Consequently, the present study investigated the impact of the type of sensory input  on cognitive map formation. To do so, early blind , late blind , and sighted control  participants were tasked to learn the layouts of mazes produced with different sensory information  and to infer routes from memory. Results show that EB manifested stronger cognitive map formation with 3D mazes, LB performed equally well with 2D and 3D tactile mazes, and SC manifested equivalent cognitive map formation with visual and 3D tactile mazes but were negatively impacted by 2D tactile mazes. 3D tactile maps therefore have the potential to improve spatial learning for EB and newly blind individuals through a reduction of cognitive overload. Installation of 3D tactile maps in public spaces should be considered to promote universal accessibility and reduce blind individuals’ wayfinding deficits related to the inaccessibility of spatial information through non-visual means.For blind individuals, tactile maps are useful tools to form cognitive maps through touch. However, they still experience challenges in cognitive map formation and independent navigation. Three-dimensional (3D) tactile information is thus increasingly being considered to convey enriched spatial information, but it remains unclear if it can facilitate  Orientation is defined as: “The knowledge of one’s distance and direction relative to things observed or remembered in one’s surroundings and the ability to keep track of these spatial relationships as they change during locomotion”5. While most humans automatically and effortlessly use visual information to establish and maintain orientation6, those living with severe visual impairments or blindness must learn to rely on less precise sensory channels, such as touch , audition , olfaction , and proprioception , to gather and encode spatial information and navigate8. To date, orientation, or wayfinding, is still one of the main daily challenges of visually impaired individuals, which can lead to decreased autonomy and confidence9, increased anxiety before and during navigation, disorientation, and ultimately to decreased professional opportunities and societal isolation11.Orientation and independent spatial navigation are intricate behaviors that depend on highly complex cognitive processescognitive maps of their surroundings2. A cognitive map is defined as an internal mental representation of an environment that preserves spatial properties  and relationships  between memorized landmarks, paths, or other environmental features13. Such cognitive maps are therefore allocentric, or viewpoint-independent, and flexible in a way that enables individuals to infer routes and detours despite barriers and regardless of their location and viewpoint14. Cognitive map formation, or spatial learning, is thus an essential skill for navigation as it enables better route planning and improves problem solving skills. For example, individuals with good cognitive maps can more easily realize if they stray away from a planned route and then correct themselves instead of becoming disoriented. Although blind individuals can form such cognitive maps and gain efficient spatial knowledge to guide navigation, spatial learning is a heavily serialized process that requires significant exposure to environmental features, and thus more time and cognitive effort for memorization16. It is therefore likely for blind individuals to be disoriented in complex spaces that require a lot of turns and non-linear movements17.In fact, orientation in the absence of vision is heavily dependent on individuals’ ability to form 1. These can be rapidly explored by the user, thus providing a relatively simultaneous aerial view of an environment while sidelining many difficulties linked with real-time exploration such as various dangers, anxiety, veering, disorientation, and the constant strain on memory20. Tactile maps can thus provide a survey, or allocentric, understanding of an environment in a more effective way than direct experience22. Because of their usefulness, there are now numerous technologies and ways to produce tactile maps23. However, early blind (EB) still experience challenges in orientation and tactile map reading27.Tactile maps are effective tools used by blind individuals to facilitate cognitive map formation in novel small- and large-scale environments. As a tactile substitution for visual maps, they only provide essential spatial information and preserve all spatial relationships between landmarks, destinations, or objects in the environment28 and have led many researchers to investigate the potential of 3D printers to automatically produce tactile maps32. Indeed, 3D scale models like those produced with 3D printing are viewed as beneficial and durable tools to convey spatial information and facilitate orientation in indoor and outdoor spaces35. Gual-Orti and colleagues39 showed that volumetric symbols produced by 3D printing can be understood, easily memorized, and used to efficiently follow routes. Holloway and colleagues41 demonstrated that completely 3D printed tactile maps, when compared to traditional 2D embossed tactile graphics, were advantageous for the memorization of information and for the comprehensibility of harder concepts such as depth and occlusion . They also showed that blind individuals preferred these models over their 2D counterparts41. However, there is less evidence on how, and if, 3D tactile information can improve cognitive map formation at a functional level.In recent years, advances in 3D printing technology have spurred interest in the field of vision rehabilitation6, we tested subjects with early- and late-onset blindness as well as sighted controls who also performed the task visually. We hypothesized that (1) mazes that carry more information in the 3rd dimension  will lead to more accurate cognitive maps than 2D mazes for all groups; and (2) the 3rd dimension would be particularly beneficial to those lacking visual experience, but that vision will still lead to better cognitive maps in the sighted.Therefore, the present study investigated, for the first time with quantitative measures, the impact of tactile 3D information on the ability of blind and sighted subjects to explore mazes and form functional allocentric cognitive maps. To do so, we compared equivalent 2D swell paper and 3D printed tactile maps in an original paradigm of “contextual mazes”. Furthermore, since age at onset of blindness and visual experience have a significant impact on brain development and performance in complex spatial tasksAs for symbol localization times (symbol detection and identification), SC took 92.25 ± 73.87 s to find all symbols in simple 2D mazes, 284.74 ± 226.71 s in complex 2D mazes, 24.31 ± 24.66 s in simple 3D mazes, 85.07 ± 42.87 s in complex 3D mazes, 2.49 ± 0.90 s in simple visual mazes, and 5.84 ± 1.28 s in complex visual mazes. LB took 50.46 ± 31.77 s in simple 2D mazes, 141.63 ± 115.66 s in complex 2D mazes, 16.75 ± 12.08 s in simple 3D mazes, and 96.08 ± 55.51 s in complex 3D mazes. Finally, EB took 31.85 ± 19.15 s in simple 2D mazes, 58.19 ± 19.05 s in complex 2D mazes, 13.12 ± 9.05 s in simple 3D mazes, and 87.39 ± 69.00 s in complex 3D mazes. These results are illustrated in Fig. p < 0.001***, p = 0.078). This indicate that age is an important factor in SC symbol localization times.When looking at SC in all conditions , it is noteworthy that SC almost immediately detected visual symbols but took longer for tactile symbols, and more so for 2D than 3D symbols. However, when performing the 2 × 3 ANCOVA (corrected for age and sex), this effect of information type was cancelled out by the effect of age. Indeed, the test revealed a significant interaction between age and information type  = 18.022, p < 0.05*, p < 0.05*, p < 0.001***, p < 0.001***) and 3D mazes  = 17.657, p < 0.001***), but post-hoc T-tests with Bonferroni correction revealed that EB took less time to find symbols in simple mazes only in the 3D tactile condition . This indicates that EB were so efficient in exploring 2D mazes that the number of symbols did not affect their symbol localization time while it did for 3D mazes , meaning that, despite the effect of complexity in 3D mazes, they were equally fast in 2D than in 3D. On the contrary for LB and SC, ANOVAs revealed that both were impacted by information type  = 6.313, p < 0.05*, p < 0.001***, p < 0.001***, p < 0.01**, p < 0.001***; SC: mean difference = 126.627 s, t = 4.177, p < 0.01**) and were faster to find symbols in 3D when compared to 2D . This last result is illustrated in Fig. p = 0.062, When analyzing symbol localization times for the three groups in the tactile 2D and 3D conditions, the 3 × 2 × 2 ANCOVA (corrected for age and sex) revealed three-way interactions between groups, information type and complexity  = 4.721, p < 0.001***, p < 0.05*), an effect that did not survive the correction for the age covariate  = 0.043, p = 0.840). This indicates that age is an important factor in SC symbol localization times. Nonetheless, we note that SC took longer times finding 2D symbols when compared to 3D symbols , an effect likely caused by the time taken by all groups to find the 3D version of the symbol  revealed two significant two-way interaction effects. The first interaction was between information type and group F1,32) = 13.289, ,32 = 13.,32 = 13.p = 0.004, 95% CI ), but no significant effect of information type . Furthermore, the grouping factor accounted for a negligible proportion of the variance. These results demonstrate that the POI recall performance did not change across groups and was only affected by complexity. Indeed, average POI recall performance was 95.73% for simple mazes and decreased to 85.73% for complex mazes.Q-Q plots revealed deviations from normality in the distributions of points of interest (POI) recall performances. Therefore, a GLMM analysis was conducted to examine the effects of information type and complexity on POI recall performance, taking into account the grouping factor as a random effect in the model. Contrary to our hypothesis, the analysis revealed only a significant effect of complexity . The test thus only revealed a significant effect of maze complexity  = 5.116, p < 0.05*, p < 0.001***).When looking at SC route performance in all conditions , we notice that SC performed equally well in the tactile 3D and the visual control conditions, while their performance decreased in the tactile 2D condition. However, when performing the 2 × 3 ANCOVA, this effect of information type was cancelled out by the effect of the age covariate  = 3.999, p < 0.01**, p < 0.001***, p < 0.001***) and SC  = 36.994, p < 0.001***), but not LB  = 0.371, p = 0.558). However, when correcting for the age covariate, the main effect of information type on EB remained  = 6.904, p < 0.05*), but disappeared for SC  = 1.714, p = 0.217). Since it was noticed that age negatively impacted SC performance in route elaboration (see next section), we further investigated the effect of information type on route elaboration performance for SC who were older than 50 years old (n = 7) compared to those who were younger than 50 years old (n = 7) with a 2 × 2 ANOVA. The test revealed no interaction between the age group and information type  = 0.419, p = 0.530), but a significant effect of information type  = 61.554, p < 0.001***). Post-hoc T-tests with Bonferroni correction revealed that 3D mazes improve performance for both SC  and EB , but not LB . This reveals that 3D tactile information can improve the cognitive mapping skills of individuals who lack either tactile experience (SC) or visual experience (EB), but not those who cumulate both (LB). All route elaboration results are illustrated in Fig. When analyzing route performance for the three groups in the tactile 2D and 3D conditions, the 3 × 2 × 2 ANCOVA (corrected for age and sex) revealed a significant maze complexity effect  = 9.263, p < 0.001***, p = 0.024*, p = 0.005**, p = 0.001**, p = 0.017*, p = 0.019*, p = 0.03*, p = 0.089, Pearson’s R tests were performed to investigate the previously identified effects of age on symbol localization time and route elaboration performance. For SC, age correlated with symbol localization time in 2D mazes  and, to some extent, LB , meaning that good navigators have better cognitive mapping skills in the context of our task  as expected, SC immediately detected visual symbols, but took a longer time to locate these symbols when presented in the tactile modality, an effect that is most likely due to the surprisingly long time they took to detect symbols in 2D mazes  facilitates exploration for tactilely untrained individuals and even reduces interindividual differences caused by age, blindness onset, and visual experience. This is consistent with previous studies that showed that 3D haptic perception is not affected by age45 and leads to equal or superior shape recognition performance in blind individuals compared to SC42. However, enhancing tactile contrast with 3D information also comes with significant caveats. While the advantage of using 3D information in tactile maps is the ability to change the elevation of important information, its disadvantage is that some information may be masked by protruding information and that, in the same way, hollowed information  becomes ineffective. Professionals developing 3D maps must therefore (1) avoid using hollowed information; and (2) avoid using high objects that mask less elevated information and, thus, impede exploration.Symbol localization time differences between SC, EB and LB are in line with previous studies showing that extensive tactile training and overreliance on tactile information in EB and LB can improve tactile sensitivity and, thus, the efficiency of tactile exploration47 and that blind individuals, regardless of visual experience, can form allocentric spatial representations of unfamiliar environments46. However, EB and SC route performance with tactile 2D mazes shed light on significant disadvantages of using tactile maps to support cognitive map formation. On the one hand, learning can be negatively impacted by tactile exploration skills and age, as shown by SC. On the other hand, learning with 2D tactile information is also limited by individuals’ prior knowledge and exposure to spatial information. This is shown by EB who experienced more challenges in forming accurate cognitive maps from 2D tactile maps compared to LB48. This difficulty may arise from EB participants’ lack of familiarity with the representational rules of visual 2D maps 53 while prior knowledge of such representational rules  benefits less experienced tactile readers like LB50. Indeed, it has been shown that this performance gap is reduced when EB become more familiar with 2D images50 or, similarly to the present study, when using 3D shapes42. Additionally, 3D tactile information surprisingly led to route performance equivalent to that with vision. This finding indicates that the tactile sensory modality can carry as much relevant spatial information as vision and further emphasizes the advantage of 3D tactile information to improve cognitive map formation for all types of individuals.All participants were able to infer routes with varying levels of complexity, supporting the idea that cognitive map formation is possible using visual and tactile sensory modalities50. This sequential exploration therefore is highly taxing for spatial working memory capacities and might subsequently delay or interfere with the integration of spatial information into higher-order representations. Furthermore, reading 2D maps relies on the memorization of different types of symbols that are abstract in nature and are thus more demanding on memory and attentional resources56. Consequently, this cognitive overload57 can negatively impact cognitive map formation. Since adding 3D information to tactile maps can benefit all participants regardless of age and blindness onset, we hypothesize that 3D information in tactile maps can reduce this cognitive overload by optimizing working memory capacity57, an advantage that resides in the following factors. First, 3D haptic perception relies less on tactile sensitivity and attention. Indeed, 2D embossed information like Braille relies mainly on one type of somatosensory receptor59 and varies according to stimuli texture60 and the individual’s attentional capacities61. On the contrary, haptic feedback from 3D tactile information involves receptor types that respond to skin stretch, muscle stretch, and articulation angle, and relies less on the individual’s ability to attend tactile stimuli at the cutaneous level45. 3D information is thus more advantageous for individuals who recently became blind or visually impaired or for older adults living with vision loss. As tactile sensitivity is known to decrease with age45 and as the prevalence of vision loss related to age and health conditions  is rising, technology that is less reliant on tactile sensitivity, like 3D printed maps, would be more universally advantageous. Second, it is known that abstract symbols in traditional 2D tactile maps can be more challenging to certain people, especially individuals living with a cognitive impairment and older adults with memory deficits62. Compared to 2D embossed information, 3D is less reliant on abstract symbols and can display more faithful representations of objects62. These are thus easier to locate, identify and even memorize39.Cognitive map formation is a cognitively demanding task. This is especially true when the individual is limited to the sense of touch for which exploration is highly sequential compared to visual explorationConsequently, a 3D map user does not need to constantly remember the meaning of abstract symbols or pay attention to the boundaries of the navigable and non-navigable areas since routes and paths become easier to follow. 3D information can thus increase the allocation of attentional resources to the spatial learning task itself and lead to more complete cognitive maps for the same exploration time. Additionally, 3D tactile maps are less affected by interindividual differences such as tactile training and age. Thus, our research demonstrates that publicly displaying well-produced 3D maps, for example in museums, malls, or airports, would enable blind and visually impaired individuals worldwide to find their destinations more efficiently and independently in novel areas.65. However, our study shows that improving the way tactile information is presented can also improve the spatial learning process and lead to equivalent cognitive map formation without the need for extensive training. The quality of the depicted information thus seems to be a more important factor than visual or tactile experience because 3D information improves spatial learning for individuals who lack one or the other. These findings are congruent with the amodality hypothesis of spatial representations according to which spatial representations, including allocentric cognitive maps, are formed independently from sensory modalities and visual experience67. However, the amodality hypothesis remains highly debated since EB often show deficits in spatial processing71 and rely more on egocentric strategies (route knowledge) rather than allocentric ones67. Nonetheless, EB remain able to form allocentric cognitive maps and to use related wayfinding strategies for which errors often depend on how spatial information was acquired74. It is therefore increasingly believed that deficits arise from flawed infrastructure design that only leaves spatial information fully accessible to sighted individuals and not to those with visual impairments67. This viewpoint is supported by recent literature that shows that, when given the same amount of spatial information, EB perform as well as SC and LB in many spatial tasks and, by doing so, they activate the same cortical areas and neural networks than their sighted counterparts66. This emphasizes the need to improve the accessibility of spatial information and to adequately adapt and standardize the tactile maps available in public spaces75. Clearer 3D maps may give rise to more opportunities for autonomous navigation, more exposure to complex environments, and hence improved cognitive mapping skills in visually impaired populations.It has been shown that EB, even those with no visual experience, can perform equally well to LB and SC at wayfinding tasks after extensive training77 or, in some cases, by adding textures78. However, textures and multimodal learning were not investigated in the context of the present study, and this may have limited the spatial information that blind participants were able to absorb. Therefore, one could assume that combining 3D information with textures, verbal descriptions, and direct exploration may improve the efficiency of tactile maps.Even though tactile maps are beneficial tools, they often provide a spatial representation from one unique aerial perspective. Indeed, to facilitate exploration and reading, blind participants often have the same viewpoint , which might lead to the formation of less flexible cognitive maps. In fact, sources of errors, especially in EB, were linked to the inability to switch between the aerial  viewpoint and the intended egocentric viewpoint. For example, participants would confuse “left” and “right” when facing south or would use vocabulary such as “going  in the map”. Furthermore, it is known that the efficiency of tactile maps can be improved by combining their use with verbal feedback and direct explorationThe present study investigated, for the first time with quantitative measures, the impact of information type on the ability of blind subjects to form allocentric cognitive maps and infer different routes from memory. It demonstrated that 3D information can lead to cognitive maps that are equivalent to those formed with vision by reducing the cognitive overload that stems from tactile exploration and abstract symbol memorization. Therefore, the present findings support that cognitive mapping deficits observed in early blind individuals may not arise from blindness per se, but from the inaccessibility—or inadequate adaptation—of the spatial information available in the environment. Installation of 3D printed tactile maps should be considered in public spaces to promote universal accessibility by facilitating orientation and promoting independence of blind and visually impaired travelers.80.Previous versions of this work were published as preprints84. Table The present study was approved by the Clinical Research Ethics Committee (CERC) of the University of Montreal (project # CERC-20-081-D) and was conducted in accordance with the Declaration of Helsinki. All participants gave informed consent to participate in this study. We included a total of 25 participants with complete blindness  including 11 early blind  and 14 late blind . Blind participants were classified according to the following criteria: if complete blindness occurred at ≤ 6 years old, participants are considered EB; if blindness onset happened after 6 years old, participants are considered LB. However, two LB (participants 12 and 13) had total blindness at 7 and 8 years old but were functionally blind since early years of life with progressive vision loss. Therefore, these participants were included in the EB group during statistical analysis, totalizing 13 participants in the EB group and 12 in the LB group. Consequently, all participants in the EB group were functionally or completely blind before the developmental period in which normally sighted children demonstrate significant improvements in spatial memory and in the use of allocentric cognitive maps and strategies86. To evaluate the influence of experience-dependent plasticity in both EB and LB, we also calculated the blindness duration index (BDI) according to the formula “(age–age onset blindness)/age”88. The BDI score can range from 0 to 1, indicating the proportion of life that participants have been blind, with low scores indicating recent blindness onset and high scores, longer blindness duration.We included 14 age- and sex-matched sighted controls (SC) with normal or corrected-to-normal vision . Participants had no associated neuropathy or other health conditions that could affect tactile sensitivity or mental spatial representations. All blind participants were Braille readers, fluent in French, and had previous experience with tactile maps. Before the start of the experiment, participants completed a questionnaire on their visual diagnosis, orientation and mobility (O&M) training, Braille reading efficiency, and spatial abilities, including the Santa Barbara Sense of Direction (SBSOD) scale89. Eight different contextual mazes (C-mazes) were produced in three different information types: tactile 2D (swell paper), tactile 3D (3D printed), and visual (pictures displayed on a computer monitor). Each C-maze was composed of a network of interconnected hallways and contained entrance halls, symbols indicating floor changes (staircases and elevators), and points of interest (POIs) consisting of business names  annotated in Canadian French Braille and randomly taken from a list of businesses found in the Montreal area. Furthermore, the C-mazes were also produced with two levels of complexity (simple and complex) according to the number of included features: the four simple mazes included one entrance hall, one elevator, one staircase (ascending), and six POIs; the four complex mazes had two entrance halls, two elevators, three staircases , and ten POIs. To avoid recall bias, POIs were different for each maze. The 3D C-mazes were 3D printed with the Prusa i3 MK2S  printer and the 2D C-mazes were produced on traditional swell-touch paper with the PIAF Tactile Image Maker . Figure The research material for this study was produced with resources from the Open Science Fabrication Laboratory OptoFABcontextual maze learning paradigm and its three phases  and four 2D  C-mazes across two experimental sessions. Blocks always alternated between 2D and 3D mazes. Participants were randomly distributed into two categories: starting with either a 2D or 3D maze. Following this system, the first session began with two simple C-maze blocks and then two complex C-maze blocks, while the second session began with two complex C-maze blocks and ended with two simple C-maze blocks (AB-BA order).SC group. SC participants were given four visual C-mazes  as visual controls and four tactile C-mazes including two 3D  and two 2D  mazes across two experimental sessions. In the first session, participants performed four blocks of visual C-mazes in the following order: two simple C-maze blocks followed by two complex C-maze blocks. In the second session, blocks alternated between 2D and 3D mazes, and participants were randomly distributed into two categories: starting with a 2D or a 3D maze. Following this system, the session started with two simple C-maze blocks and ended with two complex C-maze blocks. For tactile C-mazes, SC participants were blindfolded and only saw the mazes after completing the experiment.All steps of the experiment were preceded by a period of instructions during which the experimenter explained the task and the participant could not see or touch the maze. A complete diagram of the paradigm can be found in Fig. Phase 1: Symbol localization (detection + identification). The C-maze was either placed on the table in front of the participant (tactile 2D or 3D condition) or displayed on a monitor . Participants were asked to explore the maze either with their hands (tactile conditions) or with their eyes  with the goal of detecting and identifying all symbols  present in the maze. Symbol localization (detection + identification) times and order of localization were collected during that stage. In tactile conditions, the experimenter then made sure that the participants could read all POIs written in Braille. To do so, blind participants were asked to browse the perimeter of the maze and to read POIs aloud, whereas SC participants had their hand guided by the experimenter who verbally identified POIs for them.Phase 2: Maze exploration and learning. This stage consisted of a maze memorization task. During a limited period of either four minutes (simple mazes) or five minutes (complex mazes), participants were asked to freely explore the given maze with the goal of learning its layout as much as possible. For tactile 2D and 3D conditions, maze exploration could be done with both hands. As SC participants could not read Braille, the experimenter verbally identified POIs every time the participants touched one. Aside from this interaction, the experimenter did not make any interventions. Once the time was up, the tactile maze was removed from the participants’ reach or the monitor displaying the visual maze was turned off.Phase 3: POI recall and route elaboration. The goal of phase 3 was to test the cognitive map formed through exploration in phase 2. Participants were first asked to recall all POIs that were present in the given maze. If some POIs were forgotten, the experimenter reminded their names to the participant. Then, the route elaboration task consisted of six questions that necessitated participants to mentally represent routes related to the map elements and verbalize them. These questions were of two types: (1) route completion: the experimenter gave a route and participants were tasked to identify the POI at which the route ended; (2) route elaboration: participants were asked to elaborate a route between two POIs. To complete the route elaboration task, participants were told to roleplay and give their answers like they would if guiding someone who needed help reaching their destination in the maze.Since route elaboration is a cognitively complex task and participants were allowed to use their own strategies and vocabulary, responses were prone to various sources of errors. To circumvent this limitation, each answer was scored by four independent reviewers: three trained orientation and mobility (O&M) specialists  who were either present during the experimentation or were provided audio recordings, and who have the experience to subjectively evaluate routes according to their experience with the clientele; and one research assistant provided with anonymized audio recordings and a strict grading system to objectively grade each answer. The final score for each answer was the average of the four independent scores. More detailed information regarding the performance scoring protocol can be found in supplementary file Since a double-blind experiment was not achievable due to the nature of the tactile stimuli, the study could be subject to confirmation bias from participants and experimenters. To circumvent this limitation, all questions were formulated in neutral language that did not advantage any of the information types. Moreover, we ensured that at least two O&M specialists and the research assistant—tasked with the objective evaluation—were: (1) absent during data collection; and (2) provided with anonymized audio recordings containing no information with which they could identify the participant’s group or the information type.90. The effects of factors such as group , type of information , complexity , and symbol identity  on the dependent variables were analyzed with mixed factor ANCOVAs with age and sex entered as covariates. Greenhouse–Geisser correction to the degrees of freedom were applied on analyses where the assumption of sphericity of data was violated as detected by significant Mauchly’s tests. Normality of the data was visually checked with Q-Q plots, and a generalized linear mixed model (GLMM) analysis was performed when deviations from normality were observed. We also reported the effect of covariates (age and sex) only when they had a significant effect on the dependent variables or interacted with other factors of interest.The collected data consisted of the three following dependent variables: symbol localization times , and performance (in %) in the POI recall and route elaboration tasks. Statistical analyses of the collected data were performed using JASP version 0.14.1, an open-source graphical program developed by the University of AmsterdamWe first analyzed data from the SC group independently. Symbol localization times were analyzed using a 3 (information type) × 2 (complexity) ANCOVA and a 3 (information type) × 4 (symbol identity) ANCOVA, while performance in POI recall and route elaboration was analyzed using 3 (information type) × 2 (complexity) ANCOVAs. Then, we performed analyses to test how the three groups compared to each other in tactile 2D and 3D conditions and how they were affected by the different factors. Symbol localization times were analyzed using a 3 (group) × 2 (information type) × 2 (complexity) ANCOVA and 3 (group) × 2 (information type) × 4 (symbol identity) ANCOVA, while performance in POI recall and route elaboration was analyzed using 3 (group) × 3 (information type) × 2 (complexity) ANCOVAs. Simple main effects tests and post-hoc T-tests were then performed to locate and identify significant differences in localization times and performance. Lastly, we investigated if there were correlations between age, blindness onset, BDI, SBSOD scores, and the different dependent variables using Pearson’s R and Spearman Rho tests. For every test, the significance level of α = 0.05 was chosen in order to accept or reject the null hypothesis .Supplementary Information.


Wistar, wild-type (WT) and TgF344-AD rats (TG) were scanned for 60 min with [11C]leucine. Arterial blood activity was monitored online and with discrete whole blood and plasma samples by γ-counting in Wistar rats, WT (n = 4) and TG (n = 5). Unlabelled amino acids were measured in plasma. The sensitivity of [11C]leucine PET to measure alterations in PSR was assessed in Wistar rats by injection of PSR inhibitor anisomycin before PET acquisition. Anisomycin administration significantly reduced the net uptake rate constant (Kcplx) of [11C]leucine and PSR, proving the suitability of the method. For the longitudinal study, averaged population-based input functions were used to calculate PSR. We found a significant genotype effect on PSR (decrease in TG vs WT) only in the globus pallidus. This study suggests that [11C]leucine PET is sensitive enough to measure brain PSR in rat but that cross-sectional design with individual input function should be preferred.Long-term memory requires stable protein synthesis and is altered in Alzheimer’s disease (AD). This study aimed to implement a method to measure the cerebral protein synthesis rate (PSR) with [The online version contains supplementary material available at 10.1007/s11307-022-01796-0. Here, we have used the compartmental model from Schmidt et al. leucine and (ii) apply this method to investigate potential changes in PSR in the TgF344-AD rat model of AD [11C]leucine is technically demanding, so we initially developed our methodological approach in Wistar rats. In addition, to aid analysis, a population-based measurement of the input function and unlabelled amino acid measurement coming from the Fischer-344 strain were generated and used.The aims of the present study were therefore to el of AD , 13 at 6el of AD –15. Meas11C]leucine; all animals had online and discrete blood sampling; 6 were used for baseline scan, and 3 received a pre-injection of anisomycin (PSR inhibition) prior to PET scan, and an additional 5 rats were used for measurements of [11C]leucine and LNAA concentrations in blood and plasma on the bench only (i.e. not scanned). Two male and two female Fischer-344 (WT) and TgF344-AD (TG) rats with the APPswe and PS1Δe9 mutations  were set up as breeding pairs, housed in the Biological Services Unit at the University of Manchester for breeding purposes. Genotyping was outsourced to Transnetyx® . Male WT and TG rats were imaged via [11C]leucine PET at 6, 12 and 18 months of age  preventing scanning of the animals or leading to the animal to be culled to prevent unnecessary suffering. Whenever possible, animals that had died or were excluded before the 18 months time point were replaced by age-matched rats of the same genotype . Fourteen rats were scanned with 1Cleucine11C as previously described leucine PET to measure PSR alterations, three Wistar rats were injected i.v. with 60 mg/kg of anisomycin in saline (pH 7) leucine ratios were measured by γ-counting . For the longitudinal study, the PSR was calculated using population-based values of unlabelled leucine, a population-based AIF scaled for each rat using an image-derived input function derived from quantification of the activity in the left ventricle  were determined for each Wistar rat , written in MATLAB, was used to determine the PSR through the calculation of the three rate constants Fig. , Kcplx alx and λ . The comlx and λ 1CleucineCf is the concentration of free leucine in the precursor pool in the tissue and Cp is the concentrations of leucine in plasma (cold = unlabelled leucine). Unlabelled leucine is at steady state in tissue and:Cf for labelled leucine is equal toλ is defined asλ will be 4\documenKcplx is calculated as follows [The unidirectional uptake rate of plasma leucine into tissue  follows :5\docume11C]leucine, it is assumed that none is recycled from radioactive proteins during the experimental time of 60 min; krec = 0. Therefore, the PSR can be estimated by the following equation:PSR is in μM·min−1, Kcplx in min−1 and leucine[C] is the concentration of unlabelled leucine in arterial plasma in nmole·ml−1.For [Kcplx and PSR were analysed using a two-way ANOVA (treatment and ROIs as main factors and interaction treatment × ROIs). Two-way ANOVA  as main factors and interaction age × origin) and Šídák post hoc tests were used to compare the differences in concentration of unlabelled leucine between arterial and venous plasma samples in WT rats at 12 and 18 months. PSR and SUV in the longitudinal study were analysed using mixed effect analysis (age and genotype as main factors and interaction age × genotype) and Šídák post hoc tests.GraphPad Prism 9.4 was used to analyse the data. All data are expressed as mean ± SD. Effects of anisomycin on 11C]leucine PET is sensitive enough to detect inhibition of the PSR, we used the potent protein synthesis inhibitor Leucine uptake in WT and TG rats was first measured using standard uptake values (SUV)  and genotype × age interaction   and TG  vs 6 months in the hippocampus and in the temporal cortex of TG rats at 12  and 18 months  vs 6 months. The post hoc tests showed a significant difference between WT and TG only in temporal cortex at 12 months (p = 0.024)  Fig. . The SUVly) Fig. . More sp24) Fig. .Fig. 5Re11C]leucine has complex uptake and incorporation processes, and SUV quantification is not sufficient to accurately represent the PSR; therefore, we also performed kinetic modelling using a compartmental model as previously described by Sundaram et al. leucine PET to measure PSR and potential changes in PSR in rats. To study protein synthesis in vivo, different amino acids have been used [11C]Leucine fulfils these characteristics.The aim of this project was first to implement the use of [een used –28. For een used . Additioeen used . [11C]Le11C]leucine PET to measure changes in PSR in vivo, we administered the PSR inhibitor anisomycin at (60 mg/kg) 10 min prior to the PET acquisition. This dose induced a PSR inhibition of ~ 86% vs baseline in all brain regions analysed  a rat strain more readily available in the UK and less expensive such as Wistars and (ii) easier obtainable venous samples instead of arterial samples. Comparison of blood samples revealed significant differences in leucine and LNAA concentrations between Wistar and F344 strains and between arterial and venous samples. Differences in unlabelled leucine concentrations between species have been reported before, but to our knowledge, this is the first report showing differences between strains of a same species leucine PET with fairly large ROIs and potential issues of partial volume effect vs ex vivo microdissection of small brain region and [3H]leucine quantification and modelling).In the brain as a whole region of interest, there are known PSR values in humans  and monkh et al.  . The di11C]leucine PET as a means of measuring PSR in vivo in rats. This first study in a model of neurodegenerative disease did not find any difference in brain PSR between WT and TG rats at any age in this model of AD. However, considering the limitations of using a PBIF discussed above, this study recommends the use of individual blood samples to establish the IF and measure the concentrations of unlabelled LNAA, hence favouring a more laborious and expensive cross-sectional study design over a longitudinal setting, to refine the measure of the PSR in future studies using AD or other models of neurodegenerative diseases.Data obtained in this study confirm and support the use of [Supplementary file1 (DOCX 35 KB)Supplementary file2 (PDF 474 KB)Below is the link to the electronic supplementary material.


Escherichia coli (a Gram-negative bacterium) and Staphylococcus aureus (a Gram-positive bacterium) emphasize that the nanostructured surfaces based on nanowire arrays present excellent antibacterial activity against both type of bacteria. This study proves that functional surfaces obtained by relatively simple and highly reproducible preparation techniques that can be easily scaled to large area are very attractive in the field of water repellent coatings with enhanced antibacterial function.Nanostructured surfaces based on silver nanoparticles decorated ZnO–CuO core–shell nanowire arrays, which can assure protection against various environmental factors such as water and bacteria were developed by combining dry preparation techniques namely thermal oxidation in air, radio frequency (RF) magnetron sputtering and thermal vacuum evaporation. Thus, high-aspect-ratio ZnO nanowire arrays were grown directly on zinc foils by thermal oxidation in air. Further ZnO nanowires were coated with a CuO layer by RF magnetron sputtering, the obtained ZnO–CuO core–shell nanowires being decorated with Ag nanoparticles by thermal vacuum evaporation. The prepared samples were comprehensively assessed from morphological, compositional, structural, optical, surface chemistry, wetting and antibacterial activity point of view. The wettability studies show that native Zn foil and ZnO nanowire arrays grown on it are featured by a high water droplet adhesion while ZnO–CuO core–shell nanowire arrays (before and after decoration with Ag nanoparticles) reveal a low water droplet adhesion. The antibacterial tests carried on  Hence, metal oxide nanowires based on ZnO or/and CuO can be engineered by combining different preparation methods21 such as thermal oxidation in air, radio frequency (RF) magnetron sputtering, plasma assisted thermal vapor deposition, ultrasonic spray-assisted chemical vapor deposition, chemical spray pyrolysis, chemical synthesis, hydrothermal, etc. to yield attractive functional nanomaterials, which can be applied in field effect transistors10, diodes10, photodetectors14, solar cell15, photocatalysis16, surfaces with special wetting properties21, etc. Furthermore, multicomponent nanowires with complex architectures developed by decoration of metal oxide nanowires with metal nanoparticles is an effective route for expanding and enhancing the nanowires functionalities26.Metal oxides are highly competitive materials owed to their interesting features like abundance, low environmental impact and rich family of morphologies with controlled sizes that can be synthesized by various wet and dry aproaches17 and copper foils16, respectively. Thermal oxidation in air is a straightforward and environmental friendly preparation path, which can be adapted for large-scale production of metal oxide nanowires. This technique can be also regarded as a viable solution to micro/nanostructuring a metallic surface, specific properties and new functionalities, different from those presented by the pristine metal bulk material, being achieved by the fabricated micro/nanostructured surfaces. In the recent years, many researches have been focused on the development of such micro/nanostructured surfaces for bactericidal applications29. It is known that ZnO and CuO present antibacterial activity separately as single components30 or combined as nanocomposites33, several studies emphasizing the potential application of ZnO and CuO nanowires in the antibacterial area37. ZnO is a n-type compound with a wide band gap (~ 3.37 eV)5 while CuO is a p-type compound with a narrow band gap (~ 1.2 eV)6, the p–n junction obtained by joining these two semiconductors improving the separation of the charge carrier pairs. Thus, in a previous study we reported on the fabrication of staggered gap radial heterojunctions based on ZnO–CuxO core–shell nanowires by covering thermally oxidized ZnO nanowires with an optimum thickness of CuxO layer by RF magnetron sputtering, these nanomaterials finding applications as diodes14 or as water stable photocatalysts16. Although, the addition of Ag to composites based on ZnO and CuO can improve the separation of the electron–hole pairs, only few papers were reported on the nanocomposites such as Ag-ZnO38, Ag-CuO38, Ag and CuO impregnated on Fe doped ZnO39, Cu2O and Ag co-modified ZnO40 with an enhanced antibacterial activity, these nanomaterials being synthesized by wet chemical methods. As regards the preparation of one-dimensional multicomponent nanostructures  based on ZnO, CuO and Ag, despite the fact that the wet chemistry consume and discharge different chemicals with potentially adverse effects on the environment, many studies47 were carried on their synthesis by wet chemical paths  and on their applications in superhydrophobic coatings, photocatalysis, dye photodegradation, trace pesticide detections, etc. In comparison to the wet chemistry, the development of metal oxide nanowires by dry techniques such as thermal oxidation in air, radio frequency (RF) magnetron sputtering and thermal vacuum evaporation can be considered “clean” environmental friendly routes being solution-free preparation pathways, which do not involve hazardous raw materials and liquid solvent and do not produce harmful by-products.Metal oxide nanowire arrays based on ZnO and CuO can be easily grown by thermal oxidation in air directly on zinc foils29. For example, the water droplets can easily roll-off on superhydrophobic surfaces with water angle greater than 150° carrying away the bacteria, the nanoprotrusions can mechanically damage the bacteria cells while the use of materials well known for their pronounced antibacterial activity  can lead to a chemical contact killing of bacteria. A priori, due to the synergy effect between the individual bacteria-killing phenomena, a hydrophobic nanostructured metal surface containing sharp nanostructures like metal oxide nanowires decorated with metal nanoparticles can be an ideal candidate for inexpensive coatings with enhanced antibacterial function. Additionally, the formation of different type of junctions between the components can also favors the separation of charge carrier pairs, augmenting the generation of highly reactive oxygen (ROS), generally these species playing the central role in the mechanisms responsible for the bacteria killing.Usually, the performance of the antibacterial surfaces depends on parameters such as surface morphology, surface chemical composition and surface wettability, these being mainly responsible for the most common bacteria-killing phenomena: contact-killing surfaces, nanoprotrusions and superhydrophobic surfaces21, the thermal oxidation of zinc foils offers the main advantage of growing ZnO nanowires directly on them, these metal oxide functionalized metallic substrates being attractive for applications where properties such as anticorrosion, self-cleaning, hydrophobicity or antibacterial are demanded. Hence, ZnO nanowire arrays were grown directly on zinc foils by thermal oxidation in air, further these were coated with a CuO layer by RF magnetron sputtering, the obtained ZnO–CuO core–shell nanowires being decorated with Ag nanoparticles by thermal vacuum evaporation. The morphological, compositional, structural, optical, surface chemistry, wetting and antibacterial activity properties of the prepared samples were evaluated. The nanostructured surfaces based on nanowire arrays present exceptional antibacterial activity against both Escherichia coli (a Gram-negative bacterium) and Staphylococcus aureus (a Gram-positive bacterium). Most probably, the synergetic effect between the sharp morphology of the nanowires and the low water droplet adhesion behavior of the nanostructured surfaces is responsible for the excellent antibacterial effect.In this context, the present study is focused on the development of nanostructured surfaces based on silver nanoparticles decorated ZnO–CuO core–shell nanowire arrays by combining thermal oxidation in air, RF magnetron sputtering and thermal vacuum evaporation. To our knowledge, no attempt was made for developing multicomponent nanowires based on ZnO, CuO and Ag by combining these three dry techniques that are frequently used in the preparation of inorganic nanostructures. Furthermore, this preparation approach presents two key advantages: (i) can be applicable to large-area for obtaining multicomponent nanowire arrays with high density and good uniformity and (ii) can be regarded as a viable strategy for tuning the architecture and composition of the multicomponent nanowires in order to achieve desired properties and improved performances. Moreover, in comparison to other preparation methods of ZnO nanowires that use chemical reagents and require additional steps for the deposition of the precursor layers 2, subsequently these were cleaned in acetone and isopropyl alcohol for 5 min in an ultrasonic bath, washed in deionized water and dried under nitrogen gas flow. ZnO nanowires were prepared by thermal oxidation in air of Zn foil samples at 500 °C for 12 h. Then, the ZnO nanowires were coated with a CuO layer by RF magnetron sputtering using a copper oxide sputtering target . Before each deposition, the metal oxide target was pre-sputtered for 30 min in order to remove any possible contamination from its surface. During the deposition, the power applied on the magnetron was 100 W and the pressure in the chamber was 5.4 × 10−3 mbar in an Ar atmosphere with a purity of 9.6 (99.9999%) from Linde. Further, the ZnO–CuO core–shell nanowires were decorated with Ag nanoparticles by thermal vacuum evaporation using a silver wire , the pressure in the chamber being 3 × 10−6 mbar. The photographs of the investigated samples are presented in Fig. Zinc foil  was cut into pieces of approximately 2 × 1.5 cmexc = 350 nm), respectively. The surface chemistry was analyzed by X-Ray photoelectron spectroscopy (XPS) with an AXIS Ultra DLD  setup using Al Kα1 (1486.74 eV) radiation produced by a monochromatized X-Ray source at operating power of 225 W (15 kV × 15 mA). In the analysis chamber, the base pressure was at least 1.0 × 10–8 mbar. A flood gun operating at 1.5 A filament current, 2.7 V charge balance and 1.0 V filament bias was utilized for achieving the charge compensation. Hybrid lens mode, 40 eV pass energy and a slot aperture were applied for recording high-resolution core level spectra, Voigt profiles (singlets or doublets) being used in their deconvolution according to previously described methods48.The prepared samples were characterized by complementary techniques. The surface morphology and its elemental composition were studied using a Zeiss Merlin Compact field emission scanning electron microscope (FESEM) and a Zeiss EVO 50XVP scanning electron microscope equipped with an energy dispersive X-ray analysis (EDX) QUANTAX Bruker 200 accessory for composition. Furthermore, the morphology, atomic structure and local chemical composition structure at nanoscale level were analyzed by transmission electron microscopy (TEM), EDX including elemental mapping in scanning transmission electron microscopy (STEM) and selected area electron diffraction (SAED) using a Cs probe-corrected JEM ARM 200F analytical electron microscope. The crystalline phase was investigated using a Bruker AXS D8 Advance X-ray diffractometer (XRD) with Cu Kα radiation (λ = 0.154 nm), the source being operated at 40 kV and 40 mA. The reflectance and photoluminescence (PL) were evaluated using a Perkin-Elmer Lambda 45 UV–VIS spectrophotometer equipped with an integrating sphere for reflectance and a FL 920 Edinburgh Instruments spectrometer with a 450 W Xe lamp excitation and double monochromators on both excitation and emission for photoluminescence (λp) and dispersive (γd) components for the solid surface was evaluated using the apparent CA values measured between a test liquid and the sample surface. Water and diiodomethane were used as test liquids for assuring an accurate evaluation of the polar and dispersive components of the surface free energy of the investigated samples. Thus, a droplet from the test liquid was placed on the sample surface via a blunt-end, stainless steel needle (outer diameter of 0.5 mm) attached to a syringe pump controlled by the DSA3® software supplied with the instrument. The volume of the droplet was 1 μL for diiodmethane (DIM) while that for water (W) was increased from 1 to 5 μL . CA was measured by fitting a polynomial equation of second degree or a circle equation to the shape of the sessile drop and then calculating the slope of the tangent to the drop at the liquid–solid vapor interface line. The detailed processes of the behavior of the water droplet adhesion at the sample surface was recorded by a video camera tilted at 2–3° with respect to the plane of the sample surface supporting the droplet. Hence, the surface free energy was estimated based on the Owens–Wendt relationship γSL = γL + γS–2(γLdγSd)1/2–2(γLpγSp)1/2, where γSL is the interfacial energy between solid and liquid, γL and γS are the surface energy of liquid and solid, respectively, γLp and γSp are the polar component of surface energy of liquid and solid, respectively and γLd and γSd are the dispersive component of surface energy of liquid and solid, respectively49. The γLp, γLd γL values50 for the test liquids are: γwaterp = 51 mN/m, γwaterd = 21.8 mN/m and γwater = 72.8 mN/m and γdiiodomethanep = 2.3 mN/m, γdiiodomethaned = 48.5 mN/m and γdiiodomethane = 50.8 mN/m. The adhesion work (Wad) between the liquid droplet and the sample surface was estimated based on Young–Dupre relationship Wad = γL(1 + cosθ), where θ is the CA of liquid on investigated solid surface52. The measurements were performed in duplicate at room temperature, the CA mean value being used for evaluating the surface free energy and the adhesion work. Roll-off angles were measured with a goniometer in order to control the tilt angle, the orthoscopic images being taken with a commercial photocamera.The wetting properties were assessed by measuring the static contact angle (CA) with a Drop Shape Analysis System, model DSA100 from Kruss GmbH. The surface free energy (SFE) with its polar (γEscherichia coli (E. coli ATCC 25922) strain (Gram-negative bacteria) and non-pathogenic Staphylococcus aureus (S. aureus ATCC 25923) strain (Gram-positive bacteria), both in planktonic state. Thus, 2 ml tryptic soy broth (TSB) and 20 μl microbial suspension with 0.5 McFarland density (1.5 × 108 CFU/mL) were added on the sterilized investigated samples placed in 6-well plates. After the incubation (carried at 37 °C for 24 h), decimal dilutions obtained from the suspension recovered in sterile saline were seeded in triplicate (3 replicates of 10 µl each) on agar medium for quantifying the colony forming units (CFU)/ml. It has to be noted that each replicate was performed in triplicate, the experiments being repeated 2 times in triplicates. The antibacterial efficiency of the investigated samples was calculated in terms of percentage of bacterial cell reduction  determined as follows: R % = [(CFUc-CFUp)/CFUc] × 100; where CFUc and CFUp represent the numbers of CFU/ml for the control and each investigated sample.The antibacterial activity was evaluated against non-pathogenic The surface morphology of the metallic substrate after each functionalization step was analyzed by FESEM, the images being presented at different magnifications Figs.  and 2. AAt higher magnification, the FESEM images Fig.  indicate53. As can be seen in Fig. The cross-sectional FESEM images Fig.  emphasizThe elemental distribution and the composition of the ZnO nanowire arrays and Ag nanoparticles decorated ZnO–CuO core–shell nanowire arrays samples were assessed by EDX spectroscopy, the EDX mapping images and the corresponding spectra being given in Fig. The EDX mapping images illustrate an uniform distribution of the chemical elements on the investigated surfaces. The EDX spectrum of ZnO nanowires reveals the presence of Zn and O elements while the EDX spectrum of Ag nanoparticles decorated ZnO–CuO core–shell nanowire arrays discloses the simultaneously presence of the signals associated to Zn, Cu, Ag and O elements. Based on the EDX analysis, the atomic percentage of the elements contained in the analyzed samples was estimated at ~ 44–46% for Zn, ~ 2.5% for Cu and ~ 0.4% for Ag.6. Additionally to these decrease peaks, the reflectance spectrum of Ag nanoparticles decorated ZnO–CuO core–shell nanowire sample discloses a shoulder of ~ 550 nm, which can be associated to the Ag plasmon band55. The presence, position and intensity of this band is strongly influenced by parameters of the Ag nanoparticles 56. The photoluminescence spectrum of ZnO nanowires exhibits two emission bands: one intense and sharp in the UV domain centered at ~ 380 nm of excitonic origin57 and another one weak, broad covering almost all the visible range centered at ~ 530 nm originating from defect emission58. Although, the origin of this visible emission is still under debate, the generally accepted hypothesis considers the oxygen vacancies responsible for the appearance of this “green emission”60. It has to be noted that the presence of the oxygen vacancies can modify the wetting properties of the ZnO nanowires21. Moreover, a study focused on the origin of the “green emission” in ZnO nanostructures (including nanowires) prepared by a vapor phase transport method  emphasized that the oxygen gas concentration used during the ZnO growth influences the depth of the oxygen vacancies related defects60.The structural and optical properties of the investigated samples were evaluated from data shown in Fig. 61 and to a separation effect of the charge carriers due to the formation of type-II band alignment between the two metal oxides63.The photoluminescence spectra of ZnO–CuO core–shell nanowires with or without Ag nanoparticles show a decrease in the intensities of these two emission bands. The result can be explained considering that the CuO layer deposited on the surface of ZnO nanowires leads to a passivation effect reducing the ZnO surface oxygen related defects3/2 and Zn 2p1/2 levels of Zn2+ state in ZnO65, (ii) at ~ 933 eV and ~ 954 eV to Cu 2p3/2 and Cu 2p1/2 levels of Cu2+ state in CuO65, (iii) at ~ 368 eV and ~ 374 eV to Ag 3d5/2 and Ag 3d3/2 levels of Ag (0) state67, (iv) at ~ 529 eV to O2− state in metal oxides65 and (v) at ~ 532 eV to C=O due to a contamination of the surfaces with carbonate from the environment. Further, the deconvolution obtained by Voigt profiles of the core level spectra of Zn 2p, Cu 2p, Ag 3d and O 1S  Cu 2p3/2 core level—a broad peak with two components, one centered at 933.9 eV owed to Cu2+ state in CuO65 and other at 934.8 eV linked most probably to carbonate resulted from the slight contamination of the surface in the environment conditions and the satellite peaks at ~ 940–945 eV and ~ 964 eV characteristic only to the bivalent oxidation state of Cu, their presence proving the formation of CuO65; (iii) Ag 3d5/2 core level—a broad peak with two components, one centered at 368.1 eV associated to the free metallic silver atoms (Ag (0))67 and other centered at 368.7 eV related most probably to the positively charged silver atoms (Ag (0) + δ) resulted from the slight oxidation in air of the metallic atoms in the ambient conditions69 or from the silver interaction with a relatively high electronegative atom like oxygen)72. Consequently, the XPS results prove the presence of ZnO, CuO and Ag in the Ag nanoparticles decorated ZnO–CuO core–shell nanowires samples.The Ag nanoparticles decorated ZnO–CuO core–shell nanowires sample was investigated by XPS in order to certify the chemical composition of the shell and nanoparticles and the oxidation states of each component, the XPS spectra being presented in Fig.  1S Fig.  were anaBoth ZnO nanowires and Ag nanoparticles decorated ZnO–CuO core–shell nanowires samples were investigated by TEM measurements in order to emphasize the core–shell morphology of the ZnO–CuO nanowires and their decoration with Ag nanoparticles, the data being shown in Fig. Hence, the TEM images of a single ZnO nanowire and a single Ag nanoparticles decorated ZnO–CuO core–shell nanowire evidence a cylindrical shape for both types of nanowires with diameter of ~ 30 nm and ~ 70 nm, respectively, in agreement with the FESEM images Fig. . The SAEUsually, the wettability of a solid surface is decided by the geometrical structure of the surface as well as its chemical composition. Hence, in the present case, the micro/nano-structuration of the metallic surface and the material-dependent surface free energy have a combined and significant effect on the surface wettability, this being a key parameter related to their potential applications in surfaces with antibacterial activity. Thus, the samples were investigated by CA and SFE measurements using water and diiodomethane as test liquids in order to evaluate their wetting properties, the obtained values being given in Fig. 20 and even if the oxygen vacancy defects can modify their wetting properties21, it can be assume that the nanowire shape of the structures presented on the surface of the zinc foils plays the major role in their hydrophobicity taking into account that all samples containing nanowires have WCA of ~ 134°. Regarding the SFE components, the dispersive component is higher than the polar one for all samples. Thus, the lower polar component values  leads to high WCA value for water droplets while the higher dispersive component (P2 and P3 samples) results in small DIMCA value for diiodomethane droplets.It has to be noted that in the case of ZnO nanowires, the hydrophobic property is in accordance with that reported in the literatureWad values were estimated being presented in Table Wad. Hence, the Wad can be a measure of the contact strength between two adjacent phases, being defined as the work required to separate these two phases (ex. liquid–solid phase boundary). Thus, the micro/nanostructured effect of the metallic surfaces consisting in the presence of the nanowire structures results in a lower Wad value (~ 22 mN/m) in comparison to the smooth native metallic surface (~ 55 mN/m). It can be seen that the water droplet is highly adherent to the surface of the foils containing ZnO nanowires (similar to the water droplet behavior on the surface of Zn foil), resting stick, firmly pinned on the surface even when these were turned upside down. Instead, in the case of the foils having the surfaces covered with ZnO–CuO core–shell nanowires or Ag nanoparticles decorated ZnO–CuO core–shell nanowires, the water droplets rolled off very easily at slight tilt, the roll-off angle being evaluated at 55° for P2 or 25° for P3. The result can be explained taking into account the models concerning the three main states of a liquid droplet penetration in the textured (micro/nanostructured) surface: Wenzel, Cassie-Baxter and an intermediate state between them73.Further, the adhesion of the water droplets to the surface of the investigated samples were assessed, the images being given in Fig. Hence, the wetting regime for P1 sample can involve a stronger adhesion of the water droplet to the textured surface—the droplet is pinned to the surface as the air pockets are penetrated by the water while the wetting regime for P2 and P3 samples can presume a lower adhesion of the water droplet to the textured surface—the droplet sits on the top of the surface due to the air trapped underneath, in the gap between the nanostructures, preventing the penetration of the water. Such surfaces with hydrophobic behavior and low water droplet adhesion can be effective for antibacterial uses.E. coli and S. aureus, the data being shown in Fig. S. aureus and Ag nanoparticles decorated ZnO–CuO core–shell nanowire arrays. Hence, it can be assume that the nanowire shape plays the significant role in achieving the excellent antibacterial response, the needle tip of the nanowire structures mechanically damaging the bacteria cells like a thorn that penetrates the membrane as can be seen in the FESEM image from Fig. xO core–shell nanowires16, we evidenced that a ZnO dissolution can occurs in the aqueous solutions and the presence of a CuO layer with an adequate thickness on the surface of the ZnO nanowires protects them against the dissolution in the aqueous media. Accordingly, the zinc ions (P1 sample) or the silver ions (P3 sample) generated in the aqueous media can adhere to the cell wall, increasing its permeability and further leading to the cell membrane rupture74 while the formation of ZnO–CuO p-n junction16 (P2 sample) and Ag-CuO Schottky barrier75 (P3 sample) can favor the separation of charge carrier pair, which further can improve the generation of highly ROS involved in the mechanism of bacteria killing. Consequently, the antibacterial activity of the samples due to the mechanically damaged bacteria membrane can be assist by a chemical contact killing bacteria mechanism.In the following, the antibacterial activity of the nanostructured surfaces based on nanowire arrays was investigated against The outcome of the experiments proves that such functional nanostructured surfaces based on silver nanoparticles decorated ZnO–CuO core–shell nanowire arrays with low water droplet adhesion and excellent bactericide effect can open new insights in the field of water repellent and antibacterial coatings.E. coli and S. aureus emphasize that all nanostructured surfaces based on nanowire arrays present excellent antibacterial activity against both type of bacteria, the hydrophobic and low water adhesion behavior of the nanowire samples enhancing the antibacterial effect by limiting the contact area between the microbial suspension and the nanostructured surface. The nanowire shape plays the major role in achieving the excellent antibacterial response, the nanowire structures mechanically damaging the bacteria cells, process that can be assists by other mechanisms involving release of metal ions or formation of different type of junctions. This study emphasized that such functional surfaces can be viable candidates for water repellent surfaces with enhanced antibacterial activity.High-aspect-ratio silver nanoparticles decorated ZnO–CuO core–shell nanowire arrays were fabricated by combining low-cost and reproducible dry preparation techniques that can be relative easily scaled to large area. Hence, on zinc foils, ZnO nanowire arrays were grown by thermal oxidation in air, subsequently coated with a CuO layer by RF magnetron sputtering, the obtained ZnO–CuO core–shell nanowires being further decorated with Ag nanoparticles by thermal vacuum evaporation. The wettability measurements show that all investigated samples reveal hydrophobic behavior. Moreover, the native Zn foil and the ZnO nanowires arrays are featured by a high water droplet adhesion while ZnO–CuO core–shell nanowires arrays with or without Ag nanoparticles by a low water droplet adhesion. The antibacterial tests carried on Supplementary Information.


Instead, key predictors included total microbial biomass, 16S gene abundance, bacterial ASV membership, and abundances of specific prokaryotic taxa and functional groups . These results suggest that bacterial α diversity may be a useful indicator of soil ecosystem function and stability, but that other characteristics of bacterial communities are stronger statistical predictors of ecosystem function and better reflect the biological mechanisms by which microbial communities influence ecosystems. Overall, our results provide insight into the role of microorganisms in supporting ecosystem function and stability by identifying specific characteristics of bacterial communities that are critical for understanding and predicting ecosystem responses to global change.Ecosystem functions and services are under threat from anthropogenic global change at a planetary scale. Microorganisms are the dominant drivers of nearly all ecosystem functions and therefore ecosystem-scale responses are dependent on responses of resident microbial communities. However, the specific characteristics of microbial communities that contribute to ecosystem stability under anthropogenic stress are unknown. We evaluated bacterial drivers of ecosystem stability by generating wide experimental gradients of bacterial diversity in soils, applying stress to the soils, and measuring responses of several microbial-mediated ecosystem processes, including C and N cycling rates and soil enzyme activities. Some processes  exhibited positive correlations with bacterial diversity and losses of diversity resulted in reduced stability of nearly all processes. However, comprehensive evaluation of all potential bacterial drivers of the processes revealed that bacterial α diversity  Soils are the foundation of terrestrial ecosystems and support many ecosystem functions and services, including plant productivity, carbon storage, and nutrient cycling . These fEcosystem stability can be defined as the ability of an ecosystem to defy change or return to equilibrium following a disturbance . The staper se in accounting for variation in ecosystem functions [Indeed, rigorous evaluations of relationships between microbial communities and the ecosystem-scale processes they facilitate have only started to emerge in the past decade – these studies have shown soil microbial α diversity to be positively associated with several ecosystem functions, including C mineralization , C use eunctions , 19, 20.unctions –23, and Though it is difficult to generate specific predictions regarding microbial contributions to ecosystem stability, we note that microbial diversity and community membership are highly variable among ecosystem types , 25, andOur study includes soils from contrasting ecosystems (undisturbed prairie vs. cultivated field) that host dramatically different soil bacterial communities . The prairie site is at an elevation of 1128 m and receives 685.8 mm of precipitation per year. The second site was a cultivated field at the NRCS Plant Materials Science Center in Pullman, WA , which is at an elevation of 767 m and receives 516.9 mm of precipitation per year. This site has a legacy of N fertilization and is intensively managed via annual tillage and a winter wheat-fallow crop rotation. The cultivated soils have lower pH, higher NO3-N, lower NH4-N, and lower C:N compared with the undisturbed prairie soils , and 1 × 10−6 diluted suspension (D2). We adjusted soils to 65% of water holding capacity and incubated soils at 20 °C for six weeks to allow for microbial establishment. At the end of the six weeks, we verified that microbial activity had stabilized in all microcosms by measuring respiration using a LI-COR 8100 A . The respiration data showed that the dilution treatments within each land use had indistinguishable microbial activity  of the irradiated soils. Sterilized soils (30 g dry weight) were added to autoclaved mason jars and sterile microcosms were inoculated with suspensions originating from the same nonsterile soil. Inoculum suspensions were extracted by shaking 6 g of nonsterile soil in 50 ml sterile 1X PBS solution for 1 h, with the two soils extracted in quadruplicate and then the replicates pooled. To create a gradient of diversity, three levels of dilution were then used as inocula: undiluted suspension (D0), 1 × 10−1, which is within the range detected in soils [After community establishment, we induced stress in communities by adding oxytetracycline, an agricultural antibiotic used in livestock production and commonly found in soils , 30. We in soils . TherefoAt the conclusion of the experiment, we measured several bacterial community characteristics in soils. We measured total microbial biomass in all soils using a chloroform extraction technique  and as aWe characterized bacterial communities by amplicon sequencing of the V4 region of the 16S rRNA gene using the 515 F/806 R primer pair . Amplico2) [4-N + NO3-N) accumulation in the soils [3-N accumulation [D0 | /(C0 + | D0 | )). D0 represents the value of stressed soils while C0 is the value of controls. This index is bounded between −1 and 1 with a value of ‘1’ indicating no change in an ecosystem process following stress. To assess overall biogeochemical ecosystem function, i.e., multifunctionality, we conducted multivariate analyses considering all the microbial-mediated functions (see below).At the end of the experiment, we measured basal respiration rates  of all soils using a 24-hour static incubation method  and we s2) . To meashe soils –50 and tmulation . We alsomulation . Becausemulation . To quanmulation  where reBray dissimilarity matrix for all downstream analyses. We visualized community composition using principal coordinates analysis  and determined effects of land use, dilution, and stress using PERMANOVA (‘adonis2’ function). We visualized multifunctionality by performing principal components analysis on the scaled rates of the individual processes (‘princomp’ function) and determined effects of land use, diversity treatment, and stress on multifunctionality using PERMANOVA with Euclidean distances.All statistical analyses were performed in R . For allWe quantified the importance of microbial variables in accounting for variation in ecosystem process rates using random forest regression (randomForest R package) . The ranFor external validation, we applied the same analytical approach to data from a recent study that also examined ecosystem responses to anthropogenic stressors across microbial diversity gradients and included similar ecosystem functions  and microbial variables  to our experiment but also included fungal abundance, α diversity, and membership metrics .−3 diluted) soils had 19% lower 16S Shannon diversity than D0 soils while D2 (1 × 10−6 diluted) soils had 49% lower Shannon diversity than D1 soils and 58% lower diversity than D0 soils   Fig. . For exaD2) Fig. . AntibioD2) Fig. , while iD2) Fig. . Further2) and 25% higher enzyme activity in stressed than control treatments  was associated with lower Proteobacteria:Actinobacteria ratios , which we quantified independently of α diversity. This is striking because, at face value, our experimental results do suggest strong influences of α diversity; stress responses of ecosystem processes were only seen in low diversity treatments and we also observed significant bivariate relationships between bacterial α diversity and some processes, e.g., between Shannon diversity and C mineralization rates. Multiple prior studies have inferred mechanistic importance of microbial α diversity to ecosystem functioning on the basis of similar observations –18, 21. Some prior studies focused on plant communities have similarly found ecosystem functioning to be incorrectly attributed to plant α diversity when other characteristics of plant communities  were omitted from analyses , 65. Ind2) and genus-level taxonomy improved some models (SIR and qCO2) and suggested important C-cycle functions for Pseudomonas and Bacillus. Our results also demonstrated that information about specific functional groups will be important for predicting ecosystem functions that are performed by phylogenetically narrow groups [Though α diversity was not a strong predictor of ecosystem processes, we did observe that specific bacterial taxa only exhibited stress responses in low diversity treatments, which suggests that diversity begets compositional stability of communities under stress. This result suggests indirect influences of bacterial α diversity on ecosystem function and stability. For example, it is likely that α diversity indirectly supported soil function and stability in our experiment via increased functional redundancy in the more diverse bacterial communities , 67, 68.2) were more impacted by stress in prairie soils while N-cycle metrics  were more impacted by stress in cultivated soils. This further demonstrates that ecosystem responses to anthropogenic stress are strongly dependent upon microbial community membership – these two ecosystems hosted different initial bacterial communities and thus exhibited distinct responses to stress. The stress responses of the prairie soils were also larger in magnitude, particularly when examining overall ecosystem functioning, i.e., multifunctionality. This contrasts with our original hypothesis that the more diverse communities present in the prairie soils would confer greater ecosystem stability and suggests that historical exposure to stress  may select for a more stress-tolerant community that is more resistant to future stressors. An alternative explanation is that the historical stress applied to the cultivated soils has resulted in low biomass communities  following antibiotic application, which has also been observed in prior studies [2) we observed in these soils [Many of the stress responses we observed were expected given that the stressor we applied, oxytetracycline, is a bacteriostatic antibiotic that inhibits bacterial activity by preventing protein production. For example, stress reduced SIR and N mineralization rates, two processes that are dependent upon overall microbial activity. We also observed increased qCO studies , 70. Thise soils . InteresIt is also important to note that our study did not consider influences of eukaryotic organisms  as our experimental approach proved unsuitable for manipulation of those communities. Despite this limitation, the lack of fungal establishment in our microcosms has the benefit of allowing us to more completely isolate the influences of bacteria (the direct targets of most antibiotics) on ecosystem function and stability. However, multiple prior studies have provided evidence of eukaryotic influences on ecosystem functions, including significant statistical associations between eukaryotic α diversity and soil processes , 16. TheRegardless, the central conclusions of our study are clear – that microbial abundance and bacterial community membership, and not α diversity, emerge as the strongest predictors of soil ecosystem processes. This conclusion is supported by re-analysis of a prior microbial diversity – ecosystem stress experiment , which ySupplementary Methods


Bilateral internal carotid artery (ICA) dissection on heavy weight lifting is a very rare cause of stroke in young patients. Arterial dissection is due to a tear in the intima and internal elastic lamina which leads to extravasation of blood into the media and subintimal plane. Clinical diagnosis of carotid artery dissection is difficult with common clinical presentations like headache and neck pain. Here we present a case of a 40-year-old young man who presented to us  with headache and quadriparesis. MRI brain showed multiple acute infarcts in bilateral centrum semiovale extending up to frontal periventricular white matter with few tiny foci in bilateral medial temporal and left gangliocapsular regions and CT cerebral angiography showed bilateral ICA dissection. He was treated with low molecular weight heparin and was discharged for follow-up with regular physiotherapy. Bilateral internal carotid artery (ICA) dissection is itself a rare condition with an incidence of up to five cases per 100,000 with a slight male predilection of 55% . The patA 40-year-old gentleman with a history of lifting heavy stacks of sugarcane (approximately 100 stacks) 2 days back while working at his farm, presented with sudden onset holocranial headache with giddiness and transient blurring of vision for 2 days. He was found to have high blood pressure (180/100 mmHg). He was managed conservatively with antihypertensives (details were not available). The next day, he developed left hemiparesis with a further fall in his sensorium. At presentation to this hospital, he was conscious with quadriparesis, power was MRC  grade 4/5 on the right side and 2/5 on the left side at all joints, with plantar bilaterally extensor.MRI brain showed multiple acute ischemic infarcts in bilateral centrum semiovale extending up to frontal periventricular white matter with few tiny foci in bilateral medial temporal and left gangliocapsular regions Figures -1D.CT cerebral angiography showed ill-defined circumferential hypodensity eccentrically surrounding bilateral cervical ICA in the mid and distal segment resulting in significant stenosis and irregularity reaching up to the skull base Figures -2D.His other routine stroke workup including electrocardiogram, 2D echocardiography, transesophageal echocardiography, serum homocysteine levels, connective tissue /Antinuclear Factor (ANF), Indirect Fluorescent Antibody (IFA), Erythrocyte Sedimentation Rate (ESR), C-Reactive Protein (CRP), phospholipid antibodies panel IgG, IgM,) was normal. He was treated with low molecular weight heparin. On day 5, he showed clinical improvement and was discharged for follow-up with regular physiotherapy.Bilateral internal carotid artery dissection in young patients is extremely rare ,6. DisseMRI has replaced conventional angiography for diagnosing ICA dissection due to the improved resolution of recent MRI machines and the non-invasive nature of the scan. MRI also helps in visualizing the intra-mural hematoma. Further, it can also be diagnosed incidentally on cerebral or neck angiogram. However, the pathognomic features of imaging are seen in only 10% of patients. More frequently detected findings on imaging are irregularity of the vessel wall as seen in this patient and signs of stenosis and occlusion about 2 cm distal to the carotid bulb .The appropriate MRI protocol includes a magnetic resonance angiogram (MRA) of the neck to evaluate the lumen and axial fat-suppressed T1-weighted images to evaluate for subacute intramural haematoma. However, when the MRI protocol is not tailored accordingly, subpetrous internal carotid artery dissection with intramural haematoma can still be detected on routine brain MRI sequences . Double Our patient did not show any clinical features suggesting connective tissue disorder. Traumatic events like road traffic accidents, and sports-related trauma to the head and neck can cause ICA dissection, or mechanical stress like heavy weight lifting, strong coughing or vomiting can be an important contributing factor in ICA dissection .A case of cough-induced extracranial internal carotid artery dissection was reported by Mei-Ling Sharon Tai et al. in 2020 . A patieThis is an extremely rare incidence where routine heavy weight lifting during farming has led to bilateral carotid artery dissection. This suggests that people lifting heavy weights are at risk of ICA dissection and should be advised to carry weights in smaller batches and should keep some rest period between lifting weights.In conclusion, this is a rare case of stroke with bilateral ICA dissection in a young farmer. We would like to emphasize that ICA dissection, though rare, is an important cause of stroke in the young, which is often missed. History of farm-related heavy weight lifting and neck pain shall be carefully elicited and must raise a suspicion of dissection. Heavy weight bearing is an important trigger factor for developing carotid arterial dissection.


PARP [Poly(ADP-ribose) polymerase] proteins are important signaling enzymes in eukaryotic DNA damage repair. We investigated how PARP1 and PARP2 bind at DNA double-strand breaks (DSBs) by developing a single-molecule magnetic tweezers assay to measure protein binding at DSBs while controlling DNA tension, supercoiling, and end-chemistry. We found that PARP2 forms an extremely stable mechanical bridge across blunt DNA ends and restores DNA torsional continuity. We also discovered that PARP2 switches between end-binding and bridging modes depending on whether overhangs have 5′ or 3′. PARP1, in contrast, binds at DSB ends but does not form a bridge when opposing ends are brought together. Our results reveal fundamental mechanisms of how PARP proteins engage at DSBs, which may aid in inhibitor development. Poly(ADP-ribose) polymerases (PARPs) play key roles in DNA damage repair pathways in eukaryotic cells. Human PARPs 1 and 2 are catalytically activated by damage in the form of both double-strand and single-strand DNA breaks. Recent structural work indicates that PARP2 can also bridge two DNA double-strand breaks (DSBs), revealing a potential role in stabilizing broken DNA ends. In this paper, we have developed a magnetic tweezers–based assay in order to measure the mechanical stability and interaction kinetics of proteins bridging across the two ends of a DNA DSB. We find that PARP2 forms a remarkably stable mechanical link (rupture force ~85 pN) across blunt-end 5′-phosphorylated DSBs and restores torsional continuity allowing DNA supercoiling. We characterize the rupture force for different overhang types and show that PARP2 switches between bridging and end-binding modes depending on whether the break is blunt-ended or has a short 5′ or 3′ overhang. In contrast, PARP1 was not observed to form a bridging interaction across blunt or short overhang DSBs and competed away PARP2 bridge formation, indicating that it binds stably but without linking together the two broken DNA ends. Our work gives insights into the fundamental mechanisms of PARP1 and PARP2 interactions at double-strand DNA breaks and presents a unique experimental approach to studying DNA DSB repair pathways. A typical mammalian cell undergoes tens of thousands of DNA damage events per day . These eThe human poly(ADP-ribose) polymerase (PARP) family of proteins includes PARPs 1-3 which are DNA damage response proteins that are known to bind to a variety of types of SSBs and DSBs , 7. Bind+) access to the active site through an allosteric unfolding of regulatory alpha-helices in the catalytic domain –based linker which constrains two DNA ends and used magnetic tweezers to controllably apply tension and supercoiling to the DNA molecule while measuring its extension. This method allows us to perform highly controlled experiments which directly measure the kinetics and mechanics of PARP proteins as they form a bridge across broken DNA ends. We find that PARP2 forms an extremely stable bridge across blunt DNA ends and restores DNA torsional continuity. This bridging is specific to 5′-phosphorylated ends and is not observed when dephosphorylated ends are present. We also characterize different types of overhangs and find differences in the interaction modes for 3′ and 5′ overhangs. In comparison, PARP1 did not form a bridging link for any of the DNA end chemistries tested and competed away PARP2 bridge formation. Our results elucidate the fundamental properties of PARP protein interactions at DSBs and set a force scale for PARP binding at DNA breaks.SI Appendix, Figs. S1–S3). The DNA molecule is 1.8 kbp in length and is flanked by sections labeled with multiple digoxigenins and biotins for attachment to a coverslip surface and superparamagnetic bead, respectively. Light scattering measurements of PEG (e-e) obeys a power law dependence on molecular weight (Mw) of Me-e = 0.047*(Mw)0.588, giving a value of Me-e = 16 nm for Mw = 20 kDa . We designed the two labeled thymine bases on our synthetic DNA oligonucleotide to have a separation of 46 bp = 16 nm (given 0.34 nm/bp) in order to match the PEG mean end-to-end length. Therefore, upon formation of a DSB in between the two PEG attachment points, the PEG will hold together the two DSB ends in close proximity under conditions of low or zero force. To enable the creation of such a break, we positioned a recognition site for the restriction enzyme NruI which cuts both strands at the same position, leaving a blunt end. 28To study the mechanics of binding at a double-strand DNA break, we developed a method for making a double-stranded DNA molecule featuring a 20 kDa PEG molecule bridging across a central section A. The cos of PEG  show thaSI Appendix, Fig. S5). The DNA cleavage by NruI could be observed on multiple beads across the field of view—the time to cleavage varied from bead to bead due to the stochastic arrival of the enzyme to its recognition site . To test the correct assembly of the DNA construct, we tracked DNA extension as a function of time before and after adding NruI C. A consSI Appendix, Figs. S7 and S8).Having established a method to create two opposing double-stranded DNA breaks tethered by a PEG linker, we applied our system to study the mechanical bridging of a DNA DSB by PARP2. Fδ where F is the force and δ is the distance between the bound state and activation energy peak , and BTk is thermal energy. This equation was fitted to the measured rupture force distribution using the Levenberg–Marquardt nonlinear least squares algorithm and yielded fit parameters of ont = 1.3 × 105 s  and δ = 0.51 nm . Finally, we add in a test protein, incubate for 1 min, and repeat the magnet rotation. Having characterized the force-induced rupture of PARP2 bridging across DNA, we next studied the influence of bridging on DNA supercoiling. Magnetic tweezers enables measurement of DNA supercoiling by rotating the magnets, which applies a torque due to preferential alignment of the superparamagnetic bead along its easy axis , 35. FigTo initially test the system, we performed positive and negative controls using T4 DNA ligase. A concentration of 10 U/μL was used  and incubated for 1 min in the flow-cell before testing the percentage of beads in the field of view, which show a restoration of supercoiling. Ligase without ATP showed no restoration of supercoiling as expected C. For liSI Appendix, Fig. S9). After cleavage, we used the overhangs formed by DrdI to ligate on different synthetic duplex structures in situ in the magnetic tweezers’ flow-cell. Specifically, we made structures with 5′ and 3′ overhangs of four base lengths and with the same 5′-CGCG-3′ sequence .In SI Appendix, Fig. S11). The rupture force histograms in SI Appendix, Fig. S12). This suggests that it is unlikely that two PARP2 proteins can bind to hybridized DNA overhangs—potentially explaining why the rupture forces are lower than those observed for blunt-ended DNA. However, the exact conformational details of the two states remain to be determined.In After characterizing the binding and bridging of PARP2 across different types of DNA DSBs, we investigated the behavior of PARP1. We repeated the tests described in SI Appendix, Fig. S13)—where the lack of bridging now signifies that phosphatase can rapidly access the 5′ phosphate which is crucial for PARP2 bridging.We also performed competition experiments between PARP2 and PARP1. In a second approach, we repeated the competition assay with PARP1 but this time held the ends in close proximity before solution exchange such that the PARP2 bridge could reform C. Here, In In summary, we have used a magnetic tweezers assay to measure the end-binding and bridging of PARP1 and PARP2 at DNA DSBs. Our assay allows us to perform highly controlled experiments using single-molecule manipulation to control the distance between two DSB ends and to measure the rupture force of proteins that bridge across the break. Our results confirm recent structural work revealing that PARP2 can form a bridged structure across 5′ phosphorylated blunt DNA ends . Moreove38In addition to measuring blunt-ended DNA, we tested the effect of dephosphorylating the 5′ end as well as DNA double-strand breakage that results in 5′ or 3′ overhangs. Our experiments show that PARP2 bridging is highly specific to 5′ phosphorylated DNA. Interestingly, we measured a switch between binding and bridging for the same DNA sequence overhang depending on whether it was in the 5′ or 3′ direction. We also observed multiple peaks in rupture force histograms in the presence of PARP2 in the context of different overhang types. These measurements show a rich variety of interaction modes depending on the exact configuration of the DNA break. The observation that short 3′ overhangs also show stable PARP2 bridging indicates that it might be an important feature during end-resection processes.Our experiments also revealed that PARP1 did not form a bridge linking together the two ends and it competed away PARP2 bridge formation. This observation reveals a notable difference in activity between the two proteins at DSBs. Currently, clinically approved PARP inhibitors target both PARP1 and PARP2—with more specific PARP1 inhibitors under development . LiteratFinally, the PEG–DNA synthesis and force-spectroscopy platform we have established has potential for the study of other proteins involved in DNA DSB repair. In the context of PARP biochemistry, our approach could be extended to test for small-molecule inhibitor effects and examine how other proteins compete for binding. More broadly, the technique could be used to study a wide range of protein complexes that are known to bind across DNA ends such as those involved in nonhomologous end-joining and microhomology-mediated end joining.SI Appendix, Fig. S14 for further details). The PEG–DNA construct synthesis is described in detail in SI Appendix, Figs. S1–S3. Briefly, a 20 kDa bis-azide PEG was reacted overnight with a Dibenzocyclooctyne (DBCO) modified oligonucleotide before agarose gel purification. The full-length DNA construct was then generated in a golden-gate assembly reaction  affinity and size-exclusion chromatography  phosphine (TCEP), 1 mg/mL Bovine Serum Albumin (BSA), and 1 mg/mL β-casein. NruI and DrdI cleavage was performed in 1× CutSmart buffer New England Biolabs (NEB) supplemented with 1 mg/mL BSA, 1 mg/mL β-casein. Tests of T4 DNA ligase  were incubated with gel-purified DNA constructs before introducing into the flow-cell. The beads were imaged by collimated LED illumination  with a 40× or 60× Nikon oil-immersion objective and a monochrome camera . We used a pair of 5 mm × 5 mm × 5 mm N50 cube magnets  in one of the following two configurations: 1) with a 2-mm gap, mounted on a rotating motor to enable DNA supercoiling, and 2) with a 0.3-mm gap. The magnets used for each experiment and calibration methods are described in lgorithm  with cusA ligase  were perAppendix 01 (PDF)Click here for additional data file.


Supplementary Figures 6M, N. The sample correlation clustering was performed using “promoter activity” instead of “WGBS”. The correct material statement appears below.In the published article, there was an error in the legend for (M) Heatmap shows the correlation of three pairs of promoter activity in GSE70091.(N) Heatmap shows the better performance correlation of two pairs of promoter activity in GSE70091 after removing N3 and T3 pairs.RABGAP1L” was mistakenly written as “RABGAPL1”.In the published article, there was a typographical error. The gene name “Results,The Methylation Regulated APs Could Be Used as Tumor Diagnostic Markers, Paragraph 1. This sentence previously stated:A correction has been made to TNFRSF10, prmtr.32651 of RGS3, prmtr.36049 of CCDC150, and prmtr.5237 of RASSF1) and two downregulated mrAPs (prmtr.37640 of TACC1 and prmtr.39585 of RABGAPL1) by promoter activities .”“The six mrAPs were clustered into four upregulated mrAPs  and two downregulated mrAPs (prmtr.37640 of TACC1 and prmtr.39585 of RABGAP1L) by promoter activities .”“The six mrAPs were clustered into four upregulated mrAPs  for the publication of any potentially identifiable images or data included in this article” with the following statement.“The studies involving humans were approved by the Ethics Committee of Guangxi Medical University Cancer Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.”The authors apologize for these errors and amendments and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.