13. In Staphylococcus aureus, the ATPases ClpX and ClpC along with the proteolytic activity of ClpP are involved in the degradation of the LexA NTD and subsequent SOS activation14. Since the LexA NTD retains some of its ability to bind to SOS boxes and to repress target genes in both Gram-positive and Gram-negative bacteria16, its targeted removal by ClpXP is required for SOS activation.The bacterial SOS response is a key signal that activates the phage lytic cycle. It is triggered by DNA damage and coordinates the expression of genes involved in DNA repair, replication, and cell division17. CI repressors are present in a wide variety of temperate phage genomes of both Gram-positive and Gram-negative hosts19. Interestingly, several examples of phage CI repressor molecules have been identified that retain some and sometimes even full binding affinities with their cognate promoter DNA after autocatalytic separation of their C- and NTDs23. The retention of such levels of DNA affinity by the CI NTD therefore would require its active inactivation to ensure progression from the lysogenic to the lytic phage lifecycle. Despite its vital role in temperate phage biology, how the CI NTD repressor activity is abolished after RecA* mediated release remains elusive.Prophage induction is a fundamental process in phage biology and is controlled by the CI protein in phage λ. λ CI is a LexA-like repressor comprised of an NTD involved in DNA binding and repression and a CTD that upon recognition of activated RecA* are autocatalytically separated24. ClpP alone is only able to degrade small peptides and requires association with one of its ATPases which act as energy-dependent unfoldases, unfolding and delivering the protein into the ClpP proteolytic chamber25. The target specificity of the protease complex is determined by its ATPase, which recognises specific degron motifs, often with the assistance of additional adaptor proteins28.The proteolytic degradation of the LexA NTD together with the conserved domain architecture found in both LexA and LexA-like repressors such as CI suggest that the bacterial proteasome and specifically Clp proteases may also be involved in prophage induction. These proteases mediate ATP-dependent protein degradation and consist of a proteolytic subunit (ClpP) associated with an AAA-ATPase Here we show that the ClpX ATPase performs an essential role in prophage induction by abolishing the ability of the CI NTD to repress lytic phage genes after RecA* mediated CI cleavage and allowing the prophage to enter lytic replication. This process is distinct from the role of ClpX on the staphylococcal SOS response and acts via direct binding of ClpX to the CI NTD. By contrast, the ClpP protease and its proteolytic interaction with ClpX are not required for this process but are essential for the activation of the staphylococcal SOS response.S. aureus phages Φ11 and 80α, which have been extensively used as models for Gram-positive phages30. Both phages encode a CI-like repressor that maintains the phage in its lysogenic prophage state within the bacterial chromosome. To test whether any of the Clp proteases . Similarly, the clpX mutant showed a significant reduction in phage tires (108–109-fold) and produced very few plaques (10–100 PFU per ml)  Fig. , highligclpX and clpP genes were deleted using allelic replacement . These strains were either infected with Φ11 or 80α or lysogenised with these phages  and the phage cycle induced with MitC. In agreement with the previous results, lysis behaviour and phage titres remained unaffected during phage infection  or clpP  into the respective mutants under the control of a cadmium-inducible promoter fully restored wild-type phage lysis and titres  were inconsistent with the known roles of ClpXP in SOS response induction14, where the concerted action of ClpXP is required for LexA NTD degradation. To explore this further, we constructed a set of reporter plasmids in which the promoters of the SOS-inducible genes lexA and recA were fused to a β-lactamase reporter gene in plasmid pCN41 . These were introduced into the RN4220 wt, RN4220 ΔclpP and RN4220 ΔclpX mutant strains and the SOS response induced by MitC addition.Since prophage induction and activation of the bacterial SOS response are linked, and since in lexA and recA promoters were activated upon MitC addition in the wt S. aureus strain background  showing a 10.0- and 7.6-fold induction, respectively  was observed in the ΔclpP mutant background , showing that ClpP was essential for the activation of the SOS response. This confirmed that ClpP-mediated degradation of the bacterial LexA NTD was required for SOS induction14.Consistent with their SOS-inducibility, both lexA and recA reporter plasmids into an RN4220 derivative strain expressing an SOS-insensitive version of the LexA repressor (LexAG94E) that can no longer catalyse autocleavage and consequently can no longer induce the SOS response32 . As expected, neither reporter was inducible by MitC addition and expression levels mimicked those observed in the ΔclpP mutant background.To further validate the essential role of ClpP in SOS response activation, we introduced the lexA and recA reporter plasmids were introduced in the ΔclpX mutant background , although the overall transcriptional levels of both lexA and recA reporters were reduced  cycle, where the prophage first excises from the bacterial genome. Instead, they follow the replication, packaging, and excision (RPE) cycle, where the prophage initiates replication and packaging while still integrated into the bacterial genome and excises late in the induction cycle29. Hence, it was clear that ClpP and ClpX should control the heart of the lysogenic switch system and should control prophage derepression.Given the distinct roles of ClpP and ClpX in SOS- and prophage induction, we aimed to investigate their specific functions in prophage induction. We hypothesised that the reduced prophage titres observed in the Δ29 and Southern blotting experiments before and after MitC induction of RN450, RN450 ΔclpX (JP18031) or RN450 ΔclpP (JP18030) lysogenised with either Φ11 or 80α , the prophage left attachment site  or the phage attachment site  to the appropriate reference genomes or circular prophage sequences. MitC induction of the wt strain background reduced the proportion of bacterial chromosomes lysogenised with either prophage (Table attP) reads for Φ11 and a 257-fold increase for 80α  showed increased levels of circular, excised phage DNA compared to the wt strain prior to MitC induction (~13.2-fold for Φ11 and 3.4-fold for 80α)  , indicating full integration of the prophage in all bacterial genomes. Similar observations were made for the ΔclpP mutant Φ11 lysogen, where only 1.8% (uninduced sample) or 3.5% (induced sample) of reads were mapped across the attB sites.By contrast, the Δ0α) Fig. , where aype Fig. . NonetheclpX mutant lysogens, read coverage remained relatively constant and did not increase substantially after MitC induction (1.67-fold for Φ11 [JP18157] and 1.13-fold for 80α [JP18169] compared to uninduced), indicating no excision of the prophage . Interestingly, we still observed some reads mapping to the excised phage attP sites indicating that some excision was still possible in these strains. However, the total number of normalised reads in the ΔclpX mutant lysogens mapping to the attP sites was similar to the uninduced wt in the case of Φ11 and around 30% for 80α lysogens, always lower than those observed for either phage in the ΔclpP mutant lysogens. This suggests the possibility of spontaneous prophage induction, which will be further investigated later.Importantly, in the ΔattL read coverage compared to the uninduced strain, indicating in situ replication of the prophage prior to excision29, we did not observe any notable increase in attL coverage in either the ΔclpP or ΔclpX mutant Φ11 lysogens. Since endogenous replication does occur independently of prophage excision but requires prophage induction, this provided additional support for both ClpP and ClpX acting prior to replication and excision from the chromosome. Unfortunately, we were unable to verify this observation in the 80α lysogen backgrounds due to the slower kinetics of induction and endogenous replication, which were not covered in these experiments29.Contrary to the induced Ф11 lysogen in the wt background, where we observed a 6.2-fold increase in ori) as controls for phages capable of excision but not replication and generated ΔclpX and ΔclpP mutants in these strain backgrounds. If ClpX and/or ClpP were involved in prophage induction  or excision (excisionase is only expressed after CI degradation33), these mutants would not show any changes in prophage sequence coverage before and after SOS induction in the ori mutant lysogens. Conversely, if either ClpX or ClpP were involved in phage replication, sequence coverage in the mutant strains in the ori mutant lysogens would resemble the behaviour of the ori mutants in the wt RN450 strain background, and MitC induction would lead to the loss of sequence coverage.To fully confirm whether ClpP and ClpX targeted the core repression module rather than replication, packaging and excision, we also included two phage mutants in Φ11 and 80α defective in their origins of replication , we observed an appreciable drop in read coverage in the prophage region of the induced ori mutants in both Φ11  and 80α  , potentially indicating differences in the ability of the phage to reintegrate into the bacterial chromosome after excision. This difference was less evident in the 80α Δori mutant (63.96% compared to 69.7% for the wt strain) and might reflect differences in the speed of 80α derepression and/or initial replication compared to Φ11.In the wt RN450 strain background lysogenic for the Φ11 and 80α ori-containing gene in either Φ11 or 80α in the ΔclpP mutant background  eliminated the increased read coverage for the prophage observed with the ΔclpP single mutant prior to MitC induction. Furthermore, no difference in attL site coverage was observed after MitC induction with over 93 and 96% of the bacterial chromosomes being lysogenic for Φ11 or 80α, respectively  showed no differences in sequence coverage, with or without MitC induction . Together, these data provide strong evidence that both ClpP and ClpX are required for prophage induction and act prior to prophage in situ replication and excision.Deletion of the cro promoter was fused to a β-lactamase reporter gene , (ii) a non-degradable version  insensitive to SOS induction34, and (iii) a construct expressing only the post-cleavage N-terminal domain of CI . This last construct mimics the N-terminal fragment of CI after SOS-induced cleavage and allowed us to study the ability of this fragment to block phage induction as well as to assess the impact of the ΔclpX (JP20999) and ΔclpP (JP19795) deletions on its processing.The results presented in Figs. wt reporter plasmid (JP19910) repressed reporter gene expression, which increased by approximately 36-fold upon MitC addition  showed no induction by MitC, indicating its SOS insensitivity. Interestingly, the CIG131* reporter plasmid (JP19911) displayed higher expression levels even in the absence of MitC , suggesting that the N-terminal domain of CI alone was insufficient to repress reporter expression in the wt strain background.We introduced these reporter plasmids into the wt RN4220 strain and monitored the expression of the β-lactamase reporter gene with or without MitC induction. The CIclpP mutant background and repeated the experiment. The ΔclpP mutant carrying either the CIwt reporter plasmid (JP19918) or the CIG131E reporter plasmid (JP19920) showed no expression either with or without MitC induction. However, the CIG131* reporter plasmid in the ΔclpP background (JP19919) exhibited high β-lactamase expression regardless of MitC presence  as well as the plasmid expressing CIG131E (JP19916) were fully repressed either with or without the addition of MitC, showing only background levels of reporter gene expression  was repressed in the ΔclpX mutant background and this repression could not be relieved by MitC addition  confirming that (i) the CIG131* NTD post-cleavage fragment was still able to bind and repress the cro promoter and that (ii) ClpX was required for abrogating this repression.When we introduced the reporter plasmids into the Δwt JP1991 as well wt JP1991 as well G94E protein (JP1841). This mutant is no longer able to induce the bacterial SOS response, particularly its further amplification through derepression of RecA. RecA levels and consequently RecA* levels therefore remain under the threshold required for the initial RecA*-mediated CI cleavage step in prophage activation. As expected, the CIwt-expressing plasmid could no longer be induced by MitC induction in this strain background (JP20406), confirming that autocatalytic cleavage of CI could no longer be triggered by RecA* present in this background. Logically, this was also the case when introducing the SOS-insensitive CIG131E reporter into the LexAG94E strain background (JP20407). However, no changes compared to the wt strain were observed with the CIG131* reporter plasmid (JP20408)  into the cadmium-inducible expression vector pCN51 . These plasmids were introduced into either the RN450 wt, its ΔclpX or ΔclpP mutant derivatives and the ability of Φ11 to infect these strains was assessed  or ΔclpX (JP19536) mutant strains were equally susceptible to infection with Φ11  or the non-degradable CIG131E (JP19530), phage infection was almost completely blocked, resulting in a ~7-log reduction in phage titres. By contrast, the overexpression of the CIG131* NTD (JP19531) in the wt strain remained susceptible to phage superinfection comparable to the empty plasmid control strain.To test this, we cloned the different clpP mutant background, the overexpression of CIwt (JP19537) and CIG131E (JP19538) resulted in complete blockage of Φ11 superinfection (~7-log reduction), while the overexpression of CIG131* (JP19539) led to a 10-fold reduction in phage titres  and CIG131E (JP19534) effectively blocked superinfection (~7-log reduction). Interestingly, consistent with the central role of ClpX in the removal of the CI-NTD, the overexpression of CIG131* in the ΔclpX mutant background (JP19535) resulted in a reduction in phage titres by more than 5-log units  or the I265E substitution variant (ClpXI265E) into the cadmium-inducible expression vector pCN51 . These plasmids were then introduced into the ΔclpX mutant lysogens of Φ11  and 80α .ClpX functions both as a substrate specificity protein, directing target proteins to the ClpP protease, and as a chaperone, assisting in correct protein foldingwt) in the ΔclpX mutant fully restored wt phage titres, expression of ClpXI265E also restored phage titres to 1.5-logs below wt levels  and the ClpXwt or ClpXI265E mutant as N-terminal fusion product to the T18 fragment of the B. pertussis adenylate cyclase in plasmid pUT18c  rather than the full-length CIwt protein. Moreover, the binding of ClpX to the N-terminal fragment alone is sufficient to trigger phage induction, independent of ClpP-mediated proteolytic degradation.Our previous findings demonstrated that the CI-NTD cleavage fragment alone retained repressor functionality and could block prophage induction and infection in the absence of ClpX. Furthermore, ClpX, but not ClpP, was essential for relieving this repression, suggesting that ClpX could interact with the N-terminal CI fragment but not the full-length protein. To investigate this further, we used a bacterial two-hybrid system, where we cloned CINTD Fig. , confirm31* Fig. . CollectrecA mutations or the expression of an insensitive LexA protein, there is always some basal induction of resident prophages41. Hence, we sought to investigate whether the ΔclpP and/or ΔclpX mutants had any influence on spontaneous phage release. To assess this, we cultured the wt, ΔclpP, and/or ΔclpX mutants lysogenic for Φ11 or 80α, following the same experimental procedure as with MitC induction but without adding MitC. The released phage titres were then determined by plating on a lawn of RN4220, and the results were normalised to the phage release from the wt background to account for inter-experimental variability.The results presented in Fig. clpX mutants were the result of spontaneous, ClpX-independent induction events. Therefore, both ClpP and ClpX proteins exerted distinct effects on spontaneous prophage release, but with opposite effects.Our findings revealed that the loss of ClpP led to a significant increase in phage release for both Φ11 1.75-fold) and 80α (8.39-fold) lysogens , Lon (N-protein and Xis), and FtsH (CII and CIII)4. Presently, only the Mu repressor protein, which is different to LexA-like CI repressors, has been shown to be subject to ClpXP-mediated degradation. Even though the precise mechanism of this induction is still unknown, it is thought that a C-terminal ClpX-recognition motif that is present but conformationally inaccessible in the repressed state can be exposed by environmental triggers and thus lead to the degradation of the Mu repressor and subsequent activation of the Mu transposase48. The dual roles of the ClpX protein therefore necessitate the precise determination of whether the biological effects caused by ClpX are mediated via its chaperone or protein degradation function.ClpX specifically interacts with the CI-NTD after SOS-induced cleavage and not with the full-length CI proteins. Furthermore, the use of a ClpX protein unable to shuttle substrates for proteolytic degradation but able to perform its chaperone functionclpP mutant to induce the SOS response, we still observed higher lysis, phage replication and higher phage titres compared to a clpX mutant, where phage replication was almost completely absent. Interestingly, loss of clpP gave rise to a faint phage replication band during Southern blot analysis, to increased sequence coverage and attP circular phage site reads coverage during whole genome sequencing of the prophage region even in the absence of MitC induction. This further translated into an increased spontaneous release of phage particles without MitC induction and indicates that ClpP alone or in combination with other proteins performs (an) additional role(s) either required for full prophage stabilisation in the host’s chromosome or in preventing erroneous induction events. This could be facilitated through interaction/degradation with/of other regulatory phage proteins modulating the lytic switch. Alternatively, loss of ClpP or loss of the ability of ClpX to interact with ClpP, could liberate additional ClpX which is able to interact with CI NTD fragments generated by self-processing at low rates31. Note that each ClpXP complex contains a heptameric ClpP and hexameric ClpX ring25. This would reduce the cellular pool of available full CI proteins and NTD-CI fragments that could repress the prophage. A reduction in the cellular repressor pool concentration could then result in an increased likelihood of prophage derepression in individual cells, and consequently produce elevated spontaneous phage release. This model is supported by the Southern blot phenotypes of the ClpXI265E mutant strain which mimics that of the ΔclpP mutant and produces a faint replicating phage band even without MitC induction . Thus, cumulatively, sufficient RecA* might be produced to result in a degree of prophage derepression despite an inability to induce the SOS response. By contrast, ClpX was important for spontaneous prophage release and its deletion resulted in a > 2-log reduction suggesting that its role in spontaneous and SOS-mediated prophage induction might be analogous. Because MitC-induced phage titres and spontaneously-induced phage titres in ΔclpX mutant were identical  at 37 °C and 120 rpm or on Tryptic soy agar (TSA) plates supplemented with sodium citrate (1.7 mM). E. coli strains were grown in Lysogeny broth (LB) 37 °C and 180 rpm and on LB agar plates. Antibiotic selection was used where appropriate (10 or 2.5 μg ml−1 erythromycin (as indicated), 100 μg ml−1 ampicillin and 30 μg ml−1 kanamycin).The bacterial strains used in this study are listed in Table 540~0.15) at 37 °C and 120 rpm. 12 ml of this culture were pelleted by centrifugation  and the pellet was re-suspended in 6 ml of fresh TSB and 6 ml of phage buffer . Note that the phage buffer provides MgSO4 and CaCl2 required for phage adsorption. Equal numbers of phages (~108 PFU) were added to each culture and infected cultures were incubated for 4 h at 30 °C and 80 rpm followed by overnight incubation at room temperature. Cleared lysates were filtered using a 0.22 µm polyethersulfone syringe filter .Test strains were grown in TSB to the early exponential phase  at 37 °C and 120 rpm. Cultures were then induced by the addition of mitomycin C (MitC) (2 µg ml−1) and incubated for 4–5 h at 30 °C, 80 rpm followed by overnight incubation at room temperature before filtering using a 0.22 µm polyethersulfone syringe filter . To determine the phage titres, RN4220 cultures were grown to OD540~0.35 and 100 µl of this culture were mixed with 100 µl of serially diluted phage lysates in phage buffer . The mixtures were incubated for 5 min at room temperature, then 3 ml of molten phage top agar  was added and the mixture overlaid onto phage base agar plates . Plates were incubated overnight at 37 °C and the plaque forming unit (PFU ml−1) was determined.540~0.35 and 100 µl of this culture were mixed with 3 ml of molten phage top agar  and the mixture overlaid onto phage base agar plates . Serial dilutions of the prophage to be lysogenised in phage buffer  were spotted as 10 μl spots onto the recipient layer and dried. Plates were incubated overnight at 37 °C and a plaque forming unit (PFU ml−1) was determined. Microcolonies of recipient strains in low dilutions of phage spots were selected and streaked onto fresh TSA plates supplemented with sodium citrate (1.7 mM) and antibiotics where required. Single colonies were restreaked several times onto fresh plates and the presence of prophage lysogens was established by MitC induction (where possible) and PCR using primers Sa5-F and Sa5-R to confirm Φ11 lysogens and Sa7-F and Sa7-R for 80α lysogens49.Recipient strains were grown to ODS. aureus strains carrying the required plasmids were grown in TSB supplemented with the relevant antibiotics to an OD540~0.35 and 100 µl of this culture were mixed with 3 ml of phage top agar  and overlaid onto phage base agar plates . Phage lysates and dilutions in phage buffer  were spotted in triplicates of 10 µl each onto lawns of the specified strains, dried and incubated overnight at 37 °C prior to plaque forming unit (PFU ml−1) determination.Plasmid constructs used in this study Table  were gen50 using oligonucleotide primers described in Table −1 X-gal and 2.5 µg ml−1 erythromycin. Single crossover events were isolated (light blue colonies) and grown overnight under replication-permissive conditions  to facilitate excision and loss of the integrated plasmid. Serial dilutions of the cultures were plated on TSA plates supplemented with 80 µg ml−1 X-gal and correct deletion mutants identified by PCR followed by sequencing using oligonucleotides annealing outside of the recombination region and indicated in Table The required clean deletion mutants were constructed using allelic replacement by cloning flanking regions up- and downstream of the respective gene (0.5−1 kbp) into pMAD540~0.15) in 10 ml of TSB supplemented with antibiotics where plasmids were present. Phages were induced with MitC (2 µg ml−1) and, where pCN51 expression plasmid derivatives were present, 1 µM CdCl2 was added to induce expression as indicated. One ml samples were taken at the defined time points, pelleted by centrifugation  and pellets shock frozen on dry ice. The pellets were re-suspended in 50 µl lysis buffer  sucrose) and 2.5 µl lysostaphin [12.5 µg ml−1]) and incubated at 37 °C for 1 h. Following the incubation, 55 µl of SDS 2% proteinase K buffer  was added before incubation at 55 °C for 30 min. Samples were vortexed for 1 h with 11 µl of 10× loading dye followed by three cycles of 5 min incubations in dry ice/ethanol and at 65 °C in a water bath. Samples were run on 0.7% agarose gel at 25–30 V overnight. DNA was transferred by capillary action to a positively charged nylon membrane (Roche), processed as per the manufacturer’s instructions, and exposed using a DIG-labelled probe (see DNA methods) and anti-DIG antibody , Roche, catalogue number 11093274910) before washing and visualisation.Strains containing the defined phage and plasmids were grown to early exponential phase  system and plated on LB supplemented with ampicillin (100 µg ml−1), kanamycin (30 µg ml−1), X-gal (20 µg ml−1) and IPTG (100 µM). After incubation at 30 °C for 48 h (early reaction) to 72 h (late reaction), the protein–protein interaction was detected by a colour change. Blue colonies represent an interaction between the two clones, while white/yellow colonies are negative for any interaction.The two-hybrid assay for protein–protein interaction was performed as described previously−1 erythromycin and incubated at 37 °C, 120 rpm for 16–18 h. The cultures were then diluted 1/50 in 13 ml of fresh TSB supplemented with 10 µg ml−1 erythromycin and grown at 37 °C and 120 rpm to early exponential phase (OD540~0.15–0.2). 200 µl of culture was added directly to 800 µl of potassium phosphate buffer  and frozen on dry ice. Where SOS induction of the cro promoter was assessed, cultures were split in two (6 ml each), induced either with or without MitC (2 μg ml−1) and incubated at 30 °C, 80 rpm until sampling as described above. β-lactamase assays, using nitrocefin as substrate, were performed as described54: 50 µl of the collected sample were mixed with 50 µl of nitrocefin stock solution , and immediately reading the absorbance at 490 nm using an ELx808 microplate reader (BioTek) for 30 min. Promoter activity was calculated as Promoter activity = (dA490/dt(h))/(OD540 × d × V), where OD540 is the absorbance of the sample at OD540 at collection, d is the dilution factor, and V is the sample volume. Note that only the linear segment of the resulting absorbance readings is considered for activity calculations.For the β-lactamase assays, overnight cultures were prepared by inoculating a single colony from each strain into a 5 ml TSB supplemented with 10 µg ml(OD540~0.15–0.20) to collect samples before induction. Next, the cultures were treated with MitC (2 μg ml−1) and incubated at 30 °C and 80 rpm for 60 min prior to sample collection. One ml samples were collected, and genomic DNA was extracted using the GenElute Bacterial DNA kit (Sigma Aldrich) according to the manufacturer’s instructions. The DNA was precipitated by adding 10% (v/v) 3 M sodium acetate (pH 5.2), 2.5 volumes of 100% ethanol and incubation of the mixture for 1 h at −80 °C. The DNA was then pelleted at 16873 × g for 30 min at 4 °C and washed once with 1 ml of ice-cold 70% (v/v) ethanol. After centrifugation, the DNA pellets were air-dried for 30 min and re-suspended in 25 μl of TE buffer . Quality control of DNA samples was tested using Agilent Bioanalyzer 2100 and whole genome sequencing (WGS) was performed at the University of Glasgow’s Polyomics Facility using Illumina TruSeq DNA Nano library prep, obtaining 2 × 75 bp pair end reads with DNA PCR free libraries.Overnight cultures were prepared by inoculating a single colony from each strain into a 5 ml TSB and incubated at 37 °C, 120 rpm for 16–18 h. The desired strains were then diluted 1:50 in 13 ml of fresh TSB and grown to exponential phase https://www.ncbi.nlm.nih.gov/nuccore/87201381]) reference genome using the PATRIC Bioinformatics Resource Center55. The three prophages of NCTC8325 were deleted and any mutations identified were curated manually. Sequencing reads were then reassembled to the curated genome (GenBank Accession CP097113 [https://www.ncbi.nlm.nih.gov/nuccore/CP097113]) as described above for verification. The sequences of either Φ11 (RefSeq Accession NC_004615 [https://www.ncbi.nlm.nih.gov/nuccore/NC_004615]) or 80α (RefSeq Accession NC_009526 [https://www.ncbi.nlm.nih.gov/nuccore/NC_009526]) were inserted into attachment sites 5 and 749, respectively and correct insertion verified by assembly of genome sequencing reads for strain JP18269 (GenBank Accession CP097114 [https://www.ncbi.nlm.nih.gov/nuccore/CP097114]) or JP18270 (GenBank Accession CP097115 [https://www.ncbi.nlm.nih.gov/nuccore/CP097115]) for Φ11 or 80α lysogens, respectively. The curated genomes were next uploaded to the Galaxy web platform, and we used the public server at usegalaxy.org to analyse the data56. Genomes were reannotated using Prokka v1.14.658 .Genomic DNA of the RN450 (NCTC8325-4) reference stock in the lab (JP1250) was extracted and sequenced as described above. Next, reads were assembled to a scaffold of the deposited NCTC8325  followed by adapter trimming using Trimmomatic v0.3860  and standard setting for paired-end reads and Illumina data. Trimmed reads were then reassessed using FastQC and mapped to custom genomes of RN450 reference genome containing either prophage Φ11 or 80α using the Burrows-Wheeler Alignment Tool v0.7.17.462  with default settings and saved as BAM files. To normalise sequence coverage across experiments, we first filtered the aligned reads mapping to the bacterial chromosome and not belonging to the prophage using the BAMTools v2.4.0 Filter tool63 . The number of mapped reads for each experiment was extracted from the filtered BAM files using the SAMTools stats utility v1.964 . Average genome coverage was calculated using the following formula: average genome coverage = (number of mapped reads) × (average read length (bp))/(genome length (bp)). Next, we computed the relative coverage over 50 bp sliding windows along the entire chromosome without normalisation for each of the experiments of the unfiltered BAM files using the bamCoverage tool of the deepTools2 package v3.3.265 . These coverage files were saved in bedgraph format and further analysed using RStudio v2021.9.1.372 (Ghost Orchid)66 and R v4.1.267. Samples were normalised by dividing each coverage window by the average genome coverage calculated for each experiment. Final coverage graphs were plotted in RStudio using ggplot2 v3.3.568 and genome organisation around the plotted area extracted from the gff3 file produced by the Prokka annotation and graphed using gggplot2 and the gggenes package v0.4.169.The sequencing data were uploaded to the Galaxy web platform, and we used the public server at usegalaxy.org to analyse the data62 to determine read coverage of the bacterial attB, the prophage left attachment site attL and the excised phage attP sites, respectively. Reads around the relevant attachment site (±800 bp), which had a matching mate read were extracted using the Samtools view64 command in Galaxy. The reduced dataset was then further filtered in Rstudio (for filtering scripts, see att site by the average genome coverage calculated for each experiment. The fraction of integrated prophage was calculated by dividing the reads mapping to attL by the sum of the reads mapping to both attL and attB. The fraction of circularised and excised phage was calculated by dividing the reads mapping to attP by the sum of the reads mapping to attP and attL.Reads were mapped onto either the prophage-free, prophage-containing or circularised phage genomes using BWA10-transformed and analysed by either One-Way ANOVA followed by Tukey’s HSD post-test or using a Student’s unpaired two-tailed t test as appropriate for the relevant comparison. Promoter activity data were analysed on raw activity data by either One-Way ANOVA followed by Tukey’s HSD post test and Bonferroni correction or using a Student’s unpaired two-tailed t test as appropriate for the relevant comparison. All analysis was done using RStudio.Statistical analysis was performed as indicated in the figure legend. In general, phage titres were logFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of additional supplementary filesSupplementary SoftwareReporting SummarySource data


In particular, melting in a layer-by-layer manner rather than random nucleation at the early stage was identified as a notable finding. Emphasizing the value of direct atomistic observation, our study suggests that lattice defects inside crystals should not be overlooked as preferential nucleation sites for phase transformation including melting.Since two major criteria for melting were proposed by Lindemann and Born in the early 1900s, many simulations and observations have been carried out to elucidate the premelting phenomena largely at the crystal surfaces and grain boundaries below the bulk melting point. Although dislocations and clusters of vacancies and interstitials were predicted as possible origins to trigger the melting, experimental direct observations demonstrating the correlation of premelting with lattice defects inside a crystal remain elusive. Using atomic-column-resolved imaging with scanning transmission electron microscopy in polycrystalline BaCeO 3 crystals below the melting temperature in a layer-by-layer manner.No experimental observations have been reported to clarify how a melting transition proceeds inside a crystal. Here the authors demonstrate that melting is initiated at two-dimensional faults inside BaCeO In particular, as reviewed in ice12, this local loss of crystallinity followed by the subsequent formation of a liquid layer below the bulk melting temperature is called premelting of a crystal. While numerous studies have been reported to elucidate the notable features during crystallization of various materials, few attempts at experimental observation on melting at atomic-column resolution in inorganic crystals have been made since the early empirical work by Lindemann in 1910 based on the atomic mean square displacement as a criterion of melting13.A crystalline solid melts into a liquid phase, showing typical first-order phase transformation behavior of significant discontinuities in the thermodynamic variables, such as molar volume and entropy, at the melting point. As in other phase transformations via a heterogeneous nucleation process, it has been accepted that melting is preferentially initiated at the crystal surfaces6. As a result, experimental evidence of whether lattice defects inside a crystal indeed act as preferential sites for premelting remains elusive, even though Frenkel theoretically suggested that the formation of vacancies and interstitials is required for melting in a crystal14. Moreover, unless the defect sites in the crystal lattice are clearly visualized, it is considerably challenging to trace where the melting begins and how it evolves further. In addition, computer-aided simulations and experimental investigations on crystal melting have been largely performed in single-component materials to avoid complexity stemming from multiple compositions and sophisticated crystal structures19. Consequently, no atomistic details on the premelting have been reported in complex ionic crystals.Direct imaging and dynamic simulations of melting have several limitations. First, most observations of melting have been confined to the interfaces encompassing surfaces and grain boundaries3 as a model oxide in this work, we provide the atomic-scale experimental evidence of premelting at lattice defects inside oxide crystals. This observation thus demonstrates that the lattice defects inside a crystal are significant origins of the vibrational and elastic instability triggering melting, as suggested by both Lindemann and Born criteria20. To scrutinize the premelting aspects at an atomic level, the selection of defect sites is of great significance for successful and straightforward observation. We thus exploit two-dimensional (2D) homologous faults, which are not interfaces (such as surfaces and grain boundaries) but rather are internal lattice defects consisting of two consecutive [BaO] layers along the {001} planes inside the crystal, as will be described in more detail in Fig. 2] layers. We also identify that melting further proceeds along with the atomically rough interface between the crystal and the liquid layer as the annealing temperature is elevated. Experimentally verifying the crucial role of lattice defects in premelting inside a crystal at atomic resolution, our work highlights the value of direct probing to unveil the phase transformation behavior in sophisticated condensed-matter systems.Dealing with perovskite-type BaCeO3-type materials21, 2D homologous Ruddlesden−Popper (RP) faults of [AX]−[AX] interleaved layers along the [100] direction in a cubic framework are frequently observed in a variety of perovskite oxides and halides27. Figure 3 crystal from a polycrystalline sample sintered at 1330 °C. A pair of enlarged images for the location denoted by a white rectangle is also provided in Fig. Z) contrast between Ba (ZBa = 56) and Ce (ZCe = 58) in the HAADF mode is considerably low. Therefore, it is somewhat difficult to readily recognize the presence of two consecutive [BaO] layers in the magnified HAADF images at a first glance in Fig. 28 in STEM straightforwardly confirms the formation of RP faults. As shown in the EELS map in Fig. 32 is also provided in Supplementary Fig. As recently visualized in many ABX3. As illustrated in Fig. 3 perovskites can be considered a face-centered-cubic (FCC) derivative structure in which the large A cation ion and oxygen together form an FCC lattice. The smaller B cation occupies the octahedral interstitial sites, having six oxygen anions as the nearest neighbors, while the large A cation locates in the dodecahedral sites. Consequently, the bond length of Ba−O is larger than that of Ce−O. As can be seen in the density of states (DOS) of BaCeO3 in Fig. p (black curve) and Ce 4d (red curves) states in the {CeO6} octahedra is identified below the Fermi level (EF = 0 eV) in the valence band, whereas the Ba 5p states are located far below the O 2p states with no overlapping. This indicates a substantial degree of covalent bonding in Ce−O in contrast to Ba−O. Indeed, the significant covalency between Ce and O can be visualized when the distribution of the electron density at each atom is acquired by density functional theory (DFT) calculations. Figure 3 supercell containing an RP fault. As clarified in the magnified contour maps, there is not a notable distribution of electron density between Ba (sky blue) and O (gray) at the fault plane (lower map), while a high density of electrons between Ce and O can be found (upper map). Consequently, unless Ba atoms are surrounded by stable [CeO6] octahedra, the Ba−O bonds at the RP fault plane may be easily breakable, and thereby premelting at these defect sites is anticipated to occur.Prior to specific STEM observations, it is necessary to understand the crystal structure and the different nature of chemical bonding between Ba−O and Ce−O in BaCeO3 for application as proton conducting electrolytes37 usually ranges from 1400 to 1650 °C below the melting point, ~1740 °C39. We thus prepared samples for STEM observations via post-annealing of the polycrystals at 1400, 1500, and 1600 °C, respectively, in order to systematically track the structural variations at the RP faults with temperature . This directly indicates that the [CeO2] layers begin to melt in addition to the complete melting of the [BaO]−[BaO] layers. However, when melting proceeds further and thereby the disordered liquid layers become thicker, the distinct layer-by-layer melting behavior was no longer clearly observable. As can be seen in Image IV  is readily observable in Figs. ers Fig.  and furters Fig. . Showing42 to induce crystallization during STEM observation. Figure The disordered liquid layers at the fault planes are amorphous after quenching to room temperature  of the lattice constant13. As the vibrational amplitude of atoms at the surface should be much more substantial than that of the bulk atoms, surface melting prior to the bulk melting point was suggested. On the other hand, in 1939 Born proposed a distinct model of melting in terms of a rigidity catastrophe caused by the shear modulus vanishing20. In a milestone work in 1946, Frenkel further specified the necessity of intrinsic defects, encompassing vacancies and interstitials, for melting in addition to the Born criterion based on the elastic lattice instability14. Through a combination of ultrafast electron diffraction technique43 and molecular dynamics (MD) simulations, a recent study19 indeed showed that radiation-driven vacancy clusters in tungsten films can act as preferential sites for the melting transition below the melting point in accordance with Frenkel’s suggestion. In addition, using Lennard−Jones particles in an FCC box with MD simulations, Jin et al. in 2001 discovered that the Lindemann and Born criteria both lead to the same conclusion for the mechanism of melting when applied to the bulk crystal instead of the surface45. The report by Jin et al. has an important implication, as it clarifies that the accumulation of internal lattice instability constitutes the primary mechanism for melt nucleation inside the crystal. In this respect, we believe that the STEM images for BaCeO3 in Figs. In 1910, Lindemann proposed that melting is triggered by a vibrational instability in a crystal when the root-mean-square displacement of atoms, 〈Δ48. For a direct comparison, we carried out additional STEM analyses on more than 50-grain boundaries in a polycrystalline sample annealed at 1400 °C. As can be seen in Supplementary Fig. Although the present study focuses on premelting initiated inside a crystal, it is worthwhile to compare our observations with previous results regarding grain-boundary complexions, which encompass various transitions of faceting, order−disorder, wetting, premelting, and adsorption at grain boundaries as a general concept3-type perovskite oxides on the formation of an amorphous layer at grain boundaries. As demonstrated in SrTiO3, a small deviation from the complete stoichiometry of Sr:Ti = 1:1 results in the formation of a secondary liquid phase at high temperature49  in WSe2/WS2 moiré heterostructures; atomic-scale visualization of vortex-type ferroelectric nanodomains57 ) in PbO3 and BiFeO3 thin films; and in-situ imaging of a multi-phase transformation60  and thus dubbed the Ostwald rule of stage63) during crystallization of amorphous LiFePO4 are noteworthy examples emphasizing the significance of direct observation in the relevant fields. The STEM imaging in the present study is another impactful result that proves valuable classical predictions that have yet to be atomic-scale direct evidence in crystals.Finally, we note that experimental direct observations often play a critical role in both validating theoretically suggested physical phenomena and elucidating details that cannot be entirely covered by predictions. Recent observations on the 2D Wigner electron crystal3 crystals as a model system. In addition to identifying the thickness increment of the disordered liquid layers with annealing temperature, we successfully captured atomic-level images demonstrating the layer-by-layer behavior at the initial stage of melting. The experimental findings in this study clarify the significance of lattice defects present inside a crystal as major sources of the vibrational and elastic instability originally proposed by the Lindemann and Born criteria.We have provided the atomistic details on premelting initiated at the lattice defects of 2D homologous faults inside BaCeO3 polycrystals, powders were synthesized via a conventional solid-state reaction method by using BaCO3  and CeO2 . A small amount (5%) of an acceptor-type dopant, Dy2O3 , was also added to facilitate densification during sintering. A stoichiometric mixture of the starting materials was ball-milled in high-purity alcohol for 24 h with a zirconia jar and balls. After being dried, the slurry was calcined at 1050 °C for 12 h in the air for solid-state synthesis. The calcined powder was ball-milled again to obtain fine particles. The synthesized powder was slightly pressed into disks and isostatically pressed under 200 MPa. The disk-type pellets were sintered at 1330 °C for 5 h in the air to verify that no premelting occurs. For the post-annealing process, the sintered samples without the acceptor dopant were annealed again at 1400, 1500, and 1600 °C for 5 h, respectively, and rapidly removed from the furnace at a cooling rate of 300 deg/min to room temperature. Pellets with the dopant were sintered at 1400 and 1500 °C to observe grain-boundary premelting and measure the thickness distribution of an intergranular amorphous phase at gran boundaries as well. To examine the crystal surface, particles synthesized by the solid-state reaction were annealed at 1400 °C for 3 h.To fabricate BaCeOα peak overlaps with the Ba-Lβ1 peak in EDS. Therefore, the Ba-Lα and Ce-Lβ1,2 peaks were selected for atomic-column-resolved composition mapping by EDS. EELS analysis was performed with a Gatan Image Filter . Electron energy-loss spectra for the Ba-M5 (781 eV) and Ce-M5 (883 eV) edges were acquired for atomic-column spectrum imaging of BaCeO3 with a dispersion of 0.25 eV per channel and a collection aperture of 5 mm in diameter. To record a video clip during e-beam irradiation for recrystallization, the beam current was adjusted to be 100−150 pA with a pixel dwell time of 3 μs and thus the electron dose was estimated to be 2.8−4.2 × 105 electrons/Å2 s  in our conditions.The cross-sectional microstructure of fractured samples in addition to the overall polycrystalline microstructure was observed by scanning electron microscopy . Samples for STEM observation were prepared by mechanical grinding to a thickness of 60 μm, dimpling to a thickness of <10 μm and finally ion-beam thinning by using a precision ion polishing system  for electron transparency. STEM images were acquired with a transmission electron microscope  at 300 kV with a spherical aberration (Cs) corrector (CEOS GmbH). The optimum size of the electron probe was ~1 Å with a convergence semiangle of 19 mrad. The collection semiangles of the STEM detectors were set to 50.5−200 mrad for HAADF imaging and 0–27.5 mrad for BF imaging. Another Cs-corrected transmission electron microscope  was used at 200 kV. For phase-contrast HREM imaging of the particle surfaces, a conventional transmission electron microscope  was also utilized at 200 kV. Chemical composition mapping with EDS was performed in the Titan cubed G2 at 300 kV along with four integrated silicon-drift EDS detectors (ChemiSTEM™ technology) at a collection solid angle of 0.7 srad. The EDS maps were low-pass filtered using Bruker Esprit software after the reduction of background noise for better visualization. It is noted that Ce-L64 based on the multislice method. A beam energy of 300 kV, spherical aberration coefficients of Cs = 0 mm, C5 = 0 mm, and C7 = 0 mm without coma and astigmatism, an electron probe size (FWHM) of 1 Å, and a slice thickness of 2 Å were set during the simulations. Each of the real collection semiangles of the STEM detectors (50.5−200 mrad for HAADF imaging and 0–27.5 mrad for BF imaging) was also used for precise comparison with experimentally acquired images.STEM images were simulated by using Dr. Probe softwareSupplementary InformationDescription of Additional Supplementary FilesSupplementary Movie


Ovarian tissue transplantation can restore fertility in young cancer survivors, however the detrimental loss of follicles following transplantation of cryopreserved ovarian tissue is hampering the efficiency of the procedure. This study investigates whether needle puncturing prior to transplantation can enhance revascularization and improve follicle survival in xenotransplanted human ovarian cortex.N = 36) from 20 women aged 24–36 years were included. During the thawing process, each piece of tissue was cut in halves; one half serving as the untreated control and the other half was punctured approximately 150–200 times with a 29-gauge needle. The cortex pieces were transplanted subcutaneously to immunodeficient mice for 3, 6 and 10 days (N = 8 patients) and for 4 weeks (N = 12 patients). After 3, 6 and 10 days, revascularization of the ovarian xenografts were assessed using immunohistochemical detection of CD31 and gene expression of angiogenic factors , and apoptotic factors (BCL2 and BAX) were performed by qPCR. Follicle density and morphology were evaluated in ovarian xenografts after 4 weeks.Cryopreserved human ovarian cortex pieces , however, in the needle group this increase was not observed from day 6 to 10 (Ang2 p = 0.027). The BAX/BCL2 ratio was similar in the needle and control groups. After 4-weeks xenografting, follicle density  was higher in the needle group (5.18 ± 2.24) compared to control (2.36 ± 0.67) (p = 0.208), and a significant lower percentage of necrotic follicles was found in the needle group (19%) compared to control (36%) (p = 0.045).A significant increase in the CD31 positive area in human ovarian xenografts was evident from day 3 to 10, but no significant differences were observed between the needle and control group. The gene expression of Needle puncturing of human ovarian cortex prior to transplantation had no effect on revascularization of ovarian grafts after 3, 6 and 10 days xenotransplantation. However, needle puncturing did affect angiogenic genes and improved follicle morphology. Cryopreservation and autotransplantation of human ovarian cortex tissue has gained ground worldwide as the only fertility preserving option for prepubertal girls and patients in need of immediate cancer treatment –4. SinceThe massive follicle loss in the ovarian grafts is due to ischemia and hypoxia and the injury following reperfusion early in the post-transplantation period –24. MultN = 10), Hodgkin's lymphoma (N = 2), brain cancer (N = 1), sarcoma (N = 1), benign disease (N= 1), and other cancers (N = 5).Donated ovarian cortex tissue from 20 women aged 24–36 years  was included in this study. The ovarian cortex tissue was cryopreserved for fertility preservation using slow-freezing as previously described [Ten female immunodeficient Naval Medical Research Institute (NMRI)-NUDE mice aged 6–7 weeks were purchased from Taconic, Denmark. Mice were housed in groups, fed pellets and water ad libitum, and kept under controlled 12-h light/12-h dark cycles at 20–22 °C. One week after arrival the mice were anesthetized using Zoletil , xylazin , and butorphanol  before they were ovariectomized to increase endogenous FSH levels. Post-operative analgesia in the form of buprenorphine  and carprofen  was used. Two weeks after ovariectomy either six (4-week study) or eight pieces  of human ovarian cortex were transplanted subcutaneously to the back of each mouse. After surgery the mice were single housed for at least 1 week. Euthanasia was performed by cervical dislocation, upon graft retrieval.Thirty-six pieces of cryopreserved ovarian tissue was thawed according to standard clinical procedure . Each coN = 8; 2 cortex pieces from 8 patients, 8 xenografts per mouse). The 4-week setup was performed for follicle density evaluation because at this time point, atretic follicles has been reabsorbed and only surviving follicles permeate the ovarian xenografts . Grafts retrieved after short-term xenotransplantation on day 3, 6 and 10, were evaluated for gene expression by quantitative reverse transcription PCR (qRT-PCR) and revascularization by immunohistochemistry (IHC). Follicle counts and morphological assessment was evaluated in the 4-week xenografts.The experimental design is presented in Fig. Grafts were dissected from the mice and cleaned for surrounding murine tissue. Grafts retrieved after 3, 6 and 10 days were cut into half; one half for qRT-PCR was snapfrozen and stored at -80ºC, and the other half was fixated in 4% paraformaldehyde (PFA) for IHC analysis. The grafts retrieved after 4 weeks were fixed in Bouin’s solution for histological evaluation. The fixation was done overnight, then dehydrated and embedded in paraffin for sectioning. For IHC the samples were sectioned at 5 μm thickness, and for follicle evaluation the samples were sectioned at 10 μm thickness for Periodic-Acid-Schiff (PAS) staining.Vegfα),Angiopoietin 1 (Angpt1), Angiopoietin 2 (Angpt2), Angiopoietin-like 4 (Angptl4) and human B-Cell Lymphoma 2 (BCL2) and BCL-2 associated X protein (BAX), . Glyceraldehyde 3-phosphatdehydrogenase (Gapdh/GAPDH) was used as endogenous control for both murine and human gene expression assays [GAPDH, Hs02786624_g1; Gapdh, Mm99999915_m1). The cDNA samples were amplified in duplicates using the LightCycler®480 quantitative PCR instrument  as previously described [The human ovarian cortex tissue was homogenized, and RNA-isolation, reverse transcription and qRT-PCR was performed as previously described . In brien assays   primary antibody  diluted in 1% bovine serum albumin (1:100) was added to the slides and incubated overnight at 4 °C in a humid chamber. The following day the sections were incubated with the secondary antibody , washed and staining was visualized with 3.3′-diaminobenzidine tetrahydrochloride  and briefly dipped in diluted (1:3 water) Mayers Hematoxylin. IgG-negative controls were performed by excluding the primary and only using antibody dilution buffer. Digital pathology using Whole Slide Imaging (WSI) technology and the Visiopharm Author™ module was used to quantify the CD31-positive endothelial area and the number of vessels per mm2. The scanning of the slides was performed using the NanoZoomer S360 digital slide scanner with magnification × 40 and one focal point. Scanned WSI files were analysed in the Visiopharm Integrator System (VIS) using the APP via the Visiopharm Author™ module. The APP used has been described earlier [2), small vessels (300–1000 µm2) and large vessels (> 1000–3000 µm2). Heatmaps were generated for each section to visualize and highlight the CD31 positive areas in the ovarian tissue xenografts  . Gene expression, CD31 positive area (%), and follicle morphology data was log transformed, before analyzing data. All analyses comparing control and treatment with respect to gene expressions, CD31 positive areas, vessel densities, follicle densities and morphology were analysed with a linear mixed-effects model with patient as a random effect as each woman contributes with more than one sample in the study. Differences between individual groups were analysed using a Tukey post hoc test. 2. Furthermore, heatmaps was generated to make a visual representation of where the CD31 positive areas was located in the tissue  on day 3 was 10.26 ± 6.08 vessels/mm2 for the control group and 5.62 ± 3.47 vessels/mm2 for the needle group . There was a significant increase in vessel density between the different timepoints in both groups (p < 0.05).The vessel density (vessels/mm2), small vessels (300–1000 µm2) and large vessels (> 1000–3000 µm2). The distribution of between the subgroups was not different at any timepoint and the main proportion of vessels was micro vessels (97–88%). Table The APP subdivided the CD31 positive areas into categories according to size: micro vessels . The gene expression of murine Vegfα showed a tendency toward a higher expression in the needle group compared to the control group at all three timepoints . The expression of murine Angptl4 was similar on day 3 and 6 . The ratio of human BAX/BCL2 was similar at all three time points and there was no difference between the needle and control groups  .The xenografted ovarian tissue retrieved after 4 weeks was used to evaluate the morphology and the total number of follicles in each graft Fig. . A totalThis is the first study investigating if mechanical injury by needle puncturing could increase revascularization and hereby follicle survival in xenotransplanted human ovarian tissue. Current results showed no statistically significant differences in murine vessel density in human ovarian xenografts after needle puncturing compared to untreated control grafts. However, gene expression data did show differences in the expression patterns of angiogenic factors indicating an effect of the needle puncturing on the regulation of these genes. Furthermore, a higher follicle survival was observed in the needle group compared to control, although not statistically significant, and there was a significant difference in morphology, where the needle group had more morphological healthy follicles compared to control. Taken together our findings indicate that needle puncturing does not directly increase revascularization of ovarian xenografts, but it appears to affect the expression of angiogenic genes and has a positive effect on the follicle morphology.2 before day 3 in xenografted human ovarian tissue [BAX/BCL2, demonstrating that this is not a harmful procedure.Without vascular re-anastomosis, the tissue grafted is solely dependent on the development and invasion of new blood vessels. Xenografting studies has previously shown that the host is primarily responsible for revascularization of the xenografted human ovarian cortex . And resn tissue . After 6n tissue . These rn tissue  indicaten tissue . Our curVegf, did show a tendency toward a treatment effect on day 6, and there was a higher expression level in the needle group compared to control at the other time points. Moreover, there was a consistent and similar expression on all time points, showing that Vegfα is expressed in the human ovarian tissue early and consistently after transplantation. The expression of Ang1 and Ang2 had similar patterns, which was surprising due to their opposite nature. ANG1 and ANG2 act through the same tyrosine kinase receptor, Tie-2. They both have pro-angiogenic effects in the presence of VEGF, however they exert opposite effects upon receptor binding. ANG1 stabilizes the blood vessels formed through the actions of VEGF [Ang1 and Ang2 could be explained by the increase in angiogenic processes, however, Ang2 is also increased and important during inflammation and hypoxia [Ang2 expression from day 3, 6 and also day 10 for the control group. However, the same increase was not observed for the needle group, which is consistent with our previous results using the Er:YAG laser [Angptl4 was similarly expressed on day 3 and 6, but there was observed a decrease for both groups from day 6 to day 10, which could indicate the first pro-angiogenic period, where wound healing and revascularization is occurring stimulated by the hypoxic period, where on day 10 there may be a stabilization which leads to the decrease in Angptl4 expression. Taken together, the results indicate that the needle puncturing did affect the expression of angiogenic genes, and it illustrates the complexity of the revascularization process.The gene expression data of  of VEGF , whereas of VEGF . If VEGF of VEGF –57. More of VEGF . The obs hypoxia –61, whicAG laser . ANGPTL4AG laser –64, and AG laser . FurtherAG laser . Angptl4Current results showed significantly fewer atretic follicles and a tendency toward a higher follicle density in the needle group compared to the control group. Moreover, no signs of follicle activation were observed in the needle punctured tissue as the vast majority of follicles were primordial follicles (97%) which was similar to the control group (93%). These results is comparable to our previous study where the Er:YAG laser treatment resulted in a lower follicle survival, however, the proportion of follicles in the different stages did not differ and did not suggest follicle activation in the laser treated group compared to control . The effNeedle puncturing of thawed human ovarian tissue prior to transplantation was not able to increase revascularization in the human ovarian xenografts. However, needle puncturing did affect the expression of angiogenic genes and resulted in slightly higher follicle survival and significantly better follicle morphology in the ovarian xenografts compared to control. Current results call for further investigations of needle puncturing in the context of OTT.


By optically exciting the heterostructures with linearly polarized light close to resonance and measuring the helicity resolved photoluminescence, we obtain a degree of polarization of up to −7% and 8% in MoS2/right-handed (R-(+)-) and left-handed (S-(-)-) 1-(1-naphthyl)ethylammonium lead iodide perovskite, respectively. We attribute this to spin selective charge transfer from MoS2 to the chiral perovskites, where the perovskites act as a spin filter due to their chiral nature. Our study provides a simple, yet robust route to obtain room temperature valley polarization, paving the way for practical valleytronics devices.The two degenerate valleys in transition metal dichalcogenides can be used to store and process information for quantum information science and technology. A major challenge is maintaining valley polarization at room temperature where phonon-induced intervalley scattering is prominent. Here we demonstrate room temperature valley polarization in heterostructures of monolayer MoS Valleytronics is a portmanteau of ‘valley’ and ‘electronics’ and refers to the use of the valley degree of freedom, present in some materials, for encoding and processing information. Here, Shrestha et al demonstrate a room temperature valley polarization in heterostructures composed of molybdenum disulfide and a chiral lead halide perovskite, an important step in development of valleytronics. Two-dimensional (2D) materials with a honeycomb lattice such as transition metal dichalcogenides (TMDs) have two degenerate valleys, K and K′, at the corners of their hexagonal Brillion zone  which leads to valley-dependent optical selection rules2. Moreover, strong spin–orbit coupling due to the heavy metal atoms splits the electronic band by hundreds of meV3. Since time reversal symmetry requires that the spin splitting at the two valleys is opposite, spin and valley are coupled1 or locked. Therefore, to change an electron’s valley, a simultaneous flip in its spin is required which makes valley index in monolayer TMDs robust.Besides an electron’s charge and spin, the valley it occupies in momentum space can also be used to process and store information for classical or quantum computing. As the valleys are often well separated in momentum space, the valley index is robust against scatteringone Fig.  and are 8. The valley population in TMDs can be further manipulated using strain9, magnetic field11, or by adding plasmonic metasurfaces12. Moreover, taking advantage of the layered nature of TMDs, enhanced valley polarization due to charge transfer and other proximity effects in heterostructures consisting of TMDs14, other 2D materials15 and magnetic materials16 has also been demonstrated.Selectively populating a specific valley, or valley polarization which can be considered as writing or encoding data, is the first step towards valleytronics applications. The most common route used to obtain valley polarization in TMDs is via optical pumping with a circularly polarized light. Due to the valley-dependent optical selection rules, K and K′ valleys can be selectively populated with left (σ+) and right (σ−) circularly polarized light, respectively. Similarly, valley polarization can be read out by measuring the σ+ and σ− components of the emitted photoluminescence (PL)17. In these heterostructures, a chirality induced spin selectivity effect19 results in chiral perovskites acting as spin filters, preferentially injecting charges with one spin, depending on the handedness of the perovskite material. Since spin and valley in monolayer TMDs are coupled, the valley population is therefore controlled via selective spin injection. This elegant, yet simple way to control valley polarization does not require circularly polarized light, magnetic field, or complex device structure. However, the phenomenon was observed only at low temperature (78 K) and steadily decreased as the temperature increased, leaving no polarization at room temperature. In fact, the majority of studies mentioned here so far have been performed only at low temperatures16, as depolarization due to intervalley scattering becomes prominent at higher temperature20. Requirement of cryogenic temperatures makes these systems expensive, bulky, and complex. Achieving robust valley polarization in TMDs at room temperature is a challenge which needs to be addressed for practical applications of TMD-based valleytronics devices in quantum information science and technology.Valley polarization was recently demonstrated in heterostructures combining TMDs and 2D chiral lead halide perovskites2 and one-dimensional (1D) chiral lead halide perovskite R-(+)- or S-(-)-1-(1-naphthyl)ethylammonium lead iodide (R- or S-NEAPbI3). We obtain a degree of polarization of up to 8% by exciting MoS2 with a linearly polarized laser with energy close to A-exciton resonance, at ambient conditions. We attribute the ability to observe room temperature valley polarization to a stronger chirality of the 1D perovskite system versus earlier reported 2D perovskite together with resonant excitation conditions employed here. Our results demonstrate a simple and robust method to control room temperature valley polarization in TMDs, thus paving the way for practical valleytronics devices which could change the way we store and process information using conventional charge and spin-based electronic devices.Here we demonstrate room temperature valley polarization in heterostructures of monolayer MoS3 chiral lead halide perovskites using a slow cooling method23. Powder X-ray diffraction pattern of the crystals are consistent with literature22 -1-(1-naphthylammine). The organic chiral ligands form asymmetric hydrogen bonds with the inorganic framework and have been reported to transfer chirality via helical structural distortions22 and asymmetric electronic interactions25. In comparison to 2D chiral perovskites, where chiral ligands are located between 2D lead-halide sheets, the 1D chiral perovskites show a stronger chiroptical activity because the chiral ligands surrounding the lead-halide chains lead to a stronger distortion in the later systems27. The circular dichroism (CD) spectra of spin cast thin films of R- and S-NEAPbI3 in Fig. 26.We begin by synthesizing single crystals of R/S-NEAPbI30 (confirmation by atomic force microscopy (AFM) in Supplementary Fig. 2 flakes were mechanically exfoliated on Si/SiO2 substrate and monolayers were identified using AFM and Raman micro-spectroscopy  show that the chiral perovskites have a valence band maxima at −7.7 eV relative to the vacuum level . Therefore, in this work we focus on only MoS2 emission.We optically excited the heterostructure with a linearly polarized laser of 1.96 eV energy and measured the emitted photoluminescence (PL) in a confocal microscope (see “Methods” for details). As the chiral perovskite has a bandgap of 3.3 eV  heterostructure to the conduction band of chiral perovskite  defined as:I(σ+) and I(σ−) are the peak intensities of left and right circular polarized components of the PL emission, respectively. The DOP for monolayer MoS2, R-NEAPbI3/MoS2, and S-NEAPbI3/MoS2 are 0%, −7%, and 8%, respectively. A value of DOP of 0% obtained for monolayer MoS2 confirms that artifacts due to difference in transmission of σ+ and σ− of optical components of the confocal setup, such as mirrors and lenses, are negligible and validates our measurements. In Supplementary Fig. 3/MoS2 and S-NEAPbI3/MoS2 heterostructures which also showed DOP of −5 ± 1% and 6 ± 2%, respectively. Heterostructures transferred on hBN also demonstrated a DOP of −6%, confirming that the substrate does not affect the DOP  in comparison to the case when they are opposite (σ−σ+ or σ+σ−), as we pump and probe the same valley in the former case8. The DOP for σ+ and σ− excitation also have the same magnitude but opposite sign since the two valleys are symmetric. In R-NEAPbI3/MoS2 heterostructure, however, when K′ valley is selectively excited by σ−, σ+ emission from K valley is further reduced due to preferential transfer of spin up electrons from K valley to R-NEAPbI3. Therefore, the DOP for σ−(σ+) excitation is further enhanced while that for σ+(σ−) excitation is diminished in R-NEAPbI3(S-NEAPbI3)/MoS2 heterostructure in comparison to bare MoS2 monolayer. We note that besides the peak at 1.86 eV, the emission appears to have an additional peak at 1.89 eV. As we observed this signal also from SiO2 substrate measured under the same conditions, we attribute the peak to scattered laser leaking in through the filters  and measured the photo-induced changes in absorption of a white light continuum probe after a controlled time delay. Figure 2/S-NEAPbI3 heterostructure, with features at 1.8 and 2.0 eV corresponding to the A- and B-exciton transitions of MoS2, respectively38  and for the heterostructure MoS2/S-NEAPbI3 (τHet), respectively. We attribute the shorter carrier lifetime observed in the heterostructure to electron transfer from monolayer MoS2 to 1D chiral perovskite. Faster decays due to ultrafast charge transfer between TMDs and perovskites (as fast as 45 fs) have been previously reported43 from transient absorption and time-resolved PL measurements. We estimated a charge transfer rate, −1 and 27%45.We obtained direct evidence for a photoinduced charge transfer in the presented heterostructures by pump-probe transient absorption spectroscopy. We optically pumped the samples at 2.48 eV  electrons from K′ valley can recombine with holes radiatively, before being transferred to S-NEAPbI3; (2) electrons from K valley can be transferred to S-NEAPbI3 since the spin selectivity of S-NEAPbI3 is less than 100%; or (3) electrons from both K and K′ valleys can scatter to the opposite valley and depolarize. While depolarization due to intervalley scattering can be suppressed by lowering the temperature, requirements for cryogenic temperature would increase complexity and cost of the experiment. Alternative strategies can be adopted in the future to enable faster charge transfer and higher spin selectivity in such heterostructures. As hybrid lead halide perovskites offer large flexibility in terms of composition engineering, the energy band alignment in the heterostructure can be tuned to facilitate faster charge transfer. Similarly, more suitable chiral ligands could be designed to improve spin selectivity.In an ideal case, where there is good contact between the monolayer TMD/1D perovskite layers of the heterostructure and a 100% spin selectivity of the chiral perovskites, for a S-NEAPbI2/1D chiral perovskite heterostructures at room temperature via spin selective charge transfer. By pumping with a linearly polarized laser light close to A exciton resonance and measuring helicity resolved PL, we obtain a DOP of 0%, −7%, and 8% for monolayer MoS2, MoS2/R-, and S-NEAPbI3 heterostructures, respectively. We attribute this to the chiral perovskites which act as a spin filter and control the valley population in monolayer MoS2 via spin selective charge transfer. We show that the polarization decreases under non-resonant excitation energy and predict that by selecting appropriate excitation energy, either spin up or spin down carriers could be selectively populated in both the valleys. Moreover, using ultrafast transient absorption spectroscopy, we present direct evidence for charge transfer from MoS2 to chiral perovskite. Our results provide a simple and robust route to achieve valley polarization in monolayer TMDs without the need of low temperature, magnetic field, or complex device structure, thus paving the way for practical valleytronics devices.In summary, we demonstrate robust valley polarization in monolayer MoS23. In brief, equimolar amounts of R- and S-1-(1-naphthyl)ethylamine  and lead iodide (PbI from Sigma Aldrich) were dissolved in HI (47% in H2O from Sigma Aldrich) and H3PO2 (50% in H2O from Sigma Aldrich). The ratio of HI:H3PO2 was 5:1. The solution was heated to 100 °C under ambient conditions and continuous stirring. Once a clear solution was obtained, the solution was slowly cooled to room temperature resulting in single crystal precipitation. The single crystals were rinsed in chloroform and dried in vacuum at 60 °C for 3 h.Chiral lead halide perovskite single crystal was synthesized following protocols reported in literaturePerovskite thin films used for CD spectroscopy were made by spin coating inside an Argon filled glovebox. 10 wt% perovskite solution in Dimethylformamide (DMF) was prepared. 100 µl of the precursor solution was deposited on quartz substrates cleaned in acetone and isopropanol in an ultrasonic bath for 10 min each. The substrates were spun at 4000 rpm for 30 s. Finally, the substrates were annealed at 100 °C for 30 min.2, bulk MoS2 crystals (from HQ Graphene) were mechanically exfoliated using the standard “scotch tape” method. In brief, a small piece of bulk MoS2 was placed on a sticky tape and then the tape was folded a peeled several times to thin down the flake. Finally, the tape was pressed onto a clean Si substrate with 290 nm thick SiO2 and slowly peeled off. Monolayers transferred on the Si substrate were identified with an optical microscope and confirmed with Raman micro-spectroscopy and AFM.To produce monolayer MoS2 on sapphire substrate (from 2D Semiconductors) was used.For transmission measurements, a CVD grown monolayer of MoS31 using a commercial transfer stage from HQ graphene under ambient conditions. Perovskite flakes were directly exfoliated on a Polydimethylsiloxane (PDMS) stamp (from Gelpak). The PDMS stamp with perovskite was turned upside down and aligned on top of a monolayer MoS2. The PDMS stamp was slowly lowered and pressed on top of the target monolayer MoS2 flake. After the entire perovskite flake was in contact with MoS2, the PDMS stamp was slowly retrieved. This resulted in the perovskite flake being transferred from the PDMS stamp to the monolayer MoS2 flake on Si/SiO2 substrate. Perovskite flakes were transferred on CVD grown monolayer MoS2 on sapphire substrate (2D Semiconductors) for transient absorption measurements. For all other measurements mechanically exfoliated monolayer MoS2 on Si/SiO2 substrate were used.Heterostructures were made with dry transfer techniquePhotoluminescence (PL) was measured with a home-built confocal microscope consisting of an Olympus IX81 inverted microscope equipped with a ×60 dry objective lens, NA 0.7 (Olympus America) and delivering a spatial resolution of ~0.59 µm. Samples were excited with a continuous wave monochromatic laser light of 633 nm (He Ne Melles Griot), 594 nm (He Ne Melles Griot), or 532 nm . A polarizing beam-splitter cube was used to linearly polarize the excitation beam. Appropriate laser-line optical filters were used to clean the excitation laser. The PL emitted from the sample was collected by the same objective lens. A dichroic beam-splitter mirror and a long-pass filter were used to filter out the excitation laser from the emission. For PL spectra, the emission was directed to a monochromator (Acton SpectraPro 2750) via a multimode optical fiber. A quarter waveplate and a linear polarizer were added in front of the optical fiber to separately detect the left and the right circularly polarized PL emission for helicity resolved PL measurements. Similarly, another quarter waveplate was added after the linear polarizer to create circularly polarized laser excitation. For PL mapping, the emission was spatially filtered with a 100 µm pinhole. To simultaneously measure PL and reflection images, the emission was split by a 50:50 cube and imaged onto two single-photon-counting avalanche photodiodes . For the PL map, an additional long-pass filter was used to filter out the laser. The photodiodes were coupled to a time analyzer  and the SymPhoTime 64 software (Picoquant) was used for data acquisition and analysis.−2. All measurements were performed under ambient conditions.Transient Absorption spectroscopy (TAS) was performed with a commercial broadband transient absorption microspectrometer . The fundamental optical output from an Ytterbium-doped Potassium-Gadolinium Tungstate (Yb:KGW) laser  was split into two parts. One part was directed to an optical parametric amplifier  to generate a pump beam (500 nm). The other part was focused on a sapphire crystal to generate a super-continuum white-light probe beam. The time delay between the pump and probe beams was controlled using a computer-driven optical delay stage . The beams were combined and focused on the sample with a ×20 objective lens . The laser spot of the pump and the probe were 10 µm and 7 µm in diameter, respectively. The transmitted probe beam was collected by another ×20 objective lens and directed to the Helios spectrometer, with experiments performed at a fluence of 40 µJcm3 single crystals were crushed into fine powder using mortar and pestle. X-ray tube was operated at 40 kV and 45 mA.Power X-ray diffraction (XRD) was performed with Rigaku SmartLab Universal Diffractometer. R-SNEAPbIRaman micro-spectroscopy was performed with a WiTec Alpha 300/Apron microscope . A 532 nm laser was used for excitation in combination with a 100×objective lens.Atomic force microscopy (AFM) was performed with an Asylum Cypher AFM ES VRS in AC tapping mode.Circular dichroism (CD) spectroscopy of perovskite thin films on quartz substrates was measured with a Jasco J-815 CD Spectropolarimeter. Each sample was flipped and measured from both directions with the beam first hitting the (1) perovskite side (front), and (2) the quartz side (back). The average of the front and the back measurements were computed to obtain CD spectra.–10 Torr equipped with a SPECS Phoibos 100, MCD-5 hemispherical energy analyzer, and an ultraviolet source UVS 10/35. He(I) at 21.2 eV radiation was used. The angle between the analyzer and UV- source was 45° and photoelectrons were collected along the sample surface normal. R-SNEAPbI3 single crystals were crushed into fine powder using mortar and pestle and placed on a Flag style sample plate. The spectra were calibrated vs the Fermi edge of Au (111) crystal. The work-function of the material was determined by subtracting the ultraviolet radiation energy (21.2 eV) from the secondary electron cut-off energy of UPS. The energy gap between valence band maxima to Fermi level was determined by UPS low binding energy edge to Fermi level. Bandgap between conduction and valence bands was obtained by UV-vis. Thus, the conduction band (CB) edge, valence band (VB) edge and Fermi level relative to the vacuum level was estimated as shown in Fig. Ultraviolet photoelectron spectroscopy (UPS) was carried out in an ultrahigh vacuum system with base pressure of 2 × 102 was measured in an inverted microscope . The samples were illuminated with a broadband light source (Energetiq LDLS EQ-99) and the transmitted light was directed to a monochromator (Acton SpectraPro HRS-500) and a PIXIS Roper camera.UV-vis transmission of monolayer MoSSupplementary InformationPeer Review File