This revealed single-strandedness of the bulge, a following G residue plus the loop subsequence GUUGU as the few key determinants for priming and initiation site selection; unexpectedly, they functioned independently of a specific structure context. These data provide new mechanistic insights into avihepadnaviral replication initiation, and they imply a new concept towards a feasible in vitro priming system for human HBV.Hepadnaviruses, including human hepatitis B virus (HBV), replicate their tiny DNA genomes by protein-primed reverse transcription of a pregenomic (pg) RNA. Replication initiation as well as pgRNA encapsidation depend on the interaction of the viral polymerase, P protein, with the ε RNA element, featuring a lower and an upper stem, a central bulge, and an apical loop. The bulge, somehow assisted by the loop, acts as template for a P protein-linked DNA oligo that primes full-length minus-strand DNA synthesis. Phylogenetic conservation and earlier mutational studies suggested the highly based-paired ε structure as crucial for productive interaction with P protein. Using the tractable duck HBV (DHBV) model we here interrogated the entire apical DHBV ε (Dε) half for sequence- and structure-dependent determinants of  For extension into full-length minus-strand DNA the oligo is translocated to a complementary acceptor at the 3′ proximal direct repeat DR1*  DNA genome present in mature nucleocapsids and enveloped virions2.Hepadnaviruses, with the important human pathogen hepatitis B virus (HBV) as their prototype, are small enveloped DNA viruses that replicate via protein-primed reverse transcription of a pregenomic (pg) RNA intermediate2 Fig. . A cruci2 Fig. . ε is sp2 Fig.  in proteR1* Fig. . At leas11 and in the CRISPR/Cas9 system12, and must also encode the information for proper positioning of P protein over the initiation site at the 3′ end of the bulge; in most conventional DNA copying systems the initiation site is defined by the extendable 3′ end of a nucleic acid primer, e.g. a host tRNA in retroviruses. Current knowledge on how this is achieved in hepadnaviral P - ε complexes is limited. To date it has been impossible to isolate functional RNPs in sufficient quantity and quality for direct structural investigation. For HBV, mutational analyses were largely confined to using pgRNA packaging and viral DNA formation in transfected cells as readout. In vitro HBV RNP complex formation has been achieved but without detectable DNA synthesis activity13; we refer to this a non-productive binding. The most advanced cell-free HBV priming system relies on co-expression of tagged P protein and ε RNA in mammalian cells, with subsequent affinity purification15; however, the RNA in the isolated RNPs cannot be exchanged. Hence for functional studies using P protein and/or ε RNA mutants each new RNP must individually be generated, precluding larger scale analyses. For DHBV, in contrast, in vitro priming systems are long established16. Priming activity of recombinant full-length or near full-length P protein requires cellular chaperones, as present in rabbit reticulocyte lysate19 or added in substance18, in particular heat shock proteins Hsc70 and Hsp4020. This chaperone dependence is overcome by terminal truncations in P protein21, with the respective “miniDP” proteins providing the most feasible systems to investigate Dε-dependent protein-priming. Conversely, the impact of ε mutations can even be analyzed in the context of genuine DHBV infection of ducks22.Hence ε RNA contains elements that activate P protein, similar to the RNA components in telomerase25, and NMR investigations revealed further details, including non-canonical basepairs, in the previously assumed  hepta- and hexaloop sequences capping the upper stem, creating a tetraloop in Dε26 and a pseudotriloop in HBV ε27.Collectively, the available data indicate that both the bulge and the loop are important for a productive interaction with P protein. Bulges, loops and unpaired residues within an RNA double-helix offer a wealth of unique recognition surfaces and thus are predestined as highly specific binding sites, including for P protein. The actual presence of both elements in free HBV and DHBV ε RNA was biochemically confirmed28; hence the connecting upper stem could also act as a molecular ruler that determines the spacial distance and orientation of the two subelements, optimally positioning them for productive binding to P protein, including for proper initiation site selection  procedure revealed a subsequence of the classical heptaloop sequence, GUUGU, and a single G residue immediately following the bulge as the only crucial determinants in the apical Dε part, including for selecting the genuine 3′ terminal bulge nt (bulge position 6) as initiation site; again, however, no need for a specific structure context became obvious. We then confirmed our suspicion that the bulge region itself harbors functionally important determinants for initiation site selection via combinatorial mutations targeting bulge position 6 and the unpaired U residue opposite the bulge. Altogether, the results suggested a revised model for a productive DHBV P protein - Dε interaction whereby the critical determinants in the RNA are a single-stranded bulge region (guaranteed by the stable lower stem), a G residue following the bulge 6 position with an oppositely located pyrimidine, plus a GUUGU motif in not too far a distance but independent from a specific upper stem structure. This was verified by the priming activity and replication-competence of artificial Dε RNAs in which these primordial elements were embedded into a completely unstructured context. Hence accessibility of a few crucial RNA determinants is the main prerequisite for a functional upper stem sequence.We here sought to comprehensively interrogate the entire apical Dε part for determinants that allow for a productive interaction with P protein, using as readout in vitro priming. The methods developed in this study should provide suitable tools to test this concept.These results shed new mechanistic light on replication initiation in avian hepadnaviruses and have also implications for mammalian HBVs, including a new concept as to why human HBV ε with its highly stable upper stem structure does not support 26. To modulate this distance with minimal induction of new, alternative basepairings we used as parent a Dε variant, S1230, in which the top part of the upper stem lacks basepairing potential; however, the five basepairs on top of the bulge and the two basepairs closing the tetraloop can form . Notably, variant −2LR_II lacks 10 of the totally 27 nt in the Dε upper stem.In a first set of variants (termed “class I”) we deleted two residues from the left or the right half-stem , or both (−2LR_I); alternatively, two C residues each were inserted in the left or right half-stem , or both (+2LR_I). An analogous series of more severely altered variants (class II) were based on a derivative in which 6 nt at the base of the right half-stem (positions 2597–2602) were in vitro priming with α32P-labeled dGTP, the natural first nt of minus-strand DNA templated by the 3′ terminal C of the bulge (b6 position), either with full-length DHBV P protein in vitro translated in rabbit reticulocyte lysate (RRL) or using a truncated recombinant P protein variant (miniDP) that retains priming activity without any protein co-factors31. Reactions with wt Dε or without RNA served as controls. Reaction products were separated by SDS-PAGE and band intensities of 32P-labeled P protein were quantitated by phosphorimaging . All class I mutants produced similarly strong signals as wt Dε in either system, except for a slightly stronger reduction with the most extended variant +2LR_I. Hence adding or deleting up to 4 nt from the upper stem had little effect on priming efficiency with dGTP.We then assessed the ability of the variant RNAs to support bottom panel). To test whether the reduced class II priming signals reflected a general reduction in priming efficiency or an altered dNTP preference caused by a shift in initiation site we repeated all experiments using α32P-dATP  and also dATP utilization; however, the priming signals with dATP remained ten- to twenty-fold lower than with dGTP, corroborating a comparably strong dGTP preference of the variants as seen with wt Dε and indicating that a C residue acted as template.In contrast, only very weak signals (though still higher than in the no-RNA control) were obtained with the class II RNAs lacking additional two or four nt, whereas adding back two or four nt partially rescued higher priming activity  into the DHBV expression vector pCD16. Upon transfection into LMH cells all constructs produced similar amounts of cytoplasmic capsids , and stronger reductions for the class II variants  compared to wt DHBV; this was confirmed by direct detection of the viral DNAs in intact capsids  were indistinguishable between variants and wt DHBV. Hence also in the cellular setting were all variants able to generate oligo primers supporting formation of full-length viral DNA, in line with their using the authentic priming initiation site; this was corroborated by mapping the minus-strand DNA 5′ ends by primer extension . All variants except −2R_II and −2LR_II with the poorest in vitro performance were able to establish viremia, although with later onset and substantially lower maximal titers than the wt DHBV controls  into LMH cells, then enriched viral particles and used nuclease-resistant DNA as template for another subgenomic PCR, the products of which served to create a new vector pool. This procedure was repeated several times. For characterization, we used pool sequencing combined with cloning of individual sequences, various of which were also analyzed for replication competence. As targets for randomization we chose the bulge, the loop, and the seven nt of the upper left stem (ULS) immediately following the bulge.Given the importance of a properly ε-templated oligo primer for the subsequent replication steps, randomizing the bulge allowed to validate the procedure. Figure 23, in the context of either wt Dε or the previously described viable variant S130 in which the sequence underlying the loop cannot basepair  immediately following the bulge. A requirement for basepairing should result in selection of nt that are complementary to the right half-stem, including for clarification whether the 3′ end of the bulge needs to be closed by a stable double-stranded structure (and thus would meet the definition of a bulge). Using as recipient variant S12nce Fig. . Not theMonitoring selection by sequencing the pools Fig.  plus indAltogether, the SELEX data revealed surprisingly few specificity determinants for a productive Dε - P protein interaction in the entire upper stem, except a G residue following the initiation site and a downstream GUUGU motif, functional in various sequence and structure contexts. Thus a remaining option for proper initiation site selection was that selective recognition of the template region relies largely on the bulge region as such, including its immediate vicinity.32 the stable lower stem in wt Dε expels the 5′ end of the bulge as well as the oppositely located unpaired U26. To test whether these features contribute to defining bulge position 6 (b6) as initiation site we deleted the unpaired U (uΔ) or replaced it by the other three nucleotides . We then combined these mutations with all possible nt at the b6 position. The variants were designated as uXb6Y where X denotes the nt at the unpaired U position and Y that at the b6 position. Using the miniDP in vitro priming system we then assessed each RNA’s dNTP preference. Preferential incorporation of the dNTP complementary to b6 would indicate use of the authentic initiation site. The original priming data are compiled in Fig. Besides carrying sequence-specific determinants in its tipupper left panel). Replacement of the unpaired U or its deletion maintained this preference, although at lower efficacy . A G at b6  reduced overall priming efficiency (see below) and led, uniquely, to preferential utilization of both dC and dT when combined with the authentic unpaired U and, less pronouncedly, with an A at the unpaired U position; in all other b6G combinations signals were very low, without an evident preference for a particular nt. An A at b6  caused preferential utilization of dT, especially in combination with the genuine unpaired U or an A at this position. Lastly, with a U at b6  dA was preferentially incorporated by all unpaired U variants, except those with a G.Though complex in detail, already visual inspection of the priming signals Fig.  revealedThe graphic representation of these data in Fig. A common feature of the variants exerting strongly reduced priming efficacy, e.g. those carrying a G at b6 or the unpaired U position, was their potential to severely alter the bulge region architecture by new basepairings, as indicated for some representative examples in Fig. in vitro priming signals) produced detectable bands at the authentic RC-DNA and dsL-DNA positions; band intensities correlated well with in vitro priming efficiency. For instance, the two best-performing variants uAb6C and uCb6C  had also given the strongest in vitro priming signals, with a pronounced preference for the authentic dG  shift was seen with the b6G variants (see above), underscoring the potential importance of a G residue immediately after the initiation site.in vitro priming efficiency by ~20-fold while a preference for dG utilization was preserved  and 35% (mini-Dε2) of those generated by wt Dε RNA  at the junction to the upper stem double-helix could contribute to its specific use as initiation site. Not the least, the upper stem sequence could itself harbor direct determinants for a productive interaction with P protein.The hepadnaviral ε RNA element allows selective recognition of pgRNA amongst a plethora of other RNAs in a cell by P protein, activates P protein’s enzymatic function and provides the information for initiating DNA synthesis at a specific internal site. The intricate bi-partite ε stem-loop structure appears highly suited to fulfil all these requirements and the special importance of the unpaired bulge and apical loop regions, as in many other protein-RNA interactions, was experimentally provenThe comprehensive mutational data of this study challenge all of these notions. The only fundamental determinants identifiable in the entire apical half of Dε were the GUUGU motif in the classical Dε loop sequence and a G residue following the bulge with an oppositely located pyrimidine; yet they could exert their functions in priming and initiation site selection independently of a specific RNA structure context. Hence the structural determinants in Dε are confined to the lower stem which ensures single-strandedness of the bulge and the opposite unpaired U; in addition, the specific bulge architecture must not be altered by improper basepairing.In vitro, all variants preferred dGTP over dATP  revealed as the only strongly selected feature a G residue immediately following the bulge, with the notable absence of G from the following four positions Fig. . Multipling Fig. . Hence ting Fig.  which ising Fig. . Hence ting Fig. .In contrast to the virtual absence of specificity determinants in the upper stem (except the GUUGU motif and the G following the bulge), the distinct architecture of the bulge region was critical for priming activity and initiation site selection, as inferred from some low replicators from early SELEX rounds Fig.  and direHence single-strandedness of the bulge region for at least 6 nt from the top end of the lower stem appears crucial for efficient priming, with the sixth nt predestined as initiation site; however, its efficient utilization is strongly favored by a G residue at the following position, as confirmed by the bulge closing basepair mutant c-g which still preferred dG but at a drastically lower overall priming level Fig. . Replaci36, with the top two or three carrying sequence-specific information32. These elements suffice to activate P protein and position the template region such that the catalytic residues of the RT domain, the dNTP binding pocket, and the last nt of the bulge (the initiation site) are all properly aligned; only then can the bond to the priming Tyr-residue in the TP domain form  be reconciled with the highly structured upper stem in wt Dε? A likely scenario is outlined in Fig. orm Fig. .Figure 932. A third binding site is predicted to accomodate the GUUGU motif or parts thereof; a strong candidate for a direct contact is the first U (L4 position) where no other nt was tolerated . In this model, mini-Dε1 and mini-Dε2 represent one extreme with little or no energy input required to disrupt existing structures. Wild-type Dε with its largely basepaired upper stem would represent an intermediate case where the energy gained by productive binding to P protein is just high enough to enable the RNA rearrangement, possibly via mini-Dε-like intermediates. The tether rather than ruler function of the upper stem sequences would conform to a “fly-casting” mechanism37 whereby occupation of one site increases chances for engagement of nearby other RNA determinants. Further stabilizing the upper stem would increase the energetic barrier beyond a threshold and prevent in vitro priming6. In non-binding Dε variants the a priori accessible RNA determinants would be insufficient to sustain any stable protein interaction, in line with the dramatic influence of RNA context on protein-binding to specific RNA sequences38.Priming-active P protein - ε RNA complexes do not form via lock-and-key binding but in a dynamic multi-step process. For instance, various Dε RNA variants can bind to P protein but do not support priming30 the principal conclusions should apply to all of them. Nonetheless, there is much less natural sequence variability in the upper stem part than implicated by the in vitro priming activity of the mini-Dε variants. This probably reflects the multiple additional functions of the ε sequence22, e.g. compatibility with the mRNA function of pgRNA for core and P protein, and as part of the preC ORF from which the secreted  e antigen is produced. Notably, all natural avian ε sequences maintain two or more canonical basepairs to close the top of the bulge and the apical loop, in seeming contrast to our data. Plausibly such local basepairing constrains the ability of the relevant ε determinants to undergo irrelevant pairings elsewhere in the pgRNA. Also, while no defined loop structure is required for the GUUGU motif to function, its presentation on a properly structured loop could still facilitate initiation of direct contacts with P protein.How relevant is this model for naturally occurring ε sequences? As the identified primordial RNA determinants are invariantly present in the ε sequences of all known avian HBVs14 and/or the following A39. Otherwise, a specific architecture of the bulge region itself appears also crucial in HBV41. The classical sequence in orthohepadnaviruses is similar to that in avihepadnaviruses (CUGUGC vs. CUGUUGU) and also here the central GUG motif appears most important34. However, any deeper mechanistic understanding will require an HBV in vitro priming system, ideally comprising just P protein and ε RNA; obviously, this could also serve to screen for new antivirals that interfere with protein-priming as a highly virus-specific target.The overall similarity to Dε of HBV ε Fig.  suggestsin vitro systems that work for DHBV has yielded authentic ε-templated protein-priming activity. Intriguingly, specific though non-productive in vitro binding to HBV P protein requires most of the ε RNA but not the apical loop33, implying that failure of P protein to engage the loop sequence causes the lack of activity. One interpretation is the absence, from all systems tested, of an auxiliary loop-binding host factor13. Based on our model . Generation of the SELEX vector pools is detailed in Supplementary Methods. All construct were verified by Sanger sequencing.The parental DHBV16 expression vector pCD16 carries a 1.1 × DHBV16 genome  under control of the cytomegalovirus (CMV) immediate-early (IE) enhancer promoter. In its derivative pCD16_Δ3′ε the 3′ copy of Dε was made non-functional by a 40 nt deletion (DHBV16 positions 2568–2607). For In vitro Dε transcripts were generated from the respective pUC19T7 vectors linearized immediately after the Dε cassette30 using the T7 MEGAScript kit (Ambion). Priming assays were performed using either DHBV P protein in vitro translated in rabbit reticulocyte lysate6, or using bacterially expressed DHBV miniDP protein (which does not require chaperones for activation) and 1 µM in vitro transcribed Dε RNA plus the desired α32P-labeled dNTP  as previously described31; Covalently 32P labeled P protein resulting from successful protein-priming was detected by autoradiography and/or phosphorimaging  after SDS-PAGE separation. Band intensities were quantified by phosphorimaging, using ImageQuant software.22.Chicken LMH hepatoma cells were cultured and transfected using Mirus TransIT-LT1 reagent (Mirus) as previously described32P labeled DHBV DNA probe were all conducted as previously described22. The presence of the mini-Dε1 and mini-Dε2 sequences in nucleocapsid-associated viral DNAs from LMH cells transfected with the respective pCD16_Δ3′ε plasmid derivatives was verified by sequencing of PCR amplicons obtained using a forward primer matching DHBV positions 2474–2496 and and a reverse primer complementary to positions 2821–2844.Detection of cytoplasmic capsids by immunoblotting after native agarose gel electrophoresis (NAGE), isolation of viral nucleic acids associated with cytoplasmic nucleocapsids and extracellular viral particles after enrichment by polyethylen glycol precipitation, and Southern blotting using a8 vge of transfection-derived virus and viremia over time was monitored using a one-step qPCR with a lower detection limit of about 105 vge/ml42. For sequencing of the Dε regions, primers DR1-SpeI DHBV+  and D2738- (TTAGCATCTCTAACAAGATCATC) spanning DHBV positions 2537 to 2738 were used22.All animal experiments were approved by the Regierungspräsidium Freiburg (project G02/36) and performed in compliance with German animal welfare legislation at a registered facility of the University Hospital Freiburg under veterinary supervision. Two- to three-day-old Pekin ducklings were inoculated with a dose of 10in vivo selection, two ducklings were inoculated as described above with virions from a first round transfection-derived vector pool carrying a randomized loop sequence in the context of variant S122. Sequence analyses were performed on PCR products obtained using DNA from serum-borne virions as template at the indicated time points post inoculation as described22.A detailed account of the generation of the SELEX vector pools and the selection procedure, including the built-in precautions to minimize contamination with wt-DHBV sequences during the multiple cloning and amplification steps as well as a functional validation is given in Supplementary Methods. In brief, a ~160 bp Dε comprising DNA fragment was created via PCR using one synthetic oligonucleotide carrying the desired randomized region  as template, plus two oligos acting as forward and reverse amplification primers. The product was extended in a second PCR to generate a ~2 kb DHBV fragment which was finally cloned into a special recipient pCD16_Δ3′ε derivative, carrying a nonrelated 1.2 kb stuffer DNA instead of the 5′ proximal DHBV sequence. For the in-cell SELEX, the vector DNA pooled from ten- to twenty-thousand individual colonies was transfected into LMH cells. Three days post transfection, DNA associated with viral particles  was used as template for PCR amplification of a ~2 kb genome segment harboring the Dε region from which a new vector pool was reconstituted. This procedure was repeated from five to thirteen times. Selection progress was monitored by sequencing the resulting new vector pools plus various individual plasmid clones from each round. Functionality of the pools and of select clones with known sequence was assessed by Southern blotting, using viral DNA from cells transfected with pCD16 and/or pCD16_Δ3′ε as reference. For All data generated or analysed during this study are included in this published article and its Supplementary Information files.Supplementary Information


Controlled fabrication of metallic nanostructures plays a central role in much of modern science and technology, because changing the dimensions of a nanocrystal enables tailoring of its mechanical, electronic, optical, catalytic and antibacterial properties. Here we show direct superplastic nanoimprinting (SPNI) of crystalline metals well below their melting temperatures, generating ordered nanowire arrays with aspect ratios up to ∼2,000 and imprinting features as small as 8 nm. Surface-enhanced Raman scattering (SERS) spectra reveal strongly enhanced electromagnetic signals from the prepared nanorod arrays with sizes up to ∼100 nm, which indicates that our technique can provide an ideal way to fabricate robust SERS substrates. SPNI, as a one-step, controlled and reproducible nanofabrication method, could facilitate the applications of metal nanostructures in bio-sensing, diagnostic imaging, catalysis, food industry and environmental conservation. Rapid fabrication of well-controlled metallic nanostructures remains a challenge. Here, the authors directly nanoimprint several different crystalline metals by superplastic forming well below their melting temperatures, obtaining uniform nanostructures with small features and high aspect ratios in one step. Rapid and reproducible fabrication of metallic nanostructures is essential to much of modern science and technology12467910112131116151820232528et al.3313334Tg) and below crystallization temperatures (Tx), which has triggered broad research in exploring BMGs' applications such as electrochemical catalysts37Tm)283343728Among the variety of developed nanofabrication methods, nanoimprinting30Tm. This technique enables one step and rapid fabrication of metallic nanostructures with excellent controllability and predictability, in particular allowing the fabrication of high-aspect ratio nanostructures.Here we show direct nanoimprinting of a variety of crystalline metals  by superplastic forming well below 2O3 templates prepared by anodic oxidation. When heated the metal/mould combinations to a target temperature, the metal is compressed against the mould and nanostructures form by materials creeping surrounding pores in the mould. The target temperature is usually set as 0.5Tm≤T<Tm, with unit of absolute scale of temperature. Typically, after SPNI, the mould is dissolved to release the replicated metal nanostructures. 2O3 template at 500 °C, well below the melting temperature of bulk Au . The Al2O3 template was purchased from Hefei Pu-Yuan Nano Technilogy Ltd. with an average pore diameter of 55 nm. The uniform rust-red colour of the sample in 2O3 template in KOH solution .2O3 templates with uniform cylindrical nanopore sizes of 292±34 nm are used in this experiment. First, five Au short rods (61.8±0.5 mg) were cut from an Au wire with a uniform diameter of 2 mm. Subsequently, the five Au short rods were superplastically formed into the Al2O3 templates at 500 °C, under an applied force of 10 kN and held for 1, 4, 8, 20 and 60 min, respectively. The lengths of Au nanowires at the centre of each sample are measured under SEM, which is used to calculate the aspect ratios of prepared Au nanowires (black dots in L) under applied stress (σs=(3SijSij/2)1/2, where Sij is the stress deviator) and temperature (T) can be generally quantified asSPNI relies on direct mechanical deformation of mouldable materials and can therefore achieve great replicating precision in lateral dimensions. Now we investigate the controllability of SPNI along vertical direction. In general, the length of imprinted metal nanowire by SPNI is a function of forming pressure, processing temperature and time, which provides ways to control aspect ratio of replicated metal nanostructures. Taking nanoimprinting of Au as an example, the effect of holding time on aspect ratios of imprinted Au nanowires is first investigated. Ald and t are diameter of nanopore and forming time, respectively. Applying classic Norton–Bailey's creep power lawwhere L0 approximates the lengths of metal flowing into nanopores during the loading force increasing from zero to the maximum value, and it is in general a function of temperature, nanopore size and loading rate. R and Q are gas constant and active energy for creep, respectively. A is a constant related to nanopore size and material properties such as elastic modulus, n is the stress exponent of creep rate. For nanoimprinting at constant temperature and constant force, for example, T=500 °C and Fm=10 kN in the above experiment, the calculated mean forming pressures  for the five samples are where the constant are  MPa , which cBi  are constants related to nanopore size, material properties, processing temperature and forming pressure. Tg is limited by the crystallization time due to the metastable nature of BMGswhere L/d=4.65+1.33 × t0.36  (red line in Q=5.48 × RTm=5.48 × 8.31 × (1063+273) J mol−1=14.5 kcal mol−1, which is comparable to the measured gold surface diffusion activation energy ∼21 kcal per mole by using a scanning tunnelling microscope to monitor surface profiles decay at 125, 150 and 170 °C, respectivelyIn addition to varying holding time to control the length of replicated metal nanowires, processing temperature can also be independently varied to study its effect on the aspect ratios of prepared metal nanowires . For thi2O3 template with multiple branched nanopores in its surface layer, where the branched nanopore sizes gradually increase from ∼8 nm in the outmost surface to 200 nm in the base layer (2O3 template (see Methods section). The aggregation of Au nanostructures into bundles makes it difficult to examine the replicated hierarchical structures. The prepared Au nanostructures were herein transferred onto a transmission electron microscopy (TEM) mesh grid (see Methods section). SEM imaging of Au hierarchical nanostructures on the TEM mesh grid clearly shows the primary stems abruptly multiplied by several small branches . It is observed that Bi can completely fill the template within 36 s under the applied force of 8 kN. Beside increasing processing temperature, extending forming time can also be applied to fabricate high-aspect ratio metal nanowires  but are difficult to achieve colours : the sur−5 M crystal violet (CV) as a sensitive SERS analyte to detect the electromagnetic enhancement . Although CV molecular absorbed on the bulk Au shows rather weak Raman signals, five Raman lines located at 442 cm−1, 802 cm−1, 1,174 cm−1, 1,384 cm−1 and 1,620 cm−1 can still be recognized and these Raman shifts agree well with literature reports  flows into a channel with diameter of d, under a constant driving pressure (p0) at the entrance of the channel. Assuming the velocity of the fluid elements is so small that the fluid can be regarded as quasi-static flow, then the kinetic energy can be neglected and the energy dissipation rate from creeping-flow  andFor Hagen–Poiseuille flow, the only non-zero component is L is the length of fluid filled in the channel. Substituting where On the other hand, the power input by external forces (neglecting of body force) isBy defining an apparent strain rate asWe finally obtain2O3 template with multiple branched nanopores in its outmost surface . The demoulded sample was then characterized under SEM (A piece of bulk Au was brought into contact with an Al surface . After tnder SEM  after rinder SEM .2O3 templates with nanopore sizes of 20 (s1), 55 (s2), 90 (s3) and 200 (s4) nm, respectively. The processing temperature is 500 °C and all of the four samples are held at 2.5 kN for 4 min. The remaining one was superplastically formed between two flat platens (without surface nanostructures) and set as the reference sample. After dissolving Al2O3 templates away and cleaning the samples with distilled water and acetone for several times, the five Au samples were finally immersed in the prepared ethanol with dissolved CV (concentration of 1.0 × 10−5 mol l−1) for ∼30 min and then taken out to dry in the air.CV with analytical purity purchased from Sinopharm Chemical Reagent Co., Ltd is used as the probe molecule to study the SERS activities of Au nanowire arrays. Before the SERS measurement, five Au short rods with the same volume were cut from an Au wire, four of them were superplastic formed into Al−1. The laser beam power is 17 mW and only 15% of the power being used in the experiments to avoid fluorescence effect. Excitation wavelength of 514 nm, scan time of 30 s, field lens of 20 times and accumulation of 10 times were applied.The SERS measurements were carried out at room temperature under a microscopic confocal Raman spectrometer  by using a charge-coupled device detector with a resolution of 1–2 cmThe data that support the findings of this study are available from the corresponding author on reasonable request.How to cite this article: Liu, Z. One-step fabrication of crystalline metal nanostructures by direct nanoimprinting below melting temperatures. Nat. Commun.8, 14910 doi: 10.1038/ncomms14910 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables and Supplementary Reference.


Staphylococcus aureus (MRSA). Using an exactly solvable model, which takes into account the solvent and membrane effects, we demonstrate that drug-target interactions are strengthened by pronounced polyvalent interactions catalyzed by the surface itself. These findings further enhance our understanding of antibiotic mode of action and will enable development of more effective therapies.The alarming increase of pathogenic bacteria that are resistant to multiple antibiotics is now recognized as a major health issue fuelling demand for new drugs. Bacterial resistance is often caused by molecular changes at the bacterial surface, which alter the nature of specific drug-target interactions. Here, we identify a novel mechanism by which drug-target interactions in resistant bacteria can be enhanced. We examined the surface forces generated by four antibiotics; vancomycin, ristomycin, chloroeremomycin and oritavancin against drug-susceptible and drug-resistant targets on a cantilever and demonstrated significant differences in mechanical response when drug-resistant targets are challenged with different antibiotics although no significant differences were observed when using susceptible targets. Remarkably, the binding affinity for oritavancin against drug-resistant targets (70 nM) was found to be 11,000 times stronger than for vancomycin (800 μM), a powerful antibiotic used as the last resort treatment for streptococcal and staphylococcal bacteria including methicillin-resistant  Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus are well-known aetiological agents of a wide variety of infections caused by structural changes at a cellular target. Antimicrobial resistance (AMR) in vancomycin-resistant Staphylococcus aureus (or VRSA)While molecular recognition exhibits complementarities between a host and guest, cross-reactive binding at a single docking site is possible16The alarming increase of pathogenic bacteria that are resistant to multiple antibiotics is now recognized as a major health issueStaphylococcus aureus (MSSA) cell line (ATCC 29213).Here, we show that the mechanical forces induced by drug-target interactions, regulated by solvent interactions and membrane effects, are critical to our understanding of bactericidal activity against drug-resistant bacteria. In order to demonstrate that molecular changes within a membrane receptor can incapacitate recognition and efficacy of drugs , we usedTo probe different pathways of signal-transduction central to the efficacy of recognition mechanisms and therapeutic agents, we used cantilever force-based technology. Because of its nanometre strain sensitivity, this technology can directly measure the smallest changes in mechanical strain caused by specific antibiotic binding. The principles of force transduction and measurements are well established181921222324252632814K1, the equilibrium dissociation constant of ligand-ligand interactions). Conversely, when a near-membrane layer thickness is increased, ionic shielding and molecular charge is reduced, thus hindering molecules from close contact and decreasing the association kineticsAs proof of concept and to understand how membrane effects regulate molecular recognition to stimulate force transduction and transmission, we investigated the impact of near-membrane rather than bulk solvent effects on the antibiotic binding efficiency. This was achieved by limiting the distance at which the membrane targets are felt by molecules in solution via changing the ionic strength of the medium whilst keeping the solution pH at 7.4 to reflect physiological conditions. Assays were performed under constant flow conditions at three different ionic strengths for the same antibiotic concentrations. While constant flow experiments can induce an offset during mechanical deflection measurements depending on the position of the chip within the flow system, such variations are eliminated if a scanning laser is aligned within the middle region of the position sensitive detector (PSD). 50) of drug-receptor interactions at the surface was used as measure of membrane effects on the association kinetics and mechanical response. 50, we analyzed the data using In order to demonstrate the feasibility of membrane effects in regulating molecular recognition to enhance force transduction, we considered the interaction of drugs at the interface between bacterial surface and solution to be defined by three-dimensional (3-D) rates and affinity constants, while the induced mechanical response following drug binding is restricted to a two-dimensional (2-D) environment . In the eq is the equilibrium mechanical stress, σligand1 is the mechanical stress generated by ligand1 in the absence of a competing ligand2, σligand2 is the corresponding minimum stress generated by ligand1 in the presence of a large excess of the concentration of competing ligand2. The outcome of the fit of 50 of 15.4 ± 0.2 μM for Van and 50.7 ± 2.1 μM for Ori 23 mNm−1 . The obs23 mNm−1  the diff23 mNm−1 . We hypos, for therapeutically effective drugs such as Ori against drug-susceptible and drug-resistant bacteria. To achieve this, we used the relation s = σeq/n, where n is the number of receptor molecules per cantilever, estimated at ~1011 Thus far, we have discussed the impact of near-membrane layer on the association kinetics, mechanical force transduction, specificity of molecular recognition and microbial susceptibility. To gain mechanistic insight , we averoccupied . To calcKsurf is raised to the power n to ensure that it maintains the dimension of concentration as n varies. Setting n = 1 yields the Langmuir isotherm where the reactions occur independently, while n > 1 corresponds to positive co-operative polyvalent interactions and n < 1 is a measure of negative co-operativity. In n, Ksurf and σmax) is summarized in Ksurf for Ori binding to VSR is 40 ± 10 nM, which is 25 times stronger than for Van where Ksurf is 1.0 ± 0.3 μM. The Ksurf of Ori against VRR is 70 ± 10 nM, an astonishing 11,000 times stronger than for Van where Ksurf is 800 ± 300 μM. As a further measurement control and to confirm the accuracy of surface binding affinity, we used a commercially available SPR method  for each membrane receptor, while fixing K1, to the tabulated values (K1–K6 in the models and equations below are in the form of association constants (1/M) while the plotted and tabulated values are shown in the form of disassociation constants (M). The maximum mechanical stress σmax was fixed at 33.9 ± 3 mNm−1 for VSR and 19.2 ± 2 mNm−1 for VRR, respectively  in the half-height point of the Langmuir curves of only two orders of magnitude, while the experimental variability is four orders of magnitude.Inspection of d values . For algectively , becauseVRR data  – the “bNm and Np in the numerator. Our attempt to fit the data with this generalization was unable to account for the much larger variability in K1 of the VRR data. As an admittedly non-unique attempt to improve on Model I, but in direct response to the strongly implied importance of the surface effects in mediating cooperative processes discussed above, we included a near-membrane layer between the 2-D surface and 3-D solution, yielding Model II  of near membrane layer to relate the solution and surface reactions. Moreover, given that the molecules studied differ only in the chemical functionality of groups remote from the ligand binding site, and that the tracking of either monovalent or multivalent ligand complexes at the near-membrane layer ought not depend strongly on these groups, the equilibrium constants in Cα = (K5(K3/K2)^1) and Cγ = (K6(K3/K2)^2), which again are unlikely to depend strongly on the chemical functionality of remote groups. Consequently, α and γ in The terms Further work using more microscopic techniques such as X-ray scattering and NMR will be required to understand the individual contributions of these equilibrium constants and to validate the simplifying assumptions made above.max, Cα, Cγ, K0 and h), where K1 was fixed to the tabulated values , implies that the surface effects for VRR data cannot be neglected. In contrast, for VSR, the very small K0 (i.e. K0 ≪ hK1) suggests that it is the solution rather than the surface effects that dominate the recognition process, in good agreement with In light of Model II, we fitted d values . In Fig. targets . The verKsurf of Ori on drug-resistant targets of 70 ± 10 nM, an astonishing 11,000 times stronger than for Van where the Ksurf is 800 ± 300 μM. This finding is in agreement with solid-state NMR, which has concluded that Ori is a more potent inhibitor of the transpeptidation process than transglycosylationOur experiments have shown unequivocally the importance of surface effects in the mediation of molecular cooperativity and the understanding of antibiotic’s action, furthermore we have made significant steps in unraveling the mechanisms by which a near membrane surface layer regulates molecular association kinetics both for mechanical force transduction and microbial susceptibility. Indeed, even though it is widely accepted that vancomycin analogues inhibit peptidoglycan biosynthesis without substrate bindingThe molecular binding events occurring between antibiotics in solution and at membrane bound targets were found to enhance the binding efficiency and mechanical stress signals. Our results from intact bacterial cell assays and stress measurements suggest that bactericidal activity is linked to the extent of molecular recognition and associated mechanical force transduction. We find four orders of magnitude of bactericidal activity are strongly linked to mechanical force, consistent with the picture that following the application of a lethal dose, the insertion of antibiotic molecules into bacterium cell walls can induce a local strain, which grows as the number of reacted regions grows, until a deleterious strain is generated which the bacteria cannot ultimately withstand . Consequ46zabs was measured using a time-multiplexed optical detection system in different liquid environments . The desired flow rate was first determined before each experiment to ensure a constant flow rate for all the arrays. The data acquisition was automated using LabView  software via a 6-way valve . All cantilever bending signals were acquired under an average liquid flow rate of 30–180 μL min−1. The equilibrium differential cantilever bending signals were not found to be significantly dependent on the direction or fluid flow rates. The measurement protocol involved the following steps, (1) sodium phosphate buffer  for 5 or 10 minutes to establish a baseline; (2) injection of antibiotics in sodium phosphate buffer for 30–60 minutes; (3) to dissociate the complex, sodium phosphate buffer  wash for 10–30 minutes; (4) a further washing step using 10 mM HCl for another 30 minutes to regenerate the membrane surface targets; and (5) finally another sodium phosphate buffer step for 10 minutes to restore the baseline signal. We investigated the impact of surface regeneration by undertaking repeated regeneration measurements with more than 10 cycles using 10 mM hydrochloric acid and found that the quality of sensing layers can deteriorate if a large number of receptor regeneration steps are employed particularly when using acidic reagents, thus limiting the lifetime of the sensor itself. The detection specificity was achieved by performing measurements in differential mode, where the bending response of the reference PEG-coated cantilevers, was subtracted from VSR- and VRR-coated cantilever signals.The modified cantilever chip was mounted in a sealed liquid cell. The alignment of each light source onto the free end of each cantilever was confirmed by heating liquid chamber to 1 °C rise. All eight Au-coated cantilever arrays were found to undergo compressive downward bending because of the bimetallic effect caused by the differences in expansion rates of silicon and Au. Care was taken so that the optical alignment error was less than 5% between the minimum and maximum bending signals within the cantilever arrays. In addition, the resonant frequency of all eight Au-coated cantilever arrays was measured to ensure that the variation of spring constant between each cantilever within a chip was ≤1%. The absolute deflection at the free-end of each cantilever ∆To convert the bending signals into differential surface stress, the raw data from each set of separate cantilever chips were were analyzed off-line using automated data software to rapidly analyze large data sets and remove user bias. The bending signals were converted into differential surface stress between the upper and lower sides of the cantilever, according to the Stoney’s equationt is the thickness, E/(1 − ν) = 181 GPa is the ratio between the Young’s modulus E and Poisson ratio ν of Si (100) and Δzabs is the absolute cantilever bending deflection. The differential equilibrium surface-stress Δσeq was calculated by subtracting in-situ reference Δσabs (PEG) surface-stress signal from the absolute stress signals of Δσabs(VSR and VRR coated cantilevers). When comparing four different arrays, there was a high reproducibility of within-array measurements, and an increased variance associated with between-array measurements in agreement with our previous findingswhere L is the effective length of the cantilever, Ksurf of drug-target interactions.The SPR sensor chip functionalization procedure is summarized .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


PRKACA) were found to be causative for 30–40% of cortisol-producing adenomas (CPA) of the adrenal gland, rendering PKA signalling constitutively active. In its resting state, PKA is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits with the latter inhibiting PKA activity. The human genome encodes three different PKA catalytic subunits and four different regulatory subunits that are preferentially expressed in different organs. In normal adrenal glands all regulatory subunits are expressed, while CPA exhibit reduced protein levels of the regulatory subunit IIβ. In this study, we linked for the first time the loss of RIIβ protein levels to the PRKACA mutation status and found the down-regulation of RIIβ to arise post-transcriptionally. We further found the PKA subunit expression pattern of different tumours is also present in the zones of the normal adrenal cortex and demonstrate that the different PKA subunits have a differential expression pattern in each zone of the normal adrenal gland, indicating potential specific roles of these subunits in the regulation of different hormones secretion.Somatic mutations in protein kinase A catalytic α subunit ( With the advent of whole exome sequencing somatic mutations in the gene encoding the catalytic subunit α of protein kinase A (PRKACA) has been revealed in approximately 30–40% of CPA, associated with overt Cushing syndrome11. Also other components of cAMP/PKA signalling were often shown to be either mutated, such as the stimulating G protein (Gas) which is encoded by the GNAS gene or deregulated in different endocrine disorders13 indicating the major role played by this pathway in hormonal secretion functions.ACTH-independent Cushing syndrome is mostly due to cortisol-producing adrenocortical adenomas or carcinomas. Whereas adrenocortical cancer is a very rare tumour14. In addition, the protein kinase X was also shown to associate with the PKA regulatory subunits15. PKA has two isoforms, referred to as I and II, depending on whether type I or type II regulatory subunits bind the catalytic subunits17. Common to all regulatory subunits is the inhibitory role of the activity of the PKA catalytic subunits in the absence of cAMP18. PKA signalling is activated when two molecules of cAMP bind each regulatory subunit of the PKA heterotetramer, leading to its dissociation and releasing of the catalytic subunits, which thereby become active. Furthermore, the regulatory subunits can specifically interact with A-kinase anchoring proteins (AKAPs) that target PKA to specific subcellular compartments20. This interaction may explain the different expression patterns and roles played by the different PKA isoforms.Under basal conditions, cAMP-dependent Protein Kinase (PKA) is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits. The human genome encodes three different catalytic subunits  and four different regulatory subunits  that are each preferentially expressed in different organsPRKACA mutations identified in CPA are situated at the interface between the catalytic subunit and the regulatory subunits6. By functional characterization of the initially identified L206R substitution and 199–200 insW insertion, it was shown that the binding of the regulatory subunits to the catalytic subunits is affected by these mutations, rendering PKA constitutively active21.Interestingly, the 22, but a detailed analysis had not been performed. Thus, it remains unclear if the expression of the regulatory subunits influences the specific steroidogenic profile of the three zones of the adrenal cortex. Of note, in some CPA, reduced protein levels of the regulatory subunit IIβ have been previously observed24. Here we investigate if the PRKACA mutations leading to hyperactivity of PKA signalling in CPA are associated with the loss of RIIβ protein levels and demonstrate a clear connection between RIIβ protein levels and the mutation status of PRKACA. Furthermore, we describe a comprehensive pattern of PKA subunits expression in the different zones of the adrenal cortex and speculate about the functional consequences of these findings.In normal adrenal glands (nAG), as in other endocrine tissues, both type I and II regulatory subunits are expressed23 for mRNA and protein analyses. All patients gave informed consent and the study was approved by the Ethics Comities of the University Hospitals of Wuerzburg, Munich and Padua and the Institute Cochin Paris. The PRKACA mutation status was determined using exome sequencing 11 or Sanger sequencing of the hotspot around the amino acid L206  . In addition, GNAS mutation status was determined using exome sequencing  or Sanger Sequencing of the hotspot region between amino acids R201 and Q227 (CPAGNASWT n = 5). The methods were carried out in accordance with the approved guidelines. Detailed patient and PRKACA mutation data of all CPA are listed in Supplementary Tables Formalin-fixed paraffin embedded (FFPE) tissues from patients that underwent surgery due to adrenocortical adenomas (n = 103) or carcinomas (n = 33) were consecutively collected at the University Hospitals of Wuerzburg (n = 115), Munich (n = 9) and Padua (n = 12). In addition, 15 FFPE tissues from normal adrenal glands from patients that underwent adrenalectomy due to kidney tumours were consecutively collected at the University Hospital Wuerzburg. Furthermore, fresh frozen tissues of 34 CPA were collected consecutively at the University Hospitals of Wuerzburg (n = 15) and Munich (n = 9), and the Hôpital Cochin Paris (n = 10)25, Weiss score, Ki67 index, presence and number of distant metastasis, and detailed follow-up information were collected through the German ACC and the ENSAT Registry (www.ensat.org/registry)26. Malignancy and hormonal hypersecretion were defined according to established clinical, biochemical, and morphological criteria. The patient characteristics are listed in the Supplementary Tables Clinical parameters, such as sex, age at diagnosis, tumour size, and laboratory hormone analysis, and in case of adrenocortical carcinoma (ACC), tumour stage according to the European Network for the Study of Adrenal Tumours (ENSAT) classificationFull FFPE slides were deparaffinised and antigen retrieval was performed in 10 mM citric acid monohydrate buffer (pH 6.5) in a pressure cooker for 13 min. Unspecific binding sites were blocked with 20% human AB serum at room temperature (RT) for 1 h and primary antibodies were incubated in an appropriate dilution concentration at RT for 1 h . Signal amplification was achieved by En-Vision System Labelled Polymer-HRP Anti-Rabbit (Dako) for 40 min and developed for 10 min with DAB+ Liquid Kit (Dako). Nuclei were counterstained using Mayer’s hematoxylin for 2 min. Specificities of antibodies were determined by WB. All antibodies gave one specific band at the predicted size.28. A semi quantitative H-score was then calculated by multiplying the staining intensity grading score with the proportion score as described previously29. Where discrepancies were observed, results were jointly assessed by both investigators and the final score was formed by consensus. The Spearman r for inter-observer agreement for each staining was high (>0.85).Chromogenic staining intensities were determined by two independent investigators (I.W. & S.S.) and graded as 0 (negative), 1 (low), 2 (medium) and 3 (high). The proportion of positive tumour cells was calculated for each specimen and scored 0 if 0% were positive, 0.1 if 1–9% were positive, 0.5 if 10–49% were positive and 1 if 50% or more were positiveFull FFPE slides were deparaffinised and antigen retrieval was performed as described in the previous chapter. Unspecific binding sites were blocked with 20% human AB serum in TBS and to reduce auto fluorescence, cold water fish skin gelatine (Aurion) was added to a final concentration of 0.1% to the blocking buffer and incubated at RT for 30 min. Primary antibodies were incubated in an appropriate dilution concentration overnight at 4 °C . Fluorescence labelled secondary antibodies against rabbit  and mouse  were used at RT for 3 h. Vectashield with DAPI (Vector Laboratories #H1200) was used as nuclear counterstain and mounting medium. Optical images were acquired using a Leica SP5 confocal microscope.PRKACA (Hs00427274_m1) and all PKA regulatory subunits . Endogenously expressed β-actin (Hs9999903_m1) was used for normalization. 5 ng cDNA were used for each PCR reaction and each sample was analysed in duplicate. Transcripts were amplified using the TaqMan Gene Expression Master Mix (Thermo Fischer), the CFX96 real-time thermocycler (Bio-rad) and Bio-Rad CFX Manager 2.0 software. Cycling conditions were 95 °C for three min followed by 50 cycles of 95 °C for 30 sec, 60 °C for 30 sec, and 72 °C for 30 sec. Using the ΔCT method30, the gene expression levels were normalized to those of β-actin.RNA from fresh frozen tissues was isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen) and reverse transcribed with the QuantiTect Reverse Transcription Kit (Qiagen). qRT-PCR was performed using pre-designed Taqman gene expression probes (Thermo Fischer Scientific) for 20 mg of tumour tissue were shredded in lysis buffer containing 1% Triton-X and protease inhibitors. After sonication, cells were frozen at −80 °C, thawed and protein concentrations were determined using a bicinchoninic acid (BCA) kit (Sigma-Aldrich).31. Proteins were transferred by tank blot onto a PVDF Membrane  and membranes were blocked in 5% skimmed milk in TBS-Tween at RT for 1 h. Primary antibodies  were incubated over night at 4 °C. Membranes were washed 3 times in TBS-Tween and HRP-labelled secondary antibodies  were diluted 1:10000 and incubated at RT for 1 h. The protein-antibody complex was visualized by enhanced chemiluminescence using the Amersham ECL Prime reagent  and documented on X-ray film (Fuji). Normalization of protein levels was performed by re-probing the blot with an antibody recognizing human GAPDH (Sigma-Aldrich 1:10000). Autoradiographs were scanned, and the quantification of individual bands was performed using the FIJI software32.12 µg protein from each sample was loaded on a 10% denaturing gel and proteins were separated by SDS-PAGEThe Mann-Whitney- U test was used to determine differences between two non-parametric datasets and Kruskal-Wallis test with Dunns post-test were used to determine statistically significant differences between more than two non-parametric datasets. Statistical analyses were performed using GraphPad Prism (version 6.0). P values < 0.05 were considered as statistically significant.PRKACA), aldosterone producing adenoma = APA (n = 20), EIA (n = 25) and ACC (n = 33) was performed for all PKA regulatory subunits and the catalytic subunit α. While Cα protein levels were not altered between PRKACAmut CPA  and WT (n = 20) CPA Fig.  and PRKACPA Fig. , CPAs inCPA Fig. . RIα hadCPA Fig.  comparedCPA Fig. . OverallCPA Fig. .Figure 1PRKACAwt and PRKACAmut CPA for the regulatory subunits IIα and Iβ    Fig. . However05) Fig. . A few PPRKACAmut CPA vs PRKACAwt CPA: 0.08 ± 0.12 vs 0.86 ± 0.53, p < 0.0001) . Also Cα protein levels were reduced in mutated compared to WT samples although not to the same extent  Fig. . In geneent Fig. . Since a Fig. . IPRKACAmut and PRKACAwt CPA for all the PKA subunits we tested . Co-staining of RIIβ and several different organelle markers in adrenocortical tumour cells NCI-H295R, revealed a co-localization of RIIβ with the Golgi apparatus but not with the endoplasmic reticulum, mitochondria or lysosomes     Fig. . The pat89) Fig. . Thus, t24. There were some discrepancies between the two studies, with Mantovani et al. demonstrating that a loss of RIIβ results in a compensatory increase of RIα protein levels in Y1 cells, while Vincent-Dejean et al. failed to observe this phenomenon in tumour samples. Furthermore, the latter found decreased RIIβ protein levels only in a subpopulation of CPA with increased PKA activity. This discrepancy came as a surprise, as the compensatory up-regulation of one regulatory PKA subunit in response to down-regulation of the other has been shown in several systems34. One study demonstrated that also mutations in the regulatory subunit RIα, leading to its decreased expression, induced the compensatory mechanism leading to an increase of other regulatory subunits34. The down-regulation of RIIβ in CPA could not be explained for some time. Recently, we could prove that mutations in the Cα subunit decrease the binding potential of regulatory subunits21 and already many years ago it was demonstrated, that unbound regulatory subunits are normally degraded in the proteasome in a ubiquitin-dependent manner35. Following the discovery of mutations in the gene encoding for the catalytic subunit α of PKA as the underlying cause of CPA tumourigenesis6 we hypothesized that these mutations might be causative for the decreased RIIβ protein expression in these tumours. Therefore, we assessed the protein levels of PKA subunits by both immunohistochemistry and immunoblotting in a large series of adrenocortical tumours with known PRKACA mutation status, including 10 samples that had already been included in the study by Vincent-Dejean et al.23.In 2008 two studies had shown that protein expression of the RIIβ subunit of PKA is markedly reduced in CPA compared to other adrenocortical tumours, while no such differences were observed regarding the expression of the other regulatory subunits24 but were particularly pronounced in a subpopulation of CPA with high PKA activity23, we hypothesized, that this subpopulation is the PRKACA mutated CPA group. This hypothesis was confirmed by our immunohistochemistry results where CPA with mutations in the PRKACA gene showed reduced RIIβ protein levels compared to PRKACAwt CPA. We also observed significantly reduced RIα protein levels in PRKACAmut compared to PRKACAwt CPA but no significant differences concerning the other two regulatory subunits or the catalytic subunit itself. One possible explanation for this down-regulation could be the general differences in abundance of these two regulatory subunits in the adrenal cortex, so the degradation of unbound regulatory subunits failing to bind the mutated catalytic subunits may be stronger in the case of the subunits with higher adrenal expression.Since reduced RIIβ protein levels were found in CPA in generalPRKACAwt CPA that lacked immunoreactivity for RIIβ in immunohistochemistry was from a patient with autonomous cortisol secretion and did not reveal any somatic mutations affecting the cAMP/PKA signalling pathway. Interestingly, in this sample, by whole exome sequencing, we could identify a germline genomic variant in the GNAS gene  and involved in cAMP regulation) 11, however, the clinical significance of this SNP has never been investigated before.Surprisingly, the only et al. found decreased RIIβ protein levels in CPA compared to ACC24. In our series only the PRKACAmut CPA had significantly decreased RIIβ levels compared to ACC but not WT CPA. Neither did we observe any significant differences between the other PKA subunits between any of the adrenocortical adenomas subgroups, nor when we compared them with adrenocortical carcinomas. This previous study also showed that in the low RIIβ group there was also a significant reduction in RIIα protein and a tendency for reduced Cα and RIα subunits protein expression. Although we did not investigate RIIα with immunoblotting, we did not observe reduced RIIα protein levels in PRKACAmut CPA by immunohistochemistry. However, what we could show was that the Cα and RIα protein levels were both significantly reduced in PRKACAmut compared to PRKACAwt CPA. The discrepancy between these results and the fact that immunohistochemistry did not reveal a similar reduction for the Cα could arise from partly different samples and also from the two methods used. But also other groups found a reduced expression of the mutant Cα protein8 in the tumours.Mantovani PRKACA mutations, decrease of RIIβ protein levels was observed in only some of the other mutated tumours. Highest RIIβ expression was observed in the sample carrying the p.E32V mutation11, which lies outside the interface of regulatory and catalytic subunits and does maybe not influence the formation of a stable PKA holoenzyme. Unchanged RIIβ protein expression in western blot analysis was also found in the sample carrying the p.245_248.del mutation11, however, this tumour is from a patient without signs of clinical Cushing Syndrome, suggesting that the effects of this mutation are less severe.In contrast to the samples carrying the frequent L206R PRKACAmut samples compared to PRKACAwt CPA, therefore a post-translational regulation and/or degradation is a more likely explanation for the decrease in protein levels. In 2011, it was shown that the E3 ligase Praja2 interacts with the regulatory subunits Iα, IIα and IIβ, tagging them for proteasomal degradation in order to prolong the activated PKA signalling36. However, our results indicate a more restricted mechanism of degradation, especially for RIIβ in PRKACAmut samples, since RIIβ protein levels were completely absent in most samples. This degradation could be mediated by a RIIβ-specific A-kinase anchoring protein (AKAP), that modifies either its subcellular localization or tags it for proteasomal/lysosomal degradation. Indicative for such a degradation tagging would be the change of RIIβ protein localization between PRKACAwt and PRKACAmut samples towards a more proteasomal/lysosomal localization. As we hypothesized that the PRKACA mutations possibly changes RIIβ subcellular localization we checked if RIIβ is also co-localized with the golgi apparatus in tissues derived from PRKACAwt and PRKACAL206Rmut CPA or if this localization changes in the mutated samples. A co-localization of RIIβ with the Golgi apparatus has already been described in different cell types38. This hypothesis proved not to be true so we don’t have yet a conclusive answer for the reasons leading to low levels of RIIβ in PRKACAL206Rmut CPA. Now, additional functional studies would be necessary to answer this question but they are beyond the scope of this publication.As at mRNA level, we did not observe any downregulation of RIα and RIIβ in 39 found exactly the opposite, no to low levels of RIIβ and RIα in normal thyroid tissue but both subunits had increased levels in both benign and malignant thyroid tumours39 showing that PKA function in endocrine organs is very variable.Interestingly, other groups investigating the expression of the PKA subunits in other endocrine tissues such as thyroid and thyroid derived tumours38. Here we have demonstrated further, that some PKA subunits are not only organ specific, but that their expression is different between the different functional zones of the adrenal cortex, indicating possible specific roles in the secretion of hormones. The catalytic subunit α is most abundantly expressed in the zona glomerulosa, the zone of the adrenal cortex that secretes mainly mineralcorticoids. However, the strong expression of Cα seems to be compensated by a strong expression of all regulatory subunits, with the exception of RIβ, in this zone, suggesting a high level of regulation through formation of an inactive PKA heterotetramer. RIβ showed the most limited expression pattern in the adrenal gland being restricted to the zona reticularis, responsible for sex hormones secretion. Therefore, it could be involved in the regulation of these hormones. RIIα was the only subunit that showed expression in all zones of the adrenal cortex and additionally in the adrenal medulla, a part of the adrenal gland of neuroendocrine origin that secretes catecholamines. RIIβ, although considered to be one of the main regulatory subunits in the adrenal glands, showed only a weak to medium immunoreactivity in the zona fasciculata, which produces mainly cortisol but was abundant in the zona glomerulosa and zona reticularis. Together with the fact that low RIIβ expression is associated with an increase in cortisol production in a subset of adrenocortical adenomas, this suggests that at least in the adrenal cortex this subunit is responsible for the negative regulation of cortisol production.A differential expression of several PKA isoforms in different tissues or subcellular compartments has already been describedPRKACA mutations and reduced protein levels of two regulatory subunits of PKA in a sub-group of CPA and that specific expression overlapping the different functional zones of the adrenal cortex, could indicate distinct roles of the different PKA regulatory subunits in the secretion of different hormones.In conclusion, our study shows a direct link between Supplementary information


The results of this study provided valuable information to local administrators on the water quality of Küçükçekmece Lake, which is a large water resource of the Istanbul Metropolitan Area. Results also show that such a methodology structured by use of reflectance values provided from satellite imagery, in situ water quality measurements, and basin land use/land cover characteristics obtained from images can serve as a powerful and rapid monitoring tool for the drinking water basins that suffer from rapid urbanization and pollution, all around the world.This study focuses on the analysis of the Landsat-5 TM + SPOT-Pan (1992), IRS-1C/D LISS + Pan (2000), and Landsat-5 TM (2006) satellite images that reflect the drastic land use/land cover changes in the Küçükçekmece Lake region, Istanbul. Landsat-5 TM satellite data dated 2006 was used for mapping water quality. A multiple regression analysis was carried out between the unitless planetary reflectance values derived from the satellite image and


Soluble forms of oligomeric beta-amyloid (Aβ) are thought to play a central role in Alzheimer’s disease (AD). Transgenic manipulation of methylation of the serine/threonine protein phosphatase, PP2A, was recently shown to alter the sensitivity of mice to AD-related impairments resulting from acute exposure to elevated levels of Aβ. In addition, eicosanoyl-5-hydroxytryptamide (EHT), a naturally occurring component from coffee beans that modulates PP2A methylation, was shown to confer therapeutic benefits in rodent models of AD and Parkinson’s disease. Here, we tested the hypothesis that EHT protects animals from the pathological effects of exposure to elevated levels of soluble oligomeric Aβ. We treated mice with EHT-containing food at two different doses and assessed the sensitivity of these animals to Aβ-induced behavioral and electrophysiological impairments. We found that EHT administration protected animals from Aβ-induced cognitive impairments in both a radial-arm water maze and contextual fear conditioning task. We also found that both chronic and acute EHT administration prevented Aβ-induced impairments in long-term potentiation. These data add to the accumulating evidence suggesting that interventions with pharmacological agents, such as EHT, that target PP2A activity may be therapeutically beneficial for AD and other neurological conditions. AD is currently the 6d States , and thed States –4. Here EHT was identified in a screen for natural compounds that enhance the activity of the serine/threonine protein phosphatase, PP2A, toward phospho-protein substrates associated with AD and PD . MultiplPP2A is a heterotrimeric protein composed of a catalytic subunit (C), a structural subunit (A), and a regulatory subunit (B). Multiple isoforms exist for each of these subunits and they are assembled in a highly-regulated process . The ideAβ is a 40–42 amino acid peptide generated by proteolytic cleavage of the amyloid precursor protein (APP). Aβ is the primary constituent of the amyloid plaques that characterize AD, and soluble forms of Aβ produce AD-related impairments in cell and animal models . To examin vivo EHT administration in the current experiments. After 7–10 days of recovery from surgery, animals were then tested on a battery of behavioral tasks conducted over the course of 2 weeks in the following order: open field behavior, 2-day radial arm water maze, contextual fear conditioning, visible platform water maze and sensory threshold assessment. Prior to testing in each task, animals were infused with 1 μl per side of 200 nM synthetic oligomeric Aβ peptide or vehicle as described in [in vivo dose selected for these studies corresponds to the threshold at which maximal cognitive impairment was observed [To test the effect of EHT on Aβ-induced cognitive impairments, we shifted 3-month-old wild-type mice to diets containing either 0, 0.01 or 0.1% EHT , as wellribed in . Oligomeribed in . In earlribed in , 27, andWe found that animals treated with diets containing either 0.01 or 0.1% EHT were resistant to Aβ-induced impairments in a contextual fear conditioning task . This taTo test for possible differences in baseline activity levels among these groups that might confound our interpretation of their behavior in the contextual fear condition task, we examined their behavior in an open field environment. To test for possible differences in shock perception among these groups that might affect their performance in the contextual fear condition task, we also examined their behavioral responses to a range of shock intensities. We found that neither dietary EHT administration nor Aβ infusion 20 min prior to testing affected their ambulatory activity, as assessed by total distance travelled  and immoAs an additional test of EHT’s ability to protect against Aβ-induced cognitive impairments, we tested these animals on 2-day radial arm water maze task. This task is a test of short-term spatial memory and requires animals to learn and remember the identity of visual cues to navigate to a specific location. It is also hippocampus-dependent and sensitive to elevated Aβ levels . We founTo test for potential differences in visual perception, motivation or swimming ability among these groups that might affect their performance in the 2-day radial arm water maze task, we assessed their performance in a visible platform version of the Morris water maze. We found no significant differences among these groups in either escape latency , or swimin vitro and in vivo dose-response relationships for EHT’s protective effects, they are also consistent with the higher efficacy of 0.01% EHT-containing diets in protecting against Aβ-induced vs. α-synuclein-related impairments in that study.Activity-dependent changes in the efficacy of synaptic transmission within the hippocampus are thought to be required for particular forms of learning and memory, and interference with these changes, caused by elevated levels of Aβ is thought to contribute to AD-associated cognitive impairments . To detein vivo, we prepared acute hippocampal slices from the same vehicle-infused EHT treated animals used in the behavioral assays. We then performed extracellular field potential recordings of LTP at Schaffer collateral synapses in these preparations in the presence or absence of 100 nM Aβ. As expected, TBS produced robust potentiation of responses in vehicle-treated slices prepared from animals that received control diet that were significantly reduced by Aβ treatment .ad libidum as described in the text. Synthetic Aβ peptide corresponding to amino acids 1–42 of human amyloid precursor protein was purchased from the UCLA Biopolymer Laboratory  and oligomerized according to previously described protocols [EHT was prepared and synthesized at Signum Biosciences as described in . For acurotocols , 54  into the dorsal part of the hippocampi  [For experiments involving 1.30 mm)  under anAnimals were placed into a plexiglass chamber (27.3 cm long × 27.3 cm wide × 20.3 cm high) for 10 min on each of two successive days during which time their movements were tracked using a arrays of infrared beams and a computerized tracking system and analyzed using behavioral analysis software (Med Associates).Testing was performed in a 120 cm diameter pool containing a six arm radial maze insert and filled with opaque water as described previously . Mice weAnimals were placed into a conditioning chamber (33cm x 20cm x 22cm) made of transparent Plexiglas on two sides and metal on the other two located inside a sound-attenuating box (72cm x 51cm x 48cm) . Background white noise (72dB), was provided through a speaker installed in one of the side of the sound-conditioning chamber and illumination was provided by a 24 W bulb. A clear Plexiglas window allowed the experimenter to record and monitor freezing behavior using a video camera connected to a personal computer. Foot shocks were administered through a removable 36-bar grid floor and the entire apparatus was cleaned and deodorized between animals with distilled water and 70% ethanol. Animals were placed in the conditioning chamber once on each of two consecutive days. On the first day of exposure mice were placed in the conditioning chamber for 2 minutes before the onset of a discrete 30s, 2800Hz, 85dB tone, the last 2s of which coincided with a 0.5 mA foot shock. After the tone and shock exposure, the mice were left in the conditioning chamber for another 30s before returning to their home cages. 24 hours after their first exposure, animals were returned to the conditioning chamber for 5 min without foot shock or tone presentation. Freezing behavior during all phases of testing was calculated using FreezeFrame software (Med Associates).This task was conducted in the same 120 cm diameter pool used for the radial arm water maze task but with the partitions removed. Training for this task was carried out over 2 days with 3 morning and 3 afternoon trials on each day. Intertrial intervals were 15 to 20 min and rest periods between morning and afternoon sessions were 2–3 hrs. Each trial was a maximum of 120 sec during which time the animals were required to swim to a visible escape platform located just above the water surface. Animals that did not reach the platform within the allotted time were guided to it and allowed to sit there for 15 sec before returning to their home cage. The location of the platform was varied among 4 different locations such that it was not present in the same location on any two successive trials. Water temperature was maintained at approximately 24°C, and animals were dried and placed in a clean warmed cage after each trial to prevent hypothermia. Animal movements were recorded using a video-tracking system and time required to reach the hidden platform (latency) and swim speed were determined using Ethovision behavioral analysis software (Noldus).Animals were placed into the same apparatus used for contextual fear conditioning. A sequence of single, 1sec foot shocks were then administered at 30 sec intervals and 0.1 mA increments from 0 up to a maximum of 0.7 mA. Each animal’s behavior was monitored by the experimenter to determine their thresholds for first visible response to the shock (flinch), their first gross motor response (run/jump), and their first vocalized response.2HPO4, 25 NaHCO3, 2 CaCl2, 2 MgCl2, and 10 glucose. Hippocampi were the dissected and sliced into 400 μM sections using a tissue chopper. Slices were incubated at 29°C in an interface chamber under continuous perfusion (2 ml/min) with oxygenated ACSF and allowed to recover for a minimum of 90 min prior to recording responses in the CA1 region to stimulation of Schaffer collateral projections with a bipolar electrode. Input/output relationships were determined prior to each recording and stimulus intensities that elicited 30% of the maximal response were utilized. Stable baselines were obtained for a minimum of 15 min prior to drug or vehicle application and a theta-burst stimulation protocol consisting of 3 trains separated by 15 second intervals with each train consisting of 10 bursts at 5 Hz and each burst consisting of 5 pulses at 100 Hz was used to elicit LTP.Extracellular field potential recordings were performed on acute hippocampal slices prepared as described previously  from wilAfter 4 weeks of dietary EHT administration animals were euthanized by cervical dislocation–an AVMA approved method that allows for rapid removal of non-hypoxic brains. Hippocampi were then rapidly dissected, snap frozen and stored prior to homogenization for western blot analysis. Hippocampal homogenates were prepared by sonication at 95°C in aqueous buffer containing 2% lithium dodecyl sulfate and 50 mM Tris pH 7.5. Total protein concentrations were determined by bicinchoninic acid assay according to the manufacturer’s instructions (Pierce) and 15 μg of total protein was loaded per lane on SDS-PAGE gels. Proteins were transferred to PVDF membranes, which were then blocked with Odyssey Blocking Buff (TBS) (LI-COR) for 1 hr at rm temp. Blots were probed with primary antibodies  overnighTo demethylate PP2Ac, the following method was modified from Yu et al. . HomogenS1 FigRepresentative western blot of synthetic Aβ preparation used in behavioral and electrophysiological assays. Synthetic peptide corresponding to human Aβ 1–42 sequence was incubated for 24 hrs at 4°C in artificial cerebral spinal fluid. The incubated preparation was then separated by SDS-PAGE on a 10% NuPAGE bis-Tris gel, and probed with the monoclonal antibody 6E10 which recognizes an epitope contained within residues 1–17. The pattern of immunoreactivity observed is consistent with that originally reported in Stine et al,  and in s(TIFF)Click here for additional data file.


The chromatic mechanical response originates from the d orbitals and is related to the strength of the direct exciton resonance A and B of the few layer 2H-MoS2 affecting optical absorption and subsequent mechanical response of the nanocomposite. Applying uniaxial tensile strains to the semiconducting few-layer 2H-MoS2 crystals in the nanocomposite resulted in spatially varying energy levels inside the nanocomposite that enhanced the broadband optical absorption up to 2.3 eV and subsequent mechanical response. The unique photomechanical response in 2H-MoS2 based nanocomposites is a result of the rich d electron physics not available to nanocomposites based on sp bonded graphene and carbon nanotubes, as well as nanocomposite based on metallic nanoparticles. The reversible strain dependent optical absorption suggest applications in broad range of energy conversion technologies that is not achievable using conventional thin film semiconductors.The ability to convert photons of different wavelengths directly into mechanical motion is of significant interest in many energy conversion and reconfigurable technologies. Here, using few layer 2H-MoS Establishing an optical-mechanical interface has been attempted since 1881 when A.G. Bell used the optoacoustic effect (photophone) to produce sound in a gas with chopped beam of sun light2623458910118910117m−1 2 has an indirect band gap of ~1.29 eV2 is determined by strong covalent bonds resulting from the overlap between the 4d and 3p electron orbitals of Mo and S respectively2 is expected to be E2D/9, where E2D is the in-plane stiffness as per the theory of rupture and flow in solids25272829303132Layered transition metal di chalcogenide (TMDs) provide intriguing opportunities to develop low cost, light and wavelength tunable stimuli responsive systems that are not possible with their conventional macroscopic counterparts. TMDs are stack of triple layers with transition metal layer between two chalcogen layers. While the atoms within the layers are chemically bonded using covalent bonds, the triple layers can be mechanically/chemically exfoliated due to weak van der Waals bonding between the layers. Due to the large optical absorption ~107m−1  in these2, unique layer by layer process of nanocomposite fabrication, and strain engineering, here we present direct chromatic mechanical response in MoS2-polymer nanocomposites between 405 nm–808 nm. The advantage of our fabrication methods are as follows: (1) liquid phase exfoliation process resulting in high quality few layers with distinctive direct electron transition peaks, (2) phase separated nanocomposite due to the layer by layer processing, (3) access to the unique layer dependent optical absorption properties of MoS2 layers inside the nanocomposite, (4) the reported architectures enables the creation of spatially varying bandgap profiles of 2-D nanomaterials and study of resonance Raman scattering as a function of number of layers and (5) the design enables scalable and flexible process for developing stimuli responsive and energy conversion devices at macro, micro and nanoscopic length scales utilizing different polymeric systems. Further, the layer by layer process as reported here enables preservation of the intrinsic properties and eliminates environmental degradation effects such as oxidation. The reported photomechanical actuators based on this process not only showed strong and wavelength dependent light matter interaction in the visible region of the electromagnetic spectrum, but also showed unexpected deviations. Not only, we found that the photo-thermal energy transduction from the MoS2 crystals to the polymer chains is high resulting in high amplitudes of photomechanical actuation, it is also dependent on the wavelength of light and the photo-thermal actuation is better than graphene2 additives  compared to bulk2 show strong peak and enhanced light-matter interaction in the visible region of the electromagnetic spectrum due to the van Hove singularities in the density of statesEg~1.9 eV24 with i22 thin films −1 m−1; for intermediate layer αintermediate-layer = 1133.8 (mg/ml)−1 m−1; and αfew-layer = 2230 (mg/ml)−1 m−1. The extinction coefficient increases with decrease in number of layers. Since extinction is a measure of absorption and scattering, and since the few layer nanoparticles were small (<100 nm), the large values of extinction is a result of optical absorption in the semiconducting few layers. K pointK point2 (~1 μm) samples in solution.2 suspensions, shear mixing of PDMS base with cross-linkers, formation of layer of cross-linked poly dimethyl siloxane (PDMS) on glass substrate using spin coating, curing and post-curing of PDMS, MoS2 transfer to the PDMS and spin coating another identical second layer of PDMS on to the transferred MoS2 layer and crosslinking and final step of curing and post-cure relaxation for 12 hours. The MoS2 concentrations for all the three samples were maintained at 0.1 wt. % and the final nanocomposites are presented in 2 additives and similar levels of scattering in all the nanocomposites2 in between. The particles were seen to be along the line indicated in red  and A1g (~409 cm−1) peaks, which correspond to the in-plane and out-of-plane vibrational modes of the S atoms with respect to the Mo atoms, are dominant features of the non-resonant Raman spectrum of 2H-MoS2Significant changes in the shape of lines are observed between bulk, intermediate and few layer samples in −1 that consists of E1g + XA (~466 cm−1) peak (XA is a transverse phonon from the vicinity of M point of the Brillouin zone)441 (~441 cm−1) peak assigned to a second-order band (A1g(Γ) + E22g(Γ)), L2 (~455 cm−1) peak to a van Hove singularity between K and M and L3 (~460 cm−1) peak due to LA′ phonon from the vicinity of M point2 additives. Other significant lower-frequency lines are the “b” (~420 cm−1) peak due to a two-phonon Raman process involving the emission of a dispersive quasi-acoustic phonon and an optical E21u phonon along the c-axis, the B1u(Γ) (~404 cm−1) mode, which is the Davydov couple of the A1g (Γ) mode and the “c” (~380 cm−1) peak due to a dispersionless E21u (Γ) phonon−1 and 700 cm−1(1g(M)+2XA (~644 cm−1) peak1g(M)+LA(M) (~466 cm−1) peak, E12g(M) + LA(M) (~600 cm−1) peak and 2E1g(Γ) (~572 cm−1) peak.In the bulk nanocomposite, the strongest spectral feature is a broad line located around 460 cm 700 cm−1, includi1u (Γ) peaks are also seen to increase in intensity. Both these peaks are Raman-inactive and their appearance suggests symmetry breaking2 atoms between the layers arranged in the few layer sample.Several significant changes of the spectrum are observed with the decrease in the number of layers, especially for the few layer nanocomposite. First, the “b” peak shows higher intensity which is an indicative of the phonon involved that has a wave vector along the c-axis. The “c” and B2 crystal leads also to significant increase of the L1 and L2 peaks  inside the optically transparent PDMS suggest most of the light may be transmitted through the sample rather than scattered. Further experiments were conducted to understand how optical absorbance, scattering and photomechanical response change with increase in weight fractions of bulk MoS2. 2 additive. The transmitted power is also plotted on the same graph. An exponential decrease in transmittance is seen between 0.1 wt. % and 1 wt. % at all wavelengths. Above 1 wt. %, the power transmitted is almost zero suggesting strong light absorption. The rise in photomechanical actuation follows the same exact trend as the power absorbed by the sample. If the exponential decrease in transmittance in 2 additive. The A/l is seen to follow the photomechanical actuation stress amplitudes. The extinction, absorbance and photomechanical stress amplitudes all increase with increase in MoS2 additive even for bulk over all the wavelength (300 nm to 1100 nm). Above 1 wt. % non-linear behavior is noted and saturation of optical absorption and photomechanical response. Thus we see two regimes in bulk nanocomposites. At low weight percentage of bulk MoS2 additive (~0.1 wt. %), high light transmission through the sample and geometric scattering cause lower photomechanical response. However, as the weight fractions of bulk MoS2 additive increases in the nanocomposite, optical absorption becomes dominant resulting in significant increase in photomechanical response. These results are also validated by the presence of van Hove singularity  of the chalcogen atom couples this absorbed light due to the superposition of the 4d orbitals of Mo and 3p orbitals of S in the conduction band into an extraordinary thermal effect thus giving rise to this chromatic photomechanical effect. This is a unique mechanism not available to sp bonded materials such as carbon nanotubes and graphene, as well as plasmonic metallic gold nanoparticles.In MoS1, b1, c1, a2, b2, and c2 that is dependent on the wavelength  that resulted in significantly better photomechanical response. It should be noted that although the design enables high strains to be applied to the sample, the strain transfer to the MoS2 additives inside the sample is between 1–5% for all the strains applied2 (1–6 layers) stacked on top of one another, stretching the sample globally must result in strain gradients and differential shift in the layers resulting in spatially varying energy levels of the stacked crystals that led to increase in optical absorption. Our results also suggest one can tune the power absorbed almost all wavelengths in the visible region in a macroscopic device with strains. Thus the strains, optical absorption and photomechanical response can be continuously, rapidly and reversibly tuned in these nanocomposites at different wavelengths and offers room for development of flexible energy harvesting devices in the future with advanced designs of our nanocomposites with scalability of localized strain engineering into macroscopic response. This does not happen in carbon nanotube911Finally, another way to confirm that this is strain induced reduction in bandgap is to measure the power absorption directly at different strains . The resplied2 1– layers s2 composites to a known illumination source was evaluated−5% under quasi-static motion, about three orders of magnitude less efficient than our exfoliated MoS2-elastomer systems (~6 × 10−2%). Finally, the efficiency was twice as high as recently reported graphene-elastomer photo-mechanical actuators (0.03%)2 few-layer nanocomposite is fully reversible and robust actuation mechanism. Second, the strain based optical absorption experiments did not delaminate the interface/sample, which would otherwise cause an irreversible change in the amplitude of photomechanical actuation stress (a significant reduction in stress) due to increased scattering in these long term experiments. Third, the strain gradients on the MoS2 crystals and the change in bandgap is also completely reversible as the sample exhibits pristine-like behavior (0% strain behavior) with the same amplitude of mechanical response in these long term experiments. The strain transfer to the MoS2 additive is thus in line with previous reports2 additive. Thus the high strength and strong light-matter interactions in 2H-MoS2 giving rise to this robust chromatic mechanical response is not only a new mechanism but also a new material design for future macroscopic photo mobile polymer networks based on TMDs.2 solution was obtained by 7-hour bath sonication of MoS2 Ultrafine Powder obtained from Graphene Supermarket. The intermediate solutions were sonicated for 50 hours, and then centrifuged at 1500 rpm for different time durations (45 to 120 minutes). The few layer samples were bought from Graphene Supermarket in ethanol and used as such. The number of layers were characterized for each type of suspension using AFM. The AFM images were taken using a NaioAFM of Nanosurf in tapping mode with a cantilever resonance frequency of ~148 kHz. HRTEM was conducted using an FEI Tecnai transmission microscope (TM) (model G2 F20). FFT was obtained from the HRTEM image. The operating voltage was set at 200 kV. The sample was prepared by dropping one drop of exfoliated MoS2 in ethanol solution on lacey-carbon Cu grids . The Cu grids were dried in air to make sure there are sufficient isolated flakes to be observed. SEM images were obtained using a JEOL JSM-7000F instrument at 20 kV of power and under an ultra-high vacuum, 10−5 Pa. Secondary electron detector was utilized at 8 mm working distance to capture high-resolution images of MoS2 flakes at magnifications as high as 100,000X. The optical absorbance of the MoS2 solutions and the nanocomposites were measured using a Hitachi U–5100 ratio beam spectrophotometer in a wavelength range of 300 nm-1100 nm.The bulk MoS2 in ethanol is then used (200 μg MoS2) for vacuum filtration process resulting in a mass per area of MoS2 nanoparticles of 0.18601 g/m2 on filter membrane (Whatman Anodisc). Area is calculated based on the diameter of the deposited film. Here 47 mm diameter Anodisc membrane after vacuum filtration resulted in 37 mm diameter of deposited mass. This resulted in 0.18601 g/m2. This layer is then transferred to an 80 μm thick PDMS layer and then another layer of 80 μm thick PDMS is spun coated on top of this layer. The mass of the two layers of PDMS is then measured by cutting known areas of the neat PDMS (i.e. the clear part without any MoS2) and calculating the mass/area of PDMS layers. This process is repeated several times for each sample to get averages. The ratio of the mass per area of the MoS2 to that of the mass per area of the PDMS gives the weight percentage of the MoS2 in the composite samples (i.e. 0.1 wt. %).10.0 mg as-received ultrafine nanopowder from Graphene Supermarket is weighed and added to 100 mL of ethanol (200 proof Sigma Aldrich), and sonicated for 7 hours in a bath sonicator. Using this solution, 2 mL of MoS2 nanoparticles removed from the top parts of each vial and depositing it on filter membranes. This process is repeated for several samples to ensure repeatability. The concentration of the used centrifuged solution for the intermediate nanocomposites is also verified using the Lambert-Beer law and optical absorbance method2 in the solution. This 11.628 ml sample is then vacuum filtered on a filter membrane  arriving at a mass per area concentration of 0.18601 g/m2. The same process as presented above in bulk is accomplished to reach a 0.1 wt. % of intermediate nanocomposites.The process for intermediate samples is the same as bulk except that we used 50 hours sonication and then the solution is centrifuged at 1500 rpm. The top and bottom parts of the solution is then separated similar to reported in literature2 solution (Graphene Supermarket) is used in the fabrication process. Because of ultra-small size of few-layer nanoparticles, transferring to PDMS layer cannot be accomplished from Anodisc membrane and is done using an evaporative drop deposition method. The initial concentration of the purchased few-layer solution as per manufacturer specification was 18 mg/L. 258 μL of this solution (4.65 μg of MoS2) is then deposited directly on the 5 mm × 5 mm sample area of the PDMS to reach the same mass per area as bulk and intermediate samples (i.e. 0.18601 g/m2). The final weight percentage (0.1 wt.%) calculation is the same as the bulk and intermediate samples after the second PDMS layer is spun coated on top of the PDMS/MoS2 layers.As-received MoS2/PDMS actuators were fabricated based on a layer-by-layer (LBL) method. PDMS silicone elastomer obtained from Dow Corning (Sylgard 184) was used as the host matrix. PDMS is a two part solvent-free flexible silicone organic polymer in the form of a base compound with a separate hydrosilane curing agent that acts as a cross-linker. The term cross-linking ratio (CLR) used throughout the paper refers to the ratio of PDMS cross-linker to the base compound. The PDMS base was mixed in 1:10 ratio to the cross-linker and then deposited on a glass slide. A standard spin coating process at 750 rpm for 90 seconds produces 80 μm thick film of PDMS on the glass slides. The films are then cured at 120 °C for 20 minutes and post-cured for 12 hours at room temperature. A uniformly distributed layer of MoS2 nanoparticles deposited on Whatman Anodisc inorganic membrane using a standard vacuum filtration process is then transferred into the first layer of PDMS. Another layer of PDMS is then spin coated and cured on top of the MoS2 layer resulting in a layer-by-layer structure of about 160 μm thick. The samples are then cut in 50 mm × 5 mm strips (30 mm of active test area with 10 mm on each side for fixture mounting). All experiments were conducted in a climate-controlled laboratory. The photomechanical testing system, strain application, lasers all form an automated system that is placed inside a black box to avoid stray light from interfering the experiments. The laser sources include wavelengths from ultraviolet to near-infrared regions  all with the same laser power of 50 mW and beam diameters of ~3 mm. The laser diode was placed ~100 mm from the middle of the test strip such that the illumination impacted normal to the PDMS/MoS2 surface. More detailed information on photomechanical stress tests are availableMoS2 nanocomposite actuators with 10% increments shown in 2 layer, the incident optical power was significantly less than 1 mW, not enough to induce heating effects in line with previous reportsA custom strain gadget was developed using design and laser cutting clamps. The stretching fixture was developed with 500 μm resolution and 5 mm initial clamps’ spacing. This fixture was used to study the effect of strain on the Raman shifts of polymer/MoSMicroscopic measurements of the nanocomposite optical spectra were performed in backscattering geometry with a resonant excitation. The 632 nm line of a He-Ne laser was used for these measurements. All measurements were done using an optical filter with an incident power of ~1 mW. Since there was an 80 μm PDMS layer on top, the incident power is significantly less than 1 mW, not enough to induce local heating effect in line with previous reports on resonant Raman scatteringHow to cite this article: Rahneshin, V. et al. Chromatic Mechanical Response in 2-D Layered Transition Metal Dichalcogenide (TMDs) based Nanocomposites. Sci. Rep.6, 34831; doi: 10.1038/srep34831 (2016).