The phase component of molecular states similarly determines their nonclassical supplements, due to probability “convection”. The local-energy concept is used to examine the phase equalization in the equilibrium, phase-transformed states. Continuity relations for the wavefunction modulus and phase components are reexamined, the convectional character of the local source of the resultant gradient information is stressed, and latent probability currents in the equilibrium (stationary) quantum states are related to the horizontal (“thermodynamic”) phase. The equivalence of the energy and resultant gradient information (kinetic energy) descriptors of chemical processes is stressed. In the grand-ensemble description, the reactivity criteria are defined by the populational derivatives of the system average electronic energy. Their entropic analogs, given by the associated derivatives of the overall gradient information, are shown to provide an equivalent set of reactivity indices for describing the charge transfer phenomena.In this article, sources of information in electronic states are reexamined and a need for the resultant measures of the entropy/information content, combining contributions due to probability and phase/current densities, is emphasized. Probability distribution reflects the wavefunction modulus and generates  These physical descriptors summarize different aspects of the state electronic structure: the probability density represents the static “structure of being”, while its flux characterizes the dynamic “structure of becoming”  and I[p], due to the logarithm of the state probability density of Equation (2), constitute the classical IT concepts of Shannon’s global entropy y events ,37:I[p] S[φ] and I[φ] = I[j], due to the state phase or its gradient generating the probability velocity:S[φ] and M[φ] ≡ −I[φ] = −I[j] are negative since the current pattern introduces an extra dynamic “order” into the system electronic “organization”, compared to the corresponding classical descriptors S[p] and M[p] = I[p], thus decreasing the state overall “uncertainty” content. These generalized descriptors of the resultant uncertainty (entropy) content S[ψ] in the quantum state ψ, or of its overall (gradient) information I[ψ]  ,12,13,14ion I[ψ] ,39,40.ψ, the classical probability functionals, of Shannon’s global entropy or Fisher’s intrinsic accuracy for locality events, are supplemented by the corresponding nonclassical complements S[φ] or I[φ] = I[j], respectively, due to the wavefunction phase or the electronic current it generates. In the overall  entropy . Indeed, the presence of finite currents implies more state spatial “order”, i.e., less electronic “disorder”. The resultant measure of the state average gradient information [T[ψ] = 〈ψ|T|ψ〉 = κ−1I[ψ].Therefore, the negative nonclassical entropy effectively lowers the state classical uncertainty measure ormation ,13,14,15r} of an electron exhaust the whole physical space and constitute the complete set of elementary particle-position events. The associated infinite and continuous probability scheme of the classical mapping {r→p(r)} in indeterminacy (uncertainty). It is best reflected by Shannon’s global entropy S[p], measuring a “spread” (width) of the probability distribution, since we know only the probabilities p(r) = |ψ(r)|2 of possible definite outcomes of the underlying localization experiment in the pure quantum state ψ. Another suitable classical probe of the average information content in p(r) is provided by Fisher’s probability functional I[p]. This gradient measure of the position determinacy reflects the “compactness” (height) of the probability distribution, thus complementing the Shannon global descriptor.In both the classical IT and in position representation of QM the admissible locations {probability scheme has been completely eliminated. The average information gained by such tests thus amounts to the removed position uncertainty. The larger the uncertainty in p(r), the larger the amount of information obtained when we eventually find out which electron position has actually been detected after the experiment. In other words, the amount of information given us by the realization of the classical, probability scheme alone equals the global entropy in the classical probability scheme of The information given us by carrying out the given experiment consists of removing the uncertainty existing before the experiment . If we cwavefunction scheme {r → ψ(r)} of r → p(r)} constitutes only a part of the overall (complex) mapping. In fact, the wavefunction mapping implies a simultaneous ascription to the parametrically specified electron position of the local modulus (static) and phase/current (dynamic) arguments of the state wavefunction, or the related local probability and probability velocity descriptors. This two-level scheme in QM ultimately calls for the resultant measures of the entropy/information content in quantum states, combining classical (probability) and nonclassical (phase/current/velocity) contributions. The difference between the resultant and classical information contents can be best compared to that between the (phase-dependent) hologram and (phase-independent) ordinary photograph.In QM, however, one deals with the φ and hence gives rise to a finite current density j. This probability flux vanishes only in the stationary state of Equation (6), for the purely time-dependent stationary phase st.φ(t): jst. = Vst. = 0. For such states an experimental determination of electronic position removes completely all the uncertainty contained in the spatial wavefunction st.R(r) and the probability distribution st.p(r) = st.R(r)2. Indeed, the quantum scheme of The resultant IT measures are in principle experimentally accessible, since the local probability velocity in physical space, defined by the velocity of probability current, is uniquely specified in QM. In other words, all static and dynamic arguments of the resultant IT descriptors are all sharply specified by the corresponding expectation values of the associated observables. However, the localization experiment alone cannot remove all the uncertainty contained in a general electronic state, which exhibits a nonvanishing local phase component j(r) includes the momentum operator of an electron, P(r) = −iħ∇,r(r) = r,r and j(r) do not have common eigenstates. In other words, these quantities cannot be simultaneously defined sharply, in accordance with Heisenberg’s uncertainty principle of QM. Therefore, the position dispersion rσ cannot be simultaneously eliminated with the current dispersion jσ in a single type of experiment, e.g., that of the particle localization. Indeed, a removal of jσ ultimately calls for an additional momentum experimental setup, which is incompatible with that required for determining the electronic position. Only the repeated, separate localization and momentum experiments, performed on molecular systems in the same quantum state, can fully eliminate the position and current uncertainties contained in a general electronic state. Neertheless, both the particle position r and the local convection velocity V(r) of the probability distribution are precisely defined as expectation values of the associated Hermitian operators. Therefore, their resultant IT functionals are all uniquely specified, with their densities exhibiting vanishing spatial dispersions.Since the current operator uncertainty S[φ], proportional to the state average phase 〈φ〉ψ, effectively lowers the information received from the localization-only experiment. The removable uncertainty in ψ(r) is then less than its classical content S[ρ] or M[ρ] = I[ρ]. In other words, the nonvanishing current pattern introduces an extra (dynamic) determinacy in the system electronic structure, which diminishes its resultant uncertainty (indeterminacy) descriptors.The nonclassical phase equilibria corresponding to phase-transformed quantum states,eq.φ ≡ eq.φ(r) for the specified probability density p(r) st.= p(r) in the stationary state ψ = st.ψ of Equation (6) marks the exact cancellation of the state classical (S[p]) and nonclassical (S[eq.φ]) entropy contributions:grand ensemble. The .},9have belsewhere ,12,13,14 entropy  and the eq.ψ: eq.φ ≥ 0 (phase convention) gives:The above condition determines the equilibrium  local phase for the conserved (stationary) probability distribution,wavefunction functionals:We can also observe that writing the average functionals for resultant entropy measures as expectations of the corresponding (multiplicative) operators,grand ensemble, the statistical mixture of molecular stationary states {|Ψji〉 ≡ |Ψj(iN)〉} for different numbers of electrons {iN}, defined by the corresponding density operator,ji = |Ψji〉〈Ψji| stands for the state projector. The average entropy or information—say, the resultant IT quantity G represented by the associated operator G, possibly state-dependent, G = G[Ψji] ≡ Gji, is given by the weighted average of the property state-expectations {jiG = 〈Ψji|G|Ψji〉}:ji = jiS Oji and the expectation value of entropy in state Ψjiexternal probability jiP in the mixture, shaped by thermodynamic conditions, and is devoid of any local  content of the constituent wavefunction distributions.Consider now the mixed quantum state in the  Neumann ,〈S〉ens.ji〉 the probability of finding an electron at the specified location r is given by the state internal distribution,jiρ(r). In thermodynamic ensemble it is given by the weighted average over such internal state densities {jip(r)}, with the state  probability weights {jiP}:P represents the normalized joint probability of finding in state Ψji an electron at r, with both its factors thus acquiring the status of conditional probabilities:S[{jiP}] of von Neumann and the weighted average of “internal” state contributionsOne would expect a similar feature in the overall IT description of molecular ensembles. In the pure quantum state |Ψmixed states of the open reactive complexes and their substrate subsystems, it would be desirable that in each phase-transformed pure state,eq.φ ≡ φh)((r) , the second  contribution of Equation (45), exactly vanishes. This is indeed the case when the internal entropy of each equilibrium state is exactly zero:phase-transformed states the thermodynamic and resultant equilibrium entropies are thus consistent with one another:For a consistent IT description of the equilibrium see also , equilibion (32) ,12,13,14jiρ(r) = i pjiN(r), Ψjieq., = Ψeq.[jiρ], since both Ψji = Ψji[jiρ], by the first Hohenberg–Kohn (HK) [eqφ. = eq.φ[jip].To summarize, the equilibrium “thermodynamic”  phase is proportional to the local probability logarithm. This is very much in spirit of density-functional theory (DFT) ,44,45,46ohn (HK)  theorem,phase-transformed states for conserved (stationary) probability distribution generates the mutual consistency between the external (ensemble) and internal (resultant) entropy descriptors. It implies that for the single stationary state the resultant global and gradient uncertainty descriptors of the specified wavefunction vanish in equilibrium, as indeed does von Neumann’s , due to the moving reference (monitoring) point. This separation applies to wavefunctions, their components, and expectation values of physical observables. In In Schrödinger’s dynamical picture the state vector |p = p gives:time derivative dp/dt determines the vanishing local probability “source”: pσ = 0. It measures the time rate of change in an infinitesimal volume element of probability fluid moving with probability velocity V = rd(t)/dt, while the partial derivative ∂p/∂t refers to a volume element around the fixed point in space. The divergence of probability flux in the preceding equation,V, related to the phase Laplacian ∇2φ = Δφ:V⋅∇) and ∇2 = Δ represent the “convection” and “diffusion”, respectively. Thus, in Equation (52), the local evolution of the particle probability is governed by the density “convection”, while the preceding equation implies the vanishing “diffusion” of the phase distribution.It directly follows from the SE that the probability field is sourceless:Iσ(t) due to a finite phase source φσ. The classical contribution to Iσ(t) identically vanishes due to the probability continuity of Equation (52). These relations directly follow from the molecular SE and identify the relevant local sources of the distributions of interest. In ħR and multiplying by exp[−iφ] gives the following (complex) dynamic relation linking the wavefunction components:imaginary parts generates the time evolution of the modulus part of electronic state,real parts of Equation (57) similarly determines the phase dynamicsφσ = dφ/dt in the phase-continuity relationV of the probability current j = p V also determines the phase flux and its divergence, the convection term in the continuity Equation (61): phase source:As an example, consider continuities of the wavefunction components. When expressed in terms of the state modulus and phase parts the SE reads:The component SE (57) also allows one to identify the wave-number distributions introduced in Equations (14)–(17):probability variable of electronic states identically vanishes, while that of their nonclassical phase part remains finite. In overall descriptors of the state information or entropy contents they ultimately generate finite production terms. For example, the nonclassical information I[φ] generates the nonvanishing  source of the average resultant gradient information I[ψ]:Iσ is determined by a product of the local probability “flux” j and “affinity“ factor proportional to the gradient of the phase source. It also follows from this local information source in φσ:To summarize, the effective velocity of the probability current also determines the phase flux in molecular states. The source (net production) of the classical mixed-state description in terms of ensemble-average physical quantities  consisting of the mutually open (microscopic) molecular fragment M(v) and an external (macroscopic) electron reservoir 𝓡(μ). In the theory of chemical reactivity one adopts such populational derivatives of the system ensemble-average energy and its underlying Taylor expansion in predicting reactivity behavior of molecules (single-reactant criteria) or bimolecular reactive systems (two-reactant criteria in situ)  = E0, while the equilibrium horizontal (“thermodynamic”) phase is determined by a supplementary IT rule ( = (ħ/m)∇φh)( = p0⋅Vh)( = φh)(Vh)( = ∇φh)(⋅Vh)( and the resultant phase source is defined in Equation (108).The SE for components of ΨE was shown to shape the dynamics of wavefunction phase, while the time evolution of the state modulus was shown to be governed by the imaginary component of local energy. In QM the spatial equalization of the local wave-number or phase concepts marks the system stationary state. The resultant IT descriptors, combining the modulus/probability and phase/current contributions, were revisited and the wave-function mapping in QM was compared with the probability scheme of classical IT. The nonclassical, phase/current supplements in the resultant IT measures effectively lower the classical entropic uncertainty and increase the spatial information determinicity of quantum states.In this conceptual overview we have first examined the spatial equalization of the electronic phase in molecules, using the (complex) local-energy concept. The real component of phase-transformed (“thermodynamic”) states were introduced and their IT optimum phases were determined from the auxiliary entropic principle. These equilibrium states were shown to exhibit the exactly vanishing internal (resultant) IT descriptors of electronic states. Therefore, in statistical mixtures of such states, the overall entropy content reduces to the external ensemble entropy of von Neumann. This brings more consistency into the quantum IT description of open molecular systems. We have also summarized the local continuity relations for the wavefunction components, state physical descriptors, and information densities. They follow from the component-resolved SE and the realization that flows of molecular properties are carried by (convection) fluxes of electronic probability. Therefore, in such treatments, the electrons are carriers of densities of both the system physical and information properties.The grand potential was interpreted as an equivalent information rule. In an ensemble description of chemical reactions in the acid–base systems, the populational derivatives of the ensemble-average resultant information were shown to constitute adequate entropic criteria for diagnosing the molecular CT phenomena, fully equivalent to their energy analogs. Latent electronic fluxes in the stationary molecular states were identified. These hidden  electronic flows, along the constant-probability contours, do not affect the stationary probability distribution and generate velocity vortices in molecules. Using the SE for wavefunction components, their local velocity was related to the “thermodynamic” phase of the phase-transformed equilibrium states.The principal variational principle for the minimum of the


The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs. Jochheim et al report an alternative, dimeric form of the coronavirus SARS-CoV-2 RNA dependent RNA polymerase (RdRp), which is used to replicate and transcribe its genome. They resolve its structure at 5.5 Å resolution and speculate that the dimer facilitates template switching during production of sub-genomic RNAs. Following the structure of the RdRp of SARS-CoV6, structures of the RdRp of SARS-CoV-2 were obtained in free form7 and as a complex with bound RNA template–product duplex11. These structures revealed a monomeric RdRp with a subunit stoichiometry of one copy of the catalytic subunit nsp1212, two copies of the accessory subunit nsp813, and one copy of the accessory subunit nsp714. Two studies additionally observed monomeric RdRp lacking one of the two nsp8 subunits and nsp79. Here we show that the RdRp of SARS-CoV-2 can also adopt a dimeric form with two RdRps arranged in an antiparallel fashion.Replication and transcription of the RNA genome of the coronavirus SARS-CoV-2 rely on the viral RNA-dependent RNA polymerase (RdRp)11), we detected many particles that showed RdRps with a preferred NN distance of 80 Å and a relative orientation of 180° . We calculated nearest-neighbor (NN) distances and relative orientations of neighboring RdRp enzymes. In one of our published data sets  and the sliding pole of the remaining nsp8 subunit  2 heterotetramer17.The structure of the antiparallel RdRp dimer showed that the two polymerases interact via their nsp7 subunits, with the nsp7 helices α1 and α3  contacting each other Fig. . To our 18, coevolved with the globally dominating spike protein mutation D614G19, is often found in severely affected patients20 and is predicted based on the structure to stabilize nsp12 association with nsp8a21. In contrast, the nsp7 mutation S25L is predicted to destabilize nsp7 binding to nsp8a21 and the nsp7 mutation L71F, which is associated with severe COVID-1922, may destabilize binding of the nsp7 C-terminal region to nsp8b. We speculate that mutations in RdRp subunits can influence the relative stabilities of the RdRp monomer and dimer.Frequently occurring mutations in the SARS-CoV-2 genome are predicted to influence formation of the RdRp dimer. The nsp12 mutation P323LNeither the catalytic sites nor the RNA duplexes are involved in RdRp dimer formation. It is therefore likely that the two RdRp enzymes remain functional within the dimer structure. The two RdRp enzymes in the dimer may thus be simultaneously involved in RNA-dependent processes. Unfortunately, we could not test whether the RdRp dimer is functional because we were unable to purify it. In particular, we attempted to reconstitute the RdRp with a nsp12:nsp8:nsp7 stoichiometry of 1:1:1, but obtained preparations showed again an apparent stoichiometry of 1:2:1 that was observed in previous RdRp structures. Therefore, the functional relevance of the RdRp dimer reported here needs to be established.25. In this intricate process, positive-strand genomic RNA (gRNA) is used as a template to synthesize a set of nested, negative-strand sgRNAs that are 5′ and 3′ coterminal with gRNA. The obtained sgRNAs are later used as templates to synthesize viral mRNAs. Production of sgRNAs involves a discontinuous step, a switch of the RdRp from an upstream to a downstream position on the gRNA template26. These positions contain transcription regulatory site (TRS) sequences29, but it is enigmatic how a single RdRp enzyme could ‘jump’ between these.We hypothesize that the RdRp dimer is involved in the production of sub-genomic RNA (sgRNA)31  at the 5′-end of the template. The resulting RNA duplex could then bind to the active center of the second RdRp (RdRp 2) to continue sgRNA synthesis.Our dimer structure suggests a model for sgRNA synthesis that extends a recent proposal31 Fig. . In the 32 can be modeled on our dimer structure without clashes  at 4 °C. Peak fractions corresponding to the RdRp–RNA complex were pooled and diluted to ~2 mg/ml. Three microliters of the concentrated RdRp–RNA complex were mixed with 0.5 µl of octyl ß-d-glucopyranoside  and applied to freshly glow discharged R 2/1 holey carbon grids (Quantifoil). The grids were blotted for 5 s using a Vitrobot MarkIV (Thermo Fischer Scientific) at 4 °C and 100% humidity and plunge frozen in liquid ethane.We reused the already processed data set 3 from our previous publication11. Briefly, data was collected using SerialEM37 on a 300 keV Titan Krios transmission electron microscope (Thermo Fischer Scientific) and a K3 direct electron detector (Gatan). Inelastically scattered electrons were filtered out prior to detection using a GIF quantum energy filter (Gatan) using a slit width of 20 eV. Images were acquired at a nominal magnification of 105,000x and a calibrated pixel size of 0.834 Å/pixel. Due to previously observed preferred orientation when imaging RdRp complexes10, data was collected using a 30° tilt to obtain more particle orientations. 7043 raw micrographs were acquired in total and preprocessed on-the-fly in Warp for automatic contrast transfer function (CTF) estimation, averaging, motion correction, and particle prediction and extraction. 2.2 million individual RdRp particles were predicted and exported by Warp and imported into cryoSPARC and subjected to a Hetero Refinement job using five ‘Junk‘ classes and one class representing monomeric RdRp as described previously11. The resulting particle set was used for a 3D homogeneous refinement to obtain refined poses and positions for each RdRp monomer.Data collection and preprocessing was the same as previously describedx-axis. We chose to express relative orientation through a single angle by calculating the angle of the rotation around the eigenvector of the rotation matrix. This showed that certain distances and relative orientations between two monomers were highly prevalent  distances and the relative orientations for all neighboring RdRp complexes using the previously refined monomer poses. To account for the tilted data acquisition, we treated the influence on distances observed on the micrograph as a 30° rotation around the 38 using the monomeric RdRp–RNA structure10 filtered to a resolution of 30 Å as a 3D reference to pick further monomers that might have been missed due to their proximity to other particles. Template-based picking of monomeric RdRp however did not introduce any model bias that could influence the dimer structure. Second, we used the previously established NN distance and relative orientation to predict for each RdRp monomer the position where its partner in a dimeric particle should be located on the micrograph. From our NN search we obtained the 3D offset that the second monomer should adopt in a dimeric particle .Because the yield of dimeric particles depended on both halves of a dimer being first detected as monomer, we aimed to detect more RdRp monomers using two strategies. First, we carried out template-based picking and particle extraction in RELION39 (Supplementary Fig. 11, removal of mobile regions and fitting of domains as rigid bodies was done in UCSF Chimera40.Final dimer coordinates were used to extract particles using a box size of 300 Å and a pixel size of 1.201 Å. 2D classification in cryoSPARCFurther information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Augmented Reality (AR) is a rapidly emerging technology finding growing acceptance and application in different fields of surgery. Various studies have been performed evaluating the precision and accuracy of AR guided navigation. This study investigates the feasibility of a commercially available AR head mounted device during orthopedic surgery.Thirteen orthopedic surgeons from a Swiss university clinic performed 25 orthopedic surgical procedures wearing a holographic AR headset  providing complementary three-dimensional, patient specific anatomic information. The surgeon’s experience of using the device during surgery was recorded using a standardized 58-item questionnaire grading different aspects on a 100-point scale with anchor statements.Surgeons were generally satisfied with image quality (85 ± 17 points) and accuracy of the virtual objects (84 ± 19 point). Wearing the AR device was rated as fairly comfortable (79 ± 13 points). Functionality of voice commands (68 ± 20 points) and gestures (66 ± 20 points) provided less favorable results. The greatest potential in the use of the AR device was found for surgical correction of deformities (87 ± 15 points). Overall, surgeons were satisfied with the application of this novel technology (78 ± 20 points) and future access to it was demanded (75 ± 22 points).AR is a rapidly evolving technology with large potential in different surgical settings, offering the opportunity to provide a compact, low cost alternative requiring a minimum of infrastructure compared to conventional navigation systems. While surgeons where generally satisfied with image quality of the here tested head mounted AR device, some technical and ergonomic shortcomings were pointed out. This study serves as a proof of concept for the use of an AR head mounted device in a real-world sterile setting in orthopedic surgery.The online version contains supplementary material available at 10.1186/s12891-021-04339-w. Precise positioning of mechanical elements including screws, surgical guides, prosthetic components and anchors to restore anatomy and function remains a desirable goal in orthopedic surgery . Image-bAugmented Reality (AR) is a rapidly emerging technology providing the user with computer-generated information superimposed to real-world environment. The user’s field of view is transformed into a display where real-world objects can be complemented with virtual data. This way information including drilling axis or cutting planes can be projected directly onto the patients’ anatomy within the surgical field. Although its application in orthopedic surgery today remains limited, AR was gradually introduced in different experimental medical and surgical settings –16. ReceOrthopedic surgeons from a single Swiss university clinic where the subject of this prospective study. Surgeons were asked to wear a commercially available holographic AR headset  during surgery after receiving an introduction explaining its functionality and time to test voice commands and hand gestures.For each operation, a three-dimensional triangular surface model of the patients readily available CT data was generated using a commercial software  . Only alDuring the operation, the three-dimensional model of the patients CT-data was displayed holographically in the surgeon’s field of view in addition to routinely mounted radiological imaging on conventional screens. The displayed surface model could be moved, rotated, scaled and placed using contactless hand gestures and voice commands Fig. . The timThe device was evaluated using a standardized 58-item questionnaire administered as an electronic survey using the REDCap data capture tool . The surDescriptive analysis was performed using SPSS . Continuous variables are reported as mean ± standard deviation (SD).Thirteen orthopedic surgeons from different subspecialties  participated in this study. Four surgeons performed > 500 cases per year, 3 surgeons 251–500 and 6 surgeons 51–250 surgeries per year. Six surgeons were in the position of chief of service and 7 surgeons were attending physicians or fellows. In total, 25 surgeries  were included in this study.The general acceptance of the AR device was good Fig. . WearingConsidering usability Fig. , surgeonMost surgeons used the AR device for less than 60 min (23/25 surgeries) and no surgeon used the device for the entire surgery. All but three surgeons used the AR device only for preoperatively defined surgical steps Fig. .Fig. 7BThe most frequent reasons for early termination of use were impaired sight caused by the device (3/25) and surgical steps requiring other instruments (3/25) Fig. . HoweverThe greatest potential in the use of the AR device was found for surgical correction of deformities (87 ± 15 points), osteotomies (82 ± 17 points), revision surgery (77 ± 22 points), and tumor surgery (77 ± 21 points), whereas lowest potential was found for arthroscopic (32 ± 25 points) and reconstructive surgery (41 ± 22 points) Fig. .Fig. 9BPotential for future use within the orthopedic subspecialties was highest estimated for application in spine (86 ± 13 points) and pelvic surgery (88 ± 13 points).The greatest benefits of AR technology were expected to be increased intraoperative accuracy (78 ± 23 points), improved surgical outcome (77 ± 22 points) and reduction of exposure to radiation (72 ± 26 points) Fig. .Fig. 10Overall, surgeons were satisfied with the application of this novel technology (78 ± 20 points) and future access to it was demanded (75 ± 22 points).This prospective clinical study offers a proof of concept of the clinical feasibility of a wearable augmented reality device for using three-dimensional virtual object reconstructions of individual patients’ anatomy as an intraoperative aid during orthopedic surgical procedures. Thirteen surgeons from different orthopedic subspecialties used an AR headset as an additional display providing complementary three-dimensional, patient specific anatomic information during 25 surgical procedures. The surgeons experience with the device was recorded using a standardized questionnaire.Augmented reality is rapidly developing technology that can display a vast variety of information within the field of view of the user using a compact device with minimum infrastructure. Spatial mapping and recognition of the physical environment is a crucial difference between augmented reality and other navigations systems using conventional display methods. Projecting the required information directly into the line-of sight of the surgeon is considered the natural progression of these well-established methods mitigating the errors associated with attention shift by directly projecting the navigation guidance onto the surgical field , 23. IncThe here used iteration of the HoloLens offers a rather narrow diagonal angle of view of 34° for projection of virtual objects. However, for most surgical tasks requiring high precision, a narrow field of view is sufficient and this study, surgeons were generally satisfied with the quality of the displayed three-dimensional images.n = 2) might also be explained by the fact that participants were only supplied with supplementary information required for essential steps of the surgery. Hence, the threshold for removing the device, in particular while performing the surgical approach and during wound closure was low.The general acceptance of the device was fairly good. Considering comfort, the mostly frontloaded weight of 579 g and the size of this first iteration of the HoloLens was considered as a shortcoming. During 6 of 25 cases, the device was worn for more than 30 min, and in another 7 of 25 cases, for 15–30 min. However, despite its weight and size, most surgeons participating in this study estimated that the device could be worn for more than an hour when considering comfort. Other authors investigating the usability of the HoloLens during neurosurgical procedures graded the comfort more favorable , 26. ThiThe questionnaire also revealed technical limitations of the device, namely the voice command and gesture function. Using voice commands as a mean of interaction with the headset was less favorably graded. In most cases the recognition of voice commands was impaired by loud environment in the operation room. Other reports analyzing the usage of the HoloLens device for different surgical specialties reported more stable results using voice commands . This diSurgeons saw the biggest potential for this technology in complex surgical procedures including revision surgery, deformity correction, tumor surgery and trauma where typical anatomical landmarks are frequently not easily identifiable. However, a crucial step to provide reliable navigation in complex surgery is the registration process, the automated and precise overlay of virtual information with the real environment. Various methods of registration have been described including ultrasound-based techniques, reflective markers and non-invasive skin placed markers , 23, 27.In summary, AR is a rapidly evolving technology with large potential in different surgical settings, offering the opportunity to provide a compact, low cost alternative requiring a minimum of infrastructure compared to conventional navigation systems. While surgeons where generally satisfied with image quality of the here tested head mounted AR device, some technical and ergonomic shortcomings were pointed out. This study serves demonstrates the feasibility of the use of an AR head mounted device in a real-world sterile setting in orthopedic surgery.Additional file 1.


The second was to examine whether terazosin (TE) could abolish the vascular contractile effects of ergot alkaloids. Twelve adult female sheep were randomly placed into control and exposure groups (n = 6/group). Ergot sclerotia were collected and finely ground. The concentrations of six ergot alkaloids  were determined using HPLC/MS at Prairie Diagnostic Services Inc., . Each ewe within the treatment group received a single oral treatment of ground ergot sclerotia at a dose of 600 µg/kg BW  while each ewe in the control group received water. Animals were euthanized 12 h after the treatment, and the pedal artery  from the left hind limb from each animal was carefully dissected and mounted in an isolated tissue bath. The vascular contractile response to phenylephrine (PE) (α1-adrenergic agonist) was compared between the two groups before and after TE (α1-adrenergic antagonist) treatment. Acute exposure to ergot alkaloids resulted in a 38% increase in vascular sensitivity to PE compared to control . TE treatment resulted in a significant dose-dependent increase in EC50 in both exposure and control groups . Surprisingly, TE effect was significantly more pronounced in the ergot exposed group compared to the control group at two of the three concentrations of TE . Similar to chronic exposure, acute exposure to ergot alkaloids results in increased vascular sensitivity to PE. TE is a more potent dose-dependent antagonist for the PE contractile response in sheep exposed to ergot compared to the control group. This study may indicate that the dry gangrene seen in sheep, and likely other species, might be related to the activation of α1-adrenergic receptor. This effect may be reversed using TE, especially at early stages of the disease before cell death occurs. This study may also indicate that acute-single dose exposure scenario may be useful in the study of vascular effects of ergot alkaloids.Ergotism is a common and increasing problem in Saskatchewan’s livestock. Chronic exposure to low concentrations of ergot alkaloids is known to cause severe arterial vasoconstriction and gangrene through the activation of adrenergic and serotonergic receptors on vascular smooth muscles. The acute vascular effects of a single oral dose with high-level exposure to ergot alkaloids remain unknown and are examined in this study. This study had two main objectives; the first was to evaluate the role of α Clavicipitaceae  on ice until transport to the laboratory. Immediately upon arrival to the lab, adipose and connective tissue were carefully removed from each arterial segment, which was later sliced into four 3 to 5 mm cross sections. Each arterial section was suspended between the bases of two triangular-shaped wires within an isolated 10 mL tissue bath  containing modified Krebs–Henseleit buffer solution maintained at the above conditions. Arterial rings were allowed to equilibrate for 1 h under a resting tension of 2 g with the bath solution changed every 15 min. Each day, a fresh stock solution was prepared for phenylephrine (PE)  at a concentration of 1 M, followed by a 10-fold serial dilution to prepare the remaining working solutions. 10 µL were added from each dilution to the 10 mL incubation buffer to obtain the desired final concentration (1 × 10−9–1 × 10−4 M). Similarly, a fresh initial 1 M stock solution of terazosin  was prepared from which a 10 µM and 100 µM dilutions were made. The desired final concentrations of 30, 100, and 300 nM were prepared by adding 30 µL from the first stock or 10 and 30 µL from the second stock as appropriate. Arterial rings were treated with PE (1 × 10−4 M)  to initiate contraction and to confirm tissue viability and responsiveness. The tissues were later washed with incubation buffer until resting tension was achieved.Twelve healthy adult ewes were randomly assigned into treatment or control groups (−9 M to 1 × 10−4 M). After each PE treatment, arterial rings were allowed to achieve maximum tension which plateaued for 2 min before the next concentration was added. After the last PE treatment, arterial rings were allowed to return to resting tension with buffer replacement occurring every 15 min for 1 h. This was followed by incubating each of the three rings with 30, 100 or 300 nM TE for 20 min after which the cumulative PE contractile response was repeated in each chamber as above. Following completion of the exposure, all rings were exposed to 1 × 10−4 M PE to verify their viability.Three vascular rings from the pedal artery of each animal were used to assess the PE contractile response before and after the incubation of each ring with a different concentration of TE. Initially, a cumulative concentration-dependent contraction in response to PE was obtained by adding increasing concentrations of PE . For each PE treatment, the maximum tension in grams achieved before the 2 min plateau period was recorded and corrected for a baseline. To minimize variation due to arterial size, each contractile response from an individual ring was normalized to its maximum contractile tension induced by 1 × 10SEM of the maximum contractile effect induced by 1 × 10−4 M PE treatment. For each treatment type, a sigmoidal dose-response curve was plotted using nonlinear regression with variable slope utilizing GraphPad Prism 7 , which was later used to calculate potency presented as the concentration producing 50% of the maximum response (EC50). Results were presented as the log of the EC50 value. Statistical differences in EC50 among the different dose-response curves were calculated by the extra sum-of-squares F-test where a p-value less than 0.05 was considered significant.Contractile response data were presented as percentage means ±


Results indicated that the high concentrations of AVG and SA recorded the lowest percentage of preharvest fruit drop and, hence, the highest yield. Trees receiving either concentration of AVG showed the highest fruit firmness. High concentrations of all three ethylene inhibitors reduced fruit weight loss, total carotenoids, and soluble solid content (SSC), but increased total acidity (TA) during cold storage (2 °C). A high score of overall taste acceptability was observed with a higher concentration of SA, which was also recorded the lowest fruit malondialdehyde content (MDA) at harvest and during storage. The highest concentrations of SA and chitosan recorded no decay for 28 days of storage. Gene expression analysis reflected higher expression of PaACS1 gene with the highest concentrations of ethylene inhibitors, suggesting that SA (4 mM) is recommended for optimal yield, quality, and storability of ‘Canino’ apricot fruit grown under Egyptian conditions.Ethylene plays a pivotal role in the climacteric fruit ripening and senescence process. The effect of three ethylene inhibitors on the yield, quality, and storability of ‘Canino’ apricot fruit was studied. Foliar sprays of distilled water (control), aminoethoxyvinylglycine (AVG) (150 and 100 mg·L Prunus armeniaca L., a member of the Rosaceae family, is one of the most widely distributed deciduous fruit trees in the world that produce highly nutritional fruit  . Chitosan   was prepared according to Tezotto-Uliana et al. [v/v) as a surfactant for a total volume of 4 L per tree.Seven foliar spraying treatments were applied twice at 30 and 15 days before harvest, as follows: distilled water (control), aminoethoxyvinylglycine (AVG) at 150 and 100 mg·La et al. . All sol−1) with no pesticide or waxing treatments, and left for air-drying at room temperature (22–23 °C) for 30 min.Preharvest fruit drop (PFD) was determined daily by counting the number of dropped fruit under each tree for six consecutive days before harvest, and then PFD was calculated as a percentage in relation to the total number of harvested fruit . Fruit w−1). The same fruit samples were used for harvest date analyses. A sample of 10 uniform fruits per tree (20 fruits/replicate) was randomly collected and weighed using a bench-top digital scale Model PC-500  with an accuracy of 0.1%. Average fruit weight (g) was calculated, and then multiplied by the total number of fruit per tree to calculate total fruit yield , were collected free of mechanical injuries and decay. Each group of fruit was placed in a cardboard box and stored at 0 °C and 90% RH. At weekly intervals , one box was used to evaluate fruit characteristics during storage. Fruit WL (%) was calculated according to the following equation: 2) at harvest and at weekly intervals during storage was measured at the equatorial area on two sides of 10 fruits using a handheld Shimpo digital force gauge, Model FGV-50XY fitted with 8 mm diameter plunger tip . The firmness reduction rate (fruit softening rate) during each storage period was calculated as a percentage of the original fruit firmness at harvest using the following equation:After storage, fruit were placed at room conditions (20 ± 2 °C and 80–85% RH) for 2 days, as a shelf-life period, before assessing other fruit parameters. Average fruit firmness  was also calculated at harvest and by the end of the storage period. Color was assessed after a period of cold storage plus 2 days at room conditions, in comparison to fruit color at harvest date to calculate total color difference (ΔE), using the following formula [Fruit color was colorimetrically assessed on two opposite sides at the equatorial area of each fruit  using a  formula :ΔE = (ΔL−1) were measured at harvest and then at 7-day intervals during storage [Fruit chlorophyll and carotenoid contents (µg·mL storage . Five grExtracted juice from about 200 g of fruit was used to determine SSC (%), using a digital refractometer . Total acidity (TA) was estimated as malic acid (%)  using anA group of eight trained panelists ran a test panel to evaluate fruit sensory attributes  using a hedonic scale of 1–9 for each sensory attribute (excellent (9), very good to excellent (8), very good (7), good to very good (6), good (5), average (4), acceptable (3), unsatisfactory to acceptable (2), and unsatisfactory (1)) . Scores g for 10 min. The supernatant (2 mL) was mixed with 2 mL of 2-thiobarbituric acid (TBA) (0.5%) in a test tube, and then heated at 95 °C for 15 min in a water bath. The tube was immediately cooled in ice bath, and then centrifuged at 1800× g for 10 min. The solution was then tested using a spectrophotometer  at wavelengths of 450, 532, and 600 nm, and the amount of accumulated MDA was calculated as follows:Lipid peroxidation of the cellular membrane was determined by estimating the MDA concentration using thiobarbituric acid-reactive substances (TBARS) ,104 withPaACS1 gene expression analysis was performed by the qRT-PCR [−∆∆Ct method [PaACS1(f) 5′–ATTCAACCAGGCAAAGAAACGC–3′,PaACS1(r) 5′–GATGGAGTGGAAATGGACGAGA–3′,Pa26sRIB(f) 5′–AACGCAGGTGTCCTAAGATGAG–3′,Pa26sRIB(r) 5′–GCTGCCACAAGCCAGTTATCC–3′.The ripening-related  qRT-PCR . Total R qRT-PCR . The relt method , accordip ≤ 0.05 [Data were first analyzed for numerical normality and homogeneity of variance using Shapiro–Wilk’s and Levene’s tests, respectively. Data were then statistically analyzed, and one-way analysis of variance (ANOVA) was performed using CoStat software package . Means were expressed as the value ± standard deviation (SD) and compared using Duncan’s multiple range test (DMRT) at p ≤ 0.05 . PaACS1 gene was also more correlated to the higher concentrations of AVG, followed by SA and then chitosan. Overall, AVG treatment led to an uncommon green color, along with high rates of WL and DI during cold storage, which make the fruit unmarketable. Therefore, this study suggests the application of SA (4 mM) at 30 and 15 days before harvest for optimal yield, quality, and storability of ‘Canino’ apricot fruit grown under Egyptian conditions. Future research could include the impact of ethylene inhibitors at all three stages of fruit growth and development, as well as studying more ripening-related genes associated with ethylene biosynthesis.Managing the appearance of ‘Canino’ apricot fruit in the market, as well as extending fruit shelf life with minimal negative effects on yield and fruit quality, is a crucial issue in the Egyptian apricot industry. To address this, we carried out an investigation that revealed that the foliar spray of ethylene inhibitors such as AVG, SA, and chitosan reduced the percentage of PFD compared to the control, with the highest yield recorded for both AVG and SA. The effect of AVG on fruit firmness was more pronounced in comparison to SA and chitosan. All three compounds were effective in reducing fruit WL, total carotenoids, and SSC, along with increased TA in comparison to the control during the 28 days of cold storage at 0 °C. Fruit sensory analysis reflected the highest quality with the application of SA. In addition, this treatment also showed the lowest membrane peroxidation level at harvest and during storage. No decay was recorded for 28 days of storage with the application of SA or chitosan. The upregulation of ripening-related


When 6-mercapto-1-hexanol is used to fill the unoccupied places on a silver surface (as in experiments on standard gold substrates), a quick detachment of chemisorbed DNA from the silver surface is observed. Whereas, using ω-substituted alkanethiols with a shorter alkane chain makes it possible to easily form mixed DNA/backfilling thiol monolayers. Probably, the significantly lower desorption rate of the thiolated DNA induced by alkanethiols with shorter chains is due to the lower stabilization energy in monolayers formed from such compounds.Layers formed from single-stranded DNA on nanostructured plasmonic metals can be applied as “working elements” in surface–enhanced Raman scattering (SERS) sensors used to sensitively and accurately identify specific DNA fragments in various biological samples . Therefore, the proper formation of the desired DNA layers on SERS substrates is of great practical importance, and many research groups are working to improve the process in forming such structures. In this work, we propose two modifications of a standard method used for depositing DNA with an attached linking thiol moiety on certain SERS-active structures; the modifications yield DNA layers that generate a stronger SERS signal. We propose: (i) freezing the sample when forming DNA layers on the nanoparticles, and (ii) when forming DNA layers on SERS-active macroscopic silver substrates, using  In recent years, there has been much research activity in developing new techniques enabling genetic disorders to be diagnosed through the identification of DNA chains having a given sequence in various biological samples . Various techniques can be used for such identification ,3,4,5,6.There are many different approaches used for detecting specific DNA chains based on measurements of SERS signals, including the following:(i) A moiety with an exceptionally large cross-section for Raman scattering (known as a Raman reporter) is connected to the target DNA, and after hybridization with the capture DNA, this target DNA with a Raman reporter is immobilized on the SERS substrate and a strong SERS signal is recorded ,8.(ii) A capture DNA hairpin chain is covalently bonded via one end to the SERS substrate, and a Raman reporter is attached to the other end of the hairpin chain, which is close to the SERS substrate; hybridization between the target DNA and the capture DNA forming the hairpin disrupts the stem-loop configuration and causes a decrease in the SERS signal of the Raman reporter ,10,11.(iii) Capture DNA is adsorbed on a SERS substrate, and this then hybridizes with the target DNA; subsequently, a hybridization with a second “auxiliary DNA chain” labelled with a Raman reporter is carried out, resulting in a sandwich structure giving a strong SERS signal ,13,14,15(iv) Two different capture DNA chains are immobilized on two various sols of SERS-active nanoparticles; when the target DNA is present in the sample, an aggregation of the plasmonic nanoparticles is induced by the target DNA, which leads to a significant increase in the intensity of the measured SERS spectrum ,17,24.(v) A modification of the method described in point (iv), where instead of one modified nanoparticle, a macroscopic plasmonic substrate  or magne(vi) The application of head-flocked gold nanopillar SERS substrates modified with a capture nucleic acid; in the presence of the target nucleic acid, the substrates self-assemble and form SERS hot spots .(vii) Capture DNA is immobilized on a SERS-active surface via thiol linking moiety; hybridization with the target DNA induces a change in the conformation of the alkanethiolate linking moiety and in its SERS spectrum .In all these SERS DNA sensors, the active part of the device contains a layer of “capture” DNA attached via a thiol linking moiety to a SERS-active substrate, with additional alkanethiols that fill the “empty” spaces on the metal surface between the DNA chains. Moreover, this kind of “capture” DNA layer on a plasmonic metal is also used in sensors that utilize a surface plasmon resonance signal as an analytical response . For theAs mentioned in the introduction, we decided to analyze how certain modifications of the method for depositing thiolated DNA on plasmonic nanoparticles might affect the intensity and repeatability of the SERS spectra recorded, and how the chemisorption of various alkanethiols used to fill those places on the metal surface unoccupied by DNA could affect the structure of the obtained layer.2)6–SH and CTCT–(CH2)6–SH on gold nanoparticles. In all cases, the solution of thiolated DNA and a sol of gold nanoparticles was mixed and kept overnight; either at room temperature or in a freezer .As mentioned in the introduction, it is well-known that thiols chemisorb on a surface of gold and silver, forming strong sulphur-metal chemical bonds ,37,38,392)6–SH, CTC–(CH2)6–SH, CTCTG–(CH2)6–SH, CTCTGT–(CH2)6–SH, CTCTGTA–(CH2)6–SH, CTCTGTAG–(CH2)6–SH, CTCTGTAGC–(CH2)6–SH, CTCTGTAGCT–(CH2)6–SH, CTCTGTAGCTA–(CH2)6–SH and CTCTGTAGCTAG–(CH2)6–SH (sequences are given from 5′ to 3′). To quantitatively estimate the freezing-induced increase in the intensity of the recorded SERS spectra, in all the obtained spectra we determined the integral intensity of the band at ca. 800 cm−1, which is due to the ring breathing vibration of the pyrimidine ring of cytosine [As can be seen from cytosine   is attached to the DNA chain. In addition to the described above difference in the SERS activity of the samples prepared using a freezing step and incubated at room temperature, a significant difference in the extinction spectra of the samples prepared using these two methods was observed. 2)6–SH, CT–(CH2)6–SH, CTC–(CH2)6–SH, CTCT–(CH2)6–SH, CTCTG–(CH2)6–SH, CTCTGT–(CH2)6–SH, CTCTGTA–(CH2)6–SH, CTCTGTAG–(CH2)6–SH, CTCTGTAGC–(CH2)6–SH, CTCTGTAGCT–(CH2)6–SH, CTCTGTAGCTA–(CH2)6–SH, CTCTGTAGCTAG–(CH2)6–SH. As can be seen from this Figure, there is a significant decrease in the intensity of the measured SERS spectra when the chain of deposited thiolated DNA contains more than 4 bases . One may assume that the wider slits created by the gold nanoparticles surrounded by a thicker layer of DNA are not as efficient in enhancing the SERS spectra.The majority of SERS signals are generated in SERS hot spots ,44, whic−1 due to the cytosine ring breathing vibration; at 1030 cm−1 due to the N–sugar stretching vibration; at 1288 cm−1 due to the CN stretching vibration; at 1500 cm−1 due to the NH2 deformation vibration; at 1564 cm−1 due to the pyridine ring stretching; and at 1636 cm−1 due to C=O stretching vibrations , the most intense bands in the spectra are due to the following vibrations; at 798 cmsee also  [40,45,4We also investigated whether freezing the solution during DNA deposition had any impact on the intensity of the SERS signal recorded on the macroscopic nanostructured plasmonic substrates . In such a case, we did not observe any significant differences between the SERS spectra of the silver electrode stored at room temperature and the silver electrode after freezing. This observation also supports the hypothesis that an increase in the intensity of the SERS spectrum after freezing is caused by the agglomeration of plasmonic nanoparticles (such an agglomeration is not possible in the case of macroscopic SERS substrates).DNA attached to a metal surface via a thiol moiety tends to interact with the metal surface not only through the thiol groups, but also through the nitrogen bases. This effect hinders the operation of DNA sensors, because it disturbs the hybridization process with other DNA strains. To eliminate direct interactions of the DNA nitrogen bases with the metal surface, certain compounds can be used to “backfill the empty spaces” on the metal surface between the adsorbed DNA chains . In the case of the formation of DNA layers on gold (gold is the most widely used substrate for the formation of DNA layers for various sensors), the empty spaces are usually backfilled using chemisorbed 6-mercapto-1-hexanol (MCH) ,33. Yet,−1, which is practically identical to the SERS spectrum of MCH adsorbed on a silver surface. Moreover, even decreasing the concentration of MCH by one order of magnitude (to 1 mM) did not prevent the chemisorbed DNA from being removed from the silver surface—See Cy3 labelled thiolated DNA was adsorbed on a nanostructured silver electrode by overnight incubation. Next, the SERS spectrum of the layer of DNA formed on the silver was measured, and the substrate was then immersed in a 10 mM MCH solution (pH 4.5) for 5 min and 20 min. After soaking in the MCH solution, the SERS spectrum of the sample was measured again. In a separate experiment, a nanostructured silver substrate was immersed for 5 min in a 10 mM solution of MCH, and the SERS spectrum of the modified substrate was measured. All the obtained spectra are presented in ω-substituted alkanethiols: 3-mercaptopropionic acid and cysteamine. We chose substituted alkanethiols with very short chains in order to decrease the very large stabilization effect that takes place during the self-assembly of alkanethiols with long alkane chains, which is due to the alkane chains forming crystal-like structures.Due to the immersion of a thiolated DNA layer on silver in a 10 mM (or even in a 1 mM) MCH solution induces an efficient desorption of DNA from the metal surface, which means that this compound seems to be inappropriate for backfilling the empty spaces on the metal surface. On this basis, we decided to check whether a similar quick desorption of DNA would be also observed when soaking a layer formed by the chemisorption of thiolated DNA in solutions of two other simple −1 due to the S–C stretching vibrations of the gauche conformer of the S–C–C chain [−1 due to the vibration of the non-dissociated carboxylic group vibrations [–C chain ,38,39,47−1 (DNA) and 642 cm−1 (CYS) and the bands at 798 cm−1 (DNA) to 730 cm−1 (CYS) shows that these ratios stabilized relatively quickly (in less than 20 min). This means that semi-stable Ag/thiolated-DNA/CYS systems  are formed relatively quickly, and CYS is a very promising compound for backfilling the “empty spaces” on a silver surface between adsorbed thiolated DNA chains.As mentioned above, the second compound tested in this contribution as a possible compound for backfilling the empty spaces in DNA layers was cysteamine (CYS). We also carried out analogous experiments concerning the application of these back-fillers using plasmonic nanoparticles. Unfortunately, the adsorption of back-fillers on nanoparticles with, or even without, adsorbed DNA significantly decreased the stability of the sols, and so we were unable to obtain reliable results in these cases.2PO4, Na2HPO4, and KCl were purchased from Chempur, Piekary Slaskie, Poland. Analytical grade cysteamine hydrochloride, 97% 6-mercapto-1-hexanol, 99% 3-mercaptopropionic acid, chloroauric acid and sodium citrate tribasic dehydrate were purchased from Sigma, Poznan, Poland. The platinum plate was purchased from Mennica Polska, Warsaw, Poland. The water used in all the experiments was purified in a Millipore Milli-Q system .Analytical grade NaH2)6–SH (1), CT–(CH2)6–SH (2), CTC–(CH2)6–SH (3), CTCT–(CH2)6–SH (4), CTCTG–(CH2)6–SH (5), CTCTGT–(CH2)6–SH (6), CTCTGTA–(CH2)6–SH (7), CTCTGTAG–(CH2)6–SH (8), CTCTGTAGC–(CH2)6–SH (9), CTCTGTAGCT–(CH2)6–SH (10), CTCTGTAGCTA–(CH2)6–SH (11), CTCTGTAGCTAG–(CH2)6–SH (12), and Cy3–CGAGATTTCTCTGTAGCTA–(CH2)3–SH. All the nucleotides were dissolved in a phosphate-buffered saline with a Na+ concentration of 25 mM. The concentration of nucleotides in the final solution was 50 mM.All the single-stranded DNA used in this work was purified using HPLC (high-performance liquid chromatography) and was purchased from Genomed, Warsaw, Poland. Nucleotides having the following sequences were used : C– images of the resulting gold nanoparticles are presented in the g. The supernatant was removed, and 3 μL of the concentrated solution was deposited on Au-covered glass slides and the SERS spectra of the liquid probes recorded.Spherical gold nanoparticles with a diameter of about 35 nm were prepared using the following method: 99 mL of water was mixed with 1 mL of a 1% aqueous solution of HAuCl−1, and then the electrode was kept for 5 min at −0.4 V. All potentials are given versus the 0.1 M KCl AgCl/Ag electrode. A Scanning Electron Microscopy (SEM) image of the nanostructured silver electrode obtained is presented in The silver electrodes were roughened by three successive positive-negative polarization cycles in a 0.1 M KCl aqueous solution, from 0.3 V to −0.3 V at a scan rate of 5 mV sλ = 633 nm) was generated by an He–Ne laser, and the power at the sample was 2 mW. Spectra were recorded using an air objective (10x) with a numerical aperture of 0.25—The diameter of the laser spot at the sample was 2.5 micrometers. For the experiments on the gold nanoparticles, at least five measurements were performed for each sample, and all the SERS spectra presented in this work are the average of at least five spectra collected at different points of the sample.Raman spectra were accumulated using a Thermo Fisher DXR Raman spectrometer . The excitation radiation  in a range of 400–800 nm with a resolution of 2 nm. To place a sample, quartz cells were used.The electrochemical preparation of the silver electrodes was performed in a conventional three-electrode cell using a PGSTAT204 potentiostat/galvanostat from Metrohm Autolab BV, operated using NOVA 1.10 software.For both the labeled and label-free DNA chains, the SERS spectra obtained for the samples prepared using a freezing step were more intense than the spectra of the samples prepared by the standard method of incubation at room temperature. This was probably due to the formation of aggregates of plasmonic nanoparticles, which led to the creation of a larger number of SERS hot spots. This means that using a freezing step for preparing DNA probes on gold nanoparticles seems to be a promising way of preparing samples for SERS measurements.ω-substituted alkanethiols with significantly shorter alkane chains may be more suitable —See also 6-mercapto-1-hexanol, commonly used to fill in the “unoccupied” places on a gold surface covered with a thiolated DNA monolayer, does not seem to be an effective compound for backfilling the empty spaces between DNA chains on SERS-active silver surfaces, because it induces a quick detachment of chemisorbed DNA from the silver surface. We propose that, for DNA layers on silver,


Chiroptical properties of amino acids – expressed in circular dichroism (CD) – have been previously investigated in solid and solution phase. However, both environments distort the intrinsic charge distribution associated with CD transitions. Here we report on CD and anisotropy spectra of amino acids recorded in the gas phase, where any asymmetry is solely determined by the genuine electromagnetic transition moments. Using a pressure- and temperature-controlled gas cell coupled to a synchrotron radiation CD spectropolarimeter, we found CD active transitions and anisotropies in the 130–280 nm range, which are rationalized by ab initio calculation. As gas phase glycine was found in a cometary coma, our data may provide insights into gas phase asymmetric photochemical reactions in the life cycle of interstellar gas and dust, at the origin of the enantiomeric selection of life’s l-amino acids.Life on Earth employs chiral amino acids in stereochemical  Chiroptical properties of amino acids are challenging to investigate in the gas phase due to the low vapor pressure of these molecules. Here the authors succeed in measuring circular dichroism active transitions and anisotropies in the ultraviolet range for several gas-phase amino acids, shedding light on the interactions between molecules and circularly polarized light that lead to chiral symmetry breaking. However, the origin of the molecular asymmetry and the conformational landscape of the 21 left-handed proteinogenic amino acids that orchestrate the assembly of proteins into their unique, biologically relevant three-dimensional structures1, is still not properly understood. The importance of studying isolated pure amino acids in the gas phase becomes apparent, not only in terms of changes to their conformation due to (i) the absence of the polarizability of the surrounding medium2, (ii) intermolecular hydrogen bonding or coordination with adjacent molecules, but also (iii) because amino acids exist as zwitterions in solution and solid state3, whereas they are in the non-ionic molecular form when isolated in the gas phase5—the configuration amino acids adopt in proteins and polypeptides6 as well as in interstellar gas clouds.Evolution has led to proteins with an incredible array of structures and functions. All proteins have in common that their molecular structure exclusively uses amino acids in stereochemical 11, microwave and millimeter wave spectroscopy15, as well as photoelectron circular dichroism (PECD)17 for probing the structural properties of amino acids in the gas phase collectively aimed to map the conformational behavior of protein-embedded amino acids. However, absorption-based chiroptical measurements in the gas phase have so far been inaccessible for amino acids because the target molecules show insufficient vapor pressure leading to low gas phase density and therefore to poor signal-to-noise ratios. The very few efforts made, for instance via infrared Cavity-Ring Down Spectroscopy (CRDS)18 to probe circular birefringence and circular dichroism in the gas phase, have so far been limited to volatile organic species not including amino acids19. First attempts in the VUV on alanine, even with the very-sensitive technique of ion yield, provided only upper limits without succeeding in providing the absolute configuration20. With a specifically constructed gas cell  normalized CD spectrum, g(λ) = CD(λ)/A(λ), is directly related to the enantiomeric excess (ee) inducible by enantioselective photolysis21. Compared to the CD spectrum, the anisotropy spectrum has enhanced intensity at long wavelength, despite the much more intense CD states at shorter wavelengths. Our data show that highest enantiomeric excesses can be photochemically induced into alanine at λ = 214 nm and λ = 244 nm.The anisotropy spectrum, which is the absorbance  level was obtained applying the M06-2X/aug-cc-pVQZ basis set  transition in carbonyl compounds . Thus, besides the broad nO → π*CO band, a series of transitions belonging to the nO  →  3 sp Rydberg excitations are predictable (Table nN → 3sp) of the neutral amine group, inaccessible in solution and solid state. Theoretical approaches to assign the observed CD bands of higher lying excited states to specific orbital transitions become less accurate as significant mixing occurs. We tentatively assign the valence πC=O → 3s and πC=O → π*CO states of neutral amino acids to be located below 155 nm. Upon quantitative and qualitative examination, important differences between proline and all other amino acids come to the fore as the five-membered pyrrolidine ring introduces significant distortions to the conformational space, so that band positions, signs, and magnitudes of all electronic excitations differ considerably.While we can confirm the assignments of previously proposed states of the electronic spectrum of neutral alaninele Table . Moreove35, refs therein such as molecular parity violation, selective adsorption on minerals surfaces, and enantioselective interactions with circularly polarized light or spin-polarized electrons. The detection of l-enriched amino acids in the Murchison meteorite37 suggested the possibility of an extra-terrestrial contribution to the missing link between purely racemic molecules on prebiotic earth and homochirality of life. Substantial progress has been made in the understanding of the photochemical formation of life’s molecular building blocks in interstellar molecular clouds based on the identification of amino acids38, aldehydes39, and sugar molecules40 in simulated interstellar pre-cometary ices. Obtained results were confirmed by in situ data of ESA’s cometary Rosetta mission and its COSAC41 and Ptolemy42 instruments on board the Philae Lander. Most of the current models of comet nuclei presume that to a major extent they are the aggregates of interstellar icy grains in their evolved state in the collapsing molecular cloud. Gas and solid-state chemical reactions induced by UV photolysis (unpolarized and polarized), cosmic rays or thermal cycling form and process the complex organic inventory of interstellar dust that is later integrated in the presolar nebula from which planets and small solar system bodies formed43. Besides, Rosetta’s ROSINA instrument detected the achiral amino acid glycine in the cometary coma44 allowing the assumption that comets may contain more complex amino acids that can desorb into the comet’s atmosphere at sufficiently high temperatures.Life’s amino acid preference for left-handedness is still a central puzzle in modern biochemistry, with several theoretical and experimental causes discussed46 and given the strong evidence that the solar system originated in a high-mass star-forming region47, this has been considered to further strengthen the hypothesis of a photochemical origin of life’s homochirality. Most relevant astronomical circularly polarized light (CPL) sources are reflection nebulae in high-mass star forming regions that show high degrees of circular polarization due to dichroic extinction of linearly polarized scattered light46. All CPL sources so far exhibit a quadrupolar pattern of left-handed and right-handed CPL which is explained by the scattering of the bipolar outflow lobes where the right-handed or left-handed CP regions are situated at the opposite sides of the outflow axis as well as at the opposite sides of the central illuminating source. Each quadrant of single-handed CPL extends on very large spatial scales of up to 0.65 pc46, which is hundreds of times the size of most planetary-forming systems, including our solar system. Assuming our solar system formed in a similar high-mass star-forming region, all organic molecules in the gas phase or condensed on icy grains would have been illuminated by CPL of one helicity only during protoplanetary disk evolution49.As several sources of naturally polarized light have been detected in interstellar star-forming regions21. Corresponding experiments to understand the transfer of asymmetry from chiral photons to racemic starting material demonstrated that CPL can induce asymmetric photochemistry in alanine50 and proline17, as well as being capable of asymmetric photochirogenesis of several amino acids in interstellar analog ices49. Due to an interstellar dust cycling process43, by which organic molecules such as amino acids can reversibly and continuously desorb and condense back onto the surface of cometary and pre-cometary dust particles, chiral photon-molecule interactions are assumed to have—at least partly—taken place in the gas phase of interstellar environments. Such desorption–condensation cycles may have been crucial for an ee amplification via asymmetric photolysis of interstellar ices intimately connected with the surrounding gas, i.e., starting from 10−3g values, as reported here and 10−3 to 10−2g values in the solid state21, to a few %ee as identified in carbonaceous chondrites. In such a scenario, the origin and evolution of biomolecular asymmetry would be considered as a two-step process, with an initial mirror-symmetry breaking event in the life cycle of interstellar gas and dust during cloud evolution followed by nonequilibrium reaction networks on the early Earth driving prebiotic molecular systems toward a homochiral state52. Our gas phase chiroptical data on neutral amino acids therefore complement previous CPL-based investigations on zwitterionic amino acids in the solid state50 and the absolute asymmetric photosynthesis of amino acids in interstellar analog ices49.Anisotropy spectroscopy of chiral molecules is a direct measure of the selectivity and efficiency of circularly polarized light to induce optical activity and enrich one enantiomer over the other via asymmetric photolysis45 matching the wavelength range of the anisotropy extrema of the investigated amino acids studied here. Given the decline in UV photon flux of most stars in the far UV range (<200 nm)53, the broad anisotropy nO → π*CO band (220–250 nm) would dictate the inducible ee in almost all gas phase amino acids, except valine, where the nN → 3 s band (190–220 nm) would dominate the interstellar asymmetric photolysis processes. Because stellar CPL would induce a net eeof the same handedness in almost all amino acids, such gas phase asymmetric photochemical reactions might have originally contributed to the evolution towards an enantiomeric selection of life’s l-amino acids. The gas phase chiroptical and absorption spectra reported here are moreover crucial to trigger the UV-spectroscopic identification of chiral molecules in interstellar and circumstellar space, including their enantiomeric excess determination.Stellar CPL, which is polychromatic, exhibits a dominant UV emissiong(λ), and absorption spectra A(λ) of seven pairs of amino acid enantiomers were recorded using a temperature and pressure-controlled gas cell at the ASTRID2 synchrotron radiation source, Aarhus University, Denmark (Supplementary Methods). The cell consists of a stainless-steel tube, 500 mm in length with an 18 mm inner diameter, mounted on the AU-CD beam line. The pressure inside the gas cell is monitored using a type 631D Baratron heated manometer (MKS instruments), while the cell is evacuated using a Varian V70 turbo pump. Two all-metal valves are mounted on the main gas cell tube: one connects, via a flexible hose, to the turbo pump, while the other valve connects to the heated sample reservoir. Heating tapes and cartridge heaters were used to heat the entire gas cell up to the required temperature using PID computer control, with a maximum possible temperature of 200 °C. Differential heating of the windows to 5–10 °C higher temperatures compared to the tube avoids local condensation of analytes.Circular dichroism spectra CD(λ), anisotropy spectra T = 160 °C with gradually increased temperatures until sufficient vapor pressures with optimal optical densities were reached. The gas cell was typically operated under flow conditions, where the valves to the sample and the turbo pump were partially open, to ensure a continuous renewal of the gas in the cell.Typically, less than 1 g of a given amino acid enantiomer was placed into a glass container  and inserted into the sample reservoir heater. Absorbance and CD spectra of each amino acid were recorded starting at TM software. The source and transfer temperature were kept at 230 and 240 °C, respectively. The column set consisted of a Chirasil-L-Val column  in the first-dimension modulator-coupled to a DB Wax secondary column . Helium was used as carrier gas at a constant flow of ū = 1 mL min−1. Sample volumes (1 µL) were injected in splitless mode at an injector temperature of 230 °C. The GC primary oven was operated as follows: 40 °C (1 min), warm up to 80 °C (10 min) at 10 °C min−1, and warm up to 190 °C (15 min) at 4 °C min−1. The secondary oven used the same temperature program with a constant temperature offset of 20 °C. A modulation period of PM = 5 s was applied.The GC × GC–TOF-MS Pegasus IV D system from operated at a storage rate of 100 Hz, with a 50–400 amu mass range and a detector voltage of 1.45 kV (Supplementary Methods). Data were processed using the LECO Corp. ChromaTOF−1. The HPLC was used in isocratic mode with 95% chromatography grade water (A) (acidified with 1% formic acid) and 5% methanol (B) during 40 min.The HPLC Agilent 1200 system was composed of a quaternary pump (Agilent G1311 A) equipped with a Diode Array Detector (DAD Agilent G1315D) and an ELSD (Evaporative Light Scattering Detector). A Luna C18 column  was used at 25 °C with an injection volume set at 10 μL and a flow rate set at 1.0 mL minA thorough conformational search was achieved by a biased molecular dynamics simulation following an Umbrella Sampling protocol. A single alanine residue was described in the neutral state using the ff14SB force field. All 22 selected alanine structures were refined with Gaussian16 using a combination of different methods and a large basis set quadruple-ζ with polarization and diffuse functions: aug-cc-pvQZ. A single-point energy calculation with zero-point corrected energies including frequency calculations was done to verify their nature of true minima. Three methods were evaluated: CAM-B3LYP, ωB97X-D, and M06-2X. Finally, seven low energy conformers were further considered for calculations. TD-DFT calculations were done on the seven optimized structures using the three previous functionals combined with the aug-cc-pvQZ basis set. Two hundred excited states were calculated for each optimized conformer.27 study of alanine conformers was used as a starting point, from which all reported 13 local minimum structures were obtained and subsequently Boltzmann-weighted. A total of nine alanine conformers were included in the computation of the CD spectrum. All 13 conformer structures were energy optimized with the program package Molpro on the df-CCSD(T)-F12 level with the aug-cc-pVDZ-F12 basis set, specifically with the F12b method and the 3*C ansatz as implemented in Molpro and their cartesian coordinates in Å are provided in the Supplementary Data For the coupled cluster calculations, Csaszar’sSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


Directed motility enables swimming microbes to navigate their environment for resources via chemo-, photo-, and magneto-taxis. However, directed motility competes with fluid flow in porous microbial habitats, affecting biofilm formation and disease transmission. Despite this broad importance, a microscopic understanding of how directed motility impacts the transport of microswimmers in flows through constricted pores remains unknown. Through microfluidic experiments, we show that individual magnetotactic bacteria directed upstream through pores display three distinct regimes, whereby cells swim upstream, become trapped within a pore, or are advected downstream. These transport regimes are reminiscent of the electrical conductivity of a diode and are accurately predicted by a comprehensive Langevin model. The diode-like behavior persists at the pore scale in geometries of higher dimension, where disorder impacts conductivity at the sample scale by extending the trapping regime over a broader range of flow speeds. This work has implications for our understanding of the survival strategies of magnetotactic bacteria in sediments and for developing their use in drug delivery applications in vascular networks. Microswimmers can navigate porous environments, however the impact of their directed motility on their movement in fluid flow remains an open issue. The authors show that the motility of magnetotactic bacteria in flow through a porous constriction gives rise to nonlinear flow conductivity similar to electrical diodes. To navigate these complex geometries, cells use environmental cues to direct their motility towards favorable conditions via an array of sensing 6 and physical reorientation mechanisms 7. However, directed cell motility can be modified by ambient fluid flows11, which profoundly affects biomass distribution and productivity in the environment8. The geometric confinement of these environments regulates fluid flow and consequently, cell growth12 and swimming dynamics15. Confinement thus plays a central role in coupling directed motility to flow19. For example, in porous media flows, chemotaxis significantly augments the retention of swimming bacteria20. Directed microbial motility in porous geometries is integral to ecological, environmental, and biomedical applications21. Yet the impact of the microscopic coupling between directed cell swimming and heterogeneous flows on sample-scale cell transport properties remains elusive22.Hydraulic networks, ranging from saturated soils and sediments to human tissues and filtration media, play host to diverse swimming microbes7, enabling them to seek out microaerobic conditions in heterogeneous marine sediments23. MTBs provide an ideal physical18 and relevant ecological24 model system to probe the effects of directed motility on cell transport in heterogeneous flows: Their primary directional cue—the magnetic field—is purely mechanical, uniform, and independent of the ambient flow field. Beyond gaining insight into the evolutionary advantages of natural magnetotactism, MTBs are a promising candidate in biomedical applications for targeted drug delivery due to their potential to be directed through heterogeneous vascular network flows26.Magnetotactic bacteria (MTB) naturally biomineralize nano-scale magnets called magnetosomes, which are embedded in their cell membrane. These particles are arranged in a linear array and form a permanent magnet that mechanically and passively aligns MTBs with Earth’s magnetic field27, the transport is completely halted for a wide range of flow rates. This bacterial diode phenomenon is in stark contrast to linear fluidic network and continuum transport models29, which typically stem from a detailed understanding of pore-scale processes32. Simulations, based on single-cell motility modeling, reveal that cell trapping due to vortical localization is associated with diverging residency times and enhanced dispersion. We experimentally show that vortical trapping persists in higher dimensions (3D packed beds). Further, we extend our simulations to illustrate how disorder greatly broadens the trapping regime to impact sample scale transport, due to the dominance of a few strongly trapping pores. These nonlinear transport properties pose new challenges for modeling directed active matter33, far beyond the paradigms of classical passive scalar transport34.In this study, we use microfluidic experiments Fig.  in 1D poMagnetococcus marinus (MC-1) bacteria35  ensures that cell−cell interactions are negligible36. The channels 38 comprise semi-circular walls with alternating concavity . The restoring magnetic torque on the cells competes with stochastic and hydrodynamic body reorientations and aligns the MTB swimming direction upstream.A suspension of north-seeking rinus MC- bacteriarinus MC- bacteriarinus MC- bacteriarinus MC- bacteriaUmax/Vs  and are evident in the spatial distribution of cells  In the upstream regime (Φ = Umax/Vs < 1), the swimming speed is large compared to the flow speed  The trapping regime (1 < Φ < Φ*) is characterized by a near-zero mean bacterial advection speed, 〈Vx〉 ≈ 0, and coincides with the emergence of bacterial vortices  In the downstream regime (Φ > Φ*), the flow speed dominates cell swimming speed even in the pore region, Vs < Umin. Bacteria are swept downstream with a negative mean bacterial advection speed, 〈Vx〉 < 0, and the vortical orbits begin to dissipate. In contrast to the linear hydrodynamic conductivity of the carrier fluid , the escape rate is large owing to the dominance of upstream swimming and downstream advection, respectively. For intermediate flow speeds (1 < Φ < Φ*) in the trapping regime, the escape rate decreases to nearly zero, indicating suppressed transport. Beyond the qualitative agreement in the swimming trajectories  > 0) as they pass through low-speed pores and high-speed throats  < 0) on the lower branch of the distribution. Individual cells switch between these two states in a stochastic catch-and-release process.Retention of bacteria in porous media has been extensively studied for its environmental and ecological importanceMTB Fig. . In the ats Fig. a with noies Fig. . The restes Fig. c. In thetes Fig. . Subsequτres = 1/α, in a pore increases as the normalized flow speed approaches Φ = 1 for every geometry, corresponding to 〈Vs〉 = 0 2〉 and normalized by the effective diffusion coefficient, D0, without flow in a straight duct  is reached, D/D0 can no longer be defined in our simulations, since the trapping time is longer than the simulation time , the diffusive behavior is recovered and exhibits a strikingly large dispersion coefficient  established for the corrugated channels to two different 2D geometries: (i) a regular square lattice of cylindrical obstacles Fig. d and iie. Both gIn the disordered geometry, the same three distinct transport regimes are also observed, but the sample scale conductivity exhibits a marked five-fold increase in the range of the trapping regime  are cultivated in standard agarose gels within a chemically imposed oxygen gradient, using well-established protocols35. A stock solution of artificial sea water  is prepared in a 1 l glass bottle. 100 ml is sampled from this stock solution, to which the following are added: 0.2 g of HEPES, 0.03 g of NH4Cl, 0.5 μl of Wolfe mineral solution, 80 μl of 0.5% resazurin solution, and 300 μl of 0.01 M FeS04 solution. The solution is completed with 180 μl of 0.5 M phosphate buffer (pH 6.8) and 0.16 g of agarose. Separately, 1 g of sodium bicarbonate is inserted into a tightly screwed glass tube. Both the 100 ml solution and the sodium bicarbonate tube are autoclaved. The solution is cooled to 60 ∘C, and 560 μl of a 20% sodium thiosulfate solution, 40 mg of fresh cysteine, and 280 μl of a 10% sodium bicarbonate solution are added. Before gelation, the solution is poured into a 16 ml glass test tube, leaving 1 cm of air space, and capped. An oxygen gradient forms via diffusion and is visualized by the presence of resazurin. Cells grow at the oxic−anoxic transition zone (OATZ), where they form a heterogeneous growth front. After three days of growth, ≈1 μl of cells is sampled from the front via pipette and deposited at the microfluidic channel inlet. Cells are then directed into the corrugated microchannel via an applied magnetic field. These conditions result in a dilute cell concentration of ~106 cells/ml, which enables the tracking of individual cells. Motility media, which is a liquid version of the growth media , was used for flow experiments.Magnetotactic bacteria (Umax ≤300 μm/s ± 2 μm/s) is regulated using a precision pressure controller (Elveflow OB-1), where the dynamic range (0 ≤ P ≤ 150 mbar) is set by an additional resistor channel  using standard soft lithography techniques and plasma bonded to standard 1 mm glass slides. The flow speed in the channels , and videos of cell motility (≥400 frames each) were captured (Andor Zyla camera) at frame rates ≥30 frames/s. Cells were tracked using a predictive particle tracking algorithm implemented in a custom MATLAB codeFlow fields Fig. c in the 39 was constructed by considering the forces and torques (Fig. Vs, and local fluid velocity, u and v. The orientational dynamics are governed by hydrodynamic rotation due to vorticity, ω, magnetic field alignment due to a magnetic torque, and rotational noise that encompasses Brownian rotational diffusion and flagellar-induced orientational fluctuations of the cells (Fig. u and v, and vorticity, ω, are determined from finite element simulations in each geometry (Supplementary Information and Supplementary Fig. Dr = 0.25 ± 0.05 s−1, is measured from MTB swimming trajectories in the absence of both external flow and applied magnetic field (Supplementary Fig. M, and the hydrodynamic rotational mobility of the cells, μr, are not readily accessible individually, their product is easily obtained from cell tracking measurements in a calibrated magnetic field (Supplementary Fig. τ = tVs/wt = 20,000 with a time step δτ = 0.005. In the 2D structures, the total dimensionless simulation time was set to τ = tVs/2R = 256, 5 times the necessary time for a cell to swim from one end to the other. Steric boundary conditions prevent cells from entering the solid surfaces in our simulations. A Weeks−Chandler−Andersen repulsive potential was implemented:d is the distance to the closest solid surface, a is the bacterial radius, and ϵ is the hardness (see A Langevin model of swimming MTBues Fig. b acting entclass1pt{minimaFurther information on research design is available in the Supplementary InformationSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Reporting Summary